A caprin1 gene molecular marker related to intramuscular fat content of erhualian pigs and application thereof

Abstract

The application relates to a CAPRIN1 gene molecular marker related to intramuscular fat content of Erhualian pigs and application thereof. A variation site is located in a second intron region of a pig CAPRIN1 gene (at the 26859339th base of a second pig chromosome), and a polymorphic SNP site is provided, and the genotype of the SNP site is AA, AG or GG. The intramuscular fat content of the Erhualian pig 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 the 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 application has the beneficial effect that, by selecting 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] This invention relates to a molecular marker for the CAPRIN1 gene related to the detection of intramuscular fat content in Erhualian pigs and its application, belonging to the field of molecular biology technology. Background Technology

[0002] Intramuscular fat content in pork is a key quality attribute determining consumer acceptance and market value. It is white fat tightly bound to membrane proteins in muscle tissue. Intramuscular fat content depends on the number and size of intramuscular fat cells, and its deposition rate is closely related to muscle growth. Furthermore, breed, feeding methods, hormones, and genes also significantly influence intramuscular fat content. The Erhualian pig is characterized by its excellent meat quality and high intramuscular fat content. Exploring the reasons for the high intramuscular fat content in the Erhualian pig is of great significance for the effective protection and utilization of Erhualian pig resources and also provides important reference value for the improvement of other pig breeds. Currently, many genes have been found to be related to intramuscular fat deposition in pigs. For example, transcriptome sequencing has identified differentially expressed genes related to glycerol lipid metabolism, such as PNPLA3, and PLIN1, a key gene regulating intramuscular fat breakdown, in Enshi Black pigs between high and low intramuscular fat content groups. Differentially expressed genes related to lipid metabolism, such as SPTLC3 and NR3C2, have been found in local Huai pigs with high intramuscular fat content and Duroc pigs with low intramuscular fat content.

[0003] Cell cycle associated protein 1 (CAPRIN1) is a cyclin that regulates ATP binding, signal adaptor and RNA binding activities, and participates in the assembly of non-membrane organelles and the regulation of gene expression. CAPRIN1 is a key molecule regulating the formation and homeostasis of stress granules. It has been found that stress granules can regulate fatty acid oxidation through mitochondria, and are closely related to 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 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 pig tissues, with higher expression in white adipose tissue, spleen, and kidney. It can be seen that the CAPRIN1 gene may regulate lipid production in the body; however, research on the CAPRIN1 gene in pigs has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a primer for an SNP molecular marker to detect intramuscular fat content in pigs and its application, which facilitates rapid determination of intramuscular fat content in pork and provides a basis for obtaining better pork quality.

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

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

[0007] When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of the GA and AA genotypes. That is, the intramuscular fat content of samples with the GG genotype at the mutation site is higher than that of samples with the GA and AA genotypes at the 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] Secondly, the present invention provides the application of the molecular markers described above in the identification or auxiliary identification of intramuscular fat content in Erhualian pigs.

[0009] Thirdly, the present invention provides the application of the molecular markers described above in the preparation of products for identifying or assisting in the identification of intramuscular fat content in Erhualian pigs.

[0010] Fourthly, the present invention provides the application of the molecular markers described above in the selection or assisted selection of pigs with high intramuscular fat traits.

[0011] Fifthly, the present invention provides the application of the molecular markers described above 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 markers:

[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, where 26859339 (position 126 in the sequence) is the molecular marker site.

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

[0017] Eighthly, the present invention provides a kit containing the primer pairs or reagents described above.

[0018] In a ninth aspect, the present invention provides the application of the primer pairs, reagents, or kits described above in the identification or auxiliary identification of intramuscular fat content in Erhualian pigs.

[0019] In a tenth aspect, the present invention provides the use of the primer pairs, reagents, or kits described above in the preparation of products for identifying or assisting in the identification of intramuscular fat content in Erhualian pigs.

[0020] In the eleventh aspect, the present invention provides the application of the primer pairs, reagents, or kits described above in the selection or assisted selection of pigs with high intramuscular fat traits.

[0021] In a twelfth aspect, the present invention provides the use of the primer pairs, reagents, or kits described above in the preparation of products for breeding or assisting in the breeding of pigs with a high intramuscular fat trait.

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

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

[0024] The beneficial effects of this invention are as follows: By studying the correlation between the CAPRIN1 gene and intramuscular fat content in Erhualian pigs, a SNP locus rs346324673 related to intramuscular fat content was screened in the intron region. This yielded functional genes and molecular genetic markers related to intramuscular fat content, laying the foundation for further control of pork intramuscular fat content. This is of great significance for improving meat quality and provides a basis for conveniently and quickly determining the intramuscular fat content of pork to obtain better meat quality. Attached Figure Description

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

[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 A schematic diagram showing the relationship between different genotypes and intramuscular fat content.

[0029] Figure 5 A schematic diagram showing the mRNA expression results of the CAPRIN1 gene in individuals with different genotypes. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out in accordance with known means in the art.

[0031] Example 1

[0032] 1. Laboratory animals

[0033] The 189 Erhualian pigs used in this study were collected from the Jiaoxi Erhualian Pig Professional Cooperative in Tianning District, Changzhou City. All individuals were raised in the same environment and were sent to a designated slaughterhouse for slaughter at approximately 250-300 days of age.

[0034] Before slaughter, the number and pre-slaughter live weight of each pig were recorded. Immediately after slaughter, approximately 200g of longissimus dorsi muscle sample was collected from the last two ribs of the left carcass. A portion of the longissimus dorsi muscle sample was selected for DNA extraction and intramuscular fat content determination.

[0035] 2. Experimental Procedure

[0036] 2.1 Measurement of intramuscular fat content

[0037] (1) Distribute the quantitative filter paper in a clean enamel dish and dry it at 105°C for more than 2 hours until its weight no longer changes. Accurately weigh the dried filter paper (W1) using a precision balance (sensitivity: 0.00001g).

[0038] (2) Cut 2-3g of muscle sample into small pieces, wrap them in dried filter paper, and weigh the paper package (W2). Spread the paper packages on clean enamel trays, place them in an oven, and dry at 65℃ for at least 15 hours (or overnight) until their weight no longer changes. Weigh the dried paper packages (W3).

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

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

[0041] (5) To eliminate errors caused by uneven moisture loss during sample pretreatment, the relative content of intramuscular fat is expressed as the percentage of the total extracted fat content in the dried sample (dried at 105℃ for 15h), and the calculation formula is as follows:

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

[0043] 2.2 Genomic DNA Extraction

[0044] (1) Take about 100 mg of porcine longissimus dorsi muscle tissue stored at -80℃, carefully cut it into small pieces, add 500 μL of tissue lysis buffer and 50 μL of 10 mg / mL proteinase K, shake to mix, and incubate overnight at 55℃.

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

[0046] (3) Take the supernatant, add 1 mL of DNA extraction phenol reagent: chloroform: isoamyl alcohol (25:24:1) mixture, shake to mix, and centrifuge at 12000 rpm for 10 min at 4℃.

[0047] (4) Take the supernatant, add an equal volume of chloroform, shake to mix, and centrifuge at 12000 rpm for 10 min at 4℃.

[0048] (5) Take the supernatant, add 1 mL of anhydrous ethanol to precipitate the DNA, and centrifuge at 12000 rpm for 5 min at 4℃.

[0049] (6) Discard the liquid, add 1 mL of 75% ethanol to wash the DNA precipitate, shake gently, centrifuge at 12000 rpm for 15 min, discard the residual liquid and air dry.

[0050] (7) Add 20 μL of ultrapure water, blow until the DNA is completely dissolved, measure the concentration on NanoDrop 2000 and store at -20℃.

[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, addition of "A" letters, adapter addition, and PCR enrichment to construct the DNA library. Qualified libraries were then used for whole-genome resequencing on the BGI DNBSEQ-T7 sequencing platform. FastP was used for quality control of the data obtained after sequencing. The filtering conditions were as follows:

[0053] (1) Remove reads with connectors;

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

[0055] (3) Remove more than 50% of reads with Q≤5 bases;

[0056] (4) Remove reads with a length < 100bp.

[0057] The quality-controlled reads were aligned to the pig reference genome, version 110 (https: / / ftp.ensembl.org / pub / release-110 / fasta / sus_scrofa / dna / ), using BWA software. Samtools was used to analyze sequencing depth, genome coverage, and other information for each sample and to convert the format. GATK software was used to detect 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, ReadPosRankSum < -8.0.

[0058] 2.4 Genotype data screening

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

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

[0061] (2) Delete SNP sites that are not located on autosomes, and retain only SNP sites on chromosomes 1-18 for subsequent analysis;

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

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

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

[0065] The SNPs filtered 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 the `--pca 10` command in the PLINK software to perform principal component analysis. By decomposing the genetic variation covariance matrix, the study quantified the kinship among populations and detected potential stratification. Based on the results, the principal component values ​​that captured the main genetic variation information among populations were incorporated into the covariate matrix of the GWAS analysis to correct for the confounding effects of population structure on the association analysis results. The results of the first three principal component analyses were visualized using R 4.3.3 to intuitively reflect the population stratification patterns.

[0068] 2.6 Linear Mixture Model

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

[0070] 2.7 Population Stratification Correction Test

[0071] The effects of population stratification were corrected by adding the first three PCA components to the covariates. To examine the correction effect of population stratification and avoid false positive results caused by population stratification, the R package qqman was used to draw a QQ plot to visually represent the difference between predicted and observed values, and the genome expansion coefficient λ was calculated to evaluate the correction of population stratification.

[0072] 2.8 Candidate Gene Screening

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

[0074] 2.9 Primer Design

[0075] Primers were designed based on the porcine CAPRIN1 gene sequence (Chromosome2:26,830,267-26,875,375) from 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 Sequencing and sequence alignment of PCR products

[0083] The amplification products were subjected to agarose gel electrophoresis. 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 to collect individual genotype information.

[0084] 2.12 Tissue RNA Extraction

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

[0086] (2) Take about 100 mg of the longissimus dorsi muscle tissue and put it into a 2 mL centrifuge tube. Add 500 μL of Trizol reagent to the tube, process the longissimus dorsi muscle tissue with a homogenizer, and finally add another 500 μL of Trizol reagent. Place on ice for 5 min.

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

[0088] (4) Transfer the upper layer of liquid to a 1.5 mL centrifuge tube, add 500 μL of pre-cooled isopropanol to the centrifuge 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, gently shake, and centrifuge at 12,000 rpm for 5 min at 4°C.

[0090] (6) Discard the supernatant, retain the RNA precipitate at the bottom, remove the water at the bottom of the tube as much as possible with a pipette, and air dry in a clean bench for 25 minutes.

[0091] (7) Add 20 μL of DEPC water to a centrifuge tube to dissolve the RNA, let it stand for 15 min, and use a NanoDrop spectrophotometer to determine the RNA concentration. Calculate the OD260 / 280 ratio to judge the quality of the RNA. The OD260 / 280 ratio should be 1.8-2.0, indicating that the quality of the RNA is qualified. After determining the RNA quality, store it at -80℃ or use it directly for reverse transcription.

[0092] 2.13 Reverse transcription

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

[0094] Table 2 Reverse transcription reaction system

[0095]

[0096] The 0.2 mL PCR tube was then placed in the PCR instrument, and the reaction program was 37°C for 15 min; 85°C for 5 s; and 4°C for cooling and storage. The cDNA obtained after the reaction was completed was stored at -20°C.

[0097] 2.14 Primer Design for Quantitative Real-Time PCR

[0098] The primer sequences for quantitative real-time PCR of 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. The specific sequences are shown in Table 3.

[0099] Table 3 Primer sequences for real-time PCR

[0100]

[0101] 2.15 Quantitative Real-Time PCR

[0102] Add the components to the 96-well plate according to the reaction system in Table 4. After adding all components, mix well and centrifuge for a few seconds. Perform the detection according to the real-time PCR reaction procedure in Table 5, using the RPLLP0 gene as an internal reference gene. -ΔΔCt The method involves statistical analysis of the data.

[0103] Table 4. Real-time PCR reaction system

[0104]

[0105] Table 5. Quantitative PCR reaction procedure

[0106]

[0107] 2.16 Statistical Analysis

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

[0109] 3. Test Results

[0110] 3.1 Measurement of body weight and intramuscular fat content

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

[0112] 3.2 Quality Control of Genome Resequencing Data

[0113] This study yielded a total of 6100.59 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 obtained from sequencing were aligned to the pig reference genome version 110 using BWA software, with an average alignment rate of 99.67% and an average sequencing depth of 12.09× for all samples. Plink was then used to screen these SNPs, and after quality control, a total of 14,254,908 SNP loci remained for subsequent genome-wide association studies.

[0114] 3.3 Results of Genome-wide Association Analysis

[0115] The first three PCA principal components were used to correct the population stratification, and the correction results were verified using Q-Q plots. Figure 1 The calculated genome expansion coefficient λ was 1.044259, which is within a reasonable range. This shows that the actual value and the predicted value fit well, indicating that the population stratification effect has been effectively corrected.

[0116] A SNP locus located at chromosome 26859339 in the whole genome was found to be significantly correlated with intramuscular fat content in Erhualian pigs (P = 6.45 × 10⁻⁶). -6 ()( Figure 2 Therefore, it was named the CAPRIN1 gene g.26859339G>A site, and numbered rs346324673. Simultaneously, PCR amplification products of the three genotypes at this site were sequenced, verifying the presence of AA, AG, and GG genotypes at this site. Figure 3 This indicates that this site can also be used for primer amplification followed by sequencing and genotyping.

[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 resequencing data revealed 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 there was no significant difference in intramuscular fat content between individuals with the AG genotype and those with the AA genotype (P>0.05). Figure 4 ).

[0119] 3.5 mRNA expression analysis of individuals with different genotypes

[0120] Analysis of CAPRIN1 gene mRNA expression differences among individuals with different genotypes revealed that CAPRIN1 gene expression levels were significantly higher in individuals with the GG genotype than in those with the AG and AA genotypes (P<0.05), while there was no significant difference in CAPRIN1 gene expression between individuals with the AG genotype and those with the AA genotype (P>0.05). Figure 5 ).

[0121] 4. Results Analysis

[0122] The mutation sites screened in this invention are significantly correlated with intramuscular fat traits in pigs and can be developed into SNP molecular markers for detecting intramuscular fat content in pigs. For the g.26859339G>A mutation site in the intron region of the CAPRIN1 gene, individuals with the genotype GG have significantly higher intramuscular fat content than individuals with the genotypes AG and AA.

[0123] This invention screened a SNP locus associated with intramuscular fat content in 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 improving pork quality traits.

[0124] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. The application of primer pairs of SNP molecular markers for detecting intramuscular fat content in pigs, or reagents containing said primer pairs, or kits containing said primer pairs, in the identification or auxiliary identification of intramuscular fat content in Erhualian pigs; 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; 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; The genotype of the SNP molecular marker is AA, AG, or GG; When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA types.

2. The application of primer pairs of SNP molecular markers for detecting intramuscular fat content in pigs, or reagents containing said primer pairs, or kits containing said primer pairs, in the preparation of products for identifying or assisting in the identification of intramuscular fat content in Erhualian pigs; 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; 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; The genotype of the SNP molecular marker is AA, AG, or GG; When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA types.

3. Application of primer pairs of SNP molecular markers for detecting intramuscular fat content in pigs, or reagents containing said primer pairs, or kits containing said primer pairs in the breeding or assisted breeding of Erhualian pigs with high intramuscular fat trait; 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; 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; The genotype of the SNP molecular marker is AA, AG, or GG; When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA types.

4. The application of primer pairs of SNP molecular markers for detecting intramuscular fat content in pigs, or reagents containing said primer pairs, or kits containing said primer pairs, in the preparation of products for breeding or assisting in the breeding of Erhualian pigs with high intramuscular fat trait; 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; 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; The genotype of the SNP molecular marker is AA, AG, or GG; When the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA types.

5. A method for identifying or assisting in the identification of intramuscular fat content in Erhualian pigs, characterized in that, The procedure includes the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pair described in claim 1, wherein when the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA types.

6. A method for selectively or assistedly selectively breeding Erhualian pigs with a high intramuscular fat trait, characterized in that, The procedure includes the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pair described in claim 1, wherein when the genotype of the SNP molecular marker is GG, the intramuscular fat content of pork is higher than that of AG and AA types.

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