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.