Identification of Tanzanian saprophytic edible mushrooms by amplification and sequencing of ITS/LSU regions of ribosomal RNA operon

Abstract

In this study, ten wild saprophytic edible mushrooms samples, collected from Tanzania natural forests and planted trees, and their two domesticated forms were characterized by in-vitro/in-vivo amplification and sequencing of ITS/LSU regions. Mushroom genomic DNA was extracted by ZR Fungal/Bacterial DNA MniPrep Kit. ITS and LSU regions were amplified using ITS-4/ITS-5 and LR16/LROR primers, respectively and sequenced. The amplicons with messy sequences were cloned. For analyzing recombinant E. coli DH5α cells, colony PCR and sequencing were done using M13-F/M13-R primers. The studied mushrooms were identified as Amylosporus sp. IJ-2014, Polyporales sp., Polyporus tenuiculus, Pleurotus cystidiosus, Laetiporus sp. IJ-2014, Lentinus sajor-caju, Favolus roseus and Auricularia polytricha. The ITS-based phylogeny inferred by Neighbor-Joining method accommodated six genera under bootstrap support values of 100% with each genus consisting mushrooms of a single species. The LSU-based phylogeny inferred by Maximum Likelihood method accommodated nine genera with bootstrap support of ≥ 66% with some genera consisting mushrooms of different species. From these results, it is clear that both ITS and LSU markers successfully discriminated wild saprophytic edible mushrooms to their respective genera but ITS marker demonstrated the higher resolving power at the species level than LSU marker.

Keywords:  Saprophytic edible mushrooms, ITS, LSU, RNA operon