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Background  Molecular genotyping in  Plasmodium  serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for  Plasmodium malariae , an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of  P. malariae  population genetics.    Methods  Markers for microsatellite genotyping and  pmmsp1  gene polymorphisms were developed and validated in symptomatic  P. malariae  field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity ( H   d  ) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed.    Results  Six microsatellite markers were developed and tested in 37  P. malariae  isolates which showed sufficient heterozygosity (0.530–0.922), and absence of linkage disequilibrium ( I   A    S  =0.03,  p value  >  0.05 ) (N = 37). In addition, a tandem repeat (VNTR)-based  pmmsp1  INDEL polymorphisms marker was developed and assessed in 27  P. malariae  isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and  pmmsp1  showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10 −4  and was further reduced to 1.66 × 10 −4  when  pmmsp1  polymorphisms were included.    Conclusions  Six novel microsatellites genotyping markers and a set of  pmmsp1  VNTR-based INDEL polymorphisms markers for  P. malariae  were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.

Polymorphic markers for identification of parasite population in Plasmodium malariae