Improvement of a high throughput method for identification of T-DNA insertion events in an activation-tagged population of Populus tremula x Populus alba.
poplar sequencing populus activation-tag
Mutant populations are a cornerstone of modern studies in genetics; their usefulness in gene discovery, and in understanding the various forms of gene regulation, can not be overstated. Since the introduction of mutation in plants nearly a century ago (Stadler, 1928), mutant populations have been generated in every major crop as well as many additional species (Sikora et al., 2011). While mutant populations have become commonplace in the study of plant biology, the usefulness of each population is limited by the ability of researchers to characterise the mutations present. This limitation has led me to search for an improved method to characterise the random T-DNA insertions sites of the activation tagged Populus tremula x Populus alba population generated by the Regan lab. In this thesis, I explore the potential of using a capture-based sequencing method to construct a high-throughput sequencing method that can be used to identify the genomic flanking regions that border T-DNA insertions. This theoretical process is then used to evaluate experimental data generated in 2 sequencing runs, examining the output for quality, consistency, and accuracy. Finally, I use my method to positively confirm T-DNA insertion sites in 4 selected lines of activation tagged P. tremula x P. alba, demonstrating the viability of my method to identify random T-DNA insert locations.