Molecular marker combination and application for dairy cattle parentage identification

Abstract

The application discloses a dairy cow parentage identification molecular marker combination and application, relates to the technical field of biology, and 257 SNP site combinations provided by the application can be used for quickly and accurately identifying the parentage between a large group of dairy cows, solve the problems that the operation of dairy cow parentage identification in the prior art is complicated and the effect is poor, and can be used for low-cost and large-scale parentage identification of dairy cow groups.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the field of biological detection technology, and more specifically to the identification of SNP locus combinations personally identified by dairy cows and their applications. Background Technology

[0002] Pedigree is a crucial source of information for plant and animal genetics and breeding research and practice, influencing the reliability of results related to gene mapping, genotypic value prediction, and phenotypic value prediction. Errors or loss of pedigree records in dairy cows can lead to inbreeding depression and reduced genetic diversity, delaying genetic progress and significantly impacting dairy cow selection, mating, and genetic improvement. In actual production, pedigree recording errors are a common phenomenon, caused by various factors including farm feeding methods, production management, and personnel operation records. Currently, microsatellite DNA (SSR) is commonly used for parentage identification. SSRs are DNA sequences composed of tandem repeats of motifs consisting of several (usually 1-6) bases, widely distributed throughout the genome. Microsatellite DNA markers are a highly polymorphic genetic marker system with advantages such as rich polymorphic information, high heterozygosity, a large number of alleles, and wide distribution in the herd's genomic DNA. Microsatellite DNA markers have repeat units of only 1-6 base pairs, with a short core sequence of 2-7 bp and tandem repeats of approximately 10-60 times, hence they are also called simple sequence repeats (SSRs) or short tandem repeats (STRs). Because microsatellite loci have a large number of alleles and high accuracy, many individual genotypes can be automatically detected and results obtained through polymerase chain reaction (PCR) and the synergistic amplification of several loci, making the operation simple. Therefore, microsatellite DNA has been widely used in paternity testing of livestock, especially dairy cows. However, this method also has several problems: ① The development process is cumbersome and costly: Developing microsatellite markers requires constructing a genomic library, screening, sequencing, and designing specific primers. These steps are very cumbersome and time-consuming. In addition, specific PCR techniques and electrophoresis equipment, as well as specialized analysis software, are required, all of which increase the cost of detection. ② Presence of null alleles: Null alleles are alleles that are not amplified by PCR, often caused by point mutations, insertions, or deletions at the primer binding site. This leads to an individual's genotype being significantly inconsistent with classic Mendelian inheritance, affecting the accuracy of paternity testing. ③ Appearance of stutter bands: During PCR amplification, multiple non-specific bands or overlapping bands may lead to misinterpretation. This subjectivity may affect the accuracy and reliability of the identification results. When using denaturing polyacrylamide gel electrophoresis to detect PCR amplification products of microsatellite loci, sometimes an allele is not a single band, but consists of a main band and several additional bands. These additional bands are called stutter bands or "shadow" bands. These bands mostly appear at microsatellite loci with repeat units of 2 bases, and are usually several repeat units shorter than the main band. Their intensity decreases with increasing distance from the main band, while their probability of occurrence increases with the number of repeats.④ Instability of PCR amplification: PCR amplification is affected by many factors, such as primer design, reaction conditions, and template quality. This may result in long alleles not being amplified, i.e., "invalid genes." This affects the accuracy and reliability of paternity testing. ⑤ Complexity of microsatellite evolution: Microsatellite evolution is complex, and homologous or heterologous types may occur, increasing the uncertainty and complexity of microsatellite markers. ⑥ Imbalance in polymorphic information content: Although microsatellite DNA is highly polymorphic, the polymorphic information content may be uneven at certain sites, which can affect its application in population genetics research. Summary of the Invention

[0003] To address the issues of high error rates, poor repeatability, and difficulty in automation of microsatellite marker genotyping in paternity testing, this invention provides a rapid, accurate, low-cost, automated, high-throughput SNP locus combination and method suitable for large-scale applications in paternity testing. This method can not only be used for genetic diversity research in large herds of dairy cows (e.g., Holstein cows), accelerating the breeding process and enabling rapid and accurate breed tracing of dairy cows, but also contributes to germplasm resource protection and improvement. It is time-efficient, low-cost, and has broad market benefits.

[0004] To achieve the technical objective of this invention, the first aspect of this invention provides a molecular marker combination for identifying parentage in dairy cows, which has the SNP site information shown in Table 1:

[0005]

[0006]

[0007] Its physical location was determined based on bovine (Bos_taurus_UMD_3.1) genome sequence alignment.

[0008] To achieve the technical objective of this invention, a second aspect of this invention provides a method for identifying parent-child relationships in dairy cows, comprising detecting SNP locus information of two individuals with different birth dates, and calculating the likelihood ratio of the parent-child relationship between the two individuals and other kinship relationships based on the SNP locus information. If the likelihood ratio of the parent-child relationship is greater than a given threshold (0.5), the result indicates that the two individuals are parent-child.

[0009] Wherein, the SNP site is the SNP site described in claim 1.

[0010] In particular, of the two individuals with different birth dates, one individual's birth date is earlier than the other individual's birth date.

[0011] To achieve the technical objective of this invention, a third aspect of this invention provides a molecular probe combination for identifying parentage in dairy cows. The molecular probe combination detects SNP site combinations in the sample to be tested as shown in Table 1. The physical location information of the site combinations in Table 1 is determined based on bovine (Bos_taurus_UMD_3.1) genome sequence alignment.

[0012] In particular, the molecular probe assembly has nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.355.

[0013] To achieve the technical objective of this invention, a fourth aspect of this invention provides a gene chip for identifying parentage in dairy cows, wherein the gene chip is loaded with the aforementioned molecular probe combination.

[0014] To achieve the technical objective of this invention, a fifth aspect of this invention provides a kit for identifying parentage in dairy cows, which has the above-described combination of molecular probes or gene chip.

[0015] To achieve the technical objective of this invention, this invention further provides an application of the above-mentioned molecular probe combination, gene chip, or kit in any of the following aspects:

[0016] (1) Application in dairy cow breed traceability;

[0017] (2) Application in dairy cow breeding;

[0018] (3) Application in germplasm resource conservation and utilization;

[0019] (4) Application in germplasm resource improvement.

[0020] Beneficial effects:

[0021] 1. The SNP locus combination and method provided by this invention can quickly and accurately identify the parentage among a large group of dairy cows, solving the problem of complicated operation and poor effect of existing dairy cow parentage identification.

[0022] 2. Compared with traditional microsatellite DNA marker methods, the method provided by this invention can significantly reduce the cost of paternity testing. By calculating the likelihood ratio, the parent-child relationship between individuals can be determined, enabling low-cost and large-scale paternity testing of dairy cow populations.

[0023] 3. The liquid-phase chip technology prepared using the molecular marker combination provided by this invention is simple to operate. It can be used by computer to identify SNPs, which are usually just biselelic or bivariate genetic variations, that is, there are two different bases at this position. Therefore, the typing is very simple. The binary form can be used by computer programs to perform large-scale pairwise parentage testing, which greatly improves the identification efficiency, realizes automated high-throughput detection, reduces the occurrence of human error, saves labor and costs, and can also flexibly adjust SNP markers according to the actual population situation, increasing the flexibility and applicability of the technology.

[0024] 4. The SNP locus combinations and methods provided by this invention can rapidly and accurately detect the genotype of individual dairy cows. The average SNP detection rate is as high as 99.62%, and the individual detection rate is as high as 99.62%. Genotyping stability is good, and genotype consistency can reach 100%. Compared with microsatellite markers, the false positive rate is low. Figure 3 As shown.

[0025] 5. The method of this invention has low requirements for the amount of DNA sample. In the PCR process, only a fragment length of about 100 bp is generally needed to amplify, and the amount of DNA sample used for SNP tag genotyping only needs to reach 2.5~5 ng.

[0026] 6. The method of this invention enables efficient, rapid and accurate detection of dairy cows, reduces dependence on foreign breeding chips, and safeguards the biosecurity of dairy cow breeding in my country. Attached Figure Description

[0027] Figure 1 A map showing the distribution density of markers on chromosomes;

[0028] Figure 2 A statistical chart showing the individual detection rate of the test samples;

[0029] Figure 3 A statistical graph showing the consistency of SNP loci typing. Detailed Implementation

[0030] The present invention will be further illustrated below with reference to a detailed description of specific embodiments. However, these embodiments are merely illustrative and should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and can be referred to the third edition of the original book "Bioinformatics and Functional Genomics" or related books. The bioinformatics software and products used are also commercially available. Various processes and methods not described in detail are conventional methods known in the art. The source of materials used, trade names, and components that need to be listed are indicated upon their first appearance. Unless otherwise specified, the same reagents used thereafter are considered the same as those initially indicated.

[0031] Furthermore, it should be noted that the site combinations and applications provided by this invention are the result of the inventors' arduous creative work and optimization efforts.

[0032] The features and advantages described in the site combination section above also apply to molecular probe combinations, gene chips, kits, and their applications based on site combinations, and will not be repeated here.

[0033] The SNP referred to in this invention is Single Nucleotide Polymorphism, which mainly refers to DNA sequence polymorphism caused by a single nucleotide variation at the genomic level. The single nucleotide variation includes variations caused by a single base conversion, transversion, insertion, or deletion.

[0034] It should be noted that the molecular markers referred to in this invention are all heritable and detectable DNA sequences or proteins, including but not limited to molecular markers based on molecular hybridization, such as RFLP and Minisatellite DNA; molecular markers based on PCR technology, such as RAPD, STS, SSR, and SCAR; DNA markers based on restriction enzyme digestion and PCR technology; molecular markers based on DNA microarray technology, such as SNP; and analytical marker technologies developed based on EST databases. The molecular markers provided by this invention can be used for genome mapping, gene localization studies, map-based gene cloning, species phylogenetics, and systematic classification.

[0035] It should be noted that the probe referred to in this invention is a nucleic acid sequence (DNA or RNA) that is complementary to the target gene, has a known sequence and carries a detection marker, such as the Taqman-MGB probe.

[0036] It should be noted that the kit referred to in this invention is any kit commonly used in the art that contains reagents for detection or experimentation, freeing operators from the cumbersome process of reagent preparation and optimization. In one embodiment of this invention, it includes primers for amplifying the site information provided by this invention, molecular markers or probes or gene chips for detecting the site information provided by this invention, as well as enzymes and buffers for amplification, or fluorescent labels for detection.

[0037] Example 1: Obtaining Locus Combinations for Identifying Parent-Child Relationships in Dairy Cows

[0038] 1. Collection and organization of dairy cow pedigrees

[0039] Individual numbers, dates of birth, father numbers, father's country of origin, mother numbers, and mother's country of origin were collected from 19,556 Holstein cattle at Ranch 1 of a dairy farm in Beijing from January 1992 to May 2023, and from 953 Holstein cattle at Ranch 2 from January 2000 to September 2019.

[0040] 2. SNP screening

[0041] Blood samples from 2297 and 226 cows at a dairy farm in Beijing (farms 1 and 2, respectively) were analyzed using an Illumina Bovine SNP150K microarray. Each microarray contained 124,843 loci with clearly defined locations, distributed on both autosomes (chromosomes 1-29) and sex chromosomes. Referring to relevant research from the U.S. Department of Agriculture (http: / / cgemm.louisville.edu / USDA / index.html), candidate SNP loci were selected based on the following criteria: minimum allele frequency (MAF) greater than 0.45, meeting the Hardy-Weinberg equilibrium test (P < 0.001), loci located on sex chromosomes were removed, the locus detection rate was higher than 0.95, and the distance between adjacent SNPs on each chromosome was greater than 30 Mb. Based on these criteria, 260 candidate loci were obtained for paternity testing.

[0042] 3. Mother-child pairs with pedigree and microarray data

[0043] Based on the birth dates and cattle numbers recorded in the pedigree records, 816 and 51 parent-offspring pairs were selected from two different ranches, respectively. Searching the cattle number information in the microarray data revealed that all these parent-offspring pairs had undergone microarray analysis, indicating that the collected sample data contained genetic data.

[0044] 4. Construct a parent-child relationship prediction model

[0045] Based on the above steps, genotypic data for 816 pairs and 51 pairs of parent-child pairs of cows and their offspring calves were obtained. The probability of paternity exclusion for a particular calf was calculated using Cervus 3.0 software, as shown in the following formula:

[0046]

[0047] Where P: the exclusion probability of a single genetic marker, PE; n: the number of alleles for each marker; P i The frequency of the i-th allele. The formula for calculating the total exclusion probability of k markers is: .

[0048] To improve the reliability of paternity testing results, EasyPC 3.6 software was used. The maximum likelihood principle was applied to infer parentage among cattle with phylogenetic records indicating a parent-child relationship across all population groups. If the likelihood ratio for parentage was greater than 0.5, a parent-child relationship was determined between the two individuals. The formula for inferring parentage using the likelihood ratio (LR) is as follows:

[0049]

[0050] Where H1 represents the biological mother of the offspring, H2 represents the biological mother of the offspring from a random individual, and P represents the probability of the genetic relationship between the tested individual and the offspring under the two assumptions.

[0051] The pedigree error rates for Farm 1 and Farm 2 at a dairy farm in Beijing were 0.6% and 4% respectively, calculated using the exclusion rate method. The likelihood method yielded pedigree error rates of 14.5% and 7.8% for the two farms, with both methods excluding individuals with the same name who were not related. The exclusion rate method is suitable for initial screening, excluding genotypes that do not meet the criteria, but it cannot provide a specific probability value for parentage, potentially missing closely related individuals. The likelihood method provides a specific probability value for parentage, which is superior to the "yes / no" judgment of the exclusion rate. Therefore, the pedigree error rate results obtained using the likelihood method are considered more accurate.

[0052] 5. Construct a SNP candidate marker information database

[0053] To improve the universality and accuracy of candidate SNP loci application, integrate "customized genetic data" with "industry standards," and meet the needs of supporting transnational breeding cooperation, genetic resource assessment, and breed protection, the 260 loci screened in step 2 and the 200 validated loci published by the International Committee on Animal Recording (ICAR) were integrated and analyzed based on the SNP loci's names (location information), revealing 10 shared loci. Therefore, the 260 SNP loci screened in this invention are used as candidate markers for Holstein cattle paternity testing.

[0054] 6. Obtaining SNPs from paternity testing

[0055] To verify the accuracy of candidate SNPs in identifying parentage in dairy cows, a validation cohort was constructed, blood samples were collected to extract DNA, and DNA probes were designed and targeted capture sequencing was performed based on 260 candidate SNP sites. The probability of parentage was then calculated using the likelihood method. Details are as follows.

[0056] Blood samples were collected from 26, 20, and 38 pairs of Holstein cattle with pedigree-related parentage at a dairy farm in Shanghai, Ranch No. 3 in Beijing, and Ranch No. 4 in Beijing, respectively. DNA was extracted using the Tiangen reagent kit, and DNA probes were designed and CAGT was applied to 260 candidate SNP sites. ⓇTargeted capture sequencing was used to validate candidate parentage marker sites, ultimately leading to the development of the CAGT bovine parentage detection chip. Liquid-phase probe capture sequencing was performed by Beijing Compson Agricultural Technology Co., Ltd.

[0057] (1) Analysis of liquid-phase probe capture sequencing data

[0058] Locus genotyping and annotation were performed according to the following bioinformatics analysis workflow.

[0059] ① Data quality control

[0060] After the raw data is processed, it will contain reads with adapters or low quality. Before subsequent analysis, we need to filter the raw data. The filtering conditions are as follows: remove reads with adapters; remove paired reads when the N content in the sequencing read exceeds 10% of the bases in the read; remove paired reads when the number of low quality (Q<=5) bases in the sequencing read exceeds 50% of the bases in the read.

[0061] The original data was filtered using the steps described above, and the amount of data before and after filtering was statistically analyzed. The results are shown in Table 2.

[0062] Table 2. Statistical results of data volume (taking the first ten samples as an example)

[0063]

[0064] Note: Raw Base (G) is the number of bases in the raw data; Raw Reads is the number of reads in the raw data; Clean Base (G) is the number of bases after filtering; Clean Reads is the number of reads after filtering; EffectiveRate (%) is the ratio of clean reads to raw reads; Q20 (%) is the percentage of bases with a Phred value greater than 20 out of the total bases; Q30 (%) is the percentage of bases with a Phred value greater than 30 out of the total bases; GC Content (%) is the percentage of GC bases out of the total bases.

[0065] ① Alignment with reference genome

[0066] Sequence alignment was performed using the bovine ARS-UCD1.3 genome as a reference. The reference genome is available at ftp: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF_002263795.2_ARSUCD1.3 / GCF_002263795.2_ARSUCD1.3_genomic / Gna.gz, and the annotation file is available at ftp: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF_002263795.2_ARSUCD1.3 / GCF_002263795.2_ARSUCD1.3_genomic.gtf.gz. This version of the genome was assembled using short-read sequencing technology and contains 29 autosomes and X and Y sex chromosomes, with a total length of approximately 2.85 Gb. The gene annotation covers approximately 22,000 protein-coding genes. The high-quality clean data after quality control was aligned to the reference genome using BWA software. The average best alignment rate of all samples was 89.09%, and the average repetitive sequence ratio was 14.86%, as shown in Table 3. The alignment results are normal and can be used for subsequent variant detection and related analysis.

[0067] Table 3. Statistics of sample comparison results (taking the first ten samples as an example)

[0068]

[0069] Note: Total reads: Total number of reads aligned; Mapped reads: Number of reads mapped to the reference genome; Mapped ratio (%): Percentage of reads mapped to the reference genome out of all reads; Properly mapped: Number of reads whose paired-end sequencing sequences are mapped to the reference genome and whose distances conform to the length distribution of the sequencing fragments. Dup ratio (%): Percentage of duplicate reads out of all reads.

[0070] ② Target site analysis

[0071] The HaplotypeCaller module in GATK software was used to generate SNP and INDEL variant site files, and target sites were extracted. Then, 5X filtering was applied to mark sites that did not meet the requirements as ". / .". This sequencing included a total of 260 sites. Analysis yielded detailed information for all 260 sites in the samples, and some results are shown in Table 4.

[0072] Table 4 Genotype results at target loci (taking the first ten loci as an example)

[0073]

[0074] Sequencing depth directly reflects the site capture efficiency and uniformity, and can indirectly indicate the accuracy of genotyping. Generally, genotyping is more accurate when the depth is greater than 5X. Therefore, we statistically analyzed the sequencing depth and found that the sequencing depth of all sites met the requirements (Table 5).

[0075] Table 5 shows the statistical results of locus depth (taking the first ten loci as examples).

[0076]

[0077] ③ Target site annotation

[0078] Annotation was performed using Annovar software, and the annotation results were added to the INFO column of the VCF file. These columns, in order of appearance, showed the region where the mutation occurred (Func.refGene), gene information of the mutation location (Gene.refGene), detailed information about the gene containing the mutation location (GeneDetail.refGene), exon mutation type (ExonicFunc.refGene), and amino acid changes before and after the mutation (AAChange.refGene). The results showed that the number of SNPs and Indels annotated varied across different regions of different samples. Most SNPs were located in intergenic regions, followed by intronic regions, while fewer SNPs were located in exon regions and within a 1 kb range upstream and downstream of transcription start sites. No Indels were identified in any of the samples, as shown in Table 6.

[0079] Table 6 Statistics on the region or type of SNP (taking the first ten samples as an example)

[0080]

[0081] Note: Upstream and downstream refer to regions where the variant is located 1 kb from the transcription start site.

[0082] (2) Inference of parent-child relationship

[0083] Based on the CAGT bovine parentage detection chip developed using the above steps, the likelihood method was used to infer parentage relationships in 84 pairs of mother-cattle pairs from 168 individuals in a validation group experimental farm. The results showed that the pedigree error rates for a certain dairy farm in Shanghai, Ranch No. 3 and Ranch No. 4 in Beijing, and the validation group provided by Compson were 11.54%, 15.00%, and 20.51%, respectively. After further removing loci with a detection rate less than 0.9 and those exhibiting Mendelian conflicts, 257 SNP loci were ultimately retained for parentage detection, such as... Figure 2 As shown.

[0084] It should be noted that the genetic information referred to in this invention refers to the information passed from parent to offspring in order for an organism to replicate itself, or the information passed from cell to cell during each cell division.

[0085] It should be noted that the extraction of genetic information (e.g., DNA) from samples for high-depth sequencing can be performed by biotechnology companies, such as BGI Genomics and Illumina. The high-depth sequencing method adopts conventional methods in the field or the methods of biotechnology companies. In one embodiment of the present invention, an average sequencing depth of ~25.7× is used, and a resequencing analysis process is applied for high-depth sequencing.

[0086] Example 2: Obtaining a probe for identifying parentage in dairy cows

[0087] Those skilled in the art can design primers based on the sequence information in the SNPs provided by the present invention, and use the designed primers for secondary structure evaluation and Tm value evaluation, and finally obtain primers with good specificity, high sensitivity, and the ability to achieve the detection purpose under the same reaction conditions.

[0088] The secondary structure assessment and Tm value assessment can be performed using any method commonly used in the field. For example, the secondary structure can be assessed using DNA folding form (see http: / / unafold.rna.albany.edu / ?q=mfold / DNA-Folding-Form), and the Tm value can be assessed using the software RaW-Probe.

[0089] The above methods are all conventional methods. Based on the site information in the SNPs provided in this application, they can be obtained without any creative effort. Therefore, the primers obtained by the SNPs provided in this invention also fall within the protection scope of this invention.

[0090] Similarly, the preparation of probes using the SNPs provided by this invention, such as the Tanqman probe, also falls within the scope of protection of this invention.

[0091] In one embodiment of the present invention, site scoring and site evaluation are also used for DNA probe design, as detailed below.

[0092] (1) Locator scoring

[0093] The selected candidate sites were submitted to the probe design system of Beijing Compson Agricultural Technology Co., Ltd. for scoring. Probe design was based on the evaluation results of the upstream and downstream sequences of the target site. The evaluation mainly focused on the specificity, complexity, and GC content of the upstream and downstream sequences of the target site. Priority was given to placing the target site in the middle of the probe. The designed probes were 120 bp in length. Based on the scoring results, a total of 257 high-quality custom-designed sites for the chip were selected.

[0094] (2) Site assessment

[0095] The target locus set of the proposed chip was evaluated, primarily focusing on the SNP locus density distribution of the final chip product. The locus evaluation results indicate that the loci uniformly cover the entire genome, encompassing important trait loci, and can be applied to gene screening, gene mapping, marker-assisted breeding, and other fields.

[0096] The final probe sequences are shown in Table 7:

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] The distribution density of the above probes on the chromosome is as follows: Figure 1 As shown in the figure, the loci are evenly distributed across each chromosome, indicating that the loci contained in the chip can cover each chromosome well.

[0107] Example 3 Synthesis of a liquid-phase chip for identifying parentage in dairy cows

[0108] Those skilled in the art can use conventional methods to fix the primers or probes obtained in Example 2 onto a polymer substrate, such as a nylon membrane, nitrocellulose membrane, plastic, silicone wafer, or micro magnetic beads, or fix the probes onto a glass plate, or directly synthesize the primers or probes obtained in Example 2 on a hard surface such as glass, to obtain a gene chip.

[0109] In one embodiment of the present invention, in order to further improve ease of use, save costs, and further enhance detection accuracy, the present invention provides a liquid phase chip for identifying the parent-child relationship of dairy cows, the specific method of which is as follows:

[0110] 1. Based on the 257 SNP sites obtained in the previous screening, probes were designed for the two alleles of the SNPs respectively;

[0111] 2. Collect bovine blood, tissues, hair, and other samples, extract DNA, and ensure that the total amount of a single sample is >500ng, the concentration of a single sample is >10ng / ul, and the sample is free from macromolecular contamination and remains intact without degradation;

[0112] 3. The designed probe is bound to the target region of the bovine DNA sequence, and liquid-phase hybridization capture sequencing is performed on the target region to obtain the genotype of the target site;

[0113] 4. Use the likelihood method to infer parentage based on the differences in genotypes at the same locus between potential female and male offspring.

[0114] It should be noted that those skilled in the art can use any method to prepare gene chips for identifying the parentage of dairy cows, or they can entrust a biotechnology company to prepare them. However, SNP gene chips prepared based on the locus information provided in this application are all within the protection scope of this invention.

[0115] Example 4: Kit for Identifying Parentage in Dairy Cows

[0116] The identification kit provided in this application includes primers, probes, or gene chips obtained based on the SNPs obtained in the above embodiments. Depending on the type of use, it also includes corresponding detection reagents. For example, when the SNPs obtained in Example 1 are prepared as TaqMan probes, it also includes buffers, ligases, AceQ Universal U+ Probe Master Mix V2, TaqMan Probe, etc., commonly used in real-time PCR reactions.

[0117] Those skilled in the art can configure different kits depending on the usage method, but all kits for identifying SNP loci information of dairy cow parentage provided in this application are within the protection scope of this invention.

[0118] Example 5: Identification of Parentage in Dairy Cows

[0119] To further optimize SNP loci and improve the accuracy of SNP-based parentage prediction, 23 pairs of Holstein cattle blood samples from Beijing Compson Agricultural Technology Co., Ltd., which had been verified to be parent-child, were used as experimental subjects. DNA was extracted and subjected to CAGT bovine parentage microarray liquid-phase capture sequencing, and parentage was inferred using the likelihood method. The results showed that 22 pairs were parent-child pairs, with a prediction accuracy of over 95%.

[0120] The above description is merely a preferred embodiment to aid in understanding the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any alterations or modifications made by those skilled in the art based on this description without departing from the spirit of the present invention should also fall within the scope of the present invention.

Claims

1. Application of reagents for detecting 257 SNP loci in identifying kinship in dairy cows. The information of the 257 SNP loci is shown in the table below: Its physical location was determined based on bovine Bos_taurus_UMD_3.1 genome sequence alignment.

2. A method for identifying the kinship of dairy cows involves detecting SNP locus information of two individuals with different birth dates and calculating the likelihood ratio of the parent-child relationship between them and other kinship relationships based on the SNP locus information. If the likelihood ratio of the parent-child relationship is greater than a given threshold of 0.5, the result indicates that the two individuals are related as parents. wherein The SNP site is the SNP site described in claim 1.

3. A molecular probe array for identifying bovine kinship, wherein the molecular probe array detects the SNP sites as described in claim 1 in the sample to be tested, and the physical location information of the site arrays in the table is determined based on bovine Bos_taurus_UMD_3.1 genome sequence alignment.

4. The molecular probe assembly as described in claim 3, wherein the nucleotide sequence of the molecular probe assembly consists of SEQ ID NO.1-SEQ ID NO.

355.

5. A gene chip for identifying the kinship of dairy cows, said gene chip being loaded with the molecular probe combination as described in any one of claims 3 or 4.

6. A kit for identifying the kinship of dairy cows, comprising the molecular probe combination as described in claim 3 or 4 or the gene chip as described in claim 5.

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