A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.
Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.
Fluorescence in situ hybridization (FISH) is an effective tool for the physical mapping of single and high copy sequences at the chromosomal level. Prerequisite is the preparation of high quality chromosome spreads. There is no general chromosome preparation protocol that would be equally suitable for animal and plant cells. Preparation of plant chromosomes is particularly challenging due to the rigid cell wall and various cytoplasm consistency within different species. One of the favorable methods for the preparation of plant chromosomes is a so-called drop technique also known as drop-spreading technique and air-drying technique 1,2. This method was first introduced in 1958 by Rothfels and Siminovitch for in vitro grown mammalian cells 3. Later Martin et al. 4 and Kato et al. 5 adapted this method for plants.
More recently, a method named 'SteamDrop' was developed which used water steam for the preparation of non-overlapping chromosomes 6. Although, the positive influence of high humidity was observed earlier 7, 'SteamDrop' delivers a controlled workflow of high-quality chromosome preparations 6. The steam treatment causes stretching of chromosomes probably connected to some modifications of chromosomal proteins. The quality of resulting metaphase spreads is very high, although retaining of sufficient number of complete metaphase spreads for subsequent FISH experiments demands technical expertise.
Here we present a protocol for the preparation of mitotic cereal chromosomes suitable for the FISH detection of single and high copy probes 5,8. This method is an improved variant of the air-dry dropping method described by Kato 9 performed under relative humidity of 50%-55% (Figure 1). This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Using this protocol we obtained high-quality chromosome spreads and FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.
1. Chromosome Preparation
2. Fluorescent In Situ Hybridization (FISH)
3. Microscopic Analysis and Storage
Microscopic slides with the mitotic metaphase spreads were prepared by the fast air-dry dropping chromosome preparation method described above (Suppl. Figure 1). FISH analysis was carried out using both, repetitive and single-copy sequences. Images were obtained by a epifluorescence microscope with a set of filters enabling excitation of corresponding fluorophores and captured by a high-sensitivity CCD monochrome camera. For the image acquisition we used a computer with an image acquisition software. Results of the FISH experiments on mitotic metaphase chromosomes using 5S rDNA, [CTT]10, and single-copy probes were distinct and of a high quality for Hordeum vulgare (Figre 2A, B), H. bulbosum (Figure 2C), H. marinum, H. murinum, H. pubiflorum and Secale cereale (Figure 2D). Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. It is possible to store the cell suspension at -20 °C up to two months and to prepare the chromosome spreads on the day of the FISH experiment. Freshly prepared slides can be also stored at -20 °C in 96% ethanol, though we observed that the quality of hybridization signals on such chromosomes is reduced compared to the freshly-prepared metaphase spreads. The methods can be used to prepare high-quality chromosome spreads in cereals in an easy, efficient and reproducible way and most likely can be used in other plant species too.
Figure 1. A scheme describing the procedure of the air-dry dropping plant chromosome preparation method.
Figure 2. FISH on mitotic metaphase chromosome spreads of Hordeum vulgare, H. bulbosum and Secale cereale prepared by the air-dry dropping method. (A) H. vulgare with a single copy probe (FPct_40752) labeled with a red fluorescent dye. (B) H. vulgare with 5S rDNA probe labeled by a green fluorescent dye. (C) H. bulbosum with CTT-microsatellite labeled by a green fluorescent dye and (D) S. cereale with pSc119.2 repeat labeled by a green fluorescent dye. All chromosomes were counterstained with DAPI (in red). FISH signals are shown in yellow. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Table 1. Incubation time of enzyme treatment for different species.
Suppl. Figure 1. Phase-contrast and differential interference contrast (DIC) images of mitotic metaphase chromosome spreads of the air-dry dropping plant chromosome preparation method on the example of Hordeum vulgare. (A) Phase-contrast image taken at 200X magnification and (B) Differential interference contrast image taken at 630X magnification. Please click here to view a larger version of this figure.
The chromosome preparation experiment has been carried out using young roots of cereals belonging to the grass family (Poaceae). All analyzed species have 14 relatively long mitotic metaphase chromosomes (11-15 µm) in the diploid genome set and belong to large-genome species (5.1-7.9 Gbp).
Length of germinated roots was not more than 2 cm to obtain a maximum of meristematic tissue. Synchronization of dividing cells was achieved by a 20 hr long ice-water treatment that improved the quantity of mitotic metaphase spreads 10.
Two steps are important for the preparation of high-quality chromosome preparations: (I) the relative humidity of 50%-55% and (II) duration of the enzyme treatment. The first point was achieved by placing wet paper tissues on a hot plate in proximity of the glass slides. The relative humidity was measured with a hygrometer. The optimal humidity for the preparation of plant chromosomes was similar to the humidity reported by Kirov et al. 6. The positive effect on the chromosome quality at optimal relative humidity occurs by swelling of the cytoplasm and cell wall hydrolysis.
The duration of enzyme treatment is species dependent (Table 1). The period of enzyme treatment also depends on the time span of root fixation in ethanol/acetic acid and the size of the roots. The longer roots were stored in the fixative (up to 1 year at 4 °C), the longer it takes to digest roots to the proper grade. Insufficiently digested root material is difficult to macerate and will increase the total time of preparation as a result of long lasting maceration. Moreover, metaphase chromosomes remain embedded into cytoplasm that could hamper ensuing probe penetration during the FISH experiment. On the other hand over-digested material can influence the structure of the chromosomes themselves, and damage target DNA for the FISH analysis.
An additional factor for the improvement of the preparation is the use of the second drop of fixative (3:1, acetic acid/ethanol). High concentration of acetic acid in this mixture stimulates the digestion of cytoplasm and promotes chromosome spreading in species with large chromosomes. Cytoplasm reduction can also take place after the immobilization of the chromosomes on slides. For this purpose microscope slides carrying the chromosome spreads can be incubated in 45% acetic acid at RT for 2-10 min depending on cytoplasm level. Quality check of chromosome spreads was performed with a phase-contrast microscope without any supplementary staining (e.g., 1% aceto-carmin). Normally more than 25 slides containing high-quality chromosome spreads can be obtained from 20 roots using the method above.
Results of the FISH experiments on mitotic metaphase chromosomes using 5S rDNA, [CTT]10, and 6 kb long single-copy probe (FPct_40752) were distinct and of a high quality for all species described above (Figure 2). Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. It is possible to store the cell suspension at -20 °C up to two months and to prepare the chromosome spreads on the day of the FISH experiment. Freshly prepared slides can be also stored at -20 °C in 96% ethanol, though we observed that the quality of hybridization signals on such chromosomes is reduced compared to the freshly-prepared metaphase spreads.
Chromosome spreads prepared by the fast air-dry dropping technique were suitable for FISH and were reproduced a number of times. Combination of this chromosome preparation method with FISH could be widely applied to explore the genome organization in plants, for instance, for karyotyping11, chromosomal mapping 12, in synthetic studies, and for the integration of physical and genetic maps13.
The authors have nothing to disclose.
We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.
Hot Plate | MEDAX GmbH | 12603 | |
Cellulase R10 | Duchefa | C8001 | |
Cellulase | CalBioChem | 219466 | |
Pectolyase | Sigma | P3026 | |
Cytohelicase | Sigma | C8274 | |
Texas Red-12-dUTP | Invitrogen | C3176 | direct fluorochrome |
Fluor488-5-dUTP | Invitrogen | C11397 | direct fluorochrome |
Fluorecsence microscope | Olympus BX61 | BX61 | |
CCD camera | Orca ER, Hamamatsu | C10600 | |
4’,6-diamidino-2-phenylindole (DAPI) | Vector Laboratories | H-1200 | fluorecsent dye |