The strain CIRM-BRFM 902 originating from French Guiana was designated as reference strain for P. sanguineus (L) Murrill, Surinam (Lamark, 1783), the strain MUCL 39523 originating from Australia for P. coccineus (Fr.) Bondartsev & Singer, Polynesia (Fries, this website 1851), and the strain MUCL 30555 originating from Belgium for P. cinnabarinus (Jacq.) P. Karst, Europe (Karsten, 1881). The strain of Trametes suaveolens CBS 426.61 was used as an outgroup in phylogenetic analyses. Genomic DNA was isolated from mycelial powder (40–80 mg) as described by Lomascolo et al. (2002). The ITS region was amplified using the ITS1 and ITS4 primers as described by White
et al. (1990). The degenerate primers Bsens and Brev were adapted from primers already designed to match a 133-amino-acid conserved region in β-tubulin from Lentinula
spp. and Pleurotus spp. (Thon & Royse, 1999). In our study, β-tubulin gene from Trametes click here versicolor, Polyporus lepideus, Schizophyllum commune, Coprinus cinereus, and Pleurotus sajor-caju (NCBI accession numbers AY944859, AY944857, X63372, AB000116, AF132911, respectively) were aligned, and consensus primers Bsens [5′-ATCAC(A/T)CACTCICTIGGTGGTGG-3′] and Brev [5′-CATGAAGAA(A/G)TGIAGACGIGGG-3′] were designed. The universal genetic code was used. At degenerate positions, if three or four combinations were possible, the base was replaced by an inosine (I); otherwise, the two possible bases were kept. The two degenerate primers F2 [5′-CA(C/T)TGGCA(C/T)GG(A/G)TTCTTCC-3′] and R8 [5′-GAG(A/G)TGGAAGTC(A/G)ATGTG(G/A)C-3′] (-)-p-Bromotetramisole Oxalate were designed to match, respectively, the copper-binding domains I and IV, highly conserved in blue copper oxidases such as laccases
(Messerschmidt & Huber, 1990). The sequences of F2 and R8 were based on the alignment of the corresponding nucleotide regions of the basidiomycete laccases from P. coccineus, P. sanguineus, Lentinula edodes, Coriolus hirsutus and P. sajor-caju (NCBI accession numbers AB072703, AY458017, AB035409, AY081775 and AJ507324, respectively). The ITS1-5.8S rRNA gene-ITS2, laccase F2-R8 and β-tubulin Bsens-Brev fragments were amplified from 50 ng genomic DNA in 50 μL PCR reagent containing 1.5 U Expand™ High Fidelity PCR system (Roche, France) with a protocol adapted from Lomascolo et al. (2002). Annealing temperatures and extension times were respectively 51 °C and 1 min for ITS1/ITS4 amplification, 55 °C and 50 s for Bsens/Brev amplification and 55 °C and 2 min for F2/R8 amplification. In the case of the lacF2/R8 fragment, the PCR products were further cloned into the pGEM®-T Easy vector (Promega), following the manufacturer’s protocols. The PCR products were sequenced by GATC Biotech AG (Konstanz, Germany) or Cogenics (Meylan, France). All the nucleotide sequences were deposited in GenBank under the accession numbers given in Table 1.