虾夷扇贝微卫星标记的分离及其养殖群体的遗传结构分析

Isolation of microsatellite markers in Japanese scallop and its application in genetic structure analysis

采用新型生物素-磁珠吸附微卫星与同位素杂交相结合的方法筛选出15对微卫星引物,并对大连地区3个虾夷扇贝(Patinopecten yessoensis)养殖群体(黑石礁海区、小长山岛海区、广鹿岛海区)的遗传结构进行分析。获得175个阳性克隆并测序,得到160个含微卫星的序列,其中完美型,非完美型和混合型分别占42.69%(73)、46.20%(79)和11.11%(19),经筛选得到15个微卫星标记。虾夷扇贝3个群体的有效等位基因数在1.0000～8.8573之间;期望杂合度在0.0211～0.9111之间;观测杂合度在0.0000～0.8778之间。表明这几个群体遗传多样性水平较高。群体间遗传相似系数在0.9291～0.9624,相似性较高。聚类分析显示,黑石礁群体与小长山岛群体遗传距离最小,亲缘关系最近。基于Hardy-Weinberg平衡的卡方检验表明3个群体均在一定程度上偏离了平衡。本研究所获得的微卫星位点可以为虾夷扇贝的群体遗传多样性分析和遗传图谱的构建等研究提供参考,群体的遗传结构分析对虾夷扇贝的养殖和遗传育种具有一定的指导意义。

Patinopecten yessoensis is one of the most important cultured scallops in the world. Samples of cultured Japanese scallops were collected from Heishijiao sea area, Xiaochangshan island and Guanglu island area. Each population has about 90 individuals. Microsatellite DNAs were isolated from the Japanese scallop genome with magnetic beads in the present work. One hundred and seventy-one microsatellites were obtainded from the 160 positive clones （from 175 clones）, among which 42.39% were found to be perfect,46.24% imperfect, and the rest compound type （11.11%）. 56 pairs of SSR-primers were synthesized, and 15 polymorphic microsatellite markers were screened out, which were applied to analyze the population diversity of the three populations. Amplification was performed in PCR reactor with the 15 microsatellite loci, tested by 8% polypropylene gel eletrophoresis and analyzed by gel analysis software. The observed（Ho） and expected（He） heterozygosity values, the polymorphic information content（PIC） and the number of effective alleles（Nc） were all calculated. We found the genetic diversity level was relatively high in all three populations. Seventy-five alleles were obtained, and the number of alleles of each locus was 1 to 12, ranging from 134 to 520 bp. The average Ne of HSJ population, XCS population and GL population was 3.454 8,3.788 7 and 3.5241, respectively. The average Ho was 0.562 3,0.576 1 and 0.606 0, respectively. The average He was 0.610 2,0.619 8 and 0.635 4, respectively. The data above indicated that genetic potential level of GL population was higher than that of other the rest populations. The genetic similarity coefficient and Nei＇ S standard genetic distance were computed based on the allele frequencies. According to the data, HSJ population and XCS population were shortest in genetic distance among the three populations, followed by GL population. A dendrogram was constructed based on NJ methods using MEGA3.0 software package. The three populations were divided into two clusters, the HSJ population and XCS population were in one cluster, and GL population was another cluster. The Hardy-Weinberg equilibrium was checked by 2 test. The P value test of Hardy-Weinberg equilibrium showed that each population departed from Hardy-Weinberg equilibrium to some certain extent. These microsatellite markers are useful for genetic structure analysis and genetic linkage mapping construction of Patinopecten yessoensis. The population structure results obtained in this work should be helpful to the breeding and culture of Japanese scallops.