Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 50 (2017)

Results of whole-genome analysis of pink and ebony mutant
K. Tsuneizumi,*1 H. Ichida,*1 R. Morita,*1 and T. Abe*1
Heavy-ion-beam mutagenesis is generally recognized as aneffective method for mutation breeding.1, 2) Although thismethod was greatly successful with plants, its application islimited for animals. Therefore, we plan to acquire more basic
data to set up optimal conditions for the heavy-ion-beamirradiation system by using Drosophila melanogaster (fruitfly) as the model.
In our previous study, we determined that 1-3 Gy
irradiation using a carbon-ion beam is suitable for the largescale screening of mutant lines.3) To elucidate the biologicaleffect of heavy-ion-beam irradiation to the genome, weanalyzed the whole-genome sequencing of several mutants
established by the condition of 1-3 Gy irradiation. In thisreport, we applied the high-performance bioinformatics
pipeline, which was developed for analyzing rice exome
sequencing results obtained in our laboratory4), to fly genome
analysis. Mutants are established using the third
chromosome balancer, which is a unique genetic tool for fruit
fly. Because the third chromosome balancer is known toprevent homologous recombination only on the third
chromosome, the following results are limited to third
chromosome events.
In this report, we show the result of pink [Fig. 1] and ebony[Fig. 2] mutants. Each mutation is summarized in Fig. 1c andFig. 2c. These data suggest that 1-3 Gy dose is sufficient forseveral mutations such as single base substitution,

Fig. 1. Phenotype of the pink mutant and the result of wholegenome analysis.
a) Wildtype eye color is vivid red. b) The mutant eye color
becomes lighter than wildtype. c) The mutant is establishedby the condition with 80 keV/μm linear energy transfer at 1
Gy dose level. Whole-genome analysis revealed 11
mutations caused by heavy-ion-beam irradiations. Eachmutation was categorized into substitution, deletion andinsertion. The causal mutation of pink was classified intolarge deletion with red text.
1

deletion, insertion, rearrangement, and large deletion. These
diversities of mutations indicate an advantage in screening
by the heavy-ion-beam mutagenesis. Both mutants contain11 or 12 mutations, and then the third chromosome consistsof 5.2 x 107 base pairs (bp). This means that the heavy-ionbeam irradiation introduces a mutation into the genome at afrequency of approximately 4.5 x 106 bp.
Among 11 or 12 mutations, an alteration in protein
sequence was observed in only one gene. We purchased
several mutants of the same gene from the Drosophila stock
center. Through the complementation test known as a genetic
technique for fruit fly to determine whether two mutantsbelong to the same gene, we determined that the causal genes
are pink and ebony. Though it is thought that the fruit fly has
about 15000 genes, we could easily identify the causal genesof mutants by using whole-genome sequencing and thepipeline. These results suggest that the pipeline technique isa powerful tool for mutation analysis.

Fig. 2. Phenotype of the ebony mutant and the result ofwhole-genome analysis.
a) Wildtype body color is yellowish brown. b) The mutant
body color becomes darker than wildtype. c) The mutant isestablished by the condition with 80 keV/μm linear energy
transfer at 3 Gy dose level. Whole-genome analysis revealed
12 mutations caused by heavy-ion-beam irradiations. Eachmutation was categorized into substitution, deletion,insertion and rearrangement. The causal mutation of ebony
was classified into single base substitution with red text.
References
1) T. Abe et al., in Plant Mutation Breeding and Biotechnology,
edited by Q. Y. Shu et al. (CABI, Oxfordshire, 2012), p. 99.
2) A. Tanaka et al., J. Radiat. Res. 51, 223 (2010).
3) K. Tsuneizumi et al., RIKEN Accel. Prog. Rep. 49, 253 (2016).
4) H. Ichida et al., RIKEN Accel. Prog. Rep. 49, 254 (2016).

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