Ⅲ-4. Radiation Chemistry & Biology
RIKEN
Accel. Prog. Rep. 56 (2023)

RIKEN Accel. Prog. Rep. 56 (2023)

Identiﬁcation of causal site for an Arabidopsis C30-144-as3 mutant
A. D. Nagalla,∗1 S. Ohbu,∗1 K. Ishii,∗1,∗2 Y. Kazama,∗1,∗3 T. Hirano,∗1,∗4 and T. Abe∗1
Mutations are one of the crucial factors for natural selection and species evolution. They occur frequently innature. With the development of science and technology, we can create artiﬁcial mutagenesis and manually
search for desirable characters. Among those artiﬁcial
mutagenesis techniques, heavy-ion beam irradiation has
been established as a reliable approach in plant breeding.1) An important parameter for ion-beam mutagenesis can be identiﬁed as linear energy transfer (LET).
The LET implies the amount of energy deposited perunit length of a particle’s path (keV/µm). The C-ion irradiation (30 keV/µm) on Arabidopsis seeds has a highmutation frequency2) and results mainly in single nucleotide variants (SNVs), small indels (<100 bp), in theresponsible genes for mutant phenotypes. The numberof homozygous mutated genes per genome is approximately ﬁve, making it easy to identify the causativegene.3)
In this study, we irradiated Arabidopsis (Col-0) seeds
with 400 Gy C-ions (30 keV/µm). The resulting mutant plants were isolated, allowing further generations
to be selfed, and screened for interesting phenotypes.
The C30-144-as3 mutant was identiﬁed as later ﬂowering and round rosette leaves containing plant (Fig. 1).
After the whole genome sequencing, mutation calling
was conducted using the AMAP: A pipeline for wholegenome mutation detection.4) This analysis resulted inhomozygous SNVs, a large deletion, and intrachromosomal translocations (ITX) occurred in genic regions(Table 1). In order to identify the candidate gene, weFig. 1. Ion beam mutant C30-144-as3 shows later ﬂoweringand round rosette leaves containing plant (b) comparedto Col-0 plant (a) on 31 days after germination.
∗1
∗2
∗3
∗4

RIKEN Nishina Center
Department of Radiation Measurement and Dose Assessment, National Institute of Radiological Sciences
Faculty of Bioresources, Fukui Prefectural University
Faculty of Agriculture, University of Miyazaki

Table 1. Homozygous mutated genes found in C30-144-as3.

Chr.

Position

TypeSize

11308836013167752
19600910
1131686912608684
23056674
9981769318677

ITX

79.39Kb

Affectedgenes
AT1G06917

SNVDEL

1bp
1.29Mb

AT2G47860
111 genes

SNV

1bp
1bp
1bp

AT3G62300
AT4G02280
AT4G16550

23344SNVSNVplanned to perform a linkage analysis using a backcrossed (BC) population. The BC1 F2 population consists of 84 plants. Phenotype scoring referred to theperfect C30-144-as3 plant (M3 ) with later ﬂowering andround rosette leaves.
A total of 17 plants showed mutant phenotype. The
segregation of the F2 population ﬁtted the 3 : 1 segregation ratio (wild-type: mutant = 67 : 17, χ2 =1.02). Therefore, we hypothesize that this mutation wascaused by a recessive single gene. Linkage analysis wasperformed for all the homozygous mutated genes using
17 F2 plants, which showed the mutated phenotype.
The AMAP pipeline revealed large deletion of approximately 1.29 Mb in M3 plants. This deletion has lost a
total of 111 genes, according to the Arabidopsis genome
browser. We used two primer pairs and Sanger sequencing of PCR products for genotyping experiments. Linkage analysis resulted in 16 homozygous plants linked
with the above large deletion (one of the plants among
17 plants resulted in non-reproducible linkage analysis
result, which could be due to contaminated DNA), indicating responsible gene might be included in the deleted
fragment. Because among the homozygous mutatedgenes, only the 1.29 Mb deletion showed the linkage.
Next, we checked the gene description of all 111 genes
and found a single gene responsible for regulating ﬂowering time and leaf shape. It’s the gene AT3G30260 andalready identiﬁed as AGAMOUS-LIKE 79 (AGL79 ).5)
The non-functional mutants were reported to show later
ﬂowering and round rosette leaves. Then we presumed
AGL79 gene was the causative gene for the C30-40 mutant.
References
1) T. Abe et al., Plant mutation breeding and biotechnology(Oxfordshire UK: CABI, 2012), p. 99.
2) Y. Kazama et al., Plant Biotechnol. 25, 113 (2008).
3) Y. Kazama et al., BMC Plant Biol. 11, 161 (2011).
4) K. Ishii et al., Genes Genet. Syst. 91, 229 (2016).
5) R. Gao et al., Front. Plant Sci. 8, 2226 (2018).

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