基于EST的猕猴桃SSR标记的建立

Establishment of SSR markers based on EST database from Actinidia

利用NCBI公共数据库最新公布的猕猴桃EST(Expressed Sequence Tag)序列,开发猕猴桃的SSR(Simple Se-quence Repeats)标记。对来源于猕猴桃(Actinidia spp.)非冗余EST数据库(NCBI dbEST)的56400条基因序列使用SSRHunter1.3软件进行了SSR筛查,从中共获得了7939条SSR。应用Primer5.0软件设计合成了85对猕猴桃EST-SSR引物,从中筛选出可进行荧光标记的6对引物:Ad01、Ad02、Ad03、Ad09、Ad42、Ad72。利用该6对EST-SSR荧光标记引物在DNA测序仪上对猕猴桃种质的多态性进行了PCR检测,结果表明均能清晰地产生多态性分离。研究结果表明,通过猕猴桃EST数据库的发掘能够有效地筛选到基因水平的SSR标记。

Simple sequence repeats （SSRs） were screened in order to develop SSR markers based on the newly released EST database from Actinidia. A total of 56 400 Actinidia unigene sequences were searched from the EST database of NCBI by using the SSR Hunter 1.3 software. 7 939 SSRs were identified from the randomly selected kiwifruit EST resources. Eighty-five EST-SSR primers were designed and synthesized through Primer 5.0 software. Among which six primer pairs （Ad 01, Ad 02, Ad 03, Ad 09, Ad 42, Ad 72） flanking EST-SSR loci for fluorescent markers were designed to detect the polymorphism of Actinidia germplasms on the automated fluorescent-labeled DNA system. The PCR results with all these six primer pairs revealed the existence of polymorphism within Actinidia germplasms. The research results proved that the SSR markers at the gene level could be efficiently exploited in the Actinidia EST database and the six primers were available for EST-SSR analysis in Actinidia.