Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 47 (2014)

Detection of deletions induced by Fe-ion irradiation in Arabidopsis thaliana
using array comparative genomic hybridization†
Y. Kazama,＊1 T. Hirano,＊1 K. Nishihara,＊2 S. Ohbu,＊2 Y. Shirakawa,＊2 and T. Abe＊1,＊2
Heavy-ion irradiation mainly induces deletions. The sizeof deletions increases with increasing linear energy
transfers (LETs)1). Because large deletions are useful for
producing null mutations as well as disrupting multiple
genes arrayed in tandem, high-LET ion beams are
considered powerful mutagens in the field of genome
science. However, the determination of deleted
chromosomal region(s) in a mutant by using classical
PCR-based methods is difficult because designing primers
at both sides of a large unidentified deletion is quitedifficult. Thus, a method for rapidly detecting deletions at
the whole-genome level is desired.
Array comparative genomic hybridization (array CGH) isa powerful tool for detecting deletions at the whole-genome
level2). In the present study, we herein optimized the array
CGH experiment to develop a method for investigatinglarge heavy-ion induced deletions at the whole-genome
level. For the array CGH, we used an Arabidopsis mutant
having downward-pointing flowers (Fe-148-pg1) as an
example of a mutant harboring large deletions, which was
induced by Fe-ion beam irradiation (90 MeV/nucleon, 640
keV/μm) at a dose of 50 Gy. Through the phenotypiccharacterization and PCR confirmation, Fe-148-pg1 wasfound to have a large deletion around theBREVIPEDICELLUS (BP) gene.
Arabidopsis thaliana ecotype Columbia (Col-0) wasused as a wild-type plant. The whole-genome sequence ofCol-0 was tiled with oligonucleotides that started every 50
bp. Probe lengths were constrained to a minimum of 50 anda maximum of 75 bp. Considering this design, our array
was expected to detect deletions of more than 200 bp at thewhole-genome level.
The DNA of the wild-type and the mutant plant(Fe-148-pg1) were labelled with Cy5 and Cy3, respectively.
Hybridization, washing, and scanning were conducted by
Roche NimbleGen Inc (Madison, WI, USA). Raw
fluorescence intensity data were obtained from scanned
images of the oligonucleotide tiling arrays by using
NimbleScan 2.4 extraction software (Roche NimbleGenInc.). For each spot on the array, log2 ratios of theCy3-labelled sample to the Cy5 reference sample werecalculated.
Candidate deletions were identified as follows. First,
regions supported by more than 4 consecutive probes withlog2 ratios of over 1.0 were listed as candidate deletions.
Then, the candidate deletions were confirmed byperforming PCR. For the PCR test, 7 individual M3 plants
were tested; when some of the plants showed amplificationin the candidate deletion region, the candidate deletion wasdetermined as being heterozygous in the M2 generation.
Finally, we detected 7 deletions (Table 1). Candidate
deletions supported by the higher log2 ratio tended to behomozygous. However, candidate deletions supported bylog2 ratios lower than 1.209 were false positives. As
expected, the Fe-148-pg1 had a large deletion covering theBP gene; the size of deletion was found to be 90,307 bp byconducting PCR-based confirmation and sequencing. The
results suggest that our array platform can detect bothhomozygous and heterozygous deletions at the wholegenome level, although the estimated deletion size is notcompletely consistent with the actual one.

Table 1 List of deletion peaks estimated by array CGH in Fe-148-pg1.
Start site of Peak area
Genetic
Estimated
Detected
Chr.
Position
Signal*deletion size
deletion homogeneity in M3
1.500–6.097
45,129,711
88069
homogeneous○
47,875,508
3.375–6.047
81108
homogeneous○
1
17,030,769
2.289
467
2.076
317,831,368450
homogeneous○
48,550,408
1.992
264
homogeneous
○

5444
4

8,060,900
2,179,908
2,177,558
2,185,508
2,181,208

84816

1.744–1.930
1.777
1.767
1.743
1.209

○
○○850814723
469*When several peaks were detected in a naborhood, the peaks were combined
and the range of signal values were considered.
†Condensed from the article in Genes Genet. Syst. 88, 189-197 (2013)＊1
RIKEN Innovation Center＊2
RIKEN Nishina Center

References

heterogeneous
heterogeneous
heterogeneous

1) T. Hirano et al.: Mutat. Res. 735, 19 (2012).
2) A. J. Nagano et al.: Plant J. 56, 1058 (2008).

- 286 -