SNP molecular marker related to IMF character of Ningxiang pig and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker related to an IMF character of a Ningxiang pig and application of the SNP molecular marker. The SNP molecular marker comprises at least one of an SNP1 site, an SNP2 site, an SNP3 site, an SNP4 site, an SNP5 site and an SNP6 site. According to the scheme, the SNP molecular marker related to the IMF character is provided, Ningxiang pig breeds with different IMF contents can be efficiently and accurately identified and screened out, and the SNP molecular marker has the advantages of being rapid, accurate and low in detection cost, can be used for detection and application of different Ningxiang pig breeds and has wide application prospects. And an indispensable important tool is provided for breeding Ningxiang pig breeds with high IMF content and excellent meat quality and improving the quality of Ningxiang pork.

Description

Technical Field

[0001] The 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] Ningxiang pig is a famous pig breed in China with a history of more than 1,000 years. It is a high-quality local pig breed resource. Ningxiang pork products are 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 has been 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] At present, molecular research on economic traits related to Ningxiang pigs mainly uses SSR (simple sequence repeat) markers, ISSR markers, AFLP and other molecular marker technologies. SSR has low throughput and high R&D costs, and is not suitable for large-scale commercial identification. Although ISSR and AFLP marker technologies are simple and practical, the results are unstable and have poor repeatability. SNP markers have the advantages of convenient 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 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 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 an IMF trait is provided, wherein the SNP molecular marker comprises at least one of a SNP1 site, a SNP2 site, a SNP3 site, a SNP4 site, a SNP5 site, and a 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 41137403 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 having sequences as shown in SEQ ID NO.13 and SEQ ID NO.14 and a universal primer having a sequence as 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, the kit 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-mentioned 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 Ningxiang pigs to be tested;

[0039] S2. Performing polymorphism detection of the SNP molecular marker on the genomic DNA extracted in step S1, and determining 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 detected by SNP1 is AA, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype detected by SNP1 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 the 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;

[0042] When the SNP molecular marker is SNP3, if the genotype detected by SNP3 is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype detected by SNP3 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 detected by SNP4 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 detected by SNP4 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 detected by SNP5 is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype detected by SNP5 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 the 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 the 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 markers are 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 the 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 method to identify or assist in identifying the IMF content of Ningxiang pork, and selecting Ningxiang pigs with pork IMF content ≥4.0 or 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, 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, providing 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 SNP detection method based on KASP markers, which is used to detect the content of IMF in Ningxiang pig breeds. The KASP marker detection method is simple, has accurate detection results, good stability, and rapid detection, 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, wherein:

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

[0055] Figure 2 The genotype diagram of the SNP2 molecular marker in Example 1 of the present invention;

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

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

[0058] Figure 5 It 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 be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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 adopts 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, which are 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 11.1. 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 detected by SNP1 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 detected by SNP1 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 detected by SNP2 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 detected by SNP2 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 detected by SNP3 is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype detected by SNP3 is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and it has a low IMF trait;

[0066] When the genotype detected by SNP4 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 detected by the SNP4 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 detected by SNP5 is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype detected by SNP5 is CG, the IMF content of the Ningxiang pig to be tested is <4.0, and it 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 the 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] The GGP Porcine 50K chip was used for SNP typing. The chip has a total of 51,000 SNP sites, integrating the genetic differences of multiple Ningxiang pig breeds, including Duroc, Landrace and Pietrain Ningxiang pigs. Each evaluated breed has an average of 50,000 polymorphic sites, covering the entire genome information of Ningxiang pigs. The biochip system is mainly composed of chips, scanners and analysis software. The analysis software Genome Studio is used to visualize the chip data and obtain genotype data.

[0072] 2. Genome-wide association analysis

[0073] The material data detected by the chip were analyzed by rMVP for whole-genome association analysis (Yin L, Zhang H, Tang Zetal. 2021), and the model was calculated using a mixed linear model (MLM). The principal component was added to the model as a covariate for correction. The collected IMF-related phenotypic traits were analyzed for whole-genome association analysis. After a large number of experimental screenings, 6 SNP sites related to IMF traits were finally obtained. The specific site information and the 250bp 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 sequence (sequence shown in Table 1) was 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 commissioned to be synthesized by Invitrogen.

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

[0085] When the genotype detected by SNP1 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 detected by SNP1 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 detected by SNP2 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 detected by SNP2 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 detected by SNP3 is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype detected by SNP3 is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and it has a low IMF trait;

[0088] When the genotype detected by SNP4 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 detected by the SNP4 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 detected by SNP5 is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and it has a high IMF trait; if the genotype detected by SNP5 is CG, the IMF content of the Ningxiang pig to be tested is <4.0, and it 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 the 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 Ningxiang pork samples to be tested; the concentration of DNA samples was detected by Qubit fluorescence quantification instrument; the integrity of DNA samples was detected by 1% agarose gel electrophoresis, and samples that passed quality control were used for library preparation.

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

[0098] KASP-labeled reaction sequencing was performed using Douglas Scientific's 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, and the amplification conditions were as follows: 94°C for 15 min; 94°C for 20 sec, 65°C-57°C (annealing temperature decreased by 0.8°C each cycle) for 60 sec, 10 cycles; 94°C for 20 sec, 57°C for 60 sec, 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, cluster X, cluster Y and heterozygous genotype cluster, where cluster X means that the sample contains homozygous X alleles at this KASP marker site (marked in red in the genotyping diagram, located in the upper left corner of the figure), cluster Y means that the sample contains homozygous Y alleles at this KASP marker site (marked in blue in the genotyping diagram, located in the lower right corner of the figure), 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. Figure 1-Figure 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 6 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 actual average intramuscular fat (IMF) content of each material for three times is shown in Table 5.

[0109] Table 4. Genotyping information of six loci of Ningxiang pig

[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 as follows: when the genotype of SNP1 is AA, the genotype of SNP2 is CT / TT, the genotype of SNP3 is TC, the genotype of SNP4 is CC / CT, the genotype of SNP5 is GG, and the genotype of SNP6 is AG / GG;

[0115] The genotypes of samples with low IMF content are as follows: SNP1 site genotype is AG / GG, SNP2 site genotype is CC, SNP3 site genotype is TT, SNP4 site genotype is TT, SNP5 site genotype is CG, SNP6 site genotype is AA; which is consistent with the actual detection of IMF content grouping. The results show that the SNP molecular marker combination provided by the scheme of the present invention can effectively distinguish the level of IMF, indicating that the detection effect of this scheme is real and reliable.

[0116] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A SNP molecular marker associated with the IMF trait of Ningxiang pigs, characterized in that: The SNP molecular marker includes at least one 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 41137403 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 molecular marker as described in claim 1.

3. The primer set according to claim 2, characterized in that The primer set comprises: (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; (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; (3) a primer set for amplifying the SNP3 site, comprising specific primers having sequences as shown in SEQ ID NO.13 and SEQ ID NO.14 and a universal primer having a sequence as shown in SEQ ID NO.15; (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; (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; (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.

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

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

6. A gene chip, characterized in that: The gene chip comprises the primer set according to any one of claims 2 to 4.

7. Use of the SNP molecular marker according to claim 1, the primer set according to any one of claims 2 to 4, the kit according to claim 5 or the gene chip according to claim 6 in any one of the following: 1) Detect or assist in detecting the quality of Ningxiang pork; 2) Detect or assist in detecting the IMF content of Ningxiang pork; 3) Breeding Ningxiang pigs with pork IMF content ≥ 4.0; 4) Breeding Ningxiang pigs with pork IMF content less than 4.0; 5) Molecular marker-assisted breeding of Ningxiang pigs; 6) Ningxiang pig breeding; 7) Prepare Ningxiang pig breeding products.

8. A method for identifying or assisting in identifying the IMF content of Ningxiang pork using the molecular markers of claim 1, characterized in that: The method comprises the following steps: S1. Extracting the genomic DNA of Ningxiang pigs to be tested; S2. Performing polymorphism detection of the SNP molecular marker on the genomic DNA extracted in step S1, and determining the IMF content of the Ningxiang pig to be tested according to the genotype.

9. The method according to claim 8, characterized in that When the SNP molecular marker is SNP1, if the genotype detected by SNP1 is AA, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype detected by SNP1 is AG or GG, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait; When the SNP molecular marker is SNP2, if the genotype obtained by the 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; When the SNP molecular marker is SNP3, if the genotype detected by SNP3 is TC, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype detected by SNP3 is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait; When the SNP molecular marker is SNP4, if the genotype detected by SNP4 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 detected by SNP4 is TT, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait; When the SNP molecular marker is SNP5, if the genotype detected by SNP5 is GG, the IMF content of the Ningxiang pig to be tested is ≥4.0, and has a high IMF trait; if the genotype detected by SNP5 is CG, the IMF content of the Ningxiang pig to be tested is <4.0, and has a low IMF trait; When the SNP molecular marker is SNP6, if the genotype obtained by the 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 the SNP6 detection is AA, the IMF content of the Ningxiang pig to be tested is <4.0, and it has a low IMF trait.

10. A Ningxiang pig breeding method, characterized in that: The method comprises the following steps: using the method described in claim 8 or 9, selecting Ningxiang pigs with pork IMF content ≥4.0 or pork IMF content <4.0 for subsequent breeding.

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