Fine-tuning EMS treatments to produce large sorghum mutant populations for FIND-IT

Chemical, biological, and physical mutagens induce modifications of nucleotides in the exposed organisms, resulting in base changes in their DNA. When harnessed, mutagenesis increases genetic diversity in crops and aids in elucidating gene function through the study of mutants with altered phenotypes, particularly when combined with reverse genetic techniques such as Fast Identification of Nucleotide variants by droplet digital PCR (FIND-IT). Sorghum (Sorghum bicolor) is a prime candidate organism for mutagenesis; several mutant populations have been produced using ethyl methane sulfonate (EMS) as the mutagen. However, none of these studies address the optimisation of EMS treatment rates for large sorghum mutant populations. Here, we examined how varying EMS concentrations (0.05, 0.15, and 0.25%) plus a zero control affect agronomic traits, including plant survivability, seed yield, pest burden, and growth rates, as well as the number of induced genomic variants in the inbred sorghum variety BTx623. Whole-genome sequencing and variant analysis of M3 plants showed a positive correlation between EMS concentration and mutation load, although significant gains diminished beyond 0.15%. An EMS rate of 0.25% led to very poor seed yields, whilst 0.05% generated too few variants to achieve genome mutant saturation. Regression analyses indicate that a 0.10% EMS treatment may best balance seed yield and mutation load, and enable construction of large-scale mutant libraries for FIND-IT. This work underscores the importance of optimizing EMS concentration when producing sorghum mutant populations and provides insights into the effects of EMS on plant agronomic performance, germination, and mutation profiles.