CN111926005B - Probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome - Google Patents
Probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome Download PDFInfo
- Publication number
- CN111926005B CN111926005B CN201910392989.8A CN201910392989A CN111926005B CN 111926005 B CN111926005 B CN 111926005B CN 201910392989 A CN201910392989 A CN 201910392989A CN 111926005 B CN111926005 B CN 111926005B
- Authority
- CN
- China
- Prior art keywords
- probe set
- chromosome
- chrysanthemum
- hybridization
- oligonucleotide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides an oligonucleotide probe set designed based on a chrysanthemum plant repetitive sequence and discloses a chrysanthemum plant mitosis metaphase chromosome fluorescence in-situ hybridization method based on the oligonucleotide probe set. The method specifically comprises the following steps: preserving chromosome at-80 deg.C for 12h, removing sheet, dehydrating in anhydrous ethanol for more than 30min, air drying, denaturing in alkali denaturing solution at 44 deg.C for 5min, placing in 70.0%, and 100.0% ethanol at room temperature, sequentially dehydrating for 5min, and air drying; preparing FISH hybridization liquid, mixing uniformly, dripping the FISH hybridization liquid on a chromosome sheet, carrying out hybridization culture in an incubator at 37 ℃, and finally carrying out signal detection; the oligonucleotide probe set and the in-situ hybridization method developed by the invention can be effectively used for constructing the karyotype chart (figure 1) for chromosome analysis of chrysanthemum plants, and the detection is carried out by one-time FISH analysis through the assembled oligonucleotide probe set, so that repeated hybridization of one slide is avoided, the FISH analysis procedure is greatly simplified, and the experiment efficiency is improved.
Description
Technical Field
The invention belongs to the field of molecular cytogenetics research, and discloses a probe and a method for high-resolution fluorescence in situ hybridization of chromosomes in the metaphase of mitosis of chrysanthemum plants.
Background
Chrysanthemum morifolium (Chrysanthemum morifolium) originates from China, has various varieties, rich colors and various flower types, is one of ten traditional famous flowers in China and four cut flowers in the world, can be used for appreciation, medicine, eating and the like, and has high economic value. The chrysanthemum belongs to the chrysanthemum of the chamomile family of the Compositae (Asteraceae), and the chrysanthemum takes east Asia as a distribution center and shows higher genetic diversity.
Fluorescence In Situ Hybridization (FISH) is one of important means for analyzing ancestral genome source, cleaning interspecies evolution relation and analyzing chromosome composition, and is also an important means for identifying chromosome engineering breeding such as an ectopic line and an episome.
The conventional fluorescence in situ hybridization technology usually takes the whole genome of a macro gene, rRNA or Cot-1DNA as a probe to carry out in situ hybridization, however, because the genome relativity between different species in chrysanthemum is relatively close, the probe based on the whole genome of the chrysanthemum cannot be used for comparative genome research between chrysanthemum species with relatively close relativity and complex evolutionary relationship. The rRNA gene is useful in some degree for comparative genomic studies in chrysanthemum but provides limited information. Cot-1DNA technology is less sensitive, results of different repeats are different, and the current in situ hybridization probe based on denaturation technology has the disadvantages of complicated preparation method and process, high cost, less signal, and difficulty in distinguishing and identifying chromosome homology and structural variation from limited chromosome specific sites (Qi et al, 2015). Therefore, there is a need to develop more specific probes to differentiate the chromosomal composition and its structural variations among different species of the genus Chrysanthemum on the basis of finer sensitivity and higher genome coverage.
Disclosure of Invention
The invention aims to solve the problems that the preparation method and the process of a probe are complicated, the cost is high, signals are few and the probe is difficult to identify when the traditional fluorescence in situ hybridization technology is adopted for analysis in the molecular cytogenetics research of chrysanthemum plants at present, and provides an oligonucleotide probe sleeve developed based on chrysanthemum plant repeated sequences and a set of simple, feasible, low-cost and reliable fluorescence in situ hybridization method, so that the high-resolution mitotic chromosome karyotype of the chrysanthemum plants is obtained, and the practicability and the scientificity are outstanding.
The purpose of the invention can be realized by the following technical scheme:
the first object of the present invention is to provide an oligonucleotide hybrid probe set, which has the following sequence characteristics:
the second object of the present invention is to provide an oligonucleotide probe set hybridization solution comprising the aforementioned oligonucleotide mixing probe set, the oligonucleotide probe set hybridization solution comprising: dFA 7.5. mu.L, 20 XSSC 1.5. mu.L, Salmon sperm DNA (Salmon sperm DNA) 0.5. mu.L, 50.0% Dextran Sulfate 2.5. mu.L, oligonucleotide mixing probe set 4.0. mu.L; mixing the components in a 1.5mL centrifuge tube, shaking for centrifugation, denaturing in a heating block at 105 deg.C for 13min, taking out, and rapidly treating in-20 deg.C ice alcohol for 10 min.
Further, in the oligonucleotide mixed probe set, the concentration of each probe is HC _ CL110(0.55 ng/. mu.L); HC _ CL151(0.55 ng/. mu.L); LC _ CL173(0.55 ng/. mu.L); JH-Kmer7(0.55 ng/. mu.L); HC _ CL143(0.55 ng/. mu.L); LC _ CL263(0.55 ng/. mu.L); LC _ CL77(0.55 ng/. mu.L). The amount of each probe added was as follows: 0.5 μ L HC _ CL 110; 0.5 μ L HC _ CL 151; 1.0 μ L LC _ CL 173; 0.5 μ L JH-Kmer 7; 0.5 μ L HC _ CL 143; 0.5 μ L LC _ CL 263; 0.5 μ L LC _ CL 77.
The third purpose of the invention is to provide the application of the oligonucleotide mixed probe set or the hybridization solution of the oligonucleotide probe set in the high-resolution chromosome fluorescence in situ hybridization of the chrysanthemum plants.
Further, the oligonucleotide mixed probe set or the oligonucleotide probe set hybridization solution is applied to high-resolution mitosis metaphase chromosome fluorescence in-situ hybridization of the chrysanthemum plants.
The fourth purpose of the invention is to provide a method for carrying out high-resolution chromosome fluorescence in situ hybridization on chrysanthemum plants by using the oligonucleotide probe set hybridization solution, which comprises the following steps:
(1) treating the root tips of the chrysanthemum plants: collecting tissue culture seedling of Chrysanthemum plant, soaking 4-5 root systems with length of 2-3cm in a culture dish containing 0.2 μmol/L methyl ammoniumsulfuron solution, treating for 4 hr, washing with clear water, shearing off root systems, placing in 1.5mL centrifuge tube with small hole, and placing under 1.1Mpa N2O treatment for 40 min; adding 90% glacial acetic acid, treating for 10min, and storing in 1.5mL centrifuge tube filled with 70% ethanol at-20 deg.C;
(2) preparing chrysanthemum plant mitosis metaphase chromosome: taking the root tips of the chrysanthemum plants obtained by the treatment in the step (1) to perform chromosome flaking, observing by using a phase contrast microscope, selecting the mitosis metaphase chromosomes which have good dispersion, clear morphological structures and less overlap and can be used for fluorescence in situ hybridization, storing the mitosis metaphase chromosomes in a refrigerator at the temperature of minus 80 ℃, removing the slices overnight, dehydrating in a 100% ethanol solution for 30min, and airing for later use;
(3) fluorescence in situ hybridization: preparing the chromosome selected in the step (2) into a slide, placing the slide in an alkali denaturing solution in a water bath at 44 ℃ for denaturation for 4min, then sequentially placing the slide in 70% ethanol, 70% ethanol and 100% ethanol solutions, sequentially dehydrating the slide for 5min in a gradient manner at room temperature, and airing the slide for later use; preparing the oligonucleotide probe set hybridization solution, slowly dripping the hybridization solution into the preparation piece in a dripping amount of 16 mu L/piece, covering a cover glass, and placing the prepared hybridization solution in a wet box (placed in a 37 ℃ constant temperature box) wetted by 2 XSSC at 37 ℃ for hybridization culture for more than 6 hours;
(4) film washing and microscopic examination: removing the cover glass, and washing the slide by slow water flow for about 10 s; 2 XSSC, ddH in a water bath at 44 DEG C2Soaking and washing in O solution for 5min, air drying, and dripping 8uL of DAPI (4', 6-diamidino-2-phenylindole) sealing sheet on the sheet; and (6) microscopic examination.
Further, the alkali denatured liquid in the step (3) is prepared by dissolving and uniformly mixing NaOH and 70% ethanol in a ratio of 6 g/L.
Further, the microscopic examination in the step (4) specifically comprises the steps of placing the sliced chromosome under an Olympus BX60 type fluorescence microscope for microscopic examination, shooting the mitosis metaphase chromosome image of the chrysanthemum plant by using a SPOT CCD (SPOT coded Color Digital Camera), and processing the obtained image by Photoshop (v 6.0).
It is a fifth object of the present invention to provide a commercial dye liquor or kit comprising the aforementioned oligonucleotide mixed probe set or hybridization liquor comprising the aforementioned oligonucleotide probe set, and other products derived from the kit, such as a rapid detection reagent for chromosome of chrysanthemum.
The sixth purpose of the present invention is to provide the application of the oligonucleotide probe set or the hybridization solution of the oligonucleotide probe set or the kit or the detection reagent in the rapid identification and fluorescence in situ hybridization analysis of the chromosome of the chrysanthemum plant.
The invention has the following beneficial effects
The invention develops an oligonucleotide mixed probe set aiming at the chrysanthemum plant repetitive sequence, which comprises 7 chromosome oligonucleotide probes, wherein the probes have strong fluorescent signals, are easy to detect and have high repeatability; and an oligonucleotide probe set hybridization solution is further developed based on the mixed probe set, is used for high-resolution chromosome fluorescence in-situ hybridization of the chrysanthemum plants, can obtain high-resolution mitosis chromosome karyotypes of the chrysanthemum plants, and has outstanding practicability and scientificity.
Compared with the prior art, the high-resolution chromosome fluorescence in situ hybridization method for the chrysanthemum plants has the following advantages and positive effects:
(1) the invention has the advantages of simple and easy method, strong operability and outstanding practicability, and can be effectively used for constructing the karyotype chart for analyzing the chromosome of the chrysanthemum plant. The matched oligonucleotide probe set is used for detection through one-time FISH analysis, repeated hybridization of one slide is avoided, the preparation, labeling and analysis methods of the fluorescent in-situ hybridization probe are greatly simplified, and the experimental efficiency is improved.
(2) The invention has the advantages that the obtained FISH signal has moderate lighting brightness and few impurity signals, and the impurity signals are easy to be eluted by the elution conditions, thereby being beneficial to obtaining better FISH karyotype.
(3) When the invention carries out FISH analysis on the chrysanthemum plant chromosome, the single-chain oligonucleotide probe is adopted to replace the traditional plasmid probe, the hybridization signals which are the same as or more than the plasmid probe are displayed, the invention is beneficial to obtaining the high-resolution chrysanthemum plant mitosis metaphase chromosome karyotype, provides a new way for distinguishing the genetic relationship of the chrysanthemum plant variety chromosome, and is beneficial to the molecular cytogenetics research of the chrysanthemum plant.
Drawings
FIG. 1 shows FISH results (DAPI (blue), TAMRA (Red), FAM (Green)) for chrysanthemum haploid chromosomes with a scale size of 10 μm.
Fig. 2, FISH results for chromosome of chrysanthemum nankingense (c.natkinense), scale size 10 μm:
(a) DAPI Nuclear (b) FAM (5' end) Signal
(c) TAMRA (5' end) Signal (d) FISH Synthesis Pattern
FIG. 3 shows FISH results of 45S rDNA probe in Chrysanthemum nankingense (C.nankinense)
Detailed Description
The present invention will be described in further detail below with reference to examples and the accompanying drawings. The following description is only illustrative of the claimed embodiments of the present invention and is not intended to limit the embodiments in any way. The scope of the present invention is defined by the appended claims.
EXAMPLE 1 preparation of oligonucleotide Probe set hybridization solution
TAREAN and Kmer analysis is carried out on genome data of Chrysanthemum haploid and Chrysanthemum nankinense (Chrysanthemum nankinense), repeated sequences are clustered, oligonucleotide probes based on Chrysanthemum plant repeated sequences are developed by utilizing Oligo7, and the sequences of oligonucleotide mixed probes and the fluorescent labeling characteristics are determined as follows:
the oligonucleotide probe set hybridization solution preparation system is as follows:
in the mixed probe set, the concentration of each probe is HC _ CL110(0.55 ng/. mu.L); HC _ CL151(0.55 ng/. mu.L); LC _ CL173(0.55 ng/. mu.L); JH-Kmer7(0.55 ng/. mu.L); HC _ CL143(0.55 ng/. mu.L); LC _ CL263(0.55 ng/. mu.L); LC _ CL77(0.55 ng/. mu.L).
The adding amount of each probe in the probe sleeve is as follows: 0.5 μ L HC _ CL 110; 0.5 μ L HC _ CL 151; 1.0 μ L LC _ CL 173; 0.5 μ L JH-Kmer 7; 0.5 μ L HC _ CL 143; 0.5 μ L LC _ CL 263; 0.5 μ L LC _ CL 77.
Mixing the above components in 1.5mL centrifuge tube, shaking, centrifuging, placing in heating block at 105 deg.C, denaturing for 13min, taking out, and rapidly placing in ice alcohol at-20 deg.C for 10min to obtain oligonucleotide probe set hybridization solution.
Example 2 high resolution chromosomal fluorescence in situ hybridization of Chrysanthemum plants
Experimental materials: chrysanthemum haploid chromosome, chrysanthemum nankinense chromosome.
The test method comprises the following steps:
(1) treating the root tips of the chrysanthemum plants: respectively propagating the experimental materials in MS culture mediumTaking chrysanthemum plant tissue culture seedlings which are subcultured for 4-5 times in a constant temperature culture room at 25 ℃, removing residual exogenous hormones in vivo, selecting the chrysanthemum plant tissue culture seedlings which are subcultured for rooting observation, taking out and cleaning the tissue culture seedlings from an MS culture medium after 4-5 root systems with the length of 2-3cm appear in the tissue culture seedlings, immersing the root systems in a culture dish filled with 0.2 mu mol/L methyl ammoniumsulfuron solution for treatment for 4h, then washing the root systems with clear water, cutting the root systems, placing the cut root systems in 1.5mL centrifuge tubes with small holes, and placing the centrifuge tubes with 1.1Mpa N2O treatment for 40 min; adding 90% glacial acetic acid, treating for 10min, and storing in 1.5mL centrifuge tube filled with 70% ethanol at-20 deg.C;
(2) preparing chrysanthemum plant mitosis metaphase chromosome: taking the root tips of the chrysanthemum plants obtained by the treatment in the step (1) to perform chromosome flaking, observing by using a phase contrast microscope, selecting the mitosis metaphase chromosomes which have good dispersion, clear morphological structures and less overlap and can be used for fluorescence in situ hybridization, storing the mitosis metaphase chromosomes in a refrigerator at the temperature of minus 80 ℃, removing the slices overnight, dehydrating in a 100% ethanol solution for 30min, and airing for later use;
(3) fluorescence in situ hybridization: preparing the chromosome selected in the step (2) into a slide, placing the slide in an alkali denaturation solution (prepared by dissolving and uniformly mixing NaOH and 70% ethanol in a ratio of 6 g/L) in a water bath at 44 ℃ for denaturation for 4min, then sequentially placing the slide in 70% ethanol, 70% ethanol and 100% ethanol solutions, sequentially dehydrating the slide for 5min in a gradient manner at room temperature, and airing the slide for later use; the oligonucleotide probe set hybridization solution prepared in example 1 was slowly dropped onto the slide at a dropping amount of 16. mu.L/slide, covered with a cover glass, and placed in a wet cassette wetted with 2 XSSC at 37 ℃ in a thermostat at 37 ℃ for hybridization culture for more than 6 hours;
(4) film washing and microscopic examination: removing the cover glass, and washing the slide by slow water flow for about 10 s; 2 XSSC, ddH in a water bath at 44 DEG C2Soaking and washing in O solution for 5min, air drying, and dripping 8uL of DAPI (4', 6-diamidino-2-phenylindole) sealing sheet on the sheet;
the mounted chromosome slide is placed under an Olympus BX60 type fluorescence microscope for microscopic examination, an image of the chromosome in the metaphase of mitosis of the chrysanthemum plant is taken by utilizing a SPOT CCD (SPOTCooled Color Digital Camera), and the obtained image is processed by Photoshop (v 6.0).
In karyotyping, 3-12 cells are observed for each material, 1 cell with complete and well-dispersed chromosome number is selected from the cells and is used for karyotyping by Photoshop (v6.0), and 2 cells are selected for a few materials for karyotyping due to overlapping or incomplete chromosomes and the like.
The FISH analysis is carried out on the chromosome production of the chrysanthemum haploid mitosis metaphase by using the method, the result is shown in figure 1, and the result can be seen from the figure: the mixed probe set is adopted to carry out FISH analysis, and 27 chromosomes in the chrysanthemum haploid can be completely distinguished through DAPI (blue), FAM (green) and TAMRA (red) signals and the intensity thereof, so that the chrysanthemum haploid karyotype can be constructed.
FISH analysis of the chrysanthemum brain mitosis metaphase chromosome slides, the results are shown in FIGS. 2(a-d), FIG. 2a is DAPI stained (blue) chrysanthemum brain chromosomes; FIG. 2b shows FAM (green) signals of chrysanthemum brain chromosomes; FIG. 2c shows the TAMRA (red) signal of the chrysanthemum brain chromosome; FIG. 2d is a three-color synthesized image of chrysanthemum nankingense chromosome.
The results show that: when the method is used for observing chromosome production of chrysanthemum plants, the fluorescence signals observed in the chromosome production by the method are found to be richer and the mitotic phase is clearer compared with the fluorescence signals marked by the traditional plasmid probe (figure 3).
Sequence listing
<110> Nanjing university of agriculture
<120> probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atcgaccgtt tgtatttcat atgcttatca cacccttc 38
<210> 2
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gttgaacgtc taatgtgtaa ccataacata tcgactcatc c 41
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atattacaca tatctatact acttataatg gagggatgac 40
<210> 4
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aagcccctac tttaaaacat acaaccacca a 31
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cataagaaga tcccgtcgtt gcaattggtt 30
<210> 6
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttttatttgg cattttttag atgaattgtt at 32
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gttctcacgg atgttttgac aggtaattgg g 31
Claims (10)
1. An oligonucleotide mixing probe set, characterized in that the oligonucleotide mixing probe set has the following sequence characteristics:
。
2. The oligonucleotide probe set hybridization solution comprising the oligonucleotide mixing probe set according to claim 1, wherein the oligonucleotide probe set hybridization solution comprises: dFA 7.5. mu.L, 20 XSSC 1.5. mu.L, Salmon sperm DNA 0.5. mu.L, 50.0% Dextran Sulfate 2.5. mu.L, oligonucleotide mixing probe set 4.0. mu.L; mixing the components in a 1.5mL centrifuge tube, shaking for centrifugation, denaturing in a heating block at 105 deg.C for 13min, taking out, and rapidly treating in-20 deg.C ice alcohol for 10 min.
3. The hybridization solution for oligonucleotide probe set according to claim 2, wherein the concentration of each probe in the oligonucleotide mixed probe set is: 0.55 ng/. mu.L HC _ CL 110; 0.55 ng/. mu.L HC _ CL 151; 0.55 ng/. mu.L LC _ CL 173; 0.55 ng/. mu.L JH-Kmer 7; 0.55 ng/. mu.L HC _ CL 143; 0.55 ng/. mu.L LC _ CL 263; 0.55 ng/. mu.L LC _ CL 77;
the amount of each probe added was as follows: 0.5 μ L HC _ CL 110; 0.5 μ L HC _ CL 151; 1.0 μ L LC _ CL 173; 0.5 μ L JH-Kmer 7; 0.5 μ L HC _ CL 143; 0.5 μ L LC _ CL 263; 0.5 μ L LC _ CL 77.
4. Use of the oligonucleotide probe set of claim 1 or the oligonucleotide probe set hybridization solution of claim 2 in high resolution chromosome fluorescence in situ hybridization of Chrysanthemum.
5. The use according to claim 4, in high resolution mitotic metaphase chromosome fluorescence in situ hybridization of Chrysanthemum.
6. A method for performing high-resolution chromosome fluorescence in situ hybridization of chrysanthemum plants by using the oligonucleotide probe set hybridization solution of claim 2, which comprises the following steps:
(1) treating the root tips of the chrysanthemum plants: collecting tissue culture seedling of Chrysanthemum plant, soaking 4-5 root systems with length of 2-3cm in a culture dish containing 0.2 μmol/L methyl ammoniumsulfuron solution, treating for 4 hr, washing with clear water, shearing off root systems, placing in 1.5mL centrifuge tube with small hole, and placing under 1.1Mpa N2O treatment for 40 min; adding 90% glacial acetic acid, treating for 10min, and storing in 1.5mL centrifuge tube filled with 70% ethanol at-20 deg.C;
(2) preparing chrysanthemum plant mitosis metaphase chromosome: carrying out chromosome slide observation on the root tips of the chrysanthemum plants obtained by the treatment in the step (1), selecting chromosomes with good dispersion, clear morphological structure and less overlap, storing the mitosis metaphase chromosomes which can be used for fluorescence in situ hybridization in a refrigerator at the temperature of-80 ℃, taking off the slices overnight, dehydrating in 100% ethanol solution for 30min, and airing for later use;
(3) fluorescence in situ hybridization: preparing the chromosome selected in the step (2) into a slide, placing the slide in an alkali denaturing solution in a water bath at 44 ℃ for denaturation for 4min, then sequentially placing the slide in 70% ethanol, 70% ethanol and 100% ethanol solutions, sequentially dehydrating the slide for 5min in a gradient manner at room temperature, and airing the slide for later use; preparing the oligonucleotide probe set hybridization solution of claim 2, dripping the hybridization solution into the preparation piece at a dripping amount of 16 μ L/piece, covering a cover glass, and placing the preparation piece in a wet box wetted by 2 XSSC at 37 ℃ for hybridization culture for more than 6 h;
(4) film washing and microscopic examination: removing the cover glass, and washing the slide with slow water flow; 2 XSSC, ddH in a water bath at 44 DEG C2Soaking and washing in the O solution for 5min respectively, and dripping 8uL of DAPI sealing sheets on the prepared sheet after air drying; and (6) microscopic examination.
7. The method of claim 6, wherein the alkali-denatured liquid in step (3) is prepared by dissolving NaOH and 70% ethanol at a ratio of 6g/L and mixing them uniformly.
8. The method as claimed in claim 6, wherein the step (4) of microscopic examination is carried out by placing the chromosome slide after the mounting under an Olympus BX60 type fluorescence microscope, taking the image of the chromosome in the metaphase of mitosis of the chrysanthemum plant by using SPOT CCD, and processing the obtained image by Photoshop (v 6.0).
9. A kit or detection reagent comprising the oligonucleotide mixing probe set of claim 1 or comprising the oligonucleotide probe set hybridization solution of claim 2.
10. Use of the kit or detection reagent according to claim 9 in fluorescence in situ hybridization analysis of chromosomes of chrysanthemum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910392989.8A CN111926005B (en) | 2019-05-13 | 2019-05-13 | Probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910392989.8A CN111926005B (en) | 2019-05-13 | 2019-05-13 | Probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111926005A CN111926005A (en) | 2020-11-13 |
| CN111926005B true CN111926005B (en) | 2022-04-08 |
Family
ID=73282502
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910392989.8A Active CN111926005B (en) | 2019-05-13 | 2019-05-13 | Probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111926005B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116445645B (en) * | 2023-03-16 | 2024-01-02 | 南京农业大学 | Method for rapidly obtaining fluorescent in-situ hybridization probe based on transcriptome and probe set |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409523A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method of 5S rDNA on plant chromosome |
| CN105039542A (en) * | 2015-07-17 | 2015-11-11 | 南京农业大学 | Novel method for painting chromosomes by adopting oligonucleotide probe dye liquor |
| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
-
2019
- 2019-05-13 CN CN201910392989.8A patent/CN111926005B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409523A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method of 5S rDNA on plant chromosome |
| CN105039542A (en) * | 2015-07-17 | 2015-11-11 | 南京农业大学 | Novel method for painting chromosomes by adopting oligonucleotide probe dye liquor |
| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
Non-Patent Citations (3)
| Title |
|---|
| Identification of repetitive DNA sequences in the Chrysanthemum boreale genome;So Youn Won et al.;《Scientia Horticulturae》;20180330;238-243 * |
| Localization of 45S and 5S rDNA sites and karyotype of Chrysanthemum and its related genera by fluorescent in situ hybridization;Xiangyu Qi et al.;《Biochemical Systematics and Ecology》;20150829;164-172 * |
| 寡核苷酸探针套涂染结合基因组原位杂交和分子标记分析准确鉴定小麦－百萨偃麦草异源易位系的研究;杨骄 等;《麦类作物学报》;20180309;第38卷(第3期);253-261 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111926005A (en) | 2020-11-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102787166B (en) | Fluorescence in situ hybridization method of prunus mume chromosome | |
| CN104359734B (en) | Production method for fluorescent microscopic slices of ovule of pollinated azalea | |
| CN103205500A (en) | Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat | |
| CN111238888A (en) | Efficient sugarcane or sugarcane near-edge seed stem tip chromosome flaking method | |
| CN105954082A (en) | Wheat root tip chromosome production method | |
| CN107475390B (en) | Development and application of decaploid elytrigia elongata series repeat sequence specific probe | |
| CN111926005B (en) | Probe and method for high-resolution fluorescence in situ hybridization of chrysanthemum plant chromosome | |
| CN102960237A (en) | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology | |
| CN107604088A (en) | One cultivates peanut the initiative of Chromosome translocation and authentication method between A and 1 B gene group | |
| CN105296649B (en) | A kind of plant chromosome fluorescence in-situ hybridization method that improvement is simplified | |
| CN111175102A (en) | Method for preparing slices of root tip chromosomes of Paeonia plants | |
| CN109694902A (en) | One cultivates peanut chromosome probe staining kit and its application method | |
| CN115011724A (en) | Xinlium huashanense chromosome specific ND-FISH probe and application and kit thereof | |
| CN108913688A (en) | The separation and its application of salt sward centromere retrotransposon sequential element LTR | |
| CN113957166B (en) | Ribosome DNA oligonucleotide mixed probe sleeve based on chrysanthemum related species plant reference genome and development method | |
| CN110747287B (en) | Haynaldia villosa chromosome specific oligonucleotide probe and application thereof | |
| CN112067410A (en) | Fringe-based chromosome karyotype analysis method for Chinese fringetree | |
| CN107574229B (en) | Probe set for detecting oat plant chromosome, kit and application | |
| CN116445645B (en) | Method for rapidly obtaining fluorescent in-situ hybridization probe based on transcriptome and probe set | |
| CN117660686A (en) | Method for rapidly obtaining Bai Jingju oligonucleotide probe based on low-depth sequencing and application thereof | |
| CN117143966A (en) | BAC clone for distinguishing/identifying chamomile No.4 homologous chromosome and application thereof | |
| CN109161581B (en) | Mongolian leek chromosome fluorescence in situ hybridization method | |
| CN117187349A (en) | Identification method for lily hybrid by combining ND-FISH and GISH | |
| CN105861640B (en) | A kind of method of Rapid identification edible sunflower cenospecies SH338 authenticity and purity | |
| CN108441539B (en) | Method for detecting end of forest chromosome by using oligonucleotide sequence |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |