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:Brachypodium distachyon is a new model plant closely related to wheat and other cereals. In this study, we performed a comprehensive analysis of hormone-regulated genes in Brachypodium distachyon using RNA sequencing technology. Brachypodium distachyon seedlings were treated with eight phytohormones (auxin, cytokinine, brassinosteroid, gibberelline, abscisic acid, ethylene, jasmonate and salicylic acid) and two inhibitors, Brz220 (brassinosteroid biosynthesis inhibitor) and prohexadione (gibberelline biosynthesis inhibitor). The expressions of 1807 genes were regulated in a phytohormone-dependent manner. We compared the data with the phytohormone responses that have reported in rice. Transcriptional responses to hormones are conserved between Bracypodium and rice. Transcriptional regulation by brassinosteroid, gibberellin and ethylene was relatively weaker than those by other hormones. This is consistent with the data obtained from comprehensive analysis of hormone responses reported in Arabidopsis. Brachypodium and Arabidopsis also shared some common transcriptional responses to phytohormones. Alternatively, unique transcriptional responses to phytohormones were observed in Brachypodium. For example, the expressions of ACC synthase genes were up-regulated by auxin treatment in rice and Arabidopsis, but no orthologous ACC synthase gene was up-regulated in Brachypodium. Our results provide information useful to understand the diversity and similarity of hormone-regulated transcriptional responses between eudicots and monocots.
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: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:Wheat, barley, and rye, of tribe Triticeae in the Poaceae, are among the most important crops worldwide but they present many challenges to genomics-aided crop improvement. Brachypodium distachyon, a close relative of those cereals has recently emerged as a model for grass functional genomics. Sequencing of the nuclear and organelle genomes of Brachypodium is one of the first steps towards making this species available as a tool for researchers interested in cereals biology. FINDINGS:The chloroplast genome of Brachypodium distachyon was sequenced by a combinational approach using BAC end and shotgun sequences derived from a selected BAC containing the entire chloroplast genome. Comparative analysis indicated that the chloroplast genome is conserved in gene number and organization with respect to those of other cereals. However, several Brachypodium genes evolve at a faster rate than those in other grasses. Sequence analysis reveals that rice and wheat have a ~2.1 kb deletion in their plastid genomes and this deletion must have occurred independently in both species. CONCLUSION:We demonstrate that BAC libraries can be used to sequence plastid, and likely other organellar, genomes. As expected, the Brachypodium chloroplast genome is very similar to those of other sequenced grasses. The phylogenetic analyses and the pattern of insertions and deletions in the chloroplast genome confirmed that Brachypodium is a close relative of the tribe Triticeae. Nevertheless, we show that some large indels can arise multiple times and may confound phylogenetic reconstruction.
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: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: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:APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factors constitute one of the largest and most conserved gene families in plant, and play essential roles in growth, development and stress response. Except a few members, the AP2/EREBP family has not been characterized in Brachypodium distachyon, a model plant of Poaceae. We performed a genome-wide study of this family in B. distachyon by phylogenetic analyses, transactivation assays and transcript profiling. A total of 149 AP2/EREBP genes were identified and divided into four subfamilies, i.e., ERF (ethylene responsive factor), DREB (dehydration responsive element binding gene), RAV (related to ABI3/VP) and AP2. Tandem duplication was a major force in expanding B. distachyon AP2/EREBP (BdAP2/EREBP) family. Despite a significant expansion, genomic organizations of BdAP2/EREBPs were monotonous as the majority of them, except those of AP2 subfamily, had no intron. An analysis of transcription activities of several closely related and duplicated BdDREB genes showed their functional divergence and redundancy in evolution. The expression of BdAP2/EREBPs in different tissues and the expression of DREB/ERF subfamilies in B. distachyon, wheat and rice under abiotic stresses were investigated by next-generation sequencing and microarray profiling. Our results are valuable for further function analysis of stress tolerant AP2/EREBP genes in B. distachyon.
Project description:BACKGROUND: The local environment plays a major role in the spatial distribution of plant populations. Natural plant populations have an extremely poor displacing capacity, so their continued survival in a given environment depends on how well they adapt to local pedoclimatic conditions. Genomic tools can be used to identify adaptive traits at a DNA level and to further our understanding of evolutionary processes. Here we report the use of genotyping-by-sequencing on local groups of the sequenced monocot model species Brachypodium distachyon. Exploiting population genetics, landscape genomics and genome wide association studies, we evaluate B. distachyon role as a natural probe for identifying genomic loci involved in environmental adaptation. RESULTS: Brachypodium distachyon individuals were sampled in nine locations with different ecologies and characterized with 16,697 SNPs. Variations in sequencing depth showed consistent patterns at 8,072 genomic bins, which were significantly enriched in transposable elements. We investigated the structuration and diversity of this collection, and exploited climatic data to identify loci with adaptive significance through i) two different approaches for genome wide association analyses considering climatic variation, ii) an outlier loci approach, and iii) a canonical correlation analysis on differentially sequenced bins. A linkage disequilibrium-corrected Bonferroni method was applied to filter associations. The two association methods jointly identified a set of 15 genes significantly related to environmental adaptation. The outlier loci approach revealed that 5.7% of the loci analysed were under selection. The canonical correlation analysis showed that the distribution of some differentially sequenced regions was associated to environmental variation. CONCLUSIONS: We show that the multi-faceted approach used here targeted different components of B. distachyon adaptive variation, and may lead to the discovery of genes related to environmental adaptation in natural populations. Its application to a model species with a fully sequenced genome is a modular strategy that enables the stratification of biological material and thus improves our knowledge of the functional loci determining adaptation in near-crop species. When coupled with population genetics and measures of genomic structuration, methods coming from genome wide association studies may lead to the exploitation of model species as natural probes to identify loci related to environmental adaptation.