PhD Candidate University of Georgia Athens, Georgia
Body of Abstract: Mutator (Mu) is one of the most active transposable elements and generates frequent new insertions in Mu-active lines of Maize. We established an improved method to sequence Mu insertion sites, Museq2, which has been designed and optimized for the detection and quantification of rare de novo insertions in heterogeneous tissue samples. Museq2 has a detection threshold of 1 Mu insertion in 100,000 wild-type copies of DNA. Museq2 can measure the relative abundance of Mu insertion sites with a quantitative range of 5 orders of magnitude. We applied Museq2 to first leaf, mature pollen, and endosperm-derived tissue from multiple individual Mu-active seeds, detecting an average of 80,000 insertions per sample. Insertions present in both endosperm and embryo-derived tissue must have occurred pre-zygotically, making it possible to unambiguously distinguish inherited and de novo insertions. The de novo insertions most frequently occurred in Mu hotspots of ~1 kb in size, with thousands of specific hotspots identifiable across the Maize genome. Surprisingly, the vast majority of hotspots were shared between leaf and pollen, suggesting that the biological determinants of Mu insertion preference are consistent between these two tissue types. Overall, our innovations in Museq2 allow for previously unattainable quantification and characterization of de novo Mu insertions from multiple tissue types from a single Maize plant. The quantitative range displayed here for Mu insertions is unprecedented for any class of mutation and can be used to learn more about Mu-specific biology as well as somatic mutations as a general phenomenon.