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以牙鲆(Paralichthys olivaceusTemminck et Schlegel)为材料,基因组DNA经Sau3AⅠ进行部分酶切后,选取400~1 200 bp大小的片段连接到经BamHⅠ酶切的pUC19质粒中,连接产物转化大肠杆菌DH5α,构建牙鲆酶切片段基因组文库。采用菌落杂交的方法,以(AC)10(、AG)10为探针从文库中筛选阳性克隆16个,共得到20个微卫星序列,其中完全型13个,不完全型5个,复合型2个。对其中18个进行引物设计,有11对引物扩增出目的片段,其中8对引物在群体中具有扩增多态性。在威海野生牙鲆30个个体中,各座位上得到的等位基因数为4~19个,观测杂合度(Ho)为0.413 8~0.965 5。其中有3对引物扩增的等位基因数超过17个,表现出高度多态性。本研究旨为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提供基础依据。
Using Paralichthys olivaceusTemminck et Schlegel as the material, the genomic DNA was partially digested with Sau3A I, and the fragment of 400-1 200 bp in size was ligated into the pUC19 plasmid digested with BamHⅠ. The ligation product was transformed into E. coli DH5α to construct Paralichthys olivaceus fragment genomic library. Sixteen positive clones were screened from the library by colony hybridization and 20 microsatellite loci were screened out by using (AC) 10 (, AG) 10 as probes. Among them, 13 were complete, 5 were imperfect, 2. Among them, 18 primers were designed, and 11 pairs of primers amplified the target fragment. Among them, 8 pairs of primers had amplification polymorphism in the population. Among 30 individuals of wild flounder in Weihai, the number of alleles obtained from each locus ranged from 4 to 19 and the observed heterozygosity (Ho) was 0.413 8 to 0.965 5. Among them, three pairs of primers amplified more than 17 alleles and showed a high degree of polymorphism. This study aims to provide the basis for further analysis of genetic diversity, pedigree analysis and genetic map construction.