Optimization of ISSR marker for African edible-seeded <i>Cucurbitaceae</i> species’ genetic diversity analysis

Abstract

We applied a molecular approach using inter-simple sequence repeat (ISSR) markers on three African edible-seeded cucurbits (Citrullus lanatus L. Matsumura and Nakai, Cucumeropsis mannii L. Naudin and Cucumis melo var. agrestis L. Naudin). To obtain clear and reproducible bands on 1.5% agarose gels, we screened 21 ISSR primers and three parameters (annealing temperature, gel tray, and voltage and running time). The resolution of 11 ISSR markers was performed, with optimal annealing temperature (Ta) varying from 50 to 52°C. The best combination to obtain clear and well-distinguished band patterns was 1.5% agarose gel with a 20-lanes tray (6 mm width) at 80 V for 5 h. Applying the 11 ISSR primers on DNA extracted from an accession of C. lanatus, 66 bands with 4 to 11 bands per primer was observed.

Keywords: ISSR, Cucurbits, genetic diversity, molecular markers, optimization