A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants

Abstract

The major drawbacks of standard plant fluorescence in situ hybridization (FISH) designed for double-stranded DNA probes include requirement for experimentally determined heat denaturation of chromosomes at high temperatures and at least overnight hybridization. Consequently, processing with chromosomal preparations may easily result in heat-induced deterioration of chromosomal structural details, is time-consuming, and involves the use of toxic formamide and formaldehyde. Here, I have described a simple and appealing non-toxic procedure with ethylene carbonate (EC)-a formamide-substituting solvent and double-stranded repetitive DNA probes. Applying EC as a component of the hybridization solution at 46 °C not only allowed successful overnight hybridization but also gave a possibility to reduce the hybridization time to 3 h, hence converting the technique into a 1-day procedure. Importantly, the EC-FISH tended to preserve well chromosome structural details, e.g., DAPI-positive bands, thus facilitating simultaneous FISH mapping and chromosome banding on the same slide. The procedure requires no formaldehyde and RNA-se treatment of chromosomes, and no heat denaturation of chromosomal DNA. The key condition is to obtain high-quality cytoplasm-free preparations. The method was reproducible in all the plants studied (Allium, Nigella, Tradescantia, Vicia), giving a species-specific signal pattern together with clear DAPI bands on chromosomes. The procedure described here is expected to give a positive stimulus for improving gene-mapping approaches in plants.

Keywords: Chromosome DAPI banding; Ethylene carbonate; Fluorescence in situ hybridization; Heterochromatin; Plants; rDNA.

Fig. 1

a–e Simultaneous DAPI-banding (gray scale images) and EC-FISH (color images) of the two rDNA probes—5S rDNA (red signals and solid arrowheads) and 26S rDNA (green signals and solid arrows) to the somatic metaphase chromosomes of T. spathacea (a), A. cepa (b), N. damascena (c), and V. faba (d–e). Open arrowheads point to heterochromatic DAPI bands of M chromosome in V. faba. Chromosome pairs identified and numbered; f a fragment of the cytoplasm-free preparation of N. damacsena viewed under a phase contrast microscope. Bars = 10 μm