III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Whole genome sequencing analysis for detecting mutations induced bycarbon- and argon-ion irradiations of rice (Oryza sativa L.)
R. Morita,∗1 H. Ichida,∗1 Y. Shirakawa,∗1 K. Ichinose,∗1 T. Sato,∗1,∗2 and T. Abe∗1
A mutation analysis with high-throughput sequencing is a powerful tool for characterizing the molecular nature of mutations induced by mutagen at thewhole-genome level. We have investigated the mutation eﬀect following heavy ion-beam irradiation of rice
(Oryza sativa L. cv. Nipponbare), a monocotyledonous
model plant, using high-throughput sequencing. In thisstudy, we irradiated C-ion (LET: 30 keV/µm) or Ar-ion(LET: 290 keV/µm) to rice, and investigated numbers
and types of mutations induced by both ions at wholegenome level to examine whether the diﬀerence in theLET value of the ions inﬂuences the numbers and sizesof mutations in rice genome.
We irradiated C-ion (175 Gy, LET: 30 keV/µm) orAr-ion (10 Gy, LET: 290 keV/µm) to rice (Oryza sativa
L. cv. Nipponbare) dry seeds. The plants from irradiated seeds (M1 plants) were grown in a paddy ﬁeldand self-pollinated to obtain M2 seeds. Next year, wegrew M2 plants in a paddy ﬁeld to collect mutant plants
with visible phenotypes and harvest M3 seeds from theeach plant. For this study, we selected 11 and 4 mutant plants from C-ion and Ar-ion irradiations, respectively. Genomic DNA extraction for Whole-genome sequence was performed from the leaves of individual M3
plants. Candidate mutation extraction were performed
using “bioinformatics pipeline.” 1) We extracted candidate mutations using a combination of the gatk, pindel,
delly, and manta programs. gatk adopted the following parameters of (-stand_call_conf 50 -A Coverage -A
RMSMappingQuality -baq CALCULATE_AS_NECESSARY). pindel
was used following parameters of (-window_size 1–report_long_insertions –report_breakpoints–minimum_support_for_event 3 –min_inversion_size 10).

delly and manta were run with default parameters. Allmutations detected were visually conﬁrmed using Integrated Genomics Viewer (IGV).2)
We detected 1718 mutations from 11 C-ion-irradiatedmutants, and 426 mutations from 4 Ar-ion-irradiatedmutants. The average numbers of total mutations ina mutant genome were 156.2 and 106.5 for C-ion andAr-ion irradiations, respectively. The most abundant
mutations induced by both C-ion and Ar-ion irradiation were single-nucleotide variants (SNVs). The average numbers of SNVs in a mutant genome were 123.2
and 86.8 for C-ion and Ar-ion, respectively (Fig. 1).
The second most abundant mutations induced by bothC-ion and Ar-ion irradiation were small (<100 bp) insertions and deletions (indels). The average numbers ofindels in a mutant genome were 31.5 and 16.3 for Cion and Ar-ion, respectively. There were large deletions∗1∗2

RIKEN Nishina Center
Graduate School of Agricultural Science, Tohoku UniversityFig. 1. Average number of SNV, small Indel, Large deletion,and SV induced by C-ion (blue) and Ar-ion (red) irradiations. Error bars indicate standard error.

(≥100 bp) and structural variations (SVs), such as inversion and translocation in a mutant genome derived
from both C-ion and Ar-ion irradiations. The average
numbers of large deletions and SVs in a mutant genome
were 0.2 and 1.3 for C-ion, and 1.0 and 2.5 for Ar-ion,respectively. Comparing the mutations induced by Cion and Ar-ion, the number of small mutation, such as
SNVs and small indels, tended to be higher in the Cion irradiation than in Ar-ion irradiation. Conversely,
the number of both large deletion and SVs tended to behigher in Ar-ion than in C-ion. These tendencies corresponded to the results observed in the irradiation ofArabidopsis seeds.3) They compared the number of mutations induced by C-ion (LET: 30 keV/µm) or Ar-ion(LET: 290 keV/µm) irradiation using Arabidopsis, a dicotyledonous model plant, at the whole-genome level,and demonstrated that Ar-ions induced rearrangements(including large-deletion and SVs) more frequently thanC-ions.3) They also indicated that Ar-ions induced SNVsless frequently than C-ions. The ﬁndings obtained in thisstudy and that of Kazama et al. (2017) suggested thatC-ion irradiation tends to induce small mutations predominantly, whereas Ar-ion irradiation tends to induce
higher number of large deletions and SVs than C-ion
irradiations of both rice and Arabidopsis genomes. Because the sample size was not suﬃcient to compare thenumbers of each mutation induced by C-ion and Ar-ion
statistically in this study, we plan to increase the sample
size to perform a more detailed analysis.
References

1) H. Ichida et al., Plant J. 98, 301 (2019).
2) J. T. Robinson et al., Nat. Biotechnol. 29, 24 (2011).
3) Y. Kazama et al., Plant J. 92, 1020 (2017).

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