SNP (Single Nucleotide Polymorphism) marker primer pair related to pH 24 of pork and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to a pork pH 24 character and application of the SNP marker primer pair. The SNP marker is located on a pig chromosome 14, the site of the SNP marker is a molecular marker containing rs342386773 nucleotide site of the pig chromosome 14 in a reference sequence of an international pig genome version 11.1, the SNP marker has A / C polymorphism, and the SNP marker is significantly related to the pork pH 24 character. According to a primer pair for detecting the SNP marker, an upstream primer is SEQ ID NO: 2, and a downstream primer is SEQ ID NO: 3. The SNP marker provided by the invention is significantly related to the pH 24 character, pig strains with high muscle pH values 24 hours after slaughtering can be screened by identifying the SNP marker, and the obtained pig strains with good pork pH values have important economic benefits and social values.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a SNP marker primer pair related to pork pH24 and an application thereof. Background Art

[0002] Pork quality is a multi-indicator and complex economic trait, including meat color, pH value, shear force, drip loss, intramuscular fat content and other traits. pH24 refers to the muscle pH value measured 24 hours after slaughter, which is an important indicator for evaluating pork quality. This indicator mainly reflects the accumulation of acidic substances (mainly lactic acid) after glycogen metabolism in the muscle. When the pH decreases, myosin denaturation occurs, resulting in a light-colored appearance, which in turn affects the meat color, water retention capacity, softness and hardness, and flavor. This is an important indicator for judging normal and abnormal meat quality. Studies have shown that more than 40% of consumers use meat color to judge the freshness of meat. Pork with a low pH value tends to be lighter in color, which is usually called PSE meat; while pork with a high pH value is darker in color and harder in texture, which is called DFD meat. Consumers are more willing to buy pork with a bright red color, good water retention capacity and texture.

[0003] The change of pH value of pork is the result of the combined action of multiple factors, involving genetics, nutrition, management, stress, slaughtering and processing. Therefore, improving the quality of pork color requires comprehensive management and control from multiple aspects. After the pig is slaughtered, due to the termination of blood supply and respiration, oxygen is also exhausted, and a series of physical, chemical and biochemical changes continue. The energy metabolism of cells begins to change from aerobic respiration to anaerobic respiration, and glycogenolysis becomes the main pathway of energy metabolism after slaughter. As glycolysis proceeds, glycogen is decomposed into lactic acid and produces energy through a series of enzymatic reactions. As lactic acid continues to accumulate, pH gradually decreases, and muscle pH eventually drops from 7.0 in living muscle to about 5.7. The rate and degree of muscle pH change will directly affect other meat quality traits.

[0004] Pork quality is a complex trait that includes multiple indicators. Based on traditional QTL positioning (quantitative trait loci, QTL) and GWAS analysis (Genome wide association study, GWAS), some progress has been made in the genetic analysis of pork quality traits, but there are still a large number of QTL region causal genes and functional mutations that have not been determined. Therefore, it is of great significance to identify SNPs related to muscle pH 24 hours after slaughter and use marker-assisted selection technology to genetically improve pork pH traits. Summary of the invention

[0005] The purpose of the present invention is to provide SNP markers related to the pH24 trait of pork and develop them into molecular markers in view of the actual situation that the breeding of the pH24 trait of pork is time-consuming and labor-intensive and the breeding effect is poor.

[0006] Another object of the present invention is to provide a primer pair and a detection method for detecting the above-mentioned SNP marker.

[0007] Another object of the present invention is to provide uses of the above-mentioned SNP markers.

[0008] A method for developing molecular markers related to the pH24 trait of pork, wherein the pH24 trait of pork refers to the pH value of pork 24 hours after slaughter. A primer pair is designed based on a nucleotide sequence containing the nucleotide site rs342386773 of pig chromosome 14 in the international pig genome version 11.1 reference sequence, and PCR amplification is performed using pig genomic DNA as a template to convert the nucleotide site rs342386773 of pig chromosome 14 in the international pig genome version 11.1 reference sequence into a molecular marker.

[0009] The primer pair sequence is upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO: 3.

[0010] The molecular markers were obtained according to the above method.

[0011] The molecular marker sequence is preferably as shown in SEQ ID NO: 1. The nucleotide site rs342386773 of chromosome 14 of the international porcine genome version 11.1 reference sequence is located at position 182 in SEQ ID NO: 1, and there is an A / C polymorphism. The muscle pH value of pig individuals with the AA genotype at this site measured 24 hours after slaughter is significantly higher than that of individuals with the inferior genotype CC, and also higher than the pH24 of pork after slaughter of heterozygous AC individuals.

[0012] A primer pair for detecting a SNP marker associated with a pH24 trait in pork, wherein the upstream primer is SEQ ID NO: 2 and the downstream primer is SEQ ID NO: 3; the SNP marker associated with the pork color trait is located on the nucleotide sequence of the FRMD4A gene on pig chromosome 14, the site of the SNP marker is a molecular marker at the rs342386773 nucleotide site on pig chromosome 14 of the international pig genome version 11.1 reference sequence, and has an A / C polymorphism.

[0013] A method for detecting SNP markers associated with the pH24 trait of pork comprises PCR amplifying a sequence of the rs342386773 nucleotide site of chromosome 14 of the international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the A / C polymorphism of the site.

[0014] The specific interpretation method, according to the method of claim 6, is characterized by comprising the following steps:

[0015] (1) Extract total DNA from pig tissue samples;

[0016] (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair described in claim 5;

[0017] (3) The amplified product was sequenced, the sequencing results were analyzed, and the A / C polymorphism at position 182 of SEQ ID NO: 1 was determined.

[0018] The molecular markers and primer pairs of the present invention are used in screening a pig population with superior pork pH24 trait.

[0019] A method for screening a pig population with superior pork pH24 trait, comprising detecting the genotype of the rs342386773 nucleotide site on chromosome 14 of the international porcine genome version 11.1 reference sequence, and selecting AA-type individuals at the rs342386773 nucleotide site as breeding pigs.

[0020] Beneficial effects:

[0021] The SNP marker provided by the present invention is related to the pH24 trait of pork, and the molecular marker and primer developed based on the SNP can be used for the detection of SNP. Therefore, the strain with better pH trait of pork can be screened by identifying the SNP marker, and the obtained pig strain with better meat quality trait has important economic benefits and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the gel image of PCR amplification of rs342386773 site on chromosome 14 of Large White pig

[0023] Figure 2 This is the typing map of the rs342386773 locus on chromosome 14 of Large White pig, where Figure A is AA type, Figure B is CA type, and Figure C is CC type. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0025] Embodiment 1:

[0026] 1. Source of experimental animals

[0027] 394 Large White pigs from Jiangsu Zhengda Suken Pig Industry Co., Ltd.

[0028] 2PH24 assay

[0029] After slaughter, the pH24 value of pork was measured using a pH meter (model: MAN99163).

[0030] 3 Extraction of pig genomic DNA

[0031] Collect one pig ear tissue sample per pig for individual DNA extraction;

[0032] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Company, and the extraction is carried out in the following order:

[0033] ① First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse solution PW respectively, and mix thoroughly.

[0034] ② Collect about 140 mg of tissue sample and place it in a 2 mL EP tube. After completely mincing, add 200 μL of buffer GA and shake until completely suspended.

[0035] ③ Add 20 μL of proteinase K solution, mix well and digest overnight in a 56°C metal bath until the ear sample tissue is dissolved. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0036] ④ Add 200 μL of buffer GB, mix thoroughly by inversion, and place in a 70°C metal bath for 14 min. The solution should become clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0037] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0038] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in a collection tube, then centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0039] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube

[0040] ⑧ Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0041] ⑨Repeat step ⑧.

[0042] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0043] Transfer the adsorption column CB3 into a clean centrifuge tube, add 140 μL of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 2 minutes, collect the solution in a centrifuge tube, add the solution obtained by centrifugation to the adsorption column CB3, let it stand at room temperature for 2 minutes, centrifuge it at 12,000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

[0044] After the mass and concentration were detected by Nanodrop-140 spectrophotometer, the concentration was diluted to 50 ng / μL and stored at -20°C for future use.

[0045] 4. PCR amplification and sequencing of target fragments

[0046] Use the extracted DNA as a template and perform PCR amplification according to the designed primers: take 1 μL of DNA template, 0.4 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 14 μL of PCRMix reagent, and 7.2 μL of double distilled water; set up the PCR amplification system:

[0047]

[0048] The PCR product was detected by electrophoresis in 2% agarose gel. The amplified target fragment was about 469 bp in size. Figure 1 The remaining amplified products were sequenced, and the sequencing results were compared and analyzed with the relevant gene fragment sequences of pigs in DNAman software, and the genotype of the rs342386773 site was determined using Chromas software.

[0049] 5 Statistical analysis

[0050] The general linear model of SAS software was used to analyze the effect of genotype on phenotype.

[0051] Y ijnk =u i +G j +S n +D k +e jnk

[0052] Where: Y ijnk pH24 trait of pigs after slaughter; Gj represents the genotype fixed effect of the jth SNP; S n represents the fixed effect of gender; D k represents the fixed effect of birth year and season; e jnk is the residual.

[0053] 6 Results

[0054] Table 1 shows the effect of rs342386773 variant site A / C on pH24 trait after slaughter in the Large White pig population. As shown in Table 1, there are significant differences in pH24 value traits between the three genotypes of the rs342386773 locus (P<0.01). The pH24 of pork after slaughter in the dominant genotype AA type individuals is significantly higher than that in the inferior genotype CC type individuals (P<0.05), and also higher than that in the heterozygous AC type individuals (P<0.05). It can be seen that in the Large White pig population, the successive breeding of AA type individuals at the rs342386773 locus is conducive to breeding pigs with better pH24 after slaughter, thereby improving pork quality.

[0055] Table 1 Association analysis between rs342386773 locus on chromosome 14 and pH24 trait after slaughter in Large White pigs

[0056]

[0057] Note: Numbers with different letters in the same column indicate significant differences (P<0.05).

Claims

1. A method for developing molecular markers associated with the pH24 trait of pork, wherein the pH24 trait of pork refers to the pH value of pork 24 hours after slaughter, characterized in that Based on the nucleotide sequence of the rs342386773 nucleotide site on chromosome 14 of the international porcine genome version 11.1 reference sequence, a primer pair was designed, and PCR amplification was performed using porcine genomic DNA as a template to convert the rs342386773 nucleotide site on chromosome 14 of the international porcine genome version 11.1 reference sequence into a molecular marker.

2. The method according to claim 1, characterized in that The primer pair sequence is upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO:

3.

3. Molecular markers obtained according to the method of claim 1 or 2.

4. The molecular marker according to claim 3, characterized in that The molecular marker sequence is shown in SEQ ID NO:

1. The nucleotide site rs342386773 of chromosome 14 of the international porcine genome version 11.1 reference sequence is located at position 182 in SEQ ID NO: 1, and there is an A / C polymorphism. The muscle pH value of pig individuals with the AA genotype at this site measured 24 hours after slaughter is significantly higher than that of individuals with the inferior genotype CC, and is also higher than the pH24 of pork after slaughter of heterozygous AC individuals.

5. A primer pair for detecting SNP markers associated with pH24 trait in pork, characterized in that The upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO: 3; the SNP marker associated with pork pH24 is located on pig chromosome 14, and the site of the SNP marker is a molecular marker at the rs342386773 nucleotide site of pig chromosome 14 in the international pig genome version 11.1 reference sequence, and has an A / C polymorphism.

6. A method for detecting the SNP marker associated with the pH trait of pork as claimed in claim 5, characterized in that It comprises a PCR amplification of a section of the nucleotide site rs342386773 of the pig chromosome 14 of the reference sequence of the international pig genome version 11.1, sequencing the amplified product, and interpreting the A / C polymorphism of the site.

7. The method according to claim 6, characterized in that The following steps are involved: (1) Extract total DNA from pig tissue samples; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair described in claim 5; (3) The amplified product was sequenced, the sequencing results were analyzed, and the A / C polymorphism at position 182 of SEQ ID NO: 1 was determined.

8. Use of the molecular markers described in claim 3 or 4 and the primer pairs described in claim 5 in screening a pig population with better pork pH24 trait.

9. A method for screening a population with a higher pH24 trait in pork to assist in pork pH breeding, characterized in that This includes detecting the genotype of the rs342386773 nucleotide site on chromosome 14 of the International Swine Genome 11.1 version reference sequence, and selecting AA type individuals at the rs342386773 nucleotide site as breeding pigs.

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