Location: Grain, Forage, and Bioenergy Research2012 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.
3. Progress Report:
This work is the University of Nebraska’s component of research being performed through a Department of Energy competitive grant awarded in May 2009 with an official start date of January 1, 2010 through August 31, 2013. For FY-12, both laboratory and field experiments were conducted. University of Nebraska scientists were primarily responsible for labeling plants and their analyses and sequencing. Plant materials were labeled with 13CO2 and 15N (urea) in spring FY11 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 13C and 15N 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 2 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 was performed using the Illumina Hi-Seq 2000 instrument. The ~ 1.2 billion sequences obtained from cv Summer crowns + rhizomes using are being analyzed. In an experiment completed in late FY11, about ~200 million short read sequences were obtained through Illumina sequencing of triplicate samples of crowns + rhizomes obtained from cv Kanlow and cv Summer for a single harvest date in late September. These reads were mapped to the switchgrass draft genome released by the JGI in January 2012. These analyses have been completed and results indicate substantial and significant differences in the metabolism of the crowns and rhizomes obtained from cv Summer plants as compared to tissues obtained from cv Kanlow plants. For Objective 2, Crowns and rhizomes were collected from several plants from an octaploid nursery containing plants divergently selected for forage digestibility and exhibiting differences in winter hardiness. Crowns and rhizomes will be collected from 44 plants post-frost fro transcriptomic analyses using the Illumina Hi-Seq system. These studies will augment ongoing genotype-by-sequencing work on the same plants. For Objective 3, phenotyping of plants is proceeding and will be expected to be completed in the autumn of 2012. These plants have been processed for genotype-by sequencing. 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. All team members were cognizant of the planned experiments and had approved implementation.