Molecular marker in sixth intron of porcine PRKN gene, primer, kit, method and application

Abstract

The invention relates to a molecular marker in a sixth intron of a porcine PRKN gene, a primer, a kit, a method and application. The molecular marker comprises a single nucleotide Agt generated at the 6447587bp position of a first chromosome of a pig genome; and C is a nucleotide sequence formed by mutation. A target sequence containing the mononucleotide mutation site is subjected to PCR (Polymerase Chain Reaction) amplification by utilizing a primer, and the PRKN genotype can be quickly identified by sequencing an amplification product, so that the difference of pork quality is detected, and guidance is provided for breeding of pig varieties.

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 sixth intron of the pig PRKN gene. Background Art

[0002] China is a major pork producer and consumer. According to relevant reports and statistics, pork sales in China account for 60 to 70 percent of meat sales each year. As the most important component of the meat market, pork quality, which affects its quality, has become the most important economic quality. Pork quality is mainly composed of three parts: softness, juiciness and flavor. The corresponding meat quality detection indicators include intramuscular fat, meat color, pH value, drip loss and tenderness of pig muscles. In 1990, CAmeron first proposed that pork quality is a complex and comprehensive concept. It is difficult to judge the quality of meat with a single indicator. It requires a comprehensive analysis of multiple indicators, and the meat quality evaluation of individual phenotypes must be achieved through the slaughter of its siblings or descendants. The cost is high and the reliability is poor. Therefore, the use of traditional breeding methods to select and breed pigs with excellent meat quality has been slow. Fortunately, the heritability of pork quality is high. By combining molecular biology and statistical methods, in-depth research on the genetic mechanism of pork quality can be carried out to dig out candidate genes and molecular markers significantly related to pork quality, and then breeding can be carried out through genetic testing methods, which can provide opportunities for meat quality improvement. Currently, there is still insufficient understanding of the key genes that affect meat quality characteristics, and there are relatively few molecular markers that can be used for pork quality selection and breeding. Therefore, finding the key molecular genetic markers that control pork quality and applying them to molecular marker-assisted breeding is of great significance for improving pig meat quality characteristics and increasing economic benefits.

[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 associated with pork quality. Therefore, the present application designs specific primers based on the single nucleotide mutation site on the sixth 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 product 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 only by PCR amplification, and the difference in meat quality between different pigs can be compared without a large amount of group-scale sampling and measurement.

[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>C at 6447587bp in the sixth 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 6447587 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 at least one of muscle color value L1, muscle color value L24, drip loss 48h and intramuscular fat;

[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>C mutation at 6447587bp in the sixth intron of the pig PRKN gene. The red box in the figure is the SNP site, and from top to bottom are AA homozygous, AC heterozygous and CC homozygous.

[0022] Figure 3 It is a visual diagram of the nucleotide sequence of the sixth 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 301st 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 6447587 bp of the sixth 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>C at 6447587bp in the sixth intron of the pig PRKN gene. The pig PRKN gene has a reference 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 at least one of muscle color value L1, muscle color value L24, drip loss 48h and intramuscular fat.

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

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

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

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

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

[0032] Therefore, specific primers were designed with reference 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), PCR amplification was performed using porcine genomic DNA as a template, the amplified products were gel-recovered and sequenced and analyzed, and gene fragments as shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing were obtained.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Based on this, the present application also discloses a method for detecting pork quality, including: obtaining the genomic DNA of the pig to be tested; performing PCR amplification using the molecular marker primers; detecting the genotype at 6447587bp of the sixth 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 muscle color value L1, the muscle color value L24, the drip loss 48h and the intramuscular fat of the pig are determined according to the genotype.

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

[0038] 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 pig muscle color value L1, muscle color value L24, drip loss 48h and intramuscular fat, and then screen pig breeds to obtain excellent pig breeds.

[0039] 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:

[0040] 1) Detection and analysis of pork quality, 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;

[0041] 2) Screening and breeding of pigs.

[0042] 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).

[0043] 1. Extraction of porcine genomic DNA

[0044] 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).

[0045] 2. Obtaining the porcine PRKN gene fragment and detecting the PRKN gene SNP site

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

[0047] (1) PCR amplification

[0048] The following primer pairs were designed based on 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] 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+ )4μL, 0.5μM each of the upstream and downstream primers mentioned above, 2.5μM dNTPs, and 1UTAq DNA polymerase.

[0052] The PCR operation program was as follows: preheating at 98°C for 45s; denaturation at 98°C for 10s, annealing at 56°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.

[0053] (2) PCR product purification

[0054] The above PCR products were purified using the Gel ExtrAction Kit of Shanghai Shenggong Biotechnology Co., Ltd. (operated according to the instructions of the kit), and the specific steps were as follows: first, the gel containing the target fragment was cut from the agarose gel, placed in a 1.5 mL centrifuge tube, 400 μL of sol solution was added, and the gel was completely melted in a 50-60°C 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 12000 r / 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.

[0055] 2. Obtaining the mutation site of the sixth intron fragment of the porcine PRKN gene

[0056] 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 2), an A>C mutation located at the 6447587bp of the PRKN genome nucleotide sequence (i.e., the full sequence) (GeneID: 733673 in the GeneBank database), the mutation sites corresponding to the PRKN gene fragment of the present application are: an A>C base mutation (i.e., allele mutation) at 301bp in SEQ ID NO: 1; a C>A base mutation (i.e., allele mutation) at 301bp in SEQ ID NO: 2, such as Figure 3 shown.

[0057] 3. Molecular marker genotyping

[0058] The DNA sample of the individual to be tested was used as a template, and the sixth 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.

[0059] 3. Genetic diversity detection and analysis of its association with quality

[0060] Using the method provided in the examples of this application, 277 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 Alp Age for the SociAl Sciences, Version 26.0) was used for statistical analysis using the general linear model GLM. 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.

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

[0062] Table 1 Analysis of the association between the A>C mutation at bp 6447587 of chromosome 1 in the pig genome and pork quality

[0063]

[0064] Note: Shoulder marks are significant difference marks of the same quality in different genotypes

[0065] 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. Molecular markers of pork quality, including the nucleotide sequence formed by a single nucleotide A>C mutation at 6447587bp of chromosome 1 of the pig genome.

2. The molecular marker according to claim 1, further comprising a nucleotide sequence in which A>C occurs at 301 bp of the nucleotide sequence of the sixth intron of the PRKN gene.

3. According to the molecular marker of claim 1, the muscle color value L1 of the AA genotype individual is significantly higher than that of the CC genotype individual; Optionally, the muscle color value L24 of the AA genotype individual is significantly higher than that of the CC genotype individual; Alternatively, the drip loss of individuals with AA genotype at 48 h was significantly higher than that of individuals with CC genotype; Alternatively, individuals with AA genotype have significantly higher intramuscular fat content than individuals with CC genotype.

4. 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.

5. A nucleic acid molecule, obtained by PCR amplification using the molecular marker primers as claimed in claim 4, wherein the genotype of the nucleic acid molecule is associated with the pork quality.

6. The nucleic acid molecule according to claim 5, as shown in SEQ ID NO:1 and / or 2.

7. A kit comprising the molecular marker primers as claimed in claim 4 and other reagents required for PCR amplification.

8. Methods for testing pork quality, including: Obtaining the pig genomic DNA to be tested; Carry out PCR amplification by using the molecular marker primers as claimed in claim 4; According to the nucleotide sequence of the amplified product, the 6447587bp position of chromosome 1 of the pig genome was detected; The pork quality is determined according to the genotype.

9. A method for screening pigs, comprising the detection method as claimed in claim 8.

10. Use of the molecular marker according to any one of claims 1 to 3, the molecular marker primer according to claim 4, the nucleic acid molecule according to any one of claims 5 to 6, or the kit according to claim 7, wherein the use is selected from any one of the following: 1) Detection and analysis of pork quality, 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) Screening and breeding of pigs.

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