Development of molecular markers of ectomycorrhizal fungi based on ITS region

Four heavy-metal-tolerant isolates of ectomycorrhizal fungi namely Laccaria fraterna (Cooke & Massee) Pegler (EM-1083) and Pisolithus tinctorius (Mich. Ex Pers) Coker & Couch (EM-1290, EM-1293 and EM-1299) were selected on the basis of the previously performed experiments. DNA extraction and purification was done by following standard protocols. Subsequently, polymerase chain reaction (PCR) amplification of the ITS region was done using universal primer ITS 1 and ITS 4. The amplicons were digested with HinfI. The prominent band of the restricted fragments was excised and purified using geneEXIT. Cloning and sequencing of the excised fragment was done following the standard method. Primers were designed with sequence information using the Gene Fisher Interactive PCR Primer design software. Amplification was successfully obtained using the designed primers in Pisolithus tinctorius (EM-1293). The size of the amplicon was 200 bp.