Ⅰ. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 51 (2018)

Eﬀect of LET on mutational function revealed by whole-genome
resequencing of Arabidopsis mutants†
Y. Kazama,∗1 K. Ishii,∗1 T. Hirano,∗1,∗2 T. Wakana,∗1 M. Yamada,∗1 S. Ohbu,∗1 and T. Abe∗1
Heavy-ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Since the valueof LET aﬀects DNA lesion formation in several aspects,
including the eﬃciency and density of double-stranded
break along the particle path,1,2) mutations induced after the DNA lesion repair would also be aﬀected by
the value of LET. Whole-genome resequencing is aneﬀective way to assess the eﬀect of the value of LET
on mutation induction, and provides suﬃcient numberof mutations from each mutant to perform statisticalanalyses.3) Here, we investigated the diﬀerences in themutation type induced by irradiation with two representative ions, namely C ions (LET: 30.0 keV/µm) andAr ions (LET: 290 keV/µm), by whole-genome resequencing of the Arabidopsis mutants produced by theseirradiations.
Dry seeds of A. thaliana were irradiated with C ions(30.0 keV/µm) or Ar ions (290 keV/µm) with doses
found to induce 95% survival rates and the highest mutation frequencies.4) For the C-ion and Ar-ion irradiations, doses of 50 and 400 Gy were adopted, respectively. Eight mutants showing morphological phenotypes were screened in the M2 generation after eachirradiation. Then, the phenotypes of the mutants wereconﬁrmed in the M3 generation. In total, 16 mutants were selected, and DNA pools were extracted
from 40 plants of their individual progeny. The extracted DNA was sequenced using the HiSeq 2500 andHiSeq 4000 sequencing systems (Illumina Inc., https://www.illumina.com). The obtained reads were input
into AMAP, as described previously.5)
The rearrangements including translocations andlarge deletions (≥ 100 bp) that were induced by Ar ionswere 4.6 times more frequent than those induced by theC ions; the average number of rearrangements in a mutant genome was 10.3 and 2.3 for Ar ions and C ions, respectively (Fig. 1). These diﬀerences were statisticallysigniﬁcant (P < 0.01; two-sided Welch’s t-test). Both
Ar and C ions induce rearrangements. However, more
complicated rearrangements occurred following Ar-ion
irradiation, in which fragments of several hundred kbp
to several Mbp were produced and joined with direction
or positions diﬀerent from those of the original ones.
Conversely, Ar ions induced small mutations includingbase substitutions and small indels (< 100 bp), whichwere 2.3-fold less frequent than C ions: the average
number of small mutations in a mutant genome was
18.3 and 41.6 for Ar ions and C ions, respectively. This†∗1∗2

Condensed from the article in Plant J. 92, 1020 (2017)
RIKEN Nishina Center
Faculty of Agriculture, University of Miyazaki- 27 -Fig. 1. Mutations in each of the eight mutants induced by Cand Ar-ion irradiation. The rearrangements are plotted
as lines on the interior of the circles. Small mutations
are indicated by lines on the exterior of the circles. Themutations in each mutant are diﬀerently colored.
Table 1. Sum of homozygously mutated genes detected ineight mutants.

diﬀerence was also statistically signiﬁcant (P < 0.01;
two-sided Student’s t-test).
The eﬀects on gene mutations were also diﬀerent between C-ion and Ar-ion irradiations. The sum of homozygously mutated genes in eight individual mutants
after irradiation with Ar and C ions are shown in Table 1. After C-ion irradiation, amino-acid changes werefrequently observed, which were caused by small mutations. On the other hand, Ar-ion irradiation frequently
induced truncations of genes or losses of whole genes
caused by rearrangements.
These data demonstrate that the nature of mutations
is signiﬁcantly diﬀerent between beams with diﬀerent
LET values. Such a selective irradiation will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements in conjunction with the technics of mutation detection through high-throughput sequencing.
References
1) D. Alloni et al., Radiat. Res. 173, 263 (2010).
2) N. A. Franken et al., Radiat Oncol. 6, 64 (2011).
3) K. Shirasawa et al., Plant Biotechnol. J. 14, 51 (2016).
4) Y. Kazama et al., Plant Biotechnol. 25, 113 (2008).
5) K Ishii et al., Genes Genet. Syst. 91, 229 (2016).