Development of new genome-informed genotyping tools for Aphanomyces astaci Submitted by Diana Minardi to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Sciences In May 2017 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature: ………………………………………………………….. 1 Acknowledgements I would like to sincerely thank my supervisors Dr Mark van der Giezen, Dr David Studholme, and Dr Birgit Oidtmann for their invaluable guidance and support during these 3 years (plus a bit) of PhD, both practically and in the writing of the thesis. I would like to thank Cefas and the University of Exeter for funding the project and for giving me the opportunity to embrace this challenge and adventure. I would like to thank all of the University of Exeter “Biocat” lab and office, with whom I’ve spent most of my coffee time. But especially Mirella (for the laughs and cries) and Simone (for the sweaty runs around campus). I would also like to thank Chiara for her constant support, encouragement, and beer. Finally, I would like to thank Tony, for his patience, and my family, for their “overseas” support: I’ve finally finished “the shrimp” book! 2 Abstract Aphanomyces spp. are water moulds, eukaryotic fungus-like organisms, belonging to the class Oomycota. This genus contains primary pathogens of plants and animals as well as opportunistic and saprotrophic species. One of the animal parasites (A. astaci) is the causal agent of the crayfish plague, a disease listed by the World Organisation for Animal Health (OIE). It is believed that A. astaci was first introduced into Italy from the US in the late 19th century and rapidly spread in Europe causing the decline of native crayfish. It currently threatens to wipe out the UK native white-clawed crayfish (Austropotamobius pallipes). Random amplified polymorphic DNA (RAPD-PCR) on pure isolates of A. astaci distinguished five genotypes (A, B, C, D, and E). This distinction proved to be a useful tool for epidemiological studies aimed at understanding the history and spread of the disease in Europe; furthermore, there are differences in virulence among genotypes. No discriminatory morphological or physiological characters are available and widely used markers such as the internal transcribed spacer (ITS), the divergent domains regions (D1-D2) of nuclear large subunit (LSU) rDNA, and cytochrome c oxidase subunit I (COI) also fail to discriminate between A. astaci genotypes. There are some practical drawbacks to genotype by the currently available genotyping methods. Whole- genome sequencing (WGS) was used to catalogue DNA single nucleotide variants and genotype-unique genomic regions that could be exploited as phylogenetic markers. These newly developed molecular markers were tested both on pure cultures and historical samples derived from outbreaks and carrier crayfish available in our laboratories, validating these genotyping methods, which represent new diagnostic tools aiding the detection and prevention of crayfish plague. 3 List of Contents Acknowledgements ............................................................................................ 2 Abstract .............................................................................................................. 3 List of Contents .................................................................................................. 4 List of Tables .................................................................................................... 10 List of Figures ................................................................................................... 12 List of Abbreviations ......................................................................................... 16 1. Introduction ............................................................................................... 19 Aphanomyces, an overview ................................................................ 19 1.1.1 Life cycle of Aphanomyces species .............................................. 21 1.1.2 Taxonomy of the genus Aphanomyces and isolation difficulties ... 24 Aphanomyces astaci and crayfish plague ........................................... 25 1.2.1 Epidemiological summary of crayfish plague ................................ 25 1.2.2 Economic and environmental impact of crayfish plague ............... 26 1.2.3 Infection and symptoms of the crayfish plague ............................. 29 1.2.4 Diagnosis of crayfish plague ......................................................... 30 1.2.5 Aphanomyces astaci genotypes ................................................... 31 Genotyping techniques for Aphanomyces astaci ................................ 34 1.3.1 RAPD-PCR ................................................................................... 34 1.3.2 AFLP ............................................................................................. 34 1.3.3 Chitinase genes ............................................................................ 35 1.3.4 Microsatellites ............................................................................... 35 Next generation sequencing ................................................................ 37 Aims of the PhD project ....................................................................... 39 2. Materials and Methods .............................................................................. 40 Materials .............................................................................................. 40 2.1.1 Cultured Aphanomyces isolates ................................................... 40 4 2.1.2 Primers ......................................................................................... 40 2.1.3 Cloning PCR products in Escherichia coli ..................................... 44 2.1.4 Media and solutions for Aphanomyces cultures ........................... 44 2.1.5 Media and solutions for bacterial cultures ..................................... 46 2.1.6 Solutions for DNA work ................................................................. 47 2.1.7 Oomycetes used for specificity test .............................................. 49 2.1.8 Cefas historical samples and Italian outbreak samples used to validate the genotyping methods ............................................................... 49 Microbiological methods ...................................................................... 50 2.2.1 Aphanomyces routine culturing method ........................................ 50 2.2.2 Preparation of LB-ampicillin broth and plates ............................... 51 2.2.3 Transformation of E. coli competent cells with plasmid DNA ........ 51 2.2.4 Storage of bacterial clones ........................................................... 51 Molecular biology methods .................................................................. 52 2.3.1 Preparation of agarose gels and gel electrophoresis .................... 52 2.3.2 Isolation and quantification of Aphanomyces genomic DNA ......... 52 2.3.3 Whole-genome sequencing of Aphanomyces isolates ................. 53 2.3.4 PCR protocols for amplifications of targeted regions .................... 53 2.3.5 Purification of PCR reactions ........................................................ 61 2.3.6 Cloning into pGEM-T Easy (Promega) and pJET1.2 (Thermo Scientific) vectors ...................................................................................... 61 2.3.7 Colony screening and plasmid extraction ..................................... 62 2.3.8 Sequencing of purified PCR products and cloned PCR products . 62 2.3.9 Enzymatic digestion of PCR products for genotyping A. astaci .... 62 Bioinformatics methods ....................................................................... 63 2.4.1 Cloned sequences analysis and alignments ................................. 63 2.4.2 Phylogenetic analysis of cloned sequences ................................. 64 2.4.3 Quality control of genome-wide sequencing data ......................... 64 5 2.4.4 De novo assembly of Aphanomyces genomes ............................. 65 2.4.5 Quality checks on assemblies ...................................................... 67 2.4.6 Aphanomyces astaci mitochondrial DNA assembly ...................... 68 2.4.7 BWA alignments and SAMtools .................................................... 68 2.4.8 Detection of single nucleotide variants (SNVs) ............................. 69 2.4.9 Selection of SNVs associated with restriction sites ....................... 70 2.4.10 Identification of sequences unique to a subset of genomes ...... 70 2.4.11 Construction of SNV-based phylogenetic networks ................... 71 2.4.12 Aphanomyces astaci genotypes comparisons using dnadiff ..... 71 3. Results ...................................................................................................... 73 RAPD-PCR profiles for Aphanomyces astaci isolates, including A. astaci isolate 457, which displays an atypical RAPD-PCR profile ................. 73 3.1.1 Aims and objectives ...................................................................... 73 3.1.2 Materials and Methods ................................................................