Establishment And Application Of A Survey Method For Fish Resources In Hunan Province Based On Environmental DNA

Hunan Province locates in the south of the Yangtze River with a dense river network.There are mainly Xiang,Zi,Yuan,Li river systems through the entire province,and complex topographic features provide various habitats for fish,famous for"land of fish and rice".According to the 1977 edition of Hunan Fishes,there are more than 160 fish species in Hunan Province,belonging to 11 orders,26 families and 92 genera.However,in recent years,with the interference of human activities,fish resources have been significantly reduced,and the fisheries show a declining trend.How to quickly and accurately detect and investigate the spatial distribution and dynamics of fish resources in Hunan Province has become an urgent matter,because the depression of the fish resources requires the scientific development of fish resources protection and management strategies.Morphological taxonomic identification has always been the traditional investigation for fisheries,which is destructive to the surrounding environment.With the rapid development of modern science and technology,environmental DNA（e DNA）metabarcoding technology based on the molecular level can make up for the deficiency of traditional investigation methods.It has the characteristics of time-saving and high efficiency and can provide an alternative survey method for traditional morphological identification.This study collected the specific primers of freshwater fish,and the primers were used to amplify the fishes from Hunan province to construct the barcode database of Hunan province.According to the environment of the three generations of sequencing DNA research technology,and which was compared for fish diversity in Datong Lake.In the end,this method was employed in the study of Maili lake.The research contents of this paper are as follows:1.Construction of ichthyic DNA barcoding Database in Hunan ProvinceThe reported freshwater fish-specific primers were screened through literature reviews,such as 16S、COI、Cytb and RHO.And COI gene fragments had a good amplification effect and high discrimination after initial tests.After further sequencing,two pairs of primers of COI gene fragments were selected to amplify and sequence for 86 common fish in Hunan Province.A total of 240 sequences were obtained,the length of the fragment was 600bp,and the sequenced samples covered the common fish species in Hunan Province.Based on the obtained data,a COI gene-related DNA barcode database of common fish in Hunan Province was constructed to investigate and evaluate fish resources in Hunan Province.It could also benefit the protection of characteristic fish and the mining of hidden germplasm resources.2.Establishment of Hunan fish survey method based on environmental DNATaking Datong Lake as the study area,the fish diversity in Datong Lake was detected through environmental DNA standardized processes,including water sample collection,DNA enrichment and extraction,high-throughput sequencing and bioinformatics analysis.Comparing the applicability of different databases for annotating fish species,the results show that the local COI barcode database is more suitable for the annotation of the results of fish e DNA testing in Hunan Province.A total of 12 species of fish were identified based on e DNA technology and the local database,belonging to 4 orders,9 families and 12 genera.The main fish was the bottom Catapultiformes.The main fish species of traditional fishing data is pelagic Cypriniformes.Although the fish species detected by these two methods have some differences,they are consistent with the historical fish survey data of Datong Lake in previous years,This gap may be caused by the preference of net fishing and e DNA sampling.This study indicates that the new and non-invasive freshwater fish species survey method based on e DNA metabarcoding technology can assist in the conservation and monitoring of common fish diversity in Hunan Province.3.Study on the biodiversity of Maori Lake by e DNA techniqueIn order to further explore the application of e DNA metabarcoding technology,water samples were collected from five sites in Maoli Lake（Changde）to investigate the fish diversity in Maoli Lake.A total of 411 OTU of fish species were obtained by 3^rdgeneration sequencing with annotation by the DNA barcode library constructed in Chapter 2.Among them,8359 related sequences were annotated,and 22 fish species were identified,which belong to 4 orders and 11 families.Cypriniformes were the most common,accounting for50%of the fish species tested.The analysis of fish composition in Maoli Lake shows that there are some differences in the spatial distribution of fish composition in there,mainly caused by the differences of Hypostomus plecostomus,Squalidus argentatus,Macropodus operculari and Micropercops swinhonis.This also means that e DNA techniques can be used to monitor fish and reveal spatial differences in communities.To sum up,the survey method of common fish in Hunan Province established in this study based on e DNA is feasible and can be used to investigate and monitor the diversity of common fish resources in our province.Compared with traditional methods,it has the advantages of high efficiency and time saving.