M₂ plants derived from different tillers of a chemically mutagenized rice M₁ plant carry independent sets of mutations

Generation of mutant populations with high genetic diversity is key for mutant screening and crop breeding. For this purpose, the single-seed descent method, in which one mutant line is established from a single mutagenized seed, is commonly used. This method ensures the independence of the mutant lines, but the size of the mutant population is limited because it is no greater than the number of fertile M-1 plants. The rice mutant population size can be increased if a single mutagenized plant produces genetically independent siblings. Here, we used whole-genome resequencing to examine the inheritance of mutations from a single ethyl methanesulfonate (EMS)-mutagenized seed (M-1) of Oryza sativa in its progeny (M-2). We selected five tillers from each of three M-1 plants. A single M-2 seed was selected from each tiller, and the distributions of mutations induced by EMS were compared. Surprisingly, in most pairwise combinations of M-2 siblings from the same parent, =85.2-97.9% of all mutations detected were not shared between the siblings. This high percentage suggests that the M-2 siblings were derived from different cells of the M-1 embryo and indicates that several genetically independent lines can be obtained from a single M-1 plant. This approach should allow a large reduction in the number of M-0 seeds needed to obtain a mutant population of a certain size in rice. Our study also suggests that multiple tillers of a rice plant originate from different cells of the embryo.