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RNA-Seq Transcriptome Profiling of Ethiopian Lowland Bamboo

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RNA-Seq Transcriptome Profiling of Ethiopian Lowland Bamboo RNA-Seq Transcriptome Profiling of Ethiopian Lowland Bamboo (Oxytenanthera Abyssinica (A.Rich) Munro Under Drought and Salt Stresses and SSR Based Genetic Diversity Analysis o f Ethiopian Highland Bamboo (Arundinaria Alpina K. Schum). Muhamed Adem A Thesis Submitted to the Department of Microbial, Cellular and Molecular Biology Presented in Fulfillment of the Requirements for the Degree of Doctor of Philosophy (PhD) In Biology (Applied Genetics) Addis Ababa University Addis Ababa, Ethiopia July, 2019 ACKNOWLEDGMENTS First of all, I would like to say ALLHAMDULILLAHI RABIL ALAMIN for all. I would like to thank my advisors, Dr. Dereje Beyene, Dr. Tileye Feyissa, Prof. Jiang Tingbo and the Department Head of Microbial Cellular Molecular Biology, Dr. Gurja Belay and staff members Dr. Kifle Dagne and Dr. Kassahun Tesfaye. I also extend my gratitude to Madawalabu University and Addis Ababa University for accepting me as PhD student and monetary support. Special thanks also goes to Northeast Forestry University, Harbin, China for hosting me as a research visit student and lab members Kai Zhao, Guanbin, Dia Jian and Shuxuan Li. Ethiopian students in Harbin also deserve special thanks, especially Megersa Tsegaye, Solomon Tsegaye, Mayi Fekede, Dr. Fekede and Dingata Lemu. I am also grateful to Ethiopian Biodiversity Institute for providing bamboo seeds and export permit and Ethiopian Environment and Forestry Research Institute (EEFRI) for providing funding used for DNA sample collection and Dr. Yigardu Mulatu, Dr. Abeje Eshete and Dr. Wubalem Tadesse of the Institute for facilitating the support. The support from Ethiopian National Agricultural Biotechnology Research Institute for accessing the molecular lab and Dr. Dejene Girma as hosting advisor is much appreciated. I also wanted to extend my gratitude to my friends; Endris Mohammed, Endris Ahmed, Muhamed Yimer, Muhamed Sitotaw, Tilahun Mekonen, Oumer Abdi, Dr. Fekadu Gadissa and Dr. Getachew Melaku for their brotherly help. Last but not least, my family deserves special thanks for their all sided support. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................................................................................................ ii TABLE OF CONTENTS ...........................................................................................................................iii LIST OF FIGURES ......................................................................................................... .........................viii LIST OF APPENDICES .............................................................................................................................ix LIST OF ABBREVATIONS and ACRONYMS ......................................................................................... x ABSTRACT ................................................................................................................................................ xii CHAPTER 1 ................................................................................................................................................. 1 1. GENERAL INTRODUCTION ................................................................................................................. 1 1.1 Statement of the problem ........................................................................................................................ 4 1.2 Hypothesis ............................................................................................................................ 5 1.3 Objectives .............................................................................................................................. 6 1.3.1 General objective ............................................................................................................ 6 1.3.2 Specific objectives .......................................................................................................... 6 1.4 Organization of the thesis ...................................................................................................... 6 CHAPTER 2 ................................................................................................................................................. 8 2. LITERATURE REVIEW ......................................................................................................................... 8 2.1 Bamboos ................................................................................................................................ 8 2.1.1 Origin of bamboos .......................................................................................................... 8 2.1.2 Taxonomy ....................................................................................................................... 8 2.1.3 Distribution and area coverage ....................................................................................... 9 2.1.4 The biology of bamboos ................................................................................................ 9 2.1.5 Reproduction and growth habit .................................................................................... 10 2.1.6 Economic uses .............................................................................................................. 10 2.1.7 Ploidy level / genome size and genetics ...................................................................... 11 2.2 Bamboos in Ethiopia .......................................................................................................... 12 iii 2.2.1 Ethiopian lowland bamboo (Oxytenanthera abyssinica A.Rich Munro) .................... 12 2.2.2 Ethiopian high land bamboo (Arundinaria alpina K.Schum) ...................................... 13 2.3. RNA Sequencing ( RNA-Seq ) ......................................................................................... 15 2.4 Transcriptomics ................................................................................................................... 17 2.4.1 Bamboo transcriptome studies...................................................................................... 18 2.5 Plant abiotic stress ............................................................................................................... 19 2.6 Plant transcription factors.................................................................................................... 24 2.6.1 Basic leucine zipper (bZIP) transcription factor gene family ....................................... 25 2.7. Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) ........................................................................ 28 2.8 Molecular markers............................................................................................................... 29 2.8.1 Microsatellite (SSR) markers ....................................................................................... 30 CHAPTER 3 ............................................................................................................................................... 32 3. RNA-Seq global transcriptome profiling of Ethiopian lowland bamboo (O.abyssinica) under drought and salt stress .............................................................................................................................................. 32 3.1 Materials and Methods ........................................................................................................ 32 3.1. 1 Plant materials, growth conditions and treatment ....................................................... 32 3.1.2 RNA extraction, cDNA synthesis and sequencing ....................................................... 33 3.1.3 Data filtering and assembly ......................................................................................... 34 3.1.4 Functional annotation and classification of de novo assembled unigenes .................. 34 3.1.5 Gene expression and differentially expressed genes (DEGs) analysis ........................ 35 3.1.6 Validation of RNA-Seq data by RT-qPCR................................................................... 35 3.2 Results ................................................................................................................................ 36 3.2.1 Sequencing, de novo assembly and functional annotation of lowland bamboo transcriptome ......................................................................................................................... 36 3.2.2 GO and COG annotation analysis ................................................................................ 39 3.2.3 Metabolic pathway analysis ......................................................................................... 42 3.2. 4 Prediction of transcription factors and protein family assignment .............................. 44 3.2. 5 Gene expression and differential expression genes (DEGs) analysis .......................... 45 iv 3.2.6 Verification of RNA-Seq data by qRT-PCR ................................................................ 48 3.2.7 Data deposition ............................................................................................................. 49 3.2.8 Underlying mechanisms associated with abiotic stress tolerances of lowland bamboo49 3.3. Discussion .......................................................................................................................... 51 CHAPTER 4 ..............................................................................................................................................
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