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Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof

A Nile tilapia and molecular marker technology, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of amplification band change, sensitive reaction conditions, poor stability and repeatability, etc., and achieve The effect of reducing detection time, reducing detection cost and stabilizing reaction

Inactive Publication Date: 2011-10-26
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, isozymes are gene expression products, but the detection sites are limited, and it is not easy to distinguish varieties with close relationship or complex genetic basis; although the RAPD method is to identify different varieties at the gene level, the RAPD method is not sensitive to changes in reaction conditions. Very sensitive, a slight difference in many reaction factors will lead to changes in the amplified band pattern, poor stability and repeatability, thus limiting its application
Compared with isozyme, RAPD, RFLP and other technologies, microsatellite markers, also known as simple sequence repeat markers (SSR), have the advantages of high polymorphism frequency, easy detection, good repeatability, and co-dominant Mendelian inheritance. It is widely used in species and paternity identification, but there is no report on the rapid and accurate identification of different tilapia species by using microsatellite markers for multiplex PCR

Method used

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  • Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof
  • Molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof

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Embodiment 1

[0038] A kind of molecular marker method for distinguishing Nile tilapia, Olia tilapia and hybrid fish thereof of the present invention adopts the following technical steps:

[0039] Thirteen Nile tilapia were collected from Yixing Fishery, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences. 0.5 mL of blood was collected from the tail vein of each fish, and 30 μL of blood cells were taken to extract genomic DNA. Using the genomic DNA of Nile tilapia as a template, three primer pairs of primers GM017, UNH948 and GM440 were used for multiplex PCR amplification. The total volume of the PCR reaction was 15 μL, which contained 1.5 μL of 10× reaction buffer, MgC1 2　 2 mmol / L, dNTP 200 μmol / L, 3 pairs of upstream and downstream primers each 0.2 μmol / L, Taq Enzyme 0.4U, DNA 80 ng, make up volume with sterilized double distilled water. The PCR reaction conditions were: 94°C for 3 min; 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, 25 cycles; 72°C for 8 min. Aft...

Embodiment 2

[0045] A kind of molecular marker method for distinguishing Nile tilapia, Olia tilapia and hybrid fish thereof of the present invention adopts the following technical steps:

[0046] 8 Aoni hybrid tilapia (Nile tilapia ♀ × Olia tilapia ♂) were collected from Yixing Fishery, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences. 0.1 g of caudal fin rays were taken from each fish, and genomic DNA was extracted. Using the genomic DNA of Oni hybrid tilapia as a template, multiplex PCR amplification was performed with 3 primer pairs of primers GM017, UNH948 and GM440. The total volume of the PCR reaction was 15 μL, which contained 1.5 μL of 10× reaction buffer, MgC1 2　 2 mmol / L, dNTP 200 μmol / L, 3 pairs of upstream and downstream primers each 0.2 μmol / L, Taq Enzyme 0.4U, DNA 50 ng, make up volume with sterilized double distilled water. The PCR reaction conditions were: 94°C for 3 min; 30 cycles of 94°C for 30 s, annealing at 52°C for 30 s, and 72°C for 30 s; ...

Embodiment 3

[0052] A kind of molecular marker method for distinguishing Nile tilapia, Olia tilapia and hybrid fish thereof of the present invention adopts the following technical steps:

[0053] 12 Olia tilapia and 7 Neo hybrid tilapia (Nile tilapia♂×Olia tilapia♀) were collected from Yixing Fishery, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences. Genomic DNA was extracted from 30 μL of blood cells from each fish. Using these genomic DNAs as templates, multiplex PCR amplification was performed with 3 primer pairs of primers GM017, UNH948 and GM440. The total volume of the PCR reaction was 15 μL, which contained 1.5 μL of 10× reaction buffer, MgCl 2　 2 mmol / L, dNTP 200 μmol / L, 3 pairs of upstream and downstream primers each 0.2 μmol / L, Taq Enzyme 0.4U, DNA 70 ng, make up volume with sterilized double distilled water. The PCR reaction conditions were: 94°C for 3 min; 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, 28 cycles; 72°C for 8 min. After the PCR reaction...

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Abstract

The invention relates to a molecule marking method for identifying tilapia nilotica, oreochromis aureus and hybridized fish thereof. The method is characterized by comprising the following steps: selecting three pairs of specific microsatellite marked primer combinations; sampling blood or fins from tilapia to be detected; extracting deoxyribonucleic acid (DNA) of a gene group; performing multiple polymerase chain reaction (PCR) amplifications on the DNA of the gene group of the tilapia to be detected; performing electrophoretic separation on the PCR product with a 8.0 percent non-denatured polyacrylamide gel; dying by argentation; and photographing to record the electrophoretic results. According to the results of multiple PCR electrophoretic detection, the tilapia nilotica, the oreochromis aureus and the hybridized fish thereof can be identified. By the method, the tilapia nilotica, the oreochromis aureus and the hybridized fish thereof can be quickly and effectively identified.

Description

technical field [0001] The invention belongs to the technical field of molecular markers, and relates to a molecular marker method for identifying Nile tilapia, Olia tilapia and their hybrid fishes, and is for identifying different species of tilapias. Background technique [0002] Tilapia (Tilapia) originates in Africa and is a worldwide cultured species. The fish was introduced into my country in the 1950s and has developed rapidly. Now China is the largest tilapia producer and exporter in the world. At present, the main species of tilapia cultured in my country are Nile tilapia ( Oreochromis niloticus ), Olia tilapia ( Oreochromis aureus ) and Oni hybrid tilapia, in which the Oni tilapia is the first generation of hybridization of Nile tilapia (♀) and Olia tilapia (♂), with high male rate, fast growth, and disease resistance Strong and other advantages, but its male rate and breeding performance are closely related to the germplasm purity of the parents. Because tilap...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李建林俞菊华唐永凯李红霞徐跑
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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