Development And Utility Of EST-SSR Marker In Cabbage (Brassica Oleracea Var. Capitata L.)

Cabbage (Brassica oleracea var. capitata L.) plays a very important role on vegetable round-year supply and export trade in China. With the rapid development of molecular biology, molecular markers have been widely used in genetic map drawing, gene mapping of important agronomic traits, genetic diversity, genetic evolution, variety identification and other aspects. SSR (simple sequence repeat) is well known with its high polymorphism, easy operation, co-dominant and specificity. However, the SSR markers in cabbage for public were not too much. This study developed EST-SSR markers of cabbage using a total of 62,567 ESTs in Brassica oleracea from NCBI and applied on SSR fingerprint, hybrid purity identification, analysis of the marker linked with a dominant male sterility gene in the early round cabbage 397. Results were as follows:1. The characteristic and amplification of cabbage EST-SSR. By using the software SSRIT (http: //www.gramene.org/db/searches/ssrtool),1,219 SSRs distributed in 1,176 ESTs were identified from 19,611 non-redundant ESTs, with an average of one SSR per 11.48kb, and included 273 SSR motifs. Analysis of SSR motifs revealed that the dinucleotide (353 SSR) and tricleotide (423 SSR) were major motifs; AG/CT was the most frequent motifs and accounted for 25.59%, followed by AAG/CTT (94 SSR,7.71%), while the frequency for other repeat types were below 5%. Based on the 1,176 SSR-containing ESTs, a total of 978 primer pairs were successfully designed. These primers were screened against genomic DNA of inbreed line early round cabbage 397 and late flat cabbage 20-2-5. The results showed that 897 primer pairs yielded 1,026 amplification bands, of which 128 primer pairs exhibited polymorphism with 258 polymorphic bands accounted for 25.15%. The results above suggested that it was well applied on cabbage.2. Establishment of DNA fingerprinting on some cabbage and purity identification of Jingfeng 1. Based on the development of EST-SSR marker in cabbage, EST-SSR technology was used to establish DNA fingerprinting for 11 cabbage accessions included 4 spring F1 hybrids (Zhonggan 11,8398, Zhonggan 15,Zhonggan 21) and their parents and 18 autumn cabbage accessions included 6 F, hybrids (Jingfeng 1, Wanfeng, Zhonggan 8, Zhonggan 18, Zhonggan 19, and Zhonggan 22) and their parents. Primer BoE222 with the complementary bands was applied in hybrid purity identification of Jingfeng 1, the purity was 90%. The consistent rate of laboratory identification results and field identification results was 98%. 3. Study on the marker linked to the gene of a dominant male sterility in early round cabbage by EST-SSR and BSA. The bulks were formed by using 5 male-fertile plants and 4 homozygous male-sterile plants from F2 population 725 derived from selfing a heterozygous male-sterile plant with a few of viable pollen grain in the round cabbage 397. A codominant marker BoE332 was obtained with the distance 3.6 cM (sterile band was about 270bp, fertile band was about 290bp). Marker BoE332 was helped to screen rapidly the homozygous sterility genotype and also accelerate the breeding process of homozygous dominant male sterile line of early round cabbage 397.