Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 51 (2018)

Chromosomal rearrangement induced by high-LET heavy-ion-beamirradiation in Parachlorella kessleri
K. Ishii,∗1 M. Asano,∗2 Y. Kazama,∗1 T. Abe,∗1 and S. Kawano∗2
Heavy-ion beams are used as an eﬀective mutagen
that induces localized mutations owing to their high linear energy transfer (LET).1) Breeding with heavy-ion
beams has been attempted often on land plants and recently on microalgae. Parachlorella kessleri (Chlorellales, Trebouxiophyceae, Chlorophyta) is a type of unicellular green algae that has received much attention asa biological resource for biomass production. A P. kessleri mutant with high oil production has been produced by heavy-ion-beam irradiation.2) Recently, in aland plant Arabidopsis thaliana, it has been reported
that an Ar ion beam (LET: 290 keV/µm) signiﬁcantly
induced chromosomal rearrangements more frequently
than a C ion beam (LET: 30.0 keV/µm).3) In thisstudy, we produced P. kessleri mutants in which chromosomes were fragmented by Ar-ion-beam and Fe-ionbeam (LET: 640 keV/µm) irradiations. We performed
mutation analysis on those mutants to characterize thenature of mutations induced by the high-LET heavy-ion
beams.
P. kessleri was irradiated with the Ar-and Fe-ion
beams with a dose of 75 Gy and cultured for 16–20 hin a TAP medium at 23◦ C under the continuous lightcondition. For microscopic observation, both irradiatedand unirradiated cells were ﬁxed by glutaraldehyde andstained by SYBR Green I. A typical karyotype of thewild type indicates that P. kessleri has seven chromosomes (Fig. 1A). On the other hand, chromosome fragmentation was observed in the irradiated cells (Fig. 1B–C). Irradiated cell lines were established by single colony
isolation. Though some of these fragmented chromosomes were not inherited by descendant cells, the two
established lines (Fe75-1-3H and Ar75-1-3H) were conﬁrmed to stably possess fragmented chromosomes bypulsed ﬁeld gel electrophoresis (data not shown).
The two established lines (Fe75-1-3H and Ar75-13H) and the wild-type line were resequenced by theMiSeq sequencing system (Illumina Inc., https://www.
illumina.com). Mutation analysis was conducted by
the mutation analysis pipeline AMAP as described previously4) with some modiﬁcations: we modiﬁed AMAP
to utilize the draft genome sequence (consisting of 400
scaﬀolds and 60 Mb in total) and the gene structure information (in the GTF format) of P. kessleri. As rearrangements deﬁned previously3) as including indels
more than 100 bp and chromosomal rearrangements,
one inversion was detected in Ar75-1-2C. In Fe75-13H, two inversions and three translocations were detected. All of the three translocations were localized
in a region of approximately 2 kb of the genome, which∗1∗2RIKEN Nishina Center
Future Center Initiative, The University of Tokyo

Fig. 1. Karyotypes of P. kessleri. (A) Typical karyotypeof the wild type. (B–C) Typical karyotypes of cells after
irradiation with Ar- (B) and Fe-ion beams (C), indicatingthat chromosomes were fragmented into 17 and 39 parts,respectively.

Fig. 2. Mapped read sequences including only telomeric repeats. The gray bars indicate read sequences. The colored lines on read sequences indicate single nucleotide
polymorphisms. The blue box indicates telomeric repeats.

may reﬂect the dense ionization produced by the highLET heavy-ion beam. To detect chromosome rearrangements involving chromosome ends, reads that only include continuous (more than three units) telomeric repeat (TTTAGGG) were extracted from the resequenced
data and mapped to the draft genome sequence by
Burrows-Wheeler Aligner. One newly formed junction
with telomeric repeats in Ar75-1-2C was found (Fig. 2).
This junction possibly explains the stability of some of
the fragmented chromosomes. Alternatively, it was possibly the junction with internal telomere sequences. Fluorescence in situ hybridization that utilizes the genomic
sequence near the junction as a probe will provide information based on which a hypothesis would be plausible.
In this study, it was revealed that the Ar and Fe ion
beams induced chromosome fragmentations in P. kessleri. Mutation analysis suggested that the fragmentations were caused by localized DNA double-strand
breaks and that chromosome ends of stable fragmented
chromosomes possessed telomeric repeats. The complete
genome sequence will provide clearer information on thenature of mutations induced by the high-LET heavy-ion
beam.
References
1) T. Hirano et al., Mutat. Res. 735, 19 (2012).
2) S. Ota et al., Bioresour. Technol. 149, 432 (2013).
3) Y. Kazama et al., Plant J. 92, 1020 (2017).
4) K. Ishii et al., Genes Genet. Syst. 91, 229 (2016).

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