Wild Crop Relatives: Genomic and Breeding Resources: Forest Trees

Wild Crop Relatives: Genomic and Breeding Resources

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Chittaranjan Kole Editor

Wild Crop Relatives: Genomic and Breeding Resources Forest Trees

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

ISBN 978-3-642-21249-9 e-ISBN 978-3-642-21250-5 DOI 10.1007/978-3-642-21250-5 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 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 seven-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 volumes. 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 volumes, 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 these volumes 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 told me to forget about my original contributions and that I deserved at least the World Food Prize, if not Nobel Prize for peace like him. I felt honored but really very ashamed as I am aware of my almost insignificant contribution in comparison to his work, and was unable to utter any words for a couple of minutes! 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 the documents by fax.” Here was the ever-present child in him. 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, are you coming to Texas 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 Texas to see him, though it would have been a great honor.

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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 communications between us when he slowly succumbed to his health problems. The remaining volumes of the Genome Mapping and Molecular Breeding in Plants were published in 2007, and I sent him the volumes. I wished to learn about his views. During this period he could hardly speak and write. Julie prepared a letter on his behalf 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. 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. By that time I had a huge commitment to editing 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 did not even communicate my personal loss to Julie. But in the meantime, I formulated a ten-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 glove and hundreds of plant and agricultural scientists who tries 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 our next generations in the same way as personalities like Dr. Norman Borlaug did to me and many other science workers from around the world! During most of our conversations he used to emphasize the immediate impact of research on the society. 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. This advice elicited a change in my approach to science and since then I have been devotedly endeavored to develop crop varieties enriched with phytomedicines and nutraceuticals. Inspiration, advices, and blessings of Dr. Borlaug have influenced both my personal and professional life, particularly my approach to science, and I dedicate this series to him as a token of my regards and gratitude, and in remembrance of his great contribution to science and society and above all his personal affection for me.

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Dedication

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 expressing 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 our 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 presented, the need for reference books with comprehensive examinations of 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, etc. and/or potential as “new crops”. The most perplexing problem for me was to assign them to different volumes dedicated to crop relatives of diverse crops grouped based on their utility. ix

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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 trunculata, 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 crop relatives in a volume on the crop group to which they are taxonomically closest and to which they can make the greatest contributions. For example, I placed the chapter on Arabidopsis in the volume on oilseeds, which deals with the wild relatives of Brassicaceae crops. However, we have tried to include deliberations pertinent to the individual genera or species 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 conservation 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 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 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,

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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 create 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 and Medicago. 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 or species. 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 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 crucial aspects relevant to a particular wild genus or species 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 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

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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 roughly 150 chapters devoted to essential genera or species of wild crop relatives. However, I had to exclude a few 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 126 chapters in the ten volumes, contributed by 380 authors from 39 countries of the world. I extend my heartfelt thanks to all of 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/species. As happens with a mega-series, 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 offer my sincere apologies. Another unique feature of this series is that the authors of chapters dedicated to some genera/species have dedicated their chapters to scientists who pioneered the exploration, description and utilization of those wild genera/species. 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 seven-volume series on Genome Mapping and Molecular Breeding in Plants with Springer way back in 2005, and I have since 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

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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 graciously tolerating my sparing some time rightly deserved by them and constantly supporting me in the publication of this series. 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 Forage 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 – Forest Trees” includes 8 chapters dedicated to Alnus, Corylus, Cryptomeria, Eucalyptus, Juglans, Quercus, Santalum, and Trigonobalanus. The chapters of this volume were authored by 16 scientists from 4 countries of the world, namely Australia, India, Japan, 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 forest trees with an intention of serving science and society. Clemson, USA

Chittaranjan Kole

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Contents

1

Alnus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Brian D. Vanden Heuvel

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Corylus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas J. Molnar

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Cryptomeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoshihiko Tsumura

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Eucalyptus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert J. Henry

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Juglans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Keith Woeste and Charles Michler

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Quercus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preston R. Aldrich and Jeannine Cavender-Bares

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Santalum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Madhugiri Nageswara Rao, Jaya R. Soneji, and Padmini Sudarshana

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Trigonobalanus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Weibang Sun, Yuan Zhou, Chunyan Han, Gao Chen, and Yanling Zheng Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

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Abbreviations

ABA AFLP AMOVA ARS ATSC BGCI BTS CAPS CDF cpDNA ECM ECP/GR EFB EST ETS EUCAGEN EUFORGEN FFI FFPRI FISH FTGRCF FTSGCS GIS HAD HJG HTIRC HTS IAA IBPGR IEA INRA ISSR ITS LD MAS MDF MDS

Abscisic acid Amplified fragment length polymorphism Analysis of molecular variance Agriculture Research Service (USDA) Australian Tree Seed Centre Botanic Gardens Concervation International Big-tree structure Cleaved amplified polymorphic sequence Co-dominant forest Chloroplast DNA Ectomycorrhizal European Cooperative Program on Plant Genetic Resources Eastern filbert blight Expressed sequence tag External transcribed spacer Eucalyptus Genome Network European Forest Genetics Resources Programme Forest Frontiers Initiative Forestry and Forest Products Research Institute Fluorescence in situ hybridization Forest Tree Genetic Resources Conservation Forest Forest Tree Superior Gene Conservation Stand Geographical information systems Heavily disturbed by human activities Hydrojugloneglucoside Hardwood Tree Improvement and Regeneration Center Huge-tree structure Indole-3-acetic acid International Board of Plant Genetic Resources International Energy Agency French National Institute for Agricultural Research Inter-simple sequence repeat Internal transcribed spacer Linkage disequilibrium Marker-assisted selection Mono-dominant Forest Mono-dominant structure xvii

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Abbreviations

MDSD ML MOE MP Mya Mybp NALB NCGR NJ nptII OSU PCR PICME PR PRP QTL RAPD rbcL RBS rDNA RFLP RNAi rRNA RT-PCR RT-PCR rubisCO SCAR SMP SNP SSCP SSR STS SW TPS UCD uidA UPGMA USD USDA USDA VAM ybp

Mono-dominant structure in developing Maximum likelihood Module of elasticity Maximum parsimony Million years ago Million years before present North Atlantic Land Bridges National Clonal Germplasm Repository Neighbor joining Neomycin phosphotransferase II gene Oregon State University Polymerase chain reaction Platform for integrated clone management Population in recovery Proline rich protein Quantitative trait loci Random amplified polymorphic DNA Ribulose-bisphosphate carboxylase-L (gene) Relatively balanced structure Ribisomal DNA Restriction fragment length polymorphism RNA interference Ribosomal RNA Real-time PCR Reverse transcription PCR Ribulose-1,5-bisphosphate carboxylase oxygenase Sequence characterized amplified region Supplemental mass pollination Single nucleotide polymorphism Single-stranded DNA conformation polymorphism Simple sequence repeat Sequence tagged site Sprouting woods Terpene synthases University of California, Davis b-glucoronidase A gene Unweighted pair group method of arithmatic average Unstable structure in developing United States Department of Agriculture United States Department of Agriculture Vesicular arbuscular Years before present

Contributors

Preston R. Aldrich Department of Biological Sciences, Benedictine University, Birck Hall 341, 5700 College Road, Lisle, IL 60532–0900, USA, [email protected] Jeannine Cavender-Bares Department of Ecology, Evolution and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, USA, [email protected] Gao Chen Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China Chunyan Han Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China Robert J. Henry Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Australia, [email protected] Charles Michler USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, Pfendler Hall, 715 W. State St., West Lafayette, IN 47907, USA, [email protected] Thomas J. Molnar Department of Plant Biology and Pathology, Rutgers University, Foran Hall, 59 Dudley Road, New Brunswick, NJ 08901, USA, [email protected] Madhugiri Nageswara Rao IFAS, University of Florida (University of Florida, IFAS) Citrus Research & Education Center, University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850, USA, [email protected] Jaya R. Soneji University of Florida, IFAS, Citrus Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA, [email protected] Padmini Sudarshana Monsanto Research Center, #44/2A, Vasant’s Business Park, Bellary Road, NH-7, Hebbal, Bangalore 560092, India, padmini. [email protected] Weibang Sun Kunming Botanic Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China, [email protected] Yoshihiko Tsumura Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan, [email protected]

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Brian D. Vanden Heuvel Department of Biology, Colorado State UniversityPueblo, Pueblo, CO 81001, USA, [email protected] Keith Woeste USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, Pfendler Hall, 715 W. State St., West Lafayette, IN 47907, USA, Yanling Zheng Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China Yuan Zhou Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China

Contributors

Chapter 1

Alnus Brian D. Vanden Heuvel

1.1 Introduction Alnus Mill. (Betulaceae), often referred to by its common name alder, is a genus composed of monoecious trees and shrubs distributed throughout the northern Hemisphere and limited in the southern Hemisphere along the Andes in South America. Species of Alnus were long thought of as forest weeds; however, since the emergence of an industry for alder wood products in the 1980s, considerable attention has been paid to the ecology, management, and genetic improvement of Alnus (Xie et al. 2002; Xie 2008). In addition to their commercial value, alder species are important because they have the ability to associate with Frankia, a nitrogen-fixing soil actinomycete. Frankia strains provide their hosts with a source of fixed nitrogen, a nutrient that limits plant growth. The host plant, in turn, provides fixed carbon to the Frankia strain (Baker and Schwintzer 1990). Plants that associate with Frankia have a distinct advantage over other plants because they are not as limited by nitrogen and can inhabit nitrogen-poor soils. Therefore, beyond wood products and biomass uses, species of Alnus play an important role in ecosystem development by securing unstable surfaces and participate in the first stage of plant succession on both wet and dry land soils in forests (Mejnartowicz 2001, 2007) and promote growth of other plants by adding nitrogen into the soil (Klemmedson 1979). It is important to point out initially that, unlike other more traditional crop plant/wild relative systems, Alnus has just recently become a target of research. Therefore,

Brian D. Vanden Heuvel Department of Biology, Colorado State University-Pueblo, Pueblo, CO 81001, USA e-mail: [email protected]

we have very limited knowledge of the baseline genetic variation within and among species and populations, the genetic architecture for desired traits, the geographic pattern of genetic variation, the extent of genotype by environment interaction, and heritability (Xie et al. 2002). This chapter focuses on what we currently know about the diversity of the genus Alnus, both within the genus and within selected species, current research on genetic resource management of some alder species, and broad patterns of Frankia strain distribution and diversity as they relate to alder distribution across a geographical mosaic of environments.

1.2 Taxonomy, Morphology, Reproductive Biology, Ecology, and Geographic Distribution of the Genus Alnus 1.2.1 Basic Taxonomy Alnus Mill. (Betulaceae) lacks a consensus classification. Since it was first described, Alnus has undergone many revisions (Furlow 1979), each varying widely in the ranks assigned to taxa and the number of species. Numbers of species within Alnus have ranged from 20 to 35 (Furlow 1979; Hall and Maynard 1979; Bond 1983). Confusion about the number and circumscription of species within Alnus arises primarily from the lack of clear morphological delimitations between taxa (Hall and Burgess 1990), specifically leaf morphology. Variations in leaf morphology show a continuum within and between taxa, making it difficult to define boundaries among species (Steele 1961; Parnell 1994). Nonetheless, the genus Alnus is a well-defined

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_1, # Springer-Verlag Berlin Heidelberg 2011

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group, easily clustered by morphological similarities and separated from the genus Betula L. (Betulaceae), its closest ally, by several discontinuities. Most notably, Alnus is recognized by its woody infructescences with persistent scales, bud structure, and the number of stamens (Furlow 1979). Within the genus Alnus, three distinct evolutionary lines have been identified and are treated as subgenera. The three subgenera are distinguishable by the bud structure, whether the pistillate catkin is exposed over the winter months, and when pollination occurs. The three subgenera of Alnus are subgenus Alnus, subgenus Clethropsis, and subgenus Alnobetula. Alnus subgenus Alnus contains the majority of the species. It is distinguished by having stalked shoot buds, pistillate catkins closed over winter, and is pollinated in late winter or early spring (Furlow 1979). Members of Alnus subgenus Alnus are found across the range of the genus, throughout North America, Europe, and along the Andes southward to Argentina. Many of the species of Alnus currently used and also those species under evaluation within agriculture and lumber industries are in this subgenus, including Alnus rubra Bongard (Red Alder) A. cordata Desf. (Italian Alder), A glutinosa (L.) Gaertner (Black Alder), and A. acuminata Kunth (Andean Alder). Alnus subgenus Clethropsis is recognized by stalked shoot buds, pistillate catkins open and pollinated in fall (Furlow 1979). Only three species are found within this subgenus, including Alnus formosana Makino (Formosan Alder) found on Taiwan, A. maritima (Marsh.) Muhl. Ex Nutt. (Seaside Alder) found on the Delmarva Peninsula on the east coast of North America, and disjunct populations in Oklahoma and Georgia, and A. nitida Endl. (Himalayan Alder). Because of their aesthetic appeal and drought tolerance, these three species, specifically A. maritima, have been gaining heightened interest as horticultural crops (Schrader and Graves 2004). Alnus subgenus Alnobetula is characterized by shoot buds without stalks and pistillate catkins produced and pollinated in late spring. Currently there is only one species, Alnus viridis (Chaix)DC. (Green Alder), in this subgenus, yet authors often raise the subspecies currently circumscribed under viridis to specific level. Although the habit of A. viridis is small and is not used for a lumber source, it has attracted interest as a candidate for reforestation in temperate forest ecosystems in both North America and Europe (Roy et al. 2007).

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1.2.2 Basic Morphology Members of the genus Alnus are woody, ranging in size from large trees with a single trunk to small shrubs with many trunks. Most of the species found in warmer climates take on a “tree-like” habit while those species found in cooler climates are “shrubby”. Even those species, which attain sizes considered arboreal, often have multiple trunks. It has been postulated that the large, arborescent habit is ancestral and the shrubby habit is an adaptation in derived lineages to the cooler climate (Furlow 1979). In most species of Alnus, the bark is smooth, but in some of the larger species, it can take on the form of large plates. The presence of smooth bark linked with a shrubby habit has been postulated by Hall (1952) as a set of neotenic characters. Stem diameters range in size from 1 cm to as large a 2 m, and twigs have a characteristic triangle shaped pith (Furlow 1979). The leaves of species of Alnus are arranged alternately on the stems and the veins are conspicuously pinnate. Leaf margins vary from entire to deeply serrate to cuneate. Overall, species of Alnus vary considerably in leaf structure, both inter and intraspecifically (Furlow 1979; Lecerf and Chauvet 2008). The wood of Alnus has long interested plant anatomists due to the interesting evolutionary series displayed by the species with respect to ray morphology. Within Alnus, Bailey (1911, 1912) discusses the evolution of multiseriate rays from uniseriate rays and the apparent reversal of the trend in a few species. The evolution of ray morphology within Alnus has further been discussed by a host of other authors (see Furlow 1979). Of all of the economic uses of Alnus, wood production is by far the most important. Alnus wood is primarily made up of vessels and fiber tracheids. Alder wood has high machining properties (Malkoc¸og˘lu and ¨ zdemir 2006) and wood quality (Bekhta et al. 2009), O making it a highly sought after wood product. Species of Alnus are monecious; the unisexual flowers are borne in staminate and pistillate catkins. Staminate catkins are pendant, while pistillate catkins are erect on the stems. In most species, the pistillate catkins are clustered and inserted just below a solitary or small group of axillary staminate catkins. At maturity, the pistillate catkin becomes woody and conelike. The cones of Alnus are useful for identification of

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species and are also one of the best ways to differentiate Alnus from its sister group, Betula.

1.2.3 Reproductive Biology Alnus is pre-dominately wind pollinated, although there are a few reports that insects are sometimes attracted to staminate catkins (Furlow 1979). The age at which flowers are produced in Alnus is not known for all species. There are reports that Alnus in Alaska (A. viridis ssp. sinuata) flowered at the age of seven years or earlier (Lawrence 1958). Furlow (1979) reports that in a common garden in east Lansing, Michigan, A. maritima flowered in 3 years, A. viridis ssp. sinuata, A. incana ssp. rugosa, and A. serrulata all flowered in 5 years. The larger species of A. rubra, A. rhombifolia, and A. acuminata did not flower until they were relatively large. It might be expected that in northern or subalpine climates early reproduction, coupled with a small, shrubby habit, would be advantageous, while in more temperate climates a more tree-like habit and delayed reproduction would be favored (Hall 1952). It is hypothesized that alders are primarily outcrossing, although some studies of genetic diversity within populations indicate high coancestry of alleles within individuals as the result of consanguineous matings (Gibson et al. 2008). Alder seeds do not have wings and can only be dispersed by wind 30–60 m from the mother tree (Mejnartowicz 2007). Although the seeds have no wings, they do possess air bladders and can be dispersed by waterways great distances. If these waterways have periodic flooding, dispersal is increased even more. Given that most of Alnus species are associated with wet habitats, this seed dispersal strategy allows alders to colonize new territories and migrate into other alder populations (Mejnartowicz 2007).

1.2.4 Geographic Distribution The genus Alnus has seven major distribution centers according to Furlow (1979): (1) western North America from southern Alaska to northern Mexico; (2) coastal eastern North America from Nova Scotia to the Gulf of Mexico (absent from the Caribbean); (3) high elevation centers of Mexico, central, and South America; (4)

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coastal eastern Asia; (5) Himalayas; (6) the mountains of Iran, Asia Minor, and southern Europe; and (7) circumpolar Europe, Asia, and North America. The oldest Alnus fossil, a catkin, dates to the midEocene (33–55 Mybp), but Alnus-like pollen has been reported from the late Cretaceous (83–85 Mybp), earlier than any fossils for the other genera in the family Betulaceae (Miki 1977). Given the distribution of known fossils and the recent molecular phylogeny (see below), it appears the Betulaceae first originated in a Mediterranean climate in Laurasia during the late Cretaceous (89–65 Mya) (Laurasia was the northern supercontinent formed after Pangaea broke up during the Jurrasic and included what are now North America, Europe, Asia, Greenland, and Iceland). Fossil evidence suggests that all six genera within the Betulaceae, including Alnus, were recognizably differentiated by the early Eocene (55 mybp) (Chen et al. 1999). By the early Tertiary (65 mybp), movement between Eurasia and North America was possible, and the range of Alnus probably increased. The distribution of Alnus to Africa and to Taiwan probably occurred later, during the Pleistocene (1.8 mybp–11,000 ypb) when sea levels were lower (Chen et al. 1999).

1.2.5 Ecology Most of Alnus species are associated with wet habitats (Furlow 1979). These include standing water, stream banks, bogs, and wet montane environments. Also, Alnus species grow in full sunlight, with the exception of A. viridis ssp. crispa, which can be an understory component of some conifer woods. Alnus is unique in the Betulaceae as it is the only genus within the family to associate with Frankia, a filamentous bacteria (actinomycetes) that fixes N2 and induces N2-fixing root nodules on a broad range of “actinorhizal plants”. Actinorhizal plants, in turn, are defined by their ability to form root nodules when in symbiosis with Frankia. Within the root nodule, Frankia fixes nitrogen that is transported to the host plant in amounts sufficient to supply most of plants’ nitrogen requirements. This symbiosis allows actinorhizal plants to invade and proliferate in soils that are low in combined nitrogen. To date, all species of Alnus examined have been shown to nodulate (Benson et al. 2004).

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Given the ability to form root nodules with Frankia, species of Alnus can inhabit nitrogen-poor soils. Therefore, Alnus plays an important role in ecosystem development by securing unstable surfaces participating in the first forest stage of plant succession (Mejnartowicz 2001, 2007), and promoting growth of other plants by adding nitrogen into the soil (Roy et al. 2007).

1.3 Genetic Variation in Alnus

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2n ¼ 28 progenitor had two 5S rDNA regions (Oginuma et al. 2000). Meiosis appears irregular and pollen quality poor in the Alnus individuals with 2n ¼ 42 (Gram et al. 1942; Jaretzky 1930). These include A. cordata, A. subcordata, and A. orientalis. In comparison, those plants with 2n ¼ 56 and 2n ¼ 28, meiosis appears normal and almost all pollen formed is high quality (Gram et al. 1942). The only other reported problems with meiosis in Alnus are the putative hybrids between A. incana ssp. rugosa and A. serrulata (Woodworth 1929, 1930, 1931).

1.3.1 Chromosome Numbers Given the relatively small size of the genus Alnus, the genus demonstrates high polyploidy and an extensive chromosome number sequence. Chromosome counts from species of Alnus have yielded 2n ¼ 14, 28, 42, 56, 70, 84, and 112 (Furlow 1979; Oginuma et al. 2000). The diploid chromosome number of 2n ¼ 14 has been found in Alnus hirsute var. microphylla, A. pendula, and A. serrulatoides, suggesting the base number in Alnus is x ¼ 7. Although this is an extensive series, most of species of Alnus are 2n ¼ 28 and are considered tetraploids (Furlow 1979; Oginuma et al. 2000) In 2000, Oginuma et al. tested whether the dramatic ploidy series seen in Alnus was produced through successive allopolyploid events. It was suggested based on previous data that the amount of 5S rDNA does not change by allopolyploid condition, allowing one to compare amounts of 5S rDNA to chromosome number for correlations and additive patterns. When the Alnus chromosomal series was investigated using in situ hybridization of 5S rDNA, some Alnus taxa with 2n ¼ 28 (Alnus hirsute var. microphylla and A. pendula) were found to have two 5S rDNA signals, while A. serrulatoides (2n ¼ 28) was found to have four 5S rDNA signals (Oginuma et al. 2000). These results suggest that although 2n ¼ 28 is by far the most common chromosome number in Alnus, 2n ¼ 28 may have been arrived at by multiple pathways and involved complex genome evolutionary histories. Therefore, two species of Alnus may have the same chromosome number but arrived at that number in very different ways. Investigations of the species of Alnus with high chromosome numbers (A. japonica 2n ¼ 56, A. sieboldiana 2n ¼ 84, and A. firma 2n ¼ 112) all showed predicted additive polyploidization of 5S rDNA signal if the base

1.3.2 Hybridization The existence of interspecific hybrids in Alnus has been well documented. Hybrids in natural populations have been recorded and studied in North America, Europe, the Russian Far east, and Japan. Documented hybrids include Alnus glutinosa  incana (Hylander 1957; Parnell 1994; Banaev and Bazˇant 2007), Alnus serrulata  rugosa (Steele 1961; Furlow 1979); Alnus sinuate  crispa (Bousguet et al. 1989, 1990), and Alnus glutinosa  rubra (Hall and Burgess 1990). Natural hybrids, specifically between Alnus incana and A. glutinosa, have been reported to possess many economically valuable properties (Banaev and Bazˇant 2007). Hybrids of A. incana  glutinosa have been found to have greater drought resistance when compared to each of the parent species, less demand for fertility (Kobendza 1956; Kundzinsh 1957), higher wood quality (Pirags 1962), and better resistance to some forms of rot (Fer and Sˇedivy 1963). Hall and Burgess (1990) reported that fast growing, early flowering hybrids of Alnus incana  glutinosa and A. glutinosa  rubra bred true through the F2 generation. Further, A. glutinosa  rubra showed hybrid vigor when grown under greenhouse conditions and showed levels of resistance to the European alder leafminer, Fenusa dohrnii. Given that interspecific hybrids form between many species pairs in Alnus, hybridization may be an important strategy for species improvement. Hall and Burgess (1990) reports that Alnus as a genus has relatively poor tolerance to moister stress, especially the species (A. rubra, A. incana, and A. glutinosa), which are primarily used for biomass and wood production.

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This trait has hampered field establishment and largescale biomass production. Instead of identifying and selecting for drought-tolerant individuals within these targeted species, a better strategy may be to bring desired traits, such as tolerance to moister stress, from other species of Alnus, like A. cordata or A. maritima, through hybridization (Hall and Burgess 1990; Schrader et al. 2005).

1.3.3 Studies of Genetic Variation Within Alnus Species As a pioneer tree species, alders are included in the first forest stage of plant succession on wet, riparian sites (Mejnartowicz 2001). Yet, alders are also a component of climax forest communities on many soil types (Mejnartowicz 2007). These two life histories predict very different genetic structures. It has been theorized and found that pioneer tree species reveal much higher genetic diversity than climax tree species (Wehenkel et al. 2006). Further, it is predicted that in pioneer species, allelic variation is distributed between individuals, while in climax species, allelic variation is distributed in individuals as heterozygous loci (Mejnartowicz 2007). Given that species of Alnus can in some circumstances be pioneer species and in others be a member of a climax community, studies of Alnus population genetic structure are very interesting (Mejnartowicz 2007). Complicating the life histories of populations of Alnus, species of Alnus, like other woody species with large geographic ranges, outcrossing breeding systems, and seed dispersal using wind and water, have a relatively higher genetic diversity within species and populations, but lower genetic diversity among populations than woody plants with other traits (Hamrick et al. 1992). Studies of genetic variation within species and populations of Alnus have been studied in Alnus rubra (Hamann et al. 1999; Xie et al. 2002), A. maritima (Schrader and Graves 2002, 2004; Gibson et al. 2008), A. serrulata (Gibson et al. 2008), A. glutinosa (Prat et al. 1992; King and Ferris 1998; Mejnartowicz 2007), A. rugosa (Bousquet et al. 1988; Huh 1999), A. japonica (Huh 1999), and A. crispa (Bousquet et al. 1987, 1988).

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In a study of Alnus glutinosa, Mejnartowicz (2007) examined the genetic structure of 12 populations in Poland using isozymes. The study was designed to address the inter- and intrapopulation variation, gene flow between populations, and correlations between geography and genetic similarity. He found that 74% of the loci examined were polymorphic, a high number for plants and in agreement with the prediction that climax species have individuals with a high frequency of heterozygous loci. Also, different populations did not differ significantly in the proportion of polymorphic loci, suggesting that there is no correlation between the level of genetic polymorphism and site conditions. Although the number of polymorphic loci is high (74%), the measurements of heterozygosity appeared low. The average observed heterozygosity was 0.2 and a positive fixation index of F ¼ 0.305, indicating a 30% deficiency in heterozygotes. These results suggest either inbreeding, vegetative reproduction from root suckers, or low efficiency of both pollen and seed dispersal has led to lower than expected heterozygotes. Steiner and Gregorius (1999) observed large degrees of self-pollination in an A. glutinosa population, but did not see a drop in seed production, implying that inbreeding is possible in these populations and may be responsible for the low heterozygosity numbers. Lastly, Mejnartowicz (2007) found no significant correlation between genetic and geographic distance between populations. Only 8.9% of the total variation was due to interpopulation differences, and gene flow estimates showed that reproductive barriers do not separate populations of A. glutinosa. Very similar results were found in a study of A. japonica in East Asia by Huh (1999). He found a high proportion of polymorphic loci (76%), low heterozygosity, and a high fixation index (F ¼ 0.502), and only 9.5% of the total variation was due to interpopulation differences. These results indicate that populations of A. japonica, like populations of A. glutinosa in Poland, are highly polymorphic, yet the individuals have low heterozygosity, and there appears to be no significant correlation between genetic and geographic distance between populations. Both of the above studies are in alignment with the prediction by Hamrick et al. (1992) that species with large geographic ranges and outcrossing breeding systems will have relatively higher genetic diversity within species and populations, but lower genetic diversity among populations.

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In 2008, Gibson et al. compared the population genetic diversity using allozymes between a common, widespread species of Alnus, A. serrulata, and a rare species A. maritima in order to evaluate the influence of small population size and extreme isolation on genetic diversity. A. maritima exists in only three locations: the Delmarva Peninsula of Delaware and Maryland, South Central Oklahoma, and Northwest Georgia (Schrader and Graves 2004). They found, as might be expected, that genetic diversity was lower in A. maritima than the common, widespread cogener A. serrulata (A. maritima He ¼ 0.217, A. serrulata He ¼ 0.268) and inbreeding higher in A. maritime (A. maritima f ¼ 0.483, A. serrulata f ¼ 0.269). Further, the partitioning of the genetic variation was higher in A. maritima (Y ¼ 0.278) than A. serrulata (Y ¼ 0.197). All the three results are generally consistent with expected values for rare and widespread species of similar life history traits (Cole 2003; Gibson et al. 2008). Although the estimation of genetic diversity was low in A. maritima, the overall pattern of population structure and genetic diversity is not strikingly dissimilar from other species of Alnus in North America (Gibson et al. 2008). Estimations of genetic diversity in A. crispa (Bousquet et al. 1987) and A. rugosa (Bousquet et al. 1988) were similar to A. maritime. What is striking about the A. maritima data, when compared to other Alnus species, is the high genetic differentiation between populations. Most species of Alnus have high gene flow among networks of populations along a watercourse and therefore considerably less genetic differentiation among populations. The high genetic differentiation seen in A. maritima undoubtedly is due to the extreme isolation of the three populations, and gene flow is rare to non-existent. The three populations of A. maritima were studied by Schrader and Graves (2004) using inter-simple sequence repeat (ISSR) markers, and they concluded that morphological and ISSR variation was sufficient enough to warrant subspecies designations (subspecies maritima, oklahomensis, and georgiensis). They also concluded from their data that the Oklahoma population diverged first, and the Georgian and Delmarva populations were more closely related and diverged later. Interestingly, the later allozyme data (Gibson et al. 2008) suggest that the Oklahoma and Georgia populations are more similar to the Delmarva population. Both results, though, support the idea that the

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highly disjunct population structure of A. maritima is a result of natural range reduction and not humanmediated establishment in Oklahoma and Georgia (Gibson et al. 2008). The genetic structure of 19 populations of Alnus rubra in British Columbia was examined by Xie et al. (2002). A. rubra (red alder) is the most common hardwood tree in the Pacific northwest of North America. Although it makes up only a small proportion of the forest resources of the Pacific northwest, it has been gaining attention due to the increased demand for red alder wood products. The goal of this study was to understand the baseline genetic variation and population structure for use in understanding and harnessing the adaptive variation of economically important traits (Xie et al. 2002). They found that the number of alleles per locus were 1.72, 52% of the loci were polymorphic, and total expected genetic diversity (0.133) were all below what has been reported for long-lived woody species. Further, they found low among-population differentiation (8%), compared to other species that outcross and disperse seed by wind. The limited among-population differentiation was almost entirely attributed to island populations versus mainland populations. The authors conclude that, if saving genetic resources is the goal, selecting at least one mainland and one island population will contain the species local genetic variation (Xie et al. 2002). Overall, the species of Alnus show extensive gene flow, most likely due to their pollination strategy (wind) and seed dispersal (water), making populations show little correlation between genetic and geographic distance, with the exception of A. rubra, which displays strong genotype x environment interactions. Further, individuals show low heterozygosity, but loci in populations are highly polymorphic.

1.4 Phylogeny of Alnus The Betulaceae is composed of six genera and about 130 species (Mabberely 1988). The family is mostly distributed throughout the temperate regions of the northern Hemisphere, with the exception of species of Alnus, which are found throughout Central America south to Argentina. The genus Alnus is the only actinorhizal genus within the Betulaceae. The angiosperm rbcL phylogeny of Chase et al. (1993) strongly

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supported the classification Betulaceae within Fagales (a relationship long recognized based on morphology). More recent studies of this group (Manos and Steele 1997) have placed the family in a subclade with Casuarinaceae and Ticodendraceae. Recent molecular phylogenies for the Betulaceae suggest two clades or lineages (Chen et al. 1999). One lineage contains the four genera - Corylus, Ostryopsis, Carpinus, and Ostrya – and the other includes Alnus and Betula. These results suggest that Betula is the closest relative to Alnus, and Alnus diverged early in the evolution of the Betulaceae. The early divergence of Alnus agrees with dated fossils for Alnus (Miki 1977; Chen et al. 1999). Fossil evidence also suggests that all six genera in the Betulaceae, including Alnus, were differentiated by the early Eocene (55 mybp) (Chen et al. 1999). This observation suggests that if the ability to nodulate with Frankia was ancestral in the Betulaceae, loss of that ability occurred very early on in the evolution of the family (Benson et al. 2004). Two separate authors conducted independent phylogenies of the genus Alnus based on the internal transcribed spacer (ITS) of the nuclear ribosomal repeat. Navarro et al. (2003) examined 18 species and constructed phylogenetic trees using neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) search strategies. Overall, they detected three major clades corresponding to the three subgenera Alnus, Clethropsis, and Alnobetula (see Sect. 1.2.1). They also discovered that A. nepalensis, often positioned within subgenus Clethropsis with A. nitida, A. maritima, and A. formosana, fell within subgenus Alnus sister to A. japonica. Subgenus Clethropsis also displayed an increased substitution rate in comparison with the other clades corresponding to the other two subgenera. Based on their trees, Northeast Asia was inferred as the origin of the genus, based on the number of species from that region that were present in the basal, deep lineages identified. Further, fewer trans-continental migrations would have to be inferred based on the tree topology if Northeast Asia was the center of origin. Chen and Li (2004) examined 34 species using the ITS region. As expected, the phylogenies produced by Chen and Li (2004) are very close in topology to the previously published trees in Navarro et al. (2003), with the exception of having many more taxa sampled. Chen and Li (2004) also identify three major clades corresponding to the three subgenera. They report that subgenus Clethropsis is sister to subgenus Alnus. In a

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basal position is subgenus Alnobetula. Chen and Li (2004) also report that the central and South American Alnus taxa appear to have been derived from Asian ancestral taxa and migrated to central and South America via the Bering land bridge. Hall and Burgess (1990) postulated that in may be possible to introduce desired characteristics into economically important Alnus species through hybridization (see Sect. 1.3.2). In fact, interspecific hybrids have been reported between Alnus glutinosa  incana, Alnus serrulata  rugosa, Alnus sinuate  crispa, and Alnus glutinosa  rubra (see Sect. 1.3.2). When the parent taxa of these hybrids are examined within the topology of the trees produced by Navarro et al. (2003) and Chen and Li (2004), all are either in a sister and very near sister relationships, indicating hybrids are only formed by very closely related species. This may limit the possibility of Hall and Burgess’s (1990) recommendation of using hybridization as a tool for acquiring desired traits in Alnus species (see Sect. 1.5)

1.5 Evaluation of Alnus for Biomass Production Hall and Burgess (1990) published a summary of a workshop on alder improvement sponsored by the International Energy Agency (IEA) Forestry Energy Agreement. Alnus has attracted the interest of energy plantation systems because alders can symbiotically fix nitrogen and therefore lessen the drain on the soil nutrients after frequent harvests for biomass. Within this summary, Hall and Burgess identify three problems/objectives: (1) a limited number of Alnus species have been evaluated; (2) the availability of seed sources, especially for hybrids, is limited; and (3) there is a need for better exchange of information among groups of people working with Alnus to promote advantages and identify problems with selected Alnus species. The report attempts to summarize what we know about the first two problems ca. 1990. Hall and Burgess (1990) report on an evaluation trial, which examined 39 seed lots representing five species of Alnus. The seedlings were placed in nursery beds in four countries (Belgium, Canada, UK, and the USA) in identical planting designs, although the authors only report on the trials from Canada and USA. They first report that some seed lots had much

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lower germination rates than expected, yielding very few seedlings for study. A. acuminata was particularly hard to grow from seed because the seed lot was not cold-hardy and was quickly lost at most locations. They continue to report that fall dormancy for the seedlings was highly variable. The percentage of seedlings that were dormant for each test entry ranged from 91% to 0% and only 20 of the 39 entries had more than 50% of their seedlings dormant by the first of October. This is important because in Ames, Iowa, where the USA planting occurred, killing frosts typically occur by October 10. The delay of dormancy was unexpected and many of the seedlings in this study, from multiple seed lots and species, were lost. As a general pattern, A. glutinosa and A. rubra entries had the highest dormancy rates and therefore survival rates, followed by A. incana, A. cordata, and A. acuminata entries in that order. The A. acuminata entries showed no sign of dormancy and were dead by the end of October (Hall and Burgess 1990). Growth performance of the trees in the study by Hall and Burgess (1990) showed that A. glutinosa and A. incana had the highest growth rates and survival in the Canadian plots after a particularly hot, dry growing season. A. rubra had decent survival rates (68%), yet almost no growth, and the A. cordata entries showed the lowest growth and survival. During the same summer in Iowa, the USA plots also experienced a hot, dry growing season. In the USA plots, A. cordata entries actually had the highest survival, followed by A. glutinosa, A. incana, and A. rubra. Given the extreme water stress the seedling encountered that summer, very few individuals showed growth, independent of species. The authors comment that in the face of global warming, A. cordata and A. glutinosa show promise as biomass producers with their relatively good survival under extreme water stress (Hall and Burgess 1990). The study report by Hall and Burgess highlights the challenges and potentials of Alnus in the biomass industry. It appears that seed lots are quite variable in their germination rates. Also, the relatively poor tolerance Alnus shows to water stress will be a limitation to its inclusion in biomass plantations. Drought tolerance may be improved by selecting on drought tolerance variation within species such as A. rubra or A. glutinosa, or attempting to transfer drought tolerance from A. cordata by hybridizing it with species of Alnus better suited to biomass production.

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1.6 Development and Improvement of Genetic Stocks in Alnus rubra Alnus rubra (red alder), once regarded as a forest weed, is now gaining interest as a commercial tree and more concentration has been focused on its maintenance, growth, and genetics (Xie 2008). Given the new demand for A. rubra wood, natural stands are disappearing, and producers are turning to plantations of red alder (Xie 2008). Tanaka et al. (1997) reports that 2.5 million red alder seedlings are planted each year by a single corporation. Xie (2008) reports that over one million seedlings of red alder are expected to be planted in British Colombia alone. The increased demand for red alder wood suggests that a genetic improvement program would be worthwhile. Red alder contains sufficient variation in traits of interest to producers (i.e. height, stem volume, biomass, ecophysiological traits, etc.) (DeBell and Wilson 1978; Ager 1987; Hook et al. 1990; Dang et al. 1994; Xie and Ying 1994; Hamann et al. 1999) and using seed sources or selected genotypes ideal for local conditions can enhance productivity (Hamann et al. 2000). In order to identify ideal seed sources and appropriate genetic material of A. rubra, long-term provenance tests at multiple sites is needed, which can accurately identify areas for seed collection best for particular plantation environments (Hamann et al. 2000). Hamann et al. (2000) reports on early results from a large-scale, long-term provenance test in British Columbia. They tested the assumption that local sources of seed are optimal and used geographical information systems (GIS) to identify seed transfer zones and guidelines. They found significant genotype x environmental interactions, and seedlings grown near the site of seed collection showed superior performance, suggesting local adaptation of A. rubra populations. They generated general seed transfer guidelines and found, using GIS, that a transfer of seed 100 km in a northern or southern direction was associated with a decline of 2.5% in survival and about 5 cm in height after 2 years of growth. Xie (2008) reports on a 10-year provenance-progeny testing program for red alder. Results of the program indicate that trees planted near their site of origin performed better, and two regions (northern and southern) were identified with the boundary of 52 N. Interestingly, individual seedlings had about 5% decrease

1 Alnus

in stem volume and 6% decrease in mortality for every 1 latitude north or south the seedling was transferred from its seed source. These results suggest strong local adaptation (Xie 2008). Further, the levels of additive genetic variation showed that between 23% and 29% gains in stem volume could be gained in a rotation age of 40 years selecting the top 20 individuals, and selection of individuals could occur at age 6 (Xie 2008). The studies by Hamann et al. (2000) and Xie (2008) indicate that there is significant genotype x environmental interactions and strong local adaptation in A. rubra. Increasing yield, either height or biomass, is possible in A. rubra, but linking seed source to the local plantation environment appears to be essential.

1.7 Frankia and Alnus The actinobacterial genus Frankia contains filamentous bacteria (actinomycetes) that fix N2 and are defined by their ability to induce N2-fixing root nodules on a broad range of “actinorhizal plants”. Actinorhizal plants, in turn, are defined by their ability to form root nodules when in symbiosis with Frankia (Benson et al. 2004). Frankia fixes nitrogen in the root nodule, and the resulting ammonia is transported to the host plant in amounts sufficient to supply most of the plant’s nitrogen requirements. The host plant, in turn, provides Frankia within the nodule fixed sugar for growth (Benson et al. 2004). This symbiosis allows actinorhizal plants to colonize substrates low in nitrogen (Roy et al. 2007). The phylogeny of the genus Frankia has been elucidated using the 16S rRNA gene, the genes for nitrogen fixation (nif genes) and by other genes (Benson and Clawson 2000). All analyses done to date agree that the genus is comprised of three major clades, often referred to as groups (Groups 1, 2 and 3). Specificity of Frankia strains to host species and vice versa is not demonstrated at the “group” level; each group has different and sometimes overlapping plant specificity, physiological properties, and symbiotic interactions (Benson et al. 2004). Within each group are definable subgroups that constitute “genospecies,” as defined by DNA–DNA homology studies (An et al. 1985; Dobritsa and Stupar 1989; Fernandez et al. 1989; Normand et al. 1996; Benson and Clawson 2000). In general, Group 1 Frankia strains form nodules on members the order Fagales, including the three plant

9

families Betulaceae, Myricaceae, and Casuarinaceae (Benson et al. 2004). “Alder strains” within group 1 generally infect most species of alder tested in greenhouse experiments, with some variability in effectiveness depending on the plant/symbiont combination. To date, all alder species examined have been shown to nodulate. Alder strains are also generally able to infect members of the Myricaceae (Benson et al. 2004). A few strains of Frankia from Group 3 have also been shown to nodulate alders, but do so only rarely and are poorly effective (Bosco et al. 1992; Lumini and Bosco 1996). Estimations of nitrogen fixation rates in actinorhizal alders are comparable to those in legumes. Alnus rubra, A. glutinosa, and A. viridis have been found to fix nitrogen in the range of 40–300 kg N/ha/year as compared to Alfalfa and clover that can fix 57–300 kg N/ha/year and 104–160 kg N/ha/year, respectively (Hibbs and Cromack 1990; Zuberer 1998; Pepper 1999; Hurd et al. 2001). Nitrogen fixation rates have been found to vary with stand age, density, and alder–Frankia combinations (HussDanell 1990; Myrold and Huss-Danell 2003). Alnus spp. are often used as a “trapping plant” for Frankia strains in order to study Frankia strain distribution and diversity, largely because alder seeds are readily available and easily germinated and are infected by a wide variety of Group 1 and 3 Frankia strains (Benson et al. 2004). Except for a few environments, such as at the foot of retreating glaciers in Alaska (Kohls et al. 1994), Frankia strains infective on Alnus spp. are cosmopolitan and seem to persist independently of host plants as saprophytes. When trapping studies are done, estimates of Frankia strains infective on Alnus vary from a few per gram of soil to several thousands per gram in soils both near and removed from actinorhizal plants (Van Dijk 1979; Smolander 1990; Smolander and Sarsa 1990; Myrold et al. 1994; Markham and Chanway 1996; Maunuksela et al. 1999). Frankia strains specific to Alnus hosts seem to persist in soil long after the hosts have been removed and are also found outside of the normal geographic ranges of Alnus species, most likely due to wind action spreading Frankia spores (Wollum et al. 1968; HussDanell and Frej 1986; Smolander and Sundman 1987; Arveby and Huss-Danell 1988; Paschke and Dawson 1992; Maunuksela et al. 1999). For example, Alnus species nodulated at every site in New Zealand tested, even though Alnus is a very recent arrival to New Zealand (Benecke 1969; Benson et al. 2004).

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The nodulation capacity of soils for Alnus is affected by fertility (Kohls and Baker 1989; Sanginga et al. 1989; Thomas and Berry 1989; Myrold and Huss-Danell 1994; Myrold et al. 1994; Yang 1995), season (Myrold and Huss-Danell 1994), water availability (Schwintzer 1985; Dawson et al. 1986; Nickel et al. 1999), physiological status of Frankia strains (Myrold and Huss-Danell 1994), acidity (Griffiths and McCormick 1984; Smolander and Sundman 1987; Zitzer and Dawson 1992; Crannell et al. 1994), and by the type of plant cover (Huss-Danell and Frej 1986; Smolander and Sundman 1987; Smolander et al. 1988; Smolander 1990; Smolander and Sarsa 1990; Myrold and Huss-Danell 1994; Markham and Chanway 1996; Zimpfer et al. 1999). There is some evidence that Frankia strains infective on Alnus spp. sort by soil type. Within Frankia, it is possible to characterize strains as either sp(+) (containing sporangia) or sp() (devoid of sporangia) nodules (Schwintzer 1990). In British Columbia, sp() nodules of A. rubra dominate in near the coast, with no sp(+) nodules observed. As sampling moved inland, the proportion of sp(+) nodules increased up to 53% of the total (Markham and Chanway 1996). Also, as soil acidity rises, Frankia strains with the sp(+) phenotype become more prominent (Weber 1986; Holman and Schwintzer 1987; Kashanski and Schwintzer 1987). Overall, Frankia strains infective on Alnus are extremely cosmopolitan and have been found in soils lacking actinorhizal plants, suggesting alder Frankia strains can live independently of the association, not requiring continuous symbiotic interaction. It is likely that their wide distribution is related to the ranges of their hosts (Alnus extends throughout the northern hemisphere and into South America). Their abundance in New Zealand testifies to their ability to grow as saprophytes in the absence of Alnus, as well as their ability to spread.

1.8 Summary and Recommendations for Future Actions Alders are used primarily for their wood, either in the form of wood products or biomass. The value of alder logs has increased rapidly since the 1980s because of an emerging industry for alder wood products, which has attracted the interest of land managers, wood

B.D. Vanden Heuvel

producers, and researchers. Beyond their use in wood products, Alnus also play an important role in ecosystem development. Alnus has always been considered an outcrossing species given its life history strategy of wind pollination. Estimations of heterozygosity and numbers of heterozygotes within natural populations suggest inbreeding may be possible and common. Self-pollination has been documented (Steiner and Gregorius 1999) but did not result in a drop in seed production, implying that inbreeding is possible in these populations and may be responsible for the low heterozygosity. We know little about the possibility of using selfing as a tool for reproduction in Alnus or its possible effects in natural populations. Alnus displays a fantastic chromosome series for a genus of such small size. Most of the species are 2n ¼ 28 and are considered tetraploids. There have been reports of meiotic problems in some of the hexaploid individuals (2n ¼ 42), but other individuals with 2n ¼ 56 (octoploids) appear normal. Although many of the species of Alnus are (2n ¼ 28), species may not have arrived at this number in exactly the same way, suggesting complex chromosomal evolution. These past events may make hybridization and breeding using multiple species difficult. When Alnus sp. are targeted for genetic improvement to increase growth, stem volume, and growth rate, one strategy for improvement is to select on variation for the traits of interest present in the natural populations. Using this strategy depends on the existence of genetic variation. All estimations of genetic diversity within Alnus species are high, relative to other plant species, and have shown highly polymorphic loci. Further, species of Alnus show extensive gene flow, most likely due to their pollination strategy (wind) and seed dispersal (water), making populations show little correlation between genetic and geographic distance, with the exception of A. rubra. Xie (2008) estimated that genetic variation could be selected on and a gain in stem volume between 23 and 29% could be achieved. It is important to note that, with respect to A. rubra, there was significant genotype  environmental interactions and evidence of strong local adaptation for specific genotypes. This suggests that seed sources used in plantations should be selected from local environments and nearby natural populations and not from distant populations. Hall and Burgess (1990) suggested that hybridization may be a different

1 Alnus

strategy for genetic improvement of Alnus for commercial interests, specifically with respect to drought and water stress tolerance. Alnus, as a genus, has relatively poor tolerance to water stress, and this trait is an inhibitor for the use of Alnus in large-scale biomass plantations. Hall and Burgess (1990) argues that drought tolerance may be improved in Alnus species by selecting drought tolerance variation within species such as A. rubra or A. glutinosa, or attempting to transfer drought tolerance from A. cordata by hybridizing it with species of Alnus better suited to biomass production. Yet, when the parent taxa of published hybrids are examined within the topology of the phylogenies for Alnus, hybrids appear to be formed from parent taxa that are closely related. This may limit the possibility of Hall and Burgess’s (1990) recommendation of using hybridization as a tool for acquiring desired traits in Alnus species. The possibility of using hybridization in the improvement of Alnus still needs investigation. Alnus species are actinorhizal, meaning that they can enter into a symbiosis with Frankia, a N2-fixing filamentous bacteria. Frankia fixes nitrogen in the root nodule and the resulting ammonia is transported to the host plant in amounts sufficient to supply most of plants’ nitrogen requirements. This ability makes Alnus an attractive plant commercially because it can be grown in substandard soil conditions and does not require additional nutrients for growth. To date, all alder species examined have been shown to nodulate with Frankia. Although there are reports that Frankia infection on Alnus may sort by soil type, and nodulation capacity of soils varies, Alders appear to nodulate wherever they find themselves, precluding needing to “seed” Frankia within an alder plantation. We still know very little about alder host preference of Frankia strain, the signaling pathway(s) between Frankia and alder host for nodule formation, or if specific plant and Frankia genotype combinations yield high nitrogen fixation rates.

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13 Pirags DM (1962) Process of growth and structure of wood of hybrid alder (Alnus hybridus A.B.) in the Latvian SSR. Latvian Agricultural Academy, Elgava, p 19 Prat D, Leger C, Bojovic S (1992) Genetic diversity among Alnus glutinosa (L.) Gaertn. populations. Acta Oecologia 13:469–477 Roy S, Khasa DP, Greer CW (2007) Combining alders, frankiae, and mycorrhizae for the revegetation and remediation of contaminated ecosystems. Can J Bot 85:237–251 Sanginga N, Danso SKA, Bowen GD (1989) Nodulation and growth response of Allocasuarina and Casuarina species to phosphorus fertilization. Plant Soil 118:125–132 Schrader JA, Gardner SJ, Graves WR (2005) Resistance to water stress of Alnus maritima: intraspecific variation and comparisons to other alders. Environ Exp Bot 53:281–298 Schrader JA, Graves WR (2002) Intraspecific systematic of Alnus maritima (Betulaceae) from three widely disjunct provenances. Castanea 67:380–401 Schrader JA, Graves WR (2004) Systematics of Alnus maritima (seaside alder) resolved by ISSR polymorphisms and morphological characters. J Am Soc Hortic Sci 129:231–236 Schwintzer CR (1985) Effect of spring flooding on endophyte differentiation, nitrogenase activity, root growth and shoot growth in Myrica gale. Plant Soil 87:109–124 Schwintzer CR (1990) Spore-positive and spore-negative nodules. In: Gillings M, Holmes A (eds) Plant Microbiology. Garland Science/BIOS Scientific, London/New York, pp 177–193 Smolander A (1990) Frankia populations in soils under different tree species with special emphasis on soils under Betula pendula. Plant Soil 121:1–10 Smolander A, Sarsa ML (1990) Frankia strains in soil under Betula pendula: behavior in soil and in pure culture. Plant Soil 122:129–136 Smolander A, Sundman V (1987) Frankia in acid soils of forests devoid of actinorhizal plants. Physiol Plant 70:297–303 Smolander A, Van Dijk C, Sundman V (1988) Survival of Frankia strains introduced into soil. Plant Soil 106:65–72 Steele FL (1961) Introgression of Alnus serrulata and Alnus rugosa. Rhodora 63:297–304 Steiner W, Gregorius HR (1999) Incompatibility and pollen competition in Alnus glutinosa: evidence from pollination experiments. Genetica 105:259–271 Tanaka Y, Brotherton P, Hostetter S, Chapman D, Dyce S, Belanger J, Johnson J, Duke D (1997) The operational planting stock quality testing program at Weyerhaeuser. New Forest 13:423–437 Thomas KA, Berry AM (1989) Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth of Ceanothus griseus var. horizontalis. Plant Soil 118:181–187 Van Dijk C (1979) Endophyte distribution in the soil. In: Gordon JC, Wheeler CT, Perry DA (eds) Symbiotic nitrogen fixation in the management of temperate forests. Oregon State University Press, Corvallis, OR, USA, pp 84–94 Weber A (1986) Distribution of spore-positive and spore-negative nodules in stands of Alnus glutinosa and Alnus incana in Finland. Plant Soil 96:205–213 Wehenkel C, Bergmann F, Gregorius HR (2006) Is there a tradeoff between species diversity and genetic diversity in forest tree communities? Plant Ecol 185:151–161

14 Wollum AG II, Youngberg CT, Chichester FW (1968) Relation of previous timber stand age to nodulation of Ceanothus velutinus. J Forest Sci 14:114–118 Woodworth RH (1929) Cytological studies in the Betulaceae. II. Corylus and Alnus. Bot Gaz 88:383–399 Woodworth RH (1930) Cytological studies in the Betulaceae. III. Parthenogenesis and polyembryology in Alnus rugosa. Bot Gaz 89:402–409 Woodworth RH (1931) Polyploidy in the Betulaceae. J Arnold Arbor 12:206–217 Xie C, Ying CC (1994) Genetic variability and performance of red alder (Alnus rubra) in British Columbia. In: Proceedings of the ecological management of BC Hardwoods, Richmond, pp 110–113 Xie CY (2008) Ten-year results from a red alder (Alnus rubra Bong.) provenance-progeny testing and their implications for genetic improvement. New Forest 36:273–284

B.D. Vanden Heuvel Xie CY, El-Kassaby YA, Ying CC (2002) Genetics of red alder (Alnus Rubra Bong.) populations in British Columbia and its implications for gene resources management. New Forest 24:97–112 Yang Y (1995) The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. Plant Soil 176:161–169 Zimpfer JF, Kennedy GJ, Smyth CA, Hamelin J, Navarro E, Dawson JO (1999) Localization of Casuarina-infective Frankia near Casuarina cunninghamiana trees in Jamaica. Can J Bot 77:1248–1256 Zitzer SF, Dawson JO (1992) Soil properties and actinorhizal vegetation influence nodulation of Alnus glutinosa and Elaeagnus angustifolia by Frankia. Plant Soil 140:197–204 Zuberer DA (1998) Biological dinitrogen fixation: introduction and non-symbiotic. In: Sylvia DM, Fuhrmann JJ, Hartel PG, Zuberer DA (eds) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ, pp 295–321

Chapter 2

Corylus Thomas J. Molnar

2.1 Introduction The Corylus L. genus contains a wide diversity of deciduous shrub and tree species that are important components of many temperate forests across the Northern Hemisphere, all bearing edible nuts. Its most widely known and well-studied member, the European hazelnut (Corylus avellana L.), is also an economically valuable commercial tree nut crop, ranking fifth in world production behind cashews (Anacardium occidentale L.), almonds [Prunus dulcis (Miller) D.A. Webb], walnuts (Juglans regia L.), and chestnuts (Castanea spp.) (FAOSTAT 2010). The top hazelnut producing country in the world is Turkey, which typically produces more than 70% of the world’s crop, which was 1,052,001 tons in 2008. Turkey is followed by Italy, which produces around 15–20% of the total, and the US, which produces <5%. Other countries producing significant crops include Azerbaijan, Spain, Georgia, Iran, France, and China (FAOSTAT 2010). Commercial production is limited to regions with climates moderated by large bodies of water that have cool summers and mild winters, such as the slopes on the Black Sea of northern Turkey or the Willamette Valley of Oregon, where 99% of the US crop is produced. The demand for hazelnuts worldwide is predominated by a desire for round, highquality, well-blanching kernels for use in chocolates and other confectionaries, baked goods, spreads, and other products. Only 10% or less of the world’s crop is sold as in-shell nuts. Superior kernels of the Italian

T.J. Molnar Department of Plant Biology and Pathology, Rutgers University, Foran Hall, 59 Dudley Road, New Brunswick, NJ 08901, USA e-mail: [email protected]

cultivars “Tonda Gentile de Langhe”, “Tonda di Giffoni”, and “Tonda Romana” set quality standards of comparison for the industry. Hazelnut has a long history of utilization and production by man, likely predating the Roman era (Rosengarten 1984; Boccacci and Botta 2009). Despite this long history, hazelnut breeding is in its infancy compared to most other domesticated crops. Until only recently, world production has been based entirely on traditional selections made from local populations, whose exact origins have been largely lost with antiquity. Public breeding programs were initiated in Italy and the US in the 1960s, Spain and France in the 1970s, and Turkey in the 1980s (Thompson et al. 1996), but the Corylus genus as a whole remains essentially untouched by plant breeders. Outside of the US, traditional cultivars and local selections still represent a majority of the hazelnuts being established in production orchards today (Bozog˘lu 2005; Tombesi 2005; Tous 2005; Sarraquigne 2005). Nevertheless, over the past several decades much has been learned about hazelnut genetics, biology, and production, and very effective traditional and molecular genetic improvement techniques have been developed (Mehlenbacher 1994; Thompson et al. 1996; Chen et al. 2005; Molnar et al. 2005; Mehlenbacher et al. 2006; Go¨kirmak et al. 2009). While most work has been centered on cultivated forms of C. avellana, the interspecific hybridization potential and genetic diversity of the genus is high, and substantial opportunities exist to utilize wild species in genetic improvement and research efforts (Mehlenbacher 1994; Erdogan and Mehlenbacher 2000a, 2001). In this chapter, the history, current status, and breeding potential of wild Corylus are discussed, prioritizing the need to conserve and better study underutilized wild species. Few surveys of wild Corylus have been made in recent

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_2, # Springer-Verlag Berlin Heidelberg 2011

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years, and overdevelopment in many regions has increased the possibility that some species are experiencing unchecked genetic erosion. One species, C. chinensis Franch., is even considered endangered by the International Union for Conservation of Nature and Natural Resources (Sun 1998).

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self-incompatible and diploid; most researchers agree the chromosome number across the genus is 2n ¼ 2x ¼ 22 (Thompson et al. 1996). Corylus avellana – the European hazelnut of commerce and the most widely studied of the genus – is believed to have a relatively small genome (0.48 pg/1C nucleus, 413 Mbp) (Bennet and Smith 1991), with all other members of the genus unreported.

2.2 Botany of Corylus The Corylus genus is widely distributed across temperate regions of the Northern Hemisphere, with species found in Japan, Korea, and China, through Tibet, India, northern Iran, Turkey, the Caucuses, Europe, and in North America, with none endemic to the Southern Hemisphere (Kasapligil 1972; Thompson et al. 1996). Most taxonomists place Corylus L. in the subfamily Coryloideae of the family Betulaceae, order Fagales (Chen et al. 1999; Yoo and Wen 2002). Corylus comprises anywhere from 9 to 25 species, depending on the authority, with current revisions based on morphological, molecular, and hybridization studies suggesting around ten polymorphic species assigned to four subsections (Thompson et al. 1996; Erdogan 1999; Erdogan and Mehlenbacher 2000a, b). Subsection “Corylus” includes three species whose major similarities include leafy, overlapping involucres (husks) covering the nuts (Corylus avellana, C. americana Marshall, and C. heterophylla Fisch.); subsection “Siphonochlamys” includes three species with tubular, bristle-covered involucres (C. cornuta Marshall, C. californica Marshall, and C. sieboldiana Blume.); subsection “Colurnaea” includes three species that grow as single trunk trees (C. colurna L., C. chinensis, and C. jacqemontii Decne.); and subsection “Acanthochlamys” includes only C. ferox Wall, which has a spiny chestnut-like (Castanea L.) involucre unlike any other species in the genus (Erdogan and Mehlenbacher 2000a, b; Whitcher and Wen 2001). The little-studied paperbark tree species Corylus fargesii (Franch.) C.K. Schneid. (likely syn. C. papyraceae Hickel) has yet to be officially placed in a subsection. Species range in size from small, multi-stemmed bushes (1 m) to large trees (up to 40 m). All members are deciduous, with simple, alternate leaves and monoecious wind pollinated flowers that undergo anthesis before leaves develop in the spring. Plants are

2.2.1 Subsection Corylus Corylus avellana: European hazelnut. Plants are multi-stemmed shrubs 3–10 m tall, with a growth habit ranging from very erect to drooping. Ornamental forms also exist that have weeping or contorted branches. Plants spread by suckers, but the rate and number of suckers produced from the base of the plant varies considerably. Shoots are glandular pubescent and vary in their thickness and branching density. Leaves range from 5–10 cm in length and are elliptic to ovate to rounded in shape, slightly cordate at the base, and have doubly serrate margins. Nuts develop in clusters of 1–12, each separately enclosed in an involucre made up of two overlapping, leafy bracts that vary considerably across the species in terms of the length, constriction around the nut, indentation and serration at the apex, and thickness at the base. Nuts of cultivated forms, which may or may not be released from the involucre at maturity, are by far the largest of the genus, although they vary tremendously in size, shape (from oblate to long and tapered), shell thickness, and percent kernel (ratio of kernel to shell by weight). Commercial production is found near coastal areas of Europe, the Caucasus, Asia Minor, and the Pacific Northwest of the US where the climate is moderated by large bodies of water. However, the native range of C. avellana is quite extensive, spanning northward to nearly 68 N in Norway and 61 N in Finland, eastward through St. Petersburg to 58 N in the Ural Mountains of Russia, and southward to 32 N in Morocco, bounded in the west by the Atlantic Ocean. It typically grows as a common understory shrub and forest edge species in mixed deciduous forests. Most authorities now include the previously named species Corylus maxima Mill., C. pontica Koch., and C. colchica Alb. as members of C. avellana, exemplifying its very diverse and polymorphic

2 Corylus

nature (Mehlenbacher 1991a; Thompson et al. 1996; Erdogan and Mehlenbacher 2000a, b). Corylus avellana was one of the first species to colonize Europe after the last ice age, with pollen records and chloroplast DNA variation studies suggesting expansion from refugia in southwestern France into most of Europe, except for southern Italy and the Balkans, where expansion was from local populations (Palme and Vendramin 2002; Boccacci and Botta 2009). While it is not certain when the domestication of hazelnut began (Zohary and Hopf 2004), Boccacci and Botta (2009, 2010) suggest, based on genetic, historical, and archaeological data, that the species was independently domesticated in the Mediterranean (Spain and Italy), Turkey, and Iran. More than 400 cultivars have been described (G€ urcan et al. 2010). Descriptions were derived from Smolyaninova (1936), Kasapligil (1972), Deacon (1974), Mehlenbacher (1991a), and Thompson et al. (1996). Corylus americana: American hazelnut. Plants are small multi-stemmed shrubs, 1–3 m tall, that spread by abundant suckers. Shoots and leaf petioles are glandular pubescent. Leaves range from 5–16 cm in length and are generally broadly ovate to round in shape with the base rounded or slightly cordate. The leaf apex is acuminate with serrate to doubly serrate leaf margins. Nuts develop in clusters of 2–8 with each nut enclosed in an involucre made up of two overlapping, enlarged, glandular-pubescent leafy bracts that are typically two times the length of the nut, with deep, irregular indentations at the apex. Nuts, which are retained in the involucre at maturity, are round to round-compressed in shape and 1.0–1.5 cm in diameter. Nuts are generally smaller and thickershelled then those of C. avellana but are similar in flavor and quality with variability observed in productivity, size, and other attributes across populations and individual plants. The species is native to a wide part of eastern North America from Saskatchewan and Maine in the northeast to Minnesota and southern Manitoba in the northwest, all the way south to northern Florida and westward to eastern Oklahoma. Plants grow as a forest edge species and along roadsides, fence rows, ravines, and streams, as well as in waste places and tall- and mid-grass prairie habitats. Corylus americana is considered an important wildlife and riparian species that is used as a component of shelterbelts and as an ornamental due to some plants expressing attractive bright red and/or pink fall color.

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Nuts of C. americana have been harvested and used locally from wild plants. Several cultivars producing larger-sized nuts have been selected in the past (see Sect. 2.5.2). Descriptions were derived from Weschcke (1954), Drumke (1964), Duke (1989), Mehlenbacher (1991a), Boufford (1997), and Gleason and Cronquist (1998). Corylus heterophylla: Siberian hazel. Plants are multi-stemmed shrubs 1–3(7) m tall that spread by abundant suckers. Shoots and leaf petioles are glandular pubescent. Leaves range from 4–13 cm in length and are quite variable in shape from elliptic, ellipticobovate, broadly ovate, or obovate to suborbicular. Some plants express two distinctly different leaf shapes with some having an acuminate apex and others an abruptly acuminate apex (truncate) and somewhat bi-lobed leaves with the apex not exceeding the lateral lobes. Leaves are cordate at the base with margins irregularly or doubly serrate. Nuts generally develop in clusters of 1–7 with each enclosed in an involucre made up of two slightly pubescent to densely pubescent glandular, bell-shaped leafy bracts that are normally slightly longer in length than the nut (although some can range from twice the nut length to being equal or shorter than the nut, while others are very short and not well developed). The husk displays deep, irregular indentations at the apex. Nuts, which may or may not be retained in the involucre at maturity, show great variability in size, shape, and shell thickness, but are generally round to ovoid and 0.7–1.5 cm in diameter. The species is native to a large area of Korea, Japan, China, eastern Mongolia, and the Russian Far East where it grows as an understory shrub in open forests, on forest edges, on deforested hills, in dry river valleys, and in vast thickets on mountain slopes. The nuts of C. heterophylla are regularly harvested from the wild and sold in domestic markets for food and oil, with some cultivars and interspecific hybrids selected and grown commercially in China and Korea. Two botanical varieties are recognized that have a more southerly distribution in China, which are considered separate species by some authorities. Corylus heterophylla var. sutchuensis (syn. C. kweichowensis Hu) grows 3–7 m tall and is distributed throughout the Shangxi, Sichang, Hubei, Hunan, Jiangxi, Zhejiang, and Guizhou provinces. Corylus heterophylla var. yunnanesis Franch., which can be found growing in high density in some areas, grows 1–3(5) m tall primarily in the Yunnan and

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Sichuan provinces. Descriptions were derived from Smolyaninova (1936), Kasapligil (1972), Mehlenbacher (1991a), and eFloras (2009).

2.2.2 Subsection Siphonochlamys Corylus cornuta: Beaked hazel. Plants are multistemmed shrubs 1–3 m tall, which spread by suckers and abundant below ground stolons. Shoots and petioles can be glabrous to pubescent, but nonglandular. Leaves range from 5–12 cm in length and are ovate to obovate or narrowly elliptic in shape, with an acuminate apex. They are slightly cordate at the base with margins irregularly doubly serrate. Nuts develop in clusters of 1–6, each tightly enclosed in a tube-like involucre (beak) that is constricted beyond the nut, measures 2–4 times its length, and is densely covered with bristly, irritating hairs. The nuts are retained in the involucre at maturity and are typically 1.0–1.5 cm in diameter, very thick shelled, and ovoid. The species is native to a broad section of North America, farther north and west than C. americana, although their ranges overlap significantly. It can be found in the northeast from Newfoundland and New Brunswick, Canada, through Maine to South Carolina and the mountains of Georgia in the southeast, and west to Alabama. In its northern range, it can be found west of New Brunswick, through southern Canada, across the upper Midwest US states, and north into Manitoba, Saskatchewan, and Alberta. It grows as an understory plant in open woodlands and clearings, as well as along moist to dry roadsides, at the edges of woods, and along streams, often at higher elevations. It can sometimes form very dense thickets, due to its stoloniferous habit, and has the ability to re-grow after forest fires. Corylus cornuta is not widely harvested or cultivated for its small, thick-shelled nuts, although several interspecific hybrids have been developed in attempts to access its extreme cold hardiness. Descriptions were derived from Buckman (1964), Drumke (1964), Mehlenbacher (1991a, 2003), Boufford (1997), and Gleason and Cronquist (1998). Corylus californica (syn. C. cornuta var. californica). Plants are multi-stemmed shrubs 3–4 m tall that spread by suckers, lacking the stolons of C. cornuta. Young shoots and petioles are typically glandular pubescent, although glabrous in maturity. Leaves range from 4 to

T.J. Molnar

7 cm in length and are rounded or obovate to broadly elliptic, with an obtuse to acute apex. Leaves are typically more cordate at the base than C. cornuta, with margins coarsely doubly serrate. Nuts develop in clusters of 2–4, with each tightly enclosed in a tubelike bristly involucre that is constricted beyond the nut and is generally two times its length or shorter. The nuts are usually larger than those of C. cornuta and are retained in the involucre at maturity. The species is native to western North America, from southern British Columbia southward through western Washington, Oregon, and central California. Plants are generally found along streams, on damp rocky slopes, and in cool canyons in the coastal mountain ranges. Corylus californica is not widely harvested for its nuts. Descriptions were derived from Drumke (1964), Mehlenbacher (1991a), and Boufford (1997). Corylus sieboldiana (syn. C. mandshurica Maxim.). Plants are multi-stemmed shrubs, 2–6 m tall that spread by suckers. Young shoots and petioles are glandular pubescent. Leaves range from 5–12 cm in length and are round, broadly ovate, oblong, or oblong-obovate, with a mucronate-acuminate or caudate apex and cordate base. Leaf margins are dentate to irregularly and coarsely serrate. Nuts develop in clusters of 1–9 with each tightly enclosed in a tube-like bristly involucre that is constricted beyond the nut and is generally two times its length or longer. Nuts are typically small, thick-shelled, ovoid-globose, and pointed, with some having thin shells up to 1.5 cm in diameter. They are retained in the involucre at maturity. The species is native to Korea, Japan, northern China, and the Russian Far East (Primorsky and Khabarovsky regions), where it significantly overlaps the range of C. heterophylla but is much less abundant. It occurs in forest areas with moist, fertile soil high in organic matter. Nuts of C. sieboldiana are harvested from the wild, although harvesting is complicated by the bristly involucres. Descriptions were derived from Smolyaninova (1936), Kudasheva (1965), Mehlenbacher (1991a), and eFloras (2009).

2.2.3 Subsection Colurnaea Corylus colurna: Turkish tree hazel. Plants are large single trunk, pyramidal trees, 20–40 m tall, with trunk diameters ranging from 30–60(120) cm. Young

2 Corylus

shoots are glandular pubescent, with distinctive corky and furrowed bark that is light-gray to gray in color. Petioles are slightly pubescent glandular although sometimes glabrous. Leaves range from 7–18 cm in length and are round, oval, ovate, obovate, wideelliptical, to slightly lobed in shape with an acute apex and a cordate to deeply cordate base. Leaf margins are dentate to doubly serrate. Nuts develop in clusters of 2–10 with each enclosed in a fleshy, glandular-pubescent involucre that is 2–3 times longer than the nut, open at the apex, and deeply dissected almost to its base into numerous long-acuminate lobes. Nuts are ovoid-globose to nearly round to flat-compressed, sometimes extended-elliptical or angular and 1.0–1.5 cm in diameter, with thick shells that are connected strongly to the involucre at maturity, although selections have been described that release readily. The species is native to the Balkan Peninsula, Turkey, the Caucuses, and northern Iran, growing as scattered trees in deciduous and mixed coniferous forests. In the Caucuses, it can be found 840–1,750 m above sea level in shady, moist deciduous forests with soils high in organic matter. Nuts are harvested from the wild and used and sold locally. However, the species has been more widely used as a source of high quality timber for construction of buildings, boats, and furniture. It is grown as a low maintenance shade tree in Europe and the US and has also been used as a nonsuckering rootstock for C. avellana (with limited use today) for nut production and for ornamental Corylus, such as the contorted form C. avellana “Harry Lauder’s Walking Stick” and the weeping form C. avellana “Pendula”. Descriptions were derived from Smolyaninova (1936), Kudasheva (1965), Kasapligil (1972), Duke (1989), and Mehlenbacher (1991a, 2003). Corylus jacquemontii: Indian tree hazel. Plants are single trunk trees 12–15 m tall. Bark is thinner and less corky than C. colurna. Leaves range from 15–24 cm in length and are obovate in shape, with the base shallowly lobed and margins sharply serrate. Nuts develop in clusters of 1–5 in involucres up to three times the length of the nut, similar in appearance to C. colurna but less fleshy with non-glandular pubescence. The small (up to 1.5 cm), thick-shelled nuts are generally more easy to remove from the involucre than C. colurna. The species is distributed across Northeast Afghanistan, northern India, northern Pakistan, and western Nepal at 1,900–3,000 m above sea level.

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Nuts are harvested from the wild and sold in local markets. Descriptions were derived from Mehlenbacher (1991a, 2003), Thompson et al. (1996), and Farris (2000). Corylus chinensis: Chinese tree hazel. Plants are large, single-trunk trees, 20–40 m tall, with trunk diameters up to 2 m. Young shoots and petioles are sparsely villous, stipitate glandular to glabrescent. Bark is considerably thinner and smoother than C. colurna. Leaves range from 8–18 cm in length and are ovate, ovate-elliptic, or obovate-elliptic in shape, with an obliquely cordate base and mucronate or shortly caudate apex. Leaf margins are irregularly and doubly serrate. Nuts develop in clusters of 2–12, each in a fleshy, tube-like pubescent to glabrous involucre longer than the nut and tightly constricted after the nut with a forked or toothed apex. Nuts are ovoidglobose and 1.0–1.5 cm in diameter with thick shells that are strongly attached to the involucre at maturity. The species is distributed across southern China in parts of the Shangxi, Sichuan, Hubei, Hunan, Yunnan, and Guizhou provinces where it is found as scattered trees on moist, forested mountain slopes. Its timber has been used in China for furniture and paneling. The species is now considered threatened due to its scarcity (Sun 1998). Descriptions were derived from Duke (1989), Mehlenbacher (1991a), and eFloras 2009. Corylus fargesii: Paperbark tree hazel. Plants are single-trunk trees up to 25 m tall. Young shoots and petioles are pubescent. The peeling bark of older stems and the trunk is similar to that of river birch (Betula nigra L.), which is a major distinguishing characteristic separating the species from C. chinensis. Leaves range from 6–9 cm in length and are oblong-lanceolate, obovate-oblong, or lanceolate in shape, with the base cordate or sub-rounded and the apex acuminate. Leaf margins are coarsely and irregularly doubly serrate. Nuts develop in clusters of 2–4 each enclosed in a tubular involucre that is tightly constricted after the nut and measuring 2.0–5.0 cm long, with its apex divided into triangular-lanceolate lobes. Nuts are ovoidglobose and 1.0–1.5 cm in diameter. The species is distributed throughout mountain valleys of the Henan, Sichuan, Hubei, Shangxi, Gangsu, and Guizhou provinces of China. Descriptions were derived from Aiello and Dillard (2007), Farris (2000), and eFloras (2009). Corylus fargesii has not been officially placed in subsection Colurnaea due to poor representation in taxonomic studies. However, morphological

20

examination of recent introductions in the US suggests its likely inclusion. Also, C. fargesii may be a synonym for C. papyraceae Hickel; further investigation is needed to clarify its taxonomic position.

2.2.4 Subsection Acanthochlamys Corylus ferox: Tibetan hazel and Himalayan tree hazel. Plants are single-trunk trees 6–9 (20) m tall. Young shoots are pubescent, sometimes stipitate glandular or glabrescent. Petioles are densely pilose when young, glabrescent later. Leaves are 5–15 cm long and are ovate-oblong, obovate-oblong, obovate, or elliptic in shape, with the base obliquely rounded or subcordate and the apex long acuminate to caudate-acuminate. Leaf margins are sharply and doubly mucronate serrate. Nuts develop in clusters of 3–6 in spiny, cup-shaped involucres unlike any of the other Corylus species. The clusters are very similar to spiny chestnut (Castanea L.) burrs. Nuts are ovoid-globose to slightly compressed and 1.0–1.5 cm in diameter. The species is native to forested mountain slopes 1,700–3,800 m above sea level in the eastern Himalayan Mountains from Bhutan, Northeast India, northern Myanmar, and Nepal to parts of the Yunnan, Sichang, and Xizang provinces of China. The botanical variety, C. ferox var. thibetica (Batal.) Franch., is recognized to differ from C. ferox by having less spines (or pubescence) on the base of the involucre and lacking pubescence on vegetative buds and young shoots. It is found in the Gansu, Guizhou, Hubei, Ningxia, Shaanxi, Sichuan, Xizang, and Yunnan provinces of China in mixed forests at 1,500–3,600 m above sea level. Descriptions were derived from Kasapligil (1972), Mehlenbacher (1991a), Thompson et al. (1996), Farris (2000), and eFloras (2009).

2.3 Conservation of Wild Corylus Genetic Resources Essentially no work has been done to investigate population structure, genetic diversity, and possible genetic erosion (loss of genetic resources) of wild Corylus species. Nearly all efforts have been focused on cultivated forms of C. avellana largely to better

T.J. Molnar

understand their origin, to fingerprint germplasm accessions, and to evaluate genetic diversity present in collections (Bassil et al. 2005a, b, 2009; Boccacci et al. 2006, 2008; Boccacci and Botta 2009, 2010; Go¨kirmak et al. 2009; G€urcan et al. 2010). The most recent survey of Corylus in North America was conducted by Drumke (1964), though extensive land development since this time limits the usefulness of this work. Sun (1998) reported that C. chinensis was becoming scarce in China, leading to its threatened status. It is possible that genetic resources of other Corylus species are in danger of being lost, especially in highly populated countries or regions that have undergone widespread deforestation.

2.3.1 World Germplasm Collections In general, world Corylus germplasm collections consist primarily of cultivated forms of C. avellana and are located in regions where production occurs. Major collections include the US Department of Agriculture (USDA), Agriculture Research Service (ARS), National Clonal Germplasm Repository (NCGR) in Oregon, US, with a backup collection at Parlier, California (Hummer 2001; Bassil et al. 2009); the Hazelnut Research Institute in Giresun, Turkey; Institut de Recerca ˙I Technologia Agroalimenta`ries (IRTA) in Reus, Spain; Institut National de la Recherche Agronomique (INRA) in Ponte-de-laMaye, France; the University of Torino and the Institute Sperimentale per Frutticoltura in Italy; the (VIR) Breeding Station, Maykop, and the Russian Academy of Agricultural Sciences Institute of Floriculture and Subtropical Cultures, Sochi, Russia; and the Nikita Botanical Gardens in Yalta, Ukraine. These and a number of smaller collections are listed in Mehlenbacher (1991a), Koksal (2000), and Bacchetta et al. (2009). Throughout nearly all of these collections, wild Corylus species are very poorly represented, including uncultivated forms of C. avellana from across its native range. In recent years, the NCGR and Oregon State University have increased efforts to collect cultivated and wild accession of Corylus, and their collections now total more than 700 accessions between them representing all major Corylus species (G€urcan et al. 2010); however, a number of species are still lacking, especially when considering their wide

2 Corylus

21

Table 2.1 Species accessions held at the US Dept of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository in Corvallis, Oregon as of July 2010 (NCGR 2010). Subspecies and botanical varieties are included under the species headings Species Number of accessions Countries of origin Corylus americana 45 2 Corylus avellana 464 32 Corylus californica 49 1 Corylus chinensis 12 4 Corylus colurna 21 12 Corylus cornuta 19 2 Corylus fargesii 8 3 Corylus ferox 5 2 Corylus heterophylla 63 5 Corylus jacquemontii 6 4 Corylus sieboldiana 42 6

geographic range. Continued collection and evaluation efforts are still needed (Table 2.1). In addition to specifically designated Corylus collections, many botanical gardens and arboreta around the world hold specimens of the genus. These specimens represent a valuable, yet largely untapped resource. Unfortunately, some trees in these settings appear to be mislabeled as to species or cultivar, and diversity across institutions may be limited due to direct sharing of germplasm and/or collaborative collection expeditions. Efforts are needed to identify, better characterize, and catalog Corylus plants existing in these settings to make them available for conservation, research, and breeding efforts. In the US, small but diverse collections can be found at the Morris Arboretum in Pennsylvania, the Brooklyn Botanical Garden in New York, the Dawes and Holden Arboreta in Ohio, the Arnold Arboretum in Massachusetts, the Morton Arboretum in Illinois, and likely others, both public and private.

2.3.2 Germplasm Preservation Because Corylus seeds cannot be stored much longer than one year without losing viability, germplasm preservation is based on tree accessions grown in collection orchards. The expense of maintaining these orchards can be prohibitive, as exemplified by the land needed to grow trees of C. colurna and C. chinensis, which can reach well over 20 m tall. While a useful means to capture and preserve the genetic diversity of wild species is as trees derived from seeds of local origins,

the outcrossing, highly heterozygous nature of Corylus requires selected genotypes (cultivars) be propagated by asexual methods to retain their genetic identity. Clonal propagation adds to the expense and challenge of obtaining, accurately maintaining, and distributing Corylus germplasm. This has been traditionally accomplished by layering, an effective yet inefficient practice, and to a lesser extent grafting. Grafting presents problems due to the suckering growth habit of the rootstocks typically used, excluding C. colurna, which has been used but is not widely available, partly due to its slow seed germination and poor ability to be transplanted in a nursery setting (compared to C. avellana). This problem is especially acute in germplasm collection settings where rootstock shoots can be confused with the original scion accession. Also, grafted plants do not have the capability to re-grow after mechanical injury and disease, like those on their own roots. A micropropagation system for hazelnuts was recently developed that is effective for C. avellana, as well as interspecific hybrids of C. avellana with C. colurna, C. americana, and C. heterophylla (Yu and Reed 1995; Nas and Read 2004; Bacchetta et al. 2008; Gao et al. 2008). Micropropagation allows for very efficient clonal propagation and it is currently being used by commercial nurseries in the US. Since plants are on their own roots, they provide the same benefits as layered plants, but through a much more efficient propagation system. Micropropagation also provides a means for space-efficient in vitro preservation of germplasm. Currently, 70 accessions are backed up at the NCGR in the form of tissue culture plantlets, consisting mostly of economically important

22

C. avellana cultivars and C. avellana selections from the wild, with only a few other Corylus species available at present (Joseph Postman, personal communication). The aseptic process of tissue culture can also provide essentially disease- and virus-free plant material, which can aid in meeting quarantine regulations to facilitate sharing of genetic resources and improved cultivars. For long-term germplasm preservation, a method to store hazelnut embryonic axes in liquid nitrogen was developed. Excised, dehydrated embryos of hazelnut seeds previously treated to a period of moist-chilling (stratification) survived freezing in liquid nitrogen to be thawed and grown successfully in tissue culture (Normah et al. 1994; Reed et al. 1994). Based on this work, embryonic axes of seeds from C. americana, C. colurna, C. heterophylla, and C. sieboldiana were cryo-preserved in liquid nitrogen and are stored at the National Seed Storage Laboratory in Fort Collins, Colorado (Reed and Hummer 2001). Cryopreservation of Corylus pollen is also possible (Craddock 1987), and pollen of 53 C. avellana cultivars has been preserved in liquid nitrogen for long-term storage at the NCGR. A clonal cryo-preservation technique is still needed, however. Research is underway at the NCGR to develop a method to preserve dormant vegetative buds. A major challenge to developing the clonal cryopreservation of hazelnuts has been grafting the buds, once thawed, to successfully regenerate new plants (Joseph Postman, personal communication, 2009).

2.4 Genetic Studies and Genomic Resources of Wild Corylus Similar to the germplasm collection efforts discussed in the previous section, nearly all Corylus research – including genetic studies and genomic resources – has been focused on C. avellana. These advances have increased breeding efficiency and contributed to knowledge of pollen-stigma incompatibility (Mehlenbacher 1997), as well as helping to clarify the genetic control of many traits that should prove applicable to future studies and improvement efforts utilizing wild Corylus and interspecific hybrids. The inheritance of qualitative traits characterized for C. avellana include red leaf color (Thompson 1985), chlorophyll deficiency (Mehlenbacher and Thompson

T.J. Molnar

1991), cut-leaf habit (Mehlenbacher and Smith 1995), pollen color (Mehlenbacher and Smith 2002), style color (Mehlenbacher and Thompson 2004), contorted growth habit (Smith and Mehlenbacher 1996), non-dormancy (Thompson et al. 1985), self-compatibility (Mehlenbacher and Smith 2001, 2006), and resistance to eastern filbert blight (EFB), a destructive fungal disease caused by Anisogramma anomala Peck E. M€uller that has severely limited hazelnut cultivation in eastern North America (Fuller 1908; Thompson et al. 1996), from C. avellana “Gasaway”, “Zimmerman”, “Ratoli”, OSU 408.040, and OSU 759.010 (Mehlenbacher et al. 1991; Chen et al. 2005; Lunde et al. 2006; Sathuvalli 2007). The inheritance of numerous quantitative traits has also been examined, which should prove applicable in interspecific improvement efforts. These include EFB resistance (Coyne et al. 1998, 2000), bud mite resistance (Thompson 1977a), pellicle removal (Mehlenbacher and Smith 1988), nut and kernel defects (Mehlenbacher et al. 1993), and other morphological and developmental characteristics related to commercial nut production (Thompson 1977b; Yao and Mehlenbacher 2000). Recent studies have used sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region, 5S rRNA, chloroplast matK, and ribulosebisphosphate carboxylase (rbcL) gene regions to discern phylogenic relationships in Corylus and within the Betulaceae family (Chen et al. 1999; Erdogan and Mehlenbacher 2000b; Forest and Bruneau 2000; Whitcher and Wen 2001). Sequences of these genes for most wild species are accessioned and available through GenBank (2010); however, the numbers of genotypes from which the sequence data have been derived are limited. While these conserved gene regions are useful to distinguish between species, they tend to provide low intraspecific resolution, which limits their use in population studies. Fortunately, microsatellite or simple sequence repeat (SSR) markers that provide a higher level of resolution have recently been developed and used to successfully study genetic diversity and relationships of cultivated C. avellana (Bassil et al. 2005a, b; Boccacci et al. 2006; Go¨kirmak et al. 2009), with many markers (about 200) placed on a saturated genetic linkage map (Mehlenbacher et al. 2006; Mehlenbacher 2009; G€urcan et al. 2010). Bassil et al. (2005b) showed some SSR markers developed for C. avellana provide cross amplification in C. americana, C. heterophylla,

2 Corylus

C. chinensis, C. colurna, and C. californica, although no wild Corylus genetic diversity studies have yet to be completed. Work, however, is underway at Oregon State University, in Corvallis, Oregon, to assess the genetic diversity of C. americana accessions held in their collection, the NCGR, and other locations in the US, using SSR markers (Shawn Mehlenbacher, personal communication). Hopefully the development of SSR markers will open doors for similar studies with additional Corylus species in the near future. Marker-assisted selection (MAS) techniques have been developed to identify seedlings carrying the “Gasaway” single dominant gene for EFB resistance, as well as resistance genes from “Ratoli”, OSU 408.040, and OSU 759.010, using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), and other PCR-based DNA marker systems (Mehlenbacher et al. 2004; Chen et al. 2005; Sathuvalli et al. 2009). These tools greatly increase the efficiency of breeding for resistance and should be useful for advanced-generation interspecific hybrids derived from these breeding lines. In addition, MAS has been developed for identifying desired sporophytic self-incompatibly S-allele genotypes, although to date RAPD markers have only been identified linked to the S1 and S2 alleles (Pomper et al. 1998). With further development, this system will allow breeders to identify S-alleles at a much earlier stage, saving valuable time and resources that are currently expended on growing seedlings until they flower to test incompatibility reactions against known tester genotypes using florescence microscopy (Mehlenbacher 1997). Very recent work involves the development of a bacterial artificial chromosome (BAC) library for the new C. avellana release “Jefferson” (OSU 703.007), which is intended to be used for map-based cloning of the “Gasaway” EFB-resistance gene and the sporophytic incompatibility locus (Mehlenbacher 2009; Sathuvalli and Mehlenbacher 2009). In addition, efforts are in place to begin sequencing of the genome of C. avellana (S. Mehlenbacher, personal communication, 2010). Once complete, these critical advances will provide great opportunities to improve the understanding, utilization, and conservation of Corylus genetic resources.

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2.5 Role of Wild Corylus in Crop Improvement Plants of Corylus (despite its self-incompatibility system) are typically very amenable to breeding compared to nearly all of the other temperate nut crops of world importance. Generation times are shorter (3–5 years to maturity), the plant size is smaller in stature, female flowers stay receptive to pollen for several weeks or longer, flowers are easy to isolate from foreign pollen, pollen is readily collected and stored for up to one year, and hand pollinations can yield large numbers of hybrid seeds (Thompson et al. 1996). All Corylus species produce edible nuts of relatively similar quality, although size, shell thickness, amount of fiber on the pellicle, and other characteristics may vary. Furthermore, the interspecific hybridization potential within Corylus is high, as discussed by Mehlenbacher (1991a), Thompson et al. (1996), and Erdogan and Mehlenbacher (2000a). In general, successful hybrids can be readily created between members of the same subsection (Corylus, Siphonochlamys, and Colurnaea), with crosses between the different subsections more limited, but in many cases, also possible. It should be noted that reciprocal differences have been observed in many crosses, and crosses with C. avellana have been generally more successful when the species was used as the staminate (pollen) parent. Interestingly, when C. californica was used as a pistillate parent, it was compatible with all other Corylus species tested, suggesting that it may have value as a bridge species (Erdogan and Mehlenbacher 2000a). Erdogan and Mehlenbacher (2001) also suggest that sporophytic incompatibility may exist in wild Corylus species, similar to that reported for C. avellana, where incompatibility is controlled by a single S-locus with multiple alleles (Thompson 1979; Mehlenbacher 1997), although many undetermined S-alleles and the presence of other barriers to hybridization appear to be involved. While further work is needed to clarify the incompatibility system in wild Corylus, many desirable and economically useful characteristics are present that can be accessed through interspecific hybridization in wild species, which are lacking in cultivated C. avellana. Traits of interest include

24

non-suckering growth habits, tolerance of alkaline soils, extreme precocity, early maturing nuts, extreme cold hardiness, drought tolerance, attractive fall color and other ornamental attributes, rare incompatibility alleles, and novel sources of resistance to EFB. Most genetic improvement efforts will likely continue to be centered on cultivated forms of C. avellana due to its superior nut quality, yield, and other commercial production characteristics, with wild species used as donor parents to contribute desired traits by following some form of a modified backcross program. A number of examples are discussed later in this section where interspecific hybrids, sometimes difficult to create (as in C. colurna  C. avellana), are more easily backcrossed to either parental species, suggesting barriers to hybridization are reduced in advancedgeneration hybrids. Table 2.2 presents current breeding objectives and standards of C. avellana seedling evaluation in the OSU hazelnut breeding program, which is the largest and longest running hazelnut breeding program in the world (Mehlenbacher 2009). This table provides minimum selection criteria of seedlings that are necessary to develop improved cultivars to meet the demands of the international kernel market (Mehlenbacher 2003). Hybrid hazelnuts developed for production in new regions will not only require enhanced adaptation capabilities, including resistance to local pests and diseases – especially EFB if grown in North America – but they will also need to meet kernel quality standards to be competitive in the world market. Nearly all genetic studies of hazelnut have

T.J. Molnar

centered on cultivated C. avellana. As such, efforts are needed to characterize and better understand the inheritance of traits from wild species to enhance the efficiency of interspecific breeding efforts. It is especially important to recognize that the use of wild species in breeding may be accompanied by varying levels of undesirable traits, some which are unstudied and may be linked to characteristics of interest. Fortunately, a number of first-generation hybrids exist from early breeding efforts, as discussed below, which should prove useful in breeding more widely adapted cultivars. However, to maintain and enhance genetic diversity, breeders will also need to make additional first generation interspecific hybrids to widen the germplasm base and to better exploit the potential of Corylus. To be most effective, much more extensive collection of wild germplasm – and subsequent evaluation of these collections – must be made to further understand and best use existing genetic variability. Table 2.3 presents desirable breeding attributes, potential limitations, and known interspecific compatibilities of the 11 most widely recognized Corylus species. Their history of use, if available, and a thorough description of their possible roles in genetic improvement are discussed below.

2.5.1 Corylus avellana Due to its outcrossing, highly heterozygous nature, substantial genetic diversity can be found in the pool of existing C. avellana cultivars. This wide diversity,

Table 2.2 Objectives and standards of seedling selection in the Oregon State University hazelnut (Corylus avellana) breeding program, adapted from Mehlenbacher (2003). In addition to these requirements, all plants developed for North America should express a very high level of resistance to infection by Anisogramma anomala, which causes eastern filbert blight. For many locations, cold hardiness of plants, especially the male flowers (catkins), is also vital Objective Age at evaluation Minimum standard (cultivar) Ideal standard (cultivar) Bud mite resistance 4 ‘Casina’, ‘Clark’ (moderate resistance) ‘Barcelona’ (highly resistant) Round nut shape 4–5 ‘Tonda Gentile delle Langhe’ ‘Willamette’ High percent kernel 4–5 ‘Tonda Romana’ (48%) Casina (56%) Precocity 5 At least 35 nuts produced – Yield (total, consistency) 4–16 ‘Barcelona’ ‘Lewis’ Kernel blanching 5–8 ‘Barcelona’ ‘Negret’ Few nut and kernel defects 4–16 ‘Barcelona’ – Early maturity 5–8 ‘Barcelona’ ‘Tonda Gentile delle Langhe’ Free-falling nuts at harvest 5–8 ‘Casina’ (70% are free falling) ‘Barcelona’ (95% are free falling)

Small, thick-shelled nuts Late maturing nuts Cold sensitive Husk retains nut at maturity Involucres covered with irritating hairs Suckering growth habit Stoloniferous growth habita Susceptible to EFB Not precocious Limited germplasm

C. avellana C. americana C. heterophylla C. cornuta C. californica C. sieboldiana C. colurna

Possible limitations

Compatibility (as female parent)

•• •• • ¤ •• ¤ ••

••

•

•

•• ••

•• ••

••

••

• •

•

•• • •• •• ••

•

••

•

•

•• •• ••

•• •

• ••

••

•• •• •• •

•

••

• • •

•

•• •• ••

•

•

••

• ••

•• •

••

••

•

¤ • •

•

•

? ? ? ? ? ? ?

? • ••

•

••

••

•

• ••

•• • • ••

? ? ? ? ? ? ?

• ••

• ••

••

? ? ? ? ? ? ? (continued)

••

?

• •

•

Table 2.3 Summary of positive breeding attributes, possible limitations, and hybridization potential of the eleven most widely recognized Corylus species. Compatibility is based on Erdogan and Mehlenbacher (2000a) and Thompson et al. (1996) C. C. C. C. C. C. C. C. C. C. C. avellana americana heterophylla cornuta californica sieboldiana colurna jacquemontii chinensis fargesii ferox Positive attributes Diversity of cultivated forms •• Large nut and kernel size •• Early maturing nuts • •• •• High yield potential/ •• • • productive selections Cold hardiness • •• •• •• •• •• Drought tolerance • • • • • Heat tolerance • • • Small growth habit for high • • • density planting • Stoloniferous habita Non-suckering tree form • • • • Releases nuts from •• • involucres Precocious (produce nuts • •• at young age) Ornamental attributesb • • • • • • Resistance to EFB • •• ¤ • • ¤ • ? ¤ ¤ ?

2 Corylus 25

C. jacquemontii C. chinensis C. fargesii C. ferox

C. avellana ? •• ? ?

C. americana ? •• ? ?

C. C. C. C. C. heterophylla cornuta californica sieboldiana colurna ? ? ? ? ? •• ? ? ? ? ? ? ? ? ? ?

C. jacquemontii •• ? ? ?

C. chinensis ? •• ? ?

C. fargesii ? ? •• ?

C. ferox ? ? ? ••

• Characteristic observed (cross is compatible) •• Characteristic very prominent (high level of compatibility) ¤ Characteristic observed on rare occasion but more evaluation needed (compatibility reported but not regular) ? Unknown, has not been evaluated a Stoloniferous growth habit is listed as both a positive attribute and a potential limitation, as it can be useful for soil reclamation and planting on marginal sites, but is not advantageous for commercial production b Ornamental attributes vary between species. C. avellana: contorted and weeping stems, red/purple or yellow leaves, dissected leaves; C. americana: pink/red fall color, frilly involucres, small growth habit; C. heterophylla: lobed and truncated leaf habits; C. fargesii: attractive peeling bark

Table 2.3 (continued)

26 T.J. Molnar

2 Corylus

expressed as morphological, phenological, and DNA sequence variability, has been discussed by numerous authors, including more recently Mehlenbacher (1991a, b, 1997), Thompson et al. (1996), Erdogan (1999), Boccacci and Botta (2009, 2010), Boccacci et al. (2006, 2008), Biodiversity et al. (2008), Go¨kirmak et al. (2009), and G€ urcan et al. (2010). World germplasm collections hold numerous cultivars, many of which are well characterized and, in recent years, have become more readily available for use in breeding and research efforts (Bacchetta et al. 2009; NCGR 2010). Access to rapid international mail services makes it possible to share pollen and scion wood to facilitate germplasm exchange and breeding efforts between and within many countries. In addition, efficient breeding techniques have been developed (Thompson et al. 1996) and many traits vital to improved nut quality and nut production, such as percent kernel, kernel weight and nuts per cluster, have been shown to be moderately to highly heritable, as discussed by Thompson (1977b), Mehlenbacher (1991a), Thompson et al. (1996), Mehlenbacher et al. (1993) and Yao and Mehlenbacher (2000). These facts, in combination with the paucity of modern breeding efforts and wide genetic diversity of Corylus, have set the stage for rapid gains in the genetic improvement of hazelnut, which is exemplified by several new EFBresistant cultivars released from the Oregon State University breeding program. These improved cultivars, which are the result of systematic breeding efforts over the past 30 years, will save the US hazelnut industry considerable production costs due to their significantly reduced fungicide requirements and other disease management constraints (Julian et al. 2009a), and increased yields of high-quality kernels per hectare, compared to the traditional standard in Oregon, “Barcelona” (Mehlenbacher et al. 2007, 2008, 2009). While cultivated forms of C. avellana typically express better nut quality and production characteristics than their wild counterparts, most are not reliably yielding outside of Mediterranean-like climates. Many lack cold hardiness, especially of staminate flowers, as well as other traits necessary for wide adaptation and consistent yields of nuts. Breeders will need to search for genetic resources outside of cultivated forms when intending to significantly expand the regions where hazelnuts can be produced commercially. Fortunately, cold-hardy, wild C. avellana exist that grow as far north as coastal Scandinavia, as well as in the Ural

27

Mountains of Russia, the Carpathian Mountains of Poland, and other inland areas of Europe with continental climates periodically exposed to extreme temperatures. Through intraspecific hybridization, these cold-hardy wild forms represent a plausible means to substantially improve the climatic adaptability of cultivated hazelnuts. Select plants should provide a more rapid means to achieve this goal then by using other Corylus species. This is because, barring specific incompatibility alleles, wild C. avellana is fully compatible with the cultivated forms, which would allow for larger numbers of hybrid seed from controlled crosses, and the resulting progeny would be fully fertile, unlike some interspecific hybrids (Erdogan and Mehlenbacher 2000a). In addition, wild C. avellana exist that express traits amenable to commercial production that are not widely found in other Corylus species, such as short involucres that release the nuts on maturity, thin shells, high percent kernel, and upright growth habits. Other traits of C. avellana worthy of exploration include resistance to diseases and pests, such EFB and big bud mite (Phytoptus avellanae Nal. and Cecidophyopsis vermiformis Nal.) (Thompson et al. 1996; Lunde et al. 2000; Molnar et al. 2007; Chen et al. 2007; Sathuvalli et al. 2010). Unfortunately, wild C. avellana is poorly represented in world germplasm collections, especially from its most northern range. Collection and evaluation efforts remain necessary to access its full potential in breeding. The first breeding work using wild C. avellana to develop cultivated hazelnuts for colder regions began in the early 1900s. The most notable was done in the Michurinsk and Moscow provinces of Russia, as discussed below, with minor efforts made in Ukraine (Slyusarchuk and Ryabokon 2001, 2005), Belarus (Volovich and Chripach 1998), and Estonia (Kask 1998, 2001). Commercial hazelnut production is currently limited to southern regions of the former Soviet Union near Sochi, Russia, the Caucasus Mountains along the Black Sea, and southern Crimea, Ukraine.

2.5.1.1 I.V. Michurin In the early 1900s, under the direction of the famous Russian plant breeder I.V. Michurin, attempts at developing more cold-hardy hazelnuts were started at an independent genetics lab in the Tambov province of

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Russia, which was then part of the Agricultural Academy of Science of the Soviet Union (the current name is the All Russian Scientific Research Institute of Genetics and Breeding of Fruit Species named after I.V. Michurin). Michurin and his colleagues I.S. Gorshkov and S.K. Chaplyaev hybridized southern cultivars with cold-hardy wild C. avellana from the Tambov region of Russia, with goals of developing plants that combined the high quality and large nut size of the southern cultivars with the cold hardiness of local hazelnuts (Pavlenko 1957). The southern cultivars “Adygeisky” and “Panakhesky”, which were widely grown in the Krasnodar region of Russia at the time, were used as pistillate parents for the first generation hybrids with the northern C. avellana. From these crosses, they planted a reported 200,000 hybrid seedlings for evaluation at the breeding station in Michurinsk (Denisova 1975). They assessed this very large population for cold hardiness, nut yield, and kernel quality, and selected only cold-hardy plants that produced nuts with more than 40% kernel by weight for further evaluation. Only 1% of this large population met these criteria and these plants were then used to create a second generation of progeny with breeding objectives to combine yield and cold hardiness with resistance to weevils (likely Curculio nucum L). The southern cultivars “Gigantsky Gallsky” and “Barcelona” were used as recurrent parents for this second generation of hybrids. Significant improvement was reported from this generation, as 20% of the progeny expressed desirable characteristics. From this work, Pavlenko (1957) described ten selections that were productive in regions where temperatures drop to 34 C. In the years that followed, a third generation of seedlings was grown, largely from open-pollinated seed collected from improved selections, with 25% of the resulting plants expressing improved nut characteristics (Denisova 1975). Breeders eventually selected 53 plants that expressed improved cold hardiness and very consistent annual yields. However, even in these advanced generations, it remained a challenge to obtain high yields. Denisova (1975) describes the top two selections from the Michurinsk program as very cold hardy, productive, and pest resistant: Selection 4–24, derived from the open-pollination of an unreported parent, has nuts that are 22  15  15 mm and 45% kernel, which is 53.2% oil by weight; Selection 5–10, also derived from the open-pollination of an unreported parent, has nuts that are 20  13  12 mm

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and 50% kernel, which is 63.2% oil by weight. While breeding work has been terminated, the Michurinsk Institute hazelnut collection currently holds more than 50 cold-hardy cultivars and forms (Director N.I. Savelev, personal communication, 3 July 2009). Breeding selections from the Michurinsk breeding efforts, although not widely available, represent valuable genetic resources inherently useful for continuing efforts to develop further improved cold-hardy cultivated hazelnuts.

2.5.1.2 A.S. Yablokov A program similar to the one in Michurinsk was started in the 1930s by A.S Yablokov at the All Union Scientific Research Institute of Forestry and Mechanization in Moscow province (now called the All Russian Scientific Research Institute of Forestry and Mechanization). After unsuccessful attempts at growing seedlings of many southern cultivars in Moscow province to identify cold hardy individuals, in 1933–1935 a new approach was taken. Mother trees were selected from local wild C. avellana and were crossed with pollen collected from cultivars in Sochi (largely “Barcelona”, but also “Kudryavchik”, “Cherkesskii II”, “Yevgenia”, and “Brunsvik”). Reciprocal crosses were also made in Sochi on similar cultivars using pollen from select wild northern hazelnuts. The resulting seedlings were germinated at the agricultural experiment station in Moscow province and were field planted in 1938 and 1939 for evaluation. Open-pollinated seedlings of “Barcelona” from Sochi were planted for comparison. All of the “Barcelona” seedlings expressed poor growth and were killed by frost in their first years of life (Yablokov 1962; Kudasheva 1965). Around 50% of the hybrid progeny also perished due to the cold, although they persisted longer than the “Barcelona” seedlings. Of the remaining progeny, a small proportion continued to grow but was continually damaged by frost each year and produced little fruit, while the rest were winter hardy, vigorous, and productive. From these, several superior selections were identified as being exceptionally coldhardy, producing staminate flowers tolerant of very cold test winters in Russia with improved nut size over the wild type. Other selections were found with nuts similar in size to southern cultivars, although their hardiness was not remarked upon.

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Yablokov also collected open-pollinated seeds from select plants growing at the Michurinsk breeding station and grew them in Moscow province. From this plant material, promising hazelnuts were also selected. In 1948, several of the best hybrid plants were used in second-generation crosses, mostly with other selections from the Moscow institute. In addition, large populations were grown from seeds derived from open pollination of the best selections. From these populations, additional improved selections were identified, with a large focus on plants with red leaves (Kudasheva 1965).

2.5.1.3 R.F. Kudasheva In 1954, R.F. Kudasheva continued Yablokov’s breeding and evaluation work at the All Union Scientific Research Institute of Forestry and Mechanization. In addition to continuing evaluations of the breeding nurseries, in 1957 she made controlled crosses with additional southern cultivars growing in Azerbaijan with pollen from advanced hybrid selections from Moscow and wild Moscow plants. From 1957 to 1964, she created and evaluated more than 16,000 hybrids, with plant material grown and evaluated in Tambov, Moscow, and Krasnodar regions of Russia. Breeding efforts were largely discontinued in the late 1960s, but a number of notable cold-hardy cultivars were released, including “Tambovsky pozdniy”, “Tambovsky rannii”, “Moskovsky rubin”, “Moskovsky rannii”, and “Severnii 42”, several of which were recommended for planting on the central chernozem region of the central (former) USSR (Pavlenko 1985). Today, a collection of plant material derived from this early work remains at the All Russian Scientific Research Institute of Forestry and Mechanization. It holds around 350 wild hazelnut selections from the Tambov region, as well as around 500 hybrid selections from the past breeding efforts. Of these, 150 have red leaves (Eugene Momonov, personal communication, 2003). A number of the Moscow selections are also held at the All Russian Scientific Research Institute of Genetics and Breeding of Fruit Species named after I.V. Michurin in Michurinsk (Director N.I. Savelev, personal communication, 3 July 2009). Around 50 selections were imported from the Moscow institute in 2003 and are under evaluation at Oregon State University and Rutgers University in New Jersey.

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A number of these Moscow plants were found to be EFB-resistant in Oregon (Sathuvalli et al. 2010). Select genotypes will be used in breeding and eventually preserved at the NCGR.

2.5.2 Corylus americana The wild American hazelnut, Corylus americana, can be found growing across a wide range of climates and soils throughout much of eastern North America. Several C. americana selections have been identified and named that produce relatively large size nuts with good quality (“Rush”, “Winkler”, and “Littlepage”); however, most plants produce tiny nuts with thick shells that are of little commercial value. Wild hazelnuts were collected more widely in the past for home consumption and local sale, with few collected today. Corylus americana is the natural host of the fungus Anisogramma anomala, which causes EFB disease. While C. americana can vary in its response to the fungus, it is typically highly tolerant of the disease, with infected plants developing only tiny cankers, or none at all. Alternatively, the European hazelnut, C. avellana, is highly susceptible to this disease, which causes severe stem cankering, die back, and death of most plants within 4–10 years after exposure (Johnson and Pinkerton 2002). While the European hazelnut was likely brought to eastern North America as early as the first settlers from Europe (Rosengarten 1984), its production never became established there, largely due to EFB and compounded by the harsher climate of the northeastern US, compared to Europe (Fuller 1908; Thompson et al. 1996). Alternatively, European hazelnut production thrived in the Pacific Northwest for almost 100 years due its Mediterraneanlike climate and lack of EFB in this region. Unfortunately, this situation changed in the late 1960s, with the introduction of EFB into southwest Washington (Davison and Davidson 1973). After first devastating most of the production orchards in Washington, EFB spread south and is now found throughout the entire Willamette Valley where its control measures (ample fungicide applications, pruning, etc.) add much expense to current production (Julian et al. 2009a). Fortunately, the typically EFB-tolerant Corylus americana hybridizes readily with C. avellana (Erdogan and Mehlenbacher 2000a) and resistant progeny can be

30

recovered from the cross, although transmission of resistance has not been well studied and appears to be controlled by multiple genes, as well as single dominant genes in some genotypes (Thompson et al. 1996; Molnar et al. 2009). It should also be noted that any hazelnut cultivar developed for North America should express a high level of tolerance or resistance to EFB in order to be economically and environmentally sustainable. A number of attempts have been made in the past to exploit C. americana to develop better-adapted, cultivated forms of hybrid hazelnuts, as discussed in more detail later in this section. While progress has been made over the past century to develop improved hybrids, inadequate funding, intermittent efforts, lack of knowledge about EFB, and poor access to diverse genetic resources has limited the achievements of these breeding efforts. However, the potential for C. americana in breeding remains very promising, especially in advanced generations backcrossed to superior forms of C. avellana. Currently, germplasm collections of C. americana can be found at the NCGR and OSU, as well as the USDA National Resource Conservation Service Plant Materials Center in Elsberry, Missouri, with few accessions in other collections worldwide. Based on its extensive native range, existing collections do not fully represent the genetic diversity present in the species. Therefore, larger collection and evaluation efforts are needed to assess and utilize its full potential for breeding and the conservation of genetic resources. In addition to cold hardiness and resistance to EFB, selections made from its southern distribution may add heat tolerance or low-chill requirements needed to grow hazelnuts for production in more southern latitudes. Furthermore, select forms of C. americana and hybrids with C. avellana have shown high yield potential (Hammond 2006), and their smaller growth habit may be amenable for developing high-density plantings, which would provide earlier economic returns than more widely spaced orchards (Julian et al. 2009b). Small-statured plants also open up the possibility of mechanically harvesting crops directly from the bushes using modified versions of machines similar to those used for harvesting blueberries (Vaccinium spp.) or grapes (Vitis spp.). This method of harvesting would be opposed to collecting nuts from the orchard floor, as is now done in most commercial settings outside of Turkey, where hazelnuts remain harvested from the bushes by hand (Thompson et al. 1996).

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Mechanically harvesting the nuts directly from the bushes would reduce or eliminate the need for clean cultivation of the orchard floor, which would allow more sustainable planting on sloping land due to its reduced opportunity for soil erosion. This method of harvesting would also lessen the need for new cultivars to release their nuts cleanly from the involucre upon maturity – a trait lacking in most wild Corylus that is necessary to meet current mechanical harvest methods. Corylus americana also has value as an ornamental, as many selections express striking red and pink fall color, a trait not typically found in others of the genus. Also, their oversized, frilly involucres provide an additional ornamental attribute, especially when hybridized with purple-leaf C. avellana, as the purple color remains expressed in these tissues late into the summer even as the leaves typically fade to dark green.

2.5.2.1 J.F. Jones After a thorough study of introduced cultivars, J.F. Jones of Lancaster, Pennsylvania believed there was little chance production of European hazelnuts could succeed in the US, outside of the Pacific coast. To remedy this situation, in 1917 he began attempts to hybridize cultivars of C. avellana with a locally selected wild hazelnut, C. americana “Rush”, which was well-adapted, high-yielding, and produced relatively large-sized nuts for the species (Reed 1936; Crane and Reed 1937). Jones’ apparent goal was to combine the cold hardiness of the native species with the larger nut size and thin shell of the European cultivars. He was unsuccessful in acquiring hybrid seed from his crosses for 2 years during which he used “Rush” solely as the staminate (pollen) parent. Finally, in 1919 he used “Rush” as the pistillate parent and subsequently developed the first reported interspecific hybrids of the two species. He used C. avellana “Barcelona”, “Cosford”, “Daviana”, “Italian Red”, and “Duchilly” as staminate parents and grew and evaluated many hybrid progeny. Unfortunately, he passed away in 1928 before final evaluations of his hybrids could be made. However, two plants that stood out early as being superior were released from his estate in the 1930s. They were named “Bixby” (“Rush”  “Italian Red”) and “Buchanan” (“Rush”  “Barcelona”). While neither proved to warrant commercial

2 Corylus

planting, they were described as being productive, cold-hardy, and suitable for home cultivation (Reed 1936; Crane and Reed 1937; ASHS Press 1997). While “Bixby” appears to be lost from cultivation, C. americana “Rush” and “Buchanan” are available from the NCGR.

2.5.2.2 C.A. Reed The use of “Rush” as a parent in interspecific hybridizations was continued by C.A. Reed of the Bureau of Plant Industry, US Department of Agriculture in Beltsville, Maryland. From 1928 to 1932, Reed made hybrids with “Rush”, using C. avellana staminate parents similar to Jones’, as well as using “Hall’s Giant”, “Kentish Cob”, “Red Aveline”, and several others. He also made crosses, although to a much lesser extent, with C. americana “Winkler” from Iowa and “Littlepage” from Indiana, as well as intraspecific crosses between various C. avellana cultivars growing at the Bixby nursery in eastern Long Island, New York (Reed 1936; Crane and Reed 1937). From Reed’s breeding effort, around 2,000 plants were provided for evaluation at the USDA experiment station in Maryland. Seedlings were also sent for evaluation to the New York State Agricultural Experiment Station in Geneva, New York. No additional hybrids were made by Reed; however, the resulting progeny were evaluated for many years to follow. While the pure C. avellana plants showed little increase in adaptation over their parents, the hybrids with “Rush” showed promise. In 1951, two superior plants were selected and released: “Potomac”, a hybrid of “Rush”  “Duchilly”, and “Reed”, a hybrid of “Rush”  “Hall’s Giant”. While both were described as coldhardy and productive under eastern conditions, neither proved to be of commercial value (Crane and McKay 1951; Reed and Davidson 1958; ASHS Press 1997). “Potomac” was Later reported to be resistant to eastern filbert blight (EFB), while “Reed” was found to be susceptible (Lunde et al. 2000). Both cultivars are available from the NCGR.

2.5.2.3 G.H. Slate Hazelnut research was initiated in 1925 at the New York State Agricultural Experiment Station in

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Geneva, New York by G.H. Slate. The early goal was to collect and evaluate a wide range of C. avellana cultivars for production in New York; at its largest, the collection held about 120 cultivars imported from Europe, as well as several C. americana selections and interspecific hybrids. It became evident that most clones of C. avellana were poorly adapted to New York conditions, with staminate flowers and wood proving to be only marginally hardy (Slate 1935, 1936, 1947). Efforts to develop better adapted hazelnuts were initiated in 1930 when Slate first made crosses with “Rush” plants growing in Ithaca, New York, with pollen collected from several C. avellana cultivars held in the Geneva collection. Additional crosses with “Rush” were made in Ithaca in 1931 and 1933. In 1932, intraspecific crosses were made at Geneva using “Barcelona” and, to a much lesser extent, “Duchilly” as pistillate parents, crossed with several C. avellana cultivars that showed promise in the collection. No additional crosses were made by Slate (1936, 1947). The same year, however, 535 hybrid seedlings were planted that had been derived from crosses made by Reed of the Bureau of Plant Industry in Maryland. In total, nearly 2,000 hybrid seedlings were planted and thoroughly evaluated at the experiment station, of which 1,232 were offspring of “Rush”. Plants were evaluated for nut characteristics including size, shell color, kernel quality, and yield, as well as plant growth habit and cold hardiness. By 1947, 340 of the 2,000 seedlings showed merit and were retained for further observation, with 52 selected for propagation and testing in a new orchard. Nearly all were progeny of “Rush”, with only a few progeny of “Barcelona”, and no “Rush”  “Winkler” or “Rush”  “Littlepage” hybrids (Slate 1947). In 1961, Slate reported that the performance of the selected hybrids moved to the new orchard, which was more exposed to winds and on poorer soil, was much less satisfactory than in the original planting. Winter injury of the wood and catkins was more serious and crop yields were light, which made evaluations challenging. Furthermore, bud mites infested the orchard to further reduce crops and limit opportunities to evaluate the selections (Ourecky and Slate 1969). By 1980, only 24 of Slate’s original selections remained at the experiment station, of which 23 were progeny of “Rush” (Reich 1980). Today, nothing remains of the hazelnut breeding efforts in Geneva; however, a number of Slate’s most

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promising breeding selections are available at the NCGR where they can contribute to future breeding efforts. The drawbacks of using a limited diversity of C. americana parents are present in the body of work published by Slate. In addition to bud mite susceptibility inherited from “Rush”, most did not release the nuts from the husk on maturity, many were not sufficiently cold-hardy, and they generally produced low numbers of catkins (Slate 1961). After several decades of work, no cultivars were released from Slate’s efforts. However, useful EFB-resistant hybrids were developed and identified, and several have been propagated today for backyard use (Earnest Grimo, personal communication, 2010). Several of Slate’s selections have been used as EFB-resistant parents in private and public breeding efforts, including advanced generation hybrids at Oregon State University (Molnar et al. 2010). A number of Slate’s selections (designated with a New York identification number, i.e. NY616, NY398, etc.) are currently available from the NCGR.

2.5.2.4 S.H. Graham Also in the 1930s, S.H. Graham of Ithaca, New York continued work pioneered by J.F. Jones. He grew and evaluated hundreds of plants from open-pollinated seeds of Jones’ first generation hybrids, including seeds collected from “Bixby” and “Buchanan” growing in close proximity to one another. Graham considered these plants second-generation hybrids, expecting to see an improvement over Jones’ work in this apparent next generation. He also grew seedlings of C. americana “Winkler” and made his own interspecific hybrids using “Winkler”, “Rush”, and unnamed interspecific hybrids (Graham 1936). Graham’s planting was in a colder area of New York than Geneva, and his trees experienced significant winter injury there. His plantings were also infected by EFB, which was still not present in the research plots at Geneva as late as 1952 (Slate 1952). While Graham (1936) found that a majority of his progeny proved inferior to their parents in nut quality, and it was reported that he lost most of his hybrids to EFB (Slate 1969), two cultivars were named and released from his efforts: “Morningside” (“Rush”  C. ave. “Duchilly”) in 1945 and “Graham” (“Winkler”  C. ave. “Longfellow”) in 1950. “Morningside” is reported to have been lost

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due to EFB (ASHS Press 1997). The status of “Graham” is currently unknown.

2.5.2.5 C. Weschcke Carl Weschcke also worked to hybridize C. americana and C. avellana in the 1930s and 1940s. He was a very ambitious nurseryman in River Falls, Wisconsin, interested in northern nut trees, who got his start with hazelnuts in 1921 by ordering one hundred “Rush” hazelnut plants from J.F. Jones’ nursery in Pennsylvania. The plants ended up being seedlings of “Rush”, instead of clones, and many appeared to be hybrids between C. americana and C. avellana (Weschcke 1954). The diversity reported in this planting likely sparked Weschcke’s interest in hazelnut breeding. In 1927 he planted “Winkler” hazelnuts purchased from a nursery in Iowa, and the following year planted additional Jones hybrid hazels (seedlings of Jones’ interspecific hybrids). Then, in 1929, he planted clones of twelve different C. avellana cultivars purchased from a New York nursery. Over the next couple of years, most of the Jones hybrids and all the of cultivars of C. avellana, which included “Italian Red” and “Medium Long” reported to be cold-hardy by Slate (1959), were killed by cold temperatures, demonstrating the harsh climate of Wisconsin. To continue his project, in 1932, Weschcke planted C. avellana selections from J.U. Gellatly of West Bank, British Columbia, which also suffered from winter injury, although they survived for several years. They were of value to Weschcke when he found an exceptional wild C. americana plant growing in the woods on his farm. In 1934, he crossed the wild plant with pollen from a surviving Gellatly hazelnut; four cold-hardy hybrid plants were the result, which Weschcke called “hazilberts.” Three hybrid plants, possibly from this first cross, were released several years later named “Carlola”, “Delores”, and “Magdalene”. All were listed as having the staminate parent C. avellana “Brag” developed by J.U. Gellatly (ASHS Press 1997). None of these plants are known to exist today. In 1939, Weschcke made crosses between “Winkler” and various C. avellana parents. He also crossed his surviving Jones hybrids with pollen from coldhardy C. avellana seedlings from Gellatly. Later, Weschcke found several more exceptional wild hazelnuts in his area that expressed traits such as high

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yields, large size nuts, early maturity, and thin shells, which he used as female parents in his crosses; from 1942–1945, hundreds of hybrids between C. americana and C. avellana were produced. Pollen was obtained from other hazelnut growers in the US, or from C. avellana surviving on his farm. Staminate parents included “Barcelona”, “Duchilly”, “Red Aveline”, “White Aveline”, “Purple Aveline”, “Italian Red”, “Daviana”, and several others. By 1945, he had around 2,000 plants under evaluation and, by 1952, had accumulated extensive data on 650 of them. Weschcke (1954) described that, although there were likely several plants worthy of release in this group, he would prefer to see what is found in the next generation of 1,000 plants. Nearly 10 years later, he reported that his C. americana  C. avellana hybrids were reliable croppers under all conditions and were bred so that EFB would not be a problem. However, he stated that there was not yet any individual commercially valuable plant, but he expected to recover this in the next 10,000 seedlings he brought into bearing (Weschcke 1963). This expectation never came to fruition, as Weschcke (1970) later reported that most of his hybrids were killed by EFB after all, although he declared that not all of the plants died. He passed away in 1973. After several decades of breeding, no cultivars were released from his work, although his efforts generated better-adapted and EFB-resistant hybrid plant material that has been used in more recent breeding efforts.

2.5.2.6 P. Rutter Philip Rutter of Canton, Minnesota expanded on Weschcke’s work by collecting seeds from select trees of the thousands surviving in Weschcke’s overgrown, EFB-infected orchards in River Falls, Wisconsin. In an attempt to identify reliably productive parent plants to initiate his own mass selection program, Rutter collected seeds from EFB-resistant plants of apparent C. americana  C. avellana origin that were bearing nuts in a year when most plants did not produce crops. He grew the resulting plants on his hilly, windswept farm in south-eastern Minnesota. Rutter later added seedlings originating from various interspecific hybrids developed by G.L. Slate, J.U. Gellatly, and C. Farris, as well as some of his own collections of wild C. americana and C. cornuta.

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Inferior plants were eliminated by the harsh climate of Minnesota, the low-maintenance nurseries in which they were grown, and the presence of EFB. In addition to cold hardiness and resistance to EFB, the main breeding objectives were to select plants expressing high kernel productivity and increased cropping potential. Adapted, high-yielding seedlings were identified from these early plantings and their nuts were harvested to plant successive generations to undergo similar evaluations. Pollinations were controlled to some degree by emasculating inferior plants prior to anthesis (Rutter 1987). Today, several generations and over 50,000 seedlings have been cycled through evaluations by Rutter. From this work, he has identified plants expressing EFB resistance and cold hardiness that are segregating for increased nut yields and quality, with mass selection efforts continuing. While no cultivars have been released to date, improved seedling plants have been widely distributed. Experimental plantings of his hybrid seedlings have been established in numerous parts of Minnesota, Nebraska, Wisconsin, and other states. From this germplasm, Hammond (2006) identified several consistently high-yielding selections out of over 5,000 seedlings grown at the Arbor Day Farm in Nebraska City, Nebraska. Based on single-plant estimates, the 4-year average of the highest yielding selection was 4 ton/ha of dried, inshell nuts. While single-plant extrapolations can be misleading, Hammond’s work was done in a tightly spaced orchard with no irrigation, fertility, or pest management, which provides evidence for the potential of select C. americana hybrids to produce abundant crops in the Midwest US with little inputs. Overall, the past work hybridizing C. americana and C. avellana provided useful genetic resources that are currently available, as well as insight into the direction a plant breeding program may need to go to make the best, most efficient use of this hybrid combination. While the genetic resources used in the early work were limited, some cold-hardy, EFB-resistant plants with relatively large, high-quality nuts were developed. However, not all hybrids were resistant to EFB, and unreliable and occasionally low yields did not warrant commercial production in the east. Most breeding efforts stopped at the first generation, or putative second generation crosses were made by hybridizing within the best of the first generation hybrids, sometimes with no or little control of pollen flow. This approach is largely inefficient and may have

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further narrowed the genetic diversity present in the hybrid germplasm when plants were grown under the high selection pressure (bottle neck) of severe environmental stress and/or disease pressure. Based on the results of past efforts, a different approach is suggested here. To maintain the high-quality, large kernels, and high yields of C. avellana, with the wider adaptation of C. americana, a diversity of select C. americana parents must be used (parents that compliment their C. avellana counterparts) in a systematic, multigenerational breeding effort. Large hybrid progenies must be evaluated in the proper environment to identify the rare recombinants that express the highest levels of desirable traits of each species. The best individuals must then be clonally propagated and tested in multiple locations to identify those with the highest potential for consistent production and for use as improved breeding parents. Advanced generation hybrids must then be made by backcrossing the superior first-generation hybrids to improved, complimentary, and unrelated C. avellana. From here, the cycle will likely need to be continued for at least one to several generations to combine the wide adaptation of C. americana with the nut qualities of C. avellana – in the opinion of the author, a very laudable, but feasible goal.

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reported to be extremely precocious, sometimes flowering in only 1 or 2 years from seed (Thompson et al. 1996). Cho (1988) reports that this characteristic is also expressed in hybrids of C. heterophylla  C. avellana. In addition, selections of C. heterophylla and its hybrids have also been found to be EFB resistant (Coyne et al. 1998; Chen et al. 2007; Molnar et al. 2010), and Corylus het. var. yunannensis is adapted to alkaline soils (Thompson et al. 1996). Furthermore, many selections of C. heterophylla have distinct, truncated, and variable leaf shapes, which may enhance other ornamental attributes like purple leaf color when used to develop hybrid ornamental landscape plants. The success of the Chinese breeding program, as discussed below, suggests that C. heterophylla may be one of the more useful wild Corylus species for enhancing the climatic adaptation of commercial hazelnuts. Based on its wide native range, outcrossing nature, and adaptation to varieties of soils and stressful climates, it is expected that considerable genetic diversity exists in the species. Unfortunately, Corylus germplasm collections outside of China and Korea contain only a very limited representation of C. heterophylla.

2.5.3.1 Economic Forest Research Institute of Liaoning Province, Dalian, China

2.5.3 Corylus heterophylla The Siberian hazel, C. heterophylla, can be found growing across a wide range of climates and soils in Korea, Japan, China, and the Russian Far East. Corylus heterophylla crosses readily with C. avellana and C. americana, although success of seed set depends on the choice of parental clones (Cho 1988; Weijian et al. 1994; Erdogan and Mehlenbacher 2000a). Corylus heterophylla is analogous to C. americana in many characteristics, including its potential for breeding widely adapted hybrid cultivars, and plants exist that drop the nuts from the involucres at maturity – a rare occurrence in C. americana. Compared with cultivated C. avellana, the nut is smaller and thicker-shelled and the yield is generally lower. However, plants from its northern range are extremely cold-hardy and drought-tolerant, some being adapted to regions of northeast China that have snowless winters and temperatures dropping below 30 C. Seedlings of the species have been

Traditionally, nuts of C. heterophylla have been collected from wild stands for home consumption and local sale across its native range. In the 1960s and 1970s, forest management, including tree thinning, weeding, and pest control, was done in wild hazelnut stands in China to increase production (Weijian et al. 1994). To better meet a growing demand, openpollinated seeds of several cultivars of C. avellana were introduced from Bulgaria, Albania, and Italy between 1972 and 1975, with the resulting plants grown at the Economic Forest Research Institute of Liaoning Province in Dalian, China. The goals were to evaluate the potential of C. avellana in China and to select improved individuals suited for commercial production in Liaoning Province. A total of 203 seedlings from these collections were planted. They were quickly found to be sensitive to winter injury in Dalian, which routinely reaches 15 to 20 C during winter months, compounded by high wind and low air humidity in the winter. The poor performance of

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C. avellana turned breeders’ attentions to the prospect of improving wild C. heterophylla growing in northern China. In 1980, a program was undertaken to evaluate very large numbers of wild C. heterophylla growing in Liaoning province for their production potential and nut characteristics. Over the next 5 years, out of many thousands of plants, 31 superior C. heterophylla strains were identified that expressed qualities such as high yield, improved nut quality, and thin shells. The most useful plants were propagated and placed in the first Corylus germplasm collection in China, which by 1991 contained six species and over 100 cultivars and lines (Weijian et al. 1994). No individual C. heterophylla selection was shown to be ideal for commercial production, causing breeders to focus their efforts on creating interspecific hybrids between C. heterophylla and C. avellana. The first interspecific hybrids were made in 1980. Breeding goals were to develop high-yielding plants that produced the large nuts, high-quality kernels, and thin shells of cultivated C. avellana, while expressing the cold hardiness and adaptability of C. heterophylla. In their crosses, ten strains of select C. heterophylla and 20 of the best C. avellana seedlings identified from earlier efforts were used. The pollen of different strains within a species was mixed before making hybridizations, to help ensure seed set. While it was possible to use C. avellana as the pistillate parent, the compatibility was much higher when using C. heterophylla (Weijian et al. 1994). From 1980 to 1986 more than 2,300 hybrid progeny were produced and grown in Dalian. Over the next 10 years plants were evaluated for cold hardiness, nut quality, and yield. From these evaluations, around 40 hybrid plants were identified that were superior to the selected strains of pure C. heterophylla and were much better adapted than C. avellana (Weijian et al. 1994). In 1990, the best 12 of these plants were placed into replicated yield trials in Dalian, Anshan, and Shenyang. From these trials, five superior interspecific hybrid plants were named and released in 1999 for production: “Pingdinghuan”, “Bokehong”, “Dawei”, “Jinling”, and “Yuzui” (Ming et al. 2005). Recognizing a need for improved quality nuts, breeding efforts were continued in Dalian. In 2001 and 2003, attempts at second-generation hybrids were made between advanced hybrid selections and several select C. heterophylla plants, including collaborations

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with researchers in Italy (Ming et al. 2005). Although none of these second-generation plants have yet to be released, several more cultivars from the original crosses were released in 2007 and 2008: “Liaozhen 1”, “Liaozhen 2”, “Liaozhen 3”, and “Liaozhen 4” (Ming et al. 2007, 2008; JinLi et al. 2007, 2008). The cultivation of hazelnut in China has increased due to the success of the interspecific hybridization program. Currently, around 1,200 ha of hybrid hazelnuts have been planted in northern China, with production continuing to expand (Liang et al. 2008; FAOstat 2010).

2.5.3.2 Breeding Efforts in Korea A similar program to that in China was initiated in Korea in 1975 at the Rural Development Administration, as discussed by Mehlenbacher (1991a). Large numbers (more than 40,000) of native C. heterophylla and C. sieboldiana were evaluated for immediate production or for use in a hybridization program with C. avellana. From this work, 35 C. heterophylla and 10 C. sieboldiana selections were made, several of which are now held at the NCGR, including C. heterophylla “Ogyoo” and several numbered selections. A hybridization program was undertaken to combine the adaptation of the native species with the larger, high-quality nuts of C. avellana (Cho 1988), resulting in the release of “Poongsil” (C. heterophylla  C. avellana “Butler”), and “Gaeam No. 1” (“Ogyoo”  C. avellana “Butler”). The current status of this program is unknown. Hazelnut research, including a germplasm collection, has also been undertaken at the Institute of Forest Genetics in Korea (Mehlenbacher 1991a; Lee 2002), although recent details are lacking in Western literature.

2.5.3.3 C. Farris Cecil Farris, a private hazelnut breeder from Lansing, Michigan, was one of the first to hybridize Corylus heterophylla with C. avellana in the US. He used a single accession of the species C. heterophylla var. sutchuensis obtained from western China in crosses with pollen of C. avellana “Holder” in 1971–1973 (Farris 1974). Farris grew out several dozen seedlings, of which some were dwarf and stunted and others were

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vigorous and healthy. The progeny appeared to Farris to be true hybrids based on plant morphology; he selected the five best plants and named them Estrella hybrids, numbered 1 through 5. While some of the five selections appeared to have sterility issues, he successfully crossed “Estrella #2” with pollen from C. avellana “Royal”. All of the offspring appeared to grow normally, demonstrating the ability to backcross the hybrids to C. avellana. He also successfully crossed “Estrella #2” with pollen from “Faroka”, a C. colurna  C. avellana interspecific hybrid developed by J.U. Gellatly. “Estrella #1” and “Estrella #2” are available from the NCGR; “Estrella #1” was shown to be resistant to EFB (Chen et al. 2007).

2.5.4 Corylus cornuta Corylus cornuta is the most cold-hardy Corylus species in North America. It grows wild across much of the northern US and southern Canada into regions that reach 50 C. It is also believed to be highly EFBresistant as there are no reports of this disease occurring on C. cornuta even though its range significantly overlaps that of C. americana, the native host of A. anomala. Coyne et al. (1998) confirmed this resistance through greenhouse inoculations. Corylus cornuta also has very early maturing nuts, a trait that is beneficial to regions like Oregon where a rainy season begins in autumn that can significantly interfere with the harvest of late maturing cultivars. This trait is also needed to grow plants for production in northern regions with short growing seasons. Corylus cornuta has a very stoloniferous, spreading-growth habit and can form dense thickets, which may prove useful in soil reclamation or for providing wildlife habitat. Conversely, this trait would be a negative attribute in most orchard settings. While Erdogan and Mehlenbacher (2000a) were only able to cross C. cornuta with C. californica, C. sieboldiana, and to a limited degree C. heterophylla, Gellatly (1950, 1956) reported success making crosses with C. avellana to develop his “Filazel” hybrids. 2.5.4.1 J.U. Gellatly Gellatly (1950) collected extremely cold-hardy C. cornuta from the Peace River District of Alberta, Canada, and hybridized it with his own selections of cold-hardy

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C. avellana, including “Craig” and others. His goal was to combine the extreme cold hardiness and early nut maturity of C. cornuta with the large nut size of cultivated C. avellana. While he did not use hand pollinations to make the crosses, he was able to select hybrid offspring by identifying seedlings expressing characteristics that were intermediate between the two species (Gellatly 1950; Farris 1990; Thompson et al. 1996). Gellatly distributed seed and plants and released a number of selections of this hybrid cross that include “Peoka”, “Manoka”, “Fernoka”, “Farioka”, and “Myoka”. “Myoka” is available at the NCGR (2010), while the availability of others is unknown. Mehlenbacher (1991a) and Farris (1990) successfully crossed Filazel #45, a numbered hybrid (C. cornuta  C. avellana) selection of Gellatly’s, with pollen of C. avellana. Farris (1990) also reported that pollen of Filazel #45 set nuts on C. avellana “Ennis” and the advanced-generation C. colurna hybrid “Grand Traverse” and that the early maturity of this selection was expressed in its offspring. While Erdogan and Mehlenbacher (2000a) did not have success with crossing C. cornuta and C. avellana in either direction, it is possible that when using a wider diversity of germplasm and larger numbers of crosses, poorly understood barriers to interspecific hybridization between Corylus species may be overcome and successful hybrid offspring could be generated. In the case of C. cornuta, this could strongly assist in developing commercial quality hybrid plants adapted to very cold climates and short growing seasons.

2.5.5 Corylus californica Corylus californica can be found growing in the coastal mountains of the Pacific Northwest. It has only been utilized to a minor extent due to its small, thick-shelled nuts, which like its very close relative, C. cornuta, are very early maturing. This trait, plus its less-stoloniferous growth habit and shorter husks, may make it more useful for breeding, if cold hardiness is not a primary objective. Since the inadvertent introduction of EFB into the Pacific Northwest, it has been observed that C. californica growing adjacent to infected orchards remained free of disease. Coyne et al. (1998) confirmed the presence of resistance in the species through greenhouse inoculations. While selections of pure C. californica remained free of

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EFB, small cankers developed on several C. californica  C. avellana hybrids. Further supported by additional unpublished work, the performance of C. californica  C. avellana hybrids indicate that C. californica expresses quantitative resistance rather than complete resistance to EFB (Shawn Mehlenbacher, personal communication, 2009). Erdogan and Mehlenbacher (2000a) reported that C. californica, when used as a female parent, was able to hybridize with all other species used in their study (Table 2.3), suggesting it may have use as a bridge species to create advanced generation interspecific hybrids.

2.5.6 Corylus sieboldiana Corylus sieboldiana grows across much of eastern and northern Asia, including the Russian Far East. It is generally very cold-hardy, with many similar traits to C. cornuta and C. californica, although it has late maturing nuts (Erdogan 1999). Coyne et al. (1998) showed that accessions from Korea were resistant to greenhouse inoculations. Erdogan and Mehlenbacher (2000a) showed that it hybridized readily with C. cornuta and C. californica and to a much lesser extent with C. americana. Reports from Korea, described in Mehlenbacher (1991a), claim that it was hybridized with C. avellana to develop better-adapted cold-hardy plants with larger nuts, although little recent information is available on hybrids and few exist in the West for study. A substantial collection of C. sieboldiana is available at the NCGR (Table 2.3), which will prove useful to better study the breeding potential and genetic diversity of this species.

2.5.7 Corylus colurna Corylus colurna, the Turkish tree hazel, is found naturally occurring in the Balkan Peninsula, Turkey, and the Caucuses, but it has been grown widely as an ornamental shade tree in many parts of Europe and the US for centuries. In the landscape, C. colurna naturally forms an attractive pyramidal crown and displays interesting scaly, corky bark, and heavily textured leaves. It has been shown to be cold-hardy and exceedingly drought and stress tolerant, with Dirr

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(1998) suggesting its use as a street tree, even under city conditions. Its small, thick-shelled nuts with highquality kernels have been collected from the wild and consumed and sold in its native areas, but trees are usually more valued for their excellent timber. While C. colurna remains a single-trunk tree, the shrubby natured C. avellana produces suckers from the base of the plant throughout the growing season. To maintain grafted trees of C. avellana and to facilitate mechanized production in commercial orchards, suckers must be removed numerous times per year, which requires a significant amount of expense and time. As such, a non-suckering rootstock would be very beneficial to commercial production. Seedlings of C. colurna were used as rootstocks for C. avellana in the past, including for ornamental forms. However, poor germination rates and transplanting problems due to its less-fibrous tap root system, as well as decreased performance of older nut orchards grafted on C. colurna, have reduced its value for such applications (Lagerstedt 1976). Nevertheless, C. colurna still holds promise for use in interspecific hybridization programs, especially in terms of developing vigorously growing, stress-tolerant, non-suckering clonal rootstocks (Lagerstedt 1975, 1990). It is very difficult to hybridize C. colurna and C. avellana; however, a limited number of fertile hybrids have been created in the past, and are discussed below. More recently, Erdogan and Mehlenbacher (2000a) were able to recover a small number of hybrid seedlings when making large numbers of crosses between C. colurna and C. avellana. It was shown to hybridize readily with C. chinensis, and less successfully with C. heterophylla and C. californica. They suggest when attempting to make the cross with C. avellana, breeders should perform large numbers of pollinations and expect only a small number of hybrid seedlings. In addition to their growth habit and adaptation attributes, select C. colurna and C. colurna hybrids have also been reported to be resistant to EFB and big bud mites (Coyne et al. 1998; Farris 1978, 2000; Lunde et al. 2000; Chen et al. 2007), suggesting its direct usefulness for breeding improved Corylus hybrids.

2.5.7.1 J.U. Gellatly J.U. Gellatly of West Bank, British Columbia reported some of the first hybrids of C. colurna  C. avellana,

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naming the hybrid plants as a group “trazels” (Gellatly 1964, 1966). His plants resulted from the openpollination of C. colurna trees grown in close proximity to trees of C. avellana, reportedly “Craig”, “Holder”, and “Brag”. Gellatly grew large seedling populations of C. colurna, presumably for rootstocks for his nursery business, and he identified hybrids based on their appearance (Farris 2000). He then evaluated the apparent hybrids for cold hardiness and nut characteristics. After 30 years of work, several superior hybrids and numbered selections were named and released by this method, including “Morrisoka”, “Faroka”, “Karloka”, and “Eastoka” (Gellatly 1966; Farris 1978). These plants were considered to be true hybrids based on their morphology (Erdogan and Mehlenbacher 2000a). These “trazels”, as well as several numbered selections of Gellatly’s believed to be hybrids between C. colurna  C. avellana, are held in the collection at the NCGR. Recently, Chen et al. (2007) found several of Gellatly’s C. hybrids to be highly resistant to EFB, including Chinese Trazel Gellatly #6 and #11 and Turkish Trazel Gellatly #3. While Gellatly refers to the Chinese trazels as hybrids with C. chinensis (Gellatly 1966), Chen et al. (2007) believe they are instead C. colurna, based on morphology.

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successful crossing of C. avellana “Willamette” with pollen of “Faroka”, as well as successfully making the reciprocal cross with a mixture of C. avellana pollens. From his crosses, Farris identified a number of advanced-generation hybrids useful for further breeding or eventual release (Farris 2000). The most widely recognized was his EFB-resistant “Grand Traverse”, which he described as a cross between “Faroka”  “Royal” (Farris 1989), although the identity of the C. avellana parent is somewhat unclear, based on incompatibility alleles (Lunde et al. 2000). “Grand Traverse” was recently shown to be completely resistant to more than 12 different A. anomala isolates collected around the eastern US (Molnar et al. 2010). It was also shown to transmit its resistance to about 25% of its offspring (Molnar et al. 2009), and work is underway to better understand inheritance of the resistance. In 2008, “Grand Traverse” was successfully backcrossed to C. colurna and several C. avellana, with high cluster set and typical germination (T. Molnar, unreported). Farris (1990) later released “Lisa”, an open-pollinated selection of “Grand Traverse”, believed to be backcrossed to C. avellana. It was also shown to be resistant to EFB, as well as bud mites (Chen et al. 2007). “Grand Traverse” and several others of Farris’ selections are available at the NCGR. “Lisa” is held in the collection at OSU.

2.5.7.2 C. Farris A continuation of Gellatly’s “trazel” work was undertaken by C. Farris of Lansing, Michigan. Farris, a selftrained plant breeder, grew and evaluated most of Gellatly’s hybrid selections and considered “Morrisoka” and “Faroka” the best of the group. While the hybrid cultivars were not suitable for commercial production, he believed they would make superior breeding parents based on their cold hardiness, productivity, and high nut quality. His goals were to develop nonsuckering, EFB-resistant, cold-hardy plants that produced high-quality, large kernels. Throughout the 1970s and early 1980s, Farris, used controlled hand pollinations to successfully cross “Morrisoka” and “Faroka” with pollen of C. avellana “Royal”, which has large size nuts, to create advanced-generation hybrid progeny. Pollen of “Faroka” was also used successfully on C. colurna, demonstrating the crosscompatibility of Gellatly’s first-generation hybrids (Farris 1978, 1982). It should be noted that this fact was reinforced by Thompson et al. (1996) with the

2.5.7.3 H.B. Lagerstedt In search of non-suckering rootstocks for use in commercial hazelnut orchards in Oregon, H.B. Lagerstedt initiated a sizable rootstock evaluation and breeding program at Oregon State University, starting in 1968. Lagerstedt organized a collection of 19 of Gellatly’s hybrid trazels, as well as five non-suckering selections of possible rootstock potential from Farris. He also included apparent hybrid plants from O. Jemtegaard, who selected the open-pollinated hybrid “Filcorn” by similar means as Gellatly. Lagerstedt grew more than 1,000 open-pollinated seedlings of “Filcorn”, which was growing in a C. avellana orchard far away from other C. colurna, and from this isolation were assumed to be advanced-generation backcross hybrids. From these, he selected 70 plants for further testing (Lagerstedt 1975, 1976). Wide collections were also made from botanical gardens, arboreta, and nurseries in the US, Canada, and Europe (Thompson et al.

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1996). Seedlings were evaluated for vigor and nonsuckering growth habit. The challenge and expense of identifying an appropriate non-suckering rootstock is substantial. This is due to the need for the plant, once identified as a potential candidate, to first be reproduced on its own roots by layering, which can take several years. Then, it must be evaluated in replicated clonal yield trials, grafted to known cultivars, in comparison to those cultivars growing on their own roots to prove there is not a reduction of yield as a consequence of its use. Finally, in 1990, two rootstock cultivars were released by OSU: “Newberg” (tested as USOR 1-71) and “Dundee” (tested as USOR 15-71). They were selected from a nursery planting in 1971 that contained several thousand open-pollinated seedlings of C. colurna (Lagerstedt 1990). Unfortunately, both are highly susceptible to EFB. Lagerstedt also had a goal to incorporate red leaf color into a non-suckering rootstock that would facilitate maintenance and help differentiate the scion portion from the rootstock portion of a grafted tree. While working on this objective, he developed and released the ornamental hybrid “Ruby”. “Ruby” was derived form a controlled cross between Chinese trazel #4 (believed to be a C. colurna  C. avellana hybrid) and the red-leaf C. avellana “Fusco Rubra”. “Ruby” was selected because it retained its red color longer into the summer than other available red-leaf ornamental hazelnuts (Lagerstedt 1990). In 1984, a redleaf full-sibling of “Ruby”, USOR24-82, was crossed with a red-leaf breeding selection, C. avellana OSU A-28, by M. Thompson and D. Smith. From the resulting progeny, an advanced-generation, ornamental, interspecific hybrid “Rosita” was selected and released in 1999 (Smith and Mehlenbacher 2002).

2.5.8 Corylus jacquemontii Corylus jacquemontii is very poorly represented in western germplasm collection, research, and breeding efforts. Its genetic diversity and potential for breeding have not been evaluated. The species’ inclusion in recent taxonomic studies has been based on only one or two genotypes available in western collections (Erdogan and Mehlenbacher 2000b; Forest and Bruneau 2000; Whitcher and Wen 2001). Sharma and Kumar (2001) describe efforts to evaluate purported

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selections of C. jacquemontii growing in northwestern India, stressing its underutilized nature. Based on its growth habit, potential uses of this species are as a non-suckering rootstock and as an ornamental shade tree. The species was shown to be susceptible to EFB by Coyne et al. (1998), with all seven accessions succumbing to the disease. Farris (2000) described C. jacquemontii succumbing to EFB in Michigan; however, these findings were based on a very limited sample of plant material. In 2008, a seedling tree at the Dawes Arboretum in Newark, Ohio (accession D1997-0030.002) was observed by the author growing completely free of disease while adjacent to several heavily EFB-infected C. avellana plants.

2.5.9 Corylus chinensis Corylus chinensis is also very poorly represented in Western germplasm collections and breeding efforts, and it is considered endangered in China (Sun 1998). Based on its growth habit, it may have direct use as a rootstock or in the development of interspecific hybrids to develop improved non-suckering rootstocks. Based on experience with small seed lots imported from China, seedlings grow more vigorously and with a more fibrous root systems than C. colurna (an issue limiting the production of C. colurna in the nursery trade). However, based on its region of origin, the species is likely less cold hardy. It would also have use as an ornamental shade and timber tree due to its vigorous growth habit; large mature size; oversized, attractive leaves; and interesting involucres. Seedling C. chinensis trees growing at Rutgers University in New Jersey appear to express a high level of resistance to EFB, being disease-free after more than 6 years of exposure (T. Molnar, unpublished). Interestingly, they also appear to be highly resistant to feeding damage done by Japanese beetles (Popillia japonica Newman), a common, and often severe, pest problem of hazelnuts grown in the eastern US. While accessions of C. chinensis were limited, Erdogan and Mehlenbacher (2000a) successfully crossed it with C. colurna, C. avellana, C. americana, and C. californica. The cross with C. avellana, although only with C. avellana as the pollen parent, was notable in that it produced the most vigorous offspring out of all the various hybrid combinations

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in their comprehensive study. This finding strongly suggests that hybrids of C. chinensis and C. avellana may be useful for developing vigorous non-suckering rootstocks.

2.5.10 Corylus fargesii Corylus fargesii, also known as C. papyraceae, is native to southern China and has only recently been introduced to the West. Its genetic diversity and potential for breeding has not been fully evaluated. Wider collection efforts and preservation in germplasm repositories and botanical gardens is urgently needed. The earliest reported introduction to the US was made by Cecil Farris in 1982 from southern Gansu Province, China. Farris imported 50 seeds, but only recovered three seedlings (Farris 1995, 2000). More recent seed introductions were made by members of the North American China Plant Exploration Consortium in 1996 and 2005, leading to the establishment of C. fargesii at a number of US arboreta, the NCGR, and Rutgers University (Aiello and Dillard 2007). Its unique qualities include a single-trunk habit, vigorous growth, and very attractive peeling bark, which resembles that of river birch. Aiello and Dillard (2007) believe that, with improved propagation techniques, C. fargesii has merit to become a valuable ornamental shade tree in the central and eastern US. It may also hold value as a non-suckering rootstock, as seedlings grow very rapidly, are easily transplanted, and appear graft-compatible with C. avellana. Furthermore, Farris reports his introductions were resistant to EFB after many years of exposure (Farris 1995). Trees located at the Morris Arboretum in Philadelphia, Pennsylvania and Rutgers University in North Brunswick, New Jersey, also appear resistant to EFB, as plants are healthy and without symptoms while growing adjacent to heavily infected C. avellana for many years (Aiello and Dillard 2007; T. Molnar, unpublished). Farris reported successfully crossing C. fargesii with “Morrisoka” (C. colurna  C. avellana) in 1992, with all offspring inheriting the peeling bark characteristic (Farris 2000). No further reports of use in breeding have been made, although investigations are underway at OSU and Rutgers. Corylus fargesii is native to relatively warm areas of southern China, so its cold hardiness may be questionable. It also produces

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small, thick-shelled nuts that are tightly enclosed in the involucre. Therefore, its use in genetic improvement may be limited to developing vigorous non-suckering rootstocks and attractive ornamentals with peeling bark for warmer climates, unless cold-hardy complimentary breeding parents are used. The mode of inheritance of EFB resistance in C. fargesii has not yet been investigated.

2.5.11 Corylus ferox Corylus ferox and C. ferox var. tibetica are poorly represented in Western germplasm collections and research efforts. It is a small, single-trunk tree native to high elevations with mild climates in the eastern Himalayan Mountains across to parts of Yunnan, Sichang and Xizang provinces in China. The most distinctive feature of this species is its chestnut-like involucre. Farris (2000) suggested that the involucre could protect the nuts from bird and rodent predation until the nuts are mature and ready to fall. He grew a very limited number of C. ferox var. tibetica accessions in Michigan and found it sensitive to cold damage. He was able to maintain the plants in large pots in his garage to survive the winter. He reportedly crossed C. ferox with “Lisa”, an advanced-generation C. colurna  C. avellana hybrid. However, the fate of the hybrid seedlings is unknown. Based on rDNA ITS sequence data, C. ferox was separated from all the other Corylus taxa (Erdogan and Mehlenbacher 2000b). A much wider diversity of C. ferox needs to be collected and evaluated for breeding and research, especially for studies of genetic diversity and the origin and evolution of Corylus and the family Betulaceae, based on its possible ancestral taxonomic position.

2.6 Alternative Uses of Wild Corylus Access to an increased diversity of wild germplasm and its systematic use in an interspecific hybridization program should lead to the development of widely adapted plants that can produce crops over a much greater area. This increased production would support the development of new market applications and opportunities, including ornamental plants, feedstock

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for biodiesel or other oleochemicals, value-added health and food products, animal feed, and other potential areas like soil reclamation, biomass production, and others not yet investigated.

2.6.1 Hazelnut Oil Hazelnut kernels have a high oil content by weight, with most containing over 60% oil and some up to 70%. The oil is rich in monounsaturated fatty acids, especially oleic acid (around 75–80%), and to a lesser degree linoleic acid (Botta et al. 1994; Ebrahem et al. 1994; Benitez-Sa´nchez et al. 2003; Xu et al. 2007). Recent work at the University of Nebraska, Lincoln, has demonstrated an alternative potential of hybrid hazelnuts as a low-input feedstock for the production of biodiesel and other valuable oleochemicals. Based on the 3-year average production of their highestyielding hybrid hazelnut selections, Hammond (2006) estimated that an equivalent of 4-ton/ha of inshell nuts could be produced. Based on the kernel oil content and shelling percentage of these selections, an oil yield of 1,000 kg/ha – nearly double the yield per hectare of soybean oil (around 500 kg/ha) (FAOStat 2010) – could be realized (Xu et al. 2007; Xu and Hanna 2009, 2010). While single-plant extrapolations can be unreliable, the estimates made by Hammond (2006) and Xu et al. (2007) are not far from the oil yields that could be produced commercially in the Willamette Valley of Oregon, under current production systems, if the kernels were processed for oil. Calculations suggest that nearly 900 kg of oil per hectare could be produced, based on the past 10-year average hazelnut yield data using the cultivar “Casina”, which has a shelling percentage of 56% and kernel oil content of 65.3% (Ebrahem et al. 1994; Mehlenbacher 2003; FAOStat 2010). In addition to its high yield potential, hazelnut oil has a unique fatty acid composition (high monounsaturated fatty acids and small percentages of saturated and polyunsaturated acids), thermal stability, and low temperature properties that should increase its value over soybean oil for a number of applications (Xu et al. 2007; Xu and Hanna 2009). Xu and Hanna (2009) synthesized and characterized biodiesel from hybrid hazelnut harvested in Nebraska and found it to be an excellent feedstock for making the fuel. Similar findings

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were reported by Gumus (2008) when he synthesized hazelnut-oil based biodiesel and examined its performance in diesel engines in Turkey. While the economics need to be fully examined before it is suggested that hazelnuts be grown as a sustainable source of biodiesel, it should be mentioned that high-yielding, well-adapted, early-generation interspecific hybrids that may not have nut qualities acceptable for the kernel trade (round, high-quality, well-blanching kernels) may find a direct role in oil production where these characteristics are not important. Plus, they can be grown on sloping and marginal land not suitable to annual oil crops. Besides industrial applications, hazelnut oil, which is very similar in composition to olive oil (BenitezSa´nchez et al. 2003), can likely play a larger role in the diets of humans. A comprehensive review of hazelnut (C. avellana) kernel composition and their reported health effects can be found in Alasalvar et al. (2009a). In general, C. avellana hazelnut kernels by weight are 58–64% fat, 15–18% carbohydrate, and 10–16% protein. They also contain a total of 24 minerals (essential and non-essential), with potassium the most abundant, as well as the fat soluble vitamins A, E, and K and water-soluble vitamins thiamin, riboflavin, niacin, pantothenic acid, folate, and several others. The kernels are especially high in vitamin E, biotin, and folate, which may be attributed to their reported health-promoting effects. Their fatty acid composition contains a small amount of saturated fatty acids (7–9%) and consists primarily of the monounsaturated fatty acids – oleic acid (77–83%) and linoleic acid (7–14%). This ratio of low saturated fatty acids to high unsaturated fatty acids has also been attributed to the health-promoting effects of hazelnuts, especially on human plasma lipid profiles. In addition, Erener et al. (2007) found that the consumption of hazelnut oil by broiler chickens increased oleic acid content of the meat, compared to those fed soybean oil, and that the ratio of saturated fatty acid to mono-unsaturated fatty acid was decreased. While more study is needed, these results suggest hazelnut oil could be used as an animal feed supplement to produce healthier meat products containing a higher level of oleic acids important for human diets. Additionally, the protein meal remaining after oil extraction may add substantial secondary value to oil production, as animal feed or in other products such as baked goods or supplement bars. However, the use of hazelnut protein in this manner has not yet been evaluated.

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2.6.2 Potential Phytochemicals and Other Products There are a number of potentially valuable by-products that would become more widely available with the increased production of hazelnuts production that would become more widely available with increased production. A majority of the world’s crop (90% or more) is cracked and sold as some form of raw or roasted kernels. This leaves behind the shell (50% or more of the crop weight), as well as the leafy involucre that surrounds the nuts, especially in Turkey where nuts still in the involucre are harvested by hand. Shells are commonly used directly as a fuel source, many times to aid in the kernel drying process, and shells and involucres can also be used as a compost material. ¨ zcelik and Peks¸en (2007) demonstrated the usefulO ness of the involucres as a component of a substrate to cultivate shiitake mushrooms [Lentinula elodes (Berk.) Pegler]. Production also results in a significant amount of biomass from orchards in the form of tree prunings and leaves. Finding higher value use of the by-products of production would provide further economic incentives to grow this low-input crop. While little work has been done to examine possible phytochemicals and other useful compounds in wild Corylus, Alasalvar et al. (2009b), in a comprehensive review of existing studies, showed that C. avellana kernels, pellicle, shell, involucre, and leaves are a rich source of proanthocyanidins, phenolic compounds, total antioxidant activity, and flavonoids. The pellicle of hazelnut showed the highest level of antioxidant activity compared to other tissues examined and was considered to be a potential industrial source of antioxidants. Furthermore, Oliveira et al. (2007) demonstrated antimicrobial activities of C. avellana leaf extracts, suggesting they may be a good candidate as an agent to control bacteria that cause gastrointestinal and respiratory tract infections in humans. In addition, hazelnut by-products also contain low concentrations of paclitaxel and other taxanes (compounds in the widely used cancer drug Taxol) (Hoffman et al. 1998; Hoffman and Shahidi 2009). While concentrations of taxanes in C. avellana appear uneconomically low compared to the bark of Pacific yew (Taxus brevifolia Nutt) from which it is currently extracted, it is possible that these valuable compounds may be found in higher levels in other Corylus species and interspecific hybrids that have yet to be assayed

T.J. Molnar

for the compounds. This point can also be applied to all of the other compounds studied for C. avellana that have not yet been investigated in wild species.

2.6.3 Ornamental Landscape Plants A number of highly ornamental traits exist in C. avellana, including the weeping habit of “Pendula” and the contorted growth of “Contorta” (also known as “Harry Lauder’s Walking stick”). “Rote Zeller”, “Fusco Rubra”, “Purple Aveline”, and “Syrena” exhibit dark red/purple leaves in the spring and early summer, while the bright yellow leaves of “Aurea” and the highly dissected leaves of “Cutleaf” also have promising ornamental value. Unfortunately, all of these cultivars are highly susceptible to EFB, which significantly limits their use in North America. Excluding the weeping habit, the inheritance of these ornamental traits has been studied and most appear to be simply inherited (Thompson et al. 1996). By developing ornamental plants with EFB resistance, whether through C. avellana or other species, hazelnuts could be much more widely utilized in the landscape as ornamentals or even as ornamental garden plants as the plants could still produce nuts. Incorporating genes for extreme cold hardiness would further increase their range of usefulness. A number of wild species also express traits, which would make them directly useful as ornamentals or as parents in interspecific breeding programs. For example, some selections of C. americana express bright pink or red fall color that is absent in most other species. While the inheritance of fall color in Corylus is not well understood, some hybrids of C. americana  C. avellana express fall color, and through use of select parents, it has been possible to combine the red spring and summer leaf color of C. avellana with the EFB-resistance and bright red fall color of C. americana (T. Molnar, unpublished). The truncated and variable leaf shapes of C. heterophylla would further add to the ornamental attributes of such an interspecific hybrid. The tree hazels offer opportunities to develop improved shade trees that would add interest and diversity to current landscape designs, particularly if incorporated with attractive leaf shape and color traits. Interspecific hybridization offers a means to develop a wide diversity of plant shapes and forms, including those with attractive corky and peeling bark as in C. colurna and C. fargesii,

2 Corylus

respectively. Similar to breeding for nut production, the lack of breeding efforts in Corylus, especially for ornamental attributes, as well as wider access to genetic resources and the increased understanding of interspecific hybridization potentials and inheritance of traits, should provide opportunities to develop a wide variety of useful, multi-purpose, attractive, landscape plants well into the future.

2.7 Recommendations for Future Conservation, Research, and Genetic Improvement The diversity and richness of the Corylus genus, its usefulness to man, and its importance to natural ecosystems are substantial. Increased efforts should be made to preserve, study, and utilize Corylus genetic resources for the betterment of future generations. In general, hazelnuts are a very low-input, high-value crop adapted to a wide variety of climates and soils, the production of which has many economic and ecological benefits. Furthermore, recent epidemiological and clinical studies have provided strong evidence that frequent tree nut consumption, including hazelnuts, is associated with favorable plasma lipid profiles and a reduced risk of heart disease, cancers, strokes, inflammation, and other chronic health issues (Alasalvar and Shahidi 2009). These positive economic, environmental, and health factors are driving increased production and market demand worldwide. World production acreage has increased almost 14% over the past 10 years (Fideghelli and De Salvador 2009). The development of more widely adapted cultivars would provide greater options for farmers to help meet this increasing demand. As discussed at length in this chapter, Corylus genetic resources are highly underutilized and underrepresented in research studies and conservation efforts, germplasm collections, and breeding programs. Aside from cultivated forms of C. avellana, little is known of their genetic diversity and population structure, with possible unchecked genetic erosion occurring due to overdevelopment, deforestation, and other causes. Molecular biology tools are now available for Corylus, including a multitude of effective SSR markers (G€ urcan et al. 2010), that can be utilized to assess genetic diversity in wild

43

species and also fingerprint accessions to reduce duplication in germplasm collections and show gaps in collections. A more thorough and accessible catalog of wild Corylus germplasm existing at research institutions, botanical gardens, and arboreta – similar to that currently conducted by the “Safenut” project in Europe for cultivated C. avellana (Bacchetta et al. 2009) – should be compiled and made available to hazelnut researchers worldwide. Besides obvious species deficits in Western collections such as C. jacquemontii, C. ferox, and C. fargesii, collections are also deficient in plants of the more common species originating from their most northern and southern ranges. Collections from these areas would be extremely valuable for expanding production of cultivated hazelnuts into more stressful climates. Efforts are needed to collect and evaluate this germplasm, with the most useful selections preserved and made widely accessible to world germplasm banks. In the past, Corylus genetic improvement efforts demonstrated that significant progress can be made through breeding; moving forward, however, a multigenerational approach should be followed, and the enhancement of genetic diversity in breeding lines should be stressed. Molecular biology tools must be used in concert with breeding efforts to ensure this is the case. The lack of diversity used in the interspecific hybrids made with C. americana and C. avellana in the first half of the twentieth century exemplifies this point. While C. americana “Rush” produced large size nuts for the species, many of its negative attributes were also transmitted to its offspring, which included a lack of significant cold hardiness, high susceptibility to bud mites, nuts that are retained in the husk at maturity, and a reduced numbers of catkins (Slate 1961). Besides the lack of diversity, most crosses were also limited to first-generation hybrids. The recent breeding progress at OSU clearly demonstrates the potential for substantial improvement in the second and third generation of breeding (Mehlenbacher et al. 2007, 2008, 2009). Selecting a wide diversity of the best, complimentary C. avellana parents available is especially important when using a modified backcross program to incorporate characteristics from wild species. Fortunately, much wider access to cultivated forms of C. avellana is available now than in the past. Additionally, the ability to ship pollen overnight from almost anywhere in the world makes it is possible to use parents in breeding efforts that would not

44

typically grow in the location of the breeding program, providing a very efficient means to exchange genetic resources. For example, pollen from EFB-susceptible, C. avellana cultivars and breeding selections with high-yields and excellent nut quality is routinely shipped from OSU and the NCGR to use in controlled crosses at Rutgers University in New Jersey, to develop improved, locally adapted selections. Many of these parents would not survive long enough in New Jersey to use in crosses due to EFB and the colder climate. In addition, pollen carries few viruses or diseases, reducing concerns related to the importation of seeds or clonal material. For a description on pollen collection and handling see Thompson et al. (1996). Exciting opportunities now exist to study and more widely utilize the Corylus genus. Rapid genetic gains are expected in breeding, based on its highly heterozygous nature, the ability to hybridize numerous species, and very limited prior breeding efforts. In the opinion of the author, the wild species of most immediate value for breeding improved interspecific cultivated forms (backcrossed to C. avellana) are C. americana and C. heterophylla. These both cross readily with C. avellana and are adapted to a wide climatic range with those from the northern areas being extremely cold-hardy. Selections of C. americana and C. heterophylla are also resistant to EFB, although inheritance is not well understood in these species, and some plants are very precocious and high yielding. A number of first-generation hybrids already exist in the US and China that can play an integral role in developing the foundation for developing advanced-generation hybrids. The collection and evaluation of a larger variety of wild germplasm will likely lead to the identification of more diverse improved selections to be used in long-term breeding efforts. Other Corylus species merit much wider collection and study for the conservation of genetic resources and for use in breeding, to enhance genetic diversity in cultivated forms, and to donate specific traits of interest such as extreme cold hardiness, drought tolerance, non-suckering growth habit, ornamental attributes, disease and pest resistances, and other characteristics that arise as more is learned about the wild species and as market demands dictate. Acknowledgments I would like to gratefully acknowledge the contributions to this manuscript of J Capik, C Leadbetter, X Ming, R Funk, and S Mehlenbacher.

T.J. Molnar

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46 Go¨kirmak T, Mehlenbacher SA, Bassil NV (2009) Characterization of European hazelnut (Corylus avellana) cultivars using SSR markers. Genet Resour Crop Evol 56:147–172 Graham SH (1936) Notes on an experimental planting in central New York. Annu Rep Northern Nut Growers Assoc 27: 64–67 Gumus M (2008) Evaluation of hazelnut kernel oil of Turkish origin as alternative fuel in diesel engines. Renew Energ 33:2448–2457 G€ urcan K, Mehlenbacher SA, Botta R, Boccacci P (2010) Development, characterization, segregation, and mapping of microsatellite markers for European hazelnut (Corylus avellana L.) from enriched genomic libraries and usefulness in genetic diversity studies. Tree Genet Genomes. DOI 10.1007/s11295-010-0269-y. Published online: 10 Feb 2010 Hammond E (2006) Identifying superior hybrid hazelnut plants in southeast Nebraska. MS Thesis, University of NebraskaLincoln, Lincoln, NE Hoffman A, Khan W, Worapong J, Strobel G, Griffen D, Arbogast B, Barofsky D, Boone R, Li N, Zheng P, Daley L (1998) Bioprospecting for Taxol in angiosperm plant extracts. Spectroscopy 13:22–32 Hoffman A, Shahidi F (2009) Paclitaxel and other taxanes in hazelnut. J Funct Foods 1:33–37 Hummer KE (2001) Hazelnut genetic resources at the Corvallis repositiory. Acta Hort 556:21–24 JinLi Z, Ming X, Weijian L, Zhongguan J, daoMing W (2007) Breeding report of new hazelnut cultivar ‘Liaozhen 3’. Chin Fruits 4:5–7 (in Chinese) JinLi Z, Ming X, Weijian L, Zhongguan J, daoMing W (2008) Breeding report of new hazelnut cultivar ‘Liaozhen 4’. Chin Fruits 6:6–8 (in Chinese) Johnson KB, Pinkerton JN (2002) Eastern filbert blight. In: Teviotdale BL, Michailides TJ, Pscheidt JW (eds) Compendium of nut crop diseases in temperate zones. Am Phytopathol Soc Press, St. Paul, MN, pp 44–46 Julian J, Seavert C, Olsen JL (2009a) An economic evaluation of the impact of eastern filbert blight resistant cultivars in Oregon, U.S.A. Acta Hort 845:725–732 Julian J, Seavert C, Olsen JL (2009b) Establishing and producing hazelnuts in the Willamette Valley – standard verses double density. Acta Hort 845:769–774 Kask K (1998) European filberts in Estonia. Annu Rep Northern Nut Growers Assoc 89:155–156 Kask K (2001) Nut quality and wild European hazelnut in Estonia and attempts at hazelnut breeding. Acta Hort 556: 37–40 Kasapligil B (1972) A bibliography on Corylus (Betulaceae) with annotations. Annu Rep Northern Nut Growers Assoc 63:107–162 Koksal AY (2000) Inventory of hazelnut research, germplasm and references. REU Technical series 56. Food and Agriculture Organization of the United Nations, Rome, Italy: http:// www.fao.org/docrep/003/x4484e/x4484e00.htm#Toc Kudasheva RF (1965) Propagation and breeding of wild and cultivated hazel nuts. Lesnaya Promishlennost, Moscow (in Russian) Lagerstedt HB (1975) Filberts. In: Janick J, Moore JN (eds) Advances in fruit breeding. Purdue University Press, West Lafayette, IN, pp 456–488

T.J. Molnar Lagerstedt HB (1976) Development of rootstock for filberts. Annu Rep Northern Nut Growers Assoc 65:161–165 Lagerstedt HB (1990) Filbert rootstock and cultivar introductions in Oregon. Annu Rep Northern Nut Growers Assoc 81:60–63 Lee SW (2002) Forest genetic resources conservation in the Republic of Korea. In: Palmberg-Lerche C, Iversen PA, Sigaud P (eds) Forest genetic resources, vol 30. FAO, Rome, Italy, pp 40–43 Liang W, Dong D, Wand G, Dong F, Liang L (2008) Studies on hazelnut hybridization breeding of C. heterophylla  C. avellana in China. Abstracts of the 7th international congress on Hazelnut. ISHS, Viterbo, Italy Lunde CF, Mehlenbacher SA, Smith DC (2000) Survey of hazelnut cultivars for response to eastern filbert blight inoculation. HortScience 35:729–731 Lunde CF, Mehlenbacher SA, Smith DC (2006) Segregation for resistance to eastern filbert blight in progeny of ‘Zimmerman’ hazelnut. J Am Soc Hortic Sci 131:731–737 Mehlenbacher SA (1991a) Hazelnuts. In: Moore JN, Ballington JR (eds) Genetic Resources in temperate fruit and nut crops. Acta Hort 290, pp 789–836 Mehlenbacher SA (1991b) Chilling requirements of hazelnut cultivars. Sci Hort 47:271–282 Mehlenbacher SA (1994) Genetic improvement of the hazelnut. Acta Hort 351:23–38 Mehlenbacher SA (1997) Revised dominance hierarchy for S-alleles in Corylus avellana L. Theor Appl Genet 94:360–366 Mehlenbacher SA (2003) Hazelnuts. In: Fulbright DW (ed) A Guide to nut tree culture in North America, vol 1. Northern Nut Growers Assoc, pp 183–215 Mehlenbacher SA (2009) Genetic resources for hazelnut: state of the art and future perspectives. Acta Hort 845:33–38 Mehlenbacher SA, Thompson MM (1991) Inheritance of a chlorophyll deficiency in hazelnut. HortScience 26: 1414–1416 Mehlenbacher SA, Smith DC (1988) Heritability of ease of hazelnut pellicle removal. HortScience 23:1053–1054 Mehlenbacher SA, Smith DC (1995) Inheritance of the cut leaf trait in hazelnut. HortScience 30:611–612 Mehlenbacher SA, Smith DC (2001) Parital self-compatibility in ‘Tombul’ and ‘Montebello’ hazelnuts. Euphytica 56: 231–236 Mehlenbacher SA, Smith DC (2002) Inheritance of pollen color in hazelnut. Euphytica 127:303–307 Mehlenbacher SA, Thompson MM (2004) Inheritance of style color in hazelnut. HortScience 39(3):475–476 Mehlenbacher SA, Smith DC (2006) Self-compatible seedlings of the cutleaf hazelnut. HortScience 41:482–483 Mehlenbacher SA, Thompson MM, Cameron HR (1991) Occurrence and inheritance of resistance to eastern filbert blight in ‘Gasaway’ hazelnut. HortScience 26:410–411 Mehlenbacher SA, Smith DC, Brenner LK (1993) Variance components and heritability of nut and kernel defects in hazelnut. Plant Breed 110:144–152 Mehlenbacher SA, Brown RN, Davis JW, Chen H, Bassil NV, Smith DC, Kubisiak TL (2004) RAPD makers linked to eastern filbert blight resistance in Corylus avellana. Theor Appl Genet 108:651–656

2 Corylus Mehlenbacher SA, Brown RN, Nouhra ER, Go¨kirmak T, Bassil NV, Kubisiak TL (2006) A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers. Genome 49:122–133 Mehlenbacher SA, Azarenko AN, Smith DC (2007) ‘Santiam’ hazelnut. HortScience 42:715–717 Mehlenbacher SA, Smith DC, McCluskey RL (2008) ‘Sacajawea’ hazelnut. HortScience 43:255–257 Mehlenbacher SA, Smith DC, McCluskey RL (2009) ‘Yamhill’ hazelnut. HortScience 44:845–847 Ming X, Zheng J, Radicati L, Me G (2005) Interspecific hybridization of hazelnut and performance of 5 varieties in China. Acta Hort 686:65–67 Ming X, JinLi Z, ShuChai S, SuJuan G, ZhiXia H (2007) Breeding of a new hazelnut cultivar ‘Liaozhen 1’. Chin Fruits 5:8–10 (in Chinese) Ming X, JinLi Z, ShuChai S, SuJuan G, ZhiXia H (2008) Breeding of ‘Liaozhen 2’ hazelnut cultivar. Chin Fruits 1:11–13 (in Chinese) Molnar TJ, Goffreda JC, Funk CR (2005) Developing hazelnuts for the eastern United States. Acta Hort 686:609–618 Molnar TJ, Mehlenbacher SA, Zaurov DE, Goffreda JC (2007) Survey of hazelnut germplasm from Russia and Crimea for response to eastern filbert blight. HortScience 42:51–56 Molnar TJ, Capik JM, Goffreda JC (2009) Response of progenies from known resistant parents to Anisogramma anomala in New Jersey, USA. Acta Hort 845:73–81 Molnar TJ, Goffreda JC, Funk CR (2010) Survey of Corylus resistance to Anisogramma anomala from different geographic locations. HortScience 45:832–836 Nas MN, Read PE (2004) Improved rooting and acclimation of micro propagated hazelnut shoots. HortScience 39: 1688–1690 NCGR (2010) US Department of Agriculture ARS, National Clonal Germplasm Repository, Corvallis, Oregon, USA: http://www.ars.usda.gov/main/site_main.htm?modecode¼5358-15-00. Accessed 01 July 2010 Normah MN, Reed BM, Yu X (1994) Seed storage and cryoexposure behavior in hazelnut (Corylus avellana L. cv. Barcelona). Cryo Lett 15:315–322 Oliveira I, Sousa A, Valenta˜o P, Andrade PB, Ferreira ICFR, Ferreres F, Bento A, Seabra R, Estevinho L, Pereira JA (2007) Hazelnut (Corylus avellana L.) leaves as a source of antimicrobial and antioxidative compounds. Food Chem 105:1018–1025 Ourecky DK, Slate GL (1969) Susceptibility of filbert varieties and hybrids to the filbert bud mite, Phytoptus avellanae Nal. Annu Rep Northern Nut Growers Assoc 60:89–91 ¨ zcelik E, Peks¸en A (2007) Hazelnut husk as a substrate for the O cultivation of shiitake mushroom (Lentinula edodes). Bioresour Technol 98:2652–2658 Palme AE, Vendramin GG (2002) Chloroplast DNA variation, postglacial recolonization and hybridization in hazel, Corylus avellana. Mol Ecol 11:1769–1779 Pavlenko FA (1957) Hazelnut. In: Anonymous (ed) Culture of nut species. Gosselkhozdat, Moscow, pp 5–126, in Russian Pavlenko FA (1985) Hazelnut. In: Anonymous (ed) Nut, forest, and orchard species, 2nd edn. Agroparomidaz, 123, p 99, in Russian Pomper KW, Azarenko AN, Bassil N, Davis JW, Mehlenbacher SA (1998) Identification of random amplified polymorphic

47 DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L. Theor Appl Genet 97:479–487 Reed CA (1936) New filbert hybrids. J Hered 27:427–431 Reed CA, Davidson J (1958) The improved nut trees of North America. Devin-Adire, New York Reed BM, Normah MN, Yu X (1994) Stratification is necessary for successful cryopreservation of axes from stored hazelnut seed. Cryo Lett 15:377–384 Reed BM, Hummer KM (2001) Long-term storage of hazelnut embryonic axes in liquid nitrogen. Acta Hort 556:177–180 Reich JE (1980) Geneva filbert research – past and present. Annu Rep Northern Nut Growers Assoc 71:110–111 Rosengarten F (1984) The book of edible nuts. Walker Publishing, New York Rutter PA (1987) Badgersett research farm – plantings, projects, and goals. Annu Rep Northern Nut Growers Assoc 78: 173–186 Sarraquigne JP (2005) Hazelnut production in France. Acta Hort 686:669–673 Sathuvalli VR (2007). DNA markers linked to novel sources of resistance to eastern filbert blight in European hazelnut (Corylus avellana L.). MS Thesis, Oregon State University, Corvallis, OR Sathuvalli VR, Mehlenbacher SA (2009) A hazelnut BAC library for map-based cloning of a disease resistance gene. Acta Hort 845:191–194 Sathuvalli VR, Mehlenbacher SA, Smith DC (2009) New sources of resistance to eastern filbert blight and linked markers. Acta Hort 845:123–126 Sathuvalli VR, Mehlenbacher SA, Smith DC (2010) Response of hazelnut accessions to greenhouse inoculation with Anisogramma anomala. HortScience 45:1116–1119 Sharma SD, Kumar K (2001) Preliminary evaluation of hazelnut seedling trees native to India. Acta Hort 556:29–35 Slate GL (1935) Suggestions for the breeding of nut trees. Annu Rep Northern Nut Growers Assoc 26:36–41 Slate GL (1936) The filbert breeding project at Geneva. Annu Rep Northern Nut Growers Assoc 27:62–63 Slate GL (1947) Some results with filbert breeding at Geneva, NY. Annu Rep Northern Nut Growers Assoc 38:94–100 Slate GL (1952) Filbert varieties. Annu Rep Northern Nut Growers Assoc 43:53–62 Slate GL (1959) Winter injury of filberts at Geneva, N.Y. 1958–1959. Annu Rep Northern Nut Growers Assoc 50:75–76 Slate GL (1961) The present status of filbert breeding. Annu Rep Northern Nut Growers Assoc 52:24–26 Slate GL (1969) Filberts-including varieties grown in the east. In: Jaynes RA (ed) Handbook of North American nut trees. Northern Nut Growers Assoc, Knoxville, TN, pp 287–293 Slyusarchuk VE, Ryabokon AP (2001) Hazelnut in Ukraine. Acta Hort 556:137–140 Slyusarchuk VE, Ryabokon AP (2005) Ukrainian hazelnuts: cultivars, agrotechniques, perspectives. Acta Hort 686:603–608 Smith DC, Mehlenbacher SA (2002) ‘Rosita’ ornamental hazelnut. HortScience 37:1137–1138 Smith DC, Mehlenbacher SA (1996) Inheritance of contorted growth in hazelnut. Euphytica 89:211–213 Smolyaninova LA (1936) Hazelnut. In: Vavilov NI (ed) Nut species. Cultivated flora of the USSR, vol 17. Gosudarstvennoe Izdatelstvo Sovkhoznoi i Kolkhoznoi Literatury, Moscow-Leningrad, pp 126–205 (in Russian)

48 Sun W (1998) Corylus chinensis. In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.2: www. iucnredlist.org. Downloaded 13 July 2010 Thompson MM (1977a) Inheritance of bud mite susceptibility in filberts. J Am Soc Hortic Sci 102:39–42 Thompson MM (1977b) Inheritance of nut traits in filbert. Euphytica 26:465–474 Thompson MM (1979) Genetics of incompatibility in Corylus avellana L. Theor Appl Genet 54:113–116 Thompson MM (1985) Linkage of the incompatibility locus and red pigmentation genes in hazelnut. J Hered 76:119–122 Thompson MM, Smith DC, Burges JE (1985) Nondormant mutants in a temperate tree species, Corylus avellana L. Theor Appl Genet 70:687–692 Thompson MM, Lagerstedt HB, Mehlenbacher SA (1996) Hazelnuts. In: Janick J, Moore JN (eds) Fruit breeding, vol 3, Nuts. Wiley, New York, pp 125–184 Tombesi A (2005) World hazelnut situation and perspectives: Italy. Acta Hort 686:649–658 Tous J (2005) Hazelnut production in Spain. Acta Hort 686: 659–664 Volovich PI, Chripach PI (1998) Diversity and abundance of cultivated varieties of filberts in Belarus. Annu Rep Northern Nut Growers Assoc 89:157–158 Weijian L, Ming Z, Wanying X (1994) Hazelnut breeding in northern China. Acta Hort 351:59–66 Weschcke C (1954) Hazels and filberts. In: Growing nuts in the north. Webb, St. Paul, MN, pp 24–38

T.J. Molnar Weschcke C (1963) Forty-three years of active work in nut growing. Annu Rep Northern Nut Growers Assoc 54:63–65 Weschcke C (1970) A little nut history. Annu Rep Northern Nut Growers Assoc 61:113–116 Whitcher IN, Wen J (2001) Phylogeny and biogeography of Corylus (Betulaceae): inferences from ITS sequences. Syst Bot 26:283–298 Xu YX, Hanna MA (2009) Synthesis and characterization of hazelnut oil-based biodiesel. Ind Crop Prod 29:473–479 Xu YX, Hanna MA (2010) Evaluation of Nebraska hybrid hazelnuts: nut/kernel characteristics, kernel proximate composition, and oil protein properties. Ind Crop Prod 31:84–91 Xu YX, Hanna MA, Josiah SJ (2007) Hybrid hazelnut oil characteristics and its potential oleochemical application. Ind Crop Prod 26:69–76 Yablokov AS (1962) Selection of woody species. Selkhozgiz, Moscow, in Russian Yao Q, Mehlenbacher SA (2000) Heritability, variance components and correlation of morphological and phenological traits in hazelnut. Plant Breed 119:369–381 Yoo K, Wen J (2002) Phylogeny and biogeography of Carpinus and subfamily Coryloideae (Betulaceae). Int J Plant Sci 163:641–650 Yu X, Reed BM (1995) A micropropagation system for hazelnuts (Corylus species). HortScience 30:120–123 Zohary D, Hopf M (2004) Domestication of plants in the old world. Clarendon, Oxford

Chapter 3

Cryptomeria Yoshihiko Tsumura

3.1 Botanical and Ecological Characteristics of Cryptomeria Cryptomeria japonica D. Don (common name: sugi) is a wind-pollinated and wind-dispersed tree species native to Japan. This species is a monoecious coniferous species. It mainly regenerates by producing seedlings and occasionally by layering (Taira et al. 1997). The relative importance of sexual or vegetative reproduction depends on geographical location and environment (Taira et al. 1997). Seed can be dispersed over several hundred meters with an average distance of 86 m in a natural forest on Yakushima Island, and genetically related individuals were found within 60 m of the mother tree (Takahashi et al. 2008). Existing natural forests of the species are distributed in the moist temperate region from Aomori Prefecture (40
420 N, 140
120 E) to Yakushima Island (30
150 N, 130
300 E) in the Japanese Archipelago (Fig. 3.1; Hayashi 1960). However, the distribution of the species is now discontinuous and restricted to small areas, as a result of extensive exploitation over the past 1,000 years (Ohba 1993). Cryptomeria japonica D. Don has been classified as belonging to the family Taxodiaceae, but recent molecular studies have revealed that it actually belongs to the Cuppresaceae (sensu lato; Gadek et al. 2000; Kusumi et al. 2000). The genome size of this species is large, 22.09 pg/2C, but smaller than other conifers including some species of Pinaceae (Ohri and Khoshoo 1986; Murray 1998; Hizume et al. 2001).

Y. Tsumura Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan e-mail: [email protected]

The chromosome complement for this species is 11 pairs of metacentric or submetacentric chromosomes (Sax and Sax 1933). The secondary constrictions on the sixth and tenth chromosomes as well as the constrictions on the sixth chromosome vary from individual to individual. Polyploid plants of C. japonica were found in 1951 in a nursery; there were both tetra- and triploid plants (Jinnai and Chiba 1951). Aneuploids have been created by the artificial crossing of di- and triploid plants (Sasaki et al. 2001). There has also been an attempt to establish a trisomic line for chromosome study (T. Kondo, personal communication). The trisomic plants were used to assign chromosomes according to the quantity of DNA markers, for example derived from restriction fragment length polymorphism (RFLP). Suyama et al. (1996) assigned three linkage groups to their respective chromosomes using the dosage of RFLP in the open-pollinated progenies of triploid elite tree clones. Triploid plants have been planted in afforestation projects because triploids are sometimes found among elite trees (Sasaki 1996). Tetraploid plants have been rarely found in either artificial or natural forests (Kikuti and Mori 1985). However, the morphological traits associated with tetraploids are unsuitable for forestation because most seedlings exhibit dwarfism, tissue-specific gigantism, and low growth rate (Kikuti 1997). Some of these plants have been preserved in the Forest Tree Breeding Center, Hitachi, and the Forestry and Forest Products Research Institute, Tsukuba. Variations among natural forests of C. japonica across the species’ geographical range have been investigated using both morphological traits (needle length, needle curvature, and other features; Murai 1947) and diterpene components (Yasue et al. 1987). The results of these studies suggest that there are two

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_3, # Springer-Verlag Berlin Heidelberg 2011

49

50

Y. Tsumura

N

Japan Sea

Oki Is.

Wakasa Bay

Izu Peninsula

Shikoku Kii Peninsula

Kysushu

Pacific Ocean

Yakushima Is.

Fig. 3.1 Natural distribution of Cryptomeria japonica (shaded areas; from Hayashi 1960). The dotted line indicates the coastline ca. 18,000 years ago. Areas shaded in bold or within thin

diagonal lines are the refugia (Izu Peninsula, Wakasa Bay, Oki Is. and Yakushima Is.) and probable refugia, respectively, at that time (Tsukada 1986; Takahara 1998)

main lines, known locally as ura-sugi (C. japonica var. radicans, found near the Sea of Japan) and omote-sugi (C. japonica, found near the Pacific Ocean). The urasugi variety has slender branchlets with soft needles, whereas omote-sugi has rough branchlets with hard needles (Yamazaki 1995). As the sugi tree has a straight bole with soft wood; it can be split easily, even using primitive implements. Therefore, for hundreds of years it has been used for house construction, to build wooden ships and for wooden barrels, as well as for making many items of daily use (Ohba 1993). It is believed that humans started planting sugi more than 500 years ago in districts of Kyoto and other southern areas of Japan (Tokugawa 1974). Currently, the species has been

planted widely throughout Japan and now covers an area of 4.5 million ha, accounting for 44% of all the Japanese artificial forests. Fifteen million seedlings are supplied as planting material for forestation every year, making this species very important for Japanese forestry now, as it has been since ancient times (Forest Agency 2008).

3.2 Origin and Evolution of C. japonica As described earlier, C. japonica was originally classified as belonging to the family Taxodiaceae, but recent molecular studies using chloroplast sequence

3 Cryptomeria

51

Table 3.1 List of all species belong to Taxodiaceae and some species of Cuppresaceae Family Genus Species Chromosome Genome size Natural distribution number (pg/C) Taxodiaceae Athrotaxis cupressoides 22 11.03 Tasumania, Australia Cryptomeria

japonica

22

11.05

Cunninghamia

lanceolata

22

14.17

Honshu, Shikoku, and Kyushu Is., Japan China, Vietnam, Laos

Glyptostrobus

kawakamii konishii pensilis

22 22 22

– – 9.99

Taiwan Taiwan Southern part of China

Sequoia

sempervirens

66

32.14

Sequoiadendron giganteum

22

9.93

California and South Oregon, USA California, USA

Metasequoia

glyptostroboides

22

11.04

China

Taiwania

cryptomerioides

22

12.89

flousiana

22

13.4

distichum

22

9.95

Myanmar, Vietnam, Taiwan Northern Myanmar and China Southeast USA

mucronatum

22

8.75

Mexico Hoshu and Shikoku Is., Japan Honshu, Shikoku, and Kyushu Is., Japan Honshu, Shikoku, and Kyushu Is., Japan Honshu, Shikoku, and Kyushu Is., Japan Korea, Manchuria, and northern China Southwestern and central China Japan, Korea, China

Taxodium

Cupressaceae

Thuja

standishii

13.43

Thujopsis

dolabrata

–

Chamaecyparis

obtusa

22

10.02

pisifera

22

10.21

Platycladus

orientalis

22

10.46

Cupressus

funebris

22

10.76

Juniperus

rigida

22

12.15

chinensis

–

–

communis

–

–

data have revealed that Taxodiaceae and Cupressaceae are closely related families (Table 3.1, Fig. 3.2; Gadek et al. 2000; Kusumi et al. 2000). The morphological and chemical traits and immunological studies support a monophyletic origin for the two families (Hart 1987; Price and Lowenstein 1989). Species belong to Taxodiaceae are found in North America, East Asia, and Tasmania. Cryptomeria fortunei (C. japonica var.

References Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001)

Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Ohri and Khoshoo (1986) Hizume et al. (2001)

Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001) Hizume et al. (2001)

Japan, Korea, Taiwan, Sakharin The cool temperate Northern Hemisphere

sinensis) is described from China, but there is no difference in cpDNA sequences between C. japonica and C. fortunei (Kusumi et al. 2000). Glyptostrobus and Taxodium are the closest genera to Cryptomeria, which is successively more divergent from the following orders: Cuppresaceae, Sequoia/Sequoiadendron/ Metasequoia, Athrotaxis, Taiwania, and Cunninghamia. Chamaecyparis obtusa in the Cupressaceae

52

Y. Tsumura

99

Taxodium disticum ascendens Taxodium macronatum Gryptostrobus pensilis Cryptomeria fortunei Cryptomeria japonica Chamaecyparis pisifera 100 Chamaecyparis obutusa

76 Taxodium 100

100

100 64

Juniperus rigida

100 80

64 80

Thujopsis dolabrata Thuja standishii Sequoia sempervirens Sequoiadendron giganteum Metasequoia glyptostroboides 100

100

Athrotaxis laxifolia 98

Athrotaxis selaginoides Athrotaxis cupressoides

100

Taiwania cryptomerioides

80

100

Taiwania flousiania Cunninghamia lanceolata

100

Cunninghamia konishii

Fig. 3.2 Molecular phylogenetic tree based on chloroplast DNA sequences (Kusumi et al. 2000). This tree was constructed using the neighbor-joining method based on Kimura’s two parameter distance. Branch lengths are proportional to the scale given in nucleotide substitutions per site. Numbers at internal nodes are bootstrap values based on 1,000 replicates. This tree is rooted with Cunninghamia

family is another important conifer in the Japanese forestry. This is more closely related to C. japonica than previously thought, based on morphological data. We have applied the sequence tagged site (STS) markers that were developed in C. japonica to C. obtusa, and about 30% of STS markers in C. japonica were directly transferable to C. obtusa (Matsumoto and Tsumura 2004). According to the fossil record, C. japonica had appeared in Japan about 18 million years ago (Uemura 1981) when the formation of Japanese archipelago had started after leaving from Asian Continent (Taira 2001). A physical map of chloroplast DNA (cpDNA) in C. japonica has been constructed (Tsumura et al. 1993. A large inverted repeat was lacking, as reported for two Pinaceae species (Strauss et al. 1988), but the C. japonica structure was quite different from the Pinaceae species. A whole genome sequence of cpDNA in C. japonica was recently published, and the different structure from Pinus species was confirmed in details (Hirao et al. 2008). These results also

corroborate the evolutionary analysis of this species using nuclear genes (Kusumi et al. 2000, 2002).

3.3 Genetic Resources The natural forests of C. japonica have declined because of over-exploitation during the past 1,000 years, and natural stands of the species are now small and isolated, mainly found in mountainous regions. Some of the remaining natural forests have been designated for the conservation of genetic resources by the Forestry Agency (Forest Agency 1995). Twenty-seven C. japonica natural forests located throughout the natural distribution range have been designated as in-situ conservation forest (Forest Tree Genetic Resources Conservation Forest; FTGRCF). However, most of the conservation forests are small, around 10 ha or less. The exception is Yakushima Island, where the most southerly and the largest natural population of C. japonica covers more than 6,000 ha (Tsumura and Ohba 1993). The site is a UNESCO World Heritage Site, so the forest is strictly conserved (Figs. 3.1 and 3.3). The current natural populations of C. japonica are thought to have dispersed from refugia occupied during the last glacial period, ca. 20,000 years ago (Tsukada 1983, 1986). The locations of the refugia have been inferred from fossil pollen data and are around the Izu Peninsula, from Wakasa Bay to Oki Island, and Yakushima Island. Several small natural forests still remain in these areas. These forests possess relatively higher genetic diversity than the secondary forests established after the ice age (Takahashi et al. 2005; Tsumura et al. 2007). Thus, in-situ conservation of these areas is essential for genetic conservation and for providing future breeding material for a species that has potentially a very broad distribution. To augment in situ conservation, twigs have been collected from 31 populations and stored as cuttings in the arboretum of the Forestry and Forest Products Research Institute (FFPRI), Tsukuba. Some natural forests and plantations, especially old superior ones, have also been conserved as genetic resources (Forest Tree Superior Gene Conservation Stand; FTSGCS). A total of 101 stands have been designated as FTSGCS throughout Japan (Table 3.2). Most of these FTSGCS are private forests. FTSGCS can be logged, only when successor FTSGCS stands that are afforested using seedlings derived from seeds of the FTSGCS are delineated.

3 Cryptomeria

53

for resistance to insects such as Semanolus japonicus and Resseliella odai, disease and severe climate conditions were also selected from severely damaged sites (Tree Breeding Society 2004). These superior trees are stored as cuttings or grafted seedlings at the Forest Tree Breeding Center of FFPRI and have been supplied to local forestry offices for reforestation and for use as breeding material. The seeds and pollen of elite trees and the candidate trees for insect and disease resistance are also stored at clone bank and/or germplasm (seed and pollen) in the Forest Tree Breeding Center, from which they can be provided for research and other purposes (Table 3.1).

3.4 Genetic Studies of C. japonica

Fig. 3.3 The oldest tree of C. japonica in Yakushima Island at an elevation of 1,300 m, named, Jomon-sugi. The tree is 25.3 m tall, the trunk circumference at breast height is 16.4 m and its estimated age is more than 2,000 years

Table 3.2 Number of ex-situ and in-situ conservation forests and material from C. japonica in Japan available through the Forest Tree Breeding Center In situ Ex situ Forest Genetic resource 26 2 Gene conservation 38 63 Plus tree – 3,094 Seed – 1,964 Pollen – 811

Since 1960, more than 3,700 elite trees have been selected, mainly from the artificial forests (some trees were selected from natural populations also included somewhat), to use as material for forest planting and breeding. Selections have been based on a high tree growth rate, narrow tree crown and other superior traits (Tree Breeding Society 2004). Candidate trees

Many somatic mutants of C. japonica have been found in self-pollinated seedlings of elite trees and in some cultivars in nature and after gamma radiation (Ohba and Murai 1971). The inheritance modes of these mutants have then been determined (Table 3.3; Ohba et al. 1967, 1974; Kikuti and Ohba 1981). Mutated traits that have been found include abnormal chlorophylls, waxless seedlings, dwarfism, deformity (twisting), and abnormal pigmentation (green needles during winter). Of particular note was the discovery of the paternal inheritance of a chloroplast mutant (Ohba and Murai 1971), which was confirmed at the DNA level (Hirao et al. 2009) in other conifers by Neale et al. (1989). Most of these materials have been preserved for future research. Some male-sterile mutants have also been found in artificial plantations and among elite trees (Taira et al. 1993). These male-sterile trees have been used for breeding and as a material for afforestation, as a countermeasure against pollen allergens in C. japonica. Most of these mutants have recessive inheritance and maintain their recessive allele in the population at low frequencies. These mutants may survive rarely in nature because of their recessive phenotypes and no pollen production, but seedlings derived from artificial crossings can survive under controlled conditions. Genetic studies of the allozymes of 13 enzyme systems have been undertaken using artificial crossings between 36 families of C. japonica (Tsumura et al. 1989). These allozyme markers have been used

54

Y. Tsumura

Table 3.3 List of morphological and biochemical mutants in C. japonica Mutant Characteristics Chlorophyll Albino Light green White primary leaf Variegated Green color leaves in winter White to yellowish white shoots in spring Morphology twisted Multiple branching Biochemical Diterpen, phyllocladene Diterpen, Kaurene Diterpen, Sclarene Wax-less Allozyme Lethal

mainly to study population genetics (Tsumura and Ohba 1992, 1993; Tomaru et al. 1992, 1994). However, no clear differences between the two varieties have been detected in the previous studies using the limited number of available genetic markers (Tomaru et al. 1994; Tsumura and Tomaru 1999). Population genetic studies were also conducted using simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers. According to the result using 11 SSR markers, the genetic differentiation was low (FST ¼ 0.028); however, there was a trend toward high allelic diversity in five populations, which are very close to, or in, refugial areas of the last glacial period as defined by Tsukada based on pollen analysis data (Takahashi et al. 2005). According to the results of 148 CAPS markers, the genetic differentiation between 29 populations was also low (GST ¼ 0.050), however, populations within the same district or group of districts were generally clustered, and thus showed a clear geographic trend; omote-sugi variety (Pacific Ocean side) populations cluster together in a clade with relatively high bootstrap support (Tsumura et al. 2007). Among 148 loci, two were closely related to the putative divergence of the two varieties. The two genes are probably associated with the differentiation between populations. Populations of the ura-sugi variety are mostly homozygous at these loci, implying that they may confer a selective advantage to this variety. In accordance with the inferred evolutionary history of the species during and after the last glacial episode, genetic diversity was higher in western populations than in northern ones (Tsumura et al. 2007).

Inheritance mode Recessive Recessive Recessive Recessive Recessive Paternal Dominat Recessive Dominat Recessive Recessive Recessive Co-dominant Recessive

References Ohba et al. (1974) Ohba et al. (1967) Ohba et al. (1967) Kikuchi (1978) Chiba (1953) Ohba and Murai (1971) Ohba et al. (1974) Ohba et al. (1974) Yasue et al. (1978) Yasue et al. (1978) Yasue et al. (1978) Ohba et al. (1973) Tsumura et al. (1989) Kawasaki et al. (1985)

3.5 Tree Improvement Through Traditional and Advanced Approaches The development of clonal forestry began in 18th century in the western part of Japan (Fig. 3.4). Foresters in these areas have been selecting superior trees and maintaining these cultivars as cuttings for several 100 years (Ohba 1993). There are 32 named cultivars, each of which is made up of several clones, a major single clone with one or more minor clones. The number of clones per cultivar has been investigated using allozyme or DNA markers such as RAPD, CAPS, and SSR (Goto et al. 1999; Ieiri 2003; Hirao et al. 2006; Kusano et al. 2006). Currently, there are more than 200 selected cultivars in the western part of Japan. After selection, these elite clones and their offspring have been tested for growth performance, and resistance to disease and insects, at several different sites. The best plants were reselected on the basis of the results of further progeny testing and used to plant second-generation seed orchards. In total, 1,265 progeny test forests of this species have been established in Japan (Tree Breeding Society 2004). Genetic improvement through artificial crossing has been conducted since 1980. Controlled pollinations were conducted to establish sets of breeding populations. Each breeding population was consisted of progenies from 24 clones. Six sets of four-parent halfdiallel crosses, which aimed at improving single trait, or three sets of six-parent factorial crosses, which aimed at improving two traits simultaneously, were used. The

3 Cryptomeria

55

a

b

Fig. 3.4 Clonal forest (a) on Kyushu Island and seedling forest (b) in the Yoshino area, Nara Prefecture, a famous forestry region in Japan

a

b

Fig. 3.5 Common (a) and miniature (b) seed orchards of C. japonica. The spacings in common and miniature seed orchards are 2.5  2.5 m or 5.0  5.0 m and 1.0  1.0 m or

1.5  1.5 m, respectively. The tree heights in each orchard are 4 m and 1–2 m, respectively

targeted traits, such as growth, wood property, disease resistance, or other traits, of the controlled-pollinated progenies have been tested in progeny tests with six replications. More than 100 sites have been delineated so far. Target traits for improvement include growth performance and wood characters such as bole straightness, wood density, moisture content, strength of wood, and heartwood color. Other traits are resistance to cold wind damage, to frost damage, to snow damage, and to insects, and also shade tolerance (Tree Breeding Society 2004). Morphological data have been collected to evaluate the criteria and the necessary period of selection to maintain the second generation of elite trees. Seed orchards are important sources of seeds for reforestation programs and have been established by setting out superior clones of trees as grafts or rooted cuttings selected for desirable characteristics. The quality of the seed produced in a seed orchard depends on isolation from other potential sources of pollen contamination, low levels of self-fertilization, random mating, synchronization of flowering phenology, equal produc-

tion of female and male strobili, and equal compatibility of all crosses (Eriksson et al. 1973; Woessner and Franklin 1973). To evaluate the quality of seeds from seed orchards, we used microsatellite markers to investigate gene flow in five Cryptomeria japonica seed orchards of two different types (common and miniature), at widely spaced locations (Fig. 3.5). The quality of a seed crop is determined by many factors, including pollen contamination from outside sources, selffertilization, and the proportion of contributions from constituent clones. Contamination rates were found to vary among individual ramets per clone, both within seed orchards (10.0–76.7% in the most variable seed orchard) and among seed orchards (35.0–65.8% on average, Moriguchi et al. 2005). Among ramets, there were significant negative correlations between pollen contamination rate and their distance from the orchard edge. Among seed orchards, there were significant positive correlations between the pollen contamination rate and the C. japonica forest area nearby. Self-fertilization rates varied among seed orchards

56

Y. Tsumura

(1.4–4.4% on average), and there were significant positive correlations between self-fertilization rate and the number of ramets per clone in both types of seed orchard. Inbreeding depression in conifers is a serious problem, especially, for seed production. Contributions as pollen donors differed significantly among clones in all seed orchards (Moriguchi et al. 2004, 2005). The distance between planted ramets, flowering phenology, and relative pollen fecundity may also have contributed to observed differences in paternal contribution. The influence of these factors on genetic potential did not differ greatly between the two types of orchards. These results suggest that increasing the number of clones in an orchard may decrease the self-fertilization rate. To promote equal paternal contributions, improvements could be made by stocking orchards with clones that have similar flowering phenologies and male cone production levels. Combining supplemental mass pollination (SMP) with the enclosure of the seed orchard in plastic sheeting to reduce external pollen supply would be a good approach to improving the seed production of C. japonica orchards in Japan.

3.6 Genomic Resources and Molecular Markers Expressed sequence tags (ESTs) have been constructed using cDNA libraries from different tissues, including seedlings, inner bark, sapwood, male flowers, female flowers, and pollen (Ujino-Ihara et al. 2000, 2005; Yoshida et al. 2007). Recently, a full-length cDNA library was also constructed from male flowers and Table 3.4 Linkage maps of C. japonica Type of No. of Genetic markers pedigree plogeny F2 (selfed) 73 RFLP, RAPD, Isozyme 87 AFLP, CAPS F1

36,011 ESTs were obtained from either one or both ends of 19,437 clones derived from the cDNA library of C. japonica male strobili at various developmental stages. Approximately 80% of the transcripts resembled ESTs from Pinus and Picea, while approximately 75% had homologs in Arabidopsis (Futamura et al. 2008). Currently, more than 55,000 ESTs are available for C. japonica. Microarray analysis is being used to isolate candidate genes for male sterility (N. Futamura et al. unpublished data). Using EST information, STS, CAPS, and SNP markers have been developed for mapping and population analyses. Many molecular markers have been developed for this species based on EST data, in particular SSR (Moriguchi et al. 2003; Tani et al. 2004), STS, CAPS (Tsumura et al. 1997; Iwata et al. 2001; Nikaido et al. 2000; Tani et al. 2003), EST-SSR (Moriguchi et al. 2009), single-stranded DNA conformation polymorphism (SSCP; Ujino-Ihara et al. 2002), and SNP (Ujino-Ihara et al. 2010; Uchiyama et al. unpublished data). The database of genomic resources of C. japonica, particularly EST information, is available from http:// forestgen.ffpri.affrc.go.jp/en/index.html. DNA markers and linkage map information are also available from http://ss.ffpri.affrc.go.jp/labs/cjgenome/index.html.

3.7 Linkage Map and QTL Study The first linkage map was constructed using a threegeneration pedigree, F2 and mainly RFLP markers (Mukai et al. 1995; Table 3.4). A clustering of markers with distorted segregation was observed in six linkage

No. of Map length mapped loci 145 887.3 cm

Expected cm per coverage, % marker NA 10

91, 132

40/62

13/15

F1

72

RAPD

119, 84

F2 (a half-sib cross), F2 (selfed)

150

438

F2 (a half-sib cross)

150

CAPS, SSR, SNP, RFLP, RAPD, Isozyme CAPS, SSR, SNP, RFLP, HRM

1,266.1 cm, 1,992.3 cm 1,756.4 cm, 1,111.9 cm 1,372 cm

1,408

1,950.9 cm

50/80

16/18

96

3

99.9

1.34

References Mukai et al. (1995) Nikaido et al. (2000) Kuramoto et al. (2000) Tani et al. (2003)

Moriguchi et al. unpublished

3 Cryptomeria

groups because of inbreeding depression. Other linkage maps have since been constructed, mainly using amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers (Kuramoto et al. 2000; Nikaido et al. 2000). The consensus linkage map was constructed using a halfsib F2 and selfed F2 populations, using CAPS, SSR, and RFLP markers (Fig. 3.6; Tani et al. 2003). A total of 438 markers were assigned to 11 large linkage groups, one small linkage group, and one nonintegrated linkage group from the second crosses; their total map length was 1,372.2 cM. On average, the consensus map showed one marker every 3.0 cM. PCR-based co-dominant DNA markers, such as CAPS and SSR, were distributed across all linkage groups and occupied about half of the mapped loci. To construct a high-density map, we have mapped SNP marker using GodenGate genotyping system into a consensus map. More than 800 SNP markers were newly mapped into the consensus map, whose total length was 1,390 cM (Moriguchi et al. unpublished data). Molecular breeding tools have been applied to the development of superior plants, such as quantitative trait loci (QTL) mapping, seed orchard improvement using molecular markers, and genome mapping of the malesterile gene. Yoshimaru et al. (1998) mapped QTLs to investigate the genetic control of growth, flowering, and rooting ability in C. japonica. Growth is one of the most important traits for timber-producing woody species and also for carbon dioxide fixation to mitigate global warming. Loci that code for juvenile growth, including height and diameter of basal area, have been mapped. Flowering is essential for reproduction, but is not necessary for timber production. If the expression of flowering could be controlled, it would be useful not only for breeding but also for forestry and the environment. QTLs for male and female flowers have been mapped at two locations each, respectively. The rooting ability of this species is very important for clonal forestry in the southwestern part of Japan, especially in Kyushu Island (Ohba 1993). QTLs for rooting ability have been mapped but the trait is not highly significant in this family (Yoshimaru et al. 1998). Kuramoto et al. (2000) detected 15 QTLs associated with module of elasticity (MOE), an important indicator of wood strength. The QTLs for MOE explained about 45% of its total phenotypic variance. Accumulating of QTLs associated wood quality is very important for future molecular breeding of this species

57

because the average wood strength is not so strong comparing to that of pine species.

3.8 Current Problems with C. japonica Recently, allergic reaction to pollen (pollinosis) of this species has become a serious social problem. More than 10% of people living in Japan suffer reactions to C. japonica pollen between the end of February and early May (Taira et al. 2000). This pollen allergy was first reported in 1964 (Horiguchi and Saito 1964). Since the 1950s, C. japonica has been widely planted and now accounts for 44% of the afforested area. Many of these trees have reached maturity and are now flowering. To address this problem, several approaches have been attempted by the Forestry Agency in Japan. They have developed and supply male-sterile individuals and individuals with low pollen fecundity. The Agency also promotes selective thinning of individuals with high pollen fecundity, and the Ministry of Environment provides forecasting of pollen dispersal and real time pollen dispersal data. A male-sterile individual was first found in Toyama prefecture and the trait was shown to be expressed by a recessive homozygote (Taira et al. 1993, 1999). Research projects are working to identify the malesterile gene using mapping and microarray methods. If the gene can be isolated, elite trees with male sterility will be easily developed using the RNAi method.

3.9 Perspectives Cryptomeria japonica, as a native tree, is adapted to the Japanese climate and environment. It is a fast-growing species, useful to the Japanese forestry industry. However, further tree improvement, for growth rate, wood properties, bole straightness, and pollen fecundity, is still necessary. Cryptomeria japonica is fast growing, but if it could grow even more as rapidly like Pinus taeda (Li et al. 1999), which is one of the good examples for genetic gain of tree breeding, it would be beneficial, not only in forestry but also in mitigating the global warming. Wood density and strength are not as high as that of pine species because the water content of the wood is high (Nakai and Yamai 1982). Therefore, improvement

CC0421C CC1319C CC2752C CC0404C CC0622C

R02ak x o CJ S0333

CC1505R1

A08bk x o CC1505R2

CC1911Ck xo CC1664R CD1256Rk xo CP1379R CP1262R1 CD0352Rk xo CC1479R

CC3098C4 CC3098C3 CC0802Ck xo CC3098C2 CC1721C CC1650R2 CD0019Rk xo CC1650R1

CC3098C1

CP1112R

10.6 14.0 14.8 15.4 15.9

24.4 26.0

33.1

37.4 38.6

45.9 49.5 52.0 52.6 52.8 55.2 59.2

67.0 67.4 69.0 70.7 71.2 73.6 74.1

89.8

101.4

K06ak x o CC1698C CC2895C CC3133Ck x o CD0536Rk xo CD0622Rk xo CC1724R CD0511Rk xo CC3872C CC3330R CD0440R CD1278Rk xo CD1514Rk xo CD1852Rk xo CC1704C CD0810Rk xo CS2169 CP1510R CD0633R CD1914Rk xo CC0787C2 CC0411C CC0616C CD1617Rk xo CP1207R B11ak x o CC1747R CD1762Rk xo CC1802R A19c k xo

CC0911R

CC1884R

CC1756R

130.8

140.3

157.4

CJ G0175

CD0423R CC0846Ck x o CC1825R CC1674R1 CC0864C2 CC2123C CJ S0686 CD0857Rk xo CC1748Ck x o CC1748R O02ak xo CC0864C1 CC1730Ck x o CC3367C CD0355Rk xo CD1761Rk xo CS1413

CP1145R CP1076R

CP1286R

68.9 72.9 74.3 76.6 78.3 78.6 82.8 85.0 87.7 90.9 91.6 92.6 93.3 95.4 97.0 97.8 98.5 103.1 104.1 104.3 107.4 109.1 110.3 113.5 114.8 115.4 118.7 123.0 124.1

65.5

31.9 33.0 34.6 40.0 41.2 42.2 42.8 43.0 45.5 47.6 48.9 49.0 49.1 50.9 51.3 56.5 58.2

21.2 24.1

0.0

YA&KO-02

CC2713C CC3336C CC0460C

81.8

88.3 88.4

CP1501R CP1194R2 CC1634R CC1641Ckxo CD1712R CC1158C CD1484Rkx o CJS0268 CC1811R2 CC1811Ck xo CC2645C CD0344R CD0334R2k xo CD1712C CC1811R1 CC1673R CC2522C CC0673C CC2856C CJS0584 CS1985 CC2052C CC1620C CS2024

CC0489C CD1154Rkxo CD1924R1k xo

15.5 16.6 19.1

25.2 28.4 30.0 31.6 33.1 35.3 35.7 35.9 36.7 36.9 38.0 38.7 39.6 41.0 41.4 43.8 44.1 45.9 48.8 53.3 55.0 55.2 56.9 64.1

CC0906Ckxo CD1924R2k xo G10bkx o CC1508R

0.0 1.5 2.5 3.4

YA&KO-03 CC0342C CC1876C CD0337Rkxo CD1130Rk xo CS1737 CC3416C CC0493C CC1125C CC0520Ckxo CD1875C CD0689Rkxo CC1831R CC0734C

3.7

CC2467C CC1488R2 CD0517R CC2657C

Cry j2C

95.6 96.7 97.8 101.7

CC1902R

86.9

CD0440R

77.6 83.6

CC1488R1 CD0344R

68.5 71.5

CC0432C CC1632R

CD1946Rkxo CC0283Ckxo CC1912R CC0707C

48.7 49.7 51.7 54.3 57.6 60.1

CC0390C CC1490R CD1547Rkx o CD0682Rkx o CD1924R3k xo CC2583C

37.3 37.7 40.5 41.5 41.8

9.1 11.2 13.4 14.3 18.1 18.4 18.6 20.2 22.9 23.8

0.0

YA&KO-04

CJ S0520 CC1176C CC1928C CD0041C CJ S0955

124.4 126.5 127.4 128.7 133.0

CJ S0331

CC0803C2

87.9

99.9

CC1303C

K06bkx o GD3206k x o CC1613R CC1613C CC1274Ck x o CD1942Rk x o CC1367C CC1767R CC1287Ck x o CJG0170 CC1717C CC1674R2 N11akx o CJG0083 CS2294 MT5002k x o CD0195R CS1281

CD0461Rk x o CD0216R

CC0462C CD1091R CC1778R CD0217Rk xo CD1079Rk xo CD0515R CD0548R CJS0002 Cry j1C CC2989C CD1309Rk x o CD0785Rk x o CJG0125

CD0504R

84.8

47.4 50.6 51.7 53.1 54.0 55.2 56.4 56.5 57.3 57.8 58.7 61.0 61.7 63.8 64.4 67.2 69.7 75.2

34.9 35.8

7.1 7.9 10.5 11.3 12.2 13.9 14.5 14.8 16.9 17.5 23.6 25.1 27.5

0.0

YA&KO-05

140.6

99.5 102.1 103.5 105.5 107.4

86.3 87.2

81.0

77.2

CC3413C

CC0784C CC1654R2 CC2469C CC1829R CC1654R1

CC1521R CC1520R

CC1510R

CC1635R

CC3839C CP1262R2

64.9

D07dkxo CC3430C CD0791Rkx o CD0788Rkx o CC1727R CC0308R CD1839Rkx o CC0318C O04bkx o CP1007R

68.7

CC1814R

33.9

A11bkxo O02bkx o CJ S0665 CJG0122 CP1463R CC2731Ckx o CC0680Ckx o CC1921C

CC2781C

CD1111C1

39.5 40.2 41.0 42.1 43.9 45.4 47.7 50.6 52.5 55.8

20.6 21.4 22.6 23.2 24.5 25.2 25.9 27.8

7.2

0.0

YA&KO-06 CC1791R2

144.6

CC1511R2

CC2588C

CC1524R CC3816Ckx o

120.2

CC0757C CD2036Rk x o

102.9 105.2

CC0300Ckx o

108.9 109.6

CC1244Ckx o

94.6

CD1067Rkx o CD1658Rkx o

CD1545R

CC2939C2

J13akx o CJS0838 CC2340Ckx o CC2846Ckx o CP1203R CJS0091 CC2939C1 CC2939C3 CC2921C CC1263C CP1488R

N19ak xo CD2039C CD1317Rkx o CC2188C

90.9

85.9 87.3

81.7

77.7

57.4 58.7 59.7 64.2 65.9 66.0 68.0 69.0 70.9 72.0 73.3

43.1 43.7 46.2 46.9

CS1579 CJS0401

38.2

CC0613C CJG0078 CC1625C CC1791R1 CP1308R

CJG0013

CD0737R

35.2

25.0 26.6 26.7 31.1

19.7

6.8

0.0

YA&KO-07

121.4

62.0 64.7 68.0 71.4 77.7 79.9 81.6 83.2 84.5 85.4 86.7 87.3 91.7 92.7 93.0 93.4 95.3 96.3 97.6 99.0 100.1 101.8 102.8 103.3 105.2 106.3 108.3 109.3 111.6

43.8 46.0

31.7

15.4

0.0

CD1821Rkx o

CD1613R P12akxo CC1846R3 CC1846R1 CC2333C CC1846Ckx o T11bkxo CD0485Rkx o CC1806Ckx o CC0524Ckx o CC1806R CD1583Rkx o CD0015R CD2045Rkx o CP1221R CD1147Rkx o CP1190R CD1234Rkx o CD2043Rkx o CD0491R CD1066R CC1484R CD1237R CC0797C1 CD1301R CD1894Rk x o CC2750C CC1135Ck x o CC0428C GD3183k x o CC1651R

CC1511R1 CC0374Ckx o

CC3816C

CC1771R

CC0300R

YA&KO-08

140.2

128.3

124.0

123.4

56.5 57.8 60.0 62.0 62.4 62.7 63.1 64.1 64.3 68.1 68.2 68.8 70.2 71.0 73.5 76.9 77.4 77.6 77.7 79.6 79.7 83.4 84.3 89.5 95.2 97.0 98.9 109.4 109.9 110.7 110.8 115.9

CP1346R

CD0811R CD0177Rkx o GD3325k xo CC1298Ckx o CD1608Rkx o

CD0742Rkx o CD1195R CC3823C CC1601R CC2746C1 CJS0485 CD1747Rkx o CD0414R CD1425Rkx o CC1147C CC0924Ckx o CC1112Ckx o CC2746C2 CP1230R CC1689C CC1485R CD1747R CC0546C CD1831Rkx o CC2683Ckx o CD0470Ckx o CC0747Ckx o CD0470Rkx o Lapkxo CP1194R1 CD2055Rkx o CC1774R CC1774C CS2484 CC0337C CJS0455 CC2631Ckx o CC2702Ckx o CJS0356

CC1629C2 CC1629R CC0787C1

CC1787R CC0551C

28.9 32.4 34.2

45.2 47.0 47.1

CC2909C CD2043R

25.3

CC1476R

CC1681R2

18.0

CC1681R1

9.7

CC1786R

CC0539C2 CC0539C1

13.0

6.6

0.0 0.6

YA&KO-09

97.4 99.7

67.0 67.6 69.8 72.2 74.3 79.6 81.9 83.9 84.1 84.2 84.3 84.5 87.7 88.2

60.4

53.7

35.4

26.0

0.0 3.4 3.5 4.7 4.9 6.4 8.5 8.8 9.2

CD2026R CJG0101

CD2029Rkx o CC1545Ckx o CC2541C1 CD1325R CC1633R CJG0177 CC2081C CC0796C CC1504C CC2577C CC0625C CC0937C CD2076Rkx o CD1385Rkx o

CC2700C

CC2541C2

CD1126R1

CD1126R2

S10ck xo CD0482Rkx o CC1604R CC2676Ckx o CD0482C CC1874R CD1769Rkx o CC0285C CC2795C CC1670R

YA&KO-10

62.9 64.0 64.2 64.4 65.0 65.4 66.9 70.2 71.7 72.3 77.5 79.8 82.8 83.8 84.2 85.4 87.6 87.8 89.9 90.6 91.5 98.0

54.4

39.4 42.3 45.3 47.1

0.0 2.2 5.2 7.7 8.0 10.3 12.7 16.0 17.8 18.8 19.9 20.3 23.2 25.4 27.6 29.6

CD0526Rkx o CD2035Rkx o CC0858C CC0719C CD0526C CD2035C CD1179C2 CD0523Rkx o CS1226 CC1204C CC1015C G05bkx o CD1249Rkx o CD1569Rkx o CD1937Rkx o CC2643C R02bk xo CC1172C CC0731C CC0803C1 CC0590Ckxo CC2377C CC0803Ckxo CC1720R

CC1395Ckxo

CS1522 CS2056 CP1268R CD0569R

CC0482C CC0343Ckxo CC0501Ckxo CC1637R CD1232Rkx o CJG0193 CD0131Rkx o B17akxo B12akxo CD0167Rkx o CC1483R CD0657R CD0620Rkx o CD1706Ckx o CC2946Ckxo CC2448C CC1148C

YA&KO-11

Fig. 3.6 Consensus linkage map of C. japonica. The linkage groups were derived from integration analysis of both sets of segregation data from the YI (Yabukugiri  Iwao) and KO (Kumotooshi  Okinoyama) pedigrees with JoinMap 3.0 (Tani et al. 2003)

CC0980C CC1402C CC0344C CC0392Ck xo CC1014Ck xo

CC0536C

-2.7 -2.0 0.0 0.9 1.5

-19.6

YA&KO-01

58 Y. Tsumura

3 Cryptomeria

of wood properties, such as strength and water content, is essential (Fujisawa 1998). The stem is sometimes bent by snow pressure, especially in regions prone to heavy snow falls. Pollen of C. japonica is a severe cause of pollinosis in Japan, as described in the previous section. In commercial forests, pollen is not required and could be dispensed with. Therefore, if we could reduce pollen fecundity or eliminate pollen production from commercial plantations, not only would pollinosis be reduced, but the important conserved natural forests would be protected from genetic contamination via pollen. To achieve these improvements, information from genome-wide association studies between genotypes and traits will be useful. Genomic selection based on these results will be an effective method to improve forest tree species (Iwata et al. 2011). Genome-wide association studies have been carried out in humans to detect genes associated with diseases (Hirschhorn and Daly 2005). This method is strongly dependent on the extent of linkage disequilibrium (LD) and the number of analyzed loci. In conifers, including C. japonica, LD generally decays over a relatively short length, such as a few thousand base pairs (Kado et al. 2003; Neale and Savolainen 2004). Therefore, if a large number of single-nucleotide polymorphism (SNP) markers derived from ESTs were used, the important SNPs or genome regions associated with traits would be detected. Genetic structure, such as genetic differentiation between populations, is relatively low in conifers compared with many crop species (Hamrick and Godt 1989), because they are dominantly allogamous and wind-pollinated species, and the generation time is longer than for other plant species. Nucleotide diversity of conifers is relatively high, so genome-wide association studies to detect SNPs within genes are appropriate (Neale and Savolainen 2004). The nucleotide diversity of C. japonica was estimated to be 0.0035–0.0044 (Kado et al. 2003, 2006, 2007; Fujimoto et al. 2008), while those of crop species ranges from 0.001 in Oryza sativa to 0.0173 in Zea mays (Tenaillon et al. 2001; Zhu et al. 2007). The value is strongly related to the domestication process of each species. Even in economically important conifers, nucleotide diversity is not lost through domestication. Most advanced species, such as humans, have relatively low nucleotide diversity (Li and Sadler 1991; Cargill et al. 1999). Conifers have

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ample diversity and are therefore suitable for a genome-wide association study. For evaluation of phenotypes, data from progeny testing with many replicates can be used. The progeny test forests have already been established in different environment sites all over Japan. Precise evaluation of phenotypes is essential to detect the true association between SNPs and the traits, so large numbers of replicates are required. The results of genome-wide association studies will be used for the selection of superior trees through genomic selection. Genomic selection is the method for selecting superior phenotypes using genome-wide SNP information without phenotype data (Meuwissen et al. 2001; Heffner et al. 2009). This method has been used successfully in animal breeding. For example, superior male dairy cattle have been selected to enhance milk production (Schaeffer 2007). The advantages of this method are that selection can be undertaken without phenotype data, and minor effect genes for the target phenotype can be detected. These are difficult to detect using the QTL approach. It is not necessary to conduct controlled pollination to construct the model for selection, which means that existing materials and data, such as elite tree populations and the progeny test data, can be used for the model for selection. The approach is most effective when combined with generation acceleration. Protection and conservation of natural forest trees are important issues for future breeding material. The dominant forestry tree species tend to have a large plantation area, and sometimes they have been established close to natural forests. As a result, genetic contamination will occur, with genes spreading from plantations to the natural forests. Genetic diversity and the structure of natural populations may be disturbed by gene flow from the plantations. Even in the native forest, where there is no plantation nearby, conifer pollen flow occurs over large distances, sometimes over several tens of kilometers (Moriguchi et al. 2005). Fortunately, forest tree species are long-lived and can maintain their populations for several 100 years, even if the newly produced seedling populations cause genetic disturbance. Therefore, an ex-situ conservation program is currently the best way to conserve the genetic diversity and structure of natural populations of C. japonica. Plant material conserved in this manner could be used as future sources of breeding material and to facilitate the discovery of

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SNPs associated with important traits. Currently, the species retains an original genetic structure via the insitu and ex-situ conservation program. Acknowledgements I am grateful to M. Takahashi for providing the references and information on the breeding of C. japonica and to Y. Moriguchi for kindly supplying pictures of a clonal forest and a seed orchard. I am also grateful to M. Takahashi, T. Ujino-Ihara, A. Matsumoto, and S. Ueno for a critical reading of the earlier version of this chapter.

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61 Murai S (1947) Major forestry tree species in the Tohoku region and their varietal problems. In: Kokudo Saiken Zourin Gijutsu Kouenshu. Aomori Rinyukai, Aomori, Japan, pp 131–151 (in Japanese) Murray BG (1998) Nuclear DNA amount in gymnosperms. Ann Bot 82:3–15 Nakai T, Yamai R (1982) Properties of the important Japanese woods. The mechanical properties of 35 important Japanese woods. Bull Forestry Forest Prod Res Inst 319:13–46 Neale DB, Marshall KA, Sederoff RR (1989) Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl. Proc Natl Acad Sci USA 86:9347–9349 Neale DB, Savolainen O (2004) Association genetics of complex traits in conifers. Trends Plant Sci 9:325–330 Nikaido AM, Ujino T, Iwata H, Yoshimura K, Yoshimaru H, Suyama Y, Murai M, Nagasaka K, Tsumura Y (2000) AFLP and CAPS linkage maps of Cryptomeria japonica. Theor Appl Genet 100:825–831 Ohba K (1993) Clonal forestry with sugi (Cryptomeria japonica). In: Ahuja MR, Libby WJ (eds) Clonal forestry, vol II, Conservation and application. Springer, Berlin, Germany, pp 66–90 Ohba K, Murai M (1971) Recessive genes producing albino-and light green seedlings in sugi, Cryptomeria japonica D. Don. J Jpn Forest Soc 53:177–180 (in Japanese) Ohba K, Murai M, Sugimura G, Saito M, Okamoto K, Watanabe M, Nogichi T (1967) Studies on variation of forest trees (III): Cross-fertility between Kuma-sugi and some other cutting varieties of sugi (Cryptomeria japonica), growth of the F1 seedlings, and two single recessive genes detected in Kumasugi. J Jpn Forest Soc 49:361–367 (in Japanese) Ohba K, Maeda T, Fukuhara N (1974) Inheritance of twisted-leaf sugi, Cryptomeria japonica D. Don and linkage between the twisted-leaf gene and two recessive genes, albino and green (Midori sugi). J Jpn Forest Soc 56:276–281, in Japanese Ohri D, Khoshoo TN (1986) Genome size in gymnosperms. Plant Syst Evol 153:119–132 Price RA, Lowenstein JM (1989) An immunological comparison of the Sciadopityaceae, Taxodiaceae, and Cupressaceae. Syst Bot 14:141–149 Sasaki Y (1996) Somatic chromosome of sterile plus tree clones of Cryptomeria japonica and Chamaecyparis obtusa. Bull Ohita Pref Forest Res Center 13:1–14 Sasaki Y, Mishiba K, Mii M (2001) Estimation of polyploidy and aneuploidy of F1-progeny obtained through artificial crossings between diploids and tetraploids in Cryptomeria japonica and Chamaecyparis obtusa by flow cytometric analysis. Proc Kyushu Forest Soc Meet 54:69–70 Sax K, Sax HJ (1933) Chromosome number and morphology in the conifers. J Arnold Arbor 14:356–375 Schaeffer LR (2007) Strategy for applying genome-wide selection in dairy cattle. J Anim Breed Genet 123:218–223 Strauss SH, Palmer JD, Howe GT, Doerksen AH (1988) Chloroplast genomes of two conifers lack a large inverted repeat and are extensively rearranged. Proc Natl Acad Sci USA 85:3898–3902 Suyama Y, Mukai Y, Kondo T (1996) Assignment of RFLP linkage groups to their respective chromosomes in

62 aneuploids of sugi (Cryptomeria japonica). Theor Appl Genet 92:292–296 Taira A (2001) Tectonic evolution of the Japanese island arc system. Annu Rev Earth Planet Sci 29:109–134 Taira H, Teranishi H, Kaneda Y (1993) A case study of male sterility in sugi (Cryptomeria japonica). J Jpn Forest Soc 75:377–379 Taira H, Tsumura Y, Tomaru N, Ohba K (1997) Regeneration system and genetic diversity of Cryptomeria japonica at different growing altitudes. Can J For Res 27:447–452 Taira H, Saito M, Furuta Y (1999) Inheritance of the trait of male sterility in Cryptomeria japonica. J Forest Res 4:271–273 Taira H, Teranishi H, Kenda Y (2000) Preseasonal scattering of Cryptomeria japonica pollen in Japan, with reference to the dormancy of the male flowers. Allergol Int 49:263–268 Takahara H (1998) Distribution history of Cryptomeria forest. In: Yasuda Y, Miyoushi N (eds) Vegetation history of the Japanese archipelago. Asakura-Shoten, Tokyo, Japan, pp 207–223, in Japanese Takahashi T, Tani N, Taira H, Tsumura Y (2005) Microsatellite markers reveal high allelic variation in natural populations of Cryptomeria japonica near refugial areas of the last glacial period. J Plant Res 118:83–90 Takahashi T, Tani N, Niiyama K, Yoshida S, Taira H, Tsumura Y (2008) Genetic succession and spatial genetic structure in a natural old growth Cryptomeria japonica forest revealed by nuclear and chloroplast microsatellite markers. Forest Ecol Manag 255:2820–2828 Tani N, Takahashi T, Iwata H, Mukai Y, Ujino-Ihara T, Matsumoto A, Yoshimura K, Yoshimaru H, Murai M, Nagasaka K, Tsumura Y (2003) A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed tags. Genetics 165: 1551–1568 Tani N, Takahashi T, Ujino-Ihara T, Iwata H, Yoshimura K, Tsumura Y (2004) Development and characteristics of microsatellite markers for sugi (Cryptomeria japonica D. Don) derived from microsatellite-enriched libraries. Ann Forest Sci 61:569–575 Tenaillon MI, Sawkins MC, Long AD, Gaut RL, Doebley JF, Gaut BS (2001) Patterns of DNA sequence polymorphism along chromosome 1 of maize (Zea mays ssp. mays L.). Proc Natl Acad Sci USA 98:9161–9166 Tokugawa M (1974) Historical studies on silviculture in Edoera. Chikyu-Shutsuppan KK, Tokyo, Japan, p 373, in Japanese Tomaru N, Tsumura Y, Ohba K (1992) Allozyme variation in artificial stands and a plus-tree group of sugi, Cryptomeria japonica in Ibaraki Prefecture. J Jpn Forest Soc 74:44–48 Tomaru N, Tsumura Y, Ohba K (1994) Genetic variation and population differentiation in natural populations of Cryptomeria japonica. Plant Species Biol 9:191–199 Tree Breeding Society (2004) Tree breeding project. Tree Breeding Society, Tokyo, Japan, p 129 Tsukada M (1983) Vegetation and climate during the last glacial maximum in Japan. Quat Res 19:212–235 Tsukada M (1986) Altitudinal and latitudinal migration of Cryptomeria japonica for the past 20,000 years in Japan. Quat Res 26:135–152

Y. Tsumura Tsumura Y, Ohba K (1992) Allozyme variation of five natural populations of Cryptomeria japonica in western Japan. Jpn J Genet 67:299–308 Tsumura Y, Ohba K (1993) Genetic structure of geographical marginal populations of Cryptomeria japonica. Can J Forest Res 23:859–863 Tsumura Y, Tomaru N (1999) Genetic diversity of Cryptomeria japonica using co-dominant DNA markers based on sequenced-tagged site. Theor Appl Genet 98:396–404 Tsumura Y, Uchida K, Ohba K (1989) Genetic control of isozyme variation in needle tissues of Cryptomeria japonica. J Hered 80:291–297 Tsumura Y, Ogihara Y, Sasakuma S, Ohba K (1993) Physical map of chloroplast DNA in sugi, Cryptomeria japonica. Theor Appl Genet 86:166–172 Tsumura Y, Suyama Y, Yoshimura K, Shirato N, Mukai Y (1997) Sequence-tagged-sites (STSs) of cDNA clones in Cryptomeria japonica and their evaluation as molecular markers in conifers. Theor Appl Genet 94:764–772 Tsumura Y, Kado T, Takahashi T, Tani N, Ujino-Ihara T, Iwata H (2007) Genome-scan to detect genetic structure and adaptive genes of natural populations of Cryptomeria japonica. Genetics 176:2393–2403 Uemura K (1981) Ancestor of Cryptomeria japonica and distribution change. The Heredity 35:74–79, in Japanese Ujinof-Ihara T, Yoshimura K, Ugawa Y, Yoshimaru H, Nagasaka K, Tsumura Y (2000) Expression analysis of ESTs derived from the inner bark of Cryptomeria japonica. Plant Mol Biol 43:451–457 Ujino-Ihara T, Matsumuto A, Iwata H, Yoshimura K, Tsumura Y (2002) Single-strand conformation polymorphism of sequence-tagged site markers based on partial sequences of cDNA clones in Cryptomeria japonica. Genes Genet Syst 77:251–258 Ujino-Ihara T, Kanamori H, Yamane H, Taguchi Y, Namiki N, Mukai Y, Yoshimura K, Tsumura Y (2005) Comparative analysis of expressed sequence tags of conifers and angiosperms reveals sequences specifically conserved in conifers. Plant Mol Biol 59:895–907 Ujino-Ihara T, Taguchi Y, Moriguchi Y, Tsumura Y (2010) An efficient method for developing SNP markers based on EST data combined with high resolution melting (HRM) analysis. BMC Res Notes 3:51–55 Woessner RH, Franklin EC (1973) Continued reliance on windpollinated southern pine seed orchards: is it reasonable? In: Proceeding of the 12th southern forest tree improvement conference, Baton Rouge, LA, USA, 12–13 June, 1973, Louisiana State University, Baton Rouge, LA, USA, pp 64–73 Yamazaki T (1995) Cryptomeriaceae. In: Iwatsuki K, Yamazaki T, Boufford DE, Ohba H (eds) Flora of Japan, vol I, Pteridophyta and gymnospermae. Kodansha, Tokyo, Japan, p 264 Yasue M, Ogiyama K, Ebishigawa S, Kondo K, Nishina K (1978) Diterpene hydrocarbons in Cryptomeria japonica needles (IV), The inheritance of ()-kaurene, (+)-phyllocladene and ()-sclarene. J Jpn Forest Soc 60:345–348, in Japanese Yasue M, Ogiyama K, Suto S, Tsukahara H, Miyahara F, Ohba K (1987) Geographical differentiation of natural Cryptomeria stands analyzed by diterpene hydrocarbon constituents of individual trees. J Jpn Forest Soc 69:152–156

3 Cryptomeria Yoshida K, Nishiguchi M, Futamura N, Nanjo T (2007) Expressed sequence tags from Cryptomeria japonica sapwood during the drying process. Tree Physiol 27:1–9 Yoshimaru H, Ohba K, Tsurumi K, Tomaru N, Murai M, Mukai Y, Suyama Y, Tsumura Y, Kawahara T, Sakamaki Y (1998) Detection of quantitative trait loci for juvenile growth,

63 flower bearing and rooting ability based on a linkage map in sugi. Theor Appl Genet 97:45–50 Zhu Q, Zheng X, Luo J, Gaut BS, Ge S (2007) Multilocus analysis of nucleotide variation of Oryza sativa and its wild relatives: severe bottleneck during domestication of rice. Mol Biol Evol 24:875–888

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Chapter 4

Eucalyptus Robert J. Henry

4.1 Basic Botany of the Genus 4.1.1 Distribution The Eucalypts are a large group of species within the Myrtaceae (Byrne 2008). They are overwhelmingly restricted to Australia. A small number of species extend to New Guinea, Indonesia and the Philippines. The Eucalypts include the major genera: Eucalyptus, Angophora and Corymbia, and four smaller genera Eucalyptopsis (two species, New Guinea), Allosyncarpia (one species), Stockwellia (one species) and Arillastrum (one species, New Caledonia). The Eucalypts have spread worldwide due to human distribution in the last 200 years. The wild species and primary hybrids are widely cultivated with relatively little progress towards a distinct domesticated form in most cases.

4.1.2 Geographical Locations of Genetic Diversity Eucalypt diversity is distributed across the Australian continent. They are found in all but the extreme alpine environments. Corymbia species are more abundant in tropical and subtropical areas. The Corymbia are common trees of northern Australia. Their distribution extends to the coastal areas of the southeast and southwest but they are not found elsewhere in the south. R.J. Henry Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Australia e-mail: [email protected]

Angophora are a small group found in eastern Australia. Eucalyptus species are more abundant in the non-tropical regions of Australia. A total of 15 Eucalypt species extend outside Australia to Papua New Guinea, Timor, Sulawesi and the Philippines. Only nine of these are not found in Australia. The geographic distribution of genetic diversity within species is generally less well known.

4.1.3 Morphology The Eucalypts include trees and shrubs, the largest of which are among the tallest of the flowering plants. The tallest, Eucalyptus regnans, Mountain Ash, are almost 100 m in height and many other species are also very large. The smallest are very small shrubs. Some are multi-stemmed shrubs and these species are called mallees. The bark type (e.g., stingybark, ironbark) is often used in the common name of the species. The flowers lack petals and have prominent stamens. The flower buds are covered by an operculum or bud cap and this is the origin of the name Eucalyptus (eu well and cayuptos covered). The leaves of Eucalypts show a strong dimorphism between a juvenile form and an adult form. The timing of transition from one to the other differs between species and environments. Eucalypts often form a lignotuber (a vegetative swelling at or largely below ground level) and can often re-grow after being damaged or cut at ground level. Eucalypts are outcrossing and produce large numbers of seeds. The presence of a lignotuber and the production of large numbers of seeds provide mechanisms for Eucalypt populations to survive fire. Trees re-grow from vegetative buds or new plants are established by seedling emergence following fires.

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_4, # Springer-Verlag Berlin Heidelberg 2011

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Many insects found within the natural distribution of Eucalypts have developed an ability to consume the leaves and bark despite the high content of defensive compounds. Eucalypts grown outside this range often have noticeably different appearance without the characteristic markings left by these predators. The Angophora includes about ten species that are distinguished from other groups by having opposite rather than alternate leaves. Angophora have highly ribbed fruits and lack an operculum. The Corymbia are a much larger group of more than 100 species including the bloodwoods (red and yellow groups), spotted gums and ghost gums. The buds have opercula.

4.1.4 Cytology and Karyotype The basic chromosome number is n ¼ 11 but n ¼ 12 have been reported in some species.

4.1.5 Genome Size The genome sizes are small, varying from 300 to 700 Mbp. The genomic sequence of Eucalyptus grandis is currently being determined by an international collaboration (International Eucalyptus Genome Consortium, www.fabinet.up.ac.za/eucagen). The chloroplast genome sequence of Eucalyptus globulus was the first to be reported (Steane 2005).

R.J. Henry

molecular data that was at first interpreted as not supporting this grouping. Early analysis of ribosomal genes was complicated by the presence of multiple loci and the difficulty of comparing the same locus in different species. However, this has now been resolved and the genus is widely accepted and very strongly supported by molecular and morphological data (Ochieng et al. 2007a). Earlier efforts to divide the genus Eucalyptus into several additional genera (Pryor and Johnson 1971) have not been so widely accepted. Brooker (2000) defined 13 subgenera with Eucalyptus. This classification is shown in Table 4.1. Molecular tools are continuing to improve understanding of the higher level relationships within the Eucalypts (McKinnon et al. 2008). The definition of biological species in this large group is challenging. Taxonomists have identified a large number of taxa. The Flora of Australia (1988) lists 513 and many more have been described since this publication. The total number of species now numbers around 783 (Byrne 2008). Reticulate evolution may be common in the Eucalypts. Apparent hybrids between species (Stokoe et al. 2001) are commonly encountered and DNA sequence analysis suggests widespread gene flow between “species”. The species concept is challenging to apply to many populations. Taxonomic differences in some cases may not reflect differences that are due to reproductive isolation of the populations of the two taxa. More detailed comparisons of the genomes of some taxa may resolve these issues in the future (Fig. 4.1).

4.1.7 Agricultural Status 4.1.6 Taxonomic Position The taxonomic position of the Eucalyptus genus has been the subject of widespread discussion especially within the Australian community due to the dominance of these plants in the environment. The genus as defined in the Flora of Australia (1988) was large and included many species that have now been separated into the Corymbia genus. The Corymbia species are generally recognized as being more distinct from other Eucalypts than the Angophora that has long been recognized as a separate genus. The recent distinction of Corymbia (113 species) from Eucalyptus (Hill and Johnson 1995) was made controversial by some

Eucalypts are the most planted hardwood trees in the tropical and subtropical world (Grattapaglia and Kirst 2008). Eucalyptus grandis is probably the most widely Table 4.1 Classification of Eucalyptus according to Brooker (2000) Polytypic subgenera Monotypic subgenera Angophora Acerosa Corymbia Cruciformes Blakella Alveolata Eudesmia Cuboidea Sympomyrthus Idiogenes Minutifructa Primitiva Eucalyptus

4 Eucalyptus

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Fig. 4.1 Relationships within the Eucalypts modified from Ochieng et al. (2007a)

planted species. Eucalypts forests are harvested for their timbers, which are used extensively in construction. The solid wood properties of many species make them suitable for use in buildings and furniture. Many species produce timber with properties that suit specific uses (www.dpi.qld.gov.au/cps/rde/dpi/hs.xsl/26_5509_ENA_ HTML.htm). The pulp is used in paper production. The pulp properties of Eucalypts make them suitable for high quality paper production. Eucalyptus oil is produced from several species. The oil of some Eucalypts species is extracted and used as an essential oil. The major component of the oil from most species is 1,8-cineole. Tissue culture protocols have been developed for propagation of genotypes with desirable oil content (Goodger et al. 2008). Charcoal from Eucalypts is a significant fuel in some areas. Ornamental Eucalypts have been selected for cultivation. Eucalyptus species have been grown as ornamentals and only very recently ornamental hybrids have been produced. Limited production of flowers for use as cut flowers and fruits as ornaments has been undertaken. Eucalypts are an important source of honey. Genetic diversity in flowering time has been studied to assess value for honey production (Mora et al. 2009). More recently Eucalypts have been considered an important potential option for woody biomass for bioenergy production. Eucalypts have been an important source of firewood in Australia and in countries were Eucalypts have become established. Eucalyptus species from the most favorable, high rainfall and high nutrient regions have been favored

for cultivation. These species probably have the greatest potential for rapid growth when water and nutrients are not limiting. Species adapted to harsher environments, e.g. many of the Corymbia species may be better options in more marginal production environments. However, the potential of a very larger number of taxa has not been evaluated. The growth of Eucalypts depends very much on site and choice of germplasm. For example a study of the growth of 36 species to 10–12 years at 12 sites in Victoria (Duncan et al. 2000) gave average growth rates of 13–57 m3/ha/year. The performance of different seed lots varied greatly within sites (15- to 60-fold depending upon site). Growth rates were greatest on sites with more than 900 mm of rainfall per year. Species  site effects were common. Much of the potential of Eucalypts across environments remains to be established. The potential for mixed species plantings also needs more analysis. Performance of some Eucalyptus species has been evaluated (FAO, www.fao.org/DOCREP/004/AC121E/ ac121e04.htm). Eucalyptus camaldulensis has been reported to have growth rates of 5–10 m3/ha/year on 10–20 year rotations on dry sites ranging up to 30 m3/ha/ year on favorable sites. Eucalyptus deglupta has potential in the humid tropics with typical yields of 20–40 m3/ ha/year and maximum yields of around 90 m3/ha/year. Eucalyptus globulus yields 10–15 m3/ha/year but up to 40 m3/ha/year under the best conditions. Eucalyptus grandis and Eucalyptus urophylla may be high yielding

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(above 25 m3/ha/year). The best yields for E. grandis may exceed 100 m3/ha/year. Eucalyptus robusta yield of 10–35 m3/ha/year are reported. Eucalyptus saligna yield between 36–5 m3/ha/year are reported on 25 year rotations in Africa and South America.

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arrival around 200 years ago. The Australian national listing of rare and endangered species lists 23 Eucalyptus species as endangered and 49 species (plus 4 Corymbia) as vulnerable (Table 4.2, www.environment.gov. au/cgi-bin/sprat/public/publicthreatenedlist.pl?wanted¼ flora#FLORA_EXTINCT).

4.1.8 Model Plant 4.2.2 In Situ Conservation The Eucalypt is a second model forest tree species to complement the poplar in studies of the genomes of trees. The sequence of the poplar genome will be complemented by the sequence of the Eucalyptus genome as major tools for the analysis of tree genomes.

4.1.9 Weeds/Invasive Species Eucalypts are considered weeds in many areas because of their ability to persist in marginal environments. The characteristics of rapid growth that make these plants desirable as forest species contribute to their weediness. Eucalypts are weeds in many areas not only because they can grow rapidly even in environments were water and soil nutrition are limiting but because they are able to reproduce, increase in number and colonize new sites.

4.1.10 Tribal Use Eucalypts were widely used for medicinal purposes by Australian Aborigines (Lassak and McCarthy 1983). The dominance of this group of plants in the Australian landscape made them widely available for the exploration and development of traditional uses.

4.2 Conservation Initiatives 4.2.1 Genetic Erosion Humans have impacted the Eucalypt forests of Australia since their arrival. More recently, Eucalypt forests have been cleared extensively in Australia since European

Many species are protected in conservation areas such as National Parks. Conservation of the diversity of Eucalypt species in the wild requires an understanding of the genetics of the wild populations (Coates and Byrne 2005). Pollinators include insect, birds, bats and possums and the habits of pollinators may influence the structure of wild populations. Studies of wild populations have reveled contrasting patterns of diversity. An analysis of the widespread Eucalyptus grandis, in subtropical eastern Australia showed relatively little geographic structure in the population despite a discontinuity between northern and southern populations (Jones et al. 2006). Relatively recent separation of these populations and the potential for long distance pollen flow might explain this observation (Jones et al. 2007). Other species show distinct geographical structure. Natural hybrids, even between distantly related Eucalypts (Stokoe et al. 2001), are frequently found in the wild complicating definition of the evolutionary units or taxa that require conservation. Molecular analysis of genetic relations in Eucalypt population has been used to determine appropriate conservation strategies. Some examples follow. Eucalyptus consideniana. The impact of harvesting on genetic diversity of wild populations of Eucalyptus consideniana has been investigated using molecular techniques (Glaubitz et al. 2003). The results suggest that loss of genetic diversity is more likely when applying a seed tree system than under clear-felling with aerial re-sowing. Eucalyptus graniticola. Eucalyptus graniticola is a rare species known from one individual in western Australia (Rossetto et al. 1997). Molecular analysis suggests that this plant is probably a hybrid between Eucalyptus rudis and Eucalyptus drummondii. This suggested that ex situ conservation was probably a better option in this case.

4 Eucalyptus Table 4.2 Rare and endangered Eucalypts Endangered Eucalyptus absita Eucalyptus balanites Eucalyptus beardiana Eucalyptus burdettiana Eucalyptus brevipes Eucalyptus conglomerate Eucalyptus copulans Eucalyptus crenulata Eucalyptus crucis ssp. praecipua Eucalyptus cuprea Eucalyptus dolorosa Eucalyptus qunnii ssp.divaricata Eucalyptus imlayensis Eucalyptus impensa Eucalyptus insulares Eucalyptus leprophloia Eucalyptus morrisbyi Eucalyptus pachycalyx ssp. Banyabba Eucalyptus paludicota Eucalyptus phylacis Eucalyptus pruiniramis Eucalyptus recurva Eucalyptus species Howes Swamp Creek

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Vulnerable Eucalyptus alligatrix spp. limaaensis Eucalyptus alligatrix spp. miscella Eucalyptus aquatica Eucalyptus argophloia Eucalyptus argutifolia Eucalyptus articulata Eucalyptus beaniana Eucalyptus benthamii Eucalyptus blaxellii Eucalyptus cadens Eucalyptus caleyi spp. ovendenii Eucalyptus camfieldii Eucalyptus canobolensis Eucalyptus ceracea Eucalyptus cerasiformis Eucalyptus coronata Eucalyptus crispata Eucalyptus crucis spp. crucis Eucalyptus glaucina Eucalyptus hallii Eucalyptus infera Eucalyptus johnsoniana Eucalyptus kabiana Eucalyptus kartzoffiana Eucalyptus langleyi Eucalyptus lateritic Eucalyptus macrorhyncha spp. cannonii Eucalyptus mckieana Eucalyptus merrickiae Eucalyptus mooreana Eucalyptus nicholii Eucalyptus olivacea Eucalyptus paedoglauca Eucalyptus parramattensis spp. decadens Eucalyptus parvula Eucalyptus platydisca Eucalyptus pulverulenta Eucalyptus pumila Eucalyptus raveretiana Eucalyptus rhodantha var. rhodantha Eucalyptus robertsonii spp. hemisphaerica Eucalyptus rubida spp. barbigerorum Eucalyptus scoparia Eucalyptus steedmanii Eucalyptus strzeleckii Eucalyptus subarea Eucalyptus synandra Eucalyptus tetrapleura Eucalyptus virens Corymbia clandestine Corymbia leptoloma Corymbia rodops Corymbia xanthope

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Eucalyptus benthamii. Eucalyptus benthamii is a rare Eucalypt from New South Wales. Construction of a dam in 1933 probably destroyed much the habitat for this species. Molecular analysis of populations of this species found increased rates of selfing and interspecific hybridization in smaller fragmented populations (Butcher et al 2005). These observations suggested the need for ex situ propagation to ensure cross pollination of individuals within these threatened populations. In situ conservation is also supported by the need to provide habitat for species that depend upon wild Eucalypt populations for survival. The Koala (Phascolarctos cinereus) is a tree dwelling marsupial that feeds exclusively on the leaves of a modest number of Eucalypt species. Eucalypt species are also the major source of food for many other animals including birds. Public support for conservation of habitat for these species is high.

4.2.3 Ex Situ Conservation Seeds of many species are held in the Australian Tree Seed Centre (ATSC: www.csiro.au/places/ATSC) in Canberra. Living collections are found in botanic gardens. The propagation of Eucalypts is usually by seed. Vegetative propagation, required to conserve specific genotypes is generally more difficult but techniques for propagation by cuttings and by grafting has been developed for many species of economic interest.

4.3 Role in Elucidation of Origin and Evolution of Allied Crop Plants 4.3.1 Related Crop Plants Eucalypts are related to other species from the Myrtaceae that have been cultivated as sources of honey or essential oils. Many related species (e.g., Melaleuca species and Leptospermum species) in the Myrtaceae have been grown as ornamentals. Other species from this family are also forest species (e.g., Lophostemon confertus) used as a source of timber. Some of these are in limited cultivation. Some members of the Myrtaceae are grown for their edible fruits (e.g., Syzygium

R.J. Henry Table 4.3 Some widely cultivated ornamental species from the Myrtaceae Genus Number of species Common name Calothamnus 40 Claw flowers Callistemon 30 Bottle brush Calytrix 100 Star flowers Chamelauclum 30 Wax flowers Leptospermum 80 Tea tree Melaleuca 200 Tea tree Verticordia 100 Feather flowers

species). Other economic species in the Myrtaceae include clove (Syzygium aromaticum), guava (Psidium guajava) and allspice (Pimenta dioica). Many species from the family are cultivated as ornamentals (Table 4.3). Research on the Eucalypts will be of value in the conservation of these species and in their development as domesticated plants.

4.3.2 Morphotaxonomy A DVD (EUCLID: Eucalypts of Australia) with descriptions of the morphology of 894 Eucalypt taxa including details of habit, bark, leaves, flowers, fruits, buds, seeds and juveniles, and some geographic and ecological information has been published by the CSIRO.

4.3.3 Chemotaxonomy Chemotypes that have differing essential oil content have been defined in Eucalyptus and related plants. The content of oil varies between species and individuals within a population (King et al. 2006). The related species, Melaleuca alternifolia, tea tree, has been cultivated as a source of an essential oil that has been used as an antiseptic (Homer et al. 2000). Some of the genes determining chemotype in this species have been characterized (Shelton et al. 2002a, 2004). The chemotypes are probably largely genetically controlled (Shelton et al. 2002b) but may be associated with local adaptations. Geographic structure in the chemotype distribution in wild species may reflect local adaptation not apparent in the genome as analyzed by neutral genetic markers such as microsatellites (Rossetto et al. 1999a).

4 Eucalyptus

The distribution of cyanogenic glycosides has been survey in 420 Eucalypt species (Gleadow et al. 2008). Cyanogenic glycosides were found in 23 species mainly within the Symphyomyrtus but also in Eucalyptus and Corymbia.

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et al. 2006) and Corymbia (Shepherd et al. 2006) showing high levels of synteny between the maps of Corymbia and Eucalyptus.

4.5.2 Mapping of Genes 4.3.4 Molecular Markers The full range of molecular marker technologies (Henry 2001) has been applied to Eucalypts as they have been developed. Microsatellite markers used to study the population genetics of wild Melaleuca alternifolia populations (Rossetto et al. 1999b) were found to be transferable to Eucalypts (Rossetto et al. 2000). The analysis of microsatellite loci across species in the Eucalypts has been highly successful (Ochieng et al. 2007b) probably because of the relatively recent divergence of Eucalypt species. The diversity within species determined using molecular markers may often be greater than that between species in this group of plants (Ochieng et al. 2008). The genetic structure of Eucalypt populations suggests widespread reticulate evolution (McKinnon 2005) possibly explaining the great success of the Eucalypts in the Australian landscape.

4.4 Role in Development of Cytogenetic Stocks and Their Utility Genetic stocks are not well developed in the Eucalypts. An in-bred E. grandis was selected for the first sequencing of the genome to avoid complications due to heterozygosity. However this genotype still retained significant (much higher than expected) heterozygosity suggesting that homozygosity at some loci may be lethal.

4.5 Role in Classical and Molecular Genetic Studies 4.5.1 Molecular Genetic Linkage Maps Several molecular genetic maps have been produced for Eucalyptus species (Grattapaglia and Sederoff 1994; Bundock et al. 2000; Agrama et al. 2002; Freeman

The mapping of quantitative trait loci (QTL) for growth and wood quality traits has been successfully undertaken in Eucalyptus (Thamarus et al. 2004). Trait mapping has increasingly relied on genomics resources and been based upon association genetics (Moran et al. 2002).

4.5.3 Deciphering Favorable Alleles Association of single nucleotide polymorphism (SNP) with traits is progressively revealing the identity of favorable alleles at several loci associated with cell wall and lignin content that influence wood properties. Thumma et al. (2005) identified 25 SNPs in the Cinnamoyl CoA reductase (CCR) gene in Eucalyptus nitens. Two alleles associated with microfibral angle were identified. Many genes such as those involved in cell wall polysaccharide biosynthesis are only beginning to be characterized and evaluated.

4.6 Role in Crop Improvement Through Traditional and Advanced Tools Wild Eucalypt germplasm has potential to contribute to the development of improved forest trees for timber, paper and energy production.

4.6.1 Heterosis Breeding Interspecific hybrids have shown great potential to perform well due to the combination of desirable traits and apparent heterosis. Eucalyptus grandis  Eucalyptus urophylla crosses have been grown on a large scale. A fast growing hybrid between a tropical species, Corymbia torelliana and a subtropical species Corymbia

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variegata is currently being developed for forest production. Interspecific ornamental hybrids show traits outside the range of either parental species in relation to flower size or number. The large number of species would allow a very large number of interspecific combinations to be explored. Only a small proportion of the possible combinations have been evaluated. Genetic transformation has been developed for Eucalyptus species.

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wood traits (Sexton et al. 2010). Larger numbers of SNPs (more than 20,000) have been reported for E. grandis (Novaes et al. 2008) from 454 sequencing of cDNA from many tissues. The availability of complete genome sequences for Eucalypts will allow rapid progress in understanding and better managing the diversity of wild populations and in accelerating the domestication and genetic improvement of Eucalypts.

4.6.2 Abiotic Stress

4.8 Scope for Domestication and Commercialization Introgression of genes from salt tolerant species in to rapid growing species has been suggested as an approach to developing Eucalypts suited to saline production environments (Dale and Henry 2003).

4.7 Genomics Resources Developed Eucalypts have rapidly developing genomics tools that should allow application in tree improvement (Poke et al. 2005; Grattapaglia and Kirst 2008). The complete genome sequence of E. grandis will be a major resource (Myburg et al. 2008) produced by the US Department of Energy following a proposal submitted by the International Eucalyptus Genome Network (EUCAGEN). The role of small RNA in the control of wood formation is being investigated (Myburg et al. 2008). More than 10,000 expressed sequence tags (ESTs) from Eucalyptus gunnii have been annotated showing greatest homology to poplar genes (Keller et al 2009). Microsatellite or simple sequence repeat (SSR) markers have been applied widely in Eucalypts. Sequence data has been mined successfully to reveal large numbers of useful SSRs for Eucalypts (Yasodha et al. 2008). SNPs in genes associated with commercial traits are major research targets. QTL analysis has identified tools that can be applied for selection within families but association studies now aim to find markers that can be applied more widely in markerassisted selection (Grattapaglia 2008). New sequencing technologies (Henry and Edwards 2009) are being applied to discovery of SNP in Eucalyptus. For example, SNPs in candidate genes for wood properties have been assayed in Eucalyptus pilularis using highthroughput techniques to identify associations with

Eucalypts have great potential for domestication. Many plantations of Eucalypts cannot be considered to be domesticated as they have often been produced from seed collected in the wild or plants that have only been in cultivation for one or two generations. Hybrids may represent better examples of domesticated Eucalypts. Eucalypts have been widely distributed worldwide following European discovery in Australia in the late eighteenth century (Myburg et al. 2007). Eucalyptus have become probably the most planted hardwood trees in the world and is used extensively as a source of wood for solid timber, pulp, charcoal and essential oils. Eucalypt production and domestication is likely to be further accelerated by use of Eucalypts as bioenergy crops. The ability to produce rapid biomass accumulation rates on marginal sites makes Eucalypts an especially attractive bioenergy option in many places worldwide. The length of rotation that would be optimal for biomass production using Eucalypts has not been defined. The selection of species and genotypes for growth in these short rotation production systems (one or a few years between harvest) may differ greatly to that for more conventional production for pulp or solid timber.

4.9 Some Dark Sides and Their Addressing Eucalypts are weeds in many areas. Eucalypts are adapted to environments with low rainfall and limited soil nutrients but grow very rapidly when these resources are available. Transgenic Eucalypts with some types of transgenes may create problems in the Australian

4 Eucalyptus

environment due to the potential for gene flow into wild populations. Traits that contribute to weediness could be considered undesirable outside the native range of the species. Sterile Eucalypts could be a good innovation that could assist with this problem. Vegetative propagation could allow the establishment of forests but the lack of seed would prevent the trees becoming weeds. Some Eucalypts are considered weeds in Australia when established outside their natural distribution. Corymbia torelliana is a good example. This species is a native of the rainforests of tropical North Queensland. It was widely planted in gardens in subtropical areas such as Brisbane in the 1970s and 1980s and has been able to propagate itself in this environment becoming a weed. Hybridization with local species has also been observed making this plant a threat to the conservation of other species. Eucalypts are frequently weeds in environments outside Australia, competing very well with native vegetation. These problems can be found in Africa, South and North America, Asia and Europe.

4.10 Recommendations for Future Actions

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have been developed as ornamental genotypes and protected by Plant Breeders Rights in Australia. Germplasm used in forest establishment has been selected by public and private organizations and is considered of value by these groups.

4.10.3 Need for Ongoing Conservation Conservation of genetic resources of Eucalypts is important for many reasons. In the Australian environment, Eucalypt conservation is a key element of biodiversity conservation because of their dominance in many plant communities. Eucalypt forest plantations are likely to continue to expand worldwide and these will need to continue to draw on the genetic diversity in wild populations to supplement the ex situ resources in seed and other living collections. Conservation in protected areas such as notational parks is important but Eucalypts on private lands will also need ongoing conservation. This requires the importance of Eucalypts in Australia and internationally to be more widely promoted especially in the Australian community. The environmental and economic value of Eucalypts and as a consequence their social importance needs to more widely discussed and understood.

4.10.1 Local Conservation Knowledge of the genetic structure of Eucalypt populations is an important requirement in efforts to conserve diversity in wild populations. Planting of Eucalypts is extensive and the cultivated trees have the potential to interbreed with adjacent wild populations and alter their genetic structure. This may put at risk of extinction rare local germplasm. Awareness of this problem is increasing in Australian rural communities with the promotion of the planting of trees from local seed collections. Research on the genetic structures of many Eucalypt populations is required to allow better management of populations.

4.10.2 IPR-Related Issues Eucalypts are not the subject of any specific intellectual property rights (IPR). Recent interspecific hybrids

References Agrama HA, Georg L, Salah SF (2002) Construction of genome map for Eucalyptus camaldulensis DEHN. Silvae Genet 51:201–206 Brooker MIH (2000) A new classification of the genus Eucalyptus L’Her. Aust Syst Bot 13:79–148 Bundock PC, Hayden M, Vaillancourt RE (2000) Linkage maps of Eucalyptus globulus using RAPD and microsatellite markers. Silvae Genet 49:223–232 Butcher PA, Skinner AK, Gardiner CA (2005) Increased inbreeding and inter-species gene flow in remnant populations of the rare Eucalyptus benthamii. Conserv Genet 6:213–226 Byrne M (2008) Phylogeny, diversity and evolution of Eucalypts. In: Sharma AK, Sharma A (eds) Plant genome biodiversity and evolution, vol 1, Part E: phanerogamsangiosperm. Science, Enfield, NH, USA, pp 303–346 Coates DJ, Byrne M (2005) Genetic variation in plant populations: assessing cause and pattern. In: Henry RJ (ed) Plant diversity and evolution. CABI, Wallingford, Oxon, UK, pp 139–164

74 Dale GT, Henry RJ (2003) Biotechnology for salt tolerance and/or enhanced water use in plants – interesting science or a pathway to the future? Proceedings of the 9th National Productive Use and Rehabilitation of Saline Land (PURSL) conference, Rockhampton, Queensland, 29 September–2 October 2003 Duncan MJ, Baker TG, Appleton R, Stokes RC (2000) Growth of Eucalypt plantation species across twelve sites in Gippsland, Victoria. Natural Resources and Environment Report Number 99/056, Melbourne, Victoria, Australia, 50 p Flora of Australia (1988) Volume 19: Myrtaceae-Eucalyptus, Angophora. Australian Government Publishing Service, Canberra, Australia Freeman JS, Potts BM, Shepherd M, Vaillancourt RE (2006) Parental and consensus linkage maps of Eucalyptus globulus using AFLP and microsatellite markers. Silvae Genet 55:202–217 Glaubitz JC, Murrell JC, Moran GF (2003) Effects of native forest regeneration practices on genetic diversity in Eucalyptus consideniana. Theor Appl Genet 107:422–431 Gleadow RM, Haburjuk J, Dunn JE, Conn ME, Conn EE (2008) Frequency and distribution of cyanogenic glycosides in Eucalyptus L’Herit. Phytochemistry 69:1870–1874 Goodger JQD, Heskes AM, King DJ, Gleadow RM, Woodrow IE (2008) Micropropagation of Eucalyptus polybractea selected for key essential oil traits. Funct Plant Biol 35:247–251 Grattapaglia D, Sederoff R (1994) Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross mapping strategy and RADP markers. Genetics 137:1121–1137 Grattapaglia D (2008) Perspectives on genome mapping and marker-assisted breeding of eucalypts. South Forest 70:69–75 Grattapaglia D, Kirst M (2008) Eucalyptus applied genomics: from gene sequences to breeding tools. New Phytol 179:911–929 Henry RJ (2001) Plant genotyping: the DNA fingerprinting of plants. CABI, Wallingford, Oxon, UK, p 325 Henry RJ, Edwards K (2009) New tools for single nucleotide polymorphism (SNP) discovery and analysis accelerating plant biotechnology. Plant Biotechnol J 7:311 Hill K, Johnson L (1995) A revision of the bloodwoods, genus Corymbia (Myrtaceae), in Telopea, Volume 6: (2–3). Royal Botanic Gardens, Sydney, Australia Homer L, Leach D, Lea D, Lee LS, Henry RJ, Baverstock PR (2000) Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae). Biochem Syst Ecol 28:367–382 Jones ME, Shepherd M, Henry RJ, Delves A (2006) Chloroplast DNA variation and population structure in the widespread forest tree, Eucalyptus grandis. Conserv Genet 7:691–703 Jones ME, Shepherd M, Henry RJ, Delves A (2007) Pollen flow in Eucalyptus grandis determined by paternity analysis using microsatellite markers. Tree Genet Genomes 4:37–47 King DJ, Gleadow RM, Woodrow IE (2006) Regulation of oil accumulation in single glands of Eucalyptus polybractea. New Phytol 172:440–451 Keller G, Marchal T, SanClemente H, Navarro M, Ladouce N, Wincker P, Couloux A, Teulieres C, Marque C (2009) Development and functional annotation of an 11,303-EST

R.J. Henry collection from Eucalyptus for studies of cold tolerance. Tree Genet Genomes 5:317–327 Lassak EV, McCarthy T (1983) Australian medicinal plants. Reed, Sydney, Australia McKinnon G (2005) Reticulate evolution in higher plants. In: Henry RJ (ed) Plant diversity and evolution. CABI, Wallingford, Oxon, UK, pp 81–96 McKinnon GE, Vaillancourt RE, Steane DA, Potts BM (2008) An AFLP marker approach to lower-level systematics in Eucalyptus (Myrtaceae). Am J Bot 95:368–380 Mora F, Gleadow R, Perret S, Scapim CA (2009) Genetic variation for early flowering, survival and growth in sugar gum (Eucalyptus cladocalyx F. Muell) in southern Atacama Desert. Euphytica 169(3):335–344. doi:10.1007/s 10681009-9962-z Moran GF, Thamarus KA, Raymond CA, Qiu DY, Uren T, Southerton SG (2002) Genomics of Eucalyptus wood traits. Ann Forest Sci 59:645–650 Myburg A, Bradfield J, Cowley E, Creux N, de Castro M, Hatherell TL, Mphahlele M, O’Neill M, Ranik M, Solomon L, Victor M, Zhou H, Galloway G, Horsley T, Jones N, Stanger T, Bayley A, Edwards N, Janse B (2008) Forest and fibre genomics: biotechnology tools for applied tree improvement. South Forest 70:59–68 Myburg AA, Potts BM, Marques CM, Kirst M, Gion JM, Grattapaglia D, Grima-Pettenatti J (2007) Eucalypts. In: Kole C (ed) Genome mapping and molecular breeding in plants, vol 7, Forest trees. Springer, Berlin, Germany, pp 115–160 Novaes E, Drost DR, Farmerie WG, Pappas GJ, Grattapaglia D, Sederoff RR, Kirst M (2008) High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome. BMC Genomics 9:312 Ochieng JW, Henry RJ, Baverstock PR, Steane DA, Shepherd M (2007a) Nuclear ribosomal pseudogenes resolve a corroborated monophyly of the eucalypt genus Corymbia despite misleading hypotheses at functional ITS paralogs. Mol Phylogenet Evol 44:752–764 Ochieng JW, Steane DA, Ladiges PY, Baverstock PR, Henry RJ, Shepherd M (2007b) Microsatellites retain phylogenetic signals across genera in eucalypts (Myrtaceae). Genet Mol Biol 30:1125–1134 Ochieng JW, Shepherd M, Baverstock PR, Nikles G, Lee DJ, Henry RJ (2008) Genetic variation within two sympatric spotted gum eucalypts exceeds between taxa variation. Silvae Genet 57:249–256 Poke FS, Vaillancourt RE, Potts BM, Reid JB (2005) Genomic research in Eucalyptus. Genetica 125:79–101 Pryor LD, Johnson LAS (1971) A classification of the Eucalypts. Australian National University, Canberra, Australia Rossetto M, Lucarotti F, Hopper SD, Dixon KW (1997) DNA fingerprinting of Eucalyptus graniticola: a critically endangered relict species or a rare hybrid? Heredity 79:310–318 Rossetto M, Slade RW, Baverstock PR, Henry RJ, Lee SL (1999a) Microsatellite variation and analysis of genetic structure in tea tree (Melaleuca alternifolia Myrtaceae). Mol Ecol 8:633–643 Rossetto M, McLauchlan A, Harriss F, Henry RJ, Lee LS, Baverstock PR, Maguire T, Edwards KJ (1999b) Abundance and polymorphism of microsatellite markers in tea tree,

4 Eucalyptus (Melaleuca alternifolia – Myrtaceae). Theor Appl Genet 98:1091–1098 Rossetto M, Harris FCL, Mclauchlan A, Henry RJ, Baverstock PR, Lee LS (2000) Interspecific amplification of tea tree (Melaleuca alternifolia – Myrtaceae) microsatellite loci – potential implications for conservation studies. Aust J Bot 48:367–373 Sexton T, Henry RJ, McManus LJ, Henson M, Thomas D, Shepherd M (2010) Genetic association studies in Eucalyptus pilularis Smith (Blackbutt) 73:254–258 Shelton D, Leach D, Baverstock P, Henry RJ (2002a) Isolation of genes involved in secondary metabolism from Melaleuca alternifolia (Cheel) using expressed sequence tags (ESTs). Plant Sci 162:9–15 Shelton D, Aitken K, Doimo L, Leach D, Baverstock P, Henry RJ (2002b) Genetic control of monoterpene composition in the essential oil of Melaleuca alternifolia (Cheel). Theor Appl Genet 105:377–383 Shelton D, Zabaras D, Chohan S, Wyllie G, Baverstock P, Leach D, Henry RJ (2004) Isolation and partial characterization of a putative monoterpene synthase from Melaleuca alternifolia. Plant Physiol Biochem 42:875–882

75 Shepherd M, Kasem S, Lee D, Henry RJ (2006) Construction of microsatellite linkage maps for Corymbia. Silvae Genet 55:228–238 Steane DA (2005) Complete nucleotide sequence of the chloroplast genome from the Tasmanian blue gum, Eucalyptus globulus (Myrtaceae). DNA Res 12(3):215–220 Stokoe RL, Shepherd M, Lee DJ, Nikles DG, Henry RJ (2001) Natural inter-subgeneric hybridisation between Eucalyptus acmenoides Schauer and Eucalyptus cloeziana F. Muell (Myrtaceae) in Southeast Queensland. Ann Bot 88:563–570 Thamarus K, Groom K, Bradley A, Raymond CA, Schimleck LR, Williams ER, Moran GF (2004) Identification of quantitative trait loci for wood and fibre properties in two full-sib pedigrees of Eucalyptus globulus. Theor Appl Genet 109:856–864 Thumma R, Nolan MF, Evans R, Moran GF (2005) Polymorphisms in Cinnamoyl CoA Reductase (CCR) are associated with variation in microfibril angle in Eucalyptus spp. Genetics 171:1257–1265 Yasodha R, Sumathi R, Chezhian P, Kavitha S, Ghosh M (2008) Eucalyptus microsatellites mined in silico: survey and evaluation. J Genet 87:21–25

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Chapter 5

Juglans Keith Woeste and Charles Michler

5.1 Black Walnut Black walnut (Juglans nigra L.) is a large deciduous tree native to the hardwood forests of the eastern United States and southern Canada. Black walnut grows to a height of 125 ft (38 m), but most often reaches about 80 ft (25 m) (Duncan and Duncan 1988; Williams 1990). Black walnuts have alternate, pinnate-compound leaves with 15–23 leaflets; twigs contain prominent chambered pith. Black walnut produces large terminal, leathery fruits (botanically a false drupe) containing a single thick-shelled seed. Black walnut is a masting species; seed crops are irregular, but seed production can be abundant every 3–5 years. Trees growing in the open often begin producing seeds as early as 5 years after planting, but in forests bearing often does not begin for 15 years or more (Schlesinger and Funk 1977). Black walnut is valued chiefly for its wood, which is heavy, strong, and durable. Black walnut is normally straightgrained, it works easily with hand tools, and has excellent machining properties. When finished, the wood has a smooth, elegant surface and highly desirable grain pattern (Rink 1988). Black walnut is used primarily for the production of fine furniture and gunstocks. Black walnut veneer is used for the highest grade cabinets and panels (Marquis and Johnson 1989). The genetics, breeding, genomics, and biotechnological advances of black walnut have been reviewed extensively by Victory et al. (2004) and Michler et al. (2007, 2008) and will not be revisited here. K. Woeste (*) USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, Pfendler Hall, 715W. State Street, West Lafayette, IN 47907, USA e-mail: [email protected]

5.2 Persian Walnut: A Domesticated Relative 5.2.1 Range Juglans regia, also known as Persian or common walnut, is probably native to the mountainous regions of central Asia, including parts of Kazakhstan, Uzbekistan, Kyrgyzstan, Nepal, Tibet, Pakistan, Afghanistan, Turkmenistan, Georgia, Armenia, Azerbaijan, and Iran (Germain 2001). Cultivation of J. regia as a multi-purpose tree dates back at least several thousand years (Germain 2004), and it has been spread extensively by human activity. Fossil evidence places J. regia in Europe as long ago as the Pliocene (Manchester 1989). As a wild, feral, or semi-domesticated tree, J. regia is found in temperate regions stretching from the Iberian Peninsula across Turkey and the Caucasus to the Himalayas and south-central China. It continues to be exploited across this entire region both for its nuts and for wood, including lumber and firewood (Dandekar et al. 2005). Within the last few hundred years the species has been introduced to nearly every country situated from 30 to 50
N latitude and 30 to 40
S latitude.

5.2.2 Pests and Diseases Juglans regia is sensitive to autumn frosts that can even result in mortality, and late spring frosts that reduce nut yield and affect stem form. The most important diseases causing mortality are fungal, including Armillaria mellea, Phytophthora cinamomii, and P. cambivora, which affect roots. Bacterial diseases of walnut include Xanthomonas arboricola pv. juglandis, which damages

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_5, # Springer-Verlag Berlin Heidelberg 2011

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leaves, developing nuts and young shoots during humid periods, Erwinia, which causes cankers (Teviotdale et al. 1985), and crown gall, caused by Agrobacterium tumefaciens. Walnuts are the preferred host for root lesion nematodes (Pratylenchus vulnus), which can be an important local pest (Strand 2003). Walnuts infected by the cherry leaf roll virus decline and die, especially if the trees develop blackline disease, the result of a necrotic hypersensitive response at the graft union between susceptible and virus-resistant scions and stocks (Mircetich and Rowhani 1984). Codling moth (Cydia pomonella) and navel orangeworm (Amyelois transitella) are among the most destructive insect pests of Persian walnut (Strand 2003). Some or all of these pests and diseases (and more) affect walnuts wherever they are grown in commercial plantations; their impact on wild germplasm is not well studied, but is probably minimal.

5.2.3 Germplasm Collection and Assessment Germplasm collection, maintenance, characterization, and breeding are taking place in nearly every country where J. regia is native or cultivated (Germain 2001, 2004). The focus of nearly all these efforts is cultivar development for nut production. Germplasm collection activities for Persian walnut have emphasized evaluating the phenotypic and genetic variability of native populations of wild, feral or semi-domesticated seedling trees, broadening the narrow germplasm base found in existing cultivars (in areas where the species was recently introduced) and identifying sources of disease resistance. In Europe, J. regia is considered a noble hardwood with moderately high economic value (Eriksson 2001), and as such it is part of the EU Strategic planning for the preservation of noble hardwoods. Malvolti et al. (2002) found evidence of genetic erosion for J. regia in Europe, and Ferna´ndez-Lo´pez et al. (2007) summarized the conservation program for J. regia in Europe as a combined effort of ex situ collection with multiple collections focused on timber and nut diversity, and in situ maintenance of small local populations which should be encouraged to evolve. In many places in Europe, J. regia grows as a feral tree or in abandoned orchards of (typically) seedling trees (Ferna´ndez-Lo´pez et al. 2007). No natural populations of the species are present in any part of western Europe (Ferrazzini et al. 2007).

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Demesure (1996) found that, like most noble hardwoods in Europe, J. regia is most threatened by harvest, intensive forest management, loss of habitat, replacement by other species, poor silviculture (poor regeneration), low timber value leading to low retention by landowners, decreasing population sizes (genetic erosion), and genetic pollution (including mating with nonlocal genotypes introduced for nut production and mating with exotics). Despite these factors, J. regia does not appear to be genetically depauperate in Europe (Fornari et al. 2001) although the resources for any single country may be limited. In Italy, for example, J. regia has been the focus of substantial research since 1985, with a priority placed on preserving the remnants of the in situ genetic resource after intensive felling (Malvolti et al. 2004). Ferrazzini et al. (2007) used random amplified polymorphic DNA (RAPD) markers to characterize genetic relationships among 104 Italian walnuts. They reported relatively low levels of genetic differentiation, with only about 10% of the variability partitioned among locations. They concluded that the history of human-mediated movement led to the current genetic structure of J. regia in Europe, and that genetic diversity should be sampled based on individual phenotype rather than a sample of ecological zones. There do not appear to be any coordinated international programs for conservation of J. regia either as a nut crop or a timber species within its true native range in central Asia. As mentioned earlier, J. regia has been subject to local selection for millennia, resulting in an impressive variability in nut size and shape, but there are relatively few clear ecotypes or subspecies. The most prominent exceptions are J. v. sigillata, commonly referred to as iron walnut for its thick, rough shells, J. v. fallax, and J. v. turcomanica (McGranahan and Leslie 1990). McGranahan and Leslie (1990) concluded that “the source of genes for genetic improvement is changing rapidly”, since the collection and maintenance of germplasm has become worldwide priority. The European Forest Genetics Resources Programme (EUFORGEN), United States Department of Agriculture National Clonal Germplasm Repository (NCGR; located in Davis, CA), and the International Board of Plant Genetic Resources (IBPGR) have all made collections of Juglans that were made available to breeders. Important germplasm collections are maintained at NCGR, European Cooperative Program on Plant Genetic Resources (ECP/ GR; operating under the aegis of IBPGR and the United Nations Food and Agriculture Organization), European

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Economic Community (EEC) Clonal Germplasm Repository, and Instituto Sperimentale per la Fruticolture (Italy), and the French National Institute for Agricultural Research (INRA) – Station de Recherches Fruitie`res (Villenave d’Ornon Cedex, France).

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the most active programs in the development of J. regia as a timber species. Spain, Italy and France have active programs in the development of J.  intermedia for timber (see below). Spain

5.2.4 Agroforestry Juglans regia has long been used in agroforestry systems by traditional agrarian cultures (Zou and Sanford 1990). In the Netherlands and other European nations, research on the use of J. regia as a multi-purpose tree (nuts and timber) is focused on agroforestry plantation systems (Oosterbaan and Kuiters 2009) (Fig. 5.1). In many parts of Europe, however, agroforestry research is focused on hybrid walnut (J.  intermedia) (Aleta` et al. 2003; Chifflot et al. 2006).

5.2.5 Juglans regia Crop Improvement 5.2.5.1 Juglans regia as a Timber Species In Europe, where J. regia wood is considered among the highest in quality (Vassiliou and Voulgaridis 2005), there has been renewed interest in the development of J. regia as a timber species. The use of J. regia for timber production has been limited in Europe by a shortage suitable genotypes (Aleta` et al. 2003), so a research effort to describe the genetic variability of European J. regia for timber traits is underway (Fady et al. 2003). Spain and the United Kingdom (UK) have

Fig. 5.1 Persian walnut (Juglans regia) grown for timber production in an agroforestry planting (L) and on alluvial soils of the Padan plain in northern Italy (R). Photo credit: (L) Pierluigi

Research into the use of J. regia for wood production in Spain began in 1982 with the survey of available germplasm, selection of new varieties for wood production, selection of parents for production of hybrids, selection of clones, and development of training and irrigation systems (Aleta` 2004; Dı´az and Ferna´ndez-Lo´pez 2005). A walnut breeding program for timber production was started in Galicia (Northwestern) Spain in 1997. Phenotypic selections were made in local populations in northwestern Spain that could be considered landraces, as several authors found differentiation among populations in terms of height growth and bud burst (Aleta` and Ninot 1997). By the end of 2003, at least 5,000 acres of Juglans, mostly hybrids, had been planted in Spain under intensive management (Aleta` 2004). Dı´az and Ferna´ndez-Lo´pez (2005) collected data on 43 families of J. regia at two nurseries in northwestern Spain, finding considerable genetic differences among families for growth, phenology, and resistance to disease, but not for growth habit.

United Kingdom Although it is often called “English walnut” in the US, in fact, J. regia was an introduced species in the UK as well. Walnuts have not been widely cultivated for timber

Paris, Istituto di Biologia Agroambientale e Forestale Consiglio Nazionale delle Ricerche, (R) Prof. Gianfranco Minotta, Universita` di Torino

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or fruit production in the UK. As a consequence, initial research there for improvement of J. regia as a timber species focused on acquisition of additional germplasm. Genotypes were collected across 11 countries from both the introduced and natural ranges of the species (Hemery and Russell 2005). A program for selection of walnut for timber production in the UK was initiated in 1996. Results from a multi-location provenance trial showed provenances from more northerly latitudes and from within the introduced range of the species performed best after 5 years (Hemery et al. 2005). In 2000, plantations were established in UK to evaluate several types of mixed plantation systems, including interplanting with nurse trees and shrubs (Hemery and Russell 2005). The trial included 78 timber selections (J. regia, J. nigra, J. major, and their hybrids) and 66 J. regia from breeding programs across Europe and the United States of America (US). Research focused on the use of tree shelters, direct seeding, nitrogen fertilization, establishment success, identifying adapted trees with suitable timber form, and the design of plantations that will provide suitable silvicultural conditions (Hemery 2004). Buresti (1995) had previously shown that nurse trees (Prunus avium, Elaeagnus angustifolia, Alnus cordata, and Robinia pseudoacacia) could produce significant increases in walnut height and diameter.

5.2.5.2 Juglans regia as a Nut Species Unlike most Juglans, the fruit of J. regia often is imperfectly dehiscent; the non-sutured husk cracks and splits at maturity, releasing the nut, which is usually thin-shelled. Selection of preferred J. regia phenotypes probably dates from prehistory, but until recently J. regia was mostly propagated by seed. Within the last 100 years J. regia outside of its native range has become an important international commodity grown in large, commercial scale plantations of grafted trees. There is a well-developed international market for J. regia nuts, with production worldwide in 2001 of about 700,000 metric tons (http://www.fas. usda.gov/htp/Hort_Circular/2001/01-12/walnut.htm). The largest producers, by far, are China and the US, but most of the crop produced in China is consumed domestically (Dandekar et al. 2005). Where J. regia is grown for international markets its wood is rarely used because the most productive cultivars, the products of several generations of selection, are not architecturally suitable

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for timber, and because they are often grown in production systems that result in short, heavily branched stems of low value as timber. Thus, in general, only wild, feral and semi-domesticated trees are likely to serve as a genetic resource for improvement for timber production. There are conventional breeding programs for improvement of J. regia as a nut tree in nearly every country where the species is grown commercially (Germain 2004). The range of genetic variation of J. regia for disease resistance, adverse soil and climate conditions, phenology, flowering and fruiting habit, nut shape, size and quality, and kernel quality has been summarized by McGranahan and Leslie (1990). These traits are central to the improvement of J. regia as a nut crop, so the germplasm that has been characterized is almost entirely the breeding stocks or germplasm resources for nut breeding programs. Breeding programs for J. regia are primarily in the public sector. Among the most productive are those in France, Chile, and China, but the cultivars developed at the University of California, Davis (UCD), including “Hartley”, “Chandler”, and “Serr” have been adopted internationally and represent the standard by which other cultivars are judged. The UCD improvement program has emphasized yield, disease resistance, kernel quality, phenology of flowering, and nut maturity (Dandekar et al. 2005; McGranahan and Leslie 2009). Other Juglans species have been considered for use as rootstocks for J. regia nut cultivars to provide tolerance of biotic and abiotic stresses. For example, J. mandshurica provides cold hardiness and has been used as a rootstock in the northern regions of China. J. cathayensis is used as a rootstock in the Yangtze River region of China. J. microcarpa, a southwestern US species, has been used as a rootstock because of tolerance of high pH soils, high boron, and high chloride. Last, J. major, the Arizona black walnut, also has been tested as a rootstock because of its tolerance of high pH soils (McGranahan and Leslie 2009).

5.3 Juglans Wild Relatives 5.3.1 Ecology and Phylogeography Members of the genus Juglans (20–21 species) commonly called walnuts and butternuts, are found in North, Central, and South America and higher elevations of

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Asia. They are primarily forest trees although some species grow in riparian zones of semi-arid regions and a few species are shrubby. Walnuts and butternuts prefer deep, fertile, well drained loam soils, but will grow in other soil types with less vigor. Their growth is restricted in soils with high boron or high soluble salts, the latter may be found where walnut is under commercial cultivation. As mentioned previously, spring and fall frosts, rather than cold temperatures, tend to restrict growing ranges of walnuts in temperate regions. Early genetic studies by Woodworth (1930) demonstrated that Juglans spp. are diploid, with a karyotype of 2n ¼ 2x ¼ 32. The formal taxonomy of Juglans is controversial. The genus has been grouped into four sections (Manning 1978), i.e., Rhysocaryon Dode, Cardiocaryon Dode, Trachycaryon Dode ex. Mann., and Juglans Mann., based on morphology and phylogeography. Recent molecular studies have cast doubt on some aspects of the previous taxonomic paradigm. Abuı´n et al. (2006) analyzed regions of the chloroplast DNA regions of five Juglans spp. using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). They proposed a split within the Rhysocaryon, with J. nigra divergent from J. major, J. hindsii, and J. australis, species from the western US and S. America. Juglans regia was divergent from the other four species. Orel et al. (2003) also analyzed the Juglans chloroplast genome, but used RAPD markers to distinguish between the Old World clade (J. ailantifolia, regia and sigillata) and American or New World clade (J. australis, olanchana, and nigra). Stanford et al. (2000) and Fjellstrom and Parfitt (1994b) looked at phylogenetic and biogeographic relationships among 15 Juglans species using a chloroplast gene (matK), internal transcribed spacer (ITS) sequence data, and nuclear RFLP data. They proposed the separation of Juglans into two subclades, i.e., (1) Rhysocaryon, comprised all New World Juglans except butternut, and (2) Cardiocaryon (J. ailantifolia, mandshurica, and cathayensis, species sometimes called Asian butternuts), Dioscaryon (J. regia), and Trachycaryon (J. cinerea). Rhysocaryon was further separated into temperate (J. nigra, microcarpa, major, californica, and hindsii) and tropical black walnuts (J. australis, boliviana, neotropica, guatemalensis, and olanchana). Aradhya et al. (2006), based on data from chloroplast and ITS sequences, further divided the tropical black walnuts into tropical and subtropical subclades (J. guatemalensis, microcarpa and mollis). Their analysis placed J.

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hopeiensis into section Cardiocaryon; J. regia and sigillata were placed in the section Juglans rather than Dioscaryon.

5.3.2 Commercial Uses of Juglans 5.3.2.1 Timber Walnuts and butternuts are valued for both timber and nut production, although for most species the nuts and lumber have only a local commercial market. The most important exceptions to this generalization are J. mandschurica, J. hindsii, J. neotropica, and J. australis. In China, J. mandshurica is used in furniture. J. hindsii is limited in range to a small area in N. California, so the volume of production for this species is quite low, but logs from J. hindsii trees used as rootstocks can command high prices if they include a burl that can be sliced for veneer. J. neotropica, a mountainous species in Columbia, Ecuador, Peru, and Venezuela, is harvested for production of furniture and for carving, and J. australis is harvested commercially in Argentina.

5.3.2.2 Nuts All walnuts are wind-pollinated. Male flowers are borne in catkins on the previous year’s growth; female flowers are borne singly or in small clusters on the same branches with male flowers in the current season’s flush of growth. Fruits are false drupes containing a large nut enclosing a kernel that is sweet, edible, and oily. The nuts are commonly gathered and consumed by both humans and wildlife, but only J. regia and J. nigra are commercially cultivated for their nut crops. J. ailantifolia was introduced into the United States for nut production in the mid-nineteenth century; the variety cordiformis was particularly popular because of its striking, heart-shaped nuts, but a commercial market for J. ailantifolia never developed in the US. Selection and breeding of J. nigra for nuts began over 100 years ago, but until very recently it was performed mostly by amateurs and enthusiasts who managed nevertheless to name and propagate several hundred cultivars. Nearly all of these were only locally popular, most are now lost, and many were only nominally better than wild trees (Reid et al. 2004). Modern markets for black walnut nuts

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and nut-meats are relatively small and regional. Jones et al. (1998) concluded that selection for nut yield and quality and knowledge of J. nigra as a nut tree had advanced sufficiently that black walnut was on the threshold of becoming a commercially viable species for nut production (Fig. 5.2). Although black walnut can be grown as a dual-purpose crop tree (for both timber and nuts), the two uses can be difficult to coordinate. Maximizing timber yield can conflict with maximizing nut yields because management for timber requires closer tree spacing to produce a branch-free bole, while nut trees are managed to produce large crowns and heavy branches (van Sambeek and Rink 1981).

5.3.3 Crop Vulnerability Some Juglans species have a restricted native range, and nearly all species are under increasing pressure from expanding human populations, removal by log-

Fig. 5.2 Black walnut (Juglans nigra) grown for nut production in Missouri, USA. Photo credit, J.W. Van Sambeek, USDA Forest Service

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ging, and land clearing for agriculture or grazing (McGranahan and Leslie 1990). Three species particularly affected by these pressures are J. venezuelensis, J. pyriformis, and J. jamaicensis (Francis and Alemany 1994). Little is known about J. olanchana, J. boliviana, and J. soratensis; they are considered threatened because of their limited ranges and a lack of knowledge of their biology and ecology. Replacement of native germ plasm by modern J. regia cultivars threatens landraces of J. regia and several Juglans species. Foroni et al. (2005, 2007) found that in one of the most important nut producing regions of Italy (Sorrento), genotypes related to traditional landraces cultivated as multipurpose trees in southern Italy are being replaced by clonal varieties containing genes from advanced selections. The genetic resources of J. sigillata in China have been significantly depleted by grafting that species to J. regia (McGranahan and Leslie 1990).

5.3.4 Conservation Initiatives Conservation efforts for members of the genus other than J. regia have been limited to J. mandschurica, J. nigra, and J. cinerea. De’an et al. (1997) reported on provenance trials for J. mandschurica in China. Two trials were introduced five years apart. Based on those tests, they were able to recommend Shulan, Mao’ershan, and Baishishan provinces as seed sources for afforestation of the Mao’ershan province. The USDA Forest Service recently has initiated an effort to conserve J. cinerea (butternut) germ plasm in the United States in the wake of extensive mortality caused by butternut canker, a disease caused by Sirococcus clavigignenti-juglandacearum. Conservation efforts for butternut are also underway in Canada. Nut collections are being made from throughout the species’ range, and the seed will be grown in various nurseries for outplanting in 2010 and beyond. In the course of germ plasm exploration, it was determined that many surviving “butternuts” were hybrids with J. ailantifolia (the hybrids are known as J.  bixbyi). It is possible that the hybrids have greater resistance to butternut canker than butternut and Japanese walnut. Curiously, a small percentage of butternuts have a darkbarked phenotype similar to the bark of J. nigra, and many of these trees appear to have enhanced resistance to butternut canker. The genetic basis for the

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dark-barked phenotype has yet to be unraveled, but it is possible that J. ailantifolia has been a genetic bridge for the introgression of genes from J. nigra to J. cinerea, two species that are incompatible, but both of which can hybridize with J. ailantifolia (K. Woeste, personal communication).

5.4 Role in Traditional Crop Improvement 5.4.1 Juglans Hybrids Many Juglans species can hybridize, although the hybrids vary in their vegetative vigor and their ability to produce fruit and viable pollen (Dandekar et al. 2005). In general, hybridization among species within sections of the genus is more successful than intersectional hybridization (Williams 1990), but the only limitation in obtaining interspecific hybrids with J. regia appears to be concerted effort. Nearly every species within the genus could thus be considered a potential source of genes for improvement of J. nigra, either through direct hybridization (e.g., J. microcarpa, J. ailantifolia, J. regia) or by using J. regia as a bridge species. It seems likely that several unpublished hybrid combinations (e.g., J. nigra  J. major, J. nigra  J. australis) are possible but untried. Germain (2004) lists the hybrid germplasm resources in Europe along with those for J. regia (http://www.fao. org/docrep/007/y5704e/y5704e0l.htm). The Spanish breeding program for J. regia has pursued hybrids as a means of improving the relatively poor form of the remaining J. regia germplasm (Aleta` 2004). Aleta` identified five J. regia clones currently being used in controlled crosses for timber production; these clones were also used in controlled crosses with J. nigra, J. australis, J. hindsii, and J. major with the goal of evaluating full-sib progenies from among these crosses. The Royal hybrid (J. hindsii  J. nigra) is a large, vigorous tree that has not been thoroughly tested outside of California, but is probably not sufficiently cold tolerant to grow in the hardwood timber-producing regions of the US. The preeminent rootstock for production of J. regia as a nut crop is “Paradox” (J. hindsii  J. regia). Paradox is an exceptionally vigorous rootstock with

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potential as a timber tree (Forde and McGranahan 1996). Paradox rootstocks are most often J. hindsii  J. regia, but several other species, including J. major and J. nigra have contributed to commercial Paradox rootstocks, and some Paradox are complex, multi-species hybrids (Potter et al. 2002), providing an opportunity for selection and development of rootstocks with genetic variability for resistance to a number of important pests and diseases, including Phytophthora citricola (Browne 1998). At least two Paradox clones have been patented for use as a rootstock (VX211, US 20080320618; RX1, 20080320619). Paradox hybrids produced by crosses with J. hindsii are not cold-hardy, however, which led to a resurgence of interest in the use of J.  intermedia as a rootstock in Europe. The single greatest contribution of a wild relative to the improvement of J. nigra has come as a result of the recent interest in Europe in J. nigra  J. regia hybrids known as J.  intermedia. Hybrids between these two species have a relatively long history in the US because of interest in using J. regia to improve the yield and nut quality of J. nigra, which produces large but thick-shelled nuts that typically release their kernels in small pieces (about 19% kernel versus greater than 50% kernel for J. regia). Beginning in 1937, McKay began experiments in hybridizing these two species (McKay 1965), but the work was slow because, according to McKay (1959) “the first-generation hybrids are largely sterile and so much time is required to bring the second generation seedlings to fruiting.” McKay’s backcross breeding program was essentially discontinued upon his retirement. Beginning in the late 1980s, walnut breeders in France began a long-term program to introduce hypersensitivity to the cherry leaf roll virus into J. regia by crossing it to J. nigra and other North American walnut species. One result of this research was the identification of a series of hybrids, including NG23xRA (J.  intermedia) that were widely studied and commercialized for timber production (Burtin et al. 1998). These French hybrids are also being evaluated in Spain (Fady et al. 2003; Casal et al. 2006). Aleta` (2004) indicated at least one J. regia hybrid of unknown parentage IRTA X-80, is being tested as a clone in Spain, along with “Mj209xRA” and “Ng23xRA” for Armillaria root and other crown rot diseases. Italian researchers have focused on the production of additional J.  intermedia genotypes by identifying J. nigra that produce large numbers of

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hybrid progeny (Pollegioni et al. 2009). Commercial vendors of hybrids in Europe are beginning to provide information on their planting and care http://www. nogalhibrido.com/Cuidados-en.htm.

5.5 Juglans Genomics and Biotechnology Because of its commercial value, there are far more gene sequences available for J. regia than for any other member of the genus, although even J. regia lags far behind many commodity crops in the availability of sequence data. This situation is likely to change in the near term with the increasing availability of low cost, high-throughput sequencing technologies. As of April, 2009, GenBank (National Center for Biotechnology Information, NCBI), the public repository for DNA sequence data in the US, lists 5,087 total entries for J. regia, including nuclear and chloroplast genes and expressed sequence tags (ESTs). Most of these sequences derive from a cDNA library of midseason embryos submitted by researchers at UCD. Functional genomics for J. regia is limited. Walnuts expressing antisense chalcone synthase were deficient in the accumulation of flavonoids but had increased production of adventitious roots (El Euch et al. 1998). This result may have been observed because flavonoids can negatively regulate auxin transport, and auxins are strongly associated with root initiation (Brown et al. 2001). Naphthoquinone metabolism, including the formation of hydrojuglone glucosides (HJG), is important to walnut improvement because it was found that HJG is a critical component of heartwood color formation in J.  intermedia (Burtin et al. 1998). Gene discovery and biotechnology for J. regia generally precedes application in other Juglans species. For example, transformation and regeneration systems were first published in J. regia (Dandekar et al. 1989) before being applied to J. nigra (Bosela et al. 2004). The most significant developments in genomics and biotechnology in the past decade for Juglans were the characterization of regulation of oil biosynthesis in J. regia (Dandekar et al. 2005), and the use of antisense technology to produce Juglans rootstocks resistant to crown gall disease (Escobar et al. 2001).

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5.5.1 Propagation Walnuts can be easily propagated by grafting, and this remains the predominant method by which nurseries and breeding programs multiply selected genotypes. Rooting walnut is notoriously difficult, however. Starting about 1988 methods to propagate walnut in vitro (i.e., micropropagation) were published for walnut (McGranahan et al. 1988) and subsequently adapted for use by at least one commercial laboratory (Lopez 2004). The methods were later modified and extended to J. nigra (Long et al. 1995) and J.  intermedia (Cornu and Jay-Allemand 1989). Micropropagation of walnut was an important breakthrough because of the desire to propagate cultivars on their own roots and to develop clonal rootstocks, especially Paradox. Somatic embryogenesis was used in J. regia to produce a triploid plant (the first documented aneuploid for this species) (Tuleke and McGranahan 1988), later, a tetraploid, known as “Mitsuru” was developed using colchicine (Yajima et al. 1997). The development of methods for in vitro propagation of walnut has important implications for the development of novel hybrids, some of which may require embryo rescue (McGranahan et al. 1986).

5.5.2 Markers and Marker-Assisted Breeding Mutation breeding has not been widely practiced in forest trees, perhaps because of the large size of the organisms, the long time required to grow the trees to the size that they can be evaluated, and the belief that most traits that affect forest tree quality (height, diameter growth, straightness) are highly oligogenic. A large number of marker systems have been identified for use in J. regia, including isozymes, RFLP, RAPD, AFLP, ISSR, and microsatellites (Arulsekar et al. 1985; Fjellstrom and Parfitt 1994a; Woeste et al. 1996b, 2002; Pollegioni et al. 2003; Foroni et al 2005). Several of these systems have been employed in the development of genetic linkage maps for J. regia (Fjellstrom and Parfitt 1994b; Woeste et al. 1996b), although only low density maps have been published. DNA-based markers have also been used to fingerprint cultivars and to verify pedigrees (Dangl et al. 2005), to identify the members of

5 Juglans

a landrace (Sorrento) (Foroni et al. 2005, 2007) and to identify individuals in a backcross population of J. regia  J. hindsii that are resistant to cherry leaf roll virus (Woeste et al. 1996a).

5.6 Concerns Related to Transfer of Juglans Germplasm Large scale transfer of J. regia, J. nigra, and J. ailantifolia across most of the temperate zones of the N. and S. Hemisphere raises concerns about the introduction of exotics, the replacement of native species, and the potential weediness of Juglans or Juglans hybrids. In fact, J. ailantifolia has been classified as a weed in New Zealand, http://www.hear.org/gcw/species/ juglans_ailantifolia/, and the introduction of J. ailantifolia in N. American has resulted in widespread hybridization with the threatened species J. cinerea (butternut). The hybrid (Juglans  bixbyi) has complicated conservation of butternut (Ross-Davis et al. 2008), and there is evidence it is becoming invasive. Recent research by Woeste and colleagues is beginning to describe both the positive and negative attributes of the J. ailantifolia  cinerea hybrid. Although it has less desirable wood quality than J. cinerea, the existence of the hybrid is helping to maintain a remnant of wild J. cinerea germplasm, which otherwise would have been depleted by butternut canker. On the other hand, the hybrid, which displays hybrid vigor, is altering the ecology of the landscape through its vegetative vigor and ability to out-compete both parents. It is likely that movement of Juglans seeds and scion wood has also resulted in the dispersal of exotic pests and pathogens, possibly including Sirococcus clavigignenti-juglandacearum (Furnier et al. 1999), and this trend can only be expected to continue.

5.7 Summary Breeding programs for J. regia and J. nigra are increasingly turning toward hybrids with other members of the genus to gain access to desirable traits. New disease threats and human-mediated expansion of the range of J. regia are providing strong impetus for the

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development of new rootstocks for this species. Many of these rootstocks are likely to be derived from hybridization with other Juglans species. Hybrids with J. nigra already are widely planted in Europe for timber; J.  intermedia may have a future in US as a timber tree as well. Hybrids between J. nigra and other Juglans species often display hybrid vigor for timber traits, but at the potential cost of wood quality. The long-term cost of gene flow from non-native species into native populations of black walnut must be considered with care.

References Abuı´n M, Diaz R, Alfonsin J, Aleta N, Ninot A, FernandezLopez J (2006) PCR-RFLP analysis of Cp-DNA in the genus Juglans. Acta Hortic 705:215–220 Aleta` N (2004) Current research in Spain on walnut for wood production. In: Michler CH, Pijut PM, Van Sambeek JW, Coggeshall MV, Seifert J, Woeste K, Overton R, Ponder F Jr (eds) Black Walnut in a new century, Proceedings of the sixth Walnut Council research symposium, 25–28 July 2004, Lafayette, IN, USA. Gen Tech Rep GTR-NC-243. US Department of Agriculture, Forest Service, North Central Forest Experiment Station, St. Paul, MN, pp 153–155 Aleta` N, Ninot A (1997) Field evaluation of Juglans regia selected clones from seedlings populations of Mediterranean and Atlantic Spanish Coast. Acta Hortic 411:63–67 Aleta` N, Ninot A, Voltas J (2003) Characterization of the Agroforestry performance of 12 walnut (Juglans sp.) genotypes grown in two locations of Catalonia. Invest Agrar Sist Recur For 12:39–50 Aleta` N, Ninot A, Voltas J (2004) Retrospective evaluation of parental selection in nursery tests of Juglans regia L. using a mixed model analysis. Silvae Genet 53:26–32 Aradhya MK, Potter D, Simon CJ (2006) Cladistic biogeography of Juglans (Juglandaceae) based on chloroplast DNA intergenic spacer sequences. In: Motley TJ, Zerega N, Cross H (eds) Darwin’s harvest: new approaches to the origins, evolution, and conservation of crops. Columbia University Press, New York, USA, pp 143–170 Arulsekar S, Parfitt DE, McGranahan GH (1985) Isozyme gene markers in Juglans species. J Hered 76:103–106 Bosela MJ, Smagh GS, Michler CH (2004) Genetic transformation of black walnut (Juglans nigra). In: Michler CH, Pijut PM, Van Sambeek JW, Coggeshall MV, Seifert J, Woeste K, Overton R, Ponder F Jr (eds) Proceedings of the sixth Walnut Council research symposium. Gen Tech Rep NC-243. US Department of Agriculture, Forest Service, North Central Research Station, St. Paul, MN, pp 45–58 Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WS, Taiz L, Muday GK (2001) Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis. Plant Physiol 126:524–535

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K. Woeste and C. Michler Panetsos K, Paris P, Pisanelli A, Rumpf H (2003) Walnut demonstrates strong genetic variability for adaptive and wood quality traits in a network of juvenile field tests across Europe. New Forest 25:211–225 Ferna´ndez-Lo´pez J, Aleta N, Alı´a R (2007) Walnut (Juglans regia) genetic resources. European Forest Genetic Resources Programme. EUFORGEN Available via DIALOG. http:// www.bioversityinternational.org/Networks/Euforgen/networks/Scattered_Broadleaves/NHStrategies/JuglansSpp ConsStrategy.htm. Accessed 25 Jan 2007 Ferrazzini D, Monteleone I, Lecce F, Baelletti P (2007) Genetic variation of common walnut (Juglans regia) in Piedmont, Northwestern Italy. Forest 4:386–394 Fjellstrom RG, Parfitt DE (1994a) RFLP inheritance and linkage in walnut. Theor Appl Genet 89:665–670 Fjellstrom RG, Parfitt DE (1994b) Walnut (Juglans spp) genetic diversity determined by restriction fragment length polymorphisms. Genome 37:690–700 Forde HI, McGranahan GH (1996) Walnuts. In: Janick J, Moore J (eds) Fruit breeding. Wiley, New York, USA, pp 241–278 Fornari B, Malvolti ME, Taurchini D, Fineschi S, Beritognolo I, Maccaglia E, Cannata F (2001) Isozyme and organellar DNA analysis of genetic diversity in natural/naturalized European and Asiatic walnut (Juglans regia L.) populations. Acta Hortic (ISHS) 544:167–178 Foroni I, Rao R, Woeste K, Gallitelli M (2005) Characterization of Juglans regia L. through SSR markers and evaluation of genetic relationships among cultivars and the ‘Sorrento’ landrace. J Hortic Sci Biotechnol 80:49–53 Foroni I, Woeste K, Monti LM, Rao R (2007) Identification of ‘Sorrento’ walnut using simple sequence repeats (SSRs). Genet Resour Crop Evol 54:1081–1094 Francis JK, Alemany S (1994) Juglans jamaicensis C. DC. Nogal. Juglandaceae. Walnut family. USDA Forest Service, International Institute of Tropical Forestry, New Orleans, LA, p 4 Furnier GR, Stolz AM, Mustaphi RM, Ostry ME (1999) Genetic evidence that butternut canker was recently introduced into North America. Can J Bot 77:783–785 Germain E (ed) (2001) Proceedings of the fourth international walnut symposium, Bordeaux, France, ISHS. Acta Hort, 544 p Germain E (ed) (2004) Inventory of walnut research, germplasm and references. REU Technical Series 66, FAO Regional Office for Europe, FAO-CIHEAM Interregional Network on Nuts (ESCORENA), Food and Agriculture Organization of the United Nations, Rome, Italy, 264 p Hemery GE (2004) Genetic and silvicultural research promoting common walnut (Juglans regia) for timber production in the United Kingdom. In: Michler CH, Pijut PM, Van Sambeek J, Coggeshall M, Seifert J, Woeste K, Overton R, Ponder F Jr (eds) Black walnut in a new century, Proceedings of the sixth Walnut Council research symposium, 25–28 July 2004, Lafayette, IN, USA. Gen Tech Rep NC-243. US Department of Agriculture, Forest Service, North Central Research Station, St. Paul, MN, pp 138–145 Hemery GE, Russell K (2005) Advances in walnut breeding and culture in the United Kingdom. Acta Hortic (ISHS) 705:95–101 Hemery GE, Savill PS, Thakur A (2005) Height growth and flushing in common walnut (Juglans regia L.): 5-year results from provenance trials in Great Britain. Forestry 78:121–133

5 Juglans Jones JE, Mueller R, Van Sambeek JW (1998) Nut Production Handbook for Eastern Black Walnut. Southwest Missouri Resources, Conservation & Development, Inc. http://nrs.fs. fed.us/pubs/misc/walnut/ Long LM, Preece JE, Van Sambeek JW (1995) Adventitious regeneration of Juglans nigra L. (eastern black walnut). Plant Cell Rep 14:799–803 Lopez J (2004) Walnut tissue culture: research and field applications. In: Michler CH, Pijut PM, Van Sambeek JW, Coggeshall MV, Seifert J, Woeste K, Overton R, Ponder F Jr (eds) Black walnut in a new century, Proceedings of the sixth Walnut Council research symposium, 25–28 July 2004, Lafayette, IN, USA. Gen Tech Rep NC-243. US Department of Agriculture, Forest Service, North Central Research Station, St. Paul, MN, pp 146–152 Malvolti M, Pollegioni P, Bartoli S (2002) Genetic variation of walnut (Juglans regia) in Europe. In: Bozzano M, Rusanen M, Rotach P, Koskela J (Compilers) Reports 6th (9–11 June 2002, Alter do Cha˜o, Portugal) and 7th (22–24 April 2004, Arezzo, Italy) Meets, pp 77–82 Malvolti M, Pollegioni P, Bartoli S (2004) Genetic variation of walnut (Juglans regia) in Europe. In: Bozzano M, Rusanen M, Rotach P, Koskela J (Compilers) Reports 6th (9–11 June 2002, Alter do Cha˜o, Portugal) and 7th (22–24 April 2004, Arezzo, Italy) Meets, pp 77–82 Manchester SR (1989) Early history of the Juglandaceae. Plant Syst Evol 162:231–250 Manning WE (1978) The classification with the Juglandaceae. Ann Mo Bot Gard 65:1058–1087 Marquis DA, Johnson RL (1989) Silviculture of eastern hardwoods. In: Burns RM (Compiler) The Scientific basis for silvicultural and management decisions in the national forest system. Gen Tech Rep WO-55. US Department of Agriculture, Forest Service, Washington, DC, pp 9–15 McGranahan GH, Leslie CA (1990) Walnut (Juglans L.). In: Moore JN, Ballington JR (eds) Genetic resources of fruit and nut crops, vol 2. ISHS, Wageningen, Netherlands, pp 907–951 McGranahan G, Leslie C (2009) Breeding walnuts (Juglans Regia). In: Jain SM, Priyadarshan PM (eds) Breeding plantation tree crops: temperate species. Springer, New York, USA, pp 249–273 McGranahan GH, Tuleke W, Arulsekar S, Hansen JJ (1986) Intergeneric hybridization in the Juglandaceae: Pterocarya sp. X Juglans regia. J Am Soc Hortic Sci 111:627–630 McGranahan GH, Leslie DA, Uratsu SL, Dandekar AM (1988) In vitro propagation of mature Persian walnut cultivars. HortScience 23:220 McKay JW (1959) Techniques in breeding new varieties of orchard trees. Proceedings of the sixth northeastern forest tree improvement conference, Beltsville, MD, USA. Available via DIALOG. http://www.rngr.net/Publications/neftic/ 6th-northeastern-forest-tree-improvement-conference/ McKay JW (1965) Progress in black x Persian walnut breeding. Annu Rep North Nut Growers Assoc 56:76–80 Michler CH, Woeste KE, Pijut PM (2007) Black walnut. In: Kole C (ed) Genome mapping and molecular breeding in plants, vol 7, Forest trees. Springer, Heidelberg, Germany, pp 189–198 Michler CH, Pijut PM, Meilan R, Smagh G, Liang X, Woeste KE (2008) Black walnut. In: Kole C, Hall TC (eds) A compendium of transgenic crop plants, vol 9, Transgenic forest tree species. Blackwell, Oxford, UK, pp 263–278

87 Mircetich SM, Rowhani A (1984) The relationship of Cherry Leafroll Virus and blackline disease of English walnut trees. Phytopathology 74:423–428 Oosterbaan A, Kuiters AT (2009) Agroforestry in the Netherlands. In: Rigueiro-Rodrı´guez A, McAdam J, Mosquera-Losada MR (eds) Agroforestry in Europe: current status and future prospects. Springer, Dordrecht, Netherlands, pp 331–341 Orel G, Marchant AD, McLeod JA, Richards GD (2003) Characterization of 11 Juglandaceae genotypes based on morphology, cpDNA, and RAPD. HortScience 38(6):1178–1183 Pollegioni P, Bartoli S, Cannata F, Malvolti ME (2003) Genetic differentiation of four Italian walnut (Juglans regia L.) varieties by inter simple sequence repeat (ISSR). J Genet Breed 57:231–240 Pollegioni P, Woeste K, Mugnozza GS, Malvolti ME (2009) Retrospective identification of hybridogenic walnut plants by SSR fingerprinting and parentage analysis. Mol Breed 24:321–335 Potter D, Gao F, Baggett S, McKenna JR, McGranahan GH (2002) Defining the sources of Paradox: DNA sequence markers for North American walnut (Juglans L.) species and hybrids. Sci Hortic 94:157–170 Reid W, Coggeshall MV, Hunt KL (2004) Cultivar evaluation and development for black walnut orchards. In: Michler CH, Pijut PM, Van Sambeek JW, Coggeshall MV, Seifert J, Woeste K, Overton R, Ponder F Jr (eds) Black walnut in a new century, Proceedings of the sixth Walnut Council research symposium, 25–28 July 2004, Lafayette, IN, USA. Gen Tech Rep NC-243. US Department of Agriculture, Forest Service, North Central Research Station, St. Paul, MN, pp 18–24 Rink G (1988) Black walnut an American wood. FS-270. US Department of Agriculture, Forest Service, Washington, DC, USA, p 7 Ross-Davis A, Huang Z, McKenna J, Ostry M, Woeste K (2008) Morphological and molecular methods to identify butternut (Juglans cinerea) and butternut hybrids: relevance to butternut conservation. Tree Physiol 28:1127–1133 Schlesinger RC, Funk DT (1977) Manager’s handbook for black walnut. Gen Tech Rep NC-38. US Department of Agriculture, Forest Service, North Central Forest Experiment Station, St. Paul, MN, 22p Stanford AM, Harden R, Parks CR (2000) Phylogeny and biogeography of Juglans (Juglandaceae) based on matK and ITS sequence data. Am J Bot 87:872–882 Strand LL (2003) Integrated pest management for walnuts, 3rd edn. University of California Integrated Pest Management Program, ANR, Oakland, CA, USA, p 136 Teviotdale B, Schroth MN, Mulrean EN (1985) Bark, fruit and foliage diseases. In: Ramos D (ed) Walnut orchard management. Univ Calif Publ 21410:153–157 Tuleke W, McGranahan GH (1988) Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut, Juglans regia L. cv ‘Manregian’. Plant Cell Rep 7:301–304 Van Sambeek JW, Rink G (1981) Physiology and silviculture of black walnut for combined timber and nut production. In: Guries RP (ed) Proceedings of the second north central tree improvement conference. University of Wisconsin-Madison, Madison, WI, USA, pp 25–33 Vassiliou VG, Voulgaridis EV (2005) Wood properties and utilization potentials of walnut wood (Juglans regia L.) grown in Greece. Acta Hortic 705:535–542

88 Victory E, Woeste K, Rhodes OE (2004) History of black walnut genetics research in North America. In: Michler CH, Pijut PM, Van Sambeek J, Coggeshall M, Seifert J, Woeste K, Overton R (eds) Black walnut in a new century, Proceedings of the sixth Walnut Council research symposium, Lafayette, IN, USA, Gen Tech Rep NC-243. USDA Forest Service, North Central Research Station, St. Paul, Minnesota, pp 1–8 Williams RD (1990) Juglans nigra L. black walnut. In: Burns RM, Honkala BH (Tech Coord) Silvics of North America. Hardwoods, vol 2. Agric Handbook 654. US Department of Agriculture, Forest Service, Washington DC, pp 391–399 Woeste K, McGranahan GH, Bernatzky R (1996a) Randomly amplified polymorphic DNA loci from a walnut backcross [(Juglans hindsii  J. regia)  J. regia]. J Am Soc Hortic Sci 121:358–361

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Chapter 6

Quercus Preston R. Aldrich and Jeannine Cavender-Bares

6.1 Introduction The genus Quercus is a keystone taxon from both ecological and economic perspectives, forming an important source of food and habitat for wildlife, and wood and paper products for humans. With roughly 400 species distributed across five continents, the genus comprises a considerable portion of the standing stock of trees in Northern Hemisphere forests. Quercus has numerous herbivores, and gall-forming insects are notably diverse and well studied. The oaks tend to be deep-rooted and drought-tolerant, though overall they occupy a range of habitats. They form beneficial associations with mycorrhizal fungi, though several fungal diseases are threatening entire stands in certain parts of the range, namely oak wilt and sudden oak death. For these reasons and other demographic factors, oak reproductive failure has been widely reported and remains a concern in many places. Species of Quercus are recognized as occurring in several sections including the familiar white oaks (section Quercus, Fig. 6.1) and red oaks (section Lobatae). “Good” members of a species are readily recognized but individuals with intermediate combinations of traits are not necessarily rare. This condition has been attributed to extensive hybridization, lineage sorting, and phenotypic plasticity. Molecular genetic studies of oaks within a section show that their gene pools are diverse but poorly resolved, differing mostly by allele frequencies not diagnostic alleles. There exists a sizeable body of data on white oak phylogeography,

P.R. Aldrich (*) Department of Biological Sciences, Benedictine University, Birck Hall 341, 5700 College Road, Lisle, IL 60532-0900, USA e-mail: [email protected]

especially in Europe where the most heavily studied and domesticated of the oaks, Quercus robur and Q. petraea, reside. Oaks are not very far along the domestication path, and so wild populations still figure prominently in breeding programs. A long generation time and a lack of continuity in breeding programs have contributed to this slow progress. Although most oak cultivars are not many generations removed from their wild ancestors, there are numerous oak cultivars in existence, each with notable traits. Performance across cultivars can be compared in common garden plantings, or provenance studies, though many of these long-term plantings have been abandoned and valuable information lost in the process. A renewed interest in provenance trials may emanate from widening concerns about global climate change. Oak improvement relies on traditional, markerassisted, and transgenic breeding. Traditional silviculture provides the base of the oak breeding program. Here standard crosses and methods of quantitative genetics are used to improve traits, and studies show that oak populations typically maintain reasonably high heritabilities for traits of interest. The number of molecular genetic markers available for use in oaks is growing at a rapid pace, which permits improved selection of traits despite the long generation time. There are now a few linkage maps available for Quercus and several studies have utilized these to resolve quantitative trait loci (QTLs) for traits of interest for breeding. Unfortunately, Quercus is not as amenable to in vitro propagation as is Populus, but progress has been made along these lines in several of the oak sections. Oaks have a moderate-sized genome, though large when compared to current model organisms. Yet technology is closing the gap between model and

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_6, # Springer-Verlag Berlin Heidelberg 2011

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Fig. 6.1 The white oak, Quercus alba (image from USDA-NRCS 2009)

non-model systems, and numerous resources have arrived or are pending through several new oak genomics initiatives. In addition to the existing oak linkage maps that count as structural genomic resources, there are a rising number of resources in oak functional genomics that include large repositories of expressed sequence tags (ESTs) and the promise of oak microarrays. Much less work has been done on oak proteomics or metabolomics. However, because Quercus is a keystone genus from several standpoints, the future will likely hold more investments in this taxon.

6.2 Basic Taxonomy of the Oaks Here we review the taxonomy of the oaks, and the morphological and genetic variation as it relates to taxon boundaries. We consider the place of Quercus in the Fagaceae, and the sections of white and red oaks within the genus. We also take time to consider the species problem in the oaks since species resolution is an important aspect of gene pool management.

6.2.1 Fagaceae Our treatment of the Fagaceae follows that of Nixon (1997a) and Manos et al. (2001) who regard the group as comprised of 600–800 species in nine genera including Quercus L. (the oaks) along with Castanea L. (the chestnuts), Castanopsis Spach.,

Chrysolepis Hjelmquist, Colombobalanus (Lozano, Hdz-C. and Henao) Nixon and Crepet, Fagus L. (the beeches), Formanodendron (Camus) Nixon and Crepet, Lithocarpus Bl., and Trigonobalanus Forman. The group is complex and still subject to revisions, as both Manos et al. (2001) and Oh and Manos (2008) used molecular sequence data to update prior phylogenies, the latter concluding that Quercus likely evolved from a castaneoid ancestor. See Sect. 6.6.3.3 for a description of the comparative mapping of Quercus against Castanea by Barreneche et al. (2004). Species in the Fagaceae are evergreen or deciduous trees or shrubs, with alternative, simple pinnately veined leaves that are either lobed or unlobed. Inflorescences are unisexual and usually wind pollinated. Evolution at the 5.8S ribosomal RNA (rRNA) gene and flanking internal transcribed spacer (ITS) regions indicate that wind pollination has arisen three separate times in the group (Manos et al. 2001). Fruits in the Fagaceae are nuts, often with a subtending cupule, which is diagnostic in many cases, forming the acorn cap in Quercus and Lithocarpus. Having large, animal dispersed seeds is associated with considerable species-level diversification in the group and appears to be a derived condition (Manos et al. 2001).

6.2.2 Quercus The genus Quercus contains roughly 500 species of trees and shrubs from throughout the Northern Hemisphere ranging from tropical and temperate

6 Quercus

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Fig. 6.2 Phylogeny for the oaks showing some of the major sections, based on Manos et al. (1999) and Manos and Stanford (2001)

forests to semi-arid regions (Nixon 1993, 1997b). Most of the diversity present in the Western Hemisphere Fagaceae is concentrated within Quercus, particularly in Mexico (>160 species) and secondarily in the southeastern United States. The genus Quercus includes several major monophyletic lineages (Fig. 6.2) after Manos et al. (1999) and Manos and Stanford (2001), recognized as distinct taxonomic sections. The white oaks (section Quercus), live oaks (series Virentes), golden cup or intermediate oaks (section Protobalanus), and red oaks (section Lobatae) are present in, if not restricted to, the Americas. The white oaks extend into Eurasia along with the Cerris or black oaks (section Cerris), while the cycle cup oaks (subgenus Cyclobalanopsis) are entirely Asian. The oaks are evergreen or winter-deciduous trees or shrubs with simple, alternate leaves. Leaf blades may be lobed or unlobed, pinnately veined, margins toothed (red oaks, section Lobatae, Fig. 6.3) or not toothed (white oaks, section Quercus, Fig. 6.3). Flowers are unisexual and wind pollinated. Fruit is an acorn with subtending cupule, maturing in the first year (all North American white oaks) or second year (most North American red oaks).

6.2.3 Hybridization and the Oak Species Oaks rank among the most recognizable trees at the genus level but among the most problematic for

Fig. 6.3 Leaf and acorn morphologies typical of section Quercus (white oaks) and section Lobatae (red oaks) [Images from USDA-NRCS (2009) and Britton and Brown (1913)]

categorizing at the species level. Since gene pool management is influenced by species delineations, we will attempt to clarify some issues in this area before proceeding. The genus Quercus is ubiquitous in many regions of the globe. It has a distinctive morphology, and historically has played an important role in human culture and industry (Ciesla 2002; Logan 2005), all contributing to its recognizability. Moreover, Quercus species do tend to possess a phenotypic cohesion that allows one to readily distinguish “good” members of a species without much difficulty. Yet close inspection of groups of individuals reveals gradations and fuzzy rather than sharp boundaries between species.

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This phenomenon has been recognized for some time and widely attributed to the propensity for oak species to form hybrids in nature (Palmer 1948; Muller 1952; Burger 1975; Van Valen 1976). Controlled crosses verified that Quercus species readily form hybrids with other members of the same section, though intersectional crosses are not formed (Cottam et al. 1982). With the further aid of molecular genetic markers it is now clear that natural hybrids are common in several groups in the Fagaceae, including Quercus as well as Castanea and Lithocarpus (Nixon 1997a). In Fig. 6.4, we show the hybridization network for North American Quercus species. Each node represents a species of oak, and a link has been formed between all pairs of species for which there is a hybrid described in the United States Department of Agriculture (USDA) Silvics Manual (Burns and Honkala 1990). Note the high frequency of hybrid formation within a section (white
white oak, red
red oak), except for species with disjunct distributions such as the west coast oaks versus those common east oaks of the Rocky Mountains. Note also the absence of described hybrids between red and white oaks. The most highly connected white oak is Q. stellata (11 described hybrids) and for the red oaks Q. velutina (12 described hybrids). Two other factors, in addition to recurrent hybridization, likely contribute to overlap in oak species phenotypes, namely incomplete lineage sorting and phenotypic plasticity. Species that no longer cross with one another may nevertheless share most polymorphisms if there has been insufficient time for differences to accrue since the speciation event. Muir and Schlotterer (2005) have proposed that much of the genetic overlap between the European white oaks Q. robur and Q. petraea is due to this shared ancestral polymorphism. In either event, both hybridization and incomplete lineage sorting yield overlapping gene pools. On the other hand, phenotypic plasticity could contribute to phenotypic overlap between oaks even in the face of genetic differences. Phenotypic plasticity has been well described in Quercus, both physiological and morphological (e.g., Bostad et al. 2003; Quero et al. 2006), and it is thought that some of this variation serves an adaptive function (Abrams 1994). Oaks are notoriously variable in their leaf characters, across seasons and even within the same canopy (Blue and Jensen 1988; Bruschi et al. 2003), even though leaves are frequently used for identifications.

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Regardless of causation, the data show that the gene pools of oak species overlap considerably in their content (see next two sections), differing mainly by allele frequencies but not diagnostic alleles. Sectional gene pools are more readily resolved, where red and white oaks form distinct clusters based on nuclear markers (Guttman and Weigt 1989) and monophyletic clades according to chloroplast and ITS markers (Manos et al. 1999). But within a section the oak species tend to overlap in allelic composition within mixed stands (e.g., Aldrich et al. 2003b) and across regions (e.g., Bodenes et al. 1997b). Thus, at both the genotypic and phenotypic levels, oak species generally do not possess traits that are useful as both necessary and sufficient conditions to distinguish them from all other oak species. Nevertheless, molecular approaches that integrate across the entire genome demonstrate some promise for distinguishing species and resolving phylogenetic relationships (Pearse and Hipp 2009). All this begs the question, what species concept should be used to classify oaks? If hybridization is widespread then the biological species concept is not suitable since it defines species on the basis of reproductive isolation (Mayr 1942). Of the many other species concepts in use, actually none are fully suited to the oak problem since they each assume some form of essentialism, that members of a species should all share some basic element that distinguishes them from others, which do not belong in that group. This emphasis on shared, derived character states as diagnostic markers of a lineage, a central tenet of the cladistic approach to phylogenetics (Hennig 1979), often does not apply well at or below the species level. Instead, many researchers at this level have gravitated toward the phenetic clustering approaches developed by the early numerical taxonomists (e.g., Sneath and Sokal 1973) that emphasize shared (instead of shared-derived) characters – since traits are not fully “derived” at this stage of evolution. Taken a step further, the contemporary inclination is to fully embrace the reticulate nature of species-level gene pool dynamics and focus on network representations rather than bifurcating trees (e.g., Posada and Crandall 2001; Huson and Bryant 2006; see also Fig. 6.4). We adopt the view that oaks can be described adequately using a flexible terminology based on a network of shared traits, like the views held by the philosopher Ludwig Wittgenstein. Pigliucci (2003)

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Fig. 6.4 Hybridization network of North American Quercus species. Data from Burns and Honkala (1990)

recognized that much of the confusion surrounding the species problem, in general, was not empirical in its nature, but philosophical and linguistic. He noted the correspondence between the cluster concepts of the

pheneticists and the “family resemblance” concept developed by Wittgenstein (1958) which maintained that most categories in human language are assemblages of referents to items that share a family

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resemblance – without all of them actually sharing some defining element. Wittgentstein used the simple concept of a “game,” defying anyone to pull out a single feature that runs through all possible activities that we might label as a game (would it be a ball, bat, dice, cards, jump rope, dart. . .?). The actual uses of a word, like “game,” he claimed, form “a complicated network of similarities overlapping and criss-crossing: sometimes overall similarities, sometimes similarities of detail” (Wittgenstein 1958, }66), not unlike the oak hybridization network that we present here (Fig. 6.4). And indeed this is how oak gene pools are managed, through identifications grounded in clusters or networks of character states. Botanical keys can provide a definitive (albeit at times arbitrary) call on species identifications, provided the requisite plant characters are available for inspection, and the person has time to scrutinize that one specimen. In stand management, however, identifications are usually made in the field where foresters, scientists, and laypersons are inclined to form flash judgments of oak identities based on collections of readily observed, cooccurring traits. This field-Gestalt method is at times referred to as the whole-tree silvics approach to identification (Tomlinson et al. 2000; Aldrich et al. 2003b). When the number of key traits rises above some critical (and often unspoken) level, the individual is typed as X, if another set of traits co-occur then Y. Intermediate specimens go un-noted or may be cut down, but are certainly excluded from breeding programs – thereby reinforcing what counts as being a member of a given species. In this regard, the oak species concept, in practice, works just fine. As Pigliucci (2003) remarked, Wittgenstein conveys the proper sense in the following: But this is not ignorance. We do not know the boundaries because none have been drawn . . . We can draw a boundary – for a special purpose. Does it take that to make the concept usable? Not at all! (Wittgenstein 1958, }69).

6.2.3.1 Quercus Section Quercus Roughly 200 species of Quercus section Quercus comprise the white oak group (Nixon and Muller 1997), distributed throughout much of the Northern Hemisphere. Section Quercus includes evergreen or deciduous trees or shrubs, with leaf blades lobed or unlobed, margins usually entire but if toothed then

never bristle-tipped (Fig. 6.3). Acorns mature within a single season. The most heavily studied oaks are the European white oaks, principally Q. robur (Pedunculate Oak or English Oak) and Q. petraea (Sessile Oak or Durmast Oak). They tend to occupy different microhabitats in a stand, Q. robur preferring richer, wetter, more alkaline soils compared to Q. petraea (Becker and Levy 1990; see Saintagne et al. 2004). There exists an impressive array of studies regarding molecular genetic variation in Q. robur and/or Q. petraea, including but not limited to the following: isozymes (Zanetto et al. 1994; Gomory 2000), ribosomal DNA (rDNA) (Petit and Kremer 1993; Muir et al. 2001), chloroplast DNA (Petit and Kremer 1993; Cottrell et al. 2002; and see Sect. 6.4.4), proteins (Barreneche et al. 1996; Jorge et al. 2005; see also Sect. 6.7.4.2), and anonymous DNA markers (Moreau et al. 1994; Bodenes et al. 1996; Cervera et al. 2000; Zoldos et al. 2001; Mariette et al. 2002; and see below), along with a variety of QTL studies (see Sect. 6.6.3.4). Q. robur and Q. petraea exhibit genetic differences in allele frequencies only, not in diagnostic alleles. This conclusion is reached after numerous genotyping projects using several marker types, some covering a large fraction of the genomes. For example, Bodenes et al. (1997a) queried 2,800 random amplified polymorphic DNA (RAPD) fragments and found that only 2% displayed allele frequency differentiation between the species, and no fragments were exclusive to one species. Scotti-Saintagne et al. (2004b) compiled variation for 389 molecular markers in Q. robur and Q. petraea, collected from several different sites, and found that only 12% of the loci displayed significant species-level differentiation. Coding regions showed more differentiation than did non-coding regions. They were able to locate roughly half of the total markers on a Q. robur map (see Sect. 6.6.3.3), and the loci associated with species differences were spread across nine linkage groups. These findings show that the Q. robur and Q. petraea genomes overlap extensively in composition, and the differences are distributed in clusters across the genome. Notwithstanding these genetic results, there is ample evidence that the designations Q. robur and Q. petraea represent biological entities. In a largescale study of leaf morphology, Kremer et al. (2002) showed that Q. robur and Q. petraea maintain a stable bimodal distribution in mixed stands, despite

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overlapping distributions. Curtu et al. (2007) studied morphological and genetic markers in a natural stand from Romania containing Q. robur, Q. petraea, Q. pubescens, and Q. frainetto. Using isozymes, microsatellites, and a Bayesian method to infer genetic clusters, they found evidence that the oak gene pool was partitioned into four populations, each corresponding in a reasonable fashion with the species identifications based on morphology. Approximately 3.4% of the population appeared to be of first-generation hybrid origin. Several reports on QTL variation are available for Q. robur and Q. petraea. Since white oak species are distinguished largely based on their leaf morphologies (Gailing 2008), we will consider leaf QTL variation here (see Sect. 6.6.3.4 for other oak QTLs). Saintagne et al. (2004) studied 15 leaf traits that are typically used to classify Q. robur and Q. petraea. Species explained little of the variation in size-related traits, but species explained much of the variation observed in several other leaf characters: petiole length and ratio (80% of variation), pubescence (77%), venation (67–70%), and sinus width (59%). Overall they detected significant QTLs for 13 of the 15 leaf traits, with on average 1–3 genes controlling each. The five traits most associated with species differentiation were localized on 6–9 linkage groups. Leaf pubescence is noteworthy since Nixon (1997b) specifies that foliar trichomes are diagnostic in North American white oaks given the often-times wide variation in leaf shapes. Saintagne et al. (2004) showed that leaf pubescence was an important difference between Q. robur and Q. petraea even though 16% of the typically glabrous Q. robur leaves exhibited trichomes. More importantly, they detected two QTLs for leaf pubescence, each explaining 12.9–17.2% of the phenotypic variance, and one of these QTLs co-localized with another QTL for another species-associated trait (petiole length). Thus, the characters distinguishing Q. robur and Q. petraea are under polygenic control and distributed broadly throughout the genome in clusters. Interestingly, Gailing (2008) examined the same set of leaf traits but using a distinct cross and found that some QTLs were preserved while several were not. Gailing (2008) performed a cross between Q. robur from Germany and Croatia, whereas Saintagne et al. (2004) had used a French cross. The two independent approaches converged on the same co-localized QTLs for number and percentage of intercalary veins

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(linkage group 3) and for number of veins (linkage group 5). However, the other QTLs exhibited poor correspondence between the studies (though see Gailing et al. 2005 for successful transfer of oak flushing date QTLs across families). Gailing (2008) suggests that this outcome is likely due to dissimilar genetic backgrounds and environments. The conclusion remains that species-level traits are under polygenic control, but the fact that only two QTLs were stable across genetic backgrounds and environments attests to the complexities of species differences in the oaks. In North American white oaks, Craft and Ashley (2006) used microsatellite DNA analysis to examine population differentiation among three species, Quercus alba, Q. bicolor, and Q. macrocarpa, occurring in both pure and mixed stands in northeastern Illinois. They detected no strong genetic groupings using individual-based Bayesian clustering or principal components analyses. Using classical F statistics, they found significant but low genetic differentiation. They also found that some intraspecific comparisons were as genetically differentiated as interspecific comparisons, with the two populations of Q. alba exhibiting the highest level of genetic differentiation. Their work indicates that the three species do not represent distinct and differentiated genetic entities. In contrast, Cavender-Bares and Pahlich (2009) found clear and significant differentiation between two sympatric sister live oak species, Q. geminata and Q. virginiana, in Florida using nuclear microsatellites. These species show a 2-week difference in flowering time, which likely provides a reproductive isolating mechanism. The genetic differentiation was matched by clear physiological and ecological differentiation (CavenderBares and Pahlich 2009). Flowering time separation has been hypothesized in earlier work (Nixon 1985) to provide a leaky reproductive barrier that is sufficient to maintain distinct identities between these species.

6.2.3.2 Quercus Section Lobatae There are approximately 195 species of red oak (Quercus section Lobatae; Jensen 1997), a group indigenous to the Americas. These are trees or shrubs, evergreen or deciduous, with lobed or unlobed leaves, margins typically toothed and bristle-tipped though at times entire (Fig. 6.3). Acorns mature in 2 years, rarely one.

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Fig. 6.5 Leaf and acorn morphologies of several red oak species (Quercus section Lobatae; images from USDA-NRCS 2009; Britton and Brown 1913)

Phenotypic variation has been described and quantified in several red oak studies. Richard Jensen’s contributions are notable in this area and one should consult his work for a systematic and thorough appreciation of naturally occurring variation in the group (e.g., Jensen 1977, 1995; Jensen and Eshbaugh 1976; Jensen et al. 1984, 1993). Leaf forms take quite a range in shapes across red oak species (Fig. 6.5), and can vary considerably by position within a canopy (Blue and Jensen 1988), as in the white oaks (Bruschi et al. 2003). Hybrids with intermediate morphologies are described between many of the red oak species as noted in Jensen (1995) and depicted in Fig. 6.4. Much less information is available on the genetic basis of species differences in the red oaks compared to white oaks, but the evidence to date suggests that red oak gene pools are poorly differentiated. Guttman and Weigt (1989) examined ten red oak and eight white oak species using isozyme markers and found that the red and white oaks were readily distinguished from one another, but the red oaks were less resolved from one another compared to the white oaks. Moreover, within-species phylogeographic signal is very

weak within the broadly distributed Northern Red Oak, Q. rubra, across its native range in North America (Magni et al. 2005), compared to the strong signal resolved for European white oaks. These factors could indicate a more recent evolutionary origin for red oak species, leaving their gene pools more articulated. Trelease (1924) suggested this based on biogeography though more recent phylogenetic and paleobotanical evidence suggests otherwise (see Manos et al. 1999). Nevertheless, it is probably safe to say that species boundaries in the red oaks are at least as ragged as in the well-studied white oak section. Jensen’s group has conducted several detailed studies of red oaks of northern Wisconsin that illustrate patterns of variation in the section. Jensen et al. (1993) described clinal variation in leaf traits (17 landmarks) for Q. rubra (Northern Red Oak), Q. ellipsoidalis (Northern Pin Oak), and putative hybrids in natural populations of the Apostle Islands. Hokanson et al. (1993) appraised isozyme variation in the same system and found little differentiation among populations. Tomlinson et al. (2000) characterized leaf and isozyme variation in 30 mother trees and their progeny,

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showing that Q. ellipsoidalis could be distinguished from Q. rubra and hybrids based on overall phenotypic characters (whole-tree silvics), though Q. rubra was not separable from hybrids. Aldrich et al. (2003b) described another ramified red oak gene pool in an old-growth stand in Indiana. They quantified microsatellite variation (15 loci) in a mixed community containing three red oak species, Q. rubra (Northern Red Oak), Q. shumardii (Shumard Oak), and Q. palustris (Pin Oak). These taxa tend to retain phenotypic cohesion and partition the habitat, with Q. palustris occupying wet, poorly drained sites, Q. rubra preferring well-drained, xeric sites, and Q. shumardii occupying intermediate areas. Professional foresters and academic dendrologists typed the species using whole-tree silvic methods. Aldrich et al. (2003b) found high genetic variation within species but moderate differences among species. A Bayesian clustering approach suggested the existence of three populations comprised of (a) pure Q. rubra, (b) Q. rubra, Q. shumardii, and their hybrids, and (c) Q. rubra, Q. shumardii, Q. palustris, and their hybrids. The procedures, conditions, and results were similar to those of Curtu et al. (2007) who resolved four genetic clusters in a mixed stand of four European white oak species, though the white oaks were better resolved genetically. In Mexico, Gonzalez-Rodriguez et al. (2004a) described the extensive hybrid zone between Q. laurina and Q. affinis and found within this zone that molecular variation did not correspond with morphological variation.

6.3 Distribution and Ecology Generic diversity in the Fagaceae is centered in Southeast Asia, whereas species diversity is more uniformly distributed. Species of Quercus are especially prominent in the Northern Hemisphere where they can come to dominate a stand, such as in temperate seasonally dry forests. They are most diverse in Mexico (Valencia 2004). On a local scale, oaks occupy a variety of habitats though they tend toward well-drained upland areas. Oaks have beneficial associations with seed dispersal agents such as squirrels and with mycorrhizal fungi. Numerous herbivores feed on the oaks and a variety of gall wasps use oaks for habitation.

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6.3.1 Global Distribution Oaks are distributed on five continents including Europe, Asia, North Africa, North America, and Central and South America. There are some 255 species in the New World (Manos et al. 1993; Nixon 2006), with 162 species in Mexico (Valencia 2004). Fossil evidence indicates that oaks originated in China during the Eocene. Black oaks (section Cerris) and white oaks (section Quercus sensu latu) are thought to have diverged in Asia by the Eocene (56–35 Mya). Black oaks are further divided into two groups: a semideciduous Cerris group and an evergreen Ilex group (Manos and Stanford 2001; Manos et al. 2001). Section Quercus dispersed to the New World during the Oligocene and is hypothesized to have undergone a rapid radiation subsequently, giving rise to three major New World clades: the red oaks, white oaks, and intermediate oaks (Daghlian and Crepet 1983; Manos et al. 1999) (Fig. 6.2). Phylogenetic and paleobotanical evidence suggests that section Quercus s. s. evolved at middle latitudes in the Americas and subsequently migrated to the Old World prior to the break up of land bridges linking the northern continents (Manos et al. 1999). Migration could have thus occurred in the general time frame between the Late Eocene (ca. 40 Mya) and the Middle Miocene (ca. 15 Mya) (Tiffney 1985). White oak (section Quercus) fossils appeared in Asia in the Miocene and Pliocene (Zhou 1993). Fossil data indicate that oaks were evergreen in their ancestral state and subsequently evolved deciduousness (Manos and Stanford 2001). Phylogenetic hypotheses in the New World oaks suggest convergent evolution in various traits associated with climate and habitat specialization, including leaf lifespan, growth rates, and vulnerability to drought (Tucker 1974; Cavender-Bares et al. 2004a), whereas little to no differentiation has occurred in the morphology of flowers and fruit (Manos et al. 1999, 2001; Oh and Manos 2008).

6.3.2 Ecological Distribution Oaks tend to be distributed in well-drained upland areas and often in montane areas. There are widespread lowland oaks, however, including the live

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oaks (series Virentes), and there are oaks associated with rivers and wetlands that are able to tolerate some degree of flooding, such as Q. lyrata and Q. laurifolia. In northern central Florida, which lies at the confluence of the northern temperate flora and the southern subtropical flora, oaks are hyperdiverse. Due to the potential for close relatives to competitively exclude one another, the coexistence of multiple congeners in this region presents a challenge to explain (CavenderBares et al. 2004b), particularly given the lack of major elevational gradients that occur in other regions of high oak diversity (Whittaker 1956; Platt and Schwartz 1990). Associations of suites of ecophysiological traits and species ecological distributions indicate that oaks specialize into different niches associated with soil moisture and fire regime, thus allowing them to partition the landscape and maintain their diversity (Cavender-Bares and Holbrook 2001; Cavender-Bares et al. 2004a, b). Furthermore, they show a pattern of phylogenetic overdispersion (Fig. 6.6) in which

Fig. 6.6 Schematic of phylogenetic overdispersion (co-occurring species are less related to each other than expected by chance) in the three major oak dominated communities in Florida (adapted from Cavender-Bares et al. 2004a, b). Oaks within each of the major phylogenetic lineages occur in each

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distantly related species (those from distinct clades) co-occur together in communities rather than species from the same lineage (Cavender-Bares et al. 2004a). This phenomenon has been observed in other oak dominated systems (Whittaker 1969; Mohler 1990). The pattern may be caused by convergent evolution in ecological traits important for habitat specialization. Density-dependent mechanisms that operate at the clade level, such as resistance to disease (see Sect. 6.4.3) or to herbivores (see Sect. 6.3.7), may also prevent closely related oaks from co-occurring.

6.3.3 Acorn Properties and Dispersal The acorn fruit is comprised mostly of cotyledon that contains high levels of energy-rich lipid, making them an important food source for many mammals and birds, including squirrels, jays, woodpeckers,

community, and respective physiological traits match each environment, indicating convergent evolution. Vulnerability to different diseases or acorn properties that promote contrasting predator behavior may promote the coexistence of oaks from different lineages

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grackles, wild turkey, mice, chipmunks, deer, black bears, and more than 140 other species of vertebrates in North America (Van Dersal 1940). Tree squirrels play a notorious and well-documented roll in acorn dispersal (Steele and Koprowski 2001). Squirrels cache the seeds in the ground and then fail to recover some of these stored reserves (Van Dersal 1940). They also often only partially consume acorns, leaving the embryo intact, permitting subsequent germination and survival (Steele et al. 1993). By transporting and scatter hoarding acorns to individual sites just below the leaf litter, squirrels reduce the probability of seed predation and desiccation and increase the chances of germination, root establishment, and winter survival (Steele and Koprowski 2001). Red and white oaks have contrasting tannin and lipid levels, which influence feeding decisions at different times of the year (Smallwood and Peters 1986). Red and white oaks also differ in their dormancy and germination patterns in the temperate zone, with white oak acorns germinating immediately upon falling in autumn and red oak acorns undergoing dormancy throughout the winter period and germinating in the spring. Gray squirrels apparently distinguish between the dormant red oak acorns and rapidly germinating white oak acorns, selectively dispersing and caching the former and eating the latter. Differential acorn dispersal and caching preferences due to contrasting fat and tannin content have given rise to the differential dispersal hypothesis (Steele and Koprowski 2001) in which red oaks are thought to disperse more rapidly than white oaks. These contrasting dispersal and caching preferences are likely to contribute to forest structure and may help explain the observation that red and white oak species tend to co-occur more often than expected by chance (Fig. 6.6; Cavender-Bares et al. 2004a). Higher dispersal rates of red oak acorns also might help explain the lower genetic differentiation observed among some red oak species (see Sect. 6.2.3.2).

6.3.4 Mast Seeding Mast seeding, the synchronous production of large crops of seeds, has been frequently documented in oak species (Liebhold et al. 2004). Sork (1993) found evidence supporting the hypothesis that mast seeding has evolved as an anti-predator adaptation by which

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large seed crops during mast years satiate the seed predators and allow survival of some of the seeds. This work indicates that there should be stronger selective forces for evolution of masting in tropical oaks compared to temperate oaks. Cues for masting are likely to be linked to climatic factors. Asynchrony in masting between species, particularly those of different lineages (Mohler 1990) could help explain phylogenetic overdispersion and coexistence of distantly related oaks. Asynchrony in masting between species has been attributed to differences in seed production caused by the varying numbers of years (1 or 2) required to mature seeds in white versus red oaks, whereas intraspecific variation in the synchrony of seed production has been related to variation in habitat conditions (Liebhold et al. 2004). The timing of masting in oaks has been associated with population cycles of insects, including gypsy moths (Lymantria dispar). One hypothesis for this association is that the tissue quality of leaves changes during mast years to contain lower secondary metabolites. The higher nutritive quality of leaves increases larval survival and leads to moth outbreaks (Sela˚s 2004).

6.3.5 Physiology While oaks, in general, are characterized as drought adapted (Abrams 1990), they inhabit a wide range of environments with respect to soil moisture, hydroperiod, and fire regime. Oaks tend to have deeppenetrating root systems (Stone and Kalisz 1991), enabling them to maintain relatively high pre-dawn water potentials during drought, and they often have thick leaves, which prevent wilting. Oaks frequently maintain a higher rate of photosynthesis at low leaf water potentials and high vapor pressure deficits than co-occurring species of other genera (Abrams 1990). In general, they tend to have high epidermal resistance, minimizing water loss when stomata are shut, although such resistances vary with species and tend to be higher in evergreen species than in deciduous species (Kerstiens 1996; Cavender-Bares et al. 2007). Deciduous oaks have been reported as having notoriously long vessels (Cochard and Tyree 1990). The xylem anatomy of temperate oaks has often been characterized as ringporous given that many temperate species produce large early wood vessels that hydraulically support the

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spring flush of leaves followed by much narrower latewood vessels. Earlywood vessels embolize partway through the season (Sperry et al. 1994), while the narrower latewood vessels, which presumably have smaller pores in their intervessel membranes, are more resistant to drought-induced cavitation and can sustain water transport for the rest of the season. However, many subtropical or tropical oak species have diffuse porous xylem anatomy in which vessels size distributions do not change throughout the year (CavenderBares and Holbrook 2001). In the southeastern United States, oak diversity is very high, maintained in part by the heterogeneity in soil moisture and fire regime. Individual species show variation in physiological and life history traits that tend to match their ecological distributions (CavenderBares and Holbrook 2001; Cavender-Bares et al. 2004a, b). For example, species that are adapted to fire regimes that are severe, predictable, and occurring on the order of 25–50 years intervals have high rhizome resprouting capacity whereas species occurring in mesic to hydric resource rich habitats where fire occurs only rarely tend to achieve tall maximum heights to compete for light at the top of the canopy (Cavender-Bares et al. 2004b). Likewise, species from xeric environments tend to show resistance to droughtinduced embolism but cannot support large hydraulic fluxes. In contrast, species from mesic environments are characterized by large hydraulic fluxes (CavenderBares and Holbrook 2001). In the seasonally dry tropical forests of Costa Rica, live oaks show unusually high resistance to leaf wilting (Brodribb and Holbrook 2006).

6.3.6 Herbivory Oak leaves support a diverse herbivore community. In one study, 138 species of leaf-chewing insects were found on leaves of Quercus alba and Q. velutina, sampled in southeastern Missouri, USA (Le Corff and Marquis 1999). Reduction in herbivore load by birds has been shown to have positive fitness consequences in oaks (Marquis and Whelan 1994). Oaks produce a diverse array of sublethal plant secondary compounds, including phenolics and hydrolyzable tannins, which are thought to provide defense by reducing insect oviposition, feeding, and biomass gain of

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herbivores (Lill and Marquis 2001). In experimental and observational studies examining impacts of these secondary compounds in leaves of Q. alba and Q. velutina on herbivores, specialist herbivores were more likely than generalists to be negatively affected by condensed tannins (Forkner et al. 2004), and defoliation levels were generally lower where concentrations of phenolics were higher (Forkner and Marquis 2004). Sclerophylly, leaf pubescence, high lignin content, and other leaf traits and architectural characteristics of oaks may also serve as important plant defenses. For example, architectural traits that minimize leaf-to-leaf contact in oaks may be defensive traits against leaf-tying caterpillars (Marquis et al. 2002). In experimental manipulations of leaf pubescence, higher leaf hair density reduced generalist caterpillar densities (Lill et al. 2006).

6.3.7 Oak Galls Of the oak herbivores, the oak gallwasps (Cynipidae: Cynipini) are known to have highly specialized associations with their oak hosts. Oak gallwasps are a species-rich lineage (ca. 1,000 species) that induces structurally complex galls on oaks and other Fagaceae (Abe et al. 2007). They are able to bypass oak chemical defenses (phenolics and condensed tannins) by inducing the development of host plant tissues that have elevated nutritive value but low concentrations of toxic secondary plant metabolites (Cornell 1983). Little is known about the process of gall induction. Gallwasps feed on and reproduce in these tissues. Despite their ability to circumvent toxic secondary compounds, host shifts are very constrained (Stone et al. 2009). Oak gallwasp genera are usually specific to a single oak section, and within section Cerris, to a single species group (Stone et al. 2009). Specificity does not extend to the level of the species, and gallwasps frequently gall multiple oaks in the same section or species group. The Western Palaearctic gallwasp fauna are best studied and include 150 species in 10 genera. While most of these species are highly specialized, at least two groups of the Western Palaearctic gallwasps (including Andricus and Calirhytis genera) show obligate alternation between oak sections during their lifecycle. In the deep split between section Cerris and section Quercus,

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diversification of the gallwasps is closely associated but post-dates diversification of the oaks. Inter- and intraspecific phylogeographies suggest that gallwasps have pursued their oak hosts for at least 20 my across much of the Palaearctic (Stone et al. 2009). These data also support the hypothesis that Western Palaearctic and Nearctic gallwasps are derived from an initial radiation in ancient Asian or Beringian oak forests, and that the divide between Palaearctic Cerris and White oak gallwasps represents the deepest divide in the Cynipini. The extreme host conservatism within some of the major clades of oak gallwasps suggests metabolically intimate aspects of the plant-gall inducer interaction. Stone et al (2009) have suggested that oaks and oak gallwasps represent an example of a coevolutionary arms race between host plant susceptibility and gall inducer virulence.

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fine roots compared to other ECM host species. However, an increase in number of studies using molecular methods has demonstrated the feasibility of these methods for oak-ectomycorrhizal associations (e.g., Cooke et al. 1999; Pinkas et al. 2000; Nechwatal et al. 2001; Giomaro et al. 2002; Avis et al. 2003; Kennedy et al. 2003; Dickie and Fitzjohn 2007; Cavender-Bares et al. 2009). Lack of ectomycorrhizal symbionts has been implicated in some forest systems in restricting oak regeneration into old fields (Dickie et al. 2007) but not in others (Klemens et al. 2010). ECM fungal colonization has also been proposed as one of several competing hypotheses to cause monodominant oak stands (Boucher 1981), although evidence for this is limited.

6.4 Conservation Biology 6.3.8 Mycorrhizal Associates In ecological settings, oaks are functionally obligately associated with a diversity of ectomycorrhizal (ECM) fungi (included in the Ascomycota and Basidiomycota) and benefit from the symbiosis in terms of growth, seedling establishment, and survival (e.g., Avis et al. 2003; Smith et al. 2007a, b; Morris et al. 2008a). It is also now known that oaks associate to some extent with vesicular arbuscular mycorrhizal (VAM) fungi (Dickie et al. 2001). While a large number of ECM taxa (~250) are specific at the family or genus level (Molina et al. 1992; Ishida et al. 2007), it is not known how commonly specificity occurs below the genus level. Individual fungi can function differently on different host plants with different degrees of penetration into and between root cells depending on the host (Taylor and Bruns 1999; Villarreal-Ruiz et al. 2004). Contrasting ECM communities were found on the roots of sympatric oaks in California, demonstrating that fungal preferences at the host plant species level can be important in ECM assemblages within the oak genus (Morris et al. 2008b). Similarly, an experimental study of two containerized oak hosts across an experimental hydrologic gradient demonstrated that ECM fungal communities differed between a white oak (Q. montana) and a red oak (Q. palustris) (Cavender-Bares et al 2009). Oak ECM fungi are difficult to work with because oaks have very small

Most oaks maintain fairly high levels of genetic variation within their populations and relatively less variation among populations (e.g., Aldrich et al. 2005a). This may be due to their propensity to outcross via wind pollination and to their longevity (Hamrick et al. 1992). There remain large standing wild populations of many of the oak species, and at present there is little effort to actively manage the gene pools, particularly in the USA. Unfortunately, this does not account for mismanagement, such as high-grading and poor nursery practices, for the possible loss of rare or endemic alleles before they are detected, or for demographic failure in parts of the range and pathogen epidemics that can wipe out entire populations in short order, or global climate change. Except for these factors, the oak gene pools appear sound.

6.4.1 Conservation Initiatives Oak gene pools have not received as much attention, in part due to the ubiquity of oaks and the high variation found in those that have been studied, though see Jacobs and Davis (2005) for considerations of the genetic implications of hardwood nursery practices in the eastern United States. On the other hand, oak demography has received considerable attention in the USA over the past 50 years due to regeneration failure noted in many areas (see Sect. 6.4.2, see also a review

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on artificial regeneration of oaks by Dey et al. 2008). The European Union has funded a network of agencies under the EVOLTREE program (http://www.evoltree. eu/), which seeks to determine the impact of climate change on forest ecosystems. Some of the focus will be on gene pool and genome structure of trees, with oaks as the model organism. Several useful reviews on the genetics and genomics of adaptation in tree species are relevant here (Gonzalez-Martinez et al. 2006; Neale 2007; Savolainen et al. 2007; Neale and Ingvarsson 2008). As for ex situ management, oaks can be propagated through grafting (e.g., Kothencz et al. 2001) although oak lines immortalized by grafting are subject to reversion wherein shoots of the mutant revert to the wild type phenotype. Oak seeds do not store terribly well, though the National Seed Laboratory offers resources for management and propagation (http://www.nsl.fs.fed.us/). In addition, the US National Arboretum maintains the Woody Landscape Plant Germplasm Repository, which includes several oak accessions (http://www.usna.usda.gov/Research/ wlpgr.html).

P.R. Aldrich and J. Cavender-Bares

census of oaks in the canopy but an appraisal of the overall regenerative potential of the stands. Of course major shifts in the local abundances of mesic vs. xeric sites due to climate change could have profound effects on forest composition and oak demography. There are several other factors that contribute to oak demographic problems. In some Californian oaks, the spatial isolation of populations appears to be an issue where Sork et al. (2002a, b) found a reduction in oak neighborhood sizes over time, attributing this to the fragmentation of the habitat and decline of pollen donor density. In many areas it is the invasive plant species that threatens oak regeneration, such as Ailanthus altissima competing with oaks for highlight environments in the eastern United States (Hu 1979; Huebner 2003; Rebbeck et al. 2005). Exotic pests and pathogens pose an immediate threat to oak forests (see next section), and although we are currently surrounded by oaks, we only need look to Castanea dentata (the American chestnut, also within the Fagaceae) to see how rapidly a pathogen (chestnut blight, Cryphonectria parasitica) can sweep through and remove a canopy dominant taxon (Fig. 6.7).

6.4.2 Oak Regeneration Failure The demographic profile of oaks in the USA appears to be changing, and several causal agents have been identified (reviewed in Abrams 1992, 2003; Abrams and Nowacki 1992; Lorimer 1980, 1993). One factor appears to be a change in the disturbance regime of some forests. Oaks are good competitors on xeric sites where canopies tend to remain open, but oaks are rather poor competitors on closed-canopy, mesic sites at least in part because they are not as shadetolerant as many other taxa, such as Acer saccharum (sugar maple). Stands whose canopies and understories have been kept open for the past century or two through logging, grazing, and fires are subject to a successional shift toward shade-tolerant taxa when these disturbances are suppressed. Consequently, many of these mesic stands still contain numerous large oaks in the canopy but little if any oak regeneration, the subcanopy dominated primarily by shade tolerants like maple (Parker et al. 1985; Aldrich et al. 2005b). Seed and seedling predation by deer and rodents can exacerbate this condition. Thus, a proper accounting of a landscape is not simply the adult

Fig. 6.7 Castanea (chestnut) adult in the wild prior to chestnut blight (image from USDA-NRCS 2009)

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6.4.3 Pests and Pathogens Oaks are susceptible to a number of pathogens and pests. Oak wilt and Sudden Oak Death are two fungal diseases of primary concern.

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grafts between individuals and removing potential spore producing host trees. Triazole fungicides have been used as a means of chemical control (Koch et al. submitted).

6.4.3.2 Sudden Oak Death 6.4.3.1 Oak Wilt Ceratocystis fagacearum (Bretz) Hunt, the oak wilt pathogen, is recognized as one of the most destructive diseases to afflict oak species in the USA. Oak wilt was first recognized in North America in 1944 and is now widespread in eastern, midwestern, and southern states (Koch et al. submitted). The distribution and development of oak wilt in eastern and midwestern United States oak forests has been closely linked to changes in forest stand composition, forest management practices, and pathogen dissemination facilitated by human and vector activity, and there is concern about its increasing spread into southern and western states, exemplified by the Texas outbreak (Wilson 2001). Oak wilt is a vascular fungal disease that blocks the xylem vessels preventing water transport (Juzwik 2000). The red oaks (section Lobatae) are generally more susceptible than white oaks (section Quercus). White oaks have narrower vessels and block the spread of the disease with tyloses. However, within the white oak clade, semi-evergreen live oaks (Quercus fusiformis Small and Q. virginiana Mill., series Virentes) are also susceptible. Oak wilt has caused massive losses of live oaks in central Texas. High susceptibility and mortality in live oaks in Texas was not anticipated given the relatively limited oak mortality caused by the disease in the deciduous forests of the north, central, midwestern, and mid-Atlantic United States. The intensity of oak wilt in Texas is attributed to a number of factors related to host characteristics and the ability of the pathogen to adapt to limiting environmental conditions (Appel 1995). Wilt symptoms begin at the crown of the tree, but are manifested differently in different lineages (Fowler 1953; Juzwik 2000). The fungus spreads to susceptible trees either via dissemination of infective spores by beetles or by underground root grafts that connect the vascular systems of individual trees, of the same species or of closely related species. Mechanical methods for controlling oak wilt have focused on severing root

A recent epidemic of Phytophthora ramorum, the nonnative invasive pathogen that causes Sudden Oak Death in coastal woodlands of California, is causing severe mortality in some oak species belonging to the red oak (Lobatae) group. P. ramorum has killed tens of thousands of native coast live oak and tanoak trees in California, and the pathogen does not seem to differentially select among genotypes or closely related species of red oaks that are susceptible (Dodd and Kashani 2003). Mortality due to the disease was first observed in 1995 in tanoak trees (Lithocarpus densiflorus), although P. ramorum was not identified as the pathogen until 2000. Subsequently, the disease was observed on coast live oak (Q. agrifolia), California black oak (Q. kelloggii), and Shreve’s oak (Q. parvula var shrevii) (McCreary 2007). In California forests, bay laurel (Laurus nobilis, Lauraceae) is the primary carrier of P. ramorum because it acquires a non-lethal but highly contagious leaf infection (Rizzo et al. 2005). The pathogen also survives and produces chlamydospores in forest soils over summer, providing a possible inoculum reservoir at the onset of the fall disease cycle (Fichtner et al. 2007). Trunk bleeding, presence of beetles, presence of the fungus Hypoxylon thouarsianum, and tree mortality through time are visible signs of P. ramorum infection (Kelly et al. 2008). P. ramorum infects a very wide diversity of plants beyond bay laurel, many of which serve as foliar hosts and sources of inoculum. As a result, there is heightened concern that the pathogen could spread to the diverse oak woodlands of the eastern United States (Hong et al. 2005; Venette and Cohen 2006; SnoverClift et al. 2007; Tooley and Kyde 2007). Reconstruction of the epidemic using molecular population genetic methods indicates historical human spread (Mascheretti et al. 2008) and highlights unintended linkages between the horticultural industry and potential impacts on forest ecosystems (Rizzo et al. 2005). Stringent efforts have been undertaken to halt the spread of the pathogen by the horticultural industry outside of California. These efforts were initiated after

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two large-scale nurseries in Southern California were implicated in having transported the pathogen via infected camellia plants to 176 locations in 21 states. Over a million infected plants were destroyed and annual inspections are now mandatory in western nurseries that ship interstate (McCreary 2007).

6.4.4 Phylogeography The literature on oak phylogeography is too extensive to do justice in the space provided here. For brevity, we focus on the intensively studied European white oaks, noting only several highlights, and refer the reader to reviews. We do not treat the growing phylogeography literature on oaks in North and Central America (e.g., Grivet et al. 2008, Craft and Ashley 2007, Gonzalez-Rodriguez et al. 2004a, b, Cavender-Bares et al. 2011), and Asia (e.g., Okaura et al. 2007, Shih et al. 2006, Kanno et al. 2004) nor on the European black oaks (e.g., Cosimo et al. 2009, de Heredia 2007, Lumaret et al. 2005). The chloroplast genome is maternally inherited in oaks (Dumolin et al. 1995), and chloroplast markers have been used effectively to resolve haplotypic variation across Europe for the major white oak species (e.g., DumolinLapegue et al. 1997). Much of this work has entailed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using primers that span intergenic regions in the chloroplast (see Sect. 6.6.3.1). Petit et al. (2002a, b) reviewed the research by a consortium of 16 laboratories, which to date have detected 32 chloroplast DNA variants from 12,214 individual trees collected from 2,613 populations in 37 countries. The studies have revealed strong phylogeographic structure, compared with the weak structure detected in North American red oak (Magni et al. 2005; and see Sect. 6.2.3.2). White oak haplotypes found in northern Europe are also present in the south, though the reverse does not hold, suggesting that most mutations arose prior to the expansion out of southern refugia following the last glacial retreat. Even though regions can carry numerous haplotypes, it is common to encounter patches of several 100 kms dominated by a single haplotype, often cutting across white oak species boundaries. The authors suggest this is strong evidence for episodic, long-distance migrations out of the glacial refugia.

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This work has revealed some interesting natural history dynamics for the two most widespread and heavily studied of the European white oaks, Q. robur and Q. petraea. A variety of sources indicate that Q. robur has more pioneer tendencies including a greater capacity to disperse its seed (Petit et al. 2004). Thus, it is thought that Q. robur might have been among the first migrants out of the glacial refugia. The later successional Q. petraea would then have arrived and crossed with the established Q. robur populations, though there is an asymmetry in the mating success between the species. Q. petraea readily crosses to Q. robur, but the reverse does not hold. This has been found in controlled crosses (Steinhoff 1993; though see Steinhoff 1998) and in natural stands (Bacilieri et al. 1996b). Eventually the shadetolerant Q. petraea is able to replace the less tolerant Q. robur in mixed stands (Bacilieri et al. 1996b) – demographically and at the level of the genome. The overall dynamic has the effect of leaving the pioneer Q. robur chloroplast genome intact; however, the nuclear genes of Q. robur are gradually replaced or “swamped” by those of the late successional Q. petraea due to the asymmetric hybridization and introgression. This remarkable example lends some clarity as to why oak species can be problematic to demarcate.

6.5 Stocks and Lines Much of oak germplasm diversity remains in natural populations, and most managed lines are not far removed. There is some information available on how these natural lines perform through common garden, or provenance, studies. A limited amount of oak germplasm has been incorporated in breeding programs as improved lines. Many have been drawn directly from a wild tree that displayed superior form through transfer of acorns or cuttings (see Sect. 6.6.2). Some lineages have been subjected to rounds of selection, though typically not for many generations. A small and diverse set of lineages, drawing on all these sources, have been designated as cultivars. Other lines represent mutants that may be more of academic interest and include polyploids or single-gene mutants that affect the phenotype in some fashion that might illuminate a path for domestication.

6 Quercus

6.5.1 Provenance Studies A “provenance” is the location of a population and a “provenance study” is a common garden planting that brings together at one site a variety of germplasm from different parts of a species range. Provenance studies may prove critical in the coming years if it becomes necessary to assist oak species in their migration and adaptation to a changing climate (Ma´tya´s 1996). The US Department of Agriculture has detailed information on seed zones (or “plant hardiness zones”), which provide information on whether one could expect transplantation success (maps accessible online at http://www.usna.usda.gov/Hardzone/ushzmap.html). However, shifting climates will alter much of this hard-won information, and provenance studies will be needed to reestablish the information. Unfortunately in the USA, many hardwood provenance studies were abandoned during the transition to molecular-based breeding in the 1970s and 1980s (Woeste and McKenna 2004). Though some of the sites still exist, their lack of care has left many overgrown and of much less use for testing propositions as originally intended. Both the locations and the findings of provenance studies can be difficult to ascertain, since documents and reports often circulate through local institutions (state and local forestry, university departments) or at regional meetings (e.g., Weigel et al. 2005). Internet access is changing some of this, where for example one can find reviews of provenance work on the European white oaks Q. robur and Q. petraea (e.g., Kleinschmit and Kleinschmit 2000; see also Kleinschmit 1993) along with information on longterm oak plantings in Europe (e.g., Hubert 2005). Reports on several US oak provenance studies can now be accessed through the USDA-Forest Service website (http://www.treesearch.fs.fed.us/).

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breaks in continuity within breeding programs (Aldrich 2008). As a consequence, the cultivars cannot rival domesticates of corn or wheat. Nevertheless, there are numerous lineages that offer improved material that in some cases has been recognized among breeders for many years. McArdle and Santamour (1985a, b, 1987) summarized information on 119 valid names for cultivars of Q. robur (English oak), another 75 cultivars of white oaks (excluding Q. robur), and 59 cultivars outside of section Quercus (the non-white oaks). Included here is the “Gobbler”, a cultivar of Q. acutissima noted for its abundant acorn production and ability to satisfy wild turkeys. A more detailed and current repository of oak cultivar information is available online through the International Oak Society. Oak cultivars and groups are tracked, verified, and databased by the International Oak Society, and information can be accessed online through the Oak Name Database (Trehane 2007). Naming conventions follow the International Code of Nomenclature for Cultivated Plants (ICNCP). We retrieved information from the database on the top ten oak species based on their number of legitimate cultivars or Groups (Table 6.1). Nearly half (45.6%) of the world’s legitimate oak cultivars or groups are from either Q. robur or Q. petraea. This reflects the importance of these species in Europe and the longer time that Europeans have been breeding oaks. In North America, the white oak Q. alba is an important timber species and ranks well in number of cultivars, though the red oak (section Lobatae) cultivar pool is better developed there, at least among the top ten species. Two of the taxa in this abbreviated list are entirely of hybrid origin (Q.
hispanica and Q.
undulata). A total of 25 oak cultivars have been trademarked or have a restricted name, but 80% of these derive from species not in the top ten list, i.e., from oak taxa with fewer cultivars of any kind.

6.5.2 Cultivars

6.5.3 Polyploids and Other Mutants

Improved oak stock comes in many forms and with varied backgrounds, and includes such lines as “Argenteo-Marginata” or “Variegate English Oak.” Trees in general have a shallow domestication history because of their longevity and because of the frequent

Chromosomal mutations occur at a low rate in natural Quercus populations. Naujoks et al. (1995) found 1 in 400 Q. robur trees exhibited anomalous isozyme banding, altered leaf morphology, large stomata, and a triploid chromosome count. The authors took

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Table 6.1 Cultivar pools of the top 10 oak species ranked by number of legitimate cultivars or groups recognized by the International Oak Society Section/Species Common name Region Total Legit Hybrids Tm Cerris Q. cerris Turkey Oak E, As 37 20 0 0 Q.
hispanicaa Lucombe Oak 34 17 34 0 Lobatae Q. palustris Pin Oak NA 28 18 0 1 Q. rubra Northern Red Oak NA 31 19 0 0 Q. velutina Black Oak NA 11 9 1 1 Quercus Q. alba White Oak NA 20 14 0 1 Q. ilex Holly or Holm Oak E, Af 23 16 0 0 Q. petraea Sessile of Durmast Oak E 64 46 0 0 Q. robur Pedunculate or English Oak E, As 269 161 0 3 Q.
undulatab Wavyleaf Oak 18 11 18 0 Lesser taxa Miscellaneous species 232 123 72 20 Total 767 454 124 25 Information source: Trehane (2007) Oak name database, International Oak Society. Accessed Jan 2009 (http://www.oaknames.org/) Region, native distribution: As Asia, Af Africa, E Europe, NA North America Total: Total number of cultivars and groups listed, legitimate and illegitimate Legit: Legitimate cultivar or group name, published in literature and conforming to International Code of Nomenclature for Cultivated Plants (ICNCP) rules Hybrids, cultivars, or groups of hybrid origin noted within this taxon Tm, trademarked or restricted name a Q. cerris
Q. suber b Q. gambelii
several other species

cuttings and rooted the specimen. Zoldos et al. (1998) found two populations of Q. petraea containing several individuals with extra chromosomes. Dzialuk et al. (2007) also found ploidy variation in Q. robur and Q. petraea. To our knowledge these chromosomal variants have not been integrated into production lines. Higher chromosomal mutation rates also have been detected in “natural” Q. robur oak populations contaminated with radiation from the Chernobyl accident (Kalaev and Butorina 2006). Synthetic oak polyploids can be generated through different treatments. Bueno et al. (1997) used stress signaling, or starvation followed by heat shock, to alter the gametophytic trajectory in anther cultures, inducing doubled haploids. Alternatively, antimitotic agents such as colchicine, oryzalin, and amiprophos-methyl (APM) can be used to disrupt meiosis in anther cultures. Pintos et al. (2007) compared these chemical treatments in Q. suber (cork oak) and found superior yield with oryzalin. Sometimes ploidy instability is an unwanted by-product of the somatic embryogenesis process, such as an 8% rate of tetraploidy detected

in Q. robur clones that were cultured over 7 years (Endemann et al. 2001). Other studies of oak cultures show no evidence of ploidy instability (e.g., Q. suber, Loureiro et al. 2005). Then there are a limited number of additional described mutants in Quercus. Some have been systematically studied such as the ML mutant, a chlorophyll-deficient line of Q. petraea that exhibits enhanced resistance to powdery mildew, Erysiphe cichoracearum (Repka 2002). This mutant accumulates reactive oxygen species, enhancing the defense response.

6.6 Crop Improvement Quercus domestication is still in its infancy though great progress has been made in the past 10–15 years. Here we consider some impediments to oak domestication brought by the natural history of the organism and by some human practices. We then

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cover different breeding approaches and the progress to date in oak domestication. In particular, we consider traditional, molecular, and biotechnology-based breeding such as in vitro culturing. Because of considerable progress in the area of oak molecular breeding, we treat this subject in greater detail (see Sect. 6.6.3). Included here are resources for Quercus in molecular genetic markers, crosses and pollinations, and linkage and QTL mapping.

6.6.1 Impediments to Improvement Aldrich (2008) considered the domestication history of forest trees, and most of the generalizations made therein hold for the genus Quercus. Despite a long relationship between humans and oak trees (Ciesla 2002; Logan 2005), several factors have contributed to the slow progress of changing the oak phenotype, most notably generation time and breeding directly from natural populations. Oaks have a long generation time, some living to be hundreds of years (see Burns and Honkala 1990), which can work against the domestication process. Delayed onset of reproduction can require that a breeder wait 10–15 years before considering an artificial cross using an elite tree. Hand pollinations in Quercus are difficult, good seed crops can arise sporadically separated by several years of poor yield, and the acorns of the red oaks typically require 2 years to reach maturity. Moreover, it can take decades before traits of interest become evident, such as wood quality and growth form. All this contributes to slow progress as well as discontinuities in breeding programs. Most “domesticated” oak lineages are not many generations removed from wild populations (see Sect. 6.6.2). This has the benefit of repeatedly tapping natural reserves for genetic variation that should promote gains in breeding, except there have been serious problems in continuity of breeding efforts in oak programs and other hardwoods (Woeste and McKenna 2004). The proverbial wheel has been re-invented several times as breeding efforts and programs have been abandoned for various reasons, such as in the USA during the 1970s and 1980s when attention shifted away from traditional breeding and toward biotechnology.

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Perhaps the most pernicious aspect of human interactions with wild populations of trees has been that inadvertent selection pressures likely have worked against the improvement of timber traits over the millennia. Selection and harvest of the biggest and best trees for their wood removes these individuals from the gene pool. This practice of “high-grading” has the opposite effect one would like on the domestication process, though the magnitude of impact cannot be determined in most cases due to poor records. But with the possible exception of the long-standing European work with Q. robur and Q. petraea, it is safe to say that we have only recently begun to leave the hunter-gatherer stage of our relationship with the oaks.

6.6.2 Traditional Breeding Traditional silviculture forms the foundation of oak breeding programs. A valuable resource in this area is the The Ecology and Silviculture of Oaks (Johnson et al. 2002). There are other web-based resources on oak silviculture in the USA, such as through the USDA-Forest Service website (http://www.fs.fed.us/ publications/) and through state and local forestry extension services (e.g., http://web.extension.uiuc.edu/ forestry/publications/index.html). At Purdue University, the Hardwood Tree Improvement and Regeneration Center (HTIRC, Michler et al. 2005) is a collaborative enterprise involving federal, state, and private organizations involved in combining traditional breeding methods with biotechnology in the advancement of hardwood resources, including oaks (http:// www.agriculture.purdue.edu/fnr/HTIRC/index.htm). Traditional breeding methods borrow heavily from the classical and quantitative genetic approaches used in crop and livestock breeding. The common garden experiment, or provenance trial, still figures prominently as it serves to evaluate pre-existing adaptations in the gene pool (see Sect. 6.5.1). Phenotypic selection can occur any time from the point of selecting germplasm from a superior tree in the wild through the point of production and deployment of elite lines in plantations. Controlled crosses are possible with the oaks, though sometimes openpollinated canopies are utilized. In fact, much of the traditional oak breeding done today utilizes only slightly improved germplasm. Jacobs and Davis

108 Table 6.2 Narrow sense heritabilities for traits in Quercus Trait(s) h2 Height 0.64 (0.48–0.80) Diameter 0.62 (0.55–0.70) Height 0.60 (0.50–0.70) Vessel area 0.60 Sapwood rings 0.57 Bud burst 0.33 (0.15–0.51) Diameter 0.28 (0.09–0.46) Growth 0.14 (0.04–0.23) Mean 0.47

P.R. Aldrich and J. Cavender-Bares

Species Q. robur Q. pagoda Q. pagoda Q. petraea/Q. robur Q. petraea/Q. robur Q. robur Q. robur Q. robur

References Bogdan et al. (2004) Adams et al. (2007) Adams et al. (2007) Kanowski et al. (1991) Savill et al. (1993) Scotti-Saintagne et al. (2004a) Bogdan et al. (2004) Scotti-Saintagne et al. (2004a)

h 2, midpoint heritabilities (value or range reported)

(2005) reviewed hardwood tree nurseries in the eastern United States and found that only 6.8% of hardwood seedlings derived from improved stock, compared to 36% for conifers. Moreover, it is often the case that seed source is unknown since many nurseries accept anonymous collections and pool these before planting. These are areas worth improving if oak breeding is to keep a reasonable pace. Studies to date indicate that natural oak populations maintain sufficient additive genetic variation to serve as a source for breeding programs. Average heritabilities for several oak traits that we review here are around h2 ¼ 0.47 (Table 6.2). Adams et al. (2007) used 37 half-sib families representing eight provenance from the eastern United States to estimate heritabilities of h2 ¼ 0.55–0.70 for diameter and height in Q. pagodae (cherrybark oak). Heritabilities were high for height (h2 ¼ 0.62–0.78) and diameter (h2 ¼ 0.28–0.65) in Q. robur from an open-pollinated progeny test of 21 Slavonian plus trees (Bogdan et al. 2004). Kanowski et al. (1991) reported a high heritability (h2 ¼ 0.60) for crosssectional area of earlywood vessels in Q. robur and Q. petraea, a trait that may influence the development of fissures in wood. Savill et al. (1993) reported a high heritability (h2 ¼ 0.57) for the number of sapwood rings in Q. robur and Q. petraea. The genetic gain from selecting for volume in a Q. serrata seed orchard was estimated at 4.0–9.1% for 50% selection intensity (Kang et al. 2007). Mosedale et al. (1996) examined wood qualities in Q. petraea and Q. robur and found that heartwood ellagitannin content and wood density were both under strong genetic control though this was not the case for wood color (see Sect. 6.7.5.1).

6.6.3 Marker-Assisted Breeding The application of molecular markers is an active area in oak breeding as this method can be used to hasten the process of selection during a life cycle that is long and protracted. There are a variety of molecular marker types and a variety of applications that are relevant to breeding. Some markers are used to appraise variation in natural stands, whereas others to resolve the structure of variation within the individual genome. Chief among the latter applications are the molecular maps, and the attachment of phenotypic effects onto such a map in the form of QTL. All can be used as a handle to track the establishment of desirable traits in a lineage. Useful information on molecular marker systems and their applications in the management of plant genetic resources, including forest trees, is maintained through Wageningen UR (http://www.cgn.wur.nl/UK/CGN+Plant+Genetic +Resources/Research/Molecular+markers).

6.6.3.1 Molecular Genetic Markers A variety of molecular markers are available for Quercus. Commonly used phylogenetic sequences have been studied such as the ITS of the ribosomal DNA (rDNA; Muir et al. 2001; Bellarosa et al. 2005). An extensive program of research on oak phylogeography has been developed for the European white oaks Q. robur and Q. petraea using chloroplast and mitochondrial markers (e.g., Petit et al. 1997; Taberlet et al. 1998; Cottrell et al. 2002; see also Sect. 6.4.4). These primer sequences for the organellar genomes

6 Quercus Table 6.3 SNP and SSR markers developed for Quercus Marker type Genome N SNP/SSR mtDNA 4 SNP/SSR cpDNA 3 SNP/SSR cpDNA 9 SNP/SSR cpDNA 17 SNP/SSR cpDNA 14 SSR Nuclear 3 SSR Nuclear 17 SSR Nuclear 9 SSR Nuclear 32 SSR Nuclear 14 SSR Nuclear 16 SSR Nuclear 10 SSR Nuclear 11 EST-SSR Nuclear 20 EST-SSR Nuclear 1,328a EST-SSR Nuclear 931a

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Species Q. robur Q. robur Q. robur Q. petraea/robur Q. petraea Q. macrocarpa Q. petraea/robur Q. myrsinifolia Q. robur Q. rubra Q. rubra Q. mongolica Q. mongolica Q. mongolica Q. rubra Q. alba

References Demesure et al. (1995) Taberlet et al. (1991) Demesure et al. (1995) Deguilloux et al. (2003a) Sebastiani et al. (2004) Dow et al. (1995) Steinkellner et al. (1997) Isagi and Suhandono (1997) Kampfer et al. (1998) Aldrich et al. (2002) Aldrich et al. (2003a) Ueno and Tsumura (2008) Mishima et al. (2006) Ueno et al. (2008) Fagaceae Project Fagaceae Project

Many of the other sequences available at Molecular Ecology Resources database (http://tomato.bio.trinity.edu/) Untested sequences available at Fagaceae Genomics Web (http://www.fagaceae.org/web/db/index)

a

work in most oak species and are reported in Dumolin et al. (1995), Deguilloux et al. (2003a), and Sebastiani et al. (2004) (Table 6.3), and many are available online through the Molecular Ecology Resources database (http://tomato.bio.trinity.edu/). At the population level, single locus codominant markers are available for Quercus in the form of allozymes (e.g., Redkina et al. 2008), single-strand conformation polymorphisms (SSCPs) (e.g., Bodenes et al. 1996), though mainly as microsatellites (SSRs or simple sequence repeats). Such markers have been used in a variety of studies regarding the oak gene pool, characterizing diversity (Aldrich et al. 2005a), clonal structure (Ainsworth et al. 2003), mating system (Bacilieri et al. 1996a, b), paternity analysis, and pollen dispersal (Nakanishi et al. 2004), inferring the number of pollen donors in a canopy (Lexer et al. 2000), hybridization (Nason et al. 1992), detection of seed contamination (Lexer et al. 1999), seed dispersal (Dow and Ashley 1996), origin of trees in a stand (Lefort et al. 1998), origin of wood samples (Deguilloux et al. 2004), and criminal forensics (Craft et al. 2007). Primer sequences for microsatellite loci are reported for the white oaks in Dow et al. (1995), Kampfer et al. (1998), Steinkellner et al. (1997), Mishima et al. (2006), and Ueno and Tsumura (2008), for the red oaks in Aldrich et al. (2002, 2003a), and section Cyclobalanopsis in Isagi and Suhandono (1997) (see also Table 6.3 and the Molecular Ecology

Resources database as noted above). Note also that Lepais et al. (2006) provide a high-throughput protocol for multiplex amplification of a panel of 10 microsatellites in the white oaks (section Quercus). In addition to the random nuclear SSR markers, there are increasing resources for microsatellites associated with transcribed regions of the genome. Ueno et al. (2008) report a set of 20 primer pairs for SSR loci derived from inner bark ESTs in Q.mongolica var. crispula (Table 6.3). In addition, the Fagaceae Project has posted to its website (http://www.fagaceae.org/ web/db/index) sequence contigs from 454-based EST sequencing in oaks (see Sect. 6.7). Available there are 6,187 SSR locus-containing sequences from Q. rubra (section Lobatae, Northern Red Oak), 1,358 of which have suggested primer sequences. For Q. alba (section Quercus, White Oak), there are 4,350 SSR loci of which 931 have suggested primer sequences. Many of these loci display in silico evidence of polymorphism. Although codominant markers such as SSRs have desirable properties for many kinds of study, medium to high density genome screens often utilize dominant markers such as random amplified polymorphic DNAs (RAPDs) and various fingerprinting methods like amplified fragment length polymorphisms (AFLPs). These are readily applied to non-model organisms since they do not pre-suppose knowledge of a specific DNA sequence in the focal species. Several recent

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studies have applied RAPD markers to oaks (e.g., Song et al. 2002; Yakovlev and Kleinschmidt 2002; Schiller et al. 2006; Gonzalez-Rodriguez et al. 2005). Some of the larger RAPD studies utilize a fairly high marker density, such as Lee et al. (2007) who applied 144 primers to several white oak species, and Barreneche et al. (1998) who constructed a linkage map for Q. robur based largely on 271 RAPD markers. The latter study also applied a minisatellite marker as did Fladung and Ziegenhagen (1998) and Kumar and Rogstad (1998) in their studies of oaks. More recent fingerprinting work often involves intersimple sequence repeats (ISSRs) and AFLPs. Lopez-Aljorna et al. (2007) used both SSR and ISSR markers to fingerprint elite Q. suber trees, and there are now numerous studies of oaks using AFLPs (Q. robur and Q. petraea: Bakker et al. 2001; Coart et al. 2002; Mariette et al. 2002; Q. crispula and Q. dentata: Ishida and Kimura 2003; Q. ellipsoidalis: Hipp and Weber 2008). Note that Cervera et al. (2000) provide an AFLP protocol optimized for several tree species including oak. Pearse and Hipp (2009) used AFLPs to generate a reliable phylogeny resolved at the species level.

6.6.3.2 Crosses and Pollination Quercus is predominantly wind-pollinated, with various factors influencing pollen production including genetic, atmospheric, and biotic agents (RodriguezRajo et al. 2005). Schueler et al. (2005) describe considerable variability in pollen viability and sensitivity to sunlight. These factors can in turn affect acorn production (e.g., Cecich and Sullivan 1999). Acorns mature during the first year in all North American white oaks (section Quercus) but require 2 years in the North American red oaks (section Lobatae). Oaks are monecious but are predominantly outcrossers, seemingly through some combination of protandry (e.g., Q. alba, Burns and Honkala 1990) and/or self-incompatibility. Q. ilex is considered highly selfincompatible and Yacine and Bouras (1997) described slower pollen tube growth and reduced seed set in selfed flowers, and elevated rates of ovule abortion in mixed pollinations. Nevertheless, low levels of selffertilization have been reported (s < 5%), and biparental inbreeding or mating between relatives can be common (Schwarzmann and Gerhold 1991; Sork et al.

P.R. Aldrich and J. Cavender-Bares

2002a, b; Fernandez and Sork 2005; Ferna´ndezManjarre´s et al. 2006; Pakkad et al. 2008). Controlled crosses are described between Q. robur and Q. petraea (Aas 1991; Steinhoff 1998). Steinhoff (1998) reported highest success rates for outcrossing to other individuals of the same species (Q. robur, 11.3%; Q. petraea, 5.8%), lesser rates for interspecific hybrids (Q. robur
Q. petraea, 5.3%; Q. petraea
Q. robur, 0.8 %), and lowest for selfing (Q.robur, 1.3%; Q. petraea, 0%). In controlled crosses, introgression is feasible as Olrik and Kjaer (2007) report that a Q. petraea
Q. robur hybrid was able to backcross to both parent species, with only a slight bias toward the Q. robur genome. This stands in contrast to an earlier study (Steinhoff 1993) and to studies of natural populations that have suggested there exists a barrier to nuclear gene exchange in the Q. robur-to-Q. petraea direction (Bacilieri et al. 1996b; see Sect. 6.4.4). Advanced generation crosses are atypical in the oaks, mainly due to the generation time problem. To our knowledge there are no publicly available F2 or backcross lines. F1 crosses are feasible and reported, and in some instances publicly available (see Sect. 6.5.2 and below). Several mapping populations in Q. robur and Q. petraea are soon available from the EVOLTREE program. This entails the following single pedigrees: Q. robur (375 genotypes), Q. petraea (127 genotypes), and Q. robur
Q. petraea (151 genotypes). Populations for association mapping are also going public and will include: one Q. robur population (296 genotypes), three Q. petraea populations (1,251 genotypes), and one Q. robur
Q. petraea population (296 genotypes).

6.6.3.3 Linkage Mapping Barreneche et al. (1998) produced the first oak linkage map (in Q. robur), which has been used in a variety of structural and comparative genomic applications (see next section, also Sects. 6.7.3 and 6.7.4). The map includes 307 total markers of the following types and abundances: RAPDs (n ¼ 271), microsatellites (n ¼ 18), sequence characterized amplified regions (SCARs; n ¼ 10), isozymes (n ¼ 6), minisatellite (n ¼ 1), and 5S rDNA (n ¼ 1). This meiotic linkage map was constructed from a controlled cross between two French trees yielding a two-generation full-sib

6 Quercus

pedigree (94 progeny). The mapping design was based on the two-way pseudo-testcross (Grattapaglia and Sederoff 1994), yielding a male and female map that was then merged using intercross markers. Both the male and female maps resolved 12 linkage groups (the haploid number in oak), and only slightly different map lengths (maternal, 893.2 cm; paternal, 921.7 cm). Total genome coverage was 85–90%. The map can be viewed at NCBI using Mapviewer (http://www.ncbi.nlm.nih.gov/mapview). Since then other oak linkage maps have been produced through extension of the original French mapping population (e.g., Saintagne et al. 2004), and other maps have been generated based on distinct crosses. Porth et al. (2005b) describe linkage mapping of osmotic stress induced genes in two maps, the intraspecific Q. robur cross (3P*A4) and an interspecific cross of Q. robur
Q. petraea (11P*QS29). Gailing (2008) report a QTL leaf morphology study based on a linkage map constructed from another intraspecific Q. robur cross, though here between geographically distant parents, one from Germany and the other Croatia. Most recently, a linkage map has been produced for the red oaks through work initiated through the Hardwood Tree Improvement and Regeneration Center (HTIRC) at Purdue University (unpublished data). Other mapping of the Quercus genome has involved the cytogenetic localization of specific genes or gene families, comparative mapping of loci across species, and QTL mapping of phenotypic traits (see next section). As an example of cytogenetic mapping, Chokchaichamnankit et al. (2008) used fluorescence in situ hybridization (FISH) to map ribo-

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somal genes onto the karyotypes of 15 species from the Fagaceae, including several oaks. For a recent review of genes mapped to Quercus and Castanea (chestnut), see Kremer et al. (2007) where they describe genes for bud burst, hypoxia, osmotic stress, and developmental stage-dependent expression. A number of comparative studies of the oak genome have used the markers of Barreneche et al. (1998) map to query the genomes of other species (see also Sects. 6.6.3.4 and 6.7.4). For example, Saintagne et al. (2004) expanded the size of the original mapping population from 94 full-sibs to 278 in their study of leaf QTLs distinguishing Q. robur and Q. petraea (see Sect. 6.2.3.1). As for non-QTL comparative studies, Barreneche et al. (2004) used microsatellite markers to explore synteny between Q. robur and Castanea sativa (European chestnut). They were able to anchor 19 markers into two previously constructed linkage maps for Quercus and Castanea, and after some local sequencing resolved seven linkage groups and another two regions based each on a single matched marker. The Barreneche et al. (1998) map for Q. robur can be compared to other species using the CMap Comparative Map Viewer hosted by the Fagaceae Project website (http://www.fagaceae.org/cgi-bin/ cmap/). Additional linkage mapping information and resources can be found in the following. Aldrich (2008) reviewed approaches and progress in the mapping of forest tree species, Kremer et al. (2007) reviewed both genetic mapping and comparative genetic mapping in the Fagaceae, and Plomion et al. (2007) reported on the availability of mapping resources for oak through the EVOLTREE program,

Table 6.4 Quantitative trait locus (QTL) findings in Quercus robur Trait(s) QTLsa Bud burst 32.0 (32) Stomatal density and growth 18.0 (18) Water use efficiency 10.0 (10) Rooting ability 10.0 (10) Leaf morphology 10.0 (10) Leaf morphology 7.5 (6–9) Waterlogging tolerance 5.0 (5) Height growth 3.0 (2–4) Mean 11.9 a

%b 7.0 (3–11) 9.8 (3.6–15.9) 20.0 (20) 9.1 (4.4–13.8) 6.6 (3.6–9.6) – (–) 9.0 (>9%) 11.5 (4–19) 10.4

Midpoint number of significant QTLs detected (value or range reported) Midpoint percentage of the variance explained by the QTLs (value or range reported)

b

References Scotti-Saintagne et al. (2004a) Gailing et al. (2008) Brendel et al. (2008) Scotti-Saintagne et al. (2005) Gailing (2008) Saintagne et al. (2004) Parelle et al. (2007a, b) Scotti-Saintagne et al. (2004a)

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including sets of SSR and SNP markers. And as noted earlier, the Fagaceae Project has made available numerous EST sequences containing SSR loci, these from both Q. rubra and Q. alba.

6.6.3.4 Quantitative Trait Loci (QTL) Mapping There are now several QTL studies of oak involving Q. robur as at least one parent in the cross. Table 6.4 shows summary information for these. For a detailed consideration of leaf QTLs distinguishing Q. robur and Q. petraea, see Sect. 6.2.3.1. For a recent review of QTL detection in the Fagaceae, see Kremer et al. (2007). Comparisons across QTL studies can be interesting but must be made with the caveat that different traits are at times composites of several lesser traits, though these concerns are certainly diminished when the traits and protocols are identical (e.g., Saintagne et al. 2004 and Gailing 2008). Still, Gailing (2008) showed the importance of genetic background and environment in their influence on outcomes even when other aspects remained the same. Though the conditions of the studies reviewed here varied, the average number of QTLs that were identified was around 11.9 per trait, with an average of 10.6% of the phenotypic variation explained. This average number of QTLs is close to the total number of linkage groups in Quercus (n ¼ 12), though there is the tendency for the QTLs to cluster together such that any one trait is not necessarily effected by variation at all the chromosomes. The estimate also falls within the range noted by Orr (2001) who summarized work in a variety of species and noted that most QTLs fell between 1 and 20 loci.

6.6.4 Biotechnology Breeding Tree breeding through biotechnology includes in vitro methods of propagation that should hasten the rate of domestication and permit mass production of elite lines. Genetic transformations hopefully will contribute through enhancements in wood quality and growth rates along with improved tolerance to drought, pests, and pathogens. These areas are aptly reviewed in a variety of contexts including the biotechnology of

P.R. Aldrich and J. Cavender-Bares

hardwoods (Merkle and Nairn 2005; Pijut et al. 2007), wood biotechnology (Boerjan 2005), biotechnology and plantation forests (Fenning and Gershenzon 2002), and methods for accelerated tree improvement (Nehra et al. 2005). Wilhelm (2000) reviewed oak somatic embryogenesis, and we offer a brief update here. Unfortunately, Quercus is not as readily cultured and transformed as other tree species like poplar, but progress has been made. Most of the oak work has been published on section Quercus, particularly the European white oaks Q. robur, Q. petraea, and Q. suber. Reports of successful methods also exist for section Cerris (Q. accutissima, Q. cerris, Q. serrata) and section Lobatae (Q. rubra). Several in vitro reports exist for Q. robur (Juncker and Favre 1994; Sanchez et al. 1996; Chalupa 2000; Vidal et al. 2003; Toribio et al. 2004; Valladares et al. 2006) and Q. petraea (San-Jose et al. 1990; Cvikrova et al. 2003). Corredoira et al. (2006) provide a detailed morphohistological study of the development of somatic embryos of leaf cultures from a 100-year-old Q. robur tree. However, there have been several reports of chromosomal instability in Q. robur during somatic embryogenesis (Endemann et al. 2001; Wilhelm et al. 2005). Transformation systems are described for Q. suber as well (Romano et al. 1992; Hernandez et al. 2003; Sanchez et al. 2005; Alvarez et al. 2004; Alvarez and Ordas 2007). Somatic embryogenesis studies on this species include cyclin gene expression associated with adventitious rooting (Neves et al. 2006), effects of abscisic acid (ABA) and indole-3-acetic acid (IAA) on embryo maturation (Garcia-Martin et al. 2005), and small heat shock proteins (Puigderrajols et al. 2002). Reports on chromosomal stability suggest that Q. suber is stable in its ploidy during embryogenesis (Bueno et al. 2003; Loureiro et al. 2005; Lopes et al. 2006). In vitro work has been done with a few other oaks outside section Quercus. Transformations described for section Cerris include Q. cerris (Tsvetkov and Hausman 2005), Q. acutissima (Kim et al. 1997), and Q. serrata (Sasamoto and Hosoi 1992). In section Lobatae, Vengadesan and Pijut (2009a, b) review the work already done on Q. rubra (Northern Red Oak) and report successful methods for in vitro propagation by somatic embryogenesis and plant regeneration for this species.

6 Quercus

6.7 Genomics Resources Quercus entered the genomics age with the publication of the Barreneche et al. (1998) linkage map. Since then there have been gains in the area of oak structural genomics (described earlier), including the aforementioned publications on QTL mapping of oak traits. But much of the emphasis in oak genomics has been in the area of functional genomics. Though research in this area still lags far behind that on other model organisms such as Arabidopsis and Populus, considerable progress is anticipated through the development of major new oak genomics initiatives that we describe below before reviewing progress to date.

6.7.1 Genome Structure Several studies have described oak karyotypes and overall genome structure and content. We have already treated single-copy regions of the genome in several sections. Here we consider the basic karyotype and genome size, along with some surveys of repetitive DNAs.

6.7.1.1 Karyotype The chromosomal count of Quercus is 2n ¼ 24 (D’Emerico et al. 1995, 2000). This cytogenetic work has concentrated on the European members of section Quercus (white oaks; Q. dalechampii, Q. petraea, Q. pubescens, and Q. robur) and section Cerris (Q. cerris, Q. coccifera, Q. trojana, Q. suber), along with a few other species. Reports from other geographic regions concur with the same count (e.g., North Korea, Q. acutissima, Baranec and Murin 2003). By comparison, Populus has 2n ¼ 38 and Arabidopsis 2n ¼ 10. Cytomorphological work, cytogenetic banding methods, in situ hybridization, and other physical mapping techniques indicate a strongly conserved genome organization within Quercus (Zoldos et al. 1999, 2001; D’Emerico et al. 2000). This holds true for comparisons between Europe and North America, and across ecophysiological classes, namely evergreen or deciduous. Nevertheless, slight karyotypic variability has been detected as differences in intrachromoso-

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mal and interchromosomal asymmetry across species, noted by D’Emerico et al. (1995, 2000). For further discussion of ploidy variants in Quercus, see Sect. 6.5.3.

6.7.1.2 Genome Size and Composition Oaks have a moderate-sized genome, though large in comparison to current model genomes. Estimates of genome size in Quercus range from 1.84 to 2.00 pg/2C (Favre and Brown 1996; Zoldos et al. 1998), almost twice the size of the Populus genome (1.2 pg/2C, Bradshaw and Stettler 1993) and nearly five times the size of the Arabidopsis genome (0.41 pg/2C, Bennett and Leitch 2005). The oak estimates largely derive from the European members of sections Quercus and Cerris. The same studies reported GC content ranging from 39.9% to 42.1%. Repetitive DNA often comprises a large fraction of eukaryotic genomes, and repeats seem well represented in Quercus as well. As for tandem repeats, Zoldos et al. (1999) found 18S–5.8S–26S rRNA genes at one major and one minor locus, and the 5S rDNA at a separate single locus. This was true of all the oak species they examined, though rRNA gene copy number at each of the loci varied across species (1,300–4,000) according to a dot-blot survey. As for dispersed repeats, Zoldos et al. (2001) used representational difference analysis to subtract the Q. suber genome from the Q. robur genome, yielding a library of 400 DNA sequences representing genome differences. They examined in detail seven of these sequences and found that each had a high similarity to known retrotransposons. Hybridizations indicated that these repeats were present as 100–700 copies in the Q. robur genome.

6.7.2 Oak Genomics Initiatives Technology is advancing at such a pace that the distinction between model and non-model organism is beginning to erode. Moreover, oak is recognized as a keystone species in Northern Hemisphere ecosystems, and so is already a model organism of an ecological variety. These factors contribute to the rise of oak genomics initiatives on both sides of the Atlantic.

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6.7.2.1 European The Europeans continue to lead in the area of oak genetics, genomics, and domestication. Their focus has been on the European white oaks, mainly Q. robur, Q. petraea, and to a lesser extent Q. suber and Q. ilex. Much of the progress already achieved and described in the following sections derives from work out of INRA, the French National Institute for Agricultural Research (http://www.inra.fr/) led by or associated with Antoine Kremer’s team. He continues a leadership role in the new EVOLTREE network (Plomion et al. 2007, see also http://www.evoltree.eu/), a European Union-funded federation of research units with the goal to assess the impact of climate change on forests, using oak as a model organism. The genomics arm of the program anticipates the following research on oak: large-scale studies of adaptive genetic variation, large collections of ESTs, SSRs, and SNPs, mapping and QTL projects, BAC libraries, microarrays, and genome sequencing including full sequences for the mitochondrial and chloroplast genomes.

6.7.2.2 North American Three US-based agencies provide notable direct contributions to the oak genomics effort. Emphases here include centralized bioinformatics resources, EST libraries, applications of biotechnology to tree improvement, and integration of genomics resources within the Fagaceae with an ancillary goal for the reintroduction of Castanea (chestnut) back into the wild. The Fagaceae Project is a multi-institutional collaboration including NC State, Penn State, and Clemson Universities, the American Chestnut Foundation, the US Forest Service, and others. They are producing considerable genomics work on Castanea (chestnut) but also Fagus (beech) and Quercus (oak). They maintain the Fagaceae Genomics Web (http://www.fagaceae.org/web/db/index), which is a portal to a variety of genome-related resources for the family, particularly EST libraries. These resources are currently available to the public and we describe some of them in the following sections. The Hardwood Tree Improvement and Regeneration Center (HTIRC, http://www.agriculture.purdue. edu/fnr/HTIRC/index.htm) is another collaborative

P.R. Aldrich and J. Cavender-Bares

enterprise that includes academia (Purdue University), governmental agencies (US Forest Service and Indiana State Forestry), private foundations (American Chestnut Foundation, Walnut Council), various members of the wood products industry, and others. They are active in the development and transfer of technologies associated with tree improvement through traditional and biotechnology means for the major hardwoods of the Central Hardwoods Region of the USA, principally Juglans (both walnut and butternut), Prunus (cherry), and Quercus (oak, especially Q. rubra, Northern Red Oak). Thirdly, the Dendrome Project (http://www.fagaceae.org/web/db/index) provides a central portal for access to several databases on tree genomics and genetics, although their bias is for conifers.

6.7.3 Structural Genomics The primary structural genomic resources for oaks are the linkage maps constructed in the white oaks Q. robur and Q. petraea (Barreneche et al. 1998; Porth et al. 2005b; Gailing 2008) and the red oak Q. rubra (HTIRC, unpublished data). We describe these maps in Sect. 6.6.3.3. The white oak map can be viewed using Mapviewer at NCBI (http://www.ncbi. nlm.nih.gov/mapview) or CMap at the Fagaceae Project website (http://www.fagaceae.org/cgi-bin/cmap/). Others have probed the oak genome for content with respect to specific genes or gene families. For example, Porth et al. (2005b) located osmotic stress genes in Q. robur and in a Q. robur
Q. petraea cross. They began with 25 cDNAs derived from osmotically stressed Q. petraea callus tissue (Porth et al. 2005a) and, using SNPs, were able to position 13 of 14 genes on at least one of the two maps. Zoldos et al. (2001) used representational difference analysis to subtract the genome of Q. suber from that of Q. robur. The library of 400 clones representing differences contained a number of sequences identified as retrotransposons. They tested seven of these clones against hybridization in other oak species and found positive hybridization in Q. petraea but not in Q. cerris, C. coccifera, Q. ilex, or Q. palustris, though three clones did hybridize to Q. virginiana which is the species most closely related to Q. robur–Q. petraea.

6 Quercus

This pattern suggests that the retrotransposons arose in the common ancestor of this group. Comparative mapping has been done between Quercus and Castanea (chestnut) (Barreneche et al. 2004) using microsatellite markers. They tested for cross-species amplification of SSR markers and found 47% of Quercus markers would transfer as would 63% from Castanea. From this set of anchor markers, they were able to integrate 19 into linkage maps that had already been produced for the two species. Subsequent sequencing verified the homology of the markers. Casasoli et al. (2006) extended this comparative work, combining the SSR markers with sequence tagged site (STS) markers produced from oak EST information. This yielded a comparative map based on 55 orthologous markers. They compared QTL positions for three adaptively important traits, finding a shared position of control of bud burst but not for height or carbon isotope discrimination. Outside of linkage maps and related technologies, there are limited resources for Quercus in the area of structural genomics at this point. Its genome size (1.84–2.00 pg/2C, Favre and Brown 1996; Zoldos et al. 1998) is roughly twice that of Populus (1.2 pg/2C, Bradshaw and Stettler 1993). Given that the full sequence of popular is now available (Tuskan et al. 2006), there are useful opportunities for comparative genomic research on the tree habit and other topics that might improve the domestication of oaks. Much work is underway on restoration of the chestnut, also a member of the Fagaceae, including two BAC libraries that have been constructed through the Fagaceae Project for Castanea mollissima (Chinese chestnut). The EVOLTREE program plans to generate several key structural genomics resources for oak including a 10
BAC library and large-scale sequencing that includes the full oak chloroplast and mitochondrial genomes (Plomion et al. 2007).

6.7.4 Functional Genomics Much interesting work has been done on the functional genomics of trees. Large-scale EST studies of trees are commonplace now, and public resources are increasingly available. The public availability of the poplar chip has also promoted research. Tang et al. (2003)

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provide a general review of progress in the functional genomics of wood quality and properties. Most of the genomics research to date in Quercus entails studies of gene expression, mainly at the RNA level though more recently at the protein level as well. Most of this work has been done on the European white oaks, notably Q. robur, Q. petaea, Q. suber, and Q. ilex. Many studies have focused on expression of a single gene or gene family, using hybridizations, real-time polymerase chain reaction (RT-PCR), and other methods. Broader surveys query the full transcriptome using hybridizations, subtractive hybridizations, and macroarrays, and several EST libraries have been constructed for oaks, even a red oak (Q. rubra). As for protein expression, a proteome project is underway for the leaf of the Holm oak, Q. ilex.

6.7.4.1 Transcriptome Several oak studies have targeted the expression of a single gene or gene family. The stress response is a frequent topic, with recent work on small heat shock proteins in cork tissue and apical meristems (Pla et al. 1998) and in somatic embryos (Puigderrajols et al. 2002). Oxidative stress studies exist as well, including work on calmodulin genes in flooded seedlings (Folzer et al. 2006), type 2 metallothionein in cork tissue (Mir et al. 2004), and non-symbiotic hemoglobin in seedlings (Parent et al. 2008). Genes associated with fungal interactions are of interest, from the positive mycorrhizal association to the negative pathogenic interaction. One study targeted class III chitinases in root tissue during pre-mycorrhizal interactions (Frettinger et al. 2006) and another cinnamyl alcohol dehydrogenase defense in response to infection by Phytophthora cinnamomi (Coelho et al. 2006). Other work has been developmental stage specific, as in a differentially expressed gene in juvenile-like and mature shoots (Gil et al. 2003), or in micropropagated tissue during adventitious rooting wherein expression of a B-type cyclin gene was characterized (Neves et al. 2006). Larger surveys of oak transcriptomes have been conducted using hybridizations, RT-PCR, ESTs, and macroarrays. Kruger et al. (2004) used subtractive hybridization and differential expression to query Q. robur transcripts up-regulated in the pre-mycorrhizal phase in a micropropagation system. RT-PCR has been used successfully to examine seasonal variation

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in the transcripts involved in cork biosynthesis and regulation using candidate genes (Soler et al. 2008). It also has been applied to the transcriptome of Q. petraea bud burst (Derory et al. 2006). Several expressed sequence tag (EST) libraries have been published as well, including one for Q. petraea bud burst (Derory et al. 2006) and an inner bark EST library for Quercus mongolica var. crispula that Ueno et al. (2008) used to explore microsatellite marker development. EST-QTL maps have even been generated for osmotic stress-induced genes in Q. robur
Q. petraea (Porth et al. 2005a, b) and a broader comparison of adaptive traits including bud burst and height growth in oak versus chestnut (Quercus robur
Castanea sativa, Casasoli et al. 2006). Public availability of oak expression data is growing, with nearly half a million (n ¼ 489,780) Quercus EST sequences in public databases, as far as we know. There are 9,420 Quercus EST sequences presently listed with GenBank, all from the white oaks (section Quercus). Species representation is as follows: Q. robur (n ¼ 3,499), Q. mongolica subsp. crispula (n ¼ 3,385), Q. petraea (n ¼ 2,300), and Q. suber (n ¼ 236). A notable sequencing effort is underway at the Fagaceae Project, and public access to these sequences is provided through their website. They have pooled multiple above-ground tissues (including buds, cambium, flowers, fruit, phloem, and stems) to produce RNA, and then used 454-technology to sequence through the cDNAs. They have pursed sequencing in the two main timber species for North America, one for each oak section. Q. alba (White Oak, section Quercus) has a total of 203,206 EST sequences and Q. rubra (Northern Red Oak, section Lobatae) has 277,154 ESTs, totaling 480,360 sequences for these two species. Also available on the Fagaceae Project website are the contigs and unigene sets for these projects, along with similar resources for other members of the Fagaceae, namely Fagus grandifolia (American beech, 64,253 ETSs), Castanea mollissima (Chinese chestnut, 847,952 ESTs), and C. dentata (American chestnut, 398,783 ESTs). The French, in association with EVOLTREE, also have a forest tree genomics initiative (FOREST, http://www.genoscope.cns.fr/spip/Quercus-Forestfrench-initiative.html) producing Q. robur ESTs for differentiating xylem (n ¼ 9,529), leaves (n ¼ 7,097), and roots (n ¼ 19,177), along with Q. petraea ESTs for buds (n ¼ 9,990).

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The poplar genome array from Affymetrix represented a major step forward for tree functional genomics, and oak microarrays are in the planning stages. Oak macroarrays have already been used in research. For example, Frettinger et al. (2007, see also a review in Herrmann and Buscot 2007) used a cDNA array to explore transcriptional changes in pre-mycorrhizal roots and in ectomycorrhizae. Soler et al. (2007) used a cDNA array to explore suberin biosynthesis and cork differentiation in Q. suber, and Derory et al. (2006) to examine bud burst in Q. petraea. Public releases of oak microarrays are on the horizon. Following its participation in the Human Genome Project, the French Genoscope program (http://www.genoscope.cns.fr) has changed its focus to environmental genomics. In association with the EVOLTREE and FOREST programs, Genoscope states that a unigene set of 16,000 elements from Q. robur and Q. petraea ESTs is now ready to be printed on a microarray (as of January 15, 2009). On an annual basis, the EVOLTREE program plans to issue public releases of subsets of the ESTs in its possession in the form of microarrays, through the PICME (Platform for Integrated Clone Management; http://www.picme.at). This program of the Austrian Research Centers GmbH – ARC will make available to the scientific community the ESTs of a centralized database for a variety of species including Quercus but also Populus, Pinus, and Ipomoea.

6.7.4.2 Proteome There are numerous reports on protein variation in Quercus though most of these are isozyme studies that focus on a few select metabolic enzymes (e.g., Schnabel and Hamrick 1990; Berg and Hamrick 1993). Such studies can be very informative as to the gene pool structure, wherein members of the genus typically display a large amount of protein-based genetic variation within populations and little differentiation among populations, as is typical of longlived, outcrossing, woody species (Hamrick et al. 1992). Isozymes, as with other marker types, have not revealed fixed differences between oak species (see also Sect. 6.2.3). This is so even in high-throughput research as in Barreneche et al. (1996) in which they used two-dimensional gel electrophoresis to query protein variation in Q. robur and Q. petraea. In the

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scoring of 530 polypeptide spots, 101 were polymorphic, three spots displayed a frequency difference between species, but no spots were specific to one or the other species. More recently, Jorge et al. (2005) announced the Q. ilex (Holm oak) leaf proteome project. They report the first stages of a protocol optimized for assessing both analytical and biological variance in protein abundance. Methods include two-dimensional gel electrophoresis, tandem mass spectrometry, de novo sequencing, and sequence similarity searches against non-oak databases for identifications. Their initial report describes 35 identifications (out of 43 analyzed spots) mainly of proteins involved in photosynthesis and energy-based metabolism, with ribulose1,5-bisphosphate carboxylase oxygenase (RubisCO) especially well represented.

6.7.5 Metabolomics Most published biochemical research on oaks has targeted specific chemical groups or pathways and includes research in chemosystematics, acorn nutritive chemistry (e.g., Ozcan and Baycu 2005), isoprenoid emissions (e.g., Loreto et al. 1998), chemical deterrents to herbivory, stress physiology, and considerable work on secondary metabolites including tannins and other heartwood extractives. 6.7.5.1 Wood Chemistry There has been much attention given to the chemistry of wood formation in trees. Many of the approaches are integrative and include genomics research to identify genes participating in relevant biochemical pathways. As noted, most of this work has occurred in Populus, though Eucalyptus is a rising presence in this area along with the conifers, especially Pinus. For example, sequenced ESTs from the poplar secondary xylem have revealed genes active in lignin and cellulose biosynthesis (e.g., Sterky et al. 1998; Hertzberg et al. 2001). Recent reviews of various aspects of wood formation chemistry and genomics can be found in Tang et al. (2003) and Farrokhi et al. (2006). There has been much less work on the temperate hardwood metabolome in the area of wood chemistry.

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Robinia pseudoacacia (Black Locust) has provided insights into the chemistry of hardwood formation, wherein Yang et al. (2004) queried gene expression at the sapwood-heartwood transition zone using a cDNA microarray for 2,567 unigenes. They used this system to describe variation in 569 genes that displayed differential expression across seasons. As for oaks, Soler et al. (2007) recently combined subtractive hybridization and cDNA microarrays to explore the basis of suberin biosynthesis during cork formation in Q. suber. They isolated and identified sequences from cork tree bark, printed them on an array, and queried cork versus xylem tissue, resolving several genes specific to interfacing with suberin monomers. Much of the research to date on oak wood chemistry has focused on heartwood extractives, a diverse collection of secondary metabolites that includes the soluble polyphenolic compounds known as tannins (gallotannins and ellagitannins). These compounds have historically been used to cure or tan leather, though they also influence coloration (and value) of wood, and readily leach out of wooden casks or barrels thereby influencing the flavor of wine (reviewed in Puech et al. 1999; see also Doussot et al. 2002; and Sect. 6.8.3). Not surprisingly, much of this work has been done on Q. robur and Q. petraea in Europe, with a couple of reports from Q. laevis. Puech et al. (1999) summarized findings on the natural variation in tannin concentrations in oak heartwood, finding that there was considerable variation within and among trees, provenance, and species. But there seems to be a reasonable genetic basis to variation in the trait, even though the environment can have a strong influence. Mosedale et al. (1996) and Mosedale and Savill (1996) found evidence for strong genetic control of heartwood ellagitannin concentration in Q. petraea and Q. robur, though a large amount of the total variation was due to geographic origin, and heartwood color appeared to arise from environmental factors. Snakkers et al. (2000) describe extractive variation in Q. petraea across geography, silvicultural treatment, and stem. In Quercus laevis, Klaper et al. (2001) used microsatellites to show a significant correlation between genetic relatedness and leaf phenolics though there also appeared to be seasonal variation. Other studies support the notion that oak heartwood extractives can have a strong environmental determinant (e.g., Masson et al. 1995; Prida et al. 2006).

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6.8 Domestication and Commercialization The economic impact of wild oak populations is considerable through their collective contributions to the structure and function of forested ecosystems (see Sect. 6.3). Although most oak lines are not heavily domesticated, the commercial contributions of both wild and managed populations are large most notably as a source of wood and paper products. Lesser but notable contributions also exist in the areas of food and beverages.

6.8.1 Wood and Paper Products Oaks are an important source of hardwood lumber (Johnson et al. 2002; reviewed in Bowyer et al. 2007). Finer grades are used in veneer, furniture, flooring, and other building materials. Lesser grades are used in pallets, crates, and railroad ties. Oak species are usually lumped together at the mill and sold either as red or white oak. Red oaks play a greater economic role in the US market (i.e., in their native range), whereas the white oaks are economically important in many areas of the globe. We have already considered the selection and breeding of wood properties in other sections including general wood quality (see Sect. 6.6.3.4) as well as extractives that influence the color of wood (see Sect. 6.7.5.1). There is more to say regarding extractives in the context of wine and whiskey making (see below). Additional wood-related topics of commercial importance include recent proteomic research on wood decay fungi (Abbas et al. 2005) and a burst of interest in forensic identification of wood origins. Much can happen between the forest or plantation and the lumber yard, and sometimes buyers would like to know the provenance of the wood they are purchasing. Forest certification establishes that certain timber has been harvested from sustainably managed forests, and checking that the chain of custody is as advertised is of growing economic importance. Molecular markers can be used to type wood as to its geographic origin. This is especially true in places such as Europe where there is a geographically extensive and dense database on the composition of the gene pool for some agricultural species, such as the oaks. Chloroplast

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microsatellites have proven especially useful in the typing of oak wood to the stand-level (e.g., Deguilloux et al. 2002, 2003b). Dried oak wood and oak wood as old as 600 years have been used successfully in PCRbased assays of this type (Dumolin-Lape`gue et al. 2002). Among the forest industries benefiting from enhanced typing, the cooperage industry that makes oak barrels for the aging of wine and whiskey must know the geographic source of the wood used in barrels since this is critical to the flavor imparted to the end product (Deguilloux et al. 2004). Other applications of forensic techniques include court cases involving wood theft and criminal cases where the geographic source of botanical evidence is relevant (e.g., Craft et al. 2007). Since oaks are common one would anticipate their frequent appearance in such cases. For a more general consideration of wood typing, see Nielsen and Kjær (2008).

6.8.2 Food Products Oaks serve as a food source for a variety of organisms of economic importance to humans, including wildlife, a silkmoth, and a variety of mushrooms. Although indigenous cultures ate acorns and recipes can be found for acorn-based flour, the extensive preparations required to remove the tannins has caused acorns to fall off the human menu. Modern usage of oaks as a human food source is more often indirect. Clearly oaks serve as mast for a variety of wildlife, some of which humans eat, such as deer. Acorn chemistry and nutrition has been studied, and we know for instance their elemental composition (e.g., Ozcan and Baycu 2005) and their potentially therapeutic properties associated with free-radical scavenging (Jin et al. 2005). Oak leaves sustain a diverse fauna of herbivores, some of which are of economic interest such as the Japanese oak silkmoth Antheraea yamamai (Oishi et al. 2005). And oak wood is commonly used in the culture of mushrooms for human consumption. For example, oak wood chips are useful in growing Hericium (Ko et al. 2005) and Pleurotus (Suzuki and Mizuno 1997). But it is the truffle (genus Tuber) that fetches the highest price among edible fungi. This grows below ground near the base of oak trees, is notoriously hard to find, and expensive. Techniques are now available to detect and monitor the abundance of Tuber mycelia

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in the soil, exemplified by the real-time PCR study of an oak orchard by Suz et al. (2008).

6.8.3 Wine and Whiskey The wine and whiskey industries utilize oak wood in the production of barrels (cooperage), corks for bottles, and yeast indigenous to oak bark for flavoring. Casks and barrels used to age wine and whiskey are often made from oak heartwood wherein the wood serves a dual function as container but also contributor to the flavor of the wine and whiskey (Feuillat and Keller 1997; Puech et al. 1999; Doussot et al. 2002; see also Sect. 6.7.5.1). Secondary metabolites such as the tannins and other heartwood extractives leach from the wood during the aging, influencing the end product flavor. The wood chemistry is highly variable by species, geography, and wood treatment such as natural drying versus toasting. Quercus suber (cork oak, section Cerris) is a dominant source of cork, and recent genomic work is exploring the molecular genetic basis of cork development (Soler et al. 2007). The yeast Saccharomyces cerevisiae is used in wine making and researchers have begun exploring the genetic characteristics of different yeast strains collected from oak bark (Mesa et al. 2000; Aa et al. 2006; Wang and Bai 2008). The hope is that some of these naturally occurring yeast varieties would improve wine flavor during fermentation and aging. These are important areas in which Quercus research leads that done in Populus and Arabidopsis.

6.9 Conclusion Oaks and humans have interacted for many millennia, yet the character of the interactions has remained largely unchanged until only recently. We are moving from a state of near complete ignorance regarding the content of the oak genome to a place where we now know much of the protein coding sequence, through EST studies. It is likely that the whole genome will be sequenced not that far in the future. Domestication will advance at a much more rapid pace, building upon the numerous projects that have taken hold in the last several decades. A challenge for the immediate

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future is to take stock of the wild populations of oaks to ensure that naturally occurring biodiversity is retained for incorporation into the cultivars that will derive from domestication programs. Although the oak gene pools appear sound in many regards, there are numerous reasons to be cautious in our optimism and to manage the presently ubiquitous resource with care so that this critical species continues to play a dominant role in the planet’s ecosystems and our economies.

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6 Quercus Tomlinson PT, Jensen RJ, Hancock JF (2000) Do whole tree silvic characters indicate hybridization in red oak (Quercus section Lobatae)? Am Midl Nat 143:154–168 Tooley PW, Kyde KL (2007) Susceptibility of some Eastern forest species to Phytophthora ramorum. Plant Dis 91:435–438 Toribio M, Fernandez C, Celestino C, Martinez MT, San-Jose MC, Vieitez AM (2004) Somatic embryogenesis in mature Quercus robur trees. Plant Cell Tissue Organ Cult 76:283–287 Trehane P (2007) Oak name database. International Oak Society. http://www.oaknames.org. Accessed 15 Jan 2009 Trelease W (1924) The American oaks. Mem Natl Acad Sci 20:1–255 Tsvetkov I, Hausman JF (2005) In vitro regeneration from alginate-encapsulated microcuttings of Quercus sp. Sci Hort 103:503–507 Tucker JM (1974) Patterns of parallel evolution of leaf form in New World oaks. Taxon 23:129–154 Tuskan GA, DiFazio S, Jansson S, Bohlmann J et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604 Ueno S, Tsumura Y (2008) Development of ten microsatellite markers for Quercus mongolica var. crispula by database mining. Conserv Genet 9:1083–1085 Ueno S, Taguchi Y, Tsumura Y (2008) Microsatellite markers derived from Quercus mongolica var. crispula (Fagaceae) inner bark expressed sequence tags. Genes Genet Syst 83: 179–187 USDA-NRCS (2009) The PLANTS Database. National Plant Data Center, Baton Rouge, LA 70874-4490, USA: http:// plants.usda.gov. Accessed 19 Feb 2009 Valencia S (2004) Diversidad del genero Quercus (Fagaceae) en Mexico. Bol Soc Bot Mex 75:33–53 Valladares S, Sanchez C, Martinez MT, Ballester A, Vieitez AM (2006) Plant regeneration through somatic embryogenesis from tissues of mature oak trees: true-to-type conformity of plantlets by RAPD analysis. Plant Cell Rep 25:879–886 Van Dersal W (1940) Utilization of oaks by birds and mammals. J Wildl Manag 4:404–428 Van Valen L (1976) Ecological species, multispecies, and oaks. Taxon 25:233–239 Venette RC, Cohen SD (2006) Potential climatic suitability for establishment of Phytophthora ramorum within the contiguous United States. Forest Ecol Manag 231:18–26 Vengadesan G, Pijut PM (2009a) Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.). Plant Cell Tissue Organ Cult 97:141–149 Vengadesan G, Pijut PM (2009b) In vitro propagation of northern red oak (Quercus rubra L.). In Vitro Cell Dev Biol Plant. doi:10.1007/s11627-008-9182-6 Vidal N, Arellano G, San-Jose MC, Vieitez AM, Ballester A (2003) Developmental stages during the rooting of in-vitro cultured Quercus robur shoots from material of juvenile and mature origin. Tree Physiol 23:1247–1254 Villarreal-Ruiz L, Anderson IC, Alexander IJ (2004) Interaction between an isolate from the Hymenoscyphus ericae aggregate and roots of Pinus and Vaccinium. New Phytol 164:183–192 Wang SA, Bai FY (2008) Saccharomyces arboricolus sp nov, a yeast species from tree bark. Int J Syst Evol Microbiol 58: 510–514

129 Weigel DR, Van Sambeek JW, Michler CH (eds) (2005) Ninth workshop on seedling physiology and growth problems in oak plantings (abstr) 18–20 Oct 2004, West Lafayette, IN, USA. Gen Tech Rep NC-262 St. Paul, MN, US Department of Agriculture, Forest Service, North Central Research Station, p 28 Whittaker RH (1956) Vegetation of the Great Smoky Mountains. Ecological Monographs 26:1–80 Whittaker RH (1969) Evolution of diversity in plant communities. In: Diversity and stability in ecological systems. Brookhaven symposium biology, No 22, pp 178–195 Wilhelm E (2000) Somatic embryogenesis in oak (Quercus spp.). In Vitro Cell Dev Biol Plant 36:349–357 Wilhelm E, Hristoforoglu K, Fluch S, Burg K (2005) Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.). Plant Cell Rep 23:790–795 Wilson AD (2001) Oak wilt – a potential threat to southern and western oak forests. J Forest 99:4–11 Wittgenstein L (1958) Philosophical investigations, 3rd edn. Translated by Anscombe GEM. Macmillan, New York, USA, p 250 Woeste KE, McKenna JR (2004) Walnut genetic improvement at the start of a new century. In: Michler CH, Pijut PM, Van Sambeek JW, Coggeshall MV, Seifert J, Woeste K, Overton R, Ponder F Jr (eds) Black Walnut in a New Century, proceedings of the 6th walnut council research symposium, 25–28 July 2004, Lafayette, IN, USA. Gen Tech Rep NC-243. St. Paul, MN, US Department of Agriculture, Forest Service, North Central Research Station, pp 9–17 Yacine A, Bouras F (1997) Self- and cross-pollination effects on pollen tube growth and seed set in holm oak Quercus ilex L (Fagaceae). Ann Sci Forest 54:447–462 Yakovlev IA, Kleinschmidt J (2002) Genetic differentiation of pedunculate oak Quercus robur L. in the European part of Russia based on RAPD markers. Russ J Genet 38: 148–155 Yang J, Kamdem DP, Keathley DE, Han K-H (2004) Seasonal changes in gene expression at the sapwood–heartwood transition zone of black locust (Robinia pseudoacacia) revealed by cDNA microarray analysis. Tree Physiol 24: 461–474 Zanetto A, Roussel G, Kremer A (1994) Geographic variation of inter-specific differentiation between Quercus robur L. and Quercus petraea (Matt.) Liebl. Forest Genet 1:111–123 Zhou ZK (1993) The fossil history of Quercus. Acta Bot Yunnanica 15:21–33 Zoldos V, Papes D, Brown SC, Panaud O, Siljak-Yakovlev S (1998) Genome size and base composition of seven Quercus species:Inter- and intra-population variation. Genome 41: 162–168 Zoldos V, Papes D, Cerbah M, Panaud O, Besendorfer V, Siljak-Yakovlev S (1999) Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species. Theor Appl Genet 99:969–977 Zoldos V, Siljak-Yakovlev S, Papes D, Sarr A, Panaud O, Zoldos V, Papes D (2001) Representational difference analysis reveals genomic differences between Q. robur and Q. suber: implications for the study of genome evolution in the genus Quercus. Mol Genet Genomics 265:234–241

.

Chapter 7

Santalum Madhugiri Nageswara Rao, Jaya R. Soneji, and Padmini Sudarshana

7.1 Basic Botany of the Species The genus Santalum, commonly known as sandalwood, belongs to the family Santalaceae (Fig. 7.1). The genus is composed of approximately 16 species and their variants, many of which are geographically and probably reproductively isolated (Applegate et al. 1990). Several species, most notably S. album, are highly valued as they produce extremely aromatic heartwood and oil (Uma Shaanker et al. 2000; Nageswara Rao 2004; Nageswara Rao et al. 2007a). Though S. album species, known commonly as Indian sandalwood, is indigenous to Peninsular India (Srinivasan et al. 1992), disagreement persists as to whether it is native to India or was introduced for cultivation over 2,000 years ago from the Timor Islands of Indonesia (Roxburgh 1820; Sprague and Summerhayes 1927; Fischer 1928, 1938; Tuyama 1939; St. John 1947; Thirawat 1955; Shetty 1977; Mc Kinnell 1990; Rai 1990) or Australia. S. lanceolatum is believed to have originated in Australia, S. austrocaledonicum in New Caledonia, and S. yasi in Tonga, while S. ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum are endemic to Hawaiian Islands (Merlin et al. 2006; Thomson 2006). S. album is distributed between 30
N and 40
S from Indonesia in the West to Jaun Fernandez Islands in the East and from Hawaiian Archipelago in the North to New Zealand in the South. S. austrocaledonicum and S. yasi have limited plantings, outside of its native range, in Australia for

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

trial purposes (Thomson 2006). S. austrocaledonicum has also been planted in Fiji and the Cook Islands. S. ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum have primarily been planted inside of their natural range for economic or preservation purposes (Merlin et al. 2006). S. acuminatum is wide spread in all Australian mainland states (George 1984). Sandal grows naturally in a variety of climates from warm desert in Australia, through seasonally dry monsoon climate in India, eastern Indonesia, and Vanuatu, to subtropical climate with almost uniform rainfall in Hawaii and New Caledonia (Applegate et al. 1990). It is adaptable to most soil conditions but grows well in light to medium, well-drained soils (Merlin et al. 2006). Most sandal species are small trees or large shrubs, attaining a height of about 5–20 m or more and girth of 1–2.5 m with slender drooping and/or erect branching (Fig. 7.2a). They are slow growing root hemiparasites, with roots tapping the root systems of surrounding trees for water, minerals, and nutrients (Stemmermann 1977). Scott (1871) for the first time reported the parasitic nature of sandal. Requirement of host for proper growth of sandal was demonstrated in a field study by Ananthpadmanabha et al. (1984). Depending on the sandal species and the location, the host trees may vary though they seem to rely on nitrogen fixing trees such as Acacia and Casuarina, along with many other legumes, shrubs, herbs, and grasses. A wide range of hosts are utilized by S. album in India and occasionally it self-parasitizes (Rai 1990), while in Timor it associates with numerous species such as Eugenia, Casurina, Cassia, Schleichera, Pterocarpus, etc. (Suriamihardja and Suriamihardja 1993). In Australia, S. spicatum and S. lanceolatum parasitizes Acacia, Eremophlia, Melaleuca, etc. (Applegate and McKinnell 1993).

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_7, # Springer-Verlag Berlin Heidelberg 2011

131

132 Fig. 7.1 Taxonomic position of Santalum

M. Nageswara Rao et al. Kingdom Plantae

Division Magnoliophyta

Subdivision Magnoliophytina

Class Magnoliopsida

Subclass Rosidae

Superorder Santalanae

Order Santalales

Suborder Santalineae

Family Santalaceae

four ploidy levels, ranging from diploidy to octoploidy, and differ significantly from one another in DNA content (Harbaugh 2008). S. album is diploid with 2n ¼ 20, while other members of the genus, particularly S. acuminatum and S. macgregorii, are believed to be tetraploid (Kulkarni et al. 1998; Byrne et al. 2003a). Most of the Australian species (S. acuminatum, S. lanceolatum, S. murrayanum, S. obtusifolium, and S. spicatum) are diploids, with the exception of S. leptocladum, which is a tetraploid. S. austrocaledonicum from Vanuatu and New Caledonia and S. yasi from Fiji are also diploids, while S. macgregorii from Papua New Guinea and S. ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum from Hawaiian Islands are tetraploids. S. insulare may be a putative hexa- or octaploid (Harbaugh 2008). Incongruence between nuclear and chloroplast trees in the positions of two taxa, S. boninense and S. macgregorii, shows that hybridization and possibly allopolyploidization may have played a role in the evolution of the genus (Harbaugh and Baldwin 2007).

Subfamily Santaloideae

Tribe Santalaceae

Subtribe Santalinae Genus Santalum

The leaves of sandal are opposite and decussate, and sometimes show whorled arrangement. The tree starts flowering at an early age of 2–4 years and flowers twice a year from March to May and September to December. Flowers (Fig. 7.2b and Table 7.1) are unscented, purplish brown, and borne in axillary or terminal cymose panicles (Srinivasan et al. 1992). Petals are tetra or pentamerous, fused to the perianth (Hewson and George 1984). The ovary is semi-inferior and unilocular yielding one single-seeded drupe (Fig. 7.2c). Seeds are naked, i.e. lack testa. However, S. spicatum has dry and fibrous fruits enclosing a hard nut (Jones and Plummer 2008). S. album and S. spicatum are outcrossing insect-pollinated trees; however, self-pollination is possible (Rugkhla 1997). Birds disperse sandal fruits. Seedling establishment has been found to be positively associated with seed size (Hegde et al. 1991). Limited work has been done on the cytological features of sandal. The genus Santalum appears to include

7.2 Taxonomy and Phylogeography Santalum has been taxonomically grouped entirely on a few morphological characters such as floral tube color, ratio of corolla length to width and placement of ovary. They were divided into five sections or genus. The first section Santalum was described as usually having reddish corollas that are longer than wide and partly superior ovaries (Skottsberg 1930a; Stemmermann 1980; Wagner et al. 1999) and covered species such as S. album, S. austrocaledonium, S. boninense, S. lanceolatum, S. macgregorii, S. obtusifolium, and S. yasi occurring in Australia, Indonesia, India, New Caledonia, Vanuatu, Bonin Islands, Papua New Guniea, Fuji, or Tonga. Solenantha was the second section covering S. freycinetianum and S. haleakalae species both of which were endemic to the Hawaiian Islands and were described based on their longer perianth tubes, smaller ovaries, and absence of hair proximal to the filaments (Tuyama 1939). S. ellipticum and S. paniculatum occurring in the Hawaiian Islands formed the third section of Hawaiiensia and described as having white, green, brown, or orange corollas that are as wide as long, and inferior ovaries (Skottsberg 1930a; Stemmermann 1980; Wagner et al. 1999). Polynesica formed the fourth

7 Santalum

133

a

b

c

Fig. 7.2 (a) Sandalwood (Santalum album L.) tree in its natural habitat, (b) Fruits, and (c) Flowers

section with species such as S. fernandezianum and S. insulare occurring in Juan Fernandez Islands, Society Islands, Marquesas Islands, Pitcairn Islands, Austral Islands, or Cook Islands. Section Polynesica has characters similar to Hawaiiensia and could be distinguished from it based on their partly superior ovaries (Skottsberg 1930a). S. acuminatum, S. murrayanum, and S. spicatum occurring in Australia formed the fifth section of Eucarya (Harbaugh and Baldwin 2007). Morphological similarities between Hawaiiensian and Polynesican sections have led to the hypothesis that they may be closely related (Skottsberg 1930a, b) or may be synonyms (Fosberg and Sachet 1985). Recently, molecular phylogenetic analyses have demonstrated that sections Hawaiiensia and Polynesica are more closely related to other taxa of Santalum than to one another (Harbaugh and Baldwin 2007). Phylogeographical analysis has been carried out in sandal. It relies on interpreting patterns of congruence or lack of congruence between the geographical distribution of chlorotypes and their genealogical relationships (Avise 2000). If clades of closely related chlorotypes are geographically restricted and occur in proximity to each other, they represent a pattern of congruence, which indicates long-standing pattern of highly restricted

gene flow (Butaud et al. 2005). Harbaugh and Baldwin (2007) reported a genus-wide phylogenetic analysis for Santalum, using a combination of 18S–26S nuclear ribosomal [internal transcribed spacer (ITS) and external transcribed spacer (ETS)] and chloroplast (30 trnK intron) DNA sequences, and provided new perspectives on relationships and biogeographic patterns among the widespread and economically important sandal. Indonesia has been reported to be the possible source of S. album in India and Australia (Roxburgh 1820; Sprague and Summerhayes 1927; Tuyama 1939; St. John 1947; Mc Kinnell 1990; Rai 1990). Several specimens of S. album from India and Australia were phylogenetically identical suggesting a recent dispersal, consistent with the movement of this species by people (Harbaugh and Baldwin 2007). Another study also reported a lack of differentiation between Timorese and Indian germplasm of S. album supporting the hypothesis that S. album was introduced to India in very recent geological time, possibly through human mediation (Jones 2008). The origin of Santalum in Australia and at least five putatively bird-mediated, long-distance dispersal events out of Australia (with two colonizations of Melanesia, two of the Hawaiian Islands, and one of the Juan

Fruit color

Fruit length Fruit shape

Leaf length Leaf width Leaf shape and characteristics

Ovary

Flower size

Flower fragrance

Tree height Bole diameter at breast height Canopy diameter Flower color 2–4 m Reddish cream

2–4 m 20 cm

1,900–2,700 m Small tree

S. haleakalae Endemic to Maui

Reddish purple to black

8–24 mm Drupes

4.0–9.0 cm 1.3–7.5 cm Acute to rounded apex, green to a bit glaucous

Produce sweet fragrance Flowers are as long as wide Ovary inferior

3–7 m Greenish to tinged brown, orange

3–10 m 1m

450–2,550 m Shrubs to small tree

S. paniculatum Endemic to Hawaii

10–12 mm Drupes Black to purple black Purple to black

8–24 mm Drupes

2.5–7.5 cm 2.5–8.0 cm 2.0–6.0 cm 2.0–4.5 cm Ovate, obovate or Ovate, elliptic or orbicular, stiff to obovate, glossy coriaceous upper and dull/ surfaces pale lower

Produce weak Produce sweet fragrance fragrance Flowers are as long Flowers are as long as wide as wide Ovary partly inferior Ovary inferior

3–10 m Red to yellow

1–13 m 80 cm

250–950 m Shrubs to small tree

560–950 m Shrubs to small tree 1–12 m 30 cm

S. freycinetianum Endemic to Hawaii

S. ellipticum Endemic to Hawaii

1–5 m Greenish to tinged brown, orange, salmon Fragrant Produce fragrance Produce sweet fragrance 3.0–5.0 mm long 4.5 mm long Flowers are as long as wide Ovary semi-inferior Ovary inferior or half Ovary inferior inferior 3.0–8.0 cm 5.0–9.0 cm 2.5–6.1 cm 3.0–5.0 cm 2.0–3.5 cm 1.7–4.0 cm Opposite, decussate, Simple, obovate, Ecliptic to orbicular, ovate, or ovate glabrous, shiny at ovate, or obovate elliptical, shiny the top, dull light in shape, leathery at the top, dull green below to succulent green below 10–12 mm 10–15 mm 9–12 mm Globose to ellipsoid, Subglobose or Glaucous fleshy drupe ellipsoid Purple to black Drupe, purplish black Purple to black

5–12 m 40–50 m

4–10 m 4–8 m Greenish to purplish Greenish white to brown cream

12–20 m 1–1.5 m

Table 7.1 Botanical description of Santalum species Santalum album S. austrocaledonicum Distribution Australia, India, New Caledonia, Indonesia, New Vanuatu Zealand Elevation 650–1,200 m 5–800 m Plant size Small tree Shrubs to small tree

Reddish purple to black

12 mm Ellipsoid drupe

8–12 m Greenish to dark red, tinged brown Produce fragrance 3.0–4.5 mm long Ovary inferior or half 6.0–7.0 cm 1.5–2.0 cm Simple, narrow to broadly lanceolate, shiny green

0–300 m Shrubs to small tree 8–10 m 40–50 m

S. yasi Fiji, Niue, Tonga

134 M. Nageswara Rao et al.

7 Santalum

135

Fernandez Islands) has been supported by congruent trees based on maximum parsimony, maximum likelihood, and Bayesian methods. The phylogenetic data also provide the best available evidence for plant dispersal out of the Hawaiian Islands to the Bonin Islands and eastern Polynesia (Harbaugh and Baldwin 2007). Phylogeographical analysis of S. album using chloroplast genes showed different haplotypes for the western Ghat population, when compared with the other two major geographic regions (eastern Ghat and Deccan plateau) from Peninsular India (Nageswara Rao 2004). The geographical difference and genetic structure in the study also appeared to be consistent with the presence of three major clusters (western Ghat, eastern Ghat, and Deccan plateau) differentiated across the sandal populations in Peninsular India (Nageswara Rao et al. 2007a; Fig. 7.3). Phylogeographical analysis of S. insulare, endemic to the islands of eastern Polynesia, using chloroplast microsatellite markers separated the populations sampled into three clusters, each cluster corresponding to one geological archipelago: Marquesas Islands, Society Islands, and Cook-Austral Archipelago (Butaud et al. 2005). The diversity and phylogeographic patterns within the chloroplast genome of S. spicatum were analyzed. The chloroplast diversity structured into two main clades that were geographically sepa-

Savandurga Siddarabetta Sakrebayalu Tavrekoppa Devarayanadurga Kasaragodu Haliyala Targodu Sonda Kalasa Mananthwadi Yellapura Kivara Yercadu Tirumala Metupalyam Chitoor Chitteri

rated, one centered in the southern (semi-arid region) and the other in the northern (arid) region in Australia suggesting a fragmentation due to climatic instability. The phylogeographic pattern in the chloroplast genome was congruent with that detected in the nuclear genome (Byrne et al. 2003b).

7.3 Conservation Initiatives Despite being a renewable plant resource, sandal populations in India, Indonesia, the South Pacific, and Australia are declining due to over-harvesting (Fig. 7.4) and illegal poaching of native stands, changes in landuse patterns, grazing, poor natural regeneration, and/or spike disease (Loneragan 1990; Rai and Sharma 1990; Srinivasan et al. 1992; Meera et al. 2000; Nageswara Rao et al. 2007a, b, 2008a). Since almost all of the extraction is from natural populations, the pressure on the existing populations has been tremendous (Radomiljac et al. 1998; Nageswara Rao et al. 2001a, b; Suma and Balasundaran 2003). This overexploitation has led to a steady decrease in the availability of S. album in India (Nageswara Rao 2004; Nageswara Rao et al. 2007a, 2008c), S. insulare in eastern Polynesia (Butaud 2004), S. spicatum in

D. Plateau

W.Ghat

E.Ghat 0

2000

4000

6000 Linkage Distance

8000

10000

Fig. 7.3 Dendrogram clustering of sandal populations in peninsular India (Nageswara Rao et al. 2007a)

12000

136

M. Nageswara Rao et al. 3500

y = 2E+24e-0.0246x R2 = 0.3912

3000

Quantity (tonnes)

2500

2000

1500

1000 500

1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

0

Fig. 7.4 Extent of quantity of sandalwood extracted in Karnataka, India between 1950 and 2004 (Nageswara Rao et al. 2007a, 2008c)

0.4 BRT

Observed Heterozygosity

S.durga

T.koppa

0.36

Targodu Sonda Haliyala

0.32

S.betta

Kivara

S.bayalu D.durga

Yellapura K.godu Kalasa

0.28

Yercadu M .wadi M .palyam

Tirumala

Chitteri

0.24 Chitoor 0.2 74

75

76

77 78 Longitude (Degree East)

79

80

Fig. 7.5 Relationship between observed heterozygosity with longitude in sandalwood populations across peninsular India (Nageswara Rao 2004; r ¼ 0.51; p < 0.05)

Australia (Byrne et al. 2003a, b), and S. austrocaledonicum in Vanuatu (Bottin et al. 2005). To safeguard the natural populations of sandal, effective measures need to be taken. Critical information on the distribution and status of sandal resources throughout India have been collected and used to develop a comprehensive distribution map, which would directly help the conservation efforts and would also serve as a bench mark to further monitor

the changes in the species landscape over time (Nageswara Rao et al. 2007a, 2008d). This distribution map will be of great importance in identifying the hot-spots of genetic diversity and will be helpful in formulating management plans to conserve the genetic resources of this species (Nageswara Rao et al. 2001b, 2002, 2007a; Nageswara Rao and Soneji 2009). This study also highlighted a strong negative correlation with increasing longitude for the sandalwood populations

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in Peninsular India (Fig. 7.5). The occurrence of six biotypes of sandal in India have been reported (Kulkarni and Srimathi 1982). Efforts have been made to establish ex situ conservation gardens for S. album at different sites in India and artificial regeneration of sandal in areas where the stock of sandal growth is poor has also been suggested (Srinivasan et al. 1992; Nageswara Rao et al. 2001b, 2008c). Three clonal banks, one each at Gottipura, Bangalore Division, Karvetinagar at Chittor Division and at Kurumbapatti at Salem, have been established. Besides the clonal orchards, there have been efforts to establish a germplasm bank at Gottipura, Bangalore Division (Srinivasan et al. 1992). Eight sandal-bearing areas have been recognized as potential provenances in India (Jain et al. 1998). Using advanced Geographic Information System ecological niche modeling (DIVA-GIS), the map for possible potential occurrence of sandal genetic resources in the Peninsular India have been developed (Nageswara Rao et al. 2008d). Studies have also highlighted the possible role of protected areas in conserving the sandal resources in India (Nageswara Rao et al. 1998, 2001b, 2007b, c; Ravikanth et al. 2009). For a long-term management and conservation of sandal, “Forest Gene Bank” model has also been proposed (Nageswara Rao et al. 1999, 2008b). Conservation programs have been implemented for critically endangered S. insulare (Butaud et al. 2005). Heavy harvesting, grazing, and fires coupled with disrupted gene flow and possibly genetic drift of S. lanceolatum from Victoria and New South Wales in the mid- to late-1800s has led to its being listed as a threatened taxon under Schedule II of the Victorian Flora and Fauna Guarantee Act 1988 (Warburton et al. 2000). The restriction and isolation of the remnant S. lanceolatum populations has led to the development of a conservation strategy for the species in Victoria (Johnson 1996). Establishment of new stands of S. lanceolatum has also been proposed as a conservation objective (Warburton et al. 2000). In situ and ex situ conservation strategies have been formulated in Pacific islands for the effective conservation of S. yasi (Padolina 2007). Seed collection efforts are being made by the Forestry Department of Vanuatu to establish a gene pool collection to conserve S. austrocaledonicum (Padolina 2007). A variety of Santalum plantings have been established to ensure conservation of genetic diversity for species, such as S. album, from a range of locations in India, Timor and

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Australia, S. austrocaledonicum from New Caledonia, and S. macgregorii from Papua New Guinea (Vernes 2001). S. macgregorii seed has also been distributed for ex situ conservation in Australia (Queensland and western Australia) and the Pacific (Thomson and Bosimbi 2000).

7.4 Genetic Diversity Though a number of efforts are being made to conserve sandal, the lack of basic information on the extent of natural genetic variability of sandal populations has been a major handicap in formulating conservation plans (Nageswara Rao 2004). Studying the genetic diversity of sandal could lead to the identification of “hot-spots” of genetic variability, which can then be targeted for their efficient conservation, sustainable utilization of genetic resources, and/or genetic improvement programs. A number of studies have been carried out to understand the genetic diversity of a few species of sandal. Genetic diversity in S. album has been studied using isozymes (Nageswara Rao et al. 1998, 2007a; Angadi et al. 2003), random amplification of polymorphic DNA (RAPDs; Shashidhara et al. 2003; Suma and Balasundaran 2003), and restriction fragment length polymorphism (RFLPs; Jones 2008). Using eight populations, Angadi et al. (2003) identified eight provenances of S. album indicating that the populations used were of separate varieties or races. Suma and Balasundaran (2003) reported low degree of variability within five provenances of S. album. This might have been due to fragmentation of a previously large population, resulting in loss of genetic variation, least amount of gene flow between the provenances and differentiation of population due to random drift. RAPDs were found to be effective in distinguishing 51 genotypes of S. album (Shashidhara et al. 2003). Nageswara Rao et al. (2007a, b) used 19 populations to understand the extent of diversity remaining within the natural populations of sandal occurring in Peninsular India and have been able to identify the “hot-spots” of genetic resources of S. album. RFLP analysis of 233 S. album genotypes also revealed low levels of genetic diversity (Jones 2008). This information will be of great use in genetic improvement programs.

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RFLP analysis of S. spicatum populations collected from semi-arid and arid regions of western Australia showed differences in population structure between these regions. Populations in the arid region showed higher levels of genetic diversity and a greater number of rare alleles than those in the semi-arid region. The degree of differentiation between the populations is related to the interaction and relative influences of both drift and gene flow (Byrne et al. 2003a). These regional differences are congruent with the presence of different chloroplast lineages (Byrne et al. 2003b) and consistent with the identification of arid and semiarid ecotypes based on morphological variation (Fox and Brand 1993). Allozymes and RAPDs were used to determine the extent of clonality in remnant populations of S. lanceolatum, which is represented by only one small population of unique clones in Victoria, southeastern Australia. Both allozymes and RAPDs detected no variation within populations, suggesting that each population consisted of a single genet. The study also indicated that each of the five remnant S. lanceolatum populations existed as a single unique clone, recruiting individuals only by vegetative reproduction reflecting on the history of disturbance and fragmentation of the populations due to harvesting, clearing, grazing, and fires coupled with disrupted gene flow and possibly genetic drift. These populations also showed little or no fruit production due to pollen sterility, pollen-pistil incompatibility or pistil dysfunction (Warburton et al. 2000). Chloroplast microsatellite markers have been used to analyze 11 populations of S. insulare collected from islands of eastern Polynesia where it is an endemic. The gene flow between populations was found to be low. The gene diversity index varied among the archipelagoes but was high for the total population. Genetic structure was characterized by high levels of differentiation between archipelagoes (36% of total variation) and between islands, but differentiation between islands varied according to the archipelago (Butaud et al. 2005). Another study was carried out on the same populations of S. insulare using nuclear microsatellites. This study indicated that clonality was a frequent phenomenon in S. insulare. As a consequence of insularity, the genetic diversity within populations was also lower than the values assessed in species distributed on the mainland. This may also be due to over-exploitation of S. insulare (Lhuiller et al. 2006). S. austrocaledonicum is another insular

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tree which is confined to the archipelago of New Caledonia. Microsatellite analysis of 17 populations of S. austrocaledonicum indicated that the genetic diversity was lowest in the most recent islands when compared with the oldest ones. At both population and island levels, island age and isolation seemed to be the main factors influencing the amount of genetic diversity suggesting a strong influence of insularity on the genetic diversity and structure of S. austrocaledonicum.

7.5 Role in Crop Improvement Through Traditional and Advanced Tools Sandal is a pre-dominantly outbreeding species though its flower structure was designed for self-pollination (Sindhu Veerendra and Ananthapadmanabha 1996). Apomixis and/or parthenocarpy have not been noticed in sandal while bees, butterflies, and beetles are the pollinating agents. Although sandal is being utilized for centuries, hardly any studies have been carried out for its genetic improvement. The only tree improvement work being carried out is the selection of superior phenotypes from naturally occurring populations. The species also has a complex ecology, which has hampered field trails. Heritability studies for desirable traits such as tree form, health, early heartwood onset, high heartwood content, and essential oil yield have not been conducted extensively (Jones 2008). Studies conducted on genetic diversity will provide the researchers with information on the level of variation within and between sandal populations and help them in assessing its potential for genetic improvement. For example, RFLP analysis in S. spicatum populations collected from semi-arid and arid regions of western Australia showed polymorphisms, which may represent genotypic variation in gene structure and function that could in turn affect important phenotypic traits such as form, growth rate, and heartwood oil production (Byrne et al. 2003a, b). Clonal reproduction of S. lanceolatum has been used to advantage, as regrowth from pulled trees appears to result in healthier root suckers (Bristow et al. 2000). Conventional breeding of sandal for introgression of new genetic information can be an expensive and difficult task because of their long generation time, sexual incompatibility, and heterozygous nature (Rugkhla 1997). Also traditional crossing and selection trials would take at least

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40–50 years to evaluate, provided a suitable phenotype screen, which takes into account the environmental variability, is established. However, if genetically superior seedlings could be identified at the nursery stage, or even propagated in vitro, sandal could be planted with certainty of better long-term returns (Jones and Plummer 2008). Seeds are mainly used for natural regeneration and artificial propagation, even though the seedlings of sandal are extremely heterozygous due to outcrossing (Sanjaya et al. 2006). Dibbling of seeds in bushes, sowing of seeds on the moulds, planting of container-raised seedlings (Rai and Sharma 1986), and vegetative propagation through grafting, air layering, and root suckers (Rao and Srimati 1977) are the other artificial methods of sandal propagation. However, the production of clones is time consuming (Srimati et al. 1995). Sandal is also recalcitrant to in vivo and in vitro propagation (Sanjaya et al. 2003), though tissue culture has been used to achieve high-frequency regeneration in vitro. Various explants such as hypocotyl, endosperm, nodal, and internodal segments, protoplasts, zygotic embryo, leaves, and nodal stem segments with dormant axillary buds have been used (Bapat and Rao 1978; Lakshmi Sita et al. 1980; Bapat and Rao 1984, 1985; Rao and Bapat 1992; Rugkhla and Jones 1998; Rai and McComb 2002; Mujib 2005; Sanjaya et al. 2006). Early attempts to develop somatic embryogenesis for sandal propagation focused on indirect embryogenesis (Rao and Rangaswamy 1971; Lakshmi Sita et al. 1979, 1980; Rai 2005). However, when somatic embryos were obtained through callus, the conversion or germination of these embryos into plantlets was problematic (Rao and Bapat 1995). Rai (1996) and Rai and McComb (2002) reported the regeneration of sandal plantlets from somatic embryos developed from mature zygotic embryos of sandal through direct somatic embryogenesis. Direct somatic embryogenesis from nodal and seed explants has also been reported (Rugkhla and Jones 1998). Sandal plants have also been successfully micropropagated by in vivo methods using mature plants (Lakshmi Sita et al. 1982). Somatic embryos have been used to develop synthetic seeds in sandal (Bapat and Rao 1988), which could be germinated in vitro as well as in vivo (Fernandes et al. 1992). The synthetic seeds could also be germinated after storage at 4
C for 45 days (Bapat and Rao 1988). In vitro regeneration techniques can be used to clone

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superior lines and are needed for Agrobacteriummediated gene transfer techniques and protoplast fusion (Rugkhla and Jones 1998). However, the bottleneck is in vitro rooting, which limits the widespread application of micropropagation techniques in sandal (Sanjaya et al. 2006). As high efficiency in vitro regeneration systems are available for sandal, application of transgenic technology may offer a faster route for its genetic improvement. To date, there are only two reports of transformation of sandal. The first study reported a system for the introduction and expression of foreign genes in torpedo-cotyledonary stage embryos of sandal (Shiri and Rao 1998). These somatic embryos were inoculated by pricking and cells of disarmed Agrobacterium strains carrying b-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) genes on the binary vectors pKIWI105, pBI121, and pIG121-Hm were directed at the wound sites. Transgenic plants were regenerated from embryogenic cultures derived from these transformed somatic embryos, and the transgenic nature was confirmed by gus and nptII assays as well as polymerase chain reaction (PCR) with insert-specific primers. More recently, an efficient method for the transformation of embryogenic cell suspension cultures of sandal has been described (Shekhawat et al. 2008). Embryogenic cell suspension cultures were transformed with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 containing a gusA gene interrupted by a modified castor bean catalase intron and an hptII gene conferring resistance to hygromycin. Plantlets were regenerated from the transformed embryogenic cells. Expression of b-glucuronidase in the suspension cultures was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and gus histochemical staining and stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. These reports have opened new vistas for the transformation of sandal with gene(s) resistant to various sandal diseases and for metabolic engineering to increase and/or modify the essential oil yield.

7.6 Genomics Resources Developed As yet, no attempts have been made to map or sequence the sandal genome or transcriptome.

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However, attempts have been made to isolate and clone a proline-rich protein (PRP) cDNA from leaves of S. album (Bhattacharya and Lakshmi Sita 1998, 1999). PRPs are regarded as reactive biopolymers which are involved in maintenance of cell wall integrity and may be involved in systemic resistance to protoplasma-like pathogens. Genomic library was constructed using nuclear DNA prepared from tender leaves of S. album and screened using heterologous probes to isolate the PR1 genomic homolog. The coding region for PR1 gene was obtained by restriction mapping and hybridization (Bhattacharya and Lakshmi Sita 1999). To identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, degenerate TPS primers used amplified two genomic TPS fragments, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1,731 bp), and SasesquiTPS1 (1,680 bp) from leaf and wood tissues (Jones et al. 2008). The terpenoids produced by SamonoTPS1 and SasesquiTPS1 were not found in substantial quantities in the distilled oil of sandal, suggesting that additional TPS genes are expressed in S. album, which may contribute to the majority of heartwood essential oil. The gene structure and expression properties of TPS may be exploited through modification or selection of specific genotypes conducive to high essential oil production. The genes responsible for the production of essential oil in S. album, which is considered unique and is preferred in the preparation of various perfumes, flavors, cosmetics, etc., when identified and isolated will be of great economic importance. The gene can not only be introduced in other species of sandal but also in faster growing annual plants to provide quicker and easier route to obtaining the fragrant essential oil. Isolation and characterization of microsatellite loci in S. austrocaledonicum has been carried out (Bottin et al. 2005).

7.7 Scope for Domestication and Commercialization Heartwood and oil of sandal tree are of great commercial importance and extensively used in perfumes and medicine. The heartwood, being closely grained and amenable to carving, is one of the finest woods for making idols, boxes, and other curios of exquisite beauty. It is also used as an astringent, bitter, antipyretic, and a cooling agent. The bark when powdered is

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an important raw material for the manufacture of incense sticks. The bark extract has been reported to behave as a chemosterilant and as an insect growth inhibitor (Shankaranarayana et al. 1979). The oil is considered to be unique and used for the preparation of top-class and sophisticated scents, perfumes, flavors, cosmetics, toiletries, beauty aids, and herbal medicine (Srinivasan et al. 1992). The seeds are used for treating diuretic, hypotensive, antitumorogenic, antiviral activities, and a number of skin diseases (Kirthikar and Basu 1987; Desai and Shankaranarayana 1990). With the dwindling of natural stands of sandal, attempts have been made to replant sandal. The last 10 years have seen an increase in the establishment of tree farms of S. album and S. spicatum in Australia. Around 1,750 ha of S. album plantations have been developed on irrigated land in Ord River Irrigation Area (ORIA) in northern western Australia by managed investment scheme (MIS) companies, while the area of tree farms of S. spicatum have steadily increased to over 10,000 ha (www.tfsltd.com.au; http://www.fpc.wa.gov.au/pdfs/ industry_plans/sandalwood_idp.pdf). It is anticipated that at the current rate of planting the tree farm estate is likely to reach 50,000 ha by the year 2020. No other country has embarked on a significant program of sandal establishment. The seemingly simple process of growing seedlings, planting them in the tree farm, and maintaining trees until harvest has been very difficult. Silviculture of sandal tree farms is complex as all species of sandal are root parasites (Radomiljac et al. 1998; Brand 2002; Woodall and Robinson 2002a, b). Both S. album and S. spicatum required a short-term pot host to be grown alongside and at the time of planting into the tree farm, a medium-term host must be well established nearby and a suitable long-term host must eventually be present. It is believed that the wood with finest odor is obtained from the driest region particularly on red or stony ground (Gunther 1952) and that yield of oil will be much higher than those grown in fertile tracts (Singh 1911; Gildemeister and Hoffman 1928; Bhatnagar 1965). It has been observed that trees extracted from open fields or edges of plantation yield more heartwood than those of comparative size extracted from adjoining forests (Wilson 1915; Mitchell 1941). The hosts also seem to influence the heartwood formation besides the growth and development (Rama Rao 1911). Srimathi and Kulkarni (1980) were of the view that heartwood formation is dependent on general factors of the individual tree and

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phenotypic factors play only a secondary role. Age of sandal tree and color of heartwood influences the quality and content of sandal oil (Shankaranarayana and Venkatesan 1982; Shankaranarayana and Parthasarathi 1984). Heartwood from young trees (around 10 years of age) contains 0.2–2% of oil and that from the mature trees (around 30–50 years of age) contains 2.8–5.6% of oil. Sandal oil content markedly decreases along the length of the tree (root: 3.5–6.3% to tip; stem: 3–5%; and branches: 1–3%). Generally, there is a decrease of about 45% in oil content from root to tip and about 20% from core to periphery (Shankaranarayana and Parthasarathi 1987). It would be beneficial if the plantation industry takes into consideration these observations for obtaining a better stand of sandal population.

7.8 Conclusion Natural stands of sandal are found in Australia, Hawaii, India, eastern Indonesia, New Caledonia, and Vanuatu. Phylogeographical analysis in sandal relied on interpreting the patterns of congruence or lack of congruence between the geographical distribution of chlorotypes and their genealogical relationships. Natural sandal populations are declining due to over-harvesting and illegal poaching of native stands, changes in land-use patterns, grazing, poor natural regeneration, and/or spike disease (Nageswara Rao et al. 2007a, 2008c). To safeguard the natural populations of sandal, effective measures need to be taken. Critical information on the distribution and status of sandal resources has been collected and used to develop a comprehensive distribution map. Genetic diversity of sandal has also been mapped. Such information would directly help the conservation efforts and would also serve as a bench mark to further monitor the changes in the species landscape over time (Nageswara Rao et al. 2008d). Efforts are underway for in situ and ex situ conservation of sandal. For commercial propagation of sandal, tree farms are being established. With the plantation sandalwood industry growing rapidly in Asia and Australia, there is a substantial need for well-characterized germplasm. Knowledge and utilization of the genes involved in essential oil production may advance efforts toward sandalwood tree improvement, further development of sustainable sandalwood plantations, and thereby conservation efforts to protect

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sandalwood trees in natural forests. The establishment of high efficiency tissue culture regeneration systems will facilitate the application of transgenic technology for the genetic improvement of sandal. To identify the genes of economic importance in sandal, a genetic mapping and sequencing project needs to be initiated.

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M. Nageswara Rao et al. Johnson G (1996) Northern sandalwood, Santalum lanceolatum, flora and fauna guarantee, action statement No. 75. Department of Natural Resources and Environment, Melbourne, Australia, p 8 Jones CG (2008) The best of Santalum album: essential oil composition, biosynthesis and genetic diversity in the Australian tropical sandalwood collection. PhD thesis, Univ of Western Australia, Australia Jones CG, Keeling CI, Ghisalberti EL, Barbour EL, Plummer JA, Bohlmann J (2008) Isolation of cDNAs and functional characterization of two multi-product terpene synthase enzymes from sandalwood, Santalum album L. Arch Biochem Biophys 477:121–130 Jones CG, Plummer JA (2008) Sandalwood. In: Kole C, Hall TC (eds) A compendium of transgenic crop plants, vol 9, Transgenic forest tree species. Wiley-Blackwell, Chichester, UK, pp 309–320 Kirthikar KR, Basu BD (1987) Indian medicinal plants. International Book Distributors, Dehra Dun, India Kulkarni HD, Srimathi RA (1982) Variation in foliar characteristics in sandal. In: Khosla PK (ed) Biometric analysis in improvement of forest biomass. International Book Distributors, Dehra Dun, India, pp 63–69 Kulkarni RS, Fakrudin B, Shashidhar KS (1998) Tree improvement efforts in sandal: the need to employ novel strategies. In: Radomiljac AM, Aanathapadmanabha HS, Welbourn RM, Satyanarayana Rao K (eds) Sandal and its products. ACIAR, Canberra, Australia, pp 151–153 Lakshmi Sita G, Raghava Ram NV, Vaidyanathan CS (1980) Triploid plants from endosperm culture of sandalwood by experimental embryogenesis. Plant Sci Lett 20:63–69 Lakshmi Sita G, Ram RNV, Vaidyanathan CS (1979) Differentiation of embryoids and plantlets from shoot callus of sandalwood. Plant Sci Lett 15:265–270 Lakshmi Sita G, Vaidyanathan CS, Ramakrishnan T (1982) Applied aspects of plant tissue culture with special reference to the improvement. Curr Sci 51:88–92 Lhuiller E, Butaud JF, Bouvet JM (2006) Extensive clonality and strong differentiation in the insular Pacific tree Santalum insulare: implications for its conservation. Ann Bot 98: 1061–1072 Loneragan OW (1990) Historical review of Sandalwood (Santalum spicatum): research in Western Australia. Research Bulletin No. 4. Department of Conservation and Land Management, Perth, Australia Mc Kinnell FH (1990) Status of management and silviculture research on sandalwood in eastern Australia and Indonesia. In: Hamilton L, Conrad CE (eds) Proceedings of the Symposium in the Pacific. Honolulu, Hawaii. USDA For Serv Gen Tech Rep PSW USA, pp 19–25 Meera C, Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R, Swaminath MH (2000) Conservation of sandal genetic resources in India: I Extraction patterns and threats to sandal resources in Karnataka. My Forest 36:125–132 Merlin MD, Thomson LAJ, Elevitch CR (2006) Santalum ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum (Hawaiian sandalwood), ver. 4.1. In: Elevitch CR (ed) Specific profiles for Pacific Island agroforestry (www.traditionaltree.org). Permanent Agriculture Resources, Hawaii, USA, pp 1–21

7 Santalum Mitchell JFM (1941) Sandalwood problems, factors affecting heartwood and oil content in sandalwood. Proceedings of the fifth silviculture conference, Dehra Dun, India Mujib A (2005) In vitro regeneration of Sandal (Santalum album L.) from leaves. Turk J Bot 29:63–67 Nageswara Rao M (2004) Mapping genetic diversity of Sandal (Santalum album L.) genetic resources in peninsular India using biochemical and molecular markers: lessons for in-situ conservation. PhD thesis, Forest Research Institute (FRI), ICFRE, Dehra Dun, India Nageswara Rao M, Anuradha M, Deepali Singh BS, Uma Shaanker R, Ganeshaiah KN (1998) Sandal genetic resources at B.R.Hills: how safe are they? In: Interaction meeting on biodiversity and conservation in B. R. Hills, Sep 17–18, 1998. Institute of Wood Science and Technology, Bangalore, India Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R (2001a) Mapping genetic diversity of sandal (Santalum album L.) in south India: lessons for in-situ conservation of sandal genetic resources. In: Uma Shaanker R, Ganeshaiah KN, Bawa KS (eds) Forest genetic resources: status, threats and conservation strategies. Oxford and IBH Publ, New Delhi, India, pp 49–67 Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R (2007a) Assessing threats and mapping sandal (Santalum album L.) resources in peninsular India: identification of genetic hotspot for in-situ conservation. Conserv Genet 8:925–935 Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R (2008a) Fading fragrance? Deccan Herald, Science and Technology, Bangalore, India Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R (2008b) Mapping and conserving the genetic diversity of medicinal plant species through the establishment of Forest Gene Bank Models. The Science Advisory Board http://www.scienceboard.org/community/perspectives.208.html Nageswara Rao M, Padmini S, Ganeshaiah KN, Uma Shaanker R (1999) Sandal genetic resources of South India: threats and conservation approaches. In: National symposium on role of plant tissue culture in biodiversity, conservation and economic development. Kosi-Katarmal, Almora, UP, India, p 63 Nageswara Rao M, Padmini S, Ganeshaiah KN, Uma Shaanker R, Soneji JR (2008c) Indian sandalwood crisis. Perfum Flavor 33(10):38–43 Nageswara Rao M, Ravikanth G, Ganeshaiah KN, Uma Shaanker R (2007b) Impacts of human disturbances on genetic diversity of NTFP species. In: Plant and animal genomes XV conference, San Diego, California, USA Nageswara Rao M, Ravikanth G, Ramesha BT, Ganeshaiah KN, Uma Shaanker R (2007c) The role of protected areas in conserving the genetic diversity of forest species. In: Plant and Animal Genomes XV conference, San Diego, California, USA Nageswara Rao M, Ravikanth G, Ganeshaiah KN, Rathore TS, Uma Shaanker R (2008d) Assessing threats and identifying the ecological niche of sandal resources to identify ‘hotspots’ for in-situ conservation in southern India. In: Gairola S, Rathore TS, Joshi G, Arun Kumar AN, Aggarwal PK (eds) Proceeding of national seminar on conservation, improvement, cultivation and management of sandal. Institute of Wood Science and Technology, Bangalore, India, pp 23–31 Nageswara Rao M, Soneji JR (2009) Threats to forest genetic resources and their conservation strategies. In: Aronoff JB

143 (ed) Handbook of nature conservation: global, environmental and economic issues. Nova Publ, USA, pp 119–146 Nageswara Rao M, Uma Shaanker R, Ganeshaiah KN (2001b) Protected areas as refugias for genetic resources: are sandal genetic resources safe in our sanctuaries? In: Ganeshaiah KN, Uma Shaanker R, Bawa KS (eds) Tropical ecosystems: structure, diversity, and human welfare. Science Publ, Enfield, NH, USA, pp 121–124 Nageswara Rao M, Uma Shaanker R, Ganeshaiah KN (2002) Mapping the reek. Down to Earth 10:18 Padolina C (2007) An overview of forest genetic resource conservation and management in the Pacific. Acta Hortic 757:37–42 Radomiljac AM, Anathapadmanabha HS, Welbourn RM, Satyanarayana Rao K (1998) The effect of sandal wood availability on the craftsman community. In: Sandal and its products. ACIAR Proc (84), Publication-Australian Centre for International Agricultural Research, Canberra, Australia, p 204 Rai R (2005) Somatic embryogenesis in sandalwood. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, Dordrecht, Netherlands, pp 497–504 Rai SN (1990) Status and cultivation of sandalwood in India. In: Hamilton L, Conrad CE (eds) Proceedings of the symposium on sandalwood in the Pacific. Honolulu, Hawaii. USDA For Serv Gen Tech Rep PSW USA, pp 66–71 Rai SN, Sharma CR (1986) Relationship between height and diameter increment of sandal (Santalum album L.). Van Vigyan 24:105–138 Rai SN, Sharma CR (1990) Depleting sandalwood production and rising prices. Indian Forest 116:348–355 Rai VR (1996) Direct somatic embryogenesis from mature embryos of sandalwood. Sandalwood Res Newslett 5:4 Rai VR, McComb J (2002) Direct somatic embryogenesis from mature embryos of sandalwood. Plant Cell Tissue Organ Cult 69:65–70 Rama Rao M (1911) Host plants of the sandal tree. Indian Forest Rec 2(4):159–207 Rao PS, Bapat VA (1992) Micropropagation of Sandalwood (Santalum album L.). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, high-tech and micropropagation II, vol 18. Springer, Heidelberg, Germany, pp 193–210 Rao PS, Bapat VA (1995) Somatic embryogenesis in sandalwood Santalum album L. In: Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants. Klewer Academic Publ, Dordrecht, Netherlands, pp 153–170 Rao PS, Rangaswamy NS (1971) Morphogenic studies in tissue culture of parasite Santalum album L. Biol Plant 13:200–206 Rao PS, Srimati RA (1977) Vegetative propagation of Sandal (Santalum album L.). Curr Sci 46:276 Ravikanth G, Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R (2009) Impacts of harvesting on genetic diversity of NTFP species: implications for conservation. In: Uma Shaanker R, Joseph GC, Hiremath AJ (eds) Management, utilization, and conservation of non-timber forest products in the south asia region. Universities Press, Bangalore, India, pp 53–63 Roxburgh W (1820) Santalum. In: Carey W (ed) Flora indica, vol I. Mission, Calcutta, India, pp 442–445 Rugkhla A (1997) Intra-specific and inter-specific hybridisation between Santalum spicatum and S. album. PhD thesis, Murdoch Univ, Perth, Western Australia

144 Rugkhla A, Jones MGK (1998) Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum. J Exp Bot 49:563–571 Sanjaya BM, Anathapadmanabha HS, Rai VR (2003) In vitro and in vivo micrografting of Santalum album shoot tips. J Trop Forest Sci 15:234–236 Sanjaya MB, Rathore TS, Rai VR (2006) Micropropagation of an endangered Indian sandalwood (Santalum album L.). J Forest Res 11:203–209 Scott J (1871) Notes of horticulture in Bengal. No 2, Loranthaceae, the mistletoe order, their germination and mode of attachment. J R Hort Soc 2:287 Shankaranarayana KH, Parthasarathi K (1984) Compositional differences in sandal oils from young and mature trees and in oils undergoing color change on standing. Indian Perfum 28: 138–141 Shankaranarayana KH, Parthasarathi K (1987) On the content and composition of oil from heartwood at different levels in sandal. Indian Perfum 31:211–214 Shankaranarayana KH, Shivaramakrishnan VR, Ayyar KS, Sen PK (1979) Isolation of a compound from the bark of sandal and it’s activity against some lipidopterous and coleopterous insects. J Entomol Res 3:116–118 Shankaranarayana KH, Venkatesan KR (1982) Chemical aspects of sandalwood oil in cultivation and utilization of aromatic plants. Atakal CK, Kapoor RPL (CSIR), Jammu, India, pp 406–411 Shashidhara G, Hema MV, Koshy B, Farooqi AA (2003) Assessment of genetic diversity and identification of core collection in sandalwood germplasm using RAPDs. J Hort Sci Biotechnol 78:528–536 Shekhawat UKS, Ganapathi TR, Srinivas L, Bapat VA, Rathore TS (2008) Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. Plant Cell Tissue Organ Cult 92:261–271 Shetty RH (1977) Is sandal exotic? Indian Forest 811:804 Shiri V, Rao KS (1998) Introduction and expression of marker genes in sandal wood (Santalum album L.) following Agrobacterium-mediated transformation. Plant Sci 131:53–63 Sindhu Veerendra HC, Ananthapadmanabha HS (1996) The breeding system in sandal (Santalum album L.). Silvae Genet 45(4):188–190 Singh P (1911) Memorandum on the oil value of sandalwoods from Madras. Forest Bull No 6. FRI and Colleges, Dehra Dun, India Skottsberg C (1930a) The geographical distribution of the sandalwoods and its significance. Proc fourth Pacific Sci Congress, Java, Indonesia 3:435–440 Skottsberg C (1930b) Further notes on Pacific sandalwoods. Meddelanden fran Go¨teborgs Botaniska Tra¨dgard 5:135–145 Sprague TA, Summerhayes VS (1927) Santalum, Eucarya, and Mida. Bull Miscellaneous Information Royal Botanic Gardens, Kew 5:193–202 Srimathi RA, Kulkarni HD (1980) Preliminary findings on the heartwood formation in sandal (Santalum album L.). Report of the Sandal Research Centre, Bangalore, India, p 5 Srimathi RA, Venkateshan KR, Kulkarni HD (1995) Guidelines for selection and establishment of seed stands, seed production areas, plus trees and clonal seed orchards for sandal (Santalum album L.). In: Srimathi RA, Venkateshan KR,

M. Nageswara Rao et al. Kulkarni HD (eds) Recent advances in research and management of sandal (Santalum album L.) in India. Associated Press, New Delhi, India, pp 281–299 Srinivasan VV, Shivaramakrishnana VR, Rangaswamy CR, Anathapadmanabha HS, Shankaranarayan KH (1992) Sandal. Indian Council of Forestry Research and Education, Dehra Dun, India St. John H (1947) The history, present distribution, and abundance of sandalwood on O‘ahu, Hawaiian Islands: Hawaiian plant studies 14. Pacific Sci 1:5–20 Stemmermann RL (1977) Studies of the vegetative anatomy of the Hawaiian representative of Santalum (Santalaceae), and observations of the genus Santalum in Hawaii. Master’s Thesis, Univ of Hawaii, USA Stemmermann RL (1980) Observations on the genus Santalum (Santalaceae) in Hawai‘i. Pacific Sci 34:41–53 Suma TB, Balasundaran M (2003) Isozyme variation in five provenances of Santalum album in India. Aust J Bot 51: 243–249 Suriamihardja H, Suriamihardja S (1993) Sandalwood in Nusa Tenggara Timur. In: McKinnell FH (ed) Sandalwood in the Pacific Region. ACIAR, Honolulu, HI, USA, pp 39–43 Thirawat S (1955) Spike disease of sandal. Indian Forest 81:804 Thomson L, Bosimbi D (2000) Santalum macgregorii – PNG sandalwood. CSIRO/PNG Forest Research Institute/ACIAR project; Domestication of Papua New Guinea’s Indigenous Forest Species. Australian Tree Seed Centre, CSIRO Forestry and Forest Products, Yarralumla, ACT, Australia Thomson LAJ (2006) Santalum austrocaledonicum and S. yasi (sandalwood), ver. 2.1. In: Elevitch CR (ed) Specific profiles for Pacific Island agroforestry (www.traditionaltree.org). Permanent Agriculture Resources, Hawaii, USA, pp 1–21 Tuyama T (1939) On Santalum boninense, and the distribution of the species of Santalum. J Jap Bot 15:697–712 Uma Shaanker R, Ganeshaiah KN, Nageswara Rao M (2000) Conservation of sandal genetic resources in India: problems and prospects. In: International conference on science and technology for managing plant genetic diversity in the 21st Century, Kuala Lumpur, Malaysia, p 73 Vernes T (2001) Preliminary results from Santalum macgregorii ex situ conservation planting. Sandalwood Res Newslett 13: 6–7 Wagner WL, Herbst DR, Sohmer SH (1999) Santalum. In: The manual of the flowering plants of Hawaii, vol 2. Univ of Hawaii Press, Honolulu, Hawaii, USA, pp 1218–1222 Warburton CL, James EA, Fripp YJ, Trueman SJ, Wallace HM (2000) Clonality and sexual reproductive failure in remnant populations of Santalum lanceolatum (Santalaceae). Biol Conserv 96:45–54 Wilson CC (1915) Sandalwood (a) a parasite, (b) susceptibility to fire, (c) damage by borers, (d) spike disease. Science 188:1018–1021 Woodall GS, Robinson CJ (2002a) Direct seedling Acacias of different form and function as hosts for sandalwood (Santalum spicatum). Conserv Sci W Aust 4:130–134 Woodall GS, Robinson CJ (2002b) Same day plantation establishment of the root hemiparasite sandalwood [Santalum spicatum (R.Br.) A D.C.:Santalaceae] and its hosts. J Roy Soc W Aust 85:37–42

Chapter 8

Trigonobalanus Weibang Sun, Yuan Zhou, Chunyan Han, Gao Chen, and Yanling Zheng

8.1 Introduction The broadly circumscribed genus Trigonobalanus includes three species: T. verticillata from Sulawesi, Borneo, and the Malaya Peninsula, T. excelsa from the tropical forests of Colombia, South America, and T. doichangensis distributed in southern China and northern Thailand (Hsu et al. 1981; Nixon and Crepet 1989). Alternatively, on the basis of the unique pollen (peroblate shape and presence of endoaperature) of T. doichangensis, whorled phyllotaxy with interpetiolar stipules in T. verticillata and lack of bud scales in T. excelsa, Nixon and Crepet (1989) proposed dividing the genus into three monotypic genera: Formanodendron, Trigonobalanus, and Colombobalanus, respectively. At the time of their study, chromosome numbers (2n ¼ 42, 40, and 44) were known for T. verticillata (Hou 1971). Chen et al. (2007) determined that T. doichangensis has a chromosome number of 2n ¼ 14 and concluded that the base chromosome number of Trigonobalanus is x ¼ 7. Although recognizing the variability within the genus, Chinese experts prefer the broader systematic delineation of Trigonobalanus (Sun et al. 2004, 2006, 2007). Based on morphology and biogeography, Wu et al. (2003) proposed Fagaceae to constitute four subfamilies – Castaneoideae, Trigonobalanoideae, Fagoideae, and Quercoideae (Jones 1986; Takhtajan 1997; Li 1999). Trigonobalanus has been considered a key to an understanding of the phylogeny and biogeography of Fagaceae. The genus shares with both Fagus and

Quercus morphologies that are considered ancestral (Forman 1964; Crepet and Nixon 1989; Nixon and Crepet 1989) and has an extensive fossil record (Manos and Stanford 2001). Molecular studies have placed Trigonobalanus basal in a clade that also includes Quercus, Chrysolepis, and Castanea and have resolved this clade as sister to Fagus (Manos and Steele 1997). Biogeographical studies indicate that intercontinental variance of Trigonobalanus occurred earlier than in both Quercus and Fagus and involved dispersal across the North Atlantic Land Bridges (Manos and Steele 1997). Among the three extant species, T. doichangensis was widely recognized that it is the only one distributed in China (Hsu et al. 1981). However, recent investigations have confirmed that T. verticillata is distributed in Hainan Province (Ng and Lin 2008). Thus, China hosts two of the extant species of the genus Trigonobalanus. T. doichangensis was placed on the national Rare and Endangered Species List of China in 1984 because of its limited distribution and the destruction of its habitat within China (Fu 1992; Sun et al. 2004) and it is also China’s second-ranked taxon for priority of national protection (Anon 1999) because of its endangered status and its scientific value in providing evidence on the phylogeny and phytogeography of the Fagaceae and the biogeography of the Chinese flora. For conserving T. doichangensis, a scientifically and ecologically important plant species in Fagaceae, we have carried out comprehensive studies since 2000, and this chapter is mainly a summary of our work on T. doichangensis.

W. Sun (*) Kunming Botanic Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China e-mail: [email protected]

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_8, # Springer-Verlag Berlin Heidelberg 2011

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8.2 Geographical Distribution and Population Ecology of T. doichangensis T. doichangensis is a national rare and endangered plant of China (Fu 1992). It is restricted to four sites in southwest Yunnan, China and one site in ChiangRai, northern Thailand (Sun et al. 2006; Fig. 8.1). Our investigations revealed that four community types are currently extant in Yunnan: isolated individuals, sprouting woods, mono-dominant forest (MDF), and co-dominant forest (CDF) (Sun et al. 2004). The habitats have been severely damaged and the populations there are facing a high risk of extinction according to sun’s research (Sun et al. 2006). The adult phase of T. doichangensis is reached when a tree attains a height of about 4 m. The flowering and fruiting time varies slightly among populations and/or across the micro-habitats (Sun et al. 2004). In comparison with other Fagaceous plants in the community, T. doichangensis has an inverse flowering and fruiting period from October to May (Li 1994). At present, the vegetation destruction caused by agricultural land expansion and cuttings of the species for fuelwood are forcing endangering its habitats. The alien species Ageratina adenophra, Chromolaena odorata, and

Fig. 8.1 Geographical distribution of the five extant populations of T. doichangensis (after Sun et al. 2007). CR Chiang Rai population, ML Menglian population, LC Lancang population, XM Ximeng population, CY Cangyuan population

W. Sun et al.

Tithonia diversifolia have already invaded into communities in shrubbery layer, and they are inhibiting the species regeneration (Sun et al. 2006). Except this, according to the result of wild survey on the T. doichangensis community, Li (1994) indicated the floristic elements of the community are tropical and subtropical floristic region (90.25% of the total species). Sun et al. (2006) further indicated the population of T. doichangensis showed different characteristics in their tree-age structure and floristic composition. Four community types were recognized during their investigation (Table 8.1): (1) Type IsI (isolated individuals): Individuals of T. doichangensis were often found in secondary woods, by roadsides, in mixed woods or farmland, and occasionally within the evergreen broadleaf forests. The formation of Type IsI is due to heavy cutting and vegetation destruction. (2) Type SW (sprouting woods): Type SW is the result of fuelwood cutting by indigenous people. Investigations show that the indigenous ethnic groups of Dai, Wa, and Laku are familiar with T. doichangensis and have realized that the tree can sprout easily after top-cutting and thinning and thus they have adopted the methods of “alternate cutting or thinning cutting” for the primitive sustainable use of the tree as fuelwood. As a result of these practices, plants of T. doichangensis in these woods showed some unique characteristics in tree

8 Trigonobalanus

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Table 8.1 Population characteristics of T. doichangensis in different communities (cited from Sun et al. 2006) Communities SW MDF CDF Localities

Menglian

Lancang

Plots

1,020 N, 30 >90 800 195 24 5.1 13.5/0.2

1,450 W, 40 >90 400 140 35 5.6 9.5/0.3

59 15 26

73 21 6

45 ‚ƒ HAD

30 ‚ƒ PR

Altitude (m) Slope Coverage (%) Size (m2) TD no. in the plot TD no. per 100 m2 TD Avg. height (m) Hst/Shst of TD (m) % of TD  20 m % of TD  4–20 m % of TD 2–3 m % of TD  1.0 m % of dead TD Avg. height of DP (m) Accompanier no. Main companions (TP) Evaluation

Cangyuan BTS 1,550 SE, 40 >95 3,000 41 2 17.9 35/0.4 51 44 5

25 „… RSS

RBS 1,590 S, 30 >95 600 101 17 6.8 32/0.28 6 55 23 16 17 1.5–10 27 „… USD

MDS 1,730 S, 30 >95 400 118 30 14.4 20/0.2 0.8 92 5 3 15 3.5–18 25 „… MDSD

SW Sprouting wood, MDF Mono-dominant forest, CDF Co-dominant forest, HTS Huge-tree structure, RBS Relatively balance structure, MDS Mono-dominant structure, TD Trigonobalanus doichangensis, Hst Highest, Shst Shortest, Avg. Average, DP dead plants, TP top layer,  Vaccinium bracteatum, ‚ Castanopsis echinocarpa, ƒ Castanopsis hytrix, „ Castanopsis calathiformis, … Schima wallichii, HAD heavily disturbed by human activities, PR Population in recovery, RSS relatively stable structure, USD unstable structure in developing, MDSD mono-dominant structure in developing

shape, tree height structure, and associated floristic composition. In this community, the tallest T. doichangensis was about 13 m and the average height was around 5 m. Some 60% of T. doichangensis reached the reproductive phase and about 25.6% of the individuals were below 1 m in height. T. doichangensis was the dominant species in woods, and 50 species of accompanying higher plants were present. (3) Type MDF: Plants of T. doichangensis in type MDF are found as small mono-dominated patches scattered in the secondary evergreen broadleaf forest. The tallest plant of T. doichangensis was about 10 m and the average height was around 5–6 m. Approximately 70% of the trees in the plots were mature with a height of 4 m. Around 20% of the plants were 2–3 m in height, while seedlings and young trees represented only 6% of the total population. Accompanying higher plants were represented by some 30 species and most of the woody species were the same as in Type SW, but there were far fewer herbaceous plants and epiphytes. (4) Type CDF: Plants of T. doichangensis in Type CDF often formed a mosaic of mono-dominant patches in the primitive evergreen broadleaf forest. In this type of community, T. doichangensis grows

naturally without destructive disturbance from human activities. Based on the tree, height-grade Type CDF can be divided into three ranks. These are big tree structure (BTS), relatively balanced structure (RBS), and mono-dominant structure (MDS) of mature trees (Table 8.1).

8.3 Cytology and Karyotype 8.3.1 Base Chromosome Number of Genus Trigonobalanus Of the three species of Trigonobalanus, the base chromosome number of T. verticillata is x ¼ ca. 21 (2n ¼ 40, 42, and 44) (Hou 1971), and the five extant populations of T. doichangensis (Table 8.2) indicated that the base chromosome number of the species is x ¼ 7 (2n ¼ 14) (Chen et al. 2007; Sun et al. 2007). After carefully studying Hou’s original article and the photographs therein (his chromosome numbers are doubted due to unclear illustrations) and comparing

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all records and references of T. verticillata stored in the herbarium of the Royal Botanic Gardens, Kew, we propose that the mitotic number of 2n ¼ 42 for T. verticillata is probably the correct one. We may therefore conclude that T. verticillata is a hexaploid (2n ¼ 6x ¼ 42, x ¼ 7) derived from the ancestral T. doichagnensis. In fact, our recent cytological observations on T. verticillata populations from Fraser’s Hill in Malaysia (N 3 430 1500 , E 101 440 4000 , and altitude of 970 m) and Hainan (N 19 020 1900 , E 109 310 1500 , and altitude of 1,460 m) in China (Chen and Sun 2010) have already further confirmed the base chromosome number of x ¼ 7 for genus Trigonobalnus.

Table 8.2 Localities, geographical positions, altitudes, and voucher numbers of the five investigated T. doichangensis populations (cited from Chen et al. 2007) Population Locality Altitude Voucher (KUN) code (m) ML Menglian county, 1,100 SWB02T01-20 Yunnan, China LC Lancang county, 1,500 SWB02T21-40 Yunnan, China XM Ximeng county, 1,040 SWB02T41-60 Yunnan, China CY Cangyuan county, 1,730 SWB02T61-80 Yunnan, China 1,237 SWB02T081-100 CR Chiang Rai Province, Thailand

Fig. 8.2 Cytological features of the five T. doichangensis populations (cited from Chen et al. 2007). (a) Interphase nuclei of simple chromocenter type. (b) The prophase chromosomes of all populations in this study were of the proximal interstitial type. (c) Two B chromosomes were observed at prophase and

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8.3.2 Karyotype of T. doichangensis The interphase nuclei of the five T. doichangensis populations in China and Thailand (Table 8.2) had the same distribution pattern of chromatin, and according to Tanaka (1971, 1977), the pattern could be categorized as the simple chromocenter type (Fig. 8.2) (Chen et al. 2007). Heterochromatin and euchromatin segments were clearly observed at mitotic prophase in individuals of all the T. doichangensis populations (Chen et al. 2007). The heterochromatin segments were located in the proximal regions that were deeply stained, which indicated early condensation. While the euchromatin segments in the distal regions of chromosomes were lightly stained and extended, indicating late condensation (Fig. 8.2; Chen et al. 2007). Two B-chromosomes were commonly observed at prophase and prophase-metaphase, but rarely observed at metaphase (Fig. 8.2; Chen et al. 2007). The prophase chromosomes of all the populations belonged to the proximal interstitial type. The karyomorphological characteristics of the five populations (Fig. 8.2) are described as follows (Chen et al. 2007). The karyotype formula of five populations are 2n ¼ 14 ¼ 12m (2SAT) + 2sm + 2Bs (CR), 2n ¼ 14 ¼ 14m (2SAT) + 2Bs (LC), 2n ¼ 14 ¼ 12m (2SAT) + 2sm + 2Bs (XM), 2n ¼ 1 ¼ 14m (2SAT) + 2Bs (ML), and 2n ¼ 14 ¼ 12m (2SAT) + 2sm + 2Bs (CY) (Fig. 8.2). The ratio of the longest to the shortest

prophase-metaphase, but less at metaphase (arrows). (d–h) Mitotic metaphases. (d) CR population, 2n ¼ 14. (e) LC population, 2n ¼ 14. (f) XM population, 2n ¼ 14. (g) ML population, 2n ¼ 14. (h) CY population, 2n ¼ 14. Scale bars ¼ 10 mm

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chromosome was from 1.48 to 2.06 (Fig. 8.2). The karyotype asymmetries of five populations are classified as 1A (CR), 1A (LC), 2A (XM), 1A (ML), and 2B (CY), respectively (Fig. 8.2).

8.3.3 Implications of the Cytological Data Stebbins (1971) and Stace (2000) considered that almost all polyploids ultimately come from diploids. As all the five T. doichangensis populations are invariably diploid (2n ¼ 14) and T. verticllata is polyploid (2n ¼ 42), we suspect that T. doichangensis might represent the basal lineage of the genus Trigonobalanus, which may have originated from southeastern Asia (Zhou 1999; Manos and Stanford 2001). Trigonobalanus was probably broadly distributed (perhaps over the whole northern Hemisphere) during the Tertiary period or even earlier (Zhou 1992). However, extreme climate changes in the late Tertiary period brought on an extinction and habitat disappearance of a large number of species. Certainly, such global changes could cause species of Trigonobalanus to be diminished or even extinguished in their distribution ranges. In this way, T. doichangensis became a relict, currently restricted to some scattered populations in South Yunnan of China and North Thailand (Sun et al. 2006). As a relic species, the B-chromosome existed in T. doichangensis might be related to the strong climate changes in late Tertiary period (Zhou 1999). And the B-chromosome may contribute to the low production of the fertile nuts of the species (less that 10%) (Sun et al. 2006).

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8.4 Population Genetics of T. doichangensis 8.4.1 Genetic Diversity Five populations (ML, LC, XM, CY, and CR) were sampled that represent each of the known sites of occurrence. Within each population, 19 or 20 individuals at least 20 m apart were selected at random. Young, fully expanded leaves were harvested in January 2002 when the trees were in flower and fruit (T. doichangensis can have both flowers and fruits at the same time). Total DNA was extracted from 1–2 g of silica dried leaves using the modified CTAB method. The amplified fragments were scored for the presence (1) and absence (0) of homologous bands, the matrix of random amplified polymorphic DNA (RAPD) markers was inputted POPGENE software for further statistical analyses. UPGMA (unweighted pair group method arithmetic average) clustering was conducted according to Nei’s genetic distance using PAUP4.0b10 software. Eighty-three (52.8%) of the fragments were polymorphic across all populations (Table 8.2; Sun et al. 2007). For population XM, 34.9% of fragments were polymorphic, followed by CY, ML, and CR with 33.3, 32.8, and 20.7%, respectively (Table 8.2). Population LC had the fewest polymorphic (10.9%) and six private fragments. None of the other populations had private fragments but instead shared fragments with one or more other populations (Sun et al. 2007). Some fragments, although not unique to a population, were relatively rare and were shared between only two of the five populations (Table 8.3). Each population

Table 8.3 The percentage of polymorphic fragments (% Polym), estimated population diversity (HS), total genetic diversity (HT), number of invariant (In), polymorphic (Pl), and total fragments for each population of T. doichangensis (cited from Sun et al. 2007) Population code % Polym HT HS In Fragment (Pl) Total LC 10.9 0.033 131 16 (6.2) 147 CR 20.7 0.054 115 30 (2) 145 CY 33.3 0.104 100 50 (1) 150 ML 32.8 0.096 94 46 (1) 140 XM 34.9 0.108 97 52 (2) 149 Mean 52.8 0.160 0.079 107 39 146 The numbers of fragments that are private (bold) and rare (shared by two populations) are given in parentheses beside the number of polymorphic fragments

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possessed at least one of the eight rare fragments that were amplified (Sun et al. 2007). The estimated genetic diversity within populations (HS) also indicated that the LC population was the least diverse (HS ¼ 0.033) followed by CR (HS ¼ 0.054) (Table 8.2). The remaining three populations had similar HS values (0.108 for XM, 0.104 for CY, and 0.096 for ML). The estimated species genetic diversity of T. doichangensis was HT ¼ 0.160, the genetic diversity within populations was 0.079 (Table 8.4; Sun et al. 2007). It is apparent that genetic variation of among population was greater than that of within population.

8.4.2 Genetic Differentiation The estimated genetic differentiation among populations (FST) and that among regions (FSC) were 0.530 and 0.579, respectively (Table 8.3; Sun et al. 2007). And the genetic differentiation within populations (FCT) was 0.116 (Table 8.4). The results meant that the genetic variation among population accounted for 53.0% of the total variation and strong gene differentiation was present among populations (Sun et al. 2007). Pair-wise fixation indices indicated that LC was the most distinct population (Table 8.4). The lowest pair-wise (FST) values occurred when ML was compared with CY (0.288) or XM (0.338) (Table 8.4; Sun et al. 2007). The estimated pair-wise gene flow among populations ranged between NM ¼ 0.085 (LC and CR) and 0.618 (ML and CY)

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and averaged 0.276 across all pairs of populations (Table 8.5)

8.4.3 Genetic Distance and Cluster Analysis On the basis of individual pair-wise comparisons, the genetic identity among the five populations ranged from 0.792 to 0.952 (Table 8.6; Sun et al. 2007), with a mean of 0.888. The genetic pair-wise distances varied among populations from 0.049 to 0.234 (Table 8.5), with a mean of 0.121 (Sun et al. 2007). Gene flow (NM) was only 0.276 among populations, and gene flow among populations was very low. In agreement with the low value for the estimated gene flow between populations, all individuals from each population formed a single cluster (Fig. 8.3; Sun et al. 2007). Estimated distances between individuals within population LC (Yunnan, China) indicated that the trees sampled were quite similar in their RAPD fragment profiles (Fig. 8.3). The ML, CY, and XM populations (Yunnan, China) had relatively more divergence between individuals. The Thailand population (CR) had individuals that were intermediate in their overall similarity on the basis of RAPD fragments and genetic diversity (Table 8.3). In comparison with fagaceous species, the genetic variation of T. doichangensis was relatively lower, and over half genetic variation existed among populations (Hs ¼ 0.079). Approximately 53% of all fragments

Table 8.4 Analysis of molecular variation (AMOVA) among the two regions, northern Thailand and Yunnan Province, China, among populations and within populations for T. doichangensis (Sun et al. 2007) Source of variation Fixation value Among regions (FSC) 0.579 Among populations (FST) 0.530 0.116 Within populations (FCT)

Table 8.5 Fixation index (FST) and estimated number of migrants (NM) (below diagonal) among T. doichangensis populations (cited from Sun et al. 2007) LC CR CY ML XM LC – 0.747 0.698 0.710 0.730 CR 0.185 – 0.491 0.415 0.562 CY 0.108 0.259 – 0.288 0.354 ML 0.102 0.352 0.618 – 0.338 XM 0.092 0.195 0.456 0.491 –

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LC CR CY ML XM

LC – 0.146 0.190 0.188 0.234

CR 0.864 – 0.088 0.061 0.122

CY 0.827 0.916 – 0.049 0.070

ML 0.829 0.941 0.952 – 0.062

XM 0.792 0.886 0.932 0.940 –

XM

CY

ML

CR

LC

Table 8.6 Nei’s genetic identity (I) and genetic distance (D) (below diagonal) among T. doichangensis populations (cited from Sun et al. 2007)

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Fig. 8.3 UPGMA phenogram based on Nei’s genetic distance and 157 RAPD fragments from 99 T. doichangensis trees representing each of the five known extant populations (CY Can-

gyuan, China; XM Ximeng, China; LC Lancang, China; ML Menglian, China; CR Chiangrai, Thailand) (cited from Sun et al. 2007)

resolved in T. doichangensis were polymorphic. This level of polymorphism was low compared with T. verticillata where 79% of AFLP fragments

were polymorphic (Kamiya et al. 2002). Similar to the levels of polymorphic fragments, the estimated species genetic diversity of T. doichangensis

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(HT ¼ 0.160) was within the range of genetic diversity found in other Fagaceae species but lower than HT ¼ 0.198 for T. verticillata (Kamiya et al. 2002).

8.5 Reproductive Biology of T. doichangensis 8.5.1 Blooming and Fruit Habits, Microspore Genesis, and Development of Male Gametes T. doichangensis may flower when its height is about 4 m, and the starting date of blooming is slightly different among populations or population’s microclimatic habitants (Sun et al. 2004). In general, the plants in the higher altitude and further north latitude may have an earlier starting date of blooming. But the duration of blooming and fruiting is mostly from October until next May, and this phenological character is obviously different in comparison to that of other fagaceous species within the same populations. Observations on the microspore genesis and the development of male gametes in T. doichangensis revealed that the anther is 4-sporangiate and its anther wall formation conforms to the dicotyledonous type (Zeng and Sun 2004; Fig. 8.4). And also the tapetum is of the glandular type and its most cells are two-nucleate, and cytokinesis at meiosis of microspore mother cells is simultaneous and tetrads are tetrahedral, occasionally decussate (Zeng and Sun 2004; Fig. 8.6). Besides, mature pollen grains are two-celled. Therefore, it can be concluded that microspore genesis and development of male gametes in T. doichangensis are normal, and they are not the factors caused the lower rate of fructification (Zeng and Sun 2004; Figs. 8.5 and 8.6).

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Fig. 8.5 (a) An young anther in transverse section (T.S.) with four sporangia; (b) Secondary sporogenous cells; (c) Microspore mother cells when the middle layers begin to degenerate; (d) Microsporocyte at the meiosis and full-developed tapetum; (e) Microspore tetrads when tapetum begins to disintegrate; (f) Tapetum at the stage of uninucleate microspore; (g) Tapetum at the stage of vacuolated period of uninucleate microspore; (h) Anther wall of the time of the pollen spread; (i) Binucleate cells of tapetum; (j) Multinucleolate characters in tapetal cells. a 200; b, e 500; c, h 400; d, f, g, i, j 1,000 (cited from Zeng and Sun 2004)

c

Fig. 8.4 Inflorescences and infructescence of T. doichangensis; (a) Staminate inflorescences, (b) Pistillate inflorescences, and (c) Fructescence

8 Trigonobalanus Fig. 8.6 (a) Microsporocyte at the telophase I of meiosis; (b) Microsporocyte at the telophase II of meiosis; (c) Formation of microspore tetrads surrounded by callose; (d) Tetrahedral miscrospore tetrads; (e) Decussate microspore tetrads; (f) Young microspores just released from tetrads; (g) Microspore grows and becomes rounded, its wall begins to thicken; (h) vacuolated period of uninucleate microspore; (i) Uninucleate microspores; (j) Bicellular pollen; (k) The mature bicellular pollen grain. a–d, g–k  1,200; e, f  1,000 (cited from Zeng and Sun 2004)

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8.5.2 Ovule Abortion and Embryo Development According to our recent research, in T. doichangensis, the ovary has three locules with two ovules per locule. Observation on the ovary in transverse section showed that six ovules in each ovary were generated at an early stage, and at the developmental anaphase one of two ovules per chamber were well developed, while another one was aborted. Meanwhile, both axile and parietal placentas were observed, and thus T. doichangensis may show a transition trend from axile to parietal placentation. The ovule is anatropous and is enclosed by outer and inner integuments. The following three phenomena were also observed in the young fruits: (1) only one of the six ovules per ovary developed into an embryo and the others were aborted. The percentage of the one-ovule developed nuts was less than 10%; (2) in some fruits all the six ovules in the ovary were aborted and the ovary was lignified; and (3) the ovules in some fruits seem to be developed but the embryos

e

d

h

k

were membranous. It can be concluded that considerable embryo abortion occurs in T. doichangensis.

8.6 Propagation of T. doichangensis Producing a large number of plants through seeds could allow for conservation of the widest range of genetic diversity and would negatively affect the least its natural regeneration and population expansion. Vegetative propagation and in vitro techniques for rapid and mass propagation could offer possibilities to conserve germplasm and multiply desirable genotypes. For re-introduction planning and ex situ conservation, a holistic approach including conventional (vegetative propagation and seed germination) and in vitro techniques should be used for the propagation of T. doichangensis. Fruits (nuts) of T. doichangensis are both of the dispersal unit and germination unit. T. doichangensis

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a

b

Fig. 8.7 The anatomic feature of T. doichangensis nuts; (a) Nuts of seeds well developed (b) Nuts of seeds aborted

bears female flowers with one ovary. Each ovary has six ovules but only one has the potential to develop into a seed with an embryo. Some ovaries, however, have only aborted ovules and the fruits are either empty or lignified (Sun et al. 2006; Fig. 8.7) reported that the germination percentage of T. doichangensis was low and considerable seed abortion occurred in the species. It was observed that there was no apparent correlation between percentage of fruit fill (i.e., fruits containing seeds) and fruit morphological traits (Zheng and Sun 2008). Therefore, it is not possible to sort out fruits filled with seeds according to the fruit morphological characters. According to our observations of consecutive years, fully formed nuts collected at the same time differ obviously in color (dark and light), and dark-colored fruits have significantly higher rate of well-developed seeds (W. Sun, unpublished report). The variation in seed abortion between the two kinds of fruits may result from phenology. It could be concluded that sampling strategy must be taken into consideration when determining sample size of fruit collection for conservation or reforestation. Dark-colored fruits began to germinate after 3 days of incubation on moistened filter papers, with a peak after 5–6 days, followed by occasional germination thereafter. Percentage of fruit germination varied from 28.4  7.42 (2006) to 39.0  13.9 (2008) among years, from 0 to 59.6  3.42 among individuals (Zheng and Sun 2008). Germination percentage, germination index, and vigor index presented significant variation among populations and among individual trees within populations. The three germination-related indices had weak correlations with fruit morphological traits. Therefore, it could be concluded that the fruit size of T. doichangensis does not relate to its fruit germination capacity. Selecting fruits on the

basis of mass is not an appropriate way to enhance germination for reforestation projects. Moreover, collecting fruits from various individuals at each of the populations will be the preferred strategy to conserve the most genetic diversity of the species. So far, some 2,000 saplings propagated from seeds (botanically called nuts) (Fig. 8.8) have been conserved ex situ at the Kunming Botanic Garden, Chinese Academy of Sciences. Vegetative propagation is widely employed for multiplication of genetically superior genotypes. Although the factors affecting rooting capacity of stem cuttings have not been entirely elucidated (Dick and Leakey 2006), it has often been reported that plant age (Black 1972), pretreatment with auxin, substrates, leaf areas could affect rooting efficiency (Desrochers and Thomas 2003; Copes and Mandel 2000). According to our preliminary study, vegetative propagation of T. doichangensis with lignified cuttings is rather difficult. The leaves on cuttings will turn brown and shed within one month of culture and no rooting was occurred. Semi-lignified cuttings, with or without apical shoot, showed some rooting capacity (Fig. 8.9), however, the obtained rooting percentage was low. Further study should be conducted to maximize rooting success through selection of appropriate combination of pre-planting treatments. Micropropagation with shoots originated from 4-year-old seedlings failed. Brown substance exuded at the base of shoots and the shoots would die within 1 month. Cotyledonary nodes as one kind of explants have been used by some authors (Pradhan et al. 1998; Vengadesan et al. 2002; Jha et al. 2004). With explants of cotyledonary nodes originated from juvenile seedlings, 20–25 shoots/explants can be induced within 4 months (Fig. 8.10). And over 90% of the plantlets

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b

a

e

c

f

d

Fig. 8.8 Seeding tests of T. doichangensis; (a) Seeds (nuts); (b), (c), and (d) Tests of the mixed-medium seeding; (e) Test of perlite seeding; and six cotyledons and the first two leaves

Rooting

Fig. 8.9 The rooted semi-lignified cuttings of T. doichangensis

Rooted cuttings

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Rooted shoots

Induced shoots

Fig. 8.10 Micropropagation of T. doichnagensis, with explants of cotyledonary nodes originated from juvenile seedlings

were successfully acclimatized and established in perlite media (Zheng et al. unpublished report).

8.7 Conservation Considerations and Population Re-enforcement Program

2.

8.7.1 Conservation Consideration (Cited from Sun et al. 2006) Protection and restoration of natural habitats is the best and cheapest method of preserving the biological diversity and stability of global ecosystem. Undoubtedly, the long-term survival of T. doichangensis is dependent on habitat conservation. However, only one of the four extant populations of T. doichangensis in China has been legally preserved by government ownership and others are facing a high risk of habitat disappearance. Perhaps more than most endangered plant species in China, the unique characteristics of T. doichangensis’ population isolation and the complex community interaction exemplify the importance of habitat preservation and in situ conservation. Nevertheless, both in situ and ex situ measures are needed for preserving T. doichangensis and its genetic diversity, and following aspects should be particularly considered. 1. Preservation of the habitat and population of T. doichangensis in Canyuan must be reinforced. The Canyuan population is the only one of the four populations in China, which has been well

3.

4.

5.

protected inside a national natural reserve. Therefore, the population is a vital resource for further research on the origin and evolution, eco-biological characteristics and genetic realities of T. doichangensis. Furthermore, the population also has a great value in population restoration research. As T. doichangensis populations in Menglian, Lancang, and Ximeng are still exposed to high habitat destruction and cutting for fuelwood, it is essential that new in situ conservation sites in these area should be urgently planned. These new sites will play an important role in habitat recovery and population restoration. Although cross-planting is often controversial in plant conservation planning, it is proposed that seedling cross-planting between different populations of T. doichangensis should be considered. At least, the potential impacts of this measure should be studied to determine if low gene flow would be enhanced and if overall diversity would be increased. As young T. doichangensis plants propagated from seeds can tolerate temperatures below 2 C at Kunming (Sun et al. 2004), it may be cultivated for fuelwood and as a landscaping plant in northern parts of the Tropic of Cancer. However, the mixed planting of trees propagated from various populations is essential. Ex situ efforts need to be undertaken to preserve genetic diversity and multiply specimens. This applies, in particular, to those populations outside of the well-protected national nature reserve. Ideally, ex situ sites will be close to natural reserves or to botanical gardens. Propagation from seeds is preferred, since it would be the least detrimental

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to the extant populations and would include the widest range of genetic diversity. Optimally, seeds should be taken from many individuals from each of the populations.

8.7.2 Population Re-enforcement T. doichangensis Based on the large number of saplings ex situ conserved at the Kunming Botanic Garden, the population re-enforcement action of T. doichangensis supported by the Botanic Gardens Conservation International (BGCI), together with the governmental program of “Returning Farmland to Forest”, has been launched since 2007. Some 247 selected saplings of T. doichangensis have been planted in Banli village of Lancang, where the species occurring. With observations on growing, ecological and biological characteristics, the saplings are growing well. It can be expected that re-enforced individuals can be developed as an ecologically functional population in its natural habitat (Fig. 8.11).

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8.8 Perspective of the Genus Trigonobalanus in Future Genus Trigonobalanus has long generated considerable interest because of its hemispheric disjunction and its morphological characters, some of which are unique in Fagaceae. Because of difficulty to attain the materials of T. verticillata and T. excelsa, most of our research concentrated on T. doichangensis. Following works still need to be done.

8.8.1 Conservation Biology Protection and restoration of natural habitats is the best and cheapest method of preserving the biological diversity and stability of global ecosystem (Lande 1988). Undoubtedly, the long-term survival of T. doichangensis is dependent on habitat conservation. However, only one of the four extant populations of T. doichangensis in China has been legally preserved by government ownership and others are facing a high risk of habitat disappearance. Both in situ and ex situ

a

b

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Fig. 8.11 Propagated T. doichnagensis were re-enforced in the natural habitat; (a) Saplings from seeds, (b) Re-enforcing site, (c and d) Planting with the local people

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measures are needed for preserving T. doichangensis and its genetic diversity, fortunately, our team is doing the work through cooperation with some international organization, such as Forest Frontiers Initiative (FFI) and BGCI.

8.8.2 Pollination Ecology T. doichangensis is an endangered species in China. The genus Trigonobalanus is a transitional phase in Fagaceae. Research on polymorphism in pollination syndrome (wind vs. generalist insect) also contributes to understand the reproductive trait evolution in the family.

8.8.3 Ecological Physiology The physiological function of leaf should be measured, the seasonal modification of wax deposition, and the impact of epicuticular wax on gas-exchange as well as photoinhibition in T. doichangensis, with wax-covered leaf surfaces and the stomata also partially occluded by wax, is an interesting study. Epicuticular waxes decrease cuticular water loss? The temperature of leaves without wax is lower than that of wax-covered leaves? The wax coverage at the entrance of stomata in T. doichangensis increase resistance to gas diffusion and as a consequence decrease stomatal conductance, transpiration, and photosynthesis? These question needs to be answered in the future research.

8.8.4 Recommendation of Other Immediate Measures The following aspects are needed to be considered immediately. 1. Systematic relationships within Trigonobalanus need to be clarified. Manos and Stanford (2001) indicated that T. verticillata was sister to T. doichangensis in Asia. But, Oh and Manos (2008) indicated that T. verticillata was sister to T. excelsa.

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More molecular data are needed to support the precise relationship among the three species. 2. Chromosome numbers of T. excelsa need to be counted since of the three species of Trigonobalanus, the chromosome numbers are known for two, T. verticillata and T. doichangensis. The basic chromosome number of these taxa is x ¼ 7, which is unique in Fagaceae, even in Fagales. 3. Systematic position of Trigonobalanus in Fagaceae should be studied. The transitional characteristics of Trigonobalanus have caused a series of taxonomic problems in Fagaceae. It has been divided into different taxa in different treatments. Based on morphological observations, Jones (1986) suggested that there are four subfamilies in Fagaceae. Nixon and Crepet (1989) divided Fagaceae into two subfamilies. However, combined analysis of all sequences yielded parsimony trees identifying three basic lineages in Fagaceae: Fagus, Trigonobalanus, and the remaining genera (Li et al. 2004; Chen et al. 2008; Oh and Manos 2008). 4. Historical biogeography of Trigonobalanus should be revisited. The evergreen genus Trigonobalanus shows a disjunct continental distribution, which provides a single, yet important data point in evaluating the biogeographic history of Fagaceae. Trigonobalanus are subtropical members of the Fagaceae, and both its modern and extensive fossil distributions suggest an old biogeographical history (Manos and Stanford 2001). A 2.21% sequence divergence between the Asian species and the New World relict Trigonobalanus excelsa led Manos and Stanford (2001) to estimate a divergence time of ca. 37 million years (Manos and Stanford 2001). The combination of fossil and modern distributions with molecular dating suggests that the continuous distribution of Trigonobalanus was most likely achieved via the North Atlantic Land Bridges (NALB) during the Paleocene before the complete formation of the Turgai Strait (Manos and Stanford 2001; Tiffney and Manchester 2001). Using the cpDNA molecular clock, Kamiya et al. (2002) provided a clue about the migration routes and divergence time of T. verticillata, the divergence of the Fraser’s Hill population from the others was estimated to be 16.7 Mya. The 95% upper confidence limit indicates that T. verticillata reached the Malay peninsula not earlier than the Oligocene.

8 Trigonobalanus

Paleobotanical evidence has shown that the Oligocene and early Miocene were periods of relatively dry and cool climates in Southeast Asia (Morley 1998; Whitmore 1998). The cooler climates could have allowed the montane species to expand to lower altitudes and migrate to lower latitudes. This suggests that T. verticillata could have expanded to the south and subsequently migrated as far as Borneo during this period. Early Miocene paleogeographical reconstruction in the Sunda–Sahul region suggests that one possible migration route would have been the corridor across the Malay peninsula and Borneo (Morley and Flenley 1987). Morley (1998) also suggested that warm and moist climatic conditions prevailed during the initial parts of the middle Miocene (ca. 15 Mya) throughout large part of East and Southeast Asia. Thus, a large area of the regions was covered by tropical lowland evergreen forest species. Species of Trigonobalanus, which prefer a higher altitude in the montane zone, would have become isolated. A new distribution record for T. verticillata from Hainan Island, South China greatly expands the distribution range of this species (Ng and Lin 2008) from 7 N to 19 N. Stebbins (1971) and Stace (2000) considered that almost all polyploids ultimately come from diploids. As the population of T. verticillata in Hainan Island, China, is diploid, we suspect that T. verticillata might represent the basal lineage of the genus Trigonobalanus, which may have originated in southeastern Asia. From there, it possibly spread southwards to Celebes, Borneo, and Malaya, generating the hexaploid T. verticillata populations. To compare the genetic divergence between the Malay population and the Chinese population and to measure the origin and disjunction time of this ancient relict species, further field observations and molecular clock data are certainly required.

References Anon (1999) List of National key protected wild plants (First Group). The order of National Forestry Bureau and Agricluture Ministry of China 4:2–13 Black DK (1972) The influence of shoot origin on the rooting of Douglas-fir stem cuttings. Comb Proc Int Plant Prop Soc 22:142–157 Chen G, Sun WB (2010) Ploidy variation in Trigonobalanus verticillata (Fagaceae). Plant Syst Evol 284:123–127

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Index

5S rDNA, 4, 110 5S rRNA, 22 16S rRNA, 9 454-Technology, 116 A Abiotic stress, 72 Acorn, 94, 95, 110 chemistry, 118 properties, 98–99 Actinorhizal, 11 Adaptation, 34 Afforestation, 49 Affymetrix, 116 AFLPs. See Amplified fragment length polymorphism Agriculture Research Service (ARS), 21 Agrobacterium, 139 Agroforestry, 79 Allergens, 53 Allergic reaction, 57 Allogamous, 59 Allopolyploid, 4 Allozyme, 53 Alnus, 1–11 A. acuminata, 3 A. cordata, 4 A. firma, 4 A. glutinosa, 4 A. incana ssp. rugosa, 3, 4 A. japonica, 4 A. maritima, 2 A. nitida, 2 A. orientalis, 4 A. pendula, 4 A. rhombifolia, 3 A. rubra, 3 A. serrulata, 3, 4 A. serrulatoides, 4 A. subcordata, 4 A. viridis, 2 American hazelnut, 17 Amplified fragment length polymorphism (AFLP), 23, 57, 109 Andean Alder, 2 Angiosperm, 6 Angophora, 66 Antioxidant, 42

Antipyretic, 140 Antisense, 84 Antiseptic, 70 Antitumorogenic, 140 Antiviral, 140 Arabidopsis, 113 Arboretum, 21, 52 Aromatic heartwood, 131 ARS. See Agriculture Research Service Artificial forests, 53 Association genetics, 71 Astringent, 140 B BAC. See Bacterial artificial chromosome Backcross, 83 hybrids, 38 Bacterial artificial chromosome (BAC) library, 23 Bay laurel, 103 B-chromosome, 148 Beaked hazel, 18 Betula, 2 Bifurcating tree, 92 Biodiversity, 119 Biogeographical studies, 145 Biogeography, 96, 145 Biomass, 5, 42, 72 gain, 100 industry, 8 plantation, 8 production, 7–8 Biosynthesis, 71 Biotechnology, 84–85 Black Alder, 2 Black oak, 91 Black walnut, 77 Blooming, 152 Bridge species, 83 Butternut, 80–81 C California black oak, 103 Candidate, 2, 39 gene, 56, 115–116 tree, 53

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5, # Springer-Verlag Berlin Heidelberg 2011

161

162 CAPS. See Cleaved amplified polymorphic sequence Carpinus, 7 Castanea, 20, 90 CDF. See Co-dominant forest Cerris, 91 Charcoal, 67 Chemosterilant, 140 Chemotaxonomy, 70–71 Chemotype, 70 Chestnuts, 90 Chinese tree hazel, 19 Chlorophyll deficiency, 22 Chloroplast genome, 66, 135 Chloroplast DNA (cpDNA), 52, 72, 140 array, 116 library, 56, 84 microarrays, 117 Chromosomal stability, 112 Cleaved amplified polymorphic sequence (CAPS), 54 Clonal, 57, 82 propagation, 21 reproduction, 138 structure, 109 Clonality, 138 Coast live oak, 103 Co-dominant forest (CDF), 146 Colchicine, 84 Cold hardiness, 38 Cold-hardy, 28, 36, 83 Common walnut, 77 Comparative mapping, 115 Consensus linkage map, 57 Conservation, 20–21 biology, 101–104 efforts, 82 initiatives, 82, 101–102 Cork biosynthesis, 115–116 colurna, 37 Corylus, 7, 15–44 C. americana, 16, 17 C. avellana, 15 C. californica, 16, 36 C. chinensis, 16, 19, 39 C. colurna, 19, 37 C. colurna L., 16 C. cornuta, 18 C. cornuta even, 36 C. fargesii, 40 C. ferox, 16, 40 C. ferox var. tibetica, 40 C. heterophylla, 16–18 C. jacqemontii, 16 C. jacquemontii, 39 C. kweichowensis, 17 C. mandshurica, 18 C. papyraceae, 16 C. sieboldiana, 16, 18, 37 seed and plants, 36 Corymbia, 66, 71 Cosmetics, 140 cpDNA. See Chloroplast DNA

Index Crop vulnerability, 82 Cross-planting, 156 Cryopreservation, 22 Cryptomeria, 49–60 C. japonica, 49 C. japonica var. radicans, 49–50 C. japonica var. sinensis, 51 Cultivar, 105 Cyanogenic glycosides, 71 Cycle cup oak, 91 Cytogenetic stocks, 71 D Deciduous tree, 77 Deforestation, 20 Deformity, 53 Distorted segregation, 56–57 Diuretic, 140 Domestication, 72, 106 Dormancy, 99 Doubled haploid, 106 Drought, 37 tolerance, 8 Dwarfism, 49 E Eastern filbert blight (EFB), 22 resistance, 32, 42 Ecological, 49, 89 distribution, 97–98 physiology, 158 Ecology, 3–4 Ecosystem development, 4 ECP/GR. See European Cooperative Program on Plant Genetic Resources Ectomycorrhizal (ECM) fungi, 101 EEC. See European Economic Community Embryo development, 153 Endangered, 16, 69 plant, 146 species, 68 Essential oil, 140 ESTs. See Expressed sequence tags Eucalypts, 65 Eucalyptus, 65–73 E. benthamii, 70 E. consideniana, 68 E. drummondii, 68 E. grandis, 68, 72 E. graniticola, 68 E. rudis, 68 Eucalyptus Genome Network (EUCAGEN), 72 Euchromatin, 148 EUFORGEN. See European Forest Genetics Resources Programme European Cooperative Program on Plant Genetic Resources (ECP/GR), 78–79 European Economic Community (EEC), 78–79 European Forest Genetics Resources Programme (EUFORGEN), 78 European hazelnut, 15

Index Evergreen, 94 EVOLTREE, 102 Expressed sequence tag (EST), 56, 72, 84, 90 library, 116 sequence, 116 Ex situ collection, 78 Ex situ conservation, 60, 70, 137, 153 Ex situ propagation, 70 External transcribed spacer (ETS), 133 F Fagus, 145 Fatty acid, 41 Female map, 111 FFPRI. See Forestry and Forest Products Research Institute Firewood, 77 First-generation hybrid, 95 FISH. See Fluorescence in situ hybridization Fixation index, 5 Flavonoid, 84 Flavor, 140 Floristic composition, 146 Flowering phenology, 55 Fluorescence in situ hybridization (FISH), 111 Forest ecosystem, 102 Forestry and Forest Products Research Institute (FFPRI), 52 Forest tree genetic resources conservation forest (FTGRCF), 52 Forest Tree Superior Gene Conservation Stand (FTSGCS), 52 Fossil evidence, 77 Frankia, 1 strain, 10 Fructification, 152 FTGRCF. See Forest tree genetic resources conservation forest FTSGCS. See Forest Tree Superior Gene Conservation Stand Full-sib progenies, 83 Functional genomics, 115–117 G GenBank, 22, 84 Gene, 5, 9, 10, 22 expression, 115 flow, 5, 137 pool, 90, 91 Generic diversity, 97 Genetic, 1, 5–11 conservation, 52 differentiation, 6, 54, 59, 99, 150 distance, 150 diversity, 5, 21, 59, 65, 137–138 erosion, 68 identity, 21, 150 overlap, 92 stock, 8–9, 71 structure, 5, 59, 71 transformation, 72 Genome, 16, 23, 52 sequence, 66 size, 49, 66, 113 structure, 113

163 Genome-wide association, 59 Genomic, 56, 59 library, 140 selection, 59 Genotyping, 57 Geographical information systems (GIS), 8 Geographic structure, 70 Germplasm, 78 diversity, 104 exchange, 27 preservation, 21 Gigantism, 49 GIS. See Geographical information systems Glacial refugia, 104 Global distribution, 97 ecosystem, 156 warming, 8 Golden cup, 91 Grafting, 21, 84 Growth performance, 8 H Habitat, 3, 17, 36, 70, 89, 97 disappearance, 149 recovery, 156 specialization, 98 Hardwood, 105 Hardwood Tree Improvement and Regeneration Center (HTIRC), 107 Hazelnut, 15 kernel, 41 oil, 41 Heartwood, 140 Herbivores, 97 Herbivory, 100 Heritabilities, 108, 138 Heterochromatin, 148 Heterosis breeding, 71–72 Heterozygosity, 5 High-throughput sequencing, 84 Himalayan tree hazel, 20 HJG. See Hydrojuglone glucosides HTIRC. See Hardwood Tree Improvement and Regeneration Center Hybrid hazelnut, 24 Hybridization, 4–5, 92 Hybrid vigor, 85 Hydrojuglone glucosides (HJG), 84 Hypotensive, 140 I IBPGR. See International Board of Plant Genetic Resources ICNCP. See International Code of Nomenclature for Cultivated Plants IEA. See International Energy Agency Inbreeding, 5 depression, 56 Indian tree hazel, 19 In situ conservation, 52, 68–70 Institut de Recerca I Technologia Agroalimenta`ries (IRTA), 20

164 Institut National de la Recherche Agronomique (INRA), 79, 114 Intellectual property rights (IPR), 73 Intermediate oak, 91 Internal transcribed spacer (ITS), 7, 22, 81, 90, 133 International Board of Plant Genetic Resources (IBPGR), 78 International Code of Nomenclature for Cultivated Plants (ICNCP), 105 International Energy Agency (IEA), 7 Intersectional hybridization, 83 Inter-simple sequence repeat (ISSR), 6, 110 Interspecific, 4, 15, 22–24 breeding, 24 cross, 31 hybridization, 15 hybrid, 4, 24, 35, 71–72 Intraspecific comparisons, 95 Introgression, 72, 83 Invasive species, 68 In vitro preservation, 21 IPR. See Intellectual property rights IRTA. See Institut de Recerca I Technologia Agroalimenta`ries ISSR. See Inter-simple sequence repeat Italian Alder, 2 ITS. See Internal transcribed spacer J Juglans, 77–85 J. ailantifolia, 83 J. cinerea, 83 J.  intermedia, 79 J. nigra, 81 J. regia, 77 K Karyotype, 66, 81, 113 L Landscape plants, 42 LD. See Linkage disequilibrium Lignin content, 71 Lineage sorting, 92 Linkage, 49, 84 disequilibrium (LD), 59 group, 57 map, 56 mapping, 110–112 Lipid, 99 Live oak, 91 Local conservation, 73 Lumber, 77 M Macroarrays, 115 Male gametes, 152 Male map, 111 Male-sterile, 53 Male strobili, 56 Managed investment scheme (MIS) companies, 140 Mapping, 57, 71, 90, 107 design, 111 population, 111

Index Marker-assisted breeding, 108–112 Marker-assisted selection (MAS), 23 Mass propagation, 153 Mast seeding, 99 Maximum likelihood (ML), 7 Maximum parsimony (MP), 7 MDF. See Mono-dominant forest Melaleuca alternifolia, 70 Metabolome, 117 Metabolomics, 117 Microfibral angle, 71 Micropropagation, 21, 84, 139, 154 Microsatellites, 55, 71, 109 Microsatellite sequence repeat (SSR), 22 Microspore genesis, 152 ML. See Maximum likelihood Model forest tree, 68 plant, 68 Molecular, 7, 15, 16, 66, 68 breeding, 57 genetic map, 71 phylogeny, 3 Mono-dominant forest (MDF), 146 Mono-dominant structure (MDS), 147 Monophyletic, 92 origin, 51 Morphotaxonomy, 70 MP. See Maximum parsimony Mutant, 53, 105–106 Mutation, 104–106 breeding, 84 Mycorrhizal associates, 101 N NALB. See North Atlantic Land Bridges Naphthoquinone metabolism, 84 National Center for Biotechnology Information (NCBI), 84, 111 National Clonal Germplasm Repository (NCGR), 20, 78 Neighbor joining (NJ), 7 Nitrogen fixation, 9 North Atlantic Land Bridges (NALB), 158 Nucleotide diversity, 59 O Oak, 89 gall, 100 gallwasps, 100 microarray, 116 wilt, 103 Omote-sugi, 49–50 Open-pollination, 28 Ord River Irrigation Area (ORIA), 140 Ornamental, 30, 34, 37 hybrids, 72 traits, 42 Osmotic stress, 114 Ostrya, 7 Ostryopsis, 7 Outcrossing, 5, 65

Index Ovule abortion, 153 Oxidative stress, 115 P Paleobotanical evidence, 159 Paper products, 118 Paperbark tree hazel, 19 Perfumes, 140 Persian walnut, 77 Phenolics, 100 Phenotypic, 57, 78, 107, 111 plasticity, 92 variation, 96 Photoinhibition, 158 Phylogenetic, 7, 52, 81 analyses, 133 overdispersion, 98 relationships, 92 tree, 7 Phylogeny, 6–7, 9, 145 Phylogeographic signal, 96 Phylogeography, 80–81, 104, 132–135 Physical map, 52 Phytochemicals, 42 Phytogeography, 145 Phytophthora ramorum, 103 P. ramorum, 103 PICME. See Platform for Integrated Clone Management Plantation systems, 7 Platform for Integrated Clone Management (PICME), 116 Pollen, 23, 28, 31–39 allergy, 57 fecundity, 57 flow, 68 Pollination ecology, 158 Pollinosis, 57 Polymerase chain reaction (PCR), 139 Polyploid, 49, 105–106 POPGENE, 149 Population genetics, 149 Populus, 89, 113 Progeny testing, 59 Proline-rich protein (PRP), 140 Propagation, 84, 153 Proteome, 115–117 Provenances, 137 studies, 105 trial, 80 Pseudo-testcross, 111 Pulp, 67, 72 Q Quantitative trait loci (QTL), 57, 71, 89, 112 Quercus, 89–119, 145 Q. affinis, 97 Q. laurina, 97 Q. petraea, 89 Q. robur, 89 Q. stellata, 92 Q. velutina, 92

165 R Random amplified polymorphic DNA (RAPD), 23, 54, 57, 78, 94, 109, 137, 149 Red alder, 2, 8 Red oak, 91 Reforestation, 2, 55, 154 Regeneration, 102 Repetitive DNA, 113 Reproductive biology, 152 Restriction fragment length polymorphism (RFLP), 49, 104, 137 RNA interference (RNAi), 57 Rootstock, 21, 83 S S-allele, 23 Sandal, 131 Sandalwood, 131 Santalum, 131–141 S. acuminatum, 131 S. album, 131 S. austrocaledonicum, 131 S. boninense, 132 S. ellipticum, 131 S. freycinetianum, 131 S. haleakalae, 131 S. insulare, 132 S. lanceolatum, 131 S. leptocladum, 132 S. macgregorii, 132 S. murrayanum, 132 S. obtusifolium, 132 S. paniculatum, 131 S. spicatum, 131 Scents, 140 Secondary metabolites, 99 Seed abortion, 154 Selection, 54 Self-compatibility, 22 Self-fertilization, 55–56 Self-pollination, 10 Semi-domesticated, 77 Sequence tagged site (STS), 52, 115 Sexual incompatibility, 138 Shade tolerance, 55 Shreve’s oak, 103 Siberian hazel, 17, 34 Silviculture, 107, 140 Simple sequence repeat (SSR), 22, 54, 72 Single-nucleotide polymorphism (SNP), 59, 71 Single-stranded DNA conformation polymorphism (SSCP), 56 SMP. See Supplemental mass pollination Snow, 55 fall, 59 pressure, 59 SNP. See Single-nucleotide polymorphism Soil reclamation, 36 Somatic, 53, 84, 115 embryogenesis, 112, 139 embryo, 112, 139 Spiny chestnut, 20

166

Index

Spring flush, 99–100 SSCP. See Single-stranded DNA conformation polymorphism SSR. See Simple sequence repeat Staminate, 33 Structural, 90, 110 genomic resource, 114 genomics, 114–115 STS. See Sequence tagged site Subtractive hybridization, 115 Sudden oak death, 103–104 Sugi, 49 Supplemental mass pollination (SMP), 56 Symbiosis, 3

T. excelsa, 145 T. verticillata, 145 Triploid, 49 Turkish tree hazel, 18

T Tannin, 99, 100, 117 Tanoak tree, 103 T-DNA, 139 Terpene, 140 Terpene synthase (TPS), 140 Terpenoid, 140 Tetraploid, 49, 84 Tetraploidy, 106 Threatened, 20, 82 population, 70 taxon, 137 Tibetan hazel, 20 Timber, 57 Toiletries, 140 Transcriptome, 115–116 Transcript, 115–116 Transformation, 112 Transgenic eucalypts, 72–73 Tree-age structure, 146 Tree nut, 15 Tribal use, 68 Trigonobalanus, 145–159 T. doichangensis, 145

V Vegetation, 73 destruction, 146 Vegetative propagation, 73, 154 Vesicular arbuscular mycorrhizal (VAM) fungi, 101

U UNESCO, 52 United States Department of Agriculture (USDA), 20, 30, 82, 92, 107 Unweighted pair group method arithmetic average (UPGMA), 149 Ura-sugi, 49–50 USDA. See United States Department of Agriculture

W Weediness, 85 Weeds, 68 Whiskey, 119 White oak, 89 Wild survey, 146 Wine, 119 Winter injury, 34 Wood, 1–6 chemistry, 117 density, 57, 108 products, 118 quality, 57 strength, 57 X Xylem, 103, 116, 117 anatomy, 99