Species Identification Between Guangxi And Yunnan Tree Shrew Groups By DNA Barcode Technology

OBJECTIVE: The feasibility of applicating DNA barcode standard sequence was explored between guangxi and Yunnan tree shrew populations in classification and identification,so that a standard molecular classification model is provided for the classification of the tree shrew populations.Also for the subsequent high infection rates of tree shrews hepatitis model is set up and cultivate superior strains will provide reliable information about genetic background.METHODS: Extracted genes DNA from 43 tree shrews blood that are from Guangxi Longan populations and Yunnan Kunming populations respectively.PCR amplification of CO1 gene was used as a barcode,products purification and sequencing,then combined with Genbank database tree shrew sequences(T.belangeri modesta and T.glis)which had be downloaded,and various of biology software such as MEGA were applied to analyze the four tree shrew populations genetic associated indicators,such as the length of the CO1 gene sequence,base composition,haplotypes,conserved sites(C),variable sites(V),parsimony-informative sites(Pi),base substitution,transitions,transversions and so on.Finally,the intraspecific genetic distance and genetic distance between species of tree shrew populations was calculated,and the phylogenetic tree of tree shrew populations was builded to further validate the genetic differences among groups.RESULTS: 1.Length of 638 bp CO1 gene sequences were obtained by PCR amplication and sequencing,which no base insertions or deletions.The average content of the four bases(A,T,C,G)were 25.1%,29.3%,27.1%,18.5%,the average contents of A and T significantly higher than the bases C and G content on average.There are obviously bases A and T bias phenomenon,which conform to the characteristics of the vertebrate mitochondrial DNA base composition.2.The experimental tree shrews species belonging to 10 haplotypes,the populations in Longan,Kunming and T.belangerii modesta possess 9 haplotypes.All CO1 gene sequences were detected with 470 conserved sites(C),168 variable sites(V),97 parsimony-informative sites(Pi),31 base pairs transitions and 7 transversions.Which show the tree shrew populations more differentiation,species resources and genetic diversity.3.This study groups of four tree shrews(Longan populations,Kunming populations,T.belangeri modesta and T.glis)intraspecific genetic distance range from 0.00% to 0.79%,three populations from Tupaia belangeri interspecific genetic distance between 9.71% and 13.59%,and the genetic distance between they and T.glis range from 20.43% to 24.11%.Intraspecific genetic distance and interspecific genetic distance without overlap region,there is an obvious barcoding gap,and it shows that DNA barcode can well identify different geographical groups of tree shrews.4.Tree shrew populations were showed in phylogenetic tree that individuals gathered trend obviously,different individual branch length is shorter,branch longer between different species,and different individuals are first gathered in this population branch,branch with other tree shrew populations gathered again.Which confirmed that Guangxi Longan populations and Yunnan Kunming populations were two different groups of Tupaia belangeri.CONCLUSIONS: DNA barcode based on mitochondrial CO1 gene is effective for the classification and identification to tree shrew populations.This study analyzed the genetic differences between Guangxi Longan populations and Yunnan Kunming populations through DNA barcode and confirmed that the two groups belong to Tupaia belangeri subspecies.