THE PARTIAL MITOCHONDRIAL DNA AND PHYLOGENETIC RELATIONSHIPS OF THE ICELAND SCALLOP ( Chlamys islandica ) by Victoria Northrup Thesis submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours in Biology Acadia University April, 2008 © Copyright by Victoria Northrup, 2008 ii This thesis by Victoria Northrup is accepted in its present form by the Department of Biology as satisfying the thesis requirements for the degree of Bachelor of Science with Honours Approved by the Thesis Supervisor/ Head of the Department __________________________ ____________________ Dr. Marlene Snyder Date Approved by the Honours Committee __________________________ ____________________ Dr. Anna Redden Date __________________________ ____________________ Dr. Glenys Gibson Date __________________________ ____________________ Dr. Liza Duizer Date iii I, Victoria Northrup, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I however, retain the copyright in my thesis. _________________________________ Signature of Author _________________________________ Date iv Acknowledgments I would like to thank Duncan Bates and Dr. Mike Dadswell for supplying the scallops used in this study. Thank you to the Howard Gould Trust microbial genome sequencing initiative for funding this research. I would like to acknowledge Dr. Marlene Snyder for her input on this study. v Table of Contents Page Title page . i Approval page . ii Permission for duplication page. iii Acknowledgements . iv Table of contents . v List of Tables . vii List of Figures . viii Abstract . x Chapter 1: Introduction . 1 1.1 Chlamys islandica life history and taxonomy . 1 1.2 Mitochondria . 5 1.3 Mitochondrial Genome . 8 1.4 Secondary Structure and Compensatory Mutation . 10 1.5 Purpose of study . 13 Chapter 2: Sequence Analysis . .13 1 Introduction . 13 2 Material and Methods . 15 2.1 Sample collection . 15 2.2 DNA isolation . .15 2.3 Gene amplification . 16 2.3.1 Primers . .16 2.3.2 Polymerase chain reaction . 17 2.3.3 Gel electrophoresis, band visualization and excision . .19 2.3.4 Isolation of DNA from agarose gel . .19 2.3.5 DNA sequencing . 20 2.3.6 Sequence comparison . 21 2.3.7 Amino acid sequence . 21 2.4 Whole genome amplification . 21 2.4.1 Primer design . .22 2.4.2 Long accurate polymerase chain reaction . 22 2.4.3 Gel electrophoresis, band visualization and excision . 23 2.4.4 Isolation of DNA from agarose gel . .23 2.4.5 DNA sequencing . .23 2.4.6 Sequence comparison . 23 3 Results and Discussion . 24 3.1 Gene amplification . .24 3.2 Amplification of long accurate PCR fragments . .42 Chapter 3: Phylogenetic Analysis . 46 1 Introduction . .46 2 Material and Methods . .49 2.1 Sequence analysis . 49 2.2 Phylogenies . 50 2.2.1 Obtaining the sequences . .50 2.2.2 Creating phylogenies . 50 vi 3 Results and Discussion . 55 Chapter 4: Conclusions. 73 Chapter 5: References . 75 Appendix 1 . .82 Appendix 2 . 83 vii List of Tables Page Table 1. Primers used in the amplification of mitochondrial genes for PCR . 17 Table 2. The optimal PCR conditions for each gene . 18 Table 3. PCR cycling parameters for each gene . 19 Table 4. Primers used in successful LAPCR . 22 Table 5. The GenBank accession numbers and references for the 39 scallops and the outgroup of donkey’s foot ( Spondylus gaederopus ) . 51 Table 6. The GenBank accession numbers, classification and references for 13 bivalves with complete mitochondrial genomes sequenced and a Polyplacophora that is used as an outgroup for the phylogenies . 55 Appendix 1 Table 7. All primers used in the study, their target and primer sequence . 82 Appendix 2 Table 8. The GenBank accession numbers for genes sequenced in this study . .83 viii List of Figures Page Figure 1. 16S amplification . 25 Figure 2. CytB and 12S amplifications . 26 Figure 3. COIb amplification . 27 Figure 4. COIa amplification . 28 Figure 5. Nad1 amplification . 29 Figure 6. Nad3 amplification . 30 Figure 7. Nad4 amplification . 31 Figure 8. Atp6 amplification . 32 Figure 9. The edited partial sequence for the 16S (lrRNA) of 196 nucleotides . 32 Figure 10. The edited partial sequence for the 12S (srRNA) of 412 nucleotides . 33 Figure 11. The edited partial sequence of COIb of 234 nucleotides . 34 Figure 12. The edited partial sequence of COIa of 242 nucleotides . 34 Figure 13. The edited partial sequence of the CytB gene of 287 nucleotides . 35 Figure 14. The edited partial sequence of the Nad1 gene of 223 nucleotides . 35 Figure 15. The partial edited sequence of the Nad3 gene of 167 nucleotides . 36 Figure 16. The edited partial sequence of the Nad4 gene of 340 nucleotides . 36 Figure 17. The edited partial sequence of the Atp6 gene of 286 nucleotides . 37 Figure 18. The hypothetical amino acid sequence for COIb gene . 38 Figure 19. The hypothetical amino acid sequence for COIa gene . 39 Figure 20. The hypothetical amino acid sequence for CytB gene . 39 Figure 21. The hypothetical amino acid sequence for Nad1 gene . 39 Figure 22. The hypothetical amino acid sequence for Nad3 gene . 40 Figure 23. The hypothetical amino acid sequence for Nad4 gene . 41 Figure 24. The hypothetical amino acid sequence for Atp6 gene . 41 Figure 25. 12S-Nad1 amplification . 43 Figure 26. The edited sequence for the fragment 12S-Nad1 of 352 nucleotides . 44 Figure 27. Putative secondary structure for the tRNA for serine . 45 Figure 28. Phylogenetic relationship of.