Primer pair of FSD2 gene molecular marker related to cholesterol content of pork and application of primer pair

Abstract

The invention discloses a primer pair of an FSD2 gene molecular marker related to the cholesterol content of pork and application of the primer pair. A variation site is located in an eleventh intron region (at a No.52188710 basic group of a pig seventh chromosome) of a pig FSD2 gene and has a polymorphic SNP site, the genotype of the SNP site is TT, TC or CC, and the cholesterol content of pork can be determined according to the genotype of the variation site. According to the pig FSD2 gene disclosed by the invention, SNP loci related to the pork cholesterol content are screened in a subregion, so that a functional gene and a molecular genetic marker related to the pork cholesterol content are obtained, and a marker resource can be provided for molecular marker-assisted breeding of the pork cholesterol by preferably selecting dominant alleles of the SNP loci of the pig FSD2 gene; and a new way is provided for pork quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to substances related to cholesterol content in pork. FSD2 Primer pairs for gene molecular markers and their applications. Background Technology

[0002] Cholesterol is an essential lipid molecule in animals, participating in physiological processes such as cell membrane construction, hormone synthesis, and bile acid metabolism. The cholesterol content of pork longissimus dorsi muscle is an important indicator affecting the nutritional characteristics of meat and consumers' health perceptions; its level is influenced by breed, nutrition, and metabolic regulatory pathways. Since the human body obtains cholesterol primarily through animal-based foods, in addition to its own synthesis, and pork is my country's most important animal meat product, its cholesterol content plays a crucial role in dietary health. Therefore, breeding pigs with low-cholesterol traits is expected to provide high-quality, healthy pork products for the prevention of cardiovascular diseases. Current research on cholesterol in pigs mainly focuses on cholesterol levels in the blood. For example, mutations in the cholesterol regulatory element binding protein cleavage activator (SCAP) gene in Anhui black pigs can alter the activity of cholesterol metabolism-related enzymes, while mutations in the cholesterol ester transport protein (CETP) gene are significantly correlated with blood high-density lipoprotein cholesterol (HDL-C) and total cholesterol (TC) levels. However, research on cholesterol in pig muscle remains scarce. Identifying molecular markers and important functional genes related to cholesterol levels in pork can provide usable marker resources for gene editing and biobreeding of cholesterol traits in pork, as well as for breeding new low-fat, healthy pork varieties.

[0003] Genes containing fibronectin type III and SPRY domain 2. FSD2 It belongs to the FN3 / SPRY protein family and its main function is to interact with excitation-contraction coupling protein complexes in muscle, participating in the maintenance of normal muscle function and structure, and can participate in biological processes such as cytoskeleton junctions and cell membrane signal transduction. Current research has found... FSD2 It is mainly expressed in the pig heart, longissimus dorsi muscle, and psoas major muscle, and is correlated with intramuscular fat content and yellowness of pork. As a muscle factor, FSD2 Genes may negatively regulate lipid composition in muscle. Therefore, FSD2 Genes may also regulate cholesterol production in muscles, however FSD2 No reports have yet been found regarding the relationship between genes and pork cholesterol. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a convenient and rapid method for determining the cholesterol content of pork, thereby providing a basis for obtaining better pork quality. FSD2 Primer pairs for gene molecular markers.

[0005] A second objective of this invention is to provide a detection reagent or detection kit.

[0006] A third objective of this invention is to provide the application of the primer pairs, the detection reagents, or the kits described herein in identifying or assisting in the identification of cholesterol content in pork.

[0007] A fourth objective of this invention is to provide the application of the primer pair, the reagent, or the kit in the preparation of products for identifying or assisting in the identification of cholesterol content in pork.

[0008] A fifth objective of this invention is to provide the application of the primer pairs, reagents, or kits described herein in the selection or assisted selection of pigs or products with low cholesterol traits.

[0009] The sixth objective of this invention is to provide a method for identifying or assisting in the identification of cholesterol content in pork.

[0010] The seventh objective of this invention is to provide a method for breeding or assisting in the breeding of pigs with low cholesterol traits.

[0011] Technical solution: To solve the above technical problems, the present invention provides information related to the cholesterol content of pork. FSD2 Primer pairs for gene molecular markers, wherein the molecular marker sites are located at FSD2 The gene is located at base 52188710 on chromosome 7 of pigs in the eleventh intron region. The genotype of the molecular marker is TT, TC, or CC, and its number is rs343146619. The primer pair sequence is shown below: SEQ ID NO.3: FSD2-F: 5'-GCCATTTTGGAGCATAAGCA-3'; SEQ ID NO.4: FSD2-R: 5'-CCGCAAGTTGAGGCATAGTTT-3'.

[0012] The amplified sequence of the primer pair is shown in SEQ ID NO.1 or SEQ ID NO.2, where the molecular marker site is located at base 52188710 (position 335 in SEQ ID NO.1 or SEQ ID NO.2).

[0013] Specifically, when the genotype of the molecular marker is TC, the cholesterol content of the pork is lower than that of the TT genotype; that is, the cholesterol content of the sample with the TC genotype mutation site is significantly lower than that of the sample with the TT genotype mutation site. FSD2 The g.52188710 T>C mutation site in the intron region of the gene determines the cholesterol content of pork. The nucleotide sequence of the amplified fragment of the primer pair described in this invention is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0014] The present invention also includes a detection reagent containing the aforementioned primer pair.

[0015] The present invention also includes a detection kit, wherein the detection kit contains the primer pair or the detection kit described herein.

[0016] The present invention also includes the application of the primer pairs, the detection reagents, or the kits described herein in the identification or auxiliary identification of cholesterol content in pork.

[0017] Specifically, when the genotype of the molecular marker is TC, the pork has a low cholesterol content; when the genotype of the molecular marker is TT, the pork has a high cholesterol content.

[0018] The present invention also includes the application of the primer pairs, reagents, or kits described herein in the preparation of products for identifying or assisting in the identification of cholesterol content in pork.

[0019] The present invention also includes the application of the primer pairs, reagents, or kits described herein in the selection or assisted selection of pigs or products with low cholesterol traits.

[0020] The present invention also includes a method for identifying or assisting in the identification of cholesterol content in pork, comprising the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pair, wherein when the genotype of the SNP molecular marker is TC, the cholesterol content in pork is lower than that of the TT genotype.

[0021] The present invention also includes a method for breeding or assisting in the breeding of pigs with low cholesterol traits, comprising the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pair, wherein when the genotype of the SNP molecular marker is TC, the cholesterol content of the pork is lower than that of the TT genotype, and individuals with the TC genotype are retained as screening targets.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention studies the influence of... FSD2The correlation between genes and cholesterol content in pork was investigated. A SNP locus rs343146619, associated with cholesterol content, was identified in the intron region. This led to the acquisition of functional genes and molecular genetic markers related to cholesterol content in pork. By optimizing the dominant alleles of these SNP molecular markers, a foundation was laid for further control of cholesterol content in pork, which is of great significance for improving meat quality. This provides a basis for convenient and rapid assessment of cholesterol content in pork, thereby achieving better meat quality and is of great importance for improving pork quality traits. Attached Figure Description

[0023] Figure 1 This is a standard curve for cholesterol measurement.

[0024] Figure 2 Manhattan plot for genome-wide association analysis of muscle cholesterol content.

[0025] Figure 3 for FSD2 Sequencing maps of different genotypes of a gene.

[0026] Figure 4 This is a schematic diagram showing the relationship between different genotypes and cholesterol levels.

[0027] Figure 5 This study analyzed the correlation between the porcine FSD2 gene and porcine muscle cholesterol content. A) Comparison of pre-slaughter live weight between groups with high and low porcine muscle cholesterol content; B) Comparison of cholesterol content between groups with high and low porcine muscle cholesterol content; C) mRNA expression of the FSD2 gene in groups with high and low porcine muscle cholesterol content; D) Correlation analysis between FSD2 gene mRNA expression level and porcine muscle cholesterol content.

[0028] Figure 6 This is a schematic diagram showing the mRNA expression results of individuals with different FSD2 gene genotypes. Detailed Implementation

[0029] Unless otherwise specified, all methods used in the following examples are conventional methods, and all reagents used in the examples can be purchased commercially. The experimental data of this invention were statistically analyzed using SPSS 20.0 software, and paired Student's Law was applied between the two groups of data. t - Test analysis was performed, with Pearson correlation coefficient analysis used for correlation analysis, while one-way ANOVA was used to compare expression levels of individuals with different genotypes, and a mixed linear model was used for phenotypic association analysis between different genotypes. Y = μ + G + D + e, where Y Indicates muscle cholesterol content. μRepresents the group mean. G Indicates genotype. D The pre-slaughter live weight is used as a covariate. e Residuals are represented. Plotting was performed using GraphPad Prism version 8.0 software, and all data are represented as mean ± standard error (SEM).

[0030] 1. Experimental animals The 218 Large White pigs used in this invention were collected from the Changling Santuan Breeding Pig Farm between July and September 2024. All individuals were raised in the same environment and were sent to the Changling County Slaughterhouse for slaughter at approximately 180-200 days of age.

[0031] Before slaughter, the number and pre-slaughter live weight of each pig were recorded. Immediately after slaughter, approximately 200 g of longissimus dorsi muscle sample was collected from the last two ribs of the left carcass. The average pre-slaughter live weight of all Large White pigs was 135.79 ± 11.97 kg, with a minimum of 107.4 kg and a maximum of 172.6 kg, and a coefficient of variation of 8.82%. A portion of the longissimus dorsi muscle sample was selected for DNA and RNA extraction, as well as cholesterol content determination.

[0032] 2. Experimental Procedure 2.1 Determination of muscle cholesterol content (1) Take 200 g of longissimus dorsi muscle sample from the two ribs and homogenize the sample using a meat grinder or homogenizer.

[0033] (2) Weigh 1.000 g (accurate to 0.001 g) of the prepared sample into a 250 mL round-bottom flask, add 30 mL of anhydrous ethanol and 10 mL of 60% potassium hydroxide solution, and mix well. Saponify the sample by magnetic stirring and heating with an electric heating mantle at 100 °C for 1 h, shaking occasionally to prevent the sample from adhering to the flask wall. After saponification, rinse the inside of the flask with 5 mL of anhydrous ethanol from the top of the condenser, remove the round-bottom flask, and cool it to room temperature with running water.

[0034] (3) Quantitatively transfer all the saponified liquid into a 250 mL separatory funnel. Rinse the round-bottom flask 2-3 times with 30 mL of water, and combine the washings into the separatory funnel. Then rinse the round-bottom flask 2-3 times with a 40 mL mixture of petroleum ether and anhydrous diethyl ether (volume ratio 1:1), and combine the washings into the separatory funnel. Shake for 2 min, let stand, and allow the layers to separate. Transfer the aqueous phase, combine the three organic phases, and wash the extract with 100 mL of water each time until neutral. Gently swirl during the first water wash to prevent emulsification. Dehydrate the extract with about 10 g of anhydrous sodium sulfate and transfer it to a 150 mL flat-bottom flask.

[0035] (4) Evaporate the extract in the flat-bottomed flask to near dryness under vacuum, dissolve in anhydrous ethanol and bring the volume to 5 mL. Filter the solution through a 0.45 μm filter membrane and collect the filtrate in a sample vial. For high-performance liquid chromatography (HPLC), inject 10 μL of the sample solution into the HPLC system and measure the peak area. The peak area is then determined using a standard curve (e.g., ...). Figure 1 The concentration of cholesterol in the sample solution is obtained.

[0036] (5) The cholesterol content in the sample solution is calculated according to the formula: X = (ρ * V / m * 1000) * 100 In the formula: X — The cholesterol content in the sample, expressed in milligrams per 100 grams (mg / 100g). ρ — the concentration of cholesterol in the sample solution, in micrograms per milliliter (μg / mL). V—The final volume of the sample solution, in milliliters (mL); m — Sample mass, in grams (g); 1000, 100 — conversion factor.

[0037] In this experiment, the average cholesterol level of individuals was 39.2 ± 3.95 mg / 100g, the minimum was 30.1 mg / 100g, the maximum was 50.12 mg / 100g, and the coefficient of variation was 10.07%.

[0038] 2.2 Genomic DNA Extraction (1) Take about 100 mg of porcine longissimus dorsi muscle tissue stored at -80℃, carefully cut it into small pieces, add 500 μL of tissue lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0013B), 50 μL of 10 mg / mL proteinase K, shake to mix, and incubate overnight at 55℃.

[0039] (2) Add 500 μL DNA extraction phenol reagent (Beijing Xinhengyan Technology Co., Ltd., catalog number: J108238), shake vigorously to mix for 10 min, and centrifuge at 12000 rpm for 10 min at 4℃.

[0040] (3) Take the supernatant, add 1 mL of DNA extraction phenol reagent: chloroform: isoamyl alcohol (volume ratio 25:24:1), shake to mix, and centrifuge at 12000 rpm for 10 min at 4℃.

[0041] (4) Take the supernatant, add an equal volume of chloroform, shake to mix, and centrifuge at 12,000 rpm for 10 min at 4°C.

[0042] (5) Take the supernatant, add 1 mL of anhydrous ethanol to precipitate the DNA, and centrifuge at 12000 rpm for 5 min at 4℃.

[0043] (6) Discard the liquid, add 1 mL of 75% ethanol to wash the DNA precipitate, shake gently, centrifuge at 12000 rpm for 15 min, discard the residual liquid and air dry.

[0044] (7) Add 20 μL of ultrapure water, blow until the DNA is completely dissolved, measure the concentration on NanoDrop 2000 and store at -20℃.

[0045] 2.3 DNA Library Construction and Sequencing In this invention, the library construction and sequencing steps were completed by Novogene. DNA was randomly fragmented using Covaris technology, and end repair, addition of "A", adapter addition, PCR enrichment, and other steps were performed to construct the DNA library. Qualified libraries were re-sequencing the entire genome using the BGI DNBSEQ-T7 sequencing platform. The data obtained after sequencing were quality controlled using FASTP. The filtering conditions were: (1) removing reads with adapters; (2) removing reads with an N ratio exceeding 1%; (3) removing reads with Q ≤ 5 bases exceeding 50%; and (4) removing reads with a length < 100 bp.

[0046] The quality-controlled reads were aligned to the pig reference genome in the public database, version 110 (https: / / ftp.ensembl.org / pub / release-110 / fasta / sus_scrofa / dna / ), using BWA software. Samtools was used to analyze sequencing depth, genome coverage, and other information for each sample and to convert the format. GATK software was used to detect SNPs from the aligned data, generating a vcf file. VariantFiltration was then used to filter the data according to the following parameters: QD < 2.0, FS > 60.0, MQ < 40.0, SOR > 3.0, MQRankSum < -12.5, ReadPosRankSum < -8.0.

[0047] 2.4 Genotype Data Screening The SNPs in the VCF file were further screened using VCF Tools and PLINK software to retain SNP sites suitable for GWAS analysis. The specific screening steps are as follows: (1) SNP sites with multiple mutant bases were deleted, and only SNPs with two alleles were retained; (2) SNP sites not located on autosomes were deleted, and only SNP sites on chromosomes 1-18 were retained for subsequent analysis; (3) SNP sites with a deletion rate ≥ 0.10 were deleted; (4) SNP sites with a minimum allele frequency (MAF) ≤ 0.05 were deleted; (5) SNP sites that did not conform to Hardy-Weinberg equilibrium were deleted. P <0.01). The SNPs filtered through the above steps will be converted into plink BED format files for subsequent analysis.

[0048] Genome resequencing data quality control results: The average depth of genome resequencing of the Large White pig population in this invention was 15.15X, and a total of 20,529,172 variant sites were obtained. After excluding non-standard bases or insertion / deletion variants, 19,426,824 variant sites were finally aligned to the pig reference genome version 110 using BWA software. Then, after quality control of these SNPs using plink, 10,717,748 SNP variant sites were retained for subsequent genome-wide association analysis.

[0049] 2.5 Genome-wide association analysis (GWAS) 2.5.1 Principal Component Analysis This invention uses the --pca 10 command of the plink software to perform principal component analysis. By decomposing the genetic variation covariance matrix, it quantifies the kinship between populations and detects potential stratification. Based on the results, the principal component values ​​that can capture the main genetic variation information between populations are included in the covariate matrix of GWAS to correct the confounding effect of population structure on the association analysis results.

[0050] 2.5.2 Linear Mixed Model This invention uses the univariate linear mixed model (LMM) provided by GEMMA for GWAS analysis. The specific analysis model is: y = Wα + xβ + u + ε, where y is a 1×n phenotypic matrix (n is the number of individuals), W is an n×c fixed effects matrix, including individual live weight, sampling batch, the first three principal component values, and a column of 1s as the intercept; α is a c-dimensional coefficient vector, including the intercept term; x is the genotype vector; β is the polymorphic marker effect value; u is an n-dimensional random effects vector; and ε is an n-dimensional error vector. GEMMA performs hypothesis testing on each SNP, where the alternative hypothesis H1: β≠0, and the null hypothesis H0: β=0. The Wald test is used to obtain the estimated value of β and the corresponding p-value. The Manhattan plot of the p-values ​​of all SNP loci is plotted using the R package qqman to show the significance of the loci.

[0051] 2.5.3 Group Stratification Correction Test The correction for population stratification was assessed by adding the first three PCA components to the covariates to correct for the effects of population stratification, and the genome expansion coefficient λ was calculated.

[0052] 2.5.4 Candidate Gene Screening The significant loci obtained were aligned to the pig reference genome, version 110 annotation file in the Ensembl database using R language (version R 4.3.3) to obtain genes that may affect the cholesterol trait in pork, and the location of the loci within the genes.

[0053] Genome-wide association analysis (GWAS) results: Using the first three principal components of PCA to correct for population stratification, the calculated genome expansion coefficient λ was 1.034, within a reasonable range. This indicates a good fit between the actual and predicted values, suggesting that the population stratification effect has been effectively corrected. At the genome-wide level, a significant SNP locus located at chromosome 7 (52188710) was found to be significantly associated with cholesterol content in the longissimus dorsi muscle of Large White pigs. P =0.0000143)( Figure 2 (), numbered rs343146619.

[0054] 2.6 Primer design for mutation sites and genotyping 2.6.1 Primer Design According to the Ensembl database, pigs FSD2 The gene sequence (Chromosome 7:52144469-52194142) was used to design the following primers, which amplified a fragment of 786 bp.

[0055] SEQ ID NO.3: FSD2-F:5'-GCCATTTTGGAGCATAAGCA-3' SEQ ID NO. 4: FSD2-R: 5'-CCGCAAGTTGAGGCATAGTTT-3'.

[0056] 2.6.2 PCR Amplification Add the components to a 0.2 mL PCR tube according to the sequencing PCR reaction system in Table 1, mix well, and centrifuge.

[0057] Table 1 Sequencing PCR Reaction System

[0058] The 0.2 mL PCR tube was then placed in the PCR instrument. The reaction program was as follows: 94℃, 2 min; 94℃, 30 s, 55℃, 30 s, 72℃, 30 s, 30 cycles; 72℃, 2 min; 4℃, cooled and stored. The resulting cDNA was stored at -20℃ after the reaction.

[0059] 2.6.3 Sequencing and Sequence Alignment of PCR Products The amplification products were subjected to agarose gel electrophoresis. Single band samples that met the target fragment length and had high abundance were sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing files were viewed and compared using DNAstar 7.1 software to collect individual genotype information.

[0060] The results are as follows Figure 3 The PCR amplification products of the three genotypes at the rs343146619 locus were sequenced to verify the presence of TT, TC, and CC genotypes at this locus. Figure 3 This indicates that this locus can be used for primer amplification followed by sequencing and genotyping. Furthermore, analysis of the specific relationship between different genotypes and cholesterol levels in the resequencing data revealed that the cholesterol levels of individuals with the TC genotype at the rs343146619 locus were significantly lower than those of individuals with the TT genotype. P <0.05), while the cholesterol content of individuals with the CC genotype was not significantly different from that of individuals with the TT and TC genotypes ( P >0.05)( Figure 4 ).

[0061] 2.7 Quantitative Real-Time PCR Analysis 2.7.1 Tissue RNA Extraction (1) Clean the homogenizer rotor with DEPC water, and then clean it with Trizol reagent.

[0062] (2) Take about 100 mg of longissimus dorsi muscle tissue and put it into a 2 mL centrifuge tube. Add 500 µL of Trizol reagent to the tube, process the longissimus dorsi muscle tissue with a homogenizer, and finally add another 500 µL of Trizol reagent. Place on ice for 5 min.

[0063] (3) Add 200 µL of chloroform to the centrifuge tube, shake to mix for 30s, then place on ice for 10 min, and centrifuge at 12,000 rpm for 15 min at 4℃.

[0064] (4) Transfer the upper layer of liquid to a 1.5 mL centrifuge tube, add 500 µL of pre-cooled isopropanol to the centrifuge tube, mix thoroughly, let stand at room temperature for 10 min, and centrifuge at 12,000 rpm for 10 min at 4℃.

[0065] (5) Discard the supernatant, add 1000 µL of pre-cooled 75% ethanol, gently shake, and centrifuge at 12,000 rpm for 5 min at 4°C.

[0066] (6) Discard the supernatant, retain the RNA precipitate at the bottom, remove the water at the bottom of the tube as much as possible with a pipette, and air dry in a clean bench for 25 min.

[0067] (7) Add 20 µL of DEPC water to a centrifuge tube to dissolve the RNA, let it stand for 15 min, and use a NanoDrop spectrophotometer to determine the RNA concentration. Calculate the OD260 / 280 ratio to judge the quality of the RNA. The OD260 / 280 ratio should be 1.8-2.0, indicating that the quality of the RNA is qualified. After determining the RNA quality, store it at -80℃ or use it directly for reverse transcription.

[0068] 2.7.2 Reverse transcription Add the components to a 0.2 mL PCR tube according to the reverse transcription system in Table 2, mix well, and centrifuge.

[0069] Table 2 Reverse transcription reaction system

[0070] The 0.2 mL PCR tube was then placed in the PCR instrument, and the reaction program was 37°C for 15 min; 85°C for 5 s; and 4°C for cooling and storage. The cDNA obtained after the reaction was completed was stored at -20°C.

[0071] 2.7.3 Primer Design for Quantitative Real-Time PCR FSD2 and internal reference genes GAPDH The primer sequences for the real-time PCR were used and are published on the NCBI website. FSD2 (XM_021098895.1) andGAPDH The design was based on the reference sequence (XM_021091114.1), and the specific sequence is shown in Table 3.

[0072] Table 3 Primer sequences for real-time PCR

[0073] 2.7.4 Quantitative Real-Time PCR Add the components to the 96-well plate according to the reaction system in Table 4. After adding all components, mix well and centrifuge for a few seconds. Perform the detection according to the real-time PCR reaction procedure in Table 5. GAPDH Genes used as internal reference genes, using 2 -ΔΔCt The method involves statistical analysis of the data.

[0074] Table 4. Real-time PCR reaction system

[0075] Table 5. Quantitative Real-Time PCR Reaction Procedure

[0076] There was no significant difference in the pre-slaughter live weight among the selected individuals. P >0.05), and cholesterol levels showed significant differences ( P The longissimus dorsi muscle tissue of individuals with a length <0.0001) Figure 5 (Figures A and B in the image) Using cDNA from these two groups of samples as templates, real-time PCR analysis was performed to detect... FSD2 The expression of genes in the high-cholesterol and low-cholesterol groups indicates FSD2 The expression levels of the gene differed significantly between the two groups. P <0.01), FSD2 The mRNA level in the high-group was lower than that in the low-group. Figure 5 (See Figure C in the original text). Further statistics. FSD2 The correlation between mRNA expression levels and cholesterol levels was found. FSD2 mRNA expression in pork was significantly negatively correlated with cholesterol content. P <0.05)( Figure 5 (D diagram in the image).

[0077] Analysis of individuals with different genotypes FSD2 Differences in gene mRNA expression were observed, revealing the characteristics of individuals with the TC genotype. FSD2 Gene expression levels were significantly higher in individuals with the TT genotype than in individuals with the TT genotype. P <0.05), while individuals with the CC genotype FSD2 Gene expression did not differ significantly between individuals with TT and TC genotypes. P >0.05)(Figure 6 In view of the aforementioned findings FSD2 mRNA expression in pork was significantly negatively correlated with cholesterol content. Figure 5 D), while the cholesterol levels of individuals with the TC genotype were significantly lower than those with the TT genotype ( Figure 4 Therefore, the result that the mRNA expression level of TC genotype individuals is significantly higher than that of TT genotype individuals is consistent with the cholesterol content of each genotype population.

[0078] In summary, the mutation sites screened in this invention are significantly correlated with cholesterol traits in pork and can be developed into SNP molecular markers for detecting cholesterol content in pigs. FSD2 The g.52188710 T>C mutation site in the intron region of the gene indicates that individuals with the TC genotype have lower cholesterol levels than individuals with the TT genotype.

Claims

1. Related to the cholesterol content of pork FSD2 Primer pairs for gene molecular markers, characterized in that, The molecular marker sites are located at FSD2 The gene is located at base 52188710 on chromosome 7 of pigs in the eleventh intron region. The genotype of the molecular marker is TT, TC, or CC, and the primer pair sequence is shown below: SEQ ID NO.3: FSD2-F: 5'-GCCATTTTGGAGCATAAGCA-3'; SEQ ID NO.4: FSD2-R: 5'-CCGCAAGTTGAGGCATAGTTT-3'.

2. The method for determining the cholesterol content of pork according to claim 1. FSD2 Primer pairs for gene molecular markers, characterized in that, When the genotype of the molecular marker is TC, the cholesterol content of the pork is lower than that of the TT genotype, and the nucleotide sequence of the amplified fragment of the primer pair is shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. A detection reagent, characterized in that, The detection reagent contains the primer pair as described in claim 1 or 2.

4. A test kit, characterized in that, The detection reagent contains the primer pair as described in claim 1 or the detection reagent as described in claim 3.

5. The application of the primer pair of claim 1 or 2, the detection reagent of claim 3, or the kit of claim 4 in identifying or assisting in the identification of cholesterol content in pork.

6. In the application according to claim 5, when the genotype of the molecular marker is TC, the pork has a low cholesterol content; when the genotype of the molecular marker is TT, the pork has a high cholesterol content.

7. The use of the primer pair of claim 1 or 2, the reagent of claim 3, or the kit of claim 4 in the preparation of products for identifying or assisting in the identification of cholesterol content in pork.

8. The use of the primer pair of claim 1 or 2, the reagent of claim 3, or the kit of claim 4 in the selection or assisted selection of pigs or products with low cholesterol traits.

9. A method for identifying or assisting in the identification of cholesterol content in pork, characterized in that, The procedure includes the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pair described in claim 1, wherein when the genotype of the SNP molecular marker is TC, the cholesterol content of pork is lower than that of the TT genotype.

10. A method for breeding or assisting in the breeding of pigs with a low cholesterol trait, characterized in that, The process includes the following steps: performing PCR amplification and genotyping on the extracted genomic DNA using the primer pair described in claim 1, wherein when the genotype of the SNP molecular marker is TC, the cholesterol content of pork is lower than that of the TT genotype, and individuals with the TC genotype are retained as screening targets.

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