A genetic cross reveals the contributions of and to piperaquine drug resistance.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the () and increased copies of (). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like allele and a single copy of while KH004 has a chloroquine-resistant (Dd2-like allele with an additional G367C substitution and four copies of . We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC (LP-IC). We find that inheritance of the KH004 allele is required for PPQ resistance, whereas copy number variation in further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in . Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance.