Genome The genetics of Cannabis – genomic variations of key synthases and their effect on cannabinoids content Journal: Genome Manuscript ID gen-2020-0087.R1 Manuscript Type: Mini Review Date Submitted by the 17-Sep-2020 Author: Complete List of Authors: Singh, Aparna; University of Lethbridge, biological sciences Bilichak, Andriy; Morden Research and Development Centre Kovalchuk, Igor; University of Lethbridge Keyword: Cannabis sativaDraft L., hemp, marijuana, THCAS, CBDAS Is the invited manuscript for consideration in a Special Genome Biology Issue? : © The Author(s) or their Institution(s) Page 1 of 43 Genome 1 The genetics of Cannabis – genomic variations of key synthases and their 2 effect on cannabinoids content 3 4 Aparna Singh1, Andriy Bilichak2 and Igor Kovalchuk1* 5 6 1 – Department of Biological Sciences, University of Lethbridge, Lethbridge, AB T1K 7 3M4, Canada, 2 – Morden Research and Development Center, Agriculture and Agri-Food 8 Canada, Morden, MB R6M 1Y5, Canada 9 * Corresponding author: [email protected] 10 11 Draft 12 13 14 15 16 17 18 19 20 21 22 23 1 © The Author(s) or their Institution(s) Genome Page 2 of 43 24 Abstract 25 Despite being a controversial crop, Cannabis sativa L. has a long history of 26 cultivation throughout the world. Following recent legalisation in Canada, it is emerging 27 as an important plant for both medicinal and recreational purposes. Recent progress in 28 genome sequencing of both cannabis and hemp varieties allows for systematic analysis 29 of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single 30 nucleotide polymorphisms in the coding regions of cannabinoid synthases play important 31 role in determining plant chemotype. Deep understanding of how these variants affect 32 enzymes activity and accumulation of cannabinoids will allow breeding of novel cultivars 33 with desirable cannabinoid profile. Here we present a short overview of the major 34 cannabinoid synthases and present Draftthe data on the analysis of their genetic variants and 35 their effect on cannabinoid content using several in-house sequenced Cannabis cultivars. 36 37 Keywords: Cannabis sativa L., hemp, marijuana, THCAS, CBDAS 38 39 40 41 42 43 44 45 46 2 © The Author(s) or their Institution(s) Page 3 of 43 Genome 47 Introduction 48 Cannabis sativa L. (including marijuana and hemp) is a herbaceous plant belonging 49 to the Cannabaceae family (Vavilov and Freier 1951, Brizicky 1966). Being one of the 50 major source of medicine, oil and fibre, it has been extensively cultivated in many 51 countries (Camp 1936, Godwin 1967, Quimby, Doorenbos et al. 1973, Schultes, Klein et 52 al. 1974, Kriese, Schumann et al. 2004, Laverty, Stout et al. 2019). Since ancient times, 53 the Cannabis plant is valued for its medicinal properties and used for treating pain, 54 nausea, depression, glaucoma, asthma, insomnia, etc. (Mechoulam, Lander et al. 1976, 55 Duke and Wain 1981). Although therapeutic properties of cannabinoids have been 56 extensively studied, the role of phytocannabinoids within plants is poorly understood. 57 Cannabis is diploid and its karyotypeDraft consists of nine autosomes and a pair of sex 58 chromosomes (2n = 18+XX for female or XY for male) (Flemming, Muntendam et al. 2007, 59 Divashuk, Alexandrov et al. 2014, Vyskot and Hobza 2015). The haploid genome size of 60 female and male plants is approximately 818 Mb and 843 Mb, respectively (Sakamoto, 61 Akiyama et al. 1998). 62 The medicinal properties of Cannabis are owed to the presence of terpenophenolic 63 compounds known as cannabinoids. They can modulate the human endocannabinoid 64 system and are useful for various physiopathological processes (Izzo, Borrelli et al. 2009). 65 They are named as cannabinoids due to their typical exhibition of a C21 terpenophenolic 66 structure (Hillig 2004, Brenneisen 2007, De Meijer 2014). To date, more than 120 67 cannabinoids (class of metabolites specific to Cannabis plant), including cannabidiol 68 (CBD), tetrahydrocannabinol (THC), cannabichromene (CBC), cannabigerol (CBG) and 69 their propyl homologs CBDV, THCV, CBCV, CBGV have been identified including those 3 © The Author(s) or their Institution(s) Genome Page 4 of 43 70 that occur in plant and their derivatives (ElSohly 2007, Radwan, ElSohly et al. 2009, de 71 Meijer and Pertwee 2014). Two of the most common ones are THCA and CBDA, with 72 varying levels among cultivars. The acidic forms of these cannabinoids, THCA and CBDA, 73 are present in major quantities inside plant (De Meijer, Hammond et al. 2009, Swift, Wong 74 et al. 2013). Δ9-tetrahydrocannabinol (THC) is the main psychoactive cannabinoid 75 responsible for therapeutic and hallucinogenic effects and therefore extensively studied 76 (Brenneisen, Egli et al. 1996, Long, Malone et al. 2005, Sirikantaramas, Taura et al. 77 2007). CBG was the first compound isolated from C. sativa in a pure form and considered 78 as an intermediate precursor to most of the phytocannabinoids. Recently, seven more 79 CBG type cannabinoids have been isolated from the buds of the mature female C. sativa 80 plants (Appendino, Giana et al. 2008,Draft Flores-Sanchez and Verpoorte 2008, Radwan, 81 Ross et al. 2008, Radwan, ElSohly et al. 2009, Pollastro, Taglialatela-Scafati et al. 2011). 82 In this review, we will discuss sequence variations in synthase enzymes involved in 83 biosynthesis of cannabinoids, causes of their occurrence as well as their effect on 84 cannabinoid content leading to chemotype diversity. This review will also cover the 85 significance of these variations in distinguishing Cannabis varieties and in the 86 establishment of novel cultivars with unique chemotypes having potential to meet the 87 requirements of future pharmaceutical demands. 88 89 Historical perspective of use of Cannabis-derived products 90 Cannabis is one of the earliest cultivated plant by mankind and is native to western, 91 central and eastern Asia (Li 1974, Small 2015). It has been used traditionally as a herbal 92 medicine in ancient times by Chinese, Tibetan and Indian civilizations (Mechoulam and 4 © The Author(s) or their Institution(s) Page 5 of 43 Genome 93 Parker 2013). Cannabis has also been associated with religious practices in Southern 94 Asia, specially in India, where written records of its holy use were found (Hasan 1974). 95 The evidence of its first cultivation comes from China as early as 4000 B.C. to obtain fibre, 96 medicine and food for humans and cattle (Small and Cronquist 1976, Jiang, Li et al. 2006). 97 There were several reports supporting shamanistic uses of Cannabis suggesting ancient 98 Chinese were well aware of its psychotropic properties (Touw 1981, Farag and Kayser 99 2017). 100 Hemp, a type of Cannabis sativa plant species, is presumed as one of the oldest 101 sources of fibre and has been valued for its strength and durability, hence was used for 102 manufacturing ropes and clothes in earlier times (Allegret 2013). Nowadays, it is 103 particularly grown for industrial purposesDraft to obtain its derivatives such as oil, fibre and 104 food. In around 2000 B.C., hemp was introduced as fibre to Egypt, Europe and western 105 Asia (Schultes 1979). Hemp seeds were indeed among one of the five most important 106 grains in ancient China, where it was considered as staple food until tenth century 107 (Cheatham, Johnston et al. 2009). In the modern world, hemp is also grown for its 108 medicinal and nutritional value (Farag and Kayser 2017). 109 Apart from food, Chinese used plant extracts and seeds of Cannabis to treat various 110 illnesses including constipation, malaria, rheumatic pain and female reproductive system 111 disorders. They also used different parts of plants such as roots and foliage as a medicine 112 for various treatments (Wang and Wei 2012). Historically, hemp seeds were used for the 113 treatment of jaundice, sores pain, skin diseases, blood related illnesses and constipation 114 (Callaway 2004). A popular beverage in Scandinavia known as “Maltos-Cannabis” was 115 used in early twentieth century for treating anemia, asthenia, emaciation and pulmonary 5 © The Author(s) or their Institution(s) Genome Page 6 of 43 116 diseases (Dahl and Frank 2011). Considerable evidence of Cannabis use as medicine 117 and recreational drug in different forms (Bhang, ganja and charas) was also reported from 118 ancient India approximately 1000 years ago. It is considered as a sacred plant in Hindu 119 religion and was used in several religious rituals and ceremonies (Hasan 1974). It has 120 been also actively used as an analgesic, tranquilizer, anticonvulsant, anti-inflammatory, 121 aphrodisiac, antispasmodic and antibiotic. Cannabis was also used in Tibet for religious, 122 medicinal and meditation purposes (Touw 1981). In Africa, Cannabis is known since the 123 fifteenth century and is used for the treatment of snake bite and diseases like malaria, 124 asthma, fever and dysentery. In South America, Cannabis use presumably started for 125 recreational and medicinal purposes during seventeenth and eighteenth century (Zuardi 126 2006, Rubin 2011). Cannabis was extensivelyDraft grown for use as fibre in Europe and north 127 Asia, whereas in Africa and Southern Asia it has been mostly used as a recreational, 128 medicinal, and cultural drug. Due to its classification as narcotics, very limited research 129 related to Cannabis and its effects on human body was conducted previously. Even today, 130 Cannabis is one of the major illicitly cultivated plant in the world, but only recently it was 131 made legal in several parts around the globe, and great advances have been made to 132 understand how cannabinoids affect human brain and nervous system, to develop new 133 Cannabis-based therapeutic products. 134 135 Taxonomical Classification 136 Historically, vernacular taxonomy differentiated three different Cannabis groups - C. 137 sativa (high CBD-containing plant), C. indica (high THC-containing plant) and C.