SNP molecular marker for identifying or assisting in identifying black coat color phenotype in water buffaloes and use thereof
By using an SNP molecular marker at position 14,298,688 on chromosome 18 of the buffalo genome, accurate identification and breeding assistance for black buffalo coat color were achieved, solving the problem of black buffalo coat color identification and breeding and supporting the selection of target traits in buffalo breeding.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2026-04-16
AI Technical Summary
Currently, there is a lack of effective genetic methods to identify and assist in the selection of buffalo with black coat coloration, which affects the protection and breeding of buffalo breeds.
A SNP molecular marker is provided, located at position 14298688 on chromosome 18 of the buffalo genome. When the genotype is TT or TG, the buffalo coat color is pure black, and when the genotype is GG, the coat color is not pure black. The buffalo coat color phenotype is detected by PCR amplification and sequencing.
It enables accurate identification of black coat color in buffalo and assists in breeding, providing a convenient molecular marker method to support the selection of target traits in buffalo breeding.
Smart Images
Figure CN2024127735_16042026_PF_FP_ABST
Abstract
Description
A SNP molecular marker for identifying or assisting in the identification of buffalo black coat phenotype and its application
[0001] This application claims priority to Chinese Patent Application No. CN202411397162.3, filed on October 8, 2024, entitled "A SNP molecular marker for identifying or assisting in the identification of the black coat phenotype of buffalo and its application", the entire contents of which are incorporated herein by reference. Technical Field
[0002] This application relates to the field of animal genetics and breeding technology, and in particular to an SNP molecular marker for identifying or assisting in the identification of the black coat phenotype of buffalo and its application. Background Technology
[0003] Asian domesticated buffalo (Bubalus bubalis) are divided into two main types—river buffalo and swamp buffalo. River buffalo (Bubalus bubalis bubalis) are mainly distributed in South Asia, West Asia, and the Mediterranean region, while swamp buffalo (Bubalus bubalis kerabau) are mainly distributed in southern China and Southeast Asian countries. Common buffalo coat colors include black and non-black. Black buffalo are entirely black, such as the Mora buffalo and Mediterranean buffalo. Non-black buffalo are generally grayish-brown or black in color, with lighter grayish-white hair on the lower limbs, and light gray or white stripes on the lower neck and chest.
[0004] Research on the gene mapping and mechanism of black coat color in buffalo not only helps to reveal the molecular mechanism of coat coloration and enrich our understanding of coat color formation, but also allows for the development of molecular markers for buffalo coat color characteristics for breed purity determination and marker-assisted selection, providing scientific means for buffalo breed protection and breeding. However, to date, no reports have been found, either domestically or internationally, on the genetic mechanism of black coat color in buffalo or on marker-assisted selection methods for black coat color.
[0005] Summary of the Invention
[0006] To address the aforementioned issues, this application provides a SNP molecular marker for identifying or assisting in the identification of the black-haired phenotype in buffalo and its application. The SNP molecular marker provided in this application can be used genetically to identify or assist in the identification of the black-haired phenotype in buffalo, and can be used for assisted breeding of black-haired buffalo, showing promising application prospects.
[0007] To achieve the above objectives, this application provides the following technical solution:
[0008] This application provides an SNP molecular marker for identifying or assisting in the identification of the black coat phenotype of buffalo. The SNP molecular marker is located at the 14,298,688th base on chromosome 18 of the buffalo genome. When the base at this site is T, the buffalo coat color is pure black. The version number of the buffalo genome is UOA_WB_1.
[0009] Preferably, the SNP molecular marker is located at the 158th base of the nucleotide sequence shown in SEQ ID NO.1.
[0010] This application provides the application of the SNP molecular markers or reagents for detecting the SNP molecular markers described in the above-mentioned technical solutions in any one of 1) to 3).
[0011] 1) Prepare a kit for identifying or assisting in the identification of buffalo black hair color;
[0012] 2) To identify or assist in the identification of the buffalo's black coat phenotype;
[0013] 3) Water buffalo breeding.
[0014] Preferably, the buffalo breeding includes breeding pure black buffalo or breeding non-pure black buffalo.
[0015] Preferably, when the genotype of the SNP molecular marker is TT or TG, the buffalo's coat color is pure black; when the genotype of the SNP molecular marker is GG, the buffalo's coat color is not pure black.
[0016] Preferably, the reagent includes primer pairs.
[0017] Preferably, the primer pair includes primers with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3.
[0018] This application provides a method for identifying or assisting in the identification of the buffalo black coat phenotype, comprising the following steps:
[0019] Using the genomic DNA of the buffalo to be tested as a template, PCR amplification was performed using the primers shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification products;
[0020] The genotype at position 158 of the amplification product was detected, and the black coat phenotype of the buffalo to be tested was determined based on the genotype results.
[0021] When the genotype at position 158 of the amplification product is TT or TG, the buffalo's coat color is pure black.
[0022] When the genotype at position 158 of the amplification product is GG, the buffalo's coat color is not pure black.
[0023] Preferably, the detection method includes sequencing.
[0024] This application provides the application of the method described in the above technical solution in buffalo breeding. Beneficial effects:
[0025] This application provides a SNP molecular marker for identifying or assisting in the identification of the black coat phenotype in buffalo. The SNP molecular marker is located at position 14,298,688 on chromosome 18 of the buffalo genome. When the base at this site is T, the buffalo coat color is pure black. The version number of the buffalo genome is UOA_WB_1. The molecular marker provided in this application is an SNP molecular marker associated with the black coat phenotype in buffalo. It can be used alone or in combination to genetically identify the coat color of buffalo. Depending on the different needs of breeding buffalo for the target trait, buffalo homozygous for the black coat gene can be selected for molecular-assisted breeding, providing a convenient and accurate method for breeding buffalo with different coat colors. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0027] Figure 1 shows the detection results of dual-luciferase activity in different groups of cells in Example 2;
[0028] Figure 2 is an electrophoresis gel image of the PCR products in Example 3;
[0029] Figure 3 shows the sequencing results of the TT genotype marker g.14298688;
[0030] Figure 4 shows the sequencing results of the GG genotype marked with g.14298688;
[0031] Figure 5 shows the sequencing results of the TG gene labeled g.14298688. Detailed Implementation
[0032] This application provides a SNP molecular marker for identifying or assisting in the identification of the black coat phenotype in buffalo. The SNP molecular marker is located at base 14,298,688 on chromosome 18 of the buffalo genome. When the base at this site is T, the buffalo coat color is pure black. The version number of the buffalo genome is UOA_WB_1. In one embodiment, the SNP molecular marker is located at base 158 of the nucleotide sequence shown in SEQ ID NO.1.
[0033] SEQ ID NO.1:
[0034] This application collected samples from 215 buffalo worldwide, obtained SNP genotype information covering the entire genome through whole-genome resequencing, and then used selection signal and association analysis to find SNP molecular markers that are significantly associated with the black coat trait of buffalo, and performed functional verification through cell experiments. This application ultimately yielded one SNP molecular marker g.14298688 associated with the black coat trait in buffalo. This SNP molecular marker is located at base position 14298688 on chromosome 18 of the buffalo genome (genome version number: UOA_WB_1), within the exon region of the MC1R gene. The molecular marker has a T / G mutation at base 158 of the nucleotide sequence shown in SEQ ID NO.1. In pure black buffalo, the genotype of this molecular marker is TT or TG, while in non-pure black buffalo, the genotype is GG. Furthermore, approximately 9% of individuals with the TG genotype exhibit a non-pure black coat color. Therefore, the TG genotype can be preliminarily identified as pure black, and this genotype can be used as an auxiliary identification result.
[0035] Based on the above advantages, this application provides the application of the SNP molecular marker or the reagent for detecting the SNP molecular marker described in the above technical solutions in any one of 1) to 3).
[0036] 1) Prepare a kit for identifying or assisting in the identification of buffalo black hair color;
[0037] 2) To identify or assist in the identification of the buffalo's black coat phenotype;
[0038] 3) Water buffalo breeding.
[0039] In one implementation, the buffalo breeding can be for selecting pure black buffalo or for selecting non-pure black buffalo. The SNP molecular markers provided in this application can be used alone or in combination for genetic identification of buffalo coat color, and different coat colors (pure black buffalo or non-pure black buffalo) can be selected according to different needs of the target traits in buffalo breeding.
[0040] In one implementation, when the genotype of the SNP molecular marker is TT or TG, the buffalo's coat color is pure black; when the genotype of the SNP molecular marker is GG, the buffalo's coat color is not pure black.
[0041] As one embodiment, the reagent includes a primer pair; the primer pair can be primers with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, or other specific primers, as long as they can achieve the detection of the molecular markers described in this application.
[0042] This application provides a method for identifying or assisting in the identification of the buffalo black coat phenotype, comprising the following steps:
[0043] Using the genomic DNA of the buffalo to be tested as a template, PCR amplification was performed using the primers shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification products;
[0044] The genotype at position 158 of the amplification product was detected, and the black coat phenotype of the buffalo to be tested was determined based on the genotype results.
[0045] When the genotype at position 158 of the amplification product is TT or TG, the buffalo's coat color is pure black.
[0046] When the genotype at position 158 of the amplification product is GG, the buffalo's coat color is not pure black.
[0047] As one implementation method, the detection method is sequencing, specifically Sanger sequencing.
[0048] This application provides the application of the method described in the above technical solution in buffalo breeding.
[0049] In one implementation, the buffalo breeding can involve selecting pure black buffalo or selecting non-pure black buffalo. The method provided in this application can genetically identify the coat color of buffalo, and can select buffalo of different colors (pure black buffalo or non-pure black buffalo) according to different needs of the target traits in buffalo breeding.
[0050] To further illustrate this application, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an SNP molecular marker for identifying or assisting in the identification of the buffalo black coat phenotype and its application, but these descriptions should not be construed as limiting the scope of protection of this application.
[0051] Example 1: Obtaining molecular markers associated with the buffalo black coat phenotype
[0052] 1. Sample collection
[0053] A total of 215 buffalo samples were collected worldwide. The samples were ear epithelial tissues and placed in sample tubes containing 75% alcohol, stored at -80°C.
[0054] 2. Extraction of buffalo genomic DNA
[0055] Genomic DNA was extracted using the phenol-chloroform method.
[0056] 3. Genome sequencing and variant detection
[0057] Whole-genome resequencing was performed using the Illumina HiSeq Xten sequencing platform, with a target sequencing depth of 10×. The sequenced reads were aligned to the buffalo reference gene (UOA_WB_1) using the MEM method with BWA software (http: / / bio-bwa.sourceforge.net / ), achieving an average alignment rate of 99.31% and a coverage of 98.34%. Variation detection and quality control were performed using GATK software (https: / / software.broadinstitute.org / gatk / ), yielding 58,204,651 SNPs for subsequent analysis.
[0058] 4. Data processing and analysis
[0059] (1) Quality control of genotype data
[0060] The PLINK software (http: / / www.cog-genomics.org / plink2) was used to perform quality control on SNP sites with poor polymorphism and low detection rates. The specific quality control criteria are as follows: SNP call rate > 90%; minimum allele frequency (MAF) > 5%.
[0061] (2) Select signal analysis and correlation analysis
[0062] Use F st and θ π This study analyzed selection signals in 40 pure black buffalo (black trunk and limbs; including 20 Italian buffalo, 7 Bengal buffalo, and 13 Pakistani buffalo) and 29 wild-type buffalo (black trunk and grayish-white limbs; all Nepalese buffalo) to locate significantly selected regions and genes. SNPs within these significantly selected regions were annotated using SNPEFF software (http: / / snpeff.sourceforge.net / ), and mutation types and potential functional effects were analyzed to screen for potential candidate mutation sites.
[0063] In addition, in a sample population of 146 buffaloes with phenotypic records (including 49 Iraqi buffaloes, 45 Pakistani buffaloes, 21 Egyptian buffaloes, 20 Nepalese buffaloes, and 11 Bangladeshi buffaloes), case-control candidate locus association analysis was performed on the two coat colors using PLINK software, and the causative mutation sites were determined using the chi-square test statistical method based on the dominant genetic model.
[0064] 5. Results Analysis
[0065] (1) Selective Signal Analysis
[0066] Significant selected regions were identified in the 14160001-14470000 region of buffalo chromosome 18, with the MC1R gene region exhibiting the highest selection signal. Annotation revealed two missense mutation sites in the MC1R gene: c.14298614G>A (denoted as g.14298614) and c.14298688G>T (denoted as g.14298688), which resulted in amino acid substitutions in p.Gly104Ser and p.Met128Ile, respectively, and are potential candidate causative mutation sites.
[0067] (2) Candidate site association analysis
[0068] The genotyping results at the g.14298614 locus are shown in Table 1.
[0069] Table 1. Genotype frequency distribution of SNP locus g.14298614
[0070] Note: A - mutation, G - normal, E - 09 is 10. -9 .
[0071] The genotyping results at the g.14298688 locus are shown in Table 2.
[0072] Table 2. Frequency distribution of SNP locus g.14298688 genotype
[0073] Note: T-mutation, G-normal, E-25 is 10. -25 .
[0074] Association analysis showed a more significant association between the g.14298688 marker and black coat color. According to the statistical results, when the g.14298688 marker genotype is TT, there is a 100% confidence that the individual will have black coat; when the g.14298688 marker genotype is TG, there is a 90.9% confidence (30 / 33 × 100% = 90.9%) confidence that the individual will have black coat; and when the g.14298688 marker genotype is GG, there is a 90.3% confidence that the individual will not have black coat.
[0075] Example 2: Functional verification of SNP marker g.14298688
[0076] 1. Test Methods
[0077] Human kidney epithelial cell line (293T cells) was used for functional validation of the MC1R gene mutation sites. Recombinant plasmids were constructed based on key mutation sites (g.14298688 and g.14298614) of the MC1R gene, as detailed below:
[0078] The cDNA of the chemically synthesized wild-type (WT, SEQ ID NO.4) buffalo MC1R gene and the cDNA of two mutant buffalo MC1R genes obtained by point mutation based on the wild type were inserted between the Hind III and Xho I restriction sites of the pcDNA3.1(+) plasmid to obtain pcDNA3.1-WT plasmid, pcDNA3.1-bMC1R-1 plasmid and pcDNA3.1-bMC1R-2 plasmid; among them, the mutant buffalo MC1R with the mutation site g.14298688 was designated as bMC1R-1; the mutant buffalo MC1R with the mutation site g.14298614 was designated as bMC1R-2.
[0079] SEQ ID NO.4:
[0080] The nucleotide sequence of bMC1R-1 is shown in SEQ ID NO.5, as follows:
[0081] The nucleotide sequence of bMC1R-2 is shown in SEQ ID NO.6, as follows:
[0082] 5'-gccaccATGCCTGCACTCGGCTCCCAGAGACGGCTGCTGGGTTCCCTTAACTGCACGCCCCCAGCCACCCTCCCCCTCACTCCGGCCCCCAACCGGACGGGGCCCCAGTGCCTGGAGGTGTCCATCCCTGATGGGCTCTTTCTCAGCCTGGGGCTGGTGAGTCTCGTGGAGAACGTGCTGGTAGTGGCTGCCATCGCCAAGAACCGCAACCTGCACTCCCCCATGTACTACTTAATCTGCTGCTTGGCTGTGTCTGACCTGCTGGTGAGCGTCAGCAACGTGCTGGAGACGGCAGTCATGCTGCTGCTGGAGGCCAGTGTCCTGGCCACCCAGGCGGCCGTGGTGCAGCAGCTGGACAATGTCATCGACGTGCTCATCTGCAGCTCCATGGTGTCCAGCCTCTGCTTCCTGGGTGCCATTGCTGTGGACCGCTACATCTCCATCTTCTACGCCCTGCGGTACCACAGCGTTGTGACGCTGTCCCGAGCGTGGAGGATCATTGCGGCCATCTGGGTGGCCAGCATCCTCACCAGCCTGCTCTTCATCACCTACTACAACCACAAGGTCGTACTGCTGTGCCTCGTTGGCTTCTTCGTAGCTATGCTGGCCCTGATGGCCATCCTCTACGTCCACATGCTGGCCCGGGCCTGCCAGCATGCCCGGGGCATCGCCCGGCTCCAGAAGAGGCAGCGCCCCATTCATCAGGGCTTTGGCCTCAAGGGCGCTGCCACCCTCACCATCCTGCTGGGCGTCTTCTTCCTCTGCTGGGGCCCCTTCTTCTTGCACCTCTCGCTCATCGTCCTCTGCCCCCAGCACCCCACCTGTGGCTGCATCTTCAAGAACTTCAACCTCTTCCTGGCCCTCATCATTTGCAATGCCATTGTGGACCCCCTCATCTATGCCTTCCGCAGCCAGGAGCTCCGGAAGACACTCCAAGAGGTGCTGCAGTGCTCCTGGTGA-3';SEQ ID NO.4~SEQ ID NO.The lowercase gccacc shown in 6 is a Kozak sequence.
[0083] 0.1 μg of a mixed plasmid was co-transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). Four hours later, the transfection mixture was replaced with growth medium. The mixed plasmid consisted of MC1R plasmid, pGL4.29[luc2P-CRE-Hygro] plasmid, and pRL-TK plasmid, with a mass ratio of 2:10:1. The MC1R plasmid was either pcDNA3.1-WT plasmid, pcDNA3.1-bMC1R-1 plasmid, or pcDNA3.1-bMC1R-2 plasmid. The growth medium was DMEM medium containing fetal bovine serum.
[0084] In the absence of α-melanocyte-stimulating hormone (α-MSH) stimulation, the activity of dual-luciferase in cells expressing wild-type MC1R and mutant MC1R was detected using a dual-luciferase reporter assay kit (Promega); a concentration of 10... -7 Six hours after α-MSH of M was applied to cells expressing wild-type MC1R and mutant MC1R, the activity of dual-luciferase in the cells was detected using a dual-luciferase reporter gene assay kit (Promega), indirectly reflecting the amount of intracellular cAMP, thereby studying the activity of the cAMP pathway. All experiments were repeated three times, and data are expressed as mean ± standard error. A t-test was used to analyze differences between the two groups, with P < 0.05 as the significance threshold. The results are shown in Figure 1, where basal level represents the result without α-MSH stimulation, and α-MSH stimulation represents the result after treatment with a concentration of 10... -7 Results of α-MSH stimulation of M; ** indicates P<0.01, *** indicates P<0.001.
[0085] 2. Results Analysis
[0086] In vitro experiments were conducted on HEK293T cells expressing wild-type (WT) and mutant types. Under basal levels and α-MSH stimulation, cells with the g.14298688 mutant showed significantly higher levels of cAMP accumulation compared to WT cells (P<0.05). This indicates that the mutation at g.14298688 increases MC1R-mediated cAMP production, thereby enhancing melanin synthesis and ultimately resulting in a pure black coat color in buffalo.
[0087] Therefore, this application uses g.14298688 as a molecular marker associated with the black coat color trait of buffalo for auxiliary selection.
[0088] Example 3: Sanger sequencing detection of g.14298688
[0089] (1) Primer design
[0090] Based on the upstream and downstream sequence information of SNP markers, specific primers were designed as follows:
[0091] Upstream primer: 5'-TAATCTGCTGCTTGGCTGTG-3' (SEQ ID NO.2);
[0092] Downstream primer: 5'-ATGGAGATGTAGCGGTCCAC-3' (SEQ ID NO.3);
[0093] (2) PCR amplification
[0094] PCR reaction system: total volume 25 μL, including 2.5 μL of 10×Buffer, 2 μL of dNTP mixture (2.5 mM each), 0.5 μL of Taq enzyme (5 U / μL), 0.5 μL of forward and reverse primers (20 μM each), 1 μg of genomic DNA template and the remainder ddH2O.
[0095] PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, 35 cycles; final extension at 72℃ for 7 min.
[0096] The instrument used was an Applied Biosystems 9700 PCR instrument.
[0097] (3) Gel electrophoresis detection and sequencing of PCR products
[0098] Prepare a 2% agarose gel, and load 3 μL of PCR product from each sample onto the gel. Electrophoresis is performed in TAE buffer at 110 V for 40 min, and the electrophoresis results are observed using a gel imaging system. The PCR products are then subjected to Sanger sequencing.
[0099] (4) Sequencing of PCR products and determination of genotype
[0100] The gel image of the PCR product is shown in Figure 2. The product length is 211 bp.
[0101] The PCR products were subjected to Sanger sequencing, and the three genotypes of each SNP marker could be clearly identified based on the sequencing peak diagram. The nucleotide sequence of the amplification product of the black homozygous individual is shown in Figure 3; the nucleotide sequence of the amplification product of the non-black individual is shown in Figure 4; and the nucleotide sequence of the amplification product of the black heterozygous individual is shown in Figure 5.
[0102] (5) Statistical analysis of test results
[0103] Based on the above testing methods, this application tested a total of 44 Chinese buffalo samples, including 33 pure black buffalo (22 Murrah buffalo, 10 Binlangjiang buffalo, and 1 Dehong buffalo) and 11 non-pure black buffalo (1 Dehong buffalo, 2 Yunnan buffalo, 2 Guizhou buffalo, 2 Shanghai buffalo, and 4 Yanjin buffalo). The genotyping results are shown in Table 3. All pure black buffalo were of the TT or TG type, and all wild-type buffalo (black or gray body, grayish-white limbs) were of the GG type. The detection accuracy was 100%.
[0104] Table 3. PCR typing results of SNP site g.14298688
[0105] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative intent, and these embodiments all fall within the protection scope of this application.
Claims
1. A SNP molecular marker for identifying or assisting in the identification of the buffalo black coat phenotype, characterized in that, The SNP molecular marker is located at position 14298688 on chromosome 18 of the buffalo genome. When the base at this site is T, the buffalo's coat color is pure black. The version number of the buffalo genome is UOA_WB_1.
2. The SNP molecular marker according to claim 1, characterized in that, The SNP molecular marker is located at the 158th base of the nucleotide sequence shown in SEQ ID NO.
1.
3. The use of the SNP molecular marker or reagent for detecting the SNP molecular marker as described in claim 1 or 2 in any one of 1) to 3). 1) A kit for identifying or assisting in the identification of buffalo black hair; 2) To identify or assist in the identification of the buffalo's black coat phenotype; 3) Water buffalo breeding.
4. The application according to claim 3, characterized in that, The buffalo breeding includes the selection of pure black buffalo or non-pure black buffalo.
5. The application according to claim 3 or 4, characterized in that, When the genotype of the SNP molecular marker is TT or TG, the buffalo's coat color is pure black; when the genotype of the SNP molecular marker is GG, the buffalo's coat color is not pure black.
6. The application according to claim 3, characterized in that, The reagents include primer pairs.
7. The application according to claim 6, characterized in that, The primer pair is the primer pair for amplifying the nucleotide sequence shown in SEQ ID NO.
1.
8. The application according to claim 6 or 7, characterized in that, The primer pairs include primers with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.
3.
9. A primer pair for identifying or assisting in the identification of buffalo black coat color, characterized in that, The primer pairs include primer pairs for amplifying the nucleotide sequence shown in SEQ ID NO.
1.
10. The primer pair according to claim 9, characterized in that, The primer pair consists of primers with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.
3.
11. A kit for identifying or assisting in the identification of buffalo black coat color, characterized in that, The kit includes the primer pair as described in claim 9 or 10.
12. The kit according to claim 11, characterized in that, The kit also includes 10×Buffer, a dNTP mixture, Taq enzyme, and ddH2O.
13. A method for identifying or assisting in the identification of the black coat phenotype of buffalo, characterized in that, Includes the following steps: Using the genomic DNA of the buffalo to be tested as a template, and utilizing SEQ ID NO.2 and SEQ ID NO.3... PCR amplification was performed using primers that showed the nucleotide sequence to obtain the amplification product; The genotype at position 158 of the amplification product was detected, and the black coat phenotype of the buffalo to be tested was determined based on the genotype results. When the genotype at position 158 of the amplification product is TT or TG, the buffalo's coat color is pure black. When the genotype at position 158 of the amplification product is GG, the buffalo's coat color is not pure black.
14. The method according to claim 13, characterized in that, The detection method includes sequencing.
15. The method according to claim 13, characterized in that, The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, for 35 cycles; and finally 72℃ extension for 7 min.
16. The method according to claim 13 or 15, characterized in that, The PCR amplification reaction system consisted of: 2.5 μL of 10×Buffer, 2 μL of dNTP mixture, 0.5 μL of Taq enzyme, 0.5 μL each of the primers for the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3, 1 μg of genomic DNA template, and ddH2O to a final volume of 25 μL.
17. The method according to claim 16, characterized in that, The primer concentrations for the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 are both 20 μM.
18. The method according to claim 16, characterized in that, The concentration of the Taq enzyme is 5 U / μL.
19. The method according to claim 16, characterized in that, The concentrations of dATP, dGTP, dTTP, and dCTP in the dNTP mixture were all 2.5 mM.
20. The application of the method according to any one of claims 13 to 19 in buffalo breeding.
21. The application according to claim 20, characterized in that, The buffalo breeding includes the selection of pure black buffalo or non-pure black buffalo.