Genomic Characterization and Multiplex PCR Authentication of Commercial Triploid Oysters: Evidence for Mosaic Crassostrea gigas–C. angulata Ancestry

Triploid oysters are widely used in aquaculture, but their commercial species designation may not accurately represent their nuclear genomic composition, particularly when interspecific or polyploid broodstocks are involved. In this study, we combined whole-genome sequencing with a mitochondrial COI-based multiplex PCR assay to characterize commercial triploid Crassostrea gigas, C. angulata, and C. hongkongensis. Whole-genome allele-frequency analysis confirmed triploidy in all 15 individuals examined, comprising five individuals each from the A3N, G3N, and H3N groups. Cross-species ancestry dosage showed that G3N and H3N were predominantly assigned to their nominal species, whereas A3N exhibited mixed C. angulata–C. gigas nuclear ancestry, with approximately 2.2 and 0.7 haploid-equivalent copies assigned to these species, respectively. The minor C. hongkongensis assignment was interpreted as background cross-assignment rather than evidence of an additional ancestral contribution. Local-ancestry analysis further revealed chromosome-scale mosaics of zero, one, and two C. gigas-derived copies, with copy-weighted C. gigas contributions of approximately 0.69–0.88 haploid-equivalent copies among A3N individuals. This interspecific ancestry was supported by convergent evidence from complementary analyses of discrete dosage-state distributions, D-statistics with two alternative outgroups, and mapping-independent C. gigas-diagnostic 21-mers. All A3N individuals nevertheless carried C. angulata COI haplotypes, consistent with a C. angulata maternal lineage. The breeding history underlying the nuclear ancestry mosaics could not be resolved because the actual parental broodstock was unavailable for genomic analysis. A three-species multiplex PCR assay generated diagnostic products of 212, 359, and 169 bp for C. gigas, C. angulata, and C. hongkongensis, respectively, with no detectable cross-amplification at an optimized annealing temperature of 62 °C. Diagnostic amplification remained detectable with 1 ng of template DNA and was successfully applied to specimens from multiple Korean production sites and Dalian, China. These findings demonstrate complex nuclear ancestry in the five A3N individuals examined that was not captured by their nominal species designation or mitochondrial lineage alone. Although the observed ancestry proportions cannot be generalized to commercial A3N stocks as a whole, the results highlight the complementary roles of genome-wide ancestry analysis and rapid mitochondrial lineage authentication.