Search

CN-122016394-A - Method for monitoring estuary environment biodiversity based on eDNA

CN122016394ACN 122016394 ACN122016394 ACN 122016394ACN-122016394-A

Abstract

The invention belongs to the technical field of estuary environment biodiversity monitoring, and particularly relates to a method for monitoring estuary environment biodiversity based on eDNA. The invention forms a set of eDNA monitoring method specially aiming at complex environment of river mouth by innovatively integrating three core strategies of gradient partition point distribution, sediment leading collaborative sampling and multi-method parallel verification. The implementation result proves that the method can systematically, sensitively and noninvasively reveal the spatial pattern of the estuary biological community, and effectively solve the key technical problems of large interference, low efficiency and insufficient pertinence and poor representativeness of the general eDNA technology to benthos in the traditional method.

Inventors

  • XIE GANG
  • LI JIE
  • YU XIAOXIA
  • WANG YU
  • ZHANG NING
  • LI XINLEI
  • LI DAN
  • WANG WANTING
  • WANG GUOQIANG

Assignees

  • 山东省生态环境规划研究院
  • 山东大学

Dates

Publication Date
20260512
Application Date
20260121

Claims (10)

  1. 1. A method for monitoring the biological diversity of a estuary environment based on eDNA, which is characterized by comprising the following steps: (1) The monitoring point position layout comprises the steps of selecting a main river channel region 4 km to 30 km upstream of a river inflow sea opening as a core sampling range, and uniformly distributing sampling points along the main river channel direction at intervals of 5-5.5 km; (2) The method comprises the steps of collecting a water body sample and a sediment sample in a tide period at each sampling point synchronously, wherein the water body sample is collected by a net bag method, a membrane filtering method and a tea bag method respectively; (3) The method comprises the steps of (1) performing pretreatment on samples obtained by different methods in the step (2), extracting the eDNA of the samples, checking the concentration and quality of the extracted genome eDNA, constructing a gene library, sequencing by using an Illumina HiSeq high-throughput sequencing platform, and finally performing biological information analysis on gene data to obtain species classification and diversity information of plankton, benthonic animals and fish communities in a estuary environment.
  2. 2. The method of claim 1, wherein in the step (2), collecting sediment samples comprises collecting sediment on the surface layer of 0-5 cm of river bottom by using a sterile centrifuge tube, and freezing and preserving at-20 ℃ in situ.
  3. 3. The method according to claim 1, wherein in the step (2), the net bag method is used for collecting the water body sample, wherein the water body sample is filtered by a plankton net in the middle of a flood tide, the filtered water sample is collected by a sterile self-sealing bag, and the concentrated plankton sample is obtained and stored at-80 ℃.
  4. 4. A method according to claim 3, wherein the plankton net has a pore size of 60 to 65 μm.
  5. 5. The method according to claim 1, wherein in the step (2), the filter membrane method is used for collecting the water sample, wherein the water sample is filtered by a filter membrane in the middle of the flood tide, eDNA in the water sample is intercepted to the filter membrane, and the filter membrane is collected and stored at-80 ℃.
  6. 6. The method of claim 5, wherein the filter has a pore size of 0.4 to 0.5 μm.
  7. 7. The method according to claim 1, wherein in the step (2), the water body sample is collected by a tea bag method, wherein a filter membrane is placed in a tea bag, 3-4 magnetic particles are placed in the tea bag to enable the tea bag to be submerged in water, the tea bag is fixed, a plurality of tea bags are distributed at the same sampling site to cover a complete tide period, the filter membrane is recovered at different time intervals, the filter membrane is stored at-80 ℃, and preferably, the different time intervals are 4h, 24 h and 48 h respectively.
  8. 8. The method according to claim 1, wherein in step (3), the samples obtained by the different methods are used to extract DNA and construct a gene library independently.
  9. 9. The method of claim 1, wherein in step (3), the sequenced primer sequence is selected from any one of 18S rDNA, chloroplast (RBCL), 16S rDNA, CO1, and 12S rDNA.
  10. 10. The method of claim 1, wherein in step (3), the bioinformatics analysis comprises sequence quality control, library splitting, sequence clustering, filtering, and annotation.

Description

Method for monitoring estuary environment biodiversity based on eDNA Technical Field The invention belongs to the technical field of estuary environment biodiversity monitoring, and particularly relates to a method for monitoring estuary environment biodiversity based on eDNA. Background Environmental DNA (eDNA) refers to DNA left in the environment by organisms, possibly derived from shed tissues, mucus, secretions, excretions, etc., which have little interference to the biological community and high species detectability sensitivity, and are effective tools for monitoring the diversity of aquatic organisms. The estuary environment has obvious space-time dynamic property, unidirectional water flow and tide action in the estuary can convey the environment DNA from the source, so that the diffusion range of the estuary environment is enlarged, and compared with most fresh water and ocean environments, the turbidity of water bodies in the estuary area is higher and more frequent to change, and suspended particles possibly contain compounds for inhibiting PCR reaction. In the prior art, monitoring of the benthos diversity of estuaries mainly faces double dilemmas. Firstly, the traditional morphological investigation method has inherent defects, and the method has strong dependence on professionals, high cost and long period, samples to interfere with biological communities, and instantaneous sampling is easy to miss low-abundance species, so that the ecological reality condition cannot be comprehensively and dynamically reflected. Secondly, the effect of directly applying the conventional environmental DNA (eDNA) technology to the benthonic monitoring of the estuary is poor. The filter membrane is easily blocked by a large amount of particles in a turbid water environment, so that the water sample is small in filter volume and difficult in eDNA enrichment, and the problem is particularly prominent in estuaries (such as Huang Hekou) with high sand content. At present, a standardized, integrated and intelligent eDNA monitoring method for a complex ecosystem of a estuary is lacking. Disclosure of Invention In view of the shortcomings of the prior art, the invention aims to provide a method for monitoring the biological diversity of estuary environments based on eDNA. In order to achieve the above purpose, the present invention adopts the following technical scheme: the invention provides a method for monitoring the biological diversity of estuary environment based on eDNA, which comprises the following steps: (1) The monitoring point position layout comprises the steps of selecting a main river channel region 4 km to 30 km upstream of a river inflow sea opening as a core sampling range, and uniformly distributing sampling points along the main river channel direction at intervals of 5-5.5 km; (2) The method comprises the steps of collecting a water body sample and a sediment sample in a tide period at each sampling point synchronously, wherein the water body sample is collected by a net bag method, a membrane filtering method and a tea bag method respectively; (3) Sequencing and analyzing, namely preprocessing the samples obtained by different methods in the step (2), extracting eDNA of the samples, checking the concentration and quality of the extracted genome eDNA, constructing a gene library, sequencing by using an Illumina HiSeq high-throughput sequencing platform, and finally analyzing biological information of the gene data to obtain species classification and diversity information of plankton, benthonic animals and fish communities in the estuary environment. In the invention, a main river channel region 4 km to 30 km upstream of a river inflow sea opening is selected as a core sampling range. The range covers a downstream transition zone from the upstream river reach with the dominant fresh water to the intersection of the salty and fresh water, and completely captures the environmental gradient change from the fresh water to the salty and fresh water. Aiming at the specific environmental problems of different estuaries, the invention utilizes the complementarity of a plankton net method, a tea bag method and a filter membrane method in a sampling mechanism and a time scale to jointly construct a three-dimensional and high-resolution sampling system which can capture an instantaneous state and integrate a periodic signal. When the Membrane filtration method (Membrane) accurately records the occurrence of the target tidal phase, the composition and concentration of biological DNA in the water body reflect the biological community state at the specific moment, and the acquisition and on-site filtration of a large volume of water sample are completed within the accurate time window of the target phase calculated according to the tidal table, which is a key step for acquiring the core phase characteristic data. The plankton Net method (Net) and the filter membrane method are synchronous, which is to carry out