A comparative study of different RAPD-PCR protocols for genetic diversity analysis of Doritis germplasm

Random amplified polymorphic DNA (RAPD) is a simple, fast and cost effective method for assessing genetic diversity of plant varieties. The reproducibility of the RAPD amplification was affected by several factors, therefore causing misinterpretation. In this study, we analyzed the effect of changing the concentration of magnesium chloride, template genomic DNA and Taq DNA polymerase with the objective of determining the optimum concentration for standardization of RAPD technique for assessing genetic diversity of Doritis germplasm in the North-east of Thailand. The optimized RAPD reaction was performed by varying the concentration of MgCl2 (2.0, 2.5 and 3.0 mM), DNA template (10 and 20 ng) and Taq DNA polymerase (0.5, and 1.0 U) while fixing the concentration of RAPD primer and dNTPs at the concentration of 0.6 uM and 200 uM respectively. Reproducible amplification patterns with OPA02 primer were obtained using 2.5 mM of MgCl2, 10 ng of template DNA and 1 U of Taq DNA polymerase in 20 ul of the reaction. The optimized RAPD protocol was employed to select the suitable RAPD primers for fingerprinting of Doritis germplasm. Out of 98 RAPD primers screening, 32 primers revealed clear patterns of DNA amplifications and could be utilized as RAPD markers. These chosen primers will be useful for determining genetic diversity of these Doritis germplasm.