Construction Of DNA Barcoding Of Four Sciaenidae Species

With the development of marine fish trade and the improvement of processing level,the mislabeled and shoddy phenomenon of fish has seriously affected food safety as well as domestic and international trade order in recent years.Accurate identification of species is therefore essential in marine fish trade.DNA barcoding is based on the analysis of a short,standardized gene sequence,which has now become a new direction for biological species identification.The application of DNA barcodes to molecular identification of fish is thus of great significance.In this study,Larimichthy crocea,Larimichthy polyactis,Nibea albiflor and Collichthys lucudus were used to study the construction of DNA barcode.The main results were as follows:1.Based on the comparison results from GenBank database,mtDNA cytochrome c oxidase subunit I gene(COI),cytochrome b oxidase(Cytb),16 S rRNA and mtDNA control region(D-loop)could be used as DNA barcodes for the identification of Larimichthy crocea,Larimichthy polyactis,Nibea albiflor and Collichthys lucudus.Combining the distribution of K2 P genetic distance,interspecies genetic distance difference and molecular phylogenetic tree,meanwhile considering the application of the four genes in the database,COI gene was selected as the optimal DNA barcode for the four fish species.Using COI gene to analyze and identify the Larimichthy crocea and Larimichthy polyactis products,it was found that Larimichthy polyactis products were mislabeled and counterfeited seriously by Pennahia microcephalus and Pennahia argentata in the market.2.COI gene was used to analyze the genetic diversity of Larimichthy crocea,Larimichthy polyactis and Collichthys lucudus in four sampling points of Dongtou,Dalian,Zhoushan and Lianyungang.The results showed that the inter-and intrapopulations genetic distance of different geographic populations were not much diffenerces.The phylogenetic tree of the same species with different geographical populations was constructed.It was found that there were two different lines of Collichthys lucudus in the same species,while Larimichthy crocea and Larimichthy polyactis were not separated.3.Four kinds of restriction enzyme Vsp I,Hae II,Nhe I and Hinf I were selected to carry out the PCR-RFLP analysis of COI gene in Larimichthy crocea,Larimichthy polyactis,Nibea albiflor and Collichthys lucudus.The results showed that Vsp I,Hae II and Nhe I could identify Larimichthy crocea,Nibea albiflor and Collichthys lucudus,respectively.Hinf I could simultaneously identify four kinds of fish.The specific primers were designed for specific COI sequences of Larimichthy polyactis and Nibea albiflor.The results showed that there were specific bands in Nibea albiflor.The two methods could be used for rapid and accurate identification of these Sciaenidae species.