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Exploring the Nodule Microbiome Community Structure of Trifolium Species

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EXPLORING THE NODULE MICROBIOME COMMUNITY STRUCTURE OF TRIFOLIUM SPECIES By Prateek Shetty A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Biology - Master of Science 2016 ABSTRACT EXPLORING THE NODULE MICROBIOME COMMUNITY STRUCTURE OF TRIFOLIUM SPECIES By Prateek Shetty Plant associated microbes have been shown to increase plant growth and production drastically, yet we are just beginning to understand the parameters that impact these interactions. Rhizobia are primary bacterial symbionts of legumes and infect root hairs to form nodules, within which, the symbiotic rhizobia fix atmospheric nitrogen into biologically available forms in exchange for carbon from the host. The aim of this project is to understand the community structure and diversity of the nodule microbiome, with emphasis on the less abundant members, among coexisting clover species. North American clover Trifolium -Rhizobium communities are a good system to study host interactions with microbiomes given the high local species diversity. We analyzed the nodule microbiome of six congeneric clover plants when they were grown in soils conditioned by members of their own species and in soils conditioned by congener species by sequencing the 16s rRNA gene. The visualized microbiomes are similar, with 96% of all reads belonging to the order Rhizobiales. The rest of the OTUs belong to rarer groups of microbes. Further, the structure of the microbiome is impacted by both the host plant species and the soil in which the host is grown in, with soil explaining a larger degree of variation. There also is a strong interaction between soil and host in structuring the microbiome. The results are similar when the microbiome is analyzed with and without its most dominant order (Rhizobiales ). ACKNOWLEDGEMENTS I would like to pay my gratitude to the persons below who made my research successful and assisted me at every step of the way: My supervisor, Dr. Maren Friesen for her constant support and assistance. Her encouragement was instrumental in the completion of my thesis. My committee members, Dr. Yair Shachar-Hill and Dr. Ashley Shade, whose guidance during my research was invaluable. Dr. Alan Prather, Graduate director of the Department of Plant biology, whose reminders and constant motivation encouraged me to meet the deadlines. All the members of the Friesen Lab, interacting with whom not only improved my research but also helped me ask better questions. Finally, I would like to thank my family members and friends, without whom I would be a whole lot lesser. They not only assisted me financially but more importantly, extended moral and emotional support during the course. This work was funded by grant NSF 1342793 to Dr. Maren Friesen iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ vi LIST OF FIGURES ..............................................................................................vii KEY TO ABBREVIATIONS .................................................................................. ix Introduction ......................................................................................................... 1 Understanding factors that contribute to the coexistence ..................................... 1 Importance of microbial community on plant health .............................................. 5 Generalist vs specialist selection within plant microbiome .................................... 6 BNF: Biological nitrogen fixation ........................................................................... 7 Associative nitrogen fixation ................................................................................. 7 Current literature on nodule microbiome ............................................................... 8 Soil legacy ............................................................................................................ 8 Host species selection .......................................................................................... 9 Techniques to study microbial populations ......................................................... 10 Culture dependent methods- Before the widespread use of NGS techniques .................................................................................................. 10 Culture independent methods (Table 1) ................................................... 10 Lipid analysis ............................................................................................ 10 Gradient Gels ............................................................................................ 11 Hybridisation techniques - FISH, Microarray ............................................. 11 Targeted amplification and sequencing of a marker gene ......................... 11 Importance of studying native plants .................................................................. 12 About Trifolium species ....................................................................................... 13 Results ............................................................................................................... 16 Sample read distribution table. ........................................................................... 16 OTU picking and Taxonomic Classification ......................................................... 16 Presence/Absence matrix .......................................................................... 17 Abundance and Presence/Absence matrix .............................................. 18 Diversity estimate ............................................................................................... 18 Alpha diversity ............................................................................................ 18 Ordinations ......................................................................................................... 19 Beta diversity .............................................................................................. 19 Adonis modeling results ...................................................................................... 21 Neutral community model ................................................................................... 21 Discussion ......................................................................................................... 23 Composition of nodule microbiome ..................................................................... 23 Core microbiome ................................................................................................ 27 Alpha Diversity analysis ...................................................................................... 28 Ordinations ......................................................................................................... 28 iv a) PCoA ....................................................................................................... 28 b) CCA ......................................................................................................... 29 c) NMDS ....................................................................................................... 29 Beta diversity model comparisons ...................................................................... 29 Neutral community analysis ................................................................................ 30 Broader Impacts ................................................................................................. 31 Methods ............................................................................................................. 34 Plant experiment ................................................................................................. 34 Sequencing experiment ...................................................................................... 34 nodC ................................................................................................................... 34 16S ..................................................................................................................... 34 Microbiome community profile: Building OTU table ............................................ 35 Core microbiome ................................................................................................ 36 Ordination techniques ......................................................................................... 37 PCoA: Principal coordinate analysis ........................................................ 37 CCA: Canonical Correspondence Analysis ............................................. 38 NMDS: (Non metric MultiDimensional Scaling) ....................................... 38 Sloan neutral community analysis ...................................................................... 38 APPENDICES ..................................................................................................... 40 APPENDIX 1: List of tables .......................................................................... 41 APPENDIX 2: List of figures ......................................................................... 75 APPENDIX 3: Main script ........................................................................... 110 APPENDIX 4: Core microbiome calculations ............................................. 118 APPENDIX 5: Neutral model analysis ........................................................ 122 REFERENCES ................................................................................................
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