Abstract
Microsatellite markers are difficult to apply within lepidopteran studies due to the lack of locus-specific PCR amplification and the high proportion of “null” alleles, such that erroneous estimations of population genetic parameters often result. Herein single nucleotide polymorphism (SNP) markers are developed from Ostrinia nubilalis (Lepidoptera: Crambidae) using next generation expressed sequence tag (EST) data. A total of 2742 SNPs were predicted within a reference assembly of 7414 EST contigs, and a subset of 763 were incorporated into 24 multiplex PCR reactions. To validate this pipeline, 5 European and North American sample sites were genotyped at 178 SNP loci, which indicated 84 (47.2%) were in Hardy–Weinberg equilibrium. Locus-by-locus FST, analysis of molecular variance, and STRUCTURE analyses indicate significant genetic differentiation may exist between European and North American O. nubilalis. The observed genetic diversity was significantly lower among European sites, which may result from genetic drift, natural selection, a genetic bottleneck, or ascertainment bias due to North American origin of EST sequence data. SNPs are an abundant source of mutation data for molecular genetic marker development in non-model species, with shared ancestral SNPs showing application within closely related species. These markers offer advantages over microsatellite markers for genetic and genomic analyses of Lepidoptera, but the source of mutation data may affect the estimation of population parameters and likely need to be considered in the interpretation of empirical data.
Highlights
Species from the insect Order Lepidoptera play crucial ecological roles and impact human welfare due to feeding on cultivated food, fiber, and biofuel crops
The application of quality score criteria and removal of single nucleotide polymorphism (SNP) positions ≤150 bp from 5 and 3 ends of respective contigs resulted in the removal of 1191 SNPs (43.4%) from the pool of candidate loci, and the 1551 remaining loci were used for SNP marker development
VALIDATION OF SNP ASSAYS Single base extension assays were designed using Sequenom MassARRAY Designer Software to detect 763 of 1551 predicted SNP loci (49.2%) within 443 O. nubilalis expressed sequence tag (EST) contigs (1.73 ± 0.95 SNPs per contig; mean size of contigs containing SNPs was 637 ± 125 bp), and PCR co-amplified by 24 multiplex reactions
Summary
Species from the insect Order Lepidoptera play crucial ecological roles and impact human welfare due to feeding on cultivated food, fiber, and biofuel crops. Recent studies suggest that these difficulties in application of microsatellite marker assays may be the result of an association of lepidopteran microsatellites with transposable elements (Coates et al, 2009a, 2010, 2011; Yamamoto et al, 2006; Tay et al, 2010). This phenomenon has hindered the genetic study of endangered butterfly and agricultural pest species worldwide, and highlights a need to develop alternative genetic markers (Coates et al, 2008)
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