CYTOGENOMIC ANALYSIS OF ORGANELLAR DNA IN HIGHER PLANTS

¹ University of Wisconsin Dept. of Horticulture 1575 Linden Drive Madison, WI 53706

² University of Minnesota Dept. of Agronomy and Plant Genetics 1991 Upper Buford Circle St. Paul, MN 55108

³ USDA-ARS Vegetable Crops Research Unit 1575 Linden Drive Madison, WI 53706

Chloroplast and mitochondrial genomes of higher plants are commonly conceptualized as circular or linear molecules. A modified DNA fiber-based fluorescent in-situ hybridization(FISH) technique was used to study the structure and organization of intact organellar (chloroplast and mitochondrial) genomes. Previous experiments with BAC clones demonstrated large (300 kb) circular DNA molecules can be visualized as intact molecules on glass slides. Cytogenomic analyses revealed that the tobacco chloroplast genome (155 kb) exists as an assortment of complex oligomeric molecules. Many of these fibers contained multiple genome units, arranged as a head to tail concatamers. Circular, plasmid like DNA, molecules were observed, but existed at levels less than five percent. Differentially labeled heterologous chloroplast clones from petunia could be resolved at a minimum size of 4 kb on a single chloroplast fiber from tobacco. Our preliminary results suggest that mitochondrial DNA has a similar structure. These techniques also revealed significant transfer of organellar DNA to the nuclear genome of tobacco. Nuclear DNA fibers exhibited a large (greater than 100 kb) region of chloroplast homology. Our research demonstrates that cytogenomics is a useful technique to analyze plant organellar genome structure, organization, and evolution.