Liquid phase chip for identifying opisthopapus taihangensis and Hebei Hebei as well as preparation method and application of liquid phase chip

Abstract

The invention relates to the technical field of molecular detection, and particularly discloses a liquid chip for identifying opisthopapus taihangensis and Hebei province as well as a preparation method and application of the liquid chip. Genotyping sites of the liquid chip comprise 1140 SNP sites; the physical positions of the 1140 SNP loci are determined based on comparison of a whole genome sequence of a bovine reference genome, and the version number of the whole genome sequence of the bovine reference genome is ARSUCD 1.2; wherein the physical position information of the 1140 SNP loci is as shown in a table 1. The liquid chip can realize genetic typing of the opisthopapus taihangensis and the Hebei province cattle, has the advantage of high detection rate, and can obviously cluster the opisthopapus taihangensis and the Hebei province cattle into two classes when clustering analysis is carried out on the opisthopapus taihangensis and the Hebei province cattle, so that a marker combination of the liquid chip can represent whole genome re-sequencing genes, assists in distinguishing the opisthopapus taihangensis and the Hebei province cattle, and is high in accuracy and high in accuracy. Therefore, the variety identification of the opisthopapus taihangensis and the Hebei Hebei is realized.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, specifically to a liquid phase chip for identifying Taihang cattle and Jinan cattle, its preparation method, and its application. Background Technology

[0002] Taihang cattle and Jinan cattle, as unique local cattle breeds in Hebei Province, China, have developed distinct genetic characteristics due to their different geographical environments and climatic conditions, resulting from long-term natural and artificial selection. Genetic identification of Taihang and Jinan cattle can precisely reveal their origins and evolutionary paths, clarifying their genetic position within the bovine family and their kinship with other breeds. In terms of resource conservation, it allows for accurate assessment of their genetic diversity, the development of scientifically effective conservation strategies, and the protection of breed purity. In agricultural production, it enables the screening of markers related to key economic traits such as growth, meat quality, and reproduction based on genetic information, effectively guiding breed selection and improvement efforts. Furthermore, it allows for optimization of breeding management models based on individual genetic differences, thereby improving breeding efficiency.

[0003] With the dramatic decrease in the cost of high-throughput sequencing, SNP markers, as third-generation molecular markers, are increasingly being used in assisted breeding for phenotypic identification and evaluation. Liquid-phase chips based on SNP sites can be used for genotyping of Taihang cattle and Jinan cattle, as well as for assisted breed identification.

[0004] Currently, my country has no experience in developing identification chips for Taihang cattle and Jinan cattle breeds. Summary of the Invention

[0005] The purpose of this invention is to provide a liquid phase chip for identifying Taihang cattle and Jinan cattle, as well as its preparation method and application. This liquid phase chip can help distinguish between Taihang cattle and Jinan cattle.

[0006] This invention is achieved through the following technical solution: The liquid-phase microarray used to identify Taihang cattle and Jinan cattle includes 1140 SNP loci for genotyping. The physical locations of the 1140 SNP loci were determined based on the whole genome sequence alignment of the bovine reference genome, with version number ARS_UCD_1.2. The physical location information of the 1140 SNP loci is shown in Table 1.

[0007] The 1140 SNP loci screened in this invention can be used to genotype Taihang cattle and Jinan cattle, thereby helping to distinguish between them.

[0008] Specifically, the liquid-phase chip includes a probe array for identifying the genotypes of 1140 SNP loci.

[0009] The design method for liquid phase chips used to identify Taihang cattle and Jinan cattle includes the following steps: S1. Obtain the original loci by performing whole-genome resequencing on Taihang cattle and Jinan cattle; S2. Perform quality control on the original sites obtained in step S1 to obtain quality control sites; S3. Compare the quality control sites obtained in step S2 with the bovine reference genome, and then perform deduplication and variability detection to obtain the filter sites; S4. 1140 SNP sites for identifying Taihang cattle and Jinan cattle were selected from the filter sites obtained in step S3. S5. Based on the 1140 SNP sites selected, nucleotide probes were synthesized and liquid-phase chips were prepared.

[0010] Specifically, in step S2, the quality control conditions include: 1) Remove adapter sequences; 2) If the number of N bases in the sequencing fragment exceeds 10, remove the paired end sequences; 3) When the proportion of low-quality bases in the sequencing fragment exceeds 40% of the fragment length, remove the paired end sequences.

[0011] Specifically, in step S3, during variability detection, SNP sites with a coverage depth <5× are removed.

[0012] Specifically, in step S4, the filtering criteria include: Prioritize the identification of functional loci, MAF ≥ 0.35, ΔRAF ≥ 0.1 between Taihang cattle and Jinan cattle populations, uniform locus distribution, avoid linkage loci, and avoid complex regions of the genome.

[0013] Specifically, in step S5, the screening conditions for synthesizing nucleotide probes are as follows: The length is 110bp, the GC content is controlled between 30% and 80%, the number of homologous regions cannot exceed 5, and for each single nucleotide site, two nucleotide sequences with 60%-70% overlap covering the site must be designed as probe sequences for that site. Probes are synthesized according to the qualified sequences.

[0014] The application of the above-mentioned liquid phase chip in the genotyping of Taihang cattle and Jinan cattle.

[0015] The above-mentioned liquid phase chip is used to distinguish between Taihang cattle and Jinan cattle.

[0016] An application of a molecular marker combination consisting of 1140 SNP sites is disclosed, including its application in gene breeding chips, in the genotyping of Taihang cattle and Jinan cattle, and in the identification of Taihang cattle and Jinan cattle; wherein the gene breeding chip includes a liquid phase chip.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention screened 1140 SNP loci using 50 Taihang cattle and 50 Jinan cattle as samples, and synthesized probe combinations based on the 1140 SNP loci according to the principle of base complementarity to prepare a liquid phase chip. This liquid phase chip can be used to achieve genotyping of Taihang cattle and Jinan cattle, with the advantage of high detection rate. Moreover, when performing cluster analysis on Taihang cattle and Jinan cattle, they clearly clustered into two categories, indicating that the marker combination of this liquid phase chip can represent whole genome resequencing genes, help distinguish Taihang cattle and Jinan cattle, and thus realize the breed identification of Taihang cattle and Jinan cattle.

[0018] 2. The liquid phase chip of the present invention has the characteristics of good genotype detection quality, good stability of genotype detection, rapid detection efficiency and high throughput. This technological advancement reduces detection costs and improves the identification efficiency of Taihang cattle and Jinan cattle. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings: Figure 1 This is a diagram showing the uniform distribution of the 1140 SNP loci of this invention on the chromosome. Figure 2 This is a statistical analysis of the distribution of 1140 SNP sites in gene structure according to the present invention; Figure 3 This is a sample clustering diagram drawn based on the PCA results in Embodiment 3 of the present invention; Figure 4 This is a clustering diagram of the NJ tree constructed using MEGA-X software in Embodiment 3 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The illustrative embodiments and descriptions of this invention are for illustrative purposes only and are not intended to limit the invention. The embodiments described below are some, but not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0021] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known structures, materials, or methods are not specifically described to avoid obscuring the invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are commercially available. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art.

[0022] Example 1: Screening for molecular marker combinations used to identify Taihang cattle and Jinan cattle This embodiment uses whole-genome resequencing data from 50 Taihang cattle and 50 Jinan cattle samples to obtain raw loci (raw sequencing data). After quality control and screening, 1140 SNP loci were obtained, as shown in Table 1. The specific screening process includes the following steps: S1. The original loci were obtained by performing whole-genome resequencing on 50 Taihang cattle and 50 Jinan cattle.

[0023] S2. Preprocess the original sites obtained in step S1 to screen out invalid and low-quality data and obtain quality control sites. The specific quality control conditions are: 1) Remove adapter sequences; 2) If the N base content in the sequencing fragment exceeds 10, remove the paired end sequence; 3) When the proportion of low-quality (Q≤20) bases in the sequencing fragment exceeds 40% of the fragment length, remove the paired end sequence.

[0024] S3. Align the quality control loci obtained in step S2 with the bovine reference genome, then perform deduplication and variability detection to obtain filter loci. Specifically: Use BWA software to align the quality control loci with the bovine reference genome ARS_UCD_1.2 to locate their positions on the reference genome, generate a BAM file, sort the BAM file using samtools, and then use Picard software to remove duplicates from all PCR repeat fragments. Finally, use GATK software to detect variations in the target site SNPs, removing SNPs with a coverage depth <5× to ensure the accuracy of the genotyping results. Heterozygous mutation sites are retained only if each allele in the heterozygous genotype has at least 4 supported reads; otherwise, they are discarded.

[0025] S4. From the filter sites obtained in step S3, 1140 SNP sites were selected for identifying Taihang cattle and Jinan cattle; the specific process is as follows: ① Prioritize the identification of functional sites: Use functional sites directly as the framework of the design sites, and fill in the areas outside the functional sites; ② Appropriate allele frequency: This ensures the effectiveness of chip detection. If the SNP frequency is too low, mutations at the SNP sites are difficult to detect, and association signals cannot be detected during association analysis, resulting in wasted sites. Therefore, in this embodiment, SNPs with MAF ≥ 0.3 are selected as candidate sites. ③ Select loci with RAF ≥ 0.1 between Taihang cattle and Jinan cattle populations; ④ Uniform locus distribution: A uniform distribution of loci across the genome ensures the effectiveness of subsequent association analyses. The uniformity of the 1140 SNP loci screened in this example is as follows: Figure 1 As shown; ⑤ Avoid linkage sites: Calculate linkage blocks, and only select the first few longest linkage blocks in an interval, and only select one site in one block, in order to avoid probe waste caused by linkage sites; ⑥ Avoid complex regions of the genome: Filter out sites located in simple repetitive sequence regions, sites with more than 5 flanking homologous regions, and sites with GC content <30% or >70%, to avoid probe capture failure in complex regions. The distribution statistics of 1140 SNP sites in gene structure are as follows: Figure 2 As shown.

[0026] A total of 1140 SNP sites were obtained, as shown in Table 1: Table 1

[0027] Note: In the physical location section, the number before the underscore indicates the chromosome, and the number after the underscore indicates the physical location of the locus on the corresponding chromosome.

[0028] Example 2: A liquid phase chip for identifying Taihang cattle and Jinan cattle, the liquid phase chip includes a probe combination for identifying the genotype of 1140 SNP loci.

[0029] The purpose of this embodiment is to apply the 1140 SNP sites in Table 1 to probe design, ultimately synthesizing a probe assembly for fabricating a liquid-phase chip. The probe synthesis conditions include: ① The probe length is 110 bp; ② The GC content is controlled between 30% and 80% to ensure stability and hybridization efficiency; ③ The number of homologous regions should not exceed 5. The selected regions should avoid simple sequence repetitions and blank regions as much as possible to reduce non-specific binding; ④ Design two nucleotide sequences with 60%-70% overlap covering the selected SNP site to ensure site capture; ⑤ Based on the nucleotide sequences designed above, synthesize two DNA nucleotide sequences of 110 bp in length (both probes detect the same SNP site simultaneously) with biotinylate modification at the 5' end, which are the bovine 1K chip probes. Mix the synthesized bovine chip probes in equal molar mass and dilute with EDTA and Tris-HCl buffer to a final concentration of 3 pmol / mL to prepare a chip probe mixture for subsequent experiments.

[0030] This embodiment prepared a bovine 1K liquid phase chip, which can be used to distinguish between Taihang cattle and Jinan cattle.

[0031] Example 3: Application of liquid phase chips for identifying Taihang and Jinan cattle in genotyping of Taihang and Jinan cattle: (a) Extracting genomic DNA from the cattle to be tested: Genomic DNA was extracted from blood samples of 102 individuals, including 52 Jinan cattle and 50 Taihang cattle. The specific methods are as follows: 1) Cut an appropriate amount of ethanol-dehydrated bovine ear tissue into small pieces and place it in a 96-well plate (if the amount is small, a 2.0 mL centrifuge tube can be used), add a 5 mm steel ball, freeze with liquid nitrogen, and then grind for 1-2 minutes using a grinder. 2) Add 500 μL Buffer PL2 and 5 μL 20 mg / mL Proteinase K to the above deep-well plate, tighten the cap, and mix well using a shaker; 3) Place in a 65℃ water bath for 30 minutes, then remove and invert to mix during the middle of the water bath; 4) Centrifuge at 4000 rpm for 10 min, then transfer 400 μL of the supernatant into a new 96-well deep-well plate; 5) Add 800 μL of LPW solution and mix thoroughly; 6) Transfer the supernatant from step 15) into a 96-well centrifuge column twice and filter under vacuum; 7) Add 600 μL of WBI to a 96-well centrifuge column, incubate at room temperature for 2 minutes, and then filter under vacuum. (Confirm that anhydrous ethanol has been added to the WBI according to the volume specified on the bottle). 8) Add 600 μL of WBII to a 96-well centrifuge column and filter under vacuum. (Confirm that anhydrous ethanol has been added to the WIII according to the volume specified on the bottle). 9) Add 600 μL WBII to a 96-well centrifuge column and filter under vacuum; 10) Place the 96-well centrifuge column into an empty collection plate and centrifuge at 4000 rpm for 5 minutes. Place the 96-well centrifuge column onto a new 96-well PCR plate and allow it to air dry at room temperature. 11) Add 60-100 μL of preheated TE buffer (TE) to a 96-well centrifuge column at 65°C, let stand at room temperature for 2 minutes, centrifuge at 4000 rpm for 5 minutes, and then transfer the DNA solution into a 96-well PCR plate. Preheating the TE buffer to 65°C helps improve the elution efficiency of DNA and allows for detection of the target fragment length by glycogel electrophoresis.

[0032] (II) Construction of a high-throughput bovine DNA sequencing library 1) Prepare the following reaction system in a PCR tube with a total volume of 20 ml: 200 ng bovine DNA obtained in step 1, 4 μL GenoBaits End Repair Buffer, 3.1 μL GenoBaits End Repair Enzyme, and the remainder is ultrapure water; all reagents used in this example are from the GenoBaits DNA-seqLibrary Prep kit of Shijiazhuang Borui Biotechnology Co., Ltd. 2) After gently mixing the reaction system, briefly centrifuge to collect the reaction solution to the bottom of the tube; 3) Place the reaction tube in the PCR instrument and perform the following reactions: 82℃ hot cap: 37℃ for 20 min, 72℃ for 20 min, Hold at 4℃; 4) Add the following components directly to the above reaction tube: a total volume of 20 μL, including GenoBaits Ultra DNA Ligase 2 HL, GenoBaits Ultra DNA Ligase Buffer 8 μL, GenoBaits Adapter for MGI 2 μL, and the remainder is ultrapure water; 5) After gently mixing the above reaction system, briefly centrifuge to collect the reaction solution to the bottom of the tube; 6) Place the reaction tube in the PCR instrument, incubate at 22℃ for 2 minutes, hold at 4℃, and then remove the heat cap; 7) Add 48 μL of DNA purification magnetic beads that have been equilibrated at room temperature for more than 30 minutes to the above system, shake to mix, try not to generate bubbles, let stand for 5 minutes, and then briefly centrifuge to collect the liquid to the bottom of the tube. 8) Place on a magnetic rack for at least 3 minutes until the solution is clear, then remove the supernatant; 9) Keep the PCR tube in a magnetic rack and add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds, then remove the supernatant; 10) Keep the PCR tube in the magnetic rack, open the lid and let it air dry for 5 minutes until the ethanol evaporates. 11) Remove the PCR tubes from the magnetic rack and allow the magnetic beads to dry. All of the above steps must be completed within one day; 12) Prepare the following reaction system in a new PCR tube: the total volume is 20 μL, including 10 μL of GenoBaits PCR MasterMix, 1 μL of 15 Barcode (10 μm)-MGI, 5 μL of I7 Barcode (2 μm)-MGI, and 4 μL of ultrapure water; 13) Add the above system to a PCR tube of size 2.12, resuspend the dried magnetic beads, and then briefly centrifuge to collect the reaction solution to the bottom of the tube; 14) Place the reaction tubes in the PCR instrument and perform the reactions as shown in Table 2: Table 2 15) Add 20 μL of DNA purification magnetic beads that have been equilibrated at room temperature for more than 30 minutes to the above system, shake to mix, try not to generate bubbles, let stand for 5 minutes, and then briefly centrifuge to collect the liquid to the bottom of the tube. 16) Place the PCR tubes on a magnetic rack for at least 3 minutes until the solution is clear, then remove the supernatant; 17) Keep the PCR tube in a magnetic rack and add 100 μL of 80% ethanol. Incubate at room temperature for 30 seconds, then remove the supernatant; 18) Keep the PCR tube in the magnetic rack and let it air dry for 10 minutes with the lid off; 19) Remove the PCR tube from the magnetic rack, add 35 μL of Tris-HCl, shake to mix well, let stand for 5 min, and then briefly centrifuge to collect the liquid to the bottom of the tube. 20) Place the PCR tube on the magnetic rack and wait for the solution to become clear (about 3 minutes). Transfer the supernatant to a new tube to complete the construction of the DNA sequencing library. 21) Library quality control: Take 1 μL of the prepared library sample and quantify it using Qubit. The total DNA content of the sequencing library should be above 500 ng. Take 3 μL of the prepared library sample and perform a 1% agarose gel electrophoresis experiment. The DNA fragment range of the electrophoresis result should be 300-500 bp to ensure the effectiveness of subsequent capture experiments. The prepared library sample can be stored at 4℃ for short periods and at -20℃ for long periods.

[0033] (iii) The 1K liquid-phase chip prepared in Example 1 was mixed with a bovine DNA high-throughput sequencing library to capture DNA fragments at the target sites: 1) Equilibrate the DNA purification magnetic beads at room temperature for more than 30 minutes, mix the bovine 50 K probe and DNA purification magnetic beads in a 0.2 mL PCR tube and centrifuge briefly. 2) Mix the following reagents into the PCR tube: 500 ng / library of the above bovine DNA high-throughput sequencing library, 115 μg (5 μL) of GenoBaitsBLock, 2 μL of GenoBaitsBLock II for ILM / MGI, and 300 ng of the above probe to form a liquid-phase chip mixture containing the bovine DNA high-throughput library and the bovine 1K probe. 3) Concentrate the above PCR tubes to dryness using a vacuum concentrator at a temperature ≤60℃; 4) After concentration, the above PCR tubes need to be centrifuged at 12000 rpm for 1 min before proceeding with subsequent operations. You can pause the steps and store them overnight at room temperature (15-25℃). 5) Dissolve all the GenoBaits hybridization reagent at room temperature; if crystals appear in the GenoBaits 2X Hyb Buffer, heat to 65°C and shake periodically until completely dissolved; 6) Add the reagent system (8 μL GenoBaits 2x Hyb Buffer V3.3, 2 μL GenoBaits Hyb Buffer Enhancer V3.3, and 6 μL NucLease-Free Water) to the PCR tube completed in step S3.3.4; 7) Mix the PCR system in the tube by pipetting or vortexing, centrifuge at 12000 rpm for 1 min, let stand at room temperature for 5 min, mix by pipetting or vortexing again, centrifuge slightly, and transfer all the mixture in the PCR tube to a 0.2 mL EP tube. 8) Incubate the above LEP tubes in a PCR amplification instrument at 95℃ for 10 minutes (heat) with the cap temperature at 105℃. 9) When the PCR amplification instrument cools down to 65°C, transfer the LEP tubes to another PCR instrument with a 75°C heating cap and incubate for 1 hour. 10) Place the elution buffer requiring preheating to 65℃ into the PCR instrument beforehand; store the remaining elution buffer at room temperature. The elution buffers are shown in Table 3: Table 3 11) Prepare DNA purification magnetic beads. DNA purification magnetic beads should be prepared immediately before use. The preparation steps are as follows: place the DNA purification magnetic beads at room temperature for 10 minutes and vortex for 15 seconds to mix them before use. 12) For one reaction preparation, place 50 μL of DNA purification magnetic beads into a 0.2 mL EP tube; 13) Place the EP tube on the magnetic rack to completely separate the magnetic beads from the solution. Remove the supernatant and retain the magnetic beads; 14) Add 150 μL of GenoBaits 1X Bead Wash Buffer to the above EP tube, vortex for 10s, transfer the EP tube to the magnetic rack, completely separate the magnetic beads from the solution, and discard the supernatant. 15) Transfer 16 μL of the hybridization solution, which has been incubated at 65℃ for 1 hour, to a prepared EP tube. This can be done on a PCR instrument. Vortex for 10 seconds to mix thoroughly, and centrifuge for 5 seconds. 16) Place the above EP tubes into a PCR chamber, incubate at 65°C for 45 min, with the hot cap at 75°C to allow the DNA to bind to the magnetic beads, and shake for 5 s every 12 min. 17) Add 100 μL of GenoBaits 1×Wash Buffer I preheated at 65℃ to each EP tube, vortex briefly for 5 seconds, centrifuge for 5 seconds, then place on a magnetic rack to completely separate the magnetic beads from the solution and discard the supernatant. 18) Add 150 μL of preheated (65℃) GenoBaits 1X Stringert Wash Buffer; slowly pipette up and down 10 times (avoid generating air bubbles); after the last sample is mixed, let it stand for 2 minutes; place it on a magnetic rack, the magnetic beads will be completely separated from the solution, and the supernatant will be quickly removed with a pipette. 19) Repeat step 18). 20) Add 150 μL of room temperature GenoBaits 1X Wash Buffer I, shake for 2 min, place on a magnetic rack, and completely separate the magnetic beads from the solution. Discard the supernatant. 21) Add 150 μL of room temperature GenoBaits 1X Wash Buffer 1II, shake for 1 min, place on a magnetic rack, and completely separate the magnetic beads from the solution. Discard the supernatant. 22) Add 150 μL of room temperature GenoBaits 1XWash Buffer III, shake for 30 seconds, place on a magnetic rack, and the magnetic beads will completely separate from the solution. Discard the supernatant. 23) Remove the test tube containing the captured DNA from the magnetic rack; 24) Add 20 μL NucLease-FreeWater, pipette 10 times to ensure all magnetic beads are resuspended. Be careful not to discard the magnetic beads. Use 10 μL of the magnetic bead resuspended solution for the following PCR amplification and purification, and keep the remaining 10 μL as a backup.

[0034] (iv) Amplify and purify the captured fragments, align the sequencing results to the reference genome, and obtain the genotyping results. 1) Based on the library type, prepare the following PCR reagents in a 0.2 mL PCR tube: 15 μL GenoBaits PCR Master Mix, 1.2 μL GenoBaits Primer Mix for MGI, 10 μL magnetic beads containing captured DNA, and 3.8 μL ultrapure water, for a total volume of 30 μL. 2) Briefly rotate and centrifuge to ensure the magnetic beads remain in the solution; 3) Place the PCR tubes into the PCR instrument, set the lid temperature to 105 ℃, and perform PCR amplification according to the procedure described in Table 4: Table 4 PCR amplification products can be incubated overnight at 4°C; 4) Add 45 μL (1.5X volume) of DNA-captured magnetic beads to each PCR reaction, vortex to mix, try to avoid generating bubbles, let stand for 5 min, then briefly centrifuge to collect the liquid to the bottom of the tube; place on a magnetic rack for at least 3 min until the solution is clear, then remove the supernatant; 5) Keep the PCR tube in the magnetic rack, add 100 μL of 80% ethanol, incubate at room temperature for 30 seconds, remove the supernatant, keep the PCR tube in the magnetic rack, and let it air dry for 10 minutes. 6) Remove the PCR tube from the magnetic rack, add 35 μL Tris-HCl, vortex to mix, let stand for 5 min, then briefly centrifuge to collect the liquid to the bottom of the tube. On the magnetic rack, wait for the solution to become clear (about 3 min), then transfer the supernatant to a new tube and store at 20 degrees Celsius. The purified PCR fragment can be stored at -20 degrees Celsius for one week. 7) Measure the library using the Qubits FLuorometer and Qubit dsDNA HS Assay Kit to determine the average length of DNA library fragments captured on the digital electrophoresis system; measure the library concentration using the KAPA Library Quantification Kit; 8) Capture sequencing was performed using a sequencer, and the genotyping results of 102 samples were evaluated as follows.

[0035] a. Detection rate SNP detection rate and individual detection rate are important indicators for evaluating chip quality, generally measured using loci on autosomes and the X chromosome. In this example, the average SNP detection rate for Taihang cattle was 99.78%, with a standard deviation of 0.007; the average detection rate for Jinan cattle was 99.83%, with a standard deviation of 0.004. Test data show that the 1K liquid-phase chip prepared in Example 2 for cattle has excellent genotyping quality. The detection rates of the 102 samples in this example are shown in Table 5. Table 5 b. Stability of genotyping Stability is generally measured by the consistency and correlation coefficient of two genotype detection results for duplicate samples. Six Taihang and Jinan cattle were used to test the bovine 1K liquid phase chip prepared in Example 2. The results of two genotype detections for the six duplicate samples are as follows: genotype consistency was 99.3%, and the correlation coefficient was 99.7%, indicating that the genotype detection of the bovine 1K liquid phase chip prepared in Example 2 has excellent stability.

[0036] Example 4: Application of liquid phase chips for identifying Taihang cattle and Jinan cattle: To evaluate the performance of the bovine 1K liquid phase chip prepared in Example 2 in the actual identification of Taihang and Jinan cattle breeds, blood samples were collected from 50 Taihang cattle and 52 Jinan cattle to extract DNA. Principal component and cluster analysis were performed on the genotype results of the above samples using the bovine 1K liquid phase chip.

[0037] Principal Component Analysis (PCA) is a statistical method that transforms a set of potentially correlated variables into a set of linearly uncorrelated variables through orthogonal transformation. The transformed set of variables is called the principal components. PCA is applied in many disciplines; in genetics, it is mainly used for cluster analysis. Based on the degree of SNP differences in an individual's genome, it clusters individuals into different subgroups according to different trait characteristics, and is also used for cross-validation with other methods. This example uses GCTA software to perform PCA analysis on the genotypes of 50 Taihang cattle and 52 Jinan cattle. The results are as follows: Figure 3 As shown.

[0038] Cluster analysis is a method of classifying a group of data into relatively simple class structures based on the degree of correlation or similarity between different individuals, generating a relatively simple class structure from a complex dataset. Cluster analysis groups closely related individuals into smaller class units and relatively distant individuals into larger class units, until all samples have been clustered. Finally, the entire classification system is systematized into a pedigree chart, which displays the degree of kinship among all samples. Neighbor-Joining (NJ) is used to cluster all samples in the group. From the analysis results, it is possible to roughly infer which samples are closely related. Generally, samples from the same family lineage are grouped together, so the clustering results can roughly indicate how many families there are and which samples belong to the same family. This embodiment uses MEGA-X software to construct the NJ tree (model: p-distance; bootstrap: 1000 times), and the results are as follows. Figure 4 As shown.

[0039] pass Figure 3 and Figure 4 As can be seen, Taihang cattle (THN) and Jinan cattle (JNN) clearly cluster into two groups. Therefore, the markers in this chip can replace whole-genome resequencing to help distinguish between Taihang and Jinan cattle. This chip exhibits high stability, rapid detection efficiency, good genotype quality, and high throughput. This technological advancement reduces detection costs and improves the identification efficiency of Taihang and Jinan cattle.

[0040] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A liquid phase chip for identifying Taihang cattle and Jinan cattle, characterized in that, The liquid-phase chip genotyping sites include 1140 SNP sites; The physical locations of the 1140 SNP loci were determined based on whole-genome sequence alignment of the bovine reference genome, with version number ARS_UCD_1.

2. The physical locations of the 1140 SNP loci are shown in Table 1. Table 1 。 2. A liquid phase chip for identifying Taihang cattle and Jinan cattle, characterized in that, The liquid-phase chip includes a probe array for identifying genotypes at 1140 SNP loci.

3. The design method of the liquid phase chip for identifying Taihang cattle and Jinan cattle as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Obtain the original loci by performing whole-genome resequencing on Taihang cattle and Jinan cattle; S2. Perform quality control on the original sites obtained in step S1 to obtain quality control sites; S3. The quality control sites obtained in step S2 are compared with the bovine reference genome, and then duplicate removal and variability detection are performed to obtain the filter sites. S4. 1140 SNP sites for identifying Taihang cattle and Jinan cattle are selected from the filtering sites obtained in step S3. S5. Based on the 1140 SNP sites selected, nucleotide probes were synthesized and liquid-phase chips were prepared.

4. The design method according to claim 3, characterized in that, In step S2, the quality control conditions include: 1) Remove adapter sequences; 2) If the number of N bases in the sequencing fragment exceeds 10, remove the paired end sequences; 3) When the proportion of low-quality bases in the sequencing fragment exceeds 40% of the fragment length, remove the paired end sequences.

5. The design method according to claim 3, characterized in that, In step S3, during variability detection, SNP sites with a coverage depth <5× are removed.

6. The design method according to claim 3, characterized in that, In step S4, the filtering criteria include: Prioritize the identification of functional loci, MAF ≥ 0.35, ΔRAF ≥ 0.1 between Taihang cattle and Jinan cattle populations, uniform locus distribution, avoid linkage loci, and avoid complex regions of the genome.

7. The design method according to claim 3, characterized in that, In step S5, the screening conditions for synthesized nucleotide probes are as follows: The length is 110bp, the GC content is controlled between 30% and 80%, the number of homologous regions cannot exceed 5, and for each single nucleotide site, two nucleotide sequences with 60%-70% overlap covering the site must be designed as probe sequences for that site. Probes are synthesized according to the qualified sequences.

8. The application of the liquid phase chip as described in claim 1 or 2 in the genotyping of Taihang cattle and Jinan cattle.

9. The application of the liquid phase chip as described in claim 1 or 2 in the identification of Taihang cattle and Jinan cattle.

10. An application of a molecular marker combination, characterized in that, The molecular marker combination consists of the 1140 SNP sites shown in Table 1 of the claims, and its applications include applications in gene breeding chips, applications in the genotyping of Taihang cattle and Jinan cattle, and applications in the identification of Taihang cattle and Jinan cattle; wherein, the gene breeding chip includes a liquid phase chip.

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