A method for preparing chromosomes from root meristems of tetraploid small-bean coffee varieties

Abstract

This invention discloses a method for preparing chromosomes from root meristems of tetraploid small-bean coffee. It belongs to the fields of genetics and genomics. This invention describes a scheme for obtaining root tip meristems rich in metaphase chromosomes. The steps and parameters involved in plant cultivation before sampling, root tip sampling and fixation, and chromosome separation and diffusion are all described in detail.

Description

Technical Field

[0001] This invention relates to the fields of genetics and genomics, and more specifically to a method for preparing chromosomes from the root meristem of tetraploid small-bean coffee. Background Technology

[0002] Coffee is one of the world's three major beverages. The genus *Coffea* contains over 10,090 species, with wild-type coffee including diploid and polyploid varieties. The vast majority of commercially grown coffee varieties are closely related to two main types: the small-bean variety *Coffea arabica*, which possesses four sets of chromosomes, and the medium-bean variety *Coffea canephora* (also known as Robusta), which has two sets of chromosomes. *Coffea arabica* accounts for approximately 65% ​​of global coffee production, with each cell possessing 44 chromosomes (2n = 4x = 44). The natural hybrid 'Hibrido de Timor' (HDT), derived from *Coffea arabica* and *Coffea canephora*, was once hailed as the most effective germplasm resource for resisting coffee leaf rust (CLR). C. arabica Catimor 7963 is a resistant variety derived from the original HDT line CIFC 832 / 1. In recent years, it has become a widely cultivated superior variety in Yunnan, my country's main coffee-growing region. However, this variety currently faces the risk of losing resistance to CLR. Hybridization using these germplasms can increase the genetic base of crops and transfer useful genes from wild-related species into target crop varieties. Assessing genome assembly quality can utilize high-resolution FISH mapping, which involves chromosome banding on mitotic chromosomes. By integrating physical and genetic maps, it can reveal the pairing of genes during evolution or after gene recombination and uncover any translocations between chromosomes.

[0003] Obtaining high-quality metaphase chromosome smears is a prerequisite for cytogenetics research. Techniques such as chromosome counting, morphological analysis and mapping, chromosome banding, and in situ hybridization all rely on having clear, intact, undamaged, monolayered, and uniformly spread metaphase chromosome smears. Therefore, preparing good chromosome slides is fundamental to molecular cytogenetics research. However, although there has been some research on the preparation of root tip chromosomes, the conventional methods for preparing chromosome smears are not suitable for the varieties now widely cultivated due to differences in tissue firmness, cell wall thickness and toughness, cell size, and chromosome number between different species and even different hybrids of the same species.

[0004] In addition, the existing technology for chromosome preparation has the following problems:

[0005] Most chromosome analyses rely on metaphase of mitosis, therefore obtaining good meristematic tissue is essential. Past methods for preparing root tip chromosomes from coffee or other crops have not considered the biological characteristics of plant growth cycles and cell division, nor have they recognized the significant correlation between leaf and root growth. Previous studies have shown that coffee chromosome smears often contain residual non-meristic tissue, with uneven or overlapping chromosome distribution, or even chromosome damage, thus affecting the determination of chromosome ploidy levels and the study of genetic levels.

[0006] In summary, how to provide a method for preparing large quantities of high-quality metaphase chromosomes from tetraploid small-bean coffee is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing chromosomes from the root meristem of tetraploid small-bean coffee.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing chromosomes from root meristems of tetraploid small-seeded coffee varieties includes the following steps:

[0010] (1) Plant cultivation

[0011] Cultivate the plants into seedlings that are 20-40cm tall for later use;

[0012] (2) Root tip sampling and fixation

[0013] (21) Collect the white new root tip tissue at the end of the root 4 to 4.5 hours after sunrise each day or 4 to 4.5 hours after the lights in the greenhouse start to shine;

[0014] (22) After rinsing with sterile distilled water, take 1-1.5 cm of root tip tissue;

[0015] (23) Treat the root tip tissue with a saturated 0.05% α-bromonaphthalene aqueous solution, place it at 22-25℃ for 2h, and then place it at 4℃ for 4h;

[0016] (24) Treat with root tip fixative, place at 22-25℃ for 2 hours, then place at 4℃ for more than 2 days;

[0017] Beneficial effects achieved: Time control in this step is crucial, aiming to fix a large number of metaphase chromosomes within the root tip cells. Excessive processing time can cause chromosome damage or atrophy, hindering the acquisition of chromosomes with intact ploidy; conversely, insufficient processing time will prevent the collection of as many metaphase chromosomes as possible.

[0018] Fixed root tips can be stored at -20°C for more than 3 months. Fixed root tip tissue must be transported under sterile conditions at low temperatures, and direct sunlight or high temperatures should be avoided.

[0019] (3) Chromosome separation and diffusion

[0020] (31) Pretreatment of root tip meristem

[0021] (311) Clean the fixed root tip and then soften it with HCl;

[0022] Beneficial effects achieved: The control of HCl concentration and time is crucial in this step. Its purpose is to act as an acidic dissociation agent to soften the cell walls of the root tip tissue, dissolve the intercellular lamina, alter cell membrane permeability, and facilitate the separation of proteins and chromosomes, preparing for the subsequent infiltration and dissolution of the enzyme solution. HCl is a highly volatile strong acid; if the concentration is too low or the treatment time is too short, incomplete dissociation will occur; if the concentration is too high or the treatment time is too long, over-dissociation may occur, even damaging chromosome structure.

[0023] (312) After washing with enzyme buffer, incubate with enzyme solution at 37°C for 35 min;

[0024] Beneficial effects achieved: This step requires strict control of the time. If the treatment time is too short, the root tip tissue will not be completely digested by the enzyme solution; if the treatment time is too long, the root tip tissue will be over-digested by the enzyme solution, making the root tip too soft or breaking, resulting in the leakage of cell contents, which is not conducive to subsequent dissection.

[0025] (313) After washing with enzyme buffer, add sterile distilled water to keep the area moist;

[0026] (32) Anatomy of root apical meristem

[0027] (321) The pretreated root tip tissue was treated with 60% acetic acid as a medium for 3 min;

[0028] (322) Use a sterile needle to separate and remove non-meristic tissues more than 0.1 cm below the root cap; in this step, avoid touching or squeezing the root tip meristem, and do not leave other non-meristic tissues on the slide.

[0029] (323) Squeeze and separate individual cells to obtain a single layer of chromosomes.

[0030] Furthermore, the specific conditions for cultivation described in step (1) are as follows:

[0031] The optimal temperature for plant growth is 25–28℃;

[0032] The culture medium consists of nutrient soil and fine sand, with a volume ratio of 8:2.

[0033] Beneficial effects achieved: This ratio provides sufficient nutrition for root development and also aids in root respiration and extension. Maintain a 14-hour daytime and 10-hour nighttime light cycle, while the plant requires appropriate shading (coffee plants, especially seedlings, must avoid direct sunlight or greenhouse lighting).

[0034] The air humidity is 80-90%;

[0035] Furthermore, prepare a transparent sterilized plastic bag that matches the size of the plant in advance. Spray sterile purified water thoroughly on the inside of the plastic bag, then cover the entire plant with the plastic bag and tie the opening tightly with cotton rope. Note that you should only cover the part of the plant growing above the soil; do not cover the soil inside the bag, as this will affect root respiration.

[0036] Continue until the plant's roots grow to the sides of the pot. For plants with vigorous root branching, ensure the soil moisture content is around 20%—not too wet, not waterlogged, but not dry either.

[0037] Furthermore, the volume of the saturated 0.05% α-bromonaphthalene aqueous solution in step (23) is three times that of the plant material.

[0038] Furthermore, the fixative comprises ethanol and glacial acetic acid, with a volume ratio of 3:1;

[0039] The ethanol is a 96% or 100% (v / v) aqueous solution of ethanol.

[0040] Furthermore, in step (311), the softening treatment using HCl involves using 0.01M HCl to soften the root tip for 15 minutes (when the root tip is picked up by tweezers, the root tip head naturally droops downwards).

[0041] Furthermore, the enzyme solution includes cellulase and pectinase.

[0042] Furthermore, the specific operation of step (323) is as follows:

[0043] Dissect the root tip meristem in acetic acid, and use a needle to gently tap or squeeze to separate individual cells, spreading the mixed cells evenly.

[0044] Place a coverslip on the slide, cover one corner with a tissue or filter paper, and hold it down with one finger to prevent it from slipping. Then, gently press the coverslip vertically with another finger and tap it vertically with the eraser on the back of a pencil until the air bubbles between the coverslip and the slide disappear.

[0045] Place the glass slide with the coverslip on the folded filter paper, and press the coverslip area with your thumb or palm, slowly and carefully increasing the pressure.

[0046] Furthermore, the tetraploid small-bean coffee variety is Coffea arabica Catimor 7963.

[0047] Examine and evaluate the morphology and number of chromosomes in metaphase of mitosis under a regular optical microscope. Metaphase chromosomes should be frequently distributed on the slide (approximately 20-30% of the cells), free of cytoplasm and dirt, well-distributed, with minimal overlap, and without distortion. If further cytoplasmic removal and chromosome release are necessary, place a drop of acetic acid on the edge of a coverslip, heat the slide with an alcohol flame for a few seconds (the slide surface should not exceed 60°C), and then squeeze it again.

[0048] The Catamor7963 chromosome slides prepared using the method of this invention can be stored at 4°C in the dark for 3 to 6 months, and can be stored for a long time at -20°C.

[0049] Chromosome smear preparation is used to study the chromosome number of a species, determine its ploidy level, chromosome morphology and size, identify specific chromosomes, and the characteristic length and position of the centromere of each chromosome, aiding in species identification. This technology is particularly important as a fundamental guarantee for chromosome markers. Identifying individual chromosomes through morphological analysis or in situ hybridization with DNA probes can be used to link genetic maps in genomics with physical maps of chromosomes, and to track chromosomes in breeding programs involving hybridization and recombination. In hybridization involving a wide range of species or polyploid species, cytogenetic studies of chromosome number and morphology are of significant value in determining which hybridizations are likely to be successful and in identifying new combinations of chromosomes in hybrids and backcross derivatives. Generally, recombination between chromosomes of different species is required for genes regulating desirable agronomic traits to infiltrate the chromosomes of the target strain. Aneuploidy, involving the loss or gain of one or more chromosomes, is occasionally found, along with other types of chromosomal rearrangements such as inversion, deletion, or translocation, especially in irradiated mutagenesis materials and during some coffee tissue cultures. Therefore, chromosome genomics studies and physical localization analysis can precisely show chromosome loss or gain. Visually tracking the chromosomal location of genes and providing a cytogenetic map of chromosomes is inseparable from the preparation of chromosome smears.

[0050] Cytogenetic mapping provides an effective tool for gene localization, verifying connectivity sequences through sequence analysis, characterizing gene regions or physical genetic distances, and detecting chromosomal rearrangements (such as inversions or translocations). Coffee chromosomes in metaphase of mitosis are very small (1 μm–3 μm), and different chromosomes have similar morphologies, making individual characteristics difficult to identify. Without good chromosome smears, it is difficult to determine the precise location of small repetitive sequences or gene clusters such as BACs on these small chromosomes through in situ hybridization, and to clearly identify chromosome arms and structural domains such as chromosome ends and centromeres.

[0051] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0052] Select the best material – seedlings

[0053] Previous studies have not proposed detailed measures and schemes for obtaining the most suitable root tip material for chromosome preparation. This invention proposes that maintaining high humidity (80%–90%) in seedlings helps to activate the meristematic tissue of the root tip and promote root tip development, thereby obtaining good experimental material for chromosome preparation.

[0054] Choose the best sampling time

[0055] In many related studies, random sampling times have resulted in a low probability of obtaining chromosomes in metaphase of mitosis. This invention proposes that the optimal sampling time is 4–4.5 hours after sunrise (or after exposure to greenhouse lights). Root tips obtained during this period can yield a large number of meristematic cells in metaphase of mitosis.

[0056] To the maximum extent possible, fix chromosomes in metaphase of mitosis

[0057] This invention improves and optimizes the root tip cell fixation protocol, specifying reagent ratios and time parameters to facilitate technology promotion and application.

[0058] High-quality mitotic metaphase chromosome smears can be obtained without expensive equipment. Attached Figure Description

[0059] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0060] Figure 1This is a diagram of coffee seedlings and fresh roots in Embodiment 1 of the present invention. In Figure A, it can be seen that healthy seedlings have many freshly grown roots available for sampling. In Figure B, arrows and circles mark fresh roots, which are the best material for chromosome preparation.

[0061] Figure 2 This is a schematic diagram of enzyme solution digestion of root tips in Example 1 of the present invention;

[0062] Figure 3 The following is a description of root apical tissue under a dissecting microscope in Example 1 of the present invention: A is the root apex after enzymatic digestion; B is the non-meristic tissue of the root apex removed by a sterile needle; and C is the root cap and root apical meristem.

[0063] Figure 4 The images show chromosome morphology under a conventional optical microscope, as described in this invention. Arrows indicate several typical characteristics of chromosome preparation: A) high cell nuclear density; Arrow 1 (sampling time: 6:00 PM, other procedures as described in this invention) shows chromosomes in a single cell, but unevenly distributed; B) overly dense cells with overlapping nuclei; Arrow 2 (treated with root tip fixative, placed at 30°C for 1.5 hours, then at 10°C for 1 day) shows pre-metaphase chromosomes, unevenly distributed; Arrow 3 (over-compression) shows metaphase chromosomes, but they are over-compressed, resulting in some chromosomes being twisted or damaged; C) well-separated cells and metaphase chromosomes prepared using the method described in this invention; Arrows 4 and 5 are good examples of metaphase chromosome preparation, well-distributed with little cytoplasm (scale bar = 10 μm).

[0064] Figure 5 In Example 2 of this invention, PCR amplification of the pTa794 insert using M13 primers was performed. The figure shows the following lanes from left to right: a 2kb DNA marker (band sizes from bottom to top: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1kb, 1.25kb, 1.5kb, and 2kb), three PCR replicas of the same microprepared DNA, an empty lane, three polymerase chain reaction replicas of different microprepared DNA, and the 2kb DNA marker. The correct product is a bright band of approximately 500bp because the primers used are located outside the cloning site and increase the insertion length by 81bp from 410bp. Some background smearing and streaks are also visible. Gel extraction and purification of the PCR product is the optimal choice.

[0065] Figure 6The above is a fluorescence in situ hybridization (FISH) of chromosomes in metaphase of root tip mitosis of Catimor 7963 in Example 2 of this invention (2n = 4x = 44). In this example, A is a chromosome stained with DAPI, showing a small gap or a slightly bright band at the centromere; B is the DAPI image superimposed on the 18S rDNA signal (green); C is superimposed on the 5S rDNA signal (red); D is a pair of chromosomes with a major 5S rDNA site near the centromere and a terminal 18S rDNA site on the small chromosome arm; in addition, there is a pair of minor 18S rDNA sites (arrows) and a pair of minor 5S rDNA sites (scale bar = 5 μm). Detailed Implementation

[0066] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] The reagents, materials, and pre-fabrication steps involved in the examples are as follows:

[0068] 1. Saturated 0.05% α-bromo-naphthalene aqueous solution: Prepared by mixing 3–5 drops of α-bromo-naphthalene liquid into 500 mL of distilled water, shaking vigorously to form a saturated solution, and allowing it to precipitate. A small amount of α-bromo-naphthalene should be left at the bottom of the container to ensure saturation. This solution can be stored at room temperature for 3 months.

[0069] 2. Root tip fixative: Mix 96% or 100% (v / v) aqueous ethanol and glacial acetic acid in a 3:1 volume ratio. The fixative needs to be freshly prepared before use, and the time from preparation to use should not exceed 30 minutes.

[0070] 3. A capped, airtight tube with a capacity of 2–10 mL that can completely isolate air from the air. (e.g., EP tube or cryo-flask)

[0071] 4. Blades, dissecting needles, forceps, and scissors: These must be sterilized under high temperature and pressure before use.

[0072] 5. Enzyme buffer (10mM, pH 4.6): For a 100mM stock solution, mix 100mM citric acid and 100mM trisodium citrate at a volume ratio of 2:3 and autoclave. The stock solution can be stored at 4°C for 2 weeks. Before use, dilute the enzyme buffer stock solution to 10mM with sterile distilled water.

[0073] 6. Enzyme solution: 20 U / mL Sigma C1184 cellulase, 10 U / mL Onozuka RS cellulase, and 20 U / mL Sigma P4716 pectinase in 10 mM enzyme buffer. Store in 2–5 mL aliquots at -20°C. The enzyme solution can be reused.

[0074] 7.0.01M HCl.

[0075] 8.60% (v / v) acetic acid.

[0076] 9. Dry ice or liquid nitrogen. If dry ice or liquid nitrogen is unavailable, the slides can be frozen on a metal plate in a -80°C freezer.

[0077] 10. Microscope glass slides: Designated for microscopic examination, pre-cleaned, and if available, specially treated adhesive microscope slides (e.g., Thermofish, UK; Superfrost or Superfrostplus; Citotest, PR China). If no pre-treated slides are available, wash the slides in detergent and incubate them in 96% ethanol with 2–3 drops of HCl for better adhesion of cells and chromosomes.

[0078] 11. Coverslip: 18mm × 18mm medium thickness (No. 1) coverslip. Since the optimal cell nuclei and metaphase chromosomes are usually close to the edge of the smear preparation, using larger coverslips (e.g., 24mm × 30mm or 40mm thinner coverslips (No. 0) will help improve the observation of the oil immersion.

[0079] 12. Petri dish (9cm in diameter or smaller).

[0080] 13. Filter paper (approximately 9cm in diameter).

[0081] 14. High-temperature and high-pressure sterilizer and color-changing paper.

[0082] 15. Diamond pen: Used to scratch glass and make marks.

[0083] 16. Plastic sliding box, which can hold 20 or 50 slides.

[0084] 17. Dissecting microscope and phase contrast microscope.

[0085] Example 1

[0086] I. Preparation of Materials

[0087] 1. For the preparation of the Catimor 7963 chromosome, seedlings are preferred, and the optimal growth period is until the seedlings reach 20-40cm.

[0088] 2. Mix the potting soil and fine sand required for potted plants in a volume ratio of 8:2.

[0089] 3. Catimor 7963 plants are suitable for growth in an environment with a temperature of 25-28℃ or in a greenhouse, maintaining a light cycle of 14 hours during the day and 10 hours at night. At the same time, the plants need appropriate shading (coffee plants, especially seedlings, must avoid direct sunlight or greenhouse lights).

[0090] 4. Well-developed root systems are crucial for obtaining optimal chromosome preparation materials for the Catimor 7963 plant. We need to ensure the plant is within its optimal growth temperature range and that the air humidity is near saturation (80%–90%). To this end, prepare a transparent, sterile plastic bag that matches the size of the plant. Thoroughly spray the inside of the bag with sterile purified water, then place the bag over the entire plant and tie the opening tightly with cotton string. Note that only the part of the plant growing above the soil should be covered; avoid covering the soil inside the bag to prevent hindering root respiration.

[0091] 5. Continue sampling until the roots of the Catimor7963 plant grow to the side of the pot (for easy sampling). For plants with vigorous root branching, ensure the soil moisture content is around 20%, not too wet, not waterlogged but not dry either.

[0092] II. Fresh root sampling and fixation

[0093] 1. Sampling is conducted in a greenhouse (around 25℃). Select healthy, well-growing plants (good leaf growth indicates good root growth. Criteria: leaves without any disease spots or signs, glossy green color without yellowing, and moist surface, not dry) for sampling. Collect only the newly grown white roots at the base of the plant. Figure 1 To maximize the number of metaphase chromosomes, the optimal time to sample the root tips of Catimor 7963 plants is 4–4.5 hours after sunrise each day (or 4–4.5 hours after the greenhouse lights begin to illuminate the plant).

[0094] 2. Gently rinse the plant roots with sterile distilled water for 30 seconds to remove any remaining soil.

[0095] 3. Use high-temperature and high-pressure sterilized tweezers to pick up 1-1.5cm of the white root tip.

[0096] 4. Place the root tips in a pre-prepared saturated 0.05% α-bromo-naphthalene aqueous solution, with a volume three times that of the plant material (approximately 2 mL), ensuring complete coverage of the root tips. Incubate at room temperature (22–25°C) for 2 hours, then immediately transfer to 4°C and incubate for another 4 hours.

[0097] 5. Quickly transfer the root tip to clean filter paper, absorb any remaining solution, and then transfer the root tip to fresh fixative. Incubate at room temperature (22–25°C) for 2 hours, then immediately transfer to 4°C and incubate for at least 2 days.

[0098] 6. The fixed root tips can be stored at -20℃ for more than 3 months. They must be transported at low temperature under sterile conditions. Avoid direct sunlight or high temperature.

[0099] III. Preparation of Chromosome Smears

[0100] To obtain a large number of cytoplasmic metaphase chromosomes, we used pectinase and cellulase to remove the cell walls and then dripped acetic acid between the slide and coverslip. The meristematic tissue was dissected by squeezing with acetic acid as a medium. This step was carried out at room temperature (22-25°C).

[0101] (I) Pretreatment of root tip meristem

[0102] 1. Rinse the fixed root tip with sterile distilled water in a petri dish for 30 minutes to remove any dirt from the root, discard and cut off any unwanted impurities, leaving only a clear white tip.

[0103] 2. Soften the root tip with 0.01M HCl for 15 minutes until the root tip head naturally droops down when the root is picked up with tweezers.

[0104] 3. Wash the root tip three times with the pre-prepared enzyme buffer, 5 minutes each time.

[0105] 4. Place the root tips one by one on a clean glass slide, and fix the slide in a sterile petri dish. Figure 2 ).

[0106] 5. Add 20 μL of enzyme solution to each root tip, cover with a culture dish, and incubate at 37°C for 35 min.

[0107] 6. Use a pipette to remove the enzyme solution, and then wash the root tip three times with enzyme buffer, each time for 5 minutes.

[0108] 7. Apply a drop of sterile distilled water to the treated root tips to keep them moist.

[0109] (II) Anatomy of the root apical meristem Figure 3 )

[0110] 1. Place one drop of 60% acetic acid on a clean glass slide, then place 1-2 pretreated root tips on it and let it stand for 3 minutes.

[0111] 2. Under a dissecting microscope, use a sterile needle to separate and remove the portion of the root cap extending more than 0.1 cm below the root tip. During this step, it is crucial to avoid touching or squeezing the root apical meristem, and to prevent any other non-meristic tissue from remaining on the slide.

[0112] 3. Dissect the root tip meristem in acetic acid, and use a needle to gently tap or squeeze to separate individual cells, spreading the mixed cells evenly.

[0113] 4. Place a small coverslip on the slide, cover one corner with a tissue or filter paper, and hold it down with one finger to prevent it from slipping. Then gently press the coverslip vertically with another finger. Tap the coverslip vertically with the eraser on the back of a pencil until the air bubbles between the coverslip and the slide disappear.

[0114] 5. Place the glass slide with the coverslip in the folded filter paper, and press the coverslip area with your thumb or palm, slowly and carefully increasing the pressure.

[0115] 6. Freeze the slide on dry ice for 5-10 minutes or immerse it in liquid nitrogen for 5-10 seconds. Remove the coverslip with a blade and let the slide air dry naturally.

[0116] 7. Examine and evaluate the morphology and number of chromosomes in metaphase of mitosis under a regular optical microscope. Metaphase chromosomes should be frequently distributed on the slide (approximately 20-30% of the cells), free of cytoplasm and dirt, well-distributed, with minimal overlap, and without distortion. Figure 4 If further removal of cytoplasm to release chromosomes is required, a drop of acetic acid can be placed on the edge of the coverslip, the slide can be heated with an alcohol flame for a few seconds (the surface of the slide should not exceed 60°C), and then squeezed again.

[0117] 8. The Catamor 7963 chromosome slides prepared using this method can be stored at 4°C in the dark for 3 to 6 months, and can be stored for a long time at -20°C.

[0118] Example 2

[0119] In molecular cytogenetics research, many repetitive sequences can be used as chromosome markers to identify the location of target genes in chromosomes, thereby predicting the evolution of gene populations in tissues. They can also be used to track the distribution and recombination of chromosomes in a species after evolution and hybridization. This protocol concludes by using rDNA probes designed with repetitive DNA sequences to perform FISH fluorescence in situ hybridization experiments to verify the effectiveness of chromosome smear preparation.

[0120] In the following protocol, we use repetitive rDNA as probes to identify coffee chromosomes carrying 35S / 45S and 5S rDNA loci, which provide effective markers on the chromosomes to verify the effectiveness of chromosome smear preparation.

[0121] I. Probe Design

[0122] II. FISH Verification

[0123] 1. PCR amplification of probe DNA.

[0124] a. For 5S rDNA: Microprepared DNA from clone pTa794 with M13 primers was amplified by PCR at annealing temperature of 56°C. The expected product insert size is approximately 80 bp. Figure 5 ).

[0125] b. For 35S / 45S rDNA: Using total genomic DNA from wheat or rice and rice_18S primers, the 18S rDNA sequence was amplified by PCR at an annealing temperature of 68°C.

[0126] 2. Examine the PCR products on a 1.2% agarose gel.

[0127] a. If a single sharp band of the expected size is present, the entire PCR product can be used for labeling after purification.

[0128] b. If there are several bands, or if it is a smear, cut off the band of the expected size, extract the DNA from the band and wash it.

[0129] 3. Label the probe DNA according to the instructions of the labeling kit.

[0130] 4. Filter the labeled probe using a purification column and resuspend it in 20–30 μL of sterile ddH₂O. If a purification column is unavailable, ethanol precipitation containing sodium acetate or lithium chloride can be used.

[0131] 5. Store the probe at -20°C until use, avoiding freeze-thaw cycles. Binding of the label to the probe DNA can be checked using a simple test blot. Place a small drop onto a Southern hybridization membrane using a pipette and follow the protocol for colorimetric detection of biotin- and digoxigenin-labeled DNA using an alkaline phosphatase-linked antibody (see, for example, Roche Diagnostics; https: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / Roche / General_Information / 1 / dig-application-manual-for-filter-hybridisation-iris.pdf).

[0132] FISH Day 1: Pretreatment and Hybridization

[0133] For in situ hybridization, the protocols of Schwarzacher et al. (1989) and Schwarzacher & Heslop Harrison (2000) were used, with some protocol optimizations and parameter adjustments made to the coffee chromosome reported by Pinto Maglio et al. (2006). Unless otherwise specified, the procedures were performed at room temperature. Washing and buffer incubation were performed in Coplin jars (keeping 8 slides and 80–100 mL of solution); specific reagents were applied in small volumes of 200–300 μL per slide, covered with plastic coverslips, and incubated in a humid environment.

[0134] 1. If the slides were stored in a refrigerator or freezer, warm them to room temperature and then check the quality again. In the following steps, mark the chromosome preparation areas with a diamond pen and numbered slides for easy identification.

[0135] 2. Post-fixation of the glass slide.

[0136] a. Incubate the slide in fixative (ethanol / acetic acid volume ratio 3:1) for 10–30 min.

[0137] b. Wash twice with 100% ethanol, 5 minutes each time.

[0138] c. Air dry.

[0139] 3. RNase treatment.

[0140] a. Apply 200 μL of RNase solution to each slide and cover it with a plastic coverslip.

[0141] b. Incubate in a humid room at 37°C for 1 hour.

[0142] c. Clean twice in 2X SSC, 5 minutes each time.

[0143] 4. Pepsin treatment (optional; slides not used for this step should be stored in 2×SSC at room temperature).

[0144] a. Incubate the glass slide in 0.01M HCl for 2 min.

[0145] b. Shake off any excess solution, spread 200 μL of pepsin solution onto each slide, and cover with a plastic coverslip.

[0146] c. Incubate in a humid room at 37°C for 10 minutes.

[0147] d. Rinse in distilled water for 1 minute.

[0148] e. Clean in 2×SSC for 5 minutes.

[0149] 5. After fixation with paraformaldehyde.

[0150] a. In a fume hood, incubate the slide in a 4% paraformaldehyde solution for 10 minutes.

[0151] b. Clean in 2×SSC for 5 minutes.

[0152] 6. Dehydrate the glass slides using a series of ethanol solutions and then air-dry them.

[0153] 7. Prepare hybridization mixtures.

[0154] a. Determine the probe and dosage. Generally, the final concentration of the probe in the hybridization mixture should be 1–3 ng / μL (see Table 1). Each slide can be detected with two different probes (e.g., 5S and 35 / 45S rDNA), but each probe needs to be labeled with a different hapten (e.g., biotin and digoxigenin), so different antibodies linked to different fluorescent dyes can be used for detection.

[0155] b. Calculate and create the master mix for all slides, plus one slide, according to Table 1. Mix thoroughly and keep on ice.

[0156] c. Following the instructions in Table 1, prepare the hybridization mixture for each slide in a separate test tube by adding the master mixture, probe, and water. Mix gently but thoroughly.

[0157] d. Denature the hybridization mixture at 75°C for 10 min and stabilize it on ice for 10 min.

[0158] 8. Hybridization.

[0159] a. Apply the hybridization mixture to each slide and cover it with a plastic coverslip.

[0160] b. Degenerate chromosomes and hybridization mixture in a hybridization chamber. This step is crucial, and even when using the same species or variety, the time and temperature need to be adjusted. This typically depends on the method of chromosome preparation, the plant growth pattern, the age of the fixative used for preparation, and the storage time of the slides before FISH. As a guideline, use 72–75°C for 5–8 minutes.

[0161] c. Mix the slides overnight (approximately 16 hours) in a mixing oven or humid chamber at 37°C.

[0162] Table 1: FISH hybridization mixtures

[0163]

[0164] 1) Components of the main mixture. Add an extra slide to match the number of slides.

[0165] FISH Day 2: Detection of hybridization sites and slide mounting

[0166] The original FISH method used a 20% or 50% formamide washing step (Schwarzacher & Heslop Harrison, 2000), but to avoid using this toxic chemical, we now typically use rigorous washing under low-salt conditions, as this also reduces the background from formamide formation. Throughout the protocol, ensure the slides do not dry. If possible, wash in a shaking water bath; otherwise, gently shake by hand every 30–60 seconds. We describe the use of two probes labeled with digoxigenin and biotin, requiring two different colors for detection. We recommend using FITC for digoxigenin detection and Alexa594 for biotin detection, but other fluorescent dyes can also be used (Schwarzacher & Heslop Harrison, 2000). To visualize the chromosomes, stain the slides with DAPI (4',6-diamino-2-phenylindole) and place them in an anti-fading solution.

[0167] 1. Wash after hybridization.

[0168] a. Prepare a washing solution after hybridization and heat it in a water bath at 45°C.

[0169] b. Collect the slides from the hybridization oven, carefully inspect them for air bubbles, excess water or dried patches, and note any irregularities.

[0170] c. Place the hybridization slide in a 2×SSC at 35–40°C and let it float on the coverslip.

[0171] d. Clean the glass slide in 2×SSC at 42℃ for 2 min.

[0172] e. Clean twice in 0.1×SSC at 40-45℃ for 5 minutes each time; record the temperature.

[0173] f. Wash in 2×SSC for 5 minutes. Cool to room temperature.

[0174] 2. Testing.

[0175] a. Transfer the glass slide to the detection buffer.

[0176] b. Shake off any excess solution, spread 200 μL of blocking solution on each slide, and cover with a plastic coverslip. Incubate at room temperature or 37°C for 10 min.

[0177] c. Remove the coverslip, empty the slides, and apply 40–50 μL of appropriate detection solution to each slide.

[0178] d. Replace the coverslip and incubate at 37°C for 1 hour.

[0179] e. Wash the glass slide three times in the detection buffer solution at 40-42℃, 5 minutes each time.

[0180] 3. Empty the slide and add one drop (20-30 μL) of DAPI anti-fading solution.

[0181] 4. Place a large cover on each slide, cover it with thin paper, and press it gently but firmly.

[0182] 5. Place the slide in the dark at 4°C and observe.

[0183] 6. Microscopy and image analysis

[0184] For visualization of probe hybridization and chromosome staining, an epifluorescence microscope equipped with appropriate filters for the fluorescent dyes used in the detection steps is required. Table 2 provides a list of filter selections. In addition to a 20X or 40X lens for scanning slides, you will need top-of-the-line lenses in the 63X or 100X range for image capture; these lenses must be specified for UV fluorescence. Furthermore, ensure that an oil immersion solution specifically designed for fluorescence analysis is used. The microscope should be placed on a stable surface in a completely dark room, with a comfortable, adjustable chair, ideally a small lamp with a dimmer switch, allowing for several hours of operation.

[0185] When using DAPI and two FISH probes, images can be conveniently displayed in RGB mode, with each captured image in a separate channel. All microscope and filter manufacturers provide helpful descriptions, graphs, and frequently active visualizations of filter / fluorescent dye combinations.

[0186] Table 2 Commonly used fluorescent dyes and microscope filter sets for FISH analysis

[0187]

[0188] 1. Before you begin, ensure that your system is set up correctly, the illumination is centered, and no stray light enters the lens. Digital camera systems are now commonly used to capture images and do allow the capture of faint signals invisible to the naked eye, but no matter how expensive they are, they cannot compensate for deficiencies in microscope settings.

[0189] 2. Familiarize yourself with the camera control program, lighting, and various microscope buttons and levers so that you can operate the system quickly in the dark, as fluorescent dyes and FISH signals can be very weak and fade quickly even when viewed with a good anti-fading patch.

[0190] 3. Scan the slide under DAPI using a low magnification. This will not weaken your FISH signal, but turn off the light shutter when not viewing, primarily to avoid UV damage to the microscope lenses and filters.

[0191] 4. Once you have located the appropriate cell nucleus or metaphase chromosome, change the FISH signal filter to green or red fluorescence, and then switch to a 100X lens. This avoids UV signal attenuation but may make refocusing difficult. Only view DAPI when absolutely necessary, and then minimize the time spent.

[0192] 5. Capture images by exposing sequentially using different filter sets. Start with the FISH signal; the focus of red and green fluorescence is interchangeable, so focus on the stronger image. Generally, shoot the red or weaker image first, but it's best to do it in reverse order. Microscope filter sets are highly specific, but signal leakage is inevitable due to the broad excitation and emission spectra of fluorescent dyes, especially when one probe is very strong. Penetration is particularly strong after excitation at the correct wavelength, so shooting colors in reverse order helps identify the problem. Finally, shoot the DAPI image to ensure refocusing.

[0193] 6. Save the original image before performing image adjustments.

[0194] 7. To overlay a single image and perform image analysis, use the built-in camera or microscope software, or export as a "tif" file for use with Adobe Photoshop or NIH Image. Use only those features applied to all pixels of the image. Do not save in a lossy format (such as JPG).

[0195] Figure 6 Some examples of coffee chromosomes resulting from hybridization with 5S and 45S rDNA are given.

[0196] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0197] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing chromosomes from root meristems of tetraploid small-bean coffee, characterized in that, Includes the following steps: (1) Plant cultivation Cultivate the plants into seedlings that are 20-40cm tall for later use; (2) Root tip sampling and fixation (21) Collect the white new root tip tissue at the end of the root 4 to 4.5 hours after sunrise each day or 4 to 4.5 hours after the lights in the greenhouse start to shine; (22) After rinsing with sterile distilled water, take 1-1.5 cm of root tip tissue; (23) Treat the root tip tissue with a saturated 0.05% α-bromonaphthalene aqueous solution, place it at 22-25℃ for 2h, and then place it at 4℃ for 4h; (24) Treat with root tip fixative, place at 22-25℃ for 2 hours, then place at 4℃ for more than 2 days; (3) Chromosome separation and diffusion (31) Pretreatment of root tip meristem (311) Clean the fixed root tip tissue and then soften it with HCl; (312) After washing with enzyme buffer, incubate with enzyme solution at 37°C for 35 min; (313) After washing with enzyme buffer, add sterile distilled water to keep the area moist; (32) Anatomy of root apical meristem (321) The pretreated root tip tissue was treated with 60% acetic acid as a medium for 3 min; (322) Use a sterile needle to separate and remove the portion below the root cap that is more than 0.1 cm below the root cap; (323) Squeeze and separate individual cells to obtain a single layer of chromosomes.

2. The method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in claim 1, characterized in that, The specific conditions for cultivation described in step (1) are as follows: The optimal temperature for plant growth is 25–28℃; The culture medium consists of nutrient soil and fine sand, with a volume ratio of 8:

2. Maintain a light cycle of 14 hours during the day and 10 hours at night, while the plant needs appropriate shading; The air humidity is 80-90%.

3. The method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in claim 1, characterized in that, The volume of the saturated 0.05% α-bromonaphthalene aqueous solution in step (23) is 3 times that of the plant material.

4. The method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in claim 1, characterized in that, The fixative comprises ethanol and glacial acetic acid, with a volume ratio of 3:

1. The ethanol is a 96% or 100% (v / v) aqueous solution of ethanol.

5. The method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in claim 1, characterized in that, In step (311), the softening treatment using HCl involves using 0.01M HCl to soften the root tip for 15 minutes.

6. The method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in claim 1, characterized in that, The enzyme solution includes cellulase and pectinase.

7. The method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in claim 1, characterized in that, The specific operation of step (323) is as follows: Dissect the root tip meristem in acetic acid, and use a needle to gently tap or squeeze to separate individual cells, spreading the mixed cells evenly. Place a coverslip on the slide, cover one corner with a tissue or filter paper, and hold it down with one finger to prevent it from slipping. Then, gently press the coverslip vertically with another finger and tap it vertically with the eraser on the back of a pencil until the air bubbles between the coverslip and the slide disappear. Place the glass slide with the coverslip on the folded filter paper and press the coverslip area with your thumb or palm, slowly and carefully increasing the pressure.

8. A method for preparing chromosomes from root meristems of tetraploid small-bean coffee as described in any one of claims 1 to 7, characterized in that, The tetraploid small-bean coffee variety is Coffeaarabica Catimor 7963.

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