Establishment and application of multiplex microsatellite genotyping for parentage assignment in Manila clam Ruditapes philippinarum

The manila clam Ruditapes philippinarum has become an important bivalve species in worldwide aquaculture due to its delicious taste and high commercial value. With the rapid expansion of clam farming, there is an urgent need to obtain genetic improvement by selective breeding, such as family-based selection of improved varieties. In this study, 10 microsatellite loci were selected to establish four multiplex PCR groups using four different fluorescences (FAM, ROX, HEX, TAM). The accuracy of these loci was verified by the 100% success rate of parentage assignment in a full-sib family. Parentage assignment was carried out in the mixed-mating parents and their offspring with unknown pedigree information. The genotyping of ten microsatellite loci generated 90 alleles, having the observed heterozygosity (Ho) ranging from 0.333 to 0.729. The expected heterozygosity (He) varied from 0.358 to 0.794. The PIC ranged from 0.342 to 0.763, with a mean value of 0.620. The theoretical combined parentage exclusion (cEP) power for all ten microsatellites was 97% with known genotypes of one parent (P1) and 99% for given the genotypes of both parents (P2). The actual successful assignment rate reached 98% by using all four sets of multiplex microsatellites, serving as the effective method for parentage assignment in the clams. The development and application of multiplex microsatellite genotyping methods provide a cost-effective method for pedigree reconstruction, population genetics and selective breeding of the commercially important clams.