The molecular analysis was employed using one or more samples per species. The DNeasy 96 Plant Kit (Qiagen GmbH, Hilden, Germany) was used for this study, and the DNA was extracted by following the protocol provided by the manufacturer. 20 mg of the dried and healthy leaf tissue of each sample was utilized. These DNA samples were then used for the next steps of the experimental procedure.
The PCR master mix was prepared with a total of 15 μl of the reaction mixture, made up of 1.5 μl PCR-Buffer, 1.5 μL MgCl2 (25mM), 1.0 μl dNTPs (2.5 mM of each dNTPs), 1 μL of forward primer (5 pM/μL), 1 μL of reverse primer (5 pM/μL), 0.2 μl (5 U/μL) Hot FIREPol Taq (Solis BioDyne, Estonia) and 6.8 μL H20 with 1μL of the respective DNA sample. The reaction cycle was carried out using a program of 95°C for 15 minutes, 94°C for one minute, 50°C for one minute, 72°C for one minute (go to step two and repeat 34 times) in a Biometra TProfessional thermocycler (Jena, Germany). The samples were then electrophoretically separated on an 1% agarose gel for further quality control. The PCR products were cleaned up prior to sequencing using GENECLEAN Kit (MP Biomedicals). The PCR products were then sequenced using the Big DyeTM Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).
The PCR master mix was prepared with a total of 15 μl of the reaction mixture, made up of 1.5 μl PCR-Buffer, 1.5 μL MgCl2 (25mM), 1.0 μl dNTPs (2.5 mM of each dNTPs), 1 μL of forward primer (5 pM/μL), 1 μL of reverse primer (5 pM/μL), 0.2 μl (5 U/μL) Hot FIREPol Taq (Solis BioDyne, Estonia) and 6.8 μL H20 with 1μL of the respective DNA sample. The reaction cycle was carried out using a program of 95°C for 15 minutes, 94°C for one minute, 50°C for one minute, 72°C for one minute (go to step two and repeat 34 times) in a Biometra TProfessional thermocycler (Jena, Germany). The samples were then electrophoretically separated on an 1% agarose gel for further quality control. The PCR products were cleaned up prior to sequencing using GENECLEAN Kit (MP Biomedicals). The PCR products were then sequenced using the Big DyeTM Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).