Chromosome spatial structure variation detection method based on liquid-phase gene chip
By combining liquid-phase probe hybridization chip technology with specific probe compositions, the throughput and cost issues of detecting spatial structural variations in gene-edited wheat chromosomes have been solved, enabling efficient and low-cost detection of multiple sites and variations, thus improving detection efficiency and accuracy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-12
- Publication Date
- 2026-04-21
AI Technical Summary
Existing technologies for detecting chromosomal spatial structural variations in gene-edited wheat suffer from limitations in throughput, high cost, poor sensitivity, and low flexibility. In particular, they are difficult to achieve efficient and low-cost detection when detecting large deletion variations in the MLO gene.
By employing liquid-phase probe hybridization chip technology and combining specific probe compositions, multiple sets of probes are designed to perform high-throughput sequencing in a liquid-phase chip system, capturing multiple gene editing sites and flanking regions, and enabling parallel detection of large fragments and single-base variations.
It improves the detection efficiency of multiple sites and multiple variants, reduces analysis costs, enhances the accuracy and reliability of sequencing results, and realizes high-throughput, low-cost gene editing detection.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for detecting chromosomal spatial structural variations based on liquid-phase gene chips. Background Technology
[0002] Wheat, as one of the major food crops, feeds more than one-third of the world's population. However, it is caused by powdery mildew (… Blumeria graminis Powdery mildew (Bgt) caused by *F. sp. tritici* is one of the major diseases affecting wheat yields worldwide, with severely affected wheat fields experiencing yield reductions of over 40%, seriously threatening global food security. Successful infection by the Bgt pathogen requires the use of susceptibility genes, and mutations in these genes typically confer broad-spectrum and durable resistance. However, susceptibility genes have important physiological functions, and their mutations can lead to various negative effects on plant growth and development, significantly limiting their application in plant disease resistance breeding. Against this backdrop, the MLO (Mildew Locus O) gene has attracted considerable interest from researchers. The MLO gene is a typical susceptibility gene in plants, encoding a protein that negatively regulates the plant's defense response. This gene family is plant-specific, encoding a conserved protein with seven transmembrane domains, including a calmodulin-binding domain, potentially functioning as a membrane-bound receptor. Previous research (Li S, Lin D, Zhang Y, et al. Genome-edited powdery mildew resistance in wheat without growth penalties[J]. Nature, 2022, 602(7897): 455-460.) has reported that by editing the wheat susceptibility gene MLO, a large (304KB) deletion occurred near the TaMLO-B1 site in the wheat genome, resulting in wheat material with broad-spectrum and durable resistance to powdery mildew without affecting plant height and yield. Further analysis of the three-dimensional chromosome map of this wheat material revealed that the large deletion near the TaMLO-B1 site in the wheat genome led to a rearrangement of the chromosome's three-dimensional space, thereby altering the chromosomal spatial structure in that region.
[0003] Gene sequencing is a core method for validating and assessing the safety of gene-edited plants. It is primarily used to confirm the precise information of gene-editing sites. Commonly used techniques include NGS (next-generation sequencing), TGS (third-generation sequencing), solid-phase microarray capture, and liquid-phase microarray capture. NGS sequencing breaks the sequenced DNA into fixed-length fragments using sonication or enzymatic digestion, followed by library construction using PCR. During PCR experiments, conditions such as extremely high / low GC content and repetitive regions can cause PCR amplification bias. If base mispairing occurs during template replication, it affects the accuracy of the sequencing template, ultimately impacting sequencing quality and data volume. For large insertion / deletion variants in the genome, read length limitations prevent coverage of the entire variant region, making variant analysis impossible. Current common approaches involve increasing sequencing depth to 80-100X, providing sufficient data for detection, but significantly increasing sequencing and analysis costs. TGS sequencing, leveraging the advantage of long single-molecule sequencing lengths, effectively detects large structural variations. However, its accuracy for single-base sequencing is relatively low. While precise HiFi sequencing significantly improves accuracy, sequencing and analysis costs remain crucial considerations for large-scale, high-throughput material detection in practical applications. Solid-phase array capture can drastically increase throughput, but it lacks the ability to rapidly adjust the array size and quantity, limiting probe design flexibility and resulting in longer preparation times and higher costs. Furthermore, there are currently no effective examples for solid-phase probe capture of large fragments. Liquid-phase array capture offers greater flexibility in probe density and type, enriching sequencing data in target regions without increasing costs, significantly increasing sequencing depth by hundreds or thousands of times, providing ample evidence for variation detection. The reduction in non-target region data also lowers analysis and storage costs. Therefore, there is an urgent need for a high-throughput, low-cost, and highly sensitive sequencing method to detect large structural variations while minimizing analysis and storage costs. Summary of the Invention
[0004] The technical problem to be solved by this invention is how to detect gene-edited wheat with chromosomal spatial structure changes caused by large fragment deletions of the MLO gene.
[0005] To address the aforementioned technical problems, the present invention first provides a probe composition.
[0006] The probe composition provided by the present invention includes probe A, probe B, probe C, probe D, probe E and probe F; The nucleotide sequence of probe A includes sequence 1; The nucleotide sequence of probe B includes sequence 2; The nucleotide sequence of probe C includes sequence 3; The nucleotide sequence of probe D includes sequence 4; The nucleotide sequence of probe E includes sequence 5; The nucleotide sequence of probe F includes sequence 6.
[0007] In some embodiments, each probe in the probe composition further includes a modifying group attached to its respective end. The modifying group may be biotin, etc.
[0008] In some embodiments, the probe composition can be used to detect wheat gene-edited material; the wheat gene-edited material is wheat in which the MLO gene has been gene-edited.
[0009] To address the aforementioned technical problems, the present invention provides another chip.
[0010] The chip provided by this invention includes the above-described probe composition.
[0011] In some implementations, the chip is a liquid-phase probe hybridization chip.
[0012] To address the aforementioned technical problems, the present invention also provides new uses for the probe composition described above.
[0013] This invention provides the application of the above-described probe composition in the design, development, or preparation of chips for detecting wheat gene-edited materials; wherein the wheat gene-edited material is wheat in which the MLO gene has been edited.
[0014] In some implementations, the chip is a liquid-phase probe hybridization chip.
[0015] To address the aforementioned technical problems, the present invention also provides new uses for the probe composition or the chip described above.
[0016] This invention provides the application of the above-described probe composition or the above-described chip in any of the following M1)-M6): M1) detects whether gene editing has occurred in wheat gene-edited materials or their offspring; M2) Prepare products to detect whether gene editing has occurred in wheat gene-edited materials or their offspring; M3) distinguishes between gene-edited wheat materials and unedited wheat materials; M4) Prepare products that distinguish between wheat gene-edited materials and wheat gene-unedited materials; M5 wheat breeding; M6) is used to prepare wheat breeding products; The wheat gene-editing material is wheat from which the MLO gene has been edited; The unedited wheat material refers to wheat whose MLO gene has not been edited.
[0017] To address the aforementioned technical problems, the present invention also provides any one of the following methods (N1)-N3): N1) A method for detecting whether gene editing has occurred in wheat gene-edited material or its offspring, comprising the following steps: using the above-mentioned chip to detect the wheat to be tested, thereby detecting whether gene editing has occurred in wheat gene-edited material or its offspring; N2) A method for distinguishing between wheat gene-edited materials and wheat gene-unedited materials, comprising the following steps: using the above-mentioned chip to detect the wheat to be tested, thereby distinguishing between wheat gene-edited materials and wheat gene-unedited materials; N3) A method for wheat breeding, comprising the following steps: a step of breeding using wheat gene-edited material obtained according to the method described in N1) as a parent; The wheat gene-editing material is wheat from which the MLO gene has been edited; The unedited wheat material refers to wheat whose MLO gene has not been edited.
[0018] The wheat mentioned above can be any wheat germplasm resource, variety, strain or single plant commonly found in this technical field.
[0019] The detection of whether gene editing has occurred in wheat gene-edited materials or their offspring as described above can be performed by detecting whether gene editing of the MLO gene has occurred in the genome sequence of the wheat gene-edited materials or their offspring. Materials or their offspring in which the MLO gene has been edited are wheat with MLO gene editing; materials or their offspring in which the MLO gene has not been edited are wheat without MLO gene editing.
[0020] Wheat whose MLO gene has not been edited as described above refers to wheat whose MLO gene has been mutated compared with the Chinese Spring wheat reference genome sequence.
[0021] The wheat in which the MLO gene has been edited as described above is wheat in which the MLO gene has been mutated compared with the reference genome sequence of common wheat (Chinese spring).
[0022] The mutations described above can take the form of base substitution, base insertion, and / or base deletion (or absence). The bases include single bases and / or multiple bases and / or large segments.
[0023] The mutation sites described above include one, two, three, or more. These sites can be neighboring or non-neighboring.
[0024] The MLO genes mentioned above include the TraesCS5A02G494800 gene, the TraesCS4D02G319100 gene, and the TraesCS4B02G322700 gene.
[0025] The aforementioned TraesCS5A02G494800 gene is located at positions 663,215,692-663,218,964 on chromosome 5A in the wheat (common wheat, Chinese spring) reference genome sequence.
[0026] The aforementioned TraesCS4D02G319100 gene is located at position 483,065,495-483,068,744 on chromosome 4D in the wheat (common wheat, Chinese spring) reference genome sequence.
[0027] The aforementioned TraesCS4B02G322700 gene is located at positions 613,157,952-613,160,922 on chromosome 4B in the wheat (common wheat, Chinese spring) reference genome sequence.
[0028] Wheat with gene editing of any of the MLO genes mentioned above includes wheat with gene editing of the TraesCS5A02G494800, TraesCS4D02G319100 and TraesCS4B02G322700 genes in the wheat genome sequence.
[0029] In some embodiments, the wheat in which the MLO gene has been gene-edited is wheat in which the TraesCS5A02G494800, TraesCS4D02G319100, TraesCS4B02G322700, and TraesCS4B02G322100 genes in the wheat genome sequence have been gene-edited.
[0030] The aforementioned TraesCS4B02G322100 gene is located at positions 612,855,666-612,873,425 on chromosome 4B in the wheat (common wheat, Chinese spring) reference genome sequence.
[0031] In some implementations, the wheat in which the MLO gene has been edited includes wheat with altered chromosomal spatial structure due to a large deletion of the MLO gene. The large deletion refers to a deletion at positions 612,856,493-613,160,110 on chromosome 4B of the wheat (common wheat, Chinese spring). In this wheat, the large deletion of the MLO gene (304KB) alters the chromosomal spatial structure in that region, resulting in resistance to powdery mildew, and no significant difference in plant height and yield compared to wild-type wheat.
[0032] In some embodiments, the wheat in which the MLO gene is gene-edited is obtained by mutating the G in the TraesCS5A02G494800 gene of wheat (Yangmai 25) corresponding to position 663,217,923 on chromosome 5A of the reference genome (common wheat, Chinese spring) to GA, and mutating the A in the TraesCS4D02G319100 gene corresponding to position 483,067,668 on chromosome 4D of the reference genome (common wheat, Chinese spring) to AC, and deleting positions 612,856,493-613,160,110 on chromosome 4B of the TraesCS4B02G322100 and TraesCS4B02G322700 genes corresponding to positions 612,856,493-613,160,110 on chromosome 4B of the reference genome (common wheat, Chinese spring), while keeping other nucleotide residues unchanged.
[0033] This invention addresses several problems in current gene editing detection methods, including limited throughput, high cost, poor sensitivity, and low flexibility. It combines liquid-phase chip technology with high-throughput sequencing methods using specific probe capture to provide a method for detecting MLO gene editing in MLO gene-edited materials, along with the probe composition and liquid-phase probe hybridization chip used. Experiments demonstrate that the method provided by this invention can achieve parallel detection of large-fragment editing events and single-base editing events in wheat MLO gene-edited materials in a single detection, not only improving the detection efficiency of multiple editing events but also significantly reducing analysis costs. Specifically: 1. Enable parallel detection of multiple sites and multiple variant types. This invention designs multiple sets of specific probes in a liquid-phase chip system, enabling the simultaneous capture of multiple gene editing sites and flanking regions in a single detection, thus achieving parallel detection of multiple sites in a single sequencing run. Data capture via the liquid-phase chip can capture not only single-base variations but also large-fragment genomic deletions (304KB).
[0034] 2. Increase the accuracy and validity of data. This invention uses a high-throughput sequencing platform to sequence DNA libraries captured by liquid-phase chips, and through a standardized bioinformatics analysis process, effectively improves sequencing accuracy, increases target region coverage, and enhances the reliability of sequencing results.
[0035] 3. Improve detection efficiency and reduce analysis costs Compared to whole-genome sequencing (WGS), this invention relies on liquid-phase probes to capture target regions, enriching these regions before next-generation sequencing. This not only avoids multiple primer designs, reducing design time, but also eliminates the need to establish a PCR system. Only the test material needs to be processed and targeted region-specific probes designed, significantly improving detection efficiency and reducing reagent usage and manual design costs. Attached Figure Description
[0036] Figure 1 The distribution of the PROBE2 probe location and sequencing depth in a wheat gene editing case study.
[0037] Figure 2 The distribution of the PROBE5 probe location and sequencing depth in the wheat gene editing case.
[0038] Figure 3 The probe location distribution and sequencing depth for the wheat gene editing case DEL_303KB_L.
[0039] Figure 4 The probe location distribution and sequencing depth of DEL_303KB_R in wheat gene editing case.
[0040] Figure 5 The distribution of the PROBE13 probe location and sequencing depth in the wheat gene editing case.
[0041] Figure 6 The distribution of probe locations and sequencing depth for the PROBE17 wheat gene editing case. Detailed Implementation
[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0044] The reference genome sequences used in the following examples all refer to the common wheat Chinese spring genome sequence, version number CS v1.1, which can be obtained at the following URL: http: / / ftp.ensemblgenomes.org / pub / plants / release-53 / fasta / triticum_aestivum / dna / .
[0045] Example 1: Preparation, establishment of method, and application of liquid-phase gene chip for detecting wheat MLO gene editing materials. I. Preparation and establishment of methods for liquid-phase gene chip for detecting wheat MLO gene-edited materials The TraesCS5A02G494800 gene in this invention is located at positions 663,215,692-663,218,964 on chromosome 5A of the wheat (common wheat, Chinese spring) reference genome sequence; the TraesCS4D02G319100 gene is located at positions 483,065,495-483,068,744 on chromosome 4D of the wheat (common wheat, Chinese spring) reference genome sequence; the TraesCS4B02G322700 gene is located at positions 613,157,952-613,160,922 on chromosome 4B of the wheat (common wheat, Chinese spring) reference genome sequence; and the TraesCS4B02G322100 gene is located at positions 612,855,666-612,873,425 on chromosome 4B of the wheat (common wheat, Chinese spring) reference genome sequence.
[0046] The wheat gene-edited material was obtained by mutating the G at position 663,217,923 on chromosome 5A of the reference genome (common wheat, Chinese spring) in the TraesCS5A02G494800 gene to GA, mutating the A at position 483,067,668 on chromosome 4D of the reference genome (common wheat, Chinese spring) in the TraesCS4D02G319100 gene to AC, and deleting positions 612,856,493-613,160,110 on chromosome 4B of the reference genome (common wheat, Chinese spring) in the TraesCS4B02G322100 and TraesCS4B02G322700 genes, while keeping other nucleotide residues unchanged.
[0047] This embodiment uses the wheat variety Yangmai 25 as an example. The wheat gene-editing material is the T2 generation homozygous gene-edited wheat obtained by editing the genes TraesCS5A02G494800, TraesCS4D02G319100, TraesCS4B02G322700, and TraesCS4B02G322100 in the Yangmai 25 genome. Sequencing revealed that the TraesCS5A02G494800 gene in this T2 generation homozygous gene-edited wheat corresponds to chromosome 5A in the reference genome (common wheat, Chinese spring). The G mutation at positions 663,217,923 on the chromosome was changed to GA, and the A mutation at position 483,067,668 on chromosome 4D in the TraesCS4D02G319100 gene, corresponding to position 483,067,668 in the reference genome (common wheat, Chinese spring), was changed to AC. Furthermore, the deletions at positions 612,856,493-613,160,110 on chromosome 4B in the TraesCS4B02G322100 and TraesCS4B02G322700 genes, corresponding to positions 612,856,493-613,160,110 in the reference genome (common wheat, Chinese spring), were performed, with all other nucleotide residues remaining unchanged. This wheat gene-edited material is described in the following literature: Li S, Lin D, Zhang Y, et al. Genome-edited powdery mildew resistance inwheat without growth penalties[J]. Nature, 2022, 602(7897): 455-460. The corresponding name of this wheat gene-edited material in the literature is Tamlo-R32.
[0048] By self-pollinating T2 generation gene-edited wheat homozygotes, T5 generation homozygous offspring were obtained, denoted as FC60-1A, FC60-2A, and FC60-3A.
[0049] Unedited T1 generation wheat was self-crossed until homozygous T5 generation offspring were obtained, denoted as YM25-1A and YM25-2A.
[0050] The gene sequences TraesCS5A02G494800, TraesCS4D02G319100, TraesCS4B02G322700, and TraesCS4B02G322100 in the genome sequence of the wheat gene-edited material are recorded as the edited sequences.
[0051] The gene sequences TraesCS5A02G494800, TraesCS4D02G319100, TraesCS4B02G322700 and TraesCS4B02G322100 in the common wheat Chinese spring genome sequence are recorded as wild-type sequences.
[0052] 1. Probe design for wild-type and edited sequences. This invention compares wild-type sequences with edited sequences and designs probe sequences corresponding to the edited regions, as follows: A 21bp region was selected as the target region from the editing region of the TraesCS5A02G494800 gene on chromosome 5A (https: / / plants.ensembl.org / Multi / Search / Results?species=all;idx=;q=TraesCS5A02G494800;site=ensemblunit); and a capture probe PROBE2 was designed based on the target region. The information is shown in Table 1. The capture probe binds to the corresponding target region.
[0053] A 21bp region was selected as the target region from the editing region of the TraesCS4D02G319100 gene on chromosome 4D (https: / / plants.ensembl.org / Multi / Search / Results?species=all;idx=;q=TraesCS4D02G319100;site=ensemblunit); and a capture probe PROBE5 was designed based on the target region. The information is shown in Table 1. The capture probe binds to the corresponding target region.
[0054] Target regions of 21 bp were selected from the editing regions of the TraesCS4B02G322100 and TraesCS4B02G322700 genes on chromosome 4B (https: / / plants.ensembl.org / Multi / Search / Results?species=all;idx=;q=TraesCS4B02G322100;site=ensemblunit, https: / / plants.ensembl.org / Multi / Search / Results?species=all;idx=;q=TraesCS4B02G322700;site=ensemblunit). Capture probes DEL_303KB_L and DEL_303KB_R were then designed based on these target regions. Additionally, to increase the reliability of large DNA fragment deletions, two probes, PROBE13 and PROBE17, were added within the large fragment deletion.
[0055] Information on various probes is shown in Table 1 below.
[0056] Table 1 shows the probe design information.
[0057] 2. Liquid-phase chip fabrication and high-throughput sequencing The probes in Table 1 above were synthesized into single-stranded DNA, and biotin was added to the 5' end to modify the DNA, resulting in a liquid-phase chip for detecting wheat gene-edited materials.
[0058] Genomic DNA from the test material was subjected to sonication or enzyme digestion to form short fragments of suitable length (250 bp), constructing a DNA sequencing library with adapters. To prevent non-specific binding between the adapters and probes, blocking primers were added. Subsequently, a liquid-phase chip for detecting wheat gene-edited materials was added to the system, hybridizing with complementary target sequences in the library under suitable conditions. The complex formed by the probe and target fragment was captured by streptavidin magnetic beads due to the high affinity between biotin and streptavidin. Unbound non-target fragments were then removed by magnetic separation, and fragments that failed to hybridize with the probe were washed away, enriching the target fragment DNA. The enriched product was then sequenced using a high-throughput sequencing platform. The test materials included homozygous progeny of known wheat gene-edited materials and homozygous progeny of known unedited wheat materials.
[0059] After sequencing, the sequencing data were analyzed using a bioinformatics analysis workflow: First, quality control was performed on the sequencing data to remove low-quality reads. Then, the reads were aligned to a reference genome, with a focus on analyzing the occurrence of editing events and InDel patterns in the probe-targeted regions to determine the type and frequency of gene editing events, and to sensitively capture low-frequency variants. Simultaneous upstream and downstream validation of the target sites and coverage regions improved the sensitivity and accuracy of the detection. The sequencing results of the six probe-targeted regions were visualized using IGV visualization software.
[0060] II. Application of Liquid Phase Chip Detection of Wheat Gene Editing Materials in Wheat Gene Editing Following the method in step 2 of step one above, the prepared liquid-phase chip for detecting wheat gene-edited materials was used to detect the wheat materials to be tested, and the sequencing results of different materials were obtained.
[0061] The wheat samples tested were homozygous offspring of wheat gene-edited materials and homozygous offspring of wheat gene-unedited materials.
[0062] FC60-1A, FC60-2A, and FC60-3A are samples of T5 generation homozygous offspring of wheat gene-edited materials (mixed samples of 10 plants).
[0063] YM25-1A and YM25-2A are T5 generation homozygous progeny of unedited wheat materials (mixed samples of 10 plants).
[0064] The T5 generation homozygous progeny of the wheat gene-edited materials were all wheat materials with the following mutations: * **G** at position 663,217,923 on chromosome 5A of the *TraesCS5A02G494800* gene of Yangmai 25 genome sequence, corresponding to position 663,217,923 in the reference genome (common wheat, Chinese spring); * **A** at position 483,067,668 on chromosome 4D of the reference genome (common wheat, Chinese spring) of TraesCS4D02G319100 gene, corresponding to position 483,067,668 in the reference genome (common wheat, Chinese spring) of TraesCS4B02G322100 and TraesCS4B02G322700 genes, corresponding to positions 612,856,493-613,160,110 (304KB in size) on chromosome 4B of the reference genome (common wheat, Chinese spring) of TraesCS4B02G322100 and TraesCS4B02G322700 genes, corresponding to positions 612,856,493-613,160,110 in the reference genome (common wheat, Chinese spring) of TraesCS4B02G322100 and TraesCS4B02G322700 genes, with all other nucleotide residues remaining unchanged.
[0065] The T5 homozygous progeny of the unedited wheat materials were all wheat materials in which the genes TraesCS5A02G494800, TraesCS4D02G319100, TraesCS4B02G322700 and TraesCS4B02G322100 in the Yangmai 25 genome sequence had not been edited in any way.
[0066] 2. Efficiency verification of the capture probe The sequencing results obtained in step 1 above are used in the BWA software (0.7.17-r1198) MEM module to align the cleaned sequencing data to the reference genome; the original aligned BAM files are then merged, sorted, and indexed using the SAMTools v1.8 software to obtain basic alignment data information.
[0067] The visualization screenshots of the sequencing results of the six probe target regions are as follows: Figures 1-6 As shown in the figure, PROBE2, PROBE5, DEL_303KB_L, DEL_303KB_R, PROBE13, and PROBE17 can capture gene editing targets and are used to verify the occurrence of gene editing events.
[0068] The capture efficiency of probes at different locations is shown in Table 2. Due to the large-segment genomic deletion event in the wheat gene-edited material, the average capture depth of the probe region was about 129X, while the average depth of the probe region in the unedited wheat gene material was 228X.
[0069] Table 2. Statistics on probe capture efficiency at different locations
[0070] 3. Mutation event detection The BAM file obtained in step 2 above, combined with the BED file of the target editing region, is used to identify the SNP / INDEL of the editing region using GATK (v 4.1.2) software and bedtools software.
[0071] The results of the mutation event detection by the capture probe are shown in Table 3. FC6-1A, FC60-2A, and FC60-3A are wheat with MLO gene editing, while YM25-1A and YM25-2A are wheat without MLO gene editing. It can be seen that the probe designed in this invention can successfully detect the target gene editing products containing a large fragment (304KB) of deletion in the genome sequence.
[0072] Table 3 shows the SNP / INDEL results for probe-captured target regions.
[0073] The table above lists, from left to right, the chromatids containing the SNP / INDELs of the target regions, their physical locations, the base types of the reference genome (bases in wild-type wheat), the variant base types (bases in wheat gene-editing materials), and the variant types of the SNPs / INDELs in YM25-1A, YM25-2A, FC60-1A, FC60-2A, and FC60-3A. For each variant type, the two numbers following the variant type represent the number of reads supporting the reference genome base type and the number of reads supporting the variant base type, respectively. For example, "A / A:70,0" indicates that the base type of this material is A / A, with 70 reads supporting the reference genome base type (A) at that location; no reads support the variant base type (AC) at that location.
[0074] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A probe composition comprising probe A, probe B, probe C, probe D, probe E and probe F; The nucleotide sequence of probe A includes sequence 1; The nucleotide sequence of probe B includes sequence 2; The nucleotide sequence of probe C includes sequence 3; The nucleotide sequence of probe D includes sequence 4; The nucleotide sequence of probe E includes sequence 5; The nucleotide sequence of probe F includes sequence 6.
2. The probe composition according to claim 1, characterized in that: The probe composition is used to detect wheat gene-edited material; the wheat gene-edited material is wheat in which the MLO gene has been edited.
3. The probe composition according to claim 2, characterized in that: The wheat whose MLO gene has been edited is wheat whose chromosome spatial structure has been altered due to a large deletion of the MLO gene.
4. A chip comprising the probe composition according to any one of claims 1-3.
5. The chip according to claim 4, characterized in that: The chip is a liquid-phase probe hybridization chip.
6. The use of the probe composition according to any one of claims 1-3 in the design, development or preparation of chips for detecting wheat gene-edited materials; wherein the wheat gene-edited material is wheat in which the MLO gene has been edited.
7. The application according to claim 6, characterized in that: The chip is a liquid-phase probe hybridization chip; Alternatively, the wheat in which the MLO gene has undergone gene editing is wheat in which a large segment of the MLO gene has been deleted, resulting in changes in the spatial structure of the chromosome.
8. The use of the probe composition according to any one of claims 1-3 or the chip according to claim 4 or 5 in any one of the following M1)-M6): M1) detects whether gene editing has occurred in wheat gene-edited materials or their offspring; M2) Prepare products to detect whether gene editing has occurred in wheat gene-edited materials or their offspring; M3) distinguishes between gene-edited wheat materials and unedited wheat materials; M4) Prepare products that distinguish between wheat gene-edited materials and wheat gene-unedited materials; M5 wheat breeding; M6) is used to prepare wheat breeding products; The wheat gene-editing material is wheat from which the MLO gene has been edited; The unedited wheat material refers to wheat whose MLO gene has not been edited.
9. Any of the following methods (N1)-N3): N1) A method for detecting whether gene editing has occurred in wheat gene-edited material or its offspring, comprising the following steps: using the chip described in claim 4 or 5 to detect whether gene editing has occurred in wheat gene-edited material or its offspring; N2) A method for distinguishing between wheat gene-edited materials and wheat gene-unedited materials, comprising the following steps: using the chip described in claim 4 or 5 to detect the wheat to be tested, thereby distinguishing between wheat gene-edited materials and wheat gene-unedited materials; N3) A method for wheat breeding, comprising the following steps: a step of breeding using wheat gene-edited material obtained according to the method described in N1) as a parent; The wheat gene-editing material is wheat from which the MLO gene has been edited; The unedited wheat material refers to wheat whose MLO gene has not been edited.
10. The application according to claim 8 or the method according to claim 9, characterized in that: The wheat whose MLO gene has been edited is wheat whose chromosome spatial structure has been altered due to a large deletion of the MLO gene.