DNA barcoding mushrooms for species identification and by Ch9KyC

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									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

  • sampling: 1- many species in many genera of Agaricales
                                                Polyporales
                                                Cantharellales
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
RT-PCR
RT-PCR




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

 - Results
     > 95% success
     • primer pair developed for a broad taxonomic range
COI sequence groupings
 155 taxa with complete RNA COI                         Tricholomataceae p.p.
                                                  Schizophyllum
 barcode region or at least 200bp
 obtained from genomic DNA                         Tricholomataceae p.p.
                                                  Psathyrellaceae
                                               Lepiota
                                                 Agaricus
                                              Agrocybe, Conocybe
                                               Amanita, Lepiota

                                                                            Agaricales




                                                  Pleurotus




                                        Trametes, Grifola (Polyporales)
                                       Hygrophorus (Agaricales)
                               Cantharellus, Sistotrema (Cantharellales)
                                                                                                                 From Vilgalys
Pleurotus ostreatus clade: ITS sequences                                                                         and Sun, 1994;
                                                                                                                 Vilgalys and
                                                            II.TMI30632 JAPAN            Intersterility Groups   Moncalvo, 2000
   Alignment for                                            II.TMI30058 JAPAN
   ITS 1 - 5.8S - ITS 2 = 668 bp                         II.PDD94.077T NZ
                                                            II.PDD88.066F NZ
                                                         II.ATCC32772 INDIA
                                                         II.ATCC32771 INDIA
   Parsimony search:                                     II.Hilber1R GERMANY
   random addition sequence,                             II.Hilber4y SWEDEN
                                                         II.Hilber4x GERMANY
       1000 replicates                                   II.WC152 British Colum
                                                         II.ITCCF1725 INDIA                   II - Worldwide
   >5000 mp tree, length = 97                               II.Kumar INDIA
   CI = 0.8247                                           II.Hibbett92.107.2 PNG
                                                                            II.RV95.686 QLD
                                                            II.RV95.938 PNG
                                                                     II.RHP4203.11.SWEDEN
   Values above branches show 73                            II.RHP4203.10.SWEDEN
                                                                 II.D263 Virginia
      bootstrap frequency                                II.D352 NCarolina
      (1000 replicates)                                  II.RHP1711.13 New York
                                                         II.RHP1711.10 New York
                                                            II.RV95.773 PNG
                                                         II.RV95.522.8 Tasmania
                                                         II.Hibbett92.107.1 PNG
                                           I.Ginter31 CZECH
                                     91 I.CBS145.22 GERMANY
                                              I.Ginter55 CZECH                                  I - Europe
                                          I.GEL2206 GERMANY
                                     95 I.SJ91-070.4 SWEDEN   I.TMI30055 JAPAN
                                               I.TMI30054 JAPAN                                 I - Japan
                        68                III.EK89M2 MONTANA
                                          III.OKM7207 MARYLAND
                                     81 III.DAOM180401.2 ONTARIO
                                          III.DAOM180401.4 ONTARIO
                                          III.DAOM197110.7 ONTARIO                          III - N. America
                                80        III.DAOM197110.3 ONTARIO
                                              III.EK89M30.16 MONTANA
                                          III.EK89M30.21 MONTANA
                                                                            XIII.D327 BRAZIL XIII - Brazil
                              I.RHP3505.19 TENN
                              I.D261 TENN
                              I.D403 ARIZONA                                              I - North America
                              I.D850 CALIFORNIA
                                    94             VI.ATCC58080 AFGHAN
                           79                      VI.ATCC52666 AFGHAN
                                                   VI.ATCC62885 AFGHAN                           VI - Asia
                                    97         VI.MW85 PENNSTATE
                                                     VI.CBS130.21 EUROPE                      VI - Europe
                                                                 VI.ATCC36047 CZECH
                                                                            XII.RHP6554.6 Russia
                                   100                           XII.RHP6551.17 Russia
                                                                         XII.RHP6551.7 Russia XII - Siberia
                                                                 XII.RHP6554.17 Russia
  EF1-a (ca. 650 bp)
                                                                           D3 27 SCHY ZO gen DNAEF1PL 1 567 RA
                                                 Ostrea tu s Cze ch 33 0 and 330 b
                                                  D3 30.1 146 FR cDNA
                                                 Ostrea tu s Cze ch 33 1
                                                  Ostrea tu s Germany 334
                                                 Ostrea tu s Germany 334b
                                             Ostrea tu s Cal i fo rn ia 85 0 AND D8 50 gen D EF1P L MARCH
                                             D8 50 171 F gen DNA
                                            D8 50 151 FR cDNA
                                                                        Pu lmon ariu s NC 35 2
                                                                        Pu lmon ariu s Swe den 4 81
                                                                        D4 80 148 FR cDNA
                                                                      Pu lmon ariu s Ja pan 1 748
                                                                Pu lmon ariu s PNG 19 79
                                                                 D1 979.4 17 6F ge nDNA
                                                                  D1 979.j 1 75FR gen DNA
                                                                D1 979 15 9FR g enDNA
                                                                D1 979 ge nD EF1 PL MARCH
                                                                 19 79.4 16 9F cDNA
                                                                   D1 825 15 5FR cDNA
                                                                 D1 944 15 8F ge nDNA
                                                                Pu lmon ariu s PNG 19 80
                                                                 Pu lmon ariu s PNG 19 80b
                                                                       Pu lmon ariu s VA 26 3
      Pleurotus                                                        Pu lmon ariu s VA 26 3b
                                                                       Pu lmon ariu s BC 70 0

      ostreatus                                                       D1 728.j 1 53FR cDNA
                                                                      D2 245.19 1 62FR cDNA
                                                       Eryn gi i Europ e 625
      group                                            Eryn gi i Europ e 625 b
                                                       D1 832.1 15 6FR cDNA eryn gi i
                                                       D1 832.6 15 7FR cDNA
                                                       P.eryn gi i marke t 16 7FR cDNA
                                                       D1 820 17 3F ge nDNA e ryngi i
                                                       D1 820 ge nD EF1 PL MARCH
                                                       D1 822 17 4FR g enDNA e ryng ii
                                                       D1 822 ge nD EF1 PL MARCH
                                                                         Po pul in us US 2 212
                                                                         Po pul in us US 2 212 b
                                                                         Po pul in us Mo ntana 22 14
                                                                            Po pul in us On ta ri o 23 04
                                                                         Po pul in us Ma ryla nd 39 9
                                                                          Po pul in us Ma ryla nd 39 9b
                                                                         Po pul in us Mo ntana 76 5
                                                                       D2 230.1 16 4FR cDNA
                                              Pl genD 5 26Fdi dno two rk 165 7R MARCH
                                              Pl genD E F1PL 1 567 R MA RCH
                                           D3 32 147 FR cDNA
                                           D5 47 149 FR cDNA
                                           D5 64 150 FR cDNA
                                           D2 61.4 145 FR cDNA
                             D1 737 16 8F cDNA cornu co p
                             D1 738 15 4FR cDNA cornu cop
                                 Hyps1 031 1 52FR cDNA ZYGO BLAS T             P. conucopiae
                                pu lmo nari us D2 070 .1 16 0FR cDNA
D2 246.1 16 3FR cDNA
Pl purpurD2 342 .1 EF1 M cDNA 52 6F 15 67RMA RCH0 7
  5 changes                                                     P. purpureoolivaceus
  EF1-a (ca. 650 bp)                                                                                                               COI (ca. 550 bp)
                                                                           D3 27 SCHY ZO gen DNAEF1PL 1 567 RA
                                                 Ostrea tu s Cze ch 33 0 and 330 b                                                                 Pl eurotus28 0
                                                                                                                                                       Pl abi et D231 0a 12 F4R DNtdS TR 09FEB07 CH
                                                  D3 30.1 146 FR cDNA                                                                                  Pl abi et D231 0a 11 F2eR K C DNtd STR 09 FEB0 7 CH
                                                                                                                                                       Pl abi eticD23 30.1 KC DNtdS TR 12F4R 26Ma rch07
                                                 Ostrea tu s Cze ch 33 1                                                                               Pl abi eticD23 30.1 KC DNtdS TR 11F2eR 26 March0 7
                                                  Ostrea tu s Germany 334                                                                            Pl eryn foss D182 1a KC DNtdST R 1 1F2 eR 09FEB2 007 CH
                                                                                                                                                     Pl eryn foss D182 1a KC DNtdST R 1 2F4 R 09FE B07 CH
                                                 Ostrea tu s Germany 334b                                                                            Pl eryn foss D182 1b DNtdS TR 11F2eR 09 FEB2 007 CH
                                             Ostrea tu s Cal i fo rn ia 85 0 AND D8 50 gen D EF1P L MARCH                                            Pl eryng ma rket 1 2F4 R K CDNtdST R 09FE B07
                                                                                                                                                     Pl eryng D1 832 .6 1 1F2 eR KC DNtdST R 1 4FE B07
                                             D8 50 171 F gen DNA                                                                                      Pl eryng D1 832 .6 1 2F4 R K C DNtdSTR 14 FEB0 7
                                                                                                                                                     Pl eryng D6 25.2 12 F4R KC DNtd STR 09MA R07 CH
                                            D8 50 151 FR cDNA                                                                                         D1 932.1 2E R K C 26MARCH07
                                                                        Pu lmon ariu s NC 35 2                                                       Pl notcl ea r D62 5.2 12F4R KC DNtd STR 09 MAR07
                                                                                                                                                   Pl popu l D2 212 1 2F4R K CDNtdST R 1 4FE B07
                                                                        Pu lmon ariu s Swe den 4 81                                                  Pl ostr D174 2tu beB 1 2F4 R K C 3 1JAN07 CH
                                                                                                                                                     Pl ostr D174 2tu beA 1 1F KC 31 010 7 CH
                                                                        D4 80 148 FR cDNA                                                            Pl ostr D174 2tu beA 1 2F4 R K C 3 1JAN07 CH
                                                                      Pu lmon ariu s Ja pan 1 748                                                   Pl ostr D332 .2 30 FR NOTtdCL 12F4R 31JAN07 CH
                                                                                                                                                   Pl ostr D332 .2 10 FR DNtdS TR 11F2eR 31 JAN0 7 CH
                                                                Pu lmon ariu s PNG 19 79                                                           Pl ostr D332 .2 11 FR DNtdS TR 12F4R 31JAN07 CH
                                                                 D1 979.4 17 6F ge nDNA                                                            Pl ostr D332 .2 27 FR NOTtdCL 12F4R 31JAN07 CH
                                                                                                                                                   Pl ostr D332 .2 29 FR NOTtdCL 11F2eR 31 JAN0 7 CH
                                                                  D1 979.j 1 75FR gen DNA                                                            Pl ostr D332 .2 31 FR NOTtdCL 11F2eR 31 JAN0 7 CH
                                                                                                                                                   Pl ostr D332 .2 34 FR NOTtdCL 11F2eR 31 JAN0 7 CH
                                                                D1 979 15 9FR g enDNA                                                                Pl ostr D330 53 FR DNtdS TR 11F2eR 09 FEB0 7
                                                                D1 979 ge nD EF1 PL MARCH                                                            Pl ostr D330 55 FR DNtdS TR 12F4R 09FEB07
                                                                                                                                                   Pl ostr D547 11 F 14FEB07
                                                                 19 79.4 16 9F cDNA                                                                Pl ostr D261 .4 KC DNtd STR 12F4R 26 March0 7
                                                                                                                                                   Pl ostr D547 12 F4R KC DNtd STR 14 FEB0 7
                                                                   D1 825 15 5FR cDNA                                                              Pl ostr D850 12 F4R KC DNtd STR 14 FEB0 7
                                                                 D1 944 15 8F ge nDNA
                                                                Pu lmon ariu s PNG 19 80
                                                                                                                   Pleurotus                       Pl ostr D850 2e R KC DNtdST R 1 4FE B07
                                                                                                                                                     Pl ostreatus ma rket 12F4R KC DNtdST R 0 9FE B07
                                                                                                                                                   Pl ostr D117 1 1 1F2 eR KC DNtdS TR 14FEB07
                                                                                                                                                   Pl ostr D117 1 1 2F4 R KC DNtdSTR 1 4FEB 07
                                                                 Pu lmon ariu s PNG 19 80b
                                                                       Pu lmon ariu s VA 26 3                      ostreatus                        Pl ostr D203 8.1 0 124 FR 11 F2eR DNtd STR 09 MAR07
                                                                                                                                                   Pl ostr D203 8.1 0 135 FR 12 F4R DNtdS TR 09MAR07
      Pleurotus                                                        Pu lmon ariu s VA 26 3b
                                                                                                                                                   Pl ostr D230 8 1 02FR 12F4R DNtdST R 1 9FE B07 CH
                                                                                                                                                   Pl ostr D230 8 9 1FR 1 1F2 eR DNtdST R 1 9FE B07 CH
                                                                       Pu lmon ariu s BC 70 0
                                                                                                                   group                           Pl ostr D230 8 1 32F 1 2F DNtdS TR 09MAR07 CH
                                                                                                                                                    Pl ostr D261 .7 12 F4R KC DNtd STR 09 MAR07 CH

      ostreatus                                                       D1 728.j 1 53FR cDNA
                                                                      D2 245.19 1 62FR cDNA
                                                                                                                                                   Pl ostrD26 1.4 1 1F2 eR KC 26 MARCH0 7
                                                                                                                                                   Pl ostrD26 1.4 1 2F4 R KC 2 6MARCH07
                                                                                                                                                   Pl cornu c D172 8.j 12 F4R KC 2 6MARCH07 AMBIG
                                                                                                                                                                                        Pl pul m D197 9.j 12 2FR 11 F2e R DNtdSTR 09 MAR07 CH
                                                       Eryn gi i Europ e 625                                                                                                            Pl pul m D197 9.j 13 0FR 12 F4R DNtd STR 09MA R07 CH
      group                                            Eryn gi i Europ e 625 b
                                                       D1 832.1 15 6FR cDNA eryn gi i
                                                                                                                                                                        Pl pul m Q C2 KC1FRgr DNtd STR MAR0 7
                                                                                                                                                    Pl cf.p ul m D2 070 1 2F KC 26 MARCH0 7
                                                                                                                                                    Pl pul mon D2 070 KC DNtd STR 12 F 26March07
                                                                                                                                                     Pl pul m D352 1 26FR 1 2F4 R DNtdSTR 09 MAR07 CH
                                                       D1 832.6 15 7FR cDNA                                                                        Pl pul m D174 5 11F2eR KC DNtdS TR 09MA R07 CH
                                                       P.eryn gi i marke t 16 7FR cDNA                                                             Pl pul mon D2 245 .1 9 K C DNtdSTR 1 1F2e R 26MARCH07
                                                                                                                                                   Pl pul mon D2 245 .1 9 K C DNtdSTR 1 2F4F 2 6MARCH07
                                                       D1 820 17 3F ge nDNA e ryngi i                                                              Pl pul m saj D182 5 19FR DNtdSTR11F2 eR 31 JAN0 7 CH
                                                       D1 820 ge nD EF1 PL MARCH                                                                   Pl pul m saj D182 5 20FR DNd str12F4R 31 JAN0 7 CH
                                                                                                                                                    Pl pul m saj D182 5 39FR NOTtdCL 1 1F2e R 31JAN07 CH
                                                       D1 822 17 4FR g enDNA e ryng ii                                                             Pl pul m saj D182 5 40FR NOTtdCL 1 1F2e R 31JAN07 CH
                                                                                                                                                   Pl pul m saj D182 5 41FR NOTtdCL 1 2F4R 3 1JA N07 CH
                                                       D1 822 ge nD EF1 PL MARCH                                                                   Pl pul m saj D182 5 42FR NOTtdCL 1 2F4R 3 1JA N07 CH
                                                                         Po pul in us US 2 212                                                     Pl pul m saj D182 5 43FR NOTtdCL 1 2F4R 3 1JA N07 CH
                                                                                                                                                    Pl pul m saj D182 5 44FR NPTtdCL 1 2F4 R 3 1JA N07 CH
                                                                         Po pul in us US 2 212 b                                                   Pl pul m saj D194 4 21FR DNtdSTR 1 2F4R 3 1JA N07 CH
                                                                                                                                                   Pl pul m D198 0.3 12F4 R KC DNtdST R 1 4FE B07
                                                                         Po pul in us Mo ntana 22 14                                               Pl pul m D480 5 6FR DNtdS TR 12F4R 09FEB07
                                                                            Po pul in us On ta ri o 23 04                                          Pl pul m D174 5 12F4R KC DNtdST R 0 9MAR0 7 CH
                                                                                                                                                   Pl pul m Q C3 KC2FRgr DNtdSTR MAR07
                                                                         Po pul in us Ma ryla nd 39 9                                                      D1 932.1 12 F KC 26MA RCH0 7
                                                                                                                 Pl eryn foss D182 0 57FR DNtdSTR 1 2F4R 0 9FEB 07 CH
                                                                          Po pul in us Ma ryla nd 39 9b          Pl ostrD54 0 12FR DNtd STR 12 F4R 31 JAN07 CH
                                                                         Po pul in us Mo ntana 76 5              Pl ostrD56 4 13FR DNtd STR 12 F4R 31 JAN07 CH
                                                                                                                 Pl cornu c D173 1 17FR 12F4R DNtd 31JAN0 7 CH
                                                                       D2 230.1 16 4FR cDNA                      Pl cornu c D173 2 93FR 12F4R DNtdSTR 1 9FEB 07 CH
                                                                                                                 Pl cornu c D173 7 94FR 12F4R DNtdSTR 1 9FEB 07 CH
                                              Pl genD 5 26Fdi dno two rk 165 7R MARCH                             Pl cornu c D173 8 95FR 12F4R DNtdSTR 1 9FEB 07 CH
                                              Pl genD E F1PL 1 567 R MA RCH                                      Pl pul mon D1 824 9 6FR 12 F4R DNtd STR 19FEB07 CH
                                                                                                                 Pl pul m D197 9.4 98FR 1 2F4 R DNtdSTR 19 FEB0 7 CH
                                           D3 32 147 FR cDNA                                                     Pl pul m D352 9 2FR 12 F4R DNtd STR 19FEB97 CH
                                                                                                                 Pl pul m D197 9.2 22FR DNtd STR 12 F4R 31 JAN07 CH
                                           D5 47 149 FR cDNA                                                     Pl pul m D207 0 58FR DNtdS TR 12F4R 09FEB07
                                           D5 64 150 FR cDNA                                                     Pl purpur D22 49.4 101 FR 12 F4R DNtdS TR 19FEB07 CH
                                           D2 61.4 145 FR cDNA
                             D1 737 16 8F cDNA cornu co p
                                                                                                                 Pl purpur D22 49.4 90FR 11F2eR DNtdS TR 19FEB07 CH
                                                                                                                 Pl purpur D22 49.4 134 FR 12 F4R DNtdS TR 09MA R07 CH
                                                                                                                 Pl purpurD2 249 .4 1 23FR 1 1F2 eR DNtdST R 0 9MAR0 7 CH
                                                                                                                 Pl purpurD2 342 .1 1 2F4R M cDNA 30MARCH07
                                                                                                                                                                                                      Pleurotus
                                                                                                                 Pl purp D22 46.1 1FR 1 2F4 R 11JAN07 CH
                             D1 738 15 4FR cDNA cornu cop
                                 Hyps1 031 1 52FR cDNA ZYGO BLAS T             P. conucopiae                     Pl purp D22 46.1 KC1 T1 FR DNtdS TR 11F2eR 31 JAN07 CH
                                                                                                                 Pl purp D22 46.1 KC1 T5 FR DNtdS TR 11F2eR 31 JAN07 CH
                                                                                                                 Pl purp D22 46.1 KC2 T5 FR DNtdS TR 12F4R 31JAN0 7 CH
                                                                                                                                                                                                      ostreatus
                                pu lmo nari us D2 070 .1 16 0FR cDNA                                             Pl purp D22 46.1 KC2 T1 FR DNtdS TR 11F2eR 31 JAN07 CH
D2 246.1 16 3FR cDNA                                                                                             Pl purp D22 49.1A 11 F2e R KC 31JAN07 CH
                                                                                                                 Pl purp D22 49.1A 12 F4R KC 3 1JA N07 CH                                             group
Pl purpurD2 342 .1 EF1 M cDNA 52 6F 15 67RMA RCH0 7
  5 changes                                                     P. purpureoolivaceus                             Pl purp D22 49.1B 12 F4R KC 3 1JA N07 CH
                                                                                                                 Pl sp D2 145 .3 10 0FR 1 2F4 R DNtd STR 19 FEB0 7 CH
                                                                                                                 Pl purpurD2 342 .1 1 1F2e R M cDNA 30 MARCH0 7
                                                                                                                 1 change
Barcoding mushrooms with COI
conclusions

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

   • direct PCR: short regions only, although Long-PCR possible

   • RT-PCR: feasible, including for sporocarps
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
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
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.
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 Barcode specimens

								
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