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将牙鲆基因组DNA经限制性内切酶RsaⅠ和Bst UI双酶切后采用FIASCO法构建酶切片段基因组文库。共挑取269个克隆,177个为阳性克隆,阳性克隆率为65.79%。经测序后获得191条微卫星序列,其中完美型占74.35%;非完美型占14.66%;混合型占10.99%。用引物设计软件Primer Premier 5.0设计引物153对,挑选其中的50对合成并在32个野生牙鲆个体中进行扩增,共31个位点具有多态性,统计结果后使用POPGENE软件进行分析,平均等位基因个数为3.939 4,平均有效等位基因数为3.052 2,平均观测杂合度为0.650 1,平均期望杂合度为0.586 6,各引物的Hardy-Weinberg平衡指数在0.012 587～0.984 917变动。这些筛选出的多态性微卫星标记可应用于进一步的牙鲆遗传多样性分析、家系分析及遗传图谱的构建等工作中。
The flounder genomic DNA double restriction enzyme Rsa Ⅰ and Bst UI digestion using FIASCO method to construct digested fragment genomic library. A total of 269 colonies were picked, 177 were positive clones, the positive clone rate was 65.79%. After sequencing, 191 microsatellite sequences were obtained, of which 74.35% were perfect type, 14.66% were imperfect type and 10.99% were mixed type. A total of 153 pairs of primers were designed with primer design software Primer Premier 5.0. Fifty pairs of them were selected and amplified in 32 wild flounder individuals. A total of 31 loci were polymorphic. After statistical analysis, POPGENE software was used to analyze the results. The average number of alleles was 3.939 4, the average effective number of alleles was 3.052 2, the average observed heterozygosity was 0.650 1, the average expected heterozygosity was 0.586 6, Hardy-Weinberg equilibrium index was 0.012 587 ~ 0.984 917 change. These selected polymorphic microsatellite markers can be applied to further analysis of the genetic diversity of Japanese flounder, pedigree analysis and genetic map construction work.