Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 50 (2017)

Whole genome sequencing of 12 morphological rice mutants fromcarbon-ion irradiations
H. Ichida,∗1 Y. Shirakawa,∗1 R. Morita,∗1 Y. Hayashi,∗1 and T. Abe∗1
Heavy-ion beams are one of the physical mutagens that are classiﬁed in high-LET radiations andknown to induce double strand breaks of DNA in acell along with its track. The resulting mutations, including deletions, insertions, inversions and base substitutions, that occurr on the genome can cause theinactivation and/or temporal change of gene expressions that are necessary for morphogenesis. We previously reported the design and implementation of highperformance bioinformatics pipeline for massively parallel (aka “next generation”) DNA sequences obtained
from mutants. We irradiated a carbon-ion beam fromRRC (12 C6+ , 135 MeV/u) on dry and wet seeds of rice
(Oryza sativa ‘Nipponbare’) plants and isolated morphological mutants in the M2 generation. Of thosemutants, we selected 12 morphological mutants forcomprehensive mutation analysis by whole genome sequencing and subsequent bioinformatics analysis.
We utilized the whole-genome shotgun strategy using an Illumina HiSeq X system. Leaf blades werecollected from each M3 plant grown in a paddy ﬁeld
in September 2016, shipped in dry ice, and stored at-80◦ C until use. Genomic DNA was puriﬁed from 100
mg of bulked frozen tissue of each line using MagExtractor plant genome DNA extraction kits (Toyobo,Japan). The puriﬁed DNA was quantiﬁed and qualitychecked, follwing which it was subjected to random
fragmentation by sonication and subsequent processes
to attach the adapter sequences necessary for Illumina
sequencing. We aimed at an average insert size of 350
bp and a total of 30 billion bases of clean sequences(the sequences that passed ﬁltering by mean quality
and read length) from each mutant line. This cor-responds to approximately 80 times the equivalent ofrice genome and is expected to be suﬃcient for accurate variant calling according to the current recommendations in variant calling programs. The data wereanalyzed using our bioinformatics pipeline1) built andoperated on the Hokusai GreatWave system operatedby the Advanced Center for Computing and Communication, RIKEN.
The statistics of sequencing, mapping, and variant
calling are summarized in Table 1. We could obtaina similar amount of sequencing reads as well as othermapping metrics, indicating that the procedure is quite
stable and robust. The number of mutations identiﬁedby the Genome Analysis Took Kit (GATK)2) was alsosimilar among the mutants sequenced. Roughly 52,000
to 53,000 mutations were reported by the GATK program. However, as expected, most of those were artifacts from the intra-cultivar variations between the sequenced and our mutagenized Nipponbare cultivars of
diﬀerent origins. Our ﬁltering strategy removed suchfalse positives very eﬀectively: only 175 to 549 mutations were likely to be the actual mutations causedby heavy-ion beam irradiations. These results demonstrate that our approach using whole genome sequencing and the high-performance bioinformatics pipeline
is well suited for the mutation analysis in rice.
References
1) H. Ichida et al., RIKEN Accel. Prog. Rep. 49, 254(2016).
2) A. McKenna et al., Genome Res. 20, 1297–1303 (2010).

Table 1. Sequencing, mapping, and variant calling statistics of whole genome sequencing from 12 rice mutants
Seed
Irradiation condition
Aligned Mismatch
Indel
Num. of mutations
MutantTotal reads
condition
LET∗1
Dose (Gy)(%)(×10−3 )(×10−5 )Total
Line speciﬁc
Wet
C-249
50
15
243,240,586
99.53
3.21
5.00
53,600
191
7-80
30
50
233,349,445
99.43
3.71
5.10
52,404
175
Dry
7-84
30
50
231,042,177
99.44
3.94
5.00
52,365202(9%∗2 )
7-92
30
50
227,590,774
99.52
3.70
5.00
52,476210
14-3430150
236,257,550
98.40
3.59
5.10
53,135
276
14-55
30
150
234,303,120
99.05
4.05
5.00
52,593
193
14-64
30
150
234,084,341
99.38
3.84
5.00
53,273
273
Dry
14-70
30
150
233,143,578
99.25
3.31
4.80
53,196294(13%∗2 )
15-16
30
175
235,733,926
99.52
3.33
5.00
52,373
404
15-17
30
175
233,910,879
99.14
3.43
4.90
52,601
549
15-65
30
175
232,386,380
99.43
3.38
4.90
52,174
280
15-74
30
175
233,696,581
99.49
3.38
4.90
52,115258∗1keV/μm; ∗2 water content of the irradiated seed
∗1RIKEN Nishina Center- 271 -