Plant Molecular Biology 39: 183–188, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Identiﬁcation and expression of the kosena radish (Raphanus sativus cv.
Kosena) homologue of the ogura radish CMS-associated gene, orf138
, Nobuya Koizuka, Hideya Fujimoto, Takako Sakai and Jun Imamura
Plantech Research Institute, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Japan 227-0033 (
author for cor-
Present address: Laboratory of Cell and Developmental Biology, Faculty of Bioscience and
Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan 226-8501
Received 23 January 1998; accepted in revised form 5 August 1998
Key words: cytoplasmic male sterility (CMS), mitochondrial DNA, CMS-associated gene, radish, fertility
A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to
that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the
orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica
napus CMS cybridderived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence
is present in the 3
-ﬂanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in
the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype
in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.
Cytoplasmic male sterility (CMS) is a mitochondrial-
encoded trait, and CMS-associated mitochondrial
genes have been identiﬁed in several species .
The Shiga-Thompson, polima and ogura CMS cyto-
plasms have been identiﬁed in Brassica species and
of these polima and ogura have been studied inten-
sively . The ogura CMS cytoplasm isolated from
Japanese radish was transferred to Brassica species
by intergeneric crosses and protoplast fusion [1, 14].
Because the mitochondrial DNA (mt DNA) of the
ogura CMS cytoplasm was highly rearranged com-
pared to that of normal radish , it was difﬁcult to
identify the CMS-related region by the simple com-
parison of the genome organizations of the ogura and
normal mtDNAs. CMS cybrids derived from proto-
plast fusion are useful tools for the identiﬁcation of
CMS-related regionsbecause frequent recombinations
among the parental mtDNAs cause rearrangement of
the mt genomes in the fusion products. Young and
Hanson  have identiﬁed a unique mtDNA frag-
ment, thought to include a CMS-associated gene (pcf),
by comparing the mtDNA-digested patterns of the
CMS and fertile petunia cybrids. Bonhomme et al.
 have also isolated an ogura CMS-associated region
that included orf138 by comparing the CMS cybrid
with its fertile revertant.
A radish CMS has been isolated from a popula-
tion of the Japanese radish cultivar Kosena . Fertile
restorer lines also have been identiﬁed in the same
Kosena population. The kosena CMS cytoplasm (kos)
and its restorer gene have been transferred to Bras-
sica napus by ‘donor-recipient’ protoplast fusion .
The B. napus kos cybrid shows a stable CMS pheno-
type through several backcross generations , and
the introduced restorer gene shows single dominant
inheritance of the gene . We report the isolation of
mitochondrialgenomicclones from the kos radish and
the B. napus kos cybrid that encode the homologue of
ogura orf138. The mitochondrial region in the cybrid
differs from that in the parent radish. We used the PCR
to determine the origin of the unique region present
in the cybrid. The expression levels of the homologue
also were investigated relative to the presence of the
fertility restoration gene in the nucleus.