Molecular markers associated with pork intramuscular fat and their applications

Abstract

This application relates to molecular markers associated with pork intramuscular fat and their applications. The SNP is located at a single nucleotide polymorphism (G>A) at bp 6114468 on chromosome 1 of the porcine genome. PCR amplification of the target sequence containing the single nucleotide mutation site using primers and sequencing of the amplified product can rapidly identify PRKN genotypes, thereby detecting differences in pork quality and providing guidance for pig breed selection.

Description

Technical Field

[0001] The present application relates to the technical field of pig breeding, and in particular to molecular markers associated with pork intramuscular fat and their applications. Background Art

[0002] Pork, the world's largest meat consumer, is a key indicator of its production and quality in pig breeding. With the rapid development of molecular biology techniques, various technologies, such as high-density SNP microarrays, genotyping, and genome-wide association studies (GWAS), have been rapidly developed and applied. Through analyses of key economic traits in pigs, numerous genes associated with pork quality have been discovered. Different genotypes or expression levels of these genes can directly or indirectly influence important pork quality indicators such as backfat thickness, intramuscular fat content, and loin eye area. However, the number of major genes and loci identified remains limited. Therefore, identifying key functional genes and loci for key economic traits remains a key task in pig molecular breeding. Summary of the Invention

[0003] The Parkin RBR E3 ubiquitin protein ligase (PRKN) gene encodes the Parkin protein, an E3 ubiquitin ligase responsible for marking unused proteins in the body for ubiquitination and degradation, helping to maintain normal cellular function and health. Mutations in the PRKN gene can lead to abnormal protein accumulation, accelerating neuronal cell death and contributing to diseases such as Parkinson's disease and Alzheimer's disease. Furthermore, literature reports indicate that Parkin mediates organelle-specific autophagy, including mitochondria. This influences inflammatory diseases by eliminating damaged organelles and maintaining homeostasis, while also promoting cell survival and proliferation. However, research on the role of this gene in meat quality has not yet been reported.

[0004] The present application, through GWAS analysis of the black pork meat quality resource population in Xidu, found that there are signal sites in the PRKN gene region that are significantly correlated with meat quality. Therefore, the present application designs specific primers based on the single nucleotide mutation site on the fourth intron of the PRKN gene and performs amplification, and distinguishes the meat quality differences between individual pigs based on the polymorphism detection results of the amplified products. Primers are designed using this molecular marker, and the nucleotide sequence containing the SNP site is PCR amplified. Sequencing the amplified products can quickly identify the PRKN genotype, thereby detecting differences in pork quality and providing guidance for the breeding of pig varieties. In addition, the primers provided in the present application can specifically amplify the SNP site sequence contained in the gene intron of PRKN, and the detection method has low cost. It can be detected by PCR amplification alone, and there is no need for large-scale group sampling and measurement to compare the differences in meat quality between different pigs.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] (1): Molecular markers associated with pork intramuscular fat, including the nucleotide sequence formed by the single nucleotide mutation G>A at 6114468bp in the fourth intron of the pig PRKN gene.

[0007] (2): Molecular marker primers associated with pork intramuscular fat, including a DNA molecule as shown in SEQ ID NO: 3 and a DNA molecule as shown in SEQ ID NO: 4.

[0008] (3): A nucleic acid molecule, which is amplified by PCR using the molecular marker primer described in (2), and the genotype of the nucleic acid molecule is associated with the quality of the pork.

[0009] (4): A kit comprising the molecular marker primers described in (2) and other reagents required for PCR amplification.

[0010] (5): Methods for testing pork quality, including:

[0011] Obtaining the pig genomic DNA to be tested;

[0012] Perform PCR amplification using the molecular marker primers described in (2);

[0013] The genotype of the 6114468 bp portion of the pig chromosome 1 was detected based on the nucleotide sequence of the amplified product;

[0014] The pork quality is determined based on the genotype.

[0015] (6): A pig screening method comprising the detection method described in (5).

[0016] (7): The use of the molecular marker described in (1), the molecular marker primer described in (2), the nucleic acid molecule described in (3), or the kit described in (4), wherein the use is selected from any one of the following:

[0017] 1) Detection and analysis of pork quality, wherein the pork quality is intramuscular fat;

[0018] 2) Screening and breeding of pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the overall technical flow chart of this application.

[0020] Figure 2This is the reverse sequencing result of the complementary chain of the G>A mutation at 6114468bp in the fourth intron of the porcine PRKN gene. The red box in the figure is the SNP position. From top to bottom, they are CC homozygous, CT heterozygous, and TT homozygous, which correspond to the GG homozygous, GA heterozygous, and AA homozygous at 6114468bp in the sequence of chromosome 1 of the porcine genome (NC 010443.5 (5698508.6731132)).

[0021] Figure 3 This is a visual representation of the nucleotide sequence of the fourth intron fragment of the porcine PRKN gene (shown in SEQ ID NO: 1 or SEQ ID NO: 2). The red box represents the location of the mutation, and the mutation position is located at the 301st base of the sequence. The underlined sequence represents the primer position. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0023] The present application example sequenced the mutation site of a portion of the nucleotide sequence of the PRKN gene and found that the genotype at 6114468 bp in the fourth intron of the PRKN gene was associated with pork quality.

[0024] To this end, the present invention discloses molecular markers associated with pork intramuscular fat, including a nucleotide sequence containing a single nucleotide mutation (G>A) at bp 6114468 of the fourth intron of the porcine PRKN gene. The porcine PRKN gene is referenced in the GenBank database with GeneID: 733673, assembly number: Sscrofa11.1 (GCF 000003025.6), and position information: NC 010443.5 (5698508.6731132).

[0025] In some embodiments, the pork quality is intramuscular fat.

[0026] In some embodiments, at 6114468 bp of the fourth intron of the porcine PRKN gene, individuals with the AA genotype and the GA genotype have significantly higher intramuscular fat content than individuals with the GG genotype.

[0027] Based on this, the pork quality of individual pigs can be genetically marked according to the genotype at 6114468bp of the fourth intron of the pig PRKN gene, which is conducive to the selection of pig breeds with excellent meat quality.

[0028] Therefore, specific primers were designed with reference to the fourth intron of the porcine PRKN gene in the GenBank database (the sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:4 in the sequence listing), PCR amplification was performed using porcine genomic DNA as a template, the amplified products were gel-recovered and sequenced and analyzed to obtain the gene fragments shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing.

[0029] Based on this, the present application also discloses molecular marker primers associated with pork intramuscular fat, including a DNA molecule as shown in SEQ ID NO: 3 and a DNA molecule as shown in SEQ ID NO: 4. The "molecular marker primers" are used to amplify nucleotide sequences containing molecular markers linked to pork quality, such as SNPs, to analyze the genotype of the molecular markers and thus determine pork quality.

[0030] In another aspect, the present invention also discloses a nucleic acid molecule, which is amplified by PCR using the molecular marker primers, wherein the genotype of the nucleic acid molecule is associated with the pork quality. Further, the nucleic acid molecule is shown in SEQ ID NOs: 1-2.

[0031] On the other hand, the embodiments of the present application also disclose a kit, including the molecular marker primers and other reagents required for PCR amplification.

[0032] Based on this, the present application also discloses a method for detecting pork quality, comprising: obtaining genomic DNA of a pig to be tested; performing PCR amplification using the molecular marker primers; detecting the genotype at 6114468bp of the fourth intron of the pig PRKN gene based on the nucleotide sequence of the amplified product; and determining the pork quality based on the genotype. For example, the intramuscular fat is determined based on the genotype.

[0033] In some embodiments, the detection method further comprises sequencing the amplified product, and determining the genotype at 6114468 bp of the fourth intron of the porcine PRKN gene according to the sequencing results.

[0034] Based on this, the embodiments of the present application also disclose a method for screening pigs, including the detection method described above, to determine intramuscular fat, and then screen pig breeds to obtain excellent pig breeds.

[0035] Based on this, the embodiments of the present application further disclose applications of the molecular marker, the molecular marker primer, the nucleic acid molecule or the kit, wherein the application is selected from any one of the following:

[0036] 1) Detection and analysis of pork quality, wherein the pork quality is intramuscular fat;

[0037] 2) Screening and breeding of pigs.

[0038] The present application is now further described in conjunction with specific examples. The following examples are only intended to explain the present application but do not constitute a limitation of the present application. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels).

[0039] 1. Extraction of porcine genomic DNA

[0040] The experimental pig breed of this application is Selenium City Black Pig, and the samples come from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd. The genomic DNA of the pig was extracted using a genomic DNA kit produced by Beijing Biotech Biotechnology Co., Ltd. The extraction was performed according to the instructions of the kit. The concentration and quality of the extracted DNA were tested and stored at -20°C for later use. The remaining muscle samples were sealed in bags and stored at 4°C. They were sent to the Breeding Pig Quality Supervision and Inspection Center of the Ministry of Agriculture of Huazhong Agricultural University (Wuhan) within 4 hours, and the pork quality was determined in accordance with the Agricultural Industry Standard of the People's Republic of China "Technical Specifications for Pork Quality Determination" (Standard No.: NY / T 821-2019).

[0041] 2. Obtaining the porcine PRKN gene fragment and detecting the PRKN gene SNP site

[0042] 1. Obtaining the fourth intron fragment of the porcine PRKN gene

[0043] (1) PCR amplification

[0044] The following primer pairs were designed based on the porcine PRKN gene sequence (Gene ID: 733673 in the GenBank database):

[0045] Forward primer PRKN-F: 5'-CATATTTTCCAAGAGTTAAGCAGAG-3', SEQ ID NO: 3

[0046] Reverse primer PRKN-R: 5′-CTGGTGCCCTTTATGTCTGG-3′, SEQ ID NO: 4.

[0047] The above primers were used to perform PCR amplification in a mixed genomic DNA pool of 40 Xidu black pigs. The PCR reaction system was 50 μL, and the concentrations of each component in the system were 100 ng template DNA, 10× buffer (containing Mg 2+)4 μL, 0.5 μM each of the upstream and downstream primers mentioned above, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.

[0048] The PCR program was as follows: preheating at 98°C for 45 seconds, denaturation at 98°C for 10 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 34 cycles; extension at 72°C for 10 minutes; and storage at 4°C. PCR products were electrophoresed on 1.5% agarose gels.

[0049] (2) PCR product purification

[0050] The PCR product was purified using the Gel Extraction Kit from Shanghai Shenggong Bioengineering Co., Ltd. (according to the kit's instructions). The specific steps are as follows: first, cut the gel containing the target fragment from the agarose gel and place it in a 1.5 mL centrifuge tube. Add 400 μL of the gel solution and heat in a 50-60°C water bath until the gel is completely melted. While heating the gel, mix it every 2 minutes and cool it to room temperature. Place the centrifuge column in a collection tube and transfer the mixture to the centrifuge column. Let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute to allow the DNA to be adsorbed on the column. Discard the centrifuge column. Collect the waste liquid in the tube, place the centrifuge column in the same collection tube, add 700μL eluent, and centrifuge at 12000r / min for 1min; pour out the waste liquid in the collection tube, and centrifuge at 12000r / min for 1min; place the centrifuge column in a pre-prepared sterilized 1.5mL centrifuge tube, add 40μL eluent or double-distilled water (pH>7.0), and place at room temperature or 37℃ for 2-3min; centrifuge at 12000r / min for 1min. The liquid in the centrifuge tube is the recovered DNA fragments.

[0051] 2. Obtaining the mutation site in the fourth intron of the porcine PRKN gene

[0052] The recovered DNA fragments were sent to Wuhan AoKe DingSheng Biotechnology Co., Ltd. for sequencing using an ABI3730XL sequencer, and a single-base mutation site was found ( Figure 2 ), a G>A mutation at 6114468 bp of the PRKN genome nucleotide sequence (i.e., the complete sequence) (GeneID: 733673 in the GenBank database), corresponding to the mutation sites of the PRKN gene fragment of the present application: a G>A base mutation (i.e., an allele mutation) at 301 bp in SEQ ID NO: 1; an A>G base mutation (i.e., an allele mutation) at 301 bp in SEQ ID NO: 2, such as Figure 3 shown.

[0053] 3. Molecular marker genotyping

[0054] The DNA sample of the individual to be tested was used as a template, and the fourth intron sequence fragment of the porcine PRKN gene was amplified according to the method described in step 1 above. The obtained PCR purified product was directly sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and the genotyping results were directly read from the sequencing results, such as Figure 2 shown.

[0055] 3. Genetic diversity detection and association analysis with quality

[0056] Using the method provided in the examples of this application, genetic diversity detection and correlation analysis with quality were performed on the 274 Selenium City Black Pig Herd (from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd.). Statistical analysis was performed using the general linear model (GLM) using SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0). The model used was: Y ijklm =μ+G i +A j +X k +S l +e ijklm , where: Y ijklm represents the meat quality phenotypic value; μ represents the population mean; G i represents genotype effect; A j represents the year-season effect; X k represents the gender effect; S l represents the paternal effect; e ijklm The results are presented as the least squares mean ± standard error, and P < 0.05 was considered significant.

[0057] The results of the association analysis, shown in Table 1, revealed that the G>A locus at bp 6114468 on chromosome 1 significantly affected intramuscular fat (P<0.05). Individuals with the AA genotype had significantly higher intramuscular fat content than those with other genotypes. In pig breeding, the G>A marker at bp 6114468 on chromosome 1 could be used for early molecular marker-assisted selection of replacement pigs, prioritizing the retention of individuals with the AA genotype.

[0058] Table 1 Association analysis between the G>A mutation at bp 6114468 on chromosome 1 of the pig genome and meat quality

[0059]

[0060] Note: Shoulder marks are significant differences in the same quality of different genotypes

[0061] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. The quality testing method of Sedu Black Pig includes: Obtaining the pig genomic DNA to be tested; PCR amplification was performed using primers for SNP molecular markers associated with pork intramuscular fat; The genotype of the 6114468 bp site on chromosome 1 of the porcine genome Sscrofa11.1 version is detected based on the nucleotide sequence of the amplified product, wherein the SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:1, and there is a G / A base mutation at position 301 of the sequence shown in SEQ ID NO:1; The pork quality is determined according to the genotype, and the pork quality is the intramuscular fat content.

2. The method for detecting the quality of Xidu black pig meat according to claim 1, wherein the SNP molecular marker primers include the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO:

4.

3. The method for detecting the pork quality of Selenium City Black Pig according to claim 1, the 6114468 bp site of chromosome 1 of the pig genome Sscrofa11.1 version has AA, GA and GG genotypes, and the intramuscular fat content of individuals with AA genotype and GA genotype is significantly higher than that of individuals with GG genotype.

4. A method for screening the quality of Xidu black pig pork, comprising the detection method according to any one of claims 1 to 3, wherein the pork quality is the intramuscular fat content.

5. Application of a SNP molecular marker associated with pork intramuscular fat, a primer for a SNP molecular marker associated with pork intramuscular fat, or a kit, wherein the application is selected from any one of the following: 1) Testing and analysis of the pork quality of Xidu Black Pig, where the pork quality is measured by intramuscular fat content; 2) Screening and breeding of Sedu Black pigs with high intramuscular fat content; in, The SNP molecular marker is a nucleotide sequence as shown in SEQ ID NO: 1, wherein there is a G / A base mutation at position 301 of the sequence shown in SEQ ID NO: 1 and there are AA, GA and GG genotypes, and the intramuscular fat content of individuals with AA and GA genotypes is significantly higher than that of individuals with GG genotypes; The kit includes the SNP molecular marker primers and other reagents required for PCR amplification.

6. Use of the SNP molecular marker associated with pork intramuscular fat, the SNP molecular marker primer associated with pork intramuscular fat, or the kit according to claim 5, wherein the SNP molecular marker primer comprises the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4.

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