1. 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

2. 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

3. 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

4. RT-PCR

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

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

7. 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

8. 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

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

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

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

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

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

14. 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

15. 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

16. 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

17. 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

18. Documenting Barcode specimens