Methodological streamlining of SNP discovery and genotyping via high-resolution melting analysis (HRMA) in non-model...

The exponential growth of genetic resources is fueled by continued advances in genomic technologies and the adoption of single nucleotide polymorphisms (SNPs) for population studies. Concomitant to these developments, there is growing need for rapid screening and subsequent genotyping of SNPs in non-model organisms. Here we provide a rapid and low-cost workflow utilizing high-resolution melting analysis (HRMA) for nuclear marker development and genotyping of 774 Atlantic and Mediterranean swordfish (Xiphias gladius) that is amendable to other species. Preliminary HRMA screening of amplicons (>290bp) for 10 nuclear loci revealed the presence of nucleotide polymorphisms, however, length and variability precluded diagnostic genotyping. Two variants of HRMA were therefore utilized to provide diagnostic genotyping assays. Short-amplicon HRMA (SA-HRMA), in which primers flank closely a SNP of interest, was identified as a low cost, rapid, closed-tube diagnostic genotyping assay that could distinguish between homozygous genotypes by ΔTm, and heterozygous genotypes by heteroduplex melting curve profiles. When the patterns of sequence variation were not suitable for SA-HRMA, unlabeled probe (UP)-HRMA was utilized. UP-HRMA has the advantage of being capable of genotyping multiple linked SNPs in a single closed-tube assay without Bayesian haplotype reconstruction, and can identify new SNPs while genotyping populations. Almost 37% of the SNPs genotyped via UP-HRMA were discovered while genotyping populations and not from preliminary screening. Analysis of swordfish in the North Atlantic (NA, n=419), South Atlantic (SA, n=296), and Mediterranean (MED, n=59) found no significant linkage disequilibrium. To assess whether deviations in HWE could be the result of genotyping error rather than population admixture only swordfish from reported spawning areas in the NA (n=49), MED (n=59), and SA (n=42) were analyzed and all loci were in Hardy-Weinberg equilibrium. Significant genetic differentiation (P<0.001) was identified among populations.