Basic HTML Version
Triticeae Genomics and Genetics
TGG 2010, Vol.1, No.1
http://tgg.sophiapublisher.com
Page 2 of 7
recently diverged CMS and fertile plants become
much more reliable for our researchers to identify
CMS-associated gene regions. With this in view, the
identification of genetic differences in mtDNA
between fertile, male-sterile and restored fertile lines
under nearly constant cytoplasmic background will reveal
new strategies to locate CMS-associated gene regions.
Unlike the spontaneously occurred CMS that has been
observed in some species, CMS in wheat often results
from
interspecific
crosses,
particularly
the
interspecific cytoplasm substitution. For example, the
very famous T-type CMS wheat derived from the
interspecific cross between Triticum timopheevi and
Triticum aestivum. Such interspecific cytoplasmic
substitutions often lead to incompatibility between the
cytoplasm and the recipient nucleus, or results in
cytoplasmic dysfunction (Leaver et al., 1988). Fertility
restoration relies on the nuclear restoration gene(s)
existing in Triticum timopheevi that suppress
cytoplasmic dysfunction (Maan et al., 1984). However,
the utility of T-type CMS system in hybrid production
is limited for lack of new restorers and due to
wrinkled grain shape and pre-harvest sprouting of
developed hybrids. Besides, the application of wheat
heterosis is also impeded by other factors, including
low hybrid seed production rate, difficulty in
maintaining purity of male sterile lines, high seeding
rate etc.. In order to develop a good F1 hybrid variety
in wheat, it is necessary to explore a new male-sterile
cytoplasm and its nuclear restore gene(s). With this in
view, we developed a series of alloplasmic male
sterile lines (or called nuclear-cytoplasm substitution
lines) of wheat with Aegilops cytoplasm by
recurrently backcrossing Ae. kotschyi-Chris with a
recurrent parent 90-110 (Triticum aestivum). In
comparison with T-type CMS system, our male sterile
lines have some significant advantages, such as the
abundance of restorers, the characteristic of
easy-restoration-and-maintanence, and round grain
shape of hybrids, so it is being regarded as the most
promising among all alien cytoplasms for hybrid wheat
production (Zhang and Yang, 1989; Zhang, 1992).
In this paper, an attempt was made to study mtDNA
variation
using
RAPD
(random
amplified
polymorphic DNA) markers on the Aegilops species,
male sterile lines and fertility-restored F1 hybrids
under a nearly constant cytoplasmic background.
1 Materials and methods
1.1 Plant materials
Four Aegilops species viz. Ae. kotschyi, Ae. variabilis,
Ae. ventricosa and Ae. bicornis, and two fertile
common wheat varieties namely 90-110 and 5253,
were used in the present investigation. Four male
sterile lines viz. ms (Ae. kotschyi)-90-110, ms (Ae.
variabilis)-90-110, ms (Ae. ventricosa)-90-110 and ms
(Ae. bicornis)-90-110 were derived from recurrent
backcross of Ae. kotschyi-Chris, Ae. variabilis-Chris,
Ae. ventricosa-Chris and Ae. bicornis-Chris to
90-110 respectively, for more than 20 generations. The
wheat variety 90-110 was used as the maintainer line
while the variety 5253 was used as the restorer line to
restore fertility of the four male sterile lines.
This study was carried out with emphasis on
mitochondrial DNA variation among the Aegilops
species, their respective CMS lines and
fertility-restored F1 hybrids. Because the cytoplasmic
genomes are maternally inherited in wheat and its
relatives, the plant materials used for isolation of
mitochondrial DNA are under nearly constant
cytoplasmic background (Table 1).
1.2 DNA isolation
mtDNA was isolated from etiolated shoots as
described by Li et al. (2007). The protocol includes:
mitochondria isolation with differential centrifugation,
DNase treatment, lysis with SDS and proteinase K,
removing protein by TE-saturated phenol/chloroform
extraction and a final RNase A treatment for obtaining
mtDNA. The mtDNA samples were tested for purity
using spectrophotometry, agarose gel electrophoresis
and restriction enzyme digestions. It was proved that
the mtDNA was not contaminated by nuclear DNA,
plastid DNA, RNA and protein, and was successfully
used for PCR, cloning and southern blot analyses.
Total DNA was isolated following CTAB method as
described by Murray and Thompson (1980).
1.3 DNA amplification by RAPD
RAPD amplification of mtDNA and total DNA was