Inter simple sequence repeat (ISSR) markers as reproducible and specific tools for genetic diversity analysis of rose species

Abstract

Rose is one of the most important cultivated ornamental plants in the world. A molecular approach using inter-simple sequence repeat (ISSR) markers was applied to seven species of Rosa. To obtain clear and reproducible bands on 2% agarose gels, 9 ISSR primers and 5 parameters (annealing temperature, DNA concentrations, primer concentrations, Taq DNA polymerase and MgCl₂ concentrations) were screened. The resolution of six ISSR markers was performed, with optimal annealing temperature (Ta) varying from 45 to 50°C. A total of 66 DNA fragments were amplified, of which 50 were polymorphic. The optimal conditions for ISSR system were determined as follows: MgCl₂ concentration was 2 mM, the quantity of Taq DNA polymerase 1 U, template DNA 30 ng and the concentration of primer was 1 μM and the reaction program was: initial denaturation for 5 min at 94°C, 35 cycles of denaturation for 30 s at 94°C, annealing for 45 s at specific annealing temperature for each primer, extension for 2 min at 72°C and a final 10 min extension at 72°C.

Key words: Inter-simple sequence repeat marker, rose species, genetic diversity, optimization.