1. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Fish and Chips: microarray-based DNA- barcoding of European Marine Fishes Kochzius M 1, Antoniou A 2, Botla S 1, Campo Falgueras D 3, Garcia Vazquez E 3, Hauschild J 1, Hervet C 4, Hjörleifsdottir S 5, Hreggvidsson G 5, Kappel K 1, Landi M 6, Magoulas A 2, Marteinsson V 5, Nölte M 7, Planes S 4, Seidel C 1, Silkenbeumer N 1, Tinti F 6, Turan C 8, Venugopal MN 9, Weber H 1, Blohm D 1 1 Centre for Applied Gene Sensor Technology (CAG), University of Bremen, Germany 2 Institute of Marine Biology and Genetics, Hellenic Centre for Marine Research, Greece 3 Universidad de Oviedo, Spain 4 Université de Perpignan, France 5 Prokaria, Iceland 6 University of Bologna, Italy 7 Zentrum für Technomathematik (ZeTeM), University of Bremen, Germany 8 Mustafa Kemal University, Turkey 9 College of Fisheries, Mangalore, India

2. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de 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

3. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de 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.

4. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de The principle of DNA chips (microarrays) C A T G T C A T G T C A T G T G A G T A G A G T A G A G T A A G C T G A G C T G A G C T G Surface of the DNA Chip C A T G A T A C A T C G A C Probes C A T G A T A C A T C G A C

5. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de 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

6. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de 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

7. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Probe design (www.miconet.uni-bremen.de) Considered parameters in the design of oligonucleotide libraries: Standard features, such as length, T m, GC content Sensitivity and specificity Secondary structure of the DNA capture- molecules Avoidance of cross hybridisation Hybridisation efficiency Secondary structure of target molecules

8. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Layout of the microarry 16S (46) COI (150) cyt b (123) Block Array Microarray

9. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Hybridisation experiments Psetta maxima (16S)Psetta maxima (COI)Psetta maxima (cyt b)

10. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de COI (150) Probes cyt b (123) 16S (46) Targets 16S (34)COI (42)cyt b (42) DNA microarray experiments experiments true positive Signals missing true positive false positive

11. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de 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

12. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de DNA microarray experiments (16S) Kochzius et al., submitted

13. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de 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

14. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Srujana Chitipothu et al. (poster) Towards microarray- based DNA-barcoding of marine invertebrates

15. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Acknowledgement The “Fish & Chips” project is a Specific Targeted Research Project (STREP) funded by the European Commission under the contract no. 505491

16. Towards DNA chip technology as a standard analytical tool for the identification of marine organisms in biodiversity and ecosystem research www.fish-and-chips.uni-bremen.de Thank you very much for your attention!