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Title:   Author  item
                                            TILL, BRADLEY - UNIV OF WASH/FHCRCitem
                                            COOPER, JENNIFER - F.HUTCHINSON CANCER RESEAitem
                                            Tai, Thomasitem
                                            Colowit, Peteritem
                                            GREENE, ELIZABETH - F.HUTCHINSON CANCER RESEAitem
                                            HENIKOFF, STEVEN - F.HUTCHINSON CANCER RESEAitem
                                            COMAI, LUCA - UNIV OF WASH/UC DAVIS
  Submitted to: BMC Plant Biology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 4/11/2007
                                    Publication  Date: 4/11/2007
                                    Citation: Till, B.J., Cooper, J., Tai, T., Colowit, P.M., Greene, E.A., Henikoff, S., Comai, L. 2007. Discovery of chemically induced mutations in rice by tilling. Biomed Central (BMC) Plant Biology. 7:19.Interpretive Summary: Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics method for studying gene function.  In this study, the TILLING method was applied to rice.  Characterization of two different rice mutant populations generated using different chemical mutagenesis methods revealed a sufficient mutation rate for the feasible application of TILLING to rice.  These results indicate that a large scale rice TILLING service may be developed for the rice research community.
                                Technical Abstract: To apply TILLING to rice, we developed two mutagenized rice populations.  One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU).  To find induced mutations, target regions of 0.7-1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes.  Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population. We estimate that the density of induced mutations is 2 to 3-fold higher than previously reported rice populations (about 1/300 kb).  By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.


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