Method for rapidly identifying chromosome ploidy of cucumber horticultural variety

Abstract

The invention discloses a method for rapidly identifying chromosome ploidy of a cucumber horticultural variety. The method mainly comprises the following steps: preparing cucumber somatic cell interphase nuclear flakes; preparing a 5S rDNA (ribosomal deoxyribonucleic acid) probe / centromere sequence (Type-III) probe; and carrying out FISH hybridization. FISH hybridization signals of the 5S rDNA probe / centromere sequence (Type-III) probe are very stable in the whole cell cycle of the cucumber and are convenient to count, so that the chromosome ploidy of the cucumber variety can be accurately identified no matter whether the cell is in an interphase or a division phase. The invention provides a rapid and accurate identification method for chromosome ploidy identification of cucumber haploids or some growing point materials difficult to obtain root tips and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly identifying chromosome ploidy of cucumber horticultural varieties. Background Art

[0002] Fluorescence in situ hybridization (FISH) is a technique that uses specific DNA sequences to study genome structure, gene location, chromosome markers, and gene expression. It has been widely used in plant genetics, breeding, and genomics research. The basic principle of FISH is to use fluorescently labeled probes to specifically bind to target DNA sequences within cells, and observe the hybridization signals using fluorescence microscopy to obtain various genomic information. By fluorescently labeling chromosomal probes, FISH can help quickly and accurately determine plant chromosome number and ploidy. Currently, the main methods for determining plant chromosome ploidy are flow cytometry and chromosome counting using staining techniques. However, flow cytometry requires specialized equipment, is costly, and cannot directly visualize chromosomes. While staining chromosomes with specific probes is more convenient than these methods, it still requires phase-contrast microscopy to identify mitotic phases. This is difficult to achieve for haploid plants or for plants with difficult-to-access growth points, such as the root tip. Currently, no more rapid and effective methods for determining plant chromosome ploidy have been reported, particularly for haploid plants or plants with difficult-to-access growth points, such as the root tip. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a method for rapidly identifying the chromosome ploidy of cucumber horticultural varieties.

[0004] The present invention is achieved through the following technical solutions:

[0005] A method for rapidly identifying the chromosome ploidy of cucumber horticultural varieties comprises the following steps:

[0006] 1. Preparation of interphase nuclei of cucumber somatic cells;

[0007] 2. Preparation of 5S rDNA probe / centromere sequence (Type-III) probe;

[0008] 3. 5S rDNA probe / centromere sequence (Type-Ⅲ) probe FISH hybridization can quickly identify the chromosome ploidy of cucumber horticultural varieties based on the number of hybridization signals.

[0009] The steps for preparing cucumber somatic cell interphase nuclei in step 1 of this method are:

[0010] (1) Selecting young leaves of the cucumber material whose ploidy is to be identified;

[0011] (2) Put the young leaves into fixative solution (methanol: glacial acetic acid, volume ratio 3:1) and treat and fade green, the time required for fading green is determined according to the size of the young leaves, generally between half an hour to several hours;

[0012] (3) Transfer the young leaves into ddH20 and treat for 5 min;

[0013] (4) Cut the young leaves into blocks with an area of 2-3 mm 2 , then enzymatically digest in a 37℃ metal bath for 50 min, the enzyme solution is prepared by mixing 4% cellulase RS (Yakult), 4% pectinase Y-23 (Yakult) and 4% pectinase Pect (Aladdin) according to a volume ratio of 1:2:3;

[0014] (5) Suck out the enzyme solution, then add ddH20 and treat for 5 min;

[0015] (6) Suck out the ddH20, then add 1000 mL of fixative solution and treat for 10 min;

[0016] (7) Transfer the young leaves to a glass slide and gently knock them apart with tweezers, during which process, a drop of 45% acetic acid solution can be added to quickly break the cells. Then add a ring of fixative solution on the glass slide and immediately burn it on the flame of an alcohol lamp, see the liquid on the glass slide burning, then dry it and reserve it for use.

[0017] The steps for preparing the 5S rDNA probe / centromere sequence (Type-III) probe in step 2 of the method are as follows:

[0018] Select 60 bp sequences from the cucumber centromere sequence (Type-III) and repeat sequence 5S rDNA respectively, then let the biological company modify and prepare the probes at the 5' end with TAMRA or FAM respectively.

[0019] The steps for FISH hybridization of the 5S rDNA probe / centromere sequence (Type-III) probe in step 3 of the method are as follows:

[0020] (1) Prepare the hybridization mixture

[0021] Add 10 μL of 100% deionized formamide, 2 μL of 20×SSC, 1.5 μL of 5S rDNA probe / centromere sequence (Type-III) probe and 4 μL of 50% dextran sulfate solution to each glass slide. After mixing, place it on ice for standby use. (This step can also be completed in the dehydration link of the glass slide pretreatment)

[0022] (2) Pretreatment of the glass slide

[0023] Each slide plus 100 μL 70% deionized formamide solution, cover the coverslip, placed in 80 °C hybridization instrument for 90 s; after the end of the treatment immediately thrown off the coverslip, in turn into the ice 70%, 90%, 100% ethanol gradient dehydration 5 min.

[0024] (3) Hybridization

[0025] The slide was taken out and dried, then add hybridization mixture and cover the coverslip, and then put into the hybridization box at 37 °C for at least 4-5 h. (Before adding the hybridization mixture, the hybridization mixture should be heated in a 90 °C metal bath for 6 min, and the heated hybridization mixture should be added to the slide as soon as possible, and the hybridization mixture should be placed on ice for several minutes after heating)

[0026] (4) Washing (avoid light)

[0027] The slide was transferred to 2xSSC solution at room temperature for 1-5 min / 5-10 min (let the coverslip fall off naturally in the 2xSSC solution). Then the slide was transferred to 2xSSC solution at room temperature and shaken on a shaker for 5 min; then the slide was transferred to 2xSSC solution at 42 °C and shaken on a shaker for 10 min; then the slide was transferred to 2xSSC solution at room temperature and shaken on a shaker for 5 min; then the slide was transferred to ddH20 at room temperature and shaken on a shaker for 5 min; then the slide was transferred to 70%, 90%, 100% ethanol at room temperature in turn and gradient dehydrated for 5 min.

[0028] (5) Counterstaining

[0029] The slide was air-dried, then 15-20 μL DAPI was added to each slide.

[0030] (6) Fluorescence detection

[0031] Under the condition of light, Olympus BX51 fluorescence microscope and SenSys CDD camera were used to observe and take pictures.

[0032] Beneficial effects

[0033] The application provides a more accurate and efficient detection method for identifying the ploidy of cucumber haploid or some materials difficult to obtain growth points such as root tips. The FISH hybridization signal of the 5S rDNA probe / spindle pole sequence (Type-III) probe is very stable in the whole cell cycle of cucumber and is easy to count. Therefore, the chromosome ploidy of cucumber varieties can be accurately identified whether the cells are in the interphase or the division phase. The step of finding a relatively complete division phase in the previous determination of ploidy by chromosome smearing technology is omitted, and the material acquisition is more convenient, especially for haploid and some materials difficult to obtain growth points such as root tips. Compared with flow cytometry, it is more time-saving and labor-saving. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Distribution image of 5S rDNA probe signal sites in the interphase nucleus of the diploid cucumber variety;

[0035] Figure 2 Distribution image of 5S rDNA probe signal sites in the interphase nucleus of the haploid cucumber variety;

[0036] Figure 3 Distribution image of spindle pole sequence (Type-III) probe signal sites in the interphase nucleus of the diploid cucumber variety;

[0037] Figure 4 Distribution image of spindle pole sequence (Type-III) probe signal sites in the interphase nucleus of the haploid cucumber variety;

[0038] DETAILED DESCRIPTION

[0039] The embodiments of the application will be described in detail below, and the embodiments are implemented on the premise of the technical scheme of the application, and detailed implementation modes and specific operation processes are given, but the protection scope of the application is not limited to the following embodiments.

[0040] Comparative Example 1

[0041] A method for rapidly identifying the chromosome ploidy of a cucumber horticultural variety, comprising the following steps:

[0042] Chromosome preparation

[0043] (1) Use seed germination or cutting rooting, when the root system is 1-2 cm long, cut the root at 9:00-11:00 and put it into a solution containing 0.002M 8-hydroxyquinoline for 1h40min (avoiding light);

[0044] (2) Wash the root 2-3 times, soak it in ddH20 for 5min, and then put it in a fixing solution (methanol: glacial acetic acid, volume ratio 3:1) at 4℃ for 24h;

[0045] (3) Take out the root, wash it with ddH20 for 3 times (5 min / time), cut the root tip and put it into a 1.5 mL centrifuge tube (use the yellow gun head to suck the root tip and cut off the front end), then use the white gun head to suck out the ddH20;

[0046] (4) Add 50 mL mixed enzyme solution to each centrifuge tube, the mixed enzyme solution is mixed by 4% cellulase RS (Yakult), 4% pectinase Y-23 (Yakult) and 4% pectinase Pect (Aladdin) according to the volume ratio of 1:2:3;

[0047] (5) Enzymolysis in a 37℃ metal bath for 38 min;

[0048] (6) Suck out the enzyme solution and treat with ddH20 for 5 min;

[0049] (7) Wash the root tip with ddH20 for 1-2 times, then add 800-1000 mL fixing solution and treat for at least 10 min;

[0050] (8) Knock the enzyme-treated root tip on a slide, add a drop of 45% acetic acid solution in the middle, add a circle of fixing solution around, slightly burn the slide on an alcohol lamp for a few moments, knock off the excess liquid on paper, then dry it;

[0051] (9) Use phase contrast microscope to find better division phase and perform chromosome counting.

[0052] Example 1

[0053] A method for rapidly identifying the chromosome ploidy of a cucumber horticultural variety, comprising the following steps:

[0054] 1. Cucumber somatic interphase nuclear preparation

[0055] (1) Select tender leaves of cucumber material to be identified for ploidy;

[0056] (2) Put the tender leaves into fixing solution (volume ratio of methanol: glacial acetic acid 3:1) for treatment and greening, the time required for greening is determined according to the size of the tender leaves, generally between half an hour to several hours;

[0057] (3) Transfer the tender leaves into ddH20 for treatment for 5 min;

[0058] (4) Cut the tender leaves into blocks with an area of 2-3 mm 2 , then enzymolysis in a 37℃ metal bath for 50 min, the enzyme solution is mixed by 4% cellulase RS (Yakult), 4% pectinase Y-23 (Yakult) and 4% pectinase Pect (Aladdin) according to the volume ratio of 1:2:3;

[0059] (5) Aspirate the enzyme solution, then add ddH20 for 5 min;

[0060] (6) Aspirate the ddH20, then add 1000 mL fixative solution for 10 min;

[0061] (7) Transfer the young leaves to the slides and gently knock them apart with a pair of tweezers. A drop of 45% acetic acid solution can be added to quickly break the cells. Then add a ring of fixative solution on the slide and immediately burn it on the flame of an alcohol lamp until the liquid on the slide is burning. Then dry it and keep it ready for use.

[0062] 2. Preparation of 5S rDNA probe / Centromere sequence (Type-III) probe

[0063] Select 60 bp sequences from the cucumber centromere sequence (Type-III) and repetitive sequence 5S rDNA, respectively, and then have a biological company modify them with TAMRA or FAM at the 5' end to prepare the probes.

[0064] 3. FISH hybridization of 5S rDNA probe / Centromere sequence (Type-III) probe

[0065] (1) Preparation of hybridization mixture

[0066] Add 10 μL of 100% deionized formamide, 2 μL of 20×SSC, 1.5 μL of 5S rDNA probe / Centromere sequence (Type-III) probe, and 4 μL of 50% dextran sulfate solution to each slide. After mixing, place it on ice for standby. (This step can also be completed during the dehydration step in the slide pretreatment)

[0067] (2) Pretreatment of the slide

[0068] Add 100 μL of 70% deionized formamide solution to each slide, cover it with a cover glass, and place it in an 80°C hybridization instrument for 90 s. Immediately after the treatment is completed, remove the cover glass and sequentially place it in ice 70%, 90%, and 100% ethanol for gradient dehydration for 5 min.

[0069] (3) Hybridization

[0070] Remove the slide and dry it, then add the hybridization mixture and cover it with a cover glass. Then place it in a hybridization box and treat it at 37°C for at least 4-5 h. (Before adding the hybridization mixture, the hybridization mixture should be treated in a 90°C metal bath for 6 min. The heated hybridization mixture should be added to the slide as soon as possible, and the hybridization mixture should be placed on ice during the few minutes after heating and before adding it to the slide)

[0071] (4) Slide washing (in the dark)

[0072] Transfer the slides to 2xSSC solution at room temperature for 1-5min / 5-10min (let the coverslips fall off naturally in 2xSSC solution); then transfer the slides to 2xSSC solution at room temperature and swing on a shaker for 5min; then transfer the slides to 2xSSC solution at 42℃ and swing on a shaker for 10min; then transfer the slides to ddH20 at room temperature and swing on a shaker for 5min; then transfer the slides to 70%, 90%, 100% ethanol at room temperature for gradient dehydration for 5min.

[0073] (5) Counterstaining

[0074] Air dry the slides, then add 15-20μL DAPI to each slide.

[0075] (6) Fluorescence detection

[0076] Under the condition of light shielding, use Olympus BX51 fluorescence microscope and SenSys CDD camera to observe and take pictures.

[0077] Example 1 The method of Example 1 can be used to quickly determine the ploidy of cucumber horticultural varieties by counting the probe signals in somatic interphase nuclei. This method is not affected by chromosome morphology and division stage, and is very suitable for identifying the ploidy of haploid or some cucumber horticultural varieties that are difficult to obtain root tips and other growth points.

[0078] Example 1 The method of Example 1 can be used to quickly determine the ploidy of cucumber horticultural varieties by counting the probe signals in somatic interphase nuclei. This method is not affected by chromosome morphology and division stage, and is very suitable for identifying the ploidy of haploid or some cucumber horticultural varieties that are difficult to obtain root tips and other growth points.

[0079] Figure 1 and Figure 3 is a diploid cucumber, Figure 2 and Figure 4 is a haploid cucumber; interphase nuclei are stained with DAPI, green signals are 5S rDNA probe hybridization signals, and red signals are centromere sequence (Type-Ⅲ) probe hybridization signals. See the attached drawings for the figures.

Claims

1. A method for rapid identification of the chromosome ploidy of a Cucumis sativus horticultural variety, characterized in that, The method comprises the following steps: (1) cucumber interphase nuclear preparation; (2) 5S rDNA probe / centromere sequence (Type-III) probe preparation; (3) 5S rDNA probe / centromere sequence (Type-III) probe FISH hybridization, and the chromosome ploidy of the cucumber horticultural variety can be rapidly identified according to the number of hybridization signals.

2. The method of claim 1, wherein, The steps of the cucumber interphase nuclear preparation in step (1) are as follows: (1) selecting tender leaves of the cucumber material to be identified for ploidy; (2) placing the tender leaves into a fixing solution (methanol: glacial acetic acid, volume ratio 3:1) for treatment and decolorization, and the decolorization time is determined according to the size of the tender leaves, generally between half an hour and several hours; (3) transferring the tender leaves into ddH2O for treatment for 5 min; (4) Cut the young leaves into pieces with an area of ​​2-3mm 2 The pellets were then enzymatically hydrolyzed in a metal bath at 37°C for 50 min. The enzyme solution was a mixture of 4% cellulase RS (Yakult), 4% pectinase Y-23 (Yakult), and 4% pectinase Pect (Aladdin) in a volume ratio of 1:2:

3. (5) sucking out the enzyme solution, and then adding ddH2O for treatment for 5 min; (6) sucking out the ddH2O, and then adding 1000 mL of the fixing solution for treatment for 10 min; (7) transferring the tender leaves to a glass slide, and gently knocking them to pieces with tweezers, and a drop of 45% acetic acid solution can be added to quickly break the cells; then a circle of the fixing solution is added to the glass slide and immediately burned on the alcohol lamp flame, and the liquid on the glass slide is burned to see if the glass slide is ready for use.

3. The method of claim 1, wherein, The steps of the 5S rDNA probe / centromere sequence (Type-III) probe preparation in step (2) are as follows: 60 bp sequences are selected from the cucumber centromere sequence (Type-III) and the repeated sequence 5S rDNA, and then a biological company is asked to modify and prepare the probes at the 5' end with TAMRA or FAM.

4. The method of claim 1, wherein, The steps of the FISH hybridization in step (3) are as follows: (1) preparing a hybridization mixed solution 10 μL of 100% deionized formamide, 2 μL of 20×SSC, 1.5 μL of 5S rDNA probe / centromere sequence (Type-III) probe and 4 μL of 50% dextran sulfate solution are added to each glass slide, and the mixture is placed on ice for standby after completion of mixing; (this step can also be completed in the dehydration link in the glass slide pretreatment) (2) pretreatment of the glass slide 100 μL of 70% deionized formamide solution is added to each glass slide, a cover glass is covered, and the glass slide is placed on an 80℃ hybridization instrument for treatment for 90 s; after the treatment is completed, the cover glass is immediately thrown off, and the glass slide is sequentially placed in 70%, 90% and 100% ethanol in the ice for gradient dehydration for 5 min; (3) hybridization The glass slide is taken out and dried, the hybridization mixed solution is added and the cover glass is covered, and then the glass slide is placed in a hybridization box for treatment at 37℃ for at least 4-5 h; (the hybridization mixed solution should be treated in a 90℃ metal bath for 6 min before being added to the glass slide, and the hybridization mixed solution after heating should be quickly added to the glass slide, and the hybridization mixed solution should be placed on ice during the several minutes after heating and before being added to the glass slide) (4) washing the glass slide (in the dark) The glass slide is transferred to a 2×SSC solution at room temperature and is placed for 1-5 min / 5-10 min (let the cover glass naturally fall off in the 2×SSC solution) Then the slides were transferred to 2x SSC solution at room temperature and shaken on a shaker for 5 min; then the slides were transferred to 2x SSC solution at 42°C and shaken on a shaker for 10 min; then the slides were transferred to 2x SSC solution at room temperature and shaken on a shaker for 5 min; Then the slides were transferred to ddH20 at room temperature and shaken on a shaker for 5 min; then the slides were transferred to 70%, 90%, 100% ethanol at room temperature for gradient dehydration for 5 min; (5) Counterstaining The slides were air-dried, and then 15-20 μL of DAPI was added to each slide for sealing; (6) Fluorescence detection Under the condition of light shielding, the Olympus BX51 fluorescence microscope and SenSys CDD camera were used for observation and photography.

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