University of Bath PHD Species-specific PCR primers for the rapid and reliable identification of yeast species Harrison, Elizabeth Award date: 2006 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 08. Oct. 2021 Species-specific PCR primers for the rapid and reliable identification of yeast species Volume 1 of 1 Elizabeth Harrison A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry June 2006 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author. 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Contents List of figures i List of tables v Acknowledgements vi Abstract vii List of Abbreviations viii page 1 Introduction 1 1.1 Definition of a species 1 1.2 Traditional methods of yeast identification 2 1.3 Molecular methods of yeast identification 3 1.3.1 DNA base composition analysis 3 1.3.2 DNA homology 4 1.3.3 Ribosomal genes for identification 6 1.4 Rapid yeast identification 10 1.4.1 RAPD 11 1.4.2 AFLP 12 1.4.3 RFLP 12 1.4.4 DGGE and TGGE 13 1.4.5 Karyotype analysis 13 1.4.6 Other methods 13 1.4.7 Specific primers 14 1.4.8 Advanced methods 15 1.5 Bacterial species identification 15 1.6 Fungal species identification 16 1.7 Food spoilage yeasts 16 1.8 Aim 20 1.9 Strategy 21 1.9.1 rDNA 21 1.9.2 Housekeeping genes 22 1.9.3 Species-specific genes 22 1.9.4 Primer design strategy and parameters 22 2 Materials and Methods 24 2.1 Yeast strains and culture conditions 24 2.2 DNA preparation 24 2.2.1 Cell number quantification 24 2.2.2 Clean DNA preparation for PCR 24 2.2.3 Rapid cell lysis for PCR 28 2.3 Amplification 28 2.3.1 Amplification with new primers 28 2.3.2 Analysis of amplified PCR products 29 2.4 Optimisation 29 2.4.1 Temperature gradient PCR 29 2.4.2 Magnesium ion gradient PCR 29 2.5 Sequences 29 2.5.1 Sequences from databases 29 2.5.2 Sequencing primer design 30 2.5.3 Amplification for sequencing 32 2.5.4 Gel quantification of PCR products 32 2.5.5 PCR cleanup 33 2.5.6 Sequencing 33 2.5.7 Analysis of sequencing results 33 2.6 D1 / D2 identification of yeast species 33 2.7 Phylogeny 33 Brettanomyces / Dekkera 34 3.1 Brettanomyces 34 3.1.1 Currently accepted species 34 3.1.2 Description of the genus 34 3.2 Dekkera 34 3.2.1 Currently accepted species 34 3.2.2 Description of the genus 35 3.3 History ofDekkera / Brettanomyces 35 3.4 Results 37 3.4.1 D1 / D2 sequence alignment 37 3.4.2 Specific primer design 37 3.4.3 Specific primer testing 40 3.5 Discussion 54 3.5.1 D1 / D2 sequence alignment 54 3.5.2 Specific primer design 55 3.5.3 Specific primer testing 55 3.6 Conclusion 60 Zygosacch aromyces 61 4.1 Introduction 61 4.1.1 Currently accepted species 61 4.1.2 Description of the genus 61 4.1.3 Spoilage characteristics 61 4.1.4 History 62 4.2 Results 64 4.2.1 rDNA sequences 64 4.2.2 Housekeeping gene sequences 64 4.2.3 Specific primer design 76 4.2.4 Specific primer testing 78 4.2.5 Specific primer optimisation 84 4.3 Discussion 91 4.3.1 rDNA sequences 91 4.3.2 Housekeeping gene sequences 93 4.3.3 Specific primer design 96 4.3.4 Specific primer testing 96 4.4 Conclusion 103 Sacch aromyces 105 5.1 Introduction 105 5.1.1 Currently accepted species 105 5.1.2 Description of the genus 105 5.1.3 Spoilage characteristics 105 5.1.4 History 106 5.2 Results 108 5.2.1 Target sequences 108 5.2.2 Specific primer design 112 5.2.3 Specific primer testing 115 5.2.4 Multiplexing 147 5.3 Discussion 149 5.3.1 rDNA sequences 149 5.3.2 Specific primer design 149 5.3.3 Specific primer testing 150 5.3.4 S. cariocanus and S. paradoxus 150 5.3.5 S. pastorianus 151 5.3.6 Multiplexing 157 5.4 Conclusion 158 6 Concluding discussion 159 6.1 Primer characteristics 159 6.1.1 Total number of mismatches 159 6.1.2 3’ mismatches 159 6.1.3 Primer melting temperatures 162 6.2 Genus specific primers 165 6.3 Conclusion 165 7 References 168 List of figures Figure page 1.1 The organisation of the yeast rDNA cistron 6 3.1 ClustalW alignment of the rDNA region D1 / D2 of the type strains of genusDekkera / Brettanomyces 38 3.2 Maximum parsimony phylogenetic tree of the D1 / D2 sequences from the Brettanomyces / Dekkera type strain of each species 37 3.3 Target and closest sibling species amplified with all combinations of forward and reverse Brettanomyces nanus primers 41 3.4 B. custersianus strains amplified with combinations of the B. nanus primers 42 3.5 D. bruxellensis strains amplified with combinations of B. nanus primers 42 3.6 D. anomala strains amplified with combinations of the B. nanus primers 42 3.7 Target species amplified with all combinations of forward and reverse B. naardenensis primers 43 3.8 B. custersianus strains amplified with all combinations of forward and reverse B. naardenensis primers 43 3.9 D. bruxellensis strains amplified with all combinations of forward and reverse B. naardenensis primers 44 3.10 D. anomala and B. nanus strains amplified with all combinations of forward and reverse B. naardenensis primers 44 3.11 Species amplified with all forward and reverse combinations of B. custersianus primers 45 3.12 B. custersianus strains 1 and 2 amplified with all potentiallyB. custersianus specific combinations of forward and reverse primer 45 3.13 D. anomala strains amplified with all potentially B. custersianus specific combinations of forward and reverse primer 46 3.14 D. bruxellensis strains amplified with all potentiallyB. custersianus specific combinations of forward and reverse primer 46 3.15 B. naardenensis strains amplified with all potentiallyB. custersianus specific combinations of forward and reverse primer 47 3.16 B. nanus strains amplified with all potentiallyB. custersianus specific combinations of forward and reverse primer 47 3.17 Target species amplified with all combinations of forward and reverse D. bruxellensis primers 48 3.18 D. anomala strains amplified with all combinations of D. bruxellensis forward and reverse primer 48 3.19 B. nanus strains amplified with all combinations of D. bruxellensis forward and reverse primer 49 3.20 B. custersianus strains amplified with all combinations of D. bruxellensis forward and reverse primer 49 3.21 B. naardenensis strains amplified with all combinations of D. bruxellensis forward and reverse primer 49 3.22 D. bruxellensis and B. naardenensis strains amplified with all combinations of D. bruxellensis forward and reverse primer at 60°C annealing temperature 49 3.23 Target species amplified with all combinations of forward and reverse D. anomala primers 50 3.24 D. bruxellensis strains amplified with all combinations of D.anomala forward and reverse primer 50 3.25 B. custersianus strains amplified with all combinations of D.anomala forward and reverse primer 50 3.26 B. nanus strains amplified with all combinations of D.anomala forward and reverse primer 51 3.27 B.naardenensis strains amplified with all combinations of D.anomala forward and reverse primer 51 3.28 D.