DNA Extraction and PCR Amplification
DNA Extraction and PCR Amplification
Total genomic DNA was extracted from the young
leaves using a CTAB (hexa decyl trimethyl ammonium
bromide) method, according to the protocol described
in Saunders et al. [31]. Then, the DNA extract was
suspended in 50 μL 1× TE buffer (1 M pH 8.0 Tris
HCl; 0.5 M pH 8.0 EDTA) and kept at –20°C. The
quality and concentration of each DNA sample was
determined using a NanoDrop spectrophotometer at
260, 280 nm (ND1000. Co. USA) and running 3 μL
DNA in 0.7% (w/v) agarose gels in 0.5× TAE buffer.
30 previously SSR primer pairs that described highly
number of alleles and polymorphic alleles [8, 10, 22,
32–37] selected and synthesized by Metabion Co.
(Germany). These synthesized SSR primer pairs were
initially screened and 19 primer pairs were used in this
study. The list of primers pairs and their information
are presented in Table 2. For PCR analysis, approxi
mately 25 ng of genomic DNA was used in a 25 mL
reaction containing 1× PCR reaction buffer, 2 mM
MgCl2, 0.8 mM diNTPs, 5 pmol of each primer, 1 U
Taq DNA polymerase (F
