日本沼虾EST-SNP的筛选及多态性检测

Development of single nucleotide polymorphism markers for Macrobrachium nipponensis using expressed sequence tags

利用EST数据库开发SNP是筛选SNP标记的重要途径。本研究利用日本沼虾现有EST数据开发SNP标记—从NCBI获得8 458条EST序列;Seqclean软件去除序列中的载体、引物和接头等污染序列,得到8 453条EST序列;利用Quality SNP软件对预处理后序列中含有4条以上同源序列重叠群进行分析,在含有4条以上同源序列的1 055个重叠群中,筛选得到可靠SNP（Reliable SNPs）位点705个,较可信SNPs（High confidence SNPs）905个,潜在SNPs（Potential SNPs）1 144个。根据候选SNP位点设计28对引物,通过聚丙烯酰胺凝胶电泳分析,基因分型验证44个太湖个体,13对引物具有二等位基因多态性,观测杂合度和期望杂合度范围分别为0.259~0.745和0.255~0.728。结果表明,EST数据库中存在大量SNP位点,筛选的SNP标记可用于日本沼虾遗传学分析。

Using the EST database is an important approach to screen SNP markers. In present study, we obtained 8 453 EST sequences of Macrobrachium nipponensis from the Gen Bank after removed carriers, primers and vector sequences using Seqclean. The pre-treatment sequences contained four or more ESTs were analized by Quality SNP. Those EST sequences were clustered into contigs, of which 1 055 contained 4 or more ESTs. 705 Reliable SNPs,905 High confidence SNPs and 1144 Potential SNPs were detected from 1 055 contigs.28 of the putative SNPs were chosen for validation by allele specific PCR with SDS-PAGE with 44 individuals from Tai Lake population, and 13 of them were polymorphic with the minor allele frequency. The observed heterozygosity and expected heterozygosity were distributed from 0.259 to 0.745 and 0.255 to 0.728.The result showed that there were a large numbers of candidate SNPs from public EST data, and EST-SNPs could be development in an efficient way for molecular genetic analysis of Macrobrachium nipponensis.