Variant sites in the promoter region of the DGAT1 gene and their application in detecting intramuscular fat content in pork

Abstract

The present invention belongs to the field of molecular biology and discloses a variant site in the promoter region of the DGAT1 gene and its application in detecting the intramuscular fat content of pork. The SNP molecular marker is located 379bp upstream of the transcription start codon in the promoter region of the DGAT1 gene, and the base of this site is C or T. The intramuscular fat content of pork is determined according to the genotype of the mutation site. The present invention screens a SNP site related to intramuscular fat content in the promoter region, thereby obtaining a functional gene and molecular genetic marker related to intramuscular fat content. By optimizing the dominant allele of the SNP molecular marker and rapidly detecting the site, a reference basis can be provided for SNP molecular marker-assisted breeding related to the regulation of intramuscular fat content in pork, and at the same time, it has important significance for improving pork quality.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to a DGAT1 gene promoter region mutation site and its application in detecting pork intramuscular fat content. Background Art

[0002] In recent years, with the continuous improvement of people's living standards, the demand for pork has gradually shifted from "quantity" to "quality." Specifically, pork quality has become a growing concern, with delicious, nutritious, and healthy meat becoming a new goal. Among the many factors influencing pork quality, intramuscular fat (IMF) content plays a significant role, being highly correlated with tenderness, juiciness, and flavor, making it a key determinant of pork quality. Therefore, increasing the IMF content of pork is of great practical significance to improve pork quality, enhance its edible value, and meet the demands of consumers.

[0003] IMF is one of the main forms of fat deposition in pork. It refers to the fat deposited inside the muscle and between muscle fibers in the same part of the muscle. It is mainly located on the endomysium, perimysium or epimysium. Its main components are phospholipids and triglycerides. Studies have shown that the lower the intramuscular fat content, the lower the tenderness, juiciness, flavor and overall acceptability of the muscle. 2%-3% intramuscular fat content is an ideal standard for measuring meat quality. In addition, the intramuscular fat trait of pigs has a relatively high heritability (h 2 =0.52). Therefore, studying intramuscular fat is of great significance for improving pork quality, producing healthy meat products, and increasing the economic benefits of the pig industry. The development and utilization of molecular markers and related detection technologies have enabled rapid progress in genetic breeding and improvement of intramuscular fat traits.

[0004] Mammalian fat stores metabolic energy in the form of triacylglycerol (TG), and the synthesis of TG requires enzyme catalysis, among which the enzyme that plays a role is diacylglycerol acyltransferase (DGAT). DGAT genes include diacylglycerol acyltransferase 1 (DGAT1) gene and diacylglycerol acyltransferase 2 (DGAT2) gene. The former belongs to the acyl-CoA cholesterol acyltransferase (ACAT) gene family, and the latter belongs to the monoacylglycerol acyltransferase (MGAT) gene family. They encode the microsomal enzymes DGAT1 and DGAT2, respectively. They are transmembrane proteins located in the endoplasmic reticulum. Their topological structure has the ability to interact with other proteins and organelles, affecting fat metabolism and the deposition of lipids in tissues, and participating in the regulation of energy synthesis and catabolism in animals. The DGAT1 gene is a transmembrane protein located in the endoplasmic reticulum. Its topological structure has the ability to interact with other proteins and organelles, affecting fat metabolism and the deposition of lipids in tissues. It participates in regulating the energy synthesis and catabolism of the animal body, plays a key role in the fat synthesis process, and affects the formation and decomposition of fat in the animal body. Summary of the Invention

[0005] The purpose of the present invention is to provide a SNP molecular marker for detecting the intramuscular fat content of pigs and its application, so as to conveniently and quickly judge the intramuscular fat content of pork, thereby providing a basis for obtaining better meat quality.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A single-nucleotide polymorphism (SNP) molecular marker for detecting intramuscular fat content in pigs is located 379 bp upstream of the transcription start codon in the DGAT1 gene promoter region. The base at this site is either C or T. Therefore, this SNP is named c.-379C>T. The genotype at this site is CC, CT, or TT.

[0008] The porcine DGAT1 gene sequence is shown in Chromosome 4, NC_010446.5 (452890..463665). Additionally, the approximately 2000 bp sequence preceding the start codon (ATG) of the DGAT1 gene promoter region is listed in the sequence listing (as shown in SEQ ID NO. 1). The SNP marker site 379 bp upstream of the start codon is underlined, and its genotype is CC, CT, or TT.

[0009] A primer pair for detecting the above-mentioned SNP molecular marker, wherein the sequences of the primer pair are:

[0010] DGAT1-3F:5'-CCACAACGCACTAGGCATTTC-3'

[0011] DGAT1-3R: 5'-GGGCACCACTACCTCAGATCC-3'.

[0012] A kit for genotyping the C / T mutation site of the above-mentioned SNP molecular marker, the kit comprising the above-mentioned primer pair.

[0013] The above-mentioned SNP molecular marker, the above-mentioned primer pair, or the above-mentioned kit is used to detect the intramuscular fat content of pork. The C / T mutation site of the SNP molecular marker is genotyped, and the intramuscular fat content of pork samples with a genotype of TT or CT is higher than the intramuscular fat content of pork samples with a genotype of CC.

[0014] A method for assisting in the genetic improvement of the high intramuscular fat trait in pig breeding, wherein genotyping is performed on the C / T mutation site of the above-mentioned SNP molecular marker, and the intramuscular fat content of pork samples with the genotype TT or CT is higher than the intramuscular fat content of pork samples with the genotype CC.

[0015] As a preferred technical solution, the method specifically includes the following steps:

[0016] 1) Extracting DNA from the pork sample to be tested;

[0017] 2) performing PCR amplification on the DNA obtained in step 1) using the aforementioned primer pair or the aforementioned kit to obtain an amplified fragment;

[0018] 3) The amplified fragments were digested and typed by PCR-RFLP-PvuⅡ. The digested products of different genotypes showed different numbers of bands: CC genotype had two bands (134 bp, 581 bp), CT genotype had three bands (134 bp, 581 bp, 715 bp), and TT genotype had one band (715 bp).

[0019] 4) When the typing result is TT type or CT type, it is judged that the intramuscular fat content of the pork sample is higher than the intramuscular fat content of the pork sample of CC type.

[0020] Further preferably, in step 2), the PCR amplification reaction system of 25 μl includes: 22 μl of T3 mix, 1 μl of upstream and downstream primers, and 1 μl of DNA to be detected; the PCR reaction program is: 98°C, 3 min; 98°C, 10 s; 65°C, 10 s annealing; 72°C, 10 s; 34 cycles; 72°C, 3 min.

[0021] Further preferably, the reaction system of the PCR-RFLP-PvuⅡ method is: 17 μl of the PCR amplification product to be detected, 1 μl of PvuⅡ enzyme, and 2 μl of Tango Buffer; and the reaction conditions are: 37° C., 1 h.

[0022] Beneficial effects of the present invention:

[0023] The present invention studies the correlation between the key gene DGAT1 in triglyceride synthesis and the intramuscular fat content trait of pork, and screens out a SNP site related to the intramuscular fat content trait of pork in the promoter region, thereby obtaining functional genes and molecular genetic markers related to intramuscular fat content. This lays the foundation for further controlling the intramuscular fat content of pork and assisting in the genetic improvement of high intramuscular fat traits in pigs, is of great significance for improving meat quality, and provides a basis for conveniently and quickly judging the intramuscular fat content of pork, thereby providing a basis for obtaining better meat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Results of SNP site screening.

[0025] Figure 2 The following are the results of enzyme digestion of some PCR products.

[0026] Figure 3 The results of the correlation analysis between the mutation site and the intramuscular fat content.

[0027] Figure 4 The results of DGAT1 expression analysis in individuals with different genotypes are shown. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1

[0030] 1. Experimental Materials

[0031] 1.1 Experimental Animals

[0032] This study used 260 Duchang-Changda hybrid pigs. The experimental animals were obtained from Nantong Jialun Food Factory and raised under the same conditions. The longissimus dorsi muscle tissue was collected for DNA extraction, RNA extraction, and determination of intramuscular fat content.

[0033] 1.2 DNA extraction, RNA extraction, PCR and electrophoresis reagents

[0034] DNA extraction reagents: Tris-saturated phenol, proteinase K, chloroform, isoamyl alcohol, anhydrous ethanol

[0035] RNA extraction reagents: Trizol, chloroform, isopropanol, anhydrous ethanol

[0036] PCR and electrophoresis reagents: T3 PCRMix, DNAMarker

[0037] 10xTBE: Dissolve 108 g of Tris, 7.44 g of EDTA·2H2O, and 55 g of boric acid in deionized water and adjust the volume to 1000 ml.

[0038] 1.3 Instruments and Equipment

[0039] Real-time fluorescence quantitative PCR instrument: Applied Biosystems, USA

[0040] Low-temperature high-speed centrifuge: Eppendorf, Germany

[0041] Ultra-low temperature refrigerator, NanoDrop spectrophotometer: Thermo, USA

[0042] Adjustable micropipette: Eppendorf, Germany

[0043] GEL EQ gel imaging system: BIO-RAD, USA

[0044] Electronic balance: Yamatoz, USA

[0045] Constant temperature water bath: Beijing Medical Equipment Factory

[0046] Surgical forceps, surgical scissors, scalpels, medical gauze, vernier calipers, circular samplers, Xinhua qualitative filter paper, ziplock bags, label paper: Logistics Department of Nanjing Agricultural University

[0047] The amplified fragments were sequenced by Shanghai Sangon Biotechnology Co., Ltd.

[0048] 2. Experimental Methods

[0049] 2.1 Determination of intramuscular fat content

[0050] (1) Spread a certain amount of filter paper in a clean enamel dish and dry it at 105°C for at least 2 hours until its weight does not change. Use a precision balance (sensitivity: 0.00001g) to accurately weigh the dried filter paper (W1).

[0051] (2) Cut (mince) 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 does not change. Weigh the weight of the dried paper package (W3).

[0052] (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. (Ether is easily volatile, so seal the bottle mouth with water and regularly observe the reflux status to replenish ether in time.)

[0053] (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).

[0054] (5) Calculation formula: In this experiment, crude fat content was used to approximate the intramuscular fat content.

[0055]

[0056] To eliminate errors caused by unequal water loss during sample pretreatment, the relative intramuscular fat content is expressed as the percentage of the total fat weight extracted to the sample weight after drying (drying at 105°C for 15 hours). The calculation formula is as follows:

[0057]

[0058] 2.2 Extraction of muscle DNA

[0059] (1) Cut about 100 mg of sample, mince it, and place it in a 2.0 ml EP tube;

[0060] (2) Add 1 ml of lysis buffer and 50 μl of proteinase K to an EP tube, mix thoroughly, and digest in a water bath at 55°C for 12 h to overnight. The next day, add 900 μl of Tris-saturated phenol to a 2 ml tube, mix on a shaker for 15 min, and centrifuge at 12,000 rpm at 4°C for 15 min.

[0061] (3) Pipette 1000 μl of the upper layer into a 2 ml EP tube, add 500 μl of chloroform / isoamyl alcohol (24:1, v:v) and 500 μl of saturated phenol, mix on a shaker for 15 min, and centrifuge at 12000 rpm for 15 min at 4°C.

[0062] (4) Pipette 850 μl of the upper layer into a 2 ml EP tube, add 850 μl of chloroform / isoamyl alcohol (24:1, v:v), mix on a shaker for 15 min, and centrifuge at 12,000 rpm for 15 min at 4°C.

[0063] (5) Pipette 700 μl of the upper layer into a 2 ml EP tube, add 700 μl of chloroform, mix on a shaker for 15 min, and centrifuge at 12,000 rpm for 15 min at 4°C.

[0064] (6) Pipette 500 μl of the upper layer into a 1.5 ml EP tube, add 2 volumes of anhydrous ethanol, shake gently, and centrifuge at 10,000 rpm for 10 min at 4°C.

[0065] (7) Discard the supernatant, add 1 ml of 70% anhydrous ethanol, shake gently, and centrifuge at 10,000 rpm for 5 min at 4°C;

[0066] (8) Pour off the upper layer of liquid, remove the remaining liquid in the tube, and blow dry on the clean bench until there are no water droplets in the EP tube. The drying time is about 25 minutes;

[0067] (9) Add 50 μl of 55°C deionized water to the sample to completely dissolve the DNA, perform sample quality testing, and finally store in a -20°C refrigerator.

[0068] 2.3 DNA concentration determination and DNA sample pooling

[0069] DNA concentrations were measured in 260 pig longissimus dorsi muscle samples using a NanoDrop spectrophotometer. The quality of the DNA extraction was assessed by calculating the OD260 / OD280 ratio. If the OD260 / OD280 ratio for all samples was between 1.8 and 2.0, the DNA extraction was considered acceptable.

[0070] 2.4 Primer design and synthesis

[0071] Based on the porcine gene sequence DGAT1 (Chromosome 4, NC_010446.5 (452890..463665)) from the GenBank database, three primer pairs were designed for the 2000 bp preceding the transcription start codon in the promoter region: DGAT1-1F and DGAT1-1R, DGAT1-2F and DGAT1-2R, and DGAT1-3F and DGAT1-3R. Primer sequences are shown in Table 1.

[0072] Table 1 Pig DGAT1 gene primer design information

[0073]

[0074] 2.5 PCR-RFLP reaction system and procedure

[0075] The PCR reaction systems used in this experiment are shown in Table 2 and Table 3:

[0076] Table 2 Sequencing PCR reaction system (reaction system for sequencing to find SNPs)

[0077]

[0078] Table 3 Enzyme digestion and typing reaction system

[0079]

[0080] PCR reaction procedure:

[0081] 98℃, 3min;

[0082] 98°C, 10 s, 65°C, 10 s, 72°C, 10 s (34 cycles);

[0083] 72℃, 3min;

[0084] Store at 4℃;

[0085] The amplified PCR products were digested with enzymes according to the system in Table 3. The reaction system was: 37°C for 1 h.

[0086] 2.6 Agarose gel electrophoresis detection

[0087] Amplification products and digestion products were electrophoresed on a 1.5% agarose gel at approximately 120V for approximately 30 minutes, followed by visualization using a gel imaging system. Successfully amplified PCR products were digested with enzymes. After the digestion reaction was complete, the digestion products were subjected to agarose gel electrophoresis. Different genotypes correspond to different numbers of bands. The genotype of each sample was determined based on the number of bands observed in the electrophoresis results.

[0088] 2.7 Tissue RNA Extraction

[0089] (1) Clean the homogenizer rotor with DEPC water and then clean it with Trizol.

[0090] (2) Place approximately 100 mg of longissimus dorsi muscle tissue in a 2 ml EP tube. Add 500 μl of Trizol to the tube. Homogenize the tissue sample using a homogenizer, and finally add another 500 μl of Trizol. Incubate at room temperature for 5 minutes.

[0091] (3) Add 200 μl of chloroform to each EP tube, shake on an oscillator for 30 seconds to completely mix the liquid and tissue, and then let it stand at room temperature for 3 minutes. Centrifuge at 4°C and 12,000 rpm for 15 minutes.

[0092] (4) Take the upper layer of liquid and add it to a new 1.5 ml EP tube. Add 500 μl of isopropanol to the EP tube, mix thoroughly, let it stand at room temperature for 10 min, and centrifuge it at 4°C and 12,000 rpm for 10 min.

[0093] (6) Pour off the supernatant, paying attention to the precipitate at the bottom. Add 1 ml of pre-chilled 75% ethanol and gently shake to resuspend the precipitate. Centrifuge at 12,000 rpm for 5 min at 4°C.

[0094] (8) Pour off the upper liquid and retain the RNA at the bottom. If there is still water at the bottom, remove it with a pipette and air-dry in a clean bench for 25 minutes.

[0095] (9) Add 30 μl of 55°C DEPC water to the EP tube and let it stand for 10 min. After measuring the RNA quality, store it at -80°C or use it directly for reverse transcription.

[0096] 2.8 Reverse transcription

[0097] The reaction system is 10 μl, and the specific components are shown in Table 4:

[0098] Table 4 Reverse transcription system

[0099]

[0100] Place the PCR tube that needs reverse transcription into the PCR instrument. The reaction procedure is: first, act at 37℃ for 15 minutes, then act at 85℃ for 5 seconds, and finally store at 4℃. After the reaction, store the obtained cDNA in a -20℃ refrigerator.

[0101] 2.9 Quantitative Primer Design

[0102] The sequences of primers for DGAT1 gene quantification are shown in Table 5.

[0103] Table 5 Design information of quantitative primers for porcine DGAT1 gene

[0104]

[0105] 2.10 Real-time quantitative PCR

[0106] The experiment was completed on a fluorescence quantitative analyzer. The fluorescence quantitative PCR reaction system is shown in Table 6, and the reaction conditions are shown in Table 7. In this experiment, the RPLP0 gene was used as the internal reference gene, and 2 -ΔΔCt Statistical analysis of effectiveness data was performed.

[0107] Table 6 Fluorescence quantitative PCR reaction system

[0108]

[0109]

[0110] Table 7 Fluorescence quantitative reaction conditions

[0111]

[0112] 3. Experimental Results

[0113] 3.1 SNP screening

[0114] Twelve DNA samples were randomly selected as templates, and the DGAT1 gene promoter region was amplified using primers DGAT1-1F and DGAT1-1R, DGAT1-2F and DGAT1-2R, and DGAT1-3F and DGAT1-3R, respectively. A total of approximately 2,000 bp of fragments upstream of the transcription start codon of the porcine DGAT1 gene were obtained. The PCR products were then sequenced (provided by Sangon Biotech (Shanghai) Co., Ltd.). Sequencing results showed that only one SNP site was found in the fragment amplified by primers DGAT1-3F and DGAT1-3R, located 379 bp upstream of the transcription start codon of the DGAT1 gene, which was either cytosine C or thymine T, respectively. Figure 1 As shown, this SNP is named c.-379C>T.

[0115] 3.2 PCR-RFLP genotyping

[0116] For the above-mentioned SNP sites, primers DGAT1-3F and DGAT1-3R were used for PCR amplification, and the genotype was identified by enzymatic digestion of the PCR products using PvuⅡ restriction endonuclease. Through agarose gel electrophoresis, the enzymatic digestion products of different genotypes will show different numbers of bands: the CC genotype is two bands (134bp, 581bp), the CT genotype is three bands (134bp, 581bp, 715bp), and the TT genotype is one band (715bp). According to statistics, in the 260 Du Changda three-way hybrid population, there are 118 individuals with CC genotype, 15 individuals with TT genotype, and 127 CT heterozygous types. The enzymatic digestion results of some PCR products are shown as follows. Figure 2 .

[0117] 3.3 Correlation between mutation sites and intramuscular fat content

[0118] The number of individuals with different genotypes was counted and the correlation between SNP and intramuscular fat content was analyzed. The results showed that there was a correlation between the two. The intramuscular fat content of TT type individuals (3.198% ± 0.315) was the highest, followed by the intramuscular fat content of CT type individuals (3.188% ± 0.108), and the intramuscular fat content of CC type individuals (2.857% ± 0.112) was the lowest. The intramuscular fat content of CC type and CT type individuals was significantly different (P < 0.05). Since the number of homozygous mutant individuals in this group is extremely small and the standard deviation is large, the intramuscular fat content between mutant homozygous TT and wild-type homozygous CC individuals did not show a significant difference, but their intramuscular fat content still showed an increasing trend, as shown in Figure 2. Figure 3 .

[0119] 3.4 DGAT1 expression in individuals with different genotypes

[0120] Seven CC-type individuals and seven TT-type individuals were selected, and cDNA from these two groups of samples was used as templates for fluorescence quantitative PCR analysis. The results showed that the expression level of DGAT1 was significantly different between the two groups (P<0.05). Figure 4 .

[0121] 3.5 Transcription factor binding site prediction

[0122] According to the JASPAR database website, the transcription factor binding site at the c.-379C>T site in the DGAT1 promoter region was predicted, and the prediction results are shown in Table 8.

[0123] Table 8 DGAT1 promoter region c.-379C>T

[0124] Base type Proposed binding transcription factor C MyoD1 T ——

[0125] 4. Results Analysis

[0126] The mutation sites screened by this invention are significantly correlated with intramuscular fat content in pork and can be developed as single-nucleotide polymorphism (SNP) molecular markers for measuring intramuscular fat content in pigs. For the c.-379C>T site in the DGAT1 promoter region, pork samples with the TT or CT genotypes have higher intramuscular fat content than pork samples with the CC genotype. When the c.-379C>T site in the DGAT1 gene promoter region is C, the transcription factor MyoD1 may bind to it, regulating DGAT1 gene expression and, in turn, affecting intramuscular fat deposition.

[0127] The present invention screened a SNP site related to intramuscular fat content in the promoter region, thereby obtaining a functional gene and molecular genetic marker related to intramuscular fat content. By optimizing the dominant allele of the SNP molecular marker and rapidly detecting the site, a reference basis can be provided for SNP molecular marker-assisted breeding related to the regulation of intramuscular fat content in pork, and at the same time, it is of great significance for improving pork quality.

[0128] It can be understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the invention. However, the present invention is not limited thereto. Those skilled in the art can make various improvements and changes without departing from the essence of the invention, and these improvements and changes also fall within the scope of protection of the present invention. Sequence Listing <110> Nanjing Agricultural University <120> Variant sites in the promoter region of the DGAT1 gene and their application in detecting intramuscular fat content in pork <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2220 <212> DNA <213> Pig (Sus) <400> 1 tcccctgtct ccctcagcca caccacacct aggagcctga ggcgctaggg ctgtgggcgg 60 ggcaggtgca ggccccacct ccttcccaga ggccccgccc actattactt tcaattcccg 120 cctcctcccc aaagcaggcc ctgcgcgctg cccccccatg aggctcttcc ttatttgtgc 180 ttattagtgt gttcacagcc catgccttcc ctccattggc tccaggccct gatctctttg 240 ctctccactc cagcgccgga atcttggtcc cccaaggccc ctggtcagat tggtcctctc 300 tggtggccac ggtccactct ggtcgcctca ctcctgctgc tgcttgtcag gtgacaacag 360 cttggagcct ggacgcagac acacaggcaa ccgcctccag caagaccctt cactcaccgg 420 ctactgcgct cagcccctcc ccctactggc agcctgagcc gtcaatctta tcttctgcca 480 atcagcgtgc cagcccaaac cagccctcta gttcttggct ctatactgga gagtggcagg 540 gcctgaaggc tcacagggac ccgtgggcgc aggggttccc accagctgcc ggggagagga 600 gctgggaggg agaggcagag ctcccggagt tcactcctat gctgttggtt ctggagccga 660 ccctttcctc ggtctgacct ccctcgggat cagttctccg aaaagcctac ctctgcctgg 720 cccaggtctg aagatgaaca gatgaacttt ctggggcacc ggagaggtga cggggctggt 780 cctgacccac cgaggttgtg tgggcacctg agaggggttc tcccacccaa tcagcaggct 840 gccaggtagc ttcagcctgc cagttggggc tggcatcaac cccaagtggt ctgctgttag 900 cagaatgtat caccacgagg cccaggtgcc tgtcagtgga tgtgatagat gagggtgagc 960 ctgggaaggt ggactgacct catgctgagc cacttggttt gtttgcatag agtggaaacc 1020 ttgcaacctc ttctcaacct ttttacctca ggggagcgaa tggctagtgg gttctgctgc 1080 taggggtgtt tcctgggtaa accaggcctg tgcccccaga gaacccttgc atccggagag 1140 ccaggatgtt cacactccat gctccgtgca aaatccacgt caagcaaggt gggaagcgaa 1200 cttgcctgcc tgggagtctc accgtgcagt tcttgtggtg gattggatgc ttcgtttaac 1260 gactggatgg atgactgcac agactgtgta atggcacagg agtctctggg tccttgcgaa 1320 tccgcaccac aggatcctca agtccaggtg ggaaaccgga aagctctgcg agtccgccga 1380 ctagggagga agagggcggg gtgggggaggc agcactgtgg tcccatccca gcctgggcct 1440 gcgggggaga ggtggcggcc ctggcagtcg ttgggcagag tgggtatccc ggggatgtgg 1500 ggccttgggc ctttctcttg ctccatcctc tggcctggcg cttgtctaga cctaagccga 1560 gaacaagcgg gtgcatatag tggtaaccag agggctcgcg gagcacggct gcgcctcgag 1620 gtgggcgttc gggaaccaac gcgatgggta tcaggtgcgg cgagctctac ggacacggcg 1680 ttggcccgag acgcctggac cacaacgcac taggcatttc gtaagtaagc tacatcctca 1740 cactccgcgc ctggactgaa gcctgacgaa accctgtgtt tatagagtag gacaaggtgc 1800 aggcagcggt cagagtcagc agaggcttgc agctcctagg aagccgcgcg cgttctgcgc 1860 ggtcctgcgg gcaacagtgt ccgccgcccc gcgaccacaa ctcccagggt gcaccgcgcg 1920 ctcgcggcga ccacaattcc cagggtgcgt cgcgcgcccg cggactacaa aatgggcgcg 1980 cgccggggct ctgcgccagt tagcggcccc gggagcgacg ctgctggag cgccgcgacg 2040 accgagcggg cgcgcactga ttggaggcgc gggcaggcgc gcgcgcgcta cggggccgg 2100 caggaggcgg tggcggctgt tggccaaggg tccggaggcg gggccgcaag cctcgggcgc 2160 tgtgagcccg gcgggccacg actcggccgc ggcagggtgc aggcagaggc catgggtgac 2220 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 ccacaacgca ctaggcattt c 21 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 gggcaccact acctcagatc c 21 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 gcccactatt actttcaatt ccc 23 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <400> 5 actagccatt cgctcccct 19 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 gcctgtcagt ggatgtgata gat 23 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 gggtttcgtc aggcttcag 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tccaggcttt aggcatcacc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 ggctcccact ttgtctccag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 ctggctctga tggtctacgc 20 <210> 11 <211> twenty two <212> DNA <213> Artificial Sequence <400> 11 agtagagatc tgcagaagcg gc 22

Claims

1. Application of SNP molecular markers in detecting pork intramuscular fat content, characterized by: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and there is a C / T mutation at position 1833 of the sequence; when the genotype of the SNP molecular marker is TT type or CT type, the intramuscular fat content of pork is higher than that of the CC type genotype.

2. Application of a primer pair for detecting SNP molecular markers in detecting pork intramuscular fat content, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 1, and there is a C / T mutation at position 1833 of the sequence; the sequences of the primer pairs used to detect the SNP molecular marker are: DGAT1-3F:5'-CCACAACGCACTAGGCATTTC-3' DGAT1-3R: 5'-GGGCACCACTACCTCAGATCC-3'; Genotyping of the C / T mutation site of the SNP molecular marker was performed, and the intramuscular fat content of the pork samples with the genotype of TT or CT was higher than the intramuscular fat content of the pork samples with the genotype of CC.

3. Use of a kit for genotyping the C / T mutation site of a SNP molecular marker in detecting the intramuscular fat content of pork, characterized by: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO. 1, and there is a C / T mutation at position 1833 of the sequence; the kit comprises the primer pair described in claim 2; Genotyping of the C / T mutation site of the SNP molecular marker was performed, and the intramuscular fat content of the pork samples with the genotype of TT or CT was higher than the intramuscular fat content of the pork samples with the genotype of CC.

4. A method for genetic improvement of high intramuscular fat trait in assisted breeding pigs, characterized by: Genotyping of the C / T mutation site of the SNP molecular marker in claim 1 shows that the intramuscular fat content of pork samples with genotype TT or CT is higher than that of pork samples with genotype CC.

5. The method according to claim 4, characterized in that: The method comprises the following steps: 1) Extracting DNA from the pork sample to be tested; 2) performing PCR amplification on the DNA obtained in step 1) using the primer pair described in claim 2 or the kit described in claim 3 to obtain an amplified fragment; 3) The amplified fragments were digested and typed using the PCR-RFLP-PvuⅡ method. The digested products of different genotypes showed different numbers of bands: the CC genotype had two bands of 134 bp and 581 bp, the CT genotype had three bands of 134 bp, 581 bp, and 715 bp, and the TT genotype had a single band of 715 bp. 4) When the typing result is TT type or CT type, it is judged that the intramuscular fat content of the pork sample is higher than the intramuscular fat content of the pork sample of CC type.

6. The method according to claim 5, characterized in that: In step 2), the PCR amplification reaction system (25 μl) included 22 μl of T3 mix, 1 μl of upstream and downstream primers, and 1 μl of the DNA to be detected. The PCR reaction program was as follows: 98°C for 3 min; 98°C for 10 s; 65°C for 10 s of annealing; 72°C for 10 s; 34 cycles; and 72°C for 3 min.

7. The method according to claim 5, characterized in that: The reaction system of the PCR-RFLP-PvuⅡ method is: 17 μl of PCR amplification product to be detected, 1 μl of PvuⅡ enzyme, and 2 μl of Tango Buffer; reaction conditions: 37°C, 1 h.

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