日本沼虾微卫星引物筛选及群体遗传多样性分析

Isolation and genetic diversity analysis of microsatellite DNA in Macrobrachium nipponense

采用(CT)15、(CA)15两种生物素标记及磁珠富集法构建了太湖日本沼虾基因组微卫星富集文库。120个微卫星克隆中83个位点的核心序列重复在10个以上,长度介于191～580bp,平均为311bp;重复类型中,(CA/GT)n及(AG/TC)n分别占59.09%及20.45%,还检测到(GC)n、(AAG)n、(ACAA)n和(GGCAGA)n等17种类型,包括25.75%完美型、20.45%非完美型和53.80%复合性。用其中12条微卫星引物扩增吴江、滨湖、宜兴和吴兴4个群体240个个体的基因组DNA,各位点表现出较高的遗传多样性,除Mni86、Mni103和Mni114外,其它群体位点大都表现出显著的杂合子缺失,4个群体的遗传分化为低度分化(FST<0.05),4个群体中97.58%遗传变异存在于群体内,仅有2.42%的遗传变异来自于群体之间;吴江和吴兴群体亲缘关系最近,与宜兴和滨湖群体亲缘关系渐远。

Microsatellite-enhanced genomic library of oriental river prawn Macrobrachium nipponense of Lake Taihu was constructed using repeat-enrichment method with biotin-labeled oligos [（CA）15 and （CT）15]and streptavidin magnetic beads. From a total of randomly selected 120 clones,83 clones with more than ten motif repeats were positive. And the sequence size ranges from 191 to 580 bp with an average of 311 bp. The （CA/ GT）n and （AG/TC）n were the abundant microsatellite motifs with a percentage of 59. 09% and 20. 45% in the isolated microsatellites. Other 17 types of repeat sequences were detected,including repeats consisting of （GC）n,（AAG）n,（ACAA）n and （GGCAGA）n. Characterization of these microsatellites showed that 25. 75% of sequences were classified as perfect type,20. 45% as imperfect type and 53. 80% as compound type. Genetic diversity of 4 wild stocks of M. nipponense in 4 sites of Lake Taihu,i. e. Wujiang （WJ）,Binhu （BH）,Yixing （YX） and Wuxing （WX）,were investigated using 12 microsatellite DNA loci from the isolated primers . The allele numbers,effective allele numbers,observed heterozygosity and expected heterozygosity under Hardy-Weinberg equilibrium were estimated to characterize genetic diversity. The result showed that all the 12 loci in the 4 stocks were highly polymorphic. Tests of departures from Hardy-Weinberg equilibrium indicated the loci except Mni86、Mni103 and Mni114 in each stock with significant heterozygosity deficiency. FST and AMOVA analysis across all stocks and loci indicated that there was low level of divergence among 4 stocks. The UPGMA clustering tree based on DA genetic distance demonstrated that Wujiang and Wuxing stocks have near relationship,and they have a distant relationship with Wuxi and Yixing stocks.