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University of Florida Thesis Or Dissertation Formatting ALTERNATIVE SPLICING IN BASAL ANGIOSPERMS AND TRAGOPOGON (ASTERACEAE) By XIAOXIAN LIU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Xiaoxian Liu To my Husband and Parents ACKNOWLEDGMENTS I would like to express my most sincere gratitude to my advisors, Drs. Doug Soltis and Brad Barbazuk. Their patience, encouragement, and generosity have all been indispensable for my research. Dr. Pam Soltis has also been a true mentor who offered tremendous help in all stages of my dissertation research. I want to thank my other committee member, Dr. Mark Settles for his consistent support and guidance during my study. I am extremely grateful to my fellow graduate students and postdocs for helping me with my research, inspiring me to be a better scholar, and providing a lovely environment in which I learn and grow. Members of the Soltis and Barbazuk labs offered valuable suggestions on experimental design, data interpretation, and verbal and oral presentation. I want to give special thanks to Shengchen Shan, Qinyin Ling, Nathan Catlin and Drs. Andre Chanderbali, Mi-jeong Yoo, Miao Sun, Blaine Marchant, Srikar Chamala, Lucas Boatwright, Wenbin Mei. My work would not be possible without the hard work from several laboratory managers and assistants, Dr. Matt Gitzendanner, Dr. Evgeny Mavrodiev and Ruth Davenport. I also owe thanks to Mallory St Clair for her help in laboratory work. I appreciate Dr. Patrick Schnable from Iowa State University with his help on 10X Genomics approach. I appreciate Dr. Jim Leebens-Mark from University of Georgia for sharing data. I am extremely grateful for the financial support from National Science Foundation, including a Dissertation Improvement Grant, and research awards from the Botanical Society of America and The American Society of Plant Taxonomists. I am also 4 grateful for NSF and the Botanical Society of America for their Travel Award to attend International Botanical Congress. And last, but certainly not least, I wish to thank my husband and my family for their unconditional love and support. To them I dedicate this dissertation. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 12 ABSTRACT ................................................................................................................... 14 CHAPTER 1 INTRODUCTION .................................................................................................... 16 Whole-Genome Duplication and Plant Evolution .................................................... 16 Gene Expression Changes after Polyploidy ............................................................ 17 RNA Alternative Splicing ......................................................................................... 18 Alternative Splicing After Whole-Genome Duplication ............................................ 19 Tragopogon as a Model System for Studying Polyploidy ........................................ 20 2 DETECTION ALTERNATIVE SPLICING WITHOUT REFERENCE GENOME ...... 27 Introduction ............................................................................................................. 27 Materials and Methods ............................................................................................ 30 Amborella Genome, Annotation, and RNA-Seq Data ....................................... 30 Plant Materials and RNA Sample Preparation .................................................. 31 cDNA Synthesis, Library Construction, and Sequencing .................................. 31 Data Collection and Error Correction ................................................................ 31 Evaluation of Read Quality ............................................................................... 32 Reference-Based Transcriptome Assembly and Alternative Splicing Detection ....................................................................................................... 33 Comparisons between Iso-SeqTM Assemblies and Amborella Gene Models ... 33 De novo Detection and Validation of Alternative Splicing Events ..................... 33 Optimization of de novo Pipeline ...................................................................... 35 Results .................................................................................................................... 35 General Properties of ROIs in Iso-SeqTM .......................................................... 35 Error Correction by ICE-Quiver and Illumina Reads ......................................... 37 Mapping Full-Length ROIs and Error-Corrected Reads to the Amborella Genome ........................................................................................................ 38 Alternative Splicing Analysis Using Program to Assemble Spliced Alignments (PASA) ....................................................................................... 39 Improvements of Current Gene Models by Isoform-Level Comparison ............ 40 De novo Detection of AS Events in Iso-SeqTM Data and Comparison with PASA Results ................................................................................................ 41 AS Primer Design and RT-PCR Validation ....................................................... 42 Optimization of the de novo Pipeline Based on Decision Tree Analysis ........... 43 6 Discussion .............................................................................................................. 43 Pretreatment Strategies for Iso-SeqTM Raw Data ............................................. 43 Advantages of Iso-SeqTM in Reference-Based Transcriptome Analysis ........... 46 Iso-SeqTM and AS de novo Detection Pipeline .................................................. 48 3 CONSERVED ALTERNATIVE SPLICING IN BASAL ANGIOSPERMS ................. 61 Introduction ............................................................................................................. 61 Materials and Methods ............................................................................................ 64 Nymphaea Genome, Annotation, and Transcriptome Data .............................. 64 Nymphaea Iso-SeqTM Data QC and Error Correction ....................................... 64 Nymphaea RNA-Seq Data Assembly ............................................................... 64 Alternative Splicing Analysis in Nymphaea ....................................................... 65 Reanalysis of Amborella Alternative Splicing Events ....................................... 66 Gene Orthology Analysis .................................................................................. 67 Conserved Alternative Splicing in Basal Angiosperms, Eudicots, and Monocots ....................................................................................................... 67 Functional Annotation of Nymphaea Protein-Coding Genes ............................ 68 GO Term Enrichment ....................................................................................... 69 Principal Component Analysis (PCA) of Shared AS Events in Eight Angiosperms ................................................................................................. 69 Phylogeny and Divergence Time Estimation .................................................... 69 Results .................................................................................................................... 69 Alternative Splicing in Nymphaea ..................................................................... 69 Alternative Splicing in Amborella ...................................................................... 70 Shared AS Events in Angiosperms .................................................................. 71 Conserved AS between basal angiosperms: Nymphaea and Amborella ......... 72 Highly Conserved AS between Basal Angiosperms, Monocots and Eudicots .. 73 GO Annotation and Enrichment Analysis of the Conserved or Shared AS Events ........................................................................................................... 74 Discussion .............................................................................................................. 75 Iso-SeqTM or RNA-Seq for AS Analysis ............................................................ 75 Different AS Frequencies in Two Basal Angiosperms ...................................... 76 Dynamic AS Changes during Angiosperm Evolution ........................................ 77 Conserved AS in Basal Angiosperms ............................................................... 78 Shared AS Events in Aquatic Species (Habitat Effects) ................................... 79 4 DE NOVO ALTERNATIVE SPLICING DETECTION IN TRAGOPOGON ............... 98 Introduction ............................................................................................................. 98 Methods and Materials .......................................................................................... 100 Tragopogon dubius RNA Sample Extraction and Iso-SeqTM Sequencing ...... 100 Tragopogon dubius Iso-SeqTM Data Processing ............................................
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