基于转录组数据的凡纳滨对虾微卫星标记开发

Development of microsatellite markers from the transcriptome sequences of Pacific white shrimp(Litopenaus vannamei)

对虾遗传多样性和育种研究需要大量的分子标记。作者利用凡纳滨对虾(Litopenaeus vannamei)转录组测序数据,采用MISA软件进行微卫星序列挖掘,共获得14 767条微卫星序列。统计发现在凡纳滨对虾中微卫星出现的频率为16.76%;在所有的微卫星序列中,2碱基微卫星最多,占59.53%;其次是3碱基微卫星,占35.78%。随机选取其中74条序列设计引物,通过DNA混池扩增和分型的方法进行微卫星标记的开发,PCR扩增的显示有54对(72.97%)能扩增出清晰的目的条带,进一步分型的结果显示27条(36.49%)微卫星显示出多态性,从这些多态性的微卫星位点中选择11个位点在"科海1号"种质库材料中进行个体分型,结果显示在该群体中微卫星标记的等位基因数目为2~11个不等,期望杂合度值为0.256~0.858,观察杂合度为0.213~0.875,多态性信息含量为0.221~0.830,在11个位点中有4个位点显著偏离HWE(P<0.05)。本研究结果为后续使用微卫星标记进行对虾遗传学研究,促进对虾的遗传选育和种质资源保护提供了重要基础。

The Pacific white shrimp,Litopenaeus vannamei,is one of the most economically important marine aquaculture species in China,as well as the world.A large number of molecular markers are required for the studies of genetic diversity and breeding in shrimp.In the present investigation,14767SSRs were detected in L.vannamei unigenes derived from transcriptome sequencing.Among these,dinucleotide SSRs were the most abundant motif(59.53%),followed by trinucleotide(35.78%)SSRs.A total of74primer pairs were designed and validated using DNA pools.54pairs(72.97%)were amplified with clear band,and27pairs(36.49%)were found to be polymorphic.The genetic diversity was analyzed using11highly polymorphic SSR markers in the breeding population of“Kehai No.1,”a selected new variety of L.vannamei.Consequently,the allele number of these microsatellites ranged from2to11.Expected heterozygosity and observed heterozygosity ranged from0.256to0.858and0.213to0.875,respectively.The polymorphic information varied from0.221to0.830.Four loci deviated significantly from Hardy-Weinberg equilibrium(HWE)(P<0.05).The large number of genetic markers developed in the present study would be highly beneficial to the researches to understand genetic diversity,selective breeding,and germplasm conservation in shrimp.