Molecular characterization using rRNA genes: A simple and direct tool for identification of ectomycorrhizae from pooled root-tip samples.

Identifying ectomycorrhizal species associated with roots of higher plants had always been a laborious work mostly relying upon generation of pure cultures and morphotying. No doubt these techniques are invariable unmatched, yet these limit to address the diversity associated with soil or root samples. Here we describe a quick and reliable method for identification of ectomycorrhizae directly from root tips without compromising diversity associated with the sample. This method is speedy as there is no need of morphotyping or any anatomical study of root tips. Fine root-tips harboring ectomycorrhizae are pooled and then directly processed for molecular characterization under highly stringent conditions using overlapped rRNA gene specific primers to generate multiple bands, each band being representative of an individual fungal species. This is followed by band elution and RFLP analysis to generate individual clades. Representatives of each clade are then sequenced, assembled and subjected to global BLAST analysis for identification of ectomycorrhizal specie(s).