DISCOVERY, EXPRESSION PROFILING, AND EVOLUTIONARY ANALYSIS OF CYNODON EXPRESSED SEQUENCE TAGS by CHANGSOO KIM (Under the Direction of Andrew H. Paterson) ABSTRACT Bermudagrass (Cynodon dactylon) is a major turfgrass species for sports fields, lawns, parks, golf courses, and general utility turfs in tropical and subtropical regions. Despite its ecological importance, much of its study has been dependent upon classical approaches. Information about Bermudagrass at the molecular level has been deficient although molecular information for other plants has been accumulated for the last two dacades. In the current study, we constructed a normalized cDNA library from leaf tissue of Bermudagrass in order to expand our knowledge of its transcriptome. We sequenced and annotated 15,588 expressed sequence tags (ESTs), which were deposited in the National Center for Biotechnology Information (NCBI) to be shared with other scientists. We also conducted cDNA array hybridization (macroarray) to profile genes responding to drought stress. A total of 120 and 69 genes were identified as up- and down-regulated, respectively. BLASTX annotation suggested that up-regulated genes may be involved in osmotic adjustment, signal transduction pathways, protein repair systems, and removal of toxins, while down-regulated genes were mostly related to basic plant metabolism such as photosynthesis and glycolysis. Using the cDNA sequences, we performed a comparative genomic study to gain new insight into the evolution of Bermudagrass. Results suggested that the common ancestor of the grass family experienced a whole genome duplication event at ca. 50.0 ~ 65.4 million years ago (MYA), before the divergence of the PACC and BEP clades at ca. 42.3 ~ 50.0 MYA. This evolutionary study also provided concrete evidence that the Chloridoideae and Panicoideae subfamilies diverged from a common ancestor at ca. 34.6 ~ 38.5 MYA. However, we were not able to find any evidence of a recent whole genome duplication event in Bermudagrass, possibly due to its autopolyploid genome structure. INDEX WORDS: Bermudagrass, Cynodon dactylon, cDNA library, Expressed sequence tag, EST, Drought stress, Macroarray, Gene duplication, Genome evolution, Grass family, Poaceae, Synonymous substitution rate, Phylogenetic analysis DISCOVERY, EXPRESSION PROFILING, AND EVOLUTIONARY ANALYSIS OF CYNODON EXPRESSED SEQUENCE TAGS by CHANGSOO KIM B.S., Korea University, Seoul, Korea, 1997 M.S., Korea University, Seoul, Korea, 1999 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2007 © 2007 Changsoo Kim All Rights Reserved DISCOVERY, EXPRESSION PROFILING, AND EVOLUTIONARY ANALYSIS OF CYNODON EXPRESSED SEQUENCE TAGS by CHANGSOO KIM Major Professor: Andrew Paterson Committee: Paul Raymer Robert Carrow Russell Malmberg Wayne Hanna Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2007 iv DEDICATION I dedicate this dissertation to my respectful parents and to my loving wife, who supported me in completion of my challenge. v ACKNOWLEDGEMENTS First of all, I would like to thank Dr. Andrew Paterson, my major professor, for his guidance, patience, and assistance in completion of my doctoral dissertation. Also, I would like to thank the rest of my committee members, Dr. Wayne Hanna, Dr. Robert Carrow, Dr. Russell Malmberg, and Dr. Paul Raymer, for serving and guidance. I am also grateful to all the members in the Plant Genome Mapping Laboratory. Without their support and friendship, I could not have done what I was able to do. I would also like to express appreciation to my friend, Glenn Hawes. Over the past 4 years, Glenn has encouraged me in completion of my goal as a teacher and friend. Finally, I would like to acknowledge the United States Golf Association/Turfgrass and Environmental Research Committee for making my graduate years financially secure. vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS·············································································································v LIST OF TABLES···················································································································· viii LIST OF FIGURES ······················································································································x CHAPTER 1 INTRODUCTION·············································································································1 CHAPTER 2 REVIEW OF LITERATURE····························································································5 CHAPTER 3 CONSTRUCTION AND CHARACTERIZATION OF A NORMALIZED cDNA LIBRARY FROM Cynodon dactylon L.········································································ 55 ABSTRACT······································································································· 56 INTRODUCTION······························································································ 57 MATERIALS AND METHODS········································································ 59 RESULTS ·········································································································· 63 DISCUSSION ···································································································· 68 REFERENCES··································································································· 75 CHAPTER 4 PROFILE OF DROUGHT STRESS-RESPONSIVE GENES IN Cynodon dactylon L. ······································································································ 99 ABSTRACT·····································································································100 vii INTRODUCTION····························································································101 MATERIALS AND METHODS······································································103 RESULTS ········································································································108 DISCUSSION ··································································································114 REFERENCES·································································································125 CHAPTER 5 PHYLOGENETIC PREDATING OF GENE DUPLICATION EVENTS IN Cynodon dactylon L. AND MODEL GRASS SPECIES BY COMPARATIVE GENOMIC APPROACHES············································································································156 ABSTRACT·····································································································157 INTRODUCTION····························································································158 MATERIALS AND METHODS······································································159 RESULTS ········································································································165 DISCUSSION ··································································································170 REFERENCES·································································································178 CHAPTER 6 SUMMARY AND CONCLUSIONS···········································································198 viii LIST OF TABLES Page Table 2.1: A classification of the genus Cynodon. ···································································· 52 Table 3.1: Summary of the Cynodon dactylon normalized cDNA library.································ 84 Table 3.2: Summary of annotation of 9,414 unigenes. ······························································ 88 Table 3.3: The 100 most frequent protein signatures identified by the InterProScan application using 9,414 C. dactylon unigenes.············································································ 90 Table 3.4: 25 different protein motifs further annotated by InterProScan for 43 unigenes that were not significantly matched (No hits) in a BLASTX search.······························· 91 Table 3.5: Gene ontology mappings of 9,414 unigenes using GOblet’s plants database. ·········· 92 Table 4.1: Significantly up- or down-regulated genes during entire drought-stress treatment.·· 136 Table 4.2: Significantly up-regulated genes in at least one treatment or time point. ················· 137 Table 4.3: Significantly down-regulated genes in at least one treatment or time point.············· 141 Table 4.4: Gene ontology (GO) mappings of the 189 drought candidate genes using Goblet’s plant database.·········································································································· 144 Table 4.5: Summary of selected cis-acting regulatory elements highly represented in rice homologs of up-regulated C. dactylon genes.··························································· 150 Table.4.6: Summary of comparison between the genes from each cluster and the corresponding GO terms.················································································································· 155 Table 5.1: Number of sequences and paralogs used to analyze the distribution of Ks values in eight tested grasses.·································································································· 184 ix Table 5.2: All possible secondary Ks peaks formed by paralogous pairs for the analyzed grasses. ····················································································································