Fast neutron mutagenesis in soybean creates frameshift mutations

The mutagenic effects of ionizing radiation have been used for decades to create novel variants in experimental populations. Fast neutron (FN) bombardment as a mutagen has been especially widespread in plants, with extensive reports describing the induction of large structural variants, i.e., deletions, insertions, inversions, and translocations. However, the full spectrum of FN-induced mutations is poorly understood. We contrast small insertions and deletions (indels) observed in 27 soybean lines subject to FN irradiation with the standing indels identified in 107 diverse soybean lines. We use the same populations to contrast the nature and context (bases flanking a nucleotide change) of single nucleotide variants. The accumulation of new single nucleotide changes in FN lines is marginally higher than expected based on spontaneous mutation. In FN treated lines and in standing variation, C→T transitions and the corresponding reverse complement G→A transitions are the most abundant and occur most frequently in a CpG local context. These data indicate that most SNPs identified in FN lines are likely derived from spontaneous de novo processes in generations following mutagenesis, rather than from the FN irradiation mutagen. However, small indels in FN lines differ from standing variants. Short insertions, from 1 – 6 base pairs, are less abundant than in standing variation. Short deletions are more abundant and prone to induce frameshift mutations that should disrupt the structure and function of encoded proteins. These findings indicate that FN irradiation generates numerous small indels, increasing the abundance of loss of function mutations that will impact single genes.Significance StatementIrradiation mutagenesis is commonly viewed as a method to induce large structural variants in genomes. We also find enrichment in small insertion and deletion (indel) variants. The radiation-mutagenized lines averaged 32 indels per line, far exceeding the number estimated to occur by spontaneous processes, indicating that these arose from the irradiation treatment. Nevertheless, naturally-occurring standing variation among soybean accessions is still four orders of magnitude higher than the level of diversity introduced by mutagenesis. Induced mutations from any source are likely to constitute a relatively small portion of the genetic variation present in crop species. However, irradiation mutagenesis is useful for altering genomes by introducing small indels into single genes or disrupting gene clusters by creating structural variants.