Mutation Machine

The mutation machine is a reverse genetic screening programme using the model legume Lotus japonicus. The programme utilises the ‘TILLING’ (Targeting Induced Local Lesions In Genomes) reverse genetic methodology as developed by Colbert et. al. 2001. Seed was treated with ethylmethanesulphonate (EMS) to generate a population of plants with random point mutations. By selectively pooling the DNA and amplifying with fluorescently labelled primers, mismatched heteroduplexes were generated between wild type and mutant DNA. Heteroduplexes were incubated with the plant endonuclease CEL I, (cleaves heteroduplex mismatched sites) and the resultant products visualised on a sequencing gel. Subsequent analysis of the individual plant DNA from the pool DNA identified the plant bearing the mutation.

Genomic DNA has been extracted from 4,808 M2 lines and the M3 seed catalogued. A forward screen to identify any novel mutants, in partivular with relevance to nodulation and consequently nitrogen fixation was also undertaken. Click on http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus/ to search our mutant populations.