SNP (Single Nucleotide Polymorphism) site for identifying freezing tolerance of Chinese Holstein bull sperms

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site for identifying the freezing tolerance of Chinese Holstein bull sperms. The SNP site is nucleotide at the 161st site from the 5'tail end of SEQ ID NO: 1 in a bull genome, the sperm freezing tolerance character of the Chinese Holstein bull can be screened or screened in an auxiliary mode by detecting the genotype of the SNP site in the bull genome to be detected, and the method is simple, convenient, rapid and sensitive, and the result is reliable, stable and accurate. The method is suitable for large-group scale detection and has important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SNP site for identifying the freezing resistance of sperm of Chinese Holstein bulls. Background Art

[0002] Dairy industry is an important part of animal husbandry. It is not only closely related to people's growing material needs, but also has important strategic significance for promoting farmers' and herdsmen's income. Holstein cows are the most important dairy cattle breed in the world. At present, artificial insemination with frozen semen is the main way of dairy cow breeding. With the development of frozen semen technology, the improvement of the quality of bull frozen semen has attracted much attention, especially the vitality of semen after thawing, which has an important economic impact on the conception rate of cows and dairy production systems. The contribution rate of bulls to the genetic improvement of dairy cows can reach more than 70%. Therefore, cultivating excellent bulls with excellent semen quality is the core work of dairy cow breeding. However, the sharp decline in sperm vitality after freezing and poor sperm freezing resistance are one of the main problems facing bull frozen semen production in my country. At present, there is a significant gap between the vitality of Holstein bull frozen semen in my country and that of some developed dairy countries abroad, and it is urgent to further improve it.

[0003] Researchers found that even under the same freezing procedure, the changes in semen from different individuals to cold stress were different. Although some bulls had good sperm vitality when fresh, their sperm vitality dropped significantly after freezing. There are obvious differences in the tolerance of sperm to cold stress in different bulls, which is obviously partly due to different genetic bases. Therefore, in recent years, many studies have used multi-omics and other technical means to explore the key genes and molecular markers that affect sperm freezing resistance at the genetic level, and applied them to breeding to accelerate the selection of bull semen freezing resistance traits.

[0004] Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is widely distributed, numerous, and has high genetic stability, making it easy to perform genotyping. By exploring the important functional genes and SNPs sites of Holstein bull sperm freezing resistance and conducting molecular marker-assisted selection for bull semen quality traits, the accuracy of selection can be further improved. At the same time, individuals can be eliminated through early selection to reduce breeding costs. Therefore, the identification of molecular markers related to bull sperm freezing resistance has important economic value for the selection and breeding of Holstein bull semen quality traits, and also provides an important theoretical basis for analyzing the genetic basis of bull semen freezing resistance traits and effectively carrying out molecular breeding of bull frozen sperm traits.

[0005] As one of the core subunits of the mitochondrial ATP synthase complex, ATP5F1A plays an important role in various biological processes such as cellular energy metabolism, mitochondrial autophagy, and cell survival. Studies have shown that when the mitochondrial membrane potential is reduced or the mitochondria are damaged, the degradation of ATP5F1A is inhibited. Further studies have found that PHB2 (prohibitin 2) affects mitochondrial autophagy by regulating the degradation of ATP5F1A, revealing the key role of ATP5F1A in mitochondrial autophagy. In addition, TNK2 / ACK1 kinase can directly phosphorylate ATP5F1A and increase mitochondrial energy output, indicating that ATP5F1A plays an important role in cellular energy metabolism and survival. Summary of the invention

[0006] The purpose of the present invention is to identify the freezing resistance of sperm of Holstein bulls (especially Chinese Holstein bulls). In this application, the freezing resistance of sperm of Chinese Holstein bulls refers to: producing frozen semen in accordance with the technical regulations for the production of frozen semen of cattle in the agricultural industry standard of the People's Republic of China NY / T 1234-2018, detecting sperm motility after thawing and making the following judgment: if the sperm motility after low temperature treatment is ≥0.40, the sperm of the Chinese Holstein bull to be tested is freezing resistant; if the sperm motility after low temperature treatment is ≤0.39, the sperm of the Chinese Holstein bull to be tested is not freezing resistant.

[0007] The present invention firstly protects a kit for identifying the freezing resistance of bull sperm, which may include a substance for detecting the genotype of the bull to be tested based on a SNP site; the SNP site is the 161st nucleotide from the 5' end of SEQ ID NO:1 in the bull genome.

[0008] The "kit for identifying the freezing resistance of bull sperm" may specifically be composed of a substance for detecting the genotype of the bull to be tested based on the SNP site.

[0009] Any of the above-mentioned “substances for detecting the genotype of the bull to be tested based on the SNP site” can specifically be a primer pair consisting of an upstream primer F and a downstream primer R;

[0010] The upstream primer F may be a single-stranded DNA molecule shown in SEQ ID NO: 2;

[0011] The downstream primer R may be a single-stranded DNA molecule shown in SEQ ID NO:3.

[0012] Any of the above-mentioned substances for detecting the genotype of the bull to be tested based on the SNP site can also be used to determine the nucleotide type of the SNP site in the bull genome by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction and / or a chip based on fluorescent molecule DNA binding reaction.

[0013] The present invention also protects the molecular marker shown in SEQ ID NO:1.

[0014] The use of any of the above-mentioned kits or any of the above-mentioned molecular markers in identifying the freezing resistance of bull sperm also falls within the protection scope of the present invention.

[0015] The use of any of the above-mentioned kits or any of the above-mentioned molecular markers in screening bulls with different sperm freezing resistance also falls within the protection scope of the present invention.

[0016] The use of any of the above-mentioned kits or any of the above-mentioned molecular markers in identifying bull genotypes also falls within the protection scope of the present invention.

[0017] The application of any of the above-mentioned kits or any of the above-mentioned molecular markers in bull breeding also belongs to the protection scope of the present invention. The goal of bull breeding is to cultivate bull breeds with sperm having freeze-resistance.

[0018] The present invention also protects a method for identifying the freezing resistance of bull sperm.

[0019] The method for identifying the freezing resistance of bull sperm protected by the present invention may specifically be method 1, which may include the following steps: detecting whether the genotype of the bull to be tested based on the SNP site is CC homozygous or TT homozygous, and then making the following judgment: the freezing resistance of the sperm of the bull whose genotype based on the SNP site is CC homozygous> the freezing resistance of the sperm of the bull whose genotype based on the SNP site is TT homozygous;

[0020] The SNP site is the 161st nucleotide from the 5' end of SEQ ID NO: 1 in the bull genome.

[0021] The method for identifying the freezing resistance of bull sperm protected by the present invention may specifically be method 2, which may include the following steps:

[0022] (1) Using the genomic DNA of the bull to be tested as a template, a primer pair consisting of an upstream primer F and a downstream primer R is used for PCR amplification to obtain a PCR amplification product; sequencing the PCR amplification product, and then making the following judgment: if the 161st position of the PCR amplification product is only C, the genotype of the bull to be tested based on the SNP site is CC homozygous; if the 161st position of the PCR amplification product is only T, the genotype of the bull to be tested based on the SNP site is TT homozygous;

[0023] (2) Based on the result of (1), the following judgment is made: the freezing resistance of sperm of bulls with CC homozygous genotype based on the SNP locus is greater than the freezing resistance of sperm of bulls with TT homozygous genotype based on the SNP locus;

[0024] The SNP site is the 161st nucleotide from the 5' end of SEQ ID NO:1 in the bull genome;

[0025] The upstream primer F may be a single-stranded DNA molecule shown in SEQ ID NO: 2;

[0026] The downstream primer R may be a single-stranded DNA molecule shown in SEQ ID NO:3.

[0027] Any of the bulls described above may be Holstein bulls. The Holstein bulls described above may be Chinese Holstein bulls.

[0028] In the above, the > may specifically be a statistical >.

[0029] Experiments have shown that the method provided by the present invention can be used to detect the genotype of the bull to be tested based on the SNP site, and can screen or assist in screening the sperm freezing resistance trait of Chinese Holstein bulls. It is not only simple, rapid, and sensitive, but also the results are reliable, stable, and accurate. The present invention is suitable for large-scale group detection and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the differential expression of ATP5F1A protein in step five of Example 1 in the high sperm cryoresistant group and the low sperm cryoresistant group of Chinese Holstein bulls.

[0031] Figure 2 The Western Blot in Example 5 detected the expression level of ATP5F1A protein in sperm of bulls of different genotypes. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0033] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0034] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0035] Example 1. Differential expression of ATP5F1A in sperm protein groups of high and low sperm cryoresistant groups of Chinese Holstein bulls. I. Experimental animals and grouping

[0036] The fresh semen samples used in the present embodiment are all from Shandong Aukes bull station, and the bull breed is Chinese Holstein. All bulls are raised and managed in the same environment and are in good health. The fresh semen of Chinese Holstein bulls is collected respectively by pseudo-vaginal method, and then transferred to the laboratory using 19°C incubator, and diluted with PBS buffer in a ratio of 1:10 to obtain semen diluent. 10 μL semen diluent is dropped on a slide respectively, and sperm motility is detected by computer-assisted sperm analysis system (CASA) to obtain the fresh semen sperm motility phenotypic data of Chinese Holstein bulls, and the sperm motility before freezing of each bull semen is the mean value (i.e., pre-freezing activity) of the fresh semen sperm motility recorded in the book 10 times before the individual sampling. The sperm motility after freezing of each bull semen is the mean value (i.e., post-freezing activity) of the sperm motility after thawing of the frozen semen recorded in the book 10 times before the individual sampling. Wherein, if the number of records is less than 10 times, the mean value of all sperm motility effective values ​​is calculated.

[0037] 130 Chinese Holstein bulls were screened according to the multiple phenotypic records of fresh sperm motility and post-freezing motility, and finally a high sperm freezing resistance group (i.e., H group) consisting of 8 Chinese Holstein bulls and a low sperm freezing resistance group (i.e., L group) consisting of 6 Chinese Holstein bulls were obtained. In the H group, the fresh sperm motility of the bull semen was ≥60% and the sperm motility after thawing was ≥40%. In the L group, the fresh sperm motility of the bull semen was ≥60% and the sperm motility after thawing was ≤27%. There was no significant difference in the sperm motility before freezing between the two groups; however, the average sperm motility of the frozen semen in the H group after thawing was 43.13±2.53%, which was significantly higher than that in the L group (22.67±3.88%). It can be seen that there was a significant difference in sperm motility after freezing between the H group and the L group (P-value<0.0001, see Table 1).

[0038] Table 1. Phenotypic information of individual bulls

[0039]

[0040]

[0041] Note: Pre-freezing motility refers to the vitality of semen before freezing, which is directly measured after semen collection; post-freezing motility refers to the sperm motility after frozen semen is thawed, which is the average of 10 records of the individual; the individual numbers containing "H" belong to the high sperm freezing resistance group, and the individual numbers containing "L" belong to the low sperm freezing resistance group.

[0042] 2. Sperm separation

[0043] The fresh semen of the Chinese Holstein bull collected in step 1 was divided into 1.5 mL centrifuge tubes, and centrifuged at 19°C and 800×g for 15 min to obtain sperm precipitates. The sperm precipitates were then washed twice with PBS buffer and placed in cryopreservation tubes, which were quickly frozen in liquid nitrogen and stored at -80°C for later use to obtain sperm samples.

[0044] 3. Extraction and sequencing of total sperm protein

[0045] 1. Extraction of total sperm protein

[0046] Add 400 μL of lysis solution (solutes and their concentrations are 7 M urea, 2 M thiourea, 0.1% PMSF protease inhibitor and 65 mM DTT, and the solvent is water) to the bull sperm samples obtained in step 2 respectively, treat with ultrasound (70 W; 5 s on, 10 s off, 3-5 times) under ice bath conditions, place on ice for 40 min; then centrifuge at 4°C and 14000×g for 30 min, and collect the supernatant, which is the total sperm protein sample.

[0047] 2. Protein concentration determination

[0048] The concentration of total sperm protein in the total sperm protein sample obtained in step 1 was determined by Bradford assay (Beyotime, Shanghai, China).

[0049] The steps for determining sperm total protein concentration using the Bradford assay are as follows:

[0050] (1) Mix 10 μl of sperm total protein sample and 300 μl of staining solution to obtain sperm staining solution; mix BSA solutions of different concentrations (obtained by diluting BSA standard with water) and 300 μl of staining solution to obtain BSA staining solution;

[0051] (2) Separately place the sperm staining solution and the BSA staining solution in the dark at room temperature for 10 min, and then measure the absorbance at 562 nm using an ELISA reader;

[0052] (3) A standard curve is drawn with the concentration of the BSA solution as the horizontal axis and the corresponding absorbance as the vertical axis; then, the total sperm protein concentration in the total sperm protein sample is obtained based on the absorbance of the sperm staining solution and the standard curve.

[0053] 3. SDS-PAGE electrophoresis

[0054] 20 μg of total sperm protein was taken from each Chinese Holstein bull, and the proteins were separated using 12% SDS-PAGE. Then, the separated gel was stained with Coomassie Brilliant Blue. After staining, the gel was accurately scanned using an ImageScanner scanner to obtain detailed image information.

[0055] 4. In-gel digestion

[0056] (1) Use a scalpel to cut the film after Coomassie Brilliant Blue staining and gel destaining into pieces of about 1-2 mm 2 Small pieces of different sizes are then placed into EP tubes.

[0057] (2) After completing step (1), add 500 μl of decolorizing solution (acetonitrile and 50 mM NH4HCO3 aqueous solution mixed in a volume ratio of 1:1) to the EP tube to fully immerse the film in the liquid; then fully shake for 20 minutes and discard the waste liquid. Repeat this step 1-2 times until the film fades to colorless. Finally, add 500 μl of acetonitrile and discard the waste liquid again.

[0058] (3) After completing step (2), add 50-100 μL of DTT (concentration: 10 mmol / L) reducing solution to the EP tube and keep it at 56°C for 30 minutes. Then, discard the waste liquid and add 500 μL of acetonitrile for dehydration treatment for 5-10 minutes.

[0059] (4) After completing step (3), add 50-100 μl of iodoacetamide (concentration: 55 mmol / L) to the film and let it stand in the dark for 30 minutes (for the purpose of alkylation treatment).

[0060] (5) After completing step (4), first add 500 μl of decolorizing solution for washing for 5-10 minutes; then add 500 μl of acetonitrile and discard the waste liquid; next, dry the film under freezing conditions for 20 minutes.

[0061] (6) After completing step (5), add 50-100 μL of trypsin (concentration of 0.01 μg / μl) to the gel and place it at 4°C for 30 minutes to allow the gel to completely absorb the enzyme solution. Then add 50-100 μl of enzymatic buffer (i.e., 25 mM NH4HCO3 aqueous solution) to ensure that the gel is completely immersed in the liquid and keep it at 37°C for at least 15 hours.

[0062] (7) After completing step (6), add 100 μl of extraction solution I (5% TFA aqueous solution) to the EP tube, treat in a 40°C water bath for 1 hour (during which ultrasonic treatment is performed every 30 minutes for 3 minutes each time), and collect liquid phase 1.

[0063] (8) After completing step (7), add 100 μl of extraction solution II (containing an aqueous solution of 2.5% TFA and 50% acetonitrile) to the gel block in the EP tube, keep warm at 30°C for 1 hour (during which ultrasonic treatment is performed every 30 minutes for 3 minutes each time), and collect liquid phase 2.

[0064] (9) Liquid phase 1 and liquid phase 2 are combined to obtain an extract, which is then dried to obtain a sample.

[0065] 5. Liquid chromatography and mass spectrometry

[0066] The sample obtained in step 4 (9) was dissolved in 0.1% formic acid (FA) and loaded into LC-MS / MS liquid mass tandem mass spectrometer.

[0067] LC conditions were as follows: flow rate: 600 nL / min; chromatographic column: C18 precolumn (PepMap C18, 100 μm × 2 cm, 5 μm), analytical column (PepMap C18, 75μm×50cm, 2μm); mobile phase consists of buffer B and buffer A; buffer A is ACN-H2O-FA (1.9:98:0.1, v / v / v); buffer B is ACN-H2O-FA (98:1.9:0.1, v / v / v). Gradient elution conditions: 6-12% buffer B, 0-10min; 12-30% buffer B, 10-95min; 30-40% buffer B, 95-113min; 40-95% buffer B, 133-134min; 95% buffer B, 114-1130min; 3% buffer B, 2min, then equilibrate for 3min.

[0068] The mass spectrometry conditions are as follows: When performing mass spectrometry analysis, the mass resolution of the primary MS was first fine-tuned to 35000, and the automatic gain control value was calibrated to 1e6. To ensure the accuracy of the data, the maximum injection time limit was set to 50 milliseconds. The full scan mode was selected for mass spectrometry scanning, and the scanning range of the mass-to-nuclear ratio m / z was set between 400 and 1500. In order to deeply explore the key components in the sample, the top 10 peaks with the highest intensity in the scan results were subjected to MS / MS deep scanning. During the acquisition of the MS / MS spectrum, the data-dependent positive ion mode was adopted, and the high-energy collision fragmentation technology was applied, and the collision energy was set to 30. In addition, in order to improve the accuracy of MS / MS analysis, the resolution of MS / MS was set to 17500, and the automatic gain control value was adjusted to 2e5, and the maximum injection time was also limited to 50 milliseconds. At the same time, to avoid repeated scanning, the dynamic exclusion time was set to 30 seconds to ensure the accuracy and reliability of the results.

[0069] 4. Search database and software

[0070] Database used: The bovine Uniprot protein sequence library was used as the database (https: / / www.uniprot.org / proteomes?query=Bos+taurus), the database search software: Maxquant (1.5.2.8) software, the analysis parameters are as follows: 'Enzyme' is Trypsin / P; 'Max Missed Cleavages' is 2; 'Fixed modifications' is Carbamidomethyl (C); 'Variable modifications' is Oxidation (M), Acetyl (Protein N-term); 'Min.peptide length' is 7; 'Min.razor+unique peptides' is 1; the result filtering parameters are: Peptide FDR≤0.01, Protein FDR≤0.01.

[0071] 5. Identification of Differentially Expressed Proteins

[0072] The differential protein screening was performed with the difference fold |log2FC|>0.5 and P<0.05 as the significance threshold. The results showed that a total of 147 differentially expressed proteins were identified in the sperm of Chinese Holstein bulls, of which 93 up-regulated proteins were identified in the H group and 54 up-regulated proteins were identified in the L group. The identification results of ATP5F1A protein in the differentially expressed protein results are shown in Table 2. The expression of ATP5F1A protein in sperm of Chinese Holstein bulls is shown in Figure 1 .

[0073] Table 2. Differential expression of ATP5F1A protein in high sperm cryoresistant group and low sperm cryoresistant group

[0074] Record number Gene name Mean of group H Mean of group L <![CDATA[log2FC]]> P-value P19483 ATP5F1A 31.20 30.68 0.52 0.01

[0075] The results showed that among Chinese Holstein bulls, the content of ATP5F1A protein in the high sperm freeze-resistance group was higher than that in the low sperm freeze-resistance group. This shows that ATP5F1A protein is closely related to sperm freeze-resistance.

[0076] The above-mentioned proteomic experiments revealed that ATP5F1A protein showed significant differential expression in the sperm of Chinese Holstein bulls in the high sperm freezing resistance group and the low sperm freezing resistance group, especially ATP5F1A protein was highly expressed in the sperm of Chinese Holstein bulls in the high sperm freezing resistance group. Therefore, it is inferred that the gene encoding ATP5F1A protein (i.e., ATP5F1A gene) is an important candidate gene for the freezing resistance trait of sperm in Chinese Holstein bulls.

[0077] Example 2: Discovery of SNP sites in the genome of Chinese Holstein bulls and acquisition of primer pairs for identifying the SNP sites

[0078] 1. Discovery of SNP loci in the genome of Chinese Holstein bulls

[0079] The inventors of the present application have conducted a large number of sequence analyses, alignments and preliminary experiments on the gene encoding the ATP5F1A protein in the genome of Chinese Holstein bulls (i.e., the ATP5F1A gene), and found a SNP site in the gene encoding the ATP5F1A protein in the genome of Chinese Holstein bulls, which caused a missense mutation of an amino acid. Specifically, the SNP site is the 161st nucleotide from the 5' end of SEQ ID NO:1 in the genome of Chinese Holstein bulls, and the genotype is CC homozygous, TT homozygous or CT heterozygous.

[0080] SEQ ID NO: 1 is:

[0081] (Y is C / T)

[0082] Since genomic DNA is a double-stranded DNA molecule composed of two reverse complementary single-stranded DNA molecules, the DNA molecule encoding the protein is generally named as the sense DNA molecule; the DNA molecule that is reverse complementary to the sense DNA molecule is named as the antisense DNA molecule. In this patent, the genotypes of all sites are the genotypes of the antisense DNA.

[0083] 2. Obtaining primer pairs for identifying SNP sites

[0084] According to the above SNP site and the nucleotide sequence before and after it, a primer pair for identifying the above SNP site is designed and synthesized. The primer pair consists of an upstream primer F and a downstream primer R, and is used to amplify the target sequence of the above SNP site.

[0085] The nucleotide sequences of the upstream primer F and the downstream primer R are shown in Table 3.

[0086] Table 3

[0087] Primer name Nucleotide sequence (5'-3') and its position in the sequence listing Upstream primer F TGATGGATGGTGCTCATG(SEQ ID NO:2) Downstream primer R TTTCCATCGGTCCTATTT(SEQ ID NO:3)

[0088] Example 3: Establishment of a genotyping method for Chinese Holstein bulls based on SNP loci

[0089] 1. Extract the genomic DNA from the semen of the Chinese Holstein bull to be tested.

[0090] 2. Using the genomic DNA of the Chinese Holstein bull semen to be tested as a template, PCR amplification was performed using a primer pair consisting of an upstream primer F and a downstream primer R to obtain a PCR amplification product.

[0091] The reaction system was 25 μl, consisting of 1 μl of genomic DNA of Chinese Holstein bull semen to be tested (concentration of 100 ng / μL), 1 μl of upstream primer F aqueous solution (concentration of 10 pmol / μL), 1 μl of downstream primer R aqueous solution (concentration of 10 pmol / μL) and 22 μl of PCR mix (product of Qingke Biotechnology Co., Ltd., product number cat. TES101).

[0092] The reaction conditions were: 98°C for 2 min; 98°C for 10 s, 56°C for 10 s, 72°C for 15 s, 35 cycles; 72°C for 5 min.

[0093] 3. After completing step 2, sequence the PCR amplification product. According to the sequencing results, determine the genotype of the Chinese Holstein bull based on the SNP locus. The specific judgment principles are as follows:

[0094] If the 161st position of the PCR amplification product of the Chinese Holstein bull to be tested is only C, the genotype of the Chinese Holstein bull to be tested based on the SNP site is CC homozygous;

[0095] If the 161st position of the PCR amplification product of the Chinese Holstein bull to be tested is only T, the genotype of the Chinese Holstein bull to be tested based on the SNP site is TT homozygous;

[0096] If the 161st position of the PCR amplification product of the Chinese Holstein bull to be tested is C and T, the genotype of the Chinese Holstein bull to be tested based on the SNP site is a CT heterozygous type.

[0097] Example 4: Association analysis between genotype and sperm freezing resistance of Chinese Holstein bulls based on SNP loci

[0098] The fresh semen samples of 130 Chinese Holstein bulls in this embodiment were all provided by Shandong Aokes Bull Station and were from different Chinese Holstein bulls. The Chinese Holstein bulls providing the fresh semen samples were all raised to sexual maturity under normal nutritional levels and feeding conditions.

[0099] 1. Testing the vitality of fresh and frozen semen of 130 Chinese Holstein bulls

[0100] 1. The production process of the frozen semen of 130 Chinese Holstein bulls was carried out in full compliance with the Technical Regulations for the Production of Frozen Semen of Cattle (NY / T 1234-2018), an agricultural industry standard of the People’s Republic of China. The specific steps are as follows:

[0101] (1) Take a dilution bottle containing 30 mL of diluent (the formula is provided in Appendix C of the Technical Regulations for the Production of Frozen Semen of Cattle in the Agricultural Industry Standard of the People's Republic of China (NY / T 1234-2018)) that has been pre-warmed in a 34°C water bath, dilute and mix the semen, store it in a 34°C water bath for 10 minutes, and then add the diluent to the final dilution volume. Then proceed as follows a) or b):

[0102] a) After 10 minutes of storage, the semen can be filled, sealed and labeled on a laboratory operating table at room temperature below 20°C. The filled straws are placed in an opaque plastic box, preferably 300 straws per box. The plastic box is placed in a 3°C-5°C low-temperature cabinet for 3h-4h to balance and assess the motility.

[0103] b) After adding the diluent, use a cup to hold an appropriate amount of 34℃ water, place the dilution bottle in a 3℃-5℃ low temperature cabinet to cool and balance. After 2 hours, add ice cubes to the cup to quickly cool it to 3℃-5℃ (the straw should also be cooled to 3℃-5℃), and evaluate the activity. Fill, seal and label the straw in the low temperature cabinet.

[0104] (2) After completing step (1), freeze the semen using a programmable freezer, set the optimal freezing program, and freeze the semen strictly according to the operating procedures of the programmable freezer. After freezing, open the lid of the programmable freezer, put the frozen semen into a bucket filled with liquid nitrogen, and quickly immerse it in the liquid nitrogen to obtain a frozen semen sample.

[0105] 2. Repeat the experiment three times and take the average value of the results. Each semen sample (fresh semen sample or frozen semen sample) is tested according to the following steps:

[0106] (1) Take a fresh semen sample and dilute it with physiological saline in an appropriate ratio to obtain a fresh semen diluent; take out a frozen semen sample from liquid nitrogen, quickly immerse it in 37°C water and shake it, take it out after the frozen semen in the capillary tube is dissolved, wipe off the water droplets, cut off one end of the capillary tube, and use a special push pin to squeeze the semen into a small test tube to obtain a frozen semen diluent.

[0107] (2) 10 μL of semen diluent (fresh semen diluent or frozen semen diluent) was dropped onto a glass slide, which was then placed on the microscope stage connected to a computer-assisted sperm analysis (CASA) system to measure sperm motility, expressed as a percentage;

[0108] Sperm motility = (number of progressively motile sperm / total number of sperm) × 100%.

[0109] The sperm motility of each individual fresh sperm and frozen sperm was tested, and the average phenotypes of sperm motility and frozen sperm motility of at least 6 sperm were counted and grouped according to the following criteria:

[0110] Semen samples with fresh sperm motility ≥ 60% and sperm motility ≥ 40% after thawing of frozen semen samples constituted a high semen freeze-resistance group, namely Group H.

[0111] Semen samples with fresh sperm motility ≥ 60% and sperm motility ≤ 39% after thawing of frozen semen samples constituted the low semen freeze-resistance group, namely the L group.

[0112] The results showed that the H group consisted of semen samples from 55 Chinese Holstein bulls. The sperm motility of fresh semen samples in the H group was (68.13±1.23)%, and the sperm motility of thawed frozen semen samples was (41.62±1.48)%. The L group consisted of semen samples from 75 Chinese Holstein bulls. The sperm motility of fresh semen samples in the L group was (64.64±4.94)%, and the sperm motility of thawed frozen semen samples was (31.58±5.42)%.

[0113] 2. Detection of genotypes based on SNP loci in 130 Chinese Holstein bulls

[0114] According to the method of Example 3, the semen of the Chinese Holstein bulls to be tested was replaced with fresh semen samples of 130 Chinese Holstein bulls, and the other steps remained unchanged. A total of 101 samples were successfully tested, and thus the genotypes of the 101 Chinese Holstein bulls based on the SNP loci were finally obtained.

[0115] The quantitative statistical results and genotype frequencies of 101 Chinese Holstein bulls based on SNP loci are shown in Table 4. The allele frequency of C is 0.223, and the allele frequency of T is 0.777.

[0116] Table 4

[0117] Genotype of SNP locus Quantity(Head) Genotype frequency CC homozygous 10 0.10 CT heterozygous 25 0.25 TT homozygous 66 0.65

[0118] 3. Association analysis between genotype and sperm freezing tolerance based on SNP loci in Chinese Holstein bulls

[0119] SPSS25 software was used to conduct association analysis on the genotypes of the 101 Chinese Holstein bulls obtained in step 2 based on SNP loci, specifically the chi-square test. The test results are shown in Table 5.

[0120] Table 5

[0121]

[0122] Note: Pvalue is the significance level of the chi-square test. *This indicated that there were significant differences in sperm freezing resistance among different genotypes (P<0.05).

[0123] The results are as follows:

[0124] The SNP loci were significantly associated with sperm freezing resistance of Chinese Holstein bulls;

[0125] The freezing resistance of sperm of Chinese Holstein bulls with CC homozygous genotype based on SNP loci is greater than that of sperm of Chinese Holstein bulls with TT homozygous genotype based on SNP loci; for the freezing resistance trait of sperm of Chinese Holstein bulls, based on SNP loci, the dominant allele is C, and the excellent genotype is CC homozygous;

[0126] The above “>” are all statistically > (i.e., significantly higher at the 0.05 level).

[0127] The above results indicate that the sperm freezing resistance trait of Chinese Holstein bulls can be identified quickly and efficiently by detecting the genotype of the Chinese Holstein dairy cows based on SNP loci, which has important application value in breeding superior Chinese Holstein bulls, improving the semen quality traits of Chinese Holstein bulls, and accelerating the breeding process of Chinese Holstein bulls.

[0128] Example 5: Verify the function of the SNP site by detecting the expression level of ATP5F1A protein by Western Blot

[0129] Western Blot was used to detect the expression of ATP5F1A protein in Chinese Holstein bulls with CC homozygous genotype based on the SNP locus and Chinese Holstein bulls with TT homozygous genotype based on the SNP locus. The specific steps are as follows:

[0130] 1. Frozen semen samples were collected from three Chinese Holstein bulls with CC homozygous genotypes based on SNP loci and three Chinese Holstein bulls with TT homozygous genotypes based on SNP loci, and total protein was extracted from the frozen semen samples using a protein extraction kit (Abmart).

[0131] 2. The total protein of the frozen sperm samples was detected by SDS-PAGE and Western blot, respectively, where the primary antibody was ATP5F1A antibody (Abcam, USA) or α-Tubulin antibody (proteintech, product catalog number 14555-1-AP) (as a control).

[0132] Test results see Figure 2(CC is a Chinese Holstein bull with a CC homozygous genotype based on the SNP locus, and TT is a Chinese Holstein bull with a TT homozygous genotype based on the SNP locus). The results showed that the ATP5F1A protein expression level of the Chinese Holstein bull with a CC homozygous genotype based on the SNP locus was significantly higher than that of the Chinese Holstein bull with a TT homozygous genotype based on the SNP locus.

[0133] It can be seen that when the genotype of the SNP site mutates from CC homozygous to TT homozygous, it will lead to down-regulation of ATP5F1A protein expression in sperm. The results show that the SNP site may be an important genetic mutation that causes the reduction of freezing resistance of sperm in Chinese Holstein bulls.

[0134] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A kit for identifying the freezing resistance of bull sperm, comprising a substance for detecting the genotype of the bull to be tested based on the SNP locus; The SNP site is the 161st nucleotide from the 5' end of SEQ ID NO: 1 in the bull genome.

2. The kit according to claim 1, characterized in that: The "substance for detecting the genotype of the bull to be tested based on the SNP site" is a primer pair consisting of an upstream primer F and a downstream primer R; The upstream primer F is a single-stranded DNA molecule shown in SEQ ID NO: 2; The downstream primer R is a single-stranded DNA molecule shown in SEQ ID NO:

3.

3. The molecular marker shown in SEQ ID NO:

1.

4. Use of the kit according to claim 1 or 2 or the molecular marker according to claim 3 in identifying the freezing resistance of bull sperm.

5. Use of the kit according to claim 1 or 2 or the molecular marker according to claim 3 in screening bulls with different sperm freezing resistance.

6. Use of the kit according to claim 1 or 2 or the molecular marker according to claim 3 in identifying the genotype of a bull.

7. Use of the kit according to claim 1 or 2 or the molecular marker according to claim 3 in bull breeding.

8. A method for identifying the freezing resistance of bull sperm, comprising the following steps: detecting whether the genotype of the bull to be tested is CC homozygous or TT homozygous based on a SNP site, and then making the following judgment: the freezing resistance of the sperm of the bull whose genotype is CC homozygous based on the SNP site is greater than the freezing resistance of the sperm of the bull whose genotype is TT homozygous based on the SNP site; The SNP site is the 161st nucleotide from the 5' end of SEQ ID NO: 1 in the bull genome.

9. A method for identifying the freezing resistance of bull sperm, comprising the following steps: (1) Using the genomic DNA of the bull to be tested as a template, a primer pair consisting of an upstream primer F and a downstream primer R is used for PCR amplification to obtain a PCR amplification product; sequencing the PCR amplification product, and then making the following judgment: if the 161st position of the PCR amplification product is only C, the genotype of the bull to be tested based on the SNP site is CC homozygous; if the 161st position of the PCR amplification product is only T, the genotype of the bull to be tested based on the SNP site is TT homozygous; (2) Based on the result of (1), the following judgment is made: the freezing resistance of sperm of bulls with CC homozygous genotype based on the SNP locus is greater than the freezing resistance of sperm of bulls with TT homozygous genotype based on the SNP locus; The SNP site is the 161st nucleotide from the 5' end of SEQ ID NO:1 in the bull genome; The upstream primer F is a single-stranded DNA molecule shown in SEQ ID NO: 2; The downstream primer R is a single-stranded DNA molecule shown in SEQ ID NO:

3.

10. The kit according to claim 1 or 2, the use according to any one of claims 4 to 7, or the method according to claim 8 or 9, characterized in that: The bull is a Holstein bull.

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