Primer pair of SNP marker related to mycoplasmal pneumonia resistance trait on pig chromosome 9 and application thereof

Abstract

This invention relates to a primer pair for SNP markers on porcine chromosome 9 associated with the trait of mycoplasma pneumoniae tolerance and their application. The SNP marker is a molecular marker at nucleotide 81339640 on porcine chromosome 9 in the International Swine Genome Version 11.1 reference sequence, exhibiting A / G polymorphism. A primer pair for detecting the SNP marker is provided, with the upstream primer being SEQ ID NO: 2 and the downstream primer being SEQ ID NO: 3. The SNP markers provided by this invention can be applied to marker-assisted selection for the trait of mycoplasma pneumoniae tolerance in porcines, allowing for the screening of porcine populations or strains tolerant to mycoplasma pneumoniae by identifying the genotype of the SNP marker. Establishing such populations or strains can improve tolerance to mycoplasma pneumoniae and generate greater social and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to an SNP marker primer pair associated with the trait of porcine mycoplasma-resistant pneumonia and its 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 trait of porcine mycoplasma-resistant pneumonia on chromosome 9 of pigs are shown in SEQ ID NO: 1. These markers contain a single SNP site associated with the trait of porcine mycoplasma-resistant pneumonia. This site is the rs81339640 nucleotide site on chromosome 9 of the international pig genome version 11.1 reference sequence (identified by the team through genome-wide association analysis). The SNP site described in SEQ ID NO: 1 is located at position 320 and exhibits A / G polymorphism.

[0008] A primer pair for detecting SNP markers on pig chromosome 9 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 on porcine chromosome 9 includes PCR amplification of a sequence at nucleotide site rs81339640 on porcine chromosome 9 of the international porcine genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the A / G polymorphism at that site.

[0011] As a preferred embodiment of the present invention, the pig is a Danish Large White pig or its hybrid breed.

[0012] As a preferred embodiment of the present invention, the primer pairs described herein are used to perform PCR amplification of the genomic DNA of Danish Large White pigs or their hybrids.

[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, PCR amplification was performed 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 320 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 of the rs81339640 nucleotide site on chromosome 9 of the pig international pig genome version 11.1 reference sequence, and selecting individuals with the AA type at the rs81339640 nucleotide site as priority for breeding replacement pigs.

[0020] As a preferred embodiment of the present invention, the pig breed used is the Danish Large White pig or its hybrid breed.

[0021] As a preferred embodiment of the present invention, the method for detecting the genotype of the rs81339640 nucleotide site on pig chromosome 9 in the International Pig Genome Version 11.1 reference sequence is selected from PCR or gene sequencing.

[0022] Beneficial effects

[0023] This invention develops a SNP marker on porcine chromosome 9 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 this trait by identifying the genotype of the SNP marker. Establishing such populations or breeds can improve tolerance to porcine mycoplasma pneumoniae and generate greater social and economic benefits. Attached Figure Description

[0024] Figure 1 This is a gel image showing the PCR amplification of the rs81339640 site on chromosome 9 of Danish Large White pigs and their hybrids.

[0025] Figure 2 Example of a typing diagram of chromosome 9 rs81339640 in Danish Large White pigs and their hybrids.

[0026] Note: A has the genotype AA, B has the genotype AG, 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 records of Guangxi Haihe Breeding Pig Co., Ltd., and the percentage of lung lesion area was statistically analyzed after slaughter. The 28-point method, also known as the Madc method, is based on the degree of lesion in the seven lung lobes according to the proportion of the lung lobe area specifically damaged by porcine mycoplasma pneumonia (for accessory lobes, only the ventral surface is observed, while the ventral and dorsal surfaces of the other six lung lobes are observed) to the surface area of ​​that lung lobe. Each lung lobe is scored up to 4 points. No damage is 0 points, damage area accounting for 1%-25% of the lung lobe area is 1 point, 26%-50% is 2 points, 51%-75% is 3 points, and greater than 75% is 4 points.

[0031] 2. Extraction of pig genomic DNA

[0032] One ear tissue sample was collected from 379 Danish Large White pigs and their hybrids 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 buffer GD and 200 mL of anhydrous ethanol to the wash buffer PW, 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°C 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 seconds, 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 minutes, 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] 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°C for later use.

[0046] 3. Target fragment PCR amplification and sequencing

[0047] PCR amplification was performed using genomic DNA from Danish Large White pigs and their hybrids as templates. 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 product was subjected to agarose gel electrophoresis. The product fragment size was approximately 615 bp. The electrophoresis results are as follows: Figure 1 As shown. The remaining amplification products were sequenced, and the sequencing results were compared and verified for accuracy using DNAman software. The rs81339640 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:

[0052]

[0053] 5 Results

[0054] Table 1 shows the effects of different genotypes at the rs81339640 locus on the tolerance of Danish Large White pigs and their hybrids to mycoplasma pneumonia. The results showed that the rs81339640 genotype was significantly associated with the total score of the proportion of lobar lesions (P<0.05). Specifically, the total score of the proportion of lobar lesions in AA genotype individuals was significantly lower than that in GG genotype individuals (P<0.001), indicating that AA genotype individuals had significantly greater tolerance to mycoplasma pneumonia than GG genotype individuals (P<0.001). The total score of the proportion of lobar lesions was also significantly different between AA and AG / GG genotype individuals (P<0.05), indicating significant differences in tolerance to mycoplasma pneumonia between AA and AG / GG genotype individuals (P<0.05). Therefore, the AA type at the rs81339640 locus in Danish Large White pigs and their hybrids is beneficial to increasing the mycoplasma tolerance of Danish Large White pigs and their hybrids, thereby improving the economic benefits and disease resistance of Danish Large White pigs and their hybrids.

[0055] Table 1. Association analysis between the rs81339640 locus on porcine chromosome 9 and the area of ​​lung lesions after slaughter.

[0056]

[0057] Note: Different letters in the same row of numbers indicate significant differences (P<0.05).

Claims

1. Use of a molecular marker associated with the trait of tolerance to Mycoplasma pneumoniae in pigs in the selection of a Duroc line of Large White pigs tolerant to Mycoplasma pneumoniae, characterized in that, The molecular marker is based on the nucleotide sequence containing the rs81339640 nucleotide site on chromosome 9 of the International Pig Genome 11.1 reference sequence. Primer pairs were designed, and PCR amplification was performed using pig genomic DNA as a template to convert the rs81339640 nucleotide site on chromosome 9 of the International Pig Genome 11.1 reference sequence into a molecular marker. The rs81339640 nucleotide site exhibits A / G polymorphism, and its genotype is significantly associated with the phenotype of mycoplasma pneumoniae tolerance. Among them, individuals with the AA genotype have significantly greater tolerance to mycoplasma pneumoniae than individuals with the GG genotype.

2. Use of a primer pair for detecting a SNP marker associated with the trait of tolerance to Mycoplasma pneumoniae in pigs in the screening of a Duroc line of Large White pigs tolerant to Mycoplasma pneumoniae, characterized in that, The SNP marker is located at nucleotide rs81339640 on chromosome 9 of the pig genome in the international pig genome version 11.1 reference sequence. It exhibits A / G polymorphism, and its genotype is significantly associated with the phenotype of mycoplasma pneumonia tolerance. Among them, individuals with the AA genotype have significantly greater tolerance to mycoplasma pneumonia than individuals with the GG genotype. The upstream primer of the primer pair is SEQ ID NO: 2, and the downstream primer is SEQ ID NO:

3.

3. A method for screening a Large White pig population resistant to Mycoplasma pneumoniae, characterized in that, This includes detecting the genotype of the rs81339640 nucleotide site on pig chromosome 9 in the international pig genome version 11.1 reference sequence, and selecting individuals with the AA type at the rs81339640 nucleotide site as breeding pigs.

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