Page 8 - Triticeae Genomics and Genetics

Triticeae Genomics and Genetics 2012, Vol.3, No.4, 38-43
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3.2 Extraction of genomic DNA
Genomic DNA was extracted followed with the
instructions of the kit for extraction plant genomic
DNA developed by the Transgen company (http://
www.transgen.com.cn/uploadfile/201111/2011111109
3152454.pdf).
3.3 Primer design and PCR amplification
A pair of primers was designed based on the cDNA
sequence of Kr gene (GenBank accession No.:
AB379558.1) as a template by using the software of
Primer 5.0, the sequence of Forward Primer
5'-TCCGCGCGCCCCTAGAGAAA-3', and the Rev-
erse Primer 5'- TCGCCTCCACGCTTCACCCT-3',
which synthesized by Shanghai Sango Biological
Engineering Company.
The 50 μL PCR reaction system included as follows as:
5 μL of 10×PCR buffer (adding Mg2+), 4 μL of
2.5 mmol/L dNTPs, 1 μL of 1 μmol/L Forward Primer
and Reverse Primer each, 0.5μL of 5 U/μL DNA
Polymerase, and 50ng of the DNA template, finally by
adding sterilized ultrapure water up to 50 μL. The
above chemicals were purchased from Beijing
Transgen Company.
PCR procedures were set as follows: pre-denaturation
at 94℃ for 5 min. and then 38 cycles with the process
following denaturation at 94℃ for 45 s, annealing at
54℃ for 45 s, and extension at 72℃ for 1.5 min; final
extension at 72℃ for 10 min. The PCR products were
detected by electrophoresis with 1% agarose gel
(containing ethidium bromide), with the conditions of
1×TAE electrophoresis buffer solution, 120 V voltage
and running 25 min, the staining gel was observed
under the imaging scanner and deposited in computer.
PCR amplification product was purified by the
Shanghai Sango Biological Engineering Company to
do TA cloning prior to sequencing.
3.4 Sequence Alignment
Alignment of sequence homology comparison for the
obtained sequences was carried out by using
DNAMAN software; the high homology sequences (E
value <1e -20) were searched using the obtained Kr
gene sequence as a probe at the NCBI database.
Author contributions
CH and LQQ are the executors for the experimental design and
lab work in the study to complete data analysis, manuscript
writing and revising; CH conceived this project and took in
charge. Both authors read and approved the final manuscript.
Acknowledgements
This study were jointly funded by the Open Foundation by the
Key Laboratory of Crop Genetics and Germplasm Enhance-
ment (ZW2010002) and the Major Project of the 12th Five-Year
National Science and Technology Support Program entitled
Technology Engineering for Grain Production (2011BAD16-
B06 and 2012BAD04B09).
References
Cai H., Ma C.X., and Lu W.Z., 2006, Wheat haploid plant
produced by embryo culture of Wheat×Maize,
Nongye Shengwu Jishu Xuebao (Journal of
Agricultural Biotechnology), 14(4): 633-634
Cai H., Ma C.X., Qiao Y.Q., and Lu W.Z., 2007, Establishment
of regeneration system in wheat haploid plant
through wheat×maize, Jiguang Shengwu Xuebao
(Acta Laser Biology Sinica), 16(1): 43-48
Dao W.W., 2009, Wide hybridization: engineering the next leap
in wheat yield, J. Genet. Genomics, 36(9): 509-510
Fujimoto R., and Nishio T., 2007, Self-incompatibility,
Advances in Botanical Research, 45: 139-154
Manickavelu A., Koba T., Mishina K., and Sassa H., 2009a,
Identification of differential gene expression for Kr1
gene in bread wheat using annealing control primer
system, Mol. Biol. Rep., 36: 2111-2118
Manickavelu A., Koba T., Mishina K., and Sassa H., 2009b,
Molecular characterization of crossability gene Kr for
intergeneric hybridization in Triticum aestivum
(Poaceae: Triticeae ), Plant Systematics and
Evolution, 278(1-2): 125-131
Nou S., Watanabe M., Isogai A., and Hinata K., 1993,
Comparison of S-alleles and S-glycoproteins between
two wild populations of Brassica campestris in
Turkey and Japan, Sexual Plant Reproduction, 6(2):
79-86
Qi Z.J., Li Q.Q., and Wang H.G., 1999, Crossability of some
land wheat races from Shandong with rye, Huabei
Nongxuebao (Acta Agriculturae Boreali-Sinica),
14(2): 15-1