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Metagenomic Study of Lettuce Potting Soil

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Metagenomic Study of Lettuce Potting Soil METAGENOMIC STUDY OF LETTUCE POTTING SOIL MICROBIOME UNDER CHITIN TREATMENT AND FUNCTIONAL GENOMIC ANALYSIS OF MORTIERELLA HYALINA A PLANT GROWTH-PROMOTING, CHITIN DEGRADING FUNGUS Word count: 28125 Floris Voorthuijzen Student number: 01503491 Promoters: Prof. Dr. Peter Dawyndt, Dr. Caroline De Tender Supervisor: Dr. Annelies Haegeman A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Bioinformatics: Bioscience Engineering Academic Year: 2019- 2020 The author gives permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws, more specifically the source must be extensively specified when using results from this thesis. 06/06/2020 Floris Voorthuijzen 2 Acknowledgements I wish to express my deepest gratitude to all the friendly folks at the ILVO in Merelbeke for their help and hospitality. In particular, I would like to thank everyone in the monthly Microbiodem meetings for their input. Special thanks go to Caroline De Tender, whose vision for this thesis was of invaluable importance, Annelies Haegeman, who flawlessly took over the co-promotership in the second semester and guided me through the genome assembly, and Steve Baeyen, who was always eager to help. 3 Table of Contents Acknowledgements ................................................................................................................................. 3 Abbreviations .......................................................................................................................................... 6 Abstract ................................................................................................................................................... 8 Dutch Abstract ......................................................................................................................................... 9 Literature Research ............................................................................................................................... 10 1. Introduction ................................................................................................................................... 10 1.1 Lettuce cultivation in Flanders .......................................................................................... 10 1.2 Lettuce nutritional needs .................................................................................................. 11 1.3 Fertilizer ............................................................................................................................. 11 1.4 Chemical crop protection .................................................................................................. 12 1.5 Research Perspectives ....................................................................................................... 13 2. Chitin .............................................................................................................................................. 13 2.1 Structure, Presence & Properties ...................................................................................... 13 2.2 Effect in soil ....................................................................................................................... 15 3. Mortierella ..................................................................................................................................... 15 3.1 Presence ............................................................................................................................ 15 3.2 Functions ........................................................................................................................... 16 3.3 Genome ............................................................................................................................. 16 4. High Throughput Sequencing ........................................................................................................ 16 4.1 Second Generation Sequencing ........................................................................................ 16 4.2 Illumina sequencing ........................................................................................................... 17 4.3 Metabarcoding .................................................................................................................. 18 4.4 Whole Genome Shotgun sequencing and genome assembly ........................................... 22 5. Aim of the study ............................................................................................................................ 24 Outline ................................................................................................................................................... 25 Methods ................................................................................................................................................ 26 1. Metabarcoding of the bacterial and fungal community ............................................................... 26 1.0 Experimental setup ............................................................................................................ 26 1.1 Preprocessing .................................................................................................................... 27 1.2 Data Exploration ................................................................................................................ 28 1.3 Statistical Analysis ............................................................................................................. 30 4 2. Identification of Mortierella strain using ITS phylogeny ............................................................... 31 2.1 Preprocessing .................................................................................................................... 31 2.2 Alignment & Tree Generation ........................................................................................... 32 3. Genome Analysis ........................................................................................................................... 33 3.1 De novo assembly .............................................................................................................. 33 3.2 Genome Annotation .......................................................................................................... 35 Results ................................................................................................................................................... 37 1. Metabarcoding of the bacterial and fungal community ............................................................... 37 1.1 Preprocessing .................................................................................................................... 37 1.2 Data Exploration ................................................................................................................ 41 1.3 Statistical Analysis ............................................................................................................. 52 2. Identification of Mortierella strain using ITS phylogeny ............................................................... 56 2.1 Phylogenetic Analysis ........................................................................................................ 56 3. Genome Analysis ........................................................................................................................... 58 3.1 De novo assembly .............................................................................................................. 58 3.2 Genome Annotation .......................................................................................................... 61 Discussion .............................................................................................................................................. 65 1. Metabarcoding of the bacterial and fungal community ............................................................... 65 2. Genome Annotation ...................................................................................................................... 67 3. Importance in horticulture & conclusion ...................................................................................... 68 4. Future Perspectives ....................................................................................................................... 69 References ............................................................................................................................................. 70 Supplementary Materials ...................................................................................................................... 77 5 Abbreviations 16S rRNA Ribosomal RNA subunit of size 16 Svedberg units AA Amino Acid ABA Abscisic acid ANOVA Analysis of Variance ASV Amplicon Sequence Variant BAC Bacterial Artificial Chromosomes BAM Binary Alignment Map BLAST Basic Local Alignment Search Tool BLASTp Protein-protein BLAST BS Bulk potting soil sampling CH Chitin treatment CLI Command Line Interface CPM Count Per Million CT Control treatment Df Degrees of Freedom DNA Deoxyribonucleic acid EC Enzyme Commission (number) FWD Forward (reads) GC content Guanine-Cytosine content GFF Generic Feature Format GH Glycoside Hydrolase GlcNAc N-acetylglucosamine GLM Generalized Linear Model IAA Indole-3-acetic acid ILVO Instituut voor Landbouw en Visserij
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