1a.Objectives (from AD-416)
The objectives of this proposal are (1) To utilize novel genomic and biochemical tools to investigate molecular mechanisms underpinning nutrient partitioning and remobilization in crowns and rhizomes of switchgrass cultivars with divergent winter-hardiness. (2) To use high-throughput DNA sequencing to query transcript abundance (levels of gene expression) in specific populations of switchgrass plants during regreening and dormancy. (3) To study the genetic variation (extent of linkage disequilibrium in populations) and eventually develop genetic markers for cold-adaptation and fitness traits in switchgrass plants being developed for Central and Northern USA that show significant hybrid vigor (heterosis).
1b.Approach (from AD-416)
Five different strains of switchgrass plants that differ in their cold-hardiness and fitness parameters will be planted in the fields. Crowns and rhizomes will be harvested from these plants at specific times over two growing seasons for genetic (High-throughput pyrosequencing; 454 transcriptomics) and metabolite analyses. Additional plants will be subjected to isotope-tracer experiments using stable isotopes for carbon (C-13) and nitrogen (N-15) to query nutrient recycling over two growing seasons as affected by the genetic background of the plants. These data will permit improved insights in the molecular and physiological events that impact perenniality and fitness in switchgrass. Obj. 2. Using genomic approaches (454 pyrosequencing and bioinformatics), we will discover additional genes impacting fitness using individual plants from switchgrass populations divergently selected over ~30 years for ruminant digestibility. These plants are a unique genetic resource unavailable elsewhere, and show improved conversion to ethanol and decreased winter survival. Obj. 3. We will attempt to uncover marker-trait associations that can be used to reduce the generations, and within generation time and expense of phenotyping in the breeding process by use of marker assisted selection (based on genes uncovered in Obj.1 and Obj. 2). Over 2000 plants from various genetic backgrounds have been planted in the field for these analyses.
This work is being performed through a Department of Energy competitive grant awarded in May 2009 with an official start date of January 1, 2010 through December 31, 2012. As part of this grant, a first meeting of all project personnel was held in Lincoln, NE on July 28, 2009 to establish protocols and develop a plan of attack. A sharepoint site (Switchgrass Fitness) was established and non-ARS personnel who are direct participants of this project were given access to this site. All further communications were by telephone and email between the ADODR and appropriate project personnel at distant locations (ARS-Albany and University of Nebraska-Kearney) and by in-person meetings for project personnel located in Lincoln, NE. Action plans based on these meetings were developed and implemented in 2010. Incoming funds were disbursed according to grant-stipulated guidelines.
All field plots were successfully established in 2009. Two plexiglass chambers were constructed in May 2010 and used for labeling of plants with 13CO2 in June 2010. Plant materials will be harvested throughout the 2010 growing season. Crown materials were harvested from different plants and flash frozen in liquid nitrogen and stored at -80 C for future analyses by high-throughput sequencing. High-throughput sequencing of previously collected crown materials was performed to refine extraction protocols and data analyses parameters. Data from the first series of runs is being processed.
ADODR monitoring includes phone calls, e-mails, and personal contacts/discussions during on-site visits.