SNP molecular markers and methods for identifying the ability of Tibetan sheep to resist oxidative stress

Abstract

The present invention relates to the field of molecular marker technology, and in particular to a SNP molecular marker and a method thereof for identifying the ability of Tibetan sheep to resist oxidative stress. The SNP molecular marker used to identify the ability of Tibetan sheep to resist oxidative stress is located at the 72583723rd base on chromosome 15 of the international sheep genome Oar_v4.0 version, and the mutant base is T or C. The present invention can determine the T-AOC content of the anti-oxidative stress indicator of individual Tibetan sheep by detecting the base at the 72583723rd nucleotide site on chromosome 15 of Tibetan sheep. The present invention provides a new SNP molecular marker resource for marker-assisted selection of Tibetan sheep anti-oxidative stress traits for non-diagnostic purposes.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and in particular to a SNP molecular marker and a method thereof for identifying the anti-oxidative stress ability of Tibetan sheep. Background Art

[0002] Tibetan sheep are a vital component of the grassland animal husbandry industry in the alpine pastoral areas of the Qinghai-Tibet Plateau. The plateau is characterized by high altitude, low air pressure, strong ultraviolet rays, and hypoxia. This harsh environment triggers a series of nonspecific reactions in Tibetan sheep, primarily immune stress, cold stress, and oxidative stress, with oxidative stress being the most prominent. Stressors disrupt the dynamic balance of the body's oxidative and antioxidant systems. Antioxidants in organisms are primarily composed of two categories: antioxidant enzymes and non-enzymatic antioxidants. The antioxidant activity of these systems is primarily achieved through three pathways: eliminating free radicals and reactive oxygen species to prevent lipid peroxidation; decomposing peroxides to interrupt the peroxidation chain; and removing catalytic metal ions. Total antioxidant capacity (T-AOC) is a key indicator of the body's overall antioxidant capacity, both enzymatic and non-enzymatic. In the existing technology, there are many studies on the relationship between exercise, free radicals and antioxidant systems, but there are few studies on the adaptive regulation of Tibetan sheep, a plateau native animal, in different degrees of hypoxic environments.

[0003] By testing the total antioxidant capacity, we can assess the oxidative stress resistance and health status of Tibetan sheep. By testing the antioxidant stress indicators, we can promptly identify the oxidative stress response capacity of Tibetan sheep and take certain measures to improve their oxidative stress resistance, enabling them to better adapt to the harsh environmental conditions of the Qinghai-Tibet Plateau and provide a basis for assisted breeding of Tibetan sheep.

[0004] With the rapid development of molecular marker technology, the mutation sites in genes are searched at the gene level, and the relationship between genes and traits is discovered through correlation analysis between genes and traits, and early selection is carried out to improve the efficiency and accuracy of seed selection. It can fundamentally improve the oxidative stress capacity of Tibetan sheep and has broad application value. Therefore, molecular markers have laid the foundation for people to study the genetic mechanism of the oxidative stress capacity of Tibetan sheep at the molecular level. Screening corresponding molecular markers for more efficient use in the breeding of Tibetan sheep is an important issue in this field. There are not many studies in the prior art on the use of molecular markers related to the oxidative stress capacity of Tibetan sheep for assisted breeding. It is particularly important to provide a molecular marker related to the oxidative stress capacity of Tibetan sheep, so as to be used in the molecular marker-assisted breeding of Tibetan sheep and screen Tibetan sheep with strong anti-oxidative stress ability. Summary of the Invention

[0005] The purpose of the present invention is to provide a SNP molecular marker and a method for identifying the anti-oxidative stress ability of Tibetan sheep.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a SNP molecular marker for identifying the anti-oxidative stress ability of Tibetan sheep. The molecular marker is located at the 72583723rd base on chromosome 15 of the international sheep genome Oar_v4.0 version, and the mutant base is T or C.

[0008] Preferably, the genotype of the Tibetan sheep with the mutant base T is TT or TC;

[0009] The genotype of Tibetan sheep with the mutant base C is CC;

[0010] The anti-oxidative stress index T-AOC of Tibetan sheep individuals with TC and CC genotypes was significantly higher than that of individuals with TT genotype;

[0011] There was no significant difference in the anti-oxidative stress index T-AOC between the Tibetan sheep individuals with the TC and CC genotypes.

[0012] The present invention also provides the use of the SNP molecular marker in marker-assisted selection of the anti-oxidative stress trait of Tibetan sheep for non-diagnostic purposes.

[0013] The present invention also provides the application of the SNP molecular marker in Tibetan sheep assisted breeding.

[0014] The present invention also provides a primer pair for amplifying the SNP molecular marker, and the primer pair is shown as SEQ ID NO. 2-3.

[0015] The present invention also provides the use of the primer pair in preparing a marker-assisted selection product for the antioxidant stress trait of Tibetan sheep for non-diagnostic purposes.

[0016] The present invention also provides the use of the primer pair in preparing Tibetan sheep assisted breeding products.

[0017] The present invention also provides a method for marker-assisted selection of anti-oxidative stress traits in Tibetan sheep for non-diagnostic purposes, comprising the following steps:

[0018] (1) Extracting Tibetan sheep genomic DNA;

[0019] (2) using the Tibetan sheep genomic DNA obtained in step (1) as a template, amplifying using the primer pair to obtain an amplified product;

[0020] (3) performing genotyping analysis on the amplified products to obtain Tibetan sheep with different genotypes; correlating the genotypes of the Tibetan sheep with anti-oxidative stress indicators; the anti-oxidative stress indicators include total antioxidant capacity.

[0021] 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;

[0022] The amplification program in step (2) is: 98°C for 2 min; 98°C for 10 s, 57.5°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

[0023] The present invention also provides a kit for marker-assisted selection of Tibetan sheep anti-oxidative stress traits and / or Tibetan sheep assisted breeding for non-diagnostic purposes, comprising a reagent for detecting the SNP molecular marker or the primer pair.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The SNP molecular marker described in the present invention is located at the 72583723rd base on chromosome 15 of the international sheep reference genome Oar_v4.0 version; the variation type is T / C, named g72583723T>C, and there are three genotypes. When the 72583723rd base on the chromosome 15 is T, the genotype is TT or TC; when the 72583723rd base on the chromosome 15 is C, the genotype is CC; through the association analysis of different genotypes with the antioxidant stress index T-AOC content, it was found that the antioxidant stress index T-AOC of Tibetan sheep individuals with the TC and CC genotypes was significantly higher than that of individuals with the TT genotype (p<0.05), and the difference in the antioxidant stress index T-AOC of Tibetan sheep individuals with the TC and CC genotypes was not significant (p>0.05).

[0026] The present invention can determine the content of T-AOC, an antioxidant stress indicator, in individual Tibetan sheep by detecting the base at the 72583723rd nucleotide site on chromosome 15 of Tibetan sheep. The present invention provides a new SNP molecular marker resource for marker-assisted selection of antioxidant stress traits in Tibetan sheep for non-diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0028] Figure 1 The amplified product of the SNP site was detected by agarose gel electrophoresis, where M represents Marker; 1, 2, and 3 represent three groups of repeats;

[0029] Figure 2 Figure 3 is the peak diagram and sequence obtained after purification and sequencing of the PCR amplification product. DETAILED DESCRIPTION

[0030] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Sample Collection

[0033] The samples came from Tibetan sheep populations under natural grazing conditions, including 50 samples from Gannan Tibetan Autonomous Prefecture, Gansu Province, 50 samples from Yushu Tibetan Autonomous Prefecture, Qinghai Province, and 69 samples from Shigatse City, Tibet Autonomous Region. 5 mL of blood samples were collected from 169 fasting Tibetan sheep and placed in a clean pro-coagulant vacuum blood collection tube. The tubes were allowed to stand for 30 minutes and then centrifuged at 3500 r / min for 15 minutes. The supernatant was aspirated into a clean PE tube, sealed, and stored in a -20°C low-temperature refrigerator. Another 5 mL of blood sample was collected in a blood collection tube with EDTA-K2 anticoagulant added. After blood sample collection, the tubes were quickly mixed and temporarily placed in a sampling box containing ice packs. After being transported back to the laboratory, they were frozen in a -20°C refrigerator for genomic DNA extraction.

[0034] 2. Main reagents and instruments

[0035] EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; a blood genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; a NanoDrop 2000 spectrophotometer was purchased from Thermo Fisher Scientific (USA); DL2000 marker, agarose, and nucleic acid dye were purchased from Beijing Solebold Technology Co., Ltd.; Gold Mix (green) was purchased from Beijing Qingke Biotechnology Co., Ltd.; an electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; and a PCR instrument was purchased from BioRad. A total antioxidant capacity (T-AOC) (A015-2-1) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0036] 3. Methods

[0037] 3.1 Detection of T-AOC in serum

[0038] The ABTS method was used to determine the T-AOC detection kit from Nanjing Jiancheng Bioengineering Institute. The assay steps were as follows: (1) 10 μL of double-distilled water and 20 μL of reagent IV application solution (reagent I and peroxidase were mixed at a ratio of 9:1) were added to the blank tube, 10 μL of MTrolox solution of different concentrations and 20 μL of reagent IV application solution were added to the standard tube, and 10 μL of the serum to be tested and 20 μL of reagent IV application solution were added to the assay tube; (2) 170 μL of ABTS working solution (reagent I: reagent II: reagent III application solution = 76:5:4) was added to the blank tube, standard tube, and assay tube; (3) the reaction was carried out at room temperature for 6 min, and the OD value of each well was read using a microplate reader at a wavelength of 405 nm; (4) a standard curve was prepared using the OD of the standard sample and the curve formula was obtained. The OD measured in the sample assay tube was substituted into the calculation formula to obtain the result.

[0039] 3.2 Extraction of genomic DNA from blood

[0040] Genomic DNA was extracted from blood samples using the blood genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. The extracted genomic DNA was placed under an ultraviolet spectrophotometer to detect the concentration and purity. A concentration greater than 20 ng / μL and an OD260 / OD280 between 1.7 and 1.9 met the experimental requirements and was stored at -20°C for future use.

[0041] 3.3 Primer design

[0042] Referring to the chromosome 15 gene sequence of the international sheep genome Oar_v4.0 version (GenBank accession number: NC_019472.2), a pair of specific primers containing the g72583723T>C SNP site were designed using primerpremier5.0 software.

[0043] Primer sequences:

[0044] F: 5'-TGAAACGCCCAAAGAGTCCC-3' (SEQ ID NO. 2);

[0045] R: 5'-GTCACCTGCCTCTGTTACACC-3' (SEQ ID NO. 3).

[0046] The amplified fragment was 558 bp in length, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0047] 3.4 PCR amplification and sequencing

[0048] PCR amplification system 25 μL: Gold Mix (green) 22 μL, 1 μL each of upstream and downstream primers, and 1 μL of template.

[0049] PCR amplification program: 98°C for 2 min; 98°C for 10 s, 57.5°C for 10 s, and 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

[0050] The PCR products were detected by 1.5% agarose gel electrophoresis. After the PCR products passed the agarose gel electrophoresis test, they were 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 239 of the nucleotide sequence shown in SEQ ID NO. 1.

[0051] The bioanalysis software MEGA 6.0 was used to compare the sequencing results of PCR products, analyze the sequencing peak graphs, and complete the typing.

[0052] 4. Statistical Analysis

[0053] Based on the genotyping results, the number of individuals with different genotypes at each locus was counted. The g72583723T>C gene frequency, genotype frequency, effective number of alleles (Ne), locus heterozygosity (He), and Hardy-Weinberg equilibrium test were calculated using Popgen32 software. The polymorphism information content (PIC) calculation software was used to calculate the polymorphism information content (PIC). The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes in Tibetan sheep and the antioxidant stress marker T-AOC. The results are expressed as mean ± standard error.

[0054] 5. Results

[0055] 5.1 PCR amplification and sequencing results

[0056] The amplified product of the g72583723T>C SNP site on chromosome 15 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 PCR product fragment size was 558 bp, which was in line with the expected size, so the next experiment could be carried out.

[0057] SEQ ID NO.1

[0058] .

[0059] The peak diagram and sequence of the PCR product after purification and sequencing are shown in Figure 2 .Depend on Figure 2 It can be seen that the TC mutation occurred at the g72583723T>CSNP site, and there are three genotypes: TT, TC, and CC.

[0060] 5.2 Statistical analysis results

[0061] The genotype and allele frequencies of the g72583723T>C SNP locus on chromosome 15 of Tibetan sheep were analyzed from a population genetics perspective. As shown in Table 1, the CC genotype is the most frequent and dominant genotype at the g72583723T>C SNP locus, while the C allele is the dominant allele at a frequency of 69.2%. The chi-squared fitness test indicated that the SNP locus significantly deviated from Hardy-Weinberg equilibrium (P < 0.05) (Table 1). The expected heterozygosity and PIC of this locus are 0.426 and 0.335, respectively, with a PIC of 0.25 < 0.50, indicating moderate polymorphism.

[0062] Table 1 Polymorphism of the g72583723T>C SNP site on chromosome 15 of Tibetan sheep

[0063]

[0064] 5.3 Association analysis between different genotypes and the antioxidant stress indicator T-AOC

[0065] A general linear model was used in IBM SPSS Statistics 22 software to analyze the association between different Tibetan sheep genotypes and the antioxidant stress marker T-AOC. The results showed that T-AOC levels in Tibetan sheep with the TC and CC genotypes were significantly higher than those in those with the TT genotype (p < 0.05). There was no significant difference in T-AOC between the TC and CC genotypes (p > 0.05). This suggests that the g72583723T>C SNP on chromosome 15 is significantly associated with T-AOC in Tibetan sheep and is a T-AOC-related SNP marker. The results are shown in Table 2.

[0066] Table 2 Correlation analysis between different genotypes and the antioxidant stress index T-AOC

[0067]

[0068] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05).

[0069] In summary, the SNP molecular marker described in the present invention is located at the 72583723rd base on chromosome 15 of the international sheep reference genome Oar_v4.0 version; the variation type is T / C, named g72583723T>C, and there are three genotypes. When the 72583723rd base on chromosome 15 is T, the genotype is TT or TC; when the 72583723rd base on chromosome 15 is C, the genotype is CC; through the association analysis of different genotypes with the antioxidant stress index T-AOC content, it was found that the antioxidant stress index T-AOC of Tibetan sheep individuals with the TC and CC genotypes was significantly higher than that of individuals with the TT genotype (p<0.05), and the difference in the antioxidant stress index T-AOC of Tibetan sheep individuals with the TC and CC genotypes was not significant (p>0.05).

[0070] The present invention can determine the content of T-AOC, an antioxidant stress indicator, in individual Tibetan sheep by detecting the base at the 72583723rd nucleotide site on chromosome 15 of Tibetan sheep. The present invention provides a new SNP molecular marker resource for marker-assisted selection of antioxidant stress traits in Tibetan sheep for non-diagnostic purposes.

[0071] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a primer pair for amplifying a SNP molecular marker for identifying the ability of Tibetan sheep to resist oxidative stress in preparing a product for assisting selection of Tibetan sheep's anti-oxidative stress trait, characterized in that: The molecular marker is located at base 72583723 on chromosome 15 of the international sheep genome Oar_v4.0 version, and the mutant base is T or C; The genotype of Tibetan sheep with the mutant base T is TT or TC; The genotype of Tibetan sheep with the mutant base C is CC; The total antioxidant capacity of the anti-oxidative stress index of Tibetan sheep individuals with TC and CC genotypes was significantly higher than that of individuals with TT genotype; There was no significant difference in the total antioxidant capacity of the antioxidant stress indicators between the TC and CC genotype Tibetan sheep individuals.

2. The use according to claim 1, characterized in that The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 2-3.

3. A method for marker-assisted selection of anti-oxidative stress traits in Tibetan sheep for non-diagnostic purposes, characterized in that: The steps include: (1) Extraction of Tibetan sheep genomic DNA; (2) using the Tibetan sheep genomic DNA obtained in step (1) as a template, amplifying using the primer pair described in claim 2 to obtain an amplified product; (3) performing genotyping analysis on the amplified product to obtain Tibetan sheep with different genotypes; correlating the genotype of the Tibetan sheep with an anti-oxidative stress index; the anti-oxidative stress index is the total antioxidant capacity; The nucleotide sequence of the amplified product is shown in SEQ ID NO. 1, and the genotype is determined based on whether the 239th nucleotide of the amplified product is T or C; The total antioxidant capacity of the anti-oxidative stress indicators of Tibetan sheep with TC and CC genotypes was significantly higher than that of the individuals with TT genotype; the difference in the total antioxidant capacity of the anti-oxidative stress indicators of Tibetan sheep with TC and CC genotypes was not significant.

4. The method according to claim 3, characterized in that The amplification program in step (2) is as follows: 98°C for 2 min; 98°C for 10 s, 57.5°C for 10 s, and 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

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