A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.     

Exploring valid reference genes for gene expression studies in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>by real-time PCR

Background  

The wild grass species Brachypodium distachyon (Brachypodium hereafter) is emerging as a new model system for grass crop genomics research and biofuel grass biology. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression studies will undoubtedly follow. Therefore, stable reference genes are necessary to normalize the gene expression data.     

Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon

Background  

Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. To characterize the natural diversity of Brachypodium and provide a valuable new tool to the growing list of resources available to Brachypodium researchers, we created and characterized a large, diverse collection of inbred lines.     

Exploring the genomes: From Arabidopsis to crops

Model systems have played a crucial role for understanding biological processes at genetic, molecular and systems levels. Arabidopsis thaliana is one of the best studied model species for higher plants. Large genomic resources and mutant collections made Arabidopsis an excellent source for functional and comparative genomics. Rice and Brachypodium have a great potential to become model systems for grasses. Given the agronomic importance of grass crops, it is an attractive strategy to apply knowledge from Arabidopsis to grasses. Despite many efforts successful reports are sparse. Knowledge transfer should generally work best between orthologous genes that share functionality and a common ancestor. In higher plants, however, recent genome projects revealed an active and rapid evolution of genome structure, which challenges the concept of one-to-one orthologous mates between two species. In this study, we estimated on the example of protein families that are involved in redox related processes, the impact of gene expansions on the success rate for a knowledge transfer from Arabidopsis to the grass species rice, sorghum and Brachypodium. The sparse synteny between dicot and monocot plants due to frequent rearrangements, translocations and gene losses strongly impairs and reduces the number of orthologs detectable by positional conservation. To address the limitations of sparse synteny and expanded gene families, we applied for the detection of orthologs in this study orthoMCL, a sequence-based approach that allows to group closely related paralogs into one orthologous gene cluster. For a total of 49 out of 170 Arabidopsis genes we could identify conserved copy numbers between the dicot model and the grass annotations whereas approximately one third (34.7%, 59 genes) of the selected Arabidopsis genes lack an assignment to any of the grass genome annotations. The remaining 62 Arabidopsis genes represent groups that are considerably biased in their copy numbers between Arabidopsis and all or most of the three grass genomes.     

Distribution and characterization of more than 1000 T‐DNA tags in the genome of Brachypodium distachyon community standard line Bd21

A collection of 4117 fertile T‐DNA lines has been generated by Agrobacterium‐mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon. The regions flanking the T‐DNA left and right borders of the first 741 transformed plants were isolated by adapter‐ligation PCR and sequenced. A total of 1005 genomic sequences (representing 44.1% of all flanking sequences retrieved) characterized 660 independent T‐DNA loci assigned to a unique location in the Brachypodium genome sequence. Seventy‐six percent of the fertile plant lines contained at least one anchored T‐DNA locus (1.17 loci per tagged line on average). Analysis of the regions flanking both borders of the T‐DNA increased the number of T‐DNA loci tagged and the number of tagged lines by approximately 50% when compared to a single border analysis. T‐DNA integration (2.4 insertions per Mb on average) was proportional to chromosome size, however, varied greatly along each chromosome with often low insertion level around centromeres. The frequency of insertion within transposable elements (5.3%) was fivefold lower than expected if random insertion would have occurred. More than half of the T‐DNAs inserted in genic regions. On average, one gene could be tagged for every second fertile plant line produced and more than one plant line out of three contained a T‐DNA insertion directly within or 500 bp around the coding sequence. Approximately, 60% of the genes tagged corresponded to expressed genes. The T‐DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the BrachyTAG.org web site.     

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.     

Analysis of saccharification in <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> stems under mild conditions of hydrolysis

Background  

Brachypodium distachyon constitutes an excellent model species for grasses. It is a small, easily propagated, temperate grass with a rapid life cycle and a small genome. It is a self-fertile plant that can be transformed with high efficiency using Agrobacteria and callus derived from immature embryos. In addition, considerable genetic and genomic resources are becoming available for this species in the form of mapping populations, large expressed sequence tag collections, T-DNA insertion lines and, in the near future, the complete genome sequence. The development of Brachypodium as a model species is of particular value in the areas of cell wall and biomass research, where differences between dicots and grasses are greatest. Here we explore the effect of mild conditions of pretreatment and hydrolysis in Brachypodium stem segments as a contribution for the establishment of sensitive screening of the saccharification properties in different genetic materials.     

Brachypodium distachyon: making hay with a wild grass

Brachypodium distachyon is a wild grass with a short life cycle. Although it is related to small grain cereals such as wheat, its genome is only a fraction of the size. A draft genome sequence is currently available, and molecular and genetic tools have been developed for transformation, mutagenesis and gene mapping. Accessions collected from across its ancestral range show a surprising degree of phenotypic variation in many traits, including those implicated in domestication of the cereals. Thus, given its rapid cycling time and ease of cultivation, Brachypodium will be a useful model for investigating problems in grass biology.     

A TILLING Platform for Functional Genomics in Brachypodium distachyon

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called “BRACHYTIL”, for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.     

Comparison of a high-density genetic linkage map to genome features in the model grass <Emphasis Type="Italic">Brachypodium distachyon</Emphasis>

The small annual grass Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to study questions unique to the grasses. Many Brachypodium resources have been developed including a whole genome sequence, highly efficient transformation and a large germplasm collection. We developed a genetic linkage map of Brachypodium using single nucleotide polymorphism (SNP) markers and an F₂ mapping population of 476 individuals. SNPs were identified by targeted resequencing of single copy genomic sequences. Using the Illumina GoldenGate Genotyping platform we placed 558 markers into five linkage groups corresponding to the five chromosomes of Brachypodium. The unusually long total genetic map length, 1,598 centiMorgans (cM), indicates that the Brachypodium mapping population has a high recombination rate. By comparing the genetic map to genome features we found that the recombination rate was positively correlated with gene density and negatively correlated with repetitive regions and sites of ancestral chromosome fusions that retained centromeric repeat sequences. A comparison of adjacent genome regions with high versus low recombination rates revealed a positive correlation between interspecific synteny and recombination rate.     

Recent Progress in Model Grass Brachypodium distachyon (Poaceae)

Brachypodium distachyon, recently developed model system for temperate grasses, exhibited many traits with cereal crops and proposed to be an experimental system to access the biological approach. These traits have shown a surprised degree of phenotypic variation in many collected accessions. Like some important economical cereals, Bdistachyon also belongs to subfamily Pooideae, which make it become an unquestionable model system to research the economically important crops, such as wheat, barley and several potential biofuel plants. Recently, genome sequence and annotation of Bdistachyon has been finished. Associated with the development of the functional genomics and other experimental resources establishment, Bdistachyon will provide a key resource for improving cereal crops and facilitate the approach of sequence analyze gene expression and functional resources available for a variety of species. In this article we review and assess the current progress of Bdistachyon as a model system and then focus specifically on recent studies of comparative genomics, biological improvement, transformation and T DNA mutations.     

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.     

Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

Application of Brachypodium to the genetic improvement of wheat roots

To meet the demands of a larger and more affluent global population, wheat yields must increase faster this century than last, with less irrigation, fertilizer, and land. Modelling and experiments consistently demonstrate a large potential for increasing wheat productivity by improving root systems; however, application of research to new varieties is slow because of the inherent difficulties associated with working underground. This review makes the case for the use of the model grass Brachypodium distachyon to simplify root research and accelerate the identification of genes underlying wheat root improvement. Brachypodium is a small temperate grass with many genomic, genetic, and experimental resources that make it a tractable model plant. Brachypodium and wheat have very similar root anatomies which are distinct from rice root anatomy that is specialized to help it overcome anaerobic conditions associated with submerged roots. As a dicotyledonous plant, Arabidopsis has an even more divergent root system that features a tap root system and cambia with secondary growth, both of which are lacking in the grasses. The major advantage of Brachypodium is its small stature that allows the adult grass root system to be readily phenotyped, unlike rice and maize. This will facilitate the identification of genes in adult roots that greatly influence yield by modulating water uptake during flowering and grain development. A summary of the advantages of Brachypodium for root studies is presented, including the adult root system architecture and root growth during grain development. Routes to translate discoveries from Brachypodium to wheat are also discussed.     

Arabidopsis to rice. Applying knowledge from a weed to enhance our understanding of a crop species

Although Arabidopsis is well established as the premiere model species in plant biology, rice (Oryza sativa) is moving up fast as the second-best model organism. In addition to the availability of large sets of genetic, molecular, and genomic resources, two features make rice attractive as a model species: it represents the taxonomically distinct monocots and is a crop species. Plant structural genomics was pioneered on a genome-scale in Arabidopsis and the lessons learned from these efforts were not lost on rice. Indeed, the sequence and annotation of the rice genome has been greatly accelerated by method improvements made in Arabidopsis. For example, the value of full-length cDNA clones and deep expressed sequence tag resources, obtained in Arabidopsis primarily after release of the complete genome, has been recognized by the rice genomics community. For rice >250,000 expressed sequence tags and 28,000 full-length cDNA sequences are available prior to the completion of the genome sequence. With respect to tools for Arabidopsis functional genomics, deep sequence-tagged lines, inexpensive spotted oligonucleotide arrays, and a near-complete whole genome Affymetrix array are publicly available. The development of similar functional genomics resources for rice is in progress that for the most part has been more streamlined based on lessons learned from Arabidopsis. Genomic resource development has been essential to set the stage for hypothesis-driven research, and Arabidopsis continues to provide paradigms for testing in rice to assess function across taxonomic divisions and in a crop species.     

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.     

Exploiting the Brachypodium Tool Box in cereal and grass research

It is now a decade since Brachypodium distachyon (Brachypodium) was suggested as a model species for temperate grasses and cereals. Since then transformation protocols, large expressed sequence tag (EST) databases, tools for forward and reverse genetic screens, highly refined cytogenetic probes, germplasm collections and, recently, a complete genome sequence have been generated. In this review, we will describe the current status of the Brachypodium Tool Box and how it is beginning to be applied to study a range of biological traits. Further, as genomic analysis of larger cereals and forage grasses genomes are becoming easier, we will re-evaluate Brachypodium as a model species. We suggest that there remains an urgent need to employ reverse genetic and functional genomic approaches to identify the functionality of key genetic elements, which could be employed subsequently in plant breeding programmes; and a requirement for a Pooideae reference genome to aid assembling large pooid genomes. Brachypodium is an ideal system for functional genomic studies, because of its easy growth requirements, small physical stature, and rapid life cycle, coupled with the resources offered by the Brachypodium Tool Box.     

Website review: how to get the best from fission yeast genome data

Researchers are increasingly depending on various centralized resources to access the vast amount of information reported in the literature and generated by systematic sequencing and functional genomics projects. Biological databases have become everyday working tools for many researchers. This dependency goes both ways in that the databases require continuous feedback from the research community to maintain accurate, reliable, and upto- date information. The fission yeast Schizosaccharomyces pombe has recently been sequenced, setting the stage for the post-genome era of this popular model organism. Here, we provide an overview of relevant databases available, or being developed, together with a compilation of Internet resources containing useful information and tools for fission yeast.     

Genomics tools for QTL analysis and gene discovery

In recent years, several new genomics resources and tools have become available that will greatly assist quantitative trait locus (QTL) mapping and cloning of the corresponding genes. Genome sequences, tens of thousands of molecular markers, microarrays, and knock-out collections are being applied to QTL mapping, facilitating the use of natural accessions for gene discovery.