凡纳滨对虾(Litopenaeus vannamei)微卫星多重PCR体系的建立及其在家系亲权鉴定中的应用

Development of Multiplex PCR Systems of Microsatellite Markers for Pacific White Shrimp (Litopenaeus vannamei) and Its Application for Parentage Identification

关键词:  凡纳滨对虾  微卫星多重PCR  亲权鉴定

Key words:  Litopenaeus vannamei  Microsatellite multiplex PCR  Parentage identification

摘要:

为了在遗传分析研究中提高效率并节约成本，本研究从已报道的凡纳滨对虾(Litopenaeus vannamei)微卫星位点中选取多态性较高的位点，基于各位点的扩增片段大小及退火温度等因素进行微卫星位点的组合。通过不断优化位点组合、反应体系及反应程序，成功建立了1个五重、2个四重和1个三重PCR反应体系，并将其应用于11个凡纳滨对虾家系的亲权鉴定。结果显示，四组多重PCR体系中的16个微卫星位点在11个凡纳滨对虾家系中的平均等位基因数(Na)为6，平均多态信息含量(PIC)为0.5813，平均观测杂合度(Ho)为0.513，平均期望杂合度(He)为0.636。利用Cervus3.0软件，对已知系谱关系的11个凡纳滨对虾家系进行亲权分析，其第一亲本累积排除率(CE-1P)、第二亲本累积排除率(CE-2P)和双亲累积排除率(CE-PP)分别为0.99525487、0.99990862和0.99999986。进一步分析表明，当同时使用四组多重PCR体系进行亲权分析时，其模拟配对率和亲权鉴定准确率均为100%，全同胞和半同胞家系鉴别效果良好，表现出准确的鉴别能力。本研究所建立的四组凡纳滨对虾微卫星多重PCR体系均可为后续的凡纳滨对虾遗传多样性分析和亲权鉴定提供高效、准确的检测手段。

Abstract:

Microsatellite markers have been widely used in parentage identification, genetic linkage mapping, and diversity study of aquatic animals, due to the advantages including mendelian inheritance pattern, wide distribution, high polymorphism, high repeatability and stability, and co-dominant inheritance. This technique efficiently helps with analysis of genetic background of different populations, as well as selective breeding. Multiplex PCR could amplify many microsatellite loci simultaneously, therefore is an efficient, rapid, and economic method, and could be a powerful tool of parentage identification, pedigree management, and population genetic analysis of aquatic animals. In order to improve the efficiency and reduce the cost of genetic studies of Litopenaeus vannamei, previously reported microsatellite loci with high polymorphism were selected in developing multiplex PCR systems in this study. Four multiplex PCR systems were successfully established and optimized based on the allelic lengths and annealing temperatures of the microsatellite loci. We used these systems to perform pedigree analysis of 11 families of selected L. vannamei, and found that the average number of alleles (Na) was 6, the average polymorphism information content (PIC) was 0.5813, and the average observed heterozygosity (Ho) and expected heterozygosity (He) were 0.513 and 0.636 respectively. Together with Cervus 3.0 software, the four multiplex PCR systems were also used to verify the capacity of parentage assignment in the 11 families of L. vannamei of which the pedigree relationships were already known. 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 0.99525487, 0.99990862, and 0.99999986 respectively. Further analysis suggested that if all the multiplex PCR systems were used for pedigree analysis, the accuracy rates of both simulated assignment rate and parentage identification could reach 100%. It also indicated that the full-sib and half-sib families had great capability for precise identification. In conclusion, the four multiplex PCR systems for microsatellite markers could serve as an efficient and accurate approach in further genetic diversity and pedigree analysis of L. vannamei.