Molecular Profiling of Dried Specimens

Genomic DNA was extracted from tissue of dried specimen or fresh tissue preserved in CTAB, using a CTAB isolation procedure adapted from Doyle and Doyle (1990) . Portions of the genes atp6, cox3, rpb2, and tef1 were amplified by polymerase chain reaction (PCR). The primer pairs ATP6-1M40F/ATP6-2M (Raspé et al. 2016 (link)), COX3M1-F/ COX3M1-R (Vadthanarat et al. 2019b (link)), bRPB2-6F/bRPB2-7.1R (Matheny 2005 (link)), and EF1-983F/EF1-2218R (Rehner and Buckley 2005 (link)) were used to amplify atp6, cox3, rpb2, and tef1, respectively. PCR products were purified by adding 1 U of exonuclease I and 0.5 U FastAP alkaline phosphatase (Thermo Scientific, St. Leon-Rot, Germany) and incubated at 37 °C for 1 h, followed by inactivation at 80 °C for 15 min. Standard Sanger sequencing was performed in both directions by Macrogen with PCR primers, except for atp6, for which universal primers M13F-pUC(-40) and M13F(-20) were used. For tef1, additional sequencing was performed with two internal primers, EF1-1577F and EF1-1567R (Rehner and Buckley 2005 (link)).