Basic HTML Version
Plant Gene and Trait 2012, Vol.3, No.4, 18
-
21
http://pgt.sophiapublisher.com
21
Resources named as Lun1, Lun2, Lun3, Lun4, and
Lun5. The F
1
generations were planted with seeds
which were collected from Luntai National Research
Station of Xinjiang Fruit Germplasm Resources. The
leaves of the maternal plants and F
1
generations were
collected and stored at
-
80
℃
for DNA isolation.
3.2 SCAR primers
According to the previous report of our laboratory, we
designed a pair of primers for SCAR. They were
synthesized by Shanghai Sangon Biotechnology Co.
Ltd. (Shanghai, China). The sequences of primers
used in this experience were as follows: P1: 5'
-
ACTG
GGACTCCAATTGTATC
-
3'; P2: 5'
-
ACTGGGACT
CTCAACTAT
-
3'.
3.3 DNA isolation
Total DNA of the maternal plants and F
1
generations
were extracted by CTAB method descried by Zhang
(2003). And it was detected on the 1% agarose gel
electrophoresis and diluted to 20 ng/µL with TE, then
compared with the Marker (GM335). And then stored
at
-
20
℃
for later use.
3.4 Cloning and sequencing
The volume of the reaction system for PCR was 20 µL
as followins: 13.4 µL ddH
2
O, 2 µL 10×buffer without
Mg
2+
, 0.4 µL 10 µmol/L dNTPs, 1 µL primer, 2 µL
DNA templates and 0.2 µL
Taq
polymerase. After
mixed, the reaction solution was separated slightly in
micro centrifuge, and then was placed in PCR thermal
cycler. The PCR procedure was as follows: 94
℃
for 3 min, and then 30 cycles at 94
℃
for 30 s, 55
℃
for 30 s, and 72
℃
for 80 s, with a final extension step
at 72
℃
for 7 min, and insulation work at 4
℃
. The
PCR products were separated on 1.2% agarose gels
electrophoresis for recovery. Then cloned into PMD
19
-
T vector, and finally transformed into XL1-Blue.
After the production was induced by IPTG and X-gal,
white colonies were identified by PCR and the positive
colonies were sequenced.
3.5 Amplification of SCAR molecular marker in
maternal plants and F
1
generations
The maternal plants and F
1
generations were amplified
with P1 and P2, the amplification condition and system
of PCR as 3.4.
Authors' contributions
CXY and JXN conceived the experimental design and
objectives of all the experiments, conducted the data analyses,
and wrote the manuscript. JQL, LW, RL and HPZ conducted a
few data analyses and took an active part in experimental
design method. All authors have read and approved the
manuscript.
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (No. 30560090) and the important
National Science and Technology Specific Projects of Xinjiang
(No. 201130102-1-4)
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