Application of high-resolution melting for variant scanning in chloroplast gene atpB and atpB-rbcL intergenic spacer region of Crucifer species

Abstract

High-resolution melting (HRM) analysis is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In this study, a novel HRM assay was carried out to detect SNPs in the chloroplast gene atpB which encodes the beta subunit of the ATP synthase and atpB upstream intergenic region. The polymorphisms of the two fragments in intertribal samples from the Cruciferae family and within the species of Brassica napus were detected. Based on this results, we found that HRM were able to determine over 90% of the variants which included single or multiple variants and insertion-deletion polymorphisms (INDELs) and rendered possible genotyping of more closely spaced polymorphisms, although there were several false positives (FPs) and misclassification. Six haplotypes were identified in the intertribal materials. The analysis of 90 B. napus found five variation types and the variations were all located in the intergenic region. In conclusion, HRM analysis is a closed tube assay that is easy to perform and is a more effective approach to identify variant of chloroplast genes. This study will facilitate further functional investigations into the role of chloroplast genes in photosynthesis, phylogeny and molecular evolution.

Key words: atpB gene, chloroplast genome, crucifer, high-resolution melt curve analysis, SNP, INDEL.