Project description:The genetic transformation of monocot grasses is a resource intensive process, the quality and efficiency of which is dependent in part upon the method of DNA introduction, as well as the ability to effectively separate transformed from wildtype tissue. Agrobacterium-mediated transformation of Brachypodium has relied mainly on Agrobacterium tumefaciens strain AGL1. Currently the antibiotic hygromycin B has been the selective agent of choice for robust identification of transgenic calli in Brachypodium distachyon and Brachypodium sylvaticum but few other chemicals have been shown to work as well for selection of transgenic Brachypodium cells in tissue culture. This study demonstrates that Agrobacterium rhizogenes strain 18r12v and paromomycin selection can be successfully used for the efficient generation of transgenic B. distachyon and B. sylvaticum. Additionally we observed that the transformation rates were similar to or higher than those obtained with A. tumefaciens strain AGL1 and hygromycin selection. The A. rhizogenes strain 18r12v harboring the pARS1 binary vector and paromomycin selection is an effective means of generating transgenic Brachypodium plants. This novel approach will facilitate the transgenic complementation of T-DNA knockout mutants of B. distachyon which were created using hygromycin selection, as well as aid the implementation of more complex genome manipulation strategies which require multiple rounds of transformation.
Project description:Grain development and its evolution in grasses remains poorly understood, despite cereals being our most important source of food. The grain, for which many grass species have been domesticated, is a single-seeded fruit with prominent and persistent endosperm. Brachypodium distachyon, a small wild grass, is being posited as a new model system for the temperate small grain cereals, but little is known about its endosperm development and how this compares with that of the domesticated cereals. A cellular and molecular map of domains within the developing Brachypodium endosperm is constructed. This provides the first detailed description of grain development in Brachypodium for the reference strain, Bd21, that will be useful for future genetic and comparative studies. Development of Brachypodium grains is compared with that of wheat. Notably, the aleurone is not regionally differentiated as in wheat, suggesting that the modified aleurone region may be a feature of only a subset of cereals. Also, the central endosperm and the nucellar epidermis contain unusually prominent cell walls that may act as a storage material. The composition of these cell walls is more closely related to those of barley and oats than to those of wheat. Therefore, although endosperm development is broadly similar to that of temperate small grain cereals, there are significant differences that may reflect its phylogenetic position between the Triticeae and rice.
Project description:MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 M?, 7 M? and 2 M? MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.
Project description:BACKGROUND: A complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement. RESULTS: We have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors. CONCLUSIONS: The description of the WRKY transcription factor family in Brachypodium that we report here provides a framework for functional genomics studies in an important model system. Our database is a resource for both Brachypodium and wheat studies and ultimately projects aimed at improving wheat through manipulation of WRKY transcription factors.
Project description:To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.
Project description:Deep sequencing of Brachypodium distachyon small RNA from panicles (flowers) was done to analyze the genome-wide distribution patterns of 1) total small RNA reads and loci, 2) 21 and 24 nucleotide repeat-normalized reads and 3) 21 and 24 nucleotide phased siRNA clusters relative to gene and transposable element density. Overall design: Small RNA were extracted from total RNA by size fractionation and converted to DNA amplicons by serial adaptor ligation to both ends followed by RT-PCR. DNA amplicons were sequenced using an Ilumina Genome Analyzer. Resulting sequences were computationally trimmed to remove 3' adaptor sequences. Raw data for GSM506621 was not provided.
Project description:The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.
Project description:BACKGROUND:Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. RESULTS:Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. CONCLUSIONS:We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.
Project description:Seed storage proteins are of great importance in nutrition and in industrial transformation because of their functional properties. Brachypodium distachyon has been proposed as a new model plant to study temperate cereals. The protein composition of Brachypodium grain was investigated by separating the proteins on the basis of their solubility combined with a proteomic approach. Salt-soluble proteins as well as salt-insoluble proteins separated by two-dimensional gel electrophoresis revealed 284 and 120 spots, respectively. Proteins from the major spots were sequenced by mass spectrometry and identified by searching against a Brachypodium putative protein database. Our analysis detected globulins and prolamins but no albumins. Globulins were represented mainly by the 11S type and their solubility properties corresponded to the glutelin found in rice. An in silico search for storage proteins returned more translated genes than expressed products identified by mass spectrometry, particularly in the case of prolamin type proteins, reflecting a strong expression of globulins at the expense of prolamins. Microscopic examination of endosperm cells revealed scarce small-size starch granules surrounded by protein bodies containing 11S globulins. The presence of protein bodies containing glutelins makes B. distachyon closer to rice or oat than to wheat endosperm.
Project description: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.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.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.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.