葡萄EST-SSR标记的开发及其应用

Development of SSR markers from grape ESTs and its application

从NCBI公共数据库中随机下载葡萄表达序列标签(expressed sequence tag,EST)8 925条,利用Phrap软件对这些序列进行拼接后形成5 642条序列,利用MISA软件共发掘出含有SSR位点的序列336条,共含有367个SSR位点,候选SSR位点出现的频率为6.50%。二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复单元的SSR数目分别为79(21.53%)、83(22.62%)、29(7.9%)、60(16.35%)和116(31.61%)。利用Primer 3.0 Plus软件随机设计50对引物进行PCR扩增,用6%非变性聚丙烯酰胺凝胶电泳分析这些SSR引物的PCR扩增产物多态性。50对引物中有36对引物能在20个葡萄品种中扩增出理想的PCR产物,占总引物的72%。其中,26对引物扩增条带具有多态性。利用16对经过验证的EST-SSR引物对20个葡萄品种亲缘关系的分析获得了理想的研究结果。

A total of 8 925 grapevine EST （ expressed sequence tag） sequences were randomly downloaded from NCBI, and were further assembled into 5 642 uniform sequences using Phrap software. These sequences were screened by using MISA software to search for SSR motifs and 367 SSR loci were identified from the 336 grape EST sequences. The frequency of EST-SSR identified was 6.50%. All the 367 SSR loci included 79 （21.53%） dinucleotides, 83 （ 22.62% ） trinucleotides, 29 （ 7.9% ） tetranucleotides, 60 （16. 35% ） pentanucleotides, and 116 （31. 61% ） hexanucleotides. Fifty pairs of primers were designed against the some EST sequences by the software Primer 3.0 Plus, and the PCR products of these primers were detected by PAGE and some of them were collected for sequencing. Among the 50 pairs of EST-SSR primers, 36 ones could amplify distinct PCR bands that were the anticipated products. Twenty-six pairs of the EST-SSR primers could amplify polymorphic bands. With sequencing the PCR products, sixteen pairs of primers were verified to be able to amplify real SSR sequences, and the dendrogram of 20 grapevine cultivars generated with the polymorphic marker information from these 16 pairs of EST-SSR primers showed that these cuhivars were relatively classified well.