Use of molecular markers associated with pork flavor

Abstract

The application relates to application of a molecular marker related to pork flavor, and the molecular marker is related to alpha-nicotinamide adenine dinucleotide (NAD + ) content in a longissimus dorsi muscle of a pig. The application discloses a method for identifying pork flavor and a breeding method for breeding pigs with better pork flavor. The method is used for identifying pork flavor by detecting a nucleotide site at 131363728 of a chromosome 8 of a pig reference genome Sscrofa11.1, GCF_000003025.6 of a pig to be detected, and judging a genotype of the pig. The alpha-nicotinamide adenine dinucleotide (NAD + ) content in a longissimus dorsi muscle of a pig with a TT genotype at the site is higher than that of a pig with a CT genotype, and the alpha-nicotinamide adenine dinucleotide (NAD) content in the longissimus dorsi muscle of the pig with the CT genotype is higher than that of a pig with a CC genotype. In actual breeding work, the pig to be detected with the genotype of the site being TT can be selected as a parent for breeding, and the method has important significance for breeding pigs with better pork flavor.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker related to pork flavor and its application. The molecular marker is associated with α-nicotinamide adenine dinucleotide (NAD) in the longissimus dorsi muscle of pigs. + (Content-related) Background Technology

[0002] As the largest consumer of meat products in my country, pork production efficiency and meat quality directly affect farming profitability and consumer demand. With consumers demanding increasingly higher quality pork, traditional methods of sensory evaluation and physicochemical testing are insufficient to fully reflect its intrinsic biological characteristics. During the muscle development and post-slaughter maturation of pigs, a series of small-molecule metabolites significantly influence its flavor, color, water retention, and other quality characteristics.

[0003] α-Nicotinamide adenine dinucleotide (NAD) + As an important coenzyme, NAD+ plays a crucial role in glycolysis and fatty acid metabolism. By regulating lactic acid accumulation, fat synthesis and degradation, it influences the pH and fat content of meat. Long-chain fatty acids and unsaturated fatty acids hold significant positions in meat flavor, directly affecting its texture and aroma. Furthermore, by participating in muscle fiber metabolism, it affects the degradation of structural proteins such as collagen and elastin in muscle, thereby altering the tenderness and texture of the meat. Studies have shown that exogenous addition of NAD+ can... + Or related precursors, to increase NAD in pig muscle + The content of these compounds has a positive effect on promoting muscle metabolic activity, the generation of meat flavor compounds, and improving the tenderness and color of meat.

[0004] Currently, marker-assisted breeding technology has been widely applied in the breeding of new livestock and poultry breeds. Through genome-wide association analysis, molecular markers closely related to target traits can be obtained. Early selection of target traits using molecular markers can significantly reduce breeding costs and accelerate genetic progress. Currently, there are no molecular markers associated with the α-nicotinamide adenine dinucleotide content in pork. Summary of the Invention

[0005] The purpose of this invention is to provide the application of molecular markers related to pork flavor, wherein the molecular markers are related to the content of α-nicotinamide adenine dinucleotide in the longissimus dorsi muscle of pigs;

[0006] Another object of the present invention is to provide a method for identifying or assisting in the identification of pork flavor;

[0007] Another object of the present invention is to provide a breeding method for selecting pigs with better pork flavor.

[0008] This invention is implemented as follows:

[0009] The application of molecular markers related to pork flavor is any one of the following A1) to A8):

[0010] A1) Detection or auxiliary detection of pork flavor;

[0011] A2) Identification and auxiliary identification of pork flavor;

[0012] A3) Pig breeding;

[0013] A4) Detection or auxiliary detection of SNP polymorphisms or genotypes;

[0014] A5) Prepare products for detecting or assisting in the detection of pork flavor;

[0015] A6) Prepare products for identification and auxiliary identification of pork flavor;

[0016] A7) Preparation of pig breeding products;

[0017] A8) Prepare products for detecting or assisting in the detection of SNP polymorphisms or genotypes;

[0018] The molecular marker corresponds to nucleotide position 131363728 on chromosome 8 of the pig reference genome Sscrofa11.1, GCF_000003025.6, as shown in sequence SEQ ID NO:1, the nucleotide at position 262 bp of the DNA molecule, the nucleotide type is T or C, and the genotype of this site is related to the content of α-nicotinamide adenine dinucleotide in the longissimus dorsi muscle of pigs.

[0019] A method for identifying or assisting in the identification of pork flavor, comprising the following steps:

[0020] S1 extracts genomic DNA from the pigs to be tested as a template;

[0021] S2 designed primers targeting the 262 bp site of the DNA molecule shown in SEQ ID NO.1 for PCR amplification;

[0022] S3 detects the genotype at the 262 bp site of the pig sequence SEQ ID NO.1;

[0023] S4 uses the genotypes obtained in step S3 to identify or assist in identifying the pork flavor of the pigs to be tested. Pigs with the TT genotype have a higher α-nicotinamide adenine dinucleotide content in the longissimus dorsi muscle than pigs with the CT genotype, and their pork flavor is superior to that of pigs with the CT genotype. Pigs with the CT genotype have a higher α-nicotinamide adenine dinucleotide content in the longissimus dorsi muscle than pigs with the CC genotype, and their pork flavor is superior to that of pigs with the CC genotype.

[0024] Preferably, the primer sequences described in step S2 are as shown in SEQ ID NO.2 and SEQ ID NO.3.

[0025] A breeding method for selecting pigs with better pork flavor involves identifying the genotype of the pigs to be tested according to the aforementioned method, and selecting the pigs with the TT genotype as parents for breeding. The TT genotype is a homozygous type where the 262 bp of the SEQ ID NO.1 sequence is T.

[0026] Preferably, the aforementioned application of molecular markers related to pork flavor refers to the taste and aroma of pork, and the product is a reagent kit.

[0027] The beneficial effects of this invention are as follows: The SNP molecular marker of this invention is related to the α-nicotinamide adenine dinucleotide content trait in the longissimus dorsi muscle of pigs. It is a new molecular marker. By determining the genotype of the SNP locus in the pig to be tested, the α-nicotinamide adenine dinucleotide content trait in the longissimus dorsi muscle of pigs can be selected at an early stage, which can save production costs, improve meat quality and flavor and accelerate genetic progress, better serve pig breeding, and has great economic application value and scientific research value. Attached Figure Description

[0028] Figure 1 This is a graph showing the results of the whole genome association analysis in Example 1. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 (*) indicates a significant difference.

[0032] Example 1: Determination of the correlation between specific SNPs and the content of α-nicotinamide adenine dinucleotide in the longissimus dorsi muscle.

[0033] Experimental animals: Landrace pigs, Large White pigs, and crossbred pigs, all of which were sourced from COFCO Jiajiakang (Chifeng) Co., Ltd.

[0034] I. Determination of α-nicotinamide adenine dinucleotide content in the longissimus dorsi muscle

[0035] Under the same feeding conditions, the pigs were fed for 180 days. 521 healthy pigs were randomly selected, and 10g samples of their longissimus dorsi muscle were collected and frozen in liquid nitrogen.

[0036] Sample pretreatment: Take uniform samples, grind and mix the muscle tissue, and accurately weigh approximately 40 mg of sample (place a certain amount of sample in a mortar, add liquid nitrogen before weighing to prevent sample degradation) into a 2 mL centrifuge tube. Add 300 μL of methanol, then add a 5 mm steel bead. Homogenize the muscle tissue using a homogenizer (60 Hz, 60 s). Remove the steel bead with a magnet, add 1 mL of MTBE, vortex for 1 min, then add 250 μL of water to separate the layers and let stand for 5 min. After thorough extraction, centrifuge at 4℃ for 5 min at 5000 rpm. Take 250 μL of the lower layer and resuspend it. Reconstitute the lower layer with 200 μL of 80% methanol-water solution and analyze.

[0037] Instruments and equipment: autosampler, liquid chromatograph, mass spectrometer, liquid chromatography column (ACQUITY UPLC HSS T3 Column, 2.1mm×100mm, Waters).

[0038] Preparation of mobile phase: Mobile phase A: Dissolve 0.315 g ammonium formate in 1000 mL of deionized water (5% ammonium formate); Mobile phase B: Dissolve 0.315 g ammonium formate in 20 mL of deionized water, and then add 980 mL of acetonitrile (5% ammonium formate);

[0039] Table 1. Mobile phase gradient settings

[0040] Step Time (min) Flow rate (mL / min) ％A ％B 0 0 0.35 98 2 1 2 0.35 98 2 2 14 0.35 2 98 3 17 0.35 2 98 4 17 0.35 98 2 5 20 0.35 98 2

[0041] Table 2. Mass Spectrometer Parameter Settings

[0042]

[0043] II. Detection of SNP molecular markers

[0044] 1. Blood sample collection: Collect venous blood from the wing vein of the pig to be tested using heparin sodium anticoagulant blood collection tubes and store at -20℃ for later use.

[0045] 2. Whole blood genomic DNA extraction: Refer to the instructions for the blood genomic DNA extraction kit (Tiangen, DP319) for specific procedures.

[0046] 3. Genotyping: Genomic DNA was collected from each pig and whole-genome sequencing was performed on the Illumina HiSeq X-Ten sequencing platform. The sequencing depth for each individual was approximately 5×. The specific method followed the standard operating procedure provided by Illumina. After quality control, the data were sequenced and genotypes extracted using two bioinformatics software programs: BWA and GATK.

[0047] III. Genome-wide association analysis of α-nicotinamide adenine dinucleotide in the longissimus dorsi muscle

[0048] Genome-wide association analysis of α-nicotinamide adenine dinucleotide (NADP) and genotype in the longissimus dorsi muscle was performed using a compressed mixed linear model in EMMAX software. The results of the association analysis are shown in the figure below. Figure 1 The horizontal axis represents chromosome number, and the vertical axis represents -log. 10 (P).

[0049] A SNP significantly associated with the α-nicotinamide adenine dinucleotide trait was discovered, located at nucleotide 131363728 on chromosome 8. This SNP is named "Chr8:131363728 SNP," which corresponds to nucleotide 131363728 on chromosome 8 of the porcine genome (Sscrofa11.1, GCF_000003025.6), specifically nucleotide 262 of SEQ ID NO:1. This SNP has multiple nucleotide types at the T / C position. For simplicity, it will be referred to hereafter as a specific SNP.

[0050] A pair of primers, consisting of F and R, was designed based on a specific SNP. The target sequence of F and R in the pig genomic DNA is 383 bp, and the specific SNP is located at nucleotide 262 of the target sequence.

[0051] F (SEQ ID NO:2): 5'-TTCCCCACAATGCAACAAGC-3';

[0052] R (SEQ ID NO: 3): 5'-AATGCAAGGAGCGGAGAACG-3'.

[0053] Therefore, the genotype of the pig to be tested can be defined according to the following rules:

[0054] TT genotype: If the PCR product obtained by amplifying the genomic DNA of the pig to be tested using upstream primer F and downstream primer R contains only a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide T at position 262 of SEQ ID NO:1, and does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide T at position 262 of SEQ ID NO:1, then the aforementioned SNP genotype of the pig to be tested is TT.

[0055] TC genotype: If the PCR product obtained by amplifying the genomic DNA of the pig to be tested using upstream primer F and downstream primer R contains both a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 262 of SEQ ID NO:1 and a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 262 of SEQ ID NO:1, then the aforementioned SNP genotype of the pig to be tested is TC.

[0056] CC genotype: If the PCR product obtained by amplifying the genomic DNA of the pig being tested using upstream primer F and downstream primer R does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide T at position 262 of SEQ ID NO:1, and only contains a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 262 of SEQ ID NO:1, then the aforementioned SNP genotype of the pig being tested is CC.

[0057] Example 2: Application of specific SNPs in the genetic improvement of α-nicotinamide adenine dinucleotide content trait in the longissimus dorsi muscle of pigs.

[0058] Experimental animals: 496 Landrace pigs, Large White pigs, and crossbred pigs, sourced from COFCO Jiajiakang (Chifeng) Co., Ltd.

[0059] I. Detection of genotypes based on specific SNP loci

[0060] 1. Blood sample collection

[0061] Blood from the wing veins of experimental animals was collected using heparin sodium anticoagulant blood collection tubes and stored at -20°C for later use.

[0062] 2. Extract genomic DNA

[0063] Take the venous blood obtained in step 1 and extract genomic DNA.

[0064] 3. Genotyping

[0065] Using the genomic DNA obtained in step 2 as a template, PCR amplification was performed using primers consisting of F and R, and then the PCR amplification products were sequenced.

[0066] The results showed that PCR amplification products of 383 bp were obtained from all 496 experimental animals.

[0067] Based on specific SNPs, the 496 experimental animals were divided into three genotypes: TT genotype (393 experimental animals), TC genotype (90 experimental animals), and CC genotype (13 experimental animals).

[0068] II. Determination of α-nicotinamide adenine dinucleotide content in the longissimus dorsi muscle

[0069] Pigs were fed under the same feeding conditions for 180 days. 496 healthy pigs were randomly selected, and 40 mg samples of their longissimus dorsi muscle were collected and frozen in liquid nitrogen.

[0070] Sample pretreatment: Take a uniform sample, grind and mix thoroughly. Accurately weigh 40 mg of sample (place a certain amount of sample in a mortar, add liquid nitrogen, and weigh to prevent sample degradation) into a 2 mL centrifuge tube. Add 300 μL of methanol, then add a 5 mm steel bead. Homogenize the muscle tissue using a homogenizer (60 Hz, 60 s). Remove the steel bead with a magnet, add 1 mL of MTBE, vortex for 1 min, then add 250 μL of water to separate the layers. Let stand for 5 min. After thorough extraction, centrifuge at 4 °C for 5 min at 5000 rpm. Take 250 μL of the lower layer and resuspend it. Reconstitute the lower layer with 200 μL of 80% methanol-water for analysis.

[0071] Instruments and equipment: autosampler, liquid chromatography, mass spectrometry, liquid chromatography column (ACQUITY UPLC HSS T3 Column, 2.1mm × 100mm, Waters).

[0072] The liquid chromatography-mass spectrometry conditions are shown in Tables 1 and 2. The results in Table 3 show that the relative contents of α-nicotinamide adenine dinucleotide (α-ADN) differed significantly among the three genotypes of pigs (P < 0.001). The α-ADN content of the test pigs with genotype TT was relatively higher than that of the test pigs with genotype TC (P < 0.001) and genotype CC (P < 0.001), and the α-ADN content of the test pigs with genotype TC was relatively higher than that of the test pigs with genotype CC.

[0073] Table 3. Relative content of α-nicotinamide adenine dinucleotide in experimental animals of different genotypes

[0074]

[0075] Table 4. Genotypes and relative α-nicotinamide adenine dinucleotide content of 496 pigs tested.

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] R is either T or C.

[0089] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of molecular markers related to pork flavor, characterized in that, The application is any one of the following A1) to A8): A1) Detection or auxiliary detection of pork flavor; A2) Identification and auxiliary identification of pork flavor; A3) Pig breeding; A4) Detection or auxiliary detection of SNP polymorphisms or genotypes; A5) Prepare products for detecting or assisting in the detection of pork flavor; A6) Prepare products for identification and auxiliary identification of pork flavor; A7) Preparation of pig breeding products; A8) Prepare products for detecting or assisting in the detection of SNP polymorphisms or genotypes; The molecular marker corresponds to nucleotide position 131363728 on chromosome 8 of the pig reference genome Sscrofa11.1, GCF_000003025.6, as shown in sequence SEQ ID NO:1, the nucleotide at position 262 bp of the DNA molecule, the nucleotide type is T or C, and the genotype of this site is related to the content of α-nicotinamide adenine dinucleotide in the longissimus dorsi muscle of pigs.

2. A method for identifying or assisting in the identification of pork flavor, characterized in that, Includes the following steps: S1 extracts genomic DNA from the pigs to be tested as a template; S2 designed primers targeting the 262 bp site of the DNA molecule shown in SEQ ID NO.1 for PCR amplification; S3 detects the genotype at the 262 bp site of the pig sequence SEQ ID NO.1; S4 uses the genotypes obtained in step S3 to identify or assist in identifying the pork flavor of the pigs to be tested. Pigs with the TT genotype have a higher α-nicotinamide adenine dinucleotide content in the longissimus dorsi muscle than pigs with the CT genotype, and their pork flavor is superior to that of pigs with the CT genotype. Pigs with the CT genotype have a higher α-nicotinamide adenine dinucleotide content in the longissimus dorsi muscle than pigs with the CC genotype, and their pork flavor is superior to that of pigs with the CC genotype.

3. The method according to claim 2, characterized in that, The sequences of the primers described in step S2 are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. A breeding method for selecting pigs with better pork flavor, characterized in that, The genotype of the pig to be tested is identified according to the method of claim 2 or 3, and the pig to be tested with the TT genotype is selected as the parent for breeding, wherein the TT genotype is a homozygous type where the 262 bp of the SEQ ID NO.1 sequence is T.

5. The application of the molecular markers related to pork flavor according to claim 1, characterized in that, The pork flavor refers to the texture and aroma of pork, and the product is a reagent kit.

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