Study On Biodiversity Monitoring Of Phytoplankton Community Based On DNA Barcoding Technology

Monitoring aquatic organisms is important for protecting the water quality and ecological functions of lakes and rivers.Phytoplankton community structure changes not only reflect the changes in water quality,while the occurrence of toxic phytoplankton will also threaten water ecological security.Traditional phytoplankton identification methods are time consuming and labor intensive,and the accuracy of DNA barcoding technology can achieve the purpose of identify species rapidly and accurately.The potential of DNA barcoding technology for application in aquatic ecosystem monitoring and health assessment is significant.However,the DNA metabarcoding technology for phytoplankton monitoring in the Taihu Lake Basin has not yet been developed,and the DNA barcode database of native phytoplankton is still lacking.At the same time,how to establish the biological index method based on molecular biology to evaluate the lake water ecological health status is an unsolved problem.In this study,the phytoplankton community in Taihu Lake Basin was studied from the following aspects:(1)Specific primers suitable for the monitoring of phytoplankton biodiversity in Taihu Lake Basin were selected by comparison of single gene primers in different genomic regions;(2)To establish a database of wild-type phytoplankton DNA barcode in Taihu Lake Basin;(3)To establish a microbial barcoding technique for phytoplankton in Taihu Lake Basin based on DNA molecules and to analyze the biological diversity of phytoplankton in Taihu Lake.Based on the DNA metabarcoding technology to construct a phytoplankton index suitable for the evaluation of water health status in Taihu Lake Basin.The main results are summarized as follows:(1)Through the comparison of 14 pairs of primers,3 pairs of primers suitable for monitoring phytoplankton in Taihu Lake were screened.Then compared the monitoring results of phytoplankton diversity by 3 pairs of primers with DNA metabarcoding technology and traditional microscopic identification method.The results showed that ITS was more suitable for monitoring at the species level,and it was suitable for constructing the DNA barcode database of phytoplankton in Taihu Lake Basin.The combination of V3 and V9 primers could be used in monitoring the common phytoplankton biodiversity in Taihu Lake Basin;(2)Based on the isolation,purification and sequencing analysis of phytoplankton in Taihu Lake.A total of 95 species of algae ITS barcode database was constructed,including 11 species of Diatophyta and 84 species of Chlorophyta.20 species of 18S rRNA barcode were constructed,including 1 species of Diatophyta,1 species of Chrysophyta and 10 species of Chlorophyta(including 4 overlaps with ITS);7 species of Cyanophyta sequenced by 16S rRNA.There were 110 species of phytoplankton in the native DNA barcode database,including 7 species of Cyanophyta,12 species of Diatophyta,90 species of Chlorophyta,1 species of Chrysophyta.(3)Through the analysis of 40 evaluation parameters of Taihu Lake sample,4 parameters,PI-21(mcintosh_d-Cyanobacteria),PI-23(margalef-Cyanobacteria),PI-31(Chlorophyta density(reads%)-eukaryotic phytoplankton)and PI-37(Cyclotella density(reads%)-eukaryotic phytoplankton)which can characterize the nutritional status of Taihu Lake Basin were screened out,and the evaluation parameters were selected by the ratio method.The integrated phytoplankton index-PI(?)of Taihu Lake was established.PI(?)can be used as a new index based on molecular biological methods to evaluate the water health status of Taihu Lake,then delineated the evaluation of water quality in Taihu Lake.Through the analysis of PI(?)of Taihu Lake,the water quality of the northern division of Taihu Lake was poor.The water quality of the eastern division of Taihu Lake and the eastern division of Taihu Lake was normal.The water quality of the western division of Taihu Lake and the division part of Taihu Lake was normal.Lake Taihu overall water health status was normal.The new method of species identification based on molecular biology method provides a new opportunity for the monitoring of phytoplankton biodiversity in aquatic ecosystems.The native DNA barcode database of phytoplankton is constructed by DNA barcoding technology.It is a database of phytoplankton biomonitoring in the Taihu Lake Basin and even the freshwater ecosystem in China.The new phytoplankton index PI(?)constructed with molecular biology method,is simple and accurate,can be used as biological indicators water ecological health evaluation in Taihu Lake.