Genomic and Chemical Diversity in Cannabis, C. Lynch et al., 2015

Genomic and Chemical Diversity in Cannabis

Ryan C. Lyncha,e, Daniela Vergaraa, Silas Tittesa, Kristin Whitea, C. J. Schwartzb, Matthew J. Gibbsb, Travis C. Ruthenburgc,d, Kymron deCesarec, Donald P. Landc, and Nolan C. Kanea


Doi : 10.1080/07352689.2016.1265363



Plants of the Cannabis genus are the only prolific producers of phytocannabinoids, compounds that strongly interact with the evolutionarily ancient endocannabinoid receptors shared by most bilaterian taxa. For millennia, the plant has been cultivated not only for these compounds, but also for food, rope, paper, and clothing. Today, specialized varieties yielding high-quality textile fibers, nutritional seed oil, or high cannabinoid content are cultivated across the globe. However, the genetic identities and histories of these diverse populations remain largely obscured. We analyzed the nuclear genomic diversity among 340 Cannabis varieties, including fiber and seed oil hemp, high cannabinoid drug-types, and feral populations. These analyses demonstrate the existence of at least three major groups of diversity with European hemp varieties more closely related to narrow leaflet drug-types (NLDTs) than to broad leaflet drug-types (BLDTs). The BLDT group appears to encompass less diversity than the NLDT, which reflects the larger geographic range of NLDTs, and suggests a more recent origin of domestication of the BLDTs. As well as being genetically distinct, hemp, NLDT, and BLDT genetic groups produce unique cannabinoid and terpenoid content profiles. This combined analysis of population genomic and trait variation informs our understanding of the potential uses of different genetic variants for medicine and agriculture, providing valuable insights and tools for a rapidly emerging valuable industry.


I. Introduction

Plants of the genus Cannabis (Cannabaceae; hemp, drug- type) have been used for thousands of years for fiber, nutri- tional seed oil, and medicinal or psychoactive effects. Archeological evidence for hemp fiber textile production in China dates to at least as early as 6000 years ago (Li, 1973), but possibly as early as 12,000 years ago (Russo, 2011), sug- gesting that Cannabis was one of the first domesticated fiber plants. Archeological evidence for medicinal or shamanistic use of Cannabis has been found at Indian, central Asian, and Middle-Eastern sites (Russo, 2007), further illustrating the widespread extent of Cannabis utilization throughout the human history. A central Asian site of domestication is often cited (Schultes et al., 1974), although, genetic analyses suggest that two independent domestication events may have occurred separately (Hillig, 2005).

Cannabis plants are usually annual wind-pollinated dioecious herbs, though individuals may live more than a year in sub-tropical climates (Cherniak, 1982) and monoecious populations exist (de Meijer et al., 2003). The taxonomic composition of the genus remains unre- solved with two species (Cannabis indica and Cannabis sativa) commonly cited (Hillig, 2005); although Canna- bis ruderalis is sometimes proposed as a third species that contains northern short-day or auto-flowering plants (Small and Cronquist, 1976). Monospecific treat- ment of the genus as C. sativa L. is also common (van Bakel et al., 2011) and various alternative nomenclature schemes (e.g. C. sativa subsp. indica var. kafiristanica) are sometimes cited (Schultes et al., 1974). Even though an extensive monograph on the genus has recently been published (Small, 2015a), limited genetic and experimental data leave the questions of taxonomy unresolved (Clarke and Merlin, 2015; Small, 2015b).

The geographical and ecological range of Cannabis is unusually broad, with cultivated populations growing outdoors on every continent, except Antarctica, in a wide range of environments from sub-arctic to temperate to tropical, and from sea level to over 3000 m elevation (Clarke and Merlin, 2013; Glanzman, 2015). Feral or wild populations are also found as far north as the edge of the Arctic Circle in Eurasia, but they are most com- mon in well-drained soils of temperate continental eco- systems in Eurasia and North America, while tropical populations are absent or rare (Clarke and Merlin, 2013). The species contains extensive phytochemical diversity, particularly in cannabinoid and terpenoid pro- files (Hillig and Mahlberg, 2004; Hillig, 2005), and it also shows extensive diversity of morphological and life-his- tory characteristics, further fueling debate regarding the taxonomic status and origins of Cannabis domestication. One distinctive feature of the Cannabis genus is the production of a tremendous diversity of compounds called cannabinoids, they are so named because they are not produced in high levels in any other plant species (Bauer et al., 2008). Cannabinoids are a group of at least 74 known C21 terpenophenolic compounds (ElSohly and Slade, 2005; Radwan et al., 2008) responsible for many reported medicinal and psychoactive effects of Cannabis consumption (Poklis et al., 2010). Some estimates for the total number of phytocannabinoids range to well over a hundred (Mehmedic et al., 2010), though this number includes breakdown products as well as compounds found at extremely low levels. The plants produce a non- psychoactive carboxylic acid form of these compounds, which requires heating to convert cannabinoids into the psychoactive decarboxylated forms. Interestingly, these compounds have pronounced neurological effects on a wide range of vertebrate and invertebrate taxa suggesting an ancient origin of the endocannabinoid receptors, per- haps as old as the last common ancestor of all extant bilat- erians over 500 MYA (McPartland et al., 2006). The plant compounds thus produced have the potential to affect a broad range of metazoans, though their ecological func- tions in nature are not well understood. Indeed, the sug- gested roles for these compounds include many biotic and abiotic defenses, such as suppression of pathogens and herbivores, protection from UV radiation damage, and attraction of seed dispersers. These hypotheses about the selective benefits of cannabinoid production remain speculative, as none has been conclusively verified to date. We know more, however, about the evolutionary forces during cultivation and domestication.

In particular, high delta-9-tetrahydrocannabinolic acid (THCA) content has been selected for (Mechoulam and Gaoni, 1967). When heated, THCA is converted to delta- 9-tetrahydrocannabinol (THC), which has potent psycho- active (Volkow et al., 2014), appetite-stimulating (Berry and Mechoulam, 2002), analgesic (Zogopoulos et al., 2013) and antiemetic (Tramer et al., 2001) effects. These effects are mediated through interactions with human endocannabinoid CB1 receptors found in the brain (Di Marzo et al., 2004), and CB2 receptors, which are concen- trated in peripheral tissues (Pacher and Mechoulam, 2011). Other THC receptor binding locations are hypoth- esized as well (De Petrocellis et al., 2011). After several decades of accelerated clandestine cultivation technique and breeding improvements, some modern lines can now yield dried unpollinated pistillate inflorescence material that contains over 30% THCA by dry weight (Swift et al., 2013). However, other cannabinoids may also be present in high concentrations. In particular, high cannabidiolic acid (CBDA) plants are used in some hashish prepara- tions (Rustichelli et al., 1996; Hanus et al., 2016) and are presently in high demand as an antiseizure therapy (Devinsky et al., 2014). In contrast with THC, which acts as a partial agonist of the CB1 and CB2 receptors, CBD does not have strong psychoactive properties as THC, but instead it has antagonist activity on agonists of the CB1 and CB2 receptors (Pertwee, 2008). Thus, the two most abundant cannabinoids produced in Cannabis have, to some degree, opposing neurological effects.

THCA and CBDA are alternative products of a shared precursor, cannabigerolic acid (CBGA) (Fellermeier et al., 2001). A single locus with co-dominant alleles was pro- posed to explain patterns of inheritance for THCA to CBDA ratios (de Meijer et al., 2003; Staginnus et al., 2014). However more recent quantitative trait loci (QTL) map- ping experiments (Weiblen et al., 2015), expression studies (Onofri et al., 2015), and genomic analyses (van Bakel et al., 2011) paint a more complex scenario with several linked paralogs responsible for the various THCA and CBDA phenotypes. Other cannabinoids such as cannabi- gerol (CBG) (Borrelli et al., 2014), cannabichromene (CBC) (Izzo et al., 2012), and delta-9-tetrahydrocannabi- varin (THCV) (Mcpartland et al., 2015) demonstrate phar- macological promise, and can also be produced at high levels by the plant (de Meijer and Hammond, 2005; de Meijer and Hammond, 2016; de Meijer et al., 2008). Addi- tionally, Cannabis secondary metabolites such as terpe- noids and flavonoids likely contribute to therapeutic or psychoactive effects (Russo, 2011). For example, b-myr- cene, humulene, and linalool are proposed to produce sed- ative effects associated with specific varieties (Hazekamp and Fischedick, 2012).


Genomic and Chemical Diversity in Cannabis