CN113249495A - Sheep liquid phase chip and application thereof - Google Patents
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Abstract
The invention discloses a sheep liquid phase chip and application thereof, 935 SNP sites which can be used for chip design are discovered and screened out, and sheep genotyping can be realized by using the designed liquid phase chip through a targeted capture sequencing technology. Experimental results show that the chip designed by the invention can be used for sheep genetic diversity analysis, variety identification, genetic relationship identification, whole genome association analysis and genome selective breeding.
Description
Technical Field
The invention relates to the field of whole genome gene chips, in particular to a sheep liquid phase chip and application thereof.
Background
Molecular Marker Technology (Molecular Marker Technology) is an important tool in Molecular breeding. Traditional molecular markers, such as Restriction Fragment Length Polymorphism (RFLP) and Simple Sequence Repeat (SSR), play important roles in the field of genetic breeding. However, there are some limitations, such as small distribution number of genome, complicated operation process and low throughput, which can not meet the requirement of large-scale commercial breeding application. Single Nucleotide Polymorphism (SNP) refers to a variation of a Single Nucleotide in a genome, including a transition, transversion, insertion or deletion of a Single base pair. As a genetic marker which is more widely distributed in a genome, SNP has the characteristics of high density, high genetic stability, easiness in automatic analysis and the like, and has developed into a common molecular marker in animal genetic variation research.
At present, in the gene chip technology for SNP locus typing, the traditional solid phase chip carries out typing through a fluorescent color development signal of a marker based on complementary hybridization of a probe and a DNA sequence. The liquid phase chip is based on the re-sequencing technology, carries out specific capture on each target site, carries out high-depth re-sequencing and has the advantages of high detection accuracy and high flux. The liquid phase chip generally comprises a Biotin (Biotin) label designed for each site to be detected according to the DNA complementary principle and a probe covering target SNP, the probes are hybridized with a genome target region in a liquid state to form a double chain, the adsorption effect of streptavidin-coated magnetic beads and molecules with Biotin can be utilized, and the second-generation sequencing is carried out after elution, amplification and library establishment, so that the genotype states of the target site and the surrounding SNP are finally reduced. The liquid phase chip has already mature applications (Xuyunbi, Yang quan, Zheng hong Jiang, and so on.) in the aspects of species evolution analysis, germplasm resource evaluation and DNA fingerprint identification, molecular genetic map construction, gene/QTL positioning and gene cloning, molecular marker assisted selection, whole genome selection, and so on at present, and the technology and the application of the targeted sequencing genotype detection (GBTS) thereof, China agricultural science, 2020,53(15) 2983-.
Currently, cattle 90K Chip (IAMARTINO D, NICOLAZZI E L, VAN TASSELL C P, et al. Designation and differentiation of a 90K SNP genetic analysis for the water buffering (Bubalus libraries.) ploS, 2017,12(10): E0185220.), sheep Illumina 50K Chip (BOLORMAA S, GORE K, VAN DER WERF J H, et al. Designation of a low-density SNP Chip for the main animal skin tissue and tissue expression on genetic prediction and genetic prediction access Generation sequencing technology PLoS One,2009,4(8): e6524.), chicken 600K Affymetrix high density chip (KRANIS A, GHEYAS A, BOSCHIERO C, et al.development of a high diversity 600K SNP genetic analysis for chicken. BMC genomes, 2013,14(1):59.), has been widely used for large-scale commercial breeding, with specific applications including germplasm genetic diversity analysis, genetic and evolutionary analysis, genetic relationship identification, genome-wide association analysis and genome selection.
However, the existing solid-phase gene chip, for example, the sheep Illumina 50K chip, has the following problems in the detection process and application: firstly, the number of typing sites is large; secondly, the sheep Illumina 50K solid-phase chip can only type the SNP sites contained on the chip, and the SNP sites around the sites cannot be typed; finally, solid phase chip typing costs are high.
Disclosure of Invention
The invention aims to provide a sheep liquid phase chip and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a sheep whole genome chip, the genotyping object of the chip comprises 935 SNP sites: 1) 903 SNP loci (specifically consisting of 886 newly-found SNP loci and 17 known SNP loci) positioned on a sheep reference genome Oar4.0 can provide SNP molecular marker combinations for positioning, genetic diversity analysis, whole genome association analysis and genome selection of various sheep variety trait related genes at home and abroad, variety identification, genetic relationship identification, germplasm resource improvement and protection; 2) 26 SNP sites located on the sheep Y chromosome (NCBI accession number: CM022046.1) to enable the chip to determine the sex of the sample to be tested, or to perform secondary confirmation on the sex; 3) 6 SNP sites located on the genome of Brucella melitensis (Brucella melitensis) are identified (the involved NCBI accession numbers are: CP044341.1, CP044342.1, CP044343.1) so that the chip can simultaneously diagnose the disease in sheep. The coordinates of the 935 SNP sites on the genome are shown in Table 4.
Preferably, the chip is a liquid phase chip.
Preferably, the molecular marker is associated with a major economic trait in sheep, said economic trait relating to reproduction, growth, immunity, fat deposition, milk production, thoracic vertebrae number, tail type, tail length, tail fat, horn type, wool type, and the like.
Preferably, the detection method of the molecular marker is a liquid phase chip-based SNP site typing method, for example, a targeted capture sequencing technology.
The invention has the beneficial effects that:
according to the invention, sheep key functional sites and variety specific sites are mined from large-scale sequencing data, 935 SNP sites (namely about 1K) which can be used for chip design are discovered and screened, 886 SNP sites are newly discovered, genotyping can be realized by utilizing the designed chip, and the method has high application value in multiple fields in sheep breeding.
Furthermore, the sheep liquid phase chip related by the invention can not only carry out typing on the target site, but also accurately type the SNP in a certain range around the target site based on the targeted capture sequencing technology, thereby obtaining more SNP typing information than the marker site. Compared with the traditional solid phase chip, the flexibility is higher, and the labeling sites can be added at any time according to the application requirements; meanwhile, the liquid phase chip is based on a second-generation sequencing platform, so that the typing cost is low, and a technical means is provided for large-scale typing.
Drawings
FIG. 1 shows the distribution of 935 SNP sites of the sheep 1K liquid phase chip in the example (the number of SNPs included in each 1M window on the genome).
FIG. 2 is a Manhattan diagram of sheep tail length trait whole genome association analysis.
Detailed Description
The present invention is further described in detail below with reference to the drawings and examples, which are only used for explaining the present invention and not for limiting the scope of the present invention.
Design and preparation of sheep 1K liquid phase chip
According to the invention, by using the re-sequencing data (434 Chinese sheep are involved) of 551 sheep (table 1) and 36 wild closely related sheep (table 2) of 64 varieties in the world, SNP analysis is firstly carried out, and 99406384 SNP sites are obtained for subsequent screening after the site is filtered.
TABLE 1 sheep breed List used
TABLE 2 sample List of sheep used
The target SNP sites screened by the invention comprise the following types: chinese sheep selected region SNP, European sheep selected region SNP, Hu sheep selected region SNP, sheep domestication related region SNP, sheep variety specific SNP, important function gene related locus, sheep Y chromosome SNP locus, and sheep Brucella genome conservative locus. The specific screening process for these classes of sites is as follows:
1. screening Chinese sheep selected areas, European sheep selected areas and Hu sheep selected areas: population fixation index (Fst) and nucleic acid diversity (theta) between Chinese sheep and wild sheep, between European sheep and wild sheep, and between Hu sheep and Mongolian sheep were calculated in a 50K window, 25K step size, respectively, over the whole genomeπ) Ratio, genome-wide Fst and θπThe intersection is taken from the window with the highest ratio of the first 1 percent and respectively used as the selection areas of Chinese sheep, European sheep and Hu sheep.
2. Screening sheep domestication related areas and sheep breed specific areas: the sheep and sheep body fixed index (Fst) and the nucleic acid diversity (theta) reported in the literatureπ) Areas with high ratio, and various sheep varieties Fst and theta in domestication related areasπRegions with high ratios (LI X, YANG J, SHEN M, et al. white-genome sequencing of wild and domestic sheep identities genes associated with pathological and agronomic traits. Nat Commun,2020,11(1):2815.) were used as sheep domestication related and sheep breed specific regions, respectively.
3. Merging intervals: and merging the selected areas screened out by adopting the bedtools software, and taking out a union set of all the intervals.
4. Screening SNP sites of each region: for SNPs in each selected region, the following filtering is performed using PLINK software (merging intervals in the previous step means merging intervals from different sources, that is, only a few overlapping intervals are merged into a large interval, and most of the original non-overlapping intervals remain unchanged): retaining sites with minimum allele frequency more than 0.1 (-maf 0.1), site deletion rate less than 0.1 (-geno 0.1) and Ha-Weinberg equilibrium test P value more than 0.001 (-hwe 0.001.001); and finally, calculating the linkage condition of the region by utilizing Haploview software, and taking the tag SNP (tag SNP) in the longest linkage block in each region as a candidate site of the region.
5. Determination of important functional sites: for important functional genes reported in the literature and related to economic traits such as sheep reproduction, growth, immunity, fat deposition, milk production, thoracic vertebrae number, tail type, tail fat, horn type, wool type and the like, at least one candidate SNP site was screened on each exon of these genes (Table 3).
TABLE 3 important functional genes and number of sites
6. Screening of sheep Y chromosome locus: the invention includes 26 SNP sites on the recently released sheep Y chromosome (LI R, YANG P, LI M, et al. A Hu sheet genome with the first ovine Y chromosome genetic endogenous restriction of science China Life Sciences,2020.) for which sites they are added directly to the candidate SNP list.
7. Determination of genome site of Brucella melitensis: the sequence of VirB12 gene of Brucella melitensis was obtained from the relevant patent (for example, Chinese patent 201210190050.1), blast was performed on NCBI (https:// blast. NCBI. nlm. nih. gov/blast. cgi) using nt/nr library as the target database, and 6 sites on two strains of Brucella melitensis (Brucella melitensis str. M1981, Brucella melitensis str. RM57) were selected as candidate sites among the results.
Removing repeated sites from all the candidate sites, submitting the candidate sites to Shizhuang Boridii biotechnology Limited for evaluation, removing sites which cannot be uniquely compared on the genome, removing sites containing repeated sequences in flanking sequences, then evaluating adjacent distances of the sites again, removing sites with the adjacent site distance being less than 100bp, and finally obtaining 935 SNP sites (shown in figure 1) which are uniformly distributed on the sheep genome and the Brucella melitensis genome, wherein the coordinates of the SNP sites on the genome are shown in Table 4.
TABLE 4 SNP site IDs (NO. 001-NO. 935) and genomic POSITIONs (POSITION)
According to the positions of the 935 SNP sites and sequences on two sides of the SNP sites, primers are designed and probe synthesis is carried out by adopting a targeted capture sequencing technology by Shijiazhuang Boridi biotechnology Limited, so that the sheep 1K liquid phase chip is obtained.
(II) flow for detecting sheep DNA sample by using sheep 1K liquid phase chip
Extraction of sheep genomic DNA: blood was collected from the jugular vein of sheep, and DNA was extracted using phenol chloroform method or a blood genome extraction kit (tiangen biotechnology limited, beijing).
And (3) detecting the quality of the DNA sample: agarose gel electrophoresis with the mass fraction of 1-1.5% is used for detection, and a gel imaging system (GelDocXRSystem, American Bio-Rad company) is used for judging the electrophoresis result so as to ensure the integrity of the genome; the concentration of the genomic DNA is measured with a micro-UV spectrophotometer (Q5000, Quawell, USA) or a similar nucleic acid protein analyzer, and the concentration of the DNA is adjusted to a working concentration of 10-50 ng/. mu.L.
Detection of a sheep liquid phase chip: the procedure was followed according to the standard protocol for sheep 1K liquid phase chip detection (http:// www.molbree ding. com/index. php/Technology/genoBaits. html).
And (3) data analysis: the raw data obtained were quality-controlled using the fastp software (CHEN S, ZHOU Y, CHEN Y, et al. fastp: an ultra-fast all-in-one FASTQ prediction. bioinformatics,2018,34(17): i884-i90.), after which the sequencing data were mapped to the Sheep reference genome Oar4.0(International Sheep genome Consortium, Archibald A L, Cockett N E, et al. the Sheep genome mapping: a work in stress prediction, analysis gene, GCS 5. genome, and DNA genome of Sheep (III. genome, GCS 5. genome, III. and III. genome, III. A. and III. Bion. prediction: BWA software (LI H. Aligning sequences and analysis sequences with BWA. III. prediction: 360. III. genome of Sheep), GCF-008761615.1), SNP was detected and genotyped using the GATK software (VAN DER AUWER G A, CARNEIRO M O, HARTL C, et al. from fast Q data to high-confidence variant diameters: the genome analysis toolkit best practices peptides. Current protocols in biologics, 2013,43(1):11.0.1-. 0.33.).
Application of (III) sheep 1K liquid phase chip in sheep whole genome correlation analysis
Blood samples of 323 Hu sheep and Dongfrui sheep crossed F2 generation (Dong Hu sheep) with tail length (tail length) phenotype record (collected in Dai sheep base of Yuan Sheng farming science and technology Limited, Jinchang, Gansu, 10 months, 2020) were collected and genotyped using sheep 1K liquid chip (the specific typing method refers to the second section above). And (3) performing quality control on the obtained genotyping result, and removing individuals with the minimum allele frequency of less than 0.05, the genotype deletion rate of more than 0.1 and the sample deletion rate of more than 0.1 to finally obtain 3160 SNP markers and 317 individuals. And then, carrying out whole genome association analysis on the SNP sites obtained by screening and collected Donghu sheep tail length character data, and analyzing by adopting a linear regression model of PLINK software, wherein the marker sites which are obviously related to the Donghu sheep tail length characters are 4 marker sites comprising NO.504, NO.511, NO.526 and NO.527 and the surrounding SNP sites thereof, and respectively correspond to 4 positions comprising 11:26376293, 11:27804260, 11:42057024 and 11:42449398 and the surrounding region thereof on the sheep reference genome Oar4.0 (FIG. 2), and identifying genes such as ALOX12, HSD17B1 and RPL27 through annotation. ALOX gene polymorphism is related to osteoporosis, HSD17B1 is related to sheep placental hormone content, and affects fetal development, and the two genes are presumed to be possibly related to growth and development of sheep caudal vertebrae, and a specific regulation and control path needs more experiments to prove. The results show that even if a liquid phase chip with the extremely low density of less than 1K of the marker sites is adopted, on the basis of the designed chip (935 SNPs), the targeted capture sequencing is combined (more SNPs than the marker sites can be obtained without filling), and more accurate whole genome association analysis can be carried out. Compared with an Illumina 50K solid-phase chip of sheep, the sheep breeding work can be carried out at lower cost (the price of the 1K chip is only one fifth of that of the 50K chip).
Application of (IV) sheep 1K liquid chip in variety identification
When variety identification is performed on a sheep of unknown variety, firstly, blood samples of the sheep of known variety are collected, each variety is about 5, for example, according to the phenotype, the sheep to be detected is preliminarily judged to be Mongolian sheep, and then, 5 common Mongolian sheep varieties such as small tailed Han sheep, Wuzhu Muoqin sheep, Hu sheep, Tan sheep and the like need to be collected, and together with the sheep to be detected, a sheep 1K liquid phase chip is adopted to perform genotyping (the specific genotyping method refers to the second part above), so as to obtain an SNP site genotyping result set. And then, respectively carrying out Principal Component Analysis (PCA) and phylogenetic tree construction on the SNP typing result sets containing all the samples, and analyzing to obtain the sheep samples to be detected which are closer to the sheep of known varieties according to the clustering results of the first 3 principal components of the principal component analysis and the clustering results of the phylogenetic tree, namely judging the most possible varieties of the sheep to be detected.
(V) advantages of sheep 1K liquid phase chip
(1) Compared with the traditional solid phase chip, the invention can detect more SNP sites (4-6K) compared with the solid phase chip with the same number of probes. And the design is flexible, and interested marker sites can be added at any time in the later period.
(2) Compared with whole genome re-sequencing, the method has obvious price advantage, can be used for carrying out large-scale typing on sheep, and further promotes the breeding work of sheep.
(3) The invention contains a large number of sheep functional gene related sites, screens sites which are obviously related to properties such as reproduction, growth, immunity, fat deposition, milk production, thoracic vertebra number, tail type, tail fat, horn type, wool type and the like in the prior research, and increases the accuracy of the chip for basic research.
(4) The sample for designing the chip is from 551 of 64 varieties and 36 wild closely related species of sheep (wild sheep) all over the world, and has wide source and strong representativeness; and 434 Chinese sheep are included, namely, the sheep has more medium-high frequency SNP sites specific to Chinese local sheep varieties, is more suitable for the research of local sheep varieties, and is beneficial to the development of sheep breeding work and the research and protection of sheep germplasm resources.
Claims (10)
1. A sheep whole genome chip is characterized in that: the genotyping object of the chip comprises 886 SNP sites positioned on an sheep reference genome Oar4.0, and the positions of the SNP sites on the sheep reference genome are shown as items No.001 to No.119, No.133 to No.276, No.278, No.279, No.281 to No.312, No.314 to No.383, and No.385 to No. 903:
2. the sheep whole genome chip according to claim 1, wherein: the genotyping subjects of the chip also included one or more of the following 17 SNP sites located on the sheep reference genome oar 4.0: 2:118139291, 2:118139461, 2:118140379, 2:118141033, 2:118141972, 2:118142779, 2:118143163, 2:118144455, 2:118144976, 2:118145242, 2:118145493, 2:118145865, 2:118146119, 5:41768295, 5:41769002, 6:29315643, 7: 82534266.
3. The sheep whole genome chip according to claim 1, wherein: the genotyping objects of the chip also include one or more of the following 26 SNP sites located on the sheep Y chromosome: 168746, 265762, 317875, 466415, 473584, 507978, 535483, 565059, 601505, 651655, 671991, 681626, 683923, 753343, 808947, 850541, 990163, 1109484, 1341331, 1354321, 1383825, 1385068, 9001773, 9003921, 9009804 and 9018538.
4. The sheep whole genome chip according to claim 1, wherein: the genotyping objects of the chip also include one or more of the following 6 SNP sites located on the Brucella melitensis genome: CP044341.1:421316, CP044341.1:755521, CP044342.1:793466, CP044342.1:793758, CP044342.1:1186334 and CP044343.1: 896766.
5. The sheep whole genome chip according to claim 1, wherein: the chip is a liquid phase chip.
6. The use of the sheep whole genome chip as claimed in any one of claims 1 to 5 in sheep breeding.
7. The sheep whole genome chip as claimed in any one of claims 1 to 5, for use in sheep genome selection, sheep trait related gene localization, sheep genetic diversity analysis, sheep whole genome association analysis, sheep variety identification, sheep genetic relationship identification, or sheep germplasm resource improvement and protection.
8. The application of the sheep genome molecular marker and the detection method thereof in sheep breeding is characterized in that: the molecular marker is positioned at one or more of 886 SNP loci on an sheep reference genome Oar4.0, wherein the positions of the 886 SNP loci on the sheep reference genome are shown as items No.001 to No.119, No.133 to No.276, No.278, No.279, No.281 to No.312, No.314 to No.383, and No.385 to No. 903:
9. use according to claim 8, characterized in that: the detection method is an SNP locus typing method based on a liquid phase chip.
10. Use according to claim 8, characterized in that: among the 886 SNP sites, the molecular marker sites which are significantly related to the long shape of the tail of the east lake sheep comprise 11:26376293, 11:27804260, 11:42057024 and 11: 42449398.
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| CN113699253A (en) * | 2021-08-26 | 2021-11-26 | 青岛市畜牧工作站(青岛市畜牧兽医研究所) | Laoshan milk goat low-density liquid-phase SNP chip and application thereof |
| CN114736985A (en) * | 2022-04-21 | 2022-07-12 | 河南七度农业科技有限公司 | Tomato whole genome chip and application thereof |
| CN115232880A (en) * | 2022-05-16 | 2022-10-25 | 海南大学 | Hainan black goat liquid phase chip and application thereof |
| CN115232880B (en) * | 2022-05-16 | 2023-07-04 | 海南大学 | Hainan black goat liquid-phase chip and application thereof |
| GB2626838A (en) * | 2023-02-02 | 2024-08-07 | Univ Qingdao Agricultural | Donkey 40K liquid chip based on targeted sequencing and application thereof |
| CN115992265A (en) * | 2023-03-22 | 2023-04-21 | 中山大学 | Grouper whole genome liquid phase chip and application thereof |
| CN117051131A (en) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to sheep brucellosis resistance character, detection primer and application thereof |
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| CN118389706A (en) * | 2024-06-24 | 2024-07-26 | 中国水产科学研究院珠江水产研究所 | Marker combination for mandarin fish genotyping and whole genome liquid phase chip using same |
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| CN115029451B (en) | 2023-09-19 |
| CN115029451A (en) | 2022-09-09 |
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