A molecular marker related to the antioxidant capacity of color wool sheep, a detection primer and application thereof

Abstract

The application provides a molecular marker related to the antioxidant capacity of Seva sheep, a detection primer and application thereof, and belongs to the technical field of sheep molecular detection. The nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1. Through correlation analysis of the genotype of a Seva sheep sample and the antioxidant stress index T-AOC content, it is found that the antioxidant stress index T-AOC of Seva sheep individuals with genotypes CT and TT is significantly higher than that of individuals with genotype CC, which indicates that the genotype of the SNP site of the Seva sheep is significantly related to the T-AOC content in the serum of the Seva sheep, and is an SNP marker related to T-AOC of the Seva sheep. The molecular marker provided by the application can be applied to the assisted breeding of the Seva sheep and improve the breeding efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of sheep molecular detection, and particularly relates to a molecular marker related to the antioxidant capacity of Seva sheep, a detection primer thereof and application. BACKGROUND

[0002] In modern sheep breeding industry, the antioxidant capacity of sheep has a crucial impact on its health status, production performance and environmental adaptability. Sheep with strong antioxidant capacity can better resist oxidative stress, reduce the damage of free radicals to body cells and tissues, thereby maintaining good physiological functions and improving key production indicators such as growth rate, reproductive performance and meat quality.

[0003] Seva sheep, as a sheep breed with local characteristics in China, mainly distributes in the high-altitude areas of Bange County in Naqu region of Northern Tibet. The environment in this area is complex and changeable, with cold climate, strong ultraviolet radiation and relatively scarce feed resources. These factors make Seva sheep face severe oxidative stress challenges for a long time. In such an environment, the antioxidant capacity of Seva sheep is particularly crucial for its survival and reproduction. Seva sheep individuals with strong antioxidant capacity can better maintain a healthy state and exhibit good production performance in harsh environments, which is of great significance to the sustainable development of local sheep breeding industry.

[0004] At present, in the field of sheep breeding, in order to breed sheep breeds with excellent antioxidant capacity, researchers and breeders usually adopt traditional phenotypic selection method, that is, judging the antioxidant capacity of sheep indirectly by observing its appearance characteristics, growth performance and adaptation performance to the environment. However, this method has obvious limitations. On the one hand, antioxidant capacity is a complex physiological trait, which is jointly regulated by multiple genes and environmental factors. It is difficult to accurately evaluate by relying solely on phenotypic observation, which may lead to selection errors. On the other hand, traditional phenotypic selection requires a lot of time and effort, and needs sheep to grow to a certain stage before observation and judgment, which has a long breeding cycle and low efficiency.

[0005] With the rapid development of molecular biology technology, the use of molecular marker assisted selection technology for sheep breeding has become a research hotspot. Molecular markers can directly reflect the genetic information of individuals, and by detecting molecular markers related to target traits, accurate genetic evaluation of sheep individuals can be made at an early stage, greatly improving the efficiency of breeding. However, the current research on specific molecular markers related to the antioxidant capacity of Sewa sheep is still relatively scarce. Some existing general molecular markers are difficult to accurately establish a close relationship with the antioxidant capacity in Sewa sheep due to their unique genetic background and environmental adaptability, and cannot meet the needs of efficient breeding of Sewa sheep. Therefore, developing a specific molecular marker closely related to the antioxidant capacity of Sewa sheep and applying it to the breeding practice of Sewa sheep has urgent practical significance and broad application prospects for improving the antioxidant capacity of Sewa sheep, enhancing their adaptability to harsh environments, and improving production performance. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a SNP molecular marker related to the antioxidant capacity of Sewa sheep, a detection primer thereof and application, by detecting the SNP molecular marker in Sewa sheep, the antioxidant capacity of Sewa sheep can be quickly and accurately evaluated, and the efficient breeding of Sewa sheep can be assisted.

[0007] The present application provides a molecular marker related to the antioxidant capacity of Sewa sheep, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.

[0008] Preferably, the molecular marker comprises a SNP site, the SNP site is located at the 240th position of the molecular marker, and the SNP site mutation base is C or T.

[0009] Preferably, when the SNP site is C, the genotype is CC or CT, and when the SNP site is T, the genotype is TT.

[0010] The present application provides a primer pair for amplifying the molecular marker, comprising primer F and primer R; the nucleotide sequence of the primer F is shown as SEQ ID NO. 2, and the nucleotide sequence of the primer R is shown as SEQ ID NO. 3.

[0011] The present application provides the application of the primer pair in preparing a reagent for detecting the antioxidant capacity of Sewa sheep.

[0012] The present application provides the application of the molecular marker in the assisted breeding of Sewa sheep.

[0013] Preferably, the application comprises the following steps:

[0014] 1) extract the genome DNA of the selected color breeding sheep, use the genome DNA of the color sheep as a template, and use the primer pair to perform PCR amplification to obtain an amplification product;

[0015] 2) sequencing the amplification product to determine the genotype of the SNP site, and selecting the selected color breeding sheep with genotype TT or CT for subsequent breeding.

[0016] Preferably, the amplification system of the PCR amplification comprises 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, and 1 μL of template DNA, in 25 μL.

[0017] Preferably, the amplification procedure of the PCR amplification is as follows: 98℃ for 2min; 98℃ for 10s, 57℃ for 10s, 72℃ for 10s, a total of 40 cycles; 72℃ for 2min.

[0018] Compared with the prior art, the present application has the following beneficial effects: the present application provides a molecular marker related to the antioxidant capacity of color sheep, the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, the molecular marker comprises a SNP site, the SNP site is located at the 240th position of the molecular marker, and the SNP site mutation base is C or T; through the correlation analysis of the genotype of the color sheep sample and the antioxidant stress index T-AOC content, the results show that the antioxidant stress index T-AOC of the color sheep individuals with genotypes CT and TT is significantly higher than that of the individuals with genotype CC, which indicates that the genotype of the SNP site of the color sheep is significantly related to the T-AOC content in the serum of the color sheep, and the SNP marker related to T-AOC of the color sheep; the molecular marker provided by the present application can be applied to the assisted breeding of color sheep, and the breeding efficiency is improved. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the agarose gel electrophoresis detection result of the PCR amplification product, wherein M is a 1000bp Marker, 1-3 are the electrophoresis bands corresponding to the samples of CC, CT and TT genotypes;

[0020] Figure 2 It is the peak chart and sequence obtained after the PCR product is purified and sequenced. DETAILED DESCRIPTION

[0021] The present application provides a molecular marker related to the antioxidant capacity of color sheep, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1, which is as follows:

[0022]

[0023]

[0024] In the application, the molecular marker comprises a SNP site, the SNP site is located at the 240th position of the molecular marker (i.e. the above-mentioned bold and underlined site), the SNP site is mutated with C or T; when the SNP site is C, the genotype is CC or CT, and when the SNP site is T, the genotype is TT.

[0025] The application further provides a primer pair for amplifying the molecular marker, comprising primer F and primer R; the nucleotide sequence of the primer F is shown as SEQ ID NO. 2, and the nucleotide sequence of the primer R is shown as SEQ ID NO. 3, and the specific sequence is as follows:

[0026] Primer F (SEQ ID NO. 2): 5'-TTCAAGGCTGTCTATACCAGA-3';

[0027] Primer R (SEQ ID NO. 3): 5'-CGCTGTTTACTTCTAGACCC-3'.

[0028] The application further provides application of the primer pair in preparation of a reagent for detecting the antioxidant capacity of Seva sheep.

[0029] The application provides application of the molecular marker in assisted breeding of Seva sheep.

[0030] In the application, the application preferably comprises the following steps:

[0031] 1) extracting genomic DNA of a Seva sheep to be selected for breeding, taking the genomic DNA of the Seva sheep as a template, and performing PCR amplification on the template with the primer pair to obtain an amplification product;

[0032] 2) sequencing the amplification product to determine the genotype of the SNP site, and selecting a Seva sheep to be selected for breeding with the genotype of TT or CT for subsequent breeding.

[0033] In the application, genomic DNA of a Seva sheep to be selected for breeding is first extracted, and the genomic DNA of a blood sample is preferably extracted, and the application does not have special limitations on the extraction method of the genomic DNA, and a conventional genomic DNA extraction method in the art can be used, and in the specific implementation process of the application, a blood genomic extraction kit of Beijing Quansiji Biological Technology Co., Ltd. is preferably used, the concentration of the genomic DNA is preferably > 20 ng / μL, and OD260 / OD280 is preferably between 1.7 and 1.9, and the genomic DNA is preferably stored at -20℃.

[0034] The application obtains the amplification product after obtaining the genomic DNA, and uses the genomic DNA of the color wool sheep as a template to perform PCR amplification with the primer pair. In the application, the amplification system of the PCR amplification is preferably 25 μL, preferably including 22 μL of PCR enzyme, 1 μL of primer F1, 1 μL of primer R1, and 1 μL of template DNA. The amplification procedure of the PCR amplification is preferably as follows: 98 ℃ for 2 min; 98 ℃ for 10 s, 57 ℃ for 10 s, 72 ℃ for 10 s, for a total of 40 cycles; and 72 ℃ for 2 min.

[0035] The application determines the genotype of the SNP site after obtaining the amplification product, and selects the color wool sheep to be selected for breeding with the genotype of TT or CT for subsequent breeding. In the application, the amplification product is preferably purified before sequencing, and the purification method is not particularly limited in the application, and a conventional purification method in the art can be used. In the application, the sequencing is preferably performed by using a direct sequencing method, and the sequencing is preferably completed by Beijing Qikexing Biological Technology Co., Ltd.

[0036] The application determines the genotype of the SNP site after sequencing, preferably uses the biological analysis software MEGA6.0 to compare the sequencing results of the PCR product, analyzes the sequencing peak graph, completes typing, and determines the genotype of the SNP site, and then selects the color wool sheep to be selected for breeding with the genotype of TT or CT for subsequent breeding.

[0037] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations on the protection scope of the application.

[0038] Example 1

[0039] 1 Sample collection

[0040] The sample is from a color wool sheep group under natural grazing conditions in Bangong County Maqian Township Color Wool Sheep Characteristic Breeding Professional Cooperative, 187 blood samples of 5 mL of color wool sheep on an empty stomach are collected in a clean coagulation vacuum blood collection tube, and then centrifuged at 3500 r / min for 15 min, and the supernatant is absorbed in a clean PE tube (serum to be tested), sealed and stored in a-20℃ low-temperature refrigerator for T-AOC detection in serum. Another 5 mL of blood sample is collected in a blood collection tube with EDTA-K2 anticoagulant, and the blood sample is mixed quickly after collection, and is temporarily stored in a sampling box containing an ice bag, and is stored in a-20℃ refrigerator after being transported back to the laboratory, and is used for DNA extraction.

[0041] 2 Main reagents and instruments

[0042] The EDTA-K2 vacuum blood collection tube is purchased from Jiangsu Yuli Medical Instrument Co., Ltd.

[0043] Blood genomic extraction kit, DL1000 Marker, agarose, nucleic acid dye, PCR enzyme were purchased from Beijing Quansijing Biotechnology Co., Ltd.

[0044] NanoDrop2000 spectrophotometer was purchased from Thermo Fisher Scientific, USA.

[0045] Electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory.

[0046] PCR instrument was purchased from BioRad Company.

[0047] T-AOC(A015-2-1) detection kit was purchased from Nanjing Jiancheng Biological Engineering Institute.

[0048] 3 Method

[0049] 3.1 Detection of T-AOC in serum

[0050] The T-AOC in serum was determined according to the detection kit of Nanjing Jiancheng Biological Engineering Institute. The determination steps were as follows: (1) 10 μL of double distilled water and 20 μL of reagent four application liquid (reagent one mixed with peroxidase at 9:1) were added to the blank tube, 10 μL of different concentrations of MTrolox solution and 20 μL of reagent four application liquid were added to the standard tube, and 10 μL of the serum to be tested and 20 μL of reagent four application liquid were added to the determination tube; (2) 170 μL of ABTS working solution (reagent one: reagent two: reagent three application liquid = 76:5:4) was added to the blank tube, standard tube and determination tube; (3) reaction at room temperature for 6 min, OD value of each well was read at 405 nm wavelength using enzyme label instrument; (4) standard curve was made with standard OD and curve formula was obtained, OD of sample determination tube was substituted into the calculation formula to obtain the result.

[0051] 3.2 Extraction of genomic DNA in blood

[0052] The blood genomic extraction kit of Beijing Quansijing Biotechnology Co., Ltd. was used to extract genomic DNA from blood samples. The extracted DNA was detected for concentration and purity under ultraviolet spectrophotometer. The concentration > 20 ng / μL and OD260 / OD280 between 1.7-1.9 met the experimental requirements, and was stored at -20℃ for standby.

[0053] 3.3 Primer design

[0054] Referring to the sequence of chromosome 2 of international sheep genome Oar_v4.0 version (GenBank accession number: NC_019459.2), a pair of specific primers containing g136628517C>T SNP site was designed by primer premier 5.0 software.

[0055] Primer sequence:

[0056] Primer F: 5'-TTCAAGGCTGTCTATACCAGA-3';

[0057] Primer R: 5'-CGCTGTTTACTTCTAGACCC-3'.

[0058] The length of the amplified fragment was 498 bp, and Beijing Encke Biotechnology Co., Ltd. was commissioned to sequence the amplified fragment, and the primer was synthesized by Beijing Encke Biotechnology Co., Ltd.

[0059] 3.4 PCR amplification and sequencing

[0060] The PCR amplification system was 25 μL: PCR enzyme 22 μL, primer F and primer R each 1 μL, template DNA 1 μL.

[0061] PCR amplification program: 98℃ 2min; 98℃ 10s, 57℃ 10s, 72℃ 10s, a total of 40 cycles; 72℃ extension 2min.

[0062] The PCR product was detected by 1.5% agarose gel electrophoresis, and the PCR product was detected by agarose gel electrophoresis. After the agarose gel electrophoresis detection was qualified, the direct sequencing method was used for sequencing, and the sequencing was completed by Beijing Encke Biotechnology Co., Ltd. The amplified nucleotide sequence is shown in SEQ ID No. 1, and the SNP marker is located at position 240 of the nucleotide sequence shown in SEQ ID No. 1:

[0063]

[0064] The PCR product sequencing results were compared and analyzed by using bioanalysis software MEGA 6.0, and the peak map was analyzed to complete the typing.

[0065] 4 Statistical analysis

[0066] According to the genotyping results, the number of individuals of different genotypes at each locus was counted. The Popgen32 software was used to calculate the gene frequency, genotype frequency, effective allele number (Ne), locus heterozygosity (He), and Hardy-Weinberg balance test of g136628517C>T. The polymorphism information content (PIC) calculation software was used to calculate the polymorphism information content. The general linear model in IBM SPSS Statistics 22 software was used to analyze the correlation between different genotypes of Sora sheep and the antioxidant stress index T-AOC, and the results were expressed as "mean ± standard error".

[0067] 5 Results

[0068] 5.1 PCR amplification and sequencing results

[0069] The amplified products of the g136628517C>T SNP site of the No.2 chromosome of Seva sheep were detected by 1.5% agarose gel (see Figure 1). Figure 1 The bands were clear and specific, and the size of the PCR product was 498 bp, which was consistent with the expected size, so the next step experiment could be carried out.

[0070] The peak chart and sequence obtained after purification and sequencing of the PCR product are shown in Figure 2. Figure 2 As shown in Figure 2, C-T mutation occurred at the g136628517C>T SNP site, and there were three genotypes of CC, CT and TT. Figure 2

[0071] 2.2 Statistical analysis results

[0072] The genotype and allele frequency of the g136628517C>T SNP site of the No.2 chromosome of Seva sheep were analyzed from the perspective of population genetics. As shown in Table 1, the CC genotype frequency was the highest at the g136628517C>T SNP site, which was the dominant genotype, and the C allele frequency was 89.0%, which was the dominant allele. The χ2 fitness test showed that the SNP site significantly deviated from the Hardy-Weinberg equilibrium state (P<0.05) (Table 1). The expected heterozygosity of the site was 0.195, and the PIC was 0.176, which belonged to low polymorphism (PIC<0.25).

[0073] Table 1 Polymorphism of the g136628517C>T SNP site of the No.2 chromosome of Seva sheep

[0074]

[0075] 2.3 Association analysis of different genotypes and antioxidant stress index T-AOC

[0076] The association between different genotypes of Seva sheep and the content of antioxidant stress index T-AOC was analyzed by general linear model in IBM SPSS Statistics 22 software, and the results showed that the antioxidant stress index T-AOC of Seva sheep individuals with CT and TT genotypes was significantly higher than that of individuals with CC genotype (p<0.05), and there was no significant difference in the antioxidant stress index T-AOC of Seva sheep individuals with CT and TT genotypes (p>0.05), indicating that the base of the g136628517C>T SNP site of the No.2 chromosome of Seva sheep was significantly related to the content of T-AOC in the serum of Seva sheep, which was a SNP marker related to T-AOC of Seva sheep. The results are shown in Table 2.

[0077] ​Table 2 Correlation analysis between different genotypes and antioxidant stress index T-AOC

[0078]

[0079] Note: The same row of data marked with different lowercase letters indicates significant difference (p<0.05).

[0080] From the above examples, it can be seen that the SNP molecular marker of the present application is located at the 136628517th base of the 2nd chromosome of the international sheep reference genome Oar_v4.0 version; the variation type is C / T, named g136628517C>T, and there are three genotypes. When the 136628517th base of the 2nd chromosome is C, the genotype is CC or CT; when the 136628517th base of the 2nd chromosome is T, the genotype is TT. Through correlation analysis of different genotypes and antioxidant stress index T-AOC content, it is found that the antioxidant stress index T-AOC of the Sowa sheep individual of the CT and TT genotypes is significantly higher than that of the CC genotype individual (p<0.05), and the antioxidant stress index T-AOC of the Sowa sheep individual of the CT and TT genotypes is not significantly different (p>0.05). By detecting the base of the 136628517th nucleotide site of the 2nd chromosome of the Sowa sheep, the antioxidant stress index T-AOC content of the Sowa sheep individual can be determined, which provides a new SNP molecular marker resource for marker-assisted selection of Sowa sheep antioxidant stress traits for non-diagnostic purposes.

[0081] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. The use of primer pairs for amplifying molecular markers related to the antioxidant capacity of Sewa sheep in the preparation of reagents for detecting the antioxidant capacity of Sewa sheep, characterized by, The antioxidant capacity of the Seda sheep is evaluated by the antioxidant stress index T-AOC in serum. The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1; the molecular marker comprises a SNP site, the SNP site is located at the 240th position of the molecular marker, and the SNP site mutation base is C or T; When the SNP site is C, the genotype is CC or CT, and when the SNP site is T, the genotype is TT. The T-AOC in serum of Seda sheep individuals with genotypes CT and TT is significantly higher than that of individuals with genotype CC. The primer pair for amplifying the molecular marker related to the antioxidant capacity of Seda sheep comprises primer F and primer R; the nucleotide sequence of the primer F is shown as SEQ ID NO. 2, and the nucleotide sequence of the primer R is shown as SEQ ID NO.

3.

2. A method of assisted breeding of color plate sheep with high antioxidant capacity, characterized by, The method comprises the following steps: 1) Extracting the genomic DNA of the selected Seda sheep, using the genomic DNA of the Seda sheep as a template, and performing PCR amplification on the molecular marker in the application by using the primer pair to obtain an amplification product; The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1; the molecular marker comprises a SNP site, the SNP site is located at the 240th position of the molecular marker, and the SNP site mutation base is C or T; When the SNP site is C, the genotype is CC or CT, and when the SNP site is T, the genotype is TT. 2) Sequencing the amplification product to determine the genotype of the SNP site, and selecting the selected Seda sheep with genotypes TT or CT for subsequent breeding; the T-AOC in serum of Seda sheep individuals with genotypes CT and TT is significantly higher than that of individuals with genotype CC.

3. The color panel sheep assisted breeding method according to claim 2, characterized in that, The amplification procedure of the PCR amplification is as follows: 98℃ for 2 min; 98℃ for 10 s, 57℃ for 10 s, 72℃ for 10 s, a total of 40 cycles; and 72℃ for 2 min for extension.

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