Method for evaluating diversity of marine zooplankton on basis of macro bar code technology

A zooplankton and diversity technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that the identification method is difficult to meet the operational monitoring, the professional skills of the identification personnel are high, and the species diversity is underestimated. , to achieve the effect of eliminating product purification steps, improving sequencing quality, and increasing resolution

Active Publication Date: 2020-05-19
NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zooplankton taxa are characterized by high diversity. The traditional microscopic counting method based on morphology requires high professional skills of the identification personnel, and the results of different identification personnel are not comparable, resulting in the lack of comparability of the results between different years. These defects make it d

Method used

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  • Method for evaluating diversity of marine zooplankton on basis of macro bar code technology
  • Method for evaluating diversity of marine zooplankton on basis of macro bar code technology
  • Method for evaluating diversity of marine zooplankton on basis of macro bar code technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Sample collection

[0060] In August 2017, 22 zooplankton stations ( figure 1 ) for sampling. The shallow water type II plankton net (mesh aperture 0.160mm, net opening diameter 31.6cm, net length 140cm) was used to collect zooplankton samples according to the "Marine Monitoring Specification" (GB 17378.7-2007). The samples collected at each station were thoroughly mixed and divided into two parts: one part was fixed with 5% formaldehyde for morphological identification, and the other part was fixed with absolute ethanol after filtering out seawater with a sieve of the same aperture. molecular identification.

[0061] 2. DNA Extraction of Sample Metagenome

[0062] Draw zooplankton samples three times from each sample preserved in alcohol (total volume 100mL), 10mL each time, combine the zooplankton samples drawn three times, and filter with a 20μm sieve to remove alcohol and residual planktonic algae Wait. DNA blood and tissue extraction kit (Qiagen Canada lnc....

Embodiment 2

[0072] The primers used for PCR amplification contain an 8-base tag sequence, which is a specific sequence. 22 primer pairs (see Table 2) are provided for PCR amplification of 22 samples, and the rest are completely consistent with Example 1.

[0073] Table 2 Primer pairs used in sample PCR amplification

[0074]

[0075]

Embodiment 3

[0077] The PCR reaction system is as follows (25μL): 1x Ex Taq Buffer (20mM Mg 2+ plus, Takara, Dalian, China), 5.0mM dNTP, 0.27mM trimethylamine oxide, 0.42μM 1,6-bis(N1-p-chlorophenyl-N5-biguanide) hexane digluconate, 15pmol containing Index Primers, 100ng of genomic DNA, and PCR products were not purified, and the rest were completely consistent with Example 2.

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Abstract

The invention provides a method for evaluating the diversity of marine zooplankton on the basis of a macro bar code technology, belonging to the field of marine organism diversity research. The methodcomprises the following steps: DNA extraction of a sample metagenome, PCR amplification of a V4 hypervariable region fragment of 18S rDNA to construct a library, high-throughput sequencing, and bioinformatic analysis, wherein a PCR primer used for library construction comprises a specific tag sequence. The method provided by the invention is simple and convenient to operate, can improve the output of sequencing sequences, increases the resolution rate of a sequence with a tag, can improve the amplification efficiency of a template with high GC content, reduces the generation of repetitive sequences, improves the actual expression quantity obtained by sequencing, and increases the accuracy of evaluating the diversity of the marine zooplankton.

Description

technical field [0001] The invention belongs to the field of marine biological diversity research, in particular to a method for evaluating marine zooplankton diversity based on macro barcode technology. Background technique [0002] In order to strengthen marine ecological monitoring and strengthen marine ecological management, my country has organized the establishment of marine ecological monitoring areas and implemented operational monitoring since 2003. At present, 21 ecological monitoring areas have been established in ecologically fragile and sensitive areas of my country's coastal waters. Marine biodiversity is an important part of the operational monitoring of ecological monitoring areas. Its purpose is to grasp the distribution and changing trends of marine species and quantities. The monitoring objects include phytoplankton, zooplankton, benthic organisms and swimming animals. Zooplankton taxa are characterized by high diversity. The traditional microscopic counti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2531/113C12Q2527/125C12Q2535/122C12Q2537/165Y02A40/81
Inventor 王睿睿孙艺邵魁双王孝程林勇李冬梅张金勇李飞李宏俊
Owner NATIONAL MARINE ENVIRONMENTAL MONITORING CENTRE
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