Identification of Tanzanian Saprophytic Edible Mushrooms by Amplification and Sequencing of Its/Lsu Regions of Ribosomal Rna Operon

IDENTIFICATION OF TANZANIAN SAPROPHYTIC EDIBLE MUSHROOMS BY AMPLIFICATION AND SEQUENCING OF ITS/LSU REGIONS OF RIBOSOMAL RNA OPERON Ibrahim Juma1, Anthony Manoni Mshandete2, Donatha Damiani Tibuhwa3* and Amelia Kajumulo Kivaisi4 Department of Molecular Biology and Biotechnology, College of Natural and Applied Sciences, Uvumbuzi Road, University of Dar es Salaam, P.O. Box 35179, Dar es Salaam, Tanzania. [email protected] 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. INTRODUCTION morphological features, a task often Mushroom taxonomy is the science that complicated by inconsistencies, includes description, identification, morphological plasticity between organisms nomenclature and classification of of the same species and existence of mushroom (Simpson, 2010). The presence convergent evolution (Muruke et al., 2002). of edible and poisonous mushrooms, while In addition to that, conventional methods some of them do resemble much, entails the cannot be applied in cases where there is good scientific mushroom taxonomy in only a small amount of biological material order to avoid misidentification of available for examination or a sample mushrooms which may lead into death contains biological material from different (Tibuhwa, 2013). Mushroom taxonomy can species mixed together (Pereira et al., 2008). be done on the basis of morphological Nowadays, conventional methods are being features (conventional methods) and complemented with molecular methods such molecular methods (modern techniques) as DNA sequencing methods. The most (Tibuhwa, 2011). Conventional methods frequently sequenced genetic markers for involve observation of macro- and micro- fungi are the internal transcribed spacer Juma et al. - Identification of Tanzanian saprophytic edible mushrooms (ITS) region and nuclear ribosomal large 2002), molecular study of the genus subunit (LSU) of ribosomal RNA operon Afrocantharellus by PCR-DNA sequencing (Muruke et al., 2002). ITS/LSU regions of LSU, 5.8S-ITS2 and ATP6 (Tibuhwa et possess two salient features which make al., 2012) and molecular phylogeny of some them valuable as barcoding regions. The saprophytic edible mushrooms by Hussein et first feature is possession of highly al. (2014). This study complements the work conserved segments (common to all fungi) by Hussein et al. (2014) and it aimed at which enables researchers to design characterizing more wild/domesticated universal PCR primers that can amplify a saprophytic edible mushrooms by wide range of fungi. The second feature is combining morphological method and in- possession of variable/highly variable vitro/in-vivo amplification and sequencing regions (unique sequences) which serve as of ITS/LSU regions of ribosomal RNA signatures for different species (Pereira et operon. al., 2008). DNA sequencing methods offer data for identification that is more accurate MATERIALS AND METHODS and reproducible which portray phylogenetic Sample collection relationships among organisms (Hibbett et The study was carried out in natural forests al., 1997). The advent of gene cloning had of Lutindi, Shume-Magamba and Kieti in enabled DNA sequencing methods to Tanga region, Kazimzumbwi forest in Pwani discriminate and identify biological region and some planted trees at University materials from different species mixed in the of Dar es Salaam main campus in Dar es same sample (Pereira et al., 2008). Three Salaam (DSM) region (Figure 1). Eight wild main steps involved in any gene cloning edible saprophytic mushroom morpho- process are ligation, transformation, and species collected during rainy seasons analysis of recombinant clones (Russell and (March a an eptem er e em er Sambrook, 2001; Brown, 2006). 2011/2012) with their two domesticated counterparts were used in this study. Though Tanzania harbors natural forests rich Preliminary morphological identification in different species of wild edible was done in the field as per Tibuhwa et al. mushrooms, few studies on mushroom (2010). The identified wild edible taxonomy had been done and most of them saprophytic mushrooms were subjected to are based on conventional methods domestication trials as comprehensively (Tibuhwa et al., 2010) as well as described reported by Juma et al. (2015). The wild and single genus such as Cantharellus (Tibuhwa successfully domesticated mushrooms were et al., 2008) and Sacorscypha (Tibuhwa, dried at 50°C to constant weight and then 2011). A comprehensive study on Tanzanian preserved in silica gel for antioxidant edible and harmful mushrooms was done on potential determination, as broadly reported the basis of conventional taxonomy by by Juma et al. (2016), and molecular Hӓrkӧnen et al. (1995; 2003). Few characterization. Other fruitbodies were molecular taxonomy conducted includes dried at 55°C for herbarium deposits at the identification of mushroom mycelia by University of Dar es Salaam. PCR-RFLP of the ITS region (Muruke et al., 108 Tanz. J. Sci. Vol. 42, 2016 Figure 1: A map of Tanzania showing studied sites DNA extraction and quality/quantity et al., 1990) and LROR/LR16 (Vilgalys and check up Hester, 1990) primer pairs, respectively. DNA was extracted from dried fruiting R rea tions were arrie out in 20μ bodies by ZR fungal/bacterial DNA reaction volume using PCR master mix MiniPrep Kit (ZYMO RESEARCH) (ACCUPOWER® TAQ PCR PREMIX, a or ing to manufa turer’s instru tions. BIONEER). Preparation of PCR reaction Concentration and cleanliness of genomic mixtures and amplification were done DNA were assayed by nanodrop according to AccuPower® Taq PCR PreMix spectrophotometer(THERMO SCIENTIFIC protocol with slight modifications. The NanoDrop 2000c) whereas DNA quality reactions were heated in an initial step of 95 was checked by gel electrophoresis on 1% °C for 5 min and then subjected to 35 cycles agarose gel staine with GelRe using λ of the following program: 95 °C for 30 DNA Marker (Gόes -Neto et al., 2011). seconds, 60 °C for 30 seconds, and 72 °C for 1 min. After the final cycle, the temperature In-vitro ITS/LSU amplification and was maintained at 72 °C for 10 min and sequencing finally chilled at 10 °C to stop reaction. PCR R amplifi ation of I an regions products were analyzed by electrophoresis was performed in thermocycler (APPLIED whereby 1.8% agarose gel prepared in 0.5X BIOSYSTEMS® GENEAMP® PCR Tris-Borate EDTA buffer with addition of SYSTEMS 9700) using ITS-4/ITS-5 (White 2.5 μ gel re was use together with NA 109 Juma et al. - Identification of Tanzanian saprophytic edible mushrooms ladder (THERMO SCIENTIFIC reaction conditions were initial step of 95 °C GENERULER 1Kb+ DNA LADDER). for 3 min and then subjected to 35 cycles of Amplicons were purified using Thermo the following program: 94 °C for 1 min, 55 Scientific GeneJET PCR purification kit °C for 1 min, and 72 °C for 2 min. After the #K0701 following the manufa ture’s final cycle, the temperature was maintained proto ol. on entration of purifie at 72 °C for 15 min and finally chilled at 10 ampli ons was etermine nano rop °C to stop reaction. Analysis of colony PCR spe trophotometer ( H R I N IFI products and processing for sequencing were NAN R 2000 ) at a sor an e (A carried out as explained earlier in the 260 280). equen ing was one at at previous subsection. egoli nit of e A I RI Hu using capillary sequencer (ABI PRISM 3730 Data analysis GENETIC ANALYZER, APPLIED After quality control of sequence datasets BIOSYSTEMS) according to the using CLC Main Workbench 6.9.1 as per manufa turer’s instructions. manufa ture’s proto ol the an I sequences were interrogated on NCBI In-vivo ITS/LSU amplification (Gene GenBank database and then assembled with Cloning) and sequencing some reference sequences (Table 1) using Some mushroom DNA sequences portrayed same software, CLC Main Workbench 6.9.1. messy chromatograms thus, their purified The sequence alignments were imported PCR products were cloned by using separately in MEGA6 (Tamura et al., 2013) THERMOSCIENTIFIC InsTA CLONE that analyzed the ITS and LSU datasets and PCR CLONING KIT #K1214, following phylogenetic relationships were analyzed manufa turer’s proto ol. After ligation an using Neighbor-Joining

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