SNP molecular markers, primers and their applications for identifying pork meat color and intramuscular fat content

Abstract

The present invention relates to the technical field of pig breeding, and in particular to a SNP molecular marker, primers and applications thereof for identifying pork meat color and intramuscular fat content. The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, the N at position 140 of the molecular marker is a SNP site, and the polymorphism is C or G. The meat color and intramuscular fat content of pigs with the GG genotype at this site are both higher than those of pigs with the CC genotype and the CG genotype at this site. The present invention can be applied to continuous pig breeding and early breeding in production.

Description

Technical Field

[0001] The present invention relates to the technical field of pig breeding, and in particular to SNP molecular markers, primers and applications thereof for identifying pork meat color and intramuscular fat content. Background Art

[0002] For the past few decades, pig producers have focused on growth quality, such as growth rate, lean meat percentage, and feed conversion efficiency, primarily aiming to meet consumer demand through quantity. However, there is often a negative correlation between pig growth quality and pork quality. Improving growth quality inevitably results in a decrease in meat quality. However, consumers are increasingly seeking meat with a more refined texture and flavor. Therefore, improving meat quality has become a key research priority for pig producers.

[0003] Currently, traditional breeding methods primarily rely on phenotypic selection, such as for intuitive traits like weight and body shape. These methods are limited by slow genetic progress, long breeding cycles, and the difficulty in early prediction of complex traits like meat quality, reproductive performance, and disease resistance, leading to low breeding efficiency. Furthermore, it is difficult to accurately identify dominant individuals at an early stage, requiring the accumulation of multi-generational phenotypic data, significantly increasing both time and financial costs.

[0004] With the development of disciplines such as genomics, molecular biology, and bioinformatics, the widespread application of high-throughput sequencing, genotyping, and gene editing technologies, as well as the implementation and completion of whole-genome sequencing and haplotype planning for pigs, have greatly promoted the identification of genes with significant breeding value in pigs. SNP markers refer to polymorphisms in DNA sequences caused by single nucleotide variations in the genome. They are characterized by large numbers, high accuracy, and high polymorphism. In breeding practice, SNPs can be used to locate certain excellent genes and, combined with phenotypes, determine the association between markers and specific qualities. Molecular markers can also be verified in populations and applied in molecular breeding. However, the development of SNP molecular markers corresponding to the two phenotypes of pork color brightness and intramuscular fat content still requires further exploration and research. Summary of the Invention

[0005] The purpose of the present invention is to provide SNP molecular markers, primers and applications thereof for identifying pork meat color and intramuscular fat content, so as to solve the above technical problems.

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

[0007] The present invention provides a SNP molecular marker for identifying pork meat color and intramuscular fat content. The nucleotide sequence of the marker is shown in SEQ ID NO.1. The N at position 140 of the sequence is a SNP site, and the polymorphism is C or G. The meat color and intramuscular fat content of pigs with the GG genotype at this site are higher than those of pigs with the CC genotype and CG genotype at this site.

[0008] The present invention collects the longissimus dorsi muscle of Songlei black pigs, conducts meat quality trait analysis, uses direct sequencing to detect the polymorphism of the second exon of the FSD2 gene, analyzes the correlation between the existing polymorphic sites and meat quality traits, and obtains molecular markers related to the meat quality of Songlei black pigs, which are applied to the continuous breeding of Songlei black pigs and early selection in production.

[0009] The present invention also provides a specific primer for amplifying the above-mentioned SNP molecular marker, comprising an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

[0010] The present invention also provides a kit for detecting pork color and intramuscular fat content, wherein the kit comprises the above-mentioned specific primers.

[0011] The kit further comprises 2×Premix buffer.

[0012] The present invention also provides the use of the SNP molecular marker, the specific primer or the kit in identifying pork meat color and intramuscular fat content.

[0013] The present invention also provides the use of the SNP molecular marker, the specific primer or the kit in pig breeding or assisted pig breeding, wherein the assisted pig breeding is to cultivate pigs with high meat color and intramuscular fat content.

[0014] The present invention also provides a method for identifying pork color and intramuscular fat content, comprising the following steps:

[0015] S1, extracting the pig genomic DNA to be tested;

[0016] S2, using the pig genomic DNA to be tested as a template, amplifying using the specific primers to obtain an amplified product;

[0017] S3, identifying the genotype at position 140 of the amplified product, and determining the pork color and intramuscular fat content based on the genotype;

[0018] S4. The judgment method is: when the SNP site at position 140 of the amplified product is the GG genotype, the meat color and intramuscular fat content of the pigs are higher than those of the pigs with the CC genotype and CG genotype at the same site.

[0019] Furthermore, the amplification reaction system is as follows: 0.4-0.6 μL of upstream and downstream primers, 0.4-0.6 μL of pig genomic DNA to be tested, 9-11 μL of 2×Premix buffer, and 10-12 μL of water;

[0020] The reaction procedure is as follows:

[0021] (1) Pre-denaturation at 94-98°C for 4-6 minutes;

[0022] (2) Denaturation at 94-97°C for 25-35 seconds;

[0023] (3) Annealing at 55-65°C for 25-35 seconds;

[0024] (4) 70-74°C for 45-60 seconds;

[0025] (5) Repeat (2) to (4) for 25 to 35 cycles;

[0026] (6) Extend at 70-74°C for 4-6 minutes.

[0027] The present invention has the following beneficial effects:

[0028] For breeding pigs, genotype testing can be used to eliminate individuals with unfavorable genotypes and improve the overall meat quality of the offspring group; for piglets, their meat quality after fattening can be predicted after birth, and beneficial genotypes can be used for high-quality pork production, which has the advantages of high breeding efficiency and saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The following are the sequencing maps of different genotypes of the pig FSD2 gene SNP site. Among them, A is the sequencing map of the GG genotype, B is the sequencing map of the CG genotype, and D is the sequencing map of the CC genotype. DETAILED DESCRIPTION

[0031] 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 by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0032] Example 1: Development of SNP molecular markers for identifying pork meat color and intramuscular fat content.

[0033] 1. Experimental setup.

[0034] The experimental pig breed of this application is Songlei black pig, and the samples come from Jilin Xinghui Qipan Agricultural Technology Co., Ltd. 158 Songlei black pigs weighing about 100 kg were selected for slaughter in the experiment, and the longissimus dorsi muscle was collected 24 hours after slaughter. The thawing water loss rate was determined by measuring the water loss of the longissimus dorsi muscle before and after thawing, the initial moisture was determined by 65 ° C drying method, the meat color and pH value were determined by using a meat color meter and a pH meter, the pressurized weight method was used to determine the pressurized water loss rate, and the extraction method and cutting method were used to determine the intramuscular fat content and shear force. Blood was collected to extract DNA, specific primers were designed, and the entire sequence of the second exon of FSD2 was obtained by PCR. The SNP site was detected by direct sequencing, and the association between the SNP site and the quality traits of Songlei black pig was analyzed using SPSS26.0 software.

[0035] The porcine genomic DNA was extracted by first mincing the tissue with sterilized ophthalmic scissors, homogenizing it, and then extracting it using the genomic DNA kit (DP304) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0036] 2. Research and development of SNP molecular markers.

[0037] 1. Primer design and synthesis.

[0038] Primers were designed based on the second exon sequence of the porcine FSD2 gene in Ensemble (Ensembl No.: ENSSSCT00000002022.5) and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The primer sequence information is shown in Table 1.

[0039] Table 1: Primer sequences of porcine FSD2 gene

[0040]

[0041] 2. PCR amplification.

[0042] PCR amplification was performed using the above primers in pooled genomic DNA from 60 Songlei Black pigs. The PCR reaction system consisted of 0.5 μL of each upstream and downstream primer, 0.5 μL of pooled genomic DNA, 10 μL of 2× Premix buffer, and 11 μL of water. The PCR program was as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 50 s; and 72°C for 5 min. The SNP molecular marker sequence is shown in SEQ ID NO. 1:

[0043] SEQ ID NO. 1: AAATTGTGGATGAGTTGAGCCCAATCTAATAACCATCT.

[0044] 3. Detection of porcine FSD2 gene polymorphism.

[0045] SeqMan in DNASTAR was used to compare the Ensemble database sequence with the sequenced nucleotide sequence to screen for polymorphic sites. The fragment containing the polymorphic site was PCR amplified using the system and procedure as shown above. After amplification, the PCR product was sequenced. The SeqMan alignment sequencing results are shown in Figure 2. Figure 1As shown, a SNP site was found at 140 bp in the PCR product sequencing results, and three genotypes, GG, CG, and CC, existed.

[0046] 3. Verification of association analysis between SNP molecular markers and pork quality.

[0047] 1. Genotype frequency and gene frequency

[0048] The genotype frequency and gene frequency of this SNP locus were analyzed, and the results are shown in Table 2. GG and G are the dominant allele genotype and dominant allele, respectively. The chi-square test showed that the above SNP locus is in a state of Hartmann-Wenzhou equilibrium (P>0.05).

[0049] Table 2 Analysis of genotype frequency and gene frequency

[0050]

[0051] 2. Association analysis between SNP loci and meat quality

[0052] Meat quality traits of 158 Songlei Black pigs weighing approximately 100 kg were analyzed. T-TEST was used to analyze the significance of differences in meat quality traits among different genotype groups. As shown in Table 3, SNP molecular marker site polymorphisms were associated with brightness, yellowness, and intramuscular fat content. Individuals with the GG genotype had significantly higher brightness, significantly lower yellowness, and extremely significantly higher intramuscular fat content than those with the CC genotype. Selecting GG Black pigs can improve meat color and increase intramuscular fat content, making them suitable for early breeding, thereby accelerating the breeding process.

[0053] Table 3: Association analysis and verification of SNP loci and meat quality

[0054]

[0055]

[0056] Note: Different lowercase letters represent significant differences (P<0.05), and different uppercase letters represent extremely significant differences (P<0.01).

[0057] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0058] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. Use of a pair of specific primers or a kit containing the specific primers in identifying the meat color and intramuscular fat content of Songlei black pork, characterized in that: The specific primers are the upstream primer shown in SEQ ID NO.2 and the downstream primer shown in SEQ ID NO.

3. The genomic DNA of Songlei black pig is amplified by the specific primers, and the pig genome Sscrofa11.1 is used as a reference. When the genotype of the 140th position of the amplified product is detected to be GG genotype, it indicates that Songlei black pork has high meat color and high intramuscular fat content. When the 140th position of the amplified product is detected to be CC genotype or CG genotype, it indicates that Songlei black pork has low meat color and low intramuscular fat content.

2. A method for identifying the meat color and intramuscular fat content of Songlei black pork, characterized in that: The following steps are involved: S1. Extracting the genomic DNA of the Songlei black pig to be tested; S2. Using the genomic DNA of the Songlei black pig to be tested as a template, amplification is performed using the specific primers according to claim 1 to obtain an amplified product; S3. Using the pig genome Sscrofa11.1 as a reference, identify the genotype at position 140 of the amplified product, and determine the meat color and intramuscular fat content of Songlei Black Pork based on the genotype; S4. The judgment method is: when the SNP site at position 140 of the amplified product is the GG genotype, the pork color and intramuscular fat content of the Songlei black pig are higher than those of the Songlei black pigs with the CC genotype and the CG genotype at the same site.

3. The method for identifying the meat color and intramuscular fat content of Songlei black pork according to claim 2, characterized in that: The amplification reaction system is as follows: 0.4-0.6µL of upstream primer and downstream primer respectively, 0.4-0.6µL of Songlei Black Pig genomic DNA to be tested, 9-11µL of 2×Premix buffer, and 10-12µL of water; The reaction procedure is as follows: (1) Pre-denaturation at 94-98°C for 4-6 minutes; (2) Denaturation at 94-97°C for 25-35 seconds; (3) Annealing at 55-65°C for 25-35 seconds; (4) 70-74°C for 30-45 seconds; (5) Repeat (2) to (4) for 25 to 35 cycles; (6) Extend at 70-74°C for 4-6 minutes.

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