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The core sequence, primers and application of SNP markers related to high pH resistance of Chinese prawn

A Chinese shrimp, marker technology, applied in recombinant DNA technology, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of lack of SNP markers, and achieve the effect of speeding up the breeding process and accuracy

Active Publication Date: 2022-04-29
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The development of SNP markers in Chinese prawns is mostly found in SNP marker screening, development and population genetic diversity analysis, etc., and the number of SNP markers that can be used for germplasm resource evaluation and germplasm material mining is insufficient

Method used

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  • The core sequence, primers and application of SNP markers related to high pH resistance of Chinese prawn
  • The core sequence, primers and application of SNP markers related to high pH resistance of Chinese prawn
  • The core sequence, primers and application of SNP markers related to high pH resistance of Chinese prawn

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Experimental program
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Effect test

Embodiment 1

[0021] Example 1: Extraction of individual genome DNA of Chinese prawns

[0022] Take about 0.1g of Chinese prawn muscle tissue in a 2.0ml centrifuge tube, add 700μL of tissue lysate, add grinding beads, and homogenize the sample with a tissue homogenizer; slowly add 30μL of proteinase K with a concentration of 20mg / mL, and place in a 60°C water bath 3h, shake once every 30min, cool to room temperature after clarification; add 700μL of phenol-chloroform, shake well, centrifuge at 12000rpm for 15min; take supernatant, add 600μL of phenol-chloroform isoamyl alcohol (25:24:1), shake well , centrifuge at 12000rpm for 15min; take the supernatant and add 500μL of isopropanol, centrifuge at 12000rpm for 10min; pick out the DNA and wash it with 70% ethanol, dry it at room temperature, dilute it with sterilized water to 50ng / μL, and store it at -20℃ for later use.

Embodiment 2

[0023] Embodiment 2: Design of SNP marker amplification primers

[0024] Based on the FC1516SNP core sequence contained in the Chinese prawn transcriptome library, specific primers were designed at both ends of it to amplify the DNA fragment at this site. The specific primer sequences at both ends of the SNP core sequence of the present invention are: forward primer: 5'-ACCAATGGTCTCCCCCTTCC-3'; reverse primer sequence: 5'-AATCATCTCATCAGGACTGCTTG-3'.

[0025] The PCR amplification system is: Chinese shrimp genomic DNA 100ng, 10×PCR Buffer 2.5μL, 25mM MgCl 21.5μL, 2.5mM dNTP 2μL, 10μM positive and negative primers 2μL each; add sterilized water to 25μL; PCR reaction program: 94°C denaturation 5min, 95°C for 30sec, 60°C for 30sec, 72°C for 40sec, 30 cycles; 72°C for 10min. The PCR reaction program was: denaturation at 94°C for 5 min, 30 cycles at 95°C for 30 sec, 60°C for 30 sec, 72°C for 40 sec, and 72°C for 10 min.

[0026] After the specificity of the PCR amplification produ...

Embodiment 3

[0028] Example 3: Association analysis of Chinese prawn SNP markers and high pH tolerance traits

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Abstract

The present invention relates to a core sequence, primer and application of a SNP marker related to high pH resistance of Chinese prawns, and belongs to the technical field of shrimp molecular marker-assisted breeding. The core sequence and primer sequence of the SNP marker are respectively shown in SEQ ID NO.1 As shown in -6, the method for using the primers to carry out typing is as follows: extract the individual genomic DNA of Chinese prawns, use the SNP marker typing primers, and adopt the fluorescent quantitative PCR method to carry out SNP marker typing of Chinese prawn individuals to determine the individuality of each individual. genotype. Since the SNP marker FC1516 is closely related to the high-pH tolerance trait of Chinese prawns, and this method is a molecular marker detection method based on the DNA level, the genotype of this locus can be quickly and accurately detected for screening, Eliminate individuals that are intolerant or sensitive to high pH stress, and speed up the process and accuracy of the breeding process and accuracy of high pH stress-resistant traits in Penaeus prawns in China.

Description

technical field [0001] The invention belongs to the technical field of shrimp molecular marker assisted breeding, and specifically relates to a core sequence, primer and application of a SNP marker related to high pH resistance of Chinese prawns. Background technique [0002] Molecular marker-assisted breeding technology uses DNA molecular markers to select genetic resources or breeding materials and genetically improve important economic traits of aquatic animals. It has the advantages of being fast, accurate, and free from environmental interference. [0003] SNP, or single nucleotide polymorphism, is a genetic marker system proposed by Lander of the Massachusetts Institute of Technology in 1996. Morphology, often manifested as conversion or transversion, deletion or insertion of a single base, but mainly conversion and transversion, the ratio of the two occurrences is 2:1. As a third-generation genetic marker, SNP is widely used in the evaluation of germplasm resources o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6888C12Q1/6858C12Q2600/156C12Q2600/124C12Q2600/172C12Q2531/113C12Q2563/107
Inventor 何玉英韩旭王佳佳李健王琼
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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