Chinese cattle multi-variety identification SNP (Single Nucleotide Polymorphism) marker combination and liquid phase chip technology

Abstract

The invention relates to a Chinese cattle multi-variety identification SNP (Single Nucleotide Polymorphism) marker combination and a liquid phase chip technology. According to the present invention, the genetic characteristics of the five Chinese cattle varieties and the 38 varieties of the six districts are deeply analyzed, the specific identification sites are screened, the unique gene identification of each variety can be accurately identified, the liquid phase chip is further developed, and the chip has characteristics of rich functional sites, good pertinence and good accuracy, can be used for variety identification and specific character identification, and can be used for identifying the variety and the specific character. Low-cost and high-throughput detection is realized, and powerful support is provided for identification, protection and development of Chinese cattle varieties.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular breeding technology, and relates to SNP marker combinations and liquid phase chip technology for multi-breed identification of Chinese yellow cattle. Background Technology

[0002] Currently, there are 58 local breeds of yellow cattle in China, including the Chinese Yellow Cattle ( Bos taurus It possesses abundant and valuable local breed resources, such as Qinchuan cattle, Nanyang cattle, Luxi cattle, Yanbian cattle, Mongolian cattle, and Leiqiong cattle, which are widely distributed in different ecological zones and have formed unique adaptive characteristics (such as cold resistance, heat resistance, disease resistance, and high altitude adaptability).

[0003] For a long time, cattle breeding has faced numerous challenges. Traditional breeding methods rely on phenotypic observation and experience-based judgment, which are inefficient, time-consuming, and difficult to accurately capture desirable traits at the genetic level. Over the past decade, with the continuous support and guidance of the National Beef Cattle Genetic Improvement Program (versions 1.0 and 2.0), China's beef cattle breeding system has become increasingly sophisticated, with improvements in local breeds, introduced breeds, and the selection of cultivated breeds gradually increasing. However, for these numerous local breeds with different adaptive characteristics, the lack of rapid and accurate technical means for identification hinders the protection and utilization of breed resources. In recent years, high-throughput sequencing has developed rapidly, with second-generation and third-generation sequencing emerging, further reducing sequencing costs and increasing sequencing accuracy and speed. However, whole-genome-based analysis requires high computational power and analytical capabilities, which is not conducive to practical production. Selection signal analysis plays a crucial role in population genetics, aiming to identify the impact of natural and artificial selection on variations in the genome, compare genetic variations in different populations, and reveal the relationship between adaptive traits and selection pressure. For example, CN119710016A discloses an SNP chip for distinguishing five native Hubei yellow cattle breeds (Yiling Yellow Cattle, Dabie Mountain Yellow Cattle, Yunba Yellow Cattle, Enshi Yellow Cattle, and Sanjiaoshan Yellow Cattle). By combining whole-genome sequencing and three selection signals to obtain candidate SNP sites, and then using random forest scoring on the candidate sites to further reduce the number of required SNP sites, a breed-specific combination of SNP sites is finally obtained. Based on these SNP sites and the 60 bp sequences before and after them, probes can be designed to develop an SNP chip for distinguishing the five native Hubei yellow cattle breeds.

[0004] In conclusion, developing a high-throughput sequencing solution applicable to the identification of more Chinese cattle breeds is of great significance to the field of Chinese cattle breeding. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides SNP marker combinations and liquid phase chip technology for multi-breed identification of Chinese Yellow Cattle. It deeply mines specific SNP sites for local Chinese Yellow Cattle breeds and further develops liquid phase chips, which can be used for the identification of local Chinese Yellow Cattle breeds and traits, providing a scientific basis for precision breeding.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a combination of SNP molecular markers for the identification of cattle, as shown in Table 1.

[0007] Table 1 This invention provides an in-depth analysis of the genetic characteristics of China's five major yellow cattle breeds and 38 breeds from six regional swarms, tailoring specific identification loci for each breed to accurately identify their unique genetic markers. For example, it precisely identifies specific loci for five superior yellow cattle breeds: Qinchuan cattle, Nanyang cattle, Luxi yellow cattle, Yanbian cattle, and Jiaxian red cattle. Furthermore, for different breeds within the six regional swarms, it effectively distinguishes between: Mongolian cattle and Qaidam cattle (cold-resistant and hypoxia-tolerant from the Northwest region); 13 breeds from the East China region adapted to humid climates, such as Luxi yellow cattle; 6 breeds from the Central China region adapted to transitional climates, such as Qinchuan cattle; 6 breeds from the South China region adapted to tropical climates and resistant to insects, such as Leiqiong cattle; 11 breeds from the Southwest region adapted to high-altitude mountainous areas, such as Dianzhong cattle; and Yanbian cattle from the Northeast region, which is extremely cold-resistant. These loci, closely linked to the genetic characteristics of each breed, provide strong support for breed identification, conservation, and development.

[0008] Secondly, the present invention provides the application of the SNP molecular marker combination and / or its detection reagents for identifying cattle described in the first aspect in the preparation of cattle identification products.

[0009] Preferably, the detection reagent comprises a nucleic acid probe of the SNP molecular marker combination.

[0010] Thirdly, the present invention provides a liquid phase chip for identifying cattle, the liquid phase chip comprising nucleic acid probes for detecting the SNP molecular marker combination for identifying cattle as described in the first aspect.

[0011] This invention designs a liquid phase chip based on the discovered SNP sites, which accurately covers specific sites of Chinese Yellow Cattle, enabling breed identification and identification of specific traits. The chip can quickly identify the bloodline ratio of hybrid cattle, such as the bloodline ratio of Qinchuan cattle. Combined with GWAS analysis, it can accelerate the breeding process of traits such as disease resistance and roughage tolerance, and can achieve low-cost, high-throughput detection.

[0012] Preferably, the liquid phase chip includes a 50K liquid phase chip.

[0013] Fourthly, the present invention provides a method for preparing the liquid phase chip for identifying yellow cattle as described in the third aspect, the method comprising: Design and prepare nucleic acid probes for the SNP molecular marker combination used for the identification of yellow cattle, and use the nucleic acid probes to prepare liquid-phase chips.

[0014] Preferably, the design principles of the nucleic acid probe include: probe length of 1~150 bp, probe GC content of 20%~80%, number of homology regions ≤5, and the selected region should not contain SSR and GAP regions to the greatest extent possible.

[0015] Preferably, the 5' end of the nucleic acid probe is modified with a biotin group.

[0016] Fifthly, the present invention provides the application of the liquid phase chip for identifying scalpers described in the third aspect in the identification of scalpers.

[0017] Sixthly, the present invention provides a method for identifying scalpers, the method comprising: Obtain DNA samples from the individual cattle to be tested; Genotype data of the individual cattle to be tested are obtained based on the liquid phase chip for cattle identification as described in the fourth aspect. The genotype data is compared and analyzed with known reference genotypes to identify the individual cattle to be tested.

[0018] Preferably, the individual cattle to be tested includes at least one of Qinchuan cattle, Nanyang cattle, Luxi cattle, Jiaxian red cattle, or Yanbian cattle.

[0019] Preferably, the comparison analysis includes at least one of gene distribution frequency calculation, cluster analysis, or principal component analysis.

[0020] Preferably, the identification of the individual cattle to be tested includes pedigree identification and / or trait identification.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: This invention targets specific SNP loci in local breeds of Chinese yellow cattle and further designs liquid-phase chips, which can deeply analyze the genetic information of cattle, such as genes related to growth rate, reproductive performance, meat quality, and disease resistance, providing a scientific basis for precision breeding. Attached Figure Description

[0022] Figure 1 This is a distribution density map of SNP sites on chromosomes on a liquid-phase chip.

[0023] Figure 2 This is a statistical chart of SNP mutation types at SNP sites on a liquid phase chip.

[0024] Figure 3 This is a statistical diagram of the MAF distribution of sites on the SNP liquid phase chip.

[0025] Figure 4 This is a functional distribution diagram of the sites on the SNP liquid phase chip.

[0026] Figure 5 Phylogenetic tree diagram of the varieties tested by SNP liquid phase chip detection.

[0027] Figure 6 PCA diagrams of samples from different regions detected by SNP liquid phase chip.

[0028] Figure 7 This is a statistical chart showing the detection rate of sample sites detected by SNP liquid phase chip.

[0029] Figure 8 A statistical chart showing the consistency of genotyping in SNP liquid phase chip detection of duplicate samples.

[0030] Figure 9 Chromosome distribution map of SNP loci in Qinchuan cattle detected by SNP liquid phase chip. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0033] Example 1 This embodiment performs specific site screening.

[0034] Whole-genome resequencing was performed on 640 cattle samples. Differential loci analysis was conducted based on five core cattle breeds (Qinchuan cattle, Nanyang cattle, Luxi Yellow cattle, Jiaxian Red cattle, and Yanbian cattle) and six regional cattle populations to identify characteristic loci that effectively distinguish these populations. The 640 cattle samples consisted of 31 Mongolian cattle, 12 Qaidam cattle, 9 Luxi Yellow cattle, 10 Bohai Black cattle, 15 Guangfeng cattle, 22 Wenling Gaofeng cattle, 14 Ji'an cattle, 8 Minnan cattle, 10 Tiantai cattle, 16 Zhoushan cattle, 12 Southern Anhui cattle, 3 Anxi cattle, 4 Dabieshan cattle, 2 Eastern Anhui cattle, 159 Qinchuan cattle, 8 Nanyang cattle, 41 Jiaxian Red cattle, and 10 Enshi cattle. The following cattle breeds were included: 15 head of Xiangxi cattle, 8 head of Zaobei cattle, 18 head of Leiqiong cattle, 7 head of Hainan Yellow cattle, 14 head of Nandan cattle, 12 head of Lingnan cattle, 16 head of Weizhou cattle, 4 head of Jinjiang cattle, 12 head of Dianzhong cattle, 13 head of Xuanhan cattle, 10 head of Liangshan cattle, 12 head of Guanling cattle, 9 head of Weining cattle, 23 head of Lincang Gaofeng cattle, 18 head of Wenshan Gaofeng cattle, 10 head of Zhaotong cattle, 3 head of Zhangmu cattle, 12 head of Dengchuan cattle, 19 head of Tibetan Yellow cattle, and 19 head of Yanbian cattle. The five core cattle breeds mentioned are Qinchuan cattle, Nanyang cattle, Luxi Yellow cattle, Jiaxian Red cattle, and Yanbian cattle.

[0035] The above-mentioned cattle populations are divided into 6 regions as follows: Northwest region cattle (Mongolian cattle, Qaidam cattle), characteristics: high altitude adaptability, cold resistance, and hypoxia tolerance; East China breeds (Luxi Yellow Cattle, Bohai Black Cattle, Guangfeng Cattle, Wenling Gaofeng Cattle, Ji'an Cattle, Minnan Cattle, Tiantai Cattle, Zhoushan Cattle, Wannan Cattle, Anxi Cattle, Dabieshan Cattle, Wandong Cattle), characteristics: adapted to humid climates, with emphasis on disease resistance, moisture tolerance and insect resistance in paddy field aquaculture environments; Central China breeds (Qinchuan cattle, Nanyang cattle, Jiaxian red cattle, Enshi cattle, Xiangxi cattle, Zaobei cattle); The South China breed (Leiqiong cattle, Hainan yellow cattle, Nandan cattle, Lingnan cattle, Weizhou cattle, Jinjiang cattle) is characterized by its adaptation to hot climates and high humidity environments, with a focus on heat resistance, high temperature resistance, and insect resistance. Southwest Region Cattle (Dianzhong Cattle, Xuanhan Cattle, Liangshan Cattle, Guanling Cattle, Weining Cattle, Lincang Gaofeng Cattle, Wenshan Gaofeng Cattle, Zhaotong Cattle, Zhangmu Cattle, Dengchuan Cattle, Tibetan Yellow Cattle), Characteristics: Adapted to the variable mountain climate, with a focus on high altitude tolerance, hypoxia resistance, cold resistance and insect resistance; The Northeast region (Yanbian cattle) is adapted to the northern climate and environment, and its main characteristics are cold resistance, low temperature resistance, and strong adaptability.

[0036] (1) Genomic data preprocessing and quality control First, quality control was performed on the raw SNP genotype data. The steps included: retaining autosomal loci; removing loci with a deletion rate higher than 10%; removing loci with a minimum allele frequency (MAF) of less than 5%; and removing individuals with a sample deletion rate higher than 20%. After quality control, a total of 471 samples (23,982,697 loci) from 5 core cattle breeds and 470 samples (23,893,675 loci) from 6 regional cattle breeds were retained.

[0037] (2) Population genetic differentiation analysis (FST) Based on quality control, the FST value of each locus was calculated to assess the degree of genetic differentiation among populations: the loci were sorted from high to low according to their FST values; and highly differentiated loci with high FST values ​​were selected as characteristic SNPs.

[0038] (3) Genome-wide sliding window screening of representative loci Further sliding window analysis was performed on population genetic differentiation data of highly differentiated loci with high FST values. Combined with: SNP deletion rate, minimum allele frequency (MAF), Hardy-Weinberg equilibrium test (HWE), annotation information, and the evenness of SNP distribution in the genome, the following results were obtained: core breed cattle: 19,922 characteristic loci; regional cattle population: 23,845 characteristic loci.

[0039] (4) Constructing a background site set Based on the candidate functional site set, the missing rate, MAF, HWE test results and annotation information were further integrated to screen out 599 background sites to enhance the stability and versatility of the chip.

[0040] (5) Screening of loci related to economic traits Economic trait loci were obtained through further screening based on loci obtained from GWAS analysis in previous experiments and QTL loci, GWAS loci, and important gene loci known in public databases that are related to various economic traits (including meat quality and carcass traits, reproductive traits, production performance, health traits, variety identification, and appearance traits). Specifically, the loci of each economic trait were obtained by taking the intersection of the highly differentiated loci with the highest FST values ​​in step (2) of the above-mentioned loci set. Then, genotyping verification was performed. The ref bases were corrected by comparison with the reference genome ARS-UCD1.3, and duplicate loci were removed to establish the set of economic trait loci.

[0041] (7) Integration of various trait loci The locus sets of all the above trait categories were merged, and redundant or duplicate loci were removed while retaining representativeness and functionality to ensure the representativeness, coverage, and technical stability of the chip design. The final set of specific loci was obtained, including 16,224 variety identification loci, 19,471 regional identification loci, 8,668 meat and carcass loci, 8,802 production performance loci, 1,731 reproductive performance loci, 726 health-related loci, 112 appearance-related loci, and 598 background loci.

[0042] Example 2 This embodiment designs an SNP liquid phase chip.

[0043] Genotyping and data quality control were performed on specific loci to obtain accurate and high-quality variant loci. The quality control content is shown in Table 2, including: read coverage sequencing depth, deletion rate, kinship detection, minimum allele frequency (MAF), Hardy-Weinberg balance, outlier samples, etc., and the loci set selected was based on the chip.

[0044] Table 2 The selected candidate loci were submitted to the Compson probe design system for scoring. Probe design was based on the evaluation results of the upstream and downstream sequences of the target locus, primarily assessing the specificity, complexity, and GC content of the upstream and downstream sequences. Priority was given to placing the target locus in the middle of the probe, and the designed probes were 120 bp in length. The target locus set was evaluated, mainly including the SNP locus overview, SNP chromosomal distribution, MAF distribution, SNP type distribution, functional locus distribution, and SNP density distribution of the final microarray product. The locus evaluation results showed that the loci uniformly covered the entire genome, encompassing important trait loci, and could be applied to gene screening, gene mapping, and marker-assisted breeding.

[0045] The distribution density map of the microarray loci set on each chromosome is shown below. Figure 1 As shown, the loci are evenly distributed on each chromosome, indicating that the loci contained in the chip can cover each chromosome well.

[0046] Base transition refers to the substitution between purines or between pyrimidines; base transversion refers to the substitution between purines and pyrimidines. Due to the structure of bases, the probability of transition is usually higher than that of transversion. After detecting and filtering high-confidence SNPs, statistical analysis is performed on the SNP mutation spectrum. Point mutations include six types: A / T, A / C, A / G, G / C, G / T, and G / A. For example, A / C represents a mutation type from A to C in one chain. By classifying and statistically analyzing the mutation results of all samples, the percentage of each mutation type in each sample can be seen. The distribution chart of chip mutation types is shown below. Figure 2 As shown, the selected sites on the chip are mainly A / T and CG transition mutations, and the Ts / Tv ratio is higher than the common resequencing background value, which is consistent with the characteristics of a high-quality genotyping chip. The MAF distribution statistics are as follows: Figure 3 As shown, the MAF spectral coverage is balanced, which not only captures population differentiation and selection signals but also facilitates stable estimation and higher statistical power. The functional locus distribution results are as follows: Figure 4 As shown, the chip design sites are mainly distributed in intergenic and intronic regions, which not only ensures whole-genome LD coverage but also provides a batch of functional neighbor / potential efficacy markers, facilitating candidate gene validation.

[0047] Through conditional screening and consideration of uniform distribution, a total of 50,604 SNP sites (including 50,006 functional sites + 598 genomic background sites) were obtained for microarray synthesis.

[0048] Specifically, a bovine 50K liquid-phase chip based on targeted capture sequencing was prepared. The bovine 50K liquid-phase chip consists of individually packaged bovine 50K probe mixtures and hybridization capture reagents. The bovine 50K probes are single-stranded DNA probes, which are nucleotide sequences designed and synthesized based on screened specific sites. The design principles of the bovine 50K probes are: probe length 120 bp, probe GC content between 20% and 80%, number of homology regions ≤ 5, and the selected regions should, to the greatest extent possible, not contain SSR and GAP regions. One probe cover is designed based on the screened SNP site, and one probe cover is designed upstream and downstream of the screened INDEL site. Single-stranded nucleotides are synthesized based on the designed nucleotide sequences, and the synthesized DNA nucleotide sequence is 120 bp in length with a biotinylate group modified at the 5' end.

[0049] In addition, the identification sites can be further optimized, and one-to-many analysis can be performed: such as screening the difference sites between Qinchuan cattle and other breeds (200 specific SNPs); one-to-one analysis: such as screening 200 pedigree tracement sites for each of the 7 core breeds such as Nanyang cattle and Yanbian cattle; geographical floristic characteristic sites: screening floristic shared SNPs, economic trait sites (such as muscle growth gene MSTN, fat deposition gene FABP4) and ecological adaptation sites (such as the EPAS1 mutation related to hypoxia tolerance in Tibetan cattle) according to the 6 major ecological regions (such as the Northwest cold-resistant region and the South China insect-resistant region).

[0050] Specifically, PCA+ population differentiation index (Fst) analysis can be performed based on data of Qinchuan cattle versus other local cattle to screen 214 characteristic SNPs of Qinchuan cattle (such as the missense mutant gene ADIPOQ on chromosome 5). Analysis can be performed based on data of Mongolian cattle versus Tibetan cattle to screen SNPs that are significantly associated with altitude adaptation (P<0.01) (such as SNPs in the upstream regulatory region of the HIF1A gene).

[0051] Example 3 This embodiment provides a targeted capture sequencing detection process based on SNP liquid phase chips.

[0052] I. DNA Quality Inspection 1. Genome integrity test: Take 2 μL of the DNA sample to be tested, spot it onto a 1.5% agarose gel, electrophoresis at 150 V for 25 min, and place the gel in a UV analyzer. If there is only one band, the DNA sample is of good integrity. If slight degradation occurs (band above 5K), risky library construction can be attempted. If the degradation is severe (band below 5K), it is recommended to extract DNA again.

[0053] 2. Purity test: The purity of DNA was tested using a NanoDrop 2000 nucleic acid and protein analyzer. If the A260 / A280 ratio was between 1.7 and 2.1 and the A260 / A230 ratio was between 1.8 and 2.2, the purity of the DNA sample met the requirements for library construction.

[0054] 3. Quantitative detection: Use the dsDNA HS Assay Kit for Qubit to accurately quantify the sample with 1 μL of DNA.

[0055] II. Targeted Capture Sequencing Detection Process Compson CAGT® targeted capture sequencing products can be used. The Compson CAGT targeted capture sequencing detection process consists of three steps: genomic DNA fragmentation, whole genome library construction, and probe hybridization capture.

[0056] 1. Genomic DNA Disruption First, dilute the DNA sample: take 1.5 μg of DNA sample into a 0.6 mL labeled fragmentation tube, add TE buffer to 100 µL, vortex to mix and centrifuge.

[0057] Secondly, set the parameters for interruption: set the interruption time of the interruption device to 30 seconds, the stop time to 30 seconds, and the number of interruption cycles to 6. Place the interruption tubes symmetrically on the rotor of the interruption device to perform the interruption operation.

[0058] Finally, fragment detection: Take 10 µL of the fragmented sample into a 96-well PCR machine, add 3.5 µL of bromophenol yellow, mix well by pipetting, spot onto a 3% agarose gel, and perform electrophoresis at 170 V for 25 min. The main fragment size should be 200~300 bp (PE150 sequencing strategy).

[0059] 2. Construction of whole genome library The workflow for constructing a whole genome library is as follows: fragment selection - end repair - adapter ligation - ligation product purification - pre-PCR enrichment and purification - whole genome library quality control. 2.1 Fragment Filtering (1) Pipette 90 μL of the interrupted product into a new labeled 96-well purification plate, then add 10 μL of sterile water to 90 μL of the interrupted product, then add 85 μL of magnetic beads to 100 μL of the interrupted product, gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5 min. (2) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 min until the liquid is clear. Use a pipette to aspirate 180 μL of supernatant into a new 96-well purification plate. (3) Pipette 20 μL of magnetic beads into 180 μL of supernatant, gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5 min; (4) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Use a pipette to aspirate and discard the supernatant. (5) Keep the 96-well purification plate fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, let it stand at room temperature for 30 seconds, and then carefully discard the supernatant. (6) Repeat step (5) once, and on the last time, try to aspirate as much liquid as possible from the bottom of the plate. If a small amount of liquid remains, use a small-capacity pipette to aspirate the liquid from the bottom of the plate. (7) Keep the 96-well purification plate fixed on the magnetic rack and dry at room temperature until the magnetic beads are no longer reflective and no longer cracked; (8) Remove the 96-well purification plate from the magnetic rack, add 27 μL of sterile water to elute the DNA, gently pipette and mix thoroughly 10 times, and dissolve at room temperature for 5 min; (9) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 min until the liquid is clear. Transfer 25 μL of supernatant to a new 96-well PCR plate for the next reaction.

[0060] 2.2 End-of-trim Prepare the end-repair reaction solution on ice according to Table 3 below.

[0061] Table 3 Use a pipette to add 10 μL of end-repair reaction solution to a 6-well PCR plate, seal the plate, vortex to mix the samples, and then briefly centrifuge to collect the reaction solution to the bottom of the tube; the reaction procedure is shown in Table 4. After the reaction is completed, briefly centrifuge to collect the reaction solution to the bottom of the tube.

[0062] Table 4 2.3 Connector Connection Add 5 μL of CAGT universal adapter to the 96-well PCR plate in step 2.2; pipette 20 μL of Ligation Master Mix (connection premix) into the 96-well PCR plate, seal the plate, vortex to mix, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Perform the reaction according to the conditions in Table 5 below. Table 5 After the reaction was completed, the reaction solution was collected to the bottom of the tube by instantaneous centrifugation, and all the product was transferred to a new 96-well purification plate for purification.

[0063] 2.4 Purification of Ligation Products (1) Pipette 60 μL of magnetic beads into 75 μL of connector ligation product, gently pipette 10 times to mix thoroughly, and incubate at room temperature for 5 min; (2) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate and discard the supernatant with a pipette. (3) Keep the 96-well purification plate fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, let it stand at room temperature for 30 seconds, and then carefully discard the supernatant. (4) Repeat the above steps once. On the last time, try to absorb as much liquid as possible from the bottom of the plate. If there is a small amount of liquid remaining, use a small-capacity pipette to absorb the liquid from the bottom of the plate. (5) Keep the 96-well purification plate fixed on the magnetic rack and dry at room temperature until the magnetic beads are no longer reflective and no longer cracked; (6) Remove the 96-well purification plate from the magnetic rack, add 17 μL of TE buffer to elute DNA, gently pipette 10 times to mix thoroughly, and dissolve at room temperature for 5 min; (7) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 min until the liquid is clear. Transfer 15 μL (all of the supernatant) to a new 96-well PCR plate.

[0064] 2.5 Pre-PCR amplification, enrichment, and purification Take 15 μL of the purified ligation product and prepare the reaction system on ice according to Table 6 below.

[0065] Table 6 Use a pipette to add 35 μL of the prepared PCR amplification reaction solution to a 96-well PCR plate, seal the plate, vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Perform the PCR reaction according to the conditions in Table 7.

[0066] Table 7 After the reaction is complete, the reaction solution is centrifuged briefly to the bottom of the tube for product purification.

[0067] (1) Pipette 50 μL of magnetic beads into a 96-well purification plate containing 50 μL of PCR product, gently pipette at least 10 times to mix thoroughly, and incubate at room temperature for 5 min.

[0068] (2) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Carefully aspirate and discard the supernatant with a pipette.

[0069] (3) Keep the 96-well purification plate fixed on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, let it stand at room temperature for 30 s, and then carefully discard the supernatant.

[0070] (4) Repeat the above steps once. On the last time, try to dry the liquid at the bottom of the plate. If there is a small amount of liquid remaining, use a small-capacity pipette (0.5-10 µL) to dry the liquid at the bottom of the tube.

[0071] (5) Keep the 96-well purification plate fixed on the magnetic rack and dry at room temperature until the magnetic beads are no longer reflective and no cracks.

[0072] (6) Remove the 96-well purification plate from the magnetic rack, add 22 μL of TE buffer to elute DNA, gently pipette at least 10 times to mix thoroughly, and dissolve at room temperature for 5 min.

[0073] (7) Place the 96-well purification plate on a magnetic rack and let it stand for 2-5 minutes until the liquid is clear. Transfer 20 μL of supernatant to a new 96-well plate.

[0074] 2.6 Whole-genome library quality control The dsDNA HS Assay Kit for Qubit was used to quantify the samples (1 μL sample + 199 μL working buffer). Based on the quantified concentration, 80 ng of the purified PCR product was added to a 96-well PCR chamber, 3.5 μL of bromophenol yellow was added, and the mixture was pipetted and mixed. The mixture was then electrophoresed on a 3% agarose gel at 170 V for 25 min. The size of the qualified library fragments was approximately 350-420 bp.

[0075] The whole genome library construction is now complete.

[0076] 3. Probe hybridization and capture process The probe hybridization capture process consists of the following steps: library mixing - probe hybridization - post-hybridization elution - post-PCR enrichment and purification - capture library quality control. 3.1 Document Pooling The amount of DNA library used is referenced in Table 8 below. The total amount can exceed 1500 ng, but not more than 4 μg. Table 8 Add the prehybridization reagents listed in Table 9 to the well-mixed samples, mix well, and try to avoid generating air bubbles.

[0077] Table 9 The pre-hybridization reagents were dried at room temperature in a vacuum concentrator before proceeding with the hybridization step.

[0078] 3.2 Probe hybridization Incubate the Fast Hybridization Mix at 65°C for 10 min or until all precipitates dissolve. Quickly vortex and add 20 μL to the lyophilized sample from the previous step to resuspend the sample. Gently tap the sample with your fingertip to mix, avoiding the formation of air bubbles. Quickly centrifuge to remove air bubbles. Add 30 μL of Hybridization Enhancer to the surface of the above reagents. Place the PCR tube in a preheated PCR instrument for hybridization, as shown in Table 10. Heat the lid at 85°C.

[0079] Table 10 3.3 Washing off after hybridization (1) Shake the pre-equilibrated streptavidin magnetic beads until completely mixed, add 100 μL of magnetic beads to a 1.5 mL centrifuge tube, add 200 μL of binding buffer and mix by pipetting. (2) Place the centrifuge tube on the magnetic rack for 1 minute or until the solution is clear, discard the supernatant, and remove the centrifuge tube; (3) Repeat the above washing steps twice, for a total of three times; (4) After the final wash, add 200 μL of binding buffer and shake to resuspend and mix thoroughly. (5) After hybridization, open the PCR instrument lid and quickly transfer all the hybridization solution to the balanced magnetic beads; (6) Mix the magnetic beads with the added hybridization solution thoroughly for 30 min; (7) Remove the centrifuge tube from the mixer, centrifuge quickly, place it on the magnetic rack for 1 min, remove the supernatant, remove the tube; add 200 μL of preheated washing solution 1, mix well; incubate at 70℃ for 5 min; (8) Place the centrifuge tube on the magnetic rack for 1 min, remove the supernatant, and remove the tube; repeat the above steps, add 200 μL of preheated washing solution 1 again, mix well; incubate at 70℃ for 5 min; (9) Transfer the liquid to a new tube; place on a magnetic rack for 1 min, remove the supernatant, and remove the tube; add 200 μL of preheated elution buffer and mix with the pipette tip; incubate at 48°C for 5 min; place on a magnetic rack for 1 min, remove the supernatant, and remove the tube; (10) Repeat the above steps twice, for a total of three times; for the last time, use a 10 μL pipette tip to aspirate the washing solution. (11) Add 45 μL of water, mix well, and incubate the solution on ice; 3.4 Post-PCR enrichment and purification Mix the magnetic bead mixture from step 3.3, aspirate 22.5 μL into a 0.2 mL PCR tube, and add the PCR reaction system shown in Table 11 below.

[0080] Table 11 Mix thoroughly with a pipette tip, centrifuge quickly, and then place in a PCR instrument to execute the reaction program in Table 12.

[0081] Table 12 (1) Add 90 μL (1.8×) DNA purification magnetic beads to the amplification product and vortex to mix thoroughly; incubate at room temperature for 5 min; place the centrifuge tube on a magnetic rack for 1 min, and remove the supernatant after the solution becomes clear; (2) Add 200 μL of freshly prepared 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the washing with 80% ethanol once (2 times in total), keeping the centrifuge tube on the magnetic rack; carefully remove the residual ethanol with a 10 μL pipette tip, and place at room temperature for 5-10 min or until the magnetic beads are dry. Please be careful not to let the magnetic beads dry out. (3) Remove the tube from the magnetic rack and add 32 μL of water. Mix thoroughly by blowing with a pipette tip and incubate at room temperature for 2 min. Place the centrifuge tube on the magnetic rack for 3 min or until the solution is clear. Transfer 30 μL of supernatant to a clean 0.2 mL centrifuge tube.

[0082] 3.5 Capture Library Quality Inspection The library was initially quantified using the dsDNA HS Assay Kit for Qubit (1 μL sample + 199 μL working buffer). Based on the quantification results, the effective concentration of the library was precisely quantified using qPCR. At the same time, the size of the library fragments was evaluated using a capillary electrophoresis system. The acceptable size of the library fragments should be between 350-450 bp.

[0083] III. Sequencing After the library passes quality control, sequencing is performed according to the required effective concentration and target data volume.

[0084] Example 4 This embodiment tests an SNP liquid phase chip.

[0085] SNP loci were detected in 28 individuals from four of China's five major yellow cattle breeds—Qinchuan, Nanyang, Luxi, and Yanbian—using a liquid-phase SNP chip, yielding a total of 50,574 valid loci. Based on the information from 16,174 breed-specific loci, a phylogenetic tree was constructed to evaluate the genotyping capability of the liquid-phase chip. The results are as follows: Figure 5 As shown, this indicates that even with a large sample size, the chip still has a strong ability to classify varieties.

[0086] Microarray analysis was performed on 47 individuals from 10 geographically differentiated breeds (Central China: Qinchuan cattle, Nanyang cattle, Luxi yellow cattle, Jiaxian red cattle, Dabieshan cattle; Northern China: Yanbian cattle, Mongolian cattle; Southern China: Yiling cattle, Wuling cattle, Leiqiong cattle). Principal component analysis (PCA) was conducted based on 19,431 regional genotyping loci. The results showed that the northern breeds (Yanbian cattle, Mongolian cattle), the central breeds (5 breeds including Qinchuan cattle), and the southern breeds (3 breeds including Leiqiong cattle) formed independent and well-defined clusters. The results are as follows: Figure 6 As shown, the genotyping loci can accurately capture the geographical differentiation patterns of local Chinese yellow cattle, providing a reliable set of molecular markers for subsequent population genetic studies and breed identification.

[0087] To evaluate the detection rate of bovine 50K liquid phase microarray for production-related trait loci, SNP genotyping was performed on 13 Qinchuan cattle, and the gene frequency distribution of five traits—meat and carcass, production, reproduction, health, and appearance—was analyzed. The results showed that the average sample detection rate at 5× depth reached 98.8%. Figure 7 The consistency rate of typing in duplicate samples reached 99.8%. Figure 8 The average detection rate of all traits was higher than 96.7%, with production-related traits reaching 99.1% and meat quality and carcass-related traits reaching 98.5% (Table 13). Reference allele frequencies varied significantly among different traits, with the lowest frequency (30.3%) for the health trait and the highest (52.5%) for the meat and carcass trait; minor allele frequencies ranged from 19.6% (meat and carcass) to 25.7% (health). Excluding geographical location and breed identification loci, 18,500 loci related to the production performance of Qinchuan cattle were obtained, of which 15,998 were related to production and meat quality performance. The chromosomal distribution of these loci is shown in Table 13. Figure 9 As shown, the loci are evenly distributed on each chromosome, indicating that the loci contained in the chip can cover each chromosome well.

[0088] This indicates that the chip has good stability in detecting SNPs of various traits in Qinchuan cattle, but the allele frequency distribution shows trait specificity, which may be related to the difference in selection pressure of functional sites, providing a reliable data basis for subsequent molecular breeding.

[0089] Table 13 In summary, this invention provides an in-depth analysis of high-depth resequencing data from 38 breeds across five major Chinese cattle breeds and six families, establishing a Chinese cattle SNP reference database covering the six major families. Some breeds are categorized by geographical region, and their characteristic loci are analyzed at the familial level. These families encompass multiple breeds, compensating for the limited sample size of individual breeds and ensuring representative expression of characteristics from different families. Furthermore, a liquid-phase microarray was designed, featuring rich functional loci, strong targeting and accuracy. Based on domestically designed, detected, and analyzed processes, it significantly reduces genotyping costs. Automated detection processes can be employed, improving throughput and stability. Based on probe hybridization targeted capture sequencing technology, marker loci can be added as needed, allowing for flexible chip upgrades and expanded application scenarios, providing strong support for the identification, protection, and development of Chinese cattle breeds.

[0090] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A SNP molecular marker combination for identifying Bos taurus, characterized in that, The SNP molecular marker combination is shown in Table 1.

2. Use of the SNP molecular marker combination for yellow cattle identification and / or a detection reagent thereof in the preparation of a yellow cattle identification product.

3. Use according to claim 2, characterized in that, The detection reagent comprises a nucleic acid probe of the SNP molecular marker combination.

4. A liquid chip for identifying Bos taurus, characterized by, The liquid chip comprises a nucleic acid probe for detecting the SNP molecular marker combination for yellow cattle identification.

5. The method for preparing a liquid chip for the identification of Bos taurus according to claim 4, characterized in that, The preparation method comprises: Designing and preparing a nucleic acid probe of the SNP molecular marker combination for yellow cattle identification, and preparing a liquid chip using the nucleic acid probe.

6. The method for preparing a liquid chip for cattle identification according to claim 5, wherein, The design principle of the nucleic acid probe comprises: probe length of 100-150 bp, probe GC content of 20%-80%, and number of homologous regions ≤5, and the selected region maximally does not contain SSR and GAP regions.

7. The method for preparing a liquid chip for cattle identification according to claim 5, wherein, The 5' end of the nucleic acid probe is modified with a biotin group.

8. Use of the liquid chip for yellow cattle identification in claim 4 in the identification of yellow cattle.

9. A method of identifying a yellow cattle, characterized by, The method for identifying yellow cattle comprises: Obtaining a DNA sample of a to-be-tested yellow cattle individual; Obtaining genotype data of the to-be-tested yellow cattle individual based on the liquid chip for yellow cattle identification in claim 4; Comparing and analyzing the genotype data with known reference genotypes to identify the to-be-tested yellow cattle individual.

10. The method of identifying a yellow cattle according to claim 9, characterized in that, The to-be-tested yellow cattle individual comprises at least one of Qinchuan cattle, Nanyang cattle, Luxi yellow cattle, Jiaxian red cattle or Yanbian cattle; Preferably, the comparison and analysis comprises at least one of calculation of gene distribution frequency, cluster analysis or principal component analysis; Preferably, the identification of the to-be-tested yellow cattle individual comprises pedigree identification and / or trait identification.

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