Molecular markers of the ATP5PO gene associated with bovine sperm motility traits and their applications

Abstract

This invention relates to the field of animal genetic engineering technology, providing a molecular marker for the ATP5PO gene associated with bovine sperm motility and its application. The ATP5PO gene molecular marker is located at position 203 of the bovine ATP5PO gene fragment, where an A-G base mutation occurs, resulting in a single nucleotide polymorphism (SNP). The nucleotide sequence of the bovine ATP5PO gene fragment is shown in SEQ ID NO:1. Based on this ATP5PO gene molecular marker, genotype can be determined through sequencing, and the genotype can be associated with normal and thawed bovine sperm motility. This invention utilizes a specific primer set to obtain the bovine ATP5PO gene fragment associated with normal and thawed bovine sperm motility, and uses specific SNP sites within this fragment as molecular markers, providing a theoretical basis and practical application for marker-assisted selection in cattle.

Description

Technical Field

[0001] This invention belongs to the field of animal genetic engineering technology, and in particular relates to an ATP5PO gene molecular marker related to bovine sperm motility traits and its application. Background Technology

[0002] Artificial insemination, a cornerstone technology in modern high-efficiency breeding systems for beef and dairy cattle, is valued primarily for its efficient utilization of the genetic potential of top-performing bulls. Through this technology, the genetic material of a single outstanding bull can be distributed to tens of thousands of cows within a breeding year, significantly accelerating the targeted improvement of overall herd productivity and superior breed traits. Combined with timed insemination and efficient estrus detection protocols, it can stabilize the conception rate of cows during estrus at a high level of over 85%, thereby significantly shortening the calving interval and improving the overall reproductive output and economic benefits of the farm.

[0003] In this technological chain, the quality of sperm, especially their motility, after thawing frozen semen is crucial. During the ultra-low temperature freezing and thawing process, the cellular structure of semen is subjected to multiple stresses, including cold shock and ice crystal damage, leading to irreversible decline in sperm motility and physiological function. Typically, the percentage of sperm with linear forward motility after thawing may drop to 50%-70% of the pre-freezing level, and their ability to complete capacitation, acrosome reaction, and ultimately fertilize the egg is also weakened. Therefore, obtaining a highly motile and intact sperm population after thawing is a core prerequisite for successful artificial insemination. Relevant standards clearly stipulate that the sperm motility of ordinary bovine frozen semen used for artificial insemination should not be lower than 40% after thawing.

[0004] The final quality of frozen semen is subject to complex regulation by multiple factors. These include the bull's physiological state at the time of collection, the original quality of the semen, the formulation of the cryopreservation diluent and the freezing technique used, as well as the inherent genetic differences between individual bulls. Studies have shown that sperm from different bull individuals exhibit significantly different tolerances to the freezing process (i.e., cryoresistance), directly leading to substantial differences in sperm motility after thawing. Among various types of genetic polymorphisms, single nucleotide polymorphisms (SNPs) have become an important tool for identifying key gene loci affecting the cryoresistance of bull sperm due to their wide distribution, ease of detection, and close association with phenotype. Existing research has confirmed that specific SNP loci can significantly correlate with and affect the cryo-thaw survival rate and motility parameters of bull sperm, providing a theoretical basis for improving the quality of frozen semen from breeding bulls through marker-assisted selection.

[0005] The ATP5PO (ATP synthase peripheral stalk subunit OSCP) gene, as a component of the mitochondrial ATP synthase complex, is crucial for oxidative phosphorylation, a primary driving force for sperm motility and function. Studies have shown that abnormal function or reduced expression of the ATP synthase subunit can lead to impaired sperm motility, reduced semen quality, and even impaired testicular tissue morphology. However, current research has not shown how specific SNPs in the ATP5PO gene affect sperm cryopreservation resistance, especially post-thawing motility. Summary of the Invention

[0006] The purpose of this invention is to provide an ATP5PO gene molecular marker related to bovine sperm motility traits and its application, aiming to solve the problems raised in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The application of an ATP5PO gene molecular marker associated with bovine sperm motility traits in the preparation of a kit for detecting bovine sperm motility traits in normal and thawed conditions, wherein the bovine sperm motility traits include bovine sperm motility traits in both normal and thawed conditions; the ATP5PO gene molecular marker is located at position 203 of the bovine ATP5PO gene fragment, which has an AG base mutation, resulting in a single nucleotide polymorphism; the nucleotide sequence of the bovine ATP5PO gene fragment is shown in SEQ ID NO:1 of the sequence listing.

[0009] Another object of the present invention is to provide a specific primer set, wherein the specific primer set is used to identify the above-mentioned molecular marker of the ATP5PO gene associated with bovine sperm motility traits, and includes forward primers and reverse primers as shown in SEQ ID NO:2-3, respectively.

[0010] Another object of the present invention is to provide the application of the above-mentioned specific primer set in the preparation of a kit for detecting the normal and thawed motility traits of bovine sperm.

[0011] This invention provides a molecular marker for the ATP5PO gene associated with bovine sperm motility. Genotype determination through sequencing allows for association between genotype and normal and thawed sperm motility. Analysis results show significant differences in normal and thawed sperm motility among individuals with different genotypes. This invention utilizes a specific primer set to obtain a bovine ATP5PO gene fragment associated with normal and thawed sperm motility, and uses specific SNP sites within this fragment as molecular markers, providing a theoretical basis and practical application for marker-assisted selection in cattle. Attached Figure Description

[0012] Figure 1 The results of 1.5% agarose gel electrophoresis are shown for the ATP5PO gene amplification products in Example 1. In the figure, lane M is the standard molecular weight marker, and lanes 1-5 are 5 randomly detected PCR products with a clear and specific band at the 668bp position.

[0013] Figure 2 The image shows the sequencing peaks of the PCR products of the three genotypes of the ATP5PO gene in Example 1. In the image, the arrows indicate the mutation sites; for AA genotype individuals, the site is an A base; for AG genotype individuals, the site is an A / G base; and for GG genotype individuals, the site is a G base.

[0014] Figure 3 The results of 1.5% agarose gel electrophoresis are shown for the ATP5PO gene amplification products in Example 2. In the figure, lane M is the standard molecular weight marker, and lanes 1-6 are 6 randomly detected PCR products with a clear and specific band at the 668bp position.

[0015] Figure 4 The image shows the sequencing peaks of the PCR products of the three genotypes of the ATP5PO gene in Example 2. In the image, the arrows indicate the mutation sites; for AA genotype individuals, the site is an A base; for AG genotype individuals, the site is an A / G base; and for GG genotype individuals, the site is a G base. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] In one embodiment of the present invention, a specific mutation site is identified by cloning a bovine ATP5PO gene fragment (668 bp, nucleotide sequence as shown in SEQ ID NO:1) to serve as a method for detecting polymorphisms in genes related to normal bovine sperm motility and thawed sperm motility, providing a meaningful ATP5PO gene molecular marker for bovine marker-assisted breeding; specifically, the ATP5PO gene molecular marker is the 203rd site of the bovine ATP5PO gene fragment, which has an AG base mutation, resulting in a single nucleotide polymorphism; the nucleotide sequence of the bovine ATP5PO gene fragment is shown in SEQ ID NO:1.

[0018] In another embodiment of the present invention, a specific primer set is also provided, wherein the specific primer set is used to identify the above-mentioned molecular marker of the ATP5PO gene associated with bovine sperm motility traits, specifically including the forward primer and the reverse primer as shown in the sequence listing SEQ ID NO:2-3, respectively.

[0019] In another embodiment of the present invention, a method for screening molecular markers suitable for bovine sperm normal and thawed motility traits and for use in bovine marker-assisted selection is also provided. The ATP5PO gene molecular marker is used to screen or evaluate breeding bulls with high sperm normal and thawed motility, specifically including the following steps:

[0020] S1. Genomic DNA is extracted from the blood of individuals to be screened and amplified by PCR using the specific primer set described above to obtain PCR products. Due to the presence of an AG base mutation at position 203 of the DNA sequence of the PCR product fragment, SNP polymorphism is generated.

[0021] S2. Sequencing the PCR product to determine the base type at position 203, and obtaining the sequencing results;

[0022] S3. The genotypes of the individuals to be screened were determined based on the sequencing results, and correlation analysis was performed with the normal and thawed sperm motility traits of bovine sperm. The genotypes included AA, AG, and GG. The results showed that individuals with specific genotypes would have higher normal and thawed sperm motility. Specifically, individuals with AA genotypes had better normal and thawed sperm motility than those with AG and GG genotypes.

[0023] In this embodiment of the invention, by association analysis of the genetic polymorphism of the bovine ATP5PO gene and the normal and thawed sperm motility traits, the influence of specific SNPs of the ATP5PO gene and the genetic polymorphism at these sites on normal and thawed sperm motility was clarified. This embodiment of the invention provides an important theoretical basis and application prospect for the ATP5PO gene as a molecular marker for auxiliary selection of normal and thawed sperm motility and related reproductive performance in bovine production, and for its application in genetic improvement.

[0024] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are commercially available products; unless otherwise specified, all experimental methods used are conventional methods.

[0025] Example 1: This example uses extracted bovine genomic DNA as a template, designs a pair of specific primers, clones a partial DNA sequence of the bovine ATP5PO gene, and performs sequencing and genotyping. The association between different genotypes and normal and thawed bovine sperm motility traits is then analyzed to provide molecular markers for marker-assisted selection in cattle, as detailed below:

[0026] 1. Cloning of a partial DNA fragment of the bovine ATP5PO gene: To ensure good primer quality, the specific primers used in this embodiment of the invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences of the specific primers are as follows:

[0027] Forward primer: ATP5PO-fwd (as shown in SEQ ID NO:2 in the sequence listing): TTTCACACGCCCGAGACC;

[0028] Reverse primer: ATP5PO-rev (as shown in SEQ ID NO:3 in the sequence listing): TTACAGAATCTCCCGCATTG.

[0029] The Taq enzyme, buffer, magnesium ions, dNTPs, etc. required in the PCR reaction can be selected by the user. To obtain good results quickly, this embodiment of the invention uses the 2× chemical dye quantitative PCR premix from MonAmp ChemoHS qPCR Mix Co., Ltd. for PCR amplification. The specific reaction system is as follows: 10.0 μL of 2×MonAmp ChemoHS qPCR Mix (provided in the product packaging box), 0.5 μL each of forward and reverse primers (concentration of 10 pmol / μL), 0.5 μL of genomic DNA (containing 10-50 ng DNA), and 8.5 μL of double-distilled water. The PCR reaction conditions are: 94℃ pre-denaturation for 1 minute; 94℃ denaturation for 45 seconds, 55℃ annealing for 45 seconds, 72℃ extension for 45 seconds, for a total of 35 cycles; and a final extension at 72℃ for 5 minutes.

[0030] 2. PCR product sequencing and genotype determination: Bovine genomic DNA was amplified using primers ATP5PO-fwd (SEQ ID NO:2) and ATP5PO-rev (SEQ ID NO:3) to obtain a 668 bp specific amplified fragment. The sequence of this fragment is shown in SEQ ID NO:1 of the sequence listing, specifically:

[0031] .

[0032] Sequencing results revealed that a mutation at position 203 of the 668 bp fragment, known as AG, led to the development of different genotypes: AA, AG, and GG. Individuals with the AA genotype were homozygous for A at position 203; individuals with the AG genotype were heterozygous for A / G at position 203; and individuals with the GG genotype were homozygous for G at position 203 (e.g., ...). Figure 2 (As shown).

[0033] 3. Marker-Track Association Analysis: Using the applicant's experimental population as the experimental subjects, a marker-track association analysis was conducted. The One-Way ANOVA procedure in SPSS 22.0 software was used to establish the following statistical analysis model for the marker-track association analysis:

[0034] The statistical analysis model is: Y ij =μ+Gi +e j .

[0035] Among them, Y ij G represents the phenotypic value of the observed individual's productive performance; μ represents the least squares mean of productive performance; G i e represents the effect of genotype on production performance. j This represents the random residuals corresponding to the observed values.

[0036] 4. Cloning of partial DNA sequence of bovine ATP5PO gene and determination of different genotypes: PCR amplification products were detected by 1.5% agarose gel electrophoresis, showing them to be specific PCR products, such as... Figure 1 As shown in the image. The PCR product was recovered and sequenced, revealing a product length of 668 bp. Sequencing results showed an A and B base mutation at position 203 bp in this fragment, with some sequencing peaks as shown in the image. Figure 2 As shown.

[0037] 5. Association analysis of marker traits: Association analysis of the SNP locus at position 203 of the amplified sequence of the bovine ATP5PO gene with normal and thawed sperm motility traits showed that, among the 94 randomly selected individuals, 20 were AA type, 42 were AG type, and 32 were GG type. The results of the significant differences (mean ± standard error) between normal and thawed sperm motility among individuals with different genotypes are shown in Table 1.

[0038] Table 1. Results of the analysis of significant differences in sperm motility between individuals with different genotypes and those after thawing in Example 1.

[0039]

[0040] Note: In the same row, different letters on the shoulder labels of different groups of data indicate significant differences (P<0.05).

[0041] The analysis results showed that there were significant differences in sperm normality and post-thaw motility among individuals corresponding to different genotypes at this SNP locus. Overall, AA-type individuals had better sperm normality and post-thaw motility than AG or GG-type individuals. When selecting breeding bulls, AA-type individuals should be given priority, while GG-type individuals should be avoided as much as possible.

[0042] Example 2: Sixty-seven individuals were randomly selected from the breeding bull population at the bull station. Blood samples were collected for genomic DNA extraction, and PCR amplification was performed using the specific primers, PCR reaction system, and conditions described above. PCR amplification was performed using 2× chemical dye-based quantitative PCR premix from MonAmpChemoHS Technology Co., Ltd. The specific reaction system was as follows: 10.0 μL of 2×MonAmpChemoHS qPCR Mix, 0.5 μL each of forward and reverse primers (both at a concentration of 10 pmol / μL), 0.5 μL of genomic DNA (containing 10-50 ng DNA), and 8.5 μL of double-distilled water. The PCR reaction conditions were: 94℃ pre-denaturation for 1 minute; 94℃ denaturation for 45 seconds, 55℃ annealing for 45 seconds, 72℃ extension for 45 seconds, for a total of 35 cycles; and a final extension at 72℃ for 5 minutes.

[0043] The amplification products were detected by 1.5% agarose gel electrophoresis, and the results showed that they were specific PCR products. Figure 3 As shown, lane M represents the standard molecular weight marker, and lanes 1-6 contain randomly selected PCR products for testing. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results showed an AG base mutation at 203 bp in this fragment, with some sequencing peaks as shown... Figure 4 As shown. Of the 67 individuals examined, 13 were of type AA; 31 were of type AG; and 23 were of type GG.

[0044] Based on the sperm normality and post-thaw motility records of these 67 individuals, correlation analysis was performed using the same method as in Example 1. The correlation analysis results (least squares values ​​and standard deviations) between different genotypes and sperm normality and post-thaw motility in these 67 individuals are shown in Table 2.

[0045] Table 2. Results of analysis of significant differences in sperm motility between individuals with different genotypes and those with normal sperm and sperm motility after thawing in Example 2.

[0046]

[0047] Note: In the same row, different letters on the shoulder labels of different groups of data indicate significant differences (P<0.05).

[0048] The analysis results show that individuals corresponding to different genotypes of the SNP locus provided in this embodiment of the invention exhibit significant differences in sperm normality and post-thaw motility traits. Individuals with the AA genotype showed better sperm normality and post-thaw motility than those with the AG and GG genotypes. Therefore, this embodiment of the invention provides an important theoretical basis for using this SNP locus as a molecular marker for auxiliary selection of bull reproductive and production performance and for its application in genetic improvement, and it has promising application prospects.

[0049] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of a specific primer set for identifying molecular markers of the ATP5PO gene associated with bovine sperm motility traits in the preparation of a kit for detecting normal and thawed bovine sperm motility traits, characterized in that, The bovine sperm motility traits include normal and thawed bovine sperm motility; the ATP5PO gene molecular marker is position 203 of the bovine ATP5PO gene fragment, which has an AG base mutation, resulting in a single nucleotide polymorphism; the nucleotide sequence of the bovine ATP5PO gene fragment is shown in SEQ ID NO:1 of the sequence listing; among them, the AA type individuals have better normal and thawed sperm motility than the AG and GG types.

2. The application according to claim 1, characterized in that, The specific primer set includes forward and reverse primers as shown in the sequence listing SEQ ID NO:2-3, respectively.

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