Application of SNP (Single Nucleotide Polymorphism) labeled primer pair in porcine GFRA1 (Growth Factor Receptor 1) gene coding region in resisting mycoplasma pneumonia traits

Abstract

The invention relates to an application of an SNP (Single Nucleotide Polymorphism) labeled primer pair in a porcine GFRA1 (Growth Factor Receptor 1) gene coding region in resisting mycoplasma pneumonia traits. The locus of the SNP marker is a molecular marker of the rs329759240 nucleotide locus of a pig GFRA1 gene coding region in a reference sequence of an international pig genome version 11.1, and the SNP marker has A / G polymorphism. According to a primer pair for detecting the SNP marker, an upstream primer is SEQ ID NO: 2, and a downstream primer is SEQ ID NO: 3. The SNP marker provided by the invention can be applied to marker-assisted selection of swine mycoplasmal pneumonia resistant traits, and swine populations or strains resistant to swine mycoplasmal pneumonia traits are screened by identifying the genotype of the SNP marker. Establishment of the group or strain can improve the capability of tolerating swine mycoplasmal pneumonia and produce more social and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to a pair of SNP marker primers associated with the porcine mycoplasma-resistant pneumonia trait in the coding region of the GFRA1 gene and their application. Background Technology

[0002] Globally, porcine mycoplasma pneumoniae has caused enormous economic losses to the pig industry, severely impacting feed intake and daily weight gain, among other production indicators. The lungs are susceptible to invasion by environmental pathogens, leading to lung diseases, often accompanied by lesions, and in severe cases, even death. Mycoplasma pneumonia tolerance is a complex trait with low heritability and is influenced by many factors. Therefore, it is difficult to directly select for mycoplasma pneumonia tolerance in pig herds using traditional methods. Traditional methods are inefficient, and genetic improvement is slow. In recent years, with the development of molecular genetics and genomics, researchers have begun to utilize the association between single nucleotide polymorphism (SNP) markers and pig traits to address this problem. SNP markers are common genomic genetic markers with the advantages of high polymorphism and widespread distribution throughout the genome, thus becoming an important tool for studying pig genetic traits.

[0003] Against this backdrop, this patent proposes a method for rapidly and accurately assessing pigs' tolerance to mycoplasma pneumoniae (MPP) using SNP marker primer pairs, a trait characterized by its complexity, low heritability, and numerous influencing factors. This method utilizes genotyping technology. This technology can not only reduce production costs and improve mycoplasma pneumoniae tolerance in pig herds but also provide crucial molecular genetic evidence for pig breeding and improvement. Summary of the Invention

[0004] The purpose of this invention is to address the slow progress and limited effectiveness of traditional breeding methods for swine mycoplasma-resistant pneumonia by providing a breeding molecular marker developed from SNP markers associated with swine mycoplasma-resistant pneumonia.

[0005] Another object of the present invention is to provide primer pairs and detection methods for detecting the above-mentioned SNP markers. Another object of the present invention is to provide uses for the above-mentioned SNP markers, molecular markers, and primers.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] Molecular markers associated with the porcine GFRA1 gene coding region and the porcine mycoplasma-resistant pneumonia trait were identified in SEQ ID NO: 1. These markers contain a single SNP associated with the porcine mycoplasma-resistant pneumonia trait. This SNP is located at nucleotide rs329759240 within the porcine GFRA1 gene coding region of the international porcine genome version 11.1 reference sequence (identified by the team through genome-wide association analysis). The SNP in SEQ ID NO: 1 is located at position 498 and exhibits A / G polymorphism.

[0008] A primer pair for detecting SNP markers in the coding region of the porcine GFRA1 gene that are associated with the trait of porcine mycoplasma-resistant pneumonia, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3.

[0009] The molecular markers and primer pairs described in this invention are used in detecting the trait of mycoplasma-resistant pneumonia in pigs and in pig breeding.

[0010] A method for detecting SNP markers associated with porcine mycoplasma-resistant pneumonia in the coding region of the porcine GFRA1 gene includes PCR amplification of a sequence at nucleotide site rs329759240 in the coding region of the porcine GFRA1 gene from the international porcine genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the A / G polymorphism at this site.

[0011] As a preferred embodiment of the present invention, the pig is a Large White breed of American pig.

[0012] As a preferred embodiment of the present invention, the primer pair described herein is used to perform PCR amplification of the genomic DNA of American Large White pigs.

[0013] As a further preferred embodiment of the present invention, the method includes the following steps:

[0014] (1) Extract DNA from pig ear tissue samples;

[0015] (2) Using the extracted porcine genomic DNA as a template, perform PCR amplification using the primer pair described in this invention;

[0016] (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the A / G polymorphism at position 498 of SEQ ID NO: 1.

[0017] The application of the molecular markers described in this invention in screening for pig populations or new strains with stronger resistance to mycoplasma pneumonia.

[0018] The application of the primer pairs described in this invention in screening pig populations or new strains with stronger resistance to mycoplasma pneumonia.

[0019] A method for screening pig populations with stronger resistance to mycoplasma pneumoniae includes detecting the genotype at the rs329759240 nucleotide site in the coding region of the pig GFRA1 gene in the international pig genome version 11.1 reference sequence, and selecting individuals with the GG genotype at the rs329759240 nucleotide site as priority for breeding.

[0020] As a preferred embodiment of the present invention, the pig breed used is the American Large White pig.

[0021] As a preferred embodiment of the present invention, the method for detecting the genotype of the rs329759240 nucleotide site in the coding region of the porcine GFRA1 gene in the International Swine Genome Version 11.1 reference sequence is selected from PCR or gene sequencing.

[0022] Beneficial effects

[0023] This invention develops a SNP marker within the coding region of the porcine GFRA1 gene that is associated with porcine mycoplasma pneumoniae tolerance, and provides primer pairs and methods for detecting this marker. The SNP marker provided by this invention can be applied to marker-assisted selection for the trait of porcine mycoplasma pneumoniae tolerance, allowing for the screening of porcine populations or breeds with mycoplasma pneumoniae tolerance by identifying the genotype of this SNP marker. Establishing such populations or breeds can improve the ability to tolerate porcine mycoplasma pneumoniae and generate greater social and economic benefits. Attached Figure Description

[0024] Figure 1 shows a gel image of PCR amplification at the rs329759240 site in the coding region of the GFRA1 gene in American Large White pigs.

[0025] Figure 2 shows an example of the genotyping diagram of the rs329759240 locus in the coding region of the GFRA1 gene in American Large White pigs.

[0026] Note: A has the genotype AA, B has the genotype GA, and C has the genotype GG. Detailed Implementation Plan

[0027] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0028] Example 1

[0029] 1. Data Source

[0030] The data used came from the slaughter data records of Guangxi Haihe Breeding Pig Co., Ltd. After slaughter, the proportion of lung lesion area was used to assign scores to the seven lung lobes of the pig as phenotypes: the proportion of lesion area in each lung lobe was 0%, 1-25%, 26-50%, 51-75%, and 76-100% respectively, and 0, 1, 2, 3, and 4 points were assigned respectively, with a total score of 28 points for the seven lung lobes.

[0031] 2. Extraction of porcine genomic DNA

[0032] One ear tissue sample was collected from 327 American Large White pigs for individual DNA extraction;

[0033] Referring to the instructions for the Tissue DNA Extraction Kit from Tiangen Biotech Co., Ltd., the extraction steps are as follows:

[0034] ① First, add 68 mL of anhydrous ethanol to buffer GD and 200 mL of wash buffer PW, respectively, and mix thoroughly.

[0035] ② Collect approximately 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely cutting it into small pieces, add 200 μL of buffer GA and shake until completely suspended.

[0036] ③ Add 20 μL of proteinase K solution, mix well, and place in a 56 ℃ water bath to digest overnight until the tissue sample dissolves. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0037] ④ Add 200 μL of buffer GB, mix thoroughly by inverting, place in a 70 ℃ metal bath for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0038] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0039] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in the collection tube, then centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0040] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0041] ⑧ Add 600 μL of washing buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste liquid, and place the adsorption column CB3 into the collection tube.

[0042] ⑨ Repeat step ⑧.

[0043] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0044] ⑪ Transfer the adsorption column CB3 into a clean centrifuge tube. Add 100 μL of elution buffer TE to the middle of the adsorption membrane. Incubate at room temperature for 2-5 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube. Add the centrifuged solution back to the adsorption column CB3. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min. Collect the solution in the centrifuge tube.

[0045] The quality and concentration of DNA were determined using a Nanodrop-2000 spectrophotometer. All DNA concentrations were diluted to 50 ng / μL and stored at -20 ℃ for later use.

[0046] 3. PCR amplification and sequencing of the target fragment

[0047] PCR amplification was performed using genomic DNA from American Large White pigs as a template. The reaction system included 1 μL of DNA template, 1 μL each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR mix. The amplification program was as follows:

[0048]

[0049] The amplification products were subjected to agarose gel electrophoresis, and the product fragment size was approximately 840 bp. The electrophoresis results are shown in Figure 1. The remaining amplification products were sequenced, and the sequencing results were compared and verified for sequence accuracy using DNAman software. The rs329759240 site was genotyped using Chromas software.

[0050] 4. Statistical Analysis

[0051] The code for genotype-phenotype association analysis was run using the editor in SAS 9.4 software. The code is as follows: proc mixed data=work.import;

[0052] class SNP;

[0053] model Mhp = SNP / solution noint covb;

[0054] lsmeans SNP / tdiff pdiff adjust=bon;

[0055] estimate 'Additive effect' SNP 0.5 0 -0.5;

[0056] run;

[0057] 5 Results

[0058] Table 1 shows the effects of different genotypes at the rs329759240 locus on the tolerance of American Large White pigs to mycoplasma pneumonia. The results showed a significant association between the rs329759240 locus genotype and the total score for the proportion of lobar lesions (P < 0.05). Specifically, the total score for the proportion of lobar lesions in GG-type individuals was significantly lower than that in AA-type individuals (P < 0.05), indicating that GG-type individuals had significantly greater tolerance to mycoplasma pneumonia than AA-type individuals (P < 0.05). The total score for the proportion of lobar lesions was also significantly different between AA and GG-type individuals, and between GA and GG-type individuals (P < 0.05), indicating that GG-type individuals had stronger tolerance to mycoplasma pneumonia. Therefore, breeding for the GG genotype at the rs329759240 locus in successive generations of American Large White pigs is beneficial for increasing the mycoplasma tolerance of the American Large White population, thereby improving the economic benefits and disease resistance of the American Large White pig breed.

[0059] Table 1. Association analysis between the rs329759240 locus in the coding region of the porcine GFRA1 gene and the area of ​​lung lesions after slaughter.

[0060]

[0061] Note: Different letters in the same row of numbers indicate significant differences (P < 0.05).

Claims

1. The application of primer pairs for SNP markers associated with porcine mycoplasma pneumoniae tolerance traits in screening porcine populations resistant to mycoplasma pneumoniae, characterized in that, The SNP marker is located at nucleotide rs329759240 in the coding region of the porcine GFRA1 gene in the international swine genome version 11.1 reference sequence. This site exhibits A / G polymorphism. The genotype at the rs329759240 nucleotide site is significantly associated with the mycoplasma tolerance phenotype. Specifically, individuals with the GG genotype have significantly greater tolerance to mycoplasma pneumonia than individuals with the AA genotype.

2. The method according to claim 1, characterized in that, The primer pair sequence is: upstream primer: SEQ ID NO: 2, downstream primer: SEQ ID NO:

3.

3. The method according to claim 1 or 2, characterized in that, The pigs mentioned are American Large White pigs.

4. A method for detecting SNP markers associated with mycoplasma-resistant pneumonia in American Large White pigs as described in claim 1, characterized in that, A sequence containing a PCR amplification of a segment of the rs329759240 nucleotide site in the coding region of the porcine GFRA1 gene, based on the international porcine genome version 11.1 reference sequence, was sequenced. The A / G polymorphism at this site was determined. Individuals with the GG genotype showed significantly greater tolerance to mycoplasma pneumoniae than individuals with the AA genotype.

5. The method according to claim 4, characterized in that, Includes the following steps: (1) Collect pig tissue samples to extract total DNA; (2) Using the extracted porcine genomic DNA as a template, perform PCR amplification using the primer pair described in claim 1 or 2; (3) Sequencing of the amplified product, analysis of the sequencing results, and interpretation of the A / G polymorphism at position 498 of SEQ ID NO:

1.

6. A method for raising a pig population resistant to mycoplasma pneumonia, characterized in that, This includes detecting the genotype of the rs329759240 nucleotide site in the coding region of the GFRA1 gene of American Large White pigs (reference sequence 11.1), and selecting GG-type individuals with the rs329759240 nucleotide site as breeding pigs.

7. The method according to claim 6, characterized in that, The method for detecting the genotype of the rs329759240 nucleotide site in the coding region of the porcine GFRA1 gene in the 11.1 version reference sequence of the American Large White pig genome was selected from PCR amplification and gene sequencing.

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