SNP (Single Nucleotide Polymorphism) site combination and primer combination for identifying northern sand cattle and application of SNP site combination and primer combination
By using an identification method based on SNP molecular markers, combining 15 SNP site combinations and primer combinations with a reference group data model, the accuracy problem of traditional identification methods was solved, enabling early and efficient identification of North Sand Cattle and improving the identification accuracy.
Patent Information
- Application Number
- CN202511243340.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-12-02
AI Technical Summary
Traditional methods are difficult to accurately identify Beisha cattle in their early stages, and their reliance on experience leads to results that are not objective and accurate enough, failing to meet the needs of modern animal husbandry for rapid identification of the Beisha cattle breed.
An identification method based on SNP molecular markers was adopted, using 15 SNP site combinations and corresponding primer combinations. Through PCR amplification and fluorescence detection, combined with a reference group data model, the typing probability value was calculated to achieve early identification of North Sand Cattle.
It has achieved a high accuracy rate in identifying Beisha cattle, reaching 100% accuracy, providing technical support for the preservation and genetic breeding of Beisha cattle.
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Figure CN121046540A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a combination of SNP sites, primer combinations, their applications, and identification methods for identifying the northern sand cow. Background Technology
[0002] Beisha cattle are a unique local breed of livestock in Ningbo City, Zhejiang Province. They are a dual-purpose breed used for both draft and meat, characterized by strong adaptability, large size (adult bulls weigh approximately 500 kg), and delicious meat. Their history can be traced back to the Republican era, developed through crossbreeding of tall draft cattle introduced from Nanhui and Chuansha in Shanghai with local yellow cattle. They were primarily used for cultivating the sandy soil of coastal farmland in Ningbo. Beisha beef is firm and flavorful, and according to records, it is comparable to Kobe beef from Japan. Furthermore, the hides, horns, and bones of Beisha cattle also have high economic value; for example, the hides can be used as high-quality leather goods, and the horns can be used for handicrafts.
[0003] Since the widespread adoption of mechanization in the 1990s, the number of Beisha cattle has plummeted. In 2007, only 14 remained. Through conservation and propagation, the number increased to 29 in 2012, necessitating effective conservation measures. Traditional methods often require identification based on physical characteristics after the cattle reach adulthood, making early identification in juveniles impossible. Furthermore, traditional methods rely on the experience and subjective judgment of the assessors, making them susceptible to personal biases and resulting in less objective and accurate identification results.
[0004] With the modernization of animal husbandry, higher demands are being placed on the quality and quantity of livestock products. Constructing genetic identification systems based on SNP molecular markers allows for in-depth genetic analysis, enabling rapid breed identification and precise recognition of individual genetic information. This can provide strong support for the industrialization of Beisha cattle, meet market demand for high-quality beef, and enhance the economic value and market competitiveness of Beisha cattle. Summary of the Invention
[0005] To address the aforementioned issues, this invention provides a combination of SNP sites, primer combinations, their applications, and an identification method for identifying North Sand Cattle. The SNP site combinations provided by this invention can accurately distinguish between North Sand Cattle and non-North Sand Cattle.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] This invention provides a combination of SNP sites for identifying the northern sand cow, including SNP sites 1 to 15;
[0008] The SNP locus 1 is located at position 100712773 on chromosome 7 of the bovine genome, and its polymorphism is T / C.
[0009] The SNP site 2 is located at position 102694722 on chromosome 7 of the bovine genome, and its polymorphism is C / G.
[0010] The SNP site 3 is located at position 399177 on chromosome 8 of the bovine genome, and its polymorphism is C / T.
[0011] The SNP site 4 is located at position 630821 on chromosome 8 of the bovine genome, and its polymorphism is T / C.
[0012] The SNP locus 5 is located at position 100167170 on chromosome 7 of the bovine genome, and its polymorphism is A / G.
[0013] The SNP locus 6 is located at position 102091328 on chromosome 7 of the bovine genome, and its polymorphism is A / G.
[0014] The SNP site 7 is located at position 93038107 on chromosome 10 of the bovine genome, and its polymorphism is A / G.
[0015] The SNP site 8 is located at position 53173224 on chromosome 19 of the bovine genome, and its polymorphism is C / A.
[0016] The SNP locus 9 is located at position 837935 on chromosome 8 of the bovine genome, and its polymorphism is A / G.
[0017] The SNP locus 10 is located at position 38518575 on chromosome 28 of the bovine genome, and its polymorphism is T / C.
[0018] The SNP locus 11 is located at position 57075975 on chromosome 6 of the bovine genome, and its polymorphism is T / C.
[0019] The SNP locus 12 is located at position 172665 on chromosome 8 of the bovine genome, and its polymorphism is G / A.
[0020] The SNP locus 13 is located at position 82950081 on chromosome 6 of the bovine genome, and its polymorphism is C / T.
[0021] The SNP locus 14 is located at position 11090640 on chromosome 5 of the bovine genome, and its polymorphism is C / T.
[0022] The SNP locus 15 is located at position 114804730 on chromosome 5 of the bovine genome, and its polymorphism is A / G.
[0023] Preferably, the SNP sites 1 to 15 are determined based on the bovine whole genome standard sequence, with the reference genome version being Index of / genomes / all / GCF / 002 / 263 / 795 / GCF_002263795.1_ARS-UCD1.2(nih.gov).
[0024] This invention also provides the application of the SNP site combinations described in the above technical solution in the identification of North Sand Cattle.
[0025] This invention provides a primer combination for amplifying the SNP site combination described in the above technical solution. The nucleotide sequence of the forward primer used to amplify the SNP site 1 is shown in SEQ ID No. 1, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 2, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 3.
[0026] The nucleotide sequence of the forward primer used to amplify the SNP site 2 is shown in SEQ ID No. 4, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 5, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 6.
[0027] The nucleotide sequence of the forward primer used to amplify the SNP site 3 is shown in SEQ ID No. 7, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 8, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 9.
[0028] The nucleotide sequence of the forward primer used to amplify SNP site 4 is shown in SEQ ID No. 10, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 11, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 12.
[0029] The nucleotide sequence of the forward primer used to amplify the SNP site 5 is shown in SEQ ID No. 13, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 14, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 15.
[0030] The nucleotide sequence of the forward primer used to amplify the SNP site 6 is shown in SEQ ID No. 16, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 17, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 18.
[0031] The nucleotide sequence of the forward primer used to amplify the SNP site 7 is shown in SEQ ID No. 19, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 20, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 21.
[0032] The nucleotide sequence of the forward primer used to amplify the SNP site 8 is shown in SEQ ID No. 22, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 23, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 24.
[0033] The nucleotide sequence of the forward primer used to amplify the SNP site 9 is shown in SEQ ID No. 25, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 26, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 27.
[0034] The nucleotide sequence of the forward primer used to amplify the SNP site 10 is shown in SEQ ID No. 28, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 29, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 30.
[0035] The nucleotide sequence of the forward primer used to amplify the SNP site 11 is shown in SEQ ID No. 31, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 32, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 33.
[0036] The nucleotide sequence of the forward primer used to amplify the SNP site 12 is shown in SEQ ID No. 34, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 35, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 36.
[0037] The nucleotide sequence of the forward primer used to amplify the SNP site 13 is shown in SEQ ID No. 37, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 38, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 39.
[0038] The nucleotide sequence of the forward primer used to amplify the SNP site 14 is shown in SEQ ID No. 40, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 41, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 42.
[0039] The nucleotide sequence of the forward primer used to amplify the SNP site 15 is shown in SEQ ID No. 43, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 44, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 45.
[0040] This invention also provides the application of the primer combination described in the above technical solution in the identification of North Sand Cattle.
[0041] The present invention also provides a kit for identifying North Sand Cattle, characterized in that the kit contains the primer combination described in the above technical solution.
[0042] The present invention also provides a method for identifying the northern sand cow, comprising the following steps:
[0043] (1) Extraction of genomic DNA from the bovine samples to be tested;
[0044] (2) Using the genomic DNA of the bovine sample to be tested as a template, PCR amplification was performed using each primer pair in the SNP primers described in this invention;
[0045] (3) Perform fluorescence detection and analysis on the amplification products to obtain the genotypes of the 15 SNP loci corresponding to the test cattle sample; input the genotyping results of the 15 SNP loci into the constructed reference group data model, and calculate the probability values of "yes" and "no" corresponding to the genotyping results of each SNP locus. "Yes" means "is a North Sand Cattle", and "no" means "not a North Sand Cattle". Sum the probability values of the 15 SNP loci to obtain the total identification probability value of "yes" and "no". If the total identification probability value of "yes" is greater than the total identification probability value of "no", then the test cattle is identified as North Sand Cattle; otherwise, if the total identification probability value of "yes" is less than that of "no", then the test cattle is identified as not North Sand Cattle.
[0046] The probability value is characterized in that the probability value of the bovine breed corresponding to the genotyping result of each SNP locus in the model constructed with reference group data is the value obtained by taking the natural logarithm of P(A|B). The formula for calculating P(A|B) is P(A|B) = P(B|A) * P(A) / P(B), where P(A) is the probability that the genotyping result of the locus in the reference group is identified as a Northern Sha cattle, P(B) is the probability that the genotyping result of the locus in the reference group is identified as a non-Northern Sha cattle, P(A|B) is the probability value that the genotyping result of the locus in the model constructed with reference group data is identified as a Northern Sha cattle, and P(B|A) is a coefficient value calculated based on the genotyping data of the reference group.
[0047] In the above method for identifying cattle breeds, as a preferred embodiment, the PCR amplification conditions are as follows: pre-denaturation at 94℃ for 15 minutes, denaturation at 94℃ for 20 seconds; annealing at 61℃-55℃ for 1 minute, for a total of 10 cycles, with 61℃ being the annealing temperature for the first cycle, and the annealing temperature decreasing by 0.6℃ for each subsequent cycle; denaturation at 94℃ for 20 seconds, annealing / extension at 55℃ for 1 minute, for a total of 26 cycles.
[0048] In the above-mentioned method for identifying cattle breeds, as a preferred embodiment, the concentration ratio of the first forward primer, the second forward primer, and the universal reverse primer in the PCR system of each primer pair is 2:2:5.
[0049] In the above method for identifying cattle breeds, as a preferred embodiment, the concentration of genomic DNA in the sample to be tested is 10-30 ng / μL.
[0050] The PCR template of this invention is the genomic DNA of the sample to be tested. Its agarose gel electrophoresis band should be single and without obvious diffusion, its concentration should meet the requirement of 10-30 ng / μL, and the ratio of A260 / A280 in the ultraviolet spectrophotometric method should be greater than 1.8.
[0051] This invention is the first to propose a reference group data model for identifying the Beisha cattle breed. Using the 15 SNP loci and identification technology provided by this invention, the identification accuracy of Beisha cattle is as high as 100%, providing strong technical support for the future identification, germplasm resource protection and genetic breeding of Beisha cattle.
[0052] The beneficial effects of this invention are:
[0053] 1. This invention is the first to propose 15 representative differential SNP sites in the northern sand cow.
[0054] 2. This invention proposes for the first time a reference group data model for identifying the Beisha cattle breed. The data scale of this model can be used as a general reference group data model for identifying the Beisha cattle breed.
[0055] 3. This invention verifies the identification of the Beisha cattle breed by performing blind testing on 20 samples using gene (SNP) identification. It has determined that these 15 SNP loci can be successfully used to identify the Beisha cattle breed. Using the 15 SNP loci and identification technology provided by this invention, the identification accuracy of Beisha cattle is as high as 100%.
[0056] 4. This invention provides strong technical support for the identification, preservation, and genetic breeding of the Beisha cattle in the future. Attached Figure Description
[0057] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0058] Figure 1 A technical roadmap for screening loci for variety identification;
[0059] Figure 2 This is a flowchart of the KASP detection experiment. Detailed Implementation
[0060] This invention provides a combination of SNP sites for identifying the northern sand cow, including SNP sites 1 to 15;
[0061] The SNP locus 1 is located at position 100712773 on chromosome 7 of the bovine genome, and its polymorphism is T / C.
[0062] The SNP site 2 is located at position 102694722 on chromosome 7 of the bovine genome, and its polymorphism is C / G.
[0063] SNP 3 is located at position 399177 on chromosome 8 of the bovine genome, with a polymorphism of C / T; SNP 4 is located at position 630821 on chromosome 8 of the bovine genome, with a polymorphism of T / C; SNP 5 is located at position 100167170 on chromosome 7 of the bovine genome, with a polymorphism of A / G; and SNP 6 is located at position 102091328 on chromosome 7 of the bovine genome, with a polymorphism of A / G.
[0064] The SNP site 7 is located at position 93038107 on chromosome 10 of the bovine genome, and its polymorphism is A / G.
[0065] The SNP site 8 is located at position 53173224 on chromosome 19 of the bovine genome, and its polymorphism is C / A.
[0066] The SNP locus 9 is located at position 837935 on chromosome 8 of the bovine genome, with a polymorphism of A / G; the SNP locus 10 is located at position 38518575 on chromosome 28 of the bovine genome, with a polymorphism of T / C.
[0067] The SNP locus 11 is located at position 57075975 on chromosome 6 of the bovine genome, and its polymorphism is T / C.
[0068] The SNP locus 12 is located at position 172665 on chromosome 8 of the bovine genome, and its polymorphism is G / A.
[0069] The SNP locus 13 is located at position 82950081 on chromosome 6 of the bovine genome, and its polymorphism is C / T.
[0070] The SNP locus 14 is located at position 11090640 on chromosome 5 of the bovine genome, and its polymorphism is C / T.
[0071] SNP locus 15 is located at position 114804730 on chromosome 5 of the bovine genome, and its polymorphism is A / G. In this invention, SNP loci 1-15 are determined based on the standard bovine genome sequence, with the reference genome version being Index of / genomes / all / GCF / 002 / 263 / 795 / GCF_002263795.1_ARS-UCD1.2(nih.gov).
[0072] This invention provides the application of the SNP site combinations described in the above technical solution in the identification of North Sand Cattle.
[0073] This invention provides a primer combination for amplifying the SNP site combination described in the above technical solution. The nucleotide sequence of the forward primer used to amplify the SNP site 1 is shown in SEQ ID No. 1, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 2, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 3.
[0074] The nucleotide sequences of the forward primer used for amplifying SNP site 2 are shown in SEQ ID No. 4, the reverse primer in SEQ ID No. 5, and the extension primer in SEQ ID No. 6; the nucleotide sequences of the forward primer used for amplifying SNP site 3 are shown in SEQ ID No. 7, the reverse primer in SEQ ID No. 8, and the extension primer in SEQ ID No. 9; the nucleotide sequences of the forward primer used for amplifying SNP site 4 are shown in SEQ ID No. 10, the reverse primer in SEQ ID No. 11, and the extension primer in SEQ ID No. 12; the nucleotide sequences of the forward primer used for amplifying SNP site 5 are shown in SEQ ID No. 13, the reverse primer in SEQ ID No. 14, and the extension primer in SEQ ID No. 15; the nucleotide sequences of the forward primer used for amplifying SNP site 6 are shown in SEQ ID No. 16, the reverse primer in SEQ ID No. 17, and the extension primer in SEQ ID No. 9. The nucleotide sequence of the forward primer used to amplify SNP site 7 is shown in SEQ ID No. 18; the nucleotide sequence of the reverse primer is shown in SEQ ID No. 20; and the nucleotide sequence of the extension primer is shown in SEQ ID No. 21. The nucleotide sequence of the forward primer used to amplify SNP site 8 is shown in SEQ ID No. 22; the nucleotide sequence of the reverse primer is shown in SEQ ID No. 23; and the nucleotide sequence of the extension primer is shown in SEQ ID No. 24. The nucleotide sequence of the forward primer used to amplify SNP site 9 is shown in SEQ ID No. 25; the nucleotide sequence of the reverse primer is shown in SEQ ID No. 26; and the nucleotide sequence of the extension primer is shown in SEQ ID No. 27. The nucleotide sequence of the forward primer used to amplify SNP site 10 is shown in SEQ ID No. 28; the nucleotide sequence of the reverse primer is shown in SEQ ID No. 29; and the nucleotide sequence of the extension primer is shown in SEQ ID No. 30. The nucleotide sequence of the forward primer used to amplify SNP site 11 is shown in SEQ ID No. 31; and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 30. As shown in No. 32, the nucleotide sequence of the extension primer is shown in SEQ ID No. 33; the nucleotide sequence of the forward primer used to amplify the SNP site 12 is shown in SEQ ID No. 34, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 35, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 36.
[0075] The nucleotide sequence of the forward primer used for amplifying SNP site 13 is shown in SEQ ID No. 37, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 38, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 39; the nucleotide sequence of the forward primer used for amplifying SNP site 14 is shown in SEQ ID No. 40, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 41, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 42; the nucleotide sequence of the forward primer used for amplifying SNP site 15 is shown in SEQ ID No. 43, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 44, and the nucleotide sequence of the extension primer is shown in SEQ ID No. 45.
[0076] In this invention, the nucleotide sequences of the primer combinations are specifically shown in Table 1.
[0077] Table 1. Nucleotide sequences of primer combinations
[0078]
[0079]
[0080]
[0081] This invention also provides the application of the primer combination described in the above technical solution in the identification of North Sand Cattle.
[0082] The preferred application involves detecting the genotyping results of the 15 SNP loci in the genome of the cattle to be tested, inputting the genotyping results of the 15 SNP loci into the constructed reference group data model, and calculating the probability values of "yes" and "no" for each SNP locus. "Yes" indicates "is a North Sand Cattle", and "no" indicates "is not a North Sand Cattle". The probability values of the 15 SNP loci are summed to obtain the total identification probability value of "yes" and "no". If the total identification probability value of "yes" is greater than the total identification probability value of "no", then the cattle to be tested is identified as a North Sand Cattle; otherwise, if the total identification probability value of "yes" is less than the total identification probability value of "no", then the cattle to be tested is identified as a non-North Sand Cattle.
[0083] In this invention, the model constructed using reference group data uses Northern Sha cattle and non-Northern Sha cattle as reference groups to detect the genotyping results of the 15 SNP loci and calculate the probability value of the cattle breed corresponding to the genotyping result of each SNP locus in the reference group.
[0084] In this invention, the probability value of the bovine breed corresponding to the genotyping result of each SNP locus in the constructed reference group data model is the natural logarithm of P(A|B). The formula for calculating P(A|B) is P(A|B) = P(B|A) * P(A) / P(B), where P(A) is the probability that the genotyping of this locus in the reference group is Beisha cattle, P(B) is the probability that the genotyping of this locus in the reference group is identified as non-Beisha cattle, P(A|B) is the probability value that the genotyping of this locus in the constructed reference group data model is identified as Beisha cattle, and P(B|A) is the coefficient value calculated based on the genotyping data of the reference group.
[0085] In this invention, one of the constructed reference group data models is as follows: the probability values of each subtype of the 15 loci appearing in different varieties are shown in Table 2:
[0086] Table 2. Probability values of each subtype of the 215 loci appearing in different varieties.
[0087]
[0088]
[0089] The present invention also provides a kit for identifying North Sand Cattle, the kit containing the primer combination described in the above technical solution.
[0090] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0091] Example 1
[0092] Variety identification site screening
[0093] 1.1 Collection of experimental animals and samples
[0094] Blood samples were collected from 191 samples of four local cattle breeds in Zhejiang Province (Beisha cattle, Tiantai cattle, Wenling Gaofeng cattle, and Zhoushan cattle).
[0095] 1.2 Whole genome resequencing
[0096] Genomic DNA was extracted using a magnetic bead method, following standard extraction procedures. The extracted DNA underwent integrity and purity testing; DNA meeting the requirements was retained, while unqualified DNA was re-extracted, and samples that still did not meet the requirements after re-extraction were discarded. Experiments were performed according to the standard protocol provided by BGI Genomics. For qualified genomic DNA samples, appropriate fragment sizes were selected using gel electrophoresis, followed by PCR enrichment and library construction. After library construction, quantification and quality control were performed using Qubit. Qualified libraries were sequenced using DNBSEQ-T7, which was performed by Beijing Compson Biotechnology Co., Ltd. (Beijing, China). After DNBSEQ-T7 sequencing, base sequencing quality distribution analysis, base content distribution analysis, and filtering of the raw image data (Raw reads) obtained from high-throughput sequencing were performed. The final sequence obtained from sequencing was re-aligned to a reference genome for subsequent analysis; the technical route for this part is described in [link to technical details]. Figure 1 The quality control statistics of the sample sequencing data are shown in Table 3 below:
[0097] Table 3 Quality control statistics of sample sequencing data
[0098]
[0099] 1.3 Quality Control of Genomic Data
[0100] To obtain reliable analytical results, PLINK 1.9 software was used to perform quality control on the genotype data. The quality control conditions were as follows:
[0101] (1) Remove sites with a deletion rate greater than 10%.
[0102] (2) Remove loci with a minimum allele frequency (MAF) of less than 0.01.
[0103] After screening, a total of 31,260,109 SNP loci were finally obtained.
[0104] 1.4 Screening of differential loci among varieties
[0105] Fst analysis was performed on the 190 filtered samples and the remaining loci after quality control to screen for differentially differentiated loci among varieties. In this project, there were four varieties; therefore, during the Fst analysis, the samples were divided into two groups (e.g., D vs ND, where D represents Beisha Niu), and significantly different loci were obtained for each group. Based on the Fst ranking results from highest to lowest, the top 15 SNP loci with the greatest inter-group differences were selected.
[0106] Example 2
[0107] Authentication database construction
[0108] An identification database was constructed using the genotyping results of 15 selected loci. The probability value of each SNP locus in the Bayesian model of the reference population, corresponding to the corresponding cattle breed, was calculated using the phenotypic values of the reference population and an iterative conditional expectation algorithm. The calculation formula is: P(A|B)=P(B|A)*P(A) / P(B). Based on the detected genotyping results, the differences between different breeds for each genotype at each locus were calculated. The calculated probability value is the result of taking the natural logarithm of the actual probability value; the larger the value, the greater the probability of occurrence. Table 2 shows the constructed reference population data model.
[0109] Table 2. Locative genotyping probability
[0110]
[0111]
[0112] Example 3
[0113] Blind test verification
[0114] 3.1 Laboratory animal sample collection and DNA extraction
[0115] Blood samples were collected from 20 individuals across four breeds (5 Tiantai cattle, 5 Wenling Gaofeng cattle, 5 Beisha cattle, and 5 Zhoushan cattle). Genomic DNA was extracted from these individuals.
[0116] 3.2 Primer Design
[0117] Primers were synthesized by Beijing Compson Agricultural Technology Co., Ltd. Based on SNP site information, reaction primers were designed using Primer5 software. Primer sequences are shown in Table 1 below.
[0118] 3.3 PCR reaction
[0119] Using genomic DNA as a template, PCR amplification was performed using primer combinations. The amplification system and amplification program are shown in the table below, and the PCR amplification products were obtained.
[0120] Table 2 KASP Reaction System
[0121] KASP typing reaction system (DNA diluted to a final concentration of 5-50 ng per reaction) Components WetDNAmethod (μL) DNA* 2.5 2xMasterMix 2.5 Primermix 0.07
[0122] Table 3 KASP Reaction Procedure
[0123]
[0124] The probability value of the corresponding cattle breed for each SNP locus in the Bayesian model of the reference population was calculated using the phenotypic values of the reference population and the iterative conditional expectation algorithm; the calculation formula is: P(A|B)=P(B|A)*P(A) / P(B). Based on the detected genotyping results, the differences between different breeds for each genotype at each locus were calculated. The calculated probability value is the result of taking the natural logarithm of the actual probability value; the larger the value, the greater the probability of occurrence. Table 4 shows the statistical results of the blind test:
[0125] Table 4. Statistics of blind test sample results
[0126]
[0127] Note: Identity_breed: Identification results from the identification database, where 1 represents Northern Sha cattle and 2 represents non-Northern Sha cattle; breed: Breed: Breed information of the blind test samples provided, where 1 represents Northern Sha cattle and 2 represents non-Northern Sha cattle.
[0128] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A combination of SNP loci for identifying the northern sand cow, characterized in that, Including SNP sites 1–15; The SNP locus 1 is located at position 100712773 on chromosome 7 of the bovine genome, and its polymorphism is T / C. The SNP site 2 is located at position 102694722 on chromosome 7 of the bovine genome, and its polymorphism is C / G. The SNP site 3 is located at position 399177 on chromosome 8 of the bovine genome, and its polymorphism is C / T. The SNP site 4 is located at position 630821 on chromosome 8 of the bovine genome, and its polymorphism is T / C. The SNP locus 5 is located at position 100167170 on chromosome 7 of the bovine genome, and its polymorphism is A / G. The SNP locus 6 is located at position 102091328 on chromosome 7 of the bovine genome, and its polymorphism is A / G. The SNP site 7 is located at position 93038107 on chromosome 10 of the bovine genome, and its polymorphism is A / G. The SNP site 8 is located at position 53173224 on chromosome 19 of the bovine genome, and its polymorphism is C / A. The SNP locus 9 is located at position 837935 on chromosome 8 of the bovine genome, and its polymorphism is A / G. The SNP locus 10 is located at position 38518575 on chromosome 28 of the bovine genome, and its polymorphism is T / C. The SNP locus 11 is located at position 57075975 on chromosome 6 of the bovine genome, and its polymorphism is T / C. The SNP locus 12 is located at position 172665 on chromosome 8 of the bovine genome, and its polymorphism is G / A. The SNP locus 13 is located at position 82950081 on chromosome 6 of the bovine genome, and its polymorphism is C / T. The SNP locus 14 is located at position 11090640 on chromosome 5 of the bovine genome, and its polymorphism is C / T. The SNP locus 15 is located at position 114804730 on chromosome 5 of the bovine genome, and its polymorphism is A / G.
2. The SNP site combination according to claim 1, characterized in that, The SNP sites 1–15 are determined based on the standard bovine whole genome sequence, with the reference genome version being Index of / genomes / all / GCF / 002 / 263 / 795 / GCF_002263795.1_ARS-UCD1.2(nih.gov).
3. The application of the SNP site combination as described in claim 1 or 2 in the identification of North Sand Cattle.
4. A primer combination for amplifying the SNP site combination of claim 1, characterized in that, The nucleotide sequence of the forward primer used to amplify the SNP site 1 is shown in SEQ ID No. 1, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 2, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
3. The nucleotide sequence of the forward primer used to amplify the SNP site 2 is shown in SEQ ID No. 4, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 5, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
6. The nucleotide sequence of the forward primer used to amplify the SNP site 3 is shown in SEQ ID No. 7, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 8, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
9. The nucleotide sequence of the forward primer used to amplify SNP site 4 is shown in SEQ ID No. 10, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 11, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
12. The nucleotide sequence of the forward primer used to amplify the SNP site 5 is shown in SEQ ID No. 13, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 14, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
15. The nucleotide sequence of the forward primer used to amplify the SNP site 6 is shown in SEQ ID No. 16, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 17, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
18. The nucleotide sequence of the forward primer used to amplify the SNP site 7 is shown in SEQ ID No. 19, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 20, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
21. The nucleotide sequence of the forward primer used to amplify the SNP site 8 is shown in SEQ ID No. 22, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 23, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
24. The nucleotide sequence of the forward primer used to amplify the SNP site 9 is shown in SEQ ID No. 25, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 26, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
27. The nucleotide sequence of the forward primer used to amplify the SNP site 10 is shown in SEQ ID No. 28, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 29, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
30. The nucleotide sequence of the forward primer used to amplify the SNP site 11 is shown in SEQ ID No. 31, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 32, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
33. The nucleotide sequence of the forward primer used to amplify the SNP site 12 is shown in SEQ ID No. 34, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 35, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
36. The nucleotide sequence of the forward primer used to amplify the SNP site 13 is shown in SEQ ID No. 37, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 38, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
39. The nucleotide sequence of the forward primer used to amplify the SNP site 14 is shown in SEQ ID No. 40, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 41, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
42. The nucleotide sequence of the forward primer used to amplify the SNP site 15 is shown in SEQ ID No. 43, the nucleotide sequence of the reverse primer is shown in SEQ ID No. 44, and the nucleotide sequence of the extension primer is shown in SEQ ID No.
45.
5. The application of the primer combination according to claim 4 in the identification of North Sand Cattle.
6. A reagent kit for identifying North Sand Cattle, characterized in that, The kit contains the primer combination as described in claim 4.