The Preliminary Study Of DNA Barcode On Freshwater Crabs Genus Sinopotamon As Host Of Paragonimus In Poyang Lake Basin

Objective: To evaluate the DNA barcoding molecular markers as an auxiliary classifying tool for the genus Sinopotamon taxonomic study and provide a standardized molecular classification model for identification of freshwater crab.Methods: Based on DNA extraction effects from different material parts, we optimized the DNA extraction of freshwater crab genus Sinopotamon. According to the differences of geographic distribution and habitation pattern of crabs distributed in Poyang lake basin, our research focused on 6 representative species: S. fukienense, S. wanzaiense, S. xiushuiense, S. obliquum, S. lansi and S. zixiense. With mitochondrial COI and 16 s r DNA genes and ITS2 nuclear genes used as molecular markers, we built the DNA barcode standards base on the genetic distance and phylogenetic tree combined with the GenBank database, through standard protocol including DNA extraction, primer selection, PCR amplification, product purification and sequencing.Results: Concentration of DNA extraction by kit method could stably and reliable achieve 30 ng/μL for paraeiopod muscle. High integrity of DNA template was required when using 16 S rDNA genes as standard DNA barcoding gene for identification purposes, while DNA extracted from old samples could not guarantee sufficient PCR amplification. Considering the polymorphism existed in ITS2 gene fragments among species, 16 s r DNA and ITS2 genes were not suitable candidates as standard DNA barcoding gene. On the contrary, amplification efficiency of COI gene could stably reach 100% without insertion or deletion confirmed by sequencing. Combing with the sequencing data of a part of genus Sinopotamon from GenBank, 75% of species used in this study could be identified. Our study suggested comparing with 16 s rDNA and ITS2 genes, COI was a potentially better standard DNA barcoding gene for identification and classification of freshwater crab genus Sinopotamon.Conclusion:1. Kit method achieved stable and reliable results for DNA extraction from paraeiopod muscle.2. Comparing with 16 s r DNA and ITS2 genes, COI was a potentially better standard DNA barcoding gene for taxonomical study of freshwater crab genus Sinopotamon.3. A-T percentage in COI gene was as high as 66.0%, matching with the A-T preferential characteristics in mitochondria gene for other metazoan species.4. DNA barcoding had great advantage in discriminating implicit biodiversity of genus Sinopotamon. Crossing checking AB433578.1(S. fukienense COI gene) downloaded from GenBank, our data suggested a high likelihood to reveal hidden taxonomic unit(sub-species) in S. fukienense, based on future morphological analysis of more samples.5. The phylogenetic tree based on S. lansi COI gene database suggested that the S. lansi in east, north, central of Jiangxi province originally merged with the S. lansi in Hubei province as paraphyly, which further merged with the S. lansi in west, south, of Jiangxi province as paraphyly. The Yangtse River did not exhibited geographical disruption effect on S. lansi; instead, S. lansi species appeared to spread along the river and branches. Considering the big sized and active S. lansi were mainly found in major rivers and lakes, interlink between water systems may help diffusive travelling of freshwater crabs, rather than be served as a geographic isolation.