KASP genotyping and semi-quantitation of G275E mutation in the α6 subunit of Thrips palmi nAChR gene conferring spinetoram resistance.

Abstract

Pesticide resistance is a long-standing and growing problem in the chemical control of invertebrate pests. Molecular diagnostic methods can facilitate pesticide resistance management by accurately and efficiently detecting resistant mutations and their frequency. In this study, the kompetitive allele specific PCR (KASP) approach, a technology for high-throughput single nucleotide polymorphism (SNP) genotyping, is validated as a useful method for characterizing genotypes at a pesticide-resistance locus for the first time. We focus on the spinetoram resistance mutation of G275E in the nicotinic acetylcholine receptor alpha 6 (nAChR α6) subunit gene of Thrips palmi. Of the 341 individuals of Thrips palmi tested, 98.24% were successfully genotyped, with 100% concordance with Sanger sequencing results. We then quantitatively mixed genomic DNA of known genotypes to establish 21 DNA mixtures with a resistant allele frequency ranging from 0 to 100% at steps of 5%. The linear discriminant analysis (LDA) showed that 75.8% of original grouped cases were correctly classified; six groups had no overlap in membership (resistant allele frequency: 0%, 5%, 10-75%, 80-85%, 90-95%, and 100%). When we chose 11 pooled samples with 10% steps for LDA, 84.4% of original grouped cases were correctly classified; seven groups had no overlap in membership (0%, 10%, 20-30%, 40-70%, 80%, 90%, 100%). The results indicated that KASP applied to pooled samples may provide a semi-quantitative estimate of resistance. Our study points to the suitability of KASP for high-throughput genotyping of genotypes affecting pesticide resistance and semi-quantitative assessments of resistance allele frequencies in populations. © 2023 Society of Chemical Industry.