烟草EST-SSR位点分析(英文)

Analysis of EST-SSR Loci in Tobacco (Nicotiana tabacum L.)

利用MISA软件对烟草EST公共数据库中的简单重复序列(SSRs)进行了分析。结果表明,在133523条EST序列中,共获得81757条SSR序列,SSRs之间的距离约为0.92 kb。其中,六碱基重复丰度最大,占60.3%,而单碱基、三碱基、四碱基、二碱基和五碱基重复丰度分别为20.0%、11.0%、4.2%、2.8%和1.7%。在单碱基、二碱基、三碱基和四碱基重复模体中,丰度最大的分别是A/T、AG、AAG和AAAT,而CG在编码区内丰度很低。用CAP3软件进行冗余分析表明,在这6种类型的重复模体中,冗余与非冗余的烟草EST之间没有显著差异。在得到的SSR序列中随机选择10个序列设计引物,在7个烟草品种中进行PCR扩增。结果表明,10对引物全部扩增出PCR产物,其中8对引物扩增出预期片段。用这8组扩增出预期片段的PCR产物进行变性PAGE凝胶电泳检测,结果表明,其中有4对引物(EB4、EB5、EB6和EB8)扩增出多态性条带。

Simple sequence repeats（SSRs） of tobacco expressed sequence tags（EST） in public database were investigated by using computer program MISA（MIcroSAtellite）.Up to 81757 SSRs were found in 133523 sequences and the average distance between SSRs was approximately 0.92 kb.Among them,hexanucleotide repeats（60.3%） were the most abundance,while the monomeric,trimeric,tetrameric,dimeric,and pentameric repeats are represented in decreasing proportions of 20.0%,11.0%,4.2%,2.8%,and 1.7%,respectively.The most abundant motif was A/T,AG,AAG and AAAT in monomeric,dimeric,trimeric,and tetrameric repeats,respectively.Whereas CG rich repeats are rarely found in the coding regions.The redundancy analysis indicated that no significant differences were observed between the redundant and non-redundant set of tobacco ESTs in individual SSR motifs.Ten pairs of primers flanking EST-SSR loci were designed to detect the polymorphism in seven tobacco cultivars.The analyses on denatured PAGE by silver staining confirmed the existence of polymorphism by these four pairs of primers of EB4,EB5,EB6,and EB8 among the seven tobacco cultivars.The SSR loci reported in this study are the first molecular DNA-based genetic markers developed from public ESTs database in tobacco.The EST-SSR markers will be useful information resource for germplasm characterization and crop improvement through genetic mapping and marker-assisted selection in tobacco.