Efficient oligonucleotide fluorescence in-situ hybridization system and hybridization method for alfalfa
By applying a highly efficient oligo fluorescent in situ hybridization system on alfalfa, using the designed fluorescent modification oligo probe and liquid nitrogen freeze-sheeting method, the problem that traditional FISH technology cannot efficiently obtain chromosome pictures of alfalfa's mid-stage mitosis is solved, and efficient and clear chromosome localization and image acquisition are achieved.
Patent Information
- Application Number
- CN202510050013.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-03-18
AI Technical Summary
Traditional FISH technology cannot efficiently obtain hybrid pictures of the metaphase mitotic chromosomes on alfalfa species, resulting in inaccurate signal.
Using a highly efficient oligo fluorescent in situ hybridization system, the precise positioning of the probe and alfalfa chromosomes is achieved by designing and preparing fluorescent modifications, and combining bioinformatics tools and liquid nitrogen freeze-sheeting methods.
It has successfully achieved efficient and clear acquisition of chromosomal pictures on cells in the meso-stage mitosis of alfalfa root tip, reducing miscellaneous signal interference, and improving the application efficiency of Oligo-FISH technology.
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Figure CN119639873A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of FISH technology probe preparation and application, and specifically relates to a high-efficiency oligonucleotide fluorescence in situ hybridization system and a hybridization method for alfalfa. Background Art
[0002] Alfalfa (Medicago sativa L.) is a perennial leguminous herb. With its excellent adaptability, excellent nutritional value and extremely stable high yield, it enjoys the reputation of "king of forage". It has a wide range of planting areas around the world and plays an important role in the fields of livestock feed, edible vegetables and health-care medicine. It is also one of the most important economic crops in the world. Different alfalfa varieties have different genetic diversity, adaptability, quality and yield. One of the important factors is the polymorphic differences in their chromosomes. Chromosomes are extremely important components of organisms and carriers of biological heritage information. Their variation and recombination play an extremely important role in biological evolution. Therefore, alfalfa cytogenetics research is of great significance for enriching comparative chromosome maps, revealing chromosome polymorphism differences, and analyzing genetic diversity.
[0003] Fluorescence in situ hybridization (FISH) appeared in the 1980s. It combined traditional cytogenetics with molecular biology techniques to achieve the physical location of DNA sequences on chromosomes. The core of this technology is to use a single-stranded nucleic acid with a detectable marker as a probe. According to the principle of complementary base pairing, the probe is matched with a specific nucleic acid sequence to form an identifiable double-stranded hybrid. The emergence of FISH technology has become a powerful tool for solving important genetic problems such as chromosome engineering breeding, research on plant chromosome identification, and species origin and evolution. However, in non-model species, especially those with polyploidy or small chromosomes, it is a major challenge to establish a chromosome identification system based on FISH.
[0004] Alfalfa is a non-model plant with small chromosomes and very similar morphology. In addition, most cultivated alfalfa species are autotetraploids with complex genomes (2n=4x=32), which makes the cytogenetics research of alfalfa plants lag far behind other crops. Cytogenetics research has important reference significance for species classification, evolutionary research, chromosome engineering breeding and other fields. The probes used in traditional FISH technology are not specific enough and will cross-react with non-target sequences, resulting in inaccurate hybridization signals. It is impossible to efficiently obtain mitotic metaphase chromosome hybridization images in alfalfa species. To address the above problems, we proposed an efficient oligonucleotide fluorescence in situ hybridization system and hybridization method for alfalfa. Summary of the invention
[0005] The purpose of the present invention is to provide an efficient oligonucleotide fluorescence in situ hybridization system and hybridization method for alfalfa in view of the shortcomings of the prior art, thereby solving the problem that the traditional FISH technology cannot efficiently obtain mitotic metaphase chromosome hybridization images on alfalfa species.
[0006] The invention is realized by providing a high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa, which comprises oligo probes, alfalfa root tip mitotic metaphase cells and probe hybridization solution.
[0007] The probe hybridization solution preparation system is:
[0008] 7.5ul 100% deionized formamide, 3ul 50% dextran sulfate, 1.5ul 20xSSC, 400ng labeled oligo probe, totaling 15ul.
[0009] The preparation method of the oligo probe specifically comprises:
[0010] S101, preliminary design of oligo sequences: Obtain rice 45SrDNA sequence (GenBank: KM036294.1, 7745 bp) from NCBI database and divide it into 40-50 bp oligonucleotide sequences using bioinformatics tools;
[0011] S102, adding primers to both ends of the oligo sequence: adding the same primer sequence F1 to the 5' end and the same primer sequence R1 to the 3' end of the oligo sequence of each independent probe, the oligo sequence forms an oligo sequence library, and synthesizing primer sequences F1 and R1 with FAM modification at the 5' end;
[0012] S103, oligo probe preparation: using the synthesized oligo sequence library as a template, primer sequence F1 and primer sequence F2 form a primer pair, and amplify 45SrDNA in the oligo sequence library into a labeled oligo probe by PCR;
[0013] S104, preparation of probe hybridization solution: 7.5ul 100% deionized formamide, 3ul 50% dextran sulfate, 1.5ul 20xSSC, 400ng labeled oligo probe, totaling 15ul, incubate at 90°C for 6min and -20°C for 5min to prepare oligo probe hybridization solution.
[0014] The method for preparing alfalfa root tip mitotic metaphase cells specifically comprises:
[0015] S201, a number of alfalfa seeds, cultured at room temperature and in a moist filter paper environment for 2-3 days, took alfalfa young roots with uniform and good growth, soaked them in a 0.0025% cycloheximide solution for 2 hours, and fixed them at room temperature for 24 hours;
[0016] S202, remove the young roots of alfalfa and place them in the root cap and elongation zone, cut the meristem zone into 2-3 sections evenly, and place them in a mixture of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C for enzymatic hydrolysis for 2.5 hours;
[0017] S203, after the enzymatic hydrolysis, a fixative solution was added for fixation at room temperature for 3 days;
[0018] S204, using liquid nitrogen freezing preparation method, the alfalfa root tip that has been pretreated as above is placed in the center of the slide, 60% acetic acid is added for dissociation for a while, and then covered with a coverslip and gently tapped with a dissecting needle for several times to disperse the cells. After being evenly baked at the outer flame of an alcohol lamp for several seconds, the slice is pressed and placed in liquid nitrogen for freezing for several seconds to complete the preparation.
[0019] In step S201, the length of the alfalfa rootlets is 2-4 cm, and the alfalfa rootlets are fixed at room temperature using Carnoy's fixative;
[0020] In step S202, the volume ratio of 1% pectinase Y-23, 2% pectinase, 2% cellulase RS, and 4% cellulase R-10 at 37°C is 2:1:1:1. In step S203, the stationary liquid is a mixture of methanol and glacial acetic acid, and the volume ratio of methanol to glacial acetic acid is 3:1.
[0021] On the other hand, the present invention also provides a hybridization method for a high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa, and the hybridization method for a high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa specifically comprises:
[0022] S10, dividing the target sequence into 40-50 bp oligonucleotide sequences by bioinformatics tools for artificial synthesis, and then preparing the oligonucleotide sequences into oligo probes with fluorescent modification by PCR;
[0023] S20, soaking alfalfa rootlets in 0.0025% cycloheximide solution for 2 h to enrich the mitotic cells in the alfalfa root tips;
[0024] S30, the meristem of the alfalfa root tip was evenly cut and placed in a mixture of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C for 2.5 h;
[0025] S40, using the “liquid nitrogen freezing method” to prepare the chromosomes of the mitotic metaphase of alfalfa root tip;
[0026] S50, slides of alfalfa metaphase chromosomes, placed at 37°C, treated with 0.01% pepsin solution, denatured at 85°C in 100% deionized formamide for 5 min, and placed at -20°C in 70% and 100% ethanol gradient dehydration, added with oligo probe hybridization solution, and hybridized overnight at 37°C;
[0027] S60, wash the hybridized slides overnight in 2xSSC, 1xPBS, and distilled water, then dry them, add DAPI staining solution in the center, cover with a coverslip, and stain at low temperature in the dark;
[0028] S70, using a fluorescence microscope, observe the chromosome morphology stained with DAPI and the distribution of the labeled oligo probe on the chromosome.
[0029] Compared with the prior art, the embodiments of the present application have the following beneficial effects:
[0030] The present invention can efficiently and clearly obtain the metaphase chromosomes of the mitosis of the alfalfa root tip, can realize the physical positioning of the Oligo probe on the alfalfa chromosome, and can efficiently obtain clear cytological images of alfalfa. Thus, the efficient application of Oligo-FISH technology on alfalfa species is successfully realized, which is helpful for the analysis of alfalfa genetic diversity, molecular marker-assisted selection, and the construction of genetic maps.
[0031] The present invention is supported by FISH technology, uses bioinformatics approach to direct design into oligonucleotide (oligo) sequences, and artificially prepares oligo probes with fluorescence modification, successfully constructs an oligo-FISH system for alfalfa, can efficiently obtain alfalfa root tip mitotic metaphase cytological images with good chromosome morphology and no interference from miscellaneous signals, and can clearly show the precise positioning of the oligo probe on the alfalfa chromosome. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 It is a schematic diagram of hybridization results of the oligo probe provided by the present invention in alfalfa species. DETAILED DESCRIPTION
[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.
[0034] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.
[0035] The probes used in traditional FISH technology are not specific enough and will cross-react with non-target sequences, resulting in inaccurate hybridization signals. It is impossible to efficiently obtain hybridization images of mitotic metaphase chromosomes on alfalfa species. In view of the above problems, we have proposed a high-efficiency oligonucleotide fluorescence in situ hybridization system and hybridization method for alfalfa. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa comprises oligo probes, alfalfa root tip mitotic metaphase cells, and a probe hybridization solution. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa provided by the present invention solves the limitation that traditional FISH technology cannot efficiently obtain hybridization images of mitotic metaphase chromosomes on alfalfa species. By constructing a hybridization system, the oligo-DNA probe is physically positioned on the mitotic metaphase chromosomes of the alfalfa root tip, which greatly improves the application of oligo-FISH technology on alfalfa species and is beneficial to the molecular cytogenetics research of alfalfa species.
[0036] Example 1
[0037] The embodiment of the present invention provides a high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa, which comprises an oligo probe, alfalfa root tip mitotic metaphase cells, and a probe hybridization solution.
[0038] Wherein, the probe hybridization solution preparation system is:
[0039] 7.5ul 100% deionized formamide, 3ul 50% dextran sulfate, 1.5ul 20xSSC, 400ng labeled oligo probe, totaling 15ul.
[0040] In this embodiment, the method for preparing the oligo probe specifically includes:
[0041] S101, preliminary design of oligo sequences: Obtain rice 45SrDNA sequence (GenBank: KM036294.1, 7745 bp) from NCBI database and divide it into 40-50 bp oligonucleotide sequences using bioinformatics tools;
[0042] S102, adding primers to both ends of the oligo sequence: adding the same primer sequence F1 to the 5' end and the same primer sequence R1 to the 3' end of the oligo sequence of each independent probe, the oligo sequence forms an oligo sequence library, and synthesizing primer sequences F1 and R1 with FAM modification at the 5' end;
[0043] S103, oligo probe preparation: using the synthesized oligo sequence library as a template, primer sequence F1 and primer sequence F2 form a primer pair, and amplify 45SrDNA in the oligo sequence library into a labeled oligo probe by PCR;
[0044] S104, preparation of probe hybridization solution: 7.5ul 100% deionized formamide, 3ul 50% dextran sulfate, 1.5ul 20xSSC, 400ng labeled oligo probe, totaling 15ul, incubate at 90°C for 6min and -20°C for 5min to prepare oligo probe hybridization solution.
[0045] Example 2
[0046] The embodiment of the present invention also provides a method for preparing alfalfa root tip mitotic metaphase cells, and the method for preparing alfalfa root tip mitotic metaphase cells specifically comprises:
[0047] S201, a number of alfalfa seeds, cultured at room temperature and in a moist filter paper environment for 2-3 days, took alfalfa young roots with uniform and good growth, soaked them in a 0.0025% cycloheximide solution for 2 hours, and fixed them at room temperature for 24 hours;
[0048] It should be noted that in step S201, the length of the alfalfa rootlets is 2-4 cm, and the alfalfa rootlets are fixed at room temperature using Carnoy's fixative.
[0049] S202, remove the young roots of alfalfa and place them in the root cap and elongation zone, cut the meristem zone into 2-3 sections evenly, and place them in a mixture of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C for enzymatic hydrolysis for 2.5 hours;
[0050] In step S202, the volume ratio of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C is 2:1:1:1.
[0051] S203, after the enzymatic hydrolysis, a fixative solution was added for fixation at room temperature for 3 days;
[0052] In step S203, the stationary liquid is a mixture of methanol and glacial acetic acid, and the volume ratio of methanol to glacial acetic acid is 3:1.
[0053] S204, using liquid nitrogen freezing preparation method, the alfalfa root tip that has been pretreated as above is placed in the center of the slide, 60% acetic acid is added for dissociation for a while, and then covered with a coverslip and gently tapped with a dissecting needle for several times to disperse the cells. After being evenly baked at the outer flame of an alcohol lamp for several seconds, the slice is pressed and placed in liquid nitrogen for freezing for several seconds to complete the preparation.
[0054] Example 3
[0055] In an embodiment of the present invention, alfalfa oligonucleotide fluorescence in situ hybridization is also provided. The alfalfa oligonucleotide fluorescence in situ hybridization method specifically comprises:
[0056] S301, slides of alfalfa metaphase chromosomes were placed in 0.01% pepsin solution, reacted at 37°C for 40-90 min, soaked in 1xPBS and 2% paraformaldehyde for 5-10 min, washed several times with distilled water, and dried naturally.
[0057] S302 was denatured in 100% deionized formamide at 85°C for 5 min, and then dehydrated in 70% and 100% ethanol at -20°C. After air drying, hybridization solution was added and the tube was sealed with a coverslip and hybridized overnight at 37°C.
[0058] S303, wash the hybridized slides overnight in 2xSSC, 1xPBS, and distilled water, and then air dry. DAPI staining solution is added to the center, and a coverslip is placed on the slides. Stain the slides at low temperature and away from light for several hours.
[0059] S304, using a fluorescence microscope, respectively observe the chromosome morphology stained with DAPI and the distribution of the labeled oligo probe on the chromosome.
[0060] Example 4
[0061] In another aspect of the present invention, a hybridization method for a high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa is provided. The hybridization method for a high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa specifically comprises:
[0062] S10, dividing the target sequence into 40-50 bp oligonucleotide sequences by bioinformatics tools for artificial synthesis, and then preparing the oligonucleotide sequences into oligo probes with fluorescent modification by PCR;
[0063] It should be noted that, in this embodiment, the target sequence was divided into 40-50 bp oligonucleotide sequences for artificial synthesis using bioinformatics tools and sent to Suzhou Hongxun Biotechnology Co., Ltd. for synthesis.
[0064] S20, soaking alfalfa rootlets in 0.0025% cycloheximide solution for 2 h to enrich the mitotic cells in the alfalfa root tips;
[0065] S30, the meristem of the alfalfa root tip was evenly cut and placed in a mixture of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C for 2.5 h;
[0066] S40, using the “liquid nitrogen freezing method” to prepare the chromosomes of the mitotic metaphase of alfalfa root tip;
[0067] S50, slides of alfalfa metaphase chromosomes, were treated with 0.01% pepsin at 37°C, denatured in 100% deionized formamide at 85°C for 5 min, and dehydrated in 70% and 100% ethanol gradient at -20°C, oligo probe hybridization solution was added, and hybridization was carried out at 37°C overnight.
[0068] S60, wash the hybridized slides overnight in 2xSSC, 1xPBS, and distilled water, then dry them, add DAPI staining solution in the center, cover with a coverslip, and stain at low temperature in the dark for several hours;
[0069] S70, using a fluorescence microscope, observe the chromosome morphology stained with DAPI and the distribution of the labeled oligo probe on the chromosome.
[0070] In this embodiment, Figure 1 A schematic diagram showing the hybridization results of oligo probes in alfalfa species is shown, wherein: Figure 1 A represents the chromosome in metaphase of mitosis, B represents the oligo probe signal diagram, and C is the synthesis diagram of alfalfa oligonucleotide fluorescence in situ hybridization. Figure 1 It can be seen that the high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa constructed by the present invention can obtain dispersed, well-shaped alfalfa root tip mitotic metaphase chromosomes, and can produce clear, strong, clean-background, and non-interference fluorescent signals on the alfalfa chromosomes, proving that the high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa is effective.
[0071] In summary, the present invention provides an efficient oligonucleotide fluorescence in situ hybridization system and hybridization method for alfalfa, which can efficiently and clearly obtain the metaphase chromosomes of the mitosis of the alfalfa root tip, can realize the physical positioning of the Oligo probe on the alfalfa chromosome, and can efficiently obtain clear cytological images of alfalfa. Thus, the efficient application of Oligo-FISH technology on alfalfa species is successfully realized, which is helpful for the analysis of alfalfa genetic diversity, molecular marker-assisted selection, and the construction of genetic maps.
[0072] The present invention is supported by FISH technology, uses bioinformatics approach to direct design into oligonucleotide (oligo) sequences, and artificially prepares oligo probes with fluorescence modification, successfully constructs an oligo-FISH system for alfalfa, can efficiently obtain alfalfa root tip mitotic metaphase cytological images with good chromosome morphology and no interference from miscellaneous signals, and can clearly show the precise positioning of the oligo probe on the alfalfa chromosome.
[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also belong to the scope of protection of the present invention.
Claims
1. A highly efficient oligonucleotide fluorescence in situ hybridization system for alfalfa, characterized in that: The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa comprises oligo probes, alfalfa root tip mitotic metaphase cells and probe hybridization solution.
2. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 1, characterized in that: The probe hybridization solution preparation system is: 7.5ul 100% deionized formamide, 3ul 50% dextran sulfate, 1.5ul 20xSSC, 400ng labeled oligo probe, totaling 15ul.
3. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 2, characterized in that: The preparation method of the oligo probe specifically comprises: S101, preliminary design of oligo sequences: Obtain rice 45SrDNA sequence (GenBank: KM036294.1, 7745 bp) from NCBI database and divide it into 40-50 bp oligonucleotide sequences using bioinformatics tools; S102, adding primers to both ends of the oligo sequence: adding the same primer sequence F1 to the 5' end and the same primer sequence R1 to the 3' end of the oligo sequence of each independent probe, the oligo sequence forms an oligo sequence library, and synthesizing primer sequences F1 and R1 with FAM modification at the 5' end; S103, oligo probe preparation: using the synthesized oligo sequence library as a template, primer sequence F1 and primer sequence F2 form a primer pair, and amplify 45SrDNA in the oligo sequence library into a labeled oligo probe by PCR; S104, preparation of probe hybridization solution: 7.5ul 100% deionized formamide, 3ul 50% dextran sulfate, 1.5ul 20xSSC, 400ng labeled oligo probe, totaling 15ul, incubate at 90°C for 6min and -20°C for 5min to prepare oligo probe hybridization solution.
4. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 3, characterized in that: The method for preparing alfalfa root tip mitotic metaphase cells specifically comprises: S201, a number of alfalfa seeds, cultured at room temperature and in a moist filter paper environment for 2-3 days, took alfalfa young roots with uniform and good growth, soaked them in a 0.0025% cycloheximide solution for 2 hours, and fixed them at room temperature for 24 hours; S202, remove the young roots of alfalfa and place them in the root cap and elongation zone, cut the meristem zone into 2-3 sections evenly, and place them in a mixture of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C for enzymatic hydrolysis for 2.5 hours; S203, after the enzymatic hydrolysis, a fixative solution was added for fixation at room temperature for 3 days; S204, using liquid nitrogen freezing preparation method, the alfalfa root tip that has been pretreated as above is placed in the center of the slide, 60% acetic acid is added for dissociation for a while, and then covered with a coverslip and gently tapped with a dissecting needle for several times to disperse the cells. After being evenly baked at the outer flame of an alcohol lamp for several seconds, the slice is pressed and placed in liquid nitrogen for freezing for several seconds to complete the preparation.
5. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 4, characterized in that: In step S201, the length of the alfalfa rootlets is 2-4 cm, and the alfalfa rootlets are fixed at room temperature using Carnoy's fixative.
6. The high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 4, characterized in that: In step S202, the volume ratio of 1% pectinase Y-23, 2% pectinase, 2% cellulase RS, and 4% cellulase R-10 at 37°C is 2:1:1:
1. In step S203, the stationary liquid is a mixture of methanol and glacial acetic acid, and the volume ratio of methanol to glacial acetic acid is 3:
1.
7. A hybridization method for alfalfa using a high-efficiency oligonucleotide fluorescence in situ hybridization system, implemented using the high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 1, characterized in that: The hybridization method for the high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa specifically comprises: S10, dividing the target sequence into 40-50 bp oligonucleotide sequences by bioinformatics tools for artificial synthesis, and then preparing the oligonucleotide sequences into oligo probes with fluorescent modification by PCR; S20, soaking alfalfa rootlets in 0.0025% cycloheximide solution for 2 h to enrich the mitotic cells in the alfalfa root tips; S30, the meristem of the alfalfa root tip was evenly cut and placed in a mixture of 1% pectinase Y-23, 2% pectinase pect, 2% cellulase RS, and 4% cellulase R-10 at 37°C for 2.5 h; S40, using the "liquid nitrogen freezing method" to prepare the chromosomes of the metaphase of mitosis in the root tip of alfalfa; S50, slides of alfalfa metaphase chromosomes, were treated with 0.01% pepsin at 37°C, denatured in 100% deionized formamide at 85°C for 5 min, and dehydrated in 70% and 100% ethanol gradient at -20°C, oligo probe hybridization solution was added, and hybridization was carried out at 37°C overnight.
8. The hybridization method for the high-efficiency oligonucleotide fluorescence in situ hybridization system for alfalfa as claimed in claim 7, characterized in that: The hybridization method for the high-efficiency oligonucleotide fluorescence in situ hybridization system of alfalfa also includes: S60, wash the hybridized slides overnight in 2xSSC, 1xPBS, and distilled water, then dry them, add DAPI staining solution in the center, cover with a coverslip, and stain at low temperature in the dark; S70, using a fluorescence microscope, observe the chromosome morphology stained with DAPI and the distribution of the labeled oligo probe on the chromosome.