Application of recognition site mutation of OCT4 in breeding of cattle with number of spines and number of ribs

Abstract

The invention relates to application of recognition site mutation of OCT4 in breeding of cattle spine number and rib number. A recognition site of the OCT4 is mutated into 86142634bp of chromosome 10 of a cattle genome, a basic group is mutated into C from T, and the version number of the genome is AR-CD2.0; during breeding, individuals with the genotype of the SNP molecular marker CC are reserved, and individuals with the genotype of TC or TT are eliminated, so that the spine number and the rib number of offspring cattle are increased. The SNP molecular marker related to the spine number and the rib number of the cattle is screened and identified, during cattle breeding, the CC genotype cattle of the SNP molecular marker is selected and reserved, the spine number and the rib number of offspring cattle can be effectively increased, and a cattle strain with the character of high spine number and rib number is obtained; the genetic improvement progress and the breeding progress of the breeding cattle are accelerated.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and genetic breeding, and in particular to the application of OCT4 recognition site mutation in the breeding of vertebrae and rib numbers in cattle. Background Art

[0002] The number of thoracic vertebrae in cattle ranges from 12 to 17, while the number of lumbar vertebrae ranges from 5 to 7. Thoracic and lumbar vertebrae numbers are closely related to body length and meat yield, making them important economic traits. Selecting and improving the number of thoracic and lumbar vertebrae has significant economic value.

[0003] This research team previously investigated SNP molecular markers associated with vertebrae and rib number in pig breeding, confirming that knocking out the FOS gene increases vertebrae and rib number in pigs. This indicates that the FOS gene is a major gene associated with vertebrae and rib number in livestock (see Chinese Patent 202510513139.4). Subsequently, they confirmed that the recognition site mutation chr7-98095075: T>C in the porcine OCT4 gene significantly increases vertebrae and rib number traits, with the CC genotype having an average of 1.3 and 1.2 more ribs than the TT genotype (see Chinese Patent 202510582713.1). This research team's previous research confirmed that mutations in the OCT4 gene recognition site are associated with vertebrae and rib number in livestock. By screening for key mutations in the OCT4 gene recognition site associated with vertebrae and rib number in various livestock species, it may be possible to identify SNP molecular markers associated with vertebrae and rib number in these species, thereby guiding breeding of these livestock species.

[0004] Building on previous research by our team, we subsequently identified the recognition site chr7-84222431 in the sheep OCT4 gene: G > C. The C allele significantly increases vertebrae and rib number in sheep. Cattle are an important livestock breed in my country, but there are currently no reports of molecular markers in the Oct4 gene associated with vertebrae and rib number in cattle. Summary of the Invention

[0005] In order to solve the above problems, the present invention proposes the application of the recognition site (OCT4-Motif) mutation of OCT4 in the breeding of vertebrae and rib numbers in cattle.

[0006] The present invention provides an application of a SNP molecular marker associated with the number of vertebrae and ribs in cattle breeding. The SNP molecular marker is located at the recognition site of the cattle OCT4 gene, i.e., bp 86142634 on chromosome 10 of the cattle genome, the base is mutated from T to C, and the genome version number is ARS-µCD2.0. During breeding, the number of vertebrae and ribs in offspring cattle is increased by retaining individuals with a CC genotype of the SNP molecular marker and eliminating individuals with a TC or TT genotype.

[0007] Further, the primer combination for identifying the SNP molecular marker comprises a forward primer with the nucleotide sequence shown in SEQ ID NO. 1 and a reverse primer with the nucleotide sequence shown in SEQ ID NO. 2.

[0008] Further, the yellow cattle bone marrow mesenchymal stem cell line overexpresses the OCT4 gene.

[0009] The yellow cattle bone marrow mesenchymal stem cell line of the present application is applied in yellow cattle breeding.

[0010] The present application has the following beneficial effects: The present application screens and identifies a SNP molecular marker (chr10-86142634:T>C) related to the number of vertebrae and the number of ribs of yellow cattle, the different genotypes of the marker have extremely significant differences in the affinity of OCT4 protein, the genotype CC significantly down-regulates the expression of FOS gene, the C allele significantly increases the affinity of OCT4 protein and down-regulates the expression of FOS gene, and the CC genotype is the dominant genotype. In yellow cattle breeding, by selecting and keeping yellow cattle with the SNP molecular marker CC genotype, the number of vertebrae and the number of ribs of offspring yellow cattle can be effectively increased, yellow cattle strains with high number of vertebrae and high number of ribs are obtained, and the genetic improvement progress and breeding process of breeding yellow cattle are accelerated. The present application simultaneously provides primer sequences for PCR detection of the SNP molecular marker related to the number of vertebrae and the number of ribs of yellow cattle. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 OCT4 gene expression amount and OCT4 protein affinity experimental results of the OCT4 overexpressed yellow cattle bone marrow mesenchymal stem cells of Example 1; Figure 2 DNA pull down experiment of Example 1 to prove that the OCT4 protein of the OCT4-CBMSC cells is significantly higher than that of the wild type pLVX-CBMSC cells; Figure 3 Schematic diagram of luciferase reporter gene plasmid construction of Example 2; Figure 4 Fluorescent double reporter experiment of Example 2 to prove that the C allele of the OCT4-Motif significantly down-regulates the expression of the FOS gene compared with the T allele. DETAILED DESCRIPTION

[0012] The present application is further described below in combination with examples.

[0013] Example 1, cell function experiment to prove that the OCT4-Motif mutation can enhance the affinity of the OCT4 protein:

[0014] Previous studies by this research team have obtained two key pieces of evidence: (1) The domestic pig OCT4-Motif mutation (chr7-98095075: T>C) increases the number of vertebrae and ribs; (2) Overexpression of the Oct4 gene in mice can increase the number of thoracic and lumbar vertebrae in offspring mice by at least 6. The domestic pig OCT4-Motif mutation was compared with the µCSC database (https: / / genome.µcsc.edµ / index.html). Through homologous sequence analysis, it was found that the cattle genomic locus that is orthologous to the domestic pig OCT4-Motif genomic locus also has a mutation, which is also annotated as the cattle OCT4-Motif (using the ARS-µCD2.0 version as the reference genome, chr10-86142634: T>C). It is speculated that this OCT4-Motif mutation is one of the causal mutations that changes the number of vertebrae and ribs in cattle.

[0015] To verify this hypothesis, we obtained a primary cultured cattle bone marrow mesenchymal stem cell line (CBMSC). RT-PCR and Western blot detection showed that CBMSC hardly expressed OCT4. To verify whether there was a difference in the affinity of the OCT4-Motif mutation and the OCT4 protein, we constructed an OCT4-overexpressing CBMSC cell line (OCT4-CBMSC) based on CBMSC cells.

[0016] Experiment 1: Primary culture of cattle bone marrow mesenchymal stem cells: 1.1.1. Cattle Processing: For newly born calves, perform neck dislocation. Completely remove the tibia and femur from the hind legs using surgical instruments and soak in 75% ethanol for 5 minutes. 1.1.2. Cleaning: Use surgical instruments to remove the periosteum and muscle tissues, then wash the tibia and femur twice in DPBS containing 2% SP; 1.1.3. Culture medium flushing: Remove the cartilage from both ends of the tibia and femur. Use a sterile needle to pierce a small hole at one end of the bone. Use a 50mL syringe filled with MSC culture medium to flush out the bone marrow. Repeat the flushing process and pass the cells through a 40µm cell sieve to prepare a single-cell suspension (MSC culture medium composition: 10-20% (v / v) FBS, 0.5-2% (v / v) MEM NEAA, 0.5-2% (v / v) sodium pyruvate, 0.5-2% (v / v) glutamine, 5-20 ng / ml FGF, and α-MEM basal culture medium). 1.1.4 Centrifugation: Transfer the washed cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 10 min at room temperature. Discard the supernatant. 1.1.5 Cell culture: Culture the cells in an incubator for 24 hours, replace with fresh MSCs culture medium, remove non-adherent cells, and replace the culture medium every 2-3 days until the cells reach 80-90% confluence.

[0017] Experiment 2: Construction of OCT4-overexpressing cattle bone marrow mesenchymal stem cell (OCT4-CBMSC) cell line: 1.2.1. Synthetic sequence: Based on the CDS sequence of OCT4 or the corresponding NCBI NM number (XM_004018968.5), the sequence to be synthesized was determined to be 1083 bp in length, with Xho I and EcoR I restriction sites added at both ends.

[0018] 1.2.2 DNA Digestion: Reaction System 1: 16 μl (1 μg) of gene fragment; 2 μl of 10× Bµffer H; 1 μl (15 μg) of XhoI; 1 μl (15 μg) of EcoRI; add HO to 20 μl. Reaction System 2: 5 μl (1 μg) of pLVX-AcGFP-N1; 2 μl of 10× Bµffer H; 1 μl (15 μg) of XhoI; 1 μl (15 μg) of EcoRI; add HO to 20 μl. Incubate at 37°C overnight.

[0019] 1.2.3 DNA Ligation: Ligation reaction system: 1 μl of 10× Ligation Bµffer; 5 μl (0.2 pmol) of enzyme-digested gene fragment; 2.2 μl (0.03 pmol) of pLVX-AcGFP-N1 vector; 1 μl of PEG4000; 0.8 μl (350 μl) of T4 DNA Ligase. Incubate at 22°C for 16 h.

[0020] 1.2.4. Transformation of ligation products: Inoculate the frozen Stbl3 in glycerol and culture it upside down at 37℃ overnight; pick a single colony into a test tube containing 4ml LB and shake it at 37℃ 220rpm for 12h; pipette 1ml of bacterial solution into a 1.5ml centrifuge tube, centrifuge it at 12000g at 4℃ for 3 min, and discard the supernatant; resuspend the bacterial pellet with 400μl pre-cooled CaCl2, centrifuge it at 12000g for 3min, and discard the supernatant; resuspend the bacterial pellet again with 200μl pre-cooled CaCl2 and place it on ice overnight; add all 10μl of ligation solution to 200μl competent bacteria and place it on ice for 1h; heat shock at 42℃ for 90sec, and quickly place it on ice for 5min; add 600μl of 37℃ pre-heated LB culture medium; shake it at 37℃, 220rpm for 1h, and after centrifugation, spread the whole on a plate containing 50μg / ml Amp LB plates were incubated upside down at 37°C overnight.

[0021] 1.2.5. Identification of positive clones: Randomly select 4 single clones and transfer them to a test tube containing 4 ml of LB medium containing 100 μg / ml Amp. Shake at 37°C, 220 rpm for 4 hours. Centrifuge 100 μl of the culture, collect the bacterial pellet, resuspend it in 50 μl of ddH2O, boil it in a water bath for 5 minutes, centrifuge it, and collect 1 μl of the supernatant as a template for PCR identification. PCR system: 10× Taq Reaction Buffer 5 μl; supernatant 1 μl; F1 (20 mM) 1 μl; R2 (20 mM) 1 μl; dNTP (2.5 mM) 4 μl; Mg 2+ 3μl; Taq Polymerase 0.5μl (2.5µg); add H2O to 50μl. PCR reaction conditions: 94°C for 5 minutes; 25 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute; 72°C for 10 minutes. After the reaction, a 10μl aliquot was analyzed by 1.0% agarose gel electrophoresis.

[0022] 1.2.6. Sanger sequencing: Sequence the positive clones identified by colony PCR.

[0023] 1.2.7 Cell digestion: 293FT cells were digested and seeded into 10 cm dishes. Lentivirus Lv-pLVX and Lv-OCT4 virus solutions were packaged.

[0024] 1.2.8. Cell transfection: Terminate the infection and collect the viral fluid 48 hours later. Filter through a 0.45 µm filter, aliquot, and store at -80°C until use. Digest 293FT cells and seed 6-well plates at 2 × 105 cells / well for virus titer testing. Collect the lentiviruses Lv-pLVX and Lv-OCT4 and infect CBMSC cells. Concentrate the viral fluid and use the wild-type pLVX-CBMSC control cell line transfected with the lentivirus Lv-pLVX. Use the OCT4-CBMSC cell line overexpressing OCT4 to transfect the lentivirus Lv-OCT4.

[0025] 1.2.9 Cell culture and drug screening: pLVX-CBMSC and OCT4-CBMSC cell lines were cultured for drug screening at a concentration of 1µg / ml. The culture medium was replaced after 48 hours and the cells were passaged.

[0026] 1.2.10. RT-PCR: RNA was extracted from pLVX-CBMSC and OCT4-CBMSC cells using the Trziol method. RNA concentration and quality control were performed using a Nanodrop 2000 micro-spectrophotometer. The 260 / A280 value was 1.95. 2-5 μL RNA samples were collected for agarose gel electrophoresis. 1 μg RNA was reverse transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, RR047A) to obtain cDNA, which was immediately used for RT-PCR. The expression of the OCT4 gene in pLVX-CBMSC and OCT4-CBMSC cells was detected by RT-PCR. 2 -ΔΔCt Calculate the relative expression of genes, such as Figure 1 As shown in A, the ordinate is the relative expression level of the OCT4 gene. The relative expression level of the OCT4 gene in the wild-type pLVX-CBMSC cells is 0.01, and the relative expression level of the OCT4 gene in the OCT4-CBMSC cells is 530, indicating that the expression level of the OCT4 gene in the OCT4-CBMSC cells is significantly increased.

[0027] 1.2.11. Western blot analysis, as follows: Protein extraction and quantification: Take an appropriate amount of pLVX-CMSC and OCT4-CMSC cells, add 200-500 µl of RIPA lysis buffer, and lyse the cells using a cell disruptor. Lyse on ice for 20 minutes. After lysis, transfer the lysate to a centrifuge tube and centrifuge at 14,000 g for 15 minutes at 4°C. Transfer the supernatant to a fresh centrifuge tube. Add 25 µl of the test sample or standard to a centrifuge tube containing 200 µl of BCA protein detection reagent (Solution A:Solution B = 50:1). Protein quantification is performed using the standard curve method. Protein separation: Add 5× protein loading buffer to the test sample, mix thoroughly, and incubate at 98°C for 5 minutes. Centrifuge briefly until bubbles disappear. Load 20 µg of the treated sample onto an SDS-PAGE gel and separate the proteins according to their molecular weight using electrophoresis at 60 V for 30 minutes or 120 V for 90 minutes. Transfer: After electrophoresis, remove the stacking gel and transfer the proteins on the separation gel to the solid support (PVDF membrane). The electrophoresis program is 200mA for 1h to fix the proteins on the membrane. Blocking: After electrophoresis, use washing buffer (1×TBST) to wash at room temperature for 10min, then block the membrane with skim milk or BSA for 1 hour. After blocking, wash it 3 times with 1×TBST. This step blocks non-specific binding sites and reduces background. Antibody incubation: Incubate the membrane with a 1:1000 primary antibody (diluted in blocking buffer) at 4°C overnight, then wash it three times with 1×TBST to remove unbound antibodies. Add a 1:2000 diluted enzyme-labeled secondary antibody (such as HRP-labeled), incubate at room temperature for 1 hour, and wash it three times with 1×TBST again. Detection: Use ECL chemiluminescence reagent, HRP catalyzes the substrate to emit light, and develop it through an imaging system. Such as Figure 1 As shown in B, the OCT4 protein level in OCT4-CBMSC cells was significantly higher than that in wild-type pLVX-CBMSC cells.

[0028] Experiment 3: DNA pull-down assay to detect differences in affinity between the above OCT4-Motif mutation genotypes and OCT4 protein: 1.3.1 Annealing of the biotinylated probe into a double-stranded structure: Prepare 10× annealing buffer with the following system: 100 μl 1M Tris-HCl (pH 7.5); 20 μl 0.5M EDTA; 200 μl 5M NaCl; 100 μl ddH2O. Set up the biotinylated probe annealing reaction with the following system: F (10p) 10 μl; R (10p) 10 μl; 10× annealing buffer 10 μl; DEPC water 70 μl.

[0029] The probe sequences are: motif-WT: 5'-ATTGCTCCTCTTCACTGTGTATTACAGTGTCAATTTGGCA-3' (SEQ IDNO.3); motif-MT: 5'-ATTGCTCCTCTTCACTGTGCATTACAGTGTCAATTTGGCA-3' (SEQ ID NO. 4).

[0030] After adding all the reagents, mix the 0.2ml tube by inverting it. Centrifuge at 1500 rpm to perform the PCR reaction. PCR program: 99°C for 10 min; 99°C for 1 min, 74 cycles, each cycle at -1°C; 25°C hold.

[0031] After the biotin-labeled probe annealing reaction, store the product at -30°C until use. Quantify the annealed double-stranded probe using the Qµbit® dsDNA HS Assay Kit (Thermo, Q32854), following the manufacturer's instructions. Prepare the reagents needed for subsequent experiments, sterilize them by autoclaving, and store them at 4°C until use.

[0032] 1.3.2. Binding of biotinylated probe to magnetic beads: Pipette 50 μl of Pierce™ Streptavidin Magnetic Beads (Thermo, Cat. No. 88816) into a clean 1.5 ml centrifuge tube, place the centrifuge tube on a single-tube magnetic stand, and carefully remove the supernatant, leaving the magnetic bead pellet. Pipette 50 μl of 20 mM Tris (pH 7.5) into the 1.5 ml centrifuge tube, pipette up and down to mix with the magnetic beads, place the centrifuge tube on a single-tube magnetic stand, and carefully remove the supernatant, leaving the magnetic bead pellet. Repeat this step once. Pipette 50 μl of 1× Capµtµre Bµffer into the 1.5 ml centrifuge tube (1× Capµtµre Bµffer ingredients: Tris-HCl (pH 7.5) 20 mM; NaCl 1000 mM; EDTA 1mM), pipette up and down with the magnetic beads to mix, place the centrifuge tube on a single-tube magnetic stand, carefully remove the supernatant, and leave the magnetic bead pellet; draw up a concentration of 4μg of biotin-labeled probe and add it to a 1.5ml centrifuge tube. Pipet up and down to mix with the magnetic beads, and place the centrifuge tube on a DNA mixer, 15rpm / min, room temperature, and slowly rotate to mix for 30 minutes.

[0033] 1.3.3. Binding of transcription factors to biotin-labeled probes: Remove the 1.5ml centrifuge tube from the DNA mixer, place the tube on a single-tube magnetic stand, and carefully remove the supernatant, leaving the magnetic bead pellet. Pipette 50μl of 20mM Tris (pH7.5) into the 1.5ml centrifuge tube, pipette up and down to mix with the magnetic beads, place the tube on a single-tube magnetic stand, and carefully remove the supernatant, leaving the magnetic bead pellet. Repeat once. Pipette 100μl of 1×Binding Bµffer (1×Binding Bµffer ingredients: Tris-HCl (pH 7.5) 200mM; NaCl 500mM; MgCl2 20mM; Tween-20 1%) into the 1.5ml centrifuge tube, pipette up and down to mix with the magnetic beads, place the tube on a single-tube magnetic stand, and carefully remove the supernatant, leaving the magnetic bead pellet. Prepare the Master Mix system as follows: 10×binding bµffer 40μl; Glycerol (15% final concentration) 60μl; Protease inhibitor 4μl; Total protein 500μg; HO to 400μl; Total 400μl. Pipette 400μl of MasterMix into a 1.5ml centrifuge tube. Pipet up and down to mix with the magnetic beads. Place the centrifuge tube in a DNA mixer and rotate gently at 15 rpm at 4°C for 1 hour.

[0034] 1.3.4. Combined transcription factor elution and Western blot analysis: Remove the 1.5ml centrifuge tube from the DNA mixer and place it on a single-tube magnetic stand. Carefully remove the supernatant, leaving the magnetic bead pellet. Pipette 200 μl of 1×Wash Bµffer (1×Wash Bµffer ingredients: Tris-HCl (pH 7.5) 20mM; NaCl 10mM; Tween-20 0.1%) into the 1.5ml centrifuge tube. Pipet up and down to mix with the magnetic beads. Place the centrifuge tube on a single-tube magnetic stand and carefully remove the supernatant, leaving the magnetic bead pellet. Repeat the previous elution step three times. Pipette 30 μl of 2×Protein Loading Add Bµffer to a 1.5ml centrifuge tube and mix with the magnetic beads by pipetting up and down. Place the centrifuge tube in a metal bath at 100°C and incubate for 10 minutes. After 10 minutes, place the 1.5ml centrifuge tube on a single-tube magnetic stand and collect the supernatant in a clean 1.5ml centrifuge tube for Western blot analysis. Follow the same steps as in 1.2.11.

[0035] This study focused on the cattle OCT4-Motif mutation site chr10-86142634:T>C. A DNA probe of 41 bp, 20 bp before and after the mutation site, was synthesized and labeled with biotin. Nuclear proteins of the OCT4-CBMSC cell line were extracted and Western blot analysis was performed. The results showed that the OCT4-Motif allele (C) had a stronger affinity for OCT4 protein than its wild type (T). Figure 2 ).from Figure 2 It can be seen that motif-MT is a mutant type, motif-WT is a wild type, and the motif-MT probe captures more OCT4 protein, so the OCT4-Motif allele type (C) has a stronger affinity for OCT4 protein than its wild type (T).

[0036] Example 2: Verification that the cattle OCT4-Motif allele (C) significantly down-regulates the expression of the major gene FOS for vertebrae and rib numbers relative to its wild type (T): To verify that the OCT4-motif mutation chr10-86142634:T>C regulates the expression of the major gene FOS (approximately 350 kb from the OCT4-motif) responsible for vertebrae and rib number, a luciferase reporter assay was performed. The sequences, plasmids, and vectors used in this example were purchased commercially.

[0037] First, construct a luciferase reporter plasmid: Based on the promoter sequence of the FOS gene and the MCS site of pGL3-basic, select a 2000bp sequence upstream of the transcription start site, add Bgl II and Hind III restriction site sequences at both ends, and then ligate this sequence into the pGL3-basic vector after gene synthesis. Sequencing verifies that the inserted sequence is correct and constructs the FOSpro plasmid. The wild-type Motif fragment is cloned between Kpn I and Sac I of the FOSpro plasmid to construct the motif-WT-FOSpro plasmid. The mutant Motif fragment is cloned between Kpn I and Sac I of the FOSpro plasmid to construct the motif-MT-FOSpro plasmid ( Figure 3 ).

[0038] Then, luciferase activity was detected: OCT4-CBMSC cells were cultured at 1.5×10 5Cells were plated in 24-well plates with six replicate wells per group. Transfection was performed when the cells reached 60-70% confluency. The transfection system consisted of the following: transfection plasmid (0.5 µg per well); PRL-TK (25 ng per well); Lipo3000 (1.5 µl); and Opti-MEM (µg to 50 µl). The transfection reagents were mixed and incubated at room temperature for 15-20 minutes before being added to the cell culture plates. Samples were collected 24 hours after transfection for analysis. The plates were then washed three times with 1× PBS. 100 µl of 1× PLB lysis buffer was added to each well. The plates were shaken at room temperature for 15 minutes, centrifuged briefly, and the cell supernatant was collected. 20 µl of the supernatant was added to a dark-protected assay plate. Each well was reacted with 100 µl of LAR II for 30 seconds to measure the activity of the reporter gene, firefly luciferase. The activity of the reference gene, Renilla luciferase, was then measured with 100 µl of Stop & Glo® Reagent for 30 seconds. The relative activity value was obtained by comparing the firefly luciferase activity value to the Renilla luciferase activity value, and the data were normalized by eliminating the interference of cell number and transfection efficiency.

[0039] Finally, the difference in luciferase activity between the wild-type and mutant OCT4-Motif plasmids was analyzed.

[0040] Figure 3 This is a schematic diagram of the construction of the luciferase reporter gene experimental plasmid in this example. Figure 4 For the comparison of luciferase activity in this example, T test showed that the OCT4-Motif mutant (CC, motif-MT-FOSpro plasmid) in the experimental group significantly downregulated the expression of FOS compared with the wild type (TT, motif-WT-FOSpro plasmid).

[0041] FOS is a major gene related to the number of vertebral ribs in livestock confirmed in previous studies. When FOS is knocked out, the number of vertebral ribs in pigs increases. Therefore, the CC type of the recognition site of cattle OCT4 significantly downregulates the expression of FOS compared with the wild type (TT), which means that in actual applications, the number of vertebral ribs of the CC type of the recognition site of cattle OCT4 will increase.

[0042] Example 3: A method for genetically improving the number of vertebrae and ribs in cattle: In summary, a method for genetically improving the number of vertebrae and ribs in cattle can be obtained. The key lies in detecting the molecular marker (chr10-86142634:T>C) of individual cattle; selecting cattle with a genotype of CC and eliminating those with genotypes of TT or TC; breeding the selected cattle, and continuing to select cattle with a genotype of CC (chr10-86142634:T>C) from the offspring, and eliminating those with genotypes of TT or TC; thereby increasing the frequency of the allele C in the offspring cattle population generation by generation, thereby improving the vertebrae and rib number traits of the offspring cattle.

[0043] The molecular markers were detected by PCR amplification, and the primer sequences were: Forward primer: 5′-TTGGACCAGGAGTGTGTCAG-3′ (SEQ ID NO: 1); Reverse primer: 5′-AGCCTAACCAACCCATTGCT-3′ (SEQ ID NO: 2), PCR amplification methods include: (1) Extracting genomic DNA from the cattle to be tested; (2) Using genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NO: 1-2; (3) Analyze PCR amplification products.

[0044] The amplification system used in the PCR reaction was as follows (50 μl): 100 ng / μl template DNA 1 μl, 10 pmol / μl forward primer and reverse primer 1 μl each, 10 mmol / L dNTP Mixtμre 1 μl, 1.25μ / 25μl TaKaRa Ex Taq DNA polymerase 1 μl, 2× PCR reaction buffer 25 μl, and the balance was double-distilled water.

[0045] The PCR reaction program was as follows: PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 62°C annealing for 30 s, 72°C extension for 30 s, for a total of 32 cycles; 72°C insulation for 5 min; 4°C hold.

[0046] The molecular markers were detected by the above PCR amplification method: In cooperation with a slaughterhouse, tissue samples and vertebra number phenotypes of approximately 300 cattle were collected, and the above-mentioned PCR amplification method was used to detect the molecular marker. It was found that 10 of the 300 cattle were individuals with chr10-86142634:T>C and a genotype of CC. Among these 10 cattle, 9 had 14 vertebrae, while the other 291 individuals had 13 vertebrae. This indicates that cattle individuals with the genotype CC of the molecular marker have one more vertebrae than the wild type. In cattle breeding, it is only necessary to select cattle individuals with the genotype CC of the molecular marker (chr10-86142634:T>C), eliminate individuals with the genotype TT or TC, and gradually increase the frequency of the allele C in the offspring cattle population. This can improve the vertebrae and rib number traits of offspring cattle and accelerate the breeding process of improved cattle.

[0047] 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 embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0048] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Application of a SNP molecular marker associated with the number of vertebrae and ribs in cattle breeding, characterized in that: The SNP molecular marker is located at the recognition site of the cattle OCT4 gene, that is, at 86142634bp on chromosome 10 of the cattle genome, the base mutates from T to C, and the genome version number is ARS-UCD2.0; during breeding, the number of vertebrae and ribs of offspring cattle is increased by retaining individuals with the SNP molecular marker genotype CC and eliminating individuals with the genotype TC or TT genotype.

2. The use of the SNP molecular marker associated with the number of vertebrae and ribs of cattle in sheep breeding according to claim 1, characterized in that: The primer combination for identifying the SNP molecular marker includes a forward primer whose nucleotide sequence is shown as SEQ ID NO.1 and a reverse primer whose nucleotide sequence is shown as SEQ ID NO.

2.

3. A cattle bone marrow mesenchymal stem cell line, characterized in that: The cattle bone marrow mesenchymal stem cell line overexpresses the OCT4 gene.

4. Use of the cattle bone marrow mesenchymal stem cell line according to claim 3 in cattle breeding.

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