Inheritance of high-resolution melting profiles in assays targeting single nucleotide polymorphisms in protein-coding sequences of the Pacific oyster Crassostrea gigas: Implications for parentage assignment of experimental and commercial broodstocks

Single-nucleotide polymorphisms (SNPs) have become markers of choice in genetic studies and may be assayed by a variety of methods. For purposes of confirming pedigrees of experimental and commercial broodstocks of the Pacific oyster Crassostrea gigas, we developed PCR primers for exonic amplicons, ranging in size from 61 to 165 base pairs (average 95 bp) and high-resolution melting (HRM) assays for a set of 53 coding SNPs, which were previously identified from EST sequences and mapped in families. We define as canonical the three HRM profiles corresponding to the two homozygotes and the heterozygote expected at the target SNP (e.g. AA, GG, AG or AA, CC, AC), with variation at no other site in the PCR amplicon. We summarize data on Mendelian inheritance of HRM profiles in four, large, full-sib progenies from wild-caught parents and in 16 families with smaller numbers of progeny, a dataset of 24,065 HRM phenotypes. Not surprisingly, these family analyses confirm Mendelian inheritance of canonical HRM profiles, while showing evidence for non-amplifying null alleles and distortions of transmission ratios that can be ascribed to early viability selection. Unexpectedly, these family analyses reveal evidence for heritable, non-canonical HRM profiles, which we further describe. Non-canonical HRM phenotypes are found at 40 of the 53 markers but comprise only 5.37% of all phenotypes in our dataset (whereas technical artifacts account for only 0.4% of phenotypes and measured genotyping error from replicates is just 0.2%). Cloning and sequencing of 142, non-canonical HRM haplotypes, at 26 loci, reveal polymorphism at up to four sites besides the target nucleotide. Thus, HRM assays reveal extensive haplotype diversity within exons; interpreted correctly, this additional phenotypic variation increases the allelic diversity and power of parentage assignment for these Type-I markers.