DNA Barcoding with ITS2 and psbA-trnH

Two 5 bp tags were designed and added to the 5′ end of the universal ITS2 and psbA-trnH primers (Kress et al., 2005 (link); Chen et al., 2010 (link)) to distinguish the sequences obtained from different regions. Different primers were used to the amplify ITS2 and psbA-trnH regions in the different samples. The designed primers used in the PCR for the detection of the different samples are listed in Supplementary Tables S3, S4. PCR amplifications were performed according to the DNA barcoding protocol recorded in the Chinese Pharmacopoeia and were carried out in an Applied Biosystems Veriti^TM Thermal Cycler (Thermo Fisher Scientific, Inc., United States). For each reaction, 1 μl MgCl₂ (10 mM SBS Genetech Co., Ltd, China) and 2× Taq MasterMix (AidLab Biotechnologies Co., Ltd, China) were added to the PCR master mix. The PCR annealing temperature was increased to 58°C, and 40 cycles were run. A negative control reaction with no DNA template was included in the reactions. The DNA concentrations of the obtained amplicons were assessed via 2% agarose gel electrophoresis, and purification was performed with a QIAquick Gel Extraction kit (Qiagen). The DNA concentrations were measured on an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., United States) with the Qubit platform (Thermo Fisher Scientific, Inc., United States).