A SNP molecular marker associated with the IMF trait of Ningxiang pigs and its application

Abstract

The present invention discloses a SNP molecular marker associated with the IMF trait of Ningxiang pigs and its application. The SNP molecular marker includes at least one of the SNP1 site, SNP2 site, SNP3 site, SNP4 site, SNP5 site, and SNP6 site. The present invention provides a SNP molecular marker associated with the IMF trait, which can efficiently and accurately identify and screen Ningxiang pig breeds with different IMF contents. It has the characteristics of rapid, accurate, and low-cost detection. It can be used for the detection and application of different Ningxiang pig breeds, providing an indispensable and important tool for the breeding of Ningxiang pig breeds with high IMF content and excellent meat quality, and for improving the quality of Ningxiang pork.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a SNP molecular marker associated with the IMF trait of Ningxiang pigs and an application thereof. Background Art

[0002] The Ningxiang pig is a famous Chinese pig breed with a history of over 1,000 years. It is a high-quality local pig breed resource. Ningxiang pork products are widely popular for their unique flavor and nutritional value. As an obese breed, Ningxiang is superior to lean meat breeds in terms of intramuscular fat (IMF). Studies have shown that the intramuscular fat (IMF) content is a key factor affecting the sensory quality and nutritional value of Ningxiang pork. Properly increasing the IMF content can improve the color, tenderness, juiciness and flavor of the meat. At this stage, the use of molecular marker technology to study the economic traits of livestock and poultry is very mature. Therefore, using molecular biology methods to increase the IMF content of Ningxiang pigs is conducive to the cultivation of Ningxiang pigs with excellent meat quality and the development of the Ningxiang pork industry.

[0003] Currently, molecular research on economic traits related to Ningxiang pigs primarily uses SSR (simple sequence repeat) markers, ISSR markers, and AFLP markers. SSR markers have low throughput and high R&D costs, making them unsuitable for large-scale commercial identification. While simple and practical, ISSR and AFLP markers offer unstable results and poor reproducibility. SNP markers offer advantages such as ease of detection, low cost, and high throughput. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a SNP molecular marker related to the IMF trait.

[0005] The present invention also provides a primer set for detecting the above-mentioned SNP molecular markers.

[0006] The present invention also provides a kit.

[0007] The invention also provides a gene chip.

[0008] The present invention also proposes the application of the above-mentioned SNP molecular marker, primer set, kit and / or gene chip.

[0009] The present invention also provides a method for identifying or assisting in identifying the meat quality traits of Ningxiang pork.

[0010] The invention also provides a Ningxiang pig breeding method.

[0011] According to a first aspect of the present invention, a SNP molecular marker associated with the IMF trait is proposed, wherein the SNP molecular marker includes at least one of the SNP1 site, the SNP2 site, the SNP3 site, the SNP4 site, the SNP5 site, and the SNP6 site;

[0012] The SNP1 site is located at base 40987222 of chromosome 2 of the reference genome Sus Scrofa 11.1, and the polymorphism is A / G;

[0013] The SNP2 site is located at base 41,137,403 of chromosome 2 of the reference genome Sus Scrofa 11.1, and the polymorphism is C / T;

[0014] The SNP3 site is located at base 73007048 of chromosome 6 of the reference genome Sus Scrofa 11.1, and the polymorphism is T / C;

[0015] The SNP4 site is located at base 2164395 of chromosome 16 of the reference genome Sus Scrofa 11.1, and the polymorphism is C / T;

[0016] The SNP5 site is located at base 2620803 of chromosome 16 of the reference genome Sus Scrofa 11.1, and the polymorphism is C / G;

[0017] The SNP6 site is located at base 2709556 of chromosome 16 of the reference genome Sus Scrofa 11.1, and the polymorphism is A / G.

[0018] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular marker is proposed.

[0019] In some embodiments of the present invention, the primer set comprises:

[0020] (1) A primer set for amplifying the SNP1 site, comprising specific primers having sequences as shown in SEQ ID NO. 7 and SEQ ID NO. 8 and a universal primer having a sequence as shown in SEQ ID NO. 9;

[0021] (2) a primer set for amplifying the SNP2 site, comprising specific primers having sequences as shown in SEQ ID NO. 10 and SEQ ID NO. 11 and a universal primer having a sequence as shown in SEQ ID NO. 12;

[0022] (3) A primer set for amplifying the SNP3 site, comprising specific primers with sequences shown in SEQ ID NO. 13 and SEQ ID NO. 14 and a universal primer with sequence shown in SEQ ID NO. 15;

[0023] (4) a primer set for amplifying the SNP4 site, comprising specific primers having sequences as shown in SEQ ID NO. 16 and SEQ ID NO. 17 and a universal primer having a sequence as shown in SEQ ID NO. 18;

[0024] (5) a primer set for detecting amplification sites, comprising specific primers having sequences as shown in SEQ ID NO. 19 and SEQ ID NO. 20 and a universal primer having a sequence as shown in SEQ ID NO. 21;

[0025] (6) A primer set for detecting amplification sites, comprising specific primers having sequences as shown in SEQ ID NO. 22 and SEQ ID NO. 23 and a universal primer having a sequence as shown in SEQ ID NO. 24.

[0026] In some embodiments of the present invention, the specific primers are connected to FAM and HEX fluorescent linker sequences, respectively.

[0027] According to a third aspect of the present invention, a kit is provided, comprising the above-mentioned primer set.

[0028] According to a fourth aspect of the present invention, a gene chip is provided, wherein the gene chip comprises the above primer set.

[0029] According to a fifth aspect of the present invention, the use of the above-mentioned SNP molecular marker, primer set, kit or gene chip in any of the following is proposed:

[0030] 1) Detect or assist in detecting the quality of Ningxiang pork;

[0031] 2) Detect or assist in detecting the IMF content of Ningxiang pork;

[0032] 3) Breeding Ningxiang pigs with pork IMF content ≥4.0;

[0033] 4) Breeding Ningxiang pigs with pork IMF content less than 4.0;

[0034] 5) Molecular marker-assisted breeding of Ningxiang pigs;

[0035] 6) Ningxiang pig breeding;

[0036] 7) Prepare Ningxiang pig breeding products.

[0037] According to a sixth aspect of the present invention, a method for identifying or assisting in identifying the IMF content of Ningxiang pork using the above-mentioned molecular markers is proposed, the method comprising the following steps:

[0038] S1. Extracting the genomic DNA of the Ningxiang pig to be tested;

[0039] S2. Perform polymorphism detection of the SNP molecular marker on the genomic DNA extracted in step S1, and determine the IMF content of the Ningxiang pig to be tested according to the genotype.

[0040] In some embodiments of the present invention, when the SNP molecular marker is SNP1, if the genotype obtained by SNP1 detection is AA, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP1 detection is AG or GG, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0041] When the SNP molecular marker is SNP2, if the genotype obtained by SNP2 detection is CT or TT, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP2 detection is CC, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0042] When the SNP molecular marker is SNP3, if the genotype obtained by SNP3 detection is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP3 detection is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0043] When the SNP molecular marker is SNP4, if the genotype obtained by SNP4 detection is CC or CT, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP4 detection is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0044] When the SNP molecular marker is SNP5, if the genotype obtained by SNP5 detection is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP5 detection is CG, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0045] When the SNP molecular marker is SNP6, if the genotype obtained by SNP6 detection is AG or GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype obtained by SNP6 detection is AA, the IMF content of the Ningxiang pig to be tested is <4.0, and it has a low IMF trait.

[0046] In some embodiments of the present invention, in step S2, the SNP molecular marker is detected using KASP (competitive allele-specific PCR) technology.

[0047] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting SNP molecular markers using KASP technology is as follows:

[0048]

[0049] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is: 94°C for 15 min; 94°C for 20 s, 65°C-57°C for 60 s, 10 cycles; 94°C for 20 s, 57°C for 60 s, 33 cycles.

[0050] According to the seventh aspect of the present invention, a Ningxiang pig breeding method is proposed, comprising the following steps: using the above-mentioned SNP molecular marker identification or auxiliary identification method for the IMF content of Ningxiang pork, selecting Ningxiang pigs with a pork IMF content ≥4.0 or a pork IMF content <4.0 for subsequent breeding.

[0051] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention provides a SNP molecular marker associated with the IMF trait, which can efficiently and accurately identify and screen Ningxiang pig breeds with different IMF contents. It has the characteristics of being fast, accurate, and having low detection costs, and can be used for the detection and application of different Ningxiang pig breeds. It provides an indispensable and important tool for the breeding of Ningxiang pig breeds with high IMF content and excellent meat quality and the improvement of Ningxiang pork quality.

[0052] The present invention provides a KASP-based SNP detection method for detecting the content of IMF in Ningxiang pig breeds. The KASP-based detection method is simple, accurate, stable, and rapid, and can be applied to different detection instruments and equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0054] Figure 1 This is the typing diagram of the SNP1 molecular marker in Example 1 of the present invention;

[0055] Figure 2 This is the typing diagram of the SNP2 molecular marker in Example 1 of the present invention;

[0056] Figure 3 This is the typing diagram of the SNP3 molecular marker in Example 1 of the present invention;

[0057] Figure 4 This is the typing diagram of the SNP4 molecular marker in Example 1 of the present invention;

[0058] Figure 5 This is the typing diagram of the SNP5 molecular marker in Example 1 of the present invention;

[0059] Figure 6 This is the typing diagram of the SNP6 molecular marker in Example 1 of the present invention. DETAILED DESCRIPTION

[0060] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0061] Example 1 A SNP molecular marker combination associated with Ningxiang pork meat quality traits

[0062] The present invention uses chip detection data of 778 purebred Ningxiang Ningxiang pigs, and performs genome-wide association analysis on the material data detected by the chip and the IMF phenotype, and screens out 6 SNP molecular markers related to the IMF trait, namely SNP1-6, wherein the molecular marker SNP1 has an A / G variation at the 40987222 base of chromosome 2 of the reference genome Sus Scrofa 11.1, the molecular marker SNP2 has a C / T variation at the 41137403 base of chromosome 2 of the reference genome Sus Scrofa 11.1, the molecular marker SNP3 has a T / C variation at the 73007048 base of chromosome 6 of the reference genome Sus Scrofa 11.1, the molecular marker SNP4 has a C / T variation at the 2164395 base of chromosome 16 of the reference genome Sus Scrofa 11.1, and the molecular marker SNP5 has a C / T variation at the 2164395 base of chromosome 16 of the reference genome Sus Scrofa There is a C / G variation at base 2620803 of chromosome 16 of 11.1, and there is an A / G variation at base 2709556 of chromosome 16 of the reference genome Sus Scrofa11.1.

[0063] When the genotype obtained by SNP1 detection is AA, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype obtained by the SNP1 detection is AG or GG, the IMF content of the Ningxiang pig to be tested is <4.0, and it has a low IMF trait;

[0064] When the genotype obtained by SNP2 detection is CT or TT, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by the SNP2 detection is CC, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0065] When the genotype obtained by SNP3 detection is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by the SNP3 detection is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0066] When the genotype obtained by SNP4 detection is CC or CT, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by the SNP4 detection is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0067] When the genotype obtained by SNP5 detection is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP5 detection is CG, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0068] When the genotype obtained by SNP6 detection is AG or GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP6 detection is AA, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait.

[0069] The screening and verification methods for specific sites are as follows:

[0070] 1. SNP chip detection

[0071] SNP typing was performed using the GGP Porcine 50K microarray. This array contains 51,000 SNPs, integrating the genetic diversity of multiple Ningxiang pig breeds, including Duroc, Landrace, and Pietrain. Each evaluated breed has an average of 50,000 polymorphic sites, covering the entire Ningxiang pig genome. The biochip system primarily consists of the microarray, scanner, and analysis software. Genome Studio was used to visualize the microarray data and generate genotype data.

[0072] 2. Genome-wide association analysis

[0073] A genome-wide association analysis (rMVP) was performed on the microarray data (Yin L, Zhang H, Tang Z et al. 2021). A mixed linear model (MLM) was used to calculate the model, and the principal component was added as a covariate for correction. A genome-wide association analysis was performed on the collected IMF-related phenotypic traits. Through extensive experimental screening, six SNPs associated with IMF traits were ultimately identified. The specific loci and the 250-bp sequence before and after the SNP are shown in Table 1.

[0074] Table 1 Site information table

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] 3. Primer design

[0083] The locus sequences (sequences shown in Table 1) were extracted from the reference genome Sus Scrofa11.1 (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_000003025.6 / ) for primer design. BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ) was used to design KASP primers. A total of 6 pairs were synthesized, and the sequences are shown in Table 2. The primers were synthesized by Invitrogen.

[0084] Each KASP marker consists of three primers: two allele-specific primers, X (Primer_X) and Y (Primer_Y), and one universal primer, C (Primer_C). The 5' ends of the specific primers are linked to LGC's KASP reaction-specific fluorophores, FAM and HEX, respectively. If only FAM fluorescence is detected in a sample, the sample's genotype is homozygous for allele X (Allele_X); if only HEX fluorescence is detected, the sample's genotype is homozygous for allele Y (Allele_Y); if both FAM and HEX fluorescence are detected, the sample's genotype is heterozygous (carrying both alleles X and Y).

[0085] When the genotype obtained by SNP1 detection is AA, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype obtained by the SNP1 detection is AG or GG, the IMF content of the Ningxiang pig to be tested is <4.0, and it has a low IMF trait;

[0086] When the genotype obtained by SNP2 detection is CT or TT, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by the SNP2 detection is CC, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0087] When the genotype obtained by SNP3 detection is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by the SNP3 detection is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0088] When the genotype obtained by SNP4 detection is CC or CT, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by the SNP4 detection is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0089] When the genotype obtained by SNP5 detection is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP5 detection is CG, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait;

[0090] When the genotype obtained by SNP6 detection is AG or GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype obtained by SNP6 detection is AA, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait.

[0091] Table 2 Alleles (Allele_X, Allele_Y) and primer sequences of KASP markers used for IMF marker detection in Ningxiang pigs

[0092]

[0093]

[0094]

[0095] 4. DNA extraction and quality control:

[0096] The magnetic bead method was used to extract DNA from the Ningxiang pork samples to be tested; the concentration of the DNA samples was detected using a Qubit fluorescence quantification instrument; the integrity of the DNA samples was tested using 1% agarose gel electrophoresis, and samples that passed quality control were used for library preparation.

[0097] 5. Verification and detection of KASP labeling:

[0098] KASP-labeled reaction sequencing was performed using the Douglas Scientific Array Tape system. The Array Tape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0099] PCR reaction system: NEXAR was used to automatically assemble the PCR amplification system. The PCR reaction system is shown in Table 3 below.

[0100] Table 3. PCR reaction system for KASP marker genotyping

[0101]

[0102]

[0103] PCR amplification: PCR was performed using SOELLEX under the following conditions: 94°C for 15 min; 94°C for 20 s, 65°C-57°C (annealing temperature decreased by 0.8°C each cycle) for 60 s, 10 cycles; 94°C for 20 s, 57°C for 60 s, 30 cycles.

[0104] Signal scanning and genotyping: After the PCR reaction is completed, ARAYA is used to scan the fluorescence signal of the reaction system; then INTELLICS is used for genotyping and data analysis.

[0105] In the KASP marker genotyping test, the genotypes of the samples are divided into three clusters, namely X cluster, Y cluster and heterozygous genotype cluster. Among them, X cluster means that the sample contains homozygous X allele at this KASP marker site (marked in red in the genotyping diagram, located in the upper left corner of the diagram), Y cluster means that the sample contains homozygous Y allele at this KASP marker site (marked in blue in the genotyping diagram, located in the lower right corner of the diagram), and heterozygous genotype cluster means that the sample contains heterozygous X and Y alleles at this KASP marker site (marked in purple in the genotyping diagram). See the genotyping diagram of the 6 SNP molecular markers KASP markers. Figures 1-6 .

[0106] The results showed that the six molecular markers provided by the scheme of the present invention can be accurately typed, and the genotypes and corresponding phenotypes are consistent with the actual results.

[0107] Example 2

[0108] In order to detect the specificity and practicality of the markers in the present invention, the six molecular marker combinations obtained in Example 1 were verified using the method in Example 1 using 12 collected Ningxiang pig germplasm materials. The specific material genotype data are shown in Table 4, and the three actual average intramuscular fat (IMF) contents of each material are shown in Table 5.

[0109] Table 4 Genotyping information of six loci of Ningxiang pigs

[0110]

[0111]

[0112] Table 5 IMF values ​​of Ningxiang pigs in high and low fat groups

[0113]

[0114] As can be seen from Table 4-5, the genotypes with high IMF content in the samples are: SNP1 genotype is AA, SNP2 genotype is CT / TT, SNP3 genotype is TC, SNP4 genotype is CC / CT, SNP5 genotype is GG, and SNP6 genotype is AG / GG;

[0115] The genotypes of samples with low IMF levels were as follows: SNP1 genotype AG / GG, SNP2 genotype CC, SNP3 genotype TT, SNP4 genotype TT, SNP5 genotype CG, and SNP6 genotype AA; these patterns were consistent with the actual IMF groupings. These results demonstrate that the SNP molecular marker combination provided by the present invention is effective in distinguishing between high and low IMF levels, demonstrating the reliability of this method's detection.

[0116] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of SNP locus combination in identifying intramuscular fat traits of Ningxiang pigs, characterized in that: The SNP site combination consists of SNP1 site, SNP2 site, SNP3 site, SNP4 site, SNP5 site and SNP6 site; The SNP1 site is located at base 40987222 of chromosome 2 of the reference genome Sus Scrofa 11.1, and the polymorphism is A / G; The SNP2 site is located at base 41,137,403 of chromosome 2 of the reference genome Sus Scrofa 11.1, and the polymorphism is C / T; The SNP3 site is located at base 73007048 of chromosome 6 of the reference genome Sus Scrofa 11.1, and the polymorphism is T / C; The SNP4 site is located at base 2164395 of chromosome 16 of the reference genome Sus Scrofa 11.1, and the polymorphism is C / T; The SNP5 site is located at base 2620803 of chromosome 16 of the reference genome Sus Scrofa 11.1, and the polymorphism is C / G; The SNP6 site is located at base 2709556 of chromosome 16 of the reference genome Sus Scrofa 11.1, and the polymorphism is A / G.

2. A primer set for amplifying the SNP site combination as claimed in claim 1, characterized in that: The primer set includes: (1) A primer set for amplifying the SNP1 site, comprising specific primers with sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 and a universal primer with a sequence shown in SEQ ID NO. 9; (2) A primer set for amplifying the SNP2 site, comprising specific primers with sequences as shown in SEQ ID NO.10 and SEQ ID NO.11 and a universal primer with sequence as shown in SEQ ID NO.12; (3) A primer set for amplifying the SNP3 site, comprising specific primers with sequences shown in SEQ ID NO. 13 and SEQ ID NO. 14 and a universal primer with sequence shown in SEQ ID NO. 15; (4) A primer set for amplifying the SNP4 site, comprising specific primers with sequences shown in SEQ ID NO. 16 and SEQ ID NO. 17 and a universal primer with sequence shown in SEQ ID NO. 18; (5) A primer set for detecting and amplifying the SNP5 site, comprising specific primers with sequences as shown in SEQ ID NO.19 and SEQ ID NO.20 and a universal primer with sequences as shown in SEQ ID NO.21 and (6) A primer set for detecting and amplifying the SNP6 site, comprising specific primers having sequences as shown in SEQ ID NO. 22 and SEQ ID NO. 23 and a universal primer having a sequence as shown in SEQ ID NO.

24.

3. The primer set according to claim 2, characterized in that The specific primers are connected to FAM and HEX fluorescent linker sequences respectively.

4. A kit, characterized in that The kit comprises the primer set according to any one of claims 2 to 3.

5. A gene chip, characterized in that The gene chip comprises the primer set according to any one of claims 2 to 3.

6. Use of the SNP locus combination according to claim 1, the primer set according to any one of claims 2 to 3, the kit according to claim 4, or the gene chip according to claim 5 in any of the following: 1) Detection of intramuscular fat content in Ningxiang pork; 2) Breeding Ningxiang pigs with an intramuscular fat content of ≥4.0; 3) Breed Ningxiang pigs with an intramuscular fat content of less than 4.

0.

7. A method for identifying the intramuscular fat content of Ningxiang pork using the SNP locus combination described in claim 1, characterized in that: The method comprises the following steps: S1. Extracting the genomic DNA of the Ningxiang pig to be tested; S2. Performing polymorphism detection of the SNP site combination on the genomic DNA extracted in step S1, and determining the intramuscular fat content of the Ningxiang pig to be tested according to the genotype.

8. A Ningxiang pig breeding method, characterized in that: The method comprises the following steps: using the method according to claim 7, selecting Ningxiang pigs with pork intramuscular fat content ≥4.0 or pork intramuscular fat content <4.0 for subsequent breeding.

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