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Evolutionary Relationships Among Fungal Soybean Pathogens And Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2015 Evolutionary Relationships among Fungal Soybean Pathogens and Molecular Marker Development in the Genus Cercospora Sebastian Albu Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Plant Sciences Commons Recommended Citation Albu, Sebastian, "Evolutionary Relationships among Fungal Soybean Pathogens and Molecular Marker Development in the Genus Cercospora" (2015). LSU Doctoral Dissertations. 2140. https://digitalcommons.lsu.edu/gradschool_dissertations/2140 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. EVOLUTIONARY RELATIONSHIPS AMONG FUNGAL SOYBEAN PATHOGENS AND MOLECULAR MARKER DEVELOPMENT IN THE GENUS CERCOSPORA A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agriculture and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Plant Pathology and Crop Physiology by Sebastian Albu B.A., University of Pittsburgh, 2001 B.S., Metropolitan State University of Denver, 2005 M.S., Louisiana State University, 2012 December 2015 I would like to dedicate this dissertation to my loving wife Ruoxi Chen and daughter Vanessa Albu. ii ACKNOWLEDGEMENTS I thank my wife, Ruoxi Chen for her infinite patience, unconditional love and help with statistical analyses and formatting of this document. I am also thankful to my parents, Magdalena and Valeriu, and brother, Alex for all of their support during graduate school. I owe a great deal of gratitude to my professors, friends and family for their unwavering support during what has been a precarious, but exciting journey. I am forever grateful to Dr. Raymond Schneider, whose generosity and belief in my abilities motivated me even in the darkest hours. This work would not be what it is without the guidance and expertise of Dr. Vinson Doyle, who has provided me with new perspectives of mycology and systematics, and has pushed me to become a better scientist. I would also like to thank my committee members, Dr.Chris Clark and Dr. Cathie Aime for their mentorship and inspiration. I am obliged to Dr. Quang Cao for agreeing to serve on my committee and also to Jeremy Brown for making thoughtful suggestions during several conversations. Many thanks go out to Dr. Lawrence Datnoff for his encouragement and efforts to create a wonderfully diverse and pleasant place to call home for the last five years. I enjoyed spending time in the field with Trey Price and thank him for providing many of the isolates that were used in this study. Additional isolates and plant material used in this research were kindly provided by Dr. Guohong Cai, Dr. Susan Li, Dr. Glen Hartman, Dr. Heather Kelly and Dr. Burt Bluhm (who was also very generous to provide the resources for genome sequencing). I am very thankful for the technical help that Sandeep Sharma provided during genome sequencing in Dr. Bluhm’s lab and also to Don Nelsen who provided housing accommodations. I sincerely thank Haley Hutchins, Page Muse, Derek Clement, Caroline Baer and Charlot Amadi for their help in the lab. I have been fortunate to get to know many students and research associates during my time at LSU including my friends and lab mates, Brian Ward, Eduardo Chagas, Teddy Garcia, Elaisa Tubana, Clark Roberstson, Willie Vieira and Josiene Veloso. I will always look back fondly on my time spent with them and my other fellow graduate students and I hope our paths will cross again. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................... iii ABSTRACT ................................................................................................................................... vi CHAPTER 1. INTRODUCTION ................................................................................................... 7 1.1 History of soybean ................................................................................................................ 7 1.2 Soybean as an agricultural commodity ................................................................................. 7 1.3 Genetic diversity of soybean ................................................................................................ 8 1.4 Soybean pathogens ................................................................................................................ 8 1.5 Purple seed stain and Cercospora leaf blight ........................................................................ 9 1.6 Taxonomy of the genus Cercospora ..................................................................................... 9 1.7 Objectives ............................................................................................................................ 11 CHAPTER 2. EVOLUTIONARY RELATIONSHIPS AMONG FUNGAL SOYBEAN PATHOGENS CAUSING CERCOSPORA LEAF BLIGHT AND PURPLE SEED STAIN IN THE GULF SOUTH................................................................................................................ 13 2.1 Introduction ......................................................................................................................... 13 2.2 Materials and methods ........................................................................................................ 15 2.2.1 Collection and isolation ............................................................................................... 15 2.2.2 DNA extraction ............................................................................................................ 15 2.2.3 Marker selection, PCR and data set assembly ............................................................. 16 2.2.4 Model testing, phylogenetic analysis and species tree inference ................................. 19 2.2.5 Characterization and statistical analysis of mating type loci ....................................... 20 2.3 Results ................................................................................................................................. 21 2.3.1 Phylogenetic analyses .................................................................................................. 21 2.3.2 Species tree analyses .................................................................................................... 21 2.3.3 Ratio of MAT1-1 and MAT1-2 idiomorphs ................................................................ 23 2.4 Discussion ........................................................................................................................... 26 CHAPTER 3. PHYLOGENETIC MARKER DEVELOPMENT FROM INTERGENIC REGIONS FOR INFERRING RELATIONSHIPS AMONG SPECIES OF CERCOSPORA....32 3.1 Introduction ......................................................................................................................... 32 3.2 Materials and methods ........................................................................................................ 34 3.2.1 Taxon sampling and DNA extraction .......................................................................... 34 3.2.2 Genomic sequencing, preprocessing, assembly and annotation .................................. 34 3.2.3 Marker development .................................................................................................... 35 3.2.4 PCR amplification and sequencing .............................................................................. 35 3.2.5 Dataset assembly, alignment and model testing .......................................................... 36 3.2.6 Profiling phylogenetic informativeness and analyses of substitution saturation ......... 37 3.2.7 Phylogenetic inference and tree distance calculations ................................................. 38 3.3 Results ................................................................................................................................. 39 3.3.1 Genome sequencing ..................................................................................................... 39 iv 3.3.2 Marker development and phylogenetic informativeness profiling .............................. 40 3.3.3 Substitution saturation ................................................................................................. 46 3.3.4 Phylogenetic analyses .................................................................................................. 46 3.3.5 Pairwise comparision of individual gene trees based on matching split distances ...... 56 3.4 Discussion ........................................................................................................................... 57 CHAPTER 4. CONCLUSIONS AND PROSPECTS FOR FUTURE RESEARCH ................... 62 REFERENCES .............................................................................................................................. 65 APPENDIX 1. SPECIMEN INFORMATION FOR ISOLATES USED IN PHYLOGENETIC ANALYSES IN THIS STUDY .................................................................... 76 APPENDIX 2. HAPLOTYPES OF CERCOSPORA ISOLATES ............................................... 96 APPENDIX
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