title: Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana.
abstract: BACKGROUND
  Heavy-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.
  
  
  RESULTS
  Dry 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: RIKEN Nishina Center, Wako, Saitama, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kazama
authors.forename: Tomonari
authors.initials: T
authors.lastname: Hirano
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Sumie
authors.initials: S
authors.lastname: Ohbu
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Abe
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000528:radiation effects; D004252:DNA Mutational Analysis; D018744:DNA, Plant / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D020450:Heavy Ions; D016296:Mutagenesis; D020641:Polymorphism, Single Nucleotide
chemical_list: D018744:DNA, Plant
references.citation: Genes Genet Syst. 2009 Oct;84(5):361-70
references.pmid: 20154423
references.citation: J Radiat Res. 2010;51(3):223-33
references.pmid: 20505261
references.citation: Int J Radiat Biol. 2000 Apr;76(4):539-47
references.pmid: 10815635
references.citation: BMC Plant Biol. 2007 Apr 11;7:19
references.pmid: 17428339
references.citation: Environ Mol Mutagen. 2002;40(3):207-15
references.pmid: 12355555
references.citation: Plant Cell. 1992 Mar;4(3):333-47
references.pmid: 1354004
references.citation: Nat Biotechnol. 2000 Apr;18(4):455-7
references.pmid: 10748531
references.citation: Radiat Res. 2003 Feb;159(2):251-61
references.pmid: 12537531
references.citation: Plant Cell Physiol. 2000 Dec;41(12):1334-46
references.pmid: 11134419
references.citation: Plant J. 1995 Sep;8(3):457-63
references.pmid: 7550382
references.citation: BMC Plant Biol. 2004 Jul 28;4:12
references.pmid: 15282033
references.citation: Radiat Res. 2007 Jan;167(1):94-101
references.pmid: 17214518
references.citation: Mutat Res. 2008 Mar 1;639(1-2):101-7
references.pmid: 18068202
references.citation: Radiat Res. 2010 Mar;173(3):263-71
references.pmid: 20199211
references.citation: BMC Plant Biol. 2009 Aug 28;9:115
references.pmid: 19712486
references.citation: Plant Cell. 1993 Feb;5(2):147-57
references.pmid: 8453299
references.citation: Plant J. 1996 Oct;10(4):755-60
references.pmid: 8893551
references.citation: Nature. 1993 Nov 11;366(6451):162-6
references.pmid: 8232555
references.citation: Clin Chem. 2003 Jun;49(6 Pt 1):853-60
references.pmid: 12765979
references.citation: Genetics. 2005 Feb;169(2):881-9
references.pmid: 15371348
references.citation: BMC Plant Biol. 2008 Jan 24;8:9
references.pmid: 18218134
references.citation: Mutat Res. 1998 Jun 5;401(1-2):199-206
references.pmid: 9639705
references.citation: Mutat Res. 1982 Mar;93(1):109-23
references.pmid: 7062928
references.citation: Cell. 2000 Feb 18;100(4):469-78
references.pmid: 10693763
references.citation: Cell. 1991 Nov 1;67(3):483-93
references.pmid: 1934056
references.citation: Radiat Environ Biophys. 1995 Jun;34(2):67-72
references.pmid: 7652153
references.citation: Int J Radiat Biol. 1996 Nov;70(5):493-503
references.pmid: 8947529
references.citation: Int J Radiat Biol. 1994 Nov;66(5):427-32
references.pmid: 7983426
references.citation: Trends Plant Sci. 1999 Jul;4(7):263-269
references.pmid: 10407442
references.citation: J Exp Bot. 2005 Feb;56(412):587-96
references.pmid: 15642718
references.citation: Adv Space Res. 1996;18(1-2):127-36
references.pmid: 11538953
references.citation: Int J Radiat Biol. 1988 Nov;54(5):761-71
references.pmid: 2902170
references.citation: Plant Cell. 1999 Jul;11(7):1337-50
references.pmid: 10402433
references.citation: Plant Cell. 2001 Feb;13(2):425-36
references.pmid: 11226195
references.citation: Genetics. 2003 Jun;164(2):731-40
references.pmid: 12807792
references.citation: Genes Dev. 1994 Jun 15;8(12):1388-99
references.pmid: 7926739
references.citation: Genetics. 2001 Jan;157(1):379-87
references.pmid: 11139518
references.citation: Plant Cell. 2001 Sep;13(9):2115-25
references.pmid: 11549767
references.citation: Mutat Res. 1990 Nov-Dec;233(1-2):127-38
references.pmid: 2233794
references.citation: Genes Genet Syst. 2007 Apr;82(2):167-70
references.pmid: 17507783
references.citation: Radiat Environ Biophys. 1995 Nov;34(4):239-44
references.pmid: 8749062
references.citation: Plant Cell. 1999 Mar;11(3):335-48
references.pmid: 10072395