Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 47 (2014)

Constructing S -locus deletion mutant in common buckwheat byusing heavy-ion-beam irradiation
M. Ueno,1 T. Abe,2 Y. Hayashi,2 and Y. Yasui1
In Fagopyrum esculentum (common buckwheat), theplants exhibit short-styled or long-styled flowers, showing
distyly. The floral morphology and intra-morph
incompatibility are both determined by a single genetic
complex named S-locus. Plants with short-styled flowers
are heterozygous (S/s) and plants with long-styled flowers
are homozygous recessive (s/s) at S-locus. Previously we
discovered a new gene, S-LOCUS EARLY FLOWERING 3(S-ELF3), which is a candidate gene for short-styled
phenotypes of distyly, and its flanking region of about 500
kbp has already been sequenced1). Recombination aroundthe S-locus is supposed to be restricted, because norecombination between floral morphology and intra-morph
incompatibility was observed. Thus, genetic mapping is notpossible to determine the genomic region containing theS-locus. The purpose of this study is to construct mutants
that lack the genomic region around the S-locus by
heavy-ion-beam irradiation, in order to use the mutants fornarrowing down the S-locus in the future.

Fig. 1. Schematic diagram of the screening of S-del
haplotypes. New mutant S-del1 possessed only Y_#3
marker, which is tightly linked to S-locus, i.e., no S-ELF3
was found. The flower phenotypes of S-del1 were longstyled. Dashed line indicates the genomic region deleted byheavy-ion-beam irradiation.

For obtaining an S-locus-deletion plant, buckwheat seedswere irradiated with accelerated 12C6+ ions in doses ranging
from 100 Gy to 125 Gy. The linear energy transfer (LET)
range of 12C6+ was from 22.5 keV/µm to 30 keV/µm. The
total DNA was extracted from 1,152 plants of M2 growing
in the experimental room, and their flower types wereinvestigated. For screening of the S-locus-deletion plant,
PCR was performed using an S-haplotype specific primerset (Y_#3) obtained using cDNA-Amplified Fragment
Length Polymorphism (AFLP) analysis (Yasui et al., inpreparation). The Y_#3 PCR marker showed perfect linkage
with the S-locus in 1,400 mapping population and wasamplified only with short-styled buckwheat plants collectedfrom all over the world. S-ELF3 and Y_#3 marker werelocated physically distant to each other, because the DNA
sequence of Y_#3 marker could not be found on the 500
kbp BAC contig flanking of S-ELF3 (Yasui et al., inpreparation). Further, if short-styled plants lack genomic
region only around the S-locus, the flower type of the plantis expected to become long-styled, but must possess theY_#3 marker (Fig. 1).
In 1,152 plants investigated, one showed both positive
Y_#3 PCR amplification and long-styled flowers and wasnamed S-del1. Furthermore, S-ELF3 and six dominant PCRmarkers covering the 500 kbp sequence flanking S-ELF31)
produced no PCR products with S-del1 DNA. It isconsidered that the large genomic region (>500 kbp)
harboring the S-locus was deleted in the S-del1 mutant andthat the flower type of S-del1 changed from short-styled tolong-styled (Fig. 1). These results imply that S-ELF3 or its
flanking gene controlled short-styled phenotypes.
It is expected that combining PCR amplification withS-linked maker (Y_#3) and phenotyping of flower type on
M2 plants is effective in the screening of S-locus-deletion
plants. We are planning to screen other sets of M2
population. In the near future, we will be able to construct afine deletion map such as that of the Y chromosome ofSilene latifolia2).
In this study, we observed a phenotypic change fromshort-styled to long-styled flowers in the mutant progeny.
This makes the creating of S-del homozygous (S-del/S-del)plant possible, and the resulting plants will enable us toestimate the role of s-haplotype genes and to narrow down
the genomic region harboring these genes.
References
1) Yasui et al.: PLoS ONE 7, e31264 (2012).
2) Fujita et al.: G3 (Bethesda) 2, 271 (2012).

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Graduate School of Agriculture, Kyoto University
RIKEN Nishina Center- 293 -