SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application

A paternity test and fluorescent labeling technology, applied in the field of aquatic animal germplasm identification, to reduce time and cost, protect genetic structure, and protect genetic diversity

Active Publication Date: 2015-04-08
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the application of microsatellite molecula

Method used

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  • SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application
  • SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application
  • SSR fluorescence labeling primer for paternity test of Chinese sturgeon and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Screening of microsatellite markers for paternity testing of Chinese sturgeon:

[0049] 1) The DNA of 22 wild Chinese sturgeons in Yichang was extracted by the classic high-salt method, and diluted to 100 ng / μL for later use.

[0050] 2) Synthesize non-fluorescent labeled primers with 165 pairs of microsatellite markers designed by Primer Premier 5.0, use the DNA in step 1) as a template, and amplify under the following PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30s, and finally Anneal at the appropriate annealing temperature for 30 s, extend at 72°C for 1 min, a total of 30 cycles; then extend at 72°C for 10 min, and store at 4°C. 12% polyacrylamide gel electrophoresis was used for preliminary screening and separation, and 24 pairs of microsatellite markers with high polymorphism and stable amplification were obtained.

[0051] 3) Continue to use the DNA extracted in step 1) as a template for the screened Chinese sturgeon...

Embodiment 2

[0055] The application of SSR fluorescent marker primers for paternity identification of Chinese sturgeon in the paternity identification of Chinese sturgeon comprises the following steps:

[0056] 1) According to the routine protocol to extract the DNA of Chinese sturgeon

[0057]Collect 10 caudal fin samples of candidate parents of Chinese sturgeon (including two real parents, numbered CS-1 and CS-2), 28 offspring (both offspring of CS-1 and CS-2), absolute alcohol Save for later. Cut about 0.5g of the preserved fin ray sample, put it into a 2mL centrifuge tube, soak it in TE buffer for 12 hours to fully replace the alcohol, then take out the tissue and chop it into 352μL sterile extraction buffer, gently Mix well, add 40μL 20% SDS and 10μL 20mg / mL proteinase K, mix well, digest at 56°C for more than 4 hours, shake the centrifuge tube once every 30min during the process, until the tissue is completely digested, add 300μL 6M NaCl solution, shake vigorously immediately Centr...

Embodiment 3

[0072] A kit containing SSR fluorescently labeled primers for paternity identification of Chinese sturgeon, including the following components

[0073] Table 5: Chinese sturgeon paternity test kit

[0074]

[0075] Each component in the kit is packaged separately according to the above dosage to form a Chinese sturgeon paternity testing kit.

[0076] In summary, the Chinese sturgeon paternity identification technology and kit established in the present invention can be accurately used for the paternity identification of Chinese sturgeon, and provide technical support for the germplasm identification and family management of Chinese sturgeon. In order to reasonably guide the artificial breeding of Chinese sturgeon and protect the genetic diversity of Chinese sturgeon in the Yangtze River.

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Abstract

The invention discloses an SSR fluorescence labeling primer for the paternity test of Chinese sturgeon and application. 10 pairs of fluorescence labeling microsatellite primers are successfully screened and synthesized and can be used for establishing a Chinese sturgeon paternity test technology. The invention also discloses a kit for the paternity test. Synthesized PCR products with different fluorescence colors are mixed, and then allelic genes are tested to carry out the paternity test analysis of the Chinese sturgeon. A identification method is easy and fast to operate and low in cost, has the characteristics of high efficiency, economy, easiness, convenience for operation and the like, can be popularized and applied to the germplasm identification, family management and fine breed breeding and can be used for evaluating the manual proliferative effect and releasing 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 SSR fluorescent marker primers and applications for paternity identification of Chinese sturgeon. Background technique [0002] Chinese sturgeon (Acipenser sinensis), belonging to the order Acipenseridae, Acipenseridae, and the genus Acipenserus, is a large migratory primitive ancient fish that grows in the ocean and breeds in fresh water. It has survived for 140 million years. This species is mainly distributed in the Yangtze River and Pearl River systems. At present, the Chinese sturgeon in the Pearl River system has basically disappeared. After the construction of Gezhou Dam, the breeding and migration channels of Chinese sturgeon were blocked. In addition to overfishing, water pollution and habitat degradation, the resources of Chinese sturgeon in the Yangtze River also declined seriously. In 2010, the Chines...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895
Inventor 张书环辛苗苗危起伟
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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