Molecular marker related to oxidation resistance of colored tile sheep as well as detection primer and application of molecular marker

Abstract

The invention provides a molecular marker related to oxidation resistance of colored tile sheep as well as a detection primer and application of the molecular marker, and belongs to the technical field of sheep molecular detection. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1; through correlation analysis of the genotype of a colored tile sheep sample and the content of an anti-oxidative stress index T-AOC, it is found that the anti-oxidative stress index T-AOC of colored tile sheep individuals with genotypes of CT and TT is significantly higher than that of colored tile sheep individuals with genotypes of CC, which indicates that the genotype of the SNP site of the colored tile sheep is significantly related to the content of T-AOC in colored tile sheep serum. The SNP marker is a T-AOC related SNP marker of the colored tile sheep; the molecular marker provided by the invention can be applied to assistant breeding of the colored tile sheep, and the breeding efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of sheep molecular detection, and particularly relates to a molecular marker related to the antioxidant capacity of Sewa sheep, as well as its detection primers and applications. Background Art

[0002] In modern sheep breeding, the antioxidant capacity of sheep has a crucial impact on their 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] As a local sheep breed in China, Sewa sheep are mainly distributed in the high-altitude areas of Bangor County, Nagqu Prefecture, on the northern Tibetan Plateau. The environment in this area is complex and changeable, with cold climate, strong ultraviolet radiation, and relatively scarce feed resources. These factors have long exposed Sewa sheep to severe oxidative stress challenges. In such an environment, the antioxidant capacity of Sewa sheep itself is particularly crucial for its survival and reproduction. Sewa sheep individuals with strong antioxidant capacity can better maintain their health status in a harsh environment and exhibit good production performance, which is of great significance for the sustainable development of local sheep breeding.

[0004] Currently, in the field of sheep breeding, in order to breed sheep breeds with excellent antioxidant capacity, researchers and breeders usually adopt traditional phenotypic selection methods, that is, indirectly judge their antioxidant capacity by observing the appearance characteristics, growth performance, and environmental adaptation performance of sheep. However, this method has obvious limitations. On the one hand, antioxidant capacity is a complex physiological trait, jointly regulated by multiple genes and environmental factors. It is difficult to accurately evaluate solely based on phenotypic observation, which is prone to selection errors. On the other hand, traditional phenotypic selection requires a large amount of time and effort, and it is necessary to wait for the sheep to grow to a certain stage before observation and judgment can be made. The breeding cycle is long and the efficiency is low.

[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. By detecting molecular markers related to target traits, accurate genetic evaluation of sheep individuals can be carried out at an early stage, greatly improving the breeding efficiency. However, regarding the specific molecular markers related to the antioxidant capacity of Sewa sheep, current research is still relatively scarce. Some existing general molecular markers are difficult to accurately establish a close connection with antioxidant capacity in the specific breed of Sewa sheep due to its 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 its adaptability to harsh environments, and improving production performance. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an SNP molecular marker related to the antioxidant capacity of Sewa sheep, its detection primers and applications. By detecting this SNP molecular marker in Sewa sheep, the antioxidant capacity of Sewa sheep can be quickly and accurately evaluated, and it can assist in the efficient breeding of Sewa sheep.

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

[0008] Preferably, the molecular marker includes an SNP site, the SNP site is located at the 240th position of the molecular marker, and the mutated base of the SNP site 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 invention provides a primer pair for amplifying the molecular marker, including primer F and primer R; the nucleotide sequence of primer F is as shown in SEQ ID NO.2, and the nucleotide sequence of primer R is as shown in SEQ ID NO.3.

[0011] The present invention provides the application of the primer pair in the preparation of reagents for detecting the antioxidant capacity of Sewa sheep.

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

[0013] Preferably, it includes the following steps:

[0014] 1) Extract the genomic DNA of the selected Sewa sheep. Using the genomic DNA of Sewa sheep as a template, perform PCR amplification with the primer pairs described above to obtain an amplification product;

[0015] 2) Sequence the amplification product to determine the genotype of the SNP locus, and select the Sewa sheep to be bred with the genotype TT or CT for subsequent breeding.

[0016] Preferably, the amplification system for the PCR amplification is 25 μL in total, including 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R; and 1 μL of template DNA.

[0017] Preferably, the amplification program for the PCR amplification is as follows: 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.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a molecular marker related to the antioxidant capacity of Sewa sheep. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO.1. The molecular marker includes an SNP locus, which is located at the 240th position of the molecular marker. The mutated base of the SNP locus is C or T. Through the correlation analysis of the genotype of the Sewa sheep sample and the content of the antioxidant stress index T-AOC, the results show that the antioxidant stress index T-AOC of the Sewa sheep individuals with the genotypes CT and TT is significantly higher than that of the individuals with the genotype CC, indicating that the genotype of the SNP locus of Sewa sheep is significantly correlated with the content of T-AOC in the serum of Sewa sheep, and it is an SNP marker related to T-AOC of Sewa sheep. The molecular marker provided by the present invention can be applied to the assisted breeding of Sewa sheep to improve the breeding efficiency. Description of the Drawings

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

[0020] Figure 2 It is the peak map and sequence obtained after the PCR product is purified and sequenced. Detailed Embodiments

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

[0022]

[0023]

[0024] In the present invention, the molecular marker includes an SNP site, the SNP site is located at the 240th position of the molecular marker (i.e., the above-bolded and underlined site), and the mutated base of the SNP site 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.

[0025] The present invention also provides a primer pair for amplifying the molecular marker, including primer F and primer R; the nucleotide sequence of primer F is as shown in SEQ ID NO.2, and the nucleotide sequence of primer R is as shown in SEQ ID NO.3, specifically as follows:

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

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

[0028] The present invention also provides the application of the primer pair in the preparation of a reagent for detecting the antioxidant capacity of Se'ewa sheep.

[0029] The present invention provides the application of the molecular marker in the assisted breeding of Se'ewa sheep.

[0030] In the present invention, the application preferably includes the following steps:

[0031] 1) Extract the genomic DNA of the Se'ewa sheep to be bred, use the genomic DNA of the Se'ewa sheep as a template, and perform PCR amplification with the primer pair to obtain an amplification product;

[0032] 2) Sequence the amplification product, determine the genotype of the SNP site, and select the Se'ewa sheep to be bred with the genotype TT or CT for subsequent breeding.

[0033] In the present invention, first extract the genomic DNA of the Se'ewa sheep to be bred, preferably extract the genomic DNA of a blood sample. The present invention has no special limitation on the extraction method of the genomic DNA, and a conventional genomic DNA extraction method in the art can be used. In the specific implementation process of the present invention, it is preferably carried out using the blood genomic extraction kit of Beijing TransGen Biotech Co., Ltd. The concentration of the genomic DNA is preferably >20 ng / μL, the OD260 / OD280 is preferably between 1.7 and 1.9, and the genomic DNA is preferably stored at -20°C.

[0034] After obtaining the genomic DNA, the genomic DNA of Sewa sheep was used as a template, and PCR amplification was performed with the primer pairs described above to obtain an amplification product. In the present invention, the amplification system for the PCR amplification is 25 μL, preferably including 2.2 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, 1 μL of template DNA; the amplification program for the PCR amplification is preferably as follows: 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; 72 °C for extension for 2 min.

[0035] After obtaining the amplification product, the amplification product was sequenced to determine the genotype of the SNP locus, and Sewa sheep to be selected and bred with a genotype of TT or CT were selected for subsequent breeding. In the present invention, the amplification product is preferably purified before sequencing. The present invention has no special limitation on the purification method, and a conventional purification method in the art can be used. In the present invention, the sequencing is preferably performed by direct sequencing, and the sequencing is preferably entrusted to Beijing Qingke Biotechnology Co., Ltd. to complete.

[0036] After the sequencing is completed in the present invention, the genotype of the SNP locus is determined. Preferably, the sequencing results of the PCR product are compared using the biological analysis software MEGA6.0, the sequencing peak map is analyzed, typing is completed, and the genotype of the SNP locus is determined; then Sewa sheep to be selected and bred with a genotype of TT or CT are selected for subsequent breeding.

[0037] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] 1 Sample collection

[0040] The samples were from the Sewa sheep population under natural grazing conditions in the Sewa Sheep Special Breeding Professional Cooperative in Maqian Township, Bange County. 5 mL of fasting Sewa sheep blood samples were collected in clean coagulation-promoting vacuum blood collection tubes, left standing for 30 min, then centrifuged at 3500 r / min for 15 min, and the supernatant was aspirated into a clean PE tube (serum to be tested), sealed and stored in a -20 °C low-temperature refrigerator for the detection of T-AOC in the serum. Another 5 mL of blood samples were collected in blood collection tubes added with EDTA-K2 anticoagulant, quickly mixed after the blood sample collection was completed, placed in a sampling box containing ice packs for temporary storage, and stored frozen in a -20 °C refrigerator after being transported back to the laboratory for DNA extraction.

[0041] 2 Main reagents and instruments

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

[0043] The blood genomic DNA extraction kit, DL1000 Marker, agarose, nucleic acid dye, and PCR enzyme were all purchased from Beijing TransGen Biotech Co., Ltd.;

[0044] The NanoDrop2000 spectrophotometer was from Thermo Fisher Scientific, USA;

[0045] The electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory;

[0046] The PCR instrument was purchased from BioRad.

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

[0048] 3 Methods

[0049] 3.1 Detection of T-AOC in Serum

[0050] The T-AOC in serum was determined according to the detection kit from Nanjing Jiancheng Bioengineering Institute. The determination steps were as follows: (1) Add 10 μL of double-distilled water and 20 μL of reagent four application solution (reagent one and peroxidase were mixed at 9:1) to the blank tube, add 10 μL of MTrolox solution with different concentrations and 20 μL of reagent four application solution to the standard tube, and add 10 μL of the serum to be tested and 20 μL of reagent four application solution to the determination tube; (2) Add 170 μL of ABTS working solution (reagent one: reagent two: reagent three application solution = 76:5:4) to the blank tube, standard tube, and determination tube; (3) React at room temperature for 6 min, and use an enzyme-linked immunosorbent assay reader to read the OD value of each well at a wavelength of 405 nm; (4) Make a standard curve with the OD of the standard product and obtain the curve formula, and substitute the OD measured in the sample determination tube into the calculation formula to obtain the result.

[0051] 3.2 Extraction of Genomic DNA from Blood

[0052] The blood genomic DNA extraction kit from Beijing TransGen Biotech Co., Ltd. was used to extract genomic DNA from the blood sample. The extracted DNA was placed under an ultraviolet spectrophotometer to detect the concentration and purity. When the concentration > 20 ng / μL and OD260 / OD280 was between 1.7 - 1.9, it met the experimental requirements and was stored at -20 °C for standby.

[0053] 3.3 Primer Design

[0054] Referring to the gene sequence of chromosome 2 in the international sheep genome Oar_v4.0 version (GenBank accession number: NC_019459.2), a pair of specific primers containing the g136628517C>T SNP locus was designed using the primer premier5.0 software.

[0055] Primer sequences:

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

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

[0058] The amplified fragment length is 498 bp. Beijing Tsingke Biotechnology Co., Ltd. was commissioned to sequence the amplified fragment, and the primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0059] 3.4 PCR amplification and sequencing

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

[0061] 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; 72°C for extension for 2 min.

[0062] The PCR products were detected by 1.5% agarose gel electrophoresis. After the PCR products passed the agarose gel electrophoresis test, direct sequencing was used for sequencing, which was completed by Beijing Tsingke 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 sequencing results of the PCR products were aligned using the biological analysis software MEGA 6.0, and the sequencing peak maps were analyzed to complete genotyping.

[0065] 4 Statistical analysis

[0066] According to the gene typing results, the number of individuals with different genotypes at each locus was counted. The Popgen32 software was used to calculate the gene frequency, genotype frequency, effective number of alleles (Ne), locus heterozygosity (He), and Hardy-Weinberg equilibrium test of the g136628517C>T gene, and the PIC (polymorphism information content) 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 association between different genotypes of Semenov 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 amplification products of the g136628517C>T SNP locus on chromosome 2 of Se'ewa sheep were detected using 1.5% agarose gel (see Figure 1 ). The bands were clear without heterozygous bands, with good specificity. The size of the PCR product fragment was 498 bp, which was consistent with the expected size, and the next experiment could be carried out.

[0070] The peak maps and sequences obtained after purification and sequencing of the PCR products are shown in Figure 2 . It can be seen from Figure 2 that a C-T mutation occurred at the g136628517C>T SNP locus, and there were three genotypes: CC, CT, and TT.

[0071] 2.2 Statistical analysis results

[0072] The genotypes and allele frequencies of the g136628517C>T SNP locus on chromosome 2 of Se'ewa sheep were analyzed from the perspective of population genetics. As shown in Table 1, at the g136628517C>T SNP locus, the CC genotype frequency was the highest, being the dominant genotype, and the C allele frequency was 89.0%, showing the dominant allele. The χ2 goodness-of-fit test indicated that the SNP locus significantly deviated from the Hardy-Weinberg equilibrium state (P<0.05) (Table 1). The expected heterozygosity of this locus was 0.195, and the PIC was 0.176. PIC<0.25 belongs to low polymorphism.

[0073] Table 1 Polymorphism of the g136628517C>T SNP locus on chromosome 2 of Se'ewa sheep

[0074]

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

[0076] The general linear model in IBM SPSS Statistics 22 software was used to analyze the association between different genotypes of Se'ewa sheep and the content of antioxidant stress index T-AOC. The results showed that the antioxidant stress index T-AOC of Se'ewa sheep individuals with CT and TT genotypes was significantly higher than that of CC genotype individuals (p<0.05), and there was no significant difference in the antioxidant stress index T-AOC between Se'ewa sheep individuals with CT and TT genotypes (p>0.05). This indicated that the base at the g136628517C>T SNP locus on chromosome 2 of Se'ewa sheep was significantly correlated with the content of T-AOC in the serum of Se'ewa sheep, and it was a SNP marker related to T-AOC in Se'ewa 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: Different lowercase letters marked in the same row of data indicate significant differences (p < 0.05).

[0080] As can be seen from the above embodiments, the SNP molecular marker of the present invention is located at the 136,628,517th base on chromosome 2 of the international sheep reference genome Oar_v4.0 version; the mutation type is C / T, named g136628517C>T, and there are three genotypes. When the 136,628,517th base on chromosome 2 is C, the genotype is CC or CT; when the 136,628,517th base on chromosome 2 is T, the genotype is TT; through the correlation analysis between different genotypes and the content of antioxidant stress index T-AOC, it is found that the antioxidant stress index T-AOC of the Sewa sheep individuals with CT and TT genotypes is significantly higher than that of the CC genotype individuals (p < 0.05), and there is no significant difference in the antioxidant stress index T-AOC between the Sewa sheep individuals with CT and TT genotypes (p > 0.05). By detecting the base at the 136,628,517th nucleotide site on chromosome 2 of Sewa sheep, the content of antioxidant stress index T-AOC of Sewa sheep individuals can be judged, providing a new SNP molecular marker resource for marker-assisted selection of antioxidant stress traits of Sewa sheep for non-diagnostic purposes.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A molecular marker related to the antioxidant capacity of Sewa sheep, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.

1.

2. The molecular marker according to claim 1, characterized in that The molecular marker includes a SNP site, the SNP site is located at the 240th position of the molecular marker, and the mutant base of the SNP site is C or T.

3. The molecular marker according to claim 2, characterized in that When the SNP site is C, the genotype is CC or CT, and when the SNP site is T, the genotype is TT.

4. A primer pair for amplifying the molecular marker according to claim 1, characterized in that: It comprises primer F and primer R; the nucleotide sequence of primer F is shown as SEQ ID NO.2, and the nucleotide sequence of primer R is shown as SEQ ID NO.

3.

5. Use of the primer pair according to claim 4 in preparing a reagent for detecting the antioxidant capacity of Sewa sheep.

6. Use of the molecular marker described in claim 1 in assisted breeding of Sewa sheep.

7. The use according to claim 6, characterized in that: The following steps are involved: 1) extracting genomic DNA of Sewa sheep to be bred, using the genomic DNA of Sewa sheep as a template, and performing PCR amplification with the primer pair described in claim 4 to obtain an amplified product; 2) Sequencing the amplified product to determine the genotype of the SNP site, and selecting the Sewa sheep to be bred with a genotype of TT or CT for subsequent breeding.

8. The use according to claim 7, characterized in that: The amplification system of the PCR amplification is 25 μL, including 22 μL of PCR enzyme, 1 μL of primer F, 1 μL of primer R, and 1 μL of template DNA.

9. The use according to claim 8, characterized in that: The amplification program of the PCR amplification is as follows: 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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