Application Of DNA Barcoding In Fish Identification And Genetic Diversity Assessment In Three Gorges Reservoir Area

The Three Gorges Reservoir Area has unique natural conditions and rich species,making it a key area for biodiversity conservation in China.However,the development of water conservancy and hydropower projects has changed the ecological environment of the Three Gorges reservoir area,causing a significant impact on aquatic organisms such as fish.This study conducted a survey of fish resources in the three gorges reservoir area from 2019 to 2020,including the head(Taiping River,Xiangxi River),the middle(Xiaojiang,Wanzhou,Yunyang),and the tail(Fengdu,Jiantan River,Banan,Fujiang,Jialing River).In this study,a total of 946 DNA barcode sequences were obtained from the fish in the Three Gorges Reservoir area.The species diversity of the fish in the Three Gorges Reservoir area was evaluated at the molecular level,and the applicability of DNA barcode in the assisted species identification of the fish in the Three Gorges Reservoir area was discussed.A database of DNA barcode sequences for the fish in the Three Gorges Reservoir area was established The conclusions are as follows:(1)A total of 71 species of fish,including 5 unnamed species,were identified in the Three Gorges Reservoir area using DNA barcodes.The range of intraspecific genetic distance is 0-1.89%,with an average of 0.36%;The genetic distance between species within the genus ranged from 2.41% to 14.83%,with an average of 6.88%;The genetic distance between genera in subfamilies ranged from 4.22% to 21.63%,with an average of 13.57%.The comparison of intraspecific and interspecific genetic distance showed that the maximum intraspecific genetic distance was smaller than the minimum interspecific genetic distance,which could form obvious DNA barcode gap;Bayesian evolutionary tree shows that 71 species including 5 unnamed species have formed their own monophyletic branches,and the node support rate is high.ABGD showed that 946 samples formed 67 operational units(OTUs),among which the unnamed species of Paramisgurnu,Tachysurus,Rhodeus,Saurogobio and Pseudobagrus all separately formed OTUs;Silurus asotus constitute 2OTUs;Ancherythroculter nigrocauda,Culter alburnus and Chanodichthys mongolicus constitute one OTUs;Hemiculter tchangi and Hemiculter leucisculus constitute one OTUs;Microphysogobio kiatingensis and Microphysogobio tungtingensis form an OTUs;Rhinogobio typus and Rhinogobio cylindricus constitute an OTUs.It may be that the related species are defined as an operable unit due to the small genetic distance.The above results show that the application of DNA barcode technology in the classification and identification of fish in the Three Gorges Reservoir Area is highly effective.(2)The haplotype diversity(Hd)and nucleotide diversity(Pi)of 6 species of endemic fish,5 species of dominant fish and Coilia brachygnathus in the upper reaches of the Yangtze River were analyzed.The results showed that the haplotype diversity range was0-0.952,with an average of 0.572,and the nucleotide diversity range was 0-0.0082,with an average of 0.0027.There are 6 species of fish with Hd ≥ 0.8 and 4 species of fish with Pi ≥0.005.The genetic diversity of COI barcode in Hypophthalmichthys molitrix,Tachysurus vachellii,Saurogobio dabryi and Procypris rabaudi is 0.0082,0.0076,0.0071 and 0.0051 respectively.However,the genetic diversity of COI barcodes in Coreius guichenoti,Leptobotia elongata,Hemiculter bleekeri,Hemiculter tchangi,Tachysurus nitidus,Ancherythroculter nigrocauda,Spinibarbus sinensis,and Coilia brachygnathus is 0.0033,0.0031,0.0023,0.0021,0.0021,0.0021,0.0011,and 0.0004,respectively.Genetic diversity analysis shows that the overall genetic diversity of fish in the Three Gorges Reservoir area is relatively low.The research results provide scientific data for the protection and management of fish resources in the Three Gorges Reservoir Area.(3)Genetic structure analysis was conducted on the genera Saurogobio and Tachysurus through haplotype network diagrams.The analysis of haplotype relationship diagram shows that 112 samples of Saurogobio have produced 37 haplotypes;A total of 48 haplotypes were produced from 194 samples of the Tachysurus.Based on the analysis of sampling sites,it was found that there are unique haplotypes in the populations of Saurogobio dabryi,Saurogobio gymnocheilus,Tachysurus vachellii and Tachysurus nitidus in the reservoir head,middle,and tail of the Three Gorges Reservoir area,but no significant geographical pattern has been formed overall.