A 16-microsatellite multiplex assay for parentage assignment in the eastern oyster (Crassostrea virginica Gmelin)

We tested a high level multiplex PCR for efficient microsatellite genotyping in the eastern oyster (Crassostrea virginica). Sixteen microsatellites were selected based on polymorphism, allele size range, and reliability. They were divided into four groups according to their allele sizes and labeled with four different fluorescence dyes, so that loci in the same color did not overlap in size. A multiplex PCR protocol with eight levels of primer concentrations and four phases of touchdown reactions was developed for simultaneous amplification of all 16 loci and subsequent genotyping on a genetic analyzer. One hundred sixty progeny from a putative pool of 81 full-sib families were successfully genotyped at all 16 loci after one PCR, and all were unambiguously assigned to their perspective families. Our results show that high level multiplexing of 16 microsatellites is possible and can be used for rapid and highly efficient parentage assignment in the eastern oyster.