Specific PCR-primers for detection of Picoa lefebvrei desert truffle in Carex stenophylla roots

Abstract

<em>Picoa</em> species are hypogeous desert truffles, which can be found in semi-arid ranges of North Africa, West Asian, South of Europe and Middle East, including Iran (Moreno <em>et al</em>. 2000, Jamali & Banihashemi 2012, 2013).<em> Picoa</em> species form symbiosis mainly on roots of annual and perennial herbaceous plants of the <em>Helianthemum</em>, including <em>H. ledifolium</em> and <em>H. salicifolium</em> var. <em>salicifolium </em>(Jamali & Banihashemi 2012, 2013), <em>H. nummularium</em> (Riousset <em>et al</em>. 1989, 1996), <em>H. sessiliflorum</em> (Sbissi <em>et al</em>. 2010), <em>H. squamatum</em> and <em>H. kahiricum</em> (Moreno <em>et al</em>. 2000). In Iran, mycorrhizal association between sedge and desert truffles is little known. Ammarellou <em>et al</em>. (2007) isolated the sedge roots (<em>Kobresia bellardii</em>) directly from under zone of <em>Terfezia boudieri</em>. During 2013–14, roots of <em>Carex stenophylla</em> Wahlenb were collected from various rangeland sites in Iran. The field and anatomical studies showed that, <em>C. stenophylla</em> have ectomycorrhizal associations with <em>P. lefebvrei</em> in the studied areas. In general, external mycelium was observed around all the mycorrhizal plants. The ITS1-5.8S-ITS2 gene region was amplified with the genomic DNA extracted from roots by nested polymerase chain reaction using the universal fungal primer pair ITS1/ITS4 and specific primer pair FLE (5´GTA CCT TAC CTG TTG CTT CCG TG) and RLE (5´ATC CCT ACC TGA GCC GAG GTC AA) which were designed based on ITS1-5.8S-ITS2 gene region of <em>Picoa lefebvrei. </em>Nested PCR of ITS1/ITS4 amplified product with FLE/RLE primers resulted in amplification of 500 bp products in <em>C. stenophylla</em> roots. Sequences of PCR products amplified by FLE/RLE in two randomly root samples were similar (100%) with previously published DNA sequences of <em>P. lefebvrei</em> specimens and confirmed the existence of <em>P. lefebvrei</em> in mycorrhizal roots of <em>C. stenophylla</em> in the rangelands of Iran. Moreover, nested PCR increased the sensitivity of FLE/RLE primers to 10 fg DNA concentration. <br />lant life cycle. On the other hand, correlation coefficient between spore population and root colonization was very low for Torbat specimens, which could be related to other factors e.g. environmental and geographical conditions. <br />