INTERPHASE FLUORESCENCE IN-SITU HYBRIDIZATION MAPPING - A PHYSICAL MAPPING STRATEGY FOR PLANT-SPECIES WITH LARGE COMPLEX GENOMES

The chromatin in interphase nuclei is much less condensed than are met aphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuc lei than on metaphase chromosomes. In mammalian species it has been de monstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance betwee n the two probes. The interphase mapping strategy has never been appli ed in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) c amera system, we demonstrate that DNA probes in the 4 to 8 kb range ca n be detected on both metaphase and interphase chromosomes in maize. D NA probes pA1-Lc and pSh2.5 . SstISa1I, which contain the maize loci a l and sh2, respectively, and are separated by 140 kb, completely overl apped on metaphase chromosomes. However, when the two probes were mapp ed in interphase nuclei, the FISH signals were well separated from eac h other in 86% of the FISH sites analyzed. The average interphase dist ance between the two probes was 0.50 mu m. This result suggests that t he resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of anothe r pair of probes, ksu3/4 and ksu16, which span the Rp1 complex control ling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped gen etically approximately 4 cM apart and their FISH signals were also ove rlapped on metaphase chromosomes. These two probes were separated by a n average of 2.32 mu m in interphase nuclei. The possibility of estima ting the linear DNA distance between ksu3/4 and ksu16 is discussed.