Preliminary Study On DNA Barcode Technique Of Common Crabs Along The Zhejiang Coast

As a novel identification technique,DNA barcode could break some limitations in traditional morphological classification,and assisted morphology in species classification and identification.Since its introduction,it had been widely applied in many studies including fungi,plants,ciliates,coral,gastropods,spiders,crustaceans,insects,fish,amphibians,birds and primates.The DNA barcode based on mitochondrial CO?gene had been widely applied in the study of animal taxonomy,and gradually expanded to other genes.The classification and identification of DNA barcode technique must meet two important criterions:(1)there was a barcoding gap between the intraspecies genetic distance and interspescies genetic distance,which meaned the interspescies genetic distance was more than 10 times of the intraspecies genetic distance;(2)the species was monophyletic.This study will validate the feasibility of DNA barcode research based on the mitochondrial CO?gene,16 S rRNA gene and Cyt b gene for the crabs of Zhejiang coastal area,and to further explore the validity and accuracy based on mini-CO?mini-16 S rRNA gene.This study was divided into two parts:1.We obtained 6 kinds of crabs of the mitochondrial CO?,16 S rRNA and Cyt b gene sequences,and each kind of gene had 70 individuals.The three kinds of genes analyses showed that nucleotide composition had obvious A,T bias.Intraspecies genetic distances based on each mtDNA genes were 0.002,0.002 and 0.003,while interspecies genetic distances were 0.130,0.111 and 0.147,respectively.The average interspecies genetic distance was the average intraspecies genetic distance 65,55.5 and 49 times,they were more than 10 times,DNA barcode fragment successfully detected "barcoding gap".In the three NJ phylogenetic trees based on CO?,16 S rRNA and Cyt b gene,each kind of crab formed a monophyletic group,whichwere consistent with the two important criterions of DNA barcode classification and identification.Therefore,the DNA barcode technique bases on the mitochondrial CO?,16 S rRNA and Cyt b gene sequences can be used for theclassification and identification of crabs in this study.2.We obtained 6 kinds of crabs of the mitochondrial mini-CO?and mini-16 S rRNA gene sequences,and each kind of gene had 70 individuals.The nucleotide composition of DNA barcode generally had A,T bias.Based on mini-16 S rRNA gene,,the average intraspecies genetic distance of 6 kinds of crabs was 0.139,while the average interspecies genetic distance was 0.154,the average interspecies genetic distance species was 1.1 times of the average intraspecies genetic distance,it was less than 10 times,cannot detect the barcoding gap.Therefore,the mini-16 S rRNA gene is not suitable for the classification and identification of crabs in this study.The average intraspecies genetic distance of 6 kinds of crabs based on the mini-CO? was 0.004,the average interspecies genetic distance was 0.365,and the average interspecies genetic distances was 91 times of the average intraspecies genetic distance,it was more than 10 times,which conformed to the fist criterion for DNA barcode identification.In the NJ constructed phylogenetic tree based on the mini-CO?gene,each kind of crab formed a monophyletic group,which conformed to the second criterion for classification and identification of DNA barcode.This indicates that mini-CO?barcode is effective and accurate in classification and identification of crabs.