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

Introduction

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 re-gion- Deliver high quality sequence data- Ward(2005) -Use 2 forward and 2 reverse primers (4 combina-tions)-=> complex

2 solution1. 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)

primer design1. 159 fish mitogenome2. align: Bioedit3. anlayzing: CODEHOP4. M13 Tagging

Materials and methods

mixtureSequencing primer

DNA quality test

PCR amplification and sequencing1. 94 SPECIES, 2 negative controls( Myxini, Cephalaspidomorphi, Holocephali, Elasmobranchii,

Actinopterygii)2. 16S rRNA gene: positive controls for DNA extraction

Materials and methods

A

B

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D

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Results

Goniistius zonatus

Acipenser fulvescens

Non-specific band

Amplifying target region:96.8%Average sequencing successCOI-3: 95.2%COI-2: 93%COI-1: 86.0%Average scores and read lengthsCOI-3: 41,631bpCOI-2: 39,645bpCOI-1: 38,608bp

COI-3: Myxini, holocephalid amplifyCOI-2 & COI-3: all 93 species

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

ÞAmplify the barcode region form most taxaÞCOI-2 cocktail(for mammals): performs very well for fishÞCOI-3 cocktail(for fishes): performs very well for other taxaÞAmplified the barcode for > 3000 species

Discussion

Amplification

* 16S primers: a simple test for DNA quality as a positive con-trolÞ COI and 16S fail: DNA degradation or the presence of PCR in-hibitors

*Adding a new primer to the existing cocktailsÞ same M13 tag and same ampliconÞ considering very large number of complete mitogenomeÞ Employing degenerate or inosine-containing primers to over-come 3’ end mismatches

-> increase the chance of co-amplifying other gene regions or NUMTs (rarely problematic in fishes)

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 readsMore 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