DNA extraction, polymerase chain reaction (PCR) and sequencing
Total genomic DNA was extracted from silica gel-dried materials or herbarium specimens using the DNeasy® Plant Mini Kit (Qiagen Hilden, Germany) according to the manufacturer’s instructions. The DNA quality was checked using agarose gel electrophoresis with 1.0% agarose gels containing 0.4 x PeqGreen (VWR, Erlangen, Germany) for 40 min at 120 V, which was documented using microDOC system with UV transilluminator (Cleaver Scientific LTD, Rugby, Warwickshire, UK) using 312 nm wavelength.
Extracted DNA was 1:50 diluted with deionized water and then used for PCR. The nuclear internal transcribed spacer region (ITS) and the chloroplast trnL-trnF intergenic spacer were amplified and then sequenced from 23 samples of six species and two subspecies. For a 15 μL PCR reaction, 1 μL of diluted genomic DNA (equivalent to approx. 1–50 ng) was added to 14 μL master mix containing 1 × PCR buffer B, 2.5 mM MgCl2, 130 μM dNTP mix, 0.6 U Taq HOT FIREPol DNA Polymerase (all reagents from Solis Biodyne, Tartu, Estonia) and 300 nM forward (ITS5 [5’-GGAAGGAGAAGTCGTAACAAGG-3’; White at al., 1990] or c [5’-CGAAATCGGTAGACGCTACG-3’; Taberlet et al., 1991]) and reverse primers (ITS4 [(5’-TCCTTCCGCTTATTGATATGC-3; White et al., 1990] or f [5’-ATTTGAACTGGTGACACGAG-3’; Taberlet et al., 1991]) (Sigma Aldrich, Taufkirchen, Germany). The PCRs were performed in a MIC qPCR cycler (Biomloceular systems, Upper Coomera, Australia). PCR amplifications were performed with an initial denaturation step at 95 °C for 14:30 min, followed by 40 cycles at 95/58/72 °C for 30/30/90 s, and a final elongation step of 7 min at 72 °C. The amplified PCR fragments (2 µL of PCR products) were checked using electrophoresis in 1% agarose gels (low melting point agarose, Sigma Aldrich, Taufkirchen, Germany), using similar conditions as described above for genomic DNA.
Exonuclease I from E. coli 20 U/µl (EXO I) and Thermosensitive Alkaline Phosphatase 1 U/µl (FastAP) (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) were premixed in the ratio 1:4 and stored in the freezer. 13 µL PCR products were mixed with 1.3 µL EXO I and FastAP mixture and incubated at 37 °C for 15 min and 15 min in 85 °C. Purified PCR products were diluted with distilled water and admixed with sequencing primers according to the requirements of the sequencing company. Sequencing was performed by Microsynth Austria (Vienna, Austria) using Applied Biosystems 3730xl 96 capillary DNA analyzer (Thermo Fisher Scientific). Every Sequence was manually edited with CHROMAS vers. 2.6.6 (Technelysium, South Brisbane, Australia) and aligned with MEGA X software (Kumar et al. 2018). Edited sequences were subjected to BLAST searches for preliminary analysis (Altschul et al., 1990). Newly generated sequences were submitted to the National Center for Biotechnology Information (NCBI). GenBank accession numbers for all sequences are given in Table 1.