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CN-122020254-A - Yangtze river source river indication seed identification method based on habitat coupling eDNA monitoring

CN122020254ACN 122020254 ACN122020254 ACN 122020254ACN-122020254-A

Abstract

The invention provides a Yangtze river source river indication seed identification method based on habitat coupling eDNA monitoring, and relates to the technical field of ecological environment monitoring and biodiversity assessment. The invention focuses on the response mechanism of important indicating species of Yangtze river source to glacier ablation, fuses the environment DNA technology and the remote sensing image, and analyzes the distribution pattern of the indicating species and the response to the environment by a system. The study covers five groups of plankton, aquatic plants, benthonic animals, fishes and birds, and a life history characteristic database is constructed. Monitoring points are arranged on the main stream of the Yangtze river and typical tributaries of Chu Maer river, and the dead water area is avoided by combining the topography, hydrology and traffic conditions and is overlapped with the existing hydrologic station as much as possible.

Inventors

  • YANG FAN
  • PANG HONGXI

Assignees

  • 南京大学

Dates

Publication Date
20260512
Application Date
20260203

Claims (10)

  1. 1. The method for identifying the Yangtze river source river indication seed based on the habitat coupling eDNA monitoring is characterized by comprising the following steps of: S1, selecting and arranging sampling points in a research area, namely determining main flows and typical tributaries in a Yangtze river source area as research objects, and selecting target rivers according to topography, hydrologic water systems, traffic conditions and distribution of existing hydrologic monitoring stations, so as to avoid dead water areas, water return areas and drainage areas to arrange the sampling points; s2, sample collection and pretreatment, namely collecting surface water samples at each sampling point, and respectively carrying out on-site water quality parameter measurement, laboratory physical and chemical index detection and eDNA sample preservation; S3, eDNA extraction and gene amplification, namely extracting sample DNA by adopting an environment DNA conventional operation flow, and respectively selecting specific primer pairs for eukaryotic organisms and fishes to amplify target gene areas; S4, sequencing data processing and ASV analysis, namely merging, splitting, removing primers and clustering to remove chimeras of the original sequencing sequences to obtain Amplified Sequence Variant (ASV) information; s5, ASV annotation and quality control, namely performing species annotation on ASVs based on a special database, and removing low abundance, abnormal length sequences and wrong ASVs after supplementing classification information; S6, comprehensively screening the indication seeds, namely identifying candidate indication seeds by combining IndVal, a Molecular Ecological Network (MENs) and LEfSe; s7, verifying the life history characteristics of candidate indication seeds through historical data comparison and traditional resource investigation, and establishing an indication seed database; S8, indicating the correlation analysis of the seed and the environmental variable, namely screening the environmental variable without obvious colinear, and clearly indicating the response rule of the seed to the environmental stress through the Spearman correlation analysis.
  2. 2. The method for identifying the Yangtze river source river indication seed based on the habitat coupling eDNA monitoring of claim 1, wherein in the step S1, the target river comprises 9 main flows, wherein the main flows cover Tuo 'S river and upstream, midstream and downstream river sections of the TONG' S river, the branches comprise Chu Maer river, northern river, she Qu, buqu, mo Qu and a section at the intersection of the Golgi Qu Jibu, the arrangement density of sampling points is less than or equal to 1 site/100 km, and the sampling points are overlapped with the hydrological monitoring station as much as possible, and 17 river sampling points are arranged.
  3. 3. The method for identifying the Yangtze river source river indication seeds based on the habitat coupling eDNA monitoring, which is disclosed in claim 1, is characterized in that in the step S2, the sample collection is specifically that 3 times of surface water (0-20 cm) is collected by using a sterile sampling bag, the collection amount is 5L each time, the water is filtered by an integrated filter immediately after collection, a DNA later reagent is added for preservation at-20 ℃, a YSI water quality analyzer is adopted for measuring the on-site water quality parameters, and the measurement indexes comprise Water Temperature (WT), pH, dissolved Oxygen (DO), total Dissolved Solids (TDS) and conductivity (EC). Additional 1L surface waters were collected and brought back to the laboratory for determination of nitrogen and phosphorus nutrient elements and chemical ions according to standard methods.
  4. 4. The method for identifying the Yangtze river-origin river indicator based on the habitat coupling eDNA monitoring of claim 1, wherein in the step S3, the eukaryote comprises algae, zooplankton and benthic animals, the primer pair 1380F/1510R is adopted to amplify the V9 region of the 18S rRNA gene, and the DNA extraction, the PCR amplification and the library construction are all performed by the conventional operation steps in the field of environmental DNA.
  5. 5. The method for identifying the Yangtze river source river indication seed based on the habitat coupling eDNA monitoring of claim 1, wherein in step S4, the sequencing data processing specifically comprises the steps of merging original double-ended sequences by adopting a fastq_ mergepairs algorithm of VSEARCH, splitting data according to sample ID through a barcode_splitter script, performing de-multiplexing and primer trimming by using Cutadapt, clustering the sequences into ASVs by adopting a SWARM (d=1) algorithm, and removing a chimeric sequence by adopting a-uchime _ denovo function of VSEARCH.
  6. 6. The method for identifying the Yangtze river-origin river indication species based on the habitat coupling eDNA monitoring of claim 1, wherein in the step S5, the ASV annotation adopts a ecotag algorithm of OBITOOLs, the reference database comprises NCBI download sequences and an alga, zooplankton, benthonic animal and fish-specific bar code database, the annotation rule is that species level annotation is carried out by preferentially carrying out 100% similarity, unmatched sequences are secondarily annotated according to the following standard, namely fish Tele02 primer amplification sequences are 96-98% similarity genus level, 90-96% similarity family level, sex order level, other eukaryote V9 region sequences are 97% similarity species level, 95% similarity genus level and 90% similarity family level, low abundance (.001%) sequences and length anomaly sequences (Tele 02: 160 or >200 bp; V9: <130 or >230 bp) are removed after classification information is supplemented by TaxonKit, and error ASV is cleared by LU algorithm.
  7. 7. The method for identifying Yangtze river source river indication seed based on habitat coupling eDNA monitoring of claim 1, wherein in step S6, the IndVal screening standard is species with P <0.05 and IndVal >0.3 reserved, the Molecular Ecological Network (MENs) screening key species is a method for calculating intra-Module connectivity Z i and inter-Module connectivity P i of nodes, screening Module center points (modules hubs) of Z i > 2.5 and P i < 0.62, connecting nodes (Connectors) of Z i < 2.5 and P i > 0.62, network center points (networks hubs) of Z i > 2.5 and P i > 0.62 as key species, and the LEfSe screening standard is species with P <0.05 and LDA > 4.
  8. 8. The method for identifying the Yangtze river source river indication seed based on the habitat coupling eDNA monitoring of claim 7, wherein the calculation formula of the intra-module connectivity Z i is Z i =(K i -K_mean)/SD (K), wherein K i is the number of connections of the node i in the module to which the node i belongs, K_mean is the average value of the number of connections of all the nodes in the module, SD (K) is the standard deviation of the number of connections of all the nodes in the module, and the calculation formula of the inter-module connectivity P i is: P i =1-Σ(K is /K i ) 2 where K is is the number of connections between node i and module s and K i is the total number of connections for node i.
  9. 9. The method for identifying the Yangtze river-origin river indicator based on the habitat coupling eDNA monitoring according to claim 1, wherein in the step S7, the traditional resource investigation comprises zooplankton collection, benthonic animal collection, fish fishing and morphological observation, and the life history characteristics of the species are clarified by comparison with historical data of documents, books and the like, and finally the screened indicator comprises 9 phytoplankton (5 diatoms, 4 green algae), 6 zooplankton (3 rotifers, 3 ciliates), 4 benthonic animals (2 ring animals, 2 arthropods) and 2 fishes (1 phyllostachys, 1 nojirimis).
  10. 10. The method for identifying the Yangtze river source river indication seed based on the habitat coupling eDNA monitoring of claim 1, wherein in step S8, a variance-expansion factor (VIF) is adopted to screen environment variables, the VIF is reserved, the environment variables comprise altitudes and WT, pH, DO, EC, ca 2+ 、TN、TP、NH 4 + -N, DP, the correlation between the seed and the environment variables is clearly indicated through Spearman correlation analysis, the altitudes and TP are obviously and inversely correlated with the rhombus algae (Nitzschia linearis), and the seed is indicated to be prone to a low-altitude, oligotrophic and excellent-water environment.

Description

Yangtze river source river indication seed identification method based on habitat coupling eDNA monitoring Technical Field The invention relates to the technical field of ecological environment monitoring and biodiversity assessment, in particular to a Yangtze river source river indication seed identification method based on habitat coupling eDNA monitoring. Background Environmental DNA (eDNA) technology monitors and identifies biological species by analyzing genetic material in Environmental media (e.g., water, soil, sediment, etc.). Traditional species monitoring methods generally rely on means of capture, observation and morphological identification, which are time-consuming and labor-consuming, and have significant environmental impact, especially in difficult-to-reach areas. In addition, traditional methods often have difficulty in efficiently detecting highly occult or rare species. In contrast, the eDNA technique can non-invasively detect a variety of biological species in a body of water, particularly those species that are readily ignored in traditional monitoring, by extracting DNA from a water sample and performing amplification and sequencing analyses. The eDNA technology has the advantages of high sensitivity, low cost, non-invasiveness and the like, can develop species monitoring in a wider space range, and provides a new approach for large-scale ecological monitoring and biodiversity evaluation. However, the eDNA technology also faces challenges, one of which is how to accurately interpret the eDNA data in a body of water. Environmental factors such as water flow, temperature, sunlight irradiation, microbial degradation and the like can influence the stability and diffusion of DNA, so that certain uncertainty exists when species distribution prediction is carried out independently depending on eDNA data. Therefore, the eDNA data is combined with water habitat factors (such as temperature, dissolved oxygen, pH value, nutrient salt concentration and the like) to perform habitat coupling analysis, so that the accuracy of the identification of the indication seeds can be further improved. The research aims to solve the problems that species distribution monitoring is difficult and water quality factors are accurately analyzed in the ecological monitoring of the Yangtze river source region. By integrating the eDNA technology and the water quality factor analysis, the aquatic organism type can be monitored rapidly and at low cost on the premise of not interfering with the ecological environment. The method is particularly suitable for ecological monitoring in remote areas such as the Yangtze river source area and the like, and overcomes the defects of the traditional sampling method. The indicator species (Indicator Species) refers to a species that has a significant response to environmental changes in a particular ecological environment. The distribution and abundance of the water can reflect the nutrition status, pollution level and health status of the ecological system of the water body. By monitoring the indicator, direct indication information can be provided for aquatic ecosystem health assessment. The indicator species in the water ecology monitoring mainly comprise phytoplankton, zooplankton, benthonic animals, fish and the like, and different groups show obvious response to water quality factors (such as water temperature, dissolved oxygen, nutrient salts and the like). By analyzing the abundance and distribution of the species, ecological indexes such as water pollution condition, eutrophication degree and the like can be revealed. However, the conventional indicator seed screening method relies on a large number of field observations and captures, and is limited by space, time and human resources. To overcome these limitations, the introduction of eDNA technology provides a new means for rapid screening of indicator species. The water body indication species can be rapidly and accurately identified by collecting a water sample, extracting DNA and combining the technologies of indication value analysis (IndVal), molecular ecological network analysis (MENs), linear discriminant analysis (LEfSe) and the like, and large-scale field operation is not needed. The Yangtze river source area is located in the abdomen of Qinghai-Tibet plateau and is an important water source area of Yangtze river basin, the area is about 14.27 ten thousand square kilometers, and the average elevation is 4500-5000 meters. The regional water system mainly comprises Tuo river, tongtian river, chu Maer river, dangqu river and other rivers. The Yangtze river source region belongs to a typical plateau high Liu Xing monsoon climate, the precipitation is rare and concentrated, the annual average precipitation amount is about 362: 362 mm, the annual average air temperature is-4.4 ℃, the warm season is 5-9 months, and the cold season is 10 months to 4 months of the next year. Due to the special geographical environment and