Reduced heterozygosity at intragenic and flanking microsatellites of pfcrt gene establishes natural selection based molecular evolution of chloroquine-resistant Plasmodium falciparum in India

- New microsatellite markers identified in introns of pfcrt gene.
- Reduced heterozygosity observed in intragenic microsatellite of pfcrt gene.
- Confirmed selective sweep model in both intragenic and flanking loci of pfcrt gene.
- Differential genetic background associated with various resistant pfcrt-haplotypes.

The positive selection of a nucleotide substitution in exon 2 of Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene (mutation responsible for chloroquine resistance) causes a reduction in variation of neutral loci close to the gene. This reduction in allelic diversity around flanking regions of pfcrt gene was reported in worldwide chloroquine resistant isolates and referred as selective sweep. In Plasmodium falciparum isolates of India, the selective sweep in flanking loci of pfcrt gene is well established, however, high allelic diversity observed in intragenic microsatellites of pfcrt gene implied an ongoing genetic recombination. To understand, if molecular evolution of chloroquine-resistant P. falciparum isolates in India follow a selective sweep model, we analyzed genetic diversity at both seven intragenic and seven flanking microsatellites of pfcrt (−24 to +106 kb) gene in chloroquine sensitive and resistant parasites originating from high and low transmission areas. We observed low expected heterozygosity at all loci of resistant pfcrt-haplotypes (He = 0-0.77) compared to the wild-type (He = 0.38-0.96). Resistant SVMNT from high transmission areas showed significantly higher mean He (P = 0.03, t-test) at both intragenic and pfcrt-flanking loci (−24 to +22 kb) in comparison to low transmission areas. Our observation of reduction in variation at both intragenic and flanking loci of mutant pfcrt gene confirmed the selective sweep model of natural selection in chloroquine resistant P. falciparum isolates in India.