An ethyl methanesulfonate-induced neutral mutant-bridging method efficiently identifies spontaneously mutated genes...

Spontaneous mutants are mainly obtained from tissue culture or natural occurrences in plants. The traditional strategy for identifying spontaneously mutated genes is to continuously backcross these mutants to another variety and develop a near-isogenic F₂ population for map-based cloning or bulked segregant analysis. However, this strategy is time-consuming. Here, we have developed a new method to efficiently accelerate the identification process. The chemical mutagen ethyl methanesulfonate was first used to treat the wild type of the spontaneous mutants to induce thousands of neutral mutations. An induced individual without any statistically significant phenotypic changes which was compared with the wild type was chosen as the neutral mutant. The spontaneous mutant was then crossed with the neutral mutant to develop a pseudo-near-isogenic F₂ population in which only the induced neutral mutations and the causal mutation were segregated in the genome. This population ensures that the variation of the mutated trait is controlled only by the spontaneously mutated gene. Finally, after sequencing the neutral mutant and the mutant-type DNA pool of the F₂ population the spontaneous mutation will be identified quickly by bioinformatics analysis. Using this method, two spontaneously mutated genes were identified successfully. Therefore, the neutral mutant-bridging method efficiently identifies spontaneously mutated genes in rice, and its value in other plants is discussed.