Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 52 (2019)

Estimation of eﬃcient dose for heavy-ion beam mutagenesis bywhole-genome mutational analysis in Arabidopsis thaliana
K. Ishii,∗1 H. Ichida,∗1 S. Ohbu,∗1 and T. Abe∗1Heavy-ion beams are used as an eﬀective mutagen
that induces localized mutations due to its higher linear energy transfer (LET). The observation of the occurrence ratio of albino mutants as the mutation frequency
in the M2 generation after carbon-ion beam irradiation(30 keV/µm) at doses of 50–500 Gy showed that themutation frequency increased with increasing dose between 50–350 Gy and plateaued at 400 Gy.1) However,
the measurement of mutation frequency by visual screening of mutants from a large number of irradiated plants
is time-consuming. In this study, we tried to estimate
an eﬃcient dose for heavy-ion beam mutagenesis by performing whole-genome mutational analysis on relatively
small numbers of irradiated plants.
Dry seeds of Arabidopsis thaliana (the Col-0 strain)
were irradiated with 12 C6+ (135 MeV/nucleon) ions, atdoses of 0–350 Gy. The LET of C ion beams was controlled to 30 keV/µm. M2 seeds were harvested from selfpollinated M1 plants, and ten M3 plants were harvested
from one self-pollinated M2 plant. Genomic DNA wasextracted from the mixture of leaves of the ten M3 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, as described previously, withsome modiﬁcations.2) In short, after mapping the readsequences to the reference genome sequence (TAIR10)
with BWA (BWA-MEM) software, the mutation candidates were detected with GATK (HaplotypeCaller),
PINDEL, and BREAKDANCER software. Then, theAMAP ﬁltered out the false-positives by using its own
algorithm.
In spite of the process of ﬁltering out false-positives,
conﬁrmation of the mutation candidates in one DNApool (350-Gy irradiation) by using Integrative Genomics Viewer (IGV) software revealed that 81% of
the candidates were still false-positives (data not shown).
We conﬁgured additional criteria for the determinationof false-positives. Among the mutation candidates output from GATK, those with the ‘QUAL’ value (calculated by GATK) less than 150 or ‘MAPPING_QUAL’value (calculated by BWA) less than 60 were treated as
false-positives. Among the mutation candidates output
from PINDEL, those with the ratio of the number of readsequences supporting the mutation to that of mapped
read sequences less than 0.2 or with the number of reads
supporting the mutation less than 5 were treated as
false-positives. Among the mutation candidates output
from BREAKDANCER, those with the ratio of the number of read sequences supporting the mutation to thatof mapped read sequences less than 0.15 were treatedas false-positives. Also, among the mutation candidates∗1RIKEN Nishina Center

Fig. 1. Average number of mutations. Five DNA pools wereanalyzed at each dose.

output from PINDEL or BREAKDANCER, two candidates
from diﬀerent DNA pools overlapping one another bymore than 80% of their regions were treated as falsepositives. By using IGV software, it was revealed that
21% of the candidates in the DNA pool (350-Gy) werefalse-positives after these additional ﬁltrations and that
9% of the positive mutations were ﬁltered out by theadditional ﬁltrations. The mutation candidates in all
DNA pools were tested with the additional ﬁltrations
and checked using IGV software.
The number of mutations per genome increased monotonically as the irradiation dose increased and did notplateau until 350 Gy (Fig. 1). In this study, it wassuggested that irradiation at 350 Gy is eﬃcient in therange of 0–350 Gy. Although the means for evaluating the mutation frequency in this study was diﬀerent
from that in the previous study,1) the tendency of increase in the mutation frequency in proportion to theirradiation dose was similar. The mutation analysis
method in this study covered all mutations in the wholegenome while the mutation analysis by visual screening covered only those in genes related to the development of chloroplasts. Because this method can determine
the type (single nucleotide substitution, deletion, insertion, inversion, or chromosomal rearrangement) and thesize of each mutation, the previous study revealed theLET-dependent eﬀect for mutation induction: heavy-ion
beams with higher LET can induce chromosomal rearrangements or large deletions more frequently than thosewith lower LET.3) In this study, it was also revealed that
this method can be applied to the dose-dependent eﬀect
for mutation induction.
References

1) Y. Kazama et al., BMC Plant Biol. 11, 161 (2011).
2) K. Ishii et al., Genes Genet. Syst. 91, 4, 229–233 (2016).
3) Y. Kazama et al., Plant J. 92, 6, 1020–1030 (2017).

- 216 -