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

Picoa 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 et al. 2000, Jamali & Banihashemi 2012, 2013). Picoa species form symbiosis mainly on roots of annual and perennial herbaceous plants of the Helianthemum, including H. ledifolium and H. salicifolium var. salicifolium (Jamali & Banihashemi 2012, 2013), H. nummularium (Riousset et al. 1989, 1996), H. sessiliflorum (Sbissi et al. 2010), H. squamatum and H. kahiricum (Moreno et al. 2000). In Iran, mycorrhizal association between sedge and desert truffles is little known. Ammarellou et al. (2007) isolated the sedge roots (Kobresia bellardii) directly from under zone of Terfezia boudieri. During 2013–14, roots of Carex stenophylla Wahlenb were collected from various rangeland sites in Iran. The field and anatomical studies showed that, C. stenophylla have ectomycorrhizal associations with P. lefebvrei 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 Picoa lefebvrei. Nested PCR of ITS1/ITS4 amplified product with FLE/RLE primers resulted in amplification of 500 bp products in C. stenophylla roots. Sequences of PCR products amplified by FLE/RLE in two randomly root samples were similar (100%) with previously published DNA sequences of P. lefebvrei specimens and confirmed the existence of P. lefebvrei in mycorrhizal roots of C. stenophylla in the rangelands of Iran. Moreover, nested PCR increased the sensitivity of FLE/RLE primers to 10 fg DNA concentration.