Genetic Analyses Of Intertribal Brassica Species×Capsella Bursa-pastoris Hybrids And B. Napus Nullisomics

Wide hybridization plays an important role in crop improvement and has been used successfully to transfer desired traits from wild germplasm to large number of crop species. The Cruciferae family comprises a large number of wild species which provide rich germplasm with many useful nuclear and cytoplasmic genes for oilseed breeding. Brassica napus (2n=38, genomes AACC) was introduced into China for the first time from Korea in 1930s and subsequently from Europe in 1940s with limited genetic variability and thus the widening of its gene pool through suitable approaches including wide hybridizations is pivotal for further genetic improvement. The crucifer Capsella bursa-pastoris (L.) Medic (2n=4x=32) of tribe Lepidieae is a wild species distributed worldwide and has been used traditionally as vegetable and medicinal plants in China and some other countries for many centuries. It is a natural double-low (erucic acid <1%, glucosinolates <30μmol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Intertribal crosses were made between two Brassica species (B. rapa, 2n=20; B. napus, 2n=38) and C. bursa-pastoris with the latter as pollen parent, in order to introduce the desirable traits into the cultivated Brassica species. Progenies were investigated on morphology, cytology, molecular characteristics, fatty acid compositions and resistance to 5. sclerotiorum. On the other hand, one microspore-derived nullisomics (2n=36) without Orychophragmus violaceus (2n=24) chromosomes was obtained from the hybrid between B. napus and O.violaceus, and nulli-tetrasomics were obtained in the progenies of partial nullisomic plants. Cytogenetical analysis was applied to nullisomic and nulli-tetrasomics. The main results were as follows:1. Brassica species×C. bursa-pastoris. Majority of F₁ plants resembled female parents in morphology and only a few were morphologically intermediate between the parents. The F₁ plants expressed some characters of male parent, including small sized dark-green, deeply divided leaves, nanism, basal clustering branches and white petals. Based on cytological observation of somatic cells, the F₁ plants were classified into five types: two types from the cross with B. rapa, typeâ… had 2n=27-29; typeâ…¡had 2n=20; three types from the crosses with B. napus, typeâ…¢was haploids with 2n=19; typeâ…£had 2n=29; typeâ…¤had 2n=38. One to two chromosomes of C.bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F₁ plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in F₁ plants except for three plants (Nos. 13, 24, 32) which had no specific bands, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum.2. Male sterile hybird. The F₁ plant No. 30 from the cross between B. napus cv. Oro and C. bursa-pastoris which resembled female parent in morphology was male sterile with rudimentary stamens. The BC₁ plants of the F₁ plant pollinated by 'Oro' had good seed-set after selfing. In the selfing population of BC₁ plants, the ratio of male fertile to sterile plants was 3:1 (276 fertile, 83 sterile among 359 plants) (x_c²=0.58 < x_0.05²). Of the 109 plants from sterile F₁ x BC₁, 58 ones were male fertile and 51 male sterile, displaying a 1:1 ratio (X_c²=0.33 < x_0.05²). The result indicated that the male sterility (MS) in the hybrid was controlled by a recessive gene. Cytological observations showed that the tapetum was abnormal with multiple layers and hypertrophy since the stage of sporogenic cell, however, the meiosis was normal with chromosome pairing (19 bivalents) and segregation (19:19). At the tetrad stage, the tapetum of male sterility was vacuolated and disaggregating with multiple layers and hypertrophy, and the tetrads were extruded by tapetum. So they could not produce normal uninucleate pollen grains, the microspores began to degenerating since the stage of tetrad. In most progenies, the content of glucosinolates was reduced obviously, some reached double-low standard, together with the significantly improved resistance to S. sclerotiorum.3. B. napus nullisomics. One microspore-derived nullisomics (2n=36) was obtained before from the hybrids between B. napus and Orychophragmus violaceus (2n=24), its plant height was only 70-80 cm and flowering time was about two months earlier than B. napus. PMCs of the nullisomics had 18 bivalents at diakinesis and 18:18 segregations at anaphase I (AI); however, of 14 nullisomic plants in its selfing progenies, six plants had 31.2-43.8% PMCs with 17:19 segregations at AI, which resulted in the production of nulli-tetrasomics (2n=38). PMCs of B. rapa×nullisomics at diakinesis had 10 bivalents and 8 univalents, which indicated that the nullisomics lost one pair chromosomes of C genome from B. oleracea. The progenies of B. napus×nullisomics had plant height and flowering time similar to B. napus, but produced no nullisomic plants. Nulli-tetrasomics were only 5 cm shorter than B. napus and flowered about one month earlier than B. napus. The nulli-tetrasomics produced 59% diakinesis PMCs with 17 bivalents and one quadrivalent and the remainings with 19 bivalents and 19:19 AI segregations, and had good seed-set after selfing.Finally, it was proposed that chromosome elimination / doubling, and introgression were involved in the formation of these hybrids between Brassica species and C. bursa-pastoris during mitotic divisions of the zygotes or hybrid embryos, the Brassica -type progenies provided an opportunity to rapidly and successfully introduce useful traits of C. bursa-pastoris into Brassica species and to produce lines with improved oil quality and resistance to S. sclerotiorum. The utilization of the nullisomics and nulli-tetrasomics in plant breeding, location and functional analysis of genes was discussed.