Genome-wide association studies (GWAS) use genetic polymorphism across the genomes of individuals with distinct characteristics to identify genotype-phenotype associations. In mosquitoes, complex traits such as vector competence and insecticide resistance could benefit from GWAS use. We used the Ae. aegypti 50k SNP chip to genotype populations with different levels of pyrethroid resistance from Northern Brazil. Pyrethroids are widely used worldwide to control mosquitoes and other agricultural pests, and their intensive use led to the selection of resistance phenotypes in many insects including mosquitoes. For Ae. aegypti, resistance phenotypes are mainly associated with several mutations in the voltage-gated sodium channel, known as knockdown resistance (kdr). We phenotyped those populations with the WHO insecticide bioassay using deltamethrin impregnated papers, genotyped the kdr alleles using qPCR, and the whole genomic regions with the SNP chip. We identified single-nucleotide polymorphisms (SNPs) directly associated with resistance and one epistatic SNP pair. We also observed that the novel SNPs correlated with the known kdr genotypes, although on different chromosomes or not in close physical proximity to the voltage-gated sodium channel gene. In addition, a pairwise comparison of resistance and susceptible mosquitoes from each population revealed differentiated genomic regions not associated with pyrethroid resistance. These new bi-allelic markers can be used to genotype other populations along with kdr alleles to understand their worldwide distribution. The functional roles of the genes near the newly discovered SNPs require new studies to determine if they act synergistically with kdr alleles or reduce the fitness cost of maintaining resistant alleles.

We used the custom-designed SNP chip for Ae. aegypti to genotype all mosquitoes (Evans et al. 2015).Once we selected the mosquitoes with the desired phenotype, we removed the DNA samples from the −20 °C freezer to concentrate and purify them using Amicon^R Ultra 30k centrifugal filter devices (Millipore) according to the manufacturer instructions. We obtained approximately 23 µL of eluting. Next, we checked the genomic DNA concentration using Qubit (Invitrogen). Finally, we normalized the DNA concentrations to 20 ng/µL and sent approximately 200 ng of genomic DNA from individual mosquitoes to the Functional Genomics Core at the University of North Carolina, Chapel Hill, for hybridization with the Axiom aegypti1 SNP chip (Life Technologies Corporation CAT#550481). We used the Affymetrix Genotyping Console v.3.1.51.0 (Affymetrix) to generate and process the genotype calls (Evans et al. 2015). Briefly, we used the default parameters outlined as best practice for non-human samples, except for the call threshold set to 90%, and by using the off-target variant correction.