Total genomic DNA was extracted from dried herbarium specimens using AxyPrep Multisource Genomic DNA Miniprep Kit 50-prep (Qiagen) according to the manufacturer’s instructions. ITS (nrDNA ITS1-5.8S-ITS2) and mtSSU (mitochondrial small subunit rDNA) were amplified by polymerase chain reactions (PCR) using the primer pairs ITS1F (Gardes and Bruns 1993 (link)), ITS4 (White et al. 1990 ) and mtSSU1/mtSSU2R (Zoller et al. 1999 (link)).
Amplifications were performed in a 25 μl volume comprising 12.5 μl of 2× MasterMix (TapDNA Polymerase, 0.1 units/μl; technologies Co. Ltd), 1.0 μl of each primer, 8.5 μl ddH2O and 2 μl DNA. Conditions for the PCR were: initial denaturation at 94 °C for 4 min, 34 cycles at 94 °C for 1 min, 54 °C for 1 min and 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. PCR products were sequenced in an ABI3730X using amplification primers manufactured by Tsingke (Kunming, China).
ITS and mtSSU sequences were assembled with Seqman 7.0 (DNAStar) and manually edited using Mega6. DNA sequences were aligned with MAFFT version 7 with default parameters (Katoh et al. 2005 (link)) via the online resource (http://mafft.cbrc.jp/alignment/server/index.html ).
Amplifications were performed in a 25 μl volume comprising 12.5 μl of 2× MasterMix (TapDNA Polymerase, 0.1 units/μl; technologies Co. Ltd), 1.0 μl of each primer, 8.5 μl ddH2O and 2 μl DNA. Conditions for the PCR were: initial denaturation at 94 °C for 4 min, 34 cycles at 94 °C for 1 min, 54 °C for 1 min and 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. PCR products were sequenced in an ABI3730X using amplification primers manufactured by Tsingke (Kunming, China).
ITS and mtSSU sequences were assembled with Seqman 7.0 (DNAStar) and manually edited using Mega6. DNA sequences were aligned with MAFFT version 7 with default parameters (Katoh et al. 2005 (link)) via the online resource (
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