Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana.

Yusuke Kazama,Tomoko Abe,Hiroyuki Saito,Yoriko Hayashi,Yang Liu,Tomonari Hirano,Sumie Ohbu

doi:10.1186/1471-2229-11-161

Abstract

BackgroundHeavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis.ResultsDry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements.ConclusionsThe C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.

Highlights

Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants
We found that the linear energy transfer (LET) value affects the albino-mutant incidence in the M2 generation and that 30 keV μm-1 is the most effective LET value in Arabidopsis thaliana mutagenesis [22]
The number of ion particles could determine the number of double-strand breaks (DSBs) per cell nucleus, while the LET value might affect the efficiency of DSB induction

Summary

Introduction

Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams are accepted as a novel powerful mutagen because they are able to induce mutations with high frequency at a relatively low dose at which virtually all plants survive, and they induce a broad spectrum of phenotypes without affecting other plant characteristics [12,13]. These characteristics of heavy-ion beams are advantageous for mutation breeding. A noted physical characteristic of a heavy-ion beam is that the accelerated particles densely deposit their energy in a localized region along the particle path. When a high LET is required, a heavier and highly charged ion with a low velocity is selected

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Conclusion