Abi model 3130 genetic analyzer

Extraction of genomic DNA was carried out using the NucleoSpin Plant II Kit (Macherey-Nagel GmbH & Co. KG), according to the manufacturer’s instructions. ITS regions were amplified and sequencing with primers ITS1F-ITS4 (Gardes & Bruns 1993 (link), White et al. 1990 ) and the nrLSU with primers JS1and LR5 (Landvik 1996; http://www.biology.duke.edu/fungi/mycolab/primers.htm). PCR products were purified applying the GeneJET Gel Extraction Kit (Thermo Scientific, Thermo Fisher Scientific Inc., MA, USA). Sequencing was performed with an ABI model 3130 Genetic Analyzer (Applied Biosystems, CA, USA). Raw data were edited and assembled in MEGA v. 6 (Tamura et al. 2013 (link)). All steps of molecular studies were carried out at the Center for collective use of scientific equipment “Cellular and molecular technology of studying plants and fungi” (Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg). In case of specimens processed at the molecular lab of the mycology chair in the University of Tartu, the protocols described in Pärtel et al. (2016) , were followed.

DNA was extracted from herbarium material using a CTAB extraction buffer technique with the following steps of consecutive addition of chloroform-isoamyl alcohol mixture, then isopropyl alcohol-3M sodium acetate solution for precipitation, 70 % ethanol for washing and finally water for dissolution. The alternative method of extraction DNA was using Axy Prep Multisourse Genomic DNA Miniprep Kit (Axygen Biosciences).
The ribosomal ITS1-5.8S-ITS2 region was amplified by PCR with the fungal specific primers ITS1F and ITS4B (Gardes & Bruns 1993 (link); http://www.biology.duke.edu/fungi/mycolab/primers.htm). Sequences of nrLSU-rDNA were generated using primers LR0R and LR5 (Vilgalys & Hester 1990 (link)), and sequences of mtSSU – using primers MS1 and MS2 (http://nature.berkeley.edu/brunslab/tour/primers.html). PCR products were visualized using agarose gel electrophoresis and Gel Red staining, and subsequently purified with the kit AxyPrep PCR Cleanup Kit (Axygen Biosciences). Sequencing was performed with ABI model 3130 Genetic Analyzer (Applied Biosystems) using BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with the same primers. The raw data were processed using Sequencing Analysis 5.3.1 (Applied Biosystems).