2008 Annual Report
1a.Objectives (from AD-416)
Develop the necessary range of knowledge and technologies to use plants to produce chemical feedstocks and fuels with more renewable and more enviromentally acceptable methods than current sources such as petrochemicals. Focus initially on the cereal crops (especially wheat and rice) and other grasses and non-grass species with potential for biofuel development. Coordinate with other ARS and non-ARS projects in specific approaches to biofuel research and development.
1b.Approach (from AD-416)
To achieve these objectives, an integrated plant molecular, genomics, bioinformatics, plant transformation approach will be developed for the production of biofuels from crops. Specifically:.
1)Determine parameters underlying ectopic expression of polysaccharides and proteins in plant leaf and stem tissue, to include stability of expressed products and plant response;.
2)Determine potential for altering plant architecture and cell wall structure for altered functions such as more efficient light gathering, storage capacity, and increased efficiency of post-harvest product processing as for straw/fiber conversion;.
3)Modify polysaccharide production to enhance ethanol production, to include both existing and novel polymer structures;.
4)Develop a repertoire of tissue and development specific promoters, initially concentrating on promoters efficient in the monocotyledonous plants. Formerly 5325-21000-009-00D (8/04).
The on-going research in this project has made significant progress in the development of genetic and genomic resources for Brachypodium distachyon (Brachypodium) and switchgrass. This work furthers Component 1 (Feedstock Development) of National Program 213 - Bioenergy. Collaborative efforts within the agency and with external collaborators led to the release of a 4x draft genome sequence for Brachypodium. Continued sequencing by the DOE Joint Genome Institute to 8x coverage was completed and is being assembled and verified for release in the coming year. In order to make a high-density genetic linkage map for Brachypodium, we have identified thousands of single nucleotide polymorphisms. In collaboration with ARS scientists in St. Paul, MN, these polymorphisms will be used to create a high-density linkage map using a high-throughput genotyping platform. The first use of this genetic map will be to assemble the sequences from the genome project into chromosome-scale assemblies. To create a large, freely available germplasm collection for Brachypodium, a collaboration with researchers at Sabanci University and Namik Kemal University, both in Turkey, to collect and characterize new Brachypodium accessions was expanded. To begin the process of identifying the genes required for cell wall composition, a screen of chemically, radiation and insertional mutants of Brachypodium using near infrared spectroscopy was initiated. This technique will allow the for the rapid identification of mutants with altered cell wall composition.
For switchgrass, collaborative efforts within the agency and with external collaborators led to the sequencing and release of over 500,000 expressed sequence tags (partial sequences of expressed genes). These expressed sequence tags were assembled into unique genes and then screened for sequences that can be used as genetic markers. These new markers are being used in ongoing work to create a linkage map for switchgrass. The switchgrass genome is large and contains multiple copies (polyploid). To facilitate genomic analyses, we continued to look for switchgrass lines with reduced genomic complexity using phenotypic analysis and flow cytometry. This work led to the identification of a second switchgrass line with reduced DNA content.
We have continued to develop and use plant transformation technology within our research project for both basic and applied goals. We began making a population of Brachypodium mutants that contain a know piece of DNA randomly inserted into the genome. When this known DNA inserts in a gene it simultaneously disrupts the gene’s function and serves as a handle to identify the disrupted gene. Such ‘T-DNA’ mutants are valuable for hypotheses testing of individual gene’s specific functions and for the identification of promoters with useful expression patterns. For switchgrass we have more applied goals and, as part of a subordinate project supported by a cooperative research and development agreement, we have inserted genes predicted to increase biomass, alter cell wall composition or induce sterility into switchgrass. We began evaluation the first set of these transgenic lines for sterility.
Development of switchgrass genetic resources.
Breeding progress in switchgrass is slow and efficient breeding techniques taking advantage of genome sequence information are needed. ARS scientists in Albany CA, and Lincoln Nebraska have helped produce 500,000 switchgrass sequence tags which have been released to public through GenBank. These sequences have helped enable comparative genomics with other grass species such as sorghum and gene families associated with cell wall biosynthesis and photosynthesis have been analyzed. Assembly of the sequences into a minimally redundant set of overlapping EST produced approximately 76,000 unique genes. The sequences have been screened for common marker classes which have been tested for polymorphism and segregation in two mapping populations. These genetic resources will be used to accelerate breeding of improved switchgrass varieties through techniques such as marker assisted selection. This accomplishment furthers Component 1 (Feedstock Development) of National Program 213 - Bioenergy by addressing the research needs outlined in Problem Statement 1B: Biological and Molecular Basis of Plant Traits.
Expanding available genetic resources for Brachypodium.
A simple model for studying grass cell walls is needed to allow more rapid progress in understanding the potential to alter the properties of cellulosic biomass. ARS scientists in Albany, CA and collaborators at Namik Kemal University and Sabanci University, both in Turkey, have continued to collect and characterize new Brachypodium accessions. Over 200 inbred lines have been developed and the first set are now freely available to researchers. In addition, an inbred line with superior transformation properties was made publicly available. These lines are being used to study grass cell wall structure and other traits important for the production of cellulosic biomass. This accomplishment furthers Component 1 (Feedstock Development) of National Program 213 - Bioenergy by addressing the research needs outlined in Problem Statement 1B: Biological and Molecular Basis of Plant Traits.
Identification of additional diploid switchgrass lines.
Through a combination of phenotypic analysis, and flow cytometry ARS scientists in Albany CA have identified a second diploid switchgrass line that has been shown to have reduced DNA content in addition to the single diploid line that they have previously characterized. This line should provide another tool for analysis of genome organization in switchgrass. Working with polyploid species such as switchgrass complicates identification of beneficial genes and gene combinations that will affect biomass traits. Availability of such lines is extremely useful in several respects. Whole genome sequencing would become more feasible, genetic map construction would be simplified, and population structure with regard to origins of polyploidy could be determined. This accomplishment furthers Component 1 (Feedstock Development) of National Program 213 - Bioenergy by addressing the research needs outlined in Problem Statement 1B: Biological and Molecular Basis of Plant Traits.
Brachypodium distachyon genome resources.
A simple model for studying grass cell walls is needed to allow more rapid progress in understanding the potential to alter the properties of cellulosic biomass. ARS scientists in Albany CA are collaborating with DOE and other researchers to sequence the entire genome. A rough draft of the genome was released through the website www.brachypodium.org. Knowledge of the genome sequence of Brachypodium and the linear order of genes in the genome relative to other grasses will help to make this species useful to researchers studying important agricultural traits in energy crops and grain species. This accomplishment furthers Component 1 (Feedstock Development) of National Program 213 - Bioenergy by addressing the research needs outlined in Problem Statement 1B: Biological and Molecular Basis of Plant Traits.
5.Significant Activities that Support Special Target Populations
|Number of Active CRADAs||2|
|Number of Non-Peer Reviewed Presentations and Proceedings||8|
|Number of Newspaper Articles and Other Presentations for Non-Science Audiences||1|
Vogel, J.P., Hill, T.A. 2007. High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd 21-3. Plant Cell Reports. 27:471-478
Huo, N., Lazo, G.R., Vogel, J.P., You, F., Ma, Y., Hayden, D.M., Coleman-Derr, D., Hill, T., Dvorak, J., Anderson, O.D., Luo, M., Gu, Y.Q. 2007. Characterizing the nuclear genome structure of Brachypodium distachyon using BAC end sequences. Functional and Integrative Genomics. 8:135-147.
Vogel, J.P. 2008. Unique aspects of the grass cell wall. Current Opinion in Plant Biology. 11:301-307.
Garvin, D.F., Gu, Y.Q., Hasterok, R., Hazen, S.P., Jenkins, G., Mockler, T.C., Mur, L.A., Vogel, J.P. 2008. Development of genetic and genomic research resources for Brachypodium distachyon, a new model system for grass crop research. The Plant Genome. 48:S69-S84.