核桃BES-SSR的开发及在遗传多样性分析中的应用

Development of BAC-SSR markers in walnut and its application in genetic diversity analysis

从NCBI数据库全部已知核桃Mbo I基因组BAC文库克隆中下载22 740条末端序列（BAC end sequences,BES）。应用MISA软件搜索获得SSR位点4 732个,平均每2.8 kb出现1个SSR。在含有单个SSR的BES中,1-3个核苷酸重复的类型占SSR总数的96.86%,A/T、AT/AT、ATT/AAT分别为最丰富的单核苷酸、2核苷酸和3核苷酸重复。选择不同类型和重复次数的SSR设计合成50对引物,从中筛选出多态性引物33对,其中19对引物扩增出1或2个等位基因,无非特异扩增产物。应用这19对引物对20份核桃属不同种的基因型进行SSR分析,结果表明,每对引物检测到2-9个多态性位点,平均多态性位点5.4个;其中17个位点的多态信息含量高于0.5,总平均值为0.662,多态性较高。聚类分析显示,不同基因型按照种属分类及地理起源分组。因此,BES-SSR是一类多态性高,可用于核桃属植物遗传分析的新SSR引物。

From all known Juglans regia Mbo I genomic BAC clones in NCBI database,22 740 end sequences（ BES） were downloaded and analyzed. A total of 4 732 SSRs（ microsatellites） were found by using the MISA program. The frequency of SSRs was approximately 1 /2. 8 kb. Mono- to tri-nucleotide types accounted for 96. 86% of all the single SSRs in the BES. A / T,AT / AT and ATT / AAT motifs were the repeat categories of most abundant mono-,di- and tri-nucleotide respectively. Fifty SSR primer pairs with different types and number of repeats were designed and synthesized,and 33 highly polymorphic SSR primer pairs were screened. Nineteen out of the 33 primer pairs had 1 or 2 alleles without nonspecific amplification. Those primer pairs were used to amplify SSR for 20 genotypes in the genus Juglans. The results showed that 2- 9 polymorphic loci,5. 4 on average,were amplified per pair of primers.Polymorphism information content was greater than 0. 5 in 17 SSRs loci with a mean value of 0. 662 in all loci,which showed high polymorphism. The cluster analysis showed that the groups were clustered in accordance with species and origin. Our findings indicated that BES-SSRs identified in J. regia had high polymorphism and were new SSR primers for genetic analysis of Juglans.