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. 2022 Jan 20;18(1):e1009979.
doi: 10.1371/journal.pgen.1009979. eCollection 2022 Jan.

Detection and characterization of genome-wide mutations in M1 vegetative cells of gamma-irradiated Arabidopsis

Satoshi Kitamura  1 , Katsuya Satoh  1 , Yutaka Oono  1
Affiliations

Detection and characterization of genome-wide mutations in M1 vegetative cells of gamma-irradiated Arabidopsis

Satoshi Kitamura et al. PLoS Genet. .

Abstract

Radiation-induced mutations have been detected by whole-genome sequencing analyses of self-pollinated generations of mutagenized plants. However, large DNA alterations and mutations in non-germline cells were likely missed. In this study, in order to detect various types of mutations in mutagenized M1 plants, anthocyanin pigmentation was used as a visible marker of mutations. Arabidopsis seeds heterozygous for the anthocyanin biosynthetic genes were irradiated with gamma-rays. Anthocyanin-less vegetative sectors resulting from a loss of heterozygosity were isolated from the gamma-irradiated M1 plants. The whole-genome sequencing analysis of the sectors detected various mutations, including structural variations (SVs) and large deletions (≥100 bp), both of which have been less characterized in the previous researches using gamma-irradiated plant genomes of M2 or later generations. Various types of rejoined sites were found in SVs, including no-insertion/deletion (indel) sites, only-deletion sites, only-insertion sites, and indel sites, but the rejoined sites with 0-5 bp indels represented most of the SVs. Examinations of the junctions of rearrangements (SVs and large deletions), medium deletions (10-99 bp), and small deletions (2-9 bp) revealed unique features (i.e., frequency of insertions and microhomology) at the rejoined sites. These results suggest that they were formed preferentially via different processes. Additionally, mutations that occurred in putative single M1 cells were identified according to the distribution of their allele frequency. The estimated mutation frequencies and spectra of the M1 cells were similar to those of previously analyzed M2 cells, with the exception of the greater proportion of rearrangements in the M1 cells. These findings suggest there are no major differences in the small mutations (<100 bp) between vegetative and germline cells. Thus, this study generated valuable information that may help clarify the nature of gamma-irradiation-induced mutations and their occurrence in cells that develop into vegetative or reproductive tissues.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Anthocyanin-less sectors in triple heterozygous plants irradiated with gamma-rays.
(A) Vegetative tissues of 22–3 (top left) and 29–4 (top right) plants. Red arrows indicate anthocyanin-less sectors analyzed by NGS. A non-irradiated control plant (bottom left) is also shown. These plants were grown in 100 mm2 pots. (B) Adaxial (left) and abaxial (right) surfaces of leaves indicated by an arrow in (A). Bars indicate 5 mm.
Fig 2
Fig 2. Mutation spectrum for the DNA in the examined sectors following the gamma-irradiation of seeds.
(A) Spectrum of all mutations detected in seven anthocyanin-less sectors. (B) Characterization of SBS. Letters indicate statistical significance (one-way ANOVA with post hoc Tukey test, p < 0.05). The transition:transversion ratio (Ti:Tv) is indicated at the top right. (C) Distribution of deletion sizes. Sizes (bp) are indicated at the bottom.
Fig 3
Fig 3. Schematic representation of all SVs detected in this study.
Gray arrows indicate non-mutated genome sequences. Red and yellow arrows indicate inversions and translocations, respectively. Blue boxes indicate deletions and green triangles indicate insertions or potential duplications at the rejoined sites. Deletions with MH are indicated by blue boxes with a red outline. The types of rejoined sites are indicated by ovals with different colors: no-indel sites are gray, only-deletion sites are blue, only-insertion sites are yellow, and indel sites are green. Genomic coordinates are indicated as black letters. Abbreviations: DEL, deletion; INS, insertion; DUP, potential duplication.
Fig 4
Fig 4. Distribution of NGS reads to the reference sequences (100 kb-bin size) on chromosome 3, 4 and 5 for four M1 mutant sectors [5–3 (blue squares), 13–2 (gray triangles), 15–4 (golden circles) and 22–3 (red crosses)].
Large deletions are labeled by individual mutation names. In addition to the large deletions found by the mutation-calling algorithms (indicated by solid arrows), four additional regions (dotted arrows) showed decrease of the reads. For the regions of chromosome 5 in 5–3 (blue dotted arrow) and chromosome 3 in 15–4 (golden dotted arrow), probable junctions could not be detected from our NGS data (labeled as “undetermined”). Approximate positions of three heterozygous TT genes are labeled with pale orange bars. Distribution of NGS reads on all chromosomes for all 7 M1 mutant sectors are shown in S4 Fig.
Fig 5
Fig 5. Schematic representation of all large deletions (≥100 bp) detected in this study.
Gray arrows indicate non-mutated genome sequences. Blue boxes and green triangles indicate deletions and insertions at rejoined sites, respectively. Deletions with MH are indicated by blue boxes with a red outline. Asterisks indicate two large deletions found by dosage analysis. Genomic coordinates are indicated as black letters. Abbreviations: DEL, deletion; INS, insertion; MH, microhomology.
Fig 6
Fig 6. Comparison of the characteristics among rearrangements, medium DELs, and small DELs.
The ratio and number of rejoined sites with an insertion and without MH [INS(+) MH(−)], without an insertion and with MH [INS(−) MH(+)], and without an insertion or MH [INS(−) MH(−)] are provided in the left panel. Values in the colored boxes represent the number of detected rejoined sites. Significant differences in each category are indicated in the right panel. * p < 0.05, ** p < 0.01 (Fisher’s exact test). Abbreviations: DEL, deletion; INS, insertion; MH, microhomology.
Fig 7
Fig 7
Sequences around the junctions harboring insertions in the rearrangements (A) and small DELs (B). The longest template sequences are indicated on the right side. Inserted nucleotides are highlighted in green. Candidate templates for the insertions are labeled with a single underline (direct strand) and a double underline (reverse complement). Homologous nucleotide sequences between the template and the insertion are highlighted in yellow. Homologous nucleotide sequences between the sequences flanking the template and the insertions are highlighted in gray. Three junctions harboring insertions of ≥10 bp are labeled with asterisks. Broken and/or rejoined sites are indicated with blue triangles. Genomic coordinates are indicated at both sides of the inserted sequences. Deleted nucleotides are indicated as gray letters. Rejoined sequences with inverted direction by SVs are indicated as italic.
Fig 8
Fig 8. Characterization of mutations on the basis of the AF.
(A) Distribution of AFs of mutations [indels (orange) and SBSs (blue)] in anthocyanin-less sectors. Data were analyzed using GATK. Stars indicate the AF of the mutations responsible for the anthocyanin-less phenotypes. The mutations indicated by the black curly brackets are from the major mutation groups and are used as the representative mutations that occurred in a putative single ancestral cell in a dry seed. (B) Mutation spectrum for the representative mutations in four M1 plants (8–5, 13–2, 27–4, and 29–4) (left) and in six M2 plants analyzed in a previous study (right; [11]). The proportions of the rearrangements are indicated by dotted lines. * p < 0.05 (Fisher’s exact test).
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This work was partially supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI rant number 19K12333 to SK (https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-19K12333/). Some of the sequencing analysis was also supported by JSPS [KAKENHI grant number 16H06279 (PAGS)] (https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16H06279/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.