青石斑鱼微卫星DNA标记的筛选及群体遗传多样性分析

Isolation and population genetic diversity analysis of microsatellite DNA markers in yellow grouper (Epinephelus awoara)

以中国南海海域青石斑鱼(Epinephelus awoara)为材料,构建青石斑鱼小片段部分基因组DNA文库。以M13通用引物和设计合成的微卫星核心序列引物(CA)15,用PCR法对文库进行筛选,共获得96个微卫星序列,分别分布于28个阳性重组克隆中,其中perfect(完美型)共39个(占40.6%),imperfect(非完美型)30个(占31.3%),compound perfect(混合完美型)7个(占7.3%),compound imperfect(混合非完美型)20个(占20.8%)。同时发现(CA/GT)n序列在青石斑鱼的基因组DNA中含量非常丰富。根据微卫星侧翼序列设计28对引物扩增青石斑鱼基因组DNA,有26对引物能扩增出目的片段,选用其中13对多态性稳定的引物对19尾青石斑鱼进行遗传多样性分析。结果显示,13个位点共检测到48个等位基因,平均观测杂合度(Ho)为0.5982,平均期望杂合度(He)为0.5080,平均多态信息含量(PIC)为0.4722,平均Hardy-Weinberg遗传偏离指数(D)为0.1503。实验初步表明中国南海海域青石斑鱼的遗传多样性较为丰富,但在某种程度上受到了人为的干扰。

The microsatellites of E. awoara were isolated and the genetic diversity were analysed in this study. Firstly, a library of partial small size fractionated genomic DNA was constructed with the E. awoara from South China Sea. 96 microsatellites in 28 recombinant positive clones were obtained through PCR screening the library with M^3 universal primers and simple tandem repeats primer （CA） 15- Among these microsatellites, there were 39 perfect ones （40.6%）, 30 imperfect ones （31.3%）, 7 compound perfect ones （7.3%） and 20 compound imperfect ones （20.8%）. The results indicated that microsatellite sequences characterized by （CA/GT）n were abundant in genomic DNA ofE. awoara. Among the 28 pairs of primers which were designed to analyze genomic DNA of E. awoara according to unique microsatellite flanking sequences with the software Primer 5.0, 26 pairs could be amplified as expected bands, and 13 microsatellite markers were assessed genetic diversity for 19 individuals. As a consequence, the data showed that the average observed heterozygosity （Ho） was 0.598 2; average expected heterozygosity （He） was 0.508 0： average polymorphism information content （PIC） was 0.472 2 and average Hardy- Weinberg departure value （D） was 0.150 3 respectively. The findings indicated that although the genetic diversity orE. awoara in South China Sea was rich, it had been influenced by human behaviors to a certain extent. [Journal of Fishery Sciences of China, 2008, 15 （1） ： 22-29]