Amplification was carried out by conventional polymerase chain reaction (PCR) in 20 μl reactions containing 1× 10× DreamTaq Buffer (Thermo Fisher Scientific, USA) 0.5 μM of each primer (Table 1 ), 1U of DreamTaq (Thermo Fisher Scientific, USA), and 20 ng of template DNA. PCR was run in a PCR system 2700 (Applied Biosystems, USA): Thermocycling consisted of an initial denaturation step at 95°C (5 min), which was followed by cycles for: Universal primer 18S rRNA gene (Applied Biosystem/Ambion, USA) (95°C for 30 s, 57°C for 30 s, and 72°C for 30 s) 30 cycles; ToITS2 (95°C for 30 s, 50°C for 40 s, and 72°C for 40 s) 40 cycles; To18SA (95°C for 30 s, 54°C for 30 s, and 72°C for 30 s) 30 cycles; To18SB (95°C for 30 s, 56°C for 30 s, and 72°C for 30 s) 30 cycles; final extension step at 72°C (10 min).
All reactions were checked for amplification by gel electrophoresis.
Amplified DNA sequences were directly inserted into a pGEM-T Easy Vector System (Promega, USA). Colony PCR was performed on putatively transformed colonies using M13Forward and M13Reverse as primers. The clones that showed inserts with different molecular weights using gel electrophoresis analyses were sequenced by automated sequencing (MWG Biotech). The sequences were analyzed using BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) in order to identify them in the GeneBank.
All reactions were checked for amplification by gel electrophoresis.
Amplified DNA sequences were directly inserted into a pGEM-T Easy Vector System (Promega, USA). Colony PCR was performed on putatively transformed colonies using M13Forward and M13Reverse as primers. The clones that showed inserts with different molecular weights using gel electrophoresis analyses were sequenced by automated sequencing (MWG Biotech). The sequences were analyzed using BLASTN (