Brachypodium as a Model for the Grasses: Today and the Future

Model systems not only allow scientists to investigate complex processes that are difficult to study in nonmodel organisms but also serve to focus community efforts and resources, significantly advancing research. Arabidopsis (Arabidopsis thaliana) has served as a plant model system for almost 30 years and is widely considered the preeminent model plant. The success of Arabidopsis-related research has been driven not only by key features common to any model organism but also by the collaborative environment built by the Arabidopsis community. A decade after the Arabidopsis genome sequence was published, the development of model plants follows a different trajectory. In the past, the development of extensive resources and a large user community happened first and then sequencing the genome followed. Today, however, an organism is selected as a potential model and genome sequencing occurs prior to or concurrent with the development of experimental tools and a user community. Arabidopsis research has provided many scientific breakthroughs (Flavell, 2009). However, its utility as a model is limited to a certain extent when investigating monocot-specific processes. Within the monocots, grasses provide the vast majority of human calories and are increasingly utilized as a sustainable source of energy. Traits including cell wall composition, plant architecture, grain properties, intercalary meristems, and root architecture are best studied using grass model systems (Vogel, 2008; Watt et al., 2009). Rice (Oryza sativa) and maize (Zea mays) have numerous advantages as grass models, including sequenced genomes, large research communities, and substantial genetic resources and (http://www.gramene.org/ and http://www.maizegdb.org/). The major challenges associated with these species include the large size of the plants, long generation times, demanding growth requirements, and restricted access to germplasm due to quarantine restrictions and intellectual property concerns (Jung et al., 2008). Brachypodium (Brachypodium distachyon) was first proposed as a model system in 2001 (Draper et al., 2001). A comparison with other plants (Table I) reveals that Brachypodium’s attributes are well suited to a model system. Like Arabidopsis, Brachypodium has a small stature, short generation time, small genome, the ability to self-pollinate, and is easily grown under simple conditions (Draper et al., 2001). In addition, the phylogentic position of Brachypodium makes it a convenient model for grasses with significant genome expansions (e.g. wheat [Triticum aestivum], rye [Secale cereale], and cool season pasture grasses [http://www.umsl.edu/services/kellogg/gpwg/default.htm]). Removing a significant limitation to genetic analysis, the Vogel and Garvin laboratories have recently optimized methods to efficiently cross Brachypodium (approximately 80% efficiency; for links to the methods, see Table II). The benefits of conducting experiments rapidly in a small space are apparent when Brachypodium is compared with biomass crops like switchgrass (Panicum virgatum) and Miscanthus sinensis (Table I). Thus, significant investments have been made in developing and using Brachypodium as a model for these emerging biofuel crops. In this context, it is noteworthy that Brachypodium is a “typical” grass at the genome level, as reflected by the overall similarity in gene content and gene families when compared with the rice and sorghum (Sorghum bicolor) genomes (International Brachypodium Initiative, 2010). Therefore, for the vast majority of traits (e.g. cell wall composition, yield, stress tolerance, cell wall biosynthesis, root growth, development, and plant-pathogen interactions), Brachypodium can serve as a useful functional model for the grasses, and initial studies on cell walls, grain development, and root growth support Brachypodium’s utility as a model system (Watt et al., 2009; Larré et al., 2010; Guillon et al., 2011; Opanowicz et al., 2011; Wang et al., 2011). Comparison of select models and crops High-density planting under laboratory conditions. The difficulty of growing plants is dependent upon their size and the range of environmental conditions tolerated. Thus, small plants that tolerate varied conditions have simple growth requirements and large plants that need carefully controlled environmental conditions have demanding growth requirements. Unpublished data (T. Brutnell). Assembled genome size. Approximately 20% of the genome was not assembled because of the repetitive nature. Assembly consists of sequenced BACs many of which contain unordered genes because of the difficulty associated with assembling repetitive DNA. While rice has extensive insertional mutant resources, the availability of the resources is constrained by quarantine restrictions and intellectual property concerns. Comparison of select models and crops High-density planting under laboratory conditions. The difficulty of growing plants is dependent upon their size and the range of environmental conditions tolerated. Thus, small plants that tolerate varied conditions have simple growth requirements and large plants that need carefully controlled environmental conditions have demanding growth requirements. Unpublished data (T. Brutnell). Assembled genome size. Approximately 20% of the genome was not assembled because of the repetitive nature. Assembly consists of sequenced BACs many of which contain unordered genes because of the difficulty associated with assembling repetitive DNA. While rice has extensive insertional mutant resources, the availability of the resources is constrained by quarantine restrictions and intellectual property concerns. Internet resources for Brachypodium research Internet resources for Brachypodium research Brachypodium, like Arabidopsis, is particularly useful for basic research that requires large numbers of individual plants, carefully controlled growth conditions, multiple generations, and genetic analyses. Conversely, research on crop plants themselves should be favored if the question under study is close to creating an improved variety (translational research), if the trait under study is unique to the crop, and/or if the goal is to immediately improve the crop. In this context, it is important to note that even with the explosion of sequence information available for crop plants, model systems will continue to play an important role in gaining fundamental knowledge about genetic pathways and gene functions simply due to the ease with which experiments can be conducted with model species. Brachypodium, the small grains (e.g. wheat, oat [Avena sativa], and rye), and temperate forage grasses all belong to the Pooideae subfamily of the family Poaceae. This close phylogenetic relationship suggests that the Brachypodium genome may be useful for structural genomic studies in this group of grasses, even though the extensive rearrangements in the wheat-barley (Hordeum vulgare) lineage place limits on colinearity over large genomic regions (Opanowicz et al., 2008; International Brachypodium Initiative, 2010). Likewise, the sorghum and foxtail millet (Setaria italica) genomes will be useful as structural models for the emerging biomass crops switchgrass and Miscanthus. However, in all cases, comparison of multiple grass genomes will provide insights not available through simple pairwise comparisons. Similar to rice, wheat, and barley, Brachypodium uses the C3 photosynthetic pathway. However, maize, sorghum, and many of the emerging biomass crops use a C4 photosynthetic pathway, which is more efficient under hot, dry conditions. Thus, Brachypodium alone is not suitable to study C4 photosynthesis. Setaria viridis, another small, annual grass being developed as a model, could be particularly useful in this regard (Doust et al., 2009; Brutnell et al., 2010). We envision that these two plants will serve as dual model systems and that comparative studies may accelerate functional genomic studies in the grasses (Brutnell et al., 2010). Indeed, Brachypodium is being used as a model system to explore the feasibility of installing a C4 photosystem into C3 target organisms such as rice and wheat (T. Brutnell, unpublished data). Candidate genes identified from C4 grasses that are likely to be important for establishing major C4 traits are being introduced into Brachypodium to see if it is possible to alter photosynthetic characteristics. The high-quality draft Brachypodium sequence is available (International Brachypodium Initiative, 2010), and at 272 Mb, Brachypodium possesses one of the smallest grass genomes. The draft genome assembly is of unprecedented quality, and only 0.4% of the Brachypodium sequence reads were not contained in the final assembly. Furthermore, gaps in the Brachypodium genome assembly are predicted to amount to only 0.4% of the genome sequence. The high assembly quality is largely due to the comparatively low percentage of repetitive DNA in the genome (28%) and to the large number of bacterial artificial chromosome (BAC) end sequences used in the assembly (International Brachypodium Initiative, 2010). A recently initiated U.S. Department of Energy (DOE) Joint Genome Institute (JGI) project will close the remaining gaps and improve the sequence quality in currently ambiguous or unsequenced regions (J. Schmutz, personal communication). As a result of these efforts, Brachypodium is moving into the elite class of organisms with “finished” genome sequences. Led by the Mockler laboratory at Oregon State University, the draft genome was used to create a Brachypodium Affymetrix microarray. This array contains approximately 2.55 million expression probes covering all gene models (including exons and intron sequences) and approximately 3.95 million probes tiling intergenic sequences (described at http://arrays.brachypodium.org/). Individual exons and introns are represented by multiple probes (average, 11; median, seven), and 95% of exons or introns are targeted by at least five probes. Currently, users can order individual arrays from the Nottingham Arabidopsis Stock Centre (http://affymetrix.arabidopsis.info/) or directly from Affymetrix (part no. BradiAR1b520742; www.affymetrix.com) if large numbers are required. This array is being used to create an expression atlas for Brachypodium by the Mockler and S. Persson laboratories, the latter in collaboration with the Institut National de la Recherche Agronomique (INRA)-Versailles (T. Mockler and R. Sibout, unpublished data). This last effort is resulting in the construction of a whole-genome Brachypodium coexpression network (BrachyNet) similar to what has already been done in Arabidopsis (http://aranet.mpimp-golm.mpg.de/; Mutwil et al., 2010). BrachyNet will be useful to infer candidate genes associated with particular biological processes (Persson et al., 2005; Gu and Somerville, 2010) and incorporates expression data from a large number of different organs/tissues. The Mockler laboratory has focused on profiling gene expression in abiotic stress conditions, different light environments, and diurnal and circadian time courses. Over 100 of these data sets are available as genome viewer tracks in BrachyBase (Table II). Highly efficient Agrobacterium tumefaciens-mediated transformation methods have been developed for Brachypodium (Păcurar et al., 2008; Vain et al., 2008; Vogel and Hill, 2008). Transformation efficiencies (defined as the percentage of callus pieces cocultivated with Agrobacterium that produce a transgenic plant) now approach 50% in a production setting where hundreds of T-DNA insertion lines are generated every week (for up-to-date protocols, visit http://brachypodium.pw.usda.gov/ and http://www.brachytag.org/). Most laboratories can readily accomplish Brachypodium transformation with a modest investment in tissue culture training. At least two Brachypodium transformation services are available to the public (http://www.brachytag.org/ and http://www.agron.iastate.edu/ptf/index.aspx). With efficient Brachypodium transformation methods, it is now feasible to create large collections of sequence-indexed T-DNA mutants. Two groups have initiated large-scale projects to create T-DNA mutant collections. The BrachyTAG project at the John Innes Centre lists 5,000 T-DNA lines and has distributed mutants since 2008 (http://www.brachytag.org/; Thole et al., 2010). The U.S. Department of Agriculture Brachypodium Genome contains lines and has to create another lines In addition, the International Brachypodium was to the of T-DNA mutants by multiple laboratories, with the goal of T-DNA mutants such that is a high of an insertion in any particular laboratories from five and are currently creating T-DNA and these mutants will be into genome such as BrachyBase and (Table and is as a result of the effort of the approximately T-DNA lines will be made available to the community by A of this size has a of an insertion in any particular are another potential for creating insertional mutants. initial experiments with and systems that these are to Brachypodium unpublished data). However, other systems may for The extensive use of sequence-indexed T-DNA mutants by Arabidopsis an of the potential utility of Brachypodium T-DNA mutants. since the first of Arabidopsis T-DNA insertion lines in and T-DNA and individual lines have to the majority of the distributed to the plant community. to at the Arabidopsis has at about 80% for the The of sequence-indexed Arabidopsis lines the of this for the plant research community. use for transformation is for the of gene through or gene have been used with Brachypodium et al., and 2009; et al., 2010), and a T-DNA in Brachypodium has been with an Arabidopsis and models et al., 2011). In addition, for crop can be by and genes (e.g. cell in and have been in plant research. 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In comparison with what is available for Arabidopsis, the of resources for Brachypodium is more but it the tools and to use Brachypodium as a model system. 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The the that will sequence The the of the draft genome sequence to the community. of using Arabidopsis as a model system. The the of Arabidopsis as a model for The the of the small size of the Arabidopsis The the to use to study and processes in is a for the growth of a developing research community. The in using Brachypodium as a model system that Brachypodium is on a similar to Arabidopsis in the years The of Brachypodium as a model system can also be to and research Approximately million in has been for about 30 projects in the from the projects include targeted investments and as well as with more research but that also may have significant (e.g. small sequencing and analysis, of T-DNA mutants and cell wall and In addition, has a million to sequence In support for Brachypodium research has been provided by or by However, is an need to the U.S. effort on Brachypodium research at the to functional and genetic have the of this model system by developing Brachypodium research the investment by individual laboratories to Brachypodium as model system and its At the and the laboratories are using Brachypodium for including cell wall grain properties, and comparative genomic At the of laboratories have the Brachypodium groups include laboratories at the laboratories at the John Innes Centre and the Institute of and and groups at National and Oregon State In is developing mutants and as of their biomass In projects are under including a project at the Centre and a project to gene in wheat by using Brachypodium as a model for root architecture and traits at the and in This only a of overall the large number of projects and groups a of the growing of Brachypodium as a model system. Brachypodium has particularly useful for comparative because it is the first from the Pooideae subfamily of grasses to be This genomes from the important grass A key that from this comparison was the role of chromosome into regions as a for the of chromosome number through (International Brachypodium Initiative, 2010). While for such was et al., et al., the comparison of the Brachypodium and rice genomes provided the of the role such play in grass genome similar a comparison of the of genes in functional and regions was used to the of these regions in Brachypodium, rice, and wheat et al., 2010). research are to as now that a large of resources is available to the plant research community. since time the grasses and Arabidopsis, time can be in Brachypodium, a that be in rice, a or A comparison of time and genes in Brachypodium, rice, and Arabidopsis the for these genes in grasses with a et al., 2010). a transgenic Brachypodium was used to that a rye and its Arabidopsis could in a temperate grass et al., Furthermore, Brachypodium is particularly useful for root systems because grass root systems in and development from the root systems of rice, maize, and wheat, which are large to study under controlled conditions, Brachypodium can be readily in this (Watt et al., 2009). a model for temperate grass be and Brachypodium has recently been to be to a major wheat with being the as in wheat et al., 2011). research has also that Brachypodium for to the of and mutant have been in and lines unpublished data). provide a into the utility of Brachypodium for in plant and in emerging from Brachypodium research will The in developing Brachypodium as a model system have to the of substantial biological and investments in a are to this and that the information and biological available for the long The a role in the development of Arabidopsis as a model system. efficiently and and DNA resources, the to that biological resources developed by the Arabidopsis community are and are readily The of a available be and has been the of the success of Arabidopsis as a model system. The Brachypodium community is rapidly the where it will be feasible to on individual laboratories to all of their biological (e.g. of of T-DNA and many of DNA the has to from and T-DNA lines personal communication). 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The are available in the of this Brachypodium We for data on Brachypodium We to all were not due to space