2012 Annual Report 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. 3.Progress Report: The goal of this project is to create a population of insertional mutants in the model grass Brachypodium distachyon. This builds upon our previous work developing a high efficiency Agrobacterium-mediated transformation system for Brachypodium. This year we generated created over 5,000 T-DNA lines bringing their total T-DNA collection to about 15,000 lines. We adopted a new method based on Illumina sequencing of multidimensional DNA pools to sequence DNA flanking the insertion sites. This method allows us to sequence 10,000 lines using only 40 DNA pools which significantly reduces the labor and resources required. This resource is increasingly being used by the scientific community to study gene function and over 1,000 lines from our collection were requested in FY 12. This project relates to the parent project by providing mutants that can be screened for traits desirable in biomass crops.