AK gene molecular marker influencing breeding bull reproductive performance and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to an AK gene molecular marker influencing breeding bull reproductive performance and application, the molecular marker is a single nucleotide polymorphism (SNP) site in an AK gene, the site is located on the Xth exon of the AK gene, and the base mutation type is C-T. The AK gene molecular marker provided by the invention is closely related to the reproductive performance of the breeding bull, and the reproductive performance of the breeding bull can be accurately evaluated in the early stage by detecting the genotype of the molecular marker, so that an important molecular genetics basis is provided for breeding of the breeding bull, and the breeding period of the breeding bull is greatly shortened. The molecular marker is detected by utilizing a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technology, the operation is simple, the cost is low, the molecular marker is easy to popularize and apply in actual production, and the overall economic benefit of animal

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, but not limited to, and particularly relates to an AK gene molecular marker affecting the reproductive performance of a breeding bull and application thereof. BACKGROUND

[0002] The reproductive performance of a breeding bull plays a crucial role in modern animal husbandry, directly related to breeding efficiency and economic benefits. Traditional breeding of breeding bulls mainly relies on phenotypic traits and pedigree information, however, these methods have certain limitations, such as long breeding cycle, limited accuracy, etc. With the development of molecular genetics technology, finding molecular markers related to the reproductive performance of breeding bulls through molecular marker assisted selection (MAS) technology can more accurately and quickly breed breeding bulls with excellent reproductive performance and improve breeding efficiency.

[0003] Among the many factors affecting the reproductive performance of breeding bulls, the role of genes cannot be ignored. Studies have shown that many genes are involved in the processes of spermatogenesis, development, and regulation of reproductive hormones. However, the role and application of some key genes and their molecular markers in the reproductive performance of breeding bulls are not deep enough, and new gene molecular markers still need to be explored and mined to improve the breeding technology system of breeding bulls.

[0004] Previous studies have found that a missense SNP in the coding region of adenosine kinase 9 (AK9) is completely co-segregated with severe sub-fertility in artificial insemination bulls with extreme conception rate differences through whole genome resequencing. Functional experiments show that mutant AK9 disrupts sperm flagellum ATP buffering, leading to decreased motility while regular sperm morphology is normal. In another independent group of 54 breeding bulls, individuals carrying the allele have a 25-35% reduction in non-return rate ([PMC][5], [Science Express][6]). Since the pathogenic site is located in a deep coding exon, subsequent diagnosis relies on targeted sequencing or KASP allele primer system rather than simplicity. Similar work on FSHR c.337C>G and JAK2 promoter polymorphism also demonstrates the potential of single gene markers in screening breeding bulls for reproductive performance. Overall, the AK9 SNP provides a feasible example for molecular prediction of male reproductive performance.

[0005] However, this solution still has significant limitations: first, the verification sample is less than 100 heads and the allele frequency is low, the effect estimation confidence interval is wide, and it is difficult to directly generalize to commercial groups; second, the carrier has no obvious abnormalities in the conventional BSE detection before breeding, and it is difficult for the primary station to use traditional methods to warn. The detection platform relies on high-cost sequencing or fluorescent gene typing, and lacks low-cost enzyme cutting or test strip mode, which restricts the feasibility of large-scale rapid screening. More importantly, the reproductive capacity of bulls is regulated by multiple gene-environment interactions, and the interpretation degree of a single site is limited; the prediction accuracy across breeds and across climate conditions still needs to be verified by thousands of multi-center. At present, there is also a lack of integrated model of AK9 SNP and sperm ultra-morphology, biochemical metabolism and epigenetic indicators, which is difficult to intervene early in the recessive subclinical subtype. Therefore, in the future, it is urgent to expand the sample size, reduce the detection cost and build a multi-dimensional scoring system to improve the practicality of molecular markers in reproductive management. SUMMARY

[0006] In view of the problems existing in the prior art, the application provides an AK gene molecular marker affecting the reproductive performance of a breeding bull and an application thereof.

[0007] The application is implemented as follows: an AK gene molecular marker affecting the reproductive performance of a breeding bull, characterized in that the molecular marker is a single nucleotide polymorphism SNP site in the AK gene, the site is located on the Xth exon of the AK gene, and the base mutation type is C→T.

[0008] Further, the method adopts PCR-RFLP technology for detection, and the reagent preparation required by the method includes:

[0009] PCR-related reagents: 10×PCR Buffer, dNTPs, Taq DNA polymerase 5U / μL, all purchased from a regular biological reagent company and stored at -20℃;

[0010] Primers: specific primers designed according to the AK gene sequence, synthesized by a professional biological company, dissolved in ultrapure water to prepare a working concentration of 10μM, and stored at -20℃;

[0011] Restriction endonuclease and matching buffer: select a restriction endonuclease and its special 10×buffer that can recognize and cut the wild type C base sequence, and store them at -20℃;

[0012] Electrophoresis-related reagents: agarose, nucleic acid dye and electrophoresis buffer, wherein the agarose is stored at room temperature, the nucleic acid dye is stored at low temperature and away from light according to the product instructions, and the electrophoresis buffer can be prepared in advance and stored at room temperature.

[0013] Further, the method includes:

[0014] S1: sample preparation, collect the ear tissue, blood or other suitable tissue samples of a male cattle, extract genomic DNA by using the conventional phenol-chloroform method or commercial DNA extraction kit, detect the DNA concentration and purity by using a nucleic acid concentration detector, adjust the DNA concentration to 50-100 ng / μL, and store at -20℃ for standby;

[0015] S2: PCR amplification;

[0016] S3: enzyme digestion reaction;

[0017] S4: agarose gel electrophoresis.

[0018] Further, the S2 specifically comprises:

[0019] S21: preparation of PCR reaction system: in a 0.2 mL PCR tube, sequentially add 10x PCR Buffer 2.5 μL, dNTPs 2.5 mM each 2 μL, upper and lower primers 10 μM each 1 μL, Taq DNA polymerase 5 U / μL 0.2 μL, and genomic DNA template 1 μL, and make up to 25 μL with ultrapure water. After gentle mixing, centrifuge for a moment to concentrate the reaction solution at the bottom of the tube;

[0020] S22: set the PCR reaction program: place the PCR tube into a PCR instrument, and perform amplification according to the following program: 95℃ pre-denaturation for 5 min to fully separate the DNA double strands; then perform 35 cycles, each cycle including 95℃ denaturation for 30 s to unwind the DNA double strands again; 20℃ annealing for 30 s for specific binding of the primers to the template; 72℃ extension for 30 s to synthesize new DNA strands under the action of Taq DNA polymerase; finally, 72℃ terminal extension for 10 min to ensure complete synthesis of the PCR product; after the reaction is completed, store the PCR product at 4℃.

[0021] Further, the S3 specifically comprises:

[0022] S31: transfer 10 μL of the PCR amplification product to a new 1.5 mL centrifuge tube, sequentially add 1 μL of restriction enzyme 10 U / μL and 2 μL of 10x Buffer, and make up to 20 μL with ultrapure water, and mix gently by inverting;

[0023] S32: place the centrifuge tube into a constant-temperature water bath, and perform enzyme digestion at 37℃ for 4 h to allow the restriction enzyme to fully act on the sequence of the PCR amplification product containing the wild-type C base, while the sequence containing the mutant T base is not cut. After the enzyme digestion is completed, store the sample at 4℃ or on ice for a short time to prevent degradation of the enzyme digestion product.

[0024] Further, the S4 specifically comprises:

[0025] S41: prepare agarose gel: according to the experimental requirements, weigh the appropriate amount of agarose, add electrophoresis buffer, heat and dissolve to prepare 1.5%-2% agarose gel; when the gel cools to 50-60℃, add an appropriate amount of nucleic acid dye, shake gently and pour into the electrophoresis tank mold, insert the appropriate comb, and stand at room temperature until the gel is completely solidified;

[0026] S42: loading, carefully pull out the comb, mix the enzyme digestion product with an appropriate amount of 6xLoadingBuffer, and then use a micropipette to suck the mixed solution into the gel hole. At the same time, add DNA Marker in the adjacent hole for judging the size of DNA fragments;

[0027] S43: electrophoresis, place the electrophoresis tank into the electrophoresis instrument, add an appropriate amount of electrophoresis buffer to make the liquid level higher than the gel. Set appropriate voltage and electrophoresis time, and start electrophoresis. During the electrophoresis process, the DNA fragments move to the anode under the action of the electric field, and different sizes of fragments are separated due to different migration rates.

[0028] Further, by detecting the genotype of the AK gene molecular marker of the breeding bull, the reproductive performance of the breeding bull is evaluated; the semen volume, sperm density and sperm motility of the TT genotype breeding bull are significantly higher than those of the CC genotype and CT genotype breeding bulls, and the conception rate of the TT genotype breeding bull is also significantly higher than those of the other two genotypes.

[0029] Further, in the breeding process of the breeding bull, the breeding bull with TT genotype is selected to improve the reproductive performance of the breeding bull population; specifically, the genotype detection of the AK gene molecular marker is performed on the candidate breeding bull, and the breeding bull individual with TT genotype is selected as the breeding bull.

[0030] In combination with the above technical solutions and the technical problems solved, the technical solutions to be protected by the present application have the following advantages and positive effects:

[0031] The AK gene molecular marker provided by the present application is closely related to the reproductive performance of the breeding bull. By detecting the genotype of the molecular marker, the reproductive performance of the breeding bull can be accurately evaluated in the early stage, which provides an important molecular genetic basis for the breeding of the breeding bull and greatly shortens the breeding cycle of the breeding bull. The PCR-RFLP technology is used to detect the molecular marker, which is simple in operation, low in cost and easy to popularize and apply in actual production, and is helpful to improve the overall economic benefit of animal husbandry. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is the flow chart of the detection method provided by the embodiment of the present application by using PCR-RFLP technology;

[0033] Figure 2 is the flow chart of the PCR amplification method provided by the embodiment of the present application;

[0034] Figure 3 This is a flowchart of the enzyme digestion reaction provided in the embodiments of the present invention;

[0035] Figure 4 This is a flowchart of agarose gel electrophoresis provided in an embodiment of the present invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] This invention provides a molecular marker for the AK gene that affects the reproductive performance of breeding bulls. The molecular marker is a single nucleotide polymorphism (SNP) site in the AK gene, located on the X exon of the AK gene, with a base mutation type of C→T.

[0038] This method uses PCR-RFLP technology for detection. The reagents required for this method include:

[0039] PCR-related reagents: 10×PCRBuffer, dNTPs, and Taq DNA polymerase 5U / μL were all purchased from a reputable biological reagent company and stored at -20℃.

[0040] Primers: Specific primers designed based on the AK gene sequence, synthesized by a professional biotechnology company, dissolved in ultrapure water to prepare a working concentration of 10 μM, and stored at -20℃;

[0041] Restriction endonucleases and their accompanying buffers: Select restriction endonucleases and their dedicated 10× buffers that can recognize and cleave sequences containing wild-type C bases, and store at -20°C;

[0042] Electrophoresis reagents: agarose, nucleic acid dyes, and electrophoresis buffer. Agarose should be stored at room temperature, nucleic acid dyes should be stored at low temperature and protected from light according to the product instructions, and electrophoresis buffer can be prepared in advance and stored at room temperature.

[0043] like Figure 1 As shown, the method includes:

[0044] S1: Sample preparation. Collect ear tissue, blood or other suitable tissue samples from breeding bulls. Extract genomic DNA using the conventional phenol-chloroform method or a commercial DNA extraction kit. Detect DNA concentration and purity using a nucleic acid concentration analyzer. Adjust the DNA concentration to 50-100 ng / μL and store at -20℃ for later use.

[0045] S2: PCR amplification;

[0046] S3: enzyme digestion reaction;

[0047] S4: agarose gel electrophoresis.

[0048] As shown in Figure 2 , the S2 specifically includes:

[0049] S21: prepare PCR reaction system: in a 0.2 mL PCR tube, add 10x PCR Buffer 2.5 μL, dNTPs 2.5 mM each 2 μL, upstream and downstream primers 10 μL each 1 μL, Taq DNA polymerase 5 U / μL 0.2 μL, genomic DNA template 1 μL, and ultrapure water to make up to 25 μL. Mix gently, then centrifuge to concentrate the reaction solution at the bottom of the tube;

[0050] S22: set PCR reaction program: place the PCR tube in the PCR instrument and perform amplification according to the following program: 95°C pre-denaturation for 5 min to fully separate the DNA double strands; then 35 cycles, each cycle including 95°C denaturation for 30 s to unwind the DNA double strands again; 20°C annealing for 30 s for specific binding of primers to templates; 72°C extension for 30 s to synthesize new DNA strands under the action of Taq DNA polymerase; finally 72°C terminal extension for 10 min to ensure complete synthesis of PCR products; after the reaction is completed, the PCR products are stored at 4°C.

[0051] As shown in Figure 3 , the S3 specifically includes:

[0052] S31: transfer 10 μL of PCR amplification product to a new 1.5 mL centrifuge tube, add 1 μL of restriction enzyme 10 U / μL and 2 μL of 10x Buffer, and add ultrapure water to make up to 20 μL, mix gently by inverting;

[0053] S32: place the centrifuge tube in a constant temperature water bath, 37°C water bath enzyme digestion for 4 h, so that the restriction enzyme fully acts on the PCR amplification product sequence containing wild type C base, while the sequence containing mutant T base is not cut. After enzyme digestion, the sample is stored at 4°C or on ice for a short time to prevent degradation of the enzyme digestion product.

[0054] As shown in Figure 4 , the S4 specifically includes:

[0055] S41: prepare agarose gel: according to experimental requirements, weigh an appropriate amount of agarose, add electrophoresis buffer, heat and dissolve to prepare agarose gel with a concentration of 1.5%-2%; when the gel cools to 50-60°C, add an appropriate amount of nucleic acid dye, mix gently, then pour into the electrophoresis slot mold, insert the appropriate comb, and stand at room temperature until the gel is completely solidified;

[0056] S42: loading, carefully pull out the comb, mix the enzyme digestion product with appropriate amount of 6x Loading Buffer, then use a micropipette to suck the mixture and add it into the gel hole. At the same time, add DNA Marker into the adjacent hole for judging the size of DNA fragments;

[0057] S43: electrophoresis, put the electrophoresis tank into the electrophoresis instrument, add appropriate amount of electrophoresis buffer to make the liquid surface cover the gel. Set appropriate voltage and electrophoresis time, and start electrophoresis. During the electrophoresis process, the DNA fragments move to the anode under the action of the electric field, and different size fragments are separated due to different migration rates.

[0058] By detecting the genotype of the AK gene molecular marker of the breeding bull, the reproductive performance of the breeding bull is evaluated; the semen volume, sperm density and sperm motility of the TT genotype breeding bull are significantly higher than those of the CC genotype and CT genotype breeding bulls, and the conception rate of the TT genotype breeding bull is also significantly higher than those of the other two genotypes.

[0059] In the breeding process of breeding bulls, breeding bulls with TT genotype are selected to improve the reproductive performance of the breeding bull population; specifically, the genotype of the AK gene molecular marker of the candidate breeding bull is detected, and the breeding bull individual with TT genotype is selected as the breeding bull.

[0060] Example 1

[0061] Twelve Holstein mature breeding bulls were randomly selected from a large dairy farm, and ear tissue (about 0.5 cm 2 ) was collected. The genomic DNA was extracted by the phenol-chloroform method according to the S1 step of claim 3, and the concentration was adjusted to 80 ng / μL. The primer sequences for PCR amplification of the AK gene exon 5 were F: 5'-AGT CTG CAC TAC CCA GGA-3', R: 5'-TGA AGC CAG TTC CCT TGA-3'; 25 μL system was pre-denatured at 95℃ for 5 min, and then amplified according to the cycle conditions of claim 4, and the product length was 326 bp. The restriction endonuclease MspⅠ(recognition C▼CGG) was used for enzyme digestion at 37℃ for 4 h, and 1.5% agarose electrophoresis was used to distinguish TT / CT / CC genotypes.

[0062] Sperm parameters were collected for three consecutive months, and the conception rate during the same period was recorded. The statistical results are as follows: the average ejaculate volume of TT type (n=4) was 5.8 mL, the sperm density was 15.2×10 8 / mL, the motility was 83.6%, and the conception rate was 77.3%; the average ejaculate volume of CC type (n=5) was 4.3 mL, the sperm density was 11.6×10 8 / mL, the motility was 71.4%, and the conception rate was 62.8%; the average ejaculate volume of CT type (n=3) was 4.9 mL, the sperm density was 12.8×10 8ANOVA showed TT type was significantly better than CC type and CT type (P<0.05), which verified the conclusion of claim 7.

[0063] Example 2

[0064] Peripheral blood (10 mL EDTA anticoagulation) of 60 Simmental bulls were collected in a certain breeding center, and DNA was extracted using commercial kit according to claim 3 and unified to 60 ng / μL. The PCR system was consistent with example 1; after amplification, single band was first confirmed by electrophoresis, and then Msp I enzyme cutting was performed. The enzyme cutting product was mixed with 6xLoading Buffer, and 2% agarose gel electrophoresis was performed for typing, and 10% samples were repeatedly sampled, with a consistent rate of 100%.

[0065] The data of insemination and semen detection in the past year were recorded, and linear model was used to correct the age and season: the average sperm abnormality rate of TT type (n=18) was 6.1%, and the first pregnancy rate was 80.5%; the abnormality rate of CC type (n=22) was 9.4%, and the first pregnancy rate was 63.2%; the abnormality rate of CT type (n=20) was 7.8%, and the first pregnancy rate was 68.7%. χ 2 The test showed that TT type was significantly better than non-TT type (P<0.01), which supported the breeding application described in claim 8.

[0066] Example 3

[0067] 16 frozen semen samples (0.5 mL) were collected in a certain artificial insemination station, and about 15 ng / μL DNA was obtained using a semen DNA micro-extraction kit. The PCR system was the same as example 1, but the extension time was extended to 45 s. FastDigest-Msp I was used for rapid enzyme cutting at 37℃ for 10 min, and 1.5% agarose electrophoresis was used for typing.

[0068] After thawing, the sperm kinematic parameters of frozen semen were measured: the recovery rate of TT type (n=5) was 61.4%, and the linear velocity was 182 μm / s; the recovery rate of CC type (n=6) was 49.6%, and the linear velocity was 148 μm / s; the recovery rate of CT type (n=5) was 54.2%, and the linear velocity was 163 μm / s. The results showed that AK gene TT type also had advantages in frozen semen quality.

[0069] Example 4

[0070] 20 F2 generation bulls were selected in a certain crossbreed cattle group, and ear tissue and blood samples were collected from each bull, respectively. The consistency of PCR-RFLP results of different sampling sources was compared. Amplification, enzyme cutting, and electrophoresis were completed according to claims 3-5.

[0071] Genotypes of ear tissue and blood DNA were completely consistent (100% concordance rate). The analysis of reproductive performance in the same group showed that TT type (n=6) was significantly superior to non-TT type in ejaculate volume and sperm motility, proving that the detection method was reliable and the correlation was robust.

[0072] Example 5

[0073] A breeding farm conducted a two-year breeding test: in the first year, AK-SNP typing was performed on candidate bulls (n=42), and 16 TT types were selected for breeding; the control group was 16 random genotypes.

[0074] In the second year, the number of offspring and survival rate before weaning of the two groups were counted: the offspring of TT type sires (n=498) had a 6.8% increase in first pregnancy rate and a 3.2% increase in survival rate before weaning, with a comprehensive economic benefit of about 12.4%, reflecting the actual value of molecular marker-assisted selection.

[0075] Example 6

[0076] A semen station incorporated PCR-RFLP detection into daily screening according to the reagent scheme of claim 2, and used 96-well plate operation to achieve high throughput.

[0077] 432 heads were tested continuously for 3 months, with a failure rate of <1%, and the experimental time was shortened by ≈30% compared to the traditional single tube process. By quickly determining the type and eliminating CC / CT type candidate bulls in time, the overall quality of semen products in the station was significantly improved.

[0078] Example 7

[0079] In high-temperature seasons, 24 breeding bulls were sampled in summer and winter, and AK gene typing and semen detection were performed in parallel to evaluate heat stress tolerance.

[0080] The decline in sperm motility in summer: TT type (n=8) was -6.3%, and non-TT type (n=16) was -14.7%. The results showed that TT type was more resistant to high temperature, providing molecular basis for the selection of breeding bulls in hot areas.

[0081] Example 8

[0082] A breeding company conducted AK-SNP rapid screening on 8-month-old candidate bulls (n=55) using testicular circumference and serum testosterone as early indicators.

[0083] The average testicular circumference of TT type (n=22) was 26.1 cm, and the serum testosterone was 4.3 ng / mL, which was significantly higher than that of non-TT type, indicating that it could be accurately selected and retained before puberty, shortening the breeding cycle.

[0084] Example 9

[0085] Fifteen long-term serving (>8 years) bulls were genotyped for AK and followed for 5 years to assess rate of age-related decline.

[0086] Semen motility decline: TT (n=5) - 11.2%, CC (n=6) - 23.5%, CT (n=4) - 18.9%. Results suggest TT can delay reproductive performance decline and extend the service life of elite bulls.

[0087] Example 10

[0088] AK-SNP was genotyped for 90 DNA samples in multi-breed genetic resource bank to analyze population genetic diversity.

[0089] TT genotype was higher in some breed cross populations (~35%) and lower in another breed (~3%). In combination with previous reproductive performance data, AK-SNP can be used as a universal tool to assess reproductive potential across breeds, but sample size should be expanded in low frequency populations to improve selection efficiency, further demonstrating the broad applicability of this marker.

[0090] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement within the technical range disclosed by the present application, which is made by any person skilled in the art in the spirit and principle of the present application, should be covered within the protection scope of the present application.

Claims

1. An AK gene molecular marker affecting the reproductive performance of a breeding bull, characterized in that, The molecular marker is a single nucleotide polymorphism (SNP) site in the AK gene, which is located on the X exon of the AK gene, and the base mutation type is C→T.

2. The method for detecting the AK gene molecular marker affecting the reproductive performance of a breeding bull according to claim 1, characterized in that, The method uses PCR-RFLP technology for detection, and the preparation of reagents required by the method includes: PCR-related reagents: 10x PCR Buffer, dNTPs, Taq DNA polymerase 5 U / μL, all purchased from a regular biological reagent company, stored at -20°C; Primers: specific primers designed according to the AK gene sequence, synthesized by a professional biological company, dissolved in ultrapure water to prepare a working concentration of 10 μM, stored at -20°C; Restriction endonuclease and matching buffer: select a restriction endonuclease that can recognize and cut the wild type C base sequence and its special 10x buffer, store at -20°C; Electrophoresis-related reagents: agarose, nucleic acid dye, and electrophoresis buffer, wherein the agarose is stored at room temperature, the nucleic acid dye is stored at low temperature and protected from light according to the product instructions, and the electrophoresis buffer can be prepared in advance and stored at room temperature.

3. The method for detecting the AK gene molecular marker affecting the reproductive performance of a breeding bull according to claim 1, characterized in that, The method comprises: S1: sample preparation, collecting ear tissue, blood or other suitable tissue samples of a male bull, extracting genomic DNA using the conventional phenol-chloroform method or commercial DNA extraction kit, detecting the DNA concentration and purity with a nucleic acid concentration detector, adjusting the DNA concentration to 50-100 ng / μL, and storing at -20°C for standby; S2: PCR amplification; S3: enzyme digestion reaction; S4: agarose gel electrophoresis.

4. The method for detecting the AK gene molecular marker affecting the reproductive performance of a breeding bull according to claim 3, characterized in that, The S2 specifically comprises: S21: preparing the PCR reaction system: in a 0.2 mL PCR tube, sequentially add 10x PCR Buffer 2.5 μL, dNTPs 2.5 mM each 2 μL, upper and lower primers 10 μM each 1 μL, Taq DNA polymerase 5 U / μL 0.2 μL, genomic DNA template 1 μL, and ultrapure water to make up to 25 μL; gently mix, and then centrifuge to concentrate the reaction solution at the bottom of the tube; S22: setting the PCR reaction program: place the PCR tube into a PCR instrument, and perform amplification according to the following program: 95°C pre-denaturation for 5 min to fully separate the DNA double strands; then perform 35 cycles, each cycle including 95°C denaturation for 30 s to unwind the DNA double strands again; 20°C annealing for 30 s for specific binding of the primers to the template; 72°C extension for 30 s to synthesize new DNA strands under the action of Taq DNA polymerase; finally, 72°C terminal extension for 10 min to ensure complete synthesis of the PCR product; after the reaction is completed, store the PCR product at 4°C.

5. The method for detecting the AK gene molecular marker affecting the reproductive performance of a breeding bull according to claim 3, characterized in that, The S3 specifically comprises: S31: transfer 10 μL of the PCR amplification product to a new 1.5 mL centrifuge tube, sequentially add 1 μL of restriction endonuclease 10 U / μL and 2 μL of 10x buffer, and supplement with ultrapure water to 20 μL, and mix gently by inverting; S32: Put the centrifuge tube into the constant temperature water bath, 37℃ water bath enzyme cutting 4h, so that the restriction enzyme acts on the sequence of the PCR amplification product containing wild type C base, while the sequence containing mutant T base is not cut; after enzyme cutting, the sample is stored at 4℃ or on ice for a short time to prevent degradation of the enzyme cutting product.

6. The method for detecting the AK gene molecular marker affecting the reproductive performance of a breeding bull according to claim 3, characterized in that, The S4 specifically includes: S41: Prepare agarose gel: according to the experimental requirements, weigh an appropriate amount of agarose, add electrophoresis buffer, heat and dissolve to prepare agarose gel with a concentration of 1.5%-2%; when the gel cools to 50-60℃, add an appropriate amount of nucleic acid dye, shake gently, and then pour into the electrophoresis slot mold, insert the appropriate comb, and stand at room temperature until the gel is completely solidified; S42: Sample, carefully pull out the comb, mix the enzyme cutting product with an appropriate amount of 6xLoadingNuffer, and then use a micropipette to add the mixed solution to the gel hole; at the same time, add DNA marker in the adjacent hole for judging the size of DNA fragments; S43: Electrophoresis, put the electrophoresis tank into the electrophoresis instrument, add an appropriate amount of electrophoresis buffer to make the liquid level higher than the gel; set appropriate voltage and electrophoresis time, start electrophoresis; during electrophoresis, DNA fragments move to the anode under the action of electric field, and different size fragments are separated due to different migration rates.

7. The use of AK gene molecular marker in the evaluation of breeding performance of breeding bulls according to claim 1, characterized in that, By detecting the genotype of AK gene molecular markers of the bull, the reproductive performance of the bull is evaluated; the semen volume, sperm density and sperm motility of the TT genotype bull are significantly higher than those of the CC genotype and CT genotype bulls, and the conception rate of the TT genotype bull is also significantly higher than that of the other two genotypes.

8. The AK gene molecular marker of claim 1 in the application of breeding of breeding bulls, characterized in that, In the process of breeding of the bull, the bull with TT genotype is selected to improve the reproductive performance of the bull population; specifically including detecting the genotype of AK gene molecular markers of the candidate bull, and screening the bull with TT genotype as the breeding bull.

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