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Fish and Chips microarray-based DNA-barcoding of
European Marine Fishes Kochzius M1, Antoniou
A2, Botla S1, Campo Falgueras D3, Garcia Vazquez
E3, Hauschild J1, Hervet C4, Hjörleifsdottir S5,
Hreggvidsson G5, Kappel K1, Landi M6, Magoulas
A2, Marteinsson V5, Nölte M7, Planes S4, Seidel
C1, Silkenbeumer N1, Tinti F6, Turan C8,
Venugopal MN9, Weber H1, Blohm D1 1Centre for
Applied Gene Sensor Technology (CAG), University
of Bremen, Germany 2Institute of Marine Biology
and Genetics, Hellenic Centre for Marine
Research, Greece 3Universidad de Oviedo,
Spain 4Université de Perpignan, France 5Prokaria,
Iceland 6University of Bologna, Italy 7Zentrum
für Technomathematik (ZeTeM), University of
Bremen, Germany 8Mustafa Kemal University,
Turkey 9College of Fisheries, Mangalore, India
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Environmental problems, biodiversity, and
ecosystem functioning in European Seas

• Biodiversity and ecosystems of European Seas are
  under anthropogenic induced pressure, such as
  pollution, eutrophication, coastal construction,
  and fishery overexploitation
• Compared to terrestrial ecosystems very little is
  known about marine biodiversity and changes in
  species richness and ecosystem function
• This is mainly due to sampling difficulties and
  problems in taxonomy
• There are only few scientific specialists for
  several groups of marine organisms, including
  phytoplankton, invertebrates, as well as eggs and
  larvae of fishes

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DNA chips as a new tool for marine species
identification in biodiversity and ecosystem
research

• DNA-based identification methods are established
  as powerful tools and the following marine
  animals have been investigated (1) eggs,
  larvae and adults of fishes (2) planktonic
  copepods (3) invertebrate larvae
  (4) prey in gut content or faeces of penguins,
  whales, and fishes
• most of these methods allow to handle only single
  or a few species at the same time
• DNA microarrays are believed to have the
  potential of identifying hundreds of species in
  parallel and to differentiate them against an
  even larger number of related species.

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The principle of DNA chips (microarrays)
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Probes
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T
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G
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Surface of the DNA Chip
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The principle of DNA chips (microarrays)
Microarray (glass slide) hundreds of spots with
probes
Spots of probes with different signal intensities
after scanning with a fluorescence scanner
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Sampling, sequencing, and probe design

• Sampling all European Seas
• Sequencing of partial mtDNA genes
• COI 532 sequences, 66 species
• cyt b 434 sequences, 41 species
• 16S 479 sequences, 79 species

• Probe design
• COI 455 bp, 470 sequences, 47 species
• cyt b 404 bp, 281 Sequences, 43 species
• 16S 418-452 bp, 404 sequences, 46 species
• In silico testing against several hundred
  background sequences from sequence data bases

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Probe design (www.miconet.uni-bremen.de)

• Considered parameters in the design of
  oligonucleotide libraries
• Standard features, such as length, Tm, GC content
• Sensitivity and specificity
• Secondary structure of the DNA capture-molecules
• Avoidance of cross hybridisation
• Hybridisation efficiency
• Secondary structure of target molecules

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Layout of the microarry
COI (150)
Block
cyt b (123)
Array
16S (46)
Microarray
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Hybridisation experiments
Psetta maxima (16S)
Psetta maxima (COI)
Psetta maxima (cyt b)
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DNA microarray experiments experiments
Targets
16S (34)
COI (42)
cyt b (42)
16S (46)
Signals
true positive
COI (150)
Probes
missing true positive
cyt b (123)
false positive
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DNA microarray experiments

• 16S strong true-positive signals only a few
  false-positive signals
• COI strong true-positive signals many
  false-positive signals
• cyt b weak true-positive signals, many missing
  false-positive signals

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DNA microarray experiments (16S)
Kochzius et al., submitted
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Conclusions

• A single marker system seems to be not sufficient
  to design oligonucleotide probes for DNA
  microarrays
• Therefore, it is recommended to utilise several
  markers for the genetic identification of fishes
  with DNA microarrays
• Nevertheless, identification of fishes is
  possible with DNA microarrays, but probes have to
  be tested intensively in hybridisation experiments

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Srujana Chitipothu et al. (poster) Towards
microarray-based DNA-barcoding of marine
invertebrates
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Acknowledgement The Fish Chips project is a
Specific Targeted Research Project (STREP) funded
by the European Commission under the contract no.
505491
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Thank you very much for your attention!