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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Foundations for Bioenergy Feedstocks

Location: Genomics and Gene Discovery

2012 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.

Working with with external collaborators, 45 natural Brachypodium accessions have been re-sequenced. The data indicates that a high level of genetic variation exists within this collection and is being incorporated into public databases as it becomes available to allow researchers to mine the natural diversity found within Brachypodium.

Projects to sequence two related species, B. stacei and B. hybridum have been initiated. Both genomes have been sequenced to over 70x coverage and mapping crosses made to aid assembly. These genomes, along with the already sequenced genome of B. distachyon, will serve as a model system to study polyploidy because B. hybridum is an allotetraploid that contains sub-genomes similar to the diploids B. distachyon and B. stacei.

Our ongoing project to produce a large T-DNA mutant collection has produced over 15,000 T-DNA mutant lines. We adopted a new method based on Illumina sequencing of multidimensional DNA pools to more efficiently sequence DNA flanking the insertion sites. This resource is increasingly being used by the scientific community to study gene function and over 1,000 lines from our collection were requested in FY12.

Our ongoing collaborative project to extensively phenotype Brachypodium germplasm is running at full capacity. We completed phenotyping a collection of 100 natural accessions and noted large variation in every parameter measured including: yield, photosynthetic rate, and plant architecture. We created over 500 homozygous T-DNA lines and phenotyped the first 100. These efforts are creating a valuable genetic resource that will allow researchers to gain the information necessary to use a predictive approach to create improved crop varieties.

We have, in collaboration with other ARS collaborators in Lincoln, NE, been developing a high-throughput genotyping method in switchgrass and working to validate it on a large mapping population. This has allowed determination of true genotypic variation verses sequencing error or highly-related duplicated sequences. This method is now being used in several large switchgrass breeding populations that have been phenotyped for maturity date, cell-wall composition and yield. Together, the data and accompanying analysis techniques will help identify markers that can be used for indirect selection of these traits and make breeding switchgrass more efficient.

Other collaborative work has been directed at elucidating the role of Cg1 in switchgrass. Cg1 is a maize regulatory gene that when expressed from a strong promoter in switchgrass reprograms growth and maturity. The plants take on juvenile characteristics that include more digestible cell walls and increased starch. These are potentially valuable characteristics, however overexpression is also correlated with decreased vegetative yield. Ongoing work is attempting to understand how this happens.

1. Resequencing diverse Brachypodium accessions. A simple model for studying the biological properties of the grasses including their unique cell walls, stress tolerance and factors that affect biomass yield is needed to allow more rapid progress in developing superior cellulosic biomass crops. ARS scientists in Albany, CA, are collaborating with Department of Energy (DOE) and other researchers to resequence 54 Brachypodium accessions. Forty-five lines have been resequenced and analyzed to identify a defined set of differences between the resequenced genomes and the reference genome. DNA for an additional 9 accessions has been sent to the DOE for sequencing. 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.

2. Expanding genetic resources for Brachypodium. Brachypodium is a simple model for studying the unique properties of the grasses and has been adopted by many laboratories around the world because of its ease of use and long list of readily available resources. ARS scientists in Albany, CA, created over 5,000 T-DNA lines this year bringing their total T-DNA collection to about 15,000 lines. They have sequenced the DNA flanking the insertion sites in over 10,000 lines in order to determine which genes are inactivated by T-DNA insertions. Over 1,000 lines were requested through the project website in FY12. This resource will aid the determination of gene function which in turn will provide knowledge that can be used to create improved crops.

3. Characterizing genetic resources for Brachypodium. ARS scientists in Albany, CA, and collaborators at Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra, Australia are using a high throughput phenotyping platform (phenomics) to characterize over 150 natural accessions and 500 homozygous T-DNA lines. Extensive natural variation in phenotypes (e.g. growth rate, yield, height, growth habit and photosynthetic rate) have been noted. In addition, Albany researchers have made over 500 homozygous T-DNA lines for phenotyping. This work will identify genes that can then be targeted to create improved in bioenergy crops.

4. Cloning a Brachypodium disease resistance gene. Resistance to disease will become increasingly important as biomass crops are grown on larger and more contiguous land area. ARS scientists in Albany, CA, and St. Paul, MN in collaboration with researchers at UC Berkeley and China Agricultural University (Beijing, China) have cloned a Brachypodium gene that confers resistance to Barley Stripe Mosaic Virus (BSMV). As part of this project, a map of the recombination break points in a population of recombinant inbred lines was developed. The resistance gene is the first resistance gene cloned for BSMV and may be useful for engineering resistance into crops like barley and wheat. In addition, the recombination map and recombinant inbred lines are a valuable resource for researches seeking to clone other genes of interest.

5. Establishing a model system to study perenniality in the 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 with attributes that make it well suited for a model system (small stature, rapid seed to seed time, self-fertile, simple growth requirements and a small diploid genome). Researchers at Albany, CA, have developed and genetically characterized a collection of inbred lines from germplasm obtained from the National Plant Germplasm System. This builds upon their previous work to develop a very efficient Agrobacterium-mediated transformation method. 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.

6. Discovery of genetic variation in switchgrass. Efficient breeding of switchgrass requires many, easily measured genetic markers. ARS scientists in Albany, CA, and Lincoln, NE have implemented a system for rapid, inexpensive genotyping of 1000's of markers that can be applied for breeding purposes. These markers were verified using a switchgrass mapping population, using early-release switchgrass genome assemblies that enabled them to reduce misidentified genotypes. This work will eventually lead to enhanced breeding efficiency through a reduction in the time required to accomplish a single cycle of selection.

Review Publications
Saathoff, A.J., Tobias, C.M., Sattler, S.E., Haas, E.E., Twigg, P., Sarath, G. 2011. Switchgrass contains two cinnamyl alcohol dehydrogenases involved in lignin formation. BioEnergy Research. 4(2):120-133.

Cui, Y., Lee, M.Y., Huo, N., Bragg, J., Yan, L., Yuan, C., Li, C., Holditch, S.J., Xie, J., Luo, M.C., Li, D., Yu, J., Martin, J., Schackwitz, W., Gu, Y.Q., Vogel, J.P., Jackson, A.O., Liu, Z., Garvin, D.F. 2012. Fine mapping of the Bsrl barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon. PLoS One. 7:e38333.

Casler, M.D., Tobias, C.M., Kaeppler, S.M., Buell, R., Wang, Z., Cao, P., Ronald, P. 2011. The switchgrass genome: tools and strategies. The Plant Genome. 4:273-282.

Brkljacic, J., Grotewold, E., Scholl, R., Mockler, T., Garvin, D.F., Vain, P., Brutnell, T., Sibout, R., Bevan, M., Budak, H., Caicedo, A.L., Gao, C., Gu, Y.Q., Hazen, S.P., Holt, B.F., Hong, S., Jordan, M., Manzaneda, A.J., Mitchell-Olds, T., Mochida, K., Mur, L.A., Park, C., Sedbrook, J., Watt, M., Zheng, S., Vogel, J.P. 2011. Brachypodium as a model for the grasses: today and the future. Plant Physiology. 157(1):3-13.

Young, H.A., Lanzatella-Craig, C., Sarath, G., Tobias, C.M. 2011. Chloroplast genome variation in upland and lowland switchgrass. PLoS One. 6(8): e23980. Available:

Chochois, V., Vogel, J.P., Watt, M. 2012. Application of Brachypodium to the genetic improvement of wheat roots. Journal of Experimental Botany. 63:3467-3474.

Chuck, G., Tobias, C., Sun, L., Kraemer, F., Li, C., Arora, R., Bragg, J., Vogel, J., Singh, S., Simmons, B., Pauly, M., Hake, S.C. 2011. Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility and increases starch content of switchgrass. Proceedings of the National Academy of Sciences. 108(24).

Last Modified: 4/20/2014
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