Mutant selection from progeny of gamma-ray-irradiated rice by DNA heteroduplex cleavage using Brassica petiole extract

Selection of mutants by the reverse genetic approach is a useful tool for gene function analysis and crop improvement. TILLING (Targeting Induced Local Lesions in Genomes), in which mutants are selected from the progeny of EMS-treated plants by the SNP analysis using heteroduplex cleavage enzyme from celery, has been applied to various plant species. Since gamma-ray-induced point mutations have not been identified by the reverse genetic approach, we attempted to select mutants from a population of the gamma-ray-irradiated rice. A heteroduplex between wild-type DNA and mutant DNA was cleaved by an endonuclease extracted from petioles of Brassica rapa, and cleaved DNA was detected using agarose gel electrophoresis. M₂ plants derived from 2,130 M₁ plants irradiated by gamma rays were used for the screening of mutants having nucleotide changes in 25 regions from 1.0 kb to 1.5 kb. Six mutations were identified and the rate of mutation induced by gamma rays was estimated to be one mutation per 6,190 kb. Four of the mutations were single nucleotide substitutions and two were 2-bp and 4-bp deletions. These results suggest that the point mutation rate with gamma rays was quite lower than that with EMS, but that the rate of knockout mutations among detected mutations generated by the gamma-ray irradiation was higher than that by the EMS treatment.