NSF Award
0077737
There exist numerous genome-scale methods for obtaining mutations useful for functional analysis. In traditional genetics, random mutagenesis is followed by phenotypic analysis. The availability of large-scale genomic and cDNA sequence data, however, encourages reverse genetic approaches. In plants, reverse genetics based on transposon mutagenesis or directed anti-sense inhibition have succeeded in some cases, but the methods can be labor-intensive and unreliable. Even in Arabidopsis, where reverse genetic methodology is most advanced, directed antisense mutagenesis is typically the method of choice, in spite of the fact that it requires considerable front-end effort and frequently fails.<br/><br/>This project takes advantage of a new reverse genetic method that is especially suitable for plants. After ethylmethanesulfonate (EMS) mutagenesis and self fertilization, DNA samples from several individual plants are pooled, and pools are used as templates for PCR using primers that amplify a region of interest. To detect mutations, PCR reaction pools are heated and cooled to allow heteroduplexes to form between wild-type and mutant fragments, and a sensitive mismatch detection method is applied to each pool. Mutants identified by this "TILLING" method (for Targeting Induced Local Lesions IN Genomes) can be crossed and subjected to phenotypic analysis. In a pilot screen using mutagenized seed from Arabidopsis thaliana, transition mutations were discovered at the expected frequency for selected regions of the genome, including at least one knockout mutation in a gene of interest. Based upon this experience, it is estimated that by successively typing ~10,000 plants, a scaled-up project will be capable of generating useful mutations more efficiently than other methods used thus far in plants. Moreover, this is the only reverse genetic method that provides a range of point alleles that may be used for analysis of interactions. Given the current concern about genetically modified crop plants, there may be agricultural interest in producing variants without introducing foreign DNA of any type into a plant's genome.<br/><br/>Using the current pooling strategy for Arabidopsis and denaturing HPLC (High Performance Liquid Chromatography) as a mismatch detection method, ~100 genes per year may be TILLed. However, the use of enzymatic detection of mismatches should eliminate the need to sequence individual mutant DNAs, and this could increase productivity 10-fold or more. This means that knockouts of most genes in a plant are potentially feasible with a modest future scale-up and with future improvements in throughput.<br/><br/>Genes will be chosen for TILLING based on the needs of the community, both through co-ordination with the Plant Chromatin project and from individual requests. TILLING complements forward and other reverse genetic approaches, and collaborations are anticipated for extending this methodology from Arabidopsis to corn and other organisms.
