Multi-substrate chromosome preparations for high throughput FISH

Background: A modification of a standard method of fluorescence in situ hybridisation (FISH) is described, by which a combination of several substrates and probes on single microscope slides enables more accurate comparisons of the distribution and abundance of chromosomal sequences and improves the relatively low throughput of standard FISH methods. Results: The utility and application of multi-colour, multi-substrate FISH is illustrated by the simultaneous physical mapping of retrotransposon sequences to three species of Avena, and single locus BAC (bacterial artificial chromosome) clones and rDNA probes to three species of Brachypodium, demonstrating how this would enable better understanding of complex phylogenetic relationships among some of the species belonging to these two genera. Conclusion: The results show that use of multi-substrate chromosome preparations significantly increases the utility of FISH in comparative analyses of the distribution and abundance of chromosomal sequences in closely related plant species.