Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 48 (2015)

Rapid screening of heavy-ion-induced large deletion mutants by using
quantitative real-time PCR in Arabidopsis thaliana
Y. Kazama,＊1 T. Hirano,＊1,＊2 K. Ishii,＊1 M. Yamada,＊1 Y. Shirakawa,＊1 S. Ohbu,＊1 and T. Abe＊1
In heavy-ion irradiation, the size of deletions can bevaried according to different values of linear energy transfer(LET). In irradiation experiments on Arabidopsis thaliana,
we previously found that beams with LET of 30 keV/μm
induced small deletions (1 to 51 bp) with high frequency,
and this size is appropriate for disrupting a single gene.1)
Thus, 30-keV/μm beams are suitable for use in mutation
breeding as well as in reverse genetics to find single genedisruption lines in conjunction with SNP detection systems.
On the other hand, beams with LET of 290 keV/μm caused
larger deletions (1 kbp to several hundred kbp).2) Suchhigh-LET beams can be used for generating mutants lacking
tandemly arrayed genes (TAGs) that have functions similar
to one another, allowing us to investigate the function ofTAGs. Thus, we are now developing a deletion-mutant
correction by screening them from M3 generations after
being irradiated with 100-, 200-, or 290-keV/μm beams.
The screening is performed using array-CGH designed for
all TAGs of A. thaliana. Researchers will be able to screen
deletion mutants of interest from our stock of over 10,000of 290-keV/μm irradiated M2 lines.
However, screening deletion mutants is time-consuming.
Conventionally, it was performed by PCR in the M2
generation, in which only homozygous deletions(HomDels) could be detected unless primers were designed
to have deletions placed between them (Table 1). In casethat the HomDel mutant of interest is lethal, no candidate
would be obtained. By contrast, heterozygous deletions(HetDels) can be inherited over generations, and are foundin the M2 generation with about two times frequency
compared with HomDels (Fig.1). If a HetDel is easily
detected in the M2 generation (See Fig. 1), the screening
pool size can be reduced. Herein, we tested the quantitative
real time PCR (qPCR) for detection of a HetDel.
For our test, we selected an Arabidopsis line
(C200-84-N2) from our stock induced by carbon-ion beamirradiation (135 MeV/nucleon, 200 keV/μm) at a dose of 75
Gy. This line has a 3.5-kbp deletion at the2762232-2765771 position on chromosome 4. The 3.5-kbp
deletion is harbored as a HetDel in the M2 generation. Thus,
four individual progenies of the M3 plants of C200-84-N2
were used for qPCR, since some of them would have a3.5-kbp deletion as the HetDel.
qPCR was performed by using LightCycler and theUniversal Probe Library detection format (RocheDiagnostics, Penzberg, Germany). Relative amplification
ratios between the 3.5-kbp deleted and non-deleted regions
were calculated by using the ΔΔCp method. Sample A
RIKEN Nishina Center＊2
Faculty of Agriculture, University of Miyazaki＊1Fig. 1. Assumed deletion screening in M2 generation byqPCR. HetDels are detected more frequently than HomDels
for heritable (A) and inheritable (B) cases. No symbolindicates individuals that have no deletion.
showed the same relative amplification ratio as wild-type(Fig. 2). Samples B and C showed about half-level of theratio as compared to wild-type, indicating that they contain
HetDels. Sample D showed no amplification. These results
indicate that large deletions can be detected even if they areheterozygous. Our irradiated M2 seed stocks in conjunction
with this method will prove a powerful tool for analyzing
the functions of TAGs.

Fig. 2. qPCR in four M3 plants (A to D). Data are expressed
as fold changes compared with values in wild-type (wt).
Error bars indicate ±SD. N=3 in each plant. White
arrowheads and a black arrowhead indicate HetDel and
HomDel, respectively. *, p< 0.01 compared to wild-type.
References
1) Y. Kazama et al.: BMC Plant Biol. 11, 161 (2011).
2) T. Hirano et al.: Mutat. Res. 735, 19 (2012).

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