Natural Variation in Brachypodium Links Vernalization and Flowering Time Loci as Major Flowering Determinants.

The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon yet identified considerable ambiguity surrounding the role of VRN2 To investigate the natural diversity governing flowering time pathways in a nondomesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a single-nucleotide polymorphism-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions, and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1 Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional quantitative trait loci suggests that greater complexity underlies flowering time in this nondomesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaceae as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.

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.

Endosperm development in Brachypodium distachyon.

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.

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.

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.