SNP (Single Nucleotide Polymorphism) and InDel site combination for identifying variety of Inner Mongolia cattle, 5K targeted capture gene chip and application of 5K targeted capture gene chip

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) and InDel site combination for identifying variety of Inner Mongolia cattle, a 5K targeted capture gene chip and application of the 5K targeted capture gene chip. The SNP and InDel site combination for identifying the variety of the Inner Mongolia cattle is firstly disclosed and comprises 5000 SNP and InDel sites, and the physical position information of a cattle reference genome with the version number of AR-UCD1.2 is shown in a table 1. The invention further discloses a 5K targeting capture gene chip comprising the probe targeting the SNP and InDel site combination as well as a synthesis method and application of the 5K targeting capture gene chip. The 5K targeted capture gene chip completes genetic typing through a targeted capture probe by means of a high-throughput sequencing technology, has the characteristics of low cost, high efficiency and high accuracy, and can realize the variety identification function of Mongolian cattle, Sanhue cattle and Sunitt cattle by utilizing genotype data; important technical support and specific means are provided for identification and evaluation of Inner Mongolia cattle germplasm resources.

Description

Technical Field

[0001] The present invention relates to the technical fields of bioinformatics and molecular breeding. More specifically, it relates to a combination of SNP and InDel loci for Inner Mongolia cattle breed identification, a 5K targeted capture gene chip, and their applications. Background Art

[0002] There are diverse types of Inner Mongolia cattle, including multiple breeds such as Mongolian cattle, Sanhe cattle, Simmental cattle, Charolais cattle, Sunite cattle, Prairie Red cattle, etc. These cattle breeds each have their own characteristics and are adapted to different natural environments and livestock husbandry needs in Inner Mongolia. Mongolian cattle are strong and rough in constitution, with genetic traits such as strong adaptability, resistance to wind and sand, drought and cold tolerance, ability to tolerate roughage and extensive management, and excellent meat quality. They can maintain good viability and reproductive ability in special environments and are important excellent cattle breeds in northern China, with multiple uses including milk, meat, and draft. Other cattle breeds such as Simmental cattle are widely welcomed for their good adaptability and excellent meat quality. Breed identification helps to clarify the genetic characteristics and blood relationships of each cattle breed, providing a scientific basis for the protection and utilization of germplasm resources. This is of great significance for ensuring the diversity of cattle breeds and the sustainability of genetic resources. Through breed identification, the uses and values of each cattle breed can be more clearly defined, providing guidance for the breeding, mating, and sales of cattle. This helps to promote the high-quality development of the Inner Mongolia cattle industry, enhance industrial competitiveness and economic benefits. In summary, the types of Inner Mongolia cattle are rich and each has its own characteristics, and breed identification is of great significance for protecting germplasm resources, promoting industrial development, and enjoying policy support.

[0003] Gene chips can accurately genotype target loci and are an efficient and low-cost genotyping method. Therefore, it is extremely necessary to develop a targeted capture gene chip for Inner Mongolia cattle breed identification to detect and analyze the bloodlines of 3 breeds including Mongolian cattle, Sanhe cattle, and Sunite cattle. Summary of the Invention

[0004] One object of the present invention is to provide a combination of SNP and InDel loci and a 5K targeted capture gene chip for Inner Mongolia cattle breed identification. By performing whole-genome sequencing on different Inner Mongolia cattle breed populations, screening SNP and InDel loci for breed identification, and designing targeted capture probes based on these loci to construct a gene chip, the breed identification work of Mongolian cattle, Sanhe cattle, and Sunite cattle can be accurately completed.

[0005] The second object of the present invention is to provide the application of the above-mentioned 5K targeted capture gene chip.

[0006] To achieve the above objects, the present invention adopts the following technical solutions:

[0007] The present invention first provides a set of SNP and InDel locus combinations for Inner Mongolia cattle breed identification. The SNP and InDel locus combinations include 5,000 SNP and InDel loci, and the physical location information of the SNP and InDel loci based on the bovine reference genome with the version number ARS-UCD1.2 is shown in Table 1.

[0008] The present invention further provides a 5K targeted capture gene chip for Inner Mongolia cattle breed identification. The 5K targeted capture gene chip includes probes targeting the above-mentioned SNP and InDel locus combinations.

[0009] The present invention further also provides a synthesis method for the above-mentioned 5K targeted capture gene chip. The synthesis method includes the following steps:

[0010] S1. Perform whole-genome resequencing on Inner Mongolia cattle samples of different breeds to obtain high-quality SNP and InDel loci of the Inner Mongolia cattle.

[0011] S2. Screen purebred Inner Mongolia cattle samples of different breeds and construct a reference library containing purebred Inner Mongolia cattle samples of different breeds.

[0012] S3. Based on the reference library and high-quality SNP and InDel loci, screen to obtain a set of candidate SNP and InDel loci to be selected.

[0013] S4. Evaluate and score the set of candidate SNP and InDel loci to be selected, judge the possibility of successful probe design for the SNP and InDel loci, and incorporate the SNP and InDel loci with successful probe design into the gene chip locus set.

[0014] S5. Synthesize the probes for the SNP and InDel loci in the gene chip locus set to obtain a 5K targeted capture gene chip for Inner Mongolia cattle breed identification.

[0015] In a specific embodiment of the present invention, S1 includes the following steps:

[0016] S11. Extract genomic DNA from Inner Mongolia cattle samples of different breeds and perform whole-genome resequencing to obtain raw sequence data.

[0017] S12. Perform quality control on the raw sequence data to obtain Clean Reads, and align them to the bovine reference genome with the version number ARS-UCD1.2 for SNP and InDel locus detection.

[0018] S13. After index filtering and quality control, obtain the high-quality SNP and InDel loci of the Inner Mongolia cattle.

[0019] Further, the criteria for quality control of the original sequence data are as follows:

[0020] 1) Remove reads with adapters;

[0021] 2) When the N content in the sequencing read exceeds 1% of the read base number, remove the paired reads;

[0022] 3) When the number of low-quality (Q <= 5) bases in the sequencing read exceeds 50% of the total number of bases in the read, remove the paired reads.

[0023] Further, the index filtering includes QD, FS, MQ, SOR, MQRankSum, ReadPosRankSum, and the criteria are as follows:

[0024] 1) hardfilter_SNP and InDel Expression1 = "QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 3.0";

[0025] 2) hardfilter_SNP and InDel Expression2 = "MQRankSum < -12.5 || ReadPosRankSum < -8.0";

[0026] 3) hardfilter_indel Expression1 = "QD < 2.0 || FS > 200.0 || SOR > 10.0";

[0027] 4) hardfilter_indel Expression2 = "ReadPosRankSum < -20.0".

[0028] Further, the criteria for quality control are as follows:

[0029] 1) Only retain autosomal loci;

[0030] 2) Detection rate >= 90%;

[0031] 3) Sample detection rate >= 90%;

[0032] 4) MAF >= 0.05;

[0033] 5) P value of Hardy-Weinberg equilibrium test >= 10e-6.

[0034] In a specific embodiment of the present invention, when constructing the reference library in S2, individual likelihood values are used to calculate and screen purebred Inner Mongolia cattle samples of different breeds.

[0035] In a specific embodiment of the present invention, the candidate SNP and InDel locus set to be selected in S3 includes functional SNPs and InDels for variety identification and background SNPs and InDel markers for variety identification; wherein, the functional SNPs and InDels are obtained by performing population differentiation index analysis between varieties based on a reference library and high-quality SNPs and InDels, obtaining the Fst value of each locus, and screening SNPs and InDels with higher Fst values as functional SNPs and InDels for variety identification; the background SNPs and InDel markers are based on high-quality SNPs and InDels, and loci annotated as stopgain / loss, alternative splicing sites, and non-synonymous mutations are screened as background SNPs and InDels for variety identification.

[0036] In a specific embodiment of the present invention, the S4 evaluation and scoring is performed according to indicators such as the specificity, complexity, and GC content of the upstream and downstream sequences of the target SNP and InDel loci. Priority is given to placing the target SNP and InDel loci in the middle position of the probe, and the designed probe is 120bp in length.

[0037] The present invention further provides the application of the above SNP and InDel locus combination or the above gene chip in the genotyping, pedigree analysis, or variety identification of Inner Mongolia cattle.

[0038] The present invention further also provides a method for identifying Inner Mongolia cattle varieties, including the following steps:

[0039] S1. Based on the genomic DNA of the sample to be tested, a whole-genome library is constructed and hybridized with the probes in the above 5K targeted capture gene chip to construct a hybrid capture library;

[0040] S2. Analyze the data obtained by sequencing the hybrid capture library to obtain the genotyping results of the target SNP and InDel loci of the sample to be tested;

[0041] S3. Use the genotyping results to infer the pedigree components of the sample to be tested and complete the variety identification of Inner Mongolia cattle.

[0042] In a specific embodiment of the present invention, S2 includes the following steps:

[0043] S21. Filter the data obtained by sequencing the hybrid capture library to obtain Clean Reads;

[0044] S22. Align the Clean Reads to the bovine reference genome with the version number ARS-UCD1.2 to obtain the alignment results of the Clean Reads on the reference genome;

[0045] S23. Genotype the target SNP and InDel sites based on the alignment results of Clean Reads in the reference genome to obtain the genotyping results of the target SNP and InDel sites of the sample to be tested.

[0046] In a specific embodiment of the present invention, in step S3, inferring the ancestry components of the sample to be tested using the genotyping results is based on the reference library constructed above, and the admixture software is used to analyze the ancestry components of the sample to be tested.

[0047] In the present invention, the Inner Mongolia cattle include Mongolian cattle, Sanhe cattle, and Sunite cattle.

[0048] The beneficial effects of the present invention are as follows:

[0049] The present invention obtains the whole-genome genetic information of 149 individuals of three breeds of Inner Mongolia cattle, namely Mongolian cattle, Sanhe cattle, and Sunite cattle, through whole-genome resequencing technology. By screening, functional SNP and InDel sites and background SNP and InDel sites for breed identification are obtained, and targeted capture probes are designed based on these sites to develop a 5K targeted capture gene chip for Inner Mongolia cattle breed identification. The 5K targeted capture gene chip of the present invention captures through targeted probes and completes genotyping with the help of high-throughput sequencing technology, featuring low cost, high efficiency, and high accuracy. At the same time, the genotype data can be used to achieve the function of identifying Mongolian cattle, Sanhe cattle, and Sunite cattle, providing important technical support and specific means for carrying out the identification and evaluation of Inner Mongolia cattle germplasm resources. Description of the Drawings

[0050] The following further elaborates on the specific embodiments of the present invention in conjunction with the drawings.

[0051] Figure 1 This is the result of the principal component analysis (PCA) of the clustering of all samples in the reference library of the present invention.

[0052] Figure 2 This is the distribution map of the number of SNP and InDel sites on different chromosomes in the 5K targeted capture gene chip for Inner Mongolia cattle breed identification of the present invention.

[0053] Figure 3 This is the distribution map of the minor allele frequency (MAF) of SNP and InDel sites in the 5K targeted capture gene chip for Inner Mongolia cattle breed identification of the present invention.

[0054] Figure 4 This is the distribution of SNP and InDel sites in different functional elements in the 5K targeted capture gene chip for Inner Mongolia cattle breed identification of the present invention. Detailed Embodiments

[0055] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0056] Example 1 Development of SNP Loci and Probe Design for Inner Mongolia Cattle 5K Breed Identification

[0057] I. Selection of Inner Mongolia Cattle Breeds

[0058] Three Inner Mongolia cattle breeds were selected for population resequencing. The breed names and quantities are shown in Table 2.

[0059] Table 2 Information of Inner Mongolia Cattle Breeds for Population Resequencing

[0060] Variety Code Quantity Mongolian cattle MGN 49 Sanhe cattle SHN 50 Sunite cattle SNT 50

[0061] II. Sample Sequencing and Data Quality Control

[0062] 1. DNA Extraction and Quality Inspection

[0063] The genomic DNA (gDNA) of whole blood was extracted using a magnetic bead method DNA extraction kit (CW2361S). The integrity of the genome was detected using 1.5% agarose gel; the purity of DNA was detected using a NanoDrop 2000 nucleic acid and protein analyzer, and the A260 / A280 ratio was measured; the gDNA sample was accurately quantified using Qubit2.0.

[0064] 2. Sequencing Library Construction and Sequencing

[0065] Use A DNA universal library construction kit (for MGI) was used to construct a sequencing library. The specific steps are as follows:

[0066] The gDNA was fragmented into fragments of about 300 - 350 bp using an enzyme, followed by end repair, A-tailing, and ligation of sequencing adapters. DNA fragments of about 300 - 350 bp were screened, PCR amplification was performed, and the PCR products were purified again. Finally, a sequencing library was obtained. After the sequencing library was constructed, it was first preliminarily quantified using Qubit2.0, and then the insert fragments of the sequencing library were detected. After meeting the expectations, it was sequenced on the machine, i.e., library inspection.

[0067] After the library inspection is qualified, pooling is performed according to the effective concentration of the sequencing library and the target data volume. The DNBSEQ-T7 sequencer is used to select the PE150 sequencing strategy for on-machine sequencing. The raw image data obtained by sequencing is converted into raw sequence data (raw reads) by base calling software, and the data is stored in FASTQ file format.

[0068] 3. SNP and InDel detection and filtering

[0069] Use fastp to perform a series of quality controls (QC) on raw reads to obtain Clean Reads. The quality control standards are as follows:

[0070] 1) Remove reads with adapters;

[0071] 2) When the N content in the sequencing read exceeds 1% of the number of bases in the read, the paired reads are removed;

[0072] 3) When the number of low-quality (Q<=5) bases in a sequencing read exceeds 50% of the total number of bases in the read, the paired reads are removed.

[0073] Clean Reads were aligned to the bovine reference genome (version ARS-UCD1.2) using BWA 07.17 (men) software, and samtools 1.7 was used to sort and build an index to obtain a Bam file. The Bam file was deduplicated using the module provided by GATK 4.1.8.0 software. SNP, InDel and INEDL were detected using GATK 4.1.8.0 software, and then SNP, InDel and INEDL were strictly filtered using the VariantFiltration module to obtain filtered SNP and InDel sites. The index filtering included QD, FS, MQ, SOR, MQRankSum, ReadPosRankSum, etc. The standards were as follows:

[0074] 1)hardfilter_SNP and InDel Expression1="QD<2.0||FS>60.0||MQ<40.0||SOR>3.0"

[0075] 2)hardfilter_SNP and InDel Expression2="MQRankSum<-12.5||ReadPosRankSum<-8.0"

[0076] 3) hardfilter_indelExpression1 = "QD<2.0||FS>200.0||SOR>10.0"

[0077] 4) hardfilter_indelExpression2 = "ReadPosRankSum<-20.0"

[0078] 4. SNP and InDel Quality Control

[0079] To retain high-quality SNP and InDel sites, the SNP and InDel sites obtained after filtering were quality-controlled before analysis. After quality control, 13,582,392 SNP and InDel sites remained. The quality control criteria are as follows:

[0080] 1) Only retain autosomal sites

[0081] 2) Detection rate >= 90%

[0082] 3) Sample detection rate >= 90%

[0083] 4) MAF >= 0.05

[0084] 5) P value of Hardy-Weinberg equilibrium test >= 10e-6.

[0085] III. Construction of the Reference Library

[0086] The principle for screening reference library samples is that the sample purity is high and it can represent the genetic diversity of the breed. The individual likelihood value is used to calculate and identify purebred samples. A total of 128 purebred samples were obtained after screening 149 samples, and 122 of them were used to construct the reference library. The number of individuals of each breed in the reference library is shown in Table 3. The remaining 6 samples are 2 each of Mongolian cattle, Sanhe cattle, and Sunite cattle, which are used as positive controls to verify the accuracy of the breed identification chip. The PCA method is used to perform dimensionality reduction clustering analysis on all purebred samples in the reference library, and the results are as Figure 1 shown, indicating that the three breeds of Mongolian cattle (MGN), Sanhe cattle (SHN), and Sunite cattle (SNT) can be well distinguished.

[0087] Table 3 Purebred Samples Used to Construct the Reference Library

[0088] Variety Purebred quantity Mongolian cattle 38 Sanhe cattle 41 Sunite cattle 43

[0089] IV. Screening of SNP and InDel Sites for Breed Identification

[0090] Based on the reference library samples and the high-quality SNP and InDel sites after quality control, the fixation index (Fst) analysis was performed among three breeds to obtain the Fst value of each site. Considering that some sites would be lost during subsequent probe design, additional sites needed to be selected. Finally, 7,000 sites with higher Fst values (top7000) were screened as the functional SNP and InDel sites for breed identification.

[0091] To fill in the sparse regions of the genome, sites annotated as stopgain / loss, alternative splicing sites, and non-synonymous mutations were selected from the high-quality SNP and InDel sites after quality control as the background SNP and InDel sites for breed identification.

[0092] The above-mentioned functional SNP and InDel sites and background SNP and InDel sites were used as the candidate SNP and InDel site set to be selected.

[0093] V. Probe Design for SNP and InDel Sites for Breed Identification

[0094] The candidate SNP and InDel site set to be selected was evaluated and scored. The main evaluations included the specificity, complexity, GC content, etc. of the upstream and downstream sequences of the target SNP and InDel sites. Priority was given to placing the target SNP and InDel sites in the middle of the probe, and the designed probe was 120 bp in length. According to the evaluation and scoring results, the possibility of successful design of the target capture probe for SNP and InDel sites was judged. Finally, 4,690 functional SNP and InDel sites and 310 background SNP and InDel sites were successfully designed for the probe, as shown in Table 4.

[0095] Table 4 Composition of SNP and InDel Sites with Successful Target Capture Probe Design

[0096] Locus type Number of loci Functional locus 4690 Background locus 310

[0097] Finally, 5,000 SNP and InDel sites with successful target capture probe design were included in the final chip site set. Using the ARS-UCD1.2 version number as the bovine reference genome, the physical position information of the SNP and InDel sites is shown in Table 1.

[0098] Table 1 Physical Position Information of SNP and InDel Sites

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] Note: Taking the SNP locus numbered 1 as an example, in 1:1103859, "1" represents the chromosome where the SNP locus is located, 1103859 represents the physical position of the SNP locus on the chromosome, G represents the locus information before the mutation, and C represents the locus information after the mutation; taking the InDel locus numbered 4 as an example, in 1:2230883, "1" represents the chromosome where the InDel locus is located, indicating that the AA sequence is deleted after the 2230883rd position on chromosome 1; taking the InDel locus numbered 19 as an example, in 1:12164703, "1" represents the chromosome where the InDel locus is located, indicating that the AT sequence is inserted after the 12164703rd position on chromosome 1; in the case of mutations, with " Underline " such as A>G, representing the background SNP loci, and the others are functional SNP or InDel loci.

[0147] Probe synthesis was carried out on the targeted capture probes for the above 5000 SNP and InDel loci, thereby obtaining a 5K targeted capture gene chip for Inner Mongolia cattle breed identification.

[0148] The distribution of all SNP and InDel loci on chromosomes is as Figure 2 shown. It can be seen from the figure that the SNP and InDel loci of the present invention are distributed on different chromosomes. Therefore, the loci captured by the targeted capture probes designed according to them can cover the entire genome.

[0149] The distribution map of the minor allele frequency (MAF) of all SNP and InDel loci is as Figure 3 shown. It can be seen from the figure that the MAF values are mainly distributed between 0.4 and 0.5.

[0150] The distribution map of all SNP and InDel loci in different genomic functional elements is as Figure 4 shown. It can be seen from the figure that there are more SNP and InDel distributions in the intergenic region and introns among all SNP and InDel loci.

[0151] VI. Effect verification

[0152] For the 6 positive control purebred samples of Mongolian cattle, Sanhe cattle, and Sunite cattle identified in "III. Construction of the reference library", the genotyping results of the above 5000 loci were extracted from their sequencing data respectively. The admixture software was used to infer the genetic components of these 6 control samples, and the inference results are shown in Table 5. In Table 5, each row represents the proportion of the bloodline of each breed in a sample, and the sum is 1. Each column represents a breed bloodline. The larger the value, the higher the proportion of the corresponding bloodline in the sample. It can be seen from the table that the bloodline analysis results are basically consistent with the sample breed information, proving the accuracy of the selected breed identification loci and the reliability of the development of the 5K targeted capture gene chip for Inner Mongolia cattle breed identification.

[0153] Table 5 Bloodline composition of positive control samples

[0154]

[0155] Note: Breed is the breed to which the positive control sample belongs;

[0156] Sample number is the identification code for the sample;

[0157] The bloodlines of Sanhe cattle, Sunite cattle, and Mongolian cattle are the percentages of the corresponding breed bloodlines in the sample to be tested.

[0158] Example 2 Sequencing of a 5K Targeted Capture Gene Chip for Inner Mongolia Cattle Breed Identification

[0159] The principle of targeted capture gene chip sequencing is as follows: According to the principle of DNA complementarity, probes covering the target SNPs and InDels are designed at each target SNP and InDel locus. The biotinylated probes hybridize with the sequencing library containing the target SNP and InDel loci to form double strands. Streptavidin-coated magnetic beads are used to molecularly adsorb the biotinylated probes, thereby capturing the hybridization capture library that hybridizes with the probes. Finally, the hybridization capture library is eluted, enriched, and sequenced to ultimately obtain the genotyping results of the target SNP and InDel loci. The specific steps are as follows:

[0160] I. Construction of the hybridization capture library

[0161] 1. Genomic DNA extraction

[0162] The genomic DNA of 48 Inner Mongolia cattle samples was extracted using a magnetic bead method DNA extraction kit (CW2361S). The extracted DNA samples were detected by the following three main methods:

[0163] 1) Agarose gel electrophoresis analysis: Detect the degree of DNA degradation; determine whether there is RNA contamination;

[0164] 2) Nanodrop: Detect the purity of the DNA sample (OD260 / OD280 ratio);

[0165] 3) Qubit: Accurately quantify the DNA concentration.

[0166] 2. Genomic DNA fragmentation, end repair, and A-tailing

[0167] Add 300 ng of the DNA sample to 4 μL of Smearase Buffer and 2 μL of Smearase Enzymes, with a total volume of 24 μL. Place it in a PCR instrument and execute the program: 4°C for 1 min → 30°C for 10 min → 72°C for 20 min → store at 4°C. Fragment screening was performed on the samples that completed DNA fragmentation, end repair, and A-tailing using streptavidin-coated magnetic beads to remove overly large and overly small fragments, making the DNA fragments concentrated as much as possible between 200 - 300 bp. The final volume of the fragment screening was 25 μL and stored in a 96-well PCR plate.

[0168] 3. Adapter ligation and enrichment

[0169] Add 5 μL of CAGT Universal Adapters and 20 μL of Ligation MasterMix to a 96-well PCR plate, vortex to mix well, and centrifuge briefly to collect the reaction solution at the bottom of the tube. React at 20 °C in a PCR instrument for 15 min to complete the ligation of sequencing adapters and obtain the ligation product. After purifying the ligation product, perform PCR amplification enrichment. Take 15 μL of the purified ligation product, add 10 μL of CAGT UDIPrimer and 25 μL of Equinox Library Amp Mix (2x), mix well, and pipette 35 μL of the reaction solution into a PCR instrument for library amplification to construct a whole-genome library. Use Qubit 2.0 to quantify the whole-genome library; at the same time, detect by electrophoresis whether the main peak of the fragments of the whole-genome library is in the range of 300 - 500 bp.

[0170] 4. Chip Hybridization Capture

[0171] Pool the whole-genome libraries of each sample. After concentrating the pooled library, hybridize it with the biotinylated target capture probes on the target capture gene chip to capture the fragments in the target region from the pooled library, and remove the excess target capture probes, hybridization reagents, and other reagent components through the elution step. Finally, enrich the target region through PCR amplification after hybridization to obtain the hybridization capture library.

[0172] 5. Quality Inspection and Sequencing of the Hybridization Capture Library

[0173] After the hybridization capture library is constructed, use Qubi 2.0 to quantify the library; at the same time, detect by electrophoresis whether the main peak size of the library is in the range of 300 - 500 bp. Sequence the constructed hybridization capture library on a DNBSEQ-T7 sequencer to obtain the raw data after sequencing the hybridization capture library.

[0174] II. Data Analysis

[0175] The main processes of the analysis after sequencing the hybridization capture library are data filtering and statistics, alignment with the reference genome, and analysis of target SNP and InDel sites, which are detailed as follows:

[0176] 1. Data Filtering and Statistics

[0177] The raw data after downloading will contain reads with adapters or low quality. Before performing alignment with the reference genome and analysis of target SNP and InDel sites, it is necessary to use fastp to filter and count the data volume before and after filtering the raw data to obtain Clean Reads. Among them, the filtering conditions are as follows:

[0178] 1) Remove reads with adapters.

[0179] 2) When the N content in the sequencing read exceeds 10% of the base number of the read, the paired reads are removed;

[0180] 3) When the number of low-quality (Q<=5) bases in a sequencing read exceeds 50% of the number of bases in the read, the paired reads are removed.

[0181] The parameters set for Fastp are -u 50 -n 1 -q 5 -l 30.

[0182] 2. Comparison with the reference genome

[0183] Clean Reads were aligned to the bovine reference genome (ARS-UCD version 1.2) using BWA0.7.17 software, and samtools1.7 was used to sort and index the Bam file. The Bam file was deduplicated using the module provided by GATK 4.1.8.0 software. Then, the sequencing depth, genome coverage and other information of each sample were statistically analyzed based on the Bam file to obtain the alignment results of CleanReads on the reference genome.

[0184] 3. Analysis of target SNP and InDel sites

[0185] According to the comparison results of Clean Reads in the reference genome, the GenotypeGVCFs module in the software GATK 4.1.8.0 was used to type the target SNP and InDel sites, and then minDP=5X filtering was used, with 100% missing, and the sites that did not meet the requirements were marked as . / ., and then the site detection rate and site depth were counted, and ANNOVAR was used to annotate the genotyping results of the target SNP and InDel sites.

[0186] According to the sequencing depth of the target SNP and InDel sites, the sample detection rate and site detection rate were statistically analyzed. The sample detection rate statistics are shown in Table 6. The results show that the average sample detection rates of all samples are 98.57% (site sequencing depth greater than 5×) and 98.00% (site sequencing depth greater than 10×), respectively. The site detection rate statistics are shown in Table 7. The results show that the sites with a site detection rate of 90%-100% account for 96.64% (site sequencing depth greater than 5×) and 95.78% (site sequencing depth greater than 10×), respectively, indicating that the chip detection effect is good.

[0187] Table 6 Sample detection rate statistics

[0188]

[0189]

[0190] Note: The sample ID is the sample name;

[0191] dp0 is the number of sites with a sequencing depth greater than 0×;

[0192] dp0% is the proportion of sites with a sequencing depth greater than 0× among all sites;

[0193] And so on for other column headers.

[0194] Table 7 Statistical results of site detection rate

[0195] Locus detection rate % dp0 dp0 % dp5 dp5 % dp10 dp10 % dp15 dp15 % dp20 dp20 % (-0.001,10.0] 0 0 2 0.04 32 0.64 45 0.9 54 1.08 (10.0,20.0] 0 0 9 0.18 11 0.22 13 0.26 10 0.2 (20.0,30.0] 0 0 20 0.4 13 0.26 7 0.14 6 0.12 (30.0,40.0] 1 0.02 9 0.18 6 0.12 5 0.1 8 0.16 (40.0,50.0] 0 0 11 0.22 13 0.26 14 0.28 24 0.48 (50.0,60.0] 1 0.02 17 0.34 11 0.22 22 0.44 16 0.32 (60.0,70.0] 7 0.14 20 0.4 35 0.7 34 0.68 33 0.66 (70.0,80.0] 22 0.44 37 0.74 36 0.72 39 0.78 46 0.92 (80.0,90.0] 53 1.06 43 0.86 54 1.08 58 1.16 62 1.24 (90.0,100.0] 4916 98.32 4832 96.64 4789 95.78 4763 95.26 4741 94.82

[0196] Note: The site detection rate % is the range of site detection rate;

[0197] dp0 is the number of detected sites when the depth is greater than 0;

[0198] dp0% is the proportion of the number of detected sites when the depth is greater than 0;

[0199] And so on for other column headers.

[0200] Example 3 Application of the 5K targeted capture gene chip for Inner Mongolia cattle breed identification in breed identification

[0201] Using the sequencing method of the 5K targeted capture gene chip for Inner Mongolia cattle breed identification in Example 2, 10 samples were sequenced to detect the genotyping results of target SNP and InDel sites, and the genotyping results of target SNP and InDel sites of each sample were obtained. These samples were randomly selected from the populations of purebred Mongolian cattle, Sanhe cattle, Sunite cattle and their hybrids.

[0202] Based on the reference library constructed in Example 1, the admixture software was used to analyze the genetic composition of 10 samples to be tested. The genetic content of each sample is shown in Table 8. Each row represents the proportion of the genetic composition of each breed in a sample (the sum is 1), and each column represents a breed of genetic composition. It can be seen from this that Mongolian cattle may contain the genetic composition of Sunite or Sanhe cattle; Sanhe cattle may contain the genetic composition of Sunite cattle; Sunite cattle may contain the genetic composition of Sanhe or Mongolian cattle, which may be related to long-term crossbreeding.

[0203] Table 8 Genetic composition of samples to be tested

[0204]

[0205] Note: The sample number is the identification code for the sample;

[0206] The genetic composition of Sanhe cattle, Sunite cattle and Mongolian cattle are the proportions of the corresponding breed genetic composition in the samples to be tested, respectively.

[0207] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A set of SNP and InDel loci combination for Inner Mongolia cattle breed identification, characterized in that: The SNP and InDel site combination includes 5000 SNP and InDel sites, and the physical location information of the SNP and InDel sites based on the cattle reference genome with version number ARS-UCD1.2 is shown in the following table: 。 2. A 5K targeted capture gene chip for identification of Inner Mongolia cattle breeds, characterized in that: The 5K targeted capture gene chip comprises probes targeting the SNP and InDel site combination of claim 1.

3. A method for synthesizing a 5K targeted capture gene chip as claimed in claim 2, characterized in that: The synthesis method comprises the following steps: S1. Perform whole genome resequencing on samples of different breeds of Inner Mongolian cattle to obtain high-quality SNP and InDel sites of the Inner Mongolian cattle; S2. Screen purebred Inner Mongolian cattle samples of different breeds and construct a reference library containing purebred Inner Mongolian cattle samples of different breeds; S3, based on the reference library and high-quality SNP and InDel sites, the candidate SNP and InDel site set to be selected is screened; S4. Evaluate and score the candidate SNP and InDel site sets to be selected, determine the possibility of successful probe design of the SNP and InDel sites, and include the SNP and InDel sites with successful probe design into the gene chip site set; S5. Probes of the SNP and InDel sites in the gene chip site set are synthesized to obtain a 5K targeted capture gene chip for Inner Mongolia cattle breed identification.

4. The synthesis method according to claim 3, characterized in that The set of candidate SNP and InDel sites to be selected by S3 includes functional SNP and InDel sites for variety identification and background SNP and InDel markers for variety identification; wherein, the functional SNP and InDel sites are obtained by analyzing the population differentiation index between varieties based on a reference library and high-quality SNP and InDel sites, and the Fst value of each site is obtained, and SNP and InDel sites with higher Fst values ​​are screened as functional SNP and InDel sites for variety identification; the background SNP and InDel markers are based on high-quality SNP and InDel sites to screen sites annotated as stopgain / loss, variable splicing sites, and non-synonymous mutations as background SNP and InDel sites for variety identification.

5. The synthesis method according to claim 3, characterized in that The S1 comprises the following steps: S11, extracting genomic DNA from samples of different breeds of Inner Mongolian cattle and performing whole genome resequencing to obtain original sequence data; S12. Perform quality control on the original sequence data to obtain Clean Reads, and align them to the cattle reference genome with version number ARS-UCD1.2 to detect SNPs and InDel sites; S13. After index filtering and quality control, high-quality SNP and InDel sites of the Inner Mongolia cattle are obtained.

6. The synthesis method according to claim 3, characterized in that When constructing the reference library, the S2 uses individual likelihood value calculation to screen purebred Inner Mongolian cattle samples of different breeds.

7. The synthesis method according to claim 3, characterized in that The S4 evaluation score is evaluated based on the specificity, complexity, and GC content indicators of the upstream and downstream sequences of the target SNP and InDel sites.

8. Use of the SNP and InDel site combination of claim 1 or the 5K targeted capture gene chip of claim 2 in genotyping, pedigree analysis or breed identification of Inner Mongolia cattle.

9. A method for identifying the breed of Inner Mongolia cattle, characterized in that: The steps include: S1. Based on the genomic DNA of the sample to be tested, a whole genome library is constructed, and hybridization reaction is carried out with the probes in the 5K targeted capture gene chip according to claim 2 to construct a hybridization capture library; S2. Analyze the data after hybrid capture library sequencing to obtain the genotyping results of the target SNP and InDel sites of the sample to be tested; S3. Use the genotyping results to infer the pedigree composition of the sample to be tested and complete the breed identification of Inner Mongolia cattle.

10. The SNP and InDel site combination according to claim 1, or the 5K targeted capture gene chip according to claim 2, or the synthesis method according to claims 3-7, or the use according to claim 8, or the method according to claim 9, characterized in that: The Inner Mongolia cattle include Mongolian cattle, Sanhe cattle and Sunite cattle.

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