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Plant DNA Barcoding using  matK some work on new primer sets Dr. Alan Forrest Prof. Pete Hollingsworth Royal Botanic Garden Edinburgh Damon Little,  New York Botanic Garden Aron Fazekas,  University of Guelph Gao Lian-Ming,  Kunming Institute of Botany Sean Graham,  University of British Columbia Mehrdad Hajibabaei,  CCDB, University of Guelph Maria Kuzmina,  CCDB, University of Guelph Hollingsworth, Graham, Little (2011). "Choosing and using a plant DNA barcode." PLoSONE  6: e19254.
 
Angiosperms:   matK  baseline How good are the current “best”  matK  primers? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:   3 approaches to improve  matK  retrieval  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],matK  primer location
Angiosperms:   the test sample ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:  testing different protocols ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:   PCR  results from different primer pairs ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:   2-step  matK   PCR  amplification ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:   2-step protocol results:  xF+MALPR1 & 472F+1248R ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:   recommended work flow Dilute DNA 1:10 1 st  ROUND: all samples PCR  matK  primers xF+MALPR1 1M betaine, 0.2M trehalose, Platinum  Taq Clean successful PCR products Sequence clean PCR products 0.2M trehalose Acquire samples and extract DNA 2 nd  ROUND: all PCR and SEQ failures 3F+1R or 472F+1248R 1M betaine, 0.2M trehalose, Platinum  Taq PCR and SEQUENCE  rbcL Clean successful PCR products Sequence clean PCR products 0.2M trehalose >95%  matK  sequence success rate ALL poor quality sequences/mononucleotide motifs PCR and sequence  matK  primers xF+ERIR 1M betaine, 0.2M trehalose, Phusion  Taq
Angiosperms:   recommendations and protocols ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Angiosperms:   matK  barcode summary ,[object Object],[object Object]
 
The Guardian, 17 th  November, 2007
Gymnosperms:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Gymnosperms:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Ferns & allies:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Ferns & allies:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Liverworts:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Liverworts:   matK  barcodes
Mosses:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Mosses:   matK  barcodes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
2-step protocol =  >95% 2-step protocol =  >95% 2-step protocol =  ca. 80%  Polypodiales 1-step protocol =  100%  Cyatheales Lycophyte and early-diverging lineage primers require testing 1-step protocol =  >80% 3-step protocol =  >80% Further primer optimization required 2-step protocol =  ca. 90% Further primer optimization required
Acknowledgements ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Suppliers of data and samples: Olivier Maurin, Michelle van der Bank ACCB, University of Johannesburg Harald Schneider Natural History Museum, London Dietmar Quandt, Susann Wicke Nees Institute, University of Bonn Fay Wei Li, ChunNeng Wang, other National Taiwan University Paul Wolf Utah State University Juan Carlos Villareal University of Conneticut

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Peter Hollingsworth - Plants Plenary

  • 1. Plant DNA Barcoding using matK some work on new primer sets Dr. Alan Forrest Prof. Pete Hollingsworth Royal Botanic Garden Edinburgh Damon Little, New York Botanic Garden Aron Fazekas, University of Guelph Gao Lian-Ming, Kunming Institute of Botany Sean Graham, University of British Columbia Mehrdad Hajibabaei, CCDB, University of Guelph Maria Kuzmina, CCDB, University of Guelph Hollingsworth, Graham, Little (2011). "Choosing and using a plant DNA barcode." PLoSONE 6: e19254.
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  • 10. Angiosperms: recommended work flow Dilute DNA 1:10 1 st ROUND: all samples PCR matK primers xF+MALPR1 1M betaine, 0.2M trehalose, Platinum Taq Clean successful PCR products Sequence clean PCR products 0.2M trehalose Acquire samples and extract DNA 2 nd ROUND: all PCR and SEQ failures 3F+1R or 472F+1248R 1M betaine, 0.2M trehalose, Platinum Taq PCR and SEQUENCE rbcL Clean successful PCR products Sequence clean PCR products 0.2M trehalose >95% matK sequence success rate ALL poor quality sequences/mononucleotide motifs PCR and sequence matK primers xF+ERIR 1M betaine, 0.2M trehalose, Phusion Taq
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  • 14. The Guardian, 17 th November, 2007
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  • 23. 2-step protocol = >95% 2-step protocol = >95% 2-step protocol = ca. 80% Polypodiales 1-step protocol = 100% Cyatheales Lycophyte and early-diverging lineage primers require testing 1-step protocol = >80% 3-step protocol = >80% Further primer optimization required 2-step protocol = ca. 90% Further primer optimization required
  • 24.

Editor's Notes

  1. Success with 2 pass, with contamenents treated as fail Success with 2 pass, with contaminents treated as fail + 7 recovered with phusion Success with 2 pass, with contaminents treated missing data + 7 recovered with phusion
  2. If anyone asks about ERIR primer, it is same as MALPR1 and comparable results, but with higher annealing temp better suited to use with Phusion Taq
  3. If anyone asks about ERIR primer, it is same as MALPR1 and comparable results, but with higher annealing temp better suited to use with Phusion Taq
  4. Add gmno species number
  5. Compress to 6/8 failures failed in rbcl
  6. Number of species in those lineages