A molecular marker related to the content of glycerophosphoryl ethanolamine in pig muscle and application thereof

Abstract

The application discloses a molecular marker related to the content of glycerylphosphorylethanolamine in pig muscle and application of the molecular marker. The application provides application of a substance for detecting the genotype of a SNP site in a pig genome in at least one of the following aspects: A1) detecting or assisting in detecting the content state of glycerylphosphorylethanolamine of a pig to be tested; A2) breeding a pig with high glycerylphosphorylethanolamine content; A3) pig breeding; A4) preparing a product for detecting or assisting in detecting the content state of glycerylphosphorylethanolamine of a pig to be tested; A5) preparing a product for breeding a pig with high glycerylphosphorylethanolamine content; and A6) preparing a product for pig breeding. By detecting the genotype of the pig to be tested at the SNP site, early selection of the content of glycerylphosphorylethanolamine in pig muscle can be realized, production cost can be saved, genetic progress can be accelerated, and the application has great economic application value and scientific research value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a molecular marker related to the content of glycerophosphorylethanolamine in pig muscle and its application. 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 meat, traditional methods relying on 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] Glycerylphosphoethanolamine, a phospholipid compound, possesses certain antioxidant properties and is widely found in animal muscle tissue, particularly concentrated in cell membrane phospholipids. It can regulate fatty acid metabolism, reduce free radical generation, and slow down fat oxidation, helping to reduce rancidity and other undesirable flavors in meat during storage, thus preserving its original flavor. By regulating lipoxygenase activity, it competitively inhibits the degradation of sulfur-containing amino acids, increases aldehyde and ketone content, enhances fatty and roasted meat aromas, and reduces meaty odors. During heat processing, it reacts with reducing sugars to generate pyrazines, contributing nutty aromas. Glycerylphosphoethanolamine affects the fatty acid composition, fat oxidation process, tenderness, flavor, and nutritional value of meat through multiple pathways.

[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. However, there are currently no molecular markers associated with the glycerophosphorylethanolamine content in pork. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a molecular marker related to the content of glycerophosphorylethanolamine in porcine muscle. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To address the aforementioned technical problems, the first aspect of this invention provides the application of a substance for detecting the genotype of SNP sites in the pig genome in at least one of the following:

[0007] A1) Detection or auxiliary detection of the glycerol phosphoryl ethanolamine content status of the pig to be tested;

[0008] A2) Select pigs with high glycerol phosphoryl ethanolamine content;

[0009] A3) Pig breeding;

[0010] A4) Prepare products for detecting or assisting in the detection of glycerol phosphoryl ethanolamine content in pigs to be tested;

[0011] A5) Prepare products from pigs with high glycerol phosphoryl ethanolamine content;

[0012] A6) Preparation of pig breeding products;

[0013] The SNP site is the 193rd nucleotide of SEQ ID NO:1;

[0014] The genotype of the SNP locus is GG, CC, or GC.

[0015] In the above text, the breeding indicator is the content of glycerol phosphoryl ethanolamine.

[0016] Specifically, pig breeding involves detecting or assisting in the detection of glycerophosphorylethanolamine content in pigs to be tested, or selecting pigs with high glycerophosphorylethanolamine content.

[0017] In the above text, the pigs to be tested can be individuals, groups, or breeds.

[0018] The purpose of the breeding is to select individuals with high levels of glycerol phosphoryl ethanolamine in the longissimus dorsi muscle.

[0019] The purpose of the breeding is to select a population with high levels of glycerol phosphoryl ethanolamine in the longissimus dorsi muscle.

[0020] The purpose of the breeding is to select varieties with high content of glycerol phosphoryl ethanolamine in the longissimus dorsi muscle.

[0021] In the breeding process, individuals with the GC or CC genotypes are eliminated.

[0022] In the breeding process, individuals with the GG genotype are retained.

[0023] In the above text, the state of glycerophosphorylethanolamine content is either high or low.

[0024] Secondly, the present invention provides the application of a substance for detecting bases at SNP sites in the porcine genome in at least one of the following:

[0025] A1) Detection or auxiliary detection of the glycerol phosphoryl ethanolamine content status of the pig to be tested;

[0026] A2) Select pigs with high glycerol phosphoryl ethanolamine content;

[0027] A3) Pig breeding;

[0028] A4) Prepare products for detecting or assisting in the detection of glycerol phosphoryl ethanolamine content in pigs to be tested;

[0029] A5) Prepare products from pigs with high glycerol phosphoryl ethanolamine content;

[0030] A6) Preparation of pig breeding products;

[0031] The SNP site is the 193rd nucleotide of SEQ ID NO:1;

[0032] The bases at the SNP sites are G or C.

[0033] In the above-described applications, the substance is any one of B1-B2:

[0034] B1) Primer pair;

[0035] B2) PCR reagents or kits containing the primer pairs described in B1);

[0036] The primer pair consists of a single-stranded DNA molecule shown in SEQ ID NO: 2 and a single-stranded DNA molecule shown in SEQ ID NO: 3.

[0037] The kit may also include standard reagents for PCR amplification. The kit may also include standard reagents for sequencing.

[0038] Thirdly, the present invention provides a method for detecting or assisting in the detection of glycerophosphorylethanolamine content status in a pig to be tested, comprising the following steps: detecting the genotype of the SNP site in the genome of the pig to be tested as described in the first aspect, wherein the genotype of the SNP site is GG, CC, or GC, and the glycerophosphorylethanolamine content of the pig to be tested with the genotype of the SNP site being GG is higher than or candidate to be higher than that of the pig to be tested with the genotype of the SNP site being CC or GC.

[0039] Fourthly, the present invention provides a method for detecting or assisting in the detection of glycerol phosphorylethanolamine content in a pig, comprising the following steps: detecting the bases at the SNP sites described in the first aspect in the genome of the pig, wherein the bases at the SNP sites are G or C.

[0040] The glycerophosphorylethanolamine content of the test pigs whose SNP sites on both homologous chromosomes contain G is higher than or candidate higher than that of the SNP sites on both homologous chromosomes where the SNP sites contain C.

[0041] The glycerophosphorylethanolamine content of the pig being tested is higher than or candidate higher than that of the SNP site on only one homologous chromosome, where the base is C.

[0042] The method described above, specifically the method for detecting the genotype of the SNP sites in the first aspect of the pig genome, includes the following steps: using the genomic DNA of the pig as a template, performing PCR amplification with the primer pairs described in the second aspect to obtain PCR products, and sequencing the PCR products.

[0043] If the PCR product contains only a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is GG.

[0044] If the PCR product does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and only contains a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is CC.

[0045] If the PCR product contains both a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide G at position 193 of SEQ ID NO:1, and a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide C at position 193 of SEQ ID NO:1, then the specific SNP genotype of the pig to be tested is GC.

[0046] The method described above, specifically the method for detecting the SNP sites in the first aspect of the pig genome, includes the following steps: using the genomic DNA of the pig as a template, performing PCR amplification with the primers described in the second aspect to obtain PCR products, and sequencing the PCR products.

[0047] If the PCR product contains only a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and does not contain a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the bases at the SNP sites on the genomes of the two homologous chromosomes of the pig to be tested are all G.

[0048] If the PCR product does not contain a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and only contains a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the bases at the SNP sites on the genomes of the two homologous chromosomes of the pig to be tested are all C.

[0049] If the PCR product contains both a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being G, and a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the SNP site in the genome of the pig being tested has a base of C on only one homologous chromosome, and the SNP site in the genome of the other homologous chromosome has a base of G.

[0050] Fifthly, the present invention provides a method for breeding pigs with high glycerol phosphoryl ethanolamine content, comprising the following steps:

[0051] Pigs with the genotype GG at the SNP site described in the third aspect of the method were selected and bred to achieve the breeding of pigs with high glycerol phosphoryl ethanolamine content.

[0052] Alternatively, select pigs whose SNP sites on two homologous chromosomes in the method described in the fourth aspect are both G, and breed them to achieve the breeding of pigs with high glycerophosphorylethanolamine content.

[0053] Sixthly, the present invention provides a product comprising any one of B1-B2):

[0054] B1) Primer pair;

[0055] B2) PCR reagents or kits containing the primer pairs described in B1);

[0056] The primer pair consists of a single-stranded DNA molecule shown in SEQ ID NO: 2 and a single-stranded DNA molecule shown in SEQ ID NO: 3.

[0057] The product described above has at least one of the following applications:

[0058] A1) Detection or auxiliary detection of the glycerol phosphoryl ethanolamine content status of the pig to be tested;

[0059] A2) Select pigs with high glycerol phosphoryl ethanolamine content;

[0060] A3) Pig breeding.

[0061] In the above text, the content of glycerophosphorylethanolamine refers to the content of glycerophosphorylethanolamine in the longissimus dorsi muscle.

[0062] Those skilled in the art will understand that SEQ ID NO:1 is composed of the SNP site (nucleotide 193 of SEQ ID NO:1) and its surrounding nucleotide sequence. The amount of nucleotide sequence around the SNP site should not be used as a limiting factor for the scope of protection of this invention. It can be 25 bp, 50 bp, 70 bp, 100 bp, 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, or 1000 bp before and after the SNP site, or any other arbitrary value. Its function is to assist in locating the SNP on chromosome 3 of the pig genome.

[0063] The front and back directions mentioned in this document should be defined as front or back in a direction generally accepted by those skilled in the art, such as the 5'-3' direction.

[0064] The pigs mentioned above refer to all pig breeds.

[0065] The beneficial effects of this invention are as follows: The SNP molecular marker of this invention is related to the trait of glycerol phosphoroethanolamine content in the longissimus dorsi muscle of pigs. It is a new molecular marker. By determining the genotype of the SNP locus of the pig to be tested, the trait of glycerol phosphoroethanolamine content 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. Detailed Implementation

[0066] 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.

[0067] 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.

[0068] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0069] 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 that there is a significant difference.

[0070] Example 1: Determination of the correlation between specific SNPs and glycerol phosphorylethanolamine content in the longissimus dorsi muscle.

[0071] Experimental animals: Landrace pigs, Large White pigs, and crossbred pigs, all sourced from COFCO Jiajiakang (Chifeng) Co., Ltd.

[0072] I. Determination of Glycerol Phosphorylethanolamine Content in the Longissimus Dorsi Muscle

[0073] 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.

[0074] 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 mixture as the test sample. Perform liquid chromatography-mass spectrometry (LC-MS) to detect the glycerol phosphoroethanolamine content in the longissimus dorsi muscle (the peak area is used as a relative response value, i.e., the glycerol phosphoroethanolamine content in the longissimus dorsi muscle).

[0075] Instruments and equipment: autosampler, liquid chromatograph, mass spectrometer, liquid chromatography column (ACQUITY UPLC HSS T3 Column, 2.1 mm × 100 mm, Waters).

[0076] Mobile phase preparation: 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, then add 980 mL of acetonitrile (5% ammonium formate).

[0077] The retention time of glycerol phosphoryl ethanolamine was 0.86061 ± 0.01 min.

[0078]

[0079]

[0080] II. Detection of SNP molecular markers

[0081] 1. Blood sample collection: Collect venous blood from pigs to be tested using heparin sodium anticoagulant blood collection tubes and store at -20℃ for later use.

[0082] The pigs selected for testing were 521 pigs in good health.

[0083] 2. Whole blood genomic DNA extraction: Refer to the instructions for the blood genomic DNA extraction kit (Tiangen, DP319) for specific procedures.

[0084] 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×, following the standard operating procedures provided by Illumina. After quality control, the data were sequenced and genotypes extracted using two bioinformatics software programs: BWA and GATK.

[0085] III. Genome-wide association analysis of glycerol phosphorylethanolamine in the longissimus dorsi muscle

[0086] The genome-wide association analysis of the glycerol phosphorylethanolamine content of the longissimus dorsi muscle obtained in step one and the genotypes obtained in step two was performed using a compressed mixed linear model in EMMAX software. The association analysis revealed a SNP significantly associated with the glycerol phosphorylethanolamine trait, located at nucleotide 20767343 on chromosome 3. This SNP was named "Chr3: 20767343 SNP," which corresponds to nucleotide 20767343 on chromosome 3 of the pig genome (Sscrofa11.1, GCF_000003025.6) (corresponding to nucleotide 193 of SEQ ID NO:1). The base R at this SNP site is either G or C, and the genotype of this SNP site is GG, CC, or GC. For simplicity, this will be referred to as the specific SNP. Pigs with the specific SNP genotype GG had a higher glycerol phosphorylethanolamine content than pigs with the specific SNP genotypes CC or GC.

[0087] 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 405 bp, and the specific SNP is located at the 193rd nucleotide of the target sequence.

[0088] F (SEQ ID NO:2): 5'-CAGCAAGTACTCCCACAGGG-3';

[0089] R (SEQ ID NO:3): 5'-GCAAAGCGTTTGTTTCCGGT-3'.

[0090] Therefore, the genotype of the pig-specific SNP to be tested can be defined according to the following rules:

[0091] GG genotype: If the PCR product obtained by amplifying the genomic DNA of the pig being 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 G at position 193 of SEQ ID NO:1, and does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide C at position 193 of SEQ ID NO:1, then the specific SNP genotype of the pig being tested is GG or the SNP sites on the genomes of the two homologous chromosomes of the pig being tested both contain the base G.

[0092] 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 193 of SEQ ID NO:1 being G, and only contains a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig being tested is CC or the SNP sites on the genomes of the two homologous chromosomes of the pig being tested both contain the base C.

[0093] GC 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 193 of SEQ ID NO:1 being G, and a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is GC, or the SNP site on only one homologous chromosome has a base of C, and the SNP site on another homologous chromosome has a base of G.

[0094] IV. Establishment of a method for detecting the content of glycerol phosphoryl ethanolamine in the longissimus dorsi muscle of pigs using specific SNPs

[0095] 1. Extract genomic DNA from the blood of the pig to be tested;

[0096] 2. Using the genomic DNA described above as a template, PCR amplification was performed using the F primer shown in SEQ ID NO:2 and the R primer shown in SEQ ID NO:3 to obtain the PCR product.

[0097] The reaction system for the above PCR amplification is as follows: 2×Taq PCR MasterMix 5.00μL (Solepro, PC1120-1ml), ddH2O 3.00μL, F primer 0.5μL, R primer 0.5μL, DNA template 1.0μL.

[0098] The PCR amplification reaction program was as follows: 95℃ for 5 min; 95℃ for 15 s, 63℃ for 15 s, 72℃ for 10 s, for a total of 35 cycles; 72℃ for 5 min; and stored at 4℃.

[0099] 3. Testing

[0100] The PCR products were sent for sequencing, and the results are as follows:

[0101] If the PCR product contains only a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being G, and does not contain a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is GG, or the bases of the SNP sites in the genomes of the two homologous chromosomes of the pig to be tested are both G.

[0102] If the PCR product does not contain a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being G, and only contains a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is CC, or the bases of the SNP sites in the genomes of the two homologous chromosomes of the pig to be tested are both C.

[0103] If the PCR product contains both a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being G, and a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is GC, or the SNP site on only one homologous chromosome has a base of C, and the SNP site on the other homologous chromosome has a base of G.

[0104] Pigs with the specific SNP genotype GG have a glycerophosphorylethanolamine content greater than or greater than that of pigs with the specific SNP genotype CC or GC.

[0105] Alternatively, the glycerol phosphoryl ethanolamine content in the longissimus dorsi muscle of the tested pigs whose SNP sites on both homologous chromosomes contain G bases is higher or candidate higher than that in the genomes of the two homologous chromosomes where the SNP sites contain C bases.

[0106] The longissimus dorsi muscle of the pig being tested has a glycerol phosphoryl ethanolamine content higher than or candidate higher than that of the SNP site on only one homologous chromosome, where the base is C.

[0107] The pigs can be a group or an individual.

[0108] Example 2: Application of specific SNPs in the genetic improvement of glycerol phosphoryl ethanolamine content trait in the longissimus dorsi muscle of pigs.

[0109] Experimental animals: 491 Landrace pigs, Large White pigs, and crossbred pigs, sourced from COFCO Jiajiakang (Chifeng) Co., Ltd.

[0110] I. Determination of Glycerol Phosphorylethanolamine Content in the Longissimus Dorsi Muscle

[0111] Pigs were fed under the same feeding conditions for 180 days. 491 healthy pigs were randomly selected, and 40 mg samples of their longissimus dorsi muscle were collected and frozen in liquid nitrogen.

[0112] 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 solution for analysis.

[0113] Instruments and equipment: autosampler, liquid chromatography, mass spectrometry, liquid chromatography column (ACQUITY UPLC HSS T3 Column, 2.1 mm × 100 mm, Waters).

[0114] Solid-phase microextraction conditions and gas chromatography-mass spectrometry conditions are shown in Table 1 and Table 2.

[0115] The relative content of glycerol phosphoroethanolamine in the longissimus dorsi muscle of experimental animals with different genotypes is shown in Table 3.

[0116] II. Specific SNP Detection of Glycerol Phosphorylethanolamine Content in the Longissimus Dorsal Muscle of Pigs

[0117] 1. Blood sample collection

[0118] Venous blood was collected from experimental animals using heparin sodium anticoagulant blood collection tubes and stored at -20°C for later use.

[0119] 2. Extract genomic DNA

[0120] Take the venous blood obtained in step 1 and extract genomic DNA.

[0121] 3. Genotyping

[0122] Using the genomic DNA obtained in step 2 as a template, PCR amplification was performed using the primer pair consisting of F shown in SEQ ID NO:2 and R shown in SEQ ID NO:3, according to the method in step four of Example 1. The PCR amplification products were then sequenced.

[0123] The results showed that PCR amplification products of 405 bp were obtained from all 491 experimental animals. After sequencing, the genotype results are shown in Table 3.

[0124] Based on specific SNPs, the 491 experimental animals were divided into three genotypes, as shown in Table 4: GG genotype (372 animals), GC genotype (104 animals), and CC genotype (15 animals). The glycerophosphorylethanolamine content in the pig population with the specific SNP genotype GG was higher than that in the pig populations with the specific SNP genotypes CC or GC.

[0125] The relative glycerophosphorylethanolamine (GPO) content detected above was mapped to each genotype group, and the results are shown in Table 4. The results indicate that the relative GPO ethanolamine content differed significantly among the three genotypes (P < 0.05). The relative GPO ethanolamine content of the tested pigs with genotype GG was higher than that of the tested pigs with genotypes GC (P < 0.05) or CC (P < 0.05), and the relative GPO ethanolamine content of the tested pigs with genotype GC was higher than that of the tested pigs with genotype CC. This is consistent with the specific SNP detection of this invention, indicating that the method of this invention can detect or assist in the detection of the relative GPO ethanolamine content in pigs.

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[0141] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of a substance for detecting the genotype of SNP sites in the pig genome in at least one of the following: A1) Detection or auxiliary detection of the glycerol phosphoryl ethanolamine content status of the pig to be tested; A2) Select pigs with high glycerol phosphoryl ethanolamine content; A3) Pig breeding; A4) Prepare products for detecting or assisting in the detection of glycerol phosphoryl ethanolamine content in pigs to be tested; A5) Prepare products from pigs with high glycerol phosphoryl ethanolamine content; A6) Preparation of pig breeding products; The SNP site is the 193rd nucleotide of SEQ ID NO:1; The genotype of the SNP locus is GG, CC, or GC.

2. The application of a substance for detecting bases at SNP sites in the porcine genome in at least one of the following: A1) Detection or auxiliary detection of the glycerol phosphoryl ethanolamine content status of the pig to be tested; A2) Select pigs with high glycerol phosphoryl ethanolamine content; A3) Pig breeding; A4) Prepare products for detecting or assisting in the detection of glycerol phosphoryl ethanolamine content in pigs to be tested; A5) Prepare products from pigs with high glycerol phosphoryl ethanolamine content; A6) Preparation of pig breeding products; The SNP site is the 193rd nucleotide of SEQ ID NO:1; The bases at the SNP sites are G or C.

3. The application according to claim 1 or 2, characterized in that: The substance is any one of B1-B2: B1) Primer pair; B2) PCR reagents or kits containing the primer pairs described in B1); The primer pair consists of a single-stranded DNA molecule shown in SEQ ID NO: 2 and a single-stranded DNA molecule shown in SEQ ID NO:

3.

4. A method for detecting or assisting in the detection of glycerophosphorylethanolamine content status in a pig to be tested, comprising the following steps: detecting the genotype of the SNP site in the genome of the pig to be tested as described in claim 1, wherein the genotype of the SNP site is GG, CC, or GC, and the glycerophosphorylethanolamine content of the pig to be tested with the genotype of the SNP site being GG is higher than or candidate to be higher than that of the pig to be tested with the genotype of the SNP site being CC or GC.

5. A method for detecting or assisting in the detection of glycerophosphorylethanolamine content status in a pig, comprising the following steps: detecting the bases at the SNP sites described in claim 1 in the genome of the pig, wherein the bases at the SNP sites are G or C. The glycerophosphorylethanolamine content of the test pigs whose SNP sites on both homologous chromosomes contain G is higher than or candidate higher than that of the SNP sites on both homologous chromosomes where the SNP sites contain C. The glycerophosphorylethanolamine content of the pig being tested is higher than or candidate higher than that of the SNP site on only one homologous chromosome, where the base is C.

6. The method according to claim 4, characterized in that: The method for detecting the genotype of the SNP site described in claim 1 in the genome of the pig to be tested includes the following steps: using the genomic DNA of the pig to be tested as a template, performing PCR amplification with the primer pair described in claim 3 to obtain PCR products, and sequencing the PCR products. If the PCR product contains only a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is GG. If the PCR product does not contain a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and only contains a DNA fragment with the nucleotide sequence of SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the specific SNP genotype of the pig to be tested is CC. If the PCR product contains both a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide G at position 193 of SEQ ID NO:1, and a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide C at position 193 of SEQ ID NO:1, then the specific SNP genotype of the pig to be tested is GC.

7. The method according to claim 5, characterized in that: The method for detecting the bases at the SNP sites described in claim 1 in the genome of the pig to be tested comprises the following steps: using the genomic DNA of the pig to be tested as a template, performing PCR amplification with the primers described in claim 2 to obtain PCR products, and sequencing the PCR products. If the PCR product contains only a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and does not contain a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the bases at the SNP sites on the genomes of the two homologous chromosomes of the pig to be tested are all G. If the PCR product does not contain a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being G, and only contains a DNA fragment with the nucleotide sequence SEQ ID NO:1 and the nucleotide at position 193 of SEQ ID NO:1 being C, then the bases at the SNP sites on the genomes of the two homologous chromosomes of the pig to be tested are all C. If the PCR product contains both a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being G, and a DNA fragment with the nucleotide sequence SEQ ID NO:1 and nucleotide 193 of SEQ ID NO:1 being C, then the SNP site in the genome of the pig being tested has a base of C on only one homologous chromosome, and the SNP site in the genome of the other homologous chromosome has a base of G.

8. A method for breeding pigs with high glycerol phosphoryl ethanolamine content, comprising the following steps: Pigs with the genotype GG at the SNP site described in claim 4 were selected and bred to achieve the breeding of pigs with high glycerol phosphoryl ethanolamine content. Alternatively, select pigs whose SNP sites on two homologous chromosomes in the genome of claim 5 are both G, and breed them to achieve the breeding of pigs with high glycerophosphorylethanolamine content.

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