Molecular marker in third intron of pig prkn gene, primer, kit, method and application

Abstract

The application relates to a molecular marker, a primer, a kit, a method and application in a third intron of a pig PRKN gene. The molecular marker comprises a nucleotide sequence formed by a single nucleotide G>A mutation at 5964857 bp of a first chromosome of a pig genome. The primer is used for PCR amplification of a target sequence containing the single nucleotide mutation site, and sequencing of the amplification product can quickly identify the PRKN genotype, thereby detecting the difference of pork quality and providing guidance for pig breeding.

Description

TECHNICAL FIELD

[0001] The application relates to the field of pig breeding technology, in particular to a pig PRKN gene third intron sequence variation as a molecular marker for pork quality and application. BACKGROUND

[0002] Meat quality is an important economic characteristic in the pig breeding industry, and the level of pork quality directly affects the production and economic benefits of the breeding farm. The heritability of meat quality is between 0.25 and 0.42, and most of them belong to the medium heritability characteristic. However, it is difficult to measure pork quality in vivo, and the cost of slaughter determination is high, and the excellent individual cannot be selected and reserved due to slaughter, so it is difficult to use conventional breeding techniques for genetic improvement, and assisted selection by molecular means is an effective way. At present, the understanding of key genes affecting meat quality characteristics is still insufficient, and there are few molecular markers that can be applied to pork quality breeding, therefore, finding key molecular markers that control pork quality and applying them to molecular marker assisted breeding are of great significance for improving pork quality 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 polymorphism of DNA sequence caused by single nucleotide variation in the genome, which has the characteristics of large number, high accuracy, high polymorphism, etc. In breeding practice, SNP can be used to locate certain excellent genes, and the association between markers and specific quality can be determined by combining phenotypes, and molecular markers can be verified in populations and applied in molecular breeding. SUMMARY

[0004] Parkin RBR E3 ubiquitin protein ligase, abbreviated as PRKN gene, encodes Parkin protein, which is an E3 ubiquitin ligase responsible for marking useless proteins in the body, ubiquitination and degradation, which helps to maintain the normal function and healthy state of cells. PRKN gene mutation can cause accumulation of abnormal proteins, accelerate neuronal cell death, and cause diseases such as Parkinson's disease and Alzheimer's disease. In addition, there are documents reporting that Parkin is involved in mediating specific autophagy of organelles such as mitochondria, eliminating damaged organelles and maintaining the balance of the body to affect inflammatory diseases, and promote cell survival and proliferation. However, there is no report on the research of this gene in pork quality.

[0005] The application finds that there is a signal site significantly related to pork quality in the PRKN gene region through GWAS analysis of selenium-doped black pig pork quality resource groups. Therefore, the application designs specific primers and amplifies according to the single nucleotide mutation site on the third intron of the PRKN gene, and distinguishes the pork quality difference between pig individuals according to the polymorphism detection result of the amplification product. The primer is designed by using the molecular marker, the nucleotide sequence containing the SNP site of the primer is amplified by PCR, and the PRKN genotype can be quickly identified by sequencing the amplification product, so as to detect the difference of pork quality and provide guidance for the breeding of pig breeds. In addition, the primer provided by the application can specifically amplify the SNP site sequence contained in the intron of the PRKN gene, and the detection method has low cost. Only the PCR amplification method is needed to detect, and a large number of population sampling and determination are not needed to compare the difference of pork quality between different pigs.

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

[0007] (1) A molecular marker of pork quality, comprising a nucleotide sequence formed by a single nucleotide mutation G>A at 5964857bp of the third intron of a pig PRKN gene.

[0008] (2) A molecular marker primer of pork quality, comprising 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 formed by PCR amplification of the molecular marker primer in (2), and the genotype of the nucleic acid molecule is related to the pork quality.

[0010] (4) A kit, comprising the molecular marker primer in (2) and other reagents required for PCR amplification.

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

[0012] obtaining genomic DNA of a pig to be detected;

[0013] amplifying by the molecular marker primer in (2);

[0014] detecting the genotype of the pig at 5964857bp of the first chromosome according to the nucleotide sequence of the amplification product;

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

[0016] (6) A pig screening method comprising the detection method in (5).

[0017] (7) use of (1) the molecular marker, (2) the molecular marker primer, (3) the nucleic acid molecule or (4) the kit, the use is selected from any one of the following:

[0018] 1) detection and analysis of the pork quality, the pork quality is selected from at least one of marbling score and intramuscular fat content;

[0019] 2) screening and breeding of pigs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a general technical flowchart of the present application.

[0021] Figure 2 is a sequencing map of G>A mutation at 5964857bp of the third intron of the pig PRKN gene, and the red box in the figure is the SNP site, from top to bottom are GG homozygote, GA heterozygote and AA homozygote.

[0022] Figure 3 is a direct view of the nucleotide sequence of the third intron fragment of the pig PRKN gene (shown in SEQ ID NO: 1 or SEQ ID NO: 2), the red box represents the presence of the mutation, and the mutation position is located at the 251st base of the sequence, and the underlined sequence represents the primer position. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. The reagents not specifically described in the present application are conventional reagents, and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods, and can be known from the prior art.

[0024] The present application discloses that the genotype at 5964857bp of the third intron of the PRKN gene is associated with pork quality by sequencing the mutation site of the partial nucleotide fragment of the PRKN gene.

[0025] Therefore, the present application discloses a molecular marker of pork quality, which includes the nucleotide sequence formed by single nucleotide mutation G>A at 5964857bp of the third intron of the pig PRKN gene. Among them, the pig PRKN gene is referenced to GeneBank database Gene ID:733673, assembly number Sscrofa11.1 (GCF 000003025.6), position information: NC010443.5 (5698508.6731132).

[0026] In some embodiments, the pork quality is selected from at least one of marbling score and intramuscular fat content.

[0027] In some embodiments, the AA genotype of the pig at the position 5964857bp of the third intron of the pig PRKN gene is associated with a higher marbling score and intramuscular fat content than other genotypes.

[0028] Therefore, the pork quality of a pig can be molecularly marked based on the genotype of the pig at the position 5964857bp of the third intron of the pig PRKN gene, so as to facilitate the breeding of pigs with excellent meat quality.

[0029] Therefore, a specific primer is designed according to the third intron of the pig PRKN gene in the GeneBank database (the sequence of the primer is shown in SEQ ID NO: 3 and SEQ ID NO: 4), and the pig genomic DNA is used as a template for PCR amplification. The amplified product is recovered by gel and sequenced to obtain the gene fragment shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0030] Therefore, the pork quality of a pig can be molecularly marked based on the genotype of the pig at the position 5964857bp of the third intron of the pig PRKN gene, so as to facilitate the breeding of pigs with excellent meat quality.

[0031] In another aspect, the embodiments of the present application also disclose a nucleic acid molecule, which is obtained by PCR amplification of the molecular marker primer, 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] In another aspect, the embodiments of the present application also disclose a kit comprising the molecular marker primer and other reagents required for PCR amplification.

[0033] Therefore, the embodiments of the present application also disclose a detection method of pork quality, comprising: obtaining the genomic DNA of a pig to be detected; performing PCR amplification by using the molecular marker primer; detecting the genotype of the pig at the position 5964857bp of the third 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 intramuscular fat content of the pig are determined according to the genotype.

[0034] In some embodiments, the detection method further comprises sequencing the amplified product, and determining the genotype of the pig at the position 5964857bp of the third intron of the pig PRKN gene according to the sequencing result.

[0035] Based on the above, the application further discloses a pig screening method comprising the detection method, to determine the marbling score, intramuscular fat content and / or water content of the pig, and then screen the pig breed to obtain an excellent pig breed.

[0036] Based on the above, the application further discloses the application of the molecular marker, the molecular marker primer, the nucleic acid molecule or the kit, which is selected from any one of the following:

[0037] 1) detection and analysis of the pork quality, the pork quality being selected from at least one of the marbling score and the intramuscular fat content;

[0038] 2) screening and breeding of pigs.

[0039] The application will be further described in combination with specific examples. The following examples are only for the purpose of explaining the application, but do not constitute a limitation on the application. The test samples and test processes used in the following examples include the following contents (if the specific conditions of the experiments are not specified in the examples, the conventional conditions or the conditions recommended by the reagent company are generally used; the reagents, consumables, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified).

[0040] I. Extraction of pig genomic DNA

[0041] The test pig breed of the application is Xuedu black pig, and the sample is derived from Hubei Huajian Se Garden Animal Husbandry Technology Co., Ltd. The pig genomic DNA is extracted using the genomic DNA kit produced by Beijing Baitaike Biotechnology Co., Ltd., and the operation is performed according to the instruction manual of the kit. After the concentration and quality of the extracted DNA are detected, it is stored at -20°C for standby. The remaining muscle sample is stored in a bag at 4°C and sent to the Agricultural Ministry Pig Quality Supervision and Inspection Test Center (Wuhan) of Huazhong Agricultural University within 4 hours for determination of pork quality according to the People's Republic of China Agricultural Industry Standard "Pork Quality Determination Technical Specification" (standard number: NY / T 821-2019).

[0042] II. Obtaining of pig PRKN gene fragment and detection of PRKN gene SNP site

[0043] 1. Obtaining of pig PRKN gene third intron fragment

[0044] (1) PCR amplification

[0045] According to the sequence of pig PRKN gene (Gene ID: 733673 in GeneBank database), the following primer pairs are designed:

[0046] Forward primer PRKN-F: 5'-TAAATGTTAAATTTATTACCGTTTT-3', SEQ ID NO: 3

[0047] Reverse primer PRKN-R: 5'-AGTGAAGTATGGCAGAAAGG-3', SEQ ID NO: 4.

[0048] PCR amplification was performed using the above primers in 40 selenium all black pig mixed genomic DNA pools, and the PCR reaction system was 50 μL. The concentrations of various components in the system were 100 ng of template DNA, 10x buffer (containing Mg 2+ ) 4 μL, 0.5 μM of the above upstream and downstream primers, 2.5 μM dNTPs, 1 U Taq DNA polymerase.

[0049] The running program of PCR was as follows: 98 ℃ preheating for 45 s; 98 ℃ denaturation for 10 s, 56 ℃ annealing for 30 s, 72 ℃ extension for 30 s, a total of 34 cycles; 72 ℃ extension for 10 min; 4 ℃ preservation. The PCR product was electrophoresed by 1.5% agarose gel.

[0050] (2) PCR product purification

[0051] The above PCR product was purified by Gel Extraction Kit kit of Shanghai Shengong Bioengineering Co., Ltd. (operation 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 and placed in a 1.5 mL centrifuge tube, 400 μL of gel dissolving solution was added, and the gel was completely melted in a 50-60 ℃ water bath. When heating the gel, mix every 2 min, and cool to room temperature; place the centrifugal column in the collection tube, and move the mixed solution to the centrifugal column, and place it at room temperature for 2 min; centrifuge at 12000 r / min for 1 min, at which time the DNA is adsorbed to the column; discard the waste liquid in the collection tube, and place the centrifugal column in the same collection tube, add 700 μL of elution solution, and centrifuge at 12000 r / min for 1 min; discard the waste liquid in the collection tube, and centrifuge at 12000 r / min for 1 min; place the centrifugal column in a pre-prepared sterile 1.5 mL centrifuge tube, add 40 μL of elution solution or double distilled water (pH > 7.0), and place it at room temperature or 37 ℃ for 2-3 min; centrifuge at 12000 r / min for 1 min, and the liquid in the centrifugal tube is the recovered DNA fragment.

[0052] 2, Obtaining of the variation site of the third intron fragment of pig PRKN gene

[0053] The above recovered DNA fragment was sent to Wuhan Aokeding Sheng Biological Technology Co., Ltd. for sequencing by ABI3730XL sequencer, and one single base mutation site was found Figure 2), G > A mutation at 5964857 bp of PRKN genomic nucleotide sequence (Gene ID: 733673 in GeneBank database), corresponding to the mutation site of the PRKN gene fragment of the present application is: there is a G > A base mutation (i.e. allele mutation) at 251 bp in SEQ ID NO: 1; there is a G > A base mutation (i.e. allele mutation) at 251 bp in SEQ ID NO: 2, as shown in Figure 3 .

[0054] 3. Molecular marker genotyping

[0055] The DNA sample of the individual to be detected is used as a template, and the third intron sequence fragment of the pig PRKN gene is amplified according to the method described in step 1 above. The purified PCR product obtained is directly sent to Wuhan Aoke Dingsheng Biological Technology Co., Ltd. for sequencing, and the genotyping result is directly read from the sequencing result, as shown in Figure 2 .

[0056] III. Genetic diversity detection and correlation analysis with quality

[0057] Using the method provided in the present application, the genetic diversity detection and correlation analysis with quality of 274 Xuedou black pigs (from Hubei Huajian Selenium Garden Animal Husbandry Technology Co., Ltd.) were carried out. Statistical analysis was carried out by general linear model GLM of SPSS statistical software (Statistical Package for the Social Sciences, Version 26.0). The model used is: Y ijklm = μ + G i + A j + X k + S l + e ijklm , wherein: Y ijklm represents the phenotype value of pork quality; μ represents the population mean; G i represents the genotype effect; A j represents the annual and seasonal effect; X k represents the gender effect; S l represents the sire effect; e ijklm represents the random residual effect. The results are expressed as least squares mean ± standard error, and P < 0.05 is considered to be significantly different.

[0058] The association analysis results are shown in Table 1. It is found that the G>A site at 5964857bp of the first chromosome of the pig genome significantly affects the marbling score, intramuscular fat content and / or water quality of pork (P<0.05). In Table 1, the intramuscular fat content and marbling score of the AA genotype individual at 5964857bp of the first chromosome of the pig genome are significantly higher than those of the GG genotype, and there is a trend of AA genotype>GA genotype>GG genotype. In breeding, to select a population with better meat quality, the GG genotype individual can be eliminated first, that is, the G>A marker at 5964857bp of the first chromosome of the pig genome can be used for early molecular marker-assisted pig breeding process in the breed selection of the reserve pig population.

[0059] Table 1 Association analysis of G>A mutation at 5964857bp of the first chromosome of the pig genome with pork quality

[0060]

[0061] Note: The same quality of the same genotype of the different shoulder marks is significantly different

[0062] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be covered within the protection scope of the present application.

Claims

1.A method for detecting the pork quality of Se'du black pig, comprising: obtaining the genomic DNA of a Se'du black pig to be tested; performing PCR amplification by using a molecular marker primer, wherein the DNA sequence of the molecular marker primer is shown in SEQ ID NO: 3 and SEQ ID NO: 4; detecting the genotype at 5964857 bp of chromosome 1 of the pig genome according to the nucleotide sequence of the amplification product, wherein the assembly number of the pig genome is GCF 000003025.6 and the version is Sscrofa11.1; and determining the pork quality according to the genotype, wherein the G>A mutation exists at 5964857 bp of chromosome 1 of the pig genome, and the AA, AG and GG genotypes exist, and the intramuscular fat content and marbling score of the Se'du black pig individual with the AA genotype are higher than those of the Se'du black pig individual with the GG genotype. 2.A method for screening Se'du black pig, comprising determining the marbling score and / or intramuscular fat content of the pig according to the detection method of claim 1, so as to screen the Se'du black pig individual with excellent pork quality. 3.An application of a molecular marker associated with pork quality, a molecular marker primer or a kit for detecting the molecular marker, wherein the application is selected from any one of the following: 1) detecting and analyzing the pork quality of Se'du black pig, wherein the pork quality is at least one selected from the group consisting of marbling score and intramuscular fat content; 2) screening and breeding Se'du black pig with excellent pork quality, wherein the pork quality is at least one selected from the group consisting of marbling score and intramuscular fat content; wherein the molecular marker is the nucleotide sequence formed by the single nucleotide G>A mutation at 5964857 bp of chromosome 1 of the pig genome, wherein the assembly number of the pig genome is GCF 000003025.6 and the version is Sscrofa11.1, and the molecular marker has the AA, AG and GG genotypes, and the intramuscular fat content and marbling score of the Se'du black pig individual with the AA genotype are higher than those of the Se'du black pig individual with the GG genotype; wherein the DNA sequence of the molecular marker primer is shown in SEQ ID NO: 3 and SEQ ID NO: 4; and wherein the kit comprises the molecular marker primer and other reagents required for PCR amplification. ​ ​ ​ ​ ​ ​ wherein ​ ​ ​

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