National Research Centre on Plant Biotechnology
Indian Agricultural Research Institute, New Delhi-110012 (India)
Cytogenetical researches on Brassicas were initiated with the determination of chromosome number (1916-1930) and genome analysis by Morinaga and U (1928-1935) leading to unravelling the genetic archetecture of crop Brassicas. Spactacular progress has been achieved sence then. These include:
1. Characterization of somatic and pachytene chromosomes, 2. Artificial synthesis of alloploids through sexual and somatic hybridizations for enhancing the variability, 3. Extensive investigations on wild germplasm, classify into cytodemes, and synthesis of a large number of hybrids through sexual and somatic routes, 4. Synthesis of alloplasmics of crop specis for expression of male sterility based on very diverse cytoplasm, 5. Introgression of nuclear genes through chromosome manipulations for conferring agronomic advantages and fertility restoration for CMS, 6. Dissection of basic genomes for developing chromosome addition lines and identifying gene linkage groups and compairing gene synteny between related species, and, 7. Use of molecular markers for chromosome mapping and analyzing genomic relationships.
KEYWORDS: Genome analysis–chromosomes–hybridization–polyploidy–wild germplasm–male sterility.
Determination by Takamine, a Japanese researcher, of chromosome number for Brassica rapa in 1916 was the begning of cytogenetical researches in Brassicas. Genome analysis pioneered by Morinaga during 1928-1934 based on extensive interspecific hybridizations and analysis of chromosome pairing, and U's artifical synthesis of B. napus laid the foundations of Brassica cytogenetics. Two other researchers Karpechenko and Manton were also pioneers, the former synthesized Raphanobrassica (1924) and the latter determined the chromosome number for a large number of species. These investigations established the archetecture of crop Brassicas envisaging 3 diploids and 3 tetraploid species enolved through convergent alloploid evolution. Since then Brassica researches have achieved spectacular progress. We devide these developments in to 2 phases.
The 1st phase 1935–1970
The beginning was the investigations on somatic chromosomes by several workers (Catcheside 1934, Sikka, 1940). A major report was by Robbelen (1960) on the morphology of pachytene chromosomes proposing that diploid species are secondarily balanced polyploids with a basic chromosome number of six. Experimental synthesis of natural alloploid species were carried out extensively, since first synthesis of B. napus by U (1935). Being of academic nature in earlier years, the priorities shifted to designed enhancement of genetic variability of breeding value. Major emphasis centered around B. napus in European countries and Japan, and on B. juncea in India. Synthetics were obtained through conventional hybridizations and also through use of ovary culture and embryo rescue thanks to Japanese researchers, Nishi and Inomata. Majority of synthesis are inferior to natural cultivars in productivity, nevertheless, they represented new variations and have been utilized in breeding programms. Enormous physiological and morphological variability was obtained in B. junicea (Prakash, 1973). Several promising B. napus forms (both oil rape and swedes) were released in Sweden (Olsson, 1986), these include Svalof Panter, Norde, Brink and Jupiter. A fodder form Hakuran has been marketed in Japan.
The 2nd phase 1970 todate
The 2nd phase starts with the researches on wild germplasm. This germplasm is a repository of useful agronomic traits such as resistance to diseases; and mitochondrial genes for inducing cytoplasmic male sterility. Although Manton (1930) determined the chromosome number for many of the species, the researches, in reality, were initiated by Mizushima in 1950s. Several expeditions to Mediteranean by Japanese and Spanish scientists led to enormous collection of wild taxa. Mizushima (1950; 1968) hybridized several of these wild species and proposed his views on genomic homoeology. However, the most outstanding researches were carried out by Harberd (1972, 1976, 1980). Besides, determining the chromosome number for several of these species, hybridized them through in vitro and studied the chromosome pairing in a large number of interspecific and intergeneic hybrids. These investigations led him to classify this germplasm which he referred to as Brassica coenospecies into cytodems (crossing groups). He included 91 species belonging to 9 genera of subtribe Brassicinae of Schulz (1919) viz. Brassica, Diplotaxis, Eruca, Erucastrum, Hirschfeldiea, Hutera, Sinapis, Sinapidendron and Trachystoma and 2 genera viz., Raphanus and Enarthrocarpus from related subtribe Raphaninae.
This study was later extended by Takahata and Hinata (1984). In recent years, a new dimension has been given to taxonomic status of several species and genera, and boundaries of the subtribe Brassicinae by employing molecular techniques involving nuclear, mitochondrial and chloroplast DNA RFLPs (Song et al. 1988, 1999; Warwick and Black, 1991, 1997). These studies not only substantiated the earlier proposed taxonomic status and cytogenetical relationships, boundary of coenospecis further extended by including 3 more genera viz Moricandia, Rytidocarpus and Pseuderucaria. Majority of the taxa in coenospecies are diploids (42 cytodemes) among which every number from n=7 to n=13 is reported. Polyploidy both auto-and allo, is of limited occurrence. A large number of hybrids representing wild x wild and wild x cultivated species were obtained by overcoming pre-and post fertilization barriers for their utilization in Brassica improvement primarily in Japan and India. These include 39 inter specific, 91 intergeneric and 29 intertribe hybrids. A characteristic feature of meiosis in these hybrids was low to very low level of chromosome pairing. A partially homologus relationships was proposed among the various genomes. At the same time homoeology with in a genus was not always higher then accross the genera.
This phase also witnessed the spectacular development in somatic cell genetics. Numerous studies established Brassica as a very amenable genus for tissue culture. An array of somatic hybrids have been reported. The first report being the synthesis of intertribe hybrid Arabidobrassica (Gleba and Hoffman, 1980). Since then not only natural alloploid specis viz. B. napus, B. carinata, and B. juncea have been synthesized following proloplast fusion but also a large number of somatic hybrids between wild and cultivated species. These include 2 interspecific, 11 intergeneric and 8 intertribe hybrids. They have been characterized for morphology, chromosome number, meiosis, fertility and organelle constitution. Majoirty of them are symmetric, unfortunately they are pollen sterile. A major achievement of these researches on wild germplasm is the synthesis of a good number of alloplasmics of crop species having cytoplasm from wild species which express cytoplasmic male sterility. These are based on very diverse cytoplasm viz. Diplotaxis muralis, D. catholica, D. siifolia, D. erucoides, Brassica oxyrrhina, B. tournefortii, Moricandia arvensis, Raphanus sativus and Trachystoma ballii. Another achievement of cytogenetical researches has been the introgression of nuclear genes conferring useful agronomic traits to crops species through suitable chromosome manipulations. These traits include resistance to black leg, club rot, Phoma, beet cyst nematode, alternaria leaf spot and club root; and fertility restoring genes on CMS (Raphanus) B. napus, (Trachystoma) B. juncea and (Moricandia) B. juncea.
Ramarkable advances have been recorded since 1990 in unravelling the structure of Brassica genome. Molecular cytogenetic methods have elucidated various aspects of genome organization. All the 3 basic genomes have been dissected and chromosome addition lines have been developed for identifying gene linkage groups, assigning species specific characters to particular chromosome and compairing gene synteny between related species. Addition lines of B. nigra, B. rapa, B. oleracea and B. oxyrrhina have been characterized through genome specific markers such as isozymes, rDNA, RFLP and RAPDs.
Linkage maps based on RFLP markers for all the 3 diploid species have been generated and integrated with physical maps obtained by karyotype analysis and determining possible DNA sequences on chromosomes. Recently developed molecular cytological techniques such as Fluosescence in situ hybridization are of immense importance in physically mapping Brassica genomes and monitoring incorporation location of alien chromatin in genetic stocks.
1. Gomez-campo, C. 1999. Biology of Brassica cenospecies. Elsevier science, The Netherlands.
2. Prakash, S. and Hinata, K. 1980. Taxonomy, cytogenetics and origin of crop Brassicas a
3. review. Opera Botanica 55 : 1-57.
4. Tsunoda, S., Hinata, K. and Gomez-campo, C. 1980 Brassica crops and wild Allies—Biology and Breeding. Japan Scientific Societies Press, Tokyo.