Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Malar J. 2020 Jan 28;19(1):48. doi: 10.1186/s12936-020-3122-2.
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.
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) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed.
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=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 and was further reduced to 1.66 × 10 when pmmsp1 polymorphisms were included.
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.
疟原虫的分子基因分型有多种用途，包括为寄生虫种群遗传学研究提供工具，并区分复发与再感染。微卫星分型、插入缺失（INDEL）和单核苷酸多态性被用于基因分型，但对重要的人类疟疾物种疟原虫疟疾仅有有限的信息。本研究旨在提供一套遗传标记，以促进疟原虫疟疾种群遗传学的研究。
在来自缅甸的有症状疟原虫疟疾野外分离株（N=37）中开发和验证了微卫星基因分型和 pmmsp1 基因多态性的标记。片段分析用于确定每个位点的等位基因大小，以计算感染复数（MOI）、连锁不平衡、杂合度并构建系统发育树。评估核苷酸多样性（π）、单倍型数量和遗传多样性（H），并构建系统发育树。构建了带有新鉴定标记注释区域的全基因组微卫星图谱。
在 37 株疟原虫疟疾分离株中开发和测试了 6 个微卫星标记，这些标记显示出足够的杂合度（0.530-0.922），并且不存在连锁不平衡（I=0.03，p 值>0.05）（N=37）。此外，在 27 株疟原虫疟疾分离株中开发并评估了基于串联重复（VNTR）的 pmmsp1 INDEL 多态性标记，该标记显示核苷酸多样性为 0.0976，单倍型基因多样性为 0.698，鉴定出 14 个独特变体。采用 27 个碱基对作为等位基因的 VNTR 共识重复单元大小。标记 Pm12_426 和 pmmsp1 显示出最高的多样性，杂合度分数分别为 0.920 和 0.835。使用 6 个微卫星标记，任何两个寄生虫株具有相同微卫星基因型的可能性为 8.46×10，当包含 pmmsp1 多态性时，这一可能性进一步降低至 1.66×10。
开发并验证了 6 个新的微卫星基因分型标记和一套基于 pmmsp1 VNTR 的 INDEL 多态性标记，用于疟原虫疟疾。每个标记都可以独立或组合使用来评估寄生虫的基因分型。新开发的标记可能成为研究寄生虫多样性、种群遗传学、分子流行病学以及在药物疗效研究中区分复发与再感染的有用工具。
