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Development of Genetic Tools to Detect Three New Zealand Indigenous Freshwater Mussels in Environmental DNA

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http://researchcommons.waikato.ac.nz/ Research Commons at the University of Waikato Copyright Statement: The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). The thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: Any use you make of these documents or images must be for research or private study purposes only, and you may not make them available to any other person. Authors control the copyright of their thesis. You will recognise the author’s right to be identified as the author of the thesis, and due acknowledgement will be made to the author where appropriate. You will obtain the author’s permission before publishing any material from the thesis. Development of genetic tools to detect three New Zealand indigenous freshwater mussels in environmental DNA A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Biological Science at The University of Waikato by Xiaoxue Hu 2017 II Abstract As “ecological engineers”, freshwater mussels are key components in the freshwater ecosystem. However, surveying mussels can be challenging since mussel populations may be difficult to locate. Conventional methods for identifying mussel species are also problematic and require expertise since mussels are often morphologically cryptic. The same problems apply to New Zealand indigenous freshwater mussels. New Zealand had long been considered to have two indigenous species, Echyridella menziesii and Echyridella aucklandica, until a recent revision based on molecular evidence recognised a new species, Echyridella onekaka, which was previously mis-identified as Echyridella menziesii. Results from molecular studies have placed all three freshwater mussel species under a single genus, Echyridella, specific to New Zealand. For conservation and ecosystem management, a fast and reliable method for surveying and identifying New Zealand mussels is required. This research aims to develop molecular tools that utilise environmental DNA (eDNA) collected from freshwater environments to detect mussel species. These tools target a widely used DNA marker, cytochrome c oxidase subunit I (COI), for species identification. The COI targets in eDNA samples are detected by digital polymerase chain reaction (dPCR) using genus-specific primers and species-specific TaqMan probes. Three species-specific assays were developed, followed by assessment of III specificity and sensitivity. Assay for E. aucklandica showed high specificity and tolerance to inhibition from high levels of non-target DNA. A trial of duplexing simultaneously assays detecting E. onekaka and E. aucklandica also showed promising results. With further assessment of assays using in vivo and in situ environmental samples in the future, these techniques hold great promise for being a rapid and cost-effective method for surveying and identifying New Zealand freshwater mussels. Additionally, to increase detection rate when target concentration in eDNA is extremely low, known mitochondrial sequences for all three species were extended through PCR and de novo sequencing to allow future manufacturing of a customised commercial target DNA enrichment kit (MYbaits). This work is necessary since known mitochondrial genomic sequences for these three species are too short to generate effective MYbaits oligos. Consensus primers were designed based on available complete mitochondrial genomes of closely related mussel species. Most consensus primers successfully amplified in PCR, and about 1700bp have been sequenced so far. Once the sequencing of extended region is completed, a MYbaits kit will be manufactured for future research. IV Acknowledgements First, I would like to express my gratitude to Dr Charles K. Lee for his great guidance and patience. I must be a terrible student but you never give up encouraging me. Thanks also to Professor Craig Cary for sharing your knowledge and understanding about research. The heated discussion between you and Charles gave me a lesson about what’s the real passion of science. Also, a special thank you goes out to Roanna Richards-Babbage, Lynne Parker, Judith Burrows and all the technicians for helping me along throughout my lab work. I would also like to thank John Longmore for the help of DNA sequencing. I would like to thank Georgia Wakerley for all your help with my research and in general. I would also like to thank Caitlin Lowe, Jay Wiseman, Maria Monteiro, Rachelle Anderson, Shelly Brandt and all the past and present lab mates in TRU for the encouraging words and comradery you've given me over the last year. Thanks also to Mark Hamer from the Waikato Regional Council, Sue Clearwater from NIWA for providing pressures mussel specimens. This project cannot go on without your help. Huge thanks go out to my father Ronggui, I’d never have made it this far without you. Thank you for all the love and support throughout my study. Thank you also goes out to my friends and family for allowing me to express my feelings and V asking about my projects even when you knew you’d never have any idea what I was saying. VI Table of contents ABSTRACT ..............................................................................................................................................III ACKNOWLEDGEMENTS .......................................................................................................................... V TABLE OF CONTENTS ........................................................................................................................... VII LIST OF FIGURES ................................................................................................................................... XI LIST OF TABLES .................................................................................................................................... XV CHAPTER I INTRODUCTION .............................................................................................................. 17 CHAPTER II LITERATURE REVIEW ................................................................................................. 25 II-1 FRESHWATER MUSSELS .................................................................................................................. 25 II-1-1 Introduction .................................................................................................................... 25 II-1-2 Special physiology of freshwater mussels....................................................................... 26 II-1-3 Ecological importance of freshwater mussels ................................................................ 29 II-1-4 New Zealand indigenous freshwater mussels ................................................................ 31 II-1-5 Taxonomy of New Zealand freshwater mussels and its revision .................................... 33 II-1-6 Difficulties in studying freshwater mussels .................................................................... 35 II-2 SPECIES DETECTION USING ENVIRONMENTAL DNA .............................................................................. 37 II-2-1 Introduction .................................................................................................................... 37 II-2-2 Advantages of eDNA in species detection ...................................................................... 39 II-2-3 Marker Genes ................................................................................................................. 40 VII II-2-4 Common techniques for species identification ............................................................... 41 II-2-5 Difficulties of PCR-based species detection .................................................................... 45 II-2-6 qPCR in species detection ............................................................................................... 47 II-2-7 Advantages of dPCR in eDNA study ................................................................................ 49 REFERENCE ................................................................................................................................................ 55 CHAPTER III DESIGN AND VALIDATION OF SPECIES-SPECIFIC DPCR ASSAY .................................... 67 III-1 INTRODUCTION ............................................................................................................................. 67 III-2 MATERIAL AND METHODS ............................................................................................................... 74 III-2-1 Mussel samples............................................................................................................... 74 III-2-2 DNA extraction ............................................................................................................... 74 III-2-3 PCR .................................................................................................................................. 75 III-2-4 Gel electrophoresis ......................................................................................................... 76 III-2-5 Primer design .................................................................................................................. 76 III-2-6 Probe design ................................................................................................................... 77 III-2-7 Digital PCR .....................................................................................................................
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