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Phylogenetic Analysis of Mitochondrial Genomes of Filarial Nematodes in the Subfamily Waltonellinae

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Phylogenetic Analysis of Mitochondrial Genomes of Filarial Nematodes in the Subfamily Waltonellinae University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2015 Phylogenetic Analysis Of Mitochondrial Genomes Of Filarial Nematodes In The ubfS amily Waltonellinae Sarina Marie Bauer Follow this and additional works at: https://commons.und.edu/theses Recommended Citation Bauer, Sarina Marie, "Phylogenetic Analysis Of Mitochondrial Genomes Of Filarial Nematodes In The ubfaS mily Waltonellinae" (2015). Theses and Dissertations. 1741. https://commons.und.edu/theses/1741 This Thesis is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. PHYLOGENETIC ANALYSIS OF MITOCHONDRIAL GENOMES OF FILARIAL NEMATODES IN THE SUBFAMILY WALTONELLINAE by Sarina Marie Bauer Bachelor of Science, University of North Dakota, 2011 A Thesis Submitted to the Graduate Faculty of the University of North Dakota in partial fulfillment of the requirements for the degree of Master of Science Grand Forks, North Dakota August 2015 Copyright 2015 Sarina Bauer ii iii PERMISSION Title Phylogenetic Analysis of Mitochondrial Genomes of Filarial Nematodes in the Subfamily Waltonellinae Department Biology Degree Master of Science In presenting this thesis in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my thesis work or, in his absence, by the chairperson of the department or the dean of the Graduate School. It is understood that any copying or publication or other use of this thesis or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my thesis. Name: Sarina Bauer Date: June 23, 2015 iv TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................................... vii LIST OF TABLES ........................................................................................................................... x ACKNOWLEDGMENTS .............................................................................................................. xi ABSTRACT ................................................................................................................................... xii CHAPTER I. INTRODUCTION .............................................................................................................. 1 II. MATERIALS AND METHODS ........................................................................................ 7 Parasite materials and DNA isolation .................................................................... 7 Primer design ......................................................................................................... 7 PCR amplification and sequencing ...................................................................... 11 Assembly and annotation of mitochondrial genomes .......................................... 12 Phylogenetic analysis ........................................................................................... 13 Wolbachia detection…………………...…………………………..………………………..14 III. RESULTS ......................................................................................................................... 17 Genome Features and Organization ..................................................................... 17 Protein-coding genes ............................................................................................ 19 v Ribosomal and transfer RNA genes ..................................................................... 21 AT-rich region ..................................................................................................... 22 Phylogenetic analysis ........................................................................................... 22 Absence of Wolbachia……………………………………………………………………..27 IV. DISCUSSION ................................................................................................................... 28 Conclusions……………………………………………………………………...…………….33 APPENDIX .................................................................................................................................... 34 REFERENCES .............................................................................................................................. 50 vi LIST OF FIGURES Figure Page 1. Filarioid life cycle depicting the transmission from definitive host (vertebrate) to vector (arthropod). Modified from rowdy.msudenver.edu. ............................... 2 2. Phylogeny based on the 12 protein coding genes of the mitochondrial (mtDNA) genome sequences of nine species of filarioids. Percentages of Bayesian posterior probabilities are displayed at nodes. Black circles indicate Wolbachia-dependence while white circles indicate Wolbachia- independence17. ....................................................................................................... 5 3. Gene map of the mitochondrial DNA for Foleyellides n. sp. Protein-coding genes are shown in red with the arrows indicating direction of the coding strand. rRNA genes are shown in blue and tRNA gene are shown in green. The hypothesized positions of two tRNA genes are shown in black. The AT rich region is shown in yellow (near position 6,500). The purple rectangle (near position 11,650) represents a small 62 bp insertion not present in other species. .................................................................................................................. 19 4. Phylogeny based on mitochondrial cytochrome oxidase subunit I (Cox1) sequences. Numbers at specific nodes indicate the percentages of Bayesian posterior probabilities when ≥ 70%. Scale bar indicates branch length. (-ln likelihood score = 9407.25743) ............................................................................ 23 5. Phylogeny based on mitochondrial 12s rRNA gene sequences. Numbers at specific nodes indicate the percentages of Bayesian posterior probabilities when ≥ 70%. Scale bar indicates branch length. (-ln likelihood score = 6250.45146) .......................................................................................................... 25 6. Phylogeny based on concatenated sequences of the 12 protein-coding mitochondrial genes. Numbers at specific nodes indicate the percentages of Bayesian posterior probabilities when ≥ 70%. Scale bar indicates branch length. (-ln likelihood score = 62816.12747) ............................................ 26 7. Gel electrophoresis results from a polymerase chain reaction (PCR) using primers to detect the presence of the groEL gene of Wolbachia sp. Foleyellides n. sp. (A), Foleyellides flexicauda (B), and the negative control (-) all show negative results. The positive control (+) (Brugia pahangi adult worm) shows the optimal band size for a positive detection vii of the gene. The 100 bp ladder is shown on the left (L). ..................................... 27 8. Phylogeny based on mitochondrial encoded NADH dehydrogenase 2 gene (ND2)…………………………………………………………….……..………..35 9. Phylogeny based on mitochondrial encoded NADH dehydrogenase 4 gene (ND4)…………………………………………………………………..….……..36 10. Phylogeny based on mitochondrial encoded cytochrome c oxidase I gene (Cox1)…..…….……………………………………………………..………..….37 11. Phylogeny based on mitochondrial encoded NADH dehydrogenase 6 gene (ND6)…………………………………………………………..……..……..…...38 12. Phylogeny based on mitochondrial encoded cytochrome b gene (CytB)………….……………………………………………………..…..….…..39 13. Phylogeny based on mitochondrial encoded cytochrome c oxidase III gene (Cox3)....………………………………………………………….…..…..….…..40 14. Phylogeny based on mitochondrial encoded NADH dehydrogenase subunit gene (NDL4).………………………………………....……………..……...…....41 15. Phylogeny based on mitochondrial encoded NADH dehydrogenase 1 gene (ND1)………………………………………………………………..….………..42 16. Phylogeny based on mitochondrial encoded ATP synthase 6 gene (ATP6)…….……………………………………………………………………..43 17. Phylogeny based on mitochondrial encoded cytochrome c oxidase II gene (Cox2)……………………………………………………………..………….….44 18. Phylogeny based on mitochondrial encoded NADH dehydrogenase 3 gene (ND3)…………………………………………………………………..…….…..45 19. Phylogeny based on mitochondrial encoded NADH dehydrogenase 5 gene (ND5)…………………………………………………………………..…….…..46 20. Maximum parsimony heuristic search in PAUP based on mitochondrial cytochrome oxidase subunit I (Cox1) sequences. Numbers at specific nodes indicate the percentages of bootstrap support (>75%). Consensus of 5 trees produced (length: 1748 steps; consistency index: 0.2946; retention index:0.3575)………….……..........……..…………………………………..…..47 21. Maximum parsimony heuristic search in PAUP based on mitochondrial 12s rRNA gene sequences. Numbers at specific nodes indicate the percentages viii of bootstrap support (>75%). Consensus of 62 trees produced (length: 1139 steps; consistency index: 0.4056; retention index:0.4871)…………….…..48 22. Maximum parsimony heuristic search in PAUP based on concatenated
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