Fish Nearly half of all vertebrates species 15700: marine, 13700 freshwater species (Fish base)

2. Introduction • Fish • Nearly half of all vertebrates species • 15700: marine, 13700 freshwater species (Fish base) • FISH-BOL(Fish Barcode of Life Initiative) • Establish a reference library of DNA barcodes for all fish species • fast, accurate, cost-effective system for molecular identification • Facilitating species identification • Flagging potentially previously unrecognized species • Enabling identifications where traditional methods are not applicable • (immature stages or body fragments) • Provide powerful tool for enhanced understanding • of the natural history and ecological interactions

3. High-quality sequence records • 10 specimens per species: more than 0.5 million specimen • Ward(2005): proof-of-principle study • 200 species of Australian marine fishes • 5000 barcodes from over 2000 species • Effort • Efficient polymerase chain reaction of barcode region • Deliver high quality sequence data • Ward(2005) • Use 2 forward and 2 reverse primers (4 combinations) • => complex

4. 2 solution 1. A single primer set with degenerate sites (COI-1) 2. A Cocktail whose component primers are tailed with M13 (COI-2, COI-3) to facilitate high throughput sequencing => Test their effectiveness on a single species(94 different fish families)

5. Materials and methods primer design 1. 159 fish mitogenome 2. align: Bioedit 3. anlayzing: CODEHOP 4. M13 Tagging

6. DNA quality test Sequencing primer mixture

7. Materials and methods PCR amplification and sequencing 94 SPECIES, 2 negative controls ( Myxini, Cephalaspidomorphi, Holocephali, Elasmobranchii, Actinopterygii) 2. 16S rRNA gene: positive controls for DNA extraction

8. Results A B C D E F G H Acipenserfulvescens Goniistiuszonatus Non-specific band

9. Amplifying target region: 96.8% Average sequencing success COI-3: 95.2% COI-2: 93% COI-1: 86.0% Average scores and read lengths COI-3: 41,631bp COI-2: 39,645bp COI-1: 38,608bp COI-3: Myxini, holocephalid amplify COI-2 & COI-3: all 93 species

10. Discussion 5’ region of the COI gene: the basis for a DNA barcoding system => The availability of primers aiding from a broad range taxa => Enough variation=> Require multiple primer combination • Primer sets in this study • Amplify the barcode region form most taxa • COI-2 cocktail(for mammals): performs very well for fish • COI-3 cocktail(for fishes): performs very well for other taxa • Amplified the barcode for > 3000 species

11. Discussion • Amplification • * 16S primers: a simple test for DNA quality as a positive control • COI and 16S fail: DNA degradation or the presence of PCR inhibitors • *Adding a new primer to the existing cocktails • same M13 tag and same amplicon • considering very large number of complete mitogenome • Employing degenerate or inosine-containing primers to overcome 3’ end mismatches • -> increase the chance of co-amplifying other gene regions or NUMTs (rarely problematic in fishes)

12. Discussion • Sequencing • Some reads : obscured with background signal (5’-terminus) • COI-2, COI-3 read averaging 30bp longer than COI-1(untailed) • -> conventional primers: prone to form dimers • -> without clean up, incorporated into sequencing (5’ end) • *M13 tags: more overlap in the bidirectional reads • More overlap: more reliable and longer sequence records in the reference database, important for automating steps • Summary • Primer cocktails: high effective in generating amplicons for • diverse fish taxa and other groups of vertebrates