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Species Identification, Regulatory Agencies and DNA Barcoding David E. Schindel, Executive Secretary National Museum of Natural History Smithsonian Institution [email protected]; http://www.barcoding.si.edu 202/633-0812; fax 202/633-2938 Genomics Subgenomics Current Systematic Studies Microbes - 16S Plants - RBCL Animals - COI Species Identification Matters Basic research on evolution, ecology Endangered/protected species Agricultural pests/beneficial species Disease vectors/pathogens Invasive species (e.g., in ballast water) Environmental quality indicators Managing for sustainable harvesting Consumer protection, ensuring food quality Fidelity of seedbanks, culture collections 5 The Practice of Taxonomy Distributions of Character Variation Characters Specimens Taxonomists Taxonomic DecisionMaking The Uses of Taxonomy Socioeconomic Decisions Concerns/ Regulations Specimens Taxonomic Processes Formal Taxa Taxon Concepts Characters Specimens Growth of Biodiversity Databases Voucher Specimen Biodiversity Heritage Library Journal Publication Museum databases of associated data Species Name Authority files of taxonomic names A DNA barcode is a short gene sequence taken from standardized portions of the genome, used to identify species An Internal ID System for All Animals The Mitochondrial Genome DNA D-Loop Small ribosomal RNA Cytochrome b ND1 ND6 Typical Animal Cell COI ND5 mtDNA ND2 L-strand H-strand ND4 ND4L ND3 Mitochondrion COII COIII ATPase subunit 8 ATPase subunit 6 Non-COI regions for other taxa Land plants: – Chloroplast matK and rbcL approved Nov 09 – Non-coding plastid and nuclear regions being explored Fungi and protists: – CBOL Working Groups convened – Recommendations expected in 2010 How Barcoding Works First, build a barcode reference library: – Well-identified specimen – Tissue subsample – DNA extraction, PCR amplification – DNA sequencing – Data submission to GenBank Second, use it to identify unknowns: – Any unidentified juvenile, adult, fragment, product – Tissue sample, DNA, sequencing – Comparison with sequences in reference library Associating Life Stages, Processed Parts, Dimorphic Genders How Barcoding is Done From specimen to sequence to species C NO DI 3I I Collecting Voucher Specimen DNA extraction CO1 gene N N D 1D 2 DNA sequencing Trace file Database of Barcode Records GenBank, EMBL, and DDBJ Global, Open Access to Barcode Data http://www.insdc.org/ NBII, 25 February 2009 Current Norm: High throughput Large labs, hundreds of samples per day Large capacity PCR and sequencing reactions ABI 3100 capillary automated sequencer Emerging Norm: Table-top Labs Faster, more portable: Hundreds of samples per hour Integrated DNA microchips Table-top microfluidic systems Producing Barcode Data: 201? Barcode data anywhere, instantly Data in seconds to minutes Pennies per sample Link to reference database A taxonomic GPS Usable by nonspecialists Barcode of Life Community Networks, Projects, Organizations • Promote barcoding as a global standard • Build participation • Working Groups • BARCODE standard • International Conferences • Increase production of public BARCODE records Consortium for the Barcode of Life (CBOL) Established May 2004 with Sloan Foundation grant Secretariat opens at Smithsonian, September 2004 Now in its third two-year funding period Workshops, Working Groups, networking, representation/marketing Now an international affiliation of 200+ members in 50+ countries: – Natural history museums, biodiversity organizations – Users: e.g., government agencies – Private sector biotech companies, database providers CBOL Member Organizations: 2009 • 200+ Member organizations, 50 countries • 35+ Member organizations from 20+ developing countries BARCODE Records in INSDC Specimen Metadata Georeference Habitat Character sets Images Behavior Other genes Other Databases Phylogenetic Pop’n Genetics Ecological Voucher Specimen Barcode Sequence Trace files Primers Literature (link to content or citation) Species Name Indices - Catalogue of Life - GBIF/ECAT Nomenclators - Zoo Record - IPNI - NameBank Publication links - New species Databases - Provisional sp. Outreach Activities Cape Town, South Africa, April 2006, SANBI – Scale insects in African agriculture Nairobi, Kenya, October 2006 – Commercial fisheries in Rift Valley lakes Brazil, March 2007 – Hardwood tree species – Endangered mammals, reptiles, amphibians Taiwan, September 2007 Nigeria, October 2008 Beijing, May 2009 India, March 2010 CBOL’s Global Projects Fish Barcode of Life (FISH-BOL) – 30,000 marine/freshwater species by 2010 All Birds Barcoding Initiative (ABBI) – 10,000 species by 2010 Tephritid fruit flies – 2,000 pest/beneficial species and relatives by 2008 Mosquitoes – 3,300 species by 2008 Endangered species Trees of the world Adoption by Regulators Food and Drug Administration – Reference barcodes for commercial fish NOAA/NMFS – $100K for Gulf of Maine pilot project – FISH-BOL workshop with agencies, Taipei, Sept 2007 Federal Aviation Administration – $500K for birds Environmental Protection Agency – $250K pilot test, water quality bioassessment FAO International Plant Protection Commission – Proposal for Diagnostic Protocols for fruit flies CITES, National Agencies, Conservation NGOs – International Steering Committee, identifying pilot projects Why barcodes are short Low cost and simplicity – Single capillary reads adequate – Avoid post-sequencing IT needed for pyroseq Adequate for taxonomy – Miminizes uniformative sequence length – Limiting supply rate of identified specimens – Technology more accessible to small labs Regulatory/inspection applications easier Barcoding and Metagenomics Lots of interest for ecology, less for regulation Pyrosequencing of environmental mixtures – Construct species list from mixture – Predator-prey relations from gut contents, feces – Food-web reconstruction Relies on high accuracy (some species differ by only a few percent) Not cost-effective for single specimens What barcode providers have and are producing 770,000 records from ~100,000 species – Agricultural pests – Water quality indicators – Disease vectors – Endangered species – Commercial species of food, feed, commodities 5 million records from 500K species by 2015 International Barcode of Life Project (iBOL) Theme 1 – DNA Barcode Library WG 1.1 Vertebrates WG 1.2 Land Plants WG 1.3 Fungi WG 1.4 Human Pathogens and Zoonoses WG 1.5 Agricultural and Forestry Pest and Their Parasitoids WG 1.6 Pollinators WG 1.7 Freshwater Bio-Surveillance WG 1.8 Marine Bio-Surveillance WG 1.9 Terrestrial Bio-Surveillance WG 1.10 Polar Life What barcode providers want High PCR and sequencing success rates Bigger window into older, compromised samples Better software integration to eliminate bottlenecks Smaller labs/developing countries: – Lower equipment and maintenance costs – Simplification for techs with less training – Install anywhere without lab renovations – Willing to accept slower throughput What barcode users want Answers to specific questions: – Is this thing on this list of species or not? – Is this thing a member of this genus/family? – Which of the species on this list is this thing? – What species is this thing? Production-scale capabilities: – Hundreds to thousands of installations – Lower but constant throughput – Rapid turnaround – The right price-point and limited life cycle costs What barcode users would do with the reference libraries Inspection stations at every port and international airport for: – Agricultural pest control – Illegal trade in endangered species – Violations of trade quotas Regular Federal and State water quality surveys Federal, State and local food inspection Public health monitoring and diagnoses GenBank, EMBL, and DDBJ Global, Open Access to Barcode Data http://www.insdc.org/ NBII, 25 February 2009 Linkout from GenBank to BOLD NBII, 25 February 2009 Linkout from GenBank to Taxonomy NBII, 25 February 2009 Link from GenBank to Museums NBII, 25 February 2009