Analysis of maize mitochondrial genome structures in vivo [abstract]
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Mitochondria are organelles containing their own genomic DNA that are found in most eukaryotic cells. In contrast to those of animals, plant mitochondrial genomes are large and very complex in structure. While maize mitochondrial DNA can be mapped as a large single circle, it has not been observed as such. Thus, the in vivo organization of the mitochondrial genome is not known. Using the B37 maize inbred line, which has a 570 kilobase (kb) mitochondrial genome, observations were made regarding the possibility of sub-genomes resulting from recombination between repeats. Mitochondria were isolated from fresh B37 seedling shoots and were placed on slides to be observed using the FISH (Fluorescent in situ Hybridization) technique. Two probes were used, cosmid 6 in Alexafluor 488 (green) and cosmid 19 in Texas Red (red). These cosmids are separated in the mitochondrial genome by approximately 350kb. They also occur on separate sides of a small repeat that is known to recombine frequently. If the genome is intact the two colors should overlap and produce a yellow color. If there are sub-genomes, red and green should be seen individually. The mitochondria were observed on an Olympus Spectral Imaging Microscope and the genome appeared to be fragmented most of the time. Five slides were counted and 31,077 spots were observed. The red: green: yellow ratio was 6.44 : 2.96 :1.00. Stretching out of an intact mitochondrial genome could cause apparent sub-genomes. However, it is more likely that there are sub-genomes present in separate mitochondria because small mitochondria (about one micron) were selected during the isolation procedure. This is small enough that the microscope should have seen the signal as overlapping. Future studies could extend the analysis into other maize lines and to other types of plants to determine if the sub-genomic structures are universal.