Flow-Sorted chromosomes: a fine material for plant gene physical mapping

Abstract

Fluorescence in situ hybridization (FISH) on flow-sorted plant chromosomes is described. The protocol involves flow cytometric sorting of metaphase chromosomes, then fixing them with 4% paraformaldehyde solution, and re-sorting these chromosomes directly onto a spot on poly-lysine coated slides after stained. FISH mapping experiments were performed on the sorted chromosomes from oat and barley using 45S and 5S ribosomal DNAs as probes. The chromosome morphology was very well preserved and there were nearly no chromosomes lost after procedures of hybridization and fluorescent staining. Sorted chromosomes are advantageous over metaphase chromosomes as targets for FISH mapping studies because a large number of target chromosomes with better chromosome morphology on a small area on the slide are easy to gain by flow-sorting, background is very clear and hybridization sensitivity is enhanced. The successful and reliable location of rDNA genes in these two plant genomes, combining with the data published before in maize proves that flow-sorted chromosomes are fine materials for gene physical mapping in plants.

Key words:

• flow-sorted chromosomes
• fluorescence in situ hybridization
• gene mapping
• rDNA