The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences

Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of 10.9% of the Brachypodium genome based on 64,696 bacterial artificial chromosome (BAC) end sequences (BES). Analysis of repeat DNA content in BES revealed that approximately 11.0% of the genome consists of known repetitive DNA. The vast majority of the Brachypodium repetitive elements are LTR retrotransposons. While Bare-1 retrotransposons are common to wheat and barley, Brachypodium repetitive element sequence-1 (BRES-1), closely related to Bare-1, is also abundant in Brachypodium. Moreover, unique Brachypodium repetitive element sequences identified constitute approximately 7.4% of its genome. Simple sequence repeats from BES were analyzed, and flanking primer sequences for SSR detection potentially useful for genetic mapping are available at . Sequence analyses of BES indicated that approximately 21.2% of the Brachypodium genome represents coding sequence. Furthermore, Brachypodium BES have more significant matches to ESTs from wheat than rice or maize, although these species have similar sizes of EST collections. A phylogenetic analysis based on 335 sequences shared among seven grass species further revealed a closer relationship between Brachypodium and Triticeae than Brachypodium and rice or maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. N. Huo and G.R. Lazo contributed equally to this work.     

Molecular phylogeny of the grass genus Brachypodium P. Beauv. based on RFLP and RAPD analysis

Nuclear genome analysis using RFLPs and RAPDs has been assessed within different species of the genus Brachypodium P. Beauv. and representatives of other grasses in order to determine the characteristics of the Brachypodium genome and to establish its evolutionary position in relation to other Pooideae. Distinctive features of the Brachypodium genome are its small size, the low amount of repetitive DNA, the lack of restriction fragment length polymorphisms within the genus for the assayed probe/enzyme combinations, and the genomic variability demonstrated at species level by random DNA amplification. These molecular studies confirm Brachypodium as an isolated ancient genus best placed in its own tribe (Brachypodieae). Its relationships to other tribes Bromeae, Triticeae, Poeae are resolved, Brachypodieae being the earliest tribe to diverge from this core of pooids. Within the genus two major Old World clades are distinguishable: an annual clade, represented only by B. distachyon; and a perennial clade, represented by all the other species studied (except B. mexicanum). The perennial American species B. mexicanum appears equally attached to these two clades. RFLP data were found to be useful in obtaining phylogenies at generic and higher rank levels, whereas the highly variable RAPD data were more suitable for resolving interspecific and intraspecific evolutionary pathways.     

Genomic relationships among diploid wild perennial species of the genus Glycine Willd. subgenus Glycine revealed by crossability,meiotic chromosome pairing and seed protein electrophoresis

Summary The nomenclature of species beased on classical taxonomy can be verified from cytogenetic, biochemical and molecular studies. The objective of the study presented here was to provide further information on genomic affinities among species of the genus Glycine Willd. based on crossability, meiotic chromosome pairing of F₁ hybrids and seed-protein profiles. Meiotic chromosome pairing data revealed no genomic similarity between G. microphylla (BB) and G. falcata (FF), nor between G. tomentella (2n = 38; EE) and G. microphylla (BB). Despite morphological similarity between G. cyrtoloba (CC) and G. curvata no F₁ hybrid was obtained, although 748 flowers were pollinated. The seed-protein banding patterns showed G. latrobeana to be closer to the A-genome species than to others. Based on these results we assign genome symbol A₃A₃ to G. latrobeana. Likewise, G. curvata was allotted the designation C₁C₁ because the seed-protein banding patterns of G. curvata and G. cyrtoloba are similar. The genome designations of Glycine species based on cytogenetic investigations may be further extended by results obtained from biochemical and molecular approaches.Research supported in part by the Illinois Agricultural Experiment Station and US Department of Agriculture Competitive Research Grant 88-37231-4100     

Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat

Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecules. The analysis revealed that ~26.4% of BES have significant matches with the rice genome and 82.4% of the matches were homologous to known genes. Further analysis of paired-end BES and ~1.0 Mb sequences from nine selected BACs proved to be useful in revealing conserved regions and regions that have undergone considerable genomic changes. Differential gene amplification, insertions/deletions and inversions appeared to be the common evolutionary events that caused variations of microcolinearity at different orthologous genomic regions. It was found that ~17% of genes in the two genomes are not colinear in the orthologous regions. Analysis of BAC sequences also revealed higher gene density (~9 kb/gene) and lower repeat DNA content (~13.1%) in Brachypodium when compared to the orthologous rice regions, consistent with the smaller size of the Brachypodium genome. The 119 annotated Brachypodium genes were BLASTN compared against the wheat EST database and deletion bin mapped wheat ESTs. About 77% of the genes retrieved significant matches in the EST database, while 9.2% matched to the bin mapped ESTs. In some cases, genes in single Brachypodium BACs matched to multiple ESTs that were mapped to the same deletion bins, suggesting that the Brachypodium genome will be useful for ordering wheat ESTs within the deletion bins and developing specific markers at targeted regions in the wheat genome.     

Natural variation of drought response in Brachypodium distachyon

Brachypodium distachyon (Brachypodium) is a temperate wild grass species and is a powerful model system for studying grain, energy, forage and turf grasses. Exploring the natural variation in the drought response of Brachypodium provides an important basis for dissecting the genetic network of drought tolerance. Two experiments were conducted in a greenhouse to assess the drought tolerance of 57 natural populations of Brachypodium. Principle component analysis revealed that reductions in chlorophyll fluorescence (Fv/Fm) and leaf water content (LWC) under drought stress explained most of the phenotypic variation, which was used to classify the tolerant and susceptible accessions. Four groups of accessions differing in drought tolerance were identified, with 3 tolerant, 16 moderately tolerant, 32 susceptible and 6 most susceptible accessions. The tolerant group had little leaf wilting and fewer reductions in Fv/Fm and LWC, while the most susceptible groups showed severe leaf wilting and more reductions in Fv/Fm and LWC. Drought stress increased total water soluble sugar (WSS) concentration, but no differences in the increased WSS were found among different groups of accessions. The large phenotypic variation of Brachypodium in response to drought stress can be used to identify genes and alleles important for the complex trait of drought tolerance.     

Fine-scale spatial distribution of leaves and shoots of two chalk grassland perennials

The fine-scale spatial distribution of leaves and shoots of Brachypodium pinnatum and Carex flacca, two rhizomatous graminoids, was investigated in two chalk grasslands in South Limburg (The Netherlands). The objective was to examine whether leaves and shoots of Brachypodium, a dominant species, had a regular distribution on a small scale, as has been suggested for other clonal species that form high-density stands. Patterns were compared to Carex, which is never found to be as abundant as Brachypodium.The number of shoots and leaf contacts were counted in small quadrats, grouped in a grid. Using Moran's I analysis for autocorrelation, it appeared that leaves and shoots of both species were arranged in clumps, and that these clumps were randomly distributed across the soil surface. Shoot clumps in Carex were smaller in diameter and not as pronounced as those in Brachypodium.In most cases, patterns of leaves and shoots were positively correlated, indicating that leaves were predominantly positioned above and around the groups of quadrats where the shoots were attached. However, in dense stands of Brachypodium the positions of leaf clumps were not correlated to those of shoot clumps. This is a result of the tall growth form of this species and its high shoot densities, and it is suggested that this will be a characteristic of any species that dominates a dense stand.     

Effects of selective clipping and mowing time on species diversity in chalk grassland

Phytomass distribution and species diversity were followed in a chalk grassland with different selective clipping treatments during 4 years. Removal of the dominantBrachypodium resulted in a sharp increase in forb phytomass and in species number. The dominance-diversity curve changed in shape from geometric to log-normal. Obviously,Brachypodium restricted the growth of most other species. Removal of all species except the dominant showed, in contrast to the niche pre-emption theory, a marked increase in phytomass and reproductive effort ofBrachypodium. It seems that other mechanisms can also lead to a geometric distribution. Removal in a type of vegetation without obvious dominants and with a log-normal species distribution revealed that interspecific competition is not an important factor in this vegetation. This suggested a low intensity of species interaction and a large influence of restricting density-independent factors. Finally, to reduce the dominance ofBrachypodium we implemented a 4-year mowing experiment. Mowing earlier in the season resulted in a reduced exclusion of species byBrachypodium. This alternative mowing time may be sufficient to restore species diversity in chalk grassland. Experiments are under way to further substantiate this conclusion.     

Brachypodium distachyon as alternative model host system for the ergot fungus Claviceps purpurea

To investigate its susceptibility to ergot infection, we inoculated Brachypodium distachyon with Claviceps purpurea and compared the infection symptoms with those on rye (Secale cereale). We showed that, after inoculation of Brachypodium with Claviceps, the same disease symptoms occurred in comparable temporal and spatial patterns to those on rye. The infection rate of Claviceps on this host was reduced compared with rye, but the disease could be surveyed by fungal genomic DNA quantification. Mutants of Claviceps which were virulence attenuated on rye were also affected on Brachypodium. We were able to show that pathogenesis‐related gene expression changed in a typical manner for biotrophic pathogen attack. Our results indicated that the Claviceps–Brachypodium interaction was dependent on salicylic acid, cytokinin and auxin. We consider Brachypodium to be a suitable and useful alternative host; the increased sensitivity compared with rye will be valuable for the identification of infection mechanisms. Future progess in understanding the Claviceps–plant interaction will be facilitated by the use of a well‐characterized model host system.     

Identification of gamma irradiated Brachypodium mutants with altered genes responsible for lignin biosynthesis

Brachypodium distachyon (Brachypodium) is a novel model plant for structural and functional genomic studies of Poaceae. Brachypodium has many favorable features, such as small size, small genome, short life cycle, and easy handling. Bioethanol, as renewable resource, has been widely studied as a replacement for fossil fuels. Lignin is involved with the efficiency of energy feedstock. It is generally accepted that bioethanol production is negatively affected by lignin content. Brachypodium was irradiated with gamma irradiation, at doses of 50, 100, 150, 200, and 250 Gy, and 25 M₂ plants that showed the least staining with phloroglucinol were selected. Nucleotide alteration within genes that contribute to the lignin biosynthesis pathway was analyzed. In total, 4 INDELs and 249 SNPs which included 2 additional nonsense mutations, a mutation at the start codon, and a mutation at the 3′ splicing site were identified in the M₂ lines. The transition/transversion rate was 7.59, and single nucleotide substitutions were found every 1,143 bp. As biological resources, the M₂ populations generated in this work will contribute to functional genomics of Brachypodium and to the breeding of grass crops.     

Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon

Brachypodium distachyon (Brachypodium) has been proposed as a model temperate grass because its physical, genetic, and genome attributes (small stature, simple growth requirements, small genome size, availability of diploid ecotypes, annual lifecycle and self fertility) are suitable for a model plant system. Two additional requirements that are necessary before Brachypodium can be widely accepted as a model system are an efficient transformation system and homogeneous inbred reference genotypes. Here we describe the development of inbred lines from 27 accessions of Brachypodium. Determination of c-values indicated that five of the source accessions were diploid. These diploid lines exhibit variation for a variety of morphological traits. Conditions were identified that allow generation times as fast as two months in the diploids. An Agrobacterium-mediated transformation protocol was developed and used to successfully transform 10 of the 19 lines tested with efficiencies ranging from 0.4% to 15%. The diploid accession Bd21 was readily transformed. Segregation of transgenes in the T ₁ generation indicated that most of the lines contained an insertion at a single genetic locus. The new resources and methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.     

Infection of Brachypodium distachyon by Formae Speciales of Puccinia graminis: Early Infection Events and Host-Pathogen Incompatibility

Puccinia graminis causes stem rust, a serious disease of cereals and forage grasses. Important formae speciales of P. graminis and their typical hosts are P. graminis f. sp. tritici (Pg-tr) in wheat and barley, P. graminis f. sp. lolii (Pg-lo) in perennial ryegrass and tall fescue, and P. graminis f. sp. phlei-pratensis (Pg-pp) in timothy grass. Brachypodium distachyon is an emerging genetic model to study fungal disease resistance in cereals and temperate grasses. We characterized the P. graminis-Brachypodium pathosystem to evaluate its potential for investigating incompatibility and non-host resistance to P. graminis. Inoculation of eight Brachypodium inbred lines with Pg-tr, Pg-lo or Pg-pp resulted in sporulating lesions later accompanied by necrosis. Histological analysis of early infection events in one Brachypodium inbred line (Bd1-1) indicated that Pg-lo and Pg-pp were markedly more efficient than Pg-tr at establishing a biotrophic interaction. Formation of appressoria was completed (60–70% of germinated spores) by 12 h post-inoculation (hpi) under dark and wet conditions, and after 4 h of subsequent light exposure fungal penetration structures (penetration peg, substomatal vesicle and primary infection hyphae) had developed. Brachypodium Bd1-1 exhibited pre-haustorial resistance to Pg-tr, i.e. infection usually stopped at appressorial formation. By 68 hpi, only 0.3% and 0.7% of the Pg-tr urediniospores developed haustoria and colonies, respectively. In contrast, development of advanced infection structures by Pg-lo and Pg-pp was significantly more common; however, Brachypodium displayed post-haustorial resistance to these isolates. By 68 hpi the percentage of urediniospores that only develop a haustorium mother cell or haustorium in Pg-lo and Pg-pp reached 8% and 5%, respectively. The formation of colonies reached 14% and 13%, respectively. We conclude that Brachypodium is an apt grass model to study the molecular and genetic components of incompatiblity and non-host resistance to P. graminis.     

Comparative physiological and metabolomic responses of four <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> varieties contrasting in drought stress resistance

Brachypodium distachyon (Brachypodium) is not only a monocot grass species, but also a promising model organism of crop research. In this study, the drought resistance of four Brachypodium varieties was identified including drought stress-tolerant Bd1-1 and Bd21, drought stress-susceptible Bd3-1 and Bd18-1. Physiological assay showed that drought-tolerant varieties (Bd1-1 and Bd21) were more effective in maintenance of leaf water content, activation of catalase and peroxidase activities and accumulation of reduced glutathione, resulting in alleviated cell damage and lower reactive oxygen species level than drought-susceptible varieties (Bd3-1 and Bd18-1) in response to drought stress. In addition, 54 primary metabolites were differentially regulated among Brachypodium varieties and after drought stress treatment, indicating the complexity of Brachypodium response to drought stress. We also identified several commonly regulated metabolites especially some compatible solutes including proline and soluble sugars, which exhibited higher concentrations in the drought-tolerant varieties. Taken together, this study suggested that natural variation of Brachypodium varieties in response to drought stress might be connected with higher leaf water, enhanced accumulation of osmolyte and more effective antioxidant system, as well as the modulation of metabolic profiles under drought stress conditions.     

Identification and molecular characterization of a Brachypodium distachyon GIGANTEA gene: functional conservation in monocot and dicot plants

Developmental phase change and flowering transition are emerging as potential targets for biomass agriculture in recent years. The GIGANTEA (GI) gene is one of the central regulators that direct flowering promotion and phase transition. In this work, we isolated a GI gene orthologue from the small annual grass Brachypodium distachyon inbred line Bd21 (Brachypodium), which is perceived as a potential model monocot for studies on bioenergy grass species. A partial GI gene sequence was identified from a Brachypodium expressed sequence tag library, and a full-size gene (BdGI) was amplified from a Brachypodium cDNA library using specific primer sets designed through analysis of monocot GI gene sequences. The BdGI gene was up-regulated by light and cold. A circadian rhythm set by light–dark transition also regulated the expression of the BdGI gene. The deduced amino acid sequence of the BdGI protein shares higher than 70% of sequence identity with the GI proteins in monocots and Arabidopsis. In addition, the BdGI protein is constitutively targeted to the nucleus and physically interacts with the ZEITLUPE (ZTL) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) proteins, like the Arabidopsis GI protein. Interestingly, heterologous expression of the BdGI gene in a GI-deficient Arabidopsis mutant rescued efficiently the late flowering phenotype. Together, our data indicate that the role of the GI gene in flowering induction is conserved in Arabidopsis and Brachypodium. It is envisioned that the GI genes of bioenergy grasses as well as Brachypodium could be manipulated to improve biomass by engineering developmental timing of phase transitions.     

Assessment of genome relationships in the genus Oryza L. based on seed-protein profile analysis

Summary Cultivated and wild Oryza species belonging to different genomic groups were studied with regard to their soluble seed-protein profiles. There is an essential uniformity in the banding patterns within various genomes and the basic patterns are not species-specific but genome-specific. O. meridionalis contains a subgenome similar to the A genome of O. rufipogon. Certain specific bands present among A genome species have been found to be useful in tracing the phylogenetic affinity between the cultivated species and their presumed wild progenitors.     

Occurrence and immunological relationships of lectins in gramineous species

A survey of the occurrence of lectins in seeds from more than 100 grass species showed that all species belonging to the Triticeae tribe and the genera Brachypodium and Oryza contain lectins. All these lectins have the same sugar-binding specificity and are related to wheat-germ agglutinin, but to different degrees. Lectins from Triticeae species are immunologically indistinguishable from wheat lectin, whereas Brachypodium and rice lectins are only immunologically related to the wheat lectin. Attempts to detect lectin-deficient lines or varieties in wild and cultivated species of the three lectin-containing groups were unsuccessful. The possible use of lectins as a chemotaxonomic tool is discussed.     

Chromosome numbers in Brachypodium Beauv. (Gramineae)

A summary of previous chromosome counts of Brachypodium is given in addition to a large number of new reports. The present work does not support claims made for extensive infraspecific variation in certain species. The implication of cytological knowledge for a taxonomic and evolutionary understanding of the genus is discussed.