Development and application of fluorescence-based in situ hybridization techniques for plant genome analysis

Physical analysis of plant genomes has been a characteristically difficult pursuit due to the highly complex nature of the genomic DNA. In many plant genomes repetitive DNA sequences are the primary constituent. The complexities of the DNA structure, particularly repetitive elements, make cloning, sequencing and comparative analyses difficult, if not impossible. Fluorescent in situ hybridization (FISH) techniques hold the promise of easing many of these analyses. I describe here the development and application of FISH techniques on stretched DNA fibers that makes the physical analysis of DNA sequences tractable in many plant genomes. Using fiber-FISH, the size of two gaps on the chromosome II bacterial artificial chromosome (BAC) contig of Arabidopsis thaliana were measured and the size of several repetitive elements in A. thaliana and Oryza sativa were determined. A variant of the fiber-FISH technique, digital mapping, was developed for physical mapping on circular BAC clones. The physical structure of several repetitive elements on a centromeric BAC from O. sativa was determined using digital mapping. Because of these repetitive elements it was impossible to restriction map or assemble sequence data from this BAC, therefore digital mapping was very useful to determine the exact arrangement of the repetitive elements. These techniques have important applications in sequencing and subsequent assembly of genomic clones, subcloning of DNA elements from large insert DNA libraries, physical analysis of large segments of genomic DNA and gauging the size of gaps in physical contigs.