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A Traceable SNP Molecular Marker of Pig Amy2 Gene and Its Detection Method

A molecular marker and gene technology, applied in the field of traceable SNP molecular markers and their detection, can solve the problem of lack of SNP markers

Active Publication Date: 2015-10-28
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the long-term breeding work, researchers have accumulated a large number of SNP markers, but these SNP markers are often concentrated on certain chromosomes, such as chromosome 1, chromosome 12, etc., while other chromosomes, such as chromosome 4 and chromosome 8 Chromosomes 14, 18, etc. lack SNP markers, so in order to meet the requirements for DNA traceability of pork products, we conduct research on new SNP markers

Method used

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  • A Traceable SNP Molecular Marker of Pig Amy2 Gene and Its Detection Method
  • A Traceable SNP Molecular Marker of Pig Amy2 Gene and Its Detection Method
  • A Traceable SNP Molecular Marker of Pig Amy2 Gene and Its Detection Method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Search for Molecular Markers

[0027] (1) Construction of DNA pool (pool)

[0028] The ear tissues of 2 Duroc pigs and 2 Meishan pigs (regardless of sex) were randomly collected, and DNA was extracted, and the DNA of 4 individuals was extracted in equal amounts and put into the same centrifuge tube, mixed well and set aside.

[0029] (2) Primer design

[0030] Using the DNA sequence of the porcine AMY2 gene (Genbank: NC_010446) as a template, design primers to isolate the porcine AMY2 gene (using the DNA of a Shannon pig as a template for PCR amplification), the primers are as follows:

[0031] Forward primer: 5'-GCAAGTGGAGTGGAGAGAAG-3' (SEQ ID NO.3)

[0032] Reverse primer: 5'-ACTCGTGTGAATCCGTAAGG-3' (SEQ ID NO.4)

[0033] The total volume of the PCR reaction is 20μl, including about 100ng of pig AMY2 genomic DNA, containing 1×buffer (Promega Company), 1.5mmol / L MgCl 2 , the final concentration of dNTP (Shanghai Sangon Biotechnology Co., Ltd.) was 150 μmo...

Embodiment 2

[0045] Example 2 Distribution of alleles

[0046] (1) Design of test groups

[0047] Experimental group: collected Pietrain (21 heads), Shannon (17 heads), Dabai (43 heads), Dashen (52 heads), Changshen (16 heads), Dushen (23 heads), Pi Dashen (11 heads) DNA was extracted from the ear tissues of individuals of the head), Dachang Shen (25 heads), Du Dashen (7 heads) and Dupi Dashen (20 heads), totaling 235 DNA samples.

[0048] The purpose of the test population is to detect the distribution of SNP markers in different breeds.

[0049] (2) Genotype detection

[0050] Forward primer: 5'-GCAAGTGGAGTGGAGAGAAG-3' (sequence SEQ ID NO.3)

[0051] Reverse primer: 5'-ACTCGTGTGAATCCGTAAGG-3' (sequence SEQ ID NO.4)

[0052] Perform amplification (conditions as in Example 1), and use the same RFLP method to detect all individual genotypes in the test population.

[0053] (3) Statistical analysis

[0054] Record the genotypes of all individuals, and calculate the allele frequency and...

Embodiment 3

[0058] Example 3 SNP molecular marker usability traceability test verification

[0059] The above-mentioned SNP markers have been preliminarily confirmed to be used in traceability markers. In order to ensure the usability in traceability markers, the present invention re-samples at different locations and conducts usability traceability test verification.

[0060] Randomly collect 100 ear tissue samples and 100 muscle samples from individual pigs at Fuxing Slaughterhouse (every pig individual sample is collected at the same time as ear tissue samples and muscle samples), ear tissue samples are numbered E1-E100, and muscle samples are numbered M1-M100, respectively DNA from muscle samples and ear tissue samples was used for future use.

[0061] Detect the Eco91I SNP site of porcine AMY2 gene in 100 individual ear tissue samples and 20 randomly selected muscle samples and the genotypes of 11 SNP sites that have been reported in Table 2 below, a total of 12 SNP sites , to count...

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Abstract

The invention discloses an SNP molecular marker in a porcine AMY2 gene for tracing and a detection method thereof. The SNP molecular marker is obtained through DNA pool sequencing and has altogether 1060 bp. The SNP molecular marker contains a part of the sequence of a fifth exon, a part of the sequence of a seventh exon and the complete sequences of a fifth intron, a sixth exon and a sixth intron; a base at a position 104 mutates from 104 C to 104 T; the DNA sequence of the SNP molecular marker is represented by SEQ ID NO. 1, and an amino acid sequence encoded by the SNP molecular marker is represented by SEQ ID NO. 2. The SNP molecular marker is used in cultivation of commercial pigs and is extensively applicable to DNA tracing and detection for pork products, especially to security tracing for pork products.

Description

technical field [0001] The invention belongs to the field of food safety, and relates to a traceable SNP molecular marker of pig AMY2 gene and a detection method thereof. Background technique [0002] As an important source of protein in human diet, pork products have become a global hot issue on how to ensure their safety and sanitation and protect the health and life safety of consumers. Governments around the world have adopted a series of technical measures to strengthen the safety management of meat products in order to minimize the occurrence of food safety incidents. Major cities in my country have also established traceability systems for meat products. Through the traceability management of meat products, consumers can be provided with accurate and detailed information about products, which is conducive to timely discovery of unsafe hidden dangers in various links by producers and operators. , to provide consumers with a way to obtain effective and reliable informat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68C07K14/47
Inventor 吴潇唐雪明杨世方赵凯王金斌刘华蒋玮何建华
Owner SHANGHAI ACAD OF AGRI SCI
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