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.
At the USDA-ARS, BRL in Fargo, ND, RNA samples were isolated from roots sections of greenhouse propagated Canada thistle (monoculture NW22-1) collected at 0, 1, 2, and 3 days of incubation at 25oC under 16:8 h light:dark conditions. The RNA samples were sent to the W.M. Keck Center, University of Illinois-Urbana for the successful construction of four MID-tagged cDNA libraries. 454 next generation sequencing of the four libraries generated nearly 1 giga-base of DNA from approximately 2.5 million ESTs with an average read length of 362 bases. Bioinformatics analysis is currently underway at the W.M. Keck Center to assemble and annotate the sequence information into a unigene set that can be used for a virtual in silico analysis, and for future construction of Canada thistle oligoarrays. The ADODR actively monitors the project, reviews accomplishments, and provides technical advice with cooperating personnel through e-mail and conference calls.