Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis
Dang, Xiao-Dong; Howard-Williams, Emma; Kelleher, Colin T.; Meade, Conor V. (2012), Data from: Rapid identification of chloroplast haplotypes using High-Resolution Melting analysis, Dryad, Dataset, https://doi.org/10.5061/dryad.s904k
We have evaluated High Resolution Melt (HRM) analysis as a method for one-step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c.360 and 390bp) at the chloroplast rps16 intron (c.750bp) we applied HRM to identify haplotypes in 21 populations of two European arctic-alpine herb species Arenaria ciliata and A. norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melt-curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR-RFLP, only 7 of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMeltSM. In silico mutation analysis carried out on a 360bp wild-type rps16I amplicon determined that the expected rate of missed SNP detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. Overall, pairwise melt peak differences between haplotypes were significantly correlated with genetic distance, and in vitro HRM successfully discriminated between all amplicon templates differing by 2 or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, these differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single-reaction method for haplotype identification.