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Microsatellite primer applied to Chinese sturgeon and acipenser dabryanus species identification and application thereof

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, unreliability, and easy false detection, and achieve guaranteed purity , The effect of saving the time of identification and the cost of sequencing

Active Publication Date: 2018-09-14
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 primer applied to Chinese sturgeon and acipenser dabryanus species identification and application thereof
  • Microsatellite primer applied to Chinese sturgeon and acipenser dabryanus species identification and application thereof
  • Microsatellite primer applied to Chinese sturgeon and acipenser dabryanus species identification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Screening of SSR markers for interspecies identification of Chinese sturgeon and Dabry's sturgeon:

[0034] 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.

[0035] 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:

[0036] A. PCR reaction system:

[0037] 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

Fluores...

Embodiment 2

[0043] 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:

[0044] 1) Extract the DNA of 20 Chinese sturgeons and 20 Dabry's sturgeons according to the routine scheme;

[0045] 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:

[0046] A. PCR reaction system:

[0047] 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

[0048] B.PCR amplification program:

[0049]

[0050]

[0051] The PCR amplification ...

Embodiment 3

[0058] A kit containing SSR fluorescent primers for interspecies identification of Chinese sturgeon and Dabry's sturgeon, including the following components:

[0059] 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

[0060] Each component in the kit was packaged according to the above-mentioned doses to form the Chinese sturgeon and Dabry's interspecies identification kit.

[0061] 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 stur...

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Abstract

The invention belongs to the field of aquatic animal germplasm identification in the field of aquaculture and discloses a microsatellite primer applied to Chinese sturgeon and acipenser dabryanus species identification and application thereof. According to the microsatellite primer disclosed by the invention, a pair of fluorescent labeled microsatellite primers are successfully screened and synthesized, and the primers can be applied to establishing a Chinese sturgeon and acipenser dabryanus species identification technology; after the primers are applied to PCR amplification, allelic genes are measured to perform Chinese sturgeon and acipenser dabryanus species identification and analysis. The identification method disclosed by the invention has the characteristics of simpleness and convenience in operation, low cost, high efficiency, economical performance, convenience and practicability; the identification method can be popularized and applied to Chinese sturgeon and acipenser dabryanus species identification, germplasm identification and genetic management; furthermore, the identification method can evaluate an artificial fecundation drift effect.

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. technical background [0002] 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 some 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, ex situ prot...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 张书环杜浩危起伟邸军黄君肖新平周琼
Owner CHINA THREE GORGES CORPORATION
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