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 MgCl₂concentrations) 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.

Abbreviation

ISSR, Inter-simple sequence repeat; Ta, annealing temperature;RFLPs, restriction fragment length polymorphism; PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA; SSRs, simple sequence repeats; AFLP, amplified fragment length polymorphism; Tm, melting temperature.