HIGH-RESOLUTION PHYSICAL MAPPING IN ARABIDOPSIS-THALIANA AND TOMATO BY FLUORESCENCE IN-SITU HYBRIDIZATION TO EXTENDED DNA FIBERS

A technique to detect DNA sequences on extended DNA fibres (EDF) prepa red from interphase nuclei from tomato (Lycopersicon esculentum) and A rabidopsis thaliana leaves by fluorescence in site hybridization (FISH ) is described. Three nuclear lysis procedures have been tested for th eir ability to decondense chromatin and to generate highly extended in tact DNA fibres on microscopic slides. DNA probes of various sizes hav e been used in FISH experiments to EDFs to establish the resolution an d sensitivity of the technique. The fluorescent signals of a 5S rDNA p robe hybridized to tomato EDFs revealed continuous strings of about 20 0 mu m, that corresponded to a molecular size of about 660 kb. In A. t haliana, a contig of three cosmids spanning a genomic region with a to tal length of about 89 kb was analysed. By means of multicolour hybrid ization the physical positions of the cosmids were visualized as red a nd green fluorescence strings with overlapping regions in yellow. Comp arison of the length of the fluorescent signals with the molecular dat a revealed a stretching degree of the DNA fibres at 3.27 kb mu m(-1), which is close to the Watson-Crick DNA length estimate of 2.9 kb mu m( -1). Other experiments on small size molecular probes with both lambda clones (13.5-17 kb insert sizes) and plasmids (4.2 and 5 kb) in a con tig of A. thaliana, and the 5S rDNA region in tomato showed close agre ement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from th e 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. C onsistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance betw een these hybridization spots the size of the ribosomal unit was estim ated at 8.2 kb. This implies a drastic improvement of high-resolution physical mapping of DNA sequences by FISH on plant DNA.