Willow whole genome 40K SNP liquid phase chip and application thereof

Abstract

In order to solve the willow breeding problem with low cost, high efficiency and simple means, the willow whole genome 40K SNP liquid phase chip is provided, the willow whole genome 40K SNP liquid phase chip comprises a willow 40K site probe mixed solution and a hybridization capture reagent, the willow 40K site probe mixed solution comprises a willow SNP background site probe and a willow SNP associated site probe, and each site comprises at least two capture probes. The liquid-phase chip is used for willow genetic diversity analysis, molecular genetic map construction, whole genome association analysis, variety authenticity identification, molecular marker-assisted selective breeding and whole genome selective breeding, is applied to willow leaf detection tests, performs statistics on detection conditions, has 40188 loci, and can be used for detecting willow leaves. According to the method, the average detection rate of the original SNP sites of 16 samples is 97.39%, the site detection rate reaches 99.9% after data of the 16 samples are merged and counted, the service cycle of sample detection is greatly shortened, and the breeding efficiency of willows is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of whole genome gene chips, and relates to a willow whole genome gene chip, and specifically to a willow whole genome 40K SNP liquid phase chip and application thereof. Background Art

[0002] Genetic variation in genomic sequences, i.e. genetic markers, mainly includes single nucleotide polymorphism (SNP), microsatellite (Short Tandem Repeat, STR), copy number variation (CNV, Copy Number Variations), insertion / deletion (INDEL, insertion / deletion), structural variation (SV Structural Variation), etc. Among them, SNP is the most numerous (millions to tens of millions), the most widely distributed, the most important and the most commonly used genetic marker. SNP markers can cover the genome at high density and are low cost. SNP markers are heritable single base pair changes that occur in the DNA sequence of the genome of an organism. They occur very frequently in the human genome and are the most common genetic variation in an organism. In genetic research, SNPs are usually used as markers for genomic regions, and most of them have little effect on biological systems. However, SNPs may be functional and can cause amino acid changes, changes in RNA transcription stability, and changes in transcription factor binding affinity. Therefore, SNPs are widely used in agricultural crop and fruit tree genetic breeding due to their high density, strong specificity, good stability and easy analysis.

[0003] SNP chip technology is a high-throughput genotyping technology based on the principle of DNA hybridization. It detects the genotype of the SNP site by fixing specific probes on the surface of the chip and hybridizing with the DNA fragments in the sample to be tested. At present, the gene chip technology used for SNP site typing mainly includes solid phase chips and liquid phase chips. Solid phase chip is a chip technology based on the principle of solid phase hybridization. It fixes a series of specific DNA probes on the surface of the chip, and then hybridizes the DNA fragments in the sample to be tested with these probes to detect the SNP sites in the sample. Liquid phase chip is a chip technology based on the principle of liquid phase hybridization. It dissolves a series of specific DNA probes in the liquid phase, and then hybridizes the DNA fragments in the sample to be tested with these probes to detect the SNP sites in the sample.

[0004] Liquid-phase SNP chips have the advantages of low cost, high throughput, high sensitivity and high specificity. At the same time, they also have a wider range of applications and can be used in a variety of application scenarios such as molecular marker-assisted selection and whole genome selection. Based on resequencing technology, liquid-phase chips specifically capture each target site and perform high-depth resequencing, with the advantages of high detection accuracy, high throughput and low cost. It breaks through the technical bottlenecks of the current solid-phase custom chip platform, such as high cost, poor flexibility, and difficulty in large-scale application, and provides a non-model organism with an efficient and flexible targeted genotyping technology that is compatible with different flux levels and different marker types. Liquid-phase chips generally include a biotin-labeled probe covering the target SNP designed for each site to be tested based on the principle of DNA complementarity. These probes hybridize with the target region of the genome in the liquid to form a double strand. The adsorption of 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 status of the target site and its surrounding SNPs. Liquid phase chips currently have relatively mature applications in species evolution analysis, germplasm resource evaluation and DNA fingerprint identification, molecular genetic map construction, gene / QTL positioning and gene cloning, molecular marker-assisted selection, and whole genome selection.

[0005] Willow is an important economic forest tree species with important ecological and economic value. In recent years, with the rapid development of molecular biology technology, willow breeding has gradually entered the era of molecular breeding. As an efficient genotyping tool, SNP chip technology can provide accurate genetic information for willow breeding and accelerate the selection of excellent varieties. However, as far as reported, there are no mature genotyping chips available for willow in China. Therefore, in order to improve the efficiency of willow breeding, accelerate the breeding process of willow, and better serve the industrial application of willow trees, it is urgent to develop an economical and suitable genotyping product suitable for willow. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to apply the molecular biological method of improving germplasm resources to willow to obtain gene typing chips, so as to solve the problem of willow breeding with low cost, high efficiency and simple means.

[0007] The purpose of the present invention is to provide a willow whole genome 40K SNP liquid phase chip, which provides an important technical means for the research of willow economic traits, variety identification, whole genome selection breeding and marker-assisted breeding, and meets the needs of large-scale commercial breeding of willows and the promotion of varieties in different regions into forests.

[0008] Based on the above purpose, the present invention provides a willow whole genome 40K SNP liquid phase chip, including a willow 40K site probe mixture and a hybridization capture reagent, wherein the willow 40K site probe mixture includes a willow SNP background site probe and a willow SNP associated site probe, and each site has at least 2 capture probes.

[0009] Furthermore, the hybridization capture reagent is the GenoBaits DNA-seq Library Prep kit from Novogene Co., Ltd., including independently packaged GenoBaits Block I, GenoBaits Block II, GenoBaits 2×Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2×BeadsWashBuffer, GenoBaits 10×Wash Buffer I, GenoBaits 10XWash Buffer II, GenoBaits10×Wash Buffer III, and GenoBaits 10X Stringent Wash Buffer.

[0010] Furthermore, the willow SNP background site information and the base of the reference gene at the site are shown in Table 1:

[0011] Table 1 Information on the background SNP loci of willow and the bases of the reference genes at the loci

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[0169] The numbers / letters before the underline in the table represent the chromosome number, the following number group represents the position on the chromosome, and the last letter represents the base of the reference gene at that site.

[0170] Furthermore, the design process of the willow whole genome 40K locus probe described in the present invention includes:

[0171] (1) The probe length was 110 bp, the probe GC content was between 30% and 70%, the number of homology regions was ≤5, and the selected region did not contain SSR repeat sequence regions and genomic GAP (deletion) regions as much as possible;

[0172] (2) Design two nucleotide sequences with 60% to 70% overlap and covering the SNP sites based on the SNP sites obtained by screening;

[0173] (3) Single-stranded nucleotides were synthesized according to the designed nucleotide sequence. The two synthesized DNA nucleotide sequences were 110 bp in length and modified with a biotin group at the 5' end, which were called the willow genome 40K site probes;

[0174] (4) The two synthesized willow whole genome 40K site probes were mixed in equal molar mass, and the volume was fixed to 3 pmol / mL of the willow whole genome 40K site probe mixture using a mixture of EDTA and Tris HCl.

[0175] In addition, the present invention also provides the application of the willow whole genome 40K SNP liquid phase chip in willow breeding.

[0176] Furthermore, the willow whole genome 40K SNP liquid phase chip provided by the present invention is applied to willow genetic diversity analysis, molecular genetic map construction, whole genome association analysis, variety authenticity identification, molecular marker-assisted selection breeding, and whole genome selection breeding.

[0177] Compared with the prior art, the present invention has the following beneficial effects:

[0178] 1. A high-throughput whole-genome 40KSNP liquid phase chip for willow genotyping was developed based on targeted capture sequencing technology. Combined with the addition of associated site probes, it overcomes the high application cost of genetic diversity analysis, QTL positioning, and GWAS analysis of willow in scientific research, realizes willow genotyping, and effectively saves the sample testing service cycle, providing technical support for willow scientific research. The scientific research here includes but is not limited to willow genetic diversity analysis, molecular genetic map construction, whole-genome association analysis, variety authenticity identification, molecular marker-assisted selection breeding, whole-genome selection breeding, etc.

[0179] 2. The willow whole genome 40K SNP liquid phase chip realizes the complete localization of probe synthesis, sample testing, and detection reagents, thereby effectively avoiding the high cost of importing related equipment and reagents, reducing the risk of leakage of core material data, and avoiding trade frictions and many other inconveniences caused by sample testing.

[0180] 3. The willow whole genome 40K SNP liquid phase chip overcomes the defects of large amount of resequencing data, much useless information, and difficult analysis. On the premise of achieving the same results, it realizes advantages that are conducive to industrial development, such as rapid detection, cost reduction, and simple data analysis. In addition, compared with simplified genome sequencing (GBS), the willow whole genome 40K SNP liquid phase chip obtains high-density molecular markers representing the whole gene, avoiding the defects of excessive data volume in GBS analysis, and the difficulty in comparing and accumulating GBS marker data obtained between different materials, laboratories and platforms, which makes GBS data difficult to preserve and comprehensively utilize for a long time. The willow whole genome 40K SNP liquid phase chip has many advantages such as high consistency of data output, long-term use of data from the same material, and relatively simple data analysis.

[0181] 4. A high-throughput whole-genome 40KSNP liquid phase chip for willow genotyping was developed based on targeted capture sequencing technology. When designing the probe, the uniform distribution of the captured SNP sites in the whole genome and the polymorphism of the captured sites were taken into consideration. The minimum allele frequency (MAF) of the site was selected to be as large as possible from 0.05. At the same time, the site detection rate was taken into consideration, and SNP sites with a high missing rate were removed. At the same time, through the added SNP functional markers, genotyping can be achieved using the designed chip, which has great utilization value in willow breeding.

[0182] 5. Based on targeted capture sequencing technology, the probe has good tolerance for flanking sequences and can stably capture the target sequence when the flanking sequence variation is no more than 10%. In addition to obtaining target SNP information, it can also obtain 100bp of sequence information upstream and downstream, providing more information support for willow research and molecular breeding.

[0183] 6. Compared with traditional solid-phase chip technology, the liquid phase chip technology used has no requirements for the number of samples to be tested. A small amount of samples can be tested. There is no need to collect samples during testing and there is no limit on the number of samples. This greatly saves the service cycle of sample testing and improves scientific research efficiency and breeding efficiency. The density of the developed SNP chip can be flexibly adjusted to form application scenarios for different products such as 40K, 20K, and 10K. New sites can be added to the products as needed.

[0184] 7. It fills the embarrassing situation that there are no products available when my country's willow is moving from traditional breeding to molecular breeding. The targeted capture sequencing technology is used to design a high-throughput SNP liquid phase probe chip for willow, and the GBTS technology is promoted to the basic research and molecular breeding applications of willow, so as to better apply it to scientific research and breeding processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] Figure 1This is a design flow chart of the willow whole genome 40K liquid phase chip in the present invention;

[0186] Figure 2 The distribution map of the SNP loci of willow on chromosomes of the present invention;

[0187] Figure 3 The population structure diagram of the willow screening population of the present invention site;

[0188] Figure 4 It is a genome-wide association analysis (GWAS) diagram of the SNP site of the present invention;

[0189] Figure 5 It is a MAF distribution statistical diagram of the SNP site of the present invention;

[0190] Figure 6 This is a distribution statistical diagram of the SNP sites of the present invention in the gene structure of willow;

[0191] Figure 7 This is an example diagram of multi-omics joint analysis of SNP sites in the present invention;

[0192] Figure 8 The Next generation Sequencing analysis flow chart of the present invention;

[0193] Fig. 9 This is the flow chart of library construction and sequencing of willow of the present invention;

[0194] Fig.10 A flow chart for constructing the library of the present invention;

[0195] Fig.11 .The results of the site detection test of 16 willow samples using the willow whole genome 40K liquid phase chip. DETAILED DESCRIPTION

[0196] The technical solution of the present invention is further explained below in conjunction with specific embodiments; however, the present invention is not limited to the following embodiments.

[0197] Example 1

[0198] The present invention provides a willow whole genome 40K SNP liquid phase chip, including a willow 40K site probe mixture and a hybrid capture reagent. The willow 40K site probe mixture includes a willow SNP background site probe and a willow SNP associated site probe, and each site has at least 2 capture probes. The design flow chart is as follows: Figure 1 shown.

[0199] The high-throughput whole-genome 40K liquid phase chip for willow genotyping developed based on targeted capture sequencing technology, combined with added associated sites, realizes willow genotyping and effectively saves sample testing service cycle, providing technical support for willow scientific research.

[0200] The hybridization capture reagent was the GenoBaits DNA-seq Library Prep Kit from Novogene Co., Ltd., including independently packaged GenoBaits Block I, GenoBaits Block II, GenoBaits 2×HybBuffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2×BeadsWashBuffer, GenoBaits10×Wash Buffer I, GenoBaits 10X Wash Buffer II, GenoBaits10×WashBuffer III, and GenoBaits 10X StringentWashBuffer. The distribution of the willow SNP background site probe on the chromosome is shown in the figure. Figure 2 shown.

[0201] Example 2

[0202] The willow whole genome 40K loci provided by the present invention are obtained by the following method:

[0203] (1) Based on the high-depth resequencing sample data of 1,000 willow germplasm resources, the reference genome of the willow was aligned with the version published by Nanjing Forestry University in the internationally renowned journal "Horticulture Research" after BWA2; GATK4 was used to detect the SNP background sites of all samples and filter the sites for quality, and several sites were selected for the selection of target sites associated with traits in combination with other multi-dimensional analyses; the sites were re-selected according to the site selection and filtering standards, and the total set after selection was evaluated for the probe success rate;

[0204] (2) Willow whole genome 40K liquid phase chip. Willow SNP association sites were obtained by the following method: after aligning the willow reference genome with BWA, GATK was used to detect the union sites of all sample SNP sites, and whole genome association analysis was performed on 69 willow traits. The mixed linear model of GenoABEL software was used to obtain significantly associated SNPs, and the formula was: y = Xα + Qβ + Kμ + e; the sample population structure Q was calculated using PLINK v1.9 software and the population structure diagram was drawn, and the kinship K between samples was calculated using PLINK v1.9 software, where X was the genotype and y was the phenotype, and each SNP site in each metabolite phenotype was given an association value; the sites with a distance greater than 50 bp were filtered and selected through the threshold after Bonferroni correction to obtain significantly associated SNP sites.

[0205] (3) comparing the willow SNP functional markers with the total marker set obtained in step (1) to remove duplicates, and merging the remaining markers after removal of duplicates to obtain markers for probe evaluation and development;

[0206] (4) Select sites during probe evaluation: discard sites where the upstream and downstream 50 bp sequences on the genome are repetitive sequence regions, calculate the GC content of the remaining sites in the upstream and downstream 50 bp, retain target sites with a content greater than 30% and less than 70% as candidate sets, and select several sites containing willow SNP-associated sites;

[0207] (5) Divide the chromosome into segments of equal length, select candidate sites with MAF>0.05 for all designed sites in each segment, and obtain a number of sites based on the sites with uniform chromosome distribution and high MAF values ​​and all associated sites, and add functional markers to serve as the 50K site set for testing;

[0208] (6) Through product detection and testing, approximately 10K sites with poor typing performance were deleted to form a product collection of core sites that met the evaluation requirements and included associated sites, and a whole-genome 40K liquid phase chip was formed, and the optimal experimental process for the willow whole-genome 40K liquid phase chip was formed.

[0209] The relevant detection diagram of the willow whole genome 40K liquid phase chip obtained by Example 1 to Example 2 is as follows Figures 3 to 7 shown.

[0210] Example 3

[0211] The willow whole genome 40K SNP liquid phase chip provided by the present invention includes a willow whole genome 40K site probe design process as follows:

[0212] (1) The probe length was 110 bp, the probe GC content was between 30% and 70%, the number of homology regions was ≤5, and the selected region did not contain SSR repeat sequence regions and genomic GAP (deletion) regions as much as possible;

[0213] (2) Design two nucleotide sequences with 60% to 70% overlap and covering the SNP sites based on the SNP sites obtained by screening;

[0214] (3) Single-stranded nucleotides were synthesized according to the designed nucleotide sequence. The two synthesized DNA nucleotide sequences were 110 bp in length and modified with a biotin group at the 5' end, which were called the willow genome 40K site probes;

[0215] (4) The two synthesized willow whole genome 40K site probes were mixed in equal molar mass, and the volume was fixed to 3 pmol / mL of the willow whole genome 40K site probe mixture using a mixture of EDTA and Tris HCl.

[0216] Example 4

[0217] The application process of the willow whole genome 40K SNP liquid phase chip obtained by Examples 1 to 3 is as follows.

[0218] (1) Take 10 willow leaves, place them in the experiment to dry naturally, and use them for DNA extraction; the gene sequence extraction process is as follows Fig. 9 As shown;

[0219] (2) DNA extraction, library construction, sequencing and final SNP data were obtained according to the experimental operation steps of the willow whole genome 40K SNP liquid phase chip; the library construction process is as follows Fig.10 Shown

[0220] (3) The product detection rate is an important indicator for measuring chip quality. In plants, the detection rate is generally measured by the ratio of the number of product detection sites to the number of development sites; therefore, the detection situation was statistically analyzed (e.g. Fig.11 The chip has a total of 40,188 loci, and the average detection rate of the original SNP loci of the 16 samples is 97.39%. After the 16 sample data are combined and counted, the locus detection rate reaches 99.9%:

[0221] In summary, the willow whole genome 40K SNP liquid phase chip disclosed in the present invention has high density and high site capture efficiency. The willow whole genome 40K SNP liquid phase chip disclosed in the present invention can be applied to willow genetic diversity analysis, molecular genetic map construction, whole genome association analysis, variety authenticity identification, molecular marker-assisted selection breeding, and whole genome selection breeding.

Claims

1. A willow whole genome 40K SNP liquid phase chip, characterized in that: The method comprises a willow 40K site probe mixture and a hybrid capture reagent. The willow 40K site probe mixture comprises a willow SNP background site probe and a willow SNP associated site probe, and each site has at least two capture probes.

2. The willow whole genome 40K SNP liquid phase chip according to claim 1, characterized in that: The hybridization capture reagent is the GenoBaits DNA-seq Library Prep kit of Novogene Co., Ltd., including independently packaged GenoBaits Block I, GenoBaits Block II, GenoBaits 2×Hyb Buffer, GenoBaits HybBuffer Enhancer, GenoBaits 2×BeadsWashBuffer, GenoBaits 10×Wash Buffer I, GenoBaits 10X Wash Buffer II, GenoBaits10×Wash Buffer III, and GenoBaits 10XStringent Wash Buffer.

3. The willow whole genome 40K SNP liquid phase chip according to claim 1, characterized in that: The willow SNP background site information and the base of the reference gene at the site are shown in the following table: The numbers / letters before the underline in the table represent the chromosome number, the following number group represents the position on the chromosome, and the last letter represents the base of the reference gene at that site.

4. The willow whole genome 40K SNP liquid phase chip according to claim 1, characterized in that: The design process of the willow whole genome 40K locus probe includes: (1) The probe length was 110 bp, the probe GC content was between 30% and 70%, the number of homology regions was ≤5, and the selected region did not contain SSR repeat sequence regions and genomic GAP (deletion) regions as much as possible; (2) Design two nucleotide sequences with 60% to 70% overlap and covering the SNP sites based on the SNP sites obtained by screening; (3) Single-stranded nucleotides were synthesized according to the designed nucleotide sequence. The two synthesized DNA nucleotide sequences were 110 bp in length and modified with a biotin group at the 5' end, which were called the willow genome 40K site probes; (4) The two synthesized willow whole genome 40K site probes were mixed in equal molar mass, and the volume was fixed to 3 pmol / mL of the willow whole genome 40K site probe mixture using a mixture of EDTA and Tris HCl.

5. Use of the willow whole genome 40K SNP liquid phase chip according to any one of claims 1 to 4 in willow breeding.

6. The use according to claim 5, characterized in that: The willow whole genome 40K SNP liquid phase chip is applied to willow genetic diversity analysis, molecular genetic map construction, whole genome association analysis, variety authenticity identification, molecular marker-assisted selection breeding, and whole genome selection breeding.

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