Update on the genomics and basic biology of Brachypodium International Brachypodium Initiative (IBI)

The scientiﬁc presentations at the First International Brachypodium Conference (abstracts

The scientific presentations at the First International Brachypodium Conference (abstracts available at http:// www.brachy2013.unimore.it) are evidence of the widespread adoption of Brachypodium distachyon as a model system. Furthermore, the wide range of topics presented (genome evolution, roots, abiotic and biotic stress, comparative genomics, natural diversity, and cell walls) demonstrates that the Brachypodium research community has achieved a critical mass of tools andhas transitioned from resource development to addressing biological questions, particularly those unique to grasses.
A model for grass genome organization This report highlights recent advances made in Brachypodium research, focusing on the use of B. distachyon and related species to understand biological processes. Its experimental and genomic tractability allow B. distachyon to act as a functional genomic test-bed to accelerate the improvement of grain, forage, and biomass crops. Its strengths as a model plant (e.g., short generation time, efficient Agrobacterium-mediated transformation, and availability of mutant collections) are described in [1] and other reviews published in the past 5 years. The main web portal for Brachypodium (http://www.brachypodium.org) contains a genome browser and links to community resources. In addition, two project-specific websites (http://brachypodium. pw.usda.gov/ and http://www-urgv.versailles.inra.fr/tilling/ brachypodium.htm) provide access to T-DNA and Tilling resources, respectively.
The compact nature of the fully sequenced [2] Brachypodium distachyon genome is a major reason for the success of B. distachyon as a model system, and provides unique opportunities to study various aspects of grass genome organization and evolution. Moreover, as a monocot reference, it permits comparisons of genomic landscape dynamics with the dicot model Arabidopsis thaliana. Thus, B. distachyon has become an appealing target for plant molecular cytogenetics.
One of the most informative cytomolecular tools is chromosome painting (CP), which enables unique and unambiguous visualization of individual chromosomes or large segments, both during cell division and even at interphase, using fluorescence in situ hybridization with specific DNA probes. CP was initially applied to vertebrate systems. Whole-genome sequencing (WGS) and largeinsert genomic DNA libraries allow its application to small-genome plants such as A. thaliana. The sequencing of the B. distachyon genome [2] combined with its low (5) chromosome number and a well-developed cytogenetic infrastructure has allowed the CP of several Brachypodium species [3], a pioneering application of CP in monocots.
The chromosomes of B. distachyon can be selectively painted to address important questions about grass genome structure and evolution (in Figure 1 we demonstrate how they are arranged at interphase). CP of B. distachyon chromosome 2 (Bd2) in the nuclei of root cells revealed that Bd2 homologous chromosome territories can assume four different configurations that are observed at different frequencies ( Figure 1A). This is one example where research in Brachypodium could lead the wayin determining whether and how nuclear structure is linked to cell differentiation and tissuespecific gene expression.

A tractable model for inter-and intraspecific diversity
The genus Brachypodium contains 15-18 species with unusually variable chromosome numbers and ploidy levels. This diversity was a subject of interest long before B. distachyon became a model grass. WGS of B. distachyon [2], together with the advent of inexpensive nextgeneration sequencing (NGS) technologies, set the stage for high-resolution investigation of the genomic diversity and evolutionary relationships in the genus.
A model for abiotic stress B. distachyon, B. stacei, and B. hybridum all grow in a wide range of habitats under marked environmental gradients. Distinct genotypes are thus subject to different abiotic stresses which might have exerted, from speciation until present, different selective pressures on stress tolerancerelated traits.
Detection of adaptive variation of stress-tolerance traits in response to abiotic conditions requires the following: (i) significant genetic variation in the trait of interest, (ii) a match between adaptive genetic variation and environmental variation (e.g., local adaptation across the gradient), and (iii) positive selection for these traits in genotypes growing under abiotic stress. Progress has been made in screening for natural variation in stress tolerance among B. distachyon accessions (reviewed in [1]), the first step towards determining the heritability of adaptive traits. However, full understanding of the adaptive significance of tolerance trait variation awaits experimental evaluation of the effects of such variation on fitness in natural populations.
Progress toward understanding abiotic stress adaptation at a molecular level is being made. Promising results come from the recent characterization of a microRNA (miRNA) network controlling cell division during stress, part of a search for epigenomic regulatory mechanisms underlying drought stress [5]. Further, evidence of ancient adaptive evolution of temperate Pooideae species was inferred from nucleotide substitution rates and signatures of positive selection in genes induced by low temperature [6]. Finally, adapted genotypes of B. distachyon and B. hybridum may reveal how genomic changes such as whole-genome duplication influence ecological tolerances to abiotic stress. In fact,differential tolerance to water stress between B. distachyon and B. hybridum seems to drive the ecogeographical differentiation of these species [7].
A model pathosystem for multiple cereal diseases Pests and pathogens are major contributors to global food insecurity. Shifting climate patterns are altering disease ranges and facilitating the emergence of virulent strains.
Thus, we need a better understanding of plant-pathogen interactions to develop rapidly new and preferably durable sources of disease resistance. B. distachyon has emerged as a powerful tool to elucidate defense responses in the Poaceae. A major advance has been the demonstration that B. distachyon serves as a host for many pathogens that cause diseases such as rice blast, Fusarium head blight (FHB; Figure 1C), and barley stripe mosaic virus (BSMV) (reviewed by [4]). Studies are now exploiting the genetic and functional genomic resources available for B. distachyon to elucidate host responses to pathogens. An elegant example is the characterization of B. distachyon UDP-glycosyltransferases that can detoxify the mycotoxin deoxynivalenol produced by Fusarium graminearum, the casual pathogen of FHB [8]. As such studies progress they may identify commonalties in host responses to pathogens which could represent key defense nodes that are potential sources of durable resistance to many pathogens. Translation of this knowledge into improved crop varieties will involve identifying orthologous genes or linked molecular markers in crop germplasm. Such a strategy contributed to the targeting of Pch1 eyespot resistance in wheat (Triticum aestivum) [9]. However, the absence of an ortholog of the wheat Lr34 leaf rust resistance gene in B. distachyon [10] indicates that successful transfer of information between B. distachyon and grass crops is not guaranteed. Another powerful means of increasing crop resistance is the direct transfer of genes from B. distachyon into elite cereal germplasm through transformation. The success of this approach is enhanced by the close relationship between B. distachyon and the cereals.

A model for the grass cell wall and biomass accumulation
Similarly to other grasses, B. distachyon has a type II wall that differs markedly from the type I walls found in dicots. Until now, few studies have focused on characterizing B. distachyon cell wall polysaccharides and their biosynthetic enzymes. Detailed biochemical characterization of these polysaccharides, their distribution in different tissues and organs, and their roles during development need to be investigated. Characterization of mixed linkage glucans (a polymer unique to grasses) in B. distachyon seeds showed surprising enrichment in (1!3) linkages and that arabinoxylans were more substituted compared to wheat, barley (Hordeum vulgare), or oat (Avena sativa).
In comparison with polysaccharides, B. distachyon lignin has been studied in greater detail by genetic and biochemical characterization of some of the enzymes required for lignin biosynthesis. The enzymes are encoded by gene families, but each gene has a distinct function, and are thus good targets for mutagenesis or introgression [11]. As an elegant example of the potential of the model grass, mutation of the cinnamyl alcohol dehydrogenase (CAD1) gene has been shown to lead to a 25% decrease in lignin content, resulting in improved saccharification [12]. These results, taken together with the possibility to increase biomass by modifying polysaccharide-related metabolism, demonstrate that B. distachyon is an excellent modelforidentifying genes important for developing biomass crops with improved conversion into bioenergy or new materials (Box 1) Taken together, these advances demonstrate the wide applicability of Brachypodium as a model system and underscore the maturity of the system. Box 1. Brachypodium distachyon root and rhizosphere for underground discoveries Plant roots provide a multitude of essential functions like mechanical support, water and nutrient uptake, defense against soil pathogens and toxins. Compared to shoots, roots have been understudied, and offer important opportunities to increase global food production, and save water, land, and fossil fuels. Most molecular root research has been conducted on Arabidopsis, but its dicotyledonous root system has a different morphology, architecture, anatomy, and biochemistry from cereals such as wheat or barley. Thus, B. distachyon now has many of the tools available for A. thaliana but a root system similar to that of temperate cereals ( Figure I).
B. distachyon root system is composed of three root types ( Figure I). A single primary seminal root (PSR) emerges at germination; this is followed approximately 2 weeks later by one or two coleoptile nodal roots (CNR) from the coleoptile node located on the mesocotyl, about half way between the seed and the leaf nodes, and, finally, 3-4 weeks after germination, leaf nodal roots (LNR) start to emerge from the leaf nodes [13]. In B. distachyon the number of nodal roots, but not the seminal primary root, varies genetically and in response to water, opening the possibility of selecting root systems for specific soil conditions (V.C., unpublished). B. distachyon root variation can be studied in much smaller volumes of soil than maize or rice, permitting the characterization of the role of mature root systems during flowering and seed development [13]. Flowering and seed development are highly susceptible to drought, and knowledge of root genes at these stages can be applied to crop improvement through marker-assisted breeding.
Since the emergence of B. distachyon as a molecular model it has been applied in several fields relevant to roots, including root system architecture of cereals, response to biotic (pathogens such as Fusarium, Rhizoctonia) and abiotic (nutrient levels, drought) stresses, auxin homeostasis [14], and symbiotic root-microbe interactions such as arbuscular mycorrhizal fungi [15]. B. distachyon is an exciting new model for root research, opening the way to understanding monocotyledon root biology, and eventually leading to the improvement of major temperate crops.