AFLP fingerprinting

AFLP was used to examine the genetic diversity within 79 S. lycopersici isolates following a modified protocol as described by Al-Sadi et al. (2012a) using FAM-6-labelled EcoRI-AGX selective primers. The primer combinations EcoRI-AGA/MseI-CAT, EcoRIAGT/MseI-CAT, and EcoRI-AGT/MseI-CAA were used in this study. DNA restriction and ligation were performed as described by Al-Sadi et al. (2012c). The pre-selective amplification mix consisted of PuReTaq™ Ready-To-Go ™ PCR beads, 0.65 µl EcoRI+A (5′-GACTGCGTACCAATTCA-3′), 0.65 µl MseI+C (5′-GATGAGTCCTGAGTAAC-3′), 3.70 µl R/L mix and 20 µl Milli-Q water. The cycling profile was as described by Al-Sadi et al. (2012c). The pre-selective amplification product was diluted by adding 210 μl of TE_0.1 to the remaining amount. The selective amplification was carried out using the three primer combinations (Table 1). The PRC reaction mixtures and conditions were as described by Al-Sadi et al. (2012c) using PuReTaq™ Ready-To-Go™ PCR beads. The AFLP experiment was carried out twice for each isolate starting from the DNA extraction step.

Evaluation of the three primer pair combinations utilized to identify and assess genetic diversity within 79 Stemphylium lycopersici isolates using AFLP fingerprinting analysis

^aH = Nei’s (1973) gene diversity