采用生物素-磁珠吸附微卫星与质粒二次检测相结合的方法克隆牙鲆微卫星, 并利用所得到的引物分析野生群体遗传结构。共获得2 805个阳性克隆, 测序得到3 120个含微卫星的序列。其中完美型、非完美型和混合型分别占57.97%、7.25%和34.78%。用Primer 3.0 软件设计牙鲆微卫星引物, 从中筛选出具稳定多态性的30对, 分析中国黄海、渤海海域4个不同海区牙鲆野生群体(大连、北戴河、丹东、青岛)遗传结构。结果显示: 有效等位基因数为3.93～9.94, 平均为6.95; 观测杂合度为0.532~0.895, 平均为0.753; 期望杂合度为0.635～0.902, 平均为0.820; Hardy-Weinberg平衡指数(d)的变化范围为?0.247~0.512, 其中7个位点表现为杂合子过剩(d>0), 其余位点表现为杂合子缺失(d<0)。聚类分析显示: 4个群体聚为两类, 大连与丹东聚为一类, 北戴河与青岛聚为一类。
Magnetic bead enrich-method and plasmid detection-method were used to isolate microsatellite DNA from Paralichthys olivaceus genome. The microsatellite markers were applied to analyze natural population structure of four populations, which were collected from Dalian, Beidaihe, Dandong, and Qingdao. Two thousand eight hundred and five microsatellite sequence clones were isolated and 3120 microsatellite loci were obtained. From these sequences, 57.97% were perfect, 7.25% were imperfect and 34.78% were compound. This allowed to design 135 pairs of primers with the software Primer Premier 3.0. Seventy-eight pairs were synthesized and 30 pairs were polymorphic. The effective number of alleles ranged from 3.93 to 9.94 (6.95 in average) with observed and expected heterozygosities ranging from 0.532 to 0.895 (0.753 in average) and 0.635 to 0.902 (0.820 in average), respectively. Hardy-Weinberg equilibrium analysis in 30 microsatellite loci by genetic deviation index (d) ranged from ?0.247 to 0.512, among which 7 loci had heterozygote excess (d>0) and the rest showed heterozygote deficit (d<0). A dendrogram was constructed based on NJ methods using MEGA3.0 software package. Four populations were divided into two clusters. Dalian population and Dandong population were grouped in one cluster, while Beidaihe population and Qingdao population were classified into another cluster.
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