University of Northern Colorado Scholarship & Creative Works @ Digital UNC Dissertations Student Research 5-2019 A Multifaceted Approach to Address Variation in Cannabis Sativa Anna Louise Schwabe Follow this and additional works at: https://digscholarship.unco.edu/dissertations Recommended Citation Schwabe, Anna Louise, "A Multifaceted Approach to Address Variation in Cannabis Sativa" (2019). Dissertations. 554. https://digscholarship.unco.edu/dissertations/554 This Text is brought to you for free and open access by the Student Research at Scholarship & Creative Works @ Digital UNC. It has been accepted for inclusion in Dissertations by an authorized administrator of Scholarship & Creative Works @ Digital UNC. For more information, please contact [email protected]. © 2019 ANNA LOUISE SCHWABE ALL RIGHTS RESERVED UNIVERSITY OF NORTHERN COLORADO Greeley, Colorado The Graduate School A MULTIFACETED APPROACH TO ADDRESS VARIATION IN CANNABIS SATIVA A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Anna Louise Schwabe College of Natural and Health Sciences School of Biological Sciences Biological Education May 2019 This Dissertation by: Anna Louise Schwabe Entitled: A Multifaceted Approach to Address Variation in Cannabis sativa has been approved as meeting the requirement for the Degree of Doctor of Philosophy in College of Natural and Health Sciences in School of Biological Sciences, Program of Biological Education. Accepted by the Doctoral Committee ____________________________________________________ Mitchell McGlaughlin, Ph.D., Chair ____________________________________________________ Robert Reinsvold, Ph.D., Committee Member ____________________________________________________ Nolan Kane, Ph.D., Committee Member ____________________________________________________ Richard Hyslop, Ph.D., Committee Member Date of Dissertation Defense ______March 1, 2019___________ Accepted by the Graduate School ____________________________________________________ Linda L. Black, Ed.D., LPC Associate Provost and Dean Graduate School and International Admissions Research and Sponsored Projects ABSTRACT Schwabe, Anna Louise. A Multifaceted Approach to Address Variation in Cannabis sativa. Published Doctor of Philosophy Dissertation, University of Northern Colorado, 2019. For thousands of years, humans have cultivated and dispersed Cannabis sativa L. across the globe. Although Cannabis has been largely illegal worldwide for decades, public perceptions and attitudes are changing. Increasing interest in potential Cannabis usage worldwide and nationwide is leading to less restrictions to make way for an expanding and lucrative industry with numerous applications. Although only one species is formally recognized in the Cannabis genus, thousands of years of artificial selection for diverse phenotypes and uses have resulted in two major usage groups; hemp-types which are defined worldwide as having very low levels of THC (< 1.0%), and drug-types which exceed a specified level of THC that varies among nations. The drug-type category includes three commonly used subcategories including Sativa, Indica and Hybrid types, and newly developed high CBD varieties that have more THC than hemp-types but are not bred for high THC. The quality of federally produced Cannabis for medical studies in the U.S. has recently been brought into question, and we included samples to determine the genetic relationship to these groups. Phenotypic variation in Cannabis gives rise to commonly referenced categories, but sources of variation are unclear and understudied. Phenotypes are observable characteristics that results from a combination of both genotype and the environment. iii The preferred method of propagation for Cannabis is cloning, and therefore variation within varietals should be from differences in environmental factors. Ten microsatellite markers were developed de-novo to investigate four aims: (1) genetic variation within strains, (2) genetic relationships among the common categories, (3) if genetic variation is detectable through olfactory sensation, and (4) how genetic variation is reflected in phytochemical levels. This dissertation includes four manuscript chapters representing each aim and uses a genetic basis for a multifaceted approach to investigate variation in Cannabis sativa. Substantial genetic variation was found within strains from obtained from different facilities. Genetic divergence between hemp and drug-types was genetically supported, but the Sativa, Indica, and Hybrid subcategories were not genetically well defined. The high CBD strains appear to bridge the genetic gap between hemp and drug-types, and federally grown research grade marijuana was genetically more similar to hemp than Cannabis available through the legal cannabis market. Genetic imposters within a strain had measurable aromatic differences, but there was considerable variation in aromas among samples with identical genetic identity. Analyses of both terpene and cannabinoid profiles among individuals with identical genotypes acquired from different sources varied considerably indicating environmental variation has a substantial impact on phenotype in Cannabis. Together these results show a need for the Cannabis industry to implement regulatory checks in the form of genetic testing in order to provide consistency, especially for medical applications. These results demonstrate the need for genotyping in order for phenotypic consistency to be achieved if standard growing conditions can be established. When genetic verification and standard protocols are established, deviations iv in phenotypic changes can be identified and disclosed to consumers so they are aware that there may be abnormal effects. This investigation highlights the need for additional research to provide consistent products, which is especially important for medical marijuana flower products. In order to provide consumers consistent products, it is imperative to understand sources of variation. Consumers deserve to be provided with quality consistent products as the industry continues to grow on a global scale. v ACKNOWLEDGEMENTS I got this crazy idea to pursue a PhD after the legalization of adult recreational Cannabis use and possession in Colorado. Following discussions of variations in strains obtained from different dispensaries, it dawned on me that I could research why that would be given my extensive background in genetic relationships among closely species and plant populations of the same species. However, I knew little about Cannabis, marijuana, hemp, strains, the history of the plant etc.- so I had to learn everything from the ground up (literally), and there have been so many people who have helped me through this journey of the pursuit of knowledge, and I would like to try to thank each and every one of you. First, I would like to thank Dr. M (you know who you are) for bringing the issue of strain variation to my attention and giving me such a kickass idea and the beginning point for my research. At such a pivotal point in Cannabis reform, I was able to pitch the idea and build a case for my doctoral research to Mitchell McGlaughlin, who has been my advisor and mentor throughout this process. So, most importantly, I need to give my biggest chunk of the gratitude wheel to him. Mit has been the best research advisor and mentor I have ever had- although I have nothing to compare him to, because he was also my advisor for my master’s thesis. However, I wouldn’t have been able to follow such a crazy idea researching something that was still taboo and largely illegal around the world without support from Mit. He has been supportive and encouraging; he has been my “that’s a great idea, but let’s think about this” guy; he gave me the idea for the salad vi spinner (worked like a charm); he was always there when I called “Mit! Mit! Mit!” outside his office door for help; he was there to help field media interviews to make sure I knew what I was talking about (turns out I do); he let me use his lab and supplies, and he even paid for some stuff (Botany 2018). I also thank Susan Keenan, who not only let me come back for a third degree at UNC but also advocated for my Cannabis research. Thank you to my committee members, Rob Reinsvold, Richard Hyslop and Nolan Kane. Rob, I know you are probably sick of me, but I promise this time I am leaving ;). Richard, thank you for holding my hand and having the patience to walk me through the chemistry stuff since it has been about 12 years since I last sat in a chemistry class. Nolan Kane, I still can’t even believe you said yes to sitting on my committee. When I first learned about Nolan and the Cannabis research in his lab at the University of Colorado, I was star-struck. Sending the email to request him as a committee member felt about as promising as asking Snoop to perform at my birthday party, but he said YES! Nolan is amazing- smart and kind, and so funny- I don’t actually think he knows how funny is. Nolan encouraged me to have confidence, and not to let all the dude-bro’s in the industry talk down to me, because apparently I know what I’m talking about. Seriously though, thank you so much Thank you to Daniela Vergara who I first met at the Cannabis Research Institute Conference. I was so nervous to be presenting my work, but she followed my presentation and announced to her audience (standing room only), that we had found the same things using different tools, which was so nice of her, and helped give