Molecular marker for identifying high-fertility holstein cows and use thereof

Abstract

This invention relates to the field of biotechnology, and particularly to a molecular marker for identifying high-fertility Holstein cattle and its application. This invention, through analysis of the RBX1 gene in bovine 150K genotype microarray data, discovered SNP sites associated with the first and last mating interval trait; it also established, for the first time, a 30x resequencing database of high- and low-fertility populations, independently identifying SNP sites in high-fertility Holstein cattle; and it provides important molecular targets for the creation of new gene-edited breeding materials and for the development of domestically produced breeding microarrays and the breeding of breeding cattle in my country.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a molecular marker for identifying highly fertile Holstein cattle and its application. Background Technology

[0002] Reproductive traits in dairy cows mainly include indicators such as age at first mating, whether the first mating resulted in pregnancy, the interval between the first and last matings, the number of matings, and the calving interval. These are important indicators reflecting the reproductive capacity of dairy cows. As single-birth animals, dairy cows typically give birth to one calf per litter, resulting in lower reproductive capacity compared to multiparous animals like pigs. In recent years, the high degree of selection for milk production performance in dairy cows has led to a decline in herd reproductive capacity. This decline in reproductive capacity has resulted in an increased rate of passive culling of cows, causing significant economic losses to farms. Furthermore, reproductive traits in dairy cows are low-heritability traits controlled by numerous genes with minor effects, meaning they are significantly influenced by factors other than genetics. This indicates that conventional breeding methods are less effective at selecting for reproductive traits than for other highly heritable traits.

[0003] With the leaps in sequencing and computing technologies, molecular markers have become central to driving molecular breeding in dairy cows. Detecting tens of thousands to millions of molecular markers (such as SNPs) distributed throughout the entire genome allows for efficient scanning of all genetic variations in an individual. By establishing association models between these molecular markers and key economic trait phenotypes in a reference population, and then applying these models to candidate populations where only genotyping has been performed, genomic breeding values ​​can be accurately estimated. This method, through high-density, unbiased marker coverage of the genome, significantly improves the accuracy and efficiency of selection breeding.

[0004] RBX1 (RING box protein-1, also known as ROC1) is a RING component of the E3 ubiquitin ligase complex, mediating the direct transfer of ubiquitin from charged E2 molecules to target substrates. E3 ubiquitin ligases determine the precise substrate specificity of ubiquitination. In nematodes, RBX1 RNAi causes significant meiotic defects, abnormal chromosome condensation during mitosis, and germ cell proliferation defects; silencing its RNA in mouse embryos leads to embryonic death. Therefore, RBX1, as an important component of E3 ligases in the ubiquitination process, may play a crucial role in cell proliferation or embryonic development, while no related research reports have been found on its application in bovine reproductive traits.

[0005] Currently, there is limited research both domestically and internationally on DNA molecular markers for dairy cow reproductive traits, and potential genetic markers related to dairy cow reproductive performance are still lacking. Therefore, identifying key genetic markers affecting dairy cow reproductive traits and using them for marker-assisted breeding is of great significance for improving bovine fertility and economic benefits. Summary of the Invention

[0006] In view of this, the present invention provides an SNP molecular marker for identifying highly fertile Holstein cattle and its application.

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

[0008] In a first aspect, the present invention provides the use of the RBX1 gene in any of the following:

[0009] Screening for molecular markers for reproductive traits in dairy cows;

[0010] The study investigates the association between the molecular markers and polymorphic distribution or reproductive traits.

[0011] In some specific embodiments of the present invention, the accession number of the RBX1 gene is 518880.

[0012] The reproductive traits described are high reproductive capacity and low reproductive capacity;

[0013] The term "high reproductive capacity" refers to a first-time pregnancy or a higher first-time pregnancy rate; the term "low reproductive capacity" refers to cows that have been mated ≥3 times and are either non-pregnant or pregnant.

[0014] In some specific embodiments of the present invention, the dairy cow is a Holstein cow.

[0015] In some specific embodiments of the present invention, the molecular marker is located on chromosome 5, and the molecular marker is g.112227956C / T.

[0016] Secondly, the present invention also provides the application of SNP sites in any of the following:

[0017] Identifying or assisting in the identification of dairy cows exhibiting different reproductive traits; or

[0018] Selecting and breeding dairy cows with different reproductive traits;

[0019] The SNP site is located on chromosome 5, and the SNP site is g.112227956C / T.

[0020] In some specific embodiments of the present invention, the reproductive trait is high reproductive capacity or low reproductive capacity;

[0021] The term "high reproductive capacity" refers to a first-time pregnancy or a higher first-time pregnancy rate; the term "low reproductive capacity" refers to cows that have been mated ≥3 times and are either non-pregnant or pregnant.

[0022] In some specific embodiments of the present invention, the dairy cow is a Holstein cow.

[0023] Thirdly, the present invention also provides a set of primers for detecting SNP sites in the bovine RBX1 gene, wherein the detection primer set has the following characteristics:

[0024] The upstream primer has the nucleotide sequence shown in SEQ ID No. 5;

[0025] The downstream primer has the nucleotide sequence shown in SEQ ID No. 6.

[0026] Fourthly, the present invention also provides a detection kit for the SNP site of the RBX1 gene in dairy cows, including the aforementioned detection primer set.

[0027] Fifthly, the present invention also provides a method for detecting the RBX1 gene polymorphism in dairy cows, comprising the following steps:

[0028] S1: Using the detection primer set or the detection kit described above, amplify the bovine genomic DNA to obtain the amplification product;

[0029] S2: The amplification product is tested to obtain the test results;

[0030] The bovine RBX1 gene includes the following SNP site: the SNP site is located on chromosome 5, and the SNP site is g.112227956C / T.

[0031] Sixthly, the present invention also provides a method for identifying or assisting in the identification of dairy cows exhibiting different reproductive traits, comprising the following steps:

[0032] Step 1: Obtain the genotype of the SNP site of the RBX1 gene in dairy cows according to the method for detecting RBX1 gene polymorphism;

[0033] Step 2: Based on the genotype of the SNP locus of the RBX1 gene in dairy cows, obtain the identification results or auxiliary identification results of dairy cows with different reproductive traits;

[0034] When the genotype of the SNP locus is GG, the dairy cow is a highly fertile dairy cow;

[0035] The term "high fertility" refers to a first-time pregnancy or a higher first-time pregnancy rate.

[0036] In a seventh aspect, the present invention also provides a method for breeding dairy cows with high reproductive capacity, comprising the following steps:

[0037] Step 1: Obtain high-fertility dairy cows by using the methods described for identifying or assisting in the identification of dairy cows exhibiting different reproductive traits;

[0038] Step 2: Select high-fertility dairy cows as parent stock for breeding.

[0039] This invention is the first to discover significant SNP sites in the RBX1 gene (accession number: 518880) and its upstream and downstream 200kb regions in an association analysis of the first and last mating interval trait in dairy cows; this invention is also the first to establish a population of 200 young cattle in a national core dairy cattle breeding farm and divide them into high-breeding and low-breeding populations based on the mating status of the farm. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0041] Figure 1 This shows the expression of RBX1 in various tissues of Holstein cattle;

[0042] Figure 2 The expression of RBX1 in the uterus, ovary, endometrial epithelial cells (BEECs), and ovarian granulosa cells (GCs) of Holstein cattle is shown. The left figure shows the relative expression of RBX1 among the reproductive tissues, and the right figure shows the mRNA expression of RBX1 relative to the highly expressed internal reference gene. Note: Different lowercase letters indicate significant differences between groups (P < 0.05).

[0043] Figure 3 The electrophoresis results of the PCR products are shown; lanes 1-6 are the mixed PCR products of Holstein bovine blood DNA, M is a 100bp DNA Marker, and H2O is a blank control with water as the template.

[0044] Figure 4 The sequencing results of PCR products in lanes 1, 2, 3, 4, 5, and 6 are shown. The sequence fragment is bovine ARS-UCD1.2 reference genome chr5:112227909:112227999. Among them, 5F is the result obtained from primer pair SEQ ID No. 5, and 5R is the result obtained from primer pair SEQ ID No. 6. All samples tested were PCR products in lane 5. Detailed Implementation

[0045] This invention discloses a molecular marker for identifying highly fertile Holstein cattle and its application. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] This invention first analyzes the overall expression of RBX1 in dairy cows using the FarmGTEx big data platform, revealing its high expression in reproductive-related tissues. Further, by collecting samples from the uterus and ovary of dairy cows and isolating endometrial epithelial cells and ovarian granulosa cells, qPCR technology was used to detect the expression of RBX1 in key reproductive-related tissues, verifying its potential impact on bovine reproductive function. Subsequently, using bovine 150K SNP gene chip data, a systematic screening was conducted on candidate genes that may affect reproductive traits in Holstein cattle, including RBX1 (RING Box Proteins-1) and its upstream and downstream molecular loci within a 200kb range. A total of 15 SNPs were obtained, among which SNP1 (g.111973840A / G), SNP3 (g.112001075G / C), SNP4 (g.112039950A / G), SNP10 (g.112202530A / G), SNP11 (g.112227956C / T), and SNP15 (g.112344772A / G) were significantly associated with the first-to-last mating interval trait in young cattle. Further analysis of mating and pregnancy data from pastures led to the division of young cattle herds into high-fertility and low-fertility groups. Genotypic analysis of the six loci significantly associated with the first-to-last mating interval in young cattle using 30× high-depth genome resequencing technology revealed significant differences in the genotypic distribution of SNP11 (g.112227956C / T) between the high- and low-fertility groups. This provides a scientific basis for screening and breeding high-fertility Holstein cattle using molecular breeding methods, and offers new insights for related research on finding key molecular genetic markers that control bovine reproductive traits.

[0047] This invention, through analysis of the RBX1 gene in bovine 150K microarray data, discovered SNP sites associated with the first and last mating interval trait; established for the first time a 30x resequencing database of high and low breeding populations; and provides important molecular targets for the creation of new gene-edited breeding materials in the future.

[0048] The molecular marker for identifying highly fertile Holstein cattle provided by this invention, and the raw materials and reagents used in its application, are all commercially available.

[0049] The present invention will be further illustrated below with reference to the embodiments:

[0050] Experimental Example 1

[0051] 1. Data

[0052] This study used the cattle Genotype-Tissue Expressionatlas (cGTEx) database (https: / / cgtex.roslin.ed.ac.uk / ) in FarmGTEx as the big data platform. All gene expression-related data in the database (Gene_expression_TPM_FarmGTEx_cattle_V0.txt.gz) were downloaded. Bioinformatics analysis tools were used to analyze the expression of the RBX1 gene in all 8742 dairy cow samples, assess the expression level of the RBX1 gene in various dairy cow tissues, and preliminarily screen its potential functional role in dairy cows.

[0053] 2. RStudio was used to screen the RBX1 gene expression data from dairy cows, retaining samples originating from Holstein cattle and removing samples treated with exogenous additives or exhibiting disease conditions. All tissues were screened individually, and samples whose expression levels significantly deviated from the population mean were removed based on a factor of three. The expression levels of the RBX1 gene in different Holstein cattle tissues were visualized using the R package ggplot2. The results showed that the RBX1 gene is expressed in multiple tissues of Holstein cattle, with highly specific expression in the ovary, uterus, and embryonic development-related tissues.

[0054] Experimental Example 2

[0055] 1. Sampling of ovarian tissue and ovarian granulosa cells from cows:

[0056] (1) Wash the dairy cow ovaries collected from the slaughterhouse repeatedly with a 0.9% sodium chloride (NaCl) solution preheated at 37°C (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin);

[0057] (2) Use sterile surgical scissors to cut off the tissue around the ovary, take a portion of the tissue (0.5g / part) and freeze it in liquid nitrogen for total RNA extraction, and rinse the remaining tissue with 75% alcohol. Then, wash the ovary 4-5 times with preheated NaCl solution (containing 100U / mL penicillin and 0.1mg / mL streptomycin).

[0058] (3) Wash the ovaries once with preheated PBS. In a sterile laminar flow hood, use a 10mL syringe to draw follicular fluid from healthy, yellow, and transparent follicles with a diameter of 2-6mm. Then filter the collected follicular fluid into a new 15mL centrifuge tube using a 40μm filter sieve, wash three times with DPBS, centrifuge at 1000g for 5min, and discard the supernatant.

[0059] (4) Resuspend GCs in preheated DMEM / F12 complete medium (containing 10% PBS and 1% antibiotics) and inoculate them in a culture dish. Then, incubate at 37°C in an incubator containing 5% carbon dioxide for 24 hours and replace with fresh medium. Replace with fresh medium every 24 to 48 hours.

[0060] (5) When the GCs have merged to 80%–90%, extract their total RNA.

[0061] 2. Sampling of uterine tissue from cows:

[0062] (1) Wash the uterus of dairy cows collected from the slaughterhouse repeatedly with a 0.9% sodium chloride (NaCl) solution preheated at 37°C (containing 100 U / mL penicillin and 0.1 mg / mL streptomycin);

[0063] (2) Use sterile surgical scissors to cut a portion of the tissue and freeze it in liquid nitrogen for total RNA extraction.

[0064] 3. RNA isolation and qRT-PCR detection

[0065] (1) Total RNA extraction:

[0066] RNA was extracted from tissues and cells using the Novizan RC101 kit. The specific steps are as follows:

[0067] Tissue extraction: Carefully remove the preserved tissue from liquid nitrogen, quickly grind it into a white powder in liquid nitrogen, and immediately transfer it to a centrifuge tube. Quickly add 1000 µL of TRIzol and shake repeatedly to mix.

[0068] Cell extraction: Add 1000 µL of TRIzol directly to the cell collection tube, pipette the mixture, and shake for 15 seconds.

[0069] Add 200 µL of chloroform, cap the tube, shake vigorously for 15 s, and let stand at 4 ℃ for 5 min; centrifuge at 12,000 rpm (~13,400 × g) at 4 ℃ for 10 min; transfer the upper aqueous phase to a new RNase-free centrifuge tube; add 1.6 volumes of Buffer RL2 (with anhydrous ethanol added) to the obtained aqueous solution and mix gently. Transfer the mixture from the previous step to RNAPure Columns (already placed in the collection tube), centrifuge at 13,000 × g for 1 min, and discard the waste liquid; after discarding the waste liquid, place the RNAPure Columns back into the collection tube, add all the remaining liquid to the adsorption column, centrifuge at 13,000 × g for 1 min, and discard the waste liquid; add 500 μL of Buffer RW1 to the RNAPure Columns, centrifuge at 13,000 × g for 1 min, and discard the waste liquid; add 700 μL of Buffer RW2 (already containing anhydrous ethanol) to the RNAPure Columns, centrifuge at 13,000 × g for 1 min, and discard the waste liquid; repeat the previous step; place the RNAPure Columns back into the collection tube, centrifuge at 13,000 × g for 2 min to completely remove any residual Buffer RW2 from the RNAPure Columns; transfer the adsorption column to a new RNase-free Collection Tube 1.5. In a mL centrifuge tube, add 50-200 μL of RNase-free ddH2O dropwise to the center of the adsorption column. Incubate at room temperature for 2 min, then centrifuge at 13,000 × g for 1 min to elute RNA. Analyze RNA concentration and quality using a BioDrop-μLite ultra-micro nucleic acid analyzer and store at -80 ℃.

[0070] (2) cDNA synthesis:

[0071] RNA (1,000 ng) was reverse transcribed into cDNA using the PrimeScript™ IV 1st strand cDNA Synthesis Mix kit. The specific steps are as follows:

[0072] Prepare the mixture according to Table 1; mix gently, and follow the reaction program as follows: 30 °C, 10 min; 42 °C, 15 min; 95 °C, 5 min. Store the reverse transcription product at -20 °C.

[0073] Table 1. Preparation of cDNA synthesis reaction solution

[0074]

[0075] (3) qRT-PCR detection:

[0076] Gene expression levels were detected using Thermo Fisher Scientific PowerUp™ SYBR™ Green premixed solution and a QuantStudio™ 7 Flex System (ABI), with three technical replicates for each sample. RPL-19 was used as the reference gene in this study. The expression levels of each gene were calculated using the qRT-PCR method. The qRT-PCR reaction system is shown in Table 2. The reaction program was: 95 ℃, 30 s; then 95 ℃, 5 s and 60 ℃, 34 s cycles for 40 times; 95 ℃, 15 s; 60 ℃, 15 s; 60 ℃, 1 min; 95 ℃, 15 s. All primers used for qRT-PCR were designed online using the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ), and the primer sequences were synthesized by BGI Genomics in Beijing.

[0077] Table 2. qRT-PCR reaction system

[0078]

[0079] The qRT-PCR primers are:

[0080] qRT-PCR-RPL19 Forward: 5′-ATCGCCAATGCCAACTC-3′, as shown in SEQ ID No. 1;

[0081] Reverse: 5′-CCTTTCGCTTACCTATACC-3′, as shown in SEQ ID No. 2;

[0082] qRT-PCR-RBX1 Forward: 5′-GAATGTCAAGCCAACCAGGC-3′, as shown in SEQ ID No. 3;

[0083] Reverse: 5′-AAGCATGGTTACAGACGCCC-3′, as shown in SEQ ID No. 4.

[0084] like Figure 2As shown, compared to internal reference genes in various tissues, the expression of the RBX1 gene is relatively stable in the uterus, endometrial epithelial cells (BEECs), ovary, and ovarian granulosa cells (GCs), especially in ovarian tissue. The ovary, as the core of pregnancy, plays a decisive role in ovulation, corpus luteum formation after fertilization, and progesterone secretion, which are essential conditions for successful pregnancy. This indicates the significant potential of RBX1 in the reproductive function of dairy cows, and based on these results, subsequent screening of molecular marker sites related to dairy cow reproductive traits will be conducted.

[0085] Experimental Example 3

[0086] This study used the Illumina 150K bovine microsphere array (Illumina Inc., San Diego, CA, USA) to genotype 1578 Holstein cattle. Based on ARS_UCD 1.2 as the reference genome, polymorphic sites were searched in the RBX1 gene and within a 200kb range upstream and downstream. Then, SNP molecular markers associated with the first and last mating interval trait were identified by statistical methods.

[0087] Association analysis between each retrieved SNP and the first-to-last mating interval trait was performed using the General Linear Model (GLM) procedure in SAS 9.2 (Table 3). For SNP-based analyses, Bonferronit correction was used to control for false positives caused by multiple tests (p < 0.05). The model used for association analysis is as follows:

[0088] Y=μ+G+e

[0089] Where y is the estimated breeding value (EBV) of the first and last mating interval trait in an individual; μ is the population mean; G is the genotype; and e is the random residual effect. Multiple comparisons were performed using the Bonferroni t method, and results are expressed as "least square mean ± standard deviation". P < 0.05 was considered significant, and P < 0.01 was considered highly significant.

[0090] The impact of the seven SNP loci identified as significantly associated with the first and last mating interval trait on the reproductive performance of Holstein cattle was statistically analyzed, and the data obtained are shown in Table 3 below.

[0091] Table 3. Association analysis between SNP loci and the first and last mating interval trait in dairy cows

[0092]

[0093] Phenotypic values ​​for the interval between the first and last matings are expressed as "least square mean ± standard deviation". Data in the same column without the same letter in the subheading indicates significant differences between different genotypes at the same locus (P<0.05); data with the same letter in the subheading or without a letter in the subheading indicates no significant differences between different genotypes at the same locus (P>0.05); SNP locus genotypes are arranged in the order of reference type, heterozygous type, and mutant type.

[0094] As shown in Table 3, the seven loci SNP1 (g.111973840A / G), SNP3 (g.112001075G / C), SNP4 (g.112039950A / G), SNP10 (g.112202530A / G), SNP11 (g.112227956C / T), SNP14 (g.112319140A / G), and SNP15 (g.112344772A / G) were significantly associated with the first and last mating interval trait in Holstein cattle (P<0.05). The genotype with the smallest first and last mating interval within each SNP was the dominant genotype.

[0095] Example 4

[0096] 1. Collection of Holstein cattle samples with high and low fertility

[0097] Blood samples were collected from 200 Holstein cattle at a large-scale dairy farm. Based on the mating status of the farm, the Holstein young cattle were divided into a high-fertility group (first mating is pregnancy) and a low-fertility group (cows with ≥3 matings) for 30x resequencing analysis. The number of matings of the dairy cows is shown in Table 4.

[0098] Table 4. Number of matings at first mating for Holstein cattle in high- and low-breeding groups

[0099]

[0100] 2. Genome alignment and quality control

[0101] Trimmomatic was used to perform quality control on the raw sequencing data. Unacceptable reads were filtered according to the following criteria: 1) trimmed sequencing adapter sequences; 2) removed paired-end reads with more than 5 N bases in single-end reads; 3) removed paired-end reads with more than 40% low-quality (Q≤15) bases in single-end reads.

[0102] The quality-controlled CleanReads were aligned with the bovine reference genome (ARS-UCD version 1.2) using Sentieon software (parameter: `bwamem-k32-MR`). Then, Samtools software was used to sort the alignment results and remove duplicate reads.

[0103] 3. SNP variant detection

[0104] A gVCF file was generated based on whole-genome sequencing data. Subsequently, the GVCFtyper algorithm was used for multi-sample joint genotyping to produce the original variant dataset. The original data was then rigorously filtered according to the following conditions: `QD<2.0`, `QUAL<30.0`, `SOR>3.0`, `FS>60.0`, `MQ<40.0`, `MQRankSum<-12.5`, and `ReadPosRankSum<-8.0`. The bcftools tool was used to filter out loci that passed these criteria, resulting in the final SNP variant dataset.

[0105] For the SNP loci in Example 3 that are significantly associated with the first and last mating interval trait in Holstein cattle, the difference in genotype distribution frequency of each SNP locus between the high-breeding group and the low-breeding group was examined one by one.

[0106] Table 5. Genotype distribution frequency of SNP loci

[0107]

[0108] Table 5 shows that SNP11 (g.112227956C / T) mainly exists in two genotypes in the high-fertility group: the heterozygous genotype AG and the homozygous genotype GG. Furthermore, the frequency of the homozygous GG genotype of SNP11 (g.112227956C / T) in the high-fertility group dairy cows is significantly higher than that in the low-fertility group dairy cows, indicating that this genotype is the dominant genotype in high-fertility dairy cows. Combined with the results in Example 3, it can be seen that individuals with the GG genotype at the SNP11 (g.112227956C / T) locus in the dairy cow population also have a higher first-time pregnancy rate.

[0109] In summary, individuals with the homozygous GG genotype at SNP11 (g.112227956C / T) exhibit better reproductive performance and can be considered as a candidate locus for high fertility in Holstein cattle, thus warranting close attention.

[0110] Therefore, during the selection and breeding of cattle, the g.112227956C / T molecular marker can be used to assist in the selection of individuals with good reproductive performance. Individuals with this advantageous allele should be retained in the breeding process, which is beneficial to improving the reproductive performance of the population.

[0111] Experimental Example 5

[0112] This invention provides a genotype detection method and primer sequences for SNP11 (g.112227956C / T).

[0113] Bovine blood DNA extraction and genotyping:

[0114] (1) DNA was extracted from blood tissue using the Novizan DC112 kit. The specific steps are as follows:

[0115] 1) Take 200 μL of blood sample into a 1.5 mL centrifuge tube, add Buffer ACL to make up to 200 μL, and vortex to mix.

[0116] 2) Add 20 μL Proteinase K and 200 μL Buffer BCL sequentially and vortex to mix.

[0117] 3) Heat in a 70℃ water bath for 10 minutes, inverting the container several times during the process. There should be no particulate precipitate in the solution.

[0118] 4) Add 150 μL of anhydrous ethanol, shake to mix, and briefly centrifuge to collect the liquid on the inner wall of the tube cap.

[0119] 5) Place the FastPure gDNA Mini Columns II adsorption column into a 2 mL Collection Tube and transfer the above mixture (including the flocculent precipitate) into the adsorption column. Centrifuge at 12,000 rpm (13,400 × g) for 1 min.

[0120] 6) Discard the filtrate and place the adsorption column in the collection tube. Add 500 μL of Buffer WA along the tube wall to the adsorption column and centrifuge at 12,000 rpm (13,400 × g) for 1 min.

[0121] 7) Discard the filtrate and place the adsorption column in the collection tube. Add 600 μL of Buffer WB along the tube wall, centrifuge at 12,000 rpm (13,400 × g) for 1 min, and discard the filtrate.

[0122] 8) Repeat step 7).

[0123] 9) Place the adsorption column in the collection tube. Centrifuge the empty column at 12,000 rpm (13,400 × g) for 2 min.

[0124] 10) Transfer the adsorption column to a new 1.5 mL centrifuge tube. Add 50-200 μL of solution buffer to the center of the adsorption column membrane and incubate at room temperature for 2-5 min. Centrifuge at 12,000 rpm (13,400 × g) for 1 min.

[0125] 11) Discard the adsorption column and store the DNA product at -20°C;

[0126] (2) PCR detection: The PCR reaction system is shown in Table 6. The reaction program was as follows: 98 ℃, 45 s; followed by 30 cycles of 98 ℃, 10 s, 60 ℃, 8 s, and 72 ℃, 15 s; 72 ℃, 5 min; 4 ℃, 15 s. All primers used for PCR were designed online using the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ), and the primer sequences were synthesized by BGI Genomics in Beijing.

[0127] (3) Electrophoresis detection: Prepare a 1.5% agarose gel, mix the PCR product with the loading buffer at a ratio of 6:1, and electrophores at 120V for 25 min in TAE buffer.

[0128] Table 6. PCR reaction system

[0129]

[0130] The PCR primers are:

[0131] PCR-SNP11-1 Forward: 5′- gtgaggaaacccaggagcg-3′, as shown in SEQ ID No.5;

[0132] Reverse: 5′- aacagaagagccagtgatgct-3′, as shown in SEQ ID No. 6;

[0133] The 2% agarose gel electrophoresis analysis showed clear bands in lanes 1-6, proving that the DNA sequences of primers SEQ ID No. 5 and SEQ ID No. 6 can be used for subsequent sequencing detection and analysis of the SNP11 (g.112227956C / T) genotype.

[0134] (4) Sequencing analysis

[0135] Sequencing analysis of the PCR products revealed a significant bimodal DNA sequence in lane 5, with bases C / T (forward F) and G / A (reverse R). Sequence analysis confirmed that this sequence was SNP11 (g.112227956C / T), demonstrating that the primer set used in this invention successfully detected and classified the genotype of SNP11 (g.112227956C / T).

[0136] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of SNP sites in any of the following, characterized in that, include: Identifying or assisting in the identification of dairy cows exhibiting different reproductive traits; or Selecting and breeding dairy cows with different reproductive traits; The SNP site is located on bovine chromosome 5, and the SNP site is g.112227956C / T; When the genotype of the SNP locus is GG, the dairy cow is a high-fertility dairy cow; the reproductive trait is either high fertility or low fertility. The term "high reproductive capacity" refers to a first-time pregnancy or a higher first-time pregnancy rate; the term "low reproductive capacity" refers to cows that have been mated ≥3 times and are either non-pregnant or pregnant.

Images