草鱼EST-SSR标记及5个不同地域群体的遗传结构分析

以草鱼(Ctenopharyngodon idella)脑、肌肉、肝等组织构建cDNA文库,经测序获得unigenes序列45 318个,从中筛选微卫星序列共5 556个,据此设计EST-SSR引物118对,其中19对引物能够扩增出带型清晰且多态性较高的谱带。用这19个EST-SSR标记研究3个长江水系群体(石首、监利和长沙)和2个珠江水系群体(清远和肇庆)草鱼的遗传结构。结果表明,5个群体的平均多态信息含量(PIC)为0.415 4～0.460 4,显示草鱼群体的遗传多样性偏低;平均观测杂合度(Ho)为0.415 8～0.501 3,平均期望杂合度为(He)0.450 6～0.502 8,其中,长沙群体平均期望杂合度最高,为0.502 8,监利群体的平均观察杂合度和平均期望杂合度均最低,分别为0.415 8和0.450 6,即长沙群体的遗传多样性最高,监利群体的遗传多样性最低;对数据进行F-检验,结果表明,群体间的遗传分化程度低。Hardy-Weinberg平衡的卡方检验结果表明,5个群体均一定程度上偏离了平衡;聚类分析显示长沙群体、石首群体与监利群体聚成一支;肇庆群体与清远群体聚成一支,这与草鱼群体的流域分布一致。

Development of EST-SSR Markers and Analysis of Genetic Diversity in Five Populations of Grass Carp (Ctenopharyngodon idella)

A total of 5 556 microsatellite sequences were found from ESTs databases (45 318 unigenes) which were constructed from brain,muscle,and liver tissues of Grass Carp(Ctenopharyngodon idella).According to these microsatellite sequences,118 pairs of primers were designed with Primer Premier 5.0.Nineteen pairs of microsatellite primers could be used to successfully amplify clear and highly polymorphic products by method of PCR.These EST-SSRs primers were employed to detect the genetic diversity of three groups of Grass Carp populations from the Yangtze River System (Shishou,Jianli,Changsha) and two groups from the Pearl River System (Qingyuan,Zhaoqing).A total of 93 polymorphic loci were amplified and 2 to 8 alleles or 4.89 alleles on average were detected by each primer.The average polymorphism information content (PIC) ranged from 0.415 4 to 0.460 4,indicating that Grass Carp population had lower genetic diversity; the average observed heterozygosity (Ho) was from 0.415 8 to 0.501 3 and the average expected heterozygosity (He) was from 0.450 6 to 0.5028.Changsha group had the highest mean expected heterozygosity (0.502 8),while Jianli group had the lowest mean expected heterozygosity (0.450 6) and mean observed heterozygosity (0.415 8).These observations indicate that Changsha group has the highest genetic diversity,whereas Jianli group has the lowest; F_st value indicates that the populations are lowly differentiated.After applying Hardy-Weinberg equilibrium (HWE) test,several loci were found to be significantly deviated from HWE in five populations.The dendrogram based on genetic distance(D) showed two major clusters according to basin distribution of Grass Carp populations: the stocks from Changsha,Shishou and Jianli,which were sampled from the Yangtze River,were in one cluster; while the other cluster consisted of Zhaoqing and Qingyuan populations sampled from the Pearl River.