基于微卫星多重PCR技术的长鳍吻鮈亲子鉴定  

Parentage Assignment of Rhinogobio ventralis Using Multiplex PCR of Microsatellites

作　　者：陈亮[1,2] 汪登强[1] 何勇凤[1] 吴兴兵[1] 朱永久[1] 朱挺兵[1] 王亚龙[1,2] 杨德国[1] 

机构地区：[1]中国水产科学研究院长江水产研究所/农业部淡水生物多样性保护重点实验室,武汉430223 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《农业生物技术学报》2017年第9期1526-1537,共12页Journal of Agricultural Biotechnology

基　　金：中国长江三峡集团公司科研项目(No.0799521)

摘　　要：微卫星多重PCR技术在生物的亲子鉴定、群体遗传分析以及家系管理等方面均得到了广泛的应用。长鳍吻鮈(Rhinogobio ventralis)是我国长江上游特有珍稀鱼类,目前已达3级急切保护状,被列为低危鱼类。为了提高长鳍吻鮈微卫星分型效率,本研究从已开发的微卫星中筛选出15个多态性较高的微卫星位点进行组合扩增,并对位点组合的引物浓度、退火温度和反应体系等条件进行优化,建立了5组各含3个微卫星位点多重PCR体系。利用5组微卫星多重PCR,通过ABI 3730测序仪和Cervus v.3.0软件对136尾长鳍吻鮈进行个体基因型检测以及遗传多样性分析,结果显示,群体的平均等位基因数(number of allele,Na)为15.600,平均期望杂合度(expected heterozygosity,He)为0.825,平均观测杂合度(observed heterozygosity,Ho)为0.813,平均多态信息含量(polymorphism information content,PIC)为0.801。对40尾候选亲本和96尾子代进行亲子鉴定分析,结果显示,第一亲本的累积排除概率(combined exclusion probability of the first parent,CE-1P)、第二亲本累积排除概率(combined exclusion probability of the second parent,CE-2P)和双亲累积排除概率(combined exclusion probability of a parent pair,CE-PP)分别为0.999986 07、0.999 999 97和1.000 000 00。使用该5组微卫星多重PCR体系进行亲子鉴定准确率为100%。长鳍吻鮈微卫星多重PCR技术的建立为亲子鉴定、分析群体遗传多样性和辅助家系管理提供了一种高效的技术手段。Multiplex PCR technique of microsatellites has been wildly used in the field of parent assignment, population genetic analysis and family management. Rhinogobio ventralis is endemic and rare fish in the upper Yangtze River, China. From the species vale and threatened degreed evaluation, it has reached three-level desperate protection sate and has been classified as low risk fish. In order to develop a rapid, economically efficient and robust approach for genetic studies of R. ventralis, a technique for the parentage assignment in R.ventralis was established based on 5 multiplex PCR panels using previously reported microsatellites loci with high polymorphism. The annealing temperatures, the primer ratio and the concentrations of reaction system were optimized in this technique. With the multiplex PCR tool, the genotyping and the genetic diversity analysis on 136 individuals of R. ventralis were performed using ABI 3730 genetic analyzer and Cervus v.3.0 software. The results showed that the average number of alleles （Na） was 15.600; the average expected heterozygosity （He） was 0.825; the average observed heterozygosity （Ho） was 0.813; the average polymorphism information content （PIC） was 0.801. The parentage assignment was performed on 40 candidate parents and 96 offspring. The combined exclusion probability of the first parent （CE-1P）, the second parent （CE-2P）, and a parent pair （CE-PP） were shown to be 0.999 986 07, 0.999 999 97 and 1.000 000 00, respectively. When using multiplex PCR group A, B and C, the CE-1P was 0.998 348 13 and CE-2P was 0.999 960 49. All of offspring could correctly find their real parents. The accuracy rate of parentage assignment was 100% using 5 multiplex PCR panels. These results indicated that 15 polymorphic loci and microsatellite multiplex PCR technique were suitable for paternity test of R. ventralis. The development of microsatellite multiplex PCR system provides a high efficiency technical method for parentage assignment, genetic diversity analysis, a

关 键 词：长鳍吻鮈 微卫星 多重PCR 亲子鉴定 

分 类 号：S917.4[农业科学—水产科学]