Application of high-resolution DNA melting for genotyping in lepidopteran non-model species: Ostrinia furnacalis (Crambidae)

Abstract

Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs) discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM) curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae). Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest.

Figure 1. Details of the SNPs studied, including the primer and probe positions.

The small vertical red bars indicate the newly identified SNPs in this study using the Tpi gene (Accession number DQ204987) as the reference. The primer and probe sequences are listed in Table 1.

Figure 2. Genotyping of SNP rs84 by SA-HRM.

A, D. normalized melting curves. B, E: normalized derivative melting curves. C, F: normalized difference curves. Unknown samples were successfully genotyped by prior mixing with known controls. Arrows link genotypes with corresponding same color curves.

Figure 3. Genotyping of SNP rs577 by SA-HRM.

A, D. normalized melting curves. B, E: normalized derivative melting curves. C, F: normalized difference curves. Unknown samples were genotyped by prior mixing with known controls. Arrows link genotypes with corresponding same color curves.

Figure 4. Genotyping of SNP rs84 by UP-HRM.

A: raw melting curve data. B: derivative melting curves. C: normalization of melting curve data. D: normalized derivative melting curves. Three groups are well distinguished: A/A in blue, G/G in red, and G/A in gray.