Potato ploidy identification method and ploidy detection kit for marking and counting chromosome centromere based on potato CENH3 antibody

Abstract

The invention relates to the field of molecular cytogenetics and potato ploidy breeding, in particular to a potato ploidy identification method for marking and counting chromosome centromere based on a potato CENH3 antibody and a ploidy detection kit. According to the method, the chromosome centromere is marked by specifically recognizing the antibody of the potato centromere nucleosome core histone CENH3 and combining the immunofluorescence technology, so that the accurate counting of the chromosome centromere is realized at the single cell level, and the ploidy of potatoes is judged. The method comprises the following steps: preparing a leaf cell suspension, sequentially incubating a potato CENH3 antibody and a labeled secondary antibody after immobilization, and observing and counting by a microscope after redyeing. The method has the advantages of high species specificity, high throughput and low cost, is not limited by special material taking parts of root tips or shoot tips and cell division periods, can accurately identify the uploid and aneuploid of potatoes, and provides an efficient tool for potato germplasm resource evaluation and cross breeding.

Description

Technical Field

[0001] This invention relates to the fields of molecular cytogenetics and potato ploidy breeding, specifically to a method for identifying potato ploidy based on centromere labeling and counting using potato CENH3 antibodies, and a ploidy detection kit. Background Technology

[0002] Common potato varieties ( Solanum tuberosum L.) is tetraploid (2n=4x=48). About 75% of wild potato species and original cultivated species are diploid. These wild resources have abundant genes for resistance to biotic and abiotic stresses (insect resistance, disease resistance) and varietal traits (rich in anthocyanins, high dry matter content), but they are difficult to hybridize with tetraploid cultivated species due to ploidy barriers. Therefore, chromosome ploidy manipulation is the core key technology of potato bio-breeding. Accurate identification of ploidy is the key to tapping the potential of potato germplasm resources and overcoming hybridization barriers. High-throughput ploidy identification technology is also an important technical support for potato ploidy manipulation, such as the identification of microspore culture double haploid induced progeny and the utilization of chromosome doubling 2n gametes. However, existing technologies have significant limitations: chromosome squash counting requires special rooting induction of materials, expensive cellulose and pectinase for root tip lysis, and manual screening of high-quality metaphase cells, which is time-consuming and easily affected by slide preparation techniques; flow cytometry relies on expensive instruments and optimization of nuclear lysis buffer, which is costly for large-scale detection; traditional molecular markers (such as SSR and SNP) can only reflect differences in genomic fragments and cannot visually display chromosome structure, and are insufficient in distinguishing aneuploids (such as 2n+1) or chimeras (such as mixed tetraploid and hexaploid cells).

[0003] CENH3 is a core histone of the centromere nucleosome in eukaryotes, with its C-terminal domain being highly conserved and specifically located in the functional centromere region. Immunofluorescence labeling using potato CENH3 antibodies can produce bright, stable fluorescent signals on the chromosomes of mature leaf cells that have stopped cell division, unaffected by specific sampling sites such as root tips or shoot tips or cell division stages. However, the potato genome and ploidy level are complex, with the centromere region rich in repetitive sequences and heterochromatin, leading to low antibody recognition rates. Currently, potato CENH3 antibodies have not been used for potato ploidy identification. Developing a potato ploidy identification method using antibody-based chromosome centromere labeling and counting, and applying it to potato ploidy identification, is of great significance for determining the ploidy level of potato germplasm resources and for ploidy breeding. Summary of the Invention

[0004] To solve the above technical problems, the method provided by the application realizes the positioning and counting of the chromosome centromere by acquiring and adopting the antibody of the core histone CENH3 of the potato centromere nucleosome, the amino acid sequence of the core histone CENH3 of the potato centromere nucleosome is shown as SEQ ID NO: 1, and the immunofluorescence technology is adopted, and the ploidy of the potato is determined based on the signal statistical result, thereby providing a high-throughput ploidy identification method which is simple in operation, low in cost, accurate and reliable, and suitable for various ploidy levels of potato materials, and providing a universal technical platform for the evaluation of potato germplasm resources, ploidy operation and molecular breeding.

[0005] In one aspect, the application provides a potato ploidy identification method based on the antibody of the potato CENH3 for marking and counting the chromosome centromere, and the method comprises the following steps: The antibody of the potato CENH3 is used for staining the core histone CENH3 of the potato centromere nucleosome by the immunofluorescence technology to count the chromosome centromere.

[0006] In another aspect, the application also provides the application of the method in the potato ploidy identification.

[0007] Further, the application comprises the following steps: S1, acquiring the sequence of the core histone CENH3 of the potato centromere nucleosome, the protein sequence of which is shown as SEQ ID NO: 1; designing the amino acid sequence near the amino terminal for polypeptide artificial synthesis, and preparing the antibody through rabbit immunization experiment.

[0008] S2, preparing the cell suspension after the pretreatment of the potato leaf sample, fixing the cell suspension on the chromosome slide, and then performing the immunofluorescence reaction of the core histone CENH3 of the potato centromere nucleosome to obtain the fluorescent labeling signal; S3, locating the position of the chromosome centromere according to the fluorescent labeling signal obtained in S2, counting the number of signals, and obtaining the number of the chromosome centromere, i.e. the number of the potato chromosomes; S4, judging the ploidy of the potato according to the number of the potato chromosomes obtained in S3.

[0009] Further, in the application, the specific steps of the immunofluorescence reaction of the core histone CENH3 of the potato centromere nucleosome in S2 comprise: S201, washing and blocking the chromosome slide after fixing the cell suspension by using buffer B to obtain a primary treatment sample; S202, washing the primary treatment sample obtained in S201 by using buffer B, adding the primary antibody solution, and incubating to obtain a secondary treatment sample; S203, washing the secondary treatment sample obtained in S202 with buffer B, adding the secondary antibody solution dropwise, incubating, and obtaining a tertiary treatment sample; S204, washing the tertiary treatment sample obtained in S203 with buffer B and alcohol, drying at room temperature, and obtaining a fluorescent labeling signal.

[0010] Further, in the application, the primary antibody solution in S202 is composed of bovine serum albumin, buffer B and a primary antibody, and the volume ratio of the bovine serum albumin, buffer B and the primary antibody is 100:100:1. The secondary antibody solution in S203 is composed of bovine serum albumin, buffer B and a secondary antibody, and the volume ratio of the bovine serum albumin, buffer B and the secondary antibody is 100:100:1.

[0011] Further, in the application, the primary antibody in the primary antibody solution is a potato CENH3 antibody, and the secondary antibody in the secondary antibody solution is Goat Anti Rabbit IgG (H&L)-Alexa FLUOR 488.

[0012] Further, in the application, the step of locating the position of the centromere of the chromosome by the fluorescent labeling signal in S3 includes adding a staining solution dropwise to the fluorescent labeling substance, and observing the number of centromeres of the chromosome by sealing and observing.

[0013] Further, in the application, the staining solution is DAPI staining solution.

[0014] Further, in the application, the potato leaf sample is a diploid, triploid or tetraploid cultivated species or wild species.

[0015] Finally, the application also provides a potato ploidy identification kit, which comprises: a primary antibody, which is a potato CENH3 antibody; a secondary antibody, which is Goat Anti Rabbit IgG (H&L)-Alexa FLUOR 488; DAPI staining solution; polylysine-coated glass slides; lysis solution LB, which is composed of 15.00 mM Tris-HCl, 0.500 mM Sperminetetrahydrochloride, 79.94 mM KCl, 19.98 mM NaCl, 14.97 mM β-mercaptoethanol and 1.712 mM Triton X-100; Buffer A consisting of 20.0 mM Tris-HCl, 292 mM Sucrose, 8.54 mM MgCl2, 100 mM KCl and 0.896 mM Tween 20; Buffer B consisting of 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl 0.2 g, 1.8 mM KH2PO4; Blocking solution BT consisting of 4 wt% bovine serum albumin with 0.1 wt% Tritonex-100.

[0016] Compared with the prior art, the technical scheme provided by the present application has at least the following beneficial effects or advantages: (1) High specificity: the optimized potato CENH3 antibody marker centromere can accurately recognize and bind to the target protein, effectively avoiding non-specific background interference, making the fluorescent signal clear and explicit, providing reliable guarantee for accurate judgment of the position and number of chromosome centromere, and improving the accuracy of ploidy identification.

[0017] (2) High accuracy: the number of signals corresponds to the number of chromosomes, and the accurate correspondence makes the method not only capable of accurately identifying the ploidy of euploid potatoes, but also capable of detecting complex ploidy conditions such as aneuploidy and chimera, providing more information for fine evaluation and molecular breeding of potato germplasm resources.

[0018] (3) High efficiency: the whole identification process is clear and simple to operate, compared with traditional methods, the identification efficiency is greatly improved, which meets the needs of large-scale germplasm resource evaluation and breeding projects for ploidy identification, and accelerates the breeding process.

[0019] (4) Low cost: without using expensive large equipment such as flow cytometer, only conventional fluorescence microscope can complete the identification, reducing equipment investment and operation cost, making the technology more popular and valuable.

[0020] (5) Universality: only a small amount of mature leaves are needed for identification, which is not affected by special sampling sites such as root tips and bud tips and cell division cycle, and the operation is simple and easy to operate, which greatly improves the identification efficiency and reduces the work intensity, especially suitable for large-scale ploidy identification. Avoid the requirement of chromosome pressing and the time-consuming screening of metaphase cells.

[0021] (6) Intuitive results: the fluorescent labeling signal and chromosome centromere are observed by fluorescence microscope, the results are clear and intuitive, which can avoid the interference of chromosome overlapping and broken fragments caused by chromosome pressing, reduce human error, and improve the reliability of identification.

[0022] In summary, the application has significant advantages and broad application prospects in the field of potato ploidy identification, and provides a new technical scheme for potato germplasm resource protection, genetic improvement and molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 3 is a diagram of immunofluorescence staining and chromosome centromere number statistics and flow cytometry ploidy identification of triploid potatoes. Wherein A is the centromere signal of potato CENH3 antibody immunofluorescence reaction; B is the theoretical value and actual statistical result (average value ± standard deviation) of the number of chromosome centromeres; C is the flow cytometry ploidy identification result, wherein the abscissa is the fluorescence channel value, and the ordinate is the relative value of the number of cells determined.

[0024] Figure 2 Figure 3 is a diagram of immunofluorescence staining and chromosome centromere number statistics and flow cytometry ploidy identification of triploid potatoes. Wherein A is the centromere signal of potato CENH3 antibody immunofluorescence reaction; B is the theoretical value and actual statistical result (average value ± standard deviation) of the number of chromosome centromeres; C is the flow cytometry ploidy identification result, wherein the abscissa is the fluorescence channel value, and the ordinate is the relative value of the number of cells determined.

[0025] Figure 3 Figure 3 is a diagram of immunofluorescence staining and chromosome centromere number statistics and flow cytometry ploidy identification of triploid potatoes. Wherein A is the centromere signal of potato CENH3 antibody immunofluorescence reaction; B is the theoretical value and actual statistical result (average value ± standard deviation) of the number of chromosome centromeres; C is the flow cytometry ploidy identification result, wherein the abscissa is the fluorescence channel value, and the ordinate is the relative value of the number of cells determined. DETAILED DESCRIPTION

[0026] The technical solutions of the application will be described below in conjunction with the embodiments, but the application is not limited to the following embodiments.

[0027] In order to enable those skilled in the art to better understand the technical solutions of the application and implement them, the application will be further described below in conjunction with specific embodiments and drawings, but the embodiments are not limiting to the application.

[0028] The experimental methods and detection methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0029] Experimental instruments: 1. upright fluorescence microscope: Zeiss; model: Axio Image M2; 2. cell analyzer: Luminex; model: Guava Muse.

[0030] Experimental reagents: 1. Bovine serum albumin: purchased from SolarBio, Cat. No. A8020; 2. Potato CENH3 antibody (i.e., primary antibody): prepared by Kangtuo Antibody Technology (Wuhan) Co., Ltd.; 3. Goat Anti Rabbit IgG (H&L) - Alexa Fluor 488 (i.e., secondary antibody): purchased from ImmunoWay Biotechnology Company, Cat. No. RS3211; 4. Anti-fluorescence decay mounting medium (containing DAPI): purchased from Coolaber, Cat. No. SL1841-5mL; 5. DAPI staining solution: purchased from Solarbio, Cat. No. C0065; 6. PI dye: purchased from Beyotime Biotechnology, Cat. No. ST1569-10mg; 7. Potato leaf samples: The leaf samples used were from diploid, triploid, and tetraploid potatoes G1, G2, and G3, respectively. All potato materials were obtained from Northwest A&F University. Leaves were taken from potato tissue culture seedlings grown for 3 weeks under conditions of 22±2℃, 16h light / 8h darkness. Example 1 In this embodiment, a potato leaf sample was blindly selected from potato samples of different ploidies to be tested, named 1# potato leaf sample to be tested. The ploidy of the potato leaf sample 1# was determined by immunofluorescence reaction using an antibody against the core histone CENH3 of the potato centromere nucleosome. The specific experimental steps are as follows: Step 1: Take 10 mg of healthy potato leaf sample I to be tested in a plastic petri dish, add lysis buffer LB to the leaf, chop the leaf with a blade to lyse the cells, filter out the residue through a 300-mesh filter membrane, and mix an appropriate amount of the filtrate with buffer A at a ratio of 1:1.

[0031] Step 2: Spot the cell suspension obtained in Step 1 onto a poly-L-lysine-coated glass slide, air dry, and obtain a dried glass slide. Step 3: Place the dried glass slide described in Step 2 in buffer B and wash it twice on a decolorizing shaker for 5 minutes each time; Step 4: Add blocking solution BT to the glass slide to block non-specific antigens on the cell surface, cover the surface with sealing film, and place at 4°C for 30 minutes. Step 5: Repeat step 3; Step 6: Add primary antibody and incubate overnight at 4°C; Step seven, repeat step three; Step eight, add the secondary antibody dropwise, incubate at 37°C for 1 hour in the dark; Step nine, repeat step three; Step ten, wash the slides with 70% alcohol, 90% alcohol, and 100% alcohol for 2 minutes each, and then dry the slides at room temperature; Step eleven, add 15 μL of DAPI staining solution to each slide that has been dried at room temperature to restain the cell nucleus, and then seal the slide; Step twelve, place the slide prepared in step eleven under a fluorescence microscope at an excitation wavelength of 488 nm to observe the fluorescence signal, and randomly select ≥30 clear cells for each sample to count the number of CENH3 hybridization fluorescent spots.

[0032] Step thirteen, determine the ploidy of the material to be tested according to the ratio of the total number of fluorescence signals n to the known ploidy control (2x=24, 3x=36, 4x=48, 5x=60, 6x=72…).

[0033] The results are shown in Figure 1 Table 1. The number of kinetochore green fluorescence signals of the 1# potato leaf sample to be tested was calculated. To demonstrate the reliability of the experimental results, the first experiment was repeated 30 times. The results showed that the average kinetochore fluorescence signal count was 21.37±1.76 clear green fluorescence spots. A compliance statistical hypothesis test (χ2test) was performed on this group of data. When n=30, the freedom value =29, =42.56, and the data in each group =42.56, the null hypothesis was accepted (the actual count result of the kinetochore was consistent with the theoretical result), and the actual result was consistent with the theoretical value of diploid 2x=24, indicating that the sample ploidy was diploid. The detailed values of the χ2test are shown in Table 1.

[0034] Table 1. Immune fluorescence kinetochore counting for potato ploidy detection

[0035] When n=30, the freedom value =29, =42.56, and the data in each group =42.56, the null hypothesis was accepted.

[0036] Example 2 ​​In this embodiment, one potato leaf sample was blindly selected from the potato leaf samples and named as sample #2. The ploidy of sample #2 was determined by immunofluorescence reaction using an antibody against the core histone CENH3 of the potato centromere nucleosome. The specific experimental steps are as follows: Step 1: Take 10 mg of healthy potato leaf sample I to be tested in a plastic petri dish, add lysis buffer LB to the leaf, chop the leaf with a blade to lyse the cells, filter out the residue through a 300-mesh filter membrane, and mix an appropriate amount of the filtrate with buffer A at a ratio of 1:1.

[0037] Step 2: Spot the cell suspension obtained in Step 1 onto a poly-L-lysine-coated glass slide, air dry, and obtain a dried glass slide. Step 3: Place the dried glass slide described in Step 2 in buffer B and wash it twice on a decolorizing shaker for 5 minutes each time; Step 4: Add blocking solution BT to the glass slide to block non-specific antigens on the cell surface, cover the surface with sealing film, and place at 4°C for 30 minutes. Step 5: Repeat step 3; Step 6: Add primary antibody and incubate overnight at 4°C; Step 7: Repeat step 3; Step 8: Add secondary antibody and incubate at 37°C in the dark for 1 hour; Step 9: Repeat step 3; Step 10: Clean the slide with 70% alcohol, 90% alcohol, and 100% alcohol in sequence, for 2 minutes each time. After cleaning, place the slide to dry at room temperature. Step 11: After drying at room temperature, add 15 μL of DAPI staining solution to each slide to counterstain the cell nuclei, and then mount the slides. Step 12: Place the slide prepared in Step 11 under a fluorescence microscope at an excitation wavelength of 488 nm to observe the fluorescence signal. Randomly select ≥30 clear cells from each sample and count the number of CENH3 hybridization fluorescent spots.

[0038] Step 13: Determine the ploidy of the material to be tested by comparing the total number of fluorescence signals n with known ploidy controls (2x=24, 3x=36, 4x=48, 5x=60, 6x=72…).

[0039] The results are as follows Figure 2 As shown, the number of green fluorescent signals at the centromeres of potato leaf sample #3 was calculated. To demonstrate the reliability of the experimental results, the first experiment was repeated 30 times. The results showed that the average number of clear green fluorescent spots at the centromeres was 31.7 ± 2.08. A statistical hypothesis test (χ² test) was performed on this data. When n=30, the free values... =29, =42.56, data for each group < =42.56, accepting the null hypothesis (actual centromere count is consistent with theoretical result). The actual result is consistent with the theoretical value of tetraploid 2x=36, indicating that the ploidy of this sample is triploid. Detailed values ​​of the χ2 test are shown in Table 1.

[0040] Example 3 In this embodiment, one potato leaf sample was blindly selected from the potato leaf samples and named as sample #3 to be tested. The ploidy of sample #3 was determined by immunofluorescence reaction using an antibody against the core histone CENH3 of the potato centromere nucleosome. The specific experimental steps are as follows: Step 1: Take 10 mg of healthy potato leaf sample I to be tested in a plastic petri dish, add lysis buffer LB to the leaf, chop the leaf with a blade to lyse the cells, filter out the residue through a 300-mesh filter membrane, and mix an appropriate amount of the filtrate with buffer A at a ratio of 1:1.

[0041] Step 2: Spot the cell suspension obtained in Step 1 onto a poly-L-lysine-coated glass slide, air dry, and obtain a dried glass slide. Step 3: Place the dried glass slide described in Step 2 in buffer B and wash it twice on a decolorizing shaker for 5 minutes each time; Step 4: Add blocking solution BT to the glass slide to block non-specific antigens on the cell surface, cover the surface with sealing film, and place at 4°C for 30 minutes. Step 5: Repeat step 3; Step 6: Add primary antibody and incubate overnight at 4°C; Step 7: Repeat step 3; Step 8: Add secondary antibody and incubate at 37°C in the dark for 1 hour; Step 9: Repeat step 3; Step 10: Clean the slide with 70% alcohol, 90% alcohol, and 100% alcohol in sequence, for 2 minutes each time. After cleaning, place the slide to dry at room temperature. Step 11: After drying at room temperature, add 15 μL of DAPI staining solution to each slide to counterstain the cell nuclei, and then mount the slides. Step 12: Place the slide prepared in Step 11 under a fluorescence microscope at an excitation wavelength of 488 nm to observe the fluorescence signal. Randomly select ≥30 clear cells from each sample and count the number of CENH3 hybridization fluorescent spots.

[0042] Step 13: Determine the ploidy of the material to be tested by comparing the total number of fluorescence signals n with known ploidy controls (2x=24, 3x=36, 4x=48, 5x=60, 6x=72…).

[0043] The results are as follows Figure 3 As shown, the number of green fluorescent signals at the centromeres of potato leaf sample #3 was calculated. To demonstrate the reliability of the experimental results, the first experiment was repeated 30 times. The results showed that the average number of clear green fluorescent spots at the centromeres was 42.07 ± 2.57. A statistical hypothesis test (χ² test) was performed on this data. When n = 30, the free values... =29, =42.56, data for each group < =42.56, accepting the null hypothesis (actual centromere count is consistent with theoretical result). The actual result is consistent with the theoretical value of tetraploid 2x=48, indicating that the ploidy of this sample is tetraploid. Detailed values ​​of the χ2 test are shown in Table 1.

[0044] Example 4 This embodiment uses flow cytometry to verify the ploidy of potato leaf samples #1, #2, and #3 in Example 1.

[0045] (1) Verify the ploidy of potato leaf sample #1 to be tested Step 1: Cut 20mg of washed and vein-removed potato leaf sample #1 and place it in a plastic petri dish. Add fresh, pre-cooled lysis buffer LB to submerge the sample. Chop the sample with a double-edged blade and transfer it to a 300-mesh nylon sieve for filtration. Collect the filtrate in a 1.5mL centrifuge tube. Step 2: Add PI dye to the filtrate, mix thoroughly, and incubate at low temperature in the dark for 20 minutes; Step 3: After incubation, vortex the sample and detect it using a cytometer. Select OpenModule Yellow as the detection category and adjust the GATING PROFILE according to the target region.

[0046] (2) Verify the ploidy of potato leaf sample #2 to be tested Step 1: Cut 20mg of washed and vein-removed potato leaf sample #2 and place it in a plastic petri dish. Add fresh, pre-cooled lysis buffer LB to submerge the sample. Chop the sample with a double-edged blade and transfer it to a 300-mesh nylon sieve for filtration. Collect the filtrate in a 1.5mL centrifuge tube. Step 2: Add PI dye to the filtrate, mix thoroughly, and incubate at low temperature in the dark for 20 minutes; Step 3: After incubation, vortex the sample and detect it using a cytometer. Select OpenModule Yellow as the detection category and adjust the GATING PROFILE according to the target region.

[0047] (3) Verify the ploidy of potato leaf sample #3 to be tested Step 1: Cut 20mg of washed and vein-removed potato leaf sample #3 and place it in a plastic petri dish. Add fresh, pre-cooled lysis buffer LB to submerge the sample. Chop the sample with a double-edged blade and transfer it to a 300-mesh nylon sieve for filtration. Collect the filtrate in a 1.5mL centrifuge tube. Step 2: Add PI dye to the filtrate, mix thoroughly, and incubate at low temperature in the dark for 20 minutes; Step 3: After incubation, vortex the sample and detect it using a cytometer. Select OpenModule Yellow as the detection category and adjust the GATING PROFILE according to the target region.

[0048] Save and export the above data, process and analyze it using FlowJo software, and determine the potato ploidy based on the peak position. The results are as follows: Figures 1-3 As shown in C, the peak diagram of potato leaf sample #1 matches that of diploid with known ploidy, indicating that it is diploid and confirming that it is a leaf sample of potato G1; the peak diagram of potato leaf sample #2 matches that of triploid with known ploidy, indicating that it is triploid and confirming that it is potato G2; the peak diagram of potato leaf sample #3 matches that of tetraploid with known ploidy, indicating that it is tetraploid and confirming that it is potato G3.

[0049] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for identifying potato ploidy based on centromere labeling and counting using potato CENH3 antibody, characterized in that, The method includes the following steps: The potato CENH3 antibody and the core histone CENH3 of the potato centromere nucleosome were used to count chromosome centromeres using immunofluorescence technology.

2. The application of the method of claim 1 in potato ploidy identification.

3. The application according to claim 2, characterized in that, Includes the following steps: S1. Obtain the core histone CENH3 sequence of potato centromere nucleosome, wherein the amino acid sequence of the core histone CENH3 is shown in SEQ ID NO:1; S2. After pretreatment of potato leaf samples, cell suspensions were prepared. The cell suspensions were fixed on chromosome slides and subjected to immunofluorescence reaction of the core histone CENH3 of potato centromere nucleosomes to obtain fluorescent labeling signals. S3. Based on the fluorescent marker signals obtained in S2, locate the centromere positions of the chromosomes, count the number of signals, and obtain the number of centromeres, which is the number of potato chromosomes. S4. Determine the ploidy of the potato based on the number of potato chromosomes obtained in S3.

4. The application according to claim 3, characterized in that, The specific steps of the immunofluorescence reaction of the core histone CENH3 of the potato centromere nucleosome described in S2 include: S201. After the chromosome slides with fixed cell suspension are washed with buffer B, they are sealed to obtain a first-processed sample. S202. Wash the primary treatment sample obtained in S201 with buffer B, add primary antibody solution, and incubate to obtain the secondary treatment sample. S203. Wash the secondary processed sample obtained in S202 with buffer B, add secondary antibody solution, and incubate to obtain the tertiary processed sample. S204. The three-processed samples obtained in S203 were washed with buffer B and alcohol, dried at room temperature, and fluorescent labeling signals were obtained.

5. The application according to claim 4, characterized in that, The primary antibody solution in S202 consists of bovine serum albumin, buffer B, and primary antibody, with a volume ratio of bovine serum albumin, buffer B, and primary antibody of 100:100:

1. The secondary antibody solution in S203 consists of bovine serum albumin, buffer B, and secondary antibody, with a volume ratio of 100:100:

1.

6. The application according to claim 5, characterized in that, The primary antibody in the primary antibody solution is potato CENH3 antibody, and the secondary antibody in the secondary antibody solution is Goat Anti Rabbit IgG(H&L)-Alexa FLUOR 488.

7. The application according to claim 3, characterized in that, The step of locating the centromere position of chromosomes using the fluorescent labeling signal described in S3 includes: adding dye to the fluorescent label, mounting the slide, and observing the number of chromosome centromeres.

8. The application according to claim 7, characterized in that, The dye solution is DAPI dye solution.

9. The application according to claim 3, characterized in that, The potato leaf samples were diploid, triploid, or tetraploid cultivated or wild species.

10. A potato ploidy identification kit, characterized in that, Include: The primary antibody is a potato CENH3 antibody. The secondary antibody is Goat Anti Rabbit IgG (H&L)-Alexa Fluor 488; DAPI staining solution; Poly-L-lysine coated glass slides; LB lysate; Buffer A; Buffer B; BT sealing fluid.

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