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Accessing COI in mushrooms

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. - to begin with. - methods. > dozen of primer tested multiple PCR techniques cloning

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Accessing COI in mushrooms

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

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

  4. RT-PCR

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

  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 - Results • > 95% success • primer pair developed for a broad taxonomic range

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

  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. conucopiae P. purpureoolivaceus

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

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

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

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

  15. Barcoding mushrooms with COI conclusions • 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

  16. Barcoding mushrooms with COI conclusions • 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.

  17. Barcoding mushrooms with COI conclusions • 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?

  18. Documenting Barcode specimens

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