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Triticeae Genomics and Genetics
TGG 2010, Vol.1, No.1
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performed following Williams et al. (1990).
Ten-nucleotide primers of arbitrary sequence were
obtained from Operon Technologies and Shanghai
Sangon
Biological
Engineering
Technology.
Amplification reactions were performed in total
volumes of 25 μL containing 10 mmol/L Tris-HCl (pH
8.0), 50 mmol/L KCl, 2 mmol/L MgCl2, 100 μmol/L
of dNTPs, 0.4 μmol/L primer, 25 ng mtDNA or total
DNA as template, and 1.5 units Taq DNA polymerase.
Amplification was performed in a Bio-Rad MyCycler
Thermal Cycler programmed with an initial
denaturation step for 3 min at 95
℃
followed by 40
cycles of denaturation for 1 min at 94
℃
, annealing for
1 min at 37
℃
, primer extension for 2 min at 72
℃
, and
a final extension step of 5 min at 72
℃
. Amplification
products were analyzed by gel electrophoresis in 1.5%
agarose gel and detected by staining with ethidium
bromide. Amplification patterns were analyzed by
Gene Tools from Syngene to evaluate molecular
weight, and all the RAPD amplification reactions were
repeated more than three times.
Table 1 Materials used as sources of mtDNA in this study
Name
Type
Fertility
Cytoplasm donor
Ae. kotschyi
a
Fertile
Ae. kotschyi
ms (Ae. kotschyi)
-90-110
b
Sterile
Ae. kotschyi
ms (Ae. kotschyi)
-90-110×5253
c
Fertile
Ae. kotschyi
Ae. variabilis
a
Fertile
Ae. variabilis
ms (Ae. variabilis)
-90-110
b
Sterile
Ae. variabilis
ms (Ae. variabilis)
-90-110×5253
c
Fertile
Ae. variabilis
Ae. ventricosa
a
Fertile
Ae. ventricosa
ms (Ae. ventricosa)
-90-110
b
Sterile
Ae. ventricosa
ms (Ae. ventricosa)
-90-110×5253
c
Fertile
Ae. ventricosa
Ae. bicornis
a
Fertile
Ae. bicornis
ms (Ae. bicornis)
-90-110
b
Sterile
Ae. bicornis
ms (Ae. bicornis)
-90-110×5253
c
Fertile
Ae. bicornis
2 Results
2.1 RAPD analysis of Aegilops species and their
respective CMS lines
A total of 120 primers were used for screening RAPD
markers to distinguish the Aegilops species, their
respective CMS lines and fertility-restored F1 hybrids
using mtDNA extracted from etiolated shoots (see
supporting online material). The primer sets showing
stable polymorphisms are listed in table 2. Of therm,
eight primers were found showing stable
polymorphism in the Aegilops species and their
respective male sterile lines. In Ae. kotschyi, primer
OPY-01 produced a 600 bp band while in the male
sterile line ms (Ae. kotschyi)-90-110 a 580 bp band
was observed (Figure 1A). In Ae. variabilis, primer
S32 can amplify a 1 770 bp band while in ms (Ae.
kotschyi)-90-110 the primer can amplify 1 700 bp and
1 440 bp bands (Figure 1B). In Ae. ventricosa, primer
S22 can amplify a 2 310 bp band, while in ms (Ae.
ventricosa)-90-110 the primer can amplify a 1 770 bp
band (Figure 1C). For Ae. bicornis and ms (Ae.
bicornis)-90-110 different fingerprint patterns were
obtained based on amplification by primer pairs S202,
OPB-05, OPA-04, S153 and S21, respectively. In ms
(Ae. bicornis)-90-110, primer S202 can amplify a
specific 2 500 bp band while in Ae. bicornis the 2 500
bp band can not be amplified (Figure 2A). Also in ms
(Ae. bicornis)-90-110, primer OPB-05 can amplify a
specific 3 020 bp band, while in Ae. bicornis the 3 020
bp band can not be amplified (Figure 2B). In Ae.
bicornis, primer OPA-04 can amplify a specific 1 000
bp band while in ms (Ae. bicornis)-90-110 the 1 000
bp band can not be amplified (Figure 2C). Primer
S153 can amplify a 2 270 bp band in ms (Ae.
bicornis)-90-110, but in Ae. bicornis the specific band
was absent (Figure 2D). Similarly, premer S21 can