该研究旨在分析苹果EST中SSR位点分布规律,开发苹果EST-SSR引物,探究基于EST-SSR的苹果品种遗传差异.从NCBI公共数据库中下载63 708条苹果表达序列标签(Expressed sequence tag,EST),首先利用MI-SA软件进行SSR位点查找,筛选出符合条件的SSR位点,利用Primer 5.0 Plus软件设计49对引物,用非变性聚丙烯酰胺凝胶(PAGE)研究这些SSR引物的PCR扩增产物,总结其特点,并回收部分产物测序,以验证其真实性.结果显示63 708条苹果的EST序列中有5 423条含有总计6 153个SSR位点.二核苷酸、三核苷酸和六核苷酸重复是最主要的SSR类型,分别占总SSR的50.38％、14.85％和15.47％.电泳结果显示有33对引物能在22个苹果品种中扩增出理想的PCR产物,其中25对引物能扩增出多态性条带,测序结果显示引物能扩增出目标片段.ES7-SSR分子标记体系的聚类结果与富士系苹果家族关系基本吻合.%This experiment aimed to study the distribution of single sequence repeats (SSRs) in the expressed sequence tags (EST) of apple, develop new EST-derived SSR markers, and validate the application of EST-SSR markers in apple genetic diversity analysis. A total of 63 708 EST sequences of apple was obtained from the National Centre for Biotechnology Information (NCBI) and were screened for SSR motifs using MISA software. Forty-nine pairs of primer were designed using the Primer 5.0 Plus software and the PCR products by these primers were detected by PAGE. A summary on the characteristics of PCR products was done, and some of which were recovered for sequencing. The results displayed 6 153 SSRs identified from 5 423 apple EST sequences out of the entire apple collection of 63 708 ESTs. Dinucleotide,trinucleotide and hexanucleotide repeats were the domi-nant SSR types with proportion of 50. 38% , 14. 85% and 15.47% , respectively. Electrophoresis results showed that among the 49 pairs of EST-SSR primers, 33 could amplify distinct bands and gave expected products, and among the 33 pairs, 25 were polymorphic. The dendrogram using SSR markers from apple ESTs was consistent with Fuji apple family relationship.
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