SNP (Single Nucleotide Polymorphism) marker related to porcine No.8 chromosome and muscle shear force as well as detection method and application of SNP marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to porcine No.8 chromosome and muscle shear force as well as a detection method and application. The SNP marker is located on a pig 8 # chromosome, the site of the SNP marker is the nucleotide site of the pig 8 # chromosome rs332282301 of a reference sequence of an international pig genome version 11.1, the SNP marker has A / G polymorphism, and the SNP marker is significantly related to the pig muscle shear force. 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 related to the tenderness of the pigs, the pigs with low muscle shearing force (better tenderness) can be screened by identifying the SNP marker, and the SNP marker has important economic benefits and social values for establishing pig groups with low muscle shearing force (better tenderness).

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and relates to SNP markers related to the muscle shear force of porcine chromosome 8, a detection method and applications thereof. Background Art

[0002] With the continuous improvement of people's living standards, people not only have an increasing demand for pork, but also have higher and higher requirements for the quality of pork. Meat quality traits are indicators for evaluating the quality of pork, and meat quality traits mainly include intramuscular fat, tenderness, drip loss, pH, meat color, etc. Pork tenderness refers to the feeling when cooked pork is put into the mouth. There are many factors that can affect the tenderness of pork, mainly including the structure of various proteins in muscle, breed, muscle location, fiber diameter, etc. Generally, the shear force of cooked pork is used to measure the tenderness of pork. The shear force reflects the resistance when cutting pork; the greater the shear force, the harder the meat quality and the worse the tenderness; the smaller the shear force, the tenderer the meat quality and the better the taste.

[0003] Since the shear force of pork cannot be measured in vivo and the measurement cost is relatively high, it is difficult to efficiently improve the shear force by traditional breeding methods. In this study, phenotypic determination and gene chip analysis were carried out on the shear force of pigs, and genome-wide association analysis was used to identify candidate genes or molecular markers affecting the shear force of pigs, providing key molecular markers for molecular marker-assisted breeding and genomic selection of pig shear force. Summary of the Invention

[0004] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to pork shear force in view of the time-consuming, laborious, difficult phenotypic determination and slow breeding effect of traditional pork shear force breeding.

[0005] Another purpose of the present invention is to provide primers and a detection method for detecting the above SNP markers.

[0006] Another purpose of the present invention is to provide the use of the above SNP markers.

[0007] An SNP marker related to the muscle shear force of pigs, the SNP marker is located on porcine chromosome 8, the locus of the SNP marker is the nucleotide locus rs332282301 of porcine chromosome 8 in the reference sequence of the international porcine genome version 11.1, and has A / G polymorphism. The SNP marker is extremely significantly correlated with the tenderness of pigs. The shear force of pigs with the GG genotype at the rs332282301 locus is significantly lower than that of pigs with the AA and AG genotypes.

[0008] A method for developing molecular markers based on the SNPs described in the present invention, using the nucleotide sequence containing the SNP markers described in the present invention as the basic sequence, designing primer pairs, and performing PCR amplification using porcine genomic DNA as a template, so that the SNP markers described in the present invention are converted into molecular markers.

[0009] Among them, the sequences of the primer pairs are as follows: upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO: 3; the sequence of the molecular marker is as shown in SEQ ID NO: 1, and the SNP locus is located at the 301st position, with A / G polymorphism.

[0010] The molecular marker obtained by the method of the present invention as described above.

[0011] The preferred sequence of the molecular marker is as shown in SEQ ID NO: 1, and the SNP locus is located at the 301st position, with A / G polymorphism.

[0012] A primer pair for detecting the SNP marker described above, the upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO: 3.

[0013] A method for detecting the SNP marker described in the present invention, including PCR amplifying a sequence containing the SNP marker described in the present invention in porcine genome, sequencing the amplification product, and judging the A / G polymorphism at this locus.

[0014] The method for detecting the SNP marker described in the present invention includes the following steps:

[0015] (1) Taking a porcine muscle tissue sample and extracting total DNA;

[0016] (2) Using the extracted porcine genomic DNA as a template and performing PCR amplification using the primers described above;

[0017] (3) Sequencing the amplification product, analyzing the sequencing results, and judging the A / G polymorphism at the 301st position of SEQ ID NO: 1.

[0018] Application of the molecular marker described in the present invention in screening low muscle shear force populations or specialized strains.

[0019] Application of the primer pair described in the present invention in screening low muscle shear force populations or specialized strains.

[0020] A method for screening porcine populations with low muscle shear force, including detecting the genotypes of the nucleotide site rs332282301 on chromosome 8 of pigs in the 11.1 version of the international porcine genome reference sequence, and preferentially selecting individuals with the GG type and AG type at the rs332282301 nucleotide site as reserve breeding pigs for breeding.

[0021] As a preference of the present invention, the pig breed used is Large White pigs

[0022] As a preference of the present invention, the method for detecting the genotype of the nucleotide site rs332282301 on chromosome 8 of pigs in the international pig genome reference sequence version 11.1 is selected from PCR or gene sequencing

[0023] Beneficial effects

[0024] The SNP marker provided by the present invention is related to the shear force (tenderness) of pork. Therefore, pigs with lower pork shear force can be screened by identifying this SNP marker, and the established pig population with low muscle shear force (better tenderness) has important economic benefits and social value Description of the drawings

[0025] Figure 1 It is the distribution of the -logP values of SNP markers on the genomic chromosome obtained by the genome-wide association analysis of muscle shear force in the pig population. Among them, the information of 18 autosomes of pigs is marked on the X-axis

[0026] Figure 2 It is the electrophoresis pattern of amplifying the fragment of the rs332282301 gene locus using the primer of the present invention

[0027] Figure 3 It is the DNA sequencing result peak pattern of different genotypes of the mutation site of rs332282301, where Figure A is the AA type, Figure B is the AG type, and Figure C is the GG type Specific implementation methods

[0028] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention

[0029] Examples

[0030] 1 Experimental animals

[0031] 413 pigs and their muscle tissue samples were taken

[0032] 2 Shear force measurement

[0033] The method for measuring the shear force of pork refers to the national standard "NY / T 1180-2006 Determination of meat tenderness - Shear force measurement method". The shear force of pork was measured using a digital display muscle tenderness meter (Model C-LM3B), and each individual was measured three times, and the average value was taken, with the unit of Newton (N)

[0034] 3 Extraction of pig genomic DNA

[0035] Collect 1 muscle tissue sample from 413 pigs for individual DNA extraction;

[0036] Refer to the instruction manual of the Tissue DNA Extraction Kit from Tiangen Biotech Company. The extraction steps are as follows:

[0037] ① First, add 68 mL and 200 mL of absolute ethanol to buffer GD and wash buffer PW respectively, and mix well.

[0038] ② Collect about 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely cutting it into small pieces, add 200 μL of buffer GA and shake until thoroughly suspended.

[0039] ③ Add 20 μL of proteinase K solution, mix well, and digest overnight in a 56 °C metal bath until the tissue sample dissolves. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

[0040] ④ Add 200 μL of buffer GB, mix thoroughly by inverting, and place in a 70 °C metal bath for 10 min. The solution should become clear. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

[0041] ⑤ Add 200 μL of absolute ethanol, mix well by shaking for 15 sec. At this time, flocculent precipitates may appear. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

[0042] ⑥ Add the solution and flocculent precipitates 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 sec, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0043] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, pour out the waste liquid, and place the adsorption column CB3 in the collection tube

[0044] ⑧ Add 600 μL of wash buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, pour out the waste liquid, and place the adsorption column CB3 in the collection tube.

[0045] ⑨ Repeat operation step ⑧.

[0046] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and pour out the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual wash buffer in the adsorption material.

[0047] Transfer the adsorption column CB3 into a clean centrifuge tube. Suspendedly add 100 μL of elution buffer TE to the middle part of the adsorption membrane, place it at room temperature for 2 - 5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, add the centrifuged solution back into the adsorption column CB3, place it at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min to collect the solution into the centrifuge tube.

[0048] Use a Nanodrop - 2000 spectrophotometer to detect the quality and concentration of DNA. Dilute the DNA concentration to 50 ng / μL and store it at - 20 °C for later use.

[0049] PCR amplification and sequencing of the target fragment

[0050] Perform PCR amplification using porcine genomic DNA as a template. The reaction system includes 1 μL of DNA template, 1 μL each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 9.5 μL of ddH2O, and 12.5 μL of PCRmix; the amplification program is as follows:

[0051]

[0052]

[0053] Perform agarose gel electrophoresis on the amplification products. The size of the product fragment is approximately 458 bp, and the electrophoresis results are as Figure 2 shown. Sequence the remaining amplification products, use DNAman software to compare and verify the accuracy of the sequence, and use Chromas software to genotype the rs332282301 locus.

[0054] 5 Statistical analysis

[0055] Use the general linear model of SAS 9.4 software to perform association analysis between genotypes and phenotypes. The model is as follows: Yijk = μi + Bj + Gk + ejk

[0056] Among them, Yijk is the shear force of the individual; μ i represents the mean shear force of the population; B j represents the fixed effect of the slaughter batch; G k is the fixed effect of the SNP marker; e jk is the residual.

[0057] 6 Results

[0058] Table 1 shows the results of the effect of different genotypes at the rs332282301 locus on the muscle shear force of pigs. The results indicate that there are significant differences in shear force among individuals with the three genotypes at the rs332282301 locus (P < 0.05). Among them, the shear forces of GG and AG individuals are both significantly lower than that of AA individuals (P < 0.05), and the shear force of GG individuals is significantly lower than that of AG individuals (P < 0.05). Therefore, selecting GG and AG individuals at the rs332282301 locus during successive generations of breeding in pigs is beneficial to reducing the shear force of the pig population and thus improving the tenderness of pork quality.

[0059] Table 1 Association analysis of the rs332282301 locus on Sus scrofa chromosome 8 with shear force in Yorkshire pigs

[0060]

[0061] Note: Different superscript letters in the same row indicate significant differences (P < 0.05).

Claims

1. SNP markers on Sus scrofa chromosome 8 related to muscle shear force, characterized in that, The SNP marker is located at the nucleotide site rs332282301 on chromosome 8 of the pig in the reference sequence of the international pig genome version 11.1, and has A / G polymorphism. The muscle shear force of individuals with the GG genotype and the AG genotype is significantly lower than that of individuals with the AA genotype.

2. A method for developing molecular markers based on the SNPs described in claim 1, characterized in that Based on the nucleotide sequence containing the SNP marker described in claim 1 as the basic sequence, a primer pair is designed, and PCR amplification is performed using porcine genomic DNA as a template to convert the SNP marker described in claim 1 into a molecular marker.

3. The method according to claim 2, characterized in that The primer pair sequence is: forward primer: SEQ ID NO: 2, reverse primer: SEQ ID NO: 3; the molecular marker sequence is as shown in SEQ ID NO: 1, and the SNP site is located at position 301 of SEQ ID NO: 1, with A / G polymorphism.

4. The molecular marker obtained by the method according to claim 2 or 3.

5. The molecular marker according to claim 4, wherein The molecular marker sequence is as shown in SEQ ID NO: 1, the SNP site is located at position 301, and there is A / G polymorphism.

6. A primer pair for detecting the SNP marker according to claim 1, characterized in that The forward primer is: SEQ ID NO: 2, and the reverse primer is: SEQ ID NO:

3.

7. A method for detecting the SNP marker according to claim 1, characterized in that It includes PCR amplifying a sequence containing the SNP marker described in claim 1 in the porcine genome, sequencing the amplification product, and judging the A / G polymorphism at this site.

8. The method according to claim 7, wherein It includes the following steps: (1) Taking a muscle tissue sample of a pig and extracting total DNA; (2) Using the extracted porcine genomic DNA as a template and performing PCR amplification with the primer pair described in claim 6; (3) Sequencing the amplification product, analyzing the sequencing result, and judging the A / G polymorphism at position 301 of SEQ ID NO:

1.

9. The application of the SNP marker described in claim 1, the molecular marker described in claim 4 or 5, and the primer pair described in claim 6 in screening pigs with low muscle shear force.

10. A method for screening a pig population with low muscle shear force, characterized in that It includes detecting the genotype of the rs332282301 nucleotide site of a pig, and preferentially selecting individuals with the GG genotype and the AG genotype at the rs332282301 nucleotide site as reserve breeding pigs for breeding.

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