title: Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites.
abstract: BACKGROUND
  Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.
  
  
  RESULTS
  A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.
  
  
  CONCLUSIONS
  This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.
authors.affiliation: Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK. Paul.Hunt@ed.ac.uk
authors.forename: Paul
authors.initials: P
authors.lastname: Hunt
authors.forename: Axel
authors.initials: A
authors.lastname: Martinelli
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Modrzynska
authors.forename: Sofia
authors.initials: S
authors.lastname: Borges
authors.forename: Alison
authors.initials: A
authors.lastname: Creasey
authors.forename: Louise
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Dario
authors.initials: D
authors.lastname: Beraldi
authors.forename: Laurence
authors.initials: L
authors.lastname: Loewe
authors.forename: Richard
authors.initials: R
authors.lastname: Fawcett
authors.forename: Sujai
authors.initials: S
authors.lastname: Kumar
authors.forename: Marian
authors.initials: M
authors.lastname: Thomson
authors.forename: Urmi
authors.initials: U
authors.lastname: Trivedi
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Otto
authors.forename: Arnab
authors.initials: A
authors.lastname: Pain
authors.forename: Mark
authors.initials: M
authors.lastname: Blaxter
authors.forename: Pedro
authors.initials: P
authors.lastname: Cravo
mesh_terms: D000818:Animals; D037621:Artemisinins / Q000494:pharmacology* / Q000627:therapeutic use; D003198:Computer Simulation; D056915:DNA Copy Number Variations / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004351:Drug Resistance / Q000235:genetics*; D017125:Genes, Protozoan; D018503:Genome, Protozoan / Q000235:genetics*; D005838:Genotype; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D008288:Malaria / Q000188:drug therapy / Q000469:parasitology; D016254:Mutagenesis, Insertional / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010271:Parasites / Q000187:drug effects / Q000235:genetics; D010641:Phenotype; D010802:Phylogeny; D016788:Plasmodium chabaudi / Q000187:drug effects / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D011739:Pyrimethamine / Q000494:pharmacology; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion / Q000187:drug effects / Q000235:genetics
chemical_list: D037621:Artemisinins; C031327:artemisinin; D011739:Pyrimethamine