Oligonucleotide probe library for tracking v-chromosome in wheat disease-resistant breeding and application thereof

Abstract

The application belongs to the technical field of molecular cytogenetics and crop disease-resistant breeding, and particularly relates to an oligonucleotide fluorescence in situ hybridization probe library capable of specifically recognizing and staining all 1-7 chromosomes of an important donor of a wheat disease-resistant gene, i.e., hordeum bogdanii, wherein the probe library comprises seven oligonucleotide probes capable of specifically staining the 1-7 chromosomes of the hordeum bogdanii, respectively; and the probe library can be applied to rapidly and accurately identifying the V genome exogenous chromosome and the excellent disease-resistant gene carried by the V genome exogenous chromosome in a wheat disease-resistant breeding material.

Description

Technical Field

[0001] This invention belongs to the field of molecular cytogenetics and crop disease resistance breeding technology, specifically relating to a key donor of wheat disease resistance genes—*Triticum aestivum* (also known as tufted wheat). Dasypyrum villosum This study describes an oligonucleotide fluorescent in situ hybridization probe library for all chromosomes 1-7, the preparation method of the probe library, and its application in the rapid and accurate identification of exogenous chromosomes of the V genome and the superior disease-resistant genes they carry in wheat disease-resistant breeding materials. Background Technology

[0002] tufted wheat ( Dasypyrum villosum *L.) P. Candargy, 2n = 2x = 14, VV* is a valuable wild relative in the wheat tertiary gene pool. This species is a cross-pollinated annual with highly heterozygous chromosomes within individuals (Wu et al., 2022), which introduces unique complexity to the exploration of its genetic resources and cytogenetic analysis. *L.) Candargy is rich in superior genes, especially renowned for its excellent disease resistance. For example, the major gene for powdery mildew resistance located on its 6VS chromosome arm... Pm21 This gene has become one of the most widely used resistance sources in wheat disease resistance breeding in my country. The translocation line T6VS·6AL containing this gene has been widely used in wheat disease resistance breeding in China and has achieved remarkable results (Cao et al., 2011). However, the systematic exploration and utilization of valuable genetic resources (such as other disease resistance, stress resistance and quality-related genes) carried on other chromosomes of this species are still severely restricted.

[0003] At the cytogenetic identification level, accurately identifying and tracking superior chromosomes or fragments from *Triticum aestivum* in derived materials (such as addition lines, substitution lines, and translocation lines) after their introduction into wheat is crucial for breeding selection and gene localization. While traditional genomic in situ hybridization (GISH) can distinguish the V genome from the wheat A, B, and D genomes, its resolution is limited, making it impossible to precisely identify specific chromosome numbers (e.g., distinguishing between 1V and 6V). Furthermore, FISH probes based on conserved repetitive sequences (such as Oligo-pSc119.2) exhibit complex signal distribution patterns on *Triticum aestivum* chromosomes and lack chromosome specificity, failing to achieve clear identification of individual chromosomes. Therefore, developing a probe tool capable of high-resolution, specific visualization of each *Triticum aestivum* chromosome has become an urgent technical need for accurately tracking exogenous genetic material and accelerating the breeding process.

[0004] Oligo-Painting FISH technology offers an ideal solution to this challenge. This technology utilizes a large number of single-copy oligonucleotide sequences covering the target chromosome as probes, enabling clear and specific labeling of the entire chromosome with advantages such as strong signal, low background, and multiplex detection. The successful implementation of this technology relies entirely on a high-quality chromosome-level reference genome sequence of the target species to screen for chromosome-specific single-copy sequences. With the decoding of the chromosome-level reference genome of *Trichoderma truncatula*, the systematic development of oligonucleotide painting probe libraries covering all chromosomes 1-7 has become possible. However, there are currently no reports of single-copy oligonucleotide probe libraries for *Trichoderma truncatula*, both domestically and internationally, resulting in a lack of technical means for precise manipulation and identification of *Trichoderma truncatula* genetic material at the single-chromosome level. Summary of the Invention

[0005] The purpose of this invention is to provide an oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding and its application. Based on the chromosome-level reference genome of Triticum aestivum, it provides an oligonucleotide probe library that can specifically identify and stain all 1V-7V chromosomes, a standardized preparation method for the probe library, and its application scheme in related breeding and genetic research.

[0006] The technical solution of the present invention to achieve the above objectives is as follows.

[0007] An oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding contains seven oligonucleotide probes that can specifically stain chromosomes 1-7 of *Triticum aestivum*, respectively; the oligonucleotide sequences of the seven oligonucleotide probes are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0008] The construction principle and method of an oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding are as follows, namely, it is obtained through the following steps.

[0009] (1) Based on the high-quality chromosome-level reference genome sequence of Triticum aestivum, a set of chromosome-specific single-copy oligonucleotide sequences were screened for each chromosome, with each sequence being 45 bp in length; (2) Add non-repeating specific primer sequences to both ends of each oligonucleotide sequence, including two pairs of primers. The outer primer can specifically amplify the single copy oligonucleotide probe of the entire chromosome set, and the inner primer can specifically amplify the single copy oligonucleotide probe of a single chromosome. The primer length is 20 bp. (3) Use the corresponding fluorescently labeled primers to perform PCR amplification on the template library. The resulting product is the specific staining probe of the chromosome corresponding to the primer.

[0010] Furthermore, the set of single-copy oligonucleotide sequences corresponding to each chromosome is different from the set of corresponding sequences of the other six chromosomes in the same species, ensuring hybridization specificity.

[0011] An oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding can be applied to chromosome identification and karyotype analysis of diploid, addition, substitution, and translocation lines formed by distant hybridization of wheat and tufted wheat.

[0012] Furthermore, it can be applied to studies including the polyploid evolution of the wheat tribe, revealing the mechanisms of genome evolution by tracing the staining of the Triticum aestivum V genome chromosome against a polyploid background.

[0013] Furthermore, it can be applied to cytogenetic analysis of wheat-related materials, such as intermediate wheatgrass, which contain a wider range of V genomes.

[0014] The beneficial effects of the oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding and its application are as follows: (1) For the first time, a staining probe library of all chromosomes 1-7 of wheat was constructed, realizing the specific identification of a single chromosome of wild wheat species; (2) The probe library is designed based on chromosome-specific single-copy sequences, with high resolution and strong specificity, which can effectively overcome the bottleneck that traditional repetitive sequence probes cannot distinguish chromosomes within homologous groups. (3) The probe library adopts a modular design, and probes for any target chromosome can be flexibly prepared through a universal primer system. It is easy to operate, has good repeatability, and usually does not require additional DNA blocking during FISH detection. (4) By using this probe library, exogenous chromosomes or fragments in distant hybridization-derived materials can be quickly and intuitively identified, their identity and structure can be accurately determined, and the screening efficiency of chromosome engineering breeding can be greatly improved. (5) This technology provides a powerful cytological tool for the study of polyploid evolution in the wheat tribe, enabling visualization of the interaction and variation of different genomes during allopolyploidization at the level of a single chromosome, and deepening the understanding of the laws of genome evolution; (6) The whole system has wide applicability. It can be used not only for karyotype analysis of basic species, but also for cytogenetics research of a series of polyploid closely related species and their complex derivative materials. Attached Figure Description

[0015] Figure 1 This refers to the number of single-copy oligonucleotide probes on each chromosome in the V genome in this invention.

[0016] Figure 2This indicates the location of each single-copy oligonucleotide probe in the V genome on the chromosome in this invention.

[0017] Figure 3 The image shows the staining effect of the oligonucleotide staining probe provided for the implementation of this invention on the chromosomes of Triticum aestivum during metaphase of mitosis, specifically demonstrating the specific staining results of chromosomes 1-7 of Triticum aestivum. Detailed Implementation

[0018] Example 1: Development of a chromosome staining probe for *Triticum aestivum*.

[0019] This invention focuses on *Triticum aestivum*, an important wild relative of wheat, and develops a library of oligonucleotide staining probes that can specifically identify chromosomes 1-7 of it.

[0020] The specific development steps are as follows.

[0021] (1) Obtain the chromosome-level reference genome sequence of Triticum aestivum (Genus truncatum accession number GWHBJXA00000000) and use bioinformatics software such as Repeat Masker to mask all transposon and other repetitive sequence regions.

[0022] (2) From the masked genome sequence, a set of chromosome-specific single-copy oligonucleotide sequences were screened for each V chromosome using professional software such as Chorus2. The parameters were set as follows: oligonucleotide length 45 bp, sliding window step size 15 nt.

[0023] (3) The candidate probe sequences obtained in step (2) are strictly compared with the whole genome of Triticum aestivum to screen out oligonucleotide sequences with unique and precise matching sites in the whole genome, ensuring that each probe corresponds to only one specific position on a chromosome.

[0024] (4) The probes retained after the above screening are assigned to the corresponding chromosomes 1-7 according to their unique genomic matching positions, forming 7 preliminary chromosome-specific probe sets. To ensure that each chromosome can be uniformly stained, each chromosome is divided into several equal-length intervals, from which probes are selected evenly to ensure that the final probe density reaches no less than 100 probes per megabase (Mb) and to ensure that the entire chromosome is covered continuously without gaps.

[0025] (5) For each final selected 45 bp specific oligonucleotide sequence, add a 20 bp inner primer and an outer primer at its 5' and 3' ends, respectively. The outer primer is used to amplify single-copy oligonucleotide probes of the entire genome, and the inner primer is used to specifically amplify single-copy oligonucleotide probes of a single chromosome. The primer sequence has no significant homology with the genomes of all target species and wheat. The complete sequence set of all “outer primer-inner primer-specific sequence-inner primer-outer primer” structures constitutes the *Triticum aestivum* chromosome painting probe pool and is submitted to the company (Beijing Qingke Biotechnology Co., Ltd.) for oligonucleotide synthesis. The number of single-copy oligonucleotide probes of the V genome and their positions on the chromosome are as follows. Figure 1 and Figure 2 As shown.

[0026] The specific oligonucleotide sequences on each synthesized chromosome are shown below.

[0027] The oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 1 of *Trichoderma truncatum* are shown in SEQ ID NO:1; the oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 2 of *Trichoderma truncatum* are shown in SEQ ID NO:2; the oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 3 of *Trichoderma truncatum* are shown in SEQ ID NO:3; the oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 4 of *Trichoderma truncatum* are shown in SEQ ID NO:4; the oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 5 of *Trichoderma truncatum* are shown in SEQ ID NO:5; the oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 6 of *Trichoderma truncatum* are shown in SEQ ID NO:6; and the oligonucleotide sequences of the oligonucleotide probes that specifically recognize chromosome 7 of *Trichoderma truncatum* are shown in SEQ ID NO:7.

[0028] Example 2: Preparation of Oligo-Painting probe.

[0029] The preparation steps of the fluorescent probe are as follows: (1) Different chromosome-specific single-copy oligonucleotides were coupled with specific primers at both ends, and the oligonucleotide libraries were synthesized by Beijing Qingke Biotechnology Co., Ltd. The probe libraries were diluted to 1 ng / μL with ddH2O and stored at -80℃. The specific primer sequences added to both ends of the chromosome-specific single-copy oligonucleotide sequences are shown in the table below.

[0030]

[0031] In the table above, V is a pair of outer primers. Using this pair of primers, all probes on chromosomes 1-7 can be amplified. Theoretically, this pair of primers can make the probe library usable indefinitely.

[0032] (2) Take 10 μL of probe stock solution and dilute it with 190 μL of ddH2O to a working solution of 0.05 ng / μL. 50 μL PCR system: 25 μL KAPA HiFi HotStart ReadyMix, 3 μL probe library working solution, 3 μL each of forward and reverse primers, and 16 μL ddH2O. The amplification program is shown in the table below.

[0033]

[0034] (3) The PCR product obtained in the previous step was purified using the GeneJET PCR Purification kit. First, an equal volume of Binding Buffer was added and mixed. Then, an equal volume of isopropanol was added and mixed. 150 μL of the sample was transferred to a GeneJET purification column and centrifuged at 12000 rpm for 1 min. The waste liquid was discarded, and the adsorption column was returned to the collection tube. 700 μL of Washing Buffer was added to the adsorption column, and centrifuged at 12000 rpm for 1 min. The waste liquid was discarded, and the adsorption column was returned to the collection tube and centrifuged at 12000 rpm for 1 min. The adsorption column was placed in a 1.5 mL clean centrifuge tube, and 50 μL of Elution Buffer was added to the middle of the adsorption column. The column was centrifuged at 12000 rpm for 2 min. A probe solution that can be directly used for fluorescence in situ hybridization was obtained. The probe concentration was measured using a spectrophotometer, and the expected concentration was 300-500 ng / μL.

[0035] Example 3: Oligo-Painting.

[0036] (1) Hybridization solution preparation system: 10 μL of 100% deionized formamide, 2 μL of 20× sodium citrate buffer (SSC), 4 μL of 50% dextran sulfate (DS), and 1 μL of oligonucleotide probe. After boiling the hybridization solution in a water bath for 6 min, immediately place it on ice for at least 5 min.

[0037] (2) Fluorescent in situ hybridization: The prepared hybridization solution was dropped onto the prepared chromosome slide, covered with a coverslip, and placed in an in situ hybridization instrument at 85°C for 5 min 30 s. After sealing with mounting adhesive, the slide was placed in a humidification box and incubated overnight in a constant temperature incubator at 37°C.

[0038] (3) Slide washing and microscopic examination: Remove the coverslip, wash with 2×SSC at room temperature, 2×SSC at 42℃, 2×SSC at room temperature, 70% ethanol, and ddH2O for 5 min each. After drying with a hair dryer, add 10 μL of DAPI (4',6-diamidinyl-2-phenylindole), cover with the coverslip, and examine under a fluorescence microscope.

[0039] The Oligo-Painting probe library designed and constructed in this invention exhibits high species specificity and chromosome recognition accuracy. Using this probe library, chromosomes of Oligo-Painting can be precisely identified at the cellular level (e.g., ...). Figure 3 (As shown). This technology not only enables precise karyotype analysis of *Triticum aestivum* itself, clarifying its basic characteristics such as chromosome number and morphology, but also has wide applications in the accurate identification of genetic materials such as diploids, addition lines, substitution lines, and translocation lines created by distant hybridization with wheat. Furthermore, it provides a crucial cytogenetic tool for analyzing chromosome behavior during the polyploid evolution of the wheat tribe and accelerating chromosome engineering breeding.

[0040] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding, characterized in that, It contains 7 oligonucleotide probes that can specifically stain chromosomes 1-7 of *C. pubescens*, respectively; the oligonucleotide sequences of the 7 oligonucleotide probes are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

2. The oligonucleotide probe library as described in claim 1, characterized in that, Obtain it through the following steps: (1) Based on the high-quality chromosome-level reference genome sequence of Triticum aestivum, a set of chromosome-specific single-copy oligonucleotide sequences were screened for each chromosome, with each sequence being 45 bp in length; (2) Add non-repeating specific primer sequences to both ends of each oligonucleotide sequence, including two pairs of primers. The outer primer can specifically amplify the single copy oligonucleotide probe of the entire chromosome set, and the inner primer can specifically amplify the single copy oligonucleotide probe of a single chromosome. The primer length is 20 bp. (3) Use the corresponding fluorescently labeled primers to perform PCR amplification on the template library. The resulting product is the specific staining probe of the chromosome corresponding to the primer.

3. The oligonucleotide probe library as described in claim 2, characterized in that, The set of single-copy oligonucleotide sequences corresponding to each chromosome is different from the set of corresponding sequences of the other six chromosomes in the same species.

4. The application of an oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding as described in any one of claims 1-3, characterized in that, It is applied to chromosome identification and karyotype analysis of double diploid, addition, substitution, and translocation lines formed by distant hybridization of wheat and tufted wheat.

5. The application of an oligonucleotide probe library for V chromosome tracking in wheat disease resistance breeding as described in any one of claims 1-3, characterized in that, Used for research on the polyploid evolution process and genome evolution mechanism of the wheat tribe.

Images