Wheat generic genome 120K liquid phase chip and application thereof
By developing a wheat pan-genome 120K liquid phase chip, integrating highly specific probes and performing quality control screening, the problem of insufficient coverage and specificity of wheat genome chips in existing technologies has been solved, achieving high coverage and high throughput detection of multiple genomes, which is applicable to fields such as wheat variety typing and molecular-assisted breeding.
Patent Information
- Application Number
- CN202511129927.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-13
- Publication Date
- 2025-10-31
AI Technical Summary
Existing wheat genome chips lack sufficient coverage and specificity, making it difficult to comprehensively assess the genetic diversity of wheat. Furthermore, traditional chips are not applicable to multiple versions of the reference genome.
A wheat pan-genome 120K liquid phase chip was developed, integrating 124,265 highly specific probes. Through SNP site screening, quality control, and uniform coverage layout, it is applicable to multiple wheat genomes, including IWGSCv2.1, AK58, KN9204, and JING411, ensuring the universality and accuracy of detection.
It achieves high coverage and high throughput detection on multiple wheat genomes, significantly improving genome coverage and representativeness. The detection results are accurate and reliable, and are applicable to fields such as wheat variety typing and molecular-assisted breeding.
Smart Images
Figure CN120866565A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of crop genetics and molecular breeding, and in particular to a liquid-phase chip for detecting high-coverage regions of wheat multi-genome and genotype polymorphism and its application method. Technical Background
[0002] Wheat (Triticum aestivum) is a major global food crop with a large and complex genome. Traditional microarrays rely on a single reference genome, making it difficult to comprehensively assess genetic diversity within the species. Next-generation pan-genome research has driven the integrated application of multiple reference genome versions, while also placing new demands on genotyping microarrays, requiring high sensitivity, broad coverage, uniform distribution, and compatibility across various scenarios. Therefore, there is an urgent need to develop specialized microarrays capable of high-coverage and high-throughput detection against multiple mainstream wheat genomes, serving cutting-edge fields such as hybridization, trait mapping, and molecular-assisted breeding. Summary of the Invention
[0003] This invention aims to overcome the shortcomings of insufficient coverage and specificity in existing technologies by providing a pan-genomic liquid-phase probe chip based on multiple versions of the wheat genome. This chip integrates 124,265 highly specific probes, ensuring universality and detection effectiveness across major wheat genomes through SNP site screening, comprehensive sequence quality control, and uniform coverage of diverse sites. Its methodology is scientifically sound, with clearly defined parameters, and is easily standardized and promoted.
[0004] This invention provides a wheat pan-genome 120K liquid phase chip, which contains 124,265 oligonucleotide probes with a length of 90 bp. Each probe has a GC content of 0.3-0.65, a sequence complexity of ≥0.9, a Tm value of ≥68℃, does not contain N sites, has ≤7 single-base tandem repeats and ≤3 multi-base tandem repeats, and in whole-genome sequence alignment, the number of matching sites for a single probe does not exceed 10. It can cover multiple wheat genome versions of SNP sites such as IWGSCv2.1, AK58, KN9204 and JING411.
[0005] The chip fabrication method includes the following steps:
[0006] Step 1: SNP Quality Control: Using the SNP dataset from the Chinese Spring Reference Genome (IWGSCv2.1), bioinformatics analysis tools such as VCFtools and PLINK were used to rigorously screen the SNP variation data. High-quality SNP loci were retained as the basis for genotyping according to the following parameters: deletion rate ≤ 0.1, heterozygosity rate ≤ 0.1, and minimum allele frequency (MAF) ≥ 0.1.
[0007] Step 2: Around each SNP site that passed quality control screening, extend 20 bp to both sides of the SNP as the center, forming a candidate probe window of 41 bp in length. Using automated tools such as Python and R scripts, slide the window step-by-step in 5 bp increments to set multiple probe center positions. Using each center as a reference, extend to both sides to extract candidate probe sequences of 90 bp in length, and perform batch extraction using tools such as Biopython, BEDtools, and samtools.
[0008] Step 3: Comprehensive Quality Assessment and Determination of Candidate Probe Sequences: For each locus, the candidate probe sequences were subjected to comprehensive quality control using bioinformatics analysis tools such as Biopython and Primer3. Key parameters included GC content (0.3–0.65), absence of nitrogenous bases, sequence complexity ≥0.9, single-base tandem repeats ≤7, multi-base tandem repeats ≤3, and annealing temperature (Tm) ≥68℃. Simultaneously, whole-genome alignment tools such as BLASTN were used to determine the specificity of each probe, retaining only highly specific probes with ≤10 matching loci in whole-genome alignments. Finally, based on the comprehensive evaluation results, the probe with the best parameters and highest specificity was selected from all candidate probes for each locus as the representative probe for that locus.
[0009] Step 4: Genome-wide Uniform Coverage Optimization: Using BEDtools, R, and Python scripts, the reference genome was divided into 500 kb physical segments. High-information SNPs were selected preferentially from functional gene regions (CDS, UTR, Intron, upstream and downstream). From each segment, 3-4 probes meeting quality control standards were selected, and probes with higher MAF were prioritized when technical parameters were comparable. Ultimately, 118,403 high-quality probes were screened, achieving uniform distribution and maximizing information content across the entire genome and functional regions.
[0010] Step 5: To ensure multi-version compatibility of the chip, the same procedure was used to screen probes for other wheat reference genomes such as AK58, KN9204, and JING411. Using sequence alignment tools such as BWA and minimap2, probes obtained from the Chinese Spring (IWGSCv2.1) genome were mapped to different genomes, and additional probes were added to regions that were not effectively covered. Finally, 1,991, 2,082, and 1,789 probes were added to the AK58, KN9204, and JING411 genomes, respectively, ensuring uniform coverage of the mainstream genomes, resulting in a high-quality chip array of 124,265 probes.
[0011] Preferably, the chip, which performs multi-index quality control screening of candidate probes, is applicable to the development of pan-genomic SNP genotyping chips for other gramineous crops.
[0012] Preferably, the chip is used in wheat variety typing, molecular-assisted breeding, genome-wide association analysis, germplasm resource evaluation, genome diversity analysis and functional gene mapping.
[0013] A method for developing the wheat pan-genome 120K liquid phase chip of claim 1 includes SNP site quality control, probe candidate region generation, probe design and screening, comprehensive evaluation of probe quality, uniform coverage layout, and multi-genome completion.
[0014] Preferably, the method for developing the wheat pangenome 120K liquid phase chip of claim 1 is applicable to the development of pangenome SNP typing chips for other gramineous crops.
[0015] A method for detecting wheat genome polymorphism using the chip described in claim 1, comprising:
[0016] (1) Extract DNA from samples and construct a library;
[0017] (2) Hybridize the library with the probe pool to capture the target sequence;
[0018] (3) High-throughput sequencing: PE150 paired-end sequencing was performed using the BGI T7 platform, and SNP genotyping analysis was performed using BWA / GATK.
[0019] The present invention has the following outstanding beneficial effects:
[0020] (1) The probes are more evenly distributed on the mainstream wheat genomes, which significantly improves genome coverage and representativeness.
[0021] (2) The chip is compatible with multiple versions of genome, such as IWGSCv2.1, AK58, KN9204 and JING411, and has a wide range of applications.
[0022] (3) By independently supplementing probes, 1991, 2082 and 1789 probes were added to the genomes of AK58, KN9204 and JING411, respectively, which effectively improved the integrity of the whole chip in multiple genomes.
[0023] (4) The design process is rigorous and the quality control standards are high. The selected probes have high specificity and polymorphism, and the detection is accurate and reliable.
[0024] (5) It can be widely applied to wheat variety typing, population genetic analysis and molecular-assisted breeding, providing more efficient technical support for wheat molecular breeding. Attached Figure Description
[0025] Figure 1This is a map showing the distribution of the SNP sites of this invention on the IWGSCv2.1 genome;
[0026] Figure 2 This is a map showing the distribution of the SNP sites of this invention on the AK58 genome;
[0027] Figure 3 This is a distribution map of the SNP sites of this invention on the KN9204 genome;
[0028] Figure 4 This is a distribution map of the SNP sites of this invention on the JING411 genome;
[0029] Figure 5 This invention provides a statistical analysis of the distribution of SNP sites in gene functional regions.
[0030] Figure 6 This is a MAF distribution map of the SNP sites of this invention. Detailed Implementation
[0031] The following detailed embodiments illustrate the implementation of the present invention so that those skilled in the art can understand its technical advantages and application potential.
[0032] Example 1:
[0033] Step 1: Use bioinformatics tools such as VCFtools and PLINK to preprocess and quality control the SNP data in the Chinese Spring reference genome (IWGSCv2.1). The specific requirements are: each site has a deletion rate of no more than 0.1, a heterozygosity rate of no more than 0.1, and a MAF of no less than 0.1. These are high-quality polymorphic sites used for subsequent probe design.
[0034] Step 2: For each SNP site that passed quality control, Python and R scripts were used to automatically extract a region centered on the SNP and extending 20 bp to both sides. Multiple center points were generated with a step size of 5 bp, and then 90 bp candidate probe sequences were extracted. Bedtools and Samtools were used for batch extraction of genome sequences to improve processing efficiency.
[0035] Step 3: For all candidate probes, multi-parameter quality control was performed using Biopython, Primer3, etc., including GC content, N-base absence, sequence complexity, number of single-base tandem repeats and multi-base tandem repeats, and annealing temperature. The corresponding parameters were: GC content 0.3–0.65, complexity ≥0.9, number of single-base tandem repeats ≤7, number of multi-base tandem repeats ≤3, and Tm ≥68℃. Sequence specificity was assessed using BLASTN whole-genome alignment, with ≤10 matching sites. Finally, the probe with the optimal parameters and highest specificity for each site was selected as its representative. After the above screening, the coverage statistics of the sites in each functional region are as follows: Figure 5 As shown.
[0036] Step 4: Using BEDtools, R, and Python scripts, the reference genome is divided into 500 kb physical segments, prioritizing sites with high information content in functional gene regions (cds, utr, intron, upstream and downstream). For each segment, 3-4 probes meeting all quality control criteria are selected, and, with comparable technical parameters, the most representative high MAF probes are prioritized. Ultimately, 118,403 probes are screened, ensuring the probe set is evenly distributed throughout the genome and functional regions to maximize information.
[0037] Step 5: To accommodate different mainstream wheat genome versions such as AK58, KN9204, and JING411, the same steps described above were used to perform quality control, probe distribution, and screening on more genome sequence data. Probes selected from the Chinese Spring genome were mapped to other genome versions using sequence alignment tools such as BWA and minimap2. If mapping revealed a lack of high-quality probes in a certain region, 1-2 qualified supplementary probes were selected for that region. Through this supplementation, 1,991, 2,082, and 1,789 probes were added to AK58, KN9204, and JING411 respectively, resulting in uniform coverage of all mainstream genomes on the entire chip, with a total probe count of approximately 120K. The SNP distribution on IWGSC v2.1 is shown below. Figure 1 As shown; the distributions on AK58, KN9204, and JING411 are respectively as follows: Figure 2 , Figure 3 and Figure 4 As shown.
[0038] Step Six: Chip Fabrication and Application:
[0039] (1) Probe synthesis: The 124,265 high-quality probe sequences obtained from screening were synthesized in batches, quantitatively and equimolarly mixed to prepare a mixed probe pool.
[0040] (2) Sample DNA extraction: High-quality genomic DNA was extracted from the wheat tissue to be tested using a commercial plant genomic DNA extraction kit. The integrity and concentration of the DNA were detected by instruments such as agarose gel electrophoresis and NanoDrop to ensure the effectiveness of subsequent experiments.
[0041] (3) Library construction and probe capture: A library was prepared using a high-efficiency library construction kit, and DNA was fragmented, end-repaired, adapter-added, and amplified by PCR. The library was hybridized with a mixed probe pool in liquid phase to capture the target fragment under strict conditions. After capture, the material was eluted and purified for subsequent sequencing.
[0042] (4) High-throughput sequencing: PE150 paired-end sequencing was performed using the BGI T7 platform to obtain high-quality reads covering the target region. Sequencing data quality was evaluated and filtered using tools such as FastQC and Trimmomatic.
[0043] (5) Bioinformatics Analysis: Sequencing data is aligned with a reference genome using sequence alignment tools such as BWA and Bowtie2. SNP / Indel variant detection and genotype determination are performed using software such as GATK and samtools. Subsequently, depending on project needs, tools such as PLINK, TASSEL, and ADMIXTURE can be used for population structure analysis, genome-wide association studies (GWAS), genome diversity assessment, and various applications such as molecular-assisted breeding and variety identification. The MAF distribution of the selected loci is shown in the figure. Figure 6 As shown.
Claims
1. A wheat pan-genome 120K liquid-phase chip, characterized in that, The chip contains 124,265 oligonucleotide probes, each 90 bp in length. Each probe has a GC content of 0.3-0.65, a sequence complexity of ≥0.9, a Tm value of ≥68℃, and contains no N sites. The number of single-base tandem repeats is ≤7, and the number of multi-base tandem repeats is ≤3. In whole-genome sequence alignment, the number of matching sites for a single probe does not exceed 10, and it can cover multiple wheat genome versions of SNP sites, including IWGSCv2.1, AK58, KN9204, and JING411.
2. The chip as described in claim 1, characterized in that, The chip preparation method includes the following steps: (1) quality control of SNP sites in Chinese spring and other wheat genomes, and screening of high-quality polymorphic sites by indicators such as deletion rate, heterozygosity rate, and MAF; (2) Taking each SNP site that passed quality control as the center, extend 20 bp to both sides as candidate regions, and generate the center site with a step size of 5 bp; (3) Obtain 90bp probe sequences based on candidate centers, evaluate and select probes with the best GC content, alignment specificity and other parameters; (4) Perform multi-index quality control screening on candidate probes; (5) Distribute the genome parts and functional regions evenly and preferentially; (6) Probes for missing segments were added to different wheat genomes such as AK58, KN9204 and JING411, resulting in a high-quality probe set of 124,265 probes.
3. The chip as described in claim 2, characterized in that, Step (4) is applicable to the development of pan-genome SNP typing chips for other gramineous crops for multi-index quality control screening.
4. The application of the chip as described in any one of claims 1-3, characterized in that, The chip has applications in wheat variety typing, molecular-assisted breeding, genome-wide association analysis, germplasm resource evaluation, genome diversity analysis, and functional gene mapping.
5. A method for developing the wheat pan-genome 120K liquid-phase chip of claim 1, characterized in that, This includes SNP site quality control, probe candidate region generation, probe design and screening, comprehensive probe quality evaluation, uniform coverage layout, and multi-genome completion.
6. The application of the method as described in claim 5, characterized in that, The method is applicable to the development of pan-genome SNP genotyping chips for other gramineous crops.
7. A method for detecting wheat genome polymorphism using the chip described in claim 1, comprising: (1) Extract DNA from samples and construct a library; (2) Hybridize the library with the probe pool to capture the target sequence; (3) High-throughput sequencing: PE150 paired-end sequencing was performed using the BGI T7 platform, and SNP genotyping analysis was performed using BWA / GATK.