SNP (Single Nucleotide Polymorphism) marker primer pair related to swine chromosome 8 and swine tolerance African swine fever character and application of SNP marker primer pair

Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker primer pair related to a pig chromosome 8 and a pig tolerance African swine fever character and application of the SNP marker primer pair. The SNP marker is located at an rs344874403 nucleotide site of a pig chromosome 8, and the site of the SNP marker is a molecular marker of the rs344874403 nucleotide site of the pig chromosome 8 in a reference sequence of an international pig genome version 11.1, and has T / 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 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 invention belongs to the technical field of molecular biology and relates to a SNP marker primer pair related to the African swine fever tolerance trait of pigs and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, highly contagious pig disease caused by the African swine fever virus (ASFV), which has become a major challenge facing the global pig industry. The disease not only causes extremely high mortality in pigs, but also seriously affects the production performance of pigs, including growth rate and reproduction rate, causing huge economic losses to the pig industry. African swine fever can be transmitted in various ways, including through direct contact, airborne transmission, and contaminated feed and equipment. Therefore, it is extremely difficult to control the spread of the disease, and there is a lack of effective vaccines and treatments.

[0003] The tolerance trait of African swine fever is also a complex polygenic trait, which is 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 effect in improving the pig herd's resistance to African swine fever and progress has been slow. In recent years, with the development of molecular genetics technology, researchers have begun to explore the relationship between single nucleotide polymorphism (SNP) markers and pig herds' resistance to African swine fever, and through methods such as genomic selection and marker-assisted breeding, in order to accelerate the breeding of resistant pig breeds. 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 a SNP marker primer pair related to the pig's tolerance to African swine fever, which can quickly and accurately evaluate the pig's tolerance to African swine fever through genotyping technology. This technology can not only reduce production costs and improve the pig's tolerance to African swine fever, but also provide important molecular genetic basis for pig selection and improvement. Summary of the invention

[0005] The purpose of the present invention is to provide a breeding molecular marker developed from SNP markers related to pig tolerance to African swine fever, as the breeding of traditional pigs to tolerate African swine fever is time-consuming and labor-intensive, with slow breeding effects and little progress.

[0006] Another object of the present invention is to provide a primer pair and a detection method for detecting the above-mentioned SNP marker.

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

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

[0009] A molecular marker on chromosome 8 of pigs that is related to the pig's tolerance to African swine fever, the molecular marker sequence is shown in SEQ ID NO: 1, which contains a SNP marker site related to the pig's tolerance to African swine fever, which is the rs344874403 nucleotide site on chromosome 8 of the international pig genome version 11.1 reference sequence. The SNP marker site described in SEQ ID NO: 1 is located at position 501 and has a T / G polymorphism.

[0010] A primer pair for detecting SNP markers related to the African swine fever tolerance trait of pigs, 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 associated with the African swine fever tolerance trait of pigs, comprising PCR amplifying a sequence of the rs344874403 nucleotide site of chromosome 8 of the international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the T / G polymorphism of the site.

[0012] As a preferred embodiment of the present invention, the method comprises the following steps:

[0013] (1) Extract total DNA from pig tissue samples;

[0014] (2) Using the extracted porcine genomic DNA as a template, the primer pair described in claim 2 was used to

[0015] It performs PCR amplification;

[0016] (3) The amplified product was sequenced, the sequencing results were analyzed, and the T / G polymorphism at position 501 of SEQ ID NO: 1 was interpreted.

[0017] The molecular markers of the present invention are used in screening pig populations or new strains with stronger tolerance to African swine fever.

[0018] The primer pair of the present invention is used in screening pig groups or new strains with stronger tolerance to African swine fever.

[0019] A method for screening a pig population with a stronger ability to tolerate African swine fever, comprising detecting the genotype of the rs344874403 nucleotide site on chromosome 8 of the international swine genome version 11.1 reference sequence, and selecting GG-type individuals at the rs344874403 nucleotide site as reserve breeding pigs.

[0020] As a preferred embodiment of the present invention, the method for detecting the genotype of the nucleotide site rs344874403 of pig chromosome 8 in the international pig genome version 11.1 reference sequence is selected from PCR or gene sequencing.

[0021] Beneficial Effects

[0022] The present invention develops a SNP marker related to pig chromosome 8 and pig tolerance to African swine fever, and provides a primer pair and method for detecting the marker. The SNP marker provided by the present invention can be applied to marker-assisted selection of pig tolerance to African swine fever, and the genotype of the SNP marker can be identified to screen the pig population or strain with tolerance to African swine fever. The establishment of the population or strain can improve the ability to tolerate African swine fever and generate more social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Gel image of PCR amplification of rs344874403 site on chromosome 8 in purebred and hybrid pigs for commercial production.

[0024] Figure 2 This is an example of the genotyping map of the rs344874403 locus on chromosome 8 in purebred and hybrid pigs for commercial production.

[0025] Note: The genotype of A is TG, the genotype of B is GG, and the genotype of C is TT. DETAILED DESCRIPTION

[0026] The following examples are used to illustrate the present invention, but are not intended to 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 confidential, and the breeds are purebred pigs and hybrid pigs for commercial production

[0030] 2 Statistical methods

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

[0032] 3 Extraction of pig genomic DNA

[0033] Ear tissue samples from 330 purebred and hybrid pigs for commercial production were collected for individual DNA extraction;

[0034] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Company. The extraction steps are as follows:

[0035] ① First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse solution PW respectively, and mix thoroughly.

[0036] ② Collect about 100 mg of ear tissue sample and place it in a 2 mL EP tube. After completely mincing, add 200 μL of buffer GA and shake until completely suspended.

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

[0038] ④ Add 200 μL of buffer GB, mix thoroughly by inversion, and place in a 70°C metal bath for 10 min. The solution should become clear and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0039] ⑤ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0040] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in a collection tube, then centrifuge at 12,000 rpm for 30 seconds, 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 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube

[0042] ⑧ Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0043] ⑨Repeat step ⑧.

[0044] 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 completely dry the residual rinse liquid in the adsorption material.

[0045] Transfer the adsorption column CB3 into a clean centrifuge tube, add 100 μL of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 2 minutes, collect the solution in a centrifuge tube, add the solution obtained by centrifugation to the adsorption column CB3, let it stand at room temperature for 2 minutes, centrifuge it at 12,000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

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

[0047] 4. African swine fever virus antigen detection

[0048] The antigen detection of African swine fever virus uses the African swine fever virus constant temperature detection fluorescence kit produced by China Anpu Future Biotechnology Co., Ltd., which is suitable for the qualitative detection of African swine fever virus.

[0049] 5. African swine fever virus antibody test

[0050] The antibody test for African swine fever virus uses the African swine fever antibody detection kit produced by the French company ID-Vet. 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 porcine genomic DNA as a template. The reaction system included 1 μL of DNA template, 1 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR mix. The amplification procedure was as follows:

[0053]

[0054] The amplified product was subjected to agarose gel electrophoresis, and the product fragment size was about 525 bp. The electrophoresis results were as follows Figure 1 The remaining amplified products were sequenced, and the sequencing results were compared with DNAman software to verify the accuracy of the sequence, and Chromas software was used to type the rs344874403 site.

[0055] 7 Statistical analysis

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

[0057]

[0058] 8 Results

[0059] Table 1 shows the effects of different genotypes of the rs344874403 locus on the tolerance rate of African swine fever (ASF-SR) in purebred pigs and hybrid pigs for commercial production. The results showed that the genotype of the rs344874403 locus was significantly associated with the disease resistance performance (P<0.001). Among them, the disease resistance of the TT type was significantly lower than that of the GG type individuals (P<0.001). In addition, there were significant differences in disease resistance between the TT type and the TG type, as well as between the TG type and the GG type (P<0.001). Therefore, in the selective breeding of disease resistance traits, the G allele type of the rs344874403 locus, especially the GG type, is conducive to improving the disease resistance and economic benefits of individuals.

[0060] Table 1 Association analysis between rs344874403 locus on chromosome 8 and African swine fever tolerance rate

[0061]

[0062] Note: Numbers with different letters in the same column indicate significant differences (P<0.001).

Claims

1. A molecular marker associated with African swine fever tolerance in pigs, characterized in that The molecular marker sequence is shown in SEQ ID NO: 1, and the 501st position is a SNP marker site related to the pig's tolerance to African swine fever. The site is the rs344874403 nucleotide site on chromosome 8 of the international pig genome version 11.1 reference sequence. There is a T / G polymorphism, and the disease resistance of GG type pig individuals is significantly stronger than that of TT type individuals.

2. A primer pair for detecting the SNP marker site associated with the African swine fever tolerance trait of pigs as 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 site associated with the African swine fever tolerance trait of pigs as described in claim 1, characterized in that: It comprises a PCR amplification of a section of the nucleotide site rs344874403 of the pig chromosome 8 of the international pig genome version 11.1 reference sequence, sequencing the amplified product, and interpreting the T / G polymorphism of the site.

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

5. The method according to claim 4, characterized in that The following steps are involved: (1) Extract total DNA from pig tissue samples; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primer pair described in claim 2; (3) The amplified product was sequenced, the sequencing results were analyzed, and the T / G polymorphism at position 501 of SEQ ID NO: 1 was interpreted.

6. The application of SNP markers related to the African swine fever tolerance trait in screening African swine fever tolerant pig populations, wherein the site of the SNP marker is a molecular marker at the rs344874403 nucleotide site of chromosome 8 of the international swine genome version 11.1 reference sequence, and has T / G polymorphism.

7. Use of the molecular marker described in claim 1 in screening a population resistant to African swine fever.

8. Use of the primer pair described in claim 2 in screening a population resistant to African swine fever.

9. A method for screening a pig population tolerant to African swine fever, characterized in that This includes detecting the genotype of the rs344874403 nucleotide site on chromosome 8 of the international pig genome version 11.1 reference sequence, and selecting GG type individuals at the rs344874403 nucleotide site as reserve breeding pigs.

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