The detailed description of biological material used in this work is given in Supporting Information, Appendix S1. In this way, the species belonging to all main Tuber clades (Bonito et al., 2010), except for Gennadii, Gibbosum and Macrosporum clades, were Everolimus prepared for further analysis. Dry fruit-body material, 5 mg, was first washed in 100% ethanol, dried and extracted by NucleoSpin Plant II DNA extraction kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) as recommended by the supplier. The material was initially homogenized in 300 μL extraction
buffer PL1 using mortar and pestle pretreated by overnight soaking in 1% hydrochloric acid at room temperature, short washing with distilled water, washing in 10 mM Tris–borate–EDTA (pH 8.3), washing with distilled water and autoclaving for 25 min at 121 °C. The same procedure was used for DNA extraction from ectomycorrhizae, but 100 mg fresh material was homogenized C59 wnt order in 400 μL buffer PL1. Extraction of DNA from soil samples (250 mg) was performed using NucleoSpin Soil DNA kit (Macherey-Nagel GmbH & Co. KG) with recommended amounts of the buffer SL1 and enhancer SX. The DNA concentration in final extracts is given in Appendix S1, sheet ‘Primer_specificity’, and was measured at 260 nm using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Undiluted extracts were used directly as a template in PCR. The primers were designed on
the basis of comparison of GenBank-published ITS T. aestivum sequences with those belonging to other Tuber check details spp. The sequences are listed in Appendix S2. Fifty-one sequences that could not be successfully aligned were excluded. The remaining 130 sequences of T. aestivum (including forma uncinatum as well as 884 sequences of a further 41 Tuber spp.) were included in further analysis. The sequences of each species were aligned in bioedit software, version 220.127.116.11 (Hall, 1999), and consensus sequences were created for each species separately. Where high intraspecific variability was encountered, the sequences of the species were manually
sorted to smaller groups generating separate consensus sequences, or included in further analysis individually. Prepared consensus and individual sequences were aligned (Appendix S3) and the possible motifs that could be recognized by T. aestivum-selective primers were searched for. The selected motifs in aligned sequences of T. aestivum (including forma uncinatum; Appendix S4) were checked to exclude any possible sequence gaps. Denaturation temperature of hybridized primers, melting point of their secondary structure and homodimer stability were checked using DinaMelt tools (http://dinamelt.bioinfo.rpi.edu). Five negative controls (complex nontarget DNA) were established: A: DNA from composted spruce bark (98 ng μL−1 PCR template). All the negative controls gave strong signals in PCR with nonspecific primers amplifying the eukaryotic ITS region of the rRNA gene cassette.