2011 Annual Report
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. For FY-11, both laboratory and field experiments were conducted. 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 (ARS-Albany and University of Nebraska-Kearney) and by in-person meetings for project personnel located in Lincoln, NE.
Plant materials labeled with C13 CO2 in spring FY10 were collected throughout the growing season, separated into respective plant parts, oven dried and ground. Several samples were analyzed by mass spectrometry to gauge the level of incorporation of C13 into plant parts. Initial analysis showed that plants were labeled adequately and there was an expected dilution of label during the period of active growth. Crown + rhizomes were harvested from 4 populations at selected times over the growing season, cleaned and flash frozen in liquid nitrogen. Roots were also collected from each sample at each harvest date. All frozen plant materials were stored at -80C for future RNA analyses by high-throughput sequencing. High-throughput sequencing of previously collected crown materials from cv Summer and cv Kanlow was performed using two platforms, 454 FLX and Illumina sequencing by synthesis strategies. The ~ 1 million sequences obtained from cv Summer crowns + rhizomes using the 454 platform was used to assemble a preliminary crown + rhizome transcriptome. Analysis of these sequences is largely complete, and a manuscript is being prepared for submission to a peer-reviewed journal in FY11.
Another ~200 million short read sequences were obtained through Illumina of triplicate samples of crowns + rhizomes obtained from cv Kanlow and cv Summer for a single harvest date. These sequences were combined with those obtained from 454 sequencing to generate a hybrid “master” transcriptome assembly using open source assembly programs. Data from individual Illumina runs were then compared to the “master” assembly to detect transcripts present in all three replicates and differentiate between transcripts up/down regulated in cv Summer versus cv Kanlow. These analyses are ongoing.
In other experiments, 30 crowns+rhizome samples were analyzed for 122 metabolites through a commercial vendor. These samples were derived from cv Summer and cv Kanlow crowns + rhizomes encompassing 3 biological replicates from each population at 5 different harvest times. Initial analysis of this dataset showed potential differences in select compounds over the growing cycle as well as in response to cold. These data will serve as a background for more in-depth quantitation of key metabolites that will performed in FY11-FY12.