FISH PHYSICAL MAPPING WITH BARLEY BAC CLONES

Fluorescence in situ hybridization (FISH) is a useful technique for ph ysical mapping of genes, markers, and other single- or low-copy sequen ces. Since clones containing less than 10 kb of single-copy DNA do not reliably produce detectable signals with current FISH techniques in p lants, a bacterial artificial chromosome (BAG) partial library of barl ey was constructed and a FISH protocol for detecting unique sequences in barley BAC clones was developed. The library has a 95 kb average ba rley insert, representing about 20% of a barley genome. Two BAC clones containing hordein gene sequences were identified and partially chara cterized. FISH using these two BAC clones as probes showed specific hy bridization signals near the end of the short arm of one pair of chrom osomes. Restriction digests of these two BAC clones were compared with restriction patterns of genomic DNA; all fragments contained in the B AC clones corresponded to bands present in the genomic DNA, and the tw o BAC clones were not identical. The barley inserts contained in these two BAC clones were faithful copies of the genomic DNA. FISH with fou r BAC clones with inserts varying from 20 to 150 kb, showed distinct s ignals on paired chromatids. Physical mapping of single- or low-copy s equences in BAC clones by FISH will help to correlate the genetic and physical maps. FISH with BAC clones also provide an additional approac h for saturating regions of interest with markers and for constructing contigs spanning those regions.