Optimization of Microsatellite Multiplex PCRs for Triploidy Verification and Genetic Diversity Assessment in the Pacific Oyster, Crassostrea gigas

The ploidy detection is crucial for the oyster industry. The objective of this study was to develop a method that verifies ploidy of the triploid Pacific oyster Crassostrea gigas by analyzing the diversity of triploid through microsatellite multiplex PCRs using fluorescent universal primers. We developed four information-rich multiplex PCR panels, comprising a total of 12 genomic microsatellites located in the genome of the C. gigas, distributed across seven chromosomes with an average of 14 alleles per locus. Each panel used M13(-21) primers labeled with specific fluorochrome dyes, and the forward primers for each locus were appended with M13(-21) sequences. We validated the approach to infer ploidy using flow cytometry as a reference, finding > 95% agreement between these methods, and demonstrated its potential utility to infer aneuploidy. Genotyping of 496 triploid samples from eight populations yielded 10 or more alleles per locus in 99.63% of samples in a single capillary electrophoresis. The correct assignment of triploidy depends on the number of markers with three unique allele fragments (MNM). Using semi-strict criteria of three unique alleles at one or more loci, the detection accuracy rate was 95.26% for triploids. Using the strict criteria of three unique alleles at two or more loci, the detection accuracy rate was 98.34%. Populations with reduced genetic diversity due to selective breeding were better suited for the semistrict criterion, maximizing triploid detection. And cultured populations were more suitable for evaluation using the strict criteria, which effectively reduced false-positive diploid assignment and increased triploid detection accuracy. The markers developed in this study were highly polymorphic and effective for assessing genetic diversity and distinguishing populations, providing a reliable tool for triploid detection and analysis in oyster breeding.