Dna extraction

DNA extraction , amplification and sequencing

Genomic DNA was extracted from dried leaf material following a modified CTAB ( Doyle & Doyle 1987) procedure ( Oberlander et al. 2004). The ITS region was amplified using the primers AB101 and AB102 ( Sun et al. 1994) in PCR reagent mixtures (in 25 μl final volumes) containing 0.5 μM of each primer, 0.2 mM of each dNTP, 0.2 mM 1 × Supertherm buffer solution, 2.5 mM MgCl 2, 0.25 U Supertherm Taq polymerase and c. 40 ng template DNA . The PCR protocol (35 cycles) followed was: 94 °C denaturation for 60 s, 60 s annealing at 55 °C, 90 s extension at 72 °C and a final 7 min extension step at 72 °C. PCR products were visualised on a 2 % agarose gel and purified using a Wizard DNA Prep purification kit (Promega, Madison, Wisconsin, USA). Sequencing reactions and base calling were conducted at the Central Analytical Facility (CAF), Stellenbosch University, Stellenbosch, South Africa.