Location: Genomics and Gene Discovery
2006 Annual Report
Maintaining a sustainable fuel supply is critical for the U.S. economy and all the citizens of the U.S. Even a modest increase in domestic renewable fuel production would have major implications for the national economy, the environment, the maintenance of jobs throughout the nation, the vitality of rural America, and would contribute to enhanced national security by lessening a dependence on imported oil.
The primary aims of this project seek to improve the efficiency of biomass utilization through improved crop genetics. To accomplish this, the project is building on existing knowledge of cell wall biosynthesis to create genetically modified lines of a high-yielding energy crop Panicum virgatum (switchgrass) that are more easily converted to ethanol. To increase fundamental understanding of grass cell wall biosynthesis, a molecular genetic approach for identifying genes that alter cell wall composition has been adopted which utilizes the model temperate grass Brachypodium distachyon.
This project is aligned with National Program 307, Bioenergy and Energy Alternatives. This project will apply a variety of biotechnological approaches to the problem of producing renewable and affordable energy from plant matter. Traits that are being manipulated in potential energy crops are also important in forage grasses and legumes that are covered in National Program 205 (Rangeland, Pasture, and Forages). In addition, the scope and wide applicability of some of the molecular and genomics technologies that this project is developing will contribute directly and indirectly to all other programs that involve, or depend on, genetic or molecular data, functional genomics, gene discovery, and germplasm development; i.e., National Programs 301 (Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement), 302 (Plant Biological and Molecular Processes), 304 (Crop Protection and Quarantine), 306 (Quality and Utilization of Agricultural Products), and 107 (Human Nutrition).
Year 2 (FY2006) Objective 1: Alteration of the activity of key lignin biosynthetic enzymes - Gene cloning - Switchgrass cultivar Alamo transformation - Analysis of transgenic switchgrass for degree of silencing - Development of switchgrass transformation technology (including new accessions) Objective 2: Link transgenes to male sterility - Gene cloning - Swithgrass cultivar Alamo transformation Objective 3: Identify novel genes affecting cell wall composition - Optimize Brachypodium transformation and begin generating mutants - Optimize FTIR screen and begin screening mutants - Isolate Brachypodium promoters
Year 3 (FY2007) Objective 1: Alteration of the activity of key lignin biosynthetic enzymes - Analysis of transgenic switchgrass for degree of silencing - Development of switchgrass transformation technology (including new accessions) - Selection and propagation of lines for field trials Objective 2: Link transgenes to male sterility - Swithgrass cultivar Alamo transformation - Analyze male sterility constructs for pollen viability - Analyze transgenics for lignin composition/degree of silencing Objective 3: Identify novel genes affecting cell wall composition - Optimize FTIR screen and begin screening mutants - Isolate Brachypodium promoters - Characterize and prioritize Brachypodium mutants
Year 4 (FY2008) Objective 1: Alteration of the activity of key lignin biosynthetic enzymes - Analysis of transgenic switchgrass for degree of silencing - Development of switchgrass transformation technology (including new accessions) - Selection and propagation of lines for field trials Objective 2: Link transgenes to male sterility - Swithgrass cultivar Alamo transformation - Analyze male sterility constructs for pollen viability - Analyze transgenics for lignin composition/degree of silencing Objective 3: Identify novel genes affecting cell wall composition - Characterize and prioritize Brachypodium mutants
Year 5 (FY2009) Objective 1: Alteration of the activity of key lignin biosynthetic enzymes - Development of switchgrass transformation technology (including new accessions) - Selection and propagation of lines for field trials - Initiate field trials
Brachypodium transformation improvement. A robust and efficient transformation system is a crucial trait for a model organism. We improved our method for Brachypodium transformation by at least 10-fold. This improvement will facilitate experiments that require high-throughput transformation like T-DNA tagging and speed the adoption of Brachypodium as a model system. This accomplishment aligns with the energy crop component of National Program 307 - Bioenergy and Energy Alternatives.
Genetic markers. A genetic map is required for many experiments including the cloning of genes associated with induced mutations. We screened our EST sequences for microsatellites and designed PCR primers flanking a set of these microsatellites. Using these primers we identified 42 microsatellites that were polymorphic between at least two of the four Brachypodium accessions tested. These markers will be used in a collaborative effort to produce the first linkage map of Brachypodium. This accomplishment aligns with the energy crop component of National Program 307 -Bioenergy and Energy Alternatives.
To allow researchers to utilize Brachypodium as a modern model organism to study cell wall composition it is necessary to develop genomic resources. In collaboration with non-ARS scientists at the University of Nebraska, Kearney we constructed cDNA libraries from five different tissues and sequenced 20,440 ESTs. These sequences will be used to generate molecular markers to facilitate mapping in Brachypodium, to identify genes that may play a role in cell wall composition and to provide anchors for the development of a physical map. In addition, the cDNA libraries can be used to identify genes of interest that have not been identified during the sequencing project. To produce switchgrass with decreased lignin content, we cloned partial sequences for four lignin biosynthetic genes from switchgrass. These sequences were then used to construct transgenes designed to down-regulate the corresponding endogenous genes. These constructs were introduced into switchgrass and the resulting transgenic plants are in the process of being characterized for gene expression and lignin content. This work will lead to enhanced switchgrass lines that biomass producers, and forage breeders will use to more efficiently produce energy, or livestock. To develop Brachypodium as an effective tool to study the genetic basis of cell wall composition, we developed a high-efficiency Agrobacterium-mediated transformation protocol. This method will be used to generate a large mutant population that will be screened to identify genes involved in determining cell wall composition. In addition, this method allows Brachypodium to be used as a model to quickly test ideas before applying them to temperate grasses and cereals with longer generation times and more difficult transformation protocols.
Underpinning genetic progress and understanding of genome structure in Brachypodium and switchgrass is the need for more sequence data in these relatively uncharacterized species. We have initiated gene sequencing in these species and these efforts have already provided a large gene inventory and have helped to identify sequences for development as molecular markers. Ultimately the markers will aid in genetic characterization and breeding efforts while the genes themselves can be manipulated in biotechnological approaches to crop improvement.
Switchgrass transformation expertise developed by Drs. John Vogel and Christian Tobias has resulted in the establishment of two CRADA projects geared toward the development and commercialization of improved switchgrass varieties with value added traits. A CRADA with one technology company has been undertaken to express a heat stable cellulase in switchgrass. The resulting plants should required less purified cellulase for cellulose digestion and thus decrease the costs of saccharification. The second CRADA is with a plant biotechnology company to insert genes identified by the company as improving biomass related traits into switchgrass.
Scientists work to unlock biofuel genomes, hopes: Latest switchgrass project holds promise of tripling ethanol production, Ian Hoffman, Oakland Tribune, 7/13/2006.
Edenspace, USDA To Cooperate In Developing New Energy Crops - Press Release Edenspace Systems Corporation, September 15, 2005, http://www.edenspace.com/09-15-2005.html.
Ceres, Inc. and USDA Agricultural Research Service sign Cooperative Research and Development Agreement - Press Release, Ceres, Inc., February 1, 2006, http://www.ceres-inc.com/PressRelease_News/02_01_06.html.
Energy-rich Portfolio of New Genome Sequencing Targets for DOE JGI - Press Release, Joint Genome Institute, July 11, 2006, http://www.jgi.doe.gov/News/news_7_11_06.html.
Vogel, J.P., Gu, Y.Q., Twigg, P., Lazo, G.R., Chingcuanco, D.L., Hayden, D.M., Donze, T., Vivia-Lindsay, A., Stamova, B., Coleman-Derr, D. 2006. EST sequencing and phylogenetic analysis of the model grass brachypodium distachyon. Theoretical and Applied Genetics. 113: 186-195.
Vogel, J.P., Garvin, D.F., Leong, O.M., Hayden, D.M. 2006. Agrobacterium-mediated transformation and inbred line development in the model grass brachypodium distachyon. Plant Cell Tissue And Organ Culture. 84:199-211.
Tobias, C.M., Twigg, P., Hayden, D.M., Vogel, K.P., Mitchell, R., Lazo, G.R., Chow, E.K., Sarath, G. 2005. Analysis of expressed sequence tags and the identification of associated short tandem repeats in switchgrass. Theoretical and Applied Genetics. 111: 956-964.