Studies On The Development Of EST Markers In Chinese Cabbage And Its Applications

Based on the existing EST resource, two kinds of EST markers, EST-PCR and EST-SSR, were developed first time in Chinese cabbage. The polymorphism revealed by EST-PCR, EST-SSR and gSSR markers was compared in different varieties of Chinese cabbage. Segregation of polymorphic EST markers and gSSR markers in F2 population was analyzed. As well as the transferability to other plant species and application in resource evaluation of EST-PCR and EST-SSR markers developed in Chinese cabbage were discussed and explored. Results can be summarized as follows.Types, frequency and attribute of SSR were analyzed and clarified in Chinese cabbage ESTs. By using the software Repeatmasker, 4584 EST sequences of Chinese cabbage were screened. 434 ESTs containing the SSR were found, accounting for 9.5%. Totally, 474 SSRs accounting for 10.8% of ESTs searched, were mined out and 40 kinds of repeat motifs were detected. Dinucleotide and trinucleotide repeats, with similar frequency and accounting for 83% together in all SSRs, were dominant, while the frequency for other repeat type is below 5%.EST-PCR and EST-SSR marker were developed in Chinese cabbage. 28 pairs of EST-PCR primer and 15 pairs of EST-SSR primer were designed according to the expressed sequence tags in Chinese cabbage. After testing on the annealing temperature and the concentration of primer, dNTP and MgCl2, a suitable PCR system was established. Under the condition of reaction system developed, primers were screened against genomic DNA of inbreed line A from which the cDNA library was constructed. Among them, 18 pairs of EST-PCR primer and 15 pairs of EST-SSR primer showed the amplification. Then all the primers available to line A were subjected to PCR for DNAs from 29 Chinese cabbage varieties, 10 EST-PCR and 7 EST-SSR primer sets showing polymorphisms were found, accounting for 55.6% and 46.7% of primers tested. The results above suggest that it is an easy and effective way to develop molecular marker based on the EST.EST-PCR, EST-SSR and gSSR marker was compared in ability for polymorphism detection. 29 cabbage varieties were analyzed by using 15 pairs of gSSR primer, 18 primer sets of EST-PCR and 15 EST-SSR primer sets, and the polymorphisms frequency is 60.0%,55.6% and 46.7%, respectively. The result indicates the polymorphism revealed by gSSR marker is higher than that revealed by the marker coming from EST.