CAPRIN1 gene molecular marker related to intramuscular fat content of Erhualian pigs and application of CAPRIN1 gene molecular marker

Abstract

The invention relates to a CAPRIN1 gene molecular marker related to detection of intramuscular fat content of Erhualian pigs and application of the CAPRIN1 gene molecular marker. A variation site is located in a second intron region (a second chromosome 26859339 base of a pig) of a pig CAPRIN1 gene and has a polymorphic SNP site, the genotype of the SNP site is AA, AG or GG, and the intramuscular fat content of Erhualian pork can be determined according to the genotype of the mutation site. An SNP site related to the intramuscular fat content of the pig is screened in an intron region of the CAPRIN1 gene of the Erhualian pig, so that a functional gene and a molecular genetic marker related to the intramuscular fat content are obtained. The molecular marker has the beneficial effects that by optimizing the dominant allele of the SNP site of the pig CAPRIN1 gene, a marker resource can be provided for molecular marker-assisted breeding of the intramuscular fat content of the pig, and a new way is provided for improving the pork quality.

Description

Technical Field

[0001] The present invention relates to a CAPRIN1 gene molecular marker related to detecting the intramuscular fat content of Erhualian pigs and an application thereof, belonging to the technical field of molecular biology. Background Art

[0002] Intramuscular fat (IMF) content in pork is a key quality attribute that determines consumer acceptance and market value. It is composed of white fat tightly bound to membrane proteins within muscle tissue. IMF content is determined by the number and size of intramuscular adipocytes, and its deposition rate is closely related to muscle growth. Furthermore, factors such as breed, feeding method, hormones, and genetics also significantly influence IMF content. The Erhualian pig is characterized by excellent meat quality and high IMF content. Understanding the causes of high IMF content in the Erhualian pig is crucial for the effective conservation and utilization of the Erhualian pig resource and provides valuable insights for improving other pig breeds. Numerous genes associated with IMF deposition in pigs have been identified. For example, transcriptome sequencing identified differentially expressed genes related to glycerolipid metabolism, PNPLA3, and PLIN1, a key gene regulating lipolysis in adipocytes, between high and low IMF content Enshi Black pigs. Furthermore, differentially expressed genes related to lipid metabolism, such as SPTLC3 and NR3C2, were found between high IMF content local Huai pigs and low IMF content Duroc pigs.

[0003] Cell cycle associated protein 1 (CAPRIN1) is a cell cycle protein that regulates ATP binding, signaling adapter, and RNA binding activities, and participates in membraneless organelle assembly and gene expression regulation. CAPRIN1 is a key molecule in regulating stress granule formation and homeostasis. It has been shown that stress granules can regulate fatty acid oxidation through mitochondria and are closely associated with lipid droplet accumulation. Therefore, CAPRIN1 may regulate lipid metabolism and promote lipid accumulation through stress granules. Furthermore, mutations in the CAPRIN1 gene can lead to neurodevelopmental disorders, including symptoms such as ADHD and myasthenia gravis. Studies have also found that cyclic CAPRIN1 can inhibit lipid synthesis in colorectal cancer cells by regulating ACC1. CAPRIN1 is widely expressed in porcine tissues, with higher expression in white adipose tissue, spleen, and kidney. This suggests that CAPRIN1 may regulate lipid production in the body, but studies on CAPRIN1 in pigs have not been reported to date. Summary of the Invention

[0004] The purpose of the present invention is to address the defects of the existing technology and propose a primer and application of SNP molecular markers for detecting the intramuscular fat content of pigs, so as to conveniently and quickly determine the intramuscular fat content of pork, thereby providing a basis for obtaining better pork quality.

[0005] The present invention solves the technical problem through the following technical solutions:

[0006] In a first aspect, the present invention provides a SNP molecular marker for detecting pig intramuscular fat content, wherein the SNP molecular marker is located at base 26859339 of the pig chromosome 2 in the second intron region of the CAPRIN1 gene, and the genotype of the SNP molecular marker is AA, AG or GG, and the number is rs346324673.

[0007] When the SNP molecular marker genotype is GG, the intramuscular fat content of pork is higher than that of the GA and AA genotypes. Specifically, the intramuscular fat content of pork samples with the GG genotype at this mutation site is higher than that of samples with the GA and AA genotypes at this mutation site. The intramuscular fat content of pork is determined based on the g.26859339G>A mutation site in the intron region of the CAPRIN1 gene.

[0008] In a second aspect, the present invention provides the use of the aforementioned molecular markers in identifying or assisting in identifying the intramuscular fat content of Erhualian pigs.

[0009] In a third aspect, the present invention provides the use of the aforementioned molecular markers in the preparation of products for identifying or assisting in identifying the intramuscular fat content of Erhualian pigs.

[0010] In a fourth aspect, the present invention provides the use of the aforementioned molecular markers in the selection or auxiliary selection of pigs with high intramuscular fat trait.

[0011] In a fifth aspect, the present invention provides the use of the aforementioned molecular markers in the preparation of products for breeding or assisting in the breeding of pigs with high intramuscular fat traits.

[0012] In a sixth aspect, the present invention further provides a primer pair for detecting the above-mentioned SNP molecular marker:

[0013] CAPRIN1-F: 5′-AAGTGCTCTCTTGCGACACA-3′, as shown in SEQ NO. 3;

[0014] CAPRIN1-R: 5′-CCCCAGTAAGACCCTAGCCT-3′, as shown in SEQ NO. 4.

[0015] The amplified sequence of the primer pair is shown in SEQ NO. 1-2, wherein 26859339 (the 126th position in the sequence) is a molecular marker site.

[0016] In a seventh aspect, the present invention provides a detection reagent comprising the primer pair described above.

[0017] In an eighth aspect, the present invention provides a kit comprising the primer pair or the reagent described above.

[0018] In a ninth aspect, the present invention provides the use of the primer pair described above, or the reagent described above, or the kit described above in identifying or assisting in identifying the intramuscular fat content of Erhualian pigs.

[0019] In a tenth aspect, the present invention provides the use of the primer pair described above, or the reagent described above, or the kit described above in the preparation of a product for identifying or assisting in identifying the intramuscular fat content of Erhualian pigs.

[0020] In an eleventh aspect, the present invention provides use of the primer pair described above, or the reagent described above, or the kit described above in breeding or assisting in breeding pigs with a high intramuscular fat trait.

[0021] In a twelfth aspect, the present invention provides use of the primer pair described above, or the reagent described above, or the kit described above in the preparation of products for breeding or assisting in breeding pigs with high intramuscular fat traits.

[0022] In the thirteenth aspect, the present invention provides a method for identifying or assisting in identifying the intramuscular fat content of pigs, comprising the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pairs described above; when the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA genotypes.

[0023] In the fourteenth aspect, the present invention provides a method for breeding or assisting in breeding pigs with high intramuscular fat traits, comprising the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pairs described above; when the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of the AG and AA genotypes.

[0024] The beneficial effects of the present invention are as follows: by studying the correlation between the CAPRIN1 gene and the intramuscular fat content of Erhualian pigs, a SNP site rs346324673 related to the intramuscular fat content was screened in the intron region, thereby obtaining functional genes and molecular genetic markers related to the intramuscular fat content, laying the foundation for further controlling the intramuscular fat content of pork, which is of great significance for improving meat quality and providing a basis for conveniently and quickly judging the intramuscular fat content of pork, thereby obtaining better meat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 QQ schematic diagram of genome-wide association analysis of intramuscular fat content.

[0026] Figure 2 Manhattan plot of genome-wide association analysis of intramuscular fat content.

[0027] Figure 3 Sequencing maps of different genotypes of the CAPRIN1 gene.

[0028] Figure 4 Schematic diagram of the correlation between different genotypes and intramuscular fat content.

[0029] Figure 5 Schematic diagram of the mRNA expression results of the CAPRIN1 gene in individuals with different genotypes. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the examples. The experimental methods in the following examples without specifying specific conditions are generally based on well-known methods in the art.

[0031] Example 1

[0032] 1. Experimental Animals

[0033] This study used 189 Erhualian pigs collected from the Jiaoxi Erhualian Pig Professional Cooperative in Tianning District, Changzhou City. All pigs were raised under the same conditions and slaughtered at a designated slaughterhouse at approximately 250–300 days of age.

[0034] Before slaughter, each pig's ID and live weight were recorded. Immediately after slaughter, approximately 200 g of longissimus dorsi muscle sample was collected from the left flank of each pig. A portion of the longissimus dorsi muscle sample was used for DNA extraction and determination of intramuscular fat content.

[0035] 2. Experimental steps

[0036] 2.1 Determination of intramuscular fat content

[0037] (1) Spread the quantitative filter paper in a clean enamel dish and dry it at 105℃ for more than 2 hours until its weight does not change. Use a precision balance (sensitivity: 0.00001g) to accurately weigh the dried filter paper (W1).

[0038] (2) Chop 2-3g of muscle sample into small pieces and wrap them in dry filter paper. Weigh the paper package (W2). Spread the paper package onto a clean enamel tray and place it in an oven at 65°C for at least 15 hours (or overnight) until the weight remains unchanged. Weigh the dried paper package (W3).

[0039] (3) Place the dried filter paper bag in a Soxhlet extractor and pour in anhydrous ether to soak overnight (the ether completely submerges the filter paper bag). The next morning, open the ether reflux device and reflux at 75°C for more than 9 hours.

[0040] (4) After the extraction is completed, take out the filter paper bag and spread it on a clean enamel tray. Let the ether evaporate completely in a ventilated place for 30 minutes. Dry it at 105℃ for more than 2 hours until its weight does not change. Weigh the weight of the dried paper bag (W4).

[0041] (5) To eliminate the error caused by the unequal loss of water during sample pretreatment, the relative intramuscular fat content was expressed as the percentage of the total fat content after extraction to the dried sample (dried at 105°C for 15 h). The calculation formula is as follows:

[0042] Intramuscular fat content (%) = (W3-W4) / (W3-W1)×100%

[0043] 2.2 Genomic DNA extraction

[0044] (1) Take about 100 mg of pig longissimus dorsi muscle tissue stored at -80°C, chop it carefully, add 500 μL tissue lysis buffer and 50 μL 10 mg / mL proteinase K, shake and mix, and then incubate in a 55°C water bath overnight.

[0045] (2) Add 500 μL of DNA extraction phenol reagent, shake vigorously for 10 min, and centrifuge at 12000 rpm for 10 min at 4°C.

[0046] (3) Aspirate the supernatant and add 1 mL of a mixture of DNA extraction phenol reagent: chloroform: isoamyl alcohol (25:24:1), shake to mix, and centrifuge at 12,000 rpm for 10 min at 4°C.

[0047] (4) Aspirate the supernatant, add an equal volume of chloroform, shake to mix, and centrifuge at 12,000 rpm for 10 min at 4°C.

[0048] (5) Aspirate the supernatant, add 1 mL of anhydrous ethanol to precipitate DNA, and centrifuge at 12,000 rpm at 4°C for 5 min.

[0049] (6) Pour off the liquid, add 1 mL of 75% ethanol to wash the DNA precipitate, shake gently, centrifuge at 12,000 rpm for 15 min, pour off the remaining liquid and air dry.

[0050] (7) Add 20 μL of ultrapure water and pipette until the DNA is completely dissolved. Measure the concentration on a NanoDrop 2000 and store at -20°C.

[0051] 2.3 DNA library construction and sequencing

[0052] In this experiment, library construction and sequencing were performed by Beijing Compson Agricultural Technology Co., Ltd. DNA was randomly fragmented using Covaris technology, followed by end-repair, A-addition, adapter addition, and PCR enrichment to construct the DNA library. Qualified libraries underwent whole-genome resequencing using the BGI DNBSEQ-T7 sequencing platform. Data were quality-controlled using Fastp. The filtering conditions were:

[0053] (1) Remove reads with adapters;

[0054] (2) remove reads with N ratio exceeding 1%;

[0055] (3) remove reads where the bases with Q ≤ 5 account for more than 50%;

[0056] (4) Remove reads with length less than 100 bp.

[0057] The quality-controlled reads were aligned to the porcine reference genome, version 110 (https: / / ftp.ensembl.org / pub / release-110 / fasta / sus_scrofa / dna / ) using BWA software. Sequencing depth, genome coverage, and other information for each sample were calculated and converted to a different format using Samtools. GATK software was used to detect single-nucleotide polymorphisms (SNPs) from the aligned data, generating a vcf file. VariantFiltration was then used to filter the data according to the following parameters: QD < 2.0, FS > 60.0, MQ < 40.0, SOR > 3.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0.

[0058] 2.4 Genotype data screening

[0059] Use vcftools and plink software to further screen the SNPs in the vcf file to retain SNP sites suitable for GWAS analysis. The specific screening steps are as follows:

[0060] (1) Delete SNP sites with multiple mutant bases and only retain SNPs with two alleles;

[0061] (2) SNP sites that were not mapped to autosomes were deleted, and only SNP sites on chromosomes 1–18 were retained for subsequent analysis;

[0062] (3) Delete SNP sites with a deletion rate ≥ 0.10;

[0063] (4) Delete SNP sites with minimum allele frequency (MAF) ≤ 0.05;

[0064] (5) Delete SNP sites that do not conform to the Hardy-Weinberg equilibrium (p < 0.01).

[0065] The SNPs screened through the above steps will be converted into PLink bed format files for subsequent analysis.

[0066] 2.5 Principal Component Analysis

[0067] This study used principal component analysis (PCA) using the --pca 10 command in Plink software. By decomposing the genetic covariance matrix, we quantified the relationships between populations and detected potential stratification. Based on the results, principal component values that captured the main genetic variation between populations were incorporated into the GWAS covariate matrix to correct for the confounding effects of population structure on association analysis results. The first three principal component analysis results were visualized using R 4.3.3 to visualize the population stratification patterns.

[0068] 2.6 Linear Mixed Models

[0069] This study used the univariate linear mixed model (LMM) provided by GEMMA for GWAS analysis. The specific model is: y = Wα + xβ + u + ε, where y is a 1×n matrix of phenotypic values (n is the number of individuals); W is an n×c fixed-effect matrix consisting of individual live weight, sampling batch, the first three principal component values, and a column of 1 as an intercept; α is a c-dimensional coefficient vector containing the intercept term; x is the genotype vector; β is the polymorphic marker effect value; u is an n-dimensional random effect vector; and ε is an n-dimensional error vector. GEMMA performs hypothesis testing for each SNP, with the alternative hypothesis H1: β ≠ 0 and the null hypothesis H0: β = 0. Wald tests are used to estimate β and the corresponding p-values. Manhattan plots of the p-values for all SNPs are plotted using the R package qqman to demonstrate loci's significance.

[0070] 2.7 Population Stratification Correction Test

[0071] The first three PCA components were added as covariates to adjust for the effects of population stratification. To examine the effect of population stratification correction and avoid false positive results due to population stratification, the R package qqman was used to create QQ plots to visually represent the difference between predicted and observed values. The genomic inflation factor λ was also calculated to assess the correction for population stratification.

[0072] 2.8 Candidate gene screening

[0073] The obtained significant loci were aligned to the annotation file of the pig reference genome version 110 in the Ensembl database using R 4.3.3 to obtain genes that potentially affect intramuscular fat traits and the positions of the loci within the genes.

[0074] 2.9 Primer Design

[0075] The following primers were designed based on the porcine CAPRIN1 gene sequence (Chromosome2: 26,830,267-26,875,375) in the Ensembl database, and the amplified fragment length was 228 bp.

[0076] CAPRIN1-F: 5'-AAGTGCTCTCTTGCGACACA-3'

[0077] CAPRINA-R: 5'-CCCCAGTAAGACCCTAGCCT-3'.

[0078] 2.10 PCR amplification

[0079] The PCR reaction system used in this experiment is shown in Table 1:

[0080] Table 1 Sequencing PCR reaction system

[0081]

[0082] 2.11 PCR product sequencing and sequence alignment

[0083] The amplified products were subjected to agarose gel electrophoresis test, and single band samples that met the target fragment length and had high abundance were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing files were viewed and compared using DNAstar7.1 software, and individual genotype information was calculated.

[0084] 2.12 Tissue RNA Extraction

[0085] (1) Clean the homogenizer rotor with DEPC water and then with Trizol reagent.

[0086] (2) Take about 100 mg of longissimus dorsi muscle tissue and place it in a 2 mL centrifuge tube. Add 500 μL of Trizol reagent to the tube and treat the longissimus dorsi muscle tissue with a homogenizer. Finally, add 500 μL of Trizol reagent and place on ice for 5 minutes.

[0087] (3) Add 200 μL of chloroform to the centrifuge tube, shake and mix for 30 seconds, then place on ice for 10 minutes and centrifuge at 12,000 rpm at 4°C for 15 minutes.

[0088] (4) Transfer the upper layer of liquid to a 1.5 mL centrifuge tube. Add 500 μL of pre-cooled isopropanol to the tube, mix thoroughly, let stand at room temperature for 10 min, and centrifuge at 12,000 rpm for 10 min at 4°C.

[0089] (5) Discard the supernatant, add 1000 μL of pre-cooled 75% ethanol, shake gently, and centrifuge at 12,000 rpm at 4°C for 5 min.

[0090] (6) Pour off the supernatant and retain the RNA precipitate at the bottom. Use a pipette to remove as much water as possible from the bottom of the tube and air-dry in a clean bench for 25 minutes.

[0091] (7) Dissolve the RNA in 20 μL of DEPC water in a centrifuge tube and let it stand for 15 minutes. Measure the RNA concentration using a NanoDrop spectrophotometer and calculate the OD260 / 280 ratio to determine the RNA quality. An OD260 / 280 ratio of 1.8-2.0 indicates acceptable RNA quality. After RNA quality determination, store at -80°C or use directly for reverse transcription.

[0092] 2.13 Reverse transcription

[0093] Add the components of the reverse transcription system in Table 2 to a 0.2 mL PCR tube, mix well, and centrifuge.

[0094] Table 2 Reverse transcription reaction system

[0095]

[0096] Then, place the 0.2 mL PCR tube in a PCR instrument and run the following reaction schedule: 37°C for 15 minutes, 85°C for 5 seconds, and finally 4°C. Store the resulting cDNA at -20°C.

[0097] 2.14 Primer design for fluorescent quantitative PCR

[0098] The fluorescent quantitative PCR primer sequences for CAPRIN1 and the internal reference gene RPLP0 were designed using the reference sequences of CAPRIN1 (XM_005661021.3) and RPLP0 (NM_001098598.1) published on the NCB I website. For specific sequences, refer to the results shown in Table 3.

[0099] Table 3 Fluorescence quantitative PCR primer sequences

[0100]

[0101] 2.15 Fluorescence quantitative PCR

[0102] Add the components to the 96-well plate according to the reaction system in Table 4. After the components are added, mix them and centrifuge for a few seconds. Then, perform the test according to the fluorescence quantitative PCR reaction program in Table 5. Use the RPLP0 gene as the internal reference gene and use 2 -ΔΔCt Method to conduct statistical analysis on the data.

[0103] Table 4 Fluorescence quantitative PCR reaction system

[0104]

[0105] Table 5 Fluorescence quantitative PCR reaction procedure

[0106]

[0107] 2.16 Statistical Analysis

[0108] The experimental data were statistically analyzed using SPSS 20.0 software. One-way analysis of variance and the Bonferroni multiple comparison method were used to compare expression levels among individuals with different genotypes. Trait association analysis among different genotypes was performed using a mixed linear model: Y = μ + G + D + e, where Y represents intramuscular fat content, μ represents the population mean, G represents genotype, D represents pre-slaughter live weight as a covariate, and e represents the residual. GraphPad Prism version 8.0 software was used for plotting, and all data are presented as mean ± standard error (SEM).

[0109] 3. Test results

[0110] 3.1 Body weight and intramuscular fat content determination

[0111] The average live weight of all Erhualian pigs before slaughter was 79.74±13.16kg, with a minimum of 60kg and a maximum of 119.5kg, and a coefficient of variation of 16.50%. The average intramuscular fat content was 3.66±1.49, with a minimum of 1.37 and a maximum of 9.72, and a coefficient of variation of nearly 40.71%.

[0112] 3.2 Genome resequencing data quality control

[0113] This study generated a total of 6,100.59 Gb of data. After quality control, an average of 317,740,500 clean reads were obtained per sample, with an average Q30 of 96.14% and an average GC content of 42.31%. The clean reads were aligned to the porcine reference genome version 110 using BWA software, achieving an average alignment rate of 99.67% across all samples and an average sequencing depth of 12.09×. These SNPs were then screened using Plink. After quality control, a total of 14,254,908 SNPs remained for subsequent genome-wide association analysis.

[0114] 3.3 Genome-wide association analysis results

[0115] The first three PCA principal components were used to correct the group stratification, and the Q-Qplot was used to test the correction results of the group stratification ( Figure 1 ), the calculated genome expansion coefficient λ is 1.044259, which is within a reasonable range. It can be seen that the true value and the predicted value fit well, indicating that the population stratification effect has been effectively corrected.

[0116] The SNP locus located at 26859339 on chromosome 2 was found to be significantly associated with the intramuscular fat content of Erhualian pigs (P = 6.45 × 10 -6 )( Figure 2 ), so it was named CAPRIN1 gene g.26859339G>A site and numbered as rs346324673. At the same time, the PCR amplification products of the three genotypes of this site were sequenced to verify the presence of AA, AG and GG genotypes at this site ( Figure 3 ), indicating that this site can also be amplified by primers and then sequenced for typing.

[0117] 3.4 Correlation analysis between different genotypes and intramuscular fat content

[0118] Analysis of the specific relationship between different genotypes and intramuscular fat content in the resequencing data showed that the intramuscular fat content of individuals with the GG genotype at rs346324673 was significantly higher than that of individuals with the AG and AA genotypes (P<0.05), while the intramuscular fat content of individuals with the AG genotype was not significantly different from that of individuals with the AA genotype (P>0.05). Figure 4 ).

[0119] 3.5 Analysis of mRNA expression in individuals with different genotypes

[0120] Analysis of the differences in CAPRIN1 gene mRNA expression between individuals with different genotypes revealed that the CAPRIN1 gene expression level in individuals with GG genotype was significantly higher than that in individuals with AG and AA genotypes (P<0.05), while the CAPRIN1 gene expression level in individuals with AG genotype was not significantly different from that in individuals with AA genotype (P>0.05). Figure 5 ).

[0121] 4. Results Analysis

[0122] The mutation site screened by this invention is significantly associated with pig intramuscular fat and can be developed as a single-nucleotide polymorphism (SNP) molecular marker for measuring pig intramuscular fat content. For the g.26859339G>A mutation site in the intronic region of the CAPRIN1 gene, individuals with the GG genotype have significantly higher intramuscular fat content than those with the AG and AA genotypes.

[0123] The present invention screened a SNP site related to the intramuscular fat content of Erhualian pork, thereby obtaining functional genes and molecular genetic markers related to intramuscular fat content. By optimizing the dominant alleles of these SNP molecular markers, a reference basis can be provided for SNP molecular marker-assisted breeding related to intramuscular fat content in pigs, which is of great significance for the improvement of pork quality traits.

[0124] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A primer pair for detecting SNP molecular markers for pig intramuscular fat content, characterized in that: The SNP molecular marker rs346324673 is located at base 26859339 of the second intron region of the CAPRIN1 gene on the second chromosome of the pig, and the genotype of the SNP molecular marker is AA, AG or GG. The primer pair sequences are as follows: CAPRIN1-F: 5'-AAGTGCTCTCTTGCGACACA-3', as shown in SEQ ID NO. 3; CAPRIN1-R: 5′-CCCCAGTAAGACCCTAGCCT-3′, as shown in SEQ ID NO.

4.

2. The primer pair for detecting SNP molecular markers for pig intramuscular fat content according to claim 1, characterized in that: When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of the AG and AA genotypes. The nucleotide sequences of the SNP molecular marker are shown in SEQ ID NO.1 and SEQ ID NO.2; wherein the base at position 126 in SEQ ID NO.1 is A, and the base at position 126 in SEQ ID NO.2 is G.

3. A detection reagent, characterized in that: The reagent contains the primer pair according to claim 1.

4. A kit, characterized in that The kit contains the primer pair according to claim 1 or the reagent according to claim 3.

5. Use of the primer pair according to claim 1 or 2, the reagent according to claim 3, or the kit according to claim 4 in identifying or assisting in identifying the intramuscular fat content of pigs.

6. Use of the primer pair according to claim 1 or 2, the reagent according to claim 3, or the kit according to claim 4 in preparing a product for identifying or assisting in identifying the content of intramuscular fat in pigs.

7. Use of the primer pair according to claim 1 or 2, the reagent according to claim 3, or the kit according to claim 4 in breeding or assisting in breeding pigs with a high intramuscular fat trait.

8. Use of the primer pair according to claim 1 or 2, the reagent according to claim 3, or the kit according to claim 4 in preparing a product for breeding or assisting in breeding pigs with a high intramuscular fat trait.

9. A method for identifying or assisting in identifying the intramuscular fat content of pigs, characterized in that: The steps include: The extracted genomic DNA is amplified by PCR and genotyped using the primer pair according to claim 1. When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA genotypes.

10. A method for breeding or assisting in breeding pigs with high intramuscular fat trait, characterized in that: The steps include: The extracted genomic DNA is amplified by PCR and genotyped using the primer pair according to claim 1. When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA genotypes.

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