Catalogue Introduction ..................................................................................................................... 1 RBGE work on Phytophthora ..................................................................... 1 Overview of Oomycetes .............................................................................................. 1 Molecular Evidence of the Evolution of Oomycetes .................................. 3 Taxonomy within Oomycetes ..................................................................... 3 Species discovery ........................................................................................................ 7 Taxonomy of Phytophthora ........................................................................ 7 Morphological taxonomy review of Phytophthora ............................. 7 Molecular taxonomy and phylogeny of Phytophthora ....................... 8 DNA Barcoding analysis........................................................................... 10 Internal transcribed spacer (ITS) ...................................................... 10 Mitochondrial DNA .......................................................................... 12 Aim and Purpose ........................................................................................................... 19 Material and Methods.................................................................................................... 20 Polymorphism Evaluation of ITS, Cox2 and Cox spacer as DNA Barcodes ............ 20 Sequence editing, alignment and analysis ................................................ 21 Identification of Phytophthora Species in RBGE ...................................................... 21 Culture of Isolates ..................................................................................... 24 Sub-culturing..................................................................................... 25 Single-spore Isolation ....................................................................... 25 DNA extraction ......................................................................................... 25 PCR recipe and program ........................................................................... 26 Cox spacer ......................................................................................... 26 ITS..................................................................................................... 26 Purification ................................................................................................ 27 Sequencing Reaction ................................................................................. 27 Sequence editing, alignment and analysis ................................................ 28 Results ........................................................................................................................... 28 PCR recipe performance ............................................................................................ 28 Barcode Evaluation .................................................................................................... 30 Intraspecific and Interspecific Distance Calculation ................................ 30 Best Match and Best Close Match ............................................................ 35 Species Identification with ITS and Cox spacer ........................................................ 37 Discussion ..................................................................................................................... 40 Intra- and Interspecific Variabilities .......................................................................... 40 Barcode Evaluation, Consistency and Incongruence of Different Testing Methods..................................................................................................... 40 Species identification using different DNA barcode ................................................. 43 Conclusion ..................................................................................................................... 45 Acknowledgements ....................................................................................................... 46 Reference ....................................................................................................................... 46 Evaluation and Application of Different DNA Barcodes for Phytophthora spp. Xueyang Huang Oct. 2018 Thesis submitted in partial fulfilment for the MSc in the Biodiversity and Taxonomy of Plants. Abstract Phytophthora is a fungal genus of Oomycetes with historical and economical importance. As an expanding genus with more than 150 species currently, accuracy of species identification is necessary for diagnosis, disease control and ecology research. Therefore, the discrimination power, usability and consistency are highly required for DNA barcodes. The purpose of the project is to evaluate ITS1, ITS2, ITS and Cox 2, Cox spacer as well as their combination based on the distribution of intra- and interspecific variation and the test on identification success. By building the phylogenetic tree using sequences with species identity and those from unknown samples collected in RBGE, the identity of these unknown samples may be settled. The probability density plot, comparison of mean variation and BM/BCM test was carried out and suggested the insufficiency of using a single locus to identify species, the incongruence of different evaluating methods was revealed. The identity of unknown samples was determined with Neighbor-joining and Parsimony tree based on ITS and Cox spacer. Incongruence of sequence identity and clustering was revealed in trees based on two barcodes, which also indicated that unreliable results of barcoding could be carried with a single locus. List of Figures and Tables Figure1. Schematic illustration of flagellar apparatus in many Oomycetes…………2 Figure2. The transitional zone found in most Oomycetes…………………………...3 Figure3. Phylograms and distance histograms for ITS and COI…………………….5 Figure4. Parsimony supertree of 37 oomycete species and 6 SAR species………….6 Figure5. Matrix spreadsheet in Lucid Builder……………………………………….8 Figure6. DNA sequence search query in Lucid Builder……………………………..8 Figure7. Phylogenetic relationships for Phytophthora isolates……………………...9 Figure8. Phylogram of 47 Phytophthora taxa and 1 Peronospora species………….11 Figure9. Parsimony tree based on the Cox1 for Phytophthora species……………..13 Figure10. Maximum parsimony tree of Phytophthora species using Cox1………...14 Figure11. Maximum parsimony tree of Phytophthora species using Cox2………...17 Figure12. Phylogenetic of Pythium and Phytophthora based on partial Cox2……..18 Figure13. Diagram of ITS and Cox spacer regions in Phytophthora……………...27 Figure14. Gel electropherograms for the amplification of Cox spacer regions…….29 Figure15. Gel electropherograms for the gradient amplification of Cox spacer……29 Figure16. Gel electropherograms for the amplification of ITS……………………..29 Figure17. Probability density distribution of the intra- and interspecific variation...31 Figure18. Mean intraspecific and interspecific variation…………………………...32 Figure19. Percentage of identification success……………………………………..36 Figure20. Comparison of ITS and Cox spacer phylograms by clade………………38 Figure21. Position of P. gonapodydes in NJ tree built with ITS and Cox spacer…..42 Figure22. Comparison of the Parsimony tree built with ITS and Cox spacer……...44 Table1. Comparison of the Parsimony tree built with ITS and Cox spacer………...21 Table2. DNA regions and PCR primers…………………………………………….27 Table3. Original PCR recipe tested adjustment on forward primer.………………..28 Table4. Original PCR program based tested Annealing time and temperature…….29 Table5. Minimum, Maximum, Mean and Std of intra/interspecific variation……...33 Table6. Mean intraspecific distance and the rank of variation of different species...35 Table7. Identification success percentage of barcodes……………………………..36 Table8. Tree-based identification of samples collected from RBGE……………….40 Evaluation and Application of Different DNA Barcodes for Phytophthora spp. Introduction As one of the most important plant pathogens, the genus Phytophthora has had profound impacts on horticulture, agriculture, forestry and conservation. Since the first description in the late 1800s by Heinrich Anton de Bary, the number of species known in the genus has doubled during the past decade due to extensive surveys in previously unexplored ecosystems and made Phytophthora a genus with high-speed expansion, containing more than 150 species for now (Yang et al. 2017), costing billions of economic lost each year. RBGE work on Phytophthora Royal Botanic Garden Edinburgh has contributed to taxonomy and pathogen control basing on the systematic research in the main garden and subordinate unit. The diversity of plant species and complexity of community has also provided a great range of host for Phytophthora. In 2017, GIS map of Phytophthora species in Botanic Garden Edinburgh was accomplished by Malcolm Gibson (Gibson, 2017), combining the historic distribution and identification on both morphology and molecular (ITS2) level. The Phytophthora Database of RBGE has also been established, exporting sampling method, origin and phenotypic records which could provide considerable material for further study. Pathological research on P. kernoviae and P. ramorum based on Benmore Botanic Garden, a satellite unit of RBGE has suggested that P. ramorum was detected in spore