SNP (Single Nucleotide Polymorphism) marker primer pair related to swine tolerance African swine fever character on chromosome 6 and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to a swine tolerant African swine fever character on a chromosome 6 and application of the SNP marker primer pair. The SNP marker is located at an rs337613199 nucleotide site of a pig chromosome 6, and the site of the SNP marker is a molecular marker of the rs337613199 nucleotide site of the pig chromosome 6 in a reference sequence of an international pig genome version 11.1, and has G / A 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 the African swine fever traits tolerant to pigs, and the genotype of the SNP marker is identified to screen pig populations or strains tolerant to the African swine fever traits. Establishment of the group or the strain can improve the capacity of resisting the swine African swine fever, and more social and economic benefits can be generated.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to an SNP marker primer pair related to the trait of pigs' tolerance to African swine fever and its application. Background Art

[0002] African swine fever (ASF) is an acute and highly contagious swine disease caused by African swine fever virus (ASFV) and has become a major challenge faced by the global pig industry. This disease not only causes extremely high mortality in pigs but also seriously affects the production performance of pigs, including growth rate, reproductive rate, etc., bringing huge economic losses to the pig industry. The transmission routes of African swine fever are diverse, including through direct contact, airborne transmission, and contaminated feed, equipment, etc. Therefore, it is extremely difficult to control the spread of this disease, and there is a lack of effective vaccines and treatment methods.

[0003] The tolerance trait of African swine fever is also a complex polygenic trait, affected by multiple factors such as the environment, immune system, and genetic background. Due to the low heritability of this trait and the complex influencing factors, traditional breeding methods have limited effects and slow progress in improving the African swine fever resistance of pig populations. In recent years, with the development of molecular genetics technology, researchers have begun to explore the relationship between single nucleotide polymorphism (SNP) markers and the resistance of pig populations to African swine fever, and through methods such as genomic selection and marker-assisted breeding, in order to accelerate the cultivation of resistant pig breeds. The research in this field provides new ideas and technical support for improving the prevention and control of African swine fever.

[0004] Based on this background, this patent proposes an SNP marker primer pair related to the trait of pigs' tolerance to African swine fever, and quickly and accurately evaluates the trait of pigs' tolerance to African swine fever through genotyping technology. This technology can not only reduce production costs and improve the African swine fever tolerance of pig populations, but also provide important molecular genetics basis for the breeding and improvement of pigs. Summary of the Invention

[0005] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to pigs' tolerance to African swine fever, aiming at the time-consuming and laborious traditional breeding of pigs' tolerance to African swine fever, slow breeding effect, and small progress.

[0006] Another purpose of the present invention is to provide a primer pair and a detection method for detecting the above SNP markers.

[0007] Another purpose of the present invention is to provide the uses of the above SNP markers, molecular markers, and primers.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] A molecular marker related to the trait of pigs being tolerant to African swine fever, the sequence of the molecular marker is as shown in SEQ ID NO: 1, the 453rd position thereof is an SNP marker locus related to the trait of pigs being tolerant to African swine fever, this locus is the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, there is G / A polymorphism, and the disease tolerance ability of pigs with the AA genotype is significantly lower than that of pigs with the GG genotype.

[0010] A primer pair for detecting an SNP marker related to the trait of pigs being tolerant to African swine fever, the upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO: 3

[0011] A method for detecting the SNP marker related to the trait of pigs being tolerant to African swine fever, which includes PCR amplifying a segment of the sequence at the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, sequencing the amplification product, and judging the G / A polymorphism at this locus.

[0012] As a further preference of the present invention, the method includes the following steps:

[0013] (1) Take a pig ear tissue sample to extract DNA;

[0014] (2) Using the extracted pig genomic DNA as a template, perform PCR amplification using the primer pair of the present invention;

[0015] (3) Sequence the amplification product, analyze the sequencing result, and judge the G / A polymorphism at the 453rd position of SEQ ID NO: 1.

[0016] Application of the molecular marker described in the present invention in screening pig populations or new strains with stronger ability to tolerate African swine fever.

[0017] Application of the primer pair described in the present invention in screening pig populations or new strains with stronger ability to tolerate African swine fever.

[0018] A method for screening a pig population with a stronger ability to tolerate African swine fever, which includes detecting the genotype of the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, and preferentially selecting individuals with the GG genotype at the nucleotide locus rs337613199 as replacement breeding pigs for breeding.

[0019] As a preference of the present invention, the pig breeds used are purebred pigs and hybrid pigs for commercial production.

[0020] As a preference of the present invention, the method for detecting the genotype of the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1 is selected from PCR or gene sequencing.

[0021] Beneficial effects

[0022] The present invention has developed SNP markers on porcine chromosome 6 related to porcine tolerance to African swine fever, and provided primer pairs and methods for detecting these markers. By identifying the genotypes of these SNP markers, pig strains with stronger tolerance to African swine fever can be screened. The establishment of such strains can improve the ability of pigs to tolerate African swine fever and generate more social and economic benefits. Description of the drawings

[0023] Figure 1 PCR amplification gel diagram of the rs337613199 locus on chromosome 6 of purebred pigs and crossbred pigs for commercial production.

[0024] Figure 2 Example of the genotyping map of the rs337613199 locus on chromosome 6 of purebred pigs and crossbred pigs for commercial production.

[0025] Note: The genotype of A is GG, the genotype of B is GA, and the genotype of C is AA. Specific implementation methods

[0026] 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, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0027] Example 1

[0028] 1 Data source

[0029] The pig farm is kept confidential, and the breeds are purebred pigs and crossbred pigs for commercial production

[0030] 2 Statistical method

[0031] After the pig farm was infected with ASF, sera were collected for antigen and antibody detection of ASF virus, and divided into disease-tolerant group and disease-susceptible group. Disease-tolerant group: pigs that did not die after being infected with ASF for a period of time (antigen negative or positive, antibody positive) or pigs that were not infected with ASF (antigen negative, antibody negative); Disease-susceptible group: pigs that died acutely or within 7 days after being infected with ASF (antigen positive, antibody positive) were defined as disease-susceptible pigs.

[0032] 3 Extraction of porcine genomic DNA

[0033] One ear tissue sample of 474 purebred pigs and crossbred pigs for commercial production was collected for individual DNA extraction;

[0034] Referring to the instruction manual of the tissue DNA extraction kit of Tiangen Biotech Company, the extraction steps are as follows:

[0035] ① First, add 68 mL and 200 mL of absolute ethanol to buffer GD and wash buffer PW respectively, and mix well.

[0036] ② Collect about 100 mg of ear tissue samples and place them in a 2 mL EP tube. After completely cutting them into pieces, add 200 μL of buffer GA and shake until thoroughly suspended.

[0037] ③ Add 20 μL of proteinase K solution, mix well, and digest overnight in a 56 °C water bath until the tissue samples are dissolved. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap.

[0038] ④ Add 200 μL of buffer GB, invert and mix well, place 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.

[0039] ⑤ Add 200 μL of absolute ethanol, shake well 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.

[0040] 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.

[0041] 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

[0042] 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.

[0043] ⑨ Repeat operation step ⑧.

[0044] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, pour out the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual wash buffer in the adsorption material.

[0045] Transfer the adsorption column CB3 into a clean centrifuge tube. Suspendedly add 100 μL of elution buffer TE 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, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0046] The quality and concentration of DNA were detected using a Nanodrop-2000 spectrophotometer. The DNA concentration was diluted to 50 ng / μL and stored at -20 °C for later use.

[0047] 4 African swine fever virus antigen detection

[0048] The antigen detection of African swine fever virus used a constant-temperature fluorescence detection kit for African swine fever virus produced by China Anpu Future Biotechnology Co., Ltd. The kit is applicable to the qualitative detection of African swine fever virus.

[0049] 5 African swine fever virus antibody detection

[0050] The antibody detection of African swine fever virus used an African swine fever antibody detection kit produced by ID-Vet, France. The kit uses the indirect ELISA method to detect African swine fever virus antibodies.

[0051] 6 PCR amplification and sequencing of target fragments

[0052] PCR amplification was performed using genomic DNA of purebred pigs and hybrid pigs for commercial production 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:

[0053]

[0054] The amplification products were subjected to agarose gel electrophoresis. The size of the product fragments was approximately 818 bp, and the electrophoresis results were as Figure 1 shown. The remaining amplification products were sequenced. The sequencing results were verified for sequence accuracy using DNAman software, and the rs337613199 locus (i.e., the 453 bp locus in SEQ ID NO.1) was genotyped using Chromas software.

[0055] 7 Statistical analysis

[0056] The editor of SAS 9.4 software was used to run the code for the association analysis of genotype and phenotype. The code was as follows:

[0057]

[0058] 8 Results

[0059] Table 1 shows the results of the effect of different genotypes at the rs337613199 locus on the African swine fever survival rate (ASF-SR) of purebred pigs and crossbred pigs for commercial production. The overall analysis results show that the genotype at this locus is significantly associated with the disease tolerance ability (overall P < 0.05). Among them, the disease tolerance abilities of individuals with the GG genotype and the GA genotype are significantly higher than those of the AA genotype (P < 0.05). Therefore, in the selective breeding for disease tolerance traits, the G allele genotype at the rs337613199 locus, especially the GG genotype, is beneficial to improving the disease tolerance ability and economic benefits of individuals.

[0060] Table 1 Association analysis of the rs337613199 locus on chromosome 6 of pigs with the African swine fever survival rate

[0061]

[0062] Note: The percentage values in the table represent the mortality rate.

[0063] Different letters with superscripts in the same row of numbers indicate significant differences (P < 0.05).

Claims

1. A molecular marker related to the trait of pigs' tolerance to African swine fever, characterized in that, The molecular marker sequence described above is shown in SEQ ID NO: 1, and the 453rd position thereof is an SNP marker locus related to the African swine fever tolerance trait in pigs. This locus is the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, with G / A polymorphism. The African swine fever tolerance ability of GG-type pig individuals is significantly higher than that of AA-type pig individuals.

2. A primer pair for detecting the SNP marker related to the African swine fever tolerance trait of pigs described in claim 1, characterized in that, The upstream primer is: SEQ ID NO: 2, and the downstream primer is: SEQ ID NO:

3.

3. A method for detecting the SNP marker locus related to the African swine fever tolerance trait of pigs described in claim 1, characterized in that, It includes a sequence for PCR amplifying the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, sequencing the amplification product, and judging the A / G polymorphism of this locus.

4. The method according to claim 3, characterized in that Use the primer pair described in claim 2 to perform PCR amplification on pig genomic DNA.

5. The method according to claim 4, characterized in that It includes the following steps: (1) Take pig tissue samples to extract total DNA; (2) Using the extracted pig genomic DNA as a template, perform PCR amplification with the primer pair described in claim 2; (3) Sequence the amplification product, analyze the sequencing results, and judge the G / A polymorphism at the 453rd position of SEQ ID NO:

1.

6. The application of the SNP marker locus related to the African swine fever tolerance trait in pigs described in claim 1 in screening African swine fever-tolerant pig populations. The locus of the SNP marker is a molecular marker of the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, and has G / A polymorphism.

7. The application of the molecular marker described in claim 1 in screening African swine fever-tolerant populations.

8. The application of the primer pair described in claim 2 in screening African swine fever-tolerant pig populations.

9. A method for screening African swine fever-tolerant pig populations, characterized in that It includes detecting the genotype of the nucleotide locus rs337613199 on chromosome 6 of pigs in the reference sequence of the international pig genome version 11.1, and preferentially selecting GG-type individuals at the rs337613199 nucleotide locus as reserve breeding pigs for breeding.

Images