A SNP molecular marker for detecting oxidative stress ability of Tibetan sheep and its application

Abstract

The present invention belongs to the technical field of molecular biological detection, and in particular to a SNP molecular marker for detecting the oxidative stress ability of Tibetan sheep and its application. The present invention obtains a SNP molecular marker related to the oxidative stress ability of Tibetan sheep by screening, and the SNP molecular marker is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version, and the mutant base is G or T. The SNP molecular marker can be used to detect the content of ROS and MDA in Tibetan sheep individuals, providing a new SNP molecular marker resource for marker-assisted selection of Tibetan sheep anti-oxidative stress traits for non-diagnostic purposes, providing a basis for screening Tibetan sheep with strong anti-oxidative stress ability, and providing a new direction for assisted breeding of Tibetan sheep.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a SNP molecular marker for detecting the oxidative stress ability of Tibetan sheep and an application thereof. Background Art

[0002] Tibetan sheep are an important part of grassland animal husbandry in the high-altitude pastoral areas of the Qinghai-Tibet Plateau. The characteristics of the Qinghai-Tibet Plateau are: high altitude, low air pressure, strong ultraviolet rays, hypoxia, etc. Due to the harsh environment of the Qinghai-Tibet Plateau, a series of non-specific reactions will be caused in Tibetan sheep, mainly immune stress, cold stress and oxidative stress, among which oxidative stress is the main one. After Tibetan sheep are stimulated by stressors, the dynamic balance of the oxidation system and antioxidant system in the body is destroyed, the content of reactive oxygen species (ROS) and malondialdehyde (MDA) increases, and the content of antioxidants decreases. The body will be damaged by oxidation, thus affecting the growth performance and reproductive performance of Tibetan sheep.

[0003] Reactive oxygen species (ROS) and malondialdehyde (MDA) are important indicators for detecting oxidative stress response. The oxidative stress capacity and health status of Tibetan sheep can be evaluated by detecting the content of reactive oxygen species (ROS) and malondialdehyde (MDA). By detecting the indicators of anti-oxidative stress, the oxidative stress response capacity of Tibetan sheep can be discovered in time, and certain measures can be taken to improve their oxidative stress capacity, so that they can better adapt to the harsh environmental conditions of the Qinghai-Tibet Plateau, and provide a basis for the auxiliary breeding of Tibetan sheep.

[0004] With the rapid development of molecular marker technology, the mutation sites in genes are searched from the gene level, and the relationship between genes and traits is discovered through the association analysis between genes and traits, and early selection is carried out to improve the selection efficiency and accuracy, which 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 on molecular markers related to the oxidative stress capacity of Tibetan sheep in the prior art 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 for detecting the oxidative stress ability of Tibetan sheep and application thereof.

[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 detecting the oxidative stress ability of Tibetan sheep. The SNP molecular marker is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version, and the mutant base is G or T.

[0008] Preferably, the genotype of the Tibetan sheep with the mutant base G is GG or GT;

[0009] The genotype of Tibetan sheep with the mutant base T is TT;

[0010] The content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT was significantly higher than that in Tibetan sheep with genotype GG;

[0011] There was no significant difference in the content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT;

[0012] The anti-oxidative stress indicators include reactive oxygen species and / or malondialdehyde.

[0013] The present invention also provides the use of the SNP molecular marker in preparing a product for detecting the oxidative stress ability of Tibetan sheep.

[0014] The present invention also provides the use of the SNP molecular marker in preparing a product for Tibetan sheep auxiliary breeding.

[0015] The present invention also provides a primer pair for amplifying the SNP, and the primer pair is shown in SEQ ID NO.1-2.

[0016] The present invention also provides application of the primer pair in preparing a product for detecting the oxidative stress ability of Tibetan sheep.

[0017] The present invention also provides application of the primer pair in preparing products for auxiliary breeding of Tibetan sheep.

[0018] The present invention also provides a method for screening Tibetan sheep with strong anti-oxidative stress ability, comprising the following steps:

[0019] (1) Extracting Tibetan sheep genomic DNA;

[0020] (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;

[0021] (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, screening Tibetan sheep with a high content of the anti-oxidative stress index, and obtaining Tibetan sheep with a strong anti-oxidative stress ability;

[0022] The anti-oxidative stress indicators include reactive oxygen species and / or malondialdehyde.

[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 in step (2) is: 98°C for 2 min; 98°C for 10 s, 57°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 screening Tibetan sheep with strong anti-oxidative stress ability, comprising a reagent for detecting the SNP molecular marker or the primer pair.

[0026] The present invention provides a SNP molecular marker for detecting the oxidative stress ability of Tibetan sheep and its application. The SNP molecular marker of the present invention is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version; when the 35036005th base on the chromosome is G, the genotype of the Tibetan sheep is GG or GT; when the 35036005th base on the chromosome is T, the genotype of the Tibetan sheep is TT; through the association analysis of different genotypes with the content of ROS and MDA, it is found that the ROS and MDA of the GT and TT genotype Tibetan sheep individuals are significantly higher than those of the GG genotype individuals (p<0.05), and the ROS and MDA of the GT and TT genotype Tibetan sheep individuals are not significantly different (p>0.05). By detecting the base at the 35036005th nucleotide site on chromosome 20 of Tibetan sheep, the content of ROS and MDA, the antioxidant stress indicators of individual Tibetan sheep, can be determined. The present invention provides a new SNP molecular marker resource for marker-assisted selection of antioxidant stress traits of Tibetan sheep for non-diagnostic purposes, provides a basis for screening Tibetan sheep with strong antioxidant stress resistance, and provides a new direction for assisted breeding of Tibetan sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Agarose gel electrophoresis is used to detect the amplified products of SNP sites;

[0028] Figure 2 The peak diagram and sequence are obtained after the PCR amplification product is purified and sequenced. DETAILED DESCRIPTION

[0029] The present invention provides a SNP molecular marker for detecting the oxidative stress ability of Tibetan sheep, wherein the SNP molecular marker is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version (the gene sequence is GenBank accession number: NC_019477.2), and the mutant base is G or T. In the present invention, the genotype of the Tibetan sheep with the mutant base G is GG or GT;

[0030] The genotype of Tibetan sheep with the mutant base T is TT;

[0031] The content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT was significantly higher than that in Tibetan sheep with genotype GG;

[0032] There was no significant difference in the content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT;

[0033] The anti-oxidative stress indicators include reactive oxygen species and / or malondialdehyde.

[0034] The present invention also provides the use of the SNP molecular marker in preparing a product for detecting the oxidative stress ability of Tibetan sheep.

[0035] The present invention also provides the use of the SNP molecular marker in preparing a product for Tibetan sheep auxiliary breeding.

[0036] The present invention also provides a primer pair for amplifying the SNP, and the primer pair is shown in SEQ ID NO. 1 to 2. SEQ ID NO. 1 is an upstream primer, and the sequence is: 5'-GTCATCTATTTAAAGCCCGAT-3'; SEQ ID NO. 2 is a downstream primer, and the sequence is: 5'-CATTTATGCAGCAATAACACA-3'.

[0037] The present invention also provides application of the primer pair in preparing a product for detecting the oxidative stress ability of Tibetan sheep.

[0038] The present invention also provides application of the primer pair in preparing products for auxiliary breeding of Tibetan sheep.

[0039] The present invention also provides a method for screening Tibetan sheep with strong anti-oxidative stress ability, comprising the following steps:

[0040] (1) Extracting Tibetan sheep genomic DNA;

[0041] (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;

[0042] (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, screening Tibetan sheep with a high content of the anti-oxidative stress index, and obtaining Tibetan sheep with a strong anti-oxidative stress ability;

[0043] The anti-oxidative stress indicators include reactive oxygen species and / or malondialdehyde.

[0044] In the present invention, 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;

[0045] The amplification program in step (2) is: 98°C for 2 min; 98°C for 10 s, 57°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

[0046] In the present invention, the length of the amplified product is 536 bp, and the specific sequence is shown in SEQ ID NO.3, SEQ ID NO.3:.

[0047] The present invention also provides a kit for screening Tibetan sheep with strong anti-oxidative stress ability, comprising a reagent for detecting the SNP molecular marker or the primer pair.

[0048] 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.

[0049] Example 1

[0050] 1. Sample collection

[0051] The samples came from Tibetan sheep populations under natural grazing conditions, including 58 samples from Gannan Tibetan Autonomous Prefecture, Gansu Province, 37 samples from Yushu Tibetan Autonomous Prefecture, Qinghai Province, and 49 samples from Shigatse City, Tibet Autonomous Region. 5 mL of blood samples were collected from 144 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 and added to a blood collection tube containing EDTA-K2 anticoagulant. After the blood sample was collected, it was quickly mixed and temporarily stored in a sampling box containing an ice pack. After being transported back to the laboratory, it was frozen in a -20℃ refrigerator for extraction of genomic DNA.

[0052] 2 Main reagents and instruments

[0053] Blood collection tubes with EDTA-K2 anticoagulant were purchased from Jiangsu Yuli Medical Instrument Co., Ltd.; blood genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; NanoDrop2000 spectrophotometer was purchased from ThermoFisher Scientific, USA; DL2000 Marker, agarose, and nucleic acid dye were all purchased from Beijing Solebow 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. Reactive oxygen species ROS (E004-1-1) and malondialdehyde MDA (A003-1-2) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0054] 3 Methods

[0055] 3.1 Detection of ROS and MDA in serum

[0056] The DCFH-DA probe was used for determination according to the ROS detection kit of Nanjing Jiancheng Bioengineering Institute. The determination steps are as follows: (1) add 190 μL of serum and 10 μL of 1 mmol / L DCFH-DA solution (the final concentration of DCFH-DA is 50 μmol / L) to a 96-well ELISA plate, and mix thoroughly with a micropipette; (2) place in an ELISA reader, keep at 37°C for 30 minutes, and then detect the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 538 nm; (3) take 50 μL of serum, dilute 30 times, and take 100 μL for protein quantification; (4) The results are expressed as fluorescence intensity / mg protein.

[0057] The determination was performed using the TBA method according to the MDA detection kit of Nanjing Jiancheng Bioengineering Institute. The determination steps are as follows: (1) add 0.2mL of anhydrous ethanol and 0.2mL of reagent 1 to the blank tube, add 0.2mL of 10nmol / mL standard and 0.2mL of reagent 1 to the standard tube, add 0.2mL of the test serum and 0.2mL of reagent 1 to the test tube, and add 0.2mL of the test serum and 0.2mL of reagent 1 to the control tube, and shake to mix; (2) add 3mL of reagent 2 and 1mL of reagent 3 to the blank tube, standard tube and test tube, and add 3mL of reagent 2 and 1mL of 50% glacial acetic acid to the control tube, vortex to mix, and then place in a 95°C water bath for 40min, take out and cool in running water, and then centrifuge at 4000r / min for 10min; (3) take the supernatant and measure the absorbance value of each tube at 532nm; (4) calculate the MDA content according to the formula provided in the kit instructions.

[0058] 3.2 Extraction of genomic DNA from blood

[0059] 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.

[0060] 3.3 Primer design

[0061] Referring to the gene sequence of chromosome 20 of the international sheep genome Oar_v4.0 version (GenBank accession number: NC_019477.2), a pair of specific primers were designed using primerpremier5.0 software, and the nucleotides of the primer pair are shown in SEQ ID NO.1-2. SEQ ID NO.1 is the upstream primer, and its sequence is: 5'-GTCATCTATTTAAAGCCCGAT-3'; SEQ ID NO.2 is the downstream primer, and its sequence is: 5'-CATTTATGCAGCAATAACACA-3'.

[0062] The length of the amplified fragment was 536 bp, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0063] 3.4 PCR amplification and sequencing

[0064] PCR amplification system 25 μL: Gold Mix (green) 22 μL, upstream and downstream primers 1 μL each, template DNA 1 μL.

[0065] PCR amplification program: 98°C for 2 min; 98°C for 10 s, 57°C for 10 s, 72°C for 10 s, for a total of 40 cycles; extension at 72°C for 2 min.

[0066] 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.3, and the SNP marker is located at position 378 of the nucleotide sequence shown in SEQ ID NO.3.

[0067] 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.

[0068] SEQ ID NO.3:.

[0069] 4 Statistical analysis

[0070] According to the genotyping results, the number of individuals with different genotypes at each site was counted. Popgen32 software was used to calculate the g35036005G>T 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 the antioxidant stress indicators ROS and MDA, and the results were expressed as "mean ± standard error".

[0071] 5 Results

[0072] 5.1 PCR amplification and sequencing results

[0073] The amplified product of the g35036005G>T SNP site on chromosome 20 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 536bp, which was in line with the expected size, so the next step of the experiment could be carried out.

[0074] 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 GT mutation occurred at the g35036005G>TSNP site, and there are three genotypes: GG, GT, and TT.

[0075] 5.2 Statistical analysis results

[0076] The genotype and allele frequencies of the g35036005G>T SNP locus on chromosome 20 of Tibetan sheep were analyzed from the perspective of population genetics. As shown in Table 1, at the g35036005G>T SNP locus, the GG genotype frequency was the highest, which was the dominant genotype, and the G allele frequency was 72.0%, which was a dominant allele. The χ2 fitness test showed that the SNP locus deviated significantly from the Hardy-Weinberg equilibrium state (P<0.05) (Table 1). The expected heterozygosity of this locus was 1.680, the PIC was 0.32, and 0.25<PIC<0.50, which was moderately polymorphic.

[0077] Table 1 Polymorphism of the g35036005G>T SNP locus on chromosome 20 of Tibetan sheep

[0078]

[0079] 5.3 Correlation analysis of reactive oxygen species (ROS) and malondialdehyde (MDA) in different genotypes for antioxidant stress indicators

[0080] 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 ROS and MDA, the indicators of antioxidant stress. The results showed that the antioxidant stress indicators ROS and MDA of Tibetan sheep individuals with GT and TT genotypes were significantly higher than those of individuals with GG genotypes (p<0.05), and the antioxidant stress indicators ROS and MDA of Tibetan sheep individuals with GT and TT genotypes were not significantly different (p>0.05), indicating that the base of the g35036005G>T SNP site on chromosome 20 of Tibetan sheep was significantly correlated with ROS and MDA of Tibetan sheep, and was a SNP marker related to ROS and MDA of Tibetan sheep. The results are shown in Table 2.

[0081] Table 2 Relationship between different genotypes and anti-oxidative stress indicators ROS and MDA

[0082]

[0083] Note: Different lowercase letters between data in the same row indicate significant differences (P<0.05).

[0084] The invention provides a SNP molecular marker for detecting the oxidative stress ability of Tibetan sheep and its application. The SNP molecular marker of the invention is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version; the variation type is G / T, named g35036005G>T, and there are three genotypes. When the 35036005th base on chromosome 20 is G, the genotype of the Tibetan sheep is GG or GT; when the 35036005th base on chromosome 20 is T, the genotype of the Tibetan sheep is TT; through the association analysis of different genotypes with the content of ROS and MDA, it is found that the ROS and MDA of the GT and TT genotype Tibetan sheep individuals are significantly higher than those of the GG genotype individuals (p<0.05), and the ROS and MDA of the GT and TT genotype Tibetan sheep individuals are not significantly different (p>0.05). By detecting the base at the 35036005th nucleotide site on chromosome 20 of Tibetan sheep, the content of ROS and MDA, the antioxidant stress indicators of individual Tibetan sheep, can be determined. The present invention provides a new SNP molecular marker resource for marker-assisted selection of antioxidant stress traits of Tibetan sheep for non-diagnostic purposes, provides a basis for screening Tibetan sheep with strong antioxidant stress resistance, and provides a new direction for assisted breeding of Tibetan sheep.

[0085] 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. Use of a molecular marker reagent for detecting the oxidative stress ability of Tibetan sheep in the preparation of a product for detecting the oxidative stress ability of Tibetan sheep, characterized in that: The SNP molecular marker for the oxidative stress capacity of Tibetan sheep is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version, and the mutant base is G or T; According to the molecular marker alleles of oxidative stress capacity in Tibetan sheep, there are three genotypes of Tibetan sheep: GG, GT or TT; The content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT was significantly higher than that in Tibetan sheep with genotype GG; There was no significant difference in the content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT; The reagent is a primer pair, and the primer pair is shown in SEQ ID NO.1~2.

2. A method for screening Tibetan sheep with strong anti-oxidative stress ability, 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 a primer pair 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, screening Tibetan sheep with high levels of the anti-oxidative stress index, and obtaining Tibetan sheep with strong anti-oxidative stress ability; The anti-oxidative stress index includes reactive oxygen species and / or malondialdehyde; The primer pair is shown in SEQ ID NO.1-2; The amplification product is a fragment including a SNP molecular marker for detecting the oxidative stress ability of Tibetan sheep; the SNP molecular marker for the oxidative stress ability of Tibetan sheep is located at the 35036005th base on chromosome 20 of the international sheep genome Oar_v4.0 version, and the mutant base is G or T; The genotype analysis results show that Tibetan sheep have three genotypes: GG, GT or TT; The content of anti-oxidative stress indexes in Tibetan sheep with genotype GT or TT was significantly higher than that in Tibetan sheep with genotype GG; There was no significant difference in the contents of anti-oxidative stress indicators among Tibetan sheep with genotype GT or TT.

3. The method according to claim 2, 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 in step (2) is: 98°C for 2 min; 98°C for 10 s, 57°C for 10 s, 72°C for 10 s, for a total of 40 cycles; and extension at 72°C for 2 min.

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