麦红吸浆虫唾腺EST-SSRs的信息分析及分子标记筛选

Characterization of SSRs from the ESTs in the wheat midge, Sitodiplosis mosellana (Gehin) (Diptera: Cecidomyiidae)

昆虫EST资源库的扩充为开发新的分子标记提供了宝贵的资源。本研究对NCBI的EST数据库中来源于麦红吸浆虫Sitodiplosis mosellana唾腺的1217条EST序列进行了unigene组装、SSR信息分析和EST-SSR分子标记筛选。结果表明:在1047个unigenes中共找到141个SSR位点,分布于106个(10.12%)unigenes中,平均每3.49kb出现一个SSR位点。在1～6碱基重复基元中,1～3碱基是主要重复类型,占总SSR的97.16%以上。A/T(31.21%),AC/GT(15.60%)和AAC/GTT(9.22%)分别是单、双和三碱基中占优势的重复基元类型。利用Primer Premier5.0软件对查找的EST-SSRs进行引物设计,并以麦红吸浆虫基因组DNA为模板,对从中选出的26对SSR引物进行多态性检测。结果有20对(76.92%)引物能扩增出清晰的目的条带,并且其中9对(45%)引物表现出多态性。多态性分析结果表明,从9对EST-SSR引物中,共检测到51个等位基因,平均每个位点含有等位基因数为5.67,平均期望杂合度为0.65,平均多态信息含量为0.60。本研究能够为今后麦红吸浆虫的种群遗传结构与遗传多样性研究提供帮助。

Insect expressed sequence tags （ESTs） database provides a valuable resource for developing well-characterized molecular markers. In this study, 1 217 ESTs from the salivary glands of the wheat midge, Sitodiplosis mosellana, were downloaded from NCBI database and assembled into unigenes. The characteristics of unigene-derived SSR were analyzed and partial EST-SSRs were screened. The results showed that 141 EST-SSRs distributed in 106 （10.12%） unigene sequences were detected, with an average of one SSR in every 3.49 kb of unigene sequence. Among 1-6 nucleotide repeat types, 1-3 nucleotide repeats are the main types, accounting for 97.16% of all SSR. A/T, AC/GT and AAC/GTT account for the highest proportion in mononucleotide, dinucleotide and trinucleotide, respectively. Furthermore, EST-SSR primer pairs were designed using the Primer Premier 5.0 program and 26 pairs were selected for marker development in S. mosellana adults. Of the 26 primer pairs, 20 pairs （76.92%） produced discernable polymerase chain reaction （PCR） products, and of these 20 primer pairs, 9 pairs （45%） displayed polymorphism. Fifty-one alleles were found in the 9 primer pairs, the mean number of alleles per locus was 5.67, the average expected heterozygosity was 0.65 and the average polymorphism information content （PIC） was 0.60. The SSRs identified in this study will help explore the population genetic structure and genetic diversity of S. mosellana in the near future.