Molecular markers, primers, kits, methods and applications in the first intron of porcine PRKN gene
By designing specific primers at the first intron of the pig PRKN gene for PCR amplification and sequencing, the problem of difficult to assist breeding with molecular markers is solved, and early detection of pork quality and rapid breeding of excellent varieties are achieved.
Patent Information
- Application Number
- CN202410787845.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-18
- Publication Date
- 2025-04-01
- Estimated Expiration
- 2044-06-18
AI Technical Summary
The prior art is difficult to improve pork quality through molecular marker assisted breeding, especially because the cost of live measurement of pork quality is high, and it is difficult to improve conventional breeding.
By designing specific primers for single nucleotide mutation sites at the first intron of the pig PRKN gene, PCR amplification and sequencing, the polymorphisms of the PRKN gene were quickly and accurately identified, thereby detecting the differences in pork quality.
Early molecular marker selection for pork quality is achieved, which can accelerate the selection and breeding of pig breeds with excellent meat quality and reduce detection costs.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of pig breeding, and in particular to molecular markers, primers, kits, methods and applications in the first intron of the pig PRKN gene. Background Art
[0002] Pigs are important agricultural animals, and pork is the main source of protein for humans. With the improvement of people's living standards and the emphasis on health, high-quality pork is becoming more and more popular among consumers in the market. Therefore, the genetic improvement of pork quality is increasingly valued by breeders. Intramuscular fat (IMF) content, meat color score and marbling score are important indicators for evaluating pork quality, and are significantly correlated with pork flavor, tenderness and juiciness. Although pork quality is a medium-heritability trait, it is difficult to improve it through conventional breeding due to the high cost of in vivo determination of pork quality. Therefore, finding molecular markers that affect pork quality is of great significance for improving pork quality through molecular-assisted breeding.
[0003] Molecular markers used for auxiliary selection include protein markers, microsatellite markers, single nucleotide polymorphism (SNP) markers, etc. SNP markers refer to the polymorphism of DNA sequences caused by single nucleotide variations in the genome. They have the characteristics of large number, high accuracy, and high polymorphism. In breeding practice, SNP can be used to locate certain excellent genes, and the association between markers and specific qualities can be determined in combination with phenotypes. Molecular markers can also be verified in populations and applied to molecular breeding. Summary of the invention
[0004] Parkin RBR E3 ubiquitinprotein ligase, referred to as PRKN gene, encodes Parkin protein, an E3 ubiquitin ligase responsible for marking useless proteins in the body and degrading them by ubiquitination, which helps maintain the normal function and health of cells. PRKN gene mutations can lead to abnormal protein accumulation, accelerate neuronal cell death, and cause diseases such as Parkinson's disease and Alzheimer's disease. In addition, there are literature reports that Parkin participates in mediating organelle-specific autophagy such as mitochondria, affects inflammatory diseases by eliminating damaged organelles and maintaining homeostasis, and promotes cell survival and proliferation. However, there are no reports on the role of this gene in pork quality.
[0005] The present application, through GWAS analysis of the black pork quality resource population in Selenium City, found that there are signal sites in the PRKN gene region that are significantly correlated with pork quality. Therefore, the present application designs specific primers based on the single nucleotide mutation site on the first intron of the PRKN gene and performs amplification. After sequencing, the amplified product can quickly and accurately identify the polymorphism of the PRKN gene, thereby detecting differences in pork quality and providing guidance for the breeding of pork quality. In addition, the primers provided in the present application can specifically amplify the SNP site sequence contained in the intron of the PRKN gene, and the detection method has low cost.
[0006] To this end, the embodiments of the present application disclose at least the following technical solutions:
[0007] (1): Molecular markers of pork quality, including the nucleotide sequence formed by a single nucleotide mutation A>G at 5815150 bp in the first intron of the porcine PRKN gene.
[0008] (2): Molecular marker primers for pork quality, including a DNA molecule as shown in SEQ ID NO:3 and a DNA molecule as shown in SEQ ID NO:4.
[0009] (3): A nucleic acid molecule, which is amplified by PCR using the molecular marker primers described in (2), and the genotype of the nucleic acid molecule is associated with the quality of the pork.
[0010] (4): A kit comprising the molecular marker primers described in (2) and other reagents required for PCR amplification.
[0011] (5): Methods for testing pork quality, including:
[0012] Obtaining the pig genomic DNA to be tested;
[0013] Perform PCR amplification using the molecular marker primers described in (2);
[0014] The genotype of the 5815150 bp portion of the pig chromosome 1 was detected based on the nucleotide sequence of the amplified product;
[0015] The pork quality is determined according to the genotype.
[0016] (6): A pig screening method including the detection method described in (5).
[0017] (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:
[0018] 1) Detection and analysis of pork quality, wherein the pork quality is selected from marbling score and / or intramuscular fat content;
[0019] 2) Screening and breeding of pigs. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 It is the overall technical flow chart of this application.
[0021] Figure 2 This is the sequencing map of the A>G mutation at 5815150bp in the first intron of the porcine PRKN gene. The red box in the figure is the SNP site, and from top to bottom are AA homozygous, AG heterozygous and GG homozygous.
[0022] Figure 3 It is a visual diagram of the nucleotide sequence of the first intron fragment of the porcine PRKN gene (shown in SEQ ID NO: 1 or SEQ ID NO: 2). The red frame represents the location of the mutation, and the mutation position is located at the 417th base of the sequence. The underlined sequence represents the primer position. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.
[0024] The present application example sequenced the mutation sites of a partial nucleotide fragment of the PRKN gene and found that the genotype at 5815150 bp of the first intron of the PRKN gene was associated with pork quality.
[0025] To this end, the present application discloses a molecular marker for pork quality, including a nucleotide sequence formed by a single nucleotide mutation A>G at 5815150bp in the first intron of the pig PRKN gene. Among them, the pig PRKN gene refers to Gene ID: 733673 in the GeneBank database, assembly number Sscrofa11.1 (GCF 000003025.6), and position information: NC010443.5 (5698508.6731132).
[0026] In some embodiments, the pork quality is selected from marbling score and / or intramuscular fat content.
[0027] In some embodiments, at 5815150 bp of the first intron of the porcine PRKN gene, the marbling score and intramuscular fat content of individuals with the GG genotype were significantly higher than those of individuals with other genotypes.
[0028] Based on this, according to the genotype at 5815150bp of the first intron of the pig PRKN gene, molecular marker-assisted selection of pork quality of individual pigs can be carried out at an early stage to accelerate the breeding of pig breeds with excellent meat quality.
[0029] Therefore, specific primers were designed with reference to the first intron of the porcine PRKN gene in the GeneBank 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, and the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing were obtained.
[0030] Based on this, the present application also discloses molecular marker primers for pork quality, 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 molecular markers containing pork quality linkage, such as nucleotide sequences of SNPs, to analyze the genotype of the molecular markers, thereby obtaining pork quality.
[0031] On the other hand, the embodiment of the present application also discloses a nucleic acid molecule, which is amplified by PCR using the molecular marker primers, and the genotype of the nucleic acid molecule is associated with the pork quality. Further, the nucleic acid molecule is shown in SEQ ID NO: 1-2.
[0032] 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.
[0033] 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 5815150 bp of the first intron of the pig PRKN gene according to the nucleotide sequence of the amplified product; and determining the pork quality according to the genotype. For example, the marbling score and / or the intramuscular fat content are determined according to the genotype.
[0034] In some embodiments, the detection method further comprises sequencing the amplified product, and determining the genotype at 5815150 bp of the first intron of the pig PRKN gene according to the sequencing result.
[0035] Based on this, the embodiments of the present application also disclose a method for screening pigs, including the detection method described above, to determine the marbling score and / or intramuscular fat content of pork, and then screen pig breeds to obtain excellent pig breeds.
[0036] Based on this, the present application also discloses the application of the molecular marker, the molecular marker primer, the nucleic acid molecule or the kit, and the application is selected from any of the following:
[0037] 1) Detection and analysis of pork quality, wherein the pork quality is selected from marbling score and / or intramuscular fat content;
[0038] 2) Screening and breeding of pigs.
[0039] 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 to the present application. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial sources).
[0040] 1. Extraction of porcine genomic DNA
[0041] 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 the 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, and sent to the Agricultural Pig Quality Supervision and Inspection and Testing Center of the Ministry of Agriculture of Huazhong Agricultural University (Wuhan) within 4 hours to measure the quality of pork 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).
[0042] 2. Obtaining the nucleotide sequence of porcine PRKN and detecting the SNP sites of PRKN gene
[0043] 1. Obtaining the first intron fragment of the porcine PRKN gene
[0044] (1) PCR amplification
[0045] The following primer pairs were designed based on the porcine PRKN gene sequence (Gene ID: 733673 in the GeneBank database):
[0046] Forward primer PRKN-F: 5'-GTCACGGTCCCCTTTGAACA-3', SEQ ID NO: 3
[0047] Reverse primer PRKN-R: 5'-CCCCAACCATTTTCATTCATGC-3', SEQ ID NO: 4
[0048] The above primers were used to perform PCR amplification in a mixed genomic DNA pool of 40 Sedu 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.
[0049] The PCR operation program was as follows: preheating at 98°C for 45s; denaturation at 98°C for 10s, annealing at 62°C for 30s, extension at 72°C for 30s, for a total of 34 cycles; extension at 72°C for 10min; storage at 4°C. PCR products were electrophoresed using 1.5% agarose gel.
[0050] (2) PCR product purification
[0051] The above PCR products were purified using the Gel Extraction Kit of Shanghai Shenggong Biotechnology Co., Ltd. (according to the instructions of the kit), and the specific steps are as follows: first, the gel containing the target fragment was cut from the agarose gel, placed in a 1.5mL centrifuge tube, 400μL of sol solution was added, and the gel was completely melted in a 50-60℃ water bath. When heating the melted gel, it was mixed every 2 minutes and cooled to room temperature; the centrifuge column was placed in a collection tube, the mixed solution was transferred to the centrifuge column, and it was placed at room temperature for 2 minutes; centrifuged at 12000r / min for 1 minute, at which time the DNA was adsorbed on the column; discarded Collect the waste liquid in the tube, put the centrifuge column into the same collection tube, add 700μL of elution buffer, and centrifuge at 12000r / min for 1min; pour out the waste liquid in the collection tube, and centrifuge at 12000r / min for 1min; put the centrifuge column into a pre-prepared sterilized 1.5mL centrifuge tube, add 40μL of elution buffer or double distilled water (Ph>7.0), and place it at room temperature or 37℃ for 2-3min; centrifuge at 12000r / min for 1min, and the liquid in the centrifuge tube is the recovered DNA fragments.
[0052] 2. Obtaining the mutation site of the first intron of the porcine PRKN gene
[0053] The DNA fragments recovered above were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI3730XL sequencer, and a single-base mutation site was found ( Figure 3), an A>G mutation located at the 5815150bp of the PRKN genome nucleotide sequence (i.e., the full sequence) (GeneID: 733673 in the GeneBank database), the mutation sites corresponding to the PRKN nucleotide sequence of the present application are: an A>G base mutation (i.e., allele mutation) at 417bp in SEQ ID NO: 1; a G>A base mutation (i.e., allele mutation) at 417bp in SEQ ID NO: 2, such as Figure 3 shown.
[0054] 3. Molecular marker genotyping
[0055] The DNA sample of the individual to be tested was used as a template, and the first 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 3 shown.
[0056] 3. Genetic diversity detection and analysis of its association with quality
[0057] Using the method provided in the examples of this application, 268 Selenium City black pigs (from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd.) were subjected to genetic diversity detection and quality association analysis. SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0) general linear model GLM was used for statistical analysis. The model used was: Y ijklm =μ+G i +A j +X k +S l +e ijklm , where: Y ijklm represents the pork 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 expressed as the least squares mean ± standard error, and P < 0.05 was considered significant.
[0058] The results of association analysis are shown in Table 1. It was found that the A>G site at 5815150bp on chromosome 1 of the pig genome significantly affected the marbling score and intramuscular fat content of pork (P<0.05). The marbling score and intramuscular fat content of GG genotype individuals were significantly higher than those of other genotype individuals. The A>G marker at 5815150bp on chromosome 1 of the pig genome can be used for early molecular marker-assisted breed selection in the pig breeding process.
[0059] Table 1 Analysis of the association between the A>G mutation at 5815150bp on chromosome 1 of the pig genome and pork quality
[0060]
[0061] Note: Shoulder marks are significant difference marks of the same quality in different genotypes
[0062] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.
Claims
1. Methods for testing pork quality, including: Obtaining the pig genomic DNA to be tested; Perform PCR amplification using molecular marker primers, wherein the molecular marker primers are a DNA molecule as shown in SEQ ID NO:3 and a DNA molecule as shown in SEQ ID NO:4; The genotype at 5815150bp of chromosome 1 of the pig genome GCF 000003025.6 was detected based on the nucleotide sequence of the amplified product; The pork quality is determined according to the genotype, and the pork quality includes marbling score and intramuscular fat content. The marbling score and intramuscular fat content of individuals with the GG genotype at 5815150bp of chromosome 1 of the pig genome are significantly higher than those of individuals with other genotypes.
2. A method for screening pigs, comprising the detection method as described in claim 1; screening individuals with the GG genotype at 5815150bp of chromosome 1 of the pig genome GCF 000003025.6.