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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Foundations for Bioenergy Feedstocks

Location: Genomics and Gene Discovery

2010 Annual Report

1a.Objectives (from AD-416)
Develop switchgrass genetic resources in support of bioenergy feedstock improvement. Mine sequence resources for switchgrass and develop markers that distinguish homoeologous groups and that will allow comparative genomics among the Poaceae. Create cytogenetic landmarks for switchgrass and utilize them for karyotyping, map integration, and genetic analysis of existing diversity. Identify induced and natural variation in traits relevant to biomass crop improvement using the model grass Brachypodium. Identify mutants and natural accessions with variation in cell wall composition. Develop functional genomic resources and experimental methods that enable Brachypodium to be used as a model grass. Assess natural diversity using whole genome resequencing. Create a population of insertional mutants, sets of diverse inbred lines and improved annotation of the genome sequence. Apply knowledge gained from Brachypodium toward the improvement of switchgrass using comparative genomics and candidate genes.

1b.Approach (from AD-416)
Translational and comparative approaches that exploit relatively rapid discovery in model biological systems and the large body of knowledge from other grass taxa can be applied to energy crops. This project will enable these approaches through alignment of switchgrass genetic maps and EST collections with reference grass genomes and will be a fundamental means by which the identification of switchgrass orthologs of genes in other species can be identified. This will allow candidate gene selection for genetic studies directly in phenotypically diverse switchgrass populations for QTL and association analysis. Based on the outcome of forward and reverse genetic experiments in Brachypodium designed to elucidate cell wall composition, the prospects of applying transgenic approaches in switchgrass to manipulate these qualities can be intelligently assessed. These approaches will produce uniquely defined genetic stocks in switchgrass significantly altered with respect to digestibility, that may be subsequently assessed via several different technology platforms for conversion efficiency to useful simple sugars, heat, or combustion gases.

3.Progress Report
The on-going research in this project has made significant progress in the development of genetic and genomic resources for Brachypodium and switchgrass.

Efforts within the agency and with external collaborators led to the publication and release of the Brachypodium genome sequence and annotation. This important resource is now freely available to the research community. In addition, a project to resequence diverse natural accessions of Brachypodium was initiated. To date, four inbred lines have been resequenced and a large amount of variability identified. This data will allow researchers to mine the natural diversity found within Brachypodium.

To identify genes that affect cell wall composition, we have begun to characterize 22 Brachypodium mutants initially identified using NIR spectroscopy. As part of this characterization we have developed a simultaneous saccharification and fermentation assay based on the anaerobe Clostridium thermocellum. Significantly, the mutants deviate from wild type in this assay suggesting they affect cell wall components relevant to biofuel production.

Collaborative efforts have utilized switchgrass ESTs previously produced by this project for genetic analysis. Additional markers have been developed and used for linkage analysis and for characterization of genetic diversity. These markers allow discrimination of different ploidy levels as well as upland verses lowland ecotypes. This enables germplasm classification and tentative identification of materials based on genotype alone. As part of a collaborative project, a complete genetic linkage map of switchgrass has been produced. ARS Scientists in Albany have characterized switchgrass germplasm through controlled crosses and through analysis of mitotic chromosomes for the purposes of assigning unique identities to each chromosome and to align these with the genetic map. This work has generated several probes that hybridize to unique loci in switchgrass and has assigned BAC probes to two separate chromosomes. Chromosome arm length ratios and sizes have identified with high reliability the other chromosomes which are not yet aligned with the genetic map.

A collaborative effort has sequenced the switchgrass chloroplast genome and polymorphisms between one upland (cv. 'Summer') and one lowland (cv. 'Kanlow') genotype have been identified. Differences between upland and lowland chloroplast genomes can be used to identify different heterotic groups and may assist in explaining directional effects seen in crosses displaying heterosis. In all, 97 polymorphic sites define the difference between these individuals. These sites were distributed primarily in the non-coding sequences.

Our ongoing insertional mutagenesis in Brachypodium produced over 4,000 new T-DNA lines to bring the total collection to over 8,000 lines. We have also sequenced DNA flanking the insertion sites of more than 3,500 lines and deposited the data in a website. For switchgrass, as part of subordinate projects supported by cooperative research and development agreements, we have inserted genes predicted to increase biomass, alter cell wall composition or induce sterility.

1. Brachypodium genome sequenced. 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 collaborated with Department of Energy (DOE) and other researchers to complete the analysis and annotation of the entire genome. A paper describing the results was published in the journal Nature and the sequence and annotation is now freely available through several databases. In addition, a project to resequence additional accessions was initiated. To date, four lines have been resequenced and the analysis of the sequences has been initiated. 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.

2. Characterization of diversity in polyploid switchgrass. The genetic make-up of the energy crop switchgrass is not well understood. Using molecular markers, ARS scientists in Albany, CA have identified markers prevalent in certain populations. These have enabled distinguishing different switchgrass cultivars and populations from one another genetically. This information will be useful for preserving diversity, defining which populations are derived from common ancestors, and measuring the amount of interbreeding that has occurred between different populations.

3. Expanding and characterizing 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 CSIRO in Canberra, Australia have begun to use a high-throughput phenotyping platform (phenomics) to characterize over 100 natural accessions. To date, we have documented extensive natural variation in several traits relevant to biofuels including, cell wall composition, stem density and fermentability. In addition, Albany researchers have created over 4,000 T-DNA lines this year and have released over 4,000 T-DNA lines made in the prior year to the public through a newly established T-DNA website. This work will identify genes that can then be manipulated in bioenergy crops to improve cell wall properties for biofuel production.

4. Characterization of Brachypodium mutants with altered cell wall composition. The plant cell wall is a complex composite of polysaccharide polymers, phenolic compounds and proteins. ARS scientists in Albany, CA have begun to characterize 22 mutants with putative alterations in cell wall composition that were initially identified by near infrared spectroscopy (NIR). These mutants were backcrossed to wild type to determine segregation. They were also crossed to another accession to map the mutations. Using a simultaneous saccharification and fermentation assay, we observed significant differences between the fermentability of the mutants and wild type. This work will identify genes that can then be manipulated in bioenergy crops to improve cell wall properties for biofuel production.

5. Switchgrass chloroplast genome sequencing. Upland and lowland switchgrass represent unique ecological types (ecotypes) that also have different cellular types defined by genetic markers present in the chloroplast. To help understand the basis of hybrid vigor observed in crosses between upland and lowland switchgrass and differences observed in plant vigor, ARS researchers in Albany, CA, identified all polymorphic sites between two individual chloroplast genomes originating from different ecotypes. This work will be used to guide further analysis of individual crosses to determine which polymorphisms are positively associated with the observed directional effects, and can be used to guide analysis of biochemical variation in C4 photosynthesis and reversion to C3 photosynthesis among different grasses.

Review Publications
Vogel, J.P., Garvin, D.F., Gu, Y.Q., Lazo, G.R., Anderson, O.D., Bragg, J.N., Chingcuanco, D.L., Weng, Y., Belknap, W.R., Thomson, J.G., Dardick, C.D., Baxter, I.R. 2010. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature. 463:763-768.

Okada, M., Lanzatella-Craig, C., Saha, M., Bouton, J., Wu, R., Tobias, C.M. 2010. Complete Switchgrass Genetic Maps Reveal Subgenome Collinearity, Preferential Pairing and Multilocus Interactions. Genetics. 185:745-760.

Garvin, D.F., Mckenzie, N., Vogel, J.P., Mockler, T.C., Blankenheim, Z., Wright, J., Huo, N., Cheema, J.J., Dicks, J., Hayden, D.M., Gu, Y.Q., Tobias, C.M., Chang, J.H., Chu, A., Trick, M., Michael, T.P., Bevan, M.W., Snape, J.W. 2010. An SSR-Based Genetic Linkage Map of the Model Grass Brachypodium distachyon. Genome. 53(1):1-13.

Bevan, M.W., Garvin, D.F., Vogel, J.P. 2010. Brachypodium distachyon genomics for sustainable food and fuel production. Current Opinion in Biotechnology. 21:211-217.

Vogel, J.P., Bragg, J.N. 2009. Brachypodium distachyon, a New Model for the Grasses. p. 427-449. Genetics and Genomics of the Triticeae.

Filiz, E., Ozdemir, B.S., Budak, F., Vogel, J.P., Metin, T., Budak, H. 2010. Molecular, morphological and cytological analysis of diverse Brachypodium distachyon inbred lines. Genome. 52:876-890

Last Modified: 4/17/2014
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