Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

Abstract

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genomewide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of ''background'' mutations arising spontaneously in laboratory-grown plants, G:C >A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.[Supplemental material is available for this article.]Ionizing radiation is pervasive in the environment and acts as a natural mutagen via its DNA-damaging properties (Friedberg et al. 2006). In addition, the mutagenic property of artificial ionizing radiation has been a mainstay of genetic research since the pioneering experiments of Müller (1928). However, while the effects of ionizing radiation on individual genes are now relatively well understood, its genome-wide effects are not. We therefore undertook an analysis of the genome-wide consequences of fast neutron (FN) irradiation in Arabidopsis thaliana, comparing our findings with those of recent studies documenting the frequency and molecular spectrum of spontaneous ''background'' mutations in the genomes of laboratory-grown ''mutation accumulation'' (MA) line Arabidopsis plants (Ossowski et al. 2010) and of Arabidopsis plants regenerated in vitro from root tissue explants (Jiang et al. 2011). We found that exposure of Arabidopsis to FNs induces a broader range of mutational lesions than previously suspected, and that both the incidence and spectrum of FN-induced mutations are distinct from those of ''spontaneous'' mutations. These discoveries have important consequences for the use of FNs in experimental plant mutagenesis and provide an indication of the likely mutagenic effects of environmental ionizing radiation on organisms living in the wild (Hinton et al. 2007). Results FN irradiation, mutant generation, and isolationOur analyses began with a multiply mutant Arab...

Environmentally responsive genome-wide accumulation of de novoArabidopsis thalianamutations and epimutations

“…Finally, mutations that exhibited a heterozygous pattern (e.g., where 20%-80% of reads showed a base different from the reference base) or where there was insufficient coverage in G0 or G10 were also excluded from the final mutation lists. Our previous studies confirmed that ;100% of the mutations detected by the above described multistep variant detection pipeline were real (Jiang et al 2011;Belfield et al 2012). Using this pipeline, we identified 102 and 52 mutations in G10 saline soil and control samples, respectively (Supplemental Tables S2, S3).…”

“…The transition/transversion (Ti/Tv) ratio of single base substitutions accumulating in A. thaliana MA lineages exposed over multiple successive generations to soilsalinity stress (;1.7) is different from that of control MA lineages (;2.5) (Fig. 1C), but similar to that of mutations accumulated in in vitro regenerant or irradiated A. thaliana lineages (Jiang et al 2011;Belfield et al 2012). Intriguingly, the change in Ti/Tv ratio generated by salt stress in A. thaliana is similar to that generated by the environmental stress response in yeast (Shor et al 2013).…”

“…1D), we first determined the number of substitutions in each transition and transversion category, and then normalized these numbers by the base content (GC content 36%) of the A. thaliana Col-0 genome sequence. Ti/Tv ratios were then calculated as previously described (Ossowski et al 2010;Belfield et al 2012). …”

A Protocol for Benchtop Extraction of Single-Strand-Specific Nucleases for Mutation Discovery

“…There is a broader diversity of effects reported of plants treated with physical mutagens. For example, fast neutron irradiation of plants has been reported to generate genomic deletions ranging in size from less than 56 base pairs to nearly three million base pairs (Li et al 2001;Rogers et al 2009;Bolon et al 2011;Belfield et al 2012). The most common mutagen used in the development of the~3200 officially released mutant crop varieties curated in the IAEA's Mutant Variety Database (http://mvd.iaea.org/) is gamma irradiation.…”

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“…These mutations can now be identified at the whole-genome level using HTS. In A. thaliana, fast-neutron-induced mutations were revealed to be mainly base substitutions and small deletions (Belfield et al, 2012). We have previously identified the mutation spectrum of the heavy-ion beam in A. thaliana as comprising base substitutions, deletions, insertions and chromosomal rearrangements (Hirano et al, 2015).…”