1a.Objectives (from AD-416):
The objectives of the project are to 1. Generate 30,000 insertional mutants. 2. Sequence DNA flanking the insertion sites of all mutants to place the insertions in a genomic context. 3. Collaborate with other groups creating T-DNA lines to integrate their lines into our database.
1b.Approach (from AD-416):
Using an Agrobacterium-mediated transformation procedure we will create a collection of insertional mutants in the model grass Brachypodium distachyon. To determine what gene is disrupted in each mutant, we will sequence DNA flanking the insertion site using the known sequence of the inserted DNA as a starting point. This collection will then be used to identify mutations in genes predicted to affect biomass quality and agronomic characteristics of energy crops. The mutant collection will be made publicly available through a BLAST searchable database to allow other researchers to identify knockouts in specific genes.
Brachypodium is a simple model for studying the unique properties of the grasses and has been adopted by many laboratories around the world because of its ease of use and long list of readily available resources. This work relates to the parent project by providing experimental resources, T-DNA tagged mutants, that are used to achieve the objectives of the parent project more rapidly. In FY13 we created over 5,000 T-DNA lines bringing the total T-DNA collection to about 20,000 lines. We sequenced the DNA flanking the insertion sites in over 10,000 lines in FY13 (18,000 total) in order to determine which genes are inactivated by T-DNA insertions. Over 600 lines were requested through the project website in FY13 (>1,600 total). This resource will aid the determination of gene function which in turn will provide knowledge that can be used to create improved crops.