SNP marker for Konosirus punctatus, and screening method and application thereof

A screening method and a technique for spot sting, applied in the field of molecular biology, can solve the problems of low acquisition purity of target DNA, complicated sequencing procedures, etc., and achieve the effect of reducing the cost of tag sequencing, improving the purity, and screening and detecting at low cost.

Inactive Publication Date: 2018-12-14
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sequencing procedure of this invention is relatively complicated, and the purity of the target DNA is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A method for screening SNP markers of catfish, comprising the following steps:

[0037] Genomic DNA extraction: Cut the muscle or fin ray tissue samples of the spotted catfish into pieces, and place them in the extraction solution I: 70ml of Tris-HCl and disodium hydrogen phosphate with a concentration of 150mM, 0.5g of fatty alcohol ether ammonium sulfate, and 30ml of chlorine with a concentration of 130mM. Sodium chloride solution; add 0.5 μg plasmid DNA and 0.1 μg chitin, freeze and thaw 2-3 times, the freezing temperature is -166°C, the melting temperature is 50°C, centrifuge, collect the supernatant and add extract II: 10ml Tris-HCl with a concentration of 100mM and disodium hydrogen phosphate and 30ml of cationic polyacrylamide with a concentration of 1.6ppm were mixed upside down and centrifuged to produce a white precipitate, which was washed to obtain a DNA sample for use; circular DNA and chitin The special existence, on the one hand, can promote the combinati...

Embodiment 2

[0063]Circular DNA and chitin were not added in the process of extracting genomic DNA, and the rest were exactly the same as in Example 1; 28,730 SNPs were obtained by final filtration and screening, which indicated that the addition of circular DNA and chitin could effectively improve the integrity of the extracted DNA , which is beneficial to obtain the whole genome information of the catfish, and improve the accuracy and acquisition rate of SNP marker screening.

Embodiment 3

[0065] In the process of RAD library construction and high-throughput sequencing, chitin fluid was added during the PCR amplification system, the mass percentage of chitin was 0.16%, and the solvent was L-amino acid ethyl ester; the rest were completely consistent with Example 1; The special existence of chitin fluid has a strong adsorption force to DNA, which can improve the success rate of PCR. At the same time, chitin fluid is composed of many tiny spheres, which can wrap the reaction system, so that the PCR amplification reaction can be completed in a small independent space. , so as to improve the reaction sensitivity of PCR amplification, make the reaction more sufficient, and improve the PCR amplification effect of trace DNA; in addition, chitin fluid can increase the boiling point of the reaction system so that the system buffer is not easy to evaporate, reducing the amount of time in the PCR amplification process. The amount of reagents used, thereby reducing the cost ...

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PUM

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Abstract

The invention discloses a SNP marker for Konosirus punctatus, and a screening method and application thereof. The screening method for the SNP marker of Konosirus punctatus comprises the following steps: extraction of genomic DNAs; construction of a RAD library and high-throughput sequencing; filtering of original sequence data; and detection and screening of SNP information sites. The addition ofcircular DNAs and chitin in the extraction process of the genomic DNAs can effectively improve the screening accuracy and acquisition efficiency of the SNP marker. The SNP marker obtained by using the method can provide an important basis for related research on the analysis of the population genetic variation level and population genetic structure of Konosirus punctatus. The screening method foracquiring the SNP marker site of Konosirus punctatus has the advantages of high throughput, low cost, high screening accuracy of the SNP marker, high acquisition efficiency, etc.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a SNP marker of the catfish and its screening method and application. technical background [0002] Konosirus punctatus belongs to Clupeiformes, Clupeidae, Dorosomatinae, and Konosirus. It is a coastal warm-water pelagic fish widely distributed in China, North Korea, The coasts of South Korea and Japan are produced in my country's Bohai Sea, Yellow Sea, East China Sea, and South China Sea. They are important fishing targets for offshore fisheries in China, Japan, and the Korean Peninsula. Catfish is of great significance to the sediment resuspension in coastal ecosystems and the material exchange at the water-bottom interface; at the same time, it is the prey object of a variety of high-trophic fish, and plays a key role in connecting the preceding and the following in the structure of the marine food web. At the same time, because of its high meat content, tend...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6888C12Q2600/156
Inventor 刘炳舰江丽华柳意樊吕振明刘立芹龚理张伟男刘珠朱科桦段雯
Owner ZHEJIANG OCEAN UNIV
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