CN117701722A

CN117701722A - Cattle plateau adaptive breeding 10K liquid phase chip and application

Info

Publication number: CN117701722A
Application number: CN202310885982.6A
Authority: CN
Inventors: 苟潇; 杨舒黎; 倪彬; 高乐程; 张康; 聂泓宇; 叶泽宇; 王怡文
Original assignee: Foshan University
Current assignee: Foshan University
Priority date: 2024-01-16
Filing date: 2024-01-16
Publication date: 2024-03-15
Anticipated expiration: 2044-01-16
Also published as: CN117701722B

Abstract

The invention relates to a 10K liquid phase chip for bovine plateau adaptive breeding and application thereof, belonging to the field of genome breeding, and comprising (1) development of a 10K SNP chip in the whole genome range of cattle: identifying bovine elevation association sites by continuous elevation bovine population genomics, screening bovine plateau adaptive 10K SNP sites by marking, designing a liquid chip and designing a probe, and further obtaining a 10K liquid chip; (2) The liquid phase chip (10K) with low cost and high flux genotyping effect is provided, the accuracy and the genotyping effect of the chip are high, the plateau adaptability problem faced by plateau cattle group breeding can be solved, a rapid and accurate molecular means can be provided for breeding plateau tolerant individuals, a reliable technical platform is provided for developing relevant genetic breeding works such as cattle stress resistance character breeding, the development of the plateau cattle industry in China is promoted, and theoretical reference and method guidance are provided for breeding other plateau animals.

Description

Cattle plateau adaptive breeding 10K liquid phase chip and application

Technical Field

The invention relates to the field of genome breeding, in particular to a 10K SNP sequencing typing liquid phase chip with adaptive plateau and application thereof.

Background

The targeted sequencing genotype detection (Genotyping by target sequencing, GBTS) technology is a novel detection technology for genotyping, can be used for genetic variation and genotype detection of animals, plants, microorganisms and the like, and is widely applied to the fields of molecular marker assisted selection, germplasm resource evaluation, gene positioning and cloning, genetic map construction and the like. GBTS comprises a multi-PCR targeted sequencing genotype detection technology (Genoplexs) and a liquid-phase probe hybridization targeted genotype detection technology (Genobaits), and both technologies can realize accurate capture of any position and any length section of genome and can detect various genetic variations such as SSR, SNP, inDel. GBTS may have greater targeting, greater site coverage and sample homogeneity, and specific targeting sites are selected from a large number of genomic DNA for sequencing and genotyping.

Plateau hypoxia adaptation is a series of morphological structure and physiological and biochemical change processes of species of the world plateau, which are irreversible and stable inheritance generated by adaptation to the plateau hypoxia environment. The blood physiological mechanisms of plateau hypoxia adaptation are mainly manifested by changes in the number of erythrocytes, hemoglobin concentration, oxygen binding capacity, etc., and domestic animals such as Tibetan pigs (Kong et al.2019), tibetan sheep (Wei et al 2016; liu et al 2020), tibetan chickens (Nie et al 2005) and yaks (Wu et al 2015) have evolved unique physiological characteristics such as superior blood oxygen transport systems and high metabolic efficiency to adapt to the low-oxygen living pressure.

Tibet cattle improvement began in the 80 s of the last century, and current advanced crosses of improved Tibet cattle (Holstein Niu XTibet cattle female) have been made over the F3 generation. Through the progressive hybridization of more than 40 years, the coverage rate of 'yellow modified milk' reaches 80 percent, and the historical breakthrough is realized in the quantity and quality of the improvement. With the increase of the generation number of the improvement, the production performance of the improved Tibetan cattle is improved to a certain extent, such as growth speed, milk yield, lean meat percentage and the like. Along with the continuous progress of progressive hybridization, the proportion of the blood margin of Tibetan cattle is lower and lower, the adaptability of the hybridized offspring of high generation is obviously reduced, the death rate is about 20 percent, and the milk yield is only about 2300 kg. Therefore, no commercialized SNP chip is available in the breeding of the improved Tibetan cattle at present, the cross fixation and generation breeding of the improved population must be carried out, the gene and the locus of the hypoxia adaptation of the Tibetan cattle are excavated, the excellent blood system of the hypoxia adaptation of the Tibetan cattle is reserved, and the hypoxia tolerant individuals can be rapidly bred through a molecular breeding means.

Disclosure of Invention

The invention carries out whole genome measurement on continuous elevation cattle groups, identifies elevation association sites related to hypoxia adaptation of cattle, and carries out whole genome association analysis on blood physiology and blood rheological indexes and plateau adaptation SNP sites of third-generation individuals of improved Tibetan cattle. The liquid phase chip containing 10K loci is designed and developed preliminarily, so that efficient and reliable technical means are provided for developing genetic breeding work such as stress resistance traits of cattle, the method is convenient and quick, low in cost, the problem of long generation interval in the breeding process is reduced, and greater economic benefits are brought to farms.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

the first aspect of the invention provides a bovine plateau adaptive breeding 10K liquid phase chip, which comprises 11637 SNP loci for probe design, wherein the SNP locus information for probe design is shown in table 1, and the version number of the whole genome sequence of a reference genome is as follows: bos taurus UMD 3.1.

The SNP locus in the bovine plateau adaptive breeding 10K liquid phase chip is obtained by the following method: first, genes and loci associated with altitude are screened by continuous altitude bovine population genomics analysis, specific methods: through F _ST Three analytical methods (3500-500 m), MEMEMEA and BayPass were used for population genomics analysis of continuous-altitude bovine species, screening genes and loci associated with altitude; further preferably, the continuous altitudes refer to 500m (Yingjiang, barley land), 1500m (Tengchong), 2500m (Lijiang), 3000m (Diqing) and 3500m (Gongbujianda), and the beef species is cattle; which is a kind ofSecondly, creating a liquid breeding chip based on the association sites; finally, based on the existing backcross and cross high-generation hybridization segregation population, the system determines the index of the blood physiological series related to hypoxia, uses the chip to implement the whole genome association analysis (GWAS) case of hypoxia physiology, further digs the hypoxia physiological association site, and verifies and improves the reliability of the chip. The research result can provide a molecular approach for solving the problem of low oxygen bottle neck facing the current plateau cattle breeding.

The second aspect of the invention provides a genotyping method of a 10K liquid phase chip for adaptive breeding of cattle plateau, comprising the following steps:

(1) DNA extraction: extracting blood DNA of cattle to be detected, detecting the purity and the integrity of the DNA of the extracted DNA sample, and accurately quantifying the concentration of the DNA;

(2) GenoBaits experimental procedure: randomly and physically crushing the DNA with qualified quality inspection by using an ultrasonic crusher, controlling the peak value of the crushed fragment to be 200-300bp, and connecting the crushed DNA with the tail A after the end repair; connecting the DNA fragment added with A with a sequencing joint by using ligase, purifying the library and selecting the fragment by using carboxyl modified magnetic beads, and reserving a connecting product of the inserted fragment in 200-300 bp; adding a sequencing primer with Barcode and a high-fidelity PCR reaction system into the connection product to carry out PCR amplification, wherein different Barcode are used for distinguishing different samples; the amplified product can be used for probe hybridization experiments after being purified by carboxyl magnetic beads.

(3) Library construction and on-machine sequencing: taking the sequencing library which is completely constructed, adding a probe and a hybridization reagent after freeze-drying, and placing the mixture at 65 ℃ for 2 hours after denaturation to complete hybridization reaction; washing the hybridization product, performing a round of PCR to complete the construction of a hybridization capture library, performing preliminary quantification by using Qubit2.0, and accurately quantifying the effective concentration of the library by using a qPCR method to ensure the quality of the library; and after the library is detected to be qualified, entering an on-machine sequencing stage, and finally obtaining the genotype of the target SNP.

The third aspect of the invention provides application of the liquid chip in genotype detection, whole genome association analysis and whole genome selective breeding of Tibetan cattle.

The invention has the beneficial effects that:

the invention is based on the group genomics analysis of continuous elevation cattle, identifies genes and loci associated with elevation, digs the plateau adaptation loci of Tibet cattle, and creates a low-cost high-throughput genotyping liquid chip (10K) based on the elevation-associated loci. According to the existing backcross and cross high-generation hybridization segregation population, the system determines the blood physiological index related to hypoxia, uses the 10K SNP chip to implement the whole genome association analysis (GWAS) case of hypoxia physiology, further digs the hypoxia physiological association site, and verifies and improves the reliability of the chip. The invention can solve the problem of low oxygen bottle neck faced by plateau cattle breeding, provides a quick and accurate molecular means for breeding plateau tolerant individuals, provides a reliable technical platform for developing relevant genetic breeding works such as cattle stress resistance character breeding and the like, promotes the development of the plateau cattle industry in China, and simultaneously provides theoretical reference and method guidance for breeding other plateau animals.

Drawings

FIG. 1 is a diagram of a main component cluster and population structure of cattle; in the graph, (A) a principal component cluster graph, wherein the principal graph is a first second principal component, the sub-graph is a third fourth principal component, and the contribution degree of each principal component is in brackets; (B) Group structure diagram, ancestor presumes K value from 2 to 5, each color represents an ancestor component, each vertical bar represents a cattle individual; the length of the color represents the proportion of the corresponding ancestor component.

FIG. 2 is a radicle adjacent tree between bovine individuals.

FIG. 3 shows the distribution of SNP sites on different chromosomes.

FIG. 4 is a uniform distribution diagram of SNP sites on different chromosomes.

FIG. 5 is a Gap analysis of the target site on the genome.

FIG. 6 shows the distribution of SNP sites in a gene structure.

FIG. 7 is SNP annotation statistics.

Fig. 8 is a sample principal component analysis.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding by the skilled person.

Example 1 preparation of cattle plateau adaptive Breeding low-Density 10K SNP liquid phase chip

1. Continuous elevation bovine population sample sampling

The invention selects Tibetan cattle (3500 m) and Yunnan local cattle (3000-2500-1500-500) with different altitude gradient distribution, wherein each altitude is 100 heads, and specific sample information is 20 heads collected in the Tibetan autonomous region Gongbujiangda county (GB) with the altitude of 3500 m; 20 heads are collected in the autonomous state (DQ) of the Diqing Tibetan family of Yunnan province with the altitude of 3000 m; 10 heads are collected in the old junshan region (JS) of Wenshan in Yunnan province and the Lijiang city region (LJ) of Yunnan province with the altitude of 2500 m; 10 heads are collected in Yunnan province Tengchong City (TC) and Yunnan province and county area (WD) with the altitude of 1500 m; 10 heads are collected in the two-Bai county barley town (SB) of Yunnan province and the Yi Jiang county region (YJ) of Yunnan province at the altitude of 500 m. Jugular vein blood sampling was performed using a vacuum blood collection tube containing EDTA anticoagulant, 5ml, and DNA was extracted for whole genome resequencing.

2. Bovine whole genome resequencing

2.1DNA extraction

The procedure was performed using the blood genomic DNA extraction kit (tiangen biochemical technology, DP 348) according to the instructions to extract the genomic DNA of cattle.

2.2 detection of genomic DNA

DNA detection was performed using a 1% agarose gel, which was formulated with nucleic acid dye, using a Maker DL2000. Electrophoresis was performed in a 1 XTAE buffer at 120V for about 30 min. And after electrophoresis is completed, a gel imaging system is used for observation and photographing, and a DNA result picture is stored.

2.3 Huang Niuquan genome resequencing

Breaking the DNA sample which is qualified in detection into fragments of 300-500 bp, then carrying out terminal modification, adding an Illumina sequencing joint, selecting products of 400-500 bp through 2% gel electrophoresis, then carrying out LM-PCR amplification, and preparing a sequencing library. Library sequencing was performed using an Illumina HiSeq2500 platform for double-ended sequencing with a sequencing coverage of about 5X per individual genome, with a sequencing data volume of no less than 15G, and the entire sequencing flow was controlled by Illumina HiSeq Control Software software. Genome re-sequencing was done by beijing berui and kang biotechnology Co., ltd (Beijing, china).

2.4SNP cloning and sequence alignment

Firstly, performing quality control on sequencing original machine-down data, removing bases with sequencing quality less than 20 (namely sequencing accuracy lower than 99%) in reads, and discarding reads with length less than 35bp after trimming. For quality controlled data, the data were aligned to the corresponding reference genome (Bos taurus UMD 3.1) using the Burrows-Wheeler-Alignment (BWA) software (Li & Durbin, 2010), resulting in aligned bam files, and the samtools software (Li et al, 2009) was used to sequence and tag the repeat sequences. SNP rolling was then performed using the Genome Analysis Toolkit (GATK) v3.7 (McKenna et al 2010) standard procedure to obtain a high quality set of genetic variation information. Firstly, carrying out variants rolling by using a Haplotypeller in GATK to generate a mutation information set g.vcf file of an individual. The g.vcf files for all individuals were pooled and joint rolling was performed using GenotypeGVCFs to obtain variation information for this population and the exact genotype for each individual. The quality control of the variance information is then performed by hard filtering (hard filtering) due to the lack of a high quality set of known variances as a reference. The split initial SNP file is split SNP (single nucleotide polymorphism) and InDel (Insertion-Del) by using a SelectVariants command, and SNP information which does not meet quality conditions is filtered out by using a Variant filtration command to generate a final VCF file. The filtering parameter is "-cluster 3-window 20; QD <2.0; QUAL <30; MQ <40.0; FS >60.0; SOR >3.0; MQRankSum < -12.5; readPosRankSum < -8.0; DP <10; -missing-values-evaluation-as-training true. For VCF files, using plinkv1.90b (Chang et al, 2015) for further SNP quality control, removing SNPs with multiallelic, deletion rate greater than 10% and minimal allele frequency of 0, removing individuals with SNP deletion rate greater than 20%, and the quality controlled data will be used for subsequent analysis.

The cattle genome gff file was then downloaded from Ensemble and SNP annotated with ANNOVAR software.

3. Population structure and genetic relationship and polymorphism level assessment

To assess cattle population structure, linkage disequilibrium (linkage disequilibrium, LD) filtering was performed on the obtained SNP data using Plink software (Purcell et al, 2007), and low-linkage SNPs were retained (r 2< 0.3). The principal component analysis was performed on the filtered data using smartpca in the eigenoft (Price et al, 2006) software package. Next, group structure analysis was performed using adm ixture (Alexander et al 2009) software, where the number of groups parameter K was set from 2 to 5. To evaluate the relationships between individuals, the relationships between individuals are explored by calculating Identity By State (IBS) distances between individuals based on Plink software and constructing a proximity tree (Neighbor-joining tree) using MEGA (Kumar et al, 2016) software.

Through principal component analysis, it was found that the I.Bujiangda and Diqing populations could be completely separated from the other populations by the first and second principal components, but the Diqing populations were more dispersed, but the trends remained consistent. Other groups are mixed with each other, especially Wuding group, tengchong, barley, junshan and Lijiang, and show abnormality. However, if divided by altitude, the Wuding population is excluded, and 2500m (Lijiang, junshan) can be grouped into one class and separated from the other. Of the three remaining populations, the barley population could be singly grouped into one class, but the individual individuals in the great river were mixed, and even if the third and fourth principal components were added, two individuals in the great river were mixed with the great river, indicating that 1500m and below populations, possibly closely related, were not easily separated (fig. 1A).

To further resolve the population structure, the population structure analysis was performed using the ADMIXTURE software, assuming 2-5 ancestral components. When k=2, the cattle population is bounded by 3000m, with more than 3000m worker and diligent populations as a group, and less than 3000m populations as a group. When k=3, the junshan and lijiang at 2500m are independent, and the armed population at 1500m is not similar to TC population at the same altitude, but rather closer to junshan and lijiang populations. When k=4, the dirties at 3000m separate into one class. When k=5, the population at 3000m had more complex genetic background, the soaring and river-crossing could still be grouped into one class, the genetic background inside the remaining populations did not coincide exactly, and the genetic background of the diqing population at 3000m also appeared to be complex (fig. 1B). In combination with the results of PCA and adnixture, the population genetic relationship between the various altitudes is more similar, so that in subsequent analysis, the altitude is classified. However, since the armed population exhibits an abnormal distribution, it is excluded to reduce the interference of the later analysis. In order to further explore the relativity among individuals, an adjacent tree is constructed by utilizing the IBS distance among individuals, and the group is found to be approximately divided into three branches, one class of worker cloth, one class of Diqing and one class of group below 3000 m. Wherein the population below 3000m is mixed with each other, lijiang and Junshan are mixed together, but can be calculated as 2500m population. For the river-crossing, barley land, soaring, although each can be gathered into one branch, the groups below 1500m are still mixed by altitude division, which may be less geographically separated from the low altitude groups, and the groups are easier to communicate genetically, so that the relatedness is closer to the relatedness (fig. 2).

4. Screening method for hypoxia-tolerant sites of Tibetan cattle

4.1 Natural selection based on detection of the degree of differentiation between populations

Statistics of population genetics for measuring the degree of differentiation between populations are numerous, the most common being F _ST An index. In population genetics, F-statics is an index that measures whether the actual frequency of genotypes in a population deviates from the theoretical ratio at Hash equilibrium, F _ST The index evolved from F-statics, and represents the inbreeding coefficient of the subpopulation relative to the total population, i.e., the average inbreeding coefficient between related subpopulations. In practical use, it is assumed in advance that most genotypes are marked as neutral sites, followed by F _ST The extreme tail of the exponential empirical distribution identifies the locus of excessive differentiation. F (F) _ST There are a number of ways to calculate the index, in this study we used VCFtools v0.1.13 for F _ST Index meterThe calculation was based on the method proposed by Weir and Cockerham in 1984 to estimate F _ST An index comprising single site F _ST Index and window F _ST Index (window size 20kb, step size 5 kb). F (F) _ST The value range of the index is 0-1, and the larger the numerical value is, the larger the differentiation degree among the populations is. When F _ST When the index is 1, the alleles are fixed in the respective populations and fully differentiated; when F _ST When the index is less than 0, no specific biological explanation exists, and the index can be ignored; when F _ST When the index is 0-0.05, the differentiation degree among the populations is small and can be ignored; when F _ST When the index is 0.05-0.25, the intermediate differentiation exists among the populations; when F _ST An index greater than 0.25 indicates a large degree of inter-population differentiation, a potential positive selection site. The high-low population cattle were compared and their top1% window was set as the potential candidate window.

4.2 allele frequency and altitude association analysis

Plateau adaptation is a process of micro-evolution of animals under hypoxic conditions based on changes in allele frequency, in which animals are physiologically or ecologically directionally regulated and the adaptation of the whole species to the environment is mediated by genetic mechanisms, all of which are reflected in changes in allele frequency. The altitude is the optimal environmental variable for representing the selective pressure intensity of the low-oxygen environment, and the candidate loci responding to the environmental adaptability constraint can be detected by carrying out correlation analysis on the allele frequency and the altitude of the continuous altitude group, and meanwhile, the inherent differentiation loci caused by the group history and genetic drift are eliminated, so that the screening range of the candidate genes is effectively narrowed. In this study, we used two methods, the environmental adaptation site detection, namely the environmental adaptation mixed effect model (Mixed Effect Model for Environmental Adaptation, MEMEMEA) (Wu Fuquan, 2018) and Baypass (Gautier, 2015) developed by the subject group.

Based on the population genetics method, a MEMEMEA method is established by using likelihood estimation, and the model can be used for detecting genetic polymorphic sites subjected to environmental adaptability selection among populations under specific environmental variable gradients. The method adopts multielement normal distribution to identify SNP loci with allele frequencies selected by environmental factors, and allows adjacent loci to be subjected to different selection intensities by introducing random effect u among loci. Meanwhile, the method fully considers genetic loading effect, and a sliding window mode is used for detecting the site subjected to environmental selection pressure. In the present invention, the detection of the environmental adaptation site was performed by expanding 10kb in the front-back direction with each SNP as the center as a window.

BayPass is a piece of group genomics analysis software that, based on bayesian theory, identifies genetic markers that are undergoing natural selection or are highly correlated with target group covariates (e.g., environmental variables, quantity, or quality phenotypic characteristics). The BayPass software is based on the BayEnv2 model (loop et al, 2010) published by the loop et al (2010). The BayEnv2 model can both identify natural selection sites and further annotate the screened selection imprinting by quantifying the association with specific population covariates, with the most critical parameter being the covariance matrix across population allele frequencies. The improvement of BayPass is in three aspects: (1) The estimation of the overall covariance matrix and different relevant metrics is more accurate; (2) A posterior procedure based on XtX statistic calibration was proposed to detect sites where adaptation occurred; (3) A model with binary auxiliary variables was introduced to classify each locus as either associated or unassociated.

Both methods are based on allele frequency data, so before analysis, we first reject Gao Queshi rate SNP sites, and if the loss rate of a certain SNP is greater than 10% in any altitude population, reject the SNP sites in all altitude populations to eliminate the influence of excessive loss rate on analysis results. Both MEMEMEA and BayPass software essentially performed regression analysis to screen SNP sites that are highly correlated with environmental factor trends. The MEMEA model considers the carrying effect, effectively reduces the false positive rate, but is sensitive to the locus with the allele frequency of 0 in the subgroup, so that the locus score value is higher; baypass mainly considers the elevation trend of single sites, and the screened environment adaptability sites have higher false positives.

5. Design and development of low density liquid phase chip

5.1 Optimized screening of 10K SNP markers

In the invention, two analysis methods of MEMEMEA and BayPass are integrated, genetic polymorphic sites subjected to environmental adaptability selection among groups under continuous altitude gradient are detected, and then single site F is combined _ST The analysis results mutually verify and supplement, and candidate sites of the three analysis methods are intersected. Calculation of F by vcftools software _ST Sliding window results (20 kb window, 5kb step), combining the Fst window at top1% into a region (overlapping or < 20kb apart), combining BayPass, MEMEA, single point F _ST The intersection sites of (2) are within the region range and are used as hypoxia adaptation related SNP, the SNP is converted to the ARS-UCD1.2 of a reference genome, 11637 SNP sites related to the hypoxia tolerance character of the cattle are finally screened, and the site information is shown in Table 1.

TABLE 1SNP site information Table

Description of SNP sources: SNP sequence numbers 0001-8151 are 10K in origin; SNP sequence numbers 8152-11636 are 4K in origin; SNP sequence number 11637 is from 4K &10K.

5.2 Synthesis of probes

Screening and checking target site information, designing and synthesizing probes, testing and adjusting the probes, and determining final probes and capture sites.

According to the DNA complementation principle, one or more probes covering the target 10K SNP are designed at each site to be detected, the probes modified by Biotin (Biotin) can be hybridized with a target region in a denatured resequencing library to form double chains, and the streptavidin-coated magnetic beads are used for adsorbing molecules carrying the Biotin, and finally the genotype of the target SNP is obtained through elution, amplification and sequencing.

5.3 detection of bovine plateau adaptive breeding chip

Regarding the frozen semen improvement F3 generation of Holstein cattle as cattle to be detected.

(1) DNA extraction: 10 modified Tibetan cattle blood DNA are extracted by adopting a high-throughput DNA extraction kit. The extracted DNA samples were subjected to 2 assays: the purity and integrity of the DNA was analyzed using a 1% agarose gel electrophoresis method; the DNA concentration was accurately quantified using Qubit. The purity is OD260/280 more than or equal to 1.8, and the result shows that the DNA sample is complete and the total DNA amount is more than or equal to 0.5ug as qualified quality inspection through agarose gel electrophoresis detection.

(2) Library construction and on-machine sequencing: and (3) taking DNA qualified in quantitative quality inspection, carrying out random physical crushing by using an ultrasonic crusher, controlling the peak value of the crushed fragments to be 200-300bp, and connecting the crushed DNA with the A tail after terminal repair. The DNA fragment added with A is connected with a sequencing joint by using ligase, then the library is purified and fragment selected by using carboxyl modified magnetic beads, and the connection product of the inserted fragment between 200 and 300bp is reserved. The ligation products were added to sequencing primers with Barcode and a high-fidelity PCR reaction system for PCR amplification, and different Barcode were used to distinguish between different samples. The amplified product can be used for probe hybridization experiments after being purified by carboxyl magnetic beads.

Taking 500ng of the sequencing library which is constructed, adding a probe and a hybridization reagent after freeze-drying, and placing the mixture at 65 ℃ for 2 hours after denaturation to finish hybridization reaction. After washing the hybridization product, performing a round of PCR to complete the construction of a hybridization capture library, performing preliminary quantification by using Qubit2.0, and accurately quantifying the effective concentration of the library by using a qPCR method to ensure the quality of the library. And after the library is detected to be qualified, entering an on-machine sequencing stage.

Chip performance detection analysis: the targeted sequencing genotyping (Genotyping By Target Sequencing, GBTS) technology is a technology for deep resequencing of target sites, and has stronger targeting, higher site coverage and sample uniformity. The detection rate of 10 samples was 96.69-99.79%, the average detection rate was 99.88%, and the test performed well (FIGS. 3-6).

Example 210K evaluation of SNP liquid phase chip

1. Blood sample collection and physiological index measurement

The 1551 modified Tibetan cattle blood was collected, and within 4 hours, the normal index 11 items of blood and the viscous index 13 items of blood were measured by using a veterinary full-automatic blood analyzer (BC-30 Vet) and an automatic blood rheology tester (ZL 6000), and the detailed indexes are shown in the following table 2.

TABLE 2 blood physiological index to be measured

2. Liquid phase chip evaluation

And (3) extracting 1551 modified Tibetan cattle blood DNA by using a high-throughput DNA extraction kit, and carrying out genotyping on a quality control qualified sample by using a 10K SNP liquid-phase chip.

Gwas analysis

Filtering the raw sequencing sequence (raw reads) obtained by sequencing by using software fastp [2] (version 0.20.0, parameter: -N10-q 20-u 40), including removing the linker sequence, when the content of N contained in the sequencing read exceeds 10% of the length ratio of the strip, the pair of paired reads needs to be removed; when the number of low-mass (Q.ltoreq.20) bases contained in a sequencing read exceeds 40% of the length proportion of the strand, the pair of paired reads needs to be removed. And finally, clear reads are obtained, and the clear reads are used for subsequent analysis. Comparing the clear Reads after quality control with a reference genome (Bos taurus UMD 3.1) sequence by using software BWA, and performing mutation detection by using a UnifiedGenotoyper module of software GATK (version v3.5-0-g36282e 4) according to the comparison result of the clear Reads in the reference genome, wherein detection parameters are as follows: "-dcov 1000000-minindelfrac0.15-glm BOTH-l INFO". The filtration was performed using a VariantFiltration module with the filtration parameters of —-fileexpression "MQ0> = 4& ((MQ 0/(1.0 x DP)) > 0.1)" -filename "hard_to_valid" - -fileexpression "DP < 5|qd <2" - -filename "low_read_support". The comparison result and the variation result use self-compiled Perl script to extract the genotyping information of the SNP locus, when the covering depth of the sample at a certain SNP locus is less than 5X, the sequencing depth of the sample at the locus is insufficient, in order to ensure the genotyping result to be accurate, the locus is treated according to the deletion, and is expressed by NA in a table; when a sample has mutation in the genotype of a certain SNP locus, the mutation frequency of the locus needs to be judged, if the mutation frequency is more than or equal to 0.8 or less than or equal to 0.2, the locus is homozygous, if the mutation frequency is between 0.2 and 0.8, each allele in the heterozygous genotype is supported by at least 4 reads, the SNP locus is heterozygous mutation, otherwise, the locus is treated according to deletion. Functional annotation of SNP sites was performed using ANNOVAR (fig. 7). And carrying out principal component analysis on the detection samples subjected to subsequent association analysis, and removing the outlier group. As shown in fig. 8, the samples are basically not clustered, and the actual situation of the samples is met.

GWAS analysis was performed using a linear regression model (GLM) general linear model in Tassel software, the model being as follows:

y＝Wα+xβ+ε

in the formula, y represents the phenotype to be studied, namely, blood routine and blood rheological indexes in the study; wα is the fixed effect (age, sex and feeding level), other factors affecting y, mainly population structure; x beta is the labelling effect; epsilon represents the residual error.

The liquid phase chip for the hypoxia tolerance site is prepared to carry out whole genome association analysis on the phenotype of the blood physiological index of the Tibetan modified cow, GLM (global stem cell) of the Tassel software is adopted to carry out analysis, the number of marker sites which are obviously related to the data of the blood physiological index of the Tibetan modified cow is 1930, the number of candidate base factors is 21, and the genes are related to membrane generation, angiogenesis, development, erythrocyte shape, tumor, central nervous system, skeletal development and the like.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. Cattle plateau adaptation breeding 10K liquid phase chip, its characterized in that: the cattle plateau adaptive breeding 10K liquid phase chip comprises 11637 SNP loci for probe design, the SNP locus information for probe design is shown in table 1, and the version number of the whole genome sequence of the reference genome is: bos taurus UMD 3.1.

2. The bovine plateau adaptive breeding 10K liquid phase chip of claim 1, wherein: the chip is obtained by the following method:

s1: continuous elevation cattle were collected for whole genome sequencing, with continuous elevation distribution including 500m, 1500m, 2500m, 3000m and 3500m, through F _ST Performing group genomics analysis on continuous elevation cattle populations by using three analysis methods of MEMEMEA and Baypass, and screening 11637 loci associated with elevation;

s2: screening out the sea-tangle related sites according to the S1, designing one or more probes covering the target 10K SNP at each site to be detected according to the DNA complementation principle at the position of the binding site on the whole genome;

s3: the chip was developed using a targeted sequencing genotyping technique.

3. The use of the bovine 10K liquid chip of claim 1 in genotyping, whole genome association analysis, whole genome selective breeding of cattle.

4. The genotyping method of the bovine plateau adaptive breeding 10K liquid phase chip according to claim 1, wherein: the method comprises the following steps:

(2) GenoBaits experimental procedure: randomly and physically crushing DNA by using an ultrasonic crusher, controlling the peak value of a crushed fragment to be 200-300bp, and connecting the crushed DNA with the tail A after the end of the crushed DNA is repaired; connecting the DNA fragment added with A with a sequencing joint by using ligase, purifying the library and selecting the fragment by using carboxyl modified magnetic beads, and reserving a connecting product of the inserted fragment in 200-300 bp; adding a sequencing primer with Barcode and a high-fidelity PCR reaction system into the connection product to carry out PCR amplification, wherein different Barcode are used for distinguishing different samples; the amplified product after purification of carboxyl magnetic beads can be used for probe hybridization experiments;