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Figure 1. Map of Ladakh region of India.
assigned in a high priority for their preservation and
protection. In this study, random amplified polymorphic
DNA (RAPD) markers were used to detect variation at
the population level in samples collected from Ladakh
regions of India. The purpose of this study is to
investigate the levels of variation among populations of
Dactylorhiza hatagirea.
MATERIALS AND METHODS
Study sites and sampling
Nine populations from Ladakh regions were surveyed (Figure 1).
Young leaf tissues of 96 individuals of leaves were selected
randomly for molecular analysis. The number of samples taken in
each population was depending on geographic distribution (Table
1). The sampling stratagem was to trace several sites in different
parts of the investigated area in order to cover diverse growing
habitats which represent the richly capable yet relatively stable
primitive environment in the sampled regions.
RAPD analysis
Warghat et al. 2389
Total genomic DNA was extracted from Dactylorhiza leaves using a
cetyltrimethylammonium bromide (CTAB) method (Doyle and
Doyle, 1987) with minor modifications. The quantity and quality of
isolated total genomic DNA was determined using 0.8% agarose
gel electrophoresis in 0.5 × TAE buffer for mobility related to known
concentrations of lambda DNA. Twenty random decamer primers
from integrated device technology (IDT) Tech. USA (Table 2) were
used for RAPD amplification following the protocol of Williams et al.
(1990). Amplification reactions were performed in volumes of 17 µL
containing 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl,
200 µM of each deoxynucleotide triphosphates (dNTPs), 0.4 µM
primer, 20 ng template DNA and 0.5 unit of Taq polymerase
(Sigma-Aldrich, USA) with the following program: initial denaturation
at 94°C for 5 min, followed by 1 min denaturation at 94°C, 1 min
denaturation at specific annealing temperature (37°C), 1 min
extension at 72°C for 39 cycles, and 5 min at 72°C for a final
extension.
Amplification product were electrophoresed on 2.0% agarose gel
(Life Science technologies, USA) and run at constant voltage (80V)
in 1× TAE for 3 h, visualized by staining with ethidium bromide (0.5
µg ml -1 ) and a total of 2.5 µl loading buffer (6x) was added to