A primer pair of a snp marker related to pork ph24 and application thereof

Abstract

The application relates to a SNP marker primer pair related to a pork pH24 trait and application thereof. The SNP marker is located on a pig chromosome 14, the site of the SNP marker is a molecular marker containing rs342386773 nucleotide sites of an international pig genome 11.1 version reference sequence pig chromosome 14, and has A / C polymorphism, the SNP marker is significantly related to the pork pH24 trait. A primer pair for detecting the SNP marker, the upstream primer is SEQ ID NO: 2, and the downstream primer is SEQ ID NO: 3. The SNP marker provided by the application is significantly related to the pH24 trait, and a pig strain with higher muscle pH value after 24 hours of slaughter can be screened by identifying the SNP marker, and the obtained pig strain with better pork pH value has important economic benefits and social values.

Description

TECHNICAL FIELD

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

[0002] Pork quality traits are a multi-index 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 degree of acid substances (mainly lactic acid) after glycogen metabolism in muscle, and the pH decreases in the process, causing muscle protein denaturation, thereby producing a light-colored appearance, and further affecting meat color, water retention capacity, softness and flavor, which is an important indicator for judging normal meat quality and abnormal meat quality. Studies have shown that more than 40% of consumers use meat color to judge meat freshness. Pork with low pH tends to have lighter color, which is usually referred to as PSE meat; and pork with high pH has darker color and harder texture, which is referred to as DFD meat, and consumers are more willing to buy pork with bright red color, good water retention capacity and texture.

[0003] The change of pork pH value is the result of the joint action of multiple factors, involving genetics, nutrition, management, stress and slaughter processing and other aspects. Therefore, improving the quality of pork color needs comprehensive management and control from multiple aspects. After pig slaughter, due to the termination of blood supply and respiration of the body, oxygen is also exhausted, and a series of physical, chemical and biochemical changes still continue, the energy metabolism of cells changes from aerobic respiration to anaerobic respiration, and glycogen fermentation becomes the main way of postmortem energy metabolism. With the progress of glycolysis, glycogen is decomposed into lactic acid through a series of enzymatic reactions and energy is generated, and with the continuous accumulation of lactic acid, the pH gradually decreases, and the pH of muscle decreases 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 containing multiple indexes, and certain progress has been made in genetic analysis of pork quality traits based on traditional QTL (quantitative trait loci) mapping and GWAS (genome wide association study) analysis, but a large number of QTL regions, causal genes and functional mutations have not been determined. Therefore, it is of great significance to identify SNPs related to muscle pH value 24 hours after slaughter and use marker-assisted selection technology to genetically improve pork pH value traits. SUMMARY

[0005] The present application aims at the actual situation that breeding for pork pH24 traits is time-consuming and laborious and the breeding effect is poor, and provides a SNP marker related to pork pH24 traits and develops it into a molecular marker.

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

[0007] Another object of the present application is to provide the use of the above-mentioned SNP marker.

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

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

[0010] The molecular marker obtained by the above-mentioned method.

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

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

[0013] A method for detecting a SNP marker related to the pork pH24 trait, comprising PCR amplifying a sequence of a nucleotide site of rs342386773 on chromosome 14 of the international pig genome 11.1 version reference sequence, sequencing the amplification product, and judging the A / C polymorphism of the site.

[0014] The specific judging method comprises the following steps:

[0015] (1) extracting total DNA from a pig tissue sample;

[0016] (2) using the extracted pig genomic DNA as a template, performing PCR amplification using the primer pair of claim 5;

[0017] (3) sequencing the amplification product, analyzing the sequencing result, and judging the A / C polymorphism at position 182 of SEQ ID NO: 1.

[0018] The molecular marker and the primer pair provided by the application are used for screening a pig population with a more optimal pork pH24 trait.

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

[0020] Advantages:

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

[0022] Figure 1 PCR amplification gel map of rs342386773 site on chromosome 14 of Large White

[0023] Figure 2 Typing map of rs342386773 site on chromosome 14 of Large White, wherein 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 application, but are not used to limit the scope of the application. Modifications or replacements of the method, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.

[0025] Example 1

[0026] 1 Source of experimental animals

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

[0028] 2 PH24 determination

[0029] The pH24 value of pork was determined using a pH meter (model: MAN99163) after slaughter.

[0030] 3 Extraction of pig genomic DNA

[0031] Collect 1 tissue sample per pig from the ear for individual DNA extraction;

[0032] Refer to the instructions for the tissue DNA extraction kit from Tiangen Biosciences Co., Ltd. and perform extraction in the following order:

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

[0034] ② Collect about 140 mg of the tissue sample in a 2 mL EP tube, cut it into small pieces, add 200 μL of buffer GA, and shake until completely suspended.

[0035] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56°C metal bath for overnight digestion until the ear tissue is dissolved. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

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

[0037] ⑤ Add 200 μL of anhydrous ethanol, mix well by shaking for 15 sec. At this point, a flocculent precipitate may appear. Briefly centrifuge to remove water droplets from 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, and then centrifuge at 12,000 rpm for 30 sec. Discard the waste liquid and place the adsorption column CB3 back in the collection tube.

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

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

[0041] 9. Repeat step 8.

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

[0043] Put the adsorption column CB3 into a clean centrifuge tube, and add 140 μL of elution buffer TE to the middle of the adsorption membrane by dripping, and place it at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, and add the centrifuged solution to the adsorption column CB3, place it at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0044] After detecting the quality and concentration by Nanodrop-140 spectrophotometer, dilute the same concentration to 50 ng / μL, and store it at -20℃ for standby use.

[0045] 4. PCR amplification and sequencing of the target fragment

[0046] Using the extracted DNA as a template, PCR amplification was performed according to the designed primers: 1 μL of DNA template, 0.4 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 14 μL of PCR mix reagent, and 7.2 μL of double distilled water; the PCR amplification system was set as follows:

[0047]

[0048] The PCR product was detected by electrophoresis in a 2% agarose gel, and the size of the amplified target fragment was about 469 bp, and the electrophoresis map is shown in Figure 1 The remaining amplification product was sequenced, and the sequencing results were compared and analyzed with the related gene fragment sequence of pig in DNAman software, and the genotype of rs342386773 site was judged using Chromas software.

[0049] 5. Statistical analysis

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

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

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

[0053] 6Results

[0054] Table 1 shows the effect of the rs342386773 variation site A / C on the pH24 trait of the Large White pig population after slaughter. As shown in Table 1, there is a significant difference (P<0.01) in the pH24 value trait among the three genotypes of the rs342386773 site. The pH24 of the superior genotype AA individual after slaughter is significantly higher than that of the inferior genotype CC individual (P<0.05), and also higher than that of the heterozygous AC individual (P<0.05). Therefore, in the Large White pig population, breeding the AA individual of the rs342386773 site is beneficial to breeding a pig population with better pH24 after slaughter, and thus improving the pork quality.

[0055] Table 1 Association analysis of the rs342386773 site on chromosome 14 of pigs and the pH24 trait of the Large White pig population after slaughter

[0056]

[0057] Note: The same row numbers with different letters represent significant differences (P<0.05).

Claims

1. A method for detecting SNP molecular markers associated with pork pH traits, characterized in that: It comprises PCR amplification of a sequence of the nucleotide site rs342386773 of the pig chromosome 14 of the pig international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the A / C polymorphism of the site; The molecular marker sequence is shown in SEQ ID NO:

1. The nucleotide site rs342386773 on chromosome 14 of the International Porcine Genome Version 11.1 reference sequence is located at position 182 in SEQ ID NO: 1 and has an A / C polymorphism. The muscle pH value measured 24 hours after slaughter in pigs with the AA genotype at this site is significantly higher than that in pigs with the inferior genotype CC, and is also higher than the pH value of pork from heterozygous AC individuals after slaughter. The pig is a Large White pig.

2. The method according to claim 1, 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, PCR amplification was performed using a primer pair: upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO: 3; (3) The amplified product was sequenced, and the sequencing results were analyzed to interpret the A / C polymorphism at position 182 of SEQ ID NO:

1.

3. Use of a molecular marker or a primer pair for detecting the molecular marker in screening a pig population with a better pork pH24 trait, characterized in that: AA individuals with the rs342386773 nucleotide site are selected as breeding pigs; the pigs are Large White pigs; The molecular marker sequence is shown in SEQ ID NO:

1. The nucleotide site rs342386773 on chromosome 14 of the International Porcine Genome Version 11.1 reference sequence is located at position 182 in SEQ ID NO: 1 and has an A / C polymorphism. The muscle pH value measured 24 hours after slaughter in pigs with the AA genotype at this site is significantly higher than that in pigs with the inferior genotype CC, and is also higher than the pH value of pork from heterozygous AC individuals after slaughter. The upstream primer of the primer pair is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3.

4. A method for screening a population with a higher pH24 trait in pork to assist in pork pH breeding, characterized in that: The method includes detecting the genotype of the rs342386773 nucleotide site on chromosome 14 of the pig international pig genome version 11.1 reference sequence, and selecting AA type individuals with the rs342386773 nucleotide site as breeding pigs; the pigs are large white pigs.

Images