G C A T T A C G G C A T genes Article SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L. Andrea R. Garfinkel * , Matthew Otten and Seth Crawford Oregon CBD, Independence, OR 97351, USA; [email protected] (M.O.); [email protected] (S.C.) * Correspondence: [email protected] Abstract: The regulation of cannabinoid synthesis in Cannabis sativa is of increasing research interest as restrictions around the globe loosen to allow the plant’s legal cultivation. Of the major cannabi- noids, the regulation of cannabigerolic acid (CBGA) production is the least understood. The purpose of this study was to elucidate the inheritance of CBGA dominance in C. sativa and describe a marker related to this chemotype. We produced two crossing populations, one between a CBGA dominant cultivar and a tetrahydrocannabinolic acid (THCA) dominant cultivar, and one between a CBGA dominant cultivar and a cannabidiolic acid (CBDA) cultivar. Chemical and genotyping analyses confirmed that CBGA dominance is inherited as a single recessive gene, potentially governed by a non-functioning allelic variant of the THCA synthase. The “null” THCAS synthase contains a single nucleotide polymorphism (SNP) that may render the synthase unable to convert CBGA to THCA leading to the accumulation of CBGA. This SNP can be reliably used as a molecular marker for CBGA dominance in the selection and breeding of C. sativa. Citation: Garfinkel, A.R.; Otten, M.; Keywords: cannabinoids; cannabinoid synthesis; CBG; hemp Crawford, S. SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in 1. Introduction Cannabis sativa L. Genes 2021, 12, 228. Cannabis sativa L. is an herbaceous annual grown for its wide variety of uses includ- https://doi.org/10.3390/genes12020 ing for fiber and feed, and for its use in producing compounds with therapeutic and 228 psychoactive properties. Recent lifting of restrictions on the cultivation and sale of C. sativa in the United States has led to a proliferation of research into the use of this crop in Academic Editor: Dariusz Grzebelus pharmaceuticals. Received: 18 December 2020 C. sativa produces organic molecules known as “cannabinoids” or “phytocannabi- Accepted: 1 February 2021 noids,” of which nearly 150 have been identified [1]. These cannabinoids are of specific Published: 4 February 2021 interest for their demonstrated value in nearly all realms of medicine from the treatment of sleep disorders [2], to epilepsy [3], to cancer [4]. Publisher’s Note: MDPI stays neutral Not all C. sativa plants produce all of the known cannabinoids in equal ratios. Plants with regard to jurisdictional claims in of C. sativa have been grouped into five broad categories, or “chemotypes,” based on the published maps and institutional affil- relative ratios of three predominant cannabinoids: tetrahydrocannabinolic acid (THCA), iations. cannabidiolic acid (CBDA), and cannabigerolic acid (CBGA) [5,6]. So-called “Type I” plants comprise of individuals that contain predominantly THCA, and contain low levels of CBDA; “Type II” plants contain THCA and CBDA in approximately equal ratios; “Type III” plants have high levels of CBDA, and relatively low levels of THCA; “Type IV” plants Copyright: © 2021 by the authors. contain CBGA, which is also the precursor compound to both CBDA [7] and THCA [8], as Licensee MDPI, Basel, Switzerland. the predominant cannabinoid; “Type V” plants are characterized by undetectable amounts This article is an open access article of any of these three cannabinoids and are referred to as “cannabinoid-free” [5,6]. distributed under the terms and The inheritance of these chemotypes have been well studied in a series of papers by conditions of the Creative Commons Attribution (CC BY) license (https:// de Meijer and co-authors [5,9–12]. In these papers, the authors proposed that THCA to creativecommons.org/licenses/by/ CBDA content ratios in Types I, II, and III plants were regulated by a two-allele system 4.0/). characterized by distinct forms at a single locus: one form responsible for THCA production Genes 2021, 12, 228. https://doi.org/10.3390/genes12020228 https://www.mdpi.com/journal/genes Genes 2021, 12, 228 2 of 14 (referred to as BT) and the other CBDA production (referred to as BD)[5,9–12]. In this single-locus model, Type I plants contained a homozygous BT:BT genotype, Type II plants contained a heterozygous BT:BD genotype, and Type III plants the homozygous BD:BD genotype [5]. Genetic mapping and molecular cloning [13], followed later by whole genome sequencing of C. sativa showed that these two alleles are not at the exact same physical location as proposed, but are situated at two distinct loci on the same chromosome and are most likely linked [14]. Therefore, these genes are inherited in a fashion like the single-locus model proposed by de Meijer et al. [5]. In the plants studied by whole genome sequencing, the THCA-dominant strain assessed contained a tetrahydrocannabinol acid synthase (THCAS) and only a pseudogenic, non-functional copy of a cannabidiol acid synthase (CBDAS), whereas the CBDA-dominant plant contained a CBDAS, but no apparent copies, pseudogenic or otherwise, of a THCAS in the expected location [14]. In their model, de Meijer and Hammond [9] proposed that Type IV plants contained a third, null allele B0 that is biochemically unable to convert CBGA into THCA or CBDA, thus leading to CBGA accumulation. While de Meijer and Hammond [9] did not provide any sequence data to confirm their null locus model, they hypothesized that the null allele was a sequence variant or mutation of the BD allele. Onofri et al. [15] followed up with gene sequencing and found that out of the three CBGA-dominant cultivars, two contained copies of a gene with sequence homology to the CBDAS and one contained a gene with sequence homology to the THCAS. Onofri et al. [15] compared the gene sequences from their Type IV plants with “wild-type” alleles from Type I and Type III plants and concluded that the alleles in Type IV plants displayed unique single nucleotide polymorphism (SNP) patterns. Therefore, the authors proposed that these sequence variants represented null alleles and were given the names BDO, sequence variant of BD of which there were two types, and BT0, sequence variant of BT [15]. Although Onofri et al. [15] suspected the role of these null alleles in CBG accumulation, we do not know of any studies that have carried out crosses to confirm the inheritance of these putative null cannabinoid synthase alleles and their associations with resultant chemotypes. In this paper, we present data from Type IV cultivars containing a novel THCAS which may represent an additional BT0 allele sequence variant and provide evi- dence for its inheritance and its role as a marker in CBGA dominance. 2. Materials and Methods 2.1. Plant Material All CBG cultivars used in the trials herein were developed as a result of the self- pollination of a plant received via an order through Seedsman (Barcelona, Spain). The initial controlled cross was performed by E. Crawford in 2016 in Eugene, OR, USA. The original plant had been determined to have trace CBGA content as based on HPLC analyses (performed by OG Analytical, Eugene, OR, USA). A total of five seeds were harvested as a result of self-pollination and plants were grown up and tested for their cannabinoid composition. A total of two of the five plants were determined to be Type III and were destroyed; three plants, named TS1-2, TS1-3, and TS1-5, were determined to be Type IV and were saved. TS1-3 and CBGA-dominant cultivar FB30 were used in trials. FB30 was developed as a result of a selective breeding process (described in next section) using TS1-3. 2.2. Breeding Crosses FB30 was developed using TS1-3 as the Type IV parent in the initial F1 cross. A series of open pollinations and outcrosses resulted in a single individual, FB30, that was then selfed; a single individual from the selfed population was used in the cross described below. Throughout the breeding process for Type IV parents, mendelian recessive inheritance of the CBGA chemotype reported by de Meijer and Hammond [9] was observed. All crosses and selections were performed at Oregon CBD (Independence, OR). CBDA- and THCA-dominant plants used in test crosses were also developed by the same company or used under license. Genes 2021, 12, 228 3 of 14 A total of two segregating populations were developed for genotype and chemotype analysis in order to test the hypothesis that the novel THCAS sequence variant is a marker for CBGA dominance. For the first population, “Cross TE”, TS1-3 (Type IV) was crossed to a Type III plant, ERB, and a single individual was then selected to produce a selfed F2 population, resulting in a population of 102 individuals. For the second population, “Cross FH”, a single selfed FB30 individual (Type IV) was pollinated by a Type I plant, HO40. A single individual was then selected to produce a selfed F2 population and then a single heterozygous (BT:BT0) individual (plant #11) was then selected to produce a selfed F3(S2) population; the resultant 105 progeny were screened. All plants were genetically female (contained two X chromosomes), but one of the parents in each cross was treated three times with 750 ppm silver thiosulphate solution at five-day intervals to induce male flower production for pollination [16,17]. Segregation ratios for chemotypes were calculated for each cross and observed and expected values were compared using a Pearson’s chi-square test for independence. 2.3. Chemotyping Chemical analyses were performed according to a protocol modified from Vaclavik et al.