Use of a detection reagent for a snp molecular marker associated with pork tenderness
By developing a SNP molecular marker detection reagent for pork tenderness, and utilizing the SNP site at the 232bp position of the second intron of the Ucp3 gene, the problem of low efficiency in traditional breeding methods has been solved, enabling rapid and accurate identification and genetic improvement of pork tenderness.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JILIN AGRICULTURAL UNIV
- Filing Date
- 2025-10-24
- Publication Date
- 2026-08-04
AI Technical Summary
Current technologies for improving pork tenderness rely on traditional phenotypic selection methods, resulting in long breeding cycles, low efficiency, and dependence on post-slaughter testing, lacking effective molecular markers for rapid screening and genetic improvement.
A molecular marker detection reagent for SNPs related to pork tenderness was developed. By detecting the SNP site at 232bp of the second intron of the Ucp3 gene, PCR amplification and sequencing were performed using primer sets to identify GG-type individuals as having tender meat. This reagent can be used for pig-assisted breeding and meat quality identification.
It enables rapid and accurate identification of pork tenderness, improves breeding efficiency and genetic improvement of meat quality traits, and promotes early selection and breeding progress of high-quality pork breeds.
Smart Images
Figure CN121065360B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal breeding technology, specifically involving the application of detection reagents for SNP molecular markers related to pork tenderness. Background Technology
[0002] Meat quality is influenced by key factors such as muscle fiber type, intramuscular fat content, connective tissue distribution, and the degree of protein degradation. Among these, tenderness is one of the core evaluation indicators, directly related to the consumer's eating experience. Pork is one of the high-quality protein sources for humans. Improving the tenderness of pork can not only meet the taste requirements of different consumer groups, but also further enhance the nutritional value of pork and promote the sustainable development of the pork industry.
[0003] In current technologies, the improvement of pork quality mainly relies on traditional phenotypic selection methods. These methods have inherent limitations, including long cycles, low efficiency, and dependence on post-slaughter phenotypic testing, leading to slow breeding progress. Molecular marker technology can more accurately select pig breeds with superior tenderness traits, thereby improving breeding efficiency, farming efficiency, and product quality. However, the number of molecular markers currently developed related to pork tenderness is relatively small, and many related molecular marker loci remain undiscovered. Therefore, identifying molecular markers related to pork tenderness and developing corresponding detection reagents is essential for achieving rapid screening and genetic improvement of pork tenderness traits. Summary of the Invention
[0004] To address the aforementioned issues, this invention provides the application of detection reagents for SNP molecular markers related to pork tenderness, which can be used for pig-assisted breeding, pig selection, or pork tenderness identification, thereby accelerating the genetic breeding and early selection of high-quality meat quality pigs.
[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides an application of a detection reagent for SNP molecular markers related to pork tenderness, wherein the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP site is located at position 232 from the 5' end of the sequence shown in SEQ ID NO.1, and its polymorphism is G or A; the application is for pig-assisted breeding, pig selection, or pork tenderness identification.
[0006] Furthermore, pork with the GG genotype at the SNP locus has a higher tenderness than pork with the AA genotype.
[0007] Furthermore, the detection reagent is a primer set for detecting SNP molecular markers related to pork tenderness; the primer set consists of an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 3.
[0008] Furthermore, the pig breed is the Songlei Black Pig.
[0009] Furthermore, the pork is the longissimus dorsi muscle of Songlei black pig.
[0010] A second aspect of the present invention provides a method for identifying the tenderness of Songlei Black Pig pork, comprising the following steps: Genomic DNA was extracted from the Songlei black pigs to be tested; Using the genomic DNA of the Songlei black pig to be tested as a template, PCR amplification was performed using the primer set described above to obtain the amplification product; The amplified products were sequenced to identify the genotype of the SNP site in the sequence shown in SEQ ID NO.1. The tenderness of Songlei black pig pork was determined based on the genotype of the SNP site: the tenderness of Songlei black pig pork with the genotype of GG at the SNP site was greater than that of Songlei black pig pork with the genotype of AA.
[0011] Furthermore, each 20 μL PCR reaction system contains: 2 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, 10 μL 2x Taq Master Mix, and ddH2O to make up the difference.
[0012] Furthermore, the PCR reaction procedure is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 40 s, 34 cycles; 72℃ extension for 5 min.
[0013] Furthermore, the tenderness of the pork refers to the tenderness of the longissimus dorsi muscle of the pig.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses the application of a detection reagent for a SNP molecular marker related to pork tenderness. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1. This sequence contains an SNP site at position 232 bp from the 5' end, with a polymorphism of G / A, exhibiting three genotypes: GG, GA, and AA. The G allele is the dominant allele, indicating that individuals with the GG genotype have significantly higher meat tenderness than those with the AA genotype. This invention develops a reagent for detecting this SNP molecular marker, which can be used in pig-assisted breeding, pig selection, or pork tenderness identification. This invention also develops a method for identifying pork tenderness. This method can easily and quickly identify pigs with tender meat, facilitating the identification of meat tenderness traits in live pigs and accelerating the genetic breeding and early selection of high-quality meat quality breeds. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0016] Figure 1 To demonstrate the alignment results of some PCR product sequences, the sequences in rows corresponding to 2-136-2F_B08.abl, 2-137-2F_C08.abl, 2-138-2F_D08.abl, 2-139-2F_E08.abl, and 2-40-2F_F08.abl are sequences amplified using DNA from different Songlei black pig samples as templates, while the sequence in row corresponding to Ucp3mRNA.seq Consensus is the reference sequence.
[0017] Figure 2 Songlei Black Pig Ucp3 Sequencing diagrams of different genotypes at SNP sites in the second intron of a gene; black arrows indicate SNP sites. Figure 2 In the diagram, A represents the sequencing data of the GG genotype. Figure 2 B in the diagram represents the sequencing data of the GA genotype. Figure 2 C in the diagram represents the sequencing data of the AA genotype. Detailed Implementation
[0018] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0019] Traditional methods for improving the quality of black pork have limitations, including long breeding cycles, low efficiency, and reliance on post-slaughter testing. Discovering new molecular markers and developing corresponding detection reagents are crucial for rapid identification of pork tenderness and efficient genetic improvement of breeding pig breeds. This invention, based on the collection of longissimus dorsi muscle from Songlei black pigs, conducts meat quality trait analysis, and analyzes different Songlei black pig samples... Ucp3 After sequence alignment of the second intron of the gene, it was found that... Ucp3 A SNP site exists at 232 bp from the 5' end of the second intron gene sequence. The association between SNP site polymorphism and meat quality traits was analyzed to obtain molecular markers related to the tenderness of Songlei Black Pig pork. A reagent for detecting this SNP molecular marker and a method for identifying pork tenderness were also developed.
[0020] Example 1: Application of detection reagents for SNP molecular markers related to pork tenderness I. Development of Molecular Markers and Their Detection Reagents 1. Sample collection Longissimus dorsi muscle from 88 Songlei black pigs was collected at the Delis slaughterhouse in Jiaohe, Jilin Province. Meat quality performance was tested according to NY / T821-2019 "Specifications for Determination of Pig Muscle Quality". Simultaneously, 2g samples of the longissimus dorsi muscle were collected and flash-frozen in liquid nitrogen for later use. Both types of samples were labeled and matched, and records were kept.
[0021] 2. Genomic DNA extraction Genomic DNA was extracted from the samples using the genomic DNA extraction kit from Tiangen Biotech Co., Ltd. The concentration and purity of the DNA were detected using an ultra-micro spectrophotometer, and qualified samples were selected and stored at -80℃ for later use.
[0022] 3. Primer design and synthesis pig Ucp3 The gene's Ensembl ID in the Ensemble database is ENSSCG00105009109.1. Ucp3 The nucleotide sequence of the second intron of the gene is shown in SEQ ID NO.1. Based on this nucleotide sequence, a design for amplification was developed. Ucp3The primer set for the second intron sequence of the gene, comprising an upstream primer with nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer with nucleotide sequence as shown in SEQ ID NO.3, was synthesized by Suzhou Genewise Biotechnology Co., Ltd.
[0023] SEQ ID NO.1: .
[0024] SEQ ID NO. 2: 5'-ACCTTCCCACTGGACACG-3'.
[0025] SEQ ID NO. 3: 5'-CTTGACGGAGTCGTAGAGGC-3'.
[0026] 4. PCR amplification Using the DNA extracted in step 2 as a template, and the primer set from step 3, PCR amplification was performed according to the following PCR reaction system and procedure to obtain the amplification product. After the PCR product was identified as correct by agarose gel electrophoresis, it was sent to Suzhou Genewiz Biotechnology Co., Ltd. for Sanger sequencing.
[0027] Each 20 μL PCR reaction system contains: 2 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, 10 μL 2x TaqMaster Mix, and ddH2O to make up the difference.
[0028] PCR reaction procedure: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 34 cycles; 72℃ extension for 5 min; store at 4℃.
[0029] 5. Ucp3 Detection of gene polymorphism in the second intron The sequencing results of PCR products were compared with the pig genomic DNA sequences published in the Ensemble database using DANMAN and Chromas software. For mutation sites, the peak diagrams were analyzed using Chromas software.
[0030] like Figure 1 and Figure 2 As shown, an SNP site was found at 232 bp from the 5' end of the sequence shown in SEQ ID NO.1, with a polymorphism of G / A, and three genotypes: GG, GA, and AA.
[0031] II. Verification of the correlation between molecular markers and pork tenderness 1. Genotype frequency and gene frequency The genotype frequency, gene frequency calculation results, and chi-square goodness test results for this SNP locus are shown in Table 1.
[0032] Table 1. Statistical table of genotype frequencies and gene frequencies Table 1 shows that the GA genotype was the most numerous and was the dominant genotype; G was the dominant gene. The chi-square test of fitness indicated that the gene distribution at the C / T locus in Songlei black pigs conformed to Hardy-Weinberg equilibrium. P >0.05.
[0033] 2. Population genetic diversity Table 2 shows the genetic homozygosity, genetic heterozygosity, effective number of alleles, and polymorphism information content of SNP loci in the population.
[0034] Table 2. Genetic diversity of SNP loci The genetic heterozygosity of the SNP loci is at a moderate level; the effective number of alleles is close to 2, indicating that these alleles are evenly distributed in the population; the polymorphism information content is greater than 0.25 and less than 0.5, indicating moderate polymorphism.
[0035] 3. Correlation analysis between SNP molecular markers and pork tenderness This invention first tested the initial moisture content, pressure loss rate, pH, brightness, redness, yellowness, tenderness, and intramuscular fat content of pork samples from 88 Songlei black pigs. Then, an association analysis was performed on the three genotypes of SNP loci and meat quality traits. Among them, there were 32 samples with the GG genotype, 38 samples with the GA genotype, and 18 samples with the AA genotype. The results of the association analysis are shown in Table 3.
[0036] Table 3. Association analysis between genotype and tenderness Note: Different capital letters in the superscript of the table indicate extremely significant differences. P <0.01.
[0037] The lower the shear force value, the more tender the pork. Table 3 shows that SNP molecular marker polymorphism is significantly correlated with pork tenderness, with the shear force of GG genotype individuals being significantly lower than that of GA and AA genotype individuals. To improve pork tenderness, GG genotype individuals should be selected for future breeding.
[0038] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. The application of a detection reagent for SNP molecular markers related to pork tenderness, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1; the SNP molecular marker has one SNP site located at position 232 from the 5' end of the sequence shown in SEQ ID NO.1, and its polymorphism is G or A; the tenderness of pork with the genotype GG at the SNP site is greater than that of pork with the genotype AA at the same site; the application is pork tenderness-assisted breeding, pork tenderness selection, or pork tenderness identification; the pig is Songlei Black Pig; the pork tenderness is the tenderness of the longissimus dorsi muscle of Songlei Black Pig.
2. The application of the detection reagent for SNP molecular markers related to pork tenderness according to claim 1, characterized in that, The detection reagent is a primer set for detecting SNP molecular markers related to pork tenderness; the primer set consists of an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.
3.
3. A method for determining the tenderness of pork, characterized in that, The tenderness of the pork refers to the tenderness of the longissimus dorsi muscle of Songlei black pigs; the process includes the following steps: Genomic DNA was extracted from the Songlei black pigs to be tested; Using the genomic DNA of the Songlei black pig to be tested as a template, PCR reaction was performed using the upstream primer with the nucleotide sequence shown in SEQ ID NO.2 and the downstream primer with the nucleotide sequence shown in SEQ ID NO.3 to obtain PCR products; The PCR product was sequenced to obtain the genotype of the SNP site at position 232 from the 5' end in the sequence shown in SEQ ID NO.1; Based on the genotype of the SNP locus, the tenderness of pork is determined: pork with the genotype GG at the SNP locus has a greater tenderness than pork with the genotype AA.
4. The method for determining pork tenderness according to claim 3, characterized in that, Each 20 μL PCR reaction mixture contains: 2 μL template, 1 μL upstream primer, 1 μL downstream primer, 10 μL 2x Taq Master Mix, and ddH2O to make up the volume.
5. The method for determining pork tenderness according to claim 3, characterized in that, The PCR reaction procedure was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 40 s, for 34 cycles; 72℃ extension for 5 min.