Genetic Mapping of Resistance: <scp>ddRADseq</scp> ‐Based <scp>QTL</scp> and Associated Polymorphism Conferring Resistance to Alpha‐Cypermethrin in <i>Anopheles funestus</i>

The heavy reliance on pyrethroid-based interventions has largely driven insecticide resistance in malaria vectors, accelerating the spread of resistant strains, including resistant Anopheles funestus strains. The efficacy of insecticide-based interventions, especially insecticide-treated nets (ITNs), the cornerstone of malaria control and management, is threatened by the widespread occurrence of resistance, jeopardising malaria control efforts. Alpha-cypermethrin, a type II pyrethroid, is increasingly utilised in various ITN formulations, including those combined with piperonyl butoxide (PBO) and chlorfenapyr-based Interceptor G2 (IG2) nets, to enhance effectiveness against resistant mosquito populations. Therefore, understanding the molecular basis of resistance is essential to monitor and track resistance trends for an effective malaria control program. In this study, we identified a 1.4 Mb QTL on the telomeric end of the left arm of chromosome 2, conferring resistance to α-cypermethrin (rap1 QTL). Different crossing schemes and sequencing approaches were explored to determine the most effective strategy. Individual-based QTL mapping performed on segregating individuals from an isofemale family identified a QTL at the F₇ generation. Higher recombination density relative to the physical genome in the F₇ isofemale family, with a recombination every 240 kb, facilitated the detection of a QTL compared to the F₂ family (335 kb/cM). Additionally, we exploited bulk segregant analysis (BSA) between susceptible and resistant phenotypes from the F₇ isofemale family and an F₇ mixed cross-family to perform cost-effective and rapid QTL-mapping discovery. The strongest signal in both independent BSA analyses overlaps with the rap1 QTL, further supporting its role in α-cypermethrin resistance. The known resistant alleles of the cytochrome P450 CYP6P9a and 6.5-kb structural variant within the rap1 QTL strongly correlate with survival to α-cypermethrin. In this study, we validated that previously developed DNA-based assays, originally designed to monitor permethrin resistance, are effective for tracking resistance to α-cypermethrin as well. Additionally, we identified candidate variants that can serve as reliable markers for monitoring α-cypermethrin resistance.