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Abstract
Random amplified polymorphic DNA (RAPD) is a simple and reliable method to detect DNA
polymorphism and has been used extensively for genetic diversity studies. In the present investigation, the RAPD-
PCR reaction conditions were optimized for generating RAPD fingerprints of
Paphiopedilum thaianum
,
P. niveum
and
P. godefroya
e in southern Thailand
.
To determine the optimum conditions, concentrations of template DNA
and primers were analyzed. Excellent amplification patterns were obtained using 25 ng of template DNA, 1 ôM
primer, 20 mM Tris-HCl, 80 mM KCl, 4 mM of MgCl
2
, 0.5 mM dNTPs mix and 1 U of Taq DNA polymerase in
20 ôl of reaction volume. Cycling programs were also optimized. The optimized RAPD-PCR conditions are
suitable for further work on genetic diversity and relationships analysis of Lady’s slipper orchids
.
Keywords
: RAPD-PCR, Lady’s slipper orchid, Genetic diversity
Introduction
Lady’s slipper orchids of the genus
Paphiopedilum
features beautiful form and color flowers. They are
threatened orchids in the wild due to over-collection.
Paphiopedilum thaianum
,
P. niveum
and
P. godefroyae
are
endangered lady’s slipper orchid species. They distribute mainly in southern Thailand. There are high variation in
morphological characteristics within each species (Cribb, 1998; Koopowitz, 2008). Therefore, it is necessary to
investigate their genetic diversity and relationships for conservation planning.
Random amplified polymorphic DNA (RAPD) is a simple and reliable method to detect DNA
polymorphism. It could amplify genome DNA and investigate genetic diversities among samples with a set of
random primers. Compared with other molecular markers, RAPD marker features sensitiveness, convenience and
rapidness in detection. Thus, it has been now widely used for genetic diversity and relationships studies of orchids
(Dixon
et al
., 2003).
RAPD-PCR reaction system features tiny volume (20 ôl), many ingredients (dNTP, polymerase,
primers, template, etc) and many steps (pre-denaturing,denaturing, annealing, extension) and many cycles
(30 - 40). So little change of any parameters could affect products drastically (Diakou and Dovas, 2001; Fraga
et al
., 2002), which indicate its kinetics profile very complex. Generally, the reaction requires a lower
concentration of template DNA such as 1-100 ng for plants (Haina
et al
. 2009; Hameed
et al
. 2004; Iqbal
et al
.
2008; Kim
et al
. 1997; Padmalatha and Prasad. 2006; Ranjan
et al
. 2010), 5-100 ng for orchids (Kawinski
et al
.
2009; Lim
et al
. 1999; Miaobin
et al.
2009; Zha
et al
. 2009) and 5-50 ng for Lady’s slipper orchids
(Asawatreranakul, P. and Asawatreranakul, K. 2005; Chung
et al.
2006; Kennedy and Walker. 2007; Li
et al.
2002). This lower concentration allows the 10 mer primers to find the many sites expected from RAPD-PCR
reaction (Munthali
et al
. 1992). Therefore, the RAPD-PCR reaction system must be optimized before its
application on any kind of materials. The present investigation has been undertaken to optimize RAPD-PCR
reaction for generated the best RAPD fingerprints of
Paphiopedilum thaianum
,
P. niveum
and
P. godefroyae
.
