ADAMTS5 gene molecular marker related to motility traits of bovine sperms and application of ADAMTS5 gene molecular marker

Abstract

The invention is suitable for the technical field of animal gene engineering, and provides an ADAMTS5 gene molecular marker related to bovine sperm motility traits and application thereof.The ADAMTS5 gene molecular marker is an SNP site of the 306th site of a bovine ADAMTS5 gene segment, and the basic group of the 306th site is A or G; the nucleotide sequence of the cattle ADAMTS5 gene segment is as shown in a sequence table SEQ ID NO: 1. Based on the ADAMTS5 gene molecular marker, the genotype is judged through sequence determination, and the genotype can be associated with the normal and unfrozen activity traits of the bovine sperms. According to the invention, the specific primer group is utilized to obtain a bovine ADAMTS5 gene segment related to the normal and unfrozen activity traits of bovine sperms, and a specific SNP site in the segment is used as a molecular marker, so that a theoretical basis and specific application are provided for marker-assisted selection of cattle.

Description

Technical Field

[0001] This invention belongs to the field of animal genetic engineering technology, and in particular relates to an ADAMTS5 gene molecular marker related to bovine sperm motility traits and its application. Background Technology

[0002] Artificial insemination is currently the most commonly used technique in beef and dairy cattle breeding. This technique can significantly improve the genetic utilization efficiency of superior breeding bulls, allowing a single bull to mate with tens of thousands of cows annually, thereby accelerating the breed improvement process of the cattle herd. Simultaneously, through precise mating and the implementation of estrus synchronization techniques, the conception rate of cows can be increased to over 90%, significantly improving reproductive efficiency and economic benefits of cattle farming.

[0003] The quality of frozen semen is a key factor determining the success of artificial insemination. During the freezing and thawing process, sperm inevitably suffer some damage, leading to a decrease in their motility and fertilization capacity. Generally, after thawing, sperm motility may decrease to 60% of its pre-freezing level, and their ability to penetrate cervical mucus and the zona pellucida of oocytes will also weaken. Therefore, ensuring a sufficient number and motility of sperm after thawing is an important prerequisite for successful artificial insemination.

[0004] Numerous factors influence the quality of frozen semen, including initial sperm quality, the choice of cryoprotectant, and the freezing and thawing process. In actual production, due to differences in gene polymorphisms in their genetic makeup, different bulls exhibit natural differences in the cryoprotective capacity of their sperm, leading to significant variations in sperm motility after freezing and thawing. Single nucleotide polymorphisms (SNPs) are a class of gene polymorphisms that have received considerable attention. Studies have shown that SNPs can regulate sperm quality in animals, including cryoprotective capacity.

[0005] ADAMTS5 (ADAM Metallopeptidase with Thrombospondin Type 1 Motif 5), a protein-coding gene, plays a crucial role in tissue development, inflammation, and cell migration. Studies have shown that reduced ADAMTS5 expression is associated with the release of mature sperm from Sertoli cells in the seminiferous tubules. Decreased ADAMTS5 levels have been observed in men with non-obstructive azoospermia and asthenospermia. Since ADAMTS5 belongs to the agglutinase group of metalloproteinases, which regulates the extracellular matrix and the connection between Sertoli cells and germ cells, insufficient levels of this enzyme may lead to poor sperm development. However, no studies have yet shown how specific SNPs in the ADAMTS5 gene affect sperm cryopreservation resistance, especially post-thawing motility. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker of the ADAMTS5 gene related to bovine sperm motility traits and its application, in order to solve the problems raised in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The application of an ADAMTS5 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 ADAMTS5 gene molecular marker is the SNP site at position 306 of the bovine ADAMTS5 gene fragment, wherein the base at position 306 is A or G; and the nucleotide sequence of the bovine ADAMTS5 gene fragment is shown in SEQ ID NO:1 of the sequence listing.

[0008] Another object of the present invention is to provide a specific primer set, wherein the specific primer set is used to identify the ADAMTS5 gene molecular marker associated with bovine sperm motility traits, comprising a forward primer and a reverse primer as shown in SEQ ID NO:2-3, respectively; the bovine sperm motility traits include the motility traits of normal bovine sperm and thawed sperm; the ADAMTS5 gene molecular marker is the SNP site at position 306 of the bovine ADAMTS5 gene fragment, wherein the base at position 306 is A or G; the nucleotide sequence of the bovine ADAMTS5 gene fragment is shown in SEQ ID NO:1.

[0009] 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.

[0010] This invention provides a molecular marker for the ADAMTS5 gene, which is 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 the bovine ADAMTS5 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

[0011] Figure 1 The results of 1.5% agarose gel electrophoresis are shown for the ADAMTS5 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, which have a clear and specific band at the 664bp position.

[0012] Figure 2 The image shows the sequencing peaks of the PCR products of the ADAMTS5 gene from individuals with three genotypes 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.

[0013] Figure 3 The results of 1.5% agarose gel electrophoresis are shown for the ADAMTS5 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, which have a clear and specific band at the 664bp position.

[0014] Figure 4 The image shows the sequencing peaks of the PCR products of the ADAMTS5 gene from three genotype individuals 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 an A base. Detailed Implementation

[0015] 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.

[0016] In one embodiment of the present invention, a specific mutation site is identified by cloning a bovine ADAMTS5 gene fragment (664 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 ADAMTS5 gene molecular marker for bovine marker-assisted breeding. Specifically, the ADAMTS5 gene molecular marker is the SNP site at position 306 of the bovine ADAMTS5 gene fragment, where the base at position 306 is A or G, resulting in single nucleotide polymorphism. The nucleotide sequence of the bovine ADAMTS5 gene fragment is shown in SEQ ID NO:1.

[0017] 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 ADAMTS5 gene molecular markers related to bovine sperm motility traits, specifically including forward primers and reverse primers as shown in SEQ ID NO:2-3 of the sequence listing.

[0018] 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 ADAMTS5 gene molecular marker is used to screen or evaluate breeding bulls with high sperm normal and thawed motility, specifically including the following steps:

[0019] S1. Genomic DNA is extracted from the blood of individuals to be screened and amplified by PCR using the specific primer set mentioned above to obtain PCR products. Due to the presence of an AG base mutation at position 306 of the DNA sequence of the PCR product fragment, SNP polymorphism is generated.

[0020] S2. Sequencing the PCR product to determine the base type at position 306, and obtaining the sequencing results;

[0021] 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.

[0022] In this embodiment of the invention, by association analysis of the genetic polymorphism of the bovine ADAMTS5 gene and the normal and thawed sperm motility traits, the specific SNP locus of the ADAMTS5 gene and the influence of its genetic polymorphism on normal and thawed sperm motility were clarified. This embodiment of the invention provides an important theoretical basis and application prospect for the ADAMTS5 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.

[0023] 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.

[0024] 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 ADAMTS5 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:

[0025] 1. Cloning of a partial DNA fragment of the bovine ADAMTS5 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 these specific primers are as follows:

[0026] Forward primer: ADAMTS5-fwd (as shown in SEQ ID NO:2 of the sequence listing): CTTCCTGCCAATATGTCAA;

[0027] Reverse primer: ADAMTS5-rev (as shown in SEQ ID NO:3 in the sequence listing): GATGAGCGAGAACTGACTCAT.

[0028] 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 Biotechnology 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), 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 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.

[0029] 2. PCR product sequencing and genotype determination: Bovine genomic DNA was amplified using primers ADAMTS5-fwd (SEQ ID NO:2) and ADAMTS5-rev (SEQ ID NO:3) to obtain a 664 bp specific amplified fragment. The sequence of this fragment is shown in SEQ ID NO:1 of the sequence listing, specifically:

[0030] .

[0031] Sequencing results revealed that within this 664bp fragment, a mutation at position 306 (AG) resulted in different genotypes: AA, AG, and GG. Individuals with the AA genotype were homozygous for A at position 306; individuals with the AG genotype were heterozygous for A / G at position 306; and individuals with the GG genotype were homozygous for G at position 306 (e.g., ...). Figure 2 (As shown).

[0032] 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:

[0033] The statistical analysis model is: Y ij =μ+Gi +e j .

[0034] 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.

[0035] 4. Cloning of partial DNA sequence of bovine ADAMTS5 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 664 bp. Sequencing results showed an A and B base mutation at position 306 bp of this fragment, with some sequencing peaks as shown in the image. Figure 2 As shown.

[0036] 5. Association analysis of marker traits: Association analysis of the SNP site at position 306 of the amplified sequence of the bovine ADAMTS5 gene with normal sperm motility and post-thaw motility in bovine sperm showed that, among the 94 randomly selected individuals, there were 18 individuals with the AA genotype, 51 individuals with the AG genotype, and 25 individuals with the GG genotype. The results of the analysis of significant differences (mean ± standard error) between normal sperm motility and post-thaw motility in individuals with different genotypes are shown in Table 1.

[0037] Table 1. Results of the analysis of significant differences in sperm motility between individuals with different genotypes and those after thawing in Example 1.

[0038]

[0039] Note: In the same row, different lowercase letters in the superscript of different groups of data indicate significant differences (P<0.05).

[0040] 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.

[0041] 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 MonAmChemo HS. The specific reaction system was: 10.0 μL of 2×MonAmChemoHS 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 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.

[0042] 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 306 bp in this fragment, with some sequencing peaks as shown... Figure 4 As shown. Of the 67 individuals examined, 14 were of type AA; 37 were of type AG; and 16 were of type GG.

[0043] 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.

[0044] 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.

[0045]

[0046] Note: In the same row, different letters on the shoulder labels of different groups of data indicate significant differences (P<0.05).

[0047] 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.

[0048] 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 an ADAMTS5 gene molecular marker associated with bovine sperm motility in the preparation of a kit for detecting normal and thawed bovine sperm motility, characterized in that, The bovine sperm motility traits include normal and thawed bovine sperm motility traits; the ADAMTS5 gene molecular marker is the SNP site at position 306 of the bovine ADAMTS5 gene fragment, where the base at position 306 is A or G; the nucleotide sequence of the bovine ADAMTS5 gene fragment is shown in SEQ ID NO:

1.

2. A specific primer set, characterized in that, The specific primer set is used to identify the ADAMTS5 gene molecular marker associated with bovine sperm motility traits, and includes forward and reverse primers as shown in SEQ ID NO:2-3, respectively; the bovine sperm motility traits include the motility traits of normal and thawed bovine sperm; the ADAMTS5 gene molecular marker is the SNP site at position 306 of the bovine ADAMTS5 gene fragment, where the base at position 306 is A or G; the nucleotide sequence of the bovine ADAMTS5 gene fragment is shown in SEQ ID NO:

1.

3. The use of the specific primer set as described in claim 2 in the preparation of a kit for detecting the normal and thawed motility traits of bovine sperm.

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