| Malaria is the most significant vector-borne disease in the worldwide. The vast majority of deaths occurred not only in Africa, but also in South America and South-East Asia. The number of reported clinical cases of malaria ranges between 300 to 500 million, with one to three million fatalities each year. Approximately 90% of these deaths occur in Africa, mostly in young children. Plasmodium vivax is the most widely distributed cause of malaria. Although responsible for less mortality than Plasmodium falciparum, P. vivax causes considerable morbidity, particularly in children, due to its extended adaptability and high rate of relapse. Worldwide, Plasmodium vivax is a major public health challenge.2.6 billion people are currently at risk of infection in Central and South America, the Middle East, Central, South and Southeast Asia, Oceania and East Africa, and 70-80 million clinical cases associated with Plasmodium vivax are reported each year. Across the Amazon Basin of Brazil P. vivax has recently surpassed P. falciparum as the main cause of malaria morbidity. In China, as a result of active implementation of malaria control measure for more than 40 years, considerable success has achieved. But in recent years, there is an outbreak of P. vivax in some areas of China, and the incidence has increased gradually. In order to eradicate the malaria at all, there is an urgent need for the strategy of tracing the origin of malaria parasites. But it is rare now and understanding the population structure is essential to predict where the isolates originate.In the present study, we genotyped 23 highly polymorphic tandem repeat loci bearing 2-8bp motifs and two SNPs which were related to drug resistant gene to analyze 332 Plasmodium vivax isolates, which we collected from three different regions including Yunnan, Hainan and Central China in China, and to identify genetically diverse haplotypes or strains of these regions. Of all the microsatellites,13 loci were selected by us. The P. vivax DNA was purified from whole blood samples and from finger pricks blood spots and even from the thick smears and we use PCR to amplify all the fragments. The PCR products are detected by electrophoresis first. Then we use Genescan to achieve the repeats of the microsatellites. In the end, some statistical software may help us to know the genotypes and the variety of the populations and any things, such as the mean number of the alleles, He, LD and so on.We observed 5-33 allele per locus and high diversity (He= 0.324-0.857, mean=0.704) in all regions. Separately,for samples from Yunnan, the mean number of alleles was 14.27±1.63, and He=0.768±0.035;In Hainan, the mean number of alleles was 8.53±0.98, and He=0.707±0.042; In Central China, the mean number of alleles was 10.27±1.33, and He=0.638±0.056.In Central China populations, strong linkage disequilibrium was observed(p<0.001). We use Nei's equation to calculate the Genetic Distance within these populations. The results show isolates from Yunnan get close to Hainan (Genetic Distance=0.097), and those from Central China are further (Genetic Distance=0.161-0.197). Basing the research above, we try to construct a strategy to trace the origin for different isolates from China with the statistic method of discriminant. As the specific molecular makers, two SNPs and three microsatellites were scoring high in the process of identifying the isolates from different regions, and with SPSS software we finally observed 94.8% of original grouped isolates were correctly classified by functions. In addition,20 isolates from Central China which were not involved in the process were used to check the whole system and the results seem it was suitable for the isolates from Central China.Summarily, the population structure of P. vivax from different regions of China with high diversity has significant characters. From the genetic distance, isolates from Yunnan get close to Hainan and those from Central China are further. But with the loci we observed significant linkage disequilibrium in the isolates from Central China which may suggest high rates of inbreeding and low effective recombination rates in this region. In contrast, identical genotypes were rare and loci were randomly associated in other two populations, consistent with higher rates of out crossing and recombination. The system for tracing the origin works well with the isolates from China and we will extend the number of the isolates to improve it, make it more effective and stable. Further more, we expect it will work in other countries and areas where P. vivax still exists.
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