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.
Highlights
Insects in the order Lepidoptera, including the domesticated silkworm (Bombyx mori) and many destructive pests of agriculture and forestry, are among the most diverse and species-rich groups of insects [1]
Single nucleotide polymorphisms (SNPs) discovery by sequencing To find SNPs for genotyping, DNA samples from 30 females (10 from each population) of O. furnacalis obtained from three different populations (Of_CU, Of_CC, and Of_NC) were amplified by previously described primers [36,37] and PCR products were directly sequenced in both directions to identify the point mutation or small insertion/deletion
We first reported that High resolution melting (HRM) can provide a simple, sensitive and cost-effective technique for SNP genotyping in lepidopteran non-model species
Summary
Insects in the order Lepidoptera (moths and butterflies), including the domesticated silkworm (Bombyx mori) and many destructive pests of agriculture and forestry, are among the most diverse and species-rich groups of insects [1]. They are primarily phytophagous caterpillars, and occur in most terrestrial habitats all over the world. In non-model organisms with limited genomic information, molecular markers have proved useful as a tool for revealing the nature and extent of genetic variation [4]. Developing an ideal marker system will be invaluable for non-model lepidopteran organisms
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