A modified method using CTAB (hexadecyltrimethylammonium bromide) was applied for genomic DNA extraction, as described earlier58 (link). Species identification was performed on the basis of the sequence analysis of the Internal Transcribed Spacers of the ribosomal DNA region (ITS1-ITS2).
Polymerase chain reactions (PCRs) were performed as described earlier27 (link) using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). For the PCR amplification specific primers were used: ITS4 – forward primer (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 – reverse primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′)59 . Amplicons were separated in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described earlier60 (link). DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm against the GenBank database-deposited reference sequences.
Polymerase chain reactions (PCRs) were performed as described earlier27 (link) using DreamTaq Green DNA polymerase (Thermo Scientific, Espoo, Finland). For the PCR amplification specific primers were used: ITS4 – forward primer (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 – reverse primer (5′-GGAAGTAAAAGTCGTAACAAGG-3′)59 . Amplicons were separated in 1.5% agarose gel (Invitrogen) with GelGreen Nucleic Acid Stain (Biotium, Inc.).
For sequence analysis, PCR-amplified DNA fragments were purified as described earlier60 (link). DNA fragments were labelled using a forward primer and the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to the producer’s recommendations and precipitated with 96% ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequences were analysed using the BLASTn algorithm against the GenBank database-deposited reference sequences.
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