ABSTRACT PHYLOGENOMIC STUDY OF SELECTED SPECIES WITHIN THE GENUS Zea: MUTATION RATE ANALYSIS OF COMPLETE CHLOROPLAST GENOMES Lauren Orton, M.S. Department of Biological Sciences Northern Illinois University, 2015 Melvin R. Duvall, Director This project examines the relationships within the genus Zea using complete chloroplast genomes (plastomes). Zea mays is one of the most widely cultivated crop species in the world. Billions of dollars have been spent in the commercial agriculture sector to study and improve Z. mays. While Z. mays has been well studied, the congeneric species have yet to be as thoroughly examined. For this study complete plastomes were sequenced in four species (Zea diploperennis, Zea perennis, Zea luxurians, and Zea mays subsp. huehuetenangensis) by Sanger or next- generation methods. An analysis of the microstructural mutations, such as inversions, insertion or deletion mutations (indels) and determination of their frequencies were performed for the complete plastomes. It was determined that 197 indels and 10 inversions occurred across the examined plastomes. The most common mutational mechanism was discovered to be the tandem repeat from slipped strand mispairing events. Mutation rates were calculated to determine a precise rate over time. The mutations rates for the genus fell within the range of 0.00126 to 0.02830 microstructural mutation events per year. These rates are highly variable, corresponding to the close and complex relationships within the genus. Phylogenomic analyses were also conducted to examine the differences between species within Zea. In many cases, much of the previous work examining Zea mitochondrial and nuclear data was confirmed with identical tree topologies. Divergence dates for specific nodes relative to Zea were calculated to fall between 8,700 calendar years before present for the subspecies included in this study and 1,024 calendar years before present for the perennial species included in this study. NORTHERN ILLINOIS UNIVERSITY DE KALB, ILLINOIS AUGUST 2015 PHYLOGENOMIC STUDY OF SELECTED SPECIES WITHIN THE GENUS Zea: MUTATION RATE ANALYSIS OF COMPLETE CHLOROPLAST GENOMES BY LAUREN M. ORTON ©2015 Lauren M. Orton A THESIS SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES Thesis Director: Melvin R. Duvall ACKNOWLEDGEMENTS This project would not have been possible with out the following individuals and organizations for their help and support. First, I would like to thank Dr. Melvin Duvall, Professor and thesis advisor, for his guidance and instruction on this research project. I thank the Iowa State University Seed Science Center under the direction of Mr. Mike Stahr, and the Small Seed Department Head, Ms. Kim North, for helping in the procurement of seeds for this project. Along with Mr. Stahr and Ms. North, I would like to extend my gratitude to Mr. Mark Millard of the USDA/ARS Plant Introduction Station at Iowa State University, for providing the seed accessions used within this study. I also thank Distinguished Professor, Dr. Richard King and Assistant Professor, Dr. Wesley Swingley, both faculty members of Northern Illinois University and Graduate Committee Members for this thesis project. I would also like to thank Mr. William P. Wysocki and Mr. Sean V. Burke for their assistance. Additionally, portions of this project would not have been possible without the financial support of the National Science Foundation (NSF grant DEB1120761 to MRD). Finally, I thank the Northern Illinois University College of Liberal Arts and Sciences and the Graduate School. DEDICATION This thesis is dedicated in memory of my grandparents, George and Anna Hellmuth, who always said, “An education is the only thing that another man cannot take away from you.” I also dedicate this thesis to Adam Orton, Robert & Sherry Hellmuth, Lindsey Hellmuth, Jeffery & Mary Orton, Rachel Orton, Bob & Joan Muir, Vicky Muir, and in memory of Orville & Ardella Orton, John & Ina Asplund. TABLE OF CONTENTS Page LIST OF TABLES…………………………………………………………… vi LIST OF FIGURES….………………………………………………………. vii LIST OF ABBREVIATIONS………………………………………………… viii PREFACE………………………………………………………………….. x Chapter 1. PHYLOGENOMIC STUDY OF SELECTED SPECIES WITHIN THE GENUS ZEA: MUTATION RATE ANALYSIS OF COMPLETE CHLOROPLAST GENOMES Introduction……………………………………………… 1 Methods…………………………………………..…...... 5 DNA Extraction and Next Generation Sequencing…………………………………….... 7 Sanger Sequencing…………………………....... 7 Plastome Assembly and Annotation………….... 9 Plastome Comparisons………………………..... 11 Microstructural Mutation Analysis…………...... 11 v Page Mutation Rates and Divergence Times……….... 12 Results....................................................................... 16 Phylogenetics: Sequence Analysis...................... 16 Phylogenetics: Microstructural Mutation Analysis.... 19 Phylogenetics: Combined Sequence and Microstructural Mutation Analysis......................... 22 Divergence Time Estimation Analysis.................... 22 Mutation Rates..................................................... 24 Zea perennis Insertion........................................... 25 Discussion...................................................................... 27 Phylogenomic and Mutation Rate Analysis............. 27 Divergence Analysis (BEAST).............................. 29 Zea perennis “Insertion”....................................... 30 Conclusion..................................................................... 31 LITERATURE CITED............................................................ 32 LIST OF TABLES Table Page 1 Plastome characteristics of selected species...………………………..… 6 2 List of species-specific primers designed for Zea diploperennis………. 8 3 GenBank accessions of species for which plastomes were previously determined…………………………………………............. 10 4 Results of BEAST analysis divergence times..………………………… 15 5 ML, MP, BI analysis results for sequence, microstructural mutation (MME), and combined data sets...................... 18 6 Rare genomic changes (RGCs) characterized across the species included in this study..................................................,,,,,...... 21 7 Sequence alignment of the 83 bp insertion/deletion first identified by Doebley et al., 1987, as an insertion in the Zea perennis plastome.....................................................………… 26 LIST OF FIGURES Figure Page 1 Maximum likelihood best tree showing bootstrap support values of MP analyses………………………............................................................ 17 2 Diagram examining the relationship between the number of occurrences and lengths of MME.……………....................….…........ 20 3 Tree result of BEAST analysis with node ages shown............................. 23 LIST OF ABBREVIATIONS AA Amino Acid ACRE Anchored Conserved Region Extension BEAST Bayesian Evolutionary Analysis Sampling Trees BLAST Basic Local Alignment Search Tool bp Base pair bs Bootstrap Support value CDS Coding Sequence CIPRES Cyber Infrastructure for Phylogenetic RESearch GPWGI (II) Grass Phylogeny Working Group I (II) IR Inverted Repeat LSC Long Single Copy MCMC Markov Chain Monte Carlo ML Maximum Likelihood MME Microstructural Mutation Event MP Maximum Parsimony NGS Next Generation Sequencing NS Nucleotide Sequence PAUP * Phylogenetic Analysis Using Parsimony * and other methods RGC Rare Genomic Change ix SSC Short Single Copy SSM Slipped-strand mispairing XSEDE eXtreme Science and Engineering Discovery Environment PREFACE Maize (Zea mays subsp. mays) is a common, globally consumed and cultivated crop. In 2014 alone, it was estimated by the USDA that nearly 988.70 million metric tons of corn (maize) was produced. Along with wheat (Triticum aestivum) and rice (Oryza sativa), maize accounts for approximately 94% of all cereal grains consumed worldwide (Ranum et al., 2014). Many scholars believe that maize was domesticated some, 7,000-10,000 years ago from wild grasses or teosintes. This domestication event was a pivotal moment in humankind’s shift from hunter- gatherer to agriculturalist within a couple of thousand years (Ranum et al., 2014). Zea mays subsp. mays (Andropogoneae; Panicoideae; Poaceae), the only cultivated crop in the genus, has been well studied for its importance in agriculture and human nutrition. However, less is known about the other closely related taxa within the genus Zea. The genus is comprised of five species (Zea luxurians, Zea perennis, Zea diploperennis, Zea nicaraguaensis, and Zea mays). Zea mays is further comprised of four subspecies (subsp. huehuetenangensis, parviglumis, mexicana, and mays) (Doebley, 2003). The non-crop species and subspecies within the genus are referred to as the teosintes, as their morphologies more closely resemble wild grasses. Historical geographic distributions of the genus Zea include areas stretching from Northern Mexico into Central America, including the countries of Nicaragua and Guatemala (Hufford et al., 2012). The average range of temperatures for this region fall between 15-28°C, and average precipitation totals range from 200 mm to upwards of 2,000 mm (Hufford et al., xi 2012). However, despite being historically located in hot, humid and moderately temperate habitats, maize has readily proven that it can easily colonize the globe, given that corn fields are present in countries like China and India, across portions of Europe and North, Central, and South Americas (USDA). Despite the different global climates (some