Molecular marker for Tibetan sheep immune character identification and application thereof

Abstract

The invention relates to the technical field of molecular markers, in particular to a molecular marker for Tibetan sheep immune character identification and application of the molecular marker. According to the molecular marker for identifying the immune traits of the Tibetan sheep, the molecular marker is located at the 61720490th basic group on the 12th chromosome of the international sheep genome Oarv4.0 version, and the mutant basic group is G or A. According to the present invention, by detecting the basic group of the 61720490th nucleotide site on the 12th chromosome of the Tibetan sheep, the immunoglobulin IgA content and the immunoglobulin IgG content of the individual Tibetan sheep can be determined, and the new SNP molecular marker resource is provided for the non-diagnostic Tibetan sheep immune trait marker-assisted selection.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to a molecular marker for identifying immune traits of Tibetan sheep and an application thereof. Background Art

[0002] With the continuous development of the sheep industry, there are more and more types of sheep diseases, the complexity is increasing, and the control is becoming more and more difficult. Sheep diseases are becoming more and more harmful to sheep production. At the same time, the use of a large number of drugs will make the animal body resistant to drugs, posing a hidden danger to the safety of livestock products.

[0003] In the long run, starting from the genetic basis of disease resistance, screening resistance genes, carrying out disease-resistant breeding at the molecular level, genetically improving the resistance of sheep to pathogens, and enhancing immunity are important ways to fundamentally solve this problem. Tibetan sheep have lived in the Qinghai-Tibet Plateau for a long time, and the environment is harsh, so they need higher disease resistance to adapt to this environment. Therefore, improving the disease resistance of Tibetan sheep can make sheep more adaptable to harsh natural environments such as high altitude, low pressure, strong ultraviolet rays, hypoxia and cold season nutritional stress.

[0004] Immune indicators can reflect the disease resistance of animals. The disease resistance of animals mainly depends on the health and ability of their immune system. The main function of the immune system is to identify and eliminate pathogens that invade the body, such as bacteria and viruses. When pathogens invade, the immune system will respond quickly and initiate a series of immune response processes to fight and eliminate pathogens.

[0005] Immunoglobulin (Ig) refers to a globulin with antibody (Ab) activity or chemical structure, which is similar to antibody molecules. Immunoglobulin is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds. Immunoglobulins are divided into five categories, namely immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) and immunoglobulin E (IgE). IgG refers to immunoglobulin G, which accounts for about 75% of serum antibodies, can improve the body's immunity, and has antiviral and anti-inflammatory effects. IgA refers to immunoglobulin A, which ranks second in the body's serum, second only to IgG, and has an anti-infection effect. If the content of IgG and IgA in the body decreases, it is easy to cause low immune function. If the content of IgG and IgA in the body increases, the body's disease resistance will be enhanced. The enhancement of disease resistance can reduce the use of drugs, reduce production costs, and increase breeding benefits. Therefore, it is of great significance to screen SNP molecular markers associated with immune traits of Tibetan sheep. Moreover, immune indicators can be used as an important reference to evaluate the disease resistance and health status of animals. By monitoring the changes in immune indicators, immune problems of animals can be discovered in time, and corresponding measures can be taken to improve their disease resistance, prevent and treat diseases.

[0006] Therefore, how to use genetics and genetic engineering technology to find the variation sites at the gene level in Tibetan sheep breeding, discover the relationship between genes and traits through association analysis between them and traits, and conduct early selection, thereby improving the efficiency and accuracy of selection, has broad application value. Summary of the invention

[0007] The purpose of the present invention is to provide a molecular marker for identifying immune traits of Tibetan sheep and application thereof.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a molecular marker for identifying immune traits of Tibetan sheep. The molecular marker is located at the 61720490th base on chromosome 12 of the international sheep genome Oar_v4.0 version, and the mutant base is G or A.

[0010] Preferably, the genotype of the Tibetan sheep with the mutant base G is GG or GA;

[0011] The genotype of Tibetan sheep with the mutant base A is AA;

[0012] The immunoglobulins IgA and IgG of Tibetan sheep individuals with the AA genotype are significantly higher than those of individuals with the GG and GA genotypes;

[0013] There is no significant difference in immunoglobulin IgA and IgG between the GG and GA genotype Tibetan sheep individuals.

[0014] The present invention also provides the use of the molecular marker in the marker-assisted selection of Tibetan sheep immune traits for non-diagnostic purposes.

[0015] The present invention also provides the application of the molecular marker in Tibetan sheep assisted breeding.

[0016] The present invention also provides a primer pair for amplifying the molecular marker, and the primer pair is shown as SEQ ID NO. 2 and 3.

[0017] The present invention also provides the use of the primer pair in preparing a Tibetan sheep immune trait marker-assisted selection product for non-diagnostic purposes.

[0018] The present invention also provides application of the primer pair in preparing products for auxiliary breeding of Tibetan sheep.

[0019] The present invention also provides a method for marker-assisted selection of Tibetan sheep immune traits for non-diagnostic purposes, comprising the following steps:

[0020] (1) Extracting Tibetan sheep genomic DNA;

[0021] (2) using the Tibetan sheep genomic DNA obtained in step (1) as a template, and performing amplification using the primer pair to obtain an amplified product;

[0022] (3) Performing genotyping analysis on the amplified products to obtain Tibetan sheep with different genotypes; correlating the genotypes of the Tibetan sheep with immune indicators; the immune indicators include IgA and / or IgG.

[0023] Preferably, the amplification system in step (2) is: 22 μL of Gold Mix (green), 1 μL of upstream and downstream primers, and 1 μL of template DNA;

[0024] The amplification program of step (2) is: 98°C for 2 min; 98°C for 10 s, 56°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

[0025] The present invention also provides a kit for non-diagnostic purpose Tibetan sheep immune trait marker-assisted selection and / or Tibetan sheep assisted breeding, comprising a reagent for detecting the molecular marker or the primer pair.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The SNP molecular marker described in the present invention is located at the 61720490th base on chromosome 12 of the international sheep reference genome Oar_v4.0 version; the variation type is G / A, named g61720490G>A, and there are three genotypes. When the 61720490th base on chromosome 12 is G, the genotype is GG or GA; when the 61720490th base on chromosome 12 is A, the genotype is AA; through the association analysis of different genotypes with the content of immunoglobulins IgA and IgG, it is found that the immunoglobulins IgA and IgG of Tibetan sheep individuals with the AA genotype are significantly higher than those of individuals with the GG and GA genotypes (p<0.05), and the immunoglobulins IgA and IgG of Tibetan sheep individuals with the GG and GA genotypes are not significantly different (p>0.05).

[0028] The present invention can determine the immunoglobulin IgA and IgG content of individual Tibetan sheep by detecting the base at the 61720490th nucleotide site on chromosome 12 of Tibetan sheep. The present invention provides a new SNP molecular marker resource for marker-assisted selection of Tibetan sheep immune traits for non-diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 is the PCR amplification product; M represents Marker; 1, 2, and 3 represent three groups of repetitions.

[0031] Figure 2 The peak diagram and sequence are obtained after the PCR product is purified and sequenced. DETAILED DESCRIPTION

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] 1. Sample Collection

[0035] The samples came from Tibetan sheep populations under natural grazing conditions, including 60 samples from Gannan Tibetan Autonomous Prefecture, Gansu Province, 40 samples from Yushu Tibetan Autonomous Prefecture, Qinghai Province, and 60 samples from Shigatse City, Tibet Autonomous Region. 5 mL of blood samples were collected from 160 fasting Tibetan sheep and placed in a clean pro-coagulant vacuum blood collection tube, left to stand for 30 minutes, and then centrifuged at 3500r / min for 15 minutes. The supernatant was aspirated into a clean PE tube, which was sealed and stored in a -20℃ low-temperature refrigerator. Another 5 mL of blood sample was collected in a blood collection tube with EDTA-K2 anticoagulant added. After the blood sample was collected, it was quickly mixed and temporarily stored in a sampling box containing ice packs. After being transported back to the laboratory, it was frozen in a -20℃ refrigerator for extraction of genomic DNA.

[0036] 2. Main reagents and instruments

[0037] EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; blood genomic DNA extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; NanoDrop2000 spectrophotometer was purchased from Thermo Fisher Scientific, USA; DL2000 Marker, agarose, and nucleic acid dyes were purchased from Beijing Solebaugh Technology Co., Ltd.; Gold Mix (green) was purchased from Beijing Qingke Biotechnology Co., Ltd.; electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; PCR instrument was purchased from BioRad. Immunoglobulin A (IgA) (E027-1-1) and immunoglobulin G (IgG) (E026-1-1) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0038] 3. Methods

[0039] 3.1 Immunoglobulin IgA and IgG detection

[0040] According to the IgA and IgG detection kit of Nanjing Jiancheng Bioengineering Institute, the determination was performed using a spectrophotometer. First, a standard curve was established using a standard product; secondly, distilled water, a standard solution, and 7uL of the sample to be tested were added to the blank tube, standard tube, and measurement tube, respectively, and R1 solution was added to 900uL, incubated at 37°C for 5min, and the reading at a wavelength of 340nm was recorded as A1; then 180uL of R1 solution was added to each tube, incubated at 37°C for 5min, and the reading at a wavelength of 340nm was recorded as A2; finally, △A=A2-A1 was calculated, and △A was substituted into the standard curve equation to calculate the sample IgA and IgG concentrations.

[0041] 3.2 Extraction of genomic DNA from blood

[0042] The blood genome extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract genomic DNA from the blood samples. The extracted DNA was placed under an ultraviolet spectrophotometer to detect the concentration and purity. The concentration was greater than 20 ng / μL and OD260 / OD280 was between 1.7 and 1.9, which met the experimental requirements. It was stored at -20°C for future use.

[0043] 3.3 Primer design

[0044] Referring to the chromosome 12 gene sequence of the international sheep genome Oar_v4.0 version (GenBank accession number: NC_019469.2), a pair of specific primers including the g61720490G>A SNP site were designed using primerpremier5.0 software.

[0045] Primer sequences:

[0046] F: 5'-GACATGCCCTCACTCAGACCT-3' (as shown in SEQ ID NO: 2);

[0047] R: 5'-ATGCCATGTGTCCAGATCACC-3' (as shown in SEQ ID NO: 3).

[0048] The length of the amplified fragment was 694 bp, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0049] 3.4 PCR amplification and sequencing

[0050] PCR amplification system 25 μL: Gold Mix (green) 22 μL, upstream and downstream primers 1 μL each, template 1 μL.

[0051] PCR amplification program: 98°C for 2 min; 98°C for 10 s, 56°C for 10 s, 72°C for 10 s, for a total of 40 cycles; extension at 72°C for 2 min.

[0052] The PCR product was detected by 1.5% agarose gel electrophoresis. After the PCR product was qualified by agarose gel electrophoresis, it was sequenced by direct sequencing, and the sequencing was completed by Beijing Qingke Biotechnology Co., Ltd. The amplified nucleotide sequence is shown in SEQ ID NO.1, and the SNP marker is located at position 396 of the nucleotide sequence shown in SEQ ID NO.1.

[0053] The biological analysis software MEGA 6.0 was used to compare the sequencing results of PCR products, analyze the sequencing peak graph, and complete the typing.

[0054] 4. Statistical Analysis

[0055] According to the genotyping results, the number of individuals with different genotypes at each site was counted. Popgen32 software was used to calculate the g61720490G>A gene frequency, genotype frequency, effective allele number (Ne), site heterozygosity (He), and Hardy-Weinberg equilibrium test, and PIC (polymorphism information content, referred to as PIC) calculation software was used to calculate the polymorphic information content. The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes of Tibetan sheep and immunoglobulins IgA and IgG, and the results were expressed as "mean ± standard error".

[0056] 5. Results

[0057] 5.1 PCR amplification and sequencing results

[0058] The amplified product of the g61720490G>A SNP site on chromosome 12 of Tibetan sheep was detected by 1.5% agarose gel (see Figure 1 ), the bands were clear without any other bands, the specificity was good, and the size of the PCR product fragment was 694 bp, which was in line with the expected size, so the next step of the experiment could be carried out.

[0059] SEQ ID NO.1

[0060] .

[0061] The peak diagram and sequence obtained after the PCR product was purified and sequenced are shown in Figure 2 .Depend on Figure 2 It can be seen that the GA mutation occurred at the g61720490G>ASNP site, and there are three genotypes: GG, GA, and AA.

[0062] 5.2 Statistical analysis results

[0063] The genotype and allele frequencies of the g61720490G>A SNP site on chromosome 12 of Tibetan sheep were analyzed from the perspective of population genetics. As shown in Table 1, at the g61720490G>A SNP site, the GG genotype frequency was the highest, which was the dominant genotype, and the G allele frequency was 94.7%, which was a dominant allele. The χ2 fitness test showed that the SNP site deviated significantly from the Hardy-Weinberg equilibrium state (P<0.05) (Table 1). The expected heterozygosity of this site was 0.101, the PIC was 0.096, and the PIC was <0.25, which was a low degree of polymorphism.

[0064] Table 1 Polymorphism of the g61720490G>A SNP locus on chromosome 12 of Tibetan sheep

[0065]

[0066] 5.3 Analysis of association between different genotypes and immunoglobulins IgA and IgG

[0067] The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes of Tibetan sheep and the content of immunoglobulins IgA and IgG. The results showed that the immunoglobulins IgA and IgG of Tibetan sheep individuals with AA genotype were significantly higher than those of individuals with GG and GA genotypes (p<0.05), and the immunoglobulins IgA and IgG of Tibetan sheep individuals with GG and GA genotypes were not significantly different (p>0.05), indicating that the base of the g61720490G>A SNP site on chromosome 12 of Tibetan sheep is significantly correlated with IgA and IgG of Tibetan sheep, and is a SNP marker related to IgA and IgG of Tibetan sheep. The results are shown in Table 2.

[0068] Table 2 Correlation analysis between different genotypes and immunoglobulin IgA and IgG

[0069]

[0070] Note: Different lowercase letters between data in the same row indicate significant differences (P<0.05).

[0071] In summary, the SNP molecular marker described in the present invention is located at the 61720490th base on chromosome 12 of the international sheep reference genome Oar_v4.0 version; the variation type is G / A, named g61720490G>A, and there are three genotypes. When the 61720490th base on chromosome 12 is G, the genotype is GG or GA; when the 61720490th base on chromosome 12 is A, the genotype is AA; through the association analysis of different genotypes with the content of immunoglobulins IgA and IgG, it was found that the immunoglobulins IgA and IgG of Tibetan sheep individuals with the AA genotype were significantly higher than those of individuals with the GG and GA genotypes (p<0.05), and the immunoglobulins IgA and IgG of Tibetan sheep individuals with the GG and GA genotypes were not significantly different (p>0.05). By detecting the base at the 61720490th nucleotide position on chromosome 12 of Tibetan sheep, the immunoglobulin IgA and IgG content of individual Tibetan sheep can be determined. The present invention provides a new SNP molecular marker resource for marker-assisted selection of Tibetan sheep immune traits for non-diagnostic purposes.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A molecular marker for identifying immune traits of Tibetan sheep, characterized in that: The molecular marker is located at the 61720490th base on chromosome 12 of the international sheep genome Oar_v4.0 version, and the mutant base is G or A.

2. The molecular marker according to claim 1, characterized in that The genotype of Tibetan sheep with the mutant base G is GG or GA; The genotype of Tibetan sheep with the mutant base A is AA; The immunoglobulins IgA and IgG of Tibetan sheep individuals with the AA genotype are significantly higher than those of individuals with the GG and GA genotypes; There is no significant difference in immunoglobulin IgA and IgG between the GG and GA genotype Tibetan sheep individuals.

3. Use of the molecular marker described in claim 1 or 2 in marker-assisted selection of immune traits of Tibetan sheep for non-diagnostic purposes.

4. Use of the molecular marker described in claim 1 or 2 in Tibetan sheep assisted breeding.

5. A primer pair for amplifying the molecular marker according to claim 1 or 2, characterized in that: The primer pair is shown in SEQ ID NO. 2 and 3.

6. Use of the primer pair according to claim 5 in preparing a Tibetan sheep immune trait marker-assisted selection product for non-diagnostic purposes.

7. Use of the primer pair according to claim 5 in preparing a product for Tibetan sheep assisted breeding.

8. A method for marker-assisted selection of immune traits of Tibetan sheep for non-diagnostic purposes, characterized in that: The steps include: (1) Extracting Tibetan sheep genomic DNA; (2) using the Tibetan sheep genomic DNA obtained in step (1) as a template, and performing amplification using the primer pair described in claim 5 to obtain an amplified product; (3) Performing genotyping analysis on the amplified products to obtain Tibetan sheep with different genotypes; correlating the genotypes of the Tibetan sheep with immune indicators; the immune indicators include IgA and / or IgG.

9. The method according to claim 8, characterized in that The amplification system described in step (2) is: 22 μL of Gold Mix (green), 1 μL of upstream and downstream primers, and 1 μL of template DNA; The amplification program of step (2) is: 98°C for 2 min; 98°C for 10 s, 56°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

10. A kit for non-diagnostic purposes for marker-assisted selection of Tibetan sheep immune traits and / or Tibetan sheep assisted breeding, characterized in that: It comprises a reagent for detecting the molecular marker according to claim 1 or 2 or a primer pair according to claim 5.

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