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 genome-wide 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.

Figure 1.

Density of single nucleotide polymorphisms (SNPs) detected following alignment of (A) A. thaliana Landsberg erecta (Gan et al. 2011) and (B) progenitor reads to the Col-0 (TAIR 9) reference genome sequence. Histograms of SNPs along the length of chromosome 5 are shown. The Col-0 segment of progenitor chromosome 5 (containing the rgl3-4 T-DNA insertion) is clearly visible in (B), between 3 Mb and 6.25 Mb, where the number of variants (versus TAIR 9) falls close to baseline. The site of the RGL3 locus is shown in the Col-0 DNA segment (in gray; see inset).

Figure 2.

Distribution across chromosomes of 108 mutations detected in genomes of six FN-exposed M₃ Arabidopsis lineages. The labels indicate the type of mutation, and color their functional class or predicted consequence. Small INDELs are indicated by base-designating letters with a preceding plus or minus sign. Large deletions are indicated by a minus sign (with the number of deleted base pairs). Individual colors define intergenic region (red); intron (yellow); nonsynonymous substitution, shift of reading frame for short INDELs, or gene deletion for large deletions (blue); synonymous substitution (green); untranslated region (purple); transposable element (white). (A) E71; (B) E99; (C) E125; (D) E128; (E) E138; (F) E216. The gene symbols represent likely phenotype causal mutations conferring increased hypocotyl length: hy5 (Ang et al. 1998), hy1 (Davis et al. 1999), frs2 (Lin and Wang 2004), col2 (Ledger et al. 2001), hy2 (Kohchi et al. 2001), cry1 (Ahmad et al. 1998), and phyB (Somers et al. 1991).

Figure 3.

Estimation of original numbers of mutations in M₁ plants. M₁ seeds were irradiated with FNs, germinated, and resultant plants self-pollinated to yield M₂ seed. Elongated hypocotyl mutants were identified in the M₂ and self-pollinated to yield M₃ seed. The genomes of six mutant M₃ plants were sequenced. Mendelian segregation laws were used to estimate the number of variants, both homozygous (Homo) and heterozygous (Het), in the generations preceding the six genome-sequenced FN M₃ mutant lines. As shown, all homozygous mutations “fixed” in the M₂ (in red) will have remained homozygous in the M₃. In addition, a quarter of M₂ heterozygous mutations (144; blue text) will have segregated in a Mendelian 1:2:1 (homozygous mutation:heterozygous mutation:homozygous nonmutant) ratio, with approximately one-quarter becoming homozygous mutations (36; blue text) in the M₃. Thus, a total of 108 homozygous variants (detected by our methods) along with a further predicted 72 segregating heterozygous variants (not detected by our methods) are estimated for the M₃. To summarize, in the original FN-exposed M₁ plants, a total of 288 (black text) heterozygous mutations are estimated to have arisen. One-quarter of these mutations will have become homozygous in the M₂ (72; red text), half becoming heterozygous (144; blue text), with the remaining quarter being homozygous nonmutant. These considerations permit calculation (see Supplemental Table 5) of FN-induced mutation rates.

Figure 4.

Molecular spectrum of FN-induced mutations. (A) Frequencies and sizes of deletion mutations identified in the six FN-exposed mutant lineages. (B,C) SBS rates per A:T or G:C site and transition/transversion (Ti/Tv) ratios in (B) Col-0 MA lines (Lynch 2010; Ossowski et al. 2010) and (C) FN-exposed lineages. Complementary mutations, e.g., A>C and T>G, are pooled. The Ti/Tv ratio for each profile is shown. The mutation rate is the mutation rate weighted by the incidence of the base in the Arabidopsis Col-0 genome.