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United States Department of Agriculture

Agricultural Research Service

Research Project: Genetic Foundations for Bioenergy Feedstocks

Location: Genomics and Gene Discovery

2013 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.

In collaboration with external collaborators, we are analyzing the sequence of the genomes of 54 natural Brachypodium accessions. The analysis of the first 6 genomes has been completed and those genomes are now available to the public through the Brachypodium.org database. We noted an interesting structure to the distribution of polymorphisms within defined genomic regions. We are now using multiple independent methods including de novo genome assembly to identify sequences present in the resequenced genomes but absent from the reference genome.

Projects to sequence two related species, B.stacei and B. hybridum, as a model to study polyploidy genome evolution are continuing. Both genomes have been sequenced to over 450x coverage and mapping crosses are being sequenced to make a genetic map 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 5,000 T-DNA mutant lines in FY13 to bring the total collection to >20,000 lines. We sequenced the DNA flanking the insertion sites of 10,000 lines using a method based on Illumina sequencing of multidimensional DNA pools. Over 12,000 flanking sequence tags were added to the T-DNA database to bring the total number of unique, identified insertions sites to 15,000. This resource is increasingly being used by the scientific community to study gene function and over 600 lines from our collection were requested in FY13.

Our ongoing collaborative project to extensively phenotype Brachypodium germplasm is almost finished. We completed phenotyping a collection of 500 homozygous T-DNA lines and are currently analyzing the data. 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.

A completed collaborative effort with researchers at U.C. Davis has led to some insights about centromere evolution. Bioinformatic methods were used to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and new data. The assumption that the most abundant tandem repeat is the centromere DNA was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and in length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond ~50 million years of divergence. A set of grass centromeric repeats were painted on chromosomes to validate specificity of the repeats.


4.Accomplishments
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, California, are collaborating with Department of Energy scientists and other researchers to resequence 54 Brachypodium accessions. The last nine accessions were sequenced in FY13. The initial analysis of the first six lines was completed and the sequences are now available online (jbrowse.brachypodium.org). Analysis, including de-novo assemblies, of the genomes from the remaining 48 lines is underway. 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, California, created over 5,000 T-DNA lines this year bringing their total T-DNA collection to about 20,000 lines. They sequenced the DNA flanking the insertion sites in over 10,000 lines in FY13 (18,000 total) in order to determine which genes are inactivated by T-DNA insertions. Over 600 lines were requested through the project website in FY13 (>1,600 total). 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, California, and collaborators at Commonwealth Scientific and Industrial Research Organisation in Canberra, Australia continued to use 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. Several unique lines have been validated and are being characterized molecularly. This work will identify genes that can then be targeted to create improved in bioenergy crops.

4. Establishing a model system to study polyploidy in the grasses. Many important grass crops (e.g. wheat, oats, switchgrass, Miscanthus) have large polyloid genomes that present challenges to breeders and scientists attempting to determine the function of specific genes. A simple model polyploid system would be useful for understanding the dynamics of polyploidy genome evolution. Three related Brachypodium species, B. distachyon, B. stacei and B. hybridum can serve as such a model system because B. hybridum is an allopolyploid with genomes derived from B. distachyon- and B. stacei-like ancestors. To establish this trio as a model system, ARS scientists in Albany, California, are collaborating with the Department of Energy, Joint Genome Institute and others to sequence the genomes of B. hybridum and B. stacei. Significant progress has been made in genome sequencing and mapping populations have been produced to characterize traits and aid genome assembly. In addition, the chloroplast genomes of B. stacei and B. hybridum have been characterized.

5. Creation of a chromosome karyotype for switchgrass. Chromosome profiles or karyotypes can provide information about taxonomic relationships, genetic aberrations, and the evolutionary origins of species. However, differentiation of the tiny chromosomes of switchgrass (Panicum virgatum L.) and creation of a standard karyotype for this bioenergy crop has not been accomplished due to lack of distinguishing features. Scientists in Albany, California, conducted a cytogenetic study on switchgrass to establish a chromosome karyotype. Differences observed at specific locations on chromosome five between the upland and lowland ecotypes of switchgrass provided a basis for distinguishing these subpopulations. Collectively, the results allow the classification of switchgrass plants belonging to divergent genetic pools and have provided other insights into its genome evolution.


Review Publications
Tobias, C.M. 2013. Switchgrass Genomics. Bioenergy Feedstocks: Breeding and Genetics. 1:33-48.

Young, H., Sarath, G., Tobias, C.M. 2012. Karyotype variation is indicative of subgenomic and ecotypic differentiation in switchgrass. Biomed Central (BMC) Plant Biology. 12:117.

Lu, Y., Yang, X., Tong, C., Li, X., Feng, S., Wang, Z., Pang, X., Wang, Y., Wang, N., Tobias, C.M., Wu, R. 2012. A multivalent three-point linkage analysis model of autotetraploids. Briefings in Bioinformatics. doi:10.1093/bib/bbs051.

Melters, D.P., Bradnan, K., Young, H., Telis, N., May, M., Ruby, G.J., Sebra, R., Peluso, P., Eid, J., Rank, D., Garcia, J., Derisi, J., Smith, T.P., Tobias, C.M., Ross-Ibarra, J., Korf, I., Chan-Simon, W. 2013. Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution. Genome Biology. doi:10.1186/gb-2013-14-1-r10.

Wu, X., Ju, J., Bragg, J.N., Vogel, J.P., Anderson, O.D., Gu, Y.Q. 2013. Phylogenetic, molecular, and biochemical characterization of caffeic aicd O-methyltransferase (COMT) gene family in Brachypodium distachyon. International Journal of Plant Genomics. doi.org/10.1155/2013/423189.

Last Modified: 7/23/2014
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