Holstein cow sperm motility miRNA molecular marker selection method

Abstract

The application discloses a Holstein cow sperm activity miRNA molecular marker selection method, which comprises the following steps: (1) specific primers are designed according to a Holstein cow miRNA precursor region, and PCR amplification is carried out on to-be-detected cow genomic DNA to obtain an amplification product; (2) the PCR product is subjected to enzyme cutting by using a BspE I restriction endonuclease, and electrophoresis typing is carried out, and three genotypes, namely, a TT type, a TC type and a CC type, are obtained; and (3) the sperm activity of the CC type is significantly higher than that of the TT type, and the sperm deformity rate of the CC type is significantly lower than that of the TT type, so the CC type and the TC type are selected for breeding, and the C genotype frequency is improved. The method can improve the accuracy of sperm quality selection of the cow, is used for the selection and breeding of high-quality cows, and provides a selection and breeding tool for strengthening the excellent germplasm of Chinese Holstein cows.

Description

Technical Field

[0001] This invention relates to a method for selecting Holstein bovine sperm motility, specifically a method for selecting Holstein bovine sperm motility miRNA molecular markers, belonging to the field of molecular biology applications. Background Technology

[0002] miRNAs are a class of small, non-coding RNAs ranging from 18 to 24 nucleotides in length. They are generated by the cleavage of a 70-80 nt single-stranded RNA precursor (pre-miRNA) with a hairpin loop structure. Studies have found that miRNA precursors exhibit high evolutionary conservation across species, with strong conservation of the stem portion and a very low mutation rate, but relatively weak conservation of the loop portion. They primarily participate in regulating life activities such as individual development, apoptosis, proliferation, and differentiation by incompletely pairing with the 3'-UTR (untranslated region) of target genes, degrading the target gene mRNA or inhibiting its translation. They are crucial factors in gene expression regulation.

[0003] Different miRNA expression types and differential expression levels exist in different tissues and cells of an organism, and at different stages of tissue development. This indicates that miRNA regulation of gene expression is complex and diverse. Furthermore, the transcriptional regulation mechanisms of miRNAs themselves are also complex. Sequence variations of miRNAs have a significant impact on their biological synthesis and function. Studies have found that stem-loop miRNA precursor sequences are highly conserved across species, especially in the stem region, while the conservation in the loop region is relatively weaker. SNPs (single-mean-novel nucleotides) of miRNAs are present within primary miRNA sequences, miRNA precursor sequences, and mature miRNA sequences. These SNPs not only affect miRNA processing but also regulate the interaction between miRNAs and their target genes, further influencing target gene expression regulation and altering the biological function of miRNAs.

[0004] Holstein cattle are the most widely used dairy cattle breed worldwide. Semen quality is a crucial indicator of bull fertility and a fundamental criterion in bull breeding. Semen quality indicators include ejaculate volume, sperm density, sperm motility, sperm morphology (abnormality rate), and sperm DNA integrity. Semen quality traits are influenced by multiple factors, making them complex quantitative traits with some heritability, albeit low, and exhibiting significant differences in heritability between different breeds (0.04-0.6). Traditional phenotypic selection methods are inaccurate, and phenotypic values ​​only gradually emerge after bulls reach sexual maturity and are collected, resulting in a long selection cycle, hindering ultra-early selection, and leading to slow genetic progress. Summary of the Invention

[0005] The purpose of this invention is to provide a method for selecting miRNA molecular markers for sperm motility in Holstein cows in order to solve the above-mentioned problems.

[0006] This invention achieves the above objective through the following technical solution: a method for selecting miRNA molecular markers for sperm motility in Holstein dairy cows, comprising the following steps:

[0007] 1) 56 Holstein dairy cows were selected, and 1.5 ml of semen was artificially collected from each cow. The semen quality data were measured, and sperm DNA was extracted using the conventional phenol and chloroform method. The DNA quality was detected by electrophoresis and OD260 / 280 method.

[0008] 2) Based on the GenBank sequence NC_037350.1, a pair of specific primers were designed using Primer Premier 6 to amplify the precursor region sequence of Holstein dairy cow miR-6531 by PCR. The PCR product was detected by 1% agarose gel electrophoresis and ethidium bromide (EB) staining.

[0009] 3) A T→C mutation exists at position 73 of the miR-6531 precursor region in Holstein dairy cows, resulting in a BspE I restriction site. The amplified product was digested using BspE I restriction endonuclease. The digested products were detected by 2% agarose gel electrophoresis, stained with ethidium bromide (EB), and photographed using a gel imaging system. The 427bp fragment was defined as TT type, the three fragments of 427bp, 250bp, and 177bp were defined as TC type, and the two fragments of 250bp and 177bp were defined as CC type.

[0010] 4) One-way ANOVA was used to compare the differences in semen quality indicators among different genotypes. The results showed that the semen quality of CC-type dairy cows was significantly higher than that of TT-type cows. CC-type and TC-type individuals were selected for breeding.

[0011] Preferably, the semen quality data of the 56 Holstein dairy cows and their semen were provided by Shandong Aux Seed Industry Co., Ltd., and the semen quality data included semen volume, fresh semen motility, sperm density and sperm abnormality rate.

[0012] Preferably, the specific primer sequence in step 2) is:

[0013] P1: GCAGAGCGTGTCTTAGTTA (SEQ ID NO.1),

[0014] P2: CCGTCAGGTACTCCAACA (SEQ ID NO. 2).

[0015] Preferably, the miR-6531 precursor region 73T / C polymorphic site is obtained from the bovine SNP public database (http: / / genome.ucsc.edu / cgi-bin / hgTables; Bos_taurus_UMD3.1.1; SNP14) and screened using miRNAQTLsnp software.

[0016] Preferably, in step 3), the BspE I restriction site is TCTGGA→TCCGGA, the BspE I restriction endonuclease is purchased from ThermoFisher, and the gel imaging system is a Tanon 3500 purchased from Shanghai Tianneng Company.

[0017] Preferably, the miR-6531 precursor region sequence is NC_037350.1, 76964883-76965460 bp.

[0018] Preferably, the total PCR reaction volume is 20 μL, containing 50-100 ng of template DNA, 10 μL of 2xTaq polymerase Mix, 0.5 μL of each primer, and sterile double-distilled water to a final volume of 20 μL. The PCR reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

[0019] Preferably, the enzyme digestion reaction system consists of 20 μL of PCR product, 5 μL of PCR product, 0.5 μL of 10 U / μL BspE I enzyme, 2 μL of 10× buffer, and 12.5 μL of sterile double-distilled water; the enzyme digestion is carried out at 55°C for 3 h.

[0020] Preferably, the significant differences in semen quality among different miR-6531 genotypes of Holstein dairy cows were analyzed using the One-way ANOVA method in SPSS 10.

[0021] Preferably, it includes, but is not limited to, the molecular markers and detection methods disclosed above.

[0022] The beneficial effects of this invention are:

[0023] 1. The inventors, for the first time, screened the miR-6531 precursor region 73T / C polymorphic site in a dairy bull population using the bovine SNP public database (http: / / genome.ucsc.edu / cgi-bin / hgTables; Bos_taurus_UMD3.1.1; SNP14) and miRNAQTLsnp software. Three genotypes were found: TT, TC, and CC. The frequency of the CC genotype was lower than that of the TC and TT genotypes. Association analysis between each genotype and dairy cow semen quality showed that the 73T / C site was significantly associated with sperm motility and sperm abnormality rate in dairy cows. This method can improve the accuracy of dairy cow semen quality selection and can be used for ultra-early breeding selection.

[0024] 2. This invention uses PCR-RFLP to detect the polymorphism of the 73T / C locus in the miR-6531 precursor region and performs association analysis between genotype and semen quality traits in dairy cows. The results show that the polymorphism of the 73T / C locus in the miR-6531 precursor region is significantly correlated with sperm motility and sperm abnormality rate in Holstein dairy cows. CC-type cows have higher sperm motility than TT and TC-type cows, and a lower sperm abnormality rate than TT and TC-type cows. This polymorphic locus can be used for auxiliary selection of Holstein dairy cow semen quality, improving the accuracy of selection. Furthermore, selection can be performed at birth by detecting the genotype of male calves, accelerating the breeding process and reducing breeding costs. Attached Figure Description

[0025] Figure 1 The figure shows the electrophoretic DNA quality detection results of Holstein cow DNA extracted in this invention. M in the figure is the DL5000 marker. Figure 1 A is an electrophoresis image of the genomic DNA of dairy cows 1-48. Figure 1 B is an electrophoresis image of the genomic DNA of dairy cows 49-56.

[0026] Figure 2 The figure shows the PCR amplification results of the precursor region sequence of Holstein dairy cow miR-6531 in this invention. M in the figure is the DL2000 marker. Figure 2 Image A shows the amplified miR-6531 precursor region sequence of dairy cows 1-48. Figure 2 B is a diagram of the amplified product of the miR-6531 precursor region sequence from dairy cows 49-56.

[0027] Figure 3 This is a sequencing image of the miR-6531 precursor region 73 T / C mutation site in Holstein cows, as described in this invention.

[0028] Figure 4This is an electrophoresis image of the miR-6531 precursor region 73T / C site from Holstein dairy cows after BspEI digestion. Lane M in the image is the DL5000 marker, and TT, TC, and CC represent the TT, TC, and CC genotypes of the miR-6531 precursor region PCR amplification product from Holstein dairy cows after BspEI digestion. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example: Holstein cow sperm motility miRNA molecular marker selection method

[0031] (a) Sample and semen quality trait data

[0032] Fifty-six healthy adult Holstein dairy cows were selected. Data on the Holstein cows and their semen quality were provided by Shandong AUX Seed Industry Co., Ltd. Semen was collected artificially from each cow, placed in 1.5ml Eppendorf tubes, and stored in an ice box at -20℃ after being brought back to the laboratory. Semen quality data included semen volume, fresh semen motility, sperm density, and sperm abnormality rate.

[0033] (II) Methods for Selecting Sperm Motility miRNA Molecular Markers

[0034] 1) Sperm DNA was extracted using the conventional phenol and chloroform method, and DNA quality was assessed by electrophoresis and OD260 / 280 measurement. Figure 1 As shown;

[0035] 2) Based on the GenBank sequence NC_037350.1, a pair of specific primers were designed using Primer Premier 6. The primer sequences are as follows:

[0036] P1: GCAGAGCGTGTCTTAGTTA (SEQ ID NO.1),

[0037] P2: CCGTCAGGTACTCCAACA (SEQ ID NO.2),

[0038] The precursor region sequence of Holstein dairy bovine miR-6531 (sequence NC_037350.1, 76964883-76965460 bp) was amplified by PCR. The PCR amplification products were analyzed by 1% agarose gel electrophoresis and the amplification results were detected by ethidium bromide (EB) staining. Figure 2 As shown;

[0039] In the above steps, the total PCR reaction volume is 20 μL, template DNA is 50-100 ng, 2xTaq polymerase Mix is ​​10 μL, primers are 0.5 μL each, and sterile double-distilled water is added to a final volume of 20 μL.

[0040] The PCR reaction program in the above steps is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

[0041] 3) Based on the bovine SNP public database (http: / / genome.ucsc.edu / cgi-bin / hgTables; Bos_taurus_UMD3.1.1; SNP14) and using miRNAQTLsnp software, the miR-6531 precursor region 73T / C polymorphic site was identified. A T→C mutation exists at position 73 in the miR-6531 precursor region of Holstein dairy cows. Figure 3 As shown, a BspE I restriction site (TCTGGA→TCCGGA) was generated. The amplified product was digested with BspE I restriction endonuclease. The digested products were detected by 2% agarose gel electrophoresis, stained with ethidium bromide (EB), and photographed using a gel imaging system. The 427bp fragment was defined as TT type, the three fragments of 427bp, 250bp, and 177bp were defined as TC type, and the two fragments of 250bp and 177bp were defined as CC type.

[0042] In the above steps, the enzyme digestion reaction system consisted of 20 μL, PCR product 5 μL, 10 U / μL BspE I enzyme 0.5 μL, 10× buffer 2 μL, and sterile double-distilled water 12.5 μL; the enzyme digestion was carried out at 55℃ for 3 h.

[0043] In the above steps, the BspE I restriction endonuclease was purchased from ThermoFisher, and the gel imaging system was anon 3500, purchased from Shanghai Tianneng Company.

[0044] 4) The significance of differences in semen quality among different miR-6531 genotypes in Holstein dairy cows was analyzed using the one-way ANOVA method in SPSS 10.

[0045] (III) Results and Analysis

[0046] 1) BspE I restriction polymorphism of Holstein dairy cow miR-6531 precursor region sequence

[0047] The PCR amplification products from 56 samples were digested with BspE I, revealing three genotypes. 37 samples showed a single band (427 bp), defined as the TT genotype; 12 samples showed three bands (427 bp, 250 bp, and 177 bp), defined as the TC genotype; and 7 samples showed two bands (250 bp and 177 bp), defined as the CC genotype. The results are as follows: Figure 4 As shown.

[0048] 2) Association analysis between different genotypes of Bta-miR-6531 precursor SNPs and semen traits

[0049] One-way ANOVA was used to compare the differences in various semen quality indicators among different genotypes. The results showed that the semen volume was CC type > TC type > TT type, but the differences among the three were not significant (p>0.05); the fresh sperm motility was CC type > TC type > TT type, and the difference between CC type and TT type was significant (p<0.05); the sperm density was CC type > TC type > TT type, and the difference between CC type and TT type was significant (p<0.05); the sperm abnormality rate was CC type > TC type > TT type, and the difference between CC type and TC type and TT type was significant (p<0.05) (as shown in Table 1).

[0050] Table 1. Association analysis between different genotypes of Bta-miR-6531 precursor SNPs and semen traits.

[0051]

[0052] Note: Different shoulder letters in the same line indicate significant differences (p < 0.05).

[0053] A comprehensive analysis of the above results shows that CC-type Holstein cows have higher semen motility and sperm density than TT-type cows, while their abnormality rate is lower. Therefore, this polymorphic site can be used as a molecular marker for the selection of reproductive traits in Holstein cows. To increase the frequency of the C genotype, CC-type and TC-type individuals should be selected for breeding.

[0054] The above-mentioned Holstein dairy cow sperm motility miRNA molecular marker selection method, when applied to Holstein dairy cow selective breeding operations, can perform ultra-early selection, shorten the long genetic selection cycle, accelerate genetic progress, and is of great significance to the research on genetic breeding selection of Holstein dairy cows.

[0055] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0056] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art. Specific primer nucleotide sequence listing <110> Jiangsu Academy of Agricultural Sciences <120> A method for selecting miRNA molecular markers for sperm motility in Holstein dairy cows <140> 2021112867633 <141> 2021-11-02 <160> 2 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <220> <221> source <222> (1)...(19) <223> P1 <400> 1 gcagagcgtg tcttagtta <210> 2 <211> 18 <212> DNA <213> Artificial sequence <220> <221> source <222> (1)...(18) <223> P2 <400> 2 ccgtcaggta ctccaaca

Claims

1. An application of PCR-RFLP method for detecting the polymorphism of the 73T / C site in the precursor region of miR-6531 in Holstein dairy cow semen quality breeding selection, characterized in that: The method includes the following steps: 1) Holstein cows were selected, 1.5 ml of semen was artificially collected, the semen quality data were measured, and sperm DNA was extracted using the conventional phenol and chloroform method. The DNA quality was detected by electrophoresis and OD260 / 280 measurement. 2) Based on the GenBank sequence NC_037350.1, a pair of specific primers were designed using Primer Premier 5 software to amplify the precursor region sequence of Holstein dairy cow miR-6531 by PCR. The PCR product was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sequence of the specific primer is as follows: P1: GCAGAGCGTGTCTTAGTTA; P2: CCGTCAGGTACTCCAACA; 3) A T→C mutation exists at position 73 of the miR-6531 precursor region in Holstein dairy cows, resulting in a BspE I restriction site. The amplified product was digested using BspE I restriction endonuclease. The digested products were detected by 2% agarose gel electrophoresis, stained with ethidium bromide, and photographed using a gel imaging system. The 427bp fragment was defined as TT type, the three fragments of 427bp, 250bp, and 177bp were defined as TC type, and the two fragments of 250bp and 177bp were defined as CC type. The miR-6531 precursor region 73T / C polymorphic site was obtained from the bovine SNP public database http: / / genome.ucsc.edu / cgi-bin / hgTables;Bos_taurus_UMD3.1.1;SNP14 and screened using miRNAQTLsnp software. The BspE I restriction site is TCTGGA→TCCGGA; One-way ANOVA was used to compare the differences in semen quality indicators among different genotypes. The results showed that the semen quality of CC-type dairy cows was significantly higher than that of TT-type cows. CC-type and TC-type individuals were selected for breeding. The semen quality was defined as fresh semen motility, sperm density, and sperm abnormality rate. CC-type Holstein cows had higher fresh semen motility and sperm density than TT-type cows, while their sperm abnormality rate was lower than that of TT-type cows.

2. The application according to claim 1, characterized in that: The BspE I restriction endonuclease was purchased from ThermoFisher, and the gel imaging system was a Tanon 3500, purchased from Shanghai Tianneng Co., Ltd.

3. The application according to claim 1, characterized in that: The total PCR reaction volume was 20 μL, containing 50-100 ng of template DNA, 10 μL of 2xTaq polymerase Mix, 0.5 μL of each primer, and sterile double-distilled water to a final volume of 20 μL. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min.

4. The application according to claim 1, characterized in that: The enzyme digestion reaction system consisted of 20 μL of PCR product, 5 μL of PCR product, 0.5 μL of 10 U / μL BspE I enzyme, 2 μL of 10× buffer, and 12.5 μL of sterile double-distilled water; the enzyme digestion was carried out at 55℃ for 3 h.

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