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 DOE competitive grant awarded in August 2009 with an official termination date of August 31, 2013. This project will terminate in FY13.
One of the goals of the USDA-ARS Forage and Bioenergy project at Lincoln, Nebraska is to develop improved switchgrass plants for forage and bioenergy. The goal of this project performed through a DOE competitive grant is to understand genetic aspects of winter-survival as impacted by plant senescence and dormancy using a combination physiological and molecular tool. The ultimate aim is to develop switchgrass plants with improved winter survival and yields.
All materials required for the successful completion of all project goals were collected by November 2012. Plant materials destined for different end analyses have been extracted to obtain DNA, RNA or dried and ground for carbon and nitrogen analyses. Although significant headway has been made in many of these analyses, several samples are still in the analytical pipeline, partially due to instrument difficulties (ratio-isotope mass spectrometer) and due to long turn-around times for the next generation sequencing. Despite these challenges, several significant datasets have been obtained. These are (1) the first comparison of crown and rhizome transcriptomes for two contrasting populations of switchgrass collected at a single time point (2) complete transcriptome of the crowns and rhizomes from three switchgrass populations over the course of the growing season (3) a first analysis of gene-expression profiles in the flag leaves of cv Summer field grown plants from elongation through senescence (4) inventory of changes in 13C and 15N in shoots in several populations of switchgrass plants over the course of the growing season and (5) phenotyping and genotyping of a large number of unrelated switchgrass plants to develop trait associations.
All team members were cognizant of the planned experiments and had approved implementation. Communication was by email and telephone between the ADODR and appropriate project personnel at distant locations (University of Nebraska-Kearney) and by several in-person meetings for project personnel located in Lincoln, NE.