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

Kitamura, Satoshi; Satoh, Katsuya; Oono, Yutaka

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. Author summary: Mutations that occur in plant genome are not only related to plant evolution and speciation in nature, and also useful to identify novel gene functions and to develop new cultivars. Ionizing radiations induce various types of mutations throughout genomes in individual cells of an irradiated/mutagenized plant. However, current knowledge on radiation-induced genome-wide mutations in plants relied on the analyses of self-pollinated generations (M2 or later generations) of the mutagenized plants (M1 generation). Thus, mutations that are hardly transmitted to the next generation and those occurred in non-germline cells could not be investigated. Here, using anthocyanin pigmentation as a visible marker to reduce the genomic complexity in M1 plants, we achieved reliable detection of radiation-induced genome-wide mutations. We demonstrated that rearrangements, which were hardly detected in previous analyses using M2 genomes, occurred substantially often in gamma-irradiated M1 cells. We also revealed that mutation profile of the M1 cells was comparable with that of M2 genomes reported in previous analyses, except for the higher proportion of rearrangements in the M1 genome. Together with unique features at rejoined sites of rearrangements, medium deletions, and small deletions in the M1 genome, our findings are helpful to know the nature of genome-wide mutations induced by gamma-irradiation.

PLANT genomes; PLANT mutation; GENETIC mutation; PLANT species; IONIZING radiation; ARABIDOPSIS

PLoS Genetics, 2022, Vol 18, Issue 1, p1

1553-7390

Academic Journal

10.1371/journal.pgen.1009979