New genomic resources for an invasive mite pest help guide the development of KASP markers for pesticide resistance screening

Genomic resources provide tools for understanding the evolution of resistance and monitoring resistance as it develops, which can assist the adoption of pesticide resistance management strategies. The invasive redlegged earth mite, Halotydeus destructor, is a major pest of grain and pasture crops in Australia and is increasingly developing resistance to broad-spectrum pesticides. Here, we assembled a chromosomal-level genome of H. destructor, which comprises eight chromosomes totalling 46 Mb. We also compiled a rich genomic dataset of 190 H. destructor populations (whole-genome pooled sequencing) collected across its invasive Australian range (thousands of kilometers), in four years (2018, 2021, 2022, and 2023), and generated resistance phenotypes to multiple pesticide compounds widely used to manage this pest. These genomic resources were used to provide the most detailed characterisation of genetic structure in Australia to date, highlighting complex patterns of regional structuring and isolation-by-distance. We then screened this population genomic dataset to identify target-site mutations in the ace (acetylcholinesterase) and para (voltage-gated sodium channel) genes, which are the targets of organophosphate and pyrethroid pesticides, respectively. Genotype-phenotype associations suggest that the ace G119S mutation has a strong contribution to chlorpyrifos resistance, but not omethoate resistance. Association tests also suggest that para L1024F mutation has a strong, but weakening, contribution to pyrethroid resistance. We then developed and validated a Kompetitive allele-specific PCR (KASP) assay for the ace G1195S and para L1024F mutations linked to chlorpyrifos and bifenthrin resistance, respectively, and demonstrate how these assays can be applied to quantify allele frequencies in pooled DNA extractions. The KASP markers that we have developed will be valuable in facilitating rapid spatiotemporal screening of key resistance mutations in target genes. Our work also suggests a non-target gene basis for omethoate resistance, and a potential shift to other non-target gene mechanisms for pyrethroid resistance. To that end, our new chromosome-level assembly will be important for characterising the genetic architecture of those resistances.