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Journal of Medicinal Plants
Research Vol. 5(31), pp.
6890–6894,
23 December, 2011
ISSN 1996-0875 ©2011 Academic
Journals
DOI: 10.5897/JMPR11.1455
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Full Length
Research Paper |
DNA isolation and optimization of sequence-related amplified
polymorphism-polymerase
chain reaction
(SRAP-PCR) condition for endangered Polyporus
umbellatus
Yuejin Zhang, Yalin Qin, Zhen Wang, Lijun Guo, Xin Chen,
Zongsuo Liang and Hongbo Guo*
Shaanxi Research Center of TCM Fingerprint and NP Library,
College of Life Sciences, Northwest A & F University,
Yangling 712100, PR China.
*Corresponding author. E-mail:
zouguoge@yahoo.com.cn.
Tel/ Fax: 86-29-8709-2262.
Accepted 10 November, 2011
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Abstract |
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To provide a fast genetic diversity survey of endangered
Polyporus umbellatus (Pers.) Fries for immediate
conservation, DNA isolation and optimization of
polymerase chain reaction
(PCR) assay of sequence-related amplified polymorphism (SRAP)
were investigated. Due to high amount of polysaccharide
contained in mycelium, three special approaches were adopted to
eliminate it during DNA isolation, including adding RNAiso-mate
for Plant Tissue buffer to mycelium powder, ethanol to DNA
extraction buffer and 5% NaCl solution to the mixture of isoamyl
alcohol and DNA deposit solution. Based on screening design, the
optimal SRAP-PCR condition was a total volume of 25 μl
containing 20 ng of DNA template, forward primer 0.4 μM, reverse
primer 0.4 μM, 1× Taq MasterMix and the best annealing
temperature was 35/50°C for each primer combination. According
to our optimal SRAP-PCR condition, 49 out of 81 primer
combinations were chosen for their high clarity and repetition
in all samples.
Key words:
DNA isolation, sequence-related amplified polymorphism (SRAP),
optimal
polymerase chain reaction
(PCR) condition, chuling, Polyporus umbellatus. |
