Wild Crop Relatives: Genomic and Breeding Resources: Temperate Fruits

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Wild Crop Relatives: Genomic and Breeding Resources

.

Chittaranjan Kole Editor

Wild Crop Relatives: Genomic and Breeding Resources Temperate Fruits

Editor Prof. Chittaranjan Kole Director of Research Institute of Nutraceutical Research Clemson University 109 Jordan Hall Clemson, SC 29634 [email protected]

ISBN 978-3-642-16056-1 e-ISBN 978-3-642-16057-8 DOI 10.1007/978-3-642-16057-8 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2011922649 # Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Dedication

Dr. Norman Ernest Borlaug,1 the Father of Green Revolution, is well respected for his contributions to science and society. There was or is not and never will be a single person on this Earth whose single-handed service to science could save millions of people from death due to starvation over a period of over four decades like Dr. Borlaug’s. Even the Nobel Peace Prize he received in 1970 does not do such a great and noble person as Dr. Borlaug justice. His life and contributions are well known and will remain in the pages of history of science. I wish here only to share some facets of this elegant and ideal personality I had been blessed to observe during my personal interactions with him. It was early 2007 while I was at the Clemson University as a visiting scientist one of my lab colleagues told me that “somebody wants to talk to you; he appears to be an old man”. I took the telephone receiver casually and said hello. The response from the other side was – “I am Norman Borlaug; am I talking to Chitta?” Even a million words would be insufficient to define and depict the exact feelings and thrills I experienced at that moment!

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The photo of Dr. Borlaug was kindly provided by Julie Borlaug (Norman Borlaug Institute for International Agriculture, Texas A&M Agriculture) the granddaughter of Dr. Borlaug.

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I had seen Dr. Borlaug only once, way back in 1983, when he came to New Delhi, India to deliver the Coromandal Lecture organized by Prof. M.S. Swaminathan on the occasion of the 15th International Genetic Congress. However, my real interaction with him began in 2004 when I had been formulating a 7-volume book series entitled Genome Mapping and Molecular Breeding in Plants. Initially, I was neither confident of my ability as a series/book editor nor of the quality of the contents of the book volumes. I sent an email to Dr. Borlaug attaching the table of contents and the tentative outline of the chapters along with manuscripts of only a few sample chapters, including one authored by me and others, to learn about his views as a source of inspiration (or caution!) I was almost sure that a person of his stature would have no time and purpose to get back to a small science worker like me. To my utter (and pleasant) surprise I received an email from him that read: “May all Ph.D.’s, future scientists, and students that are devoted to agriculture get an inspiration as it refers to your work or future work from the pages of this important book. My wholehearted wishes for a success on your important job”. I got a shot in my arm (and in mind for sure)! Rest is a pleasant experience – the seven volumes were published by Springer in 2006 and 2007, and were welcome and liked by students, scientists and their societies, libraries, and industries. As a token of my humble regards and gratitude, I sent Dr. Borlaug the Volume I on Cereals and Millets that was published in 2006. And here started my discovery of the simplest person on Earth who solved the most complex and critical problem of people on it – hunger and death. Just one month after receiving the volume, Dr. Borlaug called me one day and said, “Chitta, you know I cannot read a lot now-a-days, but I have gone through only on the chapters on wheat, maize and rice. Please excuse me. Other chapters of this and other volumes of the series will be equally excellent, I believe”. He was highly excited to know that many other Nobel Laureates including Profs. Arthur Kornberg, Werner Arber, Phillip Sharp, G€ unter Blobel, and Lee Hartwell also expressed generous comments regarding the utility and impact of the book series on science and the academic society. While we were discussing many other textbooks and review book series that I was editing at that time, again in my night hours for the benefit of students, scientists, and industries, he became emotional and said to me, “Chitta, forget about your original contributions to basic and applied sciences, you deserved Nobel Prize for Peace like me for providing academic foods to millions of starving students and scientists over the world particularly in the developing countries. I will recommend your name for the World Food Prize, but it will not do enough justice to the sacrifice you are doing for science and society in your sleepless nights over so many years. Take some rest Chitta and give time to Phullara, Sourav and Devleena” (he was so particular to ask about my wife and our kids during most of our conversations). I felt honored but really very ashamed as I am aware of my almost insignificant contribution in comparison to his monumental contribution and thousands of scientists over the world are doing at least hundred-times better jobs than me as scientist or author/editor of books! So, I was unable to utter any words for a couple of minutes but realized later that he must been too affectionate to me and his huge affection is the best award for a small science worker as me! In another occasion he wanted some documents from me. I told him that I will send them as attachments in emails. Immediately he shouted and told me: “You know, Julie (his granddaughter) is not at home now and I cannot check email myself. Julie does this for me. I can type myself in type writer but I am not good in computer. You know what, I have a xerox machine and it receives fax also. Send me

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the documents by fax”. Here was the ever-present child in him. Julie emailed me later to send the documents as attachment to her as the ‘xerox machine’ of Dr. Borlaug ran out of ink! Another occasion is when I was talking with him in a low voice, and he immediately chided me: “You know that I cannot hear well now-a-days; I don’t know where Julie has kept the hearing apparatus, can’t you speak louder?” Here was the fatherly figure who was eager to hear each of my words! I still shed tears when I remember during one of our telephone conversations he asked: “You know I have never seen you, can you come to Dallas in the near future by chance?” I remember we were going through a financial paucity at that time and I could not make a visit to Dallas (Texas) to see him, though it would have been a great honor. In late 2007, whenever I tried to talk to Dr. Borlaug, he used to beckon Julie to bring the telephone to him, and in course of time Julie used to keep alive all the communications between us when he slowly succumbed to his health problems. The remaining volumes of the Genome Mapping and Molecular Breeding in Plants series were published in 2007, and I sent him all the seven volumes. I wished to learn about his views. During this period he could not speak and write well. Julie prepared a letter based on his words to her that read: “Dear Chitta, I have reviewed the seven volumes of the series on Genome Mapping and Molecular Breeding in Plants, which you have authored. You have brought together genetic linkage maps based on molecular markers for the most important crop species that will be a valuable guide and tool to further molecular crop improvements. Congratulations for a job well done”. During one of our conversations in mid-2007, he asked me what other book projects I was planning for Ph.D. students and scientists (who had always been his all-time beloved folks). I told him that the wealth of wild species already utilized and to be utilized for genetic analysis and improvement of domesticated crop species have not been deliberated in any book project. He was very excited and told me to take up the book project as soon as possible. But during that period I had a huge commitment to editing a number of book volumes and could not start the series he was so interested about. His sudden demise in September 2009 kept me so morose for a number of months that I could not even communicate my personal loss to Julie. But in the meantime, I formulated a 10-volume series on Wild Crop Relatives: Genomic and Breeding Resources for Springer. And whom else to dedicate this series to other than Dr. Borlaug! I wrote to Julie for her formal permission and she immediately wrote me: “Chitta, Thank you for contacting me and yes I think my grandfather would be honored with the dedication of the series. I remember him talking of you and this undertaking quite often. Congratulations on all that you have accomplished!” This helped me a lot as I could at least feel consoled that I could do a job he wanted me to do and I will always remain grateful to Julie for this help and also for taking care of Dr. Borlaug, not only as his granddaughter but also as the representative of millions of poor people from around the world and hundreds of plant and agricultural scientists who try to follow his philosophy and worship him as a father figure. It is another sad experience of growing older in life that we walk alone and miss the affectionate shadows, inspirations, encouragements, and blessings from the fatherly figures in our professional and personal lives. How I wish I could treat my next generations in the same way as personalities like Mother Teresa and Dr. Norman Borlaug and many other great people from around the world treated me!

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Dedication

During most of our conversations he used to emphasize on the immediate impact of research on the society and its people. A couple of times he even told me that my works on molecular genetics and biotechnology, particularly of 1980s and 1990s, have high fundamental importance, but I should also do some works that will benefit people immediately. This advice elicited a change in my thoughts and workplans and since then I have been devotedly endeavoring to develop crop varieties enriched with phytomedicines and nutraceuticals. Borlaug influenced both my personal and professional life, particularly my approach to science, and I dedicate this series to him in remembrance of his great contribution to science and society and for all his personal affection, love and blessings for me. I emailed the above draft of the dedication page to Julie for her views and I wish to complete my humble dedication with great satisfaction with the words of Julie who served as the living ladder for me to reach and stay closer to such as great human being as Dr. Borlaug and express my deep regards and gratitude to her. Julie’s email read: “Chitta, Thank you for sending me the draft dedication page. I really enjoyed reading it and I think you captured my grandfather’s spirit wonderfully. . .. So thank you very much for your beautiful words. I know he would be and is honored”. Clemson, USA

Chittaranjan Kole

Preface

Wild crop relatives have been playing enormously important roles both in the depiction of plant genomes and the genetic improvement of their cultivated counterparts. They have contributed immensely to resolving several fundamental questions, particularly those related to the origin, evolution, phylogenetic relationship, cytological status and inheritance of genes of an array of crop plants; provided several desirable donor genes for the genetic improvement of their domesticated counterparts; and facilitated the innovation of many novel concepts and technologies while working on them directly or while using their resources. More recently, they have even been used for the verification of their potential threats of gene flow from genetically modified plants and invasive habits. Above all, some of them are contributing enormously as model plant species to the elucidation and amelioration of the genomes of crop plant species. As a matter of fact, as a student, a teacher, and a humble science worker I was, still am and surely will remain fascinated by the wild allies of crop plants for their invaluable wealth for genetics, genomics and breeding in crop plants and as such share a deep concern for their conservation and comprehensive characterization for future utilization. It is by now a well established fact that wild crop relatives deserve serious attention for domestication, especially for the utilization of their phytomedicines and nutraceuticals, bioenergy production, soil reclamation, and the phytoremediation of ecology and environment. While these vastly positive impacts of wild crop relatives on the development and deployment of new varieties for various purposes in the major crop plants of the world agriculture, along with a few negative potential concerns, are envisaged the need for reference books with comprehensive deliberations on the wild relatives of all the major field and plantation crops and fruit and forest trees is indeed imperative. This was the driving force behind the inception and publication of this series. Unlike the previous six book projects I have edited alone or with co-editors, this time it was very difficult to formulate uniform outlines for the chapters of this book series for several obvious reasons. Firstly, the status of the crop relatives is highly diverse. Some of them are completely wild, some are sporadically cultivated and some are at the initial stage of domestication for specific breeding objectives recently deemed essential. Secondly, the status of their conservation varies widely: some have been conserved, characterized and utilized; some have been eroded completely except for their presence in their center(s) of origin; some are at-risk or endangered due to genetic erosion, and some of them have yet to be explored. The third constraint is the variation in their relative worth, e.g. as academic model, breeding resource, and/or potential as “new crops”. ix

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The most perplexing problem for me was to assign the chapters each on a particular genus to different volumes dedicated to crop relatives of diverse crops grouped based on their utility. This can be exemplified with Arabidopsis, which has primarily benefited the Brassicaceae crops but also facilitated genetic analyses and improvement in crop plants in other distant families; or with many wild relatives of forage crops that paved the way for the genetic analyses and breeding of some major cereal and millet crops. The same is true for wild crop relatives such as Medicago truncatula, which has paved the way for in-depth research on two crop groups of diverse use: oilseed and pulse crops belonging to the Fabaceae family. The list is too long to enumerate. I had no other choice but to compromise and assign the genera of crop relatives in a volume on the crop group to which they are taxonomically the closest and to which they have relatively greater contributions. For example, I placed the chapter on genus Arabidopsis in the volume on oilseeds, which deals with the wild relatives of Brassicaceae crops amongst others. However, we have tried to include deliberations pertinent to the individual genera of the wild crop relatives to which the chapters are devoted. Descriptions of the geographical locations of origin and genetic diversity, geographical distribution, karyotype and genome size, morphology, etc. have been included for most of them. Their current utility status – whether recognized as model species, weeds, invasive species or potentially cultivable taxa – is also delineated. The academic, agricultural, medicinal, ecological, environmental and industrial potential of both the cultivated and/or wild allied taxa are discussed. The conservation of wild crop relatives is a much discussed yet equally neglected issue albeit the in situ and ex situ conservations of some luckier species were initiated earlier or are being initiated now. We have included discussions on what has happened and what is happening with regard to the conservation of the crop relatives, thanks to the national and international endeavors, in most of the chapters and also included what should happen for the wild relatives of the so-called new, minor, orphan or future crops. The botanical origin, evolutionary pathway and phylogenetic relationship of crop plants have always attracted the attention of plant scientists. For these studies morphological attributes, cytological features and biochemical parameters were used individually or in combinations at different periods based on the availability of the required tools and techniques. Access to different molecular markers based on nuclear and especially cytoplasmic DNAs that emerged after 1980 refined the strategies required for precise and unequivocal conclusions regarding these aspects. Illustrations of these classical and recent tools have been included in the chapters. Positioning genes and defining gene functions required in many cases different cytogenetic stocks, including substitution lines, addition lines, haploids, monoploids and aneuploids, particularly in polyploid crops. These aspects have been dealt in the relevant chapters. Employment of colchiploidy, fluorescent or genomic in situ hybridization and Southern hybridization have reinforced the theoretical and applied studies on these stocks. Chapters on relevant genera/species include details on these cytogenetic stocks. Wild crop relatives, particularly wild allied species and subspecies, have been used since the birth of genetics in the twentieth century in several instances such as studies of inheritance, linkage, function, transmission and evolution of genes. They have been frequently used in genetic studies since the advent of molecular markers. Their involvement in molecular mapping has facilitated the development of mapping

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populations with optimum polymorphism to construct saturated maps and also illuminating the organization, reorganization and functional aspects of genes and genomes. Many phenomena such as genomic duplication, genome reorganization, self-incompatibility, segregation distortion, transgressive segregation and defining genes and their phenotypes have in many cases been made possible due to the utilization of wild species or subspecies. Most of the chapters contain detailed elucidations on these aspects. The richness of crop relatives with biotic and abiotic stress resistance genes was well recognized and documented with the transfer of several alien genes into their cultivated counterparts through wide or distant hybridization with or without employing embryo-rescue and mutagenesis. However, the amazing revelation that the wild relatives are also a source of yield-related genes is a development of the molecular era. Apomictic genes are another asset of many crop relatives that deserve mention. All of these past and the present factors have led to the realization that the so-called inferior species are highly superior in conserving desirable genes and can serve as a goldmine for breeding elite plant varieties. This is particularly true at a point when natural genetic variability has been depleted or exhausted in most of the major crop species, particularly due to growing and promoting only a handful of so-called high-yielding varieties while disregarding the traditional cultivars and landraces. In the era of molecular breeding, we can map desirable genes and polygenes, identify their donors and utilize tightly linked markers for gene introgression, mitigating the constraint of linkage drag, and even pyramid genes from multiple sources, cultivated or wild taxa. The evaluation of primary, secondary and tertiary gene pools and utilization of their novel genes is one of the leading strategies in present-day plant breeding. It is obvious that many wide hybridizations will never be easy and involve near-impossible constraints such as complete or partial sterility. In such cases gene cloning and gene discovery, complemented by intransgenic breeding, will hopefully pave the way for success. The utilization of wild relatives through traditional and molecular breeding has been thoroughly enumerated over the chapters throughout this series. Enormous genomic resources have been developed in the model crop relatives, for example Arabidopsis thaliana and Medicago truncatula. BAC, cDNA and EST libraries have also been developed in some other crop relatives. Transcriptomes and metabolomes have also been dissected in some of them. However, similar genomic resources are yet to be constructed in many crop relatives. Hence this section has been included only in chapters on the relevant genera. In this book series, we have included a section on recommendations for future steps to create awareness about the wealth of wild crop relatives in society at large and also for concerns for their alarmingly rapid decrease due to genetic erosion. The authors of the chapters have also emphasized on the imperative requirement of their conservation, envisaging the importance of biodiversity. The importance of intellectual property rights and also farmers’ rights as owners of local landraces, botanical varieties, wild species and subspecies has also been dealt in many of the chapters. I feel satisfied that the authors of the chapters in this series have deliberated on all the crucial aspects relevant to a particular genus in their chapters. I am also very pleased to present many chapters in this series authored by a large number of globally reputed leading scientists, many of whom have contributed to the development of novel concepts, strategies and tools of genetics, genomics and breeding and/or pioneered the elucidation and improvement of particular plant

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genomes using both traditional and molecular tools. Many of them have already retired or will be retiring soon, leaving behind their legacies and philosophies for us to follow and practice. I am saddened that a few of them have passed away during preparation of the manuscripts for this series. At the same time, I feel blessed that all of these stalwarts shared equally with me the wealth of crop relatives and contributed to their recognition and promotion through this endeavor. I would also like to be candid with regard to my own limitations. Initially I planned for about 150 chapters devoted to the essential genera of wild crop relatives. However, I had to exclude some of them either due to insignificant progress made on them during the preparation of this series, my failure to identify interested authors willing to produce acceptable manuscripts in time or authors’ backing out in the last minute, leaving no time to find replacements. I console myself for this lapse with the rationale that it is simply too large a series to achieve complete satisfaction on the contents. Still I was able to arrange about 125 chapters in the ten volumes, contributed by nearly 400 authors from over 40 countries of the world. I extend my heartfelt thanks to all these scientists, who have cooperated with me since the inception of this series not only with their contributions, but also in some cases by suggesting suitable authors for chapters on other genera. As happens with a megaseries, a few authors had delays for personal or professional reasons, and in a few cases, for no reason at all. This caused delays in the publication of some of the volumes and forced the remaining authors to update their manuscripts and wait too long to see their manuscripts in published form. I do shoulder all the responsibilities for this myself and tender my sincere apologies. Another unique feature of this series is that the authors of chapters dedicated to some genera have dedicated their chapters to scientists who pioneered the exploration, description and utilization of the wild species of those genera. We have duly honored their sincere decision with equal respect for the scientists they rightly reminded us to commemorate. Editing this series was, to be honest, very taxing and painstaking, as my own expertise is limited to a few cereal, oilseed, pulse, vegetable, and fruit crops, and some medicinal and aromatic plants. I spent innumerable nights studying to attain the minimum eligibility to edit the manuscripts authored by experts with even life-time contributions on the concerned genera or species. However, this indirectly awakened the “student-for-life” within me and enriched my arsenal with so many new concepts, strategies, tools, techniques and even new terminologies! Above all, this helped me to realize that individually we know almost nothing about the plants on this planet! And this realization strikingly reminded me of the affectionate and sincere advice of Dr. Norman Borlaug to keep abreast with what is happening in the crop sciences, which he used to do himself even when he had been advised to strictly limit himself to bed rest. He was always enthusiastic about this series and inspired me to take up this huge task. This is one of the personal and professional reasons I dedicated this book series to him with a hope that the present and future generations of plant scientists will share the similar feelings of love and respect for all plants around us for the sake of meeting our never-ending needs for food, shelter, clothing, medicines, and all other items used for our basic requirements and comfort. I am also grateful to his granddaughter, Julie Borlaug, for kindly extending her permission to dedicate this series to him. I started editing books with the 7-volume series on Genome Mapping and Molecular Breeding in Plants with Springer way back in 2005, and I have since

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edited many other book series with Springer. I always feel proud and satisfied to be a member of the Springer family, particularly because of my warm and enriching working relationship with Dr. Sabine Schwarz and Dr. Jutta Lindenborn, with whom I have been working all along. My special thanks go out to them for publishing this “dream series” in an elegant form and also for appreciating my difficulties and accommodating many of my last-minute changes and updates. I would be remiss in my duties if I failed to mention the contributions of Phullara – my wife, friend, philosopher and guide – who has always shared with me a love of the collection, conservation, evaluation, and utilization of wild crop relatives and has enormously supported me in the translation of these priorities in my own research endeavors – for her assistance in formulating the contents of this series, for monitoring its progress and above all for taking care of all the domestic and personal responsibilities I am supposed to shoulder. I feel myself alien to the digital world that is the sine qua non today for maintaining constant communication and ensuring the preparation of manuscripts in a desirable format. Our son Sourav and daughter Devleena made my life easier by balancing out my limitations and also by willingly sacrificing the spare amount of time I ought to spend with them. Editing of this series would not be possible without their unwavering support. I take the responsibility for any lapses in content, format and approach of the series and individual volumes and also for any other errors, either scientific or linguistic, and will look forward to receiving readers’ corrections or suggestions for improvement. As I mentioned earlier this series consists of ten volumes. These volumes are dedicated to wild relatives of Cereals, Millets and Grasses, Oilseeds, Legume Crops and Forages, Vegetables, Temperate Fruits, Tropical and Subtropical Fruits, Industrial Crops, Plantation and Ornamental Crops, and Forest Trees. This volume “Wild Crop Relatives – Genomic and Breeding Resources: Temperate Fruits” includes 11 chapters dedicated to Cydonia, Fragaria, Malus, Muscadiniana, Olea, Pistacia, Prunus, Pyrus, Rubus, Vaccinium, and Vitis. The chapters of this volume were authored by 27 scientists from 8 countries of the world, namely India, Italy, Japan, Portugal, Russia, Spain, UK, and the USA. It is my sincere hope that this volume and the series as a whole will serve the requirements of students, scientists and industries involved in studies, teaching, research and the extension of temperate fruit crops with an intention of serving science and society. Clemson, USA

Chittaranjan Kole

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Contents

1

Cydonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Richard L. Bell and Jose´ Manuel Leita˜o

2

Fragaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kim E. Hummer, Nahla Bassil, and Wambui Njuguna

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3

Malus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexander Ignatov and Anastasiya Bodishevskaya

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4

Muscadiniana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hemanth K.N. Vasanthaiah, D. Thangadurai, Sheikh M. Basha, Digambar P. Biradar, Devaiah Kambiranda, and Clifford Louime

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5

Olea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Rugini, C. De Pace, P. Gutie´rrez-Pesce, and R. Muleo

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6

Pistacia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 J.I. Hormaza and A. W€unsch

7

Prunus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Daniel Potter

8

Pyrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Richard L. Bell and Akihiro Itai

9

Rubus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 J. Graham and M. Woodhead

10

Vaccinium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Guo-Qing Song and James F. Hancock

11

Vitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Jaya R. Soneji and Madhugiri Nageswara-Rao

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

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.

Abbreviations

2,4-D AAMTR AAT AAT ACC ADH AMOVA Ana Ani AQP ARS AVG BA BAC BBDG BC1 BS BSA CAPS CATCN-PGR cDNA cM CoTFL1 CoTFL2 COX cpDNA DAPI DGAT DHAR DHFR DIA DN Dp-1 EMBL EMR ENP

2,4-Dichlorophenoxyacetic acid Average annual minimum temperature range Alcohol transferase Aspartate aminitransferase 1-Aminocyclopropane-1-carboxylate Alcohol dehydrogenase Analysis of molecular variance Allele for susceptibility to Alternaria alternata Allele for susceptibility to Alternaria alternate Aquaporin Agricultural Research Service (of USDA) Aminoethoxyvinyl glycine Benzyladenine Bacterial artificial chromosome The Blueberry Genomics Database First backcross generation Baron Solemacher Bulk segregant analysis Cleaved amplified polymorphic sequence Central Asian and Transcaucasian Network on Plant Genetic Resources Complementary DNA Centi-Morgan Cydonia oblonga homolog of terminal flower gene 1 Cydonia oblonga homolog of terminal flower gene 2 Cytochrome-C oxidase Chloroplast DNA 40 -6-Diamidino-2-phenylindole Diacylglycerol acyltransferase Dehydroascorbate reductase Dihydroflavonol reductase Diaphorase Day-neutral Dominant allele for resistance to Dysaphis pyri European Molecular Biology Laboratory East Malling Research Endopeptidase xvii

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Abbreviations

EST EUCPPGR EURISCO F1 F2 F3 FAO FAOSTAT FB Fe(III)EDTA FV FWt GA3 GBSSI gbssI/GBSSI GDR Gfp GM GOT GPH GRIN GSA GSI GSS Gus/GUS H Ha HiDRAS HPLC HR I IBA IBPGR IDH INRA ISSR ITS ITSR IUCN LAP LD LFY LG LTR M MAS MAT matK

Expressed sequence tag European Cooperative Program for Plant Genetic Resources European Network of ex situ National Plant Germplasm Inventories First filial generation Second filial generation Third filial generation Food and Agriculture Organization of the United Nations FAO Statistics Fragaria bucharica Ferric ethylenediaminetetraacetic acid Fragaria vesca Fresh weight Gibberellic acid-3 Granule-bound starch synthase I Granule bound starch synthase gene I Genome Database for Rosaceae Green fluorescent protein Genetic modification Glutamate oxaloacetate transaminase Gene pair haplotype Germplasm Resources Information Network (USA) Genome scanning approach Gametophytic self-incompatibility Genome survey sequences b-Glucuronidase Hapil Hectare High-quality disease resistant apples High performance liquid chromatography Hypersensitive reaction Russet-inhibiting gene in Pyrus pyrifolia Indole butyric acid International Board for Plant Genetic Resources Isocitrate dehydrogenase Institut National de la Recherche´ Agronomique Inter-simple sequence repeat Internal transcribed spacer Internal transcribed spacer region International Union for the Conservation of Nature Leucine aminopeptidase Linkage disequilibrium Leafy gene Linkage group Long terminal repeat Russet-modifying gene Marker-assisted selection Multi-auto-transformation Megakaryocyte-associated tyrosine kinase K

Abbreviations

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Mbp MFLP mRNA MT NAA NAD(P)H NCBI ndhF NPGS nptII NrITS ORAC PAT PCR PGD PGI Pgip PGM PIP PpACS PPFD PPO PpSFBB PRX/PX QTL R RAPD Rbc rbcL RefSeq RFLP RG RGA RGAP Ri Rol rolB rolC RS Rs-AFP2 RT-PCR SCAR SD Sdh SE SFBB S-genotype S-locus

Mega base pairs Microsatellite-anchored fragment length polymorphism Messenger RNA Million tons a-Naphthaleneacetic acid Nicotinamide dinucleotide phosphate (reduced) National Center for Biotechnology Information NAD(P)H dehydrogenase F National Plant Germplasm System Neomycin phosphotransferase II Nuclear internal transcribed spacer Oxygen radical absorbance capacity Phosphoinotricine acetyl transferase Polymerase chain reaction 6-Phosphogluconate dehydrogenase Phosphoglucoisomerase Polygalacturonase-inhibiting protein Phosphoglucomutase Plasma membrane intrinsic protein ACC synthase locus from Pyrus pyrifolia Photosynthetic photon flux density Polyphenol oxidase S-locus F-Box brother from Pyrus pyrifolia Peroxidase Quantitative trait loci Dominant allele for fruit russet in Pyrus pyrifolia Random(ly) amplified polymorphic DNA Ribulose 1,5-bisphosphate carboxylase large subunit Rubisco large subunit NCBI Reference Sequence database Restriction fragment length polymorphism Red Gauntlet Resistance gene analog RGA polymorphism Root-inducing plasmid Root loci Root locus gene B of Agrobacterium rhizogenes Root locus gene C of Agrobacterium rhizogenes Russian seedling Antifungal peptide 2 from Raphanus sativus Reverse transcriptase PCR Sequence-characterized amplified region Short-day Shikimate dehydrogenase Somatic embryogenesis S-locus F-box brothers Self-incompatibility genotype Self-incompatibility locus

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Abbreviations

SNP SOD SRAP S-RNase SSR STS TACy TC T-DNA TDZ TE tfGDR TFL1 TIGR TIP trnF trnL uidA USDA VIVC Vn Vnk VNTR

Single nucleotide polymorphism Superoxide dismutase Sequence-related amplified polymorphism Self-incompatibility RNase Simple sequence repeat Sequence tagged site Total anthocyanin content Tentative consensus Tumor-inducing (plasmid Ti) Thidiazuron Transposable element Tree fruit Genome Database Resources Terminal flower gene 1 The Institute for Genomic Research Tonoplast intrinsic protein Transfer RNA pseudogene F Transfer RNA pseudogene L b-Galacturonidase enzyme A United States Department of Agriculture Vitis International Variety Catalog Dominant allele for resistance to Venturia nashicola Dominant allele from the cultivar “Kinchaku” for resistance to Venturia nashicola Variable number tandem repeat

List of Contributors

Sheikh M. Basha Plant Biotechnology Laboratory, Center for Viticulture and Small Fruit Research, Florida A & M University, 6505 Mahan Drive, Tallahassee, FL 32317, USA, [email protected] Nahla Bassil USDA ARS, National Clonal Germplasm Repository, Corvallis, OR, 97333-2521, USA, [email protected] Richard L. Bell USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430, USA, [email protected] D.P. Biradar Department of Agronomy, University of Agricultural Sciences, Dharwad 580005, Karnataka, India, [email protected] Anastassia Boudichevskaia Julius Kuhn-Institut (JKI), Bundesforschungsinstitut f€ ur Kulturpflanzen, Institut f€ur Zuchtungsforschung an gartenbaulichen Kulturen und Obst, Pillnitzer Platz 3a D-01326 Dresden, Germany Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Gatersleben, Germany, [email protected] C. De Pace Dipartimento di Agrobiologia ed Agrochimica, Universita` degli Studi della Tuscia, Viterbo, Italy, [email protected] Julie Graham Department of Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK, [email protected] P. Gutie´rrez-Pesce Dipartimento di Produzione Vegetale, Universita` degli Studi della Tuscia, Viterbo, Italy James F. Hancock Plant Biotechnology Resource and Outreach Center, Michigan State University, East Lansing, MI 48824-1325, USA, [email protected] Jose I. Hormaza Instituto de Hortofruticultura Subtropical y Mediterra´nea “La Mayora”, Universidad de Ma´laga-Consejo Superior de Investigaciones Cientı´ficas (IHSM-UMA-CSIC) Estacio´n Experimental “La Mayora”, 29760 – Algarrobo-Costa, Ma´laga, Spain, [email protected] Kim Hummer USDA ARS National Clonal Germplasm Repository, Corvallis, OR, 97333-2521, USA, [email protected] Alexander Ignatov Molecular Phytopathology Group, Center ‘Bioengineering’, Russian Academy of Sciences, Moscow, 117312, Russian Federation, [email protected]

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Akihiro Itai Laboratory of Horticultural Science, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan, [email protected] Devaiah Kambiranda Plant Biotechnology Laboratory, Center for Viticulture and Small Fruit Research, Florida A & M University, 6505 Mahan Drive, Tallahassee, FL 32317, USA, [email protected] Jose´ Leita˜o BioFIG, FCT, Universidade do Algarve, Campus de Gambelas, 8005139 Faro, Portugal, [email protected] Clifford Louime College of Engineering Sciences, Technology and Agriculture, FAMU BioEnergy Group, Florida A&M University, 217 Perry-Paige Bldg, Tallahassee, FL 32307, USA, [email protected] R. Muleo Dipartimento di Produzione Vegetale, Universita` degli Studi della Tuscia, Viterbo, Italy, [email protected] Madhugiri Nageswara-Rao University of Florida, IFAS, Citrus Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA, [email protected] Wambui Njuguna Center for Ecology and Evolutionary Biology, 5289 University of Oregon, Eugene, OR 97403-5289, USA, [email protected] Daniel Potter Department of Plant Sciences, MS2, University of California, 1 Shields Avenue, Davis, CA 95616, USA, [email protected] E. Rugini Dipartimento di Produzione Vegetale, Universita` degli Studi della Tuscia, Viterbo, Italy, [email protected] Jaya R. Soneji University of Florida, IFAS, Citrus Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA, [email protected] Guo-Qing Song Plant Biotechnology Resource and Outreach Center, Michigan State University, East Lansing, MI 48824-1325, USA, [email protected] Devarajan Thangadurai Molecular Breeding Laboratory, Department of Botany, Karnatak University, Dharwad 580003, Karnataka, India, [email protected] Hemanth K.N. Vasanthaiah Plant Biotechnology Laboratory, Center for Viticulture and Small Fruit Research, Florida A & M University, 6505 Mahan Drive, Tallahassee, FL 32317, USA, [email protected] Mary Woodhead Department of Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK, [email protected] Ana W€ unsch Centro de Investigacio´n y Tecnologı´a Agroalimentaria de Arago´n, Avda, Montan˜ana 930, 50059, Zaragoza, Spain, [email protected]

List of Contributors

.

Chapter 1

Cydonia Richard L. Bell and Jose´ Manuel Leita˜o

1.1 Introduction The genus Cydonia is commonly known as quince, and the fruits are pomes, as the species is closely related to apples (Malus), pears (Pyrus), and Japanese quince (Chaenomeles). The name is derived from the name of a Greek city on the island of Crete, Cydonea, now called Canea (Sykes 1972). Quinces have been grown for their fruit for over 2,000 years, as Pliny the Elder described several cultivars in Historia Naturalis in 77 CE, and they are thought to have been cultivated in Mesopotamia (Webster 2008). The genus Cydonia is monospecific, that is, it is comprised of a single species, Cydonia oblonga Mill., and thus, almost all genomic and breeding resources are to be found in existing wild populations and cultivar forms. The exceptions are artificial intergeneric hybrids with apple (Malus domestica Borkh.) and Japanese pear (Pyrus pyrifolia [Burm. F.] Nakai). Therefore, much of the discussion of the role of crop relatives in the genomics, genetics, and breeding of quince will deal with these hybrids rather than intraspecific hybridization using Cydonia germplasm.

1.2 Basic Botany of the Genus Cydonia 1.2.1 Distribution and Geographic Locations of Genetic Diversity The genus is thought to have originated in northern Iran, Turkemenistan, and the Caucasus, including the R.L. Bell (*) USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV 25430, USA e-mail: [email protected]

countries of Armenia, Azerbaijan, and the Russian Federation. The current distribution includes the warm, temperate areas of central Asia, including the Kopet Dag and Gissar-Darvaz mountain ranges (Vavilov 1930, 1935; Bakhriddinov 1985), the Transcaucasus from Daghestan to Talysh, the Caucasus region, and throughout the Middle East, being particularly abundant and diverse in Iran (Khoshbakht and Hammer 2006; Amiri 2008) and Turkey (Ercisli 2004), with populations also in Syria (Thompson 1986), Turkmenistan, and Afghanistan (Frantskevich 1978; B€uttner 2001; Webster 2008). It is naturalized and cultivated elsewhere in western Asia and southern Europe, and is cultivated as Far North as England, but is particularly well-adapted to the Mediterranean climate and can be found up to 2,500 m above sea level (Bakhriddinov 1985). It is adapted to regions with an annual rainfall of more than 800 mm, with regular summer rains, being somewhat drought-sensitive because of a shallow root system. Optimum mean temperature should be about 15 C. The chilling requirement for bud-break is relatively low, ranging from 100 to 500 h. The species is moderately to highly tolerant of low soil pH, but high pH causes chlorosis due to poor uptake of iron.

1.2.2 Taxonomic Position Cydonia is a member of the subtribe Pyrinae, tribe Pyreae, subfamily Spiraeoideae (formerly Maloideae), family Rosaceae, order Rosales, class Magnoliopsida, and division Magnoliophyta. Sax (1931) concluded that the genera of the Maloideae form a closely related group based on the chromosome number and the presence of intergeneric hybrids. The species was formerly

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_1, # Springer-Verlag Berlin Heidelberg 2011

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R.L. Bell and J. Manuel Leita˜o

2 Table 1.1 Subspecies, botanical varieties and forma of Cydonia oblonga Mill Cydonia oblonga subsp. oblonga Cydonia oblonga subsp. integerrima Lobachev Cydonia oblonga subsp. maliformis (Mill.) Thell. Cydonia oblonga subsp. pyriformis Medik. ex Thell. Cydonia oblonga var. oblonga Cydonia oblonga var. biserrulata Kakhadze Cydonia oblonga var. integerrima Cydonia oblonga var. integerrimo-sepala Lobachev Cydonia oblonga var. integerrimoespala Kakhadze Cydonia oblonga var. lusitanica (Mill.) C. K. Schneid. Cydonia oblonga var. maliformis (Mill.) Rehder Cydonia oblonga var. obpyricarpa Lobachev Cydonia oblonga var. obpyriformis Lobachev Cydonia oblonga var. orbiculato-complanata Lobachev Cydonia oblonga var. ovalicarpa Lobachev Cydonia oblonga var. ovalis Lobachev Cydonia oblonga var. plano-cyclocarpa Lobachev Cydonia oblonga var. pomiformis Lobachev Cydonia oblonga var. rotundata Kakhadze Cydonia oblonga var. serrulata C. K. Schneid. Cydonia oblonga var. typica Kakhadze Cydonia oblonga var. urceolata Lobachev Cydonia oblonga forma lusitanica (Mill.) Rehder Cydonia oblonga forma marmorata (Dippel) C. K. Schneid. Cydonia oblonga forma pyramidalis (Dippel) Rehder Cydonia oblonga forma pyriformis (Dierb.) Rehder Source: Encyclopedia of Life (2009)

named Cydonia vulgaris Pers. and Pyrus cydonia L. Within the species, various botanical varieties, subspecies, or forma have been described (Table 1.1). Many of these subspecies, botanical varieties, and forma should probably not be considered as formal taxa but merely represent genetic variation within the species. Lobachev and Korovina (1981) divided the genus into two subspecies, C. oblonga subsp. oblonga and C. oblonga subsp. integerrima.

1.2.3 Cytology, Karyotype, and Genome Size Like apple and pear, quince is a diploid with 2n ¼ 34. The nuclear DNA content is 0.73 pg (Dickson et al. 1992). The basic number (n ¼ 17) of Cydonia is considered to be a secondary unbalanced number, and the genus is probably a secondary polyploid (Blando et al. 1992).

1.2.4 Morphology Botanical descriptions are found in Webster (2008) and the Encyclopedia of Life (2009). Quince forms a small tree or shrub, up to 8 m in height, with dense, spreading, and often pendulous tree architecture. Shoots are purplish-red when young, turning purplishbrown when mature, terete, initially densely tomentose when young but turning glabrous. Vegetative and flower buds are also purplish-brown and tomentose. Stipules are caduceus, ovate, and the petioles are 0.8–1.5 cm long and tomentose. Leaf blades are ovate to oblong 5–10 cm long and 3–5 cm wide, with veins conspicuous on the abaxial side. The adaxial side is glabrous or sparsely pubescent when young, and the abaxial side is pubescent. The leaf base is round or subcordate, with entire margins, and the apex is acute or emarginated. The flowers are 4–5 cm in diameter with caduceus, ovate bracts. They possess five styles, which are free and pubescent below, and 20 stamens, with the styles nearly as long as the stamens. The hypanthium is campanulate and abaxially tomentose. The nectaries are large in comparison with other closely related genera of the Pomoideae and possess the thickest epidermis and cuticle (WeryszkoChmiellewska and Konarska 1995). Sepals are ovate or broadly lanceolate. The petals are white or pinkish and about 1.8 cm long. The ovary is inferior and has five carpels. Floral initiation takes place immediately prior to anthesis (Webster 2008). The flowers are single and on the apices of current season shoots. The fruits are yellow when mature, densely tomentose, with persistent reflexed calyces, and can weigh over 0.5 kg. The pedicel tends to be about 5 cm long, thick, and also tomentose. The fruit is aromatic and the flesh is firm, containing many grit cells (i.e., sclerenchyma), which are large and irregular, similar to Pyrus (Aldasoro et al. 1998). Fruit shape can be either pyriform, globose, or maliform, with a ribbed contour in some genotypes.

1.2.5 Agricultural Status Quince is cultivated for fruit production all over the world. Nevertheless, most of the production is in the region where this fruit crop is supposed to have originated. In 2008, world production of quince totaled

1 Cydonia

478,813 metric tons (FAO 2010). Turkey was the most important producer, with 95,395 metric tons/year, followed by Uzbekistan (50,000), Iran (34,115), Morocco (33,133) Argentina (27,000), and Azerbaijan (26,990). Quince production is also considerable in the West Mediterranean region, where Spain (12,098) is the largest producer. This fruit crop is also produced in Mexico 6,473 mt. Compared with apples and pears, there are fewer documented cultivars (Yamamoto et al. 2004). Quinces are grown for their fruits, which need to be cooked to attain suitable texture for consumption. They have been made into marmalade, jelly, or conserves with various spices (Webster 2008). Liquors and wines can also be made. A major use for some quinces is as a rootstock for pear scion cultivars. Selected quince rootstocks impart size control, with a 40–50% reduction in tree size, precocious bearing, and improved productivity when compared to seedling and clonal pear (Pyrus communis L.) rootstocks (Lombard and Westwood 1987).

1.3 Conservation Initiatives 1.3.1 Evaluation of Genetic Erosion Cydonia has not been evaluated for inclusion on the International Union for the Conservation of Nature (IUCN) Red List. However, like most wild populations of fruit crops of the Rosaceae, it can be assumed that habitat destruction and development have caused some loss of these native populations. Avanzato and Raparelli (2005) analyzed genetic erosion of crop species as a function of the disappearance from nursery catalogs. Although the number of quince cultivars cultivated for their fruit is relatively small when compared with apples and pears, a comparatively high percentage (28%) of cultivars have apparently remained in the nursery trade during the period of 1897–2005. A relative dearth of modernization of quince cultivation, reflected in a low level of breeding activity, probably accounts for the relatively low level of genetic loss in production agriculture.

1.3.2 In Situ and Ex Situ Conservation Ex situ conservation of wild crop relative conservation has received much more emphasis than in situ

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conservation, except in a general sense of preservation of natural habitat for plants and animals. A 1989 survey listed 26 ex situ collections in 16 countries (International Board for Plant Genetic Resources 1989). Major ex situ germplasm collections resulting from plant exploration and exchange have been established in several countries, including Italy (Ianni and Mariotti 2005). USA (Postman 2008; USDA 2009), Italy (Ianni and Mariotti 2005) and UK (University of Reading 2009). Coordinated documentation of 11 collections in Italy, France, Spain, and Greece has been initiated (European Cooperative for Plant Genetic Resources 2009) as part of a European project on under-utilized fruit crop species (Bellini and Giordani 2000; European Commission 2007). At least 86% of the accessions in European collections have complete passport data and some characterization data. Ex situ collections in countries with native populations include those in India (Dhillon and Rana 2004), Iran (Amiri 2008), Turkey (Sykes 1972; K€uden 2001; K€uden and K€uden 2008), Turkmenistan (Vitkovskii and Denisov 1991), and Ukraine (Yezhov et al. 2005). A regional strategy for in situ and ex situ conservation and use of plant genetic resources for countries of central Asia and the Caucasus has been put forward (CATCN-PGR 2008). Similar plans have been devised for countries in eastern and central Europe (Alexanian 2001). The challenges of in situ conservation of wild crop relatives in general, either in protected or unprotected areas, have been discussed in detail by Heywood (2008).

1.3.3 Modes of Preservation and Maintenance Most ex situ preservation is in the form of clonally propagated trees. Clones preserved for their edible fruit are usually propagated by budding or grafting onto clonal quince rootstocks. The rootstocks can be also propagated onto specific clonal rootstocks, maintained in stool beds as self-rooted plants, or planted as self-rooted trees. In vitro methods of preservation are intended as secondary or back-up collections, established as a safeguard against loss of trees in nursery or orchard plantings due to disease, insect, or climatic hazards, e.g., due to low winter temperature. Medium-term storage of clonal propagules has been attained through

4

in vitro culture of shoots, and long-term storage has been achieved through cryopreservation of in vitro cultured apical meristems in liquid nitrogen. Mediumterm storage involves slowing growth through low temperature and medium manipulations. Viable cultures can be maintained for 12–18 months at 4 C with a 16-h photoperiod, and storage for over 2 years can be achieved using gas-permeable bags instead of glass culture tubes. Defoliated shoots have been stored for up to 4 years at 2 C on an agar medium without growth regulators (Druart 1985). The three major techniques of cryopreservation are slow freezing, vitrification, and encapsulation-dehydration (Reed and Chang 1997). Pre-treatment with cold-acclimation and abscissic acid has been shown to be very important for pear genotypes (Bell and Reed 2002), and the same is likely to be true for quince.

1.4 Elucidation of Origin and Evolution of Cydonia The most closely related genera and intergeneric hybrids are listed in Table 1.2. All species of Chaenomeles were once classified as species of Cydonia, as was Pseudocydonia sinensis and Pyronia. Classification is based on morphological and molecular genetic traits. Styles of Cydonia are free, and those of Docynia and Chaenomeles are connate; leaves are entire, as opposed to sometimes serrate. A study of pollen morphology detected considerable variation in number of pores, pollen grain shape, and exine structure, in addition to various abnormalities that were associated with fertility (Romanova et al. 1988). Pollen size, shape, and sculptural features may be useful in taxonomic studies within the Maloideae (Zhou et al. 2000). There are a few chemotaxonomic studies. Phenolic profiles for nine compounds for 36 genotypes from three geographical regions of Portugal did not reveal much polymorphism within the genus. Significant differences were found in 3-O-caffeoylquinic and 3,5O-dicaffeoylquinic acids among geographical provenance and date of leaf harvest (Oliveira et al. 2008). Within the species, early attempts at classification were strictly based on morphological traits, such as fruit shape (Hedrick 1925), or in the case of those

R.L. Bell and J. Manuel Leita˜o Table 1.2 Related crop and other plant genera and species Scientific Name Comment Chaenomeles cathayensis – (Hemsl.) C. K. Schneid. Chaenomeles japonica – (Thunb.) Lindl. Ex Spach Chaenomeles japonica f. alba – (Nakai) Ohwi Chaenomeles speciosa – (Sweet) Nakai Chaenomeles suberba C. japonica C. speciosa (Frahm) Rehder artificial hybrid Cydomalus (syn. Cydolus) Cydonia Malus pumila artificial hybrid (Rudenko 1989) Docynia delavayi (Franch.) Native to China C. K. Schneid. Docynia indica (Wall.) Decne. Native to China, IndoChina, and South Asia Pseudocydonia sinensis – (Thouin) C. K. Schneid. Pyronia veitchii (Trab.) Pyrus pyrifolia Cydonia Guillaumin oblonga artificial hybrid Pyronia veitchii var. – luxemburgiana

genotypes used as rootstocks, on the basis of plant growth habit (Tukey 1964). As with apple, pear, and other crops, early molecular studies of genetic diversity focused on isoenzyme polymorphisms. Isoenzyme analysis of acid phosphatase, esterase, peroxidase, and Polyphenol oxidase was successful in distinguishing 11 groups of Cydonia and two groups of Pyronia, but the diversity was much less than previously seen in Malus and Pyrus (Sanchez et al. 1988). Peroxidases have also been implicated in graft incompatibility between quince rootstocks and certain pear scion cultivars. Gulen et al. (2002) predicted that matching of isoperoxidase “A” in quince rootstocks in the graftcompatible “Beurre Hardy” scion may be associated with a compatible graft combination, and that the presence of isoperoxidase “A” and “B” in graft union tissues predicts compatibility between quince and “Bartlett” scions. Later, an association between rootstock/cultivar compatibility and a specific anodal isoperoxidase marker was identified by Gulen et al. (2005). Esterases and malate dehydrogenases were used by Hudina et al. (1999) to discriminate between eight European pear cultivars and the quince rootstock cultivars “Malling Quince A” and “BA29”. The authors speculate that an esterase band, common to

1 Cydonia

both quince varieties and to the pear cultivars used as interstocks, “Beurre´ Hardy” and “Vicar of Winkfield”, could be associated with rootstock/scion cultivar compatibility. Whereas Rudenko (1973) concluded that, based upon hybridization experiments, quince is closer to apple than pear, Iketani (1993) concluded on the basis of restriction fragment length polymorphisms (RFLPs) in chloroplast DNA that Cydonia was more closely related to Pyrus than to Malus and Chaenomeles. Analysis of sequences of internal transcribed spacers (ITS) and a part of the 5.8S nuclear ribosomal gene that were used to study the relationships among 19 Maloideae genera indicated that the Maloideae are not monophyletic, that there is considerable homoplasy, and that there is strong support for a close relationship between Cydonia and Pseudocydonia (Campbell et al. 1995). Supporting evidence for this close relationship was found by Kaneko et al. (2000) who confirmed by means of random amplified polymorphic DNA (RAPD) markers the closer genetic relationship between quince (Cydonia) and Chinese quince (Pseudocydonia) and their genetic distinctness from flowering quince (Chaenomeles), which could explain the failure of hybrid production between the former two species and flowering quince. No other studies using randomly amplified markers such as RAPD, inter-simple sequence repeats (ISSR), amplified fragment length polymorphisms (AFLP), etc. for quince genetic characterization have been published so far. However, Yamamoto et al. (2004) refer to the difficulty they found in discriminating between quince cultivars using RAPD markers due to the similar molecular patterns displayed by the varieties and used simple sequence repeat (SSR) markers transferred from apple and pear to discriminate between 20 quince cultivars. Fifty percent of the SSR markers derived from Japanese and European pears and 73% of the SSR markers derived from apple were successfully amplified in quince. On the whole, cultivars used as rootstock appeared as genetically distinct from varieties cultivated for fruit production, as seven of the analyzed eight rootstock clones clustered together in the resulting phenogram. Although most of the fruit cultivars were discriminated, some of them, such as “Portugal”, “Ac¸ucar”, and “Zairaishu”, showed identical molecular patterns. Using nucleotide sequence data from six nuclear (18S, gbssil, gbssi2, ITS, pgip, and ppo) and four chloroplast (matK, ndhF, rbcL, and trnL–trnF) regions,

5

Potter et al. (2007) proposed a new classification for Rosaceae in which Cydonia is included in the subfamily Spiraeoideae; supertribe Pyrodae; tribe Pyreae; subtribe Pyrinae (the long recognized subfamily Maloideae), with other fruit crops such as Malus, Pyrus, Mespilus, Eriobotrya, Crataegus, and Chaenomeles but genetically closer to Photinia and Pseudocydonia. The close genetic relationship of the genera included in the subtribe Pyrinae was already confirmed by Verbylaite˙ et al. (2006) through the analysis of the alignment of 775 nucleotide positions of the trnL–trnF chloroplast sequences. Nevertheless, there is a lack of consensus regarding the phylogeny of the subtribe Pyrinae, and the place of Cydonia in it is still the object of controversy as described by Aldasoro et al. (2005): “The group Pyrus–Cydonia–Pseudocydonia does not appear as monophyletic in the consensus tree, but is supported by a synapomorphic character: the large irregular groups of sclereids (character 6, state 2) and other: the pit surrounding the styles (character 4) which reverse in Pseudocydonia. Data from Campbell et al. (1995) showed a Cydonia–Pseudocydonia clade. Several authors suggested that Pyrus may have branched from the ancestor of Cydonia and Pseudocydonia before the latter two taxa acquired the pluriovulate condition (Iketani and Ohashi 1991; Aldasoro et al. 1998). Robertson et al. (1991) related Chaenomeles, Docynia, and Pseudocydonia to Malus, and Campbell et al. (1995) presented Chaenomeles, Heteromeles, Malus, Photinia, and Pyrus in the same clade, which is separated from that of Cydonia and Pseudocydonia. Our morphological data do not support any of these hypotheses, because Chaenomeles, Docynia, and Malus sect. Docyniopsis differ from Pyrus in having more than 40 stamens, and Docynia, Malus, and Malus sect. Docyniopsis differ from Pyrus in having phloridzin”.

1.5 Development of Cytogenetic Stocks and Their Utility There are no reports of cytogenetic stocks developed by using wild crop relatives per se in Cydonia. Triploid and tetraploid genotypes of Cydonia Malus intergeneric hybrids have been produced.

6

1.6 Classical and Molecular Genetic Studies 1.6.1 Classical Genetic Studies of Intergeneric Crosses Features of petiole anatomy in ten diploid, triploid, and tetraploid quince apple hybrids were intermediate between the two parental genera, but features of some were closer to one species than the other (Negru 1984). Flower number per inflorescence was inherited in an incompletely dominant fashion in F2 quince apple hybrids, with most having only a single blossom (Rudenko and Negru 1984). The results varied with the ploidy level of the hybrids. When a tetraploid clone was backcrossed to apple, useful sources of single-flowered inflorescences were selected. These tended to be allotriploids, which resembled quince. About 15% of the allotriploids resembled apples, had seedless fruits, and had mostly 2–3 blossoms per inflorescence (Rudenko and Rudenko 1994). In a study of 13 intergeneric hybrids, a combination of traits was found from both parental genera; transgressive segregation was found for thickness of the leaf blade, the palisade and mesophyll layers, and stomata size (Onika and Rudenko 1988). Seed anatomy was studied in diploid cultivars of apple, pear, and quince, in the allotetraploid F2 quince apple hybrids, and in the diploid pear quince hybrids (Rudenko and Rotaru 1988). The hybrids inherited testa epidermis structure from quince and sclerenchyma structure from either apple or pear. Other traits were quantitatively inherited. The content of hydrogen cyanide of F1 hybrids of quince pear was intermediate between the high cyanide quince and the low cyanide pear parents (Dilleman 1950) A quince hybrid derived by Panov et al. (1965) was shown to have seven leaf and eight shoot isoperoxidases, of which at least three of each appeared to be inherited from apple (Grushin et al. 1986).

1.6.2 Mapping of Genes and Polygenic Clusters No genetic linkage maps of intergeneric hybrids with quince have been produced. However, very dense

R.L. Bell and J. Manuel Leita˜o

genetic maps have been already constructed for apple (Liebhard et al. 2003; Naik et al. 2006) and pear (Yamamoto et al. 2007). Nevertheless, the high transferability of DNA markers from apple to pear (Yamamoto et al. 2001; Liebhard et al. 2002) and from apple and pear to Cydonia (Yamamoto et al. 2004), as well as the high level of genome synteny found between genera of the same subfamily, such as between apple (Malus) and pear (Pyrus) (Pierantoni et al. 2004; Yamamoto et al. 2007), or the relative synteny found between genomes belonging to different subfamilies, such as Prunus and Malus (Dirlewanger et al. 2004), in combination with the high intergeneric crossability of quince (e.g., Pyrus Cydonia) ensures all the conditions for the rapid construction of a reference genetic map of Cydonia. The progress registered in the construction of genomic libraries and genome physical maps of related genera as apple (Vinatzer et al. 1998; Han et al. 2007) constitute an additional resource for the rapid development of quince genomics.

1.6.3 Assessment of Gene Action The quince homologs of the Arabidopsis thaliana flower architecture-related genes Leafy (LFY) and TERMINAL FLOWER 1 (TFL1) have been isolated (Esumi et al. 2005). The LFY homologs have been shown to be expressed both in Japanese pear and quince throughout floral development and were upregulated during floral differentiation, but TFL1 homologs were initially highly expressed prior to floral differentiation and were dramatically reduced during floral differentiation (Esumi et al. 2007). In a subsequent study, the homologs of both genes were shown to be present, as expected in a genus of allopolyploid origin, in two copies (Esumi et al. 2008). Gene-specific reverse transcription-polymerase chain reaction (RT-PCR) indicated no difference between Japanese pear (P. pyrifolia Nakai) and quince or between homolog types in the expression pattern of LFY homologs. In contrast, differential expression of TFL1 homologs was evident between Japanese pear and quince. In quince, CoTFL1-1 was the only homolog detected prior to floral differentiation, but both CoTFL1-1 and CoTFL1-2 were expressed upon floral differentiation, mainly in the apical meristem. Once differentiation occurred into a single flower meristem,

1 Cydonia

expression could not be detected. There have been no similar studies in the intergeneric hybrids.

1.6.4 Synteny Because no genetic linkage maps have been constructed for Cydonia, the degree of synteny with other genera of the Rosaceae cannot yet be assessed.

1.7 Role in Crop Improvement Through Traditional and Advanced Tools 1.7.1 Intergeneric Hybridization, Traditional Breeding, and Introgression Breeding of quince for both fruit and rootstocks has been largely limited to intraspecific hybridization but with some notable exceptions. Intergeneric hybridization between C. oblonga, Pseudocydonia sinensis, and Chaenomeles sinensis has not been successful (Weber 1964), nor has an attempted cross between Cydonia and Sorbus (Kursakov et al. 1976), primarily due to failure to the pollen tubes to reach the embryo sacs. The first Pyrus Cydonia hybrid, Pyronia veitchii (Trabut) Guill; was produced before 1916 (Rudenko 1985; Trabut 1916) and presently is used as an indicator of virus diseases. An artificial hybrid of the Japanese pear Pyrus serotina and C. oblonga has been produced (Shimura et al. 1983). A clone of Pyronia veitchii var. luxemburgiana was backcrossed to pear in an attempt to produce a rootstock for pears (Rogers 1955). In addition, a number of genotypes of Cydomalus, an artificial hybrid genus of apple (Malus pumila) and quince (C. oblonga) (Rudenko and Rudenko 1994), have been produced in Russia. One hybrid set a large number of parthenocarpic fruit, which “combined” traits of the two parental species (Rudenko 1972). Pollination resulted in diploid, triploid, and tetraploid F2 hybrid seedlings (Rudenko 1974). Tetraploids in particular tended to have a low growth rate (Rudenko 1978). Two of the former were normal. The hybrids tended to have fruit similar to

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quince but with the flavor of apples (Rudenko 1983). One triploid F2 had large (230 g) fruit. Backcrossing an allotetraploid F2 with apple produced 50 mostly tetraploid hybrids with many traits of apple (Rudenko 1984). The allotetraploid “F3” hybrids were thought to be valuable in breeding for increased frost resistance and fruit quality (Rudenko 1987), presumably in a crop with quince-like fruit. Similar production of F1 hybrids was obtained in Bulgaria, where the quince cultivar “Champion” was pollinated with a mixture of five apple cultivars (Panov et al. 1965); two hybrids had fruit and other anatomical traits intermediate between the two parental species. In some cases, these hybrids were produced by pollinating with mixtures of “foreign” pollen plus pollen of the same species, which had been weakened by exposure to ionizing radiation (Semin 1961). Jakolev et al. (1968) also reported on the use of irradiation of pollen to obtain hybrids of pear quince and apple quince. Treatment of quince stigmas with 0.0005% gibberellic acid prior to a first pollination and 0.0001% boric acid prior to a second pollination resulted in improved fruit and seed set (Shcherbenev 1973); of 1,437 seedlings obtained when quince was the seed parent, 25 were later shown to be intergeneric hybrids (Shcherbenev 1974). Embryo culture and in vitro micropropagation have been used to improve the recovery and survival of Cydomalus intergeneric hybrids (Papikhin et al. 2007), the seedlings of which are generally weak and of low viability and germinability. Adventitious regeneration of plantlets from cotyledons has been attempted. Chromosome behavior varied among the hybrids, but in some crosses, normal meiosis with bivalent formation and a highly fertile hybrid was reported (Rudenko 1970), although in most seedlings, seeds are rarely formed (Rotaru et al. 1970). Among the anatomical features of these hybrids, the palisade layer of the leaves was much thinner (Rudenko 1962).

1.7.2 In Vitro Tissue Culture and Regeneration of Cydonia Although relatively timid advances have been made on molecular biology and genomic studies on quince (C. oblonga Mill.), plant biotechnology, particularly

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in vitro tissue culture and regeneration, constitutes one of the main research fields in this fruit crop. A complete review of the literature on in vitro culture of quince was compiled by Duron et al. (1989), who, based on previous work, particularly of Nemeth (1979), Druart (1980), Al Maarri et al. (1986), and their own results, established a general protocol for highly successful quince micropropagation. Aiming at studying the problem of graft incompatibility at the cellular level, Moore (1984) was able to induce callus from pear and quince. Protocols for callus induction and maintenance and subsequent shoot regeneration were delineated by Chartier-Hollis (1993) who found that as for pear, quince requires GA3 in addition to IBA and BA to sustain callogenesis. Although quince leaf protoplasts were successfully isolated by D’Onofrio et al. (1999), further studies using protoplast cultures, e.g., on somatic embryogenesis or genetic transformation, have not been performed. Very efficient in vitro adventitious shoot regeneration from cultured leaves of C. oblonga cv. Quince A was achieved in culture media containing thidiazuron (TDZ) and a-naphthaleneacetic acid (NAA) by Dolcet-Sanjuan et al. (1991). Shoots were rooted in medium containing 5 mM NAA for 1 week and then on auxin-free medium for 4 weeks. Thidiazuron (TDZ) in concentrations varying from 1.5 to 32 mM, usually in combination with NAA (0.3–2.5 mM), became a central component commonly used in the culture media for quince in vitro adventitious shoot regeneration (Dolcet-Sanjuan et al. 1991; Baker and Bhatia 1993; Staniene and Stanys 2004; D’Onofrio and Morini 2005; Erig and Schuch 2005). The role of genotype as a determinant factor in the success of shoot regeneration was observed by Aygun and Dumanoglu (2007), who obtained the highest shoot organogenesis in MS basal medium supplemented with TDZ + NAA and AgNO3. The effect of light regime on adventitious shoot regeneration of three Lithuanian quince clones was assessed by Zalunskaite et al. (2007), who found that an initial period of 4 weeks in the dark before transfer to a 16/8 h (light/ dark) photoperiod significantly increased shoot regeneration, which could be additionally augmented if leaf explants were sampled from the apical part of the shoots. Optimal temperature regimes for induction and regeneration phases were established by Morini et al. (2004), while the impact of the gaseous environment in regeneration ability was studied by

R.L. Bell and J. Manuel Leita˜o

Marino and Berardi (2004), who found that sealing petri dishes with polyvinyl chloride (PVC) transparent film resulted in much lower concentrations of carbon dioxide and ethylene and much better regeneration in comparison with petri dishes sealed with parafilm. The beneficial effect of ethylene reduction on regeneration ability via addition of aminoethoxyvinylglycine (AVG) was later found by Marino et al. (2008). The effect of NaCl and CaCl in somatic embryogenesis, root regeneration, and shoot regeneration was studied by D’Onofrio and Morini (2002a, b), who observed a favorable effect from low concentrations of NaCl in somatic embryo formation and adventitious shoot and root regeneration. CaCl apparently mitigated the adverse effect of higher concentrations of NaCl (and NaSO4) on the in vitro regeneration processes. A two-step procedure, induction in liquid media supplemented with 2,4-D for a few days followed by regeneration in gelled media for 35–40 days, was introduced by Antonelli (1995) for somatic embryogenesis induction. Using a similar induction treatment and varying the length of a subsequent kinetin + NAA treatment and the absence versus presence of growth regulators (BA, GA3, and IBA), D’Onofrio and Morini (2006) were able to modulate in vitro regeneration of somatic embryos and adventitious shoots and roots. The effect of macronutrients in the regeneration process was assayed by Fisichella et al. (2000), who tested eight different media frequently employed for in vitro plant tissue culture and observed that the most effective macroelement combination was that of MS medium. The effect of the composition of the culture vessel atmosphere on somatic embryogenesis and root formation was studied by Fisichella and Morini (2003), who concluded that some gaseous compounds other than O2 and CO2 may promote quince in vitro embryogenesis. Mingozzi and Morini (2009) observed higher embryogenesis and root regeneration in leaves from shoots previously cultured under high photosynthetic photon flux density (PPFD), evidence of the effect of in vitro culture conditions of donor shoots on subsequent morphogenesis of the leaf explants.

1.7.3 Somaclonal Variation Particularly due to its dwarfing properties, quince rootstocks are extensively used for intensive pear

1 Cydonia

production. Nevertheless, the use of quince as rootstock faces some major constraints, such as graft incompatibility with some pear cultivars, little tolerance to low temperatures, and lime-induced chlorosis due to iron deficiency, particularly important in calcareous soils. Although some in vitro studies were devoted to the selection for tolerance to stress factors as salinity (Marino and Molendini 2005), most of the in vitro selection work were dedicated to the identification of superior clones for tolerance to lime-induced chlorosis. Some studies were carried out by culturing shoots on nutrient media with low iron availability and/or supplemented with sodium or potassium bicarbonate (Dolcet-Sanjuan et al. 1990; Muleo et al. 1995, 2002); however, most of the in vitro selection research was aimed at the identification of superior clones among in vitro-obtained somaclonal variants. Two somaclonal variants, IE-1 and IE-2, were found by Dolcet-Sanjuan et al. (1992) to be more tolerant to iron-deficient in vitro conditions than the original clone “Quince A”, as evidenced by higher chlorophyll concentration and the ability of roots to reduce Fe3+ and to acidify the medium. Nevertheless, subsequent studies of selected clones showed that while young plants growing in the greenhouse confirmed the higher tolerance to iron deficiency, this tolerance was more variable under field conditions (Bunnag et al. 1996). More recently, new attempts have been made to recover lime-tolerant clones via somaclonal variation. Apparently, more tolerant somaclones have been obtained after regeneration in the presence of high medium pH (Marino et al. 2000) or under progressively lower concentrations of Fe(III)-EDTA combined with different concentrations of potassium bicarbonate (Cinelli et al. 2004). In all cases, the selected promising quince rootstock clones needed to be further confirmed in field trials as valuable alternatives to the presently used quince rootstock clones (e.g., BA 29).

1.7.4 Genetic Transformation There have been no reports of transformation of quince with genes isolated from related genera. Genetic transformation of quince is still in the early stage of development, with research concentrating on

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development or adoption of techniques. In vitro axillary shoot culture and adventitious regeneration methods are well established for Cydonia (Chevreau and Bell 2004). There have been few studies of either Agrobacteriummediated or biolistic genetic transformation. Two strains of A. tumefaciens, LBA4404 and EHA 101, were used to transform quince with the same expression vector containing the uidA (GUS) reporter gene and the nptII selectable marker (Davidson et al. 1998). GUS expression in treated leaves increased over an 8-day period following cocultivation. The intergeneric hybrid, Pyronia veitchii, was transformed with wild type Agrobacterium rhizogenes (Bassi and Cossio 1993). Chimeric plants with improved rooting were obtained. Because ease of adventitious rooting is an important trait for rootstocks, three quince rootstocks were transformed with the rolB gene isolated from A. rhizogenes using two plasmids and three gene promoters (Razanskiene et al. 2006). There was variability in the percentage of regeneration of transgenic shoots due to rootstock clone, plasmid, and concentration of thidiazuron in the regeneration medium. In another study, the percentage of rooted regenerants was increased when the rolB transgene was under the control of the native promoter (Staniene et al. 2007).

1.7.5 Trait Evaluation Different groups of quince are cultivated for their fruit and use as dwarfing rootstocks for pear. Therefore, depending on the end use, different sets of traits are important. For rootstock usage, the major traits are the ability to induce precocious bearing in the pear scion cultivar, induction of dwarf tree stature, high or efficient yields in relation to projected planting density, and large fruit size. Other abiotic and biotic stress traits include adaptation to high pH, i.e., calcareous, soils, susceptibility to the diseases pear decline, fire blight, crown gall, collar rot, the insect pest wooly pear aphid, and cold temperatures. Graft incompatibility with pear scions is also important as it leads to graft union necrosis and the resulting poor growth and graft union breakage. Rootability of softwood cuttings of F2 allotriploid and allotetraploid apple quince hybrids was reported to be high (Shcherbenev and Turovskaya

R.L. Bell and J. Manuel Leita˜o

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1977) and greater than that observed in the diploid F1 hybrids (Rudenko et al. 1984), although treatment with indole butyric acid was required for some clones (Stepanova et al. 1984). The triploid and tetraploid hybrids tended to have the most vigorous root systems

Table 1.3 Genomic resources in Cydonia Function Ammonium transporter

atpB–rbcL intergenic spacer Granule-bound starch synthase

Leafy protein NPRI-like protein

Maturase K

NADH dehydrogenase F Polyphenol oxidase 5.8S ribosomal RNA, internal transcribed spacer 1 Internal transcribed spacers 1 and 2 Ribosomal protein L16 Ribosomal protein S16 Ribulose-1,5 biphosphate carboxylkase/oxygenase large subunit TFL-like protein Transcription factor tRNA–Leu tRNA–Lys tRNA–Phe trnL–trnF intergenic spacer

(Rudenko 1981). These results suggest that the diploids may be the best rootstocks for evaluation of dwarfing potential. Pollen viability of the allotetraploid F2 hybrids varied but was shown to be 21–58% on artificial

Gene/allele Amt1-1 Amt1-2 Amt1-3 Amt1-4 Amt1-5 – c7.3F c53 GBSSI GBSSI CoLFY-1 CoLFY-2 co-42 co-44 co-45 co-46 co-48 co-49 co-50 co-1C2 co-1C4 co-2C4 co-2C6 co-2C10 matK

– – – – rpl16 rps16 rbcL

Genbank accession FJ860896 FJ860897 FJ860898 FJ860899 FJ860900 DQ860489 DQ874915 DQ874910 AF500415 AF500416 AB162031 AB162037 DQ149960 DQ149961 DQ149965 DQ149962 DQ149963 DQ149934 DQ149964 DQ146462 DQ149966 DQ149932 DQ149967 DQ152833 DQ860459 AF309195 AF309240 DQ851512 EU275350 AF186531 U16189 DQ860428 DQ848694 DQ860489

Literature reference – – – – – Campbell et al. (2007) Campbell et al. (2007) – Evans and Campbell (2002) Evans and Campbell (2002) Esumi et al. (2005) Esumi et al. (2005) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) Pilotti et al. (2008) – Robinson et al. (2001) Robinson et al. (2001) Potter et al. (2007) – Robinson et al. (2001) Campbell et al. (1995) Campbell et al. (2007) Campbell et al. (2007) Campbell et al. (2007)

CoTFL1-1 CoTFL1-2 R2R3 MYB10 trnL trnK trnF – –

AB162043 AB162049 EU153571 DQ863231 DQ860459 DQ863231 DQ863231 AM157398

Esumi et al. (2005) Esumi et al. (2005) – Campbell et al. (2007) Campbell et al. (2007) – – –

Source: National Center for Biotechnology Information (2009)

1 Cydonia

medium (Rudenko 1986). The morphology of the pollen grain and tube varied among the hybrids. The pollen grains were least uniform among the diploids, with a high incidence of sterility due to asynapsis at diakinesis, followed by the triploids (0.7–1.6% viability) and then the tetraploids (22–64% viability) (Rudenko 1987).

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1.10 Some Disadvantages of Cydonia Intergeneric Hybrids As noted above, viability, vigor, and fertility are major drawbacks to apple quince hybrids. When considering its use as rootstocks, graft compatibility may be inadequate and must also be investigated.

1.8 Genomic Resources

1.11 Recommendations for Future Action Genes and other DNA sequences isolated from Cydonia are given in Table 1.3. There are 42 entries for cloned partial or complete sequences. In addition, Genbank lists 22 corresponding mRNA sequences and 42 amino acid sequences for the corresponding proteins (National Center for Biotechnology Information 2009). A clear perspective of the state of the Cydonia genomics can be obtained by comparison of these data to the 1,040 nucleotide sequences, 777 expressed sequence tags (ESTs), and 733 proteinrelated items uploaded to NCBI database for the genus Pyrus. In addition, for both genera, there are no reported genome survey sequences (GSS), a consequence of the limited advance of the genomic studies, in general, and genome physical mapping, in particular.

1.9 Scope for Domestication and Commercialization Green (1983), as reported by Webster (2008), notes the use of the mucilaginous substance around the quince seeds in medicine and for a cosmetic. The fruit has also been used as a treatment for constipation in Iran (Khoshbakht and Hammer 2006). The apple quince hybrids may have potential as novel fruit types with some improved traits, notably cold hardiness superior to quince. Their potential as rootstocks for apple or pear has yet to be assessed adequately. The pear quince hybrid, Pyronia, has been used as an indicator for infection with quince sooty ringspot and vein yellows. It may have potential as a rootstock for pear, but this has also not been adequately investigated.

The utility of intergeneric apple quince hybrids as a novel fruit crop should be continued to be investigated. Fruit texture and acceptability of these fruit by consumers needs further research. The investigation of the utility of intergeneric pear quince hybrids as rootstocks for pear should also be continued, paying particular attention to graft compatibility.

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R.L. Bell and J. Manuel Leita˜o Pilotti M, Brunetti A, Gallelli A, Loreti S (2008) NPR1-like genes from cDNA of rosaceous trees: cloning strategy and genetic variation. Tree Genet Genom 4:49–63 Postman JD (2008) The USDA quince and pear genebank in Oregon, a world source of fire blight resistance. Acta Hortic 793:357–362 Potter D, Eriksson T, Evans RC, Oh S, Smedmark JEE, Morgan DR, Kerr M, Robertson KR, Arsenault M, Dickinson TA, Campbell CS (2007) Phylogeny and classification of Rosaceae. Plant Syst Evol 266:5–43 Razanskiene A, Staniene G, Rugienius R, Gelvonauskiene D, Zalunskaite I, vinshiene J, Stanys V (2006) Transformation of quince (Cydonia oblonga) with the rolB gene-based constructs under different promoters. J Fruit Ornament Plant Res 14(Suppl 1):95–102 Reed BM, Chang Y (1997) Medium- and long-term storage of in vitro cultures of temperate fruit and nut crops. In: Razdan MK, Cocking EC (eds) Conservation of plant genetic resources in vitro, vol 1. Science, Enfield, NH, pp 67–105 Robertson KR, Phipps JB, Rohrer JR, Smith PG (1991) A synopsis of genera in Maloideae (Rosaceae). Syst Bot 16:376–394 Robinson JP, Harris SA, Juniper BE (2001) Taxonomy of the genus Malus Mill. (Rosaceae) with emphasis on the cultivated apple, Malus domestica Borkh. Plant Syst Evol 226:35–58 Rogers WS (1955) Pomology. In: Annual report of the East Malling Research Station, 01 Oct 1954 to 30 Sept 1954, pp 20–27 Romanova GS, Duganova EA, Khrolikova AK (1988) Cytomorpological analysis of quince pollen. Byulleten’ Gosudarstvennogo Nikiskogo Botanicheskogo Sada 65:102–106 (in Russian) Rotaru GI, Rudenko IS, Dudukal GD (1970) Morphological and anatomical characters of an intergeneric quince apple hybrid. Strukturn osobennosti sochn i myasist plodov 1970:51–60 (in Russian) Rudenko IS (1962) Anatomical structure of the leaves of the intergeneric hybrid apple quince and its parents. Izv akad Nauk SSSR News Acad Sci USSR Ser Biol 1962(12):32–36 Rudenko IS (1970) Hybrids between Cydonia oblonga and cultivated apple. Otdalennaya gibridiz rast i zhivotnykh 1970(2):55–61 (in Russian) Rudenko IS (1972) Morphogenesis in the intergeneric hybridization of fruit crops, exemplified by crosses of quince with apple. Metody selekstii s kh rast v Moldavii 1972:86–97 (in Russian) Rudenko IS (1973) On the genetic relationship between apple, pear, and quince. Bul Akad stiince Mold RSS Ser Biol Khim N 1973(4):81–82 (in Russian) Rudenko IS (1974) Cytogenetic principles of intergeneric hybridization in fruit crops. Bul Akad Stiince RSS Mold ser biol i khim 1974(3):43–46 (in Russian) Rudenko IS (1978) The results of a cytogenetical study of intergeneric hybrids of fruit crops and their progeny. Selektsiya i tekhnol vyrashchivaniya plodov kul’tur 1978:53–59 (in Russia) Rudenko IS (1981) Features of root formation in F2 quince apple seedlings of different ploidy. Bul Akad stiince RSS Old Ser biol i khim n 1981(2):82–85 (in Russian) Rudenko IS (1983) New intergeneric apple quince forms (Cydolus). Sadovodstvo 1983(10):29–31 (in Russian)

1 Cydonia Rudenko IS (1984) Producing a new fruit crop, quince apple (Cydolus). Geneticheskie osnovy selektsii sel’skokhozyaistvennykh rastenii I zhivotnykh 1984:57–58 (in Russian) Rudenko IS (1985) Hybrid between pear and quince (Pyronia). Sadovodstvo Vinogradarstvo I Vinodelie Moldavii 1985(10):55–57 (in Rusian) Rudenko IS (1986) Importance of quince apple hybrids in accelerating the breeding of regularly bearing apple varieties with single-flowered infloresences. Zadachi i sovremennye mtody seleksii plodovykh I yagodnykh kul’tur Materialy Vsesoyuznogo soveshchaniya, Erevan, Armenian SSR, 4–6 Iyulya 1985, pp 25–29 (in Russian) Rudenko IS (1987) Aspects of morphology and pollen viability in F2 quince apple hybrids with different genomes in relation to disturbances in microsporogenesis. Gametnaya i zygotnaya selektsiya Respublikanskaya konferentsiya, 23 Iyunya, 1986. Stiinca. Kishinev, Moldavian SSR, pp 102–106 (in Russian) Rudenko IS (1989) Cydolus – a new pome fruit crop. Botanicheskii gibridizatsiya I ee rol’ v intensifikatsii sadovodstva 1989:18–27 (in Russian) Rudenko IS, Negru EI (1984) Patterns of inheritance of flower number/inflorescense in quince apple hybrids of different genomic composition. Geneticheskie osnovy seleksii sel’ skokhozyaistevennykh rastenii i zhivotnykh. p 58 (in Russian) Rudenko IS, Rotaru GI (1988) Morphological and anatomical features of the seeds of hybrid forms of apple, pear and quince. Izvestiya Akademii Nauk Moldavskoi SSR Biolicheskie I Khimicheskie Nauki 1988(5):15–20 (in Russian) Rudenko IS, Rudenko II (1994) Genotypic variation in apple quince progenies. Progress in temperate fruit breeding: Proc EUCARPIA fruit breeding section meet, Wadenswil/ Einsiedeln, Switzerland, 30 Sept to 3 Oct 1993. Kluwer, Dordrecht, Netherlands, pp 229–233 Rudenko IS, Stepanova AF, Yaroshenko BA (1984) A study of F2 quince apple hybrids as new rootstocks for pome fruit crops. Byulleten’ gosudarstvennogo Nikitskogo Botanicheskogo Sada 1984(54):31–36 (in Russian) Sanchez EE, Mendez RA, Daly LS, Boone RB, Jahn OL Lombard PB (1988) Characterization of quince (Cydonia) cultivars using polyacrylamide gel electrophoresis. J Environ Hortic 6:53–59 Sax K (1931) The origin and relationships of the Pomoideae. J Arn Arbor 12:3–22 Semin VS (1961) Irradiated pollen, a stimulant of fertilization in distant hybridization of fruit crops. Bjull nauc the inform, Moldav nauc issled Inst Sadov Vinograd vinodel Bull, sci tech inform Moldav Sci Res Inst Hort Vitic Wine mak 1961 (4):50–55 (in Russian) Shcherbenev GY (1973) The results of using gibberellin with boric acid in hybridizing quince with apple. Sbornik Nauchnykh rabot, Vsesoyuznyi Nacuhno Issledovatel’ skii Institut Sadovodstva imeni IV Michurin 1973(17):118–126 (in Russian) Shcherbenev GY (1974) Features of obtaining a hybrid progeny from quince and apple. Sel’ skokhozyaistvennaya Biologiya 10:308–310 (in Russian) Shcherbenev GY, Turovskaya NI (1977) Intergeneric hybrids of apple with quince and their capacity for propagation from geen cuttings. Sb nauch rabot VNII sadovod 1977 (25):45–49

15 Shimura I, Ito Y, Seiki K (1983) Intergeneric hybrid between Pyrus serotina and Cydonia oblonga. J Jpn Soc Hortic Sci 52:243–249 Staniene G, Stanys V (2004) Plant regeneration from leaves of Cydonia oblonga cultivars. Acta Universitatis Latviensis, Biology 676:231–233 Staniene G, Rugenius R, Gelvonaushiene D, Stanys V (2007) Effect of rolB transgene on Prunus cerasus P. canescens and Cydonia oblonga microshoot rhizogenesis. Biologija 53:23–26 Stepanova AF, Litchenko NA, Smykov AV (1984) Propagating fruit crops by softwood cuttings. Byllulten Gosudarstvennogo Nikitskogo Botanicheskogo Sada 1984(55):47–50 (in Russian) Sykes JT (1972) A description of some quince cultivars from western Turkey. Econ Bot 26:21–31 Thompson MM (1986) Temperate fruit crop germplasm in Syria. Plant Genet Resour Newsl 1986:29–34 Trabut L (1916) Pyronia – A hybird between the pear and quince – Produces abundance of seedless fruit of some value – Many new combinations might be made among the relatives of the pear. J. Hered 7:416–419 Tukey HB (1964) Dwarfed fruit trees. Comstock, Ithaca, NY, 561 p University of Reading (2009) National fruit collection. http:// www.nationalfruitcollection.org.uk/. Accessed 23 June 2009 USDA, ARS (2009) Quince genetic resources. http://www.ars. usda.gov/Main/docs.htm?docid¼11309. Accessed 5 June 2009 Vavilov NI (1930) Wild progenitors of the fruit trees of Turkestan and the Caucasus and the problem of the origin of fruit trees. In: Reports of the proceedings of 9th international horticulture congress 1930 Group B, pp 271–286 Vavilov NI (1935) The origin, variation, immunity and breeding of cultivated plants. In: Chester KS (ed) The Chronica Botanica 13. Waltham, MA, USA, p 176 Verbylaite˙ R, Ford-Lloyd B, Newbury J (2006) The phylogeny of woody Maloideae (Rosaceae) using chloroplast trnL-trnF sequence data. Biologija 1:60–63 Vinatzer BA, Zhang HB, Sansavini S (1998) Construction and characterization of a bacterial artificial chromosome library of apple. Theor Appl Genet 97:1183–1190 Vitkovskii VL, Denisov VP (1991) N. I. Vavilov and expeditions to study fruit crops and grape in Central Asia. Sbornik Nauchnykh Trudov po Prikladnoi Botanike, Genetike I Selektsii 140:97–111 (in Russian) Weber C (1964) The genus Chaenomeles (Rosaceae). J Arn Arbor 45:161–205 Webster AD (2008) Cydonia oblonga quince. In: Janick J, Paull RE (eds) Encyclopedia of fruit and nuts. CABI, Wallingford, UK, pp 634–642 Weryszko-Chmiellewska E, Konarska A (1995) Comparison of the nectary structure of chosen species from subf. Pomoideae (Rosaceae). Acta Agrobot 48(1):33–44 Yamamoto T, Kimura T, Sawamura Y, Kotobuki K, Ban Y, Hayashi T, Matsuta N (2001) SSRs isolated from apple can identify polymorphisms and genetic diversity in pear. Theor Appl Genet 102:865–870 Yamamoto T, Kimura T, Soejima J, Sanada T, Ban Y, Hayashi T (2004) Identification of quince varieties using SSR markers developed from pear and apple. Breed Sci 54(3):239–244 Yamamoto T, Kimura T, Terakami S, Nishitani C, Sawamura Y, Saito T, Kotobuki K, Hayashi T (2007) Integrated reference

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Chapter 2

Fragaria Kim E. Hummer, Nahla Bassil, and Wambui Njuguna

2.1 Botany 2.1.1 Taxonomy and Agricultural Status Strawberry, genus Fragaria L., is a member of the family Rosaceae, subfamily Rosoideae (Potter et al. 2007), and has the genus Potentilla as a close relative. Strawberry fruits are sufficiently economically important throughout the world such that the species is included in The International Treaty on Plant Genetic Resources, Annex 1 (http://www.planttreaty.org/). The hybrid strawberry fruit of commerce, Fragariaananassa Duchesne ex Rozier nothosubsp. ananassa, is eaten by millions of people and is cultivated from the arctic to the tropics. More than 75 countries produce significant amounts of this fruit (FAO 2010). Annual world production is increasing from 3 to more than 4 thousand MT (Fig. 2.1). About 98% of the production occurs in the Northern Hemisphere, though production is expanding in the south (Hummer and Hancock 2009). The genus Fragaria was first summarized in preLinnaean literature by C. Bauhin (1623). In Hortus Cliffortianus, Linneaus (1738) described this genus as monotypic containing Fragaria flagellis reptan; in Species Plantarum (Linneaus 1753), he described three species including varieties, though several European species now known were omitted, and one belonging to Potentilla was included (Staudt 1962). Duchesne (1766) was credited for publishing the best early taxonomic treatment of strawberries

K.E. Hummer (*) USDA ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, OR 97333, USA e-mail: [email protected]

(Hedrick 1919; Staudt 1962). Duchesne maintained the strawberry collection at the Royal Botanical Garden, having living collections documented from various regions and countries of Europe and the Americas. He distributed samples to Linnaeus in Sweden. The present Fragaria taxonomy includes 20 named wild species, three described naturally occurring hybrid species, and two cultivated hybrid species important to commerce (Table 2.1). The wild species are distributed in the north temperate and holarctic zones (Staudt 1989, 1999a, b; Rousseau-Gueutin et al. 2008). European and American Fragaria subspecies were monographed by Staudt (1999a, b), who also revised the Asian species (Staudt 1999a, b, 2003, 2005; Staudt and Dickore` 2001). Chinese and midAsian species are under study (Lei et al. 2005) but require further collection and comparison, considering global taxonomy. The distribution of specific ploidy levels within certain continents has been used to infer the history and evolution of these species (Staudt 1999a, b).

2.1.2 Geographical Locations of Species Fragaria species exist as a natural polyploid series from diploid through decaploid (Table 2.1). Diploid Fragaria species are endemic to boreal Eurasia and North America. Fragaria vesca is native from the west of the Urals throughout northern Europe and across the North American continent. However, this diploid species is not native to Siberia, Sakhalin, Hokkaido, Japan, Kamchatka, or to the Kurile, Aleutian, or Hawaiian Archipeliagos according to flora of those regions (Hulte´n 1968). It has been introduced in many of those areas.

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_2, # Springer-Verlag Berlin Heidelberg 2011

17

18

K.E. Hummer et al. World Strawberry Production

MT Production

4,200 4,000 3,800 3,600 3,400

2004

2005

2006

2007

2008

Fig. 2.1 World Strawberry production

Table 2.1 Fragaria species, ploidy, and distribution area F. bucharica Losinsk 2x F. chinensis Losinska F. daltoniana J. Gay F. iinumae Makino F. mandshurica Staudt F. nilgerrensis Schlect. F. nipponica Makino F. nubicola Lindl. F. pentaphylla Losinsk F. vesca L. F. viridis Duch. F. bifera Duch. F. corymbosa Losinsk 4x F. gracilis A. Los. F. moupinensis (French.) Card F. orientalis Losinsk F. tibetica Staudt & Dickore´ F. bringhurstii Staudt 5x (9x) F. sp. novb F. moschata Duch. 6x F. chiloensis (L.) Miller 8x F. virginiana Miller F. ananassa Duch. ex Lamarck F. ananassa subsp. cuneifolia F. iturupensis Staudt 10x F. virginiana subsp. platypetala Miller F. vescana R. Bauer & A. Bauer a

Western Himalayas China Himalayas Japan North China Southeastern Asia Japan Himalayas North China Europe, Asia west of the Urals, disjunct in North America Europe and Asia France, Germany Russian Far East/China Northwestern China Northern China Russian Far East China California China Euro-Siberia Western N. America, Hawaii, Chile North America Cultivated worldwide Northwestern N. America Iturup Island, Kurile Island Oregon, United States Cultivated in Europe

As proposed by Staudt (2008) As proposed by Lei et al. (2005)

b

Diploid strawberry species are reported on many of the islands of and surrounding Japan, in Hokkaido, on Sakhalin, and in the greater and lesser Kuriles (Makino 1940). Diploid and tetraploid species are native not only to Asia, particularly in China, but also in Siberia and the Russian Far East. Wild, naturally occurring pentaploids (2n ¼ 5x ¼ 35) have been observed in California

(F. bringhurstii) and China (Lei et al. 2005). These strawberries exist in colonies with other ploidy levels nearby. The only known wild hexaploid (2n ¼ 6x ¼ 42) species, F. moschata, is native to Europe as far east as Lake Baikal. This species is commonly known as the musk strawberry (Hancock 1999). Wild octoploid species are distributed from Unalaska eastward in the Aleutian Islands (Hulte´n 1968),

2 Fragaria

completely across the North American continent, on the Hawaiian Islands, and in South America (Chile) (Staudt 1999a, b). Wild decaploids are native to the Kurile Islands (F. iturupensis) (Hummer et al. 2009) and the old Cascades in western North America (Hummer unpublished).

2.1.3 Description of Wild Species Relatives 2.1.3.1 Diploids Fragaria vesca, a self-compatible, sympodial-runnering diploid (Staudt et al. 2003), has the largest native range (presently) among Fragaria species. It is the only diploid species with disjunct subspecies in North America. Fragaria vesca has four subspecies: the European F. vesca subsp. vesca, the American F. vesca subsp. americana, Fragaria vesca subsp. bracteata, and F. vesca subsp. californica. Fragaria vesca subsp. vesca is endemic across Europe eastward to Lake Baikal (Staudt 1989). Several forms of Fragaria vesca subsp. vesca species have been identified but more common ones include forma vesca, f. semperflorens and f. alba. Fragaria vesca subsp. americana is distributed in many US states from Virginia, to South Dakota, North Dakota, Missouri, Nebraska, and Wyoming. This subspecies is also found in Ontario, Canada, and British Columbia. Fragaria vesca subsp. americana differs from other subspecies by its slender morphological structure. Fragaria vesca subsp. bracteata occurs around the coastal and Cascade mountain ranges from British Columbia through Washington and Oregon, and the Sierra Nevada in California. Its distribution extends into Mexico where it is referred to as F. mexicana Schltdl. (Staudt 1999b). This chapter uses Staudt’s (1999b) treatment where F. mexicana is submerged under F. vesca subsp. bracteata. While the other three vesca subspecies are hermaphroditic, some genotypes of F. vesca subsp. bracteata are reported as gynodioecious (Staudt 1989). Fragaria vesca subsp. californica occurs near the Pacific Ocean from southern Oregon to California. Hybrids of F. vesca subsp. californica and subsp. bracteata have been observed in regions of overlap where subsp. bracteta approaches the coastal range distribution of subsp. californica.

19

In Europe, F. vesca subsp. vesca overlaps in distribution with another diploid, F. viridis, which has a monopodial branching system of the runners, a feature used to distinguish the two species. The fruit of F. viridis has wine red skin while the cortex and pith is yellowish–greenish and the fruit does not easily detach from the calyx (Staudt et al. 2003). In regions where F. vesca and F. viridis distributions overlap including Russia, Germany, France, Finland, and Italy, hybridization has occurred resulting in the hybrid species F. bifera. Morphological features of this hybrid species are mostly intermediate and include the stolon branching system and leaf color. The fruit, like F. viridis, does not easily detach from the calyx. In addition, the fruit has pigment only in the skin as is the case with F. viridis, and the fruits are embedded in shallow pits, a feature found in F. vesca. The triploid form of the hybrid that includes two genome copies from F. vesca seems to be similar to F. vesca in certain features such as the easy detachment of fruit from the calyx, flesh texture, smell, and taste of the fruit (Staudt et al. 2003). Fragaria mandschurica has sympodially branched runners and hermaphrodite flowers with functional stamens and fruit that shows good seed set. This diploid is distributed on the east banks of Lake Baikal and is also found in Mongolia and South Korea and spreads to northeastern China. The tetraploid F. orientalis overlaps in distribution with F. mandshurica in the Amur Valley of China and is also distributed in Russia. Fragaria nilgerrensis is a self-compatible diploid with two subspecies: subsp. nilgerrensis and subsp. hayatae Makino (Staudt 1999a). The fruit of F. nilgerrensis subsp. nilgerrensis is white to cream and is distributed in northwestern and southwestern India, East Himalaya, northeastern Burma, northern Vietnam, Southwest and central China. Despite this wide distribution of the subspecies, only limited morphological variation has been observed among different populations. The fruit of F. nilgerrensis subsp. hayatae has pink to red skin, a cream colored cortex (Staudt 1999a), and is known for its high anthocyanin levels in all plant parts including the berries (Staudt 1989). In contrast to the wide distribution of F. nilgerrensis subsp. nilgerrensis, subsp. hayatae is only recorded in Taiwan. The leaf morphology of the tetraploid F. moupinensis, distributed in Yunnan and Sichuan provinces of China and in Tibet, resembles that of F. nilgerrensis (Darrow 1966).

20

Fragaria daltoniana J. Gay is a self-compatible diploid with sympodial runners with elongate conical white to pinkish fruit. Hybridization with other diploids has been previously tested, but the results were not published and were only stated in Staudt (2006). Hybrids with F. iinumae, F. nilgerrensis, and F. nipponica Makino were morphologically intermediate. The diploid F. daltoniana is distributed in the Himalayas from India to Myanmar (Staudt 2006). Like F. daltoniana, the diploid F. bucharica is found in the Himalayan region but is self-incompatible. It has sympodial runners, a characteristic that distinguishes it from F. nubicola (Hook. f.) Lindl. ex Lacaita also found in the Himalayas. Two subspecies of F. bucharica, subsp. bucharica and subsp. darvasica, are recognized and are currently only distinguished by the size of bractlets: they are smaller in subsp. darvasica than in subsp. bucharica. Crossability tests with other diploids including F. mandshurica, F. vesca, and F. viridis resulted in mostly heterotic plants with F. bucharica morphological characters prevailing, even with reciprocal crosses. In contrast, crosses with F. nipponica produced dwarf plants. Fragaria bucharica is distributed from Tadjikistan to Afghanistan, Pakistan, and Himachal Pradesh in India (Staudt 2006). Another diploid species frequently confused with F. bucharica due to the similar morphological characteristics and also found in the Himalayas is F. nubicola. This diploid is selfincompatible with a monopodial branching pattern of the stolon, which is the only distinguishing feature separating it from F. bucharica. It is distributed along the southern slopes of the Himalayas to Southeast Tibet, and in Southwest China. Fragaria nubicola was observed to form accessory leaflets probably associated with the time of year. Fragaria pentaphylla is a self-incompatible diploid found in China. Fragaria pentaphylla f. alba Staudt and Dickore´, only known from Mt. Gyala Oeri and north of the Tsangpo Gorge in Tibet, has only been identified from a white-fruited population. Red-fruited types are expected with further exploration of this region (Staudt and Dickore` 2001). As the name “pentaphylla” suggests, this species contains accessory leaflets. However, the presence of accessory leaflets is not restricted to this species but has been seen in other strawberry species throughout the world, including F. nubicola and the tetraploid, F. tibetica. The formation of accessory leaflets has been

K.E. Hummer et al.

associated with certain times of the year as noted by Staudt and Dickore` (2001). Strawberry plants show accessory leaflets to be a common characteristic in many species including F. virginiana, F. chiloensis, and F. iturupensis. Fragaria pentaphylla is closely related to a tetraploid species, F. tibetica, which also has a white-fruited form, F. tibetica f. alba. The two species are distinguished from each other by the heteroecy, tetraploidy, larger pollen grains, and larger achenes found in F. tibetica. The distribution of the tetraploid extends from Central and Eastern Himalaya to the Chinese provinces, Yunnan and Sichuan. F. pentaphylla and F. tibetica have monopodial runners and can therefore be distinguished from Himalayan F. nubicola and F. daltoniana that have sympodial runners. Fragaria iinumae is found in the lowlands of Hokkaido in the north to the mountains of the main island Honshu in areas of heavy snow along the Sea of Japan (Hancock 1999). Fragaria iinumae is known for its unique characters not found in other Fragaria diploids such as the glaucous leaves. It has sympodial runners and its flowers have six to nine petals per flower, while Fragaria flowers commonly have five. Due to its glaucous leaves, this diploid may be a progenitor of the octoploid species, F. virginiana (Staudt 2005). The crowns of F. iinumae usually appear as rosettes, but they sometimes rise above the ground in “tufts” making this species conspicuous (Oda 2002). Fragaria nipponica, a diploid, which now includes the submerged species F. yezoensis (Naruhashi and Iwata 1988), is a self-incompatible species distributed in Honshu and Hokkaido in Japan, and, Sakhalin and Kuriles in Russia (F. nipponica subsp. nipponica), Yakushima Islands of Japan (F. nipponica subsp. yakusimensis), and in the Island of Cheju-do off the Korean mainland (F. nipponica subsp. chejuensis) (Staudt 2008). Tetraploid hybrids of F. nipponica subsp. nipponica with F. moschata (F. nipponica as the maternal parent) provided evidence of homology of the F. moschata and F. nipponica genomes (Staudt 2008). F. iinumae and F. nipponica are the only diploid species endemic to Japan and the islands north of Japan including the Kuriles. F. nipponica is confined to the Pacific Ocean side of Japan while F. iinumae is found on the Sea of Japan side (Staudt 2005). During the winter, aboveground shoots of F. iinumae die back, though the crown and roots remain alive.

2 Fragaria

2.1.3.2 Tetraploids Known named tetraploid species occur in Southeast and East Asia. Staudt (2006) proposed that four tetraploid species may have originated as the first step of ploidization from diploid species. The diploid F. pentaphylla seems to be the putative ancestor of the tetraploid F. tibetica, given their distribution and similar morphological characteristics (Staudt and Dickore` 2001). Two tetraploid species, F. corymbosa and F. moupinensis, may have been derived from the diploid F. chinensis (Staudt 2003). Similarity in morphological characters of F. mandschurica and F. orientalis and their sympatry in far eastern Russia was proposed to support F. mandshurica as the diploid ancestor of the tetraploid F. orientalis (Staudt 2003). The tetraploid F. orientalis can be distinguished from F. mandshurica by the size of its pollen grains, a characteristic related to the number of chromosomes. Though F. mandshurica is hermaphroditic, F. orientalis contains both dioecious and trioecious populations.

2.1.3.3 Hexaploid The sole hexaploid species, F. moschata, grows in forests, under shrubs and in tall grass (Hancock 1999). Like the diploids F. vesca and F. viridis, F. moschata is native to northern and central Europe. This species was extensively cultivated in Europe (France and Germany) from 1,400 to 1,850 due to its desirable flavor and aroma. The fruit only has color on the skin, while the cortex and pith are yellowish-white, with a strong, musky smell and taste (Staudt et al. 2003). The populations are dioecious (Staudt et al. 2003), which contributes to scanty yields in comparison to cultivated hermaphroditic diploid and octoploid species (Hancock 1999). Fragaria vesca, F. viridis, and F. moschata are sympatric with F. mandshurica to the east (Staudt 2003).

2.1.3.4 Octoploids Fragaria chiloensis, known as the beach strawberry, is an American octoploid. This species is divided into four subspecies. The two northerly distributed subspecies are F. chiloensis subsp. pacifica and F. chiloensis

21

subsp. lucida. These subspecies are found along sandy beaches of the Pacific Ocean from Alaska to California and have small red fruit. Fragaria chiloensis subsp. sandwicensis is distributed in mountainous regions of Hawaii and Maui (Staudt 1999b). Fragaria chiloensis subsp. chiloensis f. patagonica, also red-fruited, is distributed in coastal mountains, the central valley in Chile, and in the Andes in southern Chile with the southern limit of its distribution in Argentina. Fragaria chiloensis subsp. chiloensis f. chiloensis is cultivated in Chile, Ecuador, and Peru. White-fruited landrace of F. chiloensis was first domesticated by the Mapuche Indians. This forma has larger flower and fruit structures than other F. chiloensis subspecies. This large, white-fruited landrace with hairy petioles was imported from Chile to Europe in the early eighteenth century. It is the maternal progenitor of the cultivated strawberry (Darrow 1966; Hancock 1999). Fragaria virginiana is native to North America. This species is also known as the “scarlet” strawberry. Fragaria virginiana subsp. virginiana is the paternal progenitor of the cultivated strawberry (Hancock 1999). Wild F. virginiana is divided into four subspecies. Fragaria virginiana subsp. virginiana is found throughout eastern North America and spreads to British Columbia in the west (Harrison et al. 2000). Fragaria virginiana subsp. grayana (Vilm. ex J. Gay) Staudt is found from northwestern Texas, to Nebraska, Iowa, and Illinois. It is also found in Louisiana, Alabama, Indiana, Ohio, Virginia, and New York. The distribution of F. virginiana subsp. glauca resembles that of subsp. virginiana; however, this species spreads further west in British Columbia interacting with F. chiloensis found along the coast (Staudt 1999b). Fragaria virginiana subsp. glauca is distinguished from other subspecies by the smooth leaf surface and the dark to light bluish (glaucous) leaves. The leaves of F. virginiana subsp. platypetala are also blue green but only slightly (Staudt 1999b). Fragaria virginiana subsp. platypetala is distributed in British Columbia and extends southward to Washington, Oregon, and northern California (Staudt 1999b). Further south in British Columbia, F. virginiana subsp. glauca overlaps in distribution with subsp. platypetala (Rydb.) Staudt, and introgression has been encountered. Fragaria ananassa subsp. cuneifolia is suspected as a natural hybrid of F. chiloensis subsp. pacifica or

22

subsp. lucida and F. virginiana subsp. platypetala (Staudt 1999b). Unlike the cultivated strawberry of commerce, this hybrid has smaller leaves, flowers, and fruits. The distribution of F. ananassa subsp. cuneifolia is from the coastal regions of British Columbia (Vancouver Island) south to Fort Bragg and Point Arena lighthouse in California. Hybrids of F. ananassa subsp. cuneifolia and the two octoploids, F. chiloensis subsp. pacifica and F. virginiana subsp. platypetala, have been seen in Oregon, Washington, and California in the US (Staudt 1999b).

2.1.3.5 Decaploids Fragaria iturupensis is a polyploid strawberry distributed on the eastern slopes of Mt. Atsonupuri on Iturup, the second island in the southern section of the greater Kuril Island archipelago. This species has a limited distribution of a few colonies on the rock skree on the eastern flank of the volcano. This location might have provided a refugium from the most recent glaciations, which is reported to have come only as far south as the northern part of Iturup Island. In 1973, chromosome counts of F. iturupensis indicated that this species was octoploid (Staudt 1989). Those initial plants were lost. A return trip to Atsonupuri in 2003 obtained another sample of F. iturupensis. Chromosome counts and flow cytometry indicated this sample to be decaploid. (Hummer et al. 2009). Fragaria iturupensis resembles F. virginiana subsp. glauca (Staudt 1989) and F. iinumae (Hancock 1999) in leaf texture and color. The oblate fruit shape and erect inflorescence and flavor components of this polyploid population resemble those found in F. vesca (Staudt 2008). Staudt (1999a, b) postulated that F. iturupensis is more primitive than F. virginiana subsp. glauca. Thus far, molecular analyses have concurred (Njuguna et al. 2010).

2.1.3.6 Unusual Ploidy Fragaria bringhurstii is a hybrid species between F. chiloensis and F. vesca subsp. californica. This species is distributed near the Pacific Ocean in California in

K.E. Hummer et al.

Humboldt and Monterey counties (Staudt 1999b). Varying levels of morphological intermediacy between F. chiloensis and F. vesca were observed in the hybrid species. Genotypes of this species with different ploidy levels including pentaploid (2n ¼ 5x ¼ 35), hexaploid (2n ¼ 6x ¼ 42), and enneaploid (2n ¼ 9x ¼ 63) have been found. In 2009, plants were morphologically similar to F. virginiana subsp. platypetala but appeared decaploid based on microsatellite analysis and flow Cytometry (Wambui Njuguna and Nahla Bassil unpublished). Nathewet et al. (2009) confirmed decaploidy by chromosome counts. These plants occurred in the Oregon Cascades near the Pacific Crest Trail where it is conspecific with F. vesca subsp. bracteata. The occurrence of multiple ploidy levels in F. virginiana subsp. platypetala is suspect where its distribution overlaps with F. vesca subspecies.

2.1.4 Strawberry History of Cultivation E. L. Sturtevant, through U. P. Hedrick (1919) and Darrow (1966), describes early references for European strawberry from the Ancient Roman verses of Virgil and Ovid and the glancing mention in Pliny’s Natural History. Darrow (1966) pointed out that this fruit was not a “staple of agriculture” to explain its exclusion from Theophrastus, Hippocrates, Dioscorides, or Galen. By the 1300s, the French began transplanting F. vesca, the wood strawberry, from the wilderness into the garden. In 1368, King Charles V had his gardener, Jean Dudoy, plant no less than 1,200 strawberries in the royal gardens of the Louvre, in Paris (Darrow 1966). Written references to the strawberry in Shakespeare and his contemporaries may indicate the success of the plant in the gardens of that time. In 1530, King Henry VIII paid ten shillings for a “pottle of strawberries” (slightly less than 250 g) according to his Privy Purse Expenses (Darrow 1966). In addition to the alpine strawberry, Darrow (1966) noted that F. moschata was cultivated in Europe. Karp (2006) described this species as the most aromatic strawberry. F. viridis, the “green” strawberry, was also gathered and eaten. Between the tenth and the eighteenth centuries, in Japan, the ancient word “ichibigo” referred to many

2 Fragaria

berry crops (including Japanese strawberry species and the low-growing Rubus pseudo-japonica) gathered from the wild (Oda and Nishimura 2009). The word migrated to “ichigo”, now the term of reference for the modern day Fragaria species. The cultivated F. ananassa was first brought into Japan from the Netherlands in the early to mid-nineteenth century. The Virginia strawberries impacted the European strawberry industry of the 1800s with their high yields and deep red color, resulting in the name “scarlet strawberry”. The scarlet strawberry was cultivated in Europe, and some important cultivars included: “Oblong Scarlet”, “Grove End Scarlet”, “Duke of Kent’s Scarlet”, and “Knight’s Large Scarlet”. At the time of the reintroduction of the scarlet strawberry to the United States in the early 1700s, F. virginiana plantings were established in Boston, New York, Philadelphia, and Baltimore. “Hudson”, a vigorous, soft-fruited and high flavored F. virginiana clone, was considered the first most important American strawberry (Hancock 1999). The attractive color, large size and acceptable flavor made it favorable for making jam. It was used through the early part of the twentieth century (Fletcher 1917). Desirable horticultural traits, such as winter hardiness, frost tolerance, resistance to red stele, adaptation to diverse environmental conditions, and interfertility with the cultivated strawberry (Hancock et al. 2002), made F. virginiana a valuable genetic resource for breeders. A F. virginiana subsp. glauca clone from Hecker Pass was the primary source of the day-neutral trait in the cultivar development program of California in the 1970s and 1980s. Importation of Chilean clones to Europe in the early eighteenth century resulted in the accidental hybridization with F. virginiana subsp. virginiana from North America, forming the now cultivated F. ananassa subsp. ananassa, now known as the hybrid of commerce. Fragaria chiloensis has been used in breeding programs as a source of winter hardiness (Staudt 1999b), resistance to strawberry root disease, and virus tolerance (Lawrence et al. 1990). Fragaria ananassa, the “pineapple strawberry”, was the species name given to the accidental hybrid of F. chiloensis subsp. chiloensis f. chiloensis and F. virginiana subsp. virginiana in Europe by Duschesne in the early eighteenth century (Hancock 1999). Since the mid-1800s, breeding in Europe and United States has resulted in hundreds of cultivars from

23

35 breeding programs (Faedi et al. 2002). The F. ananassa subsp. ananassa includes these cultivated species originating from the accidental hybrids first recognized in France around 1750. Breeding work in Alaska utilized F. chiloensis to develop Sitka hybrids that were winter hardy and suited for climatic conditions in Alaska (Staudt 1999b). In North America, natural hybridization between F. ananassa subsp. ananassa, which escapes cultivation, with subspecies of F. chiloensis and F. virginiana have been observed. These hybrids are usually identified in the wild by the large berries, sometimes erratic fruit set, and fruit taste. Fragaria chiloensis populations resulting from introgression into the hybrid octoploid were observed in California (F. chiloensis subsp. lucida) and Chile (F. chiloensis subsp. chiloensis f. patagonica). Introgression of the cultivated strawberry into wild populations of F. virginiana subsp. grayana occurs in the southeastern United States.

2.1.5 Tribal Use of Primitive Forms In South America, the Mapuche (M€apfuchieu) and Huilliche Indians, the indigenous people of central and southern Chile, cultivated strawberries. Their economy was based on agriculture until the appearance of the Spanish conquistadores. They developed a landrace of the white Chilean strawberry (F. chiloensis subsp. chiloensis f. chiloensis) and cultivated this fruit, undisturbed for thousands of years until 1550–1551. The Spanish considered this fruit as a spoil of conquest. Pedro de Valdivia and his men brought this fruit to Cuzco, Peru, in 1557, where it was described as the “chili” (Darrow 1966). Spread of the Chilean berries to other countries within South America followed the Spanish invasion (Hancock 1999). Strawberry acreage found in Ecuador was reported to be largest observed in South America during the period between 1700 and 1970 (Finn et al. 1998). Despite the higher yields obtained with F. ananassa in Chile (20–70 t/ha), its flavor and aroma have been described as lower than that of F. chiloensis (Retamales et al. 2005). High-yielding F. ananassa cultivars displaced the local Chilean landrace cultivars in the twentieth century (Retamales et al. 2005).

24

K.E. Hummer et al.

2.2 Phylogeny In Fragaria, phylogenetic analysis has been attempted using chloroplast and nuclear genome sequences, but most species relationships have remained unclear. Harrison et al. (1997b) used restriction fragment length variation of chloroplast DNA from nine species, while Potter et al. (2000) used the nuclear internal transcribed spacer (nrITS) region and the chloroplast trnL intron and the trnL–trnF spacer region in 14 species. Low resolution of the phylogenetic tree from these two studies was speculated to be due to little divergence of the genome regions investigated (Rousseau-Gueutin et al. 2009). The Fragaria octoploid genome models AAA0 A0 BBB0 B0 (Bringhurst 1990), and the more recently published YYY0 Y0 ZZZZ/YYYYZZZZ models (Rousseau-Gueutin et al. 2009), suggests the contribution and close relationships, of two to four diploids to the octoploids (Fig. 2.2). The specific diploid sources of the octoploid genome are still not known but evidence indicates F. vesca, F. mandshurica, and F. iinumae (Senanayake and Bringhurst 1967; Harrison et al. 1997b; Potter et al. 2000; Davis and DiMeglio 2004; Rousseau-Gueutin et al. 2009) as the possible contributors. While some species relationships have been confirmed by crossing studies, others have never been verified. For example, the diploid F. mandshurica is

assumed to be the ancestor of the tetraploid F. orientalis (Staudt 2003). This hypothesis is based on their shared sympodially branching runners, characters absent among species found in the adjacent southwestern China, and their overlapping geographic range in northeastern China (Fig. 2.3). However, phylogenetic analysis (Rousseau-Gueutin et al. 2009; Wambui Njuguna unpublished) does not support this hypothesis. The diploid F. nilgerrensis is speculated to be a diploid ancestor of F. moupinensis (Darrow 1966). Interspecific hybridization has resulted in the formation of several species such as F. bifera (F. vesca F. viridis) (Staudt et al. 2003), F. bucharica (involving diploids, F. vesca and F. viridis) (Staudt 2006; RousseauGueutin et al. 2009), F. ananassa subsp. cuneifolia (F. virginiana, F. chiloensis) (Staudt 1989), and F. bringhurstii (F. chiloensis, F. vesca) (Bringhurst and Senanayake 1966). Limited chloroplast genome variation has created a barrier to phylogenetic resolution of the genus using standard Sanger sequencing (Harrison et al. 1997b; Potter et al. 2000). The low copy nuclear genes, granule-bound starch synthase I (GBSSI-2) or Waxy, and dehydroascorbate reductase (DHAR) were recently used to determine phylogenetic relationships based on sequence comparison in each species (RousseauGueutin et al. 2009). Previously identified relationships such as the basal position of F. iinumae in the

AA

AAA¢A¢

AAA¢A¢ BBB¢B¢/A¢A¢A¢A¢ BBBB A¢A BB

BBB¢B¢

B¢B¢

Fig. 2.2 The Fragaria octoploid genome model. An illustration of the origin of Fragaria octoploid genome modified from Bringhurst (1990) and equivalent to the YYY0 Y0 ZZZZ/YYYYZZZZ models proposed by Rousseau-Gueutin et al. (2009)

2 Fragaria

25 10x

virginiana

9x

iturupensis bringhurstii

(bringhurstii)

virginiana

ananassa

chiloensis

(iturupensis)

8x octoploid ancestor

6x

bringhurstii

moschata

5x

4x

bringhurstii

spec. nov. Changbai, China

orientalis

mandshurica

tibetica

xbifera

2x bucharica

viridis vesca subsp. vesca Clade A

iinumae vesca subsp. bracteata

moupinensis

corymbosa

pentaphylla

nubicola

chinensis

daltoniana

nipponica

nilgerensis

gracilis

vesca subsp. californica Clade B

Clade C

?

Fig. 2.3 Representation of Fragaria species relationships based on nuclear and chloroplast gene sequences and morphological characters (Harrison et al. 1997; Potter et al. 2000; Staudt 2008; Hummer and Hancock 2009; Rousseau-Gueutin et al. 2009). Clades A, B, and C refer to diploid clades deter-

mined from nuclear genes GBSSI-2 and DHAR. They correspond to possible sources of “A” and “B” genomes of the octoploid strawberry. Dotted lines indicate hypothetical relationships. Solid lines are published relationships

phylogeny and multiple polyploidization events in Fragaria (Harrison et al. 1997b; Potter et al. 2000) were confirmed. Analysis of low copy nuclear genes differentiated Fragaria diploids into three clades, X (F. daltoniana, F. nilgerrensis, F. nipponica, F. nubicola, F. pentaphylla), Y (F. mandshurica, F. vesca, F. viridis), and Z (F. iinumae) analogous to clades C, A, and B, respectively (Potter et al. 2000), with the octoploid genome originating from clades Y (A) and Z (B) based on the distribution of multiple copies of low copy nuclear genes in the octoploids. The phylogenetic study of Rousseau-Gueutin et al. (2009) is now the most extensive one in Fragaria involving a comprehensive species representation and increased phylogenetic resolution. However, there was low resolution of diploid species within clade C supporting recent divergence within the clade and placement of F. bucharica low copy genes in different clades (C and A), suggesting hybrid origin of this species or incomplete lineage sorting. The use of nuclear genes for phylogenetic analysis is complicated by polyploidy and recombination and lineage sorting, making the chloroplast genome an attractive tool for phylogenetic resolution. For the chloroplast genome to be utilized for phylogenetic

relationships in Fragaria, alternative techniques for finding species-specific identifiers and markers appropriate for phylogenetic resolution need to be explored.

2.3 Conservation Initiatives In 2008, Fragaria genebanks were located in 27 countries and, together with two genebank networks, maintained more than 12,000 strawberry accessions in about 57 locations (Hummer 2008). Roughly half of these accessions represented advanced breeding lines of the cultivated hybrid strawberry. A survey of the private sector indicated that, in addition to the public collections, global private corporations maintained another 12,000 proprietary cultivated hybrids for internal use. Unlike the public collections, however, these private collections were transitory in nature with proprietary genotypes being destroyed after intellectual property rights expire. Primary collections at national genebanks consisted of living plants, protected in containers greenhouses, or screenhouses or growing in the field. Any plant material grown outdoors cannot be certified as pathogen-

26

negative. Secondary backup collections were maintained in vitro under refrigerated temperatures. Longterm backup collections of meristems were placed in cryogenic storage at remote locations to provide decades of security. Species diversity was represented by seed lots stored in 18 C or backed-up in cryogenics. Conservation of clonally propagated material, where genotypes were maintained, was more complicated and expensive than storing seeds, where the objective is to preserve genes. The health status of both forms of storage was of primary importance for plant distribution to meet global plant quarantine regulations. Strawberries are a specialty crop. Limited world resources are available from each government for conservation of cultivated strawberries and their wild relatives. These limited resources constrain the management of strawberry resources in each country (Hummer 2008). Many genebanks are unable to perform pathogen test protocols and maintain pathogen-negative plants that satisfy quarantine requirements. Training on standard protocols for germplasm maintenance is needed for staff of genebanks in developing countries. Coordination of inventory and characterization data between genebanks is also insufficient (Hummer 2009). In situ preservation of wild strawberries has been limited. The wild species in many regions of the world would be appropriate for such conservation efforts.

2.4 Cytology and Karyotyping Longly (1926) and Ichijima (1926) performed early cytology of Fragaria. They determined that the basic chromosome set was x ¼ 7, with four main ploidy levels ranging from diploid to octoploid. Additional decaploid species were since found (Fig. 2.4) (Hummer et al. 2009). The circumpolarly distributed Fragaria vesca was diploid; some Asian species were tetraploid; the European F. moschata was the only known hexaploid; and F. chiloensis and F. virginiana subspecies were octoploid. Subsequent observations of wild and cultivated strawberries confirmed these numbers (Longly 1926; Bringhurst and Senanayake 1966; Nathewet et al. 2007). Cytologists have also studied Fragaria pollen mother cells to examine the phylogenetic relationships between parent and progeny and the genome compositions (Kihara 1930; Scott 1950; Senanayake and Bringhurst 1967: Staudt et al. 2003). Karyotype analyses

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have been conducted on the wild diploid species, F. daltoniana, F. hayatai Makino, F. iinumae, F. nipponica, F. nubicola, and F. vesca, and octoploid species F. chiloensis (Iwastubo and Naruhashi 1989, 1991; Naruhashi et al. 1999; Lim 2000; Nathewet et al. 2009). Yanagi and his laboratory team have been examining the karyotype analysis in wild strawberries (Nathewet et al. 2009). They examined phylogenetic relationships between species using cluster analysis based on karyotypic similarity. Chromosome morphology in wild diploid strawberries had greater uniformity than that in the tetraploids. Cluster analysis indicated that the diploid and tetraploid species reside in separate clades, with the exception of F. tibetica. This tetraploid clustered with the diploid species clade in their analysis. The hexaploid F. moschata clustered with the tetraploid clade. In studies with the octoploids, the size and shape of the Virginian strawberry varied more than that of the beach strawberry. Each of these octoploid species was separated into distinct clades. The Asian F. iturupensis grouped with the Virgianian strawberry clade. It is also similar in morphology to F. virginiana subsp. glauca.

2.5 Classical and Molecular Genetic Studies Many strawberry cultivars have been grown around the world and new varieties appear at frequent intervals (Nielsen and Lovell 2000). The continued introduction of strawberry cultivars to the market increases the need for reliable methods of identification and genetic diversity assessment (Degani et al. 2001). In addition, verification of strawberry cultivars is essential for growers and plant breeders to protect breeders’ rights (Garcia et al. 2002). Verification is especially important in a clonally propagated crop like strawberry where one original plant of an economically important cultivar can be easily used to produce a large number of plants (Gambardella et al. 2001). Strawberry cultivars have been identified using morphological traits (Nielsen and Lovell 2000) and molecular markers (Levi et al. 1994; Congiu et al. 2000; Degani et al. 2001; Garcia et al. 2002; Shimomura and Hirashima 2006; Govan et al. 2008; Brunnings et al. 2010). Molecular marker techniques for analysis of strawberries include isozymes

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Fig. 2.4 Chromosome separation at metaphase in a Fragaria iturupensis Staudt root tip cell (Hummer et al. 2009); bar represents 5 mm

and hybridization-based and PCR-based DNA markers and complement the use of morphological markers in germplasm characterization.

2.5.1 Morphological Identification of Strawberries Morphological characterization in strawberry involves recording variation in habit, leaf, flower, and fruit traits (Dale 1996). Morphological characters tradition-

ally identified crop species and varieties (Nielsen and Lovell 2000) and have been used in Argentina to certify cultivar identity in strawberry (Garcia et al. 2002). In the United States and Europe, morphological markers are used in addition to isozyme markers in plant patent descriptions (Nielsen and Lovell 2000). Morphological characters vary with age, time of year, production enhancement regimes, and cultivation methods (Degani et al. 2001). These characters are subjective and can vary between reports and environments (Bringhurst et al. 1981). In an identification study of strawberry cultivars from Argentina, morphological

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characters were insufficient to distinguish between three genotypes of “Pajaro” that were found to be polymorphic using molecular markers (Garcia et al. 2002). A set of morphological characters to uniquely identify strawberry cultivars (Nielsen and Lovell 2000) includes leaf morphology, leaf length and breadth, leaf base shape, teeth base shape, petal spacing, petal length and base, calyx:corolla (length ratio), fruit size, fruit length and breadth, fruit shape, band without achenes, insertion of achenes, insertion of calyx, and calyx size. In most cultivar identification cases, especially those dealing with infringement of breeders’ rights, only the fruit, and not the whole plant, is available. In a study by Kunihisa et al. (2003), strawberry imports to Japan were suspected to be mixed with Japanese varieties not licensed for production in other countries. Only the fruit was available for identity verification. Fruit processing and canning industry sales depend on marketing released varieties. Morphological markers are the traditional technique for distinguishing cultivars; however, they can sometimes result in ambiguity for identification (Chavarriaga-Aguirre et al. 1999; Dangl et al. 2001; Abu-Assar et al. 2005). This suggests the need for additional forms of identification. DNA extraction kits suitable for processed fruit are now available (for example Genetic ID, Inc. Fairfield, IA), which allow identification of cultivars using molecular markers. Despite disadvantages associated with morphological character traits, they have proved useful in breeding programs and germplasm repositories. Morphological traits help to group plants with similar qualitative and quantitative traits (Brown and Schoen 1994). However, lack of discrimination between individuals is explained by the plasticity of morphological markers (Degani et al. 2001).

2.5.2 Isozymes Isozymes are enzymes with different amino acid sequence that catalyze the same reaction. Isozymes exhibit different electrophoretic mobility, and different forms are easily distinguished. Isozyme markers were the first molecular markers to be developed. Their use in strawberry dates to the late 1970s (Hancock and Bringhurst 1979). Isozymes were used to determine adaptive strategies of 13 F. vesca (diploid) and

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19 octoploid Fragaria populations from California using two enzyme systems, phosphoglucoisomerase (PGI) and peroxidase (PX). In both the diploid and octoploid species, a high genetic differentiation was observed that depended on the site of collection. The association was attributed to variations in catalytic properties of the isozymes expressed under different environmental conditions. This illustrates the sensitivity of isozymes to the environment, even within the same species. Nevertheless, isozymes were used in strawberry for cultivar identification (Nehra et al. 1991) and in linkage analysis (Williamson et al. 1995). Like morphological markers, isozyme variation can depend on the environment or age of the plant (Hancock and Bringhurst 1979). Isozymes also exhibit low polymorphism due to the limited number of detected alleles (Khanizadeh and Be´langer 1997; Nehra et al. 1991). In a study using three enzyme assays, PGI, leucine aminopeptidase (LAP), and phosphoglucomutase (PGM), Ga´lvez et al. (2002) characterized 24 strawberry cultivars. Thongthieng and Smitamana (2003) used four enzyme systems (malate dehydrogenase, malic enzyme, leucine amino peptidase, and diaphorase) to analyze strawberry progeny from alternate crosses of four parental lines. They could not identify hybrid lines at either 90 or 95% similarity levels. They recommended using a larger number or another set of enzyme systems for fingerprinting strawberry cultivars. Ga´lvez et al. (2002) and Gambardella et al. (2001) suggested that isozymes could be more effectively applied for verification of cultivars and inferring relationships between groups of cultivars as opposed to fingerprinting.

2.5.3 DNA-Based PCR Markers 2.5.3.1 Random Amplified Polymorphic DNA Random amplified polymorphic DNA (RAPD) markers were the first PCR-based method used for cultivar identification (Williams et al. 1990). These markers are well-distributed throughout the genome, have a rapid non-radioactive detection procedure (Gidoni et al. 1994), and do not require DNA sequence information prior to primer synthesis (Williams et al. 1990; Congiu et al. 2000). RAPD markers are expressed as dominant traits; the amplification with random

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markers proceeds only in the presence of a pair of sequences homologous to that of the primer (~10 bp long) on either one or both homologous chromosomes (Zhang et al. 2003). This molecular marker was adopted as a tool that overcame limitations observed with isozymes such as sensitivity to the environment and the low number of detected alleles (Arulsekar et al. 1981; Hancock et al. 1994; Levi et al. 1994). Identification of closely related strawberry varieties is important in the protection of breeders’ rights. A perfect example of the protection of breeders’ rights using molecular markers was in the settling of a lawsuit where unambiguous identification of a cultivar, “Onebor” (MarmoladaTM), was required by court decree (Congiu et al. 2000). RAPDs were able to distinguish 13 clones of the cultivar “Onebor” (MarmoladaTM) from a group of 31 plants. The use of RAPDs was extended to distinguishing wild species populations in North and South America. These molecular markers partitioned most of the variation among plants within F. virginiana and F. chiloensis populations from North America using analysis of molecular variance (AMOVA) (Harrison et al. 2000) but were unable to discriminate among the four subspecies of F. virginiana (Harrison et al. 1997a). Morphological markers, however, distinguished among the four subspecies of F. virginiana and grouped them into different provenances. Even though RAPD markers could not distinguish between F. virginiana subsp. virginiana and subsp. glauca, they indicated a high within-population variation. In another study, RAPD-based cluster analysis separated the North American (F. chiloensis subsp. lucida and subsp. pacifica) from the South American plants (F. chiloensis subsp. chiloensis) but did not separate the two North American subspecies (Porebski and Catling 1998). These studies suggest that in strawberries, random molecular markers were better suited for discriminating between genotypes (individuals) rather than for revealing relationships among wild populations (Harrison et al. 1997a, 2000). Low levels of reproducibility within and between laboratories, a low level of polymorphism, as well as the inability to detect allelism reduces the usefulness of RAPDs for plant fingerprinting and identification. Low reproducibility results from amplification of DNA using short random primers that do not specifically bind the template (Garcia et al. 2002). Irreproducibility can also result from selecting a subset of the

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bands on agarose gels, usually the more intense ones (Gidoni et al. 1994; Hancock et al. 1994), resulting in variable scores of the same cultivars from different laboratories. Gidoni et al. (1994) observed consistent and significantly lower amplification with two primer–individual combinations that they attributed to mismatches in primer binding or presence of secondary structures in the DNA hindering PCR. Detection of polymorphism and reproducibility using RAPDs can be increased by screening a large set of random primer pairs, carrying out reactions in replicate and maintaining stringent conditions (Gidoni et al. 1994; Hancock et al. 1994; Jones et al. 1997). For example, Porebski and Catling (1998) selected 12 of 100 RAPD primers that were 100% reproducible in replicates of the 35 samples used in the genetic diversity study of North and South American F. chiloensis subspecies. Garcia et al. (2002) repeated amplifications four times with a set of 13 RAPD primers to discriminate among eight accessions to ensure reproducibility and avoid artifacts. They also used polyacrylamide gels to increase the resolution of amplified fragments, which resulted in 37 cultivar-specific bands in only three of those 13 primers. Landry et al. (1997) verified amplification profiles and polymorphism in 75 strawberry cultivars and lines using DNA from two independent microextractions, while Levi et al. (1994) ensured reproducibility by repeating reactions two or three times with eight RAPD primers to check the genetic relatedness among nine strawberry clones. Modifications of the RAPD technique in an effort to minimize disadvantages of using short random primers led to the development of two molecular markers, namely cleaved amplified polymorphic sequences (CAPS) and sequence characterized amplified regions (SCARs). CAPS markers are developed after PCR to reveal variation among individuals of interest. Following PCR amplification of a locus, restriction enzymes are used to cleave the amplified product and reveal polymorphisms resulting from mutations in restriction sites in the different individuals. In strawberry, CAPS markers were developed by Kunihisa et al. (2003) for verification of the identity of strawberry cultivars imported into Japan. Polymorphism detected was reproducible irrespective of DNA extraction method, DNA source tissue (leaves, sepals, or fruit), or laboratories (four different researchers). Six CAPS markers were developed in the study and five

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of these were sufficient to distinguish 14 cultivars from Japan. The development of CAPS markers can be expensive because it involves extensive sequencing (if sequence information is unavailable) and screening for restriction enzyme-genomic locus combinations that yield polymorphic products. In the study by Kunihisa et al. (2003), out of 156 restriction enzyme-genomic locus combinations only nine were polymorphic, a discrepancy explained by the insufficient DNA sequence information. SCARs result from cloning and sequencing a RAPD PCR product, designing longer primers (~20 bp in length) from the ends of the sequenced amplified product, and using these primers for PCR (Paran and Michelmore 1993). The SCAR primers are longer than RAPD primers and subsequently amplify a specific DNA fragment under highly stringent annealing temperatures. A high reproducibility of SCARs results from lack of mismatching in the priming site during amplification experienced when using RAPD primers (Garcia et al. 2002). One of seven RAPD markers developed by Haymes et al. (1997) linked to a red stele resistance gene, Rpf1, in strawberries was converted to a SCAR marker (Haymes et al. 2000) to increase the reproducibility of screening for resistant strawberry cultivars. Two SCAR markers were also developed that are linked to the Rca2 geneencoding resistance to anthracnose (Colletotrichum acutatum) pathogenicity group 2 (Lerceteau-Ko¨hler et al. 2005). The drawback associated with the two modifications, CAPS and SCARs, is the need for the laborious cloning and DNA sequencing for their development.

2.5.3.2 Amplified Fragment Length Polymorphism The amplified fragment length polymorphism (AFLP) technique first described by Vos et al. (1995) involves (1) restriction of DNA template (2) ligation of oligonucleotide adapters (3) pre-amplification, which involves amplification of the DNA with primers that have only one selective nucleotide thus reducing the number of DNA fragments generated and (4) selective amplification of sets of restriction fragments, which are then visualized on sequencing gels or by capillary electrophoresis. AFLP reveals polymorphisms as the presence or absence of a restriction fragment rather than length differences and is consequently scored as a dominant marker (Vos et al. 1995).

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Due to the dependence on restriction and ligation, AFLP requires a high level of DNA purity (Arnau et al. 2001), and degraded or contaminated DNA may result in incomplete restriction digestion (Perry et al. 1998) that does not reflect the true polymorphism present (Vos et al. 1995). High reproducibility and a large number of polymorphic products are the two main advantages of AFLP markers over RAPDs (Schwarz et al. 2000). The first report of the use of AFLP in strawberry was by Degani et al. (2001) who compared the genetic relationships based on pedigree, RAPD (Degani et al. 1998), and AFLP data in 19 strawberry cultivars. Nine cultivarspecific AFLP bands were identified from a total of 228 bands while 35 (15.4%) were polymorphic. These 35 polymorphic markers distinguished the 19 strawberry cultivars. A surprising result was the higher correlation of pedigree data coefficients with RAPD rather than with AFLP similarity coefficients. This result was explained by the possible even distribution of the RAPD markers used across the strawberry genome (Degani et al. 2001). The AFLP technique was also used to identify 19 strawberry genotypes from Poland (Tyrka et al. 2002). Using one restriction enzyme, PstI, they obtained a total of 129 bands of which 22 (17%) were polymorphic. As with RAPDs, AFLPs were converted to SCAR markers that were useful in strawberry breeding. By screening 179 strawberry individuals from a cross of the resistant “Capitola” and susceptible “Pajaro” with 110 EcoRI/MseI AFLP combinations, four AFLP markers were found to be linked in coupling phase to the Rca2 gene responsible for resistance to anthracnose (Lerceteau-Ko¨hler et al. 2005). Two of these markers were converted into SCARs. There was a high (81.4%) level of accuracy in the detection of resistant/susceptible genotypes from a group of 43 cultivars. These developed SCAR markers are useful in the detection of resistance in a marker-assisted selection (MAS) program since they are easier to detect as opposed to the large number of amplified products with the AFLP technique.

2.5.3.3 Microsatellites or Simple Sequence Repeats Microsatellites, also known as simple sequence repeats (SSRs), are stretches of tandemly repeated di-, tri-, or tetra-nucleotide DNA motifs that are abundantly dispersed throughout most eukaryotic genomes

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(Powell et al. 1996; Zhu et al. 2000). These short tandem repeats are found in non-coding and genic regions of the genome (Varshney et al. 2005). In strawberry, ten SSRs were first developed from genomic sequences of F. vesca “Ruegen” (James et al. 2003). Owing to the advantages associated with SSRs, including codominance, multiallelism, and high rates of polymorphism and reproducibility, the number of published Fragaria-derived SSRs has continued to increase. Over 900 Fragaria-derived SSR primer pairs are currently available for molecular studies. These SSRs were developed from genomic libraries (Ashley et al. 2003; James et al. 2003; Sargent et al. 2003; Cipriani and Testolin 2004; Hadonou et al. 2004; Lewers et al. 2005; Monfort et al. 2006; Spigler et al. 2008, 2010), GenBank sequences (Lewers et al. 2005), or EMBL sequences and expressed sequence tags (EST) (Folta et al. 2005; Bassil et al. 2006a, b; Keniry et al. 2006; Spigler et al. 2008, 2010; Zorrilla-Fontanesi et al. 2010). These published SSRs were developed from the diploid F. vesca (James et al. 2003; Cipriani and Testolin 2004; Hadonou et al. 2004; Monfort et al. 2006; Bassil et al. 2006b; Zorrilla-Fontanesi et al. 2010), diploid F. viridis (Sargent et al. 2003), octoploid F. virginiana (Ashley et al. 2003; Spigler et al. 2010), and the domestic strawberry F. ananassa (Bassil et al. 2006a; Gil-Ariza et al. 2006; Zorrilla-Fontanesi et al. 2010). Most SSR primer pairs were developed from the cultivated strawberry, F. ananassa, followed by F. vesca, F. viriginiana, and F. viridis. Each of the studies, except for two (James et al. 2003; Keniry et al. 2006), tested for cross transferability of developed SSRs to species other than the focal species. From one to 15 Fragaria species (excluding the focal species) were used to check cross species SSR transferability in the remaining publications. These studies have reported high levels of cross-species transferability within Fragaria. The highest levels of amplification were observed in the cultivated species, F. ananassa, in studies where it was the focal (Cipriani and Testolin 2004; Hadonou et al. 2004; Bassil et al. 2006a) and the non-focal (Lewers et al. 2005; Bassil et al. 2006b) species. Amplification products were observed in F. ananassa and F. chiloensis from microsatellites developed for F. virginiana (Ashley et al. 2003). Thirty-seven primer pairs developed from F. ananassa “Strawberry Festival” revealed between 89% amplification in F. vesca to 100% amplification in F. chiloensis and F. virginiana (Bassil et al. 2006a). Hadonou et al. (2004) reported

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77–100% transferability of 31 SSRs from F. vesca to other diploids and to the Fragaria octoploids, respectively. With 20 microsatellite primer pairs developed from F. vesca, 95% transferability was observed to F. ananassa (Cipriani and Testolin 2004). This transferability of SSRs between the octoploids and the diploids presents an advantage in comparative mapping and synteny studies in Fragaria (Rousseau-Gueutin et al. 2008). To date, microsatellite markers in Fragaria have been used for cultivar identification (Shimomura and Hirashima 2006), fingerprinting (Govan et al. 2008; Brunnings et al. 2010; Njuguna 2010a; Njuguna and Bassil 2008), genetic diversity analysis (Njuguna et al. 2009b, 2010), and linkage mapping (Sargent et al. 2004, 2006, 2009; Nier et al. 2006; Spigler et al. 2008, 2010). Shimomura and Hirashima (2006) were able to distinguish ten popular Japanese strawberry cultivars using two SSRs developed from “Toyonoka”. The development of SSRs to distinguish these Japanese strawberries was triggered by the infringement of Japanese strawberry breeders’ rights. The first microsatellite fingerprinting set for cultivated strawberry was developed by Govan et al. (2008) at East Malling Research (EMR) in the UK. A set of ten SSR primer pairs, flanking dinucleotide repeats, was selected from 104 that were tested. This set can be multiplexed, reducing cost and time for conducting experiments, and was evaluated in 60 octoploid accessions. The accessions included 56 F. ananassa cultivars and four wild octoploid Fragaria species representatives. The multiplex set was able to discriminate among the genotypes tested and a standard cultivar set was identified that will facilitate the harmonization of allele calling among laboratories. Nine of the ten SSRs in this fingerprinting set was used to fingerprint 26 cultivars and advanced selections from the University of Florida strawberry breeding program (Brunnings et al. 2010). More recently, a reduced fingerprinting set of four SSRs was selected from 91 primer pairs based on multiplexing ability, reproducibility in different labs, ease of scoring, high polymorphism in the domestic strawberry and its immediate octoploid progenitors, and ability to identify each accession from 22 species maintained at the Corvallis National Clonal Germplasm Repository (Njuguna and Bassil 2008; Njuguna 2010a). This reduced set consisted of three of the ten SSRs recommended by Govan et al. (2008) and an additional trinucleotide repeat-containing SSR. Public

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molecular databases of genotypic data generated with universal SSRs and using reference genotypes to harmonize genotype calling will provide a valuable resource for cultivar identification and quick detection of misidentified accessions to the strawberry breeder, grower, nursery, and research communities. The availability of SSR markers that were identified to amplify in Fragaria species of interest through cross-transference studies allows their use in population and diversity analysis. We identified 20 such primer pairs out of 91 SSRs in F. iinumae and F. nipponica and used them to evaluate genetic diversity and population structure of wild Asian diploid species collected from Hokkaido, Japan (Njuguna et al. 2009b, 2010). A model-based Bayesian clustering among accessions representing the two species groups in the program STRUCTURAMATM (Huelsenbeck and Andolfatto 2007) identified ten groups, seven of F. iinumae and three in F. nipponica, which represent the diversity of these species collected from 22 geographical locations in their native habitat. These representative groups of diploids also reflect the population structure: high population structure of the self-compatible F. iinumae is represented by seven groups while low population structure of the selfincompatible F. nipponica is captured by three groups. Preservation of this wild germplasm based on diversity (ten groups) as opposed to the traditional method according to geographical location (22 localities) is more accurate and efficient and will allow the capture and use of the available diversity in this Hokkaido collection.

2.5.4 Linkage Mapping in Strawberry A strawberry linkage map was first constructed from a F. vesca F2 population obtained from a cross between “Baron Solemacher” (BS), a highly homozygous inbred line, and WC6, a wild accession (Davis and Yu 1997). The resulting map was 445 cM in length and contained a total of 79 markers including 75 RAPD markers, an alcohol dehydrogenase locus (Adh), phosphoglucose mutase (Pgi-2) isozyme locus, shikimate dehydrogenase (Sdh) isozyme locus, and the runnering locus. An additional locus, F. vesca fruit color locus (c) that did not segregate in the F2 population in the studies of Davis and Yu (1997) was

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mapped based on its previously established linkage to the Sdh locus (Williamson et al. 1995). Among the 75 RAPD markers mapped to the F. vesca map, 11 were identified as codominant. Codominant RAPD markers were identified after detection of heteroduplex bands following PCR with mixed templates (mixed parent DNA and/or parent DNA mixed with F2 progeny DNA), a method described by Davis et al. (1995). The first genetic linkage map for the octoploid strawberry, F. ananassa, was constructed using AFLP markers (Lerceteau- Ko¨hler et al. 2003). Two putative genes, alcohol transferase (AAT) and the dihydroflavonol reductase (DHFR), were also mapped onto the octoploid map. A full-sib progeny consisting of 113 individuals obtained from a cross of “Capitola” and CF1116 (a reference from the Research and Interregional Experimentation Centre of Strawberry, Ciref, France), was used as the mapping population. Single dose restriction fragments (SDRFs) (a fragment found in only one of the parents) were used to study repulsion phase linked markers, while a pseudo-testcross configuration was used to develop two linkage maps (a female and a male linkage map). A total of 235 and 280 SDRFs were mapped on the female (1,604 cM) and male (1,496 cM) maps, respectively, covering 43 cosegregating groups in each of the maps. AFLP markers were also used to build a genetic map and identify quantitative trait loci (QTL) for day-neutrality in a population of 127 progeny of the day-neutral (DN) “Tribute” and the short-day (SD) “Honeoye” (Weebadde et al. 2008). The map was 1,541 cM in length with 43 linkage groups. Out of the eight QTLs found that were either location-specific or shared among locations, none explained >36% of the phenotypic variation, indicating that the inheritance of day-neutrality is likely a polygenic trait in strawberry. Dominant markers such as RAPDs and AFLPs are not locus-specific and are therefore not easily transferable to other related genomes of similar species or populations (Sargent et al. 2004). The low transportability of dominant markers influenced the use of transferable locus specific markers to create a linkage map to be used as a framework for future mapping studies in Fragaria. Sargent et al. (2004) mapped 68 SSRs, six gene-specific markers and one SCAR marker in an F2 population of 94 seedlings obtained from an interspecific cross of diploid F. vesca F. bucharica L (FV FB). Seventeen of the markers were scored as dominant markers (presence/absence) because they occurred in

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only one of the parents while 58 were codominant. Mapping of SSRs and gene-specific markers creates a good framework for future mapping studies, which include marker-assisted breeding and selection in the cultivated strawberry, positional cloning, and synteny studies that can transfer marker information from the diploid to octoploid relatives within a genus (Davis and Yu 1997; Sargent et al. 2004). SSRs were added to the BS WC6 (Davis et al. 2006) and to the reference map of Sargent et al. (2004), increasing its marker density by 149% (Sargent et al. 2006). To confirm the utility of the reference map as a standard in mapping studies, Nier et al. (2006) developed a reduced linkage map using SSR and gene-specific markers constructed from a wide interspecific backcross between two Fragaria species, F. vesca [F. vesca F. viridis]. In this comparative study, marker order was conserved between both maps on three of the seven linkage groups; genetic distances were similar to those on the reference map. Differences in marker order were attributed to the distant relationship of F. viridis to the diploid species F. bucharica and F. vesca as well as to the octoploid F. ananassa (Potter et al. 2000). A significant reduction in recombination frequencies between markers (and therefore mapping distances) was observed when compared to the reference map. This difference was attributed to a decrease in the frequency of chiasmata formation due to reduced homology between the homeologous chromosomes of the parental species used (Chetelat et al. 2000). Nier et al. (2006) concluded that the reference map generated by Sargent et al. (2004) was useful in generating transferable maps within the Fragaria genus. The diploid reference map (FV FB) of Sargent et al. (2004) was used to select markers for mapping in an F1 population from a cross of F. ananassa cultivars, Red Gauntlet and Hapil (RH H) (Sargent et al. 2009). The use of transferable SSR markers facilitated comparison of the two maps derived from FV FB and RH H crosses, which revealed complete synteny apart from a possible duplicated region observed in the octoploid map. The observed synteny will be useful for future comparative mapping studies. The authors attributed the possible duplicated markers in the octoploid genome to either a consequence of ancient polyploidization event or duplication in one of the diploid progenitors of the polyploids. An SSR-based linkage map was recently constructed in F. virginiana where also sex determination was mapped as two qualitative traits, male and female

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function (Spigler et al. 2008, 2010). The resultant maternal and paternal maps comprised 33 and 32 linkage groups, 319 and 331 markers, respectively. Twenty-eight chromosomes of F. virginiana were assembled into seven groups of four homeologous chromosomes by SSR commonality and comparison to the diploid Fragaria map (Sargent et al. 2006, 2008). Both sex expression traits mapped to the same linkage group (LG6C-m, p), which shared nine SSRs with the diploid LG 6, indicating autosomal origin of this “proto-sex” chromosome. Limited recombination occurs between two linked loci carrying the male and female sterility mutations that control sex determination in F. virginiana. Evidence of recombination between these two loci, an important hallmark of incipient sex chromosomes, suggests that F. virginiana might contain the youngest sex chromosome in plants and provide a novel model system for the study of sex chromosome evolution. Comparison of this map to previously published diploid strawberry maps (Davis et al. 2006; Sargent et al. 2006, 2008) that contained SSR markers in common indicated some conservation of linkages, some rearrangements in the octoploid genome between the diploid LG 1 and LG 6 to create the linkage groups present in the octoploid. Fine mapping and additional comparative analysis will allow better understanding of the evolution of octoploidy and sex determination in strawberry.

2.5.5 DNA Barcoding DNA barcoding, often referred to as barcoding, was proposed as a practical method to identify species by variation in short orthologous DNA sequences from one or a small number of universal genomic regions. In animals, a 600 bp sequence at the 50 end of the mitochondrial gene, cytochrome c oxidase 1 (COX1), was used successfully due to its rapid mutation rate in birds (Hebert et al. 2004a), fish species (Ward et al. 2005), and skipper butterflies (Hebert et al. 2004b). Limited variation in sequence and rapid change of structure in the mitochondrial genome of plants (Chase et al. 2005; Rubinoff et al. 2006) led to the exploration of other genomes for an alternative DNA barcode region. A two DNA barcode system for plants involving the nuclear internal transcribed spacer (nrITS) and the chloroplast psbA–trnH intergenic

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spacer was proposed (Kress et al. 2005). The DNA barcoding technique is simple and can be utilized for routine initial screening of species collections in genebanks. If successful, DNA barcoding could enhance the efficiency of germplasm management by providing a quick method of identification and classification of species. These two proposed DNA barcoding regions, psbA–trnH and nrITS, were tested in Fragaria species preserved at the USDA-ARS repository in Corvallis, Oregon (Njuguna et al. 2009a). The “barcoding gap”, between within species and between species variation, required for discriminating between species was absent, preventing identification of Fragaria species. DNA barcoding did not work for identifying Fragaria species and we believe that it will not identify taxa with little genetic variation.

2.5.6 Chloroplast Genome Markers The size of the chloroplast genome, its non-recombinant nature, and high sequence conservation reduces the complexity of analysis and interpretation of results. This maternally inherited genome in Fragaria was exploited for phylogenetic relationships and to resolve unanswered evolutionary questions. Chloroplast molecular markers included restriction fragment length polymorphisms (cpRFLP) (Harrison et al. 1997a, b), nucleotide sequences (Lin and Davis 2000; Lundberg et al. 2009; Mahoney et al. 2010; Njuguna et al. 2009a), simple sequence repeats (cpSSRs) (Njuguna 2010b), as well as almost complete genome and are listed in Table 2.2. Phylogenetic analysis using chloroplast RFLPs (Harrison et al. 1997a, b) and chloroplast nucleotide

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sequences (Potter et al. 2000; Njuguna et al. 2009a) have resulted in unclear relationships due to the limited variation in this genome. Harrison et al. (1997a, b) used chloroplast DNA RFLP from nine species, and Potter et al. (2000) used the nuclear internal transcribed spacer (nrITS) region and the chloroplast regions, trnL intron and the trnL–trnF spacer in 14 species. These two studies resulted in low resolution of strawberry species relationships that was speculated to result from small taxon sampling and low amount of sequence variation in the genome test regions (Rousseau-Gueutin et al. 2009). Compared to chloroplast sequences of other Rosaceae members, Fragaria seems to have limited variation. Microsatellites in the chloroplast genome (cpSSRs) mostly “A” or “T” mononucleotide repeats, though less variable than nuclear SSRs, have been used in numerous plant genetic studies. The non-recombining nature has been exploited for the design of universal primer pairs flanking chloroplast SSRs distributed across the chloroplast genome. Four universal cpSSRs, ccmp2, ccmp5, ccmp6, and ccmp7 developed in Nicotiana tabacum by Weising and Gardner (1999) were tested in 96 accessions representing 22 Fragaria species. Exploitation of these highly variable regions revealed moderate genetic diversity of these markers in strawberry (mean 0.54) (Njuguna 2010b). Sequencing of cpSSR alleles revealed lack of conservation and even loss (in ccmp6) of the microsatellite repeat in addition to size homoplasy, thus making use of size variation in determining haplotype identity of these universal markers incorrect for inferring phylogenetic inference in Fragaria. For efficient use of limited chloroplast sequence divergence, a large scale sequencing study would be required, now possible with high-throughput sequencing platforms such as Illumina 1G/Solexa (Illumina

Table 2.2 List of Fragaria chloroplast genome sequences used, the number of species from which the sequences were obtained, and the reference Chloroplast sequence # Fragaria species Reference trnL intron and trnL–trnF 14 Potter et al. (2000) rps18–rpl20 and psbJ–psbF 4 Lin and Davis (2000) trnL–trnF and trnS–trnG 3 Lundberg et al. (2009) psbA–trnH 21 Potter et al. (2000) and Njuguna et al. (2009a) 18 Njuguna et al. (2009a) YCF2/ORF2280 30 -ORF79 and ndhB 50 exon to rps7 50 end Chloroplast SSR sequences 50 to trnS, 30 to rps2, ORF77-ORF82, atpB–rbcL 18 Njuguna (2010b)

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Inc., San Diego, CA), 454 Life Sciences GS 20 (454 Life Sciences, Branford, CT) and/or SOLiD (Applied Biosystems, Foster City, CA). Sequencing multiple small genomes by taking advantage of the high sequencing depth of high-throughput sequencing platforms was recently tested (Cronn et al. 2008). Sequencing of complete plastome sequences using Illumina technology was demonstrated in pines (Cronn et al. 2008). A range of 88–94% coverage of the chloroplast genome was obtained from 36 bp single read sequencing in one lane of the Solexa flow cell of four different barcoded PCR products of pine species. Multiplexing of small organellar genomes in single lanes utilizes the sequencing depth, of up to 40 million clusters per flowcell (Morozova and Marra 2008). We used three different approaches for sequencing Fragaria chloroplast genomes with the Illumina Genome Analyzer: PCR amplification, physical chloroplast isolation, and plastome assembly from low coverage genomic sequencing (Njuguna 2010a, b). Low coverage genomic sequencing was identified as the most efficient approach for obtaining complete chloroplast genome sequences. Preliminary analysis of the sequencing data confirmed maternal inheritance of the chloroplast in Fragaria and identified F. vesca subsp. bracteata as the maternal donor to the octoploids (F. chiloensis, F. virginiana and F. ananassa subsp. cuneifolia) and the decaploid, F. iturupensis. Complete chloroplast genome sequences will be useful in revealing polymorphisms in plant species groups that have little or no detected variation such as Fragaria (Harrison et al. 1997a, b; Potter et al. 2000) facilitating species relationship resolution.

2.5.7 Mitochondrial DNA Markers Mitochondrial DNA (mtDNA) has received the least attention compared to nuclear and plastid genomes. Mahoney and Davis (2010) described the first mtDNA markers in the matR gene region in Fragaria. This marker provided evidence that mtDNA was transmitted maternally in two interspecific crosses, and that diploid F. iinumae is the likely mtDNA donor to the octoploid species F. chiloensis and F. virginiana but not to decaploid F. iturupensis. Additional Fragaria mtDNA sequences were obtained by assembling a 67 kb mtDNA contig from Illumina 36 bp paired-end reads of “Pawtuckaway”, providing

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a basis for the development of additional mtDNA markers.

2.6 Genomics Resources Developed The diploid F. vesca was adopted as a model perennial representative of the Rosaceae family. Resources were developed for two other model species for the Rosaceae, apple (Malus domestica) and peach (Prunus persica) (Shulaev et al. 2008). Advantages of F. vesca include its small genome size (~200 Mbp/1C), short generation time, transformation efficiency, self-compatibility, and abundant seed production (Shulaev et al. 2008). The following gives a brief overview of the genomic resources now available for strawberry and is not meant to be exhaustive. Since July 2005, when approximately 7,000 genomic and cDNA sequences were listed in GenBank (Davis et al. 2007), the number of Fragaria entries has increased to 60,429 nucleotide sequences in April, 2010. The majority of these sequences are ESTs, which account for 58,573, mostly from F. vesca (47,743), followed by F. ananassa (10,830). The Genome Database for Rosaceae (GDR) is an important resource for the Rosaceae research community that was initiated in response to the growing availability of genomic data for peach and has benefited the strawberry community. GDR is a curated and integrated web-based relational database that provides centralized access to Rosaceae genomics and genetics data and analysis tools to facilitate cross-species comparison and use of this data (Jung et al. 2004, 2008). Current strawberry resources enabled by initial funding by the NSF Plant Genome Program in 2003 are available at http://www.rosaceae.org/node/31 and include: Two diploid linkage maps [FV FN diploid reference map (Sargent et al. 2006) and 815 903 BC map (Nier et al. 2006)] viewed and compared through the comparative map viewer CMap; A fourth assembly of unigenes from publicly available ESTs of diploid and polyploid strawberry that contains a total of 13,896 putative unigenes; and lists and links to currently funded strawberry projects and to other public strawberry databases. One F. vesca and another F. ananassa assembly from the National Center for Biotechnology Information (NCBI) nucleotide and EST sequences are available at The Institute for Genomic Research (TIGR) Plant Transcript

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Assembly website (Childs et al. 2007) and can be downloaded at http://plantta.jcvi.org/cgi-bin/plantta_release. pl. Sequences from F. vesca were assembled into 4,825 contigs and 8,624 singlets while those from F. ananassa include 358 contigs and 4,778 singlets. Large insert bacterial artificial chromosome (BAC) libraries of strawberry have been reported as constructed or under construction (Davis et al. 2007; Shulaev et al. 2008). However, a fosmid library has been constructed from F. vesca subsp. americana “Pawtuckaway” (Davis et al. 2007) and used for GeneTrek analysis (Pontaroli et al. 2009). Assembly of the resulting ~1 Mb of the nuclear genomic DNA identified 158 genes arranged in gene-rich regions and intermixed with transposable elements (TEs). Of over 30 classified repeat families, long terminal repeat (LTR) retrotransposons were the most abundant in F. vesca and comprised ~13% of the genome sequence analyzed. This study predicted the F. vesca genome to contain at least 16% of its content in TEs, about 30,500 protein-encoding genes, and over 4,700 truncated gene fragments (Pontaroli et al. 2009). In addition to nuclear isozyme and PCR-based RAPD, SCAR, CAPS, AFLP, and SSR markers described in the previous section, a limited number of gene-specific markers exist in strawberry (Davis and Yu 1997; Deng and Davis 2001; Sargent et al. 2007). Sequence tag sites (STS) were developed for the alcohol dehydrogenase ADH gene (Davis and Yu 1997), five genes in the anthocyanin biosynthesis pathway and one associated transcription factor (Deng and Davis 2001) and 24 genes of known function based on publicly available mRNA sequences (Sargent et al. 2007). Novel markers referred to as Gene Pair Haplotype (GPH) markers are being developed in strawberry (Tom Davis personal communication) and are expected to be highly transferable from F. vesca to other strawberry species and even other genera in the Rosaceae. Many research groups are developing additional markers from the increasing sequence data available for strawberry and adding them to their diploid and octoploid linkage maps. The addition of codominant SSR, STS, gene-specific markers to these maps allows comparison among diploid and octoploid maps (Spigler et al. 2008, 2010) and assessment of colinearity among the homologous chromosomes and processes involved in the evolution of octoploidy in strawberry. Thermal asymmetric interlaced PCR (hiTAIL-PCR) was recently used to amplify the flanking region surrounding the left or right border of the T-DNA in 108

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of these unique single copy mutants. Markers (based on presence/absence, length and CAPS polymorphism) were developed to 74 of the T-DNA insertion lines and were mapped in the reference diploid F. vesca 815 F. bucharica 601 population (RuizRojas et al. 2010). Efficient transformation protocols and availability of mutants are necessary for forward and reverse approaches of elucidating gene function. Several reviews on tissue culture and transformation of strawberry were published (Folta and Davis 2006; Debnath and da Silva 2007). Efficient Agrobacterium-mediated transformation and rapid regeneration appears genotype-specific in strawberry and has been reported for F. vesca “Hawaii-4” (Oosumi et al. 2006), F. ananassa “L-9” (Folta and Davis 2006). One approach, T-DNA mutagenesis or “gene tagging”, to generate mutants is a technique used for generating loss-of-function mutations in genes by mobile or introduced DNA with a known sequence (T-DNA in this case) and was used in strawberry (Shulaev et al. 2008). These T-DNA mutants are expected to provide resources for reverse genetics in addition to novel markers as demonstrated by Ruiz-Rojas et al. (2010). A comprehensive review of functional molecular and biotechnology studies in strawberry was recently published (Schwab et al. 2009). In this genomic era, strawberry resources are expected to increase dramatically with increased federal funding and recent advances in next-generation sequencing. A Strawberry Genome Sequencing Consortium, comprised of experts in a wide array of research areas, was created in the spring of 2008 with the goal of sequencing the genome of “Hawaii-4” using nextgeneration technologies (Shulaev et al. 2010). Current support for GDR by the USDA Specialty Crop Research Initiative as part of tree fruit Genome Database Resources (tfGDR) will allow expansion of this database to include whole genome sequences and annotations for strawberry, transcript data, metacyc pathways, largescale phenotype and genotype data, breeding data, controlled vocabularies, and new analysis tools. SCRI funding for “RosBREED: Enabling Marker-Assisted Breeding in Rosaceae” promises to deliver highthroughput genome scan platforms and integrate breeding and genomic resources by implementing marker-assisted breeding primarily in four fruit crops including strawberry (Iezzoni et al. 2010). The genome

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sequence of F. vesca and bioinformatics tools to analyze such data through the GDR database, among others, will provide a valuable resource for future studies of comparative genomics in the Rosaceae, evolution of polyploidy in Fragaria and phylogenetic relationships among members of this economically important family of temperate fruits.

2.7 Functional Improvements The strawberry fruit contains thousands of metabolites, which strongly impact consumer’s senses and health (Schwab et al. 2009). Most analytical biochemical studies of strawberry fruits have relied on specific extraction/separation methods to identify and quantify specific compounds and interests. The strawberry flavor is complex. One comprehensive non-targeted metabolic analysis of strawberry identified 5,844 unique spectrophotometric peaks by analyzing fruits at four developmental stages (Aharoni et al. 2002). Many artificial strawberry flavors use only a handful of the top compounds to cheaply imitate the true constituents, and the human taste recognizes the difference. Schwab et al. (2009) summarizes the genetic work concerning volatile and polyphenolic compounds including metabolic routes and associated genetic mechanisms. Fruit firmness, a genetically complex trait, has been a focal point of many large breeding programs during the past 50 years. Though “firm” strawberries is the primary complaint of consumers of commercial strawberry fruit throughout the world, this trait has provided the strawberry industry with the capability to move fruit to the far reaches of the globe and capitalize on strawberry as a product. Breeding for firmness is a difficult task, complicated as Salentjn et al. (2003) has pointed out, because of the inverse correlation between firmness and flavor emissions. Recent breakthrough in developing fruit with flavor and firmness are the new dictum of the present commercial breeding programs. Strawberries are rich in vitamin C, ascorbic acid, and ellagic acid. Both compounds have a significant role in promoting human health. The amount of ellagic acid varies between cultivars and between different plant parts. Because of the variability of these compounds between different cultivars, molecular genetic studies will be examining major qualitative trait loci involved

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in strawberry vitamin C and ellagic acid biosynthesis to be mapped for molecular breeding efforts.

2.7.1 Allergens As in other fruits, strawberries contain proteins, which can cause allergic reaction in humans (Schwab et al. 2009). The strawberry FRA a 1 protein family is homologous to the major birch pollen allergene Bet v 1 and includes several IgE-binding peptides with small intra- and intergenotype sequence variability though subjected to post-translational modifications. Profilins and lipid transfer proteins (LTP), found in strawberries, are also represented in other cultivated crops in the rose family. Strawberry LTP and profilins are expressed in many fruit tissues and accumulate with abiotic stress (Yubero-Serrano et al. 2003). Some studies have found that strawberry LPT had lower allergenicity than apple or peach homologs. The strawberry allergens are in the range suited for immunotherapy (Zuidmeer et al. 2006).

2.8 Biotechnological Approaches to Strawberry Improvement: Benefits and Risks 2.8.1 Benefits The potential for positive application of biotechnology to strawberry, as with other fruits and vegetables, is limited by the lack of public approval of breeding through genetic manipulation (Hummer and Hancock 2009; Mezzetti 2009). The cost of research and development is high, and regulatory approval is tortuous and prohibitive. Experimentation with perennials is expensive, relative to annual crops. Thus, biotechnological application of molecular and genetic development of fruit crops through transgenes has not progressed since the early 1980s, when techniques first became available. Transformation of the octoploid strawberry has been well-documented (Mezzetti 2009), but acceptance of the products has not been given, so the industry has suppressed this research.

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If transgenes were accepted for strawberry development, many advances could be made efficiently including: • Development of glyphosate resistant cropping systems, which could help farmers who have lost methyl bromide • Improved root rot resistance – also help for the loss of methyl bromide • Promoted flowering and fruiting • Quality – maturation genes for a non-climacteric fruit • Tissue softening genes (for firmness) • Carbohydrate development for flavor and processing quality • Disease and pest – virus diseases • Cold hardiness • Parthenocarpic fruiting gene

2.8.2 Risks Several obstacles work against the acceptance of transgenic strawberries. The global economic value of this fruit crop (while high per acre) is small in total because much fewer acres are planted than that of agronomic crops. As a result, governments are not flocking to support this technology, and private stimulus is modest. The fruit industry has been reluctant to introduce products with potential negative backlash from people leery of consuming transgenic crops. A second obstacle is the tendency of strawberries to be outcrossing. Their flower is open and insect pollination is the norm. In each of the locations, where strawberries are cultivated, native relatives are widespread. These species relatives could incorporate transgenes into wild biological systems. For this reason, release of transgenic strawberries will require more scrutiny and in depth ecological surveys than have been performed in other agricultural crops. A strong influx of funds for thorough testing and environmental examination is needed before transgenic strawberries could be examined. Careful analysis of people’s perceptions regarding transgenic fruit is also required. Until this happens, transgenic strawberries will remain as a research tool without commercialization. Using marker-free transformation systems and targeted expression of transgenes will minimize

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public concern, but the fear of technology must be abated before transgenic strawberries will be commonly accepted.

2.9 Recommendations for Future Actions The studies of genetics and genomics of the Fragaria genome are proceeding at unparalleled rates. Comparative genomics and fine mapping can elucidate the processes involved in polyploidization and the evolution of sex determination in the octoploid species and are under way. Strawberry researchers are working within the rose family community and beyond to share information and relate genes and gene patterns. This newfound knowledge will be used by traditional breeders to develop improved cultivars in advanced fruit quality, expanded growing ranges, and during all seasons. Recent findings have overturned some older paradigms. Previously, Staudt (1999a, b) suggested that the diploid F. vesca, an old species, could have an origin as early as the Cretaceous period. A significant preliminary finding using Bayesian analyses of complete chloroplasts obtained by high-throughput sequencing (Njuguna 2010a, b) contradicts this timeline and indicates that Fragaria, as a genus, is young and evolved less than 5 million years ago. Also, the octoploids evolved perhaps only 2.7 million years ago. Further exploration and study of Fragaria crop wild relatives using next-generation and even third generation technology will shed light on the evolution of Fragaria and its polyploidy. Exploration has confirmed that hybridization of strawberry species in nature, such as the production of F. bringhurstii and an unnamed Chinese pentaploid (Lei et al. 2005), and decaploid F. virginiana subsp. platypetala (Davis et al. 2010) is a more frequent occurrence than suggested by Darrow (1966) or Staudt (1999a, b). Staudt (1999a, b) postulated that the first octoploid probably arose in East Asia and migrated from the west via an Alaskan–Siberian land bridge to North America. He had thought that F. iturupensis, which he first observed as the only Asian octoploid, might be a missing link. Decaploidy in F. iturupensis complicates this view. Further study of strawberries of northern Pacific Islands is needed to determine where other higher ploidy

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strawberry colonies exist and what their phylogenetic role may have been. With the finding of the clustering of F. vesca subsp. bracteata with the North American octoploid species, the possibility of an American origin for the octoploids has been suggested. Additional explorations should be taken in Asia and North America to seek potential missing links that may have contributed to the evolution of the American octoploids. This is a key time in uncovering the evolution of Fragaria and the development of the cultivated strawberry. Global interest and communication have brought the international strawberry research community together. The formation of a strawberry sequencing consortium and federal funding for many Rosaceae projects that include strawberry will lead to unprecedented discoveries for this model perennial crop. The International Treaty on Plant Genetic Resources recognized the importance of strawberry as an Annex 1 crop. The Global Crop Diversity Trust was instrumental in bringing together a scientific team to prepare a global conservation strategy for strawberry, which was completed (Hummer 2008, 2009). These activities have expanded the awareness of crop wild relatives for Fragaria in a positive fashion in most countries. Yet the strawberry conservation strategy is not being implemented due to lack of resources. Several countries, where centers for diversity of strawberry species reside, have not recognized Fragaria as a sufficiently important genus worth establishing in a national genebank. Although individual universities, institutes, and scientists continue to study the genus and provide information, the security of wild strawberry species and landraces within these countries is unrecognized and potentially vulnerable to loss. Thankfully, the conservation strategy has been formed. Local implementation, institutional support, and world recognition is paramount for the continued conservation of critical landraces and subspecies and crop wild relatives of Fragaria.

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Chapter 3

Malus Alexander Ignatov and Anastasiya Bodishevskaya

3.1 Basic Botany of the Species The apple tree belongs to the family Rosaceae that has been divided into four subfamilies by fruit type: Rosoideae, Prunoideae, Spiraeoideae, and Maloideae (Malus, Pyrus, and Cotoneaster) (Potter et al. 2002). Recent phylogenetic analyses of nuclear and chloroplast DNA provided the division of Rosaceae only into three subfamilies: Dryadoideae, Rosoideae, and Spiraeoideae (including tribe Pyreae and the genus Malus – Apples) (Potter et al. 2007). Actually, as few as eight and as many as 79 species within the genus Malus (Miller) have been recognized by morphological and biochemical studies (Ponomarenko 1992), and this number must be verified by comprehensive DNA analysis. Five to six sections within the genus were described with consensus – Malus, Sorbomalus, Eriolobus, Docyneopsis, and Chloromeles (Rehder 1940; Langenfelds 1991; Robinson et al. 2001). The section Malus includes the series of Malus, Baccatae, and Siebolbiianae. The section Sorbomalus includes the series of Yunnanenses, Kansuenses, and Florentinae (Robinson et al. 2001). All Malus species are trees or shrubs 3 to 12 m tall, with a broad, twiggy crown, deciduous or semievergreen, and usually unarmed. Leaves are alternate, simple, petiolate, and merely toothed, margin serrate or lobed. Pomes are not containing stone-cells or they are present in a few species. Fruits are 1- or 2-seeded

A. Ignatov (*) Molecular Phytopathology Group, Center ‘Bioengineering’ of Russian Academy of Sciences, Prospect 60-letia Oktyabria, bld. 7/1, Moscow 117312, Russian Federation e-mail: [email protected]

in each cell, with cartilaginous endocarp (core). Seeds are brown or black, and cotyledons plano-convex. Apple trees have long generation time, 3–8 years, from seed-to-seed and sometimes longer. One tree produces up to 700 (5–10 per fruit) seeds. Apple trees are hermaphrodites, with both sexes within the same flower, usually self-incompatible (Janick et al. 1996), with moderate to severe inbreeding depression. Alleles of self-incompatibility code proteins that prevent fertilization by pollen of a similar genotype. In Malus, incompatibility is controlled by a series of genetic sequences known as S (self-incompatibility) alleles. The first 11 S-alleles were found by F. Kobel and colleagues in 1939 (Kobel 1954), and now, up to 25 different alleles are known (Juniper and Mabberley 2006). Several researchers suggest that large herbivores could be major dispersers of seeds for apple and providers of seedbeds for germination, when Crab apples are mostly bird-dispersed (Juniper and Mabberley 2006). Most of wild apple species were found in the mountains of central and inner Asia, western and southwestern China, Far East, and Siberia (Table 3.1; Zhou 1999). It was suggested that this region could be a valuable resource of genetic diversity for important horticultural and environmentally adapted traits (Korban 1986; Dickson and Forsline 1994). Generally, southwestern China and inner and central Asia are “a sanctuary and breeding ground for . . . many thousands of plant species. . . this area today has 10 times the number of plant species of western Europe. . .” (Juniper and Mabberley 2006), and obviously, this region is the largest center of diversity of the genus Malus in general (Juniper et al. 1999; Harris et al. 2002). The cultivated apple has 34 chromosomes per diploid genome, although, some Crab apple species such

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_3, # Springer-Verlag Berlin Heidelberg 2011

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46 Table 3.1 Taxonomy of Malus species (modified from Germplasm Resources Information Network – GRIN) Section Malus 1 Malus asiatica Nakai Synonyms: Malus prunifolia var. rinkii (Koidz.) Rehder; Malus ringo Siebold ex Carrie`re; Pyrus ringo Wenz. 2 Malus chitralensis Vassilcz. Borkh. ¼ Malus pumila Mill. 3 Malus domestica Borkh. Synonyms: Malus malus (L.) Britton, nom. inval.; Malus pumila auct.; Malus pumila var. domestica (Borkh.) C. K. Schneid.; Malus sylvestris auct.; Malus sylvestris var. domestica (Borkh.) Mansf.; Pyrus malus L. 4 Malus floribunda Siebold ex Van Houtte; Synonyms: Pyrus floribunda G. Kirchn. 5 Malus kirghisorum Al. Fed. & Fed. ¼ Malus sieversii var. kirghisorum (Al. Fed. & Fed.) Ponomar. 6 Malus microcarpa var. robusta Carrie`re ¼ Malus robusta (Carrie`re) Rehder 7 Malus muliensis T. C. Ku 8 Malus niedzwetzkyana Dieck ¼ Malus pumila Mill. [¼ M. pumila “Niedzwetzkyana”] 9 Malus orientalis Uglitzk. Synonyms: Malus sylvestris subsp. orientalis (Uglitzk.) Browicz 10 Malus paradisiaca (L.) Medik. ¼ Malus pumila Mill. 11 Malus praecox Borkh. ¼ Malus pumila Mill. 12 Malus prunifolia (Willd.) Borkh. Synonyms: Pyrus prunifolia Willd. ; Malus prunifolia var. rinkii (Koidz.) Rehder ¼ Malus asiatica Nakai 13 Malus paradisiaca (L.) Medik.; Malus praecox Borkh.; Malus pumila var. niedzwetzkyana (Dieck) C. K. Schneid.; Malus pumila var. paradisiaca (L.) C. K. Schneid.; Malus sylvestris var. niedzwetskyana (Dieck) L. H. Bailey; Malus sylvestris var. praecox Ponomar.; Pyrus malus var. paradisiaca L.; Pyrus niedzwetzkyana (Dieck) Hemsl.; Pyrus praecox Pall.; Malus pumila Auct.; ¼ Malus domestica Borkh. 14 Malus pumila var. domestica (Borkh.) C. K. Schneid. ¼ Malus domestica Borkh.; Malus pumila var. niedzwetzkyana (Dieck) C. K. Schneid. ¼ Malus pumila Mill.; Malus pumila var. paradisiaca (L.) C. K. Schneid. ¼ Malus pumila Mill. ; Malus dasyphylla Borkh. 15 Malus sieversii (Ledeb.) M. Roem. Malus sieversii subsp. turkmenorum (Juz. & Popov) Likhonos ¼ Malus sieversii var. turkmenorum (Juz. & Popov) Ponomar. Malus sieversii var. kirghisorum (Al. Fed. & Fed.) Ponomar. Synonyms: Malus kirghisorum Al. Fed. & Fed. Malus sieversii var. turkmenorum (Juz. & Popov) Ponomar. Synonyms: Malus sieversii subsp. turkmenorum (Juz. & Popov) Likhonos; Malus turkmenorum Juz. & Popov 16 Malus frutescens Medik. 17 Malus spectabilis (Aiton) Borkh. Synonyms: Pyrus spectabilis Aiton 18 Malus sylvestris (L.) Mill. Malus sylvestris auct. ¼ Malus domestica Borkh.; Malus sylvestris subsp. orientalis (Uglitzk.) Browicz ¼ Malus orientalis Uglitzk.; Malus sylvestris var. domestica (Borkh.) Mansf. ¼ Malus domestica Borkh.; Malus sylvestris var. niedzwetskyana (continued)

A. Ignatov and A. Bodishevskaya Table 3.1 (continued) (Dieck) L. H. Bailey ¼ Malus pumila Mill. [¼ M. pumila “Niedzwetskyana”]; Malus sylvestris var. praecox Ponomar. ¼ Malus pumila Mill. 19 Malus zhaojiaoensis N. G. Jiang Section Sorbomalus 20 Malus bhutanica (W. W. Sm.) J. B. Phipps; Synonyms: Malus toringoides (Rehder) Hughes; Malus transitoria var. toringoides Rehder; Pyrus bhutanica W. W. Sm. 21 Malus crataegifolia (Savi) Koehne ¼ Malus florentina (Zuccagni) C. K. Schneid. 22 Malus florentina (Zuccagni) C. K. Schneid. Synonyms: Crataegus florentina Zuccagni; Malus crataegifolia (Savi) Koehne; Pyrus crataegifolia Savi 23 Malus fusca (Raf.) C. K. Schneid. Synonyms: Malus diversifolia (Bong.) M. Roem.; Malus fusca var. diversifolia (Bong.) C. K. Schneid.; Malus rivularis (Douglas) M. Roem.; Pyrus diversifolia Bong.; Pyrus fusca Raf.; Pyrus rivularis Douglas; Malus fusca var. diversifolia (Bong.) C. K. Schneid.; ¼ Malus fusca (Raf.) C. K. Schneid. 24 Malus honanensis Rehder 25 Malus kaido Dippel ¼ Malus prattii (Hemsl.) C. K. Schneid. 26 Malus kansuensis (Batalin) C. K. Schneid. Synonyms: Pyrus kansuensis Batalin 27 Malus komarovii (Sarg.) Rehder Synonyms: Crataegus komarovii Sarg. 28 Malus maerkangensis M. H. Cheng et al. 29 Malus ombrophila Hand.-Mazz. 30 Malus prattii (Hemsl.) Schneid. Synonyms: Malus kaido Dippel; Pyrus prattii Hemsl. 31 Malus sargentii Rehder. Synonyms: Pyrus sargentii (Rehder) Bean 32 Malus sieboldii (Regel) Rehder ¼ Malus toringo (Siebold) Siebold ex de Vriese; Malus sieboldii var. arborescens Rehder ¼ Malus toringo (Siebold) Siebold ex de Vriese; Malus sieboldii var. calocarpa Rehder ¼ Malus zumi (Matsum.) Rehder; Malus sieboldii var. zumi (Matsum.) Asami ¼ Malus zumi (Matsum.) Rehder 33 Malus yunnanensis (Franch.) C. K. Schneid. Synonyms: Pyrus yunnanensis Franch.; Malus yunnanensis var. Veitchii Rehder; Malus yunnanensis var. Yunnanensis 34 Malus yunnanensis (Franch.) C. K. Schneid. Synonyms: Pyrus yunnanensis Franch.; Malus yunnanensis var. Veitchii Rehder; Malus yunnanensis var. Yunnanensis. Section Chloromeles 35 Malus angustifolia (Aiton) Michx. Synonyms: Pyrus angustifolia Aiton 36 Malus bracteata Rehder ¼ Malus coronaria (L.) Mill. 37 Malus brevipes (Rehder) Rehder 38 Malus cerasifera Spach ¼ Malus mandshurica (Maxim.) Kom. ex Skvortsov 60. Malus coronaria (L.) Mill. Synonyms: Malus bracteata Rehder; Malus coronaria var. dasycalyx Rehder; Malus fragrans Rehder; Malus glabrata Rehder; Malus glaucescens Rehder; Malus lancifolia Rehder; Pyrus coronaria L.; Malus coronaria var. dasycalyx Rehder ¼ Malus coronaria (L.) Mill. (continued)

3 Malus Table 3.1 (continued) 39 Malus frutescens Medik. 40 Malus ioensis (Alph. Wood) Britton Synonyms: Malus ioensis var. texana Rehder ; Pyrus coronaria var. ioensis Alph. Wood; Pyrus ioensis var. texana (Rehder) L. H. Bailey Section Eriolobus 41 Malus trilobata (Poir.) C. K. Schneid. Synonyms: Crataegus trilobata Poir.; Eriolobus trilobata (Poir.) M. Roem.; Pyrus trilobata (Poir.) DC.; Sorbus trilobata (Poir.) Heynh. Section Gymnomeles 42 Malus baccata (L.) Borkh. Malus baccata f. gracilis Rehder ¼ Malus baccata var. baccata [¼ M. baccata var. baccata “Gracilis”]; Malus baccata f. jackii Rehder ¼ Malus baccata var. baccata [¼ M. baccata var. baccata “Jackii”]; Malus baccata subsp. himalaica (Maxim.) Likhonos ¼ Malus baccata var. himalaica (Maxim.) C. K. Schneid.; Malus baccata var. baccata Synonyms: Malus baccata f. gracilis Rehder; Malus baccata f. jackii Rehder; Malus baccata var. sibirica C. K. Schneid.; Malus pallasiana Juz.; Malus sibirica (Maxim.) Kom.; Pyrus baccata L.; Pyrus baccata var. aurantiaca Regel; Pyrus baccata var. genuina Regel; Malus baccata var. cerasifera (Spach) Koidz. ¼ Malus mandshurica (Maxim.) Kom. ex Skvortsov 43 Malus baccata var. daochengensis (C. L. Li) Ponomar. Synonyms: Malus daochengensis C. L. Li; Malus baccata var. himalaica (Maxim.) C. K. Schneid. Synonyms: Malus baccata subsp. himalaica (Maxim.) Likhonos; Malus rockii Rehder; Pyrus baccata var. himalaica Maxim.; Malus baccata var. jinxianensis (J. Q. Deng & J. Y. Hong) Ponomar. Synonyms: Malus jinxianensis J. Q. Deng & J. Y. Hong; Malus baccata var. mandshurica (Maxim.) C. K. Schneid. ¼ Malus mandshurica (Maxim.) Kom. ex Skvortsov; Malus baccata var. sibirica C. K. Schneid. ¼ Malus baccata var. baccata; Malus baccata var. xiaojinensis (M. H. Cheng & N. G. Jiang) Ponomar. Synonyms: Malus xiaojinensis M. H. Cheng & N. G. Jiang; Malus baoshanensis G. T. Deng; Malus daochengensis C. L. Li ¼ Malus baccata var. daochengensis (C. L. Li) Ponomar. 44 Malus dasyphylla 45 Malus floribunda var. spontanea Makino ¼ Malus spontanea (Makino) Makino. 46 Malus halliana Koehne 47 Malus hupehensis (Pamp.) Rehder Synonyms: Malus theifera Rehder; Pyrus hupehensis Pamp 48 Malus sikkimensis (Wenz.) Koehne ex C. K. Schneid. Synonyms: Pyrus sikkimensis Hook. f.

as M. ioensis, M. toringo, M. toringoides, and M. sikkimensis have triploid set of chromosomes (Tatum et al. 2005). Two basic hypotheses are considered (1) the basic haploid number of Malus (n ¼ 17)

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has appeared as a result of hybridization between Prunoideae (n ¼ 8) and Spiroideae (n ¼ 9) (Sax 1931; Derman 1949; Challice and Westwood 1973); (2) the basic number n ¼ 7, common in other genera of Rosaceae, was doubled, when three of the chromosomes were repeated three times, giving n ¼ 17 (Darlington and Moffett 1930). The study of DNA content by flow cytometric analysis has indicated that the difference between diploid genotypes was equal to the DNA amount of almost six chromosomes (Tatum et al. 2005).

3.2 Conservation Initiatives Ragan (1926) studied horticultural publications of the nineteenth century and found description for over 7,000 cultivars with a range of fruit quality, harvesting period, and tolerance to abiotic and biotic stresses. Profitable international trade and efficient systems for long-term storage and transportation led to dramatic changes in the assortment of apple cultivars. A few successful commercial apple cultivars (sports of “Golden Delicious/Delicious” and few others) give up to 90% of the apple production that is extensively eroding apple tree genetic resources (Hokanson et al. 1998). The loss of genetic diversity has caused significant problems with new pests, diseases, and abiotic stresses including frost, heat, salinity, and drought. To solve these problems, genetic resources of apple tree are enriched through the use of resistant genotypes obtained through interspecific hybridization with Crab apples. For instance, some new cultivars have scab resistance obtained from M. floribunda, but new races of the pathogen Venturia inaequalis can break this type of resistance (Brooks and Vest 1985; Hokanson et al. 1997). Genetic collections include many hundreds of apple varieties selected over centuries in Europe, Asia, and North America and more recently in South Africa and Chile. Unfortunately, the use of wild apple germplasm from the native population of M. sieversii was not opened for international research community until 1989 (Dickson and Forsline 1994). Only recently, seeds of the wild apple trees from forests of Kazakhstan were sampled (Hokanson et al. 1998) in order to study and distribute these valuable genetic resources with more genetic diversity than all cultivated apple

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trees have together. Wild germplasm, from the apple’s center of genetic diversity, contains a lot of variation for such important traits as tolerance to the stresses, texture, aromatics, storage life of fruits, suckering, growth vigor, tree architecture, juvenility, chilling requirement, days to flowering, and fruit maturing (Korban 1986; Forsline 1996). There are several initiatives to reintroduce Malus species into natural forests and preserve stands of wild plants in the US, Kazakhstan, and the Caucasus (Hokanson et al. 1997). Collections of apple genetic resources are maintained in a number of countries, including: the US (PGRU; Geneva, NY: ~4,000 accessions); Germany (BAZ Dresden/Pillnitz: ~1,200 accessions), Russia (Vavilov’s All Russian Research Institute of Plant Industry: ~3,800 accessions, including 3,600 M. domestica); Sweden (Nordic Genetic Resource Center: ~1,000 accessions), New Zealand (HortResearch: 650 accessions), Poland (The Botanical Garden Center For Biological Diversity Conservation: 700 accessions), and Latvia (Latvian State Institute of Fruit Growing: 351 accessions). Most of the apple-growing countries have genetic collections that maintain local and elite commercial cultivars. Thus, the Malus collections still represent mostly genotypes of current commercial value rather than the ones of high genetic diversity (Hokanson et al. 1997). The collected plants are evaluated under natural or artificial conditions for most important traits and documented. The main emphasis of such evaluation is on identifying sources of resistance to biotic and abiotic stresses and determining the inheritance type of such traits. A core subset (Frankel and Brown 1984) established for evaluation of specific genetic traits may include about a hundred to a few hundred accessions or clones. A number of strategies are possible for creating the core collections, depending on the available material and the purpose of the collection (van Hintum 1999). Wiedow et al. (2004) evaluated diversity among 1,030 seedlings, obtained from 35 wild plants of M. sieversii, using 500 amplified fragment length polymorphism (AFLP) loci and 64 morphological/phenotypic characters evaluated over several years (1998–2004), and selected representative core collection that encloses 36% genotypes (369 individuals) and reflects the overall diversity of the studied M. sieversii population. Natural hybrids (Table 3.2) and mapping populations could be a valuable source of genetic diversity

A. Ignatov and A. Bodishevskaya Table 3.2 The hybrids in genus Malus (Germplasm Resources Information Network – GRIN). No. Species 1 Malus adstringens Zabel ¼ M. baccata (L.) Borkh. M. domestica Borkh. 2 Malus arnoldiana (Rehder) Sarg.¼ M. baccata (L.) Borkh. M. floribunda Siebold ex Van Houtte 3 Malus atrosanguinea (Spaeth) Schneid. ¼ M. halliana Koehne M. sieboldii (Regel) Rehder 4 Malus dawsoniana Rehder ¼ M. fusca (Raf.) Schneid. M. domestica Borkh. 5 Malus hartwigii Koehne ¼ M. baccata (L.) Borkh. M. halliana Koehne 6 Malus heterophylla Spach ¼ M. Coronaria (L.) Miller M. domestica Borkh. 7 Malus micromalus Makino ¼ M. baccata (L.) Borkh. M. spectabilis (Aiton) Borkh. 8 Malus moerlandsii Doorenbos ¼ M. purpurea “Lemoinei” M. sieboldii (Regel) Rehder 9 Malus purpurea (Barbier) Rehder ¼ M. atrosanguinea (Spaeth) Schneid. M. sieversi F. niedzwetzkyana (Dieck) Langenf. ¼ M. atrosanguinea M. Pumila; Synonyms: Malus floribunda var. lemoinei E´. Lemoine; Malus floribunda var. purpurea A. Barbier; Malus purpurea f. eleyi (Bean) Rehder; Malus purpurea f. lemoinei (E´. Lemoine) Rehder; Malus purpurea var. aldenhamensis Rehder; Pyrus eleyi Bean; Pyrus lemoinei R. N. Notcutt ex anon.; Malus purpurea f. eleyi (Bean) Rehder ¼ Malus purpurea (A. Barbier) Rehder [¼ M. purpurea “Eleyi”]; Malus purpurea f. lemoinei (E´. Lemoine) Rehder ¼ Malus purpurea (A. Barbier) Rehder [¼ M. purpurea “Lemoinei”] Malus purpurea var. aldenhamensis Rehder ¼ Malus purpurea (A. Barbier) Rehder [¼ M. purpurea “Aldenhamensis”] 10 Malus robusta (Carriere) Rehder ¼ M. baccata (L.) Borkh. M. prunifolia (Willd.) Borkh. Synonyms: Malus microcarpa var. robusta Carrie`re; Pyrus baccata var. cerasifera Regel 11 Malus scheideckeri Spath ex Zabel ¼ M. floribunda Siebold ex Van Houtte M. prunifolia (Willd.) Borkh. 12 Malus soulardii (Bailey) Britt. ¼ M. ioensis (Wood) Britt. M. domestica Borkh. Synonyms: Pyrus soulardii L. H. Bailey 13 Malus sublobata (Dipp.) Rheder ¼ M. prunifolia (Willd.) Borkh. M. sieboldii (Regel) Rehder Synonyms: Malus ringo var. sublobata Dippel 14 Malus zumi (Matsum.)Rehder ¼ M. mandshurica (Maxim.) Komarov M. sieboldii (Regel) Rehder

because they have some recombinants with new properties. The majority of plants in the collections are clonally propagated and preserved as duplicated orchard trees. As a space-saving alternative, the accessions are stored in liquid nitrogen. Cryopreservation

3 Malus

allows the storage of plant tissues for a long time, in sanitary and genetic safety and at low cost (Forsline et al. 1998). New cryogenic techniques allow direct immersion of plant material in liquid nitrogen (“onestep freezing”) (Lambardi and de Carlo 2002).

3.3 Role in Elucidation of Origin and Evolution of Allied Crop Plants Apple tree is one of the most important fruit crops grown in the temperate regions. Many varieties have been developed for better-tasting fruits and attractive trees and flowers. Over 64 million tons of apple fruits were harvested worldwide in 2008, holding the fourth place among the most important agricultural products. Due to original adaptation to growth at highlands, including late blooming and good cold hardiness, apples can be cultivated in the cool temperate zone from about 35–50 latitude. At the same time, apples are adaptable to subtropical climates such as Brazil or South Africa. As a result, apples are produced commercially in 89 countries on almost 5 million ha (FAO 2008). On the species level, two names are generally applied by different scientific communities: Malus domestica Borkh and M. pumila P. Mill (Korban and Skirvin 1984). The name Malus domestica was suggested for all cultivated cultivars of diverse hybrid origin, but it is more probable that most of the apple varieties are related to one wild species native to central Asia – M. sieversii. Vavilov (1930) described large stands of M. sieversii in native mountain forest in Kazakhstan, Kyrgyzstan, and some other central Asian countries. Genetic diversity of those plants was wider than one of the other populations of related Malus species, and he suggested the region as a center of genetic diversity and, probably, origin for domesticated apple. The cultivated apple tree and closely related species M. sylvestris (L.) Miller, M. orientalis Uglizhikh, and M. sieversii (Lebed.) Roem. are placed at the same botanical section. M. sylvestris, and M. orientalis found in central, northern, and southern Europe and in Caucasian region and Iran (Harris et al. 2002) are suspected escapes from the central core of Malus species, migrated to west at different times (Juniper and Mabberley 2006). M. sieversii has been recognized

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as a major progenitor of cultivated apple by many researchers and by different methods (Ponomarenko 1987; Juniper et al. 1999; Forte et al. 2002). Due to the close genetic relationship to cultivated apple, M. sieversii could be a valuable genetic resource for the improvement of domesticated apple with regard to both horticultural and environmentally adapted traits (Korban 1986). Analysis of Malus chloroplast genome provides data about evolutionary relationships of the apple’s maternal line. Sequencing of 1,341 bp of matK gene showed only 16 phylogenetically informative changes within accessions of the genus Malus (Robinson et al. 2001). However, two duplications found near the end of the matK coding region, one of 8-bp, and second – a perfect 18-bp repeat, were present together in both the central Asian M. sieversii and the domesticated apple, supporting the conclusion of Watkins (1995) that the central Asian wild plant is the major maternal parent of domestic cultivars. But Coart et al. (2006) brought some new data on chloroplast matK gene diversity between M. domestica, M. sieversii, and M. sylvestris, indicating uniqueness of M. domestica cytoplasmic genome and reopened the discussion about the origin of domestic apples. Such difference in conclusions could be at least partly explained by non-representative sampling of wild apple genepool. Sequence of nuclear ribosomal internal transcribed spacer (ITS) has supported the grouping of M. sieversii with the domesticated apple, as well as with M. asiatica, M. orientalis, M. niedzwetzkyana, and M. prunifolia (all from section Malus), but variability within this group was not significant (Robinson et al. 2001). Unfortunately, molecular methods of higher taxonomic resolution such as simple sequence repeat (SSR) or random amplified polymorphic DNA (RAPD) do not always produce groups consistent with known pedigree information and/or geographic origins of the accessions of the genus Malus (Hokanson et al. 2001; Forte et al. 2002; Yan et al. 2008), although both SSR and AFLP markers were highly useful in studies of intrasection variation between accessions of M. domestica, M. sieversii, M. sylvestris, M. asiatica, M. orientalis, M. niedzwetzkyana, and M. prunifolia (Coart et al. 2003; Wiedow et al. 2004; Gharghani et al. 2009). Cultivation of apples has been known for at least 3,000 years in Greece and Persia. The ancient Greeks

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were familiar with the technique of grafting that was described by Theophrastus in the third century BC. Romans had knowledge about the techniques of grafting, rootstocks, and budding. Pliny the Elder, a Roman statesman, described about 20 different varieties of cultivated apples in his Historia Naturalis in 23 AC. By the first century AC, apples were being cultivated in every region throughout the Rhine Valley (Zohary and Spiegel-Roy 1975). Way et al. (1990) gave about 10,000 apple varieties distinguished by shape, color, texture, firmness, crispness, acidity, juiciness, sweetness, and harvesting period. A lot of them are probably sports or seedlings from smaller number of successful genotypes. Most apple cultivars mature within 120–150 days after bloom, but there are early maturing cultivars (from 70 days) and the late ones (up to 180 days). Due to cross-fertilization, the apples have a high degree of genetic heterozygosity, and all known cultivars are, in fact, somatic clones, propagated vegetatively. Seedlings are used in plant breeding and for rootstock propagation. The apple breeding involves laborious artificial self- and cross-pollination, evaluation of large segregating progenies for a number of physiological and morphological traits, and demands vast land plots and long time for field trials.

3.4 Role in Development of Cytogenetic Stocks and Their Utility There is a limited research data on apple plants with modified chromosome number. Some species of Malus include plants of triploid and tetraploid chromosome set. Tetraploid “giant” sports appear by chance on some cultivars of domestic apple (Janick et al. 1996). There is much less of such observations on Crab apple species. The first haploid plants of apple were obtained by Einset (1945) from open-pollination of a triploid variety. These plants did not survive. The first attempts of apple haploidization by anther culture in vitro gave unstable results (Nakayama et al. 1971; Milewska-Pawliczuk and Kubicki 1977), but further experiments demonstrated usefulness of in vitro androgenesis, gynogenesis, and in situ parthenogenesis (Lespinasse et al. 1999).

A. Ignatov and A. Bodishevskaya

3.5 Role in Classical and Molecular Genetic Studies In recent years, molecular biology has provided valuable tools, suitable for detailed analysis of complex genomes. Innovative molecular techniques, particularly the use of molecular markers, can considerably accelerate and improve apple breeding. This can be accomplished by looking directly into the plant’s DNA sequences at the seedling stage without waiting for the expression of the trait itself. Moreover, the use of molecular markers allows the selection of F1 plants carrying more than one gene for resistance, reducing labor and avoiding time-consuming test crossing. Use of DNA markers is beneficial for traits that are difficult to score (e.g., powdery mildew resistance), for “masked” genes revelation, in the construction of linkage maps as well as for distinguishing closely related biological accessions and accurately cataloging the germplasms. DNA (molecular) markers represent genetic differences between individual organisms or species. Generally, they do not represent the target genes themselves but act as “signs”. Such markers themselves do not affect the phenotype of the trait of interest because they are only located in close proximity to genes controlling the trait. Besides DNA markers, which reveal sites of variation in DNA, there are two other types of genetic markers: morphological (phenotypic traits) and biochemical (allelic variants of enzymes – isozymes and secondary metabolites) (Challice and Westwood 1973). Morphological and biochemical markers, which have been extremely useful to plant breeders, have, however, some disadvantages. They may be limited in number and are influenced by environment or the developmental stage of the plant that is in contrast to DNA markers (Currie et al. 2000). Molecular markers, the most widely used type of genetic markers, arise from different classes of DNA mutations such as substitution mutations (point mutations), rearrangements (insertions or deletions), or errors in replication of tandem repeated DNA (Bretting and Widrlechner 1995). A lot of markers linked to monogenic traits, mainly resistance to pathogens and pests, have been found for Malus sp. as well (Guilford et al. 1997; Oraguzie et al. 2001).

3 Malus

In Malus, RAPD markers linked to genes controlling scab resistance such as Vf gene (Koller et al. 1994; Tartarini et al. 1999); Vm gene (Cheng et al. 1998); Vx, Vr (Hemmat et al. 2002); Vb, Va (Hemmat et al. 2002), Vbj (Gygax et al. 2004), and rosy leaf curling such as Sd1 conferring resistance to rosy leaf curling aphid (Dysaphis devecta) (Roche et al. 1997) were identified. RAPD markers (Williams et al. 1990) are very sensitive to the experimental conditions that lead to poor reliability and reproducibility outside the original laboratory (Tartarini et al. 1999). More advanced AFLP technique (Vos et al. 1995) combines the efficiency of PCR-based markers, such as RAPD, with the specificity and reliability of RFLP (Powell et al. 1996). In Malus, AFLP markers were widely used to assess genetic diversity and verify plant identity (Coart et al. 2003; Wiedow et al. 2004) for marker development (James and Evans 2004; Patocchi et al. 2004) as well as for genetic mapping (Liebhard et al. 2003; Kenis and Keulemans 2005). The disadvantage of AFLP markers is their dominant nature that results either from restriction site presence or absence, not allowing differentiation between homo- and heterozygous alleles. The problem of RAPD’s reproducibility and AFLP’s dominance may be solved by development of sequence-characterized amplified regions (SCAR) or sequence-tagged sites (STS) markers derived by cloning and sequence-specific RAPD/AFLP markers. SCAR markers detect a single locus and may be codominant (Tartarini et al. 1999). For example, RAPD markers M18 and AM19 (Gianfranceschi et al. 1996; Tartarini et al. 1999) tightly linked to the Vf resistance gene were converted to robust SCARs and used for map-based cloning of Vf (Patocchi et al. 1999), resulting in isolation of the Vf-region (Vinatzer et al. 2004). RAPD markers that transformed subsequently into SCARs were identified for powdery mildew resistance genes Pl1 (AT20-SCAR; Markussen et al. 1995) and Pl2 (N18-SCAR; Seglias and Gessler 1997). To date, SCARs closely linked to scab resistance are widely used for breeding purposes, since they enable the identification of complex genetic combinations and pyramidization of different resistance sources. Identification of SSR markers linked to useful traits has been based on complete linkage maps or the

51

construction of partial maps. SSR markers have been developed more recently for major crop plants and have been found very useful for diversity study and cultivar identification of apple tree (Guilford et al. 1997; Hokanson et al. 1998; Liebhard et al. 2002; Coart et al. 2003). The SSR markers have increased the speed with which genetic maps can be constructed. The combination of new microsatellite marker information with previously generated, mostly dominant marker data allows the construction of the integrated maps (Maliepaard et al. 1998). More than 300 SSRs were developed for Malus. They are represented in HiDRAS SSR Database (http://users.unimi.it/hidras/). The majority of them have been already published (Guilford et al. 1997; Seglias and Gessler 1997; Maliepaard et al. 1998; Liebhard et al. 2002, 2003; Silfverberg-Dilworth et al. 2006). The availability of several saturated apple linkage maps sharing a high number of common SSRs (Liebhard et al. 2003; Silfverberg-Dilworth et al. 2006) has facilitated the placement of resistance genes and quantitative trait loci (QTLs) to such diseases as scab (Gygax et al. 2004; Bus et al. 2005a, b), powdery mildew (Evans and James 2003; Dunemann and Schuster 2008), and fire blight (Peil et al. 2007) to a specific linkage group. Presence of the same SSR markers across different Rosaceae genera and Malus species is an additional advantage of markers. Having a very detailed SSR map with highly polymorphic SSRs, it is possible to find a linkage between a trait and an SSR without generating a complete genetic map. This strategy was called genome scanning approach (GSA) and was proposed by Patocchi and Gessler (2003). The method consists of testing a reduced number of resistant progeny plants with a few selected and well-spaced SSRs per linkage group (Patocchi et al. 2005). GSA allows detection of a distortion of the expected 1:1 segregation ratio of the SSR alleles of the resistant parent among a small subset of progeny plants (all susceptible or resistant). Such distortion of allele segregation is due to linkage between an SSR and the resistance gene or to naturally occurring distortions linked to lethal factors (Erdin et al. 2006). GSA method allowed the mapping of apple scab resistance genes such as Vb, Vr2, and Vm (Patocchi et al. 2004, 2005; Erdin et al. 2006). SSR

52

markers linked to the scab resistance genes can be incorporated into multiplex PCR reactions and analyzed on automatic sequencers with short running times. An increased amount of available sequence information about functionally characterized target genes as well as genetic maps harboring genes of known function and QTLs have led to the development of the candidate gene approach in molecular genetics. Candidate gene analysis is based on the hypothesis that known-function genes (the candidate genes) could correspond to loci-controlling traits of interest (Pflieger et al. 2001). Candidate genes are either genes with molecular polymorphisms genetically linked to major loci or QTLs or genes with molecular polymorphisms statistically associated with variation of the studied trait. The third type of the candidate genes, named structural candidate genes, can be exemplified with the resistance gene analogs (RGAs) isolation of which is based on conserved motifs of the domains of resistance genes and not on physiological function. To select the most promising candidates from a large number of putative candidate genes, gene sequences are tested for linkage to the trait of interest. Population-based fine-mapping experiments allow precise localization of both candidate genes and the loci. Another strategy is association study, which allows testing of correlation between phenotypic variation and molecular polymorphisms within candidate genes in a set of genealogically unrelated individuals. Finally, such candidate genes, for which map cosegregation and/or statistical correlation have been found, must be validated. It means that complementary experiments such as genetic transformation must be conducted to confirm whether the candidate gene is the gene determining the trait variation. A validated candidate gene may be used for identification of varieties as well as for marker-assisted selection. Successful application of the candidate gene approach in plant genetics and breeding is reviewed in many publications and has been utilized in Malus too, for example, to detect anthocyanin biosynthesis and regulatory genes (Chagne´ et al. 2007), aroma biosynthetic genes that were ethylene regulated (Schaffer et al. 2007), and candidate genes involved in ethylene production during apple ripening (Costa et al. 2007). The candidate gene analysis as a powerful approach for identifying and isolating agronomically important genes controlling qualitative traits became a strategy

A. Ignatov and A. Bodishevskaya

of many Malus studies (Boudichevskaia et al. 2004, 2006; Patocchi et al. 2009).

3.6 Role in Crop Improvement Through Traditional and Advanced Tools Apple cultivars are highly heterozygous hybrids from intra- or interspecific hybridization that are propagated somatically. Because of the hybrid nature of plants, severe inbreeding depression in self-pollinated progenies, and problems in biotechnological plant haploidization/di-haploidization (Lespinasse et al. 1999), there are many features peculiar to apple tree breeding and genetics. Fertile hybrids are obtained from most of pollination combinations between different Malus species (Korban 1986), and interspecific hybrids were widely used for the improvement of commercial apple cultivars. Some wild apples are suggested natural hybrids (Table 3.2) and can be successfully propagated by seeds. The main ancestor of the domestic apple is M. sieversii, a species with a high degree of diversity: from almost inedible “Crabs” to fruits similar to some modern cultivars, found in mountainous areas in Southeast Kazakhstan on the border between China, Kazakhstan, Kyrgyzstan, and Tajikistan to the edge of the Caspian Sea (Hokanson et al. 1998; Juniper et al. 1999; Harris et al. 2002). Identification and integration of elite genotypes of M. sieversii into the apple breeding programs brings new alleles associated with plant resistance and desirable fruit properties into the narrowing genetic pool of modern cultivars of apple tree. Controlled pollination carried out on nine M. sylvestris clones by use of two pollen mixtures collected from five M. domestica or five M. sylvestris trees demonstrated that fruit set in interspecific (successful in 63% of crosses) and intraspecific (50%) crosses was not significantly different (Larsen et al. 2006). Breeding of a new apple variety takes as long as 15 years from the first crossing to release a commercial apple cultivar. Before crossing, the flowers of the seed parent should be emasculated, and the pollen of the known parent is transferred to female parent flowers. To prevent contamination with unknown pollen, the artificially fertilized flowers are isolated by bags. For successful germination, seeds from ripen fruits are stored in moist conditions at temperature near 5 C

3 Malus

for stratification for period up to 14 weeks. A juvenile period from 3 to 8 or more years slows down the process of pomes evaluation and sexual propagation for obtaining segregating progenies. It depends on the genotype, environmental conditions and the cultural practices, and can be shortened by grafting seedlings onto faster flowering dwarfing rootstocks (Rehder 1940). Improved fruit quality is a major objective of all fruit breeding programs, but seedlings should be first screened for basic agronomic characteristics: disease, pest, and abiotic stress resistance, tree architecture, high yield, and only the most promising lines go on to further testing. Fruit quality is a complex trait that includes size, color, texture, taste, flavor, and the shape of pomes, storage, and shelf-life. It can be evaluated in array of instrumental and expert assays. Still, a lot of resistance genes came from other species of Malus (Table 3.3). Presently, European and North American institutions are leading in the breeding of new apple varieties. In US, the disease-resistant apple breeding program is cooperative among several institutions, including Purdue University, Rutgers, State University of New Jersey, and the University of Illinois. Hopefully, the use of biotechnology tools in apple breeding programs will accelerate the development of new cultivars. The apple plants are easily transformed by argobacterial infection, and improvement of apple cultivars by genetic engineering is speeding up the breeding process (Aldwinckle et al. 2003). AppleBreed Database (Antofie et al. 2007) was developed within the framework of the European project “High-quality Disease Resistance in Apples for Sustainable Agriculture” (HiDRAS; Gianfranceschi and Soglio 2004) to support breeders in exploration of germplasm collections and genetic research. It provides information for over 2,000 apple genotypes that may assist in search for molecular markers of important agronomic traits of apple fruit quality: ripening, softening; fruit taste: acidity, sweetness, flavor, and polyphenols. It can be useful as well in selection of parental cultivars for further cross-pollination and breeding.

3.6.1 Apple Scab Apple scab, caused by the fungal pathogen V. inaequalis (Cke.) Wint., is one of the main problems in apple-growing areas worldwide and one of the most

53

costly to control. Scab lesions greatly reduce the fruits’ overall quality, decrease fruit production, and increase susceptibility to invasions by various other pathogens. Currently, the strategy for apple scab control relies on multiple applications of fungicides, often up to 20 fungicide sprays each growing season. If not controlled, the disease can cause extensive losses (70% or greater). The factors influencing the disease severity are: humid, cool weather occurs during the spring months, sanitation, topography, cultivar susceptibility, and the frequency of infection periods (Gessler et al. 2006). V. inaequalis successfully colonizes only the species of Malus. Malus species and cultivars show different levels of resistance to scab, expressed pits, chlorotic flecks, or necrotic lesions that may or may not contain conidia. All commercially important apple cultivars are susceptible to scab in the field (Koch et al. 2000). Symptoms of scab infection occur on leaf blades, fruit, petioles, sepals, blossoms, young shoots, and bud scales. The most common and obvious symptoms occur on leaves and fruit. Firstly the lower and later both surfaces of the leaves can become infected. Young lesions are velvety brown to olive green and have firstly indistinct margins, which become clear with time. The tissues adjacent to a lesion thicken, and the leaf surface becomes deformed. The number of lesions per leaf may range from one or two to several hundred. Lesions on young fruit appear similar to those on leaves, but as the infected fruit enlarges, the lesions become brown and corky. Cracks then appear in the skin and flesh, or the fruit may become deformed. When a pathogen successfully invades the host and causes disease, the pathogen is termed virulent, the host is said to be susceptible and the infection to be compatible. On the contrary, if a pathogen is unable to establish a compatible (disease) interaction in a host cultivar since it activates defense responses that suppress pathogen colonization, the pathogen is termed avirulent, the host is resistant and the interaction is incompatible. In 1899, Aderhold had already pointed out that V. inaequalis is not a single entity but can be divided into distinct physiological isolates distinguishable by their different ability to induce sporulating lesions or only flecks on various cultivars (Gessler et al. 2006).

54

A. Ignatov and A. Bodishevskaya

Table 3.3 Sources of useful traits among wild apple species (modified from Gessler et al. 2006) Pathogen, source of resistance Type of Gene name References resistancea Apple scab (Venturia inaequalis) M. atrosanguinea 804 “tree-type” M Allelic to Vf Dayton and Williams (1968) M. atrosanguinea 804 “pit-type” M Vm Dayton and Williams (1970) M. microM. 245-38 “tree-type” M Allelic to Vf Dayton and Williams (1968) M. microM. 245-38 “pit-type” M Vm Dayton and Williams (1970) Hansen’s M. baccata # 2 M Vb Dayton and Williams (1968), Williams and Kuc (1969) M. baccata jackii M Vbj Dayton and Williams (1968) M. floribunda 821 M Vf Hough (1944), Hough et al. (1953), Dayton and Williams (1968) M. pumila R12740-7A Complex Na Dayton et al. (1953) race 2 differential M Vh2 Bus et al. (2005b) race 4 differential M Vh4 Bus et al. (2005b) M. sieversii GMAL 2473 M Vr2 Patocchi et al. (2004) M. prunifolia 19651 M Allelic to Vf Shay et al. (1953), Williams et al. (1966) M. prunifolia microcarpa 782-26, Hansen’s baccata # 1 M Allelic to Vf Williams et al. (1966) M. prunifolia xanthocarpa 691-25, M.A. 4, M.A. 8, M Allelic to Vf Williams and Dayton (1968) M.A. 16 and M.A. 1255 Apple scab (Venturia inaequalis) Jonsib crab, Cathay crab M M. sargenti 843, M. sieboldii 2972-22, M. toringo 852, P M. ringo 840 M. zumi calocarpa P

Na

Powdery mildew (Podosphaera leucotricha) M. robusta MAL 59/9 M. robusta “Korea”, 24-7-7,8 M.alusx zumi MAL 68/5 White angel, crab apple M. baccata jackii

M M Complex M M

Pl1 Na Pl2 Plw Plbj

M. baccata 419 M. florentina M. hupehensis

M Nd Complex

Na Na Na

M. mandshurica M. microM.

Nd Nd

Na Na

Powdery mildew (Podosphaera leucotricha) M. platycarpa M. sieboldii 27, 722 M. sargentii 843

Nd M M

Na Na Na

M. trilobata

Nd

Na

Knight and Alston (1968) Schuster (2000) Korban and Dayton (1983), Caffier and Parisi (2007) Urbanietz and Dunemann (2005)

Crown rot (Phytophthora cactorum) M. angustafolia, M. magdeburgensis

Nd

Na

Cummins and Aldwinckle (1983)

Cedar apple rust (Gymnosporangium juniperi-virginianae) M. sieversii, M. sublobata “Novole”, M. haliana Nd M. baccata jackii Nd

Na Na

Hokanson et al. (1998)

Valsa canker (Valsa ceratosperma) M. sieboldii

Na

Bessho et al. (1994)

Nd

Na Na

Dayton and Williams (1968) Shay et al. (1962), Williams and Kuc (1969) Shay et al. (1953) Knight and Alston (1968) Gallott et al. (1985) Knight and Alston (1968) Gallot et al. (1985) Korban and Dayton (1983), Dunemann and Schuster (2008) Schuster (2000) Schuster (2000) Knight and Alston (1968), Caffier and Parisi (2007) Caffier and Parisi (2007) Fisher and Fisher (2004)

(continued)

3 Malus Table 3.3 (continued) Pathogen, source of resistance

55

Type of resistancea

Gene name

References

Fire blight (Erwinia amylovora) M. robusta “Persicifolia” M. sublobata cv “Novole”

Nd Nd

Na Na

M. floribunda, M. prunifolia, M. robusta 5 Maus atrosanguinea

M, P nd

Na Na

M. fusca

Nd

Na

M. sieversii

Nd

Na

Fisher and Fisher (2004) Aldwinckle and Beer (1976), Fisher and Fisher (2004) Fisher and Fisher (2004), Peil et al. (2007) Aldwinckle and Van der Zwet (1979), Aldwinckle and Beer (1976) Aldwinckle and Van der Zwet (1979), Aldwinckle and Beer (1976) Hokanson et al. (1998)

Wooly apple aphid (Eriosoma lanigerum) M. robusta 5 “Aotea 1”, M. Sieboldii M. halliana, M. hupehensis, M. Tschonoskii

M M Nd

Er2 Er3 Na

Knight et al. (1962) Knight et al. (1962), Bus et al. (2009) Cummins et al. (1981)

Rosy apple aphid (Dysaphis plantaginea) M. robusta MAL 59/9

M

Smh

Alston and Briggs (1970)

Rosy curling aphid (Dysaphis devecta) M. robusta MAL 59/9, M. zumi MAL68/5

M

Sd3

Alston and Briggs (1970)

Na not available; Nd no data

Shay et al. (1962) identified three physiological races of V. inaequalis, and among them, race 1 is a well-sporulating isolate on popular domestic cultivars commonly found in US and other countries. Race 2 infected Malus clones “Dolgo”, “Geneva”, and certain offspring of R12740-7A (“Russian Seedling”). Race 3 sporulated on “Geneva”. Races 4 to 7 were detected from 1968 to 1994 (Parisi et al. 1993; Benaouf and Parisi 2000). It was discussed in many studies that Vf resistance is more complex than the single Vf gene and composed of at least two functionally different forms of resistance, Vf and Vfh. The latest discovered race 8 was described for a compatible reaction with an M. sieversii host genotype carrying a new scab resistance gene named as Vh8 (Bus et al. 2005a). Gessler et al. (2006) discuss that the concept of race as a fixed genetic unit is not valid for an obligatory sexually reproducing organism, and that the term race cannot indicate more than the presence of the virulence/avirulence for which the isolate is tested. A race is, therefore, just a group of genotypes sharing the particular characteristic of being able or not to sporulate on a given set of hosts, or “differential” set. In principle, two different strategies in scab resistance breeding are currently followed. One of them is

based on polygenic scab resistance sources, preferably under the participation of QTL mapping (Liebhard et al. 2003; Calenge et al. 2004). Such quantitative resistance is not based on receptor–elicitor recognition and thus appears to work equally across all pathogen strains. Therefore, it is often hypothesized that the quantitative resistance is more durable than monogenic resistances. Polygenic resistance, however, is sensitive to environmental conditions and is difficult to handle in practical breeding and has not yet been widely taken into account in apple breeding. The other strategy favored presently by apple breeders is to pyramid several distinct major resistance genes into a single cultivar in the hope that the pathogen will not be able to undergo a sequence of mutations corresponding to each resistance gene. An overview of the position of known major scab resistance genes and scab resistance QTLs was recently summarized by Gessler et al. (2006). It was reported that at least 11 chromosomes contain scab resistance factors in different progenies (major genes or QTLs). Some of the major scab resistance genes and QTLs have been mapped to the same regions on the apple genome, suggesting the presence of gene clusters. Typically, resistance gene clusters are known in many crop-disease systems and are common in plants

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infected by (hemi-) biotrophic pathogens (Parlevliet 2002). Among the major scab resistance genes mapped onto the apple genome, there are Vf (Maliepaard et al. 1998), Va, and Vb (Hemmat et al. 2002) mapping to linkage group (LG) 1; Vbj (Gygax et al. 2004), Vh2, Vh4, and Vh8 (Bus et al. 2005a, b), Vr2 (Patocchi et al. 2004), and Vx (Hemmat et al. 2002) mapping on LG 2; Vb (Erdin et al. 2006), Vg (Calenge et al. 2004), and Vm (Patocchi et al. 2005) mapping to LG 12. The sources of the above-mentioned resistance genes are represented in A2. Almost all scab-resistant cultivars released from the different apple breeding programs in the world carry the Vf scab resistance gene derived from the small-fruited species Malus floribunda clone 821 (Laurens 1999). With the appearance of scab races 6 and 7, major attention has been focused on other scab resistance factors, particularly the “Antonovka” selections and apple genotype R12740-7A (“Russian seedling”, RS), which are currently used in several apple breeding programs. The scab resistance of the Russian cultivar “Antonovka” is often referred to as polygenic, but the evidence is not very clear-cut (Gessler et al. 2006). The reason is that the name “Antonovka” involves different genotypes, which show a marked variability and low similarity, indicating that the various “Antonovka” accessions are not clones but genetically different (Hemmat et al. 2002). There are at least several “Antonovka” selections, which are sources of monogenic scab resistance. One of them is Schmidt’s “Antonovka” PI172623. Its resistance gene designated as Va induces a hypersensitive pit-type reaction and located at a different locus from that of the Vm and Vf genes. “Antonovka” PI 172633 also shows a hypersensitive pit-type reaction, suggesting that it also has the Va gene (Lespinasse et al. 1999). This resistance gene was mapped on the same linkage group as the Vf gene but at a different position (Hemmat et al. 2002). “Antonovka Monasir”, known as a polygenic source of resistance, showed the scab resistance segregation typical for a single dominant gene (Gessler et al. 2006) in repeated field experiments . R12740-7A is another Russian source of scab resistance derived from domestic apple and identified in the United States from openpollinated apple seeds obtained from Russia in 1935. The genetics of the race-specific resistance is well but not yet completely understood. At least three major genes, one of them race-non-specific and the other two

A. Ignatov and A. Bodishevskaya

specific for races 2 and 4, respectively, are involved in this resistance (MacHardy 1996). The race-specific genes Vh2 and Vh4 (triggering a hypersensitive response, HR) were mapped by Bus et al. (2005a) on the apple linkage group LG 2 using differential segregates of R12740-7A. Bus et al. (2005a) tested the molecular marker S22-SCAR that Hemmat et al. (2002) developed for the R12740-7A scab resistance gene they called Vx (also inducing HR). Because the molecular marker was mapped close to Vh4, and both genes promote HR and derive from the same selection, Bus et al. (2005a) concluded that Vh4 and Vx are the same gene. The existence of a third (race-non-specific) resistance gene has been postulated for a long time (Shay et al. 1962), but the genetic and molecular evidence for that gene are still missing. Over the last 20 years, more than 200 scab resistance cultivars were released by apple breeders. Some of these cultivars are with proven fruit quality that appears to be commercially acceptable (e.g., “Liberty”, “Florina”, “Goldrush”). The program of apple breeding for scab (V. inaequalis) resistance was initiated at Purdue University (USA) in 1945. By the 1990s, over 1,500 selections have been made, and 44 resistant clones have been released for advanced testing (Crosby et al. 1992). In Dresden-Pillnitz (Germany), during 1972–1998, around 52,000 seeds were produced by protected crossing of parents, and 26,000 survived seedlings were resistant after artificial inoculation with scab in the greenhouse. Only 552 of those seedlings were selected after the first round of field trial, 92 of them were left after second round, resulting in only three released apple cultivars (Peil et al. 2008). Cost and putative economic profit of such programs cannot be estimated precisely, because of highly unpredictable output in both number of resulting cultivars and their market share. Growers interested in organic fruit production should strongly consider planting such varieties. However, to date, even in countries like Switzerland and Germany, where integrated and organic production systems are well developed, scab-resistant apples account for no more than 5–6% of the market; they stand at less than 1% overall in Italy , (reviewed in Sansavini 2004). Yet none of released scab-resistant cultivars could compete with standard cultivars on the global market. Sansavini (2004) emphasized on three main reasons explaining such situation (1) scab resistant cultivars are not well-known, (2) their diversity

3 Malus

has not been appropriately advertised, and, maybe the most important, (3) their sensory qualities generally are neither equal to nor better than the most popular cultivars. The authors drew an unfavorable conclusion that the organic apple industry continues to rely on “Golden Delicious” and other susceptible cultivars (which require more than 15 treatments/year) and not on those more suitable to such a market because of their reduced need for disease and pest-control sprays.

3.6.2 Powdery Mildew Powdery mildew is the second major disease of apple following scab, caused by the obligate biotrophic ascomycete fungus Podosphaera leucotricha (Ell. & Ev.) Salm. Powdery mildew occurs wherever apples are grown. Such factors as climatic conditions, cultivar susceptibility, and cultural practices have an impact on the size of economic loss from mildew. In western Europe, for example, because of mild winter temperatures and highly favorable environmental conditions during spring, 15 or more fungicide sprays are needed to control the disease. Powdery mildew attacks leaves, blossoms, and stems of apple trees, resulting in leaf abscission, shoot stunting, and an overall devitalization of the tree. Powdery mildew may infect the fruit, resulting in an unattractive apple covered with a network pattern of cork cells. Such apples are frequently used for processing rather than for fresh marketing. At present, the majority of apple cultivars of worldwide economic importance are still susceptible. Several sources (both qualitative and quantitative) of resistance to powdery mildew in apple are known (Dayton 1977). Very recently, Calenge and Durel (2006) mapped at least four genes for quantitative resistance (QTLs) to powdery mildew in an F1 apple progeny derived from “Discovery” TN10-8, which are both partially resistant to mildew. The set of stable QTLs on linkage groups 2, 8, and 13 are of interest for breeding purposes, especially if combined with other major resistance genes (Calenge and Durel 2006). Monogenic resistance to powdery mildew is generally derived from wild related species or ornamental crab apples. The major genes controlling powdery mildew resistance are listed in Table 3.3. Molecular markers, which are needed to assist gene

57

selection, are now available for all the known powdery mildew major resistance genes: Pl1 (Markussen et al. 1995), Pl2 (Seglias and Gessler 1997), Plmis (Gardiner et al. 2003), Plw (Evans and James 2003), and Plbj (Dunemann and Schuster 2008). The existence of different physiological races of P. leucotricha and the reports on the breakdown of the resistance from M. robusta and M. zumi, “White Angel” and “Mildew Immune Selection” (Korban and Dayton 1983) necessitates to concentrate on the breeding effort on cumulating (“pyramiding”) different monogenic and polygenic resistances in the same cultivar. This approach may allow breeders to create truly durable forms of genetic resistance by offering multiple resistance barriers against powdery mildew.

3.6.3 Fire Blight Another serious disease of apples and most of the Pomoideae is fire blight. This destructive bacterial disease caused by the Erwinia amylovora (Burrill) Winslow et al. affects blossoms, fruits, shoots, woody tissues, and rootstock crowns. Such factors as the diversity of host tissues susceptible to infection, sporadic nature of fire blight, and limited number of tactical options available to manage this disease well have made it difficult to stop or slow down the spread of fire blight. Once confined to the USA, fire blight has spread worldwide. In 2006, it has been reported already in 46 countries (van der Zwet 2002). Most commercially successful apple cultivars such as “Braeburn”, “Fuji”, “Gala”, “Jonagold”, and “Pink Lady” are very susceptible to fire blight (Steiner 2000). The most commonly used dwarfing apple rootstocks, Malling (M.) 9 and M.26, are highly susceptible to E. amylovora too, and in almost all cases, fire blight infection kills trees by girdling the rootstock (Norelli et al. 2003). Therefore, modern high-density orchards composed of susceptible varieties on susceptible size-controlling rootstocks have increased severity of damage caused by fire blight infection to unprecedented levels. For example, a single fire blight epidemic in Southwest Michigan in 2000 resulted in the death of over 220,000 trees and the removal of more than 240 ha of apple orchards, with a total economic loss estimated at US $42 million (Norelli

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et al. 2003). In Dresden-Pillnitz, 1,164 apple trees representing 3.3% of the collections were infected in 2003 (Peil et al. 2004) and had to be consequently discarded from the gene bank’s orchards. Currently, managing fire blight relies on an integrated approach combining several disease control strategies that continually aim at reducing the number and distribution of inoculum sources throughout the orchard, throughout the season every year.

3.7 Genomics Resources Developed The public apple genome project is developing genomic research tools for genomic studies of main phenotype and physiologic traits on a genome-wide scale. An apple expressed sequence tag (EST) project database has been established (http://titan.biotec.uiuc.edu/ apple/apple.shtml) and deposited to the Rosaceae Genome database http://www.genome.clemson.edu/ gdr/ (Jung et al. 2004). The aim of the whole genome project is shotgun sequencing of the M. domestica cv. “Golden Delicious”. The EST database from flowering and fruiting tissues and from tissues responding to pathogens E. amylovora (fire blight disease) and V. inaequalis (apple scab disease) will be created and used to generate a set of ~30,000 “unique gene” set. The apple tree has become a model fruit crop to study genetic control of such commercial traits as resistance to biotic and abiotic stress, flavor and health compound biosynthesis, etc. A substantial set of ESTs was produced for various tissues of apple cultivar “Royal Gala”. Over 150,000 ESTs have been collected from 43 different cDNA libraries obtained for 34 different tissues and treatments. The ESTs were represented by 42,938 nonredundant sequences – 17,460 tentative contigs and 25,478 singletons, together representing approximately one-half the expressed genes from apple (Newcomb et al. 2006). Several apple plant tissues from plants of cv. “Royal Gala” exposed to different stresses were used to make cDNA libraries (Wisniewski et al. 2008). The obtained ESTs were combined into three classes: control, low-temperature, and water deficit, and the annotated genes in each class were placed into 1 of 10 different functional categories. It was a distinct downregulation of genes involved in general metabolism and photosynthesis,

A. Ignatov and A. Bodishevskaya

and threefold upregulation in defense/stress-related genes, protein metabolism, and energy was observed. Genes overexpressed in the low-temperature libraries were dehydrin and metallothionein-like proteins, ubiquitin proteins, a dormancy-associated protein, a plasma membrane intrinsic protein, and an RNA-binding protein. The genes upregulated in the water-deficited plants were mainly of heat-shock proteins, dehydrins, and photosynthesis (Wisniewski et al. 2008). It is interesting that dinucleotide repeats found in 4,018 sequences had a strong bias toward AG repeats (88% of sequences) when CG was present only in 0.1% of sequences. Single nucleotide polymorphism (SNP) markers associated with gene-coding regions are highly useful tools of functional and structural genomics. They are present within most of genecoding sequences with average frequency 0.14% (Newcomb et al. 2006). A search for SNPs within an EST database of approximately 350,000 sequences developed from a variety of apple accessions was resulted in the identification of a total of 71,482 putative SNPs (Chagne´ et al. 2008). Some of the detected in silico SNPs were linked either to gene duplication or paralogous sequence variations. After verification, the set of 464 PCR primer pairs linked to allelic sequence variation was designed. The SNPs were mapped onto several apple genetic maps, including a map of “Royal Gala” A689-24 and “Malling 9” “Robusta 5” crosses. Only 93 new markers containing 210 coding SNPs were successfully mapped. This new set of SNP markers for the apple gives a new chance for understanding the genetic control of important phenotypic traits by QTL or linkage disequilibrium (LD) analysis. These also serve as a tool for aligning physical and genetic maps (Chagne´ et al. 2008). So far, there is no genomic research that has been done on wild apples or their hybrid population. The effective utilization of these genetic resources requires an integrated and centralized database for related species with associated analysis tools. The Genome Database for Rosaceae (GDR) contains comprehensive data of the genetically anchored peach physical map, an annotated peach EST database, Rosaceae maps and markers, and all publicly available Rosaceae sequences (Jung et al. 2004). Qualitative and quantitative differences of volatile components of ripe fruits in wild apple M. sieversii

3 Malus

and domestic apple (“Ralls”, “Delicious”, “Golden Delicious”, and “Fuji”) were analyzed using head space-solid phase micro extraction and gas chromatography–mass spectrometry (Chen et al. 2007). The results indicated that M. sieversii plants have considerable genetic variations in the total content of volatile components, the classes and contents of each compound, the segregation ratio, and content of main components. The results showed significant difference among seedlings and wide genetic diversity within the populations. There were 177 compounds in total belonging to 11 classes in 30 M. sieversii genotypes and 48 compounds present in M. pumila that were not detected in M. sieversii (Chen et al. 2007).

3.8 Scope for Domestication and Commercialization Crab apples are common ornamental plants used far from their homelands, and many of them are used as a stock for grafting domestic apples because of their high cold tolerance and resistance to pathogens. Rootstocks are subject of breeding programs and introduction of disease and insect resistance traits such as fire blight (E. amylovora), apple scab (V. inaequalis), and wooly apple aphid (Eriosoma lanigerum) by molecular methods (Celton et al. 2009). So far, there is no cultivar specifically developed as a source of medicines, dietary supplements, perfumes, etc. Fruits of many Crab apples contain malic and tartaric acids that inhibit fermentation in the intestines and also rich in fiber and pectin, potentially useful as therapeutic compound (Herman et al. 1996). Apple is an important source of therapeutic compounds used as herbal drugs, dietary supplements, perfumery industries; etc. As recently discovered in many fruit crops, flavonoids may play an important role in preventing many types of cancer, and may also reduce the risk of heart disease and stroke. There is an evidence that apple phenolic compounds such as quercetin, epicatechin, and procyanidin B2 may be cancer-protective and demonstrate antioxidant activity in vitro (Lee et al. 2003, 2004). Crab apples are widely grown as ornamental trees, grown for their profuse blossom and attractive fruit. Some crab apples are used as rootstocks for domestic

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apples to add beneficial characteristics. For example, Siberian crab rootstock is often used to give additional cold hardiness to the combined plant for orchards in cold northern areas. Crab apples, for example, M. sylvestris, M. fusca, and Malus pumila are an excellent source of pectin, and their juice can be processed into a ruby-colored jelly with a full, spicy flavor. Pectin is also a radiationprotector (Allardice 1993). Medical properties of M. fusca were employed by native North American Indians to treat a variety of diseases (Moerman 1998). The trunk, bark, and the inner bark of this crab have compounds with antirheumatic, astringent, blood purifier, cardiac, diuretic, laxative, and tonic activity. The ethnobotanical study of local Mediterranean food plants as medicinal resources in southern Spain (Rivera et al. 2005) revealed that fruits of M. segurensis and M. sylvestris are used for medicinal purposes as astringent. In Europe, scraped apple M. sylvestris has been used to treat infant diarrhea, dysentery, and dyspepsia. Root and bark are considered anthelmintic, hypnotic, and refrigerant, and a bark infusion is given to Indians suffering from bilious ailments and intermittent and remittent fevers. Apple leaves contain an antibacterial substance called phloretin, which is active in doses as low as 30 ppm. The fruits are astringent and laxative. They are eaten to obviate constipation (Chopra et al. 1986). The bark, and especially the root bark, is anthelmintic, refrigerant, and soporific (Duke and Ayensu 1985). An infusion is used to cure intermittent, remittent, and bilious fevers (Chopra et al. 1986).

3.9 Recommendations for Future Actions It is reasonable that preservation of wild apple trees in their natural inhabitat is the most probable way to save the current genetic variation of the species for long time. Genetic core collections are essential for research and breeding as a source of desirable properties underlined in current investigations. It takes so much effort to collect and preserve elite apple genotypes, but even more efforts will be needed to collect and preserve wild apples with genotypes representative for their natural population.

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3 Malus Patocchi A, Walser M, Tartarini S, Broggini GAL, Gennari F, Sansavini S, Gessler C (2005) Identification by genome scanning approach (GSA) of a microsatellite tightly associated with the apple scab resistance gene Vm. Genome 48:630–663 Patocchi A, Fernandez-Fernandez FK, Evans D, Gobbin F, Rezzonico A, Boudichevskaia A, Dunemann M, Stankiewicz-Kosyl F, Mathis-Jeanneteau CE, Durel L, Gianfranceschi F, Costa C, Toller V, Cova D, Mott M, Komjanc E, Barbaro L, Kodde E, Rikkerink C, Gessler C, van de Weg WE (2009) Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome. Tree Genet Genomes 5:211–223 Peil A, Garcia-Libreros T, Richter K, Trognitz FC, Trognitz B, Hanke M-V, Flachowsky H (2007) Strong evidence for a fire blight resistance gene of Malus robusta located on linkage group 3 detected by rapid genome scanning. Plant Breed 126:470–476 Peil A, Dunemann F, Richter K, Hoefer M, Kira´ly I, Flachowsky H, Hanke M-V (2008) Resistance breeding in apple at Dresden-Pillnitz. In: Boos M (ed) Ecofruit. Proceedings of 13th international conference on cultivation technique and phytopathological problems in organic fruit-growing, Weinsberg, Germany, 18–20 Feb 2008, pp 220–225 Pflieger S, Lefebvre V, Causse M (2001) The candidate gene approach in plant genetics: a review. Mol Breed 7(4):275–291 Ponomarenko V (1987) History of Malus domestica Borkh origin and evolution (In Russian). Bot J USSR 176:10–18 Ponomarenko V (1992) Critical review of the system of the genus Malus Mill (Rosaceae) species. Bulletin of applied botany genetics and plant breeding. Russ Acad Agric Sci 146:1–10 Potter D, Gao F, Bortiri PE, Oh SH, Baggett S (2002) Phylogenetic relationships in Rosaceae inferred from chloroplast matK and trnLtrnF nucleotide sequence data. Plant Syst Evol 231:77–89 Potter D, Eriksson T, Evans RC, Oh S, Smedmark JEE, Morgan DR, Kerr M, Robertson KR, Arsenault M, Dickinson TA et al (2007) Phylogeny and classification of Rosaceae. Plant Syst Evol 266:5–43 Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey SV, Rafalski AJ (1996) The comparison of RFLP, RAPD, AFLP, and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238 Ragan WH (1926) Nomenclature of the apple: a catalogue index of the known varieties referred to in American publications from 1804 to 1904. USDA Bur Plant Indust Bull 56. USDA, Washington DC Rehder A (1940) Manual of cultivated trees and shrubs exclusive of the subtropical and warm temperate regions, 2nd edn. Macmillan, New York, USA, 996 p Robinson JP, Harris SA, Juniper BE (2001) Taxonomy of the genus Malus Mill (Rosaceae) with emphasis on the cultivated apple Malus domestica Borkh. Plant Syst Evol 226:35–58 Roche P, Alston FH, Maliepaard C, Evans KM, Vrielink R, Dunemann F, Markussen T, Tartarini S, Brown LM, Ryder C, King GJ (1997) RFLP and RAPD markers linked to the rosy leaf curling aphid resistance gene (Sd1) in apple. Theor ApplGenet 94:528–533 Rivera D, Obo´n C, Inocencio C, Heinrich M, Verde A, Fajardo J, Palazo´n J (2005) The ethnobotanical study of local Mediterranean food plants as medicinal resources in southern Spain. J Physiol Pharmacol 56(1):97–114

63 Sansavini S (2004) Europe’s organic fruit industry. Chron Hortic 44(2):6–11 Sax K (1931) The origin and relationships of the Pomoideae. J Arn Arbor 12:3–22 Schaffer RJ, Friel EN, Souleyre EJ, Bolitho K, Thodey K, Ledger S, Bowen JH, Ma JH, Nain B, Cohen D, Gleave AP, Crowhurst RN, Janssen BJ, Yao JL, Newcomb RD (2007) A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway. Plant Physiol 144(4):1899–1912 Schuster M (2000) Genetics of powdery mildew resistance in Malus species. ISHS Acta Hortic 538:593–595 Seglias NP, Gessler C (1997) Genetics of apple powdery mildew resistance from Malus zumi (Pl2). Plant Pathology 54 (2):116–124 Shay JR, Dayton DF, Hough LF (1953) Apple scab resistance from a number of Malus species. Proc Am Hortic Sci 62:348–356 Shay JR, Williams EB, Janick J (1962) Disease resistance in apple and pear. Proc Am Soc Hortic Sci 80:97–104 Silfverberg-Dilworth E, Matasci CL, Van de Weg WE, Van Kaauwen MPW, Walser M, Kodde LP, Soglio V, Gianfranceschi L, Durel CE, Costa F, Yamamoto T, Koller B, Gessler C, Patocchi A (2006) Microsatellite markers spanning the apple (Malus x domestica Borkh) genome. Tree Genet Genomes 2:202–224 Steiner P (2000) Integrated orchard and nursery management for the control of fire blight. In: Vanneste JL (ed) Fire blight: the disease and its causative agent, Erwinia amylovora. CABI Publishing, Wallingford, UK, pp 339–358 Tartarini S, Gianfranceschi L, Sansavini S, Gessler C (1999) Development of reliable PCR markers for the selection of the Vf gene conferring scab resistance in apple. Plant Breed 118(2):183–186 Tatum TC, Stepanovic S, Biradar DP, Rayburn AL, Korban SS (2005) Variation in nuclear DNA content in Malus species and cultivated apples. Genome 48:924–930 Urbanietz A, Dunemann F (2005) Isolation, identification and molecular characterization of physiological races of apple powdery mildew (Podosphaera leucotricha). Plant Pathol 54:125–133 van der Zwet T (2002) Present worldwide distribution of fire blight. Acta Hortic 590:33–34 van Hintum TJL (1999) The general methodology for creating a core collection In: Johnson RC, Hodgkin T (eds) Core collections for today and tomorrow. International Plant Genetic Resources Institute, Rome, Italy, pp 10–17 Vavilov NI (1930) Wild progenitors of the fruit trees of Turkistan and the Caucasus and the problem of the origin of fruit trees. In: International horticultural congress group B, pp 271–286 Vinatzer BA, Patocchi A, Tartarini S, Gianfranceschi L, Sansavini S, Gessler C (2004) Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab-resistant accessions in Malus germplasm. Plant Breed 123(4):321–326 Vos P, Hogers R, Bleeker M, Reijans M, Van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414 Watkins R (1995) Apple and pear. In: Smartt J, Simmonds NW (eds) Evolution of crop plants. Wiley, New York, USA, pp 418–422

64 Way RD, Aldwinckle HS, Lamb RC, Rejman A, Sansavini S, Shen T, Watkins R, Westwood MM, Yoshida Y (1990) Apples (Malus). In: Moore JN, Ballington Jr JR (eds) Genetic resources of temperate fruit and nut. International Society for Horticultural Science, Wageningen, Netherlands. Acta Hortic 290:1–62 Wiedow C, Dehmer KJ, Geibel M (2004) Molecular diversity in populations of Malus sieversii (Ledeb) Roem. Acta Hortic 663:539–543 Williams EB, Dayton DF (1968) Four additional sources of the Vf locus for Malus scab resistance. Proc Am Soc Hortic Sci 92:95–98 Williams EB, Kuc J (1969) Resistance in Malus to Venturia. Annu Rev Phytopathol 7:223–224 Williams EB, Dayton DF, Shay JR (1966) Allelic genes in Malus for resistance to Venturia inaequalis. Proc Am Soc Hortic Sci 88:52–56

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Chapter 4

Muscadiniana Hemanth K.N. Vasanthaiah, Devarajan Thangadurai, Sheikh M. Basha, Digambar P. Biradar, Devaiah Kambiranda, and Clifford Louime

4.1 Introduction Muscadine grapes (Muscadiniana rotundifolia), known as American wild grapes, are among the most important Vitis species cultivated in southern United States and are popular home crop because of their natural adaptability, resistance to diseases and insects, and long vine life. Muscadine is native to the southeastern US and has been extensively cultivated since its discovery. Sir Walter Raleigh was the first to discover these grapes in 1584. Generally considered as underutilized commodity, muscadine grapes have a characteristic aroma and sweetness that make them acceptable as table wines (Talcott and Lee 2002). Muscadine is considered as the first grapes of the new world, the “United States of America”. The muscadine berries range from bronze to dark purple to black in color when ripe. Muscadines are not only eaten fresh but are also used in making pie, juice, frozen concentrated juice, jelly, and fermented as wine and brandy. Muscadine grapes are rich source of polyphenols when compared to any other fruit crops. These polyphenols (resveratrol, ellagic acid, ellagitannins, etc.) are found to be beneficial to human health. Resveratrol (3,4,5-trihydroxystilbene) is one of the important phenolic compounds found in muscadine grape. Reports have indicated that muscadine grapes have high concentrations of resveratrol

H.K.N. Vasanthaiah (*) Plant Biotechnology Laboratory, Center for Viticulture and Small Fruit Research, Florida A&M University, 6505 Mahan Drive, Tallahassee, FL 32317, USA e-mail: [email protected]

than any other crops. High performance liquid chromatography (HPLC) analysis has also indicated higher phenolic content in muscadine compared to bunch and Florida hybrid bunch grapes (Basha et al. 2004). In another study, seed extract from muscadine grape cultivars also showed genetic variation in anticancer activity (Vasanthaiah and Basha 2008). Recently, six isoforms of stilbene synthase genes have been isolated from muscadine grape cultivar cv. Regale for the first time, which may be useful for further increasing resveratrol content of grape berry (Vasanthaiah et al. 2007). Muscadine grapes are noted for having smaller clusters compared to V. vinifera (Fig. 4.1). Muscadines ripen from late July through mid-October. They will often be on the vine until the first fall frost comes. They are large, thick-skinned and seeded grapes that grow in small, loose clusters and are often harvested as individual berries. Muscadines are well-adapted to warm and humid conditions of the southeastern region. Most of the muscadines have the problem of uneven ripening. Therefore, harvesting berries at one picking is difficult. It has a range of ripeness present during the entire harvest period. This has created a problem in the harvesting of the crop. For the production of high quality grape berries, the whole cluster should ripen at one time. The lack of uniform ripening within a cluster is termed “uneven ripening” and is characterized by the presence of green berries in an otherwise mature ripe cluster (Cawthon and Morris 1983). In any fruit-growing operation, harvesting problems exist whether you pick your own or employ machine harvesters. With mechanical harvesting, scheduling and determining the proper harvest date for the various markets can be a major concern (Lanier and Morris 1979). Whatever method is used for picking, getting the fruit harvested at the right time and to the right market without losses

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_4, # Springer-Verlag Berlin Heidelberg 2011

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66 Fig. 4.1 Muscadine and bunch grape berry cluster. (a) Bunch grape cv. Blue Lake and (b) Muscadine grape cv. Regale

H.K.N. Vasanthaiah et al.

a

is not easy (Morris 1980). Uneven ripening has been linked with the variation in the bud breaking, flower opening, and fruit ripening in muscadine grape cultivars (Huang and Lu 2000). Muscadine berries have less sugar content (14–18%) when compared to V. vinifera berries, which are rich in sugars (24–26%). Therefore, muscadine requires addition of sugar to the juice to promote alcohol production during fermentation and enhance enological characteristics of wine. Muscadine grapevine is vigorous, productive, and yields large berries. The muscadine grape species have been investigated for years due to the challenges they pose to the breeders and the distinct differences in morphology, chemistry, and flavor from the commonly cultivated grape species V. vinifera. All of these characteristics make muscadine a perfect grape to grow. But the muscadine crop improvement program is still in its infancy. These unique characters can be successfully utilized in breeding programs.

4.2 Origin, Botany, and Distribution 4.2.1 Origin The muscadine grape is native to the southeastern US. Muscadine grapes are often referred to as scuppernongs.

b

Muscadines are under cultivation for more than 400 years (Poling and Fisk 2006). Native Americans preserved muscadines as dried fruit long before the Europeans inhabited this continent. Earlier varieties of muscadines were all selections from the wild. The earliest named variety was Scuppernong, which has become another name for all muscadine grapes. Commercial production of muscadine grapes is essentially limited to the southeastern US. Muscadines produce dull purple, thick-skinned musky fruits. As early as 1565, Captain John Hawkins reported that the Spanish settlements in Florida made large quantities of muscadine wine. These grapes were earlier referred as “Big White Grape” or “Bullace” referring to cow’s or pig’s eyes as the size of the fruits are big.

4.2.2 Botany Muscadine grapes belong to a separate subgenus, Muscadinia, with other grapevine species belonging to subgenus Vitis in the same genus Vitis, which has been classified within the family variously known as Vitaceae, Vitidaceae, and Ampelidaceae. Muscadine comes within the botanical order Rhamnales, which also includes the families Rhamnaceae and Leeaceae. Some taxonomists have suggested that it has a genus of its own, and some have also suggested splitting two additional species off from Vitis rotundifolia as V. munsoniana and V. popenoei.

4 Muscadiniana Taxonomic classification Kingdom Division Class Order Family Genus Species

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Plantae Magnoliophyta Magnoliopsida Vitales Vitaceae Muscadinia V. rotundifolia

V. rotundifolia and its wild relative V. popenoei represent the subgenus Muscadinia (2n ¼ 6x ¼ 40) in the genus Vitis, which typically comprises almost all of the total of about 70 taxa described including V. aestivalis, V. californica, V. labrusca, V. riparia, V. rupestris, and V. vinifera, offering great diversity in growth habit and fruit attributes (2n ¼ 6x ¼ 38). All the close relatives of V. rotundifolia are native to North America, central Asia, and West Asia as far as the Himalayas (James and Hardie 2008). Much of the taxonomic knowledge comes from studies based on morphological and molecular criteria and riddled with difficulties in establishing identities of different taxa. The confusion is due to widespread introgression among sympatric taxa resulting in clinal variation masking the taxonomic and ecological boundaries (Mallikarjuna et al. 2005; Benavente et al. 2008). Muscadines are vigorous, deciduous vines growing 60–100 ft in the wild with a tight, non-shedding bark, warty shoots, and unbranched tendrils; the foliage is slightly lobed, 2.5–5.0 in. leaves, rounded to broadly ovate with coarsely serrate edges and acuminate, dark green above and green tinged yellow beneath, glossy on both sides, firm and subglabrous; flowers are dioecious, small, short, and dense panicles; fruit is small, loose clusters of 3–40 grapes, round, 1.0–1.5 in. thick, tough skin, and contain up to five hard, oblong seeds, color range from greenish bronze through bronze, pinkish red, purple, and almost black.

4.2.3 Distribution Recognized range extends from New York south to Florida and west to Missouri, Kansas, Arkansas, Oklahoma, and Texas (Olien 2001). Muscadines are also found in the wild from Delaware to the Gulf of Mexico and near Appalachian Mountains. V. rotundifolia Michx., commonly known as muscadine, bullace, scuppernong, or southern fox grape native to the southeastern

US that has been extensively cultivated since the sixteenth Century, is extremely important today in viticulture industry as it offers resistance to many bacterial and fungal diseases and insect attacks.

4.3 Scope for Domestication and Commercialization Muscadine grapes have been in use for making commercial fine wines and port wines dating back to the sixteenth century in and around St. Augustine, Florida. Wild relatives of muscadine grape produce palatable fruits that can be eaten at once, dried to produce raisins, currants, and turned into wine. Muscadine grapes have been used for their medicinal potential since before the beginning of recorded history. For example, they are not only eaten fresh but are also used in making wine and other food products such as juice, jelly, preserves, syrup, and sauce. They are a rich source of polyphenols and other nutrients with potential health benefits (PastranaBonilla et al. 2003). Therefore, ground muscadine seeds are sold (as pills) commercially as a source of resveratrol. It has been experimentally proven that muscadine grape and wines thereof contain high concentrations of resveratrol (40 mg/L of resveratrol), a polyphenol (Le Blanc 2006) with anticancer properties against blood, colon, and prostate cancers (MertensTalcott and Percival 2005; Mertens-Talcott et al. 2006; Hudson et al. 2007). The other muscadine polyphenols include anthocyanins such as delphinidin and petunidin, tannins, quercetin, catechins and epicatechin, gallic acid, ellagic acid, ellagic acid glycosides, ellagitannins, myricetin, and kaempferol (Talcott and Lee 2002; Pastrana-Bonilla et al. 2003; Lee et al. 2005). The chromatographic analysis of headspace or solvent extracts for the volatile constituents in the fruit assumes importance to humans as sources of aroma and flavor of wine (Hardie and Obrien 1988). These include hydrocarbons, esters, aldehydes, ketones, and alcohols. It has also been reported that muscadine grape skin and pulp is an excellent source of dietary fiber, essential minerals, and carbohydrates and is low in fat than oat or rice bran. The sugar content of wild species, especially sucrose, was higher than those of cultivated varieties, breeding lines, and rootstock material.

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There is also much more potential for domesticating these grapes as an alternative crop for their phytochemical, phytopharmaceutical, and nutraceutical properties and can be a natural sweetener, an anticancer agent, and an antioxidant than as a fruit and wine crop. Even the grape leaves contain an abundant amount of vitamins and minerals. People do consume grape leaves stuffed with rice and meat, which is commonly called “Dolma”. It is very popular in Middle East and Mediterranean countries. Moreover, the cultivation and domestication of wild relatives of muscadine grapes is very promising as a source for stress tolerance and disease resistance genes.

4.4 Muscadine Crop Improvement Through Breeding Muscadine breeding is still in its infancy, even though muscadine grapes were first discovered 400 years ago. Muscadine crop improvement program is still in the early stages, with some cultivars being selected from the wild few generations ago as compared to the old world grapes (V. vinifera). Efforts to breed muscadine grapes commenced in the early 1900s and have generated a large number of cultivars and a limited number of hybrids with V. vinifera L. and other Vitis L. species. Nearly 100 years of breeding work has resulted in the release of many improved cultivars (Table 4.1). “Carlos”, “Fry”, and “Nobel” are some of the most important cultivated varieties. The main breeding objective of muscadine grape is to improve disease resistance, yield, fruit quality, and valueaddedness of the commercially important cultivars.

4.4.1 Disease and Pest Tolerance The North American muscadine grape (Muscadinia rotundifolia Small) is a valuable source of resistance to powdery mildew [Uncinula necator (Schw.) Burr], root-knot nematode (Meloidogyne Goeldi), dagger nematode (Xiphinema index Thorne and Allen), grape phylloxera (Daktulosphaira vitifoliae Fitch), and Pierce’s disease (Xylella fastidiosa) (Riaz et al. 2007). Pierce’s disease resistance is a boost to southeastern US region as this disease prevents the growth

H.K.N. Vasanthaiah et al. Table 4.1 Commercial muscadine cultivars teristics Sl. Cultivar Flower type No 1 Big Red Female (Pistillate) 2 Black Beauty Female (Pistillate) 3 Black Fry Female (Pistillate) 4 Darlene Female (Pistillate) 5 Early Fry Female (Pistillate) 6 Fry Female (Pistillate) 7 Higgins Female (Pistillate) 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Janet Jumbo Pam Scuppernong Sugargate Summit Supreme Sweet Jenny Alachua Carlos Cowart Dixieland Dixie Red Fry Seedless Granny Val Ison Janebell Late Fry Magnolia Nesbitt Noble Pineapple Pollyanna Redgate Regale Scarlet SouthLand Sterling Tara Triump Welder

Female (Pistillate) Female (Pistillate) Female (Pistillate) Female (Pistillate) Female (Pistillate) Female (Pistillate) Female (Pistillate) Female (Pistillate) Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile

and their characFruit skin color Red Black Black Bronze Bronze Bronze Pink to reddish bronze Bronze Black Bronze Bronze Black Bronze Black Bronze Black Bronze Black Bronze Light red Red Bronze Black Bronze Bronze White Black Black Bronze Black Red Black Red Black Bronze Bronze Bronze Light pink

of V. vinifera cultivars, which are highly susceptible to this disease. Nearly 10 decades of muscadine grape research has resulted in many improved cultivars, which were selections from the wild and have become standard cultivars (Poling 1996; Meredith 2001). Though muscadine grapes and many of its close relatives, viz. Summer grape (Vitis aestivalis), California grape (V. californica), American grape or Fox

4 Muscadiniana

grape (V. labrusca), River Bank grape (V. riparia), Sand grape (V. rupestris), and European grape (V. vinifera), have been used as a rootstock and breeding material in areas where the phylloxera disease is prevalent and moreover has been used in the breeding program with V. vinifera in order to impart resistance to that species. In recent times, primary disease problems are fruit rots, especially black rot, bitter rot, ripe rot, and Macrophoma rot. If severe, angular leaf spot may cause leaf abscission (shedding), resulting in smaller berries and lower sugar content than from healthy vines. Insect control is primarily limited to grape root borer, which is extremely difficult because of the subterranean feeding of larvae on root and the lack of distinct early symptoms. Early claims that muscadines were immune to grape root borer have been shown to be erroneous. Pierce’s disease is carried by sharpshooter leafhoppers, but the control of these insects is also difficult. Other pests, such as mites, aphids, grape flea beetles, grape curculios, and grape berry moths, are occasional, and severe problems can be controlled only if significant injury is observed (Meredith 2001). Research has been carried out earlier to determine the tolerance capacity of muscadine to pest and diseases (Clayton 1975; Bouquet 1980; Mortensen and Andrews 1981). It still lacks proper conclusion as most of the studies are field related.

4.4.2 Yield and Fruit Quality Yield is influenced by several factors and is locationspecific. Research has been carried out to improve and identify components influencing yield. In a study, six V. rotundifolia Michx. (muscadine) grape genotypes were measured to determine variation and simple correlations between flower and fruit number, fruit cluster and individual fruit weight, fruit set, and total vine yield (Goldy 1988). Significant variation was found for each trait, except fruit cluster weight. Ranges in mean values across selections for each trait were: 72–202 flowers/cluster; 16–26 fruits/cluster; 88.1–112.2 g/fruit cluster; 3.4–7.2 g/fruit; 11.3–23.4% fruit set; and 21.0–71.0 kg/vine for yield. Simple correlation values ranged from r ¼ 0.05 between flower number and cluster number to r ¼ 0.69 between fruit number and cluster weight. No trait was highly correlated to the total yield.

69 Table 4.2 pH and total soluble sugar content of muscadine genotypes. Sugar content is expressed in percentage (g 1001 fresh weight). Turkey’s HSD was used for mean separation. Means with same letter are not statistically significant Muscadine pH Mean separation (Turkeys genotypes HSD a ¼ 0.05) African queen 3.4 12.7075d Alachua 3.7 11.8750gf Albermarle 3.4 11.5550gh Carlos 2.9 15.0750b Cowart 3.5 13.0000cd Darlene 3.4 15.3750b Dixe Red 3.3 12.9359cd Doreen 3.4 10.0250kl Farrer 3.5 11.3000hi Fry 3.8 12.1500fe Higgins 3.3 12.5250de Jane bell 3.2 8.5100m Jumbo 3.5 10.8000ij Noble 2.8 12.0000gf Regale 2.9 9.45l Scuppernong 3.4 10.6750j Southland 3.6 10.5125jk Summit 3.2 13.2350c Sweet jenny 3.6 16.3625a Welder 3.4 12.55de

Goldy suggested selecting for total yield and not for components contributing to yield to increase yield in muscadines. Fruit shattering, seed size, and skin thickness influence the quality of fruit in muscadine along with the sugar content. Sugar levels vary among muscadine genotypes (Table 4.2). Sweet Jenny (16.3625) has the highest sugar content, and Jane bell (8.5100) has the lowest sugar content (Kambiranda et al. 2010). The variability does exist among cultivars.

4.4.3 Value-Added Characteristics While not one of the most widely marketed grape varieties are produced from muscadine, the visibility of muscadine has benefited from the discovery that it appears to provide greater amounts of antioxidants than many better-known grapes. Because they synthesize resveratrol as a defense, it has previously been claimed that the use of pesticides greatly reduces the grape’s resveratrol content; however, studies either found no correlation between pesticide use and

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resveratrol (Dugo et al. 2004) or that pesticide use has only a weak effect (Daniel et al. 1999). As one of nature’s richest sources of resveratrol and other polyphenols such as anthocyanins, tannins, quercetin, catechins, epicatechin, gallic acid, ellagic acid, ellagic acid glycosides, ellagitannins, myricetin, and kaempferol, research interest in describing and producing these phytochemicals is significant (Ector et al. 1996; Talcott and Lee 2002; Lee et al. 2005; Le Blanc 2006; Hudson et al. 2007). They have been studied for their potential health benefits, which include preliminary evidence for effects against cancer mechanisms as well as anti-inflammatory and anticlotting compounds that promote cardiovascular health (Neveln and Pelczar 2006). To date, in vitro studies have shown positive effects of muscadine phenolics against blood, colon, and prostate cancers (Mertens-Talcott and Percival 2005; Mertens-Talcott et al. 2006; Hudson et al. 2007). Fruits of selected muscadine grapes, including varieties, breeding lines, and cultivars, have been evaluated to assess the existing genetic base for these nutraceutical compounds, but the production of these health-promoting compounds was the goal of none of the works carried out by any plant/fruit breeders using both modern tissue culture approaches and other transgenic technologies. In short, there has not been much focus on improving varieties with desirable traits.

4.4.4 Cytogenetics and Interspecific Hybridization The muscadine grapes emerged in the southeastern US and are distinguished from the Euvitis species morphologically and cytologically. They are characterized by high disease and pest resistance and unique flavor (Lu et al. 2000). The major problem for gaining wider acceptance of muscadine grapes is its relatively low fruit quality as compared to the excellent fruit quality of V. vinifera. Attempts to produce V. rotundifolia V. vinifera hybrids that combine good fruit quality and disease resistance have been made by grape breeders for many years. Limited success was only reported when the V. vinifera was used as seed parents. The possible reason owes to the difference in the chromosome number between V. rotundifolia (2n ¼ 40) and

H.K.N. Vasanthaiah et al.

V. vinifera (2n ¼ 38). This has been reported by Dermen (1964). He concluded that attempts by earlier grape breeders to develop bunch grapes adaptable to southern US through hybridization between bunch and muscadine grapes have failed. The few hybrids obtained were highly or completely sterile, possibly because of the difference in chromosome numbers of the two types of grapes: bunch grapes have 2n ¼ 38 chromosomes, and muscadine grapes have 2n ¼ 40 chromosomes. Two such hybrids known as “N.C. 6-15” and “N.C. 6-16” were made fully fertile by chromosome doubling with colchicine. A small population of tetraploid seedlings was raised from the two colchicine-induced amphiploids. The tetraploid seedlings showed enough variation in percentage of good pollen and in vegetative and fruit characters to indicate that segregation of characters acquired from the two parental species, V. vinifera and V. rotundifolia, had taken place. He also reported that recently, some breeders have obtained seedlings from a few berries set on the 2x hybrid “N.C. 6-15”. Some of these plants have come to fruiting and have borne large fruit bunches, characteristic specifically of V. vinifera. Vines and fruits of these plants have shown certain characters of both parental species. As cited in Dermen et al. (1970), past attempts to hybridize V. vinifera and V. rotundifolia have resulted in a few diploid hybrids with 2n ¼ 39 chromosomes, 19 chromosomes contributed by a haploid gamete from V. vinifera and 20 chromosomes by a haploid gamete from V. rotundifolia. These diploid hybrids were self-sterile. Jelenkovic and Olmo (1968) were able to recover partial F1 hybrids and were successfully backcrossed to V. vinifera, thus making it possible to exploit Muscadinia germplasm in grapevine improvement (Olmo 1971). Here at the Center for Viticulture and Small Fruit Research, Florida A&M University, USA, research is being carried out by Dr. Lu and his team to combine good fruit quality from V. vinifera grapes and high disease resistance from V. rotundifolia grapes. More than 50 cross combinations between V. vinifera and V. rotundifolia have been made since 1993. Limited success was achieved in some crosses and only two hybrids were produced from the crosses when the muscadine grapes were used as the female parent. In these hybrids, many morphological and biological characteristics, such as leaves, stems, tendrils, time of bud break, bloom date, and ripen date, are intermediate between the parents. Some of the hybrids are

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partly fertile. Evaluation for disease resistance of these hybrids indicated that they are resistant to Pierce’s disease, anthracnose disease, and downy mildew, which are the limiting factors to growing V. vinifera in the hot, humid climate of southeastern US. Dr. Lu and coworkers suggested that these hybrids can be used as bridge to carry viticulturally important genes from V. vinifera to the muscadine grapes and vice versa, to transfer disease. Similar research is also in progress at the University of Georgia, Tifton, and North Carolina State University, Raleigh, to improve the characteristics of Muscadinia and V. vinifera grapevine through breeding.

4.4.5 Seedlessness Seedlessness has been successfully obtained in V. vinifera, whereas in V. rotundifolia, it is still unsuccessful. Research is being carried out at the Center for Viticulture, Florida A&M University, and the University of Florida to develop seedless muscadine cultivars to improve the profit of grape growers and industry in southern US. Dr. Lu et al. (1993) reported that it is difficult to use muscadines as the female parent when crossing with bunch grapes because of prefertilization barriers. Instead, they suggested using seedless bunch grapes as the female parent combined with embryo rescue. Recently at the University of Florida, researchers have demonstrated genetic transformation and discovered grape-derived genetic elements that could be utilized to regulate transgene function in embryos and seeds of muscadine grape (Li et al. 2007; Dhekney et al. 2008). They suggest that these two advances now converge, allowing evaluation of various genes to disrupt seed development in muscadine grape.

4.4.6 Biotechnology and Breeding Muscadine cultivars need to be developed with desirable characteristics such as good yield, large fruits that ripen early to mid-season, more sugar content, uniform and early ripening, increased disease and pest resistance, large clusters, good cold hardiness, high vigor, sweaty flesh with excellent and attractive

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distinctive flavor, good wine-making quality, and suitability for juice and jelly preparations (Goldy 1988). Focus also needs to be on an international program with special emphasis on seedless muscadines. Muscadine grapes can often be grown organically without insecticides/fungicides. Japanese beetles are often the most damaging insects. Selecting cultivars with some disease resistance will reduce the losses without fungicide applications. Even more, netting may be required for bird protection. In order to combine these desired characteristics, the knowledge of inheritance of these traits is very important. The knowledge regarding the general and specific combining ability of the varieties is very essential for making choice of parents in restricting the cross-combination and thereby seedling population for better selection. The conventional approach of producing inbred lines for their utilization in the production of heterosis is not being favored in muscadine grapes. They do not readily hybridize with other grape species. The crosses between muscadine and allied species are made with great difficulty as most of the resulting hybrids remain sterile (Meredith 2001). Induced mutations can be attempted when certain lines lack in one or more important characters. Without altering the whole genetic set up, mutations may occur in a particular gene responsible for that particular character (Olmo 1960). Polyploidization is also of immense importance in the improvement of muscadine cultivars, especially in increasing the size of berries (Alley 1957), but are not so economically important. The most common objectives of any muscadine grape improvement program is to produce locally adapted and high-yielding varieties with quality that is desirable for the intended use (Jindal 1985). DNA markers facilitate investigations into the origins of existing cultivars and provide powerful tools for the creation of new cultivars (Lodhi et al. 1995; Dalbo et al. 2000a, b). Microsatellite markers as the most powerful DNA markers provide unique genetic profile for every cultivar, permitting unambiguous identification that is unaffected by environment, disease, or farming methods (Riaz and Meredith 2000). Moreover, molecular marker-based mapping efforts are accelerating muscadine breeding programs by permitting early selection of promising seedlings (Stoffella et al. 1982). Genetic mapping in combination with physical mapping can lead to the isolation of

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genes that control important traits but for which the mechanism is unclear (Buck and Zyprian 2000; Grando et al. 2000). This may provide a means to reduce disease losses and pesticide usage in classical breeding without otherwise changing their quality attributes (Stoffella et al. 1982). Genetic maps assist breeders by enabling them to detect traits that are tightly linked and thus likely to be inherited together and also to determine the number and location of genetic factors controlling quantitative traits. A gene controlling flower gender has now been mapped (Dalbo et al. 2000a), and quantitative trait loci (QTL) analysis has been used to identify several genetic regions controlling resistance to fungal disease in an interspecific cross. Among other traits being mapped are seedlessness and fruit cluster characteristics (Dalbo et al. 2000b).

4.5 Genomic and Molecular Analysis Molecular approach would help utilize the unique characteristics of muscadine grape in general grape crop improvement program. A very limited research reports are available on muscadine grape compared to V. vinifera.

4.5.1 Isozyme and RAPD Markers Isozyme polymorphisms and randomly amplified polymorphic DNA (RAPD) markers have been successfully used to identify parents and hybrids among V. vinifera (bunch grapes) and V. rotundifolia (muscadine grapes) (Sawazaki et al. 1996). They reported that GOT (glutamate oxaloacetate transaminase), IDH (isocitrate dehydrogenase), and PGI (phosphoglucoisomerase) systems could differentiate the muscadine. Five allozymes of GOT, two allozymes of LAP (leucine aminopeptidase), and four allozymes of EST (esterase) have been identified. PGI and IDH systems were dimeric and had the phenotype of four allozymes in two regions and three allozymes in one region, respectively. They also reported that RAPD markers were able to differentiate among genotypes. UPGMA dendrograms obtained through isozyme and RAPD marker analysis were found to be similar. The strongest

H.K.N. Vasanthaiah et al.

linkage was found among “Italia” and “Rubi”, followed by “Patricia” and “A Dona”. Piratininga and Eugeˆnio cultivars were also closely linked to the others. Through isozymes and RAPD marker analyses, muscadine was found to be very distantly and separately related to the other groups. When RAPD was used for muscadines, the NC66C203-9 hybrid, one possible hybrid, and its female parent, the UPGMA clusters, revealed that the hybrids were located in an intermediate position for muscadine and bunch grapes; however, the possible hybrid was found similar to its female parent, while the NC66C203-9 presented bands from both muscadine and bunch grapes, confirming its hybrid origin. Ren et al. (2000) used bulk segregant analysis (BSA) to identify RAPD markers closely linked to the fruit color in muscadine grape. An F1 population of 82 progenies derived from a cross of two muscadine grape (V. rotundifolia) cultivars, “Summit” “Noble”, was used for tagging the gene determining the fruit color. They reported that segregation of red color of the berry in the F1 population is controlled by a single dominant gene. A total of 350 oligonucleotide 10-mer primers were screened for polymorphisms between the red- and white-colored DNA pools (each pool was consisted of seven individual DNA samples). Two RAPD fragments linked to the target gene were identified and one of them, a 650 bp fragment, completely co-segregated with the 56 progenies of red berries, while the white fruit progenies lacked the RAPD fragment. They suggested that RAPD can be successfully used in breeding practice and seedling screening through identification of genetic marker for the fruit color. In another study, RAPD has been productively used to analyze Vitis species and Florida bunch grapes (Wang et al. 1999). RAPD analysis was performed on 42 accessions of Vitis, representing 13 species. Inter- and intraspecific/varietal variation were observed. Principal component analysis of Nei’s and Li’s Similarity Index were used to separate V. rotundifolia from other bunch-grape species. Within the bunch-grape species, V. vinifera, the North American bunch grapes, and the East Asian bunch grapes formed three separate clusters. They concluded that RAPD analysis demonstrates its sensitivity by detecting the genetic diversity within Florida bunch-grape cultivars. RAPD analysis, together with the published morphological data, will lead to a more comprehensive

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understanding of Vitis genetic diversity. This shows the potentiality of isozymes and RAPD markers to identify genetic diversity among muscadine cultivars. Recent phylogenetic reconstructions using DNA data have disagreed with traditional classifications and other DNA-based phylogenies by grouping Vitis and Cissus species within one clade and linking V. rotundifolia Michx. (muscadine grape) more closely with these Cissus species (Timmons et al. 2007). They studied comparative developmental morphology as an independent method to investigate whether V. rotundifolia is more similar to Cissus antarctica Vent. (Kangaroo vine) than other Vitis species. Results obtained using epi-illumination light microscopy and histology were compared with those from similar studies of Vitis “Ventura”, Vitis riparia Michx., and C. antarctica. Twelve vegetative and floral characters including axillary bud dorsiventrality, uncommitted primordium shape, calyptra, and ringshaped gynoecial disk placed V. rotundifolia in with other Vitis species. Only two characters, stipule timing and simple tendrils, were common to C. antarctica and V. rotundifolia, suggesting that V. rotundifolia is more similar to Vitis spp. than to C. antarctica, and supporting traditional classifications contrary to ITS1 and trnL DNA phylogenies.

4.5.2 ISSR Markers Inter simple sequence repeat (ISSR) markers have also been used in muscadine genetic analysis studies. ISSR markers were used to assess identity, pedigree, and diversity of cultivated muscadine grapes (Riaz et al. 2008). A total of 57 accessions [39 M. rotundifolia cultivars, 3 V. vinifera cultivars, 3 Vitis spp. hybrids, and 12 V. vinifera M. rotundifolia (VR) hybrids] from collections at the USDA National Clonal Germplasm Repository and the University of California (Davis) Department of Viticulture and Enology were analyzed with 14 ISSR markers. The fingerprint profiles were used to verify published breeding records of 31 M. rotundifolia cultivars and hybrids by comparing the shared alleles of parents and progeny. Marker data indicated that four cultivars were incorrectly identified; their alleles did not match respective parent/ progeny relationships at more than five loci. Two M. rotundifolia accessions had the same fingerprint

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profile as a third accession at all 14 markers, implicating a likely planting error. They discovered that M. rotundifolia cultivars exhibited 88 unique alleles that were not present in a database of more than 600 V. vinifera cultivars. In another study, RAPD, ISSR, and simple sequence repeats (SSR) techniques were combined to study downy mildew resistance derived from muscadine grape (Merdinoglu et al. 2003). BSA was employed to identify molecular markers linked to a gene that confers downy mildew resistance to the muscadine grape. The level of resistance to downy mildew of a BC2 segregating population was estimated by visual notation after inoculation of leaves on whole plant. The broad-sense heritability of the trait was estimated at 0.88. Polymorphism revealed with RAPD (151 primers), ISSR (13 primers), and SSR (208 loci) was screened between two bulks produced by separately pooling the individual DNAs from the six most resistant and the six most susceptible plants. Using analysis of variance, they showed that one RAPD, four ISSR, and eight SSR markers have a significant effect upon the level of resistance. Twelve of these markers were mapped on the same linkage group and covered a 45 cM long region. The identification of a QTL conferring resistance was confirmed by interval mapping. This QTL accounted for 73% of the observed variation and 83% of the genetic variation. These results strongly suggested that the identified QTL corresponds to a unique major gene conditioning the muscadine grape downy mildew resistance, which they have named Rp1. Moreover, Rpv1 was shown to be tightly linked to the dominant gene conferring resistance to powdery mildew, Run1. It is clear that ISSR in combination with other marker techniques can be successfully used in muscadine to determine the unique characteristics.

4.5.3 AFLP Analysis The monogenic dominant genetic determinism of total resistance to powdery mildew, introduced from M. rotundifolia into V. vinifera, was assessed using amplified fragment length polymorphism (AFLP) analysis (Pauquet et al. 2001). A BC5 population of 157 individuals was used to select AFLP markers linked to the resistance gene, Run1. Thirteen AFLP

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markers were selected and a local map was constructed around the Run1 gene. Ten markers among the 13 were found to cosegregate with the resistance gene. The usefulness of these 13 AFLP markers for the selection of Run1-carrying genotypes was further assessed through their analysis in a set of 22 Run1carrying resistant genotypes and 16 susceptible genotypes. Three markers out of the 13 analyzed were found to be absent in all the susceptible genotypes and present in all resistant individuals and may thus represent good tools for the marker-assisted selection of grapevine varieties resistant to powdery mildew. A recombination event among the markers that were originally found to co-segregated was observed in one of the resistant individuals, showing that recombination is possible in this region and may therefore be observed in larger populations. It indicates that AFLP can also be used as a potential marker to tag important traits in muscadine.

H.K.N. Vasanthaiah et al.

4.6 Omics Resources Developed A limited number of genomic studies have been carried out in Muscadinia. Majority of the nucleotide sequences derived from these studies have been deposited in NCBI database (Table 4.3). Most of the expressed sequence tags (ESTs) are from the Center for Viticulture and Small Fruit Research, Florida A&M University. New studies have to be initiated to sequence muscadine genome in order to fully exploit its potentiality related to disease resistance and adaptability to adverse climatic conditions. Muscadine grapes have been used by grape breeders for developing disease-resistant cultivars to combat diseases, pests, and environmental stresses (Lu et al. 2008). They have selected muscadine cv. Noble for EST program for potential gene discovery and marker development. cDNA libraries were constructed from mRNA isolated from a mix of leaves, shoot tips,

Table 4.3 Nucleotide and EST sequences available for Muscadinia Sl. No Accession no. Locus type Nucleotide sequences 1 AF119174 Ribulose-1,5-bisphosphate carboxylase/oxygenase 2 FJ644942 Chalcone synthase 3 FJ598145 Resveratrol synthase 4 AJ252874 VMC4D4 microsatellite DNA 5 EF179096 tRNA–Leu (trnL) gene 6 EF141292 V096 GAI-like protein 1 (GAI1) 7 EF108325 Stilbene synthase 2 gene 8 EF080828 Stilbene synthase 9 AB235081 Chloroplast DNA, trnL–trnF IGS 10 AB234975 Chloroplast rps16 gene 11 AJ419718 Plastid partial rbcL gene for rubisco large subunit 12 AY037922 Internal transcribed spacer 1 13 AY037900 tRNA–Leu (trnL) gene 14 AB234939 Chloroplast DNA, atpB–rbcL EST sequences 16 EL930131 HSP90 17 EL784689 PHOS mRNA 18 EE663493 Chalcone synthase 19 EE297453 Stilbene synthase 20 EE297452 Stilbene synthase 21 EE297451 Stilbene synthase 22 EE297450 Stilbene synthase 23 EE297449 Stilbene synthase 24 EE297448 Stilbene synthase 25 GW392494 Sucrose Phosphate synthase 26 GW392493 Sucrose synthase 27 GW392492 Acid invertase

Source

Tissue type

V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia

Leaf Berry Berry Leaf Leaf Leaf Berry Berry Leaf Leaf Leaf Leaf Leaf Leaf

V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia V. rotundifolia

Leaf Leaf Leaf Berry Berry Berry Berry Berry Berry Berry Berry Berry

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tendril, and flowers harvested during anthesis. A total of 8,081 high-quality ESTs were produced. The ESTs were contiged into a unigene set of 4,038, which included contigs (1,935) and singletons (2,103). Functional annotation of this unigene set of muscadine ESTs was conducted. EST sequences with putative functions related to plant disease resistance and stress tolerance were selected for further analysis. These sequences were also compared with two other grape species: V. Vinifera and V. shuttleworthii. Meanwhile, ESTs were also used for SSR/single nucleotide polymorphism (SNP) marker development. They concluded that identification of the SSR/SNP markers for putative disease/defense- and stress-related genes would be very useful for further utilization of this valuable muscadine grape germplasm for improvement of the Euvitis grape varieties. The obtained results can be productively incorporated into other grape improvement program. Especially its ability to tolerate Pierce’s disease caused by bacteria Xylella fastidiosa that has restricted growing European grapes in southeastern US.

4.7 Some Dark Sides of the Muscadinia Muscadine grapevines grow wild in forest lands, uncleared lots, on the fences, green areas, and along the highways throughout the southeastern US. They are hardy, disease-tolerant, and grow vigorously, posing vegetational threat to the unused land areas. They can become invasive and cause green menace by blocking the growth of other plant species in the unattended land areas. Wild muscadine grapes are functionally known to be dioecious due to their incomplete stamen formation in female vines and incomplete pistil formation in male vines (Anderson et al. 2003). Male vines account for the majority of the muscadine grape population.

4.8 Recommendations for Future Actions The strategies for increasing muscadine productivity and berry quality should comprise of developing new varieties after germplasm evaluation. Some of the strategies that need to be adopted are discussed here.

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Germplasm evaluation by morphological, cellular, and molecular means for agronomical traits is important. Here, at the Center for Viticulture and Small Fruit Research, we have germplasm repository of more than 50 muscadine genotypes with distinct characteristics in relation to fruit color, size, sugar content, and pest and disease resistance characteristics. This resource is being utilized in muscadine crop improvement program. Development of molecular data with SSR, ESTs, cDNAs, and SNPs in muscadine is also important to identify economically important genes from muscadine cultivars. At the Viticulture Center, FAMU, we have identified several genes associated with disease tolerance and nutraceutical compounds in muscadine. Stilbene synthase expressed in muscadine grape is unique and is reported to be associated with disease resistance. These unique traits can be transferred to commercially important grape and other fruit crops through genetic engineering. Use of embryo rescue technique for interspecific hybridization between muscadine and bunch species is necessary because of the differences in their chromosome numbers. This technique acts as a bridge for transferring unique traits from muscadine into bunch grapes and vice versa. Increasing the yield and quality of berry through selection and molecular approach has to be studied thoroughly. Yield can be increased only through selections. Determination of sucrose synthesis pathway, its transport to berry, and degradation into its constituent monosaccharides in muscadine will pave way to improve muscadine fruit quality and sugar content. Apart from this, studying variation in photosynthetic efficiency among grape species will also help in improving the berry quality and its enological characteristics. Studies need to be focused on developing genotypes with desirable traits such as early to mid-time ripening, uniform ripening, high sugar content, and of course, seedlessness, which are of immediate concern to the muscadine crop/fruit breeders of today and the future.

References Alley CJ (1957) Cytogenetics of Vitis H. Chromosome behavior and fertility of some autotetraploid derivatives of Vitis vinifera L. J Hered 48:194–202 Anderson PC, Crocker TE, Breman J (2003) The muscadine grape. In: University of Florida, IFAS Extension Newsletter, publication number HS763. http://edis.ifas.ufl.edu

76 Basha SM, Musingo M, Colova VS (2004) Compositional differences in the phenolics compounds of muscadine and bunch grape wines. Afr J Biotechnol 3(10):523–528 Benavente E, Cifuentes M, Dusautoir JC, David J (2008) The use of cytogenetic tools for studies in the crop-to-wild gene transfer scenario. Cytogenet Genome Res 20:3–4 Bouquet A (1980) Differences observed in the graft compatibility between some cultivars of Muscadine grapes (Vitis rotundifolia Michx.) and European grape (Vitis vinifera L. cv. Cabernet Sauvignon). Vitis 19:99–104 Buck S, Zyprian E (2000) First approaches of molecular mapping in a model population derived from the crossing of the grapevine varieties ‘Regent ’ ‘Lemberger ’. Acta Hortic 528:203–207 Cawthon DL, Morris JR (1983) Uneven ripening of ‘Concord’ grapes. Ark Farm Res 32(1):9 Clayton CN (1975) Diseases of muscadine and bunch grapes in North Carolina and their control. NC Agric Exp Stn Bull 451:37 Dalbo MA, Weeden NF, Reisch BI (2000a) QTL analysis of disease resistance in interspecific hybrid grapes. Acta Hortic 528:215–219 Dalbo MA, Ye GN, Weeden NF, Reisch BI, Steinkellner H, Sefc KM (2000b) A gene controlling sex in grapevines placed on a molecular marker-based genetic map. Genome 43:333–340 Daniel O, Meier M, Schlatter J, Frischknecht P (1999) Selected phenolic compounds in cultivated plants: ecologic functions, health implications, and modulation by pesticides. Environ Health Perspect 107:S109–S114 Dermen H (1964) Cytogenetics in hybridization of bunch and muscadine-types grapes. Econ Bot 18(2):137–148 Dermen H, Harmon FH, Weinberger JH (1970) Fertile hybrids from a cross of a variety of Vitis vinifera with V. rotundifolia. J Hered 61(6):269–272 Dhekney SA, Li ZT, Dutt M, Gray DJ (2008) Agrobacteriummediated transformation of embryogenic cultures and regeneration of transgenic plants in Vitis rotundifolia Michx. (muscadine grape). Plant Cell Rep 77:865–872 Dugo G, Saitta M, Giuffrida D, Vilasi F, La Torre GL (2004) Determination of resveratrol and other phenolic compounds in experimental wines from grapes subjected to different pesticide treatments. Ital J Food Sci 16:305–321 Ector BJ, Magee JB, Hegwood CP, Coign MJ (1996) Resveratrol concentration in Muscadine berries, juice, pomace, purees, seeds, and wines. Am J Enol Vitic 47:57–62 Goldy RG (1988) Variation in some yield determining components in muscadine grapes and their correlation to yield. Euphytica 39(1):39–42 Grando MS, Bellin D, Madini A, Stefanini M, Pozzi C, Velasco R (2000) Construction of an AFLP and SSR genetic map of Vitis from an interspecific hybrid population. In: Proceedings of plant and animal genome VIII conference, San Diego, California, USA Hardie WJ, Obrien TP (1988) Considerations of the biological significance of some volatile constituents of grape (Vitis spp). Aust J Bot 36:107–117 Huang H, Lu J (2000) Variation and correlation of bud breaking, flowering opening and fruit ripening in muscadine grape cultivars. Proc FL State Hortic Soc 113:46–47 Hudson TS, Hartle DK, Hursting SD, Nunez NP, Wang TT, Young HA, Arany P, Green JE (2007) Inhibition of prostate

H.K.N. Vasanthaiah et al. cancer growth by muscadine grape skin extract and resveratrol through distinct mechanisms. Cancer Res 67:8396–8405 James W, Hardie L (2008) Grapevine biology and adaptation to viticulture. Aust J Grape Wine Res 6:74–81 Jelenkovic G, Olmo HP (1968) Cytogenetics of Vitis. III. Partially fertile F1 diploid hybrids between V. vinifera and V. rotundifolia Michx. Vitis 7:8–18 Jindal PC (1985) Grape. In: Fruits of India – tropical and subtropical. Naya Prokash, Calcutta, India, pp 219–276 Kambiranda D, Vasanthaiah HKN, Basha SM (2010) Relationship between acid invertase activity and sugar content in Grape species. J Food Biochem (in press) Lanier MR, Morris JR (1979) Evaluation of density separation for defining fruit maturities and maturation rates of onceover harvested muscadine grapes. J Am Soc Hortic Sci 104:166–169 Le Blanc MR (2006) Cultivar, juice extraction, ultra violet irradiation and storage influence the stilbene content of Muscadine grape (Vitis rotundifolia Michx.). PhD Dissertation, Louisiana State University, Baton Rouge, FL, USA Lee JH, Johnson JV, Talcott ST (2005) Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS. J Agric Food Chem 53:6003–6010 Li ZT, Dhekney SA, Dutt M, Van Aman M, Tattersall J, Kelley K, Gray DJ (2007) Isolation and characterization of the 2S albumin gene and promoter from grapevine. Acta Hortic 738:759–765 Lodhi MA, Daly DJ, Ye GN, Weeden NF, Reisch BI (1995) A molecular marker based linkage map of Vitis. Genome 38:786–794 Lu J, Schell L, Lamikanra S (1993) Introgression of seedlessness from bunch grapes into muscadine grapes. Proc FL State Hortic Soc 106:122–124 Lu J, Schell L, Ramming DW (2000) Interspecific hybridization between Vitis rotundifolia and Vitis Vinifera and evaluation of the hybrids. Acta Hortic 528:481–486 Lu J, Huang H, Louime C, Hunter W (2008) Identification of disease defense and stress- related genes in muscadine grape through EST analysis. In: Proceedings of plant and animal genomes XVI conference, San Diego, CA, USA Mallikarjuna A, Bernard P, Gerald D, Simon C, Stover E (2005) Genetic diversity and phylogeographic structure of the genus Vitis: implications for conservation. In: Proceedings of the international conference on crop wild relative conservation and use, Bari, Italy Merdinoglu D, Wiedeman-Merdinoglu S, Coste P, Dumas V, Haetty S, Butterlin G, Greif C (2003) Genetic analysis of downy mildew resistance derived from Muscadinia rotundifolia. Acta Hortic 603:451–456 Meredith CP (2001) Grapevine genetics: probing the past and facing the future. Agric Conspec Sci 66:21–25 Mertens-Talcott SU, Percival SS (2005) Ellagic acid and quercetin interact synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle arrest in human leukemia cells. Cancer Lett 218:141–151 Mertens-Talcott SU, Lee JH, Percival SS, Talcott ST (2006) Induction of cell death in Caco-2 human colon carcinoma cells by ellagic acid rich fractions from muscadine grapes (Vitis rotundifolia). J Agric Food Chem 54:5336–5343 Morris JR (1980) Handling and marketing of muscadine grapes. Fruit South 4(2):12–14

4 Muscadiniana Mortensen JA, Andrews CP (1981) Grape cultivar trails and recommended cultivars for Florida viticulture. Proc FL State Hortic Soc 94:328–331 Neveln V, Pelczar R (2006) The scoop on Scuppernongs. Summer gardening trends research report. Garden Writers Association Foundation, USA, pp 50–51 Olien WC (2001) Introduction to the muscadines. In: Basiouny FM, Himelrick DG (eds) Muscadine grapes. ASHS, Alexandria, VA, USA, pp 1–13 Olmo HP (1960) Plant breeding program aided by radiation treatment. Calif Agric 14:4 Olmo HP (1971) Vinifera rotundifolia hybrids as wine grape. Am J Enol Vitic 22:87–91 Pastrana-Bonilla E, Akoh CC, Sellappan S, Krewer G (2003) Phenolic content and antioxidant capacity of muscadine grapes. J Agric Food Chem 51:5497–5503 Pauquet J, Bouquet A, This P, Adam-Blondon AF (2001) Establishment of a local map of AFLP markers around the powdery mildew resistance gene Run1 in grapevine and assessment of their usefulness for marker assisted selection. Theor Appl Genet 103(8):1201–1210 Poling EB (1996) Muscadine grapes in the home garden. Horticulture Information Leaflet 8203, North Carolina Cooperative Extension Service, North Carolina State University, Raleigh, NC, USA Poling B, Fisk C (2006) Muscadine grapes in the home garden. Department of Horticultural Science, NCSU (Duplin County). http://www.ces.ncsu.edu/depts/hort/hil/hil-8203.html Ren Z, Lamikanra O, Lu J (2000) Identification of a RAPD marker closely linked to the fruit color in muscadine grapes (Vitis rotundifolia I). Acta Hortic 528:263–266 Riaz S, Meredith CP (2000) A microsatellite marker based linkage map of Vitis vinifera. In: Proceedings of plant and animal genome VIII conference, San Diego, CA, USA

77 Riaz S, Vezzulli S, Harbertson ES, Walker MA (2007) Use of molecular markers to correct grape breeding errors and determine the identity of novel sources of resistance to Xiphinema index and Pierce’s disease. Am J Enol Vitic 58 (4):494–498 Riaz S, Tenscher AC, Smith BP, Ng DA, Walker MA (2008) Use of SSR markers to assess identity, pedigree, and diversity of cultivated muscadine grapes. J Am Soc Hortic Sci 133:559–568 Sawazaki HE, Pommer CV, Passos IRDS, Terra MM, Pires EJP (1996) Identification of parents and hybrids among Vitis vinifera and Vitis rotundifolia using isozyme polymorphism and RAPD marker. Bragantia 55(2):221–230 Stoffella PJ, Mortensen JA, Hayslip NC, Brolmann JB (1982) Evaluation of muscadine grape cultivars in South Florida. Proc FL State Hortic Soc 95:90–92 Talcott ST, Lee JH (2002) Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. J Agric Food Chem 50:3186–3192 Timmons SA, Posluszny U, Gerrath JM (2007) Morphological and anatomical development in the Vitaceae. IX. Comparative ontogeny and phylogenetic implications of Vitis rotundifolia Michx. Can J Bot 85(9):850–859 Vasanthaiah HKN, Basha SM (2008) Resveratrol, a versatile natural compound. In: Thangadurai D, Tripathi L, Vasanthaiah HKN, Cantu DJ (eds) Crop improvement and biotechnology. Bioscience, Puliyur, India, pp 157–180 Vasanthaiah HKN, Katam R, Basha SM (2007) A new stilbene synthase gene from muscadine (Vitis rotundifolia) grape berry. In: Proceedings of frontiers in the convergence of bioscience and information technologies (FBIT 2007), Korea, pp 87–91 Wang Y, Chen J, Lu J, Lamikanra O (1999) Randomly amplified polymorphic DNA analysis of Vitis species and Florida bunch grapes. Sci Hortic 82(1–2):85–94

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Chapter 5

Olea E. Rugini, C. De Pace, P. Gutie´rrez-Pesce, and R. Muleo

5.1 Basic Botany of the Species 5.1.1 Taxonomic Position The genus Olea belongs to the Oleaceae family, which comprises approximately 30 genera with 600 species (Cronquist 1981), distributed in every continent as ornamental plants or in productive orchards (Fig. 5.1). Although the classification is not well defined yet, according to several modern authors, the genus Olea splits into three subgenera, Tetrapilus, Paniculatae, and Olea (cultivated olive and wild relatives), found in Asia, Australia and Asia, Africa and Europe, respectively. The subgenus Olea is divided into two sections: Ligustroides (about 10 species) and Olea (one species: O. europaea). Both these sections thrive in the mountains of East Africa and in the Pacific Islands. In particular, Olea is also found in west of the Sahara, in the Macaronesian Islands (Canary and Madeira), and the Mediterranean basin (Green 2002) (see Sect. 5.1.2). The section Olea includes the complex of O. europaea L, the Mediterranean olive tree, the only species cultivated for oil extraction and table consumption, which accounts more than 1,000 of cultivars, although many of these might be just different landraces stemmed from the same original genetic stock or different named varieties derived from the same original genetic stock. It has been reported that the cultivated olive is not a species, but rather an ensemble of different adapted ecotypes raised at the rank of sub-species (ssp.), which originated from mutations and natural hybridizations. Tropical and subtropical

E. Rugini (*) Dipartimento di Produzione Vegetale, Universita` degli Studi della Tuscia, Viterbo 01100, Italy e-mail: [email protected]

Afro-Asiatic species, such as Olea chrysophilla Lam. and Olea excelsa Ait (old nomenclature for Ligustroides species), probably contributed to the evolution of the Euro-Mediterranean olive (O. europaea L.) (Mazzolani and Altamura Betti 1976, 1977; Green and Wickens 1989; Zohary 1994). The Euro-Mediterranean olive (O. europaea L. ssp. europaea) comprises the wild oleaster (var. sylvestris) and the cultivated (var. sativa) types (Fig. 5.2). The var. sylvestris has a narrow range of distribution and it is often mistaken for olevaster (Fig. 5.3), which is a feral form derived from seeds of the cultivated type O. europaea ssp. europaea var. sativa. The feral form olevaster remains at a constant juvenile stage and never produces flowers unless it is transferred to standard cultivation (Rugini and Lavee 1992). The var. sylvestris with its very small fruits and small and almost round leaves, thorny shoots, and quadrangular branch section was used in the past as rootstock for the var. sativa. According to Green and Wickens (1989), the O. europaea L. would include also its wild relatives, i.e., the Afro-Asiatic subspecies cuspidata, laperrinei, and cerasiformis. Nonetheless, the systematic classification of the genera is still far to be defined. While some authors recognize six subspecies in O. europaea (Sect. 5.1.2), Green and Wickens (1989) distinguished four subspecies according to theirmorphology and their geographical distribution: O. europaea ssp. europaea of the Mediterranean Basin; O. europaea ssp. laperrini (Batt. and Trab.) Ciferri of the Sahara Massifs; O. europaea ssp. cerasiformis (Webb. and Berth.) Kunk. and Sund. of the Canary Islands and Madeira; O. europaea ssp. cuspidata (Wall. Ciferri) of Asia (China, India, Pakistan, Nepal, Iran, South Arabia) and Southeast Africa (Fig. 5.4). A complete review of the history of classification of olive germplasm is reported by Bartolini et al. (2002) and Ganino et al. (2006).

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_5, # Springer-Verlag Berlin Heidelberg 2011

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Fig. 5.1 World distribution of the main Olea species and subspecies

Fig. 5.2 Cultivated olive trees (Olea europaea ssp. europaea var. sativa)

Phylogenetic reconstruction based on plastid DNA data sustained a maternal origin of O. e. laperrini populations in South Algeria, where a higher allele richness was observed (Besnard et al. 2007b). Based on nuclear microsatellite data, two levels of structure were revealed: first, individuals from Niger and Algeria were separated into two distinct groups; second, four less-differentiated clusters corresponded to the four studied mountain ranges (Hoggar, Tassili n’ Ajjer, Bagzane, and Tamgak). These results provide support to the fact that desert barriers have greatly limited long distance gene flow.

Phylogenetic congruence of both ITS (ribosomal internal transcribed sequence) and plastid lineages suggested an evolutionary scenario of predominant isolation during the Plio-Pleistocene in Macaronesia, the Mediterranean, southern Africa, eastern Africa, and Asia. The clear-cut geographical distribution of chloroplast DNA haplotypes supports an early differentiation between populations from southern Africa to China (subsp. cuspidata) and the Mediterranean, Macaronesia, and the Sahara (all other subspecies) (Besnard et al. 2007a).

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Fig. 5.3 One of the oldest olevaster genotype located in central Italy has a trunk circumference of about 7.2 m, and it is able to produce up to 1 ton of olives per year

Fig. 5.4 Olea europaea ssp. cuspidata is one of the numerous wild Olea europaea sub-species, which grows also on the mountains of Nepal

Both ITS-1 and plastid DNA data indicate that most populations of O. europaea differentiated phylogenetically in five geographical areas: (1) equatorial and southern Africa (ssp. laperrini); (2) eastern Africa and southern Asia (ssp. cuspidata); (3) the eastern Medi-

terranean (ssp. europaea est); (4) the western Mediterranean (ssp. europaea west); and (5) Macaronesia and Northwest Africa (ssp. guancica, cerasifomis, maroccana). However, unexpected incongruences between plastid DNA and ITS-1 analyses, associated with

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ITS-1 intraindividual polymorphism, led Besnard et al. (2007a) to propose a more dynamic biogeographical pattern. Populations of ssp. cuspidata are present across a large area (from Ethiopia to China) and may have diverged from an ancient common ancestor that colonized this geographical area. It has been hypothesized that populations from African and Arabian coasts of the Red Sea may reflect a secondary divergence from populations from eastern Africa and southern Asia. Most of the African individuals of subsp. cuspidata display haplotypes from African and Arabian coasts of the Red Sea and eastern Africa and southern Asian populations except for one population from southern Egypt, which forms part of a new sublineage together with eastern Mediterranean (subsp. europaea) and Saharan (subsp. laperrinei) populations (Besnard et al. 2007a). This suggested an ancient hybridizone from the Sahara to northeastern African mountains, where divergent plastid and nuclear lineages still coexist. Other past hybridization events imply to all the subspecies. In fact, populations of ssp. europaea form part of three western sublineages plus a sublineage (E1), including Mediterranean and Saharan ssp. (europaea, laperrini and cuspidata), and a further lineage (M), which included the three subspecies (spp. maroccana, guanchica, cerasiformis) of Northwest Africa and Macaronesia (Besnard et al. 2007a). Therefore, there are more plastid lineages than recognized taxa, whereas two lineages (E1, M) contain the six subspecies. Cooccurrence of two divergent ITS-1 copies in an intersubspecies hybrid reported in an invasive Australian population of O. europaea (Besnard et al. 2007a), provides further evidence that mixture of divergent copies in a single lineage is the result of hybridization between genetically distant genomes. Secondary contacts in a potential, large hybrid zone consisting of the Saharan mountains (from the Hoggar to southern Egypt) when climatic conditions were favorable, and gene exchange in the Kenyan and Ethiopian highlands (Rift Valley) through a corridor connecting eastern and southern Africa, may account for limited morphological differentiation and incongruence between taxonomy, nuclear sequences, and plastid haplotypes in the olive tree complex. Overall, the differentiation of isolated populations of ssp. laperrinei, maroccana, guanchica, and cerasiformis, is considered a more recent event which occured

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after the mentioned pattern of hybridization already took place. In the late 6,000 years, the continuous olive domestication through local hybridization of cultivated trees with natural populations has brought the remarkably high genomic diversity among cultivated trees in the Mediterranean basin. Since its formation (ca. 7 Mya), the Saharan desert may have been an effective barrier, limiting significant reproductive contacts between the two major plastid lineages, which are separated in all molecular analyses (Vargas and Kadereit 2001). However, a succession of humid transitional events in the Saharan mountains may have facilitated recurrent contacts between O. europaea populations from the Mediterranean basin and Tropical Africa. Long-distance gene flow via pollen dissemination might have occurred. Palaeobotanical and palaeoclimatic evidence agrees with a deeper genetic and geographical isolation of the Saharan populations of subsp. laperrinei on high mountains as a result of aridification, particularly after the last deglaciation (Que´zel 1978; Gasse et al. 1990). The ssp. europaea originated from a pre-Quaternary Mediterranean ancestor, with no evidence for a recent hybrid origin. Common ancestry of O. europaea in Africa may have spawned new lineages in Macaronesia, Asia, and the Mediterranean long before the Quaternary (Palamarev 1989). Evolution of the Mediterranean climate in the past 3 Mya may have caused differentiation of subsp. europaea populations in the thermophilous vegetation of the Mediterranean Basin (Palamarev 1989). The occurrence of several glacial refugia in Macaronesia, Morocco, southern Iberia, and the eastern Maghreb, coupled with the introduction of multiple cultivars across the Mediterranean and consequent gene flow towards local populations, may account for recurrent contacts of olive genomes resulting in a higher genetic diversity observed in the western Mediterranean basin (Besnard et al. 2002a, b).

5.1.2 Geographical Distribution and Genetic Diversity Several evidences demonstrated the presence of some forms of olive during the last glaciation in the western and eastern Mediterranean regions 18,000 years BC (Carrion and Dupre 1996; Watts et al. 1996). Leaf fossils

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have been found in Pliocene deposits at Mongardino in Italy (between ending of Miocene 5.3 millions of years and beginning of the Pleistocene 1.8 millions years before present). Fossilized remains have been discovered in strata from the Upper Paleolithic at the Relilai snail hatchery in North Africa, and pieces of wild olive trees and stones have been uncovered in excavations of the Chalcolithic period and the Bronze Age in Spain. The existence of the olive tree, therefore, dates back to the twelfth millennium BC. Numerous studies confirmed that olives have been present for several thousands of years before its domestication in the Mediterranean Basin, particularly in the Middle East. Olive was probably domesticated in the Jordan River valley ca. 5700–5500 years BC (Zohary and Spiegel-Roy 1975), and more precisely, according to Liphschitz et al. (1991) domestication occurred during the bronze period (5200 years BC). Wild olive trees are extremely abundant and grow in thick forests in Asia Minor. It appears to have spread from Syria to Greece via Anatolia (De Candolle 1883) although other hypotheses point to Lower Egypt, Nubia, Ethiopia, the Atlas Mountains, or certain areas of Europe as its source area. Mediterranean olives comprise genuinely wild olive trees, wild-looking forms (feral olives) that are secondary derivatives produced by sexual reproduction among cultivated olives, and varieties. Past climatic conditions suitable for oleaster growth such as an increase from 1 to 1.5C recorded in southern France at the beginning of the Subatlantic chronozone of Holocene could be related to the expansion of thermophilous vegetation including Olea (Terral and Mengual 1999). Using genetic markers (alleles for isozyme) associated with characters (prolonged juvenile phase) that render plants unsuitable for domestication, Lumaret and Ouazzani (2001) showed that genuinely wild olive trees, which cannot be distinguished morphologically from feral forms, still survive in a few Mediterranean forests in southern France and Spain, North Morocco and Tunisia, Corsica, Sicily and Cyprus islands, and Palestine. In the eastern part of the Mediterranean Basin (i.e., Egypt, Syria, Turkey, Crete, or Greece), where olive trees have been extensively cultivated for longer periods, no candidate forests containing genuinely wild olive trees were found. The precise relationships of the Mediterranean olives (O. europaea L. subsp. europaea) to the other subspecies have remained elusive (Besnard et al. 2007a, b, c). As stated before, over the times, genetic

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differentiation favored by geographic barriers caused divergence within the Mediterranean olive tree populations, leading to the current assessment of the five subspecies (Green 2002): O. e. ssp. laperrinei, distributed in Saharan massifs (Hoggar, Aı¨r, Jebel Marra in Algeria); O. e. ssp. cuspidata, from Egypt to South Africa (from here it was introduced in East Australian areas and Hawaii), and from Arabia to northern India and Southwest China; guanchica in the Canary Islands; O. e. ssp. maroccana in Agadir mountains, southwestern Morocco; and O. e. ssp. cerasiformis in Madeira island (Besnard et al. 2007a). The spread of the cultivated olive to other countries of the Mediterranean region accompanied migrations of different civilizations (Egyptian, Phoenician, Greek, Etruscan, Roman, and Arab). The wild olive gene pool is considered to be indigenous to the entire Mediterranean Basin and Asia Minor, which provided the ancestral parental plants at the beginning of the domestication process that lead to the cultivated olive some six millennia ago. The Assyrians and Babylonians were the only ancient civilizations that were not familiar with the olive tree. The original home of the olive tree is the area from the southern Caucasus to the Iranian plateau, while in the Mediterranean coasts of Syria and Palestine, its cultivation developed considerably, spreading from there to the island of Cyprus and on toward Anatolia or from the island of Crete toward Egypt. When archeological olive stones are compared with stones of modern cultivars, an early and autochthonous olive domestication in northwestern Mediterranean areas is suggested. The appearance of cultivated forms at the Chalcolithic/Bronze Age seems to corroborate that farming and selective practices have been operated at least since that time. These results support hypotheses from previous bioarcheological and paleoenvironmental studies that evidence for the emergence of cultivation practices from the Neolithic and the Bronze Age in Spain (Terral et al. 2004). In the sixteenth century BC, the Phoenicians started disseminating the olive throughout the Greek islands, later introducing it into Greek mainland between the fourteenth and twelfth centuries BC, where its cultivation increased and gained importance in the fourth century BC after Solon issued decrees regulating olive planting during the Athenian democracy (600 BC), in the first written legislation of the world, prohibiting the cutting down of olive trees. From the sixth century BC onward, O. europaea ssp. europaea spread throughout the Mediterranean

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countries reaching Tripoli, Tunis, and the island of Sicily. From there, it moved to southern Italy. It is believed that the introduction of olive trees in Italy dates back to three centuries before the fall of Troy (1200 BC). Another Roman annalist (Penestrello) defends the traditional view that the first olive tree was brought to Italy during the reign of Lucius Tarquinius Priscus the Elder (616–578 BC), possibly from Tripoli or Gabes (Tunisia). Cultivation moved upwards from south to north, from Calabria to Liguria. When the Romans arrived in North Africa, they found evidences on the use, from the Berbers of the wild gene pool as scion to graft domesticate olives; the Romans really developed the grafting technology and spread its adoption throughout the territories they occupied (Zohary and Hopf 1994; Besnard et al. 2001b; Spennemann and Allen 2000; Green 2002; Terral et al. 2004). The Romans continued the expansion of the olive tree cultivation area to the countries bordering the Mediterranean, using it as a peaceful weapon in their conquests to settle the people. It was introduced in Marseilles around 600 BC and spread from there to the whole of Gaul. The olive tree made its appearance in Sardinia in Roman times, while in Corsica, the traditional belief dated the beginning of olive cultivation to a period after the fall of the Roman Empire, using specimen introduced from Liguria. Olive was introduced into Spain during the maritime domination of the Phoenicians (1050 BC) but did not develop to a noteworthy extent until the arrival of Scipio (212 BC) and Roman rule (45 BC). After the third Punic War, olives occupied a large stretch of the Baetica valley and spread toward the central and Mediterranean coastal areas of the Iberian Peninsula including Portugal. The Arabs brought their varieties with them to the south of Spain and influenced the spread of cultivation so much that the Spanish words for olive (aceituna), oil (aceite), and wild olive tree (acebuche) and the Portuguese words for olive (azeitona) and for olive oil (azeite) have Arabic roots. With the discovery of America, olive farming spread beyond its Mediterranean confines. The first olive trees were carried from Seville to the West Indies and later to the American Continent. By 1560, olive groves were cultivated in Mexico, and in the last few years, growth of olive production has expanded throughout the world to countries such as Australia, Canada, China, Peru, Chile, Argentina, and the United States (Reale et al. 2006).

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5.1.3 Genetic Diversity and Gene Flow Between Wild and Cultivated Olive The above-mentioned results of Lumaret and Ouazzani (2001) provide evidence of the survival of indigenous oleaster populations, particularly in the western part of the basin. Genetic diversity values over cultivars, feral olives, and the wild olives in ten forest areas around the Mediterranean basin were 0.286, 0.414, and 0.506, respectively, which is consistent with the interpretation that the domesticated olive represents a sample of the genetic variation in genuinely wild olive populations that persist today. Owing to their very long lifespan, these wild trees should be closely related to the Neolithic olives recognized as the crop progenitor. Other authors, using a sample of 166 oleasters taken from 20 groves of modern populations, and 40 cultivars to represent molecular diversity in the cultivated olive, evidenced that oleaster genetic diversity can be traced to the geographical diversity among seven regions that could overlay past glacial refuges (Breton et al. 2006). The gradient, or cline, of genetic diversity revealed by chloroplast and microsatellite or simple sequence repeat (SSR) molecular markers was explained by oleaster recolonization of the Mediterranean basin from refuges after the last glacial event, located in both eastern and western regions. It is likely that gene flow has occurred in oleasters mediated by cultivars spread by human migration or through trade. The observed patterns of genetic variation for amplified fragment length polymorphism (AFLP) markers suggested a clear distinction of the wild populations from the cultivated landraces and continental from insular regions (Baldoni et al. 2006). Island oleasters were highly similar to each other and were clearly distinguishable from those of continental regions. Ancient cultivated material from one island clustered with the wild plants, while the old plants from the continental region clustered with the cultivated group. A general picture (Fig. 5.5) on the past events that shaped the current oleaster distribution has emerged from mitochondrial DNA variation and random amplified polymorphic DNA (RAPD) markers (Bronzini de Caraffa et al. 2002a, b; Besnard and Berville´ 2002), chloroplast and SSR variation (Breton et al. 2006), and isozyme analysis of Lumaret et al. (2004). Oleasters from ancestors that during the last ice-age glaciation

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Fig. 5.5 Oleaster distribution based on mitochondrial DNA variation and RAPD marker (Bronzini de Caraffa et al. 2002a, b; Besnard and Berville 2000), chloroplast and SSR variation (Breton et al. 2006), and isozyme analysis (Lumaret et al. 2004). (a, d) Glacial refugia of oleaster. In the west refugium, oleaster with MOM and MCK mitotype, COM and CCK chlorotype, and western RAPD pattern were found. In the east refugium, oleaster with ME1 and ME2 mitotype, CE1 chlorotype, and eastern RAPD pattern were found. Molecular diversity is much higher in the west than in the east, supporting the hypothesis that western oleaster are genuine and differentiated earlier (Breton et al. 2006). (b) True oleasters in Sardinia and Corsica are only those plants with MOM and MCK mitotype and west RAPD pattern; oleaster with ME1 mitotype might be feral form derived

by hybridization of cultivated olives with ME1 mitotype (female) true oleaster (male) with western RAPD pattern. The reciprocal hybridization also produced feral form with MOM mitotype and east RAPD pattern. Oleaster with ME1 mitotype and east RAPD are considered feral form of the cultivated varieties and not true oleasters. Therefore, some western cultivars in those islands might have originated from western oleaters (Breton et al. 2006). (c) Cultivated olives show mostly ME1 mitotype and eastern RAPD pattern. The model support the eastern domestication of olives and the east to a west diffusion of varieties (Loukas and Krimbas 1983; Zohary and Hopf 1994), including some restricted domestication events from local genuine western oleaster occurring in Corsica and south of Spain

strived in the western Mediterranean refugium (corresponding to the territory of Algeria, Morocco, Tunisia), share the MOM and MCK mitotype and COM and CCK chlorotype that are absent in oleasters from the ancestral populations occupying the eastern refugia (Turkey, Palestine, Syria–Iran boundary). Oleasters in East Mediterranean share the ME1 and ME2 mitotype, CE1 chlorotype, and eastern RAPD pattern. Molecular diversity is much higher in the west than in the east, supporting the hypothesis that western oleaster are genuine and differentiated earlier (Breton et al. 2006). True oleasters in Sardinia and Corsica are only those plants with MOM and MCK mitotype and west RAPD pattern; oleaster with ME1 mitotype might be the feral form derived by hybridization of cultivated olives with ME1 mitotype (female) true oleaster (male) with western RAPD pattern. The reciprocal hybridization also produced feral form with MOM mitotype and

east RAPD pattern. Oleaster with ME1 mitotype and east RAPD are considered the feral form of the cultivated varieties and not true oleasters. It is not excluded that some western cultivars in those islands might have originated from western oleaters (Breton et al. 2006). Cultivated olives show mostly ME1 mitotype and eastern RAPD pattern, supporting the eastern domestication of olives and the east to a west diffusion of varieties (Loukas and Krimbas 1983; Zohary and Hopf 1994).

5.1.4 Morphology Wild and domesticated olives grow in the same areas but the wild type shows some morphological differences compared to cultivated forms, such as smaller fruit size and lower oil content in the mesocarp (Terral

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and Arnold-Simard 1996). Two distinct wild types of olives have been recognized: oleaster and feral forms. Oleaster occupies primary niches in undisturbed areas as a constituent of evergreen plant associations. Wild and cultivated varieties, representing two genetically separated complexes, have been interconnected as a result of occasional hybridizations that could have allowed the introgression of genes from the wild forms into the cultivated varieties. Due to their long life span, there has been relatively little selection and the cultivated olive gene pool is assumed to be very similar to the gene pool of their wild progenitors (Liphschitz et al. 1991). Baali-Cherif and Besnard (2005) mentioned that O. e. laperrinei in the Hoggar (Algeria) was a small multi-stemmed tree, thus suggesting that it may use a vegetative strategy for its reproduction and persistence in arid environments (Anthelme et al. 2008). In contrast, in the wetter western Darfur, O. e. laperrinei was described as a tree reaching up to 15 m high without mention of a multistemmed shape (Que´zel 1969). The rarity of sexual reproduction is one of the most remarkable life-traits of O. e. laperrinei in the Hoggar (Que´zel 1969; Baali-Cherif and Besnard 2005). The olive is a long-lived evergreen plant that adapts quite easily to many and varied environmental conditions. Its characteristic basitony gives the plants a shrub-like appearance, when allowed to grow without any pruning or training. The branches may vary from upright to pendulous according to the cultivar, and their vigor depends upon position, productivity, and the nutritional status of the tree. To develop olive trees with compact growth habit is an important breeding objective, since small olive plants would contribute to lower costs of manual labor for harvesting and pruning and may allow the use of more efficient machines than the current shaking ones. Flower buds and, less frequently, mixed buds differentiate in the same year of flowering, although flower induction occurs already by the end of the preceding summer (Proietti and Tombesi 1996). Flowers gathered during inflorescence normally are hermaphroditic, although flower anomalies frequently occur. Depending on the cultivar, flowers may be partially self-fertile or completely self-fertile (Fontanazza et al. 1990). The olive fruit is a drupe that contains a bitter component (oleuropein), a low sugar content (2.6–6%) compared with drupes of other species (12% or more),

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and high oil content (12–30%) depending on the time of year and variety. These characteristics make it a fruit that cannot be consumed directly from the tree, and it has to undergo a series of processes that differ considerably from region to region and from one cultivar to another. Some olives are, however, an exception to this rule because they sweeten right on the tree as they ripen in most cases due to fermentation. One case in point is the Thrubolea variety in Greece. Fruit-set is very low-ranging from 1 to 5% of flowers. The most crucial phases of fruit morphogenesis are the cell division and cell enlargement in the pericarp and then lipid synthesis and accumulation. They show a maximum rate, whose pattern may be strongly influenced by stresses and nutritional deficits. Apparent parthenocarpy is frequent in peculiar environmental conditions and for given cultivars, leading to very small (3–4 mm in diameter) and round drupes, which often ripen. Overcoming self-sterility, enhancing fruitsetting ability, and assuring a constant high productivity are further objectives in the improvement of the species.

5.1.5 Karyotype and Genome Size Olea is considered a genus containing diploid species, with the basic chromosome number x 23. The nuclear DNA content of olive cultivars was determined for the first time by Rugini et al. (1996), who used Feulgen cytophotometry to estimate the 2C nuclear DNA content of cvs. “Frantoio” and “Leccino”. The 1C DNA content has been estimated to be approximately 2.2 pg corresponding to a genome size of 2,200 Mb. Evidences for the presence of triploid and tetraploid mutants from the “Frantoio” and “Leccino” cultivars were given. More recently and using the same technique, Bitonti et al. (1999) estimated the genome size of cvs. “Dolce Agogia” and “Pendolino”. The results of these studies indicated a high intraspecific variation for genome size among the studied Italian cultivars. Bitonti et al. (1999) also analyzed the genome size of other Olea species and verified that it was considerably lower than the genome size of O. europaea cultivars. On the other hand, using flow cytometry, Loureiro et al. (2007) determined that the nuclear DNA content of O. europaea cultivars ranged between 2.90 0.020 pg/2C and 3.07 0.018 pg/2C and the genome size of wild olive was estimated

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as 3.19 0.047 pg/2C DNA. These results suggest a low intraspecific variation at least among the studied cultivars and between them and wild olive. The presence of polyploid plants in natural populations of O. e. ssp. cuspidata (from Iran) and maroccana was inferred from microsatellites data by Rallo et al. (2003) and Besnard et al. (2008) using flow cytometry, and nuclear microsatellite analyses provided strong evidences for polyploidy in ssp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Because polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana), it has been hypothesized that polyploidization has been favored to overcome inbreeding depression (Besnard et al. 2008). Based on previous phylogenetic analyses, Besnard et al. (2008) hypothesize also that tetraploid subsp. cerasiformis resulted after hybridization between ancestors of subspp. guanchica and europaea.

5.1.6 Agricultural Status Cultivated olive is one of the most important tree crop species of the Mediterranean basin, representing not only the 90% of the olive groves of the world but also the 90% of the olive world production. Only Spain, Italy, and Greece produced around 75% of the world’s olive oil, and together with Turkey and Tunisia are the five largest producers in the world. In 2005, world production was approximately 15,500 Mt, while in 2007, it increased to 17,500 Mt, destined to both oil and fresh consumption (FAOSTAT 2007). The world production and consumption trend of olive oil in the last 30 years have increased significantly and will continue, considering the recent introduction of its cultivation in Japan, USA, Australia, China, South America, and South Africa. An increasing interest in olive products in Australia occurred during the late 1940s and 1950s coinciding with the immigration from the Mediterranean basin. By 1959, 2,929 ha were under cultivation in Australia (Hartmann 1962), and by 1998, plantings of O. europaea had increased to more than 5,000 ha, with plants equivalent to another 7,000 ha of trees on order (Spennemann and Allen 2000). Olive orchards are now found in all the six Australian states.

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Today, there are over 100 known varieties of olives in Australia (Sweeney and Davies 1998). Australia could have the opportunity to produce organic olive oil because of the absence of the major diseases prevalent in the traditional olive-growing countries. The world olive oil production is estimated to be three million tons, ranking sixth in the world production of fluid vegetable fats exceeded by soybean, cottonseeds, peanuts, and sunflower. The olive oil production is not constant over the years, owing to the proverbial alternate-bearing tendency of this species that is genetically and environmentally determined. O. europaea ssp. cuspidata does not have great economic importance, but its wood can be used for furniture in South Africa, can be cultivated as an ornamental tree in China, and can be utilized as a rootstock and hedge plant (Spennemann and Allen 2000; Starr et al. 2003). The past history of domestication, demography (geographic isolation and ecotypic adaptation to a vast array of environmental conditions), and gene flow within and among subspecies have shaped and genetically structured the gene pool of Olea. The primary gene pool (GP1) of O. europaea ssp. europaea includes all the diploid and interfertile Mediterranean forms of O. europaea. The wild subprimary gene pool incorporates populations of the genuine oleaster and of the olevaster, and the cultivated subprimary gene pool comprises the orchard varieties. Although at the end of the last glaciations, hybridization among the ancestors of the current sub-species might have occurred, as it is suggested by Besnard et al. (2008) for the guanchica and europaea hybridization that gave rise to the ssp. cerasiformis, subsequent geographic and genetic (polyploidization) isolation events occurred, rising biological barriers to the gene flow among the six O. europaea sub-species. Therefore, those sub-species contribute to the formation of the secondary gene pool (GP2) of O. europaea ssp. europaea (Fig. 5.6). The tertiary gene pool (GP3) accounts for most of the Ligustroides species from which the gene transfer to O. europaea ssp. europaea could occur only through in vitro culture of hybrid embryos and of somatic hybrids. The quaternary gene-pool of O. europaea ssp. europaea so far includes those bacteria and plants (i.e. tobacco) from which the rol and osmotin genes, respectively, have been transferred to O. europaea ssp. europaea (Fig. 5.6) (Rugini et al. 2008).

88 Fig. 5.6 An example of Gene pools of Olea useful in a program of genetic improvement: GP1 Primary gene pool; GP2 Secondary gene pool; GP3 Tertiary gene pool; GP4 Quaternary gene pool

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GP1 genuine wild olive - oleaster O. europaea ssp europaea var sylvestris (diploid, 2n = 46) Feral forms or olevaster, often confused with oleaster

GP1 cultivated olive O. europaea ssp europaea var sativa (diploid, 2n = 46)

GP2 - O. europaea ssp europaea var cerasiformis (tetraploid)(Madera) - O. europaea ssp europaea var maroccana (hexaploid)(SW Morocco) - O. europaea ssp europaea var guanchica (diploid)(Canary islands) - O. europaea ssp europaea var laperrini (diploid; mainly veg. propag.; sexual repr. is rare)(Sahara mountains) - O. europaea ssp europaea var cuspidata (diploid) (Asia, China, India, pakistan, Iran, Arabia, East and South Africa)

GP3

O. paniculata (Australia, Sect Ligustroides China, India, Pakistan, O. exasperata (South Africa) GP4 New Caledonia) O. capensis ssp. macrocarpa (Madagascar and South Africa) O. capensis ssp. capensis (South Africa) O. woodiana (Central-southern Africa) O. lancea (Madagascar, Mauritius, Reunin)

rol genes of Agrobacterium (reduced plant vigour) Osmotin gene from Tobacco (fungi resistance)

5.2 Conservation Initiatives 5.2.1 Evaluation of Genetic Erosion The discovery of indigenous oleaster populations (Lumaret and Ouazzani 2001) along the western Mediterranean basin will encourage in those areas the preservation of environmental conditions that favor the survival of large populations of oleasters that are properly isolated from cultivated olive trees. For O. e. laperrinei, population on Mt. Hoggar (Algeria) acts as an important genetic reservoir that has to be taken into account in future conservation programs. Moreover, very isolated endangered populations (for example, Bagzane) displaying evident genetic particularities have to be urgently considered for their endemism (Besnard et al. 2007b).

5.2.2 Germplasm Banks Olive (O. europaea L.) germplasm is normally preserved in clonal collections, located in different sites of several countries. There is not a centralized service for conservation, but several institutions carry on, on their own, the recovery, propagation, and maintenance

of a number of accessions, mainly autochthonous ones. Most recently, the International Olive Oil Council promoted a campaign aimed at retrieving and also preserving accession from distant locations and countries where preservation is not provided (Fabbri et al. 2004). These field banks (ex situ) are located mainly in traditional countries including Spain, Iran (Amiri 2008), and Italy (Sicily; Fontanazza personal communication). Since few varieties are cultivated compared to a large number of known accessions, it is very important to have a large clonal collection replicated in several countries to reduce the risk of loss of valuable germplasm and at the same time to facilitate their use for genetic improvement.

5.2.3 Modes of Preservation and Maintenance During the last decades, extensive work has been done using in vitro techniques aiming at a rapid “true-totype” propagation, the production of disease-free plants, the cryopreservation of elite germplasm, and genetic improvement. In vitro propagation of the olive cultivar by axillary bud stimulation has successfully been used and is now a commercial reality in the nursery production in Italy. In addition, in Spain,

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thousands of rootstocks from seedling origin are also produced. This technique allows producing high quality and rapid growing plants. Recently, agronomic information on micropropagated plants show that the micropropagated plants flower at the same time as propagated plants by cutting. The in vitro culture for the olive is, therefore, being used in the breeding programs for the improvement of the species. In vitro culture techniques applied are discussed below.

5.2.3.1 Axillary Bud Stimulation Up to now, the major difficulties encountered for in vitro micropropagation of mature tissues of olive cultivar was the establishment of axenic cultures and subsequent initially growth of shoots. Different attempts to establish sterile cultures with meristems or shoot-tips from field-grown or greenhouse plants were unsuccessful due to the rapid oxidation of tissues after collection, even with high doses of active antioxidants. Vigorous node explants, possibly hiding subepidermal vegetative buds, and inflorescence sprouted from mixed buds, which both are easy to surface sterilize without damaging the buds, are advisable to use for starting tissue to get sterile shoots. The rapid growth is supported by OM medium (Rugini 1984, DUCHEFA catalog) with the addition of natural cytokinin, zeatin, or a mixture of them (zeatin, BAP, TDZ and metatopolin) and GA3 (20–40 mg/l), and mannitol, in place of sucrose, induced the production of tender, longer shoots more suitable for the subsequent rooting phase. Some cultivars showed beneficial effect on shoot proliferation with the use of Dikegulak (Mendoza-De Gyves et al. 2008), which increases lateral bud sprout. Reinvigoration of shoots in vitro may be essential. It can be achieved by forcing the basal lateral buds of the new shoots still attached to initial woody nodal explant, for several subcultures, by cutting the apical part at each subculture (Rugini unpublished); furthermore, grafting the buds on juvenile seedling in vitro should be advisable (Revilla et al. 1996). Rooting of explants benefits by basal etiolation (Rugini et al. 1987) or by keeping them for 5–7 days in the dark and by addition of 1 mM of putrescine to the medium (Rugini and Fedeli 1990). Putrescine contributes to increase total peroxidase activity in the basal part of the shoots in the first hours after explant preparation, allowing promoting earlier root protrusion than

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with untreated ones (Rugini et al. 1997). The micropropagation is an important procedure for cloning cultivars whose cuttings are difficult to root, as Nocellara etnea (Briccoli Bati et al. 1999). In some cultivars, it is possible to root the explants in vivo by supplying both auxin and putrescine at the basal end of the explant. 5.2.3.2 Shoot Organogenesis From Embryonal Material Organogenesis was observed under different conditions and in tissues of different origin. Direct shoot organogenesis was obtained from olive hypocotyl sections (Bao et al. 1980). Explants from mature embryos are suitable material for shoot regeneration (Rugini 1986). High shoot regeneration has also been shown by callus derived from mature cotyledon fragments of the cvs. Tanche and Picual. The regeneration was higher in calli from cotyledon segments proximal to the embryo axes than distal ones (Canas and Benbadis 1988). From Mature Tissue of Cultivars Adventitious shoots were induced only in the dark from petioles of in vitro-grown shoots of cultivars Canino, Moraiolo, Dolce Agogia, and Halkidikis. The petioles-forming shoots ranged from 10 to 40% depending on the leaf position on the shoot and the medium used (Mencuccini and Rugini 1993). The quality of shoots used to collect the petioles is essential for the subsequent success in regeneration, and the addition of gibberellic acid (GA3) in proliferation medium enhances regeneration rate. Currently, the shoot organogenesis from olive mature tissues is not an efficient method to regenerate plants from transgenic cells; however, it seems to be essential as the first necessary step to achieve somatic embryogenesis.

5.2.3.3 Somatic Embryogenesis In olive, somatic embryogenesis (SE) has been successfully achieved from both embryonal (immature and mature zygotic embryos) and somatic (seedling tissues and leaf petioles of cultivars) explants.

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SE from Embryonal Explants The first report on induction of somatic embryogenesis in olive was achieved by wounding the roots still attached to the seedlings, originated from mature embryos in vitro; the callus produced in the wounded zone produced embryos, which converted into plantlets (Rugini and Tarini 1986). However, high yield of somatic embryos can be recovered from immature (60–75 days after fertilization) zygotic embryos (Rugini 1988; Leva et al. 1995). This high capacity may be extended for at least 2 months by storing at 14–15C the whole fruitlets, collected at 75 days after fertilization. Under these conditions, although the small embryos continue to develop, they maintain the embryogenic capacity, contrary to the corresponding embryos collected from fruits left on the plants (Rugini 1995). Callus from segments of non-germinated mature embryos both of wild (Orinos and Mitrakos 1991) and cultivated olive (Mitrakos et al. 1992) could produce somatic embryogenesis. Calli derived from rootlets give the highest somatic embryogenesis. The difference in the amount of embryogenesis depends on the origin of the explant (proximal, medium, and distal part of the embryo) and on the last of the period of permanence of explants on callus induction medium. Somatic embryogenesis is not influenced by the salt concentration of the medium or by the light or dark conditions (Orinos and Mitrakos 1991).

SE from Somatic Mature Tissues of Cultivars SE from mature tissues is still difficult to achieve. However, for two cultivars, Canino and Moraiolo, it has been done using one strategy, named later in a similar work on apple by Rugini and Muganu (1998) as the “double regeneration system”. It consists of regenerating first adventitious buds from leaf petioles of in vitro grown shoots, then subculturing the small leaflets in a proper medium until pro-embryo masses appear. The embryos differentiate from its surface, recognizable by porous and smooth epidermis and yellowish in color. Cyclic embryogenesis both from normal embryos and from teratoma can be obtained indefinitely (Rugini and Caricato 1995). The embryos differentiate mainly from epidermal surface with mainly unicellular origin (Lambardi et al. 1999). The

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continuous production of long-term cycles of embryos from epidermal cells could be a great advantage in regenerating plants from transgenic cells, because it avoids the callus formation, which could be a wide source of undesirable genetic variability. In addition, it avoids wounding of the tissues that could be the cause of browning and reducing the efficiency of Agrobacterium-mediated transformation. The aim with somatic embryos is to use in transformation work but at the same time to produce “synthetic seeds”. A first successful attempt of encapsulation was done by Micheli et al. (1998) but using in vitro-proliferated short apical shoots in place of embryos. Capelo et al. (2010) described a protocol for SEinduction from an adult wild olive tree (O. europaea ssp. europaea var. sylvestris). The protocol used confirmed for the first time that there is no need to use juvenile or rejuvenated material for SE induction. It opens perspectives for using this strategy in SE protocols both for the wild and commercial genotypes. For SE induction, petiole and leaf (proximal, intermediary, and distal zones) explants were grown on Murashige and Skoog (MS) medium or Olive medium (OM) with different combinations of plant growth regulators (PGR): a-naphthaleneacetic acid (NAA), Zeatin (Zea), indole-3-butyric acid (IBA), 2-isopentyl adenine (2iP), thidiazuron (TDZ), and 6-benzylaminopurine (BAP).

5.3 Elucidation of Origin and Evolution of Olea Species 5.3.1 Related Species Recently, three taxons have been described as new allied species of O. europea. Chionanthus greenii Lombardi, a new species of Oleaceae, is distinguished by the unique combination of strigose indument, short congested inflorescences, small corolla lobes, and reniform anthers, which do not match to any described South American species (Lombardi 2006). Chionanthus amblirrhinus sp. nov. is described from Thailand, and C. decipiens sp. nov. from Burma and Thailand. The following new combinations are made: C. eriorachis (from Thailand), C. malaelengi subsp. linocieroides (from S. India) and subsp. terniflorus

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(from NE India, Burma, Thailand and Indo-China), C. microbotrys (from Thailand), C. microstigma (from Thailand and Indo-China), C. sutepensis (from Thailand), C. thorelii (from Thailand and Indo-China), and C. velutina (from Thailand) (Green 1996).

5.3.2 Biochemical and Molecular Markers Employed for Phylogenetic and Polymorphism Studies Within Olea Several genes encoding for key enzymes in fatty acid and antioxidant biosynthesis, modification, and triacylglycerol storage have been isolated (Hatzopoulos et al. 2002). The gene expression during fruit growth and seed development as well as their transient and temporal expression in different tissues allowed identification of those most important for storage of fatty acids and to the provision of signaling molecules important in plant defense mechanisms and reproduction.

5.3.2.1 Molecular Markers to Infer Evolutionary and Domestication Events Patterns of Evolution and Domestication of Olive Cultivated and wild forms have the same chromosome number (2n 46) (Green and Wickens 1989) and are fully interfertile. Studies on olive pollen flow by wind (Griggs et al. 1975), on seed dispersal of wild olive by birds in Spain (Alcantara et al. 2000), and from local reports about the extensive use of seeds from olive groves for forestation in several Mediterranean countries (Chevalier 1948) suggested that genuinely wild olive populations may be restricted to very few isolated forest areas. Consequently, at the present time, oleasters are absent from many regions (Ouazzani et al. 1993) and are limited to restricted areas along the shores of the Mediterranean (Zohary and Spiegel-Roy 1975). However, the study of oleasters appears of real interest since it might constitute a gene pool useful for olive improvement programs, i.e., for disease and stress resistance (Fig. 5.5).

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Using Molecular Markers to Validate the Patterns of Evolution and Domestication of Olive Genetic diversity within and between plant populations results from a combination of geographical distance, population size, type of mating system (selfing or outcrossing), mode of dispersal of pollen and seed, and rate of gene flow (Loveless and Hamrick 1984). The mating system of plants appears to play a major role in genetic diversity by determining the rate of exchange of genes (Darmency 1997). Species that are predominantly outcrossing are reported to show lower interpopulation and higher intrapopulation differences in genetic variation compared to species where self-fertilization predominates (Maguire and Sedgley 1997). O. europaea L. is a predominantly allogamous species showing a high degree of outcrossing. Most pollen transfer is by wind with insect involvement occurring to a small extent (Lavee 1996). Progenies are readily derived from crosses between cultivated varieties (cultivars) as well as between cultivars and both feral (escaped) and wild (oleaster) olives (Angiolillo et al. 1999). The degree of outcrossing varies between cultivars and environments (Lavee 1996). Different techniques have been used to evaluate olive diversity. Cantini et al. (1999) used morphological characters such as leaf, fruit, pit, and growth form to evaluate genetic variation within and between different accessions of known and unknown olive cultivars. Isozyme analysis has also been used to examine the genetic diversity in wild and cultivated olives (Ouazzani et al. 1993). The RAPD markers successfully identifies olive cultivars (Weisman et al. 1998; Mekuria et al. 1999; Gemas et al. 2000) and, together with the analysis of molecular variance (AMOVA) (Excoffier et al. 1992), it has been used to study population genetics in many plants (Gillies et al. 1997; Maguire and Sedgley 1997). This technique was used to evaluate the level of genetic variation within an isolated feral olive population to better understand the dynamics of spread of O. europaea L. in an isolated population of 45 trees within an area of about 1 km2. Based on visual observation, three putative groups were identified: nine trees that appeared to be an original grove, 12 trees that were assumed to be planted progenies of the original grove, and 24 feral trees in the surrounding hills and valleys. The AMOVA showed high genetic variation within each of the three

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putative groups of the population, but there were no significant genetic differences among them. A dendrogram showed the presence of three clusters significantly different from one another that persisted when the data were subjected to multidimensional scaling. Each cluster consisted of at least one or more trees from the original grove and others from the population. The overall trend supported the derivation of feral trees from the original grove. The distribution of these clusters within the population suggests that the predominant mode of feral spread was by fruit drop close to the trees and in lesser extent by animals or birds (Mekuria et al. 2002).

Informative Molecular Markers Genetic studies based on allozyme polymorphism (Lumaret et al. 1997), DNA tandem repeated sequences (Katsiotis et al. 1998), AFLP (Angiolillo et al. 1999), RAPD profiles and mitochondrial restriction fragment length polymorphism (RFLP) (Besnard and Berville 2000; Claros et al. 2000; Besnard et al. 2001a, b; Bronzini de Caraffa et al. 2002a), SSR (Rallo et al. 2000), as well as inter simple sequence repeat (ISSR) markers (Vargas and Kadereit 2001) helped to validate the patterns of domestication envisaged on the basis of palynological and archeological remnants. Molecular analyses are extremely useful when the morphological distinction between wild and feral olive forms cannot be determined because they have similar characters, such as a smaller fruit size, lower oil content in the drupe, and a typical wild aspect. They are also important diagnostic tools for the correct identification of chromosomes (Minelli et al. 2000) and cultivars and, because of their high polymorphism and discerning power, SSR or microsatellite markers have been developed and used with success for discrimination of olive cultivars (Rallo et al. 2000; Sefc et al. 2000; Carriero et al. 2002; Cipriani et al. 2002; Sabino et al. 2006).

5.3.2.2 Sub-species Differentiation Using Biochemical and Molecular Markers Tandem-repeat sequences isolated from an olive Sau3AI partial genomic library have been used as molecular markers to provide new insights in genome organization and in identifying superior olive tree

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genotypes. These repeated sequences were detected at DAPI-stained heterochromatic regions having a subtelomeric or interstitial location in Olea chrysophylla, O. e. e. var. sylvestris (syn. O. oleaster), and O. e. cuspidata (syn. O. africana) but were absent from other Oleaceae genera (Katsiotis et al. 1998). Sequence analysis of these repeated elements from different cultivars could also contribute to cultivar identification and discrimination. Angiolillo et al. (1999), using AFLP profile data, argue that Olea accessions of the same species but from different places of East Africa and Asia may be assigned to different species. Because of the ancient olive domestication, the cultivated forms, which have extended considerably over the natural populations, have caused the regression of oleasters (Bronzini de Caraffa et al. 2002b). Consequently, at the present time, oleasters are absent from many regions (as first deduced from isozyme analyses by Ouazzani et al. 1993) and are limited to restricted areas along the shores of the Mediterranean (Zohary and Spiegel-Roy 1975). The nuclear genetic variation of oleasters was analyzed over the Mediterranean Basin using isoenzymes (Lumaret et al. 2004), RAPD markers (Besnard et al. 2001a, b), and in more restricted areas, using RAPDs (Bronzini de Caraffa et al. 2002a, b), AFLPs (Angiolillo et al. 1999), and allozyme polymorphism (Lumaret et al. 1997; Lumaret and Ouazzani 2001). Allozymes at several independent loci analyzed in numerous cultivated olive trees (Ouazzani et al. 1993, 1996; Lumaret et al. 1997) were considered as appropriate codominant markers to characterize oleaster genetic variation. Using isozyme markers on multiple samples from wide areas over the Mediterranean Basin, a clear nuclear genetic differentiation was observed between oleasters from the eastern and western parts of the Mediterranean Basin. Substantial genetic differentiation was observed between the eastern oleaster populations (genetically close to most olive clones cultivated in the Mediterranean Basin) and the western oleaster populations that are related to the wild Canarian populations (Lumaret et al. 2004). Lumaret et al. (2004) studied allozyme variation at ten loci in 31 large and 44 small oleaster populations distributed in various habitats of the Mediterranean Basin and in two populations of the wild subspecies O. europaea ssp. guanchica, endemic to the Canary

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Islands and closely related to oleasters. Three isozyme alleles were shared by the Canarian populations of O. europaea ssp. guanchica and by the most occidental oleaster populations (more particularly those from the selected forests of Andalusia and Morocco), suggesting that the populations growing in the most westerly part of the species distribution have a common origin. The most western populations of the Mediterranean Basin are genetically close to the Canarian populations of ssp. guanchica, which supports previous results obtained from plant material collected in the same areas and analyzed using RAPD and ISSR markers (Hess et al. 2000). Genetic evidence that non-domesticated oleasters still survive locally was provided by the occurrence of four and one isozyme alleles shared exclusively by the eight western and two eastern oleaster populations, respectively, which were collected in forests potentially containing genuinely wild forms. Except for those four very rare isozyme alleles, oleaster populations possess all the alleles already identified in cultivars. This result is consistent with the interpretation that the domesticated olive represents a subsampling of the genetic variation in genuinely wild olive, which persists today in the Mediterranean Basin (Lumaret et al. 2004). The oriental populations are very close genetically to the two groups of olive clones cultivated in the eastern and in the western parts of the Basin, respectively, suggesting that most cultivars of the Mediterranean Basin originated from plants selected in the Middle East, as envisaged previously from the analyses of cpDNA, mtDNA, and RAPD (Besnard and Berville 2000; Besnard et al. 2001a, 2002a, b). However, in the western group, a few cultivars (particularly from Sicily, Corsica, and Andalusia) showed allozyme characteristics similar to those found in the western oleaster populations, supporting the previous evidence of a multilocal selection of cultivars in olive (e.g., Besnard and Berville 2000; Besnard et al. 2001a; Bronzini de Caraffa et al. 2002a, b; Contento et al. 2002). Feral olive-trees display an oleaster phenotype and are characterized by very close molecular relationships with varieties and not with oleasters (Angiolillo et al. 1999; Besnard and Berville 2000). Therefore, the identification of the feral forms is done a posteriori with molecular markers since generally they display close relationships with the varieties from which they are derived (Angiolillo et al. 1999; Besnard and Berville 2000). The presence of feral forms, resulting

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from cultivars escaping cultivation and also hybridizations between varieties and oleasters, have also been suggested by molecular marker analyses in Corsica and Sardinia accessions and other geographic centers in eastern places. RAPD polymorphism in oleaster displayed a gradient between east and west of Mediterranean Basin. This gradient was less visible in cultivars due to diffusion and selection (Besnard and Berville 2000). The analysis of 290 polymorphic AFLP bands allowed the detection of two major clusters of similarity: one cluster consisted mainly from cultivars, wild olives, and Northwest African species, while the other included species from East Africa and Asia. The first major group was further subdivided into (a) cultivars originating mostly from Italy, (b) cultivars dispersed in different Mediterranean countries, (c) wild olives, and (d) Olea species from Morocco (Angiolillo et al. 1999). The authors argued that Olea accessions of the same species but from different places of East Africa and Asia might be assigned to different species. RFLP of chloroplast DNA was first applied to differentiate 70 olive cultivars, about 90 old trees, and 101 oleasters. The analysis showed five distinct chlorotypes. The type I was predominant in both cultivated olive trees and oleasters, and types II, III, and IV were observed exclusively in oleasters and were restricted mainly to isolated forest populations (Amane et al. 1999). Chlorotype V was more widely distributed in both cultivated and wild olives even on distant populations. This chlorotype was correlated exclusively to male-sterile trees, suggesting that it may be related to the high yields of fruits usually observed on malesterile trees (Amane et al. 1999). Fifty-six olive genotypes from the Malaga province in Spain using RAPD analysis were distinguished into 22 varieties and clustered into three main groups. Group I contains both wild type and introduced varieties, group II consists of mostly native olive trees, and group III is a heterogeneous cluster including varieties originating from Andalusia (Claros et al. 2000). In Corsica and Sardinia, the oleasters carry inside either the MOM or MCK mitotype (Bronzini de Caraffa et al. 2002a), whereas most of the varieties carried the ME1 mitotype, characteristic of olives in the East Mediterranean. Therefore, the true oleasters are characterized by a western mitotype and a western RAPD pattern. Feral forms originate either from varieties or from hybridization between a variety and an oleaster.

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Consequently, as expected, some of them aggregated with the varieties from which they were derived. The other feral forms are clustered with the oleasters and were detected only by their mitotype determination. These results showed that the majority of the varieties displayed an eastern mitotype. This is in agreement with the history of olive domestication, which suggests an east to a west diffusion of varieties. The mtDNA RFLPs provide, thus, a sensitive and reliable method to assess intraspecific cytoplasmic diversity within the economically important olive tree cultivars in Italy and provide new insights into the maternal lineages involved in the evolution of the EuroMediterranean olive trees.

5.3.2.3 Molecular Markers for Differentiating Within Cultivated Forms The pioneering work on distinction of olive cultivars using RAPD molecular markers was published by Fabbri et al. (1995). In that work, olive cultivars were clustered into two main groups, one consisting of small-fruited cultivars grown mainly for oil production and the other including large-fruited cultivars. Using RAPD analyses, Hatzopoulos et al. (2002) showed that most of the Greek cultivars were clustered into two main groups and a third minor group. The first major group included mainly table olive cultivars while the other mostly of olive oil cultivars. The third minor group consisted mainly of the wild type olive cultivars. Using the same methodology to discriminate olive oil genotypes from Cyprus, it was possible to demonstrate that these genotypes could be grouped according to their fruit size into large or small fruit clusters (Hatzopoulos et al. 2002). RAPD methodology could provide an acceptable resolving power for many cultivar identification projects (Belaj et al. 2001). Bronzini de Caraffa et al. (2002a) were able to differentiate, using a combination of mitochondrial and nuclear RAPD markers, two populations of cultivated olives in Corsica: one with close relationships with the Italian varieties (influenced by the east) and one selected from local oleasters probably due to a better local adaptation than foreign varieties. All of the varieties from mainland Italy and Sicily displayed the ME1 eastern mitotype. The ME2 mitotype was revealed only in three Sicilian varieties.

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Substantial polymorphism has been detected at SSR loci developed from a genomic library of O. europaea L. (Sabino Gil et al. 2006). Twelve out of 19 loci revealed to be polymorphic, each showing from 2 to 14 alleles. Those loci were informative for characterizing genetic differentiation among 33 cultivars from the Cordoba Germplasm Bank (9 Italian, 4 Portuguese, 2 Tunisian, 9 Spanish, 3 Greek, 3 French, 1 Moroccan, 2 Turkish, and 1 Algerian). Structural details of genetic diversity within cultivated olive gene pool has been revealed from analysis of 119 polymorphic AFLP fragments in olive germplasm, including 65 economically important accessions cultivated in the eastern Mediterranean Basin (Owen et al. 2005). A factorial correspondence analysis (FCA) plot indicated that the cultivars clustered into two relatively modestly defined groups. The first broad group was not only dominated by cultivars from Turkey but also included genotypes originating from the Middle East (Syria and Lebanon) that collectively formed a tight subcluster. The second group comprised the Greek cultivars and those originating from the western Mediterranean. A significant genetic distance value between the Greek and Turkish cultivars was provided by an AMOVA. There was also evidence of substructure here, with an apparent separation of most Spanish and Italian clones. These findings are, in general, in accordance to previous suggestions of an east–west divergence of olive cultivars, although the dichotomy is less extensive than reported previously and complicated by regional variation within each group. Assessment of genetic variability of olive varieties by microsatellite and AFLP markers has been performed by Bandelj et al. (2004). The results of clustering analysis with both molecular systems showed the common genetic background of the Tuscan varieties (Frantoio, Leccino, Leccione, Maurino, and Pendolino) and genetic divergence within the Slovene olive germplasm. The Slovenian varieties, “Buga”, “Sˇtorta”, and “Samo”, might represent regionally selected olives, while “Zelenjak” and “Cˇrnica” are probably derived from the central Italian region. The predominant local “Istrska belica” was introduced to Slovenia independently from the other regional varieties and showed the lowest genetic similarity with the other regional varieties. Recently, applying the AFLP technology on the most relevant and old varieties cultivated in Abruzzo Region (central Italy), Albertini

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et al. (2010) have clearly distinguished eight cultivars within seven clusters. The obtained data suggest that both sexual and clonal propagation have played an important role in the evolution of olive cultivars, and the authors hypothesized that some ancestral population spread in central Italy with a relevant role of seed propagation followed by a selection of superior clones from which more traditional varieties originated. The mtDNA, generally maternally inherited in dicotyledonous species, has been studied in olive in order to determine the geographical origin of the cultivated accessions. Four mitotypes have been distinguished in the Mediterranean olives: ME1 (the mitotype from the eastern Mediterranean, no. 1) and ME2 (the mitotype from the eastern Mediterranean, no. 2) characteristic of olives in the eastern Mediterranean (Egypt, Greece, Near East and Turkey), and MOM (the mitotype from the western Mediterranean) and MCK (the mitotype characteristic of the cultivar Chemlal de Kabylie from the western Mediterranean) found in olives from the west (France, Italy, Maghreb, Spain, Yugoslavia). However, an eastern influence on some western regions, namely Italy and Libya, as well as France and Maghreb, has been shown for cultivated olives (Besnard and Berville 2000). The mtDNA RFLPs provide, thus, a sensitive and reliable method to assess intraspecific cytoplasmic diversity within the economically important olive tree cultivars in Italy. Our results also provide new insights into the maternal lineages involved in the evolution of the Euro-Mediterranean olive trees. Analysis of the Italian O. europaea L. population by mtDNA RFLPs allowed the identification of three diagnostic probe cyclooxygenase (cox3)/enzyme (EcoRI and HindIII) combinations that can be used as markers of cytoplasmic diversity to detect additional significant mtDNA RFLPs in the Euro-Mediterranean O. europaea L. complex (Cavallotti et al. 2003). The mtDNA RFLPs allowed the assignment of the analyzed 37 olive tree mitochondrial genomes to three distinct mitotypes: MIT1, MIT2, and MIT3. MIT1 mitotype is the most frequently represented and comprises 33 elite cultivars currently grown for oil, edible fruits, or both purposes all over the Italian continental territory and islands without any geographical dependence. Most of these elite cultivars are reported as self-sterile and some like the French Lucques are totally male-sterile (Besnard et al. 2000). The MIT2 mitotype comprises two Italian olive trees culti-

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vated for oil (Canino and Vallanella) and the wild O. sylvestris. The MIT3 mitotype consists of just the cultivar Ceraso`la from West Sicily showing the greatest diversity detectable with a single probe (cox3) and two different restriction enzymes (EcoRI and HindIII). It has been hypothesized that a duplication event of the cox3 locus occurred in the progenitor cytoplasm, leading to the cytoplasm observed in the cultivar Ceraso`la (MIT3). In 1952, Baldini and Guccione reported the cultivar Ceraso`la as a male-sterile cultivar for its inability to develop pollen on the basis of microscopic observations, mtDNA RFLPs, sequencing data, and expression behavior of the cox3 locus in the olive cultivar Ceraso`la that suggest a possible correlation between the presence of duplicated sequences from the cox3 locus within the mitochondrial genome of this cultivar with the male-sterile phenotype. Besnard et al. (2000) mention two major mtDNA RFLPs detected with the cox3/HindIII combination: one with only one hybridizing fragment and the other one with two fragments pattern detected in the French cultivar Olivie`re. Interestingly, this polymorphic HindIII fragment is associated with the so-called MCK mitotype, which is found strictly related to a male-sterile phenotype. Although no size of these HindIII fragments is given, it is presumed that the HindIII pattern observed in cultivar Olivie`re is the same as that observed with the cox3 probe in the cultivar Cerasola. The mtDNA RFLPs seems to provide a sensitive and reliable system in probing genetic diversity and identifying cytoplasms possibly associated with a CMS phenotype. The mtDNA RFLP markers can be also helpful for the management of the genetic and phenotypic resources in conservation and breeding programs. The redundancy level of tandem repeated DNA sequences can be used to differentiate and identify cultivars. Three tandem repeated DNA sequences have been described in O. europaea ssp. sativa (the cultivated olive tree) until now: the pOSE218 family isolated from the cultivar “Koroneiki” (Katsiotis et al. 1998) also present in other species of the genus Olea; the new family described here, present in the cultivars “Picual”, “Koroneiki”, “Hojiblanca”, “Manzanilla”, “Arbequina”, “Frantoio”, and “Mastoides”; and the 81 bp family (Katsiotis et al. 1998) with significant similarity with OetTaq80 repeats (Bitonti et al. 1999) and with the second family of repetitive DNA described here (Lorite et al. 2001). This last repetitive

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DNA is present in several cultivars and other species of the genus Olea, although with very different frequencies even between the olive cultivars studied (Bitonti et al. 1999). The authors suggest that the redundancy levels of the given repeated DNA sequences might provide suitable parameters for varietal identification within cultivated olives.

5.3.2.4 Molecular Markers for Differentiating Cultivated Forms from Diverse Geographic Origin RAPD markers have been used for cultivar identification and identity typing of olive trees (Wiesman et al. 1998; Barranco et al. 2000; Bandelj et al. 2002). In some studies, no apparent clustering of olive cultivars according to their geographic origin was evident (Fabbri et al. 1995). Using the same methodology, other workers could discriminate nine olive genotypes from central Italy (Cresti et al. 1996), or 11 genotypes from Italy that can be hardly distinguished on the basis of morphological traits and are thus easily mistaken for each other (Vergari et al. 1996). Cultivars from restricted areas were grouped according to geographical origin within Valencia, and there was no apparent clustering according to fruit size or other morphological characters using RAPD primers (Sanz-Cortez et al. 2001). Nuclear ribosomal internal transcribed spacer 1 (ITS-1) sequences and ISSR and RAPD analyses were conducted to describe the colonization history of O. europaea in Macaronesia, showing a strong support for two independent dispersal events following an east to west direction (Hess et al. 2000). ISSR polymorphisms were also used in determining the wild status of isolated populations of this species in the Euro-Siberian north of the Iberian Peninsula (Vargas and Kadereit 2001). One hundred and two RAPD profiles from around the Mediterranean Basin were correlated with the use of the olive fruits and the country or region of origin, suggesting a selection scheme from different genetic pools in different areas (Besnard et al. 2001a). Fifty-one cultivars could be distinguished using 46 random primers into three major groups (1) Cultivars from eastern or northern Spain, (2) Turkish, Syrian, and Tunisian cultivars, and (3) the common olive cultivars in Spain (Belaj et al. 2001).

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Besnard and Berville (2000), using mitochondrial DNA, were able to distinguish four mitotypes, ME1, ME2, MOM, and MCK. From these, only ME2 was unique to some cultivars while the predominant mitotype ME1 marks the Near Eastern origin of olive. The authors also argue that these different mitotypes mark independent cultivated olive origins. The spread of olive from a few centers of origin and further multilocal selection (Besnard et al. 2001a; Terral et al. 2004), together with the continuous interchange of plant material between the different regions, have contributed to the confused pattern of geographic distribution. With SSR markers, it was possible to discriminate 96% of the pairwise comparisons among 118 cultivars from Italy (61 samples), Spain (22 samples), Greece (ten samples), Turkey (six samples), France (five samples), and two samples each from Croatia, Syria, Egypt, Israel, Algeria, Tunisia, and Morocco (Sarri et al. 2006). A combination of banding patterns for three SSR loci practically allowed distinction of all the entries, and a selection of six loci allowed correct reassignment of 75.4% of the entries to their country of origin. Hannachi et al. (2008) compared cultivars and oleasters from North Tunisia to determine their relationships based on morphological traits, oil composition, and SSR genotyping at seven loci. Gas chromatography was used to determine fatty acid composition of 30 cultivar trees and 13 oleaster trees. Based on morphology, oleaster trees from agroecosystems clustered broadly in an intermediate position between cultivars and oleasters from natural ecosystems. SSR revealed that the feral and genuine oleasters plus cultivars are always overlapping. Oil composition was similar between cultivar and oleaster trees. Oil composition as fruit descriptors and drupe size appeared inefficient to discriminate between olive and oleaster trees, in comparison to SSR.

5.4 Use of the Wild Gene-Pool for Olive Improvement Through Traditional and Advanced Tools The domestication process that led to the current varieties can be traced to a small number of desirable genotypes for fruit size identified in oleaster population by our ancestors few millennia ago. It is,

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therefore, expected that the available cultivated gene pool underrepresent the alleles at functional loci of the original wild population used for selection. Therefore, the necessary alleles for further improving the mentioned traits might not be present in the genome of the cultivated forms. In this case, efforts should be devoted to (a) identification of the desired alleles in the wild forms using the genomic tools described in Sect. 5.5 and (b) developing efficient and rapid gene transfer strategies of the selected alleles from the wild form to the defective cultivars. For this second strategy, sexual gene transfer is well known to be efficient but not rapid unless the offsprings have suitable genetical response to the management practices that reduce juvenility period. Breeding in olive is difficult to carry out not only due to the very long juvenile period (>10 years) but also due to the different flower fertility expressed in the breeding populations, which range from full malesterility (Tombesi 1978; Fontanazza 1993; Bartolini and Guerriero 1995) to partial self-fertility, up to selffertility (Fontanazza et al. 1990). Therefore, while no emasculation is necessary before hybridization when the female plant is male-sterile, the labor-intensive and costly emasculation process is required for the tiny olive floret, which expresses also a low percentage (1–2%) of fruit-setting. Genes controlling the length of juvenility period by anticipating the flower initiation phase have been identified in Arabidopsis (Yanotsky 1995; Simpson et al. 1999), and two of them, LEAFY (LFY) and APETALA1 (AP1), have been used to transform Citrus (Pena et al. 2001) with the resulting effect of flower induction in 1-year-old plantlets. However, similar experiments have not been performed in olive and other fruit tree species, and in those cases, the juvenile period can be shortened by attempting methods such as plant training by continuous illumination and the choice of right cultivars as pollinators. It has been observed that the length of juvenility is genotype-dependent. In fact, the progenies obtained from cvs. Leccino (used as mother plant) compared to the progenies of cvs. Coratina, Picholine, and Tanche showed a reduced juvenility period (Bellini 1990; Fontanazza and Bartolozzi 1998). A similar effect was observed in the offspring from cv. Verdale. Up to now, only a few cultivars have been obtained by dedicated breeding programs, producing interesting plants in F2 generation. Two cultivars, one for table and one for oil extraction, the latter being resistant also to

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peacock spot, have been selected. It seems that some characters as vigor, leaf size, and fruit shape are dominant in F1 progenies (Rugini and Lavee 1992; Bellini et al. 1995). Other characters such as carpological traits of fruit, blossoming intensity, fruit set, period of fruit ripening, and yield have been considered (Bellini 1993; Parlati et al. 1994; Bellini et al. 1995). Other interesting genotypes have been selected by Prof. G. Fontanazza and coworkers such as O. europaea cultivars FS17, Don Carlo, and Giulia. Those cultivars exhibit medium plant vigor, erect branch structure, grayish green fruit, and superior olive oil. The cultivars FS17 (Patent IROCNR 1165/nv) and Don Carlo (US Patent PP13077) demonstrated satisfactory oil composition too (Cipriani et al. 2006). Nowadays, there is an increasing demand in terms of quantitative and qualitative olive production, and the olive crop improvement can have a key role in guiding production agriculture toward sustainability. Modern objective for olive breeding point to a new plant model in which reduced size, reduced apical dominance, and constant and high productivity in terms of fruit and oil are the main features. In addition, it is desired that cultivar express multiple innovative traits such as self-fertility, fruit with a suitable composition of fatty acids, high content of polyphenol and compounds with therapeutic property, a correct pattern of degradation of phenolic and pectic compounds during fruit ripening as well as tolerance to cold, salinity, drought, diseases, and pests, low plant vigor, high fruit-set capacity, and canopy architecture should be attempted. Actually, although there are more than 1,000 cultivars under cultivation originated mostly from selections made by growers over many centuries, none of them possesses all the desirable traits that could be introgressed into cultivars by conventional breeding or by gene transfer. For these reasons, to reach the mentioned goals, (see Point b before), it will be necessary to develop suitable procedures to find proper breeding materials with the promising alleles and for their transfer. This will require research-intensive analysis of the olive genetic resources to study inheritance of traits and identifying the target breeding materials. On the other end, conventional and biotechnology procedure based on in vitro culture applied to the wild gene pool could help breeding programs. These methods will be applied at different gene pool levels. They will be useful in inducing new genetic variation by mutation breeding, in transferring genes from the wild

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to cultivated primary gene pool by hybridization and clonal selection, to induce somaclonal variation, to change the ploidy level, and to obtain homozygous plants (dihaploids) for producing mapping populations and inbreds for hybrid programs. The embryo rescue will be the biotechnological tool to be chosen when the gene transfer implies a wild species of the secondary gene pool of the cultivated olive. Protoplast technology and genetic transformation will help transferring genes from more distant wild genera to olive cultivars. In fact, the role of wild gene pool in fruit breeding is promising, particularly when wild plants are used as rootstocks, with the aim to reduce growth of the scion and improve resistance to biotic and abiotic stresses of the root system. In this case, gene transfer by Agrobacterium to the wild Olea spp. will be of great importance. Studies in the above directions have been carried out so far in cultivated olive, and the methods and objectives are reviewed briefly for the strong implication on the use of genetic resources of the wild Olea gene pool.

5.4.1 Mutation Breeding Stable mutation rarely occurs in olive, while chimeric plants of cv. Frantoio (Roselli and Donini 1982) and Canino (Parlati et al. 1994) have been observed. On the contrary, dwarf plants from artificially irradiated rooted cuttings of cv. Ascolana Tenera have been obtained by Roselli and Donini (1982) and they are used as ornamental. Other compact mixoploid mutants have been obtained following irradiation of “Frantoio” and “Leccino” plantlets, resulting in the selection of dwarf “Leccino”, which is self-sterile and late-blooming and seems to be a promising rootstock (Pannelli et al. 1990). The mixoploid mutants allowed us to select triploid and tetraploids plants (Rugini et al. 1996).

5.4.2 Hybridization and Clonal Selection The selection of new clones has not been completely explored, so it is important to search new “accessions” to solve the present lack of suitable cultivars (Morettini

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1972; Berenguer 1978; Fontanazza 1987). More recently, clonal rootstocks have been selected with high rooting ability and the ability to control scion vigor (Baldoni and Fontanazza 1990) and many accessions, particularly for small tree size are under observation (Pannelli et al. 1993). The criteria for the selection of rootstocks include good rooting potential, control of scion vigor, resistance to drought, saline, and heavy soils, resistance to root diseases, and graftcompatibility with the scion. Very few studies have addressed the selection of clonal rootstocks. Preliminary work was concerned with the influence of rootstocks on scion performance. Some rootstocks are able to control scion vigor and confer cold tolerance (Pannelli et al. 2002).

5.4.3 Somaclonal Variation In order to increase the spontaneous somaclonal variation, it should be advisable to create a selective pressure conditions with a selective agent (biotic or abiotic origin, such as fungal filtrate, purified toxin of some pathogens, high osmotic pressure, etc.) or additional mutation with ionizing radiation. Reports on somaclonal variation in olive have not been reported yet following plant production through the three methods of plant production. Some phenotypic variations of in vitro grown shoots or whole plants have been observed, but they were only of temporary effect of juvenility induced by the in vitro culture. Today, this technique can be exploited by using the potent cyclic somatic embryogenesis of some cultivars as Canino, since other efficient methods of regeneration from mature tissues of valuable cultivar has not reported yet.

5.4.4 Changes in Ploidy Level Tetraploid and triploid plants of the cultivar Leccino and Frantoio have been produced (Rugini et al. 1996). First, mixoploid shoots were obtained by in vitro axillary bud stimulation of mixoploid buds collected from adult mixoploid plants raised from rooted cuttings previously treated with g-rays. Then, the group of in vitro

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mixoploid shoots spontaneously split into two cytotypes: 2n and 4n. Tetraploid shoots were recognizable by a wider, longer, and thicker leaves than diploid ones. Subsequently, the 2n and 4n shoots have been rooted and transferred to the field. In field trial, 4n plants compared to 2n plants, showed smaller plant size, more flexible branches, absence of a juvenile phase, and larger leaves and fruits, while the intensity of blossom resulted scarce in some years. Both cytotypes, when tested as rootstocks, reduce the growth of the scions. The original mixoploid plants produced both large and small fruits. Triploid seedlings were obtained from seeds of the largest fruits.

5.4.5 Haploids and Homozygotic Plants Production of homozygous olive plants by self-fertilization seems very improbable considering the selfsterility characteristic and the long juvenile phase of the species. Homozygous plants would be of great interest for isolation of mutants and recessive traits. Anthers and ovary or pollen and ovule cultures should be explored with the aim to produce dihaploid plants by doubling the chromosome number. Much effort has been taken in the past to recover haploids of olive but not with clear results (Mule´ et al. 1992; Perri et al. 1994b). Recently, promising results were obtained employing a new method of isolated microspore culture, which led to cell division and pro-embryos formation in the cultivar Arbequina (Bueno et al. 2006).

5.4.6 Embryo Rescue Recently, the embryo rescue technique has been applied with successful results in hybrid plants between a cultivar Mary and an Iranian local variety (Hossein Ava and Hajnajari 2006). In Spain, several crossing among the main Spanish cultivars, by using different methodologies for the reduction of the juvenile period of seedlings, revealed interesting characteristics of the offspring regarding oil content and yield efficiency (Leo`n et al. 2008). In Italy, field evaluation of selections derived from crossing among several local varieties showed a different vegetative and productive behavior

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of the genotypes (Bartolini et al. 2006; Pannelli et al. 2006; Ripa et al. 2006).

5.4.7 Protoplast Technology Protoplast techniques are useful for several studies including fusion of them in an attempt to produce hybrids from cross-incompatible varieties and species. Furthermore, protoplasts allow genetic modifications by introducing exogenous DNA by liposome or cellular organelles. Viable olive protoplasts from hypocotyls, cotyledons, and leaves of micropropagated shoots were isolated and cultured, and in some cases, microcalli have been achieved (Rugini 1986; Can˜as et al. 1987; Mencuccini 1991; Perri et al. 1994a). Since regeneration is not possible yet in olive from derived protoplast callus, this technology cannot be applied successfully at the moment. In addition, one should not expect good results, since somaclonal variation and chromosome rearrangements heavily change the expected results substantially, but anyway asymmetric fusion should be attempted.

5.4.8 Genetic Transformation for Higher Yield, Biotic and Abiotic Stresses Tolerance, and Quality Attributes Very few reports have been presented on biotechnological approaches for using genetic resources of Olea taxa. Genetic studies in olive have been targeted to change some traits including self-fertility, oil content and composition, parthenocarpy, abiotic stress tolerance, plant habit, fruit ripening, regular cropping, and resistance to pathogens and pests. However, the low efficiency of both conventional and modern techniques for genetic improvement suggests that genetic transformation by alien gene transfer could be a promising technique to speed up the development of new cultivars. Several genes of different origins that govern these characters have been identified; however, development of efficient regeneration methods from mature tissue of elite selections remains the limiting factor for using this technology. In olive, two procedures have been used to accomplish

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gene transfer into plant cells: Agrobacteriummediated gene transfer (Rugini et al. 2000) and bombardment of particles coated with DNA (Lambardi et al. 1999). Most of the reports on transformation and isolation of transgenic plants of olive have involved Agrobacterium tumefaciens or A. rhizogenes. These bacteria transfer a segment of the T-DNA known as tumorinducing plasmid Ti and root-inducing plasmid Ri into the nuclear genome of the plants (Chilton et al. 1982). The efficiency of transformation depends on several factors such as bacterial strains, plant cell competence, transgenic cell selection, and plant regeneration methods. In olive, the best strain of A. rhizogenes is “1855” (Rugini Eddo personal communication). Large availability of genes is essential to start a good program of genetic improvement by this technology, both from Olea taxa and from other species or genera. The transformation experiments enlisted in the following paragraphs have been carried out by using somatic embryos of cultivars because an efficient protocol for regeneration via cyclic somatic embryogenesis is available. Both an efficient in vitro regeneration method and the availability of suitable genes are essential factors.

5.4.8.1 Increasing Rooting Ability In vitro grown shoots can be rooted by inoculating in the middle or in the basal part of the stem, with a scalpel infected with A. rhizogenes (Rugini 1986, 1992); the roots emerging from both the inoculation points on the shoots rarely resulted in transformed roots (Rugini and Fedeli 1990; Rugini 1992; Rugini and Mariotti 1992). Putrescine, added to the hormonefree medium, in combination with the agrobacteria, promoted an earlier root differentiation and an increase of the rooted explant frequency. Although we have no data yet on the growth habit in the field of the rooted plants with agrobacteria, with transformed root system, we expect, on the basis of observations done on plum trees rooted with the same agrobacterium treatment, a drastic reduction of plant size. Plant size reduction has been observed also in cherry plants when grafted on transgenic for ri-T-DNA rootstock (Biasi et al. 2003).

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5.4.8.2 Modifications of Vegetative Habits and Plant Architecture Dwarf and semi-dwarf new varieties are required in the high-density olive groves. Overexpression of rol (root loci) genes in plants has demonstrated a drastic effect on the plant architecture (Rugini et al. 1991; van der Salm et al. 1997, 1998; Pe´rez and Ochoa 1998; Gutie´rrez-Pesce et al. 1998; Mercuri et al. 2001; Zhu et al. 2001), although reproduction system in some woody plants is modified (Eddo Rugini personal communication), and scarce is the knowledge on the interaction of these genes with the genome of the plants. Moreover, the development of plants with an extensive root system and/or with reduced water consumption as demonstrated in kiwi plants transgenic for rolABC genes (Gutie´rrez-Pesce and Rugini 2008) could be an advantage in areas with scarce water availability. The rolABC genes of A. rhizogenes and the marker gene nptII were transferred to somatic embryos of the cv. Canino by using the A. tumefaciens strain LBA4404. Transgenic somatic embryos selected in liquid medium under light conditions converted into plantlets, which were transferred to the experimental field (Rugini and Caricato 1995; Rugini et al. 1999). Those transgenic plants still show by RT-PCR analysis the stable integration of the rol genes and their correct transcription. In addition, those plants showed a different expression of each gene in the organs studied (stem, leaf, root), although they are under the same promoter. The genes influenced plant architecture: leaves had a wider angle of insertion on the stem and they showed changes in leaf blade shape with a reduction of internode length. In some cases, (in transgenic clone-5) showed an increase of chlorophyll content in the leaves. The transgenic clones showed different plant development and growth patterns as revealed by the significant increase of the number of internodes, wider angle of insertion of the leaves with a reduced area and less apical dominance as demonstrated by the outgrowth of lateral shoots giving them their bushy structure. An overexpression of glycosyl-transferase elongation factor, psbB, psbC, and psbD genes in the transgenic clones compared to control plants were also observed by molecular analysis. Furthermore, molecular analysis showed that not all the transcript of the rol genes is polyTail. The levels of polyTail transcript of all rol genes are different in the diverse tissue of plantlets.

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The less amount of transcript, independently of the rol gene, has been detected in the leaf tissue. This behavior is not related with the culture conditions because the same trend has been found both in vitro and in vivo. Total amount of transcript did not differ among the tissue, as seen by rolA transcript, but it is different in rolB and rolC (Miano et al. 2004). Untransformed cultivars have been grafted on transgenic olive rootstocks, and the preliminary results seem to modify the architecture of scion. The modification of plant receptors in order to change the light perception is another possibility for modifying the growth and reproductive behavior. Phytochrome genes (phyA, and/or phyB, sense or antisense), which, together with other photoreceptors, control plant development such as circadian rhythms, apical dominance, blossom, growth and fruit ripening, photosynthesis product distribution, development of photosynthetic systems, transpiration control and hormone synthesis, may contribute to develop plants with high agronomic value and suitable for very high density planting (Tucker 1976; Vince-Prue and Canham 1983; Baraldi et al. 1992; Muleo and Morini 2008; Muleo et al. 2009).

5.4.8.3 Increasing Resistance to Biotic Stress The challenge of using biotechnology in this area is to generate broad resistance mechanisms that have been difficult to achieve with classical breeding approaches. The use of antifungal genes or a pool of genes may be effective for improving resistance to fungal diseases, particularly Verticillum wilt and Spilocea oleagina. Recently, studies of a plant defense PR-5 protein called osmotin, which belongs to a large, diverse family of proteins that defend plants from fungal pathogens, revealed its expression only under stress conditions, including pathogenic attacks (Zhu et al. 1993, 1995; Grillo et al. 1995). Osmotin gene as well as stilbene synthase gene (Hain et al. 1993), hydrolytic enzymes such as chitinase and glucanase (Broglie et al. 1991; Yoshikawa et al. 1993), glucose oxidase gene (Wu et al. 1995), and polygalacturonase that inhibit the activity of endopolygalacturonases released by fungi on invasion of the plant cell wall could improve the olive defense system. The tobacco osmotin gene has been extensively employed for plant transformation in some species

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including grape (Salzman et al. 1998) and potato (Liu et al. 1994; Zhu et al. 1995). Four days of exposure to Phythophthora infestans induces the accumulation of both mRNA and its protein (Zhu et al. 1995), while a longer exposure seems to be necessary to induce the response to low temperature or osmotic stress (NaCl) as elicitors. Moreover, in transgenic potato and tobacco plants osmotin, overexpression is able to delay the development of Phythophthora disease symptoms (Liu et al. 1994). However, its mRNA induction did not always lead to protein accumulation (Grillo et al. 1995), suggesting transcription and posttranslation controls. In olive, transgenic plants for the osmotin gene under CaMV35S promoter have been recovered from somatic embryos derived from petioles of cultivar Canino (Rugini et al. 1999). The in situ localization of osmotin was performed by using a polyclonal primary antibody against osmotin jointed with a secondary antibody with alkaline phosphatase (AP) activity. The AP reaction in presence of NBT/BCIP substrate caused a purple color into the plant tissues. In transections of 1-year-old stems, coming from the apical internodes of field-grown plants, osmotin labeling was observed in epidermal and subepidermal tissues and, rarely, also in the most superficial layers of the cortical parenchyma. At cellular level, the protein was mainly localized around the vacuole. The signal was also present in the phloem and, mainly, in the cambium, but to a lesser extent than in the superficial tissues. In some cases, immature deuteroxylem cells resulted were also labeled (D’Angeli et al. 2001). Inoculation tests for the presence of Spilocea oleagina spots showed that one transgenic genotype seems to possess higher susceptibility than control plants whereas the other ones show a lower susceptibility. Both osmotin-transgenic self-rooted plants and grafted onto non-transgenic rootstock are under evaluation in field trials for their level of resistance to Spilocea oleagina (Rugini et al. 2000). In unstressed plants grown in the field, osmotin labeling was observed in the epidermal and subepidermal tissues and, rarely, also in the most superficial layers of the cortical parenchyma of the apical third internode of 1-year-old twigs. At cellular level, the protein was mainly localized around the vacuole. The signal was also present in the phloem and, mainly, in the cambium but at lower levels than in the superficial tissues. In some cases, immature deuteroxylem

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cells were also labeled. No signal was ever observed in the unstressed leaves. Our results confirm the organ specificity of the protein accumulation controlled by a post-translation pathway able to negatively interfere with the gene constitutive expression of 35S promoter (Gutie´rrez-Pesce and Rugini 2008). Regarding bacterial diseases, it is not so easy to introduce this kind of resistance into plants because the product of the gene should act in the intercellular space; however, some species with enhanced resistance to bacteria have been obtained by introducing genes encoding bactericidal polypeptide, i.e., thionin (Carmona et al. 1993), attacin E (Norelli et al. 1994), the synthetic analog MB39 of cecropine (Mills et al. 1994), and cecropine (Huang et al. 1997). Particular attention should be paid to human lysozyme, which confers resistance to both fungi and to bacterium P. syringae in tobacco plants (Nakajima et al. 1997). The isolation of homologous genes from olive cultivars with low susceptibility to the fruit-fly (Bactrocera oleae) is a potential way to achieve resistance to this pest in susceptible cultivars. Although, in the battle against insect attack, the Bt gene from Bacillus thuringensis (Vaeck et al. 1987) has successfully been introduced in other species with encouraging results and could be an alternative way to prevent olive fly damage. In addition, gene or gene isolation from more tolerant genotype, such as “Bianca di Tirana” should be attempted. Against olive fly (Bactrocera oleae), a group of researchers have developed a Minos-based transposon vector carrying a self-activating cassette, which overexpresses the enhanced green fluorescent protein (EGFP). The self-activating gene construct combined with transposase mRNA present a system with potential for transgenesis in very diverse species (Koukidou et al. 2006). In the following paragraphs are summarized the main objectives and studies on genes useful for improving oil quality in Olea taxa by applying genetic transformation.

5.4.8.4 Fruit Ripening and Oil Quality Pattern of fruit ripening and the increase of oil content and quality are of much interest in an attempt to delay fruit ripening and to reduce early fruit drop. This pattern may be corrected by generating transgenic

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plants with a reduced ethylene biosynthesis, by using an antisense gene (Oeller et al. 1991) or for reducing polygalacturonase activity (Smith et al. 1988). With regard to the oil quality, two molecular strategies can be used to modify the oil composition and content (1) the alteration of the major fatty acid levels by suppressing or expressing a specific key enzyme in lipid biosynthesis and/or (2) by creating an unusual fatty acid. The oleic acid is the main component of olive oil and is responsible for its high dietary value (Baldoni et al. 1996). Since the stearoyl-acyl carrier protein denaturase is the key enzyme for the conversion of saturated stearic acid (C18:0) to monounsaturated oleic acid (C18:1), by antisense suppression or cosuppression of oleate denaturase, it is possible to increase oleic acid (C18:1) from 24% to 80% in the transgenic soybean (Kinney 1995, 1996a, b, 1997; Yadav 1996) and in Brassica seed by the antisense expression of a stearoyl-ACP denaturase gene (Knutson et al. 1992). Temporal and transient expression of the S-ACP denaturase gene has been studied during fruit development (Haralampidis et al. 1998), and its expression is developmentally regulated with earlier expression in embryos than in mesocarp. The same strategy was adopted to increase stearate acid (C18:0) by up to 30% both in canola and rapeseed (Auld et al. 1992; Falco et al. 1995). Unusual fatty acids can be produced in one plant by transferring a gene encoding the specific biosynthetic enzyme. An example can be seen in canola that naturally does not produce laurate (C12:0), while a new transgenic genotype does contain laurate. The oxidative stability of the oleic acid in soybean oil can be also improved as reported by Ellis et al. (1996).

5.4.8.5 Parthenocarpy The alternate bearing and often the lack of pollination are the main causes of low yield in olive; particularly, the second problem happens often in areas with less intensive olive cultivation. Under some environmental conditions, olive shows some tendency to natural parthenocarpy, but the fruits remain very small. Genes inducing parthenocarpy would permit the development of parthenocarpic fruits with regular size and would overcome the problem related to self- and intersterility among olive cultivars. Self-sterility is under the control of the placental-ovule-specific defh9 gene

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regulator sequence, expressed during early flower development. Production of transgenic olive plants with the parthenocarpy gene of Arabidopsis may allow olive fruit development as already successfully experienced in eggplant (Rotino et al. 1997). However, in olive, attention should be paid because it produces hundred thousand flowers per plant, and normally fruit set is around 1%, so parthenocarpy could represent a problem, if fruits are produced from most of the flowers.

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cold-hardiness, and Ascolona, Gemlik, and Hojoblanca had moderate cold-hardiness, while Samanli, Meski, Uslu, and Manzanilla were more sensitive. Furthermore, total soluble proteins were higher in the coldacclimated stage than in the non-acclimated stage. The tissues of cvs. Domat, Lecquest, Ascolona, Hojoblanca, and Gemlik enhanced the structural stability of cellular membranes in the cold-acclimated stage by increasing both the activity of CAT, APX, and NADPH oxidase to activate the antioxidative systems and the expression of 43 kDa dehydrins.

5.4.8.6 Abiotic Stress Tolerance 5.4.8.7 Other Useful Genes Olive is a species sensitive to chilling; at 7C is subject to frost damage in leaves. For this reason, genetic improvement for cold-resistance is one of the main important objectives for olive, since few genotypes are slightly tolerant to frost. Genetic transformation with the antifreeze protein (Hightower et al. 1991) or the overexpression of the superoxide dismutase gene (McKersie et al. 1993; Van Camp et al. 1994), and the overexpression of the Arabidopsis CBF1 gene (Jaglo-Ottosen et al. 1993), which enhances freezing tolerance by inducing genes associated with cold acclimatization, could also be attempted in olive. Recent studies on transgenic olive plants for the osmotin gene obtained by Rugini et al. (2000) reported that osmotin is positively involved in (a) the acclimationrelated programmed cell death (PCD), (b) blocking the cold-induced calcium signaling, and (c) affecting cytoskeleton in response to cold stimuli (D’Angeli and Altamura 2007). Additionally, osmotin, a pathogenesis-related (PR5) protein that exhibits cryoprotective functions, is a potential target to enhance cold tolerance. This has been proven by a study done by D’Angeli and Altamura (2007), which showed that osmotin is positively involved in the acclimationrelated programmed cell death (PCD), in blocking the cold-induced calcium signaling, and in affecting cytoskeleton in response to cold stimuli (D’Angeli and Altamura 2007). Cansev et al. (2008) found that patterns of antioxidative enzymes catalase (CAT: EC 1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase varied depending on the cold-acclimation stage and the cold-hardiness level of the cultivars. Domat and Lecquest were found to have the highest

Potentially useful genes have already been isolated from several species and could be introduced into olive. The selection procedure should be improved by replacing traditional selection markers (nptII) with other markers (gfp, lecI, ipt, pmi, xyla), although the percentage of escapes is quite high (>40%) in fruit crops (May et al. 1995; Pena et al. 1995, 1997; Mourgues et al. 1998; Perl and Eshdat 1998). Endo et al. (2001) suggested that the ipt gene from A. tumefaciens might be a suitable selectable gene. Moreover, a multiauto-transformation (MAT) vector, which combines the use of genes that stimulate growth and morphogenesis for positive selection of transformed cells with an excision mechanism to remove the markers and allow recovery of plants with normal phenotypes, could be very useful. The vector ipt and maize transposable element Ac for removing the ipt gene seem to be promising (Ebinuma et al. 1997). Other approaches might involve xyla (xylose isomerase) (Haldrup et al. 1998) and pmi (phosphomannose isomerase) genes that confer the capacity to use non-metabolizable substrates. For highly regenerative cultures, it could be possible to eliminate the marker gene and select on the basis of physiological or morphological parameters, such as the response to toxins, culture filtrates, and salt/drought resistance (Graham et al. 1996).

5.5 Development of Genomic Resources To develop proper genomic resources, it is necessary to include all the gene pools that represent the genetic diversity of all the olive species, and hence the wild

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gene pools became an integral part of all the materials to be explored in order to answer questions related to the evolution, domestication, natural selection effects, and other driving forces that shaped the genetical structure of the olive population. In general, the available datasets and information on functional genomic, proteomic, and metabolomic tools for studying olive tree development, tolerance to biotic and abiotic stresses, and fruit development are still scarce. Therefore, it should be necessary to enlarge both the representativeness of the current population in the olive genomic databases and the molecular tools available for approaching the mentioned aspects. Currently, olive genome has not been sequenced yet, and functional genomics of the most important traits have been restricted to single genes. For this reason, it should be given priority to all genomic resources that, in absences to the sequenced genome, could allow innovative molecular studies for the olive species. Different genomic approaches, therefore, have been used to increase the information concerning plant signaling, transcriptional networks, and regulatory circuits involved in important physiological and developmental processes. In particular, fruit development was the most biological process studied with different innovative molecular technology as expressed sequence tags (ESTs), large-scale microarrays, deep transcriptome profiling, etc. Currently, only few olive genes have been identified and annotated, and as of the fifth of May 2010, just 6,504 as a total of nucleotide sequences (4,860 ESTs, 130 genes, 1,514 nucleotide sequences) have been deposited in the public genome databases (i.e., The National Center for Biotechnology Information, NCBI) as compared to 376,006 nucleotide sequences for apple (335,398 ESTs, 38,091 genes, 2,517 nucleotide sequences), 204,214 for Prunus (98,536 ESTs, 103,153 genes, 2,625 others), 606,348 for Citrus (594,172 ESTs, 252 genes, 11,924 nucleotide sequences), 547,064 for grape (395,454 ESTs, 33,948 genes, 117,662 nucleotide sequences), and 506,332 for poplar (307,985 ESTs, 45,032 gene, 153,315 nucleotide sequences). Recently, a website named Olea ESTdb (http:// 140.164.45.140/oleaestdb/), containing over 60,000 transcripts that were isolated from olive fruit, covering all the developmental stages from the beginning to the end, has been launched (Alagna et al. 2009). The EST reads were generated through 454 pyrosequencing

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technology. The database is constructed by tentative consensus (TC) sequences and singletons (sESTs). All the 130 genes were generated from complete sequencing of chloroplast genome of the cultivar Bianchera. ESTs were generated from three differential libraries, two of them from fruitlets and leaves and the third from infected leaves with the fungus Spilocaea oleagina. Most of 30% of the nucleotide sequences are non-coding sequences (e.g., microsatellite 190, SRAP 26, intergenic space 102) that are used as molecular markers. A large amount of chloroplast and mitochondrial genes are also deposited, together with a very high number of ribosomal RNA genes, up to 109 sequences. Most of the olive ESTs are of the allergen coding sequences (208), while the number of ESTs of all other biological plant process is reduced (164). Many genes encoding key enzymes for fatty acid biosynthesis and modification, triacylglycerol synthesis, and storage have been isolated. These are enoyl-Acyl carrier protein reductase (ear), stearoylACP desaturase, o6 plastidial desaturase (fad6), o3 plastidial desaturase (fad7), cytochrome b5 (cyt b5), o6 cytoplasmic desaturase (fad2), o3 cytoplasmic desaturase (fad3), acyl CoA diacylglycerol acyltranferase (DGAT), and oleosin (Giannoulia et al. 2007). Stearoyl-ACP desaturase (Baldoni et al. 1996) is the key enzyme for the conversion of saturated stearic acid (C18:0) to mono-unsaturated oleic acid (C18:1), the main component of olive oil and responsible for its high dietary value. A cDNA has been isolated encoding a fatty acid desaturase, which is responsible for biosynthesis of a trienoic acid, linolenic acid, a major component of chloroplast membranes, and a precursor of the oxylipins i.e., methyl jasmonate. The latter are important in signal transduction pathways relating to plant development and responses to stress and have been studied in leaves, anthers, and embryos (Poghosyan et al. 1999). Two cytochrome b5 genes and their spatial and temporal patterns of expression have been characterized during floral and fruit development (Martsinkovskaya et al. 1999). The expression of oleosin gene is strongly embryonic-stage-dependent and the transcript reaches maximum levels at mid-torpedo stage and thereafter declines, coinciding the stages of most oil accumulation in embryo tissues, while in mesocarp tissues oleosin gene is not expressed (Giannoulia et al. 2007). A triterpene synthase cDNA, cloned from olive leaves by PCR amplification using

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primers designed from oxidosqualene cyclases, codes for the lupeol synthase protein (Shibuya et al. 1999). The differential expression of diacylglycerol acyltransferase (DGAT) genes has been evaluated in different olive tissues (Giannoulia et al. 2000). The sequence of a partial cDNA clone (1,402 bp) of the chloroplast ribulose 1,5-bisphosphate carboxylase large subunit (rbcL) gene of olive has been compared with that of other genera in order to establish the systematic position of the Oleaceae family (Wallander and Albert 2000). Similarly, the chloroplast ndhF gene has been sequenced for phylogenetic studies (Olmstead et al. 2000). Olive trees are persistent (up to 1,000 years) evergreens, tolerate drought and salinity (Bongi and Palliotti 1994), and are generally cultivated in areas where water is the main limiting factor in agricultural production. Recently, the molecular bases of water transport in olive have been studied, and three aquaporin (AQPs) genes have been isolated from tissues of the cv. Leccino (Secchi et al. 2007a). In plants, AQPs are classified in different groups according to their sequence identity or to their structural features (e.g., sizes or N- and C-termini). The main groups include plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like intrinsic proteins (found in the symbiosome membrane surrounding nitrogen-fixing bacteria of soybean), and small basic intrinsic proteins, a recently formed group that includes various AQPs whose subcellular localization is generally unknown (Johanson and Gustavsson 2002). The plant PIP group is divided into two groups (PIP1 and PIP2), which are characterized by specific amino acid residues at the N- and C-terminals and around the conserved NPA motifs (Schaffner 1998). A phylogenetic analysis of the corresponding polypeptides, and expression assays in Xenopus laevis oocytes, confirmed that they were part of water channel proteins localized in the plasma membrane and in the tonoplast. In well-hydrated plants, the highest expression level of OeTIP1.1 was detected in twigs, while OePIP2.1 mRNAs were very abundant in roots and moderate in twigs, and at lower levels were detectable in leaf extracts. The accumulation of OeTIP1.1 mRNA was detected in twigs, while in roots and leaves, the expression was low. In plants subjected to drought stress, the three AQPs expression genes, in root, shoot, and leaf, were downregulated (Secchi et al. 2007b), evidencing that lower gene expression in

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drought shoots is associated with higher hydraulic resistance. The downregulation of AQP genes can be considered a negative effect of water stress (Smart et al. 2001), but can also play the role of protecting the plant from excessive water loss in conditions of severe water stress and intense transpiration requirements (North et al. 2004; Vandeleur et al. 2005). During recovery after drought, the OePIP2.1 gene expression was upregulated. This biological process would imply an increase in cell conductivity and a high water availability in vessel-surrounding living cells that may enhance the xylem refilling capacity of parenchyma cells (Secchi et al. 2007b). The expression of AQP genes in olive resulted also regulated by intrinsic plant factors dependent from development of plant architecture. Dwarfing genotypes of cv. Leccino (D), inducing strongly reduced growth when used as rootstock (Rugini et al. 1996), probably due to its lower root hydraulic conductance (Nardini et al. 2006), have higher expression levels of OePIP1.1 and OePIP2.1 aquaporins in root, stem, and leaf than in Leccino plants carrying a vigorous (M) phenotype when used as rootstock. This expression behavior is in agreement with a higher hydraulic conductance of D plant roots and leaf plants than that of M plants, when these physiological parameters are scaled by unit root DW and unit leaf surface area (Lovisolo et al. 2007). Aquaporin genes are members of a large family of genes coding for membrane intrinsic protein (MIP). Plant genomes include a large number of MIP genes, e.g., in Arabidopsis thaliana 35 different AQP transcripts are found (Johanson et al. 2001), the genome of Zea mays encodes 33 AQP homologs (Chaumont et al. 2001), and in Oryza sativa, 33-like AQP-like genes have been identified (Sakurai et al. 2005); therefore, most of the olive AQP genes should be identified. Large-scale EST sequencing projects are in progress to obtain better insight into the molecular mechanisms of flower induction and development, fruit growth, and ripening processes in olives, and to prepare probes for the identification of several transcription factors and genes related to defense and stress response. In the last 2 years, a high abundance of transcripts have been analyzed using two main approaches (1) suppression subtractive hybridization (SSH) for identifying differentially expressed genes (Galla et al. 2009) and (2) 454 pyrosequencing technology for

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high-throughput DNA sequencing, allowing gene discovery and evaluation of expression patterns in olive fruit (Alagna et al. 2009). The investigation of the expression pattern for a large set of genes is one of the most important objectives of functional genomics; accordingly, in our laboratory, several cDNA libraries underlying both the induction and development of flower in Leccino and Frantoio olive cultivar have been developed.

5.6 Therapeutic Properties and Compounds Used as Herbal Drugs Fruits and oil from semi-domesticated olive trees were widely used in nutrition and medicine, during the Etruscan and Roman civilizations, Middle Ages, and Renaissance. Because most of wild Olea spp. contain small amount of extractable oil, and samples of their fruits are difficult to collect from the original growing sites (often inaccessible or remote), and scientific knowledge about chemical compounds with therapeutical properties have been acquired from the widely available fruits, leaves, and oil of O. europaea subsp europaea. Through these studies, it has been discovered that the well-known healthy effects of olive oil, which delay aging, must be attributed to all its metabolite components and not to a single compound. They act through the reduction of the risk factor leading to coronary heart disease and several types of cancer (Colomer et al. 2007) and to the modification of immune and inflammatory responses. Among the various important chemicals, particular relevance assume oleic acid (essential for its nutritional effect and stability during cooking at high temperature) and polyphenols (with their antioxidant properties; Boskou 1996; Servili et al. 2004). Some olive oil polyphenols are rare in other plant species, such as hydrophilic phenols (Shahidi 1996), and some others, such as biophenols and secoiridoids (oleuropein) (Iwai et al. 2005), are present only in the species of the Oleaceae family. Oleuropein, the most abundant biophenols, protects membrane lipid oxidation for preventing cardiac disease (Mercier 1997), acts on coronary dilation through antirhythmic action (Petkov and Manolov 1978), improves lipid metabolism and obesity-related problems (Iwai et al. 2005), prevents hypertensive cell death in cancer patients (Bonoli et al. 2004), and

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exhibits antiviral properties (Kubo et al. 1995; Uccella 2000). In addition, olive oil contains high content a-tocopherol (Psomiadou et al. 2000; Rotondi et al. 2004) and many other compounds; some of them are mentioned below. Verbascoside (hydroxycinnamic acid derivative) has been tested as a repairing substance in oxidative damage caused by heroin consumption (Qiusheng et al. 2005). Hydroxytyrosol (derivative of oleuropein and verbascoside) shows better antioxidant capacity than vitamins C and E or 2,6-di-tertbutyl-4-methylphenol (BHT) (Visioli et al. 1998; Fabiani et al. 2002), providing protective effects from cardiac diseases and cancer (Luque de Castro and Japo`n Luja`n 2006). Oleocanthal has shown to be a COX inhibitor (Beauchamp et al. 2005). The various health benefits exerted by the olive oil compounds are part of large benefit endowed by the Mediterranean diet (Tulp et al. 2006). Leaves of Olea taxa have been used for the treatment of wounds, fever, diabetes, gout, arterioscleroses, and hypertension since ancient times (Janicke et al. 2008), and olive leaf extract (EFLA®943) has been demonstrated to be a prophylactic for lowering blood pressure in humans (Perrinjaquet-Moccetti et al. 2008). Only recently, it has been scientifically demonstrated that leaf extracts of Oleaceae family, particularly O. europaea subsp. europaea and O. lancea (syn. O. europaea (subsp. cuspidata, syn. O. africana)) and Ligustrum vulgare, have the expected antimicrobial activity (Pereira et al. 2007) due to the presence of oleuropein, and the positive effect in reducing blood pressure due to the presence of oleacin (secoiridoid 2-(3,4dihydroxyphenyl)ethyl 4-formyl-3-(2-oxoethyl)-4Ehexenoate), which is an ACE (angiotensin converting enzyme)-inhibitor (Perrinjaquet-Moccetti et al. 2008). The total oil content of the fruit (rarely exceeding 25% of fresh weight), the peculiar concentration of oleic acid (C18:1 mono-unsaturated fatty acid, 40–80% of the total triacylglycerides) and linoleic acid (about 10%), and total amount of polyphenols (50–800 mg/kg) could change according to variety and environmental conditions (Cahoon and Shanklin 2000; Bruner et al. 2001; Rahman et al. 2001). All these parameters of quality in virgin olive oils make the studies of gene regulation of oil metabolism and oil accumulation of principal interest. Leo`n et al. (2008) suggested that new olive cultivars with fatty acid

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composition fulfilling consumer and market demands could be obtained through cross-breeding because a quite different fatty acid composition in the oil of 15 advanced selections and their three progenitors was observed with the percentages of C18:1, C18:2, and saturated fatty acids being the main contributors to the total variation.

5.7 The Dark Sides of Olea and Ways to Address Them The problems related with the wild olive genetic resources, as deduced from the information in the previous paragraphs, can be synthesized in the following three aspects (a) release of pollen-causing allergies, (b) colonization events leading to invasiveness, and (c) extinction of populations due to a narrow geographical distribution. Pollen from Olea taxa produce respiratory allergy in the human settlings nearby where those plants are widely present, especially in the Mediterranean countries, Australia, and North and South America (Wheeler 1992; Liccardi et al. 1996), due to the content of up to 10 allergens (Ole e 1 to Ole e 10) (Rodrı´guez et al. 2002; Barral et al. 2004). These proteins have been indexed like members of the denominated “pollen proteins of the Ole e 1 family” (Bateman et al. 2004). Olea taxa display wide differences in the expression levels of many allergens and in the number and molecular characteristics of the allergen isoforms expressed. These differences are maintained over the years and are dependent on the genotype of each taxa. Quantitative differences in the content of Ole e 1 have been described in the pollen of several Olea taxa (Castro et al. 2003). The presence of nucleotide substitutions at the locus encoding Ole e 1 proteins result in many cases in amino acid changes (Villalba et al. 1993). The molecular variability of Ole e 1 allergen was studied throughout a number of Olea taxa, and it was concluded that the varietal origin of olive pollen is a major factor determining the diversity of Ole e 1 variants. The presence of an extremely wide germplasm in the Olea genus (Bartolini et al. 1994) clearly points to this genetic variability as a putative cause of polymorphism for Ole e 1 sequence (Alche´ et al. 2007). This information is useful for the identification of the Ole

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e 1 genetic variants and for future genetic engineering applications for modifying pollen allergen composition in Olea ssp. pollen (Hamman-Khalifa et al. 2008). The wide range of geographical sites, where the different wild olive subspecies originated (Green 2002), is frequently considered as an explanation for the invasive performance in some places where they have been introduced, such as in the Pacific islands and East and South Australia (West 2002; Starr et al. 2003; Cooke et al. 2005; Bass et al. 2006; Breton et al. 2006). In the nineteenth century, both O. europaea L. subsp. europaea and subsp. cuspidata were first introduced in Sydney territory for economic purposes, and naturalized populations (i.e., self-reproducing trees not planted for domestic or commercial use) were first recorded in the Norfolk Island, Adelaide, and across a wide range of habitats, predominantly within the 400–600 mm average annual rainfall range (Spennemann and Allen 2000; Mekuria et al. 2002; Bass et al. 2006). Since the 1960s, cuspidata naturalized populations have been found in Hawaii Archipelago (Starr et al. 2003). In the sites where Olea taxa have been introduced, the olive fruit is highly attractive to avian fauna dispersal agents and have few predators. Spennemann and Allen (2000) suggest that foxes may disperse O. europaea seed for 40–50 km from the fruit source. Since 1836, hundreds of Olea taxa have been introduced into South Australia, mainly from olive-growing regions in the Northern Hemisphere. In the 1870s, olive production in South Australia was considered to be a more viable enterprise than wine production. This led to the establishment of a 1.2 ha orchard in 1874, which was expanded to over 40.5 ha by 1882 (Reichelt and Burr 1997). By the early 1900s, however, the olive industry in South Australia was in decline. Cross-breeding from abandoned groves over 160 years has resulted in populations of feral olives that grow in all Australian regions (Spennemann and Allen 2000; Bass et al. 2006) and now feral olive trees are found in western Australia, Victoria, New South Wales, Southeast Queensland, and Tasmania (Spennemann and Allen 2000; Bass et al. 2006), and in northern New Zealand (Heenan et al. 1999). These feral populations may present ecological problems, as the olive is thought to compete successfully with native Australian vegetation, particularly in disturbed habitats. Diversity of native flora was found to be at

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least 50% lower in eucalypt woodland heavily invaded by O. europaea, when compared to similar woodland relatively free of O. europaea (Crossman 2002). This view has led some local councils to proclaim the olive tree as a weed and instigate eradication programs. Removal of feral forms of olive requires careful planning before doing so in order to avoid problems in fragile ecosystems (Crossman et al. 2002). The phylogeographic approach (Amsellem et al. 2000; Milne and Abbott 2004) was used by Besnard et al. (2007c) for reconstructing a detailed distributional range of the O. europaea complex based on DNA polymorphism from the nuclear (bi-parentally inherited) and plastid (maternally inherited) genomes. This study provided powerful data to test hypotheses about the origins of naturalized olive populations and evidenced that subspecies europaea and cuspidata is likely to hybridize or introgress when in sympatry to other Olea taxa (Besnard et al. 2001a; Rubio de Casas et al. 2006). This phenomenon could on one side alleviate the loss of genetic diversity due to bottlenecks arising from small initial founder populations during colonization events (Husband and Barrett 1991; Lee 2002), but on the other side contribute to the ecological success of colonizing populations. Using the mentioned molecular tools, it was determined that East Australian and Hawaiian populations of subsp. cuspidata have originated from southern Africa, while South Australian populations of subsp. europaea have mostly derived from western or central Mediterranean cultivars. Invasive populations of subsp. cuspidata showed significant loss of genetic diversity in comparison to a putative source population, and a recent bottleneck was evidenced in Hawaii. Conversely, invasive populations of subsp. europaea did not display significant loss of genetic diversity in comparison to a native Mediterranean population. One hybrid (cuspidata europaea) was identified in East Australia. The importance of hybridizations in the future evolution of the olive invasiveness remains to be investigated (Besnard et al. 2007a, b, c). A relationship between polyploidy and narrow endemic taxa is observed for O. europaea ssp. maroccana (hexaploid) and O. europaea ssp. cerasiformis (tetraploid) (Besnard et al. 2008). As stated earlier, these two taxa are endemic to subtropical areas of the Agadir Mountains and Madeira, respectively, and their populations are endangered (Besnard et al. 2008). Particularly, O. europaea subsp. maroccana is consid-

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ered to be one of the ten most threatened trees in the Mediterranean Basin (Me´dail et al. 2001). Also, the current populations of O. e. laperrinei in the Aı¨r mountain range of Niger are endangered due to the absence of an efficient sexual reproductive strategy coupled with the high fragmentation of very small populations and a narrow altitudinal range of distribution (Anthelme et al. 2008). To ensure adequate protection for the three Olea ssp. and their habitat, recommendations to facilitate appropriate mitigation in the form of revegetation and restoration for enhancing territorial stability and conservation of habitat complexity, while reducing longterm maintenance costs, should be addressed.

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E. Rugini et al. formed plants of Rosa hybrida L. and characterization of their rooting ability. Mol Breed 3:39–47 van der Salm TPM, Bouwer R, Van Dijk AJ, Keiser LCP, Ten Cate ChHH, Van der Plas LHW, Dons JJM (1998) Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L. J Exp Bot 49:847–852 Vandeleur R, Niemietz C, Tilbrook J, Tyerman SD (2005) Roles of aquaporins in root responses to irrigation. Plant Soil 274:141–161 Vargas P, Kadereit JW (2001) Molecular fingerprinting evidence (ISSR, inter-simple sequence repeats) for a wild status of Olea europaea L. (Oleaceae) in the Eurosiberian North of the Iberian Peninsula. Flora 196:142–152 Vergari G, Patumi M, Fontanazza G (1996) Utilizzo dei marcatori RAPDs nella caratterizzazione del germoplasma di olivo. Olivae 60:19–22 Villalba M, Batanero E, Lo´pez-Otı´n C, Sa´nchez LM, Monsalve RI, Gonza´lez de la Pen˜a MA, Lahoz C, Rodrı´guez R (1993) The amino acid sequence of Ole e I, the major allergen from olive tree (Olea europaea) pollen. Eur J Biochem 216:863–869 Vince-Prue D, Canham AE (1983) Horticultural significance of photomorphogenesis. In: Shropshire W, Mohr H (eds) Encyclopedia of plant physiology. Springer, Berlin, Germany, pp 518–544 Visioli F, Bellomo G, Galli C (1998) Free radical-scavenging properties of olive oil polyphenols. Biochem Biophys Res Commun 247:60–64 Wallander E, Albert VA (2000) Phylogeny and classification of Oleaceae based on rps 16 and trnL-F sequence data. Am J Bot 87:1827–1841 Watts WA, Allen JRM, Huntley B (1996) Vegetation history and palaeoclimate of the last glacial period at Lago Grande di Monticchio, Southern Italy. Quaternary Sci Rev 15:133–153 Weisman Z, Avidan N, Lavee S, Quebedeaux B (1998) Molecular characterisation of common olive varieties in Israel and the West Bank using random amplified polymorphic DNA (RAPD) markers. J Am Soc Hortic Sci 123:837–841 West CJ (2002) Eradication of allien plants on Raoul Island, Kermadec Islands, New Zealand. In: Veitch CR, Clout MN (eds) Turning the tide: the eradication of invasive species. IUCN SSC Invasive Species Specialist Group, Cambridge, pp 365–373 Wheeler AW (1992) Hypersensitivity to the allergens of the pollen from olive tree (Olea europaea). Clin Exp Allergy 22:1052–1057 Wiesman Z, Avidan N, Lavee S, Quebedeaux B (1998) Molecular characterization of common olive varieties in Israel and the West Bank using randomly amplified polymorphic DNA (RAPD) markers. J Am Soc Hortic Sci 123:837–841 Wu G, Shott BJ, Lawrence EB, Levine EB, Fitzsimmons KC, Shah DM (1995) Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in trangenic potato plants. Plant Cell 7:1357–1368 Yadav NS (1996) Genetic modification of soybean oil quality. In: Verma DPS, Shoemaker RC (eds) Soybean genetics: molecular biology and biotechnology. CABI, New York, pp 165–188 Yanotsky M (1995) Floral meristems to floral organs: genes controlling early events in Arabidopsis flower development. Annu Rev Plant Physiol Mol Biol 46:167–188

5 Olea Yoshikawa M, Tsuda M, Takeuchi Y (1993) Resistance to fungal diseases in transgenic tobacco plants expressing the phytoalexin elicitor-releasing factor, -1, 3-endoglucanase from soybean. Naturwissenschaften 80:417–420 Zhu B, Chen TH, Li PH (1993) Expression of an ABAresponsive osmotin-like gene during the induction of freezing tolerance in Solanum commersonii. Plant Mol Biol 21:729–735 Zhu B, Chen TH, Li PH (1995) Expression of three osmotin like protein genes in response to osmotic stress and fungal infection in potato. Plant Mol Biol 28:17–26

117 Zhu LH, Holefors A, Ahlman A, Xue ZT, Welander M (2001) Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth. Plant Sci 160:433–439 Zohary D (1994) The wild genetic resources of the cultivated olive. Acta Hortic 356:62–65 Zohary D, Hopf M (1994) Olive: Olea europaea. In: Domestication of plants in the old world, 2nd edn. Oxford Claredon, Oxford, UK Zohary D, Spiegel-Roy P (1975) Beginnings of fruit growing in the old world. Science 187:319–327

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Chapter 6

Pistacia J.I. Hormaza and A. W€ unsch

6.1 Botanical and Taxonomic Description The genus Pistacia belongs to the Anacardiaceae, a cosmopolitan family in the Sapindales/Rutales (Wannan and Quinn 1991) that comprise about 70 genera and over 600 species (Mitchell and Mori 1987). The most widely accepted classification divides the family into five tribes: Anacardieae, Rhoeae, Semecarpeae, Spondiadeae, and Dobineae (Mitchell and Mori 1987; Wannan and Quinn 1991), with Pistacia assigned to the tribe Rhoeae (Mitchell and Mori 1987). The Anacardiaceae family includes other species of horticultural interest (Janick and Paull 2008) such as Anacardium occidentale L. (cashew), Buchanania lanzan Spreng. (chironji), Mangifera indica L. (mango), Sclerocarya birrea (A. Rich.) Hochs. (marula), Semecarpus spp. (marking trees and tar tree), Spondias cythera Sonn. (ambarella), Spondias mombin L. (yellow mombin), Spondias purpurea L. (red mombin), or Tapirira guianensis Aublet. (wild mombin). The species of the genus Pistacia are evergreen or deciduous shrubs and small trees with alternate, pinnately compound leaves, with cavities that secrete resinous compounds. Pistacia species are wind-pollinated and dioecious, although a few exceptions have been described (Ozbek and Ayfer 1958; Crane 1974; Kafkas et al. 2000; Gercheva et al. 2008). Seven species of the genus are distributed from the Mediterra-

J.I. Hormaza (*) Instituto de Hortofruticultura Subtropical y Mediterra´nea “la Mayora,” - CSIC, 29750 Algarrobo-Costa, Ma´laga, Spain e-mail: [email protected]

nean Basin to central Asia (P. atlantica Desf., P. integerrima Stew ex Brandis, P. khinjuk Stocks, P. palaestina Bois., P. lentiscus L., P. terebinthus L., and P. vera L.), two species in eastern Asia (P. chinensis Bunge and P. weinmannifolia Poisson), and two species from the southwestern United States to Central America (P. mexicana H.B.K. and P. texana Swingle) (Table 6.1; Zohary 1952; Kafkas 2006a; Yi et al. 2008). Pistachio (P. vera) is the only species of the genus cultivated commercially and the rest of the species are mostly used as rootstocks for pistachio. Products of other species of the genus (mainly fruits and resins) have been traditionally used locally for a wide range of applications, from medicinal uses to fuel production. Chromosome counts have been performed in some species of the genus (Table 6.1); 2n ¼ 30 has been reported for P. atlantica (Vogt and Aparicio 1999; Ila et al. 2003), P. integerrima (Mehra 1976; Sandhu and Mann 1988), P. lentiscus (Natarajan 1977, 1978), P. terebinthus (Ila et al. 2003), and P. vera (Boczantseva 1972; Ila et al. 2003), whereas 2n ¼ 24 has been reported for P. chinensis (Huang et al. 1989) and P. khinjuk (Ghaffari and Fasihi Harandi 2002). The evolutionary history of the genus Pistacia and the taxonomic relationships among the different species are still subject to controversy. Hybridization is common among several Pistacia species (Zohary 1952; Crane and Iwakiri 1981; Morgan et al. 1992), suggesting a close phylogenetic relationship, and their classification has changed over time (Table 6.2). Morphological as well as molecular data have been used to analyze and classify the species of the genus (see Kafkas 2006a, for a review). Initially, using morphological characters, Zohary (1952) considered the genus to comprise 11 species divided into four sections (Table 6.2): Lentiscella (P. mexicana and

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_6, # Springer-Verlag Berlin Heidelberg 2011

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J.I. Hormaza and A. W€ unsch Table 6.1 Pistacia species distribution and chromosome number Species Common name P. atlantica Atlas Pistachio, Pistachier de l’Atlas P. integerrima P. khinjuk P. lentiscus Lentisque, Mastic-tree P. terebinthus Terebinth, Turpentine-tree P. vera Pistachio P. chinensis Chinese pistachio P. weinmannifolia P. mexicana Mexican pistachio P. texana American pistachio

Table 6.2 Classification of Pistacia species according to: a Zohary (1952), bParfitt and Badenes (1997)

Sectiona Lentiscella Eu Lentiscus

Eu Terebinthus

Butmela

Distribution Mediterranean–Asia Mediterranean–Asia Mediterranean–Asia Mediterranean–Asia Mediterranean–Asia Mediterranean–Asia East Asia East Asia North and Central America North and Central America

Speciesa P. mexicana P. texana P. wienmannifolia P. saportae P. lentiscus P. terebinthus P. palaestina P. khinjuk P. vera P. chinensis P. chinensis (var. integerrima) P. atlantica

P. texana), Eu Lentiscus (P. lentiscus, P. saportae, and P. winmannifolia), Butmela (P. atlantica), and Eu Terebinthus (P. chinensis, P. khinjuk, P. palaestina, P. terebinthus, and P. vera), although some authors recognize as many as 15 species (Whitehouse 1957). One of these species (P. saportae) was later suggested to be an interspecific hybrid (Zohary 1972), which has been supported by molecular data where P. lentiscus would be the female and P. terebinthus the male parent (Yi et al. 2008). P. aethiopica was proposed as a new species in 1980 (Kokwaro and Gillett 1980), but its status has not been confirmed. Similarly, P. integerrima was proposed as a diverged subspecies of P. chinensis (Zohary 1952) but, based on plastid restriction analyses and flowering phenology, Parfitt and Badenes (1997) defended the species status of P. integerrima; this has been recently confirmed with additional molecular studies (Yi et al. 2008). Other works have used a range of morphological data such as leaf (Lin et al. 1984; Al-Saghir et al. 2006) and wood (Grundwag and Werker 1976) anatomy and stomatal distribution (Al-Saghir and Porter 2005) to classify

Sectionb Lentiscus

Terebinthus

Ploidy 2n ¼ 30 2n ¼ 30 2n ¼ 24 2n ¼ 30 2n ¼ 30 2n ¼ 30 2n ¼ 24

Speciesb P. mexicana P. texana P. wienmannifolia P. lentiscus P. terebinthus P. khinjuk P. vera P. chinensis P. integerrima P. atlantica

Pistacia species, but more reliable results can be obtained from molecular studies. On the basis of plastid restriction site analysis and morphological characters, Parfitt and Badenes (1997) suggested the division of the genus into two sections, Lentiscus and Terebinthus (Table 6.2); section Lentiscus would include the sections Lentiscella and Eu Lentiscus of Zohary (1952), whereas section Terebinthus would include sections Butmela and Eu Terebinthus of Zohary (1952). The species in the Lentiscus group are evergreen with paripinnate leaves, and the species in the Terebinthus group, including P. vera, are deciduous with imparipinnate leaves (Table 6.2). This work confirmed the hypothesis of Zohary (1952) that, based on morphological observations, P. vera and P. khinjuk are the most primitive Pistacia species and that central Asia is the origin of diversity of the genus. The interspecific relationships described by Parfitt and Badenes (1997) using chloroplast DNA have been later confirmed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers

6 Pistacia

(Kafkas and Perl-Treves 2001; Katsiotis et al. 2003; Golan-Goldhirsh et al. 2004). Other studies based on genetic similarity from RAPD data (Kafkas and Perl-Treves 2002) grouped the genus into single-trunked tree species (P. atlantica, P. eurycarpa, P. integerrima, P. khinjuk, and P. vera) and shrubs or small trees (P. lentiscus, P. mexicana, P. palaestina, P. texana, and P. terebinthus). In a later study using AFLP markers, Kafkas (2006b) confirmed the differentiation of P. therebintus not only from P. vera, P. khinjuk, P. integerrima, and P. atlantica but also from P. mexicana and P. lentiscus. Recent studies using molecular data from nuclear and plastid DNA (Yi et al. 2008) defined new phylogenetic relationships within the genus, showing evidence for reticulate evolution. P. atlantica would be nested within the Therebintus section as suggested by Parfitt and Badenes (1997), whereas Parfitt and Badenes’ Lentiscus group would not be monophyletic and Zohary’s Lentiscella group species (P. texana and P. mexicana) and P. wienmannifolia would group in two parallel clades. An alternative approach to study phylogenetic relationship in the genus was undertaken by Inbar (2008) who used the very close relationships between gall-forming aphids (Homoptera: Fordinae) and species of Pistacia. He could differentiate New World Pistacia species (P. mexicana and P. texana) from the rest of the species where two groups could be observed: “Vera” (P. vera, P. atlantica, P. mutica) and “Khinjuk” (P. khinjuk, P. chinensis, P. integerrima, P. palaestina, and P. terebinthus). In spite of all the advances made using different approaches, some confusion is still present in the classification of the Pistacia genus, and further work is clearly needed to finally have an overall perspective of the phylogenetic relationships between the different Pistacia species.

6.2 Diversity and Conservation The valuable genetic resources of the different species of the genus Pistacia are suffering a severe genetic erosion that can significantly narrow down their genetic diversity in central and West Asia and Mediterranean countries (Padulosi and Hadj-Hassan 2001; Khanazarov et al. 2009; Ozden-Tokatli et al. 2010).

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Different efforts have been made in various countries to study the extant diversity in order to develop appropriate conservation strategies. In order to optimize the management of the genetic resources of different Pistacia species, two different morphological descriptors’ lists have been published; one just for P. vera (IPGRI 1997) and the other for other species of the genus Pistacia excluding P. vera (IPGRI 1998). Several studies have focused on understanding the genetic diversity of natural populations of Pistacia species using morphology and/or molecular markers. Initial studies were performed in P. vera, the most commercially important species of the genus (Hormaza and W€unsch 2007). Thus, molecular identification of pistachio using DNA markers was carried out by Hormaza et al. (1994a) and Dollo et al. (1995). They examined pistachio cultivars with RAPD markers, and the resulting cluster analysis grouped the cultivars according to their geographical origin distinguishing two major clusters. One cluster includes cultivars originated in the Mediterranean countries and the other group’s cultivars from Iran and the Caspian Sea. Further RAPD analysis of additional genotypes closer to the pistachio’s center of origin were also grouped into the Iranian–Caspian cluster (Hormaza et al. 1998). Similar results have been recently obtained with AFLP markers (Shanjani et al. 2009). RAPD markers have also been used more recently to study the diversity of local pistachio germplasm in Turkmenistan (Barazani et al. 2003). AFLPs have also been used to identify Afghan (Kafkas et al. 2006a), Syrian (Basha et al. 2007), and Iranian (Shanjani et al. 2009) P. vera genotypes, and a combination of AFLP, ISSR (inter simple sequence repeat), and RAPD markers has been used to identify 69 P. vera cultivars from different countries (Kafkas et al. 2006b). More recently, microsatellite or simple sequence repeat (SSR) markers developed in P. vera (Ahmad et al. 2003) have been used to characterize pistachio cultivars and to establish a true-to-the-type assay based on the DNA extracted from kernels and shells. Microsatellites have also been used to analyze genetic diversity in Iranian P. vera cultivars (Pazouki et al. 2010); the results showed gene flow between cultivated and wild genotypes of P. vera. The genetic diversity of P. lentiscus populations in southern Spain and northern Africa was also evaluated using RAPD markers (Werner et al. 2002).

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The populations of Morocco were found to be genetically different from the Spanish populations, while the southeastern and southern Spanish plants were genetically similar. Barazani et al. (2003) also used RAPD markers to analyze the genetic variability of P. lentiscus genotypes. Cluster analysis of the RAPD data divided the genotypes into two groups that correlated with their geographical origin. The authors also analyzed essential oil content and morphology of the same genotypes and identified high levels of variation; however, cluster analysis of oil content did not correlate with geographical origin or with the RAPD results. Barazani et al. (2003) concluded that high morphological and chemical variability together with genotypic polymorphism provide ecological advantages that might explain the wide distribution of P. lentiscus. Nahum et al. (2008) studied the morphological and genetic variation of Israeli and Cypriot populations of P. lentiscus. The authors found a correlation of the morphological variation with environmental conditions like precipitation and temperature. Since no significant genetic differentiation or isolation of the populations was detected, the authors suggested that morphological differentiation of P. lentiscus is probably due to the species’ phenotypic plasticity. Morphological diversity in P. atlantica has been studied in populations of Algeria (Belhadj et al. 2008a), identifying significant variation within and among populations. Furthermore, the Algerian populations were differentiated from populations of other origins by the presence of wax in the leaves. In Turkey, Kafkas et al. (2002) have also analyzed the morphological and phenotypic diversity of local P. atlantica, P. eurycarpa, and P. terebinthus genotypes identifying a high intraspecific diversity. More recently, microsatellites were used to analyze intraspecific genetic diversity in P. vera, P. atlantica, and P. khinjuk in Iran (Pazouki et al. 2010); the results showed a lower genetic diversity in P. atlantica as compared to the other species. RAPD markers have also been used to identify specific markers for distinguishing genotypes of Pistacia saportae Burnat, a hybrid that is difficult to differentiate morphologically but that is widely used as a rootstock for P. vera (Werner et al. 2001). Additionally, Kafkas and Perl-Treves (2001) were able to separate P. vera from P. khinjuk and established species-specific RAPD markers for the identification of unknown Pistacia germplasm. SSRs and the

J.I. Hormaza and A. W€ unsch

sequence-related amplified polymorphism (SRAP) technique have also been used to identify commercial pistachio rootstocks including genotypes of the species P. atlantica, P. integerrima, and P. atlantica P. integerrima hybrids (Ahmad et al. 2005). Adequate conservation strategies require the development of appropriate in situ and ex situ germplasm management protocols. Several countries have ex situ germplasm collections of different Pistacia species, although most of the collections are biased towards P. vera accessions. According to the Bioversity Directory of Germplasm Collections (Bioversity International 2010) and the WEIS database (FAO 2010), the most important collections of germplasm from species of the genus other than P. vera are maintained in Israel, Spain, US, Syria, and Turkey. The collection in Israel established at BIDR (The Jacob Blaustein Institute for Desert Research) holds live and seed material of Mediterranean Pistacia germplasm: P. atlantica, P. chinensis, P. khinjuk, P. palaestina, P. vera, P. lentiscus, and P. terebinthus (Golan-Goldhirsh and Koustiukovsky 1998). This collection is being characterized phenotypically and using molecular markers (Barazani and Golan-Goldhirsh 2004). Data on the characterization of the accessions of the collection are available online (Ben-Gurion University 2010). In Spain, genotypes of six Pistacia species (P. atlantica, P. integerrima, P. khinjuk, P. palaestina, P. vera, and P. terebinthus), mainly from Greece, Spain, Syria, Turkey, and USA are conserved in the IRTA Mas Bove´ research station. This Pistacia germplasm bank includes 219 accessions of the different species with new seedlings being introduced (Batlle et al. 2006). Additional small collections are also conserved in several central Asian countries (Khanazarov et al. 2009).

6.3 Breeding and Genetics The main breeding programs have been developed in P. vera where the most advanced breeding programs are now evaluating advanced selections from breeding crosses (Hormaza and W€unsch 2007). This is the case of the program initiated at the University of California at Davis in the USA (Parfitt et al. 1995; Chao et al. 1998, 2003), at the IRTA Mas Bove´ in Spain (Vargas et al. 1987, 1993, 2002; Batlle et al. 2001), and at the

6 Pistacia

Pistachio Research Institute in Gaziantep in Turkey (Mehlenbacher 2003). In other countries, such as Iran, Israel, or Australia, current pistachio genetic improvement involves evaluating cultivars, local seedling populations, and species (Mehlenbacher 2003). In Iran, the breeding potential of P. atlantica local genotypes for oil production has been determined (Arefi et al. 2006). In Turkey, the performance of different genotypes of P. vera, P. atlantica, P. therebintus, and P. eurycarpa are being evaluated to be used as P. vera rootstocks (Kafkas et al. 2006c), and crosses of P. vera and P. khinjuk are also being used for rootstock breeding (Atli and Kaska 2002). Pistachio rootstocks are mainly produced from seed. Important advances have taken place in micropropagation (Parfitt and Almehdi 1994; reviewed in Ozden-Tokatli et al. 2010) and micrografting (Onay et al. 2004a; reviewed in Ozden-Tokatli et al. 2010). Clonal propagation from leaf cuttings of the rootstock UCB-1 (hybrid between P. atlantica and P. integerrima) widely used in California has been reported (Almehdi et al. 2002). Mound layering has been shown as an appropriate method to multiply P. chinensis (Dunn and Cole 1995) and a shoot micropropagation protocol of P. mutica has also been reported (Ghoraishi 2006). Similarly, efficient somatic embryogenesis protocols have been reported for P. vera (Onay et al. 1995, 1996, 2000, 2004b) and they could be useful for other species of the genus. In vitro seed germination protocols for P. vera, P. atlantica, P. terebinthus, and P. lentiscus were reported by Molina and Trujillo (1999) and for seed and shoot in vitro and in vivo propagation of P. lentiscus (Mascarello et al. 2007). Pistacia species are dioecious and have a long juvenile period; in the case of P. vera 5–8 years are needed to reach maturity. Dioecy requires the interplanting of male and female trees in commercial pistachio orchards reducing the profitability of the crop and, thus, research is being performed to find monoecious Pistacia genotypes. Monoecious P. atlantica genotypes are being used to determine the mechanism and inheritance of sex determination in the species (Kafkas 2002), and a transsexual population of P. terebinthus found in Bulgaria is also being studied to be used as a rootstock and as a donor of monoecy (Gercheva et al. 2008). Sex-linked markers have been searched for markerassisted selection in order to discriminate between

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male and female genotypes at an early stage of development. Since morphological markers do not allow distinguishing female from male plants prior to flowering, determination of the plant gender at an early vegetative stage will greatly facilitate breeding, selection, and management of the different Pistacia species. In fact, sex determination is the only character that has been investigated at the molecular level for markerassisted selection in the genus. Initially, a RAPD marker linked to sex determination in P. vera was developed by Hormaza et al. (1994b) using bulked segregant analysis (BSA). Subsequently, this marker has proved useful for sex identification in a large number of genotypes of P. vera (Yakubov et al. 2005). A similar approach was followed by Kafkas et al. (2001) to identify markers linked to sex determination in P. eurycarpa, P. atlantica, and P. therebintus. Two markers linked to sex determination in P. eurycarpa and one in P. atlantica were identified. In both works (Hormaza et al. 1994b; Kafkas et al. 2001), hybridization signals of the identified sexrelated RAPD markers were found in repetitive sequences, and a low frequency of sex-related polymorphisms was observed. These results suggest that sex determination in Pistacia species must be restricted to a small region of the genome comprising one or few genes, surrounded by repetitive sequences.

6.4 Genomic Resources Specific microsatellite markers have been developed for two species of the genus, P. vera and P. lentiscus (Table 6.3). Eight polymorphic microsatellites have been identified in P. lentiscus (Albaladejo et al. 2008) and 14 in P. vera (Ahmad et al. 2003). The SSR markers developed by Albaladejo et al. (2008), and evaluated in P. lentiscus populations, revealed high levels of variation, allowing their utility for gene flow and conservation studies in these species (Albaladejo et al. 2008). Twelve of the markers developed in P. vera have been used to identify commercial pistachio rootstocks including P. atlantica, P. integerrima, and P. atlantica P. integerrima hybrids (Ahmad et al. 2005), and 10 of them have also been used to characterize P. atlantica subsp. kurdica and P. khinjuk (Pazouki et al. 2010). An additional group of SSR markers developed in mango (Mangifera

124 Table 6.3 SSR loci identified and screened in Pistacia species

J.I. Hormaza and A. W€ unsch SSRs (Number) Species origin Reference Ptms (14) P. vera Ahmad et al. (2003)

Pislen (8) LMMA (16)

P. lentiscus M. indica

indica), a species in the Anacardiaceae, by Viruel et al. (2005), has been screened for transferability in the Pistacia genus. The study revealed that 44% of the SSRs developed in mango are conserved in four Pistacia species studied (P. vera, P. atlantica, P. terebinthus, and P. lentiscus) (Hormaza and Viruel unpublished results) and they can be added to the set of microsatellite markers available for studies in Pistacia species.

6.5 Scope for Domestication and Commercialization The pistachio (P. vera) is the only commercially important species in the genus and the only one that has been domesticated while the rest of the species are mostly used as rootstocks and, in some cases, for fruit consumption, oil extraction, or as a source of food for domestic animals. Pistachio is cultivated for its edible fruit and is based on clonally propagated scion cultivars grafted onto seedling rootstocks of the same species or of other Pistacia species or hybrids (Hormaza and W€unsch 2007). Different rootstocks are used in the different growing areas. Thus, P. atlantica, P. integerrima, and P. atlantica P. integerrima hybrids are the main rootstocks in California; P. vera, P. atlantica, P. therebintus, and P. eurycarpa seedlings are used in Turkey (Ila et al. 2003), while P. mutica, P. khinjuk, as well as seedlings of P. vera are the main rootstocks used in Iran. In areas where wild forms exist, wild P. vera individuals are occasionally grafted in situ with selected genotypes (Zohary and Hopf 1988). The resin of different species of Pistacia was a highly valued commodity in old cultures such as ancient Egypt (Stern et al. 2008). Today, the biological properties of these oleoresins are being investigated

Transferability P. atlantica P. integerrima P. atlantica P. khinjuk Albaladejo et al. (2008) – Viruel et al. (2005) P. vera P. atlantica P. therebintus P lentiscus

References Ahmad et al. (2005) Pazouki et al. (2010) – –

due to their potential for a wide range of uses, from medicinal to cosmetic and perfumery applications (reviewed in Assimopoulou and Papageordiou 2007). Carbonized remains of small nuts of different wild pistachios, mainly P. atlantica, appear in Neolithic and Bronze Age eras in the Near East, suggesting that fruits of this wild species were collected in those periods. Resin and fruits of P. atlantica are still collected today in different countries such as Iran (Pourreza et al. 2008), Algeria (Belhadj et al. 2008b), or Afghanistan (Said 2008) and are occasionally sold in local markets, although there is no sign that P. atlantica was ever domesticated (Zohary and Hopf 1988). P. atlantica subsp. mutica resin has been traditionally used for chewing gum and medicinal uses in Iran (Delazar et al. 2004). In Algeria, P. atlantica leaves and fruits are used to feed cattle (Belhadj et al. 2008b). In Turkey, P. atlantica, P. therebintus, and P. eurycarpa (syn. P. atlantica subsp. kurdica) are locally used for food, and the resins for oil and soap production (Ila et al. 2003). Several extracts of P. lentiscus (the resin trunk exudates, called “mastic gum,” the cold pressed oil extracted from the fruits, and the essential oils) have been used since antiquity in traditional medicine mainly as anti-inflammatory, antiseptic, and in the treatment of various diseases such as gastralgia and dyspepsia (Assimopoulou and Papageordiou 2007) and also for painting and staining (Avanzato et al. 2008). The mastic gum from this species is important in some specific areas, such as the Chios Island in Greece where it is commercially exploited (Browicz 1987; Hagidimitriou and Zografos 2008). Recently, antioxidant properties and cosmetic usage (Barra et al. 2007) as well as anticarcinogenic properties (Balan et al. 2007; Magkouta et al. 2009) have also been proposed for this species. In Spain, P. lentiscus is used for reforestation, and traditionally, the fruit oil

6 Pistacia

has been used as fuel and the fruit as cattle feed (Werner et al. 2002). P. chinensis is widely used as an ornamental plant, and some advances have been performed in China in order to produce biofuel from the high oil-containing seeds (Hong-Lin et al. 2007). P. terebinthus has been mainly used traditionally as a source of turpentine (Assimopoulou and Papageordiou 2007), whereas P. integerrima is also used in traditional medicine, and both leaves and galls could have potential for control of hyperuricemia (Ahmad et al. 2008). The fruits of P. palaestina are used as food in the Middle East and its resins have also been used in local medicine (Assimopoulou and Papageordiou 2007). Similarly, the kernels of P. khinjuk are eaten either roasted or raw by local people in Afghanistan (Said 2008). However, most of the alternative local uses of the different parts of several Pistacia species are becoming less popular and restricted to remote rural areas and, consequently, they are endangered traditions.

6.6 Recommendations for Future Actions Significant advances have been obtained using classical breeding and selection approaches in P. vera. However, although the species of the genus Pistacia are considered of great importance across the central and West Asia and the Mediterranean region, with the exception of P. vera, the genus is still neglected by scientific research even if most of the wild species are ecologically interesting forest species adapted to poor soil and severe drought conditions. Consequently, an important role for these species can be envisaged for the recuperation of marginal and degraded land, especially under the forecast of increasing temperatures and irregular precipitation due to global climatic change, as well as for ornamental purposes. The use of these species could also represent a source of additional income for local farmers, especially if additional uses, such as for biofuel, medicine, or cosmetics, are promoted. However, the valuable genetic resources of the different species of the genus are suffering a severe erosion risk that can significantly narrow their genetic diversity (Padulosi and Hadj-Hassan 2001). A clear example is pistachio (P. vera), where the planting of a few selected varieties and the

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destruction of the natural forests are causing a major diversity loss. Although significant efforts have taken place to study and preserve ex situ the genetic resources of this species (Padulosi and Hadj-Hassan 2001), there is still much work to do especially in developing strategies for in situ conservation and additional in vitro conservation methods (see Ozden-Tokatli et al. 2010 for review) or in the use of molecular tools to efficiently use the genetic diversity present in species of the genus. Examples of the use of the genes from the crop wild relatives to improve crop performance are abundant (see Hajjar and Hodgkin 2007 for a review), and the still unexploited genetic variation among Pistacia species could be added to this list.

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J.I. Hormaza and A. W€ unsch Vargas FJ, Romero M, Rovira M, Batlle I (1993) Pistachio cultivar improvement at IRTA-Mas Bove´. IX GREMPA Meeting-Pistachio, Agrigento, Italy Vargas FJ, Romero MA, Vargas I (2002) Flowering precocity in pistachio progenies. Acta Hortic 591:297–303 Viruel MA, Escribano P, Barbieri M, Ferri M, Hormaza JI (2005) Fingerprinting, embryo type and geographic differentiation in mango (Mangifera indica L., Anacardiaceae) with microsatellites. Mol Breed 15:383–393 Vogt R, Aparicio A (1999) Chromosome numbers of plants collected during Iter Mediterraneum IV in Cyprus. Bocconea 11:117–169 Wannan BS, Quinn CJ (1991) Floral structure and evolution in the Anacardiaceae. Bot J Linn Soc 107:349–385 Werner O, Sanchez-Gomez P, Guerra J, Martinez JF (2001) Identification of Pistacia saportae Burnat (Anacardiaceae) by RAPD analysis and morphological characters. Sci Hortic 91:179–186 Werner O, Sanchez-Gomez P, Carrion-Vilches MA, Guerra J (2002) Evaluation of genetic diversity in Pistacia lentiscus L. (Anacardiaceae) from the southern Iberian Peninsula and north Africa using RAPD assay. Implications for reforestation policy. Isr J Plant Sci 50:11–18 Whitehouse WE (1957) The pistachio nut – a new crop for the western United States. Econ Bot 11:281–321 Yakubov B, Barazani O, Golan-Goldhirsh A (2005) Combination of SCAR primers and touchdown-PCR for sex identification in Pistacia vera L. Sci Hortic 103:473–478 Yi T, Wen J, Golan-Goldhirsh A, Parfitt DE (2008) Phylogenetics and reticulate evolution in Pistacia (Anacardiaceae). Am J Bot 95:241–251 Zohary M (1952) A monographical study of the genus Pistacia. Palest J Bot 5:187–228 Zohary M (1972) Pistacia L. Flora Palest 2:297–300 Zohary D, Hopf M (1988) Domestication of plants in the old world. Clarendon, Oxford, UK

Chapter 7

Prunus Daniel Potter

7.1 Description and Distribution Prunus L. (Table 7.1) comprises roughly 200 species, including all of the economically important crop species known as stone fruits – almonds, apricots, cherries, peaches, and plums – as well as many ornamental species and species cultivated or harvested from the wild for timber and medicinal purposes. Morphological descriptions are provided by Rehder (1940), Kalkman (1965), Wilken (1996), and Bortiri et al. (2006). Members of the genus are deciduous or evergreen trees or shrubs with alternate, simple leaves with toothed or entire margins and deciduous stipules. Nearly all species bear glands on the leaves, but the details of their morphology vary considerably among species. These are generally present in one to several pairs, but are occasionally solitary or absent, they may be found on the petiole or on the undersurface or the margin of the blade, usually near the base, they range from quite prominent to relatively inconspicuous, and they may be flat, hollow, or cushion-shaped (Kalkman 1965). The function of these glands has not been determined. The inflorescence in Prunus varies from a solitary flower to an umbel-like cluster or a raceme, which may or may not bear leaves on the peduncle. The radially symmetrical flowers have a well-developed hypanthium, whose shape varies from campanulate to tubular, with five sepals, five petals that vary in color from white to pink or red, 15 or more stamens, and a single simple pistil (composed of one carpel) with a superior

ovary. The fruit is a drupe. The base haploid chromosome number for Prunus is x ¼ 8 (Raven 1975). Like many other members of Rosaceae, species of Prunus produce significant amounts of both the sugar alcohol sorbitol, which serves as the primary transport carbohydrate in these plants (Zimmermann and Ziegler 1975; Moing et al 1997), and cyanogenic glycosides, which impart a characteristic acrid odor to crushed vegetative portions and toxicity to the seeds of many species (Wilken 1996). Members of the genus exhibit a range of breeding systems; gametophytic selfincompatibility has been documented for several species, and polyploidy and interspecific hybridization are both common. Prunus occurs in a variety of habitats, from forests to deserts, and across altitudinal ranges from sea level to alpine zones. The genus is most abundant in the temperate zone of the Northern Hemisphere and is widely distributed in North America, Europe, and northern Asia. This, combined with the fact that all of the cultivated species of global economic importance originated and are primarily grown in temperate regions, has led to the perception, even among many botanists, that Prunus is an exclusively north temperate genus. In fact, however, about 75 species have tropical and subtropical distributions, including about 45–50 species in South and Southeast Asia, about 25 in Central and South America, and one or two in subSaharan Africa (Kalkman 1965).

7.2 Classification and Phylogeny D. Potter (*) Department of Plant Sciences MS2, University of California, 1 Shields Avenue, Davis, CA 95616, USA e-mail: [email protected]

Prunus has been variously lumped and split by different taxonomists over the last several centuries (reviewed by Wen et al. 2008), and as many as seven

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_7, # Springer-Verlag Berlin Heidelberg 2011

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Table 7.1 Summary of Rehder’s (1940) classification of the genus Prunus L. and subsequent modifications thereof, with placements of representative cultivated and wild species, including all species discussed in this chapter, indicated Subgenus Prunus (treated as Prunophora Focke by Rehder): plums and apricots Section Prunus (treated as Euprunus Koehne by Rehder): Eurasian plums Representative species: P. cerasifera Ehrh., P. domestica L., P. insititia L., P. salicina Lindl., P. simonii Carr., P. spinosa L. Section Piloprunus Masona Representative species: P. texana Dietr. Section Prunocerasus Koehne: North American plums Representative species: P. alleghaniensis Porter, P. americana Marshall, P. geniculata R. M. Harper, P. maritima Marshall Section Armeniaca (Lam.) Koch: apricots Representative species: P. armeniaca L., P. mandshurica (Maxim.) Koehne, P. mume Siebold & Zucc. Section Penarmeniaca Masona Representative species: P. andersonii Gray Subgenus Amygdalus (L.) Focke: peaches and almonds Representative species: P. davidiana (Carr.) Franch., P. dulcis (Mill.) D. A. Webb., P. fenzliana Fritsch, Prunus ferganensis (Kost. & Rjab.) Y.Y.Yao, P. kansuensis Rehder, P. mira Koehne, P. persica (L.) Batsch, P. tenella Batsch, P.webbii (Spach) Vierh. Subgenus Cerasus Pers.: cherries Section Cerasus (treated as Eucerasus Koehne by Rehder) Representative species: P. avium L., P. cerasus L., P. fruticosa Pall. Section Microcerasus Webb. Representative species: P. glandulosa Thunb., P. tomentosa Thunb. Section Pseudocerasus Koehne: flowering cherries Representative species: P. canescens Bois., P. incisa Thunb., P. lannesiana E. H. Wilson, P. nipponica Matsum., P. serrulata Lindl., P. yedoensis Matsum. Section Lobopetalum Koehne Representative species: P. dielsiana Schneid. Section Phyllocerasus Koehne Representative species: P. pilosiuscula Koehne Section Mahaleb Focke Representative species: P. mahaleb L., P. pennsylvanica L. Section Phyllomahaleb Koehne Representative species: P. maximowiczii Rupr. Subgenus Emplectocladus (Torr.) Sargenta Representative species: P. fasciulata Gray Subgenus Padus (Moench) Koehne: bird-cherries Representative species: P. maackii Rupr., P. napaulensis (Ser.) Steud., P. padus L., P. serotina Ehrh., P. virginiana L. Subgenus Laurocerasus Koehne: laurel-cherries Section Laurocerasusb (continued)

Table 7.1 (continued) Representative species: P. africana (Hook. f.) Kalkm., P. laurocerasus L., P. lusitanica L Section Mesopygeum (Koehne) Kalkm.b Representative species: P. arborea (Bl.) Kalkm. Unnamed section – primarily tropical America, some North Americab Representative species: P. ilicifolia (Nutt.) Walp., P. integrifolia (C. Presl) Walp a

Following Mason (1913) Following Kalkman (1965)

b

different genera have been recognized in this group (e.g., Takhtajan 1997). Currently, however, the most widely accepted classification of Prunus is that of Rehder (1940), who adopted a broad interpretation of the genus and divided it into five subgenera that are further split into sections, most of which correspond to Old World and New World groups. Additional infrageneric taxa proposed by Mason (1913) and Kalkman (1965) have also been widely accepted (Table 7.1). Many of the tropical Old World species are sometimes classified under the genus Pygeum, but these were all transferred to Prunus subgenus Laurocerasus by Kalkman (1965; see Table 7.1). To date, infrageneric classifications have emphasized morphological characters such as presence or absence of a sulcus on the fruit, the number of axillary buds on twigs, and features of the inflorescence. Molecular phylogenetic analyses of the genus conducted over the last decade (e.g., Bortiri et al. 2001, 2006; Lee and Wen 2001; Wen et al. 2008), however, have revealed that many previously recognized subgenera and sections are not supported as monophyletic (Fig. 7.1) and many of the taxonomically important characters exhibit considerable homoplasy. In other words, traits considered diagnostic for particular groups have evolved more than once within the genus and some of them likely arose multiple times as adaptations to special habitats, such as the presence of dry fruits in species of arid regions (Bortiri et al. 2006). These findings suggest that a new infrageneric classification for Prunus is needed. The overall consensus is that there are several major clades within Prunus: one comprising species of Maddenia and subgenera Padus and Laurocerasus, another comprising the most members of subgenera Cerasus, and yet another comprising primarily members of subgenera Prunus and Amygdalus (Fig. 7.1).

Fig. 7.1 Schematic representation of current understanding of phylogenetic relationships within Prunus, based on several recently published studies (Lee and Wen 2001; Bortiri et al. 2001, 2006; Wen et al. 2008). Polytomies indicate cases in which analyses to date have not been able to resolve the branching order among lineages. Subgeneric names refer to those listed in Table 7.1

131 Other Rosaceae

7 Prunus Maddenia, some Laurocerasus some Laurocerasus (incl. some Pygeum), (incl. some Pygeum), some Padus some Padus

Many relationships within the genus remain poorly resolved to date, however, due to a combination of limited taxon sampling, especially for the tropical species, and the lack of strong support for some nodes (Fig. 7.1). The last phenomenon, in turn, results from a combination of lack of sufficient variation in sequence data, homoplasy within individual data sets, and conflict among data sets, especially nuclear ribosomal DNA internal transcribed spacer (ITS) vs. chloroplast DNA (cpDNA) regions, which suggest different placements for most members of subgenus Cerasus (Bortiri et al. 2001, 2006; Lee and Wen 2001; Wen et al. 2008). Some analyses have demonstrated support for particular infrageneric groups, including subgenera Amygdalus and Emplectocladus and section Prunocerasus (Shaw and Small 2005), but differences in taxon and sampling and relationships among the different studies conducted so far preclude definitive decisions about the status of these taxa. Because of its widespread distribution, Prunus provides an excellent system in which to examine historical biogeography of temperate and tropical regions of both the Old and New Worlds. Analyses to date (e.g., Bortiri et al. 2006; Wen et al. 2008) indicate multiple New World – Old World disjunctions within the genus, but, again, more thorough sampling and better resolved phylogenies are needed to provide a full understanding of these patterns.

Most Cerasus

Prunus, Amygdalus, Emplectocladus, sect. Microcerasus

The position of Prunus within Rosaceae has varied among taxonomic treatments over the last 50 years (reviewed in Potter et al. 2007). In what was until recently the most widely used classification of the family, Schulze-Menz (1964) recognized four subfamilies; Prunus sensu lato was treated as the largest genus in subfamily Amygdaloideae, which also included the genera Maddenia Hook. f. Thomson, with 4–5 Asian species, Prinsepia Royle, with 3–4 Asian species, and the monotypic Oemleria Reichenb, from western North America, the members of all of which produce drupes. All but the last of these were also included in tribe Pruneae by Hutchinson (1964), who did not recognize subfamilies within Rosaceae. Takhtajan (1997) recognized 12 subfamilies in Rosaceae; his Amygdaloideae included the aforementioned genera plus Exochorda Lindley, with 1–5 Asian species that produce capsules, but, like the other genera mentioned, have a base chromosome number of x ¼ 8 (Raven 1975). Recent phylogenetic studies at both the generic (see Fig. 7.1) and familial (see Fig. 7.2) levels have resulted in modifications of these schemes, however. The combination of a unicarpellate gynoecium that develops into a drupe and the base chromosome number of x ¼ 8 are synapomorphies for Prunus (Bortiri et al. 2006). Both Maddenia and Pygeum are nested within Prunus and, while Exochorda, Oemleria, and Prinsepia

132 Rosaceae

form a clade, it is not the sister clade to Prunus sensu lato. As a result, in the latest infrafamilial classification for Rosaceae (Potter et al. 2007; Table 7.2), based on phylogenetic analyses of sequences from multiple chloroplast and nuclear genes and incorporating nonmolecular characters, Prunus (including Maddenia) was placed by itself in tribe Amygdaleae, while the other three aforementioned genera were classified in tribe Osmaronieae; both of these tribes were, in turn, classified within an expanded subfamily Spiraeoideae. In summary, while recent phylogenetic analyses support Rehder’s (1940) broad circumscription of Prunus – indeed, they favor an even broader concept that includes Maddenia – they have not supported monophyly of all of the currently recognized infrageneric taxa. Because a robust and thoroughly sampled phylogeny for the genus is not yet available, however, it is premature to propose a new, phylogenetically based, infrageneric classification for Prunus. Such a phylogeny will also be required to gain a complete understanding of patterns of historical biogeography and character evolution across the genus (Bortiri et al. 2006). The thorough and careful morphological studies of past workers and the resulting classifications (e.g., Mason 1913; Rehder 1940; Kalkman 1965) provide an excellent framework and a solid foundation for future classifications, in which modifications can be made to recognize only groups strongly supported as monophyletic.

Pyrus Malus

Gillenia

Sorbaria

Spiraea

Exochorda

Kerria

Prunus

Neillia

Spiraeoideae Lyonothamnus

Rosa Fragaria

Rubus

Rosoideae

Dryadoideae

Dryas

Other Rosales (Moraceae, Rhamnaceae, etc.)

Fig. 7.2 Schematic representation of current understanding of phylogenetic relationships in Rosaceae, based on results presented by Potter et al. (2007) with the circumscriptions of the three subfamilies included in their infrafamilial classification indicated. Polytomies indicate cases in which analyses to date have not been able to resolve the branching order among lineages

D. Potter

7.3 Diversity of Wild and Cultivated Species of Prunus Not surprisingly, given the large size and wide distribution of the genus and the fact that many of the species exhibit one or more features of potential value to people (i.e., high quality timber, beautiful flowers, and/or edible fruit), Prunus includes species with varying degrees of economic importance, from exclusively wild species that are not used by people through wild species that are sometimes cultivated and are currently, or were historically, locally important as sources of food, timber, or medicine, to true domesticates that are major crop plants. In addition to their uses for food and timber and as ornamentals, medicinal uses are reported for a number of Prunus species. The major cultivated species of Prunus are almond (P. dulcis), peach (P. persica), sweet cherry (P. avium), sour cherry (P. cerasus), European plum (P. domestica), Japanese plum (P. salicina), and apricot (P. armeniaca). Most ornamental flowering cherries belong to section Pseudocerasus. Together, these species represent a broad cross-section of the phylogenetic diversity of Prunus (Table 7.1; Fig. 7.1). Each of the major domesticated species of Prunus shares its basic common name with a number of wild and minor cultivated species – e.g., “desert almond” (P. fasciculata); “desert peach” (P. andersonii), “Manchurian apricot”

7 Prunus Table 7.2 Summary of infrafamilial classification of Rosaceae by Potter et al. (2007) Subfamily Rosoideae No tribal placement: Filipendula Adans., Rubus L., Rosa L. Tribe Colurieae Representative genera: Fallugia Endl., Geum L. Tribe Potentilleae Representative genera: Fragaria L., Potentilla L. Tribe Sanguisorbeae Representative genera: Agrimonia L., Sanguisorba L. Subfamily Dryadoideae Representative genera: Cercocarpus H. B. & K., Dryas L., Purshia DC. Subfamily Spiraeoideae No tribal placement: Gillenia Moehcn., Lyonothamnus A. Gray Tribe Amygdaleae Representative genus: Prunus L. Tribe Kerrieae Representative genera: Kerria DC., Rhodotypos Sieb. & Zucc. Tribe Osmaronieae Representative genera: Exochorda Lindl., Oemleria Reichenb. Tribe Neillieae Representative genera: Neillia D. Don, Physocarpus Maxim. Tribe Pyreae Representative genera: Lindleya H. B. & K., Malus Mill., Pyrus L. Tribe Sorbarieae Representative genera: Adenostoma Hook. & Arn., Sorbaria A. Braun Tribe Spiraeeae Representative genera: Aruncus Adans., Spiraea L.

(P. mandshurica), “ground cherry” (P. fruticosa), “black cherry” (P. serotina), “beach plum” (P. maritima) – which may or may not be closely related to the major crop (Table 7.1). Although they may be very clear in the case of some major domesticates, the distinctions among wild, cultivated, and domesticated taxa are often at least somewhat ambiguous, and Prunus exhibits several features that make it especially challenging to draw these distinctions. First, in woody perennials with long generation times, the effects of human selection are not always as dramatic and obvious as they are in many annual crop plants. Second, as noted above, due to the large number of species in the genus, many of which share basic features that are of interest to humans, and its widespread distribution, there exists a

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continuum of conditions from fully wild populations to fully domesticated forms, not just across the genus, but sometimes within a single species; the use of many species and hybrids as rootstocks also contributes to this phenomenon. Third, cross-compatibility among species, especially closely related ones, has allowed interspecific hybridization to play an important role in breeding efforts, such that some cultivars include genetic contributions from more than one naturally occurring species. Pandey et al. (2008) surveyed the wild and cultivated species of Prunus available in India, where considerable genetic diversity of the genus is found in the Himalayan region and, to a lesser extent, at higher elevations farther south (peninsular India). They documented the presence of 29 species used for food, 12 used as rootstocks, and 14 used medicinally; they also mentioned the uses of several species as ornamentals. These lists included a large number of native and introduced wild species as well as the major cultivated species of Prunus. They concluded that valuable genetic diversity was present in all of the following categories of material: cultivated species with high regional and local diversity (e.g., P. persica), native species that already exist in semi-domesticated forms in some areas (e.g., P. napaulensis), and native wild species with potential for domestication and worthy of further investigation (e.g., P. tomentosa). In some cases, multiple stages of domestication may be observed within a single species. For example, cultivated sweet cherry is Prunus avium, a species that also occurs wild in Europe and North Africa and is highly valued as a timber tree (Vaughan et al. 2007). Browicz and Zohary (1996) explored the effects of domestication on species of Amygdalus, using morphologically based taxonomic studies. They noted, as many other authors have, the high frequency of naturally occurring hybrids within the group and pointed out the potential value this has for transferring valuable traits from wild to cultivated species via classical breeding methods. They further examined infraspecific variability in P. dulcis (which they treated as A. communis L.), which they designated a crop complex because it comprises multiple categories of materials, including truly wild populations in the eastern Mediterranean region, domesticated forms distinguished by sweet, non-poisonous seeds, and fruits that are larger and have thinner endocarps than wild forms and related wild species, and escapes from

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cultivation from the Mediterranean into Southwest and central Asia. As discussed above, there have been several recent molecular phylogenetic studies aimed at resolving relationships across Prunus (e.g., Aradhya et al. 2004; Bortiri et al. 2006; Wen et al. 2008). These studies have, in general, confirmed ideas based on morphology and crossing studies about the wild relatives of the major cultivated species. Lack of thorough sampling, on the one hand, and the lack of phylogenetic resolution, on the other, have, however, precluded definitive tests of hypotheses about specific wild progenitors to individual domesticated species. Thus, because the aforementioned studies sampled only one accession each of five or fewer species other than P. dulcis and P. persica, and none of them included P. fenzliana, it was not possible to test Ladizinsky’s (1999) hypothesis that P. fenzliana is the most likely wild ancestor to cultivated almond. However, a recent study based on nuclear and chloroplast simple sequence repeat (SSR) markers (Zeinalabedini et al. 2009), which focused on subgenus Amygdalus, did point towards a close relationship between P. fenzliana, represented by four accessions, and P. dulcis, represented by 39. Zohary (1992) hypothesized, based on cytogenetic and morphological evidence, that the hexaploid P. domestica is an autopolyploid derived solely from P. cerasifera, which exhibits several ploidy levels, rather than an allopolyploid derived from hybridization between diploid P. cerasifera and tetraploid P. spinosa (Weinberger 1975). Subsequently, however, restriction site analyses of ribosomal RNA genes suggested that P. spinosa itself is a hybrid, of which P. cerasifera is one parent (Reynders-Aloisi and Grellet 1994; Okie and Hancock 2008). Phylogenetic analyses of nuclear and cpDNA sequence data indicate a very close relationship among all three species, also supporting the allopolyploid origin hypothesis (Bortiri et al. 2001). Interspecific hybrids, including a variety of simple and complex hybrids between wild and cultivated Prunus species, have also been important as rootstocks for the various Prunus fruit crops (Bouhadida et al. 2007). Bouhadida et al. (2007) used a polymerase chain reaction – restriction fragment length polymorphism (PCRRFLP) approach with several regions of cpDNA to confirm the identity of the maternal parents of many of these hybrids.

D. Potter

7.4 Use of Wild Species in Crop Improvement Efforts There is considerable variation in the degree to which wild species have been important in the history of breeding of particular crop species within Prunus. Wild species have probably been most important in the history of development of plums (Okie and Hancock 2008). Okie and Hancock (2008) described Luther Burbank’s use of the Chinese species P. simonii and several native North American plum species (section Prunocerasus) to develop new cultivars of diploid Japanese plum (P. salicina) for the California plum industry and subsequent use of local species native to the northeastern and southeastern US, respectively, to develop varieties adapted to growth in those areas, though they note that most of those are no longer available due to the demise of industries outside of California. Okie and Hancock (2008) also discuss the limited use of diploid P. cerasifera, a progenitor of hexaploid European plum (P. domestica), in genetic improvement of the latter. At the other extreme, nearly all modern peach cultivars were derived from a small number of P. persica cultivars, all of which trace their parentage back to “Chinese Cling”, introduced to the United States from China via England in 1850 (Hancock et al. 2008). This narrow genetic base has stimulated interest in modern use of wild species in peach breeding (Foulongne et al. 2003a). In the case of cherries (subgenus Cerasus), there are two major species that are cultivated for fruit: diploid sweet cherry, P. avium, and tetraploid sour cherry, P. cerasus L. (Iezzoni 2008). A third closely related species, the tetraploid ground cherry P. fruticosa Pall., hybridizes freely with P. cerasus, contributing to genetic and morphological diversity, as well as reduced fertility, in sour cherry (Iezzoni 2008). Dirlewanger et al. (2004) discussed the potential value of several wild species closely related to P. persica – namely P. davidiana, P. kansuensis, and P. mira – as possible sources of resistance to several important pests and diseases of peach. They further pointed out that, although there is great potential for transfer of traits among the many intercompatible species within Prunus, realization of that potential has been limited, due primarily to the slowness of traditional breeding methods, but that recently developed

7 Prunus

genomic methods may allow greater use of the genetic variability available in the genus and already conserved in the many germplasm collections for Prunus. At the same time, Gradziel (2003) has demonstrated that interspecific hybridization and backcrossing can provide an effective means of introgressing desirable genes from wild species into both peach and almond cultivars. Wild species have been important in a few cases as sources of disease resistance for Prunus crop species, e.g., resistance to plum pox virus in P. armeniaca derived from P. mandshurica (Ledbetter 2008), and resistance to cherry leaf spot in P. cerasus derived from P. maackii and P. canescens (Iezzoni 2008). Hybrids resulting from a cross between P. persica and P. davidiana were used to select for resistance to powdery mildew, peach green aphid, and plum pox virus (Kervella et al. 1998). As a contrast to their usefulness as sources of disease and pest resistance, wild species may also serve as hosts for diseases of cultivated species and thus contribute to the continued presence of diseases in areas where crops are grown. Carraro et al. (2002) reported that several wild species of Prunus – P. spinosa, P. cerasifera, and P. domestica – serve as hosts for the phytoplasma that causes European stone fruit yellows (EFSY), a disease of many cultivated Prunus species, and the psyllid vector (Cacopsylla pruni) that transmits the disease. Similarly, P. virginiana serves as an alternate host for X-disease, a phytoplasma disease of cherries transmitted by leafhoppers (Iezzoni 2008). Damsteegt et al. (2007) showed that 40 species and varieties of Prunus, including many wild species native to the US, were susceptible to infection by plum pox virus, which causes Shakra disease, considered the most important viral disease of stone fruits in Europe, and maintained infections through repeated cycles of cold-induced dormancy over 4 years. The results suggested that many native and introduced species may serve as reservoirs for this serious disease, presenting a significant challenge to eradication efforts. The efficiency of transferring desirable traits can be greatly enhanced by modern genetic and genomic methods, including comparative mapping and marker-assisted selection. Hybrids with wild species have been important for genetic mapping studies in Prunus, including mapping studies of cherry (“Emperor Francis” P. nipponica and P. incisa; also P. avium cv. “Napoleon” and P. nipponica;

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Iezzoni 2008), peach (P. persica P. ferganensis), and peach and almond (P. cerasifera (P. dulcis P. persica); Dirlewanger et al. 2003). Dirlewanger et al. (2004) reviewed the status of Prunus genetic maps as of 2004. At that time, the Prunus reference map, constructed in a peach–almond hybrid, included 562 markers covering 519 cM. Using anchor markers from the reference map, 13 additional maps were constructed for other species of Prunus, including two additional cultivated species (P. armeniaca and P. avium) and three wild species (P. cerasifera, P. davidiana, and P. ferganensis). The availability of these maps has allowed mapping of 28 major genes affecting horticulturally important characters in the different species, including genes involved in the determination of fruit quality, phenological traits, and pest and disease resistance traits. Comparison of these maps (Dirlewanger et al. 2004) revealed essential colinearity among these diploid Prunus species, all of which are members of one of the three subgenera Amygdalus, Cerasus, or Prunus (Table 7.1). The fact that the degree of synteny observed between the Prunus genome and both component genomes of Malus (apple), a member of the polyploid tribe Pyreae, was quite high, albeit lower than within Prunus (Dirlewanger et al. 2004), suggests that, not surprisingly, gene order and overall chromosomal structure has been conserved within subfamily Spiraeoideae (Table 7.2), with the degree of rearrangement correlated to phylogenetic distance (Fig. 7.2). A general problem with the use of wild species as sources of desirable traits in breeding programs is the concomitant introgression of unfavorable traits (Quilot et al. 2004). Thus, in Prunus, as in other fruit crops, transfer of disease resistance genes from wild relatives may result in decreased fruit quality. Foulongne et al. (2003a) demonstrated the potential value of the Chinese species Prunus davidiana as a source of genes that could be introgressed into the peach genome using comparative genetic mapping of RFLP, SSR, and amplified fragment length polymorphism (AFLP) markers in F1, F2, and BC2 generations resulting from a cross between the two species. Subsequently, Foulongne et al. (2003b) found quantitative trait loci (QTL) for resistance to powdery mildew in hybrid and backcross generations derived from a cross between the commercial peach variety Summergrand and a member of the closely related wild species P. davidiana. For nine of the 13

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QTLs detected, the favorable allele was derived from the wild species. Quilot et al. (2004) reported the results of QTL analyses of fruit quality in P. persica (peach) based on an advanced backcross population derived from a cross between the commercial peach variety Summergrand and a member of the closely related wild species P. davidiana. They found QTLs for 24 physical and biochemical fruit quality traits and identified some horticulturally desirable alleles in the wild species. They identified three primary genomic regions where QTLs with negative effects are located. They proposed that future breeding efforts using P. davidiana should focus on suppressing those chromosomal regions and on finemapping of regions in which QTLs with beneficial resistance and negative fruit quality effects are colocated.

7.5 Population and Evolutionary Genetic Studies of Wild Species While wild species have been valuable in the improvement of cultivated species, on one hand, the existence of genetic tools for characterizing cultivated species has facilitated evolutionary and population genetic studies of wild species, on the other. Cross-species transportability of molecular markers, such as SSR primers, within Prunus, including both cultivated and wild species, has been reported by multiple workers. These include Vendramin et al.’s (2007) report of 21 expressed sequence tag SSRs (EST-SSRs) isolated from the peach fruit transcriptome that successfully amplified PCR products in six other Prunus species, five cultivated (P. dulcis, P. armeniaca, P. avium, P. salicina, P. domestica) and one wild (P. ferganensis), Rohrer et al.’s (2004) use of SSR markers from 15 primer pairs originally developed in P. persica and P. avium to examine phylogenetic relationships among 13 known wild species and several undetermined wild accessions of North American plums (subgenus Prunus, section Prunocerasus), and Pairon et al.’s (2008) use of microsatellite markers originally developed for various cultivated species to identify genome-specific markers for the allotetraploid wild species P. serotina. Wild species of Prunus have been the subject of numerous studies aimed at understanding the evolutionary histories and dynamics of populations. Jordano

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and Godoy (2000) used random amplified polymorphic DNA (RAPD) markers to study population genetic structure in Prunus mahaleb among seven populations across an area of about 100 km2 in Parque Natural de las Sierras de Cazorla in southeastern Spain. They found evidence both for extensive gene flow among populations and for a degree of isolation by distance, which they attributed to the combined effects of efficient long-distance dispersal by frugivorous birds and mammals and local fragmentation resulting from vicariant factors including demographic bottlenecks due to high post-dispersal seed and seedling mortality. Mohanty et al. (2002) examined cpDNA diversity, using a PCR-RFLP approach, among 25 wild populations of P. spinosa from forests across Europe. They found 32 haplotypes, of which 10 were shared by multiple populations and 22 were private. Overall, no clear phylogeographic structure was detected, but higher haplotype diversity in southern than northern Europe was attributed to glacial refugia in the more southerly locations. Roh et al. (2007) used inter-SSR (ISSR) markers and sequences of two cpDNA regions to clarify the distinction between wild Korean plants referred to as P. yedoensis and cultivated hybrid ornamental “Yoshino” cherries from Japan, referred to as P. yedoensis. They concluded that the two are sufficiently distinct that they should be treated as separate taxa. Several studies have focused on wild populations of P. avium in Europe. Frascaria et al. (1993) examined isozyme variation among four populations of the species in France. They found no significant genetic structure within the populations and no significant differentiation among them. They attributed these results to the effects of human dispersal, perhaps combined with the limited time since the last glaciation in the areas studied. Mohanty et al. (2001) surveyed variation PCRRFLP patterns of cpDNA among 23 wild populations of Prunus avium from across Europe and found a total of 16 haplotypes, six of which were shared by two or more populations and ten of which were unique. They found no genetic structure among wild populations, which they attributed to long-distance gene flow among populations mediated by birds, mammals, and humans. Subsequently, Panda et al. (2003) expanded upon this study by surveying a total of 96 cultivars. In their study, they found 16 haplotypes among wild

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populations and only three among cultivars, which represented the most common three in the wild populations, indicating higher cpDNA diversity in wild populations than in the cultivars, and providing information useful for developing germplasm conservation strategies for the species. Schueler et al. (2003), using seven microsatellite markers originally developed in peach (Prunus persica), examined genetic structure in a natural population of wild P. avium in Germany, and found sufficient variability in the markers to allow identification of individual trees. They also demonstrated that genotyping of endocarps with their markers could be used to identify the mother tree of dispersed seeds. Vaughan and Russell (2004) developed primers for 14 microsatellite loci in cultivated P. avium. Genetic mapping studies of their seven most polymorphic loci with four from a previous study (Clarke and Tobutt 2003) revealed that the 11 loci are genetically unlinked, providing powerful tools for use in studies of population structure of wild forms of the species. Subsequently, Vaughan et al. (2007) used 13 of these loci to examine patterns of spatial-genetic structure in two wild populations of P. avium, one managed and one unmanaged, in Britain. They found evidence of significant clonal reproduction and restricted gene dispersal via both pollen and seed, leading to two recommendations that should help maintain genetic diversity of the species: selective removal of mature trees from particular areas and establishment of minimum distances (they suggested 100 m) between trees to be used as sources of seeds for propagation.

7.6 Evolutionary Studies of SelfIncompatibility Genes Prunus is one of the several genera in Rosaceae that exhibits gametophytic self-incompatibility (GSI), in which specificity of self-pollen rejection is determined by a stylar component known to be an S-RNase (Ushijima et al. 1998) and its genetically linked pollen component known to be an F-box protein, which, in Prunus, has been named SFB (Ushijima et al. 2003). Because of its importance in breeding and production of fruit crops, considerable attention has been directed to understanding in detail the mechanism and genetics of GSI. Numerous S-RNase/pollen–SF-box protein gene

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pairs have been identified and sequenced from species of Prunus, including both cultivated (P. armeniaca, P. avium, P. cerasifera, P. cerasus, P. domestica, P. dulcis, P. mume, P. salicina) and wild (P. lannesiana var. speciosa Makino, P. spinosa, P tenella, P. webbii) taxa. As a result of these efforts, Prunus has become a model system in which to examine the evolution of self-incompatibility at the molecular level. Sˇurbanovski et al. (2007) examined sequences of SFB and S-RNase alleles in wild Prunus tenella, native to the Balkan Peninsula. They found evidence for positive selection on the sequences of S-RNase alleles of this species, in contrast to results obtained for P. lannesiana (Kato and Mukai 2004), P. dulcis, and P. avium (Ma and Oliveira 2002). In addition, they found that the amino acid sequence of the S-RNase encoded by one of the alleles from P. tenella was identical to one from P. avium, but that the corresponding SFB alleles showed many differences between the two species. They discussed their results in terms of the models for evolution of GSI specificities in Prunus. Specifically, their results show that the same pistil determinant (S-RNase) can tolerate variability in the pollen determinant (SFB), suggesting that the evolution of new GSI specificities is initiated by mutations in the pollen-determinant genes. Vieira et al. (2008) used phylogenetic analyses coupled with models of sequence evolution and estimates of the age of Prunus based on calibrated molecular phylogenies (Wikstron et al. 2001) to develop hypotheses about the evolution of GSI in Prunus. Their results suggested that extant Prunus harbor only about a third of the GSI specificities that would have been present in their common ancestor, suggesting one or more evolutionary bottlenecks during the evolution of the genus, perhaps resulting from processes associated with speciation and/or domestication. Several models have been proposed to explain the generation of new alleles and, correspondingly, new specificities, in a two-gene system of self-incompatibility. These include models that require selfcompatible intermediates (Uyenoyama et al. 2001), dual-specificity intermediates (Matton et al. 1999), and gradual accumulation of mutations while maintaining self-incompatibility (Chookajorn et al. 2004). Implicit in all of these models is the tight linkage and coevolution between the two loci involved, such that mutations in one must be followed by compensatory mutations in the other in order to restore or maintain

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self-incompatibility. Intriguingly, none of these models has been completely supported by empirical data from Prunus. In particular, lack of correspondence between the phylogenies for the pistil and pollen determinants has suggested a role for recombination in the evolution of new specificities (Nunes et al. 2006; Tsukamoto et al. 2008). The phylogenies presented in Fig. 7.3 illustrate several striking and related features that have been noted in recent studies of the evolution of S-RNase and SFB genes in Prunus (Nunes et al. 2006; Suther-

S-RNase

land et al. 2008; Tsukamoto et al. 2008; Vieira et al. 2008). First, the two phylogenies have some similar characteristics: homoplasy is high (consistency indices are low) in both data sets, support for the relationships among lineages of both genes, especially the deeper internal branches, is generally weak, and many of those branches are quite short, suggesting that early diversification of these genes may have occurred rapidly in ancestral species, with subsequent lineage sorting and/or recombination giving rise to the extant alleles, as suggested by Tsukamoto et al. (2008).

PaS1 100 99 PdS11 PtS8 56 PaS13 62 PspS12 PdSb PaS5 63 PmS1 PweS1 PspS10 PsSe ParS4 PsSh 71 PaS2 PspS12 ParS1 59 ParS1 66 PdSd PaS2 100 PsSg PaS4 100 PcsfS10 PdoS9 76 PaS5 100 PsSd 64 PweS1 PcsfS9 PaS7 52 PspS7 1 100 PsSa 100 ParS17 100 PdoS6 PspS3 1 99 PspS7 1 86 PspS3 2 100 PaS4 PsS7 PdoS9 PaS6 100 PsSd PaS7 PcsfS9 100 100 PaS12 PsSf PdSk 77 PaS6 PdS12 76 PspS8 79 PcsfS3 PspS9 74 PaS13 PaS12 PdSd PdSk 97 100 100 96 PcS26 PdS12 PmS7 PcsfS3 PsSf PcS26 ParS4 PmS7 PspS8 PaS3 PsSc 100 PcS34 PspS9 PdSb PdoS5 100 PsSa PsSb ParS17 100 PdoS6 PspS3 1 PdSc 51 PaS3 100 PspS3 2 94 PcS33 PsS7 53 100 PmS1 PcS33 ParS2 PspS10 ParS2 PsSe 100 PsSg PsSh PdSc PcsfS10 PsSc PsSb PcS35 PdSa 50 changes PaS1 100 71 PdS11 98 PtS8 100 PcS34 PdoS5

SFB

*

*

*

100

PcS35 PdSa 10 changes

Fig. 7.3 Comparison of relationships among S-RNase and SFB alleles from wild and cultivated species of Prunus. Left: relationships among S-RNase alleles. Single most parsimonious tree (2002 steps, ci excluding autapomorphies ¼ 0.3860, ri ¼ 0.5065); based on alignment of 50 published sequences with 747 characters, of which 239 were constant, 129 variable but uninformative, and 379 parsimony-informative. Right: relationships among SFB alleles. One of five most parsimonious trees (2,816 steps, ci excluding autapomorphies ¼ 0.4047, ri ¼ 0.4886); based on alignment of 50 published sequences with 1,158 characters, of which 343 were constant, 227 vari-

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able but uninformative, and 588 parsimony-informative. Numbers on branches represent bootstrap support values. In the tree at right, nodes marked with an asterisk were not present in the strict consensus tree for the five most parsimonious trees. Analyses were conducted as in Tsukamoto et al. (2008), where Genbank accession numbers are listed for all sequences except Pwe S1 RNase (DQ993660) and Pwe S1 SFB (DQ993667). Pa P. avium; Par P. armeniaca; Pc P. cerasus; P csf P. cerasifera; Pd P. dulcis; Pdo P. domestica; Pm P. mume; Ps P. salicina; Psp P. spinosa; Pt P. tenella; Pwe P. webbii

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Second, both genes show a pattern known as transspecific evolution (Richman et al. 1996), in which alleles from individual species do not form monophyletic groups; i.e., the closest relatives of many alleles are alleles from other species. This pattern may reflect the role of balancing selection in the evolution of selfincompatibility specificities (Richman and Kohn 2000), resulting in retention of alleles through evolution over long periods of time and multiple speciation events, although it has been shown that in Prunus, in contrast to Solanaceae, the pattern of trans-specific evolution may not be interpretable as evidence for great age of specificities (Vieira et al. 2008). A related phenomenon is that neither gene’s phylogeny is congruent with species phylogenies in Prunus (e.g., Fig. 7.1), and this lack of congruence has been shown to be significant at all taxonomic levels within the genus (Tsukamoto et al. 2008), while at least for the S-RNase locus, alleles of Malus and Pyrus (both members of tribe Pyreae) are phylogenetically distinct from those of Prunus (Igic and Kohn 2001). This pattern, like that of trans-specific evolution discussed above, results from incomplete lineage sorting (Lu 2001) and indicates that, for the members of Rosaceae sampled to date, coalescence of alleles has not occurred below the level of the genus at either locus. The third notable pattern is that the phylogenies of the two genes are incongruent with one another, which may reflect a role of intragenic recombination in the evolutionary histories of the two genes, which otherwise would be expected to show congruent patterns of relationship (Tsukamoto et al. 2008). Future studies incorporating S-RNase and SFB sequences from additional wild species of Prunus, especially members of subgenera Laurocerasus and Padus (Table 7.1; Fig. 7.1) and representatives of other tribes in Rosaceae (Table 7.2; Fig. 7.2), are required to gain a more thorough understanding of patterns and processes of evolution of self-incompatibility in the genus and the family.

7.7 Issues of Concern: Conservation As is to be expected for such a large, diverse, and widely distributed genus, Prunus species exhibit a range of conservation statuses, from widely distributed taxa that have become invasive following human dis-

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persal to new environments to those with very restricted distributions that are considered rare or threatened, including one that is now endangered due to overharvesting. Due to their considerable economic importance, many collections of cultivated and wild Prunus germplasm exist throughout the world. A search of the Biodiversity International’s Biodiversity Directory of Germplasm Collections (Biodiversity International 2009) on June 30, 2009, retrieved 3,982 accessions of Prunus classified as “wild species” at 40 institutions. When the type of germplasm was not restricted to accessions classified as wild, the numbers were 60,168 accessions and 168 institutions. Inspection of some of these records revealed that many wild taxa were not explicitly designated as such and so were not recovered by the first search. The largest single institution housing Prunus germplasm is the United States Department of Agriculture (USDA) National Clonal Germplasm Repository at Davis, CA; the Germplasm Resources Information Network (GRIN) database lists 108 taxa of Prunus for which accessions are preserved there. The GRIN database (USDA, ARS, National Genetic Resources Program 2009) lists four taxa of Prunus as rare and endangered. They are: P. africana (African cherry), widely distributed in sub-Saharan Africa, listed in CITES Appendix II and with one accession preserved in the US National Germplasm System, P. alleghaniensis (Allegheny plum), distributed in the eastern US (Rehder 1940), listed by the Center for Plant Conservation (CPC) and with four accessions preserved in the US National Germplasm System, P. geniculata (scrub plum), with a limited distribution in Florida, listed by the CPC and on the Endangered list of the US Fish and Wildlife Service and with one accession preserved in the US National Germplasm System, and P. maritima var. gravesii (Small) G. J. Anderson, with a very limited distribution in Connecticut, listed by the CPC and with no accessions preserved in the US National Germplasm System. In addition to the species listed above, concern has been raised about the conservation status of some other taxa, including wild species as well as local varieties of cultivated species, in certain regions. Vivero et al. (2001) described ecology and ethnobotany of six species of Prunus that occur wild in Andalusia, Spain, and proposed strategies to conserve germplasm of wild populations and local varieties for

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the three most economically important of those species, P. avium, P. mahaleb, and P. insititia. Among their recommendations were designation of an area in the Sierra Nevada for in situ germplasm conservation and raising awareness of the importance of these species and their conservation among forest workers and managers and the general public. P. lusitanica ssp. azorica was one of three taxa identified by Ferreira and Eriksson (2006) as a target for conservation in their proposed plan for conservation of forest tree genetic resources in the Azores. This species was selected due to its status as one of the most threatened in the archipelago. Perhaps of greatest concern purely from the point of view of biodiversity conservation are the tropical species of Prunus, which have received relatively little attention from researchers to date and are poorly represented in germplasm collections, many of which occur in areas where their habitats are threatened by anthropogenic factors such as logging, expansion of agriculture, and/or urbanization. In western New Guinea (Papua Province, Indonesia), rapid deforestation is threatening the habitats of several of the endemic species of Prunus (D. Potter, pers observ) and it is likely that the same situation exists for many of the paleoand neotropical species. A recently initiated taxonomic revision of Prunus for Colombia has so far revealed three new species (Pe´rez-Zabala 2007), all considered by the author to merit conservation concern, two as “endangered” and one as “near threatened” following IUCN criteria (International Union for Conservation of Nature and Natural Resources 2001). One of the most interesting cases of an endangered species of Prunus is P. africana, which is widely distributed in montane regions of sub-Saharan Africa, has been used traditionally by people throughout its range for multiple purposes (Stewart 2003), and, at least in some areas, is an important food source for wildlife, including some rare and endangered species of primates and birds (Fashing 2004). The discovery, in the late 1960s, that bark extracts from this species were effective in treating benign prostatic hyperplasia (Bombardelli and Morazzoni 1997) led to extensive international trade of the bark and herbal remedies prepared from it, which in turn resulted in overharvesting of wild trees (Cunningham and Mbenkum 1993), ultimately leading to the listing of the species in CITES Appendix II. Several recent studies (e.g., Dawson and Powell 1999)

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have examined the distribution of genetic diversity in this species throughout its range, resulting in recommendations for conservation strategies. In addition, several studies (e.g., Cunningham et al. 2002; Stewart 2003; Fashing 2004) have called attention to the need for establishing plantations of the species in order to reduce pressure on wild populations. P. africana presents an extremely challenging problem for conservation. The medicinal value of the species, the economic situations of local human inhabitants throughout much of its range, and the large gap between the price paid for raw bark and that paid for the final medicinal preparations tend to encourage unsustainable wild-harvesting by local people, even where this practice is in violation of local regulations (Stewart 2003). Synthesis of the therapeutically active components of the bark extracts has not been attempted and is likely to be complicated and expensive (Stewart 2003), due to the fact that synergistic interactions of multiple compounds are indicated in the effectiveness of the extracts (Bombardelli and Morazzoni 1997). Prospects for cultivation are poor in many areas due to limited availability of appropriate land (Stewart 2003). Studies of genetic diversity, based on RAPD (Dawson and Powell 1999; Muchugi et al. 2006) and SSR (Farwig et al. 2008) markers, have revealed significant variation within populations and have provided tools for identifying especially diverse populations that should be prioritized for conservation, but in situ conservation efforts may be undermined by a paradox pointed out by Fashing (2004): while P. africana appears to require disturbance for successful regeneration (Kiama and Kiyiapi 2001), disturbance can be detrimental to the species, either directly because of overharvesting that often occurs when disturbance causes or results from increased human access to an area, or indirectly due to reduced genetic diversity resulting from forest fragmentation caused by human activity (Farwig et al. 2008). Recent efforts to include P. africana as a model agroforestry species for “participatory domestication” (reviewed by Simons and Leakey 2004), in which local small-scale farmers are engaged in the process of identifying, cultivating, and improving valuable germplasm selected from wild trees, thereby alleviating pressure on wild populations, are encouraging and may represent the best hope for conservation of genetic diversity of this species.

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7.8 Issues of Concern: Invasive Species At the other end of the spectrum from rare and endangered species are those that are relatively common in their native ranges and have also become invasive in areas to which they are not native. Examples from Prunus include P. laurocerasus, native to southeastern Europe and Asia Minor, in western Europe (H€attenschwiller and Ko¨rner 2003) and in the Pacific Northwest of North America (Evergreen 2010) and North American P. serotina in Europe (e.g., Godefroid et al. 2005). P. cerasifera, native to southeastern Europe and naturalized in California, is included by the California Invasive Plant Council in its Invasive Plant Inventory but rated as “limited” because its ecological impacts are considered minor on a statewide level (California Invasive Plant Council 2009). Concerns have also been raised about northern European P. padus in Alaska (Alaska Natural Heritage Program 2006). H€attenschwiller and Ko¨rner (2003) studied the effects of elevated CO2 levels on growth rates of P. laurocerasus, whose abundance in the understoreys of Swiss forests, where the species is not native, had raised concern about its potential to become invasive. They found that P. laurocerasus seedlings grown in elevated CO2 concentrations for three growing seasons showed an average of 56% greater biomass than plants grown at ambient CO2 levels, while native species showed a range of responses to elevated CO2. They concluded that increases in atmospheric CO2 levels, an element of current and projected future global change, may facilitate naturalization and spread of this non-native species, thereby contributing to another component of global change, biotic invasions. P. serotina (black cherry), an allotetraploid species native to North America, was introduced to Europe, especially Germany, Belgium, and the Netherlands, for various purposes beginning several hundred years ago. Starfinger et al. (2003) provided a fascinating account of the history of the introduction of this species to Europe and how perceptions of it have changed over the centuries. First introduced as an ornamental in the seventeenth century, it was later widely planted as a forest timber tree beginning in the late eighteenth century; when its value as a timber tree was shown to be questionable, it began to be used for non-timber purposes, such as improving litter due to the low C/N ratio in leaves of the species.

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It had become naturalized in western Europe by the mid-twentieth century, and by the late twentieth century, it was widely considered an invasive forest pest (Starfinger et al. 2003; Pairon et al. 2006; ClossetKopp et al. 2007). Recent studies have been undertaken to investigate and characterize the factors that contribute to the invasiveness of this species in European forests; these studies have included investigations of the effects of landscape structure (Deckers et al. 2005), ecological variables (Godefroid et al. 2005; Verheyen et al. 2007), reproductive traits (Pairon et al. 2006), disturbance history (Chabrerie et al. 2008), and propagule pressure (Vanhellemont et al. 2009) on the ability of P. serotina to invade forests. Godefroid et al. (2005) investigated the ecological factors that affect the abundance of P. serotina in forests in Belgium. Species richness in the herb layer was negatively correlated with the abundance of P. serotina in the shrub layer. Slope and light intensity were the only abiotic factors measured that explained significant portions of the variation in P. serotina abundance. The light intensity results suggested that P. serotina’s response to light intensity changes as the tree matures: seedlings showed a positive response to 58–80% of full light and a negative response to lower light intensities, while saplings showed the reverse trend. In addition, further growth of saplings to maturity and seed production again requires high light intensities, but saplings can adopt a “sit-and-weight strategy”, forming a long-lived seedling bank until a canopy light gap occurs (ClossetKopp et al. 2007). Thus, the establishment and persistence of P. serotina depends on opening of light gaps in the canopy. Studying the same system, Pairon et al. (2006) investigated sexual regeneration traits of P. serotina growing in a Belgian pine plantation, in order to gain a better understanding of how those traits might affect the invasiveness of the species. They found that fruit production was high in spite of low fruit/flower ratio, because of the large number of flowers produced per tree. Seeds fell into two size classes: large (the majority) seeds, which are gravity-dispersed, and smaller, bird-dispersed seeds, resulting in thorough coverage of the area by seeds. While seed germination and seedling survival rates were low, the high seed density means that each year, the entire forest floor is covered with seedlings; the high survival rate of saplings helps

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ensure maintenance of the population. Thus, the ecological and reproductive characteristics of P. serotina seem to have pre-disposed it to be highly successful as an invader in European forests. Vanhellemont et al. (2009), noting that most studies of the invasiveness of P. serotina in western Europe, including those discussed above, had been conducted in areas where the species had been intentionally introduced, which were subject to considerable anthropogenic disturbance, and where propagule pressure was high, undertook a study to address the question of whether or not P. serotina acts as an aggressive invader in areas within its potential range that had not yet been heavily invaded. They focused on a forest reserve in central Belgium that met those criteria. They found that the spread of P. serotina in this reserve had slowed since the first establishment of the species there around 1970s and subsequent further spread in the 1980s, presumably from seedlings produced by the first arrivals. They assumed that the slowdown was due to lack of disturbance creating light gaps needed for seedling establishment. At the same time, they found no evidence that P. serotina was inhibiting the regeneration of native understorey species in this forest. They concluded that P. serotina could not be considered an aggressive invader in the study area, but they pointed out that future disturbance events opening up the canopy could result in accelerated spread and invasion of the species.

7.9 Summary and Conclusions Prunus is a large genus of tremendous economic and ecological importance worldwide. The group includes several major fruit crop species, a large number of minor cultigens and species collected from the wild for a range of uses, and many wild species that have been used as rootstocks for cultivated taxa and in their genetic improvement. The economically important species represent several phylogenetic lineages within the genus. Phylogenetic studies have confirmed some aspects of past taxonomic treatments and hypotheses about the origins and placement of particular crop species and challenged others. To date, poor sampling of some lineages, especially those including the approximately 75 species native to the New and Old World tropics, and weak resolution of some relationships across the genus, have precluded generation of a new phylogenetically

D. Potter

based classification. Nonetheless, ongoing efforts in multiple labs throughout the world, some focusing on relationships across the entire genus, others on particular species and their closest relatives, are leading to a thorough understanding of phylogeny of Prunus, which will allow robust investigations of the historical biogeography of the genus, the evolution of particular genes and traits, and the interplay of natural and human selection in shaping the extant variation in this group. Wild species of Prunus have been important in the histories of several cultivated species, and modern methods such as comparative genetic mapping and marker-assisted selection should help to facilitate the transfer of desirable traits and to minimize the concomitant transfer of undesirable traits, from wild to cultivated species. At the same time, there is considerable interest in wild species in their own right, and Prunus provides an excellent example of a system in which a complementary and synergistic relationship exists between studies of cultivated species and those of wild relatives. Tools developed for characterizing cultivated taxa have been tremendously useful in ecological and evolutionary genetic studies of wild species, while the results of the latter have provided valuable information for crop improvement efforts, and for understanding the economically significant issues associated with the spread of invasive species and the conservation of rare and potentially valuable taxa. Future efforts in all of the aforementioned areas should continue. Particular attention should be paid to the tropical species, which have received relatively little attention from researchers to date and which may be among the most threatened in terms of conservation status.

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7 Prunus reproductively isolated species of Prunus have different SFB alleles coupled with an identical S-RNase allele. Plant J 50:723–734 Sutherland BG, Tobutt KR, Robbins TR (2008) Trans-specific S-RNase and SFB alleles in Prunus self-incompatibility haplotypes. Mol Genet Genom 279:95–106 Takhtajan A (1997) Diversity and classification of flowering plants. Columbia University Press, New York Tsukamoto T, Potter D, Tao R, Vieira CP, Vieira J, Iezzoni AF (2008) Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.). J Exp Bot 59:3169–3185 USDA, ARS, National Genetic Resources Program (2009) Germplasm Resources Information Network – (GRIN). National Germplasm Resources Laboratory, Beltsville, Maryland. http://www.ars-grin.gov/cgi-bin/npgs/html/ taxgenform.pl. Accessed 30 June 2009 Ushijima K, Sassa H, Tao R, Yamane H, Dandekar AM, Gradziel TM, Hirano H (1998) Cloning and characterization of cDNAs encoding S-RNases in almond (Prunus dulcis): primary structure features and sequence diversity of the S-RNases in Rosaceae. Mol Gen Genet 260:261–268 Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H (2003) Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism. Plant Cell 15:771–781 Uyenoyama MK, Zhang Y, Newbigin E (2001) On the origin of self-incompatibility haplotypes: transition through selfcompatible intermediates. Genetics 157:1805–1817 Vanhellemont M, Verheyen K, De Keersmaeker L, Vandekerkhove K, Hermy M (2009) Does Prunus serotina act as an aggressive invader in areas with a low propagule pressure? Biol Invas 11:1451–1462 Vaughan SP, Russell K (2004) Characterization of novel microsatellites and development of multiplex PCR for large-scale population studies in wild cherry, Prunus avium. Mol Ecol Notes 4:429–431 Vaughan SP, Cottrell JE, Moodley DJ, Connolly T, Russell K (2007) Distribution and fine-scale spatial-genetic structure in British wild cherry (Prunus avium L.). Heredity 98:274–283

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.

Chapter 8

Pyrus Richard L. Bell and Akihiro Itai

8.1 Basic Botany of Pyrus Pear species belong to the genus Pyrus, the subtribe Pyrinae, the subfamily Maloideae (Pomoideae) in the family Rosaceae. There are at least 26 widely recognized primary species and 10 naturally occurring interspecific hybrid taxa (Table 8.1), which are distributed in Europe, temperate Asia, and the mountainous areas of northern Africa (Bell et al. 1996). Many of the known species are native to Asia. All species of Pyrus are intercrossable, and there are no major incompatibility barriers to interspecific hybridization in spite of the wide geographic distribution of this genus (Westwood and Bjornstad 1971). Dispersal is believed to have followed the mountain chains both east and west (Bell et al. 1996). Speciation probably involved geographic isolation and adaptation to colder and drier environments (Rubzov 1944). Kikuchi (1946) classified Pyrus species into three groups, small fruited species with two carpels, large fruited species with five carpels, and their hybrids with 3–4 carpels. Small fruited species, sometimes known as Asian pea pears, P. calleryana Decne., P. fauriei Schneid., P. betulifolia Bunge, and others are used for ornamental purpose or rootstocks. Of large fruited species with five carpels, there are four major cultivated species, P. communis L. (European pear), P. bretschneideri Rehd. (syn. P. pyrifolia (Burm.) Nakai var. sinensis Kikuchi), P. ussuriensis Maxim. and P. pyrifolia, which are

R.L. Bell (*) United States Department of Agriculture, Agricultural Research Service, Appalachian Fruit Research Station, Kearneysville, WV 25430, USA e-mail: [email protected]

commercially cultivated in the temperate zone. P. communis is native to Europe and is the main commercial species in Europe, North America, South America, Africa, New Zealand and Australia. P. bretschneideri is the main species in northern and central China. P. pyrifolia is the main species in Japan, southern and central China, Taiwan, and Korea. P. nivalis Jacq., the snow pear, is cultivated in Europe for making perry, an alcoholic pear cider. P. pashia P. Don. is cultivated in northern India, Nepal, and southern China. The genome size of P. communis has been estimated by using flow cytometry (Arumuganathan and Earle 1991). According to their report, DNA content of P. communis is 1.03 pg/2C, compared to 0.54 pg/ 2C in peach, and the genome size is approximately 496 Mbp/haploid nucleus. Asian pears are thought to have been domesticated in pre-historic times and to have been cultivated in China for at least 3,000 years (Lombard and Westwood 1987). European pears are thought to have been cultivated in Europe as early as 1000 BC. Homer referred to a large orchard with pears in the Odyssey, written in between 900 and 800 BC. The earliest written records of Japanese pears date back to the ancient manuscript of the Emperor Jito in AD 693 (Kajiura 1994). Pear is the third most important temperate fruit species after grape and apple, with a 2009 world production estimated at 21.9 metric tons (FAO 2010). Asia produced the most (16.4 million t), followed by Europe (3.9 million t), North and Central America (885,321 t), and South America (744,032 t). The European pear (P. communis) production is concentrated in five production areas: Europe, North America, South America, South Africa, and Oceania, while the production of Asian pears (P. bretschneideri, P. pashia, and P. pyrifolia) is concentrated in Asia.

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_8, # Springer-Verlag Berlin Heidelberg 2011

147

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R.L. Bell and A. Itai

Table 8.1 Pyrus species, interspecific hybrids, and geographic distributiona Geographic group and species Centers of diversity European European P. communis L.b Western and Southeast Europe, Turkey P. cordata Desv. France, Spain, Turkey P. nivalis Jacq. South central Europe, Ukraine, France P. canescens Sprach P. complexa Rubtzov Caucasus P. salvifolia DC Europe (Crimea) Circum-Mediterranean P. elaeagrifolia Pall. P. syriaca Boiss. P. cossonii Redher P. gharbiana Trab. P. mamorensis Trab. P. spinosa Forssk.(syn. P. amygdaliformis Vill.)

Southeast Europe, Ukraine, Turkey Tunisia, Libya, Middle East, Armenia Algeria Morocco, Algeria Morocco Southeast Europe, Turkey

Mid-Asian P. glabra Boiss. P. korshinskyi Litv. P.longipes Coss. & Dur. P. pashia D. Don. P. regelii Rehd. P. salicifolia Pall.

Iran Afghanistan, Kyrgyzstan, Tajikistan, Uzbekistan Algeria Pakistan, India, Nepal, Bhutan, Afghanistan, China, Indochina Afghanistan, central Asia Northern Iran, Turkey, Caucasus

East Asian P. armeniacifolia Yu P. betulifolia Bunge P. bretschneideri Rehd. P. calleryana Decne. P. dimorphophylla Makino P. fauriei Schneid. P. hondoensis Nak. & Kik. P. hopeiensis T. T. Yu P. koehnei C. K. Schneid. P. phaeocarpa Rehd. P. pseudopashia T. T. Yu P. pyrifolia (Burm.) Nak. P. serrulata Rehd. P. sinkiangensis T. T. Yu P. taiwanensis Iket. & Ohashi P. ussuriensis Maxim. P. uyematsuana Makino P. xerophila T. T. Yu

Northwestern China, Kazakhstan Central and northern China, Laos southern Manchuria China Central and southern China, Japan, Korea, Taiwan, Vietnam Japan Korea Japan China (Hebei, Shandong) South China, Taiwan Northern China China (Guizhou, Yunnan, Kansu) China, Japan, Korea, Taiwan Central China Northwestern China Taiwan Siberia, Manchuria, northern China, Korea Northwestern China

a

Source: USDA (2009b) Includes subspecies and several taxa listed as valid species names for which species status is uncertain. Also includes several taxa not recognized as valid species

b

8.2 Conservation Initiatives 8.2.1 Evaluation of Genetic Erosion Information on Pyrus is limited. The International Union for the Conservation of Nature (IUCN 2008) Red List currently contains only nine Pyrus taxa

(Table 8.2). Included in the “Low risk/near threatened” category are P. salicifolia Pall., and four taxa of uncertain taxonomic status: P. anatolica Browicz, P. asia-mediae (Popov) Maleev, P. oxyprion Woronow, and P. serikensis G€uner & Duman. Two taxa, P. asia-mediae and P. hakkiarica Browicz, are listed as “data deficient” but are included because of concern. G€uner and Zielinski (1996), in their account of the status of Turkish woody

8 Pyrus

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Table 8.2 Conservation status of Pyrus taxa listed on the IUCN red lista Taxon Native range Status Pyrus anatolica Browicz Turkey Lower risk/near threatened Pyrus asia-mediae (Popov) Maleev Kazakhstan Data deficient Kyrgyzstan Uzbekistan Pyrus cajon Zapryagaeva Tajikistan Endangered

Pyrus hakkiarica Browicz Pyrus korshinskyi Litv.

Turkey Kyrgyzstan

Data deficient Critically endangered

Pyrus oxyprion Woronow Pyrus salicifolia Pall. Pyrus serikensis G€uner & Duman Pyrus tadshihkistanica Zapryagaeva

Tajikistan Uzbekistan Turkey Turkey Turkey Tajikistan

Lower risk/near threatened Lower risk/near threatened Vulnerable Critically endangered

a

Comment Probably synonymous with P. communis Not seen since originally described. Taxonomic status not clear Rare endemic, declining due to agricultural expansion. May be synonymous with P. lindleyi Probably P. syriaca Threatened by overgrazing and overharvesting

Taxonomic status not clear

Rare endemic population reduced by cutting

Citation: IUCN (2008)

flora, also list P. elaeagrifolia ssp. bulgarica and P. yaltirikii as low risk/near threatened and P. elaeagrifolia Pall. ssp. elaeagrifolia and spp. kotschyana, P. pyraster ssp. caucasica (syn. P. caucasica Fed.) and ssp. pyraster (syn. P. communis ssp. pyraster), P. spinosa (syn. P. amygdaliformis Vill.), and P. syriaca Boiss. as “low risk/least concern”. According to Browicz (1972), P. anatolica is probably P. elaeagrifolia subsp. kotschyanae or an interspecific hybrid of P. elaeagrifolia, P. communis, and P. amygdaliformis. G€ uner and Zielinski (1996) state that P. serikensis is “known until recently as P. boissieriana subsp. crenulata”, which Aldasoro et al. (1996) recognize as a synonym of P. cordata. The taxon P. hakkiarica is probably P. syriaca (Davis 1972). The International Dendrological Society also lists P. magyarica (probably a synonym of P. communis ssp. pyraster, and only described in Hungary) as either endangered, vulnerable, or rare (Lear and Hunt 1996). In Germany and the Czech Republic, wild populations of P. pyraster are threatened (Endtmann 1999; Sindela´rˇ 2002), although there are efforts to maintain those genetic resources by in situ and ex situ preservation (Kleinschmit et al. 1998; Wagner 1999; Paprsˇtein et al. 2002). Other such efforts at in situ conservation have been planned for the Middle East (Amri et al. 2002). Wild populations in Kyrgyzstan (Blaser 1998), the Kopet Dag woodlands of Turkmenistan (World Wildlife Fund 2001), and elsewhere in central Asia are

threatened by logging, fuelwood gathering, and other activities in previously protected forests. Among Asian species, P. calleryana is listed as a vulnerable endemic species in Japan (Ohba 1996), and P. kawakamii (syn. P. calleryana) is listed as vulnerable in Taiwan (Lear and Hunt 1996). Throughout the world, there are most likely wild populations of various other species, which are also threatened by deforestation. In addition, indigenous landrace cultivars are being replaced by more modern cultivars.

8.2.2 In Situ and Ex Situ Conservation Ex situ conservation of wild crop relatives of the major cultivated Pyrus species has received much more actual work than in situ conservation. A 1989 survey listed 34 collections in 22 countries that probably contained at least one crop relative species (IBPGR 1989). Major ex situ germplasm collections resulting from plant exploration and exchange have been established in several countries, including the USA (Postman 2008; USDA 2009a), the United Kingdom (University of Reading 2009), and the People’s Republic of China (Aldwinckle et al. 1986; Xu et al. 1988; Zhang 2002). Smaller collections exist in many countries with native Pyrus populations. The European Cooperative Program for Plant Genetic Resources lists 32 noncultivated taxa in 11 European collections in eight

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countries (EUCPPGR 2009a). The EURISCO database has accession level information for close to 1,000 accessions of 48 non-cultivated taxa in numerous national collections (EUCPPGR 2009b).

dehydration (Reed and Chang 1997). Pre-treatment with cold-acclimation and abscisic acid has been shown to be very important for pear genotypes (Bell and Reed 2002).

8.2.3 Modes of Preservation and Maintenance

8.3 Origin and Evolution of Pyrus

Most ex situ preservation is in the form of clonally propagated trees. Clones preserved for their edible fruit are usually propagated by budding or grafting onto seedling or clonal pear rootstocks of various species or clonal quince rootstocks. The rootstocks can be also propagated onto specific seedling or clonal rootstocks, maintained in stool beds as self-rooted plants, or planted as self-rooted trees. In many cases, clones are indexed for viruses and phytoplasmas, treated by thermotherapy if needed, and maintained as “disease tested” or certified disease-free germplasm (Postman and Hummer 1988). The genetic diversity of wild populations is best preserved by systematic collection of seed. Preservation of these seed collections of Pyrus can be accomplished by drying to a relative humidity of 20% and storing in a desiccator at the temperature of 4–6 C for long-term storage (2–10 years or more), or even at 20 C. For subfreezing temperatures, drying to lower a moisture content of 2–5% may be preferred (Roberts 1975). In vitro methods of preservation are intended as secondary or back-up collections, established as a safeguard against loss of trees in nursery or orchard plantings due to disease, insect, or climatic hazards, e.g., due to low winter temperature. Medium-term storage of clonal propagules has been attained through in vitro culture of shoots, and long-term storage has been achieved through cryopreservation of in vitro cultured apical meristems in liquid nitrogen. Medium-term storage involves slowing growth through low temperature and medium manipulations. Viable cultures can be maintained for 12–18 months at 4 C with a 16-h photoperiod, and storage for over 2 years can be achieved using gas-permeable bags instead of glass culture tubes. Defoliated shoots have been stored for up to 4 years at 2 C on an agar medium without growth regulators (Druart 1985). The three major techniques of cryopreservation are slow freezing, vitrification, and encapsulation–

Maloideae (Pyrinae) contains the 25 genera, including Pyrus (pear), Malus (apple), Eriobotrya (loquat), Cydonia (quince), and Chaenomeles (Chinese quince). The basic chromosome number of the Maloideae (x ¼ 17) is high compared to other Rosaceae subfamilies (x ¼ 7–9), indicating a polyploid origin. Classical biochemical studies on leaf phenolic compounds, isozyme studies, and botanical data support the hypothesis of an allopolyploid origin (Chevreau et al. 1997). It has been suggested that the Maloideae arose as an amphidiploid of two primitive forms of Rosaceae, crossing a basic chromosome number of 8 and 9 (Sax 1931; Zielinski and Thompson 1967). These were possibly primitive members of the Prunoideae (x ¼ 8) and Spiraeodeae (x ¼ 9). A molecular study of the chloroplast gene rbcL suggests that Spiraeodeae is the maternal ancestor of Maloideae (Morgan et al. 1994). But phylogenetic analyses of granule-bound starch synthase gene (GBSSI) sequences for a broad sampling of taxa with the different chromosome numbers across the Rosaceae family did not support the wide hybridization between basic chromosome number of 8 and 9 (Evans and Campbell 2002). Instead, sequences of GBSSI genes resolved Gillenia (x ¼ 9) to the clade including taxa with x ¼ 15 or 17. Evans and Campbell (2002) suggest that the higher chromosome number of the Maloideae arose via hybridization and polyploidization among closely related species of an ancestral lineage (the lineage that also gave rise to Gillenia), followed by aneuploid reduction. The majority of cultivated pears are functional diploids (2n ¼ 34). A few polyploid (triploid and tetraploid) cultivars of P. communis and P. bretschneideri exist. Speciation in Pyrus has proceeded without a change in chromosome number (Zielinski and Thompson 1967). This genus is considered to have originated in the mountainous area of western and southwestern China during the Tertiary period (65–55 million years ago) and to spread to the east and west. Three subcenters of diversity for the genus have been identified (Vavilov 1951): (1) the Chinese center, where forms of

8 Pyrus

P. pyrifolia and P. ussuriensis are grown; (2) the Central Asiatic center, consisting of Tajikistan, Uzbekistan, India, Afghanistan, and western Tian-Shan mountains, where forms intermediate between P. communis and P. bretschneideri occur, and where P. communis is thought to have hybridized with P. heterophylla, and the putative species, P. korshinski and P. boisseriana; (3) the Near Eastern center comprising the Caucasus Mountains and Asia Minor, where domesticated forms of P. communis occur. Using sequences of non-coding regions of chloroplast DNA, Kimura et al. (2003) conducted a phylogenetic analysis of 24 cultivars belonging to P. bretschnedieri, P. calleryana, P. communis, P. pyrifolia, and nine interspecific hybrids. A cladogram based on mutations defined one European group and three Asian pear groups. Analysis of trnL–trnF divided Asian pears into six groups, while all European pears had identical sequences. These chloroplast DNA genotypes were useful for verifying the species ancestry of the interspecific hybrids. In a study of the nuclear ribosomal DNA internal transcribed spacer region of 44 accession representing 19 mostly Asian Pyrus species, Zheng et al. (2008) concluded that certain types of pseudogenes were more useful than functional ITS copies in resolving phylogeny, especially among Asian species.

8.4 Role in Development of Cytogenetic Stocks and Their Utility Cytogenetic stocks, other than polyploids and haploids, have not been developed in Pyrus, and species related to the major cultivated species have played no role in their development. Haploids of ‘Doyenne´ du Comice’, ‘Bartlett’ (syn. ‘William’s’), and ‘Harrow Sweet’ pear have been obtained by selection of seedlings based on morphological traits as well as pollination with irradiated P. communis pollen, followed by in vitro culture of the resulting embryos (Bouvier et al. 1993), and doubled haploids were subsequently obtained by either spontaneous doubling or treatment with oryzalin (Bouvier et al. 2002). Triploids have been obtained by anther culture of diploid P. pyrifolia cultivars (Kadota and Niimi 2004). In a series of crosses involving diploid, triploid, and tetraploid

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P. pyrifolia and P. communis pear cultivars, Cao et al. (2002) reported aneuploid seedlings at a frequency of 6%, mostly from intraspecific crosses of tetraploids by triploids and diploids by triploids. The only reports of the generation of polyploids and anueploids by wide crosses involve intergeneric hybridization of Pyrus with Cydonia, Malus, or Sorbus (Rudenko 1974) (see Sect. 8.6.2).

8.5 Role in Classical and Molecular Genetic Studies 8.5.1 Classical Genetic Studies Most classical genetic studies have been performed within single cultivated species, specifically P. communis, P. pyrifolia, and P. bretschneideri. Most of the interspecific studies are also between improved genotypes of these cultivated species rather than wild clones or other Pyrus species.

8.5.1.1 Productivity of Scions Studies in China indicated that clones and seedlings of P. pyrifolia were more precocious in fruit bearing than those of P. bretschneideri and P. communis, although no conclusions on heritability or mode of inheritance were made.

8.5.1.2 Fruit Quality Crosses between the European pear and the Asian pear, P. ussuriensis, were initially made either to improve fire blight resistance or for cold-hardiness for the northern regions of North America and Europe. Lantz (1929) characterized the mode of inheritance of fruit flavor as quantitative, with “dominance” of poor flavor. Pu et al. (1963) reported that the aromatic quality of some P. ussuriensis cultivars appeared dominant to the sweet, bland flavor of P. pyrifolia and P. bretschneideri. Subjective sensory scores for flavor in P. communis P. pyrifolia hybrid progenies and intercross progenies were found to have low narrowsense heritability (Bell and Janick 1990).

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In crosses between European and Asian species, perhaps the most striking differences among parents arise in flesh texture. Modern European cultivars are characterized by melting or buttery flesh, while P. pyrifolia and P. bretschneideri are characterized by finely grained, crisp flesh. Very little is known about the inheritance of flesh texture in pears. The differences between crisp flesh and melting flesh are likely due to a combination of enzymes affecting cell wall adhesion. Graininess is often difficult to determine independently from grit cell content. In some populations, however, grit may be confined primarily to the skin and core, and grain can be rated relatively independent of grit. Westwood and Bjornstad (1971) reported that in interspecific hybrids of wild forms of Pyrus, gritless flesh was dominant to gritty flesh. Golisz et al. (1971), on the other hand, found that in crosses of cultivars of P. communis with P. ussuriensis, the majority of offspring had numerous stone cells typical of the P. ussuriensis cultivars used as parents. Pu et al. (1963) also reported that the coarse texture of P. ussuriensis is dominant to fine grained texture of P. pyrifolia or P. bretschneideri. Zielinski et al. (1965) presented data indicating that presence of stone cells is dominant to their absence among certain crosses of P. communis cultivars. Thompson et al. (1974) concluded that in crosses involving species that contain grit, the content was controlled by a minimum of four loci, acting in an independent and additive fashion. Bell and Janick (1990) computed narrow-sense heritabilities of 0.14 and 0.48 for texture and grit scores in P. communis progenies, respectively. Heritability of texture within backcrosses of P. pyrifolia P. communis to P. communis as well as intercrosses of the hybrids was low. Heritability of grit within backcrosses of P. pyrifolia P. communis to P. communis as well as intercrosses of the hybrids was moderate (0.48 and 0.55, respectively). These values and the relatively large ratios of general to specific combining ability variance indicate that moderate genetic gains in grit, but not texture, in interspecific backcrosses should be possible in these traits through mass selection. Some researchers report that it is difficult to recover pure crisp-flesh selections in crosses between Asian species and P. communis, and fruit grain tends to be coarse (Wang 1990; Hough personal communication). However, White et al. (2000b) reported high narrow-sense heritabilities for both firmness (0.62) and crispness (0.89) in five crosses involving

R.L. Bell and A. Itai

P. pyrifolia, P. bretschneideri, and P. communis. They also found that skin grit was highly heritable, russet and aroma were moderately heritable, and juiciness, sweetness, sourness, astringency, and attractiveness were of low heritability (0.05–0.21). White and Selby (1994) concluded that all of these traits, plus skin thickness, are inherited in an additive fashion in P. pyrifolia P. communis progenies, based on segregation patterns. Flesh firmness was also highly heritable in breeding populations of P. pyrifolia (Machida and Kozaki 1976; Kajiura and Sato 1990). In crosses between major Japanese P. pyrifolia cultivars with ‘Max Red Bartlett’, Sansavini et al. (2002) noted a higher frequency of nashi-type texture, fruit shape, and sugar content. Various fruit shape parameters in crosses of P. pyrifolia or P. bretschneideri with P. communis had narrow-sense heritabilities ranging from 0.55 to 0.75, indicating that breeding for specific fruit shape in this interspecific hybridization project was possible (White et al. 2000a). Corking of the skin produces a brown russet, which is seen on many cultivars. Many people find the appearance attractive when the russet is smooth, uniform, and light tan, and for the fresh market trade, russeted cultivars such as ‘Beurre´ Bosc’, ‘Angelys’, and ‘Taylor’s Gold’ are acceptable. But when the fruit is processed whole for puree, russeting results in a flecked or brown colored product, which is unacceptable. The presence of large and dark colored lenticels on the skin is also objectionable for the same reason. Wellington (1913) proposed that russeting in P. communis was controlled by a single gene, with smooth skin incompletely dominant to russet. Kikuchi (1930), however, concluded that in P. pyrifolia two loci, R and I, are involved. Genotypes that are RR are entirely russeted, while Rrii genotypes are partially russeted. I partially inhibits cork formation so that russet does not extend over the entire fruit, but under humid conditions, RrM- genotypes are more russeted than under dry conditions. Wang and Wei (1987) also found that russeting was recessive to non-russeted skin. All of these workers based their conclusion on the segregation within progenies that involved P. pyrifolia or hybrids of P. pyrifolia and P. communis. In the P. communis progenies studied by Zielinski et al. (1965) and those studied by Crane and Lewis (1949), the inheritance of russeting appeared to be more complex, suggesting control by several genes. Narrow-sense heritability in

8 Pyrus

P. communis progenies was moderate (0.51), and similar (0.57) in progenies of P. communis P. pyrifolia backcrossed to P. communis, but very high (0.94) in progenies derived from intercrossing P. communis P. pyrifolia hybrids (Bell and Janick 1990).

8.5.1.3 Host Resistance to Disease Fire blight, incited by the bacterium Erwinia amylovora (Burrill) Winslow et al., is the most serious disease affecting pears in North America, Europe, and the Middle East (van der Zwet and Beer 1999). It is difficult to control even when recommended prophylactic measures are followed. No true immunity to fire blight in the seedlings or clones has been encountered, although high levels of resistance exist. Resistance in P. communis is relatively rare, with only 5–10% being rated as at least moderately resistant (Oitto et al. 1970; van der Zwet and Oitto 1972; Thibault et al. 1987; van der Zwet and Bell 1990). All other European, circum-Mediterranean, and midAsian species are generally susceptible. A high level of resistance occurs in greater frequency among the East Asian species, especially P. calleryana and P. ussuriensis. The evergreen pear, P. kawakamii, is susceptible. With the exception of a few seedlings selected by Reimer, P. betulifolia is also susceptible. Other species are variable, with a range of levels present. Hartman (1957) listed resistant clones within P. calleryana, P. betulifolia, P. phaeocarpa, P. fauriei, and P. variolosa (syn. P. pashia). The inheritance of resistance/susceptibility to fire blight is complex. Layne et al. (1968) found that when most of the factors affecting phenotypic expression of fire blight resistance were controlled, segregation for resistance in artificially inoculated seedling progenies was continuous, regardless of the resistance phenotypes of the parents or the species sources of resistance being tested. The proportion of offspring in each class was significantly influenced by parental phenotypes, however, and to a lesser extent by the species source of resistance. Because most segregation distributions were continuous and a number of them showed an approximately normal distribution, fire blight resistance was concluded to be polygenically inherited. Because a specific type of inheritance pattern characteristic of any of the three species studied (P. communis, P. ussuriensis, and P. pyrifolia) was not detected, it

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was concluded that the same or similar genes for resistance may be present in each species. In a few cases, with each species source of resistance, they found some seedling progenies with a segregation pattern that suggested major gene inheritance, with dominance of resistance. Thompson et al. (1962) also found evidence for major gene and polygenic inheritance in studies with the same Pyrus species, although they thought that monogenic resistance was present only in P. ussuriensis. Thompson et al. (1975) postulated the presence of a dominant gene conferring sensitivity in some genotypes. This conclusion, however, was based upon classifying ratings of natural blight into discrete classes; no bimodal distribution was demonstrated. The consensus developed from all other studies is that host response is inherited in a quantitative fashion, and is due to predominantly additive genetic effects, with dominance or epistasis playing only a minor role in differences among parents in the ability to transmit resistance to their offspring. Heritability within pure P. communis populations estimated from offspringmidparent regression was 0.52 for epiphytotic fire blight severity in mature trees, and was similar in populations involving P. communis P. pyrifolia hybrids (Bell et al. 1977). Heritability ranged from 0.30 to 0.51 when based upon artificial inoculation of parents in the orchard and of 6-month old seedlings in the greenhouse (Quamme 1981). General combining ability has been found to be significant, while specific combining ability was not significant (Quamme et al. 1990). More recent studies involving molecular markers have further elucidated the inheritance of resistance and suggest the involvement of a few quantitative traits loci (QTLs) (See Sect. 8.6.4.1.1 Disease Resistance). Pear leaf spot, caused by the fungus Fabraea maculata Atk. (anamorph: Entomosporium mespili (DC.) Sacc.), occurs in most areas of the world where pears are grown under warm, humid conditions. Susceptible cultivars are often defoliated by mid-summer, resulting in weak trees and a reduction in fruit buds. Infected fruits are disfigured, cracked, misshapen, and unmarketable as fresh fruit. In the nursery, the disease can be serious, causing defoliation and stunted growth. Most major cultivars of the European pear, P. communis, for which data are available, are considered susceptible (Bell 1990). Genotypes of a number of East Asian species, P. ussuriensis, P. pyrifolia, P. calleryana, P. fauriei, and P. dimorphophylla, have been reported as resistant (Wisker 1916; Tukey and Braese 1934;

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Kovalev 1940; Beck 1958). Zalaski et al. (1959) reported that seedlings of P. caucasica were more resistant than interspecific hybrids of P. salicifolia or P. amygdaliformis with P. communis. A multi-year study of 207 Pyrus genotypes (Bell and van der Zwet 1988) demonstrated variability within and among species and interspecific hybrids. Pure species or interspecific hybrids involving P. calleryana, P. pyrifolia, and P. ussuriensis were generally more resistant than P. communis genotypes. In a later study, Bell and van der Zwet (2005) found that the least susceptible genotypes were the P. communis cultivars ‘Beurre Fouqueray’ and ‘Bartlett’, the P. pyrifolia cultivar ‘Imamura Aki’, and the P. ussuriensis P. pyrifolia hybrid NJ 477643275. As species groups, the P. ussuriensis P. pyrifolia hybrids and the pure P. pyrifolia cultivars were most resistant. Drain (1954) noted resistance in the P. communis P. pyrifolia hybrids, ‘Mooers’ and ‘Hoskins’. Lombard and Westwood (1987) listed seedlings of P. caucasia and P. cordata Desv. as moderately tolerant, those of the CircumMediterranean species P. amygdaliformis, P. elaeagrifolia, and P. syriaca Boiss., and seedlings and clones of P. betulifolia as having high tolerance, P. calleryana seedlings and clones as very tolerant, and P. pashia D. Don. as susceptible. In contrast to some of the other studies, Lombard and Westwood (1987) list P. pyrifolia and P. ussuriensis as having only low tolerance. Since sources of resistance to this disease have been identified, breeding for resistance should be possible. The inheritance of resistance in crosses involving P. communis, P. pyrifolia, and P. ussuriensis indicates that the high level of resistance in P. calleryana is transmitted in an additive manner to its offspring (Bell and van der Zwet 1988). The principal fungal pathogen of P. pyrifolia and P. bretschneideri cultivars in Asia is Alternaria alternata (Fr.) Keissler pv. kikuchiana (formerly A. kikuchiana Tanaka), which causes black spot. Although the major Japanese cultivar, ‘Nijisseiki’, is susceptible, many others are resistant (Hiroe et al. 1958; Kanato et al. 1982). Many of the European pear cultivars are resistant. Susceptibility is controlled by a single dominant gene, and most of the susceptible cultivars are heterozygous at the locus (Kozaki 1973). Pear decline is caused by a phytoplasma transmitted by the pear psylla, Cacopyslla spp. (Hibino and Schneider 1970). Infection results in sieve-tube necro-

R.L. Bell and A. Itai

sis in susceptible trees. The severity of symptoms depends on the age and vigor of trees, the species of scion and rootstock, and environmental conditions, and can vary from leaf curl of young trees on tolerant rootstocks to premature reddening of foliage and reduced growth, or sudden wilting and death. The disease is particularly severe when cultivars of a generally tolerant species such as P. communis are grafted onto the sensitive species, P. pyrifolia, P. ussuriensis, and to a lesser extent, P. calleryana (Lombard and Westwood 1987). Most P. communis rootstocks are tolerant, and seedlings of P. betulifolia are the most tolerant. Resistance was found to be inherited in a quantitative fashion, with seedlings intermediate between the resistant and susceptible parents (Westwood 1976). Seedlings of intraspecific crosses of P. communis, P. betulifolia, and P. calleryana exhibited a low frequency of severe decline symptoms, and 75% of the seedlings of P. ussuriensis cv. Chieh Li and P. pyrifolia cv. Japan Golden Russet were resistant in spite of the fact that unselected clones of these species are usually susceptible. European pear scab, incited by the fungus Venturia pirina Aderh., is a serious disease of European pear. Host resistance of European pears is somewhat confusing because V. pirina has at least five biotypes, which appear to have a fairly narrow range of distribution (Shabi et al. 1973). Thus, cultivars that are reported resistant in one region may be susceptible in another where they are exposed to another biotype. Vondracek (1982) points out that differences among various reports in the degree of resistance could be due to differential races, environmental influences, and differences in the definition of “resistance”. Several European cultivars, including ‘Bartlett’, ‘Conference’, and ‘Dr. Jules Guyot’, are at least moderately resistant (Bell 1990). Kovalev (1963) reported that P. pyrifolia and P. ovoidae (P. ussuriensis var. ovoidae Rehder) are generally resistant, while genotypes of P. ussuriensis observed were susceptible. Westwood (1982) indicated that clones or seedling populations of P. caucasica and P. communis were susceptible, while P. pyrifolia and P. nivalis were variable. Postman et al. (2005) found that 27 of 31 P. pyrifolia, P. ussuriensis, or P. bretschneideri cultivars were highly resistant to leaf infection, and 20 of 30 were resistant to fruit infection. Species rated as resistant to fruit infection included P. betulifolia, P. calleryana, P. dimorphophylla, P. fauriei, P. pashia, P. pseudopashia, and P. regelii,

8 Pyrus

while P. salicifolia, P. syriaca, and P. nivalis rated as having low susceptibility. Only the single clone of P. cordata was highly susceptible. Resistance to fruit infection in interspecific Asian European progenies was moderately heritable (White et al. 2000a, b).

8.5.1.4 Host Resistance to Arthropod Pests Pear psylla is the major insect pest of pears in North America and western Europe. While Cacopsylla pyricola Fo¨erster is the only species that exists in North America, C. pyri L. and C. pyrisuga Fo¨erster are also endemic to Europe, and C. bidens is endemic to the Middle East. Other species or putative species exist in Asia. Damage occurs directly as a result of nymphal feeding in the phloem of leaves, which can cause leaf necrosis and defoliation, and indirectly can result in poor fruit size and reduced flower bud development. The nymphs excrete large amounts of honeydew which support the growth of sooty mold, both on leaves and fruit. Honeydew on young developing fruit can lead to russeting, and sooty mold on the fruit can further reduce crop value. Foliage feeding also suppresses root growth and reduces tree vigor. In addition to the damage pear psylla causes by itself, it also transmits the pear decline phytoplasma (Jensen et al. 1964). Pear species vary considerably in their resistance to pear psylla (Bell 1990). The East Asiatic species are generally more resistant than those from Asia Minor or Europe. Resistance to pear psylla was first discovered in the East Asian species, P. betulifolia, P. calleryana, P. fauriei, P. ussuriensis, and P. bretschneideri (Westigard et al. 1970; Quamme 1984) and in hybrids of P. communis and P. ussuriensis (Harris 1973; Harris and Lamb 1973; Quamme 1984). Among European species, resistance has been reported in some genotypes of P. nivalis and Sorbopyrus (Westigard et al. 1970; Bell and Stuart 1990; Bell 1992). Within P. communis, moderate to high degrees of resistance has been reported in genotypes of relatively poor fruit quality, specifically an old Italian cultivar, ‘Spina Carpi’ (Quarta and Puggioni 1985; Briolini et al. 1988), and in 15 cultivars from Yugoslavia and Hungary (Bell and Stuart 1990; Bell 1992, 2003). Harris and Lamb (1973) reported the use of P. ussuriensis as a source of psylla resistance. They

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found that 60% of one progeny of P. communis P. ussuriensis was resistant to psylla, concluding that resistance was quantitative and controlled in an additive genetic fashion. The narrow-sense heritability of resistance to pear slug, the larva of the sawfly Caliroa cerasi (L.), in two populations of interspecific crosses involving interspecific hybrid parents (P. pyrifolia P. communis and P. communis P. bretschneideri) were found to be high (0.79 and 0.87) (Brewer et al. 2002). Particularly low frequencies and severities of infestation were found in progenies of the P. bretschneideri cultivars ‘Yali’, ‘Shiyueli’, and ‘Huobali’.

8.5.2 Molecular Genetic Studies Linkage maps and molecular markers would be useful in traditional cross-breeding programs for perennial crops such as fruit tree species. However, genetic studies in pear, as in many fruit trees, have been rare. There is little information on genetic linkage maps and development of molecular markers on pears despite much research on mapping and molecular markers in apple. The long juvenile periods, the space necessary to manage large number of progenies, and the high level of heterozygosity due to a gametophytic incompatibility have limited inheritance studies to a few morphological characters (Chevreau et al. 1997).

8.5.2.1 Development of Molecular Markers Isozymes The first report on the use of isozymes in pears was in 1980 by Santamour and Demuth to identify six ornamental cultivars of P. calleryana by peroxidase patterns. Peroxidase diversity has also been studied in several species of Pyrus (Merendez and Daley 1986) and in 172 cultivars of P. pyrifolia (Jang et al. 1991, 1992). Isozymes variability in pollen was reported by Cerezo and Socias y Company R (1989). However, these approaches are used for cultivar identification and to differentiate genetic sports. Chevreau et al. (1997) examined the inheritance and linkage of isozyme loci in P. communis cultivars. They analyzed the polymorphisms of 11 enzymes (AAT:

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aspartateaminitransferase, ENP: endopeptidase, EST: esterase, LAP: leucineaminopeptidase, PRX: peroxidase, SOD: superoxide dismutase, ADH: alcohol dehydrogenase, DIA: diaphorase, PGD: 6-phosphogluconate dehydrogenase, PGI: phosphoglucoisomerase, PGM: phosphoglucomutase) in 11 progenies from controlled crosses. According to their report, 22 loci were identified and segregation was scored for 20 loci. Three pairs of duplicated loci-forming intergenic hybrid bands were detected and these were found to correspond to equivalent duplicated genes in apple. They identified 49 active alleles and one null allele and revealed three linkage groups, which could all be related to existing groups on the apple map. Conservation of isozyme patterns, duplicated genes, and linkage groups indicates a high degree of synteny between apple and pear. No linkage map for pears was constructed based on isozyme analysis.

R.L. Bell and A. Itai

restriction and PCR amplification. AFLP has several advantages over the RAPD technique, including a higher number of loci analyzed and a higher reproducibility of banding patterns. Monte-Corvo et al. (2000) investigated the genetic relationships among 39 cultivars including 35 P. communis and four P. pyrifolia cultivars using AFLP and RAPD markers. They confirmed that AFLP markers were five times more efficient in detecting polymorphism per reaction. Although some differences can be noticed between the dendrograms resulting from AFLP and RAPD analyses, both techniques produced similar results. Yamamoto et al. (2002b) also made 184 and 115 polymorphic AFLP fragments using 40 primer combinations in the F1 population originating from ‘Bartlett’, and ‘Hosui’, respectively. They reported that the average number of polymorphic fragments per primer combination was 4.6 in ‘Bartlett’ and 2.9 in ‘Hosui’.

Random Amplified Polymorphic DNAs Intersimple Sequence Repeats Random amplified polymorphic DNAs (RAPDs) have been widely used in pear genetic studies because RAPDs have the advantages of being readily employed and requiring small amount of genomic DNA. RAPD markers have been successfully used for identification and genetic relationships of pear. Oliveira et al. (1999) investigated molecular characterization and phenetic similarities between several cultivars of P. communis and P. pyrifolia and several wild species by RAPD markers. A total of 118 Pyrus spp. and cultivars native mainly to East Asia were analyzed by RAPD markers to evaluate genetic variation and relationships among the accessions (Teng et al. 2001, 2002). According to their reports, RAPD markers specific to species were identified, and the grouping of the species and cultivars by RAPD largely agrees with morphological taxonomy. RAPD markers have also been used to identify parentage (Banno et al. 2000). Banno et al. (1999) also identified an RAPD marker linked to the gene conferring susceptibility to black spot disease (A. alternata Japanese pear pathotype).

Amplified Fragment Length Polymorphism Amplified fragment length polymorphism (AFLP) technology is a powerful tool that combines DNA

Intersimple sequence repeat (ISSR) means a genomic region between SSR loci. The complementary sequences to two neighboring simple sequence repeats (SSRs) are used for PCR primers. Polymorphism diversity is lower than in SSR, but ISSR has the advantages of being readily employed and no knowledge of the DNA sequence for the targeted gene is required. Monte-Corvo et al. (2001) reported that ISSR analysis was used for cultivar identification and the determination of phylogenetic relationship in P. communis.

Simple Sequence Repeats Simple sequence repeats (SSRs) or microsatellites are excellent sources of polymorphisms in eukaryotic genomes. The development of SSRs is labor-intensive. However, SSRs have been very useful in studying diversity in Pyrus. Yamamoto et al. (2002a) constructed a genome library enriched with (AG/TC) sequences from ‘Hosui’ Japanese pear using the magnetic bead method. They obtained 85 independent sequences containing 8–36 microsatellite repeats. Out of the 85 sequences, 59 contained complete (AG/TC) repeats. Thirteen primer pairs could

8 Pyrus

successfully amplify the target fragments and showed a high degree of polymorphisms in the Japanese pear. Kimura et al. (2002) identified 58 Asian pear accessions from six Pyrus species using these nine SSR markers with a total of 133 putative alleles. They obtained a phenogram based on the SSR genotypes, showing three major groups corresponding to the Japanese, Chinese, and European groups. Moreover, nine apple SSRs were intergenetically applied to the characterization of 36 pear accessions (Yamamoto et al. 2001). All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear species and accessions. The differences in fragment size are mostly due to the differences in repeat number. A total of 79 alleles were detected from seven SSR loci, and thus pear and apple varieties could be differentiated (Yamamoto et al. 2001). This data show that Pyrus has a close genetic relationship with Malus. Wunsch and Hormaza (2007) also reported that the use of seven SSRs developed in apple could distinguish 61 of 63 European pear cultivars and revealed the usefulness of this set of SSR primers for cultivar identification. They found that the variability detected with SSRs in European pear varieties was low when compared with the variability detected in other fruit crops in the Rosaceae (Wunsch and Hormaza 2007). To date, more than 100 SSRs have been developed from European and Japanese pears (Yamamoto et al. 2002a, b; Bassil et al. 2005; Fernandez-Fernadez et al. 2006; Inoue et al. 2007; Terakami et al. 2007). These SSR markers have been used for cultivar identification, the evaluation of genetic diversity, and the construction of genetic linkage maps.

Restriction Fragment Length Polymorphisms and Other Markers Restriction fragment length polymorhisms (RFLPs) have been used to identify Japanese pears, including the parentage of 10 cultivars, with two minisatellite probes from human myoglobin DNA (Teramoto et al. 1994). Similar attempts have been made to distinguish Pyrus species with RFLPs of chloroplast DNA (Iketani et al. 1998; Katayama and Uematsu 2003). However, these markers were used for cultivar identification and investigating genetic relationships among Pyrus

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species. Sequence characterized amplified region (SCAR) markers were developed from RAPDs to evaluate and identify P. communis and P. pyrifolia cultivars (Lee et al. 2004). Another unique marker, copia-like retrotransposons, have been identified in pears by Shi et al. (2002). They suggest that the transposition of retrotransposons takes place during evolution, leading to diversification. However, no data on the inheritance of these markers have yet been reported.

8.5.3 Constructing Linkage Maps The first linkage maps in Pyrus species were developed for ‘Kinchaku’ and ‘Kousui’ Japanese pears using RAPD markers (Iketani et al. 2001). Black spot and pear scab are the most severe diseases of Japanese pear. Only a few cultivars are susceptible to black spot. On the other hand, most cultivars of Japanese pear are susceptible to pear scab. A survey of P. pyrifolia germplasm has identified ‘Kinchaku’ as the only cultivar having resistance. Iketani et al. (2001) used the pseudo-testcross method (Grattapaglia and Sederoff 1994) and constructed two separate maps from segregation data of 82 F1 individuals. The reason for using the pseudo-testcross method is that it is very difficult to make F2 or backcross populations in pears because of self-incompatibility and the long juvenility period of seedlings. The linkage map for ‘Kinchaku’ consisted of 120 loci in 18 linkage groups (LG) spanning 768 cM, while that for ‘Kousui’ contained 78 loci in 22 linkage groups extending over 508 cM. This was the first report of a linkage map of pear species. The resistance allele of Asian pear scab (Vn) and the susceptibility allele of black spot were mapped in different linkage groups in ‘Kinchaku’. However, in both maps, the number of linkage groups did not converge into a basic chromosome number (x ¼ 17). Therefore, the total map length is still not sufficient for covering the complete genome. The length of the apple genome was reported to be 1,200 cM or a little more (Conner et al. 1997). Pear has the same basic chromosome number as apple. In addition, the nuclear DNA content of pear species is estimated at 75 or 80% of that of apple (Dickson et al. 1992). These two pear maps are estimated to cover at least about a half of the total genome (Iketani et al. 2001).

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The second linkage map was constructed using 63 F1 individuals obtained from an interspecific cross between the European pear ‘Bartlett’ and the Japanese pear ‘Hosui’ by Yamamoto et al. (2002b, 2004). They constructed maps based on AFLP and SSR markers from pear, apple, and Prunus; isozymes; and phenotypic traits (leaf color and S-genotype). The map of ‘Bartlett’ consisted of 256 loci including 178 AFLPs, 76 SSRs (32 pear, 39 apple, 5 Prunus), one isozyme, and a self-incompatibility locus on 19 linkage groups over a total length of 1,020 cM. The average distance between each pair of loci was 4.0 cM. The size of linkage groups ranged from 88 cM (LG 4) to 11 cM (LG 18). The segregation of many markers on LG 14 was largely distorted. The self-incompatibility locus (S-locus) was in the bottom of LG 17. The map of ‘Hosui’ contained 180 loci including 110 AFLPs, 64 SSRs (29 pear, 29 apple, 6 Prunus), two phenotypic traits, and four other markers on 20 linkage groups encompassing a genetic distance of 995 cM. Genetic linkage maps of these cultivars were aligned using 37 co-dominant markers that showed segregating alleles in both the cultivars (Yamamoto et al. 2002b, 2004). They also found that of the 80 SSRs obtained from apple, more than four-fifths could produce discrete PCR bands in pear. Similar findings were observed in European pears by another research group (Pierantoni et al. 2004). Yamamoto et al. (2004) reported that 38 apple SSR markers showed 39 segregating loci on the linkage map of ‘Bartlett’, and that 27 SSRs produced 29 loci on that of ‘Hosui’. Moreover, the authors considered synteny between pear and apple linkage maps. A total of 36 SSRs originating from apple were mapped on the genetic linkage maps of ‘Bartlett’ and apple. Only two SSR loci were aligned to different linkage groups of pear and apple. Another 34 apple SSR loci were positioned in presumably homologous linkage groups of pear. All pear linkage groups were successfully aligned to the apple consensus map by at least one apple SSR, indicating that positions and linkages of SSR loci were well-conserved between pear and apple. Their trials were the first major effort in comparing maps of apple and pear. Other maps were developed for two European pear cultivars ‘Passe Crassane’ and ‘Harrow Sweet’ using SSRs, MFLPs, AFLPs, resistance gene analogs (RGAs), and AFLP-RGAs markers in 99 F1 individuals (Dondini et al. 2004). Different levels of susceptibility to fire blight, one of the most destructive diseases, exist among European pear culti-

R.L. Bell and A. Itai

vars. This suggests that it is possible to identify quantitative trait loci (QTL) related to fire blight resistance in pear germplasm. ‘Passe Crassane’ is susceptible to fire blight, and ‘Harrow Sweet’ is resistant. The ‘Passe Crassane’ map consists of 155 loci including 98 AFLPs, 37 SSRs, 6 MFLPs, 4 RGAs, and 10 AFLPRGAs for a total length of 912 cM organized in 18 linkage groups. The average distance between each pair of loci is 5.8 cM. The size of each linkage group ranges from 7.0 to 92.9 cM. The ‘Harrow Sweet’ map consists of 156 loci including 101 AFLPs, 35 SSRs, 3 MFLPs, 3 RGAs, and 14 AFLPs-RGA for a total length of 930 cM organized in 19 linkage groups. Pierantoni et al. (2007) also reported the genetic linkage maps using 99 seedlings derived from the cross ‘Abbe Fetel’ ‘Max Red Bartlett’. The total length of the two maps is 908.1 cM (with 123 loci) for ‘Abbe Fetel’ and 897.8 cM (with 110 loci) for ‘Max Red Bartlett’, divided into 18 and 19 linkage groups with an average marker density of 7.4 cM and 8.0 cM, respectively. However, four linkage groups in both the maps were not denominated because SSR markers originating from apple were not mapped on these linkage groups. More recently, Yamamoto et al. (2007) constructed integrated high density genetic linkage maps of the European pear cultivars ‘Bartlett’ and ‘La France’ based on more SSRs, AFLPs, isozymes, and phenotypic traits. The map of ‘Bartlett’, constructed by using an F1 population derived from cross between ‘Bartlett’ and ‘Hosui’, consisted of 447 loci, including 58 loci by pear SSRs, 60 by apple SSRs, and 322 by AFLPs. This map covered 17 linkage groups over a total length of 1,000 cM with an average distance of 2.3 cM between markers. The map of ‘La France’, which was constructed using an F1 population derived from a cross between ‘Shinsei’ and selection 282-12 (‘Hosui’ ‘La France’), consisted of 414 loci, including 66 loci of pear SSRs, 68 of apple SSRs, and 279 of AFLPs on 17 linkage groups encompassing a genetic distance of 1,156 cM. These are the first maps that converged into the basic chromosome number (n ¼ 17) of pear. A total of 66 SSR markers derived from apple were mapped on pear maps and showed genome synteny with the saturated reference map of apple. These maps could cover the entire genome of pear and should be useful as pear reference maps. In the future, more SSRs and other molecular markers for agronomically important characters could be

8 Pyrus

developed to construct the fine linkage maps useful for marker-assisted selection.

8.6 Role in Crop Improvement Through Traditional and Advanced Tools 8.6.1 Traditional Breeding Efforts Using Interspecific Hybridization Although interspecific hybridization has a limited role in scion and rootstock cultivar development, there have been some traits for which it has become increasingly important and novel breeding efforts are underway. These efforts involve crosses between the major cultivated species and target a variety of traits. Breeding for many traits has been reviewed by Bell et al. (1996) and Hancock and Lobos (2008). In general, there are no barriers to interspecific hybridization within Pyrus (Bell and Hough 1986). Sources of many economically important traits exist within the genus, although not all within the major cultivated species. General responses of Pyrus species to various diseases are given in Table 8.3, responses to arthropod pests and nematodes are given in Table 8.4, and responses to abiotic stresses are furnished in Table 8.5. 8.6.1.1 Adaptation to Low Chilling Hours Adaptation to low-chilling regions utilizing hybrids of the high chilling hour requiring P. communis and low chilling hour requiring P. pyrifolia has been a goal of the pear breeding program at the University of Florida in the USA and has resulted in the release of “Flordahome” pear (Sherman et al. 1982). Similar efforts have begun in Mexico (Rumayor et al. 2005) and Brazil (Barbosa et al. 2007; Instituto Agronomico Campinas Brazil 1987; Rasseira et al. 1992). In India and Pakistan, “semi-soft” interspecific hybrids or hybrids with P. pashia are also being grown (Sandhu et al. 2005).

8.6.1.2 Novel Fruit Texture and Flavor Perhaps the most ambitious program is being conducted in New Zealand, which seeks to combine the flavor of European pears with the crisp flesh of the best

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Japanese P. pyrifolia cultivars (i.e., Nashi) and genotypes of P. bretschneideri with long storage potential (White and Brewer 2002). Red-skinned cultivars of the latter species are being employed as parents. The program maintains intraspecific populations of P. communis, P. pyrifolia, and P. bretschneideri, as well as interspecific populations. From the P. communis P. pyrifolia populations, ‘Maxie’ and ‘Crispie’ have been released. Similar efforts with European Nashi progenies have been reported from Germany (Kaim et al. 2006) and Italy (Sansavini et al. 2002; Musacchi et al. 2005) and have been undertaken in the USA (Bell unpublished data). Additional goals of the latter two programs include improved resistance to fire blight and pear psylla.

8.6.1.3 Precocity Studies in China indicated that clones and seedlings of P. pyrifolia were more precocious than P. bretschneideri and P. communis (Department of Horticulture, Zhejiang Agricultural University 1978).

8.6.1.4 Cold Hardiness Pears are grown in many parts of the world where the winter temperatures are sufficiently severe to cause cold injury to shoots, fruit spurs, trunks, and roots, and may even cause death of whole trees. In northern parts of Europe, Asia, Canada, and the United States, the need for cold hardy cultivars is especially important. Spring frost during bloom is also a constant threat in many pear growing regions, even in those regions where winter injury is not a problem. Thus, resistance of blossoms to frost injury is also an important consideration in the adaptability of pear cultivars to particular regions. The greatest efforts in breeding and selecting for cold hardiness in pears were made in the former Soviet Union and North America, especially in the steppe and prairie regions where the winters are severe. Stushnoff and Garley (1982) reviewed the early work in the United States and Canada to improve cold hardiness of pears, especially the work in Minnesota, North Dakota, and Iowa where P. ussuriensis seedlings imported from northern Russia and the hybrids of P. ussuriensis P. communis were tested. Several pear cultivars, including ‘Patten’, ‘Harbin’, and ‘Bantam’,

VS-MS S-MR R MS MR-R MR-R S MR MS-MR R

S S S

S-MS S-MS – VS VS S

MR – MS MS MS – MS – MS MS

MR S S

MR MR – MR – MR

MR – MR MR – – – – VS –

– – –

– – – – – –

MR – MR – MS – – – – –

MS – – MR – R MR – – – – S S-R

VS – –

MR MR – – – MR

MS MS-MR MS –

MS MS – – – MR – – – MS

Fungal Fabrea spot

Collar rot

– – – – – – – MR – –

– MR MR

– – – MR – –

– S-R – –

White spot

MR – – MR MR MR MR – S-R S-R

– – –

MR MR – – MR –

MS S-R MR MS-MR

European pear scab

– S-MR – – – – – – S S

MR – –

– – – – – –

– R – –

Asian pear scab

MR – MR R MR – – – MS MR

MS – –

MR MR – MS – –

MS MS MR MR

Powdery mildew

b

References: Bell and van der Zwet (1988), Hancock and Lobos (2008), Lombard and Westwood (1987), van der Zwet and Keil (1979), Westwood (1982) Degrees of response: VS very susceptible, S susceptible, MS moderately susceptible, MR moderately resistant, R resistant

a

P. betulifolia P. bretschneideri P. calleryana P. dimorphophylla P. fauriei P. hondoensis P. kawakamii P. pseudopashia P. pyrifolia P. ussuriensis

East Asian

P. pashia P. regelii P. salicifolia

Mid-Asian

P. amygdaliformis P. elaeagrifolia P. gharbiana P. longipes P. mamorensis P. syriaca

Circum-Mediterranean

Table 8.3 Response of Pyrus species to diseasesa,b Bacterial Species Fire blight Blossom blast Crown gall European P. caucasica S MS S P. communis VS-R S-MR S-MS P. cordata VS – – P. nivalis S MS –

MR S MS-MR MR MS – – – S S

MR – –

MR MR – – – –

MS MR-R MR MR

Phytoplasma Pear decline

160 R.L. Bell and A. Itai

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Table 8.4 Response of Pyrus species to arthropod pests and nematodesa,b Species Pear psylla Codling moth Blister mite European

Wooly pear aphid

Root lesion nematode

P. caucasica P. communis P. cordata P. nivalis

MS S-R MS MS-R

S S S-R MS

MS MS MR –

MS-MR MS-MR VS MR

VS VS MS-MR –

MS MS-MR S MS – MS-MR

S-R S-R – MR – –

MR MR MR MR – MR

MS-MR MR VS – S MR

S S – – – MR

MS-MR R MS

MR – MR

MR MR MR

MS-MR VS –

S – –

R R R R R MR MR MR MS MS-R

R MS R R R MS R S-R MS MS

MR MR MR MR MR MR MR MR MR MR

MR – R VS MS-MR – R – MS-MR MS-MR

S – R R MS-MR – MS-MR – – –

Circum-Mediterranean P. amygdaliformis P. elaeagrifolia P. gharbiana P. longipes P. mamorensis P. syriaca Mid-Asian P. pashia P. regelii P. salicifolia East Asian P. betulifolia P. bretschneideri P. calleryana P. dimorphophylla P. fauriei P. hondoensis P. kawakamii P. pseudopashia P. pyrifolia P. ussuriensis a

References: Bell (1990), Bell et al. (1996), Hancock and Lobos (2008), Lombard and Westwood (1987), Westwood (1982) Degrees of response: VS very susceptible, S susceptible, MS moderately susceptible, MR moderately resistant, R resistant, – no data

b

have been developed that are sufficiently cold hardy to survive in the harsh prairie winter, but none of them are grown elsewhere to any extent. Recently, the cultivars ‘Luscious’ (Peterson et al. 1973) and ‘Gourmet’ (Peterson and Waples 1988) were released from the South Dakota Agricultural Experiment Station, and ‘Summercrisp’, from the Minnesota State Agricultural Experiment Station (Luby et al. 1987). These cultivars are recommended for northern regions and have higher quality than cultivars developed earlier at those stations. In Canada, breeding and testing for cold hardiness in pear has been done by public and private plant breeders in Ontario, Manitoba, Saskatchewan, and Alberta. The most promising cultivars that combine cold hardiness with size and quality are hybrids of P. communis P. ussuriensis. The most promising cultivars for the prairies include ‘Golden Spice’, ‘Olia’, ‘David’,

‘John’, ‘Peter’, ‘Philip’, ‘Pioneer 3’, ‘Tait Dropmore’, and ‘Tioma’ (Morrison 1965). The cultivar ‘Ure’ was released from the Research Station at Morden (Ronald and Temmerson 1982). None of these cultivars is as high quality as the P. communis cultivars grown in the main pear growing regions. Nevertheless, they are valuable sources of germplasm for improving cold hardiness in pear, because they are able to withstand winter temperatures, which are often as low as 30 to 40 C, and their fruit quality is superior to their P. ussuriensis parents. Cold hardiness of pear cultivars, species, and interspecific hybrids has been assessed in various European countries, especially after unusually severe winters (Ludin 1942; Anjou 1954; Enikeev 1959; Zavoronkov 1960; Matjunin 1960; Sansavini 1967). In general, the cultivars of P. communis are less hardy than those of P. ussuriensis. Greatest progress in breeding for cold

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Table 8.5 Adaptation of Pyrus species to abiotic stressa,b Species Climatic factors Edaphic factor Low chill Cold hardiness Low pH High pH European P. caucasica P. communis P. cordata P. nivalis

Wet soils

Dry soils

Sandy soils

Clay soils

L M-H H L

H M-H L H

H M L –

H M H –

H M-H L –

H M H –

H H H M-H

H L-H L M-H

VH L M M M M

L H L H L L

L M – M H L

VH VH M – – M

L M – H – –

H H H H – H

H H H – – M

M H M – – –

VH – –

S M M

VH – –

S M M

– – –

– – M-H

M-H M M

H – –

H – VH L H S VH L S L

M-H – L M H M S H M VH

H – H H H M M – M M

L – L L S-L M – – L L

H-VH – H-VH H H-VH – M – L L

VH – H L L – – – – M

H – M-VH – M – M – H M

VH – H-MH – H-VH – M – L M

Circum-Mediterranean P. amygdaliformis P. elaeagrifolia P. gharbiana P. longipes P. mamorensis P. syriaca Mid-Asian P. pashia P. regelii P. salicifolia East Asian P. betulifolia P. bretschneideri P. calleryana P. dimorphophylla P. fauriei P. hondoensis P. kawakamii P. pseudopashia P. pyrifolia P. ussuriensis a

References: Bell (1990), Bell et al. (1996), Hancock and Lobos (2008), Lombard and Westwood (1987), Westwood (1982) Degrees of response: S susceptible, L low, M moderate, H high, VH very high, – no data

b

hardiness has been made by crossing hardy selections of P. ussuriensis with good quality cultivars of P. communis.

8.6.1.5 Fire Blight Several pear breeding programs have used Asian species to breed for fire blight resistance. These programs have been reviewed by van der Zwet and Keil (1979). Breeding for fire blight resistance in pear began in the nineteenth century after the introduction of Chinese sand pears (P. pyrifolia) to the eastern United States (Hedrick et al. 1921). The cultivars ‘Le Conte’, ‘Kieffer’, and ‘Garber’ were derived from interspecific hybridization of P. pyrifolia with P. communis and were grown because they were substantially more resistant to fire blight than the European cultivars of

P. communis, but they were inferior in terms of fruit quality. During 1925–1960, a major pear breeding program was conducted in Tennessee. About 1933, McClintock discovered a resistant pear seedling and later named it ‘Late Faulkner’ (Drain 1943). Since 1925, the work consisted mainly of crossing resistant species, primarily P. pyrifolia, and also included P. calleryana and P. ussuriensis, with the more resistant cultivars of P. communis. From 1945 to 1966, several pear cultivars were introduced from this program, including ‘Ayres’, ‘Dabney’, ‘Carrick’, ‘Hoskins’, ‘Mericourt’, ‘Mooers’, ‘Morgan’, and ‘Orient’. A review of this breeding program was prepared by Deyton and Cummins (1991). Between 1942 and 1968, another large pear breeding program was conducted at the University of Illinois at Urbana. Pear species and cultivars had been

8 Pyrus

tested since 1919 (Anderson 1920). Hough (1944) used primarily cultivars and selections of Chinese species, such as P. bretschneideri cv. Pai Li and P. ussuriensis 76, and crossed them with several P. communis cultivars. The pear breeding program at Rutgers University was initiated by Bailey and Hough (1961, 1962) in 1948. It provided some very interesting and important blight-resistant selections, many with P. pyrifolia and P. ussuriensis parentage. An apparently blight-resistant P. pyrifolia seedling, NJ 1, was used extensively in this breeding program. In 1968, Hough and Bailey (1968) introduced three new blight-resistant pear cultivars for the fresh market, named ‘Star’ and ‘Lee’ (both from a cross of ‘Beierschmitt’ NJ 1) and ‘Mac’ (‘Gorham’ NJ 1). A few years later in Maryland, ‘Star’ and ‘Lee’ were found to be susceptible, whereas ‘Mac’ was confirmed to be moderately resistant (Oitto et al. 1970; van der Zwet et al. 1974). The program at Cornell University utilized another selection from the Illinois program, P. ussuriensis 65, as a source of fire blight resistance. No cultivars have been derived from those crosses, but many selections have proven to have resistance to fire blight and pear psylla and have been used in other programs for that combined purpose. Like most breeding programs for fire blight resistance, the program of the US Department of Agriculture (USDA) initially emphasized on P. communis sources of resistance, with less use of other species. For the past 20 years, advanced backcross selections derived from NJ1, ‘Pai Li’, and P. ussuriensis 76, and more recently P. bretschneideri cultivars have been employed as parents. In 2006, ‘Sunrise’, an early maturing cultivar, which has both ‘Seckel’ and NJ1 in its pedigree as sources of resistance, was released. The program at Purdue University has also used P. ussuriensis 76 as a source of resistance, but no cultivars derived from this selection have been released. The only cultivar derived from an interspecific (P. pyrifolia) ancestry is ‘Green Jade’ (Janick 2004). The Romanian pear breeding program at ClujNapoca, Pitesti-Maracineni, and Voinesti have utilized a P. pyrifolia clone as a source of moderate tolerance to fire blight (Andreies¸ 2002; Branis¸te et al. 2008). The program in Cluj-Napoca has identified sources of resistance in P. pollveria, P. lindeyi, P. malifolia, P. persica, P. ussuriensis, and P. variolosa (Sestras et al. 2008).

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8.6.1.6 Fungal Leaf and Fruit Pathogens Resistance to Fabraea leaf and fruit spot has been at least a secondary objective of a few breeding programs. Drain (1954) noticed resistance in ‘Mooers’ and ‘Hoskins’, and resistance to this disease was an added benefit of the effort to breed for fire blight resistance. The USDA program has surveyed and studied resistance to Fabraea in its germplasm and breeding populations, identified sources of resistance in P. communis and hybrids with P. ussuriensis (Bell and van der Zwet 2005), and incorporated some of these genotypes into its main program for fire blight and pear psylla resistance. It is unfortunate that one of the most promising selections for pear psylla resistance, NY 10353, is very susceptible to Fabraea. Resistance to both Fabraea and Mycosphaerella sentina (Fckl.) Schroet from P. pyrifolia has been incorporated into the Romanian pear breeding program (Andreies¸ 1983). Resistance to European pear scab has been a primary or secondary objective of European pear breeding programs (Bellini and Nin 2002), including those in Germany and Australia, although most programs use P. communis as source of resistance, One exception is the Romanian program at Voinesti-Dimbovita, which has utilized resistance from P. pyrifolia (Andreies¸ 1983). Pear leaf spot, caused by M. sentina, is a minor disease of pear. Resistance has been reported in at least 15 P. communis cultivars (Bell et al. 1996), based on epiphytotic conditions, and resistance of other species has apparently not been reported.

8.6.1.7 Pear Psylla Resistance to pear psylla has been a major objective of the USDA program, which has utilized P. ussuriensisderived resistant selections obtained from the Cornell and Rutgers University programs as well as P. bretschneideri in addition to P. communis cultivars (Bell and van der Zwet 1998). The programs of the University of Bologna, Agriculture and Agri-Food Canada and the Institut National de la Recherche´ Agronomique (INRA) have also utilized the Cornell selections. The Romanian program has utilized its P. pyrifolia selection as a source of resistance as well as local cultivars or P. communis (Branis¸te et al. 2008).

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8.6.1.8 Pear Rootstocks Lombard and Westwood (1987) reviewed the utilization of Pyrus species as rootstocks throughout the world. In addition to P. communis, they list P. betulifolia, P. calleryana, P. caucasica, P. communis var. pyraster, P. elaeagrifolia, P. kawakamii, P. pashia, P. pyrifolia, and P. xerophila as used for rootstocks. In Syria, P. syriaca is also used as a rootstock, where it is adapted for low pH soils and drought (Al Maarri et al. 2007). Most breeders of rootstocks for European pear have utilized P. communis or Cydonia oblonga L. germplasm. However, the INRA program in France has utilized P. nivalis and a clone listed as P. heterofolia (Simard et al. 2004). The latter species name is not a recognized taxon, and the clone appears to be an interspecific hybrid with characteristics of P. pyrifolia or P. bretschneideri and P. betulifolia. In addition, a joint program of INRA and the Spanish agency IRTA is breeding for low pH soils by using P. amygdaliformis, P. cordata, and P. elaeagrifolia as parents (Bonany et al. 2005). Low pH soils cause limeinduced chlorosis in pear trees on Cydonia rootstocks favored for their ability to induce reduced stature and precocious bearing.

8.6.2 Intergeneric Hybrids Karpov (1966) and Rudneko (1978) reviewed works on intergeneric hybridization at the I. V. Micurin Central Genetical Laboratory that included crosses between Malus baccata and pear and crosses between mountain ash (Sorbus spp.) and European pear. In general, such wide crosses are incompatible, for example, due to the degeneration of pollen tubes in the upper third of the style of the Sorbus parent (Panfilkina 1976). Putative intergeneric hybrids were produced by pollinating the apples ‘Kassel Reinette’ and ‘Golden Winter Pearmain’ with mixtures of European pear, quince, and Amelanchier pollen (Nikolenko 1962). Although most seedlings resembled the apple parent, three with intermediate morphological features bore pear-like fruit. A few seedlings derived from hybridizing a tetraploid clone of ‘Fertility’ pear with the tetraploid apple selection BM2812 have also been obtained in Sweden (Nybom 1957). In China, the intergeneric cultivar ‘Ganjin’ was produced from a cross of ‘Red Delicious’ apple and the

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pear ‘Pingouli’ (P. pyrifolia) (Zhang et al. 1991). In Korea, compatible mentor pollen was used to produce apple pear hybrids, with a 75% germination rate with apple as the seed parent and a 17% germination rate for the reciprocal cross (Shin et al. 1989). Experiments by Kim et al. (2004) indicated that using P. pyrifolia as the seed parent resulted in higher seed set and germination than the reciprocal cross. Three S-RNase alleles were screened by PCR-RFLP to confirm hybrid status. In addition to true hybrids, intergeneric crosses of Pyrus communis with Chaenomeles japonica (Thunb.) Lindl. ex Spach (Japanese quince) and Malus pumila have produced apomictic seedlings (Dolmatov et al. 1998), as have crosses between P. pyrifolia and Malus pumila (Inoue et al. 2002). Although intergeneric hybrids have generally been artificially produced, a naturally occurring apple European pear hybrid has been reported (Dimitrov and Delipavlov 1976) and Sorbopyrus auricularis is apparently naturally occurring. Hybridization of Japanese pear (P. pyrifolia) and Malus pumila resulted in production of some small seeds, which germinated at the reduced frequency of 71%, but all seedlings died within 6 months (Shimura et al. 1980). Sokolova (1970) reported that using either young (presumable immature) or old pistils should be pollinated rather than mature pistils to increase the chances of overcoming incompatibility in intergeneric crosses. Methods of enhancing the success of intergeneric hybridization include pollen irradiation (Jakovlev et al. 1968), and in vitro culture of seeds or embryos (Jakovlev et al. 1971; Banno et al. 2003; Papikhin et al. 2007; Sun and Leng 2008). The treatment of stigmas with a low concentration of boric acid, gibberellin, or succinic acid prior to pollination has improved hybridization of mountain ash with pear (Shcherbenev 1973, 1975). Growing hybrid seedlings at high temperature (34 C) overcame symptoms of hybrid lethality, but the high temperature eventually killed the seedlings (Inoue et al. 2003). Intergeneric hybrids between Sorbus aria and P. communis are at least partially sterile, and meiosis is irregular (Sax and Sax 1947). Pakhomova (1971) noted a failure of pairing of most chromosomes, formation of univalents and multivalents, unequal and asynchronous separation, and irregular tetrads and polyads in Malus baccata P. communis hybrids. However, gamma-irradiation of a hybrid induced chromosome doubling in the microspores, albeit with

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some chromosome abnormalities (Pakhomova 1974). Gonai et al. (2006) also used gamma-irradiation of shoots of putative hybrids to overcome hybrid lethality. Marker analysis using SSRs confirmed the hybrid nature of the lone survivor. Rudenko (1974) reported that apple pear F2 hybrids had either diploid, triploid, or tetraploid chromosome complements. Pistil cutting has been reported to improve hybridization success between apple and P. pyrifolia (Li et al. 1997). Fruits from intergeneric apple European pear crosses have been small and irregular in shape with few seeds per fruit (Inozemtsev 1972). The seeds were generally of low viability. The structure of the pericarp of hybrids between Malus baccata and the pear ‘Michurin’s Winter Beurre’ (‘Bere Zimnyaya Michuriina’) was intermediate between the apple and pear parents, and grit cell content was much reduced (Gorshkova 1980). Hybrids of Malus baccata P. communis have been shown to be highly resistant to apple scab, Venturia inaequalis (Cooke) G. Wint. (Gorshkova and Vanin 1973). Hybrids between ‘Fuji’ apple and ‘Oharabeni’ pear generally resembled the pear parent. The five hybrids showed resistance to apple blotch, apple scab, pear scab, and pear rust. Rudenko (1985) reported that Pyrus Cydonia hybrids were produced as early as 1916. Later work in Moldvia resulted in additional hybrids. The chromosome number was diploid (2n ¼ 34), and inflorescences were intermediate between Pyrus and Cydonia in that they had 2–3 flowers. An artificial hybrid of the European pear Pyrus pyrifolia and Cydonia oblonga, Pyronia veitchii, has been produced (Shimura et al. 1983). The clone of Pyronia veitchii var. luxemburgiana was backcrossed to pear in an attempt to produce a rootstock for pears (Rogers 1955). In addition to the production of novel hybrids, intergeneric crosses have been used to develop maternal pear haploids using irradiated apple pollen (Inoue et al. 2004). The authors also attempted to obtain apomictic seedlings through crossing ‘Gold Nijisseiki’ Japanese pear and apple using non-irradiated pollen. Only one of the 53 seedlings survived.

8.6.3 Sources of Other Desirable Traits Westwood (1982) reviewed the general ratings of Pyrus species for a large number of biotic and abiotic

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stresses and adaptations. Additional extensive literature reviews have been compiled by Bell (1990), Bell et al. (1996), and Hancock and Lobos (2008). Levels of resistance of Pyrus species to diseases and arthropod pests and adaptation to various climatic and edaphic conditions are given in Tables 8.3–8.5, respectively.

8.6.4 Role in Crop Improvement Through Advanced Tools 8.6.4.1 Pear Breeding Through MarkerAssisted Breeding A long juvenile period and high level of heterozygosity due to a strict gametophytic incompatibility have limited the parental combinations in pear breeding programs. Marker-assisted selection (MAS) is considered to be a powerful tool for increasing selection efficiency by identifying favorable genetic combinations in fruit trees as documented in other crops. The major advantage of MAS is the ability to evaluate many traits at the seedling stage in fruit trees that have a long juvenile phase. Especially, MAS in pear breeding programs can be particularly important for traits that are difficult to evaluate. However, available markers for MAS are limited in Pyrus.

Disease Resistance Fire blight caused by Erwinia amylovora is the most harmful disease in North America and Europe. Fire blight continues to spread throughout western, central and southern Europe despite quarantine measures adopted (Jock et al. 2002). Different levels of susceptibility to fire blight exist in European pear cultivars. Fire blight resistance in pear is known as a quantitative trait (Dondini et al. 2002). Dondini et al. (2004) constructed two genetic linkage maps of the parental lines ‘Passe Crassane’ (susceptible) and ‘Harrow Sweet’ (resistant) using SSRs, MFLPs, AFLPs, RGAs, and AFLP-RGAs markers and conducted QTL analysis for fire blight resistance. QTL analysis identified four regions (LGs 2, 4 and 9: 2 QTLs in LG2) of ‘Harrow Sweet’ associated with fire blight resistance, while no

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QTLs related to resistance were found in susceptible ‘Passe Crassane’. About 50% of the total variance was due to four QTLs, and two QTLs on LG2 showed large LOD values (Dondini et al. 2004). Pear scab, caused by two species of Venturia, Venturia nashicola and Venturia pirina, is one of the most important diseases of Asian and European pears. V. nashicola is pathogenic only on Asian pears, not on European pears, while V. pirina is pathogenic only on European pears, not on Asian pears (Bell et al. 1996; Ishii et al. 2002). The positions and linkage groups (LGs) of the genes for resistance to scab were identified in Asian and European pears (Terakami et al. 2006; Pierantoni et al. 2007). The major resistance gene Vnk of the Japanese pear cultivar ‘Kinchaku’ against V. nashicola was identified in the central region of LG1 (Terakami et al. 2006). Six markers (one SSR: Hi02c07 and five STSs derived from AFLP and RAPDs: STS-OPW2, STS-OPAW13, STS-OPO9, STS-CT/CTA, and STS-OPAQ11) showed tight linkages to Vnk, being mapped with distance ranging from 2.4 to 12.4 cM. Gonai et al. (2009) reported that STS-OPW2 and STS-OPO9 could be useful for MAS for pear scab resistance by introducing Vnk from ‘Kinchaku’. In contrast, while a single dominant gene for V. pirina resistance has not been found in European pear cultivars, there is evidence of polygenic resistance (Chevalier et al. 2004). Pierantoni et al. (2007) reported the position of two putative QTLs related to scab resistance in two F1 population maps (‘Abbe Fetel’ ‘Max Red Bartlett’) being located on LG3 and 7. The two QTLs explained 88% of the phenotypic variance and the LOD values were higher than 10, suggesting the involvement of these two major genes in V. pirina resistance (Pierantoni et al. 2007). Black spot disease, which is caused by Alternaria alternata Japanese pear pathotype, is one of the most serious diseases in Japanese pear cultivation. The susceptibility to black spot disease is controlled by single dominant gene designated as A (Kozaki 1973). Banno et al. (1999) tested 250 RAPD primers to screen a pair of bulked DNA samples derived from open-pollinated progenies of Japanese pear ‘Osa Nijisseiki’ to identify markers linked to the susceptible A gene of black spot disease. The CMNB41 primer generated a 2,350 bp fragment, which was present in the susceptible bulk but not in the resistant one. This RAPD marker, CMNB41/2350, was at a distance of about 3.1 cM

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from the susceptible A gene. They found that the frequency of occurrence of the CMNB41/2350 marker was 96% in susceptible cultivars and progenies of ‘Osa Nijisseiki’ ‘Oharabeni’. More recently, the exact positions and linkage groups of the genes for susceptibility to black spot were identified in two Japanese pear cultivars, ‘Osa Nijisseiki’ (designated as Ani) and ‘Nansui’ (Ana) (Terakami et al. 2007). Ani and Ana were located at the top region of LG11 and linked to two SSR markers CH04hO2 and CH03d02.

Insect Resistance Dysaphis pyri is an important aphid pest of P. communis, and no cultivaris are currently reported to be resistant. Evans et al. (2008) screened microsatellite markers with a progeny of ‘Doyenne du Comice’ an accession of P. nivalis to identify markers linked to the major gene (Dp-1) for resistance to D. pyri. They found that Dp-1 is flanked by NII006b and NII014 on linkage group 17, 2.3 and 3.6 cM away, respectively.

Fruit Quality Ethylene production by cultivated Japanese pear fruits varies from 0.1 to 300 nl g1 h1 during fruit ripening, suggesting that there are both climacteric and non-climacteric cultivars. Climacteric-type fruits exhibit a rapid increase in ethylene production and have a low storage potential, while non-climacteric fruits show no detectable ethylene production and fruit quality maintained for over a month in storage. Fruit storage potential is closely related to the maximum level of ethylene production in Japanese pear. Itai et al. (1999, 2003b) have cloned three ACC (1-aminocyclopropane-1-carboxylate) synthase genes (PpACS1, 2, 3), and studied their expression during fruit ripening. PpACS1 was specifically expressed in cultivars of high ethylene production, while PpACS2 was specifically expressed in cultivars of moderate ethylene production. Moreover, they have identified RFLP markers linked to the ethylene evolution rate of ripening fruit using RFLP analysis with two ACC synthase genes (PpACS1 and PpACS2). RFLPs were designated as A (2.8 kb of PpACS1) linked to high levels of ethylene (>10 nl g1 h1) and B (0.8 kb of

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PpACS2), linked to moderate levels of ethylene (0.5–10 nl g1 h1), when the total DNA was digested by HindIII. These markers (A and B) are useful for the selection of Japanese pear cultivars with enhanced post-harvest keeping ability. These markers were converted to more convenient and easier PCR-based CAPS markers (Itai et al. 2003a). Using this CAPS system, a total of 152 cultivars were categorized into four marker types (AB, Ab, aB, ab); types AB and Ab show high levels, aB a moderate level, and ab a low level of ethylene production during fruit ripening (Itai and Fujita 2008). Furthermore, linkage analysis of these two markers were conducted in the F2 populations derived from selfpollinated OT16, an F1 of ‘Osa Nijisseiki’ (a selfcompatible mutant of ‘Nijisseiki’)’ ‘Cili’, which revealed that the recombination frequency between the two markers was 20.8 3.6%. F2 populations in Pyrus have not been reported so far because of a strict gametophytic self-incompatibility. These are the first populations of self-pollinated F2 in Pyrus species. Pears are mainly marketed and served as fresh fruit and must have an attractive appearance. The fruit color is the most important factor for the fruit appearance. There are wide variations in skin colors. In Japan, yellow-green and brown russet pears are preferred. Inoue et al. (2006) reported the RAPD markers linked to major genes controlling the fruit skin color in Japanese pear. Two F1 progenies from the cross of ‘Kousui’ ‘Kinchaku’ and ‘Niitaka’ ‘Chikusui’ segregated by fruit skin color were used for segregant analysis. They tested 200 RAPD primers against four bulks and the OPH-19 primer generated a 425 bp fragment, which is present in the green-skin bulk. This RAPD marker (OPH-19425) had a recombination frequency of 7.3% from the green skin phenotype. This marker could select green fruit with probability as high as approximately 92%. In European pears, red-colored fruits have considerable eye appeal for consumers. Efforts have been made to select red-skinned sports and seedlings. The inheritance of the red color character was studied in seven progeny obtained by using cultivars with red skin and non-red skin (Dondini et al. 2008). One of these progenies (derived from the cross ‘Abbe Fetel’ ‘Max Red Bartlett’, a red bud sport of ‘Bartlett’) was used to construct two linkage maps and red color was confirmed as a monogenic dominant trait, being located on LG 4 of ‘Max Red Bartlett’ (Dondini et al. 2008). The Red gene is

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positioned between two AFLP markers, E31M56-7 and E33M48-5, at a distance of 13.5 cM and 18.2 cM, respectively.

Self-incompatibility Most pear cultivars have been classified as selfincompatible. Therefore, the presence of pollinizers interplanted in the orchard is a requirement to get an economic crop from most of the cultivars (Sanzol and Herrero 2002). The progression of our understanding of incompatibility in Pyrus has accelerated greatly since the mid-1990s. In Pyrus, gametophytic selfincompatibility is controlled by a single polymorphic gene locus, represented by the S-locus. The S-locus harbors a multi-allelic gene, which encodes for S-RNase that blocks incompatible-tube growth through the style (Ushijima et al. 1998). In Japanese pear, cDNAs encoding S1- to S10-RNase have been isolated and sequenced (Sassa et al. 1997; Ishimizu et al. 1998; Takasaki et al. 2004; Kim et al. 2006). Based on the nucleotide sequences, Ishimizu et al. (1999) established a PCR-RFLP (S1- to S7-) system for S-genotype assignments in Japanese pear. Takasaki et al. (2004) modified this system and established the system for discriminating S1- to S9-allele in Japanese pear. Kim et al. (2007) also established a new PCR-RFLP system for the determination of S-genotypes (S1- to S10-) of Japanese pear. In Japanese pear, a naturally occurring self-compatible mutant cultivar, ‘Osa Nijisseiki’, was found as a bud sport mutant of an self-incompatible cultivar ‘Nijisseiki’. The S-genotype of ‘Osa Nijisseiki’ was referred to as S2S4sm, with compared to S2S4 of ‘Nijisseiki’ (Sassa et al. 1997). Recent molecular analysis suggested that the mutation of ‘Osa Nijisseiki’ was due to the lack of S4-RNase gene expression in the style and was caused by a large deletion of a 236-kb region around S4-RNase (Okada et al. 2008). The pollen-S determinant was a long-standing puzzle of the S-RNase-mediated self-incompatibility in the Rosaceae. Recently, the pollen determinant of S-specificity in the Rosaceae was found to be an F-box protein (Ushijima et al. 2003). Sassa et al. (2007) isolated multiple F-box genes (PpSFBBs) in a genome region in ‘Osa Nijisseiki’ pear. These PpSFBBs are good candidates for the pollen-S determinant in pear. Based on the S-allele-specific sequence polymorphism of PpSFBB-genes, the most conserved SFBB in Japanese

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pear, a new S-genotyping system, has been constructed (Kakui et al. 2007). Both S-alleleic constitution and cross-incompatibility groups have been determined for many Japanese pear cultivars, although the situation contrasts with the scarce information available in European pear. In Asian countries, artificial pollination is often used for stable production, therefore knowing Sgenotype of commercial cultivars is very important, in comparison with open-pollination in Europe. Recently, molecular techniques have been developed for the identification of S-genotypes in European pears (Sanzol and Herrero 2002; Zuccherelli et al. 2002; Zisovich et al. 2004; Takasaki et al. 2006; Moriya et al. 2007). Six S-alleles (Sa- to Sh-) were identified using 10 cultivars by Zuccherelli et al. (2002), four S-alleles (S1- to S4-) were identified using seven cultivars by Sanzol and Herrero (2002), and seven S-alleles (Si- to So) were identified by Zisovich et al. (2004). Takasaki et al. (2006) isolated the full length cDNAs of nine S-RNases (Sa- Sb-, Sd-, Se-, Sh-, Sk-, Sl-, Sq-, and Sr-) and established a CAPS marker system for genotyping European pear cultivars harboring these nine alleles. Moriya et al. (2007) found new S-alleles (Sg- Ss-, and St-) and modified the CAPS marker system for S-genotyping of cultivars harboring 17 S-alleles. Using the CAPS analysis, they have assigned a total of 95 cultivars to 48 genotypes. Both the methods and the determination of S-genotypes will facilitate stable pear production.

8.6.4.2 Genetic Engineering Agrobacterium-mediated pear transformation was first demonstrated by Mourgues et al. (1996) and since then has been demonstrated by several groups around the world using several different pear cultivars (Reynoird et al. 1999; Malnoy et al. 2000, 2003, 2005a, b; Lebedev et al. 2002a, b, c; Bell et al. 1999; Gao et al. 2007; Tang et al. 2007; Wen et al. 2008). This research dealt mostly with European pears. Agrobacterium-mediated transformation was used to introduce various transgenes largely aimed at providing in fire blight resistance, including attacin E in ‘Passe Crassane’ (Reynoird et al. 1999), T4 lysozyme in ‘Passe Crassane’ (Malnoy et al. 2000), defensin Rs-AFP2 in ‘Burakovka’ (Lebedev et al. 2002a), phosphinotricine acetyl transferase (PAT) in the rootstock GP217 (Lebedev et al. 2002b), lactoferrin in ‘Passe Crassane’

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(Malnoy et al. 2003), EPS-depolymerase in ‘Passe Crassane’ (Malnoy et al. 2005a), and Harpin-Nea in ‘Passe Crassane’ (Malony et al. 2005b). Other studies have focused on plant architecture by introducing rolB and rolC genes from Agrobacterium rhizogenes (Bell et al. 1999; Zhu et al. 2003), insect resistance (Tang et al. 2007), taste improvement by the thaumatin II gene (Lebedev et al. 2002c), fruit ripening by the ACC oxidase gene (Gao et al. 2007), abiotic stress tolerance by spermine synthase (Wen et al. 2008), and human health benefit by stilbene synthase (Flaishman et al. 2005). While potentially improved forms of existing elite cultivars have been produced, years of field trials, product testing, and public acceptance are still required before genetically engineered pears reach the marketplace. In spite of many obstacles, transgenic studies will bring useful tools to assist the creation of cultivars better adapted to the future requirements for stable production in this species.

8.7 Genomic Resources Developed Information on genomic resources of Pyrus is limited when compared to Malus and Prunus. To date, over 1,200 sequencing data entries have been recorded on GenBank, EMBL, and RefSeq. These include a collection of expressed sequence tags (ESTs), expressed genes, and molecular markers. GenBank currently lists 1,215 nucleotide sequences derived from 22 taxa and one Malus domestica P. communis hybrid (National Center for Biotechnology Information 2009). The most entries are found for P. communis and P. pyrifolia. Entries for ESTs currently number 888, with most derived from P. pyrifolia (606), followed by P. communis (244). There are 132 probe sequences published in GenBank. Recently, the National Institute of Fruit Tree Science in Japan has initiated an EST project with the goal of developing the unique expressed gene set for Japanese pear. Current efforts have centered on sequencing over 25,000 cDNAs from libraries of developing and ripening fruit, flowers, leaves, buds, and shoots. These efforts have resolved into 10,350 unigenes (Nishitani et al. 2009). Di-nucleotide and tri-nucleotide repeats, which should be useful as genetic markers, were found. As an additional potential marker type, singlenucleotide polymorphisms (SNPs) among P. communis,

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P. bretschneideri, and P. pyrifolia were discovered. Sequencing of other sources is in progress. This research will provide the potential to speed up the process of gene discovery and characterization. Most of these genomic resources have been developed in P. communis or P. pyrifolia. Most studies of gene expression have dealt with fruit physiology. In addition, a cDNA library of genes expressed during infestation of pear psylla-susceptible (P. communis cv. Bartlett) and pear psylla-resistant (P. ussuriensis P. communis BC1 hybrid NY10355) pears was constructed by suppression subtractive hybridization (Salvianti et al. 2008). Fourteen genes were differentially expressed in ‘Bartlett’ and 27 were differentially expressed in NY10355, many of which are known to be involved in response to biotic and abiotic stresses. Further research is needed to determine whether any of these can be used to develop markers for resistance.

8.8 Scope for Domestication and Commercialization Aside from the major species cultivated for fruit – P. communis, P. pyrifolia, P. bretschneideri – and perhaps P. pashia, other Pyrus species will likely only be used as sources of specific donor traits in breeding rather than as cultivated crops in their own right. One major exception to that statement is that other species may have direct utility as rootstocks for the cultivated species or as sources of specific traits in breeding rootstocks. Another exception is the use of selected clones and seedlings of P. calleryana, P. fauriei, and P. salicifolia as ornamental tree cultivars. The importance of Pyrus species as sources of important dietary and therapeutic compounds has not been thoroughly investigated. A few studies of the level of phenols and total antioxidant activity of P. communis, including variation in total phenolics among cultivars, have been reported (Imeh and Khokhar 2002; Sun et al. 2002), but comprehensive data on other species is apparently lacking. Pear ranks lower than apple, but higher than orange, in total antioxidant activity. Hou et al. (2003) reported that Pyrus taiwanensis was relatively low in free radical scavenging activity.

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8.9 Some Disadvantages of Related Species The invasive potential of P. calleryana, either through intraspecific (Culley and Hardiman 2009) or interspecific (Vincent 2005) hybridization is becoming welldocumented. The use of clones of P. calleryana as ornamental trees in the United States has lead to escape from cultivation through open-pollination between cultivars with differing S-allele genotypes and with other species such as P. communis. Presumably, use of P. fauriei, the Korean pea pear, as a dwarf ornamental could lead to similar invasiveness for that species. Even though these species are not used in breeding for fruit bearing scion cultivars, their extensive use in the landscape poses a problem. The potential for invasiveness may be alleviated by genetic engineering with gene systems, which result in pollen or seed sterility. Pears can cause allergic responses in sensitive people. This response has been documented for fruit of European pear, P. communis, and the gene responsible for the allergen, Pyr c 1, has been cloned (Karamloo et al. 2001). However, there are, to date, no reports of other Pyrus species having been assayed for the occurrence of this gene and allergen. However, pollen of Pyrus pyrifolia has been shown to cause an allergic reaction, known as pollenosis, in Japanese pear growers, especially those workers involved in handpollination (Teranishi et al. 1988).

8.10 Some Recommendations for Future Action More extensive information on the current distribution, status, and genetic diversity of wild populations of Pyrus species is needed to assess the risk of genetic erosion in the potential gene pool for pear improvement. Greater efforts for in situ and ex situ preservation must follow, as must efforts to characterize and evaluate collected and native germplasm for economically important traits. Greater international cooperation and access to genetic resources would be desirable, while taking into consideration the perspectives and needs of both germplasmrich and germplasm-deficient countries.

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Studies of the mode of inheritance to define narrowand broad-sense heritabilities for these traits, as well as to determine breeding values for prospective parents, should be undertaken for those traits for which such information is lacking. Interdisciplinary studies of the underlying physiological mechanisms of important traits are also needed. This is especially true of rootstock breeding, in which the mechanism(s) controlling tree size, precocity, yield, and tree architecture are not known. The foregoing types of studies should be combined with developing genomic technologies and resources within Pyrus to define the genetic architecture of traits and generally advance genetic improvement within the domesticated species. Doubled haploid lines will prove valuable in sequencing and gene isolation, and further work to characterize existing aneuploid genotypes may prove to be valuable additions to quantitative trait loci (QTLs) and association mapping to locate and isolate important genes and chromosome regions for important traits. Expressed sequence tag (EST) libraries in Pyrus are not yet comparable in scope or number with many other crop species, even when compared to other Rosaceae. These need to be developed for several tissues, developmental stages, and major species, and cultivars.

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.

Chapter 9

Rubus J. Graham and M. Woodhead

9.1 Basic Botany of the Species The genus Rubus is composed of a highly heterozygous series of some 500 species, with a range of ploidy levels from diploid to duodecaploid (Jennings 1988; Meng and Finn 2002). Hundreds of species are divided botanically under 15 subgenera, many of which have been used in breeding (Jennings et al. 1991; Knight 1993; Finn 2001; Finn and Knight 2002; Finn et al. 2002). Members of the genus can be difficult to classify into distinct species for a number of reasons, including hybridization between species and apomixes (Robertson 1974; Dickinson et al. 2007; Evans et al. 2007). Cytological data are being generated and used in an attempt to gain insight into the relationships of the genus (Wang et al. 2008). The subgenus Rubus is divided into 12 sections with most of the cultivated blackberries being derived from the Allehgeniensis, Arguti, Flagellares, Rubus, Ursini, and/or Verotrivialis (Gustafsson 1943; Finn 2008). Red and black raspberries along with many of the wild harvested species from around the world are in the Idaeobatus subgenus. Commercially, the most important raspberries are the European red raspberry, R. idaeus L. subsp. idaeus, the North American red raspberry R. idaeus subsp. strigosus Michx, and the black raspberry (Rubus occidentalis L.). Rubus subgen. Idaeobatus is distributed principally in Asia as well as in East and South Africa, Europe, and North America. In contrast, subgen. Eubatus is mainly distributed in South America, Europe, and North America (Jennings 1988). The members

J. Graham (*) Scottish Crop Research Institute, Dundee DD2 5DA, UK e-mail: [email protected]

of subgenus Idaeobatus sp. are distinguished by the ability of their mature fruits to separate from the receptacle. The subgenus is particularly well represented in the Northern Hemisphere. The place of origin of raspberry has been postulated to be the Ide Mountains of Turkey (Jennings 1988). The center of diversity is considered to be in China, where there are 250–700 species of Rubus depending on the taxonomists (Thompson 1997). Jennings (1988) and Roach (1985) have given extensive accounts of early domestication. Records were found in the fourth century writings of Palladius, a Roman agriculturist, and seeds have been discovered at Roman forts in Britain; hence, the Romans probably spread cultivation throughout Europe. Rubus species are prostrate to erect, generally thorny shrubs producing renewal shoots from the ground (called canes). They are perennials only because each bush consists of biennial canes, which overlap in age. Leaves are compound with 3–5 leaflets, the middle one being the largest; margins serrate to irregularly toothed. Small (0.5–1.5 cm), white to pink flowers are initiated in the second year of planting. The gynoecium consists of 60–80 ovaries, each of which develops into a drupelet. There are 60–90 stamens. The flowers of Rubus are structurally rather similar to those of strawberries, with five sepals, five petals, a very short hypanthium, many stamens, and an apocarpous gynoecium of many carpels on a cone-like receptacle. They produce an aggregate fruit, composed of individual drupelets, held together by almost invisible hairs. In Rubus, each carpel will develop into a small drupelet, with the mesocarp becoming fleshy and the endocarp becoming hard and forming a tiny pit that encloses a single seed. Each drupelet usually has a single seed, though a few have two. Canes grow 1 year and fruit the next, but there are also primocane

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varieties, which fruit in the first year. The biennial growth cycle of stems begins when a bud from below soil level develops and elongation of the internodes carries the growing point, protected by leaf scales, to the soil surface. At the surface, leaves expand to form a tight rosette around the growing point. Elongation of the shoot starts in spring and continues until autumn, by which time the shoot will have attained a height of 2–3 m. In red raspberries (R. idaeus L.), shortening days and falling temperatures in late summer cause shoot elongation to cease and dormancy to set in. This is a gradual process extending over several weeks, and once a stage of complete dormancy is reached, it is not readily reversible. Black raspberries (R. occidentalis L.) or purple raspberries (hybrids between red and black raspberries) and most blackberries differ from red raspberries both in time when dormancy begins and intensity of dormancy attained. In these fruits, growth continues well into autumn. The initiation of flower buds usually starts at the same time as the canes begin to acquire dormancy. In the spring of the second year, vegetative primocanes become fruiting canes. The fruit is composed of a large number of one-seeded drupelets set together on a small conical core (Jennings 1988). Commercial blackberries are classified into three categories based on cane type: trailing, semi-erect, and erect (Strik 1992). Trailing types and semi-erect habit blackberries are crown-forming, and the primocanes trail on the ground surface until lifted and staked. Erect blackberries grow upright but less vigorously than the semi-erect types, and instead of being crown-forming, they sucker beneath the soil line. The genomic number of Rubus is seven and species representing all ploidies from diploid to duodecaploid are found in nature. The range in size is from 1 to 4 mm (Jennings 1988). The diploid genome has been estimated to be 275 Mbp. Self-incompatibility systems occur in some Rosaceous species and are common among many of the diploid Rubus species (Keep 1968). In contrast, all polyploid species are self-compatible as are the domesticated forms of the diploid raspberries. Rubus species are an important horticultural source of income and labor. In most countries, fruit from Rubus species is produced for the fresh market. Fruit for processing is usually used in the food and beverage industries where it is used to produce wine, beer, soft drinks, preserves, and desserts. Fruits may also be

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frozen or canned. Blackberry production is rapidly increasing (Strik 1992; Clark 2005; Clark et al. 2007; Strik et al. 2007) with an estimated 140,292 MT commercially harvested from 20,035 ha in 2005. Europe leads in the world in acreage (7,692 ha), while North America has the highest production (59,123 MT). Serbia (69%) dominates European production; however, a number of countries have significant production. In North America, the US, particularly the Oregon State, is the major producer. However, Mexican production has been rapidly increasing. California and Arkansas are the only other states in the US with over 1,000 MT production. Central American production (1,620 ha) is predominantly from Costa Rica and Guatemala where in addition to harvest from managed stands, a great deal is harvested from feral stands. South American production (1,597 ha) is predominantly from Ecuador and Chile. Asian production has been rapidly increasing with over 1,550 ha of new plantings, predominantly in China. The production in Oceania is mainly in New Zealand, although the area planted is small with only about 259 ha. African production is only reported in South Africa but has been initiated in Morocco, Algeria, Kenya, and possibly others. The bulk of the fruit is grown for processing applications in the Pacific Northwest US, Serbia, and China, whereas fresh market sales are the focus of the industry elsewhere. Raspberry is an important high-value horticultural industry in many parts of the world, providing employment directly in agriculture and indirectly in food processing and confectionary. Production is estimated at 482,763 MT (2005) (http://FAOSTAT. FAO.ORG). Europe is estimated to produce around half of all production of Rubus idaeus L. Most raspberry production is concentrated in the northern and central European countries, although there is an increasing interest in growing cane fruits in southern Europe, e.g., in Greece, Italy, Portugal, and Spain. The major production areas of red raspberries in North America are the Pacific Northwest (Oregon, Washington, and British Columbia), California, the eastern US (New York, Michigan, Pennsylvania, and Ohio), and a rapidly expanding industry in Mexico. While adapted to many of the same areas as the other cultivated Rubus, black raspberry (R. occidentalis) cultivation is concentrated in Oregon in the western US, in Ohio, Pennsylvania and New York in the eastern US, and in Korea.

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9.2 Conservation Initiatives A number of studies have been carried out to characterize the levels of genetic variation in wild species and to examine the turnover of wild populations. In the Tayside region of Scotland, a study of wild raspberry populations had been carried out over a 10-year period. Initially, Graham et al. (1997) examined the spatial genetic diversity in wild accessions of red raspberry from four sites. Most of the variability detected using random amplified polymorphic DNA (RAPD) markers was observed between the collection sites. Within sites, increasing diversity coincided with greater spatial separation. None of the wild populations were closely related to any of the commonly grown commercial cultivars and were all spiny. A larger study examined a wider range of wild R. idaeus from 12 sites across a greater area and compared the accessions to the cultivar “Glen Moy”. Again, greater genetic similarity was found within each population collected, which indicates a hindrance to gene movement across geographic locations. This barrier to gene flow was partly explained by a separation of flowering period, with altitude proving to be important here (Graham et al. 2003). Marshall et al. (2001) examined some of the wild accessions studied by Graham et al. (1997) to determine whether these populations were adaptively differentiated from each other. Plants were brought into a common environment and 20 traits assessed. A consistent north–south trend was identified confirming substantive differentiation between populations. Similar studies using phenotypic characteristics have been carried out on 12 wild raspberry populations in Russia (Ryabova 2007), where wild populations were examined for characteristics, which may be useful in cultivated raspberries. Ten years after these initial Scottish studies had been carried out, these wild red raspberry plants at 12 sites were reexamined for changes in population size and to address an earlier finding, which demonstrated significant population differentiation over a small scale (Graham et al. 2009a). Reductions in plant numbers was observed at almost all sites. Given that each population had unique alleles, which could be identified even in a small number of plants, this loss of plants also equates to a loss of alleles. The studies of Marshall et al. (2001) reinforce the value of this wild germplasm having demonstrated plants from

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these locations (spanning an altitudinal cline from 5 to 600 m) exhibit significant variation in flowering and fruiting period in a common environment. In the light of current climate change implications, these populations represent a huge genetic resource that could be utilized within commercial red raspberry breeding programs to develop new cultivars better adapted to a changing natural environment. R. idaeus wild accessions from a Lithuanian germplasm collection were examined for genetic diversity using RAPD loci (Patamsyte˙ et al. 2004). Soil acidity rather than geographic distance was significantly correlated to observed polymorphisms, indicating an environmental effect on diversity within populations. DNA probes from two variable number tandem repeat (VNTR) loci were utilized to examine diversity in wild populations of R. moluccanus L. in the Philippines (Busemeyer et al. 1997). The results were similar to that of Graham et al. (1997, 2003), finding greater similarity present within populations at each location than between locations. Additionally, apomictic reproduction was ruled out in these populations because no identical VNTR patterns were identified. Research on natural populations of arctic raspberry has shown genetic diversity at levels near 50% for among and within population estimates (e.g., LindqvistKreuze et al. 2003). Genetic diversity has been examined in natural populations of black raspberry (R. coreanus) in Korea using inter simple sequence repeat (ISSR) markers (Hong et al. 2003), and overall genetic relationships among populations were associated with geographic location. Black raspberry (R. leucodermis) populations have also been evaluated for traits of importance for use in red and black raspberry breeding (Finn et al. 2003). A study on 63 natural populations of Rubus strigosus across North America (Marking 2006) using chloroplast sequence and ISSR found the majority of the variation to be within populations (79.5%). Cloudberry (Rubus chamaemorus) is a highly valued berry in Scandinavia and northern Russia and has potential for domestication (Korpelainen et al. 1999). It reproduces primarily through clonal growth (Makinen and Oikarinen 1974) and although sexual reproduction occurs rarely, this is obviously important for colonizing new habitats. R. chamaemorus exhibits large morphological differences, but genetic studies on Finnish populations indicate that the levels of genetic diversity

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within populations were quite low, comprising 2–4 clonal genotypes per population (Korpelainen et al. 1999). This has implications for domestication and breeding programs because the clear morphological variability observed may be largely influenced by environmental conditions. Therefore, plants for breeding programs should be selected from populations located some significant distance apart (Korpelainen et al. 1999). A number of formal and informal gene bank collections exist around the world. These include a Rubus collection of over 140 species and 302 named cultivars and selections with seed or clones available on request, housed in the USDA-ARS National Clonal Germplasm Repository (Hummer and Finn 1999). Another Rubus collection of over 140 accessions is maintained in a field collection and in protected culture at the Canadian Clonal Genebank (Luffman 1993). A gene bank inventory is published annually. In the UK field, collections of over 150 accessions exist at SCRI in Scotland and at East Malling Research in England (Dolan personal communication). Collections resulting from botanical surveys in Columbia consist of ten Rubus species recorded in open and/or disturbed habitats (Rivera et al. 1997). Plant material and seeds from exploration trips in Sakhalin territory are stored in gene banks including an orange R. chamaemorus and a dark purple cloudberry (R. pseudochamaemorus) (Sabitov et al. 2007). Genotypes from seven Chinese provinces have been established in Jiangsu province and evaluated for a range of characteristics (Gu Yin et al. 1996). In Europe, efforts are being made to conserve the biodiversity of berries (Bartha-Pichler 2006) with an interest in the conservation of genetic resources. The “GENBERRY” project, partly funded by the European Community, has been designed to ensure that agricultural biodiversity of small berries is preserved, characterized, and used to improve varieties adapted to local European regions. Strawberry (Fragaria ananassa) and raspberry (R. ideaus) represent the two main cultivated small berries. The project is focusing on the construction of core collections, the development of a passport data list, the selection and definition of appropriate primary and secondary descriptors, characterization of genotypes using molecular markers, identification of health nutritional compounds and diseases evaluation for a large subset of the collections, and the establishment of the European

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small berries database sustained by a continuous long term network (Denoyes-Rothan et al. 2008). Romanian (Rusu et al. 2006a) and Bulgarian red raspberries (Badjakov et al. 2006) have been studied to determine their similarity with European and American germplasm using simple sequence repeat (SSR) markers. Badjakov et al. (2006) analyzed 28 raspberry genotypes from the Bulgarian germplasm collection including 18 Bulgarian cultivars and breeding lines, eight accessions from outside Bulgaria, and two wild species accessions, R. occidentalis and R. adiene, using RAPD markers. They created a genetic similarity tree with two clusters, which corresponded to two pedigree groups among the Bulgarian genotypes. They also analyzed the 28 accessions with four SSR loci, demonstrating high levels of diversity within the collection (Badjakov et al. 2006). Weber (2003) analyzed genetic diversity in cultivars of black raspberry (R. occidentalis) and red raspberry using RAPD markers and found that black raspberry genotypes showed on an average 81% genetic similarity. This compared well to the 70% similarity measured among red raspberry cultivars in Europe (Graham et al. 1994). Of the 16 genotypes investigated, five cultivars accounted for 58% of the observed variability in black raspberry, and none of the black raspberry cultivars were more than two generations from at least one wild ancestor.

9.3 Role in Elucidation of Origin and Evolution of Allied Crop Plants Blackberries and raspberries have a relatively short history of less than a century as cultivated crops that have been enhanced through plant breeding and they are only a few generations removed from their wild progenitor species. The improvements that have allowed these plants to be commercial cultivated crops are well documented: including increased yield, improved harvest efficiency, abiotic and biotic stress tolerance, increased fruit quality for fresh and processed markets, altered plant architecture, etc. Roach (1985) and Jennings (1988) gave accounts of the early domestication of red raspberry (R. idaeus L). During the nineteenth century, the North American red raspberry (R. idaeus subsp. strigosus Michx) was introduced into Europe and subsequently crossed with the European subspecies (R. idaeus subsp. vulgatus

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Arrhen.). Five parent cultivars dominate the ancestry of red raspberry; “Lloyd George” and “Pynes Royal” entirely derived from R. idaeus var. vulgatus, and “Preussen”, “Cuthbert”, and “Newburgh” derived from both subspecies. Controlled crossing began slightly earlier in the US than in the UK with the introduction of “Latham” in 1914 (McNicol and Graham 1992). There are many excellent reviews on blackberry and raspberry breeding, genetics, and germplasm including (Darrow 1937; Daubeny 1996; Waldo 1968; Oydvin 1970; Sherman and Sharpe 1971; Jennings 1988; Hall 1990; Jennings et al. 1991; Moore 1984; Clark and Finn 2008). The development and application of molecular markers has allowed improvements in taxonomical classification to be made as well as providing tools for the development of genetic linkage mapping, fingerprinting, and assessments of diversity to be undertaken in raspberry. Marker development has been reviewed by Antonius-Klemola (1999), Hokanson (2001) and Skirvin et al. (2005). As well as the deployment of anonymous DNA markers such as RAPDs (Graham et al. 1994, 1997; Weber 2003) and amplified fragment length polymprphisms (AFLPs) (Graham et al. 2006), SSR, EST-SSR, and single nucleotide polymorphism (SNP) markers have been developed (Graham et al. 2002, 2004, 2006; Stafne et al. 2005; Lewers et al. 2005; Lopes et al. 2006; Woodhead et al. 2008; McCallum et al. 2010) and can be used to characterize Rubus accessions. Alice and Campbell (1999) produced a Rubus phylogeny of 57 species including multiple raspberry species based on ribosomal internal transcribed spacer region (ITSR) sequence variation. The Rubus subgenus Idaeobatus of the Pacific region was studied in comparison with species from other subgenera to evaluate biogeographic and phylogenetic affinities of R. macraei, using chromosome analysis and chloroplast gene ndhF sequence (Morden et al. 2003). Their results showed that R. macraei is most similar to blackberry species of the subgenus Rubus. Moreover, they discovered that R. macraei and R. hawaiensis are derived from separate colonizations from North America and that similarities between them are due to convergent evolution in the Hawaiian environment. Trople and Moore (1999) calculated genetic similarities among 43 Rubus species and raspberry genotypes based on marker profiles from six RAPD primers. The similarity indices were relatively low

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between the species (0.15–0.52) with much higher indices for multiple accessions within species (0.62–0.82) (Trople and Moore 1999). In another study, 40 species of Rubus were analyzed, including many raspberry types, using RAPD markers and showed that molecular classification of species agreed with the traditional classification of Rubus in most cases, except for three species in the subgenus Malachobatus that clustered with the raspberry types in subgenus Idaeobatus (Pamfil et al. 2000). However, their RAPD-based taxonomy could not explain differential success of interspecific hybridization within each subgenus.

9.4 Role in Development of Cytogenetic Stocks and Their Utility A wealth of useful germplasm exists within and between the different Rubus species, but the complex ploidy series that exists is an obstacle to its efficient introduction into commercially important species. In order to facilitate the exploitation of existing genetic diversity, several strategies have been examined. The ploidy level of diploid Rubus species has been increased using colchicine on germinating seeds to generate tetraploids (Jennings and McNicol 1989; Jamieson and McLean 2008). The resulting tetraploid black and purple raspberries were reported to have larger fruit and set fruit more uniformly than diploids in cold conditions at flowering (Jennings and McNicol 1989), and increased ploidy influenced flower size, flower fertility drupelet numbers, seed size, and leaf morphology (Jamieson and McLean 2008). Colchicine has also been used on tissue-cultured shoot tips to generate non-chimeral autotetraploids in R. allegheniensis and R. rusticanus (Gupton 1989). Allopolyploids were made between colchicine-doubled raspberry autotetraploids and different blackberry polyploids to make hybrid berries (Knight and Rosati 1994). This material has been analyzed to confirm the nature of the hybrids using genomic and fluorescent in situ hybridization (Lim et al. 1998). A similar approach has been used with primocane fruiting, diploid R. idaeus cultivars and the resulting regenerant autotetraploids used as female parents with tetraploid and octaploid blackberries or with hexaploid hybrid berries to produce allotetraploid hybrids (Lim and

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Knight 2000). Although it is technically feasible to incorporate genetic diversity between different Rubus species, few of the hybrids were considered to have commercial potential – most produced poor, astringent fruit, which would be commercially unacceptable (Lim and Knight 2000). Blackberry breeding would also benefit from the efficient introduction of wild intra- and interspecific germplasm into breeding programs, and this would be assisted if the ploidy level of blackberries could be reduced to the diploid level (Naess et al. 1998). Ploidy reduction can happen spontaneously in plants, but it occurs rarely and methods have been developed to increase the incidence of this process, including interspecific hybridization, interploidy crosses, improved selection techniques, chemical and physical treatments of pollen and seed parent, and in vitro culture of male and female gametophytes (reviewed by Kimber and Riley 1963; Magoon and Khanna 1963; Lacadena 1974; Chu 1982; Yang and Zhou 1982). Diploid progeny of blackberries have been observed (Yarnell and Blackhurst 1947; Crane and Thomas 1949; Einset and Pratt 1954; Jennings et al. 1967), and more recently, strategies to produce dihaploids from tetraploid blackberry cultivars using selection of twin seed, interspecific hybridization, and pollen irradiation techniques have been reported (Naess et al. 1998). Of these, pollen irradiation was the most successful technique (Naess et al. 1998) and paved the way for incorporating wild Rubus germplasm into commercial cultivar breeding.

9.5 Role in Classical and Molecular Genetic Studies Domestication has resulted in a reduction of both morphological and genetic diversity in red raspberry (Haskell 1960; Jennings 1988) with modern cultivars being genetically similar (Dale et al. 1993; Graham and McNicol 1995). Similar work on the genetic relatedness of black raspberries using RAPD markers was carried out and raised similar concerns with the need for greater incorporation of more diverse germplasm into black raspberry breeding (Weber 2003). Relatedness in blackberries has also been examined using pedigree analysis with similar findings recommending the diversification of the gene pool (Stafne and Clark 2004). This restricted genetic diversity is of serious

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concern for the future of Rubus breeding, especially when seeking durable host resistance to intractable pests and diseases for which the repeated use of pesticides in some regions is ineffective, unsustainable, or unacceptable for certain selected markets, such as “organic production”. The gene base can and is being increased by the introduction of unselected raspberry clones and species material (Knight et al. 1989). However, the time required to produce finished cultivars from this material can be considerable, particularly if several generations of backcrossing are required to remove undesirable traits. Breeding in Rubus is carried out by hybridizations between cultivars and/or species with desirable characteristics for multiple generations. Each cycle of crossing involves a cycle of glasshouse screening and field observation. Prior to the advent of molecular markers, inheritance and genetic mapping studies were limited to simple morphological traits (Ourecky 1975; Jennings 1988). These studies generally utilized phenotypes that are deleterious in the recessive form so that they are undesirable to maintain in breeding programs. The advent of biotechnology has resulted in a fundamental shift in the development of genetic linkage maps and their use in variety development. Classical breeding, which selects parents and their desirable offspring based on an observable phenotype, is being integrated with techniques that can identify and manage genetic variability at the molecular level (protein or DNA). The ability to detect genome-wide variability has led to the characterization of genetic variation not only within coding regions (i.e., genes and their morphological manifestations) but also in non-coding regions as well, which make up large portions of plant genomes. These developments have enabled the construction of genetic linkage maps of red raspberry containing numerous genetic markers that are phenotypically neutral, which have been used to identify genomic regions associated with phenotype. Corresponding mapping in blackberry and other Rubus species has lagged due to their complex genetic make up and/or low economic importance. Early work on linkage analysis of morphological traits by Crane and Lawrence (1931) and Lewis (1939) documented aberrant segregation ratios among populations segregating for fruit color (T) and pale green leaves (g or ch1) in red raspberry (Crane and Lawrence 1931; Lewis 1939). Further work showed genetic linkage among five genes (waxy bloom b, apricot or

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yellow fruit t, pale green leaf g, red hypocotyl and pollen tube inhibitor w), producing the first genetic linkage group for Rubus (Lewis 1939, 1940). Sepaloid sx3 was later added to the linkage group between b and t (Keep 1964). Crane and Lawrence (1931) and Lewis (1939, 1940) also postulated on a linkage between a semi-lethal allele with the unlinked h gene. Jennings (1967) added further evidence to this linkage, proposing the symbols wt for the locus linked to the fruit color t locus and wh linked to the hairy locus (h) in place of w that Lewis (1939) used (Jennings 1967). Subsequent work in red raspberry has further elucidated the inheritance of hairiness and fruit color as well as numerous other traits. Associations between the H allele for cane hairiness and resistance to spur blight, cane Botrytis, and cane blight have been recognized (Knight and Keep 1958; Jennings 1988). This same gene also has been associated with susceptibility to cane spot, powdery mildew, and western yellow rust (Jennings and McGregor 1988; Jennings and Brydon 1989). Similarly, the recessive gene s for spine-free canes and the dominant B for waxy bloom on canes can reduce spur blight incidence (Jennings 1982, 1988). No other linkage groups based solely on morphological traits have been proposed. Daubeny (1996) lists 72 individual loci or alleles that have been identified, many of which are part of an allelic series for aphid resistance (Daubeny 1996). Corresponding work in blackberry and other Rubus species has been largely absent, probably due to the complex genetics of blackberry and the relatively unimportant economic impact of other species. The first genetic map of raspberry using markers was developed by Graham et al. (2004) utilizing SSR and AFLPs for a population of “Latham” “Glen Moy”. SSR markers were developed from both genomic and cDNA libraries from the cultivar “Glen Moy”. QTL analysis for variability in spine density identified two associated regions on linkage group 2. Graham et al. (2006) later added 20 SSR markers to the “Latham” “Glen Moy” map along with analyzing data on the H gene for cane hairiness and resistance to multiple fungal pathogens. The H gene was mapped to linkage group 2 and associated closely with resistance to cane Botrytis and spur blight. Unlike previous reports, no association between resistance to cane spot or yellow rust and gene H was identified. Raspberry root rot caused by Phytophthora fragariae var. rubi is probably the most destructive disease in

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raspberry plantations (Wilcox et al. 1993; Wilcox and Latorre 2002). Resistance to Phytophthora root rot (PRR) is found in a number of wild Rubus species including R. coreanus, R. crataegifolius (Jennings 1988), and Rubus idaeus strigosus, the native North American red raspberry, but less so in those derived from Rubus idaeus vulgatus, the European red raspberry (Pattison and Weber 2005). Generating crosses between R. strigosus and R. idaeus can facilitate the identification of the genes underpinning this resistance. Using a “Latham” (R. idaeus strigosus) “Glen Moy” (R. idaeus vulgatus) mapping population two regions, one on each of two linkage groups have been identified, and further research aimed at confirming these in a second population through glasshouse and field trials has been completed (Graham, Smith and Tierney unpublished data). BAC clones have been mapped into the resistance regions and are being sequenced to identify the genes in the region (Graham and Smith unpublished data). Using an RAPD-based linkage map from a cross between “Latham” and “Titan”, Pattison and Weber (2005) also identified markers clustered on two linkage groups, which were associated with disease score QTL for PRR using bulk segregant analysis. Recently, this work has been extended to include AFLP, RAPD, and resistance gene analog polymorphism (RGAP) markers in other “Latham” and “Titan” populations (Pattison et al. 2007). Considerable progress toward identifying markers and ultimately the gene(s) responsible for R. strigosus-based resistance to this disease is being made and this can be incorporated into raspberry breeding programs, allowing the rapid identification and selection of durable resistant genotypes. Aphids, particularly Amphorophora idaei (Borner) and Aphis idaei (van der Goot), are one of the most damaging arthropod pests in raspberry (Gordon et al. 1997) due to direct feeding damage to susceptible cultivars and because they act as vectors for virus transmission (McMenemy et al. 2009). Over the past 40 years, breeding for host plant resistance to raspberry aphids has reduced the need for pesticides and controlled the spread of aphid-borne viruses (Birch et al. 2005). However, insect pests are constantly adapting and overcoming plant resistance genes. Several types of aphid resistance genes, minor/multi-gene, and single major genes, e.g., A1 and A10, with different mechanisms have been used against A. idaei in sequence by raspberry breeders, but in the UK, each

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type of major gene resistance has been broken. To date, minor gene-based aphid resistance remains durable in raspberry, but it provides only partial resistance (Birch et al. 2005). Efforts to identify new sources of aphid resistance from wild species and other cultivars is underway, as is the development of molecular markers to speed up the selection of promising genotypes (Birch et al. 2005). This will be facilitated by efforts to map aphid resistance genes by anchoring marker data from appropriate segregating populations to the published raspberry genetic maps (Sargent et al. 2007). Determining the map location of a number of aphid resistance genes from various sources will allow the discrimination of different genes and gene pyramiding in new raspberry cultivars. Attempts to develop markers for viral resistance genes have been carried out for raspberry leaf spot and raspberry vein chlorosis utilizing the “Latham” “Glen Moy” cross of Graham et al. (2004). Field screening was carried out to measure symptom production of leaf spot and vein chlorosis in two different environments. These traits were analyzed for significant linkages to mapped markers, and resistance loci were found on linkage groups 2 and 8 (Rusu et al. 2006b). Mapping health-related compounds is a major goal in raspberry research. The emergence of metabolomics makes possible the simultaneous analysis of multiple metabolites at specific time points. In Rubus, a metabolomic approach has been used to identify bioactive compounds in a segregating mapping population planted under two different environments (Stewart et al. 2007). As a greater understanding of the relative importance and bioavailability of the different antioxidant compounds is achieved, it may become possible to develop and identify those raspberry genotypes with enhanced health-promoting properties from breeding programs (Beekwilder et al. 2005). Progress in mapping anthocyanins has been made by Kassim et al. (2009). Quantitative trait loci (QTL) for eight antioxidants mapped to the same chromosome region on linkage group 1 of the map of Graham et al. (2006) across both years and from fruits grown in the field and under protected cultivation. QTL for seven antioxidants also mapped to a region on LG 4 across years and for both field and protected sites. Candidate genes including a basic helix–loop–helix (bHLH) (Espley et al. 2007), a no apical meristem (NAM/CUC2_- like protein) (Ooka et al. 2003), and a basic leucine zipper (bZIP)

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transcription factor (Holm et al. 2002; Mallappa et al. 2006) underlying the mapped anthocyanins were identified (Kassim et al. 2009). A similar approach is underway for other Rubus antioxidants allowing the QTL and the underlying genes to be determined, with the ultimate aim of identifying favorable Rubus alleles to be used in breeding programs.

9.6 Role in Crop Improvement Through Traditional and Advanced Tools Rubus breeding is hampered by several genetic problems including polyploidy, apomixes, pollen incompatibility, and poor seedling germination. The highly heterozygous nature of the germplasm requires evaluation of large seedling populations. Breeding is based on a generation by generation improvement in breeding stock through selection and intermating of individuals showing promise of producing superior progeny. This average improvement in the progeny of breeding stock resulting from intermating selected parents is called response to selection (see review Hansche 1983). Several excellent reviews of blackberry and raspberry breeding have been written in the past few years including Finn and Knight (2002a, b), Clark et al. (2007), Finn (2008), Finn and Hancock (2008). The incorporation of novel resistance/tolerance to pests and diseases is regarded as essential for the development of cultivars suitable for culture under integrated pest management (IPM) systems. Sources of resistance in diverse Rubus spp. to many pests and diseases have been identified and exploited in conventional cross-breeding (Keep et al. 1977; Jones et al. 1984; Jennings 1988; Knight 1991; Williamson and Jennings 1992). However, germplasm bearing single resistance genes, when planted over extensive areas, can eventually be overcome by the rapid evolution of new biotypes of pests, so that new types of host resistance are required to sustain plant protection (Birch et al. 2002; Jones et al. 2002). Pest and diseases of raspberry in Europe have been extensively reviewed in Gordon et al. (2006). Pattison et al. (2007) combined generational means analysis with molecular markers and QTL analysis to map resistance to Phytophthora root rot in a BC1 population of NY00-34 (“Titan” “Latham”) “Titan”.

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Separate genetic linkage maps of NY00-34 and “Titan” were developed using RAPD, AFLP, and RGAP and analyzed for QTL associated with various parameters of root rot resistance assayed in a hydroponic system (Pattison et al. 2004). Agrobacterium-mediated genetic transformation techniques for Rubus have been described in the literature (Graham et al. 1995; Mathews et al. 1995; Kokko and K€arenlampi 1998), and transgenic raspberry plants have been generated that are resistant to raspberry bushy dwarf virus (Malowicki et al. 2008) and that contain a modified auxin synthesizing gene to enhance fruit productivity (Mezzetti et al. 2004). Should potentially favorable alleles conferring desirable characters be identified in wild Rubus species, it may be technically feasible for these to be incorporated into existing commercial cultivars. Whether genetic modification is a commercially acceptable way to producing improved Rubus cultivars remains to be seen.

9.7 Genomics Resources Developed The advances in genomics technologies have led to a massive increase in the numbers of DNA sequences held in public databases, and the numbers of Rubus sequences are very likely to increase rapidly as efforts are under way to sequence EST libraries generated from different tissues and developmental stages. At SCRI, cDNA libraries have been generated from leaves (approximately 6,500 clones), canes (approximately 8,000 clones), and roots (approximately 7,300 clones), and new generation sequencing has been used to identify gene transcripts in ripening fruit (Graham, Smith, Woodhead and McCallum unpublished data). Besides providing sequence information on genes expressed in these tissues, these resources are being used to identify gene-based markers for use in the genetic mapping programs. A project to characterize bud dormancy phase transition in woody perennial plants at a molecular level generated a total of 5,300 ESTs from endodormant (true dormancy) and paradormant (apical dominance) raspberry meristematic bud tissue (Mazzitelli et al. 2007). PCR products from these cloned cDNA fragments have been spotted onto glass slides and have been used in microarray experiments to identify genes that show differential

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expression. At present, approximately 380 clones exhibit up- or downregulation during the endodormancy–paradormancy transition. Some of these ESTs, including one encoding a MADs-box gene, a MYB gene, and several containing SSRs, have been identified and mapped in the “Latham” “Glen Moy” mapping population, and these underlie ripening QTL (Graham et al. 2009b). Genes upregulated during fruit ripening have been identified using classical plus/minus screening of cDNA libraries (Jones et al. 1998) and RNA fingerprinting techniques (Jones et al. 2000). Among the genes identified were cell wall hydrolases involved in fruit softening and ACC oxidase (Jones et al. 2000) involved in the ethylene biosynthetic pathway. The phenylpropanoid pathway is important in raspberry as end products contribute to the color and aroma of the fruit and are involved in other processes such as lignin production. Aroma and color in raspberry fruit are partly derived from the polyketide derivatives benzalacetone and dihydrochalcone, which are formed during fruit ripening as a result of the action of several enzymes, polyketide synthases (PKS), benzalacetone synthase, and chalcone synthase (CHS) during fruit development. A number of PKS genes have been characterized from raspberry (Zheng et al. 2001; Kumar and Ellis 2003a). Kumar and Ellis (2003a) reported that the PKS gene family in Rubus consists of at least 11 members, and the expression analysis of three cDNAs showed that they exhibited tissue-specific and developmental patterns of expression, with two cDNAs upregulated during fruit ripening. More recently, the cloning of a raspberry benzalacetone synthase (PKS4) has been reported (Zheng and Hrazdina 2008). Genes encoding 4-coumarate: CoA ligase, an enzyme that activates cinnamic acid and its derivatives to thioesters, which then serve as intermediates for the production of phenylpropanoid-derived compounds that influence fruit quality have also been studied. Kumar and Ellis (2003b) have characterized the 4-coumarate: CoA ligase (4CL) genes in raspberry found there are three genes, which are differentially expressed in various organs and during fruit development and ripening. Based on the expression patterns and substrate utilization profiles of the recombinant proteins, they suggest that 4CL1 is involved in the biosynthesis of phenolics in leaves, 4CL2 in cane lignification and 4CL3 in the flavonoid and/or flavor

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pathway in fruit. These genes are also being targeted in the Rubus mapping program (Woodhead, Graham and Smith personal communication). The first publicly available red raspberry BAC library from the European red raspberry, “Glen Moy”, has been constructed comprising over 15,000 clones with an average insert size of approximately 130 kb (6–7 genome equivalents). Hybridization screening of the BAC library with chloroplast (rbcL) and mitochondrial (nad1) coded genes revealed that contamination of the genomic library with chloroplast and mitochondrial clones was very low (>1%) (Hein et al. 2005). Initial screening of the BAC library employed probes for chalcone synthase, phenylalanine ammonia lyase, and a MADS-box gene involved in bud dormancy (Hein and Williamson personal communication). More recently, the library has been probed with genes involved in fruit quality genes (Woodhead and McCallum personal communication) and a peach ever-growing gene (Abbott personal communication) and with markers underlying QTL for Phythopthora root rot resistance.

9.8 Scope for Domestication and Commercialization Berries are extremely high in antioxidants, exhibiting up to four times more antioxidant capacity than nonberry fruits, 10 times more than vegetables and 40 times more than cereals (Halvorsen et al. 2002). They contain high levels of the antioxidant vitamins A, C, and E and very high levels of non-essential but strongly antioxidant phenolic compounds. Phenolics can account for 90% or more of the overall antioxidant capacity found in berry fruit (Deighton et al. 2000), the most readily visible of which are the anthocyanin pigments. These pigments impart the deep, vibrant colors of berries and can be found at concentrations of up to 500 mg 100 g FW 1. Berries represent a significant dietary source of anthocyanins, as only 24 out of 100 common foods contain anthocyanins, and non-berry anthocyanin-containing foods typically contain less than 100 mg 100 g FW 1 (Wu et al. 2006). The shift in focus from vitamin C and micronutrients toward the polyphenolics causes something of a challenge for any breeding effort, since the polyphenolics are chemically diverse and the content of individual

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health-promoting compounds varies in raspberry fruit due to both developmental and genetic factors (Beekwilder et al. 2005). However, with the emergence of metabolomics, the simultaneous analysis of multiple metabolites at specific time points is now feasible. In Rubus, a metabolomic approach has been used to identify bioactive compounds in a segregating mapping population planted under two different environments (Stewart et al. 2007). As a greater understanding of the relative importance and bioavailability of the different antioxidant compounds is achieved, it may become possible to develop and identify those raspberry genotypes with enhanced health-promoting properties from breeding programs (Beekwilder et al. 2005) and to mine for favorable alleles from wild Rubus accessions for inclusion into breeding programs. The traditional medicinal uses of different Rubus species are well known and have recently been reviewed (Patel et al. 2004). R. idaeus leaves and roots are anti-inflammatory, astringent, decongestant, ophthalmic, oxyctocic, and stimulant (Grieve 1971; Triska 1975; Foster and Duke 1990; Chevallier 1996). A tea made from them is used to treat diarrhea and as a tonic for pregnant women (only in the last 3 months of pregnancy) to strengthen the uterus in preparation for childbirth (Foster and Duke 1990). Externally, the leaves and roots can be used to treat tonsillitis, mouth inflammations, and as a poultice to treat sores, conjunctivitis, minor wounds, and burns (Bown 1995; Moerman 1998). The Kiowa and Apache made a tea from the roots of R. occidentalis species to treat stomach ache and blackberry root tea was part of the treatment for hemorrhaging and hemophilia (http://www.biosurvey.ou.edu/shrub/rubu-occ.htm). Rubus fructicosus roots infused in water with pennyroyal (Mentha pulegium) were used in the treatment of bronchitis and asthma and the leaves used as an astringent against bacterial infections (Beith 1995). Raspberry leaf tea is probably the most widely known herbal product associated with Rubus, but the value of natural components within Rubus are still being realized. Fruits from Rubus are antiscorbutic (i.e., prevent scurvy) and diuretic (Chiej 1984), and recent evidence suggests that fruit polyphenol components may affect activities of digestive tract enzymes and may provide a means for controlling diseases such as type 2 diabetes (McDougall and Stewart 2005). The antioxidant

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compounds present in black raspberry berries are being evaluated for the topical treatment of human pre-malignant oral lesions (Mallery et al. 2007, 2008), and freeze dried black raspberries and raspberry extracts are being assessed for the prevention of esophageal and colon cancer (Stoner et al. 2007). Raspberry fruits contain complex phenolic compounds, e.g., ellagitannins, which are strong antimicrobial agents against, for example, Salmonella and Staphylococcus (Puupponen-Pimi€a et al. 2005) and may have applications in both medicine and the food industry in the future. It has been reported that topical application of raspberry ketone (4-(4-hydroxyphenyl)butan-2-one), the compound that gives raspberry fruit their characteristic aroma, can promote hair re-growth in some humans with alopecia and increase skin elasticity (Harada et al. 2008) and, in mice, prevents and improves obesity (Morimoto et al. 2005). Patel et al. (2004) recently reviewed the volatile components occurring in a number of Rubus species. Not only do different Rubus species produce different types of volatile, the relative proportions of the volatiles produced can change during plant growth in raspberry and those emitted during flowering act as attractants to pollinating insects (Robertson et al. 1995). The total amount and type of volatiles produced from ripe blackberry fruit varies significantly between cultivar (Qian and Wang 2005) and in raspberry, although ethyl acetate at 12–18% was found to be the major detectable volatile product of ripe raspberry fruit (Robertson et al. 1995), it is not the major aroma compound. This is attributed to raspberry ketone, a compound widely used in perfumery, in cosmetics, and as a food additive to impart a fruity odor. This natural compound (also known as 4-(4hydroxyphenyl)butan-2-one) is a derivative of the phenolic pathway, and although it is the primary aroma compound of red raspberries (Hradzina 2006), it is found in low quantities in plants, between 1 and 17 mg/100 g FW (Borejsza-Wysocki et al. 1992). Demand for raspberry ketone is growing considerably, and although it can be produced by organic synthesis, work is underway to better understand how this compound is produced in planta. A raspberry gene encoding a benzalacetone synthase (or polyketide synthase 4, PKS4) has recently been reported (Zheng and Hrazdina 2008) and paves the way for producing this flavor compound using alternative stra-

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tegies such as microbial fermentation. Due to the sensory importance of this compound in fresh and processed raspberry products (Larsen et al. 1991), it would be desirable to screen raspberry germplasm for genotypes containing naturally high levels of raspberry ketone to improve the sensory characteristics of fruit through breeding.

9.9 Some Dark Sides and Their Addressing Rubus species are typically found as early colonizers of disturbed sites such as pastures, along forest edges, in forest clearings, and along roadsides. Blackberries are typically much more tolerant of drought, flooding, and high temperatures, while red raspberries are more tolerant of cold winters. Most species are polyploid, facultatively apomictic, and pseudogamous (i.e., pollination is required to trigger endosperm development in the seed (Gustafsson 1943)). Additionally, they exhibit vigorous vegetative reproduction by either tip layering or root suckering, permitting Rubus genotypes to cover large areas (Werlemark and Nybom 2003). The attractiveness of the fruits to frugivores, especially birds, means that seed dispersal can be widespread with the result that Rubus genotypes can very easily be spread to new sites. The overall effect – Rubus spp. are very effective, high-speed invaders (Greimler et al. 2002; Baret et al. 2004). Like many plant species that have since become invasive weeds, Rubus spp. typically moved around the world by humans who introduced them as food crops or as a result of trading activities (Ellison and Barreto 2004). Certain Rubus species have become very problematic in some regions of the world (Daehler 1998) where they produce very dense, impenetrable thickets, which make it impossible for native flora to germinate and establish, and they can also form hybrids with native species. As well as contributing to the obvious loss in biodiversity, these weeds pose a serious financial problem to agriculture, and efforts to find effective solutions to control them continue. There are many alien Rubus species and here we present several examples and the problems they pose and, in some cases, the strategies employed to control them.

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Blackberry (R. fruticosus L. aggregate) is an important weed in both agricultural and natural ecosystems in Australia, covering some 8.8 million ha. It is regarded as one of the worst weeds in Australia because of its invasiveness, potential for spread, and economic and environmental impacts (http://www.weeds.gov.au/ publications/guidelines/wons/pubs/r-fruticosus.pdf). At least 15 different but closely related species of blackberry are naturalized in Australia (Evans et al. 2005). Accurate taxonomic keys are important to determine which taxa are contributing to the problem, and DNA fingerprinting is important as a tool in this area (Evans et al. 1998, 2007). This is particularly relevant for applying biological control measures because blackberry leaf rust (Phragmidium violaceum) is ineffective against some European blackberry species and all American blackberry species (Scott et al. 2008). An additional biological control agent, the redberry mite, Acalitus essigi (Hassan), is under evaluation to aid control of blackberry (Scott et al. 2008), but other measures such as herbicides, mechanical removal, and burning are still required to control this weed. In Chile, two weedy species of blackberry, R. constrictus Lef. & M. (native to central Europe) and R. ulmifolius Schott (native to the Mediterranean), both introduced in the second half of the nineteenth century, have become naturalized and were estimated to cover 5 million ha by 1973 (Ellison and Barreto 2004). As in Australia, the use of P. violaceum has proved effective in controlling these species, particularly R. constrictus, the more problematic of the two weeds, without affecting the commercial species R. ideaus L. and R. loganobaccus (loganberry). The rust hastens normal defoliation, and infected stems do not lignify properly, which increases susceptibility to infection by other pathogens and to frost damage (Oehrens and Gonzalez 1977). Such weakened plants are less competitive and are displaced by native species (Oehrens 1977). In Hawaii, there are two endemic Rubus species: R. hawaiensis A. Gray, a major component of the forest ecosystem above 200 m elevation, and R. macraei A. Gray, which is less widely distributed. Both have North American ancestry (Howarth et al. 1997; Morden et al. 2003) and are partly sympatric with seven alien Rubus species that are naturalized in the Hawaiian islands (Randell et al. 2004) including Rubus rosifolius, a native to Australia. Apart from the

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threat of these Rubus species to invade and dominate existing forest, hybrids between R. rosifolius and R. hawaiensis have been found (Randell et al. 2004). Studies have shown the hybrids to be sterile, so although this may preclude genetic assimilation of R. hawaiensis by R. rosifolius and the acquisition of favorable, adaptive alleles by R. rosifolius from R. hawaiensis, nevertheless, the production of nonviable seed by R. hawaiensis represents a loss of reproductive effort and may have a negative impact on the species, and the hybrids may well have an advantage and pose a competitive threat (Randell et al. 2004). Rubus armenicus is now a serious invasive weed in the USA and Australia. Native to Armenia in Southwest Asia, it was introduced to Europe in 1835 and Australasia and North America in 1885. It was valued for its large, sweet fruit, similar to that of common blackberries (R. fruticosus) and attractive for domestic and commercial fruit production (cultivars “Himalayan Giant” and “Theodore Reimers” are particularly commonly planted (Ceska 1999)). R. alceifolius Poir., a bramble, is native to southeastern Asia and Malaysia and has been introduced to the Indian islands of Madagascar, Mayotte, La Reunion, and Mauritius where it is a serious weed, and to Queensland, Australia (Amsellam et al. 2001a, b). Reductions in the level of genetic diversity of the populations in areas of introduction were found and within the Indian islands, each population examined was characterized by a single clone, which was closely related to individuals from Madagascar (Amsellam et al. 2000). Amsellam et al. (2001a, b) suggest that there is a switch in the reproductive biology in this species. In its native range, R. alcefolius produces seed sexually, the plants in Madagascan populations are hybrids between R. alceifolius and native populations of R. roridus and produce seed mostly apomictically, while plants from Reunion Island (where R. alcefolius was introduced in 1850) produce seed exclusively apomictically. Considerable variation in fertility and vegetative growth in this species on Reunion Island has been described; fruit set is decreased in plants at increasing elevations, but this may be compensated for by greater vegetative growth (Baret et al. 2004). Gene flow between distantly related Rubus species has been demonstrated by the presence of naturally occurring hybrids between R. caesius (a facultatively agamous tetraploid blackberry) and diploid R. idaeus

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in Europe (Alice et al. 2001), and the presence of hybrids between R. alceifolius and R. roridus in Madagascar (Amsellam et al. 2001a, b). Luby and McNicol (1994) surveyed wild and feral Rubus populations in Scotland for evidence of the escape of two genes, L1 (for fruit size and plant morphology) and s (for spinelessness), introduced into raspberry cultivars by traditional breeding in the previous 20–30 years. The L1 gene was not found and very low frequencies of the s gene (0.004) were found in wild R. ideaus populations within the locale of the commercial planting area but not in populations remote from commercial production. Thus, should transgenic Rubus crops be deployed, there is potential for escape into sympatric wild populations, although this was considered to probably be infrequent (Luby and McNicol 1994). More recently, Graham et al. (2009a) demonstrated that limited gene flow into a wild R. ideaus population occurred but that pollen movement was hindered between populations at different altitudes, probably because of differences in flowering time. This gene flow into one population was identified by the gain of one new allele into progeny at the site; however, work also showed that three alleles were lost from parents to progeny, highlighting the flux in genetic diversity in natural populations.

9.10 Recommendations for Future Actions With the narrowing genetic base of our cultivated fruits, coupled with the increasing demands from consumers, new breeding methods are required to meet demands. Concern over the environmental impact and sustainability of agricultural and horticultural practices is leading to a greater emphasis on pest and disease resistance, as well as the ability of plants to withstand local environmental stresses. The changes in environmental, cultural, and agronomic practices within the industry will impact strongly on the nature of the germplasm required for the future. Greater conservation of genetic resources and utilization of diverse, locally adapted germplasm will be required for the future viability of Rubus production. Nevertheless, the development of molecular and genetic tools to link genotype to phenotype in Rubus mapping populations segregating for key characteristics and the

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identification of favorable alleles from diverse germplasm may allow for more rapid and targeted deployment of genes controlling these important traits, whether by marker assisted breeding (MAB) or through other means such as genetic modification (GM). The changing climate is already a major consideration for soft fruit growing due to the succession of mild winters, leading to poor bud break in some fruit species, and this may be addressed by employing a locally adapted germplasm.

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Chapter 10

Vaccinium Guo-Qing Song and James F. Hancock

10.1 Basic Botany of Vaccinium 10.1.1 History, Origin, and Distribution Vaccinium is a genus of terrestrial shrubs in the family Ericaceae (Syn. Heath) (Vander Kloet 1988). It consists of approximately 450 species with a wide geographic distribution in the Northern Hemisphere and also in the mountains of tropical Asia and Central and South America. A few species are also found in Africa and Madagascar, as well as 92 species (51 endemic) in China (Luby et al. 1991; Fang and Stevens 2005). High densities of Vaccinium species are distributed in the Himalayas, New Guinea, and the Andean region of South America (Luby et al. 1991; Hancock et al. 2008). The majority of species are found on open mountain slopes in the tropics (Camp 1942a, b, 1945). Southeast Asia (Malayan, Archipelago, New Guinea, India, China, and Japan) is the origin of almost 40% of the Vaccinium species. About 35% of the species are native to America including 25% in North America and 10% in South and Central America. The rest, about 25%, are widely scattered across the world (Luby et al. 1991). V. uliginosum L. is likely the most widely distributed Vaccinium species. Many of the Vaccinium species are invaluable ornamental plants due to their colorful leaves, flowers, and fruits (Galletta, and Ballington 1996). Three major Vaccinium fruit crops (blueberry, cranberry, and lingonberry) have been domesticated in the twentieth century (Galletta and Ballington 1996;

J.F. Hancock (*) Plant Biotechnology Resource and Outreach Center, Michigan State University, East Lansing, MI 48824-1325, USA e-mail: [email protected]

Hancock et al. 2003; Lyrene et al. 2003). Bilberry and the fruits of a number of other non-cultivated Vaccinium species also show great potential as new crops (Vorsa 1997). The most important Vaccinium crop species are found in the sections Cyanococcus, Oxycoccus, Vitis-Idaea, Myrtillus, and Vaccinium (Table 10.1). The taxonomy of these sections has been difficult to resolve due to complex polyploidy series (x ¼ 12) and a general lack of chromosome differentiation and crossing barriers within sections. The primary mode of speciation has been through unreduced gametes (Camp 1945; Darrow and Camp 1945; Hancock et al. 2008). Most production comes from species in section Cyanococcus including cultivars of Vaccinium corymbosum L. (highbush blueberry) and Vaccinium ashei Reade (rabbiteye blueberry; syn. Vaccinium virgatum Ait.) and native stands of Vaccinium angustifolium Ait. (lowbush blueberry). Highbush cultivars are further separated into northern or southern types depending on their chilling requirements and winter hardiness. Vaccinium macrocarpon Ait. (large cranberry), a member of section Oxycoccus, is also an important domesticated species. Vaccinium myrtillus L. (bilberry, whortleberry), Vaccinium membranaceum Douglas. Ex Torr. (tall bilberry, big huckleberry), Vaccinium deliciosum Piper (Cascade bilberry or huckleberry), and Vaccinium ovalifolium Sm. (oval-leaved huckleberry) in section Myrtillus and Vaccinium vitis-idaea (lingonberry) in section Vitis-Idaea are collected primarily from the wild.

10.1.1.1 Blueberries The blueberries in the section Cyanococcus occur naturally only in eastern and northcentral North America

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_10, # Springer-Verlag Berlin Heidelberg 2011

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G.-Q. Song and J.F. Hancock

Table 10.1 Important Vaccinium species (2n ¼ 2x ¼ 24) Section Species Common name Batodendron V. arboreum Farkleberry; Marsh Sparkleberry.

Cyanococcus

Ploidy 2x

Location S.E. North America

Lowbush Blueberry; Blueberry; Late Sweet Blueberry; Low Sweet Blueberry; Sweethurts; Upland Lowbush Blueberry Rabbiteye Blueberry

4x

N.E. North America

6x

S.E. North America

V. boreale Hall & Aald. V. constablaei Gray

Northern Blueberry

2x

Hillside Blueberry

6x

V. corymbosum L.

V. corymbosum L.

2x Highbush Blueberry; American Blueberry; Blueberry; Swamp blueberry; Aairelle d’Ame´rique; Amerikanische Blueberry; Ara´ndano americano Highbush Blueberry 4x

N.E. North America Mountains of SE North America S.E. North America

V. darrowii Camp

Darrow’s Blueberry

2x

S.E. North America

V. fuscatum Ait.

Black Highbush Blueberry; Thickleaf Blueberry; Downy Swamphuckleberry Velvetleaf Huckleberry

2x

Florida

2x

Central North America

V. angustifolium Ait.

V. ashei Reade. (syn. V. virgatum Aiton)

V. myrtilloides Michx.

E. North America

Potentially useful traits Drought tolerance, adaptation to basic mineral soils, open flower clusters, upright bush habit, stem blight resistance, resistance to sharpnosed leafhopper Winter hardiness, early ripening, blossom frost tolerance, adaptation to high pH, stem blight and Phytophthora root rot resistance, light blue fruit color, small scar, high soluble solids and low acidity Drought tolerance, low chilling requirement, upright plant habit, late ripening, long flowering to ripening period, fruit firmness, small scar, loose fruit cluster, cane canker, stem blight and Phytophthora root rot resistance, resistance to sharp-nosed leafhopper Winter hardiness, blossom frost tolerance Winter hardiness, high chilling requirement, light blue fruit color Low chilling requirement, upright plant habit, early ripening, light blue fruit color, small fruit scar

Low chilling requirement, upright plant habit, light blue and firm fruit color, small fruit scar, excellent flavor, stem canker resistance Low chilling requirement, heat tolerance, resistance to mummy berry, adaptation to high pH, tolerance to mineral soils, late flowering, late ripening, long flowering to ripening period, fruit firmness, excellent complex flavor, small scar, light blue fruit color, fruit hold well in heat, high soluble solids and low acidity, loose fruit cluster Very low chilling requirement, upright plant habit, vigorous

Winter hardiness, early ripening, blossom frost tolerance, resistance to mummy berry, small scar, high soluble solids and low acidity (continued)

10 Vaccinium

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Table 10.1 (continued) Section Species V. pallidum Ait.

V. tenellum Ait.

V. elliottii Chapm. Elliott’s Blueberry

2x

V. hirsutum Buckley V. myrsinites L.

Hairy Blueberry

4x

Shiny Blueberry; Evergreen Blueberry Upland Highbush Blueberry

4x

V. simulatum Small Oxycoccus

V. macrocarpon Ait.

V. oxycoccos L.

V. erythrocarpum Michx.

Vitis-Idaea

Myrtillus

Common name Ploidy Blue Ridge Blueberry; 2x; 4x Lowbush Blueberry; Hillside Blueberry; Blueridge Blueberry; Hillside Lowbush Blueberry; Upland Low Blueberry Small Black Blueberry 2x

V. microcarpum (Turcz. Ex Rupr.) Schmalh. V. vitis-idaea L.

V. cespitosum Michx.

4x

Location Mid-Atlantic North America

Potentially useful traits Adaptation to mineral soils, early ripening, small scar, high soluble solids and low acidity

S.E. North America S.E. North America

Adaptation to mineral soils, late ripening, firm fruit Drought tolerance, adaptation to high pH, tolerance to mineral soils, low chilling requirement, upright plant habit, late flowering, early ripening, upright habit, small fruit scar, excellent flavor, cane canker, stem blight and Phytophthora root rot resistance, resistance to sharpnosed leafhopper

S.E. North America S.E. North America S.E. North America

Low chilling requirement, small scar, low acidity, firm fruit Large fruit, winter hardiness, adaptation to mineral soils, deep root system Large leaf, large fruit and seed, high antioxidant content

2x North Large Cranberry; America Cultivated Cranberry; American Cranberry 2x; 4x; 5x; Circumboreal Cold hardiness Cranberry; Small 6x Cranberry; European Cranberry; Mossberry; Moosbeere; Tsurukokemomo; Bog Cranberry; Swamp Cranberry; Wild Cranberry – Southern Mountain 2x S.E. North Cranberry America and E. Asia Small Cranberry 2x Circumboreal –

Lingonberry; Cowberry; 2x Foxberry; Mountain Cranberry; Red Whortleberry; Lowbush Cranberry; Partridgeberry Dwarf bilberry; Dwarf 2x Blueberry; Dwarf Huckleberry 4x

Circumboreal

High benzoic acid, resistance to bacterial fruit rots, highly ornamental value for colorful, evergreen leaves

North America

Cold hardiness, late bloom, early ripening

(continued)

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Table 10.1 (continued) Section Species V. deliciosum Piper

Polycodium

Common name Cascade Bilberry; Alpine Blueberry; Cascade Blueberry; Blueleaf Huckleberry; Rainier Blueberry V. membranaceum Mountain Bilberry; Black Mountain Dougl. Ex Huckleberry; Black Hook. Huckleberry; Twinleaved Huckleberry; Leaf Huckleberry; Tall Huckleberry V. myrtillus L. Bilberry; Dwarf Bilberry; Myrtle Blueberry; Myrtle Whortleberry; Whortleberry V. ovalifolium Sm. Oval-leaved Bilberry; Oval-leafed Blueberry; Mountain Blueberry; Highbush Blueberry V. parvifolium Sm. Red bilberry; Red Huckleberry Grouseberry; SmallV. scoparium leaved Huckleberry; Leiberg ex Dwarf Red; and Red Coville Alpine Blueberry V. stamineum L. Deerberry; Highbush Huckleberry; Squaw Huckleberry: Southern Gooseberry

Pyxothamnus V. consanguineum Klotzch

V. ovatum Pursh

Bracteata

V. bracteatum Thunb. V. uliginosum L.

Ploidy

Location N.W. North America

Potentially useful traits Winter hardiness, blossom frost tolerance, light blue fruit color, excellent flavor

4x

W. North America

Cold hardiness, drought tolerance, high pH tolerance internal fruit pigmentation, large fruit size, excellent flavor

2x

Circumboreal

Winter hardiness, blossom frost tolerance, internal fruit pigmentation, excellent flavor

4x

Circumboreal

Large and firm fruit, small picking scar, light blue fruit color

2x

N.W. North America N.W. North America

Drought tolerance

2x

2x

Costa Rican Blueberry; 2x Deerberry; Squaw Huckleberry; Gooseberry; California Huckleberry; 2x Evergreen Huckleberry; Box Huckleberry; Evergreen Blueberry; Shot Huckleberry; California Huckleberry; Box Blueberry; Black Huckleberry Sea Bilberry 2x

Drought tolerance

Central and E. Upland adaption, drought tolerance, adaptation to mineral soils, late North ripening, very high soluble solids America and low acidity, large and firm fruit size, small stem scar, excellent flavor, resistance to sharp-nosed leafhopper S. Mexico and Blossom frost tolerance Central America N.W. North America

Adaptation to mineral soils, late ripening, ornamental value

E. Asia; China Tolerance to high pH and Japan 2x; 4x; 6x Circumboreal Cold hardiness, blossom frost tolerance, Fusicoccum canker resistance? tolerance to heavy metals

Bog Bilberry; Bog Blueberry; Bog Whortleberry; Bog Huckleberry; Northern Bilberry Source: from Ballington (1990), Luby et al. (1991), Galletta and Ballington (1996), Jacquemart (1997), Vander Kloet and Dickinson (1999), Suda (2003), Hancock et al. (2008)

Vaccinium

10 Vaccinium

(Hancock and Draper 1989). Prior to 1916, all blueberries were harvested from the wild. Today, blueberries are the most popular berry crop in Vaccinium and almost all of the commercial blueberries are harvested from three species (1) highbush (V. corymbosum L.), (2) rabbiteye [V. ashei Reade (syn. V. virgatum Ait.)], and (3) lowbush (V. angustifolium and V. myrtilloides). Highbush plants are crown-forming shrubs and generally 1.8–2.5 m tall. They are found in wetlands and drier upland wooded slopes from Nova Scotia west to Wisconsin, south to Georgia, and Alabama. The most winter hardy cultivars can be grown as Far North as plant hardness Zone 3 with 40 to 34.5 C average annual minimum temperature range (AAMTR). Most northern highbush blueberries do not grow well in the southern US because they require more than 700 chill hours to break dormancy in the spring. Lowbush blueberries, 0.30–0.60 m tall, include the low sweet blueberry (V. angustifolium) and the sour-tasting, velvet-leaf blueberry (V. myrtilloides). The low sweet blueberries are found from the Arctic to Minnesota and the mountains of New York and New Hampshire; the sour-tasting velvet-leaf blueberries are distributed wild throughout New England and west through plant hardness zone 2 with 45.6 to 40 C AAMTR. These cold-hardy bushes cannot tolerate too much summer heat, and their limit is about zone 7 with 17.8 to 12.3 C AAMTR. Rabbiteye blueberries (V. ashei) are crown-forming shrubs and generally 2.0–4.0 m tall. They are most adapted to regions with mild-winters including the southeastern US. Rabbiteye blueberries tolerate dry periods better than other blueberries. However, they are only cold hardy to the zone 6 or 7 with 23.3 to 12.3 C AAMTR (Encyclopedia of Plants 2008). Many of the wild, edible Vaccinium species have been harvested for thousands of years by indigenous people (Moerman 1998). Native Americans in western and eastern North America intentionally burned native stands of blueberries and huckleberries to renew their vigor. Highbush and rabbiteye blueberries were domesticated at the end of the nineteenth century. Plants were initially dug from the wild and transplanted into New England and Florida fields. Most of the commercial production of blueberry now comes from highbush and lowbush types, although rabbiteyes are important in the North American southeast, and hybrids of highbush lowbush (half-highs) have made a minor impact in the Upper Midwest of the

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USA. Rabbiteye cultivars are beginning to be grown in the Pacific Northwest and Chile for their very late ripening fruit. Highbush blueberries are grown in 37 states in the USA, in six Canadian provinces, and in Australia, Chile, Argentina, New Zealand, and a number of countries in Asia and Europe (Strik 2005; Strik and Yarborough 2005). The largest acreages of northern highbush are in Michigan, New Jersey, North Carolina, Oregon, and Washington in the USA, and British Columbia in Canada. The greatest amount of southern highbush acreage is in Florida, California, and Georgia. Commercial production of lowbush blueberries is mainly in Maine, Quebec, New Brunswick, and Nova Scotia (Strik 2005; Hancock et al. 2008). While the half-high blueberries are not a major contributor to the fruit market, they are very widely used as an ornamental plant for landscaping.

10.1.1.2 Cranberries Cranberries are widespread throughout the cool temperate Northern Hemisphere and their native habitat is principally in wetland areas. There are four species of cranberry (Table 10.1). The domesticated large cranberry or the American cranberry, Vaccinium macrocarpon (Ait.) Pursh. is an endemic of eastern North America and is thought to be the most primitive species in section Oxycoccus (Camp 1945). Large cranberry plants, 10.2–15.2 cm tall, are woody perennials adapted to acid soils and a temperate climate, requiring approximately 1,200 h of chilling to break winter dormancy (Polashock and Vorsa 2002a). The first cultivation of large cranberries was reported in 1810 in Cape Cod, Massachusetts (Camp 1945; Polashock and Vorsa 2002a; McCown and Zeldin 2005). Common cranberry (V. oxycoccos L.) is widespread throughout northern Europe, northern Asia, and northern North America. Small cranberry (V. microcarpum (Turcz. Ex Rupr.) Schmalh.) occurs in northern Europe and northern Asia. Southern mountain cranberry, V. erythrocarpum (Michx.) Pers., is native to southeastern North America at high altitudes in the southern Appalachian Mountains and also in eastern Asia. Currently, V. macrocarpon Ait. is grown as a commercial crop on approximately 40,000 acres across the northern United States and Canada, primarily in Wisconsin, Massachusetts, New Jersey, Washington, and Oregon, with limited plantings in British Columbia,

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Michigan, Nova Scotia, and Quebec. Chile and Germany also grow V. macrocarpon Ait.; Russia and eastern Europe are the two major regions for commercial production of V. oxycoccos L. (Roper and Vorsa 1997; McCown and Zeldin 2005). The small cranberry and southern mountain cranberry are seldom cultivated.

10.1.1.3 Lingonberries V. vitis-idaea (common known as lingonberry) is a perennial, evergreen dwarf shrub in Vaccinium section vitis-idaea (Moench) Koch (Table 10.1) (Luby et al. 1991; Galletta and Ballington 1996; Gustavsson 2001). It is thought to be a cranberry–blueberry intermediate (Galletta and Ballington 1996). This species is distributed in circumboreal regions and prefers acid soils. It has been divided into a coarser, lowland subsp. vitis-idaea (L.) Briton and a circumpolarArctic Montane subsp. minus (Lodd.) Hult (Hulte´n 1949). The plant height of V. vitis-idaea (L.) Briton may exceed 30 cm, whereas the plant height for V. vitis-idaea spp. minus rarely exceeds 20 cm (Gustavsson 2001). The subsp. vitis-idaea (L.) grows in Europe and Asia. In Europe, it is restricted to the north, ranging from the British Isles, Fennoscandinavia and North Russia, south to the Pyrenees, northern Italy, the Balkans, and Caucasus. In Asia, it is distributed from the Urals to Okhotsk and Kamchatka, south to northern Mongolia, Korea, and Japan. The subsp. minus (Lodd.) is found mainly in North America, parts of Asia, and the alpine region of Scandinavia. In North America, the subsp. minus (Lodd.) ranges from eastern Kola Peninsula and Nova Zemlia to the Chukch Peninsula and Kamchatka and across North America to Greenland and Iceland (Luby et al. 1991). Lingonberry plants are extremely hardy, tolerating 40 C or lower, but grow poorly where summers are hot. They are found in diverse habitats, ranging from lowland to upland and mountain areas, in largely acidic soils to pure peat bog (Gustavsson 1997). Lingonberry has a long history of being commercially harvested from native stands in Europe and parts of northern North America (Gustavsson 2001; Finn and Mackey 2006). The fruits are traditionally used in jams, jellies, juices, sauces, pies, and wines.

G.-Q. Song and J.F. Hancock

10.1.1.4 Berry Fruits in Genus Vaccinium, Section Myrtillus The Vaccinium section Myrtillus Dumortier includes seven species (Table 10.1) (Vander Kloet 1988). This section is restricted to the Northern Hemisphere within a radius of 100 km of 50o N latitude, 110o W longitude. It has its center of diversity along the Pacific Rim from Japan to Guatemala (Vander Kloet and Dickinson 1999). A secondary center of diversity occurs in the mountains of Honshu, Japan (Vander Kloet 1983). The origin and habitats of the seven species were summarized in Table 10.2. The common name bilberry is derived from the Danish word bollebar, which means dark berry (Grieve 1979). It is most often referred to as V. myrtillus L. There is an increasing demand for the berries of V. myrtillus due to their high nutritional value (Barney 2003; Martinussen et al. 2009).

10.1.1.5 Other Sections in Vaccinium The section Vaccinium includes V. uliginosum L., which is an Arctic and boreal circumpolar species occurring in cool temperate regions of the Northern Hemisphere, at low altitudes in the Arctic and at high altitudes south to the Pyrenees, the Alps, and the Caucasus in Europe, the mountains of Mongolia, northern China and central Japan in Asia, and the Sierra Nevada mountains in California and the Rocky Mountains in Utah in North America (Tables 10.1 and 10.3) (Luby et al. 1991). This species grows on wet acidic soils on heathland, moorland, open tundra, and in the understory of coniferous forests, from sea level in the Arctic up to 3,400 m altitude in the south of the range (Hanson 1953; Young 1970; Vander Kloet 1988; Kelso 1989). The edible berries were the most popular fruit of the Native Americans in the Fort Yukon region (Holloway and Alexander 1990). They are usually used for jam, juice, pie, jelly, and wine making (Iwagaki et al. 1977; Rui 1982). Leaves can be used for tea (Robuck 1989). In addition, both the leaves and fruits are consumed by many species of wildlife (LeResche and Davis 1973; Wolff 1978; West 1982; MacHutchon 1989). In Section Pyxothamnus, at least three species produce edible berry fruits (Table 10.3) (Luby et al. 1991; Finn 1999). These evergreen species have similar berries

In the Pacific Northwest of North America

Northern West America

An ancient circumboreal group

V. ovalifolium Sm.

V. parvifolium Sm.

V. scoparium Leiberg ex Coville

Source: adapted from Luby et al. (1991)

An ancient circumboreal group

In the early Tertiary and migrated east across North America in the early Pleistocene A hybrid between V. cespitosum Michx. and V. ovalifoliu Smith Derivative of several diploid species in section Myrtillus

V. myrtillus L.

V. membranaceum Dougl. Ex Hook.

V. deliciosum Piper

V. cespitosum Michx.

Clear cut tracks, mountain slopes at 900–2,000 m, and dry, open sites in coniferous forests (1) In lowland to alpine open pine or spruce forests in northern Europe and Asia; (2) At 1,800–3,000 m in moist, open sites, mountains of subalpine communities in the Pacific Northwest and rock mountains of the USA On raw humus in moist, coastal, coniferous, forests from seal level to 1,000 m (1) At low to intermediate elevations on the west slopes of coastal ranges from Alaska to northern California; (2) Inland to southern–eastern British Columbia At 1,500–3,000 m in open to partially open sites or in alpine meadows above the timberline in the Cascades

Wet meadows, mountain slopes, moist rocky ledges, subalpine forests, and alpine tundra at 2,000–3,000 m Open areas in subalpine forest and meadows and alpine tundra at 1,400–3,000 m

Table 10.2 Vaccinium species in section Myrtillus Dumortier Species Origin Habitat

2–5

7–9

8–12

5–10

6–20

5–6

Fruit diameter (mm) 3–7

Pink to dark red

Pink to dark red

Light blue

Dark blue

Purple or red blue

Light blue

Blue

Fruit color

Fair, acidic

Fair

Fair

Good, tart

Fair to good

Excellent

Good

Fruit flavor

Drought tolerance

Drought tolerance

Large, firm fruit, small picking scar

Cold hardiness, high fruit quality

Cold hardiness, drought tolerance, high pH tolerance

Cold tolerance, blossom forest resistance

Cold hardiness, late bloom, early ripening

Other traits of interest for breeding

Camp (1942a), Schultz (1944), Szczawinski (1962), Ballington et al. (1988b)

Schultz (1944); Vander Kloet (1988), Ballington et al. (1988b) Schultz (1944), Vander Kloet (1988)

Vander Kloet (1983), Ritchie (1956), Schultz (1944), Szczawinski (1962), Flower-Ellis (1971)

Camp (1942a), Schultz (1944), Szczawinski (1962)

Camp (1942a), Schultz (1944), Szczawinski (1962)

Camp (1942a), Schultz (1944), Szczawinski (1962)

References

10 Vaccinium 203

Eastern Asian

The smaller diploid is On wet or moist, common at organic or latitudes over inorganic soils 60 N or at high elevations compared to the coarser tetraploid. Hexaploid forms are known from Japan

V. bracteatum Thunb.

V. uliginosum L.

Bracteata

Vaccinium

Light woodland, moist soil with low pH

Mainly in high alpine regions.

Southern Mexico, Central America

In cooler, subtropical climates or in part-shade in warmer climates. Coastal coniferous forests

V. floribundum Kunth.

V. consanguineum Klotzch.

Pyxothamnus

Dry uplands

The North Pacific coast

Central and E. North America Southern Mexico, Central America

V. stamineum L.

Polycodium

Habitat

V. ovatum Pursh.

Origin

Species

Section

Table 10.3 Species in other Vaccinium sections

6–15

6

5–8

4–7

5–6

10

Fruit diameter (mm)

Considerable variation in flavor, tart Pleasantly flavored

Fair

Good, tart

Fruit flavor

Purplish Good black with bloom Dark blue to Good glaucous

Blue–black

Purplishblack

Green to yellow Reddish to blackish purple

Fruit color

Cold hardiness, Fusicoccum canker resistance?, Tolerance to heavy metals

Frost and freeze resistance during bloom Resistant to honey fungus

Drought tolerance

Upland adaption, drought tolerance Frost and freeze resistance during bloom

Camp (1945), Schultz (1944), Szczawinski (1962), Vander Kloet (1988), Young (1970), Iwagaki et al. (1977), DiLabio and Rencz (1980), Finn (1999)

Wilbur and Luteyn (1978), Popenoe (1924), Macbride (1959) Iwagaki et al. (1977), Huxley (1992a,b)

Schultz (1944), Camp (1945), Szczawinski (1962)

Camp (1945), Wilbur and Luteyn (1978)

Vander Kloet (1988)

Other traits of interest References for breeding

204 G.-Q. Song and J.F. Hancock

10 Vaccinium

(Finn 1999). Vaccinium floribundum Kunth is distributed in Costa Rica, Venezuela, Colombia, Ecuador, and Peru. Vaccinium consanguineum Klotzch is native to Costa Rica and W. Panama. The range of V. ovatum spans the Pacific northwest of North America, from British Columbia, Washington, Oregon, and Nevada, south to California (Camp 1945). V. consanguineum Klotzch is found in Costa Rica, Honduras, and Panama.

10.1.2 Botanical Features, Cytology, and Germplasm Resources As a large genus, Vaccinium is taxonomically complex. Although Sleumer (1941) divided the genus into 33 sections based on morphological phylogenetics, sectional species composition and evolutionary relationships have been the subject of much debate (Powell and Kron 2002). Many of the characters traditionally used to delimit genera based on flower, fruit, seeds, and vegetative parts fail to adequately distinguish among taxa (Stevens 1972; Kron et al. 2002). Hundreds of the Vaccinium species that are native to tropical highlands are little-known, although many have potential as ornamentals or in fruit production. More on-site botanical studies are critically needed to provide information and broad-based seed. More collections should be made to safeguard genetic resources in these species until they are better understood.

10.1.2.1 Blueberry Species delineation has been difficult to resolve in Cyanococcus due to polyploidy, overlapping morphologies, continuous introgression through hybridization, and a general lack of chromosome differentiation. In the first detailed taxonomy of the group, Camp (1945) described nine diploid, 12 tetraploid, and three hexaploid species, but Vander Kloet (1980, 1988) reduced this list to six diploid, five tetraploid, and one hexaploid taxa. He included all the crown-forming species into V. corymbosum with three chromosome levels. Most horticulturists and blueberry breeders feel that the variation patterns in V. corymbosum are distinct enough to retain Camp’s diploid Vaccinium elliottii Chapm. and Vaccinium fuscatum Ait., tetraploid

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Vaccinium simulatum Small and hexaploid V. constablaei A. Gray and V. ashei Reade, which is more properly denoted V. virgatum Ait (Ballington 1990, 2001; Galletta and Ballington 1996; Lyrene 2006). All the polyploid Cyanococcus are likely of multiple origins, and active introgression between species is ongoing. The tetraploid highbush blueberry V. corymbosum has been shown to be genetically an autopolyploid (Draper and Scott 1971; Krebs and Hancock 1989), as well as an interspecific tetraploid hybrid of V. darrowii Camp and V. corymbosum (Qu and Hancock 1995; Qu et al. 1998). Wenslaff and Lyrene (2003) found considerable chromosome homology in tetraploid southern highbush V. elliottii hybrids. The lowbush blueberry, V. angustifolium, appears to be a direct descendant of V. pallidum Ait. V. boreale Hall & Aalders, but introgression with V. corymbosum may have also influenced its subsequent development (Vander Kloet 1977). The primary mode of speciation in Vaccinium has been through unreduced gametes, as there is a strong but not complete triploid block (Lyrene and Sherman 1983; Vorsa and Ballington 1991). The unreduced gametes are produced primarily through first division restitution (Qu and Hancock 1995; Qu and Vorsa 1999), although some second division restitution occurs (Vorsa and Rowland 1997). Embryo culture was not successful in recovering triploids of V. elliottii tetraploid highbush (Munoz and Lyrene 1985). Interspecific hybridization within Vaccinium section Cyanococcus has played a major role in the development of highbush blueberries (Ballington 1990, 2001). Most homoploids freely hybridize, and interploid crosses are frequently successful (Lyrene et al. 2003). Genotypes have been found in many blueberry species that produce unreduced gametes (Ballington et al. 1976; Cockerman and Galletta 1976; Ortiz et al. 1992), and colchicine can be used to produce fertile genotypes with doubled chromosome numbers (Perry and Lyrene 1984). Even pentaploid hybrids of diploid hexaploid crosses have been shown to cross relatively easy to tetraploids (Jelenkovic 1973; Chandler et al. 1985a, b; Vorsa et al. 1987). Numerous interspecies crosses have been made by breeders within section Cyanococcus including (1) tetraploid V. corymbosum tetraploid V. angustifolium (Luby et al. 1991), (2) tetraploid V. myrsinites L. tetraploid V. angustifolium and V. corymbosum (Darrow 1960; Draper 1977), (3) colchicine-doubled

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diploid hybrids of V. myrtilloides Michx. tetraploid V. corymbosum (Draper 1977), (4) diploid V. darrowii hexaploid V. ashei (Darrow et al. 1954; Sharp and Darrow 1959), and (5) diploid V. elliottii tetraploid highbush cultivars (Lyrene and Sherman 1983). Probably the most widely employed interspecific hybrid has been US 75, a tetraploid derived from the cross of diploid V. darrowii selection Fla 4B the tetraploid highbush cultivar Bluecrop. In spite of being a hybrid of an evergreen, diploid species crossed with a deciduous, tetraploid highbush, US 75 is completely fertile and is the source of the low chilling requirement of many southern highbush cultivars (Draper and Hancock 2003). Many of the highbush types now being released are complex hybrids. Some of the most dramatic examples are “O’Neal”, which contains genes from four species (V. corymbosum, V. darrowii, V. ashei, and V. angustifolium), and “Sierra”, which possesses the genes of five species (V. corymbosum, V. darrowii, V. ashei, V. constablaei, and V. angustifolium). “Biloxi” contains the genes from five taxa [V. corymbosum (diploid and tetraploid), V. darrowii, V. ashei, and V. angustifolium] and has fewer V. corymbosum than non-V. corymbosum genes in its genome. Intersectional crosses have generally proved difficult, although partially fertile hybrids have been derived from V. tenellum Ait. and V. darrowii (section Cyanococcus) V. stamineum L. (section Polycodium) (Lyrene and Ballington 1986), V. darrowii and V. tenellum V. vitis-idaea (section Vitis-Idaea) (Vorsa 1997), V. darrowii V. ovatum Pursh (section Pyxothamnus), V. arboreum Marshall (section Batodendron) and V. stamineum (section Polycodium) (Ballington 2001), and tetraploid V. uliginosum (section Vaccinium) highbush cultivars (Rousi 1963; Hiirsalmi 1977; Czesnik 1985). Genes of V. arboreum have also been moved into tetraploid southern highbush using V. darrowii as a bridge (Lyrene 1981; Brooks and Lyrene 1998a, b). Genes from V. ovatum have been incorporated into ornamental highbush selections in the US Department of Agriculture (USDA), Agricultural Research Service (ARS), Oregon, program via NC 3048. There are several important collections of native blueberry germplasm and hybrids (Ballington 2001). The most extensive is held at the USDA-ARS’s National Clonal Germplasm Repository at Corvallis Oregon, where representatives of most species can be

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found along with almost all named non-patented cultivars. James Ballington at North Carolina State University has a particularly large collection of southern species material. Paul Lyrene at the University of Florida and James Hancock at Michigan State University also have large collections of southern and northern adapted material, respectively.

10.1.2.2 Cranberry Cranberries have the basic chromosome number of 12 and a polyploid series of 2x, 4x, or 6x (Table 10.1). Large cranberries, V. macrocarpon (Ait) Pursh. (2n ¼ 24), are the most closely related to diploid V. oxycoccus (Camp 1944). They are low, creeping perennial shrubs or vines up to 2 m long and 5–20 cm in height. Natural polyploid populations of V. macrocarpon have not been reported (Zeldin and McCown 2002). Gene exchange between V. macrocarpon and diploid V. oxycoccus is now severely limited due to a disjunctive distribution and a flowering date difference of 3 weeks (Vander Kloet 1988). Compared to V. macrocarpon, V. oxycoccus plants have small (5–10 mm) leaves. The fruit is a small pale pink berry, with a refreshing sharp acidic flavor. The largest collections of cranberry germplasm are held by Nicholi Vorsa at Rutgers University and Eric Zeldin and Brent McCown at the University of Wisconsin. In addition, the USDA National Clonal Germplasm Repository in Corvallis, OR, has 123 accessions of V. macrocarpon (large-fruited cranberry), three accessions of V. microcarpon (small-fruited cranberry), and 41 accessions of V. oxycoccus (2x/4x wild cranberry) (National Plant Germplasm 2005).

10.1.2.3 Bilberry and Lingonberry Compared to sections Cyanococcus and Oxycoccus, the section Myrtillus has drawn little attention from plant breeders. Bilberries (V. myrtillus L.) are closely related to blueberries and are diploid (2n ¼ 24) (Table 10.1; Vander Kloet 1988). The plants grow 10–60 cm tall. Their fruit is dark blue and is smaller, softer, and juicier than that of the blueberry (Table 10.2). The easiest way to distinguish the bilberry from the blueberry is that it bears one to three individual berries instead of clusters of berries.

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The lingonberry (V. vitis-idaea L.) is a diploid (2n ¼ 24), low-growing (15–30 cm tall), evergreen groundcover (Sorsa 1962; Rousi 1967). Lingonberries thrive in acidic soils (pH 3.5–5) and reproduce vegetatively through rhizomes and sexually by seeds. The ripe fruit is bright red, 6–10 mm in diameter, tart and smaller than cranberries but with a finer flavor. Wild lingonberry populations are variable in nearly all important horticultural traits (Gustavsson 2001; Persson and Gustavsson 2001; Paal 2006).

10.1.3 Economic Importance Vaccinium fruits are perceived by the public to be a health-promoting food. According to the latest ARS report from the USDA, cranberries and blueberries rank number one and two, respectively, in antioxidant values of 19 common fruits (USDA-ARS 2007). Demand for berries from the various Vaccinium species (blueberries, cranberries, bilberry, and lingonberry) will likely continue to grow due to their nutritional and therapeutic properties (Prior et al. 1998; Sun et al. 2002; Ferguson et al. 2006; Neto 2007a, b; Neto et al. 2008).

10.1.3.1 Blueberry Blueberry has become the second most important berry crop in the USA. The highbush blueberry is by far the most important commercial crop in Vaccinium. Over 110,000 t of highbush fruit are produced annually in the US on over 20,000 ha (USDA Agricultural Statistics). The estimated area of rabbiteye production is currently about 3,000 ha, with half the surface planted in Georgia. The total annual production is over 5,500 t. Half-high production is restricted to a few hundred hectares in Minnesota and Michigan. Annual production of lowbush blueberries ranges from 40,000 to 55,000 t on about 40,000 ha in primarily Maine and the Maritime provinces of eastern Canada. From 1995 to 2007, worldwide blueberry acreage grew by 254%, from 23,116 ha to 58,601, and most of that growth was in the western Hemisphere, including 20,315 additional hectares in South America and 312,950 more hectares in North America, primarily

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the US and British Columbia (Lehnert 2008). The estimated area of blueberry production in China increased from 24 ha in 2001 to 1,363 ha in 2007 (Li and Yu 2009). About 50% of the highbush crop is marketed fresh and the remainder is processed. Blueberries are used primarily in pie fillings, yogurts, ice cream, and prepared muffin and pancake mixes. Syrups, jams, and preserves are also produced but in limited quantities. The juice of blueberries is rarely consumed directly due to its strong flavor and dark color. Blueberries are one of the richest sources of antioxidant phytonutrients among the fresh fruits, with a total antioxidant capacity ranging from 13.9 to 45.9 mmol Trolox equivalents/g fresh berry (Prior et al. 1998; Conner et al. 2002a, b; Zheng and Wang 2003). Many factors including genetics, growing condition, fruit maturity at harvest, and other variables affect nutritious levels in blueberry fruits. General information on nutritional value indicates that blueberries are a source of vitamin A, vitamin C, potassium, and foliate. An average blueberry fruit is composed of approximately 83% water, 0.7% protein, 0.5% fat, 0.5% ash, and 15.3% carbohydrate (Hancock et al. 2003). The overall acidic content of Vaccinium fruit is relatively high. Ripe blueberries range from 1 to 2%, and the primary organic acid is citric acid (1.2%). They also contain significant amounts of ellagic acid, a compound thought to reduce the risk of cancer (Maas et al. 1991). Total anthocyanins in blueberry fruit range from 85 to 270 mg per 100 g, and species in the subgenus Cyanococcus carry the same predominant anthocyanins, aglycones, and aglycone-sugars, although the relative proportions vary (Ballington et al. 1988). The predominant anthocyanins were delphinidin-monogalactoside, cyanidin-monogalactoside, petunidin-monogalactoside, malvidin-monogalactoside, and malvidin-monoarabinoside. Anthocyanin is responsible for the blue color of blueberries and has been shown to be among the most powerful antioxidants that are known to reduce urinary tract infections and protect against cancer, diabetes, heart and vascular diseases, and neurodegenerative diseases in humans (Ehlenfeldt and Prior 2001; Seeram et al. 2006). The major volatiles contributing to the characteristic aroma of blueberry fruit are trans-2-hexanol, trans-2-hexanal, and linalool (Hancock et al. 2003). The predominant volatiles in the bilberry

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are trans-2-hexanal, ethyl-3-methyl butyrate, and ethyl2-methyl butyrate.

10.1.3.2 Cranberry Humans have consumed cranberry fruits for several centuries (McCown and Zeldin 2005). Since the seventeenth century, cranberries have been used for an assortment of medicinal purposes: stomach ailments, liver problems, and blood disorders. In North America, cranberry consumption has been associated with the holidays of Thanksgiving and Christmas when cranberry sauce is served with roast turkey meat. However, cranberry consumption has become a year round activity, largely through juice products. Cranberry production in North America is about 380,000 t annually on 21,700 ha. About 95% of cranberries are processed into products such as juice drinks, sauce, and sweetened dried cranberries. The remaining 5% is sold fresh to consumers and used in baking (muffins, scones, and cakes). Unlike many other berries, cranberries are normally too tart to be eaten unaccompanied. Cranberries have medium levels of vitamin C, dietary fiber and the essential dietary mineral, manganese, and other essential micronutrients (Table 10.3). The overall acidic content in ripe cranberries ranges from 2 to 3%. The cranberry contains high levels of several organic acids, including quinic (1.3%), citric (1.1%), malic (0.9%), and benzoic (0.6%). Additionally, Cranberries have long been prized for their brilliant red fruit. The deep-colored pigments are made up of anthocyanins, which are a subclass of flavonoids. Therefore, raw cranberries are excellent food sources of the anthocyanidin flavonoids, cyanidin, peonidin, and quercetin (Duthie et al. 2006). When 19 common fruit crops were compared, cranberries had the highest antioxidant capacity with the Oxygen Radical Absorbance Capacity (ORAC) of 9,584 units per 100 g of fresh fruit (USDA-ARS 2007). Antioxidants reduce the effect of free radical oxidants, which weakens the immune system and is linked to several diseases, by binding with them and decreasing their destructive power and repairing damage. A high antioxidant activity could potentially make this berry crop a candidate for cancer chemoprevention and treatment (Sun et al. 2002; Wang et al. 2005; Sun and Liu 2006; Ferguson et al. 2006; Neto 2007a, b; Neto et al. 2008). Ingestion of cranberries leads to increased acidity of the urine

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through conversion of its high quinic and benzoic acid contents to hippuric acid by the body. The high acidity and possible antibacterial effects of hippuric acid or proanthocyanidins may relieve urinary tract infections and reduce some types of kidney stones (Kessler et al. 2002; Linsenmeyer et al. 2004; Jepson and Craig 2007).

10.1.3.3 Bilberry V. myrtillus fruits are an ancient food in northern Europe (Kardell 1980; Raatikainen and Raatikainen 1983; Kostov and Stojanov 1985). The berries contain antioxidants and other compounds beneficial to human health. They are harvested commercially from the wild in Finland and other European countries. In Europe, the berries are used for fruits, pies, tarts, syrups, jellies, and wine making (Tyler 1994). In North America, native people historically ate the fruit fresh or dried and continue to use it today (Turner 1997). Herbalists and physicians have used bilberry fruit in traditional European medicine for nearly 1,000 years (Morazzoni and Bombardelli 1996). Traditionally, bilberry preparations have been used as an astringent for treatment of diarrhea and dysentery (Bone and Morgan 1997). In addition, they are used to help stop the flow of breast milk and to relieve scurvy, dysentery, and vascular disorders (Grieve 1979; Bruneton 1995). As herbal medicines, bilberry has shown vasoprotective, antiedematous, antioxidant, anti-inflammatory, and astringent actions (Bone and Morgan 1997).

10.1.3.4 Lingonberry In Europe, lingonberries have long history of medicinal uses for treatments of inflammatory diseases, wounds, gastric distress, and rheumatism. In addition, the antioxidant compounds in lingonberries may play an important role in cancer chemoprevention and treatment (Wang et al. 2005). Lingonberry leaves have been used as medicine for curing kidney and bladder diseases (Paal 2006). Lingonberries are rich in active antioxidants such as anthocyanin and phenolic compounds (Zheng and Wang 2003; Wang et al. 2005) and are rich in benzonic acid. In comparison with blueberries (cv. Serra) and cranberries

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(cv. Ben Lear), lingonberries (cv. Amberland) had a significantly higher antioxidant activity that was associated with a higher content of anthocyanin and phenolic compounds (Zheng and Wang 2003). When Wang et al. (2005) evaluated fruits of 11 selected lingonberry cultivars, he found fruit soluble solids, titratable acids, anthocyanins, phenolics, and antioxidant activities to vary greatly among the cultivars. To date, commercial production of lingonberry from cultivated fields is far less common than from wild plants (Hendrickson 1997; Gustavsson 2001; Paal 2006). However, there is clearly an increasing demand for lingonberry products due to its rich antioxidant content, medicinal properties, and expanding fresh market (Galletta and Ballington 1996; Penhallegon 2003; Wang et al. 2005; Finn and Mackey 2006). In Sweden, lingonberry is called “red gold” due to its economic importance (Persson and Gustavsson 2001). Major lingonberry exporting countries are Sweden, Finland, and counties of the former Soviet Union (Paal 2006).

10.2 Breeding and Genetics 10.2.1 History of Breeding 10.2.1.1 Blueberry Highbush breeding began in the early 1900s in New Jersey, with the first hybrid being released in 1908 by Frederick Coville of the USDA. He conducted the fundamental life history studies of the blueberry that served as the basis of cultivation such as soil pH requirements, cold and day-length control of development, pruning strategies, and modes of propagation. Working with Elizabeth White and others, he collected several outstanding wild clones of V. corymbosum and V. angustifolium, which he subsequently used in breeding improved types. Over 75% of the current blueberry acreage is still composed of his hybrids, most notably “Bluecrop”, “Jersey”, “Weymouth”, “Croatan”, “Blueray”, “Rubel”, and “Berkeley” (Mainland 1998). George Darrow assumed the USDA program after Coville died in 1937 and made important contributions on the interfertility and phylogeny of the native Vaccinium species in cooperation with the taxonomist W.H. Camp (Hancock 2006a). He formed a large

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collaborative testing network that encompassed private growers and Agricultural Experiment Station scientists in Connecticut, Florida, Georgia, Maine, Massachusetts, Michigan, New Jersey, and North Carolina. From 1945 to 1961, he sent out almost 200,000 seedlings to his cooperators for evaluation. Arlen Draper followed Darrow and focused on incorporating the genes of most wild Vaccinium species into the cultivated highbush background (Draper 1995; Hancock 2006b). He maintained and strengthened Darrow’s collaborative network and released a prodigious number of southern and northern highbush cultivars, with improved fruit color and firmness, smaller pedicle scars, and higher productivity (Hancock and Galletta 1995). His “Duke” and “Elliott” have been major successes, along with the newer release “Legacy”. Mark Ehlenfeldt assumed the USDA-ARS program in 1998. Ralph Sharp began working in the 1950s in Florida on the development of southern highbush types in collaboration with Darrow (Sharp and Darrow 1959; Lyrene 1998). He was the first collector of V. darrowii for breeding, and until very recently, all southern highbush cultivars contained genes from his wild clones. Sharp and his colleague Wayne Sherman developed several successful cultivars, including “Sharpblue”, which was grown commercially until very recently. Paul Lyrene took over the breeding work in Florida in 1977 and has released a number of important cultivars including “Star” and “Jewel”. Stanley Johnson at Michigan State University spent a considerable amount of time in the 1950s and 1960s improving the cold tolerance of highbush by crossing it with V. angustifolium. Out of this work came the “half-high” cultivar Northland and the mostly pure highbush type “Bluejay”, which was released by his successor James Moulton. The program was abandoned in 1978 but was renewed in 1990 by James Hancock. He has released three important cultivars, “Aurora”, “Draper”, and “Liberty”. In the Pacific Northwest, Joseph Eberhart, in Olympia, Wash., released three cultivars, Pacific, Olympia, and Washington, in the 1920s and 1930s. “Olympia” is still widely grown today. Outside of the USA, blueberry breeding work was conducted in Australia, Germany, and New Zealand. Johnston sent open-pollinated seed to D. Jones and Ridley Bell in Australia in the 1960s that generated the important cultivar “Brigitta Blue” along with

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several others. Narandra Patel at HortResearch in New Zealand released the cultivars Nui, Puru, and Reka from breeding material initially provided by the University of Arkansas and the USDA at Beltsville in the 1960s and 1970s. Walter Heermann in Germany, working with seed provided by Frederick Coville, released several varieties in the 1940s and 1950s including “Blauweiss-Goldtraube”, “BlauweissZukertraube”, “Heerma”, “Rekord”, “Ama”, and “Gretha”. Rabbiteye breeding was initiated in 1939 by George Darrow in collaboration with Otis J. Woodard at the Georgia Coastal Plain Experiment Station (Tifton, Ga.) and Emmett B. Morrow at the North Carolina Experiment Station, although a collection of wild selections from Florida and Georgia had been planted at Tifton in the 1920s (Austin 1994). This work was continued by Max Austin and then Scott NeSmith in Georgia, Gene Galletta followed by James Ballington in North Carolina, and Ralph Sharp, Wayne Sherman, and then Paul Lyrene in Florida (Lyrene 1987). These breeding programs have resulted in significant improvements in fruit color, size, texture, and appearance over the original wild selections. The most important cultivars have been “Tifblue” (1955) and “Brightwell” (1971) from Georgia, “Bluegem” (1970) and “Bonita” (1985) from Florida, and “Powderblue” and “Premier” (1978) from North Carolina. Rabbiteye cultivars were also bred in the New Zealand HortResearch, Inc. program of Narandra Patel. Several releases came from this program in the 1990s including “Maru” and “Rahi”. Lowbush blueberry breeding has generally received little attention. The primary effort has been centered with Agriculture and Agri-Foods Canada (Kentville, NS), currently overseen by Andrew Jamieson. At this station, wild selections from Maine and the Maritime Provinces were tested and crossed, resulting in a number of releases including “Augusta”, “Blomidon”, “Brunswick”, “Chignecto”, and “Fundy”. Recently, a seed-propagated lowbush cultivar, “Novablue”, was released by Andrew Jamieson from the cross of “Fundy” “Brunswick”. The hybrids have unusually large berries and spread more rapidly by rhizomes than the parent clones. Lowbush blueberries have been hybridized with V. corymbosum to produce “halfhigh” cultivars (Finn et al. 1990). The major releases of this type were “Northland” developed by Stanley Johnston in Michigan and

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“Northblue”, “Northsky”, “Northcountry”, “St. Cloud”, “Polaris”, and “Chippewa” released by James Luby in Minnesota. The “half-highs” have much higher yields and larger fruit than lowbush but have low enough stature to be protected by snow in areas with extreme winter cold.

10.2.1.2 Cranberry Breeding of cranberries has been sporadic since the mid-1900s. However, during the last decade of the twentieth century, much of the acreage previously planted to native selections “Early Black”, “Howes”, “McFarlin”, and “Searles” has been renovated with first generation hybrids (Galletta and Ballington 1996), the cultivar Stevens being the most widely planted. In 1929, the USDA began a major cooperative cranberry-breeding project with the New Jersey, Massachusetts, and Wisconsin Agriculture Experiment Stations to develop varieties resistant to false blossom disease, a phyoplasma (Chandler et al. 1947). Resistance to false bottom was based on developing varieties that would be less attractive to the blunt-nosed leaf hopper, the vector of the false blossom agent. The majority of the seedlings were planted in New Jersey because of the severity and prevalence of false bottom in the state. Out of this program came “Pilgrim”, “Wilcox”, and “Stevens”. “Pilgrim” was released for improved productivity, size, color (purplish red), keeping quality, productivity, and resistance to the blunt-nosed leafhopper. “Stevens” was selected and released for its improved productivity, color (deep red), firmness, and resistance to softening (Dana 1983). “Crowley” was introduced from the Washington Agriculture Station in the 1960s as a better pigmented replacement for “McFarlin” but has lost favor due to variable and generally low productivity.

10.2.1.3 Bilberry Although commercial prospects for medicinal and nutritional supplement products may be promising, bilberry (V. myrtillus L.) has not been commercially cultivated. In 1994, evaluations of bilberry selections obtained from a wide area of western North America and northern Europe was begun at the University of Idaho. To date, 13 bilberry selections

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have been selected for advanced testing, although no bilberry cultivars have been released.

10.2.1.4 Lingonberry Beginning in the 1960s, domestication of lingonberry was initiated in Sweden, Finland, Lithuania, Germany, and Latvia (Te€ar 1972; Luby et al. 1991; Gustavsson 2001; Paal 2006). In the USA, cultivation of lingonebrry started in 1987 (Stang et al. 1993). From the 1960s–1990s, lingonberry breeding focused on selection and evaluation of wild clones and seedlings. To date, 24 cultivars have been released (Table 10.4), with some selections still under field trails (Finn and Mackey 2006).

10.2.2 Breeding Objectives In general, the major breeding objectives for Vaccinium species are either for improved fruit production (high productivity, fruit quality, and resistance to pests, diseases, and abiotic stresses) or for modified ornamental characteristics (colorful flowers and leaves, wide adaptability of habitat, and distinguished plant architectures).

10.2.2.1 Blueberries The current goals of southern highbush breeders are to obtain early ripening cultivars with high plant vigor, improved disease resistance, and later flowering dates (particularly in the southeastern USA, where late freezes are a problem). Higher yields, better flavor, and characteristics favorable for mechanical harvest are also being sought. Cultivars and advanced breeding lines are being used to breed southern highbush, along with hybrids derived from native, lowchill highbush selections from Florida and Georgia (V. ashei, V. elliottii, and V. darrowii). Because of their low chill requirement and the influence of genes from V. darrowii, many southern highbush cultivars can be grown as evergreens that avoid dormancy in areas with mild winters, with a harvest season that extends for several months through the winter and early spring (Darnell and Williamson 1997). Rabbiteye breeders hope to expand harvest dates, improve berry size and fruit quality, reduce susceptibility to rain cracking, and extend storage life. Southern highbush cultivars are being developed at several locations, including Arkansas, California, Florida, Georgia, Mississippi, Australia, Chile, and Spain. Paul Lyrene at the University of Florida has the most active program dealing with very low chill genotypes and has released many high impact cultivars including

Table 10.4 Lingonberry cultivars Source Cultivar (year of release) Dutch cultivars “Koralle” [1969]; “Red Pear” [1981] German cultivars

Swedish cultivars

Norwegian cultivar Polish cultivars Canadian cultivars Russian cultivars Latvian cultivars American cultivars selected from Finnish seeds

“Erntedank” [1975]; “Erntekrone” [1978]; “Erntesegen” [1981]; “Ammerland” and “Erzgebirgeperile” [1993] “Sussi” [1986]; “Sanna” [1988]; “Ida” and “Linnea” [1999] “Scarlet” [unknown] “Masovia” [1985]; “RunoBielawskie” [1996] “Utopia” [1998] “Kostromskaya rozovaya” and “Kostromichka” [1996]; “Rubin” [1998] “Salaspils 1”, “Salaspils 2”, “Salaspils 4”, and “Salaspils 5” “Splendor” and “Regal” [1987]

References Liebster (1977), Gustavsson (1999), Pliszka and Kawecki (2000) Zillmer (1985), Pliszka and Kawecki (2000)

Gustavsson (1993, 1999), Gustavsson and Trajkovski (1999), Trajkovski and Sjo¨stedt (1986), Eckerbom (1988) Gustavsson (1997) Pliszka and Kawecki (1985, 2000) Estabrooks (1998) Tyak et al. 2000 Audrinja (1992) Stang et al. (1994)

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“Emerald”, “Jewel”, “Misty”, and “Star”. James Ballington in North Carolina has the most significant program operating at the interface between northern and southern highbush types and has generated a number of important cultivars including “Lenore”, “New Hanover”, “O’Neal”, “Reveille”, and “Sampson”. James Moore and now John Clark at the University of Arkansas have focused on mixing southern wild species with northern types and recently released “Ozarkblue”, a very high quality late type. Scott NeSmith at the University of Georgia has generated several new early varieties including “Rebel”, “Camellia”, and “Palmetto”. He also has an active rabbiteye breeding program and his late season cultivar Ochlockonee has generated considerable interest. Steven Stringer, Arlen Draper, and James Spiers at the USDA-ARS in Mississippi have developed a number of southern highbush types including “Biloxi”, “Gupton”, and “Magnolia”. Several private breeding programs have also emerged that are developing southern highbush types including Atlantic Blue in Spain (Ridley Bell), Berry Blue in Michigan and Chile (Edmond Wheeler), Driscoll Associates in California (Brian Caster), Mountain Blue Orchard in Australia (Ridley Bell), and Vital Berry in Chile (James Ballington). Berry Blue is also devoting some effort to rabbiteye types. Northern highbush breeders are concentrating on flavor, longer storing fruit, expanded harvest dates, disease and pest resistance, and machine harvestability. Established breeding lines are being used in these efforts along with complex hybrids made up of V. darrowii, V. angustifolium, V. constablaei, and most of the other wild species. Even though it has limited winter hardiness, V. darrowii has proven to be an interesting parent in colder climates because it passes on a powder blue color, firmness, high flavor, heat tolerance, and upland adaptation (Hancock 1998). Northern highbush blueberries are currently being bred in New Jersey, Michigan, Oregon, and Chile. James Hancock at Michigan State University is focusing on late maturing, long storing genotypes and has released three new northern highbush cultivars that show high promise, “Aurora”, “Draper”, and “Liberty”. Mark Ehlenfeldt of the USDA program in New Jersey is focusing on identifying genotypes with high disease resistance and tolerance to winter cold and has released several cultivars including “Chanticleer” and “Hannah’s Choice”. Nicholi Vorsa at the Cranberry

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and Blueberry Research Station of Rutgers University has begun a program in New Jersey to develop locally adapted highbush cultivars with machine harvestability and high fruit quality. Chad Finn of the USDA in Oregon is active in identifying genotypes that are well suited to the Pacific Northwest. Other worldwide northern highbush breeding projects include “Berry Blue” in Michigan and Chile, Fall Creek Farm and Nursery in Oregon, Driscoll Associates in California and Washington, and the University of Talca and Vital Berry in Chile. Danny Barney at the University of Idaho, and to a lesser extent the USDA-ARS (Ore.), is selecting superior genotypes of V. membranaceum, V. ovalifolium, and V. deliciosum that may have potential as commercial “huckleberry” cultivars, and some of these are in commercial trial. They have also attempted to cross these species with highbush blueberry with very limited success. Rabbiteye breeders hope to expand harvest dates and improve flavor and storage life. The northern highbush breeders are concentrating on flavor, longer storing fruit, expanded harvest dates, disease and pest resistance, and machine harvestability. Established breeding lines are being used by northern highbush breeders along with complex hybrids made up of V. darrowi, V. angustifolium, V. constablei, and most of the other wild species. Even though it has limited winter hardiness, V. darrowii has proven to be an interesting parent in colder climates, because it passes on a powderblue color, firmness, high flavor, heat tolerance, and potential upland adaptations.

10.2.2.2 Cranberry Cranberry breeding efforts are being focused on early maturing fruit, uniform large size, intense color (total anthocyanin content – TACy), keeping quality, high productivity, disease resistance, and plant vigor. The greatest emphasis is being placed on productivity and resistance to fruit rot organisms. Cranberries are currently being bred by Nicholi Vorsa at Rutgers University in New Jersey and Eric Zeldin and Brent McCown at the University of Wisconsin. The Wisconsin team recently released the first new cranberry cultivar in over 30 years – “HyRed”, which is distinguished by its earliness and deep red color (McCown and Zeldin 2003). The Rutgers program released three cultivars in

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2006, “Crimson Queen”, “Mullica Queen”, and “Demoranville”. “Crimson Queen” and “Demoranville” have tested for high TACy, large fruit size, and productivity. “Mullica Queen” is being released for high production potential and improved TACy relative to “Stevens”.

10.2.2.3 Bilberry Bilberry domestication efforts started with evaluation of elite selections. Danny Barney at the University of Idaho has made a number of advanced selections of bilberry (V. myrtillus), Cascade huckleberry (V. deliciosum), mountain huckleberry (V. membranaceum), and oval-leaved bilberry (V. ovalifolium, a.k.a. Alaska blueberry, V. alaskaense) since 1994. The selection criteria include canes (numerous, vigorous, upright, many fruiting laterals), physiology (late-blooming, self-fruitful, suitable ripening period), leaves (thick, tough, leathery), fruit (flavor, color, high anthocyanin and antioxidant capacity, and size), and disease resistance (mummy berry, blight, twig blight, and bacterial canker) (Danny Barney personal communication).

10.2.2.4 Lingonberry Lingonberry breeding efforts are being focused on broadly adapted cultivars with high productivity, increased fruit size, insect- and disease-resistance, tolerance to abiotic stresses, suitability for mechanical harvesting, high flavor and aroma retention, and enhanced polyphenolic (flavonoid) profiles beneficial for human health (Galletta and Ballington 1996). The first and best known cultivar, “Koralle”, was initially selected as an ornamental plant by H. Van der Smith in Holland in 1969 (Liebster 1977). It was first cultivated in Germany as a fruit crop because of its suitability for machine harvesting and fall cropping habit (Gustavsson 1999). In terms of overall agronomic worth, “Koralle” is still one of the best cultivars for commercial cultivation (Finn and Mackey 2006). A number of additional cultivars have been released in Europe. “Red Pearl” selected by Blanke in Boskoop and “Ammerland” selected by Kr€ uger in Westerstede are similar to “Koralle” in plant growth

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and berry yield but have larger fruits (Dierking and Beerenobst 1993). The now closed Balsga˚rd Research Station released a number of cultivars and selections (Trajkovski and Sjo¨stedt 1986; Eckerbom 1988; Gustavsson and Trajkovski 1999). Of these, “Ida” and 8723-10 showed the best characteristics for commercialization (Finn and Mackey 2006). In Germany, “Erntekrone”, “Erntedank”, and “Erntesegen” were released by Albert Zillmer in 1970s. “Erntesegen” has particularly large and aromatic berries (Zillmer 1985). “Ammerland” and “Erzgebirgeperle” were selected by Kr€uger in 1993 (Pliszka and Kawecki 2000). In Latvia, four cultivars “Salaspils 1”, “Salaspils 2”, “Salaspils 4”, and “Salaspils 5” were selected from natural habitats by Ripa and Audrinja in the 1980s (Audrinja 1992). “Salaspils 2” has particularly high yield (about 900 g m2) and large fruits (4.0 g) (Paal 2006). In Poland, Kawecki selected two cultivars “Masovia” and “Runo Bielawskie” (Pliszka and Kawecki 1985, 2000). In Russia, three one-time blooming cultivars “Kostromskaya rozovaya”, “Kostromichka”, and “Rubin” were selected by Tyak and Cherkasov in 1990s (Tyak et al. 2000). In Estonia, a number of lingonberry selections are currently in field trials (Paal 2006). In North America, “Regal” and “Splendor” were selected from Finnish seeds in Wisconsin by Stang et al. (1994). They showed high productivity and adaptability to North American climatic conditions (Stang et al. 1994; Galletta and Ballington 1996). In Canada, selections of V. minus, including F91-3, F91-5, and F91-1 (“Utopia”), have shown promise (Estabrooks 1998).

10.2.3 Traditional Breeding Techniques Vaccinium are crops propagated through cuttings and micropropagation, so elite genotypes can be directly utilized without the need to develop pure lines. The breeding of Vaccinium species has evolved, as with other woody crops, from casual selection of elite wild clones to the use of controlled crosses and rigorous field selection. Techniques involving marker-assisted selection are just beginning to emerge to maximize the efficiency of plant breeding. Currently, Vaccinium cultivars are obtained exclusively through traditional breeding approaches.

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10.2.3.1 Crosses Within Species Self-pollinations are rarely used in Vaccinium breeding due to reduced seed set, germination, and because seedlings from selfing tend to be weak. Most breeding programs have relied primarily on pedigree breeding where elite parents are selected each generation for intercrossing. However, the Florida southern highbush and rabbiteye breeding programs have utilized recurrent selection (Lyrene 1981, 2005). About 150 different genotypes are used in the Florida program each year, in random pairwise combinations. Blueberries are all primarily outcrossing with varying levels of self-fertility, depending on species and genotype. In general, northern highbush blueberries have the highest levels of self-fertility, followed by southern highbush and then rabbiteye. Cultivars that are not highly self-fertile display reduced fruit set and berry size when self-pollinated (Morrow 1943; El-Agamy et al. 1981; Rabaey and Luby 1988; Gupton and Spiers 1994; Ehlenfeldt and Prior 2001). Highbush are generally planted in solid blocks, although having a pollinizer would be beneficial for most cultivars. All rabbiteye cultivars need pollinizers and alternate row plantings are recommended. Lowbush fruit is harvested from highly variable native stands, with abundant opportunity for cross-pollination. Self-infertility in blueberries has been shown to be the result of late-acting inbreeding depression (Krebs and Hancock 1988, 1990; Hokanson and Hancock 1998). Harrison et al. (1993) found that parental self-fertility was not predictive of the self fertility of progeny in segregating families of half-high and highbush genotypes. Cranberries are generally self-fertile, but crosspollination can enhance seed production (Sarracino and Vorsa 1991; Galletta and Ballington 1996). For lingonberries, cross-pollination gives twice the fruit and seed set of self-pollination (Fernqvist 1977; Lehmushovi 1977).

Interspecific Hybridization To enhance levels of genetic variability within a species (primary genepool), interspecific hybridization followed by backcrossing is commonly used to introgress desirable genes from related species within the genus (secondary genepools) to commercial cultivars

G.-Q. Song and J.F. Hancock

(Ballington 2009; Vorsa et al. 2009). Interspecific hybridization has played a significant role in development of blueberry cultivars for fruit production (Brevis et al. 2008; Ballington 2009). Ballington (2009) has recently summarized the successful role of interspecific hybridization in blueberry improvement as following (1) For highbush blueberry improvement, the species used in interspecific hybridization include V. angustifolium (lowbush blueberry), V. darrowii (Darrow’s evergreen blueberry), V. ashei or V. virgatum (rabbiteye blueberry), V. tenellum (southern lowbush blueberry), V. elliottii (Mayberry), and V. constablaei (Constable’s blueberry). (2) For improvement of hexaploid rabbiteye blueberries (V. virgatum), interspecific hybridization has only played a minor role to date. (3) Cultivar improvement in tetraploid lowbush blueberries has been confined exclusively to the primary gene pool of V. angustifolium to date. (4) V. pallidum holds promise for contributing to future highbush and half-high blueberry improvement. (5) V. constablaei and hexaploid southern highbush will probably contribute significantly to rabbiteye blueberry improvement in the future. (6) Intersectional crosses among tetraploid species also may be promising for future blueberry cultivar improvement.

Genetics and Marker-Assisted Selection (MAS) A wide array of markers have been utilized in blueberry for fingerprinting and linkage mapping including proteins (Bruederle et al. 1991; Hokanson and Hancock 1998), restriction fragment length polymorphisms (RFLPs) (Haghighi and Hancock 1992), random amplified polymorphic DNA (RAPD) (Aruna et al. 1993; Levi et al. 1993; Qu and Hancock 1997), simple sequence repeat (SSR), and express sequence tag-polymerase chain reaction (EST-PCR) (Rowland et al. 2003a, b; Boches et al. 2005, 2006). More limited numbers of marker studies have been conducted in cranberry, although isozymes were used to measure diversity patterns in native V. macrocarpon (Bruederle et al. 1996), and RAPDs were utilized to determine cultivar identity and heterogeneity in commercial beds (Novy et al. 1994). Polashock and Vorsa (2002a, b) used the sequence-characterized amplified region (SCAR) technique to fingerprint over 500 accessions and to estimate the degree of genetic similarity.

10 Vaccinium

Most recently, blueberry markers, 39 EST-SSRs and 10 genomic SSRs, have been tested for the ability to amplify a polymorphic marker in American cranberry accessions. Sixteen SSRs resulted in informative and polymorphic primer pairs and were used to fingerprint 16 economically important cranberry cultivars (Bassil et al. 2009). Rowland and Levi (1994) developed the first blueberry map using a diploid population segregating for chilling requirement. Their population was a cross between an F1 interspecific hybrid (V. darrowii V. elliottii) and another clone of V. darrowii. They have continued to periodically add markers and at the last report, the map had 72 RAPD markers on 12 linkage groups, which is in agreement with the basic chromosome number of blueberry (Rowland and Hammerschlag 2005). Later, Rowland et al. (1999, 2003b) constructed RAPD-based maps of diploid V. corymbosum (V. caesariense Mack.) V. darrowii hybrids crossed with other V. darrowii and V. corymbosum selections. The goal was to develop populations that were segregating for chilling requirement and cold tolerance. First RAPD and more recently ESTPCR markers were added to this map and a quantitative trait loci (QTL) was identified that explained about 20% of the genotypic variance associated with cold hardiness (Rowland et al. 2003a, b, c; Rowland and Hammerschlag 2005). Qu and Hancock (1997) constructed an RAPDbased genetic map of a tetraploid population resulting from the cross of US 75 tetraploid V. corymbosum, “Bluecrop”. One hundred and forty markers were mapped to 29 linkage groups. The map was essentially that of V. darrowii, as US 75 was produced from an unreduced gamete of V. darrowii and only unique markers for Fla 4B were used. Fla 4Bwas one of the V. darrowii clones used by Rowland and Levi (1994) and Rowland et al. (1999). As was previously noted, Fla 4B hybrids (in particular US 75) have been used extensively in breeding to produce low-chilling types. SSR markers are powerful tools for fingerprinting blueberry cultivars. Thirty SSRs were derived from either EST or genomic DNA libraries of highbush blueberry cv. Bluecrop (Boches et al. 2005, 2006). One or just two (NA-1040 þ CA421) selected SSRs allowed identification of each of the 75 tested cultivars (Hinrichsen et al. 2009). In addition, the EST-SSRs were also very effective at estimating genetic relationship as well as at distinguishing closely spaced

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lowbush blueberry cultivars (Bell et al. 2008; Brevis et al. 2008). Most recently, Brevis and Hancock at Michigan State University used these SSRs to develop a linkage map of the tetraploid cross “Jewel” (southern highbush) “Draper” (northern highbush). The ultimate goal is to identify QTL for the chilling requirement. Polashock and Vorsa (2006) are using bulked segregant analysis to tag genes for mummy berry resistance in segregating blueberry populations with V. darrowii as the source of resistance. Kreher et al. (2000) found that 15 RAPD markers distinguished 67 genets of 99 total samples of deerberry (V. stamineum L.) from 22 patches in a 1-ha site. There was genetic diversity within individual patches (Kreher et al. 2000). For V. vitis-idaea L., genetic and genotypic diversity of four Swedish populations was investigated using automated image analysis of leaf shape and RAPD analysis (Persson and Gustavsson 2001). Forty-three RAPD allowed for the identification of 29 different genotypes among 129 plants from two populations. Most of the variation could be attributed to within-population variation. RAPDs were also used to determine genetic diversity in 15 lingonberry (V. vitis-idaea L.) populations in Sweden, Finland, Norway, Estonia, Russia, Japan, and Canada (Gustavsson et al. 2005).

10.2.4 Genomic Resources The rapid advance in DNA sequencing technology has accelerated the accumulation of plant genome sequence data, including whole genome sequencing, genome survey sequencing, and ESTs of genomic resources. For Vaccinium species, collection of genomic resources began with the generation about 1,300 ESTs (Dhanaraj et al. 2004). These ESTs were subsequently demonstrated to be a reliable genomic resource for effective analysis of gene expression associated with cold acclimation. cDNA microarrays were also used for gene expression studies under field and cold room conditions (Dhanaraj et al. 2007). Based on these ESTs and microarray data, the blueberry genomics database (BBDG) was developed (Alkharouf et al. 2007). This database is presently focusing on identification of genes associated with cold acclimation and freeze tolerance in blueberry (Alkharouf et al. 2007).

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More recently, a fruit ripening-related EST library has been generated for bilberry (V. myrtillus L.), and the ESTs will be used to characterize genes involved in fruit development and ripening (Jaakola et al. 2009).

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219 revealed by RAPDs and leaf-shape analysis. Mol Ecol 10: 1385–1397 Pliszka K, Kawecki L (1985) ‘Masovia’ – a new Polish selection of lingonberries. Acta Hortic 165:273 Pliszka K, Kawecki L (2000) A new selection of lingonberries (Vaccinium vitis-idaea L.) in Poland. In: Proceedings of conference on problems of rational utilization and reproduction of berry plants in boreal forests on the eve of the 21st century, Gomel-Glubokoye, Belorus, pp 197–199 Polashock J, Vorsa N (2002a) Cranberry transformation and regeneration. In: Khachatourians GG, McHughen A, Scorza R, Nip WK, Hui YH (eds) Transgenic plants and crops. Marcel Dekker, New York, NY, pp 383–396 Polashock J, Vorsa N (2002b) Breeding and biotechnology: a combined approach to cranberry improvement. Acta Hortic 574:171–174 Polashock J, Vorsa N (2006) Segregating blueberry populations for mummy berry fruit rot resistance. In: New Jersey annual vegetation meeting proceedings, Atlantic City, NJ, USA Popenoe W (1924) Economic fruit-bearing plants of Ecuador. Contrib US Nat Herb, vol 24, Pt 5. Smithsonian Institute, Washington DC, USA, pp 101–134 Powell EA, Kron KA (2002) Hawaiian blueberries and their relatives-A phylogenetic analysis of Vaccinium sections Macropelma, Myrtillus, and Hemimyrtillus (Ericaceae). Syst Bot 27:768–779 Prior RL, Cao G, Martin A, Sofic E, McEwen J, O’Brien C, Lischner N, Ehlenfeldt M, Kalt W, Krewer G, Mainand CM (1998) Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. J Agric Food Chem 46:2686–2693 Qu L, Hancock JF (1995) Nature of 2n gamete formation and mode of inheritance in interspecific hybrids of diploid Vaccinium darrowi and tetraploid V. corymbosum. Theor Appl Genet 91:1309–1315 Qu LP, Hancock JF (1997) Randomly amplified polymorphic DNA-(RAPD-) based genetic linkage map of blueberry derived from an interspecific cross between diploid Vaccinium darrowi and tetraploid V. corymbosum. J Am Soc Hortic Sci 122:69–73 Qu L, Vorsa N (1999) Desynapsis and spindle abnormalities leading to 2n pollen formation in Vaccinium darrowii. Genome 42:35–40 Qu L, Hancock JF, Whallon JH (1998) Evolution in an autopolyploid group displaying predominantly bivalent pairing at meiosis: genomic similarity of diploid Vaccinium darrowi and autotetraploid V. corymbosum (Ericaceae). Am J Bot 85:698–703 Raatikainen M, Raatikainen T (1983) Mustikan sato, poiminta ja markkinointi Pihtiputaalla. Silva Fenn 17:113–123 Rabaey A, Luby J (1988) Fruit set in half-high blueberry genotypes following self and cross pollination. Fruit Var J 42: 126–129 Ritchie JC (1956) Biological flora of the British Isles Vaccinium myrtillus L. J Ecol 44:291–299 Robuck OW (1989) Common Alpine plants of Southeast Alaska. Misc. Publ. Juneau, AK, US Department of Agriculture, Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, 207 p

220 Roper TR, Vorsa N (1997) Cranberry: botany and horticulture. Hortic Rev 21:215–249 Rousi A (1963) Hybridization between Vaccinium uliginosum and cultivated blueberry. Ann Agric Fenn 2:12–18 Rousi A (1967) Cytological observation on some species and hybrids in Vaer. Zuechter 36:352–359 Rowland LJ, Hammerschlag FA (2005) Vaccinium spp. blueberry. In: Litz RE (ed) Biotechnology of fruit and nut crops. CABI, Cambridge, MA, pp 222–246 Rowland LJ, Levi A (1994) RAPD-based genetic linkage map of blueberry derived from a cross between diploid species (Vaccinium darrowi and V. elliottii). Theor Appl Genet 87: 863–868 Rowland LJ, Ogden EL, Arora R, Lim CC, Lehman JS, Levi A, Panta GR (1999) Use of blueberry to study genetic control of chilling requirement and cold hardiness in woody perennials. HortScience 34:1185–1191 Rowland L, SmritiM DA, Ehlenfeldt M, Ogden E, Slovin J (2003a) Development of ESTPCR markers for DNA fingerprinting and mapping in blueberry (Vaccinium, section Cyanococcus). J Am Soc Hortic Sci 128:682–690 Rowland LJ, Dhanaraj AL, Polashock JJ, Arora R (2003b) Utility of blueberry-derived EST-PCR primers in related Ericaceae species. HortScience 38:1428–1432 Rowland LJ, Mehra S, Arora R (2003c) Identification of molecular markers associated with cold tolerance in blueberry. Acta Hortic 625:59–69 Rui H (1982) Germplasm resources of the wild fruits in the Chang-Bai-Shan mountain region (In Chinese). Acta Hortic Sin 9:9–18 Sarracino J, Vorsa N (1991) Self- and cross-fertility in cranberry. Euphytica 58:129–136 Schultz JH (1944) Some cytotaxonomic and germination studies in the genus Vaccinium. PhD Dissertation, Washington State University, Pullman, WA, USA Seeram NP, Adams LS, Zhang Y, Lee R, Sand D, Scheuller HS, Heber D (2006) Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. J Agric Food Chem 54:9329–9339 Sharp RH, Darrow GM (1959) Breeding blueberries for the Florida climate. Proc FL State Hortic Soc 72:308–311 Sleumer H (1941) Vaccinioidee-Studien. Botanische Jahrb€ ucher 71:375–510 Sorsa V (1962) Chromosomenzahlen finnischer Kormophyten. I Ann Acad Scient Fenn 58:1–14 Stang EJ, Anderson SP, Klueh J (1993) Lingonberry cultural management research in Wisconsin, USA. Acta Hortic 346:327–333 Stang EJ, Klueh J, Weis GG (1994) ‘Splendor’ and ‘Regal’ lingonberry – new cultivars for a developing industry. Fruit Var J 48:182–184 Stevens PF (1972) Notes on the infrageneric classification of Agapetes with four new taxa from New Guinea. Notes R Bot Gard Edinb 32:13–28 Strik B (2005) Blueberry – an expanding world berry crop. Chron Hortic 45:7–12 Strik B, Yarborough D (2005) Blueberry production trends in North America, 1992 to 2003 and predictions for growth. HortTechnology 15:391–398

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221 Young SB (1970) On the taxonomy and distribution of Vaccinium uliginosum. Rhodora 72:439–459 Zeldin EL, McCown BH (2002) Towards the development of a highly fertile polyploid cranberry. Acta Hortic 574: 175–180 Zheng W, Wang SY (2003) Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries. J Agric Food Chem 51:502–509 Zillmer A (1985) Account of my three types of Vaccinium vitisidaea: ‘Erntedank’– ‘Erntekrone’–‘Erntesegen’. Acta Hortic 165:295–297

.

Chapter 11

Vitis Jaya R. Soneji and Madhugiri Nageswara-Rao

11.1 Basic Botany of the Species With a total world production of 60.9 million tons (FAO 2007), grapes are grown in more than 80 countries with acreage in excess of 7.8 million hectares (Torregrosa et al. 2002a, b). Grapes are the world’s third largest fruit crop, one of the oldest cultivated plants (Olmo 1976) and are also the most widespread fruit crop (Perl and Eshdat 2007). They are used as fresh fruit or processed into juice, jelly, raisins, wine, and organic compounds. Conversion of grape berries into wine is the most value-added use among all fruit crops (Gray et al. 2005). Italy, China, and the USA are the top three grape producers in the world (FAO 2007; Fig. 11.1). Grapes (Vitis) belong to the family Vitaceae. There are about 60 Vitis species in the world, with the greatest concentration in the USA and Asia, particularly China (Owens 2008). The number of Vitis species is in taxonomic dispute due to the interfertility of all the species, their sympatric nature, and the resulting high degree of hybridity (Riaz et al. 2007). The Asian Vitis species are not well described outside of the Chinese literature and the germplasm is often unavailable outside of Asia, while the Vitis species of Mexico, Central America, and the extreme northern portions of South America are poorly characterized (Comeaux et al. 1987). Among the Asian species, only V. amurensis has been domesticated and used for fresh fruit, juice, wine, and jelly production

J.R. Soneji (*) IFAS, Citrus Research & Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USA e-mail: [email protected]

(Huang 1980). The North American species, including V. aestivalis, V. cinerea var. helleri, V. labrusca, V. riparia, and V. rupestris, have been extensively used to produce rootstocks and fruiting cultivars with fungal resistance (Owens 2008). V. vinifera is the sole native species in Europe, the Near East, and northern Africa and is the most widely cultivated species (Owens 2008). The genus Vitis is divided into two subgenera, viz., Euvitis Planch. and Muscadinia Planch. Euvitis (2n ¼ 38) contains all bunch grape species and is divided into three major groups of species, an American group of 18–28 species, an Asian group of 10–15 species and an European or central Asian group containing the widely cultivated V. vinifera L. species (Einset and Pratt 1975). Muscadinia (2n ¼ 40) contains V. rotundifolia Michx., V. munsoniana Simpson ex Munson, and V. popenoei Fennell, a native to the southeastern USA and Central America (Winkler et al. 1974; Einset and Pratt 1975). V. vinifera subsp. sylvestris is regarded as the wild ancestral grape due to its close morphological resemblance and free gene flow within themselves (Heywood and Zohary 1991). Extant, isolated patches of V. vinifera subsp. sylvestris can be found from western Europe to central Asia and North Africa (Owens 2008). Grapes are woody, climbing, or trailing perennial vines with simple, serrate to sinuate, petiolate leaves. They have coiled tendrils opposite leaves, which appear in an alternate phyllotactic pattern (Pratt 1974). The inflorescence is also borne opposite to the leaves. Wild grapes (V. vinifera subsp. sylvestris) are dioecious, while domesticated grapes (V. vinifera subsp. sativa) are hermaphrodite (excluding rare exceptions of female cultivars) (Di Vecchi et al. 2009). They have five sepals, a petal, and a stamen jointly arising from each of five primordial, and the pistil appears as a ring from the inner

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8_11, # Springer-Verlag Berlin Heidelberg 2011

223

224

J.R. Soneji and M. Nageswara-Rao

Fig. 11.1 Top ten grape-producing countries of the world with production in metric tons FAO (2007)

Chile, 2,350,000

South Africa, 2,813,003

Italy, 8,519,418

Argentina, 2,900,000

Iran, 3,000,000 China, 6,787,081 Turkey, 3,612,781

USA, 6,384,090

Spain, 5,995,300 France, 6,044,900

sides of which the placentae develop. The upper part of the ring grows to cover the ovules and forms a short style and disk-like stigma. The petals are free at first but later join along their tips to form a cap or calyptra, which covers the flower bud. The ovary is bilocular with each locule containing two ovules (Reisch and Pratt 1996). The pollen is disseminated by wind, rain, insects and animals. The fruit is a true berry, grows in clusters of 6–300, and can be crimson, black, dark blue, yellow, or green in color. Grapes have a small genome size of 475–500 Mb (approximately four times the size of Arabidopsis and one sixth the size of corn) (Thomas et al. 1993; Lodhi and Reisch 1995). A few cytogenetic studies have been carried out in grapes, the small size of the chromosomes being the limiting factor. These studies, using karyotyping of homogenously stained metaphase plates, have been restricted to the determination of the ploidy level and/or determination of the chromosome length and did not provide any significant information on useful chromosome characteristics, such as the chromosome structure (Alley 1957; Raj and Seethaiah 1969, 1973; Bouquet 1978; Patil and Yadhav 1985; Martens and Reisch 1988; Patil and Patil 1992). Recently in situ hybridization technique with rDNA probes has been used to mark specific chromosome regions (Haas and Alleweldt 2000). Haas and Alleweldt (2000) reported that in

V. vinifera karyotype there is only one pair of satellite chromosomes leading to the assumption that V. vinifera is not allotetraploid; however, they did not rule out the possibility of a Robertsonian translocation. Grapes have tremendous genetic diversity and an extremely wide range of variants have been selected over the millennia. They are generally grown in the Northern Hemisphere between 20 N and 51 N latitude with Germany’s Rhine Valley and British Columbia, Canada being the most northern extent while the southern range extends to India. In the southern hemisphere, grapes are grown between 20 S and 40 S latitude (Reisch and Pratt 1996). Major questions regarding grapevine domestication concern the number of domestication events and the geographic locations where they took place (Arroyo-Garcia et al. 2006). This has led to the formulation of two divergent hypotheses. The first one hypothesizes a restricted origin in which domestication took place from a limited wild stock in a single location, with those cultivars subsequently being transplanted to other regions (Olmo 1976). On the other hand, the second one hypothesizes a multiple origin in which domestication could have involved a large number of founders recruited during an extended time period and along the entire distribution range of the wild progenitor species (Mullins et al. 1992).

11 Vitis

11.2 Conservation Initiatives Wild-growing grape individuals have been identified in France (Lacombe et al. 2002), Spain (Benitez and Ocete-Rubio 1992; Arroyo-Garcia et al. 2006), Italy (Anzani et al. 1990; Grando et al. 1995), Germany, Switzerland, Austria, Romania (Grassi et al. 2003), and Tunisia (Perret et al. 1998), as well as many other European countries (Arroyo-Garcia et al. 2006). However, it is needed to be confirmed whether these individuals are real V. vinifera subsp. sylvestris that have never undergone cultivation or “escaped” individuals from vineyards or hybrids between wild and cultivated forms (This et al. 2006). Habitat loss and the ease with which the wild species can cross with cultivated forms have led to a sharp decline in the number of V. vinifera subsp. sylvestris present and have also led to an increase in the existence of complexes of feral and wild forms (Owens 2008). This loss pointed out the urgency and necessity of international collaboration and cooperation for germplasm collection, maintenance, and preservation of Vitis species, cultivars, and clones in repositories for their characterization, maintenance, evaluation, and free exchange of genetic material (Dettweiler 1990). For this purpose, the Institute for Grapevine Breeding at Geilweilerhof is engaged in the inventory of Vitis species, varieties, and genotypes existing in the grapevine collections worldwide since 1983 and a database, Vitis International Variety Catalog (VIVC), which is accessible via Internet since 1996, was established. To date more than 18,500 prime names of Vitis species, cultivars, and genotypes have been registered (Dettweiler and Eibach 2003). Another database, The European Vitis Database, was established to preserve the old and abandoned grape varieties for their consideration for breeding purposes and contains 27,000 accessions collected from the grape collections of the 18 European project partners involved in the setting up of this database (Dettweiler and Eibach 2003). Nearly every wine producing country has its own grapevine germplasm collection, owing to quarantine restrictions and the need to maintain the material in the field as living plants (This et al. 2006). These databases will enable the curators of germplasm collections, breeders, researchers, and wine producers to access and acquire germplasm. The National Plant Germplasm System (NPGS) of USDA-ARS has established an

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ex situ grape germplasm collection that comprises 3,225 accessions of which 18.8% are wild American grapes (Pavek et al. 2003). In Vietnam, four organizations, viz., Cotton Research Center, Hanoi Vegetable and Fruit Research Institute, Ninh Thuan Domestic Animal and Crops Breeding Center, and Binh Thuan Socio-Economic Development Center, are maintaining and researching on 61, 26, 33, and 42 grapes, respectively. The Spanish grape germplasm bank contains a total of 1,066 V. vinifera accessions (Ortiz et al. 2003). The National Repository of Grape Germplasm in India contains 112 indigenous and 288 exotic accessions of grapes (Mundankar and Karibasappa 2008). The knowledge of the amount and pattern of distribution of genetic variation is central to the development of effective conservation strategies and efficient use of Vitis germplasm (Aradhya et al. 2003). For in situ conservation, sweet mountain grape and Calloosa grape have been examined (Pavek et al. 2001). A sixstep strategy for identifying populations of rock grape (V. rupestris Scheele) to serve as complements to existing ex situ grape germplasm collections has been described, and seven in situ conservation sites for rock grape have been proposed (Pavek et al. 2003). Ex situ conservation techniques, such as the use of slow growth storage and cryopreservation, have also been developed for grape. Slow growth storage techniques of in vitro plants have been used in various genebanks as a backup to grape field collections (Torregrosa et al. 2000). Cryopreservation of grape apices sampled from in vitro plantlets using encapsulation– dehydration techniques has been achieved (Plessis et al. 1991, 1993). The encapsulation–dehydration protocol combined with a slow freeze–dehydration to 40 C has also been used to cryopreserve three cultivars of grape (Zhao et al. 2001). Cryopreservation of in vitro-cultured shoot tips by vitrification has been successfully applied to ten Vitis cultivars or species (Matsumoto and Sakai 2003). Long-term in vitro storage of grapes at low temperatures has been reported (Galzy and Compan 1988; Miaja et al. 2000). Slow growth storage and cryopreservation of the grape rootstock “Kober 5BB” (V. berlandieri V. riparia) have been achieved (Benelli et al. 2003). Ultralow temperature cryopreservation of suspension-grown embryogenic cultures from different cultivars of grape has been demonstrated using a two-step gradual cooling procedure (Dussert et al. 1991) as well as

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encapsulation–dehydration procedure involving a single-step cooling procedure (Wang et al. 2002). A simple noncryogenic technique for long-term storage of viable mature somatic embryos has also been demonstrated (Jayasankar et al. 2005).

11.3 Origin, Evolution, and Diversity of Grape Legend and tradition favor ancient Armenia as the home of the first grape (Olmo 1976). The domestication of V. vinifera began ca. 8000–6000 BC in Transcaucasia in central Asia between the Black Sea and the Caspian Sea (Mullins et al. 1992) extending to Asia Minor, the Fertile Crescent and the Nile Delta by 4000 BC (Dutt 2006). It is likely that prehistoric huntergatherers used wild grapes as a food source (Zohary 1996). Grapes were introduced in China in second century BC during the Han Dynasty (Huang 1980). Greeks and Phoenicians introduced grapes into Europe and northern Africa. The first vineyards in the Marseille region of France were planted by Greeks around 600–500 BC (Levadoux 1953), while Cortez introduced grapes to the Americas (Mullins et al. 1992). There are several morphological and biochemical traits associated with the domestication of V. vinifera that was derived from the progenitor species V. vinifera subsp. sylvestris. Based on geographical origin and morphological characteristics, V. vinifera is further subdivided into three morphotypes, viz., occidentalis, pontica, and orientalis (Negrul 1938). The occidentalis group is characterized by small berries, small clusters, highly fruitful shoots and is associated with cultivars of western European origin. The orientalis group consists of large berried, loose clustered cultivars from West Asia. The pontica group comprises an intermediate grouping of cultivars from eastern Europe and the basin of the Black Sea. Debate exists concerning the number of domestication events and the location of their occurrence, as V. vinifera ssp. sylvestris had a wide geographic range. A wide range of biochemical and molecular markers have been utilized to characterize and classify grape germplasm collections (Calo et al. 1989; Tschammer and Zyprian 1994; Bowers et al. 1996; Cervera et al. 1998; Tessier et al. 1999). For cultivar identification and genetic characterization of grape,

J.R. Soneji and M. Nageswara-Rao

isoenzyme analyses have widely been used by using peroxidase, glucose phosphate isomerase, phosphoglucomutase, catechol oxidase, and esterase (Schwennesen et al. 1982; Bachmann and Blaich 1988; Parfitt and Arulsekar 1989; Scienza et al. 1994; Crespan et al. 1999). For 48 grapevine varieties, the isoenzyme patterns of four enzyme systems (catecholoxidase, glutamate-oxalacetate-transaminase, acid phosphatase, and peroxidase) were analyzed (Jahnke et al. 2009). In recent years, various DNA-based molecular markers have been used for genetic diversity studies, cultivar characterization, and identification. Random amplified polymorphic DNA (RAPD) analyses were applied to investigate the genetic relationships among 14 Muscat grapevines in Apulia (Fanizza et al. 2000) and to analyze Hungarian cultivars and hybrids (Bisztray et al. 2003; Halasz et al. 2004), and rootstock cultivars (Kocsis et al. 2005a, b). RAPD has also been utilized to study the genetic diversity within a group of 19 cultivated grapevine cultivars and a set of 18 accessions of the wild European V. vinifera subsp. sylvestris (Jahnke et al. 2009). RAPD was successfully used to identify 31 grape accessions in two germplasm banks and also to solve identification problems (Bowers et al. 1993; Moreno et al. 1995; Stavrakakis et al. 1997; Ye et al. 1998). Restriction fragment length polymorphism (RFLP) analysis was used for the identification of 16 commercialized rootstock cultivars (Bourquin et al. 1992). Characterization of diverse germplasm collections using microsatellite (SSR) markers has been achieved (Lopes et al. 1999; Sefc et al. 2000; Aradhya et al. 2003; Martin et al. 2003). SSR analysis at six loci was used to analyze and identify 101 Hungarian grapevine varieties (Halasz et al. 2005). Wine-making cultivars of Portugal were genotyped using six SSR loci and all the 51 cultivars were distinguished (Almadanim et al. 2007). The SSR profiles in six loci (VVS2, VVS16, VVMD7, VMC4A1, VMC4G6 and VrZag79) of 48 grapevine varieties were analyzed (Jahnke et al. 2009). SSR marker analysis of 70 accessions originating from Malvasia, the Domaine de Vassal grape germplasm repository allowed the discrimination of 49 varieties and described synonyms for 37 varieties (Lacombe et al. 2007). SSR analysis has also been utilized to identify Spanish (Gonzalez-Andres et al. 2007; Fernandez-Gonzalez et al. 2007), Italian (Vignani et al. 1996), and Hungarian (Cseh et al. 2006; Jahnke et al. 2009) accessions. However, the cross-correlation

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between all these studies remains to be performed. This has led to the proposal of a coding strategy to compare the data between laboratories easily (This et al. 2004). Reference data for six SSR markers and a set of internationally recognized cultivars and rootstocks are now available at http://www.montpellier. inra.fr/vassal (This et al. 2006). SSRs have also been employed to trace the geographic origin of cultivars that have been introduced to areas outside the region of initial cultivation (Maletic et al. 2004). The main cultivars used for wine making in Portugal were genotyped with six SSR loci, which made it possible to distinguish all of the 51 cultivars (Almadanim et al. 2007). Only a few investigations have made use of molecular markers to study genetic structure and differentiation in V. vinifera (Sefc et al. 2000; Dangl et al. 2001). Recently, chloroplast molecular markers study supported the presence of at least two major domestication centers, approximately corresponding with Negrul’s occidentalis and orientalis group (ArroyoGarcia et al. 2006). Attempts made to identify genetic relationships between cultivars have provided only weak discrimination among geographic groupings and the presence of secondary domestication centers (Aradhya et al. 2003; Grassi et al. 2003).

11.4 Linkage Mapping Genetic linkage maps are a prerequisite to studying the inheritance of both qualitative and quantitative traits and to integrating molecular information that

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is necessary for marker-assisted selection (MAS) and map-based gene cloning techniques (Morgante and Salamini 2003). Thus, a key resource in support of classical genetics and genomics of Vitis spp. is the construction of a dense genetic map based on well characterized, gene-specific molecular markers (Troggio et al. 2007). Grapes are extremely heterozygous and hence their mapping populations are usually F1, and the pseudo-testcross mapping strategy is used to construct genetic linkage maps of both the parents, which can be then integrated into a consensus map with the use of multiallelic codominant markers with alleles that segregate in both the parents (Grattapaglia and Sederoff 1994). Several genetic linkage maps of grapes have already been published (Table 11.1) since the 1980s using isozymes, RFLP, RAPD, SSR, cleaved amplified polymorphic sequences (CAPS), amplified fragment length polymorphism (AFLP), sequence characterized amplified region (SCAR), single-strand conformation polymorphism (SSCP), etc. (Weeden et al. 1988; Mauro et al. 1992; Lodhi et al. 1995; Dalbo´ et al. 2000; Grando et al. 2003; Doucleff et al. 2004). These maps represent interspecific crosses utilizing V. vinifera (Grando et al. 2003), and other Vitis species or more complex interspecific hybrids (Lodhi et al. 1995; Doucleff et al. 2004; Fischer et al. 2004; Lowe and Walker 2006; Mandl et al. 2006). These maps would be useful for both quantitative trait loci (QTL) analysis and for anchoring bacterial artificial chromosome (BAC) and the genomic sequence.

Table 11.1 Summary of genetic linkage maps developed for grape Mapped parent Type of cross Population Markers used size Cayuga White Aurora Double pseudo60 Isozymes, RAPD, testcross RFLP Horizon Illinois 547-1 Double pseudo58 AFLP, CAPS, testcross RAPD, SSR Moscato bianco Pseudo-testcross 81 AFLP, SSCP, V. riparia SSR V. rupestris Pseudo-testcross 116 AFLP, ISSR, V. arizonica hybrids RAPD, SSR Regent Lemberger Psuedo-test cross 153 AFLP, CAPS, RAPD, SCAR, SSR 98 SSR Storgozia F2 *

M maternal; #P paternal

Total markers M* 214 P# 255 M 153 P 179 M 338 P 429 M 160 P 144 M 265 P 164

Map length (cM) M* 1,196 P# 1,477 M 1,199 P 1,470 M 1,639 P 1518 M 756 P 1,082 M 1,277.3 P 1,157.7

Linkage groups M* 20 P# 22 M 20 P 20 M 20 P19 M 17 P 19 M 20 P 26

References

92

692

19

Hvarleva et al. (2009)

Lodhi et al. (1995) Dalbo´ et al. (2000) Grando et al. (2003) Doucleff et al. (2004) Fischer et al. (2004)

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In grape, it is very difficult to use reverse genetic approach to tag and isolate genes. Multiple genes control most horticultural traits and no information is available about gene function and their expression. With the availability of molecular markers, it became possible to map traits of interest on genetic linkage maps of segregating populations. Several traits of horticultural importance such as powdery mildew resistance (Bouquet 1986; Pauquet et al. 2001), dagger nematode resistance (Doucleff et al. 2004; Lowe and Walker 2006), Pierce’s disease (Doucleff et al. 2004; Krivanek et al. 2006), downy mildew resistance (Luo et al. 2001), locus controlling flower type (Dalbo´ et al. 2000), and major gene for berry color (Fischer et al. 2004) have been tagged. Attempts have also been made to position phylloxera resistance on a genetic linkage map from a cross between the resistant V. rupestris and the susceptible V. vinifera (Roush et al. 2004). QTL analysis for tagging regions containing genes that influence powdery and downy mildew resistance, seedlessness, berry size, and veraison and axillary shoot growth have been carried out (Dalbo´ et al. 1997; Zyprian et al. 2002; Fischer et al. 2004). These molecular maps are very useful in genomic manipulation, map-based cloning, and marker-assisted breeding programs.

11.5 Role in Crop Improvement Through Traditional and Advanced Tools 11.5.1 Traditional Breeding Efforts Production of locally adapted, high-yielding quality cultivars adapted to abiotic and biotic stresses are the common objectives of most grape breeding programs. As grapes are produced for different purposes such as table, raisin, and wine production, achieving these objectives is difficult and other desirable qualities are considered when breeding rootstocks (Riaz et al. 2007). The East Asian grape, V. amurenis, was introduced into grape breeding programs as a source for fungal and frost resistance (Burr et al. 2003). V. labrusca and V. amurensis did not form tumors following inoculation with pathogenic Agrobacterium strains (Hoerner 1945; Tamm 1954). Selection of Asian and American wild grapes for crown gall resistance revealed several promising sources for breeding resis-

J.R. Soneji and M. Nageswara-Rao

tant rootstocks, as well as table or wine grapes (Burr et al. 2003). The use of the crown gall resistant V. riparia cv. Gloire de Montpellier as a rootstock in field experiments significantly reduced the severity of this disease as compared with the susceptible V. berlandieri V. riparia Teleki 5C (S€ule and Burr 1998). V. vinifera has been bred with native Florida bunch grapes, V. smalliana, to produce Florida hybrid bunch grapes, which are tolerant to Pierce’s disease (Halbrooks and Mortensen 1989). The cultivation of grapes cannot be effectively done in North India as the ripening season coincides with the monsoon rains causing the berries to split. To overcome this, two wild species of grapes, viz., V. lanata Roxb., which can withstand rain, and V. himalayana Br., which is a late ripening cultivar, were hybridized with V. vinifera cultivars to obtain grape cultivars with fruit quality of V. vinifera and able to withstand the subtropical monsoon season (Parmar 1988). V. rotundifolia has been identified as a source of dominant resistance to the powdery mildew. Crosses between V. rotundifolia and V. vinifera have yielded breeding lines and genetic resources that have been useful in determining the nature of this resistance (Bouquet 1986; Doligez et al. 2002; Donald et al. 2002). Variability of powdery mildew resistance has been studied in two hybrid families, in (Muscadinia V. vinifera) BC4 (FrancoAmerican hybrid V. vinifera V. amurensis) and in (Muscadinia V. vinifera) BC4 (V. amurensis V. vinifera) BC2 combinations (Kozma and Dula 2003). North American Vitis species have been used for more than a century to obtain phylloxera resistant rootstocks. “French American hybrid” wine grapes, resistant to phylloxera, have been produced by hybridization of V. vinifera with Native American species (Olmo 1976). Phylloxera resistance has also been transferred into the susceptible V. vinifera cultivars from resistant Native American grape species (Galet and Morton 1990). Crosses between SV12375 Alfold 100 (V. vinifera V. amurensis BC1) gave rise to two candidate varieties, which had fungus disease resistance, frost and winter hardiness, as well as were early ripening (Kriszten 1990). The hybrids of V. vinifera V. rotundifolia were immune to phylloxera and had good grafting affinity with V. vinifera varieties. These hybrids were hybridized with V. champinii, V. rupestris, and V. riparia to combine phylloxera and nematode resistance in the same stock (Olmo 1980). V. latifolia, a close relative of V. vinifera, is

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resistant to mildews and has a natural tendency to form tubers, which is an advantage for vegetative propagation. Use of V. latifolia in breeding programs of grapes would yield interesting hybrids (Salunkhe et al. 1999). Ramming et al. (2000) were the first to obtain a stenospermic hybrid (C41-5) derived from 19 plants of the interspecific cross V. vinifera V. rotundifolia. This allowed the introgression of disease-resistant traits with seedlessness. However, crossing incompatibility caused by different chromosome numbers of the two species led to low breeding efficiency. Wild Chinese Vitis species, which exhibited high resistance to fungal diseases, have also been hybridized with seedless V. vinifera (Wang et al. 1995). There are several main constraints to grape breeding and genetic improvement. Grape is a long-lived perennial with a long juvenile period and requires time and space for adequate evaluation. It was estimated by Wagner and Bronner (1974) that evaluation of a single seedling in a grape breeding program tied up 3–4 m2 of prime viticultural land for at least 7 years. Most grape cultivars are extremely heterozygous, and old varieties carry deleterious alleles that exhibit pronounced inbreeding depression after selfing or sibling mating. Moreover, characters that make a good cultivar are usually polygenic in their inheritance, with only few traits being controlled by single genes with dominant alleles. The screening methods used for fruit quality, yield, disease resistance, winter hardiness, and tolerance to other abiotic stresses determine the efficiency of grape breeding. Evaluation of new wine grape cultivars is more complex as single seedling vines produce very small amounts of fruit, adding to the difficulty of judging wine making potential (Riaz et al. 2007). Thus, it is difficult to produce desirable resistant cultivars via conventional breeding within a short period of time.

11.5.2 Ploidy Manipulation Anthers of grape have the capacity for regeneration by organogenesis as well as embryogenesis (Popescu et al. 1991), and plants derived by anther culture that are haploid/induced dihaploid are valuable in conventional breeding (Popescu et al. 1995). There is a single report of haploidy in Vitis (Zou and Li 1981), but it

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was unconfirmed (Hollo and Misik 2000). Anther culture is the most commonly used method for inducing somatic embryogenesis in grapevine, and it is generally accepted that embryos obtained from in vitrocultured anthers arise from the diploid cells of the connective tissue and are therefore of somatic origin (Faure et al. 1996). Anther-derived calli of hybrid grape contained a mixoploid population of cells, one diploid of somatic origin, and the other haploid and of pollinic origin (Rajasekaran and Mullins 1983). Cytological examinations of root tips of plantlets regenerated from V. latifolia anther culture showed cells to have chromosome numbers of 19 and 38 (Salunkhe et al. 1999). It has also been postulated that, in addition to microspore division, callus induction and growth may also bring about a reduction in the number of chromosomes (D’Amato 1985). Anther culture has also been carried out for Madchentrauben (Leankya), Rupestris du Lot and Rhein Riesling varieties of grape. The success of producing haploid plants is strongly influenced by the developmental stage of microspore besides the genotype, in vitro cultural conditions, etc., and therefore the ability to stain and recognize microspores of all developmental stages is critical first step in developing the in vitro androgenesis (Hollo and Misik 2000). Consumer demand for seedless grape is increasing in all parts of the world. Triploid grape breeding is one of the effective approaches to obtain new seedless cultivars. However, it is difficult to get triploid progenies by conventional methods due to poor compatibility, low fruit potential, hybrid embryo abortion at early stage of endosperm breakdown and low vitality of triploid seeds (Zhao et al. 2009). Reciprocal crosses between 2x 4x as well as 4x 2x have been attempted in grapes. However, very few seeds obtained from 2x 4x crosses had good germinability, while those from 4x 2x had lost their germinability. To overcome these problems, ovules and embryos have been cultured in vitro after hybridization to obtain triploid plants in an efficient way. Triploid grape plants have been produced by ovule embryo culture from cross between “Muscat Hamburg” (V. vinifera) tetraploid “Kyoho” (V. vinifera V. labrusca) (Zhao et al. 2009). Twenty-three crosses between 2x and 4x cultivars were carried out using four diploid (“Muscat Bailey A”, “Delaware”, “Rizamat”, and “Sekirei” which are all hybrids of V. vinifera and North American Vitis species) and four tetraploid

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(“Red Pearl”, “Yufu”, “Cannon Hall Muscat”, and “Kyoho”, which are all V. vinifera cultivars) grape cultivars, and triploid hybrids were obtained (Wakana et al. 2003). Four diploid varieties of Fenghuang51, Jingxiu, Muscat Hamburg, and Exotic and three tetraploid varieties of Fujiminori, Kyoho and Horizon were selected as parents and 2x 4x crosses were carried out. A total of 41 hybrid progenies from eight combinations were obtained, of which only 1 was triploid, 17 were diploid, and 14 aneuploid (Yang et al. 2007). Tetraploid forms, obtained from chance sports on normal diploid vines of grapes or from colchicine treatment, produce large-sized berries but exhibit poor fruitfulness, lack vigor, and have brittle shoots due to lack of tri- and tetra-allelic forms (Olmo 1952). A number of tetraploid clones have been obtained by chromosome doubling using colchicine and may be used as male parents in breeding, since they do not develop seeds, to produce other seedless grapes with large berries (Dermen and Scott 1962). A tetraploid clone of “Himrod Seedless” was obtained by in vitro chromosome doubling of diploid “Himrod Seedless”. A 2x 4x cross carried out between “Black Olympia” the tetraploid clone of “Himrod Seedless” gave rise to “Adumasizuku”, a tetraploid seedless table grape cultivar (Sato et al. 2009). Efforts to breed tetraploid hybrids have also focused on crosses between known tetraploids (Olmo 1942). Unreduced (2n) pollen would be useful for breeding highly heterozygous tetraploid grapes and triploid seedless grapes (Park et al. 2002). Diploid pollen grains in Vitis have been observed in F1 plant of an interspecific cross between “Almeria” (2n ¼ 2x ¼ 38) and “Trayshed” (2n ¼ 2x ¼ 40) (Jelenkovic and Olmo 1969). Twenty 2x 4x and 20 4x 2x crosses were carried out, using “Delaware”, “Muscat of Alexandria”, “Muscat Bailey A”, “Neo Muscat”, “Rizamat 2x”, and “Sekirei” as the diploid parent and “Cannon Hall Muscat”, “Kyoho”, “Red Pearl”, “Yufu”, and “Rizamat 4x” as the tetraploid parents, to study the origin of unreduced (2n) gametes in grapes. Two tetraploid hybrids were obtained in 4x 2x combination, while no hybrids were obtained in 2x 4x crosses (Park et al. 2002). Of the 41 hybrid progenies obtained from crosses between four diploid varieties of Fenghuang51, Jingxiu, Muscat Hamburg, and Exotic and three tetraploid varieties of Fujiminori, Kyoho, and Horizon, only nine were tetraploids (Yang et al. 2007).

J.R. Soneji and M. Nageswara-Rao

11.5.3 Tissue Culture Regeneration Grapes are among the first plants to be cultured in vitro (Morel 1944a, b). They can be regenerated in vitro via somatic embryogenesis, adventitious organogenesis, and micropropagation. Somatic embryogenesis in grape was first reported using nucellar tissue of unfertilized ovules of grape (Mullins and Srinivasan 1976) followed by anther culture (Mauro et al. 1986; Popescu 1996; Salunkhe et al. 1999). Thereafter, research on grape somatic embryogenesis was reported with a variety of grape explants such as leaves (Krul and Worley 1977; Robacker 1993), petiole and stem segments (Krul and Worley 1977), ovaries (Gray and Mortensen 1987; Martinelli et al. 2003), ovules (Gray 1989), zygotic embryos (Emershad and Ramming 1994), tendrils (Salunkhe et al. 1997), stigma and style (Morgana et al. 2004), and inflorescence tissues (Carmi et al. 2005). Direct somatic embryogenesis has also been demonstrated from epidermal cells of larger embryos (Margosan et al. 1994). Success in regeneration of grape has been limited to a relatively small number of cultivars, but the list of successful source material has been steadily increasing (Torregrosa 1998; Perrin et al. 2004). Many modifications and improvements have been reported (Perl et al. 1995; Perl and Eshdat 1998; Iocco et al. 2001; Perrin et al. 2001; Wang et al. 2004). Highly reliable protocols for optimizing initiation and maintenance of embryogenic cultures were reported for 29 Vitis species and varieties, including 18 V. vinifera varieties, V. riparia, V. rupestris, V. champinii and eight Vitis hybrids (Dhekney et al. 2009). Somatic embryogenesis has also been established from ovary explants of V. x labruscana cultivars (Motoike et al. 2001). Organogenesis was first reported by both Favre (1976) and Hirabayashi et al. (1976). Direct organogenesis via adventitious bud production without intermediate callus production has been produced from leaves, petioles (Stamp and Meredith 1988; Cheng and Reisch 1989; Stamp et al. 1990a, b), hypocotyls, and cotyledons (Vilaplana and Mullins 1989) of grapes. Indirect organogenesis via callus formation has been reported from leaves (Clog et al. 1990), petioles (Tang and Mullins 1990) and seedlings (Rajasekaran and Mullins 1981) of grapes. Micropropagation of grapes has been achieved by culturing axillary buds and shoot apices (Barlass and Skene

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1978). Mother plant vigor is of primary importance in micropropagation with rapidly expanding shoots from healthy, stress-free vines providing the best explants for grapes (Barlass and Skene 1978; Chee and Pool 1982; Hu and Wang 1983). Protocols for initiation of micropropagation cultures are well established for a large number of grape species such as V. vinifera, V. labrusca, V. rotundifolia, V. vinifera V. rotundifolia hybrids, and other Vitis hybrids as compared with somatic embryogenesis (Monette 1983; Chee and Pool 1985, 1987; Lee and Wetzstein 1990; Gray and Benton 1991; Zlenko et al. 1995; Torregrosa and Bouquet 1995; Mhatre et al. 2000). In vitro meristem, shoot apex cultures, one-node explant culture, and fragmented shoot apex cultures were developed to produce virus-free grape plants (Barlass et al. 1982; Barlass 1987; Hatzinikolakis and Roubelakis-Angelakis 1993; Staudt and Kassemeyer 1994). Recently, it was shown that micrografting of scion meristems on hypocotyls of germinating embryos resulted in simultaneous virus indexing (Tanne et al. 1993, 1996). Tissue culture has also been used to aid breeding efforts in grapes. Ramming et al. (2000) were the first to utilize embryo rescue to obtain a stenospermic hybrid (C41-5) derived from 19 plants of the interspecific cross V. vinifera V. rotundifolia. This allowed the introgression of disease-resistant traits with seedlessness and has resulted in significantly high frequency of seedlessness in offspring compared with the traditional methods (Ramming et al. 1990). Tissue culture procedures offer new opportunities to augment clonal variation and to provide new raw material for clonal selection (Mullins 1990). Somaclonal variation has been primarily observed on those genotypes of grape which are highly regenerative in vitro such as V. vinifera V. rupestris hybrid, Gloryvine, which has exhibited abnormalities like dwarfism, albinism, variations in leaf shape, sex expression, etc. (Valat and Rives 1973; Galet 1979; Rajasekaran and Mullins 1983). Although protoplast culture has been attempted in grapes, plant regeneration has not been reported (Skene 1975; Marino 1990; Ui et al. 1990; Phosang et al. 1994; Xu et al. 2007).

11.5.4 Genetic Engineering Genetic transformation technology potentially bypasses the limitations of traditional breeding by

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allowing useful characteristics to be introduced into a crop plant, including resistance to diseases and pests and improved product quality, without changing the unique features of the cultivar (Iocco et al. 2001). In grapes, genetic transformation is particularly useful for adding one trait to a cultivar, since, in wine grapes, phenotype is particularly important and varietal fidelity is of paramount concern (Gray et al. 2002). It also allows significant opportunities for improvement of grapes, while allowing the continued use of economic significance (Gray et al. 2005). In grapes, genetic engineering has been achieved using Agrobacteriummediated transformation and biolistics. Early attempts to transform grape using Agrobacterium met with difficulty despite the bacterium being a naturally occurring pathogen of the species (Owens 2008). Huang and Mullins (1989) and Mullins et al. (1990) were the first to report the successful transgenic plant production in grape. A number of grape cultivars have been stably transformed using Agrobacteriummediated transformation and most progress has been achieved using embryogenic cell masses (Gray et al. 2002, 2005). In grapes, Agrobacterium-mediated transformation has also been used to transform meristematic tissues such as adventitious buds (Mullins et al. 1990), in vitro internode explants (Levenko and Rubtsova 2000), and meristematic bulk tissue (Mezzetti et al. 2002). Biolistics has been used to transform a few Vitis cultivars, but to a much lesser extent than Agrobacterium-mediated transformation. Gray et al. (1993) were the first to report transient GUS expression using biolistics in V. vinifera “Thompson Seedless” somatic embryos. However, stable GUS expression in somatic embryos of the Vitis interspecific hybrid “Chancellor” was reported by Hebert et al. (1993). Transgenic plants have been produced from the embryogenic suspension cultures of “Chancellor” (Kikkert et al. 1996, 2000). Genetic transformation of grape has focused on acquiring pathogenic resistance to a number of major diseases and pests in a number of Vitis species (Table 11.2). The pathogenic fungi downy mildew, botrytis, and powdery mildew are the main targets for production of fungal-tolerant transgenic grapes. Alien genes such as stilbene synthase gene have been expressed in the grape genome (Coutos-Thevenot et al. 2001). Research has also been focused on introducing specific virus-resistant genes into grapes. Genes

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Table 11.2 Examples of names of genes, their origin, properties, and the species/cultivars of grapes in which they have been incorporated Name of the gene Origin Property Plant in which References incorporated Coat protein of Viral Grapevine chrome mosaic virus 110R Le Gall et al. (1994) grapevine chrome resistance mosaic virus Coat protein of grape Viral Grape fanleaf virus resistance 110R, 41B, SO4, Krastanova et al. fanleaf virus V. rupestris (1995), Mauro et al. (1995) GNA Plant (Galanthus Homopeteran insect resistance Freedom Viss and Driver (1996) nivalis) Stilbene synthase gene Plant (grape) Tolerance to Botrytis cinerea 41B Coutos-Thevenot et al. (1998) Antifreeze protein – Freeze tolerance V. rupestris, 110R Tsvetkov et al. (2000) Movement protein Viral Grapevine virus A and B V. rupestris Martinelli et al. (2000) resistance Replicase Viral Grapevine fanleaf virus 110R Barbier et al. (2000) resistance Vr-ERE Plant (grape) Resistance to Eutypa lata 110R Legrand et al. (2003) Truncated virE2 Bacterial Resistance to crown gall 110R Holden et al. (2003) Ferritin Plant (Medicago Freeze tolerance 110R Zok et al. (2009) sativa)

encoding the viral coat protein or movement protein have been used. Coat protein genes of grapevine virus A, grapevine virus B, grapevine chrome mosaic virus, and grapevine fanleaf virus (Le Gall et al. 1994; Barbier et al. 2000; Krastanova et al. 2000; Martinelli et al. 2000), and movement protein of grapevine virus A and B, (Martinelli et al. 2000) have been introduced into grape. For obtaining bacterial resistance in grapes, a truncated virE2 gene has been introduced (Holden et al. 2003). To make the plants more tolerant to oxidative damage induced by freezing temperatures, ferritin gene derived from Medicago sativa has been introduced into grape (Zok et al. 2009). A number of gene promoters have been isolated and may find application in regulating gene expression in transgenic grapes. A fusion reporter marker construct composed of the enhanced green florescent protein and kanamycin resistance genes was developed for effective monitoring of transformation of grape (Li et al. 1999, 2001a, b). The synthetic gibberellins promoter regulating expression of the yeast invertase gene (sucII) in transgenic grape plants is an example where application of a gibberellins spray to berries may not only increase berry size but also activate the invertase gene to modify sugar accumulation. Although a number of transgenic grape lines are under evaluation, their release is a concern con-

sidering the ease with which pollen flow can occur and the existence of wild grape species in many production regions.

11.6 Conclusion Grape is the third most important fruit crop in the world. It is vegetatively propagated by cuttings and grafting. Conventional breeding and advances in viticulture and enology have been used to optimize vine growth and wine production. However, its long juvenile period poses severe constraints and is land and labor intensive. Tissue culture regeneration has opened new vistas for grape genetic improvement. Efforts have been made to produce haploids via anther culture and to produce seedless grapes by triploid breeding, with and without the use of ovule/embryo culture. Tetraploid production has expanded the range of germplasm available for grape breeders. Integrating conventional technologies with those based on molecular biology and genomics could make this objective possible. Genetic linkage maps focused on biotic and abiotic stress resistance and tolerance, and fruit quality improvement have been developed. These will be

11 Vitis

useful for identifying QTLs for use in MAS breeding programs. Grape genetic transformation has largely focused on production of transgenic plants resistant to fungal, bacterial and viral disease. Efforts have also been made to produce transgenic plants by the incorporation of genes for increasing the sugar content in the berries and tolerance to oxidative damage, and seedless grapes have been produced and are already in field trials. Genomic technologies have been developed and applied in grapes. An Affymetrix-based platform for microarray analysis containing more than 15,000 unique Vitis features is currently available of which 1,700 transcripts are from Vitis species other than V. vinifera (This et al. 2006). The precise knowledge of all the genes influencing quality and resistance traits would be highly beneficial for future breeding and genetic engineering programs as it will provide new information and gene targets for manipulation.

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Index

A AAMTR. See Average annual minimum temperature range Abiotic stress, 182, 232 tolerance, 103 Adaptability, 211 Adaptation, 159 AFLP. See Amplified fragment length polymorphism Agrobacterium, 9, 90, 99–100, 168, 187, 228, 231 A. rhizogenes, 9, 100 A. tumefaciens, 9, 100 Allergen, 37, 107, 169 Allergy, 107 Allotetraploid, 9–10, 136, 183–184 Allotriploid, 9–10 Allozyme, 92 Almond, 129 American grape, 68–69 AMOVA. See Analysis of molecular variance Amphidiploid, 150 Amplified fragment length polymorphism (AFLP), 5, 30, 48, 73–74, 84, 120–121, 135, 156, 183, 227 Amygdaloideae, 131 Anacardiaceae, 119 Analysis of molecular variance (AMOVA), 91–92 Androgenesis, 229 Aneuploid reduction, 150 Anther culture, 151 Anthocyanidin, 208 Anthocyanin, 67, 188, 207 Anticancer properties, 67 Anticlotting, 70 Antiedematous, 208 Anti-inflammatory, 70, 124, 188, 208 Antioxidant, 69, 186, 189, 208 activity, 208 Antiscorbutic, 188 Antiseptic, 124 Aphid, 166, 185 Apomictic, 189

Apple, 47 scab, 53 Apricot, 129 Aquaporin, 105 Arabidopsis, 6 Arctic raspberry, 181 Arthropod, 155 Asian pea pears, 147 Asian pears, 147 Astringency, 152 Astringent, 188, 208 Autotetraploid, 183 Average annual minimum temperature range (AAMTR), 201 Axillary bud stimulation, 89 B BAC. See Bacterial artificial chromosome Bacterial artificial chromosome (BAC), 36, 227 library, 188 Basic helix-loop-helix (bHLH), 186 Basic leucine zipper (bZIP), 186 BBDG. See Blueberry Genomics Database bHLH. See Basic helix-loop-helix (bHLH) BIDR, 122 Bilberry, 206–207 Bioavailability, 188 Biogeography, 131 Biolistics, 231 Biotic stress, 101–102, 182, 232 Blackberry, 180 Black cherry, 141 Black raspberry, 180 Black spot, 166 Blossom frost tolerance, 200 Blueberries, 197, 201, 205–206 Blueberry Genomics Database (BBDG), 215 BSA. See Bulked segregant analysis Bulked segregant analysis (BSA), 123 Bushy dwarf virus, 187 bZIP. See Basic leucine zipper

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Temperate Fruits, DOI 10.1007/978-3-642-16057-8, # Springer-Verlag Berlin Heidelberg 2011

241

242 C California grape, 68–69 Candidate gene, 52 Cane spot, 185 CAPS. See Cleaved amplified polymorphic sequence cDNA. See Complementary DNA Center for Plant Conservation (CPC), 139 Chemotaxonomic, 4 Cherrie, 129 Chilling, 159 requirement, 1, 198 Chionanthus, 90 C. decipiens, 90 C. eriorachis, 90–91 C. malaelengi, 90–91 C. microbotrys, 90–91 C. microstigma, 90–91 C. sutepensis, 90–91 C. thorelii, 90–91 C. velutina, 90–91 Chloroplast DNA (cpDNA), 131, 134 genome, 25, 34–35 Chlorotype, 93 Chromosome differentiation, 205 doubling, 230 homology, 205 Cleaved amplified polymorphic sequence (CAPS), 29, 227 Clonal selection, 98 Cloudberry, 181 C/N ratio, 141 Cold hardiness, 151, 159, 161, 162, 199 Complementary DNA (cDNA), 104, 168, 187 library, 58, 74–75, 169 microarrays, 215 sequences, 35 Conservation, 140, 148–150 CPC. See Center for Plant Conservation Crab apples, 47 Cranberries, 201–202, 206 Cryopreservation, 48–49, 88, 150, 225 Cydonia, 1–11 C. oblonga, 2 Cytogenetic stocks, 5, 50, 151, 183–184 D Decaploid, 22 Decongestant, 188 Dehydration, 225–226 Di-haploidization, 52 Dioecious, 123 Disease resistance, 135 tolerance, 68–69 Diuretic, 188

Index DNA barcode, 33–34 barcoding, 33–34 content, 147 fingerprinting, 190 Domestication, 59, 67–68, 124–125, 169, 184, 188–189 Dormancy, 180 Drought, 164 tolerance, 198 Duodecaploid, 179 E EFSY. See European stone fruit yellows Embryo, 90 culture, 7 rescue, 98, 99 Embryogenesis, 8 Embryonal explants, 90 Encapsulation, 150, 225–226 Encapsulation-dehydration, 4 Endangered, 140, 149 Enneaploid, 22 Ericaceae, 197 Erwinia, 57 E. amylovora, 57 EST. See Expressed sequence tag Ethnobotanical, 59 Ethylene, 166 EUCPPGR. See European Cooperative Program for Plant Genetic Resources EURISCO. See European Network of ex situ National Plant Germplasm Inventories European Cooperative Program for Plant Genetic Resources (EUCPPGR), 149–150 European grape, 68–69 European Network of ex situ National Plant Germplasm Inventories (EURISCO), 149–150 European pears, 147 European pear scab, 154, 163 European stone fruit yellows (EFSY), 135 Evolutionary scenario, 80 Expressed sequence tag (EST), 11, 58, 74, 104, 168 library, 216 Ex situ conservation, 3, 149–150, 225 preservation, 150 F Field banks, 88 Fire blight, 57, 153, 162–163, 165 resistance, 151 Fox grape, 68–69 Fragaria, 17–39 F. bifera, 19 F. bringhurstii, 18 F. bucharica, 20 F. chiloensis, 21 F. daltoniana, 20

Index F. iinumae, 20 F. iturupensis, 19, 22 F. mandshurica, 19 F. mexicana, 19 F. moschata, 18 F. nilgerrensis, 19 F. nipponica, 20 F. nubicola, 20 F. pentaphylla, 20 F. tibetica, 20 F. vesca, 19 F. virginiana, 20 F. viridis, 19 F. yezoensis, 20 Fruit flavor, 159 ripening, 102 spot, 163 texture, 159 G Gametophytic self-incompatibility (GSI), 137 GDR. See Genome Database for Rosaceae GenBank, 11, 35 GENBERRY project, 182 Gene expression, 91 flow, 84–85, 123, 190–191 pool, 184 primary, 87, 214 secondary, 87, 214 tertiary, 87 Genebank, 26, 225 Gene pair haplotype (GPH), 36 Genetic assimilation, 190 diversity, 4, 82–84, 121, 133, 181 engineering, 168, 231–232 erosion, 3, 88, 148–149 linkage map, 227 map, 215 modification, 191 stock, 79 transformation, 9, 99, 231 Genome size, 86–87 Genome Database for Rosaceae (GDR), 35, 58 Genome scanning approach (GSA), 51 Genome survey sequences (GSS), 11 Geographical distribution, 82–84 Germplasm, 122, 225 bank, 88 repository, 226 Germplasm Resources Information Network (GRIN), 139 GPH. See Gene pair haplotype Graft incompatibility, 8 Grapes, 224

243 GRIN. See Germplasm Resources Information Network GSA. See Genome scanning approach GSS. See Genome survey sequences H Haploid, 99, 151 Haploidization, 52 Haplotype, 136 Herbal drugs, 106–107 Heritability, 152 Hexaploid, 21, 22 HiDRAS. See High-quality disease resistant apples Highbush, 211 High performance liquid chromatography (HPLC), 65 High-quality disease resistant apples (HiDRAS), 53 Homoplasy, 130, 138 Homoploid, 205 Homozygotic, 99 HPLC. See High performance liquid chromatography Hybrid, 10 I IBPGR. See International Board for Plant Genetic Resources Infrageneric, 131, 132 INRA. See Institut National de la Recherche´ Agronomique In situ conservation, 3, 149–150 Institut National de la Recherche´ Agronomique (INRA), 163 Integrated pest management (IPM), 186 Intergeneric cross, 6 hybrid, 5, 164 hybridization, 164 Internal transcribed spacer (ITS), 5, 49, 131 Internal transcribed spacer region (ITSR), 183 International Board for Plant Genetic Resources (IBPGR), 149 International Union for the Conservation of Nature (IUCN), 3, 148 Red List, 3 Intersectional crosses, 206 Inter-simple sequence repeat (ISSR), 5, 73, 181, 186 Interspecific hybrid, 52, 134 hybridization, 70–71, 129, 159, 183, 205, 214 Introgression, 7, 205 Invasive species, 141–142 weed, 190 IPM. See Integrated pest management Isoenzyme polymorphisms, 4 Isozyme, 28, 72–73, 92, 136, 155 ISSR. See Inter-simple sequence repeat ITS. See Internal transcribed spacer ITSR. See Internal transcribed spacer region IUCN. See International Union for the Conservation of Nature

244 J Japanese pears, 147 Japanese plum, 134 Juvenility period, 97 K Karyotype, 26, 224 L Landraces, 79 LD. See Linkage disequilibrium Leaf spot, 163 Lingonberries, 202, 206–207 Linkage group (LG), 157 map, 32, 155, 157 mapping, 32, 227–228 Linkage disequilibrium (LD), 58 Lipid transfer protein (LTP), 37 LTP. See Lipid transfer protein M MAB. See Marker-assisted breeding Maddenia, 132 Maloideae (Pomoideae), 5, 45, 147 Malus, 6, 45–59 M. asiatica, 49 M. domestica, 48 M. floribunda, 47 M. ioensis, 47 M. niedzwetzkyana, 49 M. orientalis, 49 M. prunifolia, 49 M. sieversii, 49 M. sikkimensis, 47 M. sylvestris, 49, 52 M. toringo, 47 M. toringoides, 47 Mangifera, 123–124 Mango, 123–124 Marker-assisted breeding (MAB), 191 Marker-assisted selection (MAS), 165, 227 MAS. See Marker-assisted selection Medical properties, 59 Messenger RNA (mRNA), 74–75 Microarray, 187 Micropropagation, 7, 89 Microsatellites, 30–32, 121, 156, 226 Mitochondrial DNA (mtDNA), 35, 95 Mitotype, 85, 94 Mixoploid, 98 Monophyletic, 132 Morphological diversity, 122 mRNA. See Messenger RNA mtDNA. See Mitochondrial DNA Muscadine grape, 65 Muscadinia, 223 Muscadiniana, 65–75

Index Mutation, 98 N National Center for Biotechnology Information (NCBI), 11, 35–36 National Plant Germplasm System (NPGS), 225 NCBI. See National Center for Biotechnology Information Near threatened, 140 Nematode resistance, 228–229 New World, 130 NPGS. See National Plant Germplasm System Nutraceutical, 68 O Octoploid, 21–22 Oil quality, 102 Old World, 130 Olea, 79–108 O. europaea, 79 O. europaea ssp. cerasiformis, 79 O. europaea ssp. cuspidata, 79 O. europaea ssp. europaea, 79 O. europaea ssp. laperrini, 79 Oleaceae, 79 Oleuropein, 86 Olive, 79 Ophthalmic, 188 Organogenesis, 230 Oxyctocic, 188 P Parthenocarpy, 102–103 Participatory domestication, 140 PCR. See Polymerase chain reaction Peache, 129 Pear, 147 Pear leaf spot, 153, 163 Pear psylla, 155, 163 Pear scab, 166 Pentaploid, 22 Pest tolerance, 68–69 Phenogram, 5 Phenolics, 209 content, 65 Phenology, 120 Phenylpropanoid pathway, 187 Phylloxera resistance, 228 Phylogenetic, 25 congruence, 80 distance, 135 reconstruction, 80 relationships, 132 Phylogeny, 24–25, 129–132, 139 Phythophthora, 101 Phytochemical, 68

Index Phytopharmaceutical, 68 Pistachio, 123 Pistacia, 119–125 P. aethiopica, 120 P. atlantica, 119 P. chinensis, 119 P. integerrima, 119 P. khinjuk, 119 P. lentiscus, 119 P. mexicana, 119 P. palaestina, 119 P. saportae, 120 P. terebinthus, 119 P. texana, 119 P. vera, 119 P. weinmannifolia, 119 Ploidy level, 98–99 manipulation, 229–230 Plum, 129 Plum pox virus, 135 Podosphaera, 57 P. leucotricha, 57 Pollen flow, 232 Polymerase chain reaction (PCR), 134 Polyphenolics, 188 Polyphenols, 65 Polyploid, 151 Powdery mildew, 57, 185 Precocity, 159 Proanthocyanidins, 208 Profilins, 37 Protoplast culture, 8 technology, 99 Prunoideae, 45 Prunus, 6, 129–142 P. andersonii, 132–133 P. armeniaca, 132 P. avium, 132 P. canescens, 135 P. cerasifera, 135 P. davidiana, 134, 135 P. domestica, 132 P. dulcis, 132 P. fasciculata, 132–133 P. ferganensis, 135 P. kansuensis, 134 P. maackii, 135 P. mandshurica, 135 P. mira, 134 P. napaulensis, 133 P. persica, 132 P. salicina, 132 P. serotina, 136, 141 P. tomentosa, 133 Pseudocydonia, 5 Pseudogamous, 189 Pseudo-testcross, 32 Pyrinae, 147

245 Pyrus, 6, 147–170 P. amygdaliformis, 149 P. anatolica, 148 P. asia-mediae, 148 P. betulifolia, 147 P. boissieriana, 149 P. bretschneideri, 147 P. calleryana, 147 P. communis, 147, 149 P. cordata., 149 P. elaeagrifolia, 149 P. fauriei, 147 P. hakkiarica, 148, 149 P. oxyprion, 148 P. pyraster, 149 P. pyrifolia, 147 P. salicifolia, 148 P. serikensis, 148, 149 P. spinosa, 149 P. syriaca, 149 P. ussuriensis, 147 P. yaltirikii, 149 Q QTL. See Quantitative trait loci Quantitative trait loci (QTL), 32, 51, 72, 135, 153, 158, 186, 215, 227 Quince, 1 R Rabbiteye, 210 Randomly amplified polymorphic DNA (RAPD), 28–30, 49, 72–73, 84, 120–121, 136, 156, 181, 214, 226 RAPD. See Randomly amplified polymorphic DNA Raspberry, 180 Red raspberrie, 180 Reforestation, 124–125 Resistance gene analog (RGA), 52 Resistance gene analog polymorphism (RGAP), 185 Restriction fragment length polymorphism (RFLP), 157, 214, 226 Resveratrol, 65 RFLP. See Restriction fragment length polymorphism RGAP. See Resistance gene analog polymorphism Ribosomal DNA, 151 River Bank grape, 68–69 RNA finger printing, 187 Root rot, 185 Rootstock, 4, 123, 164 Rosaceae, 7, 17, 35, 131, 132, 147 RosBREED, 36 Rosoideae, 17, 45 Rubus, 179–191 R. allegheniensis, 183 R. chamaemorus, 181 R. hawaiensis, 183

246

Index

Rubus (cont.) R. idaeus, 180 R. leucodermis, 181 R. macraei, 183 R. moluccanus, 181 R. occidentalis, 180 R. rusticanus, 183

Strawberry, 17 Summer grape, 68–69 Sweet cherry, 134 Synapomorphic, 5 Synteny, 7 Synthetic seed, 90

S Sand grape, 68–69 Scab resistance, 51 SCAR. See Sequence-characterized amplified region Scion, 151 SCRI, 182 SDRFs. See Single dose restriction fragments Secondary polyploid, 2 Second filial generation (F2) hybrid, 10–11 Seedlessness, 71 Self-fertility, 214 Self-incompatibility, 129, 167–168, 180 Self-incompatibility RNase (S-RNase), 137, 167 Self-infertility, 214 Self-pollination, 214 Self-sterility, 102–103 Sequence-characterized amplified region (SCAR), 29, 51, 157, 214–215, 227 Sequence-related amplified polymorphism (SRAP), 122 Sex determination, 123 linked marker, 123 S-genotyping, 167–168 Shoot organogenesis, 89 Simple sequence repeat (SSR), 5, 30–32, 49, 84, 121, 134, 156, 182, 214 Single dose restriction fragment (SDRF), 32 Single nucleotide polymorphism (SNP), 58, 75, 168–169, 183 Single-strand conformation polymorphism (SSCP), 227 Slow freezing, 4, 150 SNP. See Single nucleotide polymorphism Somaclonal variant, 9 variation, 8–9, 97–98, 231 Somatic embryo, 231 embryogenesis, 89, 230 Sour cherry, 134 Speciation, 147 Spiraeoideae, 45 Spurblight, 185 SRAP. See Sequence-related amplified polymorphism S-RNase. See Self-incompatibility RNase SSCP. See Single-strand conformation polymorphism SSR. See Simple sequence repeat Stimulant, 188 Stone fruits, 129

T T-DNA. See Tumor-inducing plasmid Ti TE. See Transposable element Tetraploid, 21, 98 tfGDR. See Tree fruit Genome Database Resources The Institute for Genomic Research (TIGR), 35–36 Therapeutic properties, 106–107 TIGR. See The Institute for Genomic Research Transgene, 9 Transgenic, 187, 232 shoot, 9 Transposable element (TE), 36 Tree fruit Genome Database Resources (tfGDR), 36 Triploid, 98, 151 Tumor-inducing plasmid Ti (T-DNA), 36 U USDA. See US Department of Agriculture USDA-Agricultural Research Service (USDA-ARS), 182, 207, 225 US Department of Agriculture (USDA), 36, 139, 163, 206 V Vaccinium, 197–216 V. angustifolium, 201 V. ashei, 201 V. consanguineum, 205 V. corymbosum, 201, 205 V. darrowii, 206 V. erythrocarpum, 201 V. macrocarpon, 206 V. microcarpum, 201 V. myrtilloides, 201 V. myrtillus, 202 V. ovatum, 205 V. oxycoccos, 201 V. oxycoccus, 206 V. uliginosum, 197, 202 V. virgatum, 201 V. vitis-idaea, 202 Variable number tandem repeat (VNTR), 181 Vasoprotective, 208 Venturia V. inaequalis, 53 Verbascoside, 106 Vitis, 65, 223–233 V. aestivalis, 67, 223 V. amurensis, 223, 229 V. californica, 67 V. cinerea, 223 V. labrusca, 67, 223, 229

Index V. munsoniana, 66, 223 V. popenoei, 66, 223 V. riparia, 67, 223 V. rotundifoli, 223 V. rotundifolia, 67 V. rupestris, 67, 223 V. vinifera, 65, 67, 223 Vitis international variety catalog (VIVC), 225 Vitrification, 4, 150 VIVC. See Vitis international variety catalog VNTR. See Variable number tandem repeat Vulnerable, 149

247 W Western yellow rust, 185 Wide hybridization, 150 Wild gene-pool, 96–103 germplasm, 48, 181 species, 135 Wine-making quality, 71 Winter hardiness, 198