Molecular Markers And Utilization In Resynthesized Yellow-seeded Brassica Napus

Rapeseed is one of the major oilseed crops, Brassica napus is the most important in the world. A primary objective in the breeding of Brassica napus is to increase the oil yield per unit area which is determined by the seed yield potential and oil content in the seeds. A common strategy to enhance the yield potential is to develop hybrid varieties i.e. through the utilization of heterosis. Polima cytoplasmic male sterility (cms) is currently the most important hybrid system for hybrid breeding in China.The approach to enhance oil content in the seeds is to increase the oil content in embryos or to reduce the hull proportion of seeds. At the same genetic background, compared with black seeds, yellow seeds of Brassica napus have significantly thinner seed coat leading to a lower hull proportion and thereby higher oil content. Some other advantages of yellow seeds include a clearer oil, higher protein and lower fiber contents. However, there is lack of stable yellow-seeded germplasm.To transfer the yellow-seeded gene into Pol cms restorer lines, and breed hybrids would be an ideal approach to increase the oil yield.The resynthesized yellow-seed Brassica napus No.2127-17 is purely yellow and shows steadily genetic behavior. In this research, we study inheritance mode of the No.2127-17 seedcoat colour, identify molecular markers closely linked to its yellow-seeded gene, and transfer its yellow-seeded gene into the Hui5148-2 by MAS to breed yellow-seeded restorer lines. The main results are as follow:In microspore culture, we analyzed the frequency of embryogenesis at different sampling time and the doubling efficiency of different treatments with colchicines, when the donor plants were grown in field, the results indicated that the best sampling time was 7:00 Am and the best inflorescence age was 6-20 days after the first bud flowering, resulted in 4.31 embryo/bud, 4.08 embryo/bud-4.34 embryo/bud ratio of embryogenesis respectively. In the doubling experiment, microspores were treated with colchicines at the first step incubation and the highest doubling efficiency of 76.47% was obtained from the treatment at 50mg/L concentration.The inheritance mode of seed colour in No.2127-17 was investigated in the F2, BC1 and F1-derived DH progenies of the two crosses (Hui5148-2, 99Yu42 and No.2127-17). It was indicated that seed colour was under the control of maternal genotype and yellow seed was partially dominant over black, segregation analysis revealed a single gene locus for the partial dominance of the yellow seed colour.Bulked segregant analysis (BSA) combined with RAPD and AFLP techniques would quickly identify markers linked to the yellow seed coat gene. A total of 810 RAPD primers, 240 (29.6%) revealed polymorphisms between the parents, among the 240 RAPD primers and 512 AFLP primer pairs, 4 RAPD and 16 AFLP pairsshowed polymorphisms between the bulks, furthermore, two RAPD and eight AFLP markers were identified in the vicinity of the seed colour gene locus using a 127 individuals DH progeny of the cross (Hui5148-2xNo.2127-17). Seven (2 RAPD and 5 AFLP) of the 10 markers were linked to the allele for yellow seed whereas the other 3 markers to the allele for black seed. The linkaged distance covered a map length of 53.8cM with an average of 5.4 cM per marker. The seed colour gene locus was bracketed by two tightly linked markers EA02MG08 (2.4 cM) and S1130 (3.9 cM).RAPD is sensitive to reaction conditions resulting in poor reproducibility and AFLP technique is generally expensive to employ. We recycled, cloned and sequenced 6 markers region (including 2 RAPD, 2 AFLP in coupling and 2 AFLP in repulsing), designed specific PCR primers (20bp-25bp) based on the sequence, and then successfully developed S1130 into SCAR (SCS1130) and EA05MC12 digested with Bshl2S6 I into CAPS (SCA1) makers associated with yellow seed coat gene, they tightly flanked (3.2cM and 3.8cM, respectively) to the gene. The resulting SCAR and CAPS markers were evaluated in backcross breeding population, and demonstrated to be more accurate, easier to score and valuable in the large-scale MAS, to facilitate and accelerate the yellow seedcoat breeding in Brassica napus.By MAS as well as phenotype selection and quality analysis, we successfully transferred No.2127-17 yellow seed coat gene into the Hui5148-2 using cross-backcross-selfed methods. We used S1129 and S1130 to screen the yellow-seeded plants in BC1F1 whereas SCS1130 together S1130 and S1129 in BC2F1. For the positive yellow-seeded plants, we adopted two-step method to select the background, 88 RAPD primers (30+58) in BC1F1 whereas 60 AFLP pairs (20+40) in BC2F1. We selected 3 individuals backcrossed with Hui5148-2 every generation, however, we selected one cross for MAS after quality analysis and observation of the appearance in the field. In BC2F2, we used co-dominant marker SCA1 to identify the individual genotypes are homozygous or not, selected 7 homozygous yellow-seeded Pol cms restorer lines with high oil content, low erucic acid and middle glucosinolate.