Plant, Animal & Microbe Genomes X Conference Plant, Animal & Microbe Genomes X ConferenceJanuary 12-16, 2002
Town & Country Convention Center
San Diego, CA
Workshop: Arabidopsis
A new reverse genetics method has been developed to identify and isolate deletion mutants for targeted plant genes. Deletion mutant libraries are generated using fast neutron bombardment. DNA samples extracted from the deletion libraries are used to screen for deletion mutants by polymerase chain reaction (PCR) using specific primers flanking the targeted genes. By adjusting PCR conditions to preferentially amplify the deletion alleles, deletion mutants were obtained for greater than 80% of targeted loci from an Arabidopsis population of 51,840 M2 families. A large number of deletion mutants have been identified and multiple deletion alleles are often recovered for targeted loci. By isolating deletion mutants for genes with a wide range of sizes, we demonstrated that the method is very useful for targeting small genes. In addition, we have showed that it is possible to find deletion mutants mutating two or three tandem homologous genes. Data on molecular and phenotypic analysis of these mutants will be presented. We also showed that it is possible to apply this method to plant species other than Arabidopsis by isolating a deletion mutant for a rice gene using a similar approach. Since fast neutron mutagenesis is highly efficient, it is practical to develop deletion mutant populations with more complete coverage of the genome than methods based on insertional mutagenesis. Because fast neutron mutagenesis is applicable to all plant genetic systems, this method has the potential to enable reverse genetics for a wide range of plant species.