Sheep 3K liquid phase chip and application thereof

Abstract

The invention belongs to the technical field of gene chips, and discloses a sheep 3K liquid chip and application thereof. The method comprises the following steps: collecting blood samples of 15 different varieties of sheep, screening and identifying 3000 SNP sites, extending each SNP site for 60bp before and after a sheep reference genome, and taking a reverse complementary sequence including the SNP sites and totally 121bp as a probe sequence; synthesizing the probe sequence to obtain a liquid phase chip; and synthesizing probe sequences of other sites by adopting the same method to obtain the sheep 3K liquid phase chip. The sheep 3K liquid chip can realize sheep genetic typing through a targeted capture sequencing technology, and has important contribution significance in researches of sheep genetic diversity analysis, variety identification, genetic relationship identification, germplasm resource improvement and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of gene chips and relates to a sheep 3K liquid phase chip and an application thereof. Background Art

[0002] As an important livestock, sheep have undergone long-term natural selection and artificial domestication, forming unique physiological and behavioral characteristics. Sheep breeds in Xinjiang, such as Duolang sheep, Kirgiz sheep, Altay sheep, Balchuk sheep and Bayinbuluke sheep, are famous for their unique geographical indication product protection and excellent production performance. For example, Altay sheep are famous for their large body and high meat and fat production performance; Balchuk sheep are respected for their meat and wool dual-purpose local breed and excellent production performance; Bayinbuluke sheep are known for their advantages such as early maturity, tolerance to roughage, strong adaptability, and high meat production. These different breeds show the diversity of sheep in nature and under human domestication, from wild sheep adapted to extreme environments to breed sheep optimized in specific regions, reflecting the different effects of natural selection and artificial selection.

[0003] In the research work of sheep germplasm genetic diversity analysis, genetic and evolutionary analysis, kinship identification, whole genome association analysis and genome selection, single nucleotide polymorphism (SNP) as a widely distributed genetic molecular marker has the characteristics of high density, strong genetic stability and easy automated analysis, and is therefore increasingly used.

[0004] Gene chips prepared based on SNP mainly include solid-phase chips and liquid-phase chips. Solid-phase chips are based on complementary hybridization between probes and DNA sequences, and typing is performed through fluorescent color development signals of markers. Liquid-phase chips are based on resequencing technology, which specifically captures each target site and performs high-depth resequencing, with the advantages of high detection accuracy and high throughput. Based on the principle of DNA complementarity, liquid-phase chips are designed for each site to be tested, with a biotin-labeled probe covering the target SNP. These probes hybridize with the target region of the genome in the liquid phase to form double strands. The adsorption between streptavidin-coated magnetic beads and biotin-containing molecules can be used. After elution, amplification, and library construction, second-generation sequencing is performed to finally restore the genotype of the target site and its surrounding SNPs.

[0005] Although liquid phase chips have many of the above advantages, there is still a lack of mature commercial liquid phase chips that can be used in sheep breeding and other work. The main reasons are low density and uneven distribution of SNP molecular markers and high detection costs, which have a great impact on the efficiency of research work. This requires the development of sheep SNP liquid phase chips with high density, good accuracy and commercial applicability. Summary of the invention

[0006] In order to speed up the sheep breeding process and identify the genotypes of batch samples efficiently and accurately, the inventors initially analyzed and studied sheep samples from 15 different breeds, obtained 3,000 SNP sites located in the sheep genome, and prepared them into liquid phase chips in order to achieve rapid identification of different sheep breeds.

[0007] In one aspect, the present invention provides a group of sheep SNP sites, wherein the SNP sites are shown as NO.1 to NO.3000:

[0008]

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] The SNP site is located in the sheep reference genome ARS-UI_Ramb_v2.0.

[0032] Further, the SNP sites include:

[0033] 1) SNP loci of Altay sheep;

[0034] 2) SNP loci of Balchuk sheep;

[0035] 3) SNP loci of Bashibai sheep;

[0036] 4) SNP loci of Bayinbuluke sheep;

[0037] 5) SNP loci of Chinese Merino sheep;

[0038] 6) SNP loci of Cele Black Sheep;

[0039] 7) SNP loci of Kazakh sheep;

[0040] 8) SNP loci of Hetian sheep;

[0041] 9) SNP loci of Kirgiz sheep;

[0042] 10) SNP loci of Robe sheep;

[0043] 11) SNP loci of Turpan black sheep;

[0044] 12) SNP loci of Taxkorgan sheep;

[0045] 13) SNP loci of Yecheng sheep;

[0046] 14) SNP loci of Duolang sheep;

[0047] 15) SNP loci of Karakul sheep.

[0048] Further, the SNP site is screened by the following steps:

[0049] 1) Sequencing: Blood samples from 15 different sheep breeds were subjected to genome resequencing (Xinjiang Compson Biotechnology Co., Ltd.) to obtain fastq.gz offline data for each sample.

[0050] 2) Format conversion: The fastq.gz data was subjected to quality control by fastqc software, sequence alignment by bwa software, sorting and duplication removal by samtools software, and variant calling by gatk software to obtain a vcf.gz format file.

[0051] 3) Filtering: Use gatk, bcftools and plink software to filter and obtain sites with minor allele frequency (MAF) greater than 0.1, missing rate (missing rate) less than 0.1 and Hardy Weinberg equilibrium (HWE) P value greater than 0.001; use plink software to remove linked sites, with the conditions: window size of 50 SNPs, step size of 10 SNPs and LD threshold of 0.2.

[0052] 4) Preliminary screening: The population fixation index (Fst) was calculated using plink software, and the top 1% SNP sites with the highest Fst for each breed were screened in the whole genome of different sheep breeds; the top 1% SNP sites were merged, and duplicate sites were removed;

[0053] 5) Rescreening: The best sites were screened using the random forest model and support vector machine learning method in Python. The SNP site data set was divided into a training set and a test set at a ratio of 8:2, and stratified sampling was performed to ensure that the distribution ratio of each variety in the training set and the test set was consistent. The feature importance of the random forest model was used to select the most important SNP features, and the number of selected features was controlled, with 200, 500, 1000, 2000, 3000, 4000, 5000 and 10000 features selected; the number of decision trees was set to 1000; the random number seed was equal to 0; the parameter settings of the support vector classifier (SVC): C = [0.01, 0.1, 1, 5, 10, 100]; the number of cross-validation folds was set to 5.

[0054] 6) Elimination: All the above-mentioned candidate SNP sites that meet the conditions are removed from the SNP sites that cannot be uniquely matched on the genome and the SNP sites that contain repetitive sequences in the flanking sequences. Then, the spacing between the SNP sites is evaluated to make the distance between any two adjacent SNP sites greater than 60bp to achieve probe synthesis.

[0055] On the other hand, the present invention also provides the use of the SNP site in the preparation of a sheep liquid phase chip: each SNP site is extended by 60 bp before and after the sheep reference genome, and a total of 121 bp of reverse complementary sequence including the SNP site is used as a probe sequence; the probe sequence is synthesized to obtain a liquid phase chip; the probe sequences of the remaining sites are synthesized using the same method to obtain a sheep 3K liquid phase chip.

[0056] Based on the above application, the present invention also provides a sheep 3K liquid phase chip, and the sheep 3K liquid phase chip is prepared using the above SNP sites.

[0057] The invention of this law also requests protection for the application of the above-mentioned sheep 3K liquid phase chip in sheep gene typing detection, variety identification, breeding and genetic diversity analysis.

[0058] Compared with the prior art, the present invention "a sheep 3K liquid phase chip and its application" has the following beneficial effects:

[0059] 1) The present invention mines key functional sites and breed-specific sites of sheep from large-scale sequencing data, and screens out 3,000 SNP sites, all of which are newly discovered SNP sites. The liquid phase chip prepared using these SNP sites can accurately realize genotyping, and has high application value in multiple research fields of sheep.

[0060] 2) The sheep 3K liquid phase chip provided by the present invention, based on targeted capture sequencing technology, can not only type the target SNP site, but also accurately type the SNP sites within a certain range around the target SNP site, so more SNP site information (4-6K) can be obtained. Compared with traditional solid phase chips, it is more flexible and other SNP sites can be added according to application requirements.

[0061] 3) Compared with whole genome resequencing, the present invention has obvious cost advantages and can be used for large-scale genotyping of sheep, thus promoting the progress of related work.

[0062] 4) The sheep samples of the present invention are derived from Xinjiang local breeds, including 15 different breeds of sheep. SNP sites are obtained through deep resequencing, which have more reliable SNP information and are more suitable for the research of Xinjiang local sheep breeds. It is beneficial to the development of Xinjiang local sheep breed selection and breeding work and the protection of germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Phylogenetic tree based on genetic distance for 15 Xinjiang local sheep breeds.

[0064] Figure 2This is the distribution of 3000 SNP sites in the whole genome (the number of SNPs contained in every 1M window on the genome). DETAILED DESCRIPTION

[0065] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all 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.

[0066] Example 1

[0067] This example provides screening of sheep SNP sites.

[0068] Based on the evolutionary tree of 15 breeds in Xinjiang (Chinese Merino sheep, Altay sheep, Balchuk sheep, Bashbai sheep, Bayinbuluke sheep, Cele black sheep, Kazakh sheep, Hotan sheep, Kirgiz sheep, Karakul sheep, Lop sheep, Turpan black sheep, Taxkorgan sheep, Yecheng sheep, Duolang sheep), the evolutionary tree is shown in Figure 1. Figure 1 ) were used for SNP analysis of the resequencing data, and 9,959,424 SNP sites were initially obtained for subsequent screening.

[0069] The specific screening process is as follows:

[0070] 1) Sequencing: The blood samples were subjected to genome resequencing (Xinjiang Compson Biotechnology Co., Ltd.) to obtain fastq.gz data for each sample.

[0071] 2) Format conversion: The fastq.gz data was subjected to quality control by fastqc software, sequence alignment by bwa software, sorting and duplication removal by samtools software, and variant calling by gatk software to obtain a vcf.gz format file.

[0072] 3) Filtering: Use gatk, bcftools and plink software to filter and obtain sites with minor allele frequency (MAF) greater than 0.1, missing rate (missing rate) less than 0.1 and Hardy Weinberg equilibrium (HWE) P value greater than 0.001; use plink software to remove linked sites, with the conditions: window size of 50 SNPs, step size of 10 SNPs and LD threshold of 0.2.

[0073] 4) Preliminary screening: The population fixation index (Fst) was calculated using plink software, and the top 1% SNP sites with the highest Fst for each breed were screened out in the whole genome of different sheep breeds; the top 1% SNP sites were merged, and duplicate sites were removed.

[0074] 5) Rescreening: The best sites were screened using the random forest model and support vector machine learning method in Python. The SNP site data set was divided into a training set and a test set at a ratio of 8:2, and stratified sampling was performed to ensure that the distribution ratio of each variety in the training set and the test set was consistent. The feature importance of the random forest model was used to select the most important SNP features, and the number of selected features was controlled, with 200, 500, 1000, 2000, 3000, 4000, 5000 and 10000 features selected; the number of decision trees was set to 1000; the random number seed was equal to 0; the parameter settings of the support vector classifier (SVC): C = [0.01, 0.1, 1, 5, 10, 100]; the number of cross-validation folds was set to 5.

[0075] 6) Elimination: Remove duplicate sites, SNP sites that cannot be uniquely matched on the genome, and SNP sites containing duplicate sequences in the flanking sequences from all the candidate SNP sites that meet the conditions above. Then evaluate the spacing of SNP sites so that the spacing between any two adjacent SNP sites is greater than 60bp to achieve probe synthesis.

[0076] After the above screening steps, a total of 3000 SNP sites that met the conditions were obtained. The distribution of these SNP sites on the sheep reference genome ARS-Ul Ramb v2.0 is shown in Figure 2 , see Table 1 for specific locations.

[0077] Table 1. Specific locations of 3000 SNP sites in the sheep genome

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Note: The format of mutation type is: reference allele / mutant allele.

[0102] According to the position of the above 3000 SNP sites, the reverse complementary sequence of 121bp including the target SNP site was extended by 60bp before and after the sheep reference genome, and the reverse complementary sequence of 121bp including the target SNP site was used as the probe sequence of the site. For example, for the first site located at 100499325 on chromosome 1 in Table 1, according to the NCBI database, it can be found that the site is extended by 60bp before and after the sheep genome ARS-Ul Ramb v2.0 version, and sequence A is obtained. Then, its reverse complementary sequence A' is obtained according to sequence A, and sequence A' is used as the probe sequence of the site. After the probe sequences of the remaining sites are determined in the same way, the probes are synthesized (Xinjiang Compson Biotechnology Co., Ltd.) to obtain the sheep 3K liquid phase chip.

[0103] Example 2

[0104] This embodiment provides the workflow of the sheep 3K liquid phase chip.

[0105] Extraction of sheep genomic DNA: Blood was collected from the jugular vein of sheep, and DNA was extracted using the phenol-chloroform method or a blood genomic extraction kit (Tiangen Biotechnology Co., Ltd., Beijing).

[0106] DNA sample quality detection: Use agarose gel electrophoresis with a mass fraction of 1-1.5% to detect, and use a gel imaging system (GelDocXRSystem, Bio-Rad, USA) to judge the electrophoresis results to ensure the integrity of the genome. Use a micro-ultraviolet spectrophotometer (Q5000, Quawell, USA) or a similar nucleic acid protein analyzer to measure the concentration of genomic DNA and adjust the DNA concentration to the working concentration (10-50ng / μL).

[0107] Data analysis: The obtained raw data were quality controlled using fastp software (refer to "Fastp: an ultra-fast all-in-one FAS TQ preprocessor."), and then the sequencing data were aligned to the sheep reference genome ARS-Ul Ramb v2.0 (refer to "The complete mitochondrial DNA sequence of the domestic sheep (Ovis aries) and comparison with the other major ovine haplotype.") using bwa software (refer to "Aligning sequencing reads, clone sequences and assembly contigs with BWA-MEM."). The standard process of GATK software (refer to "From FastQ data to high confidence variant calls: the genome analysis toolkit best practices pipeline.") was used to detect SNPs and perform genotyping.

[0108] Example 3

[0109] This example provides the prediction accuracy of the sheep 3K liquid phase chip.

[0110] A total of 1486 sheep samples from 15 sheep breeds (Chinese Merino sheep, Altay sheep, Balchuk sheep, Bashbai sheep, Bayinbuluke sheep, Cele black sheep, Kazakh sheep, Hotan sheep, Kirgiz sheep, Karakul sheep, Lop sheep, Turpan black sheep, Taxkorgan sheep, Yecheng sheep, and Duolang sheep) were genotyped, and breed prediction was performed based on the genotyping results. The prediction results are shown in Table 2.

[0111] Table 2. Breed prediction results of 1486 sheep

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Note: In Table 2, “D” or “Brola” means Chinese Merino sheep, “ALT” means Altay sheep, “BECK” means Balchuk sheep, “BSB” means Bashbai sheep, “BYBLK” means Bayinbuluke sheep, “CLH” means Cele black sheep, “HSK” means Kazakh sheep, “HT” means Hotan sheep, “KEKZ” means Kirgiz sheep, “KLKE” means Karakul sheep, “LB” means Lop sheep, “TLFH” means Turpan black sheep, “TSKEG” means Taxkorgan sheep, “YC” means Yecheng sheep, and “DLZ” means Duolang sheep.

[0131] In the theoretical results and predicted results in Table 2, different numbers represent different breeds of sheep. The theoretical results of Chinese Merino sheep are represented by "0", the theoretical results of Altay sheep are represented by "1", the theoretical results of Balchuk sheep are represented by "2", the theoretical results of Bashbai sheep are represented by "3", the theoretical results of Bayinbuluke sheep are represented by "4", the theoretical results of Cele black sheep are represented by "5", the theoretical results of Kazakh sheep are represented by "6", the theoretical results of Hotan sheep are represented by "7", the theoretical results of Kirgiz sheep are represented by "8", the theoretical results of Karakul sheep are represented by "9", the theoretical results of Lop sheep are represented by "10", the theoretical results of Turpan black sheep are represented by "11", the theoretical results of Taxkorgan sheep are represented by "12", the theoretical results of Yecheng sheep are represented by "13", and the theoretical results of Duolang sheep are represented by "14". If the numbers of the predicted results and the theoretical results are consistent, it means that the prediction is correct, otherwise it is wrong.

[0132] As shown in Table 2, among the prediction results of the sheep 3K liquid phase chip for 1486 sheep, 6 were inconsistent with the theoretical results, and the prediction accuracy was (1486-6) / 1486×100%=99.60%.

[0133] Example 4

[0134] This example describes the application of sheep 3K liquid phase chip in breed identification.

[0135] Blood samples of the sheep to be tested were collected and genotyped using the sheep 3K liquid phase chip (genotyping was the same as in Example 2). The SNP sets obtained by genotyping were then subjected to principal component analysis and phylogenetic tree construction, and the breed of the sheep to be tested was determined based on the results of the principal component analysis and phylogenetic tree.

[0136] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A set of sheep SNP loci, characterized in that: The SNP sites are shown as NO.1 to NO.3000: The SNP site is located in the sheep reference genome ARS-UI_Ramb_v2.0 version.

2. The SNP site according to claim 1, characterized in that: include: 1) SNP loci of Altay sheep; 2) SNP loci of Balchuk sheep; 3) SNP loci of Bashibai sheep; 4) SNP loci of Bayinbuluke sheep; 5) SNP loci of Chinese Merino sheep; 6) SNP loci of Cele Black Sheep; 7) SNP loci of Kazakh sheep; 8) SNP loci of Hetian sheep; 9) SNP loci of Kirgiz sheep; 10) SNP loci of Robe sheep; 11) SNP loci of Turpan black sheep; 12) SNP loci of Taxkorgan sheep; 13) SNP loci of Yecheng sheep; 14) SNP loci of Duolang sheep; 15) SNP loci of Karakul sheep.

3. The SNP site according to claim 2, characterized in that: The SNP sites are screened by the following steps: 1) Sequencing: Blood samples of different sheep breeds were resequenced to obtain fastq.gz data for each sample; 2) Format conversion: The fastq.gz data is subjected to quality control by fastqc software, sequence alignment by bwa software, sorting and duplication removal by samtools software, and variant calling by gatk software to obtain a vcf.gz format file; 3) Filtering: Use gatk, bcftools and plink software to filter and obtain sites with allele frequency greater than 0.1, site missing rate less than 0.1 and Hardy-Weinberg equilibrium test P value greater than 0.001; use plink software to remove linked sites, with the conditions: window size of 50SNPs, step size of 10SNPs, and LD threshold of 0.2; 4) Preliminary screening: The population fixation index was calculated using plink software, and the top 1% SNP sites with the highest Fst of each breed were screened out in the whole genome of different sheep breeds; the top 1% SNP sites were merged, and duplicate sites were removed; 5) Rescreening: Using the random forest model and support vector machine learning method in Python, the SNP locus data set was divided into a training set and a test set at a ratio of 8:2, and stratified sampling was performed to ensure that the distribution ratio of each variety in the training set and the test set was consistent; The feature importance of the random forest model was used to select the most important SNP features, and the number of selected features was controlled, with 200, 500, 1000, 2000, 3000, 4000, 5000, and 10000 features selected; Set the number of decision trees to 1000; The random number seed is equal to 0; the parameter setting of the support vector classifier is: C = [0.01, 0.1, 1, 5, 10, 100]; the number of cross-validation folds is set to 5; 6) Elimination: All the candidate SNP sites that meet the conditions mentioned above are removed, including those that cannot be uniquely matched on the genome and those that contain repetitive sequences in the flanking sequences. The spacing between SNP sites is then evaluated to ensure that the spacing between any two adjacent SNP sites is greater than 60 bp.

4. Use of the SNP site according to claim 1 in preparing a sheep liquid phase chip, characterized in that: Each SNP site was extended by 60 bp before and after the sheep reference genome, and a total of 121 bp of reverse complementary sequence including the SNP site was used as a probe sequence; the probe sequence was synthesized to obtain a liquid phase chip; the probe sequences of the remaining sites were synthesized using the same method to obtain a sheep 3K liquid phase chip.

5. The use according to claim 4, characterized in that: The sheep reference genome is ARS-Ul Rambv2.

0.

6. A sheep 3K liquid phase chip, characterized in that: The sheep 3K liquid phase chip is prepared using the SNP site described in claim 1.

7. Use of the sheep 3K liquid phase chip according to claim 6 in sheep genotyping detection.

8. Use of the sheep 3K liquid phase chip according to claim 6 in sheep breed identification.

9. Use of the sheep 3K liquid phase chip according to claim 6 in sheep breeding.

10. Use of the sheep 3K liquid phase chip according to claim 6 in sheep genetic diversity analysis.

Images