Genetic Diversity Analysis of Chinese Leishmania Isolates and Development of L. donovani Complex Specific Markers by RAPD

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background</jats:bold>: Leishmaniasis is one of the most neglected tropical diseases in the world and is still endemic in some underdeveloped regions including western of China. The phylogeny and classification of Chinese <jats:italic>Leishmania</jats:italic> has not yet been clarified, especially within <jats:italic>Leishmania </jats:italic>(<jats:italic>L.</jats:italic>) <jats:italic>donovani</jats:italic> complex, although phylogenetic analyses based on a series of gene markers were carried out. More different analytic method and data still be needed. Random amplified polymorphic DNA (RAPD) technology could identify slight intraspecific differences sensitively, and it always be a powerful tool to seek the species-specific markers. This work aimed to identify Chinese <jats:italic>Leishmania</jats:italic> isolates from diverse geographic regions at genomic level. Meanwhile, the specific markers of <jats:italic>L. donovani</jats:italic> complex were also developed by RAPD.<jats:bold>Methods</jats:bold>: The RAPD was applied on 14 Chinese <jats:italic>Leishmania</jats:italic> isolates from diverse geographic regions and 3 WHO reference strains. The polymorphic sites of amplification were transformed into data matrix, based on which genetic similarity were calculated and UPGMA dendrogram were constructed to analyze genetic diversity of these <jats:italic>Leishmania</jats:italic> isolates. Meanwhile, the specific amplification loci of <jats:italic>L. donovani</jats:italic> complex were TA-cloned, sequenced and converted into sequence characterized amplified regions (SCAR) markers, which were validated preliminarily in available 17 <jats:italic>Leishmania</jats:italic> strains in this study and analyzed by bioinformatics.<jats:bold>Results: </jats:bold>The cluster analyses showed that the three <jats:italic>Leishmania</jats:italic> sp. isolates SC10H2, SD and GL clustered together and apart from others, and the strains of <jats:italic>L. donovani</jats:italic> complex clearly divided into two clades and the three isolates Cy, WenChuan and 801 formed a subclade. Three specific SCAR markers of <jats:italic>L.</jats:italic> <jats:italic>donovani</jats:italic> complex, i.e. 1-AD17, 2-A816 and 3-O13, were successfully obtained and validated on available 17 <jats:italic>Leishmania</jats:italic> strains in this study. Through bioinformatics analyses, Marker 1-AD17 may have more specificity on PCR detection of VL and Marker 3-O13 has the potential of encoding protein.<jats:bold>Conclusions:</jats:bold> the RAPD result verified that the undescribed <jats:italic>Leishmania</jats:italic> species causing visceral Leishmaniasis (VL) in China was a unique clade distinguished from <jats:italic>L. donovani</jats:italic>, and revealed that there was genetic differentiation among Chinese <jats:italic>L. donovani</jats:italic>. The development of <jats:italic>L.</jats:italic> <jats:italic>donovani</jats:italic> specific markers may provide the foundation for developing new specific diagnostic markers of VL and the research of specific gene function.</jats:p>

Application of RAPD Molecular Technique to Study the Genetic Variations of Citrus in South Sulawesi, Indonesia

“…Molecular markers such as RAPD, RFLP, AFLP, ISSR, and SSR have been used to research germplasm characterization, genetic diversity, and systematic and phylogenetic analysis [25], [26]. Random Amplified Polymorphism DNA (RAPD) has the advantage that with a simple procedure, relatively inexpensive price, and a small amount of DNA for analysis, it can produce highly polymorphic DNA representative of the entire genome [27]- [29]. RAPD has been widely applied to citrus plants, among others, for studying the genetic variations of citrus germplasm [5], [30].…”