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Brachypodium Genomics

Brachypodium Genomics Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2008, Article ID 536104, 7 pages doi:10.1155/2008/536104 Review Article 1 2 1 1 Bahar Sogutmaz Ozdemir, Pilar Hernandez, Ertugrul Filiz, and Hikmet Budak Biological Science and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University Orhanli, 34956 Tuzla-Istanbul, Turkey Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Alameda del Obispo s/n, Apartado 4084, 14080 Cordoba, Spain Correspondence should be addressed to Hikmet Budak, budak@sabanciuniv.edu Received 19 July 2007; Accepted 25 November 2007 Recommended by P. K. Gupta Brachypodium distachyon (L.) Beauv. is a temperate wild grass species; its morphological and genomic characteristics make it a model system when compared to many other grass species. It has a small genome, short growth cycle, self-fertility, many diploid accessions, and simple growth requirements. In addition, it is phylogenetically close to economically important crops, like wheat and barley, and several potential biofuel grasses. It exhibits agricultural traits similar to those of these target crops. For cereal genomes, it is a better model than Arabidopsis thaliana and Oryza sativa (rice), the former used as a model for all flowering plants and the latter hitherto used as model for genomes of all temperate grass species including major cereals like barley and wheat. Increasing interest in this species has resulted in the development of a series of genomics resources, including nuclear sequences and BAC/EST libraries, together with the collection and characterization of other genetic resources. It is expected that the use of this model will allow rapid advances in generation of genomics information for the improvement of all temperate crops, particularly the cereals. Copyright © 2008 Hikmet Budak et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. INTRODUCTION genomics research conducted in this model grass species is briefly summarized in this review. Brachypodium P. Beauv (from the Greek brachys “short” and 2. BRACHYPODIUM GENOMICS AS A MODEL SYSTEM podion “a little foot,” referring to its subsessile spikelets, [1]) is a genus representing some temperate wild grass species. 2.1. Desirable attributes In particular, Brachypodium distachyon (L.) Beauv., also de- scribed as “purple false broom,” has recently emerged as a B. distachyon has many attributes that make it a suitable new model plant for the diverse and economically impor- model for conducting functional genomics research not only tant group of temperate grasses and herbaceous energy crops among cereal crops like wheat and barley, but also for biofuel [2]. Temperate crops such as wheat, barley, and forage grasses crops like Switchgrass [2]. Due to its small haploid genome are the basis for the food and feed supply. However, the size (∼355 Mbp) and availability of a polyploid series with a and complexity of their genomes are major barriers to ge- basic chromosome number of x = 5, (2n = 2x = 10), the nomics research and molecular breeding. Similarly, although diploid race of B. distachyon can be used as a model for the the herbaceous energy crops (especially grasses) are becom- much larger polyploid genomes of crops such as bread wheat ing novel target sources of renewable energy, very little is (16979 Mbp, 2n = 6x = 42), durum wheat (12030 Mbp, 2n known about the biological basis underlying their bioenergy = 4x = 28), and barley (5439 Mbp, 2n = 2x = 14) (all C- traits. Therefore, there is a growing need for a temperate values from [3]). Besides its small genome size, other de- grass model to address questions directly relevant both for sirable attributes include a small physical stature (approxi- improving grain crops and forage grasses that are indispens- mately 20 cm), self-fertility, lack of seed-shattering, a short able to our food/feed production systems, and for develop- lifecycle that is normally completed within 11–18 weeks de- ing grasses into superior energy crops. The present status of pending on the vernalization requirement [2](mightbeas 2 International Journal of Plant Genomics fast as 8 weeks under optimized conditions, [4]), and simple nuclear ITS to reconstruct phylogeny among these selected growth requirements with large planting density and easy ge- species within the genus [8]. Similarly, RFLPs and RAPDs netic transformation [4, 5]. This combination of desirable at- were used for nuclear genome analysis to establish the evo- tributes, together with the biological similarities with its tar- lutionary position of the genus. The genus Brachypodium get crops, is responsible for the recent research interest in this constitutes morphologically more or less closely resembling species. A few ecotypes of this taxon collected from diverse species that are native to different ecological regions such that geographic regions of Turkey are shown in Figure 1, indicat- B. distachyon is the Mediterranean annual, nonrhizomatous ing a high level of variation among different accessions. B. mexicanum is from the New World, B. pinnatum and B. Brachypodium species range from annuals to strongly sylvaticum are Eurasian, and B. rupestre is a European taxon rhizomatous perennials that exhibit breeding systems rang- [7]. In view of the above, B. distachyon has been proposed ing from strictly inbreeding to highly self-incompatible [6]. as an alternative model for functional genomics of temperate Some of the characteristic features of the genus Brachy- grasses [2]. podium include the following [17]: (i) hairy terminal ovary appendage, (ii) the single starch grains, (iii) the outermost 2.3. Brachypodium and the tribe Triticeae thick layer of the nucellus, (iv) the long narrow caryopsis, (v) spicate or racemose inflorescences, and (vi) hairy nod- The tribe Triticeae Dumort belongs to the grass family, ules [7]. Poaceae, and constitutes one of the economically most im- portant plant groups. It includes three major cereal crops, wheat, barley, and rye (belonging to the genera Triticum, 2.2. Brachypodium as a model system: a comparison Hordeum,and Secale, resp.), which are traditionally culti- with Arabidopsis and Oryza vated in the temperate zone. The tribe has basic chromosome The available genome sequences of the model plants Ara- number of seven, and contains taxa ranging from diploids bidopsis [9]and rice [10] are considered to be the major (2n = 2x = 14) to duodecaploids (2n = 12x = 84), including resources for plant genomics research. Nevertheless, these all intermediate ploidy levels [14]. Polyploidy (the most im- model species are not suitable for the functional genomics portant cytogenetic process in higher plants, [15]) and more studies of temperate grasses. Arabidopsis has all the desirable specifically, allopolyploidy, has played and plays a main role attributes for a model plant: it is small in size, grows eas- in the tribe’s evolution. With around 350 species, crossabil- ily and quickly (reaching maturity in 6 weeks), has a small ity barriers are poorly understood, and it is remarkable how diploid genome, is self-compatible and easily transformable. its species, even species in different genera, can be made to Its utility as a model system has been proven by the wealth hybridize even if they do not hybridize naturally. Therefore, of genomic discoveries, useful for a broad range of crops (in- the tribe also includes man-made crops such as ×Triticosecale cluding cereals) it has generated. However, as a dicot species, (triticale) and ×Tritordeum (an amphiploid of Triticum aes- it does not share with grass crops most of the biological tivum ×Hordeum chilense [16, 17]and Triticum aestivum × features related to agricultural traits and in this sense, rice Leymus arenarius [18]). It has also been possible to apply would provide a better alternative. The rice plant, however, interspecific and intergeneric hybridization to increase the does not fulfill the requirements of short size, rapid life cy- genetic variability of crops belonging to this tribe (mainly cle, inbreeding reproductive strategy, simple growth require- wheat, [19, 20]). ments, or easy transformation, thus imposing practical lim- As mentioned earlier, the chromosome numbers within itations. As a tropical species, it does not display all agro- B. distachyon accessions range from 10 to 30 [21], and the nomic traits that are relevant to temperate grasses, especially haploid genome size in diploid Brachypodium (2n = 2x = to forage grasses; these agronomic traits include resistance to 10) varies from 172 Mbp to 355 Mbp [2, 22], although the specific pathogens, freezing tolerance, vernalization, peren- former value may be an underestimate [22]; the genome niality, injury tolerance, meristem dormancy mechanisms, size is thus assumed to be approximately 355 Mbp. There- fore, within Poaceae, B. distachyon carries one of the small- mycorrhizae, sward ecology, or postharvest biochemistry of silage [2]. Moreover, rice is phylogenetically distant from the est genomes, which is intermediate between the genomes of Pooidae subfamily that includes wheat, barley, and temperate Arabidopsis thaliana with 157 Mbp, and rice with 490 Mbp grasses [11], whereas Brachypodium diverged from the an- (All C-values from [3]). These data are consistent with pre- cestral Pooidae clade immediately prior to the radiation of vious reports that species of Brachypodium have the small- the modern “core pooids” (Triticeae, Bromeae,and Avenae), est 5S rDNA spacer among the grasses, and contain less than which include majority of important temperate cereals and 15% highly repetitive DNA [7]. Additionally, GISH analy- forage grasses [8]. Based upon cytological, anatomical, and sis of somatic chromosomes has shown preponderance of physiological studies, Brachypodium is placed into its own repetitive DNA in the pericentromeric regions, reflecting the tribe Brachypodieae of the Poaceae family [12]. In fact, the compactness and economy of this genome [12]. This study perennial outbreeding species, B. sylvaticum (2n = 18) was also revealed structural uniformity of the diploid accessions considered suitable for study of archetypal grass centromere ABR1 and ABR5, confirming their status as model geno- sequences, which allowed detection of repetitive DNA se- types, from which two BAC libraries have recently been pre- quences that are conserved among wheat, maize, rice, and pared for functional genomics analysis [23]. Recent analysis Brachypodium [13]. Several species of this genus were stud- of BAC end sequences (BESs) also corroborates this unusu- ied using combined sequences of chloroplast ndhFgeneand ally compact genome [24, 25]. The other accessions having Hikmet Budak et al. 3 (a) (b) (c) (d) Figure 1: The Brachypodium distachyon lines grown under greenhouse conditions. (a) Seed heads are covered to prevent crosspollination in case it persists. (b), (c), and (d) seeds were collected from a diverse geographic region of Turkey. chromosome numbers in multiples of 10 suggested that this the establishment of phylogenetic relationships among dif- species has evolved as a polyploid series based upon 2n = 2x ferent species of the tribe. The large and complex genomes = 10, and that ecotypes that deviate from multiples of 10 of some members of the tribe are a main constraint for ge- evolved due to aneuploid or dysploid changes in chromo- nomics research within this tribe, which would be greatly fa- some number [21]. A cytotaxonomic analysis of the mem- cilitated with the availability of a suitable model species like bers of the polyploid series has revealed hybrid origin of sev- B. distachyon. eral of the polyploid genotypes, suggesting a complex evo- lution of this species that is not entirely based on chromo- 3. CURRENT STATUS OF BRACHYPODIUM GENOMICS some doubling [12]. For example, allotetraploid artificial hy- brids between B. distachyon and B. sylvaticum exhibited ir- 3.1. Development of inbred lines regular meiosis and infertility [6], although allotetraploids were fertile, one of them (ABR100) showed normal meio- Inbred lines make an important resource for genomics re- search. Keeping this in view, diploid inbred lines have been sis [12]. This indicates that either the constituent diploids of developed in B. distachyon by selfing [4]aswellasthrough this allotetraploid are more compatible in hybrids, or the hy- brids themselves have evolved pairing control mechanisms selection from segregating populations derived from crosses among diploid ecotypes [26]. similar to those of wheat and other allopolyploids. It has also been shown using GISH that the genomes of the con- stituent diploids remain separated in the allotetraploid, and 3.2. Development of transformation and that there is no recombination between homoeologous chro- regeneration protocols mosomes. These features make the natural polyploid hybrids within the genus Brachypodium a suitable material for the An efficient transformation procedure and an optimized isolation and characterization of diplodizing genes. plant regeneration protocol have been developed in B. dis- For the reasons stated above, the whole tribe Triticeae is tachyon. For instance, in a study reported in 1995, callus considered to be an enormous gene pool for crop improve- induction and plant regeneration from mature embryos, as ment, deserving efforts not only for its morphological, phys- well as in vitro clonal propagation of shoots were success- iological, genetic, and genomic characterization, but also for fully achieved in B. distachyon [27]. In our own studies also, 4 International Journal of Plant Genomics 3.4. Mutagenesis Mutagenesis with sodium azide was also successful in B. dis- tachyon although response to this mutagen differed among different accessions [30]. The results obtained were com- parable with those earlier obtained in barley and rice un- der higher concentrations of mutagens. Application of ethyl- methane sulphonate (EMS) is currently on the way in diploid Brachypodium accessions. 4. BRACHYPODIUM GENOMES: ADVANCES ON THE WAY Figure 2: Callus induction of mature Brachypodium distachyon em- bryos supplemented with 5 mg/L 2,4-D (2,4-dichlorophenoxyacetic 4.1. BAC-based physical maps acid). A BAC-based physical map of B. distachyon is being devel- oped at the John Innes Centre (Norwich, UK) as an aid to the international effort to make BAC-based physical maps of efficient callus formation from mature embryos of B. dis- the genomes of Chinese Spring bread wheat [31]. Since es- tachyon was successfully achieved using MS basal medium tablishing a physical map of the genome of bread wheat, one supplemented with sucrose and 2,4-dichlorophenoxyacetic of the most important crops worldwide, is a major challenge (2,4-D) acid at a concentration ranging from 2.5 to 5 mg\L due to the enormous size of the genome and its hexaploid (see Figure 2). The results are suggesting that in Brachy- constitution, it is expected that the availability of a Brachy- podium species, higher rates of callus induction can be podium physical map will greatly facilitate this task. The close achieved through the use of (i) MS basal medium [43] rather phylogenetic relationship of Brachypodium to wheat leads to than LS basal medium [44] (ii) sucrose rather than maltose, high similarity in gene sequences. Unambiguous hybridiza- and (iii) higher concentrations of auxin as a plant growth tion signals are also generated, when Brachypodium probes regulator (unpublished data). are used on wheat BAC filters and southern blots. Prelimi- Agrobacterium-mediated transformation involving inser- nary experiments have also shown that it is feasible to an- tions of single genes has also been achieved in several geno- chor Brachypodium BACs to the rice genome by BES to cre- types of B. distachyon (including diploid and polyploid taxa), ate an outline physical map. An outline physical map of B. giving T transgenic plants [4]. These transformation studies distachyon genotype, Bd3-1 using BES and fingerprinting, is also involved the diploid genotype Bd21, which has also been being established and will be used to start assembling contigs used for construction of BAC libraries [28] and for gener- in wheat chromosome groups [31]. ating 20440 ESTs [29]. Agrobacterium-mediated transforma- Another B. distachyon physical map is being developed tion was successful in 10 out of the 19 lines, with efficiencies at the University of California and US Department of Agri- ranging from 0.4% to 15% [4]. Embryogenic calli derived culture (USDA) [25] by using two BAC libraries constructed from immature embryos were also transformed through bi- from B. distachyon genotype, BD-21. These BACs are be- olistic transformation leading to transgene expression in T ing fingerprinted using snapshot-based fingerprinting. This progeny [2, 5]. In later study, transformation with an av- physical map of B. distachyon will also be integrated with erage efficiency of 5.3% was achieved. In this study, testing BES, again providing genome-wide Brachypodium resources of T as well as T generations and seed production in T 0 1 2 for sequence assembly, comparative genome analysis, gene was achieved within one year due to the short life cycle of B. isolation, and functional genomics analysis. distachyon [5], confirming its importance as a model plant species. 4.2. B. distachyon genome and retrotransposons 3.3. BAC libraries and expression sequence tags (ESTs) The genome of B. distachyon is also being examined for the presence, diversity, and distribution of the major BAC libraries of two diploid ecotypes of B. distachyon,ABR1 classes of plant transposable elements, particularly the retro- and ABR5, have also been constructed and have been used transposons [32], since retrotransposons comprise most to determine synteny among rice, Brachypodium, and other of the existing DNA between genes in the large cereal species of Poaceae family. For this purpose, BACs were genomes. The compact genome of B. distachyon contains marker-selected (BAC landing) using primers designed ac- relatively few retrotransposons, which include copia, gypsy, cording to previously mapped rice and Poaceae sequences. TRIM, and LARD groups of elements. The availability Most BACs hybridized as single loci in known Brachy- of retrotransposon sequences will facilitate the develop- podium chromosomes, whereas contiguous BACs colocalized ment of retrotransposon-based molecular markers like IRAP, on individual chromosomes, thus confirming conservation REMAP, SSAP, and RBIP markers, which have a variety of ap- of genome synteny [23]. plications. Hikmet Budak et al. 5 4.3. Genetic linkage maps useful in identifying additional markers for specific regions of wheat chromosomes. Agenetic mapof B. distachyon genotype, Bd21 is being devel- A 371-kb region in B. sylvaticum has already been se- oped by the International Brachypodium Initiative [33]. Ge- quenced was also compared with orthologous regions from netic maps will provide anchor points linking the genome of rice and wheat genomes [36]. In this region, Brachypodium Brachypodium with those of rice, wheat, and some biofuel and wheat showed perfect macrocolinearity, whereas rice crops, and will establish chromosome-scale physical maps contains an approximately 220-kb inversion. Using con- of BACs for whole genome sequencing. In order to develop served genomic and EST sequences, divergence between these genetic maps, mapping populations are being devel- Brachypodium and wheat was estimated to be 35–40 million oped, which currently comprise several hundred F lines de- years, which is significantly more recent than the divergence rived from the cross Bd21 × Bd3-1. Thesewillbeadvanced of rice and wheat, which is estimated to have occurred ap- to F6 to establish RILs that can serve as a common map- proximately 50 million years [37]. ping resource for the community. Several approaches have Chosen target loci from Brachypodium genome are also been used to identify polymorphisms between parents of the being sequenced and compared with genomic sequences mapping population. First, conserved orthologous sequence from a variety of plant species including the following: (i) (COS) markers derived from wheat and millet were used to wheat species (Triticum and Aegilops)withdifferent ploidy identify a set of 80 confirmed polymorphisms between these levels, (ii) rice, and (iii) B. sylvaticum, for which a BAC li- two parental lines (Bd21, Bd3-1). Another strategy was the brary is available [38, 39]. This comparison revealed that use of ESTs derived from Bd21 in order to identify addi- there is a better conservation of microcolinearity between tional polymorphisms [29]. The most productive approach wheat and Brachypodium orthologous regions than between has been to predict introns in Brachypodium genes, based on wheat and rice, as was also shown in an earlier study [36]. a comparison of Bd21 ESTs with the annotated rice genome For instance, sequence comparison at the grain hardness lo- sequence; nearly all primers designed from predicted introns cus shows that genes responsible for grain hardness/softness, gave amplified products in PCR reactions. Most markers de- which is seed quality trait in wheat, are absent from the rice veloped thus were polymorphic among the 5 diploid inbred orthologous region, but present in the B. sylvaticum ortholo- lines used for testing, and thus proved to be useful markers gous region. The gene density found in B. sylvaticum genome for genetic mapping. is comparable to that of rice (one gene per 8 kb). These re- sults illustrate that Brachypodium species may represent an intermediate model for wheat genome analysis. 4.4. Whole genome sequencing To test the potential of Brachypodium as a model for The Brachypodium nuclear genome is currently being se- the functional analysis of ryegrass (Lolium perenne)flower- quenced within a project that was funded in early 2006 by ing genes, expression of two Terminal Flower 1 orthologs, the US Department of Energy (DOE). A draft genome se- namely, LpTFL 1 (from L. perenne)and TFL 1 (from Ara- quence is expected to be completed by the end of 2007. bidopsis), was examined in two different B. distachyon acces- This project is generating a whole-genome shotgun se- sions [40]. Both these repressors significantly delayed head- ing date. The short life cycle of Brachypodium and the rapid quence of B. distachyon genotype, Bd21 genome, and is coupled with another project aimed at generating nearly transformation system allowed heading date scoring of T1s 250.000 ESTs. Data from both projects will be made pub- within the first year after transformation, thus demonstrat- ing the potential of Brachypodium as a model for ryegrass (L. licly available through an online database (BrachyBase at http://www.brachybase.org) and a community-dedicated perenne) also. portal (http://www.brachypodium.org). BrachyBase will en- Brachypodium is also being explored as a model for the able efficient exploitation of genome and transcriptome se- genomics research involving study of cereals-pathogen inter- quences to identify genes underlying traits and will facilitate actions. For instance, varying degrees of susceptibility and resistance to Magnaporthe grisea (economically destructive comparisons with other grass genomes [34]. Generation and analysis of over 60 000 BES from large- pathogen and casual agent of Rice Blast disease that can insert BAC clones has provided the first view of Brachy- also infect temperate cereals and forage grasses) have been found in several Brachypodium accessions. Aetiology of fun- podium genome composition, structure, and organization [35]. In this study, ∼10% of the BES show similarity gal development and disease progression in Brachypodium to known repetitive DNA sequences in existing databases, closely resembled those of rice infections; an overexpres- sion of genes that were homologous with barley genomic whereas ∼40% matched sequences in the EST database, which suggests that a considerable portion of the Brachy- probes was also observed [41]. Recent advances in Brachy- podium genome is transcribed. Gene-related BESs that were podium genomics also involved use of metabolic profiling identified for the Brachypodium genome were also aligned in using Fourier-transform infrared spectroscopy (FT-IR) for silico to the rice genome sequences. On the basis of gene co- high-throughput metabolic fingerprinting and electrospray ionization mass spectrometry (ESI-MS). These metabolomic linearity between Brachypodium and rice, conserved and di- verged regions were identified. BES with significant matches approaches have shown considerable differential phospho- to wheat ESTs that have been mapped to individual chromo- lipids processing of membrane lipids during M. grisea-B. dis- tachyon accessions ABR1 (susceptible) and ABR5 (resistant) some and bin positions were also identified. These BACs rep- resent regions that are colinear with mapped ESTs and will be interactions [42]. 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Davey, andP.T.Lynch,“Plantregen- and phosphatidyl glycerol phospholipids are major discrim- eration and micropropagation of Brachypodium distachyon,” inatory non-polar metabolites in responses by Brachypodium Plant Cell, Tissue and Organ Culture, vol. 42, no. 1, pp. 97–107, distachyon to challenge by Magnaporthe grisea,” The Plant Jour- 1995. nal, vol. 46, no. 3, pp. 351–368, 2006. [28] N. Huo, Y. Q. Gu, G. R. Lazo, et al., “Construction and charac- [43] T. Murashige and F. Skoog, “A revised medium for rapid terization of two BAC libraries from Brachypodium distachyon, growth bioassays with tobacco tissue cultures,” Physiologia a new model for grass genomics,” Genome,vol. 49, no.9,pp. Plantarum, vol. 15, no. 3, pp. 473–497, 1962. 1099–1108, 2006. [44] E. M. Linsmaier and F. Skoog, “Organic growth factor re- [29] J. P. Vogel, Y. Q. Gu, P. 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Trick, et al., “Developing a ge- netic map of Brachypodium distachyon Bd21,” in Plant & An- imal Genomes XV Conference, San Diego, Calif, USA, January [34] T. C. Mockler, S. Givan, C. Sullivan, and R. Shen, “Bioinfor- matics and genomics resources for Brachypodium distachyon,” in Plant & Animal Genomes XV Conference,San Diego, Calif, USA, January 2007. [35] Y. Q. Gu,N.Huo,G.R.Lazo, et al., “Towards Brachypodium genomics: analysis of 60,000 BAC end sequences and sequence comparison with cereal crops,” in Plant & Animal Genomes XV Conference, San Diego, Calif, USA, January 2007. [36] E. Bossolini, T. Wicker, P. A. Knobel, and B. Keller, “Compar- ison of orthologous loci from small grass genomes Brachy- podium and rice: implications for wheat genomics and grass genome annotation,” The Plant Journal, vol. 49, no. 4, pp. 704– 717, 2007. [37] A. H. 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Jensen, et al., “Analysis of two heterol- ogous flowering genes in Brachypodium distachyon demon- strates its potential as a grass model plant,” Plant Science, vol. 170, no. 5, pp. 1020–1025, 2006. [41] A. P. M. Routledge, G. Shelley, J. V. Smith, N. J. Talbot, J. Draper, and L. A. J. Mur, “Magnaporthe grisea interactions with the model grass Brachypodium distachyon closely resem- ble those with rice (Oryza sativa),” Molecular Plant Pathology, vol. 5, no. 4, pp. 253–265, 2004. [42] J. W. Allwood, D. I. Ellis, J. K. Heald, R. Goodacre, and L. A. J. 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Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2008, Article ID 536104, 7 pages doi:10.1155/2008/536104 Review Article 1 2 1 1 Bahar Sogutmaz Ozdemir, Pilar Hernandez, Ertugrul Filiz, and Hikmet Budak Biological Science and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University Orhanli, 34956 Tuzla-Istanbul, Turkey Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Alameda del Obispo s/n, Apartado 4084, 14080 Cordoba, Spain Correspondence should be addressed to Hikmet Budak, budak@sabanciuniv.edu Received 19 July 2007; Accepted 25 November 2007 Recommended by P. K. Gupta Brachypodium distachyon (L.) Beauv. is a temperate wild grass species; its morphological and genomic characteristics make it a model system when compared to many other grass species. It has a small genome, short growth cycle, self-fertility, many diploid accessions, and simple growth requirements. In addition, it is phylogenetically close to economically important crops, like wheat and barley, and several potential biofuel grasses. It exhibits agricultural traits similar to those of these target crops. For cereal genomes, it is a better model than Arabidopsis thaliana and Oryza sativa (rice), the former used as a model for all flowering plants and the latter hitherto used as model for genomes of all temperate grass species including major cereals like barley and wheat. Increasing interest in this species has resulted in the development of a series of genomics resources, including nuclear sequences and BAC/EST libraries, together with the collection and characterization of other genetic resources. It is expected that the use of this model will allow rapid advances in generation of genomics information for the improvement of all temperate crops, particularly the cereals. Copyright © 2008 Hikmet Budak et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. INTRODUCTION genomics research conducted in this model grass species is briefly summarized in this review. Brachypodium P. Beauv (from the Greek brachys “short” and 2. BRACHYPODIUM GENOMICS AS A MODEL SYSTEM podion “a little foot,” referring to its subsessile spikelets, [1]) is a genus representing some temperate wild grass species. 2.1. Desirable attributes In particular, Brachypodium distachyon (L.) Beauv., also de- scribed as “purple false broom,” has recently emerged as a B. distachyon has many attributes that make it a suitable new model plant for the diverse and economically impor- model for conducting functional genomics research not only tant group of temperate grasses and herbaceous energy crops among cereal crops like wheat and barley, but also for biofuel [2]. Temperate crops such as wheat, barley, and forage grasses crops like Switchgrass [2]. Due to its small haploid genome are the basis for the food and feed supply. However, the size (∼355 Mbp) and availability of a polyploid series with a and complexity of their genomes are major barriers to ge- basic chromosome number of x = 5, (2n = 2x = 10), the nomics research and molecular breeding. Similarly, although diploid race of B. distachyon can be used as a model for the the herbaceous energy crops (especially grasses) are becom- much larger polyploid genomes of crops such as bread wheat ing novel target sources of renewable energy, very little is (16979 Mbp, 2n = 6x = 42), durum wheat (12030 Mbp, 2n known about the biological basis underlying their bioenergy = 4x = 28), and barley (5439 Mbp, 2n = 2x = 14) (all C- traits. Therefore, there is a growing need for a temperate values from [3]). Besides its small genome size, other de- grass model to address questions directly relevant both for sirable attributes include a small physical stature (approxi- improving grain crops and forage grasses that are indispens- mately 20 cm), self-fertility, lack of seed-shattering, a short able to our food/feed production systems, and for develop- lifecycle that is normally completed within 11–18 weeks de- ing grasses into superior energy crops. The present status of pending on the vernalization requirement [2](mightbeas 2 International Journal of Plant Genomics fast as 8 weeks under optimized conditions, [4]), and simple nuclear ITS to reconstruct phylogeny among these selected growth requirements with large planting density and easy ge- species within the genus [8]. Similarly, RFLPs and RAPDs netic transformation [4, 5]. This combination of desirable at- were used for nuclear genome analysis to establish the evo- tributes, together with the biological similarities with its tar- lutionary position of the genus. The genus Brachypodium get crops, is responsible for the recent research interest in this constitutes morphologically more or less closely resembling species. A few ecotypes of this taxon collected from diverse species that are native to different ecological regions such that geographic regions of Turkey are shown in Figure 1, indicat- B. distachyon is the Mediterranean annual, nonrhizomatous ing a high level of variation among different accessions. B. mexicanum is from the New World, B. pinnatum and B. Brachypodium species range from annuals to strongly sylvaticum are Eurasian, and B. rupestre is a European taxon rhizomatous perennials that exhibit breeding systems rang- [7]. In view of the above, B. distachyon has been proposed ing from strictly inbreeding to highly self-incompatible [6]. as an alternative model for functional genomics of temperate Some of the characteristic features of the genus Brachy- grasses [2]. podium include the following [17]: (i) hairy terminal ovary appendage, (ii) the single starch grains, (iii) the outermost 2.3. Brachypodium and the tribe Triticeae thick layer of the nucellus, (iv) the long narrow caryopsis, (v) spicate or racemose inflorescences, and (vi) hairy nod- The tribe Triticeae Dumort belongs to the grass family, ules [7]. Poaceae, and constitutes one of the economically most im- portant plant groups. It includes three major cereal crops, wheat, barley, and rye (belonging to the genera Triticum, 2.2. Brachypodium as a model system: a comparison Hordeum,and Secale, resp.), which are traditionally culti- with Arabidopsis and Oryza vated in the temperate zone. The tribe has basic chromosome The available genome sequences of the model plants Ara- number of seven, and contains taxa ranging from diploids bidopsis [9]and rice [10] are considered to be the major (2n = 2x = 14) to duodecaploids (2n = 12x = 84), including resources for plant genomics research. Nevertheless, these all intermediate ploidy levels [14]. Polyploidy (the most im- model species are not suitable for the functional genomics portant cytogenetic process in higher plants, [15]) and more studies of temperate grasses. Arabidopsis has all the desirable specifically, allopolyploidy, has played and plays a main role attributes for a model plant: it is small in size, grows eas- in the tribe’s evolution. With around 350 species, crossabil- ily and quickly (reaching maturity in 6 weeks), has a small ity barriers are poorly understood, and it is remarkable how diploid genome, is self-compatible and easily transformable. its species, even species in different genera, can be made to Its utility as a model system has been proven by the wealth hybridize even if they do not hybridize naturally. Therefore, of genomic discoveries, useful for a broad range of crops (in- the tribe also includes man-made crops such as ×Triticosecale cluding cereals) it has generated. However, as a dicot species, (triticale) and ×Tritordeum (an amphiploid of Triticum aes- it does not share with grass crops most of the biological tivum ×Hordeum chilense [16, 17]and Triticum aestivum × features related to agricultural traits and in this sense, rice Leymus arenarius [18]). It has also been possible to apply would provide a better alternative. The rice plant, however, interspecific and intergeneric hybridization to increase the does not fulfill the requirements of short size, rapid life cy- genetic variability of crops belonging to this tribe (mainly cle, inbreeding reproductive strategy, simple growth require- wheat, [19, 20]). ments, or easy transformation, thus imposing practical lim- As mentioned earlier, the chromosome numbers within itations. As a tropical species, it does not display all agro- B. distachyon accessions range from 10 to 30 [21], and the nomic traits that are relevant to temperate grasses, especially haploid genome size in diploid Brachypodium (2n = 2x = to forage grasses; these agronomic traits include resistance to 10) varies from 172 Mbp to 355 Mbp [2, 22], although the specific pathogens, freezing tolerance, vernalization, peren- former value may be an underestimate [22]; the genome niality, injury tolerance, meristem dormancy mechanisms, size is thus assumed to be approximately 355 Mbp. There- fore, within Poaceae, B. distachyon carries one of the small- mycorrhizae, sward ecology, or postharvest biochemistry of silage [2]. Moreover, rice is phylogenetically distant from the est genomes, which is intermediate between the genomes of Pooidae subfamily that includes wheat, barley, and temperate Arabidopsis thaliana with 157 Mbp, and rice with 490 Mbp grasses [11], whereas Brachypodium diverged from the an- (All C-values from [3]). These data are consistent with pre- cestral Pooidae clade immediately prior to the radiation of vious reports that species of Brachypodium have the small- the modern “core pooids” (Triticeae, Bromeae,and Avenae), est 5S rDNA spacer among the grasses, and contain less than which include majority of important temperate cereals and 15% highly repetitive DNA [7]. Additionally, GISH analy- forage grasses [8]. Based upon cytological, anatomical, and sis of somatic chromosomes has shown preponderance of physiological studies, Brachypodium is placed into its own repetitive DNA in the pericentromeric regions, reflecting the tribe Brachypodieae of the Poaceae family [12]. In fact, the compactness and economy of this genome [12]. This study perennial outbreeding species, B. sylvaticum (2n = 18) was also revealed structural uniformity of the diploid accessions considered suitable for study of archetypal grass centromere ABR1 and ABR5, confirming their status as model geno- sequences, which allowed detection of repetitive DNA se- types, from which two BAC libraries have recently been pre- quences that are conserved among wheat, maize, rice, and pared for functional genomics analysis [23]. Recent analysis Brachypodium [13]. Several species of this genus were stud- of BAC end sequences (BESs) also corroborates this unusu- ied using combined sequences of chloroplast ndhFgeneand ally compact genome [24, 25]. The other accessions having Hikmet Budak et al. 3 (a) (b) (c) (d) Figure 1: The Brachypodium distachyon lines grown under greenhouse conditions. (a) Seed heads are covered to prevent crosspollination in case it persists. (b), (c), and (d) seeds were collected from a diverse geographic region of Turkey. chromosome numbers in multiples of 10 suggested that this the establishment of phylogenetic relationships among dif- species has evolved as a polyploid series based upon 2n = 2x ferent species of the tribe. The large and complex genomes = 10, and that ecotypes that deviate from multiples of 10 of some members of the tribe are a main constraint for ge- evolved due to aneuploid or dysploid changes in chromo- nomics research within this tribe, which would be greatly fa- some number [21]. A cytotaxonomic analysis of the mem- cilitated with the availability of a suitable model species like bers of the polyploid series has revealed hybrid origin of sev- B. distachyon. eral of the polyploid genotypes, suggesting a complex evo- lution of this species that is not entirely based on chromo- 3. CURRENT STATUS OF BRACHYPODIUM GENOMICS some doubling [12]. For example, allotetraploid artificial hy- brids between B. distachyon and B. sylvaticum exhibited ir- 3.1. Development of inbred lines regular meiosis and infertility [6], although allotetraploids were fertile, one of them (ABR100) showed normal meio- Inbred lines make an important resource for genomics re- search. Keeping this in view, diploid inbred lines have been sis [12]. This indicates that either the constituent diploids of developed in B. distachyon by selfing [4]aswellasthrough this allotetraploid are more compatible in hybrids, or the hy- brids themselves have evolved pairing control mechanisms selection from segregating populations derived from crosses among diploid ecotypes [26]. similar to those of wheat and other allopolyploids. It has also been shown using GISH that the genomes of the con- stituent diploids remain separated in the allotetraploid, and 3.2. Development of transformation and that there is no recombination between homoeologous chro- regeneration protocols mosomes. These features make the natural polyploid hybrids within the genus Brachypodium a suitable material for the An efficient transformation procedure and an optimized isolation and characterization of diplodizing genes. plant regeneration protocol have been developed in B. dis- For the reasons stated above, the whole tribe Triticeae is tachyon. For instance, in a study reported in 1995, callus considered to be an enormous gene pool for crop improve- induction and plant regeneration from mature embryos, as ment, deserving efforts not only for its morphological, phys- well as in vitro clonal propagation of shoots were success- iological, genetic, and genomic characterization, but also for fully achieved in B. distachyon [27]. In our own studies also, 4 International Journal of Plant Genomics 3.4. Mutagenesis Mutagenesis with sodium azide was also successful in B. dis- tachyon although response to this mutagen differed among different accessions [30]. The results obtained were com- parable with those earlier obtained in barley and rice un- der higher concentrations of mutagens. Application of ethyl- methane sulphonate (EMS) is currently on the way in diploid Brachypodium accessions. 4. BRACHYPODIUM GENOMES: ADVANCES ON THE WAY Figure 2: Callus induction of mature Brachypodium distachyon em- bryos supplemented with 5 mg/L 2,4-D (2,4-dichlorophenoxyacetic 4.1. BAC-based physical maps acid). A BAC-based physical map of B. distachyon is being devel- oped at the John Innes Centre (Norwich, UK) as an aid to the international effort to make BAC-based physical maps of efficient callus formation from mature embryos of B. dis- the genomes of Chinese Spring bread wheat [31]. Since es- tachyon was successfully achieved using MS basal medium tablishing a physical map of the genome of bread wheat, one supplemented with sucrose and 2,4-dichlorophenoxyacetic of the most important crops worldwide, is a major challenge (2,4-D) acid at a concentration ranging from 2.5 to 5 mg\L due to the enormous size of the genome and its hexaploid (see Figure 2). The results are suggesting that in Brachy- constitution, it is expected that the availability of a Brachy- podium species, higher rates of callus induction can be podium physical map will greatly facilitate this task. The close achieved through the use of (i) MS basal medium [43] rather phylogenetic relationship of Brachypodium to wheat leads to than LS basal medium [44] (ii) sucrose rather than maltose, high similarity in gene sequences. Unambiguous hybridiza- and (iii) higher concentrations of auxin as a plant growth tion signals are also generated, when Brachypodium probes regulator (unpublished data). are used on wheat BAC filters and southern blots. Prelimi- Agrobacterium-mediated transformation involving inser- nary experiments have also shown that it is feasible to an- tions of single genes has also been achieved in several geno- chor Brachypodium BACs to the rice genome by BES to cre- types of B. distachyon (including diploid and polyploid taxa), ate an outline physical map. An outline physical map of B. giving T transgenic plants [4]. These transformation studies distachyon genotype, Bd3-1 using BES and fingerprinting, is also involved the diploid genotype Bd21, which has also been being established and will be used to start assembling contigs used for construction of BAC libraries [28] and for gener- in wheat chromosome groups [31]. ating 20440 ESTs [29]. Agrobacterium-mediated transforma- Another B. distachyon physical map is being developed tion was successful in 10 out of the 19 lines, with efficiencies at the University of California and US Department of Agri- ranging from 0.4% to 15% [4]. Embryogenic calli derived culture (USDA) [25] by using two BAC libraries constructed from immature embryos were also transformed through bi- from B. distachyon genotype, BD-21. These BACs are be- olistic transformation leading to transgene expression in T ing fingerprinted using snapshot-based fingerprinting. This progeny [2, 5]. In later study, transformation with an av- physical map of B. distachyon will also be integrated with erage efficiency of 5.3% was achieved. In this study, testing BES, again providing genome-wide Brachypodium resources of T as well as T generations and seed production in T 0 1 2 for sequence assembly, comparative genome analysis, gene was achieved within one year due to the short life cycle of B. isolation, and functional genomics analysis. distachyon [5], confirming its importance as a model plant species. 4.2. B. distachyon genome and retrotransposons 3.3. BAC libraries and expression sequence tags (ESTs) The genome of B. distachyon is also being examined for the presence, diversity, and distribution of the major BAC libraries of two diploid ecotypes of B. distachyon,ABR1 classes of plant transposable elements, particularly the retro- and ABR5, have also been constructed and have been used transposons [32], since retrotransposons comprise most to determine synteny among rice, Brachypodium, and other of the existing DNA between genes in the large cereal species of Poaceae family. For this purpose, BACs were genomes. The compact genome of B. distachyon contains marker-selected (BAC landing) using primers designed ac- relatively few retrotransposons, which include copia, gypsy, cording to previously mapped rice and Poaceae sequences. TRIM, and LARD groups of elements. The availability Most BACs hybridized as single loci in known Brachy- of retrotransposon sequences will facilitate the develop- podium chromosomes, whereas contiguous BACs colocalized ment of retrotransposon-based molecular markers like IRAP, on individual chromosomes, thus confirming conservation REMAP, SSAP, and RBIP markers, which have a variety of ap- of genome synteny [23]. plications. Hikmet Budak et al. 5 4.3. Genetic linkage maps useful in identifying additional markers for specific regions of wheat chromosomes. Agenetic mapof B. distachyon genotype, Bd21 is being devel- A 371-kb region in B. sylvaticum has already been se- oped by the International Brachypodium Initiative [33]. Ge- quenced was also compared with orthologous regions from netic maps will provide anchor points linking the genome of rice and wheat genomes [36]. In this region, Brachypodium Brachypodium with those of rice, wheat, and some biofuel and wheat showed perfect macrocolinearity, whereas rice crops, and will establish chromosome-scale physical maps contains an approximately 220-kb inversion. Using con- of BACs for whole genome sequencing. In order to develop served genomic and EST sequences, divergence between these genetic maps, mapping populations are being devel- Brachypodium and wheat was estimated to be 35–40 million oped, which currently comprise several hundred F lines de- years, which is significantly more recent than the divergence rived from the cross Bd21 × Bd3-1. Thesewillbeadvanced of rice and wheat, which is estimated to have occurred ap- to F6 to establish RILs that can serve as a common map- proximately 50 million years [37]. ping resource for the community. Several approaches have Chosen target loci from Brachypodium genome are also been used to identify polymorphisms between parents of the being sequenced and compared with genomic sequences mapping population. First, conserved orthologous sequence from a variety of plant species including the following: (i) (COS) markers derived from wheat and millet were used to wheat species (Triticum and Aegilops)withdifferent ploidy identify a set of 80 confirmed polymorphisms between these levels, (ii) rice, and (iii) B. sylvaticum, for which a BAC li- two parental lines (Bd21, Bd3-1). Another strategy was the brary is available [38, 39]. This comparison revealed that use of ESTs derived from Bd21 in order to identify addi- there is a better conservation of microcolinearity between tional polymorphisms [29]. The most productive approach wheat and Brachypodium orthologous regions than between has been to predict introns in Brachypodium genes, based on wheat and rice, as was also shown in an earlier study [36]. a comparison of Bd21 ESTs with the annotated rice genome For instance, sequence comparison at the grain hardness lo- sequence; nearly all primers designed from predicted introns cus shows that genes responsible for grain hardness/softness, gave amplified products in PCR reactions. Most markers de- which is seed quality trait in wheat, are absent from the rice veloped thus were polymorphic among the 5 diploid inbred orthologous region, but present in the B. sylvaticum ortholo- lines used for testing, and thus proved to be useful markers gous region. The gene density found in B. sylvaticum genome for genetic mapping. is comparable to that of rice (one gene per 8 kb). These re- sults illustrate that Brachypodium species may represent an intermediate model for wheat genome analysis. 4.4. Whole genome sequencing To test the potential of Brachypodium as a model for The Brachypodium nuclear genome is currently being se- the functional analysis of ryegrass (Lolium perenne)flower- quenced within a project that was funded in early 2006 by ing genes, expression of two Terminal Flower 1 orthologs, the US Department of Energy (DOE). A draft genome se- namely, LpTFL 1 (from L. perenne)and TFL 1 (from Ara- quence is expected to be completed by the end of 2007. bidopsis), was examined in two different B. distachyon acces- This project is generating a whole-genome shotgun se- sions [40]. Both these repressors significantly delayed head- ing date. The short life cycle of Brachypodium and the rapid quence of B. distachyon genotype, Bd21 genome, and is coupled with another project aimed at generating nearly transformation system allowed heading date scoring of T1s 250.000 ESTs. Data from both projects will be made pub- within the first year after transformation, thus demonstrat- ing the potential of Brachypodium as a model for ryegrass (L. licly available through an online database (BrachyBase at http://www.brachybase.org) and a community-dedicated perenne) also. portal (http://www.brachypodium.org). BrachyBase will en- Brachypodium is also being explored as a model for the able efficient exploitation of genome and transcriptome se- genomics research involving study of cereals-pathogen inter- quences to identify genes underlying traits and will facilitate actions. For instance, varying degrees of susceptibility and resistance to Magnaporthe grisea (economically destructive comparisons with other grass genomes [34]. Generation and analysis of over 60 000 BES from large- pathogen and casual agent of Rice Blast disease that can insert BAC clones has provided the first view of Brachy- also infect temperate cereals and forage grasses) have been found in several Brachypodium accessions. Aetiology of fun- podium genome composition, structure, and organization [35]. In this study, ∼10% of the BES show similarity gal development and disease progression in Brachypodium to known repetitive DNA sequences in existing databases, closely resembled those of rice infections; an overexpres- sion of genes that were homologous with barley genomic whereas ∼40% matched sequences in the EST database, which suggests that a considerable portion of the Brachy- probes was also observed [41]. Recent advances in Brachy- podium genome is transcribed. Gene-related BESs that were podium genomics also involved use of metabolic profiling identified for the Brachypodium genome were also aligned in using Fourier-transform infrared spectroscopy (FT-IR) for silico to the rice genome sequences. On the basis of gene co- high-throughput metabolic fingerprinting and electrospray ionization mass spectrometry (ESI-MS). These metabolomic linearity between Brachypodium and rice, conserved and di- verged regions were identified. BES with significant matches approaches have shown considerable differential phospho- to wheat ESTs that have been mapped to individual chromo- lipids processing of membrane lipids during M. grisea-B. dis- tachyon accessions ABR1 (susceptible) and ABR5 (resistant) some and bin positions were also identified. These BACs rep- resent regions that are colinear with mapped ESTs and will be interactions [42]. Brachypodium distachyon, being a host for 6 International Journal of Plant Genomics M. grisea and other disease-causing pathogens of Pooid ce- Plant Systematics and Evolution, vol. 220, no. 1-2, pp. 1–19, reals [42], is a suitable model for conducting functional ge- [9] The Arabidopsis Genome Initiative, “Analysis of the genome nomics research involving study of M. grisea pathology and sequence of the flowering plant Arabidopsis thaliana,” Nature, plant responses [41]. vol. 408, no. 6814, pp. 796–815, 2000. [10] International Rice Genome Sequencing Project, “The map- 5. CONCLUSIONS based sequence of the rice genome,” Nature, vol. 436, no. 7052, pp. 793–800, 2005. With the small genome size and simple growth requirements, [11] E. A. Kellogg, “Evolutionary history of the grasses,” Plant Phys- Brachypodium provides us with a genome, which is a model iology, vol. 125, no. 3, pp. 1198–1205, 2001. for in-depth understanding of functional genomics of tem- [12] R. Hasterok, J. Draper, and G. 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