Arabidopsis 2010: Tools and Technologies to Enable Genome-Wide Screens in Arabidopsis

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This project specifically addresses an important aim of the 2010 project - to identify genetically stable loss-of-function mutations in all Arabidopsis genes. Computational analysis of the initial genome sequence of Arabidopsis thaliana in 2000 provided evidence for the existence of approximately 25,500 genes. Intensive efforts during the past six years to experimentally annotate this genome sequence have identified many additional protein-coding and non-coding RNA genes, expanding the gene list to 31,128 genes. Importantly, even the most current version of the genome annotation does not include many hundreds of unannotated non-coding (large and small) RNA genes. The identification of T-DNA/transposon insertion mutations in all Arabidopsis genes has been an on-going aim of worldwide functional genomics programs. Analysis of the current set of sequence-indexed integration sites reveals that mutations for ~6,000 "new" genes have not yet been identified. This project will use specific gene-directed PCR to identify two insertion mutations for each of these 6,000 genes, thereby allowing completion of the "uni-mutant" set for annotated Arabidopsis genes. In addition, the project will provide for the genotyping of these mutations which segregate for these T-DNA insertions to identify homozygous mutants in all genes that do not lead to lethality or infertility. In addition, large-scale genotyping of the known Salk, SAIL, Wisconsin and GABI-KAT T-DNA insertion mutant lines will be done as part of the effort to obtain homozygous insertion mutants in the 25,000 genes initially identified by the Arabidopsis Genome Initiative. Combined, these efforts will result in the production of a "unimutant" set of homozygous insertion mutations for the core set of plant genes. When completed, this resource will enable the initiation of whole genome phenotypic screens, allowing the identification of genes important for understanding basic plant biology as well those genes whose functions are important to further improvement of food, biomass and energy production.Broader Impacts. The genomic resources developed by this project will be widely available to a large number of researchers, will provide the basis for a variety of research projects that rely upon whole genome information and will enable genome-wide mutant screens for any visible phenotype of interest. All of the mutant plants will be available to the broader research community as soon as they are produced through the Arabidopsis Biological Resource Center (ABRC). All DNA sequence data (T-DNA insertions and genome sequences) will be deposited in GenBank, and TAIR (http://www.arabidopsis.org). The data will also be made available via the SIGnAL database (http://signal.salk.edu/cgibin/tdnaexpress), allowing browsing/retrieval of these data types, integration with the genome annotation, transcription units and DNA methylation, cDNA/ORF clones, and with all public insertion mutant sequences. An equally important aspect of the project is the hands-on training in plant genome research that will be provided at a variety of levels, including outreach to minority high school and undergraduate students.