Pcrbio taq mix red

A total of eight candidate DNA barcode loci were amplified and sequenced from the total genomic DNA of the reference samples. Established primers were used to amplify four coding cpDNA loci, matK, rbcL, rpoB, and rpoC1, two non-coding cpDNA intergenic spacer loci, psbA-trnH and trnL-trnF, and the nDNA loci, ITS and ITS2. For agarwood samples, the trnL-trnF locus was amplified using additional internal primers: 1) primer e was coupled with the primer E-Aq-rev-1, and 2) primer f was coupled with primer F-forw-2 [17 ]. Details on the primers are listed in Table 3. PCR was conducted in a final reaction volume of 25 μL, containing 12.5 μL of 2x PCRBIO Taq Mix Red (PCRBiosystems, UK), 10 mM of each primer, and 25 ng of genomic DNA as template. PCR amplification was conducted on a SpeedCycler² Thermal Cycler (Analytik Jena, Germany). Successful PCR amplification was inspected through electrophoresis on 1% agarose gel, before DNA sequencing on an ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, USA).

Approximately 100 mg of fungal mycelia was used for fungal genomic DNA extraction. Fungal genomic DNA was extracted as previously described by Landum et al. (2016 (link)), in accordance to the manufacturer's instructions, using the DNeasy Plant Minikit (Qiagen, Germany). The nuclear ribosomal DNA internal transcribed spacer (ITS) of the fungal isolates were amplified using the forward primer, ITS1-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′ and the reverse primer, ITS4 (5′-CTTGGTCATTTAGAGGAAGTAA-3′) (White et al., 1990 (link)). The final reaction volume was 25 μL, containing 12.5 μL of 2X PCRBio Taq Mix Red (PCR Biosystems, UK), 0.4 μM of forward and reverse primers, and 10 ng of genomic DNA template. For negative control, the DNA was replaced with distilled water to verify absence of contamination. PCR was carried out using MyCycler™ (Bio-Rad, USA), programmed for 1 min 95°C; 35 cycles for 15 s at 95°C, 15 s at 55°C, and 1 min at 72°C; and a final 10 min extension at 72°C. The PCR products were separated using 1% agarose gel in 1X TAE buffer (90 mM Tris-acetate and 2 nM EDTA, pH 8.0), stained with ethidium bromide (0.5 μg/mL) and documented using FluorChem™ (Alpha Innotech, USA). PCR products were sent for direct bi-directionally sequencing using ABI PRISM 3730 × 1 Genetic Analyzer (Applied Biosystems, USA) at the First BASE Laboratory Sdn. Bhd., Selangor, Malaysia.

Genomic DNA was extracted using the Favorprep^TM Plant Genomic DNA Extraction Mini Kit (Favorgen, Taiwan) according the manufacturer’s suggested protocol. The isolated DNA was quantified using a Nanophotometer (Implen, Germany). A total of five chloroplast DNA (cpDNA) regions—two coding cpDNA loci, matK and rbcL; the trnL intron; and two non-coding cpDNA intergenic spacer loci, trnL-trnF and psbC-trnS—and an nrDNA ITS region were amplified (Table 2). PCR was conducted in a final reaction volume of 25 μL, containing 12.5 μL of 2x PCRBIO Taq Mix Red (PCRBiosystems, United Kingdom), 10 mM of each primer, and 20 ng of genomic DNA as a template. PCR amplification was conducted in a MyCycler^TM thermal cycler system (Bio-Rad, United States). PCR conditions (Lee et al., 2016 (link)) and annealing temperatures for each primer set are shown in Supplementary Table S1. PCR products were visualized in 1% agarose gel prior to direct DNA sequencing (ABI PRISM 3730xl Genetic Analyzer, Applied Biosystems, United States), performed by the First Base Laboratory Sdn. Bhd., Malaysia. In the case where targeted sequences were available in the GenBank database through previous studies of the same plant specimen, the records were included in this study without performing additional PCR and sequencing (Table 3).