Rhinogobio ventralis Sauvage et Da bry paternity testing method based on micro-satellite fluorescence multiplex PCR

A technology for paternity identification and albacore, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., and can solve the problems of paternity identification of albacore and achieve the protection of genetic diversity , improve the accuracy of qualitative, improve the effect of efficiency and speed

Active Publication Date: 2018-08-17
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, there is no paternity test method specifically for albacores

Method used

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  • Rhinogobio ventralis Sauvage et Da bry paternity testing method based on micro-satellite fluorescence multiplex PCR
  • Rhinogobio ventralis Sauvage et Da bry paternity testing method based on micro-satellite fluorescence multiplex PCR
  • Rhinogobio ventralis Sauvage et Da bry paternity testing method based on micro-satellite fluorescence multiplex PCR

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Experimental program
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Effect test

Embodiment 1

[0030] The steps of polymorphic microsatellite marker screening and multiplex PCR primer combination selection of albacores are as follows:

[0031] 1. Extraction of DNA from individuals of the longfin snout

[0032] Use the Tissue DNA Kit (D3396) kit of Omega Bio-Tek Company to extract the genomic DNA of the sample. The operation steps are as follows: Take 12 wild albacores, cut about 30 mg of fin rays or other tissue samples, wash with double distilled water , cut it thoroughly with small scissors, put it into a 1.5mL centrifuge tube, add 200μL TL Buffer and 25μL OB ProteaseSolution, digest at 55℃ for 1-1.5h, until the tissue is completely digested; centrifuge at 12000rpm for 5min to precipitate insoluble tissue fragments, and aspirate the supernatant Transfer the solution to another 1.5mL centrifuge tube; add 220μL BL Buffer, mix well, and place it in a constant temperature water tank at 70°C for 10min; add 220μL absolute ethanol, mix well, and briefly centrifuge to remove ...

Embodiment 2

[0045] The method for paternity identification of albacores based on microsatellite fluorescence multiplex PCR, the steps are:

[0046] 1. DNA Extraction of Longfin snout individuals

[0047] Collect 6 parental fin ray samples of albacore (3 females and 3 females, reproduced at a ratio of 1:1) and 96 individual samples of the corresponding offspring, and 34 fin ray samples of other breeding parents at the same time, using genomic DNA extraction reagents Genomic DNA was extracted using the kit, and the sample DNA concentration was adjusted to 100ng / μL for storage.

[0048] 2. Microsatellite fluorescent multiplex PCR amplification

[0049] According to the primer sequences and fluorescent labels shown in Table 1, synthesize 15 pairs of albacore microsatellite primers, and label the 5' end of the forward primer of each pair of primers with fluorescent substances, and the genomic DNA in step 1 according to Table 3 The indicated reaction system and annealing temperature were used...

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Abstract

The invention discloses a Rhinogobio ventralis Sauvage et Da bry paternity testing method based on micro-satellite fluorescence multiplex PCR. The Rhinogobio ventralis Sauvage et Da bry paternity testing method comprises: 1, extracting the genomic DNA of a to-be-tested sample; 2, carrying out a multiplex PCR amplification reaction by using a primer combination, and carrying out allele typing by using a sequencer; and 3, detecting the correlation between a to-be-tested subject and a parental genotype through likelihood ratio, and determining the paternity. Compared to the traditional PCR method, the method of the present invention has the following advantages that the detected microsatellite loci are increased by about 3 times so as to substantially improve the efficiency and the speed of the paternity testing, the detection cost is only about 1 / 3 of the original detection cost, the allele size interpretation error and other problems in the typing using the acrylamide gel electrophoresis are overcome, the accuracy of the genotype data is improved, and the analysis results show that 100% of the to-be-tested individuals can correctly find the parents.

Description

technical field [0001] The invention belongs to the technical field of fish molecular markers, and in particular relates to a microsatellite fluorescence multiplex PCR parentage identification method of the longfin gobi. Background technique [0002] Rhinogobio ventralis Sauvage et Dabry, belonging to Osteichthyes, Cypriniformes, Cyprinidae, Gobioninae, Rhinogobio, commonly known as ocean Fish and soil consumption are mainly distributed in the upper reaches of the Yangtze River, the Jinsha River, the Yalong River, the Minjiang River, the Tuojiang River, the middle and lower reaches of the Jialing River and the lower reaches of the Wujiang River. It likes to live in the bottom of the river, and it is a rare and unique fish in the upper reaches of the Yangtze River with high nutritional value and economic value. In recent years, due to human activities such as overfishing, water pollution, habitat destruction, and construction of water conservancy projects, the amount of reso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/6869C12Q1/6888C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122
Inventor 陈亮杨德国朱永久何勇凤吴兴兵
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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