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Materials and methods
Plant materials
Forty-seven lines of alloplasmic or NC hybrids which have plasmons
derived from various Triticum and Aegilops species
combined with a single nuclear genome from Triticum aestivum
L. em Thell. cv. Chinese Spring (CS) were used in this study. For
some NC hybrids, their cytoplasms were replaced by that of CS after
reverse crosses of the hybrids as male parents with CS as a female
parent (see Results and discussion). The original hybrids were
provided by Prof. K. Tsunewaki, Kyoto University (now Fukui
Prefectural University). CS served as a euplasmic control.
RAPD-PCR detection of DNA polymorphisms
Total DNAs were extracted according to Liu et al. (1990) from one to
five plants obtained after self-fertilization of fertile NC hybrids
and from single progeny plants of male-sterile NC hybrids after
backcrosses with the paternal CS. DNAs from the reverse F1
hybrids were also extracted in the same way. RAPD-PCR was performed
in a reaction mixture (10 micro-liter) containing 10 ng of total DNA,
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.001%
gelatin and 0.32 microM random decamer primer (Operon Technologies).
PCR program was as follows: a pre-denaturation step for 30 s at 93C;
and 40 cycles of 1 min at 93C, 1 min at 36C, 1.5 min at 72C; followed
by postextension for 2 min at 72C. Amplified fragments were resolved
by electrophoresis through 1.4 % agarose gels and visualized by
staining with ethidium bromide.
Results and discussion
Polymorphic DNA fragments were detected by RAPD-PCR analysis
using total DNAs extracted from 47 NC hybrid lines. Only 31 random
primers were used in this study and seven were found to give
polymorphic fragments among them. The polymorphisms detected were
either amplification or non-amplification of DNA fragments unique in
single lines or in several lines mostly with phylogenetically related
cytoplasms.
Six among seven polymorphic fragments were assigned to the
cytoplasmic genomes (Table 1). Three types
of cytoplasmic polymorphisms were detected as amplification of single
fragments. A primer OPA02 amplified a 1.6 kbp fragment
(A021,600) in three NC hybrids with M plasmon of Ae.
comosa thessalica (Kyoto University code number C05),
Mh plasmon of Ae. herdreichii (C06) and N plasmon
of Ae. uniaristata (C07) (Fig. 1A).
The polymorphism in the male-sterile M and Mh plasmons was
detected in the progeny plants obtained after backcrosses of the NC
hybrids with the paternal CS and thus considered to be derived from
the plasmons. The cytoplasmic origin of this polymorphism was
confirmed by RAPD-PCR analysis using DNAs from the reverse hybrids in
which their cytoplasms were replaced by that of CS. As expected the
fragment A021,600 could not be amplified in the reverse
hybrids (Fig. 2A). Another primer OPB19
amplified a 1.3 kbp fragments (B191,300) in the NC hybrids
with Sb (C12, Ae. bicornis), T (C13, Ae.
mutica) and T2 (C14, Ae. mutica) plasmons
(Fig. 1B). OPE07 amplified a 3.2 kbp fragment
(E073,200) in three NC hybrids with Sl (C10,
Ae. sharonensis), Sb (C12, Ae. bicornis )
and Sv(C33, Ae. kotschyi ) but did not in NC
hybrids with S (C08, C17, Ae. speltoides) and other
S-derivative plasmons. They were assigned to the cytoplasmic
variations in the same way using the reverse hybrids. The polymorphic
fragment A021,600 detected in M and two M-related plasmons
was absent in three NC hybrids with T and T2 plasmons of
Ae. mutica and Mo plasmon of Ae. ovata.
Mo plasmon of Ae. ovata is thought to have
evolved from T plasmon of Ae. mutica after hybridization with
Ae. umbellulata as a pollen parent followed by
amphidiploidization (Tsunewaki 1995). Therefore, as far as this
polymorphism is concerned, T plasmon of Ae.mutica can be
considered to have remained unchanged in Mo, plasmon of
Ae. ovata after its evolution. On the other hand,
B191,300 was absent in a NC hybrid with Mo
plasmon of Ae. ovata. This polymorphism thus can be considered
to have evolved after amphidiploidization of Ae. ovata. A
reason why this same polymorphism was present in Sb
plasmon of remains unknown.
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