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Identifying Untapped Microbial Resources in the Marine Sponge Microbiome

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Identifying Untapped Microbial Resources in the Marine Sponge Microbiome Identifying untapped microbial resources in the marine sponge microbiome A thesis submitted for the award of Doctor of Philosophy at School of Medicine, Faculty of Medicine, Nursing and Health Sciences, Flinders University Qi Yang December 2016 i TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES....................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................ x SUMMARY .................................................................................................................... xii DECLARATION ........................................................................................................... xiv ACKNOWLEDGEMENTS ............................................................................................. xv Chapter 1: Introduction ................................................................................................. 1 1.1 Sponge molecular taxonomy ......................................................................................... 1 1.1.1 Sponge taxonomy: from morphology to molecular ...................................................... 3 1.1.2 Various DNA markers for sponge molecular taxonomy .............................................. 5 1.1.3 Phylogenetic classification: update of Demospongiae (class) sponge ........................ 9 1.2 Abundant and valuable sponge-associated microorganisms ................................... 10 1.2.1 Sponge-associated microorganisms ......................................................................... 10 1.2.2 Sponge-associated actinobacteria: diverse and pharmaceutically valuable bacteria 14 1.2.3 Sponge-associated microorganisms as promising source of natural bioactive compounds ....................................................................................................................... 19 1.3 Sponge microbiome revealed by Next Generation Sequencing ................................ 21 1.3.1 Next Generation Sequencing technology ................................................................. 21 1.3.2 Sponge microbiome revealed by 16S rRNA gene based metagenomic sequencing . 23 1.3.3 Sponge specific microbial community ....................................................................... 30 1.4 Research plan ............................................................................................................... 33 1.4.1 Hypotheses .............................................................................................................. 33 1.4.2 Aims of the project ................................................................................................... 34 1.4.3 Applied methods and techniques ............................................................................. 35 1.4.4 Project framework .................................................................................................... 36 1.4.5 Thesis structure and contributions ............................................................................ 36 References .......................................................................................................................... 38 Chapter 2 Development of a multilocus-based approach for sponge (Porifera) identification: refinement and limitations ................................................................. 62 2.1 Introduction .................................................................................................................. 62 2.2 Materials and methods ................................................................................................. 64 2.2.1 Sponge collection and morphological classification .................................................. 64 ii 2.2.2 DNA extraction ......................................................................................................... 67 2.2.3 PCR amplification and sequencing ........................................................................... 67 2.2.4 Data processing ....................................................................................................... 68 2.2.5 Development of a Sponge Identification Protocol (SIP) ............................................ 70 2.2.6 Phylogenetic analysis ............................................................................................... 71 2.2.7 Morphological re-examination and final identification ............................................... 71 2.3 Results .......................................................................................................................... 72 2.3.1 Initial morphological classification ............................................................................ 72 2.3.2 Valid sequences ....................................................................................................... 73 2.3.3 Identification using the proposed Sponge Identification Protocol (SIP) ..................... 75 2.3.4 SIP reliability validated by phylogenetic analysis ...................................................... 77 2.3.5 Re-examination of morphological identifications ....................................................... 80 2.3.6 Approach to the final identity and the improved discrimination ................................. 83 2.3.7 SIP reliability evaluated by morphological identification ............................................ 84 2.3.8 Suitability and capacity of three DNA markers for sponge identification ................... 85 2.3.9 Limited entries in database ...................................................................................... 86 2.4 Discussion .................................................................................................................... 88 2.5 Conclusion .................................................................................................................... 90 References .......................................................................................................................... 90 Chapter 3 Sponge-associated actinobacterial diversity: Validation of the methods of actinobacterial DNA extraction and optimisation of 16S rRNA gene amplification ................................................................................................................ 96 3.1 Introduction .................................................................................................................. 96 3.2 Materials and methods ................................................................................................. 98 3.2.1 Specimens ............................................................................................................... 98 3.2.2 Sample preparation ................................................................................................ 100 3.2.3 DNA extraction ....................................................................................................... 100 3.2.4 PCR amplification of 16S rRNA gene ..................................................................... 100 3.2.5 PCR optimisation for 16S rRNA gene ..................................................................... 100 3.2.6 PCR amplification of COI mtDNA ........................................................................... 101 3.3 Results ........................................................................................................................ 102 3.3.1 DNA extraction efficiency of sponge spiked with actinobacteria ............................. 102 3.3.2 Highly-diluted DNA required for successful PCR amplification ............................... 102 3.3.3 Primer selection and PCR optimisation required for successful PCR amplification of individual sponges .......................................................................................................... 106 3.4 Discussion .................................................................................................................. 108 3.4.1 Comparable DNA recovery from sponge spiked with actinobacterial spores or mycelia iii ....................................................................................................................................... 108 3.4.2 Identification and elimination of interferences in the sponge DNA .......................... 109 3.4.3 Individual optimisation of PCR conditions required for successful 16S rRNA gene amplification .................................................................................................................... 110 3.5 Conclusion .................................................................................................................. 110 References ........................................................................................................................ 111 Chapter 4 Combination of different region-specific primers of 16S rRNA gene: essential for sponge microbiome analysis using Illumina platform ..................... 116 4.1 Introduction ................................................................................................................ 117 4.2 Materials and methods ............................................................................................... 119 4.2.1 Sponge collection and community DNA extraction ................................................. 119 4.2.2 454 GS FLX amplicon library
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