Expanding the mutagenic code: characterizing the ethylnitrosourea (ENU) mutational spectrum in Arabidopsis and barley

Chemical mutagenesis is a cornerstone of plant breeding and functional genomics, with ethyl methanesulfonate (EMS) as the most commonly used agent. As EMS induces predominantly G→A and C→T transition mutations, its use restricts the range of mutagenic variation accessible. To assess codon limitations imposed by EMS, we performed a principal component analysis of amino-acid physicochemical properties, mapping how transitions and transversions shift amino-acid choice in physicochemical space. Informatics-based evaluation of the genetic code revealed an underlying bias that buffers codon usage against transitions but not transversions. Further analysis of amino-acid properties and codon usage showed that transversions encode more disruptive missense changes than transitions. We demonstrate that ethylnitrosourea (ENU) broadens the mutational spectrum to include transversions and A→G/T→C transition mutations, enabling access to allelic variants that EMS cannot generate. Whole-genome resequencing in Arabidopsis and targeted TILLING in barley showed that ENU-induced mutations comprise a higher proportion of transversions, along with novel missense and nonsense A→G and T→C alleles that are less readily accessed under the EMS signature class. Together, these findings support ENU as a complementary mutagen to EMS for plant genetics where access to A→G/T→C transitions and transversions is desired.