Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice

BackgroundClassical genetic crosses in malaria parasites involve isolation, genotyping, and phenotyping of multiple progeny parasites, which is time consuming and laborious. Bulk segregant analysis (BSA) offers a powerful and efficient alternative to identify loci underlying complex traits in the human malaria parasite, Plasmodium falciparum. MethodsWe have used BSA, which combines genetic crosses using humanized mice with pooled sequencing of progeny populations to measure changes in allele frequency following selection with antimalarial drugs. We used dihydroartemisinin (DHA) drug selection in two genetic crosses (Mal31xKH004 and NF54xNHP1337). We specifically investigated how synchronization, cryopreservation, and the drug selection regimen of progeny pools impacted the success of BSA experiments. FindingsWe detected a strong and repeatable quantitative trait locus (QTL) at chr13 kelch13 locus in both crosses, but did not detect QTLs at ferredoxin (fd), the apicoplast ribosomal protein S10 (arps10), multidrug resistance protein 2 (mdr2). QTLs were detected using synchronized, but not unsynchronized pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools. InterpretationOur results provide proof-of-principal of the utility of BSA for rapid, robust genetic mapping of drug resistance loci. Use of cryopreserved progeny pools expands the utility of BSA because we can conduct experiments using archived progeny pools from previous genetic crosses. BSA provides a powerful approach that complements traditional QTL methods for investigating the genetic architecture of resistance to antimalarials, and to reveal new or accessory loci contributing to artemisinin resistance. FundingNational Institutes of Health (NIH); Wellcome trust. O_TEXTBOXResearch in contextO_ST_ABSEvidence before this studyC_ST_ABSGenetic crosses have been immensely successful for determining the genetic basis of drug resistance in malaria parasites, but require laborious cloning, characterization of drug resistance and genome-wide genotyping of individual progeny. This is a major limitation given that genetic crosses can now be conducted efficiently using humanized mice, rather than chimpanzees. Bulk segregant analysis (BSA) provides an attractive alternative approach because (i) large numbers of uncloned recombinant progeny can be analyzed, increasing statistical power (ii) phenotyping is not required, because we identify QTLs by treatment of progeny bulks, and identifying genome regions that show skews in allele frequency after treatment (iii) genome sequencing of bulk samples provides a rapid, accurate readout of genome-wide allele frequencies. This approach has been effectively leveraged in yeast, rodent malaria and several model organisms. Added value of this studyHere we validate and optimize this approach for P. falciparum genetic crosses, focusing on resistance to dihydroartemisin (DHA) a central component of the first-line antimalarial combination. Mutations in kelch13 are known to confer resistance to DHA, but several additional candidate loci have also been suggested to contribute. Our results confirm involvement of kelch13, but did not identify linkage with other putative candidate loci. We optimized methodology, showing that synchronization is critical, and that BSA can be successfully applied to cryopreserved progeny pools. Implications of all the available evidenceBSA combined with recent advances in rapidly generating genetic crosses provides a powerful approach to investigate the genetic basis of drug resistance in P. falciparum. C_TEXTBOX