Estimation of original numbers of mutations in M1 plants. M1 seeds were irradiated with FNs, germinated, and resultant plants self-pollinated to yield M2 seed. Elongated hypocotyl mutants were identified in the M2 and self-pollinated to yield M3 seed. The genomes of six mutant M3 plants were sequenced. Mendelian segregation laws were used to estimate the number of variants, both homozygous (Homo) and heterozygous (Het), in the generations preceding the six genome-sequenced FN M3 mutant lines. As shown, all homozygous mutations “fixed” in the M2 (in red) will have remained homozygous in the M3. In addition, a quarter of M2 heterozygous mutations (144; blue text) will have segregated in a Mendelian 1:2:1 (homozygous mutation:heterozygous mutation:homozygous nonmutant) ratio, with approximately one-quarter becoming homozygous mutations (36; blue text) in the M3. Thus, a total of 108 homozygous variants (detected by our methods) along with a further predicted 72 segregating heterozygous variants (not detected by our methods) are estimated for the M3. To summarize, in the original FN-exposed M1 plants, a total of 288 (black text) heterozygous mutations are estimated to have arisen. One-quarter of these mutations will have become homozygous in the M2 (72; red text), half becoming heterozygous (144; blue text), with the remaining quarter being homozygous nonmutant. These considerations permit calculation (see Supplemental Table 5) of FN-induced mutation rates.
