A liquid phase chip for beef cattle pedigree identification and its design method and application

Abstract

The invention discloses the field of molecular detection technology, and specifically relates to a liquid phase chip and a design method and application for beef cattle pedigree identification, wherein the genotyping object of the liquid phase chip includes 16690 SNP sites, and the liquid phase chip includes a set of nucleotide probes for detecting the 16690 SNP sites, and the physical positions of the 16690 SNP sites are determined based on the whole genome sequence alignment of the Hereford cattle reference genome; the version number of the whole genome sequence of the Hereford cattle reference genome is GCF_002263795.3; the 16690 SNP sites are shown in s00001-s16690 in Table 1; beef cattle include the following varieties: Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle. The liquid phase chip of the present invention can realize pedigree identification of beef cattle.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and in particular to a liquid phase chip for beef cattle pedigree identification and a design method and application thereof. Background Art

[0002] Pedigree analysis, also known as ancestral analysis, is mainly used to estimate the proportion of different genetic sources (or ancestral groups) in an individual's genome. A common research method is to infer the genetic structure of an individual through cluster analysis, which is often used to study the genetic structure and gene exchange of human or animal populations. Through the results of pedigree analysis, it can be inferred whether the sample is purebred or hybrid, and if it is a hybrid, the approximate proportion of different ancestral bloodlines.

[0003] With the sharp decline in the cost of high-throughput sequencing, SNP markers, as third-generation molecular markers, are increasingly being used for phenotypic identification and evaluation in assisted breeding. In the process of beef cattle breeding, pedigree identification of beef cattle can clarify their genetic background, determine their breed origin based on different ancestral components, and thus can be used for breed identification, optimize breed structure, standardize breeding order, protect beef cattle breed resources, and promote the sustainable development of the beef cattle industry. Summary of the invention

[0004] The purpose of the present invention is to provide a liquid phase chip and a design method and application for beef cattle pedigree identification. The liquid phase chip can realize the pedigree identification of beef cattle and can infer whether the sample is purebred or hybrid. If it is a hybrid, the approximate proportion of different ancestral bloodlines.

[0005] The present invention is achieved through the following technical solutions:

[0006] A liquid phase chip for beef cattle pedigree identification, wherein the genotyping objects of the liquid phase chip include 16690 SNP sites, and the liquid phase chip includes a set of nucleotide probes for detecting the 16690 SNP sites, and the physical positions of the 16690 SNP sites are determined based on the whole genome sequence alignment of the Hereford cattle reference genome; the version number of the whole genome sequence of the Hereford cattle reference genome is GCF_002263795.3;

[0007] The 16,690 SNP loci are shown as s00001-s16690 in Table 1 ;

[0008] Beef cattle include the following breeds: Angus, Chinese Grassland Red, Hereford, Red Angus, Simmental, Limousin, Yanbian, Mongolian and Yanhuang.

[0009] Table 1

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[0258] The number before the “:” in the genomic position represents the chromosome, and the number after the “:” represents the physical position of the site on the corresponding chromosome.

[0259] The 16690 SNP sites of the present invention are obtained by resequencing and screening the DNA samples of nine varieties of beef cattle, namely Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle, respectively. The 16690 SNP sites obtained can be used as the basis for pedigree identification of nine varieties of beef cattle, namely Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle. The liquid phase chip designed according to the 16690 SNP sites not only has the advantage of high accuracy when performing pedigree identification of beef cattle, but also has the advantages of simple and rapid operation, high sensitivity, high accuracy, etc. when conducting research on beef cattle population genetics, genome selection, etc., and can realize genotyping of target SNP sites quickly and at low cost.

[0260] A method for designing a liquid phase chip comprises the following steps:

[0261] S1. Obtain high-quality SNP sites in the whole genome of beef cattle through whole genome resequencing and literature;

[0262] S2. Conduct relevant bioinformatics analysis based on beef cattle pedigree identification to complete functional SNP site mining; screen out 16,690 SNP sites;

[0263] S3. Design nucleotide probes based on the 16,690 SNP sites screened and prepare liquid phase chips.

[0264] In a preferred embodiment, step S1 comprises the following steps:

[0265] S11. Sample collection:

[0266] DNA samples were collected from Angus cattle, Chinese Grassland Red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle; samples were collected in the form of ear tissue, blood or semen;

[0267] S12, resequencing the DNA sample obtained in step S11 to obtain original sequencing data;

[0268] S13. Perform quality control on the original sequencing data to obtain high-quality SNP sites in the whole genome after filtering.

[0269] In a preferred embodiment, in step S13, quality control includes filtering the original sequencing data containing sequencing fragments with connectors or low quality and data quality control related to molecular markers, wherein the data quality control related to molecular markers sequentially performs the following steps: comparison with the reference genome to remove duplicates and filtering SNP sites.

[0270] In a preferred embodiment, the filtering conditions of the original sequencing data include:

[0271] 1) Remove the sequencing fragments with adapters;

[0272] 2) When the N content in the sequencing fragment exceeds 1% of the number of bases in the sequencing fragment, the sequencing fragment is removed;

[0273] 3) When the number of low-quality (Q≤5) bases in a sequencing fragment exceeds 50% of the total number of bases in the sequencing fragment, the sequencing fragment is removed.

[0274] In a preferred embodiment, the filtering conditions of SNP sites include: SNPcluster filtering (there cannot be 2 SNPs within 5bp), SNP filtering near Indel (SNPs within 5bp of Indel are filtered out) and adjacent Indel filtering (the distance between two Indels cannot be less than 10bp); it also includes filtering out sites with GQ (Genotype Quality) less than 20.0, that is, marking sites with GQ less than 20.0 in the typing quality in the sample as lowGQ and filtering them out.

[0275] An application of a liquid phase chip, including application in beef cattle genotyping or beef cattle pedigree identification.

[0276] In a preferred embodiment, the liquid phase chip is used for beef cattle pedigree identification, which specifically comprises the following steps:

[0277] Step A, obtaining a DNA sample of an individual beef cattle to be tested;

[0278] Step B, obtaining genotype data of the individual beef cattle to be tested based on the above liquid phase chip;

[0279] Step C: Based on the constructed reference sample library, pedigree identification is performed by comparing the genotype data of the individual beef cattle to be tested with the gene frequency of the reference population corresponding to the reference sample library.

[0280] In a preferred embodiment, in step C, pedigree identification includes:

[0281] The ancestral composition of the individual beef cattle to be tested is estimated by comparing the genotype data of the individual beef cattle to be tested with the gene frequency of the reference population corresponding to the reference sample library;

[0282] The maximum likelihood estimation method is used to optimize the model to maximize the probability of the observed genotype data under a given ancestral population combination. This method makes the estimation results more reliable, especially when the reference sample is of high purity and the family coverage is complete. Through the pedigree analysis results, it can be inferred whether the sample is purebred or hybrid, and if it is a hybrid, what proportion of different ancestral pedigrees each accounts for.

[0283] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0284] The liquid phase chip designed according to 16690 SNP sites has high detection efficiency and simplifies the bioinformatics analysis process when performing beef cattle pedigree identification. It can quickly and at low cost realize the genotyping of target SNP sites; and it is specific to nine breeds of beef cattle, namely Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle. It can quickly realize the genotype analysis and pedigree identification of Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle, so as to infer whether the sample is purebred or hybrid, and if it is a hybrid, the approximate proportion of different ancestral bloodlines. BRIEF DESCRIPTION OF THE DRAWINGS

[0285] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0286] Figure 1 This is the evolutionary tree diagram of 16690 SNP sites of the present invention;

[0287] Figure 2 This is the evolutionary tree diagram of the whole genome of the present invention. DETAILED DESCRIPTION

[0288] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0289] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to those of ordinary skill in the art that these specific details do not have to be employed to practice the present invention. In other embodiments, in order to avoid confusing the present invention, known structures, materials or methods are not specifically described. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.

[0290] Embodiment 1:

[0291] The SNP molecular marker information of nine breeds of beef cattle, namely, Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle, is obtained, specifically including the following steps:

[0292] 1) Obtain high-quality SNP sites in the whole genome of beef cattle through whole genome resequencing and literature:

[0293] The whole genome resequencing data of 153 samples from nine breeds of beef cattle (Angus, Chinese Grassland Red, Hereford, Red Angus, Simmental, Limousin, Yanbian, Mongolian and Yanhuang) were used to obtain the SNP molecular marker information of the nine breeds of beef cattle through data analysis and quality control. The breeds and numbers of the samples used are shown in Table 2.

[0294] Table 2

[0295]

[0296] Requirements for screening samples of Angus cattle, Chinese Grassland Red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle: screen individuals that meet the appearance characteristics of the corresponding cattle breeds and have relatively complete information records, and then conduct sampling according to family distribution and gender, avoiding the collection of individuals with close bloodlines.

[0297] DNA sample requirements and genetic testing strategy:

[0298] a) Type: DNA sample; no degradation or slight degradation; no contamination;

[0299] b) Required quantity: 1.5ug of sample is required for each sample preparation. If multiple sample preparations are required, the total amount of sample required is equal to the number of sample preparations multiplied by 1.5ug;

[0300] c) Sample concentration: sample concentration ≥ 50 ng / μl, recommended concentration is 100~200 ng / μl;

[0301] d) Genetic testing strategy: BGI sequencing platform, individual sequencing depth is recommended to be 10X.

[0302] The basic process of resequencing is as follows:

[0303] First, the CWseq Universal DirectFast DNA Library Prep Kit was used to construct a resequencing library for the DNA that passed the quality inspection. After the library construction was completed, the library was sequenced using the BGI-T7 sequencing platform after the quality inspection was qualified, and the sequencing mode was PE150 mode.

[0304] The raw sequencing data obtained by sequencing needs to be quality controlled, which includes filtering the raw sequencing data containing sequencing fragments with adapters or low quality and quality control of data related to molecular markers.

[0305] The original sequencing data (sequenced reads or raw reads) obtained by sequencing contain low-quality sequencing fragments (reads) with adapters. In order to ensure the quality of information analysis, raw reads must be filtered to obtain filtered data (clean reads), and clean reads are used for subsequent analysis. Specifically, the software fastp (version 0.23.4, parameters: -n1-q5 -u 50-l 100) is used to filter raw reads. The steps of data processing are as follows:

[0306] 1) Remove the sequencing fragments with adapters;

[0307] 2) When the N content in the sequencing fragment (reads) exceeds 1% of the number of bases in the sequencing fragment, the sequencing fragment is removed;

[0308] 3) When the number of low-quality (Q≤5) bases in a sequencing fragment exceeds 50% of the total number of bases in the sequencing fragment, the sequencing fragment is removed.

[0309] Data quality control related to molecular markers:

[0310] 1) Align with the reference genome and remove duplicates; Specifically, the clean reads after quality control were aligned with the reference genome sequence using the software BWA (-mem v0.7.17, parameter: -t10-MR). The clean reads on the Hereford reference genome were located by alignment to obtain the BAM file; the version number of the whole genome sequence of the reference genome is GCF_002263795.3. The aligned BAM files were sorted using SAMtools (-sort v1.20), indexed using SAMtools (-index v1.20), and deduplicated using Picardv2.17.5 to obtain the final BAM file.

[0311] 2) Use the HaplotypeCaller module of GATK (4.18) to perform mutation detection on multiple samples with the processed alignment files, and obtain a total of 55,040,410 variant sites (SNP + Indel);

[0312] 3) Use GATK (4.18) VariantFiltration module to perform hard filtering on SNP and INEDL, with the following parameters: QD<2.0, FS>60.0, MQ<40.0, SOR>3.0, MQRankSum<-12.5, ReadPosRankSum<-8.0;

[0313] 4) Strictly filter SNPs: SNP cluster filtering (no two SNPs within 5bp), SNP filtering near Indel (filter out SNPs within 5bp of Indel) and adjacent Indel filtering (the distance between two Indels cannot be less than 10bp); it also includes filtering out sites with GQ (Genotype Quality) less than 20.0, that is, marking sites with GQ less than 20.0 as lowGQ in the typing quality of the sample and filtering them out.

[0314] Using the above filtering conditions, 11,035,051 sites were finally obtained for subsequent chip site selection.

[0315] 2) Based on the identification of beef cattle pedigree, relevant bioinformatics analysis was performed to complete the mining of functional SNP sites; 16,690 SNP sites were screened:

[0316] The mining process is as follows:

[0317] 1. Only autosomal loci are retained, with a detection rate ≥ 95% and MAF ≥ 0.01;

[0318] 2. The remaining sites were subjected to LD-based redundancy removal (r² value 0.3), after removing redundant linkage sites;

[0319] 3. The remaining sites were subjected to population differentiation index analysis among the nine varieties to obtain the FST value of each site. The sites with FST values ​​in the top 25,000 were screened and probes were scored for them, and ultimately 16,690 sites were successfully selected.

[0320] Phylogenetic tree and PCA analysis were constructed based on 16,690 SNP loci. The phylogenetic tree results are as follows: Figure 1-Figure 2 As shown, the 16690 SNP loci are highly consistent with the results calculated based on the whole genome SNP data. At the same time, it can be seen that the use of 16690 SNP loci can clearly distinguish each variety, and the variety identification effect is obvious.

[0321] Embodiment 2:

[0322] Acquisition of Nucleotide Probe Combinations:

[0323] Using GenoBaits Probe Designer software, probe design is performed based on the evaluation results of the upstream and downstream sequences of the target site. The complexity of the upstream and downstream sequences of the target site, GC content, etc. are mainly evaluated. The target site is placed in the middle of the probe first, and the designed probe is 120 bp in length. Generally, the performance of the probe is comprehensively evaluated from the aspects of GC content (20%-80%), Tm value (60-80℃), number of alignments on the reference genome (≤5), etc. The probe is 120bp in length, with a DNA nucleotide sequence modified with a biotin group at the 5' end.

[0324] The probe combination obtained above was adjusted with the 16690 SNP sites in Table 1, and finally a nucleotide probe combination capable of capturing 16690 SNP sites was obtained.

[0325] According to the principle of DNA complementarity, probes covering the target SNP are designed at each site to be tested, and the target probes are modified by biotin labeling. Then, the biotin-modified probes are hybridized with the target region of the genome in a liquid state to form a double strand. Subsequently, the biotin-modified probes are molecularly adsorbed by streptavidin-coated magnetic beads to capture the target hybridized with the probe. Finally, the captured target sequence is eluted, amplified and sequenced to obtain the genotype of the target SNP.

[0326] Embodiment 3:

[0327] A liquid phase chip for beef cattle pedigree identification, wherein the genotyping objects of the liquid phase chip include 16690 SNP sites, and include a set of nucleotide probes for detecting the 16690 SNP sites, wherein the physical positions of the 16690 SNP sites are determined based on the whole genome sequence alignment of a Hereford cattle reference genome; the version number of the whole genome sequence of the Hereford cattle reference genome is GCF_002263795.3;

[0328] The 16,690 SNP loci are shown as s00001-s16690 in Table 1 ;

[0329] Beef cattle include the following breeds: Angus, Chinese Grassland Red, Hereford, Red Angus, Simmental, Limousin, Yanbian, Mongolian and Yanhuang.

[0330] The above chip sites were used for genome sequencing and alignment, and the mapping ratio was above 99.9%, and the dp5 detection rate (the number of detected sites when the depth was greater than 5X) was above 99%, indicating that the chip detection effect was good. As shown in Table 3:

[0331] Table 3

[0332]

[0333]

[0334]

[0335]

[0336] Embodiment 4:

[0337] The method for performing genotype detection on the 16690 SNP sites in Table 1 using the liquid phase chip developed in Example 3 comprises the following steps:

[0338] 1. Genomic DNA Extraction

[0339] The sample DNA was extracted using a magnetic bead DNA extraction kit (CW2361S, Kangwei Century).

[0340] Liquid capture library construction

[0341] 2. Genomic DNA fragmentation

[0342] Take 300 ng of DNA sample, add 4 μL of Smearase Buffer and 2 μL of Smearase Enzymes, with a total volume of 24 μL, and then place the reaction plate in the PCR instrument to execute the program: 4℃ 1 min→30℃ 10 min→72℃ 20 min→store at 4℃.

[0343] 3. Fragment screening and end repair and A addition

[0344] First, the range of the fragments was screened by magnetic beads to remove oversized and undersized fragments, so that the DNA fragments were concentrated in 200-300bp as much as possible; secondly, 40μL of fragmented DNA / cfDNA, 6μl of End Repair&A-TailingBuffer, 4μl of End Repair&A-Tailing Enzyme were taken, and the total reaction system was 50ul. After mixing, 10μL was taken in a 96-well PCR plate, incubated at 20℃ for 30min, incubated at 65℃ for 30min, and stored at 4℃.

[0345] 4. Adapter Linking and Enrichment

[0346] Add 5μL CAGT Universal Adapters and 20μL LigationMaster Mix to the 96-well PCR plate in step 3, vortex and mix, centrifuge briefly to collect the reaction solution to the bottom of the tube, react in a PCR instrument at 20℃ for 15min to complete the sequencing adapter connection. After purifying the connection product, perform PCR amplification and enrichment, take 15μL of the connection purification product, add 10μL CAGT UDI Primer, 25μL Equinox Library Amp Mix (2x), mix well, and draw 35μL of the reaction solution in a PCR instrument for library amplification. After the whole genome library is constructed, use the dsDNA HS Assay Kit for Qubit to quantify the library; at the same time, use electrophoresis to detect whether the main peak of the library fragment is in the range of 300-500 bp.

[0347] 5. Liquid chip hybridization capture

[0348] The whole genome library of each sample was pooled, and the total amount of the final hybrid capture library was 4 μg. The pooled library was concentrated and probe hybridized to capture the fragments of the target region from the whole genome library. The excess probes, hybridization reagents and other reagent components were removed through the elution step. Finally, the target region was enriched by PCR amplification after hybridization to obtain the machine library.

[0349] 6. Library quality inspection and sequencing

[0350] After the hybrid capture library was constructed, the dsDNA HS Assay Kit for Qubit was used to quantify the library; at the same time, electrophoresis was used to detect whether the main peak size of the library was in the range of 300-500bp. The constructed library was sequenced on a DNBSEQ-T7 sequencer.

[0351] Embodiment 5:

[0352] Example 3 Application of the developed liquid phase chip in beef cattle genotyping.

[0353] A sample of Chinese Grassland Red Cattle was genotyped using the above-mentioned liquid phase chip. The successful mapping ratio of the sequencing data to the genome was 99.97%, and the dp5 detection rate was 99.44%, with good detection results. The data after typing of this individual was compared with the reference pedigree library, and the results showed that this individual accounted for 98.35% of the pedigree of Chinese Grassland Red Cattle, and it was confirmed that this breed was Chinese Grassland Red Cattle.

[0354] Embodiment 6:

[0355] The application of the liquid phase chip developed in Example 3 in beef cattle pedigree identification includes the following steps:

[0356] Step A, obtaining a DNA sample of an individual beef cattle to be tested;

[0357] Step B, obtaining genotype data of the individual beef cattle to be tested based on the above liquid phase chip;

[0358] Step C: Based on the constructed reference sample library, pedigree identification is performed by comparing the genotype data of the individual beef cattle to be tested with the gene frequency of the reference group corresponding to the reference sample library; specifically: the ancestral composition of the individual beef cattle to be tested is estimated by comparing the genotype data of the individual beef cattle to be tested with the gene frequency of the reference group corresponding to the reference sample library; at the same time, the maximum likelihood estimation method is used to optimize the model so that the probability of the observed genotype data under a given ancestral group combination is maximized. This method makes the estimation results more reliable, especially when the reference sample purity is high and the family coverage is complete; through the pedigree analysis results, it can be inferred whether the sample is purebred or hybrid, and if it is a hybrid, the approximate proportion of different ancestral bloodlines.

[0359] In this example, the pedigree components of 18 samples to be tested were inferred based on the purebred reference library constructed when preparing the liquid phase chip.

[0360] The inference results are shown in Table 4:

[0361] Table 4

[0362]

[0363] In Table 4, each row represents the proportion of each breed pedigree in a sample, and each column represents a breed pedigree. Among them, Angus is Angus cattle, Hereford is Hereford cattle, Limousin is Limousin cattle, Mongolian is Mongolian cattle, Red_Angus is Red Angus cattle, Simmental is Simmental cattle, Yanbian is Yanbian cattle, Red_Steppe is Chinese grassland red cattle, and Yanhuang is Yanhuang cattle.

[0364] From the data in Table 4, we can see that:

[0365] By using the liquid phase chip of the present invention, it is possible to infer whether the sample is purebred or hybrid through the results of pedigree analysis. If it is a hybrid, what is the approximate proportion of different ancestral pedigrees:

[0366] For example, samples numbered 15-17 are all purebred, among which the probability that the ancestor of sample numbered 15 is Limousin is 99.99%, and the probability that the ancestor of samples numbered 16-17 is Red_Angus is 99.99%.

[0367] For example: Sample number 18 is a hybrid.

[0368] Under the premise that the two samples are known to be Red Angus cattle, the present invention uses the liquid phase chip prepared in Example 3 to perform genotyping and pedigree ratio detection. The results are shown in Table 5:

[0369] Table 5

[0370]

[0371] From the data in Table 5, we can see that:

[0372] Comparison of the test results of the two samples using the liquid chip with the Red Angus pedigree library showed that the test results of the liquid chip reached 99.99%, further demonstrating that the liquid chip results of the above invention are accurate.

[0373] After liquid chip detection and analysis, the results were accurate to 99.99%.

[0374] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method 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 in the scope of protection of the present invention.

Claims

1. A liquid phase chip for beef cattle pedigree identification, characterized in that: The invention comprises a set of nucleotide probes for detecting 16,690 SNP sites, wherein the physical positions of the 16,690 SNP sites are determined based on the whole genome sequence alignment of the Hereford cattle reference genome; the version number of the whole genome sequence of the Hereford cattle reference genome is GCF_002263795.3; The 16,690 SNP loci are shown as s00001-s16690 in Table 1 ; The beef cattle include the following breeds: Angus cattle, Chinese grassland red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle; Table 1 ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； ； 。 2. The method for designing a liquid phase chip according to claim 1, characterized in that: The steps include: S1. Obtaining high-quality SNP sites in the whole genome of the beef cattle through whole genome resequencing and literature; S2. Conduct relevant bioinformatics analysis based on beef cattle pedigree identification to complete functional SNP site mining; screen out 16,690 SNP sites; S3. Design nucleotide probes based on the 16,690 SNP sites screened and prepare liquid phase chips.

3. The design method according to claim 2, characterized in that: Step S1 includes the following steps: S11. Sample collection: DNA samples were collected from Angus cattle, Chinese Grassland Red cattle, Hereford cattle, Red Angus cattle, Simmental cattle, Limousin cattle, Yanbian cattle, Mongolian cattle and Yanhuang cattle; S12, resequencing the DNA sample obtained in step S11 to obtain original sequencing data; S13, performing quality control on the original sequencing data to obtain the filtered high-quality SNP sites of the whole genome.

4. The design method according to claim 3, characterized in that: In step S11, the sample collection form includes ear tissue, blood or semen.

5. The design method according to claim 3, characterized in that: In step S13, the quality control includes filtering the original sequencing data containing sequencing fragments with connectors or low quality and data quality control related to molecular markers, wherein the data quality control related to molecular markers sequentially performs the following steps: comparison with the reference genome to remove duplicates, and filtering the SNP sites.

6. The design method according to claim 5, characterized in that: The filtering conditions of the raw sequencing data include: 1) Remove the sequencing fragments with adapters; 2) When the N content in the sequencing fragment exceeds 1% of the number of bases in the sequencing fragment, the sequencing fragment is removed; 3) When the number of bases with Q≤5 in a sequencing fragment exceeds 50% of the total number of bases in the sequencing fragment, the sequencing fragment is removed.

7. The design method according to claim 5, characterized in that: The filtering conditions for filtering SNP sites include: SNP cluster filtering, SNP filtering near Indel, and adjacent Indel filtering; and also include filtering out sites with GQ less than 20.

0.

8. The use of the liquid phase chip as claimed in claim 1, characterized in that: This includes applications in genotyping of beef cattle or applications in pedigree identification of beef cattle.

9. The use according to claim 8, characterized in that: The liquid phase chip is used for beef cattle pedigree identification and specifically comprises the following steps: Step A, obtaining a DNA sample of an individual beef cattle to be tested; Step B, obtaining the genotype data of the individual beef cattle to be tested based on the liquid phase chip as claimed in claim 1; Step C: Based on the constructed reference sample library, pedigree identification is performed by comparing the genotype data of the beef cattle individual to be tested with the gene frequency of the reference group corresponding to the reference sample library.

10. The use according to claim 9, characterized in that: In step C, the pedigree identification includes: Estimating the ancestral composition of the beef cattle individual to be tested by comparing the genotype data of the beef cattle individual to be tested with the gene frequency of the reference population corresponding to the reference sample library; The maximum likelihood estimation method was used to optimize the model to maximize the probability of the observed genotype data under a given ancestral population combination.

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