III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Detection of structural variations in three responsible genes induced byrelatively high-LET ion beams in rice
Y. Shirakawa,∗1 R. Morita,∗1 Y. Hayashi,∗1 K. Ichinose,∗1 T. Sato,∗1,∗2 and T. Abe∗1
The molecular characteristics of mutations induced bya heavy-ion beam with a relatively high LET in rice,a model plant of monocotyledonous, has not yet been
revealed. In this study, we irradiated dry seeds of rice(Oryza sativa L. ‘Nipponbare’) with Fe- or Ar-ion beams,isolated 3 mutants for which the responsible gene has
been clarified, and characterized the structure of the mutated gene using polymerase chain reaction (PCR) analysis.
A pla1 mutant (Ar7-165, Fig. 1) was isolated from239 M1 lines irradiated with Ar ions (7.5 Gy, LET:289 keV/µm). In this mutant, no PCR amplification
occurred when primers designed for amplification of thecentral area of the PLA1 gene (primer pairs F4 and R20,
F6 and R21, and uF5 and R21, as shown in Fig. 2) wereused, suggesting that structural variations (SVs) such as
inversion or translocation occurred in the gene (Fig. 2).
A dwarf mutant (Fe15-235, Fig. 1) that showed a defect in gibberellin biosynthesis was isolated from 236 M1lines irradiated with Fe ions (15 Gy, LET: 650 keV/µm).
We conducted PCR analysis with the primers for gibberellin biosynthesis-related genes and detected a deletion in the region including the 1st to 6th exon of theOsKS1 gene (Fig. 2). Additional PCR and sequencing
analyses revealed that Fe15-235 harbors a 9,843 bp deletion (Chr04: 31018475 - 31028317) in the OsKS1 gene.
We isolated the brd1 mutant (WAr30-204), whichshowed a dwarf phenotype with a defect in brassinosteroid biosynthesis from 242 M1 lines irradiated withFig. 2. Schematic representation of mutated genes. Purple
boxes indicate protein coding regions (exons), and yellow
marks indicate the position of the DNA double-strand
breaks induced by heavy-ion beams. In the case of theAr7-165 mutant, the position of the primers used in PCR
are shown. Red arrows indicate primers for which DNA
amplification occurs in PCR, whereas blue arrows indicate
primers for which DNA amplification did not occur inPCR.

Ar ions generated by the WACAME line1) (30 Gy,
LET:184 keV/µm). PCR analysis revealed that this mutant harbors a 27 kb deletion involving the entire OsBRD1 gene (Fig. 2). Another deletion of approximately
5 kb was also detected 18 kb downstream from the 27kb deletion. It suggested that a complex mutation withlarge deletions occurred in the OsBRD1 gene region.
Our findings suggest that a heavy-ion beam with a relatively high LET may predominantly induce large deletions or SVs in the responsible gene region in rice. Thistendency was also observed in the PCR and sequence
analysis for Arabidopsis mutants induced by Ar-ion irradiation on dry seeds2) and rice mutants induced byAr-ion irradiation on imbibed seeds.3)
Fig. 1. Morphological comparison of (a) Nipponbare, (b)Ar7-165, and (c) Fe15-235 plants 24 days after sowing.
Scale bar = 5 cm.
∗1∗2

RIKEN Nishina Center
Graduate School of Agricultural Science, Tohoku University

References

1) N. Fukunishi et al., Proc. 13th Heavy Ion Accelerator
Technology Conference (HIAT2015), 42 (2016).
2) T. Hirano et al., Mutation Res. 735, 19 (2012).
3) S. Kogure et al., RIKEN Accel. Prog. Rep. 47, 289 (2014).

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