III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Dose-dependent mutagenic eﬀects of 160-MeV/nucleon-argon beamin Arabidopsis thaliana
K. Ishii,∗1 S. Ohbu,∗1 Y. Shirakawa,∗1 and T. Abe∗1
Heavy-ion beams are used as an eﬀective mutagens that induce localized mutations owing to theirhigh linear energy transfer (LET). We previously reported a dose-dependent mutagenic eﬀect of a 135MeV/nucleon-carbon beam (30 keV/µm) in Arabidopsis thaliana in which the number of mutations per
genome was dose-dependently increased in the rangeof 150–350.1) In this study, we investigated the dosedependent mutagenic eﬀect of a 160-MeV/nucleonargon beam and its diﬀerence to that of a 135MeV/nucleon beam.
Dry seeds of Arabidopsis thaliana (the Col-0 wildtype strain or CS16118 strain, which possesses a heterozygous null mutation in the APG3 gene for measuring the mutation rate in M1 generation2) ) were irradiated with 160-MeV/nucleon-argon ions in doses of0–120 Gy. The LET of the argon-ion beams was controlled to 188 keV/µm. Survival and mutation rates
in M1 generation were measured as previously described.2) Randomly selected ten M2 plants were harvested from one self-pollinated M1 plant. Genomic
DNA was extracted from a mixture of the leaves of
the ten M2 plants. Five DNA pools were sequenced
for each dose using HiSeq X-Ten sequencing systems(Illumina Inc.). The read sequences obtained were input into the mutational analysis pipeline, AMAP, asdescribed previously,3) with some modiﬁcations. Themutation candidates were detected with gatk (HaplotypeCaller), pindel, and breakdancer software.
The number of mutations was counted as follows: ForFig. 1. Mutation rate in M1 generation after irradiation of160-MeV/nucleon-argon beam (n ≥ 500).
∗1

RIKEN Nishina Center

Fig. 2. Number of mutations in M2 generation induced after irradiation of 188 keV/µm argon-ion beam (blackcircles) and 30 keV/µm carbon-ion beam (whiterhombi).

small mutations that included single nucleotide substitutions and <100 bp indels (insertions and deletions),
each mutation was counted as one. For large mutations, that included ≥100 bp indels and chromosome
rearrangements), the number of junctions in each mutant line were counted. Total number of these mutations per DNA pool was envisaged as the number ofmutations per genome.
In the M1 generation, the average survival rate (ﬂowering rate one month after cultivation2) ) was 94% at
80 Gy-irradiation and decreased to 74% at 100 Gyirradiation (data not shown). The mutation rate (appearance ratio of white sector mutation2) ) indicatedan increasing tendency with the increasing dose in therange of 10–80 Gy (Fig. 1). The highest mutation rate
was 7.7% at 60 Gy-irradiation, which is as high as that
previously reported (7.0% at 400 Gy with 30 keV/µmof LET; p = 0.54 by chi-square test).2) The decreased
mutation rate at 100 Gy-irradiation may reﬂect theradiation overdose, as seen in the survival rate.
The number of mutations per genome in the M2 generation after 188 keV/µm argon ion beam irradiationcompared to that after 30 keV/µm carbon ion beamirradiation previously reported1,4) is shown in Fig. 2.
After 400-Gy irradiation of the 30 keV/µm carbonion beam, the mutation rate in the M1 generation wasapproximately 7%2) and the average number of mutations in the M2 generation was 59 (Fig. 2). In contrast,
after the 60-Gy irradiation of the 188 keV/µm argonion beam, the mutation rate in the M1 generation was- 176 -1

III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

7.7% (Fig. 1) but the average number of mutations in
the M2 generation was 29 (Fig. 2). The diﬀerence inthe average number of mutations in the M2 generation
under between the two irradiation conditions can be
attributed to the content of the mutations. Among thenumber of mutations in the M2 generation induced by
the 30 keV/µm carbon-ion beam, only 4% were derived
from large mutations, whereas 32% were obtained by
188 keV/µm argon-ion beam. Further analysis, for example, to compare the number of genes aﬀected by themutations including inversions that possibly cause thechromosomal position eﬀect of the 30 keV/µm carbonion and the 188 keV/µm argon-ion beam may provide insight into the mutagenic characteristics of eachbeam.
References

1) Ishii et al., RIKEN Accel. Prog. Rep. 52, 216 (2019).
2) Y. Kazama et al., Plant Biotechnol. 29, 441 (2012).
3) K. Ishii et al., Genes Genet. Syst. 91, 229 (2016).
4) Y. Kazama et al., Plant J. 92, 1020 (2017).

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