Microsatellite primers and their application for interspecies identification of Chinese sturgeon and Dabry's sturgeon

A technology of Chinese sturgeon and Dabry's sturgeon, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as cumbersome operation, easy false detection, unreliability, etc., and achieve guaranteed purity , easy to judge, save the time of identification and the cost of sequencing

Active Publication Date: 2021-06-01
CHINA THREE GORGES CORPORATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, testing this result is unreliable
The patent applicant discovered 10 microsatellite (simple sequence repeats, SSRs) site combinations in the previous research, which can be used for paternity identification of Chinese sturgeon and interspecies identification of Chinese sturgeon and Dabry's sturgeon. However, This combination has the defects of many primers, cumbersome operation and easy false detection in the actual operation process
Therefore, up to now, there is no more convenient and effective method to quickly identify the two species of sturgeon

Method used

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  • Microsatellite primers and their application for interspecies identification of Chinese sturgeon and Dabry's sturgeon
  • Microsatellite primers and their application for interspecies identification of Chinese sturgeon and Dabry's sturgeon
  • Microsatellite primers and their application for interspecies identification of Chinese sturgeon and Dabry's sturgeon

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Screening of SSR markers for interspecies identification of Chinese sturgeon and Dabry's sturgeon:

[0035] 1) The classic high-salt method was used to extract DNA samples from 6 Chinese sturgeon and 6 Dabry's sturgeon, diluted to 100ng / ul, and set aside.

[0036] 2) 332 pairs of microsatellite primers will be designed by primer premier 5.0 software, wherein the forward primer (F primer) of each pair of primers has a universal M13 linker sequence (TGTAAAACGACGGCCAGT) added. In addition, M13 fluorescent linker primers with TAMRA fluorophore were synthesized. After the primer is synthesized, use the DNA in step 1) as a template, and use the three primer method to carry out PCR amplification, as follows:

[0037] A. PCR reaction system:

[0038] reagent Volume (μl) 2×Taq PCR Master Mix 5 Template (genomic DNA) 1 Upstream primer (concentration 10μmol / μl) 0.1 Downstream primer (concentration 10μmol / μl) 0.4 Fluorescent M13 primer (co...

Embodiment 2

[0044] The application of the SSR fluorescently labeled primers for the interspecific identification of Chinese sturgeon and Dabry's sturgeon in the interspecific identification of Chinese sturgeon and Dabry's sturgeon includes the following steps:

[0045] 1) Extract the DNA of 20 Chinese sturgeons and 20 Dabry's sturgeons according to the routine scheme;

[0046] 2) After the primer is synthesized, use the DNA in step 1) as a template, and use the three-primer method to carry out PCR amplification, as follows:

[0047] A. PCR reaction system:

[0048] reagent Volume (μl) 2×Taq PCR Master Mix 5 Template (genomic DNA) 1 Upstream primer (concentration 10μmol / μl) 0.1 Downstream primer (concentration 10μmol / μl) 0.4 Fluorescent M13 primer (concentration 10μmol / μl) 0.4 ddH 2 O

3.1 total capacity 10

[0049] B.PCR amplification procedure:

[0050]

[0051]

[0052] The PCR amplification product was subjected to ag...

Embodiment 3

[0059] A kit containing SSR fluorescent primers for interspecies identification of Chinese sturgeon and Dabry's sturgeon, including the following components:

[0060] reagent Volume (μl) 2×Taq PCR Master Mix 5 Template (genomic DNA) 1 Upstream primer (concentration 10μmol / μl) 0.1 Downstream primer (concentration 10μmol / μl) 0.4 Fluorescent M13 primer (concentration 10μmol / μl) 0.4 wxya 2 o

3.1 total capacity 10

[0061] Each component in the kit was packaged according to the above-mentioned doses to form the Chinese sturgeon and Dabry's interspecies identification kit.

[0062] In summary, the Chinese sturgeon and Dabry's sturgeon interspecific identification technology and test kit established by the present invention can be accurately used for the interspecific identification of Chinese sturgeon and Dabry's sturgeon which are the closest relatives, and are Chinese sturgeon and Dabry's sturgeon. Germplasm identificat...

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Abstract

The invention belongs to the field of germplasm identification of aquatic animals in the field of aquaculture, and discloses microsatellite primers and applications for interspecific identification of Chinese sturgeon and Dabry's sturgeon. The invention successfully screens and synthesizes a pair of fluorescently labeled microsatellite primers. The primers can be used to establish the interspecies identification technology of Chinese sturgeon and Dabry's sturgeon. After the primers were used for PCR amplification, the alleles were determined, and the interspecies identification analysis of Chinese sturgeon and Dabry's sturgeon was carried out. The identification method described in the present invention is simple, quick, and low in cost, and has the characteristics of high efficiency, economy, and simplicity, and can be popularized and applied in interspecific identification, germplasm identification and genetic management of Chinese sturgeon and Dabry's sturgeon, and can be applied to The effect of artificial proliferation and release was evaluated.

Description

technical field [0001] The invention belongs to the field of germplasm identification of aquatic animals in the field of aquaculture, and in particular relates to microsatellite primers and applications for interspecific identification of Chinese sturgeon and Dabry's sturgeon. [0002] technical background [0003] Chinese sturgeon (Acipensersinensis) and Dabryanus sturgeon (A.dabryanus) are the first-class key protected aquatic wild animals in my country. They belong to the Acipenseridae, Acipenseridae, and Acipenser genus. They are large fishes distributed in the Yangtze River Basin. The ecological niches of the two fish species overlap to a certain extent. Due to the interference of human activities, such as the construction of cascade hydropower stations, overfishing, habitat destruction, etc., the two species of fish are in danger of extinction. At present, a series of protection measures have been taken for these two kinds of fish, such as establishing nature reserves,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 张书环李翀杜浩危起伟邸军黄君肖新平周琼吴兴华李媛
Owner CHINA THREE GORGES CORPORATION
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