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

Analysis of carbon ion-induced mutations by exome sequencing of an
unselected rice population
H. Ichida,∗1 R. Morita,∗1 Y. Shirakawa,∗1 Y. Hayashi,∗1 and T. Abe∗1mutations was 9.06 ± 0.37 (average ± standard error) per line. There were a total of 997 mutations,which consisted of 573 base substitutions, 372 deletions, 36 insertions, 13 substitutions, and 3 inversions,
identiﬁed from the irradiated M2 lines. The percentageof deletions and insertions, against all detected mutations, was 40.9%, which was consistent with the previously described characteristics of mutations induced byhighly accelerated heavy-ion beams, which often cause
nucleotide substitutions and deletions and insertionsof less than 100 bp.1) In contrast, no mutation wasidentiﬁed in non-irradiated Nipponbare pools, indicating that the mutations detected from the carbon-ionirradiated samples were likely to be induced by themutagenesis.
Based on an interpolation from the proportion oftarget exon regions against the entire genome (the total length of the target exon regions is 9.12% of thelength of the entire genome), roughly 100 mutations
are expected to be induced in the entire genome. Thismight be an underestimation due to the diﬀerence inbiological signiﬁcance between protein-encoding exonsand other genomic regions, which are mostly intergenicregions and repetitive elements; however, this estimation was of the same order as our previous results (175
to 549 mutations per genome in the 12 mutants analyzed) obtained from the whole genome sequencing ofmorphological mutants in rice.2)

Massively parallel sequencing technology has beenutilized in many areas of biology including mutation
analysis, by using a large amount of gene and genome
sequencing information to achieve a comprehensive
and genome-wide analysis. Heavy-ion beams are oneof the physical mutagens that are classiﬁed as highLET radiation and are known to induce double strand
breaks of DNA in a cell along with its track. The resulting mutations, including deletions, insertions, inversions and base substitutions, that occur on thegenome can cause the inactivation and/or temporalchange of gene expressions that are necessary for morphogenesis. We have developed a custom-designed
oligonucleotide probe library to capture entire exons
within the rice genome, which targets a total of 300,746
genomic loci at the same time. We also developed abioinformatics pipeline to map the sequencing reads to
the reference Nipponbare genome sequence and identify reliable mutations in a highly paralleled way byusing the “HOKUSAI” parallel computing system operated by the Advanced Center for Computing andCommunication, RIKEN. In addition to these previous
eﬀorts to make the genome-based mutation detections,
we developed a pre-mixed target capture procedure tofurther reduce the usage of the custom-designed target capture oligonucleotide probes, which takes nearly
half of the overall cost in whole exome sequencing, by
mixing multiple libraries with diﬀerent index sequences
prior to the target capturing: this reduces the persample oligonucleotide probe usage to 1/8th of that of
the original protocol.
In the present study, we analyzed a total of 110 independent M2 lines from carbon-ion beam irradiations(12 C6+ , 135 MeV/u, LET: 30 keV/µm, 150 Gy) toNipponbare rice seeds, the water content of which wasadjusted to 13%. The irradiated seeds were grown ina paddy ﬁeld, and the M2 seeds were harvested fromeach line. In each line, 10 to 15 plants were grownin soil, and an equal amount of leaf blades were collected from each plant and subjected to genomic DNA
extraction and sequencing library preparation. As acontrol, 8 pools of non-irradiated Nipponbare plants
were also processed in the same maner. A total of 8libraries were mixed together prior to the target capturing and then sequenced in a half lane on a HiSeq
4000 instrument. The obtained sequencing dataset wasprocessed using our bioinformatics pipeline describedabove. As a result, the number of mutations within thetarget region of whole exome capturing was between 3
and 26 in each line (Fig. 1). The average number of∗1

The number of mutations detected

30
20
100

12345678

910111213

1415

161718

1920

21222324

25

262728

2930

3132

33

3435

3637383940

4142

43

4445

46

47

48

49

50

5152535455

5657

58596061626364302010030201003020100LineFig. 1. Number of mutations detected from thecarbon-ion irradiated rice M2 lines (Nos. 1–64).

References
1) Y. Kazama et al., BMC Plant Biol. 11, 161 (2011).
2) H. Ichida et al., RIKEN Accel. Prog. Rep. 50, 271(2017).

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