Providing the link between physical and cytogenetic map of barley - a case study on chromosome 2H

The physical map of barley chromosome 2H in its current state comprises 867 genetically anchored BAC contigs derived by high information content fingerprinting (HICF) and FPC assembly. The relative order of the BAC contigs anchored to the same genetic position remains to be resolved by additional measures. We were interested to explore the potential of cytogenetic anchoring especially for the pericentromeric regions of the barley chromosomes which are characterized by long physical distances corresponding to short genetic intervals. Therefore, an efficient approach of developing single-copy FISH probes from genomic sequence information had to be established. The sequence information linked to 9 different BAC contigs (FP contigs) of the physical map genetically anchored to the distal region of chromosome 2HL was extracted. Two levels of repeat masking including conventional masking with annotated repeat sequences and a mathematically defined repeat (MDR) analysis was performed. Corresponding PCR products (range 1.3 - 3 kb) of the putative single-copy sequences of the nine FP contigs were combined to produce 9 probe pools (range 4.2 - 8.7 kb). All but one probe pools produced clear and specific signals on mitotic metaphase chromosomes 2H of barley pinpointing their cytogenetic locations. Six of the FP contigs co-localized to the same chromosomal position, thus their genetic/physical order could not be resolved at the cytogenetic level. The long distance resolution of the more proximally anchored contigs, however, is a promising result indicating that physical ordering of genetically non-resolved contigs will be feasible and can be efficiently supported by FISH mapping.