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.
