DISSERTATION MOLECULAR GENETICS OF HERBICIDE RESISTANCE IN PALMER AMARANTH (AMARANTHUS PALMERI): METABOLIC TEMBOTRIONE RESISTANCE AND GEOGRAPHIC ORIGIN OF GLYPHOSATE RESISTANCE Submitted by Anita Küpper Department of Bioagricultural Sciences and Pest Management In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2018 Doctoral Committee: Advisor: Todd A. Gaines Franck E. Dayan Scott J. Nissen Anireddy S. N. Reddy Copyright by Anita Küpper 2018 All Rights Reserved ABSTRACT MOLECULAR GENETICS OF HERBICIDE RESISTANCE IN PALMER AMARANTH (AMARANTHUS PALMERI): METABOLIC TEMBOTRIONE RESISTANCE AND GEOGRAPHIC ORIGIN OF GLYPHOSATE RESISTANCE Palmer amaranth (Amaranthus palmeri) is a major weed in U.S. cotton and soybean production systems, partly because it evolved resistance to five different herbicide modes of action. Resistance to the 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibitor tembotrione in a population from Nebraska (NER) is due to enhanced metabolism. This type of non-target-site resistance is especially troublesome because of its potential for cross-resistance. Tembotrione-susceptible (NES) and NER formed the same tembotrione metabolites but NER exhibited faster 4-hydroxylation followed by glycosylation. The T50 value (time for 50% production of the maximum 4-hydroxylation product) was 4.9 and 11.9 h for NER and NES, respectively. Hydroxylation is typically catalyzed by cytochrome P450 monooxygenases (CYPs). Metabolism differences between NER and NES were most prominent under 28°C conditions and herbicide application at the four-leaf stage. An RNA-Seq transcriptome analysis was conducted with Pseudo-F2 tembotrione-resistant and -susceptible individuals originating from three separate NER x NES crosses that were sampled before, six, and twelve h after treatment (HAT). Differential gene expression analysis identified CYP72A219 and CYP81E8 as strong candidates for metabolic resistance. The contigs were constitutively expressed in resistant plants, as were the contigs for several glycosyltransferases (GTs), oxidase, and glutathione-S-transferase (GST). Exposure to tembotrione further increased their expression in both resistant and susceptible plants. Originally native to the Southwest, A. palmeri has spread throughout the country. In 2004 a population was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant crop systems. Glyphosate resistance in the species is now highly ii prevalent in USA and was also discovered in Brazil in 2015. This was confirmed by species identification with a genetic marker, dose-response studies, shikimate accumulation assay, and EPSPS copy number assay. The Brazilian population was also resistant to sulfonylurea and imidazolinone ALS inhibitor herbicides conferred by two different alleles for target-site mutations in the ALS gene (W574L and S653N). The degree of genetic relatedness among eight different populations of glyphosate-resistant (GR) and – susceptible (GS) A. palmeri from various geographic regions in USA was investigated by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimate accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri. iii ACKNOWLEDGEMENTS I want to thank Stephen Duke who encouraged me to apply for a PhD in the weed research laboratory of Colorado State University. I would also like to thank my committee, Franck Dayan, Scott Nissen, and Anireddy Reddy. I am deeply indebted to Todd Gaines who has been a phenomenal advisor. He provided an educational, safe, and supportive environment with continuous guidance and allowed me to explore any scientific area I wanted to pursue. I also learned a tremendous amount about herbicide resistance from Roland Beffa who has taken on a role equivalent to a co-advisor. He has been a dedicated and excellent brainstorming partner and mentor over the years. I would also like to thank Patrick Tranel, Christopher Preston, Philip Westra, Harish Manmathan, Paul Neve and his lab, Dale Shaner, and William McCloskey for their advice. I am grateful to have studied alongside many great graduate students in the weed lab of which many have helped me along the way: Darci Giacomini, Eric Westra, Dean Pettinga, Olivia Todd, John Coyle, Tom Getts, Kallie Kessler, Christopher van Horn, Mirella Ortiz, Neeta Soni, Derek Sebastian, Raven Bough, Rachel Seedorf, Kristen Tanz, Shannon Clark, Adrien Quicke, Abigail Barker, and Hudson Takano. I especially want to thank Karl Ravet and Eric Patterson for teaching me lab techniques and bioinformatic tools, as well as Marcelo Figueiredo for many discussions on resistance, help in the radioactivity lab and shouting out “Courage!” whenever I needed to hear it. Also, I would like to thank the people from other labs, John Long, Graham Tuttle, Jessica Warren, Stacy Endriss, Taylor Person, Craig Beil, Paul Tanger, Federico Martin, Michael Friedman, Stephen Cohen, Becky Gullberg, Margret Fleming, Justin Lee, and Tammy Brenner for their support. Many hourly workers have helped me with labor-intensive planting, harvest and seed cleaning of Palmer amaranth over the years, I want to especially acknowledge Tyler Hicks, Tyler Todd, Dillon Thompson, Jessica Scarpin, Colton Hankins, Hailey Meiners, Nicholas McKenna, Mitch Hoffman, Henrique Scatena, Bryna Burns, Beatrice Bachur, Rachel Chayer, and Crystal Sparks. I would also like to thank Susana Gonzales, Veronika Brabetz, Julia iv Unger, Thomas Schubel, Rebecka Dücker, Johannes Hermann, Ragnhild Paul, Bodo Peters, Harry Strek and especially Falco Peter from Bayer’s Herbicide Resistance Competence Center for all their advice and help during my visit. Furthermore, I want to thank Janet Dill for always having a smile on her face and guiding me through the bureaucracy of a PhD program as well as Elden Pemberton for his friendly reminders. Funding was generously provided by Bayer CropScience, Dow AgroSciences and the USDA National Institute of Food and Agriculture Hatch fund. I am grateful to my “American parents” Billie and David Novy for their visits and support from Minnesota as well as my actual parents Luzie and Anton Küpper for supporting my decision to go to graduate school on the other side of the globe, visiting me in the U.S. three times, regularly sending big packages with German candy and skyping with me almost every Saturday morning for the past four years. I want to especially thank my father for showing an interest in my research and for having read about the devices I use and science I do to be able to have challenging discussions with me. Finally, I want to thank Curtis Hildebrandt who, no matter the circumstances, has been my rock throughout the entire PhD program. v TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................................... iv 1. INTRODUCTION ................................................................................................................................ 1 FIGURES ................................................................................................................................................ 16 REFERENCES ........................................................................................................................................... 19 2. TEMBOTRIONE DETOXIFICATION IN HPPD-INHIBITOR RESISTANT PALMER AMARANTH (AMARANTHUS PALMERI S. WATS) .............................................................................. 24 INTRODUCTION .................................................................................................................................. 24 MATERIALS AND METHODS ............................................................................................................ 26 RESULTS ............................................................................................................................................... 30 DISCUSSION ........................................................................................................................................