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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Foundations for Bioenergy Feedstocks
2011 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 ongoing 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 collaborators led to the genotyping and analysis of replicated field trials for detecting genetic loci controlling many switchgrass traits. This important field data is now being analyzed and markers flanking desirable QTL will be used in selection. The result will be a method to rapidly increase desirable genotypes within populations of improved switchgrass without needing to rely solely on phenotypic data. To date several QTL for dry matter yield have been identified.

Analysis of sequence data for switchgrass has identified the extent of chloroplast variation in different cultivars and ecotypes by alignment of short reads derived from large sequence samples to a reference chloroplast sequence. Differences between chloroplast genomes can be used to identify different heterotic groups and may assist in explaining directional effects seen in crosses displaying hybrid vigor. These data indicate that the variation within switchgrass chloroplasts is similar to that found between indica and japonica subspecies of rice.

ARS scientists in Albany, CA, have characterized switchgrass genome structure through a process of chromosome painting and microscopy. This work has demonstrated divergence of repetitive genetic loci between the multiple switchgrass subgenomes. This directly demonstrates that the subgenomes are functionally differentiated and may evolve independently of one another. It also has the potential to shed light on the role genome duplication plays in adaptation to environmental stress, competition in natural environments, and productivity of natural ecosystems.

Efforts within the agency and with external collaborators are underway to re-sequence 56 Brachypodium accessions. The analysis of the first 6 lines is nearly complete and DNA for 19 lines has been submitted for sequencing. This data will be incorporated into public databases as it becomes available to allow researchers to mine the natural diversity found within Brachypodium.

To identify genes that affect cell wall composition, we are continuing to characterize 30 Brachypodium mutants initially identified using NIR spectroscopy. We were able to follow the NIR phenotype in first genetic crosses examined indicating that a genetic approach to identifying the underlying gene is possible. These efforts will lead to a better understanding of the control of biomass accretion, composition, and degradation in grasses.

Our ongoing collaborative project to produce mutant populations and extensively phenotype natural accessions has so far produced over 9500 well characterized mutant lines. These efforts have been described in a web site accessible to the public and phenotyping efforts have moved from optimization to full scale characterization producing a wealth of data on the growth of Brachypodium under a variety of environmental conditions. These efforts will eventually enable modeling in a predictive sense of the role of genetic factors in controlling response to environmental stimuli.


4.Accomplishments
1. Establishment of a switchgrass chromosome karyotype. Switchgrass is under development as a feedstock for biofuels. The individual chromosomes of switchgrass are not characterized in relation to the genome or genetic map. Using molecular markers, microscopy and chromosome staining techniques, ARS scientists in Albany, CA, have identified the sizes, condensation-patterns and centromeric regions of individual chromosomes that allow some to be distinguished. This work will provide a basic understanding of the separate chromosomes that can be eventually be used to identify all individual chromosomes in different distinct switchgrass populations containing multiples of the base chromosome number and which in many cases are missing one or more chromosomes. This information will be useful for preserving germplasm diversity, defining separate breeding populations, and assigning specific traits to genes on individual chromosomes.

2. Enhanced switchgrass digestibility. Difficulty converting plant biomass to sugars and liquid fuels is due in part to the presence of lignin in the plant cell wall. To provide new routes toward efficient conversion of plant biomass ARS researchers in Albany, CA, in collaboration with scientists at the Plant Gene Expression Center, have demonstrated two unique strategies for increasing cell wall digestibility. The first strategy is to use a plant regulatory factor identified in maize that alters the plant's juvenile to adult transition. The second approach is to reduce levels of a lignin synthetic enzyme. Both resulted in structural alterations to the plant cell wall that enhanced digestibility. This work has guided the researchers to pursue second generation strategies designed to eliminate impacts on yield potential to produce lines that are more commercially viable.

3. Resequencing diverse Brachypodium accessions. 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 Department of Energy (DOE) and other researchers to resequence 56 Brachypodium accessions. Six lines have been resequenced and analyzed to identify a defined set of differences between the resequenced genomes and the reference genome. DNA has been prepared for an additional 19 accessions and has been sent to the DOE for sequencing. Sequence has been produced for 9 of these accessions. Knowledge of the genetic diversity of Brachypodium will help to make this species useful to researchers studying important agricultural traits in energy crops and grain species.

4. 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 Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra, Australia have begun to use a high-throughput phenotyping platform (phenomics) to characterize over 100 natural accessions. Experiments were carried out to evaluate the growth of Brachypodium under a range of environmental conditions in order to understand more of environmental effects on cell walls. In addition, Albany researchers have created over 2,000 T-DNA lines this year bringing the total T-DNA collection to about 10,000 lines. This work will identify genes that can then be manipulated in bioenergy crops to improve cell wall properties for biofuel production.

5. Characterization of Brachypodium mutants with altered cell wall composition. Understanding cell wall structure is important for developing biomass/biofuel crops. The plant cell wall is a complex composite of polysaccharide polymers, phenolic compounds and proteins. ARS scientists in Albany, CA, have continued to characterize 30 mutants with putative alterations in cell wall composition that were initially identified by near infrared spectroscopy (NIR). Significantly, using the same NIR technique, Albany researchers were able to identify mutant individuals in the F2 progeny from the first backcross and a mapping crosses examined. Thus, a genetic approach to identifying the mutated gene can be used. This work will identify genes that can then be manipulated in bioenergy crops to improve cell wall properties for biofuel production.

6. Perenniality of biofuel grasses. A perennial lifecycle is favorable for energy crops because it minimizes fertilizer and other energy requiring inputs while at the same time reducing the erosion often associated with establishing annual crops. Brachypodium sylvaticum is a perennial grass closely related to the annual model grass Brachypodium distachyon. Therefore, B. distachyon resources can be leveraged to establish B. sylvaticum as a system to test perenniality-related topics. Researchers at Albany, CA, have developed a very efficient Agrobacterium-mediated transformation system for B. sylvaticum, establishing a key requirement for a model system. This work will allow researchers to more easily study the basis of perenniality in the grasses and rapidly test biotechnological approaches in a perennial grass.


Review Publications
Saathoff, A.J., Sarath, G., Chow, E.K., Dien, B.S., Tobias, C.M. 2011. Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment. PLoS One. 6(1):e16416. DOI: 10.1371/journal.pone.0016416.

Li, J., Das, K., Fu, G., Lie, Y., Tobias, C.M., Wu, R. 2010. EM Algorithm for mapping quantitative trait loci in multivalent tetraploids. International Journal of Plant Genomics. Volume 2010 (2010), Article ID 216547. Available: http://www.hindawi.com/journals/ijpg/2010/216547/.

Mur, L., Allainguillaume, J., Catalan, P., Hasaterok, R., Jenkins, G., Lesniewski, K., Thomas, I., Vogel, J.P. 2011. Exploiting the Brachypodium tool box in cereal and grass research. New Phytologist. DOI: 10.1111/j.1469-8137.2011.03748.x.

Tyler, L., Bragg, J.N., Wu, J., Yng, X., Tuskan, G., Vogel, J.P. 2010. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon. Biomed Central (BMC) Genomics. 11:600.

Huo, N., Garvin, D.F., You, F., McMahon, S.A., Luo, M., Gu, Y.Q., Lazo, G.R., Vogel, J.P. 2011. Comparison of a high-density genetic linkage map to genome features in the model grass Brachypodium distachyon. Theoretical and Applied Genetics. 123(3):455-464.

Last Modified: 11/27/2014
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