Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oi...

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines.. High [r]

PLANT BREEDING REVIEWS

Volume 31

American Society of Horticultural Science International Society for Horticultural Science

Editorial Board, Volume 31

PLANT BREEDING REVIEWS

Volume 31

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Library of Congress Cataloging-in-Publication Data:

ISBN 978-0-470-38762-7 (cloth) ISSN: 0730-2207

Contents

Contributors ix

1 Dedication: Anthony H D Brown

Conservation Geneticist 1

Reid G Palmer and Jeff J Doyle

I Biographical Sketch

II Research Accomplishments

III The Man 10

IV Honors and Awards 12

Selected Publications of Anthony H D Brown 12

2 Brassica and Its Close Allies: Cytogenetics

and Evolution 21

Shyam Prakash, S R Bhat, C F Quiros, P B Kirti, and V L Chopra

I Introduction 24

II Cytogenetics 26

III Genome Manipulation 56

IV Wide Hybridization 71

V Cytoplasmic Substitution and Male Sterility 95 VI Genome Dissection and Development

of Chromosome Addition Lines 104

VII Mitochondrial Genome 110

VIII Plastid Genome 113

IX Potential Role of Arabidopsis thaliana

in Brassica Improvement 114

X Chloroplast Genomes and their Phylogenetic

Implications 123

XI Evolution of Morphological Characters 137

XII Concluding Remarks 142

Literature Cited 146

3 Genetic Enhancement for Drought

Tolerance in Sorghum 189

Belum V S Reddy, S Ramesh, P Sanjana Reddy, and A Ashok Kumar

I Introduction 189

II Breeding for Drought Tolerance 190 III Selection among Cultivars and Landraces 194

IV Breeding for Drought Escape 197

V Growth Stage–Specific Screening Techniques 199 VI Physiological Response Traits for Drought Tolerance 207 VII Marker-Assisted Breeding for Drought Tolerance 210

VIII Outlook 212

Literature Cited 214

4 Breeding for Resistance to Stenocarpella

Ear Rot in Maize 223

Johannes D Rossouw, Z A Pretorius, H D Silva, and K R Lamkey

I Introduction 224

II Distribution and Importance 225

III Pathogen 229

IV Epidemiology 232

V Disease Management 233

VI Summary and Conclusion 240

Literature Cited 241

5 Cassava Genetic Resources: Manipulation

for Crop Improvement 247

Nagib M A Nassar and Rodomiro Ortiz

I Introduction 248

II Wild ManihotSpecies: A Botanical Review 252

III Interspecific Hybrids 253

IV Cassava Diversity as Revealed by DNA Markers

and Genetics 257

V Trait Transfer 262

VI Outlook 267

6 Breeding Roses for Disease Resistance 277

Vance M Whitaker and Stan C Hokanson

I Introduction 277

II Causal Pathogens 279

III Resistance Screening 288

IV Breeding 298

V Molecular Tools 305

VI Future Prospects 313

Literature Cited 316

7 Plant Breeding for Human Nutritional Quality 325

Philipp W Simon, Linda M Pollak, Beverly A Clevidence, Joannne M Holden, and David B Haytowitz

I Introduction 327

II Sources of Nutrients 328

III Progress in Breeding for Nutrient Content

and Composition 350

IV Plant Breeding Strategies for Increasing Intake

of Shortfall Nutrients 374

Literature Cited 377

Subject Index 393

Cumulative Subject Index 395

Contributors

S R Bhat National Research Centre on Plant Biotechnology, Indian Agricul-tural Research Institute, New Delhi 110012 India

V L Chopra National Research Centre on Plant Biotechnology, Indian Agri-cultural Research Institute, New Delhi 110012 India

Beverly A Clevidence Food Components and Health Laboratory, United States Department of Agriculture—Agricultural Research Service, Beltsville Agri-cultural Research Center, Beltsville, Maryland 20705 USA

Jeff J Doyle L H Bailey Hortorium, Department of Plant Biology, Cornell University, Ithaca, New York 14853 USA

David B Haytowitz Nutrient Data Laboratory, United States Department of Agriculture—Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 USA

Stan C Hokanson University of Minnesota, Department of Horticultural Science, 1970 Folwell Avenue, St Paul, MN 55108 USA

Joannne M Holden Nutrient Data Laboratory, United States Department of Agriculture—Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 USA

P B Kirti Plant Science Department, University of Hyderabad, Hyderabad, 500046 India

A Ashok Kumar International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India

K R Lamkey 2101 Agronomy Hall, Iowa State University, Ames, Iowa, USA Nagib M A Nassar Departamento de Genetica e Morfologia, Universidade de

Brasilia, 70919 Brasilia, Brazil

Rodomiro Ortiz Centro Internacional de Mejoramiento de Maiz y Trigo (CIM-MYT), El Batan, Texcoco, Apdo Postal 6-641, 06600 Mexico, D.F Mexico Reid G Palmer United States Department of Agriculture, Agricultural

Research Service, Corn Insects and Crop Genetics Research Unit, Department of Agronomy, Iowa State University, Ames, Iowa 50011 USA

Linda M Pollak Corn Insects and Crop Genetics Research Unit, United States Department of Agriculture, Agricultural Research Service, Department of Agronomy, Iowa State University, Ames, Iowa 50011 USA

Z A Pretorius Department of Plant Sciences, University of the Free State, Bloemfontein, 9300 South Africa

Shyam Prakash National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012 India

C F Quiros Department of Vegetable Crops, University of California, Davis, California 95616 USA

S Ramesh International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India

Belum V S Reddy International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India

P Sanjana Reddy International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India

Johannes D Rossouw Monsanto Singapore Co (PTE) Ltd., 151 Lorong Chuan 06-08 New Tech Park, Singapore

H D Silva Monsanto Brazil, Rodovia Uberlaˆndia-Araxa´, Uberlandia, MG, Brazil

Philipp W Simon Vegetable Crops Research Unit, United States Department of Agriculture, Agricultural Research Service, Department of Horticulture, Uni-versity of Wisconsin, Madison, Wisconsin 53706 USA

1

Dedication: Anthony H D Brown Conservation Geneticist

Reid G Palmer

United States Department of Agriculture Agricultural Research Service

Corn Insects and Crop Genetics Research Unit Department of Agronomy

Iowa State University Ames, Iowa 50011 USA Jeff J Doyle

L H Bailey Hortorium Department of Plant Biology Cornell University

Ithaca, New York 14853 USA

I BIOGRAPHICAL SKETCH II RESEARCH ACCOMPLISHMENTS

A Conservation Genetics

B Plant Mating Systems and Population Structure III THE MAN

IV HONORS AND AWARDS ACKNOWLEDGMENT

SELECTED PUBLICATIONS OF ANTHONY H D BROWN

This volume of Plant Breeding Reviews is dedicated to Anthony (Tony) H D Brown, known internationally for his research in conservation and population genetics and plant breeding Dr Brown’s primary contributions in the area of conservation genetics followed two major themes: optimum sampling strategies and core collections His life’s

Plant Breeding Reviews, Volume 31 Edited by Jules Janick Copyright& 2009 John Wiley & Sons, Inc

activities in this area were inspired primarily by his long friendship and close working relationship with Sir Otto Frankel His research in population genetics focused on the estimation of mating systems and their impact on plant population structure while his research in breeding was on the use of wild relatives in crop improvement Dr Brown started with the Commonwealth Scientific and Industrial Research Organization (CSIRO) in 1972 as a research scientist and retired as a chief research scientist in 2006 He is now an Honorary Research Fellow in the Centre for Plant Biodiversity Research, CSIRO Plant Industry, Canberra, Australia

I BIOGRAPHICAL SKETCH

Tony Brown was born on November 25, 1941, in Waverley, Sydney NSW, Australia It was wartime and his father, Arthur Brown, was in Darwin, Australia, serving as Squadron Leader in the Royal Australian Air Force Arthur was from three or more generations of Australian stock Tony’s maternal grandfather, Hugh Milligan, son of Scottish immigrants, was an eminent primary school headmaster Hugh’s task was to register Tony’s birth, the agreed name being Anthony Hugh Dean Brown Hugh urged that the last two names be hyphenated because plain ‘‘Brown’’ was insufficiently distinguished for a future Macquarie Street specialist doctor However, Tony’s mother Joyce intervened and said, ‘‘Plain Brown is good enough for me, it should be for my son.’’ This ensured that name hyphenation could await future needs Yet Hugh had other major influences on Tony, inspiring a love of plants, of arithmetic shortcuts, and of parsing sentences That three initials were an encumbrance emerged later in the United States, where names and official forms were triplet coded, allowing only one middle initial And the inevitable inversion happened after publications on alcohol dehy-drogenase, when the AHD became ADH, which spawned a growing list of mutant miscitations in the Science Citation Index

student He shaped the honors course, and infused enthusiasm as the DNA era was unfolding Jim had spent some time in Adelaide University, and brought back insights from meeting Sir R A Fisher and the team of population genetics students there Jim devised an unforgettable experiment for the honors genetics class with 16 blue and 16 yellow plastic beads in a jar to simulate genetic drift theory The jar was shaken and inverted 16 times and the color of the first two beads noted After 16 repeats, the jar was opened and its contents adjusted to the new observed gene frequency Over 50 population replications were run over tens of generations, or until fixation blissfully occurred Why was fixation happening faster than predicted? Sampling with or without replacement? Late into the night the rattle of balls in the jar resounded down the college corridors, until crash .extinction: The neck of the jar wore through and broke However, the experiment had sown the seed of a lifelong interest in sampling issues

On graduating (in 1963), Tony was assigned by the Colonial Sugar Refining Company to its sugarcane experiment station in Lautoka, Fiji This was a major transition, from collegiate to colonial life, and he was fully briefed at the head office in Sydney on how to behave toward the local population The company itself was in transition, hiring local staff as officers, and the country was preparing for independence Tony’s immediate boss was Joe Daniels, a sugarcane breeder respected around the world and a scholarly and imaginative leader All communications were directed through the mill manager, including scientific reports Tony had an early lesson in communication when management enrolled him in an in-house training course on report writing The fact that management chose Tony’s report on fiber content to be one anonymous example of bad writing firmly made a point It was an object lesson in the ‘‘Gunning fog index,’’ which is a function of the average length of sentences and the number of words with more than two syllables The index is intended to equate to the number of years of education that a reader requires to understand the writing Clearly no one in the head office had sufficient schooling to read Tony’s report

to track their movement in cane fields in retrospect seem incongruous in a small island colony

After nearly four years in Fiji, Tony returned in 1966 to academia and graduate school He chose to work with Dr Robert W Allard at the University of California, Davis, primarily because of his classic plant breeding book and his research on quantitative genetics On arrival, however, Tony found that Professor Allard was convinced that the new isozyme technique would open the door to empirical population genetics in plants Professor Allard recommended that the PhD project should not be on quantitative genetics but on isozyme variation in Zea mays This would fit better with his assigning Tony as half-time research assistant to implement an isozyme lab In so many ways, this was a opportune moment to arrive in Davis and share the excitement and friendship of the Allard lab (particularly Drs M T Clegg, S K Jain, D R Marshall, and B S Weir) The scientific collaborations begun at Davis continued in projects for several decades and led later to sabbatical visits at Stanford University and the University of California, Riverside UC Davis was thus a watershed in Tony’s science and life including marriage

With the completion of his PhD in 1969, Tony was appointed as a lecturer in Biology at the University of York, England A seminar by Professor Warren Ewens in Leeds on the sampling theory of neutral genes had a lasting influence on Tony’s research After three years, Tony returned to Australia to CSIRO Plant Industry as a research scientist in Canberra in 1972 There, two sons, Laurence and Christopher, were born At CSIRO, Tony collaborated with Dr Don Marshall, who had preceded him from UC Davis, and with Dr Bruce Weir then at Massey, New Zealand One early project was on the charge-state model of electrophoretic variation, from which a number of experiences flowed One experience was to have their joint work scooped by Drs T Ohta and M Kimura On another occasion, a manuscript by Tony and Don was being subjected to the internal CSIRO editorial process and was sent by the panel for review to Professor P A P Moran at Australian National University Professor Moran was intrigued by the problem and concerned about some aspects of the existence and convergence properties of the distribution He not only submitted his review, but more important also phoned Tony after hours to discuss this paper This led Professor Moran to write a series of theoretical papers, and this thinking was referred to by Dr J F C Kingman in the history of coalescent that he wrote for Genetics in 2000

to think critically on this subject Later he promoted their strategy in international meetings and used it to challenge conventional collecting practice, particularly when he thought sampling was excessive In the field, such theoretical strategies are but a guide, requiring adjustment to reality This is particularly so for sampling the diversity of wild relatives, where one is deliberately seeking populations in diverse habitats and of greatly varying size

Along with the excitement of discovering variation new to science in its native setting came the experience of diverse human situations Tony’s first real germplasm collecting mission was an object lesson in adjusting theory to field reality This was a frenetic mission to Iran with Israeli professors Dani Zohary and Eibi Nevo The trip went from Mehran near the border with Iraq, across the Zagros Mountains and the southern Caspian shores to Gonbad-e-Qabus, just two years before the 1979 revolution With portraits of the shah’s family in every hotel room, the future course of events was not evident At the hotel in Andimesk, the grim faces of the hotel staff were unforgettable as they examined the scientists’ passports Although the target of the trip was wild cereals, particularly wild barley, the diversity being grown by farmers in the many barley fields was inspiring This led to a sampling deliberately aimed at testing the allozyme diversity and genetic structure of these landraces, particularly to see whether the richness of diversity so apparent to the eye was just a mixture of a few genotypes That research ultimately led to Tony’s principal commitment as Honorary Research Fellow with the International Plant Genetic Resources Institute in their project on the significance of crop genetic diversity still present on farms in traditional agroecosystems (with Drs Toby Hodgkin and Devra Jarvis) The research focus was to develop a scientific basis of the use and conservation in situ of this diversity

that the reserve concept had dulled his original challenge to gene-bank managers to prune their holdings

II RESEARCH ACCOMPLISHMENTS A Conservation Genetics

Sir Otto Frankel was a member of the FAO Expert Panel on Plant Exploration and Introduction, and was preparing for the 1973 FAO/IBP Technical Conference on Crop Plant Genetic Resources in Rome He felt that previous papers on plant collecting were strongly biased toward the practical details of collecting expeditions and that little emphasis had been given to the science of plant exploration The original paper presented at the technical conference by Drs Don Marshall and Tony Brown entitled ‘‘Optimum Sampling Strategies in Genetic Conserva-tion’’ (subsequently published in the book Crop Genetic Resources for Today and Tomorrow edited by O H Frankel and J G Hawkes) was controversial but has since been widely accepted and expanded to cover other issues, such as sampling in biological control programs

Tony followed his early work on sampling strategies by extensive work on developing the concept of core collections This concept, first introduced in 1984, was to facilitate the use of genetic resources in the major crops By the mid-1980s it was felt that many collections, especially in the major crops, had grown so large that their mere size was likely to deter their extensive use by individual scientists, breeders, or students, except for a few characters that could be readily and rapidly discerned on single plants It was proposed that giving priority in evaluation to a smaller number of accessions would faci-litate greater use of germplasm collections, particularly for a range of characters In a series of papers over the last 20 years, Tony has pro-vided much of the underlying scientific rationale for the establishment and use of core collections When first introduced, the core collection concept, because it challenged accepted dogma, was controversial, but it now has become widely applied in practice

B Plant Mating Systems and Population Structure

One of his earliest papers with Professor Allard, which was based on his PhD research, reported the use of isozyme polymorphisms to esti-mate mating system parameters in open-pollinated maize populations Over his career Tony developed procedures not only for the estimation of mating system parameters in both predominantly inbreeding and outbreeding populations but for also for apomictic species Tony also worked with a wide range of colleagues in applying these techniques in species as diverse as Eucalyptus, Lupinus, wild Hordeum, and a number of colonizing weed species (with Drs Jeremy Burdon and Spencer Barrett)

Tony’s work on population structure was focused on genetic polymorphism, heterozygosity, multilocus associations, and population differentiation A theoretical project with Dr Marc Feldman, which is enjoying renewed attention with the burgeoning DNA sequence data, dealt with the measuring and testing of multilocus associations Another example is the analysis of published isozyme data undertaken with Dr Dan Schoen, which showed that not only inbreeding and outbreeding species differ in overall levels of genetic diversity, but they also differ in the amount of among population variance of gene diversity Inbreeding species exhibited much greater variation in how their populations are structured than the populations of individual outbreeding species We have to be clear that the comparison is the variability between the populations of one species; that is, populations and of species A, not population of species A with population of species B

This work led to the release of a commercial cultivar (Tantangara) carrying a known scald resistance gene from wild barley An allied project, also conducted with Dr Dave Garvin, was the use of molecular markers in breeding adapted proanthocyanidin-free barley

Glycine Research The legume genus Glycine includes G max (soybean) and its wild progenitor, G soja These annual species are native to northern Asia and so would seem to have little or nothing to with Australia Yet, surprisingly, their closest relatives, and the only other members of the genus Glycine, are native to Australia This group of wild perennial species, Glycine subgenus Glycine, represents the tertiary gene pool for the soybean and is thus of potential economic importance Collecting and characterizing these perennials has been a major focus of Tony’s work

The potential of this uniquely Australian resource was recognized at CSIRO by Dr Don Marshall, working initially with Paul Broue` and Jim Grace Subsequent staff changes led to Tony taking over the program in 1982 At that time there were fewer than 10 species recognized in subgenus Glycine, but that has changed dramatically In 1982, the International Board of Plant Genetic Resources (IBPGR) held a workshop on soybean genetic resources at Urbana, Illinois, where Tony met those who were already, or would become, among the key figures in soybean diversity research, including Drs Theodore Hymowitz, Reid Palmer, Randy Nelson, Christine Newell, and Duncan Vaughn At the time of this workshop, papers on crosses between soybean and perennial Glycine species by the Hymowitz group and by the CSIRO group (Broue` and Marshall) were in draft, and there was tremendous excitement about the potential of the perennials for plant breeding, particularly as sources of drought- and disease-resistance genes

It was through Tony’s role as curator of the perennial Glycine seed collection that he began a longtime collaboration with Dr Jeff Doyle (Cornell University) and his wife, Jane Doyle, when Dr Doyle contacted CSIRO requesting seed for systematic studies in 1982 Tony’s detailed knowledge of Glycine has guided their collaboration, which has produced numerous papers on the molecular phylogenetics of the subgenus The chloroplast phylogeny of Glycine corroborated the existence of the genome groups that were based on cytological data amassed by the CSIRO and the University of Illinois groups, and offered the first hypothesis of relationships among these groups of species The availability of a phylogeny based on defined molecular markers shared among all species also allowed the affinities of newly described species to be determined without recourse to the painstaking studies of chromosome pairing in difficult-to-produce artificial F1 hybrids

con-ducted by Tony and colleagues in the 1980s and by the group at Illinois Phylogenies based on nuclear markers subsequently showed some incongruence with chloroplast sequences, and some relationships in Glycine remain unresolved Despite these limitations, molecular systematic approaches have replaced artificial hybridization as the standard method for categorizing new species in the subgenus

to solve biological problems and are a tribute to his congenial and collegial personality

In the two and a half decades since Tony assumed responsibility for CSIRO’s perennial Glycine research, he published papers on a number of other topics, including disease resistance, seed size, floral biology, several on population genetics, and the distribution of calcium oxalate crystals in Glycine and allies And through all of this, Tony drew on his Glycine work as a complement to his studies on Hordeum and other plants, to refine and illustrate his views on germplasm contributions, the area to which he has dedicated himself for many years

III THE MAN

Dr Brown has a passion for conservation genetics, from his formative years with sugarcane to the present with Glycine species His admiration for Otto Frankel, his diligent research at CSIRO, and his affiliation with the International Plant Genetic Resources Institute (now Bioversity International), Rome, Italy (1982–present), are evident in his many contributions CSIRO Plant Industry as his home base has been an excellent and supportive research environment, where Tony worked jointly with many outstanding colleagues, including Drs Jeremy Burdon (who is the current chief of Plant Industry), Curt Brubaker, Andrew Young, Jake Jacobsen, and several others Indeed, these characteristics of Plant Industry owe much to Sir Otto who, as a former chief of division, instilled a vision of excellence in plant research

The discovery of taxa new to science is the unique reward for the collector of wild species related to important crops Each of his many trips had memorable incidents for Tony, and three are mentioned here If you happened to be one of the few vehicles driving the remote dirt Peninsula ‘‘highway’’ in Cape York, north Queensland, in July of 1983, you may have seen three collectors (Ted Hymowitz from Illinois and Jim Grace and Tony from CSIRO) sprawled on the lawn outside the Lakeland pub below the billboard saying ‘‘Ice Cold Beer.’’ This was no early knock off; they actually were sampling rare, tiny Glycine tomentella plants The billboard had nothing to with site selection; a collector must check all habitats The roadside pub, a lone building in the rural landscape, was a haven for the thirsty traveler, and it surrounds a haven for wild plants that grazing animals would otherwise decimate Thus, sampling strategies for germplasm collection adapt to reality

the Carnarvon Gorge of central Queensland, which proved a special site, rich in diverse new taxa Excitement died, however, when halfway along the 150-km return trip, it became clear there was insufficient fuel to reach home at Injune There followed a long silent drive, meeting no other vehicles Despite eking out the last drop of fuel, the vehicle slowed to a stop 15 km north of their destination Tony and Jim remained with the vehicle; Bill and Michael chose to jog and walk to town for help, guided by moonlight and the smell of road-killed cattle and kangaroos During the long wait at the vehicle, the silent darkness was broken by another vehicle, the first sighted since Carnarvon and, luckily, approaching the road to town, from the property right where the vehicle had stopped When they apprised the driver of the pickup of their situation, he pointed to the rifle above his rear window and replied in a rural Texas accent, ‘‘Just as well you told me If I’d been forced to stop on the road in the dark by two desperados on foot, looking for a ride to town, I’d answer with this.’’

Meeting the wildlife is a feature of any field trip in Australia A trip to collect wild Australian Gossypium species, and to evaluate the risks to them of GM cotton, with botanist Professor Herbert Hurka from Osnabrueck, Germany, brought them to a remote Corona farm 70 km north of Broken Hill, western NSW Herbert was intrigued by the caged talking sulphur-crested cockatoo The farmer’s wife had warned them that the bird had lived in a hotel in the ‘‘silver city’’ but was banished because of bad language Clearly the garrulous bird enjoyed the attention of the team of rare visitors, and Herbert lingered to converse with it while the CSIRO team sampled When he turned to leave, the bird had a fail-safe method to retrieve attention To the visiting professor, it screeched ‘‘A***hole’’—a fully effective way to grab Herbert’s notice

Sir Otto Frankel was one of the major influences of Tony’s science and life His unyielding insistence on high standards and exactness led to many legendary stories Memorable for Tony was a Christmas Eve lunch at which Tony hosted Sir Otto and Lady Margaret, along with Professor Herbert and Ute Hurka and family members At one point, Tony introduced a wine he was particularly enjoying, and asked who would like some of this excellent Orlando chardonnay, Otto’s response was immediate and emphatic: ‘‘That wine is good, but is certainly NOT excellent!!’’ Silence fell; then he asked, ‘‘Which vintage?’’ Stunned, Tony checked the label and replied, ‘‘1988.’’ Back came the riposte: ‘‘1987 is better!!’’ Those quips have often proved useful, not only when recalling Otto’s outspokenness

in migration history in the Department of History, University of Manchester, UK; and Chris is an investment banker with the Mergers and Acquisitions Section of UBS, New York, USA Tony has a new younger family of three stepchildren who are themselves embarking on diverse careers

IV HONORS AND AWARDS

Dr Brown has been extensively recognized for his contributions and achievements to conservation genetics To further broaden his expertise, Tony has been a visiting professor at Stanford University and the University of California, Riverside, visiting research fellow at Haifa University, Israel, and a visiting scientist at the Universitaet Osnabrueck, Germany Tony has excelled in his editorial duties for the journals Genetics, Molecular Biology and Evolution, and Conservation Genetics as well as serving as editor or coeditor of 10 books, and conference and symposia proceedings Of the eight plant collecting missions, Tony has been leader or coleader of six in Australia, one in Israel, and one in Iran Tony has been the International Plant Genetic Resources Institute (IPGRI) technical advisor and on the organizing committee of 18 international workshops in 10 different countries Perhaps the most rewarding honor was the award as Honorary Research Fellow by the IPGRI, Rome, Italy The initial award was in 1997 and Tony has been reappointed three times, most recently with Bioversity International, IPGRI’s new name

ACKNOWLEDGMENT

The authors thank Dr Don Marshall of Plant Breeding Solutions Pty Ltd., Hamilton, NSW, Australia, for his contributions to the text and for his critical review of this chapter

SELECTED PUBLICATIONS OF ANTHONY H D BROWN

Brown, A.H.D., J Daniels, and B.D.H Latter 1968 Quantitative genetics of sugarcane I Analysis of variation in a commercial hybrid sugarcane population Theor Appl Genet 38:361–369

Brown, A.H.D 1970 The estimation of Wright’s fixation index from genotypic frequen-cies Genetica 41:399–406

Brown, A.H.D and R.W Allard 1970 Estimation of the mating system in open–pollinated maize populations using isozyme polymorphisms Genetics 66:133–145

Brown, A.H.D 1971 Isozyme variation under selection in Zea mays Nature 232:570 Brown, A.H.D and R.W Allard 1971 Effect of reciprocal recurrent selection for yield on

isozyme polymorphisms in maize (Zea mays L.) Crop Sci 11:888–893

Marshall, D.R and A.H.D Brown 1973 Stability of performance of mixtures and multi-lines Euphytica 22:405–412

Brown, A.H.D., D.R Marshall, and L Albrecht 1974 The maintenance of alcohol dehydrogenase polymorphism in Bromus mollis L Aust J Biol Sci 27:545–559 Marshall, D.R and A.H.D Brown 1974 Estimation of the level of apomixis in plant

populations Hered 32:321–333

Brown, A.H.D., A.C Matheson, and K.G Eldridge 1975 Estimation of the mating system of Eucalyptus obliqua L Herit using allozyme polymorphisms Aust J Bot 23:931–949 Brown, A.H.D 1975 Efficient experimental designs for the estimation of genetic

para-meters in plant populations Biometrics 31:145–160

Brown, A.H.D 1975 Sample sizes required to detect linkage disequilibrium between two or three loci Theor Pop Biol 8:184–210

Brown, A.H.D., D.R Marshall, and L Albrecht 1975 Profiles of electrophoretic alleles in natural populations Genet Res Camb 25:137–143

Brown, A.H.D., D.R Marshall, and B.S Weir 1975 Population differentiation under the charge state model Genetics 81:739–748

Marshall, D.R and A.H.D Brown 1975 The charge state model of protein polymorphism in natural populations J Molec Evol 6:149–163

Marshall, D.R and A.H.D Brown 1975 Optimum sampling strategies in genetic conservation pp 53–80 In: O.H Frankel and J.G Hawkes (eds.), I.B P.2 Crop Genetic Resources for Today and Tomorrow Cambridge Univ Press, Cambridge

Brown, A.H.D., D.R Marshall, and J Munday 1976 The adaptedness of variants at an alcohol dehydrogenase locus in Bromus mollis L (Soft Bromegrass) Aust J Biol Sci 29:389–396

Weir, B.S., A.H.D Brown, and D.R Marshall 1976 Testing for selective neutrality of electrophoretically detectable protein polymorphisms Genetics 84:639–659

Brown, A.H.D., E Nevo, and D Zohary 1977 Association of alleles at esterase loci in wild barley Hordeum spontaneum Nature 268:430–431

Brown, A.H.D 1978 Isozymes, plant population genetic structure and genetic conserva-tion Theor Appl Genet 52:145–157

Brown, A.H.D., E Nevo, D Zohary, and O Dagan 1978 Genetic variation in natural populations of wild barley (Hordeum spontaneum) Genetica 49:97–108

Brown, A.H.D., D Zohary, and E Nevo 1978 Outcrossing rates and heterozygosity in natural populations of Hordeum spontaneum Koch in Israel Hered 41:49–62 Brown, A.H.D 1979 Enzyme polymorphism in plant population Theor Pop Biol

15:1–42

Nevo, E., D Zohary, A.H.D Brown, and M Haber 1979 Genetic diversity and environmen-tal associations of wild barley, Hordeum spontaneum, in Israel Evolution 33:815–833 Doll, H and A.H.D Brown 1979 Hordein variation in wild (Hordeum spontaneum) and

cultivated (H vulgare) barley Can J Genet Cytol 21:391–404

Brown, A.H.D and L Albrecht 1980 Variable outcrossing and the genetic structure and predominantly self-pollinated species J Theor Biol 82:591–606

Brown, A.H.D., M.W Feldman, and E Nevo 1980 Multilocus structure of natural popu-lations of Hordeum spontaneum Genetics 96:523–536 Corrigendum May 1981, p 238A Green, A.G., A.H.D Brown, and R.N Oram 1980 Determination of outcrossing rate in a breeding population of Lupinus albus L (White Lupin) Z Pflanzenzuchtg 84:181–191 Brown, A.H.D and M.W Feldman 1981 Population structure of multilocus associations

Proc Natl Acad Sci U.S 78:5913–5916

Brown, A.H.D and D.R Marshall 1981 Evolutionary changes accompanying coloniza-tion in plants pp 351–363 In: G.G.E Scudder and J.L Reveal (eds.), Evolucoloniza-tion Today, Proc Second Int Congr Syst and Evol Biol Univ British Columbia, Vancouver Hunt Institute for Botanical Documentation, Pittsburgh

Marshall, D.R and A.H.D Brown 1981 The evolution of apomixis Hered 47:1–15 Marshall, D.R and A.H.D Brown 1981 Wheat genetic resources pp 21–40 In:

W J Peacock and L.T Evans (eds.), Wheat Science, Today and Tomorrow Cambridge Univ Press, Cambridge

Brown, A.H.D and J.V Jacobsen 1982 Genetic basis and natural variation of alpha– amylase isozymes in barley Genet Res Camb 40:315–324

Brown, A.H.D and J Munday 1982 Population genetic structure and optimal sampling of land races of barley from Iran Genetica 58:85–96 Erratum 60:237

Nevo, E., E Golenberg, A Beiles, A.H.D Brown, and D Zohary 1982 Genetic diversity of environmental associations of wild wheat, Triticum dicoccoides in Israel Theor Appl Genet 62:241–254

Brown, A.H.D 1983 Barley, pp 57–77 In: S.D Tanksley, and T.J Orton (eds.), Isozymes in plant genetics and breeding, Part B Elsevier, Amsterdam

Brown, A.H.D and J.J Burdon 1983 Multilocus diversity in an outbreeding weed, Echium plantagineum L Aust J Biol Sci 36:503–509

Brown, A.H.D and M.T Clegg 1983 Analysis of variation in related DNA sequences pp 107– 132 In: B.S Weir (ed.), Statistical analysis of DNA sequence data Marcel Dekker, New York Brown, A.H.D and B.S Weir 1983 Measuring genetic variability in plant populations pp 219–239 In: S.D Tanksley and T.J Orton (eds.), Isozymes in plant genetics and breeding, Part A Elsevier, Amsterdam

Burdon, J.J., D.R Marshall, and A.H.D Brown 1983 Demographic and genetic changes in populations of Echium plantagineum L J Ecology 71:667–679

Brown, A.H.D 1984 Multilocus organization of plant populations pp 159–169 In: K Wohrmann and V Loeschcke (eds.), Population biology and evolution Springer Verlag, Berlin

Clegg, M.T., A.H.D Brown, and P.R Whitfeld 1984 Chloroplast DNA diversity in wild and cultivated barley: Implications for genetic conservation Genet Res Camb 43: 339–343 Hanson, A.D and A.H.D Brown 1984 Three alcohol dehydrogenase genes in wild and cultivated barley: characterization of the products of variant alleles Biochem Genet 22:495–515

Grant, J.E., A.H.D Brown, and J.P Grace 1984 Cytological and isozyme diversity in Glycine tomentella Hayata (Leguminosae) Aust J Bot 32:665–677

Grant, J.E., J.P Grace, A.H.D Brown, and E Putievsky 1984 Interspecific hybridization in Glycine subgenus Glycine Willd (Leguminosae) Aust J Bot 32:655–663

Schroeder, H.E and A.H.D Brown 1984 Inheritance of legumin and albumin contents in a cross between round and wrinkled peas Theor Appl Genet 68:101–107

Brown, A.H.D., J.E Grant, J.J Burdon, J.P Grace, and R Pullen 1985 Collection and utilization of wild perennial Glycine pp 345–352 In: R Shibles (ed.), Proc World Soybean Research Conference III Westview Press, Boulder, Colorado

Doyle, M.J and A.H.D Brown 1985 Numerical analysis of isozyme variation in Glycine tomentella Biochem Syst Ecol 13:413–419

Brown, A.H.D., J.E Grant, and R Pullen 1986 Outcrossing and paternity in Glycine argyrea by paired fruit analysis Biol J Linn Soc 29:283–294

Burdon, J.J and A.H.D Brown 1986 Population genetics of Echium plantagineum L.—a target weed for biological control Aust J Biol Sci 39:369–378

Doyle, M.J., J.E Grant, and A.H.D Brown 1986 Reproductive isolation between isozyme groups of Glycine tomentella (Leguminosae), and spontaneous doubling in their hybrids Aust J Bot 34:523–535

Grant, J.E., R Pullen, A.H.D Brown, J.P Grace, and P.M Gresshof 1986 Cytogenetic affinity between the new species Glycine argyrea and its congeners J Hered 77: 423–426

Brown, A.H.D and J.J Burdon 1987 Mating systems and colonizing success in plants pp 115–131 In: A.J Gray, M.J Crawley, and P.J Edwards (eds.), Colonization, succession and stability 26th Symposium of British Ecol Soc Blackwell Scientific, Oxford Henry, R.J and A.H.D Brown 1987 Variation in the carbohydrate composition of wild

barley (Hordeum spontaneum) grain Z Panzenzuăchtung 98:97–103

Hoffman, N.E., D Hondred, A.D Hanson, and A.H.D Brown 1988 Lactate dehydrogen-ase isozymes in barley: Polymorphism and genetic basis J Hered 79:110–114 Brown, A.H.D., J Munday, and R.N Oram 1988 Use of isozyme-marked segments from

wild barley (Hordeum spontaneum) in barley breeding Plant Breed 100:280–288 Brown, A.H.D 1989 The case for core collections pp 136–156 In: A.H.D Brown, O.H

Frankel, D.R Marshall, and T Williams (eds.), The use of plant genetic resources Cambridge Univ Press, Cambridge

Brown, A.H.D 1989 Core collections: A practical approach to genetic resources manage-ment Genome 31:818–824

Brown, A.H.D 1989 Genetic characterization of plant mating systems pp 145–162 In: A.H.D Brown, M.T Clegg, A.L Kahler, and B.S Weir (eds.), Plant population genetics, breeding and genetic resources Sinaeuer Associates, Sunderland, Massachusetts Brown, A.H.D., J.J Burdon, and A.M Jarosz 1989 Isozyme analysis of plant mating

systems pp 73–86 In: D Soltis and P Soltis (eds.), Isozymes in plant biology Dioscorides Press, Portland, Oregon

Brown, A.H.D., G.J Lawrence, M Jenkin, J Douglass, and E Gregory 1989 Linkage drag in backcross breeding J Hered 80:234–239

Doyle, J.J and A.H.D Brown 1989 5S nuclear ribosomal gene variation in the Glycine tomentella polyploid complex Syst Bot 14:398–407

Hurka, H., S Freunder, A.H.D Brown, and U Plantholt 1989 Aspartate amino transferase isozymes in the genus Capsella (Brassicaceae): Subcellular location, gene duplication and polymorphism Biochem Genetics 27:77–90

Kenworthy, W.J., A.H.D Brown, and G.A Thibou 1989 Variation in flowering response to photoperiod in perennial Glycine species Crop Sci 29:678–682

Brown, A.H.D 1990 The role of isozyme studies in molecular systematics Aust Syst Bot 3:39–46

Brown, A.H.D., J.J Burdon and J.P Grace 1990 Genetic structure of Glycine canescens, a perennial relative of soybean Theor Appl Genet 79:729–736

Doyle, J.J., J.L Doyle, and A.H.D Brown 1990 A chloroplast DNA phylogeny of the wild perennial relatives of soybean (Glycine subgenus Glycine): Congruence with morpho-logical and crossing groups Evolution 44:371–389

Doyle, J.J., J.L Doyle, and A.H.D Brown 1990 Chloroplast DNA phylogenetic affinities of newly described species in Glycine (Leguminosae: Phaseoleae) Syst Bot 15:466–471 Doyle, J.J., J.L Doyle, and A.H.D Brown 1990 Chloroplast DNA polymorphism and

phylogeny in the B genome of Glycine subgenus Glycine (Leguminosae) Amer J Botany 77:772–782

Doyle, J.J., J.L Doyle, A.H.D Brown, and J.P Grace 1990 Multiple origins of polyploids in the Glycine tabacina complex inferred from chloroplast DNA polymorphism Proc Natl Acad Sci USA 87:714–717

Doyle, J.J., J.L Doyle, J.P Grace, and A.H.D Brown 1990 Reproductively isolated polyploid races of Glycine tabacina (Leguminosae) had different chloroplast genome donors Syst Bot 15:173–181

Feuerstein, U., A.H.D Brown, and J.J Burdon 1990 Linkage of rust resistance genes from wild barley (Hordeum spontaneum) with isozyme markers Plant Breed 104:318–324 Schoen, D.J and A.H.D Brown 1991 Intraspecific variation in population gene diversity and effective population size correlates with the mating system in plants Proc Natl Acad Sci USA 88:4494–4497

Abbott, D.C., J.J Burdon, A.M Jarosz, A.H.D Brown, W.J Muller, and B.J Read 1991 The relationship between seedling infection types and field reactions to leaf scald in Clipper barley backcross lines Aust J Agric Res 42:801–809

Brown, A.H.D and J.D Briggs 1991 Sampling strategies for genetic variation in ex situ collections of endangered plant species pp 99–119 In: D.A Falk and K.E Holsinger (eds.), Genetics and Conservation of Rare Plants Oxford Univ Press, Oxford Lagudah, E.S., R Appels, A.H.D Brown, and D McNeil 1991 The molecular-genetic

analy-sis of Triticum tauschii, the D-genome donor to hexaploid wheat Genome 34: 375–386 MacLeod, L.C., R.C.M Lance, and A.H.D Brown 1991 Chromosomal mapping of the Glb

1 locus encoding (1!3), (1!4)–ß–D–glucan 4–glucanohydrolase EI in barley J Cereal Sci 13:291–298

Schoen, D.J and A.H.D Brown 1991 Whole and part-flower self-pollination in Glycine clandestina and G argyrea and the evolution of autogamy Evolution 45:1651–1664

Abbott, D.C., A.H.D Brown, and J.J Burdon 1992 Genes for scald resistance from wild barley (Hordeum vulgare ssp spontaneum) and their linkage to isozyme markers Euphytica 61:225–231

Brown, A.H.D 1992 Genetic variation and resources in cultivated barley and wild Hordeum Barley Genetics 6:669–682

Brown, A.H.D 1992 Human impact on plant gene pools and sampling for their con-servation Oikos 63:109–118

Schoen, D.J., J.J Burdon, and A.H.D Brown 1992 Resistance of Glycine tomentella to soybean leaf rust Phakopsora pachyrhizi in relation to ploidy level and geographic distribution Theor Appl Genet 83:827–832

Schoen, D.J and A.H.D Brown 1993 Conservation of allelic richness in wild crop relatives is aided by assessment of genetic markers Proc Natl Acad Sci USA 90:10623–10627

Brown, A.H.D and D.J Schoen 1994 A revised measure of association of gene diversity values Hereditas 120:77–79

Guerin, J.R., R.C.M Lance, A.H.D Brown, and D.C Abbott 1994 Mapping of malt endopeptidase, diaphorase and esterase loci on barley chromosome 3L Plant Breed 112:279–284

Prober, S.M and A.H.D Brown 1994 Conservation of the grassy white box woodlands I Population genetics and fragmentation of Eucalyptus albens Benth Conservation Biol 8:1003–1013

Abbott, D.C., E.S Lagudah, and A.H.D Brown 1995 Identification of RFLPs flanking a scald resistance gene on barley chromosome J Hered 86:152–154

Brown, A.H.D 1995 The core collection at the crossroads pp 3–19 In: T Hodgkin, A.H.D Brown, T.J.L van Hintum, and E.A.V Morales (eds.), Core collections of plant genetic resources John Wiley, Chichester

Brown, A.H.D and D.R Marshall 1995 A basic sampling strategy: Theory and practice pp 75–91 In: L Guarino, V Ramanatha Rao, and R Reid (eds.), Collecting plant genetic diversity technical guidelines CAB International, Wallingford

Frankel, O.H., A.H.D Brown, and J.J Burdon 1995 The conservation of plant bio-diversity Cambridge Univ Press, Cambridge

Brown, A.H.D., D.F Garvin, J.J Burdon, D.C Abbott, and B.J Read 1996 The effect of combining scald resistance genes on disease levels, yield and quality traits in barley Theor Appl Genet 93:361–366

Young, A., G.T Boyle, and A.H.D Brown 1996 The population genetic consequences of habitat fragmentation for plants Trends in Ecology and Evolution 11:413–418 Young, A.G and A.H.D Brown 1996 Comparative population genetic structure on the

rare woodland shrub Daviesia suaveolens and its common congener D mimosoides Conservation Biol 10:1220–1228

Brown, A.H.D., C.L Brubaker, and J.P Grace 1997 The regeneration of germplasm samples: Wild versus cultivated species Crop Sci 37:7–13

Brown, A.H.D., C.L Brubaker, and M.J Kilby 1997 Assessing the risk of cotton transgene escape into wild Australian Gossypium species pp 83–94 In: G.D McLean, P.M Waterhouse, G Evans, and M.I Gibbs (eds.), The commercialisation of transgenic crops: Risk, benefit and trade considerations Bureau of Resource Sciences, Kingston, ACT, Australia

Garvin, D.F., A.H.D Brown, and J.J Burdon 1997 Inheritance and chromosome locations of novel scald resistance genes derived from Iranian and Turkish wild barleys Theor Appl Genet 94:1087–1091

Roulin, S., P Xu, A.H.D Brown, and G.B Fincher 1997 Expression of specific (1 !3)-b-Glucanase genes in leaves of near-isogenic resistant and susceptible barley lines infected with the leaf scald fungus (Rhynchosporium secalis) Phys Mol Plant Path 50:245–261 Garvin, D.F., J.E Miller-Garvin, E.A Viccars, J.V Jacobsen, and A.H.D Brown 1998 Identification of molecular markers linked to ant28, a mutation that eliminates proanthocyanidin in barley seeds Crop Sci 38:1250–1255

Prober, S.M., L.H Spindler, and A.H.D Brown 1998 Conservation of the grassy white box woodlands: Effects of remnant population size on genetic diversity of the outcrossing, allotetraploid herb, Microseris lanceolata Conservation Biol 12:1279–1290

Young, A.G and A.H.D Brown 1998 Comparative analysis of mating systems in the rare woodland shrub Daviesia suaveolens and its congener D mimosoides Hered 80: 374–381 Brown, A.H.D 1999 The genetic structure of crop landraces and the challenge to conserve them in situ on farms pp 29–48 In: S.B Brush (ed.), Genes in the field: Conserving plant diversity on farms Lewis Publishers, Boca Raton, FL

Burdon, J.J.P.H Thrall, and A.H.D Brown 1999 Resistance and virulence structure in two Linum marginale—Melampsora lini host-pathogen metapopulations with different mating systems Evolution 53:704–716

Doyle, J.J., J.L Doyle, and A.H.D Brown 1999 Incongruence in the diploid B-genome species complex of Glycine (Leguminosae) revisited: Histone H3-D alleles vs chlor-oplast haplotypes Molec Biol Evol 16:354–362

Doyle, J.J., J.L Doyle, and A.H.D Brown 1999 Origins, colonization, and lineage recombination in a widespread perennial soybean polyploid complex Proc Nat Acad Sci USA 96:10741–10745

Marshall, D.R and A.H.D Brown 1999 Sampling wild legume populations pp 78–89 In: S.J Bennett and P.S Cocks (eds), Genetic resources of Mediterranean pasture and forage legumes Kluwer Acad Press, Dordrecht

Young, A.G., A.H.D Brown, and F.C Zich 1999 Genetic structure of fragmented populations of the endangered grassland daisy Rutidosis leptorrhynchoides Conserva-tion Biol 13:256–265

Young, A.G and A.H.D Brown 1999 Paternal bottlenecks in fragmented populations of the grassland daisy Rutidosis leptorrhynchoides Genet Res 73:111–117

Abbott, D.C., J.J Burdon, A.H.D Brown, B.J Read, and D Bittisnich 2000 The incidence of barley scald in cultivar mixtures Aust J Agric Res 51:355–360

Brown, A.H.D and C.L Brubaker 2000 Genetics and the conservation and use of Australian wild relatives of crops Aust J Bot 48:297–303

Brown, A.H.D and C.M Hardner 2000 Sampling the gene pools of forest trees for ex situ conservation pp 185–196 In: A Young, T Boyle, and D Boshier (eds.), Forest conservation genetics: Principles and practice CSIRO, Melbourne, Australia

Brown, A.H.D and A.G Young 2000 Genetic diversity in tetraploid populations of the endangered daisy Rutidosis leptorrhynchoides and implications for its conservation Hered 85:122–129

Doyle, J.J., J.L Doyle, A.H.D Brown, and B.L Pfeil 2000 Confirmation of shared and divergent genomes in the Glycine tabacina polyploid complex (Leguminosae) using histone H3-D sequences Syst Bot 25:437–448

Garvin, D.F., A.H.D Brown, H Raman, and B.J Read 2000 Genetic mapping of the barley Rrs14 scald resistance gene with RFLP, isozyme and seed storage protein markers Plant Breed 119:193–196

Brown, A.H.D and C.L Brubaker 2001 Indicators for sustainable management of plant genetic resources—how well are we doing? pp 249–262 In: J.M.M Engels, V Ramanatha Rao, A.H.D Brown, and M T Jackson (eds.), Managing plant genetic diversity CAB International, Wallingford, Oxon, UK

Lin, J.-Z., A.H.D Brown, and M.T Clegg 2001 Heterogeneous geographic patterns of nucleotide sequence diversity between two alcohol dehydrogenase genes in wild barley (Hordeum vulgare ssp spontaneum) Proc Nat Acad Sci USA 98:531–536

Teshome, A., A.H.D Brown, and T Hodgkin 2001 Diversity in landraces of cereal and legume crops Plant Breed Rev 21:221–261

Brown, A.H.D., J.L Doyle, J.P Grace, and J.J Doyle 2002 Molecular phylogenetic relationships within and among diploid races of Glycine tomentella (Leguminosae) Aust Syst Bot 15:37–47

Doyle, J.J., J.L Doyle, A.H.D Brown, and R.G Palmer 2002 Genomes, multiple origins, and lineage recombination in the Glycine tomentella (Leguminosae) polyploid complex: histone H3-D gene sequences Evolution 56:1388–1402

Rauscher, J.T., J.J Doyle, and A.H.D Brown 2002 Internal transcribed spacer repeat– specific primers and the analysis of hybridization in the Glycine tomentella (Legumi-nosae) polyploid complex Molec Ecol 11:2691–2702

Brubaker, C.L and A.H.D Brown 2003 The use of multiple alien chromosome addition aneuploids facilitates genetic linkage mapping of the Gossypium G genome Genome 46:774–791

Genger, R.K., A.H.D Brown, W Knogge, K Nesbitt, and J.J Burdon 2003 Development of SCAR markers linked to a scald resistance gene derived from wild barley Euphytica 134:149–159

Genger, R.K., K.J Williams, H Raman, B.J Read, H Wallwork, J.J Burdon, and A.H.D Brown 2003 Leaf scald resistance genes in Hordeum vulgare and Hordeum vulgare ssp sponta-neum: parallels between cultivated and wild barley Aust J Agric Res 54:1335–1342 Doyle, J.J., J.L Doyle, J.T Rauscher, and A.H.D Brown 2003 Diploid and polyploidy

reticulate evolution throughout the history of the perennial soybeans (Glycine subg Glycine) New Phytologist 161:121–132

Murray, B.R., A.H.D Brown, and J.P Grace 2003 Geographic gradients in seed size among and within perennial Australian Glycine species Aust J Bot 51:47–56 Rau, D., A.H.D Brown, C.L Brubaker, G Attene, V Balmas, E Saba, and R Papa 2003

Population genetic structure of Pyrenophora teres Drechs., the causal agent of net blotch in Sardinian landraces of barley complex (Hordeum vulgare L.) Theor Appl Genet 106:947–959

Doyle, J.J., J.L Doyle, J.T Rauscher, and A.H.D Brown 2004 Evolution of the perennial soybean polyploid (Glycine subgenus Glycine): A study of contrasts Biol J Linnean Soc 82:583–597

Joly, S., J.T Rauscher, S.L Sherman-Broyles, A.H.D Brown, and J.J Doyle 2004 Evolu-tion of the 18S-5.8S-26S nuclear ribosomal gene family and its expression in natural and artificial Glycine allopolyploids Molec Biol Evol 21:1409–1421

Murray, B.R., A.H.D Brown, C.R Dickman, and M.S Crowther 2004 Geographical gradients in seed mass in relation to climate.J Biogeography 31:379–388

Rauscher, J.T., J.J Doyle, and A.H.D Brown 2004 Multiple origins and nrDNA internal transcribed spacer homoeologue evolution in the Glycine tomentella (Leguminosae) allopolyploid complex Genetics 166:987–998

Cervantes-Martinez, T., H.T Horner, R.G Palmer, T Hymowitz, and A.H.D Brown 2005 Calcium oxalate crystal macropatterns in leaves of species from groups Glycine and Shuteria (Glycininae; Phaseoleae; Papilionoideae; Fabaceae) Can J Bot 83:1410–1421

Genger, R.K., K Nesbitt, A.H.D Brown, D.C Abbott, and J.J Burdon 2005 A novel barley scald resistance locus: Genetic mapping of the Rrs15 scald resistance gene derived from wild barley, Hordeum vulgare ssp spontaneum Plant Breed 124: 137–141

Rau, D., F.J Maier, R Papa, A.H.D Brown, V Balmas, E Saba, W Shafer, and G Attene, 2005 Isolation and characterization of the mating-type locus of the barley pathogen Pyrenophora teres frequencies of mating-type idiomorphs within and among fungal populations collected from barley landraces Genome 48:855–869

Brown, A.H.D and T Hodgkin 2007 Measuring, managing and maintaining crop genetic diversity on-farm pp 13–33 In: D Jarvis, C Paddoch, and D Williams (eds.), Managing biodiversity in agricultural ecosystems Columbia University Press, New York Jarvis, D.I., A.H.D Brown, V.I Imbruce, J Ochoa, M Sadiki, E Karamura, P Trutmann,

and M.R Finckh 2007 Managing crop disease in traditional agroecosystems: The benefits and hazards of genetic diversity pp 292–319 In: D Jarvis, C Paddoch, and D Williams (eds.), Managing biodiversity in agricultural ecosystems Columbia University Press, New York

Triono, T., M.D Crisp, A.H.D, Brown, and J.G West 2007 A phylogency of Pouteria (Sapotaceae) from Malesia and Australasia Aust Syst Bot 20:107–118

Rau, D., G Attene, A.H.D Brown, L Nanni, F.J Maier, V Balmas, E Saba, W Schaefer, and R Papa 2007 Phylogeny and evolution of mating-type genes from Pyrenophora teres, the causal agent of barley ‘‘net blotch’’ disease Current Genetics 51:377–392 Jarvis, D.I., A.H.D Brown, et al 2008 A global perspective of the richness and evenness of

2

Brassica and Its Close Allies: Cytogenetics and Evolution

Shyam Prakash

National Research Centre on Plant Biotechnology Indian Agricultural Research Institute

New Delhi 110012 India S R Bhat

National Research Centre on Plant Biotechnology Indian Agricultural Research Institute

New Delhi 110012 India C F Quiros

Department of Vegetable Crops University of California

Davis, California 95616 USA P B Kirti

Plant Science Department University of Hyderabad Hyderabad, 500046 India V L Chopra

National Research Centre on Plant Biotechnology Indian Agricultural Research Institute

New Delhi 110012 India

I INTRODUCTION II CYTOGENETICS

A Cytogenetic Architecture of Brassica Coenospecies B Crop Species

Plant Breeding Reviews, Volume 31 Edited by Jules Janick Copyright& 2009 John Wiley & Sons, Inc

1 Nature of Diploid Species Nature of Alloploid Species Nuclear DNA

4 Karyotypes

5 Pachytene Chromosomes

6 Satellite Chromosomes and rDNA Loci Archetype and Evolution of Genomes III GENOME MANIPULATION

A Resyntheses of Natural Allopolyploid Brassica spp B Agronomic Potential of Synthetics

C Diploidization of Allopolyploid Species D Raphanobrassica

E Higher Allopolyploids in U Triangle Species through Protoplast Fusion IV WIDE HYBRIDIZATION

A Sexual Hybrids B Somatic Hybrids C Introgression of Genes

V CYTOPLASMIC SUBSTITUTION AND MALE STERILITY

VI GENOME DISSECTION AND DEVELOPMENT OF CHROMOSOME ADDITION LINES

VII MITOCHONDRIAL GENOME A Organization

B Gene Content

C Mitochondrial Plasmids VIII PLASTID GENOME

IX POTENTIAL ROLE OF ARABIDOPSIS THALIANA IN BRASSICA IMPROVEMENT A A thaliana as a Model Crucifer

B Cytology and Possible Origin of the A thaliana Genome C Synteny Conservation

D Synteny-Based Gene Discovery and Cloning

E Arabidopsis Knowledge–Based Gene Discovery and Brassica Improvement Understanding Domestication

2 Understanding Metabolism

3 Testing for Gene Function by Complementary Transformation X CHLOROPLAST GENOMES AND THEIR PHYLOGENETIC IMPLICATIONS

A Subtribe Brassicinae Brassica

2 Diplotaxis Erucastrum Sinapis Trachystoma Hirschfeldia incana Sinapidendron Coincya Eruca

XI EVOLUTION OF MORPHOLOGICAL CHARACTERS A Cotyledons

B Adult Leaves C Fruits

D Isthmus Concept XII CONCLUDING REMARKS ACKNOWLEDGMENTS LITERATURE CITED

ABBREVIATIONS

ACO Aconitase

ADH Alcohol deshydrogenase

AFLP Amplified fragment length polymorphism Ag-NOR Silver-stained nucleolus organizer region BACs Bacterial artificial chromosomes

BTL Binary trait loci

CAGs Conserved Arabidopsis genome sequences cp Chloroplast

CP Condensation pattern CMS Cytoplasmic male sterility Cytodeme Crossing group

DAPI 40,6-diamidino-2-phenylindole ESTs Expressed sequence tags

FISH Fluorescence in situ hybridization GISH Genomic in situ hybridization GOT Glutamate oxaloacetate transaminase GSL Glucosinolate

IDH Isocitric dehydrogenase ISSR Inter-simple sequence repeats ITC Isothiocynanates

ITS Internal transcribed spacers of nuclear ribosomal DNA genes LAP Leucine amino-peptidase

MDH Malate dehydrogenase mt Mitochondria

NOR Nucleolus organizer region PrBn Pairing regulator Brassica napus PGD 6-phosphogluconase dehydrogenase PGDH 6-phosphogluconate deshydrogenase PGI Phosphoglucoisomerase

RAPD Randomly amplified polymorphic DNA RFLP Restriction fragment length polymorphism rDNA Ribosomal DNA genes

rRNA Ribosomal RNA

SDH Shikimic acid dehydrogenase SSR Simple sequence repeats TE Transposable element TPI Triose phosphate isomerase

I INTRODUCTION

Brassica species, Brassicaceae (Cruciferae), provide an important com-ponent of human diet as major sources of edible oil and vegetables (Table 2.1) The antiquity of crops belonging to the genus Brassica is manifest from references in ancient literature of the Indian, Chinese, Greek, and Roman civilizations (Prakash and Hinata 1980; Go´mez-Campo and Prakash, 1999) A number of taxonomic treatments of this family are available since 1700 Prominent among these are by Tournefort (1700), Linnaeus (1753), deCandolle (1821), Hooker (1862), Baillon (1871), and Prantl (1891) However, the most comprehensive one is by Schulz (1919, 1936), a German schoolteacher (Hedge 1976; Prakash and Hinata 1980; Gomez-Campo 1999b) A recent molecular account of the family has been provided by Beilstein et al (2006) Brassiceae is one among the 19 tribes recognized by Schulz in the family and is divided into to subtribes (Go´mez-Campo 1980, 1999b) Brassica is the core genus in the subtribe Brassicinae Several members of related subtribes, such as Raphaninae and Moricandiinae, exhibit close genetic affinities with Brassica However, morphological distintiveness of these three subtribes is not well substantiated and molecular data provide scanty support for their independent status A majority of the species related to Brassica are wild and weedy They possess, however, useful genes that may confer agronomic advantages and/or enhance the quality and utility of crop species In fact, genetic enrichment of crop species with genes from wild allies is a major approach for many crop improvement programs Such gene transfer can be achieved both by conventional plant breeding methods and through biotechnology

and not being amenable to pachytene investigations were major deterrants to cytogenetical analyses Advances in tissue culture techni-ques, including ovary and embryo rescue and protoplast fusion, since the 1950s made varied cytogenetic material available to investigate genome homologies and facilitated introgression of useful nuclear genes even across conventional generic boundaries Such investigations require reliable markers for chromosome identification A significant step toward this development has been the extensive use of molecular markers Molecular biology in Brassica started with the determination of female parents of allopolyploid species using chloroplast DNA RFLPs by Palmer et al (1983a) Use of genomic and fluorescence in situ hybridization (GISH and FISH respectively) methodology, in combination with ribosomal DNA markers have given new directions in genome analysis

Table 2.1 Taxonomic components of Brassica and related genera and their usage Botanical name Common name Usage

B nigra black mustard condiment (seed) B oleracea

var acephala kale vegetable, fodder (leaves) var capitata cabbage vegetable (head) var sabauda savoy cabbage vegetable (terminal buds) var gemmifera brussels sprouts vegetable (head) var gongylodes kohlrabi vegetable, fodder (stem) var botrytis cauliflower vegetable (inflorescence) var italica broccoli vegetable (inflorescence) var fruticosa branching bush kale fodder (leaves)

var alboglabra Chinese kale vegetable (stem, leaves) B rapa

spp oleifera turnip rape oilseed var brown sarson brown sarson oilseed var yellow sarson yellow sarson oilseed var toria toria oilseed

ssp rapifera turnip fodder, vegetable (root) ssp chinensis bok choi vegetable (leaves) ssp pekinensis Chinese cabbage vegetable, fodder (head) ssp nipposinica — vegetable (leaves) ssp parachinensis — vegetable (leaves) B carinata Ethiopian mustard vegetable, oilseed B juncea mustard oilseed, vegetable B napus

spp oleifera rapeseed oilseed spp rapifera rutabaga, swede fodder

Eruca sativa rocket, taramira vegetable, nonedible oilseed Raphanus sativus radish vegetable, fodder

and characterization of parental genome components as well as precise identification of the individual chromosomes and location of gene sequences directly on the chromosomes These investigations led to the generation of first FISH-based molecular karyotypes (Fukui et al 1998) At the same time, chloroplast and mitochondrial DNA RFLPs have been used extensively to elucidate phylogeny of Brassica and related genera Molecular markers also have been identified to tag important agronomic traits This research has not only substantiated some of the already existing concepts but also proposed several new ones Potential sources of germplasm have been identified outside of the conventional boundaries, thus increasing the range of available germplasm relevant to Brassica improvement

Genomic studies on Arabidopsis, a crucifer and closely related to brassicas, has given a new direction in evolutionary studies of the family Brassicaceae and in particular the members of the genus Brassica Inferences from comparative genomics between Arabidopsis and Brassica species have elucidated evolutionary processes Arabi-dopsis has become a model plant in the field of experimental biology because of its several unique features: short life span, autogamy, and ease of tissue culture Its entire genome has recently been sequenced (Arabidopsis Genome Initiative 2000)

Biologically, Brassica and allied taxa have been grouped collectively and referred to as Brassica coenospecies (Harberd 1972) This review is an attempt to synthesize available literature and developments in Brassica coenospecies from classical to molecular cytology and applica-tion of genomic informaapplica-tion to throw light on genome organizaapplica-tion, genome manipulation, and phylogeny in Brassica and related genera Informative reviews dealing with some of these aspects are given in Biology of Brassica Coenospecies (Go´mez-Campo 1999a) and Biotech-nology in Agriculture and Forestry: Brassica (Pua and Douglas 2004)

II CYTOGENETICS

A Cytogenetic Architecture of Brassica Coenospecies

Brassicinae of Schulz comprising of 11 genera and also on a part of related subtribe Raphaninae His investigations involved studies on chromosome pairing and extent of fertility of hybrids Harberd proposed that nine genera from subtribe Brassicinae—Brassica, Coincya, Diplotaxis, Eruca, Erucastrum, Hirschfeldia, Sinapis, Sinapidendron, and Trachystoma—and two genera from subtribe Raphaninae—Enarthrocarpus and Raphanus—constitute Brassica coenospecies Harberd (1972) involved a wide spectrum of species in his hybridization program and studied this germplasm biologically rather than taxonomically to classify it into cytodemes or crossing groups to resolve the confusion about their species and generic status A cytodeme is defined as a group consisting of any number of species or genera that have the same chromosome number, and crosses between them always yield fertile hybrids Harberd (1972) established 38 cytodemes in the coenospecies This number was further extended by Takahata and Hinata (1983) In fact, Harberd’s results revealed, for the first time, extensive genome homoeology across species and generic boundaries, implying that Brassica coenospecies constitutes a large gene pool and thus opening the possibilities of transferring agronomi-cally desirable traits to crop species The boundaries of coenospecies have further expanded with developments in molecular biology that have resulted in massive incongruities with established taxonomy Chloroplast DNA RFLP studies on members of other related subtribes also suggest that delemitation of genera and species by Schulz does not fully reflect the natural boundaries (Warwick and Black 1991; Pradhan et al 1992) These investigations strongly support the inclusion of not only Raphanus and Enarthrocarpus in the coenospecies as suggested by Harberd (1972, 1976), but also of three more genera—Moricandia, Pseuderucaria, and Rytidocarpus—from the related subtribe Morican-diinae (Warwick and Black 1997) At present, 63 cytodemes are recognized in coenospecies that spread over 14 taxonomically defined genera, as shown in Fig 2.1 and Table 2.2 (Prakash et al 1999)

Table 2.2 Cytodemes in Brassica coenospecies

Chromosome no (n) Principal species Brassica deflexa Boiss

Diplotaxis erucoides (L.) DC Erucastrum virgatum C Presl Erucastrum varium Durieu

Sinapis aucheri (Boiss.) O.E Schulz Hirschfeldia incana (L.) Lagreze-Fossat Pseuderucaria spp O.E Schulz Brassica nigra (L.) Koch

Brassica fruiticulosa Cyr (ỵ maurorum ỵ spinescens) Diplotaxis siettiana Maire

Erucastrum abyssinicum (A Rich.) O.E Schulz

Erucastrum nasturtiifolium (Poiret) O.E Schulz (ỵ leucanthum) Erucastrum strigosum (Thunb.) O.E Schulz

Trachystoma spp

9 Brassica oleracea L and wild Mediterranean allied species Brassica oxyrrhina Coss

Diplotaxis assurgens (Del.) Gren Diplotaxis catholica (L.) DC Diplotaxis tenuisiliqua Del Diplotaxis virgata (Cav.) DC

Diplotaxis berthautii Braun-Blanq and Maire Erucastrum cardaminoides Webb and Berth

(ỵ canariense þ ifniense)

Raphanus L all species and subspecies Sinapis arvensis L (ỵ allioni)

Sinapis pubescens L 10 Brassica tournefortii Gouan

Brassica barrelieri (L.) Janka Brassica gravinae Ten

Brassica repanda (Willd.) DC (ỵ desnottesii) Brassica rapa L (ỵ many cultivated subspecies) Diplotaxis siifolia G Kunze

Diplotaxis viminea (L.) DC Enarthrocarpus spp Sinapidendron spp 11 Brassica souliei Batt

Diplotaxis acris (Forsk.) Boiss Brassica elongata Ehrh

Diplotaxis tenuifolia (L.) DC (ỵ pitardiana) Eruca spp Mill

12 Coincya spp (syn Hutera and Rhynchosinapis) Sinapis alba L

Sinapis flexuosa Poir

13 Diplotaxis harra (Forsk.) Boiss (ỵ several subsps.) 14 Erucastrum virgatum C Presl (subsp pseudosinapis)

Moricandia arvensis (L.) DC

Table 2.2 (Continued)

Chromosome no (n) Principal species 15 Erucastrum gallicum (Willd.) O.E Schulz

Erucastrum elatum (Ball.) O.E Schulz 16 Brassica cossoniana (Boiss & Reut.) (4x)

North African subspecies Brassica balearica Pers

Erucastrum nasturtiifolium (Poiret) O.E Schulz (4x) Erucastrum abyssinicum (A Rich.) O.E Schulz (4x) 17 Brassica carinata A Braun

18 Brassica juncea (L.) Czern & Coss 19 Brassica napus L

20 Brassica gravinae Ten (4x) 21 Diplotaxis muralis (L.) DC 22 Brassica dimorpha Coss & Dur 24 Coincya spp (4x)

28 Moricandia suffruticosa (Desf.) Coss & Dur 42 Moricandia spinosa Pomel

80? Brassica repanda (Willd.) DC (High Atlas)

Source: From Prakash et al 1999; C Go´mez-Campo, personal communication

Family Brassicaceae

Brassicaceae Tribe

Subtribe Brassicinae Raphaninae Moricandiinae

Genera Brassica (20) Enarthrocarpus (1) Moricandia (1) Coincya (1) Raphanus (1) Pseuderucaria (1) Diplotaxis (13) Rytidocarpus (1) Eruca (1)

Erucastrum (11) Hirschfeldia (1) Sinapis (5) Sinapidendron (1) Trachystoma (1)

series of publications Extensive taxonomical investigations on wild germplasm have been carried out by Go´mez-Campo (1999b)

The lowest chromosome number in coenospecies, n¼ 7, is character-istic of seven cytodemes Harberd (1972) was of the view that cytodeme with n¼ 14 or higher chromosome numbers should be attributed to polyploidy According to this view, 43 cytodemes are diploids where every chromosome number from n¼ to n ¼ 13 is represented However, variations in isozyme numbers of a vast range of taxa in the tribe Brassiceae suggest that genera with n¼ 14 to 18 are not necessarily polyploids of n¼ to 13 genomes (Anderson and Warwick 1998) Around 50% of the cytodemes have gametic chromosome number n¼ and n¼ 10 Polyploidy also played a role as both auto- and allo-polyploids are represented by 20 cytodemes (Table 2.3) The majority are tetraploids This polyploidy level is exceeded only in some accessions of Moricandia spinosa (2n¼ 84, x ¼ 6) and Brassica repanda (2n¼ 160, x ¼ 8) (Prakash et al 1999) The genus Moricandia seems to be exclusively polyploid (Al-Shebaz 1984)

B Crop Species

Genome analysis in crop species, pioneered by Morinaga (1928; 1929a, b,c; 1931; 1933; 1934a,b) was based on hybridizing high-chromosome species with low-chromosome species and interpreting the chromo-some pairing behavior of the hybrids This research led Morinaga (1934) to propose that crop brassicas comprise six species Of these, three are low-chromosome monogenomic diploids—B nigra (n¼ 8), B oleracea (n¼ 9), and B rapa (syn B campestris, n ¼ 10)—and three are high-chromosome digenomics—B carinata (n¼ 17), B juncea (n¼ 18), and B napus (n ¼ 19), which evolved in nature through convergent alloploid evolution between any two of the diploid species Morinaga also assigned genome symbols to these species U (1935) represented this cytogenetical relationship diagramatically, in what is now commonly referred to as U triangle (Fig 2.2) These relationships have, in recent years, been substantiated by cytogenetics, molecular analysis of nuclear and chloroplast DNA, and by genomic and fluorescence in situ hybridization (Snowdon et al 2003; Snowdon 2007) This complex of diploids and allopolyploids is now considered a model system for investigations on polyploidy in crop plants (Lukens et al 2006; Pires et al 2006)

(Manton 1932) and are regarded as secondary polyploids Evidence for this conclusion were adduced from chromosome associations at meiosis in their respective haploids (Thompson 1956; Prakash 1974b; Armstrong and Keller 1981) Investigations on pachytene chromosome analysis (Roăbbelen 1960; Venkateswarlu and Kamla 1971), isozyme markers, and rDNA genes (Quiros et al 1987) suggested that these species originated from a now-extinct archetype with a probable basic chromosome number of x¼ It was believed that selective doubling of some chromosomes in this archetype led to the evolution of the three diploid genomes However, results of recent investigations on nuclear,

Table 2.3 Polyploid cytodemes in Brassica coenospecies

Allopolyploids Diploid progenitors Reference Brassica carinata, n¼ 17 B nigra, B oleracea U 1935 Brassica juncea, n¼ 18 B rapa, B nigra U 1935 Brassica napus, n¼ 19 B oleracea, B rapa U 1935

Brassica balearca, n¼ 16 B oleracea group Snogerup and Persoon another species 1983

Diplotaxis muralis, n¼ 21 D viminea, D tenuifolia Harberd 1976; Mummenhoff et al 1993: Ueno et al 2006 Erucastrum gallicum, n¼ 15 E leucanthum sp.? Harberd 1976

Erucastrum elatum, n¼ 15 Hirschfeldia incana Go´mez-Campo 1983; Erucastrum littoreum Sa´nchez-Ye´lamo 1992 Tentative autopolyploids Diploid homolog Reference Moricandia arvensis, n¼ 14 unknown Harberd 1976 Moricandia moricandiodes,

n¼ 14 unknown Harberd 1976 Rytidocarpus moricandiodes,

n¼ 14 unknown Harberd 1976 Erucastrum virgatum (subsp

pseudosinapis), n¼ 14 E.virgatum Harberd 1976 Brassica cossoneana, n¼ 16 B maurorum, n¼ Pradhan et al 1992 Erucastrum abyssinicum, n¼ 16 E abyssinicum, n ¼ Harberd 1976 Erucastrum nasturtiifolium, E nasturtiifolium, n¼ Harberd 1976

n¼ 16

Brassica gravinae, n¼ 20 B gravinae, n¼ 10 Takahata and Hinata 1983

Brassica dimorpha, n¼ 22 B soullei, n¼ 11 Go´mez-Campo 1980 Coincya spp., n¼ 24 Coincya sp., n¼ 12 Harberd 1976

Moricandia suffruticosa, n¼ 28 Moricandia sp., n¼ 14 Sobrino-Vesperinas 1980 Moricandia spinosa, n¼ 42 Moricandia sp., n¼ 14 Sobrino–Vesperinas

mitochondrial, and chloroplast DNA (Palmer 1988; Song et al 1988a; Warwick and Black 1991; Pradhan et al 1992) have discounted this theory of monophyletic origin and have instead suggested their origin from two linages: B oleracea and B rapa originating from one archetype and B nigra evolving from the other Nevertheless, these genomes share close homologies, as revealed by cytogenetical (Mizush-ima 1950a; Prakash and Hinata 1980; Attia and Roăbbelen 1986) and molecular studies (Hosaka et al 1990; Teutonico and Osborn 1994; Truco et al 1996; Parkin et al 2003) Cytogenetical investigations in digenomic and trigenomic interspecific hybrids involving the three basic species showed high frequency of bivalents and multivalents A good number of these were suggested to arise due to allosyndesis GISH analysis confirmed three allosyndetic bivalents between B and A/C (Ge and Li 2007) and five bivalents between A and C genomes (Liu et al 2006) All three genomes contain similar genetic information with many duplications (Slocum et al., 1990; Chyi et al 1992; Jackson et al 2000; Parkin et al 2003); just the organization and distribution on chromosomes is different (Truco et al 1996) Chromosome differentia-tion and repatterning has occurred mainly through duplicadifferentia-tions and translocations (Quiros et al 1988; Hosaka et al 1990; McGrath et al 1990; Truco and Quiros 1994) and also deletions (Hu and Quiros 1991) These changes were tolerated and adjusted because of the secondary

B carinata

n = 17, bc

B oleracea

n = 9, c

B nigra

n = 8, b

B napus

n = 19, ac

B juncea

n = 18, ab

B rapa

n = 10, a

balanced nature of these genomes (Kianian and Quiros 1992a) Also, a large number of rearrangements separated the B genome from the A or C genome In comparison, A and C genomes are less differentiated (Lagercrantz 1998) Genomes A and C are also cytogenetically very close (Mizushima 1950a; Olsson 1960b), a fact substantiated by: (1) FISH mapping of two families of repetitive DNA that are common to pericentromeric regions of most chromosomes of A and C genomes but are absent in the B genome (Harrison and Heslop-Harrison 1995); (2) structural analysis of rDNA intergenic spacers (Bhatia et al 1996); (3) colinearity between them as revealed by comparative analysis (Scheffler et al 1997); and (4) extent of homoeologous pairing detected by GISH (Snowdon et al 1997a; Ge and Li 2007) and FISH and molecular markers (Nicolas et al 2007) Interestingly, RFLP analysis of rDNA reveals, on the contrary, closer affinities between B and C genomes (Hasterok and Maluszynska 2000a) Also, as detected by microsatellites (Bornet and Blanchard 2004), the C genome is more conserved than A or B Among the three basic species, two types of cytoplasm exist: the B type found in B nigra and the A/C type occurring in B rapa and B oleracea The A and B types are quite distinct although they retain homology to a large extent (Palmer et al 1983a; Yanagino et al 1987; Warwick and Black 1991; Pradhan et al 1992)

3 Nuclear DNA Nuclear DNA content and nuclear volume were first estimated by Yamaguchi and Tsunoda (1969) in B rapa, B oleracea, and naturally occurring and synthetic strains of B napus They observed that the values for synthetic B napus were the sum of the constituent parents However, there was an appreciable reduction in total DNA content in natural forms These authors proposed that nuclear DNA had been lost subsequent to evolution of allotetraploids Verma and Rees (1974) further investigated this problem by estimating the amount of DNA in diploids and their allotetraploid derivatives in root meristem nuclei at the GI phase No significant intraspecific variation in nuclear DNA amount was observed However, differences exist at the inter-specific level In spite of the fact that values for allotetraploids were very close to the sum values of their constituent parents, reduction from the expected values for every species was observed They postulated that the lower values in tetraploids result from underestimation of DNA due to higher nuclear density It was also suggested that values observed by Yamaguchi and Tsunoda (1969) were based on dense nuclei and were underestimations; thus, when corrected, the values showed no sig-nificant deviations from those anticipated Therefore, decrease in the amount of DNA was not associated with allopolyploidy One significant observation by Verma and Rees (1974) was a remarkable reduction in nuclear size in natural allotetraploids, which suggested condensation of chromosomal material that probably reflected an adaptive switching off of redundant gene copies In several recent investigations, DNA values have been estimated afresh (Arumugunathan and Earle 1991; Narayan 1998; Bennet and Leitch 2005; Johnston et al 2005) A general observation is the evolution of DNA content from low to high in the genus These studies also support the earlier observations of Yamaguchi and Tsunoda (1969) that there has been a decrease in DNA content in the present-day alloploid species A decrease of 6% was observed by Narayan (1998), and the values for B napus, B juncea, and B carinata are 0.095, 0.094, and 0.049 pg less respectively than the sum of their parental species (Johnston et al 2005, Table 2.4)

nucleoli in different genomes They distinguished chromosomes into long, medium, small, and very small with median, subterminal and terminal constrictions In recent years, karyotypes, particularly of diploid species, based on mitotic (Olin-Fatih and Heneen 1992; Cheng et al 1995a; Fukui et al 1998; Hasterok and Maluszynska 2000a; Hasterok et al 2005a) and meiotic chromosome (Cheng et al 1994b; Mackowiak and Heneen 1999; Koo et al 2004) phenotypes, have been constructed using different staining Mitotic prometaphase and meiotic diakinesis offer better possibilities for characterizing individual chromosomes and constructing karyotypes Since early 1990s, use of FISH with ribosomal DNA probes has further helped in generating chromosome markers Molecular karyotypes based on FISH have been generated, enabling more reliable identification of individual chromo-somes Maluszynska and Heslop-Harrison (1993), Snowdon et al (1997a) and Fukui et al (1998) employed FISH with a 45S rDNA probe for individual chromosome identification However, simultaneous probing with 45S rDNA and 5S rDNA in B napus, Sinapis alba and Raphanus sativus (Schrader et al 2000) and in all the six species of U triangle (Hasterok et al 2001) proved to be more informative as they provided numerous signals on somatic chromosomes revealing new landmarks Fukui (1998) developed a system for computer imaging of plant chromosomes that led to the definition of a new parameter, the ‘‘condensation pattern’’ (CP) for chromosome analysis It is an effective and reproducible parameter and very useful in identification of small chromosomes It is a general observation that somatic chromosomes of A and C genomes are morphologically very similar and difficult to distinguish (Olin-Fatih and Heneen 1992) although their condensation patterns differ in prometaphase chromosomes (Cheng et al 1995a) However, making use of FISH and GISH, it has been possible to identify individual chromosomes of A, B, and C genomes and also to match chromosomes with corresponding counterparts in alloploid species

Table 2.4 1c nuclear DNA content and genome size in Brassica species

Species 1c nuclear DNA content (pg se) Genome size (1 x) (Mbp) B nigra 0:647  0009 632

with considerable reliability (Snowdon et al 1997b, 2002; Kamisugi et al 1998; Maluszynska and Hasterok 2005) Also, these investigations are very helpful in integrating genetical maps, emerging from analysis of molecular markers, with physical maps based on morphometric analysis

Although many papers describing the karyotypes of these species have appeared since 1937, overall conclusions can be summarized in this way: Chromosomes of A genome are morphologically most diverse, B genome chromosomes are much more uniform and difficult to identify individually, and C genome chromosomes are poorly differentiated in morphology and size, and undergo variable degree of condensation of hetero- and euchromatin in the chromosome arms (Olin-Fatih 1994) Discrepencies in nomenclature and numbering of chromosomes have occurred due to polymorphisms in the rDNA sites and contraction rates of the chromosomes

Brassica nigra Hasterok and Maluszynska (2000a) observed that B nigra chromosomes are more or less similar in size, ranging from 2.47 to 3.57 mm, and are morphologically undistinguishable Only two types of chromosomes are present: median (no 1–4) and submedian (no 5–8) Chromosomes and contain secondary constriction and satellite on the short arm Earlier, Mackowiak and Heneen (1999) pre-sented a karyotype based on diakinesis bivalents wherein each chro-mosome exhibited a specific pattern of chromatin condensation or darkly stained regions The eight bivalents were classified into three groups Group 1, comprising pairs and 2, has darkly stained median position signifying pericentric chromatin Pair was the smallest Group comprised pairs to with a submedian-subterminal, darkly stained region that represent pericentric chromatin Group compri-sed pairs to with relatively large-size subterminal-terminal darkly stained region Pair was larger than pairs and Pairs to were the satellited nucleolar chromosomes

Brassica oleracea Cheng et al (1995a) described a karyotype for B oleracea where the absolute length ranged from 2.8 to 4.5 mm The genome is comprised of three median group (1–3), four submedian group (4–7), and two subterminal group chromosomes with a non-satellite pair (8) and a non-satellite pair (9)

the latter one being a nucleolus chromosome including the satellite and NOR The short arm of this chromosome possessed 45S and 5S rDNA sites Chromosomes 1, 3, 4, and had 45S rDNA loci in their long arm Chromosome 10 was the shortest (2.85 mm), and its short arm occupied a 5S rDNA site The number of rDNA sites in the interphase nuclei varied from to 10

The three high-chromosome allotetraploid species have numerous chromosomes, making karyotype formation very difficult when based just on morphometric features In recent investigations, Hasterok and Maluszynska (2000b) and Kulak et al (2002) have presented karyotypes of B carinata, B juncea, and B napus on features combining morphometric information and multicolor FISH

Brassica carinata Its karyotype consists of fairly uniform chromo-somes, both in morphology and in length, ranging from 1.56 to 2.40 mm Two groups of chromosomes can be distinguished: median (1–6) and submedian (7–15) There are two pairs of satellite chromosomes (16–17) with distinct secondary constrictions (Kulak et al 2002)

Brassica juncea The chromosome length in B juncea ranges from 1.38 to 3.25 mm, and the karyotype comprises of median (1–6) and submedian groups (7–15).Two chromosomes (17–18) are NOR-bearing with prominent secondary constrictions in the short arm Chromosome 16, although NOR bearing, does not have a distinct secondary constriction/satellite region (Kulak et al 2002) The extent of variations in chromosome size and morphology is due to A genome chromosomes

chromosomes can be helpful in this regard For example, A genome chromosomes are characterized by pericentromeric localization, and C genome chromosomes have terminally distributed rRNA genes (Has-terok and Maluszynska 2000b) These were clearly identified using rDNA hybridization and DAPI staining by Snowdon et al (2002) Another B napus karyotype has been constructed based on Cot-1 DNA FISH banding patterns by Wei et al (2005) Their results agreed with the earlier reports It was demonstrated that this technique can be used with precision to identify individual chromosomes and would be very helpful in recognizing homologous and nonhomologous chromosome pairing

5 Pachytene Chromosomes The cytologically difficult nature of material has restricted investigation on pachytene chromosome mor-phology to only a few attempts Roăbbelen (1960), for the rst time, analyzed pachytene chromosomes in the three basic species: B nigra, B oleracea, and B rapa The chromosomes revealed differentiation into proximal heterochromatic and distal euchromatic segments Individual chromosomes within the genomes were identified by the number, size, and distribution pattern of the heterochromatic segments near the centromeres The chromosomes were classified on the basis of their absolute length and were distinguished into five different types: very short (up to 20 mm); short (20–25 mm); medium (25–30 mm); long (30–40 mm); and very long (more than 40 mm)

regions and nucleolus organizer regions were observed Two NOR-associated chromosomes were acrocentric, containing heterochroma-tin blocks at the ends of their short arm, and were designated C4 and C7 A prominent chromomere was present on the long arm of a submetacentric chromosome Submetacentric chromosome C2 dis-played three 5S rDNA loci on the same arm with medium (M), strong (S) and weak (W) FISH intensities While locus M was very close to centromere, the two adjacent loci, S and W, were more distal These three loci offer prominent landmarks for C2 chromosome of B oleracea Pachytene chromosome karyotype of B rapa generated by Koo et al (2004) was based on multicolor FISH and comprised of two metacentric (nos 1, 6), five submetacentric (nos 3, 4, 5, 9, and 10), two subtelocentric (nos and 8), and one acrocentric (no 2) chromosomes Their corresponding centromeric index ranges were 38.8% to 41.0%, 29.5% to 36.7%, 17.4% to 20.2%, and 9.38% respectively The mean lengths varied from 23.7 to 51.3 mm with a total of 385 mm As compared to mitotic metaphase chromosome length (1.46–3.30 mm), it is 17.5-fold higher DAPI staining revealed variable length of heterochromatic blocks in the pericentromeric regions of all the chromosomes Also, small heterochromatic regions, with a total length of 38.2 mm and approximately 10% of the total length of pachytene chromosomes, were observed on the long arm of chromo-somes 3, 4, 5, and FISH indicated 5S rDNA loci on pericentromeric regions of the short arms of chromosomes and 10 and the long arm of chromosome Similarly, 45S rDNA loci were observed on pericen-tromeric regions of short arms of chromosomes 1, 2, 4, and and the long arm of chromosome A 5S rDNA locus, observed on the long arm of bivalent no 7, had not been detected on mitotic metaphase chromosomes in any earlier investigations

Roăbbelen (1960) recognized six basic types of chromosomes in each genome based on absolute length, symmetry of arms, and shape of heterochromatic centromeric region These six types are:

‘‘A’’ with a distal heterochromatic satellite involved in nucleolus organization

‘‘B’’ with two heterochromatic segments of equal size near the centromere

‘‘C’’ with a small chromomere and two heterochromatic segments near the centromere

‘‘E’’ with four or more heterochromatic segments ‘‘F’’ with two unequal heterochromatic segments

Based on these observations, Roăbbelen (1960) proposed the genetic constitution of the three basic species B rapa has two chromosome types, A and D, in tetrasomic and type F in hexasomic condition and the constitution AABCDDEFFF B nigra is tetrasomic for chromo-somes D and F and has the constitution ABCDDEFF B oleracea is a triple tetrasomic for three chromosome types B, C, and E, with the constitution ABBCCDEEF

Venkateswarlu and Kamala (1971) arrived at a conclusion very similar to that of Roăbbelen (1960) They also identified six basic types of chromosomes However, their observations regarding the type of chromosomes present in disomic or tetrasomic condition differed According to them, the A genome has the genetic constitution AABCDDEFFF; B genome has the constitution ABCDEEFF; and C genome has the constitution ABCCDDEEF These authors opined that basic genomes originated from loss of different sets of chromosomes from an allotetraploid (2n¼ 20) rather than from duplication of different chromosomes, as proposed by Roăbbelen (1960)

Generating karyotypes based on meiotic chromosome preparations rather than mitotic ones has a number of advantages although the clumping of pericentromeric heterochromatin makes the resolution of individual chromosomes difficult Thus, combining pachytene and metaphase chromosome analysis for efficient physical mapping by FISH would be advantageous (Ziolkowski and Sadowski 2002)

observed in B nigra in early and late telophase The two Ag-NORs in B rapa represent one pair of active rDNA loci Maluszynska and Heslop-Harrison (1993), Snowdon et al (1997a), and Fukui et al (1998), on the contrary, reported five pairs of rDNA loci following in situ hybridization These represent both active and inactive rDNA sites while silver staining reveals only the sites with active rDNA It appears that four pairs of these sites are inactive in nucleolus formation in B rapa (Cheng and Heneen 1995) The observations on B nigra having six pairs not correspond with the earlier investigations of Sikka (1940) and Lan et al (1991), where only four satellite chromosomes were observed Roăbbelen (1960) also observed that only four chromosomes were associated with nucleoli at pachytene of meiosis in B nigra This et al (1990) also assigned rDNA markers to two pairs of B nigra

Table 2.5 Satellite chromosomes in Brassica and allied genera No satellite

Species chromosomes Reference

B nigra Sikka 1940; Roăbbelen 1960; This 1990; Lan et al 1991; Hasterok and Maluszynska 2000a

6 Cheng and Heneen 1995; Mackowiak and Heneen 1999; Hasterok et al 2005a B oleracea Sikka 1940, Wang and Luo 1987;

Olin-Fatih and Heneen 1992; Cheng et al 1995a; Armstrong et al 1998; Hasterok and Maluszynska 2000a; Ziolkowski and Sadowski 2002; Hasterok et al 2005a,b

B rapa Sikka 1940; Nishibayashi 1992; Olin-Fatih and Heneen 1992; Cheng and Heneen 1995; Cheng et al 1995a; Hasterok and Maluszynska 2000a; Hasterok et al 2005a,b; Lim et al 2005

B carinata Kulak et al 2002

B juncea Sikka 1940; Maluszynska and Hasterok 2005

4 Kulak et al 2002

B napus Olin-Fatih and Heneen 1992; Olin-Fatih 1994, 1996; Skarzhinskaya et al 1998 Snowdon et al 1997a; Hasterok and

Maluszynska 2000b; Kulak et al 2002 Raphanus sativus Mukharjee 1979

location of these rDNA sites, B rapa has 25S rDNA loci near the centromere of metacentric chromosomes 1, 4, 5, and Chromosome bears NOR and contains the fifth largest 25S rDNA locus extending over NOR and satellite Chromosome has a large 25S locus located interstitially and colocalized with a large 5S rDNA locus Short arms of chromosome and 10, the largest and smallest acrocentric chromo-somes, respectively, in B rapa genome have two more 5S loci (Snowdon et al 2002) Koo et al (2004) and Hasterok et al (2005a) also observed the same number of 45S and 5S rDNA loci at the same locations Of these 10 rDNA loci, only are active, distributed on the secondary constriction of chromosome 10 (Hasterok and Maluszynska 2000a) Koo et al (2004) studied pachytene bivalents and observed 5S rDNA loci on pericentromeric region of short arm of chromosomes and 10 and the long arm of chromosome The long arm of chromosome exhibited another 5S rDNA site, which was not detected in mitotic metaphase These authors believe that two closely linked 5S rDNA loci could not be detected in earlier investigations because of lower resolution of FISH on mitotic chromosomes Localization of 45S rDNA loci was revealed on pericentromeric regions of the short arm of chromosomes 1, 2, 4, and and the long arm of chromosome

Brassica oleracea genome has two 18S-5.8S-25S rDNA sites subtelomerically on the short arms of two satellited acrocentric chromosomes (nos and 7) The third one occurs adjacent to the centromere on the short arm of chromosome 2, which is submeta-centric On the long arm of this chromosome, 5S rDNA sequences are located with closely adjacent major and minor loci (Armstrong et al 1998) These results match those of Hasterok et al (2001, 2005a) and Snowdon et al (2002), who observed 5S rDNA genes in two closely adjacent loci on the long arm of a single large submetacentric chromosome Two acrocentric satellite-possessing chromosomes (nos and 7) have 25S loci at the terminal ends of their short arm, which extends over the satellite A novel 5S rDNA locus was also detected by Ziolkowski and Sadowski (2002) B nigra has three pairs of 25S loci; one pair is located on the short arm of chromosome and two pairs are located at the secondary constriction and satellite of chromosome pairs and Only these two pairs of loci are transcriptionally active (Hasterok and Maluszynska 2000c)

Chromosomes 4, 10, and 16 have colocalized both the gene sites (Kulak et al 2002) Employing imaging methods in combination with FISH, Kamisugi et al (1998) detected 25S/18S rDNA loci in the centromeric and distal regions of seven chromosomes in B napus While eight 5S rDNA loci were observed on five chromosomes mainly in the centromeric regions, two chromosomes carried both 25S/18S and 5S rDNA loci in close proximity Regarding their localization, according to Snowdon et al (1997a, 2000a), the largest site covers the satellite and short arm of the largest NOR-carrying chromosome The second largest is located on a telomeric NOR-like structure on the short arm of a large subtelocentric pair, and the smallest locus is at the telomere on the short arm of a smaller submetacentric chromosome The three other loci are localized at or near the centromeres of metacentric chromosomes About the origin of these rDNA sites carrying chromo-somes, Snowdon et al (1997a) inferred that the two largest noncen-tromeric signal blocks and the NOR-carrying chromosomes closely resemble those of B rapa and B oleracea Similarly, the three largest centromerically located loci in B napus match to those of B rapa Schrader et al (2000) indicated that in B napus, one of the pair with 5S rDNA gene sites belongs to B oleracea Additionally, two submetacentric chromosomes having two closely adjacent 5S DNA clusters belong to B oleracea The other four pairs probably derived from the B rapa progenitor A comparison of chromosome sets of B napus, B oleracea and B rapa revealed that B napus chromosomes carrying rDNA loci could be matched with those of constituent parents (Snowdon et al 2002) Chromosomes possessing rDNA loci could be identified based on size and centromere position The chromosomes belonging to A and C genomes could clearly be distinguished with minor discrepancies In general, these observations closely correspond to those of Kamisugi et al (1998) Maluszynska and Hasterok (2005), using two-color GISH, successfully discriminated partaking genomes in B juncea and assigned chromosomes to A and B genomes Molecular analysis also indicated that in allopolyploids, B nigra rRNA genes are dominant over those of B rapa, which are in turn dominant over B oleracea (Chen and Pickard 1997; Ge and Li 2007) However, according to Hasterok and Maluszynska (2000c), the number of Ag NORs in the alloploid species is equal to the sum of active NORs in diploid parental species, clearly indicating an absence of nuclear dominance in root meristematic cells

et al 1995) However, B napus is of very recent origin, and we also have the conflicting views that the partaking A and C genomes are unaltered to a large extent (Parkin et al 1995) In fact, Delseny et al (1990) have reported that rDNA-carrying chromosomes of B oleracea have not undergone any major structural changes since the evolution of B napus Earlier, Bennet and Smith (1991) suggested that there has been a large reduction in copy number of rDNA in present-day B napus as compared to ancestral forms, which is due to a reduction in B oleracea–type rDNA in existing B napus forms from a total copy number of 1,500 to one of 800, while B rapa–type rDNA is unaltered Maluszynska and Heslop-Harrison (1993) are of the view that a C-genome locus has been lost in B napus due to reduction in B oleracea–type rDNA However, Snowdon et al (1997a, 2000a) believe that both ancestral B oleracea loci are still present, with reduced rDNA copy numbers, as suggested earlier by Bennet and Smith (1991) They also proposed that the smallest and relatively insignificant rDNA locus from B rapa is absent in B napus On the whole, since substantial C-genome rDNA has been lost, it appears that A-genome rDNA is of greater genetic importance than C-genome rDNA in B napus

A comparative analysis clearly revealed the occurrence of poly-morphism in number and chromosomal distribution of rDNA loci among different ecotypes of a species and their population (Hasterok et al 2006) Inter- and/or intravarietal polymorphism was evident in B oleracea, B rapa, B carinata, B juncea, B napus, and Raphanus sativus It was also observed that Brassica species carrying A genome—B rapa, B juncea, and B napus—are highly polymorphic and contain high numbers of rDNA sites (Hasterok et al 2001)

and (3) three copies of the gene encoding acyl-CoA-binding protein in B rapa and B oleracea (Hills et al 1994) It is inferred that the number of three chromosome pairs carrying the 25S rDNA gene is basic for the family Brassicaceae The earlier view was that the three basic diploid species evolved from a common archetype following duplication of whole chromosomes (i.e., aneuploidy) accompanied by differentiation following structural changes The mapping data, in contrast, clearly discounts the role of polysomy or duplication of whole chromosomes (Quiros 1999)

Recent information emerging from use of molecular markers firmly disproves the theory of monophyletic origin and instead suggests a biphyletic origin of the diploid species It concludes that B oleracea/ B rapa originated from one archetype while B nigra originated from the other (Song et al 1990; Warwick and Black 1991; Pradhan et al 1992) Cytogenetical investigations preceeded in predicting this genetic divergence between B nigra and B oleracea/B rapa based on chromosome pairing in hybrids (Mizushima 1950a; Prakash and Hinata 1980) Subsequently, molecular analysis has been very revealing The first evidence came from nuclear RFLPs by Song et al (1988a), which was substantiated by other investigations Information from nuclear, chloroplast, and mitochondrial DNA RFLPs has established that the primitive genome diversified into two lineages and all the taxa in subtribe Brassicinae fall in these two lineages (Warwick and Black 1991; Pradhan et al 1992) This view also gets support from a comparative study of molecular markers (Lagercrantz 1998) and rDNA intergenic spacer (Bhatia et al 1996) The evolu-tionary divergence is also reflected in their cytoplasm (Palmer 1988; Warwick and Black 1991; Pradhan et al 1992) B oleracea and B rapa cytoplasms are closer to each other than either is to B nigra (Palmer 1988) Evidence also indicates that the A genome was derived in the distant past from an already existing C genome, as these two genomes have extensive genomic regions of conserved homology (Slocum 1989)

genomic number of the family, followed by tetraploidization before the separation of the Brassica and Arabidopsis lineages An interesting hypothesis proposed by Lagercrantz and Lydiate (1996), Lagercrantz (1998), and O’Neil and Bancroft (2000) envisages that (i) Arabidopsis shares common ancestry with Brassica crop species, and (ii) three ancestral species with x¼ and whose genomes were similar to Arabidopsis genome gave rise to a hexaploid following hybridization between them This was the ancestral archetype of A, B, and C genomes from which the basic genomes evolved through reduction in chromosome number by extensive chromosome fusion This view, known as the triplication theory, gets support from the fact that some loci are triplicated as detected by molecular markers (Cavell et al 1998; O’Neil and Bancroft 2000; Parkin et al 2002, 2003, 2005; Rana et al 2004; Lysak et al 2005, 2007; Park et al 2005; Yang et al 2005; Lim et al 2006; Matthew and Lydiate 2006; Nelson and Lydiate 2006; Ziolkowski et al 2006; Yang et al 2006) and also that diploid Brassica genomes contain approximately three times the DNA of Arabidopsis genome (Arumugnathan and Earle 1991) The event of hexaploidy occurred around 7.9 to 14.6 million years ago (Lysak et al 2005) However, the occurrence of a large proportion of heterochromatin, repetitive DNA (Gupta et al 1990, 1992; Iwabuchi et al 1991), transposable elements (TE) (Zhang and Wessler 2004; Gao et al 2005; Lim et al 2007), and the ancestral role of Arabidopsis would argue against this hypothesis Lukens et al (2004) also found no strong evidence of the role of the ancestral hexaploid genome Furthermore, considering that the ancestral species to the Brassicaceae was a tetraploid of 2n¼ 4x ¼ 16 (Henry et al 2006), it already explains the origin of species such as B nigra, also with 2n¼ 16, without the need to invoke another round of polyploidization or hexaploidy

Lim et al 2007) Surprisingly, the genome size of these basic species has remained practically unaltered in spite of changes in chromosome numbers and structure

Based on marker arrangement conservation, Truco et al (1996) proposed a model of genome evolution and phylogenetic relationships among the chromosomes of the three basic species considering two assumptions: that (1) A and C genomes are closely related, and possibly C genome is the predecessor of A genome; and (2) the genus Brassica is of biphletic origin It envisages that the ancient genome possessed at least five and no more than seven chromosomes B and/or C genome chromosomes evolved from six ancestral chromosomes (W1 to W6) (Fig 2.4) C genome chromosomes also gave rise to A genome chromosomes Two intermediate chromosomes Bx and Cx originated from W1 Bx produced B1, B2, B4, and B8 chromosomes and the Cx

Chromosomal changes Geographic isolation

Derived genomes Chromosomal changes

x=4, 5

TE 1st cycle of

allopolyploidy Aneuploidy

Diploid species genomes Structural changes 2nd cycle of allopolyploidy

Cultivated allopolyploids

Ancestral genome x=4 , ?

Z

Z Z Z

Z n

B C A

AC BC

AB

chromosome gave rise to A7 Chromosomes Bx and C1 were similar in their genetic content Chromosomes B7 and C9 might have originated from W6 or independently, one from W6 and other from a seventh ancestral chromosome, W7 These two chromosomes, B7 and C9, not share homology with any other group

In spite of their biphyletic origin, the three basic genomes still share regions of homology, as determined by Truco et al (1996), expressed in cM by adding the distance of chromosome segments sharing homology between the two genomes following the comparison of linkage maps of these species The lowest homology is between A and B genomes, which share 92.7 and 219.5 cM of their genomes respectively and results in up to six bivalents in hybrids between them (Prakash 1973a,b) Homology between B and C genomes is intermediate with 223 and 365.7 cM respectively and form up to four bivalents between them (Mizushima

C5 A5 A10

A4

C1

A1

C7

A7

C3

C8

B1

B8

B4

B2

C2 A9

A2 B7

C9 A8 A3 C6 A6 C4

B5 B3

B6

C×

B× W5 W3

W4

W6 W1

W2

1950a; Song et al.1993) The highest homology is observed between A and C genomes where they share 337.2 and 487.2 cM respectively and form up to nine bivalents (Olsson 1960b) ISSR data also reflected these relationships, as observed by Liu and Wang (2006) The average genetic distance between B rapa and B oleracea is 0.499, indicating close homology; between B rapa and B nigra, 0.528; and between B oleracea and B nigra, 0.615 showing clearly the divergence between A/C and B genomes In fact, the genomic contents of A and C genomes are equivalent, and rearrangements are the cause of difference in their chromosome number (Parkin et al 2003)

To summarize, basic Brassica genomes evolved and differentiated from an originally smaller genome Chromosome arrangements due to homoeologous recombination and hybridizations were the major factors in their stabilization These three species are, in fact, secondary polyploids with regions of shared ancestry As expected, duplications are widespread in these genomes

III GENOME MANIPULATION

A Resyntheses of Natural Allopolyploid Brassica spp

Table 2.7 Major investigations on artificial synthesis of natural allopolyploid species B carinata, B juncea, and B napus through sexual hybridization

Species Reference

B carinata (B nigra B oleracea Frandsen 1943; Mizushima 1950b; Pearson 1972; and reciprocals) Prakash et al 1984; Song et al 1993

B juncea (B rapa B nigra Frandsen 1943; Ramanujam and Srinivasachar and reciprocals) 1943; Olsson 1960a; Prakash 1973a,b; Campbell

et al 1990, 1991; Song et al 1993; Srivastava et al 2001, 2004; Se´guin-Swartz et al 2004 B napus (B rapa B oleracea

and reciprocals)

Oil rape U 1935; Karpechenko and Bogdanova 1937; Frandsen 1947; Rudorf 1950; Hoffmann and Peters 1958; Olsson 1960b; Gland 1982; Pra-kash and Raut 1983; Chen et al 1988a,b; Chen and Hennen 1989; Akbar 1989; Hossain et al 1990; Mithen and Magrath 1992; Song et al 1993; Ozminkowski and Jourdan 1994a,b; Beschorner et al 1995; Heath and Earle 1996; Girke et al 1999; Lu et al 2001; Rahman et al 2001; Zhang et al 2002; Happastadius et al 2003; Luhs et al 2003; Seyis et al 2003; Niu et al 2004; Zhang et al 2004; Abel et al 2005; Rahman 2005; Zhou et al 2007; Wen et al 2008 Forage rape Hosoda 1950, 1953, 1961; Feng 1955; Sarashima

1967, 1973; Hosoda et al 1969; Nishi et al 1970 Rutabaga Olsson et al 1955; Olsson 1960b; Hosoda et al

1963, 1969; Namai and Hosoda 1967, 1968; Kato et al 1968

Heading form Shinohara and Kanno 1961

Table 2.8 Major papers on synthesis of natural allopolyploid species through protoplast fusion

Brassica species Reference

carinata Narasimhulu et al 1992; Jourdan and Salazar 1993 juncea Campbell et al 1990, 1991; Se´guin-Swartz et al 2004;

Bhat et al unpubl

The objectives in these allopolyploid syntheses vary, from purely academic (e.g., Song et al 1993; Srivastava et al 2001, 2004), to developing agricultural forms that include early and productive B carinata forms for Indian conditions (Prakash et al 1984), high-seed-yielding B juncea (Olsson 1960a; Prakash 1973a), early-maturing B napus suitable for the Indian subcontinent (Prakash and Raut 1983; Akbar 1989), productive oil seed B napus (Olsson 1960b; Seyis et al 2003), fodder forms of B napus (Namai and Hosoda 1967, 1968; Ellerstroăm and Sjoădin 1973), root-forming sweeds or rutabagas (Olsson et al 1955; Kato et al 1968; Namai and Hosoda 1968), and a new head-forming vegetable form (Shinohara and Kanno 1961; Takeda 1986) A major objective in B napus syntheses in recent years has been the modification of oil and meal quality (Lu et al 2001; Luăhs et al 2003; Seyis et al 2005) and incorporation of yellow seed coat color (Shirzadegan and Roăbbelen 1985; Liu and Gao 1987; Chen et al 1988; Tang et al 1997; Meng et al 1998; Baetzel et al 1999; Rahman 2001; Wen et al 2008) Initially these studies were carried out chiefly in Japan, Sweden, Germany, and India, and later in other countries In fact, resynthesis has been widely attempted for improvement of B napus (Olsson and Ellerstroăm 1980; Chen and Hennen 1989b; Luăhs et al 2002; Friedt et al 2003)

enlarging nuclear variability, this methodology generates novel combinations of cytoplasmic organelles and has received considerable attention in recent years Synthesis of somatic hybrids of B napus by Schenck and Roăbbelen (1982) was the earliest success Most reports on somatic hybridization relate to synthesis of B napus Ozminkowski and Jourdan (1994a,b) and Heath and Earle (1996) reconstructed B napus both sexually and following somatic cell fusion B napus allohaploids have also been synthesized by fusing pollen protoplasts of B oleracea var italica and haploid mesophyll protoplasts of B rapa; Fan et al (2007) present the first report about a hybrid formation between two haploid protoplasts Hybrids could be obtained faster through somatic fusion because of avoidance of chromosome doubling in F1 sexual hybrids for restoring fertility Somatic hybrids have also been obtained involving B carinata and B juncea (Table 2.8) These investigations on somatic hybridizations represent significant break-throughs in interspecific hybridizations

Synthetics obtained following sexual hybridization and chromo-some doubling have the sum of the parental chromochromo-some number The somatic hybrids also have, in general, these summations However, in some somatically produced B napus, there were deviations, where the plants possessed variable chromosome number ranging from 33 to 57 (Table 2.9) These resulted from triple fusions, such as one B oleracea and two B rapa protoplasts (2n¼ 58) or vice versa, resulting in digenomic hexaploid AAAAACC (2n¼ 58) and AACCCC (2n ¼ 56) plants (Terada et al 1987; Heath and Earle 1996) Aneuploids with somatic chromosome number 33, 49, 54, 57 were also recorded These probably originated by chromosome elimination during regeneration and subsequent development of plants Somatic hybrid plants of B carinata had the normal chromosome number of 34 (Narasimhulu et al 1992; Jourdan and Salazar 1993) Campbell (1993), Se´guin-Swartz et al (2004), and Bhat et al (unpublished) also observed the normal somatic chromosome number in B juncea somatic hybrid plants (Table 2.9)

advancing generations, and meiotic stabilization with regular bivalent formation was achieved by the amphidiploid (A3) generation

Sexually obtained synthetics had reduced pollen and seed fertility in early generations, sometimes as low as 6% in B juncea (Olsson 1960a) With stabilization of meiosis and selection, fertility improved considerably By the A5 generation, the attained fertility was much higher A7 generation plants had fertility comparable to naturally occurring forms (Table 2.10) Somatic hybrids also had very low pollen fertility and seed set Pollen was ineffective in producing seeds on selfing or on pollinations to natural forms of B carinata (Jourdan and Salazar 1993) Similar observations were recorded for B napus by Rosen et al (1988), Sundberg et al (1987), and Heath and Earle (1996, 1997) Since the plants had more or less regular meiosis, the reasons for a high degree of sterility are unknown

The organellar constitution of somatic hybrids does not follow any pattern; all possible combinations of mitochondria and chloroplast genomes are observed in addition to frequent intergenomic mitochon-drial recombination A majority of B napus somatic hybrids contain B rapa chloroplast; some have both chloroplast types (heteroplasti-dic); and only a few have B oleracea chloroplasts They contain mostly B rapa and recombinant mitochondria Some plants also have a mix of mitachondria and chloroplast genomes of B rapa and B oleracea Most B carinata plants contain both chloroplast and mitochondrial genomes from B nigra, but some combine these from both the parents A similar phenomenon is observed with in B juncea, where combinations of chloroplast and mitchondria parental genomes have been obtained as shown in Table 2.9 (Bhat et al unpublished)

B Agronomic Potential of Synthetics

modified fatty acid composition, particularly low erucic acid, have been created through resynthesis in B napus (Chen and Hennen 1989b; Lu et al 2001; Luăhs et al 2002, 2003; Seyis et al 2005) Synthetic rapeseed with high erucic acid content for industrial use has also been produced (Chen et al 1989a; Luăhs and Friedt 1994, 1995a,b; Weir et al 1997; Luăhs et al 1999a,b; Han et al 2001) Heath and Earle (1996, 1997) introduced a nonshattering trait and large-size seeds in somatic hybrids of B napus These authors also obtained B napus somatic hybrids that were low in linolenic acid (Heath and Earle 1997) and high in erucic acid content (Heath and Earle 1995) Synthetic self-incompatible B napus lines have recently been obtained through sexual hybridizations for developing commercial F1hybrids (Rahman

2005)

Fodder forms of B napus have been synthesized in Japan and Sweden using leafy and root-forming forms of B rapa—ssp chinensis, pekinensis, narinosa, nipposinica, and rapa Hosoda (1950) bred a fodder rape ‘CO’, which was very popular in Japan because of its vigorous growth and winter hardiness A novel synthetic head-forming vegetable type has been developed in Japan from the cross B oleracea var capitata B rapa ssp pekinensis It is a popular vegetable form released in 1968 under the name ‘Hakuran’ It has soft leaves, fewer fibers, tastes like heady lettuce, and possesses high degree of resistance to soft rot (Shinohara and Kanno 1961; Takeda 1986) Such a type has also been produced using the same parents through protoplast fusion (Taguchi and Kameya 1986)

be integrated into high-yielding varieties either by developing semisynthetic forms or in backcross breeding programs (Kraling 1987; Friedt et al 2003) Many studies have suggested that heterosis for seed yield in intervarietal hybrids is positively correlated with genetic distance (Jain et al 1994; Ali et al 1995; Diers et al 1995; Seyis et al 2003; Shen et al 2003; Burton et al 2004) The variability and genetic distance of synthetics from cultivars in cultivation can be usefully exploited for generating both highly productive hybrids and genetically enhanced cultivars Seyis et al (2006) demonstrated the potential of synthesized B napus for developing experimental hybrids having high yields

C Diploidization of Allopolyploid Species

pairing However, this view was later discounted (Busso et al 1987) By observing frequent intergenomic recombination in a B rapa–B alboglabra monosomic addition line but not in trigenomic AAC hybrids, Chen et al (1992) proposed that it could be due to a pairing control mechanism Jenczewski et al (2002) postulated a pairing regulator gene for diploidlike meiotic regime in an induced autote-traploid of B oleracea Later, a hypothesis that envisions the presence of a major gene PrBn (Pairing regulator Brassica napus) in alloploid B napus was proposed by Jenczewski et al (2003) These authors studied the chromosome pairing in low- and high-bivalent-forming haploids of B napus and observed that chromosome pairing patterns are inherited in a Mendelian way, indicating the presence of a major gene for restricting the homoeologous pairing It was also suggested that since regular bivalents are observed in all B napus accessions, regardless of bivalent frequency in their haploids, PrBn could contribute to the regularity of chromosome pairing It could be ineffective at hemizygous stage or at least less efficient as compared to at the diploid state (Jenczewski and Alix 2004) PrBn gene has been mapped on a C genome chromosome and displays complete pene-trance Additionally, three to six minor QTL/BTL have slight additive effect on pairing without any interaction with PrBn However, a number of other loci interact epistatically with PrBn (Liu et al 2006)

pressure on alien nuclear genome and brings about a harmoneous interaction between cytoplasm and both the nuclear genomes in the new environment To understand the process of changes in the genomes, Song et al (1993) developed a series of synthetic alloploids of B carinata, B juncea, and B napus following reciprocal hybridizations and characterized them for RFLP patterns of nuclear and cytoplasmic genomes It was observed in subsequent generations that frequency of genome changes are associated with genetic divergence of constituent diploid parents: the more the genetic divergence, the higher the frequency of changes (Song et al 1995) These changes could have resulted from chromosome rearrangements, point mutations, gene conversion, and DNA methylation Interge-nomic homologous recombination could lead to chromosome rearran-gements and provide opportunities for gene conversion–like events (Osborn 2004; Pires et al 2004, 2006) It has been suggested that extensive genome changes occur during early generations of poly-ploidy, and this accelerates the evolutionary processes (Song et al 1995; Lukens et al 2006) Also, intergenomic heterozygosity and epigenetic changes give rise to new variations crucial to their ecological success (Schranz and Osborn 2004) Another factor in stabilizing the chromosome pairing may be the role of rRNA genes It has been reported in a number of allopolyploids that rRNA genes from only one parent are transcribed while the transcription of such genes of the other parent is suppressed: a phenomenon referred to as nucleolar dominance A hierarchy of nucleolar dominance has been demonstrated to be B nigra > B rapa > B olerace in three Brassica allotetraploids (Chen and Pikaard 1997; Pikaard 2000; Ge and Li 2007) These results suggest that nucleolar dominance may contribute decisively in preferential stabilization of chromosomes from rRNAs-donor parent

D Raphanobrassica

quality of fodder radish with winter hardiness and high productivity of B oleracea In fact, several superior lines designated as Radicole were developed at Svaloăf, Sweden (Olsson and Ellerstroăm 1980) and the Scottish Plant Breeding Institute (McNaughton 1982) Some of the strains exceeded forage rape in fresh weight and dry matter yield by 20% They also have resistance to clubroot and downy mildew Somatic hybrids were generated to introgress clubroot resistance from radish to B oleracea, and these did in fact posses high degree of resistance to clubroot (Hagimori et al 1992; Yamanaka et al 1992) Another synthetic alloploid involving radish is Raparadish (Brassicor-aphanus, 2n¼ 38) It was obtained from the cross B rapa  R sativus primarily for determining the homoeology between the two genomes (Terasawa 1932; Mizushima 1950b) Later the synthesis aimed at developing a fodder type (Tokumasu and Kato 1976, 1988) and transferring resistance to beet cyst nematode from Raphanus to B rapa (Dolstra 1982) Raparadish grows vigorously, combining the rapid growth with resistance to beet cyst nematode and clubroot (Lange et al 1989) A detailed cytogenetical study on Brassicoraphanus synthe-sized for fodder has been carried out by Tokumasu and Kato (1976) and Matsuzawa et al (2000), who recorded 1519 IIỵ 08 I at M1 of meiosis, with occasional occurrence of a tri- or quadrivalent However, the pollen fertility was low (0–89%) and the seed set was 0.01 to 0.1 seeds per siliqua after self- and open pollinations, respectively Some of the A3 generation plants with yellow flowers showed considerably improved fertility It was suggested that the genes for flower color are closely linked with those controlling embryo development The genetic reconstitution due to intergenomic segmental exchange pro-motes development of embryos leading to higher fertility in yellow-flowered plants (Kato and Tokumasu 1976; Tokumasu and Kato 1988) Matsuzawa et al (2000) reported that two Raparadish lines had potential to be used as fodder Further, they also obtained it from the reciprocal cross R sativus  B rapa, which showed mostly regular meiosis with 19 bivalents

E Higher Allopolyploids in U Triangle Species through Protoplast Fusion

genomes (see Prakash and Hinata 1980) In recent years, the three genomes have been brought together through protoplast fusion (Table 2.11) to make use of them as bridge species for transferring traits of agronomic importance, particularly resistance to fungal diseases, such as blackleg and clubroot caused by Phoma lingam and Plasmodiophora brassiceae, respectively These are serious diseases on B napus in Europe, Australia, and Canada Genes conferring resistance are available in B nigra and natural alloploid species containing B nigra genome, specically B carinata and B juncea (Sacristan and Gerdeman-Knoărck 1986; Sjoădin and Glimelius 1989a,b; Zhu and Spanier 1991) The other objectives are incorporation of herbicide resistance and cytoplasmic male sterility (Kao et al 1992; Hansen and Earle 1995; Arumugam et al 1996)

IV WIDE HYBRIDIZATION

Hybridization in brassicas goes back to early 19th century when Sageret (1826) obtained intersub-tribal hybrid Raphanus sativus B oleracea and Herbert (1847) produced interspecific hybrid B napus B rapa Cytogenetical interest following determination of chromosome numbers gave a boost to wide hybridization Initial attempts at hybridizations were for elucidating genomic homoeology Later, attention shifted to utilizing wide hybridization for expanding genetic variability, introgressing nuclear genes that conferred desirable agro-nomic traits or cytoplasmic genes for inducing male sterility Chromo-some addition lines have also been generated to locate genes on specific chromosomes and for construction of genetic maps During the last 30 years, in vitro techniques such as ovary and embryo culture and protoplast fusion have been employed successfully to obtain a large number of sexual and somatic hybrids

A Sexual Hybrids

Limited investigations have been undertaken to determine the details of postfertilization barriers Lack of a functional endosperm or its early degeneration appear to be the major reasons for abortion of hybrid embryos

Ways devised to overcome these hybridization barriers include grafting, mixed pollination, bud pollination, and stump pollination (Hosoda et al 1963; Sarashima 1964; Namai 1971) Kameya and Hinata (1970) succeeded in performing in vitro fertilization and obtained inter-specific hybrids A modified technique of placental pollination was used by Zenkteler (1990) Embryo rescue technique has been an effective technique for overcoming postfertilization barriers and is used exten-sively to obtain wide hybrids Japanese scientists, particularly Nishi and his group (1959), pioneered it in Brassica in the late 1950s (Nishi et al 1959) Sequential culture, which involves successive culture of ovaries, ovules, and seeds/embryos, is more effective than simple ovary or ovule culture (Shivanna 1996; Wen et al 2008)

Although wide hybridizations in Brassica have been carried out for a long time, here we define it in terms of hybridizing species of secondary and tertiary gene pools A pioneer in this area was Mizushima (1950a,b, 1968) who attempted such hybridizations involving wild germplasm Subsequent extensive investigations were by Harberd and McArthur (1980), who reported nearly 50 distant hybrids in which a majority were intergeneric At present, hybridization between wild and crop species has become a routine The last 20 years have witnessed a large number of sexual hybrids comprising interspecific, intergeneric, intersubtribal, and intertribal combinations These hybrids and their meiotic behavior are listed in Table 2.12

Sexual hybrids are characterized by a highly disorganized meiosis, particularly when both parents are diploid Chromosomes, due to the absence of a homologous partner, remain mostly as univalents but occasionally undergo pairing and also form bivalents in a very low fre-quency Bivalents, when they occur, are mostly rod-shape monochias-mates and rarely ring shape with multiple chiasmata Multivalents in diploid hybrids occur only rarely However, a variable number of bivalents and frequent trivalent/quadrivalents are formed in triploid (tetraploid  diploid) and tetraploid (tetraploid  tetraploid species) combinations Harberd and McArthur (1980) observed a close relation-ship between mean chromosome number and mean bivalent frequency at three ploidy levels (Table 2.13)

Quiros et al 1988), B fruticulosa B nigra (2n ¼ 16, II, Mizushima 1968), B nigra Hirschfeldia incana (2n ẳ 15, III ỵ II, Quiros et al 1988), Erucastrum canariense B oleracea (2n ¼ 18, II, Harberd and McArthur 1980), E cardaminoides B oleracea (2n ẳ 18, IV ỵ III ỵ II, Mohanty 1996), and Enarthrocarpus lyratus B rapa(2n ¼ 20, III ỵ II, Gundimeda et al 1992) The triploid and tetraploid hybrids where higher associations have been observed include B juncea Diplotaxis virgata (1 IV/2 III, Inomata 2003), B napus Hirschfeldia incana (1 IV, Kerlan et al 1993), Diplotaxis viminea B napus (2 IV, Mohanty 1996), and Diplotaxis erucoides B napus (1 IV, Delourme et al 1989)

Hybrids between the diploids were absolutely pollen and seed sterile while triploid and tetraploid hybrids had a little pollen and seed fertility Bivalents and higher associations may be interpreted to result from archaic homology within the chromosomes of the same genome (autosyndesis) or because of intergenomic homoeology (allosyndesis) However, it is difficult to interpret the pairing precisely in terms of auto- or allosyndesis since there is little information on the extent of autosyndesis observed through chromosome pairing in haploids Mizushima (1950a, 1968, 1980) made some observations on the extent of allosyndesis between a limited number of genomes; Harberd and McArthur (1980) could not arrive at any definite conclusion What can be stated safely is that intrageneric homoeology is not always higher than intergeneric homoeology With the progress in GISH techniques, the degree of auto-and allosyndesis can be ascertained precisely in wide hybrids

An interesting cytological phenomenon was observed in hybrids between Brassica species and Orychophragmus violaceus (2n¼ 24) O violaceus is cultivated in China as an ornamental plant and has desirable oil quality Hybrids with all the six crop species have been obtained with O violaceus always the pollen parent Chromosomes remain unpaired as univalents in hybrid cells, and separation of parental genomes occurs regularly during mitotic and meiotic

Table 2.13 Mean chromosome number and bivalent frequency at three ploidy levels in the tribe Brassiceae

divisions (Li et al 1995, 1996, 1998a,b, 2003; Li and Heneen 1999) During mitosis, any of three situations may occur, and subsequently the chromosomes are doubled following chromosome duplications in daughter cells:

1 Complete separation of parental genomes results into cells with haploid and diploid complements of the two parents

2 Partial separation leads to inclusion of some chromosomes of one parent with the haploid complement another genome producing hypo- and hyperdiploid cells

3 During partial separation, chromosomes of either parent are included in the genomes resulting into substitution lines Hybrids B oleracea  O violaceus had the sum of parental chromosomes (2n¼ 21) in mitotic and meiotic cells B rapa  O violaceus hybrids were mixoploid with somatic chromosome number ranging from 23 to 42 but cells with 2n¼ 34 predominating Partial separation of parental genomes occurred during mitosis, leading to the addition of some Orychophragmus chromosome to the B rapa complement Hybridization with B nigra produced a majority of maternal type F1 plants (2n¼ 16) and some mixoploids Hybrids with the three tetraploid species showed variable chromosome numbers: B carinata  O violaceus (2n ¼ 12 34), B juncea  O violaceus (2n¼ 30 42), and B napus  O violaceus (2n¼12–38) Partial and complete separation was more frequent in B juncea  O violaceus and B carinata O violaceus hybrids respectively Somatic cells and PMCs with additional O violaceus chromosomes often occurred in B juncea  O violaceus and not in other two combinations It was proposed that differences in the duration of somatic cell cycles of two parents cause partial or complete genome elimination Based on cytological observations, Li and Heneen (1999) and Li et al (2003) proposed that B genome accounts for complete and partial genome separation in B carinata; both A and B genomes contribute to this separation in B juncea; and A genome is more influential than C genome in B napus during mitosis and meiosis Genetic information from Orychophragmus has been introgressed into Brassica genomes as demonstrated by GISH (Hua and Li 2006) Employing these hybridiza-tions, it may be possible to produce Brassica aneuploids and haploids and subsequently homozygous lines (see review by Li and Ge 2007)

B Somatic Hybrids

improvement As mentioned earlier, barriers to sexual hybridization are easily overcome through the somatic route Recent years have seen spectacular developments in protoplast fusion technology, particularly in Brassicaceae Also, Brassica and related genera are very amenable to tissue culture techniques Cell fusion allows cytoplasmic substitutions and generation of novel cytoplasmic variability through organellar reassortment and DNA recombination, a phenomenon not possible during sexual hybridization Because of these advantages, cell fusion has become a promising methodology for introgressing desirable alien genes in crop cultivars (see reviews by Glimelius 1999a; Christey 2004; Navra´tilova´ 2004; Liu et al 2005) The first successful report of cell fusion in Brassicacea was by Kartha et al (1974) and involved protoplasts of B napus and Glycine max A major breakthrough was made by Gleba and Hoffmann (1979, 1980) when, following fusion of B rapa and Arabidopsis thaliana protoplasts, an intertribal hybrid was successfully regenerated This event achieved the distinction of first somatic hybrid in Brassicaceae, although no offspring could be obtained from it Subsequently, a large number of somatic hybrids have been obtained that combine crop species with taxonomically divergent wild germ pools (Tables 2.14, 2.15) These represent inter-specific, intergeneric, and a substantial number of intertribal combina-tions from six different tribes— Sisymbrieae (Arabidopsis thaliana, Camelina sativa); Arabideae (Armoracia rusticana, Barbarea stricta, B vulgaris); Drabeae (Lesquerella fendleri); Lepidieae (Capsella bursa-pastoris, Lepidium, Thlaspi caerulescens, T perfoliatum); Lunarieae (Lunaria annua); and Hesperideae (Matthiola incana)—and a few subtribes— Raphananiae (Raphanus, Trachystoma), and Moricandii-neae (Moricandia) Another species, Orychophragmus violaceus, pre-viously included in subtribe Moricandiinae but now excluded from tribe Brassiceae (Go´mez-Campo, personal communication), has also been hybridized The priorities have shifted to practical utilization and efforts are toward introgressing nuclear and cytoplasmic genes from wild relatives to crop species The desirable traits targeted include:

 C3–C4 intermediate photosynthetic system (from Moricandia arvensis and M nitens)

 Resistance to: club root (from Raphanus sativus); alternaria leaf spot (from Sinapis alba, Camelina sativa, Capsella bursa-pastoris); beet cyst nematodes (from Sinapis alba, C bursa–pastoris); blackleg (from B tournefortii, Sinapis arvensis, Arabidopsis thaliana)

 High lesquerolic acid content (from Lesquerella fendleri)

 High linoleic and palmitic acid content (from Orychophragmus violaceus)

 Cold tolerance (from Barbarea vulgaris)

 Zinc and cadmium hyperaccumulation (from Thlaspi carulesens)  Cytoplasmic genes for inducing male sterility from a number of

wild species

Somatic hybrids in several combinations—for example, Camelina sativa þ B carinata (Narasimhulu et al 1994), Camelina sativa þ B oleracea (Hansen 1998) and Barbarea vulgarisỵ B napus (Fahleson et al 1994b)—could not be established to viable field plants It appears that although protoplast fusion removes fertilization barriers, genetic incompatibilities due largely to phylogenetic distances still prevail at the somatic level, affecting differentiation, growth, and development of normal plant parts, particularly floral organs, thus leading to sterility However, other distant hybrids, particularly with Arabidopsis thaliana, probably could be established successfully, due to its small genome and also with little repetitive DNA, which promotes greater homoeology between the partaking genomes (Hansen 1998) Lunaria annua ỵ B napus hybrid has been reported only up to callus stage (Craig and Millam 1995) Somatic hybrids have been identified and characterized by a range of techniques including morphological attributes, chromosome number, meiotic behavior, fertility, DNA content estimation, isozyme analysis, RFLP, AFLP, and cytoplasmic constitution However, there are not many reports on chromosome cytology, and in several studies, the ploidy status has been determined by estimating DNA content

Somatic hybrids, in general, are intermediate in morphology between the fusion partner species This expression is particularly relevant for leaves and frequently for flower characteristics Floral abnormalities are also observed and include to petals and multiple carpellike structures in A thalianaỵ B napus (Bauer-Weston et al 1993); or petals in Thlaspi perfoliatum þ B napus (Fahleson et al 1994a); enlarged, distorted or globular pistils, and reduced or missing stamens in L fendleriỵ B napus (Skarzhniskaya et al 1996); stamens with stunted filaments in R sativus ỵ B napus (Lelivelt et al 1992); and shorter, thicker pistils in D harraỵ B napus (Klimaszewska and Keller 1988)

cybrids (Navara´tilova´ et al 1997; Hu et al 2002a) Sometimes the regenerants from the same fusion events have different chromosome numbers (Hoffman and Adachi 1981) Meiotic studies have been carried out in some of these hybrids which include intergeneric and a few intertribal combinations Besides occurrence of normal bivalents as the sum of parental chromosomes at M1, higher associations such as tri- and quadrivalents, in addition to univalents, were also encountered (Table 2.15) Interestingly, the intersubtribal hybrid Moricandia arvensisỵ B juncea exhibits up to three quadrivalents (Kirti et al.1992b) Such higher associations suggest intergenomic chromosome homoeology Post-metaphase-1 stages have not been investigated, but it appears that meiosis proceeds normally, as can be inferred from normal pollen formation in many of the hybrids Intergenomic chromosome recombination due to allosyndesis has been documented in some somatic hybrids, such as Moricandia arvensisỵ B juncea, D catholica þ B juncea, Trachystoma ballii þ B juncea Genomic in situ hybridization has been used effectively to determine the alien chromosome status at mitosis in some somatic hybrids and their progeny, for example, in Eruca sativaỵ B napus (Fahleson et al 1988, 1997), Lesquerella fendeleri ỵ B napus (Skarzhniskaya et al 1996), Crambe abyssinicaỵ B napus (Wang et al 2004a,b), and Sinapis albaỵ B napus (Wang et al 2005a) GISH was also employed to dectect intergenomic homoeologous recombi-nation in these hybrids

A majority of somatic hybrid plants were seed sterile when selfed However, some fertile hybrids were also obtained, including inter-tribal hybrids Arabidopsis thalianaỵ B napus (Forsberg et al 1994), Thlaspi perfoliatum ỵ B napus (Fahleson et al 1994a), Capsella bursa-pastorisỵ B oleracea (Sigareva and Earle 1999b), Orychophra-gmus violaceusỵ B napus (Hu et al 2002b), and Moricandia arvensis ỵ B oleracea (Ishikawa et al 2003), and a few intergeneric and interspecific ones Wherever pollen fertility was observed, it was quite low in A1generation With a few exceptions seed fertility was

1998) and Orychophragmus violaceusỵ B napus (Hu et al 2002b) In many instances, to produce reasonably fertile hybrids, irradiated protoplasts from wild species have been used, eliminating substantial amounts of alien DNA to obtain asymmetric hybrids that contain varying amounts of alien DNA from donor species

Somatic hybrids present three possibilities with respect to their cytoplasmic genomes: (1) parental genomes segregate to homogeneity during cell division, (2) both the parental genomes occur as a mixed population, and (3) novel genome constitution is generated when parental genomes undergo recombination Segregation of chloroplasts is independent of mitochondrial segregation In Brassiceae, mitochon-drial recombination has been observed to occur frequently and is very well documented (Glimelius 1999a) In sharp contrast, intergenomic chloroplast recombination is rare Two chloroplast types occurring in mixture is also rare, and there is no information about whether this mixture persists in subsequent generations, wherever it does occur

It is observed in interspecific, intergeneric, and intertribal somatic hybrids that chloroplast from crop species are generally favored This biased segregation is attributed to genetic divergence, ploidy level differences between the parental species, and rate of chloroplast division (Sundberg and Glimelius 1991) Also, plastome-genome incompatibility may be a factor A higher ploidy level of one of the parental species contributes a larger number of chloroplasts per cell (Butterfass 1989) Since in most of the hybrid alloploid species B napus or B juncea has been one of the parents, they contribute more chloroplasts to the fusion products than the wild diploid parent However, it is not possible to predict which parental chloroplast will establish in hybrids The intertribal somatic hybrid Lesquerella fendleri ỵ Brassica napus has been reported to have mixed chloroplasts (Skarzhinskaya et al 1996), as has intergeneric hybrid Diplotaxis catholica ỵ B juncea (Mohapatra et al 1998) A report documented the occurrence of intergeneric chloroplast recombination in the somatic hybrid Trachystoma balliỵ B juncea (Baldev et al 1998) where the recombination has occurred in a single copy region and remains stable over the generations Also, it caused no imbalance in the recombinant plastomes in terms of chloroplast-related functions In addition, choroplast recombination was also indicated in the somatic hybrid B oleraceaỵ Raphanus sativus (Kanno et al 1997)

parents or entirely new and unique ones not found in parental types (Belliard et al 1979) While investigating seven sets of interspecific, intergeneric, and intertribal combinations, Landgren and Glimelius (1994b) observed that 43% to 95% of the hybrids had mt DNA rearrangements Recombination hot spots have also been found; for example, Mohapatra et al (1998) suggested that intergenomic recombi-nation is preferred at specific sites in somatic hybrids Diplotaxis catholicaỵ B juncea The cox2 coding region may serve as an active site for inter- or intragenomic recombination (Stiewe and Roăbbelen 1994; Liu et al 1995) Conflicting views are reported regarding the segregation of mitochondria in somatic hybrids According to Landgren and Glimelius (1990, 1994a,b), crop types are favored In cybrids where CMS line is one of the parents, the mt segregation was slightly biased toward the CMS parent (Mukhopadhyay et al 1994; Liu et al 1996) However, many of the somatic hybrids have recombinant mitochondrial genomes, although a lack of recombination has also been documented, for example, S albaỵ B napus (Lelivelt et al 1993), Lesqurella fendleri ỵ B napus (Skarzhinskaya et al 1996), and Moricandia arvensis ỵ B oleracea (Ishikawa et al 2003)

Somatic hybridization in Brassicaceae has crossed all the intergeneric and intertribal barriers However, the results are not too encouraging because of a general high degree of sterility or severe intergenomic incompatibilities leading to many abnormalities Asymmetric hybrids in such instances appear to be more promising as crop species, tolerating only a fraction of alien genetic content rather the whole genome for integrated functioning of the system Such asymmetric fusions have been obtained by irradiating donor (wild) protoplasts to induce double-strand DNA breaks Most of intertribal hybrids are asymmetric and show improved fertility One of the limiting factors in gene transfer from wild to crop species is the very low level or complete absence of intergenomic chromosome pairing, which implies that overall genome structures interfere with free gene flow across the generic boundaries Never-theless, several traits of agronomic importance have been observed in somatic hybrids and in some cases, the genes have been introgressed to crop species, as revealed by progeny plant analysis

Examples include:

1 Raphanus sativus ỵ B napus: express resistance to beet cyst nematode—Heterodera schachtii (Lelivelt and Krens 1992) Sinapis albaỵ B napus: possess high level of beet cyst nematode

(Heterodera schachtii) resistance (Lelivelt et al 1993)

4 B tournefortiiỵ B napus: express resistance to blackleg (Phoma lingum) (Liu et al 1995)

5 Moricandia arvensisỵ B napus: C3-C4 character is expressed at both the physiological and anatomical level (O’Neill et al 1996)

6 Moricandia nitensỵ B oleracea: C3C4 character expressed as transition between the parents (Yan et al 1999)

7 Sinapis alba ỵ B oleracea: exhibit resistance to Alternaria brassicicola and Phoma lingam (Ryschka et al 1996; Hansen and Earle 1997; Sigareva et al 1999)

8 Capsella bursa-pastoris ỵ B oleracea: exhibit high degree of resistance to Alternaria brassicicola (Sigereva and Earle 1999b) Thlaspi caerulescensỵ B napus: accumulate high levels of zinc and cadmium, which would have been toxic to B napus (Brewer et al 1999)

10 Camelina sativaỵ B oleracea: possess resistance to Alternaria (Sigareva and Earle 1999a )

11 Lesquerella fendleriỵ B napus: contain high amount of erucic acid for industrial purpose (Glimelius 1999b; Schroăder-Pontok-pidan et al 1999)

12 Arabidopsis thaliana ỵ B napus: possess resistance to Lepto-sphaeria maculans (Bohman et al 2002)

13 Orychophragmus violaceusỵ B napus: contain high content of palmitic and linoleic acid expressed in the progeny plants (Hu et al 2002b; Ma and Li 2007)

14 Sinapis avensisỵ B napus: possess resistance to blackleg in the hybrids and progeny (Hu et al 2002a)

15 Crambe abyssinicaỵ B napus: progeny contain high amount of seed erucic acid (Wang et al 2004b)

Several CMS systems in B napus and B juncea have been obtained following protoplast fusion These are based on Arabidopsis thaliana, Brassica tournefortii, Diplotaxis catholica, Eruca sativa, Moricandia arvensis, Orychophragmus violaceus, Raphanus, Sinapis arvensis, and Trachystoma ballii

C Introgression of Genes

alternaria leaf spot (Alternaria spp.), white rust (Albugo candida), black rot (Xanthomonas campestris pv campestris), soft rot (Erwinia carotovora), and sclerotinia stem rot (Sclerotinia sclerotiorum) are important Nuclear genes conferring resistance to these diseases have been transferred from related species and alien wild germplasm Other desirable traits, particularly the fertility restoration for several CMS systems, have also been incorporated (Table 2.16) These genes have been introgressed, taking advantage of nonhomologous allosyndetic recombination in early backcross generations following sexual/somatic hybridizations and also through generating chromosome addition lines In recent years, efforts have been made to identify alien introgres-sions to specific chromosomes through GISH and molecular markers Results of GISH are not very encouraging primarily due to unusually low copy number of repeat sequences in chromosome arms, which form the basis of GISH signals Nevertheless, examples of detecting intro-gressions include B napus from Lesquerella fendleri (Skarzhinskaya et al 1998), Raphanus sativus (Voss et al 2000), Sinapis arvensis (Snowdon et al 2000b), Crambe abyssinica (Wang et al 2004b), and Orychophragmus violaceus (Li and Ge 2007)

V CYTOPLASMIC SUBSTITUTION AND MALE STERILITY

During the last 50 years, several investigations have reported the expression of a high degree of heterosis for seed yield in intervarietial hybrids of B rapa, B juncea, and B napus (see Fu and Yang 1998) However, in earlier years, full potential of heterosis could not be exploited in B juncea and B napus as these are predominantly self-fertilized crops A suitable pollination control mechanism is required to produce commercial hybrid seed A cytoplasmic male sterility (CMS) fertility restoration system is an excellent potential means to facilitate hybridization because it is easy to maintain CMS, a maternally inherited inability to produce fertile pollen, is encoded in the mitochondrial genome and can arise spontaneously due to mutation in the genome (autoplasmy) or can be expressed following cytoplasmic substitutions due to nuclear-mitochondrial incompatibility (alloplasmy)

(B oleracea var italica) by placing its nucleus in B nigra cytoplasm A large number of alloplasmics have been reported since Brassica coenospecies is a rich repository of diverse mitochondrial genomes, as revealed by RFLP studies (Pradhan et al 1992) By combining these cytoplasms with crop nuclei, a spectrum of alloplasmic lines of diverse origin expressing male sterility has been obtained, particularly in B juncea (Table 2.17) (see reviews by Delourme and Budar 1999; Prakash 2001; Budar et al 2004)

Cytoplasmic male sterile lines have been developed following backcrossings of either sexually synthesized allopolyploids or somatic hybrids between wild and crop species Somatic hybridization for the synthesis of an alloplasmic was attempted for the first time by Kameya et al (1989) when they combined the nucleus of B oleracea with Raphanus cytoplasm Subsequently, it has been employed extensively to obtain new combinations As expected, CMS originating from sexual hybridizations possess unaltered organellar genomes because of exclusive maternal inheritance Since organelle assortment and intergenomic mitochondrial recombinant is of frequent occurrence in Brassiceae, the cytoplasmic constitution is entirely different in those originating from somatic hybrids, and various possible combinations of mitochondrial and chloroplast genomes have been reported in different CMS lines (Table 2.17)

and Roăbbelen 1994; Liu et al.1996), (Tournefortii) B juncea (Arumu-gam et al 1996), and (Arabidopsis) B napus (Leino et al 2003)

VI GENOME DISSECTION AND DEVELOPMENT OF CHROMOSOME ADDITION LINES

Chromosome addition lines have a major role in revealing genome organization and evolution, identifying gene linkage groups, assign-ing species-specific characters to a particular chromosome, and comparing gene synteny between related species Localization of specific markers on individual chromosomes facilitates construction of genetic and cytogenetic maps Their practical utilization lies in introgressing characters of agronomic value, particularly from alien species to crop cultivars Several Brassica and related genomes— B nigra, B oleracea, B rapa and B oxyrrhina, Diplotaxis erucoides, Raphanus sativus, Sinapis alba, S arvensis, Moricandia arvensis, Crambe abyssinica, Orychophragmus violaceus, and Arabidopsis thaliana—have been dissected using a series of monosomic addition lines (Table 2.18) Disomic additions have also been generated but only in a few instances for a specific chromosome as in (A thaliana) B napus–A thaliana (Leino et al 2004), B napus–S alba (Wang et al 2005b) and B napus–C abyssinica (Wang et al 2006a) The recently developed full set of nine disomic B napus–R sativus addition lines by Budhan et al (2008) is the first complete disomic alien chromo-some addition series in Brassicaceae B oleracea was the first genome to be dissected and is the most extensively studied Addition lines generally not show specific morphological phenotypes associated with a particular chromosome and are rarely distinguishable from one another, thus requiring additional markers for identification It may well be that the recipient nuclear background masks the effect of added chromosome, or its effect is negated by homoeologous chromosome as these genomes evolved from a common archetype Nevertheless, the added chromosomes sometimes exhibit peculiar morphological characters For example, a radish chromosome addi-tion in B napus exhibits white flower color (Sernyk and Stefansson 1982) Chromosome of Diplotaxis erucoides in B napus was distinguished by light yellow color of their flowers (Chevre et al 1994b) Also all additions of Sinapis alba in B napus background possessed a long beak characteristic of S alba (Wang et al 2005b)

univalent at metaphase of meiosis However, it underwent pairing also and formed a trivalent as in R sativus–B oleracea (Kaneko et al 1987), B rapa–B oleracea (Chen et al 1992; Heneen and Jrgensen 2001; Hasterok et al 2005b), B rapa–B oxyrrhina (Srinivasan et al.1998), B napus–S alba (Wang et al 2005b) and B napus–Crambe abyssinica (Wang et al 2006a) These associations reflected inter-genomic homoeology between the added chromosome and recipient genome chromosomes Using GISH, Wang et al (2005b) observed homoeologous associations between S alba and B napus chromo-somes, and in some cases recombinant chromosomes could clearly be identified Hasterok et al (2005b) identified B oleracea chromosomes undergoing pairing with B rapa chromosomes including chromosome C5 with an intercalary 5s rDNA locus and chromosomes C8 and C9 involving the regions occupied by 18S-5.8S–25S rRNA genes On the contrary, in B nigra additions on B napus, only occasional chromosome pairing was observed (Jahier et al 1989; Struss et al 1991), reflecting the genetic distance between B nigra and B napus (AC) genomes as proposed earlier by several investigators

Transmission frequency of added chromosomes through male and female gametes does not follow a Mendelian pattern Many factors, such as meiotic behavior of added chromosomes and their integrity (intact or recombined), genotype, and ploidy level of the donor and recipient species, affect transmission Transmission frequency is generally far higher through the ovules than the pollen Reduction in transmission frequency of added chromosomes due to competition with normal gametes was a common feature, leading to production of normal euploid type Transmission of B nigra additions was assessed using isozyme markers carried by different chromosomes It was on an average 14% to 23% through ovules while the male transmission values ranged from 27% to 39% (Chevre et al 1997b) and 8% to 30% (This et al 1990) However, in Oxyrrhina addition lines, there was a decrease in ovule transmission frequency (Srinivasan et al 1998) Addition lines Raphanus sativus–B rapa, R sativus–B oleracea and R sativus–Moricandia arvensis were generally stable, and predomi-nant formation of gametes with added chromosomes might explain these observations (Kaneko et al 1991; Bang et al 2002; Kaneko et al 2003)

in D erucoides background Monosomic R sativus additions to alloplasmic (R sativus) B napus showed disturbed stamen develop-ment with very poor pollen production (Budhan et al 2008) Some of the monosomic additions have been observed to restore fertility to alloplasmics, and four such examples are reported Synteny group of B oxyrrhina to (B oxyrrhina) B rapa (16% pollen fertility, Srinivasan et al 1998), an unspecified chromosome of Moricandia arvensis to (M arvensis) B juncea (53% pollen fertility, Prakash et al 1998), chromosome c of Moricandia arvensis to (M arvensis) R sativus (85.6 pollen fertility, Bang et al 2002), chromosome III of Arabidopsis thaliana to (A thaliana) B napus (Leino et al 2004), and Raphanus chromosome f to (R sativus) B napus (Budhan et al 2008)

Due to small size of chromosomes and nonavailability of precise cytological landmarks in the earlier years, addition lines were characterized either through rare association with specific morpholo-gical characters, such as flower color, male sterility, or disease resistance; in recent years, isozyme and DNA markers are widely employed to characterize them Markers employed include RFLP, RAPD, SSR, and GISH and FISH By making use of these techniques, substantial information has been accumulated Isozymes were initially used to characterize addition lines B rapa-oleracea was identified using such enzyme systems, such as 6PGD, PGI, LAP, and PGM (Quiros et al 1987); PGD-1, PGM-1, and GOT-5 (McGrath and Quiros 1990); and PGM-2, PGDH-1, and PGDH-2 (Hu and Quiros 1991) B nigra chromosome additions in the background of B napus genome were characterized extensively using a large number of isozymes, such as MDH, IDH, LAP, 6-PGDH, ACO, PGI, TPI, GOT, PGM, and ADH (Chevre et al 1991; Struss et al 1996) Monosomic additions of Diplotaxis erucoides–B nigra revealed synteny associations for loci coding for isozyme markers GOT-2, 6PGD-2, MDH-2, LAP-2, and TPI-1 (Quiros et al 1987) This et al (1990) located these synteny associations on four different B nigra chromosomes using six isozyme loci and confirmed the observations of Quiros et al (1987) Also a Diplotaxis erucoides chromosome was observed to carry three isozyme alleles (Chevre et al 1994b)

be identified The markers revealed extensive intergenomic recombi-nation, presence of duplicated loci, and synteny rearrangements of chromosomes GISH has been one of the major tools to identify alien chromosomes and was employed in addition lines for Sinapis arvensis (Snowdon et al 2000b), Arabidopsis thaliana (Leino et al 2004), S alba, Crambe abyssinica (Wang et al 2005b, 2006a), and Orycho-phragmus violaceus chromosomes (Li and Ge 2007) in the background of the B napus genome Recently FISH has been used to identify the addion lines For example, Peterka et al (2004) identified chromosome d of Raphanus sativus carrying a gene imparting resistance to beet cyst nematode Hasterok et al (2005b) characterized three of the nine B oleracea var alboglabra chromosome additions using double target FISH

Chromosome addition lines as such are commercially unacceptable because of their unstable nature, reduced fertility, and expression of undesirable traits due to alien chromosomes However, these lines are of academic interest and important genetic stocks for introgressing alien genetic material that might ultimately confer agronomic or horticultural advantages For achieving gene introgression, homoeolo-gous recombination between the alien chromosome and its homo-logous counterpart of the recipient genome should occur

Enarthrocarpus lyratus to CMS (Lyratus) B rapa (Deol et al 2003) and B juncea (Banga et al 2003a) RAPD markers linked with the genes for erucic acid and seed color on B oleracea var alboglabra chromosomes have been established (Jrgensen et al 1996; Chen et al 1997b) It was obsereved that chromosome carries the gene for seed color and exerts its control embryonically Chromosome carries a gene that controls seed color maternally (Heneen and Brismar 2001)

Hasterok et al (2005b) are of the view that precise identification of extra chromosome in addition lines could be accomplished by using chromosome-specific or even arm-specific sets of BAC clone-based probes, as has been demonstrated by Howell et al (2002), Ziolkowski and Sadowski (2002), and Koo et al (2004)

VII MITOCHONDRIAL GENOME A Organization

example, the B napus mt genome has two large repeats of to 10 kb and 37 of 0.1 to 1.0 kb A thaliana has four repeats of to 10 kb and 90 repeats of 0.1 to 1.0 kb These repeats are involved in homologous recombination Intra- and intermolecular recombination in the repeat region is believed to generate multipartite subgenomic circular molecules Such recombination events have been implicated in substoichometric shift in mitochondrial genome in different tissues and accessions, and creation of novel open reading frames (orfs) S alba carries only a single copy of the repeat found in B rapa and thus is the only species known to lack any large direct repeats (Palmer and Herbon 1987) The repeat sequences contain protein coding sequences; hence such genes are duplicated The repeat sequences including the protein coding genes are different in different species For example, in B napus, a part of the cox2 gene is found in the repeat region whereas in A thaliana atp6 gene is duplicated (Handa 2003)

Detailed restriction profiles of mitochondrial genomes of Brassica species have revealed very limited intraspecific variation within species Intraspecific variations in the form of two short deletions (100 and 700 bp in B nigra) and one inversion (in S alba) were detected (Palmer 1988) Considerable variation is found among species in both mt-DNA restriction and RFLP patterns (Palmer and Herbon 1987; Palmer 1988; Pradhan et al 1992) However, most of the variation appears to be restricted to noncoding regions (Palmer and Herbon 1986, 1987) Based on comparative restriction analysis of different mt-genomes, it was found that inversions and small deletions are mainly responsible for the observed variation in mt-genomes among species For example, mt-genome restriction profiles of S alba and B rapa differ significantly However, most of the mt-genome can be divided into 11 regions; sequences within each region have the same arrangement in the two genomes, but the relative orientation and order of these regions differ between the species (Palmer and Herbon 1987) Similarly, B rapa and B oleracea differ by three large inversions whereas B rapa and Raphanus differ by 14 inversions (Palmer 1988)

B Gene Content

following the availability of complete mitochondrial genome sequences of A thaliana and B napus (Handa 2003) Plant mitochondrial genomes contain about 50 genes coding for various functions such as transcription, protein synthesis and transport, oxidative phosphoryla-tion, and so on In addiphosphoryla-tion, dozens of orfs of unknown function are also found in sequenced mitochondrial genomes of plants The overall Gỵ C content of the B napus genome is 45.2%, which is comparable to other plant mitochondrial genomes The gene content of mitochondrial genomes of B napus and A thaliana is summarized in Table 2.19

The only major difference in gene content between mitochondrial genomes of B napus and A thaliana is with respect to rps14 gene, which is a nuclear gene in A thaliana (Figueroa et al 1999) Although A thaliana contains 22 tRNA species (five more than B napus), both the species can specify only 15 amino acids Thus a complete set of t-RNA genes is lacking in Brassica and Arabidopsis mitochondrial genomes Some of the sequences (about 3.6%) present in the B napus mt-genome appears to be of plastid origin, including some tRNA species

Mitochondrial genes of B napus share many features, such as the presence of introns and RNA editing with mt-genes of other species Despite wide evolutionary divergence between A thaliana and B napus, there is a high degree of conservation at the functional level The size and number of introns are identical between the two species Similarly, the RNA editing sites (441 in Arabidopsis versus 427 in B napus) are highly conserved (Handa 2003)

Table 2.19 Number of genes in mitochondrial genomes of B napus and A thaliana

Genes B napus A thaliana Respiratory Complex I 9 Complex II — — Complex III 1 Complex IV 3

Complex V 5

Cytochrome biogenesis 4 Transcription 1 Translation

Transport 1

t-RNA 17 22

r-RNA 3

It is now clear that during the course of evolution, much of the mitochondrial genome has been transferred to the nucleus The complete genome sequencing of A thaliana and rice have shown this more clearly A 620-kb segment of mt genome is found on chromosome of A thaliana (Stupar et al 2001) Similarly, a 190-kb sequence of rice mitochondrial genome is present on chromosome 12 (Ueda 2005) Therefore, it is not unexpected that other large segments of mt DNA will be found in nuclear genomes of Brassica species

C Mitochondrial Plasmids

Small autonomously replicating linear plasmids are also found in some accessions of Brassica Palmer et al (1983b) observed a 11.3-kb plasmid in B rapa whose copy number varied 100-fold among accessions containing the plasmid Its nucleotide sequence was found to differ from other known sequences Further, the presence of plasmid was associated with cytoplasmic male sterility Since this plasmid was absent in the cytoplasm donor species (R sativus), its transmission from the male side was suspected Handa et al (2002) also reported a 11.6-kb linear plasmid in B napus, which was capable of transmission through both maternal and paternal route This plasmid contains six orfs (two coding for phage-type DNA polymerase and one coding for phage-type RNA polymerase) All six orfs were found to be transcribed, and proteins of at least three orfs are found at high levels in flower buds of B napus

VIII PLASTID GENOME

Palmer et al (1983a) compared restriction patterns of six U-triangle species along with S alba and R sativus Small insertions or deletions (indels, 50–400 bp) seem to be the cause of most of the variations observed among species Total sequence variation among Brassica species was estimated to be about 2.4% Low level (0–0.01%) of intraspecific variation was also reported by Warwick’s lab based on cp-DNA RFLP and restriction analyses A majority (53–80%) of restriction site mutations recorded were found between species These studies have been extremely useful in identifying the maternal parents of the allotetraploid species Availability of the complete cp-DNA sequence of A thaliana (Sato et al 1999) may provide further opportunity for more incisive investigation of cp-genome evolution in Brassiceae

IX POTENTIAL ROLE OF ARABIDOPSIS THALIANA IN BRASSICA IMPROVEMENT

A A thaliana as a Model Crucifer

The fact that Arabidopsis and Brassica are in the same family is of great advantage to Brassica researchers who are benefiting from the information generated by the completed Arabidopsis thaliana genome sequence Although the taxonomic distance between the two genera is large, with approximate divergence of 15 to 20 million years (Yang et al 1999; Wroblewski et al 2000), there is a great deal of conservation The genomes of diploid brassicas are three to four times larger than that of Arabidopsis (157 Mb, Bennett et al 2003), ranging from 468 Mb for B nigra to 662 Mb for B oleracea (Arumuganathan and Earle 1991) In spite of these differences, sequence conservation and synteny are large enough in most cases to use the genome of A thaliana as a guide to find genes of interest in Brassica species

B Cytology and Possible Origin of the A thaliana Genome

ribosomal (18S, 26S, and 5S rDNAs), pericentromeric, centromeric, and telomere repeats (reviewed in Koornneef et al 2003; Lysak et al 2003) Both mitotic and meiotic chromosomes have been investigated, but better resolution was achieved with meiotic prophase complements, and heterochromatic and centromeric regions could be clearly differ-entiated Using BAC contigs as probes in FISH, Fransz et al (1998) presented a comprehensive pachytene bivalents karyotype Accord-ingly, the mean total length of pachytene bivalents is 331 mm The major part is euchromatin, with heterochromatin regions comprising of only 7.1 %, confined mostly in pericentromeric regions and NOR Chromo-somes and are the longest and metacentric with average length of 80.76 and 76.32 mm respectively Chromosome 5, the second largest, carries a major and a minor 5S rDNA loci The major locus is in the pericentromeric heterochromatin region of the upper arm and the minor locus is in the opposite arm Chromosomes and are acrocentric and carry NOR Their average length is 52.12 and 52.65 mm respectively Chromosome contains a 5S rDNA locus in the pericentromeric heterochromatin region of the short arm Chromosome 3, a submetacentric with an average length of 69.34 mm, contains a major 5S rDNA in the middle of the long arm Polymorphism for 5S rDNA loci was also observed in different ecotypes However, all of them possess chromosomes and in the short arms Earlier investigations documented 45S rDNA on NOR of chromosomes and and 5S rDNA on chromosomes and and polymorphic sites on chromosome (Murata et al 1997)

be n¼ A thaliana evolved from the hypothetical tetraploid species approximately million years ago by reduction in chromosome number caused mostly by chromosome fusions and also by translocations and inversions (Henry et al 2006; Schranz et al 2006) These chromosomal rearrangements were accompanied by substantial DNA losses in A thaliana (Town et al 2006), when compared to A lyrata and other related species (Schranz et al 2006)

C Synteny Conservation

D Synteny-Based Gene Discovery and Cloning

Based on genomic shotgun sequences covering close to half of the B oleracea genome, Ayele et al (2006) estimated that 84% of the A thaliana genes have a match in B oleracea They called these regions CAGs (conserved Arabidopsis genome sequences) and found that the highest sequence alignments occur near the centromeres of the Arabidopsis chromosomes Sequence conservation is high in exons, ranging from 70% to 90% with the majority having similarities higher than 80%, whereas for introns it is <70% Protein similarity or orthologs is often above 95% (Gao et al 2006) These high similarity values along with synteny conservation make it possible, in most cases, to find Brassica orthologs based on A thaliana gene models with ease

Sadowski et al (1996) exploited the genetic map of A thaliana (Hauge et al 1993) to probe the Brassica genomes with an A thaliana gene complex carrying five genes within a 20-kb span (Gaubier et al 1993) This complex comprises a well-characterized Em-like protein coding gene and other four flanking genes on chromosome Although the five-gene complex array from A thaliana was conserved on a single chromosome of each Brassica genome, additional copies for most of the genes were found in one or two other chromosomes A similar situation was observed for a six-gene complex on A thaliana chromosome 4, including the disease resistance gene RPS2 (Sadowski and Quiros 1998) In this case, besides the conserved array in one Brassica chromosome, four other chromosomes contained copies for some of the genes

The benefit of synteny conservation for gene discovery in Brassica is well demonstrated in studies on genes coding for glucosinolates (GSL), which are secondary metabolites synthesized by many species of the order Capparales, including Brassica and Arabidopsis Breakdown products of GSLs, particularly isothiocynates, have been found to be anticarcinogenic (Talalay and Zhang 1996) Therefore, consumption of some of the brassica crops, such as broccoli, has been reported to exert cancer-protecting effects due to the formation of sulforaphane, an aliphatic glucosinolate-derived ITC (Fahey et al 1997)

(Mikkelsen et al 2002; Wittstock and Halkier 2002), and knockout mutants for most of these genes are available

The colinearity between A thaliana and B oleracea has been explored for three chromosomal regions carrying three glucosinolate genes Two of them are involved in side-chain elongation and belong to a gene family of major genes encoding methylthioalkylmalate synthase enzymes (MAM) In A thaliana, three loci are duplicated in tandem (MAM1, MAM2 and MAM-L) on chromosome 5, and their presence depends on the ecotype; MAM-L is always present, but MAM1 and MAM2 are dispensable A functional allele of MAM1 results in the presence of GSL with side chains containing four carbons (4C-GSL), whereas the presence a MAM2 in the absence of MAM1 results in the presence GSL with side chains containing three carbons (3C-GSL) The function of MAM1 is dominant to that of MAM2, because when both are present, the plants produce 4C-GSL (Kryomann et al 2003) It was found that in B oleracea, the BoGSL-ELONG gene corresponds to MAM1 in A thaliana, which results in plants with 4C-GSL (Li and Quiros 2002) Comparing the sequence of a 96.7-kb-long BAC clone (B19N3) from Brassica oleracea (broccoli) harboring the BoGSL-ELONG gene with its equivalent regions in A thaliana disclosed these breaks in synteny:

 B19N3 contains eight genes and six TEs

 The first two genes in this clone, Bo1 and Bo2, have its corresponding region at the end of chromosome of Arabidopsis (24 Mb)

 The third gene, Bo3, corresponds to an ortholog at the opposite end (2.6 Mb) of the same chromosome

 The other five genes, Bo4 to Bo8, also have a equivalent region on the same chromosome but at 7.7 Mb Bo5 is a tandem duplicate of BoGSL-ELONG (Bo4) and was named BoGSL-ELONG-L, which is equivalent to MAM-L in A thaliana

The region was further expanded by constructing a contig primer walking and BAC-end sequencing, revealing general gene colinearity beyond the segment harboring the BoGSL-ELONG gene (Gao et al 2005)

Two other B oleracea BAC clones were surveyed for colinearity The second BAC clone contained gene BoGSL-PRO, which is also a homolog of the MAM A thaliana gene family This gene has its homolog at the top of chromosome I in A thaliana (At1g18500, MAM4) A duplicate member of this gene is located in the opposite arm of the same chro-mosome (At1g74040, MAM3) This gene is likely orthologous to BoGSL-PRO-L, another member in the family also at a different location in B oleracea

genome surveyed so far, is the lower gene density found in the three BAC B oleracea clones This is due mostly to the insertion of TE in intergenic spacers and introns As a consequence of these changes and breaks in colinearity, especially the frequent absence of genes in corresponding segments of A thaliana, using this species as a guide to find a corresponding Brassica gene is not a trivial task The tandem duplicates often found in the latter species require further experimenta-tion to determine the correct gene based on its funcexperimenta-tionality and expression

E Arabidopsis Knowledge-Based Gene Discovery and Brassica Improvement

Brassica and Arabidopsis genomes share a high degree of homology (>80%), particularly in the exon regions, and most of the genes present in Brassica are represented in Arabidopsis Hence knowledge gained from Arabidopsis is highly transferable to Brassica, and is providing valuable insights into various aspects of Brassica, including domes-tication and speciation, growth and development, and metabolism Various approaches and resources currently are being employed to accomplish the goal of assigning functions to all the genes in Arabidopsis by 2010 Brassica improvement is expected to get a boost from the availability of complete functional genomic information of Arabidopsis Once the key genes responsible for expression of a given trait are identified in Arabidopsis, they can be used to engineer the trait in Brassica The examples discussed next highlight the significance of Arabidopsis functional genomics to Brassica

Arabidopsis, which negatively regulate SHATTERPROOF (Roeder et al 2003) Based on this information, stergaard et al (2006) developed nonshattering B napus lines Genes governing vernalization response and flowering time have also been well characterized in Arabidopsis Robert et al (1998) isolated four orthologues of Arabidopsis CONSTANS gene from B napus lines differeing in flowering time and showed that their function is conserved between Arabidopsis and Brassica FLC is a major gene responsible for suppression of flowering in Arabidopsis and is downregulated upon exposure to cold temperature Kole et al (2001) found that the major QTL, VFR2 responsible for winter type B rapa cosegregated with FLC orthologues These studies illustrate how Arabidopsis could serve as a reference for Brassica improvement Understanding Metabolism Fatty acid metabolism has been exten-sively studied in Arabidopsis, and genes encoding key enzymes involved in fatty acid synthesis, elongation, and modification have been cloned and characterized Analysis of QTLs for oil quality in Brassica crops have revealed that, in a majority of cases, these QTLs correspond to the known Arabidopsis genes involved in fatty acid metabolism For example, FAE1 gene encodes the enzyme responsible for erucic acid biosynthesis in Arabidopsis Mutations in the othrolo-gues of the FAE1 gene have been found to be responsible for low-erucic acid in seed oils of B rapa and B oleracea (Das et al 2002) Similarly, in B juncea, FAE1.1 and FAE1.3 genes have been shown to cosegregate with QTLs, which account for 60% and 38% varaince for erucic acid content (Mahmood et al 2003)

Vitamin E (a-tocopherol) synthesis is restricted to photosynthetic organisms Molecular analysis of Arabidopsis mutants has helped unravel the genes involved in tocopherol biosynthesis Shintani and Della Penna (1998) cloned the gene encoding the enzyme g-tocopherol methyltransferase, which catalyzes the final step of vit E biosynthesis Seed-specific overexpression of this gene resulted in elevated accumulation of vitamin E in seeds of Arabidopsis Transgenic B juncea lines accumulating vitamin E have been generated through ectopic expression of A thaliana gene (Yusuf and Sarin 2007)

opportunities for developing yellow-seeded Brassica varieties (Debeaujon et al 2003; Gruber et al 2007; Lu et al 2007; Wei et al 2007) These examples amply demnonstrate the usability of genetic information from Arabidopsis in Brassica molecular biology and improvement

3 Testing for Gene Function by Complementary Transformation The most common and straightforward method to demonstrate that a cloned candidate gene is in effect the correct gene searched for a specific function is by in planta complementary transformation Unfortunately, transformation is not always an easy task in Brassica species, which is largely genotype dependent However, A thaliana is easily and efficiently transformed (Clough and Bent 1998) Furthermore, a series of knockout stocks are available in these species covering many of the major genes of interest Therefore, a routine approach to test for Brassica gene function is to introduce these genes by Agrobacterium transformation to various A thaliana ecotypes and knockout mutants, depending on the gene under scrutiny Following phenotypic changes predicted by the introduced gene by gain in function often demon-strates that the candidate gene is indeed the right gene An example of this approach is illustrated by Li and Quiros (2003) who tested the function of the BoGSL-ALK genes described in the previous section In this study, they introduced a functional allele of BoGSL-ALK into A thaliana ecotype Columbia, which has a nonfuctional allele for this gene By doing so, they were able to change the GSL profile of the Arabidopsis ecotype, which normally produces 4-methylsulfinylbutyl and 3-methylsulfinylpropyl GSL The transformants had a profile including three new additional compounds, 2-hydroxy-3-butenyl, 2-propenyl glucosinolate, and 3-butenyl glucosinolate, resulting from the conversion by desaturation of 4-methylsulfinylbutyl GSL precursor into 3-butenyl glucosinolate and the 3-methylsulfinylpropyl GSL precursor into 2-propenyl glucosinolate The third compound resulted from hydroxylation of 3-butenyl glucosinolate, which is the next step on the side chain modification pathway and mediated by another gene in the AOP family

X CHLOROPLAST GENOMES AND THEIR PHYLOGENETIC IMPLICATIONS

into seven subtribes chiefly on fruit characters (Go´mez-Campo 1980) However, the morphology-based taxonomy is considered highly artificial by many taxonomists as the chromosome homology across the subtribes is often higher than within the subtribe Possibilities of genetic exchange have been demonstrated

Molecular markers, particularly the chloroplast DNA restriction site variation, have been employed to infer phylogeny of subtribe Brassici-nae and related subtribes, RaphaniBrassici-nae and MoricandiiBrassici-nae, and also to clarify the status and relationships among various species and genera Such investigations were initiated by Warwick and Black (1991) and Pradhan et al (1992) who studied chloroplast DNA RFLPs in a number of taxa These studies were subsequently extended to other related subtribes encompassing a wider spectrum by Warwick and her colleagues in a series of articles (Warwick and Black 1991, 1993, 1994, 1997a; Warwick et al 1992; Warwick and Sauder 2005)

Phylogenetic analysis clearly revealed a vertical division of these subtribes into two lineages referred to as Rapa/Oleracea and Nigra lineages (Warwick and Black 1991; Pradhan et al 1992) Earlier invest-igations on species relationships involving morphology and cytology had not suggested such dichotomy However, the separation of the three cultivated diploid Brassica species into two lineages had earlier been suggested from cp DNA studies (Palmer et al 1983; Erickson et al 1983; Yanagino et al 1987) and molecular DNA RFLP data (Song et al 1988a,b, 1990) The smaller genera are monophyletic, while polyphyly is evident in large genera—Brassica, Diplotaxis, Erucastrum, and Sinapis, as these have taxa in both the lineages (Table 2.20) Recent investigations using ITS, trnL and combined ITS/trnL sequence data also supported it (Warwick and Sauder 2005) Interestingly, a high congruence is observed between genetically estabilished cytodemes and the clusters defined by cp DNA Chloroplast genome information may form the basis for future taxonomic realignment and generic and specific delimitation along with morphological, cytogenetical, geographical and other molecular data for a more natural classification of the coenospecies We will discuss the status of different genera separately

A Subtribe Brassicinae

subspecies Harberd (1972) established 10 cytodemes to which two more were added by Takahata and Hinata (1983) Cp DNA-based phylogenetic analysis and phenetic clustering separates the genus into two lineages (Warwick and Black 1991; Pradhan et al 1992) Earlier cp DNA studies by Erickson et al (1983), Palmer et al (1983a), and Yanagino et al (1987), and nuclear RFLP investigations by Song et al (1988a,b, 1990) also suggested a vertical division Based on cp DNA variations, B rapa, B oleracea, B deflexa, B oxyrrhina, B repanda, B gravinae, B elongate, and B barrelieri belong to Rapa/Oleracea lineage The Nigra lineage includes B nigra, B fruticulosa, and

Table 2.20 Genera and species of Brassica coenospecies in Nigra and Rapa/Oleracea lineage

Nigra lineage n Rapa/Oleracea lineage n GROUP I

Brassica nigra Sinapis arvensis Diplotaxis ibicensis Diplotaxis siettiana Sinapis alba 12 Brassica fruticulosa Erucastrum littoreum 16 Trachystoma balii GROUP II

Brassica tournefortii 10 Sinapis pubescens Brassica procumbens Diplotaxis brachycarpa Erucastrum varium Erucastrum virgatum Hirschfeldia incana GROUP III

Erucastrum canariense Diplotaxis assurgens Diplotaxis siifolia 10 Sinapidendron spp 10 Diplotaxis berthautii 10 Diplotaxis virgata Diplotaxis catholica Erucatrum brevirostre GROUP IV

Coincya spp 12

GROUP I

Brassica rapa 10 Brassica oleracea Diplotaxis cossoneana Diplotaxis erucoides Erucastrum abyssinicum 16 Erucastrum strigosum Erucastrum nasturtifolium Brassica deflexa Sinapis aucheri Enarthrocarpus lyratus 10 Raphanus spp Brassica barrelieri 10 Brassica oxyrrhina GROUP II

Diplotaxis harra 13 Eruca spp 11 Diplotaxis tenuifolia 11 Rytidocarpus moricandiodes 14 GROUP III

Moricandia arvensis 14 Moricandia moricandiodes 14 Moricandia suffruticosa 28 GROUP IV

Brassica gravinae 10 Brassica repanda 10 Diplotaxis viminea 10 GROUP V

B tournefortii (Warwick and Black 1991; Pradhan et al 1992) There are subgroups in both the lineages: three in Rapa/Oleracea and two in Nigra A high level of congruence was found between cytodemes and the groups defined by chloroplast DNA restriction site variations Rapa/Oleracea Lineage There are three subgroups in the Rapa/ Oleracea lineage:

1 B elongata (n¼ 11) constitutes a very distinct group, which is reflected in its characteristic morphological traits: torulose pods with an inconspicuous seedless beak It is endemic to south-eastern Europe, western Russia, and the Near East

2 Another group comprises three species: B repanda, B gravinae, and B desnotesii (all n¼ 10) Of these, B repanda and B desnotesii have very similar cp and are placed in the same cytodeme (Takahata and Hinata 1983) B desnotesii is endemic to Morocco, and B gravinae and B repanda overlap in their distribution in northwestern Africa All these species were ascribed to subgenus Brassicaria and have recently been trans-ferred to a separate genus, Guenthera, based on a set of distinctive characters including seedless beak (Go´mez-Campo 2003)

3 Five species—B rapa, B oleracea, B oxyrrhina (n¼ 9), B barrelieri (n¼ 10), and B deflexa (n ¼ 7) constitute the third group B rapa and B oleracea form one subgroup, B oxyrrhina and B barrelieri another, and B deflexa forms the third subgroup Within B oleracea, various wild taxa of the complex, including cretica, montana, insularis, incana, drapenensis, macrocarpa, and villosa, show a high degree of chloroplast genome similarity with cultivated forms, thus substantiating the proposals that these belong to B oleracea (Snogerup 1980; La´zaro and Aguinagalde 1998a,b) A close relationship between B rapa and B oleracea is reflected in both possessing very similar chloroplast genomes, a fact supported from serological analysis of seed proteins (Vaughan 1977), isozyme patterns (Takahata and Hinata 1986), a high degree of chromosome affinities between their genomes (Olsson 1960b), and considerable similarities in size and morphology of their chromosomes and nuclear RFLPs (Song et al 1988a,b, 1990; Hosaka et al 1990)

in Flora Europea (Tutin et al 1964), but it is now recognized as a separate cytodeme by Harberd (1972) This separate status is confirmed by cp DNA studies (Warwick and Black 1991; Pradhan et al 1992) B oxyrrhina is proposed to have evolved from a loss of one pair of chromosomes from B barrelieri (Harberd 1976) Both are identical in the vegetative stage, forming a rosette of leaves B deflexa shows strong homology with Sinapis aucheri They have many similarities— for example, cp DNA, ITS/trnL sequence data, an overlap in distribution in the eastern Mediterranean, and pendant, torulose pods—but they form separate cytodemes (Warwick and Sauder 2005) Interestingly, the three species—B oxyrrhina, B barrelieri, and B deflexa—close cp DNA homologies with Raphanus and S aucheri and represent a unique trend in the evolution of pod morphology in the tribe Although Raphanus with strong heteroarthrocarpic fruits (where the valvar portion is represented by vestigial scales and is formed entirely by the beak) represents an extreme, Brassica has a well-developed unsegmented portion B oxyrrhina and B barrelieri represent an intermediate condition having disproportionally devel-oped beaks

species of Brassica: B nigra, B fruticulosa, and B tournefortii (Warwick and Black 1991; Pradhan et al 1992)

2 Diplotaxis This genus contains about 27 species (Martı´nez-Laborde 1993; Go´mez-Campo 1999c), which are mainly distributed in Central Europe and the Mediterranean region, particularly northwest Africa It has been separated from other members of subtribe Brassicinae primarily in having biseriate, small, generally ovoid or ellipsoidal seeds (Schulz 1919; Tutin et al 1964; Al-Shehbaz 1985) Interestingly, many primitive morphological characters for the tribe Brassiceae are present in Diplotaxis (Go´mez-Campo 1980) The leaves are generally pinnatifid or pinnatisect Schulz (1936) recognized 22 species and grouped them into four sections: Rhynchocarpum, Catocarpum, Anocarpum, and Hesperidium The different species have a continuous series of chromosome numbers from n¼ to n ¼ 13, also high-chromosome allopolyploids with n¼ 21, and have been grouped into 13 cytodemes (Harberd 1976; Takahata and Hinata 1983) Chloroplast DNA investigations clearly indicated a division into two lineages and the suggested level of divergence and taxon groupings are highly congruous with the cytodeme status (Warwick et al 1992; Pradhan et al 1992) However, the morphologically based delimitation of the species is not always consistent with these studies All the species are separated into six groups, three each in both the lineages (Table 2.21) Interestingly, the boundaries of the sections established by Schulz (1919, 1936) correspond closely to the group defined by cp DNA For example, groups B and C in Rapa/Oleracea and group F in Nigra lineages corresponds to sections Catocarpum, Anocarpum, and Rhyncocarpum, respectively

Rapa/Oleracea Lineage The different species in the lineage not form a single group but are separated into three major groups (Warwick et al 1992) Diplotaxis erucoides (n¼ 7) with two subspecies (subsp erucoides and subsp longisiliqua) form a distinct cp DNA entity in group A The distinction between both subspecies is based on petal color, nervation patterns on petals, and fruit size (Schulz 1919; Maire 1965; Go´mez-Campo 1981; Martı´nez-Laborde 1988) Both are also separated by strong breeding barriers In areas of sympatric distribu-tion, hybrids between the two are rare and completely sterile Cp DNA data also substantiate this fact and might justify a specific rank (subsp longisiliqua! Diplotaxis cossoniana) and separate cytodeme status

and Takahata and Hinata (1983) in one cytodeme D tenuifolia and D cretacea are morphologically very similar (Martı´nez-Laborde 1988) While D tenuifolia has a very wide distribution in Europe, D cretacea is a narrow endemic in Eastern Europe and adjacent Russia (Tutin et al 1964) Diplotaxis simplex has more similarities than differences in the other species; however, its distribution is different, as it occurs in Algeria, Tunisia, Libya, and Egypt These facts coupled with low levels of chloroplast divergence not warrant a separate specific status for these species and constitute a single cytodeme Diplotaxis harra (n¼ 13) has a wide distribution across northern Africa and the Middle East It has several subspecies: harra, crassifolia, and lagascana Two species— D viminea (n¼ 10) and D muralis (n ¼ 21)—constitute group C Diplotaxis viminea is assigned a separate cytodeme status while D muralis is a naturally evolved allopolyploid between D viminea D tenuifolia (Harberd and McArthur 1980) Close similarities of cp and mitochondrial DNA between D muralis and D viminea suggest the latter as maternal parent and also indicate that D muralis is of recent origin (Pradhan et al 1992) D simplex—a part of D tenuifolia cytodeme—is morphologically very similar to D muralis (Schulz 1936; Martı´nez-Laborde 1988) and is more likely the other parent (Warwick et al 1992) Nigra Lineage Three major groups have been recognized by cp DNA data in this lineage (Warwick et al 1992) Four species, all n¼ 8—D siettiana, D ibicensis, D brevisiliqua, and D ilorcitana—are included

Table 2.21 Species of the genus Diplotaxis in Rapa/Oleracea and Nigra lineages

Rapa/Oleracea lineage Nigra lineage GROUP A GROUP D D erucoides, n¼ D siettiana, n¼ D cossoneana, n¼ D brevisilique, n¼ GROUP B D Gomez-campoi, n¼ D tenuifolia, n¼ 11 D ibicensis, n¼ D cretacea, n¼ 11 GROUP E

D simplex, n¼ 11 D brachycarpa, n¼ D harra, n¼ 13 GROUP F

GROUP C D assurgens, n¼ D viminea, n¼ 10 D tenuisiliqua, n¼ D muralis, n¼ 21 D virgata, n¼

in one group (D) Each occupies a narrow region in the western Mediterranean Genetically and morphologically all four taxa are very close (Martı´nez-Laborde 1988) This closeness is also reflected in their cp DNA (Warwick et al 1992) In fact, all four species constitute one cytodeme: D siettiana Diplotaxis brachycarpa (n¼ 9) possesses a chloroplast genome very different from other species of Diplotaxis, and no information is available on its cytodeme status It is placed in group E Group F includes three subgroups: (1) comprising D assurgens (n¼ 9), D tenuisiliqua (n ¼ 9), and D siifolia (n ¼ 10); (2) comprising D virgata, D berthautii (n¼ 9); and (3) D catholica (n ¼ 9) Separate cytodeme status to D assurgens, D tenuisiliqua, D virgata, D berthautii, and D catholica have been recognized (Prakash et al 1999) The three species in subgroup occur along the coast of Portugal and Morocco The cp DNA data strongly supports the separate species and cytodeme status for D virgata and D berthautii in subgroup

Using intersimple sequence repeat nuclear DNA markers, Martin and Sa´nchez-Ye´lamo (2000) investigated 10 Diplotaxis species and observed that five species—D tenuifolia, D cretacea, D simplex, D viminea, and D muralis—constitute one group Morphologically, Prantl (1891) grouped them in section Anocarpum Crossability and chromosome pairing in their hybrids also reflect high homologies among these five species (Harberd 1972; Takahata and Hinata 1983) One of the common shared characteristics is presence of glucosinolates giving a strong odor Biochemical markers such as flavonoid (Sa´nchez-Ye´lamo and Martı´nez-Laborde 1991; Sa´nchez-(Sa´nchez-Ye´lamo 1994), seed proteins and isozymes (Sa´nchez-Ye´lamo and Martı´nez Laborde 1991), and cp and mt DNA analysis (Pradhan et al 1992) also suggested such a close relationships D virgata, D catholica, D siettiana, D harra, and D erucoides constitute the second group These are all odorless because of very low amount of glucosinolates (Sa´nchez-Ye´lamo 1994) The cp and mt DNA analysis shows close relationships among D virgata, D catholica, and D siettiana (Pradhan et al 1992)

D siifolia’s placement in Brassica and of D assurgens in Diplotaxis, although the two species have very similar cp DNA (Pradhan et al 1992) D siifolia shares cp DNA homologies with D tenuisiliqua, D catholica, D virgata, E cardaminoides, and Hirshfeldia (Pradhan et al 1992) As no Brassica species is placed in this group indicating the remoteness between the taxa of this group and Brassica D siifolia has been reported to possess strong isolation barriers with Brassica species, which are mostly postfertilization Although intergenomic homoeology between chromosomes of D siifolia and B rapa and B nigra has been observed (Batra et al 1990), placement of D siifolia in the genus Diplotaxis rather than in Brassica seems appropriate

This genus is morphologically unique, having both types of taxa: some with seedless beaks and others with seeded beaks Species in two of the subgenera—Diplotaxis and Hesperidium—always show seed-less beak Seeded beak (heteroarthrocarpic fruits) is also present in subgenera Rhynchocarpum and Heterocarpum Go´mez-Campo (1999b) believed that much of the molecular heterogeneity is associated with beak duality

3 Erucastrum The genus Erucastrum comprises 21 species and is traditionally considered close to Brassica and Diplotaxis (Go´mez-Campo 1999c) It has a distribution in the western Mediterranean and eastern and southern Africa Polyphyly is evident in this genus, as indicated by placement of its species in both the lineages

Rapa/Oleracea Lineage Five species form three subgroups in this lineage:

1 E leucanthum and E nasturtiifolium (both n¼ 8) have close affinities and both belong to the same cytodeme Morphologically they are similar E leucanthum has white flowers while E nasturtiifolium is characterized by the retrorse lower segments of its leaves

2 E abyssinicum and E strigosum (both n¼ 8) are aligned together They form a small group and both represent separate cytodemes

Nigra Lineage Erucastrum species form three subgroups in this lineage:

1 E canariense and E cardaminoides (both n¼ 9), endemic to Canary islands, have very similar cp genome and constitute one cytodeme Both are morphologically very similar

2 E virgatum (n¼ 7) and E elatum (n ¼ 15) show close affinities in cp DNA and morphological attributes The latter is an allopoly-ploid between E virgatum (n¼ 8) and Hirschfeldia incana (n ¼ 7) (Go´mez-Campo 1983; Sanchez-Yelamo 1992; Warwick and Black 1993)

3 E brevirostre (n¼ 9) forms a small group with Diplotaxis catholica It is endemic to central and western Morocco However, its cytodeme status is unknown Go´mez-Campo (1982) suggested a close affinity with the Canarian species of group 1, supported by cp DNA analysis (Warwick and Black 1993)

(Tsukamoto et al 1993; Simonsen and Heneen 1995), karyotypes (Yuan et al 1995), RAPD patterns (Wu et al 1996), nuclear sequence of S-locus related gene SLR1 (Inaba and Nishio 2002), and ITS/trnL sequence data (Warwick and Sauder 2005)—substantiate this close-ness S pubescens (n¼ 9) deserves a specific rank and separate cytodeme status However, the close cp DNA affinities between S pubescens and Hirschfeldia incana is intriguing, which is reflected morphologically also where only the degree of sepal erectness separates them (Schulz, 1919; Tutin et al 1964)

Sinapis aucheri has been placed in the annual section Chondrosi-napis by Schulz (1936) Unlike other SiChondrosi-napis species, which have multilocular pods and typical beak, S aucheri has highly heterocarpic pods with long torulose, corky, and 6- to 10-seeded beak Its distribution is confined to western Iran and eastern Iraq; all other Sinapis species are distributed in the Mediterranean region (Schulz 1936; Al-Shehbaz 1985) Chloroplast DNA analysis (Warwick and Black 1991; Pradhan et al 1992) indicates close relationship between S aucheri and Raphanus sativus S aucheri is often confused with Raphanus aucheri of section Hesperidopsis in taxonomy and nomen-clature (Schulz 1936) It has strong heterocarpy like R aucheri and has narrow endemism in western Iran Considering its distribution and pod morphology, it would be justified to transfer S aucheri to Raphanus

5 Trachystoma Trachystoma includes three species—labasi, ballii, and aphanoneurum—and all have similar chloroplast genomes in Nigra lineage and have been placed into one cytodeme (Harberd 1976) It has variably been treated under subtribes Brassicinae and Raphani-nae (Go´mez-Campo 1980) One of its characteristic features is strongly heteroarthrocarpic silique Chloroplast DNA studies supports its inclusion in subtribe Brassicinae and also suggest the close affinities with B nigra and S arvensis (Warwick and Black 1997) All the three taxa are endemic to Morocco Its spontaneous hybridization with Ceratocnemum challenges the presently defined limits of coenospecies (Al-Shehbaz 1985)

DNA has close homology with an allotetraploid species Erucastrum elatum (n¼ 15) and is one of the components of it (Go´mez-Campo 1983) It also bears close genetic relationship with Sinapis pubescens (Warwick and Black 1991) In fact, Hirshfeldia is an Erucastrum with specialized fruits (Go´mez-Campo 1999c)

7 Sinapidendron Three species—S angustifolium, S frutescens and S rupestre—are endemic to Atlantic islands (Madeira, Canarias, and Cabo Verde) and regarded as Miocenic relic The cotyledons exhibited by this genus (broad lamina and shallow notch) represent an ancestral type (Go´mez-Campo and Tortosa 1974) All three species constitute a single cytodeme, which is reflected in close cp DNA affinities and placed in Nigra lineage (Warwick and Black 1993)

8 Coincya This is a highly heteroarthrocarpic genus with maximum variability in the Iberian peninsula and is placed in Nigra lineage It was variously been described under different genera, such as Brassi-cella, Coincya, Hutera, and Rhynchosinapis (Go´mez-Campo 1980) Earlier, six species were recognized by Leadlay and Heywood (1980) However, cytological (Harberd and McArthur 1972) and molecular studies indicate a homogenous group (Warwick and Black 1991) Eruca This is a monotypic genus placed in the Rapa/Oleracea lineage All the three species—vesicaria, sativa, and pinnatifida—are now treated as subspecies of sativa and constitute one cytodeme and possess similar cp DNA Although partial sterility was observed in ssp sativa vesicaria hybrids (Sobrino-Vesperinas 1995) E sativa ssp vesicaria is unique with nonheteroarthrocarpic silique and is widely distributed in the Mediterraneanregion; subspecies pinnatifida is endemic to southern Spain, Algeria, Morocco, and Tunisia It has a very short life cycle and is well adapted to harsh drought conditions Subspecies sativa is cultivated in many parts of the world, particularly in drier habitats, for its oil (Tsunoda 1980; Go´mez-Campo 1999c) Its seeds are a common source of industrial oil in India Ibn al-Awam, a Spanish Moor in the 12th century, mentioned its cultivation in Spain in his book Kitab-al-Falaha (Gomez-Campo and Prakash 1996) It is very popular as a pungent salad in Italy while nonpungent ones are grown in Turkey and Egypt

B Subtribe Raphaninae

the core genus of this subtribe Harberd (1972) placed two genera from this subtribe—Raphanus (n¼ 9) and Enarthrocarpus (n ¼ 10)—in coenospecies Nuclear DNA RFLP investigations by Song et al (1990) also strongly supported the inclusion of Raphanus in subtribe Brassi-cinae Both the genera are placed in Rapa/Oleracea lineage by Warwick and Black (1991, 1997) and Pradhan et al (1992) Go´mez-Campo (1980) believed that Raphanus and Enarthrocarpus are intermediate between subtribes Raphaninae and Brassicinae, but are more closely related to Brassicinae This closeness is also reflected in hybridization and chromosome pairing in hybrids In fact, the intersubtribal hybrid Raphanus B oleracea was obtained as early as 1927 by Karpechenko, and it exhibits high chromosome homologies (1 IIIỵ II, 2n ẳ 18, RC, McNaughton 1973) Similar high chromosome affinities were observed in hybrids E lyratus B oleracea, (2n ẳ 19, III ỵ II) and E lyratus  B rapa (2n¼ 20, III ỵ II, Gundimeda et al 1992) Warwick and Black (1997) were of the view that five more genera of the subtribe— Cordylocarpus, Otocarpus, Guiraoa, Kremeriella, and Ceratocne-mum—all North African endemics that fall in Nigra lineage, might also be considered for their inclusion into Brassica coenospecies Ceratocnemum (n¼ 8) shows close cp DNA and ITS/trnL sequence homology with Trachystoma (Warwick and Black 1991; Warwick and Sauder 2005) Both are also genetically close, as supported by the observation that an intergeneric hybrid Trachycnemum mirabile Maire and Samuels (Trachystoma ballii Ceratocnemum rapistroides) occurs in nature (Maire and Samuelsson 1937; Maire 1965; Al-Shehbaz 1985)

C Subtribe Moricandiinae

(all n¼ 14)—are easily hybridized (Sobrino-Vesperinas 1997), have very similar cp DNA profiles, and are included into one cytodeme, M arvensis Their seed protein profiles also show large similarities (Sa´nchez-Ye´lamo et al 2004) Earlier, Maire (1967) also treated these species as subspecies of M arvensis The other species, M morican-dioides (also n¼ 14), has cp genome and seed protein profile distinct from the M arvensis complex and is also included in Rapa/Oleracea lineage Taxa of M arvensis complex are widely distributed in the Mediteranean region and appear to be exclusively polyploids (Al-Shehbaz 1984) Close genetic affinities between M arvensis and Brassica species is evidenced by the fact that sexual and somatic hybrids between M arvensis / nitens and several Brassica species show a high degree of chromosome pairing (Takahata 1990; Takahata and Takeda 1990; Kirti et al 1992b; Takahata et al 1993; Meng et al 1997, 1999; Meng 1998) The monotypic Moroccan genus Rytidocarpus is very close to Moricandia (Go´mez-Campo 1980) in morphology as it has Moricandia-like cotyledon with an almost absent notch, succulent and entire leaves, purple flower, and the same chromosome number n¼ 14 Another genus, Pseuderucaria (n¼ 14), earlier assigned to Moricandii-nae (Schulz 1936; Go´mez-Campo 1980), has a weak relationship with Moricandia and Rytidocarpus but deserves a place in the coenospecies (Warwick and Black 1994)

D General Considerations

(B desnottessi), G repanda (B gravinae, B repanda), G nivalis (B jordanoffii, B nivalis), G setulosa (Eruca setulosa), and G loncholoma (B loncholoma syn Eruca loncholoma) Although ITS data does not provide support for such status because Guenthera itself, as defined, appears to be polyphyletic (Warwick and Sauder 2005), it is also certain that polyphyletism is still present in the remaining taxa of Brassica

The separate status of three subtribes—Brassicinae, Raphaninae, and Moricandiinae— has been questioned as morphological distinc-tiveness does not provide sufficient basis for it (Al-Shahbaz 1985; Warwick and Black 1994) Brassicinae and Moricandiinae have elongated dehiscent fruits while Raphaninae has reduced indehiscent fruits Recent hybridization studies and phylogenetic analysis based on S-locus related gene SLR1 (Inaba and Nishio 2002) and cp DNA and ITS, trnL and ITS/trnL data also not support separate recognition of subtribes (Warwick and Sauder 2005)

The genus Orychophragmus was placed in the tribe Brassiceae sub-tribe Moricandiinae by Schulz (1936), but its position is not very clear (Go´mez-Campo 1980; Al-Shehbaz 1985) It has been excluded by Go´mez-Campo (1980) because it lacks the key tribal morphological features However, several studies that include isozymes (Anderson and Warwick 1998), easy hybridization with cultivated Brassica species, and exchange of genetic material (Li et al 2003; Li and Ge 2007), and ITS sequences and cp trnL intron information (Warwick and Sauder 2005) strongly support its inclusion and also of two more genera, Calepina and Conringia, in the tribe Brassiceae However, Beilstein et al (2006) placed Conringia and Calchanthus in a separate well-supported clade

XI EVOLUTION OF MORPHOLOGICAL CHARACTERS

It has been suggested that the Himalayan region is the prime center of variation for several Crucifer tribes, where the area of dispersion extends from the region up to the Atlantic Ocean across vast regions of the Mediterranean, Irano-Turanian, and Saharo-Sindian phytochoreas (Hedge 1976) However, the maximum variability in Brassiceae occurs in the southwest Mediterranean area comprising chiefly Morocco, Algeria, and Spain This can be regarded at least the secondary center of origin if not the primary one from which vigorous evolutionary radiations occurred

position in the cp lineage It appears that these morphological characters evolved much after lineage differentiation We consider here three such characters: cotyledon, adult leaf, and fruit shape We exclude flowers as these are rather homogeneous in the coenospecies and members of the tribe Brassiceae Those interested in floral characters are referred to Clemente-Mun˜oz and Herna´ndez-Bermejo (1980)

A Cotyledons

An extensive investigation on cotyledonary characters has been carried out by Go´mez-Campo and Tortosa (1974, Fig 2.5) In general, expanded

cotyledons in the coenospecies are wide to oblong and variably notched Diplotaxis species have small, slightly longer than wide and slightly notched cotyledons These together with those present in Guenthera and Sinapidendron (wider but with shallow notch) probably represent the primitive type Then heteroarthrocarpic genera (Brassica, Rapha-nus, Coincya, and Sinapis) undergo a progressive tendency toward wider cotyledons with deeper notches Erucastrum, Eruca, Hirschfeldia, Enarthrocarpus, and Trachystoma represent intermediate steps be-tween Diplotaxis and Brassica However, there are some exceptions: D siifolia, and Erucastrum cardaminoides show cotyledons that are very similar to Brassica Conversely, cotyledons of Moricandia and Rytidocarpus have an almost absent notch and a short petiole, succulent appearance, and glaucous color representing xerophytic features The only deviation from such types within Brassica coenospecies is in Pseuderucaria, which, like other psammophylls, have thick notchless cotyledons

B Adult Leaves

Adult leaves in the coenospecies are of four types, as observed by Go´mez-Campo (1980) The names of leaf silouettes are here adapted to a more correct and updated nomenclature These are:

1 Simple, entire to shallowly lobed

2 Lobed to pinnatifid (sinuses not reaching the midnerve) Pinnatisect (divided with sinuses reaching the midnerve) Pinnatisect with reduced number (vestigial to two pairs) of lateral

segments

the most heterogenous and has leaves of every kind; for example, pinnatifid in B repanda, B elongate (Guenthera);  pinnatisect in B barrelieri, B oxyrrhina, and B tournefortii; lyrate-pinnatisect with variable reduction in segments in all the cultivated species Xeromor-phous species such as Moricandia and Rytidocarpus have simple entire leaves Simple entire leaves might be a basic type from which others evolved, but the habit of the species (annual, biennial, perennial, etc.) has probably been determinant for a rapid evolution of the different types

C Fruits

Many authors have studied the fruit characters in a wide range of taxa of the coenospecies The siliqua consists of two separate cavities

The distal cavity is formed by the substylar region and is empty in most crucifers Only in a part of the tribe Brassiceae it can have seeds—a phenomenon referred to as heterocarpy or, more correctly, heteroarthrocarpy The valvar portion is, in general, the seed-bearing cavity, dehiscent by separation of the valves In Raphanus and some species of Enarthrocarpus, the beak is highly developed and is dehiscent by fragmentation Raphanus is an extreme case where the valvar portion is only vestigial Trachystoma, Enarthrocarpus, Sinapis aucheri, and Coincya may also exhibit strong heteroarthro-carpy A moderate reduction in fruit size may also occur in some cases (such as some Diplotaxis, Erucastrum, or Brassica species), but it is much stronger in some Raphaninae Most of the genera have pods that are  erecto-patent However, adpressed pods also occur in Hirschfeldia, B nigra, and some Erucastrum species while Coincya longirostra and Diplotaxis harra have reflexed or pendulous fruits

Heteroarthrocarpy and fruit reduction plus some additional char-acters, such as ribs, rugosities and wings, have resulted into a diversity of pods and have been assigned a major importance in taxonomy

D Isthmus Concept

characters on the east side Evolution of heteroarthrocarpic fruits might have occurred with the origin of the subgenus Rhynchocarpum of Diplotaxis (D assurgens, D virgata, D tennuisiliqua, D catholica, D berthautii, and D siifolia) Thus, Diplotaxis occurs on both sides of the ‘‘isthmus’’ or ‘‘bridge’’ between both radiations The second radiation involves Erucastrum, Hirschfeldia, Sinapis, Coincya, Eru-caria, Trachystoma, Raphanus, Enarthrocarpus, and Brassica (exclud-ing Guenthera), which have heteroarthrocarpic fruits with vary(exclud-ing degrees of beak development sometimes accompanied with fruit reduction A set of predominantly west Mediterranean genera with reduced fruits such as Rapistrum, Ceratocnemum, Otocarpus, Guiraoa, and so on might be not far, phylogenetically, from Brassica coenos-pecies Other genera, such as Crambe, Crambella, Kremeriella, and so on, are more distant and represent extreme situations of globose beaks with null or vestigial valvar portions The heteroarthrocarpic radiation may not be completely monophyletic as both cp lineages seem to occur at both sides of the isthmus

XII CONCLUDING REMARKS

The genus Brassica with its vast diversity of forms and uses has been subjected to intensive investigations by researchers and has served as a model for studies on cytogenetics, speciation, and domestication The choice of Arabidopsis as a model eudicot plant for genomics investiga-tions has given new impetus to Brassica research Brassica and allied genera constitute a potential germplasm pool possessing many desir-able horticultural traits The last few decades have witnessed a spectecular progress in cytological, in vitro, and molecular techniques Thus, classical cytogenetics has given way to molecular cytogenetics As Brassica chromosomes are relatively small and lacking distinctive physical landmarks, their precise identification and generating reliable karyotypes is difficult In situ hybridization techniques (GISH, FISH) and a spectrum of molecular markers allow identification of individual chromosomes through direct localization of DNA probes on chromo-somes and are very helpful for structural and functional chromosome analysis

lines of B napus, B juncea, and B carinata have become available during the last 60 years, this added variability is still inadequate More synthetic genetic variants are yet to be obtained by utilizing the enormous morphological, physiological, and geographical variability of the diploid constituent parents As many of these current diploid variants have evolved after the natural syntheses of the allopolyploids, they are likely to produce new useful variability Hybridization in nature was always unidirectional Synthetics with new cytoplasms as com-pared to the natural ones and also new combinations of cytoplasmic organelles following protoplast fusion can be obtained easily at present, generating further variability

As in any crop improvement program, wild germplasm always plays a pivotal role Nuclear genes conferring desirable traits as well as cytoplasmically controlled characters, such as male sterility, herbicide resistance, and photosynthetic activity, are frequently distributed in the related wild germplasm in the tribe Brassiceae Enriching con-ventional germplasm with alien genetic diversity is a much-desired goal Introgression of traits can be achieved successfully in view of the advances made via in vitro protoplast fusion methodology In recent years, a large number of wild species have been combined with crop species, overcoming even intertribal barriers However, introgression of traits across generic boundaries has not been very successful in a majority of instances due to a general lack of intergenomic chromo-some homoeology It is necessary to devise ways to induce such homoeologous pairing to facilitate alien gene transfer One such approach might be a chromosome-5B-like manipulative system used in wheat Although the occurrence of a pairing regulator gene has been proposed in B napus and B juncea based on indirect evidence, it remains to be clearly demonstrated

present Molecular investigations indicate enormous variability for chloroplast and mitochondrial genomes This offers opportunity for generating novel cytoplasmic male sterile lines for use in hybrid seed production As discussed in earlier sections, mitochondrial genome rearrangement/recombination are a rule rather than an exception in somatic hybrids, particularly in Brassicaceae It has also been demonstrated that mitochondrial genome organization and its expres-sion in synchrony with the nuclear gene expresexpres-sion control flower morphology Different flower types could be produced by developing cybrid lines for correcting the defects in flower morphology It has been demonstrated time and again that some CMS systems in Brassicaceae were associated with defects in floral morphology Although intensive efforts in the past three decades have made avai-lable an array of CMS and restorer lines through convetional methods, the challenge is to develop better ones using the in vitro bio-technological methods These include rectifying developmental and floral abnormalities in the traditionally developed CMS lines following protoplast fusion Protoplast fusion techniques can also remove exces-sive alien mitochondrial DNA through intergenomic mitochondrial recombination, which makes restoration easier

With the availability of genetic systems for controlled pollination, hybrids are likely to become popular in most countries in the near future Given the current status of Brassica genomics and recombinant technology, it is worth exploring the possibility of fixing heterozy-gosity and hybrid vigour through apomixis Some species of the genera Boechera and Draba, both crucifers, reproduce through diplosporous apomixis (Sharbel and Mitchell-Olds 2001; Richards 2003) Investiga-tions are under way to unravel the genetic and molecular mechanisms that cause apomixies expression Introgression of this trait will have significant impact on Brassica production

emerging from these investigations will contribute to unraveling the structure and origin of Brassica genomes

Traditional classifications of the Brassicaceae are mostly based on flower and fruit characters and also geographical distribution However, the subdivision of the family into tribes and subtribes and also generic delimitation have been contentious issues Molecular phylogeny, in recent years using molecular markers, specifically the maternally inherited cpDNA and biparently inherited ITS sequences (internal transcribed spacers of nuclear ribosomal DNA and 5.8S rRNA gene), strongly suggest massive incongruities in the generic and specific delineations Chloroplast DNA, ITS, and cp trnL intron information not support the separate recognition of subtribes Brassicinae, Morican-diinae, and Raphaninae Surprisingly, the information from ITS and ITS/trnL data does not provide evidence of cp lineages in Nigra and Rapa/Oleracea as suggested and discussed earlier, but clearly indicates the polyphyletc origins for the larger genera: Brassica, Diplotaxis, and Erucastrum However, as Go´mez-Campo (1999b) suggests, it is pre-mature to disturb their current

Rapid-cycling Brassica (RcBr) developed by Paul Williams of Wisconsin University (Williams and Hill 1986) have become model organisms for basic and applied research primarily because of their short life span, small size, and absence of seed dormancy Rapid-cycling plants of all the six crop species are available with life spans ranging from 35 days for B rapa to 60 days for B oleracea These stocks have been used in protoplast fusion for resynthesis of alloploid B napus, developing cytoplasmic male sterility systems, and transfer-ring cp genome encoded characters Another major application of Rc is in undergraduate research and education related to plant breeding, genomics, and ecology where one of the goal is to have undergraduates independent research projects

ACKNOWLEDGMENTS

We thank Robert Hasterok, Silician University, Poland; Xiaoming Wu, Oil Crop Research Institute, Wuhan, China; and Y P.Wang, Yangzhou University, China, for providing publications Special thanks are due to Professor C Go´mez-Campo, University Polytechnica, Madrid, Spain, Professor K Hinata, Tohoku University, Sendai, Japan, and Dr R.K Downey, AAFC-Saskatoon Research Centre, Saskatoon, Canada for their valuable comments and suggestions on this chapter Financial assistance from the Indian National Science Academy, New Delhi, to Shyam Prakash in the form of a senior scientist position is gratefully acknowledged

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