Plant & Animal Genomes XIII Conference Plant & Animal Genomes XIII ConferenceJanuary 15-19, 2005
Town & Country Convention Center
San Diego, CA
Valentina Talamé , Silvio Salvi , Maria Corinna Sanguineti , Roberto Tuberosa
TILLING (Targeting Induced Local Lesions IN Genomes) is a generally applicable reverse-genetics strategy first described by McCallum et al. (2000, Plant Phys. 123: 439-442) providing an allelic series of induced point mutations from a population of chemically mutagenized individuals.
We choose to realize a TILLING platform in barley because this species is both of significant economic importance and a good model for all the Triticeae. A mutagenized barley (variety “Morex”) population has been obtained by sodium azide (10 mM NaN3) seed treatment. During the season 2004, about 10,000 M2 seeds isolated from single M1 plants were sown in the field and ca. 5700 M2 plants reached maturity and set seeds. Phenotypic screening on M2 plants grown in the field showed a high frequency of morphological alterations (ca. 19% of germinated plants), which have been classified. A screening system based on fluorescently-labelled PCR primers, digestion with CELI nuclease and detection with a Licor automatic sequencer has been implemented and a sub-set of DNA samples isolated from 900 M2 seeds was analyzed to verify the effectiveness of the mutagenized population. The genes Dhn4 and Rpg1 have been considered for screening the test sample resulting in one mutation identified over 360 kb analyzed. We are currently extracting high quality DNA from individual M2 plants; we plan to be ready to till barley genes by spring 2005.