2010 Annual Report 1a.Objectives (from AD-416) 1. Implement 454 sequencing technology to compare gene expression impacted by ecological factor affecting invasiveness. 2. To use this technology to also identify regulatory regions and potential targets for future manipulation of invasive perennial weeds. 1b.Approach (from AD-416) Ecological factors have an impact on invasiveness of perennial weeds of the Northern plains; for example, Canada thistle and leafy spurge. These ecological factors cause changes in gene expression that are regulated by cis-acting elements. Advances in 454 sequencing technology now make it uniquely suited for the rapid and efficient quantification of both. 1)gene expression, and. 2)the identification of the cis-acting elements that control gene expression. Quantification of gene expression will be accomplished by 454 sequencing of biological samples collected from various ecological settings. This information will be analyzed and compared to obtain a dataset of genes linked to invasive traits. Further, 454 sequencing will be used to characterize genomic DNA to identify the specific cis-acting elements regulating the genes from our dataset. 3.Progress Report A Canada thistle EST-database produced from a 454 sequencing project conducted in collaboration with the W.M. Keck Center, University of Illinois-Urbana, generated nearly 2.5 million reads. After assembly, 67,358 sequences were identified of which 42,549 had hits to known Arabidopsis homologues while 24,810 had no hits. Annotation of the Canada thistle sequence data against the Arabidopsis database indicated the presence of at least 15,232 unique transcripts. Preliminary MIPs analysis indicated that transcripts belonging to functional categories involved in cell cycle and DNA processing, biogenesis of cellular components, and systemic interaction with environment were over-represented (p<0.05) and up-regulated (>3-fold) in paradormant root sections, while amino acid metabolism, energy, protein fate and binding, cellular transport and communication, cell rescue/defense, and subcellular localization were over-represented among up-regulated transcripts during paradormancy release. Sub-network analysis further help to identify physiological processes, expression targets and binding partners for genes, and pathways involved in paradormancy release. Data from this project were presented at the 2010 WSSA Annual Meeting in Denver, CO, and a manuscript related to the project is being drafted. The ADODR actively monitors the project, reviews accomplishments, and provides technical advice with cooperating personnel through e-mail and conference calls.