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.     

Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics.

Brachypodium is well suited as a model system for temperate grasses because of its compact genome and a range of biological features. In an effort to develop resources for genome research in this emerging model species, we constructed 2 bacterial artificial chromosome (BAC) libraries from an inbred diploid Brachypodium distachyon line, Bd21, using restriction enzymes HindIII and BamHI. A total of 73,728 clones (36,864 per BAC library) were picked and arrayed in 192,384-well plates. The average insert size for the BamHI and HindIII libraries is estimated to be 100 and 105 kb, respectively, and inserts of chloroplast origin account for 4.4% and 2.4%, respectively. The libraries individually represent 9.4- and 9.9-fold haploid genome equivalents with combined 19.3-fold genome coverage, based on a genome size of 355 Mb reported for the diploid Brachypodium, implying a 99.99% probability that any given specific sequence will be present in each library. Hybridization of the libraries with 8 starch biosynthesis genes was used to empirically evaluate this theoretical genome coverage; the frequency at which these genes were present in the library clones gave an estimated coverage of 11.6- and 19.6-fold genome equivalents. To obtain a first view of the sequence composition of the Brachypodium genome, 2185 BAC end sequences (BES) representing 1.3 Mb of random genomic sequence were compared with the NCBI GenBank database and the GIRI repeat database. Using a cutoff expectation value of E<10-10, only 3.3% of the BESs showed similarity to repetitive sequences in the existing database, whereas 40.0% had matches to the sequences in the EST database, suggesting that a considerable portion of the Brachypodium genome is likely transcribed. When the BESs were compared with individual EST databases, more matches hit wheat than maize, although their EST collections are of a similar size, further supporting the close relationship between Brachypodium and the Triticeae. Moreover, 122 BESs have significant matches to wheat ESTs mapped to individual chromosome bin positions. These BACs represent colinear regions containing the mapped wheat ESTs and would be useful in identifying additional markers for specific wheat chromosome regions.     

Plant regeneration and micropropagation of Brachypodium distachyon

Brachypodium distachyon (L.) P. Beauv. has several features of its genome and growth habit reminiscent of Arabidopsis thaliana (L.) Heyn. that may allow it to be developed as a model molecular genetic system representative of the temperate grasses. In order for B. distachyon to be exploited in this way, it will be necessary to develop tissue culture procedures. This report details initial studies of the characteristics of mature seed-derived callus and the production of fertile plants from callus of three ecotypes of B. distachyon. Optimum development of embryogenic callus occurred on LS (Linsmaier & Skoog 1965) and N6 (Chu et al. 1975) media containing 3.0% w/v sucrose and 11.25 M (2.5 mg l^-1) 2,4-dichlorophenoxyacetic acid. Plants were recovered at a high frequency from embryogenic callus of ecotype B200 maintained on growth regulator-free N6 medium and were easy to establish in compost. A method was also developed for the in vitro clonal propagation of shoots using MS (Murashige & Skoog 1962) medium supplemented with 4 to 13 M (1.0 to 3.0 mg l^-1) benzyladenine. It was concluded that B. distachyon performed well in tissue culture and was suitable for further studies aimed at genetic transformation and its continued development as a model molecular genetic system.Abbreviations BA benzyladenine - 2,4-d dichlorophenoxyacetic acid - LS Linsmaier and Skoog (1965) - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - MSO growth regulator-free Murashige & Skoog (1962)     

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.     

以色列二穗短柄草遗传多样性分析

采用ISJ(Intron-Splice Junction,内含子切接点引物)分子标记方法对29份源自以色列的二穗短柄草(Brachypodium distachyon)及7份其他地区的二穗短柄草材料进行遗传多样性分析.结果表明:8个ISJ引物和10个引物组合共扩增出稳定条带145条,其中93条为多态性条带,多态性比率为64.58%.聚类分析将36份供试材料分成3大类,二倍体类型的二穗短柄草,六倍体类型源自以色列以外地区的二穗短柄草及来自以色列的六倍体二穗短柄草且以色列二穗短柄草材料进一步聚成不同生态环境下的两个亚类.主坐标分析结果与聚类分析结果基本吻合.研究结果为二穗短柄草的起源与分布提供了一定的理论依据,并为分子标记技术在二穗短柄草种质资源收集、评价和鉴定等方面的利用奠定了基础.     

短柄草染色体酶解法制片技术

以六倍体短柄草为研究材料,对短柄草的染色体制片方法进行了优化,建立了一种改进的短柄草染色体酶解制片方法。试验结果表明,以45%醋酸固定液固定根尖、酶解时间2h可以获得最佳的根尖染色体制片。此方法不仅可以得到分散良好的有丝分裂中期分裂相,而且还缩短了酶解的时间,提高了制片的效率。     

Brachypodium distachyon grain: characterization of endosperm cell walls

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ～52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.     

Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and ??-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 ??g of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence, the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular, molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.     

A rapid and efficient transformation protocol for the grass Brachypodium distachyon

A fast and efficient microprojectile bombardment-mediated transformation protocol is reported for the grass species Brachypodium distachyon, a proposed alternative model plant to Oryza sativa for functional genomics in grasses. Embryogenic calli derived from immature embryos were transformed by a construct containing the uidA (coding for beta-glucuronidase) and bar (coding for phosphinothricin acetyl transferase) genes, and bialaphos, a non-selective herbicide, was used as the selection agent throughout all phases of the tissue culture. Average transformation efficiencies of 5.3% were achieved, and for single bombardments transformation efficiencies of up to 14% were observed. The time frame from the bombardment of embryogenic callus to the harvesting of transgenic T1 seeds was 29 weeks and 25 weeks for the diploid and two tetraploid accessions used, respectively. Since the seed-to-seed life cycle is 19 weeks for the diploid and 15 weeks for the tetraploid accessions, our B. distachyon transformation system allows testing of both the T0 and the T1 generation as well as production of T2 seeds within 1 year.     

Evolution and taxonomic split of the model grass Brachypodium distachyon

Background and Aims

Brachypodium distachyon is being widely investigated across the world as a model plant for temperate cereals. This annual plant has three cytotypes (2n =  10, 20, 30) that are still regarded as part of a single species. Here, a multidisciplinary study has been conducted on a representative sampling of the three cytotypes to investigate their evolutionary relationships and origins, and to elucidate if they represent separate species.

Methods

Statistical analyses of 15 selected phenotypic traits were conducted in individuals from 36 lines or populations. Cytogenetic analyses were performed through flow cytometry, fluorescence in situ hybridization (FISH) with genomic (GISH) and multiple DNA sequences as probes, and comparative chromosome painting (CCP). Phylogenetic analyses were based on two plastid (ndhF, trnLF) and five nuclear (ITS, ETS, CAL, DGAT, GI) genes from different Brachypodium lineages, whose divergence times and evolutionary rates were estimated.

Key Results

The phenotypic analyses detected significant differences between the three cytotypes and demonstrated stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirmed that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents the allotetraploid derived from them. Phylogenetic analysis demonstrated that the 2n = 20 and 2n = 10 cytotypes emerged from two independent lineages that were, respectively, the maternal and paternal genome donors of the 2n = 30 cytotype. The 2n = 20 lineage was older and mutated significantly faster than the 2n = 10 lineage and all the core perennial Brachypodium species.

Conclusions

The substantial phenotypic, cytogenetic and molecular differences detected among the three B. distachyon sensu lato cytotypes are indicative of major speciation processes within this complex that allow their taxonomic separation into three distinct species. We have kept the name B. distachyon for the 2n = 10 cytotype and have described two novel species as B. stacei and B. hybridum for, respectively, the 2n = 20 and 2n = 30 cytotypes.     

Lipid profiling of the model temperate grass, Brachypodium distachyon

Lipids are essential metabolites in cells and they fulfil a variety of functions, including structural components of cellular membranes, energy storage, cell signalling, and membrane trafficking. In plants, changes in lipid composition have been observed in diverse responses ranging from abiotic and biotic stress to organogenesis. Knowledge of the lipid composition is an important first step towards understanding the function of lipids in any given biological system. As Brachypodium distachyon is emerging as the model species for temperate grass research, it is therefore fundamentally important to gain insights of its lipid composition. We used HPLC-coupled with tandem mass spectrometry to profile and quantify levels of sphingolipids and glycerophospholipids in shoots and undifferentiated cells in suspension cultures of B. distachyon. A total of 123 lipids belonging to 10 classes were identified and quantified. Our results showed that there are differences in lipid profiles and levels of individual lipid species between shoots and undifferentiated cells in suspension cultures. Additionally, we showed that 4-sphingenine (d18:1^??4) is the main unsaturated dihydroxy-long chain base (LCB) in B. distachyon, and we were unable to detect d18:1^??8, which is the main unsaturated dihydroxy-LCB in the model dicotyledonous species, Arabidopsis thaliana. This work serves as the first step towards a comprehensive characterization of the B. distachyon lipidome that will complement future biochemical studies.     

Brachypodium distachyon promoters as efficient building blocks for transgenic research in maize

The biotechnological approach to improve performance or yield of crops or for engineering metabolic pathways requires the expression of a number of transgenes, each with a specific promoter to avoid induction of silencing mechanisms. In maize (Zea mays), used as a model for cereals, an efficient Agrobacterium tumefaciens-mediated transformation system has been established that is applied for translational research. In the current transformation vectors, the promoters of the 35S gene of the cauliflower mosaic virus and of the ubiquitin gene of maize are often used to drive the bialaphos-selectable marker and the transgene, respectively. To expand the number of promoters, genes with either constitutive or seed-specific expression were selected in Brachypodium distachyon, a model grass distantly related to maize. After the corresponding Brachypodium promoters had been fused to the β-glucuronidase reporter gene, their activity was followed throughout maize development and quantified in a fluorimetric assay with the 4-methylumbelliferyl β-D-glucuronide substrate. The promoters pBdEF1α and pBdUBI10 were constitutively and highly active in maize, whereas pBdGLU1 was clearly endosperm-specific, hence, expanding the toolbox for transgene analysis in maize. The data indicate that Brachypodium is an excellent resource for promoters for transgenic research in heterologous cereal species.     

Brachypodium distachyon as a model for defining the allergen potential of non-prolamin proteins

Epitope databases and the protein sequences of published plant genomes are suitable to identify some of the proteins causing food allergies and sensitivities. Brachypodium distachyon, a diploid wild grass with a sequenced genome and low prolamin content, is the closest relative of the allergen cereals, such as wheat or barley. Using the Brachypodium genome sequence, a workflow has been developed to identify potentially harmful proteins which may cause either celiac disease or wheat allergy-related symptoms. Seed tissue-specific expression of the potential allergens has been determined, and intact epitopes following an in silico digestion with several endopeptidases have been identified. Molecular function of allergen proteins has been evaluated using Gene Ontology terms. Biologically overrepresented proteins and potentially allergen protein families have been identified.