DNA Barcoding Of Genus Sinopotamon From Qinling Mountains And Preliminary Analysis Of Mitochondrial Genome Of Freshwater Crabs

Objective: 1.Try to use DNA barcode to identify Sinopotamon freshwater crabs from Qinling Mountains to explore the applicability of DNA barcodes in freshwater crabs,and to reveal that large mountains have a barrier effect in the distribution of freshwater crabs or act as a corridor.2.To obtain the whole mitochondrial genomes of Sinopotamon xiushuiense and Huananpotamon lichuanense in China.The whole mitochondrial genome data of Brachyura crabs were used for phylogenetic analysis,to clarify the taxonomic status and phylogenetic relationship of freshwater crabs in the Brachyura,the differentiation time and the selection pressure in the evolutionary process.Methods: 1.The samples were collected from the north and south of Qinling Mountains and morphological identification was carried out.The COI and ITS2 genes were selected as the DNA barcode candidate standard sequences.The total DNA of the samples was extracted and amplified by PCR and sequenced.The experimental data were combined with the existing data on GenBank to constitute a data set.After data screening and testing,genetic distance and phylogenetic analysis were performed.2.Retrieve the GenBank database and download the existing mitochondrial genome data of the Brachyura.The mitochondrial genomes of Sinopotamon xiushuiense and Huananpotamon lichuanense were obtained,the characteristics of the base composition,gene composition and arrangement and codon utilization were compared with those of other Brachyuran crabs.The product of protein-coding genes perform different functions in electron transfer reactions,so we divided the 13 protein genes into 4 groups(Complex I,Complex III,Complex IV,Complex V),two rRNA genes were added as another group,phylogenetic analysis were performed respectively.Using BEAST software to estimate the divergence time of each group in Brachyura.The average selection pressure of 13 protein coding genes were calculated by using PAML software,the selection pressure of freshwater crabs and other Brachyura crabs was calculated by using the branch model respectively.LRT(Likelihood Ratio Test)was carried out to test the significance.Conclusion: 1.The phylogenetic tree constructed by COI gene could identify S.depressum,S.fukienense,S.davidi davidi,S.lansi and S.yangtsekiense yangtsekiense.2.The difference of "DNA barcode gap" in Sinopotamon in this study still exists,S.changanense and S.acutum formed a clade,the interspecies genetic distance between the two species was overlapped,that is,the barcode gap between the two species disappeared.S.yangtsekiense tongbaiense,S.yangtsekiense shanxianense and S.shensiense formed a clade,and the interspecies genetic distance were overlapped.3.S.changanense and S.acutum were divided into two groups,north and south,based on the location of the sampling site,suggesting that the Qinling Mountains had a blocking effect on the migration of some Sinopotamon species.4.The extraction efficiency of ITS2 gene was low and the bimodal sequence rate was higher(37.97%).This study do not support it as DNA barcode standard sequence.5.The total length of the mitochondrial genomes of Sinopotamon xiushuiense and Huananpotamon lichuanense are 18460 bp and 15380 bp,encoding 37 genes common in metazoan.Compared with the original arrangement of Pancrustacean,two tRNA rearrangements(t RNAHis and tRNAGln)were found in both freshwater crabs,which were previously only found in Geothelphusa dehaani,supporting the monophyly of Potamidae at the genomic level.6.The phylogenetic tree constructed by using complex V(ATP6 and ATP8)and complex III(CYTB)is quite different from the currently accepted taxonomic system,and the branch confidence were very low,which are not suitable for higher taxa phylogeny analysis.The other three groups showed consistent results with the current accepted taxonomic system,the branch confidence were higher,suggesting that molecular markers of higher taxa phylogeny can be found in these three sets of data in the future.7.Estimation of divergence time based on 13protein-coding genes showed that Brachyura occurred in the early Triassic(about254.41 Ma),whereas Potamidae differentiated in the early Cretaceous(about133.58 Ma).8.Contrast with complex I(ND1 ~ 6,ND4L)and complex V(ATP6,ATP8),complex IV(COX1 ~ III)and complex III(CYTB)were under stronger purifying selection pressure.Except ATP6 and ND4 L genes,the dN / dS values of 11 protein genes in freshwater crabs were greater than those in other marine crabs(thedifference was significant),the accumulation of more non-synonymous substitutes prompted the evolution of freshwater crabs to adapt to the terrestrial environment.