Sika deer whole-genome high-density SNP (Single Nucleotide Polymorphism) breeding chip and application thereof
By developing a sika deer genome 100K high-density SNP breeding chip, using targeted capture sequencing technology to screen and design SNP sites, the technical problem that existing chips cannot meet the whole genome selection is solved, and high-throughput and low-cost gene segmentation are achieved and breeding applications significantly improve breeding efficiency and industrial benefits.
Patent Information
- Application Number
- CN202411562683.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2024-12-17
AI Technical Summary
The existing sika deer 40K liquid-phase SNP chip cannot meet the requirements of whole genome selection and breeding. The site design only comes from the Northeast region, and the sample resequencing depth is low, which cannot meet the high-throughput, low-cost genotyping and whole genome selection of Sika deer demand.
Developed a sika deer whole genome 100K high-density SNP breeding chip based on targeted capture sequencing technology. By screening the whole genome resequencing data of 196 individuals, a chip containing 100,000 SNP sites was designed, and the probe design was modified with biotin groups. DNA nucleoglycerol sequence covers the chromosome group, has strong representativeness, high polymorphism and high specificity, and is suitable for high-throughput genotyping, gene localization and genome-wide selection.
Provides a wider genetic background coverage, higher density and flexibility, and can quickly detect relevant SNP markers at low cost, significantly improves the breeding and selection process of sika deer, meets the application needs of whole genome selection, and improves breeding efficiency and industry. income.
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Figure CN119144734A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of gene chip technology and gene molecular breeding technology, and specifically relates to a sika deer whole genome high-density SNP breeding chip and application thereof. Background Art
[0002] With the development of genetics and high-throughput sequencing technology, molecular markers, especially SNPs, have been widely used in a variety of plants and animals. Gene chips composed of dense whole-genome SNPs have become a valuable tool for analyzing genomic information. One of the main advantages of SNP chip technology is that it can obtain more information than earlier methods at a relatively low cost. In addition, it has accelerated the development and application of genomic selection in animal and plant breeding, bringing revolutionary changes to the animal genetics industry and research, and has brought great changes to livestock and poultry breeding.
[0003] Sika deer ranks first among special livestock and poultry. It is one of the deer species with important economic and cultural value, and its whole body is a treasure. my country is a country with abundant sika deer resources and breeding power, and it is also the world's largest consumer and importer of deer products. In 2020, sika deer was included in the "National Catalogue of Livestock and Poultry Genetic Resources" for the first time, becoming one of the 33 livestock and poultry species. However, there is a lot of room for technological improvement in the entire industrial chain of my country's sika deer industry from breeding to product development. In particular, the sika deer pedigree is confusing and the breeding technology remains at the phenotypic selection stage, which seriously restricts the development of the sika deer industry. In order to better promote the innovation of the sika deer seed industry and the sustainable and healthy development of the industry, make full use of modern molecular breeding technology, accelerate the improvement of the production performance of my country's sika deer, increase industry benefits and reduce breeding costs, it is urgent to carry out sika deer genome selection breeding.
[0004] Whole genome selection is based on genomic breeding value (GEBV). By detecting molecular markers covering the whole genome, genetic information at the genome level is used to perform genetic evaluation on individuals to obtain higher breeding value estimation accuracy. Selecting traits that are difficult to measure in the early stage shortens the generation interval and accelerates the breeding process, thereby saving a lot of breeding costs. Whole genome selection breeding requires the selection of high-density SNP chips, and medium and low-density chips cannot meet the requirements. Therefore, the existing sika deer 40K liquid phase SNP chip in the field cannot meet the requirements of whole genome selection breeding. Its site design is only derived from sika deer in Northeast China, and the sample resequencing depth is low. Therefore, it is urgent to develop a sika deer whole genome breeding chip with a wider genetic background and higher density to meet the application of sika deer whole genome selection. Summary of the invention
[0005] In order to address the deficiencies in the above-mentioned prior art, and to meet the needs of high-throughput and low-cost genotyping of sika deer, and the application of whole genome selection of sika deer, as well as large-scale commercial breeding, the present invention provides a sika deer whole genome 100K high-density SNP breeding chip developed based on targeted capture sequencing technology. The SNP sites used evenly cover the chromosome set, and have strong representativeness, high polymorphism, high specificity, and strong versatility, providing an indispensable and important tool for molecular breeding research such as high-throughput genotyping, gene positioning, fingerprint mapping, whole genome selection, etc. of sika deer.
[0006] The present invention provides the following technical solutions:
[0007] In a first aspect, the present invention provides a whole-genome SNP molecular marker combination for sika deer, wherein the whole-genome SNP molecular marker combination comprises 100,000 SNP molecular markers, and the positions of the site information of the 100,000 SNP molecular markers in the sika deer reference genome MHL_v1.0 are shown in Table 1.
[0008] In a second aspect, the present invention provides a sika deer whole genome SNP targeted capture probe, comprising the 100,000 SNP molecular marker site probes, wherein the probe is a DNA nucleotide sequence modified with a biotin group at the 5' end synthesized based on the SNP molecular marker.
[0009] In a third aspect, the present invention provides a sika deer whole genome 100K high-density SNP chip, wherein the chip comprises the sika deer whole genome SNP targeted capture probe.
[0010] Preferably, the chip is a liquid phase chip.
[0011] Furthermore, the design and preparation method of the sika deer whole genome 100K SNP liquid phase chip of the present invention is as follows:
[0012] 1. Screening of 100K SNP loci in sika deer
[0013] The 100K SNP loci of sika deer were obtained by screening using the following method:
[0014] 1. Based on the whole genome resequencing data of 196 individuals, the data were aligned to the sika deer reference genome (MHL_v1.0) to construct a set of SNP sites on the genomes of all samples for the selection of target SNP sites;
[0015] 2. The SNP site set was screened under the following conditions: (1) dimorphic sites with missing rate < 0.1; (2) no tandem sequences adjacent to the target site; (3) conforming to the Hadwin equilibrium; (4) minimal allele frequency MAF > 0.05; (5) nucleotide polymorphism pi ≥ 0.25; (6) high specificity of each 60 bp flanking the target site, with no more than 7 SNPs within the 120 bp flanking range.
[0016] 2. Design and preparation of 100K SNP locus probes for sika deer
[0017] The design and synthesis method of the sika deer whole genome SNP targeted capture probe is:
[0018] The candidate sites to be selected are typed and checked, the ref bases are compared with the reference genome, duplicates are removed, and the correctly typed sites are retained for probe design and scoring. The site set is submitted to the probe design system for scoring, and the probe is designed according to the evaluation results of the upstream and downstream sequences of the target site. The main evaluation items include the complexity of the upstream and downstream sequences of the target site, the target site is in the middle position of the probe, the GC content of the probe is 20%-80%, the Tm value is 60-80℃, the number of comparisons on the reference genome (≤5), and other comprehensive evaluations. The probe length is 120bp, and the 5' end is modified with a biotin group DNA nucleotide sequence.
[0019] The present invention also provides applications of the sika deer whole genome 100K SNP chip, which includes but is not limited to: (1) sika deer SNP site typing; (2) sika deer genetic background analysis; (3) sika deer molecular genetic map construction; (4) sika deer whole genome association analysis; (5) sika deer variety identification; (6) sika deer molecular marker-assisted selection breeding; and (7) sika deer whole genome selection breeding.
[0020] The present invention also provides a kit for detecting the trait of saw-antler weight of sika deer, and the kit comprises a 100K high-density SNP chip of the whole genome of sika deer.
[0021] The applications of the sika deer antler weight trait detection kit include but are not limited to: (1) application in sika deer variety screening; (2) application in sika deer variety identification; (3) application in sika deer breeding; (4) application in sika deer germplasm resource protection; and (5) application in sika deer germplasm resource improvement.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] 1. The SNP marker combination of the present invention has strong representativeness, high polymorphism, high specificity and strong versatility.
[0024] The liquid phase chip described in the present invention is a whole genome resequencing of 196 sika deer individuals from 8 sika deer subspecies at home and abroad: Northeast subspecies, Taiwan subspecies, South China subspecies, Sichuan subspecies, Hokkaido subspecies, Honshu subspecies, Yakushima subspecies, and nominate subspecies and 7 sika deer breeds (strains): Dongda sika deer, Shuangyang sika deer, Dongfeng sika deer, Siping sika deer, Xingkai Lake sika deer, Changbai Mountain sika deer, and Aodong sika deer. It has a wide range of site sources, full variety coverage, and wide variety applicability, and has relatively rich polymorphism in the sika deer population.
[0025] 2. The liquid phase chip described in the present invention is based on targeted capture sequencing technology, which can not only type the target site, but also type the SNPs within a certain range near the target site, so more typing information can be obtained.
[0026] 3. The 100K high-density SNP liquid phase chip of the present invention can adjust the target SNP site by directly adding or removing probes, and has better flexibility compared with the solid phase chip.
[0027] 4. The liquid phase chip described in the present invention can detect relevant SNP markers quickly and at low cost, which is of great significance in the breeding and selection of sika deer. It makes it possible to popularize the whole genome selection molecular breeding of sika deer through the 100K high-density SNP liquid phase chip of the whole genome of sika deer, which will greatly improve the breeding and selection process of sika deer in my country. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is the distribution map of the SNP sites of the present invention in Example 1 on the chromosomes of the sika deer genome.
[0029] Figure 2 This is the MAF distribution diagram of the SNP site of the present invention in Example 1.
[0030] Figure 3 This is the SNP site mutation frequency distribution diagram of Example 1 of the present invention.
[0031] Figure 4 This is a statistical chart of the site detection of the test samples in Example 3. Note: The horizontal axis is the sample name, and the vertical axis is the number of SNP detections and the detection rate in different samples.
[0032] Figure 5 Example 3 Repeat sample typing consistency rate.
[0033] Figure 6 Example 4 Phylogenetic tree analysis results of four varieties.
[0034] Figure 7 Example 4 PCA analysis results.
[0035] Figure 8 Example 5 Line graph of percentiles of weight of different sawdust varieties.
[0036] Fig. 9 Example 5 Results of genome-wide association analysis. DETAILED DESCRIPTION
[0037] The embodiments of the technical solution of the present invention are described in detail below. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs.
[0038] Example 1 Design and preparation of sika deer 100K SNP liquid phase chip
[0039] 1. Establishment of SNP background database of sika deer
[0040] The present invention utilizes the whole genome resequencing data of 196 individuals from 8 sika deer subspecies and 7 sika deer breeds (lines) at home and abroad, and obtains highly reliable SNP sites for subsequent screening through sequencing analysis.
[0041] S1. The experimental population was derived from 196 sika deer individuals from 8 sika deer subspecies at home and abroad: Northeast subspecies, Taiwan subspecies, South China subspecies, Sichuan subspecies, Hokkaido subspecies, Honshu subspecies, Yakushima subspecies, and nominate subspecies and 7 sika deer breeds (lines): Dongda sika deer, Shuangyang sika deer, Dongfeng sika deer, Siping sika deer, Xingkai Lake sika deer, Changbai Mountain sika deer, and Aodong sika deer (Table 2). First, blood was collected from 196 individuals, whole genome DNA was extracted, and whole genome resequencing was performed. The average genome coverage of each sample was nearly 30X. The raw data obtained by sequencing was quality controlled to obtain high-quality clean reads, which were then compared with the reference genome (MHL_v1.0).
[0042] S2. The initial SNP site set was screened under the following conditions: (1) dimorphic sites with missing rate < 0.1, 31,320,156 sites remaining; (2) no tandem sequence adjacent to the target site, 30,819,938 sites remaining; (3) conforming to Hadwinberg equilibrium, 30,388,507 sites remaining; (4) minimal allele frequency MAF> 0.05, 22,752,771 sites remaining; (5) nucleotide polymorphism, pi ≥ 0.25, 13,288,503 sites remaining; (6) high specificity of each 60 bp flanking the target site, no more than 7 SNPs within the 120 bp flanking range, 11,543,014 sites remaining. The remaining sites were retained for subsequent analysis.
[0043] 2. Screening of background sites
[0044] Based on the remaining sites in the resequencing background database, 115K sites were retained according to the principle that the selected SNP sites were evenly distributed in the physical distance of the chromosomes on the genome.
[0045] The 115K sites obtained by the above steps were scored, and the sites were selected based on the scoring results and the priority sliding window 1x, and finally 100K sites remained. The specific genomic coordinates of the 100K sites obtained are shown in Table 1. The 100,000 SNP molecular markers in Table 1 are in the database https: / / ngdc.cncb.ac.cn / National Genome Science Data Center. The distribution of all sites in the chromosomes of the sika deer genome can be found in Figure 1 As shown; the MAF distribution of sites is shown in Figure 2 The mutation frequency distribution of the sites is shown in Figure 3 According to the location of 100,000 SNP sites and the sequence information on both sides, primers were designed and probes were synthesized using targeted capture sequencing technology to obtain the sika deer 100K SNP liquid phase chip.
[0046] Specific method: perform typing check on the candidate sites to be selected, compare the ref base with the reference genome, remove duplicates, retain the correctly typed sites for probe design and scoring. Submit the site set to the Compson probe design system for scoring, and design probes based on the evaluation results of the upstream and downstream sequences of the target site. The complexity of the upstream and downstream sequences of the target site, the GC content, etc. are mainly evaluated. The target site is placed in the middle of the probe, and the designed probe is 120bp in length. Generally, the performance of the probe is comprehensively evaluated from the aspects of GC content (20%-80%), Tm value (60-80℃), and the number of alignments on the reference genome (≤5). According to the design results, a total of 100K high-quality chip customized sites were selected. After the chip site is confirmed to be correct, it is submitted to Compson for chip probe synthesis. The probe length is 120bp, and the 5' end is modified with a DNA nucleotide sequence with a biotin group.
[0047] Table 1 Positions of 100,000 SNP molecular markers
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[0252] Table 2. Sources of chip site data
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[0254] Example 2 Process of detecting sika deer DNA samples using 100K liquid-phase breeding chip for the whole genome of sika deer
[0255] 1. Sample collection and DNA extraction
[0256] The chip detection sample can be a sika deer jugular vein blood or tissue sample, etc., and the sika deer genomic DNA can be extracted using a genomic DNA extraction kit.
[0257] 2. DNA quality inspection
[0258] Detection of genome integrity by agarose gel electrophoresis: Take the DNA sample to be tested, spot it on 0.8% agarose gel, and perform electrophoresis at 170V for 25 minutes.
[0259] NanoDrop detection of DNA purity: The purity of DNA was detected using NanoDrop2000 nucleic acid protein analyzer. The A260 / A280 ratio was in the range of 1.7-2.1, and the A260 / A230 ratio was between 1.8-2.2, which met the requirements for library construction.
[0260] 3. Construction of Sika Deer DNA Library and Chip Detection
[0261] The genomic DNA samples of sika deer are used to prepare library samples according to the high-throughput library construction reaction kit process. After quality control experiments, the library samples can be used for chip detection, hybridization with chip probes and samples, capture target site regions, sequence on the machine and complete basic bioinformatics analysis, complete target interval variation information analysis of the samples to be tested, and perform genotyping.
[0262] Example 3 Quality evaluation of sika deer 100K whole genome breeding chip genotyping
[0263] According to the steps of Example 2, 32 sika deer were subjected to genotyping detection using the sika deer 100K whole genome breeding chip provided by the present invention, and the following quality evaluation was performed on them.
[0264] 1. Detection rate
[0265] In this example, the capture efficiency of the sample sites of the 32 sika deer tested was between 95.77% and 97.67%, and the average sample detection rate reached 97.23%. Figure 4 This indicates that the sika deer 100K whole genome breeding chip sample detection effect of the present invention is good.
[0266] 2. Genotyping stability
[0267] Three randomly selected sika deer samples were tested and typed twice independently, showing an average typing consistency of 99.51%. Figure 5 This indicates that the sika deer 100K whole genome breeding chip of the present invention has good genotyping stability.
[0268] Example 4 Application of the 100K Sika Deer Whole Genome Breeding Chip in Genetic Background Analysis of Sika Deer
[0269] In this example, 843 sika deer DNA samples (including four large-scale sika deer breeding farms: Aodong 133, Siping 276, Tonghua 337, Zuojia 97) were tested using the sika deer 100K whole genome breeding chip, and the phylogenetic tree and principal component analysis (PCA) were performed on the obtained liquid phase chip target SNP analysis results. The clustering results of the phylogenetic tree are shown in Figure 1. Figure 6 The clustering results of principal component analysis are shown in Figure 7 As shown, the liquid phase chip of the present invention can distinguish the population structure of four species of Northeastern Sika deer, which shows that the chip of the present invention has the function of identifying and distinguishing the species of Sika deer.
[0270] Example 5 Application of the 100K whole genome breeding chip for sika deer in whole genome association analysis of important economic traits of sika deer
[0271] In this example, 98 male sika deer DNA samples were genotyped using the sika deer 100K whole genome breeding chip. A total of 98 individuals were analyzed based on 1-6 saws, and samples with 3-5 saws accounting for the top 25% of all samples were selected as high-yield samples, and the bottom 20% of samples were selected as low-yield samples. The line chart of antler weight percentiles of different saw types is shown in Figure 8 .
[0272] Finally, 14 high-yield and 14 low-yield samples were subjected to genome-wide association analysis. Fig. 9 As shown, the SNP loci significantly associated with the important economic trait velvet weight were found, which shows that the use of the chip of the present invention for genotype detection can obtain relatively accurate genome-wide association analysis results. The SNP heritability was estimated using GCTA software, h2 = V (G) / Vp = 0.684342. It was proved that almost 70% of the variation of the phenotype (velvet weight) was attributed to genetics, which illustrates the importance of genomic selection breeding. The present invention can provide more possibilities for genomic selection breeding.
[0273] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A genome-wide SNP molecular marker combination of sika deer, characterized in that: The whole genome SNP molecular marker combination includes 100,000 SNP molecular markers, and the positions of the site information of the 100,000 SNP molecular markers in the reference genome MHL_v1.0 are shown in Table 1 of the specification.
2. A sika deer whole genome SNP targeted capture probe, characterized in that: The method comprises the 100,000 SNP molecular marker site probes as claimed in claim 1, wherein the probes are DNA nucleotide sequences with a biotin group modified at the 5' end synthesized according to the SNP molecular markers.
3. A sika deer whole genome 100K high-density SNP chip, characterized in that: The chip comprises the sika deer whole genome SNP targeted capture probe according to claim 2.
4. The sika deer whole genome 100K high-density SNP chip according to claim 3, characterized in that: The chip is a liquid phase chip.
5. A method for designing and preparing a 100K high-density SNP chip for the whole genome of sika deer, characterized in that: Here are the steps: Screening of 100K SNP loci in sika deer 1. Based on the whole genome resequencing data of 196 individuals, the SNP sites on the genomes of all samples were detected by comparing them with the sika deer reference genome MHL_v1.0 for the selection of target SNP sites; 2. The alignment results were screened according to the following conditions: (1) missing rate of dimorphic sites < 0.1; (2) no tandem sequences adjacent to the target site; (3) conforming to Hardweinberg equilibrium; (4) minimum allele frequency MAF > 0.05; (5) nucleotide polymorphism, pi ≥ 0.25; (6) high specificity of each 60 bp flanking the target site, with no more than 7 SNPs within the 120 bp flanking range; 2. Design and prepare sika deer 100K SNP targeted capture probe; The candidate sites to be selected are typed and checked, the ref bases are compared with the reference genome, duplicates are removed, and the correctly typed sites are retained for probe design and scoring; Probe design is performed based on the evaluation results of the upstream and downstream sequences of the target site.
6. The method for designing and preparing the 100K high-density SNP chip of the whole genome of sika deer according to claim 5, characterized in that: The main evaluation items for the probe design scoring include the complexity of the upstream and downstream sequences of the target site, the target site being in the middle of the probe, the GC content of the probe being 20%-80%, the Tm value being 60-80°C, the number of alignments on the reference genome, etc. Through comprehensive evaluation from multiple aspects, a DNA nucleotide sequence with a probe length of 120bp and a biotin group modified at the 5' end was obtained.
7. A kit for detecting the weight of antlers of sika deer, characterized in that: The kit comprises the sika deer whole genome 100K high-density SNP chip according to claim 3.
8. Use of the 100K high-density SNP chip of the whole genome of sika deer as described in claim 3 in any of the following applications: (1) SNP locus typing of sika deer; (2) genetic background analysis of sika deer; (3) construction of molecular genetic map of sika deer; (4) whole genome association analysis of sika deer; (5) identification of sika deer varieties; (6) molecular marker-assisted selection breeding of sika deer; (7) whole genome selection breeding of sika deer.
9. Use of the sika deer antler weight trait detection kit according to claim 7 in any of the following applications: (1) in the screening of sika deer varieties; (2) in the identification of sika deer varieties; (3) in the breeding of sika deer; (4) in the protection of sika deer germplasm resources; (5) in the improvement of sika deer germplasm resources.