DNA Barcodes And Characteristic Diagnostic Sites Of Schizothoracinae Fishes From Qinghai Province

There are at least 20 species of schizothoracine fish in Qinghai Province,accounting for more than 40% of the native fish species,among which 5 species are endangered and 8 species are vulnerable.Accuracy species identification is essential for protection of these species.The species identification of schizothoracine fish through classical taxonomic method is complicated and even prone to errors.Therefore,it is necessary to develop a method for rapid identification of Schizothoracinae species.DNA barcoding is a convenient molecular technology for rapid species identification.The Self-Organized Map(SOM)could be used to predict the specific diagnostic sites that differentiate different taxa.Based on the COI gene sequences,DNA barcode and its characteristic diagnostic sites analysis of Schizothoracinae fish in Qinghai Province was carried out in this study,in which Cyt b was used as molecular reference.The main results were as follows:1.A total of 159 COI sequences(belong to 8 genera 15 species / subspecies)of schizothoracine fishes from 24 sampling sites in Qinghai Province were obtained in this study.98% as successful identification ratio was obtained through Blast.The average interspecies genetic distance of schizothoracine fish in Qinghai Province was 9.17% based on Kimura-2-Parameter(K2P)model,which was about 42 times over the average intraspecies genetic distance(0.22%).However,there were only 5spcies whose intraspecies K2 P distance was less than 2% and over 10 times value was observed in interspecies/intraspeices ratio,suggesting that overlapping regions existed between interspecies genetic distance and intraspecies genetic distance.The same results were observed based on sequence analysis of Cyt b.Besides,11 groups were classified out of all specimens by the Automatic Barcode Gap Discovery System(ABGD),of which 8 were groups with single species,indicating that several species could not be classified via barcode gap.2.Five samples at most per species were used to construct phylogenetic trees via three methods(the Maximum Likelihood method,the Bayesian algorithm method and the Maximum Parsimony method).The results showed that schizothoracine fishes were classified into three major branches by COI gene with strong nodal support,which were identical with three grades of Schizothoracinae.The phylogenetic topology of COI trees was almost consistent of that of Cyt b(1080 bp)trees using the same individuals,and species in genus Gymnocypris,Schizopigopsis pylzovi and Chuanchia labiosa could not be clustered into single branches alone.Moreover,the nodal support of the highly specialized grade schizothoracine fish in Cyt b trees was significantly higher than corresponding nodal support in COI trees.3.A total of 34 characteristic diagnostic sites from 7 schizothoracine species were predicted from 156 variable sites from 15 species / subspecies based on SOM model,among which 7 characteristic diagnostic sites were from Gmnodiptychus pachycheilus,3 from Platypharodon extremus,9 from Ptychobarbus kaznakovi,2 from Schizopygopsis anteroventris,2 from Schizopygopsis malacanthus chengi and 2 from Schizothorax lantsangenesis.The results indicated that the SOM model was feasible in the prediction of the characteristic diagnostic sites of the Schizothoracinae fish in Qinghai Province at species level.Above all,COI barcode could be used in schizothoracine species identification of Qinghai Province except for species in genus Gymnocypris,S.pylzovi and C.labiosa,and the use of COI gene as a barcode for species identification was more economical due to its shorter length.Meanwhile,the SOM model and those characteristic diagnostic sites predicted in this study were proposed to be applied to the rapid species identification of Schizothoracinae fish and to guide the development of the protection work of carps in Qinghai Province.