SORTED CHROMOSOMES: A FINE MATERIAL FOR PLANT GENE PHYSICAL MAPPING

Center for Biotechnology University of Nebraska - Lincoln Lincoln, NE 68588-0665 U.S.A

A major interest of our laboratory is the gene localization on plant chromosomes collected by flow cytometric sorting. We can sort chromosomes from varius plants including barley, maize, oat and wheat. Sorted chromosomes have many important applications for plant genome research, one of which is for gene mapping in plants using fluorescent in situ hybridization (FISH). Fixation of sorted chromosomes is a prerequisite for application in situ hybridization or storage for later use. However, chromosomes isolated from fixed plant materials (especially cereals) are not suitable for chromosome sorting (Lee et al., 1996). In this study, we developed and tested a method for FISH on sorted chromosomes. Chromosomes are first sorted into a fixative (4% paraformaldehyde solution), then stained and re-sorted directly onto a spot on poly-lysine coated slides. Fluorescent in situ hybridization experiments were performed on the sorted chromosomes using various DNA probes. The chromosome morphology was very well preserved with minimal chromosome loss after procedures of hybridization and fluorescent staining. We can have large number of target chromosomes on a small area on the slide compared to metaphase spreads. The ability to detect clear hybridization signals on sorted chromosomes allows us to consider sorted chromosomes as a fine material for physical mapping of genes in plants.