Transcript Seifertetal.Barcoding.IMC9

Promoting fungi
in the DNA barcoding movement
Keith A. Seifert1 , Ursula Eberhardt2 , Conrad L. Schoch3
1Biodiversity,
2 CBS
3 National
Agriculture & Agri-Food Canada, Ottawa, Ontario
Fungal Diversity Centre, Utrecht, the Netherlands
Centre for Biotechnology Information, Bethesda, MD (GenBank)
MSA symposium 2010
Politics!
1:00 Advances in DNA barcoding for fungi. Conrad L. Schoch, Keith A. Seifert
Specific Genes
1:30 DNA barcoding using cytochrome c oxidase I (COI): A valuable addition to oomycete
molecular taxonomy. Gregg Robideau et al.
2:00 Barcoding the Yeasts – Which Genes? Cletus P. Kurtzman
2:30 Coffee Break
Applications
3:00 Barcoding the Dothideomycetes and Sordariomycetes. Andy N. Miller
3:30 Barcoding agaric fungi in the Great Smoky Mountains National Park: What have we
learned? Karen W. Hughes et al.
4:00 Challenges and successes in ITS barcoding of fungal communities in Alaskan boreal
forest soil. L. Taylor
4:30 Discussion
Aftermath
A chat over beer
IMC Symposium U5 – Fungal Barcoding
Chairs: Ursula Eberhardt & Keith Seifert
Promoting fungi in the DNA barcoding movement KA Seifert*, U Eberhardt, CL Schoch
Practice towards DNA barcoding of the nectriaceous fungi P. Zhao, J. Luo, W.-Y. Zhuang*
DNA Barcoding of the mycobiota in indoor environments R A Samson*
The ITS region as barcoding for medical fungi W Meyer*, C Serena, S Chen, M Arabatzis, A
Velegraki
DNA barcoding of arbuscular mycorrhizal fungi (Glomeromycota)
A. Schuessler*, H.
Stockinger, M. Krueger
DNA barcoding of fungal endophytes from a Eucalyptus grandis tree in South Africa
K Pillay, M Gryzenhout*, B Slippers, MJ Wingfield
QBOL - Barcoding organisms of quarantine importance to Europe
Quaedvlieg, P.J.M. Bonants, N. Boonham, P.W. Crous
J.Z. Groenewald*, W.
Overview
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The politics of DNA barcoding.
What happened to cox1?
Should ITS be the official Fungal Barcode?
Data standards for the barcode keyword.
Data resources for DNA barcoding.
International barcoding projects
Our goal: Motivate mycological participation in DNA barcoding!
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Formalize the fungal barcode.
Establish Fungal Working Group
Develop new fungal barcoding projects.
Participate in multi-kingdom projects.
“DNA, you know, is Midas’ gold. Everybody who touches it goes mad.”
Maurice Wilkins, quoted in The Eighth Day of Creation
What is DNA barcoding?
Pre-2002 (or whenever)
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2.
Trichocladium asperum
Humicola grisea
Pileus comex to comparmulate, lemellae ascending-adnote, peleipellis of clonate cells,
chilocystidia ventruiore to eubryludinal, basidiospores with pronviant apical gum pore
.……………………………………………………………………………………….Panaeolus mallochii
Pileus glalions to finely primrose, lemellae dark blocked umpher, peleipellis hymenform,
cheilocystidia a continuous timule margin, bandospins conspeniously compressed
………………………………………………………………………………….. Panaeolus summerbellii
Post-2002 (or whenever)
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2.
CATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAT
CGAATAAACTGGGTGGGTTGTTGCTGTCCCTCTCGGGGGAACTGTGCACGCCTTACCTTTTT
TGTTTTTCCACCTGTGAA ………………………………………………………Panaeolus mallochii
CATTATTGAATAAACTTGGTTAGGTTGCTGCTGGCTCCTTGGAGCATGTGCACACCTAGCACC
NTTTTTACCACCTGTGCACCCTTTGTAGACCTGGATACCTCTCGAGGAAACTCGGTTTGAGGA
C ………………………………………………………………………………. Panaeolus summerbellii
OR
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…………………………………..……………………………………………………Panaeolus mallochii
….………………………………………………………………………………. Panaeolus summerbellii
Consortium for the Barcode of Life (CBOL)
• Regulates the ‘barcode’ keyword for GenBank
• Organization
– Secretariat
• Smithsonian Institution, Washington, D.C., USA
• 5 people
www.barcodeoflife.org
– Executive Committee
• 7 members, including Pedro Crous
– Implementation Board
• 19 members, including Conrad Schoch
– Members
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200 Member Organizations, 50 countries
Natural history museums, biodiversity organizations
Users: e.g., government agencies
Private sector biotech companies, database providers
• Facilitates barcoding but does not fund research
The Barcode Keyword in GenBank
The Barcode Data Standard
• Sequence records with…
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Voucher specimens
On-line metadata
Reliable standard of taxonomic identification
Agreed gene region
• sanctioned by peer review by CBOL Implementation Board
– Sequence traces
• Identification of unknowns of ALL kingdoms (except bacteria)
– GenBank
– Barcode of Life Database (BOLD)
What is DNA Barcoding? Part 2.
• All Kingdoms, All Species, One gene (or two or three genes?)
• The purpose is identification … NOT phylogeny
• In animals, the barcode gene is cytochrome oxidase 1 (cox1 or CO1)
• A single copy mitochondrial gene, AT rich
• 648 bp region is the core for animals
• In plants, the barcode genes are matK and rbcL
– Hollingsworth et al., 2009, PNAS 106: 12794–12797, 2009.
• In fungi, the barcode gene has not been formalized
– By default it is cox1 until an alternative is accepted
Barcode
What is DNA Barcoding? Part 3.
• Big science
– More than $100,000,000
• Biodiversity Institute of Ontario
(Guelph)
• Netherlands Centre for Biodiversity
Naturalis (Leiden) – € 30 million
• High throughput sequencing
– Near genome centre volumes
– Immediate data release policy
(based on public genome model) is
controversial
– Primer mixes often used for PCR
– M13 tagged sequencing primers
– Data release papers
What is DNA Barcoding? Part 4.
• Taxon specific ‘campaigns’
• Development of multikingdom databases
– For use by all scientists and the public…
not just mycologists
• Multi-kingdom projects
– QBOL – Quarantine Barcode of Life
• Ecological projects
– IM-Bol Indoor Mycota Barcode of Life
• Geographic Projects
– Moorea
www.barcodingbirds.org
www.lepbarcoding.org
www.fishbol.org
mooreabiocode.org
Barcode of Life Database – BOLD
www.boldsystems.org
Mirrored in China, negotiations with ECBol, Australia, Mexico
•Accepts ITS sequences as barcodes
•Submission open to anyone
•Few fungal sequences
•Spreadsheets for metadata
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No specific fungal format
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Process can be tedious
•Images of specimens
•Descriptions
•‘Automated’ GenBank submission
Barcode Submission Tool
www.ncbi.nlm.nih.gov/WebSub/index.cgi?tool=barcode
•Familiar submission process
•Trace archive available
www.ncbi.nlm.nih.gov/Traces/home/
•ITS sequences not yet accepted as barcodes
•Submission open to anyone
•Few fungal barcode sequences are now cox1
•Metadata must be stored off-site
RefSeq Targeted Loci
www.ncbi.nlm.nih.gov/RefSeq/
GenBank
RefSeq
Not curated
Curated
Author submits
NCBI creates from existing data
Only author can revise
NCBI revises as new data emerge
Multiple records for same loci common
Single records for each molecule of major
organisms
Records can contradict each other
Data exchanged among INSDC members
Exclusive NCBI database
Akin to primary literature
Akin to review articles
• Bacteria: all type sequences
• Fungi: 200 sequences from AFToL, 28S, 18S, just beginning
INSD
submission
(International Nucleotide
Sequence Databases)
DDBJ
EMBL
GenBank
selection &
some curation
(NCBI)
model organisms
reference organisms
genomic level
RefSeq
16S rRNA*
other molecular markers
other RNAs
RefSeq Accession #
Project number
Additional references
RefSeq references
Expanded qualifiers
Criticisms of DNA Barcoding
• Not science (or bad science)
• A threat to classical taxonomy
• Removes funding from
classical taxonomy
• Standardization of markers
impossible
• Oversimplifies delimiting
species
• It is not phylogeny
• Distrust and questioning of
CBOL’s mandate
• National pride
• Disciplinary pride
www.ibolproject.org
iBOL Member Nations
Each central node should
have a high-throughput
DNA barcoding facility
Scientific Steering Committee (mycologists): Pedro Crous, Keith Seifert
Now Canada US France Poland
ECBOL- European Consortium for the Barcode of Life
Mission: To unleash the potential of European expertise and collections to
contribute towards identifying life on earth.
Plan:
Establish a Network of European Leading Laboratories (NELL)
Formalise National DNA barcoding campaigns in each European country
Establish new projects and barcoding campaigns,
Functioning as the European node for international initiatives such as iBOL
Participating countries: Belgium, Finland, France, Germany, Italy, Lithuania,
Netherlands, Norway, Poland, Portugal, Spain, Switzerland, U.K.
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www.ecbol.org
ECBOL2 meeting
June 2010, Braga, Portugal
Lorenzo Lombard
[email protected]
Selecting barcode genes
• International Nucleotide Sequence Database Collaboration
– INSDC, GenBank, the European Molecular Biology Laboratory and
the DNA Data Bank of Japan
– CBOL Implementation Board decides by peer review which gene
regions receive BARCODE status
• Possible reasons for rejection of cox1
– PCR problems (amplification or primer problems)
– Patterns of inter- and infraspecific variation
– Resolving power
• Selection of new genes
– Patterns of inter- and infraspecific variation
– Prefer a ‘barcode’ gap
– Universality of primer pair etc.
between species
within species
Percent of strains
Comparison of barcodes in Oomycetes (250 species)
ITS
cox1
LSU
n= 1179
n=1179
n=375
Min
Mean
Max
0
0.297
0.539
Min
Mean
Max
0
0.102
0.307
Min
Mean
Max
0
0.098
0.201
Min
Mean
Max
0
0.004
0.062
Min
Mean
Max
0
0.004
0.037
Min
Mean
Max
0
0.002
0.037
Average pairwise distance
Courtesy Gregg Robideau & André Lévesque
0 introns
1 intron
2 introns
3 introns
7 introns
cox1 Barcoding of
Eumycota & Oomycota
Species resolution
in Penicillium
cox1
B-tub
ITS
67.1 %
81.2 %
24.5 %
Fusarium. Low resolution.
Multiple copies.
F. oxysporum genome 1
F. circinatum DAOM235753
F. sacchari DAOM 235795
F. verticillioides genome **
F. circinatum DAOM 235752 b1
F. circinatum DAOM 235752 b5
F. graminearum DAOM 235624
F. graminearum DAOM 235800
F. oxysporum genome 2
3 spp./3 major
clades. Identical
barcodes.
All Fungi Barcode of Life Planning Workshop
Smithsonian Conservation and Research Center
Front Royal, Virginia
13-15 May 2007
Zasmidium nocoxi Crous
Rossman, AY. 2007. Report of the Planning Workshop for All Fungi DNA Barcoding. Inoculum 58(6): 1-5.
The ITS reality… Hurray!
• Large reference database
– most not barcode data standard compliant
• Robust primers
• Strong demand from many mycologists that this be the
fungal barcode
– Especially ecologists studying environmental metagenomics
The ITS Reality – BOO!
• Multiple copies within species
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At this conference…
Dan Lindner – Laetiporus, this conference
Uwe Simon & M. Weiss – Ascomycetes
Of concern for cloning based and metagenomic studies
wanting to use barcode ID databases
• Serious lack of resolution in Ascomycetes
– Possibly too short
– 500-700 bp optimum barcode but subtract 150 bp 5.8S…
• Chimera problems for some methodological approaches
The Ascomycete Barcode Problem
Dikarya
– TEF1-α
– RPB1 or RPB2
– Mcm7
Schmitt et al. 2009. Persoonia 23: 35-40.
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– FG1565
– MS204 [email protected]
– FG1093
V. Robert @ CBS/ C. Lewis @ AAFC
• Ascomycete Barcode
Working Subgroup?
Courtesy J. Spatafora
Fungi
RPB1
RPB2
TEF
RPB1
RPB2
TEF
per net
• ITS (and cox1) lack resolution in
many groups
• A second barcode is needed
Action
Establish Fungal DNA Barcoding Working Group
• Chair: Conrad Schoch
• International Membership
• 10-15 members
• Establish Ascomycete subgroup (or other subgroups?)
Establish Working Plan
• Prepare Fungal Barcoding proposal
• For peer reviewed publication
• For CBOL implementation board approval
• CBOL will support meetings of WG as necessary
Possible approaches
• Volunteers needed for Fungal Working Group
• Collaborators to provide data
• Mine data from existing publications
• Collaborators to provide DNA
• Multiple strains per species
• Multiple species per genus
• Authorship open to all who contribute
• AFTOL model
• Sequencing of other markers
• part of main proposal
• or as separate activity
• CBOL agreement with Life Tech
CONTACT:
Conrad Schoch:
[email protected]
Fungal DNA Barcoding – Proposed Proposal
• General Barcode for Fungi
– rDNA internal transcribed spacer (ITS)
• Secondary barcodes
– Yeasts
• rDNA large subunit (28S, LSU) D2 region
– Ascomycetes
• Second barcode required, subsequent proposal
• Oomycetes
– A separate proposal
– Data on ITS, cox1 and LSU now being prepared for publication
– 2 barcode system
• rDNA internal transcribed space (ITS)
• rDNA large subunit (28S, LSU) D2 region
• Other groups?
CBOL Implementation Board
Peer review of barcode marker proposals
Chairs
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Robert Hanner, University of Guelph, Databases
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Peter Hollingsworth, Royal Botanic Gardens Edinburgh, Plant WG
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Line Le Gall, Muséum National d’Histoire Naturelle, Paris; Protist WG
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Neil Sarkar, Marine Biol. Lab., Woods Hole, MA; Data analysis WG
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Conrad Schoch, NCBI, GenBank Taxonomy, Fungal WG
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Lee Weigt, Smithsonian Inst., Washington, DC; Leading Lab Network
CBOL Campaign and Project Leaders
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George Amato, American Museum of Natural History, Conservation
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Damon Little, NY Botanical Garden, TreeBOL
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Marc De Meyer, Royal Mus. Central Africa, Belgium; Tephritid Barcoding Initiative
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Yvonne Linton, NHM, London; Mosquito Barcoding Initiative
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Dirk Steinke, University of Guelph, MarBOL
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Mark Stoeckle, Rockefeller University, ABBI
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Pablo Luis Tubaro, Museum of Natural Sciences, Argentina, ABBI
Liaisons for Associated Initiatives
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Paul De Barro, CSIRO Agriculture Australia, invasive species
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Scott Federhen, NCBI, GenBank Taxonomy
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Paul Hebert, University of Guelph, iBOL
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Daniel Masiga, ICIPE, Nairobi, Kenya, East Africa
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Chris Meyer, Smithsonian Institution, Washington, Moorea/BioCode
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Sujeevan Ratnasingham, Biodiversity Institute of Ontario, BOLD
Taxonomic group
# spp.
Volunteers for data
Pezizomycotina
60 000
Saccharomycotina
1000
Taphrinomycotina
150
Agaricomycotina
21 000
Ustilaginomycotina
1700
Pucciniomycotina
8300
Glomeromycotina
170
Mucoromycotina
325
Kickxellomycotina
265
Zoopagomycotina
190
Entomophthoromycotina
275
Blastocladiomycotina
180
Chytridiomycotina
700
Neocallimastigomycotina
20
Microsporidia
Rozella
1300
22
Websites
www.barcodeoflife.org
www.ibolproject.org
Thanks for images:
Pedro Crous
André Lévesque & Gregg Robideau
Joey Spatafora
Keith Seifert: [email protected]
Ursula Eberhardt: [email protected]
Conrad Schoch: [email protected]
www.boldsystems.org
www.qbol.org
www.ecbol.org
www.lepbarcoding.org
www.barcodingbirds.org
www.fishbol.org