A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA]n microsatellite, the [TTTAGGG]n telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.

Laboratory of Molecular Cytogenetics and Cytometry Institute of Experimental Botany Olomouc Czech Republic

References provided by Crossref.org

Flow Cytometric Analysis and Sorting of Plant Chromosomes

Chromosome analysis and sorting

Characterization and Dynamics of Repeatomes in Closely Related Species of Hieracium (Asteraceae) and Their Synthetic and Apomictic Hybrids

Integration of Genetic and Cytogenetic Maps and Identification of Sex Chromosome in Garden Asparagus (Asparagus officinalis L.)

Putative interchromosomal rearrangements in the hexaploid wheat (Triticum aestivum L.) genotype 'Chinese Spring' revealed by gene locations on homoeologous chromosomes

Extensive pericentric rearrangements in the bread wheat (Triticum aestivum L.) genotype "Chinese Spring" revealed from chromosome shotgun sequence data

Mapping nonrecombining regions in barley using multicolor FISH

Chromosomes in the flow to simplify genome analysis

Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley

Chromosome-based genomics in the cereals

Dissection of the nuclear genome of barley by chromosome flow sorting

Chromosome sorting in tetraploid wheat and its potential for genome analysis