采用富集文库-菌落原位杂交法分离了30个三疣梭子蟹(Portunus trituberculatus)的微卫星标记.三疣梭子蟹基因组DNA经HaeⅢ酶切连接后.用固定了((AC)15和(AG)15探针的尼龙膜((Hybond N+)捕捉含有微卫星的片段.转化至大肠杆菌DH5α中.构建三疣梭子蟹微卫星富集文库.菌落原位杂交后.任意选取150个克隆进行测序.阳性克隆率为85.48%.利用软件设计了105对微卫星引物.筛选到稳定扩增标记56对.采用30个三疣梭子蟹个体进行多样性评价.30个位点均表现出多态性.观测杂合度(Ho)、期望杂合度(He)及多态性信息含量(PIC)的范围分别为0.2222～1.000 0、 0.4367～0.9099和0.350～0.892.本研究筛选的微卫星位点将为下一步三疣梭子蟹遗传多样性分析、家系分析、遗传图谱构建和QTL定位等研究提供基础依据.%Thirty microsatellite markers of swimming crab Portunus trituberculatus were isolated and characterized by screening SSR-enriched library. Genomic DNA were digested by HaeⅢ, then DNA fragments containing microsatellites were captured by a piece of nylon membrane(Hybond N+) bounded with the probe combinations of (AC)15 and (AG)15. After transformation to Escherichia coli strain DH5α, the clones were regularly re-arrayed in a new agar plate with the density of about 300 clones per plate and were screened with (AC)15 and (AG)15 probes labeled by DIG luminescent Detection Kit system(Roche). One hundred and fifty clones were randomly selected for sequencing. One hundred and five primer pairs were designed using the software Primer Premier 5.0, of which 56 pairs could amplify distinct PCR products. Polymorphism of these distinct loci was assessed using 30 swimming crab individuals, and the results showed that the 30 loci were all polymorphic. The values of Ho, He and PIC varied from 0.222 2 to 1.000 0, from 0.436 7 to 0.909 9 and from 0.350 to 0.892, respectively. These microsatellite markers would be useful for the studies of genome mapping, QTL, parentage determination and population genetics for this species. [Journal of Fishery Sciences of China, 2011, 18(1): 194-201]
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