Exploiting the Brachypodium Tool Box in cereal ... - Wiley Online Library

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Tansley review Exploiting the Brachypodium Tool Box in cereal and grass research Author for correspondence: Luis A. J. Mur Tel: +44 (0)1970 622981 Email: [email protected] Received: 4 March 2011 Accepted: 25 March 2011

Luis A. J. Mur1, Joel Allainguillaume1, Pilar Catalań2, Robert Hasterok3, Glyn Jenkins1, Karolina Lesniewska3, Ianto Thomas1 and John Vogel4 1

Institute of Biological, Environmental and Rural Sciences, Aberystwyth, Wales SY23 3DA, UK;

2

Department of Agriculture, University of Zaragoza, High Polytechnic School of Huesca, Ctra.

Cuarte km 1, ES–22071 Huesca, Spain; 3Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia, PL–40-032 Katowice, Poland; 4USDA ARS Western Regional Research Center, Albany, CA 94710 USA

Contents Summary

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V.

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Introduction

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VI. Whence for the Brachypodium Tool box? Primus inter pares?

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A challenge for the Brachypodium Tool Box: the legacy of cereal domestication

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Opening the Brachypodium Tool Box: what is Brachypodium?

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IV. The Brachypodium Tool Box: where are we now?

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III.

Targets for the Brachypodium Tool Box: key traits

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Acknowledgements

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References

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Summary New Phytologist (2011) 191: 334–347 doi: 10.1111/j.1469-8137.2011.03748.x

Key words: Brachypodium, comparative genomics, functional genomics, genome sequencing, model grass.

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It is now a decade since Brachypodium distachyon (Brachypodium) was suggested as a model species for temperate grasses and cereals. Since then transformation protocols, large expressed sequence tag (EST) databases, tools for forward and reverse genetic screens, highly refined cytogenetic probes, germplasm collections and, recently, a complete genome sequence have been generated. In this review, we will describe the current status of the Brachypodium Tool Box and how it is beginning to be applied to study a range of biological traits. Further, as genomic analysis of larger cereals and forage grasses genomes are becoming easier, we will re-evaluate Brachypodium as a model species. We suggest that there remains an urgent need to employ reverse genetic and functional genomic approaches to identify the functionality of key genetic elements, which could be employed subsequently in plant breeding programmes; and a requirement for a Pooideae reference genome to aid assembling large pooid genomes. Brachypodium is an ideal system for functional genomic studies, because of its easy growth requirements, small physical stature, and rapid life cycle, coupled with the resources offered by the Brachypodium Tool Box.

2011 The Authors New Phytologist 2011 New Phytologist Trust

New Phytologist I. Introduction The publication of the entire genome sequence for Brachypodium distachyon (which we will subsequently referred to Brachypodium, except when we describe intrageneric variation) accession Bd21 early in 2010 (IBI, 2010) is a fitting end to a decade that started with the serious proposal of Brachypodium as a model grass for temperate cereals and forage grasses (Draper et al., 2001). Equally importantly, the same decade has seen the development of next generation sequencing (NGS) technologies offering the capability of generating unprecedented amounts of genomic sequence information (Metzker, 2010). When coupled with improved bioinformatic approaches allowing genomes to be more readily assembled (Cantacessi et al., 2010), the consequence is that large genome cereal species are now tractable for studies that would previously have been the preserve of model species. Even wheat is now within the sights of genome sequencers and thoughts are now focusing on how to exploit these sequence data in crop breeding programmes (Edwards & Batley, 2010). In the wake of these major technological developments, we will reconsider in this review the value of a Brachypodium model to cereal biology and to crop breeding programmes. In doing so, it is not the intention of the authors to describe the details of the Brachypodium genomic sequence (IBI, 2010).

II. A challenge for the Brachypodium Tool Box: the legacy of cereal domestication Temperate cereals were domesticated by humanity from wild grassland species in a multi-episodic process between 10 000 and 3000 yr ago (Brown et al., 2009). However, domestication resulted in population bottlenecks – a loss of genetic diversity compared with wild ancestors – among cultivated cereal varieties, with perhaps only 10–20% of the wild variation being used in modern wheat varieties (Langridge et al., 2006). This lack of variation thwarts attempts to improve elite germplasm, such as increasing its yield (e.g. possibly by 71% between 1997 and 2050; Rosegrant & Cline, 2003) during a period of potential environmental change and population increase. In order to meet this challenge, a number of approaches are available to cereal scientists. Agricultural practices, particularly in the developing world, could be improved to increase the areas under cultivation and maximize production but this should be also coupled with significant improvements in the cereal gene pools (Rosegrant & Cline, 2003). The latter could come about via screens of relatively restricted collections of elite germplasm or through exploitation of cereal landraces – semidomesticated variants of crop lines that have been locally maintained through traditional farming and often are excellent sources of stress tolerance (Newton et al., 2010). More innovative processes


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may involve the rehybridization of cereal ancestors to form new ‘synthetic’ progeny. However, ‘linkage drag’ – the association of a desired genetic trait with nonoptimal traits – is a problem, as to avoid ‘linkage drag’ a genetic modification (GM) strategy can be adopted where the focus is squarely upon the transfer of well-defined genetic element(s). However, at least within a European context, this strategy has met with considerable opposition (Schenkelaars, 2001; Hartl & Herrmann, 2009). Against such a background, what are the roles for a model species such as Brachypodium, which has no agricultural value and is not easily introgressed into other cereals (Khan & Stace, 1999)? It is our contention that Brachypodiumbased research will play a vital role on two fronts. First, the Brachypodium genome will act as a reference for the assembly of large temperate cereal genomes. Classically, plant genomes are currently sequenced either by a global shotgun sequencing approach or by sequencing of large-insert overlapping genomic clones (Venter et al., 1996). The prevalence of repeated sequences in the genomes of plants, especially the cereals, makes the assembly of a completed genome very difficult. This problem is aggravated by the plasticity of repeated sequences within the intragenic regions, which is greater than that observed in primates (Dubcovsky & Dvorak, 2007). This results in intragenic regions with large numbers of very similar nucleotide sequences, which has effectively prevented the use of the shotgun genome sequencing approach for diploid Triticeae species (Luo et al., 2010). Newer approaches such as highinformation-content-fingerprinting (HICF) of bacterial artificial chromosome (BAC) clones (Luo et al., 2003) are undoubtedly aiding the assembly of large, complex genomes. However, comparative genomic strategies using a smaller low-repetitive DNA reference genome, such as Brachypodium, still has value. An incidental, albeit not insignificant, additional benefit will be that Brachypodium will contribute towards the supply of markers for mapping difficult regions of large genome cereals. The latter role, the provision of markers to fine map traits in (for example) heterochromatic areas of crop plants, has already been demonstrated (Turner et al., 2005; Griffiths et al., 2006; Spielmeyer et al., 2008). Second, its role as a functional genomic model to rapidly determine gene function is likely to be much more valuable. In this context, the innate biological traits of Brachypodium make it a useful model for all the grasses. Platform infrastructure (The Brachypodium Tool Box: Fig. 1b–c) is being developed with which function can be assigned to hitherto intractable genes. Thus, within the context of breeding programmes, Brachypodium could be seen as a tool by which gene function, could be rapidly assigned by alignment with other genomes, and by which tightly-linked markers to useful alleles could be developed. Further, in those areas of the world where GM approaches are widely accepted by the general public,

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(a)

(b)

Biological infrastructure • Small stature • Rapid seed-to-seed cycle • Undemanding growth conditions • Growing availability of germplasm • Important traits

(c)

Genomic infrastructure

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• Genome sequence • EST libraries • Genomic markers for forward genetic screens • Syntenic relationships with major cereals • T-DNA tagged populations • EMS/fast neutronmutated populations • Cytogenetic markers • Oligo-microarrays • Bioinformatic resources

Brachypodium sylvaticum Brachypodium pinnatum Brachypodium phoenicoides Brachypodium rupestre Brachypodium retusum Brachypodium arbuscula Brachypodium mexicanum Brachypodium distachyon Triticeae Poaceae Oryza

Fig. 1 Components of the Brachypodium Tool Box for genomic and post genomic analyses. (a) Brachypodium distachyon growing in the wild (Castillo de Mur, Lleida, Catalunya, Spain: N42.09763 ⁄ E0.87750); bar, 1 cm. The Brachypodium toolbox contains considerable (b) biological and (c) genomic resources. (d) Phylogenetic relationships within the Brachypodium genus and with the Triticeae, Poaceae and Ehrhartoideae (adapted from Catalan & Olmstead, 2000).

Brachypodium genes, or their cereal orthologues, could be used to generate transgenic crops.

III. Opening the Brachypodium Tool Box: what is Brachypodium? Brachypodium distachyon, commonly called purple false brome, is native to the countries around the Mediterranean basin, the Middle East, south-west Asia and north-east Africa (Schippmann, 1991). It has been widely naturalized beyond its natural range with a notable recent colonization in North (Mexico, USA) and South (Argentina, Ecuador, Peru, Uruguay) America, Australia and South Africa (Schippmann, 1991; Catalań 2003) and even the UK (England and Scotland) (Stace, 2010). Although in the wake of the completed B. distachyon genome sequence (IBI, 2010), this species has received much of the attention, the Brachypodium genus encompasses between 15 and 18 species distributed worldwide (Catalań et al., 1997; Catalań & Olmstead, 2000). The various Brachypodium species have different geographical ranges. B. pinnatum and B. sylvaticum are wide-ranging and found throughout Eurasia, and B. retusum across the Mediterranean, while B. rupestre is predominantly found in western Europe, and B. phoenicoides in the western Mediterranean. Other Brachypodieae are more geographically restricted, B. arbuscula being endemic to the Canary Islands, B. boissieri to southern Spain and B. kawakamii to Taiwan while B. mexicanum is found in Central and South America, B. flexum in eastern and western tropical Africa and


South Africa, and B. bolusii in South Africa (Schippmann, 1991; Catalań & Olmstead, 2000). The Brachypodieae have different chromosome base numbers (x = 5, 7, 8 and 9). Brachypodium distachyon has a chromosome base number of x = 5 and has been described as having different ploidies (2n = 10, 20 and 30; Robertson, 1981). As a rule, the 2n = 10 cytotypes tend to be much smaller and many accessions require vernalization to induce flowering. By contrast, B. distachyon 2n = 20, 30 tend to be taller, have a lesser requirement for vernalization and almost invariably (at least from our observations) exhibit prominent anthesis and larger seeds (Schwartz et al., 2010). Much early work initiated by Professor C. A. Stace (Leicester University, UK), focused on establishing the phylogenetic relationships between the Brachypodieae (Fig. 1d). Perhaps unsurprisingly, the long-rhizomatous perennial species (B. arbuscula, B. retusum, B. rupestre, B. phoenicoides, B. pinnatum and B. sylvaticum) are the most closely related, being distinct from both the geographically isolated short-rhizomatous B. mexicanum and the widespread annual B. distachyon (Catalań et al. 1995; Catalan & Olmstead, 2000). Furthermore, the Brachypodieae have been shown to be sister to the recently evolved core pooid’ grasses, which include the temperate cereals and forages of tribes Triticeae–Bromeae and Aveneae–Poeae, in all the molecular phylogenetic studies conducted so far. Based on sequence variation (mean synonymous substitution rates, Ks) between orthologous genes from B. distachyon, sorghum (Sorghum bicolor), rice (Oryza sativa) and wheat (Triticum aestivum) genomes, it was estimated that B. distachyon diverged from a common wheat ancestor 32–39 million yr ago (Mya), from the common rice ancestor 40–53 Mya and from that common ancestor to sorghum 45–60 Mya (IBI, 2010). Until recently, it was assumed that B. distachyon had an autopolyploid series with diploid (2n = 10), tetraploid (2n = 20) and hexaploid (2n = 30) chromosomal races (Robertson, 1981). However, cytogenetic studies using fluorescence in situ hybridization (FISH) with total nuclear DNA (genomic in situ hybridization; GISH) and ribosomal probes indicated that the 30 chromosome accessions of B. distachyon were in fact allotetraploids (Hasterok et al., 2004). Subsequently, FISH analysis with BAC-based probes from a genomic library of B. distachyon (2n = 10), coupled with genome size analysis, has further confirmed that diploid B. distachyon (2n = 10; 0.631 pg ⁄ 2C) genotypes may have hybridized with another diploid (2n = 20; 0.564 pg ⁄ 2C) (of which only a single accession – ABR114 – currently exists) to form the allotetraploid (2n = 30; 1.265 pg ⁄ 2C) (Hasterok et al., 2006; E. Wolny pers. comm.). Given these observations and clear morpho-anatomical differences between the cytotypes, it is entirely appropriate to consider each B. distachyon chromosome class as different species. Some of the authors of this review will be formally presenting these new Brachypodium species in the near future. Forthwith, we will


New Phytologist refer to B. distachyon as the diploid 2n = 10 form. The Wolny & Hasterok (2009) study has been extended to examine other species within the genus and has provided some insights into the organization, phylogeny and evolution of Brachypodium genomes at the chromosomal level. Genomic in situ hybridization studies revealed inter alia that B. retusum (2n = 38) is likely to be an allopolyploid comprising the genome of B. distachyon or a close relative. Similarly, two other species with 2n = 28, B. pinnatum and B. phoenicoides, are in fact allotetraploids, each containing the B. distachyon genome and either B. pinnatum (2n = 18) or B. sylvaticum (2n = 18), respectively. This complex evolution of the Brachypodium genus should be seen as an opportunity to study the factors governing genome polyploidization using powerful functional genomic tools that have been added to the Brachypodium Tool Box. Many important cereal species are also allopolyploids and elements such the Ph1 locus, which diploidizes allohexaploid wheat, are of interest. Given this, the genus Brachypodium represents an ideal system with which to study the value of synthetic hybrids which exploit genomic interactions to deliver beneficial traits not necessarily present in the parental diploid or diploid-like lines.

IV. The Brachypodium Tool Box: where are we now? Given the relatively short period since it was first suggested as a model species, the genomic resources comprising the Brachypodium Tool Box have been very rapidly established. Most recently, a very high quality draft genome sequence was published (IBI, 2010). The final genome assembly was remarkably complete, with 99.6% of all the sequences incorporated into the final assembly and only 0.4% of the sequence predicted to be missing based on paired end information. Similarly, the initial annotation was of high quality and was facilitated by a large number of ESTs (Vogel et al., 2006). Thus, the genome sequence is now in place to serve as a foundation for a myriad of applications. For example, a combination expression and tiling Affymetrix microarray has been created for Brachypodium (T. Mockler, pers. comm.). Other genomic resources include BAC libraries (Hasterok et al., 2006; Huo et al., 2008), BAC end sequences and a physical map based on contigs derived from a BAC library using the SNaPshot high-informationcontent-fingerprinting (HICF) method (Huo et al., 2009). However, other challenges, which are considered later in this text, must be overcome for the Brachypodium Tool Box to achieve its full potential. 1. Comparative genomics: Brachypodium as a Pooid reference genome? The value of Brachypodium was first seen in its ability to act as a bridge species (Jenkins et al., 2005) to aid the clon-


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ing of genes in temperate cereals with extremely large genomes. A bridge species is necessary because larger genomes have many DNA repeats that effectively isolate genes or ‘gene islands’ in map-based cloning approaches (Devos, 2010). By contrast, small genome grasses have gene-rich euchromatic regions with one gene per 5–15 kb, on average. Brachypodium and rice have similar proportions of retrotransposon sequences (21.4% and 26%, respectively), which is much less than sorghum (54%) or bread wheat (over 80%) (IBI, 2010). Even before the genome sequence was available, the chromosomal pairing locus Ph1 was accurately mapped to a cluster of kinase genes within a heterochromatic segment of wheat chromosome 5B using markers derived from a much smaller orthologous region in B. sylvaticum (Griffiths et al., 2006). Interestingly, although the rice genome sequence was available, its sequence was too divergent from wheat in this region to provide markers and it could not be used to fine map the Ph1 locus. In other examples, markers from Brachypodium were used to identify the Lr34 ⁄ Yr18 wheat rust resistance gene (Spielmeyer et al., 2007) and the barley Ppd-H1 photoperiod response gene (Turner et al., 2005). The availability of the Bd21 genomic sequence, EST collections and resequencing data from other accessions (soon to be available) means that more genomic sequence data and markers are now coming on line to facilitate genomic comparisons. Simple sequence repeat (SSR) microsatellite markers are widely used as anchor markers in genetic mapping, and in marker-assisted breeding. A large number of SSR markers have been derived from B. distachyon (Azhaguvel et al., 2009; Vogel et al., 2009; Garvin et al., 2010) offering the possibility of rapidly identifying genetic loci associated with a trait and translating this to other cereals. Garvin et al. (2010) populated the Brachypodium genome with a total of 139 SSR markers derived from ESTs, BAC end sequences (BES) and conserved orthologous sequences (COS) from a range of grass species. In macrosyntenic comparisons, 13 out of 20 Brachypodium linkage groups have equivalents in the rice genome sequence. Azhaguvel et al. (2009) developed and used 160 EST- and 21 derived genomic microsatellite markers to evaluate genetic diversity among the Brachypodium accessions and to relate the observed genetic races to the feeding preferences of the wheat greenbug (Schizaphis graminum Rondani) and the Russian wheat aphid (RWA) (Diuraphis noxia). Further, phylogenetic analysis suggested that Brachypodium is closer to Aegilops tauschii (the D genome donor of common wheat) than to rice. When EST contigs were compared, Brachypodium exhibited orthology with wheat EST contigs from all 21 of the wheat chromosomes (Kumar et al., 2009). Some astonishing examples of orthology between Brachypodium and the temperate cereals at a single gene level have been reported. In Triticeae genomes, a tandem


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duplication occurred in a globulin gene leading to the selection of a high molecular weight glutenin gene – an essential trait in bread wheat. This globulin duplication was present in Brachypodium but not in any tropical grass genome (Gu et al., 2010). The Eps-A (m) 1 locus, which controls morphological changes, such as spiking in Brachypodium and in one wheat genome progenitor (A. tauschii), is very similar to orthologues at this locus, including, crucially, Mot1 and FtsH4, which are tightly linked to the earliness per se phenotype (Faricelli et al., 2010). Nevertheless, some studies have suggested other genomic relationships. Yu et al., (2009) associated the Hessian fly resistance gene H26 (mapped to the wheat 3DL distal region 3DL3-0.81-1) with a Brachypodium supercontig. Encouragingly, 14 of the 15 ESTs were collinear between the distal region of wheat 3DL and Brachypodium supercontig 13. However, of 46 STS (sequence tagged site) primer markers derived from this supercontig, only one could be mapped to wheat chromosome 3D. The apparent lack of conservation was such that the authors advised caution when using the Brachypodium genomic sequence for molecular mapping and gene cloning in wheat. Reconciliation of these seemingly contradictory data came about with the publication of the Brachypodium genome sequence. On a global level, Brachypodium and rice exhibit a high degree of conservation of gene order such that entire rice chromosomes or chromosome arms can be mapped to their Brachypodium counterparts. By contrast, the alignment of diploid wheat ancestor (A. tauschii) and barley genetic maps to the Brachypodium genome is more fragmentary, although there are still large segments of conserved alignment. This indicates that there were a large number of genomic rearrangements in the lineage containing wheat and barley after the divergence from the Brachypodium lineage. Thus, instead of simply using Brachypodium as a roadmap for the wheat and barley genomes through simple pairwise comparisons, it will be more effective to use multiple comparisons of gene order in sequenced grass genomes. However, because Brachypodium shares a higher degree of nucleotide sequence conservation with the temperate grains, markers created using Brachypodium sequence have a much higher conversion rate. When colinearity of genes within seven large gene families was examined four exhibited a high degree of conservation in gene order between cereal genomes, but this was not the case for the nucleotide binding site (NBS)leucine-rich repeat (LRR) and F-box gene families (IBI, 2010). This could indicate rapid diversification owing to strong natural selection driven by pathogen pressure in the case of NBS-LRR and by the regulation of both developmental and stress responsive traits in the case of F-boxes (Meyers et al., 2003; Xu et al., 2009). Thus, the degree of synteny between Brachypodium and wheat in any particular region will vary owing to both the historical rearrangements


New Phytologist after the divergence of the lineage and evolutionary history of the gene, which may be accelerated in specific gene families. Interestingly, when only gene sequence is compared, the overwhelming majority of gene families are highly conserved between rice, sorghum, Brachypodium, wheat and barley; only 265 out of 16 215 gene families were specific to the Pooideae (Brachypodium, wheat and barley). Thus, there are considerable genomic similarities between the Ehrhartoideae (rice), Panicoideae (Sorghum) and Pooideae (B. distachyon, T. aestivum and Hordeum vulgare), undoubtedly reflecting their relatively recent evolutionary divergence. Thus, in addition to serving as a structural model for the large genomes of the temperate grains, the overall similarity at the gene level indicates that, for the majority of traits, Brachypodium can serve as a functional genomic model for all the grasses. As a corollary to this, depending on the trait, Brachypodium might not be the best source of comparative genomic information for the Triticeae and a comparative approach using all available genomes is advised. This may be more important for traits that have come under selection pressure during rapid evolutionary diversification, and are therefore specific to the Triticeae, such as traits conferred by the NBS-LRR and F-box’s genes (IBI, 2010). 2. Transformation and reverse genetic tools within the Brachypodium Tool Box Efficient transformation is a prerequisite for a modern model organism and we are fortunate that Brachypodium has been very amenable in this regard. Biolistic bombardment-based transformation of a polyploid line was first reported in 2001 (Draper et al., 2001). A more efficient method that worked on a diploid line was later reported in 2005 (Christiansen et al., 2005). However, Agrobacterium is the preferred transformation method, where it is important to have simple, low copy integration events and methods with efficiencies up to 30% have been published (Vain et al., 2008; Vogel & Hill, 2008). Today, the average efficiency in a production setting where the emphasis is on minimizing the labour per transgenic line rather than maximizing the efficiency of each transformation is c. 45% (up-to-date methods are available at http://brachypodium.pw.usda.gov/). With these improvements Brachypodium is arguably one of, if not the, most easily transformed grasses. The efficiency of Brachypodium transformation makes feasible the creation of collections of sequence indexed TDNA mutants. An excellent example of the power of a T-DNA population to reveal gene function is the SALK T-DNA tagged Arabidopsis resource (Ecker, 2002). Such a resource is a crucial component of the Brachypodium Tool Box. Two groups have established projects to create Brachypodium T-DNA mutants. Researchers at the John Innes Centre have reported the generation of a collection of 4500 T-DNA lines. Analysis of 741 accessions showed


New Phytologist that 660 T-DNA loci could be assigned to a unique location in the Brachypodium genome sequence (Thole et al., 2010). Of these, c. 60% could be associated with ESTs. The T-DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the web site (http://www.brachytag.org/). Similarly, researchers at the USDA-ARS Western Regional Research Center are generating thousands of tagged lines. At the time of writing > 10 000 have been created, and over 6000 flanking sequence tags (FSTs), have been assigned to a unique location in the genome. Rather than concentrating only on T-DNA tagging, this effort is also using gene-trapping vectors to identify promoters with useful expression patterns, and activation tagging vectors that contain transcriptional enhancers to ‘activation tag’ nearby genes. Details and ordering instructions can be found at http:// brachypodium.pw.usda.gov/TDNA. In addition to insertional mutants, a method to mutagenize seeds with ethyl methanesulphonate (EMS) has been optimized (http://brachypodium.pw.usda.gov), and irradiation with fast neutrons has also been used to create mutant collections (D. Laudencia-Chingcuanco and M. Byrne, pers. comm.). These EMS mutants are very useful for forward genetic screens because of the large number of mutants per plant. In addition, EMS mutants can also be used to identify mutations in particular genes for reverse genetic studies by a TILLING (Targeted Induced Local Lesions in Genomes) approach (McCallum et al., 2000) which employs a mismatch specific endonuclease to identify mutated PCR amplicons. The authors are aware of the creation of one TILLING population (http://www-ijpb. versailles.inra.fr/en/institut/actualite.htm) which currently consists of c. 6000 individuals. Although these emerging mutagenic approaches are impressive, it will take some time before sufficient genome coverage is achieved for most Brachypodium genes to have a corresponding mutant. Given this, the development of a targeted gene disruption strategy based on virus-induced gene silencing (VIGS) represents a significant advance as it represents an immediately applicable strategy through which the expression of targeted genes can be disrupted. Barley stripe mosaic virus (BSMV) is a single-stranded tripartite RNA virus where infection of the host can occur following simple rubbing of leaves with naked (nonenveloped or capsidated) genome. Derivatives of this virus, including fragments of a targeted gene, have been used to suppress gene expression in barley and wheat (Holzberg et al., 2002; Scofield et al., 2005). Recently, two studies have shown the efficacy of BSMV-induced VIGS in Brachypodium. Demircan & Akkaya (2010) silenced a phytoene desaturase gene and Pacak et al. (2010) suppressed the expression of genes IPS1, PHR1 and PHO2 known to participate in phosphate (Pi) uptake and reallocation.


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Following these successes, we expect VIGS to be widely employed in Brachypodium research. 3. Cytogenetic tools for genome analyses An important outcome of Brachypodium research has been the rapid development of cytogenetic tools. Routine FISH of BAC clones is able to identify and delimit specific chromosomes, chromosome arms, and particular chromosome regions (Hasterok et al., 2006; Jenkins & Hasterok, 2007). This has provided unprecedented insights into the genomic relationships within the Brachypodium genus, and should help to reveal evolutionary relationships between members of the Pooideae and more distant grass species (Wolny et al., 2010). From a more practical viewpoint, the use of ordered, labelled BAC clones representing the definitive Brachypodium nuclear sequence has allowed the construction of supercontigs covering large regions of the B. distachyon chromosomes, which has proved invaluable for the validation of linkage group assembly (Febrer et al., 2010; IBI, 2010). In addition, these resources enabled robust chromosome ‘painting’, which is now at our disposal for investigating with greater resolution the structural relationships between related genomes and the early association of chromosomes during meiosis (Fig. 2). As an example of the application of such tools, the Jenkins and Hasterok teams are actively utilizing these cytogenetic resources to understand and manipulate meiosis and recombination in grasses. A major obstacle to plant breeding programmes is not so much sexual and genomic

Fig. 2 Pachytene in Brachypodium (2n = 10) with physically mapped alternating clusters of red and green bacterial artificial chromosome (BAC) probes hybridizing to the short arm of chromosome Bd1. Bar, 5 lm (courtesy of Dr Dominika Idziak, Department of Plant Anatomy and Cytology, University of Silesia, Katowice, Poland).


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incompatibility, as the retention over many backcross generations of undesirable wild germplasm. Purging potential new cultivars of this material is costly and time consuming, and largely governs the speed at which new cultivars are released. These negative effects of linkage drag are inversely proportional to the extent of genetic recombination in successive backcross generations. Recombination is assayed traditionally by direct cytological scoring of chiasmata at metaphase I of meiosis, which has shown that the chromosomes of temperate, largegenome cereals and grasses, such as wheat, barley, rye and ryegrass, each have regularly two or more chiasmata. However, cytological inspection shows that the vast majority of these are confined to distal regions, meaning that interstitial and proximal chromosome segments seldom engage in recombination events. This uneven distribution of crossovers can also be inferred from measurements of recombination frequencies between genetic markers in mapping populations, which are then correlated to physical chromosome positions using deletion and introgression mapping or recombination nodules. Such studies in, for example, wheat (Erayman et al., 2004; Saintenac et al., 2009) have revealed that there is a common gradient of increasing recombination from proximal to distal regions of chromosomes. Because it is now known that the interstitial and proximal regions of cereal and grass chromosomes harbour a sizeable proportion of the genes of the genome (albeit at a lower density), the inevitable conclusion is that a large part of the genome of these species is not regularly involved in recombination events, effectively consigning many genes to recombination backwaters. This limits the potential of genetic variation in these crops, it prolongs linkage drag in introgression programmes, and it confounds the scope of map-based cloning approaches. Indeed, it has been acknowledged that ‘> 30% of wheat genes are in recombination-poor regions and thus are inaccessible to map-based cloning’ (Erayman et al., 2004). Clearly, it would be desirable to crack open these recombination coldspots in order to release new genetic variation which could be exploited in advanced breeding programmes. However, even if sites of recombination were shifted, map-based cloning strategies would still be constrained by the inordinately large genome sizes of these species. Preliminary studies of meiosis in a diploid and an allotetraploid accession of Brachypodium (Jenkins et al., 2005) have shown that chiasmata are not distally localized as in the other members of the Poaceae mentioned above. Thus, if there is variation in recombination among Brachypodium germplasm there is a real prospect of establishing the genetic basis of chiasma location and frequency. Variation in genomic recombination has been noted in Arabidopsis (Sanchez-Moran et al., 2002), rye (Rees, 1961), Lolium and Festuca (Jones & Rees, 1966; Rees & Dale, 1974), and barley (Gale & Rees, 1970).


New Phytologist 4. Looking into the Brachypodium Tool Box: bioinformatic tools In the multinational effort required to develop Brachypodium as a model, parallel development of appropriate bioinformatic tools allows the collection, curation and interrogation of genomic and post-genomic data. Readers unfamiliar with Brachypodium are recommended to use the bioinformatic tools available through the http://www.brachypodium.org website. From this website, it is possible to access the Brachypodium genome browser Brachybase (http://www.brachybase.org), where the 8X Assembly genome can be viewed, downloaded it in its entirety, and related to EST databases and Affymetrix (BdArray) array probes. A BLAST tool (http://www.brachybase.org/blast/) allows nucleotide and amino acid sequence comparisons. For excellent cross-species genomic comparisons and as a repository of much information on Brachypodium, the Gramene website (http://www.gramene.org/Brachypodium_ distachyon/) is also a good place to start. Other databases (http://mips.helmholtz-muenchen.de/ plant/brachypodium/) allow whole genome, protein sequence ⁄ structures and motifs to be investigated and promoter regions to be accessed. http://www.modelcrop.org/ has many of the functions of these other sites but also allows the Brachypodium physical map to be displayed and easily compared with the rice and sorghum genomes. With http:// www.phytozome.net it is possible to search for orthologous or homologous genes among all sequenced plant genomes based on name or sequence. Matches may be extracted and alignments compared by progressive alignment algorithms (dynamic programming) while relationships can be displayed using phylogenetic approaches or multivariate principal component analysis. One result of all of these bioinformatic data and tools has been the development of an Affymetrix array based on the Bd21 genome sequence and EST databases (S. E. Fox et al., unpublished data). The derived oligonucleotides were used to generate the array representing unique single copy sequences, with mean probe spacing of 42 bases with 95% of probe pairs < 126 nucleotides apart enabling studies of gene-specific expression. S. E. Fox et al. (unpublished) are using this array resource to characterize Bd21 expression during development, diurnal and circadian cycling as well as abiotic or biotic stress to represent a ‘Bd21 expression atlas’. It is expected that these data will be made available via a web portal analogous to the Arabidopsis genomic site (e.g. TAIR Microarray Experiments). 5. Diversity within the Brachypodium Tool Box: germplasm collections If Brachypodium is to act as functional genomic model for the Pooideae and other grasses, germplasm collections must


New Phytologist contain accessions with economically relevant traits and encompass sufficient variation for mapping projects to succeed. Thus, an essential part of the Brachypodium Tool Kit must be well-characterized germplasm collections (Filiz et al., 2009). Sequence data from comprehensive germplasm collections will yield markers linked to specific loci and help identify gene function, for example, improved stress tolerance or yields, enabling the isolation of corresponding alleles in grasses and cereals. Brachypodium genes could also be used directly to generate GM cereal derivatives. Beyond cereal ⁄ forage grass improvement strategies, a well-curated Brachypodium germplasm collection will also allow macro and micro level evolutionary trends to be modelled and related to past or present selection pressures. It should also be possible to predict losses in genetic diversity by genetics and ecological niche modelling, due to environmental change or human activities. Until recently, a salient feature of Brachypodium research has been the relative paucity of available germplasm. Much early work focused on only seven inbred lines (Bd1-1, Bd23, Bd3-1, Bd18-1, Bd21, Bd21-3 and Bd29) developed from USDA collections (http://www.ars-grin.gov/npgs) and another small collection of ABR accession (ABR1 through ABR7) originating mainly from Spain (Stace and Catalań collection, Leicester, UK). Now, however, there is a large collection of Turkish germplasm available: 195 diploid lines collected from 53 locations in Turkey (Filiz et al., 2009; Vogel et al., 2009). Within this large collection considerable variation in flowering time, seed size, and plant architecture was noted. Sixty-two wild accessions (first generation) were analysed with 43 SSR markers and the vast majority of individuals were homozygous, despite the presence of multiple alleles in the local population. Under laboratory conditions, intimately grown lines failed to outcross. This reflects near-cleistogamy in the diploid lines which, while facilitating genotype conservation, poses a considerable barrier to full exploitation of the Brachypodium Tool Box as this makes crosses and derivation of mapping families quite difficult. However, following on from an early demonstration of successful crossing of two Brachypodium accessions (Routledge et al., 2004), recombinant inbred lines have been derived (Garvin et al., 2008) and simple, very efficient step-by-step crossing protocols are now available (see http://www.ars.usda.gov/ SP2UserFiles/person/1931/BrachypodiumCrossing.pdf and http://brachypodium.pw.usda.gov). These relatively simple protocols represent an essential component of the Brachypodium Tool Box and we expect the populations of recombinant inbred lines developed by Garvin et al. (2008) will soon be added to by those from other groups. We are now greatly expanding the germplasm collection with numerous collections from Northern Spain (Fig. 3a). In this collection, we have concentrated on developing inbred lines from various environments. Thus, we have


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sampled individual seeds from 46 localities, from high altitudes in the foothills of the Pyrenees, to lowland areas around the flood plain of the Ebro river basin, coastal areas around Catalunya and a Balearic Island population collected from Ibiza (Table 1). Between 10 and 30 individuals were sampled from each site, with each sample separated from the others by at least 1 m to avoid sampling close relatives. This collection is being developed specifically to search for intrapopulation and interpopulation variation and to relate this genetic diversity to potential adaptation to environmental selection pressures. We also are seeking to determine the relationships between sympatric 2n = 10 and 2n = 30 Brachypodium populations, and we have at least one site where the two ploidy-level species are sympatric (Table 1, Bierge, Huesca, Spain). Preliminary assessment of genotypic variation in a total of 38 wild individual samples, representative of most of the Spanish Brachypodium germplasm localities (Table 1, Fig. 3a), involved comparison with five individual samples representing different Turkish Brachypodium lines (Vogel et al., 2009) and Bd21. The number of alleles surveyed per locus ranged from 2 to 14, with an average of 6.85 across the 44 samples studied. Six out of 14 screened loci showed total or predominant levels of homozygosity across the 44 germplasm lines assessed (with observed heterozygosity values ranging from 0 to 0.159). Relationships among individual samples were evaluated through Nei et al.’s (1983) Da distance-based neighbour-joining (NJ) reconstruction. The unrooted NJ phenogram (Fig. 3b) showed: a large Spanish clade formed by samples from medium to relatively high altitude Pyrenean and Prepyrenean localities (Fig. 3a ‘highland’) and their close low altitude Ebro river valley localities (Fig. 3a ‘lowland’); a Turkish–Iraqi clade formed by most of the samples surveyed from different altitudinal localities of Turkey, including the sequenced line Bd21 and a line developed from the same location, Bd21-3; and a ‘Miscellaneous’ clade formed by the coastal Balearic isles (Ibiza: Fig. 3a ‘island’) samples plus one sample from low altitude Tekirdag (Tek10, Turkey) and four samples from both highland and lowland Iberian localities. Although only a small number of Turkish–Iraqi germplasm representatives were tested, a greater genetic diversity was observed in wild Brachypodium individuals from the western Mediterranean region compared with those from the eastern. Variation in DNA sequences within other temperate Mediterranean grasses (e.g. Hordeum marinum, Jakob et al. 2007), has similarly suggested greater diversity in the lines from the western part of the region, which may have contained a larger number of glacial refugia during the last ice age from which a larger number of ‘bottlenecked’ populations would have been derived. More exhaustive comparative phylogeographical studies of Brachypodium populations across its native Mediterranean distribution area are currently underway by Catalań’s group to test this hypothesis.


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(a)

(b) Turkey- Iraq Spain

33

Miscellaneous

29

0.05

26

29

V. Targets for the Brachypodium Tool Box: key traits When Brachypodium was first suggested as a model, some key traits were highlighted which were suggested to be of relevance to temperate cereals and grasses (Draper et al., 2001). Subsequently, a large number of groups have begun to define the molecular basis of these traits and, as a consequence, assess to what extent these are of relevance to cereals, grasses and biofuel crops. 1. Pathogen attack Given the interests of the research groups which first suggested Brachypodium, many early papers focused on responses to plant–pathogen interactions (Draper et al.,


Fig. 3 The development of an ecotypic Brachypodium germplasm collection. (a) Sites of Brachypodium sampling in north-east Spain: ‘Lowland’, light blue circles; ‘Highland’, orange circles; ‘Coastal’, green circles; ‘Island’, dark blue circles. Insets are example images of a ‘lowland’ (Alfranca, Zaragoza, Spain), ‘highland’ (Puerto del Perdoń, Navarra, Spain), ‘coastal’ (Port Lligat, Cadaque´s, Girona, Spain) and ‘Island’ (Port des Torrents, Ibiza, Spain) region. (b) Preliminary assessment of genotypic variation of this new germplasm collection has been undertaken using 14 bacterial artificial chromosomes end sequences (BES) -derived tetranucleotide- trinucleotide- and dinucleotide-based repeats simple sequence repeat (SSR) markers described at the USDA Brachypodium web site (http:// brachypodium.pw.usda.gov/SSR/) (loci DB069I22, DB078E03, DB088E23, DB080E01, DH044F02, DB078M18, DH038F15, DB060E14, DB092H16, DB082M05, DB009L08, DB071F17, DH048N18, DH038H13, DB088J19). Descriptions of the germplasm together with either confirmed or tentative estimations of ploidy are given in Table 1. Neighbourjoining phenogram from 96 shared SSR alleles based on Nei & Tajima (1983) Da distance between 44 Spanish and Turkish– Iraqi Brachypodium wild-type germplasm lines. Bootstrap values on branches are based on 10 000 replications. Symbols: blue circle, Spanish highlands; red circle, Spanish lowlands; dark green square, Turkish–Iraqi (the Bd21 whole sequenced-genome sample is encircled); bright green square, Tekirdag 10 (Turkey); purple triangle, Balearic coastal.

2001). Following on from preliminary pathogen screens which included examining cereal-adapted fungal rust and mildew pathogens (Draper et al., 2001), the first well-characterized Brachypodium pathogen was Rice Blast (Magnaporthe grisea) (Routledge et al., 2004). Subsequent metabolomic analyses have suggested that phospholipid processing and jasmonate accumulation play important roles in the interaction of Brachypodium with M. grisea (Allwood et al., 2006). However, it is questionable how far Brachypodium should be pursued as a model for Rice Blast disease, given the availability of a genome sequence for rice and its excellent functional genomic infrastructure (Leung & An, 2004). Instead, there is a clear need for greater efforts to develop Brachypodium interactions for pathogens of temperate cereals. Perhaps most pressingly well-characterized interactions involving rust fungal pathogens are required (Ayliffe et al., 2008). Following


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Table 1 Inbred Brachypodium distachyon lines Accession name Bie2 Bie3 Bie10 Bie13 Bou2 Bou13 Bou18 Cel4 Foz1 Gal2 Men5 Men7 Men14 Men19 Men30 Mig3 Mon3 Mur3 Rei7 Sar2 Tor2 Tor7 Tor8 Uni2 Yas3

Site

Latitude

Longitude

Alt

Bierge, Sierra de Guara, Huesca, Spain

42.17305 N

0.09075 W

730

Cala De Bou, San Antonio, Ibiza, Spain

38.96774 N

1.26819 E

Santa Cilia, Sierra de Guara, Huesca, Spain Foz de Lumbier, Navarra, Spain Murillo de Gallego, Zaragoza, Spain Menarguens, Lleida, Spain

42.23836 42.63651 42.34552 41.72018

0.16599 1.30484 0.74299 0.72452

San Miguel de Foces, Ibieca, Huesca, Spain Puerto de Pallaruelo, Castejoń de Monegros, Zaragoza, Spain Castillo de Mur, Lleida, Spain Puente de la Reina, Huesca, Spain Laguna de Sarin˜ena, Huesca, Spain Port des Torrents, San Antonio, Ibiza, Spain

Escuela Politećnica Superior, Huesca, Spain Yaso, Sierra de Guara, Huesca, Spain

42.117408 N 42.20237 N

N N N N

3

W W W E

791 434 515 234

42.14799 N 41.65132 N

0.19497 W 0.21042 W

572 515

42.09763 42.56319 41.78619 38.96740

0.87750 E 0.78688 W 0.18278 W 1.2818 E

487

0.445046 W 0.12236 W

480 731

N N N N

292 6

Chromosome number (2n)

Genome size 2C (pg)

Standard deviation

10 10 30 30 30 30 30 10 10 10 30 30 30 30 30 10 10

0.681 0.662 1.314 1.316 1.319 1.293 1.336 0.693 0.694 0.670 1.287 1.278 1.275 NM NM 0.671 0.687

0.009 0.008 0.016 0.02 0.028 0.018 0.015 0.009

10 10 10 30 30 30 10 10

0.682 0.669 0.668 1.291 1.293 1.307 0.675 0.662

0.013 0.011 0.016 0.02 0.011 0.02 0.009 0.008

0.016 0.011 0.009 0.013 NM NM 0.008 0.019

NM, not measured.

from our early work on wheat and barley adapted yellow (Puccinia striiformis) and brown (Puccinia recondita) rusts, we have focused on crown rust (Puccinia coronata; J. V. R. Smith et al., unpublished). A model interaction between Brachypodium and the more important stem rust (Puccinia graminis) has also been established (David Garvin, USDA, St Paul, MN, USA, pers. comm.). However, (to the authors’ knowledge) no model interactions between Brachypodium and other major cereal pathogens, including Blumeria (powdery mildew), Septoria (the cause of leaf blotch), Fusarium graminearum (the cause of ear blight) and Rhizoctonia solani or Gaeumannomyces sp. (the cause of take all) have been established. We note that a model interaction between aphid feeding Schizaphis graminum and Diuraphis noxia and Brachypodium has been established (Azhaguvel et al., 2009) but others involving biting rather than piercing insects need to be developed. Of relevance to plant pathology are the comparative studies that have been made with defined resistance genes within the Brachypodium genome. Drader & Kleinhofs (2010) used genetic markers for barley chromosomes to construct a synthetic map with Brachypodium but with a particular focus on rust resistance genes. The stem rust resistance genes Rpg1 and Rpg4 have very similar orthologues but with Rpg5 key motifs, consisting of the -lLRR


and a serine ⁄ threonine protein kinase domain that is separately encoded on two Brachypodium contigs. In a wider genomic comparison Li et al. (2010) examined NBS-LRR genes in maize, sorghum, Brachypodium and rice. They observed considerable variation between species, with only 3.93% of NBS-LRR conforming to a conserved family. This compared with 96.1% conservation in selected housekeeping genes (Li et al., 2010). Although these comparisons were with nontemperate grasses, the lack of conservation was striking and most likely reflects rapid gene diversification as a result of pathogen-mediated selection pressures. 2. Cell wall and bioenergy crop characteristics Much of the funding for sequencing the Brachypodium genome originated from the US Department of Energy who foresaw its value as a model for energy grasses (DOE, 2006). Biofuel crops can contribute to the energy economy by serving as either direct combustion inputs into power stations or representing substrates for biofermentation processes leading the production of bioethanol or biodiesel (Chang, 2007; Gomez et al., 2008a,b). In particular, moving beyond simply fermenting easily extracted starch and sugar, the substantial resources captured within the lig-


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nocellulosic cell wall is proving to be an attractive target (Gomez et al., 2008a). Emerging biofuel crops such as Miscanthus and switchgrass (Panicum virgatum) therefore represent good sources of biomass with considerable potential. However, the conversion of lignocellulose to biofuel is a scientifically and economically demanding process whereby cell walls must be efficiently hydrolysed and the released sugars fermented. It is imperative to develop our understanding of the construction of the cell wall and its degradation and saccharification (Wyman, 2007). Alfalfa mutants with altered lignin levels have already proved useful in such studies (Chen & Dixon, 2007) but, with its genomic infrastructure, a focus on Brachypodium represents a cost effective strategy to rapidly gain greater insights. Indeed, a characterization of saccharification of Brachypodium stem segments under different pretreatment and hydrolysis conditions has already been published (Gomez et al., 2008b). Crucially, the composition of the Brachypodium cell wall was more similar to that of biomass grasses and cereals than that of Arabidopsis, confirming it to be a more suitable model species (Gomez et al., 2008b; Opanowicz et al., 2008). Christensen et al. (2010) compared the cell walls of Brachypodium, H. vulgare and T. aestivum, focusing particularly on hemicelluloses. This study showed that the presence of (1,3;1,4)-beta-D-glucans correlated with cell wall elongation but this could not be related to the transcriptional regulation of genes involved in (1,3-1,4)beta-D-glucan synthesis (cellulose synthase-like F family (CSLF)). Similarly, in all three species arabinoxylans increased during growth but in Brachypodium subsequent crosslinking via ferulic acid dimers and ester-linked pcoumaric acid were more prominent. In a key study, Van Hulle et al. (2010) demonstrated that by modulating genes involved in the Brachypodium cell wall biosynthesis the extractability of sugars could be improved. This important study validates Brachypodium as a biofuel crop model. This early work will surely be followed up by many. Senescence is also an important trait in a biofuel crop, in terms either of its delay to prolong plant greenness and thus increase sugar levels for fermentative processes, or of improving nutrient mobilization to overwintering organs in perennial crops. Brachypodium has been used along with switchgrass to study fatty acid mobilization and the initiation and development of senescence (Yang & Ohlrogge, 2009). 3. Abiotic stress tolerance Somewhat surprisingly, given its preference for growth on marginal ground or at higher altitudes, the potential of Brachypodium as a model to understand abiotic stress tolerance has yet to be addressed. There has been some limited work in this area using B. pinnatum (Hurst & John, 1999) or B. rupestre (Liancourt et al., 2005). Although focusing on


switchgrass, Matts et al. (2010) used high-throughput sequencing and cross-genomic data mining to identify 27 conserved miRNAs with possibly an additional 129 in Brachypodium. Some of the miRNAs proved to be modulated by cold, the vast majority of which were not conserved, suggesting some unique aspects to the response to cold in Brachypodium. As stresses such as drought and flooding, are important limiting factors to cereal crop yields (Cassman, 1999), we fully expect other studies into abiotic stress responses and tolerance to be published in the near future. 4. Grain characteristics One surprising feature of Brachypodium is its large grain size, relative to its stature. As has been frequently pointed out (Draper et al., 2001; Garvin et al., 2008; Opanowicz et al., 2008), in its length if not its width, Brachypodium seeds are similar to those of rice and wheat. Furthermore, its large seed head is also very prominent in natural environments (Fig. 1a). Thus, even though B. distachyon is an undomesticated grass species, workers have examined its seed storage accumulation and localization (Gu et al., 2010; Larre et al., 2010). Proteomic analyses have revealed the predominance of globulins which, based on their solubility, appeared to be similar to the rice seed storage protein glutelin (Laudencia-Chingcuanco & Vensel, 2008).It appears that some Brachypodium species may have been among the first grains to be processed by Palaeolithic hunter gatherers c. 30 000 yr ago (Revedin et al., 2010). Brachypodium has also been exploited in studies examining flowering. One key study demonstrated that the flowering transition gene GIGANTEA functions in a similar way in Brachypodium as in Arabidopsis (Hong et al., 2010). In another bioinformatic screening of existing B. distachyon EST databases, Unver & Budak (2009) detected 26 miRNA which were predicted to target as many as 246 mRNAs. Most of the putative target mRNAs encoded transcription factors, which were predicted to regulate plant development and flowering time (Unver & Budak, 2009). The potential practical application of such studies was shown by Olsen et al. (2006) who expressed Lolium and Arabidopsis Terminal Flower 1 genes in Brachypodium accessions to delay heading.

VI. Whence for the Brachypodium Tool box? Primus inter pares? It is very likely that, in the very near future, whole genomic sequence data will be available for all major cereal and forage crops. Some such as Lolium and barley (H. vulgare) have many attributes that make them good candidates for a model species. Lolium is physically small and easy to grow while barley is easy to grow and has functional genomic resources such as a TILLING population (Talame et al., 2008).


New Phytologist Further, Lolium has enormous value as a forage grass and barley as a cereal, neither of which is the case for Brachypodium. However, such considerations betray a monolithic view where one model species dominates research efforts, possibly influenced by the dominance that Arabidopsis has exerted on plant science. With the genomic difficulties of using actual crop species rapidly disappearing, we suggest a pluralistic approach where research employs model, semimodel and crop species as required. Such a strategy would also augment comparative genomic approaches whereby functions assigned in Brachypodium can be used to annotate as well as assemble grass and cereal genomes. The intermediate evolutionary position of Brachypodium between the core Pooideae (including Triticeae and Aveneae–Poeae) and the Ehrhartoideae (including Oryzeae) makes it particularly valuable for such approaches. Clearly, one species cannot adequately serve as a model of all biological traits (e.g. photosynthesis in C4 grasses) but, as outlined above, Brachypodium comes close. It can serve as a host for some important cereal pathogens, act as a biofuel crop model as a way of gaining insight into cell wall structures and biosynthesis, and can be used to study grain development. Even more importantly, its ease of growth and small stature make it a low (logistical) cost but high gain (in terms of insights) species to work on. When these inherent traits are combined with the established infrastructure (e.g. extremely efficient transformation, excellent genome sequence, growing T-DNA collections, microarrays, expanding sequence resources, etc.) it becomes clear that Brachypodium will remain an important model system. This stated, the Brachypodium Tool Box is not complete and the plant research community will benefit from continued investment, particularly in additional reverse genetic tools. The burgeoning number of germplasm collections also represents both an opportunity and a challenge. Harvesting genotypic variation will be essential to the functional exploitation of the Brachypodium genomic sequence(s); the germplasm needs to be available to all. Precedents set by TAIR (http://www.arabidopsis.org) and NASC (http:// arabidopsis.info/) demonstrate the need to have at least two geographically disparate sites through which germplasm can be maintained and distributed. This must be associated with a suitable web accessible interface, and http:// www.brachypodium.org is clearly developing into such a facility. However, the infrastructure and resources (human as well as computational) should not be underestimated; they are a challenge, especially as funds for such projects are becoming scarcer (Abbott, 2009). For those of us working with Brachypodium, further efforts are needed to develop the Brachypodium Tool Box so that it could fulfil its potential. That potential could be as a model that is ‘first amongst equals’ (i.e. a preferred model species but complementary to others).


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Acknowledgements We thank Mrs Dorota Siwinska (University of Silesia, Katowice) for her technical assistance with flow cytometry. This work was partially supported by the Polish Ministry of Science and Higher Education (grant N N303 570738). Pilar Catalań was funded by a Spanish Research Grant Project CGL2009-12955-C02-01. The establishment of the Mur-Catalań Spanish germplasm collection was supported by the Genetics Society (UK).

References Abbott A. 2009. Plant genetics database at risk as funds run dry. Nature 462: 258–259. Allwood JW, Ellis DI, Heald JK, Goodacre R, Mur LAJ. 2006. Metabolomic approaches reveal that phosphatidic and phosphatidyl glycerol phospholipids are major discriminatory non-polar metabolites in responses by Brachypodium distachyon to challenge by Magnaporthe grisea. Plant Journal 46: 351–368. Ayliffe M, Singh R, Lagudah E. 2008. Durable resistance to wheat stem rust needed. Current Opinion in Plant Biology 11: 187–192. Azhaguvel P, Li W, Rudd JC, Gill BS, Michels GJ Jr, Weng Y. 2009. Aphid feeding response and microsatellite-based genetic diversity among diploid Brachypodium distachyon (L.) Beauv accessions. Plant Genetic Resources Characterization and Utilization 7: 72–79. Brown TA, Jones MK, Powell W, Allaby RG. 2009. The complex origins of domesticated crops in the Fertile Crescent. Trends in Ecology & Evolution 24: 103–109. Cantacessi C, Jex AR, Hall RS, Young ND, Campbell BE, Joachim A, Nolan MJ, Abubucker S, Sternberg PW, Ranganathan S et al. 2010. A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing. Nucleic Acids Research 38: e171. Cassman KG. 1999. Ecological intensification of cereal production systems: yield potential, soil quality, and precision agriculture. Proceedings of the National Academy of Sciences, USA 96: 5952–5959. Catalan P, Kellogg EA, Olmstead RG. 1997. Phylogeny of Poaceae subfamily Pooideae based on chloroplast ndhF gene sequences. Molecular Phylogenetics and Evolution 8: 150–166. Catalan P, Olmstead RG. 2000. Phylogenetic reconstruction of the genus Brachypodium P-Beauv. (Poaceae) from combined sequences of chloroplast ndhF gene and nuclear ITS. Plant Systematics and Evolution 220: 1–19. Chang MCY. 2007. Harnessing energy from plant biomass. Current Opinion in Chemical Biology 11: 677–684. Chen F, Dixon RA. 2007. Lignin modification improves fermentable sugar yields for biofuel production. Nature Biotechnology 25: 759–761. Christensen U, Alonso-Simon A, Scheller HV, Willats WGT, Harholt J. 2010. Characterization of the primary cell walls of seedlings of Brachypodium distachyon – a potential model plant for temperate grasses. Phytochemistry 71: 62–69. Christiansen P, Andersen CH, Didion T, Folling M, Nielsen KK. 2005. A rapid and efficient transformation protocol for the grass Brachypodium distachyon. Plant Cell Reports 23: 751–758. Demircan T, Akkaya MS. 2010. Virus induced gene silencing in Brachypodium distachyon, a model organism for cereals. Plant Cell Tissue and Organ Culture 100: 91–96. Devos KM. 2010. Grass genome organization and evolution. Current Opinion in Plant Biology 13: 139–145. DOE, ED. 2006. Breaking the biological barriers to cellulosic ethanol: a joint research agenda. US Department of Energy, Office of Science and Office of Energy Efficiency. URL http://genomicscience.energy.gov/


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Tansley review

biofuels/2005workshop/2005low_lignocellulosic.pdf [accessed 29 April 2011]. Drader T, Kleinhofs A. 2010. A synteny map and disease resistance gene comparison between barley and the model monocot Brachypodium distachyon. Genome 53: 406–417. Draper J, Mur LAJ, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R, Routledge APM. 2001. Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiology 127: 1539–1555. Dubcovsky J, Dvorak J. 2007. Genome plasticity a key factor in the success of polyploid wheat under domestication. Science 316: 1862–1866. Ecker JR. 2002. A sequence-indexed library of insertion mutations in the Arabidopsis genome. Plant Physiology (Rockville) 129: 405–406. Edwards D, Batley J. 2010. Plant genome sequencing: applications for crop improvement. Plant Biotechnology Journal 8: 2–9. Erayman M, Sandhu D, Sidhu D, Dilbirligi M, Baenziger PS, Gill KS. 2004. Demarcating the gene-rich regions of the wheat genome. Nucleic Acids Research 32: 3546–3565. Faricelli ME, Valarik M, Dubcovsky J. 2010. Control of flowering time and spike development in cereals: the earliness per se Eps-1 region in wheat, rice, and Brachypodium. Functional & Integrative Genomics 10: 293–306. Febrer M, Goicoechea JL, Wright J, McKenzie N, Song X, Lin J, Collura K, Wissotski M, Yu Y, Ammiraju JS et al. 2010. An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research. PLoS One 18: e13461. Filiz E, Ozdemir BS, Budak F, Vogel JP, Tuna M, Budak H. 2009. Molecular, morphological, and cytological analysis of diverse Brachypodium distachyon inbred lines. Genome 52: 876–890. Gale MD, Rees H. 1970. Genes controlling chiasma frequency in Hordeum. Heredity 25: 3 93–410. Garvin DF, Gu YQ, Hasterok R, Hazen SP, Jenkins G, Mockler TC, Mur LAJ, Vogel JP. 2008. Development of genetic and genomic research resources for Brachypodium distachyon, a new model system for grass crop research. Crop Science 48: S69–S84. Garvin DF, McKenzie N, Vogel JP, Mockler TC, Blankenheim ZJ, Wright J, Cheema JJS, Dicks J, Huo NX, Hayden DM et al. 2010. An SSR-based genetic linkage map of the model grass Brachypodium distachyon. Genome 53: 1–13. Gomez LD, Bristow JK, Statham ER, McQueen-Mason SJ. 2008a. Analysis of saccharification in Brachypodium distachyon stems under mild conditions of hydrolysis. Biotechnology for Biofuels 1: 15. Gomez LD, Steele-King CG, McQueen-Mason SJ. 2008b. Sustainable liquid biofuels from biomass: the writing’s on the walls. New Phytologist 178: 473–485. Griffiths S, Sharp R, Foote TN, Bertin I, Wanous M, Reader S, Colas I, Moore G. 2006. Molecular characterization of Ph1 as a major chromosome pairing locus in polyploid wheat. Nature 439: 749–752. Gu YQ, Wanjugi H, Coleman-Derr D, Kong X, Anderson OD. 2010. Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins. Functional & Integrative Genomics 10: 111–122. Hartl J, Herrmann R. 2009. Do they always say no? German consumers and second-generation GM foods. Agricultural Economics 40: 551–560. Hasterok R, Draper J, Jenkins G. 2004. Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.) Beauv. Chromosome Research 12: 397–403. Hasterok R, Marasek A, Donnison IS, Armstead I, Thomas A, King IP, Wolny E, Idziak D, Draper J, Jenkins G. 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. Holzberg S, Brosio P, Gross C, Pogue GP. 2002. Barley stripe mosaic virusinduced gene silencing in a monocot plant. Plant Journal 30: 315–327. Hong SY, Lee S, Seo PJ, Yang MS, Park CM. 2010. Identification and molecular characterization of a Brachypodium distachyon GIGANTEA


New Phytologist gene: functional conservation in monocot and dicot plants. Plant Molecular Biology 72: 485–497. Huo NX, Lazo GR, Vogel JP, You FM, Ma YQ, Hayde DM, ColemanDerr D, Hill TA, Dvorak J, Anderson OD et al. 2008. The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences. Functional & Integrative Genomics 8: 135–147. Huo NX, Vogel JP, Lazo GR, You FM, Ma YQ, McMahon S, Dvorak J, Anderson OD, Luo MC, Gu YQ. 2009. Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat. Plant Molecular Biology 70: 47–61. Hurst A, John E. 1999. The biotic and abiotic changes associated with Brachypodium pinnatum dominance in chalk grassland in south-east England. Biological Conservation 88: 75–84. International Brachypodium Initiative (IBI). 2010. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463: 763–768. Jakob SS, Ihlow A, Blattner FR. 2007. Combined ecological niche modelling and molecular phylogeography revealed the evolutionary history of Hordeum marinum (Poaceae) – niche differentiation, loss of genetic diversity, and speciation in Mediterranean Quaternary refugia. Molecular Ecology 16: 1713–1727. Jenkins G, Hasterok R. 2007. BAC ‘landing’ on chromosomes of Brachypodium distachyon for comparative genome alignment. Nature Protocols 2: 88–98. Jenkins G, Mikhailova E, Phillips D, Hasterok R, Draper J, Jones N. 2005. Models and meiosis in the ‘omics era. In: Zwierzykowski Z, Kosmala A, eds. Recent advances in genetics and breeding of grasses. Poznan, Poland: Institute of Plant Genetics PAS, 97–104. Jones N, Rees H. 1966. Chiasma frequencies and potential genetic variability of Lolium populations. Nature 211: 432–433. Khan MA, Stace CA. 1999. Breeding relationships in the genus Brachypodium (Poaceae: Pooideae). Nordic Journal of Botany 19: 257–269. Kumar S, Mohan A, Balyan HS, Gupta PK. 2009. Orthology between genomes of Brachypodium, wheat and rice. BMC Research Notes 2: 93. Langridge P, Paltridge N, Fincher G. 2006. Functional genomics of abiotic stress tolerance in cereals. Briefings in Functional Genomics & Proteomics 4: 343–354. Larre C, Penninck S, Bouchet B, Lollier V, Tranquet O, Denery-Papini S, Guillon F, Rogniaux H. 2010. Brachypodium distachyon grain: identification and subcellular localization of storage proteins. Journal of Experimental Botany 61: 1771–1783. Laudencia-Chingcuanco DL, Vensel WH. 2008. Globulins are the main seed storage proteins in Brachypodium distachyon. Theoretical and Applied Genetics 117: 555–563. Leung H, An GL. 2004. Rice functional genomics: large-scale gene discovery and applications to crop improvement. Advances in Agronomy 82: 55–111. Li J, Ding J, Zhang W, Zhang YL, Tang P, Chen JQ, Tian DC, Yang SH. 2010. Unique evolutionary pattern of numbers of gramineous NBS-LRR genes. Molecular Genetics and Genomics 283: 427–438. Liancourt P, Corcket E, Michalet R. 2005. Stress tolerance abilities and competitive responses in a watering and fertilization field experiment. Journal of Vegetation Science 16: 713–722. Luo MC, Ma Y, You FM, Anderson OD, Kopecky´ D, Simkova´ H, Safa´r J, Dolezel J, Gill B, McGuire PE, Dvorak J. 2010. Feasibility of physical map construction from fingerprinted bacterial artificial chromosome libraries of polyploid plant species. BMC Genomics 11: 122–130. Luo MC, Thomas C, You FM, Hsiao J, Ouyang S, Buell CR, Malandro M, McGuire PE, Anderson OD, Dvorak J. 2003. High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis. Genomics 82: 378–389.


New Phytologist Matts J, Jagadeeswaran G, Roe BA, Sunkar R. 2010. Identification of microRNAs and their targets in switchgrass, a model biofuel plant species. Journal of Plant Physiology 167: 896–904. McCallum CM, Comai L, Greene EA, Henikoff S. 2000. Targeted screening for induced mutations. Nature Biotechnology 18: 455–457. Metzker ML. 2010. Sequencing technologies – the next generation. Nature Reviews Genetics 11: 31–46. Meyers BC, Kozik A, Griego A, Kuang HH, Michelmore RW. 2003. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15: 809–834. Nei M, Tajima F. 1983. Maximum likelihood estimation of the number of nucleotide substitutions for restriction sites data. Genetics 105: 207–216. Nei M, Tajima F, Tateno Y. 1983. Accuracy of estimated phylogenetic trees from molecular data. Journal of Molecular Evolution 19: 153–170. Newton AC, Akar T, Baresel JP, Bebeli PJ, Bettencourt E, Bladenopoulos KV, Czembor JH, Fasoula DA, Katsiotis A, Koutis K et al. 2010. Cereal landraces for sustainable agriculture. A review. Agronomy for Sustainable Development 30: 237–269. Olsen P, Lenk I, Jensen CS, Petersen K, Andersen CH, Didion T, Nielsen KK. 2006. Analysis of two heterologous flowering genes in Brachypodium distachyon demonstrates its potential as a grass model plant. Plant Science 170: 1020–1025. Opanowicz M, Vain P, Draper J, Parker D, Doonan JH. 2008. Brachypodium distachyon: making hay with a wild grass. Trends in Plant Science 13: 172–177. Pacak A, Geisler K, Jørgensen B, Barciszewska-Pacak M, Nilsson L, Nielsen TH, Johansen E, Grønlund M, Jakobsen I, Albrechtsen M. 2010. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat. Plant Methods 6: 26–33. Rees H. 1961. Genotypic control of chromosome form and behaviour. Botanical Review 27: 288–318. Rees H, Dale PJ. 1974. Chiasmata and variability in Lolium and Festuca populations. Chromosoma 47: 335–351. Revedin A, Aranguren B, Becattini R, Longo L, Marconi E, Lippi MM, Skakun N, Sinitsyn A, Spiridonova E, Svoboda J. 2010. Thirty thousand-year-old evidence of plant food processing. Proceedings of the National Academy of Sciences of the United States of America, 107: 18815–18819. Robertson IH. 1981. Chromosome-numbers in Brachypodium Beauv (Gramineae). Genetica 56: 55–60. Rosegrant MW, Cline SA. 2003. Global food security: challenges and policies. Science 302: 1917–1919. Routledge APM, Shelley G, Smith JV, Talbot NJ, Draper J, Mur LAJ. 2004. Magnaporthe grisea interactions with the model grass Brachypodium distachyon closely resemble those with rice (Oryza sativa). Molecular Plant Pathology 5: 253–265. Saintenac C, Falque M, Martin OC, Paux E, Feuillet C, Sourdille P. 2009. Detailed recombination studies along chromosome 3B provide new insights on crossover distribution in wheat (Triticum aestivum L.). Genetics 181: 393–403. Sanchez-Moran E, Armstrong SJ, Santos JL, Franklin FCH, Jones GH. 2002. Variation in chiasma frequency among eight accessions of Arabidopsis thaliana. Genetics 162: 1415–1422. Schenkelaars P. 2001. Uncertainty and reluctance: Europe and GM foods. Biotechnology and Development Monitor 47: 16–19. Schippmann U. 1991. Revision of the European species of the genus Brachypodium Palisot De Beauvois Poaceae. Boissiera 45: 1–250. Schwartz CJ, Doyle MR, Manzaneda AJ, Rey PJ, Mitchell-Olds T, Amasino RM. 2010. Natural variation of flowering time and vernalization responsiveness in Brachypodium distachyon. Bioenergy Research 3: 38–46.


Tansley review

Review

Scofield SR, Huang L, Brandt AS, Gill BS. 2005. Development of a virusinduced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiology 138: 2165–2173. Spielmeyer W, Singh RP, McFadden H, Wellings CR, Huerta-Espino J, Kong X, Appels R, Lagudah ES. 2008. Fine scale genetic and physical mapping using interstitial deletion mutants of Lr34 Yr18: a disease resistance locus effective against multiple pathogens in wheat. Theoretical and Applied Genetics 116: 481–490. Stace CA. 2010. New flora of the British Isles, 3rd edn. Cambridge, UK: Cambridge University Press. Talame V, Bovina R, Sanguineti MC, Tuberosa R, Lundqvist U, Salvi S. 2008. TILLMore, a resource for the discovery of chemically induced mutants in barley. Plant Biotechnology Journal 6: 477–485. Thole V, Worland B, Wright J, Bevan MW, Vain P. 2010. Distribution and characterization of more than 1000 T-DNA tags in the genome of Brachypodium distachyon community standard line Bd21. Plant Biotechnology Journal 8: 734–747. Turner A, Beales J, Faure S, Dunford RP, Laurie DA. 2005. The pseudoresponse regulator Ppd-H1 provides adaptation to photoperiod in barley. Science 310: 1031–1034. Unver T, Budak H. 2009. Conserved microRNAs and their targets in model grass species Brachypodium distachyon. Planta 230: 659–669. Vain P, Worland B, Thole V, McKenzie N, Alves SC, Opanowicz M, Fish LJ, Bevan MW, Snape JW. 2008. Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis. Plant Biotechnology Journal 6: 941. Van Hulle S, Roldan-Ruiz I, Van Bockstaele E, Muylle H. 2010. Functional analysis of genes involved in cell wall biosynthesis of the model species Brachypodium distachyon to improve saccharification. Sustainable Use of Genetic Diversity in Forage and Turf Breeding 5: 479–482. Venter JC, Smith HO, Hood L. 1996. A new strategy for genome sequencing. Nature 381: 364–366. Vogel J, Hill T. 2008. High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Reports 27: 471–478. Vogel JP, Gu YQ, Twigg P, Lazo GR, Laudencia-Chingcuanco D, Hayden DM, Donze TJ, Vivian LA, Stamova B, Coleman-Derr D. 2006. EST sequencing and phylogenetic analysis of the model grass Brachypodium distachyon. Theoretical and Applied Genetics 113: 186–195. Vogel JP, Tuna M, Budak H, Huo NX, Gu YQ, Steinwand MA. 2009. Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon. BMC Plant Biology 9: 88–99. Wyman CE. 2007. What is (and is not) vital to advancing cellulosicethanol. Trends in Biotechnology 25: 153–157. Wolny E, Hasterok R. 2009. Comparative cytogenetic analysis of the genomes of the model grass Brachypodium distachyon and its close relatives. Annals of Botany 104: 873–881. Wolny E, Lesniewska K, Hasterok R, Langdon T. 2011. Compact genomes and complex evolution in the genus Brachypodium. Chromosoma 120: 199–212. Xu GX, Ma H, Nei M, Kong HZ. 2009. Evolution of F-box genes in plants: different modes of sequence divergence and their relationships with functional diversification. Proceedings of the National Academy of Sciences, USA 106: 835–840. Yang ZL, Ohlrogge JB. 2009. Turnover of fatty acids during natural senescence of Arabidopsis, Brachypodium, and switchgrass and in Arabidopsis beta-oxidation mutants. Plant Physiology 150: 1981–1989. Yu GT, Cai XW, Harris MO, Gu YQ, Luo MC, Xu SS. 2009. Saturation and comparative mapping of the genomic region harboring Hessian fly resistance gene H26 in wheat. Theoretical and Applied Genetics 118: 1589–1599.


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