Biologia 68/5: 816—820, 2013Section Cellular and Molecular BiologyDOI: 10.2478/s11756-013-0228-9Genetic variability in Fomes fomentarius reconf i rmed by translationelongation factor 1-α DNA sequences and 25S LSU rRNA sequencesPeter Pristas 1,2 , Svetlana Gaperova 2 , Jan Gaper 3,4 & Jana Judova 21 Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4–6, SK 04001 Košice, Slovakia; e-mail:pristas@saske.sk2 Department of Biology and Ecology, Faculty of Natural Sciences, Matej Bel University, Tajovského 40, SK 97401 BanskáBystrica, Slovakia3 Department of Biology and General Ecology, Faculty of Ecology and Environmental Sciences, Technical University, T. G.Masaryka 24, SK 96053 Zvolen, Slovakia4 Department of Biology and Ecology, Faculty of Sciences, University of Ostrava, Chittussiho 10, CZ 71000 Ostrava, CzechRepublicAbstract: The existence of two cryptic species within strains of the wood-decaying fungus Fomes fomentarius was revealedrecently based on the internal transcribed spacer (ITS) sequence variability. In this study for the f irst time the sequencesof another molecular markers, partial translation elongation factor 1-α (efa) region and partial 25S large subunit ribosomalRNA gene were obtained and used to evaluate genetic variability of F. fomentarius. Congruent phylogeny was observedfor all three markers used conf irming the presence of two cryptic species within F. fomentarius. Surprisingly, ITS sequencevariability within F. fomentarius was signif icantly lower compared to the variability of efa sequences (0.023 versus 0.036nucleotide substitutions per site) questioning the discriminatory power of ITS sequences for fungal species identif ication.Key words: Polyporaceae; cryptic species; Fomes fomentarius; molecular phylogeny; variability.Abbreviations: efa, translation elongation factor 1-α; ITS, internal transcribed spacer; LSU, 25S large subunit ribosomalRNA.IntroductionThe wood-decaying fungus Fomes fomentarius (L.) J.Kickx f. is widely distributed throughout the range ofits hosts in the northern hemisphere. It infects a verylarge range of deciduous and (rarely) coniferous trees.Moreover, in healthy beech (Fagus sylvatica) trees itcan also operate as an endophyte (Baum et al. 2003).F. fomentarius is commonly known as the Tinder Poly-pore, Hoof Fungus, Ice Man Fungus or Tinder Conk.Its basidioma is also known as the “Mudi”in Chinese,which has been used as a traditional Chinese medicinefor many centuries in China for the treatment of variousdiseases (Chen et al. 2008).Until recently, no signif icant genetic variability wasobserved in F. fomentarius. In our previous work (Ju-dova et al. 2012) we observed the existence of two geno-types in this species based on comparison of internaltranscribed spacer (ITS) sequences. Two genotypes dif-fer from each other by 3% at the nucleotide level andthe presence of 7 bp indel in the ITS2 region is the mostremarkable dif ference. A rapid method was developedfor discrimination of these genotypes and analyses sug-gest that one of genotypes is prevalent on beech (F.sylvatica), while other prefers other broadleaves trees(Judova et al. 2012).The aim of the present study was to re-evaluategenetic variability in F. fomentarius seen at the ITSlevel using other molecular methods based on sequencecomparison of both the partial translation elongationfactor 1-α (efa) region and partial 25S large subunitribosomal RNA (LSU) gene as well as to compare thediscriminatory power of three molecular markers used.Material and methodsDNA isolation and analysisAll DNA techniques used were in principle as already de-scribed (Judova et al. 2012). Shortly, total genomic DNA ofF. fomentarius strains was prepared using microwave treat-ment according to Goodwin & Lee (1993) with small modif i-cations. Released DNA was purif ied by repeated extractionsusing chloroform-isoamylalcohol (24:1) mixture and DNAwas precipitated using isopropylalcohol. Purif ied DNA wasvisualized after electrophoresis on 0.8% agarose gels.PCR amplif i cations and primersFor PCR amplif ication, about 50 ng of total DNA was am-plif ied using EF595F (5’-CGTGACTTCATCAAGAACATG-3’) and EF1160R (5’-CCGATCTTGTAGACGTCCTG-3’) primer pair for the partial translation elongation fac-tor 1-α (efa) region (Kauserud & Schumacher 2001) orLR0R (5’-ACCCGCTGAACTTAAGC-3’) and LR7 (5’-TACTACCACCAAGATCT-3’) primer pair for the partialc ?2013 Institute of Molecular Biology, Slovak Academy of Sciences
