ISSR

The ISSR fingerprinting was performed using a protocol, developed by Sigma (Biometera Uno thermal cycler, Germany), and does not involve DNA extraction. The procedures recommended by the manufacturer have been followed. In brief, a small disc of fresh leaves taken from actively growing seedlings using a 50-mm Harris Uni-core puncher supported by cutting mat. The disc was added directly into 25 μl PCR reaction mix containing 25 mM MgCl2, 1X PCR buffer, 200 μM dNTPs (Applied Biosystems), 1 U of Taq DNA polymerase (Applied Biosystems, Ampli-Taq Gold), 2 pmole of each primer, and the leaf disc. Polymerase chain reaction was made for amplification of ISSR fingerprinting. The PCR amplification was performed using Bio-Rad thermo-cycler according to the following cycle profile: initial denaturation at 95 °C for 10 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C, and 5 min at 72 °C for final product extension. A total of 16 ISSR primers were used and only 10 primers produced a clear readable profile (Table 3) The PCR products of RAPD were separated in 1.5% agarose gel containing 0.5 μg/ml ethidium bromide using a submarine EC370 Minicell and an EC105 power supply (EC Apparatus Corporation, USA). The DNA size was calibrated against Hyper-Ladder I (GE Health care UK Limited, Amersham, UK). Meanwhile, the ISSR PCR products were separated in 1.8% agarosegel. The DNA size was calibrated against 100 bp ladder (Thermo scientific). The Lab image program version 2.7 produced by Kapelan Bio-Imaging GmbH was used for DNA size determination.

The sequence of primers assayed in ISSR-PCR

The presence or absence of protein, RAPD, and ISSR bands was scored as 1 for presence or 0 for absence of markers respectively for estimating genetic variation. Euclidian distance Romesburg [32] was calculated and used for measuring the similarity between the 14 samples using the software program, Community Analysis Package 4.0 (CAP) developed and was used according to Seaby and Henderson [33]. The dendrogram was constructed based on the similarity matrix data using the unweighted pair-group method with arithmetic averages (UPGMA) clustering and Free Tree software [34].