Plant Breeding Reviews (Volume 26)

PLANT BREEDING REVIEWS Volume 26

Plant Breeding Reviews is sponsored by: American Society for Horticultural Science Crop Science Society of America Society of American Foresters National Council of Commercial Plant Breeders International Society for Horticultural Science

Editorial Board, Volume 26 M. Gilbert I. L. Goldman C. H. Michler

PLANT BREEDING REVIEWS Volume 26

edited by

Jules Janick Purdue University

John Wiley & Sons, Inc.

This book is printed on acid-free paper. Copyright © 2006 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 7508400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 7486008, e-mail: [email protected]. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: ISBN 13: 9780471732150 ISBN 10: 0-471-73215-X ISSN: 0730-2207 Printed in the United States of America 10

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Contents

List of Contributors 1. Dedication: George P. Rédei

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Arabidopsis Geneticist and Polymath Csaba Koncz

2. Developing Papaya to Control Papaya Ringspot Virus by Transgenic Resistance, Intergeneric Hybridization, and Tolerance Breeding

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Dennis Gonsalves, Ariadne Vegas, Vilai Prasartsee, Rod Drew, Jon Y. Suzuki, and Savarni Tripathi I. II. III. IV. V. VI. VII.

Introduction Papaya and Papaya Ringspot Virus Development of Transgenic Papaya for Hawaii Development of Transgenic Papaya for Other Regions Breeding Through Intergeneric Hybridizations Development of PRSV-Tolerant Papaya Future Aspects for Developing PRSV-Resistant Papaya VIII. Summary Comments Literature Cited

3. Rol Genes: Molecular Biology, Physiology, Morphology, Breeding Uses

37 38 40 55 63 67 70 73 73

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Margareta Welander and Li-Hua Zhu I. II. III. IV. V.

Introduction The Hairy Root Disease Ri T-DNA and Its Effect on Transgenic Plants Synergistic Effect of Rol Genes Individual Effect of Rol Genes

80 80 82 84 85 v

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VI. Discussion and Conclusions Literature Cited

4. Terminology for Polyploids Based on Cytogenetic Behavior: Consequences in Genetics and Breeding

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Domenico Carputo, Elsa L. Camadro, and Stanley J. Peloquin I. Introduction II. Role of 2n Gametes and Endosperm in the Origin of Polyploids III. Terminology for Polyploids IV. Bases of the New Terminology V. Conclusions Literature Cited

5. Breeding Barley for Resistance to Fusarium Head Blight and Mycotoxin Accumulation

106 108 111 114 121 121

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Thin Meiw Choo I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction Fusarium Species Fusarium Toxins Losses in Yield and Quality Sources of Genetic Resistance Traits Associated with FHB Resistance Breeding Strategies Mutation and In vitro Selection Genetic Transformation Conclusions and Prospects Literature Cited

126 127 129 134 136 139 144 154 155 157 158

6. Using Genomics to Exploit Grain Legume Biodiversity in Crop Improvement

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Sangam L. Dwivedi, Matthew W. Blair, Hari D. Upadhyaya, Rachid Serraj, Jayashree Balaji, Hutokshi K. Buhariwalla, Rodomiro Ortiz, and Jonathan H. Crouch I. Introduction II. Available Genetic Resources of Key Legume Crops

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III. Management and Utilization of Legume Genetic Resources IV. Impact of Genetic Resources in Conventional Legume Breeding V. Molecular-Enhanced Strategies for Manipulating Novel Genetic Variation for Legume Breeding VI. Advanced Applications in Legume Molecular Breeding VII. Conclusions and Future Prospects Acknowledgments Literature Cited

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Subject Index

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Cumulative Subject Index

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Cumulative Contributor Index

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List of Contributors

Jayashree Balaji, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, A.P., India Matthew W. Blair, Centro internacional de Agricultura Tropical (CIAT), A.A. 6713 Cali, Colombia Hutokshi K. Buhariwalla, International Crops Research Institute for the SemiArid Tropics (ICRISAT), Patancheru, PO 502324, A.P., India Elsa L. Camadro, Estación Experimental Agropecuaria Balcarce, Instituto Nacional de Teconología Agropecuaria (INTA)-Facultad de Cs. Agrarias, Universidad Nacional de Mar del Plata (UNMdP) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), C.C. 276, 7620 Balcarce, Bs. As., Argentina Domenico Carputo, DISSPA—Department of Soil, Plant and Environmental Sciences, University of Naples “Federico II”, Via Università 100, 80055 Portici, Italy, [email protected] Thin Meiw Choo, Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario K1A 0C6, Canada, [email protected] Jonathan H. Crouch, CIMMYT, Apdo. Postal 6-421, 066 Mexico, D.F., Mexico, [email protected] Rod Drew, School of Biomolecular and Biomedical Science, Griffith University, Nathan Q4111, Australia Sangam L. Dwivedi, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, A.P., India, [email protected] Dennis Gonsalves, USDA-ARS Pacific Basin Agricultural Research Center, 99 Aupuni Street, Suite 204, Hilo, Hawaii 96720, USA, [email protected] .usda.gov Csaba Koncz, Max-Planck Institute for Plant Breeding Research, Carl-von-LinnéWeg 10, D-50829 Cologne, Germany, [email protected] Rodomiro Ortiz, CIMMYT, Apdo. Postal 6-421, 066 Mexico, D.F., Mexico, [email protected] Stanley J. Peloquin, Department of Horticulture, University of Wisconsin–Madison, 1575 Linden Drive, Madison, Wisconsin, 53706, USA Vilai Prasartsee, Office of Agricultural Research and Development, Region 3, Horticulture Section, Khonkaen, 40260, Thailand Rachid Serraj, Soil and Water Management and Crop Nutrition Section, Joint FAO/IAEA Division, Wagramer Strasse, Room A-2273, A-1400 Vienna, Austria Jon Y. Suzuki, USDA-ARS Pacific Basin Agricultural Research Center, 99 Aupuni Street, Suite 204, Hilo, Hawaii 96720, USA

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Savarni Tripathi, USDA-ARS Pacific Basin Agricultural Research Center, 99 Aupuni Street, Suite 204, Hilo, Hawaii 96720, USA Hari D. Upadhyaya, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, PO 502324, A.P., India Ariadne Vegas, Instituto Nacional de Investigaciones Agrícolas, Centro Nacional de Investigaciones Agropecuarias, Unidad de Biotecnología Vegetal, Apartado 588, Maracay 2101, Estado Aragna, Venezuela Margareta Welander, Department of Crop Science, Swedish University of Agricultural Sciences, P.O. Box 44, 230 53 Alnarp, Sweden, margareta.welander@ vv.slu.se Li-Hua Zhu, Department of Crop Science, Swedish University of Agricultural Sciences, P.O. Box 44, 230 53 Alnarp, Sweden

George P. Rédei in 1992.

1 Dedication: George P. Rédei Arabidopsis Geneticist and Polymath Csaba Koncz Max-Planck Institute for Plant Breeding Research Carl-von-Linné-Weg 10, D-50829 Cologne, Germany

It was a sunny afternoon in early autumn of 1985. I was in the middle of processing my regular plasmid preps, when a distinguished gentleman accompanied by his wife entered our lab at the Max-Planck Institute in Cologne. He wore a moustache that accented his smile and had a neatly trimmed round beard, a splendid grey suit and an attractive necktie. He asked me in formal, polite English about the availability of my boss and friend, the late Professor Jeff Schell. Upon introducing himself, he probably noticed a familiar accent in my answer because he immediately turned the conversation to Hungarian, my mother tongue. Indeed, I was astonished. George Rédei, the legendary geneticist of Arabidopsis, stood in front of me saying that he would spend his sabbatical with us, fulfilling the invitation of Jeff Schell. As a young undergraduate, I saw him once at a congress in Szeged (Hungary), where in 1974 he disproved Ledoux’s infamous DNA transformation studies, which caused much controversy in plant science during the early 1970s. Ledoux, a well-known researcher at that time, reported in Nature that he accomplished genetic complementation of Rédei’s thiamine auxothrophic Arabidopsis mutants with transducing lambda phage DNAs carrying the thi locus of E. coli (Ledoux et al. 1974). Rédei et al. (1976)

Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 1

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provided clear and ultimate genetic evidence that Ledoux’s transformed lines were as thiamine auxotrophic as his original Arabidopsis mutants. Using biochemical methods, F. P. Lurquin (1976) demonstrated at the same time that Ledoux’s transformations did not result in the integration of exogeneous foreign DNA into either cytoplasmic or nuclear genomes of plants. This event terminated an uncertain, but luckily short, period in plant science and opened the way to the development of new powerful transformation technologies. Prominently, this included the use of transferred DNA (T-DNA) of Agrobacterium tumor-inducing Ti plasmids, which was pioneered by Jeff Schell, and later also became my favorite work subject. In 1975, Rédei published an outstanding review highlighting the unique values of Arabidopsis as model organism for genetics. Like many other young fellows interested in plant genetics, I read this review many times. Being motivated to learn more about Arabidopsis and the emerging molecular genetic techniques, I often paid as much as a third of my monthly income to have papers copied in the library despite the justified criticism of my wife. That afternoon in 1985, our discourse with George Rédei and his wife Magdi lasted until late evening. For the first time, I heard many happy and sad stories about their life before and during the 1956 uprising in Hungary, and after their immigration to the USA. To my surprise, I also learned that George’s widely known genetic studies with Arabidopsis did not gain much support in the form of grants for nearly two decades in the USA. In fact, the bitterness over his situation led him to look for help and new ideas in Cologne. Lucky coincidence! We were just about to exploit the integration of Agrobacterium T-DNA into plant chromosomes to generate insertion mutations and fusions between plant genes and T-DNA encoded reporter genes. That evening I decided to shift the focus of our project from haploid Nicotiana plumbaginifolia to Arabidopsis. In retrospective, it was a wise decision. Today the Arabidopsis genome is sequenced and carries over half a million of T-DNA and transposon insertions. Knockout mutations can be identified in any gene, which is not essential for both female and male meiosis. In the following days, George (64 years old in 1985!) and Magdi started to prepare media and germinated seed. In a month, the first experiments yielded Agrobacterium-transformed calli expressing the T-DNA encoded antibiotic resistance markers. The subsequent months, which linked us closely together, were spent on designing methods for regeneration of fertile plants from the transformed tissues, interspersed with enjoyable conversations about various subjects in genetics. From these I quickly grasped that my knowledge of genetics, which I thought was acceptable,

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had some gaps and holes. Thus, as George often said, I realized that there is indeed an essential difference between knowing things and knowing about them. He taught me genetics and I explained to him molecular biology, and no disagreement disturbed this marvelous relationship until his departure. The farewell was cheered by the fact that we saw our first T-DNA tagged Arabidopsis plants regenerating! During the subsequent months, we raised over 8,000 T-DNA transformed plants from tissue culture. However, as Arabidopsis was a novelty in the hands of our gardeners in Cologne, we collected seed from only about 900 plants. My bitterness about this failure turned to a happy smile when I received some news from George again. Upon his return to Columbia, Missouri, he equipped some of his old bookshelves with lamps and converted his air-conditioned old laboratory to a tissue culture facility. He spent his salary buying clay pots and soil, repaired his glasshouse and, despite his heavy teaching load, helped Magdi to regenerate and plant hundreds of plants every week. By the end of 1986, they obtained seed from over 3,000 T-DNA mutagenized plants, and we nearly accomplished the analysis of the first T-DNA tagged gene in the CH42 locus of Arabidopsis chromosome 4. This breakthrough was accompanied by a success of Arabidopsis transformation and characterization of mutant genes also in other laboratories, and brought longawaited NSF support for George. He gained permission to continue working for five more years at the University of Missouri. The success vitalized him. Full of energy and optimism, we continued hunting for new mutants and new principles. He was 70 when he became Professor Emeritus, but he could barely accept that he had to stop all experiments in the lab. He was very bitter. It was my turn to cheer him up. Together with Jeff Schell and Nam-Hai Chua (Rockefeller University, New York), we edited a handbook entitled Methods in Arabidopsis Research. Then, with the help of the European Molecular Biology Organization (EMBO), we organized a training course on Advanced Arabidopsis Molecular Genetics in honor of George’s extraordinary efforts and contributions, which led to the acceptance of Arabidopsis as the model plant of genetics and molecular biology during the early 1990s. Many who contributed to the start of the first international coordinated Arabidopsis project wrote chapters in the book and attended the course as lecturers. I was glad to see him again in the greenhouse teaching students, telling anecdotes, and discussing science with the new generation of Arabidopsis researchers. When visiting him three years later, having in hand our freshly printed Cell paper on the recognition of the essential hormonal function of brassinosteroids, he told me: “Now I see that the new generation does

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this job well and I’m too old to compete. But I think that I found something to do, which might be useful.” George learned computing from his daughter Mari, and started a huge project based on his extraordinarily wide knowledge in genetics and other fields of biology. For 1998 he assembled over 18,000 genetic concepts and terms, 600 illustrations, and over 1,000 references to books and databases on 1,142 pages of a Genetics Manual, which is an extremely useful encyclopedic handbook, the first of its kind. Last, we met personally in 1996, when George as Fulbright Lecturer trained Ph.D. students at the Universities of Keszthely and Budapest in Hungary. Since then and from time-to-time, we communicated only through the Internet. I received few letters from him and I thought that he spent his well-deserved free time with his beloved granddaughters Paige, Grace, and Anne. This turned out to be only partially true. Some days ago, a sweating mailman brought a heavy parcel from him with no letter as usual, but a book inside: George P. Rédei, Encyclopedic Dictionary of Genetics, Genomics and Proteomics. I found his message in the Preface, where he cited the Nobel-laureate geneticist H. J. Muller: “Must we geneticist become bacteriologists, physiological chemists and physicists, simultaneously with being zoologists and botanists? Let us hope so.” Then he continues: “The vision of genetics today is not less then the complete understanding of how cells and organisms are built, how they function metabolically and developmentally, and how they evolved. This requires the integration of previously separate principles based on diverse concepts and tongues.” I fully share George’s optimism and realize that he is “only” 82, yet full of energy. Therefore, I’m looking forward to see the third edition of his book, this universal treasure of genetics!

FROM CHILDHOOD THROUGH WAR AND STALINISM TO IMMIGRATION George Rédei was born in 1921 in Vienna, but he grew up in Hungary, where his father received a job as an agronomist. The large estate on which his father was employed practiced progressive agriculture involving crops, animals, and industrial processing of products. George’s father read Hungarian and German professional journals and during the 1920s contributed to them on various topics, including the hybrid vigor in crosses of maize. He regularly invited scientific consultants, among them Carl Fruwirth, a polyhistor professor of Technische Hochschule in Vienna, who published a Handbuch of Pflanzüchtung, the first modern handbook of plant breeding. Fruwirth, who regularly visited their home,

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was a reserved old man with a long beard and was greatly admired by young George. Perhaps this was the first motivation that induced George to become a polymath. George attended the “Benedictine Realgymnasium” (i.e., high school) in Pápa, where he graduated with an “A” average in 1939. Although his interest was attracted to the humanities, languages, literature and law, his family objected to his pursuing college studies in these fields because of economic reasons. His brother was a highly regarded young painter and illustrator. Thus, his parents felt that George had better start working on their small farm in order to secure financial means for both of them. To find some satisfaction, George performed some breeding experiments with angora rabbits and soon won blue ribbons with several of his animals at national exhibitions. In 1941, he sold his rabbits, making enough money to pay for his further education in the College of Agriculture at Magyaróvár, the alma mater of his father. The war interrupted his studies. He had to work as a lumberjack and later as a forced laborer in the hellish “cinder space” of a large coal-fired power plant. The war took away his brother, who died in a Soviet prisoners’ camp, and also his beloved parents, leaving him completely alone. When he managed to return home with great difficulties in 1945, he found an empty house, but enough grain to start farming. Thanks to his good sense of agronomy, he survived the after-war famine and managed to make enough money to finish college. In 1948, George was classified “kulak,” his farm was confiscated and merged into a cooperative (i.e., the Hungarian version of a Russian “kolhoz”). Accidentally, he learned that the National Institute of Plant Breeding launched a special course to prepare students for a research career in civil service. When he decided to make a trip to Budapest, he found that there was no longer a vacant training spot available. Fortunately, the Chief of the sponsoring agency, the Research Division of the Ministry of Agriculture, was willing to interview him and gave him permission to enroll without stipend. When telling me this story, George added a remark to characterize conditions of life in Stalinistic Hungary: “I was extremely lucky to acknowledge the acceptance, because on the next day my benefactor was arrested by the state security police on the basis of a false accusation and thrown into jail for several years without any court procedure.” George completed the course with the highest grade among his peers and was offered a job with a regular stipend of “excellence” that was almost equal to a monthly salary. One of the greatest influences in George’s life was his acquaintance with Professor Barna Györffy, an excellent geneticist, during the course. As George recalls: “He has been

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the most knowledgeable person I have ever met. He was also the kindest person. It was the most fortunate event in my professional life when Professor Györffy accepted me as a graduate student in 1953 in the Institute of Genetics of National Academy of Sciences in Budapest.” Until 1953, George was employed as research assistant at an experimental station. During the days, he worked in the nurseries under the guidance of an eminent plant breeder, Vilmos Teichmann, who was a student of Erich von Tschermak-Seysenegg, one of the rediscoverers of Mendelian laws. In the evenings, he turned into an accountant and a payroll clerk. Although he disliked it, he did his job very well, which caught the attention of his supervisors. Against his will, soon he was promoted to be a Program Director of Plant Breeding and Genetics in the Ministry of Agriculture with the expectation that he would follow the official doctrines of Lysenkoist genetics. To keep his brain trained, during the weekends he worked as a translator of English, German and French journal articles, and took a day off weekly to escape and read science in the library. Although the power of Hungarian dictator Rákosi had weakened by 1953, George had to carefully select his first research subjects, because modern genetics was banned in Hungary as in the Soviet Union, and Lysenko’s opponents were effectively persecuted. His first research efforts were concerned with the inheritance of fruit weight in tomato. He observed that the average of segregating F2 displayed a geometric mean of parents and constructed a heuristic model, assuming multiplicative effects of alleles and additivity of contributions of loci examined. In his first paper, he showed that this model explained remarkably well his experimental data in about two dozen crosses (Rédei 1949). As a practical breeder at that time, he was advised to work on wheat-rye hybrids and, to his surprise, succeeded in creating some hybrids with Triticum turgidum. These initial breeding studies were continued by his good friend, Dr. Árpád Kiss, an exceptionally skilled research worker, who later produced hundreds of agronomically useful hexaploid and octaploid Triticale lines (Kiss and Rédei 1952; Bona and Kiss 2002). George was interested in practical and theoretical aspects of heterosis, which proved to be important in maize production. Outcrossing in rye was considered not advantageous (Roemer and Rudorf 1939; Kress 1951), but George demonstrated in 60 combinations of 17 diploid rye cultivars that the best hybrids surpassed the parentals by up to 35% (Rédei et al. 1954). In 1953, George was instructed by the Ministry to reproduce the reports of T. D. Lysenko, who claimed that spring planting stably converts winter wheat into a new cultivar, which does not need vernalization anymore. The results showed that only 2 of 387 winter wheat

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cultivars responded to this type of selection with an annually decreasing extent. Both of these cultivars originated from crosses between winter and spring wheats many years before. Therefore, it became obvious that commercial wheat cultivars are genetically not pure lines and thus the Lysenkoist theory failed under exact tests (Rédei, Györffy, Makó and Váróczy 1953). At the request of I. E. Glushchenko, George agreed to a Russian translation and publication of his paper in the Izvestiya Akademii Nauk SSSR. Upon back-translation of the Russian article, it turned out that the editor, Gluschenko, altered the text to fit to the Lysenkoist ideology. After joining Professor Györffy, George continued his studies on the hybridization of distantly related species. With the aid of embryo culture, he could raise hybrids from crosses between Triticum durum and rye (Rédei 1955). As such distant crosses led to endosperm degeneration, he started cytological and physiological studies on embryo and karyopsis development (Rédei and Rédei 1955a). For the first time, he obtained mature wheat plants by a combination of ovary and embryo cultures (Rédei and Rédei 1955b, 1955c). He also made considerable attempts to identify growth factors critical for endosperm development (Rédei 1955c). Some earlier reports indicated that the milky maize endosperm (Nétien and Beauchesne 1953) and coconut milk (Schantz and Steward 1952) contain some growth promoting substances. Therefore, George and his wife Magdi, who worked as a technician in the same institute, started to search for growth promoting substances by fractionation of maize, wheat and barley endosperms, as well as carrot tissue cultures (Györffy et al. 1955). Their ultimate goal was to proceed from single cells to regeneration of fertile plants in vitro, in order to do genetics with plant somatic cells. Roger J. Gautheret (1959) cited these early research efforts at length in the 2nd edition of his plant tissue culture monograph. Even upon discovery of kinetin by Folke Skoog (Miller 1961), G. Doby (1965) referred to George’s thesis and publications in his plant biochemistry book as pioneering achievements in tissue culture research. In 1955, George completed his thesis work and started to look for a new experimental tool that would facilitate research in classical and biochemical genetics. He read some papers of F. Laibach, who successfully regenerated flax from embryo cultures, and thereby also became familiar with Laibach’s cytological studies on a weed called Arabidopsis (at that time Stenophragma thalianum). Laibach favored this plant for genetic and developmental studies because he observed that Arabidopsis has only a few chromosomes (n=5), a short life cycle, high seed yield, and can be easily crossed and cultivated in Petri dishes and test tubes. Prof. Laibach’s collaborator, Erna Reinholz (1947), at the University of

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Frankfurt also succeeded in inducing mutations by X-radiation in Arabidopsis. Given these advantages, George recognized that Arabidopsis is an ideally suited model plant for genetic studies. With the help of Prof. Györffy, he obtained seed samples from Friedrich Laibach in 1956 and had just initiated some radiation experiments, when an uprising broke out in the country. At that time, he worked at the Agricultural Research Institute in Martonvásár, close to Budapest. When the Russian tanks circled the capital in November, many of the staff decided to departure to a sugarbeet-breeding institute at Sopronhorpács, in the vicinity of the Austrian border. After the last dramatic call for international help by the prime minister, Imre Nagy, was broadcasted, George and others decided to cross the border. They lived in a refugee camp for a couple of months until January 1957, when George got permission to immigrate to the USA and work on a temporary basis as an assistant professor at the University of Missouri. As he was passing through the agricultural safety check, he honestly declared that he carried in his pocket a vial with Arabidopsis seeds. Fortunately, the officer never heard about this unimportant plant and did not find it in the list of prohibited materials.

ARABIDOPSIS: A STRANGER IN THE NEXUS OF PLANT GENETICS IN COLUMBIA, MISSOURI At the University of Missouri, Dean John H. Longwell assigned the former laboratory space of Barbara McClintock to George and generously permitted him to conduct research in any field of genetics of his choice. George continued his experiments from the point where he had finished in Hungary. Radiation mutagenesis offered a possibility to gain insight into the nature of the gene as anticipated by Timoféeff-Ressovsky, Zimmer and Delbrück (1935). Now, in addition to chemical mutagens, which became popular at that time (Westergaard 1957), radiomimetic agents and carbonyl compounds discovered by Auerbach and Robson (1944) and Rapoport (1946), respectively, were all within his reach. Next to his door were located L. J. Stadler’s old X-ray machine and greenhouse. With his appointment, he received an annual salary of $5,000 and a research budget of $300 to equip the lab and cover all operating expenses for a year. In a storage area, he found a few pieces of glassware and test tube racks, and some clay pots in the greenhouse. He began to work within days. Once, he told me the following story from those days: “Shortly after my appointment, a new department chairman arrived. Everybody forgot to introduce me to him, as I looked very busy. About four months later the chairman, Emmett Pinnell, came over to my

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George P. Rédei and Barbara McClintock in 1978.

lab. I had to admit not knowing who he was! He actually enjoyed my response and quipped: ‘I must be a damn good chairman if you do not know about me even after 4–5 months.’ And he was! He had been always most helpful whenever I needed him, but never imposed any burden on me. Although very few people occupied the building, I found a very vibrant research activity. I remain indebted to Ernie and Lotti Sears for the intellectual atmosphere secured around them during the years and until their death.” George’s primary aim was to induce auxotrophic mutations in Arabidopsis, although several of his colleagues warned him that he might follow a mirage. After screening over 2,000 irradiated single families, he found some interesting mutations, but none of them responded to complete nutrition. The July temperature was merciless in the greenhouse. Thus, on his wife’s urging, he took a week of vacation in the Rockies. Upon return, anticipating the worst, he rushed to look at his plants growing in test tubes on minimal medium. One of the families segregated several bleaching plants, but with an odd ratio, which was puzzling. When planting the same family on various vitamins, he observed that thiamin restored normal growth of the bleaching mutants. Further analysis revealed that the auxotrophs were deficient in the synthesis of 2,5dimethyl-4-amino pyrimidine. Within weeks, he solved the rest of the

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puzzle. The pyrimidine auxotrophy locus was closely linked to an unusual female gametophytic factor mutation, which caused approximately 1:1 segregation (Rédei 1960). Within a year, he identified a mutation that caused auxotrophy for 4-methyl-5-β-hydroxyethyl thiazole, another precursor of Vitamin B1 (Rédei 1962a). By 1963, he characterized five additional thiamin auxotrophs and submitted a manuscript to the American Journal of Botany (Rédei 1965b). As George remembers: “This paper was lost in the editorial office and its publication was delayed by two years. One of the reviewers commented that there are numerous auxotrophic mutations in higher plants. Upon my request of naming at least one of them, the Editor informed me that the reviewer made a mistake, as he confused Arabidopsis with Neurospora. Actually, even 40 years later, these and other, subsequently identified thiamin mutants of Arabidopsis are the only obligate auxotrophs in angiosperms except tomato.”

EXPLOITING ARABIDOPSIS MUTANTS TO UNCOVER NOVEL GENE FUNCTIONS AND GENETIC PRINCIPLES In 1965, Steve Li joined Rédei’s laboratory. By 1967 they had accumulated over 60 thiamin auxotrophs, but failed to find auxotrophy in any other metabolic pathway suggesting a functional redundancy of corresponding genes (Li et al. 1967). The thiamin mutants occurred at several loci and ranged from a few leaky mutants to obligate auxotrophs (Rédei 1965b; Li and Rédei 1969e). They provided very useful tools for the study of reversion (Rédei 2003), estimation of mutation rate (Li and Rédei 1969c; Rédei and Li 1969b), the first direct observation of glucose effect in a higher plant (Li and Rédei 1969d), as well as for the study of allelic complementation (Li and Rédei 1969b), and analysis of overdominance (Li and Rédei 1969a; Rédei 1962b). Li and Rédei continued to characterize the female gametophyte factor GF1. The gf1 mutation showed linkage with the pyrimidine (py1) mutation. By that time, George identified five mutations in the same linkage group. This was the first reported case, when linkage was used in Arabidopsis for genetic analysis. The segregation ratio for py1 in crosses with wild type was 12923:4290, but in crosses with the gf1 mutant it was 5726:5968. All other linked markers in the same chromosome displayed distorted ratios according to their map distance from the GF1 locus, yielding the first ever constructed Arabidopsis chromosomal linkage map with the gf1–er1–py1–as1–su1 markers. It was expected that the fruits, in which the segregation of gf1 mutation was

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examined, should show 50% seed set due to total absence of female transmission. By examining over 400 fruits with more than 12,400 seeds, however, George found that the total seed set exceeded 60%. This appeared an impossible outcome for Mendelian segregation. Sectioning the ovules revealed twin megaspores and embryo sacs in some of the plants, suggesting that the higher than 50% seed set must have been brought about by megaspore selection. The megaspore tetrads with wild type GF1 constitution in the basal position were preferentially selected to contribute to the formation of the egg (Rédei 1965a,c). During the early 1960s, George identified some X-ray mutants that displayed late onset of flowering and altered photoperiodic responses. Some of the mutants produced 20 times higher dry weight and 10-fold higher seed yield than the wild type. All late flowering mutants (gi1, gi2, ld1 and co1) were recessive under long-day conditions, but co1 was unusual, as it displayed a change of dominance under short day. As late flowering ecotypes of Arabidopsis are predominant in natural ecological conditions, George performed a population genetic analysis to test their selective value under laboratory conditions. In segregating families started with F1 hybrids of wild type and late flowering mutants, the wild type was practically eliminated (i.e., about 99% of the survivors were mutants) within about ten generation cycles. The estimated selective advantage of three independent late-flowering mutations was 1.3, 2, or >2, respectively. This was a very unusual finding for any mutation, particularly for X-ray induced mutations (Rédei 1962c). Consequently, George became interested to determine the nature of genetic change in these mutants. He could not detect larger deletions by cytological analysis, and the transmission of mutant alleles appeared perfectly normal (Rédei and Steinitz-Sears 1961). However, he observed that 8-azaadenine (Hirono and Rédei 1966b) and 5-bromodeoxycytidine or 5bromodeoxyuridine dramatically promoted the onset of flowering in some late-flowering mutants under short days (Hirono and Rédei 1966c). Arabidopsis is not an obligate long-day plant, but the onset of flowering is much delayed under short-day conditions. By characterizing the physiology of flowering response, George found that flowering readily took place in about 7 weeks in complete darkness in glucose supplemented liquid medium. After 9 weeks of culture in total darkness, some plants even developed fruits with seeds, which however failed to germinate. The time to develop flower primordia is usually characterized by the number of rosette leaves. According to George’s observations, the lateflowering mutants did not significantly differ from the wild type. This observation on the ld1/ld1 homozygote was particularly surprising because this mutant never developed flower primordia under short days

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(i.e. 9 hours daily illumination). Therefore, George concluded that light is not the epigenetic factor required for the synthesis of flower-inducing substance. Rather, he proposed that prolonged cycles of daily illumination are conducive to the inactivation or breakdown of an inhibitory protein or metabolite synthesized under short-day conditions. As the response of wild type and late-flowering mutants in dark cultures were similar, he argued that an early step of epigenesis, leading to initiation of flowering, is probably controlled by the length of illumination, and not by locus-specific florigens. George tried to exploit his observation that halogenated nucleoside analogs dramatically shortened the time required to induce onset of flowering in all late-flowering mutants, but not in ld1, which did not respond to these analogs under short days. He performed extensive biochemical genetic studies with radioactive bromodeoxyuridine (BrDU) by fractionating methanol-acetone water extracts from wild type and mutants using thin layer and radioscanning gas chromatography. Although he failed to detect differential metabolism of BrDU in the lateflowering mutants, he found that BrDU could replace about 18–26% of thymidine residues in the DNA without causing a substantial mutagenic effect. Upon feeding the plants with BrDU and both 14C-arginine and 14C-valine, he found an increased incorporation of radioactive carbon into a non-histone protein fraction (Rédei et al. 1974). Based on our current knowledge, of course, the limitations of these experiments are evident. However, one should consider that these studies were pursued in the early 1970s before the birth of molecular biology. Nonetheless, the granting agencies did not appreciate George’s excursions to biochemical genetics. As he recalls: “In retrospect, I have no doubt that we were on the right track. The information on eukaryotic transcription factors was very limited at that time. The 1975 edition of the classic biochemistry book from L. Stryer did not even mention this term. In 1969 the NSF terminated the support of my research based on the recommendation of the Genetics Panel, which was convinced that it is not worthwhile to develop Arabidopsis because prokaryotes are more likely to contribute significantly to new knowledge. During the following decades I relied on the $1,800 annual combined research and teaching support of the University of Missouri.”

STUDIES ON VARIEGATION By 1964, George identified several chlorina (ch) mutations, which resulted in reduced chlorophyll b production (Hirono and Rédei 1963b;

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Rédei and Hirono 1964). One of these ch mutations was mapped to chromosome 4 in a close distance from another mutation, which caused dwarfism with dark green leaf color. X-irradiation of plants heterozygous for these and other linked markers revealed the presence of single and twin spots on the leaves. In a few cases, seed-bearing shoots could be obtained from the mutant sectors, facilitating classical genetic tests on the sectors. This was of considerable interest because evidence for mitotic recombination did not exist in plants at that time, except for a single case suggesting somatic translocation in maize (Jones 1938). Using the ch and dwarf mutations in linkage with proper flanking markers, George demonstrated that during premeiotic recombination the genetic exchange generally, but not always, involved a reduced transmission of chromosome strands participating in the exchange (Hirono and Rédei 1965b). Thus, Arabidopsis was the first higher plant with verified Xray–induced premeiotic recombination (Hirono and Rédei 1965a). In the early 1960s, George also studied a recessive mutation, immutant (im), which caused an extremely high variegation depending on the intensity and duration of illumination (Rédei 1963b). Initially, he assumed that this phenotype was attributed to an active transposable element. However, this hypothesis turned out to be invalid because the progeny of green and white sectors were not different. When predominantly white mutant lines were grown in test tubes on 6-azauracil–containing medium, the chlorophyll and carotenoid contents of leaves increased. Exposure of seeds or plants to X-irradiation also conspicuously increased the extent of green sectors (Rédei 1967b). The white-green variegation persisted through mitotic divisions, but vanished after meiosis (Rédei 1967h). As expected, electron microscopic studies showed that chloroplasts in white cells lacked grana stacks of thylakoid membranes, but also the green sectors displayed abnormal thylakoid differentiation (Chung et al. 1974; Rédei 1975a). Further studies detected increased ribonuclease activity in the nuclear and chloroplast fractions of variegated leaf tissues, but not in plants that were grown in vitro in the presence of 6azauracil (Rédei 1967a,b). In analogy with the 6-azauracil effect on the hereditary human syndrome orotic acidurias (Pinsky and Kroth 1967), George observed that 6-azauracil elevated the orotidine-5′-monophosphate decarboxylase activity in the variegated im leaves (Chung and Rédei 1974). Today, we know that IM1 encodes a chloroplast homolog of mitochondrial alternative oxidase, which likely serves as a redox component in phytoene desaturation (Aluru et al. 2001; Joët et al. 2002). How IM1 participates in the compensation of photooxidative damage is an intriguing question, which still awaits explanation, as does George’s 35-yearold observations on the compensatory effect of 6-azauracil (Rédei 2003a).

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PLASTID MUTATOR Inspired by Barbara McClintock’s work on transposable elements, George aimed to activate silent transposons by chemical and X-ray mutagenesis, and thus continued to analyze several other variegated Arabidopsis mutants. In maize, Rhoades (1943) described an intriguing nuclear mutation, iojap, which causes variegation by affecting chloroplast differentiation. Röbbelen (1966) found a similar mutation (am) in Arabidopsis, which as iojap was characterized by alterations in chloroplasts development. It was conceivable that mutation of a nuclear gene could result in either activation of a transposable element or defective DNA repair in the chloroplasts. As transposable elements were already well studied in prokaryotes and chloroplast was considered of prokaryotic origin (Thorsness and Weber 1996; Millen et al. 2001; Traven et al. 2001), this assumption appeared to be worth testing. By screening for variegated mutants, George isolated three independent mutant alleles of a gene in chromosome 3, which he named CHM, chloroplast mutator (Rédei 1973e). He described the first instance of testing allelism between mutations affecting chloroplast biogenesis. The classical allelism test is based on non-complementation/complementation of recessive nuclear mutations. However, as the chm mutation affected an organellar (i.e., plastid) phenotype, the conventional cross did not provide an unambiguous answer. Therefore, George combined one of the alleles, chm2, with an unlinked as1 mutation affecting leaf shape and then crossed chm2, as1 plants with wild type. The resulting F1 was then crossed with plants, which carried the second allele, chm1, in linkage with the gl1 mutation blocking leaf trichome differentiation. In case the two independent chm mutations would have been non-allelic, the testcross progeny would not have displayed variegation. However, half of the testcross progeny was variegated, indicating allelism of chm1 and chm2 mutations. The chm mutation resulted in inhibition of chloroplast biogenesis at various stages, yielding white leaf sectors (Rédei and Plurad 1973). George demonstrated that after removal of the nuclear chm mutation and several cycles of selfing, homoplastidic mutants could be sorted out, which synthesized very low amounts of chlorophyll, but could be propagated on sugar-supplemented media. Despite intensive efforts, George could not detect rearrangements and point mutations of chloroplast DNA in the homoplastidic chm mutants. Today we know that the CHM gene encodes an Arabidopsis homolog of E. coli MutS protein that is involved in mismatch repair and recombination in bacteria. Remarkably, chloroplast defects of chm lines show co-inheritance with specific rearrangements in the mitochondrial DNA, which highlights a

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yet-unknown aspect of coordinated regulation of chloroplast and mitochondrial biogenesis in higher plants (Martinez-Zapater et al. 1992; Abdelnoor et al. 2003).

FRUCTOSE EFFECT During his studies of chlorophyll deficient mutants (i.e., many of which could only be maintained on sugar-containing media), George observed that wild type Arabidopsis, Hylandra, and Cardaminopsis plants showed retarded growth on media containing 3% fructose (Rédei 1974e). As he was aware of hereditary fructose intolerance in humans (Froesch 1972), which was dramatically revealed by increased application of fructose as low caloric value sweetener, he made a short excursion to study the observed fructose effect. He found that fructose underwent substantial degradation during autoclaving, and that plants grown on autoclaved fructose media exhibited reduced amounts of chlorophylls, carotenoids, phospholipids, pyruvic and glutaric acid. By monitoring the activity of various enzymes involved in fructose metabolism, he found a significant reduction in the condensation reaction of fructose-1,6 diphosphate aldolase. He showed that fructose-1-phosphate aldolase, which is defective in human hereditary fructose intolerance, had no detectable activity in the studied crucifers, but the observed growth defect correlated with a suppressed synthesis of fructose-1-6 diphosphate aldolase in plants grown on autoclaved fructose media (Rédei 1973f,g, 1974e).

MUTAGENESIS STUDIES When choosing Arabidopsis as a genetic model, George realized that this plant is a better organism for studies of the mutation process in the diploid germ line than the majority of plant species. The major advantage of Arabidopsis is that it has a short life cycle and small size, which facilitates screening of very large populations. Being a good geneticist whose research budget was severely limited, George paid particular attention to developing procedures that yielded the largest number of independent mutations at the lowest cost. Therefore, he worked out a simple mathematical procedure to optimize the size of M1 and M2 populations in mutation experiments. He showed that theoretically it was most effective if only a single offspring was tested in M2 from each cell of the germline. This was seemingly a controversial idea, because if a recessive mutation segregates in a Mendelian manner, only 0.25% of the

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tested sample would display the sought-after phenotype. By contrast, testing 24 M2 progeny would increase the probability of detection of a recessive mutation to 0.999. Although this logic is of course correct, with simple mathematics George showed that examining a large number of offspring in the segregating M2 generation is actually deceptive under experimental conditions. He argued that the frequency of induced mutation at a particular gene is low (e.g., ~10–3), thus only a small fraction of M1 individuals will carry the mutation desired. He stressed therefore that it is most important to treat with mutagen as large a number of germline cells as required by the expected rate of mutation and the expected rate of recovery of that mutation. Because in each germline cell only one mutation per gene is expected, and a second one is unlikely to occur more frequently than the product of the two independent mutation events (e.g., at the above assumed frequency at ~10–6), it is best to use a large M1 and examine only a single individual per each cell of the germline in the M2. Since the production cost of a plant in the M1 and M2 generations may not be identical, he worked out the principles of how to make decisions in planning the optimal size of M1 and M2 in mutation experiments (Rédei 1974h, 1981; Rédei et al. 1984b; Rédei and Koncz 1992). Till today, plant researchers follow these guidelines to ensure the efficiency of low-cost mutant selection and screening experiments. George demonstrated that these procedures can suitably be applied not only for induction of genetic variations to obtain chromosomal markers and mutations for plant breeding purposes, but also for screening of T-DNA knockout mutations (Feldmann and Marks 1987), and testing chemical, physical, and environmental mutagens (Réde et al. 1984b). In a test series, he studied 42 compounds, including mutagens, carcinogens and genetically inactive chemicals. The efficacy of mutagenic tests with Arabidopsis was compared with other prokaryotic and eukaryotic mutagen assays conducted within an international project by 62 laboratories. The outcome indicated a remarkable performance in the sensitivity and accuracy of the Arabidopsis test (Rédei et al. 1984c). Based on the principle of fluctuation test developed by Luria & Delbrück (1943), which is generally credited as one of the most important contributions of bacterial genetics (Stent and Calendar 1978), George worked out a modified fluctuation test for Arabidopsis. In this mutagen test, George used random sampling of an average of 2 progeny per M1 plants yielding several hundred M2s, which along with wild type controls were planted in hundreds of small pots in the greenhouse. The distribution of low-frequency-induced and spontaneous mutations observed in the individual pots was evaluated by fitting to the Poisson and expanded negative binomial distributions. The latter procedure

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proved to be better, as the negative binomial reflected a joint outcome of separate Poisson series. The detection of mutations depended on the occurrence of mutations, sampling of the mutant cell progeny, the collection of seed output of M1 plants, and other random events. The difference between control and mutagen-treated series was ascertained by the log-likelihood method. The application of the fluctuation test permitted the determination of extremely low mutation frequencies with an unprecedented precision at high statistical significance (Rédei et al. 1984b; Acedo and Rédei 1984; Rédei and Koncz 1992). In addition, using half of the probability of Hardy–Weinberg distribution in the populations, George was able to estimate the frequency of undetected mutations (Rédei and Koncz 1992). Based on accurate calculation of induced mutation rates (i.e., either per locus or per genome), George estimated in 1982 that the Arabidopsis genome encodes 27,813 genes (Rédei 1982c). Sequencing the genome 18 years later suggested that Arabidopsis has 25,498 protein coding genes (Arabidopsis Genome Initiative 2000). However, reannotation of the genome by April 2003 (ftp://ftp.tigr .org/pub/data/a_thaliana/ath1/Arabidopsis_release.v4.0.README) revealed that Arabidopsis contains 27,170 protein-coding genes, only a few hundred less than George estimated by measuring the mutation rates. As only a few biological assays are available for detection of aneuploidy caused by environmental factors, George worked out a genetic test to detect chromosome disjunction. He took advantage of recessive mutations characteristic in the Neatby’s virescent gene v1 in the short arm of chromosome 3B of hexaploid wheat. E. R. Sears has earlier identified a centromere defect in this stock leading to nondisjunction. The v1 allele is a recessive suppressor of pigmentation and displays hemizygous ineffective condition. The homozygotes express cream color, the hemizygotes are normal green, and the trisomics are white. On this basis, both losses and duplications of a locus at that critical chromosomal location are easily detectable. The appearance of single or twin sectors indicate the recovery of one or both products of nondisjunction, as well as deletions or duplications, in response to mutagenic treatments (Rédei and Sandhu 1998).

GENETIC TRANSFORMATION In the absence of grants, George had only $3,500 of research support from the Missouri Agricultural Experiment Station during the early 1980s, which was obviously insufficient to perform modern research. In 1983, Jeff Schell, the Director of the Max-Planck Institute (Cologne), visited his

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laboratory and invited him to Germany. In 1985, he spent six months in Cologne establishing with us the Agrobacterium-mediated transformation and T-DNA insertion mutagenesis techniques, which provided a simple tool for isolation and functional characterization of mutagenized Arabidopsis genes (Rédei et al. 1988). In 1989, we showed that the TDNA is preferentially integrated into potentially transcribed chromosomal loci of transformed plants, indicating that it is a very efficient mutagen (Koncz et al. 1989a). In 1990, we published the first characterized T-DNA insertion mutation in the CH-42 locus that encodes the ATP-binding subunit of protophorphirine IX Mg2+-chelatase involved in chlorophyll biosynthesis. George has located the T-DNA tagged gene, which he named CS, to chromosome 4 by trisomic analysis and allelism test with the X-ray-induced ch42 mutation. Subsequent mapping involved scoring over 80,000 seeds in F2 fruits to identify the yellow cs progeny, which provided a resolution of about 10 to 25 kb in terms of physical distance (Koncz et al. 1990a). Our further experiments concentrated on the study of T-DNA integration mechanism. Sequence analysis of T-DNA junctions and genomic target sites indicated that the T-DNA is integrated into plants’ chromosomes by illegitimate recombination. The T-DNA recombination model suggested that the VirD2 protein, which is covalently attached to the 5′-end of single-stranded T-DNA intermediate (i.e., the integrating T-strand), plays a chief role in the recognition of chromosomal target sequences (Mayerhofer et al. 1991; Koncz et al. 1994). Although it took some time to explain this observation, recently we could demonstrate that the VirD2 protein interacts with the TATA-box binding TBP protein and with a protein kinase (CAK2) component of the RNA polymerase II associated TFIIH general transcription factor complex. As these proteins are involved in transcription-coupled DNA repair, the interaction of VirD2 with TBP and TFIIH well correlates with the observed frequent integration of T-DNA into transcribed chromosomal domains (Bakó et al. 2003). Subsequently, we searched with George for insertion mutations causing so-called genetic dwarfism, which is characterized by cell elongation defects that cannot be corrected by any known plant hormone. We have found a beautiful dwarf mutant that showed constitutive photomorphogenesis in the dark (i.e., except for chlorophyll biosynthesis, the mutant developed as light-grown plants in the dark). The analysis of TDNA tagged cpd (CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARF) locus revealed that the gene encoded a cytochrome P450 enzyme, which showed remarkable homology to animal steroid hydroxylases. As it was known for a while, primarily from the work of Japan-

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ese scientists, that crucifers and other plants produce growth-promoting steroids, called brassinosteroids, we performed some chemical complementation experiments. These studies showed that the CPD gene encoded P450 enzyme, CYP90A1, is a steroid C23-hydroxylase, which is essential for conversion of cathasterone to teasterone in the biosynthesis pathways of brassinosteroids (Szekeres et al. 1996). These results indicated that brassinosteroids are essential plant hormones and opened a new research field aiming at the study of regulatory functions of plant steroid hormones. As we were primarily interested in regulatory mutants that affect multiple qualitative and quantitative traits, we started to characterize a TDNA-tagged gene that we called PRL1 (PLEIOTROPIC REGULATORY LOCUS 1). The prl1 mutant displayed many alterations in normal metabolism, development, hormonal regulation and gene expression, which all were caused by inactivation of a nuclear regulatory protein carrying seven WD-40 (tryptophan-asparagine) repeats (Németh et al. 1998). From the breeders’ point of view, it was highly interesting to learn why this single gene mutation causes altered leaf development, overproduction of chlorophyll and anthocyanins, increased production of free glucose, fructose and sucrose, and accumulation of starch, as well as hypersensitivity to cold stress and plant hormones auxin, cytokinin, ethylene and abscisic acid. A key to understand the regulatory function of PRL1 derived from the observation that many stress-regulated genes, which are either positively or negatively modulated by glucose repression, showed highly increased expression in the prl1 knockout mutant. PRL1 was found to interact with several important signaling proteins, among them with AMP-activated protein kinases (AMPKs) that play a central and conserved role in the regulation of glucose repression and stress responses in eukaryotes (Bhalerao et al. 1999). PRL1 turned out to be an inhibitor of plant AMPKs, which were found in so-called transcription co-activator complexes. In these co-activator complexes, AMPKs interact with histone arginine methylases, which are also PRL1-binding regulatory proteins. In addition to modulating transcription, PRL1 also affects another function of AMPKs. In plants, the AMPK kinases occur in stable association with the 26S proteasome and SCF (Skp1-cullin-Fbox) E3 ubiquitin ligases that control ubiquitination dependent proteasomal degradation of important regulatory factors (Farrás et al. 2001). By inhibiting the functions of AMPKs, PRL1 plays a role in the regulation of stability of several transcription factors involved in hormonal and metabolic signaling. To identify T-DNA mutagenized genes on a large scale, in 1999 we started to sequence the junctions of T-DNA tags isolated from George’s

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mutant population. We surveyed 1000 T-DNA insertions regarding their chromosomal locations. The data showed that only 4.7% of the insertions landed in interspersed, centromeric, telomeric regions, and rDNA repeats. By contrast, open reading frames contained 35.4 % of the inserts, and of these 62.2% landed in exons and 37.8% in introns. In accordance with our previous observations, we found preferential integration of the T-DNA in 5' and 3' regulatory domains of genes. In summary, almost half of the T-DNA insertions caused knockouts in Arabidopsis (Szabados et al. 2002). To reach our ultimate goal, we completed a saturation T-DNA mutagenesis by generating 92,600 transgenic plants that carry over 220,000 insertions in the Arabidopsis genome. This collection now allows the identification of a gene mutation with an estimated probability of 0.77 (Rios et al. 2002), which was one of our ultimate goals when we started the T-DNA mutagenesis project with George in 1985.

AFTER “RETIREMENT” In 1991, George retired and donated approximately 6,700 mutant stocks and genetic constructs to the Arabidopsis Resource Center at Ohio State University. The Columbia wild type and Landsberg erecta (Ler) stocks from his laboratory are the most widely used standard types for research. The former became the first higher plant with completed genome sequence. Besides experimental genetics, George has always been interested in the history of the emergence of genetic ideas and their experimental foundations. As he said: “I always felt that I need some familiarity with the developmental course of genetics as a whole and not only with the history of research of my objects, to be able to make decisions regarding my goals. Scientific research is distinguished from crafts by the originality of its approaches and ideas. Good science does not repeat its facts, but aims at the unexplored. Historical retrospect guides the research workers toward new horizons.” In 1973, George composed an annotated list of 477 papers on genetics that, according to his judgment, have made the most important contributions to the development of the field (Rédei 1974a). As he recalls the fate of this manuscript: “Two journals rejected it by editorial comments that such a paper would not be of interest to the readers. Eventually, I submitted it to Professor Hans Stubbe, who himself was a highly regarded historian of genetics. He accepted it for Biologisches Zentralblatt, which did not have a particularly high impact factor (i.e. 0.224). Yet, after it appeared in print, I received more

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than 400 requests for reprints, indicating that not all editorial judgments are perfect.” Recently, in two invited papers George has surveyed some of the historical oddities of genetics along with some of the current developments (Rédei 2002) and controversies, as well as the future potential of longterm selection for quantitative traits (Rédei 2003b). His first comprehensive review on biology and genetics of Arabidopsis appeared in print in 1970 (Rédei, 1970c) and this was followed by updated reviews (Rédei 1975a,c,d). The latter paper in Annual Review of Genetics attracted to Arabidopsis a new generation of research workers, who made major contributions to the field (Pennisi 2000). The major historical papers on genetics of Arabidopsis were summarized in Rédei (1992c), whereas a comparative review on the historical development of Arabidopsis genetics within the context of relevant milestones of genetics was published in 1994 (Koncz and Rédei 1994). He has also written tributes to L. J. Stadler (Rédei 1971a), A. Sturtevant (Rédei 1971d), B. Györffy (Rédei 1986) and E. R. Sears (Rédei 1992) analyzing the impact of their contributions to genetics.

TEACHER AND EDITOR George is not only an excellent geneticist but, as I also experienced, a warm-hearted, precise and honest teacher. Through his career at the University of Missouri, he always provided detailed lecture notes to students in all classes he taught. He taught formal one-semester courses of Basic Plant Genetics, Analytical Genetics, Genetic Engineering, Evolution of Genetic Concepts, Plant Cell and Tissue Culture, and Genetic Bases of Physiological Responses. He lectured for three years in a graduate-level course at the ELTE University of Budapest, and was a Fulbright Lecturer for a semester in Keszthely, Hungary. His book Basic Plant Genetics (1,191 pages) was published in five editions, each time revised and updated. His textbook Genetics (736+36 pages) was translated into Hungarian and Chinese, and during the past decades has been used by over 50,000 students worldwide. In 1998, he published his Genetics Manual. Current Theory, Concepts and Terms (1,141 pages). The Encyclopedic Dictionary of Genetics, Genomics, and Proteomics (1,392 pages), one of the most comprehensive books of its kind, appeared in print in 2003. For four years, George was the founder and editor of the Hungarian plantbreeding journal Növénytermelés, and served as co-editor of the now defunct Arabidopsis Information Service, the first and only journal of Arabidopsis researchers during the pioneering time. In 1969, George

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initiated with Gordon Kimber the annual Stadler Genetics Symposia, and then organized its meetings and edited its proceedings for 15 years in Columbia, Missouri.

IMPACT For nearly twenty years, George Rédei was almost alone in the USA to appreciate the extraordinary value of his beloved genetic model organism Arabidopsis. Today more than 12,000 people work in the field of Arabidopsis research, contributing to progress in plant biology, genetics, breeding and biotechnology (Somerville and Koornneef 2002). By 2003, more than twice as many molecular genetics papers were recorded in Medline using Arabidopsis rather than maize, another higher plant with a much longer tradition in genetics. In one of his papers (Rédei 1992c) about science history, George says: “History has a meaning only in shaping the future.” This is exactly what he achieved through the history of his scientific career. And the use of Arabidopsis in plant genetics will continue to provide tribute to his pioneering contributions. Personally, I remain ever indebted for his help as friend, teacher, and mentor.

PUBLICATIONS OF GEORGE P. RÉDEI Rédei, G. 1949a. Inheritance of fruit weight in tomatoes. Botanikai Közlemények. Budapest (Abstr.). Rédei, G. 1949b. Elements of biometry, p. 10–45, In: Györffy, B. (ed.), General Biology, Univ. Agricult., Budapest. Kiss, Á., and G. Rédei. 1952. Experiments to produce wheat-rye hybrids (Triticale). Növénytermelés 1:67–84. Rédei, G. P. 1952. Quis non malarum haec inter obliviscitur. Növénytermelés 1:265–266. Kiss, Á., and G. Rédei. 1953. Experiments to produce rye-wheat hybrids (Triticale). Acta Agr. Acad. Sci. Hung. 3:257–276. Rédei, G., B. Györffy, J. Makó, and E. Váróczy. 1953. Producing spring wheat out of winter wheat. Növénytermelés 2:227–237. Rédei, G. 1954. In vitro culture of cereal embryos. Thesis. Institute of Genetics, Hung. Acad. Sci., Budapest. Rédei, G., B. Györffy, J. Makó, and E. Váróczy. 1954. Prevrashchenye ozmoi pshenyci v yarowiyou. Izvestiya. Akad. Nauk S.S.S.R. Ser. Biol. 5:46–54. Rédei, G. P., E. Váróczy, and G. Rédei. 1954. Experiments on crossing rye populations. Növénytermelés 3:181–202. Györffy, B., G. P. Rédei, and G. Rédei. 1955. Substance de croissance du maize laïteux. Acta Bot. Acad. Sci. Hung. 2:57–76.

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Rédei, G. 1955. Producing hybrids of Triticum durum abyssinicum × Secale cereale by the aid of embryo culture. Növénytermelés 4:365–367. Rédei, G. P., and G. Rédei. 1955a. On the development of wheat caryopses. Növénytermelés 4:133–140. Rédei, G. P., and G. Rédei. 1955b. Developing wheat embryos excised from ovaries cultured in vitro. Experientia (Basel) 11:387–388. Rédei, G. P., and G. Rédei. 1955c. Rearing wheats from ovaries cultured in vitro. Acta Bot. Acad. Sci. Hung. 2:183–186. Rédei, G. 1956a. Lelley János—Rajháthy Tibor: A búza nemesítése. Növénytermelés 5:117– 118. Rédei, G. 1956b. Techniques in plant tissue culture. p. 15. In: I. Törö (ed.), Laboratory handbook of clinical medicine. Budapest. Rédei, G. 1958. General biology (about 200 entries). In: Encyclopedia of agriculture. Mezo"gazdasági Kiadó, Budapest. Rédei, G. 1960. Genetic control of 2,5-dimethyl-4-aminopyrimidine requirement in Arabidopsis thaliana. Genetics 45:1007 (Abstr.). Rédei, G., and L. M. Steinitz-Sears. 1961. X-ray mutants with high selective advantage in Arabidopsis. Genetics 46:892 (Abstr.). Rédei, G. P. 1962a. Genetic block of “vitamin thiazole” synthesis in Arabidopsis. Genetics 47:979 (Abstr.). Rédei, G. P. 1962b. Single locus heterosis. Z. Vererbungslehre 93:164–170. Rédei, G. P. 1962c. Supervital mutants of Arabidopsis. Genetics 47:443–460. Hirono, Y., and G. P. Rédei. 1963a. Somatic recombination in Arabidopsis. Genetics today, Proc. XI. Int. Congr. Genet., The Hague, Vol. 1, p. 15 (Abstr.). Hirono, Y., and G. P. Rédei. 1963b. Multiple allelic control of chlorophyll b level in Arabidopsis thaliana. Nature 197:1324–1325. Rédei, G. P. 1963a. A genetic oscillatory mechanism in Arabidopsis. Genetics today, Proc. XI. Int. Congr. Genet., The Hague, Vol. 1, p. 41 (Abstr.). Rédei, G. P. 1963b. Somatic instability caused by a cysteine-sensitive gene in Arabidopsis Science 139:767–769. Hirono, Y., and G. P. Rédei. 1964a. Somatic recombination induced by X-rays. Arabidopsis Inf. Serv. 1:15–16. Hirono, Y., and G. P. Rédei. 1964b. Somatic screening technique for alterations at specific loci Arabidopsis. Inf. Serv. 1:21. Rédei, G. P. 1964a. Megaspoore selection in Arabidopsis thaliana. Genetics 50:278–279 (Abstr.). Rédei, G. P. 1964b. A pollen abortion factor. Arabidopsis Inf. Serv. 1:10–11. Rédei, G. P. 1964c. A female gametophyte factor. Arabidopsis Inf. Serv. 1:11. Rédei, G. P. 1964d. An episome resembling factor in Arabidopsis. Arabidopsis Inf. Serv. 1:11. Rédei, G. P. 1964e. Crossing experiences with polyploids. Arabidopsis Inf. Serv. 1:13. Rédei, G. P. 1964f. An attempt to analyze the genetic fine structure of chromosomes. Arabidopsis Inf. Serv. 1:14. Rédei, G. P. 1964g. D-alanine, a specific inhibitor. Arabidopsis Inf. Serv. 1:23–24. Rédei, G. P. 1964h. New bioassay for vitamin B1. Arabidopsis Inf. Serv. 1:24. Rédei, G. P. 1964i. Additional thiamine requiring mutants. Arabidopsis Inf. Serv. 1:25. Rédei, G. P. 1964j. Prescient. Science 146:717. Rédei, G. P., and Hirono, Y. 1964. Linkage studies. Arabidopsis Inf. Serv. 1:9–10. Hirono, Y., and G. P. Rédei. 1965a. Induced premeiotic exchange of linked markers in the angiosperm Arabidopsis. Genetics 51:519–526.

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Németh, K., K. Salchert, P. Putnoky, R. Bhalerao, Z. Koncz-Kálmán, B. StankovicStangeland, L. Bakó, L. Mathur, L. Ökrész, S. Stable, P. Geigenberger, M. Stitt, G. P. Rédei, J. Schell, and C. Koncz. 1998. Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis. Genes Devel. 12:3059–3073. Rédei, G. P. 1998a. Genetics manual of current theory, concepts and terms. World Scientific, Singapore. Rédei, G. P. 1998b. A tanult ember felelo"ssége. p. 594–600. In: Á. Fasang and A. Fodor (eds.), Hivatás és hitvallás. Mundus Publ., Budapest. Rédei, G. P., and C. Koncz. 1999. Episztázis, pleiotrópia és a nukleinsav-elmélet. IV. Magyar Genetikai Kongr. p. 24–25 (Abstr.). Rédei, G. P. 2001. Semmelweis and the battle against infection. Nature 409:761. Rédei, G. P. 2002a. Happy 50th Birthday Növénytermelés. Növénytermelés 50:5–6. Rédei, G. P. 2002b. Vignettes of the history of genetics. p. 1–20. In: M. S. Kang (ed.), Quantitative genetics, genomics and plant breeding. CABI Wallingford, Oxon, UK. Rios, G., A. Lossow, B. Hertel, F. Breuer, S. Schaefer, M. Broich, T. Kleinow, J. Jásik, J. Winter, A. Ferrando, R. Farrás, M. Panicot, R. Henriques, J.-B. Mariaux, A. Oberschall, G. Molnár, K. Berendzen, V. Shukla, M. Lafos, Z. Koncz, G. P. Rédei, J. Schell, and C. Koncz. 2002. Rapid identification of Arabidopsis insertion mutants by nonradioactive detection of T-DNA tagged genes. Plant J. 32:243–253. Szabados, L., I. Kovács, A. Oberschall, E. Ábrahám, I. Kerekes, L. Zsigmond, R. Nagy, M. Alvarado, I. Krasovskaja, M. Gál, A. Berente, G. P. Rédei, A. Ben Haim, and C. Koncz. 2002. Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. Plant J. 32:233–242. Rédei, G. P. 2003a. Encyclopedic dictionary of genetics, genomics and proteomics. WileyLiss, Hoboken, NJ. Rédei, G. P. 2003b. Genes and selection: Retrospect and prospect. Plant Breed. Rev. 24(1): 11–40.

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2 Developing Papaya to Control Papaya Ringspot Virus by Transgenic Resistance, Intergeneric Hybridization, and Tolerance Breeding Dennis Gonsalves USDA-ARS Pacific Basin Agricultural Research Center 99 Aupuni Street, Suite 204 Hilo, Hawaii 96720 USA Ariadne Vegas Instituto Nacional de Investigaciones Agrícolas Centro Nacional de Investigaciones Agropecuarias Unidad de Biotecnología Vegetal Apartado 588 Maracay 2101, Estado Aragna Venezuela Vilai Prasartsee Office of Agricultural Research and Development Region 3 Horticulture Section Khonkaen, 40260 Thailand Rod Drew School of Biomolecular and Biomedical Science Griffith University Nathan Q4111 Australia Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 35

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Jon Y. Suzuki USDA-ARS Pacific Basin Agricultural Research Center 99 Aupuni Street, Suite 204 Hilo, Hawaii 96720 Savarni Tripathi USDA-ARS Pacific Basin Agricultural Research Center 99 Aupuni Street, Suite 204 Hilo, Hawaii 96720 We thank Ms. Leone Drew for reviewing and editing the manuscript.

I. INTRODUCTION II. PAPAYA AND PAPAYA RINGSPOT VIRUS A. Papaya B. Papaya Ringspot Virus III. DEVELOPMENT OF TRANSGENIC PAPAYA FOR HAWAII A. PRSV in Hawaii B. Attempts to Control PRSV by Cross Protection C. Development of Transgenic Papaya for Hawaii 1. Parasite-Derived Resistance 2. Development of Transgenic Papaya 3. Transgenic T1 Plants of Line 55-1 Are Resistant to Hawaii Strains but Susceptible to Strain Outside Hawaii 4. Field Trials with T0 Plants Show Line 55-1 Is Effective for Controlling PRSV in Hawaii 5. ‘SunUp’ and ‘Rainbow’ Cultivars 6. PRSV Invades Puna in 1992 7. Establishment of Transgenic Field Trial in Kapoho 8. Deregulation and Commercialization of Transgenic Papaya D. Impact of Transgenic Papaya 1. Controlling PRSV 2. Increased Production of Papaya 3. Help in Production of Nontransgenic ‘Kapoho’ in Puna 4. Expanding Papaya Production Areas and the Diversification of Cultivars Available for Hawaii E. Challenges Facing Hawaii’s Papaya Industry 1. Canadian and Japanese Markets 2. Achieving Durable Resistance 3. Guarding Against Large-Scale Resurgence of PRSV in Nontransgenic Papaya in Puna IV. DEVELOPMENT OF TRANSGENIC PAPAYA FOR OTHER REGIONS A. Brazil B. Jamaica C. Thailand D. Venezuela

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VI.

VII.

VIII.

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E. Taiwan F. Australia G. Hongkong, China H. Papaya Biotechnology Network of Southeast Asia I. Bangladesh and Africa J. Florida, USA BREEDING THROUGH INTERGENERIC HYBRIDIZATIONS A. Resistance and Susceptibility of Vasconcellea Species to PRSV B. Intergeneric Hybridizations between Carica papaya and Vasconcellea Species DEVELOPMENT OF PRSV-TOLERANT PAPAYA A. Screening for PRSV Tolerance and Recurrent Selection Scheme B. Recommendation and Availability of Seeds C. Maintenance of PRSV-Tolerant Papaya Lines FUTURE ASPECTS FOR DEVELOPING PRSV-RESISTANT PAPAYA A. Segmented Gene Approach B. Synthetic Gene Approach SUMMARY COMMENTS LITERATURE CITED

I. INTRODUCTION The report by Abel et al. (1986) that transgenic tobacco expressing the coat protein (CP) gene of Tobacco mosaic virus (TMV) was resistant to TMV spurred the development of virus-resistant transgenic crops. Since then, the initial hope that pathogen-derived resistance (Sanford and Johnston 1985) might be a practical way to control plant viruses has been firmly established and applied to many viruses and crops (Beachy 1997). Efficient protocols have resulted from technical improvements in transgene engineering, in transformation of a variety of crops, and in the understanding of the mechanism of resistance. However, the commercial potential has not yet been realized. Only three virus-resistant crops have reached the marketplace in the U.S., the last of these being the Hawaiian solo transgenic papaya back in 1998 (C. Gonsalves et al. 2004; D. Gonsalves et al. 2004). This review focuses on the development and impact of transgenic papaya resistant to the Papaya ringspot virus (PRSV) in Hawaii, on the development of similar technology in other parts of the world, the development of PRSV-tolerant plants in Thailand, and the current status of resistance breeding of papaya using genes from Vasconcellea species. It will be shown that the technology to control PRSV via transgenic papaya is well established and workable. However, the current situation also points out that it is now apparent that other factors, mainly the controversy over deployment of transgenic plants in places outside of the U.S., have important bearing on whether this technology goes beyond academic

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curiosity. This review provides a basis for readers to analyze the papaya case as a model for deployment of transgenic papaya crops as a solution to potentially devastating viruses. Lastly, readers will be familiarized with the tools of transgenesis, resistance breeding, and tolerance breeding for the control of the worldwide problem of PRSV.

II. PAPAYA AND PAPAYA RINGSPOT VIRUS A. Papaya Papaya (Carica papaya L., Caricaceae) is an important fruit crop in tropical and subtropical regions due to its economic, nutritional, industrial, pharmaceutical and medicinal values, for local and export markets (Badillo 2000). Its origin is probably Southern Mexico and Northern Central America (Badillo 1993). The papaya was disseminated into Asian tropics during the 1600s through seeds taken to the Malay Peninsula, India and Philippines. Documents show wide distribution in the Pacific Islands by the 1800s (Nakasone 1975). Papaya is a large herbaceous, dicotyledonous plant that grows up to 3–8 m in height. The papaya plant usually has a single erect stem and a crown of alternate large palmate lobed leaves. The inflorescences are borne in leaf axils (Plate 2.1). Cultivated plants are polygamous, with staminate, pistillate, and hermaphrodite flowers, while wild plants are frequently dioecious. Domesticated plants show different sexual types, including hermaphrodite flowers with different masculinity grades described by Storey in 1976 (Badillo 1993). Staminate flowers are long, pendulous, many-flowered cymose inflorescences. The corolla of the flower forms a slender tube two-thirds of its length and terminates in five free petals. The flower is unisexual, having functional stamens and lacking a functional pistil. Pistillate flowers are inflorescences with few flowers. The flowers have large functional pistils but entirely lack stamens or even vestiges of stamens. The petals are free from one another. Hermaphrodite or flowers are bisexual with pistil and stamens on a relatively short inflorescence. The petals are fused for one-half to threefourths of their lengths, forming a rigid tube (Giacometti 1987). The pistillate tree is extremely stable, always producing pistillate flowers. Conversely, the staminate and hermaphrodite trees can be sensitive to fluctuations of environmental factors, and can go through seasonal sex-reversals (Hofmeyr 1939). Fruit from pistillate trees are spherical whereas those from hermaphroditic trees are pyriform, oval or elongate. Fruit size ranges from 255 g to 5–6 kg. Fruit color

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ranges from pale to bright yellow-orange to red. Dioecious cultivars are more stable and vigorous, and grow better in the subtropical areas of Florida, Australia, and South Africa when compared to hermaphrodite cultivars, which are not adapted to cool winters (Giacometti 1987). In tropical countries, where production is designed for export, the Hawaiian Solo types are grown. The small fruit (450–500 g) produced by these cultivars are easier to pack and ship (Gonsalves 1994). Papaya fruit is most commonly eaten fresh, peeled and seeded, or it is processed for making fruit salad, juice, jam, jelly, pie, or ice cream flavoring. Unripe fruit can be eaten raw in salad, cooked in syrup and eaten as a dessert, and the boiled leaves are used as a vegetable. The fruit aids the digestive process due to the presence of a digestive enzyme papain (Sturrock 1940) and has nutritional value with high contents of vitamin A, vitamin C, calcium, potassium and iron (USDA-ARS 2001). The latex of unripe fruit is a fluid of milky appearance that contains proteinases, including papain. Papain is extracted by making incisions on the surface of green fruit, and is used to tenderize food and in pharmaceutical industries. Approximately 350 tonnes (t) of crude papain are exported annually from production areas (Jones and Mercier 1974). For the majority of producer countries, papain is a by-product of fruit production (Madrigal et al. 1980) from about 0.2% of the total harvested area (FAO 2000). Papaya is widely planted in home gardens because it is relatively easy to grow from seed, the first mature fruit can be harvested nine months after sowing seeds, and fruit is produced year-round. Commercially, when trees are grown at a density of 1500 to 2500 trees/hectare, annual production can range from 57 to 136 t/ha. Fruit are harvested for 1 to 2 years, after which the trees are usually too tall for efficient harvesting. The FAO (2000) estimates that about 6 million t of fruit were harvested in 2002, almost doubling the 1980 harvest. Brazil (25.2%), Nigeria (12.6%), India (11.8%), Mexico (11.6%) and Indonesia (8.6%) are the largest producers of papaya. B. Papaya Ringspot Virus This section on PRSV is largely taken from a review by Gonsalves (1998) and it describes important features on the biology of the virus. PRSV is by far the most widespread and damaging virus that infects papaya (Purcifull et al. 1984). The name of the disease, papaya ringspot, was taken from the ringed spots that develop on the fruit of infected trees (Jensen 1949). Trees infected with PRSV develop a range of symptoms: mosaic and chlorosis of leaf lamina, water-soaked oily streaks on the petiole and

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upper part of the trunk, and distortion of young leaves that resembles mite damage. Infected plants lose vigor and become stunted. When infected at the seedling stage or before flowering, trees do not normally produce mature fruit. Production of fruit by trees infected at progressively later stages is severely reduced and of poor quality, due to the presence of ringspots and generally lower sugar concentrations. PRSV is transmitted by numerous species of aphids in a non-persistent manner to a limited host range of cucurbits and papaya. PRSV also produces local lesions on Chenopodium quinoa and C. amaranticolor. A number of investigations have failed to demonstrate that PRSV is transmitted through seeds of papaya or cucurbits, except for one study that showed that 2 of 1355 papaya seedlings from PRSV-infected fruit showed PRSV symptoms (Bayot et al. 1990). However, seed transmission is not a significant means for spreading PRSV. PRSV is grouped into type P (PRSV-p), which infects cucurbits and papaya, and type W (PRSV-w), which infects cucurbits but not papaya (Purcifull et al. 1984). The latter type was previously referred to as WMV-1. Although both types are serologically closely related, observations suggest that papaya is the most important primary and secondary source for the spread of PRSV in large plantations and small orchards alike. Hereafter, we will designate PRSV-p as PRSV. Much progress has been made in the molecular characterization of PRSV. The genomic RNA consists of 10,326 nucleotides and has the typical array of genes found in potyviruses (Shukla et al. 1994). The genome is monocistronic and is expressed via a large polypeptide that is subsequently cleaved to yield all functional proteins. There are two possible cleavage sites, 20 amino acids apart, for the N terminus of the CP (Yeh and Gonsalves 1985; Quemada et al. 1990). These two sites may be functional; the upstream site for producing a functional NIb protein (the viral replicase), and the other, to produce the CP present in aphidtransmissible virions. It is impossible to segregate PRSV-p and PRSV-w types by their CP sequences. Within the p-types, however, the CP nucleic acid sequences can diverge by as much as 12%.

III. DEVELOPMENT OF TRANSGENIC PAPAYA FOR HAWAII Transgenic papaya resistant to PRSV was commercialized in the U.S. in 1998 (D. Gonsalves et al. 2004). It represents one of three virus resistant crops to be commercialized, with the first being the transgenic squash that was resistant to WMV II and Zucchini yellow mosaic virus. How-

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ever, virus resistant crops have not been commercialized since the papaya in 1998 and potato in 1999. Thus, in this review, we will cover the technical aspects but also relate them to the problem that occurred in Hawaii because the approach we took for developing and commercializing the transgenic papaya was to a large extent governed by the PRSV papaya situation in Hawaii at that time. It is hoped that lessons might be learned from this case such that more virus resistant plants may be commercialized given the fact that this approach has proven extremely effective for many viruses. The PRSV and transgenic papaya in Hawaii has been reviewed by one of the authors; much of this section is taken from parts of previous reviews. (Gonsalves 1998; Fermin, Tennant et al. 2004; C. Gonsalves et al. 2004; Gonsalves and Fermin 2004; D. Gonsalves et al. 2004). A. PRSV in Hawaii The papaya industry started in the 1940s on the island of Oahu, on about 200 ha (Ferreira et al. 1992). PRSV was discovered in 1945, and its name was coined by Jensen (1949). By the 1950s, production on Oahu was affected and the industry subsequently moved to Hawaii Island into the area of Puna, which had no previous commercial production. Crop area increased to 260 ha by 1960 and to 900 ha in 1990. In contrast, the area on Oahu fell to less than 20 ha by 1990 (Ferreira et al. 1992). Remarkably, despite the presence of PRSV in Hilo and Keaau, communities only 19 miles away, Puna remained free of PRSV for over 30 years, thanks to an effective physical barrier of lava rocks and diligence by the Hawaii Department of Agriculture (HDOA) in surveying and rouging infected trees in the Hilo and Keaau areas. In 1978, one of the authors (D. Gonsalves) started research towards developing control methods for PRSV, since it was probable that PRSV would one day be found in Puna, where Hawaii was growing 95% of its papaya. B. Attempts to Control PRSV by Cross Protection In 1979, investigations began on the possibility of using cross protection as a control measure. Cross protection is the phenomenon whereby plants that are systemically infected with a mild strain of a virus are protected against the effects of infection by a more virulent related strain (Yeh and Gonsalves 1984; Fulton 1986; Yeh and Gonsalves 1994). This practice had long been known and has been used to control citrus tristeza (Costa and Muller 1980), Tobacco mosaic (Rast 1975), and Zucchini

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yellow mosaic viruses (Walkey et al. 1992). The key component in crossprotection programs is the availability of a mild strain that effectively protects against the target virus. Two mild strains (Yeh and Gonsalves 1984), designated PRSV HA 51 and PRSV HA 6-1, were selected following nitrous acid treatment of leaf extracts of squash infected with PRSV-HA, a severe strain from Hawaii that had been recently characterized (Gonsalves and Ishii 1980). Greenhouse experiments showed that both strains were mild on papaya and afforded protection against PRSV HA. The various trials and experiments with HA 5-1 have been reviewed (Yeh and Gonsalves 1994). Briefly, the mild strain was only marginally effective in Taiwan where it was tried extensively. In Hawaii, it was effective in protecting against the severe strains, but the mild strain produced quite severe symptoms on certain cultivars, such as ‘Sunrise’, and thus did not receive widespread use (Mau et al. 1989; Ferreira et al. 1992; Pitz et al. 1994). However, the mild strain HA 5-1 was later used as the source of CP for the transgenic papaya. C. Development of Transgenic Papaya for Hawaii 1. Parasite-Derived Resistance. The concept of parasite-derived resistance (PDR), conceived in the mid-1980s (Sanford and Johnston 1985), offered a new approach for controlling PRSV. Parasite-derived resistance is a phenomenon whereby transgenic plants containing genes or sequences of a parasite (in the Hawaiian case, the CP gene of a PRSV) are protected against detrimental effects of the same or related pathogens. The application of this concept, first demonstrated by Beachy’s group (Abel et al. 1986) has been successfully applied to developing virus resistant crops to numerous viruses. This subject has been reviewed (Lomonossoff 1995; Goldbach et al. 2003). Recent information suggests that the mechanism of resistance is RNA mediated (Tennant et al. 1994; Tennant et al. 2001); however, when we started the work, we assumed that the CP had to be expressed since that was the thinking at the time. 2. Development of Transgenic Papaya. We began the utilization of the PDR concept in 1986 by cloning the CP gene of PRSV HA 5-1 in joint efforts with the then Asgrow Seed Company. The aim was to develop transgenic vegetables with resistance to PRSV. The research team assembled to develop transgenic papaya consisted of D. Gonsalves, Jerry Slightom of Upjohn Company, Richard Manshardt of the University of Hawaii, and Maureen Fitch, an ARS/USDA employee who was at that time a graduate student of Richard Manshardt. We were also very fortunate to tap the expertise and services of John Sanford at Cornell Uni-

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versity, who had recently co-invented the gene gun. Two strengths of the team were the blend of expertise that included molecular biology, virology, tissue culture, and horticulture, and our expressed goal of developing a practical solution in a timely manner. Our target gene of PRSV was the CP gene of PRSV HA 5-1, the mild mutant of PRSV HA that had been cloned recently and sequenced. The CP gene of PRSV HA 5-1 had a 97.7% identity to PRSV-w from Florida (Quemada et al. 1990). Because of various technical difficulties and the requirement that the gene be expressed as a protein, the gene was engineered as a chimeric protein containing 17 amino acids of cucumber mosaic virus at the N terminus of the full-length CP gene of PRSV HA 5-1 (Ling et al. 1991). Whether this would enhance or decrease the chances of obtaining resistant plants was unclear. However, in tobacco this gene construct had expressed high levels of the CP as measured by ELISA (Ling et al. 1991). The task of transforming papaya was taken up in 1987 by M. Fitch. The target cultivars were the red-fleshed ‘Sunrise’, ‘Sunset’ (a sib selection of ‘Sunrise’), and the yellow-fleshed ‘Kapoho’, which was by far the most grown cultivar in Puna. Numerous efforts to develop a papaya regeneration system via organogenesis failed. However, a technique to develop transgenic walnuts by transforming embryogenic cultures had recently been reported (McGranahan et al. 1988). Papaya research progressed rapidly once the decision was made to shift to transforming embryogenic tissue. A technique to produce highly embryogenic tissue starting from immature zygotic embryos was developed (Fitch and Manshardt 1990). In 1988–1989, embryogenic tissue was bombarded with tungsten particles coated with DNA of the PRSV HA 5-1 CP gene using the gene gun in Sanford’s laboratory. Fifteen months later, transgenic plants had been obtained and were growing in the greenhouse (Fitch et al. 1990). Clones of nine T0 transgenic lines (clones of the original transformants), six ‘Sunset’ and three ‘Kapoho’, were sent to Cornell for inoculation tests with PRSV HA, which is the severe parent of the mild strain PRSV HA 5-1. T0-micropropagated plants of the first line, designated 551, that were tested showed excellent resistance to PRSV HA (Plate 2.2, Fitch et al. 1992). Three other lines showed varying degrees of delay in the onset of symptoms, while the other lines developed symptoms at the same time as control plants. Line 55-1 was pistillate and thus progenies could not be obtained directly from the T0 plants, as would be the case for a hermaphrodite. A two-pronged approach was instituted to move the research ahead aggressively and to determine whether line 55-1 would be resistant to PRSV under field conditions and have suitable horticultural characteristics. First, a decision was made to conduct a field trial using T0 plants instead of waiting a year to obtain Tl plants. Second, Tl plants were obtained by crossing line 55-1 with nontransgenic ‘Sunset’

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under greenhouse conditions both at Cornell University and the University of Hawaii. These plants were screened in the greenhouse for resistance to PRSV isolates from around world. Analysis clearly showed that 50% of the progenies had one insert of the nptII gene and, presumably, the CP gene with the rest being nontransgenic. 3. Transgenic Tl Plants of Line 55-1 Are Resistant to Hawaii Strains but Susceptible to Strains Outside Hawaii. The resistance of Tl plants of line 55-1 against 3 PRSV isolates from Hawaii and 13 isolates from different parts of the world was tested (Tennant et al. 1994). It should be noted that the transgenic T1 plants were hemizygous for the transgenes. The results of the tests clearly showed that T1 plants were resistant to PRSV HA and other Hawaiian isolates of PRSV. Rather surprisingly, however, the plants were largely susceptible to the strains of PRSV from other regions (Australia, Bahamas, Brazil, China, Ecuador, Florida, Guam, Jamaica, Mexico, and Okinawa). Strains from Bahamas, Mexico, and Florida infected 24 to 72% of the inoculated plants; however, the symptoms were not as severe as the control plants and symptom expression was delayed as compared to the nontransgenic controls. Guam, Brazil, Thailand, Ecuador, and Okinawa isolates infected all of the inoculated plants with severe symptoms and milder symptoms were caused by the Australia, China, and Jamaica isolates. In parallel experiments, the effectiveness of PRSV mild strain HA 51 for protecting nontransgenic papaya against infection by the above strains was also tested. Interestingly, the effectiveness of cross protection was similar to the results obtained with the transgenic line 55-1. That is, cross protection was complete against the Hawaiian PRSV strains, not complete against the strains that produced milder symptoms on the transgenic papaya, and did not give any protection against those strains that produced severe symptoms on the transgenic papaya. Overall, the results with transgenic papaya reinforced the information previously obtained with cross protection: T1 plants of line 55-1 would not be effective against all isolates of PRSV. However, the results clearly showed that line 55-1 had potential to control PRSV in Hawaii. 4. Field Trials with T0 Plants Show Line 55-1 Is Effective for Controlling PRSV in Hawaii. In 1991, Animal Plant Health Inspection Service (APHIS) issued permission for a field trial at the University of Hawaii’s experimental farm at Waimanalo, on Oahu Island. The importance of a field evaluation at an early stage cannot be overemphasized because it allowed an early appraisal on the resistance and horticultural characteristics of line 55-1, demonstration of the long-term resistance of a transgenic fruit crop to infection, and provided the initial and subse-

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quent plantings in the field that were used to develop the papaya cultivars ‘Rainbow’ and ‘SunUp’. Plants were set in the field by the end of June 1992. The field trial was designed to determine the resistance of T0 plants to mechanical and aphid inoculations of PRSV. Nontransgenic plants in border rows of the plot were also inoculated with a PRSV isolate from Oahu Island to create a high virus inoculum pressure for the field plot. Data were taken on total soluble solid levels of fruit, growth characteristics, and virus symptoms. The transgenic papaya showed excellent resistance throughout the twoyear trial (Lius et al. 1997). Nearly all (95%) of the nontransgenic plants and those of a transgenic line that lacked the CP gene showed PRSV symptoms by 77 days after the start of the field trial, whereas none of the line 55-1 plants showed symptoms. Virus was not recovered from line 55-1 plants except for two plants, which showed virus symptoms on side shoots but none on the leaves of the main canopy. Plants of line 55-1 grew normally and fruit appearance and total soluble solids of about 13% were within the expected range. Thus, by mid-1993, the trial had provided convincing evidence that line 55-1 would be useful for controlling PRSV in Hawaii, or at least on Oahu Island. 5. ‘SunUp’ and ‘Rainbow’ Cultivars. Our efforts had focused on transferring virus resistance into commercial papaya cultivars suitable for Hawaii. Fortunately, the commercial Hawaiian solo cultivars (‘Kapoho’, ‘Sunrise’, ‘Sunset’) are isogenic and thus breed true to type. As noted above, line 551 was transformed from embryos of ‘Sunset’, which is a commercial redfleshed cultivar that is a sib of ‘Sunrise’. The dominant cultivar growing in Puna, however, was the yellow-fleshed ‘Kapoho’. Thus, it was anticipated that the growers would want a yellow-fleshed cultivar that would be comparable to ‘Kapoho’. Using the same field trial location, during 1992–1994, crosses and selections were made to bring line 55-1 to homozygosis for resistance, which was accomplished by the T3 generation. The homozygous transgenic line 55-1 was then crossed with ‘Kapoho’, anticipating a yield of a yellow-fleshed hybrid (because yellow is dominant over red) to substitute for Kapoho (Manshardt 1998). The homozygous line 55-1 was later named ‘SunUp’ (Plate 2.3). The hybrid made from the cross of the transgenic ‘SunUp’ and the nontransgenic ‘Kapoho’ was named ‘Rainbow’ (Plate 2.4). Thus, the proposed field trial became a test not only of virus resistance but also of fruit quality. This effort became timely because PRSV was discovered in the main Hawaiian production area of Puna within a month of initiating the field trial. 6. PRSV Invades Puna in 1992. The inevitable entry of PRSV into the Puna district on Hawaii Island was discovered during the first week of

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May in a papaya field in Pahoa, 1–3 miles from the major papaya growing areas in Puna (Isherwood 1994). By late 1994, nearly all papaya of ‘Kapoho’ was infected by the virus (Plate 2.5 & 2.6). In October 1994, the HDOA declared that PRSV was uncontrollable and stopped the practice of marking trees for rouging. In less than three years, a third of the Puna papaya area was infected. By 1997, Pohoiki and Kahuawai were completely infected. Kalapana was the last place to become heavily infected. Five years after the onset of the virus in Pahoa, the entire Puna area was severely affected. 7. Establishment of Transgenic Field Trial in Kapoho. The rapid spread and severity of PRSV in Puna suggested that the only practical solution was the transgenic virus resistance approach. By late 1994, an application for a field trial in Puna was submitted to APHIS. The field trial was allowed with several stipulations: (1) the field must be sufficiently isolated from commercial orchards to minimize the chance of transgenic pollen escaping to nontransgenic material outside of the field test, (2) all abandoned trees in the area must be monitored for the introgression of the transgene into fruit of these trees, and (3) all fruit had to be buried on site. Approval by APHIS was obtained in early 1995 and the field trial was set up in Kapoho in October 1995, under the leadership of S. Ferreira, who had been working with the investigators on cross protection. The trial was set up on the farm of a grower who had ceased growing papaya because of PRSV. One part of the trial consisted of replicated blocks to compare virus-resistance performances of ‘SunUp’ and ‘Rainbow’, ‘Kapoho’ cross protected with PRSV HA 5-1, a transgenic ‘Sunrise line’ (63-1) that had been identified to be resistant to PRSV (Ferreira et al. 2002), and PRSV-tolerant lines that were being developed in Thailand (described in this review). Another part of the trial was established to simulate commercial conditions. A solid block of ‘Rainbow’ (0.4 ha2) was planted adjacent to the replicated blocks. Several rows of nontransgenic ‘Sunrise’ were planted on the perimeter of the replicated and solid blocks. An abandoned papaya field alongside the field plot was used as a primary source of the virus. The results of the field trial clearly demonstrated the potential value of the transgenic ‘SunUp’ and ‘Rainbow’ papaya for reclaiming papaya land in Puna (Plate 2.7, Ferreira et al. 1997). Of the nontransgenic controls plants, 50% showed virus symptoms within four and a half months after transplanting, and all were infected by seven months. The growth differences between the transgenic and nontransgenic trees were remarkable; transgenic plants grew vigorously, with dark green leaves and full fruit columns, whereas nontransgenic plants were stunted, with yellow

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and mosaic leaves and very sparse fruit columns. In the solid block planting, ‘Rainbow’ averaged about 112 t/ha of marketable fruit annually, whereas nontransgenic plants averaged about 5.6 t/ha. The transgenic papaya performed much better than the PRSV-tolerant lines and the cross-protected plants. Despite their excellent resistance, there was no certainty that the farmers would accept these cultivars as an acceptable substitute for ‘Kapoho’, a mainstay of the Hawaiian papaya industry for several decades. The performance of ‘Rainbow’ was especially critical because it was targeted as the alternative to ‘Kapoho’. Taste, production, color, size, and packing and shipping qualities of ‘Rainbow’ were analyzed. In addition to tests by research personnel, field days were held to allow farmers, packers, politicians, and University personnel to observe the field and fruit at the test site. The consensus was that ‘Rainbow’ was a more than adequate substitute for ‘Kapoho’. 8. Deregulation and Commercialization of Transgenic Papaya. To develop a transgenic papaya with virus resistance and excellent fruit qualities represents one hurdle, but to achieve deregulation and commercialization is another critical hurdle. The process to deregulate the transgenic papaya was taken up by the researchers, while the process to commercialize it was taken up by the Papaya Administrative Committee (PAC). The latter consisted of a group of growers formed under a USDA marketing order. They collected an assessment per 0.454 kg of papaya that was sold. Prior to the outbreak of PRSV in Puna, funds obtained by the PAC were used primarily for advertising and marketing of papaya. The governmental agencies involved with deregulation of a transgenic product are APHIS, Food and Drug Administration (FDA), and Environmental Protection Agency (EPA). APHIS was largely concerned with the potential risk of transgenic papaya to the environment. Two main risks were of heteroencapsidation of the incoming virus with CP produced by the transgenic papaya and of recombination of the transgene with incoming viruses. The former might allow nonvectored viruses to become vector transmissible, whereas the latter might result in the creation of novel viruses. A third concern was that the escape of the transgenes to wild relatives might make the relatives more weedy or even make papaya more weedy because of resistance to PRSV. The concern proved to be of no consequence since there are no papaya relatives in the wild in Hawaii, nor is papaya considered to be a weed, even in areas where there is no PRSV. In November 1996, transgenic line 55-1 and another recently tested line 63-1 (Tennant 1996) and their derivatives were deregulated by APHIS (Strating 1996). This action greatly

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increased the efficiency of the ongoing field trial because fruit no longer had to be buried at the test site, which allowed us to sample and send fruit for analyses by various laboratories and a packing house. According to the EPA, the CP transgenes are considered to be pesticides because they confer resistance to plant viruses. A pesticide is subjected to tolerance levels in the plant. In the permit application, we petitioned for an exemption from tolerance levels of the CP produced by the transgenic plant. We contended that the pesticide (the CP gene) was already present in many fruit consumed by the public, since much of the papaya eaten in the tropics is from PRSV-infected plants. In fact, we had earlier used cross protection (the deliberate infection of papaya with a mild strain of PRSV) to control PRSV. Fruit from these trees was sold to consumers. Furthermore, there is no evidence to date that the CP of PRSV or other plant viruses are allergenic or detrimental to human health in any way. Finally, measured amounts of CP in transgenic plants were much lower than those of infected plants. An exemption from tolerance to lines 55-1 and 63-1 was granted in August 1997. The FDA is concerned with food safety of transgenic products. This agency follows a consultative process whereby the investigators submit an application with data and statements corroborating that the product is not harmful to human health. Several aspects of the transgenic papaya were considered: the concentration range of some important vitamins, including vitamin C; the presence of gus and nptll genes; and whether transgenic papaya had abnormally high concentrations of benzyl isothiocyanate, a highly toxic compound. This latter compound has been reported in papaya (Tang 1971). FDA approval was granted in September 1997. In the U.S., a transgenic product cannot legally be commercialized unless it is fully deregulated and until licenses are obtained for the use of the intellectual property rights for processes or components that are part of the product or that have been used to develop the product. The processes in question for the transgenic papaya were biolistic transformation and parasite-derived resistance, in particular, CP-mediated protection. The components were translational enhancement leader sequences and genes (nptll, gus, and CP). License agreements were obtained from all parties in April 1998, allowing the commercial cultivation of the papaya or its derivatives in Hawaii only. Fruit can be sold outside Hawaii, provided that the importing state or country allows the importation and sale of transgenic papaya. Fruit derived from licensed transgenic papaya grown outside of Hawaii cannot be sold commercially. Even before license agreements had been obtained, the PAC had commissioned the Hawaii Agricultural Research Center to produce ‘Rain-

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bow’ seeds so that they would be available when needed. Distribution of seeds started in May 1998. Seeds were given free-of-charge to growers who satisfied two conditions: watching a video describing the transgenic papaya and signing a sub-license agreement not to sell seeds of ‘Rainbow’ or ‘SunUp’ outside of Hawaii. Basically, ‘SunUp’ and ‘Rainbow’ could be grown in Hawaii and fruit sold worldwide wherever the transgenic papaya was legally accepted. In 1998, the mainland U.S. was the only country to deregulate the transgenic papaya and thus allow sales of the product. D. Impact of Transgenic Papaya 1. Controlling PRSV. In 1998, PRSV was widespread in Puna where numerous abandoned fields of PRSV-infected papaya dotted the landscape. The newly planted transgenic papaya showed excellent resistance under very severe disease pressure. It allowed farmers to directly reclaim their farms without first clearing their land of all infected papaya trees. Common practices were to grow transgenic plants next to mature papaya plants, which were subsequently cut after the transgenic plants were established or to simply grow transgenic papaya among abandoned fields. In all cases, the transgenic papaya remained virus-free. Within a year of the release of the seeds, it was common to see fields of healthy ‘Rainbow’ (Plate 2.8). This remained the case in 2004. The field resistance of the transgenic papaya in Puna proved to be durable, a result that was not necessarily predictable given that greenhouse tests had shown ‘Rainbow’ to be resistant to the several PRSV isolates from Hawaii but susceptible to a range of isolates from outside of Hawaii (Tennant et al. 1994). Thus, ‘Rainbow’ plantings are constantly being monitored for evidence of breakdown of resistance. Fortunately, in Puna and Oahu, the resistance has endured under diverse conditions of plantings and disease pressure. 2. Increased Production of Papaya. The release of the transgenic papaya resulted in an increase in papaya production in Hawaii and Puna. The transgenic ‘Rainbow’ is widely sold in Hawaii (Plate 2.9) and exported to mainland U.S. and Canada. The following observations were made for Puna up to the year 2002 (Table 2.1), the most recent year for which statistics are available from National Agricultural Statistics Service (NASS). In 1992, Puna produced 24,000 t of the state’s 25,300 t of fresh papaya. The production remained high for two years following the discovery of PRSV in Puna due to massive efforts to control the spread of the virus. However, by 1995 papaya production in Puna had dropped to 17,800 t

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Table 2.1. Fresh papaya production in state of Hawaii and in Puna district, 1992–2002.z Puna

Year

Total (t)

(t)

(%)

Comments

1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

25316 26405 25497 19010 17149 16197 16151 17875 22798 23592 19373

24050 25084 25191 17791 15514 12617 12136 11619 15403 18279 16278

95 95 99 94 90 78 75 65 68 77 84

Virus in Puna

Transgenic seed Released

z

Data were compiled from USDA Statistical Reports of Papaya grown in Hawaii (www.usda.gov/hi).

and was down to 12,100 t in 1998 when transgenic seeds of cultivars were released to farmers. Increase in production was evident in 2000 and peaked at 18,300 t in 2001; 16,300 t were produced in 2002. The effect of PRSV on papaya production in Puna can also be seen by the drop in the total percentage of Hawaii’s fresh papaya production that was produced in Puna. In 1992, Puna accounted for 95% of the total production, but this figure subsequently dropped to 65% in 1999 and has since risen to 84% in 2002. The impact of the transgenic papaya on papaya production in Puna is also evident in figures showing relative bearing area of ‘Rainbow’ and the nontransgenic ‘Kapoho’ (Table 2.2). In 1998, Puna’s production was 11,900 t from 656 ha of bearing ‘Kapoho’ (since ‘Rainbow’ had not yet produced mature fruit). In 2000, production had increased to 15,400 t from 476 bearing hectares, with ‘Kapoho’ comprising 32% and ‘Rainbow’ comprising 50% of the area. In 2001, 18,300 t were produced from 670 bearing hectares with ‘Kapoho’ and ‘Rainbow’ accounting for 39 and 41% of the area, respectively. In 2002, the bearing area dropped and the amount of ‘Kapoho’ rose to 49% while ‘Rainbow’ remained steady at 37%. Production dropped from 18,300 t in 2001 to 16,300 t in 2002. These data suggest that ‘Rainbow’ accounts for at least half of the fresh fruit production in Puna. Furthermore, production of similar amounts of papaya can be obtained with less area. This latter observation is attributed to the higher yield of ‘Rainbow’ compared to nontransgenic ‘Kapoho’.

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Table 2.2. Bearing area in Puna of nontransgenic ‘Kapoho’ and transgenic ‘Rainbow’ and its relationship to production of fresh fruit utilized.z

Year

Bearing area (ha)

Kapoho (%)

Rainbow (%)

Production (t)

1998 2000 2001 2002

656 476 670 554

100 32 39 49

0 50 41 37

11909 15403 18279 16278

z

Data were compiled from USDA Statistical Reports of Papaya grown in Hawaii (www.nass.usda.gov/hi).

3. Help in Production of Nontransgenic ‘Kapoho’ in Puna. One might ask the logical question: Why doesn’t Hawaii produce only transgenic papaya? In fact, it is critical that Hawaii continues to produce nontransgenic papaya to supply the Japan market, as will be discussed below. Arguably, one of the major contributions that the transgenic papaya has made to the papaya industry is that of helping in the economic production of nontransgenic papaya (D. Gonsalves and Ferreira 2003). This has occurred in several ways. Firstly, the initial large-scale planting of transgenic papaya in established farms, along with the elimination of abandoned virus infected fields, drastically reduced the virus inoculum. The reduction in virus inoculum allowed for strategic planting of nontransgenic papaya in areas that were free of infected plants and were not surrounded by areas of infected plants, such as had been present in 1992. In fact, HDOA instituted a plan in 1999 to ensure the production of nontransgenic papaya in the Kahuawai area of Puna. Kahuawai was isolated from established papaya fields and prevailing winds in Kahuawai came from the ocean that borders the area (Gonsalves and Ferreira 2003). Furthermore, growers were to monitor for infection and rogue infected plants quickly. Growers who followed the recommended practices were able to economically produce ‘Kapoho’ without major losses from PRSV. Secondly, although definitive experiments have not been carried out, it seems that transgenic papaya can provide a buffer zone to protect nontransgenic papaya that are planted within the confines of the buffer. The reasoning is that viruliferous aphids will feed on transgenic plants and thus be purged of virus before traveling to the nontransgenic plantings within the buffer. This approach also has the advantage that it allows the grower to produce transgenic and nontransgenic papaya in relatively close proximity. Timely elimination of infected trees would need to be practiced to delay large-scale infection of the nontransgenic plants.

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4. Expanding Papaya Production Areas and the Diversification of Cultivars Available for Hawaii. The availability of PRSV-resistant papaya provided options for papaya growers on Oahu island. Prior to the release of transgenic papaya, Oahu growers farmed only small plots of papaya due to the effect of PRSV on production. Growers on Oahu enjoy a niche market, growing ‘Rainbow’ papaya for residents in Honolulu and other urban areas of the island. The transgenic papaya has also allowed for the development of new cultivars to meet niche market needs on Oahu island. The new transgenic cultivar ‘Laie Gold’, which is a hybrid between ‘Rainbow F2’ (selfed Rainbow) and the nontransgenic ‘Kamiya’, also serves a niche market on Oahu island. Since ‘Rainbow F2’ is not homozygous for the CP gene, ‘Laie Gold’ needs to be micropropagated to achieve uniformity of production. Micropropagation of the papaya also has the added benefit of ensuring the production of only hermaphrodite plants that is demanded by the market, of earlier and lower bearing trees with initially higher yields, and of providing selected, superior clones that could result in improved quality and yield. Since the introduction of transgenic papaya, the cultivars available to papaya growers in Hawaii has actually increased. As noted earlier, ‘Kapoho’ accounted for 95% of Hawaii’s papaya market in 1992. Now ‘Rainbow’ and ‘Kapoho’ are dominant and the transgenic ‘SunUp’, ‘Laie Gold’, and the nontransgenic ‘Sunrise’ contribute a small but significant part of Hawaii’s papaya production. In addition to the cultivars mentioned, newer ones developed for niche markets include large-fruited, firm cultivars used as green papaya in South and Southeast Asian cuisine and a red-fleshed ‘Laie Gold’ progeny called ‘Red Kamiya’ that is gaining in popularity in the Oahu market. In addition, initial hybrids that were made with ‘Rainbow’ and nontransgenic ‘Kamiya’ have been backcrossed four times to nontransgenic ‘Kamiya’. Selected lines will be field trialed (M. Fitch and S. Ferreira, unpubl.). E. Challenges Facing Hawaii’s Papaya Industry Although a major constraint to papaya production in Hawaii was eliminated with the introduction of PRSV-resistant transgenic plants, Hawaii’s papaya industry still faces a number of challenges: penetrating the Canada and Japan markets, growing of nontransgenic papaya, and the durability of the resistance in transgenic papaya. 1. Canadian and Japanese Markets. Japan and Canada are large markets for the Hawaii papaya industry. Currently, Japan accounts for 20% of Hawaii’s export market, while Canada accounts for 11%. Canada approved

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the import of ‘SunUp’ and ‘Rainbow’ transgenic papaya in January 2003, and transgenic papaya shipments are continuing to Canada. However, the application process for sale of transgenic papaya in Japan has not yet been approved. Meanwhile, it is critical that papaya shipments to Japan are not contaminated with transgenic papaya fruit. Several steps are being taken to minimize contamination. At the request of Japanese importers, HDOA adopted an Identity Preservation Protocol (IPP) that growers and shippers must adhere to in order to receive an IPP certification letter from HDOA that accompanies the papaya shipment. This is a voluntary program. Papaya shipments with this certification can be distributed in Japan without delay during the time Japanese officials are conducting spot testing to detect contaminating transgenic papaya. In contrast, papaya shipments without this certificate must remain in custody at the port of entry until Japanese officials complete their spot checks for transgenic papaya. Completing the tests may take several days or a week, during which time fruit lose quality and marketability. Some significant features of the IPP are that the nontransgenic papaya must be harvested from papaya orchards that have been approved by HDOA. To get approval, every tree in the proposed field must be tested for the transgenic reporter gene (1,3-β-glucoronidase) that is linked to the virus resistance gene, and found negative. Trees (nontransgenic) must be separated by at least a 4.5 m papaya-free buffer zone, and new fields to be certified must be planted with papaya seeds that have been produced in approved nonGMO fields. Tests for detecting transgenic papaya trees in the field are monitored by HDOA and conducted by the applicant, who must submit detailed records to HDOA. Before final approval of a field, HDOA will randomly test one fruit from 1% of papaya trees in the field. If approved by HDOA, fruit from these fields can be harvested. Additionally, the applicant must submit the detailed protocols that will be followed to minimize the chance of contamination of nonGMO papaya by GMO papaya. This includes a protocol by the applicant on the random testing of papaya before they are packed for shipment. If the procedures are followed and tests are negative, a letter from HDOA will accompany the shipment stating that the shipment is in compliance with a properly conducted IPP. The above procedure represents a good faith effort by HDOA and applicants to prevent transgenic papaya contamination in shipments of nontransgenic papaya to Japan. It also illustrates meaningful collaboration between Japan and HDOA resulting in continued shipment of nontransgenic papaya to Japan with a minimum of delay once they arrive in Japan, while adhering to the policy that transgenic papaya will not

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commercially enter Japan until it is deregulated by the Japanese government. These efforts, along with the effectiveness of the transgenic papaya in boosting production of nontransgenic papaya, have allowed Hawaii to maintain significant shipments of the latter to Japan. Obviously, deregulation of transgenic papaya in Japan will circumvent much of the concern about accidental introduction of transgenic papaya into Japan. To this end, efforts to allow the transgenic papaya into Japan were initiated by the PAC soon after the transgenic papaya was commercialized in Hawaii. Again, the researchers took the lead in developing the petition. Approval of the transgenic papaya in Japan requires the approval of the Ministry of Agriculture Fisheries and Forestry (MAFF) and the Ministry of Health Labor Welfare (MHLW). The petition to the MAFF was approved in December 2000. The petition process for approval by MHLW is still in progress. An initial petition was submitted to MHLW in April 2003. MHLW requested more information, which is currently being generated by the researchers. 2. Achieving Durable Resistance. Finally, the issue of durability of resistance should be considered. Studies have shown that ‘SunUp’ papaya has broader resistance than ‘Rainbow’ (Tennant et al. 1994; Tennant et al. 2004), but the reality is that ‘Rainbow’ is the dominant transgenic papaya grown in Hawaii. So far, no breakdown of resistance has been observed of ‘Rainbow’ in Puna or on Oahu. However, vigilance is required. The possibility of new virulent strains developing from recombination of PRSV strains in Puna with the CP transgene of ‘Rainbow’ is remote. A more realistic danger is the introduction of PRSV strains from outside of Hawaii. We have shown that ‘Rainbow’ or hemizygous 55-1 is susceptible to many strains of PRSV from outside of Hawaii, including strains from Guam, Taiwan, and Thailand (Tennant et al. 1994; Tennant et al. 2001). Goods imported or in transit through Hawaii increase the opportunity for the introduction of PRSV strains into Hawaii. Technically, ‘SunUp’ should be resistant to many strains of PRSV that might be introduced into Hawaii. However, as noted above, the red-fleshed ‘SunUp’ is not the preferred cultivar in Hawaii. A potential solution is to develop transgenic ‘Kapoho’ that is resistant to a wide range of strains. This could be used as a stand-alone cultivar, or it could serve as a transgenic parent for creating a new type of ‘Rainbow’ by crossing the transgenic ‘Kapoho’ with ‘SunUp’. This F1 hybrid should have the horticultural characteristics of current Rainbow but would likely have much wider resistance than the current Rainbow due to increase in CP gene dosage. Transgenic Kapoho that is resistant to a range of PRSV strains has in fact been developed (D. Gonsalves unpubl.).

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However, the time required to commercialize this transgenic Kapoho may be longer than the time it took to commercialize line 55-1. These circumstances highlight the need to carefully guard against the introduction of PRSV strains into Hawaii, and to maximize the usefulness of our existing transgenic cultivars. As an alternative, Rainbow F2 plants that were backcrossed four times with ‘Kapoho’ and subsequently self-pollinated. Homozygous plants yielding BC4 fruit nearly identical to ‘Kapoho’ were developed (M. Fitch and S. Ferreira, unpubl.). These could possibly be used as a transgenic ‘Kapoho’ parent to cross with ‘SunUp’, hopefully producing horticultural characteristics equal to those of ‘Rainbow’, but with increased gene dosage and presumably stronger resistance than ‘Rainbow’. Lastly, given the fact that ‘Rainbow’ has narrow resistance, PRSV isolates from throughout Hawaii need to be continually screened for their ability to overcome the PRSV resistance of ‘SunUp’ and ‘Rainbow’. 3. Guarding Against Large-Scale Resurgence of PRSV in Nontransgenic Papaya in Puna. Despite the above efforts to protect nontransgenic ‘Kapoho’ from PRSV, observations suggest that PRSV infections are increasing in nontransgenic papaya in Puna. As virus inoculum builds up in Puna, it will become more difficult to economically produce nontransgenic papaya. Strict attention needs to be paid to planting nontransgenic papaya in as much isolation as possible, to timely elimination of infected trees, and to plow under nontransgenic plantings that are no longer in production. The latter will reduce PRSV inoculum source.

IV. DEVELOPMENT OF TRANSGENIC PAPAYA FOR OTHER REGIONS Since PRSV threatens papaya production worldwide, other countries have shown interest in developing this technology for their own use. Thus, a program was established by one of the authors (DG) to develop and transfer the technology to interested countries. This section summarizes the status of the program and the results obtained in other laboratories. Starting in 1992, a technology transfer program was implemented with various agencies in Brazil, Jamaica, Venezuela, and Thailand. The program involved students or scientists coming to the host institution (initially Cornell University) to develop a transgenic papaya that would be used in their country. With the exception of Venezuela, initial characterization of the transgenic plants was done at Cornell University before desired lines were transferred back to their own country for

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further testing and eventual deregulation and commercialization. Since the Hawaiian work had shown that resistance of transgenic ‘Rainbow’ papaya was largely limited to PRSV strains from Hawaii, the transgenic papaya was targeted for resistance to local viral strains. The CP gene from country of origin was used and transformation was done on papaya grown in country. The technology transfer program has pointedly reaffirmed that it is relatively straightforward to develop transgenic papaya in a timely manner and to return them to the collaborating country. However, progress toward commercialization has been vague and slower than the program that was followed in Hawaii. Failure to progress toward commercialization has been partly due to the differing state of governmental programs to deal with transgenic crops, relative to the U.S. system. Undoubtedly, generally unsettled attitudes throughout the world toward GMOs has also had a significant impact. A. Brazil In 1992, two researchers, Dr. Osmar Nickel and then researcher Manoel Teixeira Souza, Jr., supported by the Brazilian government [Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, and Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA)] spent time at Cornell University working to develop transgenic papaya. The CP gene was obtained from a PRSV isolate from the southeast region of the state of Bahia. Manoel Souza continued his work as a Ph.D. student at Cornell in 1993. Translatable or nontranslatable CP gene constructs were used in the transformation experiments. The resulting T0 plants appeared to be resistant to the homologous virus as well as to the Hawaii strain PRSV and HA to an isolate from Thailand (Souza Jr. and Gonsalves 1999). Transformants were also challenged by the Brazilian strain of PRSV. Candidate resistant lines were sent to Brazil in 1999, where they were subsequently analyzed up to the third generation. Thirty-two transgenic papayas were tested in the field in Brasilia in a 900 m2 plot where they showed good resistance. The main purpose of the initial tests was to produce seeds from which plants could be tested, in producing areas in the states of Ceara, Espirito Santo, and Bahia. Unfortunately, the program has come to a halt because of the strict regulations imposed on GMOs by the home regulatory body, the Brazilian Technical Committee for Biosafety (CTNBio). Currently, a small field trial is being conducted at the experimental station at Cruz das Almas. Recently, Brazil passed legislation to allow further testing of transgenic plants and it is hoped that more intensive work will be resumed to identify and eventually commercialize the resistant line.

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B. Jamaica Collaborative work with Cornell University to control PRSV by cross protection was initiated in 1990 by Paula Tennant, who came to Cornell as a visiting scientist. Soon after, efforts were started on developing transgenic papaya for resistance to PRSV, with Ms. Tennant enrolling as a graduate student at Cornell University in 1991. The project has been a collaborative effort between the University of the West Indies (Mona campus), Cornell University, and the Jamaica Agricultural Development Foundation (JADF). A virus isolate from the island of Cayman was used in the construct. Two versions of the transgene were made: one with a translatable CP gene and the other a non-translatable CP gene. Transgenic plants were obtained following bombardment into ‘Sunrise’ (solo type) somatic embryos. Under greenhouse conditions with manual inoculation, high (78%) resistance was found for the translatable CP construct compared to only 10% for the non-translated construct. However, even for the sensitive plants, a delayed recovery was observed in which initial sensitive phenotype disappeared in subsequent new growth. T0 plants were transferred to Jamaica in 1998. Field trials in Jamaica on primary transformant (T0) plants were delayed for two years to 1998. During this time the National Biosafety Committee, a subcommittee of the National Commission of Science and Technology responsible for importation, was established, and the necessary regulations were passed allowing such experiments to take place. Resistance to field sources of PRSV of the homologous type was similar to mechanical inoculation in the greenhouse with 80% of the transgenic papaya carrying the translatable CP gene compared to 44% for the non-translated CP construct. Field trials conducted in 1999 on R1 plants showed a similar trend to parental lines with 58% resistance (Tennant et al. 2002). Thus, in the case of the transgenic papaya in Jamaica, greater resistance was correlated to translatability of the CP. The horticultural, nutritional, and other components fell within the range documented for non-transgenic papaya. Results of rat feeding trials showed no adverse health affects attributable solely to transgenic papaya fruit. Taken together, these data indicated that transgenic papaya was safe for consumption. As in other studies, one goal of the project in Jamaica was the development of local resistant cultivars by crossing the resistance transgene into locally favored cultivars. One molecular trait deemed useful for such development is the identification of transgenic lines with simple genetics (i.e., a single transgene insert) that can be maintained by selfing and the availability of local cultivars with attributes that are attractive to the local market. In Jamaica, these include the large-fruited ‘Santa Cruz giant’ and ‘Cedro’ cultivars. The release of the Jamaica transgenic

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cultivars, initially targeted for 2002, is currently on hold due to the lack of support for a third and final field trial. A further complication is that Europe is a significant market for Jamaican papaya. Unless the papaya is deregulated by the European Union, transgenic papaya will not be able to be exported to Europe or elsewhere. At this moment, there is still a “GMO controversy” with the deregulation of transgenic products in Europe. Conceivably, this could hold back the commercialization of transgenic papaya in Jamaica for fear that the transgenic papaya from Jamaica may somehow be mistakenly shipped to Europe before it is deregulated in Europe. C. Thailand This program is an offshoot of a USAID funded effort initiated in 1986 with Ms. Vilai Prasartsee to help subsistence farmers of Northeast Thailand control PRSV by cross protection and by breeding for papaya lines that are tolerant to PRSV. In 1994, the government of Thailand (initially the Ministry of Agriculture and Cooperatives and continued to the present time by the Department of Agriculture) started a program for developing PRSV-resistant transgenic papaya for the farmers of Northeast Thailand. In 1995, the Department of Agriculture sent scientist Dr. Nonglak Sarindu to Cornell with the mission of learning and applying the skills necessary to develop and identify potential transgenic papaya lines that would be candidates for use in Northeast Thailand. Two cultivars, the popular ‘Khakdum’ and ‘Khaknuan’, were targeted for transformation using the nontranslatable CP gene of a Thailand isolate of PRSV from Northeast Thailand. She was aided by Dr. Suchirat Sakuanrungsirikul, who later came to Cornell to provide interim help. The project in Cornell worked well and several transgenic resistant lines of CP transgenic ‘Khakdum’ and ‘Khaknuan’ were identified after greenhouse inoculation at Cornell. A number of potential T0 lines were delivered to Thailand in July 1997. Work was immediately started by Nonglak Sarindu and Vilai Prasartsee on the propagation, seed increase, and testing for resistance of the potential lines; Prasartsee has headed the work since 1997. By 1999, field trials of the T1 generation (Plate 2.10) showed excellent results. By the year 2002, a T3 line of ‘Khaknuan’ had been selected and showed excellent PRSV resistance (Plate 2.11) and horticultural characteristics. In comparative tests, the transgenic line showed that 97% of the progeny were resistant under intense disease pressure and yielded 63 kg fruit per tree in the first year, whereas nontransgenic papaya yield only 0.7 kg per tree per year. Crosses between independent lines of ‘Khakdum’ have recently shown good resistance under greenhouse and field conditions (Prasartsee, unpubl.).

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Concurrently, molecular characterization, biosafety experiments, and analysis of transgenic fruit for food properties and food safety, and intellectual property rights were initiated using material that had been selected for eventual deregulation and commercialization. Nearly all biosafety experiments that are mandated by the national committee on biosafety have been completed. Tests on food safety and other characteristics such as vitamin, amino acids, soluble solids and other profiles are being done and should be completed in the near future. However, recent controversial events regarding GMOs will very likely slow down the process of deregulating the transgenic papaya. D. Venezuela In 1992, the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICIT, now FONACIT) through a joint venture with the Interamerican Development Bank approved a grant for University of Los Andes (ULA) to develop PRSV-resistant transgenic papayas in collaboration with Cornell University. Gustavo Fermin came from ULA as a visiting scholar and then to pursue a Ph.D. Two local Venezuelan isolates (LA and EV) of PRSV were collected from domestically and commercially grown papaya. The CP gene of the above isolates was cloned in a plant transformation vector in the sense/translatable, sense/untranslatable and antisense forms (Fermin 1996). The plant transformation vectors with the cloned genes were sent to Venezuela, where they were used to transform a local papaya ‘Thailandia Roja’ via A. tumefaciens. A few putative transgenic lines were recovered in 1997. While a student at Cornell, Dr. Fermin analyzed these few transgenic lines molecularly and for their resistance against PRSV strains from Venezuela, Hawaii, and Thailand (Fermin et al. 2004). The resistance appeared to be RNA mediated, and R1 and R2 plants showed a promising level of resistance not only to local isolates but also to different geographic isolates of PRSV, such as isolates from Thailand and Hawaii (Fermin et al. 2004). Two hermaphrodite plants showing a high level of resistance from the T2 generation were identified and kept for further multiplication and testing. A small field test was conducted at Lagunillas, Merida, with a special permit from the Ministry of Health of Venezuela (MHV). The field was planted with T1 individuals previously selected in the greenhouse as PRSV resistant. Subsequently, the plants performed well in the field under local pressure of virus. When the transgenic PRSV-resistant papaya set flowers, unexpected problems started to emerge with GMO activists, who set fire to the plants in December 2000 (Fermin et al. 2004). Currently, Dr. Fermin has seeds of resistant plants from T2 generations but their use in field trials is still in doubt.

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E. Taiwan Transgenic papaya resistant to PRSV were successfully developed by Dr. Shyi-Dong Yeh’s team of the National Chung Hsing University following an approach similar to that used for developing the cultivars ‘Rainbow’ and ‘Sunup’ for Hawaii; i.e., by use of CP-mediated resistance. The CP gene of a local PRSV isolate, YK was inserted as a transgene in the Taiwanese papaya cultivar, ‘Tainung No. 2’. Unlike HA 5-1, the source of the CP gene used for developing the Hawaiian transgenic papaya, the PRSV isolate YK is a severe virus strain found in Taiwan. Transgenic ‘Tainung No. 2’ papaya were obtained by Agrobacterium-mediated transformation rather than by the biolistic method used for developing ‘Rainbow’ and ‘Sunup’. Transgenic, resistant papaya lines carrying the CP gene of PRSV YK were generated and four transgenic lines resistant in greenhouse experiments were evaluated from 1996 to 1999 under field conditions. Both resistance properties and fruit production were investigated. Performance of the transgenic lines in the field trials was found to be similar to that of ‘Rainbow’ and ‘Sunup’. None of the transgenic lines showed severe symptoms of PRSV infection, whereas 100% of the nontransgenic plants were severely infected 3 to 5 months after planting (Bau et al. 2004). However, 20–30% of the transgenic plants exhibited mild symptoms in the first and second field trials but fruit yield and quality were not affected. The transgenic lines were not only protected from virus infection, but also produced 11–56% more marketable quality papaya compared to nontransgenic papaya (Bau et al. 2004). In another transformation experiment, 45 putative transgenic lines were obtained in the laboratory of S.-D. Yeh that had exhibited PRSV resistance ranging from delay of symptom development to complete immunity following inoculation in the greenhouse. Similar to ‘Rainbow’ and ‘Sunup’, molecular analysis of nine selected lines revealed that the expression level of the transgene was negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. Segregation analysis showed that the transgene in immune line 18-0-9 had an inheritance of two dominant loci and the other four highly resistant lines had single dominant loci. Seven selected lines were tested further for resistance to three heterologous PRSV strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, whereas one line (19-0-1) was immune not only to the homologous YK strain but also to the three heterologous strains (Bau et al. 2003). So far none of the transgenic plants from Taiwan have been deregulated or commercialized.

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F. Australia PRSV was described in Australia as late as 1991 and as yet has not caused major limitations to production of papaya there. Strict quarantine measures in Southeast Queensland, where PRSV was first discovered, has prevented its spread to the major production region in tropical North Queensland. Nevertheless, mindful of the history of the virus in other parts of the world, researchers in Australia have been active in the production of transgenic papaya resistant to Australian PRSV isolates. Transformation and regeneration techniques for papaya were developed. Based on CP nucleotide sequence, data comparisons of isolates from within and outside Australia have shown that domestic PRSV isolates vary by only 2%. The PRSV isolate used for transgenic studies was obtained from Southeast Queensland. The transgene was designed with a premature stop codon in the PRSV CP sequence, thus it was expected that a functional CP would not be expressed. Transformation was facilitated by biolistic transformation of secondary somatic embryos of cultivars GD3-1-19 and ER6-4, local cultivars that are also used for production and breeding. Only two resistant lines were regenerated for each cultivar, each with multiple inserts, and interestingly both were male plants. According to RNA blot analysis, the plants with the best resistance exhibited the least detectable message, strongly suggestive of the involvement of an RNA silencing mechanism for viral resistance. Copy number also appeared to play a role in the level of resistance, as those with single copies were more susceptible. This observation of gene copy number dependence is consistent with that found for RNA-mediated silencing and PRSV resistance of the original Hawaiian transgenic papayas (Lines et al. 2002). G. Hongkong, China Researchers in China reported the first use of the PRSV replicase gene for the production of PRSV-resistant papaya. The replicase gene was cloned by PCR from RNA from pumpkin leaves infected with PRSV. For the papaya replicase construct, the 3′ end of the gene was deleted and additional codons were added to the 5′ end of the gene. Transformants were obtained by Agrobacterium-mediated transformation of embryogenic calli of the cultivar ‘Tainung No. 2’. The resulting transformants showed varying levels of resistance in response to mechanical inoculation, including apparent complete immunity in the greenhouse (Chen et al. 2001). The mechanism of resistance, whether protein or RNA-mediated, is unknown. The greenhouse tests were encouraging but the fate of these transgenic lines under natural field conditions are as yet unknown.

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H. The Papaya Biotechnology Network of Southeast Asia Recently, the International Service for the Acquisition of Agri-biotech Application (ISAAA) performed a collaborative, comprehensive analysis of the needs of Southeast Asian countries. The virus-resistant papaya was identified by ISAAA as a high-priority need. Its introduction could improve the lives of subsistence farmers throughout the region. ISAAA and its collaborators concluded that a regional project to transform papaya with PRSV resistance and delayed-ripening characteristics could help meet Southeast Asia’s urgent food needs and enhance the region’s capacity to develop and deploy other transgenic crops in the future. In 1998, ISAAA initiated The Papaya Biotechnology Network of Southeast Asia through the partnership and collaborative effort of five target countries (Indonesia, Malaysia, The Philippines, Thailand, and Vietnam) and two private sector companies (Monsanto and Syngenta) to improve papaya for Southeast Asia’s resource-poor farmers. Scientists from the respective Network laboratories produced three plant expression vectors containing genes from PRSV isolates from Thailand, Vietnam, Philippines and Malaysia during their research internships at Monsanto. PRSV sequence diversity in Southeast Asia is much higher than in other locations, thus the practice of using genes from local virus isolates for the production of resistant transgenic papaya is a necessity. Currently the vectors are being used in the transformation of papaya somatic embryos as the first step in the generation of transgenic papaya plants (www.isaaa.org). I. Bangladesh and Africa Considering the seriousness of the PRSV problem in Bangladesh and African countries, efforts have been extended to restore the papaya industry in those countries. The laboratory of Dr. Gonsalves is taking an active role in attempting to solve the severe PRSV problems of Bangladesh and African countries in a timely manner, through USAID technology transfer programs. An extensive survey was conducted to collect and characterize PRSV isolates from all around Bangladesh and several regions in Africa. Activity is underway to molecularly characterize and study the sequence diversity of these isolates and to develop a transgenic resistant papaya for these countries. J. Florida, USA Surveys of the PRSV CP gene sequences of Florida isolates indicate a close relationship to PRSV sequences from Puerto Rico and Mexico compared to those isolated from more distant locations. Thus, study on

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transgenic papaya carrying the PRSV CP of a Florida isolate was done with the intention of producing cultivars resistant to PRSV of the Caribbean region. The source of the CP was the Florida PRSV H1K isolate. Four different types of constructs—a sense, antisense, frame shift and stop codon mutation of the CP—were made. The construct was transformed into immature zygotic embryos of the experimental cultivar F65, which is an ancestor of the PRSV-tolerant cultivar ‘Red Lady’, by Agrobacterium-mediated transformation. None of the resulting plants were immune when inoculated in the greenhouse with the homologous PRSV at 10 weeks, but moderate to highly resistant individuals were identified from among each construct type. Interestingly, the lines derived from sense and antisense constructs were found to be infertile. The remaining stop codon and frame shift mutation constructs lines were fertilized with pollen from ‘Red Lady’ and experimental No. 15 and cultivars grown at the University of Puerto Rico including ‘Puerto Rico 6-65’, ‘Tainung No. 5’, ‘Solo 40’, and ‘Sunrise’. The ‘Puerto Rico 6-65’ and ‘Tainung No. 5’ are PRSV-tolerant cultivars whereas ‘Solo 40’ and ‘Sunrise’ are highly sensitive. Resistance of the T1 progeny in the field seemed to be influenced to some extent by the particular cross, with progeny from the tolerant ‘Tainung No. 5’ and sensitive ‘Sunrise’ being more sensitive than that of other combinations (Davis and Ying 2004).

V. BREEDING THROUGH INTERGENERIC HYBRIDIZATIONS An ideal approach for controlling PRSV is through incorporation of PRSV-resistant genes via breeding. However, numerous efforts have failed to identify PRSV-resistant genes in C. papaya. Early on, researchers identified PRSV-resistant genes in other Carica species, which subsequent work has shown is grouped into the genus Vasconcellea. A major barrier has been the incompatibility between crosses of C. papaya and Vasconcellea species. However, recent events provide more hope for this approach, as will be described below. A. Resistance and Susceptibility of Vasconcellea Species to PRSV There have been variable reports on the susceptibility or resistance of Vasconcellea, which was formerly considered a wild Carica species (Badillo 2000), to local strains of PRSV from Florida, Hawaii, Venezuela, Mexico, and Australia. Conflicting findings are probably due to genetic differences in local virus strains and plant material (Horovitz and Jimenez 1967), environmental conditions, and/or deficient diagnostic methods.

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V. cauliflora has been reported resistant in Australia (Magdalita et al. 1988), Mexico (Alvizo and Rojkind 1987), and Venezuela (Horovitz and Jimenez 1967), but susceptible in Florida (Conover 1962) and, more recently, also in Venezuela (Gonzalez 2000). V. quercifolia was susceptible in Venezuela and resistant in Florida, Hawaii, and Australia. V. parviflora and V. goudotiana were susceptible in Venezuela (Horovitz and Jimenez 1967) and Australia (Magdalita et al. 1988) and Venezuela. Other susceptible species included: V. microcarpa, V. monoica, V. horovitziana, V. ¥heilbornii nm chrysopetala. Other reported resistant species were: V. stipulata, V. pubescens, V. candicans and V. ¥heilbornii nm pentagona in Venezuela (Horovitz and Jimenez 1967). Some of the species that showed susceptibility or resistance to PRSV are closely related species. B. Intergeneric Hybridizations between Carica papaya and Vasconcellea Species Extensive intergeneric hybridization studies have been carried out in Venezuela (Jiménez and Horovitz 1958; Horovitz and Jimenez 1967), India (Padnis et al. 1970), Hawaii (Manshardt and Wenslaff 1989a,b), Brazil (Gama et al. 1985), and more recently in Taiwan (Chen et al. 1991), Australia (Drew et al. 1998), and Venezuela (Vegas et al. 2003). These intergeneric crosses were made in an attempt to transfer resistant genes from wild species to papaya and to understand the mechanism of resistance involved in Vasconcellea. Evidence had indicated that this resistance could be based on a dominant gene (Micheletti 1962). Some hybrid plants have been obtained by embryo rescue after crossing C. papaya with V. cauliflora, V. cundinamarcesis (V. pubescens), V. querciflolia, V. stipulata, V. goudotiana, and V. parviflora. A large degree of sterility and levels of vigor were displayed in the hybrids. Although a number of relative successes have been reported in hybridizations between the wild species (Mekako and Nakasone 1975), incompatibility between papaya and Vasconcellea species has been a major limitation to production of useful intergeneric hybrids for 50 years. A comprehensive study of reproductive barriers that limited crosses between papaya and its wild relatives demonstrated that prezygotic barriers were unimportant. However, significant postzygotic barriers included embryo abortion and lack of endosperm development (Manshardt and Wenslaff 1989a,b). In view of recent genetic analyses, it is unfortunate that so much effort has been directed to crosses between papaya and V. cauliflora, as they have been shown to be the most genetically distant (Jobin-Décor et al. 1996). Hybrids between these two species lack vigor, rarely survive till flowering, and if they do, are infertile (Manshardt and Wenslaff 1989b). Simi-

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larly, hybrids between papaya and V. stipulata were reported to lack vigor and viability (Horovitz and Jimenez 1967). By contrast, in an extensive breeding program in Australia, crosses between C. papaya and the Vasconcellea species V. pubescens, V. quercifolia, V. parviflora, and V. goudotiana were vigorous both in the glasshouse and field (Drew et al. 1998). Sex expression varied between crosses. Pubescens crosses were all pistillate, quercifolia hybrids had a sex ratio of 2:49:49 of staminate: hermaphrodite: pistillate, and V. parviflora and V. goudotiana demonstrated normal dioecious behavior with a 1:1 ratio of staminate and pistillate plants (Drew et al. 1998). Most plants were infertile; however, some staminate plants of hybrids between papaya and V. quercifolia, and papaya and V. parviflora, produced small quantities of viable pollen that germinated on Brewbaker and Kwak (1963) medium (Drew et al. 1998). Some backcross generations were obtained in Hawaii but were sterile (Manshardt and Zee 1994). Most of the work on intergeneric hybridization has been aimed at introgressing genes for PRSV resistance from the wild species related to papaya. PRSV resistance has been reported often in crosses between C. papaya and V. cauliflora (Moore and Litz 1984; Vegas et al. 2003); however, the infertility or the lack of vigor of these hybrids has prevented further backcrossing. In stark contrast to other published results, Khuspe et al. (1980) reported production of viable F1 and F2 populations that were resistant to PRSV in the F1 population and segregated for PRSV resistance in the F2 population with a 3:1 ratio. However, no confirmation of hybridity was demonstrated and no resistant papaya genotypes have resulted from this work. Seeds of a subsequent generation were obtained from these crosses in India and were field-tested in Australia. Morphologically they were similar to papaya genotypes, the fruit were characteristic of papayas, and they were susceptible to the Australian strain of PRSV (R. A. Drew, unpubl.). Interspecific hybrids between C. papaya and other Vasconcellea species have also demonstrated resistance to PRSV. All V. pubescens hybrids were resistant to PRSV when manually inoculated three times at two-weekly intervals in a glasshouse (Drew et al. 1998). A large population (>300) of C. papaya × V. quercifolia hybrids were manually inoculated using the same procedure. Of those tested, 75% were resistant and 25% produced symptoms of the virus (Drew et al. 1998). These latter results suggest that PRSV resistance in V. quercifolia is not controlled by a single dominant gene. Progressing past intergeneric F1 hybrids has been very difficult, and the only successes have resulted from backcrosses from C. papaya × V. quercifolia to C. papaya. Embryo culture has produced no F2 progeny to date and only limited progenies in backcrosses with papaya. In Hawaii, F1

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hybrids contributed only unreduced gametes in backcrosses, yielding sequidiploid plants that were very sterile, indicating that meiosis did not function normally in those hybrids (Manshardt and Drew 1998). Recently, in Australia, with the aid of funding from the Australian Centre for International Agricultural Research, an extensive crossing program has resulted in fertile backcross plants being produced (Drew and O’Brien 2001). Pollen from C. papaya × V. quercifolia male plants has been used to successfully pollinate C. papaya flowers. Initially, a backcross population (BC1) of 50 plants was generated, and one of these, a staminate, demonstrated some PRSV resistance and produced fertile pollen. Seventy clones of this plant were inoculated with PRSV at two-weekly intervals and only one showed symptoms of PRSV. Initially, they appeared resistant in the field; however, one clone developed PRSV symptoms after 12 months in the presence of aphids and PRSV-infected plants. Pollen from this symptomatic BC1 plant was used to produce 200 plants of a second backcross (BC2) population. These were inoculated with PRSV 5 times in a glasshouse over a 12-month period. Twenty-six plants remained free of symptoms, and when planted in the field with infected papaya trees and exposed to natural inoculation by aphid vectors, progressively produced symptoms over a 9-month period. Time to develop symptoms and symptom severity varied between plants. In 2004, clones of another 50 plants of a BC1 generation (C. papaya × V. quercifolia backcrossed to C. papaya) were field tested in Southeast Queensland. Again, PRSV symptom severity varied from severe to mild and one clone has shown no symptoms after 6 months. A much larger BC1 population (up to 500 plants) is currently being produced and will be fieldtested for PRSV resistance in Australia and the Philippines. These results show promise for further work with C. papaya and V. quercifolia. Five factors have contributed to the successes with intergeneric hybridization. Firstly, some papaya genotypes are more compatible with the wild species and more likely to produce embryos when crossed (Magdalita et al. 1998). Selection of papaya genotypes more compatible with wild species is critical in crossing programs. Secondly, the use of embryo rescue before embryo abortion occurs, and subsequent plantlet production in vitro, is an essential step in the procedure. Embryo rescue and culture was attempted as early as 1967 and refined by Micheletti (1962), Horovitz and Jimenez (1967), and Manshardt and Wenslaff (1989a,b). Thirdly, recent advances in molecular biology have facilitated advanced taxonomic analysis of Carica and Vasconcellea species to determine which wild species are genetically the most similar and dissimilar to papaya. Analyses of RAPD profiles and comparison of DNA sequences from nuclear and mitochondrial genes all show that V. quercifolia is the most similar and V. cauliflora the most dissimilar to papaya

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(Manshardt and Drew 1998). Consequently, an extensive crossing program between V. quercifolia and papaya led to the first fertile backcross plants. Fourthly, because of the difficulties in making these wide crosses, success has been achieved when large numbers of crosses have been attempted and large field populations evaluated (Drew et al. 1998). This was made possible by the development of improved and efficient protocols for large-scale crossing, in vitro culture, and plantlet production (Magdalita et al. 1996; Magdalita et al. 1998). Fifthly, these large populations have allowed selection of a few male interspecific hybrids with both PRSV resistance and some fertility that has allowed backcrossing to a female papaya. It is possible that mitochondrial DNA is important in obtaining fertility. Fertility has been obtained in backcross plants when papaya was the female parent, but not when the interspecific hybrid was the female parent (Manshardt and Wenslaff 1987). It is expected that the genome of papaya would be more stable than that of the interspecific hybrid.

VI. DEVELOPMENT OF PRSV-TOLERANT PAPAYA Although most papayas are highly susceptible to PRSV, some selections have shown tolerance. Tolerance to PRSV is inherited in a quantitative manner (Conover and Litz 1981) and has been incorporated into some newer cultivars by recurrent selection programs using papaya lines with moderate to high levels of tolerance crossed with local cultivars, followed by screening for resistant seedling progeny by virus inoculation. In such a way, ‘Cariflora’, which is tolerant to virus found in South Florida and the Caribbean, was developed (Conover et al. 1986). This selection is dioecious and produces round fruit of yellow-orange flesh. Trees bear ripe fruit about 5 months after planting in the field. This Florida tolerant papaya does become infected by PRSV but the leaves are symptomless or show only mild mosaic phenotype and are not distorted. The fruit does not show any virus-induced distortion, although some fruit develop ringspots. Florida tolerant papaya produces acceptable amounts of fruit despite being infected with PRSV. However, the small round fruit and yellow flesh along with the dioeciousness are not ideal characteristics for papaya grown in tropical areas. Nevertheless, it has been used as parental genetic material for a number of breeding programs. In Thailand, a series of papaya lines developed by crossing the Florida tolerant and local variety ‘Khakdum’ followed by recurrent selections is the result of an on-going breeding program since 1987 (Prasartsee et al. 1998). ‘Khakdum’ is a popular Thai cultivar with desirable fruit characteristics, but it is very susceptible to PRSV (Nopakunwong et al. 1993).

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Plants of ‘Khakdum’ are hermaphrodite or pistillate with the hermaphrodite producing oblong fruit of orange-red flesh. The first ripe fruit from this cultivar is produced about 10 months after planting. Previous trials at the Khonkaen Horticultural Experiment Station (KKHS) (Prasartsee et al. 1998) showed that Florida tolerant papaya produced acceptable amounts of fruit despite being infected with PRSV. Thus, reciprocal crosses were made between the Florida tolerant variety and ‘Khakdum’ in an effort to produce hybrid lines that are PRSV-tolerant and have acceptable horticultural characteristics. Seedlings are under evaluation. A. Screening for PRSV Tolerance and Recurrent Selection Scheme All plants were screened with a severe PRSV isolate from Thapra in Northeast Thailand. Papaya seedlings were exposed to PRSV by mechanical inoculation in the greenhouse or by aphid inoculations under field conditions where incidence of PRSV was high. Greenhouse-grown and inoculated plants with mild or no symptoms were transplanted to the field for further evaluation of PRSV tolerance and horticultural characteristics. Since PRSV tolerance is inherited quantitatively, a recurrent selection scheme was used to maintain tolerance and incorporate desired horticultural characteristics. Thus, only one cross was made initially between ‘Florida Tolerant’ and ‘Khakdum’. Subsequent crosses were made from within the progeny population. The first priority was to maintain a high level of PRSV tolerance followed by selection of desirable horticultural characteristics. The plants derived from seeds of papaya fruit (lines) resulting from reciprocal crosses were evaluated under field conditions. Five lines resulting from those crosses showing good PRSV tolerance were selected in the first cycle. Progeny from these promising lines were evaluated by recurrent selections through the fifth cycle, resulting in the successful development of three papaya lines named ‘Thapra 1’, ‘Thapra 2’, and ‘Thapra 3’ showing excellent PRSV- tolerance and horticultural characteristics. In local field trials, the papaya cultivars ‘Thapra 1’, ‘Thapra 2’, and ‘Thapra 3’ that showed good tolerance under heavy PRSV disease pressure at KKHS, Thapra, and Khon Kaen were subsequently tested in other areas in Northeastern, Central, and Southern Thailand. Data collected over 18 months from the three regional PRSV infection sites showed that ‘Thapra 1’ and ‘Thapra 2’ had better tolerance to virus disease under severe virus pressure compared to ‘Thapra 3’. ‘Khakdum’ papaya showed the most severe symptoms under severe virus pressure. According to the results of local field trials, ‘Thapra 2’ generally showed the most promise. It grew vigorously, had excellent PRSV tolerance, and was productive even when grown under severe PRSV pressure. Trees

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Table 2.3. Some characteristics of PRSV-tolerant papaya cultivars ‘Thapra 1’, ‘Thapra 2’, and ‘Thapra 3’ compared to non-tolerant ‘Khakdum’. Papaya cultivars Characteristics

Thapra 1

Thapra 2

Thapra 3

Khakdum

Tree height at first ripe fruit (m)

1.7

1.3

1.5

2.0

First ripe fruit after planting (mo.)

7

6

8

9

1.5

1.2

1.3

Shape of hermaphrodite

Avg weight of fruit (kg)

oblong, long

lengthened cylindrical

pear shape

lengthened cylindrical

Flesh color

yellow

yellow

red

red

Brix (%)

1.4

11.2

11.2

11.2

13.5

150,080

159,360

182,880

100,200

z

Average annual yields (kg/ha) z

Provinces (Khon kaen, Srisaket, Nongbualumpoo, Nongkai)

produced fruit as early as three months after transplantation into the field and the first fruit ripened at 6–7 months. Trees were short, with an average height of 1.3 m. The flesh of ripe fruit is light to deep yellow with a mild aroma. It is medium to large in size with long, cylindrical-shaped fruit. The average fruit weight is 1.5 kg with a Brix level of 11.2. ‘Thapra 2’ is good for flesh consuming and desirable for canning as well. The general characteristics of these papaya are given in Table 2.3. B. Recommendation and Availability of Seeds In 1997, the Department of Agriculture (DOA) released ‘Thapra 2’ as a recommended cultivar (Plate 2.12). Its name was changed from ‘Thapra 2’ to ‘Khakdum Thapra’ and has been planted out for large-scale seed production since 1998 and distributed to farmers in all regions of Thailand. ‘Thapra 3’ showed good characteristics although relatively less PRSV tolerance. It bears a small-size fruit averaging 1.2 kg weight and is pyriform in shape; it is suitable for table consumption and for export. The ripe flesh is reddish orange color, which is the preferred color for Thai consumers. Since 2000, selection has continued to obtain fruit ≤1 kg, reddish orange color flesh, and good taste with high sugar content. The selection of lines more tolerant to PRSV has also been considered in parallel. The selection program has been conducted based on pure line selection. In the first year (year 2000), 10 lines showing good characteristics were identified. The selected plants had been self- pollinated to

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obtain seeds of the progenies for the next selection. It is now in the third round of selection. The papaya lines will be evaluated for another cycle before local field trials will begin. ‘Thapra 3’ papaya is expected to be released for table consumption and export in the near future. C. Maintenance of PRSV-Tolerant Papaya Lines Since the tolerance to PRSV found in ‘Khakdum Thapra’ is a quantitative trait, it can be lost if that trait is not continually evaluated and selected for in subsequent sexual generations. During large-scale production of seed, the seed source plants must have acceptable levels of tolerance and horticultural properties consistent with the line. To maintain the tolerance and qualities of the line, papaya seeds used for establishment of the plants for seed production should always be obtained from specific ‘Khakdum Thapra’ trees identified as tolerant and having good horticultural characteristics. The tolerance program has enabled farmers to grow papaya with reasonable fruit production despite plants becoming infected with PRSV, and has provided farmers with good quality, reasonably priced seed. Other tolerant lines have also been developed. In Taiwan, Lin et al. (1989) reported the development of the hybrid ‘Tainung No. 5’ from the cross of FL 77-5 (from Florida) and ‘Costa Rica Red’, with a good level of tolerance and horticultural characteristics. It has a strong trunk and shows early fruit bearing and ripening. The height of the first fruit from the base of trunk is about 56–60 cm. The use of tolerant papayas has not resolved the virus problem in the long term and development of genetically resistant cultivars is considered the only reliable solution to PRSV control.

VII. FUTURE ASPECTS FOR DEVELOPING PRSV-RESISTANT PAPAYA Pathogen-derived resistance (PDR) based on exploiting different genes coding for viral proteins has been successful for controlling plant virus diseases, but the CP gene is, by far, the most widely used to engineer transgenic resistance (Lomonossoff 1995). The CP-mediated transgenic papaya in Hawaii is a successful example of the utilization of PDR in controlling PRSV. It is now conclusive that transgenic resistance in papaya is RNA-mediated through post transcriptional gene silencing (PTGS) (Tennant et al. 1994; Tennant et al. 2001). In fact, the prevailing mechanism for transgenic resistance is via PTGS (Goldbach et al. 2003). As shown by our work in Hawaii, ‘Rainbow’, which is hemizygous for the CP gene, had a much more narrow base of resistance than ‘SunUp’,

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which is homozygous for the same CP gene. Thus, our early approaches for developing countries have been to use CP from PRSV strains of the target countries. In our quest to develop transgenic papaya with a broader range of resistance, we took advantage of our work on tospoviruses, which is described below. A. Segmented Gene Approach Evidence that viral transgenes could only confer resistance against closely related viruses stimulated new approaches to create transgenic plants that could be simultaneously resistant to different viruses. The first approach involved the use of multiple CP or nucleoprotein (N) transgenes regulated by independent promoters and terminators (Fuchs and Gonsalves 1995; Prins et al. 1995; Tricoli et al. 1995). Two main disadvantages precluded the extensive use of this approach: concerns about the introduction of increasingly higher amounts of foreign DNA into crop plants, and limitation on the number of CP genes that could be simultaneously transformed into the plant. A second, more feasible approach toward multiple virus resistance was developed when it was realized that virtually any segment of DNA derived from the N gene of Tomato spotted wilt virus (TSWV) conferred resistance to TSWV in transgenic tobacco (Pang et al. 1997). These N gene fragments could be as short as 100 bp if they were fused to a longer fragment of non-related DNA, such as the 720 bp green fluorescent protein (GFP) gene (Jan et al. 2000a). As a consequence of this finding, chimeric transgenes consisting of the CP genes of Turnip mosaic virus fused to a 217 bp segment of the N gene of TSWV were designed and shown to confer resistance to both viruses in Nicotiana benthamiana (Jan et al. 2000b). The strategy has been extended to make transgenic N. benthamiana plants resistant to three different tospoviruses (Jan 1998). Experiments are underway to test the strategy of pasting multiple 240 bp long fragments of CP genes from different geographical isolates of PRSV in different combinations and orientations for their efficacy in PRSV resistance (Chiang and D. Gonsalves, unpubl.). Resistance is predicted to be specific for all those isolates that share a high degree of similarity to any of the fragments in the chimeric gene. The downside of this approach is that even though segments of the chimeric gene are short, there is still a limitation on the number of PRSV segments that can be engineered together and consequently there is still a limitation against which PRSV isolates a given transgene construct will be effective. Even so, this is the first rational approach to widen transgenic resistance without increasing the amount of foreign DNA delivered to the target plant. Papaya plants transformed with multiple PRSV CP genes fragments are

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still under evaluation but the first virus challenges indicate that we have been able to widen transgenic resistance for the viruses whose transgenes are involved in the system described here. B. Synthetic Gene Approach A new approach is currently being tested to widen the range of resistance involving the creation of transgenes with a single, short DNA segment (ca. 250 bp) able to confer resistance to multiple viruses. This approach does not involve the use of native CP genes, chimeras, or multiple transformations with selected sequences and would reduce the amount of foreign DNA inserted into the plant. Instead of searching for a natural variant of the CP gene able to confer a broad-spectrum resistance, it was created by rational design (Fermin 2002; Fermin and Gonsalves 2004). To test the feasibility of this approach, we used N. benthamiana and the transgenes encoding the third fourth of the TSWV N gene that was previously shown to confer resistance to TSWV (Pang et al. 1997) as a model system (Fermin and Gonsalves 2001; Fermin 2002). Based on the nucleotide sequence of the N gene fragment, we designed and synthesized a novel sequence from oligo nucleotides that was highly similar (90% in this case) to the corresponding fragments of three distantly related tospoviruses, TSWV, groundnut ringspot virus (GRSV), and tomato chlorotic spot virus (TCSV) that are normally only ca. 78% similar at the nucleotide level. The aim was to introduce nucleotide changes at specific locations that would create a synthetic gene equally distant to all sequences used for its creation. Importantly, the nucleotide changes were designed to create short stretches of 20 nucleotides or more of total identity to the different genes at different points along the synthetic gene, which is a prerequisite for targeting RNA degradation by PTGS. This synthetic sequence allowed us to obtain transgenic N. benthamiana plants resistant to two different tospoviruses, GRSV (24% resistance) and TSWV, and potentially to a third one (TCSV, that could not be used in our lab). We have designed a PRSV-derived single sequence aimed at targeting different isolates of the virus via PTGS (Fermin and Gonsalves 2004). The engineered sequence is equally distant at the nucleotide level to all PRSV isolate sequences that we compiled and used for its design. We targeted the variable (5′) and conserved regions (3′) of the PRSV CP. The two synthetic genes were each independently cloned in tandems of three, and transcriptionally fused to a silencer gene (Chiang and Wang, unpublished results), which in this case was half the N gene of Tomato spotted wilt virus (Jan et al. 2000a). The two different constructs have already been cloned in a plant expression cassette and somatic papaya

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embryos have been transformed by the biolistic method. Infection experiments will be performed in the near future. Results derived from these experiments might lead to the design and creation of a synthetic gene in which we would be able to experiment not only with sequence similarity, but also with the secondary and tertiary structures of the protecting transgene. The long-term goal of these experiments is to create a short, synthetic sequence able to confer universal and durable resistance to PRSV.

VIII. SUMMARY COMMENTS Major breakthroughs to control PRSV have occurred in the last decade, especially with the development of PRSV-resistant transgenic papaya. Recent advances in intergeneric hybridizations provide hope that resistant genes from genera related to Carica papaya may be incorporated into papaya for subsequent efforts to develop PRSV-resistant commercial cultivars. And, the successful program to develop PRSV-tolerant papaya for Northeast Thailand is another example that shows progress in controlling PRSV. The impact of the transgenic papaya in controlling PRSV in Hawaii is a good example of the development of a control measure in a timely manner. Other transgenic papaya have also shown great promise in controlling PRSV, especially in Thailand and Taiwan. Given the success of the transgenic papaya in Hawaii, one would expect that more transgenic papaya would be commercialized outside of Hawaii. Our observations, however, suggest that the deployment of these papaya will not occur anytime soon; not because of inadequate technology but more because of the political climate and controversy over the commercialization of transgenic crops (Fermin, Tennant et al. 2004). It is hoped that these technologies would be evaluated in a timely manner to determine if they can be deployed in areas where PRSV is causing severe damage, especially in developing countries.

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Manshardt, R. M., and T. F. Wenslaff. 1989a. Interspecific hybridization of papaya with other Carica species. J. Am. Soc. Hort. Sci. 114:689–694. Manshardt, R. M., and T. F. Wenslaff. 1989b. Zygotic polyembriony in interspecific hybrids of Carica papaya and C. cauliflora. J. Am. Soc. Hort. Sci. 114:684–689. Manshardt, R. M., and F. T. P. Zee. 1994. Papaya germplasm and breeding in Hawaii. Fruit Var. J. 48:146–152. Mau, R. F. L., D. Gonsalves, and R. Bautista. 1989. Use of cross protection to control papaya ringspot virus at Waianae. Proc. 25th Annu. Papaya Industry Assoc. Conf., Sept. 29, 1989, at Hilo, Hawaii. McGranahan, G. H., C. A. Leslie, S. L. Uratsu, L. A. Martin, and A. M. Dandekar. 1988. Agrobacterium-mediated transformation of walnut somatic embryos and regeneration of transgenic plants. BioTechnology 6:800–804. Mekako, H., and H. Nakasone. 1975. Interspecific hybridization among 6 Carica species. J. Am. Soc. Hort. Sci. 100:237–242. Micheletti, D. 1962. Descripcion morfologica y citologica de una planta segregante de un cruce entre Carica cauliflora Jacq and C. monoica Desf. Agron Tropical (Maracay) XI:193–200. Moore, G., and R. E. Litz. 1984. Biochemical markers for Carica papaya, C. cauliflora, and plants from somatic embryos of their hybrid. J. Am. Soc. Hort. Sci. 109:213–218. Nakasone, H. Y. 1975. Papaya development in Hawaii. HortScience 10:198. Nopakunwong, U., S. Sutchapongse, P. Anupunt, S. Prompan, and S. Rattanakusol. 1993. Papaya selections tolerant to papaya ringspot virus disease. Srisaket Horticultural Research Center Yearly Rep. p. 172–175. Padnis, N. A., N. D. Budrukkar, and S. N. Kaulgud. 1970. Embryo culture technique in papaya (Carica papaya L.). Poona Agricultural College Mag. 60:101–104. Pang, S.-Z., F.-J. Jan, and D. Gonsalves. 1997. Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proc. Nat. Acad. Sci. (USA), 94:8261–8266. Pitz, K., S. Ferreira, R. Mau, and D. Gonsalves. 1994. Papaya cross protection: The nearcommercialization experience on Oahu. Proceedings: 30th Annu. Hawaii Papaya Industry Assoc. Conf., Sept. 23–24, 1994, Maui, Hawaii. p. 4–6. Prasartsee, V., S. Chiakiatiyos, K. Palakorn, P. Cheuychoom, W. Kongpolprom, S. Wichainum, A. Fungkiatpaiboon, C. Gonsalves, and D. Gonsalves. 1998. Development of papaya lines that are tolerant to papaya ringspot virus disease. New Technologies for the Development of Sustainable Farming in Northeast. Proc. of JIRCAS-ITCAD Seminar, pp. 27–33, March 24, 1998, at Khon Kaen, Thailand. Prins, M., P. De Haan, R. Luyten, M. Van Veller, M. Q. J. M. Van Grinsven, and R. Goldbach. 1995. Broad resistance to tospoviruses in transgenic tobacco plants expressing three tospoviral nucleoprotein gene sequences. Mol. Plant Microbe Interactions 8:85–91. Purcifull, D., J. Edwardson, E. Hiebert, and D. Gonsalves. 1984. Papaya ringspot virus. CMI/AAB Descriptions of Plant Viruses. 292. (No. 84 Revised, July 1984). Quemada, H., B. L’hostis, D. Gonsalves, I. M. Reardon, R. Heinrikson, E. L. Hiebert, L. C. Sieu, and J. L. Slightom. 1990. The nucleotide sequences of the 3'-terminal regions of papaya ringspot virus strains w and p. J. General Virol. 71:203–210. Rast, A. T. B. 1975. Variability of tobacco mosaic virus in relation to control of tomato mosaic in glasshouse tomato crops by resistance breeding and cross protection. Agr. Res. Rept. (Versl. landbouwk. Onderz.) 834. p. 1–76. Inst. Phytopath. Res., Wageningen, The Netherlands. Sanford, J. C., and S. A. Johnston. 1985. The concept of parasite-derived resistance: Deriving resistance genes from the parasite’s own genome. J. Theor. Biol. 113:395–405. Shukla, D. D., C. W. Ward, and A. A. Brunt. 1994. The Potyviridae. CAB Int. Wallingford, Oxon, UK.

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Souza Jr., M. T., and D. Gonsalves. 1999. Genetic engineering resistance to plant virus diseases, an effort to control Papaya ringspot virus in Brazil. Fitopatol. Brasileira 24: 485–502. Strating, A. 1996. Availability of determination of nonregulated status for papaya lines genetically engineered for virus resistance. Fed. Regist. 61 (48663). Sturrock, D. 1940. Tropical fruits for southern Florida and Cuba and their uses. Arnold Arboretum of Harvard Univ. Jamaica Plain, MA. Tang, C. S. 1971. Benzyl isothiocyanate of papaya fruit. Phytochemistry 10:117–120. Tennant, P., G. Fermin, M. M. Fitch, R. M. Manshardt, J. L. Slightom, and D. Gonsalves. 2001. Papaya ringspot virus resistance of transgenic Rainbow and SunUp is affected by gene dosage, plant development, and coat protein homology. European J. Plant Path. 107:645–653. Tennant, P., C. Gonsalves, K. Link, M. M. Fitch, R. M. Manshardt, J. L. Slightom, and D. Gonsalves. 1994. Transgenic and classically cross protected papaya show limited protection against papaya ringspot virus isolates from different geographical regions. Phytopathology 84:1375. Tennant, P. F. 1996. Evaluation of the resistance of coat protein transgenic papaya against papaya ringspot virus isolates and development of transgenic papaya for Jamaica. Ph.D. thesis, Depart. Plant Pathology, Cornell Univ., Ithaca, NY. Tennant, P. F., M. H. Ahmad, and D. Gonsalves. 2002. Transformation of Carica papaya L. with virus coat protein genes for studies on resistance to Papaya ringspot virus from Jamaica. Trop. Agri. (Trinidad) 79:105–113. Tennant, P. F., C. Gonsalves, K. S. Ling, M. Fitch, R. Manshardt, J. L. Slightom, and D. Gonsalves. 1994. Differential protection against papaya ringspot virus isolates in coat protein gene transgenic papaya and classically cross-protected papaya. Phytopathology 84:1359–1366. Tricoli, D. M., K. J. Carney, P. F. Russell, J. R. Mcmaster, D. W. Groff, K. C. Hadden, P. T. Himmel, J. P. Hubbard, M. L. Boeshore, and H. D. Quemada. 1995. Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini yellow mosaic virus. Bio/Technology 13:1458–1465. USDA-ARS. 2001. USDA Nutrient Data Base for Standard Reference. Release 13 Nutrient Data Laboratory Home Page (online). Available from http://www.nal.usda.gov/fnic/ foodcomp. Vegas, A., G. Trujillo, Y. Sandrea, and J. Mata. 2003. Obtención, Regeneración, y Evaluación de híbridos intergenéricos entre Carica papaya y Vasconcellea cauliflora. Interciencia 28:710–714. Walkey, D. G. A., H. Lecoq, R. Collier, and S. Dobson. 1992. Studies on the control of zucchini yellow mosaic virus in courgettes by mild strain protection. Plant Pathol. 41:762–771. Yeh, S. D., and D. Gonsalves. 1984. Evaluation of induced mutants of papaya ringspot virus for control by cross protection. Phytopathology 74:1086–1091. Yeh, S. D., and D. Gonsalves. 1985. Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindricalinclusion protein, and amorphous-inclusion protein. Virology 143:260–271. Yeh, S. D., and D. Gonsalves. 1994. Practices and perspective of control of papaya ringspot virus by cross protection. In: K. F. Harris (ed.), Adv. Disease Vector Research. New York: Springer-Verlag.

3 Rol Genes: Molecular Biology, Physiology, Morphology, Breeding Uses Margareta Welander and Li-Hua Zhu Department of Crop Science Swedish University of Agricultural Sciences P.O. Box 44, 230 53 Alnarp, Sweden

I. II. III. IV. V.

INTRODUCTION THE HAIRY ROOT DISEASE RI T-DNA AND ITS EFFECT ON TRANSGENIC PLANTS SYNERGISTIC EFFECT OF ROL GENES INDIVIDUAL EFFECT OF ROL GENES A. The rolA Gene 1. Molecular Studies 2. Physiological Modifications 3. Morphological Traits 4. Use in Plant Breeding B. The rolB Gene 1. Molecular Studies 2. Physiological Modifications 3. Morphological Traits 4. Use in Plant Breeding C. The rolC Gene 1. Molecular Studies 2. Physiological Modifications 3. Morphological Traits 4. Use in Plant Breeding D. The rolD Gene and Other ORFs of the TL-DNA VI. DISCUSSION AND CONCLUSIONS LITERATURE CITED

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I. INTRODUCTION Since it was demonstrated that the rol (root loci) genes from Agrobacterium rhizogenes in combination or individually were able to modify morphological, physiological, and developmental processes in plants (Shen et al. 1988; Dehio et al. 1993; Nilsson et al. 1993), several attempts have been made to use these genes in plant breeding to obtain desired agronomic traits (Rugini et al. 1991; Van der Salm et al. 1998; Welander et al. 1998; Giovanni et al. 1999; Zhu et al. 2003). These genes induce specific modifications such as dwarfism (Sinkar et al. 1988; Van Altvorst et al. 1992), increased branching (Schmülling et al. 1988; Zuker et al. 2001), early flowering (Kurioka et al. 1992; Mercuri et al. 2001) and improved rooting (Cardelli et al. 1987; Frugis et al. 1995; Zhu et al. 2001b, 2003). The fact that the protein products from these genes appear to be involved in either the metabolism of plant hormones or the response of plant cells to those hormones has provided scientists with an excellent system to exploit regulatory mechanisms of gene expression relevant to morphogenetic and developmental processes. Due to the great interest in the function of the rol genes among molecular and developmental biologists, our knowledge in this respect has increased over the years, although full comprehension of the fundamental mechanisms of these genes is still lacking. However, due to the pleiotropic effects of the rol genes in transformed plants, it is of great importance to overcome undesired side effects. One alternative is to apply better gene constructs and organ or tissue specific promoters, leading to a more defined expression of the rol genes. For these reasons, we have chosen to review the basic knowledge of the rol genes, as well as their use so far in agricultural practice.

II. THE HAIRY ROOT DISEASE Riker (1930) was the first to demonstrate that Agrobacterium rhizogenes was the causal agent of the hairy root disease on apple trees. Agrobacterium rhizogenes, a gram-negative soil bacterium, has the capability to infect wounds of dicotyledonous plants and thereby causes proliferation of roots at the infection site. Hairy root disease has been found under natural conditions mainly on perennial plants belonging to the Rosaceaes (De Cleene and De Ley 1981). However, infection trials have revealed a much broader host-range. A. rhizogenes can actually infect wounds on hosts from 49 families distributed in all dicot subclasses (Porter 1991). After infection, neoplastic cells produce and secrete opines, unusual amino acids derivates, which can be utilized by Agrobacterium as a car-

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bon and nitrogen source (Guyon et al. 1980). The structure of the opine depends on the Agrobacterium strain. The A. rhizogenes strains are classified according to the principal opine found in transformed tissue such as mannopine, agropine, or cucumopine (Petit et al. 1983; Petit and Tempé 1985; Petit et al. 1986; Binns and Costantino 1998). The rhizogenicity is correlated with the presence of a large plasmid named the Ri (root inducing) plasmid. During infection, a specific segment of the plasmid, the T-DNA, is transferred from the bacterium to the plant cell followed by its integration into the plant genome and subsequent expression (Costantino et al. 1984). The Ri plasmid carries the oncogenes that lead to neoplastic outgrowth and the genes encoding enzymes for opine production. However, in contrast to the Ti (tumor inducing) plasmid from Agrobacterium tumefaciens, the Ri plasmid induces root formation with very little or no undifferentiated cell proliferation (Moore et al. 1979). Subsequent characterisation of the Ri plasmids from different strains has shown that the Ri T-DNA from a mannopine strain (pRi8196) or cucumopine strain (pRi2659) is composed of a single continuous segment 15-30 Kb (Byrne et al. 1983; David and Tempé 1987). In contrast, the agropine strains pRi1855, pRiA4, and pRi15834 consist of two segments TL (left) and TR (right) separated by a 15kb sequence that is not integrated into the plant genome (Huffman et al. 1984; De Paolis et al. 1985). The TR region from the Ri plasmid has a significant homology to the T-DNA genes of the Ti plasmid from Agrobacterium tumefaciens. Both contain the auxin synthesis genes iaaM (tms1) and iaaH (tms2), as well as genes, encoding enzymes involved in opine synthesis. Interestingly, the mannopine and cucumopine T-DNAs lack auxin biosynthesis genes but induce hairy roots indistinguishable from the agropine strains. On the TL region from the agropine producing strain pRiA4, four loci were identified by transposon-tagging genetic analysis (White et al. 1985). These loci could affect induction and growth of hairy roots and were designated root loci A, B, C, and D. The rol genes do not share sequence homology with the Ti-DNA (Huffman et al. 1984). Later, Slightom et al. (1986) completed sequence analysis of the 21kb TL-DNA of the agropine strain A4, corresponding to 18 open reading frames (ORFs). Of these, ORFs 10, 11, 12, and 15 coincide with the rolA B, C, and D, respectively. A number of the Ri ORFs and all rol genes were cloned into plant vectors either individually or in various combinations in order to identify their functions. Recombinant Agrobacterium strains containing the rol genes were utilised for infections on different plant hosts for the production of transgenic plants. Plants containing the complete Ri T-DNA from pRiA4 develop a series of characteristically morphological alterations

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denominated the “ hairy root syndrome” (Tepfer 1984). The most striking phenotypic alterations are profound root system, wrinkled leaves, altered internode length, altered flower morphology, and reduced seed production. Changes in root gravity are also associated with the entire Ri T-DNA from pRiA4 (Legué et al. 1994). Genetic analysis of hairy root induction showed that a Ri T-DNA segment encompassing only rolA, B, and C genes was as effective in inducing roots as the whole Ri T-DNA (Capone et al. 1989). Even more interesting was that the rolB gene alone was capable of inducing roots on all the plants tested (Cardarelli et al. 1987; Spena et al. 1987). Transgenic plants containing the rolA gene have wrinkled leaves and reduced internode length (Sinkar et al. 1988). The rolC gene caused reduced apical dominance, altered leaf morphology, optimal growth capacity of newly formed roots, and reduced seed production (Schmülling et al. 1988). The rolD gene has been shown to influence the transition from the vegetative to reproductive phase in transgenic tobacco plants (Mauro et al. 1996). Thus, each of the rol genes affects plant development and morphogenesis in its own characteristic and conspicuous way. For this reason, these genes are of particular interest in plant breeding to create novel characteristics and to develop desired phenotypes. III. RI T-DNA AND ITS EFFECT ON TRANSGENIC PLANTS Wild-type Agrobacterium rhizogenes strains have been considered as useful tools to improve rooting on cuttings of recalcitrant species as they cause abundant root formation at the site of infection. The use of A. rhizogenes to improve rooting of woody species, by simple inoculation of the stems, has resulted in production of transformed roots in almond (Rugini 1984; Strobel and Nachmias 1985), olive (Rugini 1986), and apple rootstocks (Lambert and Tepfer 1991; Pawlicki-Jullian et al. 2002). Lambert and Tepfer (1991) found that the strain pRiA4 was able to induce roots in all genotypes of apple tested, whereas the strain pRi8196 produced roots only in shoots of genotypes that are easy to root in vitro. Pawlicki-Jullian et al. (2002) showed that the most efficient strain in the apple rootstock ‘Jork 9’ was the mannopine strain pRi8196 and the weakest the cucumopine strain (unknown) (Fig. 3.1). However, these plants are chimeric, having the Ri T-DNA only in their roots, but not in the aerial parts. Thus, cuttings taken from these chimeric plants have no improved rooting. On the other hand, transformed whole plants can be obtained from genetically transformed roots. However, it was necessary that the roots still be attached to the mother plant in order to obtain shoots (Lambert and Tepfer 1992; Pawlicki-Jullian et al. 2002). The percentage rooting of transgenic shoots was 100% with a more developed

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Fig. 3.1. Adventitious root formation in micropropagated shoots of the apple rootstock ‘Jork 9’ after inoculation with different wild-type strains of Agrobacterium rhizogenes. (A) a mannopine producing strain (pRi8196), (B) an agropine producing strain (pRiA4), (C) an agropine producing strain (pRi15834), and (D) a cucumopine-producing strain (unknown). Kluwer Academic Publishers book/Plant Cell, Tissue and Organ Culture 70(2): 163–171, 2002, Pawlicki-Jullian, Sedira, and Welander, Fig. 2: with kind permission of Springer Science and Business Media.

root system compared to untransformed plants. However, shoot regeneration efficiency was extremely low. About 15 species, mainly herbaceous, have been successfully regenerated from A. rhizogenes transformed roots. These transgenic plants containing the complete Ri T-DNA developed remarkably similar morphological alterations. They varied in growth rates, size, and leaf and flower morphology. The transformed genotype and phenotype were also sexually transmitted (Tepfer 1984; Durand-Tardif et al. 1985; Birot et al. 1987). Successful plant regeneration has also been obtained from hairy root- derived protoplasts of Hyoscyamus muticus. Transformed roots have also been obtained for a number of species that have been used to produce secondary metabolites (Rhodes et al. 1986; Jung and Tepfer 1987; Ferreira and Janick 2002; Ayadi and TrémouillauxGuiller 2003). In some of the clones, the rol genes could not be detected although the morphology was altered, indicating that other genes from the Ri T-DNA were still present. The clones lacking the rol genes had similar alkaloid production as the controls, whereas the alkaloid production

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was clearly reduced in the clones containing the rol genes (Sevón et al. 1997). The fact that plants regenerated directly from hairy roots had similar alkaloid production as the control plants (Oksman-Caldebty et al. 1987, 1991) indicates a different function of the rol genes and other genes present on the Ri T-DNA at least for some characteristics. A thorough analysis of silver birch plants derived from pRiA4 transformed roots was performed by Piispanen et al. (2003). The regeneration of shoots from the hairy roots varied considerably among the different clones. Although plants were derived from six hairy root clones only two clones produced numerous shoots. Plants derived from these two clones were grouped according to the genes present. Plants carrying either rolC plus rolD or all rol genes (A+B+C+D) were shorter, bushier, had smaller leaves and greater root formation than plants lacking both rol genes and auxin synthesis genes. It was concluded that the rolC plus rolD induced typical “rol phenotype.” Transgene mRNA was most abundant in the phloem/cambium and xylem samples in the stem. Higher expression of the rolC was noted in the phloem, whereas the rolD signals were stronger in the xylem.

IV. SYNERGISTIC EFFECT OF ROL GENES Since it was shown that only a fragment of the RiT-DNA containing the rolABC genes was sufficient to produce the hairy root syndrome ( Jouanin et al. 1987), several studies have been undertaken to elucidate the effects of rol genes under their own promoters on different growth characteristics. Rugini et al. (1991) showed that plants of Kiwi fruit (Actinidia deliciosa) transformed with the rolABC genes were more compact, had shorter internodes, and higher rooting percentage and root number compared to control plants. Transgenic plants also had wrinkled leaves. However, after potting, some of the hairy root characteristics were lost. Transformed rolABC plants of Rosa hybrida L. ‘Moneyway’ were smaller and had shorter internodes and increased lateral branching and rooting compared to control plants. However, considerable variation was obtained among independently transformed plants. One of the transgenic rootstocks showed that the architecture of the untransformed scion was altered by the rootstock by forming more basal shoots and larger leaf area, resulting in improved growth performance (Van der Salm et al. 1998). Contradictory results were obtained in rolABC transformed walnut rootstocks where the phenotype of the grafted scion was unaffected. Both transformed hardwood cuttings and microshoots rooted significantly less efficiently than untransformed ones (Vahdati et al. 2002.).

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Valuable ornamental traits were observed on rolABC, rolAB, and 35S rolC transformed plants of Osteospermum ecklonis. The phenotypic alterations caused by rolABC included an increased number of flowers, and erect plants with an increased number of branches. These plants also produced profuse root systems when grown in vitro (Giovanni et al. 1999). Modification of plant architecture was also induced in rolABC transgenic plants of Limonium spp. such as dwarfness and early flowering (Mercuri et al. 2001). On the other hand, phenotypes of rolABC transformed plants of Begonia tuberhybrida resulted in a number of clones with highly undesirable characters including dwarfness, delay of flowering, and wrinkled leaves and petals (Kiyokawa et al. 1996). Transgenic lines of Rhododendron species with rolABC under control of the native promoters showed different morphological alterations regarding branching, shorter internodes and smaller slightly wrinkled leaves. Some lines that exhibited increase in apical dominance lacked the rolC fragment. All rolABC transgenic lines exhibited increased rooting both in vitro and ex vitro. Interestingly, some lines also showed increased tolerance to lime stress (Dunemann et al. 2002). RolAB lettuce plants exhibited reduced stem and inflorescence heights, leaf area, and internodal length. Some of the transformed lettuce plants also showed aberrant flower development. Leaf wrinkling was absent in transgenic plants, but appeared in some of the seedlings derived from those plants (Curtis et al. 1996).

V. INDIVIDUAL EFFECT OF ROL GENES A. The rolA Gene 1. Molecular Studies. The predicted translation product of the rolA gene is a small (11.4 kDa) basic protein that has no significant similarity to sequences in the database (Vilaine et al. 1998). Although the function of the rolA gene is not known, very interesting information has been obtained concerning its expression. Carneiro and Vilaine (1993) showed that the rolA mRNA in transgenic tobacco plants was most abundant in the stem, 5-fold lower in the leaf, and 50-fold lower in the roots. The transgenic rolA plants showed wrinkled leaves, shortened internodes (dwarfism), and delayed flowering, but also induction of roots in leaf segments from transgenic plants. Characterisation of promoter regions of genes whose expression is developmentally regulated is an important tool for unravelling molecular events leading to plant cell differentiation. Carneiro and Vilaine

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(1993) made a series of successively increasing deletions of the promoter region of the rolA gene. The resulting genes (Bra1-4), when inserted in the tobacco genome, resulted in new patterns of rolA transcripts in leaves, stems, and roots as well as in the phenotypic appearance of the transgenic plants. Bra1 gene corresponding to the intact rolA promoter had all the features of the rolA phenotype. Bra2, with the smallest deletion, accumulated higher rolA transcripts in the roots compared to Bra1 but displayed the entire rolA phenotype, except that the capability to induce roots on leaf segments was lost. In Bra3 plants, rolA transcripts were only detected in the stem. These transgenic plants showed the same phenotype as control plants except for shortened internodes. Bra4 plants had no detectable mRNA transcripts and were entirely normal in appearance. Guivarc’h et al. (1996) investigated the spatial and temporal activity of the entire and individual promoter domains of the rolA gene. They found that the rolA promoter domain responsive to dwarfism without wrinkled leaves was mainly expressed in the stem vascular bundles and in the internal phloem of the leaves. 2. Physiological Modifications. Several attempts have been made to relate physiological changes of rolA transformed plants with phenotypic alterations. The rolA gene has been implicated in both changes in hormone physiology and polyamine metabolism. Dehio et al. (1993) investigated phenotypic traits and hormonal status in transgenic rolA tobacco plants with either the rolA promoter or the 35S promoter. The transgenic plants with the 35S promoter showed enhanced morphological alterations. The most striking results were the reduction of GA1 content that could be indirectly involved in dwarfism. Similar results were obtained by Moritz and Schmülling (1998) using also transgenic rolA tobacco plants with either the rolA promoter or 35S promoter. They found that the levels of gibberellins increased in the apical stems and leaves of both transgenic rolA clones compared to the wild type, but were less pronounced in the transgenics with the rolA promoter. In contrast, the concentrations of both GA20 and GA1 decreased significantly in transgenic clones compared with controls and again less pronounced in the clone with the rolA promoter. These results indicate a metabolic block at the conversion of GA19 to GA20, a precursor of GA1. Although there is strong evidence that reduction of the active GA content is correlated with reduced stem elongation, it cannot explain all aspects of the rolA phenotype since stem elongation is not completely restored by exogenous application of GA. Prinsen et al. (1994) found that tobacco plants transformed with the rolA gene had reduced indole acetic acid levels in the shoot apices and consequently reduced basipetal auxin gradient compared to untrans-

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formed plants. The decrease in free IAA was observed from week 6 to 12, a period when leaf wrinkling was most pronounced. The rolA gene product seemed to modulate auxin sensitivity of the ATPase-mediated proton pump on the plasmalemma membrane (Vansuyt et al. 1992), especially during flowering. This is consistent with auxin induced membrane hyperpolarisation detected by Maurel et al. (1991). There is also some evidence that changes in polyamine metabolism could be found in rolA phenotypes. RolA plants have reduced content of putrescine and spermidine during the entire vegetative phase (Burtin et al. 1991), whereas lack of polyamine conjugation occurs first after anthesis (Sun et al. 1991). Expression of the rolA gene leads to profound modifications in plant development. Although changes in both hormone levels and polyamine metabolism have been reported, it has been difficult to prove the direct effects of the rolA protein in the cascade of events leading to phenotypic modifications. 3. Morphological Traits. The rolA gene is pleiotropic and can influence almost all plant organs and at all stages of development. The phenotype expressing the rolA gene shows wrinkled leaves, shortened internodes (dwarfism), delayed flowering, and reduced fertility. These traits have been observed in tobacco (Schmülling et al. 1988; Sinkar et al. 1988), tomato (Van Altvorst et al. 1992), and rice (Lee et al. 2001). RolA transgenic plants of the apple rootstock ‘M26’ showed reduced stem length and shortened internodes. In two clones, the leaf area was also reduced. However, wrinkled leaves were only observed occasionally during subculturing in vitro, but not in greenhouse-grown plants (Holefors et al. 1998). Transformation of the vigorous apple rootstock ‘A2’ with the rolA gene resulted in two clones with reduced plant height and shorter internodes, whereas no wrinkled leaves or reduced leaf area were observed (Zhu et al. 2001a). 4. Use in Plant Breeding. Since the rolA gene can produce several undesired characteristics such as reduced number of flowers, flower size, and fertility, it may not be useful to introduce this gene into seed propagated flowering plants. However, for vegetatively produced plants such as apple rootstocks the gene has been successfully used to introduce dwarfism. It remains to be clarified whether rol characteristics from the rootstocks would be transferred to grafted scions. Growth experiments using the method by Ingestad and Lund (1986) showed reduced relative growth rate on ‘Gravenstein’ apple grafted on the rolA transformed ‘M26’ compared to those grafted onto the non-transformed rootstocks (Zhu and Welander 1999). Although the entire rolA gene is not useful, it has been shown that the rolA gene with a truncated promoter (Bra3 gene) can

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abolish several of the undesired traits. Thus, the use of the Bra3 gene could be useful in controlling the plant size of agricultural and horticultural plants. B. The rolB Gene 1. Molecular Studies. The rolB gene present on the TL-DNA of the agropine type strain contains 777bp encoding a protein with a molecular weight of 30 kDA. Recently, an ORF region similar to the rolB has been detected on the TR-DNA (rolBTR), but no functional studies have been performed (Bouchez and Camilleri 1990). No proteins homologous to rolB have been reported outside Agrobacterium. The rolB gene has been shown to be particularly efficient in triggering root induction in different plant species (White et al. 1985; Cardarelli et al. 1987; Spena et al. 1987; Capone et al. 1989). Characterization of the promoter region of the rolB gene has been performed by Capone et al. (1991, 1994). They analysed the pattern of expression of different GUS constructs with deletions of the rolB promoter. Carrot disc transformation and analysis of transgenic tobacco plants containing these different constructions verified the presence of distinct regulatory domains of the rolB promoter. Two regions controlled the level of expression, but not tissue specificity. Further deletions drastically influenced tissue specificity, showing that distinct domains were responsible for root meristematic and differentiated (vascular) expression. Using the same approach, five regulatory regions were identified on the rolB promoter, when pattern of expression was analysed in the root apex of transgenic tobacco plants. The presence of all domains (A to E) resulted in gene expression in the root cap, protoderm, and tissues within the meristematic region including initials to root cap and protoderm, ground meristem (cortex), and procambium (immature vascular tissue). Deletion of domain A suppresses expression in the root cap and protoderm; domain D suppresses gene expression in all tissues except the procambium (inner part of the meristematic region). Both domains B and E are indispensable for expression in all tissues of the root apex, as deletion of either one causes total loss of activity. Indeed, one plant regulatory factor binding to domain B (NtBBF1) (De Paolis et al. 1996) and another one binding to domain A (RBF1) (Filetici et al. 1997) have been identified. The domains of B, C, D, and E also seem to be critical for auxin responsiveness in mesophyll protoplasts. It has also been shown that during zygotic embryogenesis in transgenic tobacco plants the rolB promoter is activated simultaneously in all cells of the embryo at the end of the globular stage when polarised growth begins (Chichiriccò et al. 1992). This indicates that the transition from globular stage to the heart

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stage is the period when embryonic cells acquire the capability to respond to auxin. The rolB protein has now been isolated and it is posited to have a tyrosine-phoshatase activity (Filippini et al. 1996), suggesting a direct role for rolB in the auxin signal-transduction pathway. 2. Physiological Modifications. The mechanism of action of the rolB protein is not known, but the morphogenetic alterations observed in rolB transgenic plants indicate changes in hormone responsiveness. This is supported by numerous reports on increased sensitivity to auxin in excised organs (Shen et al. 1988; Spanò et al. 1988; Shen et al. 1990) protoplasts (Barbier-Brygoo et al. 1991; Maurel et al. 1991), as well as intact shoots (Sedira et al. 2001). The increased sensitivity is not due to modified levels of free IAA or conjugates since it has been shown that the rate of IAA metabolism and IAA conjugation are unchanged in transgenic tobacco plants (Nilsson et al. 1993; Delbarre et al. 1994). Instead, the increased auxin sensitivity can be explained by altered auxin binding capacity since it has been shown that the auxin binding activity was substantially higher in rolB transformed tobacco cells compared to untransformed ones (Filippini et al. 1994). It has also been shown that oligogalacturonides, which prevent rhizogenesis in rolB transformed leaf explants of tobacco, strongly inhibit auxin-induced expression of the rolB promoter (Bellincampi et al. 1996). Altamura et al. (1998) claimed that the rolB gene enhanced shoot formation in leaf explants and thin cell layers (TCL) of rolB transformed tobacco plants compared to control ones. In hormone free medium, rolB leaf explants produced only roots and no organ formation in control leaf explants. By addition of a low concentration of benzyl adenine (BA) in the medium, the rolB explants produced both roots and shoots and no organs in control ones. Higher concentrations of BA produced similar percentages of bud formation in both explants, although the number of shoot buds was higher for rolB explants. Increased bud formation in rolB plants might be a result of cytokinins produced in the roots. The different results from TCL explants might be due to lack of vascular tissue and impaired transport of hormones. 3. Morphological Traits. The rolB gene induced rooting in most of the plants tested. However, other morphogenetic traits seem to vary among different plant species. Transgenic tobacco plants expressing rolB with its own promoter displayed alterations in leaf and flower morphology, besides increased rooting (Schmülling et al. 1988). When the rolB gene is fused to the 35S promoter, root induction in transgenic tobacco leaf discs is less efficient, indicating that localized rolB activity is important for stimulation of root induction.

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In apomictic Hieracium plants, the rolB gene induced meristems with variable developmental potential. For example, when ectopic meristems formed on the inflorescence stems were immersed in water, they developed into roots. Under greenhouse conditions, the ectopic meristems developed mainly into vegetative rosettes. These data suggest that the primary effect of rolB is to induce meristems and that they are initially indeterminate (Koltunow et al. 2001). The expression of the rolB gene fused to different flower specific promoters alters growth and development of flower organs. Tobacco plants transformed with the rolB gene fused with the anther specific tap1 promoter from snapdragon showed shrivelled anthers, reduction in elongation of peduncle of inflorescences, and reduced flower size. Such growth alterations were correlated with an increase in free IAA content and reduced gibberellin activity (Spena et al. 1992). Recently, it was shown that when the rolB gene is driven by the promoter of the meiosis specific gene (DMC1) from Arabidopsis, the expression occurred earlier in androecium than in gynoecium. As a result, self-pollination is prevented in plants carrying the pDCM1:rolB. The use of the promoter of the petunia gene FBP7 (floral binding protein 7) resulted in concomitant delay of both anther dehiscence and pistil development without affecting self-pollination. The delay in anther dehiscence and pistil development could be phenocopied by exogenously applied auxin. These results suggest that auxin is involved in both male and female organ development (Cecchetti et al. 2004). The influence of the rolB gene on the coordinated processes of flower organ development can be very useful in plant breeding. 4. Use in Plant Breeding. The rolB gene has been used in a number of economical plants to improve agricultural and horticultural traits. Productivity-related phenotypical modifications were observed in rolB transgenic plants of the forage legume Medicago sativa, including significant increases in root mass and in number of stems per plant. RolB plants also showed a clear uniformity in stem regrowth after cutting (Frugis et al. 1995). RolB tomato plants were characterised by reduced internode length and apical dominance besides induced root formation (Van Altvorst et al. 1992). In apple and pear rootstocks, the rolB gene has been very useful to improve rooting (Fig. 3.2). Rooting percentage, root number, and auxin sensitivity were increased for the rolB transformed plants of the apple rootstocks ‘M26’ and ‘Jork 9’ (Welander et al. 1998; Sedira et al. 2001). RolB transformed ‘M9/29’ apple rootstock and the pear rootstock ‘BP10030’ also exhibited reduced stem length and node number (Zhu et al. 2001b, 2003). Fig. 3.2 shows the improved rooting induced by the introduction of the rolB gene in the apple rootstock

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Fig. 3.2. From left to right: (A) two rolB transformed clones and one control of the apple rootstock ‘M9/29’; (B) one control plant and four transformed clones of the pear rootstock ‘BP10030’; and (C) four transformed clones and one control of the pear rootstock ‘BP10030’. Reprinted from Plant Science, Vol 165, Zhw et al, The rooting ability . . ., pp. 829–835, 2003, with permission from Elsevier.

‘M9/29’ and the pear rootstock ‘BP10030’ and the reduced stem length in the pear rootstock. However, the rolB transformed rose rootstock ‘Moneyway’ showed altered morphological traits different from pear and apple rootstocks (Van der Salm 1996). The rolB plants resembled control plants in size, but exhibited round-edged leaf form instead of lanceolate as in control plants. The apical dominance was strongly increased, which was also reflected in decreased total dry weight and reduced total leaf area. Meanwhile, the root-system was reduced in the transgenics. Since this phenotype was based on a single transformant, the observations must be treated with caution. On the other hand, leaflets and stem explants from rolB micropropagated rose plants rooted in an auxin-free medium clearly showed that the rolB gene promotes rooting. C. The rolC Gene 1. Molecular Studies. The rolC gene (ORF 12) contains 540 bp encoding a protein with a molecular weight of 20kD. No homologous protein has been reported outside Agrobacterium. Analysis of the rolC promoter region fused to the gus reporter gene showed an expression pattern parallel to somatic embryogenesis in carrot (Fujii and Uchimiya, 1991). Using embryo cells in different stages, Fujii et al. (1994) found that the highest GUS activity was associated with the heart and torpedo stages. Deletion analysis of the rolC promoter revealed three regions containing putative regulatory elements. Retention and deletion of any one region revealed interaction and synergism between the different regions. 2. Physiological Modifications. It is assumed that the pleiotropic alterations of rolC activity results from both direct and indirect effects on levels of various hormones. In both tobacco and hybrid aspen (Populus

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tremula × P. tremuloides) (Nilsson et al. 1993, 1996), GA metabolism was down regulated, due to an inhibition of the conversion of GA19 to GA20. The reduced content of GA1 could be responsible for reduced stem length. However, GA applications could only increase internode length but not restore the other rolC traits. Analysis of endogenous immunoreactive hormone concentrations in rolC transgenic potato and tobacco plants revealed increased content of cytokinins (Schmülling et al. 1993), whereas the IAA content remained constant. In tobacco plants, only the isopentenyladenosine (iPA) content increased in stem tissue, whereas for potato plants an increase of free cytokinins including iPA, dihyrozeatin riboside was noted for all tissues investigated. Estruch et al. (1991) suggested that the increase in level of active cytokinins was due to a release of free cytokinins from inactive conjugates by rolC induced β-glucosidase activity. However, more refined data from analysis of both free and conjugated cytokinins in shoot apical regions, as well as upper leaves of rolC transformed aspen plants, does not support this hypothesis (Nilsson and Olsson 1997). Instead, an increase in cytokinin conjugates was associated with an elevation of free cytokinin zeatin riboside. Apart from changes in hormone levels rolC seedlings turned out to have an altered sensitivity to phytohormones. The seedlings had a higher tolerance to auxins and ABA and higher sensitivity to cytokinins compared to control plants. These results indicate that rolC does not have a direct effect on hormone metabolism. Transgenic tobacco plants expressing the rolC gene also seem to have altered polyamine metabolism, similar to plants expressing the rolA gene (Martin-Tanguy 1997). 3. Morphological Traits. Transgenic tobacco plants transformed with the rolC gene under its own promoter are shorter, more highly branched, have an increased ratio of leaf length to width, reduced flower size and pollen production, and flower earlier compared to wild type (Oono et al. 1987; Schmülling et al. 1988). When rolC is expressed from a 35S promotor, the morphological alterations are exaggerated, resulting in dwarfed plants with short internodes. Transgenic hybrid aspen plants expressing 35S:rolC displayed varying degrees of phenotypic alterations (Fig. 3.3) (Nilsson et al. 1996). Some plants were extremely stunted and had small light-green leaves. At the early stage of development, plants with high levels of expression also showed reduced apical dominance with more side shoots produced. In some of the transgenic plants, the shoot apices enlarged and became fasciated. The severity of the alterations was correlated with the level of rolC expression (Nilsson et al. 1996). These authors also showed that the induced phenotype was maintained during the second growing period. Fladung et al. (1996, 1997) obtained similar results

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Fig. 3.3. (A) wild type on left, rolC on right and (B) six-week-old rolC hybrid aspen plants in the growing period. (C and D) dormant shoots after 2 months at 4°C. (E and F) plants in the second growing period. Printed with permission from the American Society of Plant Biologists.

for 35S:rolC transgenic aspen (Populus tremula) and hybrid aspen plants showing reduced stem height and an increased number of light-green leaves. In addition, a strong promotion of root growth was observed. Furthermore, transgenic plants were released from dormancy earlier than control plants. The onset of dormancy varied between transgenic plants and the controls depending on environmental conditions. The expression of the 35S:rolC gene in diploid and tetraploid potato plants induced dwarfism and increased tillering. However, undesired traits such as changes in tuber shape and increased number of tubers with decreased weight per tuber were also obtained. This shows that the 35S promoter was probably too strong in this crop (Fladung 1990).

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Phenotypic alterations induced by the 35S:rolC in Atropa belladonna were increased root growth, reduced apical dominance, small flowers, earlier flowering, and pale and slender leaves. None of these characters were observed in transgenic rolC plants with its own promoter (Kurioka et al. 1992). The differences in phenotype were due to either the level of expression and/or differential expression pattern in the tissues. 4. Use in Plant Breeding. The rolC gene under its own promoter has been used to improve growth characteristics in ‘Beurre Bosc’ pear. The rolC transformants displayed reductions in total height, number of nodes, leaf area, and internode length compared to untransformed plants. Since the natural growth habit of this pear cultivar is characterised by extensive yearly shoot growth, a reduction in vigor can be of great commercial value as long as productivity is not adversely affected (Bell et al. 1999). Similar results were obtained for rolC transformed plants of the apple rootstock ‘Marubakaidou’ (Malus prunifolia), although the observed phenotypes differed among different transgenic clones. These studies also confirmed improved rooting of transformed plants (Igarashi et al. 2002). The rolC gene has also been used to induce dwarfism in the citrus rootstock (Poncirus trifoliata) (Kaneyoshi and Kobayashi 1999) and the Japanese persimmon (Diospyros kaki) (Koshita et al. 2002). In the study on citrus, both 35S and rolC promoters were used. The 35S:rolC transgenic plants were shorter than rolC:rolC plants. RolC transgenic plants also rooted more readily than control plants. The rolC gene has also been used to generate carnations with novel agronomic and ornamental traits (Zuker et al. 2001). Transgenic 35S:rolC plants exhibited improved rooting and production yield in terms of number of cuttings and number of flowering stalks per mother plant. Moreover, these traits were stable after 2 years testing in the greenhouse. Interestingly, the negative traits observed in many transgenic plants using rolC with the 35S promoter were not seen in transgenic carnation. The introduction of the rolC gene also influences secondary metabolites that could be of great interest for the pharmaceutical industry. In Atropa belladonna, transformed with rolC, the hairy root growth was increased as well as biosynthesis of tropane alkaloids that were 12-fold times higher in transgenic roots than untransformed roots (Bonhomme et al. 2000). In ginseng (Panax ginseng) roots transformed with rolC, the ginsenoside content was about three times higher than untransformed roots (Bulgakov et al. 1998). In Rubia cordifolia, the rolC and rolB genes increased the anthraquinone production, especially after treatment with cantharidin and methyl jasmonate (Bulgakov et al. 2002).

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D. The RolD Gene and Other ORFs of the TL-DNA The transcriptional level of rolD and other ORFs of the TL-DNA have been much less investigated. Mauro et al. (1996) reported that the rolD gene stimulated flowering in transgenic tobacco plants. Phenotypic alterations were also obtained by overexpression of ORF13 in transgenic tobacco (Hansen et al. 1993). Lemcke and Schmülling (1998) tested the morphogentic potential of 15 open reading frames of the TL-DNA of the agropine strain HRI. Expression of three loci, ORF3n, ORF8 and ORF13 altered plant morphogenesis or the response of transgenic tissues to plant hormones. ORF13 retarded flowering, and altered internode elongation and leaf shape in transgenic tobacco plants. ORF3n and ORF8 reduced sensitivity to auxin and cytokinin in combination or auxin alone. It was concluded that TL-DNA harbours genetic information that is important for pathogenicity apart from the rol genes.

VI. DISCUSSION AND CONCLUSIONS The rol genes offer great opportunity to manipulate plant architecture such as plant height, branching, leaf form and colour, flowering, fruiting, and root system for the horticultural, agricultural, and forestry industries. The rol genes have been introduced either by using the wild type of A. rhizogens containing all the rol genes plus auxin synthesis genes or A. tumefaciens containing a recombinant plasmid including the rol genes separately or in combination. The wild type of A. rhizogenes has been used to induce hairy roots in several plant species. These roots have been used to increase the content or the production of secondary metabolites or to generate new plants. Plants obtained from hairy roots will contain single or different combinations of the rol genes as well as the auxin synthesis genes and therefore the outcome of such plants will be difficult to predict. Although valuable modifications have been obtained using the rolABC genes for some species both regarding secondary metabolites and morphological characteristics, the risk of obtaining undesired characters increases using a combination of all the rol genes. Obviously, the use of single rol genes is more straightforward when aiming at improving a specific trait. The rolB gene has been particularly useful in woody species to improve rooting regarding both the percentage and number of roots. Since the rolB gene usually causes formation of abundant roots with increased fine roots, the uptake of nutrients and water would be improved for the transgenic plants. Thus, the

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use of rolB transgenic rootstocks in combination with untransformed scions would allow plants to adapt to various soil conditions with no or limited transgene spreading. Besides improved rooting, the rolB gene also reduces plant height, which is a valuable character for commercial production of horticultural plants. Since the rolB plants root easily, cuttings might be an alternative method for producing fruit tree rootstocks instead of layering and stooling. However, the long-term effect of the rolB gene on growth and development of scions needs to be evaluated before any commercialisation. In Sweden, a field trial was established in 2001 using rolB transformed ‘M26’ and ‘M9/29’ grafted with 5 cultivars. In the coming years, we will be able to obtain data regarding tree anchorage, growth, flowering, fruit setting, and fruit quality. The rolC gene has also been used successfully to induce dwarfism in fruit tree rootstocks. Meanwhile, improved rooting was also reported, but mainly rooting percentage, not root number. The promoters used seem to influence the degree of the rolC effect. In citrus, transformants obtained with 35S:rolC were much shorter than those from rolC:rolC. In carnation, the rolC gene under the 35S promoter generated plants with improved rooting and increased number of cuttings and flower stalks per mother plant. However transgenic aspen expressing the 35S:rolC resulted in severe growth alterations such as extremely stunted plants and enlarged shoot apices that became fasciated. In potato, 35S:rolC gene induced dwarfism and increased tillering but at the same time changed tuber shape and increased the number of tubers with decreased weight. Thus, it can be concluded that a strong promoter like 35S can be harmful in some plant species but not in others. The rolA gene can also reduce plant height but it may not be useful in plant species where flowers are essential since the rolA gene could reduce the number of flowers, flower size, and fertility. However, using a truncated promoter (Bra3 gene), several of these undesired traits could be abolished. The apple ‘Gravenstein’ grafted on rolA transgenic ‘M26’ did not show reduced number of flowers, size, or fertility (unpublished results). This might indicate that the rolA gene can be used to dwarf rootstocks in horticultural crops. Transgenic lines obtained after transformation with either of the rol genes often show high phenotypic variability. These differences can be associated with copy number of the inserted genes or where in the plant genome the transgenes are integrated. This means that quite a number of independent transgenics must be evaluated to select the most appropriate ones. The prerequisites for release of improved plants are the genetic and phenotypic stabilities, which require several cycles of clonal propagation for vegetatively produced plants under commercial conditions.

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There is no doubt that the rol genes from A. rhizogenes are very suitable for improvement of both agricultural and horticultural crops. However, practical application might be hampered by the many pleiotropic side effects. The undesired characteristics might be overcome by using specific gene constructs and organ or tissue specific promoters leading to a more defined expression of the rol genes. Therefore, increased collaborations with breeders, physiologists, and developmental biologists are needed in order to successfully implement these new technologies.

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Igarashi, M., H. Ogasawara, Y. Hatsuyama, A. Saito, and M. Suzuki. 2002. Introduction of rolC into marubakaidou [Malus prunifolia Borkh. Var. ringo Asami Mo 84-A] apple rootstock via Agrobacterium tumefaciens. Plant Sci. 163:463–473. Ingestad, T. and A. B. Lund. 1986. Theory and technique for steady state mineral nutrition and growth of plants. Scandinavian J. Forest Res. 1:439–453. Jouanin, L., P. Guerche, N. Pamboukdjian, C. Tourneur, F. Casse-Delbart, and J. Tourneur. 1987. Structure of T-DNA in plants regenerated from roots transformed by Agrobacterium rhizogenes strain A4. Mol. Gen. Genet. 206:387–392. Jung, G., and D. Tepfer. 1987. Use of genetic transformation by the Ri T-DNA of Agrobacterium rhizogenes to stimulate biomass and tropane alkaloid production in Atropa belladonna and Calystegia sepium roots grown in vitro. Plant Sci. 50:145–151. Kaneyoshi, J., and S. Kobayashi. 1999. Characteristics of transgenic trifoliate orange (Poncirus trifoliate Raf.) possessing the rolC gene of Agrobacterium rhizogene Ri plasmid. Japan. Soc. Sci. 68:734–738. Kiyokawa, S., Y. Kikuchi, H. Kamada, and H. Harrada. 1996. Genetic transformation of Begonia tuberhybrida by Ri rol genes. Plant Cell Rep. 15:606–609. Koltunow, A. W., S. D. Johnson, M. Lynch, T. Yoshihara, and P. Costantino. 2001. Expression of rolB in apomictic Hieracium piloselloides Vill. Causes ectopic meristems in planta and altered ovule formation. Planta 214:196–205. Koshita, Y., Y. Nakamura, S. Kobayashi, and K. Morinaga. 2002. Introduction of the rolC gene into genome of the Japanese persimmon causes dwarfism. Japan. Soc. Hort. Sci. 71:529–531. Kurioka, Y., Y. Suzuki, H. Kamada, and H. Harada. 1992. Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid. Plant Cell Rep. 12:1–6. Lambert, C., and D. Tepfer. 1991. Use of Agrobacterium rhizogenes to create chimeric apple trees through genetic grafting. Bio/Technology. 9:80–83. Lambert, C., and D. Tepfer. 1992. Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones. Theor. Appl. Genet. 85:105–109. Lee, S.-H., N. W. Blackhall, J. B. Power, E. D. Cocking, D. Tepfer, and M. R. Davey. 2001. Genetic and morphological transformation of rice with the rolA gene from the Ri TLDNA of Agrobacterium rhizogenes. Plant Sci. 161:917–925. Legué, V., F. Vilaine, M. Tepfer, and G. Perbal. 1994. Modification of the gravitropic response of seedling roots of rapeseed (Brassica napus) transformed by Agrobacterium rhizogenes A4. Physiol. Plant. 91:559–566. Lemcke, K., and T. Schmülling. 1998. Gain of function assays identify non-rol genes from Agrobacterium rhizogenes TL-DNA that alter plant morphogenesis or hormone sensitivity. Plant J. 15:423–433. Martin-Tanguy, J. 1997. Conjugated polyamines and reproductive development: biochemical, molecular and physiological approaches. Physiol. Plant. 100:675–688. Maurel, C., H. Barbier-Brygoo, A. Spena, J. Tempé, and J. Guern. 1991. Single rol genes from Agrobacterium rhizogenes TL-DNA alter some of the cellular responses to auxin in Nicotiana tabacum. Plant Physiol. 97:212–216. Mauro, M. L., M. Trovato, A. D. Paolis, A. Gallelli, P. Costantino, and M. M. Altamura. 1996. The plant oncogene rolD stimulates flowering in transgenic tobacco plants. Dev. Biol. 180:693–700. Mercuri, A., S. Bruna, L. De Benedetti, G. Burchi, and T. Schiva. 2001. Modification of plant architecture in Limonium spp. Induced by rol genes. Plant Cell, Tissue, Organ Culture 65:247–253.

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Moore, L. W., G. Warren, and G. A. Strobel. 1979. Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes. Plasmid 2:617–626. Moritz, T., and T. Schmülling. 1998. The Gibberellin content of rolA transgenic tobacco plants is specifically altered. J. Plant Physiol. 153:774–776. Nilsson, O., A. Crozier, T. Schmülling, G. Sandberg, and O. Olsson. 1993. Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene. Plant J. 3:681–689. Nilsson, O., T. Moritz, B. Sundberg, G. Sandberg, and O. Olsson. 1996. Expression of the Agrobacterium rhizogenes rolC gene in a deciduous forest trees alters growth and development and leads to stem fasciation. Plant Physiol. 112:493–502. Nilsson, O., and O. Olsson. 1997. Getting to the root: The role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol. Plant. 100: 463–473. Oksman-Caldebty, K-M., and H. Vuorela, A. Strauss, and R. Hiltunen. 1987. Variation in the tropane alkaloid content of Hyoscyamus muticus plants and cell culture clones. Planta Med. 53:349–354. Oksman-Caldebty, K-M., O. Kivelä, and R. Hiltunen. 1991. Spontaneous shoot organogenesis and plant regeneration from hairy root cultures of Hyoscyamus muticus. Plant Sci. 78:129–136. Oono, Y., T. Handa, K. Kanaya, and H. Uchimiya. 1987. The TL-DNA gene of Ri plasmids responsible for dwarfness of tobacco plants. Japan. J. Genet. 62:501–505. Pawlicki-Jullian, N., M. Sedira, and M. Welander. 2002. The use of Agrobacterium rhizogenes transformed roots to obtain transgenic shoots of the apple rootstock Jork 9. Plant Cell, Tiss. Org. Cult. 70:163–171. Petit, A., C. David, G. Dahl, G. J. Ellis, P. Guyon, F. Casse-Delbart, and J. Tempé. 1983. Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol. Gen. Genet. 190:204–214. Petit, A., A. Berkaloff, and J. Tempé. 1986. Multiple transformation of plant cells by Agrobacterium may be responsible for the complex organization of T-DNA in crown gall and hairy root. Mol. Gen. Genet. 202:388–393. Petit, A., and J. Tempé. 1985. The function of T-DNA in nature. p. 625–636. In: L. van Vloten-Doting, G. Groot, and T. Hall (eds.), Molecular form and function of the plant genome. Plenum Publ. Corp., New York. Piispanen, R., T. Aronen, X. Chen, P. Saranpää, and H. Häggman. 2003. Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology, xylem structure and chemistry. Tree Physiol. 23:721–733. Porter, J. R. 1991. Host range and implications of plant infection by Agrobacterium rhizogenes. CRC Crit. Rev. Plant. Sci. 10:387–421. Prinsen, E., D. Chriqui, F. Vilaine, M. Tepfer, and H. van Onckelen. 1994. Endogenous phytohormones in tobacco plants transformed with Agrobacterium rhizogenes pRi TL-DNA genes. J. Plant Physiol. 144:80–85. Rhodes, M. J. C., M. Hilton, A. J. Parr, J. D. Hamill, and R. J. Robins. 1986. Nicotine production by “hairy root” cultures of Nicotiana rustica: fermentation and product recovery. Biotechnol. Lett. 8:415–420. Riker, A. J. 1930. Studies on infectious hairy root of nursery apple trees. J. Agri. Res. 41:507–540. Rugini, E., A. Pellegrineschi, M. Mencuccini, and D. Mariotti. 1991. Increase of rooting ability in the woody species kiwi (Actenidia deliciosa A. Chev.) by transformation with Agrobacterium rhizogenes rol genes. Plant Cell Rep. 10:291–295. Rugini, E. 1984. Progress in studies on in vitro culture of almond. Proc. 41st Conf. on Plant Tissue Culture and its Agricultural Applications. Nottingham. 17–21 Sept. p. 73.

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Rugini, E. 1986. Olive. p. 253–267. In: Y. P. S. Bajaj (eds.), Biotechnology in agriculture and forestry trees. Springer-Verlag, Berlin. Schmülling, T., J. Schell, and A. Spena. 1988. Single genes from Agrobacterium rhizogenes influence plant development. EMBO J. 7:2621–2629. Schmülling, T., M. Fladung, K. Grossmann, and J. Schell. 1993. Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T-DNA. Plant J. 3:371–382. Sedira, M., A. Holefors, and M. Welander. 2001. Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting. Plant Cell Rep. 20:517–524. Sevón, N., B. Dräger, and R. Hiltunen. 1997. Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus. Plant Cell Rep. 16:605–611. Shen, W., A. Petit, J. Guern, and J. Tempé. 1988. Hairy roots are more sensitive to auxin than normal roots. Proc. Natl. Acad. Sci. (USA) 85:3417–3421. Shen, W. H., E. Davioud, D. Chantal, H. Barbier-Brrigoo, J. Tempé, and J. Guern. 1990. High sensitivity to auxin is a common feature of hairy root. Plant Physiol. 94:554–560. Sinkar, V. P., F. Pythoud, F. F. White, E. W. Nester, and M. P. Gordon. 1988. rolA locus of the Ri plasmid directs developmental abnormalities in transgenic tobacco plants. Genes Devel. 2:688–697. Slightom, J. L., M. Durand-Tardif, L. Jouanin, and D. Tepfer. 1986. Nucleotide sequence analysis of the TL-DNA of Agrobacterium rhizogenes agropine type plasmid. J. Biol. Chem. 261:109–121. Spanò, L., D. Mariotti, M. Cardarelli, C. Branca, and C. Costanino. 1988. Morphogenesis and auxin sensitivity of transgenic tobacco with different complements of Ri T-DNA. Plant Physiol. 87:479–483. Spena, A., T. Schmülling, C. Koncz, and J. S. Schell. 1987. Independent and synergistic activity of rolA, B and C loci in stimulating abnormal growth in plants. EMBO J. 6:3891–3899. Spena, A., J. J. Estruch, E. Prinsen, W. Nacken, H. Vanonckelen, and H. Sommer. 1992. Anther-specific expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and alters anther development and whole flower growth. Theor. Appl. Genet. 84:520–527. Strobel, G., and A. Nachmias. 1985. Agrobacterium rhizogenes promotes the initial growth of bare root stock almond. J. Gen. Microbio. 131:1245–1249. Sun, L. Y., M. O. Monneuse, J. Martin-Tanguy, and D. Tepfer. 1991. Changes in flowering and accumulation of polyamines and hydroxycinnamic acid-polyamine conjugates in tobacco plants transformed by the rolA locus from the Ri TL-DNA of Agrobacterium rhizogenes. Plant Sci. 80:145–146. Tepfer, D. 1984. Transformation of several species of higher plants by Agrobacterium rhizogenes: Sexual transmission of the transformed genotype and phenotype. Cell 47:959–967. Vahdati, K., J. R. McKenna, A. M. Dandekar, C. A. Leslie, S. L. Uratsu, W. P. Hackett, P. Negri, and G. H. McGranahan. 2002. Rooting and other characteristics of a transgenic walnut hybrid (Juglans hindsii × J. regia) rootstock expressing rolABC. J. Am. Soc. Hort. Sci. 127:724–728. Van Altvorst, A. C., R. J. Bino, A. J. van Dijk, A. M. J. Lamers, W. H. Lindhout, F. van der Mark, and J. J. M. Dons. 1992. Effects of the introduction of Agrobacterium rhizogenes rol genes on tomato plant and flower development. Plant Sci. 83:77–85. Van der Salm, T. P. M. 1996. Introduction of Rol genes in Rosa hybrida L. for improved rootstock performance. PhD thesis, Wageningen, The Netherlands.

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Van der Salm, T. P. M., R. Bouwer, A. J. van Dijk, L. C. P. Keizer, Ch. H. Hänisch ten Cate, L. H. W. van der Plas, and J. J. M. Dons. 1998. Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L. J. Expt. Bot. 49:847–852. Vansuyt, G., F. Vilaine, M. Tepfer, and M. Rossignol. 1992. RolA modulates the sensitivity to auxin of the proton translocation catalyzed by the plasma membrane H+-ATPase in transformed tobacco. FEBS Lett. 298:89–92. Vilaine, F., J. Rembur, D. Chriqui, and M. Tepfer. 1998. Modified development in transgenic tobacco plants expression a rolA::GUS translational fusion and subcellular localization of the fusion protein. MPMI 11:855–859. Welander, M., N. Pawlicki, A. Holefors, and F. Wilson. 1998. Genetic transformation of the apple rootstock M26 with the rolB gene and its influence on rooting. J. Plant Physiol. 153:371–380. White, F. F., B. H. Taylor, G. A. Huffman, M. P. Gordon, and E. W. Nester. 1985. Molecular and genetic analysis of the transferred DNA regions of the root-inducing plasmid of Agrobacterium rhizogenes. J. Bacteriol. 164:33–44. Zhu, L. H., and M. Welander. 1999. Growth characteristics of the apple cultivar Gravenstein plants grafted onto the transformed rootstock M26 with the rolA and rolB genes under non-limiting nutrient conditions. Plant Sci. 147:75–80. Zhu, L. H., A. Ahlman, X. Y. Li, and M. Welander. 2001a. Integration of the rolA gene into the genome of the vigorous apple rootstock A2 reduced plant height and shortened internodes. J. Hort. Sci. Biotech. 75:758–763. Zhu, L. H., A. Holefors, A. Ahlman, Z. T. Xue, and M. Welander. 2001b. Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth. Plant Sci. 160:433–439. Zhu. L. H., X. Y. Li, A. Ahlman, and M. Welander. 2003. The rooting ability of the dwarfing pear rootstock BP10030 (Pyrus communis) was significantly increased by introduction of the rolB gene. Plant Sci. 165:829–835. Zuker, A., E. Shklarman, G. Scovel, H. Ben-Meir, M. Ovadis, I. Neta-Sharir, Y. Ben-Yephet, D. Weiss, A. Watad, and A. Vainstein. 2001. Genetic engineering of agronomic and ornamental traits in carnation. Acta. Hort. 560:91–94.

4 Terminology for Polyploids Based on Cytogenetic Behavior: Consequences in Genetics and Breeding Domenico Carputo DISSPA—Department of Soil, Plant and Environmental Sciences, University of Naples “Federico II”, Via Università 100, 80055 Portici, Italy Elsa L. Camadro Estación Experimental Agropecuaria Balcarce, Instituto Nacional de Teconología Agropecuaria (INTA)-Facultad de Cs. Agrarias, Universidad Nacional de Mar del Plata (UNMdP) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), C.C. 276, 7620 Balcarce, Bs. As., Argentina Stanley J. Peloquin Department of Horticulture, University of Wisconsin–Madison, 1575 Linden Drive, Madison, Wisconsin, 53706, USA

I. INTRODUCTION II. ROLE OF 2n GAMETES AND ENDOSPERM IN THE ORIGIN OF POLYPLOIDS III. TERMINOLOGY FOR POLYPLOIDS A. The Old Terminology B. The Need for a New Terminology IV. BASES OF THE NEW TERMINOLOGY A. Cytogenetics B. Genetics C. Reproductive Behavior D. Breeding V. CONCLUSIONS LITERATURE CITED

Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 105

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I. INTRODUCTION Polyploidy is the common term used to denote the presence of more than two complete chromosome sets in somatic cells, tissues, or individuals. It is a widespread phenomenon in plants, where at least 30–35% (Stebbins 1947) and perhaps up to 80% (Masterson 1994) of angiosperms are polyploid. Even some diploid species are considered the product of an ancient polyploid evolution whose tracks have been lost due to genomic rearrangements. For example, maize (Zea mays) is considered a diploid species with 2n=2x=20. However, its genome is replete with repetitive DNA and chromosome duplications resulting from an ancient polyploidization event that probably occurred 11 × 106 years ago after its divergence from sorghum, which was followed by genomic rearrangements and diploidization (Gaut and Doebley 1997). Likewise, the soybean genome (2n=2x=40) presents extensive duplication, giving credence to its status as an ancient polyploid (Soltis et al. 1993; Schoemaker et al. 1996). About 50% of economically important species are polyploid. Examples of polyploidy in species of agronomic and ornamental value are presented in Table 4.1. Among them, there are species used for food and/or feed such as strawberries, wheat, oats, potato, forage grasses, and legumes; species used as fibers such as cotton, and as ornamentals such as cyclamen and impatiens; and species used for production of oil, such as sesame and oilrape, and species used for sugars such as sugarcane. Due to the prominence of polyploidy within the most important plant species, there has always been a great interest in this phenomenon in Table 4.1.

Examples of polyploidy in species of agronomic and ornamental value

Common name Cereals Barley meadow squirrel tail Oats cultivated hull-less red cultivated wild Wheat bread durum emmer spelt

Scientific name

Ploidy

Type of inheritance

Hordeum nodosum H. jubatum

6x=42 4x=28

Avena sativa A. nuda A. byzantina A. fatua

6x=42 6x=42 6x=42 6x=42

disomic

Triticum aestivum T. durum T. dicoccum T. spelta

6x=42 4x=28 4x=28 4x=42

disomic disomic disomic disomic

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(continued)

Common name Forage grasses Dallis grass Johnson grass Kentucky bluegrass Orchard grass Quackgrass Switch grass

Scientific name

Ploidy

Type of inheritance

Paspalum dilatatum Sorghum halepense

4x=40 4x=40

Poa pratensis Dactylis glomerata Agropyron repens Panicum virgatum Festuca arundinacea Paspalum urvillei

4x=28 to 18x 4x=28 4x=28; 6x=42 4x=36; 6x=54; 8x=72; 10x=90; 12x=108 6x=42; 10x=70 4x=40; 6x=60

Wheat grass bluestem crested intermediate tall

Agropyron smithii A. cristatum A. intermedium A. enlongatum

6x=42; 8x=56 4x=28; 6x=42 4x=28; 6x=42 8x=56; 10x=70

Legumes Alfalfa (common) Birdsfoot trefoil Lupine Peanuts Soybean White clover

Medicago sativa Lotus corniculatus Lupinus alba Arachis hypogaea Glycine max Trifolium repens

4x=32 4x=24 4x=40 4x=40 4x=40 4x=32

Fiber plants Cotton egyptian upland

Gossypium barbadense G. hirsutum

4x=52 4x=52

Sugar plants

Saccharum officinalis

8x=80

Coffea sp. Camelia sinensis var. macrophylla Nicotiana tabacum

4x=44; 6x=66; 8x=88 3x=45; 4x=60 4x=48

disomic

Oil plants Oilseed rape Sesame

Brassica napus Sesamum indicum

4x=38 4x=52

disomic

Vegetables Leek Potato Pumpkin Rhubarb Squash Sweet Potato Yams

Allium porrum Solanum tuberosum Cucurbita pepo Rheum rhaponticum C. moschata Ipomea batatas Dioscorea sativa

4x=32 4x=48 4x=40 4x=44 4x=40 6x=96 6x=60

Tall fescue Vaseygrass

Stimulants Coffee Tea Tobacco

tetrasomic

polysomic

tetrasomic tetrasomic disomic disomic

disomic disomic

tetrasomic

(Continues)

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Table 4.1.

(continued)

Common name Fruits Apple Banana Blueberry highbush rabbiteye Cherry (tart) Grapes Kiwifruit Lime Pineapple Plum (European) Raspberry Black Red Strawberry Ornamentals Cyclamen New Guinea Impatiens Impatiens

Scientific name

Ploidy

Malus × domestica Musa sp.

2x=34; 3x=51 3x=33; 4x=44

Vaccinium corymbosum V. ashei Prunus cerasus Vitis vinifera Actinidia deliciosa Citrus aurantifolia Ananas comosus Prunus domestica

2x=24; 4x=48

Rubus occidentalis

Fragaria × ananassa

2x=14; 13x+1=92, 13x+ 2= 94, 14x=98 2x=14, 12x=84, 13x=91, 14x=98 8x=56

Cyclamen persicum

4x=56

Impatiens schlecteri Impatiens mooreana

4x=32 6x=66

R. idaeus

Type of inheritance

in 4x: tetrasomic

6x=72 4x=32 3x=57; 4x=76 2x=58; 4x=116; 6x=174 3x=27 3x=75, 4x=100 6x=48

various fields of the plant sciences. Although several scientists recognized the significance of the type of inheritance of polyploids, the terminology is still based on their origin and genome differentiation. In this review, we discuss the problems associated with the old, classical terminology, and provide evidence that polyploid classification based on the genetic and cytogenetic behavior is more suitable.

II. ROLE OF 2n GAMETES AND ENDOSPERM IN THE ORIGIN OF POLYPLOIDS Polyploidy in plants is considered an on-going, dynamic process producing genetic novelty. Recently, Otto and Whitton (2000) estimated that 2–4% of speciation events in angiosperms involve polyploidization. There is strong circumstantial evidence that most polyploids originated through mechanisms of sexual polyploidization via gametes with unre-

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duced chromosome numbers (2n gametes), and that speciation of polyploids from diploids is highly influenced by endosperm barriers (Ramsey and Schemske 1998; Carputo et al. 2003). 2n gametes can result from random pre- or post-meiotic chromosome duplication events or from the expression of inherited mutations affecting all aspects of nuclear and cytoplasmic events during micro- and megasporogenesis. They have been extensively studied in a number of genera, including Solanum, Medicago, Manihot, Malus, Arachis, Lolium, and Agropyrum (Bretagnolle and Thompson 1995), and have been generally attributed to the action of single recessive genes (Mok and Peloquin 1975; Werner and Peloquin 1990). These genes exhibit incomplete penetrance and variable expressivity, and their phenotypic expression is significantly modified by genetic, environmental, and developmental factors (Peloquin et al. 1999). The products of the meiotic modifications leading to 2n gamete formation are genetically equivalent to those formed by first division restitution (FDR) and second division restitution (SDR) mechanisms, which transmit different levels of intra- and inter-locus interactions (Peloquin et al. 1999). The genetic structure of a tetraploid family with respect to one locus— i.e., the proportion of tetraallelic, triallelic, balanced diallelic, unbalanced diallelic and monoallelic individuals—depends on the genotypic constitution, degree of relationship, ploidy level, and modes of 2n gamete formation in the two parents and the position of the locus with respect to the centromere (Mendiburu et al. 1974). Consequently, polyploid progenies deriving from the functioning of FDR and SDR 2n gametes are expected to be genetically different. For heterozygous loci in the parents, it has been estimated that with FDR mechanisms all loci from the centromere to the first crossover and one-half of those beyond the first crossover will be heterozygous in the gametes. By contrast, with SDR mechanisms all loci from the centromere to the first crossover will be homozygous in the gametes, and all loci past the first crossover will be heterozygous. FDR gametes are expected to strongly resemble each other and the parental genotype they come from. By contrast, SDR mechanisms are expected to produce a heterogeneous population of highly homozygous gametes. Hybridization events involving 2n gametes from both parents, as well as those involving n gametes from one parent and 2n gametes from the other, can give rise to new polyploid populations. Due to the genetic consequences associated with the mode of 2n gamete formation, various levels of variability, fitness, and heterozygosity are expected in these newly formed polyploids. The fact that 2n gamete production can be genetically determined ensures repeated events of sexual polyploidization as well as the incorporation of genetic diversity of more than one diploid species at higher ploidy levels. Natural selection can act upon these

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novel phenotypic, ecological and physiological characteristics, establishing the conditions for the origin new species. There is evidence that speciation of polyploids from diploids is highly influenced by endosperm barriers. The endosperm balance number (EBN) hypothesis was first proposed in Solanum (Johnston et al. 1980) to explain unusual results from interspecific crosses. According to the EBN hypothesis, each species has an EBN value, and interploidy/intra- and interspecific crosses have normal endosperm development only when there is a 2:1 maternal to paternal EBN ratio in the hybrid endosperm. This means that successful crosses occur only when the male and female gametes have the same EBN. In all the other cases the endosperm degenerate. Clearly, EBN differences between parents prevent hybridization within and between different ploidies within and between species. The EBN is a number experimentally assigned to each species following crosses with a tester species whose EBN was arbitrarily established, and assuming the 2:1 ratio as a prerequisite for normal endosperm development. All species that successfully crossed with the tester were assigned its same EBN. In fact, when species with the same EBN are crossed, the maternal to paternal EBN ratio in the developing endosperm will always be 2:1, regardless of the parental ploidies and the direction of the cross. EBN have been assigned in various genera: from 1 to 4 in Solanum and Avena (Hanneman 1994; Katsiosis et al. 1995); from 2 to 4 in Impatiens (Arisumi 1982); from 2 to 8 in Trifolium (Parrott and Smith 1986). Genetic studies demonstrated that the EBN in potato is under the control of a few genes with additive effects (Ehlenfeldt and Hanneman 1988; Camadro and Masuelli 1995). For a review on the EBN and its genetics and breeding implications, see Carputo et al. (1999). The EBN represents a powerful screen for 2n gametes during sexual polyploidization events leading to polyploid evolution. Indeed, if fertilization involves parents with the same EBN, a new population arises and a new species could eventually be formed. By contrast, when parents differ in their EBN value, the 2:1 EBN requirement favors the functioning of 2n gametes from the parent with the lower EBN. In Solanum, for example, a 4x (2EBN) species producing 2n gametes can cross with a sympatric 4x (4EBN) species resulting in a hexaploid population, as experimentally proved (Camadro and Espinillo 1990). It is clear that both endosperm and 2n gametes play a combined role in maintaining and breaking the ploidy integrity of ancestral species, while providing the flexibility for continuous sexual polyploidization events. It should be pointed out that this complementary role of EBN and 2n gametes has an important meaning not only because it facilitates interspecific gene introgression, but also because it maintains the ploidy integrity of the two parental species.

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III. TERMINOLOGY FOR POLYPLOIDS A. The Old Terminology Are the terms auto- and allopolyploid obsolete? The original terminology of polyploidy started with the use of the words auto- and allopolyploidy to describe individuals with more than two of the monoploid chromosome set characteristic of the species (autopolyploids), and those that arise after natural or artificial crossing of two (or more) species or genera and that contain the different chromosome sets of the particular parents (allopolyploids) (Kihara and Ono 1926). Kostoff (1939) proposed the additional term autoallopolyploid to designate polyploids higher than tetraploids (i.e., 6x Heliantus tuberosus, 6x Phleum pratense) that combine the characteristics of the two types, i.e., hexaploids that are autopolyploids with respect to one genome, but allopolyploids with respect to the other genome that they contain. The main concept was that the genomes were identical in autopolyploids and different in allopolyploids. However, it was evident that the degree to which genomes differ from each other varies among species and, thus, this classification system was not adequate. Stebbins (1947) indicated three main reasons for the inadequacy of the original terminology: (1) genic and chromosomal differentiation are independent of each other, thus a polyploid may contain genomes that are different in genic content but similar in chromosome structure; (2) polyploids higher than tetraploids may contain genomes that differ at various degrees; (3) only polyploids originated by somatic chromosome doubling of a homozygous parent have genomes completely identical to each other. He also added a new type of polyploids, segmental allopolyploids. They were defined as allopolyploids in which genomes are not totally dissimilar, thus allowing partial synapsis and crossing over between chromosomes of different genomes. He gave several examples of segmental allopolyploids, such as Primula kewensis, Allium cepa-A. fistulosum, Lycopersicon esculentum-L. peruvianum, Solanum douglasii-S. nodiflorum. However, it should be pointed out that for both autoallopolyploids and segmental allopolyploids, the emphasis was still on origin and genomic differentiation. B. The Need for a New Terminology The terminology used for polyploidy that is based on origin and genome differentiation is inadequate, especially in light of the fact that the origin of many polyploid species has not been elucidated yet and that genome differentiation as revealed by chromosome pairing at meiosis (multivalent vs. bivalents) cannot be used as an indicator of type of polyploidy. Indeed, species with short chromosomes have the potential

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for only one chiasmata per chromosome, making the multiple chiasmata needed for an association of multivalents impossible. Consequently, chromosomes pair mainly as bivalents. There are several examples in the literature in which this terminology often created confusion. Solanum tuberosum ssp. tuberosum has been proposed to be alternatively an auto- and allo-polyploid (see Matsubayashi 1991). Although there are recent strong evidences of a likely hybrid origin, deriving from the diploid species S. tarijense as the maternal ancestor (Hosaka 2003) and S. stenotomum (Hawkes 1990), this species behaves as a tetrasomic tetraploid. Another example is given by the segregation ratios of different families in Phleum pratense (2n=6x=42). Hexasomic inheritance was the most likely form of segregation and, despite the fact that multivalents were rarely formed, the three genomes were more or less homologous so that chromosomes paired as random bivalents (Nordenskiöld 1953). In a synthetic autohexaploid Phleum nodosum, obtained by colchicine treatment of diploid P. nodosum, only bivalent pairing was observed. However, in 6x hybrids between the two species, having 35, 42, and 48 chromosomes, the three chromosome sets from Phleum pratense were able to pair reciprocally as well as with the sets from P. nodosum (Nordenskiöld 1949). Similar results were reported by Qu and Hancock (1995) and Qu et al. (1998) in tetraploid Vaccinium darrowi × V. corymbosum hybrids (2n=4x=48). The authors found that, despite the fact that chromosome pairing occurred mainly as bivalents, inheritance patterns of RAPD markers was tetrasomic. Lotus corniculatus (2n=4x=24) has been classified as both an autotetraploid and an allotetraploid. However, the inheritance of RFLP markers in an F2 population from a cross between two diverse accessions indicated that, although bivalent pairing predominated in the two parental lines and the F1 hybrids, the results could only be explained by assuming tetrasomic inheritance (Fjellstrom et al. 2001). Molecular markers were also used to detect the tetrasomic inheritance of Solanum tuberosum-S. commersonii hybrids (2n=4x=48) (Barone et al. 2002), and to study disomic inheritance patters of tetraploid Agrostis palustris (2n=4x=28) (Warnke et al. 1998). As pointed out by Dvorák during the Symposium on Classical and Molecular Cytogenetic Analysis held at Kansas State University in 1994 (Raupp and Gill 1995), “. . . autopolyploidy and allopolyploidy are terms that are important in describing the nature of inheritance, either disomic inheritance or polysomic inheritance. Stebbins went wrong in putting all emphasis on the origin rather than in the genetic consequences.” Thus, polyploid classification based on the genetic and cytogenetic behavior is more suitable to describe the type of polyploidy.

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There are several cytogenetic findings that contributed to alter our view of the terminology based on origin and emphasis on genome differentiation. 1. Tetrasomic-nullisomic Compensation in Wheat. Triticum aestivum is an allohexaploid (2n=6x=42). Its chromosomes were placed into seven groups of three chromosomes each, given that the nullisomic for any of the three can be compensated by extra doses of either of the other two (Sears 1954; Sears and Okamoto 1958). This is an evidence of partial homology (homoeology) between the three chromosomes of each group that were assigned to three genomes: A, B and D. However, only bivalents are formed in meiosis due to the genetic control of pairing in this and other polyploid wheats. 2. Genetic Control of Bivalent Pairing. The diploid-like meiotic behavior of polyploid wheats at metaphase I is influenced by the action of several loci, the two most important being Ph1 (Sears and Okamoto 1958; Riley and Chapman 1958) and Ph2 (Martínez et al. 2001). These loci suppress pairing of homoelogous chromosomes. Weak alleles of the Ph1 gene are also expressed in intergeneric hybrids of Triticum and Aegilops peregrina (Ozkan and Feldman 2001). A similar genetic system has been proposed for hexaploid oats (Gauthier and McGinnis 1968). In allotetraploid oilseed rape, Brassica napus (2n=4x=38), clear bivalent pairing is observed at metaphase I but the extent of pairing in haploids is variable, indicating a genetic control. It has been proposed that a major gene, Pr Bn, mainly controls the extent of diploid pairing in this species, similarly to the Ph genes (Jenczewski et al. 2003). These are examples of disomic allopolyploids that can, however, exhibit polysomic inheritance if the genes controlling bivalent pairing are removed or their expression is altered. 3. Synaptic Mutants Affecting Pairing and Crossing Over. In all sexually reproducing organisms, homologous chromosomes synapse longitudinally during zygotene and pachytene to form bivalents in diploids and bivalents or higher configurations in polyploids. This synapsis is a prerequisite for crossing over and chiasmata formation and, like other events in meiosis, is under genetic control. The presence of univalents at the first meiotic division could be due to incomplete homology of chromosomes, environmental influences, or gene mutations. Lack of pairing has been observed in interspecific and intergeneric hybrids, apomictic plants, aneuploids, plants under stress, and individuals carrying mutant synaptic (asynaptic and desynaptic) genes. In asynaptic

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mutants, chromosomes fail to pair completely or incompletely, whereas in desynaptic mutants, chromosomes pair during zygotene and remain so at pachytene but fall apart during diplotene or diakinesis. Synaptic mutants have been described in over 100 plant species belonging to 93 genera; many of them are polyploids that have been classified as either auto- (i.e., Dactylis glomerata) or allo-polyploids (i.e., Avena sativa, Gossypium hirsutum, Triticum aestivum, T. durum) (for a review, see Katayama 1964 and Koduru and Rao 1981). In view of the above findings, we think that the terms polysomic polyploid and disomic polyploid are more logical and appropriate, and give a more precise picture of the nature of polyploidy. These terms are not based on origin (rarely known) or on genome differentiation, but on genetic behavior at meiosis. Polysomic polyploids are made up of homologous chromosomes that segregate from multivalents or random bivalents. By contrast, disomic polyploids are made up of two or more sets of homoeologous chromosomes that segregate from bivalents formed only between homologous chromosomes. The terms allo- and autopolyploid should be used only when the origin is known. But even in this situation, it will be more helpful for geneticists and plant breeders to refer to the genetic and cytogenetic behavior in addition to the origin (for example, disomic autohexaploid Phleum nodosum), because it is more meaningful from cytogenetic, genetic, and breeding standpoints.

IV. BASES OF THE NEW TERMINOLOGY The basic differences between disomic and polysomic polyploids can be summarized by considering some major features of bread wheat and potato, two typical polyploids (Table 4.2). This seems appropriate since, together with rice and maize, they represent the most important world food crops. In particular, wheat is a disomic hexaploid (2n=6x=42); potato is a polysomic tetraploid (2n=4x=48). A. Cytogenetics Disomic polyploids have bivalent pairing and, therefore, disomic inheritance as does any diploid species. They can also have duplicate and triplicate factor inheritance due to the presence of similar loci in homeologous chromosomes. The most important aspects of functional diversification of duplicated genes in disomic polyploids have been recently reviewed by Wendel (2000). The author reported that there is evidence that duplicated genes may retain their original or similar function, undergo diversification in protein function or regulation, or one copy

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Basic differences between disomic and polysomic polyploids. Disomic (wheat, 2n=6x=42)

Character

Tetrasomic (potato, 2n=4x=48)

Genomes

AABBDD

PPPP

Cytogenetics

Only bivalents

Multivalents

Genetics

Disomic

Tetrasomic

Reproductive behavior

Autogamous

Allogamous

Fertility

High

Reduced

Breeding

Homozygosity at each locus; inter-locus interactions

Heterozygosity at each locus; intra- & inter-locus interactions

may become silenced. It is also possible that duplicated genes may interact through inter-locus recombination, gene conversion, or concerted evolution. The genetics of polysomic polyploids is very complex. Three types of segregation have been proposed or recognized for this type of polyploids: chromosome segregation, random chromatid segregation, and maximum equational segregation (see Burnham 1962). Chromosome segregation is the type in which chromatids derived from a particular multivalent belong to different chromosomes in that multivalent. It occurs for genes genetically completely linked to the centromere. For example, in a polysomic tetraploid with the duplex genotype AAaa, AA, Aa, and aa gametes are formed in a 1:4:1 ratio. If either of the other two types of segregation occurs, aa gametes derived from sister chromatids are also expected. This behavior has been termed double reduction, post-reduction, or equational segregation and its total frequency is represented by alfa (∝). Its underlying cytological bases include multivalent pairing, one crossing over between the locus and the centromere, the two pairs of chromatids from that crossing over passing to the same pole in Anaphase I, and random separation of chromatids in Anaphase II. The theoretical extreme values for alfa varies between 0 for genes genetically completely linked to the centromere and 1/6 when maximum equational segregation occurs, a condition that is not likely to be observed experimentally given the extreme requirements for its occurrence. Therefore, for the same duplex genotype mentioned before, AA, Aa, and aa gametes are formed in a 2:5:2 ratio with maximum equational segregation, in a 3:8:3 ratio with random chromatid segregation if ∝=0, and in a 1+2∝/6: 4-4∝/6: 1+2∝/6 ratio with both types of chromatid segregation if ∝ is different from 0 (Burnham 1962).

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Between these two extremes—complete bivalent pairing in disomic polyploids and complete multivalent pairing or random chromosome bivalent pairing in polysomic polyploids—there are intermediate situations when the genomes of a species are partially homologous (homoeologous) and can, therefore, pair, or when pairing between chromosomes of the same genome occurs. In both situations, multivalents may also be formed, leading to the formation of gametes with genetic ratios for the loci involved similar to those expected under polysomic inheritance (Burnham 1962). In hexaploids and higher polyploids carrying two or more genomes in different dosages (for example, AABBBB), both disomic and polysomic inheritance could be expected, depending on the factors previously considered. B. Genetics There are four main genetic differences between a polysomic and a disomic polyploid. Taking a polysomic tetrasomic and a disomic tetraploid as examples, they can be summarized as follows: 1. At a given locus with two alleles, five genotypes: aaaa, Aaaa, AAaa, AAAa, and AAAA, are possible vs. three genotypes: AA, Aa, and aa. Thus, the terms homozygote and heterozygote do not have the same meaning in both types of polyploids. A specific terminology has to be applied according to the number of dominant alleles per locus: nulliplex, simplex, duplex, triplex, and quadruplex. 2. With two alleles at a locus, the probability of obtaining a completely recessive genotype upon selfing is 1/36 with chromosome segregation in a duplex genotype (AAaa) vs. 1/4 in a diploid heterozygote (Aa). 3. There could be up to four alleles per locus (A1A2A3A4) vs. two. The higher number of alleles per locus provides the opportunity for a larger number of intra-locus interactions: a total of 11 of first, second and third order (see next section) vs. one of first order, as well as inter-locus interactions. Thus, maximum heterozygosity can be achieved. For traits whose genetic variance is almost entirely nonadditive, this is a very important plant breeding issue, for the breeder must strive to obtain maximum heterozygosity to optimize heterotic combinations (Mendiburu et al. 1974; Dunbier and Bingham 1975). 4. Gametes can be homozygous or heterozygous (AA, Aa, and aa) vs. gametes that are hemizygous (A and a). This implies that polysomic polyploids can display gametophytic heterosis (Simon and Peloquin 1976; Groose and Bingham 1991).

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C. Reproductive Behavior Most polyploids with disomic inheritance (i.e., Triticum, Brassica) have been found in connection with autogamy, and those with polysomic inheritance (i.e., Lotus, Solanum, Medicago) in connection with allogamy. In disomic polyploids with partially differentiated genomes, autogamy provides maximum genetic fixation for alleles at homologous chromosomes and heterozygosity for alleles at homoelogous chromosomes, a condition known as “built-in heterozygosity” (i.e., AA.aa in a disomic tetraploid). In addition, if homozygosity for corresponding genomes is high, fitness and genetic flexibility for the survival of the species are also high (MacKey 1970). By contrast, polysomic polyploids have advantages in their reproductive efforts when they are outcrossers. In this type of polyploids, individuals can have more than two alleles per locus (i.e., up to four in a tetrasomic tetraploid), providing the opportunity for rich interaction patterns. For example, in tetrasomic tetraploids such as potato and alfalfa, 11 interactions are possible at a locus with four alleles: six of first order (between two alleles), four of second order (between three alleles), and one of third order (between four alleles) (Mendiburu et al. 1974). Multiple allelism is common in potato. Leonard-Schippers et al. (1994) detected four alleles per locus in 31% of 111 RFLP loci analyzed in an F1 population from non-inbred parents, whereas Jacobs et al. (1995) detected multiple alleles at more than one third of RFLP loci in a diploid BC1 mapping population. Consequently, self-fertilization would not confer any advantage to polysomic polyploids because it will reduce the number of potential intra- and interlocus interactions. The type of inheritance strongly influences the fertility of polyploids. Indeed, the occurrence of regular bivalent pairing at meiosis in disomic polyploids results in gametes with balanced chromosome numbers and, thus, high male and female fertility. By contrast, multivalent pairing in polysomic polyploids results in gametes with unbalanced chromosome numbers and sterility problems. When endosperm barriers are operating, the reproductive system of polyploids may influence 2n gamete production. Indeed, the autogamous disomic tetraploid Solanum species require an identical EBN in the male and female gametes for normal endosperm development. Thus, a high frequency of either 2n pollen or 2n eggs in these species would not be an advantage because it would result in an incompatible EBN ratio and, therefore, seed abortion. On the contrary, in allogamous polysomic polyploids, mutant genes controlling 2n gamete production may be favored for they will allow genetic flow between ploidy levels. This is

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the case of 4x(4EBN)-2x(2EBN) crosses involving tetrasomic tetraploid Solanum species and related diploid species that produce 2n gametes. The EBN system acts as a screen for 2n gametes, allowing them to transmit high values of parental heterozygosity to the polyploid offspring. D. Breeding The type of polyploidy has a significant impact on plant breeding goals, methods, and strategies. Only appropriate approaches that take the type of polyploidy (disomic vs. polysomic) into account can result in successful breeding efforts, such as those based on chromosome or genome manipulations. In polysomic polyploids, a drastic decline in yield and fertility occurs upon self-fertilization and in progenies involving closely related parents (Howard 1970; Jones and Bingham 1995; Kimbeng and Bingham 1998). It can be attributed not only to increased homozygosity but also to loss of intra- and interlocus interactions. In tetraploid alfalfa, Busbice and Wilsie (1966) studied the basis of the discrepancy between the inbreeding depression predicted by the coefficient of inbreeding and that which actually occurs. They developed a system to predict inbreeding depression based upon the loss of allelic interactions, and postulated the presence of multiple allelic series at many loci as the basis for the rapid loss of vigour following inbreeding. The frequency of these highly heterozygous loci diminished rapidly upon inbreeding. They concluded that tetra- and triallelic loci are important in inbreeding depression and that, by contrast, they are responsible for heterotic effects. In potato, Mendiburu et al. (1974) hypothesized that the decrease in performance following haploidization (haploid depression) is not only due to strict inbreeding but also to the loss of favorable allelic intralocus interactions and of favorable epistatic combinations. The importance of epistatic interactions has been pointed out also by Comstock et al. (1958) in strawberry, another clonally propagated polyploid. Since heterosis for several traits in polysomic polyploids is based on non-additive genetic variance (Mendoza and Haynes 1974; Jones and Bingham 1995), maximum heterosis is expected with maximum intralocus interactions and epistatic effects. Hence, heterozygosity at each given locus of interest is the primary important genetic effect that plant breeders have to take into account. In tetrasomic tetraploids like alfalfa and potato, tri- and tetra-allelic loci are produced if genetically diverse parents are used in controlled crosses. In designing breeding schemes aimed at maximizing heterozygosity, breeders need to be very careful in the appropriate use of parents. As reported by Dunbier and Bingham (1975), four randomly mated unre-

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lated duplex genotypes (A, B, C, D) at equilibrium will have the following population structure: 0.19% monoallelic, 9.67% diallelic, 49.22% triallelic, 41.02% tetrallelic. By contrast, if they are mated two by two (A×B, C×D), and the resulting hybrids are crossed (AB × CD), the population structure will change, with a percentage of tetrallelic loci as high as 79%. Alternatively, high levels of heterozygosity can be reached when polysomic polyploids are synthesized through crossing schemes involving 2n gametes. Indeed, as reported by Peloquin et al. (1999), 2n gametes derived by first division restitution and second division restitution mechanisms transmit about 80% and 40% of the parental heterozygosity, respectively. They also maintain and transmit a large amount of inter-locus epistatic interactions, which are important in maximizing heterosis. In modern potato breeding, haploids (2n=2x=24) produced from commercial cultivars (2n=4x=48) are crossed to 2x species to broaden the genetic diversity through the introduction of new alleles for traits of interest and allelic diversity to maximize heterozygosity. Diploid hybrids that produce 2n gametes are then used in unilateral (4x × 2x and 2x × 4x), and bilateral (2x × 2x) sexual polyploidization crossing schemes to yield highly heterotic 4x progenies. In the 4x-2x schemes, the direction of the crosses will depend on the mode of 2n gamete production available and the fertility of the parents involved. The asexual reproduction system and the presence of 2n gametes in potato provide the unique possibility of accumulating non-additive genetic effects at the tetraploid level and the immediate fixation of heterosis. The extent of conservation of allelic interactions depends mainly on the mode of 2n gamete production [first division restitution (FDR) vs. second division restitution (SDR)], whereas the creation of new interactions depends mainly on the allelic diversity of the parents involved. The value of these approaches in maximizing allelic interactions is well documented in potato, where highly heterotic tetraploid progenies have been produced between cultivated S. tuberosum and tuber-bearing Solanum species (Tai and de Jong 1991; Werner and Peloquin 1991; Darmo and Peloquin 1991; Peloquin and Ortiz 1992; Ortiz 1998). Ortiz et al. (1997) evaluated eight FDR-2n pollen producing diploid hybrids with different genetic background. They found that their breeding value was much higher than that of 4x clones, and also suggested the 4x × 2x approach to combine earliness and large tuber size from the tetraploid parents and allelic diversity and specific gravity from the diploid parent. Diploid selected hybrids involving S. tuberosum haploids and either S. phureja or S. chacoense diploid species were used by Buso et al. (1999) to produce 4x offspring. Evaluations in two different environments provided evidence of heterosis values for total tuber yield of 48% and 28% over the mean of the 4x parents. Ortiz et al. (1991) reported

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that besides the allelic diversity transmitted, the 4x × 2x breeding scheme is more effective than the traditional 4x × 4x scheme also because fewer replications and locations are necessary to evaluate the progenies. Disomic polyploids usually display high levels of homozygosity due to their autogamous reproduction systems, and homoallelic duplexes are the primary unit of allelic function within each set of homologous chromosomes. However, disomic polyploids may also possess non-segregating heteroallelic multiplexes stabilized by homologous pairing within each genome. This may result in high levels of heterozygosity between homeologous loci. Thus, self-fertilizing diploid Triticum species can have only one fixed allele at each locus, whereas their self-fertilizing tetraploid and hexaploid related species (T. durum and T. aestivum, for example), can have simultaneously two and three fixed alleles, respectively. The presence of such heterozygosity is a unique and important feature in disomic polyploid breeding that deserves special attention. Indeed, it will introduce a fundamental characteristic of heterozygosity (the presence of more than one allele simultaneously at a locus) in an otherwise homozygous genotype. This permits as many alleles to occur simultaneously in homozygous conditions as there are reduplications of the somatic chromosome set (MacKey 1970). Breeding work in disomic polyploids is essentially based on the production of homozygous lines. In order to maximize heterozygosity, it should lead to the simultaneous selection for heteroallelism between homeologous loci in the genomes involved (i.e., A, B and D in wheat). Wendel (2000) reported that in disomic polyploids genetic diversity in homoeologous genes is expected due to the fact that duplicated genes may be subjected to pressures that lead to differential rates of sequence evolution. The B genome of polyploid wheat, for example, harbors more diversity than the other genomes (Siedler et al. 1994). In studying 14 isozymic loci, Allard et al. (1993) found that much of the greater heterosis and wider adaptation observed in the disomic tetraploid Avena barbata, another self-fertilizing species, in comparison with its diploid ancestor A. hirtula, could be attributed to favorable intra- and inter-locus interactions among alleles at loci that form heteroallelic quadruplexes. Patterns of quadruplex formation differed for most of the 14 loci studied. Three loci (Mdh2, Pgm1, and Got1) were nearly completely homoallelic. The others formed homoallelic as well as several heteroallelic quadruplexes. The authors pointed out that in disomic polyploids there are two types of interactions to be taken into account: (1) interactions among alleles of the same locus, which is stabilized by the “diploidized” genetics of disomic polyploids, and (2) interlocus interactions among alleles of different loci, which is stabilized by the mating system of selfing.

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One final comment on breeding aspects of disomic polyploids is related to the convenience of F1 hybrid production for commercial purposes. Since disomic polyploids can have built-in heterozygosity in homoeologous genomes as discussed previously, heterotic effects in F1 hybrids are not expected to be of the same magnitude as those obtained in crosses between inbred lines. V. CONCLUSIONS The terminology of polyploids based on the type of inheritance (disomic vs. polysomic) is genetically more meaningful than the terminology based on origin with emphasis on genome differentiation (allo- vs. autopolyploidy), although the former terms do not negate the latter. The origin of polyploid species is rarely known and pairing behavior is determined not only by genome similarities, but also by chromosome size and the action of mutant genes affecting the meiotic process. The type of inheritance, which is closely associated to the reproductive behavior, has important genetic consequences in both evolution and breeding. Thus, emphasis should be given to the terms disomic polyploid and polysomic polyploid. ACKNOWLEDGMENTS This is contribution no. 93 from DISSPA. ELC had a leave of absence from INTA and UNMdP. LITERATURE CITED Allard, R. W., P. García, L. E. Sánez-de-Miera, and M. Pérez de la Vega. 1993. Evolution of multilocus genetic structure in Avena hirtula and Avena barbata. Genetics 135:1125–1139. Arisumi, T. 1982. Endosperm balance numbers among New Guinea-Indonesian Impatiens species. J. Hered. 73:240–242. Barone, A., J. Li, A. Sebastiano, T. Cardi, and L. Frusciante. 2002. Evidence for tetrasomic inheritance in a tetraploid Solanum commersonii (+) S. tuberosum somatic hybrid through the use of molecular markers. Theor. Appl. Genet. 104:539–546. Bretagnolle, F., and J. D. Thompson. 1995. Tansley review No. 78. Gametes with somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytol. 129:1–22. Burnham, C. R. 1962. Discussions in cytogenetics. Burgess Publ. Co., Minneapolis, MN. Busbice, T. H., and C. P. Wilsie. 1966. Inbreeding depression and heterosis in autotetraploids with application to Medigago sativa L. Euphytica 15: 52–67.

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5 Breeding Barley for Resistance to Fusarium Head Blight and Mycotoxin Accumulation Thin Meiw Choo* Eastern Cereal and Oilseed Research Centre Agriculture and Agri-Food Canada Ottawa, Ontario K1A 0C6 Canada

I. INTRODUCTION II. FUSARIUM SPECIES III. FUSARIUM TOXINS A. Natural Occurrence B. Toxicity IV. LOSSES IN YIELD AND QUALITY V. SOURCES OF GENETIC RESISTANCE A. Cultivated Barley B. Wild Species VI. TRAITS ASSOCIATED WITH FHB RESISTANCE A. Two-Row Spike B. Lax Spike C. Nodding Spike D. Infertile Lateral Spikelets E. Hulless Kernels F. Black Lemma and Pericarp G. Purple Lemma and Pericarp H. Long Glume Awn I. Closed (Cleistogamous) Flowering

*I dedicate this article to my teacher the late Mr. Lau Yuke Choi, formerly of Pei Yuen High School, Kampar, Malaysia. I thank Dr. Richard Martin for his unpublished data, Professor Weizhong Lu and Dr. Junmei Wang for their assistance, and four anonymous reviewers for their constructive comments. Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 125

126

VII.

VIII. IX.

X.

T. CHOO J. Heading Date K. Plant Height and Lodging Resistance L. Phenolic Acids M. Flavonoids N. Lignin BREEDING STRATEGIES A. Heritability B. Crossing Schemes C. Production of Homozygous Lines D. Screening Methods E. Selection Strategies F. Marker-Assisted Selection G. Active Resistance H. Passive Resistance I. Off-Season Nurseries MUTATION AND IN VITRO SELECTION GENETIC TRANSFORMATION A. Pathogenesis-related Protein Genes B. Trichothecene Genes C. Pleiotropic Drug Resistance Genes D. Zearalenone Detoxifying Gene E. Antifungal Protein Genes CONCLUSIONS AND PROSPECTS LITERATURE CITED

I. INTRODUCTION Fusarium head blight (FHB) or scab is one of the most destructive and widespread diseases of barley (Hordeum vulgare L.). This disease not only reduces grain yield and lowers grain quality, but also produces toxins in the grain. The toxins, in turn, are harmful to animal and human health. During epidemic years of FHB, crop losses and human suffering can be tremendous. In the United States, the economic losses for barley due to FHB were estimated to be $200 million in North Dakota from 1993 to 1997 (United States General Accounting Office 1999), and $136 million in the Northern Great Plains from 1998 to 2000 (Nganje et al. 2001). These FHB outbreaks represent the most recent ones in the United States; however, FHB has been present in the region for years. Between 1900 and 1930, for example, the barley crop in Iowa went through FHB disease cycles at least six times (Weaver 1950). FHB has caused severe outbreaks in other countries as well. In South Korea, at least half of the 1963 barley crop was destroyed because of a severe epidemic of FHB, causing not only heavy economic losses but also mycotoxicoses in animals and humans (Vestal 1964; Kim et al. 1993). In Anhui (China) in 1991, approximately 130,000 people suffered from gastrointestinal dis-

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orders after they consumed food products made from Fusarium-infected wheat and barley (Li et al. 1999b). In view of the substantial economic losses associated with FHB, barley researchers in many countries are actively working toward a better understanding of the genetic resistance to FHB and the development of FHB-resistant cultivars. The objective of this article is to review the profile of Fusarium species on barley, natural occurrence of toxins, impact of FHB on yield and quality, sources of genetic resistance, traits associated with resistance, breeding strategies, and biotechnological methods for FHB resistance. A book entitled “Fusarium head blight of wheat and barley” was published very recently. In it, three chapters are relevant to FHB of barley. Steffenson (2003) reviewed the impact, epidemics, management, and strategies for identifying and utilizing genetic resistance. Schwarz (2003) described the effect of Fusarium-infected barley on malting and brewing, while Muehlbauer and Bushnell (2003) discussed the transgenic approaches to FHB resistance. Previously, Tekauz et al. (1999) reviewed the development of FHB in Western Canada. This article emphasizes the breeding aspects for FHB resistance in barley.

II. FUSARIUM SPECIES At least 23 Fusarium species have been isolated from barley kernels (Table 5.1). Among these, F. graminearum, F. culmorum, F. avenaceum, F. sporotrichioides, and F. poae are most prevalent. The Fusarium species profile, however, is different from region to region and from country to country. F. graminearum is most prevalent in North America and the Far East, while F. culmorum is most prevalent in Europe. In South Africa, F. acuminatum is most common (23% in 1994 and 65% in 1995). The profile can change over time in one country. In Canada, for example, F. graminearum was rarely isolated from barley seed in the past but it has now become the most prevalent Fusarium species in barley in recent years. Gordon (1952) did not isolate F. graminearum from any of the 529 barley seed samples collected in Western Canada from 1939 to 1943, and isolated F. graminearum from only 27 (5%) of the 513 barley samples collected in Eastern Canada. By contrast, all of the 117 barley samples collected in Manitoba in 1993–1994 (Abramson et al. 1998 ) and 79 of the 91 barley samples collected in Eastern Canada (Martin et al. 2003) were infected by F. graminearum. F. graminearum, F. culmorum, F. avenaceum, F. sporotrichioides, and F. poae are pathogenic on barley, and all can produce toxins under artificial inoculation conditions (Table 5.2). F. graminearum produces

128 Table 5.1.

T. CHOO Fusarium species isolated from seed of naturally infected barley.

Country

Fusarium speciesz

Reference

Canada

po, av, ac, eq, cu, gr, ox, sp, sa, so, se gr, av, po, sp, ac, eq, ox, tr, cu, sa gr, av, sp, ac, po, eq, tr, cu, ox, ni gr, po, sp, av, ac, cu, eq, fl, ox, sa, se, tr gr, po, sp, av, ac, cu, eq, mo, sa, sc, tr gr, av, eq, mo, ca, cu, se, so av, ar, tr, sp, cu, sa, po po, av, tr, cu, eq, ox cu, av, gr cu, av, gr, po, sp ac, eq, gr, ox, ch, po, re, se, sa, sc, so, su mo, su cu, gr, fu, av, po, tr, sp, ni, mo, ta, se, sa gr, po, sp, av, eq, ac, sc, mo, ox, su, cu

Gordon 1952 Clear et al. 1996 Abramson et al. 1998 Clear et al. 2000 Martin et al. 2003 Wang et al. 1986 Eskola et al. 2001 Marasas et al. 1979 Elen et al. 1997 Perkowski et al. 1995 Ackermann 1998 Castellá et al. 1999 Hacking et al. 1976 Salas et al. 1999

China Finland Germany Norway Poland South Africa Spain U.K. U.S.A. z

The most prevalent species is listed first followed by less prevalent ones in decreasing order: ac = F. acuminatum; ar = arthrosporioides; av = F. avenaceum; ca = F. camptoceras; ch = F. chlamydosporum; cu = F. culmorum; eq = F. equiseti; fl = F. flocciferum; fu = F. fusaroides; gr = F. graminearum; mo = F. moniliforme; ni = F. nivale; ox = F. oxysporum; po = F. poae; re = F. reticulatum; sa = F. sambucinum; sc = F. scirpi; se = F. semitectum; so = F. solani; sp = F. sporotrichioides; su = subglutinans; ta = F. tabacini; tr = F. tricinctum.

Table 5.2. plants.

Toxins produced by Fusarium species following inoculation on barley

Fusarium species F. avenaceum F. culmorum

F. graminearum

F. poae F. sporotrichioides

z

Toxinz

Reference

DON MON ZEN NIV, DON, 15-ADON DON, 3-ADON, NIV, ZEN DON, 3-ADON, 15-ADON, ZEN, NIV DON, 15-ADON DON, NIV, 15-ADON, 3-ADON DON, 15-ADON, 3-ADON DON, 15-ADON, ZEN NIV, MAS, DON, STO, T2-tetraol, HT-2 NIV, DON HT-2, T2-tetraol, T-2, DON, MAS, NEO T-2, HT-2, NEO T-2, HT-2

Salas et al. 1999 Abramson et al. 2002 Gross and Robb 1975 Mirocha et al. 1994 Perkowski et al. 1995 Perkowski et al. 1996 Mirocha et al. 1994 Perkowski et al. 1995 Salas et al. 1999 Schwarz et al. 2001 Salas et al. 1999 Schwarz et al. 2001 Salas et al. 1999 Perkowski et al. 1995 Abramson et al. 2004

The most predominant toxin is listed first followed by less predominant ones in decreasing order: 3-ADON = 3-acetyldeoxynivalenol; 15-ADON = 15-acetyldeoxynivalenol; DON = deoxynivalenol; MAS = 15-monoacetyoxyscirpenol; MON = moniliformin; NEO = neosolaniol; NIV = nivalenol; ZEN = zearalenone; STO = scirpentriol.

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Table 5.3. Toxins produced on various culture media by Fusarium species isolated from barley seeds. Fusarium species F. acuminatum F. avenaceum F. culmorum F. equiseti F. graminearum

F. moniliforme F. oxysporum F. tricinctum

Medium

Toxinz

Reference

maize maize maize rice barley barley maize rice maize liquid rice

T-2, DAS MON ZEN, DON, ADON ZEN, DON ZEN DON, ZEN ZEN, NIV, 4-ANIV ZEN, DON, 15-ADON FB1, FB2 ZEN ZEN, DAS, 4-ANIV, NIV

Rabie et al. 1986 Marasas et al. 1979 Marasas et al. 1979 Holmberg and Pettersson 1986 Neish et al. 1982 Neish and Cohen 1981 Logrieco et al. 1988 Piñeiro et al. 1996 Castellá et al. 1999 El-Kady and El-Maraghy 1982 Holmberg and Pettersson 1986

z

The most predominant toxin is listed first followed by less predominant ones in decreasing order: ADON = acetyldeoxynivalenol; 15-ADON = 15-acetyldeoxynivalenol; 4-ANIV = 4-acetylnivalenol (fusarenon X); DAS = diacetoxyscirpenol; DON = deoxynivalenol; FB1 = fumonisin B1; FB2 = fumonisin B2; MON = moniliformin; NIV = nivalenol; ZEN = zearalenone.

deoxynivalenol (DON), nivalenol (NIV), and their acetylated derivatives. This species has been classified into two chemotypes: DON-producer and NIV-producer (Yoshizawa 1997). The former chemotype produces DON, 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and 3,15-diacetyldeoxynivalenol (3,15-DADON), while the latter produces NIV, 4-acetylnivalenol (4-ANIV), and 4,15-diacetylnivalenol (4,15DANIV). The same classification can be applied to F. culmorum. When inoculated with F. culmorum, barley grains accumulated much more NIV than DON in some studies (Mirocha et al. 1994; Chelkowski et al. 2000) but also accumulated much more DON than NIV in other studies (Perkowski et al. 1995, 1996). F. sporotrichioides produces mainly T-2 and HT-2 toxins. Some Fusarium species isolated from barley kernels can produce toxins in vitro (Table 5.3).

III. FUSARIUM TOXINS A. Natural Occurrence Fusarium toxins contaminate barley crops all over the world (Table 5.4). The Fusarium toxins detected so far include DON, NIV, zearalenone (ZEN), T-2, T-2 tetraol, HT-2, 3-ADON, 15-ADON, 3,15-DADON , 4-ANIV, 4,15-DANIV, 15-monoacetyoxyscirpenol (MAS), 4,15-diacetoxyscirpenol (DAS), scirpentriol (STO), and fumonisin B1 (FB1) (Table 5.4). In addition

130

T. CHOO

Table 5.4. Fusarium toxins detected in barley seed samples from 22 countries across five continents. Country Asia China

Fusarium toxinsz

Reference

NIV, DON, ZEN DON, ZEN, NIV ZEN, T-2, DON, FB

Tanaka et al. 1988 Li et al. 1999b Haubruge et al. 2003

Japan

NIV, DON, ZEN DON, NIV, 3-ADON, 4-ANIV, ZEN, 15-ADON

Tanaka et al. 1988

Korea

NIV, DON, ZEN NIV, DON, ZEN NIV, DON, ZEN, 4-ANIV, 3-ADON, 4,15-DANIV NIV, DON, ZEN, 3-ADON, 4-ANIV, 15-ADON NIV, DON, 3-ADON

Tanaka et al. 1988 Park et al. 1992

Yoshizawa and Jin 1995

Kim et al. 1993 Lee et al. 1995 Ryu et al. 1996

Nepal

ZEN, NIV

Tanaka et al. 1988

Saudi Arabia

DON, NIV

Al-Julaifi and Al-Falih 2001

Yemen

ZEN, DON, NIV

Tanaka et al. 1988

Europe Finland

DON, 3-ADON, T-2, NIV DON, NIV, HT-2

Hietaniemi and Kumpulainen 1991 Eskola et al. 2001

France

DON, NIV, HT-2, ZEN

Malmauret et al. 2002

Germany

ZEN, DON, NIV DON, ZEN, 3-ADON, NIV, T-2, HT-2

Tanaka et al. 1988 Müller and Schwadorf 1993

Hungary

DON, ZEN, T-2, NIV

Rafai et al. 2000

Italy

DON, ZEN, NIV

Tanaka et al. 1988

Lithuania

NIV, DON, HT-2, T-2, STO, MAS, DAS

Keblys et al. 2000

Netherlands

ZEN, DON, NIV

Tanaka et al. 1990

Norway

DON, 3-ADON, NIV, 4-ANIV HT-2, DON, NIV, T-2, 3-ADON

Langseth and Elen 1996 Langseth and Rundberget 1999

Poland

NIV, DON DON, NIV, T-2, HT-2, 3-ADON, 15-ADON DON, NIV, T-2, HT-2, DAS, T-2 tetraol

Tanaka et al. 1988

DON NIV ZEN, DON, NIV

Pettersson et al. 1986 Pettersson et al. 1995 Tanaka et al. 1988

Sweden U.K.

Perkowski et al. 1997 Perkowski et al. 2003a

5. BREEDING BARLEY FOR RESISTANCE TO FUSARIUM HEAD BLIGHT Table 5.4.

131

(continued) Fusarium toxinsz

Country North America Canada

Reference

DON, ZEN, DAS, T-2 DON, HT-2, NIV, T-2, 15-ADON DON, 15-ADON, 3-ADON, 3,15-DADON DON, NIV, ZEN, 3-ADON, 15-ADON, T-2 DON, 15-ADON

Stratton et al. 1993 Abramson et al. 1997

DON, 3-ADON, 15-ADON, ZEN DON, ZEN

Schwarz et al. 1995a Jones and Mirocha 1999

Oceania New Zealand

DON, NIV, ZEN

Lauren et al. 1991

South America Argentina

DON, NIV, ZEN

Tanaka et al. 1988

Venezuela

DON

Contreras et al. 2000

U.S.A

Abramson et al. 1998 Campbell et al. 2000 Clear et al. 2000

z The most common toxin is listed first followed by less common ones in decreasing order: 3-ADON 3-acetyldeoxynivalenol; 15-ADON = 15-acetyldeoxynivalenol; 3,15-DADON = 3,15-diacetyldeoxynivalenol; 4-ANIV = 4-acetylnivalenol (fusarenon X); 4,15-DANIV = 4,15-diacetylnivalenol; DAS = diacetoxyscirpenol; DON = deoxynivalenol; FB1 = fumonisin B1; MAS = 15-monoacetyoxyscirpenol; NIV = nivalenol; ZEN = zearalenone; STO = scirpentriol.

to these 15 toxins, fusaric acid can also be present in barley samples (Smith and Sousadias 1993). Neosolaniol has been detected in artificial inoculation tests of barley (Table 5.2), but not under natural conditions (Perkowski et al. 1997; Campbell et al. 2000; Clear et al. 2000; Al-Julaifi et al. 2001). The same is true for moniliformin (Table 5.2). Until very recently the natural occurrence of fumonsins have been limited to corn samples (Lombaert et al. 2003); however, Haubruge et al. (2003) recently reported the detection of FB1 in barley samples. Globally, DON, NIV, and ZEN are the most common Fusarium toxins found naturally in barley, followed by 3-ADON, 15-ADON, T-2, and HT-2. The remaining eight toxins are relatively minor. There are, however, some geographical differences in the incidence of Fusarium toxins. DON is predominant in many parts of the world, but NIV is predominant in the Far East. Barley samples frequently are contaminated by two or more Fusarium toxins. In Eastern Canada, almost 30% of the samples surveyed by Campbell et al. (2002) were contaminated by two to four toxins. In Japan, Yoshizawa and Jin (1995) found that seven of 17 samples contained at least five

132 Table 5.5.

T. CHOO Fusarium toxins detected in samples of beer made from barley malt.

Country Argentina Canada Germany Korea

Fusarium toxinsz

Reference

DON DON, NIV DON DON, NIV

Molto et al. 2000 Scott et al. 1993 Niessen et al. 1993 Shim et al. 1997

z

The most common toxin is listed first followed by the less common one in decreasing order: DON=deoxynivalenol; NIV=nivalenol.

toxins. Likewise, Lee et al. (1995) found one of the 59 Korean barley samples they analyzed contained at least five toxins. Both DON and NIV have been detected in beer samples (Table 5.5) and both DON and ZEN have been found in barley-based infant cereals (Lombaert et al. 2003). The presence of DON, NIV, and ZEN in beer and infant cereals suggests that at least part of these toxins can survive the brewing or food preparation processes. The maximum levels of DON, 3-ADON, NIV, ZEN, and fusaric acid as reported in the literature are extremely high, while those of the other Fusarium toxins are relatively low (Table 5.6). The DON level in barley samples varies from country to country and from year to year. In Eastern Canada, approximately 5% of the samples surveyed from 1991 to 1998 contained more than 5 mg/kg with a maximum of 9.1 mg/kg (Campbell et al. 2000). In Minnesota, 80% of the barley samples collected during the epidemics of 1993 and 1994 contained more than 4 mg DON/kg with a maximum of 39.7 mg/kg, but none of the 100 Minnesota samples were contaminated with NIV (Jones and Mirocha 1999). Severe DON contamination also occurred in Manitoba in 1993 and 1994. The mean and maximum DON content of the 117 Manitoba samples were 2.6 and 15.8 mg/kg, respectively (Abramson et al. 1998). B. Toxicity Fusarium metabolites can be harmful to animal and human health. The 50% lethal doses (mg/kg) of selected trichothecenes to mouse are as follows: 4-ANIV 3.4, NIV 4.1, T-2 5.2, HT-2 9.2, 4,15-DANIV 9.6, DAS 23.0, 3-ADON 49.0, and DON 70.0 (Ueno 1983). DON can cause emesis and feed refusal in swine (Forsyth et al. 1977). Swine can ingest only up to 2 mg DON/kg of feed without lowering their feed intake and weight gain (Trenholm et al. 1984; House et al. 2002), while both poultry and dairy cattle can tolerate at least 5 mg DON/kg in feed (Trenholm et al. 1984). Similarly, NIV can slow feed intake and weight gain in swine (Williams and Blaney 1994) and broiler chicks (Hedman et al. 1995).

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Table 5.6. Maximum levels of Fusarium toxins in barley seed samples and their origin as reported in the literature. Level (mg/kg)

Origin

Trichothecene B DON 3-ADON 15-ADON 3,15-DADON NIV 4-ANIV 4,15-DANIV

70.50 18.70 0.52 0.40 26.00 2.47 0.03

Japan Japan Japan Canada Japan Japan Korea

Yoshizawa and Jin 1995 Yoshizawa and Jin 1995 Yoshizawa and Jin 1995 Abramson et al. 1998 Yoshizawa and Jin 1995 Yoshizawa and Jin 1995 Kim et al. 1993

Trichothecene A DAS HT-2 MAS STO T-2 T-2 tetraol

0.22 0.44 0.03 0.08 2.40 0.04

Canada Sweden Lithuania Poland Poland Poland

Stratton et al. 1993 Langseth and Rundberget 1999 Keblys et al. 2000 Perkowski et al. 2003b Perkowski et al. 1997 Perkowski et al. 2003a

Toxinz

Reference

Fusaric acid

13.25

Canada

Smith and Sousadias 1993

ZEN

15.30

Japan

Yoshizawa and Jin 1995

FB1

1.10

China

Haubruge et al. 2003

z

3-ADON = 3-acetyldeoxynivalenol; 15-ADON = 15-acetyldeoxynivalenol; 3,15-DADON = 3,15-diacetyldeoxynivalenol; 4-ANIV = 4-acetylnivalenol (fusarenon X); 4,15-DANIV = 4,15-diacetylnivalenol; DAS = diacetoxyscirpenol; DON = deoxynivalenol; FB1 = fumonisin B1; MAS = 15-monoacetyoxyscirpenol; NIV = nivalenol; ZEN = zearalenone; STO = scirpentriol.

Although NIV reduces a feed intake in laying hens, diets containing up to 5 mg NIV/kg have no effect on body weight, egg production, and egg quality (Garaleviciene et al. 2001). NIV can cause diarrhea in swine (Williams and Blaney 1994). T-2 is more toxic than DON, and it reduces the feed intake of swine at 0.5 mg/kg or even lower (Rafai et al. 1995). The estrogen ZEN can cause infertility, small litter size, and other reproductive disorders in swine (Chang et al. 1979). FB1 can cause leukoencephalomalacia in horses (Kellerman et al. 1990), pulmonary edema and hydro thorax in swine (Harrison et al. 1990), and may be associated with esophageal cancer in humans (Sydenham et al. 1990; Yoshizawa et al. 1994). Fusaric acid affects the nervous, cardiovascular, and immune systems in animals (Wang and Ng 1999). Because of the concerns over the harmful effects of mycotoxins on animal and human health, 77 countries have established maximum tolerance levels for mycotoxins in foodstuffs, dairy products, and animal feed (FAO 1997). In Canada, the tolerance levels in feed recommended by the

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Canadian Food Inspection Agency are as follows: DON < 1 mg/kg for swine, young calves, and lactating dairy animals, DON < 5 mg/kg for cattle and poultry, ZEN < 1 mg/kg for swine, ZEN < 10 mg/kg for cattle, T2 < 1 mg/kg for swine and poultry, HT-2 < 0.025 mg/kg for dairy animals, HT-2 < 0.1 mg/kg for cattle and poultry, DAS < 1 mg/kg for poultry, and DAS < 2 mg/kg for swine (Charmley and Trenholm 2000). Table 5.6 indicates that the reported levels of DON, 3-ADON, NIV, and ZEN can exceed the maximum tolerance levels by many folds; however, reported levels of other Fusarium toxins were low and less than the maximum tolerance levels (Table 5.6). Although the levels of these minor toxins are relatively low, they could have an additive or synergistic effect on the toxicity of the sample if this is contaminated with two or more Fusarium toxins. Quite often, barley samples are not only contaminated by Fusarium toxins but also by ochratoxin A, which is a metabolite of Aspergillus ochraceus and Penicillium verrucosum (Campbell et al. 2000; Rafai et al. 2000; Garaleviciene et al. 2001). These incidences of multiple contamination can increase toxicity of mycotoxins on animal and human health (Rotter et al. 1989; Garaleviciene et al. 2001). If the level of mycotoxin(s) is low to intermediate, barley grain may still be used as feed for less-sensitive animals such as cattle and poultry. However, Fusarium toxins can remain in animal tissues, milk, and eggs (Galtier 1998). As such, there is a potential for the carryover of toxins into animal-derived food products. Contaminated barley can be diluted by blending this with toxin-free grains. Grain can also be detoxified by physical, chemical, and biological methods (Scott 1998). Eliminating small kernels (<2.5 mm) from contaminated samples by sieving can reduce the level of DON by up to 80% (Perkowski 1998). Pearling can reduce DON, 15-ADON, NIV, and ZEN content in a contaminated barley, but the resulting bran fraction then may contain a much higher concentration of the toxins than the original (Lee et al. 1992; Clear et al. 1997; House et al. 2003). All these remedial measures, however, are costly and may be ineffective. Therefore, the best measure for reducing toxin accumulation is to prevent FHB development in the field.

IV. LOSSES IN YIELD AND QUALITY In addition to toxin accumulation, infection of barley by F. graminearum, F. culmorum, or F. sporotrichioides reduces the number of grains per spike, seed weight, and ultimately grain yield (Perkowski et al. 1995). In an extreme case, yield reduction approaching 40% can occur if the DON concentration reaches 2.3 to 5.3 mg/kg or if the T-2 concentration reaches 6.0 mg/kg (Perkowski et al. 1995, 1996). Normally, yield

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reduction, if any, may be somewhat lower. For example, despite a severe infection of FHB (with an average DON content of 4.7 mg/kg) in the Maritime Six-Row Barley Registration-Recommendation Test at Charlottetown (Prince Edward Island) in 2003, its average yield reached 3.65 t/ha, which was still higher than that (3.45 t/ha) of the same test at the same location in 2002 under light infection conditions (with an average DON content of 0.7 mg/kg) (T. M. Choo, unpubl.). Although the two tests are not directly comparable because of a slight difference in test entries and a difference in the two growing seasons, it does provide an indication that yield reduction, if any, due to FHB infection may not be very severe. Both test weight and seed weight were found to be negatively correlated with DON content (Jones and Mirocha 1999). Likewise, seed weight was negatively correlated with both FHB incidence and DON content in both two-row and six-row doubled-haploid lines (Vigier et al. 2004). Therefore, barley producers could face two major impacts from Fusarium outbreaks: (1) they not only may have to sell their crops at a discounted price because of toxin contamination, and (2) they could also have less grain to sell because of yield reductions. This is what happened to barley producers in Fukuoka Prefecture of Japan in 1998 because of poor emergence and an FHB outbreak (Yamamoto et al. 1999). The yield of two-row barley was only 1.73 t/ha in comparison to 3.34 t/ha during a normal year and only 29% of the harvested grain was of Grade 1 Class. Fusarium infection has negative impacts on grain and malt quality. Barley grain can be infected by Fusarium species at various growth stages from heading through maturity (Prom et al. 1999). Infected, mature kernels may not show any characteristic visual symptoms. Pinkish-red mycelium, orange sporodochia, or black perithecia may be present on a few Fusarium damaged kernels. Fusarium and other fungi, however, also can cause kernel discoloration in barley. Therefore, a chemical test is required to confirm that kernels are contaminated with mycotoxins. Fusarium damaged kernels have lower percent plumpness and a lower germination rate than healthy kernels. They produce increased levels of wort soluble nitrogen, free amino nitrogen, and wort color during malting (Schwarz et al. 2001). Steeping may not completely remove the mycelia and conidia because some are present under the husk of seeds. Because of this, fungal growth and accompanying DON accumulation can occur later on during germination (Schwarz et al. 1995b). Steeping can reduce the DON content by at least 89%, but subsequent seed germination allows re-accumulation of DON from new fungal growth (Schwarz et al. 1995b). DON is heat stable and is not destroyed by kilning or brewing. Consequently, DON can survive the malting process and remains in beer. The higher the DON content in a bottle of beer, the more likely the bottle will gush (Schwarz et al. 1996). The effects of F.

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graminearum on both grain and malt quality are more pronounced than those of F. poae because the former produce higher amounts of Fusarium toxins than the latter (Schwarz et al. 2001). DAS lowers α-amylase activity and α-amino nitrogen levels more than DON during malting (Schapira et al. 1989).

V. SOURCES OF GENETIC RESISTANCE A. Cultivated Barley Japanese and Chinese researchers have searched for sources of genetic resistance to FHB since the 1960s (Table 5.7). Thousands of accessions have been screened for FHB resistance; however, none of the accessions were immune to F. graminearum. Those with relatively low FHB symptoms in the screening test were defined as resistant accessions. The frequency of FHB-resistant accessions was very low (less than 0.1%). The frequency of accessions classified as resistant in a more recent study (Zhang et al. 2001) was high, because the 191 accessions used in the study were the elite of the 16,240 accessions stored in the National Genebank of China (Table 5.7). Seemingly, many of the resistant accessions identified have not been used extensively in barley breeding programs outside the Far East; the exceptions are ‘Mimai 114’, ‘Gobernadora’, and ‘Clho 4196’. ‘Mimai 114’ was developed by the Zhejiang Academy of Agricultural Sciences in 1973 and was widely grown in Zhejiang, Jiangsu, and Shanghai in the early 1980s. It is a hulless two-row barley derived from a (‘Xiaoshan Ciwangerleng’ × ‘Xiaoshan Lixiahuang’) × ‘Aibaiyang’ cross (Zhao 1989a). Its FHB resistance (Zhao 1989b; Zhou et al. 1991; Wan et Table 5.7. Total number of barley accessions screened for Fusarium head blight (FHB) resistance and number of FHBresistant accessions identified by Japanese and Chinese scientists.

Reference

Total

FHB-resistant accessions

Heta and Hiura 1963 Wang et al. 1987 Takeda and Heta 1989 Zhou et al. 1991 Chen et al. 1993 Zhao et al. 1998 Zhang et al. 2001

1,515 2,486 4,881 7,944 5,058 3,271 191

23 2 23 27 14 9 59

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al. 1997) most likely is derived from the two-row covered cultivars ‘Xiaoshan Ciwangerleng’ (Zhou et al. 1991) and ‘Aibaiyang’ (Wan et al. 1997) that are resistant to F. graminearum. The other parent, ‘Xiaoshan Lixiahuang’ (six-row hulless), was susceptible to FHB (Zhao 1989b). ‘Mimai 114’, however, is susceptible to FHB infection in Eastern Canada (T. M. Choo, unpubl. data). ‘Mimai 114’ was used by Choo et al. (2001) to develop the hulless two-row barley ‘AC Alberte’ (pedigree: (‘Mimai 114’ × ‘Rodeo’) × ‘Rodeo’) for the Maritime Region of Canada. ‘AC Alberte’ is moderately resistant to F. graminearum and DON accumulation (Choo et al. 2004a) and has high test weight, high seed weight, and excellent threshability (Choo et al. 2001). ‘Shenmai No. 1’ (aliases ‘Gobernadora’ and ‘Zhenmai-1’) was developed jointly by ICARDA/CIMMYT in Mexico and the Shanghai Academy of Agricultural Sciences in 1988 and it was widely grown in the Shanghai area in the 1990s (Vivar 1987; Wang et al. 1996). ‘Shenmai No. 1’ is a two-row covered barley with the following pedigree: ((‘OC640’ × ‘Mari’) × ‘Pioneer’) × ‘Maris Canon’ (Vivar 1987). This cultivar is reported to be resistant to the spread of F. graminearum (or Type II resistance) in China (Wang et al. 1996) and in Mexico (Zhu et al. 1999; Gilchrist 2000). ‘Shenmai No. 1’, however, was susceptible to FHB infection in the midwest United States (Prom et al. 1997). The source of FHB resistance is unknown; however, it might have come from the three European cultivars: ‘Pioneer’, ‘Mari’, and ‘Maris Canon’ (Vivar 1987). ‘Shenmai No. 1’ is also tolerant to barley yellow mosaic virus. ‘Shenmai No. 1’ was used as a parent to develop FHB-resistant ‘Shenmai No. 3’ (pedigree: (‘Shenmai No. 1’ × ‘Humai No. 10’) × ‘Shenmai No. 1’) for the Shanghai area (Chen 1996). The two-row cultivar ‘Clho 4196’ is a local strain from Henan, China (local name: ‘Honan wang ta mai’). It is resistant to F. graminearum (Heta and Hiura 1963; Takeda and Heta 1989; Prom et al. 1997; Urrea et al. 2002b; Buerstmayr et al. 2004) and is being used extensively as a parent by the six-row barley breeding program at North Dakota State University (Urrea et al. 2002b). A six-row germplasm line (‘6NDRFG-1’) with partial resistance to F. graminearum was developed from a ‘Foster’ × ‘Clho 4196’ cross by Urrea et al. (2002a). In addition to ‘AC Alberte’, ‘Island’, another two-row commercial cultivar for Eastern Canada, shows partial resistance to F. graminearum. ‘Island’ was derived from the ‘TBR635-25’ × ‘Symko’ cross (Choo et al. 2003). Its level of resistance to FHB and DON accumulation is as good as ‘Chevron’ (Choo et al. 2004a). ‘Island’ also compares favorably with ‘Clho 4196’ for DON accumulation. When both were tested in Ottawa (Ontario) in 2002 and 2003, the mean DON content for ‘Island’ and

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‘Clho 4196’ , based on seven replicates, were 15.3 and 16.6 mg/kg, respectively (T. M. Choo, unpubl. data). In Eastern Canada, ‘Island’ heads about 7 days earlier and is about 10 cm shorter than ‘Clho 4196’. Therefore, ‘Island’ could be a useful source of FHB resistance for tworow barley breeding programs. Its resistance traces back to introductions from Europe. To date, the six-row spring cultivar with the best resistance to FHB is ‘Chevron’ (‘Clho 1111’). ‘Chevron’ was originally selected in California from an unimproved domestic cultivar obtained by the U.S. Department of Agriculture from Switzerland in 1914. The same Swiss cultivar also gave rise to ‘Peatland’ (‘Clho 5267’) in a selection program in Minnesota. ‘Peatland’ performed well on peat land and was grown commercially in northern Alberta and Quebec from 1933 to 1955 (Hamilton et al. 1955). ‘Chevron’ and ‘Peatland’ were found to be resistant to FHB by Shands (1939) and Immer and Christensen (1943), respectively. In comparison with ‘Chevron’, ‘Peatland’ is about 5 cm shorter in height, earlier, and less resistant to FHB (Åberg and Wiebe 1946). Both cultivars have been used extensively as a source of resistance to multiple diseases for many sixrow barley breeding programs. Many cultivars have either one in the parentage, this includes the recently released ‘MNBrite’, which has an intermediate level of resistance to FHB (Rasmusson et al. 1999) Other FHB-resistant cultivars include ‘Svanhals’ (Heta and Hiura 1964; Takeda and Heta 1989; Prom et al. 1997; Gilchrist 2000; Buerstmayr et al. 2004), ‘Zhedar 1’ (Prom et al. 1997; Gilchrist 2000), ‘Zhedar 2’ (Gilchrist 2000), ‘Fredrickson’ (Takeda and Heta 1989; Prom et al. 1997; Buerstmayr et al. 2004), and ‘Shyri’ (Gilchrist 2000; Buerstmayr et al. 2004). ‘Svanhals’ (‘Clho 187’) was developed by the Svalof Plant Breeding Association in Sweden and was tested extensively across the United States and Canada from 1903 to 1915 (Harlan and Martini 1925). It may be a source of resistance in many European cultivars. Both ‘Svanhals’ and ‘Zhedar 2’ carry the vrs1.d (two-row spike with large sterile laterals) allele (Dahleen et al. 2003). B. Wild Species Resistance to FHB in wild species has also been the subject of a number of studies that aim at transferring FHB resistance from wild species to cultivated barley. Hordeum bogdanii, H. bulbosum, and H. chilense appeared to be resistant to F. graminearum, while H. parodii, H. procerum, and H. violaceum were moderately resistant; and H. depressum, H. gussoneanum, H. leporinum, H. marinum, and H. murinum were susceptible (Wan et al. 1997) . Rubiales et al. (1996) suggested that H. chilense could be a valu-

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able source of resistance to F. culmorum because its average DON content (23 mg/kg) was lower than X Tritordeum (27 mg/kg) and Triticum (43 mg/kg). Weng and Liu (1989), however, found that the H. bulbosum accession they tested was susceptible to F. graminearum. Both R. ciliaris (Agropyron ciliare) and Roegneria kamoji (Agropyron tsukushiense) were reported resistant to F. graminearum (Weng and Liu 1989). Li et al. (1999a) obtained 73 F3 and 140 BC1F4 lines from intergeneric hybrids between R. ciliaris and H. vulgare (cv. ‘Arupo’). Of the 60 of these tested, seven of the 60 lines tested were resistant to F. graminearum. Zhou and Wan (1994) produced an F1 hybrid between R. kamoji and H. vulgare with an intermediate reaction to F. graminearum. These wild species, however, are unlikely to help solve our immediate need for FHB-resistant cultivars because they all have poor agronomic and quality traits.

VI. TRAITS ASSOCIATED WITH FHB RESISTANCE Many morphological, physiological, and chemical traits are reported to be associated with low FHB infection and low mycotoxin in barley. A. Two-Row Spike Barley cultivars can be classified into two types based on spike morphology: six-row and two-row. In six-row barley, the three spikelets at each node are fertile. The fertile lateral spikelets can overlap in some cultivars, particularly at the upper part of the spike. In two-row barley, however, only the central spikelets are fertile and the lateral florets are reduced or absent. The two-row vs. six-row phenotypes are controlled by alleles at the vrs1 locus on chromosome 2HL. It is widely recognized that two-row accessions are often more resistant to FHB (Heta and Hiura 1963; Takeda and Heta 1989; Takeda 1990; Zhou et al. 1991; Chen et al. 1993; Chelkowski et al. 2000; Urrea et al. 2002b; Mesfin et al. 2003; Choo et al. 2004b; Buerstmayr et al. 2004). The following factors may contribute to the reduced level of FHB infection observed in two-row barley. First, two-row spikes have only two rows of fertile spikelets that do not have overlap, and thus they likely dry faster and are less favorable for Fusarium infection and spread. Second, the larger amount of starch in plumper two-row kernels (Kong et al. 1995) could dilute the DON concentration because DON is predominantly concentrated in the hull portion of the grain (Clear et al. 1997; House et al. 2003). Third, the two-row type is taller and more resistant to lodging (Choo et al. 2004b).

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B. Lax Spike Lax spikes may be associated with FHB resistance because this architecture retains less moisture and probably takes longer for the fungi to spread than dense spikes (Zhu et al. 1999). But Urrea et al. (2002b) found that lax spike was associated with FHB susceptibility and Ma et al. (2000) reported no association between lax spike and FHB reaction. C. Nodding Spike For some barley cultivars, the upper part of their stems may bend down in the field at maturity and thus their spikes appear to be nodding. The nodding spike was associated with FHB resistance in one study (Urrea et al. 2002b) but not in another (Ma et al. 2000). Water could run off easier in the nodding spike than in the erect spike, providing less favorable conditions for Fusarium infection. D. Infertile Lateral Spikelets In two-row barley, the lateral spikelets of some plants become fertile and can set small kernels. Fertile lateral spikelets are rarely found in nature, but are common among the progeny derived from two-row × six-row crosses. The intermedium spike (or partially fertile lateral spikelets) is controlled by both alleles at the vrs1 locus and at the Int-c locus on chromosome 4H. Zhu et al. (1999) found that resistance to FHB and DON accumulation was associated with small lateral spikelets. In contrast, Mesfin et al. (2003) did not detect any correlation between FHB resistance and lateral floret size. The lateral florets are absent in the deficiens (Vrs1.t) type of two-row barley. Vivar et al. (1997) found that 27 of the 30 FHB-resistant lines from a ‘Gobernadora’ × ‘Atahualpa’ cross were of the deficiens type. Seemingly, the deficiens type is associated with FHB resistance. E. Hulless Kernels Barley cultivars can also be classified into two types based on hullcaryopsis adherence: covered and hulless. In hulless barley, the hull is easily separated from its caryopsis when the crop is mechanically harvested, because its pericarp epidermis fails to produce a cementing substance. Hullessness is controlled by the nud1 gene on chromosome 7HL. In a survey for mycotoxin, Park et al. (1992) found that the average DON content was lower in hulless cultivars compared to covered cultivars,

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but that the average NIV content was higher in hulless cultivars. In an inoculation test, Legzdina and Buerstmayr (2004) observed a lower content of DON, 3-ADON, and 15-ADON in hulless cultivars. Although the NIV content was higher in hulless barley, the difference between hulless and covered types was not statistically significant (Legzdina and Buerstmayr 2004). Dehulling barley grain during harvest can reduce the DON content by as much as 59% (Clear et al. 1997). Therefore, hulless barley could be used as a means of reducing DON content in barley. F. Black Lemma and Pericarp The lemma and pericarp of some barley cultivars are black in color because they contain melanin-like pigments. Black barley is grown in many developing countries or regions (Choo et al. 2005). Expression of black lemma and pericarp is controlled by the Blp1 gene on chromosome 1HL. Black barley appears to be more resistant to F. graminearum. In an FHB screening study, Zhou et al. (1991) found that 20% of the 151 black accessions tested were resistant, compared to only 5% of the 5,560 yellow accessions. A preliminary study has shown that black barley contains more lignin than yellow barley (Choo et al. 2005). Whether or not a high lignin content is associated with FHB resistance in black barley remains to be determined. G. Purple Lemma and Pericarp The lemma and pericarp of some barley cultivars are purple because they contain a considerable amount of anthocyanin, delphinidin, and cyanidin, plus a small amount of pelargonidin (Mullick et al. 1958; JendeStrid 1978). Expression of purple lemma and pericarp is controlled by the Pre2 gene on chromosome 2HL. Zhou et al. (1991) did not find any FHB-resistant accessions among the 419 purple barleys they studied. Choo et al. (2004b), on the other hand, found that purple barley was associated with a reduced level of FHB incidence. H. Long Glume Awn Long glume awn (Lga) was found to be associated with FHB resistance in a DH population derived from a ‘Léger’ × ‘Clho 9831’ cross (Choo et al. 2004b). The association most likely is due to genetic linkages between the Lga gene and an FHB resistance gene on chromosome 7HS. Further studies in other populations are needed to ascertain if this morphological marker can be used in marker-assisted selection for FHB resistance.

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I. Closed (Cleistogamous) Flowering Additionally, barley cultivars can be classified into two types based on their flowering mechanism: open (chasmogamous) or closed (cleistogamous). In the open flowering type, the lodicules swell at anthesis and push the palea and lemma apart. This allows anthers and stigmata to be extruded. In the closed flowering type, however, lodicules are small and anthers may be pushed only to the level of the palea tip by the filaments. Anthers can support massive growth of F. graminearum by making choline and betaine readily available to the fungi under high humidity conditions (Strange et al. 1978). Closed flowering may provide a mechanical barrier to the entry of fungal spores. It has been used to select for ‘pseudo-resistance’ to loose smut (Ustilago nuda) in barley (Pedersen 1960). Closed flowering is reported to be associated with FHB resistance (Heta and Hiura 1963; Zhai 1981;Weng 1994; Vivar et al. 1997). J. Heading Date The association between heading date and FHB resistance is inconsistent from test to test, possibly as a result of different environmental conditions and inoculation methods. Heading date was either negatively, positively, or not correlated with resistance to FHB and DON accumulation ( Heta and Hiura 1963; de la Pena et al. 1999; Ma et al. 2000; Urrea et al. 2002b; Mesfin et al. 2003; Choo et al. 2004b). The inconsistent results may be caused by the large number of loci that respond to photoperiod differences in barley. K. Plant Height and Lodging Resistance Many researchers have observed negative correlations between plant height and DON content in their experimental populations (de la Pena et al. 1999; Ma et al. 2000; Urrea et al. 2002b; Choo et al. 2004b). As pointed out by Ma et al. (2000), tall plants are further away from the Fusarium inoculum on the ground and further away from the soil moisture. In small plot nurseries, spikes of tall plants have little protection from wind and thus likely dry faster than those of short plants. Consequently, the micro-environment surrounding the spikes of tall plants are less favorable for Fusarium infection and spread. Lodged plants have more diseased kernels and accumulate more DON in their kernels (Choo et al. 2004b). Thus, the advantage of plant height is likely lost when lodging occurs.

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L. Phenolic Acids Ferulic and π-coumaric acids are the two most predominant phenolic acids in the outer layers (husk, pericarp, testa, and aleurone cells) of barley seed (Nordkvist et al. 1984). The influence of these phenolic acids on the level of FHB resistance has not been studied in barley. In wheat, several studies have indicated that the rate and extent of phenolic acid synthesis are associated with resistance to Fusarium infection (Bhaskara Reddy et al. 1999; McKeehen et al. 1999; Siranidou et al. 2002). Ferulic and π-coumaric acids can inhibit the growth of F. graminearum and F. culmorum in vitro (McKeehen et al. 1999). Resistant wheat synthesizes more ferulic acid or π-coumaric acid than susceptible wheat (McKeehen et al. 1999; Siranidou et al. 2002) and wheat kernels, when treated with chitosan, produce more ferulic acid and are infected less by F. graminearum than those left untreated (Bhaskara Reddy et al. 1999). Note that π-coumaric acid is a precursor of ferulic acid and flavonoids, while both ferulic and π-coumaric acids are precursors of lignin. M. Flavonoids As indicated earlier, purple barley was reported to be more resistant to FHB than yellow barley (Choo et al. 2004b). The reason why is not clear, but may relate to the high levels of flavonoids such as anthocyanin, delphinidin, cyanidin, and pelargonidin in purple barley. Studies in barley have suggested that flavonoids play a role in the level of FHB resistance. According to Riis et al. (1995), non-pigmented (i.e., lack of anthocyanins) lines are significantly more susceptible to Fusarium infection than pigmented lines. Barley that can accumulate small amounts of dihydroflavonol (dihydroquercetin) in the testa show extreme resistance to F. graminearum, F. culmorum, and F. poae (Skadhauge et al. 1997). The flavonoids 4′-methylflavone and 2′,6′-dichloroflavone were found to severely inhibit the growth of F. graminearum, F. culmorum, F. poae, F. avenaceum, and F. nivale on culture medium (Silva et al. 1998). N. Lignin Lignin or lignification may create some physical barriers to inhibit Fusarium growth. Wheat seedlings from seeds treated with chitosan contained more lignin and were less infected with F. graminearum than those from untreated seeds (Bhaskara Reddy et al. 1999). The FHB-resistant wheat cultivar Frontana showed a higher level of lignin accumulation in host

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cell walls at the infection sites than the FHB-susceptible cultivar Agent (Siranidou et al. 2002). Black barley is resistant to biotic and abiotic stresses (Ma 2000) and it may contain more lignin than yellow barley (Choo et al. 2005). Further studies on the lignin content and FHB resistance in black barley could shed light on the role of lignin as one FHB resistance factor. On the other hand, some morphological traits have not been associated with FHB resistance. These traits include awn barbing (Urrea et al. 2002b; Choo et al. 2004b) and long rachilla hairs (Choo et al. 2004b). All of the above findings suggest that the ideotype of barley for regions with a long history of FHB infection is two-row, lax and nodding spike, hulless, purple or black, early maturity, medium stature, lodging resistant, and polyphenol-rich cultivars. Early maturity cultivars could avoid Fusarium infection and have a shorter period of DON accumulation than late cultivars. Tall cultivars could be prone to lodging, while spikes of dwarf cultivars would be close to a potential source of Fusarium inoculum at the soil surface and close to soil moisture. Polyphenol-rich cultivars might not only result in reduced FHB infection, but they may also contain antioxidants that contribute to human health in barley used for food (Bu et al. 2002).

VII. BREEDING STRATEGIES A. Heritability Resistance to FHB is quantitatively inherited in barley (Takeda 1990). Although additive gene action is predominant, dominance gene action may also be present (Takeda and Wu 1996). The estimates of heritability for FHB severity range from low to moderately high depending on genetic backgrounds and test environments (Table 5.8). Note that the heritabilities on a line basis were relatively high in the study of Capettini et al. (2003), because the F5:7 lines were evaluated in 3 to 5 tests each with 2 replicates. Estimates of heritability for resistance to DON accumulation have been reported in only three studies and these estimates were either low or moderate (Table 5.8). The heritability estimates for both FHB and DON from a ‘Leger’ × ‘Clho 9831’ cross varied from year to year at Ottawa (Table 5.8), and they were similar to the one for grain yield at the same location (h2 = 0.51) (Jui et al. 1997). The results suggest that the effects of environment and genotype × environment interactions are important to the expression of resistance to FHB and DON accumulation.

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Table 5.8. Heritability estimates for resistance to Fusarium head blight (FHB) and deoxynivalenol (DON) accumulation in barley.

Cross

FHB

DON

CI3725 × France Naked 1 CI3725 × Gun-eki

0.02/0.63

—

0.27/0.14

—

CI3725 × Shirohadaka 1

0.32/0.53

—

CI3725 × Hozoroi

0.25/0.62

—

Russia 6 × HES 4

0.21/0.74

—

8 × 8 diallel

0.45

—

6 × 6 diallel

0.42

—

Gobernadora × CMB643

0.50–0.81

—

Chevron × Stander

0.31

0.25

Foster × CIho4196 Chevron × M69

0.65 0.48

0.46 —

Gobernadora × Excel

0.68

—

GD2-27 × M79

0.76

—

(Zhedar 1 × Stander) × Foster Leger × CI9831

0.48

—

0.39, 0.71y

0.64, 0.37y

Estimation method Selection/ correlationz Selection/ correlation Selection/ correlation Selection/ correlation Selection/ correlation Variance components Variance components Variance components Variance components Correlation Variance components Variance components Variance components Variance components Variance components

Reference Takeda 1990 Takeda 1990 Takeda 1990 Takeda 1990 Takeda 1990 Takeda and Wu 1996 Takeda and Wu 1996 Zhu et al. 1999 Ma et al. 2000 Urrea et al. 2002b Capettini et al. 2002 Capettini et al. 2003 Capettini et al. 2003 Capettini et al. 2003 Choo, T. M., unpublished

z

Estimated by the selection response/parent-offspring correlation method. Heritability on a plot basis was estimated at Ottawa in 2001 and 2002, respectively. See Choo et al. (2004b) for materials and methods.

y

B. Crossing Schemes Most of the FHB-resistant accessions have poor agronomic traits, which include low yield, low test weight, low seed weight, susceptibility to lodging, late maturity, and/or poor malting quality. With so many undesirable alleles associated with FHB-resistant accessions, the probability of obtaining FHB-resistant lines with desirable agronomic performance and malting quality is extremely low from any susceptible × resistant

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crosses. One way to increase the frequency of desirable alleles in the segregating populations is to cross the F1 hybrids to the FHB-susceptible and agronomically superior parents (i.e., backcrosses or three-way crosses). The backcross and three-way cross also allow one extra round of recombination to break up linkages of genes. For example, in an attempt to transfer the hulless gene (nud1) from the very low yielding Chinese cultivar ‘Mimai 114’ to three Canadian cultivars “Rodeo’, ‘Albany’ and ‘AB79-17’, we crossed ‘Mimai 114’ with each of the three Canadian cultivars and also backcrossed the F1 to each of the three cultivars. A new cultivar, ‘AC Alberte’, was selected from a backcross (Choo et al. 2001) and none from the other five crosses. As indicated earlier, the FHB-resistant cultivar ‘Shenmai No. 3’ was also developed from a backcross (Chen 1996). There are ample examples of rapid transfer of disease-resistance gene(s) from one cultivar to another by the backcross method. The backcross method works best if the disease resistance is controlled by a single major gene. It should work well for FHB resistance, particularly where the FHB-resistant accessions carry many undesirable alleles of agronomic and quality traits. A single-cross hybrid may be used to produce new recombinants if both parents have some levels of FHB resistance and acceptable levels of agronomic performance. For example, ‘Island’ barley may be used to hybridize with ‘AC Alberte’ with a hope of accumulating FHB-resistant and high-yielding alleles in lines of both covered and hulless background. Selfing of the F1 hybrids derived from single crosses offers more opportunity for recombination than backcrossing because effective crossing over can take place in both male and female parents. Three-way crosses and double crosses may be used also but these crossing schemes will require an extra round of crossing. Their population size needs to be increased also to enhance the probability of obtaining the best recombinants from the segregating generations. C. Production of Homozygous Lines One way to rapidly achieve homozygosity is through recurrent backcrossing. After five backcrosses, 73% of the BC5F1 plants may be homozygous for the ten loci of the recurrent parent. Recurrent backcrossing can be carried out in the greenhouse by regular technical personnel and BC5F1 plants can be obtained within two years. Recurrent backcrossing is very useful to derive homozygous lines to meet the immediate needs in emergency situations. Furthermore, fully backcrossed lines do not require as extensive testing for yield or quality as those derived by selfing (Allard 1960). What we need to do is to screen

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them for resistance in FHB nurseries. Once resistant lines are identified, seeds of those fully backcrossed lines can be bulked, increased, and released for commercial production. For single crosses, one short-cut to rapidly produce homozygous lines is by the haploid or single seed descent method. Different methods can be used to produce doubledhaploid lines in barley (see Choo et al. 1985 for review). In Canada, at least 20 barley cultivars have been developed by the bulbosum method and anther culture (Choo 1999). Doubled-haploid lines are completely homozygous and their genetic make-up should remain the same for generations. Near homozygous lines can be obtained through single seed descent in the greenhouse in two years after the cross is made. Doubledhaploid lines derived by the bulbosum method and single-seed-descent lines have been shown to be a random sample of lines from the F1 hybrid of single crosses (Choo et al. 1982). D. Screening Methods The natural occurrence of FHB fluctuates from year to year. Even in an epidemic year, disease development varies from location to location. Therefore, for effective screening extra sources of inoculum and high humidity conditions must be provided to ensure good infection and disease development. Takeda et al. (1995) reported that there were little interactions for FHB severity between 12 Fusarium strains (5 F. graminearum, 3 F. acuminatum, 2 F. avenaceum, 1 F. culmorum, and 1 F. sporotrichioides) and 10 barley cultivars tested. Therefore, any aggressive strain can be used to screen for FHB resistance. Three inoculation techniques can be used to apply inoculum to barley plants: grain spawn, spray inoculation, and point inoculation. The grain spawn technique was developed in China in the 1950s (Chinese Academy of Agricultural Sciences-Jiangsu Academy, 1961) and has been used extensively in many barley breeding programs. Typically, wheatbarley mixtures are first soaked in water for one to two days to facilitate Fusarium colonization. Then, the grain mixture is sterilized in an autoclave for one hour at 120°C. Subsequently, the grain mixture is inoculated with one or more Fusarium isolates and incubated at 20°C in the dark for at least two weeks to allow mycelium to develop in the mixture. Finally, the colonized grain mixture, or grain spawn, is broadcast throughout the FHB nursery at a rate of from 7.5 to 50 g/m2 when barley plants are at the booting stage. The application of grain spawn inoculum can be repeated in a weekly interval. At 25–28°C, only seven days are required for perithecia to form on the colonized grains and for ascospores to be released from perithecia (Ye

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1980). Both ascospores and conidia appear most frequently and most abundantly near the soil surface. They gradually decrease in frequency and in density as the height increases (Chinese Academy of Agricultural Sciences-Jiangsu Academy, 1961). Perithecia formation and ascospore release require high humidity or rain. As such, the disease nursery ideally should be watered with a sprinkler or misting system on a regular basis to ensure high-humidity conditions. The forcibly released ascospores are carried by wind to the barley spikes to initiate infection. Disease symptoms appear as early as 5 days after heading, but DON can accumulate to detectable levels as early as 36 hours after infection (Evans et al. 2000). Disease symptoms can be rated in terms of FHB incidence and FHB severity: FHB incidence being the percentage of infected spikes and FHB severity the average percentage of infected kernels per spike. An FHB index can be expressed as follows: (FHB incidence × FHB severity)/100. DON content can be quantified by gas chromatography-mass spectrometry, enzymelinked immunosorbent assay, or other analytical methods (Schaafsma et al. 2003). A susceptible check cultivar should be included in the nursery to measure the level of disease development. Resistant check cultivars are also be included to compare and measure the relative level of FHB resistance of other cultivars and breeding lines in the test. Barley plots in the field and plants in the greenhouse can also be inoculated by spraying the spikes with an aqueous conidial suspension. For field screening, it is appropriate to apply a conidial suspension of 5 × 104 macroconidia/mL at a rate of 125 liters/ha at the heading stage (Choo et al. 2004a). This application can be repeated once or twice. For greenhouse screening, 4 mL of a 5 × 104 macroconidia/mL suspension can be applied to the entire spike at two weeks after heading and then the plants placed in a humidity chamber at 100% relative humidity for 24 hours (McCallum and Tekauz 2002). Infection can also be achieved by injecting 5 μL of a 5 × 104 macroconidia/mL suspension into a single spikelet with a syringe (Evans et al. 2000; McCallum and Tekauz 2002). The point inoculation technique is best suited to determine the level of resistance to FHB spread within a spike. McCallum and Tekauz (2002), however, concluded that point inoculation may not be particularly useful in screening barley for resistance because variation in FHB spread within the spike among barley cultivars is rather small. The reliability of these inoculation techniques for screening purposes, however, needs to be evaluated in barley. In view of the complexity of FHB, it is imperative that we determine whether the cultivar response to artificial inoculation correlates well with that under natural infection in the field. Heta and Hiura (1964), using the conidial suspension

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method in a growth chamber, found that the levels of FHB severity of 12 barley cultivars under artificial inoculation agreed with those they observed under field conditions. These 12 barley cultivars basically consisted of two groups of cultivars (susceptible and resistant) in terms of FHB resistance. Therefore, their results may or may not apply to routine screening tests because differences in FHB severity among breeding lines mostly likely are quite minor. de la Pena et al. (1999) reported that the FHB severities of 101 F4-derived lines from a single cross in the naturally infected test correlated with those in only one of the three artificially inoculated tests. In a preliminary study using the data from Charlottetown in the epidemic year of 2003, Martin (R. A. Martin, unpubl. data) found that the cultivars in his four artificial inoculation tests, in which the plots were sprayed with a conidial suspension of F. graminearum (see Choo et al. 2004a for details), did not correlate in DON content with those in naturally infected tests. The grain spawn technique may provide a better assessment of the FHB development under natural infection conditions than the spray inoculation technique. Whenever FHB is an epidemic, every effort should be made to collect data from the cultivar performance trials to develop a profile of actual reactions of the cultivars to FHB infection and DON accumulation under natural infection conditions. Once the profile is available, then we can use it for comparative studies to refine the artificial inoculation procedures for screening purposes. E. Selection Strategies Selection for FHB resistance can be carried out during the production of homozygous lines as well as after the homozygous lines are produced. The BC1F1 plants can be screened for FHB resistance in the greenhouse and only FHB-resistant plants are selected for further backcrossing. Likewise, the F2 plants from single crosses can be screened for FHB resistance in the greenhouse for FHB resistance and only FHB-resistant F2 plants are selected for doubled-haploid production. Early selection would enhance the frequency for FHB-resistant alleles in later generations, but it would also delay the production of homozygous lines by one generation. The F1-derived families from a series of single crosses can be evaluated for FHB resistance in replicated trials in multiple environments. As Takeda and Wu (1996) reported, the gene action for FHB resistance is predominantly additive. Therefore, selection for FHB resistance can be practiced in early generations. The levels of FHB resistance among the F2 bulks can be compared and those crosses with a high level of FHB resistance in their F2 bulks will be retained for further selfing,

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selection, and field evaluation. In so doing, available resources can be spent only on the selection and testing of the most promising crosses. Cooper (1990) developed a modified early generation testing procedure for yield selection in soybean. His procedure may be adopted for selection of FHB-resistant lines in barley. Testing the early generation material in the FHB nursery for several years offers two advantages. First, by the time the breeding materials reach a near homoyzgosity, they would have already been exposed to several years of selection cycles and thus their levels of FHB resistance are reasonably assured. Second, lines from the same family or cross can be considered as replications for assessing the merits of families or crosses. The effectiveness of early generation testing for FHB resistance needs to be evaluated in barley. If not tested for FHB resistance in the early generations, homozygous lines should be evaluated for resistance to FHB and DON accumulation in replicated trials in multiple site-years to obtain an accurate assessment of their resistance levels. Both kernel weight and test weight are negatively correlated with DON content in barley (Jones and Mirocha 1999; Vigier et al. 2004). Also, resistance to kernel discoloration is positively correlated with resistance to DON accumulation (de la Pena et al. 1999). This suggests that selecting for high kernel weight, high test weight, and kernel brightness may result in low DON content. F. Marker-Assisted Selection Resistance to FHB has a low heritability and thus requires multiple locations and/or years to separate the resistance levels among breeding lines. If molecular markers can be identified and are associated with QTLs for resistance to FHB and DON accumulation, selection could first be carried out for these molecular markers and only the selected lines or plants would subsequently be evaluated in the greenhouse or the field tests. In so doing, the selection efficiency for FHB resistance would be increased. Attempts to identify QTLs for FHB resistance have been published for five crosses: ‘Gobernadora’ × ‘CMB643’ (Zhu et al. 1999), ‘Chevron’ × ‘M69’ (de la Pena et al. 1999), ‘Chevron’ × ‘Stander’ (Ma et al. 2000), ‘Fredrickson’ × ‘Stander’ (Mesfin et al. 2003), and (‘Zhedar 2’ × ‘ND9712’) × ‘Foster’ (Dahleen et al. 2003). Of these parents, ‘Gobernadora’, ‘CMB643’, ‘Fredrickson’, and ‘Zhedar 2’ are two-row barley, while the others are six-row barley. QTLs for resistance to FHB and DON accumulation have been found on all seven barley chromosomes. Chromosome 1H carried minor QTLs for low FHB severity or DON accumulation in all five of the above

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crosses. Only one of the QTLs on chromosome 1H, flanked by HVM20 and Bmac0090, was associated with a QTL for heading date (Mesfin et al. 2003). Major QTLs for FHB severity or DON accumulation were found on chromosome 2H in all five crosses, and they were associated with the vrs1 locus, QTLs for heading date, and plant height. Schmierer et al. (2003) designated the three QTLs on chromosome 2H in the ‘Chevron’ × ‘M69’ cross as FHBqtl1, FHBqtl2, and FHBqtl3, and the two QTLs in the ‘Chevron’ × ‘Stander’ cross as FHBqtl3 and FHBqtl4. They suspected that the one found in the ‘Gobernadora’ × ‘CMB643’ cross was FHBqtl3 (Schmierer et al. 2003). In addition to the those near the vrs1 locus, other QTLs for FHB resistance also were detected on chromosome 2H in the two-row × six-row crosses (Mesfin et al. 2003; Dahleen et al. 2003). In view of the importance of chromosome 2H in FHB resistance, Schmierer et al. (2003) are in the process of developing a saturation map for this chromosome to dissect its QTLs for FHB resistance. Chromosome 3H carried detectable QTLs for FHB severity or DON accumulation in four of the five crosses, with some of the QTLs associated with those for plant height and heading date. Chromosome 4H carried QTLs for FHB severity or DON accumulation in two of the five crosses. Chromosome 5H carried minor QTLs for FHB severity and DON accumulation in all five crosses and minor QTLs for heading date or plant height in four crosses. Chromosome 6H carried QTLs for FHB or DON in four crosses. Only one of such QTLs on chromosome 6H was associated with that for heading date (Dahleen et al. 2003). Lastly, chromosome 7H carried minor QTLs for FHB severity or DON accumulation in four of the five crosses. Some of these QTLs were also associated with those for heading date or plant height. These studies indicate that resistance to FHB and DON accumulation in barley is governed by multiple genes. Many of the QTLs for FHB or DON are associated with those for heading date or plant height. The QTLs, except those on chromosome 2H, are detected only in some environments and in some crosses. Consequently, the usefulness of these QTLs for marker-assisted selection in barley is very limited. This is in contrast to wheat, for which chromosome 3BS carries a QTL with a major effect on FHB spread (Kolb et al. 2001). The associations between morphological traits and FHB resistance could be caused by linkages on chromosome 2H because several critical loci are located in the centromeric region of 2H. These critical loci include vrs1 for spike type, Pre2 for purple lemma and pericarp, Eam6 for early maturity, hcm1 for plant height, lin1 for spike length (Franckowiak 2001), and cly1 and cly2 for cleistogamy (Turuspekov et al. 2004).

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G. Active Resistance Natural disease resistance mechanisms occurring in higher plants can be classified as either active or passive resistance mechanisms (Jones and Clifford 1983). When resistant cultivars are attacked by fungus, their defense response genes encode pathogenesis-related proteins or phytoalexins to inhibit fungal growth, or encode enzymes to lignify cell walls, or activate the hypersensitive response of cell death around the invading haustorium. These resistance reactions are called active resistance. In contrast, passive resistance mechanisms are simply structural, morphological, or chemical barriers that act against the infection process or the subsequent spread of the pathogen. According to Mesterházy (1995), there are five active resistance mechanisms in FHB resistance: (1) resistance against initial infection, (2) resistance to spread within the spike, (3) resistance to kernel infection, (4) tolerance, and (5) resistance to mycotoxin accumulation. The resistance mechanism for each of these components could be different. Vivar et al. (1997) found that ‘Shenmai No. 1’, ‘Shyri’, and ‘Atahualpa’ are resistant to both initial infection and spread. Zhu et al. (1999) discovered a major QTL for resistance to spread, which is coincided with one for resistance to DON accumulation, on chromosome 2 in the ‘Gobernadora’ × ‘CBB643’ cross. Among these five resistance components, resistance against initial infection is most desirable because a barley crop, once contaminated with mycotoxins, may still be rejected by maltsters even though it has high yield. Pyramiding the resistance alleles for these components into one cultivar should help minimize the production risk.

H. Passive Resistance Fusarium head blight has drastically changed the rural landscape in Zhejiang and Jiangsu provinces of China in the early seventies (Gao and Fen 1991; Shen et al. 1991; Fu et al. 1993). Prior to 1970, six-row barley cultivars are predominant in the two provinces. The FHB epidemic in 1973 resulted in severe infection, poor yield, and poor quality of six-row cultivars. The two-row cultivars, on the other hand, were not as severely infected by the FHB epidemic. Since 1974, six-row cultivars have gradually been replaced by two-row cultivars in the two provinces. For example, 97% of barley acreage in Zhejiang in 1979 were sown to the two-row cultivar ‘Zaoshu No. 3’ (original name: ‘Kantou Nijou 3’), and most of commonly grown cultivars in Zhejiang in 1986 were the two-row type (Gao and Fen 1991). This drastic change from six-row to two-row cultivars in southern China clearly demonstrates that a passive resis-

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tance mechanism has been used successfully as a means of reducing FHB infection. Commercial two-row cultivars, in general, are weak in straw strength in comparison with commercial six-row cultivars. Lodging increases the chance of FHB infection and it also may reduce yield and lower quality. Therefore, breeding for resistance to lodging should help reduce FHB infection. Efforts have been made to shorten the height of ‘Zaoshu No. 3’ and to increase its lodging resistance by radiation and hybridization (Fu et al. 1993). Recent barley cultivars in southern China are shorter with stiff straw and are more resistant to lodging than ‘Zaoshu No. 3’ (Ding et al. 1993; Chen 2004). The level of FHB resistance of these cultivars is as good as that of ‘Zaoshu No. 3’, if not better. The rapid adoption of ‘Zaoshu No. 3’ in southern China was partly due to its medium lax spike and closed flowering (Zhai 1981). Chinese scientists found that closed flowering occurs only in two-row cultivars but not in six-row cultivars, and that six-row plants derived two-row × sixrow crosses were all open flowering type. Therefore, they hypothesized that closed flowering is closely linked with the Vrs1 locus (Zhai 1981). Very recently, Japanese scientists mapped the two cleistogamy loci on chromosome 2HL (Turuspekov et al. 2004). Zhai (1981) recommended that development of closed flowering, two-row barley cultivars should constitute one of the breeding goals for FHB resistance. As indicated earlier, closed flowering barley may provide control of loose smut as well (Pederson 1960). Early maturity may be used as a passive resistance mechanism for barley to escape primary and/or secondary infection of F. graminearum. In the Maritime Region of Canada, winter wheat was less contaminated with DON than barley and spring wheat during the FHB-epidemic years of 2003 and 2004 (R. A. Martin, unpubl. data). Therefore, breeding for early maturity may help reduce the risk of severe infection of FHB in most years. Use of hulless barley may also be a way to reduce DON contamination because dehulling at harvest can reduce concentrations of F. graminearum and DON by 95% and 59%, respectively (Clear et al. 1997). Hulless barley has been used in many countries as human food and livestock feed. It may be useful for whisky production also, because it produces higher alcohol yield than covered barley (Brosnan et al. 2004). Black and purple barley may provide some chemical barriers against the invasion of Fusarium species. Black and purple barley, however, is unlikely to be used as malting barley because polyphenols can lead to the formation of beer haze. In general, barley traits associated with passive resistance mechanisms are simply inherited. As such, genetic manipulation of these barley traits should be relatively easy to

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do. Fully exploiting these passive resistance mechanisms may provide another approach to develop FHB-resistant cultivars. I. Off-Season Nurseries In southern China, where FHB is one of the major diseases, barley is grown as a winter crop. Several barley researchers in North America are using locations in southern China for off-season nurseries (Steffenson 1998; de la Pena et al. 1999; Zhu et al. 1999; Ma et al. 2000). Generally, barley is seeded in southern China in November and FHB severity is rated the following late April to early May. FHB-resistant lines identified in the off-season nursery can thus be seeded for re-evaluation in North America in May. By so doing, one can test two generations a year and hasten the evaluation and selection process for FHB resistance. The effectiveness of this shuttle breeding for FHB resistance is dependent on a similar response of barley lines to FHB infection between the environments of North America and southern China. Choo et al. (2004a) found that three cultivars (‘AC Alberte’, ‘Island’, and ‘Chevron’) were consistently low in FHB infection when grown in Ottawa, Charlottetown, and Hangzhou (Zhejiang) despite differences in Fusarium isolates, inoculation methods, and growing conditions at the three locations. Therefore, it appears that shuttle breeding can be used for FHB resistance. Currently, North American barley researchers use Hangzhou and Shanghai as sites for their off-season nurseries in China. However, FHB severity must be rated at these locations in late April, while seeding in North America normally begins in late April or early May. This allows only for a very short time period for seed preparation, transport, and planting. An off-season nursery in the southernmost provinces of China would allow more time, particularly if seeds need to be shipped to North America for re-planting; for example, at Putian (Fujian) where ‘Zaoshu No. 3’ and ‘Dulihuang’ can mature 34 to 37 days earlier than at Hangzhou (Lu et al. 1991). However, shipment of FHB-contaminated grain between North America and China should be avoided to minimize the risk of introduction of foreign strains of F. graminearum.

VIII. MUTATION AND IN VITRO SELECTION Mutation can be used as a breeding method for FHB resistance because new defense response genes or modifications of genes encoding different enzymes may be induced during the mutation process. Skadhauge et al. (1997) discovered a mutant that is more resistant to FHB infection than any of the wild types tested. Attempts to use in vitro selection to

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obtain novel genes for FHB resistance have been made in barley. Chawla and Wenzel (1987) obtained eight somaclonal lines from calli that were cultured for 4 selection cycles on medium with 0.8 mM fusaric acid. Unfortunately, none of these eight lines were resistant to fusaric acid (Wenzel and Foroughi-Wehr 1990). By contrast, results from in vitro selection studies in wheat were more positive (Zhang et al. 1991; Lu et al. 1998). Two FHB-resistant somaclonal lines ‘Shengkang No. 1’ and ‘Shengxuan No. 3’ have been released as commercial wheat cultivars in China (Lu et al. 2001; Lu et al. 2003). It is quite possible that FHBresistant mutants can be induced through somaclonal variation. In barley, a high-yielding somaclonal line has been released as ‘AC Malone’ (Choo et al. 2000), which is on the cultivar recommended list for Quebec province.

IX. GENETIC TRANSFORMATION Another strategy that can be used to increase the level of FHB resistance is to introduce potentially FHB-resistant genes from other plant or microbial species into barley by genetic transformation. Note that direct gene transfer can be done on barley. For example, Jensen et al. (1996) were able to transfer a Bacillus (1,3-1,4)-β-glucanase gene into the barley cultivar ‘Himalaya’. Research on barley transformation for FHB resistance is being carried out in several laboratories (Dahleen et al. 2001), but this research is still in its infancy. Several types of candidate genes could be used for transformation of barley. A. Pathogenesis-related (PR) Protein Genes In response to attack by fungi, host plants can synthesize PR proteins as part of their defense mechanisms. To date, five groups of PR protein genes (PR-1 to PR-5) have been shown to have antifungal activities (Yun et al. 1997). PR-2 (β-(1-3)-glucanases) and PR-3 (chitinase) proteins inhibit fungal growth by degrading β-(1-3)-glucan and chitin in fungal cell walls, respectively. PR-5 (thaumatin-like) proteins exhibit sequence homologies to thaumatin, a protein from fruits of Thaumatococcus daniellii. PR-5 proteins may bind β-(1-3)-glucan of fungal cell walls and then alter the plasma membrane (Trudel et al. 1998). Barley β-(1-3)glucanases are encoded by two genes and chitinases by six to eight genes (Leah et al. 1991). In barley, these PR proteins are abundant in aleurone layers during late seed development (Leah et al. 1991; Skadsen et al. 2000). At their current levels in barley, these proteins do not provide full protection from Fusarium invasion. Therefore, enhanced

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expression of such PR protein expression could enhance FHB resistance in barley. In wheat, Chen et al. (1999) obtained a transgenic line (‘BT-14-18’) expressing a rice thaumatin-like protein gene. In greenhouse tests, they found a reduced number of infected spikelets per spike in the transgenic line in the T1, T2, and T3 generations compared with the control plants. Anand et al. (2003) obtained a transgenic line (32A) from ‘Sumai 3’ wheat co-expressing chitinase and β-(1-3)-glucanase genes. This line showed a delay in FHB spread in greenhouse tests. Unfortunately, neither of these two transgenic lines was resistant to FHB under field conditions, possibly because both are susceptible to initial infection (Anand et al. 2003). Experiments are under way to cross the two lines and further evaluate the resistance level of the progeny (Anand et al. 2003). B. Trichothecene Genes Trichothecene genes encode the enzymes that are required for the biosynthesis of trichothecenes. These enzymes play a role in Fusarium self-protection against trichothecenes through a metabolism to reduce toxicity or via transport by toxin efflux pumps. TRI101 in F. sporotrichioides encodes trichothecene 3-O-acetyltransferase, an enzyme essential for T-2 biosynthesis (McCormick et al. 1999). The trichothecene 3-O-acetyltransferase also mediates a self-defense mechanism by Oacetylation of the trichothecene ring specifically at the C-3 position, thus converting toxic trichothecenes to less toxic derivatives (Kimura et al. 1998). Therefore, if the TRI101 gene is inserted into the host plant, then the transgenic plant could modify trichothecenes to less toxic compounds. This line of research has been undertaken in wheat and tobacco. In greenhouse tests, Okubara et al. (2002) found that one transgenic wheat plant exhibited an increased level of TRI101 enzyme activity as well as an increased level of resistance to F. graminearum. In a preliminary field trial, however, this transgenic wheat did not have better resistance to FHB than its parental cultivar (Okubara et al. 2002). In another study, transgenic expression of the TRI101 gene increased resistance of tobacco seed to DAS inhibition in germination tests (Muhitch et al. 2000). C. Pleiotropic Drug Resistance (PDR) Genes PDR genes found in the yeast (Saccharomyces cerevisiae) are one group of genes that could be useful for genetic transformation. The PDR5 gene encodes a protein of 1,511 amino acids, which is responsible for pumping drugs out of the cell (Balzi et al. 1994). When the gene is inserted into

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tobacco, it increases resistance of tobacco seed to DAS inhibition in germination tests (Muhitch et al. 2000). D. Zearalenone Detoxifying Gene The zearalenone detoxifying gene (zdh101) was recently isolated from the fungus Clonostachys rosea (Takahashi-Ando et al. 2002). Its enzyme is able to detoxify zearalenone by cleavage of the lactone ring. Five transgenic rice plants have been obtained and will be used for in planta detoxification experiments (Higa et al. 2003). E. Antifungal Protein Genes Once F. graminearum or F. culmorum successfully invades barley plants, they synthesize alkaline proteinases, which in turn degrade storage proteins in infected grains (Pekkarinen et al. 2003). The role of alkaline proteinases in the growth of Fusarium species in kernels is not clear, but they may make nutrients more readily available for the growing mycelium. In defense, barley produces proteins that inhibit the alkaline proteinases of Fusarium species to slow or prevent the degradation of storage proteins during fungal infection (Pekkarinen and Jones 2003). These inhibitor proteins, however, do not totally prevent fungal invasion. Recently, an alkaline proteinase inhibitor has been isolated from Streptomyces sp. (Vernekar et al. 1999). Another antifungal protein, similar to phospholopase A2, has been isolated from the mold Aspergillus giganteus (Nakaya et al. 1990), which can inhibit the growth of F. moniliforme and other fungi in vitro (Vila et al. 2001). The gene(s) encoding these antifungal proteins have not been identified. These gene(s), if identified, could be useful for genetic transformation of barley. X. CONCLUSIONS AND PROSPECTS FHB is a widespread and destructive disease of barley. At least 23 Fusarium species have been isolated from and at least 15 Fusarium toxins have been detected in infected barley kernels. F. graminearum, F. culmorum, F. avenaceum, F. sporotrichioides, and F. poae are pathogenic on barley. They can reduce grain yield and lower malting quality. Sources of FHB resistance are available, but transferring FHB resistance genes from these exotic genotypes to adapted cultivars is difficult. Several morphological, physiological, and chemical traits appeared to be associated with FHB resistance. In general, cultivars with two-row spike, purple (or black) lemma, tall stature, and/or lodging resistance have low FHB and DON content. FHB resistance is controlled by many minor

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genes and its heritability is low to moderately high. Most of the QTLs for FHB resistance are associated with plant height, heading date, and/or specific morphological traits. Genetic transformation offers the potential to enhance the FHB resistance in barley but this research is in its preliminary stages. As the understanding of biochemical response of barley to Fusarium improves, genetic transformation efforts can be more directed toward specific genes. Barley researchers have many challenges ahead as they attempt to develop FHB-resistant cultivars. The sources of resistant germplasm are very limited, particularly in six-row barley. The level of resistance currently available is only partial and can be overcome by severe infection. No major QTLs directly responsible for FHB resistance have been identified. As well, many of the QTLs identified for FHB resistance are associated with QTLs for other traits. Therefore, marker-assisted selection for FHB resistance will be inefficient until the genetics of FHB-resistance are better understood. The absence of major QTLs requires that the size of breeding populations be large enough to recover the best recombinants. Evaluating large populations in multiple environments is time consuming, labor intensive, and costly. Ultimately, chemical tests must be done to determine the DON content in grains to determine the level of susceptibility to DON accumulation. All tests for the DON content are expensive in terms of seed grinding, flour sampling, and chemical analysis. Analysis for DON would be considerably simplified if near-infrared spectroscopy could be used to accurately measure the DON content of barley grains (Ruan et al. 2002). Genetic transformation offers the potential to improve the level of FHB resistance in barley, but the level of improvement that genetic transformation might bring may not justify the huge cost associated with the regulatory process. Acceptance of transgenic barley by consumers is another issue to be addressed. For the present, increased international cooperation, shuttle breeding, and a free exchange of resistant material should be pursued to hasten the development of FHB-resistant barley cultivars worldwide.

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6 Using Genomics to Exploit Grain Legume Biodiversity in Crop Improvement* Sangam L. Dwivedi, Hari D. Upadhyaya, Jayashree Balaji, and Hutokshi K. Buhariwalla International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru, PO 502324, A.P. India Matthew W. Blair Centro internacional de Agricultura Tropical (CIAT) A.A. 6713 Cali Colombia Rodomiro Ortiz and Jonathan H. Crouch CIMMYT Apdo. Postal 6-641, 0660 Mexico, D.F., Mexico Richard Serraj Soil and Water Management & Crop Nutrition Section, Joint FAO/IAEA Division, Wagramer Strasse 5, A-1400 Vienna, Austria

I. INTRODUCTION A. Phylogeny and Taxonomy B. Production and Uses C. Major Constraints 1. Diseases and pests 2. Environmental stress D. Variation in Legume Genomes *Generation Challenge Program, Cultivating Plant Diversity for the Resource Poor (www.generationcp.org). Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 171

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II. AVAILABLE GENETIC RESOURCES OF KEY LEGUME CROPS A. Tropical Legumes B. Temperate Legumes C. Model Species Related to Grain Legumes III. MANAGEMENT AND UTILIZATION OF LEGUME GENETIC RESOURCES A. Collection and Enhancement B. Regeneration and Conservation C. Characterization, Evaluation, and Documentation D. Core Collection E. Elite Germplasm, Genetic Stock, and Cultivar F. Wild Species Germplasm IV. IMPACT OF GENETIC RESOURCES IN CONVENTIONAL LEGUME BREEDING A. Germplasm Distribution B. Impact of Domesticated Germplasm on Breeding Gains C. Use of Wild Germplasm D. Conclusions from Conventional Manipulation of Genetic Resources V. MOLECULAR-ENHANCED STRATEGIES FOR MANIPULATING NOVEL GENETIC VARIATION FOR LEGUME BREEDING A. Interspecific Hybridization B. Linkage Mapping and QTL Detection C. Linkage Disequilibrium and Association Mapping D. Dissection and Manipulation of Legume Physiology VI. ADVANCED APPLICATIONS IN LEGUME MOLECULAR BREEDING A. Comparative Genomics and Allele Mining B. Functional Genomics and Gene Discovery C. New Technologies for Marker-Assisted Selection 1. High-throughput SSR marker genotyping 2. Simple markers from fingerprinting assays 3. Gel-free diagnostics 4. Trait-specific SNP markers 5. Array-based genotyping D. Successful Applications of Molecular Breeding in Legumes VII. CONCLUSIONS AND FUTURE PROSPECTS LITERATURE CITED

LIST OF ABBREVIATIONS ACCase AFLP AM ASAPs ASH AVRDC BAC BAMNET

Acetyl-CoA carboxylase Amplified Fragment Length Polymorphism Arbuscular mycorrhiza Allele specific associated primers Allele specific hybridization Asian Vegetable Research and Development Centre Bacterial Artificial Chromosome The International Bambara Groundnut Network, Germany

6. GENOMICS TO EXPLOIT GRAIN LEGUME BIODIVERSITY IN CROP IMPROVEMENT

BCMNV BCMV BLAST CAPS CBB CE CGIAR CIAT CIMMYT CMS COS CRSP CSIRO cDNA DAGAT DArT DH DNA ELISA EST FAO FASTA IARCs IBPGR ICARDA ICRISAT IITA ILRC ILRI INIA IPGRI IPK IRRI IVR LIMS LD LG LRR

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Bean common mosaic necrosis virus Bean common mosaic virus Basic Local Alignment Search Tool Cleaved Amplified Polymorphic Sequences Common bacterial blight Capillary electrophoresis Consultative Group on International Agriculture Centro Internacional de Agricultura Tropical International Maize and Wheat Improvement Centre Cytoplasmic male sterility Conserved ortholog sequence Collaborative Research Support Project Commonwealth of Scientific and Industrial Organization Complementary Deoxyribonucleic acid Diacylglycerol acyltransferase Diversity Arrays Technology Double haploids Deoxyribonucleic acid Enzyme-Linked Immunosorbent Assay Expressed sequenced tags Food and Agricultural Organization FAST Algorithm for sequence alignment International Agriculture Research Centers International Bureau Plant Genetic Resources International Center for Agricultural Research in the Dry Areas International Crops Research Institute for the Semi Arid Tropics International Institute of Tropical Agriculture Inverted repeat-loss clade International Livestock Research Institute O Instituto Nacional de Investigação Agrária, Portugal International Plant Genetic Resources Institute Institut fur Pflanzengenetik und Kulturpflanzenforschung International Rice Research Institute Vavilov Institute of Plant Industry Laboratory Information Management Systems Linkage disequilibrium Linkage Groups Leucine-rich repeat

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MADRP MAS NARSs NBPGR NBS NILs NSF ODAP QTL RAPD RFLP RILs RNA SAGE SCAR SCN SNF SNP SSR TAGs TAM TCS TIGR TILLING TIR USDA–ARS

Ministério da Agricultura Desenvolvimento Rural e Pescas, Portugal Marker-assisted selection National Agricultural Research Systems National Bureau of Plant Genetic Resources Nucleotide-binding sites Near isogenic lines National Science Foundation β-N-oxalyl-L-α, β-diaminopropionic acid Quantitative trait loci Random Amplified Polymorphic DNA Restriction fragment length polymorphism Recombinant inbred lines Ribonucleic acid Serial analysis of gene expression Sequenced characterized amplified region Soybean cyst nematode Symbiotic nitrogen fixation Single Nucleotide Polymorphism Simple Sequence Repeats Triacylglycerols Tagged Microarray marker Tentative consensus sequences The Institute for Genomic Research Targetting induced local lesions in genomes Toll/Interleukin-1 cytoplasmic receptor United States Department of Agriculture–Agricultural Research Service

I. INTRODUCTION A. Phylogeny and Taxonomy Legumes represent the second largest family of higher plants, second only to grasses in agricultural importance (Doyle and Luckow 2003). The legume family or Leguminosae, also known as Fabaceae, consists of about 20,000 species across 700 genera that have traditionally been divided into three subfamilies (Caesalpinoideae, Mimosoideae, and Papilionoideae) based largely on floral characteristics. The Papilionoideae subfamily is the largest of the three subfamilies with 476 genera and about 14,000 species, whereas the Mimosoideae subfamily

6. GENOMICS TO EXPLOIT GRAIN LEGUME BIODIVERSITY IN CROP IMPROVEMENT

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contains 77 genera and 3000 species and the Caesalpinoideae subfamily contains 162 genera and 3000 species (Lewis et al. 2003). There are two major groups of cultivated species in the Papilionoideae: the tropical or ‘phaseoloid’ legumes (including Phaseolus, Vigna, Glycine, and Cajanus) and the temperate or ‘galegoid’ legumes (including Melilotus, Trifolium, Medicago, Pisum, Vicia, Lotus, Cicer, Lens, and Lathyrus). Phylogenetically, the temperate legumes can be differentiated by the absence of one copy of an approximately 25 kb inverted repeat, commonly found in the chloroplast genomes of most angiosperms, and therefore are known collectively as members of the inverted repeat-loss clade (IRLC). Lupins and peanuts are somewhat distinct from the phaseoloid and galegoid groups of grain legumes. The phenotypic similarities between many of the grain legumes have led to a plethora of English common names with pea or bean suffixes for the various crop species. All legumes, from giant Caesalpinoid rainforest trees to tiny Papilionoid annual herbs, are united by descent from a single common form (Doyle et al. 2000; Kajita et al. 2001; Doyle and Luckow 2003). Molecular phylogenetic relationship studies within the Leguminoseae (Wojciechowski 2003) have revealed that the Papilionoideae diverged from other legumes as early as 45 to 50 million years ago, and that the Papilionoideae and Mimosoideae are both monophyletic (i.e., the clades include an ancestor plus all its descendents and no extraneous, unrelated taxa), while the Caesalpiniodeae are paraphyletic (i.e., the clade comprises a diverse assemblage of unrelated lineages lacking the distinctive floral features used to group genera into the other two subfamilies). Given the taxonomic distinctness and importance of the legume family, two model species have already emerged: Medicago truncatula (www.noble.org/medicago) and Lotus japonicus (http://cryo.naro.affrc.go .jp/sakumotu/mameka/lotus-e.htm). A number of laboratories across the world adopted one or other of these two species as a model system for the study of symbiotic nitrogen fixation but they are now model species for the whole legume biology and genomic community. Scientists now study these species to investigate a range of questions from disease resistance to environmental tolerance and from bacterial and fungal symbiosis to complex secondary metabolism. Indeed, it is expected that the large-scale sequencing of these legume genomes will greatly synergize legume crop biology and molecular breeding just as the full sequencing of the Arabidopsis genome has revolutionized fundamental plant research. As both Medicago and Lotus are temperate forage species, there is still a need to study other species for characters uniquely associated with grain development and to better represent the tropical phaseoloid clade. Soybean is receiving by far the largest research investment

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amongst the legumes due to its pre-eminent global economic value. However, the large degree and complexity of genome duplication in soybean confounds any attempts at developing it as a model genome. Genome sizes vary considerably between members of the Phaseoloid clade (tropical legume species): common bean, mung bean, and cowpea (574 to 637 Mbp), pigeonpea (784 to 882 Mbp), and soybean (1115 Mbp). Thus, common bean is probably the strongest candidate as a hub species for the phaseoloid clade in view of a relatively small genome and good progress in the development of genomics and germplasm resources. In a recent survey of grain legume genomics research in advanced labs (Vandenbosch and Stacey 2003), ten groups were highlighted for M. truncatula, five for L. japonicus, four for soybean, and two for Phaseolus. These had an emphasis on marker development, genetic linkage maps, genome sequences, ESTs, expression arrays, genetic transformation, gene cloning, functional genomics, plant-microbe interactions, molecular genetics and breeding, and conservation of genetic resources of these legumes. International genomics consortia have also been established for Medicago, Lotus, Phaseolus, soybean, chickpea, and pea. It is expected that the genetic and genomic resources developed through these consortia will be freely available to those engaged in enhancing the genetic potential of these legumes. B. Production and Uses The importance of legumes is paramount for world agriculture. Although legumes account for just 15% of arable farming land worldwide, they play a vital role in agroecosystem health. Legumes improve soil health through biological nitrogen fixation and enhance human nutritional well-being through their role as a major source of protein among poor consumers and subsistence farming communities of the developing world (Crouch et al. 2004; Ortiz 2004a) (Table 6.1). Legumes fix substantial quantities of biological nitrogen by virtue of their symbiotic association with Rhizobium bacteria (Schultze and Kondorosi 1998; Serraj 2004), ranging from potential rates of 20 to 260 kg ha–1 N2 for soybean [Glycine max (L.) Merr.], 73 to 80 kg ha–1 for cowpea [Vigna unguiculata (L.) Walp.], 72 to 240 kg ha–1 for peanut (Arachis hypogaea L.), 40 to 350 kg ha–1 for alfalfa (Medicago sativa L.) and clover (Trifolium ssp.) (Yamada 1974); to 69 kg ha–1 for pigeonpea (Cajanus cajan L.) (Kumar et al. 1983). The capacity for biological nitrogen fixation is particularly important in developing countries where legumes can and do reduce the dependency of resource-poor farmers on expensive petroleum-based, chemical fertilizers, while simultaneously improving soil and water

Table 6.1.

Legume food supply statistics for ASIA, Latin America and sub-Saharan Africa from 1961 to 2000.z 1961

Commodity

Supply (kg year–1)

Asia Total food Crops Animals

Calorie

2000 Protein

Fat

(per caput daily) 1893 1783 110

48.8 41.6 7.2

24.5 17.0 7.5

12.0 2.7 2.1 7.2

113 25 19 69

7.0 1.6 1.3 4.1

0.8 0.1 0.1 0.7

Oil crops Soybean Peanut

6.8 2.6 0.6

49 24 9

2.9 2.3 0.4

Vegetable oil Soybean Peanut

2.5 0.3 0.8

61 7 19 2297 2013 284

Pulses Beans Peas Others

Central America Total Crops Animal

Supply (kg year–1)

Calorie

Protein

Fat

(per caput daily)

Growth rate Within diet (40 year %)y

2708 2340 368

71.1 49.8 21.3

65.7 37.5 28.2

5.1 1.3 0.5 3.2

48 12 4 32

2.9 0.8 0.3 1.9

0.4 * * 0.3

–57.5 –51.9 –76.2 –55.6

2.9 0.8 0.7

10.4 2.9 1.6

81 37 23

4.8 3.5 1.0

5.1 1.3 1.9

52.9 11.5 62.5

* 0.1 —

6.9 0.8 2.1

9.0 1.9 0.9

* * —

24.0 5.1 2.6

260.0 533.3 12.5

62.5 45.9 16.6

50.8 29.9 20.9

81.3 48.1 33.2

78.2 42.4 35.7

21.5 45.0 23.0 2934 2425 509

177 (continued )

178 Table 6.1.

(continued )

Legume food supply statistics for Asia, Latin America and sub-Saharan Africa from 1961 to 2000.z 1961

Commodity Pulses Beans Peas Others

Supply (kg year–1)

Calorie

2000 Protein

Fat

(per caput daily)

Supply (kg year–1)

Calorie

Protein

Fat

(per caput daily)

Growth rate Within diet (40 year %)y

16.6 15.4 1.0 1.2

159 147 1 11

9.0 8.3 0.1 0.6

0.8 0.7 0.1 0.1

12.7 11.0 0.1 1.6

121 105 1 15

7.2 6.0 0.1 1.1

0.6 0.5 0.1 0.1

–23.5 –28.6 –90.0 33.3

Oil crops Soybean Peanut

2.7 * 0.9

21 * 14

0.7 * 0.6

1.7 * 1.1

3.2 0.1 0.9

27 1 14

1.0 0.1 0.7

2.3 * 1.2

18.5 ≈ 00 0.0

Vegetable oil Soybean Peanut Peanut

5.1 0.1

123 1

* 0.1

13.9 0.2

9.0 3.9

218 94

* 0.1

24.7 10.6

76.5 3800.0

0.1

*

0.2

0.1

3

0.4

0.0

South America Total food Crops Animals Pulses Beans Peas Others

13.5 11.9 0.6 1.0

—

2322 1909 413

62.6 36.4 26.2

49.0 19.9 29.2

125 110 6 9

8.2 7.2 0.4 0.6

0.6 0.5 * 0.1

10.9 9.4 0.6 0.9

—

2850 2263 586

76.1 73.4 38.7

82.1 41.3 40.8

101 87 6 8

6.6 5.7 0.4 0.6

0.5 0.4 * 0.1

–19.3 –21.0 0.0 –10.0

Oil crops Soybean Peanut

3.2 * 0.5

24 * 13

0.9 * —

2.1 * 1.5

2059 1919 141

59.6 42.0 10.6

40.5 31.5 9.0

10.2 3.4 0.9 5.9

94 32 8 55

6.2 2.1 0.5 3.6

0.5 0.1 * 0.3

Oil crops Soybean Peanut

6.7 0.2 3.5

79 2 52

3.1 0.2 2.2

Vegetable oil Soybean Peanut

5.4 * 1.0

129 1 25

0.1 * —

Sub-Saharan Africa Total food Crops Animals Pulses Beans Peas Others

8.0 1.2 0.1

38 6 2

1.6 0.8 —

2.8 0.1 0.3

250.0 ≈ 00 –80.0

2226 2087 140

54.2 43.7 10.5

44.4 35.4 9.0

9.5 2.9 0.5 6.1

88 27 5 57

5.7 1.7 0.3 3.7

0.5 0.1 * 0.3

–6.9 –14.7 –44.4 3.4

6.6 0.1 4.3

5.3 0.8 2.5

63 9 37

2.8 0.7 1.6

4.9 0.4 3

–20.9 300.0 –28.6

14.7 0.1 2.8

7.5 0.6 1.9

181 14 45

0.1 * —

20.5 1.6 5.1

38.9 ≈ 00 90

*Traces Population (million) in each continent in 1961: 1697 for Asia, 51 for Central America, 153 for South America, 208 for sub-SAHARAN AFRICA. Population (million) in each continent in 2000: 3659 for Asia, 125 for Central America, 345 for South America, 605 for subSAHARAN AFRICA. y Changes (%) in the food per caput and legume supply of each commodity for 40-year period (from 1961 to 2000). Ortiz (2004a). z

179

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quality. In addition, many legumes are also able to release soil-bound phosphate through their symbiotic relationships with mycorrhizal fungi (Sanders and Tinker 1973; Hayman 1974, 1983). In terms of human nutrition, grain legumes are a rich source of protein, lysine, and essential vitamins and minerals (Duranti and Gius 1997; Welch et al. 2000; Grusak 2002). They also contain beneficial secondary compounds with significant health-promoting properties that are reported to provide protection against human cancers and to reduce the risk of high serum cholesterol (Kennedy 1995; Stark and Madar 2002). Legumes can also be milled into flour to make breads, doughnuts, tortillas, chips, spreads, extruded snacks, milk substitutes, yogurts, and infant food (Graham and Vance 2003). Novel uses of specific legumes include popping of some varieties of common bean (Phaseolus vulgaris L.) (Popenoe et al. 1989), production of licorice from Glycyrhiza glabra (Kindscher 1992), and elaboration of soybean candy (Genta et al. 2002). In addition, soybeans are processed into many food products such as tofu and tempeh, and provide 26.6% of the world’s (96.3 M t) processed vegetable oil with a resulting by-product that is a rich source of dietary protein for human food and feed industries (FAO 2002). Meanwhile, forage legumes provide the basis for many meat and dairy industries across the world (Russelle 2001). There are also many industrial uses of both grain and forage legumes including biodegradable plastics, oils, gums, dyes, and inks (Graham and Vance 2003). Global legume production (225 million t from 132 million ha) is very much dominated by soybean (79.8%), bean (Phaseolus sps.) (8.1%), pea (Pisum sativum L.) (4.4%), and chickpea (Cicer arietinum L.) (3.5%) (Table 6.2) (FAO 2002). The most commonly grown legume species vary by continent and according to use. For example, chickpea and pigeonpea are important in Asia and bean in Latin America, whereas cowpea and peanut are among the most important food legumes in sub-Saharan Africa. Soybean is an important source of oil in Asia and Latin America, while peanut serves a substantial portion of the vegetable oil demand in sub-Saharan Africa. Thus, the Americas produce 85% of the world’s soybean (180 million t) followed by Asia (13.2%). Meanwhile, for common bean (Phaseolus vulgaris L.) Asia and the Americas each produced 42% of the total production (18.3 million t). Conversely, Europe is the largest producer of pea (56.4% of 9.8 million t total produced) followed by Asia (21.8%) and the Americas (16.0%). Asia is the largest producer of chickpea (87.4% of 7.8 million t), lentil (Lens culinaris Medik.) (72.5% of 2.9 million t), pigeonpea (92.3% of 3.0 million t), and broad bean (Vicia faba L.) (44.4% of 3.7 million t) (Table 6.3).

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Table 6.2. Global area and production statistics for soybean, beans, peas, chickpea, cowpea, broadbeans, lentil, and pigeonpea in 2002 (http://www.apps.fao.org/page/collections?subset=agriculture).

Crop Soybean Beans Peas Chickpea Cowpea Broadbean Pigeonpea Lentil World z

Crop area (000 ha)

Production (000 t)

79,410 (55.9)z 26,837 (18.9) 5,812 (4.4) 9,894 (7.0) 9,828 (6.9) 2,446 (1.7) 4,157 (2.9) 3,623 (2.6) 142,007

179,917 (79.8) 18,334 (8.1) 9,872 (4.4) 7,808 (3.5) 3,728 (1.6) 3,728 (1.6) 2,994 (1.3) 2,938 (1.3) 229,319

% of world area and production.

Table 6.3. Regional contribution to the global production of soybean, beans, peas, chickpea, broadbean, lentil, cowpea, and pigeonpea in 2002 (http://www.apps.fao.org/page/collections?subset=agriculture). Share of the major grain legumes to the global production (%)

Region Africa Asia Europe North Central America South America Oceania

Soybean

Beans

Peas

Chickpea

Broadbean

Lentil

Cowpea

Pigeonpea

0.5 13.2 1.0

13.2 41.8 3.1

3.0 21.8 56.4

4.5 87.4 1.1

30.4 44.4 14.7

3.1 72.5 1.4

90.1 7.1 0.9

6.9 92.3 —

42.7 42.5 0.1

21.6 20.0 0.3

16.0 0.9 1.9

5.2 0.1 1.7

1.1 2.7 6.7

16.0 0.7 6.3

1.2 — 0.1

0.1 0.1 —

National productivity of grain legumes varies greatly between and within regions (Tables 6.4 and 6.5). Average regional legume productivity is substantially higher in the Americas than in Asia and Africa. However, yields of soybean and field bean in China, for example, are at least twice that achieved in India. The prevalence of biotic and abiotic stresses and the level of adoption of technological innovations at the farm level are probably the major sources of yield variation observed across regions and between countries within regions.

Table 6.4. Top five producers and their average national yield for soybean, beans, peas, chickpea, broadbean, lentil, cowpea, and pigeonpea in 2002 (http://www.apps.fao.org/page/collections?subset=agriculture).

Crop

Country

Production (000 t)

Average yield (t ha–1)

Beans

Brazil India Mexico Myanmar China USA

3,017 (16.5)z 3,000 (16.4) 1,648 (9.0) 1,467 (8.0) 1,356 (7.4) 1,360 (7.4)

0.73 0.33 0.80 0.79 1.12 1.95

Broadbean

China Egypt Ethiopia France Australia

1,550 (41.6) 440 (11.8) 385 (10.3) 309 (8.3) 250 (6.7)

1.55 3.14 0.96 3.96 1.40

Chickpea

India Turkey Pakistan Iran Mexico

5,320 (68.1) 590 (7.6) 362 (4.6) 250 (3.2) 240 (3.1)

0.87 0.91 0.39 0.33 1.60

Cowpea

Nigeria Niger Burkina Faso Myanmar Mali

2,174 (58.3) 400 (10.7) 330 (8.8) 250 (6.7) 88 (2.4)

0.43 0.11 0.66 0.92 0.28

Lentil

India Turkey Canada Australia Nepal

983 (33.5) 480 (16.3) 354 (12.0) 185 (6.3) 148 (6.9)

0.71 0.96 0.83 1.48 0.82

Peas

France Russian Federation Canada China India

1,665 (16.9) 1,578 (16.0) 1,366 (13.8) 1,200 (12.2) 730 (7.4)

4.93 3.51 1.26 1.50 1.00

Pigeonpea

India Myanmar Malawi Uganda Tanzania

2,440 (81.5) 300 (10.0) 79 (2.6) 78 (2.6) 47 (1.6)

0.73 0.62 0.64 1.00 0.71

Soybean

USA Brazil Argentina China India

74,291 (41.3) 41,903 (23.3) 30,000 (16.7) 16,900 (9.4) 4,270 (2.4)

2.54 2.56 2.63 1.79 0.75

z

% of global production

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Table 6.5. Regional and world average yields of soybean, beans, peas, chickpea, broadbean, lentil, cowpea, and pigeonpea in 2002 (http://www.apps.fao.org/page/collections?subset=agriculture). Average yield (t ha–1)

Region World Africa Asia Europe North Central America South America Oceania

Soybean

Beans

Peas

Chickpea

Broadbean

Lentil

Cowpea

Pigeonpea

2.27 0.91 1.38 1.88

0.68 0.67 0.54 1.48

1.70 0.55 1.18 2.99

0.79 0.72 0.78 0.83

1.52 1.36 1.58 2.39

0.81 0.58 0.77 0.86

0.37 0.35 0.93 2.95

0.72 0.76 0.72 —

2.54 2.56 2.12

1.07 0.76 1.12

1.32 1.07 0.57

1.30 1.09 0.68

0.79 1.02 1.39

0.92 0.95 1.48

0.78 — 0.43

— 0.77 —

International trade of grain legumes also varies considerably between and within regions, both in terms of market share (Table 6.6), and the relative importance of different commodities (Tables 6.7 and 6.8) (FAO 2001). The Americas are the largest exporter of grain legumes (89% of the 66 million t total), whereas Asia and Europe are net importers (84% of the 65 million t) despite contributing together 62% of the 55 million t of global grain legume production. Soybean is the single most imported grain legume in international trade, with the bulk of soybean imports Table 6.6. Contribution of six regions to the world trade of grain legumes in 2001. (http://www.apps.fao.org/page/collections?subset=agriculture). Import Import Region Africa Asia Europe North Central America South America Oceania World z

Export Quantity (t)

Export Value (000 US $)

Quantity (t)

Value (000 US $)

1,491,141 (2.26)z 31,692,800 (48.03) 23,662,961 (35.86)

492,285 (2.88) 8,793,422 (51.44) 5,039,576 (29.48)

211,269 (0.32) 2,987,635 (4.49) 3,107,013 (4.67)

113,233 (0.83) 1,171,084 (8.58) 795,172 (5.83)

6,332,799 (9.60)

1,879,334 (10.99)

33,320,116 (50.06)

6,743,577 (49.43)

2,773,543 (4.20) 30,551 (0.05) 65,984,227

826,899 (4.84) 64,123 (0.37) 17,093,739

25,802,554 (38.77) 1,145,230 (1.72) 66,553,045

4,518,207 (33.12) 301,348 (2.21) 13,643,063

% of total world trade.

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Table 6.7. Regional import statistics for seven grain legumes in 2001 (http://www.apps.fao.org/page/collections?subset=agriculture). % contribution to the import of grain legumes Total import valuez

Europe

North Central America

Africa

Asia

South America

Oceania

2,015,079 3,005,236

7.21 1.96

34.08 61.00

26.50 3.50

19.53 20.74

11.98 11.08

0.70 1.78

Beans (green)

293,840 314,509

1.17 0.76

9.07 5.46

70.92 74.32

17.91 18.75

0.30 0.08

0.63 0.63

Broadbean (dry)

567,212 157,733

47.07 53.62

10.00 13.51

41.98 30.65

0.89 2.07

0.04 0.13

0.01 0.02

Broadbean (green)

50,034 32,172

4.32 3.55

41.76 43.77

50.43 50.53

2.43 0.93

0.86 0.60

0.10 0.61

Chickpea

1,087,859 495,885

10.60 14.71

71.95 60.27

13.47 19.88

2.13 2.47

1.82 2.61

0.03 0.63

Lentil

1,097,771 435,878

21.01 21.00

41.48 42.62

18.77 19.65

5.33 5.38

13.18 10.77

0.22 0.58

Peas (dry)

3,363,995 703,095

2.59 5.01

45.33 49.16

44.42 35.30

2.19 4.23

5.16 5.62

0.31 0.68

234,774 153,683

4.22 2.19

39.56 33.68

42.65 43.18

11.16 19.78

2.20 0.77

0.21 0.40

2,431 1,296

78.98 70.22

1.03 8.33

8.51 3.70

11.48 17.75

— —

— —

57,271,232 11,794,253

1.09 1.19

48.97 51.09

36.16 35.07

9.95 9.30

3.82 3.33

0.00 0.00

Crop Beans (dry)

Peas (green) Pigeonpea Soybean z

Figures in bold are tons and in nonbold are US $ (000).

going to Asia and Europe (85% of the total world-wide imports of 57 million t) from the Americas, which together produce 96% of the total world-wide production of 57 million t. For chickpea, Asia is the largest producer (87% of 7.8 million t total world-wide production) but imports substantial quantities of the crop as well (72% of the 1.1 million t in world-wide imports), mostly from North America and Oceania. C. Major Constraints 1. Diseases and Pests. Major biotic constraints to grain legume production include anthracnose in soybean, cowpea, and common bean; ascochyta blight in broadbean, chickpea, lentil, and pea; bacterial wilt in common bean, cowpea, and pea; fusarium wilt in broadbean, chickpea,

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Table 6.8. Regional export statistics for seven grain legumes in 2001 (http://www.apps.fao.org/page/collections?subset=agriculture). % contributions to the export of grain legumes Total export valuez

Europe

North Central America

Africa

Asia

South America

Oceania

3,005,236 1,235,643

1.96 2.12

61.00 49.91

3.45 6.09

20.73 26.14

11.08 13.75

1.78 1.98

Beans (green)

238,893 217,252

18.03 23.44

12.30 9.08

43.20 40.05

25.53 26.43

0.39 0.34

0.55 0.66

Broadbean (dry)

446,239 120,435

1.77 3.66

11.81 25.71

31.04 23.49

1.71 2.64

0.16 0.73

53.50 43.75

Broadbean (green)

74,235 51,935

10.08 4.75

36.27 33.58

29.94 42.86

22.56 16.52

0.26 0.39

0.88 1.88

996,519 462,252

1.51 1.28

30.62 35.83

2.32 2.85

38.74 42.35

— —

26.74 17.58

Lentil

1,168,640 462,302

2.24 1.50

26.82 41.30

1.74 2.25

50.47 40.10

0.02 0.02

18.70 14.82

Peas (dry)

3,494,945 619,592

0.78 1.48

0.33 0.65

28.90 29.19

59.40 56.85

0.31 0.58

10.27 11.24

120,629 77,356

3.18 2.93

17.89 21.30

51.92 36.62

26.04 36.70

0.95 2.40

0.01 0.05

213 290

7.51 3.1

1.41 4.83

22.06 21.38

69.01 70.69

— —

— —

57,007,496 10,396,010

0.04 0.05

0.64 1.05

2.86 3.33

51.80 53.76

44.65 41.76

0.01 0.02

Crop Beans (dry)

Chickpea

Peas (green) Pigeonpea Soybean z

Figures in bold are tons and in nonbold are US $ (000).

common bean, cowpea, lentil, pea, and pigeonpea; phytophthora root rot in chickpea, pigeonpea, and soybean; rust in broadbean, common bean, cowpea, lentil, and soybean; and web blight in common bean and cowpea (Table 6.9). Many of these diseases are widespread, as in the case of anthracnose, angular leaf spot, bean common mosaic virus (BCMV), and bean common mosaic necrosis virus (BCMNV), common bacterial blight, rust and web blight in common bean (Coyne et al. 2003); ascochyta blight, rust and vascular wilt in lentil (Erskine et al. 1994); ascochyta blight and fusarium wilt in chickpea (Singh et al. 1994); broomrape and chocolate spot in broadbean (Bond et al. 1994); and fusarium wilt in pigeonpea (Reddy et al. 1990). Some diseases, especially those caused by viral pathogens, are more crop and location specific. Meanwhile, among pests, nematodes (cyst, root-knot, and stem nematodes) are common in broad-

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Table 6.9. Diseases that cause substantial losses to production of soybean, beans, peas, chickpea, broadbeans, lentil, and pigeonpea. Crop

Disease

Broadbean

Fungal

Viral Parasitic weed

Common/scientific name Chocolate spot (Botrytis fabae Sardina); ascochyta blight (Ascochyta fabae Speg.); root rot (Fusarium spp.); rust [Uromyces fabae (Grev.) Fuckel = U. viciae-fabae Pers.:Pers.) J. Schrot] Bean yellow mosaic virus (BYMV); bean leaf roll virus (BLRV) Broomrape (Orobanche crenata Forsk.)

Chickpea

Fungal

Fusarium wilt [Fusarium oxysporum Schlecht. Emend. Snyd. F.sp. ciceris [Padwick)Snyd. & Hans.]; ascochyta blight [Ascochyta rabiei (Pass.) Lab.]; botrytis grey mold (Botrytis cinerea Pers. ex Fr.); dry root rot [Rhizoctonia batanicola (Taub) Butler]; phytophthora root rot [Phytophthora megasperma Drechs.]

Common bean

Fungal

Angular leaf spot (Phaeoisariopsis griseola (Sacc.) Ferraris); anthracnose [Colletotrichum lindemuthianum Sacc. and Magn.) Scrib.]; rust (Uromyces appendiculatus (Pers) Unger var. appendiculatus); root rot (Fusarium spp.); web blight [Thanatephorus cucumeris (Frank) Donk (anamorph Rhizoctonia solani Kuhn)] Common bacterial blight [Xanthomonas campestris pv phaseoli (Xap)]; halo blight (Pseudomonas syringae pv. Phaseolicola) Bean common mosaic virus (BCMV); bean common mosaic necrosis virus (BCMNV); bean golden yellow mosaic virus (BGYMV)

Bacterial

Viral

Cowpea

Fungal

Bacterial Viral Parasitic weed Lentil

Fungal

Anthracnose [Colletotrichum lindemuthianum (Sacc. and Magnus) Lams.-Scrib)]; septoria leaf spot (Spetoria vignae P. Henn and S. vignicola Vasat Rao, S. Kozopolzanii Nikolajeva); cercospora leaf spot (Cercospora canescens Ellis and G. Martin); web blight (Rhizoctonia solani Kuhn); Fusarium wilt [Fusarium oxysporium Schlechtend.:Fr. F. sp. Tracheiphilum (E.F. Smith) W.C. Snyder and Hansen)]; rust [Uromyces appendiculatus (Pers.: Pers.) Unger] Bacterial blight [Xanthomonas campestris pv vignicola (Burkholder) Dye] Cowpea yellow mosaic virus (CYMV); Cowpea aphid-born mosaic virus (CABMV) Striga [Striga gesneroioides (Wild.) Vatke)]; alectra (Alectra vogelii Benth.) Rust [Uromyces fabae (Pers.) de Bary) ( = U. vicia-fabae)]; ascochyta blight [Ascochyta fabae Speg. F. sp. lentis Gossen et al. (A. lentis Vassiljevsky)]; fusarium wilt [Fusarium oxysporum f. sp. lentis (Vasudeva + Srinivas) Gordon]

6. GENOMICS TO EXPLOIT GRAIN LEGUME BIODIVERSITY IN CROP IMPROVEMENT Table 6.9.

187

(continued)

Crop

Disease

Lentil (cont.)

Viral Parasitic weed

Pea seedborn mosaic virus (PSbMV) Broomrape (Orobanche spp.)

Pea

Fungal

Ascochyta blight (Ascochyta pisi Lib.); powdery mildew [Erysiphe polygoni Syd. (= E. polygoni DC.)]; downy mildew [Peronospora viciae (Berk.) Casp.]; Fusarium wilt (Fusarium oxysporum f. sp. pisi); common root rot (Aphanomyces euteiches Drechs.); fusarium root rot (Fusarium solani Kuhn) Bacterial blight (Pseudomonas syringae Van Hall pv. pisi) Pea seedborn mosaic virus (PSbMV); Pea common mosaic virus (PCMV); Pea enation mosaic virus (PEMV)

Bacterial Viral Pigeonpea

Fungal

Viral Soybean

Fungal

Bacterial Viral

Common/scientific name

Fusarium wilt (Fusarium udum Butler); phytophthora blight [Phytophthora dreschsleri Tucker f. sp. cajani (Pal, Grewal and Sarbhoy) Kannaiyan, Riberio, Erwin and Nene] Sterility mosaic virus (SMV) Frogeye leaf spot (Cercospora sojina); sudden death syndrome [Fusarium solani (Mart.) Sacc. f. sp. glycines]; anthracnose (Colletotrichum truneatum); phytophthora rot (Phytophthora sojae Kaufmann and Gerdemann); brown stem rot (Philalophora gregata); white mold (Sclerotinia sclerotiorum); stem canker [(Diaporthe phaseolorum var. sojae Athow.)]; rust (Phakopsora pachyrhizi H. Sydow and Sydow); purple seed stain (Cercospora kikuchii) and phomopsis seed decay (Phomopsis spp. and Diaporthe phaseolorum var. sojae) Bacterial blight (Pseudomonas syringae pv. Glycinea) Soybean mosaic virus (SMV); Peanut mottle virus (PMV); Yellow mosaic virus (YMV); soybean leaf crinkle virus (SLCV)

bean, chickpea, pea, pigeonpea, and soybean; legume pod borer is particularly troublesome in chickpea, pigeonpea, and soybean; aphids in broadbean, chickpea, cowpea, peas, and lentil; maruca pod borers are major constraints in cowpea, lentil, and pigeonpea; bruchids in chickpea and common bean; and weevils in common bean, cowpea, and pea (Table 6.10). Most of these pests are not as widely distributed as some of the diseases cited above. Apart from pests and diseases, parasitic weeds are found in cowpea, broad bean, and lentil and include striga, alectra, and broomrape. The diseases and pests are reported to cause substantial losses to production. For example, the total losses due to disease for the

Table 6.10. Insect pests that cause substantial losses to production of soybean, beans, peas, chickpea, broadbean, lentil, and pigeonpea. Crop

Insect pests

Broadbean

Field pest Nematode

Aphids (Aphis fabae Scop.) Stem nematode [Ditylenchus dipsaci (Kuhn) Filipjev]; pea cyst nematode (Heterodera goettingiana Liebscher)

Chickpea

Field pest

Pod borer (Helicoverpa armigera Hubner); leaf miner (Liriomyza cicerina Rond.); aphids (Aphis craccivora Koch.) Bruchid beetles (Callosobruchus chinesis L.) Cyst nematode (Heterodera ciceri Vovlas, Greco and Di Vito); root-knot nematode [(Meloidogyne arneria (Neal) Chitwood; M. incognita (Kofoid and White) Chitwood; M. javanica (Treub) Chitwood]

Storage pest Nematode

Common/scientific name

Common bean

Field pest Storage pest

Leaf hoppers (Empoasca spp) Bruchids (Zabrotes subfasciatus Boheman and Acanthoscelides obtectus (Say)

Cowpea

Field pest

Aphids (Aphis crassivora Koch); thrips (Megalurothrips sjostedti Trybom); maruca pod borer (Maruca testulalis Geyer); pod sucking bug (Clavigralla spp, Anoplocnemis curvipes Fabricius, Riptortus dentipes Fabricius, and Nizara viridula L.) Weevil (Callosorbruchus maculates Fabricius) Root-knot nematode [Meloidgyne incognita Kofoid and White; M. javanica (Treub) Chitwood)]

Storage pest Nematode Lentil

Field pest

Aphids (Aphids craccivora Koch. and Acyrthosiphon kondoi Shinji); pod borer (Etiella zinkenella Treritschke); Bruchids (Callosobruchus chinensis Linn. and C. maculates Fabricius)

Pigeonpea

Field pest

Pod borer [Helicoverpa armigera and Maruca vitrata Geyer]; pod fly (Melanagromyza obtuse Malloch); pod wasp (Tanaostigmodes cajaninae La Salle) Root-knot nematode [Meloidogyne javanica (Treub) Chitwood and M. incognita (Kofoid and White) Chitwood]; reniform nematode (Rotylenchulus reniformis Linford and Oliveira); cyst nematode (Heterodera cajani Koshy)

Nematode

Pea

Field pest Nematode

Pea weevil (Bruchus pisorum L.) Pea cyst nematode (H. goettingiana Liebscher); rootknot (Meloidogyne spp.) nematode

Soybean

Field pest

Mexican bean beetle [Epilachna varivestes (Mulsant)]; corn earworm (Helicoverpa zea Boddie); soybean looper [Pseudoplusia includens (Walker)]; velvetbean caterpillar [Anticarsia gemmatalis (Hubner)]; Bean fly (Melenagromyza sojae) Soybean cyst (Heterodera glycines Ichinohe), root-knot (Meloidogyne spp)

Nematode

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1994 soybean harvested in the 10 highest country producers were estimated to be 15 million tons valued at US $3.3 billion (Wrather et al. 1997). Soybean cyst nematode caused the greatest reduction in yield followed by stem canker, brown spot, and charcoal rot. Cowpea is a major legume grown in West African semi-arid areas, and striga is reported to cause an average 30% reduction in crop yield in susceptible varieties (Aggrawal and Ouedraogo 1989), whereas attacks from Maruca pod borer can result in up to 80% yield losses (Singh et al. 2003b). Diseases are also a constraint to the production of good-quality seeds in legumes. In soybean, for example, phomopsis seed decay reduces seed germination, seed weight, and oil quality (Bradley et al. 2002; Wrather et al. 2003); soybean mosaic virus or peanut mottle virus infection reduces seed germination and vigour, also further increasing susceptibility to phomopsis seed decay (Koning et al. 2001; Gore et al. 2002); fusarium spp. infection reduces seed weight and volume, increasing total oil content but decreasing the linoleic and linolenic acid composition (Wilson et al. 1995; Meriles et al. 2002); and sclerotinia stem rot reduces seed germination, seed weight, and also changes oil quality (Hoffman et al. 1998). In pea, ascochyta blight induces a premature water loss from hulls and leaves, accelerates seed desiccation, alters carbohydrate metabolism and content, as well as protein remobilization and free amino acid translocation, and reduces seed weight and total carbohydrate and nitrogen contents (Garry et al. 1996). Similarly, in chickpea, ascochyta blight also reduces seed weight and protein content (Gaur and Singh 1996). 2. Environmental Stress. The major abiotic factors affecting common bean, cowpea, lentil, and soybean production are drought and high temperature stress, while low temperatures are an additional constraint in chickpea and lentil production areas. Other abiotic constraints of importance are ozone stress in common bean, non-availability of iron in calcareous soils in soybean, while aluminium (Al) toxicity and low availability of phosphorus in acid soils are a common limiting factor for a range of grain legumes. Many tropical soils, including those of the acid savannas of Africa and Latin America, are low in available phosphorus due to high compositions of iron and aluminum oxides (Weir 1972, 1977). Some legume crops have adapted to these soils by becoming more nutrient efficient, often with specialized root traits that contribute to a superior ability to acquire nutrients (Sanginga et al. 2003). For example, piscidic acid and its derivatives exuded from pigeonpea roots enhance the ability of this legume to access phosphorus that is bound to iron (Ae et al. 1990; Ishikawa et al. 2002). This phosphorus-solubilizing activity has been associated with superior growth of pigeonpea and peanut under deficient conditions (Ae and Shen 2002).

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D. Variation in Legume Genomes Legumes form a coherent taxonomic group with frequent and widespread macro- and microsynteny. However, comparative legume genomics is far behind that of the cereals but is beginning to provide an insight into genome size, gene clustering, genome duplication, distribution of repetitive elements (Young et al. 2003) as well as gene order and sequence conservation across genera (Young et al. 2003; Choi et al. 2004a). There is huge variation in nuclear genome size of legume species, ranging from 370 million base pair (Mbp) in Lablab niger to 13,000 Mbp in Vicia faba (Arumugunathan and Earle 1991; http://www.rbgkew.org.uk/cval/homepage.html). Meanwhile, the two model legumes, L. japonicus and M. truncatula, have relatively compact genomes of approximately 470 Mbp. In terms of the genome size variation for the major legume crops focused upon in this review, urd bean or black gram [Vigna mungo (L.) Hepper], mung bean [Vigna radiata (L.) R. Wilczek], common bean, lima bean (Phaseolus lunatus L.), tepary bean (Phaseolus acutifolius A. Gray), and cowpea have the smallest genomes (574 Mbp to 647 Mbp); pigeonpea (784, 882 Mbp) and chickpea (738 Mbp) have slightly larger genomes; soybean has a relatively large genome (1,115 Mbp); while pea and lentil (4,063 Mbp to 4,397 Mbp) and broad bean (12,603 Mbp) have massive genome sizes. The largest legume genomes are characterized by extensive abundance of retroelements (Murray et al. 1978, 1981; Pearce et al. 1996; Flavell et al. 1992; Neumann et al. 2001). When compared to the genome of Arabidopsis thaliana, whole genome duplication and segmental duplications appear to have played a significant role in creating new diversity in higher plants, including the legumes (Arabidopsis Genome Initiative 2000; Vision et al. 2000). The case of the soybean genome is of particular interest as a polyploid model system because it allows the study of both palaeopolyploidy and neopolyploidy. ‘Diploid’ Glycine species are all 2n = 4x = 38 or 40, in contrast to its allies in the legume tribe Phaseoleae, most of which are all 2n = 20 or 22 (Goldblatt 1981). The genome duplication that led to this change in chromosome number is evident in the modern soybean genome (Zhu et al. 1994; Shoemaker et al. 1996), and is estimated to have taken place around 15 Mya (Schlueter et al. 2004). Doyle et al. (2004) have shown that the various polyploid taxa known from the subgenus Glycine are all part of a single large allopolyploid complex, linked by shared diploid genomes. Many elements of the complex have arisen recently, and most show evidence of recurrent origins. However, there are also many dissimilarities among even closely related polyploids.

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Polyploids differ from one another in terms of the number of original polyploidization events, the amounts of allelic diversity harboured at different loci, bidirectional vs. unidirectional origins, retention of ribosomal gene homoeologues, success as measured by geographical range and abundance, and patterns of gene expression. Comparative mapping has provided a means for detecting and mapping large numbers of duplicated loci and integrating maps generated using different populations. In Glycine soja, RFLP mapping data from nine mapping populations has been used to reveal that large portions of the soybean genome seem to have undergone duplication in more than one round of duplication events (Shoemaker et al. 1996). In that study, the size of the homoeologous segments ranged from 1.5 to 106.4 cM, with an average size of 45.3 cM, and segments were present in as many as six copies, with an average of 2.55 duplications per segment. The presence of nested duplications suggests that at least one of the original genomes may have undergone an additional round of polyploidization, thus accounting for the highly duplicated nature of the G. soja genome. More detailed background information has been reviewed elsewhere for genetic markers and plant genetic resources (Bretting and Widrlechner 1995; Haussman et al. 2004); legume phylogeny, genetic transformation, nutritional quality, product development and utilization (Anonymous 2003; Wojciechowski 2003); model legumes (Anonymous 2003); and on genetic resources, breeding, and genomics of individual legume crops: soybean (Hymowitz et al. 1998; Boerma and Specht 2004; Stacey and Nguyen 2004); common bean (Singh 2001; Broughton et al. 2003; Kelly et al. 2003; McClean et al. 2004); cowpea (Hall et al. 1997; Kelly et al. 2003); chickpea (Kumar and Abbo 2001; Weeden and Muehlbaur 2004); pea (Myers et al. 2001; Weeden and Muehlbaur 2004); lentil (Weeden and Muehlbaur 2004); grasspea (McCutchan 2003); and peanut (Dwivedi et al. 2003; Holbrook and Stalker 2003; Paterson et al. 2004).

II. AVAILABLE GENETIC RESOURCES OF KEY LEGUME CROPS A. Tropical Legumes 1. Phaseolus vulgaris (Common Bean). Common bean is the most widely grown Phaseolus species in the world (Singh 1992) and among all grain legumes is used for direct human consumption at the highest level (Broughton et al. 2003). Common beans are grown worldwide in many different countries and regions over a wide range of latitudes (from 40°

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S in Chile to 60° N in Scandanavia) and altitudes (up to 3000 masl = meter above sea level). The crop is found in many agroecosystems, from the tropical highlands of Central and South America to the subtropical belts of South-east Brazil, Southern Africa, and India to the temperate regions of Europe and North America (Debouck 1991; Singh 1992). Common beans were domesticated from wild populations of P. vulgaris that were annual climbing vines growing at mid-altitudes (1500–2000 masl) in forest clearings or disturbed environments from northern Mexico to northern Argentina (Toro et al. 1990). The primary gene pool of common bean comprises both cultivars and wild populations, and hybrids between these two groups are fully fertile (Singh et al. 1995). Domestication occurred in two separate centers of origin, giving rise to two distinct gene pools: the Andean gene pool from the Andean region of South America and the Mesoamerican gene pool from Central America. These differ particularly in terms of seed size, Andean cultivars being large seeded (>40 g 100-seed weight–1) and Mesoamerican cultivars being small seeded (<25 g 100-seed weight–1) (Evans 1973, 1980). Growth habit, photoperiod insensitivity, pod fiber, seed dormancy, and seed weight were also influenced during the domestication of common beans (Evans 1980; Smartt 1988; Gepts and Debouck 1991). The Andean and Mesoamerican gene pools can be divided into six races based on agro-ecological adaptation and seed types (Singh et al. 1991), including the Mesoamerican races, Durango, Jalisco, and Mesoamerica, and the Andean races, Chile, Nueva Grenada, and Peru. There is also additional diversity within Mesoamerican races, especially a group of Guatemalan climbing bean accessions that are not grouped with any of the previously defined races (Beebe et al. 2000). In addition, the gene pools and races overlap in many regions. During pre-Colombian times in northern South America, both Andean and Mesoamerican beans grew together, allowing some hybridization to occur between the genepools (Amirul-Islam et al. 2004). The crop’s dispersal into other regions of the world, especially Europe and Africa, started with Spanish and Portuguese colonization of the New World and allowed even further diversification along with additional opportunities for genepool mixing. Significant adaptative evolution also occurred in the Middle East, South Asia, Southeast Asia, and China. Common bean is grown for a variety of purposes and is harvested as a dry grain or as a fresh vegetable (either as snap beans or as greenshelled beans). Dry beans have a huge range of commercial classes based on seed size, color, and pattern and there are many established varieties for each class. Dry bean cultivars differ in growth habit (determinate bush to indeterminate climbing bean), phenological traits such as growth cycle (60 to 330 days), seed shape and size (10 to 100 g per 100 seed

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weight), seed coat color (from white, creams, yellows, pinks, reds, purples to blacks with many different mottled, speckled, or two-tone patterns), and canning and cooking qualities (Voysest and Desert 1991; Singh 1992). Snap beans, in contrast, possess a thick succulent mesocarp and low or absent fiber in the green pod walls and sutures (Myers and Baggett 1999; Myers 2000). The different market classes of the snap bean cultivars are largely determined on the basis of pod shape (flat, cylindrical, or oval), color (dark green, light green, yellow, or purple), and length. Among snap bean cultivars, there can be substantial variation in growth habit and adaptation traits, although snap beans are postulated to be a more recent selection from dry beans based on the stringless pod character and other horticultural traits (Singh 1992). 2. Phaseolus coccineus (Scarlet Runner Bean) and Phaseolus polyanthus (Year-long Bean). Scarlet runner bean and year-long bean are closely related domesticates and, along with the wild relative P. costaricensis Freytag and Debouck, belong to the secondary gene pool of common bean. Scarlet runner bean and year-long bean have a more limited distribution than common bean and are grown mostly in mid-elevation areas of Central America and northern South America and small parts of the Caribbean (Jamaica, Puerto Rico), Europe (Portugal, Spain, and the United Kingdom), and Africa (Ethiopia, Kenya, and South Africa). They are all vigorous climbing beans adapted to cooler highlands above 800 masl except for some improved varieties that can be shrubs growing at lower elevations in cool climates. Traditionally, both species were intercropped with maize but they are also grown on trellises, fences, or walls. White seeded types of P. coccineus are preferred in most commercial settings, but the seed coat color and pattern can vary substantially in this species, although most are large-seeded (60 or more g per 100 seed). The seed of P. polyanthus is often yellow, tan, white, or reddish brown. Scarlet runner bean was domesticated from wild accessions of P. coccineus, which grew in a region from Mexico to Colombia. In contrast, wild accessions of year-long bean are only found in a small region of Guatemala and Mexico (Freytag and Debouck 2002). Out-crossing of both scarlet runner bean and year-long bean has introgressed large amounts of genetic diversity (Hawkins and Evans 1973). Crosses with common bean are also successful but only when common bean is used as the female parent (Ibrahim and Coyne 1975; Manshardt and Bassett 1984; Singh et al. 1997). Scarlet runner beans and year-long beans are good sources of disease resistance that can be readily transferred to common bean given their cross-compatibility. However, they also convey poor vine growth habit, late maturity, low pod set, and reduced yield.

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3. Phaseolus acutifolius (Tepary Bean). Tepary bean belongs to the tertiary gene pool of common bean and is a minor crop grown sporadically in the dry regions of Northwest Mexico, Southwestern United States, and Central America (Singh 1992). Tepary beans are very drought tolerant and are planted in either desert washes or late in the rainy season to rely on residual soil moisture (Pratt and Nabhan 1988). Domestication was thought to have occurred about 5000 years ago in or near the Sonoran desert of Northwest Mexico, from where the crop spread north and south as recently as 1200 years ago (Pratt and Nabhan 1988; Debouck 1991). Tepary beans are divided into two clades, P. acutifolius var. acutifolius and P. acutifolius var. tenuifolius; however, the exact source of the domesticate is unknown (Garvin and Weeden 1994). The most closely related wild species is P. parvifolius Freytag. The two cultivated forms are distinguished on the basis of leaflet shape: var. acutifolius accessions have obovate to subovate leaflets whereas var. tenuifolius accessions have linear leaflets (Pratt and Nabhan 1988). Allozyme and phaseolin analyses failed to reveal diversity at the molecular level (Schinkel and Gepts 1988, 1989), which may suggest a narrow genetic base in tepary bean. Indeed, Garvin and Weeden (1994) have observed that cultivated tepary beans are less diverse than common beans and that there appears to have been little introgression from wild relatives since domestication. Tepary beans are known to have a very low natural crossing rate that limits the creation of new diversity within the crop and consequently its genetic potential. Almost all cultivated tepary beans have similar seed size (10 to 20 g per 100 seed) and growth habits, usually a sprawling indeterminate semi-prostrate habit (Debouck 1991). In addition, the range of seed colors in the cultigen is limited to white, cream, and yellow, with or without black or purple flecking (Debouck 1991). Tepary beans are cross compatible with common bean but require embryo rescue to produce viable offspring (Mejiá-Jiménez et al. 1994). 4. Phaseolus lunatus (Lima Bean). Lima bean is fairly distinct from the rest of the cultivated Phaseolus species and is grown mainly in dry regions under irrigation or as a horticultural crop. As with common bean domestication, lima beans were first cultivated in both the Andean and Mesoamerican regions, leading to large-seeded and small-seeded types of gene pools, respectively (Maquet et al. 1997; Fofana et al. 1997). Although not a major commodity in international trade, lima beans are an important horticultural crop in several countries in the Americas and Europe, although, as a grain crop, lima beans are grown on a very small scale as a backyard crop in Latin America (Debouck 1991). The presence of cyanogenic glucosides in some cultivars of lima beans has prevented

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their wide use as a dry grain except when properly processed. The growth habit of lima beans varies from a short-season indeterminate bush to perennial vigourous climbers. Some cultivars of lima beans are relatively tolerant to heat, drought, and salinity stresses (Bayuelo-Jimenez et al. 2002). Lima beans are considered as diverse as common beans given that landraces are found over a wide area of the Neo-tropics; however, diversity is highest in the large seeded types and lower in the small seeded types (Gutierrez-Salgado et al. 1995). Although they are predominantly an inbreeding crop, lima beans can undergo moderate outcrossing, and have introgressed substantial diversity through crosses between the gene pools and between large-seeded types and wild forms in South America (Maquet et al. 1997). However, similarly to common beans, overall diversity may be greater in wild than in cultivated lima beans (GutierrezSalgado et al. 1995). In addition, like common beans, lima beans have a great range of seed colors and patterns, some of which are very attractive and unusual. Lima beans vary widely in seed size (from 16 to 280 g per 100 seed), with Andean ‘Big Lima’ types larger than Mesoamerican ‘Sieva’ or Caribbean ‘Potato’ types (Gutierrez-Salgado et al. 1995). Horticultural varieties within the determinate Fordhook (Andean) and Henderson (Mesoamerican) classes are generally closely related and well established in the United States (Nienhuis et al. 1987). The closest wild relatives to lima beans are P. filiformis, P. augusti, and P. angustissimus. Neither lima beans nor any of these wild species have been successfully crossed with common beans, therefore these species would be considered to be in the quarternary gene pool of the common bean (Debouck 1991). Centro Internacional de Agricultura Tropical (CIAT) at Cali (Colombia) holds, under an agreement with the Food and Agricultural Organization of the United Nations (FAO), the largest number of Phaseolus germplasm (a total of 41,061 accessions, including 26,500 accessions of cultivated common beans, 1,300 of wild common beans, 1,000 from the secondary gene pool, and 350 from the tertiary gene pool) (Debouck 1999). Other institutions holding sizeable Phaseolus germplasm collections are the United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Western Regional Plant Introduction Station, Pullman, WA, United States (with 11,809 primary, 475 secondary, and 121 tertiary gene pool accessions); the Instituto Nacional de Investigaciones Forestales y Agropecuarias, Pecuar, Mexico (10,570 cultivated and 600 wild accessions of common bean); the Vavilov Institute of Plant Industry (VIR), St. Petersburg, Russia (9,762 accessions); the Institut fur Pflanzengenetik und Kulturpflanzenforschung (IPK), Gaterslaben, Germany (7,609 accessions); the National Biological Institute,

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Bogor, Indonesia (3,846 accessions); and the Punjab Agricultural University, Ludhiana, India (3,000 accessions); as well as other national programs in Latin America, Africa, Europe, and Asia. The European Phaseolus database contains 31,717 accessions, the largest from Phaseolus vulgaris (http://www.agrobio.bmlf.gv.at/phaseolus). 5. Vigna radiata (Mung Bean). Mung bean was domesticated in India (Vavilov 1926) from its wild ancestral form V. radiata var. sublobata (Chandel et al. 1984), a widely distributed species found in areas stretching from Central and East Africa, Madagascar, through Asia, New Guinea, to Northern and Eastern Australia (Tateishi 1996). There are three botanical varieties: V. radiata var. radiata, the cultivated form (mung bean); var. sublobata (Roxb.), the wild ancestral form of mung bean; and var. setulosa, another wild form distributed in India, Indonesia, and southern China (Marechal et al. 1978). Compared to its wild relatives, mung bean has pale yellow flowers, smaller pockets on the keel, longer pods, and pods that attach at the side or bottom of the peduncle. Cultivars exhibit diverse colours of mature pods (black, brown, or pale gray) and seeds (yellow, greenish yellow, light green, shiny green, dark green, dull green, black, brown, and green mottled with black). Var. sublobata has many desirable attributes such as resistance to bruchid and yellow mosaic virus (Singh and Ahuja 1977), high methionine content in the seed (Babu et al. 1988), higher photosynthesis efficiency, and tolerance to drought (Ignacimuthu and Babu 1987), and high tolerance to saline and alkaline soils (Lawn et al. 1988). The Asian Vegetable Research and Development Centre (AVRDC) in Taiwan maintains 5,108 mung bean accessions from 51 countries. Other institutions holding sizeable collections are the USDA–Southern Regional Plant Introduction Station, Griffin, GA, United States (3,494 accessions); and the National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India (2,789 accessions). 6. Vigna mungo (Urd Bean or Black Gram). Urd bean was domesticated in India from its wild ancestral form, Vigna mungo var. silvestris Lukoko, Marechal and Otoul (Zeven and de Wet 1982), and is confined to cultivation in South Asia and adjoining regions (India, Pakistan, Afghanistan, Bangladesh, the Philippines, and Myanmar). There are two botanical varieties: var. mungo (the cultivated form) and var. silvestris (the wild ancestor) (Lukoki et al. 1980). Compared to its wild relative, black gram has bright yellow flowers, longer pockets on the keels, shorter pods, pods attached upright to the peduncle, and dull black seed color. However, shiny black and shiny green seeded black gram has also been reported from Nepal.

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The National Bureau of Plant Genetic Resources (NBPGR) maintains about 2,100 accessions of which 829 are active collections, while the AVRDC maintains a collection of around 200 accessions. 7. Vigna angularis (Adzuki Bean). Adzuki bean is an important crop in East Asia. It consists of two botanical varieties: var. angularis (Willd.) Ohwi and Ohashi, the cultivated form; and var. nipponensis Ohwi and Ohasi, the presumed wild form of adzuki bean (Marechal et al. 1978). An intermediate weedy or semi-wild adzuki also exists (Yamaguchi 1992). The wild and weedy adzuki beans are believed to be widely distributed in Asia (Yamaguchi 1992). The greater genetic variation in the wild and weedy relatives of adzuki bean (Xu et al. 2000a,b) suggests they may be useful for crop improvement. V. angularis is primarily autogamous but a significant percentage of cross pollination has been reported (Lumpkin and McClary 1994). Wild adzuki bean is fully fertile in crosses with the cultigen (Siriwardhane et al. 1991), thus it can be directly used in breeding programs. However, introgression of desirable traits from wild germplasm is usually confounded by deleterious linkage drag. Intermediate weedy or semi-wild adzuki beans are, therefore, better alternatives for breeding programs than wild adzuki beans because they closely resemble the cultigen, while offering greater variation than the cultigen. The world collection of adzuki bean consists of a large number of landraces maintained in national gene banks, mainly in China, Japan, and Korea, while NBPGR in India maintains 194 accessions of adzuki bean. 8. Vigna umbellata (Rice Bean). Rice bean was domesticated in Southeast Asia, and wild forms are distributed across northern India, Burma, Thailand, Laos, and Vietnam (Ohashi et al. 1988). There are two recognized botanical varieties in rice bean: Vigna umbellata var. umbellata (the cultivated type) and V. umbellata var. gracilis (the wild ancestor). Morphologically, rice bean is similar to adzuki bean. It has golden yellow flowers, pods attached to the peduncle downward, and slender seeds with protruding hilums. There are many agricultural types and varieties of rice bean, with seed color ranging from ivory to greenish ivory, red violet, and black (Chatterjee and Dana 1997). NBPGR in India holds 1,081 rice bean accessions. 9. Vigna aconitifolia (Moth Bean or Meat Bean). Moth bean is a native to India, Pakistan, and Burma (Rachie and Roberts 1974). It has a short, compact plant habit, and is widely grown for food in the arid and semiarid regions of India. NBPGR in India maintains 702 accession of moth bean.

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10. Vigna unguiculata (Cowpea). Cowpea was domesticated in Africa, and this is the only continent where wild forms exist and it is the location of the greatest genetic diversity. Pasquet (1993a,b, 1997) divides cowpea into 10 perennial subspecies and one annual subspecies (ssp. unguiculata). The annual subspecies is differentiated into two botanical varieties: var. unguiculata (cultivated cowpea) and var. spontanea (Schweinf.) Pasquet (annual wild cowpea). Accessions of the 11 subspecies have been classified into three groups according to their breeding systems: perennial outcrossing accessions, perennial outcrossing and inbreeding accessions, and annual inbreeding accessions (Pasquet 1994). The perennial outcrossing accessions look primitive and are more remote from each other and from inbreeding taxa. The perennial outcrossing taxa include ssp. baoulensis (A. Chev.) Pasquet, ssp. aduensis Pasquet, and ssp. pawekiae Pasquet. The perennial outcrossing and inbreeding taxa include ssp. dekindtiana (Harms) Verdic. sensu stricto; ssp. stenophylla (E. Mey.) Verdc.; ssp. tenuis (E. Mey.) Marechal, Mascherpa and Stainier; ssp. alba (G. Don) Pasquet; and ssp. pubescens (R. Wilczek) Pasquet. The annual inbreeding taxa include ssp. unguiculata var. spontanea (Schweinf.) Pasquet and var. unguiculata. Within this large gene pool, mainly made of perennial taxa, cultivated cowpea (ssp. uniguiculata var. unguiculata) form a genetically coherent group and are closely related to annual cowpeas (ssp. unguiculata var. spontanea), which may include the most likely progenitor of cultivated cowpea (Pasquet 1999). The International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria holds, under an agreement with FAO, 14,816 cultivated and 1,651 wild cowpea germplasm accessions from 84 countries characterized for up to 30 agro-botanical traits revealing diverse growth habit, variable pod length, pod shape, seed size, and seed-coat colors (Ng and Singh 1997). More than 50 wild Vigna species from more than 50 countries are represented in this collection. NBPGR in India holds 2,499 cowpea accessions in its gene bank. 11. Vigna subterranea (Bambara Groundnut). Bambara groundnut is an indigenous grain legume from West Africa (Hepper 1963) that is grown by subsistence farmers in drier parts of sub-Saharan Africa where the soils are too poor for cultivation of other favored legumes such as common beans and peanut. It is tolerant to drought, and its seeds are highly nutritious. Bambara cultivars are differentiated by the ratio of petiole length to internode length: bunch (8.1–11.0), semi-bunch (7.0–8.0), and open (4.4–6.5) (Doku 1969; Karikari 1972) or by the canopy diameter at 100 days after planting (bunch— < 40 cm, semi-bunch—40–80 cm, and open—> 80 cm) (Ezedinma and Maneke 1985). Pasquet and Fotso (1997)

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classified the domesticated Cameroonian landraces based on seeds per pod: single seeded pods (northern group) and two to four seeds per pod (southern group). The domesticated bambara groundnut has a compact growth habit, whereas wild types produce long vines (Hepper 1963). The freshly dug fruits of domesticated bambara groundnut possess thick fleshy pods that wrinkle upon drying, but the fruits of wild germplasm have thin pods that do not wrinkle upon drying. Seeds of wild accessions are small, uniform in size, and 9–11 mm long, while the seeds of domesticated accessions are 11–15 mm in length. Petioles of leaves of wild plants are much shorter and not as erect or closely tufted, and more slender than those of domesticated types (Hepper 1963). The wild and domesticated bambara groundnuts are characterized by a low total genetic diversity and a comparatively high intra-population diversity, which suggests that they are predominantly autogamous. High genetic identity between wild and domesticated forms suggests that wild bambara groundnuts are the true progenitor of the domesticated bambara groundnut (Pasquet et al. 1999). IITA maintains 2,029 bambara groundnut accessions in its gene bank following an agreement with FAO. The European Vigna database contains 4,000 accessions (http:// www.agrobio.bmlf.gv.at/vigna); the largest collection is from VIR, St. Petersburg, Russia. 12. Glycine max (Soybean). The genus Glycine is divided into two subgenera: Glycine and Soja. The subgenus Glycine contains 16 wild perennial species (Burridge and Hymowtiz 1997) that are indigenous to Australia with diverse morphological features and genomes, variable chromosome numbers (13 wild species 2n = 40; G. tabacina: 2n = 40 or 80, and G. tomentella: 2n = 38, 40, 78, or 80) but representing an invaluable source of economically important traits such as resistance to biotic and abiotic stresses. The subgenus Soja is composed of G. max (L.) Merrill, the cultivated soybean (2n = 40), and its wild annual counterpart, G. soja Sieb. and Zucc. (2n = 40). The genetic base of soybean is extremely narrow (Gizlice et al. 1993, 1994, 1996; Burton 1997). Harlan and de Wet (1971) catagorised the genetic resources into three main gene pools, based on cross compability with the cultigen and value in plant breeding. The primary gene pool (GP1) consists of soybean cultivars and landraces (G. max) as well as their wild annual progenitor (G. soja), all of which readily intercross producing vigorous fertile hybrids that exhibit normal meiotic behavior and gene segregation. GP1 is divided into two subspecies: subspecies A (cultivated races) and subspecies B (spontaneous races). The secondary gene pool (GP2) has yet to be fully defined but the tertiary gene pool (GP3) comprises very diverse germplasm including the 16 wild perennial species of the subgenus Glycine that are

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geographically isolated from G. max and G. soja. Crosses between primary and tertiary gene pools fail to produce viable progeny so gene transfer between these two gene pools is not possible or requires radical techniques (Harlan and de Wet 1971). Soybean genetic resources in Japan are classified into eight groups based on days to flowering and days from flowering to maturity, whereas soybean genetic resources in North America are classified into 13 maturity groups based on their relative time of maturity. There are 100,000 G. max accessions held in germplasm collections around the world, of which less than 10,000 are G. soja accessions, and 3,500 are accessions of wild perennial Glycine species (Palmer et al. 1995). The institutions holding large numbers of soybean germplasm are the Institute of Crop Germplasm Resources, Beijing, China (15,334 accessions); the National Seed Storage Laboratory, Fort Collins, CO, United States (14,379 accessions of G. max and 1,102 G. soja accessions); the AVRDC, Taiwan (12,916 accessions); IITA, Nigeria (2,500 accessions), and the Commonwealth Scientific and Industrial Research Organization (CSIRO), Canberra, Australia (2,000 accessions). The European Glycine database consists of 11,915 accessions from eight countries, the largest from Russia and Germany (Vishnyakova and Omelchenko 2001). 13. Cajanus cajan (Pigeonpea). The genus Cajanus has six sections: Cajanus, Atylia Benth, Fruticosa van der Maesen, Cantharospermum (W. & A.) Benth, Volubilis van der Maesen, and Rhyncosoides Benth, comprising a total of 32 species (van der Maesen 1985). Taxonomically, the genera Atylosia, Dunbaria, and Rhynchosia are very close to cultivated pigeonpea. Morphological, cytological, and chemo-taxonomical data revealed that these three genera are congeneric, therefore all 28 species of Atylosia and one species each of Rhynchosia (R. acutifolia) and Dunbaria (D. heynei) have been recently merged into Cajanus. Further taxonomic studies revealed that A. cajanifolia is the closest species to pigeonpea compared to the successively more distant species: A. lineata, A. scarabaeoides, A. sericea, A. albicans, A. volubilis, A. platycarpa, and R. Rothii (Pundir and Singh 1985). The pigeonpea gene pool consists of the cultigens in GP1; C. acutifolius, C. albicans, C. cajanifolius, C. lanceolatus, C. latisepalus, C. lineatus, C. reticulatus, C. scarabaeoides, C. sericeus, and C. trinervius in GP2; and C. goensis, C. heynei, C. kerstingii, C. mollis, C. platycarpus, C. rugosus, C. volubilis, and other Cajaninae (e.g., Rhynchosia, Dunbaria, and Eriosema) in GP3 (Smartt 1990). The International Crops Research Institute for the Semi Arid Tropics (ICRISAT), Patancheru, India holds under an agreement with FAO 13,548 pigeonpea germplasm accessions including 555 wild accessions from 74

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countries. Other institutions holding substantial amounts of pigeonpea germplasm include the NBPGR (5,454 accessions) in India and the USDA, Southern Regional Plant Introduction Station (4,116) in USA. 14. Arachis hypogaea (Peanut or also known as groundnut). The genus Arachis has been divided into nine sections: Arachis, Caulorhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae (Krapovickas and Gregory 1994) which include diploid (2n = 2x = 20), tetraploid (2n = 4x = 40), and aneuploid (2n = 2x = 18) species. There are four gene pools in peanut. The primary gene pool consists of landraces of Arachis hypogaea and its wild form A. monticola; the secondary gene pool consists of diploid species from section Arachis that are cross-compatible with A. hypogaea; the tertiary gene pool includes species in section Procumbentes that are weakly cross-compatible with A. hypogaea; and the quaternary gene pool comprises all remaining wild Arachis species across seven other sections that are completely incompatible with the cultigen (Singh and Simpson 1994). Cultivated peanut germplasm is classified into two main subspecies: hypogaea (no flowering on the main stem and alternate branching) and fastigiata (flowering on the main stem and sequential branching). The subsp. hypogaea contains two botanical varieties: hypogaea (common name: Virginia) and hirsuta. The subsp. fastigiata contains four botanical varieties: fastigiata (common name: Valencia), peruviana, aequatoriana, and vulgaris (common name: Spanish). All six botanical varieties have unique morphological characteristics that separate them from one another (Krapovickas and Gregory 1994). ICRISAT holds, under an agreement with FAO, 14,966 accessions of cultivated peanut and 453 accessions of wild Arachis species (representing nine sections and 44 species) from 93 countries. Other institutions holding a large number of peanut accessions are the National Research Center for Groundnut, Junagadh, India (7,935 accessions) and the USDA Southern Regional Plant Introduction Station, USA (6,233 accessions). In the United States, wild Arachis species are maintained at North Carolina State University, Raleigh (250 accessions) and at the Texas Agricultural Experiment Station, TAMU, Texas (300 accessions). B. Temperate Legumes 1. Pisum sativum (Pea). The genus Pisum is comprised of two species: P. sativum and P. fulvum (Polhill and van der Maesen 1985). P. sativum is then divided into the subspecies ssp. sativum and spp. elatius. Cultivated P. sativum ssp. sativum has been further divided into var. sativum (that contains the horticultural types), and var. arvense (that contains

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fodder and winter pea types). Subspecies elatius is comprised of var. elatius, var. pumilio, and var. brevipedunculatum (Smartt 1990). Crosses between P. sativum and P. fulvum are successful but only when the latter is used as the pollen parent. The major gene banks holding pea germplasm are the John Innes Center, Norwich, UK (5,000 accessions); the Nordic Genebank, Alnarp, Sweden (5,000 accessions); the Vavilov Institute of Plant Industry, St. Petersburg, Russia (5,500 accessions); the USDA Regional Plant Introduction Station, Geneva, NY, United States (2,800 accessions); the USDA National Seed Storage Laboratory, Fort Collins, CO, United States (2,213 accessions); the Institute de Germoplasma, Bari, Italy (4,090 accessions); the Tropical Forage Crop Genetic Resources Center, Queensland, Australia (3,300 accessions); and NBPGR in India (2,721 accessions). The European Pisum database consists of 35,775 accessions from 18 countries, the largest from Russia and Italy (Ambrose 2001). 2. Vicia faba (Broad/Faba Bean). The genus Vicia is a large genus comprised of more than 130 species (Smartt 1990). Vicia faba, the cultivated species, is assigned to the sub-genus Vicia and placed together with V. narbonensis L., V. hyaeniscyamus Mouterde, V. galilaea Plitm. and Zohary, V. johannis Tamamschian, and V. bithynica L. in section Faba of that sub-genus (Ladizinsky et al. 1988; Smartt 1990). Its wild progenitor has not yet been discovered but V. faba var. paucijuga, smallseeded types still grown in Afghanistan and northwest Kashmir, seems the closest to the wild form. However, V. faba appears to be reproductively isolated from other Vicia species. V. faba has two subspecies: ssp. faba L. and ssp. paucijuga Murat. V. faba subsp. faba has three varieties: var. minor Beck, var. equina Pers., and var. faba L. (Maxted 1993). The International Center for Agricultural Research in the Dry Areas (ICARDA) maintains two types of germplasm collections for this crop: the international legume faba bean (ILB) collection (original germplasm accessions being heterogeneous populations maintained as composite bulks) and the faba bean pure line (BPL) collection (derived from the ILB collection but maintained by pure-breeding of single-plant progeny rows). The ICARDA gene bank holds, under an agreement with FAO, 4,453 ILB and 5,248 BPL accessions from 30 countries (Robertson 1997). A catalogue containing 840 BPL lines describes useful accessions for various traits (Robertson and El-sheerbenny 1988). Other institutions holding large amounts of broadbean germplasm are the Institute de Germoplasma, Bari, Italy (3,671 accessions); the Vavilov Institute of Plant Industry, St. Petersburg, Russia (2,525 accessions); the Institute of Crop Genetic Resources, Beijing, China (1,999 accessions); the Institut

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fur Pflanzengenetik und Kulturpflanzenforschung (IPK), Gaterslaben, Germany (1,300 accessions); and the Ethiopian Genebank, Addis Ababa, Ethiopia (1,298 accessions). The European Vicia database has 13,000 accessions, with 52% of European origin (Duc et al. 2001). 3. Lens culinaris (Lentil). The genus Lens is comprised of four species: L. culnaris, L. odemensis, L. nigricans, and L. ervoides. L. culnaris has two subspecies: ssp. culinaris, the cultivated lentil, and ssp. orientalis, the closest wild relative (Ladizinsky 1993). Subspecies culinaris has two varieties: var. microsperma (small-seeded lentils) and var. macrosperma (large-seeded lentil) (Barulina 1930). Based on cross-compatibility, the genus Lentil forms two groups: L. culinaris / L. odemensis and L. ervoides / L. nigricans. Of the wild lentils, the putative ancestor of the cultigen L. culnaris ssp. orientalis is a member of the crop’s primary gene pool, whereas L. odemensis and L. ervoides constitute the secondary gene pool (Ladizinsky 1993). ICARDA holds, under an agreement with FAO, 7,477 germplasm accessions from 64 countries, and published a catalogue containing 4,550 accessions (Erskine and Witcombe 1984). Other institutions maintaining substantial amounts of lentil germplasm include the Vavilov Institute of Plant Industry, St. Petersburg, Russia (2,358 accessions); the USDA Western Regional Plant Introduction Station (2,259 accessions); and NBPGR (2,212). The European Lens database consists of 1,675 accessions from seven countries, the largest from Turkey and Spain (Acikgoz 2001). 4. Cicer arietinum (Chickpea). The genus Cicer is comprised of 34 wild perennial, eight wild annual, and one cultivated annual (Cicer arietinum) species (van der Maesen 1987). Ladizinsky and Adler (1976) grouped six annual Cicer species into three distinct groups based on cross-compatibility with cultivated chickpea (Cicer arietinum): The primary gene pool (GP1) consists of Cicer arietinum, C. reticulatum, and C. echinospermum; the secondary gene pool (GP2) consists of C. judaicum, C. pinnatifidium, and C. bijugum; and the tertiary genepool (GP3) consists of only one species, C. cuneatum. Hybridizations within the groups are possible but with variable success, while crosses between members of different groups are not successful. There is no barrier to gene flow between C. arietinum and C. reticulatum of GP1, while it is much more difficult to produce hybrids with C. echinospermum. Two distinct forms of chickpeas have evolved since domestication: “desi” types characterized by small seeds that are angular and pigmented and “Kabuli” types characterized by large seeds that have a rounded

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appearance and lack pigmentation. The desi types are primarily grown in South Asia and the kabuli types in the Mediterranean region. ICRISAT, under an agreement with FAO, holds 17,188 cultivated and 58 wild accessions of Cicer species, whereas ICARDA, following the same FAO agreement, maintains 9,628 cultivated and 263 wild accessions. Other institutions holding chickpea germplasm are the NBPGR, India (14,566 accessions); CLIMA (4,351 accessions) and AusPGRIS (7922 accessions) in Australia; USDA (4,662 accessions); and the Seed and Plant Improvement Institute, Keraj, Iran (4,925 accessions). The European Cicer database consists of 3,700 cultivated accessions from 11 countries, the largest from Turkey and 75 wild accessions from 13 Cicer species (Pereira et al. 2001). 5. Lathyrus sativus (Grasspea). Grasspea is an important pulse crop in South Asia and China that is very tolerant to drought. It has a very hardy and penetrating root system that enables it to grow on a wide range of soil types (Campbell et al. 1994). There are two main groups in the genus Lathyrus: Group 1 consists of blue flowered types from South-west Asia, South Asia, and Ethiopia, while Group 2 consists of white, or white and blue flowered types distributed around the Mediterranean basin (Jackson and Yunus 1984). The genus Lathyrus has 160 species and 45 subspecies (Allkin et al. 1986). The species are separated into 13 sections based on morphological traits (Kupicha 1983), and L. sativus is grouped in the section Lathyrus with 33 other species. The other sections in the genus Lathyrus are Clymenum and Linearicarpus. In taxonomic studies, Lathyrus species clustered into three distinct groups, which correlated with the three sections. L. gorgoni and L. cicera from the section Lathyrus are most similar to the cultigen L. sativus (Croft et al. 1999). The species in section Lathyrus include both annual and perennial forms. Lathyrus sativus has been placed in the primary gene pool, L. amphicarpos and L. cicera in the secondary gene pool, and the remaining species in the tertiary gene pool (Yunus and Jackson 1991). Excessive consumption of grasspea causes a neurological disorder in humans and livestocks called lathyrism, a non-reversible paralysis of the lower limbs that is due to β-N-oxalyl-L-α, β-diaminopropionic acid or ODAP (Bell 1964; Murti et al. 1964). Genetic variation in ODAP content is reported among lathyrus germplasm accessions (Kaul et al. 1986). Institutions holding Lathyrus germplasm are Jawaharlal Nehru Agricultural University, Jabalpur, India (503 accessions); ICARDA (100 accessions); and the USDA Western Regional Plant Introduction Station, USA (300 accessions). Grain legume collection in Hungary and Bulgaria contains 509 (Holly 2001) and 320 (Angelova 2001) Lathyrus accessions, respectively.

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C. Model Species Related to Grain Legumes 1. Medicago truncatula Gaertn. (Barrel Medic). Barrel medic is a forage legume commonly grown in Australia, which originated in the Mediterranean basin, and is closely related to the extensively grown forage legume, alfalfa (M. sativa L.). The National Plant Germplasm System in the United States holds 320 accessions of barrel medic assembled from the Mediterranean basin, Eastern Europe, and the Caucasus. The Australian Medicago Genetic Resource Center has assembled 5,284 accessions from 38 countries, and INRA (Institut National de Recherche Agronomique), France, has assembled more than 300 natural populations collected from 9 countries (http://www.naaic.org/Publications/ 1998Proc/abstracts/Prosperi2.html). M. truncatula has emerged as a model plant for legume genomic studies (see section VI A). 2. Lotus japonicus (Miyakogusa). Miyakogusa (also known as capital weed) was first recognized at the ancient capital city of Kyoto, in Japan. A number of accessions have been collected from northernmost Hokkaido island to the southernmost Miyakojima island in Japan, and these are conserved at Miyazaki University, which has the mandate to collect, conserve, and distribute L. japonicus genetic resources. Like M. truncatula, the L. japonicus has also emerged as a model plant for legume genomic studies (see section VI A). III. MANAGEMENT AND UTILIZATION OF LEGUME GENETIC RESOURCES Plant genetic resources are the basic raw materials required to meet the current and future needs of crop improvement programs. For effective legume genetic resource management, several steps are needed: (A) enriching the genetic resources through collections or new germplasm and creation of new genetic variability, (B) conserving and regenerating genetic resources, and (C) characterizing, evaluating, and documentating genetic diversity. The development of core collections has been shown to be a particularly powerful strategy for providing crop breeding programs with a systematic yet manageable entry point into global germplasm resources. A. Collection and Enhancement Botanical and ethno-botanical prospecting and collecting have been the principal methods by which legume genetic resources have been accumulated in gene banks around the world. Collecting expeditions have

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usually targeted major production regions (markets and farmers’ fields) and/or primary and secondary centers of diversity (natural environments where wild relatives are found). Eco-geographical, social, or economic targeting, along with local interviews, together with an appreciation of biological factors (crop maturity, seed set) have been used to help decide on the most propitious areas and times for collecting. Passport data and expedition numbers are usually assigned while collecting and germplasm entries are cleaned, checked for seed viability, and placed in medium-length to long-term storage. In addition to collecting expeditions, germplasm exchange between gene banks and with local organizations has also provided an effective mechanism for the accumulation of genetic resources. B. Regeneration and Conservation The need for regeneration may arise any time after a collection is made. Sample size and reproduction system (self or cross pollination) are the key factors that have influenced the genetic integrity of original samples. Increasing the size of the original sample used for multiplication usually ensures better genetic integrity of the accessions. Since leguminous crops have papilionaceous flowers (i.e., the male and female parts are enclosed within petals), cleistogamy is commonly forcing a high level of self-pollination in many legume species. However, flower structures of crops such as broadbean and pigeonpea are relatively looser and when insects visit the flowers cross-pollination can be common. To ensure selfing in these crops during regeneration, plants are covered with thin cloth bags or individual germplasm lines are grown in isolation. Especially in mixed samples of self-pollinated crops or in populations of cross-pollinated crops, every effort should be made to conserve the entire genetic variability found in the original sample. In this respect, retaining larger seed samples is likely to preserve a greater proportion of the genetic variation. Since legumes have orthodox seeds (Roberts 1973) that can be dried to low seed moisture content (about 5–7%) and conserved effectively, they are usually easy to store under controlled conditions. However, it is important to ensure phytosanitary health and long-term germination. The seeds are dried in cool and dry conditions to reduce the moisture (5 ± 1%) to a desired level and then stored as active (0 to 4°C, 20–30% RH) and base (–18° to –20° C) collections (IBPGR 1976). Extremely dry conditions (below 5%) can have a more detrimental effect on the large-seeded legumes species (such as common bean, faba bean) than on small-seeded legume species and should be avoided (Suzuki 2003). Seeds with high oil content generally have lower

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and shorter viability in storage than those with low oil content, thus requiring more frequent regeneration (Ellis et al. 1990). C. Characterization, Evaluation, and Documentation Characterization of germplasm is a critical factor for the efficient management and utilization of genetic resources. Precise assessment of the genetic relationships among ex situ conserved accessions should allow gene bank curators to eliminate duplicates, form a core collection, acquire new germplasm, initiate genetic and evolutionary studies, and efficiently manage and conserve genetic resources. The presence of duplicate accessions within collections is a burden to gene banks and their users. The increasing size of collections and the decreasing available resources have stimulated gene banks to identify and remove redundant germplasm in order to increase the efficiency of conservation and utilization (van Hintum and Visser 1995; van Hintum and Knupffer 1995; van Hintum et al. 1996; van Treuren et al. 2001). The first stage of characterization is the evaluation of descriptor traits that are diagnostic, generally highly heritable, and usually easily scored in discrete classes. The second stage of characterizing germplasm is evaluation of other traits considered desirable by the breeders, farmers, and consumers of that particular crop. Characters such as plant height, flowering and maturity time, number of branches, number of fruits or yield can all indicate agronomic worth of an accession. Descriptors for chickpea (IBPGR, ICRISAT, and ICARDA 1993), peanut (IBPGR and ICRISAT 1992), broadbean (IBPGR 1983b), grasspea (IPGRI 2000), lentil (IBPGR 1985a), pigeonpea (IBPGR and ICRISAT 1993), Phaseolus (USDA 1998), common bean (IBPGR 1982; INIA, IPGRI, and MADRP 2001a), lima bean (INIA, IPGRI, and MADRP 2001b), scarlet runner bean (IPGRI 2003), tepary bean (IBPGR 1985b), soybean (IBPGR 1984), cowpea (IBPGR 1983a), mung bean (IBPGR 1980), and bambara groundnut (IPGRI, IITA, and BAMNET 2000), as well as multi-crop passport descriptors as listed (http://www.ipgri.cgiar.org), provide an important framework for characterization of legume genetic resources for various morphophysiological, reproductive, and biochemical traits. The passport, characterization, and evaluation data should be easily accessible to the users in a searchable format that readily assists the selection of desired germplasm. The use of the SQL software package has been very helpful for streamlining entry, storage, and retrieval of information on genetic resources. However, the development of complex query systems and decision support tools will greatly enhance the future utilization of the germplasm collection.

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While a germplasm curator or botanist can readily evaluate germplasm for basic descriptors, further evaluation for grain quality, resistance to pests and diseases, tolerance to abiotic stresses, and molecular genetic characterization requires diverse specialized skills. The estimates of genetic relationships can be useful for organizing germplasm for conservation of genetic resources, for the identification of cultivars, for selection of parents for hybridization, for predicting favorable heterotic combinations, and for reducing the number of accessions needed to ensure sampling of a broad range of genetic variability. Molecular characterization of germplasm is a particularly useful tool for assisting gene bank curators to better manage genetic resources, helping them to identify redundant germplasm and to provide users with the most diverse germplasm for applications in research and breeding (Bretting and Widrlechner 1995; Virk et al. 1995; Brown and Kresovich 1996; van Treuren et al. 2001). Accessions with the most distinct DNA profiles are likely to contain the greatest number of novel alleles. It is in these accessions that one is likely to uncover the largest number of unique and potentially agronomically useful alleles. This strategy has resulted in the identification of a significant number of new and agronomically useful quantitative trait loci (QTL) alleles in wild germplasm of rice and tomato (Tanksley and McCouch 1997). Molecular markers also facilitate the genetic mapping of important traits that plant breeders can then use to enhance the power and efficiency of their selection (see section V B). Marker-assisted introgression of agronomic traits from germplasm to elite cultivars offers powerful new mechanisms for efficient use of genetic resources in crop breeding programs (Haussmann et al. 2004). Morphological traits, isozymes, and DNA-based genetic markers are routinely used to measure genetic relatedness among germplasm. However, the conflicting results obtained from different assays illustrate that these relationships are far from simple (Moser and Lee 1994; Powell et al. 1996; Tatineni et al. 1997; Dillmann et al. 1997; Schut et al. 1997; Crouch et al. 2000; Jordan et al. 2003; Soleimani et al. 2002; Hamza et al. 2004). This is not surprising, as the traits and genomic regions reflected by various morphological and molecular markers can be under very different profiles and intensities of natural and artificial selection. However, in contrast to phylogenetic analysis, for effective germplasm utilization diversity assessments need to capture variation in as many loci of potential agronomic importance as possible. Thus, characterizing germplasm with a variety of both morphological and molecular markers is likely to provide the best estimate of genetic diversity if a precise level of differentiation is required for selection of germplasm.

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D. Core Collection Yield improvement in most legume crops has lagged behind the cereals, even where yield increases have occurred, as in the case of chickpea productivity in India, which steadily increased from 482 to 780 kg ha–1 from 1951 to 2000 (Ali and Kumar 2003). Similarly, soybean yield increased in the USA from 1690 to 2561 kg ha–1 from 1961 to 2000 (FAO 2003). Thus, legume yield appears to have reached a plateau and the lack of further significant progress is a cause for concern. One reason for the stagnation or complete absence of progress is that legume breeders have tended to confine themselves to crosses within their working collection, consisting largely of highly adapted materials, and rarely use more diverse germplasm sources. For instance, in 24 years (1978 to 2001) the chickpea breeders at ICRISAT involved only 801 germplasm but 11,383 pre-bred materials in hybridization that resulted in 3430 advanced breeding lines. Similarly, in India’s chickpea program, a nursery of 184 breeding lines evaluated in 2001 included 284 cultivars and breeding lines in their pedigrees but only 13 gene bank accessions (mostly for stress resistance) as parents. This is obviously only a small fraction of the available germplasm diversity of over 17,000 chickpea accessions in ICRISAT’s gene bank. Core collections present a manageable and cost-effective entry point into germplasm collections for identifying parental genotypes with new sources of disease and pest resistance or abiotic stress tolerance. Screening of core collections is usually the most efficient and reliable means of carrying out an initial search of the germplasm collections. Evaluation of larger amounts of germplasm through multi-location trials is both very expensive and time consuming, while large-scale generation of accurate and precise evaluation data from such trials is generally not possible to dramatically reduce the probability of identifying desirable material. Core collections are usually constituted from the 10% of the entire germplasm collection that represents 90% of the collections variability (Brown 1989). These representative subsample collections are developed from the entire collection, using all available information on accessions including the origin and geographical distribution plus characterization and evaluation data. Ten percent of most crop germplasm collections are a much more feasible amount of material for intensive and precise evaluation. In this way, the development of a core collection has the advantage of displaying much of the variability conserved in the gene bank in a limited number of accessions, allowing a researcher to identify new sources of resistance to new isolates or biotypes of diseases and pests at a substantially lower cost than evaluating the full

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collection. In some cases this approach will not lead to the identification of many new sources of favourable genetic variation. Nevertheless, evaluation of the cluster representatives captured by the core collection then provides an efficient means of identifying which areas of the entire germplasm collection warrant more extensive evaluation in the search for more genetic variation associated with the target trait. Core collections also serve as an excellent mechanism for transferring genetic diversity from the primary centers of diversity of a crop to secondary centers. Core collections are available for chickpea, pigeonpea, peanut, common bean, mung bean, pea, lentil, and cowpea (Table 6.11). Most core collections have been designed from global or regional collections held within international agricultural research centers or national program gene banks, while a few have also been developed for wild accessions (Tohme et al. 1996). In legume crops with over 10,000 accessions, even a core collection could be unmanageably large, so a further reduction is also valuable, providing it is not associated with losing too much of the spectrum of diversity. Thus, Upadhyaya and Ortiz (2001) developed a strategy for sub-sampling a core collection to develop a mini-core collection, again based on selecting 10% of the accessions representing 90% of the variability of the larger collection. In this process, the core collection is evaluated for various morphological, agronomic, and quality traits to select a subset of 10% of accessions from this core subset (i.e., 1% of the entire collection) that captures a large proportion (i.e., more than 80% of the entire collection) of the useful variation. Selection of core and minicore collections is based on standard clustering procedures used to separate groups of similar accessions, combined with various statistical tests to identify the best representatives. The mini-core collection for chickpea consisted of 211 accessions, while the peanut mini-core consists of 184 accessions (Table 6.11). Core or mini-core germplasm collections have been used for identifying a range of germplasm with beneficial traits for use in breeding programs. Both germplasm curators who manage gene banks as well as plant breeders who use germplasm in improvement programs have benefited from the development of legume core collections that represent the large variability in the germplasm collections of any given gene bank. Some examples of the benefits of using core collections are described below: 1. Peanut. When 20 agronomic traits were evaluated on 504 accessions of the Asian peanut core collection in multi-location environments, 60 diverse accessions were identified that could be used to broaden the

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Table 6.11. Description of core collections of chickpea, common bean, cowpea, peanut, mungbean, pigeonpea, pea, and lentil. Size and extent of characterization of original collection Crop Chickpea

Common bean

Cowpea Lentil

Core collection

Accessions (No.)

Traits (No.)

16,991

13

Core

1,956

22

Minicore

211

3,315

—

Core

505

Upadhyaya et al. 2001a Upadhyaya and Ortiz 2001 Hannan et al. 1994

388 157 975

47 14 —

52 31

Rodino et al. 2003 Zeven et al. 1999

114

Tohme et al. 1996

24,000

—

Iberian core Netherlands core CIAT wild species core CIAT cultivated core

1,420

Tohme et al. 1995

Core Core

2,078 287

IITA 2002 Simon and Hannan 1995

Description

Accessions (No.) 1,956

Reference

10,227 2,390

— —

Mungbean

1,532

38

Core

152

Bisht et al. 1998

Pea

2,886

—

Core

505

Simon and Hannan 1995

14,310

14

Core

1,704

1,704

31

Minicore

184

4,738

15

Asian core

504

7,432

24

USDA core

831

12,153

14

Core

Peanut

Pigeonpea

1,290

Upadhyaya et al. 2003 Upadhyaya et al. 2002 Upadhyaya et al. 2001c Holbrook et al. 1993 Reddy et al. 2004

genetic base of cultivars (Upadhyaya et al. 2004). In addition, 10 new diverse sources of early maturity (landraces) that were as early as the earliest maturing control (Chico) but produced 25 to 36% more pod yield than in the early maturing, widely adapted cultivar JL 24 (ICRISAT 2002b). Further evaluation of the peanut core identified five accessions that showed tolerance to low temperature but produced higher pod yield (Upadhyaya et al. 2001b). Incorporation of cold tolerance and early maturity into improved genetic background would extend peanut cultivation

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in the Indian north plains. Evaluation of the USDA peanut core collection (Holbrook et al. 1993) identified two accessions that showed 90% less root-knot nematode damage (Holbrook et al. 2000). 2. Chickpea. Evaluation of a chickpea core collection resulted in identification of accessions that had maturity dates similar to those of the early maturing control cultivar but higher seed yields than commercial controls (ICRISAT 2002b). Accessions showing drought (Krishnamurthy et al. 2003) and salinity (Serraj et al. 2004) tolerance have also been identified in the chickpea core and chickpea mini-core collections, respectively. The drought-tolerant accessions had deeper roots than drought-tolerant control cultivar (Krishnamurthy et al. 2003). 3. Pea. Screening of a pea core collection for Fusarium wilt resistance identified 62 accessions with resistance to race 2, 39 accessions with resistance to race 1, and one of wild progenitors with resistance to both races (McPhee et al. 1999). Fusarium root rot (Fusarium solani f. sp. pisi) is another economically important fungal disease of pea in most peagrowing areas around the world. Screening of the pea core collection identified 44 accessions with partial resistance to Fusarium root rot, with a disease severity rating of 2.5 or less on a 0 to 5 scale (where 5 = completely rotted) (Grunwald et al. 2003). 4. Lentil. When the lentil core collection of 577 germplasm accessions was evaluated for resistance to vascular wilt disease, six accessions showed ≤ 5% wilted plants in comparison to 100% wilted plants in the susceptible control (Sarker et al. 2001). From the USDA lentil core collection of 287 accessions (Simon and Hannan 1995), accessions with high grain and fodder yields were identified for use in breeding programs (Tullu et al. 2001) 5. Common Bean. Eleven new sources of resistance to white mold were reported when Miklas et al. (1999) evaluated a subset of the USDA common bean core collection. Similarly, Mahuku et al. (2003) identified 32 accessions that were resistant to angular leaf spot in the CIAT common bean core collection, which had also been screened by Islam et al. (2002) for anthracnose and common bacterial blight resistance. E. Elite Germplasm, Genetic Stock, and Cultivar It is beyond the scope of this review to provide a detailed summary of the literature related to improved grain legumes breeding lines and cul-

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tivars. However, analysis of 24 years (1980–2003) of scientific literature published in the journal Crop Science revealed 570 articles on new grain legume genetic resources, including the registration of grain legume germplasm (102 publications), genetic stocks (12 publications), and cultivars (456 publications). Among the species represented, soybean ranked first with 325 publications, followed by common bean (120), chickpea (38), cowpea (27), pea (24), lentil (17), and pigeonpea (9). Two recent review articles provide a detailed description of the genetic resources of peanut and their utilization in crop improvement programs (Dwivedi et al. 2003; Holbrook and Stalker 2003). Grain legume germplasm and cultivars possess many desirable attributes that have been introgressed into improved genetic backgrounds. For example, germplasm releases or cultivars exist for soybean with variation in seed size (large or small seeded types) and seed composition (high protein content, low oil content, altered fatty acids, low in oligosaccharide, deficient in lipoxygenase isozyme and trypsin inhibitor); for chickpea, pigeonpea, and pea germplasm with high protein content; for pea and lentil with varying cotyledon color (yellow, green and/or red); for common bean and pea with excellent canning quality; for soybean, cowpea, chickpea, pigeonpea, pea, lentil, and common bean with varying degrees of resistance to pests (defoliators, aphids, leaf minor, and nematodes) or diseases (fungal, bacterial, and viruses) and tolerance to drought and/or high temperature; for soybean with resistance to lodging, iron chlorosis, and pod shattering; and for soybean and common bean with adaptation to wide and narrow planting systems. In addition, herbicide tolerant (wild perennial Glycine species) and special-purpose soybeans (for sprouts and fermented products); cold and salinity tolerant chickpea; winter-hardy peas; double-podded chickpeas and peas; leafless peas; genetic or cytoplasmic male sterile pigeonpea and soybean; and extra early-maturing chickpea and common bean are now available that may be used in breeding programs to incorporate these beneficial traits into new genetic backgrounds. F. Wild Species Germplasm Wild species of grain legumes harbor beneficial alleles and genes for improvement of grain quality and yield, resistances to pests and diseases, and tolerance to environmental stresses (Table 6.12). For example, wild relatives have provided resistance to nematodes in pigeonpea, chickpea, and soybean; to Ascochyta blight in chickpea, lentil, and pea; to weevils in common bean, rice bean, and pea; to bruchids in cowpea, chickpea, mung bean, and rice bean; to Fusarium wilt, leaf miner, botrytis gray

214

Table 6.12.

Wild species relatives of grain legume crops reported to possess agronomically beneficial traits.

Crop Chickpea

Trait

Wild species

Pest and disease resistance Fusarium wilt C. bijugum, C. judiacum, C. reticulatum, C. echinospermum, C. pinnatifidum, C. canariense, C. chorassanicum, C. cuneatum, and C. montbreti Cyst nematode C. bijugum, C. pinnatifidum, and C. reticulatum Root-knot nematode Leaf miner Bruchid Bortytis gray mold Ascochyta blight and Fusarium wilt Ascochyta blight Phytophthora Agronomic characters Cold tolerance Drought tolerance High seed protein

C. judiacum, C. pinnatifidum, C. chorassanicum, and C. cuneatum C. reticulatum, C. echinospermum, C. pinnatifidum, C. chorassanicum, C. bijugum, C. judiacum, and C. cuneatum C. reticulatum, C. echinospermum, C. pinnatifidum, C. bijugum, C. judiacum, and C. cuneatum C. bijugum and C. pinnatifidum C. judiacum, C. bijugum, C. pinnatifidum

Reference

Infantino et al. 1996; Croser et al. 2003; Rao et al. 2003 Sharma et al. 1994; Vito et al. 1996; Rao et al. 2003 Sharma et al. 1994 Croser et al. 2003 Croser et al. 2003 Stevenson and Haware 1999; Rao et al 2003 Stamigna et al. 2000

C. reticulatum, C. echinospermum, C. pinnatifidum, C. bijugum, C. judiacum, C. cuneatum, and C. montbreti C. echinospermum

Croser et al. 2003; Rao et al. 2003 Croser et al. 2003; Knights et al. 2003

C. reticulatum, C. echinospermum, C. pinnatifidum, C. bijugum, C. judiacum, and C. microphyllum C. microphyllum C. bijugum and C. reticulatum

Croser et al. 2003 Croser et al. 2003 Rao et al. 2003

Common bean

Cowpea

Lentil

Pest resistance Mexican bean weevil Agronomic characters Salinity Pest resistance Cowpea mottle carmo virus Bruchid

Wild P. vulgaris

Acosta-Gallegos et al. 1998

P. micranthus, P. mcvaughii, P. lunatus, P. filiformis, and P. vulgaris

Bayuelo-Jimenez et al. 2002

V. vexillata

Ogundiwin et al. 2002

V. riukiuensis resistant to Callosobruchus maculates and V. reflexo-pilosa to C. chinensis

Tomooka et al. 1992

Disease resistance Ascochyta blight Vascular wilt Agronomic characters Winter hardiness

Lens ervoides and L. odemensis L. nigricans subsp. ervoides and L. culinaris subsp. orientalis

Ahmad et al. 1997 Bayaa et al. 1995

L. culnaris subsp. orientalis

Hamdi et al. 1996.

Mungbean

Pest resistance Bruchid

Vigna sublobata

Kaga and Ishimoto 1998

Pea

Pest and disease resistance Aschochyta blight P. fulvum and P. humile Weevil P. fulvum Agronomic characters Cold tolerance

Peanut

P. elstius (JI 2055) and P. elstius (JI 1398)

215

Pest and disease resistance Rust, leaf spots, nemaSeveral accessions from secondary and tertiary gene pool todes, defoliators, and virus

Ali et al. 1994a; Wroth 1998 Hardie and Clement 2001; Clement et al. 2002 Ali et al. 1994b Holbrook and Stalker 2003; Dwivedi et al. 2003

(continued )

216 Table 6.12. Crop Rice bean Soybean

(continued) Trait Pest resistance Bruchid

Wild species V. umbellata

Pest and disease resistance Sclerotinia stem rot G. tomentella and sudden death syndrome Rust G. tomentella Soybean mosaic G. canescens, G. clandestine, and G. tomentella virus (SMV) Brown spot G. clandestina (PI 255745) and G. tabacina (PI 319697 and PI 321392) Cyst nematode G. soja and G. gracilis G. soja Agronomic characters High linolenic fatty G. soja acid High protein content G. soja and G. gracilis Salinity tolerance G. argyrea (accession 1626), G. clandestina (accession1388 and 1389), G. microphylla (accessions 1143 and 1195) Herbicide (2,4-D) G. latifolia and G. microphylla resistance

Reference Tomooka et al. 2000 Hartman et al. 2000

Schoen et al. 1992 Zhuang et al. 1996 Lim and Hymowitz 1987 Lin 1996 Wang et al. 2001 Pantalone et al. 1997b Lin 1996 Pantalone et al. 1997a Hart et al. 1991

Pigeonpea

Pest and disease resistance Pod fly and pod wasp C. scarabaeoides, R. bracteata, C. albicans, and F. stricta Phytophthora blight C. platycarpus and C. serieus Pod borer C. acutifolius, C. albicans, C. platycarpus, C. reticulatus, C. scarabaeoides, and C. serieus Pod wasp C. albicans and C. scarabaeoides Pod fly and pod borer C. scarabaeoides Sterility mosaic virus (SMV) Pod fly Root-knot and reniform nematodes Cyst nematode Reniform and cyst nematodes Agronomic characters Salinity tolerance Drought tolerance Photoperiod insensitivity High seed protein

Sharma et al. 2003 Rao et al. 2003

C. scarabaeoides, R. densiflora, and Flamingia spp. C. platycarpus

Rao et al. 2003 Rao et al. 2003 Verulkar et al. 1997; Romeis et al. 1999 Kulkarni et al. 2003; Rao et al. 2003 Saxena et al. 1990; Rao et al. 2003 Sharma et al. 1993b; Rao et al. 2003 Sharma et al. 1993a Sharma 1995

Atylosia platycarpa, Rynchosia, Dunbaria, A. albicans, A. cajanifolia, C. platycarpus C. reticulates C. platycarpus and C. sericeus

Subbarao et al. 1991; Rao et al. 2003 Rao et al. 2003 Rao et al. 2003

C.albicans, C. mollis, and C. scarabaeoides

Rao et al. 2003

C. scarabaeoides, C. albicans, C. crassus, C. lineatus, and C. sericeus A. scarabaeoides, C. acutifolius, C. albicans, C. lineatus, C. scarabaeoides, and C. sericeus C. scarabaeoides, R. reniformis, and C. reticulatus

217

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mold, and phytophthora rot in chickpea; to pod fly, pod wasp, and sterility mosaic virus in pigeonpea; and to Sclerotinia stem rot, sudden death syndrome, rust, soybean mosaic virus, and brown spot in soybean. Wild species are also reported to possess salinity tolerance in pigeonpea, soybean, and common bean; drought tolerance in pigeonpea, chickpea, and pea; cold tolerance in chickpea; herbicide tolerance in soybean; and winter hardiness in lentil and pea. Wild species have also been reported with high seed protein content in chickpea, pigeonpea, and soybean; high linoleic fatty acid in soybean; and photoperiod insensitivity in pigeonpea. For a detailed summary of peanut wild genetic resources possessing beneficial traits, the reader is referred to Dwivedi et al. (2003) and Holbrook and Stalker (2003). These examples convincingly demonstrate the potential of wild genetic resources to contribute agronomic traits that are suboptimum or entirely absent in cultivars (see section V A).

IV. IMPACT OF GENETIC RESOURCES IN CONVENTIONAL LEGUME BREEDING Plant breeding has contributed to increased crop productivity by systematically creating new high yielding and better adapted genotypes. Crop genetic resources have played an important role in providing novel genetic variation that legume breeders have used in improvement programs to develop these new genotypes. However, it is generally agreed that integrated approaches are necessary to continue to increase agricultural production and profitability by capturing the full benefit of plant biodiversity. The Global Plan of Action (FAO 1996) proposed a number of measures to foster greater utilization of genetic resources in crop improvement, including the expanded creation, characterization, and evaluation of core collections; increased genetic enhancement and base-broadening efforts; development and commercialization of underutilized species; development of new markets for local varieties and “diversity-rich” products and concomitant efficient seed production and distribution; comprehensive information systems for PGR; and promoting public awareness of the value of PGR for food and agriculture. There are already many success stories related to the use of both elite and exotic gene pools of legume genetic resources in the development of improved genotypes that are better adapted to diverse environments, that possess resistance to abiotic and biotic stresses, and that are expected to increase legume production in target production systems across the globe.

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A. Germplasm Distribution There are more than one million accessions of legume genetic resources documented in gene banks around the world, of which less than 20% (187,418) are held by international agricultural research centers (IARCs), which, as a result, have been major sources of seed distribution to researchers in the developing and developed worlds (FAO 1998). Germplasm distribution from CIAT, CIMMYT, ICARDA, ICRISAT, ILRI, and IRRI was tracked during a period from 1973 to 2001 and over 80% of the one million samples distributed from these gene banks during this period were sent to organizations in developing countries (mostly to universities and national agricultural research institutes). Thus, much of the germplasm (73%) that emerged from those countries through germplasm collections flowed back as distributed seed (Raymond 2001). 1. Chickpea. At ICRISAT, the pattern of germplasm distribution from 1973 to 1998 was analyzed; it was found that 112,818 samples of 16,311 chickpea accessions had been supplied to scientists in 81 countries, with countries in Asia receiving the maximum number of accessions followed by countries in the Americas, Europe, and Oceania. A maximum of 302 requests was received for a single accession, ICC 4973 (L 550), a kabuli cultivar from India. Shannon-Weaver diversity index (H′) (Shannon and Weaver 1949) analysis of the accessions distributed was similar to the diversity in the entire collection. Chickpea germplasm distribution has resulted in the release of 15 varieties in 12 countries up until 1999 (Table 6.13). 2. Peanut. Up until 1998, ICRISAT distributed 14,180 accessions of peanut (94.7% of the full collection), with only 794 accessions (5.3%) having never been requested. Countries in Africa received the maximum number of accessions (92.3%) followed by countries in Asia (76.6%), Europe (5.6%), the Americas (5.3%), and Oceania (2.2%). A maximum of 297 requests from 73 countries were received for the accession ICG 799 (Kadiri 3), a hypogaea cultivar from India. Most requests were received for accessions belonging to subsp. hypogaea followed by subsp. vulgaris, and subsp. fastigiata. The diversity index H′ of the accessions distributed was similar to the diversity of the entire collection, indicating that the diversity available in the entire collection has been well sampled by the users. Eleven varieties have been released in 12 countries from the peanut germplasm distributed from ICRISAT (Table 6.13). 3. Pigeonpea. ICRISAT distributed 65,747 seed samples of 10,648 pigeonpea accessions during the period 1974 to 2003, revealing that

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Table 6.13. List of chickpea, pigeonpea, peanut, broadbean, lentil, common bean, cowpea, and soybean genetic resources released as cultivars in different parts of the world. Crop

Accession number and country of release

Broadbean

BPL 710 (Icarus) in Australia; ILB 1270 (Giza Blanka) in Egypt; ILB 938(Giza 461) in Egypt; ILB 1269 (Barkat) in Iran.

Chickpea

ICC 237 in Oman; ICC 552 (Yezin1) in Myanmar; ICC 3274 (Barichhola 7) in Bangladesh; ICC 14880 (Hira) in Australia; ICC 4923 (Jyothi) in India; ICC 4951 in Myanmar; ICC 4998 (Bina Sola 2) in Bangladesh; ICC 6098 (Radha) in Nepal; ICC 8521 (Aztec) in USA; ICC 8649 (Shendi) in Sudan; ICC 11879 in Turkey, Algeria, Morocco, and Syria (Ghab 1); ICC 13816 in Algeria (Yialousa), Italy (Sultano), and Syria (Ghab 2); ICC 14559 (Barichhola 5) in Bangladesh; and ICC 14911 in Turkey and Morocco; ICC 4944 (Keyhman) in Myanmar.

Common bean

G76 in Cuba, Chile (as Redkloud) and Peru (Rojo Mollepata); G685 in Burundi, Kenya and Rwanda (Vunikingi); G858 in Rwanda (Muhondo 6), G1753 in Argentina; G2331 in Burundi (Muhondo) and Congo (Kihembe); G233 in Congo (Aliya), Kenya, Rwanda and Uganda (Umabano); G2579 in Panama (Renacimiento); G2816 in Burundi (Mavutaninka) and Ethiopia (Gofta); G2829 in Peru (Gloriabamba); G2858 in Congo (Maharagi Soja); G3410 in Peru and Rwanda (Puebla); G3645 in Peru (Jamapa); G3680 in Brazil (Ouro Negro), G3807 in Ecuador (Bayito); G4017 in Bolivia, Peru and Swaziland (Carioca); G4445 in Canada, China, Zimbabwe (Ex Rico) and Ethiopia (Awash/Bunsi); G4450 in Peru (Royal Red); G4494 in Burundi, Malawi, Mozambique , Panama (Calima) and Tanzania (Lyamungu 90); G4495 in Costa Rica (Porillo Sintético); G4523 in Panama (ICA Palmar), Peru (INIA 17), Rwanda (Rubona 5); G5476 in Tanzania (SUA90); G5773 in Bolivia, Costa Rica, Cuba, Mozambique, Peru (ICA Pijao), Guatemala (Suchitán) and Venezuela (Tenerife); G5853 in Peru (Cristál Blanco); G7930 in Peru (Alubia); G7951 in Bolivia (Araona) and Burundi; G11239 in Ethiopia (Mexican 142); G11780 (INIAP 416-Canario); and G12488 (ICA Llanogrande) in Colombia; G13369 and G13374 in Tanzania; G13614 in Rwanda (de Celaya); G13625 in Burundi; G14013 in Cuba (Guamá 23); G17702 in Bolivia (Carioca 80) (Voysest 2000).

Cowpea

IITA bred and shared a range of cowpea lines combining multiple resistances to diseases and pests, early maturity and preferred seed types to over 65 countries.

Lentil

ILL 4400 in Algeria; ILL 481 (Indian head) in Canada; ILL 5523 (Centinela) in Chile; ILL 4605 (Precoz) in Egypt and Morocco and Pakistan (Manserha 89); ILL 358 and NEL 2704 in Ethipoia; ILL 5582 in Iraq, Jordan and Libya; ILL 4402 in Nepal; ILL 813 (Rubatab) in Sudan; ILL 942 (Erzurum 89), ILL 1384 (Malazgirt 89), and ILL 854 (Sazak 91) in Turkey; and ILL 784 (Crimson) in USA.

6. GENOMICS TO EXPLOIT GRAIN LEGUME BIODIVERSITY IN CROP IMPROVEMENT Table 6.13.

221

(continued)

Crop

Accession number and country of release

Peanut

ICG 7886 (Cordi payne) in Jamaica; ICG 7827 in Phillipines, Myanmar (Sinpadetha 2), and Sierra Leone; ICG 2974 in Myanmar (Sinpadetha 3), Tanzania (Johari), and Gambia; ICG 273 (Sedi) in Ethiopia; ICG 221 in Swaziland; ICG 1697 (Singa) and ICG 1703 (Panter) in Indonesia; ICG 2271 in Nepal; ICG 7794 in Ethiopia; ICG 12991 in Uganda (Serenut 4T) and Malawi (Baka); ICG 7898 in Mauritius.

Pigeonpea

ICP 14770 (Abhaya) in India; ICP 14056 in Australia and Fiji (Royes); ICP 8863 (Maruti) in India; ICP 11384 (Bageswari) in Nepal; ICP 9145 (Nandolo wa nswana) in Malawi; ICP 7035 (Kamica) in Fiji; and ICP 6997 (Rampur Arhar) in Nepal.

Soybean

TGX 306-036C and TGX 536-02D in Nigeria and Ghana; TGX 297192C and TGX 813-6D in Ghana; TGX 814-76D and TGX 849-294D in D.R. Congo; TGX 849-313D, TGX 923-2E, TGX 1019-2EB, TGX1019-2EN, and TGX1830-20E in Nigeria; TGX 1440-1E, TGX 1448-2E, and TGX 1740-2F in Nigeria, Togo and Benin; TGX 14851D in Nigeria, Benin, Togo and Uganda.

78.6% of the pigeonpea accessions stored in the gene bank have been accessed. The predominant users have been scientists in India followed by those in Kenya, Uganda, Malawi, Venezuela, and Australia. The germplasm accession ICP 7035 has been in greatest demand as it belongs to the vegetable type, possesses large seeds, and is resistant to sterility mosaic virus disease. Based on the ICRISAT-supplied pigeonpea germplasm, seven cultivars have been released in five countries (Table 6.13). 4. Broadbean and Lentil. ICARDA distributed 2,716 accessions of broadbean, 6,230 accessions of lentil, and 638 accessions of wild Lens species during 1990–94, most of them to developing countries. The greatest use of this material has been for identifying new sources of resistance to rust, Ascochyta blight, chocolate spot, and striga. Many broadbean-breeding programs in various countries were established using ICARDA germplasm resources. Thirteen lentil accessions and four broadbean accessions have been released in 14 and 3 countries, respectively (Robertson 1997; Robertson and Erskin 1997) (Table 6.13).

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5. Common Bean. CIAT distributed 80,000 Phaseolus bean germplasm samples during the period from 1979 to 1994. Fifty-five of the 203 common bean cultivars released in 37 countries are derived from genebank accessions that were included in CIAT international bean nurseries distributed worldwide during those years (Hidalgo and Beebe 1997; Voysest 2000) (see also Table 6.13). B. Impact of Domesticated Germplasm on Breeding Gains The distribution of grain legume genetic resources has had a profound impact particularly on the enhancement of biotic stress resistance and abiotic stress tolerance. For example, in common bean, CIAT and partners have used gene-bank accessions in the development of improved germplasm/cultivars with resistance to bean golden yellow mosaic virus, bean common mosaic necrosis virus, bean pod weevil, common bacterial blight, rust, web blight, as well as greater tolerance to high temperatures for Central America (Beebe et al. 1993, 1995; Beaver et al. 2003). In the case of cowpea, IITA and partners have developed unique germplasm with characteristics such as snap-type pods, green manure/cover crop capabilities, heat and chilling tolerance, delayed leaf senescence, differences in carbon isotope discrimination, harvest index, rooting and plant water- and nutrient-relations traits, resistance to root-knot nematodes, fusarium wilt, Striga, thrips, aphid, lygus bug, and cowpea weevil, and for quality traits including all-white and sweet grains. This has produced a valuable resource for all future breeding of cowpea cultivars for Africa and the USA (IITA 2001; Hall et al. 2003; Singh et al. 2003a). An improved Striga-resistant cowpea variety IT97K-499-38 yielded 50% to 300% higher than the local varieties in Striga infested fields in Benin Republic that also caused high percentage of suicidal germination of Striga hermonthica seeds (IITA 2001). Early maturing chickpea germplasm (ICCV 2 and ICCV 96029) has enabled chickpea to escape damage due to drought, cold, and Helicoverpa at flowering and podding stages and thus opened up new possibilities for growing chickpea in semiarid and arid regions globally (Kumar and Abbo 2001). IITA cowpea breeding lines combine multiple resistances to diseases and pests, early maturity and preferred seed types (Singh et al. 2003a). This material bred initially at IITA stations in Nigeria (Ibadan and Kano) was sent for testing worldwide and 65 countries released cultivars. In 2002, 409 sets of cowpea trials including 140 cultivars and breeding lines were shared by IITA with 105 partners in 24 countries (IITA 2002). In 2003, the farmer-to-farmer diffusion of an improved

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223

cowpea cultivar included more than 27,000 farmers in Kano State of Nigeria (IITA 2003). IITA and African national programs are making concerted efforts to breed resistant soybean cultivars for home and industrial uses, which are driving intensive cultivation of soybean in several regions, particularly in Nigeria where about 80 soybean-based agro-processing businesses are flourishing (Singh et al. 2004). The resultant cultivars combine disease resistance with seed longevity, promiscuous nodulation, early maturity, and resistance to pod shattering. After thorough testing in mutilocation trials, many of these cultivars were released in Nigeria, Ghana, D.R. Congo, Benin, Togo, and Uganda. C. Use of Wild Germplasm There has been very limited use of exotic gene pools in legumes in comparison to the cereals or horticultural crops, in which the introduction of alien germplasm in breeding programs has been shown to have promise in widening the germplasm base of these crops (reviewed by Tanksley and McCouch 1997). The few successful examples of transfer of beneficial traits from wild legume accessions or species to legume breeding lines or cultivars include genes for resistance to rust, leaf spots, and nematodes in peanut (reviewed by Dwivedi et al. 2003; Holbrook and Stalker 2003); for cleistogamous flowers (Saxena et al. 1990a), high protein (Saxena et al. 1987), and cytoplasmic nuclear male sterility (Saxena and Kumar 2003) in pigeonpea; for resistance to cyst nematode and phytophthora root rot (Malhotra et al. 2002; Knights et al. 2003) as well as cold tolerance in chickpea (ICARDA 1996); and for arcelin-based bruchid resistance in common bean (Osborn et al. 1988; Acosta-Gallegos et al. 1998). The effectiveness of these novel traits has often been dramatic. For example, pigeonpea natural outcrossing species suffers from rapid genetic deterioration among germplasm accessions, genetic stocks, and cultivars but breeding has led to the development of a few elite partially cleistogamous lines with tenfold lower rates of outcrossing than the typical pigeonpea cultivars in India (Saxena et al. 1993). Similarly, cytoplasmic male sterility (CMS) lines originating from Cajanus scarabaeoides have opened up the possibility of producing the CMSbased hybrids in pigeonpea (Saxena and Kumar 2003). Arcelins are abundant, lectin-like seed storage proteins that are present in wild P. vulgaris. Seven allelic variants of arcelin, designated as arcelin 1 to arcelin 7, have been reported (Osborn et al. 1986; Lioi and Bollini 1989; Santino et al. 1991; Acosta-Gallegos et al. 1998). High levels

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of resistance to the bean weevil in wild P. vulgaris populations of Mexican origin (Schoonhoven et al. 1983) are associated with the arcelin (arcelin 1 or arcelin 5) locus itself or a factor linked to it (Osborn et al. 1988; Goossens et al. 2000). Breeding lines derived from a cross between a wild accession (G02771) and cultivated P. vulgaris showed high levels of resistance to weevil (Kornegay et al. 1993). The genes for promiscuous nodulation and seed longevity that led to the success of soybean cultivars in West Africa were introgressed from ‘wild’ sprawling soybean accessions (TGm 737, 719 618, 579, 577), mostly from Indonesia (Ortiz 2004b). Of course, the most difficult task for IITA soybean breeders was to recover the agronomic background of elite cultivars while maintaining the introgressed traits (E. Kueneman, FAO, pers. commun.). D. Conclusions from Conventional Manipulation of Genetic Resources There is a great abundance of useful genetic variability across the primary, secondary, and tertiary gene pools of most important legume crops. However, it is the elite breeding lines and landraces of the cultigen that breeders continue to focus on while the vast resources of the wild species remain largely untapped despite often containing the best sources of pest and disease resistance or tolerance to environmental stresses. The underlying reasons for the underutilization of crop related biodiversity are complex, varied, and often crop specific. Nevertheless, there are three main limiting factors that appear to be common to most crops: the lack of accurate and precise multilocational characterization of germplasm, the lack of rational systematic entry points into the vast international collections, and the lack of robust cost-effective tools to facilitate the efficient utilization of exotic germplasm in plant breeding programs. The development of core germplasm collections offers an important strategic solution to the first two constraints, even though there is some indication that current core and mini-core collections may be somewhat confounded by the type of phenotype data used in their assignments. Nevertheless, their existence has facilitated intensive phenotypic evaluation of diverse germplasm, which provides an essential foundation for future multidisciplinary efforts and refinements. This has already resulted in the identification of countless new sources of pest and disease resistance or tolerance to environmental stresses that had been overlooked by previous more extensive (but necessarily less intensive) screening processes. Clearly, the current challenge is to now define genetic mini-core collections that represent the total genetic diversity of gene banks that can equally serve current breeding criteria and as yet

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undetermined new breeding goals. This will then provide a highly valuable systematic entry point to germplasm collections for the entire research and breeding community. Meanwhile, rapid progress is being made in our knowledge and ability to manipulate these novel sources of economically important genetic variation. The opportunities offered in this respect by recent advances in legume genomics is the focus of the remaining sections of this paper.

V. MOLECULAR-ENHANCED STRATEGIES FOR MANIPULATING NOVEL GENETIC VARIATION FOR LEGUME BREEDING A. Interspecific Hybridization Inter-specific hybridization is an important method for expanding the gene pool available to grain legume breeders by introgressing genes from wild relatives of each crop (see Table 6.12 and also section III F). However, reproductive incompatibility mechanisms between species, embryo mortality, hybrid sterility, and limited genetic recombination present major barriers to greater use of wild germplasm from the secondary and tertiary gene pools (Muehlbauer et al. 1994). Reproductive isolation between a cultigen and its tertiary gene pool can be the result of crossing barriers both at the pre- and post-zygotic levels. Pre-zygotic barriers include biochemical incompatibilities that stop pollen germination, restrict pollen tube growth in the style or ovary, or prevent growth of the pollen tube towards the micropyle or embryo sac (Stalker 1980). Post-zygotic barriers include abnormal endosperm or embryo development, and chromosome elimination or aberration due to inconsistencies in parental genomes often resulting from ploidy level differences or cytoplasmic incompatibilities (Cooper and Brink 1940). A list of the successful crosses made between grain legume cultigens and their wild species is provided in Table 6.14 along with an indication of the beneficial traits available from these inter-specific hybridization programs. Inter-specific crosses have been useful for incorporating resistance to nematodes in chickpea and groundnut; resistance to rust and leaf spots in groundnut; resistance to bruchid in mung bean and cowpea; resistance to ascochyta blight in pea; resistance to common bacterial blight in common bean; tolerance to drought and cold temperature in chickpea; tolerance to salinity in pigeonpea; and improved agronomic traits and seed quality in soybean, chickpea, and pigeonpea. Inter-specific hybridization in pigeonpea has generated progeny with unique characteristics such as dwarf stature, new types of cytoplasmic

226 Table 6.14.

Examples of successful gene introgression from wild species to cultivated grain legume crops.

Crop

Source of introgressed trait

Description of interspecific derivatives

Reference

Successful trait introgression from wild species to the cultigen Chickpea

C. reticulatum and C. echinospermum

Resistance to cyst nematode, drought and cold tolerance, high biomass, and earliness.

Malhotra et al. 2003; Singh and Ocampo 1997

Common Bean

P. acutifolius

Introgression of common bacterial blight resistance into common bean.

Rava et al. 1996

Mungbean

V. sublobata

Kaga and Ishimoto 1998

V. glabrescens

Bruchid resistance incorporated into cultivated mungbean (V. radiata). Pest resistance incorporated into V. radiata.

Pea

P. fulvum (JI1006)

Ascochyta blight resistance incorporated.

Wroth 1998

Peanut

Several species

Resistance to rust, leaf spots, nematodes, insect pests, and peanut bud necrosis virus disease.

Dwivedi et al 2003; Hoolbrook and Stalker 2003

Soybean

G. soja

Introgressed lines carrying the PI407305 haplotype at QTL locus demonstrated 9.4% yield advantage over control genotypes. Selected introgressed lines showed profuse pod production and branching, large seeds and strong stems, high protein and fat content.

Concibido et al. 2003; Wang et al. 2004

G. soja and G. gracilis

Chen et al. 1989

Lin 1996

Viable hybrids produced from crosses between wild species and cultivated crops Cowpea

V. vexillata

F1 hybrid produced between V. vexillata × V. unguiculata using in vitro embryo rescue procedure.

Gomathinayagam et al. 1998; Ogundiwin et al. 2002

Lentil

L. orientalis, L. odemensis, L. ervoides, and L. nigricans

Viable hybrids produced between L. culinaris and the four wild species.

Ahmad et al. 1995

Pigeonpea

C. platycarpus

Using the embryo rescue technique, F1 hybrids were produced but these were completely pollen sterile. F1 hybrids between C. cajan and C. reticulatus produced.

Mallikarjuna and Moss 1995

F1 and BC1 hybrids produced. Genetic introgression of salinity tolerance from A. albicans to C. cajan demonstrated in the F1.

Mallikarjuna and Saxena 2002 Subbarao et al. 1990

Fertile plants produced from backcross introgression into G. max from tertiary gene pool species G. tomentella.

Singh et al. 1993

C. reticulatus var. grandifolius C. acutifolius A. albicans Soybean

G. tomentella

Reddy et al. 2001

227

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male sterility, and cleistogamous flowers (Reddy 1990). Products of inter-specific hybridization are not usually released directly as varieties due to deleterious linkage drag usually associated with introgression of traits from wild species. However, ‘Coan’ and Nema TAM in peanut (Simpson and Starr 2001; Simpson et al. 2003) and Tara and Jules in common bean (Muñoz et al. 2004) are notable exceptions, where deleterious linkage drag to a large extent has been overcome. They have been released in the United States. In general, inter-specific derivatives are treated as pre-breeding products that are useful for introducing new sources of variation into the initial cycles of a breeding program. The most useful tertiary gene pool species for common bean breeders has been P. acutifolius or tepary bean, which offers many sources of resistance to diseases and pests and tolerance to environmental stresses (Pratt and Gordon 1994; Mejía-Jiménez et al. 1994). Congruity backcrossing has been shown to help in the transfer of desirable quantitative traits between the two species by raising recombination, although total rates of introgression still remain low (Mejía-Jiménez et al. 1994; Muñoz et al. 2004). Attempts to incorporate useful genes of rice bean (V. umbellata) into adzuki bean (V. angularis) have been even more difficult due to reproductive barriers between the two cultigens. However, another wild relative of adzuki bean, V. riukiuensis, is cross compatible with both adzuki and rice beans and therefore may serve as a bridging species (Siriwardhane et al. 1991). In the case of broadbean, no wild progenitors are known and there are no reported cases of interspecific hybridization between cultivated broadbean and any of the wild Vicia species.

B. Linkage Mapping and QTL Detection 1. Molecular Markers and Genetic Linkage Maps. High-density genetic linkage maps are a useful basis for identifying markers tightly linked to quantitative trait loci (QTL) that contribute to economically important traits (Paterson et al. 1988; Lander and Botstein 1989), for cloning gene(s) by chromosome walking (Wicking and Williamson 1991), and for developing marker-assisted selection of desirable genes in breeding programs (Burr et al. 1983; Tanksley et al. 1989). A wide range of marker techniques have been used for linkage mapping and QTL detection in legumes, including those based on morphological and biochemical (isozymes and proteins) assays, hybridization assays such as RFLP (restriction fragment length polymorphism), and polymerase chain reaction (PCR)-based assays such as RAPD (random amplified polymorphic

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229

DNA), AFLP (amplified fragment length polymorphism), and SSR (simple sequence repeats) or microsatellites. The high polymorphism, high reproducibility, easy automation, and codominant nature of microsatellite markers have led to them becoming the assay of choice for marker-assisted selection. The AFLP marker system remains the most powerful and cost-effective assay for background selection in marker-accelerated backcross programs. In many crop plants, including some of the legumes, expressed sequence tags (ESTs) from tissue specific cDNA libraries are being increasingly used as candidate gene markers. However, EST markers generally detect a low level of polymorphism in intra-specific mapping populations and thus need to be converted to CAPS (cleaved amplified polymorphic sequences) (Caranta et al. 1999) or SNP (single nucleotide polymorphism) markers before routine use in mapping and molecular breeding. Large-scale SNP marker development in legumes has been inspired by the large-scale EST sequencing and development of high-density genetic linkage maps in humans (1.42 million markers) (The International SNP Map Working Group 2001) and in model systems such as rice (Oryza sativa) (Nasu et al. 2002) and Arabidopsis thaliana (Cho et al. 1999; Drenkard et al. 2000; Jander et al. 2002; Schmid et al. 2003; Torjek et al. 2003). SNP development has also been initiated in the model legumes, M. truncatula and L. japonicus (Table 6.15). SNP markers offer several key advantages over conventional genetic markers; they are biallelic, codominant, highly abundant, capable of high throughput genotyping, have low mutation rates, and are often linked to genes (Kwok and Gu 1999). Soybean is the most advanced legume crop with regard to marker technologies, already having a large set of SNP markers, while most other legume crops have only progressed to routine application of SSRs and/or ESTs with a few proof-of-concept SNP markers in chickpea, common bean, cowpea, peanut, mung bean, pea, and pigeonpea (Table 6.15). Thus, there is an urgent need to accelerate marker development in most of the legumes, particularly the research-neglected species. Genetic linkage maps are available for both model and crop legumes with linkage groups aligned to the haploid chromosome complement of the two species (Table 6.16). These maps have an average marker density of 4.24 cM for M. truncatula, and 0.6 to 2.6 cM for L. japonicus. Genetic linkage maps for the legume crops are generally much less saturated and have been derived from both (i) inter-specific populations as in chickpea and lentil (Table 6.17) or mung bean, cowpea, adzuki bean, and peanut (Table 6.18) and (ii) intra-specific populations as in pea, chickpea, broadbean, and grasspea (Table 6.19), or soybean, common bean, and cowpea (Table 6.20). Most of the initial genetic maps reported

230

Table 6.15. Marker

Overview of SSR, EST, SNP, and CAP markers reported in model species and grain legume crops. Summary of the marker information

Reference

Crop Legumes Broadbean SSR Chickpea SSR

EST Common bean SSR

EST SNP Cowpea SSR

12 SSR located on chromosome 1.

Pozarkova et al. 2002

10 SSR from genomic library of C. arietinum cultivar Pusa 362. 218 SSR primers designed from 389 microsatellite containing clones.

Sethy et al. 2003 Winter et al. 1999 Huttel et al. 1999 Winter et al. 1999

43 of the 53 clones from chickpea genomic libraries selected for sequencing showed the presence of microsatellites. 2,860 EST sequences from subtracted root library.

ICRISAT 2002

57 SSR from coding and non-coding sequences. Isolated 21 SSR (GA)n from a highly microsatellite-enriched library. Isolated, cloned, and sequenced genomic DNA fragments containing 68 microsatellite loci from 3 Phaseolus vulgaris genomic libraries, and number of alleles ranged from 1–14 alleles per locus when tested on 21 diverse genotypes. 49 SSRs from common bean and 12 from the genus Vigna. Forty-four primer pairs derived from cowpea microsatellite-enriched libraries constructed from the DNA of the breeding line IT84S-2264-2; one primer pair each derived from the sequences of the 1-amino-cycloprane-1-carboxylate oxidase cDNA of mungbean and the protein kinase cDNA of mothbean. 728 EST sequences submitted to dbEST. SNPs detected between the homologous sequences of the 1150-bp DNA fragments on COK-4 locus from anthracnose resistant (SEL 1308) and susceptible (Black Magic) genotypes.

Blair et al. 2003 Yaish and Vega 2003 Gaitan-Solis et al. 2002

Forty-four SSR isolated from cowpea microsatellite-enriched libraries constructed from the DNA of a breeding line IT84S-2264-2, and one SSR each from sequences of the 1-aminocycloprane-1-carboxylate oxidase cDNA of mungbean (V. radiata) and the protein kinase cDNA of mothbean (V. aconitifolia).

Li et al. 2001

Yu et al. 1999 Li et al. 2001

Hernandez et al. 2004 Melotto and Kelly 2001

SSR

29 SSR primer pairs generated from defense-related ESTs derived from L. sativus cDNA library.

Skiba et al. 2003

Mungbean SSR

23 microsatellite loci and six cryptically simple sequence repeats.

Kumar et al. 2002

171 SSR from 663 sequences retrieved from genebank/EMBL databases: CT/AG and TCT/AGA most frequent nucleotides. 318 SSR (http://www.agrogene.com/ssrdevelopment.htm). 15 SSR

Burstin et al. 2001

110 SSR from genomic cDNA libraries of peanut cultivar, Florunner. Six SSR. Several SNP detected while comparing the coding sequences from the high and low oleic acid genotypes: two (at 442 and 448 bp) associated with the high O/L oil trait.

Ferguson et al. 2004 Hopkins et al. 1999 Lopez et al. 2000

20 SSR.

Burns et al. 2001

600 SSR 120,000 EST from more than 50 cDNA libraries, coalesced into 16,928 contigs and 17,336 singletons. On average, each contig composed of 6 ESTs and spanned 788 bp. 308,582 EST 29,540 EST obtained by sequencing a cDNA library constructed from salicylic acid treated soybean seedlings. 216 SNP detected from 116 gene-derived STSs. Eight SNP detected from six-converted AFLP markers representing 996 bp sequences from alleles of each of Forrest (resistant to soybean cyst nematode) and Essex (susceptible to cyst nematode). Two SNPs (A519-1SNPs) reported within approximately 400 bp of the sequence of RFLP locus A519-1.

Cregan et al. 1999a Shoemaker et al. 2002

Pea SSR

Peanut SSR SNP Pigeonpea SSR Soybean SSR EST

SNP

231

Scarlet runner bean EST 20,120 ESTs from Phaseolus coccineus embryo development

— Ford et al. 2002

Rudd 2003 Tian et al. 2004 Zhu et al. 2003a Meksem et al. 2001b

Coryell et al. 1999

Rudd 2003 (continued )

232

Table 6.15. Marker

(continued) Summary of the marker information

Reference

Model legumes Medicago truncatula SSR Five SSRs obtained from microsatellite-enriched genome libraries and four from sequences available in GenBank. EST 899 ESTs, of which 603 have homology to known genes, from root-hair-enriched cDNA library (http://bio-SRL8.stanford.edu). EST 10,500 EST markers from 28,000 cDNAs obtained from 5- to 13-day old immature seeds. 40% of these ESTs have no match in the public sequence databases suggesting that many represent mRNAs derived from genes specifically expressed in seeds (www.plantphysiol.org). EST Over 140,000 ESTs sequences from 30 cDNA libraries representing various vegetative and reproductive organs. Of these, 340 putative gene products or tentative consensus sequences expressed solely in root nodules, and are represented by two to 379 ESTs (http://www.tigr.org/tdb/mtgi). EST ESTs to characterize the sets of genes expressed in roots during Rhizobial and/or mycorrhizal symbiosis: 21,473 5′—and 3′—ESTs grouped into 6359 clusters, corresponding to distinct virtual genes, along with 52,498 other M. truncatula ESTs available in the dbEST database. EST 36,976 of 189,919 unique EST sequences. Lotus japonicus EST 110 ESTs EST 93,000 5′ and 3′ ESTs obtained from normalized and size-selected cDNA libraries constructed from seven different organs, and 70,137 of these 3′ ESTs clustered into 20,127 nonredundant groups (http://www.kazusa.or.jp/en/plant/lotus/est/). EST 2397 ESTs from roots carrying root nodule primordial appearing after inoculation with Mesorhizobium loti bacteria.

Baquerizo-Audiot et al. 2001 Covitz et al. 1998 White et al. 2000

Fedorova et al. 2002

Journet et al. 2002

http://www.medicago.org Szczyglowski et al. 1997 Asamizu et al. 2000 Poulsen and Podenphant 2002

Table 6.16.

Overview of genetic and cytogenetic linkage maps generated for Lotus japonicus and Medicago truncatula.

Marker and mapping population details Medicago truncatula 313 markers (72 RAPD + 220 AFLP + 19 known genes + 2 isozymes) 124 RILs from Jemalong 6 × DZA315.16

Lotus japonicus 15 markers (3 morphological + 12 DAF) 100 F2 plants from B-129-S9 Gifu × B-581 Funakura

Description of the genetic, cytogenetic or chromosomal map

289 uniformly distributed markers mapped on 8 linkage groups with a total map length of 1225 cM, and average map density of 4.24 cM. Eight M. truncatula LG are homologous to those of diploid alfalfa (M. sativa) implying a good level of macrosynteny between the two genomes. Molecular cytogenetic map constructed based on a pachytene DAPI karyogram that enabled the identification of all chromosomes based on chromosome length, centromere position, heterochromatin patterns and position of three repetitive sequences.

Reference

Thoquet et al. 2002

Kulikova et al. 2001

233

This was the first molecular linkage map of this model legume with 11 linkage groups.

Jiang and Gresshoff 1997

605 markers (524 AFLP + 3 RAPD + 39 gene-specific + 33 SSRs + 6 recessive symbiotic mutant loci) F2 populations from L. japonicus × L. filicaulis

Genetic map consisting of 6 linkage groups corresponding to the 6 chromosomes in L. japonicus. The total map length is 367 cM and the average marker distance is 0.6 cM.

Sandal et al. 2002

F2 population from L. japonicus Gifu × L. filicaulis

Chromosomal map developed using DNA clones from 32 genomic regions that enabled the assignment of linkage groups to chromosomes, the comparison between genetic and physical distances throughout the genome, and partially characterized different repetitive sequences. Nineteen of these clones were also mapped genetically; that makes the L. japonicus map one of the most extensive correlations of the genetic and chromosomal maps in plants, enabling the determination of physical and genetic distance regions along the whole chromosome complement. The map spans 572.1 cM comprising 6 linkage groups: three (LG 2, 3, and 4) well defined; two (LG 5 and 6) poorly discriminated due to apparent marker duplication between them, and one (LG 1) was poorly defined because of high degree of differentiation between its homologous members.

Pedrosa et al. 2002

217 markers (single-dose RFLP, RAPD, ISSR, STS, isozyme, and 5 duplex RFLP) Tetraploid Lotus corniculatus population

Fjellstrom et al. 2003

234

Table 6.17.

Overview of genetic linkage maps generated from interspecific crosses in temperate grain legume crops.

Marker and population size Chickpea (n = 8) 47 gene specific markers integrated into an existing map based on SSR, AFLP, DAF, and other anonymous markers (Winter et al. 1999, 2000) 159 RILs from ICC 4958 × PI 489777

Summary of the genetic linkage map

Reference

The map consists of 296 markers and covers 2483.3 cM in 8 large and 4 small linkage groups. The gene-specific markers derived from sequences of protein known to be involved in plant defense responses are distributed throughout the whole map but particularly on linkage groups 3–5.

Pfaff and Kahl 2003

51 markers (one RGA and 50 STMS) 142 RILs from FLIP 84-92C × PI 599072

The map consists of 167 markers and covers 1174.5 cM with 9 linkage groups, with an average marker distance of 7.0 cM.

Tekeoglu et al. 2002

354 markers (118 STMS, 96 DAF, 70 AFLP, 37 ISSR, 17 RAPD, 8 isozyme, 3 cDNA, 2 SCAR) 130 RILs from ICC4958 × PI489777

303 markers cover 2077.9 cM in 8 large and 8 small linkage groups with an average distance of 6.8 cM between markers. A clustering of markers observed in central regions of linkage groups. The map includes 3 loci contributing to Fusarium resistance.

Winter et al. 2000

120 SSR markers 90 RILs from ICC 4958 × PI 489777)

120 markers grouped into 11 linkage groups with a total map length of 613 cM and an average distance of 5.47 cM between markers.

Winter et al. 1999

91 markers (9 morphological + 27 isozyme + 10 RFLP + 45 RAPD) 3 F2 populations

The map consists of 10 linkage groups with a total distance of 550 cM, and average marker density of 6.04 cM.

Simon and Muehlbauer 1997

144 markers (1 morphological + 11 isozyme + 111 RAPD + 21 ISSR) 142 RILs from FLIP84-92C × PI 599072

The 116 markers grouped into 9 linkage groups with a total map length of 981.6 cM and average marker density of 8.4 cM.

Santra et al. 2000

Lentil (n = 7) 200 markers (71 RAPDs, 39 ISSRs, 83 AFLPs, 2 SSRs, and 5 morphological) 113 F2 plants from L. culinaris ssp. culinaris × L. culinaris ssp orientalis

At a LOD score of 3, 161 markers were grouped into 10 linkage groups covering 2,172.4 cM, with an average distance between markers of 15.87 cM. There were six linkages with 12 or more markers each, and four small groups with two or three markers each.

Duran et al. 2004

177 markers (89 RAPD + 79 AFLP + 6 RFLP + 3 morphological) 86 RILs from ILL 5588 × L692-16-1 (s)

The map comprises of 177 markers grouped into 7 linkage groups with a total map distance of 1073 cM and average marker density of 6.0 cM.

Eujayl et al. 1998a

Table 6.18.

Overview of genetic linkage maps generated from interspecific crosses in tropical grain legume crops.

Marker and population size Adzuki bean (n = 11) 132 markers (108 RAPD + 19 RFLP + 5 morphological) F2 population from Adzuki bean and its wild relative V. nakashimae Cowpea (n = 11) 171 markers (RAPD, SSR, AFLP, and morphological) RIL population from improved cowpea cultivar × wild relative 80 markers (77 RAPD + 3 morphological) RIL population from IT84S-2246-4 × TVNu 110-3A

Mungbean (n = 11) 255 RFLP loci 80 RILs from Berken × ACC 41

Peanut (n = 20) RFLP markers BC1 from TxAG 6 × Florunner

Summary of the genetic linkage map

Reference

The map consists of 14 linkage groups and covers a distance of 1250 cM, with an average between marker density of 9.47 cM.

Kaga et al. 1996

The map consists of 12 linkage groups with a total map length of 2269 cM and average marker density of 13.27 cM.

Ortiz 2003

The map spanned 669.8 cM, and 12 linkage groups that ranged in size from 14.0 to 175.4 cM. The distribution of interval sizes between adjacent markers ranged from 0.7 to 26.7 cM with an average distance of 9.9 cM.

Ubi et al. 2000

The map consists of 13 linkage groups with a total distance of 737.9 cM and average marker density of 3.0 cM. The linkage groups vary in length from 8.7 cM to 100.7 cM.

Humphry et al. 2002

370 RFLP loci mapped into 23 linkage groups with a total map length of 2210 cM and average marker density of 5.97 cM.

Burow et al. 2001

235

236 Table 6.19.

Overview of genetic linkage maps generated from intra-specific crosses in temperate grain legume crop genotypes.

Marker and population size Broadbean (n = 6) 84 markers (3 enzyme + 76 RAPD + 2 seed protein genes + 3 SSRs) 196 F2 plants from Vf 6 × Vf 136 71 markers (9 isozyme + 45 RAPD + 5 seed protein genes + 12 SSR) 11 F2 populations sharing Vf6 as female parent Chickpea (n = 8) 66 markers (51 STMS + 3 ISSR + 12 RGA) 85 F2 plants from ICC12004 × Lasseter

Grasspea (n = 7) 75 markers (71 RAPD + 3 isozyme + 1 morphological) 100 F2 individuals

64 markers (47 RAPD + 7 STMS + 13 STS/CAPS) 92 backcross derived individuals from ATC 80878 × ATC 80407

Summary of the genetic linkage map

Reference

The map consists of 16 linkage groups covering 1445.5 cM, with an average marker density of 13.77 cM.

Roman et al. 2002

192 loci arranged into 14 linkage groups with a total map distance of 1559 cM, and average marker density of 8 cM.

Roman et al. 2004

The map consists of 8 linkage groups with a total map length of 534.5 cM and average distance between markers of 8.1 cM. LG I represents the largest and LG VIII the smallest linkage group. SSR markers are distributed throughout genome while RGA markers cluster along with ISSR makers on LGs 1, 2, and 3.

FlandezGalvez et al. 2003

Sixty-nine markers (one morphological + 3 isozyme + 65 RAPD) assigned to 14 linkage groups with a total map length of 898 cM and average distance between markers was 17.2 cM.

Chowdhury and Slinkard 1999

Sixty-four markers assigned to 9 linkage groups with a total map length of 803.1 cM, and the average spacing between markers was 15.8 cM.

Skiba et al. 2004

Pea (n = 7) 69 markers (3 morphological + 4 RGA + 56 RFLP + 4 SSR + 2 RAPD) 174 F2 plants from Erygel × 661

69 markers mapped across 12 linkage groups with a total map distance of 550 cM, and average distance between markers of about 8.0 cM.

Dirlewanger et al. 1994

240 markers (164 AFLP + 33 RAPD + 12 ISSR + 5 CAPs + 1 STS + 11 isozymes + 14 morphological) 104 RILs from Wt 10245 × Wt 11238

204 markers mapped across 9 linkage groups spanning a total of 2416 cM, with an average distance between markers of 12 cM. The size of the linkage groups ranged from 34 cM to 503 cM but around half of the map intervals are shorter than 10 cM and only 1.5% intervals longer than 30 cM.

Irzykowska et al. 2001

206 markers (192 AFLP + 13 RAPD + 1 STS) 88 RILs from Carneval × MP 1401

206 markers assigned to 10 linkage groups spanning a total of 1274 cM of pea genome, with an average distance between markers of 6.2 cM. Fourteen markers were common to the previous pea genetic maps (Gilpin et al. 1997; Laucou et al. 1998) that allowed six (I, II, III, IV, VI, and VII) of these linkage groups to be aligned to the previous pea linkage maps, whereas four linkage groups (A to D) remained unassigned.

Tar’an et al. 2003b

209 markers (RFLP/RAPD/AFLP/ RGA) 102 F2 plants from Prima × OSU442-15

The map consists of 14 linkage groups and covers 1330 cM distance, with an average distance between markers of 6.36 cM.

Gilpin et al. 1997

240 RAPD markers 139 RILs from Terese × K 586

The map consists of 9 linkage groups and covers 1139 cM distance, with an average marker density of 4.75 cM. The size of the 9 LGs ranges from 21.1 cM to 187.1 cM.

Laucou et al. 1998

355 RFLP markers 71 RILs from JI 281 × JI 399

The map consists of 7 linkage groups covering 1881 cM, with an average distance between markers of 5.29 cM.

Ellis et al. 1992

237

Table 6.20.

Overview of genetic linkage maps generated from intra-specific crosses in tropical grain legume crop genotypes.

238

Marker and population size Common bean (n = 11) 246 markers (78 SSR, 48 RFLP, 102 RAPD and 18 AFLP) on 87 RILs (DOR364 × G 19833) as well as 22 microsatellites on 75 additional RILs from BAT93 × Jalo EEP558

Summary of the genetic linkage map

A genome-wide anchored map consists of 246 markers covering 11 linkage groups with a total map length of 1720 cM and average SSR marker density of 19.5 cM.

Reference

Blair et al. 2003

SSR loci were distributed across each of the 11 chromosomes, with the number per chromosome ranging from 5 to 17 with an average of 10 SSRs. The map consists of 11 linkage groups with a total map length of 1226 cM and average marker density of 2.17 cM.

Freyre et al. 1998

563 markers (120 RFLP + 430 RAPD + few isozyme/ morphological) 75 RILs from BAT93 × Jalo EEP558 152 markers (112 RFLP, 7 isozyme, 8 RAPD, 15 known genes) F2 population from BAT93 × Jalo EEP558 244 markers (224 RFLP + 9 each isozyme and seed protein + 2 morphological) Back cross population from XR-235-1-1 × Calima

Total map length of 827 cM in 15 linkage groups. Average interval between markers of 6.5 cM.

Nodari et al. 1993

The 244 markers assigned to 145 loci/locus across 11 linkage groups with a total map length of 960 cM, and average marker density of 3.93 cM.

Vallejos et al. 1992

Cowpea (n = 11) 181 markers (133 RAPD + 19 RFLP + 25 AFLP + 3 morphological and classical + 1 biochemical) 94 RILs from IT84S-2049 × 524B

The 181 loci grouped into 12 linkage groups spanning 972 cM with an average marker distance of 6.4 cM. Linkage groups ranged from 3 to 257 cM in length and included 2 to 41 markers.

Menendez et al. 1997

The genetic map consists of 11 linkage groups with a total map length of 2670 cM and average marker density of 6.43 cM. A large contiguous portion (580 cM) of LG 1, that had been undetected in previous mapping work, was discovered and is composed of entirely AFLP markers. An extraordinary variation in the size of linkage groups was observed: > 300 cM (LG1); 200–300 cM (LG2 to LG6); 100–200 cM (LG7 and LG8); <100 cM (LG9 to LG11).

Ouedraogo et al. 2002a

423 markers (242 AFLP + 181 previously mapped RAPD, RFLP, AFLP, and biochemical) 94 RILs from IT84S-2049 × 524B

70 RAPD markers 75 F2 plants from GSC01 × GSC02

The map spanned 474.1 cM across 11 linkage groups, with an average marker distance of 6.87 cM. The six linkage groups have 40 cM or more whereas the remaining 5 linkage groups ranged from 4.9 to 24.8 cM. The number of markers per linkage group ranged from 2 to 32. The longest (LG 1) spans 190.6 cM while the shortest (LG 1) is 4.9 cM.

Shim et al. 2001

Integrated genetic map consisting of 20 linkage groups with a total map length of 2523.6 cM containing 1849 markers (1015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, 6 AFLPs, 10 isozymes, and 12 others), with an average marker density of 1.36 cM. The number of new SSR markers added to each linkage group ranged from 12 to 29 but the ratio of SSR marker number to linkage group map distance did not differ among 18 of the 20 linkage groups; however, the SSRs were not uniformly spaced over a linkage group. These clusters of SSRs may be indicative of gene-rich regions of soybean, indicating a significant association of genes and SSRs.

Song et al. 2004

452 markers (RFLP + SSR + EST) 184 RILs from Kefeng No.1 × Nannong 1138-2

The map consists of 21 linkage groups with a total map length of 3595.9 cM, and average marker density of 7.9 cM. All the linkage groups except LG F were consistent with those of the consensus map of Cregan et al. (1999).

Zhang et al. 2004

436 markers (329 RAPD + 103 SSR + 4 other) 76 RILs from PI 437088A × Asgrow A3733

The 2943 cM genetic linkage map consists of 35 linkage groups that, on the basis of SSR homology, aligned with the 20 known soybean linkage groups. 606 SSR loci mapped in one or more of three populations. Each SSR mapped to a single locus in the genome, with a map order that was essentially the same in all the three populations. The 20 linkage groups derived from each of the three populations could be consolidated into a consensus set of 20 homologous linkage groups presumed to correspond to the 20 pairs of soybean chromosomes. The 3441 cM map consists of 840 markers grouped into 28 LGs, with an average between marker density of 4.09 cM.

Chung et al. 2003

Soybean (n = 20) 420 new SSR markers Five populations

SSR markers 2 F2 populations from USDA/Iowa State G. max × G. soja and Univ. Nebraska Clark × Harosoy) and RIL population from Univ. Utah Minsoy × Noi 1

840 markers (165 RFLP + 25 RAPD + 650 AFLP) 239

300 RILs from BSR101 × P1437.654

Cregan et al. 1999a

Keim et al. 1997

240

DWIVEDI, BLAIR, UPADHYAYA, SERRAJ, BALAJI, BUHARIWALLA, ORTIZ, CROUCH

for legumes were based on interspecific crosses, usually due to the practical need to use mapping populations with a maximum level of genetic polymorphism when the number of markers available in a crop is relatively low and the cultigen has a low level of genetic variability. An exception to this has been in crops with diverse intra-specific gene pools such as common bean, in which inter-specific crosses were not necessary. Trait-linked markers identified by using inter-specific populations may be useful for introgression/pre-breeding programs but may not be highly valuable for marker-assisted selection in narrow crosses used to develop breeding populations in which the linkage or polymorphism may be lost. This means markers must be generated or validated in a panel of lines/populations representing improved varieties and including the donor genotypes before routine application in a breeding program. Unfortunately, there are very few reports in the literature of marker validation or application in diverse breeding populations. Clearly, a large proportion of legume linkage maps, particularly in lesserstudied crops, are not truly representative of the cultigen genome, although they do offer valuable starting points for the species or genus of interest. There is a large variation in map length both within and between legume species; for example, total map distance ranges from 550 to 2416 cM in pea, 534 to 2483 cM in chickpea, 960 to 1720 cM in common bean, and 474 to 2670 cM in cowpea. This is generally likely to result from differences in the number of chromosomes and total size of the genomes as well as the use of different numbers of markers (increasing the number of markers will generally, until a certain threshold is reached, give larger total map lengths), inclusion of skewed markers (that tend to exaggerate map distances), and use of different mapping softwares (which vary in their estimates of map distances). In addition, many published maps report more linkage groups (LGs) than the haploid chromosome number of that species, for example 9 to 14 LGs in pea (n = 7), 9 to 16 LGs in chickpea (n = 8), 14 to 16 in broadbean (n = 6), and 20 to 35 LGs in soybean (n = 20). This is frequently the result of insufficient marker density, as most saturated maps can be directly aligned with the haploid chromosome complement (Tekeoglu et al. 2002). Segregation distortion introduces another problem to the development of genetic maps, which is particularly common when using populations from interspecific crosses. This distortion can be due to (i) pre- or postzygotic selection bias due to reduced fitness or lethal effect of certain allelic combinations, (ii) chromosome inversion leading to reduced recombination, and (iii) suppression of recombination at meiosis caused by a non-/or partial homology between chromosomes. This

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distortion can be exacerbated during the tissue culture phase of double haploid (DH) population development or during selection of individuals at each generation of single seed descent during the development of recombinant inbred lines (RILs) mapping populations (Rick 1966; Cheng et al. 1981; Paran et al. 1995; Flandez-Galvez et al. 2003). Skewed segregation in mapping populations can affect both the establishment of linkage groups and the estimation of recombinant frequency. These problems can be minimized by decreasing the recombination fraction used to reject linkage and by limiting the ability to detect linkage among closely co-segregating markers (Wang et al. 1994). Another reason for reduced recombination is the presence of heterochromatin, which due to its more condensed structure has less opportunity for synapsis compared to euchromatin (Tanksley et al. 1992). However, a significant proportion of genes are to be found in these regions which have (especially areas proximal to the centromeres) meiotic crossovers occurring at a rate 5- to 10-fold less than other regions. High-resolution genetic maps will be much easier to generate in regions of higher recombination. For regions of suppressed recombination, much larger progeny sizes will be needed to detect the crossovers necessary for constructing dense genetic maps. Suppression of recombination is also likely to enhance the effects of deleterious “linkage drag” where target genes are introgressed along with genes that convey negative effects on agronomic performances (Stam and Zeven 1981). Excess heterozygosity is also reported to contribute genetic map distances (Knox and Ellis 2002). The generation of reliable, dense biallelic maps will allow rapid identification of markers for an array of monogenic and polygenic traits, thus facilitating rapid, intensive, and cost-effective study and manipulation of a range of economically and biologically important processes (Cho et al. 1999). Therefore, there is clearly an urgent need to develop saturated genetic linkage maps particularly of the research-neglected crops. The quickest and most cost-effective means of doing this is with co-dominant PCR-based markers (preferably SSRs and SNPs). Increased marker density around genes of interest will provide starting points for chromosome walking, which is the first step in map-based cloning (Tanksley et al. 1992) and will provide sufficient markers for routine marker-assisted breeding. The use of large-insert genomic libraries, especially bacterial artificial chromosomes (BAC), for chromosome walking and for physical mapping of the genome becomes more feasible as the marker density increases (Woo et al. 1994). These libraries are also useful for positional cloning of genes (Arondel at al. 1992) and targeted development of microsatellite markers (Cregan et al. 1999; Meksem et al. 2000).

242

DWIVEDI, BLAIR, UPADHYAYA, SERRAJ, BALAJI, BUHARIWALLA, ORTIZ, CROUCH

Genome-wide physical maps are important tools for genome sequencing, targeted marker development, efficient positional cloning, and highthroughput EST mapping (Marra et al. 1999; Mozo et al. 1999; Chang et al. 2001; Tao et al. 2001; Chen et al. 2002). Amongst the legumes, whole genome physical maps are only available in soybean (Wu et al. 2004a,b), for which a 10x BAC library and a plant-transformation-competent binary large-insert plasmid clone (BIBAC)-based physical map (based on 2905 BAC/BIBAC contigs spanning 1408 Mb) have been developed (Wu et al. 2004a). When successfully integrating this physical map with the existing soybean composite genetic map using 388 DNA markers, the ancient tetraploid origin of soybean was also confirmed. This provides an important platform for advanced genome research of soybean and other legume species. Wu et al. (2004b) have also developed a 5x largerinsert BAC library in a smaller vector (pECBAC1), using EcoRI. This BAC library contains 38,400 clones; about 99% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp) than in the library created for physical mapping, thus providing a much more useful resource for positional cloning. Mudge et al. (2004) used 683 soybean BAC contigs anchored with RFLPs to explore microsyntenic relationships among duplicated regions and examine the physical organization of hypomethylated (gene rich) genomic regions. This study has revealed that RFLPs are physically clustered in less than 25% of the genome; most of the genes are clustered in less than 275 Mbp of the genome; and approximately 40%-50% of this gene rich portion is associated with RFLP-anchored contigs. BAC libraries have also been reported for common bean, pea, chickpea, and peanut. The common bean BAC library, from a Sprite snap bean-derived genotype, consists of 33,792 clones and estimated 3- to 5fold coverage of the common bean genome, with an average insert size of the library of around 100 kb (Vanhouten and MacKenzie 1999). Coyne et al. (2000) constructed a BAC library from a pea germplasm line PI 269818 that represents one genome equivalent and contains 50,000 BAC clones with an average insert size of 110 kb and packages another 20,000 mini-BACs (≤60 kbp) that may be useful in filling gaps in a 5- to 6-fold coverage library. Thus, the final library consists of approximately 250,000 clones representing 5- to 6-fold haploid genome equivalents, depending on the average insert size of the BAC clones. In chickpea, a BAC library from germplasm line FLIP 84-92C has 23,780 clones representing approximately 3.8 haploid genome equivalents, with an average insert size of 100 kb (Rajesh et al. 2004). A 10x BAC library in chickpea is also under construction (Cook, pers. commun.). The BAC library in

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243

peanut contains 182,784 clones representing 6.5 haploid genome equivalents, with an average insert size of 104 kb, and thus allowing the isolation of virtually any single-copy locus (B. Yuksel, pers. commun.). 2. Molecular Markers for Genes Contributing to Important Agronomic Traits. Genetic linkage maps have been used for identifying QTL (quantitative trait loci) markers for a wide range of biochemical, physiological, and morphological traits in the model species and major legume crops (Ane et al. 2002). Amongst the crop legumes, soybean, common bean, and pea are the most extensively studied, with QTL markers reported for a number of important agronomic traits including resistance to pests and diseases, abiotic stress tolerance, nitrogen fixation potential, and grain quality (Tables 6.21 and 6.22). Rapid progress is now also being seen in a number of lesser-studied crops, with markers reported for resistance to fusarium wilt in pea, chickpea, broadbean, common bean, and lentil; for ascochyta blight in pea, chickpea, broad-bean, and lentil; for common bacterial blight in chickpea and common bean; for powdery mildew in common bean and mung bean; for rust in broadbean and common bean; for broomrape in broadbean and cowpea; and for anthracnose in common bean (Tables 6.21 and 6.22). C. Linkage Disequilibrium and Association Mapping Conventional linkage mapping relies on the development of defined genetic populations, which can be used for molecular marker analysis, and identification of genes underlying traits by marker-phenotype correlations (see section V B). The effective mapping of complex traits requires large recombinant inbred line (RIL) (Burr et al. 1988), advanced backcross (Tanksley and Nelson 1996) or double haploid (Kasha and Kao 1970; Chen and Hayes 1989) populations or, alternatively, near-isogenic lines (NILs) (Muehlbauer et al. 1988). These genetic populations or stocks (unlike F2 mapping populations) usually can be readily evaluated in several locations and seasons thus greatly improving the quality of phenotype data to be used for identifying marker associations. However, the development of such populations is time consuming and takes several years. Moreover, any marker-trait associations identified in this way must be validated in independent populations before routine application in breeding programs (Sun et al. 2001). Another drawback to linkage mapping is that it is based on genetic distance and thus is confounded by different recombination patterns within a given physical distance between target loci and marker in different populations. Given the

Table 6.21.

Overview of markers associated with beneficial agronomic traits in tropical grain legume crops.

244

Trait Common bean Angular leaf spot (ALS)

Summary of the QTL identified

Reference

A single dominant gene confers resistance to ALS in MAR-2, and a RAPD marker (OPE-4) has been identified that is linked to the resistance gene at a distance of 5.8 cM. A single dominant gene confers resistance to ALS pathogen race 61.41 in BAT 332. Identified two RAPD markers, OPAA01950 (5.1 cM) and OPAO12950 (5.8 cM), linked with resistance to ALS.

Ferreira et al. 2000 Caixeta et al. 2003

AFLP markers (ECAG/MACC-1, EACA/MAGA-2, EAGG/MAAC-8) linked to the Co-1 locus associated with resistance to anthracnose.

Mendoza et al. 2001

Six RAPD markers linked to the Co-4 gene: 4 in coupling and 2 in repulsion phase. Marker OPY20830C is tightly linked (0.0 cM) to Co4.

de Arruda et al. 2000

RAPD marker (OPAZ20940) linked in coupling phase at 7.1 cM of the Co-6 gene in the F2 populations Rudá × AB136.

Alzate-Marin et al. 2000

RAPD marker (ROH20450) linked to the Mesoamerican Co-2 anthracnose resistance gene, was transformed into a SCAR marker (SCH20) and tested in different genetic backgrounds.

Geffroy et al. 1998

Two RAPD markers linked to the Co-42 allele were identified (OAS13950 and OAL9740) and one dominant SCAR (SAS13) was developed.

Young et al. 1998

Four RAPD markers (OF10530 at 1.9 from the Co1(A) allele and in repulsion-phase; OAB3450 at 5.9 cM from the Co5(Mexique3) allele and in coupling-phase; and OAH1780 and OAK20890 at 12.3 and 7.3 cM from the Co6 allele and in coupling and repulsion phase, respectively) were developed for differentials.

Young and Kelly 1997

Bacterial brown spot (BBS)

A genomic region on LG 2 was significantly associated with QTL for BBS resistance.

Jung et al. 2003

Bean common mosaic virus (BCMV)

A RAPD marker, tightly linked (0 cM) with bc-12 gene conferring resistance to BCMV, was converted into a SCAR marker (SBD1300) that mapped on LG B3.

Miklas et al. 2000

A marker for the bc3 gene was obtained from the co-dominant RAPD band ROC11/350/420 and confirmed in different genetic backgrounds.

Johnson et al. 1997

A SCAR marker (SW13) was developed from the corresponding RAPD primer (OW13690) linked to the I gene, at between 1.0 and 5.0 cM in three F2 populations.

Melotto et al. 1996; Haley et al. 1994

Anthracnose

Bean golden mosaic virus (BGMV)

One codominant RAPD marker (R2570/530) tightly linked at 4.2 cM to the recessive resistance gene bgm-1.

Urrea et al. 1996

Fourteen RAPDs linked to 7 QTL conferring resistance to BGMV and/or common bacterial blight.

Miklas et al. 1996

Common bacterial blight (CBB)

Major gene located on LG G5, and one QTL each on LG G2, G3, and G5 explained 36.5%, 10.2% and 42.2% of the variation for reaction to CBB, respectively.

Tar’an et al. 2001

SCAR marker BC420 created and analyzed in RIL (HR67 × W1744d) and breeding populations.

Yu et al. 2000

QTL study with 78 F9 RILs derived from Great Northern Belneb RR-1 × A 55 for mapping resistant to common bacterial and halo blight as well as bean common mosaic necrosis virus (BCMNV). The linkage map spanned 755 cM and 11 LGs, and contained 87 RAPD markers.

Ariyanthne et al. 1999

A RAPD marker (BC409.1250) significantly associated with CBB resistance in 3 crosses and all three Xcp species, indicating that it might be tightly linked to the CBB resistance genes.

Jung et al. 1999

Two genes for resistance to CBB from XR-235-1-1 were detected, one linked to Bng40 in LG A, one linked to Bng139 in LG F and the last one linked to Bng154 in LG J. Effect of two Calima-derived resistance genes, linked to Bng25a and Bng154 in LG J, were also detected

Yu et al. 1998

The two previously identified RAPD markers (R7313 and R4865) located on the same linkage group explained 22% of the variation in response to CBB in the current mapping population.

Ta’ran et al. 1998

QTL study based on a RAPD marker map for the cross PC50 × XAN159 spanning 426 cM based on 70 RILs and 168 RAPD markers.

Jung et al. 1997

Four RAPD markers (R7313, RE416, RE49, and R4865) associated with resistance to CBB.

Bai et al. 1997

QTL study based on partial linkage map for the cross BAC 6 × HT 7719 spanning 545 cM and based on 84 markers and 128 RILs analyzed for CBB, web blight and rust.

Jung et al. 1996

245 (continued )

246

Table 6.21.

(continued)

Trait

Summary of the QTL identified

Common bean (continued) Fusarium wilt A RAPD marker (U20.750) tightly linked with QTL on LG 10 accounting for 63.5% of the variation.

Reference

Fall et al. 2001

Powdery mildew and angular leaf spot

RAPD markers linked with resistance to powdery mildew were OPRO2-832, OPDO8-759, and OPN10851 and for angular leaf spot were OPNO2-436 and OPNO7-1072.

Melo et al. 2002

Rust

Six RAPD markers in coupling phase linkage, and marker OAB18.650 closely linked (7.6 cM) to Ur-7 gene. All the linked markers detected in F2 also segregated in RILs, and located on LG 11.

Park et al. 2003

Two RAPD markers flanking the rust resistance gene block identified: one at 5.8 cM (OX11630) and the other at 7.7 cM (OF101050) from the gene.

Faleiro et al. 2000

Two SCAR markers SCARBA08 at 4.3 cM and SCARF10 at 6.0 cM from the rust resistance locus.

Correa et al. 2000

A RAPD marker OA4.1050 closely linked to the Ur-9 gene at a distance of 8.6 cM.

Park et al. 1999

Two RAPD markers linked to the gene block of interest were identified (OF10970 at 2.15 cM and OI19460 at 0.0 cM in a 97 individual BC6F2 population.

Haley et al. 1993

A RAPD marker OA141100 linked to the dominant Ur2 gene in an 84 individual BC6F2 population.

Miklas et al. 1993

2 QTL conferring resistance to white mold, located on LG B6 and B8, contributing 13% and 26% of the disease reaction in the field, respectively.

Miklas et al. 2003

White mold

QTL affecting partial physiological resistance, partial field resistance, and porosity over furrow were reported. Park et al. 2001 Drought

4 and 5 QTLs identified for drought tolerance in two recombinant inbred populations (Sierra/AC1028 and Sierra/Lef-2RB), respectively grown under stress and non-stress conditions at eight locations.

Schneider et al. 1997

Nodulation and common bacterial blight (CBB)

Four QTL associated with increased number of nodules and resistance to Xanthomonas, suggesting a common genetic control for response to bacterial infection in common bean.

Souza et al. 2000

Four QTL identified in the same genomic region for resistance to CBB and nodulation which accounted for 75% and 50% phenotypic variation for CBB and nodulation, respectively.

Nodari et al. 1993

QTL analysis of nodule number (NN) and CBB resistance in a 70 individual F2-derived F3 families of BAT93 × Jalo EEP-558 identified 8 QTLs for NN of which 4 were shared for CBB resistance.

Tsai et al. 1998

Cooking time

A RAPD marker (UNAM-16) of 300 bp explained 23% of the variation in cooking time of the lines studied.

Jacinto-Hernandez et al. 2003

Nutritional traits

Five putative QTL significantly associated with seed mass, two with Ca, two with Fe, one with Zn, and four with tannin content in the seed, explaining 42%, 25%, 25%, 15%, and 41% of the phenotypic variation, respectively.

GuzmanMaldonado et al. 2003

Agronomic traits

Twenty QTL identified for 14 traits: the number ranged from one to three, and accounted for 11.3–43.1% variation for the traits.

Tar’an et al. 2002

Canning quality

Degree of splitting (SPLT) and overall appearance (APP) determines the canning quality. Major QTL identified on two linkage groups: one QTL linked with 7 RAPD markers on B8 LG of the core map; the second QTL linked with 4 markers but not assigned to core LGs.

Posa-Macalincag et al. 2002

Domestication syndrome

Three genomic regions are reported to have major effect on domestication traits (loss of seed dispersal, seed dormancy, determinate growth habit, fruit size, and seed weight) in common bean: one region greatly affects growth habit and phenology; the other seed dispersal and dormancy; and a third, the size of fruit and seed.

Koinange et al. 1996

Earliness and seed weight

A gene for earliness mapped on LG 2 in an interval spanning 26 cM around RFLP marker locus D1301a, accounting for 21% of the phenotypic variation. A RAPD marker OB6a on LG 5 accounted for 9% of the phenotypic variation in seed weight.

Menendez et al. 1997

Striga

Three and six AFLP markers tightly linked to Rsg2-1 and Rsg4-3 loci conferring resistance to S. gesnerioides race 1 from Burkina Faso and to race 3 from Nigeria, respectively. Two markers linked to both Rsg2-1 and Rsg4-3.

Ouedraogo et al. 2001

Cowpea

247 (continued )

248

Table 6.21.

(continued)

Trait

Summary of the QTL identified

Reference

Cowpea Striga (cont.)

Seven AFLP markers linked to Rsg3, the gene conferring resistance to race 1 in Gorom, with two markers (E-AGA/M-CTA460 and E-AGA/M-CAG300) flanking Rsg3 at 2.5 and 2.6 cM, respectively. Five AFLP markers linked to the race 1 resistance gene 994-Rsg present in IT81D-994. The two markers showing the tightest linkage to the 994-Rsg locus are E-AAG/M-AAC450 and E-AAG/MAAC150 at 2.1 and 2.0 cM, respectively. Two of the markers linked to 994-Rsg, E-AGA/M-CAG300 and E-AGA/M-CAG450, are also linked to Rsg3.

Ouedraogo et al. 2002b

Four AFLP markers (E-ACT/M-CTC115, E-ACT/M-CAC115, E-ACA/M-CAG108, and E-AAG/M-CTA190) mapped at 3.2, 4.8, 13.5, and 23.0 cM, respectively, from the Rsg1 gene that confers resistance to Striga race 3 in IT93K-693-2. A SCAR marker (SEACTMCAC83/85) was also developed from an AFLP fragment from E-ACT/M-CAC that may be useful in breeding programs.

Boukar et al. 2004

Agronomic traits

QTL detected for leaf length and width, primary leaf length and width, leaf area, days to flowering, maturity, pod length, and seed/pod weight. Several regions of the genome affected more than one trait.

Ubi et al. 2000

Aphid

A tightly linked RFLP marker (bg4D9b) associated with aphid resistance gene (Rac1) and several flanking markers in the same linkage group reported.

Myers et al. 1996

Cucumber mosaic virus (CMV)

Cry gene in Kurodane-Sanjaku confers resistance to CMV. RAPD markers D13/E14-350 (5.2 cM), WA3850 (11.5 cM), OPE3-500 (24.5 cM) and RGA (CRGA5 at 0.7 cM) were found associated with cry locus.

Chida et al. 2000

A major QTL accounted for 64.9% of the variation in resistance to powdery mildew.

Chaitieng et al. 2002

Three genomic regions together contributed 58% of the total phenotypic variation associated with resistance to powdery mildew.

Young et al. 1993

Mungbean Powdery mildew

Bruchid

A single dominant gene, Br, confers resistance to bruchid in TC1966. Eight RAPD-based markers, one mungbean and four common bean genomic clones effectively integrated around Br within a 3.7 cM interval. Br gene mapped to a 0.7 cM segment between a cluster consisting of 6 markers and a common bean RFLP marker, Bng110.

Kaga and Ishimoto 1998

A RAPD marker, OPC-11 (600 bp) associated with male sterility in 288A and 67A but absent in the maintainer (288B and 67B) and restorer (TRR 5 and TRR 6) lines.

Souframanien et al. 2003

Yield

Yield QTL located on LG B2 (U26) of the G. max genetic map, and individuals carrying the PI 407305 haplotype at the QTL locus demonstrated to have a 9.4% yield advantage.

Concibido et al. 2003

Seed size

For seed size across three environments, 12 SSR markers individually accounted for 8.1 to 14.9% variation in population 1; 16 markers accounted for 7.8 to 36.5% variation in population 2; and 22 markers accounted for 8.6 to 28.8% variation in population 3.

Hoeck et al. 2003

Seed yield, and oil and protein content

A seed protein, oil, and yield QTL mapped close to RAPD marker (OPAW13a) in a small LG-I interval flanked by the SSR markers Satt496 and Satt239.

Chung et al. 2003

Seed weight

Three and five markers significantly associated with seed weight variation in the F2 and F3 populations and together they explained 50% and 60% of the phenotypic variation, respectively.

Maughan et al. 1996

Sprout yield

Four QTL associated with sprout yield located in the same genomic region as the QTL for seed weight. This reveals that either QTL for sprout yield are genetically linked to seed weight QTL or else that seed weight QTL pleiotropically condition sprout yield.

Lee at al. 2001

Seed Isoflavone

Six QTL significantly associated with seed isoflavone content.

Meksem et al. 2001a; Kassem et al. 2004

Pigeonpea Cytoplasmic male sterility (CMS) Soybean

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(continued )

Table 6.21.

(continued)

250

Trait

Summary of the QTL identified

Reference

Water use efficiency (WUE) and leaf ash (LASH)

A total of 4 and 6 independent RFLP markers linked with WUE and LASH together explained 38% and 53% of the variability, respectively. A major QTL on USDA LG J explained 13.2% of the variation in WUE. LASH was negatively correlated with WUE and two QTL were associated with both WUE and LASH.

Mian et al. 1996

WUE

Two independent RFLP markers (A063E and A489H) associated with WUE. A063E was also linked with WUE in the Young × PI416937 population. Marker A489H explained 14% of the variation in WUE.

Mian et al. 1998

Drought tolerance

Major QTL detected for yield beta, yield, and carbon isotope discrimination.

Specht et al. 2001

Salt tolerance

A major QTL for salt tolerance close to the Sat_091 SSR marker on LG-N accounted for 41%, 60%, and 79% of the total genetic variation in the field, greenhouse, and combined environments, respectively.

Lee et al. 2004

Soybean mosaic virus (SMV) and peanut mottle virus (PMV)

6.8 cM region around Rsv1 and Rpv1, resistant genes, mapped using over 20 RFLP, RAPD, and microsatellite markers. The Rsv 1 and Rpv 1 are tightly linked at a distance of 1.1 cM.

Gore et al. 2002

Corn earworm

One major and two minor QTL associated with resistance to corn earworm. The major QTL is linked with RFLP marker 584 on LG M of the USDA/Iowa soybean genetic map, and contributes 37% of the total variation for resistance.

Rector et al. 1998

Cyst nematode

A major QTL conferring resistance to cyst nematode mapped to the region containing rhg1 on LG G and to the region containing Rhg4 on LG A2. Mapping of many different sources of resistance revealed QTL in this same region suggesting that these diverse sources may share common genes for resistance. Candidate genes for rhg1 and Rhg4 have also been cloned.

Concibido et al. 2004

Soybean (cont.)

Root-knot nematode

Two QTL associated with resistance to root-knot nematode were located on two LGs: RFLP marker B212-1 on LG F and A725-2 on LG D1 accounted for 46% and 13% of the variation in gall number, respectively.

Tamulonis et al. 1997a

Peanut root-knot nematode

Two QTL conferring resistance to peanut root-knot nematode identified: one mapped at 0-cM from RFLP marker B212V-1 and accounted for 32% variation in gall number, whereas another mapped between B212D-2 to A111H-2 and accounted for 16% of the variation.

Tamulonis et al. 1997b

Sudden death syndrome (SDS)

Two QTL identified by RAPD markers, OO05250 and OC01650, associated with mean SDS response and together contributed 34% of the total phenotypic variation.

Hnetkovsky et al. 1996

Agronomic traits

63 QTL that had LOD>3 for 9 agronomic traits mapped on 12 LGs. Seven ESTs linked closely with or located at the same loci as the QTL. EST marker, GmK.F0590, accounted for 20% of the total variation for 4 agronomic traits.

Zhang et al. 2004

Major QTL identified for plant height, lodging, flowering, reproductive period, maturity, yield, seed weight, and seed oil and protein contents.

Mansur et al. 1996; Brummer et al. 1997; Orf et al. 1999

Iron deficiency chlorosis

QTL with minor effects detected on six linkage groups of the Pride B216 × A15 population, whereas one major QTL detected in population Anoka × A7.

Lin et al. 1997

Flowering, maturity, and photoperiod insensitivity

Time to flowering, maturity, and photoperiod insensitivity is controlled by a major QTL with large effect, modified by several minor QTL, all three QTL are located in the same region on LG C2 in both populations.

Tasma et al. 2001

251

252

Table 6.22. Trait

Overview of markers associated with beneficial agronomic traits in temperate grain legumes. Summary of the QTL identified

Reference

Broadbean Broomrape

Three QTL confer resistance to broomrape, and together accounted for 74% of the variation. One QTL explained more than 35% of the phenotypic variance whereas the others accounted for 11.2 and 25.5%, respectively.

Roman et al. 2002

Seed weight

Several QTL for seed weight; the most important of which, located on chromosome 6, explained about 30% of the total phenotypic variation.

Vaz-Patto et al. 1999

Rust

Three RAPD markers in coupling phase (OPD13736, OPL181032, and OPI20900) and two in repulsion phase (OPP021172 and OPR07930) mapped to the resistance gene for race 1 (Uvf-1). No recombination detected between OPI20900 and Ufv-1.

Avila et al. 2003

Ascochyta blight

Two putative QTL, Af1 and Af2, identified on LGs VIII and IVa and together explained 46% of the total phenotypic variation for resistance to Ascochyta blight.

Roman et al. 2003

Two QTLs conferring resistance to Ascochyta blight accounted for 45.0% and 50.3% phenotypic variation in 1997 and 1998, and mapped to LG 6 and LG 1, respectively. Two RAPD markers flanking the first QTL were 10.9 cM apart while one ISSR and enzyme marker flanking the second QTL were 5.9 cM apart.

Santra et al. 2000

A major locus, ar1 mapped on LG 2, conferring resistance to Ascochyta blight pathotype 1 and two independent recessive major loci, ar2a on LG2 and ar2b on LG4, conferring resistance to pathotype 2. ar2a is tightly linked to ar1 indicating a clustering of resistance genes in that region of the chickpea genome.

Udupa and Baum 2003

Resistance to Ascochyta blight is encoded by 2-3 QTL. OPS06-1 and OPS03-1, located on LG 4, were linked to markers UBC733B and UBC181A flanking the major Ascochyta blight locus. The former mapped at the peak of QTL between UBC733B (4.1 cM) and UBC181A (9.6 cM) while the latter mapped 25.1 cM away from UBC733B. Three of these markers closely linked to the major QTL.

Rakshit et al. 2003

A major QTL for resistance to Ascochyta blight, located close to the locus of the OPAC04/1200 marker, explained 20 to 23.7% of the total phenotypic variation.

Millan et al. 2003

Chickpea Ascochyta blight

Fusarium wilt

Resistance to Fusarium oxysporum f. sp. Ciceris race 3 is controlled by single gene, designated as foc-3 that has been mapped 0.6 cM from SSR markers TA96 and TA27 and STMS marker CS27A. Another SSR marker, TA194, at 14.3 cM, flanked the gene on the other side. Established the linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h1) and foc-4. Foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM.

Sharma et al. 2004

A RAPD marker (OPJ20600) linked with resistance to fusarium wilt.

Rubio et al. 2003

Linkage analysis indicated that the genes for resistance to races 4 and 5 were on the same LG and were separated by 11.2 cM. The gene for resistance to race 0 was not linked to the race 4 and 5 resistance genes. An allele-specific marker (CS-27R/CS-27F) was located between the two-resistance genes and was 7.2 and 4 cM from the genes for resistance to races 4 and 5, respectively.

Tekeoglu et al. 2000

Two RAPD markers (CS 27700 and UBC 170550) located 9 cM from the race 4-resistance locus, were on the same side of the resistance gene. The genes for resistance to race 1 and 4 are 5 cM apart.

Tullu et al. 1998

An ISSR marker (UBC-855500) linked to fusarium wilt race 4 resistance gene at a distance of 5.2 cM. It co-segregated with CS 27700, a RAPD marker previously shown to be linked to fusarium wilt resistance race 1 gene, and mapped to LG 6.

Ratnaparkhe et al. 1998

Double podding

A gene that confers double podding, gene symbol “s”, is important for breeding high yielding chickpea cultivars. A SSR marker, TA-80, was located at 4.84 cM from the “s” locus.

Rajesh et al. 2002

Agronomic traits

Four QTL identified for 100-seed weight (on LG 4 and LG9), seed number per plant (LG 4), and days to 50% flowering (LG 3). A double podding gene mapped to LG 6 was linked to Tr44 and Tr34 at a distance of 7.8 cM and 11.5 cM, respectively.

Cho et al. 2002

Nodulation and common bacterial blight (CBB)

For each trait, at least four putative QTL identified, which accounted for 50% and 75% of the phenotypic variation in nodule number and CBB resistance.

Nodari et al. 1993

QTL1 located on LG 1 and QTL2 located on LG2 contributed 12% and 9% of the phenotypic variation for resistance to Ascochyta blight, respectively, in the backcross population of the cross ATC 80878 × ATC 80407.

Skiba et al. 2004

Grasspea 253

Ascochyta blight

(continued )

254

Table 6.22.

(continued)

Trait

Summary of the QTL identified

Reference

Lentil Anthracnose

A major dominant gene, LCt-2, confers resistance to anthracnose in PI 320937, and 2 flanking RAPD and 3 AFLP markers linked to the LCt-2 locus.

Tullu et al. 2003

Ascochyta blight

A single recessive gene, ra12, confers resistance to Ascochyta blight in Indianhead cultivar. Two flanking RAPD marker, UBC2271290 and OPD-10870, linked in repulsion phase to the gene ra12 at 12 and 16 cM, respectively.

Chowdhury et al. 2001

Frost injury

Tolerance to frost is monogenic, and a RAPD marker (OPS 16750) linked to the locus for radiation-frost tolerance (Frt) trait at 9.1 cM.

Eujayl et al. 1999

Fusarium wilt

A single dominant gene (Fw) confers resistance to fusarium wilt, and a RAPD marker (OPK 15900) located at a distance of 10.8 cM. Two other RAPD markers in coupling (OP-B17800 and OP-D15500) and another in repulsion (OP-C04650) phase were found associated with resistance to Fusarium wilt.

Eujayl et al. 1998b

Resistance to lodging and mycospharella blight

Two QTL associated with resistance to lodging together contributed 58% of the phenotypic variation. Three QTL each for plant height and mycospharella blight resistance accounted for 65% and 36% of the total phenotypic variation, respectively. These QTL were relatively consistent across environments. The AFLP marker associated with the major locus for lodging resistance was converted into a SCAR marker that corresponds well with the lodging reaction of 50 pea varieties.

Tar’an et al. 2003b

Fusarium wilt race 1

Single dominant gene confers resistance to fusarium wilt race 1, and an AFLP marker (ACG:CAT_222) has been located within 1.4 cM of the Fw gene.

McClendon et al. 2002

Fusarium wilt, powdery mildew, and pea common mosaic virus (PCMV)

Molecular markers linked with resistance to fusarium wilt (6 cM from Fw), powdery mildew (11 cM from er), and PCMV (15 cM from mo) have been reported, and three QTL explained most of the variation associated with resistance to Ascochyta blight race C.

Dirlewanger et al. 1994

Pea

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Aphanomyces root rot

A major QTL, Aph1, that explained up to 47% of the variation, and few minor QTL associated with resistance to aphanomyces root rot.

Pilet-Nayal et al. 2003

Ascochyta blight and/or plant maturity

Six QTL detected at the seedling stage together explained up to 74% of the variance and 10 QTL detected at the adult plant stage in the field accounted for 56.6–67.1% of the variance. Four QTL were identified under both growth chamber and field conditions suggesting that these loci were not plant growth-stage specific. A few QTL for flowering and plant height were co-located with QTL for resistance.

Prioul et al. 2004

Eleven and fourteen QTL detected for resistance to Ascochyta blight in A26 × Rovar and A88 × Rover populations, respectively. Of these, six QTL were associated with the same genomic regions in both the populations that reside on LG-II, III, IV, V, and VII (2 QTL). For plant maturity, six QTL were detected in the A26 × Rovar while five QTL mapped in the A88 × Rovar populations. QTL for plant maturity coincide with Ascochyta blight resistance QTL in four genomic regions: LG II (two regions), III, and V, linked either in repulsion or coupling phase.

Timmerman-Vaughan et al. 2004

Eight of 13 QTL associated with resistance to Ascochyta blight were detected in multiple environments.

Timmerman-Vaughan et al. 2002

Pea seed born mosaic virus (PSbMV)

An STS marker (sG05_2537) located approximately 4 cM from the sbm1 gene that confers resistance to PSbMV.

Frew et al. 2002

An RFLP marker (GS185) about 8 cM from the sbm-1, the gene for resistance to PSMV.

Timmerman-Vaughan et al. 1993

Powdery mildew

Three RAPD primers in coupling (OPO-18, OPE-16, and OPL-6) and two in repulsion phase (OPE161600 and OPL-61900) linked to powdery mildew resistance gene, er-1.

Tiwari et al. 1998

Green seed color

Two major QTL affecting seed color: QTL 1 and QTL 2 account for 61% and 56% variation of the variation for seed lightness and seed hue, respectively.

McCallum et al. 1997

Seed weight

A common seed weight QTL mapped to the same region of LG III in two crosses.

Timmerman-Vaughan et al. 1996

Grain yield, seed protein, and maturity

Across 13 environments, four QTL identified each for grain yield and maturity and 3 QTL for seed protein concentration that accounted for 39%, 45%, and 35% of the total phenotypic variation, respectively.

Tar’an et al. 2004

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limitations of conventional linkage mapping, there has been considerable interest recently in the development of methodologies that do not require the creation of mapping populations for each trait and that generate markers that can be more immediately applicable in diverse breeding programs. Linkage disequilibrium (LD) and genetic association mapping analyses are means of identifying a close association between genes contributing to target traits and marker loci using a structured collection of diverse germplasm (Thornsberry et al. 2001). LD mapping relies on population-level associations between target loci and nearby markers and has been extensively employed for mapping disease traits in mammals in which genetic populations are more difficult to develop. LD itself typically arises when all or most of the target alleles in a population share a common ancestral origin and a species or crop has undergone an evolutionary bottleneck. Most importantly, the LD approach is based on the use of natural or human-selected populations of plants rather than genetic populations. Moreover, LD mapping has the advantage that resultant markers tend to be both genetically and physically close to the gene of interest and therefore more readily applicable in a diverse range of breeding programs. The fundamentals of LD mapping have been reviewed in detail elsewhere (Boreck and Suarez 2001; Nordborg and Tavare 2002; Flint-Garcia et al. 2003; Rafalski and Morgante 2004). In contrast to the numerous linkage disequilibrium (LD) studies in humans and other mammals, there are very few publications on this topic in agriculturally important crops including legumes (Virk et al. 1996; Beer et al. 1997; Pakniyat et al. 1997; Forster et al. 1997; Igartua et al. 1999; Remington et al. 2001; Thornsberry et al. 2001; Turpeinen et al. 2001; Hansen et al. 2001; Sun et al. 2001, 2003; Skot et al. 2002; Ivandic et al. 2002, 2003; Amirul Islam et al. 2004; Zhu et al. 2003a; Simko et al. 2004; Sabharwal et al. 2004). Traditionally, the plant community has been reticent to use LD mapping, believing that it can lead to spurious and non-functional associations due to mutation, genetic drift, population structure, breeding systems and selection pressure (Hill and Weir 1994; Pritchard et al. 2000). However, most of these limitations are being overcome in recent mammalian studies by following precautions that minimize circumstantial correlations and maximize the accuracy of association statistics. Unfortunately, the real value of LD mapping in plants remains to be demonstrated, as most of the reports to date are based on small population sizes or a limited number of markers and generally lack validation.

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The most powerful LD analyses take account of two types of recombination: (i) unique or rare recombination between members of different populations or species (between different parts of the genome); and (ii) repeated recombination within a population between homologous sites. The analytical methods appropriate in the former context are inappropriate in the latter because they depend on recognizing the existence of runs of nucleotides with similar ancestry (Smith 1999). It is also important to determine the optimum number of markers required, which depends on the extent of disequilibrium and on how finely loci are to be mapped (Altshuler et al. 2000). In general, LD studies have tended to use a large number of markers selected from high-density linkage maps available in many mammalian systems. This number of markers is not available in most legumes, so a compromise number must be determined. Initial LD mapping studies have been conducted in the model plant Arabidopsis thaliana, which is characterized by a high level of polymorphism and extensive haplotype structure and thus provides a highly appropriate scenario for association mapping (Hagenblad and Nordborg 2002). In a study of the FRI locus, Nordborg et al. (2002) found that LD decays at a distance of roughly 1 cM or 250 kbp in Arabidopsis compared to one to a few kbp in maize. Meanwhile, a study on patterns of local and genome-wide linkage decay around six genes in maize inbreds using SSR and SNP loci revealed that intra-genic linkage decay generally declined rapidly with distance (r2 <0.1 within 1500 bp), but that rates of decline were highly variable among genes. This rapid decline of LD in maize probably reflects the large effective population size during maize evolution and high levels of recombination within genes. Provided the effects of population structure are effectively controlled, this research suggests that association studies can help to identify the genetic basis of important traits in maize (Remington et al. 2001). Association mapping between DNA markers and agronomic characters in a collection of plant genetic resources would allow (i) assessment of the genetic potential of specific genotypes prior to phenotypic evaluation, (ii) identification of superior trait alleles in germplasm collections, (iii) high resolution QTL mapping, and (iv) validation of candidate genes responsible for quantitative agronomic characters (Gebhardt et al. 2004). In a recent large-scale study in potato, association mapping was carried out using a gene bank collection of 600 cultivars of potato (Solanum tuberosum ssp. tuberosum) bred between 1850 and 1990 (Gebhardt et al. 2004). Highly significant associations were identified with QTL for resistance to late blight (Phytophthora infestans) and plant maturity. They also

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traced introgression of marker alleles associated with increased resistance and late maturity from the wild species S. desmissum. Other studies have detected associations between quantitative traits and RAPD markers, with Virk et al. (1996) predicting the performance of rice accessions. Similarly, RFLP markers were associated with quantitative traits in oats (Beer et al. 1997); however, most of these markers were not validated during QTL mapping of a population derived from a cross between ‘Kanota’ and ‘Ogle’. Using association mapping, Sabharwal et al. (2004) identified 15 AFLP markers obtained from eight AFLP primer combinations associated with seed-coat colour in 39 Brassica juncea lines, two of which (E-ACA/M-CTG350 and E-AAC/M-CTC235) were validated in recombinant inbred populations associated with yellow and brown seed-coat color, respectively. Kraakman et al. (2004) reported association between AFLP markers and complex quantitative traits such as mean yield, adaptability (Finlay-Wilkinson slope), and stability (deviations from the regression) in a collection of 146 modern two-rowed spring barley cultivars trial data. Regression of those traits on individual marker data disclosed marker-trait associations for mean yield and yield stability. In addition, many of the associated markers were located in the regions where earlier QTL were found for yield and yield components, thus demonstrating that association mapping can be a viable alternative to classical QTL analyses based on crosses between inbred lines, especially for complex traits with costly measurements. LD mapping has also been carried out using SNP haplotype analysis of 25 diverse soybean accessions that revealed a low level of linkage decay and a deficiency in the number of haplotypes (Zhu et al. 2003a). This lack of genome-wide linkage decay coupled with the low haplotype diversity suggested that the cultivated soybean genome was a mosaic of a limited number of haplotypes that may have resulted from recombination among three or four ancestral haplotypes. A recent study in common bean by Amirul Islam et al. (2004) showed that about 10% of the accessions in the CIAT core collection exhibited evidence of introgression between the Andean and Mesoamerican gene pools and that RAPD markers associated with these introgressions were associated with several morphological, economic, and nutritionally important characteristics. D. Dissection and Manipulation of Legume Physiology The public release of the entire genome sequence of A. thaliana and rice, and the rapid progress being made in sequencing the gene space of other major crops (including maize and soybean) and model legumes (M. trun-

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catula and L. japonicus) provide the basis for new approaches to plant research and breeding that may now be largely anchored to specific gene sequences and their putative functions. Because of the similarities between genes that code for the same trait in different species, comparative genomics will become an essential tool for technological leapfrogging from major crops and model systems to many related crops. Meanwhile, transgenic technologies have the potential to facilitate quantum leaps in improvements in economically important metabolic pathways that can be drastically influenced by a small number of genes. The current adoption, success and failures, and future prospects of transgenic crop varieties has been reviewed in detail elsewhere (Chandra and Pental 2003; Halford 2004; Popelka et al. 2004). To date, the most widely grown transgenic crops are soybean (33.3 m ha) and maize (9.8 m ha) with herbicide tolerance or insect pest resistance (James 2003). The products from these new crops are now incorporated into a wide range of processed foods that include altered protein, starch, or oil quality; and improved micronutrient or vitamin content (Dunwell 1998). There has also been large-scale adoption of cotton (6.8 m ha) and canola (2.7 m ha) with the same herbicide tolerance or insect pest resistance transgenes (James 2003). Six countries grew 99% of the global transgenic area (67.7 m ha) in 2003: USA (42.8 m ha), Argentina (13. 9 m ha), Canada (4.4 m ha), Brazil (3 m ha), China (2.8 m ha), and South Africa (0.4 m ha) (James 2003; http://www.isaaa.org/kc). Recent developments in legume genomics are also providing an array of molecular breeding opportunities to legume breeders. The critical advances include the availability of a large number of robust PCR-based markers such as SSR, ESTs, and SNPs and the generation of high-density genetic maps. At the same time, the generic advances in automation technologies now offer real possibilities for the efficient development and application of marker-assisted selection techniques at a scale and unit cost that is finally of relevance to plant breeding programs and international germplasm collections. Meanwhile, developments in microarray technology promise a new paradigm of simultaneous selection for a diverse range of complex traits. Among the traits of interest, the exploitation of genomics, transgenics, and molecular breeding will allow legume breeders to enhance pest and disease resistance, increase environmental stress tolerance, improve symbiotic nitrogen fixation, and develop nutritional quality (biofortification) in the future. Plant breeders have been successful in creating new high-yielding cultivars in many crops by incorporating just a few genes, i.e., those

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involved in plant height and photoperiodism (Miflin 2000). However, continued improvement has been increasingly complex and difficult. Manipulating components of yield is confounded by the highly complex genetic nature of this trait in which there is substantial interaction between underlying genes (epistasis), and significant environmental dependency of gene expression (Ribaut and Hoisington 1998). Many of the most agronomically and economically important traits (i.e., drought, salinity, yield, and nutritional quality) in most crops have quantitative phenotypic variation, are under polygenic control, and are significantly affected by the environment. These traits are, therefore, very difficult and time consuming to manipulate in conventional breeding programmes. These factors are also at least as troublesome during the identification of trait-based genetic markers. Moreover, the expertise, the time, and expense required to generate appropriate phenotype data for QTL mapping should never be underestimated. However, once effective and reliable markers have been identified, substantial progress can be made in the speed and precision of manipulating these traits in breeding programs. Whole plant physiology modeling is becoming an increasingly important tool for aiding improvement in breeding efficiency of complex traits. These models help define crop ideotypes for different environments. QTL mapping is carried out for physiological components that are more likely to relate directly to gene expression. Modeling can then be used to extrapolate information to a new environment (Podlich and Cooper 1998; Cooper et al. 2002; Chapman et al. 2002, 2003; Yin et al. 2003, 2004). Using the ideotype approach, Chinese researchers have developed superhybrid rice by incorporating a few morpho-physiological traits such as long, narrow, and erect top leaves and large panicles that hang close to the ground to enhance the efficiency of crop light capture (Setter et al. 1995). In field trials at four separate locations in China, these superhybrids produced 15% to 20% higher yields than existing hybrids that already generated 10.5 t ha–1 (Normile 1999). Based on the complimentary aspects of modeling and mapping, Yin et al. (2003) proposed an approach that integrates marker-assisted selection (MAS) into the model-based ideotype framework to support breeding for improved crop yield. For this approach to be effective, there is a need to develop crop models capable of predicting yield differences among genotypes in a population under various environmental conditions. System biology attempts to discover and understand biological properties that emerge from the interaction of many system elements (Ideker et al. 2001; Kitano 2002). It examines the structure and dynamics of cellular and organismal function, rather than the characteristics of isolated

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parts of a cell or organism. The substantial progress in molecular biology, particularly in genomics, proteomics, and high-throughput measurements, has facilitated researchers to collect comprehensive datasets on the mechanisms underlying plant growth and plant responses to perturbations. A plant requires information about its environment and its interaction with that environment and uses that information to dictate its adaptive responses that result in the plant phenotype. Significant endeavors in the field of whole-plant modeling are now being directed at understanding genetic regulation and aiding crop improvement (Hammer et al. 2002; Cooper et al. 2002; Chapman et al. 2003; Yin et al. 2003, 2004). Crop models with generic approaches to underlying physiological processes (Wang et al. 2002) provide a means to link phenotype and genotype through simulation analysis to provide what is being termed the “in silico plant.” Modern genomics has enormous potential for generating huge amounts of information about individual components in the genotype to phenotype pathway. Whole plant physiology modeling offers a means of reconstructing the entire pathway and thereby providing a critical link between molecular genetics and crop improvement. Important progress in this area has already been made for leaf growth in maize (Zea mays L.) in response to temperature and water deficit (Raymond et al. 2003), photoperiod responses in sunflower (Helianthus annuus L.) (Leon et al. 2001), stay green trait in sorghum (Sorghum bicolor L.) (Borell et al. 2001), specific leaf area in barley (Hordium vulgare L.) (Yin et al. 1999), and tolerance to water deficit in various crops (Tardieu 2003). 1. Enhancing Stress Tolerance. Drought and salinity are major abiotic factors limiting legume productivity. In this context, the development of enhanced tolerance or avoidance mechanisms is a critical element in ensuring yield stability in many target environments. Despite intensive efforts over the past 50 years or more, the identification and utilization of drought and/or salinity tolerance traits in conventional breeding programs has been generally unsuccessful. New knowledge and tools from the rapid progress in legume genomics and functional genomics are identifying a cascade of regulatory genes that open new possibilities for altering plant architecture and metabolism that will undoubtedly contribute to breaking the current impasse in stress tolerance breeding. Drought Tolerance. The necessary tools and methods for characterizing drought environments, sources of drought tolerance, and drought tolerant traits (root characteristics, photosynthate partitioning, water use efficiency, etc.), in addition to our ability to rapidly advance generations

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using marker-assisted selection, are now available to help us embark upon a focused program of genetic enhancement for drought tolerance using trait-based molecular breeding. For instance, an ideotype approach was adopted at ICRISAT for genetic improvement of drought tolerance in chickpea (Saxena 2003). Concurrent selection for drought-tolerant traits, disease resistance, and yield in early segregating generations is essential to develop viable new varieties. Several gene transfer approaches have been attempted with the objective of improving the stress tolerance of plants both based on single genes or a product of a regulatory gene that activates a whole cascade of other gene products in the plant stress response pathway (Holmberg and Bulow 1998). Genetically engineered plants for single gene products include those encoding for enzymes required for the biosynthesis of osmoprotectants (Hayashi et al. 1997), modified membrane lipids (Ishizaki-Nishizawa et al. 1996), a LEA (late embryogenesis abundant) protein (Xu et al. 1996), and detoxification enzymes (Mckersie et al. 1996). Similarly, many genes that are thought to be involved in stress response can be simultaneously regulated by using a single gene encoding, a stress-inducible transcription factor (Kasuga et al. 1999). DREB/CBF genes are a small family of transcription factors that bind to the drought-responsive elements (DRE) found in the promoters of many drought-responsive genes of Arabidopsis (Liu et al. 1998; Gilmour et al. 1999) and rice (Dubouzet et al. 2003). This offers the possibility of manipulating the regulation of plant response to multiple stresses including drought, salinity, and freezing. Preliminary evaluation of peanut, wheat, and rice transformants containing the DREB1A gene has shown enhanced tolerance to drought stress (Dubouzet et al. 2003; Mathur et al. 2004; Pellegrineschi et al. 2004). However, expression of DREB1A gene under constitutive promoter CaMV 35s induces growth retardation under normal growth conditions (Liu et al. 1998). In contrast, plants produced using the stress-regulated rd29A promoter demonstrated increased tolerance to freezing, salt and water limited stresses, without producing changes in the normal phenotype of the transformed plants (Kasuga et al. 1999). Transgenic plants carrying coda and P5CSF: 129A genes for drought tolerance in chickpea (ICRISAT 2004) and a gene for aluminium tolerance in soybean (Ermolayev et al. 2003) have also been developed that are in various stages of characterization under containment glasshouses/field trials. Polyamines are small, ubiquitous, nitrogenous compounds that have been implicated in a variety of stress responses in plants (Bajaj et al. 1999). Plant polyamine content has been modulated by the overexpression/down-regulation of arginine decarboxylase (adc), ornithine decar-

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boxylase (odc), and S-adenosylmethionine decarboxylase (samdc) (Lepri et al. 2001; Mehta et al. 2002; Thu-Hang et al. 2002; Trung-Nghia et al. 2003). Overexpression of heterologous adc or odc cDNAs in plants generally results in the production of high levels of putrescine that protected the plants from environmental stresses (Capell et al. 1998). Capell et al. (2004) produced transgenic rice plants containing adc gene (maize Ubi1 promoter) from Datura stramonium that produced much higher levels of putrescine under stress, promoting spermidine and spermine synthesis. This study demonstrated that manipulation of polyamine biosynthesis in plants could produce drought-tolerant germplasm. Thus, both DREB and non-DREB genes are now available to breeders to use in enhancing the drought-tolerance profile of their legumes. However, the complex nature of this trait and the negative yield effects of many stressrelated transgenes suggests that a combination of transgenic and markerassisted selection technologies will be required to create effective new varieties. This would most likely involve the pyramiding of transgenes from outside the crop species together with drought-tolerance and yieldrelated genes from within the crop species. Salinity Tolerance. Transgenic technologies have also been recently used to develop salt-tolerant plants by over-expressing a single gene. Glenn et al. (1999) successfully engineered transgenic Arabidopsis plants to overexpress AtNHX1, a vacuolar Na+/H+ antiport, which allowed the plants to grow in 200 mM NaCl. Meanwhile, the same genetic modification expressed in tomato plants enabled them to grow in 200 mM NaCl solution as well as the wild type (Zhang and Blumwald 2001). A similar strategy was also used to genetically modify rapeseed (Brassica napus), one of the most important oilseed crops cultivated worldwide (Zhang et al. 2001). The resulting plants could grow in salt concentrations that were 50 times higher than normal producing conditions. Although the transgenic plants grown in high salinity under greenhouse conditions accumulated sodium up to 6% of their dry weight, growth of these plants was only marginally affected by the high salt concentration. Moreover, the seed yields and oil quality were not affected by the high salinity of the soil. Such plants are not only tolerant to higher levels of salinity, but can also remove a high proportion of salts from soil, thus offering bioremediation of saline soils. These examples demonstrate that it should be possible in the near future to bio-engineer a broad spectrum of legume crops to be productive on saline soils or under poor water quality irrigation. 2. Increasing Symbiotic Nitrogen Fixation. Legumes have the ability to establish symbioses with rhizobia and mycorrhizal fungi, a characteris-

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tic that makes them of interest for plant nutrition research (Udvardi 2002). Caetano-Anolles and Gresshoff (1991) identified many genetic loci that are essential for symbiotic nitrogen fixation (SNF) in different legume species. Some of these genes are required for early events, such as bacterial signal recognition or events that allow colonization of the root by rhizobia. Other plant genes play essential roles in later stages of nodule development, in biochemical processes that support nitrogen fixation, and in feedback control of nodulation (Colebatch et al. 2002). Recent developments in reverse genetics and functional genomics promise to accelerate the isolation of important symbiotic genes in legumes, including the model legumes M. truncatula and L. japonicus. Approximately 40 symbiosis (Sym) mutants have already been identified in various legume species, including pea, soybean, M. truncatula, and L. japonicus (Udvardi 2002) and several have been cloned by genetic and physical mapping. These Sym genes are essential for nodulation and/or symbiotic nitrogen fixation in legumes. Similarly, the discovery of a gene controlling the proliferation of induced nodule primordia in soybean (NARK) opens a new field of functional genomics of symbiosis (Gresshoff 2003). Since increasing information and resources are becoming available from the model legumes and major crops such as pea and soybean, these resources can be harnessed for SNF research in other legumes as well. Transfer of knowledge from the model legumes to lesser-studied legume crops is a multifaceted process, which should include a systematic analysis of existing legume SNF mutants, large-scale generation and physiological characterization of new SNF mutants, and analysis of mutations focusing on known NOD and ENOD cDNAs of model legumes. Functional genomics of mutant, normal, and high-performance SNF lines could also be performed by hybridization of their nodulated and non-nodulated root cDNAs to gene chips available for the model legumes (Winter et al. 2003). 3. Biofortification. Legume crops are extremely important in human and animal diets, supplying 33% of human protein globally. Legume seeds are rich in protein, oil (peanut and soybean), minerals (iron and zinc), and vitamins. Isoflavones that are produced exclusively in legumes have drawn much attention because of their benefits to human health, and for their roles in plant defense and root nodulation. The potential to produce legume-based foods that have altered composition (both changes in nutritional properties and in anti-nutrients) is now a reality, and will further enhance the benefits from legumes in human diets. Potential biotechnology modifications of the three major seed components of nutritional and commercial significance, namely of seed oil fatty acids, proteins, and carbohydrates, is now possible. For example, modifications of fatty acid composition, especially for high oleic acid

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content have been realised. These have been achieved through induced mutation and rDNA modifications (Downey and Taylor 1996). Soybean oil can also be changed in terms of the degree of unsaturation and the length of fatty acid chains (Topfer et al. 1995). The reduction in polyunsaturated fatty acids reduces the need for hydrogenation and thereby reduces the incidence of trans-fatty acids, which have detrimental effects on indices of coronary heart disease risk. Increasing the level of essential amino acids in oil seed meals as well as legume grains is also the subject of considerable research. For example, Kinney (1995) was able to increase the level of lysine by 7.5% to 40% of the total amino acid content of soybean and canola meals by inserting the gene cordap A, from Corynebacterium glucamicum, that encodes one of the enzymes insensitive to lysine feedback (dihydrodipecolinate synthase). Molving et al. (1997) introduced a chimeric gene with seed-specific expression of a sulfur-rich sunflower seed albumin into narrow-leafed lupin (Lupinus angustifolius L.). In feeding trails with rats, the transgenic seeds (0.94% increase in methionine content and 12% reduction in cystein content) gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization compared with wild type seeds. Transgenic plants have also been developed that carry a gene encoding for a methionine-rich storage albumin protein from Brazil nut to enhance methionine in common bean and soybean (Muntz et al. 1998; Aragao et al. 1999); a gene that co-suppresses an enzyme (Grierson et al. 1996) that converts oleic acid to linoliec acid and thus improves oil quality in soybean (Mazur et al. 1999); tumor-associated, embryonic protein antibody in pea for in vitro immuno-diagnosis and in vivo imaging of human cancers (Perrin et al. 2000); and Arabidopsisbased MPBQ/MSBQ methyltransferase gene (VITAMIN E 3; VTE3) to engineer increased vitamin E activity in soybean (Sattler et al. 2004). The feasibility of using genetic engineering to improve the nutritive value of grain legume has thus been demonstrated. These genes are candidates for the recently launched CGIAR global challenge program on ‘Biofortified Crops for Improved Human Nutrition’ (http://www.ifpri.org/ themes/grp06/paper/biofort.pdf) (also known as Harvest Plus CP), which will support research and development of nutrient-dense cultivars of 17 crops (including beans, peanut, lentil, cowpea, and pigeonpea). 4. Bioinformatics. There are many publicly available bioinformatic resources for legume researchers (Table 6.23). These include sequence repositories, gene expression databases, gene identification and structure databases, databases containing genetic and physical maps, genomic databases, metabolic pathways, protein and structure databases, and their associated tools. All of these resources have been developed by

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Table 6.23.

Bioinformatic resources for legume genomics and molecular breeding.

Database

Analysis and retrieval tool

Database content

URL

TIGR Medicago Gene Indices

Blast, query pages and sequence downloads

Sequence resource permitting searches based on sequence similarity, keywords or tissue origin, functional annotation, analysis and comparisons of EST expression between different libraries or tissues

http://www.tigr.org/tigrscripts/tgi/T_index.cgi?specie s=medicago

M. truncatula genomic resources

Genscan, Diogenes for ORF finding, and Blast tools

Provides access to M. truncatula cDNA and BAC libraries, chromosome marker maps, protocols and links to other databases like the Oybase, TAIR, Gramene, Beangenes, and UKCropnet.

http://www.medicago.org

M. truncatula genome database

Blast

Integrated database containing physical mapping data, genetic mapping data and BAC sequence data

http://mtgenome.ucdavis.edu/db/

Medicago EST Navigation System (MENS)

PATSCAN, BLAST, FrameD, gene finding programmes, electronic northerns, sequence analysis tools, protein family search

Genomic, EST, proteomic, and expression data from Medicago have been integrated in this database and can be mined.

http://medicago.toulouse.inra.fr /mt/EST

SNRF Plant 2DPAGE database for Medicago truncatula

Query pages

2D PAGE gel archives.

http://www.noble.org/2Dpage/s earch.asp

Proteomics of Medicago truncatula

Query pages

2D MaldiTOF experimental data, Tandem Mass spectrometry and 2D electrophoresis experiments

http://www.mtproteomics.fr.st/

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L. japonicus Genomics database

Blast, keywords search, searching the Lotus japonicus EST index through query pages, and the Legume genome Scanner

Provides access to LegumeBase, a database containing genetic resource information from L. japonicus and G. max. Besides, EST, marker, mapping data and access to leguminous genes from Lotus and Medicago is provided.

http://www.kazusa.or.jp/lotus/

TIGR Lotus Gene Indices

Blast, query pages and sequence downloads

Sequence resource, permitting searches based on sequence similarity, keywords or tissue origin, functional annotation, analysis and comparisons of EST expression between different libraries or tissues and analysis.

http://www.tigr.org/tigrscripts/tgi/T_index.cgi?specie s=l_japonicus

TIGR soybean Gene Indices

Blast, query pages and sequence downloads

Sequence resource, permitting searches based on sequence similarity, keywords or tissue origin, functional annotation, analysis and comparisons of EST expression between different libraries or tissues.

http://www.tigr.org/tigrscripts/tgi/T_index.cgi?specie s=soybean

Soybase

Blast and query pages to search EST collection and annotations

Provides access to comprehensive genetic maps, 25 single population molecular marker maps, marker information, allele data, information on agronomic traits, pathways data, germplasm data, proteins mapped, etc.

http://soybase.org

Soybean Gbrowse database

Gbrowse

Soybean genomics information.

http://bioinformatics.siu.edu/

Soybean genomics and microarray database

Database query tools

Sequence and microarray experiment data.

http://psi081.ba.ars.usda.gov/S GMD/Default.htm (continued )

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Table 6.23.

(continued)

Database

Analysis and retrieval tool

Database content

URL

Soybase Pathways Database

Metabolic component of soybase, contains diagrams for viewing pathways

Metabolic pathways, descriptions, and diagrams for basic pathways.

http://soybase.ncgr.org/cgi-bin/ ace/generic/search/soybase

Legume Information system (LIS)

Blast LIS

EST, genomic, map, pathway, proteomic resources from multiple legume species, enabling cross species comparisons.

http://www.comparativelegumes.org/

Legume DB

Blast, Fasta, Fastacmd, Repeat Masker, ClustalW, EMBOSS tools, Xcompare, MultiBlast, CMAP and BioDAS map visualization tools

Permits comparative genomics, candidate gene mining, PCR primer design. Map visualization tools allow highlighting of areas of macro and micro synteny across closely related target species.

http://cbbc.murdoch.edu.au/ projects/legumedb/

ILDIS-International legume database and Information service

Web based query tool-LegumeWeb and litchi software

Permits searching of the world’s legume species diversity catalog. The litchi software enables merging of taxonomic data from different sources.

http://www.ildis.org/

Consensus Legume DB

Provides links to several databases and their associated sequence analysis and mapping tools.

Integrates EST, unigene, HTGS, markers, maps, etc. from multiple sources: the M. truncatula consortium DB, TIGR resources, Soybase, Arabidopsis Information resource (TAIR) and Gramene.

http://www.legumes.org

KEGG pathways Database

Query pages, visualization tools

Information on metabolic pathways.

http://www.genome.jp/kegg/ metabolism.html

Sputnik

Query interface

Functional predictions, comparative analysis and annotation for 500,000 plant EST derived peptides.

http://sputnik.btk.fi/

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researchers focusing on M. truncatula, L. japonicus, and G. max. However, the information is valuable for comparison and extrapolation to other legumes. There are also increased efforts toward providing a single portal/or consensus database with links to individual crop-specific databases to provide a single platform for the comparative genomics of legumes. Most databases provide access to several bioinformatic tools, comparative mapping, and data visualization tools. These include sequence comparison methods such as various versions of BLAST, gene prediction tools, pattern searching and mapping programs, etc. Databases generated specifically for legume researchers are more eclectic, combining varieties of tools for the analysis of several types of data— sequence, proteomic, or expression data (such as the MENS database). Since data analysis methods are dynamic, varying with the nature of data and hypothesis tested, database systems are now being designed that insulate the analysis methods from the data itself. One such system is DOME (http://medicago.vbi.vt.edu/index.html), a relational database that allows merging of microarray, proteomic, and metabolic profile data in Medicago. While these represent a new generation of databases, still the most frequently used resources are those individual model legume repositories such as the TIGR Gene Indices. These databases are curated, containing the results and analysis of worldwide projects on the target species; the data is non-redundant and annotated. Information on gene expression and protein family is also available and retrievable, making this a highly useful resource for legume research. The molecular maps for legume crops of the semi-arid tropics (cowpea, groundnut, and pigeonpea) are not very dense compared to the other related legume crops such as soybean, common bean, and pea. However, data from related species could be mined to develop maps and markers. Hence, nucleotide sequences of the legume model species M. truncatula, available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), have been downloaded in FASTA format and the repeat patterns in the sequence were located using the tandem repeat finder program at http://c3.biomath.mssm.edu/trf.html (Mahalakshmi et al. 2002). The sequences with potential repeat motifs were then analysed to determine possible potential flanking regions around the repeat motifs, which might yield product sizes of about 200 bp using the PRIMER 3 program (http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi). The gene indices database from TIGR (http://www.tigr.org/tdb/tgi/mtgi/) was also downloaded, and a local database containing the entire sequences in FASTA format, the repeat motif, the potential primer, and the gene indices was created in a relational database (SQL7.0TM). The resultant database of repeat motifs was

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analyzed to classify the patterns, and their occurrence and abundance (Mahalakshmi et al., submitted). Of about 156,000 sequences that were searched, 7325 sequences were found to contain a repeat motif and may yield SSRs with amplification product sizes of around 200 bp. Of these, the most abundant repeats were the tri-nucleotide (5210) group. Except for a very small proportion (436), these link to the gene annotation database at TIGR. To facilitate further exploration of this resource, a dynamic database with options to search and link to other resources is being developed. Such an approach may lead to the development of microsatellite markers for the same species or closely associated species within the same genus. Modern comparative and functional genomics coupled with new marker technologies as well as bioinformatic, biometric, and modeling tools are becoming a major force for an emerging revolution in crop improvement that seeks to identify and define the structure and function of key genes, and reveal when and how these genes generate desirable phenotypes that can be used in crop improvement. Combining this knowledge with recent advances in genomics technologies provides the mechanism for “Breeding by Design” (Peleman and van der Voort 2003) where new varieties and new crop products can be designed in a more rational way than has been previously possible through conventional plant breeding. Having documented here the progress in studying and manipulating key individual traits, we are now poised for a new paradigm in plant breeding in which entire profiles of specifically selected genes can be manipulated to provide a complement of desirable traits. High-value crop ideotypes will be sequentially generated combining target product profiles for any specific cropping system environment. These “designer legumes” will transform and merge agricultural and industrial production to an extent only limited by mankind’s imagination and motivation to continue to innovate.

VI. ADVANCED APPLICATIONS IN LEGUME MOLECULAR BREEDING A. Comparative Genomics and Allele Mining In this section, we describe the comparative genomic relationships between model species, major crops, and lesser-studied but economically important legume crops. In particular, we focus on the potential of comparative mapping and gene sequence homology to benefit structural and functional genomics, and ultimately molecular breeding of

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legume crops. In this context, we consider structural genomics the construction of genetic and physical maps, and functional genomics the comparison of gene expression across tissues, species, and environments. Both of these fields are being fueled by rapid access to new genetic and sequence information, and are driving the important area of comparative biology. 1. Model Systems. As discussed in the introduction, two model legumes, L. japonicus and M. truncatula, have been studied intensively with a particular initial emphasis on symbiotic relationships with nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi as well as the synthesis of secondary metabolites such as isoflavonoids that are not found in A. thaliana (Barker et al. 1990; Handberg and Stougaard 1992; Jiang and Greshoff 1997; Cook et al. 1997). Along with A. thaliana, these species have emerged as the genomic hubs for comparative mapping and genomic studies in the dicots. The key attributes of all model plants are a small diploid genome, rapid reproductive cycle, autogamous nature, prolific seed production, and numerous genetic and genomic resources available for the molecular investigation of diverse traits of biological and economic importance (Table 6.24). M. truncatula is a forage legume commonly grown in Australia and is a close relative of lucern (alfalfa: M. sativa), the most important forage crop worldwide. In addition, it belongs to the same phylogenetic group (galegoid) as Pisum (pea), Vicia (vetch, broadbean, and cowpea), Cicer (chickpea), Lens (lentil), and Trifolium (clover) (Doyle et al. 1996). The M. truncatula genome is organized into two distinct regions: the pericentromeric heterochromatin, which is rich in repeated sequences but contains a low density of expressed genes and the extensive gene-rich euchromatic regions. Gene density is approximately one gene per 6–10 kilobase pairs (kbp) in M. truncatula, on par with that of A. thaliana (Nakamura et al. 2002; Young et al. 2003). In contrast, L. japonicus is phylogenetically distant from the galegoid phylum and less closely related to most economically important legumes except for groundnut and Lupin. Both model legumes are distant from the Phaseoleae tribe that includes many important legumes such as soybean (Glycine), common bean (Phaseolus), and pigeonpea (Cajanus). Researchers have made substantial progress toward creating numerous genetic and genomics resources in the two model legumes: M. truncatula has a dense genetic linkage map (Thoquet et al. 2002), numerous mutants (Sagan et al. 1995; Penmetsa and Cook 1997), tissue specific cDNA libraries (Gamas et al. 1996; Covitz et al. 1998; Gyorgyey et al. 2000), EST collections (Journet et al. 2002), isolated resistant gene analogues (Cannon et al. 2002; Zhu et al. 2002), large-insert BAC libraries

272 Table 6.24.

Key attributes of the legume model systems Medicago truncatula and Lotus japonicus compared with Arabidopsis thaliana.

Key attributes

Medicago truncatula

Lotus japonicus

Arabidopsis thaliana

Ploidy level/ chromosome # (n)

Diploid, n = 8

Diploid, n = 6

Diploid, n = 5

Genome size and sequencing information

500–550 Mbp; sequencing of the entire gene space ongoing

400 Mbp; large-scale genome sequencing initiated

120 Mbp; entire genome sequenced

Breeding behavior

Self fertile and short generation time

Self fertile and short generation time

Self fertile and short generation time

Reproduction

Prolific seed production

Prolific seed production

Prolific seed production

DNA markers

PCR (RAPD, AFLP, SSRs, and ESTs)based markers reported (Cho et al. 1999; Drenakrd et al. 2000; Jander et al. 2002; Torjeck et al. 2003; Schmid et al. 2003)

PCR-based markers (RAPD, AFLP, SSRs, and ESTs) reported (Covitz et al. 1998; White et al. 2000; Baquerizo-Audiot et al. 2001; Fedorova et al. 2002; Journet et al. 2002; www.medicago.org)

Both RFLP and PCR (RAPD, AFLP, SSRs, ESTs, and SNPs)-based markers reported (Asamizu et al. 2000; Poulsen and Podenphant 2002)

Genetic linkage map

A genetic linkage map with an average map density of 4.24 cM reported (Chang et al. 1988, 2001; Nam et al. 1989; Reiter et al. 1992; Lieu et al. 1996; Choi et al. 2004)

A high density genetic map with an average map distance of 0.6 cM reported (Kulikova et al. 2001; Thouquet et al. 2002; Sandal et al. 2002; Hayashi et al. 2001); Chromosome map integrating the position of BAC and plasmid clones from 32 genomic regions (Pedrosa et al. 2002)

Genetic maps with average map density between 1.88 to 5.24 cM are reported (Jiang and Gresshoff 1997; Sandal et al. 2002; Pedrosa et al. 2002; Fjellstrom et al. 2003)

Genome sequencing

111 Mbp of the 454–526 Mbp genome sequenced; 190,000 ESTs submitted to Genbank of which about 37,000 are Unigenes http://www.medicago .org/genome/stats.php

A total of 320 TAC clones covering 32.5 Mb sequenced (Sato et al. 2001; Nakamura et al. 2002; Kaneko et al. 2003; Asamizu et al. 2003; Kato et al. 2003)

115.4 Mb of the 125 Mb genome sequenced (AGI 2000)

Genetic transformation

Efficient transformation protocol established

Efficient transformation protocol established

Efficient transformation protocol established

Symbiotic relationship

Symbiotic relationships with Sinorhizobium and mycorrhizae; a model legume to study indeterminate nodulation

Symbiotic relationships with Rhizobium and mycorrhizae; a model legume to study determinate nodulation

No symbiotic relationships either with Rhizobium or with mycorrhizae

Genetic and genomic resources

BAC clones; large ESTs; FISH for analysis pachytene chromosome and gene TILLING to identify point mutation; efficient transformation system; high density genetic map; numerous ecotypes and RILs; high degree of synteny with diploid alfalfa and pea

Linkage maps, BAC library (Men et al. 2001); expression arrays both for plant and endosymbiont genes; efficient transformation system; cDNA libraries constructed and large number of ESTs developed; gene TILLING to identify point mutations; genome sequencing initiated; tools for rapid map-based cloning of genes; diverse ecotypes and mutants and RILs (Gifu B-129 × Miyakojima MG-20)

BAC/BIBAC library; large numbers of RFLP, RAPD, AFLP, SSRs, ESTs, and SNPs; efficient transformation system; high density of genetic map; numerous ecotypes and RILs

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(Nam et al. 1999), an efficient transformation system (Chabaud et al. 1996; Trinh et al. 1998; Trieu et al. 2000), and a well-characterized nitrogen-fixing symbiont, Sinorhizobium meliloti (Galibert et al. 2001). A database of over 170,000 M. truncatula (Mt) ESTs has been assembled based on in-depth sampling from various developmental stages and pathogen-challenged tissues (http://www.medicago.org/MtDB) and can be queried through a series of interfaces and filters. A relational database provides a wide range of user-defined data mining options, allowing researchers to quickly and independently identify sequences that match specific research interests through user-defined criteria (Lamblin et al. 2003). The L. japonicus genomic resources include diverse ecotypes, a large number of DNA markers, a high-density genetic linkage map, expression arrays, cDNA and BAC libraries, a partially sequenced genome (320 TAC clones covering 32.5 Mb), an efficient transformation protocol, and well-studied symbiotic relationships with Rhizobium and Mycorrhizae (Table 6.24). 2. Comparative Mapping Synteny between Dicot Families. Comparative genomics is defined as the study of similarities and differences in the structure and function of gene(s) across taxa (Paterson et al. 2000). It facilitates the mapping of orthologous loci across plant species, genera, and families. Orthologous loci show common vertical descent and function as opposed to paralogs that are genes that have arisen within the same genome through duplication and may have evolved different functions (Fitch 1970). Comparative mapping in monocots has demonstrated that the order and sequence of genes is highly conserved between different cereal crop genomes (Devos and Gale 1997; Gale and Davos 1998). More recently, nearly six thousand Triticaea ESTs that had been physically mapped using wheat (Triticum aestivum L.) deletion lines and segregating populations were compared with the first draft of the public rice genome sequence. A rice genome perspective on the homoeologous wheat genome based on sequence analysis shows general similarity to the previously published comparative maps based on RFLP analysis. For most rice chromosomes, there is a preponderance of wheat genes. However, some wheat ESTs with multiple wheat genome locations is associated with genomic regions not well conserved between rice and wheat. Conversely, comparing the wheat deletion map with the rice genome sequence revealed a breakdown of gene content and order synteny. This suggests there is an abundance of rearrangements, insertions, and duplications that differentiate the wheat and rice genomes. These differences may complicate the use of rice as a hub for cross-species transfer of infor-

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mation in non-conserved regions (La Rota and Sorrells 2004). Comparative mapping amongst dicots has revealed an even a more complex pattern but comparative genomics still offers a range of important potential impacts (Bennetzen 2000; Paterson et al. 2000). Initial comparative mapping in the Solanaceae family (like that in cereals) was based on restriction fragment length polymorphism (RFLP) analysis. More recently, a large number of conserved ortholog sequence (COS) markers have also been identified for comparative genomics between Arabidopsis and members of the Solanaceae (Fulton et al. 2002). COS markers can be used directly as hybridization probes in RFLP mapping or, alternatively, the COS sequences can be used to BLAST search EST databases or to design PCR markers to amplify the orthologous locus in the target species. Significant synteny between Arabidopsis and soybean has been demonstrated along the entire length of Arabidopsis chromosome 1 and soybean linkage group A2 (Grant et al. 2000). There are also several examples of synteny mapping between Arabidopsis and the model legume species (Table 6.24). Synteny amongst Legumes. The phylogenetic relationships between different legume genera suggests that a comparative genomics approach will be useful in translating knowledge and tools from the model legumes to other legume species within the Papilionoideae subfamily. Legumes are seen to form a coherent taxonomic group with frequent and widespread macro- and micro-synteny (Young et al. 2003), as evidenced by an increasing number of studies showing the relationships between Medicago/Lotus and various grain legume crops (Table 6.25) and between different food legumes (Table 6.26). There are a number of reports concerning the cross species and genera amplification of SSR markers. Eujayl et al. (2004) demonstrated that a high proportion of M. truncatula ESTs-based SSRs markers amplified in species throughout the Medicago genus. Similarly, a high proportion of SSR markers from pea amplified in vetch (39%) and lentil (60–75%), although a lower proportion of chickpea SSR markers amplified in pea (18%) (Pandian et al. 2000). However, Peakall et al. (1998) reported limited cross-genera SSR amplification (3% to 13%) within the Papilionoideae, although cross species amplification was more successful. More recently, the transferability of SSR markers between Medicago, soybean, cowpea, and peanut revealed a high proportion (30.78%) of the SSR primers generating reproducible and cross-genus amplicons (117 polymorphic bands) that could be used as DNA markers for characterization and evaluation of legume germplasm (Wang et al. 2004a). Twentythree universal primers [SNP-containing sequence tagged sites (STS)] in soybean, common bean, cowpea, chickpea, and barrel medic are reported

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Table 6.25. Examples of comparative mapping between dicot model systems (Arabidopsis, Medicago or Lotus) and legume crops (chickpea, pea and soybean). Plant species Arabidopsis and chickpea Arabidopsis and soybean

Comparative mapping result

Reference

Synteny between the Fusarium resistance gene cluster of chickpea (19 markers on linkage group 2) and the corresponding regions in the Arabidopsis genome (short segments of chromosome 1 and 5). Mapping of 82 tentative orthologous genes reveals a lack of extended macrosynteny between the two genomes, although localized synteny has been frequently reported over small genetic intervals. The genetically linked loci in M. truncatula often share multiple points of synteny with Arabidopsis. The two genomes are related by network of microsynteny that is often highly degenerate either due to selective gene loss from duplicated loci or due to the absence of close homologs of M. truncatula genes in Arabidopsis. 27 of 78 sequences on soybean molecular linkage group (MLG)-G showed significant similarity to Arabidopsis. The conserved microsynteny in Bng122–Bng173 region of soybean with Arabidopsis extends over several hundred kbp. Soybean linkage group A2 (soyA2) and Arabidopsis chromosome 1 showed significant synteny over almost their entire lengths, with only 2–3 chromosomal rearrangements required to bring the map into substantial agreement. Smaller blocks of synteny were identified between soyA2 and Arabidopsis chromosome 4 and 5 (near the RPP5 and RPP8 genes) and between soyA2 and Arabidopsis chromosomes 1 and 5 (near the PhyA and PhyC genes).

Benko-Iseppon et al. 2003 Zhu et al. 2003b

Arabidopsis, Lotus, and pea

LjSYM2 gene from L. japonicus and PsSYM19 from P. sativum are required for the formation of nitrogenfixing root nodules and arbuscular mycorrhiza. Local colinearity in the region around LjSYM2/PsSYM19 identified in all the three species; LjSYM2/PsSYM19 corresponds to two duplicated segments of the Arabidopsis chromosomes AtII and AtIV.

Stracke et al. 2004

Arabidopsis, Medicago, and soybean

27 of the 50 soybean contig groups showed microsynteny with Medicago. Substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. Seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.

Yan et al. 2003

Medicago and pea

M. truncatula and pea genomes at SYM2 region share a conserved gene content.

Gualtieri et al. 2002

Medicago, Lotus, and soybean

Apyrases are suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. A phylogenetic analysis of apyrase homologs from M. truncatula, Glycine max, and Lotus japonicus identified a potentially legume-specific clade that contains a well-characterized soybean (Glycine soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus, Medicago, and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae, and Fabaceae.

Cannon et al. 2003

FosterHartnett et al. 2002 Grant et al. 2000

Table 6.26.

Examples of comparative mapping amongst tropical and temperate grain legumes, 1992 to 2002.

Plant species Azuki bean and rice bean

Cowpea and mungbean

Mungbean, common bean, and soybean Cowpea, mungbean, and soybean Mungbean and lablab

Level of synteny Comparison between two adzuki interspecific maps, the UA linkage map (V. umbellata × V. angularis) and the AN map (V. angularis × V. nakashimae), revealed that UA linkage groups 1, 3, 4, 6, 7, 10, and 11 correspond to AN linkage groups 4, 3, 6, 1, 5, 7, and 11, respectively. Sixteen conserved linkage blocks were found in the interspecifc map of V. angularis × V. nakashimae and V. radiata. Genomic regions in linkage group 2 with greatest effect on seed weight spanned the same RFLP markers that are collinear in arrangement on homologous linkage groups both in cowpea and mungbean. The hybridization analysis revealed that the genic complement between mungbean and cowpea appears to be similar at the nucleotide level, although the copy number of some loci changed between them. The comparative map demonstrated that some linkage blocks remained highly conserved in both the species but that the linear arrangement of the markers has changed. Other linkage groups in cowpea consists of segments from different linkage blocks in mungbean. Mungbean and common bean exhibited a high degree of linkage conservation and preservation of marker order. On average, the length of conserved genetic blocks between these species is approximately 36.6 cM and the longest is 103.5 cM. Only short and scattered linkage blocks are conserved between mungbean or common bean and soybean, with an average length of 12.2 cM for conserved blocks between mungbean and soybean and 13.9 cM for the conserved block between common bean and soybean. Soybean and cowpea share an orthologous seed-weight gene, and the genomic region significantly associated with seed weight spanned the same RFLP markers (sgA816, sgK024, and sgA226) in the same linkage order, but not in mungbean. However, the linear order of these marker loci is conserved in all the three species. Comparison of mungbean and lablab genetic linkage map, using common 65 RFLP probes, reveals highly conserved marker order between the two genomes. However, the two genomes have apparently accumulated a large number of duplicates/deletions after they diverged.

Reference Kaga et al. 2000

Fatokun et al. 1993 MenancioHautea et al. 1993

Boutin et al. 1995

Maughan et al. 1996

Humphry et al. 2002

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Pea and lentil

Conserved linkage relationships exist for 250 cM regions on linkage groups 1, 2, 4, 5, and 6 between lentil and pea genetic map.

Weeden et al. 1992

Pea, chickpea, and lentil

Five regions of the chickpea map have gene orders similar to those found in the pea genome. However, the degree of similarity is somewhat less than that found between pea and lentil.

Pea, lentil, and broadbean

One of the 11 linkage groups in broadbean contains two isozyme loci, EST and Tpi-p, which share some homology with chromosome 4 of pea. Another isozyme loci, Prx-1, is conserved in pea, broadbean, and lentil.

Simon and Muehlbauer 1997 Torres et al. 1993

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when screening 1204 soybean-derived STS (http://www.embrapa.br/ labex/download/perry-cregan-group-poster.pdf). Similarly, a comparison of sequenced regions in M. truncatula, L. japonicus, and G. max revealed high conservation between the genomes of M. truncatula and L. japonicus but a lower level of conservation between M. truncatula and G. max. This suggests that comparative mapping may have considerable utility for basic and applied research in the legumes, although its predictive value is likely to decline with increasing phylogenetic distance and genome duplication (Choi et al. 2004a). Thus, an ongoing collaboration of Aarhus University (Denmark) with EMBRAPA and UCB (Brazil) is integrating Arachis into a single unified legume genetic framework using “legume family anchor markers.” This project has identified 867 evolutionary conserved sequences (ECSs) from M. truncatula, G. max, and L. japonicus that have a high probability of being conserved in less well-characterized legumes. The comparison of map positions of ECS markers in different legumes should allow the development of a preliminary comparative map across an extensive range of legume crops and model systems (David Bertioli, EMBRAPA, unpublished data). 3. International Initiatives Harnessing Comparative Genomics for Crop Improvement. The revolution in biology, data management, and communications has provided the scientific communities with tremendous opportunities for solving some of the world’s most serious agricultural and food security issues. This has led to the formation of the Generation Challenge Program (GCP) “Unlocking Genetic Diversity in Crops for the Resource-Poor” (www.generationcp.org). Generation Challenge Program (GCP) is based on harnessing comparative biology to release the value of global germplasm collections through the development of improved molecular breeding systems with a particular focus on drought tolerance. The development of gene-based markers in cereals, legumes, and clonal crops through comparative analysis with the model systems is a key component of this program. The GCP is creating a strong coalition of institutions dedicated to alleviating poverty through the application of recent advances in biological sciences. This alliance aims to harness powerful tools of the genomics revolution to unlock the genetic potential within crop germplasm to address the needs of the resource-poor in developing countries. The key feature of the platform is the creation of a platform that is applicable to any crop and any trait. Preliminary proofof-concept activities focus on the 22 mandate crops of the CGIAR (Consultative Group on International Agricultural Research) system and environmental stress tolerance traits. The development goal of this challenge program is to increase food security and improve livelihoods in

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developing countries by enhancing the use of freely available genetic resources in plant breeding programs. This requires a concerted focus on the generation, management, dissemination, and application of comparative biological knowledge. The GCP contributes to this goal by creating an integrated platform for dissecting genetic diversity in crop plant genetic resources by identifying important genes to reduce the impacts of environmental stresses on crop productivity. Complementary efforts will also be captured to enhancing yield through improved pest and disease resistancee and improve nutritional quality of crop products. Beyond this, the challenge program will identify, manipulate, and validate gene expression, resulting in plants with potential value far beyond the present-day crops. New traits (and the tools to manipulate them) will be transferred, through seeds or vegetative propagules, to breeding programs across the developing world. The challenge program is divided into five subprograms—genetic diversity of global genetic resources; comparative genomics for gene discovery; trait capture for crop improvement; genetic resource, genomic, and crop information systems; and capacity-building and product delivery. Medicago Genomics Consortium. The Center for Medicago Genomics Research, established at the Samuel Roberts Noble Foundation, USA (http://www.noble.org/medicago/index.htm), is engaged in the study of genetic and biological events associated with growth, development, and environmental interactions in M. truncatula using large-scale EST sequencing, gene expression profiling, and high throughput metabolite and protein profiling. The center is interfacing these multiple technologies to produce an integrated set of tools that can address fundamental questions about (a) biosynthesis of natural products affecting forage quality and human health, (b) cellular and molecular basis for the gravitropic root growth and the role of cytoskeleton in this process, (c) legume root development and molecular mechanisms of polar auxin transport, (d) non-host pathogen resistance, (e) RNA silencing pathway, and (f) arbuscular mycorrhizal (AM) symbiosis. Other global genomics projects in M. truncatula include an inventory of gene function; BACbased sequencing of gene-rich euchromatic regions; application of bioinformatic tools to functional genomics; high-throughput TILLING (targetting induced local lesions in genome) mutant analysis; meristem proteomics; molecular interactions between rhizobium, phytohormones, and nodulation mutants; and R gene isolation and defense gene expression profiling (VandenBosch and Stacey 2003). Lotus Genomics Consortium. The Miyakogusa foundation, a nonprofit organization in Japan, initiated research on L. japonicus in 1999 and

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develops public resources such as linkage maps, expression arrays (of both plant and endosymbiont genes), and transformation techniques. The consortium brings together 30 laboratories spread throughout Japan. A seed center has also been established at the National Agricultural Research Center, Hokkaido (http://cryo.naro.affrc.go.jp/sakumoto/mameka/lotus-e.htm) and a large set of ESTs from normalized and size-selected cDNA libraries, obtained from seven different organs, is available from the Kazuza Institute (http://www.kazusa.or.jp/en/plant/lotus/est). Soybean Genomics Consortium. Soybean is a major source of edible oil and high-quality protein, and a leading commercial crop in the United States, where soybean commodity boards (notably the North Central Soybean Research Program and United Soybean Board) have supported genomics as a way of encouraging genetic enhancement in the crop through the development of DNA markers, transformation techniques, structural and functional genomics, as well as bioinformatics. Soybean has a complex genome structure but a rich repertoire of genomic tools and resources that include a vast EST collection (more than 300,000 ESTs from approximately 80 cDNA libraries mostly from the cultivars Williams and Williams 82) and 400 SNP markers developed from ESTs, a densely populated genetic map (over 1000 SSRs and 700 RFLP markers on a consensus map), a developing physical map (consisting of 3,000 contigs anchored to about 500 genetic markers), microarray resources, and an efficient transformation system. The array of resources available would be improved by finishing the physical map and by developing a better understanding of the gene space and chromosomal topography of the species (Shoemaker et al. 2003; http://www.agbioforum.org; http:// www.soybeangenome.org/soybeansIntlminutes.html). The successful application of biotechnology-assisted breeding of soybean is expected to provide considerable direct and indirect support for similar progress in other legume crops. Common Bean Consortium. Common bean genomics scientists formed a consortium to establish the necessary framework of knowledge, resources, and tools required to generate disease-resistant, stresstolerant, high-quality, and high-yielding common bean varieties (http:// www.phaseolus.net/phaseomics; Broughton et al. 2003). Chickpea Genomics Consortium. This consortium was formed in early 2003 to produce a consensus genetic linkage map of the Cicer genome, to make bacterial artificial chromosome (BAC) libraries of chickpea, and to establish a chickpea consortium website. A consensus map for Cicer arietinum and Cicer species is planned as one of the first products

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of the consortium followed by physical mapping and sequencing of important regions of the chickpea genome (http://www.icgc.wsu.edu). Pea Microsatellite Consortium. This consortium, led by AgroGene, has 21 members worldwide, has cloned 882 sequences and designed 318 primer pairs, for the detection of polymorphism in leading pea genotypes (http://www.agrogene.com/ssrdevelopment.htm). 4. Genome Sequencing in Legumes. Genome sequencing has begun in L. japonicus (450 Mb) and M. truncatula (525 Mb) and is the basis for comparisons to the completed sequences for Arabidopsis (128 Mb) and rice (425 Mb) (indica and japonica subspecies), which has facilitated the assignment of putative functions of thousands of genes. The Medicago genome sequencing efforts also include a physical map and over 170,000 ESTs. Common bean and cowpea have small genomes, chickpea and pigeonpea have relatively large genomes, and soybean, peanut, pea, lentil, and broad bean have large to massive and complex genomes (Bennett and Leitch 2003; see also section I D). Hence, there is little likelihood in the immediate future of genome-wide sequencing projects in other legume crops. Thus, legume scientists and breeders must devise strategies to make maximum use of the complete genome sequence of Arabidopsis and the partial genome sequences of Medicago and Lotus. The development of tissue and stress-specific subtractive EST libraries in legume crops is likely to be a highly efficient means of capturing advances from model systems for these lesser-studied crops. 5. Allele Mining. Sequence information and DNA markers form the foundation of genetic linkage mapping and marker-assisted breeding, while also providing a tool for the comparison of genomes between species and for allele mining of germplasm collections. Linked SSR and AFLP markers are currently the most abundant type of mapped marker in most legume crops; however, they are not always appropriate for gene mining due to recombination between these neutral markers and the gene of interest. For this reason, the use of gene-based markers (developed from ESTs) provides a more direct path for the identification of new allelic variation from germplasm collections and a quick and inexpensive route to a short list of germplasm accessions with potentially new traits compared to phenotypic screening. Fortunately, a wealth of EST information exists in soybeans, Medicago, and several other legumes. To be useful, gene-based markers derived from ESTs need to be associated with traits, a process called candidate gene discovery. Thereafter, the new candidate gene alleles must be evaluated to determine which ones actually provide

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a beneficial effect on the trait of interest under field conditions. Associating allelic polymorphisms with phenotypic variation is confounded in plants by population structure (Buckler and Thornsberry 2002). The presence of subgroups with an unequal distribution of alleles can lead to spurious associations. This problem can be corrected by estimating population structure and by conducting appropriate association tests. This approach has been effectively used for the identification of candidate gene-based markers through association mapping of flowering time in maize (Thornsberry et al. 2001). Mining for R-gene Alleles. The candidate gene approach has been particularly useful for the investigation of pest and disease resistance. A large group of plant resistance genes encode cytoplasmic receptor-like proteins that contain leucine-rich repeat (LRR) and nucleotide-binding site (NBS) domains. As a group, these genes have been called resistance gene analogs (RGAs). The high degree of sequence conservation among the NBS-LRR class of resistance genes has permitted the design of degenerate oligonucleotides for use in PCR for gene isolation and subsequent development of molecular markers. Defense-related genes associated with broad-spectrum resistance have been targeted to identify effective alleles in rice (Ramalingam et al. 2003). The same approach has been used in chickpea in which C. arietinum RGAs were used to isolate the orthologous alleles from C. reticulatum and where alleles were found to cluster into distinct classes, each associated with a known resistance phenotype (Huettel et al. 2002). Other examples of RGA cloning include the use of candidate RGAs to discover alleles for resistance to the soybean mosaic virus Rsv1, Rsv3, and Rsv4 (Liao et al. 2002) and the use of a kinase RGA to mine for alleles for the Co-4 anthracnose resistance locus in common bean (Melotto and Kelly 2001). Divergence of RGA sequences at the Co-y, Co-z, Co-9 locus were studied in common bean and were found to be correlated with the molecular evolution of the anthracnose pathogen at the population level (Geffroy et al. 1999; Ferrier-Cana et al. 2003). The LRR domains are considered the major determinants of recognition specificity for Avr (avirulence) factors and pathogen elicitors, often through direct protein-protein interactions (Leister and Katagiri 2000). Other Examples of Candidate Gene Allele Mining. There is a rapidly increasing number of functionally annotated sequences associated with stress tolerance, including genes for dehydrin, catalase, glycolate oxidase, and thioredoxin peroxidase that are induced or suppressed as a result of the combined effect of drought and heat shock (Rizhsky et al. 2002). This has led to reports of the mining of desiccation-induced transcripts that may have a role in anthocyanin biosynthesis (Gopalakrishna

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et al. 2001). Another area in which allele mining has been useful is in the explanation of morphological and phenological variation. For example, El-Assal et al. (2001) identified the EDI locus as a QTL partly accounting for the difference in flowering response to photoperiod between two Arabidopsis accessions. Positional cloning of the EDI QTL showed it to be a novel allele of CRY2, encoding the blue-light photoreceptor cryptochrome-2 that has previously been shown to promote flowering in long-day photoperiods (Guo et al. 1998). Mapping of the vernalization trait to the FRIGIDA locus (FRI) led to mining Arabidopsis ecotypes for FRI alleles in an attempt to identify the underlying genetic differences between the late-flowering and early-flowering ecotypes. An extensive set of FRI alleles was surveyed and the polymorphisms in the form of deletions (31–376 bp) were associated with early flowering (Johanson et al. 2000). Amongst legumes, the dehydrin candidate gene (Dhn, LEA-D11) has been used in association studies in cowpea to determine the genetic variation underlying chilling tolerance (Ismail et al. 1999). Two types of allelic variation were identified at the protein level (one deletion and two amino acid substitutions), which were confirmed to be functionally significant in inheritance studies. In another study, the molecular nature of the high oleate trait in peanut was examined using the oleoyl-PC desaturase candidate gene ahFAD2B wherein a single residue mutation co-segregated with the trait (Jung et al. 2000b). In a similar approach in pea, degenerate primers based on the amino acid sequences of a 49 kDa apyrase (EC 3.6.1.5) were used to screen for alleles in a pea stem cDNA library (Shibata et al. 2001). The cultivated gene pool in a number of legume species (pea, common bean, and cowpea) has been identified as genetically diverse, whereas other legumes (chickpea, soybean, and peanut) possess a narrow genetic base for the cultigen. Sequencing of alleles in one or more of the genetically diverse crops may provide important diversity for crop improvement if allelic differences confer an improved phenotype. In such an approach, the objective is to identify a sequence change that is associated with the improved phenotype. This sequence change can then become the basis of a SNP marker for that allele that can be used in subsequent MAS applications using those new alleles. Variation at the sequence level can also help infer relationships within the legume family from trees that describe the phylogenetic relatedness between the sequences of different alleles. B. Functional Genomics and Gene Discovery Functional genomics refers to the association between sequence and functional phenotypes. Knowing the entire sequence and location of

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genes underlying a target trait is only the first step in determining the relative function and importance of those genes. Functional genomics provides the means to determine the key roles and interactions of these genes that then become the targets for molecular breeding. Different specializations within functional genomics include transcriptomics, proteomics, and metabolomics. The interface between these different areas of functional genomics allows the unequivocal assignment of functions to new plant genes (Holtorf et al. 2002). Plant functional genomics has greatly benefited from technology developments driven by the genomics communities working on humans, yeast, fruit flies, mice, and nematodes. These developments now allow researchers to simultaneously profile vast numbers of different genes or proteins. Gene discovery has benefited tremendously from developments in high throughput EST sequencing and DNA microarrays. Large-scale analysis of gene expression in Arabidopsis using cDNA and/or oligonucleotide arrays has given new insights into photosynthesis, biotic and abiotic stresses, nitrogen assimilation, and organ development (Desprez et al. 1998; Ruan et al. 1998; Maleck et al. 2000; Schenk et al. 2000; Wang et al. 2000; Girke et al. 2000; White et al. 2000; Zhu and Wang 2000; Bohnert et al. 2001; Seki et al. 2001). Similarly, gene expression analysis in M. truncatula and L. japonicus is beginning to elucidate many details of traits that are not well represented in Arabidopsis, including plantmicrobe symbiotic associations with N-fixing rhizobia and with mycorrhizal fungi, defense response, and other agronomic traits such as secondary metabolism and pod development (Penmetsa et al. 2003; Cook 2004; Yahyaoui et al. 2004; Kouchi et al. 2004; Arimura et al. 2004; Naoumkina and Dixon 2004). Among the grain legumes, soybean has the most EST sequences and cDNA libraries developed from a broad range of genotypes, developmental and reproductive stages, organs, tissues, and abiotic and biotic stresses (Shoemaker et al. 2002). In this section, we review examples of different approaches to gene discovery in legumes. 1. Discovering Candidate Genes Based on Conserved Functional Domains. Multi-gene families of related genes control most agronomic traits. These gene families generally evolved through duplication of the original gene followed by sequence divergence of the different copies of that gene. Presumably, during the first phase of such evolution, the generation of allelic variation altered the level of trait expression or conferred expression on different parts of the plant. However, following continued evolution, the different families have often diverged substantially to create new genes encoding new traits affecting adaptation to different environmental stresses or production of new metabolites. In the following section, we give examples of gene families encoding dis-

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ease resistance and affecting nodulation. Clearly, the discovery of genes that have evolved through different evolutionary pathways to influence the same trait requires a different set of approaches than that described here for allele mining. Using NBS Domains to Discover Disease Resistance Genes in Legumes. Most disease resistance (R) genes in plants share significant homologies in terms of DNA and amino acid sequences as well as structural motifs (Hammond-Kosack and Jones 1997; Dangl and Jones 2001). Nucleotidebinding sites (NBS) and leucine-rich repeats (LRRs) are the most common domains found in plant R-genes where NBS are involved in signal transduction cascades through phosphorylation and dephosphorylation events (Dangl and Jones 2001) and LRRs are a conserved domain involved in ligand binding and pathogen recognition (Hammond-Kosack and Jones 1997). Degenerate oligonucleotide primers have been used to amplify NBScontaining sequences in near isogenic lines or segregating populations of soybean (Yu et al. 1996; Kanazin et al. 1996; Pennuela et al. 2002), chickpea (Huettel et al. 2002), common bean (Rivkin et al. 1999), cowpea (Gowda et al. 2002), peanut (Bertioli et al. 2003), and alfalfa (Cordero and Skinner 2002). In M. truncatula, over fifty sequences with homology to the NBS domain were obtained using both PCR amplification and database searches and were found to be representative of the NBS sequences of other legumes within the Papilionoid subfamily, establishing M. truncatula as a potentially useful model for resistance gene organization (Zhu et al. 2002). In the same study, the genomic locations of RGAs in M. truncatula correlated with those of resistance gene loci in soybean and pea. Mining Genes Involved in Nodulation. Using molecular genetic technologies such as genome sequencing and gene knock-outs, many of the bacterial genes needed for nodulation (Nod and Nol) and fixation (Fix) have been identified. Similarly, plant genes involved in the nodulation process are also being identified using insertion mutagenesis and positional cloning from the model legumes L. japonicus and M. truncatula. The first legume nodulation gene (Nin) was isolated from L. japonicus using transposon mutagenesis with an Ac/Ds transposon system (Schauser et al. 1999). Later, two genes encoding non-nodulation mutants (LjsymRK and MsNORK) were positionally cloned in both L. japonicus and M. sativa (Stracke et al. 2002; Endre et al. 2002). Alterations in this gene were revealed when the same gene was cloned from non-nodulating mutants in M. truncatula (dmi2) and pea (sym19). Meanwhile, map-based cloning was useful in soybean for the isolation of a nodule auto-regulation receptor kinase (GmNARK), which as a missense

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mutation has a weak nodulation phenotype, while as a nonsense mutation causes extensive super nodulation (Searle et al. 2003). In addition, the lotus LjNin gene has been used to isolate PsNin, an orthologous gene in pea (Borisov et al. 2003). In an approach using EST sequencing, the genome-wide analysis of nodule-specific transcripts in M. truncatula revealed 340 tentative consensus sequences (TCs) that were expressed solely in root nodules and that could be grouped into nine categories based on the predicted function of their protein products (Fedorova et al. 2002). In a similar study using ESTs, Szczyglowski et al. (1997) identified a range of novel ESTs associated with late developmental events during nodule organogenesis in L. japonicus. Parallel experiments have also been initiated in common bean through comparative analysis of 14,000 ESTs from nodule, root, pod, and leaf (Hernandez et al. 2004). The isolation of these candidate genes and the whole field of comparative biology of symbiosis is a role model for comparative genomics studies of other agronomic traits in model legumes and related crops. 2. Studying Candidate Genes Using Microarrays. DNA microarrays, which consist of thousands of gene sequences spotted in high-density array on glass or nylon membranes, are being used for gene expression analysis, polymorphism detection, DNA re-sequencing, and large-scale, whole-genome genotyping (Schena et al. 1995; Chee et al. 1996; Lemieux et al. 1998). The use of microarrays in plants creates extensive databases of quantitative information about changes in gene expression in various tissues or organs in response to developmental processes or changes in the level of pathogens, pests, drought, cold, salt, photoperiod, and other environmental variables. An alternative to DNA spotting of microarrays is the use of photolithography to generate gene chips with tens of thousands of oligonucleotides synthesized directly onto a glass slide (Lockhart et al. 1996; Lipshutz et al. 1999). These gene chips have the advantage that since they are based on short sequences they do not suffer from cross-hybridization of structurally related genes (Wodicka et al. 1997). Although the chips are currently too costly for routine use in many breeding programs, it seems likely that technical innovations and efficiencies associated with expanded use will drive their cost down. While microarray-based characterization of plant genomes has the potential to revolutionize plant breeding and agricultural biotechnology, there are still technical limitations associated with the currently available microarray technology, including high background intensities that obscure signals for weakly expressed transcripts and the difficulty of distinguishing homologous genes due to sequence similarity (Duggan et al. 1999; Gibbings et al. 2003).

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Among the legumes, Maguire et al. (2002) constructed cDNA microarrays for soybean containing 4100 unigene ESTs derived from axenic roots and evaluated their applicability and utility for functional genomics of organ differentiation in legumes. In this study, several ESTs showed high levels (a 50-fold) of differential expression in either root or shoot tissues of soybean. There was a linear correlation (r2 = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data when a small number of physiologically interesting and differentially expressed sequences found by microarray were verified by both quantitative real-time RT-PCR and Northern blot analysis. Thus microarray analysis of legume has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also for studying the expression of previously characterized genes, gene networks, and gene interaction in wild–type, mutant, or transgenic plants. Somatic embryos constitute a model system to study basic aspects of embryogenesis, as well as a tool for efficient transformation. Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, Thibaud-Nissen et al. (2003) identified 495 cDNA clones that are differentially expressed during the development of somatic embryos. Clustering the clones according to expression profile data allowed them to determine the timing of the molecular events taking place during the embryogenesis in soybean. Soybean Genomics and Microarray Database (SGMD) is a public database that provides an integrated view of the interaction of soybean with the soybean cyst nematode. SGMD contains genomic, EST, and microarray data with embedded analytical tools allowing correlation of soybean ESTs with their gene expression profiles. It also has analytical tools to facilitate the rapid mining of microarray data by integrating many analytical methods within the data itself (http://psi081.ba.ars.usda.gov/ SGMD/default.htm; Alkharouf and Matthews 2004). A purified yeast elicitor that contains polysaccharides composed entirely of mannose, has been shown to be a strong inducer of the isoflavonoid pathway and to lead to accumulation of phenylpropanoidderived natural products in M. truncatula. Using DNA arrays consisting of 16,000 70-mer oliginucleotides designed from a unigene set of M. truncatula EST sequences, Naoumkina and Dixon (2004) observed rapid increases in mRNA levels of specific members of gene families encoding the enzymes L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), caffeoly coenzyme A 3-O-methyl-transferase (CCOMT), isoflavone reductase (IFR), and several other defense genes. Hierarchical cluster analysis revealed a group of these genes with distinct expression patterns. There is a growing interest in genes involved in the

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phenylpropanoid pathway, and thus these genes serve as an internal control for global expression profile analysis. Temperature stress has a dramatic effect on crop productivity throughout the world. Using cDNA microarrays, Chen et al. (2004) profiled gene expression of mung bean that responded to temperature stress during the germination, and identified many stress-responsive ESTs using the Eisen Cluster and TreeView software (http://www.life.nthu.edu.tw/~islty/). cDNA microarray technology has also been used to study the molecular mechanisms intrinsic to reproductive organ development. Genes specifically expressed during the development of anther and pistil in L. japonicus have been isolated. Cluster analysis of the mircoarray data revealed 21 and 111 independent cDNA groups that were specifically expressed in immature and mature anthers, respectively (Endo et al. 2004). Similarly, Kuster et al. (2004) have reported candidate genes for nodule and arbuscular mycorrhiza development, amongst them different nodulespecific leghaemoglobin and nodule genes as well as a mycorrhizaspecific phosphate transporter gene. This study demonstrates that the Mt6k-RIT arrays serve as useful tools for an identification of genes relevant for legume root endosymbioses. A comprehensive profiling of such candidate genes will provide a new set of gene targets for molecular breeding of crops better adapted to improved cultivation practices. 3. Identifying Candidate Genes Using Serial Analysis of Gene Expression (SAGE). SAGE is a powerful technique that provides absolute measures of gene expression based on sequencing of mRNA-derived fragments, or SGAT tags. The technique has been developed to quickly and efficiently survey genome-wide transcript expression. It is best applied to organisms whose genomic sequences are known or that have a substantial cDNA sequence database. Because SAGE tags tend to originate from the 3′ portion of transcripts, they are less effectively screened against most cDNA libraries, which are generally sequenced only from their 5′ ends (Donson et al. 2002). Both SAGE and cDNA microarray techniques have the potential to produce vast amounts of data that must be carefully organized and analyzed in order to be able to draw meaningful conclusions. This becomes a special challenge when data is generated from experiments conducted under many different conditions or locations. To address this issue, Lash et al. (2000) constructed a public gene expression data repository (SAGEmap) and online data access and analysis site (http://www.ncbi.nlm.nih.gov/sage) to maintain SAGE data and to help in gene assignments and statistical testing. The National Science Foundation (NSF), USA, is supporting a project (http://soybean.ccgb.umn.edu/) to stimulate basic and applied research on functional genomics of soybean through the development of tools for

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global gene expression analysis. The main objectives are to build a soybean “unigene” set defined by 5' and 3' sequence data, to construct and use microarrays for global expression, and to generate and sequence SAGE-tagged libraries. To date, this project has constructed 20 cDNA libraries and generated 132,992 SAGE tags, of which 40,121 are unique, in soybean (http://soybean.ccgb.umn.edu). Similarly, over 70,000 3′ ESTs, have been obtained from normalized and size-selected cDNA libraries made from different organs of L. japonicus, and these cluster into over 20,000 unigenes (Asamizu et al. 2000). Substantial developments have recently been made to create the SuperSAGE methodology (Matsumura et al. 2003), which uses the type III restriction endonuclease EcoP15I, to isolate tags of 26 bp in length from defined positions of cDNAs. This approach will be especially useful for transcriptome profiling of two or more interacting organisms like host and pathogens. SuperSAGE has been applied to Maganporthe grisea (blast)-infected rice leaves to simultaneously study gene expression profiles of both the rice host and blast fungus. This was possible because the genomes of both organisms have been fully sequenced. However, the methodology can also be used with organisms that do not have whole genome sequence databases. 4. Dissecting Entire Metabolic Pathways. Metabolites are the final products of gene expression, and the comprehensive large-scale analysis of metabolites is termed metabolomics. Metabolite profiling is usually achieved through the use of gas chromatography or mass spectrometry. These qualitative and quantitative analyses provide a holistic view of the biochemical status or biochemical phenotype of an organism. The use of metabolite profiling is an important tool for comparative display of gene function that has the potential not only to provide deeper insight into complex regulatory processes but also to directly determine metabolic phenotypes (Fiehn et al. 2000). In this section, we provide some key examples in which the metabolic pathways underlying important agronomic traits in legumes have been sufficiently well dissected to see likely practical impacts. Isoflavonoid Biosynthesis. Isoflavonoids are a specialized form of flavonoids, synthesized exclusively in legumes. They are reported to reduce the occurrences of certain types of cancers, reduce postmenopausal symptoms, prevent coronary heart disease, and have positive effects on neurobehavioral activities. The isoflavonoid synthesis pathway is probably the best-characterized natural product pathway in plants (Dixon and Steele 1999). Roessner et al. (2001) developed a metabolic profiling technique based on gas chromatography–mass spectrometry technology that

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allows detection of a wide range of hydrophilic metabolites within a single chromatographic run. This approach was used to study potato sucrose metabolism, demonstrating the use of metabolic profiling in conjunction with data-mining tools as a powerful technique for the comprehensive characterization of a plant genotype. For details, the readers may refer to a review article on isoflavone biosynthesis pathways that focuses on key structural enzymes and transcription factors, and on progress in metabolic engineering of isoflavone biosynthesis in both legume and non-legume plants (http://www.oardc.ohio-state.edu/soydefense/silencing/yupubs/ metabengineer.pdf). Isoflavone synthase catalyzes the first committed step of isoflavone synthesis, a branch of the phenylpropanoid pathway. Soybean contains the highest level of isoflavones, roughly more than 100-fold higher than many other legumes (http://www.nal.usda.gov/fnic/foodcomp/data/ isoflav/isoflav.html). Jung et al. (2000a) identified two soybean genes encoding isoflavone synthase that they then used to isolate homologous genes from other leguminous species, including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine. Identification of isoflavone synthase genes should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes. Dhaubhadel et al. (2003) detected isoflavonoids in all organs of soybean plants, but the amount varied depending on the tissue and development stage; the greatest concentrations were found in mature seeds and leaves. Using 2-hydroxyisoflavonone synthase genes (IFS1 and IFS2), they determined patterns of expression in different tissues and developmental stages. The highest expression of IFS1 was in the root and seed coat, while IFS2 was mostly expressed in embryos and pods and in elicitor-treated or pathogen challenged tissues. Developing soybean embryos have an ability to synthesize isoflavonoids de novo but transport from maternal tissues may also contribute to the accumulation of these natural products in the seed. Thiol Tripeptides Synthesis Pathway. The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. Matamoros et al. (2003) characterized three genes involved in the synthesis of GSH and hGSH in the model legume L. japonicus, where they are present as single copies. The y-glutamylcysteine synthetase (yecs) gene mapped on the long arm of chromosome 4 (70.0 cM), whereas the glutathione synthase (gshs) and homoglutathione synthase (hgshs) genes mapped on the long arm of chromosome 1 (81.3 cM) arranged in tandem with a separation of approximately 8 kb. The promoter regions of yecs,

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gshs, and hgshs contain regulatory elements related to a plant’s response to light, hormones, and stress conditions. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that yecs and hgshs are expressed in all organs, whereas gshs is functional at significant levels only in nodules, strongly suggesting an important role of GSH in the rhizobia-legume biosymbiosis. Triacylglycerol Synthesis Pathway in Lupin. Oil content and quality are fundamental agronomic traits of oil-seed legumes such as soybean, peanut, and lupine. Oil quality is determined by triacylglycerols (TAGs) that are stored as seed energy reserves. In a comparative study of two lupin species, L. mutabilis (high oil species) and L. angustifolius (low oil species), Francki et al. (2002) cloned a differentially-expressed glucose dehydrogenase-like gene and compared expression levels of this gene and other key enzymes of the lipid biosynthetic pathway, including acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), in an attempt to explain differences in TAG accumulation. Symbiosis with Rhizobium and Mycorrhiza. Root protein profiles have been studied during root development and nodule formation in M. truncatula. Hundreds of proteins are induced during nodule formation (Mathesius et al. 2001; Bestel-Corre et al. 2002). In experiments with different arbuscular mycorrhiza (AM) species, Burleigh et al. (2002) found that different species vary widely in their expression of root-specific genes involved in response to phosphorous starvation. For example, Glomus mosseae colonization of M. truncatula resulted in the greatest reduction in MtPT2 and Mt4 gene (phosphorous starvation-inducible plant genes) expression and the highest level of phosphorous uptake and growth. At the other extreme, Gigaspora rosea, whose colonization resulted in the highest level of MtPT2 and Mt4 gene expression, had the lowest phosphorous uptake and growth. In addition, other legumes may have a differential response to mycorrhiza at the level of colonization, nutrient uptake, and growth both in terms of profile and level of gene expression. Seed Development and Germination. Seed germination is a complex physiological process but a highly important component of crop productivity. In farming systems, seed priming is often used to accelerate germination and improve uniformity of crop establishment. Proteomic analysis of Arabidopsis seed germination and priming revealed changes in the abundance (up- and down-regulation) of the 74 proteins observed during germination and the specificity of certain proteins with certain phases of seed germination (Girke et al. 2000; Gallardo et al. 2001).

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More recently, advances have also been made in our understanding of how metabolic networks are regulated at the protein level during reserve deposition in seeds in M. truncatula (Gallardo et al. 2003). This information should be useful for the analysis of seed development in legumes that may help in the engineering of legume seed composition for increased yield and enhanced end-user value. These discoveries highlight the power of proteomics and metabolomics to unravel specific features of complex developmental processes and to provide candidate protein markers for traits of commercial importance. It is expected that in turn the underlying genetic markers will be identified that can be used as candidate gene-markers in molecular breeding programs. C. New Technologies for Marker-Assisted Selection 1. High-Throughput SSR Marker Genotyping. The throughput potential and unit cost of virtually all PCR-based assays can be substantially increased with readily available semi-automated technologies developed largely to serve the mass sequencing market. Although this requires significant investment in equipment, protocol development, and optimization, this is nevertheless appropriate even for lesser-studied crops, as ICRISAT has achieved for chickpea, groundnut/peanut, and pigeonpea. One of the most compelling reasons for adopting this approach is the opportunity to scale-down reaction volumes. For example, moving from a total reaction volume of 10 μl to 5 μl, moving from 96 well to 384 well format, and pooling PCR products prior to electrophoresis detection has substantially reduced unit costs. Further reductions in PCR costs can be achieved through optimization of the individual component concentrations. Although the optimization process is laborious, it also improves the efficiency of the PCR, resulting in fewer spurious amplification products. A major constraint to large-scale genotyping is resolving the resultant bottlenecks at the DNA extraction stage and data management and analysis steps. The genotyping process invariably starts with DNA sample preparation and it is important that the DNA be of the highest quality, as this has a huge impact on whether robust, easily callable data is generated. Low-cost high-throughput DNA extraction protocols must be developed for each specific crop of interest, as has been achieved for ICRISAT mandate crops (Mace et al. 2003). Although commercial kit options are available, these are too expensive for routine use in molecular breeding programs. Conversely, although there are many reports of large-scale low-cost DNA extraction systems, these rarely provide the

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quality of DNA required for trouble-free reliable applications in highthroughput genotyping systems. Similarly, Laboratory Information Management Systems (LIMS) and Decision Support Systems must be developed to serve the unique demands and processes of each lab (Ermolaeva et al. 1998). The M.S. Swaminathan Applied Genomics Lab at ICRISAT uses 96well DNA extraction, 8-tip liquid handling robotics, 96-capillary semiautomated genetic analysis, and high-throughput data management systems tailored to applications in all five mandate crops: sorghum, pearl millet, chickpea, groundnut/peanut, and pigeonpea (ICRISAT 2002a). This lab is almost entirely dedicated to germplasm diversity analysis, trait mapping, and marker-assisted breeding with less than 5% of throughput used for sequencing or functional genomics research. This type of genotyping potential is particularly valuable for QTL mapping of complex traits, in which a large number of markers (n > 100) need to be screened across a range (n > 3) of large populations (n > 250). The use of high-throughput semi-automated genotyping systems for PCR-based markers, such as SSR markers, dramatically increases the efficiency of data collection while reducing the cost per data point compared to manual systems. It was not uncommon in the early 1990s, particularly in lesser-studied crops, to see genotyping costs of US $5 per sample (including DNA extraction, RFLP analysis, and data collection). During the mid-1990s there were several reports of US $2 or even US $1 per sample. For example, Concibido et al. (1996) estimated a cost of US $2.00 per data point for RFLP compared to US $1.50 per data point for SSR fingerprinting for cyst nematode resistance using marker-assisted selection. Similarly, the high-throughput human genotyping project estimated unit costs of US $1.00 per SSR data point (Hall et al. 1996). However, $1 per sample is prohibitively high for many breeding programs. Most recently, the high-throughput genotyping facility at the University of Georgia reported developing a soybean genotyping pipeline capable of generating 7500 SSR data points per week at an estimated cost of around US $0.50 per data point (http://www.gsf99.uiuc .edu/invited/2_2_01.pdf). So costs are clearly falling, but unfortunately it is not possible to compare directly most cost analyses presented in the literature, as these often do not include all relevant costs such as DNA extraction and data collection, labour, and equipment depreciation. Moreover, once a particular marker has been reliably associated with an important trait of interest, there are then a wide range of options for optimizing, refining, or even transforming that marker to reduce the unit costs of large-scale screening.

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2. Simple Markers from Fingerprinting Assays. The conventional marker assays including RFLP, RAPD, AFLP, and SSR have greatly contributed to our current understanding of genome organization and genetic variation. However, they are constrained by their dependence on gel electrophoresis, resulting in low throughput through manual systems and limited scalability even if detection can be automated. Moreover, these methods are based on size separation of multiple DNA fragments and thus suffer from difficulties in precisely correlating bands on gels with allelic variants. 1. Sequence Characterized Amplified Region (SCAR) Markers. RAPD markers belong to the dominant class of genetic markers, have poor reproducibility, and their application in marker-assisted selection (MAS) is severely limited. Transforming a RAPD marker into a SCAR marker generally improves reproducibility and increases throughput potential in MAS programs (Paran and Mitchelmore 1993) and offers the possibility of eliminating the use of gel electrophoresis (Gu et al. 1995). However, developing a SCAR marker is laborious and tight linkage of the RAPD marker to a gene (or QTL) of interest must be confirmed before converting RAPD bands into SCAR markers. Tightly linked RAPD markers are reported for resistance to common bacterial blight, fusarium wilt, and bean common mosaic virus in common bean (Miklas et al. 2000; Yu et al. 2000; Fall et al. 2001) and for ascochyta blight resistance in lentil (Chowdhury et al. 2001). The utility of SCAR markers in MAS has been demonstrated for the selection of resistance to common bacterial blight in common bean (Yu et al. 2000) and for resistance to ascochta blight in lentil (Chowdhury et al. 2001). However, development of SCAR markers may also be associated with a loss of detectable polymorphism, as was seen when a RAPD fragment (UBC2271290) was converted into a SCAR marker in lentil (Chowdhury et al. 2001). Single Locus or Allele-Specific Assays from AFLP. Amplified fragment length polymorphism (AFLP) technology is based on the PCR amplification of selected restriction fragments of a total genomic digest. Separation of labeled amplified products is then achieved through denaturing polyacrylamide gel electrophoresis (Vos et al. 1995). Unlike SSR and RFLP, a priori knowledge of genome structure is not required for AFLP assays, which also have the added advantage of generating a large number of amplification products. Thus, AFLP analysis is particularly valuable for diversity analysis and background genome selection in marker-accelerated backcross programs. However, AFLP assays are less suitable for allele frequency studies, marker-assisted selection, or mapbased cloning, as many AFLP markers are redundant and hence the

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assay becomes too expensive and too laborious for large-scale single locus screenings (Liu et al. 1998). On this basis, the use of AFLP analysis in linkage mapping must be followed by the conversion of specific AFLP markers into single locus PCR markers, such as cleaved amplified polymorphic site (CAPS) markers (Konieczny and Ausubel 1993) or sequenced characterized amplified region (SCAR) markers (Paran and Mitchelmore 1993). However, conversion of AFLP markers from complex fingerprints into simple single locus assays can be problematic, as DNA sequence information is required to design new locus-specific PCR primers, and single locus polymorphism information is required to design an allele-specific assay. Nevertheless, Brugmans et al. (2003) describes a procedure demonstrating a high success rate for the conversion of AFLP markers (from rather small 131 bp to large 359 bp size fragments) into locus-specific markers in tomato (Lycopersicum esculentum) and flax (Linum ussitatisimum). There are few reports of the successful conversion and application of SCAR-based AFLP markers. Five AFLP markers were converted to CAP and SCAR markers associated with resistance to clubroot disease in Brassica (Piao et al. 2004). Similarly, AFLP-derived SCAR markers have been utilised in fine mapping of the Vf region controlling resistance to fungal disease in apple (Huaracha et al. 2004), for seed coat colour in Brassica (Negi et al. 2000), for development of sorghum downy mildew resistance gene markers in maize (Agrama et al. 2002), and for resistance to lodging in pea (Tar’an et al. 2003b) and Striga gesneriodes in cowpea (Boukar et al. 2004). 3. Gel-free Diagnostics. TaqMan technology relies on the use of a 20–30 bp probe labeled with a fluorescent reporter dye and a fluorescent quencher dye, which detect specific sequence polymorphisms in PCR products. The TaqMan probe hybridizes to a complementary region within the PCR product; during the process of amplification, the TaqMan probe is degraded due to the 5'→3' exonuclease activity of Taq DNA polymerase. This leads to a release of the quenching effect on the reporter component of the probe, resulting in an increase of fluorescence intensity of the reporter dye. The light emission increases exponentially through subsequent PCR cycles and can be measured by spectrophotometry during or at the end of the PCR. The TaqMan assay is useful in diagnostic applications, such as the screening of samples for the presence or incorporation of favorable traits and the detection of pathogens and diseases. The TaqMan assay allows high sample throughput, because no gelelectrophoresis is required for detection, and short analysis time, as the

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reaction can be stopped as soon as a significant amplification difference is detected. The major disadvantages with this technology are: requirement for sequence data for primer and probe construction, high unit costs due to the requirement for the fluorogenic probes/quencher, and potential inefficiencies from single allele specific tests if no allied approach is employed to distinguish false negatives (PCR failures) from true negatives (absence of allele). Indeed, it has been reported that TaqMan screening for detection of a candidate gene Rhg4 conferring resistance to soybean cyst nematode was accurate in only 90% of RIL progeny compared to 95% accuracy during conventional electrophoresis (Meksem et al. 2001c). Clearly, this is unacceptable for large-scale application of a capital and operationally expensive assay. Fortunately, it is likely that in most cases precision and accuracy can be readily increased to more acceptable levels through intensive optimization. A rapid TaqMan assay has been developed to assess the dosage of the dihydroflavonol 4-reductase (dfr) allele in cultivated potato, which is associated with red-skinned cultivars (Jong et al. 2003). Through the use of fluorogenic allele-specific probes, one for red allele and the other for “not-red” dfr alleles, a TaqMan allelic discrimination assay clustered the diploid clones tested into three distinct groups (homozygous for the red allele, heterozygous for the red allele, and homozygous for the notred allele), based on the relative amount of two different dyes released. The assay has been reported to successfully discriminate allelic dosage in autopolyploid potato (Jong et al. 2003). The development of microplate or filter-based detection systems is likely to be an important component intervention for the more widespread adoption of MAS technologies in developing countries. In particular, the availability of allele-specific gene-based marker systems will have a substantially higher throughput potential than gel-based system assays, while requiring less expertise and expense. Clearly the development of low-cost high-throughput DNA extraction and PCR amplification systems is a parallel requirement toward providing a complete appropriate package for tropical breeding programs. The Generation Challenge Program is currently addressing these issues on behalf of the wider community. 4. Trait-Specific SNP Markers. Recent technological advances in DNA sequence analysis and the establishment of DNA-chip technologies have provided the framework for large-scale discovery and analysis of DNA variation. Single nucleotide polymorphisms (SNPs) are markers that focus on variation at the nucleotide level within the genome. However, this approach does require a prior knowledge on the allelic nature of the variations within the genome for the genetic population under study.

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There are a number of approaches used for the development of SNPs: screening ESTs for polymorphic sites (Zhu et al. 2003a; Choi et al. 2004b) or through an enriched shotgun genome sequencing methodology involving multiple individuals (Altshuler et al. 2000). There is much interest in SNP markers due to their highly multiplex nature and ease of automated detection, resulting in the generation of more data points per unit time than with any other marker system. Indeed, the rationale of the HapMap project in humans is to focus on SNPs associated with disease traits. The eventual aim is to be able to scan the entire genome by genotyping fewer than 500,000 SNPs as opposed to 10 million common SNPs (International HapMap Consortium 2004). Research in plants lags far behind humans and animals. However, there are a few examples that suggest the plant community is moving in a similar direction. For example, several of the multinational breeding companies are highly focused on SNP-based markers in their soybean molecular breeding programs (Cahill 2000). In the public sector, soybean unigenes ESTs have been used for SNPs discovery in soybean as well as several other legume species including common bean, cowpea, chickpea, pea, peanut, and Medicago truncatula. For example, when 1,204 soybean-derived sequence tagged sites (STS) were amplified in other legume species, 15.3, 13.8, 6.1, 5.6, 2.7, and 2.7% of the 1204 primer sets were able to generate sequencable products in genomic DNA of P. vulgaris, V. unguiculata, C. arietinum, M. truncatula, P. sativum, and A. hypogea, respectively (http://www.Embrapa.br/labex/download/perry/-cregan-groupposter.pdf). The frequency of SNPs in soybean is somewhat low: between 1.98 SNPs per kbp (coding DNA) and 4.68 SNPs per kbp (noncoding DNA), as estimated from the analysis of 25 soybean genotypes (Zhu et al. 2003a). Trait specific SNPs have also been identified in barley for the Mlo gene and have been used by breeders as a routine assay for marker-assisted selection for mlo-mediated resistance to powdery mildew in barley at the seedling stage (Paris et al. 2003). 5. Array-Based Genotyping. There is no doubt that with the use of robotics and capillary electrophoresis (CE), the cost of SSR genotyping will continue to fall as marker sets are further optimized, multiplex PCR and CE co-loading sets and conditions elucidated, and high-throughput genotyping systems further refined. Similarly, the comparative advantage of SSR markers as a research tool for linkage analysis and QTL mapping is likely to remain for some time to come. However, to reach a compelling costbenefit threshold for large-scale routine molecular breeding applications, there is significant need to reduce genotyping costs below $0.10 per sample (including costs from DNA extraction to data analysis). At the same time, there is significant pressure to provide a unified system capable of

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simultaneous foreground selection of multiple complex traits and background selection of the recurrent parent genome. Achieving this level of scale-up and or cost effectiveness will require the application of an entirely different type of genotyping platform. Although hybridization technologies gave way to PCR-based markers during the 1990s in the quest for automation, in the current decade hybridization approaches (now miniaturized through array technology) are returning as the best solution for cost-effective scaling-up of marker-aided selection. However, before that can happen, substantial genomic resources must be generated in the crop of interest and a range of technical issues must be resolved to ensure reliable DNA-DNA or cDNA-cDNA hybridization of hundreds or thousands of DNA templates on a single tiny matrix. The quest for genome-wide analysis of expression led to the development of a number of array-based approaches, including macroarrays (Desprez et al. 1998) and microarrays (Schena et al. 1995). DNA chips are based on binding random or known DNA fragments or oligonucleotides onto a microscope slide, displaying up to 409,000 spots in an area of 1.28 cm2 (Fodor 1997), and detecting mRNA at levels of 1/100,000 or 1/500,000 (Gerhold et al. 1999). Diversity arrays technology (DArT) (Jaccoud et al. 2001; http:// www.cambia.org.au/main/diversityarrays.htm) is a novel methodology for genotyping a large number of arbitrary genome-wide markers using array technology. It is a low-cost high-throughput robust system, a sequence-independent form of genotyping, and requires a minimal DNA sample to provide comprehensive genome coverage. Although this approach has been successfully applied in Arabidopsis, rice, cassava, wheat, barley, apple, and a forage grass (Jaccoud et al. 2001; Peng et al. 2002; Patarapuwadol et al. 2004), it has been difficult to establish routine applications in the lesser-studied crops in which its use is the most justified. Conventional microarray analysis interrogates the target or template RNA or DNA in solution using DNA probes on the array slide. This is well suited to linkage mapping studies in which thousands of markers must be screened across hundreds of individuals. However, more recently the reverse arrangement has been employed such that amplified PCR products from thousands of individuals are spotted on an array slide and screened with a small number of labeled probes. The approach is known as Tagged Microarray Marker (TAM) (Flavell et al. 2003). TAM is highly suitable for large-scale analysis with a small number of codominant molecular markers based on retrotransposon insertion sequence polymorphisms or SNPs. The cost for TAM is estimated at 6

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cents per assay, with an initial capital equipment cost of approximately $100,000 for the microarrayer and scanner (Flavell et al. 2003). Low-cost high-throughput assay technologies will be a critical element of regional shuttle genotyping hubs where breeders from NARS partners and indigenous seed companies can achieve large-scale cost-effective MAS using state-of-the art facilities. Building molecular breeding success stories without substantial capital investment is likely to be an important intermediate step for more widespread adoption of MAS technologies in developing countries. However, it is far from clear which technology offers the best opportunity for MAS applications in these settings. Thus, multi-site and multi-application evaluation of a range of these technologies is an urgent priority that the Generation Challenge Program is currently addressing on behalf of the wider community. D. Successful Applications of Molecular Breeding in Legumes Marker-assisted selection (MAS) is most useful for traits where phenotypic evaluation is expensive or difficult, particularly for those polygenic traits with low heritability that are highly affected by the environment (Nienhuis et al. 1987). Indirect selection based on marker genotype rather than phenotype can be used to accelerate the speed and increase the precision of genetic progress, as well as reducing the number of generations and in turn lowering costs. Marker-assisted breeding can also break linkages between the target traits and undesirable genes (Young and Tanksley 1989). MAS is accomplished through the positive selection of markers tightly linked to (or within) genes of interest (often referred to as foreground selection) while marker-accelerated backcross (MAB) involves the concomitant negative ‘negative background selection’ of marker alleles elsewhere across the donor parent genome (Tanksley et al. 1989). The efficiency of MAS and MAB depends on the size of the population, the number of markers used, the distance between marker loci, the genomic region containing the desired quantitative trait loci (QTL), and the experimental design used in the marker association studies (number of replications, locations, and seasons plus size and type of population). The effectiveness of MAS decreases as the distance between the marker and target QTL increases because of the increased probability of recombination between marker and trait loci and thus the increased probability of false positives or false negatives. Similarly, selective power may be lost due to differences in recombination patterns in the breeding population as compared to the genetic population used for the original marker-trait association. Thus, validation of QTL markers is a critical precursor to routine use in applied breeding programs.

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At least four levels of validation can be envisaged for use: a different population from the same cross, a half-sib population, a population from one or more closely related parental genotypes, and a population from distantly related parental genotypes. These populations should also be phenotyped in a number of different environments to simultaneously detect environmental (E) effects and QTL × E interactions for the putative QTL. Trait heritability, the proportion of additive genetic variance explained by the marker loci affecting the trait, and the selection method used all influence the selection efficiency of both conventional and marker-assisted breeding programs. Deleterious linkage drag is the most frequent reason given by plant breeders for not making extensive use of exotic germplasm for the introgression of novel traits. The extent of linkage drag depends upon the population size, the number of meiotic generations before selection is applied, the size of the donor genome segments retained, and the genomic location of the locus of interest (Hanson 1959; Stam and Zeven 1981; Young and Tanksley 1989). However, in most cases, MAS can provide a substantial improvement over conventional introgression and backcross breeding. Of course, MAS cannot help when the perceived linkage drag turns out to be deleterious pleiotropy of the target gene. The literature now contains hundreds of research articles reporting the development of markers, identification of polymorphisms amongst cultivated and wild germplasm, constructing of genetic linkage maps, and QTL mapping of economically and agronomically important traits (see Tables 6.15 to 6.22; also see section V B). MAS is now routinely used in the breeding of many major cereal crops (Ahmadi et al. 1992; Yoshimura et al. 1995; Ribaut et al. 1997; Huang et al. 1997; Tuvesson et al. 1998; Hittalmani et al. 2000; Chen et al. 2000; Sanchez et al. 2000; Robert et al. 2001; Thomas 2003; Xu et al. 2004). In contrast, there are relatively few reports on the application of MAS in legume crops. The ratio of MAS and MAB reports compared with the number of research articles on the mapping of agronomic traits is surprisingly low in both cereal and legume crops. In this section, we briefly discuss the few documented cases in soybean, common bean, lentil, and pea that have demonstrated the effectiveness of MAS in legume breeding. 1. Soybean Nematodes. The soybean cyst nematode (SCN) is one of the most economically destructive pests of soybean (Noel 1992). Conventional breeding for SCN resistance is difficult because multiple genes control this characteristic (Caviness 1992) and nematode populations are geneti-

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cally heterogeneous (Niblack 1992). One major locus conferring partial resistance to SCN has been identified that controls more than 50% of variation in resistance against several races of the nematode (Concibido et al. 1994, 1996, 1997; Web et al. 1995). Marker-assisted selection using the SSR marker Satt309 (located only 1–2 cM away from the rhg1 locus) now forms the basis of most public breeding efforts (Cregan et al. 1999b; Mudge et al. 1997). Indirect selection with Satt309 was 99% accurate in predicting lines that were susceptible in subsequent greenhouse assays (Cregan et al. 1999b). Wang et al. (2001a) reported two major QTL that confer resistance to race 3 of the soybean cyst nematode in an F2 population (A81-356022 × G. soja) and they further confirmed these QTL in a population of 100 BC1F2 plants developed by crossing A81-356022 to a line from the F2 population that carried the two resistance QTL from G. soja. Earworm. MAB approaches have also been used to pyramid a QTL conditioning corn earworm resistance in the soybean line PI 229358 together with cry1Ac transgene from the recurrent parent Jack-Bt (Walker et al. 2002). When BC2F3 plants with or without the cry1Ac transgene were subjected to leaf feeding bioassays with corn earworm and soybean looper larvae, few larvae of either species survived on leaves expressing the cry1Ac protein. Though not as great as the effect of cry1Ac, the PI 229358-derived QTL also had a detrimental effect on larval weights of both pest species. The combined deployment of transgene and QTLmediated resistance to a lepidopteran pest may be the most viable strategy for control of insect pests. Seed Weight. This is an important component of yield, as well as an important aspect of market preference in soybean. Mian et al. (1996a) identified seven and nine independent RFLP loci associated with seed weight in population 1 (Young × PI 416937) and population 2 (PI 97100 × Coker 237), respectively. Together these loci explained at least 73% of the variability in seed weight with a heritability of at least 90%. The six marker loci associated with seed weight in each population were highly consistent across environments and years, which is a critical precursor to the development of an effective MAS program. Hoeck et al. (2003) identified SSR markers associated with QTL for seed size and compared the effectiveness of phenotypic selection and MAS for seed size in three populations of soybean. Population 1 had 12 markers that individually accounted for 8.1% to 14.9% of the variation for seed size combined across environments, population 2 had 16 markers that individually accounted for 7.8% to 36.5% of the variation, and population 3 had 22 markers that individually accounted for 8.6% to 28.8% of the

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variation. In this study, phenotypic selection for seed size was at least as effective as and less expensive than MAS. The lack of added value for MAS of this trait is an important example of the need for establishing clear justification prior to the development of MAS systems. This trait has high heritability in soybean (0.45 to 0.93) and is relatively easy to accurately and precisely phenotype and, thus on this basis at least, does not present a compelling case for needing marker-aided intervention. 2. Common Bean. The development of integrated consensus linkage maps in common bean, including the map locations of disease and insect pest resistance genes, has provided a sound basis for the development of MAS systems for disease resistance in bean that are now routinely carried out in many bean breeding programs (Kelly et al. 2003). Bacterial Blight. Breeding for common bacterial blight (CBB) resistance in common bean is complicated by pathogen variability (Schuster et al. 1983), linkage of resistance with undesirable traits (Beebe 1989), and different genes conditioning resistance in leaves, pods, and seeds (ArnaudSantana et al. 1994). Two RAPD markers (R7313 and R4865) linked to genes conferring resistance to CBB have been reported in Phaseolus vulgaris (Bai et al. 1997). Tar’an et al. (1998) examined the use of these markers for selecting CBB resistant material from F5:6 recombinant inbred lines (RILs). The two markers located on the same linkage group accounted for 22% (P = 0.0002) of the variation for resistance to CBB. Seventy percent of the lines that possessed both markers were observed to be resistant, whereas 73% of the lines that had neither of the RAPD markers were susceptible. This indicates that these disease resistance markers are stable and of potential value for plant breeding programs. Yu et al. (2000) reported a RAPD marker, BC420900, significantly associated with a major QTL that accounted for approximately 62% of the phenotypic variation for resistance to CBB in HR67. When converted into a sequence characterized amplified region (SCAR) marker and used for selection in a different population (Envoy × HR67), the prediction of resistance was 94.2% accurate. The estimated cost for using SCAR and RAPD markers to analyse 100 bean lines is around US $4.50 per data point, respectively, whereas conventional greenhouse screening costs nearly $7.00 per data point. The greenhouse test requires more than 30 days, whereas MAS can be completed in about one week. Anthracnose. Resistance to anthracnose (Colletotrichum lindemuthianum) in the cultivar ‘TO’ is monogenic and controlled by a dominant resistance gene, Co-4 (Bassett 1996), which is different and independent of other anthracnose resistance genes, Co-1, Co-2, Co-3, Co-5, and Co-6 (Fouilloux 1979; Young et al. 1998). The gene Co-4 is reported to be

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effective against 22 of the 25 C. lindemuthianum pathotypes identified in Brazil (Rava et al. 1994), and is an important source of resistance used by several breeding programs (Fouilloux 1979; Pastor-Corrales et al. 1994; Young and Kelly 1996, 1997). Six RAPD markers linked to the Co-4 genes have been reported: four in coupling and two in repulsion phases. The combined use of both markers allows the differentiation of homozygous and heterozygous resistant plants with selection efficiencies of 100% and 98%, which shows that it is possible to develop codominant assays from RAPD markers providing both repulsion and coupling phase markers are available for the gene of interest (de Arruda et al. 2000). Rust. The Guatemalan black bean (Phaseolus vulgaris L.) genotype PI 181996 is resistant to all known races of the bean rust. Johnson et al. (1995) investigated the value of two RAPD markers (OAC20490 in coupling and OAE10890 in repulsion phases) linked to rust resistance in PI 181996, using a diverse group of common bean cultivars and breeding lines. All the cultivars into which PI 181996 resistance was introgressed had the RAPD OAC20490. Drought. Breeding for a highly complex quantitative trait like drought tolerance could be substantially assisted by the development of MAS systems capable of identifying tolerant genotypes in early generations. Schneider et al. (1997) identified four RAPD markers that were consistently and significantly associated with yield under stress, yield under optimum irrigation, and geometric mean yield across a broad range of environments. Using these markers in breeding populations from the same parental genotypes as the mapping populations generated individuals with 11% increase in yield under drought stress, whereas conventional selection based on yield performance failed to increase performance in the USA. However, when a parallel validation was carried out in Mexico, the markers were not found to offer any advantage over conventional selection. Seed Yield. A procedure for MAS of complex traits in common bean using an index based on QTL-linked markers and genetic distances between lines and a target parent has been reported (Tar’an et al. 2003a). A comparison of the mean seed yields of the top five lines selected by different schemes demonstrated that the highest-yielding group was selected on the basis of a combination of phenotypic performance and a high QTL-based index, followed by groups identified by a high QTLbased index, conventional selection, and a low QTL-based-index. This study also showed that the use of the QTL-based index in conjunction with genetic distance to the target parent would enable a plant breeder to select lines that retain important QTL in a desirable genetic background.

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Thus, it is increasingly clear that the greatest gains will be achieved through the best combination of molecular and conventional approaches and not through either alone. 3. Lentil Ascochyta and Anthracnose. Ascochyta blight and anthracnose are the two most destructive diseases of lentil. Two genes are believed to confer resistance to Ascochyta blight (ral1 and AbR1), while a single gene is reported to control resistance to anthracnose (95B36 isolate). Tar’an et al. (2003c) pyramided the two genes for resistance to Ascochyta blight together with the gene for resistance to anthracnose in F6:7 RILs (CDC Robin × 964a-46). More than 82% of the lines that had either or both of the Ascochyta blight resistance (18% false negatives) markers were resistant, while 80% of the lines that had neither marker were susceptible (20% false positives). When a parallel validation was carried out using different ascochyta blight isolates, the selective power was slightly lower with 26% false negatives and 21% false positives. Similarly, screening with the anthracnose marker correctly identified 85% of the resistant lines. These validation studies suggest good potential for marker-assisted disease resistance breeding in lentil. Although the selective power of these markers does not appear especially high, it is likely that this is partly due to the innate problems associated with the reproducibility of this assay. Thus, the development of SCAR or allele-specific assays from these markers would surely increase the robustness and power of selection of marker-assisted disease resistance breeding in lentil. 4. Pea Mycosphaerella Blight. Tar’an et al. (2003b) identified two major QTL that together explained 58% of the total phenotypic variation associated with lodging resistance in RILs evaluated over 11 environments in Canada. They converted the most important AFLP band into a SCAR marker (A001). The presence or absence of A001 corresponded well with the known lodging reaction of 50 commercial pea cultivars; thus demonstrating that selection for lodging resistant genotypes can be done indirectly using the A001 marker. The validation case studies described in this section show that although there are few examples in the public domain literature, nevertheless, where time and effort are invested, MAS can be an effective tool in public legume plant breeding programs, as indeed has long been appreciated

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and extensively utilized in the private seed sector. Thus, marker technology can assist the transfer of beneficial traits into otherwise elite genetic backgrounds, and can be used for the characterization and exploitation of biodiversity in gene banks. However, a number of difficulties remain for the development and application of molecular breeding technologies. Most critical is the need to develop better phenotypic evaluation methodologies for marker development. All too often, plant breeders use standard 1–5 or 1–9 scores during the marker development phase. Generally, such scoring systems are a good compromise for genetic progress in the context of conventional crop improvement. However, they are rarely the most appropriate methodology for the identification of marker-trait associations, particularly for traits with a complex genetic basis, low heritability, and high environmental interaction. For these traits, a more intensive phenotyping approach is required that provides highly quantitative data for component traits of the target character. In addition to generating accurate and precise phenotype data, it is equally important that field screening programs be based on multiple replications, locations, and seasons. Finally, there is a critical need for basing marker development on mapping population sizes substantially larger than those commonly used in academic situations, together with validation in multiple diverse populations (Young 1999). For successful, large-scale, cost-effective MAS screening, there is generally a need for assay technology development that reduces the cost of DNA extraction and PCR, and, where possible, eliminates the need for data collection through electrophoresis. For example, there are several options for direct staining of DNA once allele-specific associated primers (ASAPs) have been identified that will specifically amplify only a DNA fragment tightly linked to one allele at a locus of interest (Gu et al. 1995). Although once such a plus-minus assay is available, one must also develop multiplex controls for false negatives. Recent developments in legume genomics are rapidly providing an array of molecular breeding opportunities to legume breeders. This includes the availability of a large number of robust PCR-based markers such as SSR, ESTs, and SNPs, the generation of high-density genetic maps, and the progress being made in sequencing the genomes of the two model legumes, M. truncatula and L. japonicus. At the same time, generic advances in automation technologies now offer real possibilities for the efficient development and application of marker-assisted selection techniques at a scale and unit cost that is finally of relevance to plant breeding programs and international germplasm collections. However, for the next magnitude increase in throughput and decrease in unit

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costs, we will have to look to micro-array technology to finally provide the required scale and cost for simultaneous selection of a diverse range of complex traits.

VII. CONCLUSIONS AND FUTURE PROSPECTS Resource-poor farmers across the developing world depend on grain legumes to sustain the health of their families and livestock, and to enhance their economic well-being. Invariably, they grow these crops under rain-fed low-input systems. These factors create a multiplicity of demands and stresses on these crops that can only be effectively addressed through holistic agricultural research and development programs that are now tasked with increasing productivity, yield stability, and profitability. Traditional agricultural systems across the world have depended on the rotation of cereal and legume crops. However, with increasing intensification of agriculture during the twentieth century, there has been a substantial emphasis on cereals as the pre-eminent food commodity in national production and international trade. In turn, this has been reflected by a continuous and cumulative increase in funding for research and breeding of cereal crops (Goff and Salmeron 2004) that has resulted in the state-of-the-art in legumes falling further and further behind. Nevertheless, a renaissance is in sight thanks to the designation of two legume species as model genomes. Progress in the genomics of Medicago and Lotus offers the potential for real technological leapfrogging amongst legume crops. Our newfound ability to dissect the genetics and biology of complex traits influencing the full range of agronomic characters is innately complementary and synergistic to ideotype breeding and should drive the long-awaited appearance of knowledge-led breeding systems. So-called ‘molecular-enhanced plant breeding’ will facilitate a new generation of seed-based products emerging from a much more target-orientated process both in terms of ecoregional adaptation and end-user preference. However, substantial advances in bioinformatics and whole plant physiology modeling will be required to enable the largely reductionist approaches of the genomics community to be effectively re-engineered into a meaningful crop plant. In addition, considerable increases in our knowledge and ability to positively manipulate genotype-byenvironment interaction and epistasis will be required to ensure that crop architecture performs as envisaged. The evolving science of simulating breeding systems is likely to be a tremendously important tool in

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this respect (Chapman et al. 2003; Wang et al. 2004c). Similarly, technologies that readily allow research scientists to work alongside plant breeders (Podlich et al. 2004) on real populations, grown in realistic environments (where possible in farmers’ fields) using appropriate largescale experimental designs, will be increasingly critical. This will require breeders to augment and adjust their trait assessment profiles. Thus, in addition to scoring plant characteristics, breeders will need to assess agro-ecozone parameters such as water productivity and nutrient use efficiency. The critical issue here will be the development of systemic multidisciplinary and multi-sector teams that integrate researchers, growers, processors, traders, consumers, and other stakeholders. Only then will it be possible to effectively design holistic solutions to complex multifaceted problems that can be evaluated and refined early in the product development pathway. The increasing absence of academic (public sector) critical mass in this fundamentally important domain between research outputs and product development is surely one of the most pervasive reasons for insufficient impact in farmers’ fields from investments in tropical plant science research and breeding. There is an urgent need for the academic community to value intellectual endeavour in this area and to populate it with some of the best minds in the field. Only then will we begin to see the promise of research for development again reaching the dramatic levels of impact realized during the green revolution. Plant breeding during the twentieth century has been characterized by continuous incremental changes such as improvements in genetics and biometrics, plus more infrequent revolutionary changes including the automation of breeding trials and the computerization of phenotyping. In this context, the molecularization of plant breeding is a natural and inevitable next step. Beyond the increased potential power of selection, marker-assisted breeding offers additional advantages in the area of economics of scale both in terms of cost and time, as very different traits can be manipulated using the same technology. Most importantly, as with previous revolutionary changes, the integration of genomics will require a fundamental redesign of breeding systems in order to maximize the value of this new tool. Nowhere is this need more intense than in the effective merging of genetic resources, biotechnology, and pre-breeding strategies. Conventional approaches to identifying and utilizing genetic resources have tended to be highly inefficient. Genomics and bioinformatics now offer highly targeted mechanisms for providing plant breeders with structured and systematic entry points into vast global crop-related germplasm collections. Moreover, tools are emerging from these disciplines that will offer highly powerful approaches for rapid

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screening for target alleles, genes, and traits. However, again the promised impact will require radical changes in the design and implementation of trials, not least in the way traits are screened. A number of model plant species have emerged that will each likely play a different role in the advancement of science and technology: Arabidopsis (metabolic pathways of fundamental importance to the functioning of plants), Medicago and Lotus (nitrogen fixation and other physiological processes of agronomic importance), and rice and maize (components of productivity, plus biotic and abiotic stresses important for yield stability). However, the extent to which genetic knowledge from model systems will be readily translated into economic impact in related crops remains to be empirically demonstrated (Thro et al. 2004). Dramatic progress in comparative mapping of cereals during the 1990s (Gale and Davos 1998) gave much impetus to an excessive focus on a few species based on the premise that advances in model systems could be directly translated into related crop species for immediate application and agronomic progress. More recently, there has been a shift to more gene-based (sequence) information and the ability to reverse engineer target traits into their genic components has become more routine. This has led to a partial re-evaluation of the comparative genomics doctrine, as unique gene level variation within the target crop has been assigned increasing importance (Doust et al. 2004). We must expect that the answers to many questions about crop productivity will be found in these crops and their related germplasm (Thro et al. 2004). Recent studies show a strong correlation between the degree of synteny and phylogenetic distance in legumes. There appears to be a high level of conservation between Medicago and pea but the synteny with soybean is difficult to characterize (Choi et al. 2004a). In addition, genome size, ploidy, and structural reorganization are seen as significant confounding factors for the effective utilization of comparative genomics in molecular breeding. Moreover, as the whole genome sequence of rice opens up huge new possibilities for studying economic traits, it has also raised fundamental questions about the validity of current standard operating procedures. Most critically, it appears that the cross genera assignment of gene function deserves a more cautionary approach (Bennetzen et al. 2004). Functional validation of new genes within the species of interest is clearly becoming an essential component of the process. Conversely, different genes may be responsible for the same phenotype in different species (Havey 2004). Thus, some traits will require de novo research in the target crop or crop group. These types of findings will almost certainly lead to the emergence of a whole new generation of model crops, each valued for the intensive study of one or more specific trait(s) within their respective taxonomic clades: for exam-

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ple, pearl millet, cowpea, and cassava for drought tolerance; wheat, barley, and rice for disease resistance; and so forth. These studies will provide fundamentally important information for trait, gene, and allelemining of germplasm collections. However, it seems clear that there will be few major shortcuts or silver bullets. Instead, a continuous cumulative but iterative build-up of information from germplasm through breeding populations to new varieties will be required for consistent impact from unlocking crop-related genetic diversity. Genomics research in legume crops and model systems will soon routinely define the location of loci controlling a target trait as well as identify underlying candidate genes and their sequences (through mapping, mutation, and transcriptional-based investigations). Based on this new knowledge, it will be possible to develop highly precise DNA markers for assisted-selection or aided-introgression. However, the efficacy of these markers will depend largely on the stringency of the marker development process and the tailoring of the breeding system. Where these criteria are fulfilled, we should expect to see new crop types with increased productivity, enhanced yield stability, and reduced input requirements. These new crops will have grossly altered architectures and/or physiological profiles. Yet, to see these advances have an impact beyond the experiments of crop physiologists, it will be necessary for the research and breeding community to adopt very different perspectives on their roles in product development, testing, refinement, and delivery. Most importantly, perhaps, the academic community must become more favourably inclined to publish negative results and inconsistencies that will allow the plant breeding community to more rapidly and efficiently identify and move ahead with best bet technologies and approaches.

ACKNOWLEDGMENTS The authors wish to thank Rolf Folkertsma and Hari Sharma (ICRISAT) for their critical review of the manuscript, and three anonymous reviewers (Plant Breeding Reviews) for their extensive suggestions. We also thank the staff of ICRISAT library for their tireless efforts in conducting literature searches and arranging for reprints, and KDV Prasad for text editing, references, and tables as well help in obtaining rare publications. Sangam Dwivedi and Jonathan Crouch are partially funded by the Generation Challenge Program. Legume molecular breeding research at ICRISAT has benefited from substantial contributions from the Asian Development Bank, the Department for International Development, the European Union, and the Government of Japan.

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Subject Index Volume 26 A Arabidopsis, history, 1–33 B Barley, resistance breeding, 125–169 Biography, George P. Rédei, 1–33 Breeding barley resistance, 125–169 legumes, 171–357 papaya, 35–78 rol genes, 79–103

Genomics, grain legumes, 171–357 Germplasm, legumes, 171–357 Grain breeding, barley resistance, 125–169 I Intergeneric hybridization, papaya, 35–78 L Legume breeding, genomics, 171–357

C

M

Carica papaya, see Papaya Cytogenetics, polyploidy terminology, 105–124

Molecular biology legumes, 171–357 papaya, 35–78 rol genes, 79–103

D Diseases and pest resistance barley, 125–169 papaya, 35–78 Diversity, legumes, 171–357 F Fruit breeding, papaya, 35–78 Fungal diseases, Fusarium head blight, 125–169 Fusarium head blight (barley), 125–169 G Genes, rol in breeding, 79–103 Genetic engineering legumes, 171–357 papaya, 35–78 rol genes, 79–103 Genetics, polyploidy terminology, 105–124

P Papaya, breeding, 35–78 Polyploid terminology, 105–124 R Rédei, George P. (biography), 1–33 Rol genes, 79–103 S Selection, marker assisted, 292–299 T Transformation and transgenesis: barley, 26:155–157 papaya, 35–78 V Virus diseases, papaya, 35–78

Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 359

Cumulative Subject Index (Volumes 1–26) A Adaptation: blueberry, rabbiteye, 5:351–352 durum wheat, 5:29–31 genetics, 3:21–167 testing, 12:271–297 Aglaonema breeding, 23:267–269 Alexander, Denton, E. (biography), 22:1–7 Alfalfa: honeycomb breeding, 18:230–232 inbreeding, 13:209–233 in vitro culture, 2:229–234 somaclonal variation, 4:123–152 unreduced gametes, 3:277 Allard, Robert W. (biography), 12:1–17 Allium cepa, see Onion Almond: breeding self-compatible, 8:313–338 domestication, 25:290–291 transformation, 16:103 Alocasia breeding, 23:269 Alstroemaria, mutation breeding, 6:75 Amaranth: breeding, 19:227–285 cytoplasm, 23:191 genetic resources, 19:227–285 Animals, long term selection 24(2):169–210, 211–234Aneuploidy: alfalfa, 10:175–176 alfalfa tissue culture, 4:128–130 petunia, 1:19–21 wheat, 10:5–9 Anther culture: cereals, 15:141–186 maize, 11:199–224

Anthocyanin maize aleurone, 8:91–137 pigmentation, 25:89–114 Anthurium breeding, 23:269–271 Antifungal proteins, 14:39–88 Antimetabolite resistance, cell selection, 4:139–141, 159–160 Apomixis: breeding, 18:13–86 genetics, 18:13–86 reproductive barriers, 11:92–96 rice, 17:114–116 Apple: Domestication, 25:286–289 genetics, 9:333–366 rootstocks, 1:294–394 transformation, 16:101–102 Apricot: domestication, 25:291–292 transformation, 16:102 Arachis, see Peanut in vitro culture, 2:218–224 Artichoke breeding, 12:253–269 Avena sativa, see Oat Avocado domestication, 25:307 Azalea, mutation breeding, 6:75–76

B Bacillus thuringensis, 12:19–45 Bacteria, long-term selection, 24(2):225–265 Bacterial diseases: apple rootstocks, 1:362–365 cell selection, 4:163–164 cowpea, 15:238–239 potato, 19:113–122

Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 361

362 Bacterial diseases (cont.) raspberry, 6:281–282 soybean, 1:209–212 sweet potato, 4:333–336 transformation fruit crops, 16:110 Banana: breeding, 2:135–155 domestication, 25:298–299 transformation, 16:105–106 Barley: anther culture, 15:141–186 breeding methods, 5:95–138 diversity, 21:234–235 doubled haploid breeding, 15:141–186 gametoclonal variation, 5:368–370 haploids in breeding, 3:219–252 molelcular markers, 21:181–220 photoperiodic response, 3:74, 89–92, 99 vernalization, 3:109 Bean (Phaseolus): breeding, 1:59–102; 10:199–269; 23:21–72 breeding mixtures, 4:245–272 breeding (tropics), 10:199–269 heat tolerance, 10:149 in vitro culture, 2:234–237 long-term selection, 24(2):69–74 photoperiodic response, 3:71–73, 86–92; 16:102–109 protein, 1:59–102 rhizobia interaction, 23:21–72 Beet (table) breeding, 22:357–388 Beta, see Beet Biochemical markers, 9:37–61 Biography: Alexander, Denton E., 22:1–7 Allard, Robert W., 12:1–17 Bringhurst, Royce S., 9:1–8 Burton, Glenn W., 3:1–19 Coyne, Dermot E., 23:1–19 Downey, Richard K., 18:1–12 Dudley, J.W., 24(1):1–10 Draper, Arlen D., 13:1–10 Duvick, Donald N., 14:1–11 Gabelman, Warren H., 6:1–9 Hallauer, Arnel R., 15:1–17 Harlan, Jack R., 8:1–17 Jones, Henry A., 1:1–10 Laughnan, John R. 19:1–14 Munger, Henry M., 4:1–8, Rédei, George, P., 26:1–33 Peloquin, Stanley J., 25:1–19

CUMULATIVE SUBJECT INDEX Ryder, Edward J., 16:1–14 Sears, Ernest Robert, 10:1–2 Simmonds, Norman W., 20:1–13 Sprague, George F., 2:1–11 Vogel, Orville A., 5:1–10 Vuylsteke, Dirk R., 21:1–25 Weinberger, John H., 11:1–10 Yuan, Longping, 17:1–13 Biotechnology politics, 25:21–55 Birdsfoot trefoil, tissue culture, 2:228–229 Blackberry, 8:249–312 mutation breeding, 6:79 Black walnut, 1:236–266 Blueberry: breeding, 13:1–10 domestication, 25:304 rabbiteye, 5:307–357 Brachiaria, apomixis, 18:36–39, 49–51 Bramble: domestication, 25:303 transformation, 16:105 Brassica, see Cole crops Brassica: napus, see Canola, Rutabaga rapa, see Canola Brassicaceae: incompatibility, 15:23–27 molecular mapping, 14:19–23 Breeding: Aglaonema, 23:267–269 alfalfa via tissue culture, 4:123–152 almond, 8:313–338 Alocasia, 23:269 amaranth, 19:227–285 apomixis, 18:13–86 apple, 9:333–366 apple rootstocks, 1:294–394 banana, 2:135–155 barley, 3:219–252; 5:95–138; 26:125–169 bean, 1:59–102; 4:245–272; 23:21–72 beet (table), 22:357–388 biochemical markers, 9:37–61 blackberry, 8:249–312 black walnut, 1:236–266 blueberry, rabbiteye, 5:307–357 bromeliad, 23:275–276 cactus, 20:135–166 Calathea, 23:276 carbon isotope discrimination, 12:81–113 carrot, 19:157–190

CUMULATIVE SUBJECT INDEX cassava, 2:73–134 cell selection, 4:153–173 chestnut, 4:347–397 chimeras, 15:43–84 chrysanthemum, 14:321–361 citrus, 8:339–374 coffee, 2:157–193 coleus, 3:343–360 competitive ability, 14:89–138 cowpea, 15:215–274 cucumber, 6:323–359 cytoplasmic DNA, 12:175–210 diallel analysis, 9:9–36 Dieffenbachia, 271–272 doubled haploids, 15:141–186; 25:57–88 Dracaena, 23:277 drought tolerance, maize, 25:173–253 durum wheat, 5:11–40 Epepremnum, 23: 272–2mn epistasis, 21:27–92 exotic maize, 14:165–187 fern, 23:276 fescue, 3:313–342 Ficus, 23:276 Flower color, 25:89–114 foliage plant, 23:245–290 forest tree, 8:139–188 fruit crops, 25:255–320 gene action 15:315–374 genotype x environment interaction, 16:135–178 grapefruit, 13:345–363 grasses, 11:251–274 guayule, 6:93–165 heat tolerance, 10:124–168 Hedera, 23:279–280 herbicide-resistant crops, 11:155–198 heritability, 22:9–111 heterosis, 12:227–251 homeotic floral mutants, 9:63–99 honeycomb, 13:87–139; 18:177–249 hybrid, 17:225–257 hybrid wheat, 2:303–319; 3:169–191 induced mutations, 2:13–72 insect and mite resistance in cucurbits, 10:199–269 isozymes, 6:11–54 legumes, 26:171–357 lettuce, 16:1–14; 20:105–133 maize, 1:103–138, 139–161; 4:81–122; 9:181–216; 11:199–224; 14:139–163, 165–187, 189–236; 25:173–253

363 mitochondrial genetics, 25:115–238 molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37, 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174 mosaics, 15:43–84 mushroom, 8:189–215 negatively associated traits, 13:141–177 oat, 6:167–207 oil palm, 4:175–201; 22:165–219 onion, 20:67–103 papaya, 26:35–78 palms, 23:280–281 pasture legumes, 5:237–305 pea, snap, 212:93–138 peanut, 22:297–356 pearl millet, 1:162–182 perennial rye, 13:265–292 persimmon, 19:191–225 Philodendron, 23:273 plantain, 2:150–151; 14:267–320; 21:211–25 potato, 3:274–277; 9:217–332; 16:15–86; 19:59–155, 25:1–19 proteins in maize, 9:181–216 quality protein maize (QPM), 9:181–216 raspberry, 6:245–321 recurrent restricted phenotypic selection, 9:101–113 recurrent selection in maize, 9:115–179; 14:139–163 rice, 17:15–156; 23:73–174 rol genes, 26:79–103 rose, 17:159–189 rutabaga, 8:217–248 sesame, 16:179–228 snap pea, 21:93–138 somatic hybridization, 20:167–225 sorghum male sterility, 25:139–172 soybean, 1:183–235; 3:289–311; 4:203–243; 21:212–307 soybean hybrids, 21:212–307 soybean nodulation, 11:275–318 soybean recurrent selection, 15:275–313 spelt, 15:187–213 statistics, 17:296–300 strawberry, 2:195–214 sugar cane, 16:272–273

364 Breeding (cont.) supersweet sweet corn, 14:189–236 sweet cherry, 9:367–388 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:313–345 Syngonium, 23:274 tomato, 4:273–311 transgene technology, 25:105–108 triticale, 5:41–93; 8:43–90 Vigna, 8:19–42 virus resistance, 12:47–79 wheat, 2:303–319; 3:169–191; 5:11–40; 11:225–234; 13:293–343 wheat for rust resistance, 13:293–343 white clover, 17:191–223 wild rice, 14:237–265 Bringhurst, Royce S. (biography), 9:1–8 Broadbean, in vitro culture, 2:244–245 Bromeliad breeding, 23:275–276 Burton, Glenn W. (biography), 3:1–19 C Cactus: breeding, 20:135–166 domestication, 20:135–166 Cajanus, in vitro culture, 2:224 Calathea breeding, 23:276 Canola, R.K. Downey, designer, 18:1–12 Carbohydrates, 1:144–148 Carbon isotope discrimination, 12:81–113 Carica papaya, see Papaya Carnation, mutation breeding, 6:73–74 Carrot breeding, 19: 157–190 Cassava: breeding, 2:73–134 long-term selection, 24(2):74–79 Castanea, see Chestnut Cell selection, 4:139–145, 153–173 Cereal breeding, see Grain breeding Cereal diversity, 21:221–261 Cherry, see Sweet cherry domestication, 25:202–293 Chestnut breeding, 4:347–397 Chickpea, in vitro culture, 2:224–225 Chimeras and mosaics, 15:43–84 Chinese cabbage, heat tolerance, 10:152 Chromosome, petunia, 1:13–21, 31–33 Chrysanthemum: breeding, 14:321–361 mutation breeding, 6:74

CUMULATIVE SUBJECT INDEX Cicer, see Chickpea Citrus: domestication, 25:296–298 protoplast fusion, 8:339–374 Clonal repositories, see National Clonal Germplasm Repository Clover: in vitro culture, 2:240–244 molecular genetics, 17:191–223 Coffea arabica, see Coffee Coffee, 2:157–193 Cold hardiness: breeding nectarines and peaches, 10:271–308 wheat adaptation, 12:124–135 Cole crops: Chinese cabbage, heat tolerance, 10:152 gametoclonal variation, 5:371–372 rutabaga, 8:217–248 Coleus, 3:343–360 Competition, 13:158–165 Competitive ability breeding, 14:89–138 Controlling elements, see Transposable elements Corn, see Maize; Sweet corn Cotton, heat tolerance 10:151 Cowpea: breeding, 15:215–274 heat tolerance, 10:147–149 in vitro culture, 2:245–246 photoperiodic response, 3:99 Coyne, Dermot E. (biography), 23:1–19 Cranberry domestication, 25:304–305 Crop domestication and selection, 24(2):1–44 Cryopreservation, 7:125–126,148–151,167 buds, 7:168–169 genetic stability, 7:125–126 meristems, 7:168–169 pollen, 7:171–172 seed, 7:148–151,168 Cucumber, breeding, 6:323–359 Cucumis sativa, see Cucumber Cucurbitaceae, insect and mite resistance, 10:309–360 Cybrids. 3:205–210; 20: 206–209 Cytogenetics: alfalfa, 10:171–184 blueberry, 5:325–326 cassava, 2:94

CUMULATIVE SUBJECT INDEX citrus, 8:366–370 coleus, 3:347–348 durum wheat, 5:12–14 fescue, 3:316–319 Glycine, 16:288–317 guayule, 6:99–103 maize mobile elements, 4:81–122 maize-tripsacum hybrids, 20:15–66 oat, 6:173–174 pearl millet, 1:167 perennial rye, 13:265–292 petunia, 1:13–21, 31–32 polyploidy terminology, 26:105–124 potato, 25:1–19 rose, 17:169–171 rye, 13:265–292 Saccharum complex, 16:273–275 sesame, 16:185–189 triticale, 5:41–93; 8:54 wheat, 5:12–14; 10:5–15; 11:225–234 Cytoplasm: breeding, 23: 175–210; 25:115–138 cybrids, 3:205–210; 20:206–209 incompatibility, 25:115–138 male sterility, 25:115–138,139–172 molecular biology of male sterility, 10:23–51 organelles, 2:283–302; 6:361–393 pearl millet, 1:166 petunia, 1:43–45 sorghum male sterility, 25:139–172 wheat, 2:308–319 D Dahlia, mutation breeding, 6:75 Date palm domestication, 25:272–277 Daucus, see Carrot Diallel cross, 9:9–36 Dieffenbachia breeding, 23:271–272 Diospyros, see Persimmon Disease and pest resistance: antifungal proteins, 14:39–88 apple rootstocks, 1:358–373 banana, 2:143–147 barley, 26:135–169 blackberry, 8:291–295 black walnut, 1:251 blueberry, rabbiteye, 5:348–350 cassava, 2:105–114 cell selection, 4:143–145, 163–165 citrus, 8:347–349

365 coffee, 2:176–181 coleus, 3:353 cowpea, 15:237–247 durum wheat, 5:23–28 fescue, 3:334–336 herbicide-resistance, 11:155–198 host-parasite genetics, 5:393–433 induced mutants, 2:25–30 lettuce, 1:286–287 papaya, 26:161–357 potato, 9:264–285, 19:69–155 raspberry, 6:245–321 rutabaga, 8:236–240 soybean, 1:183–235 spelt, 15:195–198 strawberry, 2:195–214 virus resistance, 12:47–79 wheat rust, 13:293–343 Diversity: land races, 21:221–261 legumes, 26:171–357 DNA methylation, 18:87–176 Doubled haploid breeding, 15:141–186; 25:57–88 Downey, Richard K. (biography), 18:1–12 Dracaena breeding, 23:277 Draper, Arlen D. (biography), 13:1–10 Drought resistance: durum wheat, 5:30–31 maize, 25:173–253 soybean breeding, 4:203–243 wheat adaptation, 12:135–146 Dudley, J.W. (biography), 24(1):1–10 Durum wheat, 5:11–40 Duvick, Donald N. (biography), 14:1–11 E Elaeis, see Oil palm Embryo culture: in crop improvement, 5:181–236 oil palm, 4:186–187 pasture legume hybrids, 5:249–275 Endosperm: balance number, 25:6–7 maize, 1:139–161 sweet corn, 1:139–161 Endothia parasitica, 4:355–357 Epepremnum breeding, 23:272–273 Epistasis, 21:27–92 Escherichia coli, long-term selection, 24(2):225–224

366 Evolution: coffee, 2:157–193 fruit, 25: 255–320 grapefruit, 13:345–363 maize, 20:15–66 sesame, 16:189 Exploration, 7:9–11, 26–28, 67–94 F Fabaceae, molecular mapping, 14:24–25 Fern breeding, 23:276 Fescue, 3:313–342 Festuca, see Fescue Fig domestication, 25:281–285 Flavonoid chemistry, 25:91–94 Floral biology: almond, 8:314–320 blackberry, 8:267–269 black walnut, 1:238–244 cassava, 2:78–82 chestnut, 4:352–353 coffee, 2:163–164 coleus, 3:348–349 color, 25:89–114 fescue, 3:315–316 garlic: 23:211–244 guayule, 6:103–105 homeotic mutants, 9:63–99 induced mutants, 2:46–50 pearl millet, 1:165–166 pistil in reproduction, 4:9–79 pollen in reproduction, 4:9–79 reproductive barriers, 11:11–154 rutabaga, 8:222–226 sesame, 16:184–185 sweet potato, 4:323–325 Flower color, 25:89–114 Forage breeding: alfalfa inbreeding, 13:209–233 diversity, 21:221–261 fescue, 3:313–342 perennials, 11:251–274 white clover, 17:191–223 Foliage plant breeding, 23:245–290 Forest crop breeding: black walnut, 1:236–266 chestnut, 4:347–397 ideotype concept, 12:177–187 molecular markers, 19:31–68 quantitative genetics, 8:139–188 Fragaria, see Strawberry

CUMULATIVE SUBJECT INDEX Fruit, nut, and beverage crop breeding: almond, 8:313–338 apple, 9:333–366 apple rootstocks, 1:294–394 banana, 2:135–155 blackberry, 8:249–312 blueberry, 13:1–10 blueberry, rabbiteye, 5:307–357 breeding, 25:255–320 cactus, 20:135–166 cherry, 9:367–388 citrus, 8:339–374 coffee, 2:157–193 domestication, 25:255–320 ideotype concept, 12:175–177 genetic transformation, 16:87–134 grapefruit, 13:345–363 mutation breeding, 6:78–79 nectarine (cold hardy), 10:271–308 origins, 25:255–320 papaya, 26:35–78 peach (cold hardy), 10:271–308 persimmon, 19:191–225 plantain, 2:135–155 raspberry, 6:245–321 strawberry, 2:195–214 sweet cherry, 9:367–388 Fungal diseases: apple rootstocks, 1:365–368 banana and plantain, 2:143–145, 147 barley, Fusarium head blight, 26:125–169 cassava, 2:110–114 cell selection, 4:163–165 chestnut, 4:355–397 coffee, 2:176–179 cowpea, 15:237–238 durum wheat, 5:23–27 Fusarium head blight (barley), 26:125–169 host-parasite genetics, 5:393–433 lettuce, 1:286–287 potato, 19:69–155 raspberry, 6:245–281 soybean, 1:188–209 spelt, 15:196–198 strawberry, 2:195–214 sweet potato, 4:333–336 transformation, fruit crops, 16:111–112 wheat rust, 13:293–343 Fusarium head blight (barley), 26:125–169

CUMULATIVE SUBJECT INDEX

G Gabelman, Warren H. (biography), 6:1–9 Gametes: almond, self compatibility, 7:322–330 blackberry, 7:249–312 competition, 11:42–46 forest trees, 7:139–188 maize aleurone, 7:91–137 maize anthocynanin, 7:91–137 mushroom, 7:189–216 polyploid, 3:253–288 rutabaga, 7:217–248 transposable elements, 7:91–137 unreduced, 3:253–288 Gametoclonal variation, 5:359–391 barley, 5:368–370 brassica, 5:371–372 potato, 5:376–377 rice, 5:362–364 rye, 5:370–371 tobacco, 5:372–376 wheat, 5:364–368 Garlic breeding, 6:81, 23:211–244 Genes: action, 15:315–374 apple, 9:337–356 Bacillus thuringensis, 12:19–45 incompatibility, 15:19–42 incompatibility in sweet cherry, 9:367–388 induced mutants, 2:13–71 lettuce, 1:267–293 maize endosperm, 1:142–144 maize protein, 1:110–120, 148–149 petunia, 1:21–30 quality protein in maize, 9:183–184 Rhizobium, 23:39–47 rol in breeding, 26:79–103 rye perenniality, 13:261–288 soybean, 1:183–235 soybean nodulation, 11:275–318 sweet corn, 1:142–144 wheat rust resistance, 13:293–343 Genetic engineering: bean, 1:89–91 DNA methylation, 18:87–176 fruit crops, 16:87–134 host-parasite genetics, 5:415–428 legumes, 26:171–357 maize mobile elements, 4:81–122 papaya, 26:35–78.

367 rol genes, 26:79–103 salt resistance, 22:389–425 transformation by particle bombardment, 13:231–260 transgene technology, 25:105–108 virus resistance, 12:47–79 Genetic load and lethal equivalents, 10:93–127 Genetics: adaptation, 3:21–167 almond, self compatibility, 8:322–330 amaranth, 19:243–248 Amaranthus, see Amaranth apomixis, 18:13–86 apple, 9:333–366 Bacillus thuringensis, 12:19–45 bean seed protein, 1:59–102 beet, 22:357–376 blackberry, 8:249–312 black walnut, 1:247–251 blueberry, 13:1–10 blueberry, rabbiteye, 5:323–325 carrot, 19:164–171 chestnut blight, 4:357–389 chimeras, 15:43–84 chrysanthemums, 14:321 clover, white, 17:191–223 coffee, 2:165–170 coleus, 3:3–53 cowpea, 15:215–274 cytoplasm, 23:175–210 DNA methylation, 18:87–176 domestication, 25:255–320 durum wheat, 5:11–40 flower color, 25:89–114 forest trees, 8:139–188 fruit crop transformation, 16:87–134 gene action, 15:315–374 history, 24(1):11–40 host-parasite, 5:393–433 incompatibility, 15:19–42 incompatibility in sweet cherry, 9:367–388 induced mutants, 2:51–54 insect and mite resistance in Cucurbitaceae, 10:309–360 isozymes, 6:11–54 lettuce, 1:267–293 maize aleurone, 8:91–137 maize anther culture, 11:199–224 maize anthocynanin, 8:91–137 maize endosperm, 1:142–144

368 Genetics (cont.) maize male sterility, 10:23–51 maize mobile elements, 4:81–122 maize mutation, 5:139–180 maize seed protein, 1:110–120, 148–149 male sterility, maize, 10:23–51 mapping, 14:13–37 markers to manage germplasm, 13:11–86 maturity, 3:21–167 metabolism and heterosis, 10:53–59 mitochondrial, 25:115–138. molecular mapping, 14:13–37 mosaics, 15:43–84 mushroom, 8:189–216 oat, 6:168–174 organelle transfer, 6:361–393 overdominance, 17:225–257 pea, 21:110–120 pearl millet, 1:166, 172–180 perennial rye, 13:261–288 petunia, 1:1–58 photoperiod, 3:21–167 plantain, 14:264–320 polyploidy terminology, 26:105–124 potato disease resistance, 19:69–165 potato ploidy manipulation, 3:274–277; 16:15–86 quality protein in maize, 9:183–184 quantitative trait loci, 15:85–139 quantitative trait loci in animals selection, 24(2):169–210, 211–224 reproductive barriers, 11:11–154 rhizobia, 21–72 rice, hybrid, 17:15–156, 23:73–174 rose, 17:171–172 rutabaga, 8:217–248 salt resistance, 22:389–425 selection, 24(1):111–131, 143–151, 269–290 sesame, 16:189–195 snap pea, 21:110–120 soybean, 1:183–235 soybean nodulation, 11:275–318 spelt, 15:187–213 supersweet sweet corn, 14:189–236 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:327–330 temperature, 3:21–167 tomato fruit quality, 4:273–311 transposable elements, 8:91–137

CUMULATIVE SUBJECT INDEX triticale, 5:41–93 virus resistance, 12:47–79 wheat gene manipulation, 11:225–234 wheat male sterility, 2:307–308 wheat molecular biology, 11:235–250 wheat rust, 13:293–343 white clover, 17:191–223 yield, 3:21–167 Genome: Glycine, 16:289–317 Poaceae, 16:276–281 Genomics, grain legumes, 26:171–357 Genotype x environment, interaction, 16:135–178 Germplasm, see also National Clonal Germplasm Repositories; National Plant Germplasm System acquisition and collection, 7:160–161 apple rootstocks, 1:296–299 banana, 2:140–141 blackberry, 8:265–267 black walnut, 1:244–247 cactus, 20:141–145 cassava, 2:83–94, 117–119 chestnut, 4:351–352 coffee, 2:165–172 distribution, 7:161–164 enhancement, 7:98–202 evaluation, 7:183–198 exploration and introduction, 7:9–18,64–94 genetic markers, 13:11–86 guayule, 6:112–125 isozyme, 6:18–21 grain legumes, 26:171–357 legumes, 26:171–357 maintenance and storage, 7:95–110,111–128,129–158,159–182; 13:11–86 maize, 14:165–187 management, 13:11–86 oat, 6:174–176 peanut, 22:297–356 pearl millet, 1:167–170 plantain, 14:267–320 potato, 9:219–223 preservation, 2:265–282; 23:291–344 rights, 25:21–55 rutabaga, 8:226–227 sesame, 16:201–204 spelt, 15:204–205 sweet potato, 4:320–323

CUMULATIVE SUBJECT INDEX triticale, 8:55–61 wheat, 2:307–313 Gesneriaceae, mutation breeding, 6:73 Gladiolus, mutation breeding, 6:77 Glycine, genomes, 16:289–317 Glycine max, see Soybean Grain breeding: amaranth, 19:227–285 barley, 3:219–252, 5:95–138; 26:125–169 diversity, 21:221–261 doubled haploid breeding, 15:141–186 ideotype concept, 12:173–175 maize, 1:103–138, 139–161; 5:139–180; 9:115–179, 181–216; 11:199–224; 14:165–187; 22:3–4; 24(1): 11–40, 41–59, 61–78; 24(2): 53–64, 109–151; 25:173–253 maize history, 24(2): 31–59, 41–59, 61–78 oat, 6:167–207 pearl millet, 1:162–182 rice, 17:15–156; 24(2):64–67 sorghum, 25:139–172 spelt, 15:187–213 transformation, 13:231–260 triticale, 5:41–93; 8:43–90 wheat, 2:303–319; 5:11–40; 11:225–234, 235–250; 13:293–343; 22:221–297; 24(2):67–69 wild rice, 14:237–265 Grape: domestication, 25:279–281 transformation, 16:103–104 Grapefruit: breeding, 13:345–363 evolution, 13:345–363 Grass breeding: breeding, 11:251–274 mutation breeding, 6:82 recurrent selection, 9:101–113 transformation, 13:231–260 Growth habit, induced mutants, 2:14–25 Guayule, 6:93–165 H Hallauer, Arnel R. (biography), 15:1–17 Haploidy, see also unreduced and polyploid gametes apple, 1:376 barley, 3:219–252

369 cereals, 15:141–186 doubled, 15:141–186; 25:57–88 maize, 11:199–224 petunia, 1:16–18, 44–45 potato, 3:274–277; 16:15–86 Harlan, Jack R. (biography), 8:1–17 Heat tolerance breeding, 10:129–168 Herbicide resistance: breeding needs, 11:155–198 cell selection, 4:160–161 decision trees, 18:251–303 risk assessment, 18:251–303 transforming fruit crops, 16:114 Heritability estimation, 22:9–111 Heterosis: gene action, 15:315–374 overdominance, 17:225–257 plant breeding, 12:227–251 plant metabolism, 10:53–90 rice, 17:24–33 soybean, 21:263–320 Honeycomb: breeding, 18:177–249 selection, 13:87–139, 18:177–249 Hordeum, see Barley Host-parasite genetics, 5:393–433 Hyacinth, mutation breeding, 6:76–77 Hybrid and hybridization, see also Heterosis barley, 5:127–129 blueberry, 5:329–341 chemical, 3:169–191 interspecific, 5:237–305 maize high oil selection, 24(1):153–175 maize history, 24(1): 31–59, 41–59, 61–78 maize long-term selection, 24(2):43–64, 109–151 overdominance, 17:225–257 rice, 17:15–156 soybean, 21:263;-320 wheat, 2:303–319 I Ideotype concept, 12:163–193 In vitro culture: alfalfa, 2:229–234; 4:123–152 barley, 3:225–226 bean, 2:234–237 birdsfoot trefoil, 2:228–229 blackberry, 8:274–275

370 broadbean, 2:244–245 cassava, 2:121–122 cell selection, 4:153–173 chickpea, 2:224–225 citrus, 8:339–374 clover, 2:240–244 coffee, 2:185–187 cowpea, 2:245–246 embryo culture, 5:181–236, 249–275 germplasm preservation, 7:125,162–167 introduction, quarantines, 3:411–414 legumes, 2:215–264 mungbean, 2:245–246 oil palm, 4:175–201 pea, 2:236–237 peanut, 2:218–224 petunia, 1:44–48 pigeon pea, 2:224 pollen, 4:59–61 potato, 9:286–288 sesame, 16:218 soybean, 2:225–228 Stylosanthes, 2:238–240 wheat, 12:115–162 wingbean, 2:237–238 zein, 1:110–111 Inbreeding depression, 11:84–92 alfalfa, 13:209–233 cross pollinated crops, 13:209–233 Incompatibility: almond, 8:313–338 molecular biology, 15:19–42 pollen, 4:39–48 reproductive barrier, 11:47–70 sweet cherry, 9:367–388 Incongruity, 11:71–83 Industrial crop breeding, guayule, 6:93–165 Insect and mite resistance: apple rootstock, 1:370–372 black walnut, 1:251 cassava, 2:107–110 clover, white, 17:209–210 coffee, 2:179–180 cowpea, 15:240–244 Cucurbitaceae, 10:309–360 durum wheat, 5:28 maize, 6:209–243 raspberry, 6:282–300 rutabaga, 8:240–241 sweet potato, 4:336–337

CUMULATIVE SUBJECT INDEX transformation fruit crops, 16:113 wheat, 22:221–297 white clover, 17:209–210 Intergeneric hybridization, papaya, 26:35–78 Interspecific hybridization: blackberry, 8:284–289 blueberry, 5:333–341 citrus, 8:266–270 pasture legume, 5:237–305 rose, 17:176–177 rutabaga, 8:228–229 Vigna, 8:24–30 Intersubspecific hybridization, rice, 17:88–98 Introduction, 3:361–434; 7:9–11, 21–25 Ipomoea, see Sweet potato Isozymes, in plant breeding, 6:11–54 J Jones, Henry A. (biography), 1:1–10 Juglans nigra, see Black walnut K Karyogram, petunia, 1:13 Kiwifruit: domestication, 25:300–301 transformation, 16:104 L Lactuca sativa, see Lettuce Landraces, diversity, 21:221–263 Laughnan, Jack R. (bibliography), 19:1–14 Legume breeding, see also Oilseed, Soybean: cowpea, 15:215–274 genomics, 26:171–357 pasture legumes, 5:237–305 Vigna, 8:19–42 Legume tissue culture, 2:215–264 Lethal equivalents and genetic load, 10:93–127 Lettuce: breeding, 16:1–14; 20:105–133 genes, 1:267–293 Lingonberry domestication, 25:300–301 Linkage: bean, 1:76–77

CUMULATIVE SUBJECT INDEX isozymes, 6:37–38 lettuce, 1:288–290 maps, molecular markers, 9:37–61 petunia, 1:31–34 Lotus: hybrids, 5:284–285 in vitro culture, 2:228–229 Lycopersicon, see Tomato M Maize: anther culture, 11:199–224; 15:141–186 anthocyanin, 8:91–137 apomixis, 18:56–64 breeding, 1:103–138, 139–161 carbohydrates, 1:144–148 cytoplasm, 23:189 doubled haploid breeding, 15:141–186 drought tolerance, 25:173–253 exotic germplasm utilization, 14:165–187 high oil, 22:3–4; 24(1):153–175 history of hybrids, 23(1): 11–40, 41–59, 61–78 honeycomb breeding, 18:226–227 hybrid breeding, 17:249–251 insect resistance, 6:209–243 long-term selection 24(2):53–64, 109–151 male sterility, 10:23–51 marker-assisted selection. 24(1):293–309 mobile elements, 4:81–122 mutations, 5:139–180 origins, 20:15–66 origins of hybrids, 24(1):31–50, 41–59, 61–78 overdominance, 17:225–257 physiological changes with selection, 24(1):143–151 protein, 1:103–138 quality protein, 9:181–216 recurrent selection, 9:115–179; 14:139–163 RFLF changes with selection, 24(1):111–131 selection for oil and protein, 24(1):79–110, 153–175 supersweet sweet corn, 14:189–236 transformation, 13:235–264

371 transposable elements, 8:91–137 unreduced gametes, 3:277 Male sterility: chemical induction, 3:169–191 coleus, 3:352–353 genetics, 25:115–138, 139–172 lettuce, 1:284–285 molecular biology, 10:23–51 pearl millet, 1:166 petunia, 1:43–44 rice, 17:33–72 sesame, 16:191–192 sorghum, 139–172 soybean, 21:277–291 wheat, 2:303–319 Malus spp, see Apple Malus _× domestica, see Apple Malvaceae, molecular mapping, 14:25–27 Mango: domestication, 25:277–279 transformation, 16:107 Manihot esculenta, see Cassava Medicago, see also Alfalfa in vitro culture, 2:229–234 Meiosis, petunia, 1:14–16 Metabolism and heterosis, 10:53–90 Microprojectile bombardment, transformation, 13:231–260 Mitochondrial genetics, 6:377–380; 25:115–138 Mixed plantings, bean breeding, 4:245–272 Mobile elements, see also transposable elements: maize, 4:81–122; 5:146–147 Molecular biology: apomixis, 18:65–73 comparative mapping, 14:13–37 cytoplasmic male sterility, 10:23–51 DNA methylation, 18:87–176 herbicide-resistant crops, 11:155–198 incompatibility, 15:19–42 legumes, 26:171–357 molecular mapping, 14:13–37; 19:31–68 molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174, 26:292–299 papaya, 26:35–78 quantitative trait loci, 15:85–139

372 Molecular biology (cont.) rol genes, 26:79–103 salt resistance, 22:389–425 somaclonal variation, 16:229–268 somatic hybridization, 20:167–225 soybean nodulation, 11:275–318 strawberry, 21:139–180 transposable (mobile) elements, 4:81–122; 8:91–137 virus resistance, 12:47–79 wheat improvement, 11:235–250 Molecular markers, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174 alfalfa, 10:184–190 apomixis, 18:40–42 barley, 21:181–220 clover, white, 17:212–215 forest crops, 19:31–68 fruit crops, 12:195–226 maize selection, 24(1):293–309 mapping, 14:13–37 plant genetic resource mangement, 13:11–86 rice, 17:113–114, 23:73–124 rose, 17:179 somaclonal variation, 16:238–243 wheat, 21:181–220 white clover, 17:212–215 Monosomy, petunia, 1:19 Mosaics and chimeras, 15:43–84 Mungbean, 8:32–35 in vitro culture, 2:245–246 photoperiodic response, 3:74, 89–92 Munger, Henry M. (biography), 4:1–8 Musa, see Banana, Plantain Mushroom, breeding and genetics, 8:189–215 Mutants and mutation: alfalfa tissue culture, 4:130–139 apple rootstocks, 1:374–375 banana, 2:148–149 barley, 5:124–126 blackberry, 8:283–284 cassava, 2:120–121 cell selection, 4:154–157 chimeras, 15:43–84 coleus, 3:355 cytoplasmic, 2:293–295 gametoclonal variation, 5:359–391 homeotic floral, 9:63–99

CUMULATIVE SUBJECT INDEX induced, 2:13–72 long term selection variation, 24(1):227–247 maize, 1:139–161, 4:81–122; 5:139–180 mobile elements, see Transposable elements mosaics, 15:43–84 petunia, 1:34–40 sesame, 16:213–217 somaclonal variation, 4:123–152; 5:147–149 sweet corn, 1:139–161 sweet potato, 4:371 transposable elements, 4:181–122; 8:91–137 tree fruits, 6:78–79 vegetatively-propagated crops, 6:55–91 zein synthesis, 1:111–118 Mycoplasma diseases, raspberry, 6:253–254 N National Clonal Germplasm Repository (NCGR), 7:40–43 cryopreservation, 7:125–126 genetic considerations, 7:126–127 germplasm maintenance and storage, 7:111–128 identification and label verification, 7:122–123 in vitro culture and storage, 7:125 operations guidelines, 7:113–125 preservation techniques, 7:120–121 virus indexing and plant health, 7:123–125 National Plant Germplasm System (NPGS), see also Germplasm history, 7:5–18 information systems, 7:57–65 operations, 7:19–56 preservation of genetic resources, 23:291–34 National Seed Storage Laboratory (NSSL), 7:13–14, 37–38, 152–153 Nectarines, cold hardiness breeding, 10:271–308 Nematode resistance: apple rootstocks, 1:368 banana and plantain, 2:145–146 coffee, 2:180–181 cowpea, 15:245–247

CUMULATIVE SUBJECT INDEX soybean, 1:217–221 sweet potato, 4:336 transformation fruit crops, 16:112–113 Nicotiana, see Tobacco Nodulation, soybean, 11:275–318 O Oat, breeding, 6:167–207 Oil palm: breeding, 4:175–201, 22:165–219 in vitro culture, 4:175–201 Oilseed breeding: canola, 18:1–20 oil palm, 4:175–201; 22:165–219 sesame, 16:179–228 soybean, 1:183–235; 3:289–311; 4:203–245; 11:275–318; 15:275–313 Olive domestication, 25:277–279 Onion, breeding history, 20:57–103 Opuntia, see Cactus Organelle transfer, 2:283–302; 3:205–210; 6:361–393 Ornamentals breeding: chrysanthemum, 14:321–361 coleus, 3:343–360 petunia, 1:1–58 rose, 17:159–189 Ornithopus, hybrids, 5:285–287 Orzya, see Rice Overdominance, 17:225–257 Ovule culture, 5:181–236 P Palm (Arecaceae): foliage breeding, 23:280–281 oil palm breeding, 4:175–201; 22:165–219. Panicum maximum, apomixis, 18:34–36, 47–49 Papaya: Breeding, 26:35–78 domestication, 25:307–308 transformation, 16:105–106 Parthenium argentatum, see Guayule Paspalum, apomixis, 18:51–52 Paspalum notatum, see Pensacola bahiagrass Passionfruit transformation, 16:105 Pasture legumes, interspecific hybridization, 5:237–305

373 Pea: breeding, 21:93–138 flowering, 3:81–86, 89–92 in vitro culture, 2:236–237 Peach: cold hardiness breeding, 10:271–308 domestication, 25:294–296 transformation, 16:102 Peanut: breeding, 22:297–356 in vitro culture, 2:218–224 Pear: domestication, 25:289–290 transformation, 16:102 Pearl millet: apomixis, 18:55–56 breeding, 1:162–182 Pecan transformation, 16:103 Peloquin, Stanley, J. (biography), 25:1–19 Pennisetum americanum, see Pearl millet Pensacola bahiagrass, 9:101–113 apomixis, 18:51–52 selection, 9:101–113 Pepino transformation, 16:107 Peppermint, mutation breeding, 6:81–82 Perennial grasses, breeding, 11:251–274 Perennial rye breeding, 13:261–288 Persimmon: breeding, 19:191–225 domestication, 25:299–300 Petunia spp., genetics, 1:1–58 Phaseolin, 1:59–102 Phaseolus vulgaris, see Bean Philodendrum breeding, 23:273 Phytophthora fragariae, 2:195–214 Pigeon pea, in vitro culture, 2:224 Pineapple domestication, 25:305–307 Pistil, reproductive function, 4:9–79 Pisum, see Pea Plant breeders; rights, 25:21–55 Plant breeding: politics, 25:21–55 prediction, 15–40 Plant introduction, 3:361–434; 7:9–11, 21–25 Plant exploration, 7:9–11, 26–28, 67–94 Plantain: breeding, 2:135–155; 14:267–320; 21:1–25 domestication, 25: 298 Plastid genetics, 6:364–376, see also Organelle

374 Plum: domestication, 25:293–294 transformation, 16:103–140 Poaceae: molecular mapping, 14:23–24 Saccharum complex, 16:269–288 Pollen: reproductive function, 4:9–79 storage, 13:179–207 Polyploidy, see also Haploidy alfalfa, 10:171–184 alfalfa tissue culture, 4:125–128 apple rootstocks, 1:375–376 banana, 2:147–148 barley, 5:126–127 blueberry, 13:1–10 gametes, 3:253–288 isozymes, 6:33–34 petunia, 1:18–19 potato, 16:15–86; 25:1–19 reproductive barriers, 11:98–105 sweet potato, 4:371 terminology, 26:105–124 triticale, 5:11–40 Pomegranate domestication, 25:285–286 Population genetics, see Quantitative Genetics Potato: breeding, 9:217–332, 19:69–165 cytoplasm, 23:187–189 disease resistance breeding, 19:69–165 gametoclonal variation, 5:376–377 heat tolerance, 10:152 honeycomb breeding, 18:227–230 mutation breeding, 6:79–80 photoperiodic response, 3:75–76, 89–92 ploidy manipulation, 16:15–86 unreduced gametes, 3:274–277 Protein: antifungal, 14:39–88 bean, 1:59–102 induced mutants, 2:38–46 maize, 1:103–138, 148–149; 9:181–216 Protoplast fusion, 3:193–218; 20: 167–225 citrus, 8:339–374 mushroom, 8:206–208 Prunus: amygdalus, see Almond avium, see Sweet cherry Pseudograin breeding, amaranth, 19:227–285 Psophocarpus, in vitro culture, 2:237–238

CUMULATIVE SUBJECT INDEX Q Quantitative genetics: epistasis, 21:27–92 forest trees, 8:139–188 gene interaction, 24(1):269–290 genotype x environment interaction, 16:135–178 heritability, 22:9–111 maize RFLP changes with selection, 24(1):111–131 mutation variation, 24(1): 227–247 overdominance, 17:225–257 population size & selection, 24(1):249–268 selection limits, 24(1):177–225 statistics, 17:296–300 trait loci (QTL), 15:85–139; 19:31–68 variance, 22:113–163 Quantitative trait loci (QTL), 15:85–138; 19:31–68 animal selection, 24(2):169–210, 211–224 selection limits: 24(1):177–225 Quarantines, 3:361–434; 7:12, 35

R Rabbiteye blueberry, 5:307–357 Raspberry, breeding, 6:245–321 Recurrent restricted phenotypic selection, 9:101–113 Recurrent selection, 9:101–113, 115–179; 14:139–163 soybean, 15:275–313 Red stele disease, 2:195–214 Rédei, George P. (bibliography), 26:1–33 Regional trial testing, 12:271–297 Reproduction: barriers and circumvention, 11:11–154 foliage plants, 23:255–259 garlic, 23:211–244 Rhizobia, 23:21–72 Rhododendron, mutation breeding, 6:75–76 Rice, see also Wild rice: anther culture, 15:141–186 apomixis, 18:65 cytoplasm, 23:189 doubled haploid breeding, 15:141–186 gametoclonal variation, 5:362–364 heat tolerance, 10:151–152 honeycomb breeding, 18:224–226

CUMULATIVE SUBJECT INDEX hybrid breeding, 17:1–15, 15–156; 23:73–174 long-term selection 24(2): 64–67 molecular markers, 73–174 photoperiodic response, 3:74, 89–92 Rosa, see Rose Rose breeding, 17:159–189 Rubus, see Blackberry, Raspberry Rust, wheat, 13:293–343 Rutabaga, 8:217–248 Ryder, Edward J. (biography), 16:1–14 Rye: gametoclonal variation, 5:370–371 perennial breeding, 13:261–288 triticale, 5:41–93 S Saccharum complex, 16:269–288 Salt resistance: cell selection, 4:141–143 durum wheat, 5:31 yeast systems, 22:389–425 Sears, Ernest R. (biography), 10:1–22 Secale, see Rye Seed: apple rootstocks, 1:373–374 banks, 7:13–14, 37–40, 152–153 bean, 1:59–102 garlic, 23:211–244 lettuce, 1:285–286 maintenance and storage, 7:95–110, 129–158, 159–182 maize, 1:103–138, 139–161, 4:81–86 pearl millet, 1:162–182 protein, 1:59–138, 148–149 rice production, 17:98–111, 118–119, 23:73–174 soybean, 1:183–235, 3:289–311 synthetic, 7:173–174 variegation, 4:81–86 wheat (hybrid), 2:313–317 Selection, see also Breeding bacteria, 24(2): 225–265 bean, 24(2): 69–74 cell, 4:139–145, 153–173 crops of the developing world, 24(2):45–88 divergent selection for maize ear length, 24(2):153–168 domestication, 24(2):1–44 Escherichia coli, 24(2): 225–265 gene interaction, 24(1):269–290

375 genetic models, 24(1):177–225 honeycomb design, 13:87–139; 18:177–249 limits, 24(1):177–225 maize high oil, 24(1):153–175 maize history, 24(1):11–40, 41–59, 61–78 maize long term, 24(1):79–110, 111–131, 133–151; 24(2):53–64, 109–151 maize oil & protein, 24(1):79–110, 153–175 maize physiological changes, 24(1):133–151 maize RFLP changes, 24(1):111–131 marker assisted, 9:37–61, 10:184–190; 12:195–226; 13:11–86; 14:13–37; 17:113–114, 179, 212–215; 18:20–42; 19:31–68, 21:181–220, 23:73–174, 24(1):293–309; 26:292–299 mutation variation, 24(1):227–268 population size, 24(1):249–268 prediction, 19: 15–40 productivity gains in US crops, 24(2):89–106 quantitative trait loci, 24(1):311–335 recurrent restricted phenotypic, 9:101–113 recurrent selection in maize, 9:115–179; 14:139–163 rice, 24(2): 64–67 wheat, 24(2): 67–69 Sesame breeding, 16:179–228 Sesamum indicum, see Sesame Simmonds, N.W. (biography), 21:1–13 Snap pea breeding, 21:93–138 Solanaceae: incompatibility, 15:27–34 molecular mapping, 14:27–28 Solanum tuberosum, see Potato Somaclonal variation, see also Gametoclonal variation alfalfa, 4:123–152 isozymes, 6:30–31 maize, 5:147–149 molecular analysis, 16:229–268 mutation breeding, 6:68–70 rose, 17:178–179 transformation interaction, 16:229–268 utilization, 16:229–268 Somatic embryogenesis, 5:205–212; 7:173–174 oil palm, 4:189–190

376 Somatic genetics, see also Gametoclonal variation; Somaclonal variation: alfalfa, 4:123–152 legumes, 2:246–248 maize, 5:147–149 organelle transfer, 2:283–302 pearl millet, 1:166 petunia, 1:43–46 protoplast fusion, 3:193–218 wheat, 2:303–319 Somatic hybridization, see also Protoplast fusion 20:167–225 Sorghum: male sterility, 25:139–172 photoperiodic response, 3:69–71, 97–99 transformation, 13:235–264 Southern pea, see Cowpea Soybean: cytogenetics, 16:289–317 disease resistance, 1:183–235 drought resistance, 4:203–243 hybrid breeding, 21:263–307 in vitro culture, 2:225–228 nodulation, 11:275–318 photoperiodic response, 3:73–74 recurrent selection, 15:275–313 semidwarf breeding, 3:289–311 Spelt, agronomy, genetics, breeding, 15:187–213 Sprague, George F. (biography), 2:1–11 Starch, maize, 1:114–118 Statistics: advanced methods, 22:113–163 history, 17:259–316 Sterility, see also Male sterility, 11:30–41 Strawberry: biotechnology, 21: 139–180 domestication, 25:302–303 red stele resistance breeding, 2:195–214 transformation, 16:104 Stress resistance: cell selection, 4:141–143, 161–163 transformation fruit crops, 16:115 Stylosanthes, in vitro culture, 2:238–240 Sugarcane: mutation breeding, 6:82–84 Saccharum complex, 16:269–288 Sweet cherry: Domestication, 25:202–293

CUMULATIVE SUBJECT INDEX pollen-incompatibility and selffertility, 9:367–388 transformation, 16:102 Sweet corn, see also Maize: endosperm, 1:139–161 supersweet (shrunken2), 14:189–236 Sweet potato breeding, 4:313–345; 6:80–81 T Tamarillo transformation, 16:107 Taxonomy: amaranth, 19:233–237 apple, 1:296–299 banana, 2:136–138 blackberry, 8:249–253 cassava, 2:83–89 chestnut, 4:351–352 chrysanthemum, 14:321–361 clover, white, 17:193–211 coffee, 2:161–163 coleus, 3:345–347 fescue, 3:314 garlic, 23:211–244 Glycine, 16:289–317 guayule, 6:112–115 oat, 6:171–173 pearl millet, 1:163–164 petunia, 1:13 plantain, 2:136; 14:271–272 rose, 17:162–169 rutabaga, 8:221–222 Saccharum complex, 16:270–272 sweet potato, 4:320–323 triticale, 8:49–54 Vigna, 8:19–42 white clover, 17:193–211 wild rice, 14:240–241 Testing: adaptation, 12:271–297 honeycomb design, 13:87–139 Tissue culture, see In vitro culture Tobacco, gametoclonal variation, 5:372–376 Tomato: breeding for quality, 4:273–311 heat tolerance, 10:150–151 Toxin resistance, cell selection, 4:163–165 Transformation and transgenesis alfalfa, 10:190–192

CUMULATIVE SUBJECT INDEX barley, 26:155–157 cereals, 13:231–260 fruit crops, 16:87–134 mushroom, 8:206 papaya, 26:35–78 rice, 17:179–180 somaclonal variation, 16:229–268 white clover, 17:193–211 Transpiration efficiency, 12:81–113 Transposable elements, 4:81–122; 5:146–147; 8:91–137 Tree crops, ideotype concept, 12:163–193 Tree fruits, see Fruit, nut, and beverage crop breeding Trifolium, see Clover, White Clover Trifolium hybrids, 5:275–284 in vitro culture, 2:240–244 Trilobium, long-term selection, 24(2):211–224 Tripsacum: apomixis, 18:51 maize ancestry, 20:15–66 Trisomy, petunia, 1:19–20 Triticale, 5:41–93; 8:43–90 Triticosecale, see Triticale Triticum: Aestivum, see Wheat Turgidum, see Durum wheat Tulip, mutation breeding, 6:76 U United States National Plant Germplasm System, see National Plant Germplasm System Unreduced and polyploid gametes, 3:253–288; 16:15–86 Urd bean, 8:32–35 V Vaccinium, see Blueberry Variance estimation, 22:113–163 Vegetable breeding: artichoke, 12:253–269 bean, 1:59–102; 4:245–272, 24(2):69–74 bean (tropics), 10:199–269 beet (table), 22:257–388 carrot 19: 157–190 cassava, 2:73–134; 24(2):74–79 cucumber, 6:323–359

377 cucurbit insect and mite resistance, 10:309–360 lettuce, 1:267–293; 16:1–14; 20:105:133 mushroom, 8:189–215 onion, 20:67–103 pea, 21:93–138 peanut, 22:297–356 potato, 9:217–232; 16:15–86l; 19:69–165 rutabaga, 8:217–248 snap pea, 21: 93–138 tomato, 4:273–311 sweet corn, 1:139–161; 14:189–236 sweet potato, 4:313–345 Vicia, in vitro culture, 2:244–245 Vigna, see Cowpea, Mungbean in vitro culture, 2:245–246; 8:19–42 Virus diseases: apple rootstocks, 1:358–359 clover, white, 17:201–209 coleus, 3:353 cowpea, 15:239–240 indexing, 3:386–408, 410–411, 423–425 in vitro elimination, 2:265–282 lettuce, 1:286 papaya, 26:35–78 potato, 19:122–134 raspberry, 6:247–254 resistance, 12:47–79 soybean, 1:212–217 sweet potato, 4:336 transformation fruit crops, 16:108–110 white clover, 17:201–209 Vogel, Orville A. (biography), 5:1–10 Vuylsteke, Dirk R. (biography), 21:1–25 W Walnut (black), 1:236–266 Walnut transformation, 16:103 Weinberger, John A. (biography), 11:1–10 Wheat: anther culture, 15:141–186 apomixis, 18:64–65 chemical hybridization, 3:169–191 cold hardiness adaptation, 12:124–135 cytogenetics, 10:5–15 cytoplasm, 23:189–190 diversity, 21:236–237 doubled haploid breeding, 15:141–186

378 Wheat (cont.) drought tolerance, 12:135–146 durum, 5:11–40 gametoclonal variation, 5:364–368 gene manipulation, 11:225–234 heat tolerance, 10:152 hybrid, 2:303–319; 3:185–186 insect resistance, 22:221–297 in vitro adaptation, 12:115–162 long-term selection, 24(2):67–69 molecular biology, 11:235–250 molecular markers, 21:191–220 photoperiodic response, 3:74 rust interaction, 13:293–343 triticale, 5:41–93 vernalization, 3:109

CUMULATIVE SUBJECT INDEX White clover, molecular genetics, 17:191–223 Wild rice, breeding, 14:237–265 Winged bean, in vitro culture, 2:237–238 Y Yeast, salt resistance, 22:389–425 Yuan, Longping (biography), 17:1–13. Z Zea mays, see Maize, Sweet corn Zein, 1:103–138 Zizania palustris, see Wild rice

Cumulative Contributor Index (Volumes 1–26) Abdalla, O.S., 8:43 Acquaah, G., 9:63 Aldwinckle, H.S., 1:294 Alexander, D.E., 24(1): 53 Anderson, N.O., 10:93; 11:11 Aronson, A.I., 12:19 Ascher, P.D., 10:93 Ashri, A., 16:179 Baggett, J.R. 21:93 Balaji, J., 26:171 Baltensperger, D.D., 19:227 Barker, T., 25:173 Basnizki, J., 12:253 Beck, D.L., 17:191 Beebe, S., .23:21–72 Beineke, W.F., 1:236 Bell, A.E., 24(2): 211 Below, F.E., 24(1):133 Berzonsky, W.A., 22:221 Bingham, E.T., 4:123; 13:209 Binns, M.R., 12:271 Bird, R. McK., 5:139 Bjarnason, M., 9:181 Blair, M.W., 26 Bliss, F.A., 1:59; 6:1 Boase, M.R., 14:321 Borlaug, N.E., 5:1 Boyer, C.D., 1:139 Bravo, J.E., 3:193 Brenner, D.M., 19:227 Bressan , R.A., 13:235; 14:39; 22:389 Bretting, P.K., 13:11 Broertjes, C., 6:55 Brown, A.H.D., 21:221 Brown, J.W.S., 1:59 Brown, S.K., 9:333,367

Buhariwalla, H.K., 26:171 Bünger, L., 24(2):169 Burnham, C.R., 4:347 Burton, G.W., 1:162; 9:101 Burton, J.W., 21:263 Byrne, D., 2:73 Camadro, E.L., 26:105 Campbell, K.G., 15:187 Campos, H., 25: 173 Cantrell, R.G., 5:11 Carputo, D., 25:1; 26:105 Carvalho, A., 2:157 Casas, A.M., 13:235 Cervantes-Martinez, C.T., 22:9 Chen, J., 23:245 Chew, P.S., 22:165 Choo, T.M., 3:219; 26:125 Christenson, G.M., 7:67 Christie, B.R., 9:9 Clark, R.L., 7:95 Clarke, A.E., 15:19 Clegg, M.T., 12:1 Condon, A.G., 12:81 Cooper, M, 24(2):109; 25:173 Cooper, R.L., 3:289 Cornu, A., 1:11 Costa, W.M., 2:157 Cregan, P., 12:195 Crouch, J.H., 14:267; 26:171 Crow, J.F., 17:225 Cummins, J.N., 1:294 Dana, S., 8:19 De Jong, H., 9:217 Dekkers, J.C.M., 24(1):311 Deroles, S.C., 14:321

Plant Breeding Reviews, Volume 26, Edited by Jules Janick ISBN 0-471-73215-X © 2006 John Wiley & Sons, Inc. 379

380 Dhillon, B.S., 14:139 Dickmann, D.I., 12:163 Ding, H., 22:221 Dodds, P.N., 15:19 Dolan, D., 25:175 Donini, P., 21:181 Draper, A.D., 2:195 Drew, R., 26:35 Dudley, J.W. 24(1):79 Dumas, C., 4:9 Duncan, D.R., 4:153 Duvick, D.N., 24(2):109 Dwivedi, S.L., 26:171 Echt, C.S., 10:169 Edmeades, G., 25:173 Ehlers, J.D., 15:215 England, F., 20:1 Eubanks, M.W., 20:15 Evans, D.A., 3:193; 5:359 Everett, L.A., 14:237 Ewart, L.C., 9:63 Farquhar, G.D., 12:81 Fasoula, D.A., 14:89; 15:315; 18:177 Fasoula, V.A., 13:87; 14:89; 15:315; 18:177 Fasoulas, A.C., 13:87 Fazuoli, L.C., 2:157 Fear, C.D., 11:1 Ferris, R.S.B., 14:267 Flore, J.A., 12:163 Forsberg, R.A., 6:167 Forster, B.P., 25:57 Forster, R.L.S., 17:191 Fowler, C., 25:21 Frusciante, L., 35:1 Frei, U., 23:175 French, D.W., 4:347 Gai, J., 21:263 Galiba, G., 12:115 Galletta, G.J., 2:195 Gepts, P., 24(2):1 Gmitter, F.G., Jr., 8:339; 13:345 Gold, M.A., 12:163 Goldman, I.L. 19:15; 20:67; 22:357; 24(1):61; 24(2)89 Gonsalves, D., 26:35 Goodnight, C.J, 24(1):269 Gradziel, T.M., 15:43 Gressel, J., 11:155; 18:251

CUMULATIVE CONTRIBUTOR INDEX Gresshof, P.M., 11:275 Griesbach, R.J., 25:89 Grombacher, A.W., 14:237 Grosser, J.W., 8:339 Grumet, R., 12:47 Gudin, S., 17:159 Guimarães, C.T., 16:269 Gustafson, J.P., 5:41; 11:225 Guthrie, W.D., 6:209 Habben, J., 25:173 Haley, S.D., 22:221 Hall, A.E., 10:129; 12:81; 15:215 Hall, H.K., 8:249 Hallauer, A.R., 9:115; 14:1,165; 24(2):153 Hamblin, J., 4:245 Hancock, J.F., 13:1 Hancock, J.R., 9:1 Hanna, W.W., 13:179 Harlan, J.R., 3:1 Harris, M.O., 22: 221 Hasegawa, P.M. 13:235; 14:39: 22:389 Havey, M.J., 20:67 Henny, R.J., 23:245 Hill, W.G., 24(2):169 Hillel, J., 12:195 Hodgkin, T., 21:221 Hokanson, S.C., 21:139 Holbrook, C.C., 22: 297 Holland, J.B: 21: 27; 22:9 Hor, T.Y., 22:165 Hunt, L.A., 16:135 Hutchinson, J.R., 5:181 Hymowitz, T., 8:1; 16:289 Janick, J., 1:xi; 23:1, 25:255 Jansky, S., 19:77 Jayaram, Ch., 8:91 Jenderek, M.M., 23:211 Johnson, A.A.T., 16:229; 20:167 Johnson, R., 24(1):293 Jones, A., 4:313 Jones, J.S., 13:209 Ju, G.C., 10:53 Kang, H., 8:139 Kann, R.P., 4:175 Karmakar, P.G., 8:19 Kartha, K.K., 2:215, 265 Kasha, K.J., 3:219 Keep, E., 6:245 Kleinhofs, A., 2:13

CUMULATIVE CONTRIBUTOR INDEX

381

Keightley, P.D., 24(1):227 Knox, R.B., 4:9 Koebner, R.M.D., 21:181 Kollipara, K.P., 16:289 Koncz, C., 26:1 Kononowicz, A.K., 13:235 Konzak, C.F., 2:13 Krikorian, A.D., 4:175 Krishnamani, M.R.S., 4:203 Kronstad, W.E., 5:1 Kulakow, P.A., 19:227

Moose, S.P., 24(1):133 Morrison, R.A., 5:359 Mowder, J.D., 7:57 Mroginski, L.A., 2:215 Muir, W.M., 24(2):211 Mumm, R.H., 24(1):1 Murphy, A.M., 9:217 Mutschler, M.A., 4:1 Myers, J.R., 21:93 Myers, O., Jr., 4:203 Myers, R.L., 19:227.

Lamb, R.J., 22:221 Lambert, R.J., 22: 1; 24(1):79, 153 Lamborn, C., 21:93 Lamkey, K.R., 15:1; 24(1):xi; 24(2):xi Lavi, U., 12:195 Layne, R.E.C., 10:271 Lebowitz, R.J., 3:343 Lee, M., 24(2):153 Lehmann, J.W., 19:227 Lenski, R.E., 24(2):225 Levings, III, C.S., 10:23 Lewers, K.R., 15:275 Li, J., 17:1,15 Liedl, B.E., 11:11 Lin, C.S., 12:271 Lovell, G.R., 7:5 Lower, R.L., 25:21 Lukaszewski, A.J., 5:41 Lyrene, P.M., 5:307

Namkoong, G., 8:139 Navazio, J., 22:357 Neuffer, M.G., 5:139 Newbigin, E., 15:19 Nyquist, W.E., 22:9

Maas, J. L., 21: 139 Maheswaran, G., 5:181 Mackenzie, S.A., 25:115 Maizonnier, D., 1:11 Marcotrigiano, M., 15:43 Martin, F.W., 4:313 Matsumoto, T.K. 22:389 McCoy, T.J., 4:123; 10:169 McCreight, J.D., 1:267; 16:1 McDaniel, R.G., 2:283 McKeand, S.E., 19:41 McKenzie, R.I.H., 22: 221 McRae, D.H., 3:169 Medina-Filho, H.P., 2:157 Mejaya, I.J., 24(1): 53 Mikkilineni, V., 24(1):111 Miles, D., 24(2):211 Miles, J.W., 24(2):45 Miller, R., 14:321 Mondragon Jacobo, C., 20:135

Ohm, H.W., 22:221 O’Malley, D.M., 19:41 Ortiz, R., 14:267; 16:15; 21:1; 25:1, 139; 26:171 Palmer, R.G., 15:275, 21:263 Pandy, S., 14:139; 24(2):45 Pardo, J. M., 22:389 Parliman, B.J., 3:361 Paterson, A.H., 14:13 Patterson, F.L., 22:221 Peairs, F.B., 22:221 Pedersen, J.F., 11:251 Peiretti, E.G., 23:175 Peloquin, S.J., 26:105 Perdue, R.E., Jr., 7:67 Peterson, P.A., 4:81; 8:91 Polidorus, A.N., 18:87 Porter, D.A., 22:221 Porter, R.A., 14:237 Powell, W., 21:181 Prasartsee, V., 26:35 Proudfoot, K.G., 8:217 Rackow, G., 18:1 Raina, S.K., 15:141 Ramage, R.T., 5:95 Ramesh, S., 25:139 Ramming, D.W., 11:1 Ratcliffe, R.H., 22:221 Ray, D.T., 6:93 Reddy, B.V.S., 25:139 Redei, G.P., 10:1; 24(1):11 Reimann-Phillipp, R., 13:265

382 Reinbergs, E., 3:219 Rhodes, D., 10:53 Richards, R.A., 12:81 Roath, W.W., 7:183 Robinson, R.W., 1:267; 10:309 Rochefored.T.R., 24(1):111 Ron Parra, J., 14:165 Roos, E.E., 7:129 Ross, A.J., 24(2):153 Rotteveel, T., 18:251 Rowe, P., 2:135 Russell, W.A., 2:1 Rutter, P.A., 4:347 Ryder, E.J., 1:267; 20:105 Samaras, Y., 10:53 Sansavini, S., 16:87 Saunders, J.W., 9:63 Savidan, Y., 18:13 Sawhney, R.N., 13:293 Schaap, T., 12:195 Schaber, M.A. 24(2):89 Schussler, J., 25:173 Schneerman, M.C. 24(1):133 Schroeck, G., 20:67 Scott, D.H., 2:195 Seabrook, J.E.A., 9:217 Sears, E.R., 11:225 Seebauer, J.R., 24(1):133 Serraj, R., 26:171 Shands, Hazel L., 6:167 Shands, Henry L., 7:1, 5 Shannon, J.C., 1:139 Shanower, T.G., 22:221 Shattuck, V.I., 8:217; 9:9 Shaun, R., 14:267 Sidhu, G.S., 5:393 Simmonds, N.W., 17:259 Simon, P.W., 19:157; 23:211 Singh, B.B., 15:215 Singh, R.J., 16:289 Singh, S.P., 10:199 Singh, Z., 16:87 Slabbert, M.M., 19:227 Sleper, D.A., 3:313 Sleugh, B.B., 19:227 Smith, J.S.C., 24(2):109 Smith, S.E., 6:361 Snoeck, C., 23:21 Sobral, B.W.S., 16:269 Socias i Company, R., 8:313 Soh, A.C., 22:165

CUMULATIVE CONTRIBUTOR INDEX Sondahl, M.R., 2:157 Spoor, W., 20: 1 Stalker, H.T., 22:297 Steadman, J.R., 23:1 Steffensen, D. M., 19:1 Stevens, M.A., 4:273 Stoner, A.K., 7:57 Stuber, C.W., 9:37; 12:227 Sugiura, A., 19:191 Sun, H. 21:263 Suzaki, J.Y., 26 :35 Tai, G.C.C., 9:217 Talbert, L.E., 11:235 Tan, C.C., 22:165 Tarn, T.R., 9:217 Tehrani, G., 9:367 Teshome, A., 21:221 Thomas, W.T.B., 25:57 Thompson, A.E., 6:93 Tiefenthaler, A.E. 24(2):89 Towill, L.E., 7:159, 13:179 Tracy, W.F., 14:189; 24(2):89 Tripathi, S., 26:35 Troyer, A.F., 24(1):41 Tsaftaris, A.S., 18:87 Tsai, C.Y., 1:103 Ullrich, S.E., 2:13 Upadhyaya, H.D., 26:171 Uribelarrea, M., 24(1):133 Vanderleyden, J., 23:21 Van Harten, A.M., 6:55 Varughese, G., 8:43 Vasal, S.K., 9:181; 14:139 Vegas, A., 26:35 Veilleux, R., 3:253; 16:229; 20:167 Villareal, R.L., 8:43 Vogel, K.P., 11:251 Volk, G.M., 23:291 Vuylsteke, D., 14:267 Wallace, D.H., 3:21; 13:141 Walsh, B. 24(1):177 Wan, Y., 11:199 Waters, C., 23:291 Weber, K., 24(1):249 Weeden, N.F., 6:11 Wehner, T.C., 6:323 Welander, M., 26:79 Wenzel, G. 23:175

CUMULATIVE CONTRIBUTOR INDEX Westwood, M.N., 7:111 Whitaker, T.W., 1:1 White, D.W.R., 17:191 White, G.A., 3:361; 7:5 Widholm, J.M., 4:153, 11:199 Widmer, R.E., 10:93 Widrlechner, M.P., 13:11 Wilcox, J.R., 1:183 Williams, E.G., 4:9; 5:181, 237 Williams, M.E., 10:23 Wilson, J.A., 2:303 Wong, G., 22: 165 Woodfield, D.R., 17:191 Wright, D., 25:173 Wright, G.C., 12:81 Wu, L., 8:189 Wu, R., 19:41

383 Xin, Y., 17:1,15 Xu, S., 22:113 Xu, Y., 15:85; 23:73 Yamada, M., 19:191 Yan, W., 13:141 Yang, W.-J., 10:53 Yonemori, K., 19:191 Yopp, J.H., 4:203 Yun, D.-J., 14:39 Zeng, Z.-B., 19:41 Zhu, L.-H., 26:79 Zimmerman, M.J.O., 4:245 Zinselmeier, C., 25:173 Zohary, D., 12:253

1991 Plate 2.1. Hawaiian solo papaya ‘Kapoho’. Photo from D. Gonsalves.

Plate 2.2. Transgenic papaya line 55-1 (left) showing resistance to PRSV while nontransgenic papaya is infected and stunted. Photo from D. Gonsalves.

Plate 2.3. Transgenic papaya ‘SunUp’ is red fleshed and homozygous for CP gene. Photo from University of Hawaii.

Plate 2.4. Transgenic papaya ‘Rainbow’ is yellow fleshed and hemizygous for the CP gene. It is a F1 hybrid between ‘SunUp’ and nontransgenic ‘Kapoho’. Photo from University of Hawaii.

Puna: 1992 Plate 2.5. Healthy field of papaya growing in Kapoho of Puna district in 1992. Photo from D. Gonsalves.

Puna: 1994 Plate 2.6. Papaya field completely infected with PRSV in Kapoho of Puna district in 1994. Photo from Steve Ferreira.

May 1997, 19 months after planting

1999: Commercial Planting

Plate 2.7. Field trial showing resistance of transgenic ‘Rainbow’ surrounded by severely infected nontransgenic ‘Sunrise’ papaya. Field trial started on October 1995, with photo taken in May 1997. Photo from Steve Ferreira.

Plate 2.8. Field of transgenic ‘Rainbow’ growing in Kapoho under severe disease pressure. Photo from D. Gonsalves.

Plate 2.9. Transgenic ‘Rainbow’ on supermarket shelves in Hawaii. Photo by Carol Gonsalves.

Plate 2.10. T1 fruit from transgenic ‘Khaknuan’ that is resistant to PRSV in Thailand. Photo from V. Prasartsee.

Plate 2.11. Field Trial of T3 transgenic ‘Khaknuan’ showing resistance to PRSV in Thailand. Infected row of papaya on left. Photo from V. Prasartsee.

Plate 2.12. ‘Thapra 2’ papaya that was selected for tolerance to PRSV in Thailand. It is commercially used in Thailand. Photo from V. Prasartsee.