Plants containing engineered chromosome breakage constructs will be used as a study system to investigate the nature of inaccurate repair of double strand DNA breaks (DSB), and the genetic or environmental factors that affect the nature of inaccurate repair. DSB repair, in all organisms from bacteria to plants and animals, is highly accurate. This accuracy is necessary to allow chromosomes to be replicated and passed on to subsequent generations of progeny because these breaks are so frequent and are lethal if unrepaired. However, inaccurate repair of DSB does occur, leading to small deletions, insertions or other outcomes at the break site. Some previous studies have suggested that the relative sizes and frequencies of the inaccurate DSB repair events may vary from species to species, and that this variance might be responsible for differences in the rate at which genomes change size and for differences in their rate of sequence change. This project will use a regulated DSB system, involving the enzyme I-SceI expressed off a transgenic construct in plants that are then crossed to other plants that have a cutting site for I-SceI. The progeny of this cross have been shown, in preliminary experiments, to undergo DSB at the I-SceI site, and to sometimes have inaccurate repair at that site. The I-SceI site in many of these progeny plants will be DNA sequenced by the investigators to see what kind of deletions, insertions, et cetera, are found at the cleavage site. This work is being done in four grass species (maize, pearl millet, rice, and sorghum), with very different genomic sizes and genomic stability, to see if this influences the nature or frequency of inaccurate DSB repair. The I-SceI sites are also integrated at various different chromosomal locations in different plants, so one can investigate chromosome position effects on repair outcomes. Finally, the investigators will determine the nature of inaccurate DSB repair at different times in development, under different environmental stresses (e.g., high UV light intensity or high ozone levels) and in genetic backgrounds that have known mutations in DNA repair processes or chromatin structure. Taken together, these studies will provide insights into how plant genomes evolve, and particularly how DSB repair participates in this process, to generate the natural diversity on which natural selection acts.
This research will provide a powerful approach, and numerous novel insights, for understanding how genomes change. This will lead to a better understanding of how genetic diversity can be interpreted and influenced by biologists. The many direct participants in the project, especially the postdoctoral fellow, graduate student and undergraduate students, will receive direct training in a multi-disciplinary approach to a core question in modern genetics: how do genomes change, and what effect does that change have on current and future function of genes in that genome. The description of the project and of the results will be available at a web site that will be created for this project, with a presentation style that makes the information digestible for all levels of interested parties, from K-12 students, to the general public, to active research scientists. Raw data and the most advanced results will also be provided in public-accessible databases (e.g., GenBank for sequence information and peer-reviewed publications for detailed analysis), including the project web site. Biological materials (seeds of transgenic plants, constructs for the I-SceI approach, etc.) will be available from the PI's lab until the time that some more permanent maintenance resource can be identified. Understanding DNA variation, particularly the mechanisms and rates/outcomes, is vital for any mature understanding of genetics or genomes, and is shared by animal, human, plant, fungal, viral and microbial researchers, so it is expected that the training, outreach and data enrichment components of this project will benefit the broadest scientific community and general public.