Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana

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.

Effect of high-LET Fe-ion beam irradiation on mutation induction in <i>Arabidopsis thaliana</i>

“…When the albino mutant incidence was investigated in the M 2 generation after irradiation treatment with heavy-ion beams with LETs ranging from 22.5 to 640 keV·μm -1 , the most effective LET was found to be 30.0 keV·μm -1 (Kazama et al, 2008). This irradiation treatment showed similar mutagenic efficiency to that of ethyl methanesulphonate (Koornneef et al, 1982;Kazama et al, 2011). The LET of 30.0 keV·μm -1 was also efficient for causing a high mutation frequency in the M 1 generation .…”

“…From the M 2 generation, hy and gl mutants were screened, whose phenotypes were long-hypocotyl (Koornneef et al, 1980) and non trichome (Walker et al, 1999), respectively. The putative responsible genes of hy and gl mutants (HY1, HY2, HY3, HY4, and HY5 for hy mutants; GL1, GL2, and TTG1 for gl mutants) were amplified by performing PCR, and then the amplified fragments were sequenced as described previously (Kazama et al, 2011;Hirano et al, 2012). When the whole region or a part of the coding region of the putative responsible genes could not be amplified, flanking sequence analysis by using TAIL-PCR was performed (Liu et al, 1995).…”

“…Following this, they were washed 5 times with sterilised water, and then incubated on 0.7% agar-containing Murashige and Skoog (MS) medium that was supplemented with MS vitamins and 3% sucrose at 4°C in the dark for 4 d to induce vernalisation. The M 2 seeds were collected as described previously (Kazama et al, 2011;Hirano et al, 2012). In short, eleven seedlings were grown in a plastic tray, from which M 2 seeds of all plants were corrected and were treated as one batch.…”

“…This highly efficient LET was designated as LETmax. The value of the LET also affects the size of the induced deletion: it becomes large with increasing LET Kazama et al, 2011;Hirano et al, 2012). Taken together, it is possible to select the LET value depending on the experimental purpose or target genes.…”

“…Taken together, it is possible to select the LET value depending on the experimental purpose or target genes. LETmax is suitable for inducing single gene mutations, because it provides a high mutation frequency and mainly causes deletions with the size range of 1-53 bp (Kazama et al, 2011). LET at 290 keV·μm -1 is useful for disrupting tandemly arrayed multiple genes, since it causes large deletions, the extent of which disrupt more than 2 tandemly arrayed genes .…”