牙鲆CA/GT微卫星标记的筛选

采用生物素选择杂交法与放射性同位素杂交法相结合的技术,成功地从牙鲆(Paralichthys olivaceus)基因组中分离出含有CA/GT重复类型的微卫星序列。通过两轮淘选,共获得526个阳性菌落。测序其中的119个菌落,结果获得133个含有微卫星座位的序列。除了两个复合型微卫星外(1.5%),完美型63个(47.37%),非完美型68个(51.13%)。设计并合成22对微卫星引物,对8个人工雌核发育家系的亲本进行遗传背景分析。PCR结果表明,4对引物无扩增带或者扩增带不是目的条带,1对引物表现为单态,其余17对引物均呈多态性,平均每个座位产生5.2个复等位基因,杂合度为0.375～0.846,多态信息含量为0.305～0.823。结果表明,所筛选的大部分微卫星标记能够用于牙鲆群体遗传学研究。

Isolation of CA/GT Microsatellites from the Paralichthys olivaceus Genome

A library rich in CA/GT microsatellites was constructed from the Paralichthys olivaceus genome by combining biotin capture method and radioactive labeling hybridization. Five hundred and twenty six positive clones were obtained through twice screens. Sequencing confirmed 133 microsatellite loci (number of repeats≥5) in 119 positive clones. Of these microsatellites, two (1.5%) had compound repeat motifs, 63 (47.37%) had perfect motifs and 68 (51.13%) had imperfect motifs. Primer pairs were designed in the flanking regions of 22 microsatelites and subjected to PCR amplification. In 8 artificial gynogenesis families, four pairs failed to amplification, one pair was monomorphic, and the rest were polymorphic with an average of 5.2 alleles per locus. Heterozygosities ranged between 0.375 and 0.846, PIC ranged between 0.305 and 0.823. The results suggested that most of the microsatellites we isolated were qualified to be applied to the population genetic studies of P. olivaceus.