SNP (Single Nucleotide Polymorphism) marker primer pair related to porcine tolerance mycoplasmal pneumonia character on porcine chromosome 9 and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to porcine tolerance mycoplasma pneumonia traits on a porcine chromosome 9 and application of the SNP marker primer pair. The locus of the SNP marker is a molecular marker of a nucleotide locus rs81339640 of a pig chromosome 9 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] The present invention belongs to the technical field of molecular biology, and relates to a SNP marker primer pair related to the trait of pigs tolerant to Mycoplasma hyopneumoniae and its application. Background Art

[0002] Worldwide, Mycoplasma hyopneumoniae has caused huge economic losses to the pig industry. Mycoplasma hyopneumoniae seriously affects production indicators such as feed intake and daily weight gain of pigs. The lungs are easily invaded by environmental pathogens, leading to lung diseases, usually accompanied by lung lesions, and even causing the death of pigs in severe cases. The trait of tolerance to Mycoplasma hyopneumoniae is a complex trait with low heritability and many influencing factors. Therefore, it is difficult to directly select and breed pigs for the trait of tolerance to Mycoplasma hyopneumoniae through traditional methods. Traditional methods are inefficient and the progress of genetic improvement is slow. In recent years, with the development of molecular genetics and genomics, researchers have begun to use the association between single nucleotide polymorphism (SNP) markers and pig traits to solve this problem. SNP markers are a common type of genomic genetic marker, which has the advantages of high polymorphism and wide distribution in the genome, so it has become an important tool for studying pig genetic traits.

[0003] Based on this background, this patent proposes a SNP marker primer pair related to the trait of tolerance to Mycoplasma hyopneumoniae, which is a complex trait with low heritability and many influencing factors. The trait can quickly and accurately evaluate the trait of pigs' tolerance to Mycoplasma hyopneumoniae through genotyping technology. This technology can not only reduce the production cost and improve the tolerance of pigs to Mycoplasma hyopneumoniae, but also provide important molecular genetics basis for the selection and improvement of pigs. Summary of the Invention

[0004] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to pigs' tolerance to Mycoplasma hyopneumoniae in view of the slow effect and small progress of traditional pig breeding for tolerance to Mycoplasma hyopneumoniae.

[0005] Another purpose of the present invention is to provide a primer pair and a detection method for detecting the above SNP markers. Another purpose of the present invention is to provide the uses of the above SNP markers, molecular markers, and primers.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] Molecular markers on porcine chromosome 9 related to the trait of porcine tolerance to Mycoplasma hyopneumoniae. The sequence of the molecular marker is as shown in SEQ ID NO: 1, which contains an SNP marker locus related to the trait of porcine tolerance to Mycoplasma hyopneumoniae. This locus is the nucleotide locus rs81339640 on porcine chromosome 9 in the reference sequence of the international porcine genome version 11.1 (this locus was identified by the team through genome-wide association analysis). In SEQ ID NO: 1, the SNP marker locus is located at the 320th position, with A / G polymorphism.

[0008] A primer pair for detecting the SNP marker on porcine chromosome 9 related to the trait of porcine tolerance to Mycoplasma hyopneumoniae. The upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO: 3.

[0009] The application of the molecular marker and the primer pair of the present invention in detecting the trait of porcine tolerance to Mycoplasma hyopneumoniae and pig breeding.

[0010] A method for detecting the SNP marker on porcine chromosome 9 related to the trait of porcine tolerance to Mycoplasma hyopneumoniae, which includes PCR amplifying a segment of the sequence of the nucleotide locus rs81339640 on porcine chromosome 9 in the reference sequence of the international porcine genome version 11.1, sequencing the amplification product, and judging the A / G polymorphism of this locus.

[0011] As a preference of the present invention, the pig is Danish Large White pigs and their crossbred pig breeds.

[0012] As a preference of the present invention, the genomic DNA of Danish Large White pigs or their crossbred pig breeds is subjected to PCR amplification using the primer pair of the present invention.

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

[0014] (1) Taking a porcine ear tissue sample to extract DNA;

[0015] (2) Using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair of the present invention;

[0016] (3) Sequencing the amplification product, analyzing the sequencing results, and judging the A / G polymorphism at the 320th position of SEQ ID NO: 1.

[0017] The application of the molecular marker of the present invention in screening pig populations or new strains with stronger ability to tolerate Mycoplasma hyopneumoniae.

[0018] The application of the primer pair of the present invention in screening pig populations or new strains with stronger ability to tolerate Mycoplasma hyopneumoniae.

[0019] A method for screening a pig population with stronger ability to tolerate Mycoplasma pneumoniae, including detecting the genotype of the nucleotide site rs81339640 on chromosome 9 of pigs in the reference sequence of the international pig genome version 11.1, and preferentially selecting individuals with the AA genotype at the nucleotide site rs81339640 as reserve breeding pigs for breeding.

[0020] As a preference of the present invention, the pig breed used is Danish Large White pigs or their crossbred pig breeds.

[0021] As a preference of the present invention, the method for detecting the genotype of the nucleotide site rs81339640 on chromosome 9 in the reference sequence of the international pig genome version 11.1 is selected from PCR or gene sequencing.

[0022] Beneficial effects

[0023] The present invention has developed SNP markers on chromosome 9 of pigs related to the tolerance of pigs to Mycoplasma pneumoniae, and provided primer pairs and methods for detecting these markers. The SNP markers provided by the present invention can be applied to marker-assisted selection of the trait of pigs' tolerance to Mycoplasma pneumoniae, and a pig population or strain with the trait of tolerance to Mycoplasma pneumoniae can be screened by identifying the genotype of this SNP marker. The establishment of this population or strain can improve the ability of pigs to tolerate Mycoplasma pneumoniae and generate more social and economic benefits. Description of the drawings

[0024] Figure 1 It is the PCR amplification gel diagram of the rs81339640 site on chromosome 9 of Danish Large White pigs and their crossbred pig breeds.

[0025] Figure 2 It is an example of the genotyping diagram of the rs81339640 site on chromosome 9 of Danish Large White pigs and their crossbred pig breeds.

[0026] Note: The genotype of A is AA type, the genotype of B is AG type, and the genotype of C is GG type. Specific implementation examples

[0027] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0028] Example 1

[0029] 1 Data source

[0030] The data used are from the slaughter data records of Guangxi Haihe Breeding Swine Co., Ltd. After slaughter, the proportion of the lung lesion area was statistically analyzed. The 28-point method, namely the Madec method, is to evaluate according to the proportion of the lung area with specific lesions of Mycoplasma hyopneumoniae in 7 lung lobes (only the ventral surface of the accessory lobe is observed, and both the ventral and dorsal surfaces of the other 6 lung lobes need to be observed) in the surface area of the lung lobe. The maximum score for each lung lobe is 4 points. Among them, no damage is 0, the damaged area accounts for 1%-25% of the lung lobe area is 1 point, 26%-50% is 2 points, 51%-75% is 3 points, and more than 75% is 4 points.

[0031] 2 Extraction of porcine genomic DNA

[0032] Collect 1 ear tissue sample from 379 Danish Large White pigs and their hybrid pig breeds for individual DNA extraction;

[0033] Refer to the instruction manual of the tissue DNA extraction kit of Tiangen Biotech Co., Ltd. The extraction steps are as follows:

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

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

[0036] ③ Add 20 μL of proteinase K solution, mix well and place it in a 56 °C water bath for digestion overnight until the tissue sample is dissolved. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

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

[0038] ⑤ Add 200 μL of absolute ethanol, mix well by shaking thoroughly for 15 sec. At this time, flocculent precipitates may appear. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

[0039] ⑥ Add the solution and flocculent precipitates obtained in the previous step into an adsorption column CB3. Place the adsorption column in a collection tube, then centrifuge at 12,000 rpm for 30 sec, pour out 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 sec, pour out the waste liquid, and place the adsorption column CB3 in the collection tube

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

[0042] ⑨ Repeat the operation steps in ⑧.

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

[0044] Transfer the adsorption column CB3 into a clean centrifuge tube, suspend and add 100 μL of elution buffer TE dropwise to the middle part of the adsorption membrane, place at room temperature for 2 - 5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into the centrifuge tube, add the centrifuged solution back into the adsorption column CB3, place at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min to collect the solution into the centrifuge tube.

[0045] Use a Nanodrop - 2000 spectrophotometer to detect the quality and concentration of DNA. Dilute the DNA concentration to 50 ng / μL and store it at - 20 °C for later use.

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

[0047] Use the genomic DNA of Danish Large White pigs and their crossbred pig breeds as templates for PCR amplification. The reaction system includes 1 μL of DNA template, 1 μL of each primer shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR mix; the amplification program is as follows:

[0048]

[0049] The amplification products are subjected to agarose gel electrophoresis. The size of the product fragments is approximately 615 bp, and the electrophoresis results are as Figure 1 shown. Sequence the remaining amplification products, verify the accuracy of the sequence using DNAman software, and genotype the rs81339640 locus using Chromas software.

[0050] 4 Statistical analysis

[0051] Use the editor of SAS 9.4 software to run the code for the association analysis of genotypes and phenotypes. The code is as follows:

[0052]

[0053] 5 Results

[0054] Table 1 shows the results of the influence of different genotypes at the rs81339640 locus on the ability of Danish Large White pigs and their hybrid pig breeds to tolerate Mycoplasma pneumonia. The results show that the genotype at the rs81339640 locus is significantly associated with the total score of the proportion of lung lobe lesion area (P<0.05). Among them, the total score of the proportion of lung lobe lesion area of individuals with the AA genotype is significantly lower than that of individuals with the GG genotype (P<0.001), indicating that the ability of individuals with the AA genotype to tolerate Mycoplasma pneumonia is significantly greater than that of individuals with the GG genotype (P<0.001); the total score of the proportion of lung lobe lesion area is significant between AA and AG, GG individuals (P<0.05), indicating that there are significant differences in the ability of AA and AG, GG individuals to tolerate Mycoplasma pneumonia (P<0.05). Therefore, continuous selection of the AA genotype at the rs81339640 locus in Danish Large White pigs and their hybrid pig breeds is beneficial to increasing the ability of Danish Large White pigs and their hybrid pig breeds to tolerate Mycoplasma, thereby improving the economic benefits and disease tolerance of Danish Large White pigs and their hybrid pig breeds.

[0055] Table 1 Association analysis of the rs81339640 locus on chromosome 9 of pigs with the area of post-slaughter lung lesions

[0056]

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

Claims

1. A method for developing molecular markers related to the trait of pigs tolerant to Mycoplasma hyopneumoniae, characterized in that, Using the nucleotide sequence of the nucleotide site rs81339640 on chromosome 9 of the international pig genome version 11.1 as the basic sequence, a primer pair was designed, and PCR amplification was performed using porcine genomic DNA as a template, converting the nucleotide site rs81339640 on chromosome 9 of the international pig genome version 11.1 into a molecular marker. The genotype of the rs81339640 nucleotide site is significantly associated with the phenotype of the ability to tolerate Mycoplasma. Among them, the ability of AA-type individuals to tolerate Mycoplasma pneumonia is significantly greater than that of GG-type individuals.

2. The method according to claim 1, characterized in that The primer pair sequences are as follows: forward primer: SEQ ID NO: 2, reverse primer: SEQ ID NO:

3.

3. The molecular marker obtained by the method according to claim 1 or 2.

4. The molecular marker according to claim 3, wherein The molecular marker sequence is as shown in SEQ ID NO:

1. The nucleotide site rs81339640 on chromosome 9 of the international pig genome version 11.1 is located at position 320 in SEQ ID NO: 1, with A / G polymorphism. The ability of AA-type individuals to tolerate Mycoplasma pneumonia is significantly greater than that of GG-type individuals.

5. A primer pair for detecting SNP markers related to the porcine trait of tolerance to Mycoplasma hyopneumoniae, characterized in that, The forward primer is: SEQ ID NO: 2, the reverse primer is: SEQ ID NO: 3; the SNP marker related to the trait of porcine tolerance to Mycoplasma pneumonia is the molecular marker of the nucleotide site rs81339640 on chromosome 9 of the international pig genome version 11.1, and has A / G polymorphism. The SNP marker genotype is significantly correlated with the trait of porcine tolerance to Mycoplasma pneumonia.

6. A method for detecting the SNP marker related to the trait of pigs resistant to Mycoplasma hyopneumoniae described in claim 5, characterized in that, It includes amplifying a sequence of the nucleotide site rs81339640 on chromosome 9 of the international pig genome version 11.1 by PCR, sequencing the amplification product, and determining the A / G polymorphism of this site.

7. The method according to claim 6, characterized in that, It includes the following steps: (1) Taking a porcine tissue sample to extract total DNA; (2) Using the extracted porcine genomic DNA as a template, performing PCR amplification using the primer pair described in claim 5; (3) Sequencing the amplification product, analyzing the sequencing results, and determining the A / G polymorphism at position 320 of SEQ ID NO:

1.

8. The application of the molecular marker according to claim 3 or 4 and the primer pair according to claim 5 in screening a porcine population tolerant to Mycoplasma pneumonia.

9. A method for a pig population resistant to Mycoplasma pneumoniae, characterized in that, It includes detecting the genotype of the nucleotide site rs81339640 on chromosome 9 of the international pig genome version 11.1 in pigs, and preferentially selecting AA-type individuals at the rs81339640 nucleotide site as breeding pigs.

10. The method according to claim 9, characterized in that, The pigs are Danish Large White pigs and their crossbred pig breeds.

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