pBrachyTAG vector system: New tools for Brachypodium functional genomics

In Europe, cereals such as wheat and barley are particularly important crops for food and animal feed. These plants, as well as other grasses, are also potential sources of biomass for the production of fuel and biomaterials. There is a continuing need to produce better varieties, to increase seed production, improve quality and to provide resistance to a range of pests and diseases. There are now new molecular techniques available that can be used to help breeding new improved cereal varieties. These new techniques allow us to discover the role of each plant gene. This is generally done by switching-off each gene by inserting some foreign DNA in the middle of it. When a gene stops working properly, then we can find out what it normally would do. In addition, it is also possible to monitor the expression of plant genes by attaching a fluorescent marker gene to them. Unfortunately, these molecular techniques cannot yet be used directly for wheat and barley as it is difficult to insert foreign DNA into these plants and interesting wheat or barley genes are scattered in vast quantities of repetitive junk plant DNA. As a consequence, the function of each gene needs to be studied in a closely related plant species serving as a model. In the past, rice has been used as a model plant for wheat and barley; however rice is a tropical cereal and is very different from wheat and barley. Recently, a new model species closely related to wheat and barley, the purple false brome (Brachypodium distachyon), has been identified. Brachypodium is a small grass plant which can grow in the UK. It contains less junk DNA than wheat and barley and has a smaller number of genes. Our laboratory is one of the first to have developed efficient ways to introduce foreign DNA into Brachypodium and we are now ready to use this system to switch-off Brachypodium genes. This project aims at constructing and testing new improved tools to switch off genes and to study their expression in Brachypodium. Existing tools fail, in a third of the cases, to identify the gene that has been switched-off. Addressing this problem is very important as scientists worldwide will have to introduce around 300,000-times foreign DNA into Brachypodium to be sure to hit and switch-off all of its genes at least once. Tens of thousands of unproductive DNA insertions could be avoided using this new technology, speeding up research in this area. All the tools constructed during this project will be made available to all scientists in the UK and worldwide using the internet and scientific publications. These new technologies will help scientists to understand how genes are working in Brachypodium and to apply this knowledge to plants and crops that matter to us.

Brachypodium distachyon has recently emerged as a new model system for bridging research into wheat and barley, and for promoting research in novel biomass crops such as Miscanthus and switchgrass. The genome of B. distachyon (inbred line Bd21) is currently being sequenced. This has raised considerable interest, both within the UK and internationally, in using this species as a functional genomic resource to understand temperate grass biology. In the past, T-DNA insertion mutant lines have been invaluable resources for gene mining in model plants such as Arabidopsis thaliana and rice. The aim of this project is to develop new tools and technologies facilitating the development of comparable tagged mutant resources in B. distachyon. In this project, we will develop an improved binary vector system, pBrachyTAG, for T-DNA tagging and trapping in B. distachyon. Binary vectors previously used in large-scale T-DNA tagging programmes in rice or Arabidopsis frequently led to T-DNA insertions where Flanking Sequence Tags (FSTs) could not be retrieved (~30%). We will address these limitations by designing improved binary vectors combining features known to reduce backbone transfer, to limit the introduction of superfluous DNA sequences, to improve transformation efficiency and to enable FST retrieval using either an adaptor-ligation or a tail-PCR approach. These concepts will be combined and tested, for the first time, for T-DNA tagging using a highly efficient Agrobacterium-mediated transformation protocol and FST identification procedure recently developed at the John Innes Centre for the community standard line Bd21. The improved tools developed during this project have the potential to significantly promote and accelerate the development of community resources for functional genomics in B. distachyon.