Oligonucleotide-coated fluorescence in-situ hybridization probe for specifically recognizing Chr1-4 chromosome of tartary buckwheat and application of oligonucleotide-coated fluorescence in-situ hybridization probe

Abstract

The invention discloses an oligonucleotide-coated fluorescence in-situ hybridization probe for specifically recognizing a tartary buckwheat Chr1-4 chromosome and application, and belongs to the field of cytological markers. The method comprises the following steps: screening a single-copy oligonucleotide sequence of Chr1-4 from a tartary buckwheat whole genome by using Chorus2 software; filtering and removing repetitive sequences to obtain a Chr1-4 chromosome probe sequence library; screening a specific single copy oligonucleotide sequence of the Chr1-4 chromosome; and adding specific primers at the two ends of each single copy oligonucleotide sequence of Chr1-4, and combining the probes into a buckwheat Chr1-4 Oligo-Painting probe mixing pool. By utilizing the constructed Oligo-Painting probe mixing pool and a fluorescence in-situ hybridization technology, accurate identification and karyotype analysis of the buckwheat chromosomes can be realized, the number, morphology, heritable variation and other structural characteristics of the chromosomes can be known, and an important technical support is provided for cytological analysis of buckwheat crossbreeding.

Description

Technical Field

[0001] The present invention relates to the field of cytological markers, and in particular to an oligonucleotide-painted fluorescent in situ hybridization probe for specifically identifying tartary buckwheat Chr1-4 chromosomes and its application. Background Art

[0002] Buckwheat, a genus of the Polygonaceae family (Fagopyrum), has a short growth period, strong adaptability, and high nutritional and medicinal value. Rich in flavonoids, it possesses free radical scavenging, antioxidant, anti-inflammatory, and anti-cancer properties (He et al., 2024). The main cultivated species are sweet buckwheat, tartary buckwheat, and golden buckwheat, which is widely cultivated as a medicinal plant (Zhang et al., 2023). my country is a major producer and consumer of buckwheat, with annual tartary buckwheat production ranking first in the world (FAO, 2016).

[0003] Buckwheat is a microchromosome species, with chromosome lengths ranging from 1 to 3 μm. Homologous chromosomes 1–8 show little variation. Furthermore, due to its dense cytoplasm, obtaining clear chromosome specimens is challenging, making traditional karyotype analysis difficult to provide accurate cytological information (Zhang Jiupan et al., 2019; Chen Qingfu, 2010). Current cytogenetic research on buckwheat is primarily limited to basic chromosome counting and traditional karyotype analysis. These methods reveal only basic information about the number, morphology, and size of buckwheat chromosomes, but are unable to effectively and deeply analyze more complex genetic characteristics, such as genomic inheritance and variation.

[0004] Fluorescence in situ hybridization (FISH) has been widely used in plant genome research due to its efficient cytogenetic properties, particularly in wheat, cotton, and sugarcane (Wang et al., 2013; Li et al., 2021; Yu et al., 2024). Using specially designed probes, specific locations on chromosomes can be precisely located, enabling accurate analysis and identification of plant chromosome structural characteristics, chromosome group classification, karyotype, and the presence of exogenous chromosomes (Chen et al., 2023; Meng et al., 2024). Implementing this technique for plant chromosome analysis requires the development of specific and identifiable fluorescent probes, which are essentially physical markers of chromosomes. However, physical markers for buckwheat chromosomes are currently scarce, with few available, aside from 45S rDNA and 5S rDNA (Sheng Maoyin, 2013; Wang Tian, ​​2007). Due to the lack of effective identification markers, the identification of buckwheat chromosomes faces major challenges. Compared with cultivated crops with large chromosomes (greater than 12μm) such as wheat, the progress of buckwheat chromosome research lags significantly. In the absence of appropriate chromosome physical markers as an auxiliary means, accurate identification of buckwheat chromosomes becomes extremely difficult. It can be said that the lack of buckwheat chromosome physical markers has become a key technical problem hindering the development of buckwheat plant cell genetics. Therefore, the successful development of probes that can identify single chromosomes will greatly promote the development of buckwheat chromosome-related research.

[0005] Oligonucleotides refer to short nucleotide sequences that are usually in the range of tens of bases in length. Oligo-FISH technology uses single-copy oligonucleotides as probes and designs an Oligo probe library based on sequenced genomes or unsequenced second-generation sequencing data. Based on the experimental objectives, probes covering the entire chromosome or specific regions of the chromosome are selected to effectively identify and distinguish homologous chromosome groups from different species or subspecies (Li et al., 2021; Chen et al., 2024; Meng et al., 2024). During the design process, Oligos probes strictly remove repetitive sequences and genomic homologous sequences to ensure specific recognition of chromosomes or chromosome segments. The probe design is flexible and can be designed to cover the entire chromosome, partial fragments of the chromosome, multiple regions of the chromosome, or different regions of different chromosomes according to research needs. Among the species of the genus Fagopyrum, the reference genomes of Fagopyrum tataricum (approximately 0.48 Gb), Fagopyrum serrata (approximately 1.33 Gb), and Fagopyrum chinense (approximately 1.08 Gb) have been sequenced and assembled (Zhang et al., 2018; He et al., 2022; He et al., 2023). Despite the comprehensive genome data, an oligo-painting FISH system capable of specifically identifying chromosomes has not yet been established. Summary of the Invention

[0006] The purpose of the present invention is to provide an oligonucleotide painting fluorescence in situ hybridization probe for specifically identifying tartary buckwheat Chr1-4 chromosomes and its application, so as to solve the problems existing in the above-mentioned prior art. By screening a specific single-copy oligonucleotide sequence from the whole genome of tartary buckwheat as a probe, and adding specific primers at both ends to construct a buckwheat oligo-painting probe pool, the buckwheat chromosome identification and karyotype analysis can be achieved by using this probe pool and fluorescence in situ hybridization technology.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an oligonucleotide-painted fluorescent in situ hybridization probe for specifically identifying tartary buckwheat chromosomes. The fluorescent in situ hybridization probe is prepared by the following method:

[0009] Based on the tartary buckwheat reference genome, single-copy oligonucleotide sequences specific to each chromosome 1-4 were screened, and specific primers were added to both ends of each single-copy oligonucleotide sequence to construct a probe library;

[0010] amplifying and fluorescently labeling the probe library to obtain oligonucleotide-stained fluorescent in situ hybridization probes that specifically identify buckwheat chromosomes;

[0011] The single copy oligonucleotide sequence is located on each chromosome 1-4 of the whole buckwheat genome, including a segment of 0-62023384 bp on chromosome 1, a segment of 0-60833585 bp on chromosome 2, a segment of 0-58607543 bp on chromosome 3, and a segment of 0-56693299 bp on chromosome 4. Specific single copy oligonucleotide sequence probes with a length of 45 bp were designed using the software Chorus2 and the buckwheat genome, respectively. The specific gene information of the specific single copy oligonucleotide sequence probes on each chromosome 1-4 of the buckwheat reference genome is as follows:

[0012] The designed Chr1-specific single copy oligonucleotide sequence probes include, but are not limited to, 5'-GGCAAGCATCACATTTCAAGCATTCTGACATTGACCGCAGACTTC-3' (SEQ ID NO. 9) or 5'-GTCTTCCACCATTATCAGCTTGATGTGTACTATATGTTGATATTA-3' (SEQ ID NO. 10);

[0013] The designed Chr2-specific single-copy oligonucleotide sequence probes include, but are not limited to, 5'-TGCGCTCGGATGTCGGAACGGGGCGGACGATACACGGAAACGACC-3' (SEQ ID NO. 11) or 5'-AAAGATAGAAGAAGCTCAAAGGAATCCTAGTTTCGCAAGGGATCA-3' (SEQ ID NO. 12);

[0014] The designed Chr3-specific single-copy oligonucleotide sequence probes include, but are not limited to, 5'-AAGGCTAGAAAATCTAACATTATATTCATCACCGTATTCCAATAC-3' (SEQ ID NO. 13) or 5'-AGTACATCAACTGAATAATGTCAAAACATGCTCGAGTATCTGCAA-3' (SEQ ID NO. 14);

[0015] The designed Chr4-specific single copy oligonucleotide sequence probes include, but are not limited to, 5'-ATTGTACATGGGAAGCATCAAGATCCAAATGTATGTATTCCTGGG-3' (SEQ ID NO. 15) or 5'-GGTATAATATGCGTAGATTATTCGTTTATGAAATCATTTTATTAT-3' (SEQ ID NO. 16).

[0016] Optionally, the accession number of the tartary buckwheat reference genome in Genbank is PRJNA381676.

[0017] Optionally, the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 1 are as shown in SEQ ID NOs: 1-2; the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 2 are as shown in SEQ ID NOs: 3-4; the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 3 are as shown in SEQ ID NOs: 5-6; the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 4 are as shown in SEQ ID NOs: 7-8.

[0018] Optionally, the single copy oligonucleotide sequence specific to each chromosome is different from the single copy oligonucleotide sequences of the other three chromosomes.

[0019] Optionally, the length of each chromosome-specific single copy oligonucleotide sequence is 45 bp.

[0020] Optionally, the specific primer does not overlap with the single copy oligonucleotide sequence specific to each chromosome and meets the requirements of PCR primers, and the sequence length of the specific primer is 15 to 24 bp.

[0021] The present invention also provides an application of the fluorescent in situ hybridization probe in buckwheat chromosome identification or karyotype analysis.

[0022] Optionally, the buckwheat comprises tartary buckwheat.

[0023] The present invention also provides a product for buckwheat chromosome identification or karyotype analysis, comprising the fluorescent in situ hybridization probe.

[0024] Optionally, the product comprises chips; and / or the buckwheat comprises tartary buckwheat.

[0025] The present invention discloses the following technical effects:

[0026] The present invention screens single-copy oligonucleotide sequences specific to each chromosome as candidate probes based on the whole genome of tartary buckwheat. Specific primers are then added to both ends of each chromosome candidate probe to construct a probe library. Through DNA amplification and fluorescent labeling, oligonucleotide-based fluorescent in situ hybridization probes that specifically identify tartary buckwheat chromosomes are constructed. The probe library provided by the present invention is simple to construct, and probes from the probe library are used to perform in situ hybridization on tartary buckwheat mitotic metaphase chromosomes. After staining and counterstaining, the results show that the fluorescent signal is clear and bright, easy to detect, and highly reproducible. High-resolution cytogenetic karyotypes of tartary buckwheat chromosomes can be obtained, demonstrating strong applicability.

[0027] The oligonucleotide probes disclosed in this invention are highly specific and accurate, capable of precisely identifying specific sequences on buckwheat chromosomes and enabling accurate chromosome identification. Using oligonucleotide probe technology, buckwheat karyotypes can be analyzed to understand structural characteristics such as chromosome number, morphology, inheritance, and variation, providing important technical support for cytological identification in buckwheat hybrid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The chromosome painting results of the Oligo-Painting probe library on buckwheat are shown in Figure 2. The scale size is 2 μm. A shows the chromosome painting results of Chr1 (green) and Chr2 (red) on buckwheat. B shows the chromosome painting results of Chr3 (green) and Chr4 (red) on buckwheat. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The English and Chinese interpretations of this invention:

[0033] Oligonucleotide: Oligo;

[0034] Oligo-Painting FISH: Oligo-Painting FISH.

[0035] Example 1 Development of Oligo-Painting Probe

[0036] (1) Using Pinku 1 as the reference genome (Genbank accession number PRJNA381676), an oligo probe library was constructed using Chorus2 software. Oligo sequences of Chr1-4 were screened from the whole genome sequence of Tartary Buckwheat. All the selected oligos had more than 75% homology with the Tartary Buckwheat genome.

[0037] (2) The tartary buckwheat Chr1-4 genomic oligonucleotide sequences extracted from the results of step (1) were compared with the Jiujiang tartary buckwheat second-generation sequencing data to screen out oligonucleotide sequences with 100% homology.

[0038] (3) All repeated sequences are filtered from the oligonucleotide sequences of step (2), and the obtained probe sequences are grouped by chromosomes and compared with each other in pairs to obtain a single copy oligonucleotide sequence library specific for Chr1-4 chromosomes.

[0039] (4) Each chromosome is divided into 10 regions, and single-copy oligonucleotide sequences are evenly selected from the 10 regions to maintain a consistent density and ensure that the single-copy oligonucleotide sequences can evenly cover the entire chromosome.

[0040] (5) Compare the single copy oligonucleotide sequence library of each chromosome with each primer to ensure that the primer sequence is not identical to any single copy oligonucleotide sequence of any chromosome.

[0041] (6) Oligos probe library was designed on chromosomes 1-4 of buckwheat. The probe was labeled to form a double-stranded probe. After high-temperature denaturation and hybridization with the metaphase chromosomes of the buckwheat root tip, red or green fluorescence was produced on chromosomes 1-4 as a signal. The red and green colors can change depending on the different fluorescent groups carried by the primers. The four pairs of chromosome-specific Oligo-Painting probes were composed of 7125, 6989, 6733, and 6513 single-copy oligonucleotides, respectively, for a total of 27,359 single-copy oligonucleotides. There are about 115 single-copy oligonucleotide sequences per 1 Mb. The regional position and length of the single-copy oligonucleotides on the chromosome are shown in Table 1.

[0042] Table 1 Regional location and length of single copy oligonucleotides on chromosomes

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[0044]

[0045] (7) Specific primers are added before and after each selected single-copy oligonucleotide sequence to combine the probes into a mixed pool of buckwheat oligo-painting probes.

[0046] Each single copy oligonucleotide sequence is 45 bp, and the starting position of each sequence in the pool in the buckwheat genome is as follows;

[0047] Table 2 Starting position of Chr1Oligos sequence

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[0065] Table 3 Starting position of Chr2Oligos sequence

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[0083] Table 4 Starting position of Chr3Oligos sequence

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[0100] Table 5 Starting position of Chr4Oligos sequence

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[0117] Example 2 Amplification of Oligo-Painting Probe Library

[0118] The amplification method includes the following steps:

[0119] (1) Single-copy oligonucleotides specific for different chromosomes have specific primers at both ends. Single-copy oligonucleotide libraries were synthesized in batches by GenScript BiotechCorp (Nanjing, China) to obtain probe library chips. Oligo-Painting probe library chips were diluted into a 2 ng / μL stock solution and stored in a -20°C refrigerator. TAMRA or FAM was used to fluorescently modify the 5' ends of the front and back primers. PCR amplification was performed according to the following system and procedure. The front and back primer sequences were set based on the principle that the F end was consistent and the R end was reverse complementary, as shown in Table 6 below.

[0120] Table 6 Probe primer sequence information

[0121]

[0122] (2) 50 μL amplification system: 2 μL of oligonucleotide probe library working solution (2 ng / μL), 2 μL of each primer (100 μM / L), 25 μL of KAPA HiFi HotStart ReadyMix enzyme, and 19 μL of ddH2O. The amplification program is shown in Table 7.

[0123] Table 7 Amplification program

[0124]

[0125] (3) Use Gene JET PCR Purification kit to purify the PCR product and use it as a probe. First, add an equal volume of Binding Buffer and mix it with 50 μL of PCR amplification product. Then add an equal volume of isopropanol (50 μL) to the PCR amplification product and mix it. Aspirate all the samples into the adsorption column and centrifuge for 50 seconds at 13,000 rpm. Discard the waste liquid and put the adsorption column back into the collection tube.

[0126] (4) Add 700 μL of Wash Buffer to the adsorption column and centrifuge for 60 seconds at 13,000 rpm. Discard the waste liquid, return the adsorption column to the collection tube, and spin for 1 minute at 13,000 rpm.

[0127] (5) Place the adsorption column in a clean 1.5 mL centrifuge tube. Add 50 μL of Elution Buffer to the center of the adsorption membrane and centrifuge for 1 min at 13,000 rpm. Measure the probe concentration using spectrophotometry; the expected probe concentration is between 400 and 800 ng / μL.

[0128] Example 3 Fluorescence in situ hybridization and signal detection

[0129] (1) Preparation of single copy oligonucleotide probe hybridization solution: The system is 3 μL each of the red and green probes prepared in Example 1, 2 μL of 20× sodium citrate buffer (SSC), 4 μL of 50% dextran sulfate, and 10 μL of deionized formamide. Mix by gently pipetting and centrifuge.

[0130] (2) Place in a 100°C water bath for 6 minutes, then immediately place in an ice box filled with ice for 5 minutes.

[0131] (3) Fluorescence in situ hybridization: Take a good chromosome preparation, add the prepared single copy oligonucleotide probe hybridization solution on the preparation, cover with a coverslip, place in an 85℃ hybridization oven for high temperature hybridization for 5 min 30 s, immediately remove the slide, seal the slide with mounting glue and quickly place the slide in a hybridization wet box, place in a moistened wet box and incubate in a 37℃ constant temperature box for hybridization culture for 20-24 h.

[0132] (4) Washing and microscopic examination: Remove the coverslip and wash the slides in 42°C 2×SSC, room temperature 2×SSC, ddH2O, and anhydrous ethanol for 5 min each. After drying, add 10 μL of LDAPI (4',6-diamidino-2-phenylindole) to the slides and seal them. Cover with a coverslip and observe them under a microscope after 5 min. Use an Olympus BX60 fluorescence microscope to examine and photograph mitotic metaphase chromosomes. The images are then processed using Photoshop.

[0133] The above method was used to detect single copy oligonucleotide probe signals on buckwheat mitotic metaphase chromosome preparations. The results are summarized as follows: Figure 1 As shown. The probes were applied to the tartary buckwheat cultivar Xiqiao 2. FISH results showed that all four chromosome-specific staining probes produced clear, bright fluorescence signals on two homologous chromosomes, confirming that these probes can specifically identify tartary buckwheat chromosomes (2n=2x=16). To validate the probe results, chromosomes 1-4 were found to be uniformly covered by the fluorescence signal. The probes were designed and developed based on tartary buckwheat genome data. Cytological results showed consistency with the tartary buckwheat genome assembly, indirectly confirming the correctness of the assembly results. Eight chromosomes were identified in the cells, and each of the four probes produced a distinct, specific hybridization signal on each of the four pairs of homologous chromosomes, demonstrating that the probes are chromosome-specific.

[0134] The oligo probe designed in this invention has high specificity and accuracy, accurately identifying specific sequences on buckwheat chromosomes and enabling accurate chromosome identification. Using oligo probe technology, buckwheat karyotype analysis can be performed to understand structural characteristics such as chromosome number, morphology, inheritance, and variation.

[0135] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A fluorescent in situ hybridization probe specifically identifying tartary buckwheat chromosomes, characterized in that: The fluorescent in situ hybridization probe is prepared by the following method: Screening single-copy oligonucleotide sequences specific to chromosomes 1-4 based on the tartary buckwheat reference genome, and adding specific primers at both ends of each single-copy oligonucleotide sequence to construct a probe library; amplifying and fluorescently labeling the probe library to obtain oligonucleotide-stained fluorescent in situ hybridization probes that specifically identify buckwheat chromosomes; The gene information of the single copy oligonucleotide sequence on chromosomes 1 to 4 of the tartary buckwheat reference genome is as follows; Single-copy oligonucleotide sequence probes that specifically recognize chromosome 1 include: 5′-GGCAAGCATCACATTTCAAGCATTCTGACATTGACCGCAGACTTC-3′ or 5′-GTCTTCCACCATTATCAGCTTGATGTGTACTATATGTTGATATTA-3′; The single-copy oligonucleotide sequence probes that specifically recognize chromosome 2 include: 5′-TGCGCTCGGATGTCGGAACGGGGCGGACGATACACGGAAACGACC-3′ or 5′-AAAGATAGAAGAAGCTCAAAGGAATCCTAGTTTCGCAAGGGATCA-3′; Single-copy oligonucleotide sequence probes that specifically recognize chromosome 3 include: 5′-AAGGCTAGAAAATCTAACATTATATTCATCACCGTATTCCAATAC-3′ or 5′-AGTACATCAACTGAATAATGTCAAAACATGCTCGAGTATCTGCAA-3′; The oligonucleotide sequence probes specifically identifying single copies on chromosome 4 include: 5'-ATTGTACATGGGAAGCATCAAGATCCAAATGTATGTATTCCTGGG-3' or 5'-GGTATAATATGCGTAGATTATTCGTTTATGAAATCATTTTATTAT-3'.

2. The fluorescence in situ hybridization probe according to claim 1, wherein The accession number of the tartary buckwheat reference genome in Genbank is PRJNA381676.

3. The fluorescence in situ hybridization probe according to claim 1, wherein The specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 1 are shown in SEQ ID NOs: 1-2; the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 2 are shown in SEQ ID NOs: 3-4; the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 3 are shown in SEQ ID NOs: 5-6; and the specific primers added to both ends of the single copy oligonucleotide sequence that specifically recognizes tartary buckwheat chromosome 4 are shown in SEQ ID NOs: 7-8.

4. The fluorescence in situ hybridization probe according to claim 1, wherein The single copy oligonucleotide sequence specific to each chromosome is different from the single copy oligonucleotide sequences of the other three chromosomes.

5. The fluorescence in situ hybridization probe according to claim 1, wherein The length of each chromosome-specific single-copy oligonucleotide sequence is 45 bp.

6. The fluorescence in situ hybridization probe according to claim 1, wherein The specific primer and the single copy oligonucleotide sequence specific to each chromosome do not repeat each other, and the sequence length of the specific primer is 15 to 24 bp.

7. Use of the fluorescent in situ hybridization probe according to any one of claims 1 to 6 in buckwheat chromosome identification or karyotype analysis.

8. The use according to claim 7, characterized in that The buckwheat includes tartary buckwheat.

9. A product for buckwheat chromosome identification or karyotype analysis, characterized in that: The method comprises the fluorescent in situ hybridization probe according to any one of claims 1 to 6.

10. The product according to claim 9, characterized in that The product comprises chips; and / or the buckwheat comprises tartary buckwheat.

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