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Brachypodium distachyon: making hay with a wild grass
Review
Brachypodium distachyon: making hay with a wild grass Magdalena Opanowicz1, Philippe Vain1, John Draper2, David Parker2 and John H. Doonan1 1 2
John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK Institute of Biological Sciences, Aberystwyth University, Aberystwyth, SY23 3DA, UK
Brachypodium distachyon is a wild grass with a short life cycle. Although it is related to small grain cereals such as wheat, its genome is only a fraction of the size. A draft genome sequence is currently available, and molecular and genetic tools have been developed for transformation, mutagenesis and gene mapping. Accessions collected from across its ancestral range show a surprising degree of phenotypic variation in many traits, including those implicated in domestication of the cereals. Thus, given its rapid cycling time and ease of cultivation, Brachypodium will be a useful model for investigating problems in grass biology. Introduction: a new model for temperate grasses A small, fast-growing species of grass, Brachypodium distachyon, has recently emerged as an attractive experimental model for the study of small-grain temperate cereals and related grasses (Figure 1) [1]. As a group, the temperate grasses underpin much of agriculture. Their seeds are used directly to make human foods such as flour, starch, sugar, syrup, oils, and malt for the production of alcoholic beverages, and are used indirectly as animal feed. Crop residues (e.g. straw and processing waste) have been used as a minor energy source [2], but recently there has been an upsurge of interest in using these residues and developing new crops for ‘biofuel’ production [3]. The agronomically important grasses tend to be physically large, and to have relatively long life cycles and large, complex genomes, characteristics that are inconvenient and expensive for research purposes. Brachypodium, however, possesses many of the characteristics required of a tractable experimental model – short life cycle, small plant size, resilient, and easy to cultivate. Moreover, it has one of the smallest genomes of any grass, a factor that has led to it being chosen as a candidate for genome sequencing. A genome sequencing project is already well underway, with a draft sequence released in 2007 and a more complete annotated sequence expected in 2008 (International Brachypodium Initiative, http://www.brachypodium.org/node/8). Other resources are rapidly being developed and made available to the community (Table 1). Although the track record of Brachypodium as a model genetic system is currently limited, genetic variation can be induced easily by classical or transgenic mutagenesis, and there is already a tremendous resource in terms of natural variation. When the Corresponding author: Doonan, J.H. ([email protected]).
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genomic tools are in place, it will be possible to unlock these sources of variation and to address a wide range of important issues in grass biology (Table 2). In this review, we consider some of the attractions of Brachypodium as an emerging model system for the temperate grasses, and we highlight the surprising degree of variation for agronomic traits found in natural accessions. Geographical range and phylogenetic position of Brachypodium distachyon The natural range of B. distachyon spans the Middle East (Figure 2), largely overlapping the ancestral range of cultivated small grain cereals [4]. Diploid B. distachyon is an annual and has a basal chromosome number of n = 5 [1,5], with a c-value originally estimated as 0.21 pg/haploid genome [1]. However, the currently accepted value is 0.36 pg/haploid genome [6–8]. This is a relatively small genome for a grass and a highly desirable feature for any model species. Other species in the genus have also been used for molecular studies, particularly Brachypodium sylvaticum, but these tend to be perennial and their genomes vary in chromosome number and DNA content. It seems likely that efforts in the immediate future will focus on B. distachyon. As with wheat, different natural accessions of B. distachyon have variable genome size (Figure 2), owing to recent hybridisation events. Some of the variant distachyon cytotypes resemble that of a closely related species (2n = 20). Genomic in situ hybridisation revealed that other variant cytotypes are actually hybrids (2n = 30) between this species and B. distachyon [5]. In general, the small grain cereals tend to have large and variable polyploid genomes derived from relatively recent interspecies hybridisation and genome doubling events. This suggests that these species have an innate ability to tolerate dramatic changes in ploidy, and this ability might in turn confer selective advantage and facilitate speciation [9]. Consistent with this idea, the 2n = 30 accessions of Brachypodium tend to be physically larger, generally lack vernalisation requirements, and might have an ability to tolerate a broader range of environmental conditions than their diploid progenitors, as observed in other polyploid plants [10–12]. Genome sequencing projects have revealed that the Brachypodium genus is more closely related to wheat, barley and forage grasses than it is to rice. For example, comparison of a 370 kb region from the B. sylvaticum
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Figure 1. Phylogenetic relationships between Brachypodium and the small grain cereals. Grasses are a large, morphologically diverse and successful family of plants comprising 10 000 species. Classically, grasses are characterized by a single-seeded starch-rich fruit called a caryopsis or grain (examples are all shown at same scale, bar = 5 mm). Furthermore, they all have a spikelet style of floral structure (groups can be distinguished by the number and pattern of these spikelets in the inflorescence [58]). The Pooideae subfamily includes forage grasses, Brachypodium and many economically important small grain cereals adapted to cool or dry climates, such as wheat (Triticum aestivum), rye (Secale cereale), barley (Hordeum vulgare) and oats (Avena sativa). Rice (Oryza sativa) and maize (Zea mays) belong to distinct subfamilies [59] and have many adaptations for tropical climates.
genome with an orthologous region from rice [13] supported the idea that Brachypodium diverged from the Pooideae approximately 35–40 Mya [14]. The full genome sequence of B. distachyon should provide additional insight into the evolution of genome structure with the grasses. Natural variation for disease resistance One of the original attractions of Brachypodium was, and remains, its capacity to help us understand plant– pathogen interactions in wild plant populations. As cereals became domesticated, their pathogens co-evolved to exploit the new plant populations. Combating plant disease is a major focus for many cereal breeding programmes [15] and the agrochemical industry [16]. The detrimental effects of
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disease on yield, and the desire to reduce chemical inputs are such that plant breeders wage a never-ending battle to breed new varieties with resistance to the current prevalent strains of pathogens. Different Brachypodium ecotypes varied in their responses to a range of fungal plant-pathogens that cause significant crop loss [1]. Although all tested ecotypes reacted similarly to Blumeria graminis, there was greater variability in the response to Puccinia and Magnaporthe grisea infection, suggesting that Brachypodium will be a useful model to study these important plant-pathogens. One out of seven ecotypes was resistant to M. grisea, an economically important fungal pathogen that is the casual agent of rice blast and which, in addition, infects temperate and forage grasses [17,18]. Different accessions show variable levels of resistance and susceptibility to the different host-limited forms (rice- and grass-adapted) of M. grisea [19]. One ecotype of B. distachyon was resistant to both of the M. grisea strains, whereas another was susceptible specifically to the grass-adapted strain. Genetic crosses between these ecotypes suggested that a single dominant resistance gene conferred resistance to the rice-adapted form of M. grisea. A genome sequence is already available for M. grisea [20,21]; the Brachypodium genome sequence should aid in the identification of factors (e.g. defence proteins [22] and metabolites [23]) that control the balance of resistance and susceptibility alleles in wild populations. In doing so, this will provide insight into how plant populations co-evolve with their natural diseases. Standing variation for domestication traits Domestication can lead to dramatic changes in plant form as humans select for particular traits and produce cultivars more suited to their purposes (reviewed by [4]). The origin of such traits has been widely debated, and the relative importance of pre-existing (or standing) variation in the wild progenitors is unclear. As a wild grass not under direct human selection, Brachypodium might provide some insight into this process. Recently, investigators surveyed available accessions from the United States Department of
Table 1. Public resources available for Brachypodium research Resource International Brachypodium Initiative The Brachypodium Newsgroup Brachypodium BrachyBase Prototype Genome Viewer USDA-ARS Germplasm Search Genomics and Gene Discovery bEST Resource Brachypodium genome browser Brachypodium T-DNA tagging (BrachyTAG)
Website http://www.brachypodium.org/ http://mailman.cgrb.oregonstate.edu/mailman/listinfo/brachy-info http://www.aber.ac.uk/plantpathol/brachyomics.htm http://www.brachybase.org/ http://www.ars-grin.gov/ http://brachypodium.pw.usda.gov/ http://www.modelcrop.org/ http://www.jic.ac.uk/staff/philippe-vain/brachypodium.htm
Table 2. Comparison of Brachypodium with other cereals and Arabidopsis Arabidopsis thaliana
Triticum aestivum
Zea mays
Oryza sativa
Hordeum vulgare
Number of chromosomes Genome size (1C) Reproductive strategy Life cycle (weeks)
Brachypodium distachyon 10 (2n) 300 Mb Self-fertilizing 10–18
10 (2n) 164 Mb Self-fertilizing 10–11
20 (2n) 2400 Mb Cross-pollination 10+
24 (2n) 441 Mb Self-fertilizing 20–30
14 (2n) 5000 Mb Self-fertilizing 16+
Height at maturity (m) Transformation Growth requirements
0.3 Facile Very simple
0.2 Facile Very simple
42 (2n) 16 700 Mb Self-fertilizing 12 (spring wheat) 40+ (winter wheat) Up to 1 Possible Simple
Up to 2 Facile Simple
1.2 Facile Specialized
Up to 1.2 Facile Simple 173
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and J.H. Doonan; unpublished). Analysis of spikelet and grain morphology (Figure 3) illustrates the degree of variation available in what is, to date, a relatively small number of accessions. Generation time, partly as a result of variable vernalisation requirements, is also variable in diploid accessions (http://www.brachypodium.org/node/8). A key feature of domesticated grains is the retention of the ripe grain within the dry inflorescence, but in such a way that it can be easily removed post-harvest by threshing. The manner in which the grain breaks free (or disarticulates) from the spike varies owing to preferential breakage of different tissues in different species of grass [24]. However, the timing and extent of disarticulation is crucial for domestication; when disarticulation occurs too early, harvesting becomes inefficient and therefore not cost-effective; when disarticulation is too difficult, grains are not released easily and cleanly, leading to a reduction in the net energy value of the crop. Thus, the development and function of the abscission zone is a focus of interest in cereals. The sh4 gene plays an important role in establishment of the abscission layer at an early stage of flower development [25]. It is thought that the development of a tough rachis was one of crucial changes that took place during the domestication of wheat [26]. Variation in the extent of disarticulation is clearly present in Brachypodium (Figure 3) and must have evolved for reasons quite distinct from those of human convenience.
Figure 2. Geographic origins of Brachypodium, and selected adaptive traits. The genus Brachypodium is currently distributed throughout most temperate regions of the world, but its ancestral range lies in the Middle and Near East. Accessions have been collected from various regions of both the ancestral range and the New World. Different accessions vary for several traits, including generation time (in the range of 12 to >18 weeks under greenhouse conditions), flowering time, plant size and ploidy level. Some traits such as metabolic composition (d) and disease resistance (e) are likely to be adaptive and related to the geographic origins of the species in question. (a) The geographic location of different accessions from the old world. Presumptive diploids are indicated by red circles, whereas accessions with higher ploidy levels are shown in blue. Green circles represent possible interspecific hybrids. (b) Ploidy has a dramatic effect on plant size. Flow cytometry confirmed that Bd21 and JIB05, two accessions of differing sizes, were diploid and allopolyploid, respectively (data not shown). (c) Higher ploidy accessions (such as ABR113) might be derived from interspecific crosses with a closely related species (ABR114; 2n = 20) [5]. These images show in situ hybridisation using probes made from the genomes of ABR1 (purple) or ABR114 (blue–green) and hybridised against spread chromosome preparations of the accessions as shown (bar = 5 mm). The chromosome spread of accession ABR113 contains chromosomes of both types, indicating that it is an allopolyploid. The cytotypes for many of the higher ploidy accessions have yet to be characterised. (d) Metabolite composition of six diploid (2n = 10) accessions of B. distachyon, as assessed by linear discriminant analysis of metabolite fingerprints using flow injection electrospray mass spectrometry. The two major discriminants are plotted. The different accessions cluster according to accession and to geographic region of origin. (e) Differential responses to rice blast infection [19] in two diploid (2n = 10) accessions of B. distachyon from Turkey (ABR1, chlorotic leaves with large black lesions indicate susceptibility) and Spain (ABR5, the small black flecks indicate resistance).
Agriculture (USDA) and Aberystwyth University (Wales, UK), and recently collected accessions from Turkey. The survey indicated the presence of extensive variation for numerous traits of agronomic importance (M. Opanowicz 174
Applications to bioenergy Diverse grass species are being evaluated as biofuel crops because they grow rapidly and, in theory, can be grown on land that is too marginal for other agricultural purposes. As with wheat, candidate biofuel species tend to have large genomes, and attempts to identify relevant quantitative trait loci (QTL) will benefit from knowledge gained in model species with smaller and syntenic genomes. Brachypodium has the cellular and subcellular makeup of a typical grass and will be a useful additional tool in the effort to improve these species for biomass and biofuel production. For example, tillering plays a significant role in biomass production of the forage grasses, and the number and quality of tillers can be affected by many environmental factors, including grazing, temperature, level of soil nutrients, day length, light intensity and plant density [27–30]. In Brachypodium accessions, the extent of tillering is related in part to flowering time and varies from a low of 6–7 tillers per plant in an early-flowering accession (USDA-ARS number PtdIns 255334) to 24 in a late-flowering accession (USDA-ARS number PtdIns 250647). The extent of variability in tillering and its genetic control in Brachypodium could benefit efforts to breed bioenergy crops. Cell wall composition has direct implications for biofuel feedstock. As the bulk of the harvested material, cell walls represent an important feedstock for bioenergy; however, grasses have distinctive cell wall structure and composition [31] that differs from the better studied cell walls found in dicotyledonous species. Although the cellulose microfibril structure is similar in both types of cell wall, the cross-linking matrix in the Poales is pectin-poor and rich in phenylpropaniods and silica, which can have
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implications for processing and use of the product. Genetic analysis of cell wall composition and deposition in Arabidopsis has provided a wealth of information on the dicotyledonous cell wall [32], and similar approaches will be possible in Brachypodium. The comparison between the two model species will be very interesting, both academically and for crop improvement. Metabolite fingerprinting indicated that there were significant differences in the spectrum of metabolites between different accessions of Brachypodium. These differences were associated with variation in the geographic origins of different Brachypodium species (Figure 2e) and might reflect adaptations to local conditions. Core metabolomic resources specific for B. distachyon and other Poaceae are being developed [23,33], including spectral libraries for gas chromatography mass spectrometry (GC-MS) analyses and signal annotation tools for liquid chromatography mass spectrometry (LCMS) data (http://www.armec.org/).
Figure 3. Brachypodium inflorescence architecture and morphological variation. (a) Inflorescence architecture of wheat (left), Brachypodium (centre) and rice (right). Inflorescence architecture in cereals depends on the branching pattern of the inflorescence meristem and varies from a single determinate spike in wheat to an indeterminate unbranched spike in barley, to more complex branched systems such as those of rice, in which there is a multiplicity of distinct meristems (for review, see [60]). (b) Morphological variation in inflorescence morphology between Brachypodium accessions. In the left panel, a newly collected Turkish accession with reduced inter-spikelet spacing. The middle panel shows the primary spike from accession JIB02 (John Innes Brachypodium 2) and right panel illustrates three different spikes from the same Bd21 plant where the number of spikelets per inflorescence varies between one and more than seven. In all accessions, the primary shoot tends to have the most complex inflorescence (leftmost spike in right panel), whereas subsequent shoots have smaller and less branched inflorescences. Several genes involved in controlling rice inflorescence architecture have been cloned [61], and synteny has enabled the identification of equivalent genes in maize [60]. A comparison between Brachypodium and other grasses using these genes as candidates might help to explain some of the differences in their inflorescence architectures [62]. (c) Level of lemma hairiness in two distinct accessions, JIB05 (top panels) and JIB01 (bottom panel). The Brachypodium caryopsis has a typical morphology for the Poaceae. It is surprisingly large, an average of 8 mm long by 2 mm wide. The caryopsis in Brachypodium is surrounded with two unequal-sized husks, a smaller palea and a larger lemma that terminates in a long awn. When examined under scanning electron microscopy (SEM) (left panels in (c) and (d)), the lemma and awn showed varied levels of hairiness, which probably reflects adaptation to local habitats. For example, hairs on plants growing in windy locations can break-up the flow of air across the plant surface, reducing evaporation. Alternatively dense coatings of hairs reflect solar radiation, protecting the more delicate tissues underneath in hot, dry, open habitats [63]. Hairs can also modify the function of the awns, which have been implicated in grain dispersal [64]. (d) Shatter zones in Brachypodium
Brachypodium – a stepping stone to the giants The small Brachypodium genome has already been invaluable as an aid for cloning wheat and barley genes of agronomic importance. Early studies had indicated that gene order (or synteny) is largely conserved between Brachypodium and the small grain cereals [34–36], and this synteny was instrumental in the characterization of the wheat gene Ph1 [37] and the barley gene Ppd-H1 [38]. Ph1 is particularly interesting in the context of genome evolution. This complex locus is required for correct pairing of homologous chromosomes in hexaploid and tetraploid wheats, but the absence of allelic variation at the Ph1 locus precluded conventional mapping. Markers (based on synteny between the wheat genome and the smaller genomes of rice and B. sylvaticum) were used to map deletion lines and to pinpoint functional aspects of the Ph1 locus [36,37]. Sequence analysis of the region revealed a complex of repeated versions of a novel cyclin-dependent protein kinase, the functional implications of which are currently being investigated. Molecular genetics and genomics in Brachypodium To develop B. distachyon as a link between rice and temperate grasses, Hasterok et al. [39] constructed a bacterial artificial chromosome (BAC) library of 9100 clones, representing more than two genome equivalents. In situ hybridization [40] between defined BACs and the 10 chromosomes of B. distachyon revealed strong synteny between B. distachyon and Poaceae species, with most of the BACs hybridizing at single loci on defined chromosomes. BAC libraries with three times coverage have been employed to construct physical maps for the Brachypodium genome [41,42]. The US Department of Energy Joint accessions (as described in (c)). Grain dispersal mechanisms vary tremendously among grass species. The dispersal module can vary from naked isolated grains to intact spikes in different species, and various selective advantages have been proposed for each different strategy [58]. Surprisingly, 8 out of 13 Brachypodium accessions examined retain their grain within the spike after ripening, and SEM revealed that the structure of the abscission zone reflects these differences. Therefore, the grain dispersal module varies in different accessions and indicates that a considerable degree of variation for this important agricultural trait already exists in a wild population of grass with no known history of cultivation.
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Review Genome Institute (JGI) has released a Brachypodium whole genome shotgun sequence that covers the genome to an approximate 4x depth (http://www.Brachypodium.org/). JGI is continuing sequencing to achieve 8x coverage, and a draft genome sequence of the community standard inbred line Bd21 (2n = 10) is expected in 2008 (International Brachypodium Initiative http://www.brachypodium.org/node/8). In addition, expressed-sequence tag (EST) libraries have been sequenced [8], and an additional 180 000 ESTs are expected as part of the genome sequencing project. A complete genome sequence will enable us to define both induced and natural variation, and will facilitate transcriptomics and association mapping. Association mapping might become a powerful tool for identifying alleles and loci responsible for natural variation in Brachypodium. With the use of chemical, radiation or T-DNA tagging approaches, genetic resources are also being developed for B. distachyon, with the aim of conducting forward and reverse genetic screens. Genetic transformation technologies are crucial for the development of these resources and for the validation of gene function. Biolistic- [1,43] and Agrobacterium-mediated transformation [7,44–46] systems have been developed for a wide range of Brachypodium genotypes. Cytotypes with 30 chromosomes are generally more amenable to genetic engineering than diploid lines (2n = 10). BDR018 was the first diploid line to be transformed by particle gun bombardment, with 5% efficiency [43]. Bombardment transformation was used to compare the effects of flowering control gene Terminal flower 1 (TFL1) from different species in Brachypodium [47]. Past studies in Arabidopsis showed that expression of TFL1 delays flowering and prevents expression of the floral meristem identity genes LEAFY and APETALA1. In monocotyledons, however, the orthologue LpTFL1 (Lolium perennae TFL1) seems to completely prevent flowering [48]. Overexpression of LpTLF1 or TLF1 in transgenic Brachypodium plants resulted in a significant delay in flowering compared with that in controls [47]. Large-scale transformation efforts now use Agrobacterium-based approaches. The community standard strain, Bd21, has been transformed using Agrobacterium, with 17% efficiency [44]. Another line, Bd21–3, which is genetically distinct from Bd21 but which was also produced by recurrent self-pollination of the same USDA accession (PtdIns 254867), produces transformation efficiencies of up to 36% [7]. Line BDR018 [45] shows 55% efficiency, indicating a high degree of diversity for this ‘trait’. Bd21 TDNA insertion lines have been produced, and the flanking regions are currently being defined. Flanking-sequence tags (FSTs) of the T-DNA inserts and the seeds from the first 2000 insertion lines are available for dissemination to the science community [44]. T-DNA mutants have been an essential factor in the success of more established model systems such as Arabidopsis thaliana [49] and rice [50,51]. Extensive populations potentially enable researchers to obtain insertional activation or inactivation of any gene of interest, so the production of large characterised populations containing defined insertions will be one of the crucial next steps in establishing Brachypodium as an attractive experimental system. 176
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Perspectives Brachypodium is, therefore, an attractive model system for the elucidation of grass biology. It exhibits extensive biodiversity and holds great potential for genetic analyses and engineering. Furthermore, the Brachypodium genome will soon be fully annotated, and this powerful combination of factors promises to facilitate the dissection of many traits of agronomic and ecological importance. Many of these traits will probably be multigenic in makeup and therefore best analysed as quantitative characteristics. Previously, such traits were difficult to study, but the combination of genetic and genomic resources available for plants such as Arabidopsis, rice or maize now make this feasible (e.g. [52– 57]), and Brachypodium should make a valuable contribution towards this goal. Its comparative ease of cultivation, small size and rapid life cycle are likely to recommend Brachypodium as a model system for cereal and grass biology in the developed and developing world. Acknowledgements M.O. was supported by a Short Term Fellowship from the Human Frontier Science Program and Marie Curie Intra-European Individual Fellowship; PV, DP and JHD were supported by the Biotechnology and Biological Sciences Research Council. The chromosome images in Figure 2c were kindly supplied by Robert Hasterok (University of Silesia, Poland) and Glyn Jenkins (Aberystwyth University, UK). Photography was undertaken by Andrew Davies (John Innes Centre photography unit). We thank Mary Byrne, Simon Griffith, John Snape and Sinead Drea for critical reading of the manuscript, and the reviewers for helpful comments.
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39 Hasterok, R. et al. (2006) Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization. Genetics 173, 349–362 40 Jenkins, G. and Hasterok, R. (2007) BAC ‘landing’ on chromosomes of Brachypodium distachyon for comparative genome alignment. Nat. Protoc. 2, 88–98 41 Huo, N. et al. (2006) Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics. Genome 49, 1099–1108 42 Huo, N. et al. (2007) The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences. Funct. Integr. Genomics 43 Christiansen, P. et al. (2005) A rapid and efficient transformation protocol for the grass Brachypodium distachyon. Plant Cell Rep. 23, 751–758 44 Vain, P. et al. (2007) Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis. Plant Biotechnol. J. (Epub ahead of print) 45 Pa˜curar, D.I. et al. (2007) A high-throughput Agrobacterium-mediated transformation system for the grass model species, Brachypodium distachyon L. Transgenic Res. (Epub ahead of print) 46 Vogel, J.P. et al. (2006) Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss. Org. Cult. 84, 199–211 47 Olsen, P. et al. (2006) Analysis of two heterologous flowering genes in Brachypodium distachyon demonstrates its potential as a grass model plant. Plant Sci. 170, 1020–1025 48 Ratcliffe, O.J. et al. (1998) A common mechanism controls the life cycle and architecture of plants. Development 125, 1609–1615 49 Alonso, J.M. et al. (2003) Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 100, 2992–2997 50 Jeon, J.S. et al. (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22, 561–570 51 Sallaud, C. et al. (2004) High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. Plant J. 39, 450– 464 52 Alonso-Blanco, C. and Koornneef, M. (2000) Naturally occurring variation in Arabidopsis: an underexploited resource for plant genetics. Trends Plant Sci. 5, 22–29 53 Borevitz, J.O. and Nordborg, M. (2003) The impact of genomics on the study of natural variation in Arabidopsis. Plant Physiol. 132, 718–725 54 Jia, Y. et al. (2003) Natural variation at the Pi-ta rice blast resistance locus. Phytopathology 93, 1452–1459 55 Koornneef, M. et al. (2004) Naturally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172 56 Maloof, J.N. (2003) Genomic approaches to analyzing natural variation in Arabidopsis thaliana. Curr. Opin. Genet. Dev. 13, 576–582 57 Upadyayula, N. et al. (2006) Genetic and QTL analysis of maize tassel and ear inflorescence architecture. Theor. Appl. Genet. 112, 592–606 58 Soderstrom, T.R. et al. (1987) Grass systematics and evolution, Smithsonian Press 59 Salamin, N. et al. (2002) Building supertrees: an empirical assessment using the grass family (Poaceae). Syst. Biol. 51, 136–150 60 Kellogg, E.A. (2007) Floral displays: genetic control of grass inflorescences. Curr. Opin. Plant Biol. 10, 26–31 61 Malcomber, S.T. et al. (2006) Developmental gene evolution and the origin of grass inflorescence diversity. Adv. Bot. Res. 44, 423–479 62 Bommert, P. et al. (2005) Genetics and evolution of inflorescence and flower development in grasses. Plant Cell Physiol. 46, 69–78 63 Valkama, E. et al. (2004) Changes in leaf trichomes and epicuticular flavonoids during leaf development in three birch taxa. Ann. Bot. (Lond.) 94, 233–242 64 Elbaum, R. et al. (2007) The role of wheat awns in the seed dispersal unit. Science 316, 884–886
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Brachypodium distachyon: making hay with a wild grass Magdalena Opanowicz1, Philippe Vain1, John Draper2, David Parker2 and John H. Doonan1 1 2
John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK Institute of Biological Sciences, Aberystwyth University, Aberystwyth, SY23 3DA, UK
Brachypodium distachyon is a wild grass with a short life cycle. Although it is related to small grain cereals such as wheat, its genome is only a fraction of the size. A draft genome sequence is currently available, and molecular and genetic tools have been developed for transformation, mutagenesis and gene mapping. Accessions collected from across its ancestral range show a surprising degree of phenotypic variation in many traits, including those implicated in domestication of the cereals. Thus, given its rapid cycling time and ease of cultivation, Brachypodium will be a useful model for investigating problems in grass biology. Introduction: a new model for temperate grasses A small, fast-growing species of grass, Brachypodium distachyon, has recently emerged as an attractive experimental model for the study of small-grain temperate cereals and related grasses (Figure 1) [1]. As a group, the temperate grasses underpin much of agriculture. Their seeds are used directly to make human foods such as flour, starch, sugar, syrup, oils, and malt for the production of alcoholic beverages, and are used indirectly as animal feed. Crop residues (e.g. straw and processing waste) have been used as a minor energy source [2], but recently there has been an upsurge of interest in using these residues and developing new crops for ‘biofuel’ production [3]. The agronomically important grasses tend to be physically large, and to have relatively long life cycles and large, complex genomes, characteristics that are inconvenient and expensive for research purposes. Brachypodium, however, possesses many of the characteristics required of a tractable experimental model – short life cycle, small plant size, resilient, and easy to cultivate. Moreover, it has one of the smallest genomes of any grass, a factor that has led to it being chosen as a candidate for genome sequencing. A genome sequencing project is already well underway, with a draft sequence released in 2007 and a more complete annotated sequence expected in 2008 (International Brachypodium Initiative, http://www.brachypodium.org/node/8). Other resources are rapidly being developed and made available to the community (Table 1). Although the track record of Brachypodium as a model genetic system is currently limited, genetic variation can be induced easily by classical or transgenic mutagenesis, and there is already a tremendous resource in terms of natural variation. When the Corresponding author: Doonan, J.H. ([email protected]).
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genomic tools are in place, it will be possible to unlock these sources of variation and to address a wide range of important issues in grass biology (Table 2). In this review, we consider some of the attractions of Brachypodium as an emerging model system for the temperate grasses, and we highlight the surprising degree of variation for agronomic traits found in natural accessions. Geographical range and phylogenetic position of Brachypodium distachyon The natural range of B. distachyon spans the Middle East (Figure 2), largely overlapping the ancestral range of cultivated small grain cereals [4]. Diploid B. distachyon is an annual and has a basal chromosome number of n = 5 [1,5], with a c-value originally estimated as 0.21 pg/haploid genome [1]. However, the currently accepted value is 0.36 pg/haploid genome [6–8]. This is a relatively small genome for a grass and a highly desirable feature for any model species. Other species in the genus have also been used for molecular studies, particularly Brachypodium sylvaticum, but these tend to be perennial and their genomes vary in chromosome number and DNA content. It seems likely that efforts in the immediate future will focus on B. distachyon. As with wheat, different natural accessions of B. distachyon have variable genome size (Figure 2), owing to recent hybridisation events. Some of the variant distachyon cytotypes resemble that of a closely related species (2n = 20). Genomic in situ hybridisation revealed that other variant cytotypes are actually hybrids (2n = 30) between this species and B. distachyon [5]. In general, the small grain cereals tend to have large and variable polyploid genomes derived from relatively recent interspecies hybridisation and genome doubling events. This suggests that these species have an innate ability to tolerate dramatic changes in ploidy, and this ability might in turn confer selective advantage and facilitate speciation [9]. Consistent with this idea, the 2n = 30 accessions of Brachypodium tend to be physically larger, generally lack vernalisation requirements, and might have an ability to tolerate a broader range of environmental conditions than their diploid progenitors, as observed in other polyploid plants [10–12]. Genome sequencing projects have revealed that the Brachypodium genus is more closely related to wheat, barley and forage grasses than it is to rice. For example, comparison of a 370 kb region from the B. sylvaticum
1360-1385/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.01.007 Available online 17 March 2008
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Figure 1. Phylogenetic relationships between Brachypodium and the small grain cereals. Grasses are a large, morphologically diverse and successful family of plants comprising 10 000 species. Classically, grasses are characterized by a single-seeded starch-rich fruit called a caryopsis or grain (examples are all shown at same scale, bar = 5 mm). Furthermore, they all have a spikelet style of floral structure (groups can be distinguished by the number and pattern of these spikelets in the inflorescence [58]). The Pooideae subfamily includes forage grasses, Brachypodium and many economically important small grain cereals adapted to cool or dry climates, such as wheat (Triticum aestivum), rye (Secale cereale), barley (Hordeum vulgare) and oats (Avena sativa). Rice (Oryza sativa) and maize (Zea mays) belong to distinct subfamilies [59] and have many adaptations for tropical climates.
genome with an orthologous region from rice [13] supported the idea that Brachypodium diverged from the Pooideae approximately 35–40 Mya [14]. The full genome sequence of B. distachyon should provide additional insight into the evolution of genome structure with the grasses. Natural variation for disease resistance One of the original attractions of Brachypodium was, and remains, its capacity to help us understand plant– pathogen interactions in wild plant populations. As cereals became domesticated, their pathogens co-evolved to exploit the new plant populations. Combating plant disease is a major focus for many cereal breeding programmes [15] and the agrochemical industry [16]. The detrimental effects of
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disease on yield, and the desire to reduce chemical inputs are such that plant breeders wage a never-ending battle to breed new varieties with resistance to the current prevalent strains of pathogens. Different Brachypodium ecotypes varied in their responses to a range of fungal plant-pathogens that cause significant crop loss [1]. Although all tested ecotypes reacted similarly to Blumeria graminis, there was greater variability in the response to Puccinia and Magnaporthe grisea infection, suggesting that Brachypodium will be a useful model to study these important plant-pathogens. One out of seven ecotypes was resistant to M. grisea, an economically important fungal pathogen that is the casual agent of rice blast and which, in addition, infects temperate and forage grasses [17,18]. Different accessions show variable levels of resistance and susceptibility to the different host-limited forms (rice- and grass-adapted) of M. grisea [19]. One ecotype of B. distachyon was resistant to both of the M. grisea strains, whereas another was susceptible specifically to the grass-adapted strain. Genetic crosses between these ecotypes suggested that a single dominant resistance gene conferred resistance to the rice-adapted form of M. grisea. A genome sequence is already available for M. grisea [20,21]; the Brachypodium genome sequence should aid in the identification of factors (e.g. defence proteins [22] and metabolites [23]) that control the balance of resistance and susceptibility alleles in wild populations. In doing so, this will provide insight into how plant populations co-evolve with their natural diseases. Standing variation for domestication traits Domestication can lead to dramatic changes in plant form as humans select for particular traits and produce cultivars more suited to their purposes (reviewed by [4]). The origin of such traits has been widely debated, and the relative importance of pre-existing (or standing) variation in the wild progenitors is unclear. As a wild grass not under direct human selection, Brachypodium might provide some insight into this process. Recently, investigators surveyed available accessions from the United States Department of
Table 1. Public resources available for Brachypodium research Resource International Brachypodium Initiative The Brachypodium Newsgroup Brachypodium BrachyBase Prototype Genome Viewer USDA-ARS Germplasm Search Genomics and Gene Discovery bEST Resource Brachypodium genome browser Brachypodium T-DNA tagging (BrachyTAG)
Website http://www.brachypodium.org/ http://mailman.cgrb.oregonstate.edu/mailman/listinfo/brachy-info http://www.aber.ac.uk/plantpathol/brachyomics.htm http://www.brachybase.org/ http://www.ars-grin.gov/ http://brachypodium.pw.usda.gov/ http://www.modelcrop.org/ http://www.jic.ac.uk/staff/philippe-vain/brachypodium.htm
Table 2. Comparison of Brachypodium with other cereals and Arabidopsis Arabidopsis thaliana
Triticum aestivum
Zea mays
Oryza sativa
Hordeum vulgare
Number of chromosomes Genome size (1C) Reproductive strategy Life cycle (weeks)
Brachypodium distachyon 10 (2n) 300 Mb Self-fertilizing 10–18
10 (2n) 164 Mb Self-fertilizing 10–11
20 (2n) 2400 Mb Cross-pollination 10+
24 (2n) 441 Mb Self-fertilizing 20–30
14 (2n) 5000 Mb Self-fertilizing 16+
Height at maturity (m) Transformation Growth requirements
0.3 Facile Very simple
0.2 Facile Very simple
42 (2n) 16 700 Mb Self-fertilizing 12 (spring wheat) 40+ (winter wheat) Up to 1 Possible Simple
Up to 2 Facile Simple
1.2 Facile Specialized
Up to 1.2 Facile Simple 173
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and J.H. Doonan; unpublished). Analysis of spikelet and grain morphology (Figure 3) illustrates the degree of variation available in what is, to date, a relatively small number of accessions. Generation time, partly as a result of variable vernalisation requirements, is also variable in diploid accessions (http://www.brachypodium.org/node/8). A key feature of domesticated grains is the retention of the ripe grain within the dry inflorescence, but in such a way that it can be easily removed post-harvest by threshing. The manner in which the grain breaks free (or disarticulates) from the spike varies owing to preferential breakage of different tissues in different species of grass [24]. However, the timing and extent of disarticulation is crucial for domestication; when disarticulation occurs too early, harvesting becomes inefficient and therefore not cost-effective; when disarticulation is too difficult, grains are not released easily and cleanly, leading to a reduction in the net energy value of the crop. Thus, the development and function of the abscission zone is a focus of interest in cereals. The sh4 gene plays an important role in establishment of the abscission layer at an early stage of flower development [25]. It is thought that the development of a tough rachis was one of crucial changes that took place during the domestication of wheat [26]. Variation in the extent of disarticulation is clearly present in Brachypodium (Figure 3) and must have evolved for reasons quite distinct from those of human convenience.
Figure 2. Geographic origins of Brachypodium, and selected adaptive traits. The genus Brachypodium is currently distributed throughout most temperate regions of the world, but its ancestral range lies in the Middle and Near East. Accessions have been collected from various regions of both the ancestral range and the New World. Different accessions vary for several traits, including generation time (in the range of 12 to >18 weeks under greenhouse conditions), flowering time, plant size and ploidy level. Some traits such as metabolic composition (d) and disease resistance (e) are likely to be adaptive and related to the geographic origins of the species in question. (a) The geographic location of different accessions from the old world. Presumptive diploids are indicated by red circles, whereas accessions with higher ploidy levels are shown in blue. Green circles represent possible interspecific hybrids. (b) Ploidy has a dramatic effect on plant size. Flow cytometry confirmed that Bd21 and JIB05, two accessions of differing sizes, were diploid and allopolyploid, respectively (data not shown). (c) Higher ploidy accessions (such as ABR113) might be derived from interspecific crosses with a closely related species (ABR114; 2n = 20) [5]. These images show in situ hybridisation using probes made from the genomes of ABR1 (purple) or ABR114 (blue–green) and hybridised against spread chromosome preparations of the accessions as shown (bar = 5 mm). The chromosome spread of accession ABR113 contains chromosomes of both types, indicating that it is an allopolyploid. The cytotypes for many of the higher ploidy accessions have yet to be characterised. (d) Metabolite composition of six diploid (2n = 10) accessions of B. distachyon, as assessed by linear discriminant analysis of metabolite fingerprints using flow injection electrospray mass spectrometry. The two major discriminants are plotted. The different accessions cluster according to accession and to geographic region of origin. (e) Differential responses to rice blast infection [19] in two diploid (2n = 10) accessions of B. distachyon from Turkey (ABR1, chlorotic leaves with large black lesions indicate susceptibility) and Spain (ABR5, the small black flecks indicate resistance).
Agriculture (USDA) and Aberystwyth University (Wales, UK), and recently collected accessions from Turkey. The survey indicated the presence of extensive variation for numerous traits of agronomic importance (M. Opanowicz 174
Applications to bioenergy Diverse grass species are being evaluated as biofuel crops because they grow rapidly and, in theory, can be grown on land that is too marginal for other agricultural purposes. As with wheat, candidate biofuel species tend to have large genomes, and attempts to identify relevant quantitative trait loci (QTL) will benefit from knowledge gained in model species with smaller and syntenic genomes. Brachypodium has the cellular and subcellular makeup of a typical grass and will be a useful additional tool in the effort to improve these species for biomass and biofuel production. For example, tillering plays a significant role in biomass production of the forage grasses, and the number and quality of tillers can be affected by many environmental factors, including grazing, temperature, level of soil nutrients, day length, light intensity and plant density [27–30]. In Brachypodium accessions, the extent of tillering is related in part to flowering time and varies from a low of 6–7 tillers per plant in an early-flowering accession (USDA-ARS number PtdIns 255334) to 24 in a late-flowering accession (USDA-ARS number PtdIns 250647). The extent of variability in tillering and its genetic control in Brachypodium could benefit efforts to breed bioenergy crops. Cell wall composition has direct implications for biofuel feedstock. As the bulk of the harvested material, cell walls represent an important feedstock for bioenergy; however, grasses have distinctive cell wall structure and composition [31] that differs from the better studied cell walls found in dicotyledonous species. Although the cellulose microfibril structure is similar in both types of cell wall, the cross-linking matrix in the Poales is pectin-poor and rich in phenylpropaniods and silica, which can have
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implications for processing and use of the product. Genetic analysis of cell wall composition and deposition in Arabidopsis has provided a wealth of information on the dicotyledonous cell wall [32], and similar approaches will be possible in Brachypodium. The comparison between the two model species will be very interesting, both academically and for crop improvement. Metabolite fingerprinting indicated that there were significant differences in the spectrum of metabolites between different accessions of Brachypodium. These differences were associated with variation in the geographic origins of different Brachypodium species (Figure 2e) and might reflect adaptations to local conditions. Core metabolomic resources specific for B. distachyon and other Poaceae are being developed [23,33], including spectral libraries for gas chromatography mass spectrometry (GC-MS) analyses and signal annotation tools for liquid chromatography mass spectrometry (LCMS) data (http://www.armec.org/).
Figure 3. Brachypodium inflorescence architecture and morphological variation. (a) Inflorescence architecture of wheat (left), Brachypodium (centre) and rice (right). Inflorescence architecture in cereals depends on the branching pattern of the inflorescence meristem and varies from a single determinate spike in wheat to an indeterminate unbranched spike in barley, to more complex branched systems such as those of rice, in which there is a multiplicity of distinct meristems (for review, see [60]). (b) Morphological variation in inflorescence morphology between Brachypodium accessions. In the left panel, a newly collected Turkish accession with reduced inter-spikelet spacing. The middle panel shows the primary spike from accession JIB02 (John Innes Brachypodium 2) and right panel illustrates three different spikes from the same Bd21 plant where the number of spikelets per inflorescence varies between one and more than seven. In all accessions, the primary shoot tends to have the most complex inflorescence (leftmost spike in right panel), whereas subsequent shoots have smaller and less branched inflorescences. Several genes involved in controlling rice inflorescence architecture have been cloned [61], and synteny has enabled the identification of equivalent genes in maize [60]. A comparison between Brachypodium and other grasses using these genes as candidates might help to explain some of the differences in their inflorescence architectures [62]. (c) Level of lemma hairiness in two distinct accessions, JIB05 (top panels) and JIB01 (bottom panel). The Brachypodium caryopsis has a typical morphology for the Poaceae. It is surprisingly large, an average of 8 mm long by 2 mm wide. The caryopsis in Brachypodium is surrounded with two unequal-sized husks, a smaller palea and a larger lemma that terminates in a long awn. When examined under scanning electron microscopy (SEM) (left panels in (c) and (d)), the lemma and awn showed varied levels of hairiness, which probably reflects adaptation to local habitats. For example, hairs on plants growing in windy locations can break-up the flow of air across the plant surface, reducing evaporation. Alternatively dense coatings of hairs reflect solar radiation, protecting the more delicate tissues underneath in hot, dry, open habitats [63]. Hairs can also modify the function of the awns, which have been implicated in grain dispersal [64]. (d) Shatter zones in Brachypodium
Brachypodium – a stepping stone to the giants The small Brachypodium genome has already been invaluable as an aid for cloning wheat and barley genes of agronomic importance. Early studies had indicated that gene order (or synteny) is largely conserved between Brachypodium and the small grain cereals [34–36], and this synteny was instrumental in the characterization of the wheat gene Ph1 [37] and the barley gene Ppd-H1 [38]. Ph1 is particularly interesting in the context of genome evolution. This complex locus is required for correct pairing of homologous chromosomes in hexaploid and tetraploid wheats, but the absence of allelic variation at the Ph1 locus precluded conventional mapping. Markers (based on synteny between the wheat genome and the smaller genomes of rice and B. sylvaticum) were used to map deletion lines and to pinpoint functional aspects of the Ph1 locus [36,37]. Sequence analysis of the region revealed a complex of repeated versions of a novel cyclin-dependent protein kinase, the functional implications of which are currently being investigated. Molecular genetics and genomics in Brachypodium To develop B. distachyon as a link between rice and temperate grasses, Hasterok et al. [39] constructed a bacterial artificial chromosome (BAC) library of 9100 clones, representing more than two genome equivalents. In situ hybridization [40] between defined BACs and the 10 chromosomes of B. distachyon revealed strong synteny between B. distachyon and Poaceae species, with most of the BACs hybridizing at single loci on defined chromosomes. BAC libraries with three times coverage have been employed to construct physical maps for the Brachypodium genome [41,42]. The US Department of Energy Joint accessions (as described in (c)). Grain dispersal mechanisms vary tremendously among grass species. The dispersal module can vary from naked isolated grains to intact spikes in different species, and various selective advantages have been proposed for each different strategy [58]. Surprisingly, 8 out of 13 Brachypodium accessions examined retain their grain within the spike after ripening, and SEM revealed that the structure of the abscission zone reflects these differences. Therefore, the grain dispersal module varies in different accessions and indicates that a considerable degree of variation for this important agricultural trait already exists in a wild population of grass with no known history of cultivation.
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Review Genome Institute (JGI) has released a Brachypodium whole genome shotgun sequence that covers the genome to an approximate 4x depth (http://www.Brachypodium.org/). JGI is continuing sequencing to achieve 8x coverage, and a draft genome sequence of the community standard inbred line Bd21 (2n = 10) is expected in 2008 (International Brachypodium Initiative http://www.brachypodium.org/node/8). In addition, expressed-sequence tag (EST) libraries have been sequenced [8], and an additional 180 000 ESTs are expected as part of the genome sequencing project. A complete genome sequence will enable us to define both induced and natural variation, and will facilitate transcriptomics and association mapping. Association mapping might become a powerful tool for identifying alleles and loci responsible for natural variation in Brachypodium. With the use of chemical, radiation or T-DNA tagging approaches, genetic resources are also being developed for B. distachyon, with the aim of conducting forward and reverse genetic screens. Genetic transformation technologies are crucial for the development of these resources and for the validation of gene function. Biolistic- [1,43] and Agrobacterium-mediated transformation [7,44–46] systems have been developed for a wide range of Brachypodium genotypes. Cytotypes with 30 chromosomes are generally more amenable to genetic engineering than diploid lines (2n = 10). BDR018 was the first diploid line to be transformed by particle gun bombardment, with 5% efficiency [43]. Bombardment transformation was used to compare the effects of flowering control gene Terminal flower 1 (TFL1) from different species in Brachypodium [47]. Past studies in Arabidopsis showed that expression of TFL1 delays flowering and prevents expression of the floral meristem identity genes LEAFY and APETALA1. In monocotyledons, however, the orthologue LpTFL1 (Lolium perennae TFL1) seems to completely prevent flowering [48]. Overexpression of LpTLF1 or TLF1 in transgenic Brachypodium plants resulted in a significant delay in flowering compared with that in controls [47]. Large-scale transformation efforts now use Agrobacterium-based approaches. The community standard strain, Bd21, has been transformed using Agrobacterium, with 17% efficiency [44]. Another line, Bd21–3, which is genetically distinct from Bd21 but which was also produced by recurrent self-pollination of the same USDA accession (PtdIns 254867), produces transformation efficiencies of up to 36% [7]. Line BDR018 [45] shows 55% efficiency, indicating a high degree of diversity for this ‘trait’. Bd21 TDNA insertion lines have been produced, and the flanking regions are currently being defined. Flanking-sequence tags (FSTs) of the T-DNA inserts and the seeds from the first 2000 insertion lines are available for dissemination to the science community [44]. T-DNA mutants have been an essential factor in the success of more established model systems such as Arabidopsis thaliana [49] and rice [50,51]. Extensive populations potentially enable researchers to obtain insertional activation or inactivation of any gene of interest, so the production of large characterised populations containing defined insertions will be one of the crucial next steps in establishing Brachypodium as an attractive experimental system. 176
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Perspectives Brachypodium is, therefore, an attractive model system for the elucidation of grass biology. It exhibits extensive biodiversity and holds great potential for genetic analyses and engineering. Furthermore, the Brachypodium genome will soon be fully annotated, and this powerful combination of factors promises to facilitate the dissection of many traits of agronomic and ecological importance. Many of these traits will probably be multigenic in makeup and therefore best analysed as quantitative characteristics. Previously, such traits were difficult to study, but the combination of genetic and genomic resources available for plants such as Arabidopsis, rice or maize now make this feasible (e.g. [52– 57]), and Brachypodium should make a valuable contribution towards this goal. Its comparative ease of cultivation, small size and rapid life cycle are likely to recommend Brachypodium as a model system for cereal and grass biology in the developed and developing world. Acknowledgements M.O. was supported by a Short Term Fellowship from the Human Frontier Science Program and Marie Curie Intra-European Individual Fellowship; PV, DP and JHD were supported by the Biotechnology and Biological Sciences Research Council. The chromosome images in Figure 2c were kindly supplied by Robert Hasterok (University of Silesia, Poland) and Glyn Jenkins (Aberystwyth University, UK). Photography was undertaken by Andrew Davies (John Innes Centre photography unit). We thank Mary Byrne, Simon Griffith, John Snape and Sinead Drea for critical reading of the manuscript, and the reviewers for helpful comments.
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