Salt marsh wetland benthic organism environment DNA monitoring method giving consideration to biological invasion early warning

Abstract

The invention belongs to the field of ecological environment evaluation and treatment, and particularly relates to a salt marsh wetland benthic organism environment DNA monitoring method giving consideration to biological invasion early warning. Comprising the following steps: 1) collecting a sample; 2) indoor analysis; 3) environment DNA extraction, PCR amplification and sequencing; and 4) data analysis. Wherein in the step (3), sediment DNA is extracted by adopting a CTAB (cetyltrimethyl ammonium bromide) method, then the purity and the concentration of the DNA are detected by utilizing agarose gel electrophoresis, a purified environment DNA sample is subjected to PCR (Polymerase Chain Reaction) amplification by using a Barcode specific primer pair environment DNA template, and the sequences of the four primers are shown as SEQ ID NO.1-8. According to the invention, salt marsh wetland benthos species lists detected by the four primers are compared, and in combination with synchronously collected morphological identification results, the method can be applied to salt marsh wetland benthos diversity monitoring and biological invasion molecule detection in various coastal provinces and cities.

Description

Technical Field

[0001] The invention belongs to the field of ecological environment evaluation and management, and specifically relates to a salt marsh wetland benthic organism environmental DNA monitoring method taking into account biological invasion early warning. Background Art

[0002] Biological invasion refers to the phenomenon that organisms invade another new environment from their original habitat through natural or artificial means, causing adverse effects on the ecosystem of the invaded area. The impact of alien species invasion on ecosystems is multifaceted, such as oppressing the living space of native species, changing the nutritional structure, changing the genetic structure, etc., which ultimately leads to fundamental changes in natural communities and habitat loss. Habitat loss, destruction, fragmentation and habitat degradation will directly threaten the biodiversity of the original ecosystem. Protecting key habitats is the most critical means to protect regional biodiversity. Salt marsh wetlands generally refer to areas with intertidal vegetation coverage ≥30%. Because they are located in the land-sea transition zone, salt marsh wetlands are not only an important part of the "blue carbon sink" while exporting organic matter, but also play an important role in maintaining and promoting regional biogeochemical balance, biodiversity and ecosystem balance. Compared with other ecosystems, they are more susceptible to human disturbance and biological invasion.

[0003] It is costly and difficult to achieve the desired results if invasive species are controlled after they have established populations in the invaded areas. Therefore, early monitoring and detection of invasive species is conducive to making a quick and effective response and formulating corresponding prevention and control decisions, so as to promptly curb the spread of invasive species.

[0004] Environmental DNA metabarcoding (eDNA Metabarcoding) technology is a new method for detecting biodiversity internationally. Its technology originates from DNA barcoding technology and has great application potential in biological monitoring. The DNA material containing species diversity information released by organisms in the surrounding environment when they are active in the environment is called environmental DNA. Environmental DNA metabarcoding technology uses different primers to perform polymerase chain reaction (PCR) amplification on environmental DNA samples, combined with second- and third-generation high-throughput sequencing technology, to generate millions of sequences reflecting species diversity. By analyzing the sequences, diversity information such as species composition and community structure in the environment can be identified. Compared with traditional monitoring methods, environmental DNA metabarcoding technology can identify subtle changes in biological communities in ecosystems more quickly, more accurately, and more economically.

[0005] At present, biological invasion monitoring and detection mainly adopts morphological identification or molecular markers. Morphological identification requires high experience and professional ability of personnel, poor recognition of young invasive species, and high requirements for sample representativeness and quality. Molecular markers can make up for the shortcomings of morphological identification, but commonly used molecular techniques interfere with or harm the environment and biological samples, and it is difficult to monitor hidden invasive species. Summary of the invention

[0006] In order to solve the above technical problems, the present invention uses environmental DNA-based benthic biodiversity monitoring in salt marsh wetlands, uses biological environmental DNA macrobarcodes combined with a list of alien invasive species, and warns of alien invasive species while investigating biodiversity. Simultaneously, morphological methods are combined to compare and screen primers for benthic organisms in salt marsh wetlands, and primer combinations with high species detection efficiency are obtained, providing a feasible and effective method for monitoring benthic organisms in salt marsh wetlands using environmental DNA technology that takes into account early warning of biological invasion.

[0007] The first object of the present invention is to provide a method for monitoring benthic environmental DNA in salt marsh wetlands with early warning of biological invasion, including four primer sequences and reaction conditions thereof, as shown in the following table:

[0008]

[0009] The second object of the present invention is to provide a salt marsh benthic environmental DNA monitoring method taking into account early warning of biological invasion, comprising the following steps:

[0010] 5) Sample collection;

[0011] 6) Indoor analysis;

[0012] 7) Environmental DNA extraction, PCR amplification and sequencing;

[0013] 8) Data analysis.

[0014] In step 3), the CTAB method is used to extract sediment DNA, and then the purity and concentration of the DNA are detected by agarose gel electrophoresis. The purified environmental DNA sample uses a barcode-specific primer to perform PCR amplification on the environmental DNA template. The sequences of the four primers are shown in the table below:

[0015]

[0016] Furthermore, the step 4) is specifically as follows: splitting according to the barcode to obtain the raw data of each sample, removing the barcode and primers, splicing the sequence data by FLASH software, and then using fastp software to perform quality control on the spliced ​​Raw Tags to obtain Clean Tags, and then filtering the chimeras to obtain effective data (Effective Tags) that can be used for subsequent analysis. Primers ①②: Use the DADA2 module in the QIIME2 software to perform sequence quality control and noise reduction on the effective data, and filter out sequences with an abundance of less than 5, so as to obtain the final ASVs, and use the pre-trained Naive in the classify-sklearn algorithm of QIIME2 The Bayes classifier was used to compare the nucleic acid sequence database in NCBI. Primers ③④: The Uparse algorithm was used to cluster all valid data of all samples, and the sequences were clustered into OTUs with 97% consistency. Representative sequences of OTUs were selected for species annotation. The sequencing results of the four primers ASV / OTU sequences were annotated with species using the NT database BLAST best value. The biological functional group of benthic was defined as benthic organisms through the WoRMS official website.

[0017] Furthermore, step 4) also includes comparing the salt marsh benthic species annotation results with the "China's Alien Invasive Species" and the "List of Alien Invasive Species in China's Natural Ecosystems" jointly issued by the Ministry of Environmental Protection and the Academy of Sciences.

[0018] The beneficial effects of the present invention are: 1) the environmental DNA macrobarcoding technology is applied to the investigation of benthic organisms in salt marsh wetlands and early warning monitoring of biological invasion, and the environmental DNA primer combination, PCR reaction conditions, screening of benthic organisms in sequence species, and biological invasion determination technology suitable for monitoring benthic organisms in salt marsh wetlands are clarified; 2) the present invention compares the list of benthic organisms in salt marsh wetlands detected by four primers, and combines the morphological identification results collected simultaneously to explore the consistency and specificity of environmental DNA technology and traditional methods of monitoring at the phylum and genus levels, and screens out an environmental DNA primer combination suitable for the investigation of benthic biodiversity in salt marsh wetlands, which can be used by coastal provinces and cities for monitoring benthic biodiversity in salt marsh wetlands and molecular detection of biological invasion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the ecological survey map of Dingzi Bay

[0020] A, B, and E in the figure are sections of salt marsh wetlands;

[0021] Figure 2 The composition diagram of benthic organisms detected by environmental DNA primers ①;

[0022] Figure 3The composition diagram of benthic organisms detected by environmental DNA primers ②;

[0023] Figure 4 The composition map of benthic organisms detected by environmental DNA primers ③;

[0024] Figure 5 The composition diagram of benthic organisms detected by environmental DNA primer ④;

[0025] Figure 6 This is a morphological classification diagram of the benthic phyla. DETAILED DESCRIPTION

[0026] Example 1 Sample Collection

[0027] like Figure 1 As shown in the figure, an ecological survey of Dingziwan salt marsh wetland was carried out. A total of 15 stations in 3 sections (A, B, and E) were set up to collect environmental DNA sediment samples and morphological classification of large benthic animals, and record the types of salt marsh vegetation. When collecting morphological classification samples, 3 sample plots were randomly selected at each sampling station to quantitatively collect large benthic animals. The specification of the tidal flat quantitative sampler was 25cm×25cm, and the sampling depth was 30cm. The collected mud samples were sorted on site with a 0.5mm aperture sampler. All the samples left on the sieve were collected and bottled, and fixed and preserved with 5% formaldehyde solution. A sterilized plastic spoon was used on site to take environmental DNA sediment samples from the surface layer of 0 to 3cm. Impurities (shells, gravel) were removed and about 20g of samples were placed in 50mL sterile enzyme-free cryopreservation tubes. Three parallel samples were taken at each station and stored in a -20℃ vehicle refrigerator and transported to the laboratory. The sediment samples were homogenized in the laboratory and transferred to a -80℃ ultra-low temperature refrigerator for storage until environmental DNA extraction.

[0028] Example 2 Indoor Analysis

[0029] Morphological classification samples were classified, identified and counted under a stereo microscope, and weighed using an electronic balance with a sensitivity of 0.001g. The standard species names were derived from the WoRMS official website. All sample collection, processing, storage and testing were carried out in accordance with the Marine Monitoring Specifications.

[0030] Example 3 Environmental DNA extraction, PCR amplification and sequencing

[0031] Sediment DNA was extracted using the CTAB method, and then the purity and concentration of the DNA were detected by agarose gel electrophoresis. The purified environmental DNA samples were amplified by PCR using barcode-specific primers. The sequences of the four primers are shown in the table below:

[0032]

[0033]

[0034] The reaction system was 30 μL, including Phusion Master Mix 15 μL, PrimerF 0.2 μL (1 μM), PrimerR 0.2 μL (1 μM), gDNA 10 μL (about 10 ng), and ddH2O to make up to 30 μL system. Each PCR reaction used ddH2O. 2 O was used as a template for PCR negative control. Each sample was amplified three times, and the PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The qualified PCR products were purified by magnetic beads, quantified by enzyme labeling, and mixed in equal amounts according to the concentration of the PCR products. After sufficient mixing, the PCR products were detected by 2% agarose gel electrophoresis, and the target bands were recovered using the gel recovery kit provided by Qiagen. The library was constructed using the Truseq DNA PCR-Free Library Preparation Kit of Illumina. The constructed library was quantified by Qubit and detected by library detection. The PCR products of each sample were separately constructed. After the library was qualified, the PE250 sequencing was performed on the Illumina NovaSeq6000 sequencing platform.

[0035] Example 4 Data Analysis

[0036] The raw data of each sample was obtained by splitting according to the barcode. After removing the barcode and primers, the sequence data was spliced ​​by FLASH software. Then, the spliced ​​Raw Tags were quality controlled by fastp software to obtain Clean Tags, and then the chimeras were filtered to obtain effective data (Effective Tags) that can be used for subsequent analysis. Primer ①②: The DADA2 module in the QIIME2 software was used to perform sequence quality control and noise reduction on the effective data, and sequences with an abundance of less than 5 were filtered out to obtain the final ASVs. The pre-trained Naive Bayes classifier in the classify-sklearn algorithm of QIIME2 was used to align the nucleic acid sequence database in NCBI. Primer ③④: The Uparse algorithm was used to cluster all the valid data of all samples, and the sequences were clustered into OTUs with a consistency of 97%. At the same time, the representative sequences of OTUs were selected for species annotation. After the sequencing results of the four primers ASV / OTU sequences were annotated by species using the best BLAST value of the NT database, the biological functional group was defined as benthic organisms through the WoRMS official website.

[0037] The annotation results of benthic species in salt marsh wetlands were compared with the "List of Alien Invasive Species in China" and the "List of Alien Invasive Species in China's Natural Ecosystems" jointly issued by the Ministry of Environmental Protection and the Chinese Academy of Sciences. The abundance of detected alien invasive species sequences and sample collection areas were analyzed and assessed, their hazard risks were assessed, early warnings and forecasts were issued in a timely manner, response measures were proposed, and prevention and control work was guided.

[0038] In high-throughput sequencing, the number of ASV sequences amplified by primers ① and ② was the largest, 14981 and 18537, respectively, while the number of OTU sequences amplified by primers ③ and ④ was relatively small, 5863 and 8151, respectively. Figure 2 As shown in Figure 1, environmental DNA primer ① detected 232 genera of benthic organisms from 41 phyla, such as Figure 3 As shown in Figure 2, primer ② detected 39 phyla and 177 genera. Figure 4 As shown in Figure 3, primer ③ detected 100 genera from 23 phyla. Figure 5 As shown, primer ④ detected benthic organisms belonging to 27 phyla and 159 genera.

[0039] Morphological classification revealed 49 species of large benthic animals in salt marsh wetlands, belonging to 46 genera in five phyla, namely, annelids, molluscs, brachiopods, arthropods and chordates. Figure 6 As shown in the table, a total of 523 genera belonging to 49 phyla were detected by the primers of environmental DNA, of which 18 phyla were detected in the sequences obtained by the four primers, including amoeba, annelid, arthropoda, ascomycetes, basidiomycetes, chordates, chytrids, cnidarians, cryptophytes, gastrochaetes, anisoflagellates, molluscs, nematodes, brown algae, platyhelminthes, porifera, red algae and rotifers, as shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043] 15 morphological classification genera were detected in environmental DNA samples, and primers ③ and ④ had a high coverage of morphologically identified species. Comparative analysis of four environmental DNA primers showed that primers ① and ② of 18S rDNA detected 102 common genera, primers ③ and ④ of COⅠ detected 59 common genera, and 20 common genera were detected in the two amplification regions. It can be seen that environmental DNA technology can detect more abundant species and species numbers compared with traditional morphological methods, but due to the large number of marine benthic organisms, the amplification preferences of each phylum for single primers vary greatly. The 18S sequence has a better amplification effect on fungi, algae, annelids, mollusks and bryozoans in salt marshes, while the COⅠ gene has a higher ability to identify arthropods and echinoderms.

[0044] Since there is significant complementarity between the two molecular markers, a single marker can capture up to about 43% of the species in environmental DNA and morphological samples (primer ①). Using at least two pairs of primers at the same time is a method to reduce labeling bias and improve the efficiency of species diversity detection in salt marsh wetland environmental samples. This protocol recommends the combination of 18SrDNA primer ① and COⅠ primer ④ as the environmental DNA macrobarcoding primers for benthic organisms in salt marsh wetlands. The two primers detected a total of 383 genera of benthic organisms from 44 phyla, basically covering 90% of the phyla and 74% of the genera in the sediments.

[0045] The species obtained from environmental DNA and morphological tests of salt marsh wetlands were compared with the "List of Alien Invasive Species in China" and the "List of Alien Invasive Species in China's Natural Ecosystems". No alien invasive species were found, indicating that no biological invasion has occurred in the survey area. The monitoring results can provide baseline biological data for early monitoring and detection of invasive species in the region in the future.

Claims

1. A method for monitoring benthic environmental DNA in salt marsh wetlands with early warning of biological invasion, characterized by: Including four primer sequences and their reaction conditions, as shown in the following table:

2. A method for monitoring benthic environmental DNA in salt marsh wetlands with early warning of biological invasion, characterized in that: The following steps are involved: 1) Sample collection; 2) Indoor analysis; 3) Environmental DNA extraction, PCR amplification and sequencing; 4) Data analysis; In step 3), the CTAB method is used to extract sediment DNA, and then the purity and concentration of the DNA are detected by agarose gel electrophoresis. The purified environmental DNA sample is PCR amplified using barcode-specific primers on the environmental DNA template, and the four primer sequences and reaction conditions are as shown in the table of claim 1.

3. The method for monitoring benthic organisms in salt marsh wetlands based on environmental DNA and taking into account early warning of biological invasion as claimed in claim 2, characterized in that: The step 4) is specifically as follows: splitting according to the barcode to obtain the raw data of each sample, removing the barcode and primers, and splicing the sequence data through FLASH software, then using fastp software to perform quality control on the spliced ​​RawTags to obtain Clean Tags, and then filtering the chimeras to obtain effective data (Effective Tags) that can be used for subsequent analysis, primers ①②: using the DADA2 module in the QIIME2 software to perform sequence quality control and noise reduction on the effective data, and filtering out sequences with an abundance of less than 5, so as to obtain the final ASVs, using the pre-trained Naive Bayes classifier in the classify-sklearn algorithm of QIIME2 to align the nucleic acid sequence database in NCBI, primers ③④: using the Uparse algorithm to cluster all effective data of all samples, clustering the sequences into OTUs with a consistency of 97%, and selecting representative sequences of OTUs for species annotation, and annotating the sequencing results of the four primers ASV / OTU sequences by the NT database BLAST best value, and querying the biological functional group of benthic as benthic organisms through the WoRMS official website.

4. A method for monitoring benthic organisms in salt marsh wetlands based on environmental DNA and taking into account early warning of biological invasion as claimed in claim 3, characterized in that: Step 4) also includes comparing the salt marsh benthic species annotation results with the "China's Alien Invasive Species" and the "List of Alien Invasive Species in China's Natural Ecosystems" jointly released by the Ministry of Environmental Protection and the Academy of Sciences.

Images