High-density mutation tracks are associated with proton-beam irradiation patterns in Sorghum bicolor

Induced mutagenesis is a cornerstone of crop functional genomics, yet the extent to which distinct radiation sources reshape the spatial distribution of mutations remains difficult to evaluate in reduced-representation datasets. Here, we analyze a published genotyping-by-sequencing (GBS) panel (192,040 loci) to compare proton-beam and gamma-ray mutagenesis in Sorghum bicolor. Because GBS sampling is nonuniform, all analyses were conducted within an explicitly defined GBS-callable sequence space. Within this callable space, 96-channel trinucleotide spectra were broadly similar between radiation types, whereas spatial summaries differed. Macroscale analysis using the Gini coefficient indicated that proton-treated lines exhibit a highly unequal, spike-like distribution of mutations, whereas gamma-treated lines show a more diffuse window-level distribution. Microscale spatial statistics were consistent with clustering patterns that were more prominent in proton-treated lines, including an aggregation scale of ∼500 kb, with a substantial fraction of the mutational burden falling into high-density tracks. Within the callable locus set, coding- and promoter-proximal categories were not depleted of induced mutation events (single-nucleotide variants) across treatments. Furthermore, we did not detect a negative association between total mutational load and the coding-region mutation fraction in this dataset. These findings suggest that, within this dataset, proton mutagenesis is characterized not by unique chemical signatures but by a distinct spatial geometry that concentrates detectable mutation events. Because proton irradiation was represented by a single dose, whether proton treatment produces stronger clustering than gamma irradiation at equal mutational burden remains to be directly tested.