BACKGROUND
despite advances in vector control and attempts to develop effective vaccines, chemotherapy remains a principal mode of malaria control.
unfortunately malaria parasites resistant to drugs such as chloroquine  have arisen by gene mutation; their prevalence increasing by subsequent selection and transmission.
these drug resistant parasites seriously compromise efforts to treat and control malarial disease both in individual cases and in communities.
the ability to describe, understand and respond to these evolutionary processes continues to depend upon the identification of the precise genetic mutations which underlie the resistance phenotypes.
also, an identification of the genes involved may lead to insights regarding the mechanisms of drug action and resistance, and the design of improved drugs and treatment strategies.
for chloroquine resistance , genetic linkage studies  <cit> , other experimental approaches  <cit>  and phenotype/genotype associations in parasites from natural infections  <cit>  have mapped and identified the k76t mutation in the chloroquine resistance transporter, pfcrt, as the dominant genetic determinant in the most important human parasite plasmodium falciparum.
this protein mediates the export of cq from the parasite digestive vacuole   <cit> , its presumed site of action  <cit> .
additionally, specific point mutations in the multidrug resistance gene  encoding an abc transporter  have also been shown to modulate the level of resistance in cq-r parasites in transfection experiments  <cit>  and in association studies using parasites from natural infections  <cit> .
however, these two genes neither account for the full variation of in vitro cq responses, including high-level cq-r   <cit>  nor the appearance of cq-r in another major human pathogen, p. vivax  <cit> .
understanding the genetic basis of cq-r in the rodent malaria parasite, p. chabaudi could illuminate both of these questions; firstly, because, as in p. vivax, the orthologues of the pfcrt and pfmdr <dig> genes are not involved  <cit> , at least in an existing lineage  of parasites ; secondly because this same lineage contains parasites  with different levels of cq-r  <cit> .
previous classical linkage analysis of a genetic cross between the cq-r mutant as-3cq and a genetically distinct sensitive strain, aj, defined a region of  <dig> kb on chromosome  <dig>  as that containing the mutation conferring the first level of cq-r  <cit>  but the critical gene or mutation was not identified.
for p. chabaudi cq-hir in as-30cq, there has been no previous quantitative description of higher level cq-r or cq-hir phenotypes, nor a systematic mapping of the genetic loci containing the critical mutations.
indeed, even the number of genes and mutations involved, and the sizes of their effect have remained undefined  <cit> .
for drug resistance in malaria, classical linkage analysis of genetic crosses has proved effective in mapping single genes conferring clear phenotypes  <cit> .
also, quantitative trait loci  approaches  <cit>  or genome wide association studies  <cit>  have identified multiple major loci contributing to drug-resistance phenotypes.
however, because these methods require the individual characterisation of many parasite lines or clones, they can be time-consuming or may fail to reveal the specific identity of a critical gene.
accordingly, linkage group selection   <cit>  or qtl analysis were previously combined with illumina® whole genome re-sequencing  to specify the mutations conferring resistance to artemisinin   <cit> , sulphadoxine   <cit> , or mefloquine and lumefantrine resistance   <cit> .
here, we define the basic and high-level cq-r phenotypes, extend the lgs strategy to map the multiple loci contributing to increasing levels of cq-r, and use quantitative wgs of > <dig>  single nucleotide polymorphisms  to improve their resolution.
we propose that, within these loci, mutations occur in the p. chabaudi as-lineage that confer cq-r and cq-hir.
we identify these and additional mutations using wgs.
RESULTS
the p. chabaudi as lineage contains parasites with increasing levels of cq-r
in order to quantitate the cq-r phenotypes in the as lineage, the clones as-sens, as-3cq and as-30cq  <cit>   were passaged in mice treated with either  <dig>   <dig> or  <dig> mg cq kg- <dig> day- <dig>  the growth of these parasites  demonstrated that there is an increasing level of resistance to cq within the lineage.
as-sens parasites grew only in untreated animals.
as-3cq grew at  <dig> and  <dig> mg cq kg- <dig> day- <dig> but not at  <dig> mg cq kg- <dig> day- <dig> while as-30cq was able to survive  <dig> mg cq kg- <dig> day- <dig>  we therefore denoted the cq responses of these clones as cq sensitive , cq-r or cq-hir, respectively.
these data are consistent with a previous proposal that multiple mutations confer cq-hir  <cit>  in this lineage, and suggest a suitable range of cq doses for dissecting the critical genetic loci in lgs experiments, below.
for example, we expected that parasites surviving  <dig> mg cq kg- <dig> day- <dig> would be enriched with parasites having cq-r  phenotypes, while those surviving  <dig> mg cq kg- <dig> day- <dig> would be preferentially enriched with cq-hir parasites only.
improved lgs strategies resolve multiple large-effect genes
in the case of drug resistance, lgs uses drug treatment to select the uncloned progeny of a genetic cross  before measuring the proportions of parental alleles in the surviving parasites  <cit> .
it generates a genome-wide scan of selection, revealing 'selection valleys' that are regions of the genome where the proportion of alleles from the drug-sensitive parent is greatly reduced  and where the genes conferring resistance are located.
in the present study, an uncloned backcross  between the cq-hir clone as-30cq and the genetically different cq-s parasite, aj was generated and treated with different cq doses  to map progressively the signatures of increasing cq selection.
firstly, the proportions of parental alleles in all populations were measured in the surviving parasites, using a library of ~ <dig> pyrosequencing assays  <cit>  .
secondly, we developed a novel approach to improve the resolution and confidence of lgs mapping ), thus.
we defined an expanded set of genome-wide parental as/aj snps by wgs of the sensitive parent aj .
50-base paired-end reads  were mapped against the  <dig>  mb wellcome trust sanger institute  as reference sequence .
92% of the reads mapped uniquely.
by filtering, we identified  <dig>  high stringency snps in aj relative to as-wtsi at a mean frequency of ~ <dig>  substitutions/nucleotide, similar to previous estimates of genetic diversity between the parental strains  <cit> .
at these snp positions, by counting short sequencing reads containing the as or aj base variant in populations of the lgs parasites surviving  <dig> or  <dig> mg cq kg- <dig> day- <dig> , we quantitated the proportions of as and aj alleles, and investigated  the statistical significance of the difference between the allele proportion after each of the two treatments.
the proportions of alleles  in the lgs populations surviving  <dig> mg cq kg- <dig> day- <dig>  revealed by lgs-illumina and by lgs-pyro were remarkably similar  suggesting that the experimental errors incurred by either methodology were small.
lgs-pyro revealed progressively distinct selection valleys on chr <dig>  chr <dig> and chr <dig> as the cq dose increased .
lgs-illumina confirmed selection valleys on chr <dig>  and chr <dig>  at  <dig> mg cq kg- <dig> day- <dig>  these data suggest that cq-r phenotypes in the as lineage are conferred by the action of a major effect gene on chr <dig>  confirming previous linkage analysis  <cit> , and for cq-hir, major effect genes on chr <dig>  and chr <dig>
mutation in aminoacid transporter  is predicted to confer cq-r
lgs-pyro identified a partial selection valley on chr <dig> at  <dig>  mg cq kg- <dig> day- <dig> which proved to be dominant at  <dig> mg cq kg- <dig> day- <dig> : for example, the proportion of an aj allele of marker pcpf06- <dig> decreased from  <dig> % in untreated infections to  <dig> % under cq selection.
importantly, an independent genetic backcross between the mefloquine- and cq-resistant clone as-15mf  <cit>   and aj also showed a similar distinct selection valley on chr <dig> at  <dig> mg cq kg- <dig> day- <dig> .
both selection valleys coincided with the  <dig> kb region previously mapped by classical linkage analysis  <cit>  .
lgs-illumina confirmed the selection valley  on chr <dig> at high resolution and statistical significance .
here, a region at nucleotide ~ <dig> , <dig> where the proportion of aj alleles reached a minimum < 3%, was flanked on either side by regions of increasing aj-allele proportion.
the gradual and regular change in aj allele proportion suggested the presence of many independent recombinant clones in the cross progeny.
the differences in the gradients of the two slopes forming the selection valley may reflect different local recombination rates along the chromosome.
these data resolved the presence of a mutation that confers cq-r, close to nucleotide  <dig> , <dig> on chr <dig>  this locus corresponds to the base of the selection valley as defined by lgs-pyro  and to the  <dig> kb locus previously mapped  <cit> , confirming that the gene bearing the mutation conferring cq-r lies toward the right-hand end of chr <dig>
wgs ) identified a total of  <dig> point mutations  in as-30cq relative to as-sens , four of which are shared between as-30cq and as-15mf  <cit> .
of these four mutations, only one maps to chr11; a non-synonymous mutation  in a gene  encoding a predicted aminoacid transporter .
it is found at base  <dig>   coincident with the floor of the chr <dig> selection valley .
we concluded that the probability of failing to identify a genuine point mutation  in this region is very small, for three reasons.
firstly, > ~  <dig> - 98% of the as-wtsi genome was covered by uniquely mapping short-reads  employed here  <cit>  .
secondly, the read coverage is high: for  <dig> kb upstream and downstream of aat <dig> on chr <dig>  only  <dig> % or  <dig> % of bases showed a read coverage of <  <dig> or <  <dig>  respectively .
thirdly, we identified a very low overall genome-wide substitution frequency  in as-30cq  relative to as-sens.
the pyrimethamine  and cq responses of clones of the as lineage are shown along with all mutations detected in as-30cq  relative to as-sens.
<dig> dhfr, dihydrofolate reductase; aat, aminoacid transporter; cir, chabaudi interspersed repeat; ubp, ubiquitin specific protease .
<dig> refers to genes pchas_ <dig> or pchas_ <dig>   <dig> mixed genotype and phenotype, see additional file  <dig>   <dig> note that as-15mf carries an alternative allele of pchas_ <dig>  namely i102del.
single-letter amino acid code used: a, alanine; e, glutamate; f, phenylalanine; h, histidine; i, isoleucine; n, asparagine; s, serine; t, threonine; v, valine; y, tyrosine; del, codon deleted.
summary of the high confidence mutations proposed in clone as-30cq .
read depth and quality scores fro mutations were according to ssaha <dig>  start and finish nucleotides for deletions are approximate only.
dideoxysequencing confirmed that the a173e aat <dig> mutation first appeared in the as lineage in as-3cq, along with the cq-r phenotype .
we therefore propose that aat <dig> a173e is the determinant of cq-r in this particular p. chabaudi lineage.
the a173e aat <dig> mutation shares some properties with the determinant  of cq-r in p. falciparum.
for example, like pfcrt, aat <dig> is predicted to encode a 10-transmembrane  helix transporter  and its p. falciparum orthologue  is targeted to the membrane of the dv .
the wild-type function of pfcrt is uncertain but amino acid transport has been suggested  <cit> .
both k76t and a173e mutations result in negative charge shifts.
residue  <dig> in aat <dig> is at the start of a highly conserved region  close to the start of the first tm-helix : in pfcrt, residue  <dig> lies at the start of tm <dig>  predicted to be internal to the dv where cq is thought to act.
these data suggest that aat <dig> and crt may share some structure/function relationships impacting on their physiological function in the absence and/or presence of cq.
mutation in another transporter  is predicted to confer intermediate cq-r
lgs-pyro experiments showed that as markers on chr <dig> were selected at  <dig>   <dig> or  <dig> but not at  <dig>  mg cq kg- <dig> day- <dig> .
on chr <dig>  the proportion of the aj allele of marker pcpf02- <dig> decreased from  <dig> %  to  <dig> % at  <dig> mg cq kg- <dig> day- <dig>  lgs-illumina analysis confirmed that aj alleles are reduced across the whole of chr03; from about 82% in untreated parasites to about 16% in cq-treated population .
details of the selection profile on chr <dig> are consistent with a focus of selection close to base ~ <dig> .
the gradients of this selection valley are not dissimilar to those observed on chr <dig> when observed on a genome-wide scale .
wgs of as-30cq revealed  a non-synonymous mutation  at base  <dig>  on chr <dig>  and dideoxysequencing confirmed that this mutation arose between as-3cq and as-30cq .
however, this mutation does not appear in clones as-15mf and as-atn  - these two clones being selected from as-15cq  using mefloquine and artesunate, respectively.
instead, as-15mf and as-atn bear a  <dig> base deletion  in the same gene .
we therefore suggest that both the t719n and i102del mutations were partially selected by intermediate levels of cq in as-15cq prior to fixation during treatment with cq , mefloquine  or artesunate  and subsequent cloning .
pchas_ <dig> is predicted to encode a  <dig> tm-helix protein  and its p. falciparum orthologue pfb0675w is also predicted  to target the dv membrane .
the t719n substitution occurs in a large loop between tm <dig> and tm <dig>  a highly conserved region of the gene .
the i102del mutation is predicted to locate to the centre of tm <dig> and to change the overall prediction of tm domain structure .
the general transmembrane domain structure and dv localisation of pchas_ <dig> are therefore similar to other proteins  identified as conferring or modulating cq-r in p. falciparum or p. chabaudi.
these data suggest that the pchas_ <dig> t719n mutation confers an increased cq-r phenotype.
interestingly, the p. yoelii orthologue  of pchas_ <dig> lacks sequence corresponding to tm <dig> and tm <dig> that are otherwise present in other plasmodium spp.
.
wild-type p. yoelii  had not been exposed to antimalarial drugs but was reported to be highly cq-r  <cit> .
we suggest the possibility that p. yoelii cq-r resistance might be related to this structural variation.
wgs of as-30cq also revealed a non-synonymous mutation t707n in pchas_ <dig> at base  <dig>  on chr <dig> , predicted to encode a member of the p. chabaudi-specific variant gene family   <cit> .
this mutation maps to the left-hand of chr <dig>  the detailed lgs-illumina profile of aj allele proportion  supports the possibility that this mutation too may contribute to an augmented  cq-r. dideoxysequencing confirmed that this mutation is specific to as-30cq and does not appear in either as-15mf or as-atn.
mutation in de-ubiquitinating enzyme  is predicted to confer the highest level cq-r
lgs-pyro shows that as markers on chr <dig> are selected at  <dig> and  <dig>  but not at  <dig>   <dig>  or  <dig> mg cq kg- <dig> day- <dig> : e.g.
at  <dig> mg cq kg- <dig> day- <dig> on chr <dig>  the percentage of aj alleles of marker pcpf01- <dig> decreased from  <dig> %  to  <dig> %.
further resolution within the chromosome was not possible because parasites surviving  <dig> or  <dig> mg cq kg- <dig> day- <dig> were not analysed by lgs-illumina.
wgs had previously identified a single mutation  on chr <dig>  <cit>  in both as-30cq and as-15mf.
it is the only mutation detected on chr <dig> and occurred between as-3cq and as-15cq .
we conclude therefore that ubp <dig> v2728f confers cq-hir.
this mutation was predicted to reduce the activity of a de-ubiquitinating enzyme  <cit>  and also to confer artemisinin resistance in as-30cq, without previous exposure to this drug  <cit> .
it is therefore predicted to affect the responses of malaria parasites to multiple drugs with diverse chemical structures and modes of action.
dideoxysequencing confirms that this mutation appears in as-30cq and as-15mf but not in as-atn.
instead an alternative mutation v2697f  ubp <dig> appears in as-atn  <cit> .
as with the alternative mutations in pchas_ <dig>  we suggest that these two alternative ubp <dig> mutations are partially selected  in the uncloned parasite as-15cq.
their differential selection and fixation in clones as-30cq, as-15mf and as-atn derived from as-15cq after selection by cq, mefloquine or artesunate are fully discussed along with a complete resolution of apparent contradictions regarding their linkage  with alternative mutations in pchas_ <dig> .
other mutations in as-30cq
nine mutations are identified in as-30cq relative to as-sens; seven point mutations and two deletions .
four point mutations  and chr02) are associated with signatures of cq selection and were discussed above.
they all first appeared in the p. chabaudi as lineage  in as-3cq or as-30cq .
a fifth point mutation was identified in as-30cq, as predicted, on chr <dig>  this mutation, s106n dhfr  was confirmed by dideoxysequencing to have first appeared in as-pyr <dig>  it was previously shown to confer resistance to pyrimethamine  <cit> .
four mutations , identified in as-30cq, were not associated with signatures of drug-selection.
three were confirmed by dideoxysequencing; a non-coding point mutation on chr <dig>  a  <dig> bp non-coding deletion on chr <dig>   and a non-synonymous point mutation on chr <dig>  namely y162h pchas_ <dig>  arising first in as-30cq .
a fourth mutation could not be confirmed by dideoxysequencing: extensive low or zero-coverage and/or a small cluster of poor quality snp calls in as-30cq, .
other studies will be required to evaluate whether these  <dig> point mutations and  <dig> deletions are consistently neutral , or whether they play a minor role in drug  resistance.
such roles could include a weak selective advantage in the presence of drugs or compensation for fitness costs incurred by the 'drug resistance' mutations .
the low probability of failing to identify point mutations  on chr <dig> was discussed in the aat <dig> section above.
similar arguments and data may be applied equally to the whole genome  and are addressed more fully here  and previously  <cit> .
our conclusion is that the probability of a false negative point mutation in central regions of a chromosome is low .
for regions of chromosomes closer to the telomeres where p. chabaudi-specific genes are located, we suggest that the probability of a false negative is higher but not easily quantified.
however, with the exception of possible selection at the left hand end of chr <dig>  our experiments show no evidence of cq selection in these regions.
genome-wide scan of selection - other observations
both lgs-pyro and lgs-illumina data indicated that aj allele proportions were high  in the untreated lgs population but were reduced after drug treatment  at many loci genome-wide, including chromosomes other than chr <dig>  chr <dig> or chr <dig>  for example across chr <dig>  aj allele proportions were reduced from ~86% to ~65%, after drug treatment .
these data may reflect high aj proportions in the backcross and the loss of aj parental parasites  after cq treatment.
additionally, or alternatively, aj alleles may have been positively selected during growth without drugs, reflecting the possible action of multiple  genes that underlie the faster growth of aj compared to as parasites, observed routinely in previous experiments  <cit> .
the lgs-pyro data showed that the selection valleys on chr <dig>   <dig> and  <dig> were produced progressively at increasing cq doses .
thus, low doses resulted in the selection of as alleles on chr <dig>  and increasing doses resulted in selection of as alleles on chr <dig> and then on chr <dig>  we note that the maximum depth of the chr <dig> selection valley was reached at a lower cq dose than that required to achieve maximum selection at chr <dig> .
these data may be interpreted by invoking two possible factors.
firstly, we suggest that the mutations conferring cq-hir on chr <dig> and on chr <dig> may incur 'fitness costs': i. e. that in the absence of a sufficiently high concentration of cq, these mutations may reduce the growth of parasites.
this would mean that, at lower cq concentrations, parasites with cq-r  would be selected to a greater degree than cq-hir parasites bearing multiple mutations.
secondly, the effects of the mutations on chr <dig> and/or chr <dig> may be epistatic to the a173e aat <dig> mutation, because mutated as alleles at these loci  only show signs of selection  after the selection of mutated as alleles at the aat <dig> locus  .
according to this interpretation, parasites bearing only the mutations on chr <dig> or chr <dig>  are not selectable by lower doses of cq.
the lgs-illumina analysis revealed an abrupt discontinuity of aj proportion at the right hand end of chr <dig>  and similar changes on chr <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> , often in both untreated and drug-treated parasites.
these are described and discussed in additional file  <dig>  section  <dig>  these discontinuities are also observable in the lgs-pyro data.
our conclusion is therefore that they are not artefacts of lgs- illumina.
also, they did not arise by natural genetic or selection phenomena.
they are most likely to arise from differences  between as-wtsi  and our parental strains as-sens and/or aj.
lgs-pyro and lgs-illumina revealed regions showing possible weak drug selection but where mutations were not detected; for example, the left-hand end of chr <dig> .
further studies are required to investigate whether these represent reproducible regions of selection or arise from random variation.
discussion
we have described in vivo cq-r and cq-hir phenotypes in the genetically related p. chabaudi as parasites as-3cq and as-30cq, mapped the underlying genetic loci on chr <dig> , chr <dig> and chr <dig>  and, by wgs, have identified a small number of mutations proposed to confer these phenotypes.
a173e aat <dig>  is proposed to confer cq-r. t719n pchas_ <dig>  and v2728f ubp <dig>  are proposed to confer cq-hir.
we also suggest that t707n pchas_ <dig>  may also contribute to cq-hir.
mutations conferring cq-r are identified by integrating genetic and genomic data
the genetic  and genomic  approaches employed showed a remarkable concordance on chr <dig>  chr <dig> and chr11: i.e.
these major selection valleys contain  <dig> of the  <dig> mutations that arose in the lineage under cq selection .
the genome-wide scans do not reveal selection around the other two mutations arising during cq selection  nor around those  arising during previous pyrimethamine selection ; i.e.
<dig> bp deletion and s106n dhfr) , non-coding point mutation .
the correspondences between the genetic and genomic data and the arguments deployed previously  <cit>  and above  together suggest that only 3- <dig> major effect genes conferring cq-r and cq-hir were fixed by strong selection  during experimental evolution from as-sens to as-30cq.
furthermore, only a small number of weakly selected or effectively neutral mutations were fixed stochastically during population bottlenecks such as those occurring during transmission of parasite lines, or experimental cloning.
previous similar investigations in the p. chabaudi as lineage demonstrated that one mutation conferred resistance to each of pyrimethamine, sulphadoxine  <cit> , mefloquine  <cit>  and artemisinin  <cit>  and that ≤  <dig> mutations were fixed stochastically each time during the selection and cloning of as-50s/p, as-15mf and as-30cq respectively.
the low rate of nucleotide substitution, even during multiple passages and bottlenecks, makes this system well adapted for investigations of resistance to other drugs.
the genetic architecture of cq-r
cq-hir was previously generated in as-30cq of the p. chabaudi as-lineage by drug selection over about  <dig> passages  <cit> , leading to the suggestion that the cq-hir phenotype arose by a series of small steps and was mediated by several mutations at different loci, although the number of mutations was not predicted.
actually, for cq-r, the present data strongly support the contribution of aat <dig>  as a single major effect gene, confirming previous classical genetic linkage analysis  <cit> .
for cq-hir, two  major effect genes  and ubp <dig>  on chr <dig> and chr <dig>  respectively) are strongly supported.
in this respect, the architecture of cq-r in p. chabaudi may be similar to that of p. falciparum where pfcrt and pfmdr <dig> alleles appear to generate high levels of cq resistance epistatically  <cit> .
the hypothesis that cq-hir in p. chabaudi is mediated by a large number of small-effect mutations is rejected by the data presented here.
the genetic determinants of cq-r
the concordance between the phenotypic, genetic and genomic data strongly supports the identification of mutations in aat <dig> as the key determinant of cq-r. we have also revealed shared properties of pfcrt and aat <dig> and their mutations , such as putative function , subcellular location, tm-helix topology, type and position of mutation.
genome-wide transcription profiling of the cq-s p. falciparum parasite  and cq-r transfectants identified the aat <dig> orthologue pff <dig> as one of a small number of differentially expressed genes  <cit>  which also included genes predicted to encode transporters, proteases and components of the trafficking pathways.
this suggests future transfection and functional studies to address questions regarding the role of these mutations in amino acid or cq transport and cq-r in p. falciparum and p. chabaudi.
the involvement of aat <dig>  another  dv transporter and ubp <dig> in cq-hir also provoke questions regarding the relationship between haemoglobin digestion, protein turnover, amino acid transport/availability and cq action, export and resistance phenotypes.,
interestingly, the v2728f ubp <dig> mutation has now been shown to confer resistance to both artemisinin  <cit>  and chloroquine .
this resolves a previously unexplained result - that artemisinin resistance mediated by mutations in ubp <dig> appeared before parasites were exposed to artemisinin.
this data also suggests that there must be some commonality in the molecular mechanisms of resistance to the two drugs, at least in the p. chabaudi as lineage.
the rodent model and public health
p. chabaudi and p. falciparum have similar or identical genetic bases for resistance to pyrimethamine  and to mefloquine   <cit> .
for cq-r, both parasite species share key features .
now, notwithstanding the evidence supporting the proposal that different genes confer cq-r in the two species, the data presented here suggest that some features of the molecular mechanism of resistance acquisition may be similar.
this supports the use of the rodent model to identify candidate genetic markers of resistance to future antimalarial drugs.
furthermore, the orthologues of aat <dig>  and pchas_ <dig>  should now be evaluated as candidate molecular markers of cq-r in p. vivax and modulators of cq responses in p. falciparum.
the experiments reported here share some features previously exploited by genome-wide selection scans in yeast  <cit> , genome-wide association  <cit>  and high-resolution identification of mutated genes  <cit>  in p. falciparum.
the present study demonstrates how similar approaches can be used to identify genes conferring complex selectable phenotypes such as drug-resistance in experimental systems.
CONCLUSIONS
previously, increasing chloroquine resistance phenotypes were experimentally selected in a lineage of the rodent malaria, plasmodium chabaudi.
here, these phenotypes were measured, the underlying genetic loci mapped and mutations specified using a novel quantitative genetics and genomics approach.
this approach analysed genetic crosses by selecting progeny en masse at different drug concentrations.
the frequencies of parental alleles in the surviving parasites were measured using ~  <dig> pyrosequencing single nucleotide polymorphism  assays, and for ~ <dig>  single nucleotide polymorphisms, by illumina short-read sequencing.
this defined 'selection valleys' on chromosomes  <dig>   <dig> and  <dig>  where genes conferring resistance were expected to be located.
whole genome re-sequencing of the chloroquine resistant mutant parasite and the sensitive progenitor wild-type parasite showed that only  <dig> point mutations in the whole genome had arisen in the lineage.
the specific isolated mutations within the selection valleys were identified.
a mutation in a putative aminoacid transporter  encoded on chr <dig> confers chloroquine resistance.
mutations in another transporter  and a deubiquitinating enzyme  confer higher level chloroquine resistance.
orthologues of these genes in p. falciparum and p. vivax can now be studied for their contribution to chloroquine resistance in human infection.
these data will generate insights of the mechanism of chloroquine resistance in human and rodent malaria parasites.

 despite advances in vector control and attempts to develop effective vaccines, chemotherapy remains a principal mode of malaria control. unfortunately malaria parasites resistant to drugs such as chloroquine have arisen by gene mutation; their prevalence increasing by subsequent selection and transmission. these drug resistant parasites seriously compromise efforts to treat and control malarial disease both in individual cases and in communities. the ability to describe, understand and respond to these evolutionary processes continues to depend upon the identification of the precise genetic mutations which underlie the resistance phenotypes. also, an identification of the genes involved may lead to insights regarding the mechanisms of drug action and resistance, and the design of improved drugs and treatment strategies. for chloroquine resistance, genetic linkage studies, other experimental approaches and phenotype/genotype associations in parasites from natural infections have mapped and identified the k76t mutation in the chloroquine resistance transporter, pfcrt, as the dominant genetic determinant in the most important human parasite plasmodium falciparum. this protein mediates the export of cq from the parasite digestive vacuole, its presumed site of action. additionally, specific point mutations in the multidrug resistance gene encoding an abc transporter have also been shown to modulate the level of resistance in cq-r parasites in transfection experiments and in association studies using parasites from natural infections. however, these two genes neither account for the full variation of in vitro cq responses, including high-level cq-r nor the appearance of cq-r in another major human pathogen, p. vivax. understanding the genetic basis of cq-r in the rodent malaria parasite, p. chabaudi could illuminate both of these questions; firstly, because, as in p. vivax, the orthologues of the pfcrt and pfmdr genes are not involved, at least in an existing lineage of parasites; secondly because this same lineage contains parasites with different levels of cq-r. previous classical linkage analysis of a genetic cross between the cq-r mutant as-3cq and a genetically distinct sensitive strain, aj, defined a region of kb on chromosome as that containing the mutation conferring the first level of cq-r but the critical gene or mutation was not identified. for p. chabaudi cq-hir in as-30cq, there has been no previous quantitative description of higher level cq-r or cq-hir phenotypes, nor a systematic mapping of the genetic loci containing the critical mutations. indeed, even the number of genes and mutations involved, and the sizes of their effect have remained undefined. for drug resistance in malaria, classical linkage analysis of genetic crosses has proved effective in mapping single genes conferring clear phenotypes. also, quantitative trait loci approaches or genome wide association studies have identified multiple major loci contributing to drug-resistance phenotypes. however, because these methods require the individual characterisation of many parasite lines or clones, they can be time-consuming or may fail to reveal the specific identity of a critical gene. accordingly, linkage group selection or qtl analysis were previously combined with illumina® whole genome re-sequencing to specify the mutations conferring resistance to artemisinin, sulphadoxine, or mefloquine and lumefantrine resistance. here, we define the basic and high-level cq-r phenotypes, extend the lgs strategy to map the multiple loci contributing to increasing levels of cq-r, and use quantitative wgs of > single nucleotide polymorphisms to improve their resolution. we propose that, within these loci, mutations occur in the p. chabaudi as-lineage that confer cq-r and cq-hir. we identify these and additional mutations using wgs.  the p. chabaudi as lineage contains parasites with increasing levels of cq-r in order to quantitate the cq-r phenotypes in the as lineage, the clones as-sens, as-3cq and as-30cq were passaged in mice treated with either or mg cq kg- day- the growth of these parasites demonstrated that there is an increasing level of resistance to cq within the lineage. as-sens parasites grew only in untreated animals. as-3cq grew at and mg cq kg- day- but not at mg cq kg- day- while as-30cq was able to survive mg cq kg- day- we therefore denoted the cq responses of these clones as cq sensitive, cq-r or cq-hir, respectively. these data are consistent with a previous proposal that multiple mutations confer cq-hir in this lineage, and suggest a suitable range of cq doses for dissecting the critical genetic loci in lgs experiments, below. for example, we expected that parasites surviving mg cq kg- day- would be enriched with parasites having cq-r phenotypes, while those surviving mg cq kg- day- would be preferentially enriched with cq-hir parasites only. improved lgs strategies resolve multiple large-effect genes in the case of drug resistance, lgs uses drug treatment to select the uncloned progeny of a genetic cross before measuring the proportions of parental alleles in the surviving parasites. it generates a genome-wide scan of selection, revealing'selection valleys' that are regions of the genome where the proportion of alleles from the drug-sensitive parent is greatly reduced and where the genes conferring resistance are located. in the present study, an uncloned backcross between the cq-hir clone as-30cq and the genetically different cq-s parasite, aj was generated and treated with different cq doses to map progressively the signatures of increasing cq selection. firstly, the proportions of parental alleles in all populations were measured in the surviving parasites, using a library of ~ pyrosequencing assays. secondly, we developed a novel approach to improve the resolution and confidence of lgs mapping ), thus. we defined an expanded set of genome-wide parental as/aj snps by wgs of the sensitive parent aj. 50-base paired-end reads were mapped against the mb wellcome trust sanger institute as reference sequence. 92% of the reads mapped uniquely. by filtering, we identified high stringency snps in aj relative to as-wtsi at a mean frequency of ~ substitutions/nucleotide, similar to previous estimates of genetic diversity between the parental strains. at these snp positions, by counting short sequencing reads containing the as or aj base variant in populations of the lgs parasites surviving or mg cq kg- day-, we quantitated the proportions of as and aj alleles, and investigated the statistical significance of the difference between the allele proportion after each of the two treatments. the proportions of alleles in the lgs populations surviving mg cq kg- day- revealed by lgs-illumina and by lgs-pyro were remarkably similar suggesting that the experimental errors incurred by either methodology were small. lgs-pyro revealed progressively distinct selection valleys on chr chr and chr as the cq dose increased. lgs-illumina confirmed selection valleys on chr and chr at mg cq kg- day- these data suggest that cq-r phenotypes in the as lineage are conferred by the action of a major effect gene on chr confirming previous linkage analysis, and for cq-hir, major effect genes on chr and chr mutation in aminoacid transporter is predicted to confer cq-r lgs-pyro identified a partial selection valley on chr at mg cq kg- day- which proved to be dominant at mg cq kg- day-: for example, the proportion of an aj allele of marker pcpf06- decreased from % in untreated infections to % under cq selection. importantly, an independent genetic backcross between the mefloquine- and cq-resistant clone as-15mf and aj also showed a similar distinct selection valley on chr at mg cq kg- day-. both selection valleys coincided with the kb region previously mapped by classical linkage analysis. lgs-illumina confirmed the selection valley on chr at high resolution and statistical significance. here, a region at nucleotide ~, where the proportion of aj alleles reached a minimum < 3%, was flanked on either side by regions of increasing aj-allele proportion. the gradual and regular change in aj allele proportion suggested the presence of many independent recombinant clones in the cross progeny. the differences in the gradients of the two slopes forming the selection valley may reflect different local recombination rates along the chromosome. these data resolved the presence of a mutation that confers cq-r, close to nucleotide, on chr this locus corresponds to the base of the selection valley as defined by lgs-pyro and to the kb locus previously mapped, confirming that the gene bearing the mutation conferring cq-r lies toward the right-hand end of chr wgs ) identified a total of point mutations in as-30cq relative to as-sens, four of which are shared between as-30cq and as-15mf. of these four mutations, only one maps to chr11; a non-synonymous mutation in a gene encoding a predicted aminoacid transporter. it is found at base coincident with the floor of the chr selection valley. we concluded that the probability of failing to identify a genuine point mutation in this region is very small, for three reasons. firstly, > ~ - 98% of the as-wtsi genome was covered by uniquely mapping short-reads employed here. secondly, the read coverage is high: for kb upstream and downstream of aat on chr only % or % of bases showed a read coverage of < or < respectively. thirdly, we identified a very low overall genome-wide substitution frequency in as-30cq relative to as-sens. the pyrimethamine and cq responses of clones of the as lineage are shown along with all mutations detected in as-30cq relative to as-sens. dhfr, dihydrofolate reductase; aat, aminoacid transporter; cir, chabaudi interspersed repeat; ubp, ubiquitin specific protease. refers to genes pchas_ or pchas_ mixed genotype and phenotype, see additional file note that as-15mf carries an alternative allele of pchas_ namely i102del. single-letter amino acid code used: a, alanine; e, glutamate; f, phenylalanine; h, histidine; i, isoleucine; n, asparagine; s, serine; t, threonine; v, valine; y, tyrosine; del, codon deleted. summary of the high confidence mutations proposed in clone as-30cq. read depth and quality scores fro mutations were according to ssaha start and finish nucleotides for deletions are approximate only. dideoxysequencing confirmed that the a173e aat mutation first appeared in the as lineage in as-3cq, along with the cq-r phenotype. we therefore propose that aat a173e is the determinant of cq-r in this particular p. chabaudi lineage. the a173e aat mutation shares some properties with the determinant of cq-r in p. falciparum. for example, like pfcrt, aat is predicted to encode a 10-transmembrane helix transporter and its p. falciparum orthologue is targeted to the membrane of the dv. the wild-type function of pfcrt is uncertain but amino acid transport has been suggested. both k76t and a173e mutations result in negative charge shifts. residue in aat is at the start of a highly conserved region close to the start of the first tm-helix: in pfcrt, residue lies at the start of tm predicted to be internal to the dv where cq is thought to act. these data suggest that aat and crt may share some structure/function relationships impacting on their physiological function in the absence and/or presence of cq. mutation in another transporter is predicted to confer intermediate cq-r lgs-pyro experiments showed that as markers on chr were selected at or but not at mg cq kg- day-. on chr the proportion of the aj allele of marker pcpf02- decreased from % to % at mg cq kg- day- lgs-illumina analysis confirmed that aj alleles are reduced across the whole of chr03; from about 82% in untreated parasites to about 16% in cq-treated population. details of the selection profile on chr are consistent with a focus of selection close to base ~. the gradients of this selection valley are not dissimilar to those observed on chr when observed on a genome-wide scale. wgs of as-30cq revealed a non-synonymous mutation at base on chr and dideoxysequencing confirmed that this mutation arose between as-3cq and as-30cq. however, this mutation does not appear in clones as-15mf and as-atn - these two clones being selected from as-15cq using mefloquine and artesunate, respectively. instead, as-15mf and as-atn bear a base deletion in the same gene. we therefore suggest that both the t719n and i102del mutations were partially selected by intermediate levels of cq in as-15cq prior to fixation during treatment with cq, mefloquine or artesunate and subsequent cloning. pchas_ is predicted to encode a tm-helix protein and its p. falciparum orthologue pfb0675w is also predicted to target the dv membrane. the t719n substitution occurs in a large loop between tm and tm a highly conserved region of the gene. the i102del mutation is predicted to locate to the centre of tm and to change the overall prediction of tm domain structure. the general transmembrane domain structure and dv localisation of pchas_ are therefore similar to other proteins identified as conferring or modulating cq-r in p. falciparum or p. chabaudi. these data suggest that the pchas_ t719n mutation confers an increased cq-r phenotype. interestingly, the p. yoelii orthologue of pchas_ lacks sequence corresponding to tm and tm that are otherwise present in other plasmodium spp.. wild-type p. yoelii had not been exposed to antimalarial drugs but was reported to be highly cq-r. we suggest the possibility that p. yoelii cq-r resistance might be related to this structural variation. wgs of as-30cq also revealed a non-synonymous mutation t707n in pchas_ at base on chr, predicted to encode a member of the p. chabaudi-specific variant gene family. this mutation maps to the left-hand of chr the detailed lgs-illumina profile of aj allele proportion supports the possibility that this mutation too may contribute to an augmented cq-r. dideoxysequencing confirmed that this mutation is specific to as-30cq and does not appear in either as-15mf or as-atn. mutation in de-ubiquitinating enzyme is predicted to confer the highest level cq-r lgs-pyro shows that as markers on chr are selected at and but not at or mg cq kg- day-: e.g. at mg cq kg- day- on chr the percentage of aj alleles of marker pcpf01- decreased from % to %. further resolution within the chromosome was not possible because parasites surviving or mg cq kg- day- were not analysed by lgs-illumina. wgs had previously identified a single mutation on chr in both as-30cq and as-15mf. it is the only mutation detected on chr and occurred between as-3cq and as-15cq. we conclude therefore that ubp v2728f confers cq-hir. this mutation was predicted to reduce the activity of a de-ubiquitinating enzyme and also to confer artemisinin resistance in as-30cq, without previous exposure to this drug. it is therefore predicted to affect the responses of malaria parasites to multiple drugs with diverse chemical structures and modes of action. dideoxysequencing confirms that this mutation appears in as-30cq and as-15mf but not in as-atn. instead an alternative mutation v2697f ubp appears in as-atn. as with the alternative mutations in pchas_ we suggest that these two alternative ubp mutations are partially selected in the uncloned parasite as-15cq. their differential selection and fixation in clones as-30cq, as-15mf and as-atn derived from as-15cq after selection by cq, mefloquine or artesunate are fully discussed along with a complete resolution of apparent contradictions regarding their linkage with alternative mutations in pchas_. other mutations in as-30cq nine mutations are identified in as-30cq relative to as-sens; seven point mutations and two deletions. four point mutations and chr02) are associated with signatures of cq selection and were discussed above. they all first appeared in the p. chabaudi as lineage in as-3cq or as-30cq. a fifth point mutation was identified in as-30cq, as predicted, on chr this mutation, s106n dhfr was confirmed by dideoxysequencing to have first appeared in as-pyr it was previously shown to confer resistance to pyrimethamine. four mutations, identified in as-30cq, were not associated with signatures of drug-selection. three were confirmed by dideoxysequencing; a non-coding point mutation on chr a bp non-coding deletion on chr and a non-synonymous point mutation on chr namely y162h pchas_ arising first in as-30cq. a fourth mutation could not be confirmed by dideoxysequencing: extensive low or zero-coverage and/or a small cluster of poor quality snp calls in as-30cq,. other studies will be required to evaluate whether these point mutations and deletions are consistently neutral, or whether they play a minor role in drug resistance. such roles could include a weak selective advantage in the presence of drugs or compensation for fitness costs incurred by the'drug resistance' mutations. the low probability of failing to identify point mutations on chr was discussed in the aat section above. similar arguments and data may be applied equally to the whole genome and are addressed more fully here and previously. our conclusion is that the probability of a false negative point mutation in central regions of a chromosome is low. for regions of chromosomes closer to the telomeres where p. chabaudi-specific genes are located, we suggest that the probability of a false negative is higher but not easily quantified. however, with the exception of possible selection at the left hand end of chr our experiments show no evidence of cq selection in these regions. genome-wide scan of selection - other observations both lgs-pyro and lgs-illumina data indicated that aj allele proportions were high in the untreated lgs population but were reduced after drug treatment at many loci genome-wide, including chromosomes other than chr chr or chr for example across chr aj allele proportions were reduced from ~86% to ~65%, after drug treatment. these data may reflect high aj proportions in the backcross and the loss of aj parental parasites after cq treatment. additionally, or alternatively, aj alleles may have been positively selected during growth without drugs, reflecting the possible action of multiple genes that underlie the faster growth of aj compared to as parasites, observed routinely in previous experiments. the lgs-pyro data showed that the selection valleys on chr and were produced progressively at increasing cq doses. thus, low doses resulted in the selection of as alleles on chr and increasing doses resulted in selection of as alleles on chr and then on chr we note that the maximum depth of the chr selection valley was reached at a lower cq dose than that required to achieve maximum selection at chr. these data may be interpreted by invoking two possible factors. firstly, we suggest that the mutations conferring cq-hir on chr and on chr may incur'fitness costs': i. e. that in the absence of a sufficiently high concentration of cq, these mutations may reduce the growth of parasites. this would mean that, at lower cq concentrations, parasites with cq-r would be selected to a greater degree than cq-hir parasites bearing multiple mutations. secondly, the effects of the mutations on chr and/or chr may be epistatic to the a173e aat mutation, because mutated as alleles at these loci only show signs of selection after the selection of mutated as alleles at the aat locus. according to this interpretation, parasites bearing only the mutations on chr or chr are not selectable by lower doses of cq. the lgs-illumina analysis revealed an abrupt discontinuity of aj proportion at the right hand end of chr and similar changes on chr and, often in both untreated and drug-treated parasites. these are described and discussed in additional file section these discontinuities are also observable in the lgs-pyro data. our conclusion is therefore that they are not artefacts of lgs- illumina. also, they did not arise by natural genetic or selection phenomena. they are most likely to arise from differences between as-wtsi and our parental strains as-sens and/or aj. lgs-pyro and lgs-illumina revealed regions showing possible weak drug selection but where mutations were not detected; for example, the left-hand end of chr. further studies are required to investigate whether these represent reproducible regions of selection or arise from random variation. discussion we have described in vivo cq-r and cq-hir phenotypes in the genetically related p. chabaudi as parasites as-3cq and as-30cq, mapped the underlying genetic loci on chr, chr and chr and, by wgs, have identified a small number of mutations proposed to confer these phenotypes. a173e aat is proposed to confer cq-r. t719n pchas_ and v2728f ubp are proposed to confer cq-hir. we also suggest that t707n pchas_ may also contribute to cq-hir. mutations conferring cq-r are identified by integrating genetic and genomic data the genetic and genomic approaches employed showed a remarkable concordance on chr chr and chr11: i.e. these major selection valleys contain of the mutations that arose in the lineage under cq selection. the genome-wide scans do not reveal selection around the other two mutations arising during cq selection nor around those arising during previous pyrimethamine selection; i.e. bp deletion and s106n dhfr), non-coding point mutation. the correspondences between the genetic and genomic data and the arguments deployed previously and above together suggest that only 3- major effect genes conferring cq-r and cq-hir were fixed by strong selection during experimental evolution from as-sens to as-30cq. furthermore, only a small number of weakly selected or effectively neutral mutations were fixed stochastically during population bottlenecks such as those occurring during transmission of parasite lines, or experimental cloning. previous similar investigations in the p. chabaudi as lineage demonstrated that one mutation conferred resistance to each of pyrimethamine, sulphadoxine, mefloquine and artemisinin and that ≤ mutations were fixed stochastically each time during the selection and cloning of as-50s/p, as-15mf and as-30cq respectively. the low rate of nucleotide substitution, even during multiple passages and bottlenecks, makes this system well adapted for investigations of resistance to other drugs. the genetic architecture of cq-r cq-hir was previously generated in as-30cq of the p. chabaudi as-lineage by drug selection over about passages, leading to the suggestion that the cq-hir phenotype arose by a series of small steps and was mediated by several mutations at different loci, although the number of mutations was not predicted. actually, for cq-r, the present data strongly support the contribution of aat as a single major effect gene, confirming previous classical genetic linkage analysis. for cq-hir, two major effect genes and ubp on chr and chr respectively) are strongly supported. in this respect, the architecture of cq-r in p. chabaudi may be similar to that of p. falciparum where pfcrt and pfmdr alleles appear to generate high levels of cq resistance epistatically. the hypothesis that cq-hir in p. chabaudi is mediated by a large number of small-effect mutations is rejected by the data presented here. the genetic determinants of cq-r the concordance between the phenotypic, genetic and genomic data strongly supports the identification of mutations in aat as the key determinant of cq-r. we have also revealed shared properties of pfcrt and aat and their mutations, such as putative function, subcellular location, tm-helix topology, type and position of mutation. genome-wide transcription profiling of the cq-s p. falciparum parasite and cq-r transfectants identified the aat orthologue pff as one of a small number of differentially expressed genes which also included genes predicted to encode transporters, proteases and components of the trafficking pathways. this suggests future transfection and functional studies to address questions regarding the role of these mutations in amino acid or cq transport and cq-r in p. falciparum and p. chabaudi. the involvement of aat another dv transporter and ubp in cq-hir also provoke questions regarding the relationship between haemoglobin digestion, protein turnover, amino acid transport/availability and cq action, export and resistance phenotypes., interestingly, the v2728f ubp mutation has now been shown to confer resistance to both artemisinin and chloroquine. this resolves a previously unexplained result - that artemisinin resistance mediated by mutations in ubp appeared before parasites were exposed to artemisinin. this data also suggests that there must be some commonality in the molecular mechanisms of resistance to the two drugs, at least in the p. chabaudi as lineage. the rodent model and public health p. chabaudi and p. falciparum have similar or identical genetic bases for resistance to pyrimethamine and to mefloquine. for cq-r, both parasite species share key features. now, notwithstanding the evidence supporting the proposal that different genes confer cq-r in the two species, the data presented here suggest that some features of the molecular mechanism of resistance acquisition may be similar. this supports the use of the rodent model to identify candidate genetic markers of resistance to future antimalarial drugs. furthermore, the orthologues of aat and pchas_ should now be evaluated as candidate molecular markers of cq-r in p. vivax and modulators of cq responses in p. falciparum. the experiments reported here share some features previously exploited by genome-wide selection scans in yeast, genome-wide association and high-resolution identification of mutated genes in p. falciparum. the present study demonstrates how similar approaches can be used to identify genes conferring complex selectable phenotypes such as drug-resistance in experimental systems.  previously, increasing chloroquine resistance phenotypes were experimentally selected in a lineage of the rodent malaria, plasmodium chabaudi. here, these phenotypes were measured, the underlying genetic loci mapped and mutations specified using a novel quantitative genetics and genomics approach. this approach analysed genetic crosses by selecting progeny en masse at different drug concentrations. the frequencies of parental alleles in the surviving parasites were measured using ~ pyrosequencing single nucleotide polymorphism assays, and for ~ single nucleotide polymorphisms, by illumina short-read sequencing. this defined'selection valleys' on chromosomes and where genes conferring resistance were expected to be located. whole genome re-sequencing of the chloroquine resistant mutant parasite and the sensitive progenitor wild-type parasite showed that only point mutations in the whole genome had arisen in the lineage. the specific isolated mutations within the selection valleys were identified. a mutation in a putative aminoacid transporter encoded on chr confers chloroquine resistance. mutations in another transporter and a deubiquitinating enzyme confer higher level chloroquine resistance. orthologues of these genes in p. falciparum and p. vivax can now be studied for their contribution to chloroquine resistance in human infection. these data will generate insights of the mechanism of chloroquine resistance in human and rodent malaria parasites. 
a knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. to address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite p. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions. a mutation in the gene encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in p. falciparum, but we lack a full account of higher level chloroquine resistance. quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. the identities of three genes conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. furthermore, the determinants of resistance in the other major human malaria parasite, p. vivax, are not known. importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias p. vivax and p. falciparum. the critical mutation conferring the first level of chloroquine resistance is found in aat a putative aminoacid transporter. loci containing the critical genes were mapped by linkage group selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. drug resistance in the malaria parasite plasmodium falciparum severely compromises the treatment and control of malaria. they are required to design better therapies and limit drug resistance. quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection.
 drug resistance in the malaria parasite plasmodium falciparum severely compromises the treatment and control of malaria. a knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. they are required to design better therapies and limit drug resistance. a mutation in the gene encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in p. falciparum, but we lack a full account of higher level chloroquine resistance. furthermore, the determinants of resistance in the other major human malaria parasite, p. vivax, are not known. to address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite p. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.  loci containing the critical genes were mapped by linkage group selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. a novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr chr and chr that appear progressively at increasing drug doses on three chromosomes. whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. the critical mutation conferring the first level of chloroquine resistance is found in aat a putative aminoacid transporter.  quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. the identities of three genes conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias p. vivax and p. falciparum. 