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.
Highlights
Because plants are sessile organisms, they are constantly exposed to harmful factors, including pathogens, toxic chemicals, ultraviolet light, and ionizing radiation
Mutations that are hardly transmitted to the generation and those occurred in nongermline cells could not be investigated
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
Summary
Because plants are sessile organisms, they are constantly exposed to harmful factors, including pathogens, toxic chemicals, ultraviolet light, and ionizing radiation These factors can damage genomic DNA in various ways, leading to oxidized bases, abasic sites, single-strand breaks, and double-strand breaks (DSBs). DSBs, which are the most serious type of DNA damage, are repaired by two main pathways, namely homologous recombination, which is possible if there are homologous sequences around the DSBs, and non-homologous end-joining (NHEJ), which may occur at any point in the cell cycle [1] Despite these intrinsic repair systems, DNA lesions are often incompletely repaired, and those that remain may result in permanent genomic mutations. Identifying and characterizing mutations is important for clarifying fundamental mechanisms broadly related to plant biology
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