采用人工合成的生物素标记(AG)₁₅探针及磁珠富集法构建了脊尾白虾基因组微卫星富集文库。将脊尾白虾基因组DNA经Hae Ⅲ酶切后,收集400~1 200 bp片段,连接特定接头,与生物素标记(AG)₁₅探针杂交并捕获含有微卫星的DNA片段,然后连接到pMD18-T载体中,构建脊尾白虾微卫星富集文库。随机挑取947个克隆,经菌落PCR检验,其中667个为阳性克隆。从阳性克隆中随机选取184个克隆进行测序,有150个克隆含有微卫星序列,共获得199个微卫星序列,其中完美型158个(79.4%),非完美型27个(13.6%),复合型14个(7.0%)。根据微卫星序列,设计了119对微卫星引物,64对扩增出稳定的具有特异性的条带,经30个脊尾白虾个体进行多样性评价后,有26个位点表现出多态性。26个微卫星位点获得的等位基因数从3~16个不等,平均每个位点扩增得到6.5个等位基因。观测杂合度(H_o)、期望杂合度(H_e)及多态性信息含量值(PIC)的范围分别为0.111~0.929、0.246~0.932、0.231~0.909。本研究开发的微卫星位点将为脊尾白虾种群多样性研究、家系识别和种质鉴定提供有效的工具。

A microsatellite-enriched library of the ridgetail white prawn(Exopalaemon carinicauda)was constructed using the method of magnetic bead hybridization enrichment.Genomic DNA of ridgetail white prawn was digested with the restriction endonuclease Hae Ⅲ.DNA fragments from 400 to 1 200 bp were recycled and ligated to Hae Ⅲ adaptors.The ligated DNA fragments were hybridized with biotinylated probe(AG)₁₅.Then,enriched DNA fragments were ligated to pMD18-T vector.947 clones were randomly picked and screened by colony PCR.667 clones were positive.184 clones were randomly selected and sequenced from positive clones.150 clones were found to contain 199 microsatellite sequences.Among these microsatellites,79.4% belonged to perfect type,13.6% belonged to imperfect type and 7.0% belonged to compound type.From the primers designed for the 119 microsatellite loci,64 could amplify expected PCR products and 26 were found to be polymorphic.The number of alleles at each locus ranged from 3 to 16.The observed and expected heterozygosities varied from 0.111 to 0.929 and from 0.246 to 0.932,respectively.The PIC value ranged from 0.231 to 0.909.These new loci will be useful in the study of population genetic structure and conservation in this species.

国家自然科学基金项目(30871933,40806067);科技部农业科技成果转化资金项目(2010GB23260589)