Accessing COI in mushrooms Jean-Marc Moncalvo and Maryna Didukh Department of Natural History Royal Ontario Museum Department of Ecology and Evolutionary Biology University of Toronto

Direct PCR > dozen of primer tested multiple PCR techniques cloning sampling: 1- many species in many genera of - to begin with... Agaricales Polyporales Cantharellales - methods

Direct PCR > dozen of primer tested multiple PCR techniques cloning sampling: 1- many species in many genera of - to begin with... Agaricales Polyporales Cantharellales - methods - results < 5% success

RT-PCR

Images from soumon-mori.com, and bugs.bio.usyd.edu.au/Mycology/ RT-PCR

Accessing COI sequence data - RT-PCR RNA isolation create cDNAs using Reverse-Transcriptase PCR amplification using cDNAs as template

Accessing COI sequence data - RT-PCR RNA isolation create cDNAs using Reverse-Transcriptase PCR amplification using cDNAs as template > 95% success primer pair developed for a broad taxonomic range - Results

COI sequence groupings 155 taxa with complete RNA COI barcode region or at least 200bp obtained from genomic DNA Pleurotus Cantharellus, Sistotrema (Cantharellales) Trametes, Grifola (Polyporales) Agaricales Hygrophorus (Agaricales) Amanita, Lepiota Agaricus Lepiota Psathyrellaceae Schizophyllum Tricholomataceae p.p. Agrocybe, Conocybe

Pleurotus ostreatus clade: ITS sequences From Vilgalys and Sun, 1994; Vilgalys and Moncalvo, 2000

EF1-a (ca. 650 bp) Pleurotus ostreatus group P. purpureoolivaceus P. conucopiae

EF1-a (ca. 650 bp) Pleurotus ostreatus group P. purpureoolivaceus P. conucopiae COI (ca. 550 bp) Pleurotus ostreatus group Pleurotus ostreatus group

Barcoding mushrooms with COI direct PCR: short regions only, although Long-PCR possible conclusions

Barcoding mushrooms with COI direct PCR: short regions only, although Long-PCR possible RT-PCR: feasible, including for sporocarps conclusions

Barcoding mushrooms with COI direct PCR: short regions only, although Long-PCR possible RT-PCR: feasible, including for sporocarps tissue storage in “RNALater” feasible conclusions

Barcoding mushrooms with COI direct PCR: short regions only, although Long-PCR possible RT-PCR: feasible, including for sporocarps tissue storage in “RNALater” feasible multiple copies; with / without introns conclusions

Barcoding mushrooms with COI direct PCR: short regions only, although Long-PCR possible RT-PCR: feasible, including for sporocarps tissue storage in “RNALater” feasible multiple copies; with / without introns intra and interspecies variation? documenting COI in fungi will serve the universal COI barcode database, but ITS and EF1-alpha are more easily accessible and offer better species-level resolution at least in mushrooms. conclusions

Barcoding mushrooms with COI direct PCR: short regions only, although Long-PCR possible RT-PCR: feasible, including for sporocarps tissue storage in “RNALater” feasible multiple copies; with / without introns intra and interspecies variation? conclusions

Documenting Barcode specimens