Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice
Introduction
Ion beams are charged particles produced by particle accelerators that use electromagnetic fields. As with other ionizing radiations, ion beams cause damage to DNA molecules in living organisms and have been used as physical mutagens in plant and microbe breeding [[1], [2], [3], [4]]. Ion beams are characterized by the deposition of a high energy transfer per unit length (linear energy transfer, LET) and are believed to induce mutations as a consequence of biological effects distinct from low LET radiation such as gamma-rays and electrons. Indeed, during the screening of mutants from irradiated explants of carnations, ion beams induced a wider variety of mutants with respect to flower color and shape than gamma-rays and X-rays [5]. A qualitative difference in flower color variations in an ion-beam-irradiated population has also been observed in chrysanthemums [1]. In contrast, Yamaguchi et al. reported no remarkable differences in the mutation spectra between ion-beam and gamma-ray irradiation [6].
Characterization of ion beam-induced mutations in plant DNA was conducted using several approaches in Arabidopsis, a model plant for plant molecular genetics. The most common approach for characterizing germline mutations is the isolation of mutants deficient in well-characterized marker genes responsible for visible phenotypes such as seed color (tt) and leaf trichome morphology (gl), followed by analysis of the DNA sequence of the marker gene [[7], [8], [9], [10]]. The isolation of mutants or tissue sectors of well-characterized marker genes is also useful for characterizing ion-beam-induced somaclonal mutations by detecting the loss of heterozygosity [[11], [12], [13]]. An alternative method that can eliminate the isolation of mutant plants is the use of the Escherichia coli ribosomal protein small subunit S12 (rpsL) mutation detection system [14,15]. In this system, plants containing the rpsL transgene are irradiated, and genomic DNA from irradiated transgenic plants are introduced into E. coli. Mutated nonfunctional rpsL DNA fragments can be recovered from drug-resistant E. coli colonies. Previous experiments using these methods suggest that 1) ion beams induce various types of mutations into plant DNA, such as base substitutions, DNA insertions and deletions (InDels), inversions (INVs), and chromosomal translocations [8,10]; 2) ion beams induce germline mutations with a higher ratio of rearrangements such as InDels greater than 100 bp, inversions, translocations, and total deletions of the marker gene than electron beams, which induce mostly point-like mutations such as base substitutions and InDels less than100 bp [8]; 3) the size of the deletion induced by ion beams positively correlates with the degree of LET [12,16,17]; and 4) most mutants obtained with a pollen-irradiation method have large deletions > 6 Mb, most of which are not transmittable to the next generation [11].
Recent advances in genome analysis technologies have allowed for more quantitative evaluation of mutations at the genomic level without bias arising from genomic positioning and of the functional significance of DNA sequences of a specific marker gene. A mutation-accumulating experiment combined with whole genome analysis with next-generation sequencing revealed an estimated spontaneous mutation rate in Arabidopsis of 7 × 10−9 base substitutions per site per generation [18]. Whole genome sequencing analysis of ion beam-irradiated Arabidopsis DNA suggested that 200-Gy carbon ion beams increased the mutation rate nearly 47-fold compared to spontaneous mutations [19]. In addition, Arabidopsis whole genome sequencing has demonstrated that the frequency and type of mutations induced by ion beams are affected by the LET of ion beams [20] and the physiological status of irradiated tissues [21].
Rice is one of the most important crops for humans and is a major target for ion-beam breeding strategies [[22], [23], [24], [25], [26]]. Several genes that cause mutant phenotypes were successfully cloned by map-based cloning from ion-beam-induced rice mutants, and the mutation sites in the genes were characterized [23,26]. However, due to a relatively large genome size compared to Arabidopsis, the characterization of ion beam-induced mutations at the genomic level was not achieved in rice until very recently [27,28]. In the present work, we used a whole-exome sequencing procedure to analyze the properties of induced mutations in selected rice mutants generated with carbon ion beams accelerated using the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) azimuthally varying field (AVF) cyclotron at TARRI, QST. We demonstrated that a relatively small number of induced mutations with a potentially high impact on protein function quickly narrowed down the candidate genes responsible for the mutant phenotypes.
Section snippets
Plant materials and ion beam irradiation
Rice seeds (Oryza sativa L. cv Nipponbare, NPB) used in this work were harvested in RBD, NICS, NARO (Hitachi-ohmiya, Japan). Since the water content of a seed affects its radiation sensitivity [29], the water content was adjusted to 12–13 % by keeping the rice seeds in a plant growth chamber (BIOTRON LPH200, NK system, Japan) at 24 °C and 60 % RH for 4 d just before irradiation. Then, the seeds were irradiated with 10–150 Gy (at 10 Gy intervals) of 25.9 MeV/u 12C6+ ions (LET on surface:
Preparation of an ion-beam irradiated rice population and mutant selection
To estimate the biological effectiveness of the carbon ion beam on the NPB seeds, a dose response for the survival rate and plant height were determined (Fig. 1A). A dose at a shoulder (shoulder dose) or slightly lower dose in the survival curve was empirically considered to be optimal for obtaining a large number of mutants [1,6,22] and therefore, the absorbed dose chosen for the genetic screen in this work was 40 Gy, which was approximately 2/3 of the shoulder dose (approximately 60 Gy) for
Author contributions
Y.O and Y.H designed the experiment. S.N. and Y.H. conducted ion-beam irradiation, mutant screening, and characterization of mutants in the green house. Y.O., S.N., K.S., A.S., H.K., and Y.H. performed mutant screening and characterization of mutants in the paddy field. Whole exome analysis was done by H.I., R.M., and T.A. Y.O. and H.I. analyzed the data and Y.O. wrote the manuscript with input from H.I. and Y.H. All authors approved the final manuscript.
CRediT authorship contribution statement
Yutaka Oono: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft. Hiroyuki Ichida: Investigation, Methodology, Software, Formal analysis, Writing - review & editing. Ryouhei Morita: Methodology, Investigation. Shigeki Nozawa: Methodology, Investigation. Katsuya Satoh: Investigation. Akemi Shimizu: Resources, Investigation. Tomoko Abe: Investigation, Funding acquisition. Hiroshi Kato: Investigation, Resources. Yoshihiro Hase: Conceptualization, Methodology,
Acknowledgements
The authors thank Shoya Hirata, Eenzen Mungunchudur, Toshihiko Sanzen, Hiroki Arai, Shogo Ozawa, Satoshi Kitamura, and members of the Research Planning Office, Quantum Beam Research Directorate, QST for their help in harvesting rice seeds and characterizing the rice mutants, Dr. Feng Li from RBD, NICS, NARO for his valuable comments on the manuscript. The bioinformatics analysis was performed using the HOKUSAI supercomputing system, operated by the Information Systems Division, RIKEN, under the
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Current address: Genetic Resources Center, NARO, Tsukuba, Ibaraki, Japan.