Abstract
A DNA fiber-based fluorescence in situ hybridization (fiber-FISH) technique was developed to analyze the structure and organization of a large number of intact chloroplast DNA (cpDNA) molecules from Arabidopsis, tobacco, and pea. Using this cytogenomic approach, we determined that 25 to 45% of the cpDNA within developing leaf tissue consists of circular molecules. Both linear and circular DNA fibers with one to four copies of the chloroplast genome were present, with monomers being the predominant structure. Arabidopsis and tobacco chloroplasts contained previously unidentified multimers (>900 kb) consisting of six to 10 genome equivalents. We further discovered rearranged cpDNA molecules of incomplete genome equivalents, confirmed by both differential hybridizations and size estimations. The unique cpDNA organization and novel structures revealed in this study demonstrate that higher plant cpDNA is more structurally plastic than previous sequence and electrophoretic analyses have suggested. Additionally, we demonstrate how the fiber-FISH-based cytogenomic approach allows for powerful analysis of very rare events that cannot be detected by traditional techniques such as DNA gel blot hybridization or polymerase chain reaction.