Primer for monitoring DNA of fish environment at middle and upper reaches of Yarlung Zangbo River

A fish and upstream technology, applied in the field of molecular ecology, can solve problems such as the inability to monitor the level of fish species in the middle and upper reaches of the Brahmaputra, and achieve the effects of easy operation, accurate and comprehensive detection, and simple steps.

Pending Publication Date: 2022-01-04
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to develop a set of environmental DNA monitoring for fishes in the middle and upper reaches of the Yarlung Zangbo River by using molecular marker technology to solve the problems that the existing environmental DNA universal primers cannot accurately and completely monitor the species level of the fish in the upper and middle reaches of the Yarlung Zangbo River. The primers, namely YZR_Cytb-F and YZR_Cytb-R

Method used

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  • Primer for monitoring DNA of fish environment at middle and upper reaches of Yarlung Zangbo River
  • Primer for monitoring DNA of fish environment at middle and upper reaches of Yarlung Zangbo River
  • Primer for monitoring DNA of fish environment at middle and upper reaches of Yarlung Zangbo River

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Embodiment 1

[0032] Acquisition of primers for environmental DNA monitoring of fish in the middle and upper reaches of the Yarlung Zangbo River:

[0033] The applicant collected the fish list in the middle and upper reaches of the Yarlung Zangbo River through field investigation and literature review, including 15 indigenous species and 14 exotic species (Table 1), belonging to 4 orders and 10 families. Species of different purposes, but also closely related species of the same genus.

[0034] The primers used in environmental DNA technology need to be relatively conservative, so as to ensure effective amplification in all species; and the amplicon sequence needs to be diverse, so as to ensure the identification effect on all species. The mitochondrial 12S, COI and Cytb gene sequences of all fish in the middle and upper reaches of the Yarlung Zangbo River were collected by DNA sequencing and NCBI database. The average K2P genetic distances within species were 0.06%, 0.20% and 0.34%, respec...

Embodiment 2

[0040] Application of primers in monitoring fish species diversity in the middle and upper reaches of the Yarlung Zangbo River:

[0041] (1) 1 L of water was collected in the Lalu Wetland in Lhasa (sample point A) and the Yarlung Zangbo River in Zha Nang County (sample point B), and the DNA was collected on a mixed cellulose filter membrane by a vacuum filter pump, and Store cryopreserved in liquid nitrogen.

[0042] (2) Extract the water sample eDNA using the Powerful Water Sample DNA Extraction Kit, and the water sample DNA concentrations in sample points A and B are 10.5ng / ul and 6.6ng / ul, respectively.

[0043] (3) Using water sample eDNA as a template, edm_YZR_Cytb-F and edm_YZR_Cytb-R as primers, and using NEB Q5 ultra-fidelity DNA polymerase for the first PCR amplification, the reaction system is: 12.5ul Q5 ultra-fidelity 2× Master Mix, each 1.0ul forward and reverse primers, 5.0ul DNA template, 5.5ul ddH 2 O. The reaction parameters were: pre-denaturation at 95°C fo...

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Abstract

The invention relates to a primer for monitoring DNA (Deoxyribose Nucleic Acid) of a fish environment in the middle and upper reaches of the Yarlung Zangbo River and an application method thereof, and belongs to the field of molecular ecology. The primers comprise the following components of YZR-Cytb-F:TGRCTTGAARAACCACCGTTGT and YZR-Cytb-R: AARAAGAAWGADGCKCCRTTDGCRTG. The method comprises the following steps of (1) collecting a water sample and extracting eDNA; (2) slightly transforming the primer and then carrying out primary PCR (Polymerase Chain Reaction) on eDNA (Deoxyribose Nucleic Acid); (3) carrying out secondary PCR on the purified product of the primary PCR by using an Illumina library building primer; (4) performing high-throughput sequencing on the purified product of the second PCR; and (5) analyzing the fish species composition in the water sample according to the sequencing data. The primer has an identification effect on the species level of all fishes in the middle and upper reaches of the Yarlung Zangbo River, and the method can be used for accurately and completely monitoring the species diversity of the fishes in the middle and upper reaches of the Yarlung Zangbo River on the premise of not damaging the environment and fish populations.

Description

technical field [0001] The invention belongs to the field of molecular ecology, and in particular relates to a primer for monitoring the environmental DNA of fish in the middle and upper reaches of the Yarlung Zangbo River and an application thereof. Background technique [0002] Freshwater fish diversity is facing serious threats from climate change and human activities, such as habitat destruction, overfishing and biological invasion. Long-term monitoring of biodiversity can provide basic data for the protection of biodiversity. At present, the monitoring methods of freshwater fish diversity mainly include net capture and electric capture, which are time-consuming, laborious, inefficient, unfriendly to the environment and organisms, and difficult to capture rare species. [0003] Environmental DNA (eDNA) refers to DNA extracted from environmental samples such as water, soil or sediment, which can reveal biodiversity information in the environment. In freshwater ecosystem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/11
CPCC12Q1/6888C12Q1/6869C12Q2600/124C12Q2531/113C12Q2539/10
Inventor 冯秀隋晓云朱仁贾银涛李冰陈毅峰
Owner INST OF AQUATIC LIFE ACAD SINICA
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