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Downloaded from Genbank (Refer to Table COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION o Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. o NonCommercial — You may not use the material for commercial purposes. o ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original. How to cite this thesis Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujdigispace.uj.ac.za (Accessed: Date). ` The assessment of DNA barcoding as an identification tool for traded and protected trees in southern Africa: Mozambican commercial timber species as a case study By Ronny Mukala Kabongo Dissertation presented in the fulfilment of the requirements for the degree MAGISTER SCIENTIAE in BOTANY in the FACULTY OF SCIENCE DEPARTMENT OF BOTANY AND PLANT BIOTECHNOLOGY at the UNIVERSITY OF JOHANNESBURG SUPERVISOR: PROF MICHELLE VAN DER BANK CO-SUPERVISOR: DR. OLIVIER MAURIN January 2014 I hereby declare that this dissertation has been composed by me and work contained within unless stated otherwise, is my own. Signed: Ronny Mukala Kabongo Date: 30 January 2013 Table of Contents Table of Contents .......................................................................................................................... iii Abstract ......................................................................................................................................... vi Acknowledgements ...................................................................................................................... vii List of abbreviations .................................................................................................................... viii Chapter 1 ....................................................................................................................................... 1 1. General introduction and objectives ...................................................................................... 1 1.1 Illegal logging and the global timber market .................................................................. 1 1.2 Effects of Illegal logging ................................................................................................. 2 1.3 The fight against illegal logging ..................................................................................... 5 1.4 Convention on International Trade in Endangered Species ............................................ 6 1.5 Other technical means of timber identification ............................................................... 8 1.6 Scale and effectiveness of global response ................................................................... 10 1.7 Molecular genetics tool for wood identification ........................................................... 12 1.8 DNA extraction from wood materials ........................................................................... 13 1.9 DNA Barcoding ............................................................................................................ 16 1.10 Diagnostic markers for land plants.............................................................................. 18 1.11 Tree-BOL Africa initiative .......................................................................................... 21 1.12 Study site: Mozambican forest .................................................................................... 23 1.13 State of Mozambican Forests ...................................................................................... 26 1.14 Research objectives ..................................................................................................... 28 Chapter 2 ..................................................................................................................................... 30 2. Materials and Methods ........................................................................................................ 30 Page | iii 2.1 Specimen collection and reference samples. ................................................................. 30 2.2 DNA extraction and sequencing. .................................................................................. 31 2.3 Sequence editing, alignment and broad analysis. .......................................................... 33 2.4 Assessment of core DNA barcodes identification efficiency. ....................................... 34 2.5 Phylogenetic analysis .................................................................................................... 36 2.6 Correspondence of query sequences to the database..................................................... 37 Chapter 3 ..................................................................................................................................... 58 3. Results ................................................................................................................................. 58 3.1 Summary statistics ........................................................................................................ 58 3.2 Genetic divergence and barcode gap analyses .............................................................. 59 3.3 DNA barcode identification success rates using distance-based analysis ..................... 60 3.4 Cumulative error and threshold optimization ................................................................ 61 3.5 DNA barcode identification success rates using tree-based analysis ............................ 62 3.6 DNA barcode query assignment ................................................................................... 63 Chapter 4 ................................................................................................................................... 100 4. Discussion ......................................................................................................................... 100 4.1 Development of DNA barcode reference library ........................................................ 100 4.2 Genetic divergence and implication for identification ................................................ 102 4.3 Identification success rates .......................................................................................... 105 4.4 DNA barcoding: practical considerations ................................................................... 107 Chapter 5 ................................................................................................................................... 110 5. Conclusion ............................................................................................................................ 110 6. References ............................................................................................................................. 115 7. Supplementary information ................................................................................................... 131 Page | iv Figure 7-1 Specimen illustrations as submitted on BOLD Systems database, an additional scan of the herbarium voucher specimen is available for every specimen sampled. ........ 138 ........................................................................................................................................... 138 Page | v Abstract Global efforts to protect the world’s forests from unsustainable and inequitable exploitation have been undermined in recent years by rampant illegal logging in many timber-producing countries. A prerequisite for efficient control and seizure of illegally harvested forest product is a rapid, accurate and tamper proof method of species identification. DNA barcoding is one such a tool, relatively simple to apply. It is acknowledged to bring about accuracy and efficiency in species identification. In this study a DNA barcode reference library for traded and protected tree species of southern Africa was developed comprising of 81 species and 48 genera. Four primary analyses were conducted to assess the suitability of the core barcodes as a species identification tool using the R package Spider 1.2-0. Lastly, to evaluate this identification tool, query specimens independently sampled at a Mozambican logging concession were identified using DNA barcoding techniques. The nearest neighbour (k-NN) and best close match (BCM) distance based parameter yielded 90% and 85% identification success rate using the core plant barcodes respectively. DNA barcoding identification of query specimens maintained a constant 83% accuracy over the single marker dataset and the combined dataset. This database can serve as a backbone to a control mechanism based on DNA techniques for species identification and also advance the ability of relevant authorities to rapidly identify species of timber at entry and exit points between countries with simple, fast, and accurate DNA techniques. Page | vi Acknowledgements The work presented in this document would not have been possible without the contribution of a small group of people; therefore, I would like to take this opportunity to acknowledge them. First and foremost I would like to express my appreciation to my research supervisor prof. Michelle Van der Bank for her constant guidance and enthusiastic
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