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Lines in T-DNA collection 18000 T-DNA lines sequenced 7864 FSTs assigned to genome 17275 Genes tagged 5784
T2 seed harvested 7702 Bulk pools available coming soon
The goal of this project (funded by the DOE Feedstock Genomics Program through interagency agreement # 60-5325-7-573) is to add to the growing collection of genomic resources available for Brachypodium by creating a large collection of T-DNA lines. These lines are indexed through flanking sequence tags (FSTs) that facilitate mapping of the T-DNA insertions within the Brachypodium genome. The collection can therefore serve to identify mutations in genes predicted to affect biomass quality and agronomic characteristics of cereal and energy crops. Mutant lines are publicly available through this site, allowing any interested researchers to identify knockouts in specific genes of interest.
Download Bragg et al. 2012. PLoS ONE 7:e41916
BLAST search of Brachypodium genes/regions with T-DNA insertions (at this time there are 2203 genes tagged by insertions in an exon, intron, or 1,000 bases upstream or 500 bases downstream of a predicted gene)
The pJJ2LBA and pJJ2LBA2 vectors contain transcriptional enhancers within the T-DNA sequence. These "activation tagging" constructs are designed to increase the transcription of nearby genes. Importantly, the transcriptional enhancers are designed to give overexpression with the same expression pattern, rather than constitutive expression, of affected genes. Activation tagging is particularly well suited to assign function to genes with redundant functions where knockouts in an individual family member do not produce a phenotype.
In generating this collection, we have done an extensive evaluation of vectors built with different promoters, reporter genes, and selectable markers in order to optimize transformation efficiency. We have observed considerable variation in efficiency and plant survival and fertility depending on the construct used. Our observations include the following: Transformations employing hygromycin selection yielded consistently higher efficiency and survival over those using BASTA. The promoter driving the selectable marker greatly affects transformation efficiency (maize ubiquitin > CaMV 35S with a 5' intron > CaMV 35S without a 5' intron >> rice tubulin). Considering time and ease of evaluation, screening transformed tissue for GUS staining was more efficient than checking for GFP or RFP fluorescence. T-DNA vectors containing two left border sequences produce transformants that yield a higher rate of successfully recovering sequence flanking the T-DNA insertion sites. Ac/Ds and En/Spm transposons function in Brachypodium, but are lethal, possibly because they are too active.
Our final vectors are available upon request to researchers interested in creating T-DNA insertional mutants. We welcome all collaborators who share a desire to create a large, freely available Brachypodium T-DNA collection. (Note: because of problems pJJ2LBA2, this will not be distributed.) See Bragg et al. 2012. PLoS ONE 7:e41916 for more details.