Genetic adaption at the cPRS locus accounts for acquisition of halofuginone resistance after generation 32 in HFGRII and HFGRIII. (A,B) Quantitative PCR copy number and allele type of uncloned HFGRII (A) and HFGRIII (B) revealed parasites with mutant cPRS alleles that failed to proceed to fixation in either population in favor of clones with wild-type amplified loci. In HFGRII, mutant parasite clones reached a maximum allele frequency of 0.57 and were competed out by those with amplified wild-type loci. In HFGRIII, parasite clones with mutant cPRS loci were undetectable. Neither cPRS mutation nor amplification reached sufficient allele frequency before the 34th (HFGRII) or 32nd (HFGRIII) generation after selection with 42 nM halofuginone (60× EC50). (C) Though HFGRII and HFGRIII have different amplification breakpoints as illustrated by the next-generation sequencing read pileups, both include wild-type cPRS (PF3D7_1213800) alleles. The HFGRII 41st generation pileup confirms that the cPRS locus is unamplified and reflects a mixture of wild-type and mutant haploid parasites. (D) The natural allelic series of HFGRIII generation 58.5 clones with 1, 2, and 3 copies confirms that amplification of the cPRS locus confers resistance to halofuginone. Copy number variation determined by quantitative PCR of three clones investigated for sensitivity to halofuginone using the 3′ cPRS assay. SYBR growth dose-response assay confirms that more cPRS copies desensitize parasites to halofuginone. Relative copy number in (A,B,D) was determined with SerRS as an endogenous control to a single cPRS copy Dd2 parasite. Allele frequencies were determined from whole-genome sequencing. Read pileups in (C) were generated from aligned reads generated on an Illuminia HiSeq 2000 and visualized with IGV v 2.3.32. Error bars in (D) denote standard deviation.