Molecular Marker, Primer, Kit, Method and Application in the Sixth Intron of Porcine PRKN Gene

Abstract

This application relates to a molecular marker, primer, kit, method and application in the sixth intron of the porcine PRKN gene. The molecular marker includes a nucleotide sequence formed by a single nucleotide A>C mutation at position 6,447,587 on chromosome 1 of the porcine genome. Using a primer pair to perform PCR amplification on a target sequence containing this single nucleotide mutation site, sequencing the amplification product can quickly identify the PRKN genotype, thereby detecting differences in pork quality and providing guidance for the breeding of pig breeds.

Description

Technical Field

[0001] This application relates to the technical field of pig breeding, and specifically relates to molecular markers, primers, kits, methods and applications in the sixth intron of the pig PRKN gene. Background Art

[0002] China is a major producer and consumer of pork. According to relevant reports and statistics, in China, the annual sales volume of pork accounts for sixty to seventy percent of the sales of meat products. As the most important part of the meat product market, pork quality, which affects its quality, has become the most important economic quality. Pork quality is mainly composed of softness, juiciness and flavor. The corresponding meat quality detection indicators include intramuscular fat, meat color, pH value, drip loss and tenderness of pig muscle, etc. In 1990, CAmeron first proposed that pork quality is a complex comprehensive concept. It is difficult to judge the quality of meat with a single index. Multiple indicators need to be comprehensively analyzed. And the meat quality evaluation of individual phenotypes must be achieved through the slaughter determination of its siblings or descendants, with high costs and poor reliability. Therefore, the progress of breeding excellent meat pigs using traditional breeding methods is slow. Fortunately, the heritability of pork quality is relatively high. By combining molecular biology and statistical means, deeply studying the genetic mechanism of pork quality, excavating candidate genes and molecular markers significantly related to pork quality, and then selecting through gene detection means, opportunities can be provided for meat quality improvement. At present, the understanding of the key genes affecting meat quality characteristics is still insufficient, and there are few molecular markers that can be applied to the breeding of pork quality. Therefore, finding the key molecular genetic markers controlling pork quality and applying them to molecular marker-assisted breeding is of great significance for improving the meat quality characteristics of pigs and increasing economic benefits.

[0003] Molecular markers for assisted selection include protein markers, microsatellite markers, single nucleotide polymorphism (SNP) markers, etc. SNP markers refer to the polymorphisms of DNA sequences caused by single nucleotide variations on the genome. They have the characteristics of large quantity, high accuracy, high polymorphism, etc. In breeding practice, SNPs can be used to locate certain excellent genes, determine the association between markers and specific qualities in combination with phenotypes, and molecular markers can also be verified in populations and applied in molecular breeding. Summary of the Invention

[0004] The Parkin RBR E3 uBiquitin-protein ligase, abbreviated as the PRKN gene, encodes the Parkin protein, which is an E3 ubiquitin ligase responsible for marking useless proteins in the body, ubiquitinating and degrading them, and helping to maintain the normal functions and healthy state of cells. Mutations in the PRKN gene can lead to the accumulation of abnormal proteins, accelerate the death of neuronal cells, and cause diseases such as Parkinson's disease and Alzheimer's disease. In addition, there are also literature reports that Parkin is involved 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 is no report on the research of this gene in pork quality.

[0005] In this application, through GWAS analysis of the selenium-rich black pork quality resource population, it is found that there are significant signal loci related to pork quality in the PRKN gene region. Therefore, in this application, specific primers are designed and amplified based on the single nucleotide mutation site in the sixth intron of the PRKN gene, and the meat quality differences between pig individuals are distinguished according to the polymorphism detection results of the amplification products. Using this molecular marker to design primers, PCR amplification is performed on the nucleotide sequence containing this SNP site, and sequencing of the amplification products can quickly identify the PRKN genotype, thereby detecting the differences in pork quality and providing guidance for the breeding of pig breeds. In addition, the primers provided in this application can specifically amplify the SNP site sequence contained in the gene intron of PRKN, and the detection method has a low cost. Only through the PCR amplification method can it be detected, and it is not necessary to conduct large-scale population sampling and determination to compare the meat quality differences between different pigs.

[0006] Therefore, the embodiments of this application at least disclose the following technical solutions:

[0007] (1): A molecular marker for pork quality, including a nucleotide sequence formed by a single nucleotide mutation A>C at 6447587bp in the sixth intron of the pig 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 amplified by PCR with the molecular marker primers described in (2), and the genotype of the nucleic acid molecule is associated with the pork quality.

[0010] (4): A kit, including the molecular marker primers described in (2) and other reagents required for PCR amplification.

[0011] (5): A detection method for pork quality, including:

[0012] Obtain genomic DNA of the pig to be tested;

[0013] Perform PCR amplification using the molecular marker primers described in (2);

[0014] Detect the genotype at position 6447587bp on chromosome 1 of the pig according to the nucleotide sequence of the amplification product;

[0015] Determine the pork quality according to the genotype.

[0016] (6): A pig screening method including the detection method described in (5).

[0017] (7): The application 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), and the application is selected from any of the following:

[0018] 1) Detection and analysis of the pork quality, and the pork quality is selected from at least one of muscle color value L1, muscle color value L24, drip loss at 48h, and intramuscular fat;

[0019] 2) Screening and breeding of pigs. Brief Description of the Drawings

[0020] Figure 1 is the overall technical flow chart of this application.

[0021] Figure 2 is the sequencing map of the A>C mutation at position 6447587bp in the sixth intron of the pig PRKN gene. The red frame in the figure is the SNP site. From top to bottom are the AA homozygous type, the AC heterozygous type, and the CC homozygous type.

[0022] Figure 3 is the intuitive diagram of the nucleotide sequence of the sixth intron fragment of the pig PRKN gene (shown in SEQ ID NO:1 or SEQ ID NO:2). The red frame represents the position where the mutation exists. The mutation position is at the 301st base of this sequence, and the underlined sequence represents the primer position. Detailed Description of the Embodiments

[0023] In order to make the purpose, technical solution and advantages of this application clearer, the following further details this application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application. The reagents not described in detail and separately in this application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail and specifically are all conventional experimental methods and can be learned from the existing technology.

[0024] In the embodiments of the present application, by sequencing the mutation sites of partial nucleotide fragments of the PRKN gene, it is found that the genotype at 6447587 bp in the sixth intron of the PRKN gene is associated with pork quality.

[0025] For this reason, the embodiments of the present application disclose molecular markers for pork quality, including the nucleotide sequence formed by a single nucleotide mutation A>C at 6447587 bp in the sixth intron of the porcine PRKN gene. Among them, the reference of the porcine PRKN gene is Gene ID: 733673 in the GeneBank database, the assembly number is SscrofA11.1 (GCF 000003025.6), and the position information is: NC010443.5 (5698508.6731132).

[0026] In some embodiments, the pork quality is selected from at least one of muscle color value L1, muscle color value L24, drip loss at 48 h, and intramuscular fat.

[0027] In some embodiments, at 6447587 bp in the sixth intron of the porcine PRKN gene, the muscle color value L1 of individuals with the AA genotype is significantly higher than that of individuals with the CC genotype, and there is a trend of AA genotype > AC genotype > CC genotype.

[0028] In some embodiments, at 6447587 bp in the sixth intron of the porcine PRKN gene, the muscle color value L24 of individuals with the AA genotype is significantly higher than that of individuals with the CC genotype, and there is a trend of AA genotype > AC genotype > CC genotype.

[0029] In some embodiments, at 6447587 bp in the sixth intron of the porcine PRKN gene, the drip loss at 48 h of individuals with the AA genotype is significantly higher than that of individuals with the CC genotype, and there is a trend of AA genotype > AC genotype > CC genotype.

[0030] In some embodiments, at 6447587 bp in the sixth intron of the porcine PRKN gene, the intramuscular fat content of individuals with the AA genotype is significantly higher than that of individuals with the CC genotype, and there is a trend of AA genotype > AC genotype > CC genotype.

[0031] Based on this, according to the genotype at 6447587 bp in the sixth intron of the porcine PRKN gene, the pork quality of pig individuals can be genetically marked to facilitate the breeding of pig breeds with excellent meat quality.

[0032] Therefore, specific primers were designed referring to the sixth 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). Using porcine genomic DNA as a template, PCR amplification was carried out. The amplified product was subjected to gel recovery and sequencing analysis, and gene fragments shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing were obtained.

[0033] Based on this, the embodiments of the present application also disclose molecular marker primers for pork quality, including the DNA molecule shown in SEQ ID NO:3 and the DNA molecule shown in SEQ ID NO:4. Among them, the "molecular marker primers" are used to amplify nucleotide sequences containing molecular markers linked to pork quality, such as SNPs, to analyze the genotypes of the molecular markers, so as to know the pork quality.

[0034] On the other hand, the embodiments of the present application also disclose a nucleic acid molecule amplified by PCR using the above-mentioned molecular marker primers. 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.

[0035] On the other hand, the embodiments of the present application also disclose a kit, including the above-mentioned molecular marker primers and other reagents required for PCR amplification.

[0036] Based on this, the embodiments of the present application also disclose a detection method for pork quality, including: obtaining genomic DNA of the pig to be tested; performing PCR amplification using the above-mentioned molecular marker primers; detecting the genotype at 6447587bp of the sixth intron of the porcine PRKN gene according to the nucleotide sequence of the amplified product; determining the pork quality according to the genotype. For example, determining the muscle color value L1, muscle color value L24, drip loss at 48h, and intramuscular fat of the pig according to the genotype.

[0037] In some embodiments, the detection method further includes sequencing the amplified product and judging the genotype at 6447587bp of the sixth intron of the porcine PRKN gene according to the sequencing result.

[0038] Based on this, the embodiments of the present application also disclose a screening method for pigs, including the above-mentioned detection method, to determine the muscle color value L1, muscle color value L24, drip loss at 48h, and intramuscular fat of the pigs, and then screen the pig breeds to obtain excellent pig breeds.

[0039] Based on this, the embodiments of the present application also disclose the applications of the above-mentioned molecular markers, molecular marker primers, nucleic acid molecules or kits, and the applications are selected from any of the following:

[0040] 1) Detection and analysis of the pork quality, where the pork quality is selected from at least one of muscle color value L1, muscle color value L24, drip loss after 48 hours, and intramuscular fat;

[0041] 2) Screening and breeding of pigs.

[0042] The present application will be further described in conjunction with specific examples. The following examples are only for explaining the present application and do not constitute a limitation to the present application. The test samples and test procedures used in the following examples include the following content (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, unless otherwise specified, can be obtained from commercial channels).

[0043] I. Extraction of porcine genomic DNA

[0044] The test pig breed in the present application is Xidu Black Pig, and the samples are from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd. The genomic DNA of pigs was extracted using a genomic DNA kit produced by Beijing Bioteke Corporation, and the extraction was carried out according to the instructions of the kit. After detecting the concentration and quality of the extracted DNA, it was stored at -20 °C for later use. The remaining muscle samples were sealed in bags and stored at 4 °C, and were sent to the Quality Supervision and Inspection Test Center for Breeding Swine of the Ministry of Agriculture (Wuhan), Huazhong Agricultural University within 4 hours, and the pork quality was determined in accordance with the Agricultural Industry Standard of the People's Republic of China "Technical Specification for Determination of Pork Quality" (Standard No.: NY / T 821-2019).

[0045] II. Obtaining of porcine PRKN gene fragment and detection of SNP sites of PRKN gene

[0046] 1. Obtaining of the sixth intron fragment of porcine PRKN gene

[0047] (1) PCR amplification

[0048] The following primer pairs were designed according to the porcine PRKN gene sequence (Gene ID: 733673 in the GeneBank database):

[0049] Forward primer PRKN-F: 5’-ACAGTAAGACAGGAGCAAAGTTTTC-3’, SEQ ID NO: 3

[0050] Reverse primer PRKN-R: 5’-GCTCTACAGCAGGATCTCATT-3’, SEQ ID NO: 4.

[0051] Using the above primers, PCR amplification was performed in a pool of genomic DNA from 40 Se-du black pigs. The PCR reaction system was 50 μL, and the concentrations of each component in the system were 100 ng of template DNA, 4 μL of 10× Buffer (containing Mg 2+ ), 0.5 μM each of the above upstream and downstream primers, 2.5 μM dNTPs, and 1 U Taq DNA polymerase.

[0052] The running program of PCR was as follows: preheating at 98 °C for 45 s; denaturation at 98 °C for 10 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, for a total of 34 cycles; extension at 72 °C for 10 min; storage at 4 °C. The PCR products were electrophoresed on a 1.5% agarose gel.

[0053] (2) Purification of PCR products

[0054] The above PCR products were purified using the Gel ExtrAction Kit from Shanghai Sangon Biotech Co., Ltd. (operated according to the instructions of this kit). The specific steps were as follows: First, cut the gel containing the target fragment from the agarose gel, put it into a 1.5 mL centrifuge tube, add 400 μL of solubilization solution, and incubate in a water bath at 50 - 60 °C until the gel completely melts. When heating the melted gel, mix it every 2 min, and cool it to room temperature; place the centrifugal column into the collection tube, transfer the mixed solution to the centrifugal column, and let it stand at room temperature for 2 min; centrifuge at 12000 r / min for 1 min, and at this time the DNA is adsorbed onto the column; pour out the waste liquid in the collection tube, put the centrifugal column into the same collection tube, add 700 μL of elution solution, and centrifuge at 12000 r / min for 1 min; pour out the waste liquid in the collection tube, and centrifuge at 12000 r / min for 1 min; place the centrifugal column into a pre-prepared sterilized 1.5 mL centrifuge tube, add 40 μL of elution solution or double-distilled water (Ph > 7.0), and let it stand at room temperature or 37 °C for 2 - 3 min; centrifuge at 12000 r / min for 1 min, and the liquid in the centrifuge tube is the recovered DNA fragment.

[0055] 2. Obtaining the variant sites of the sixth intron fragment of the porcine PRKN gene

[0056] The recovered DNA fragments obtained above were sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing using an ABI3730XL sequencer, and 1 single-base mutation site was found ( Figure 2) An A>C mutation at position 6,447,587 bp of the PRKN genomic nucleotide sequence (i.e., the full sequence, GeneID: 733673 in the GeneBank database) corresponds to the mutation sites of the PRKN gene fragment in this application as follows: There is an A>C base mutation (i.e., allelic gene mutation) at position 301 bp in SEQ ID NO: 1; there is a C>A base mutation (i.e., allelic gene mutation) at position 301 bp in SEQ ID NO: 2, as Figure 3 shown.

[0057] 3. Molecular marker genotyping

[0058] Using the DNA sample of the individual to be tested as a template, amplify the sixth intron sequence fragment of the porcine PRKN gene according to the method described in step 1 above. Send the obtained PCR purified product directly to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing, and directly read the genotyping results from the sequencing results, as Figure 2 shown.

[0059] III. Genetic diversity detection and correlation analysis with quality

[0060] Using the method provided in the embodiment of this application, perform genetic diversity detection and correlation analysis with quality on 277 Xidu black pigs (from Hubei Huajian Selenium Garden Agriculture and Animal Husbandry Technology Co., Ltd.). Use the general linear model GLM of SPSS statistical software (StAtisticAl PAckAge for the SociAl Sciences, Version 26.0) for statistical analysis. The model used is: Y ijklm = μ + G i + A j + X k + S l + e ijklm , where: Y ijklm represents the phenotypic value of pork quality; μ represents the population mean; G i represents the genotype effect; A j represents the year-quarter effect; X k represents the gender effect; S l represents the paternal effect; e ijklm represents the random residual effect. The results are expressed as least squares mean ± standard error, and P < 0.05 is determined as significantly different.

[0061] The results of the association analysis are shown in Table 1. It was found that the A>C locus at position 6447587 bp on chromosome 1 of the pig genome significantly affected the pork muscle color value L1, muscle color value L24, drip loss at 48 h, and intramuscular fat. The results showed that the muscle color value L1, muscle color value L24, drip loss at 48 h, and intramuscular fat of individuals with the AA genotype were all significantly higher than those of the CC genotype, and there was a trend of AA genotype > AC genotype > CC genotype. During the pig breeding process, the A>C marker at position 6447587 bp on chromosome 1 of the pig genome can be used for early molecular marker-assisted breeding of the reserve pig population, and individuals carrying the AA genotype should be preferentially selected and retained.

[0062] Table 1 Association analysis of the A>C mutation at position 6447587 bp on chromosome 1 of the pig genome and pork quality

[0063]

[0064] Note: The shoulder mark is the significant difference marker of the same quality for different genotypes

[0065] The above is only the preferred specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in this application should be covered by the protection scope of this application.

Claims

1. A method for detecting the pork quality of Selenium City Black Pig, comprising: Obtaining the pig genomic DNA to be tested; Performing PCR amplification using molecular marker primers, wherein the molecular marker primers include a DNA molecule as shown in SEQ ID NO:3 and a DNA molecule as shown in SEQ ID NO:4, and the nucleic acid molecule amplified by the molecular marker primers is as shown in SEQ ID NO:1 and / or 2; Detecting the genotype of a molecular marker associated with the pork quality according to the nucleotide sequence of the nucleic acid molecule, wherein the molecular marker is located at 301 bp of the nucleic acid molecule, and there is an A>C base mutation at 301 bp in SEQ ID NO:1, and there is a C>A base mutation at 301 bp in SEQ ID NO:2; The pork quality is determined according to the genotype, and the molecular marker has AA, CC and AC genotypes. The muscle color value L1, muscle color value L24, drip loss 48h and intramuscular fat content of the Selenium City Black Pig individuals with AA genotype are significantly higher than those with CC genotype.

2. A method for screening Selenium City black pigs, comprising the detection method as described in claim 1, screening the Selenium City black pig individuals according to the genotype of molecular markers associated with pork quality, and retaining the Selenium City black pig individuals carrying the AA genotype.

3. Application of the molecular marker primer or a kit containing the molecular marker primer, wherein the application is selected from any one of the following: 1) Detection and analysis of pork quality of Xidu black pigs, wherein the pork quality is selected from at least one of muscle color value L1, muscle color value L24, drip loss 48h and intramuscular fat; 2) Breed selection and breeding of Sedu Black Pig; in, The molecular marker primers include a DNA molecule as shown in SEQ ID NO:3 and a DNA molecule as shown in SEQ ID NO:

4. The nucleic acid molecule amplified by the molecular marker primers is as shown in SEQ ID NO:1 and / or 2. The molecular marker associated with the pork quality is located at 301 bp of the nucleic acid molecule. There is an A>C base mutation at 301 bp in SEQ ID NO:1, and there is a C>A base mutation at 301 bp in SEQ ID NO:

2. The molecular markers have AA, CC and AC genotypes; when detecting and analyzing the quality of the pork, the muscle color value L1, muscle color value L24, drip loss 48h and intramuscular fat content of the Selenium City Black Pig individuals with the AA genotype are significantly higher than those of the CC genotype individuals; during variety screening and breeding, the Selenium City Black Pig individuals carrying the AA genotype are retained.

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