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The biosynthetic pathway of the most common cannabinoids in Cannabis plants. For each step, the relative enzyme has been indicated (if known), and the state of the alleles at the B locus is proposed, accounting for the chemical phenotype. The inset shows the B190 / B200 markers, obtained amplifying Cannabis DNA with the RAPD-deriving SCAR primers. Note the codominancy of this marker.
Source publication
The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also...
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... presently at GW Pharmaceuticals (UK). Different segregating F2s were obtained from initial crosses between inbred lines with contrasting chemotypes (I and III, i.e. almost pure THC and almost pure CBD); the genetic analysis of the gas-chromatographic data demonstrated that the F1 offspring was completely hybrid (chemotype II), while all three chemotypes were again present in the F2 generations, in a 1:2:1 proportion (pure THC:mixed THC + CBD:pure CBD) within each progeny; this finding was in agreement with the hypothesis of one gene and two codominant alleles ( B D and B T ) for chemotype determination. This hypothesis is not the only possible, but it is the simplest explaining of the presently available data. The F2 segregating groups were screened by RAPD markers using the bulk segregant approach, and several CBD- or THC-associated markers were identified. All these markers behaved as dominant, except one (named B190/B200 ; Figure 4 in- sert), deriving from a CBD-associated RAPD fragment, that once transformed into a SCAR marker, turned out to be codominant, and therefore able to genotype completely at the B locus the plants. The efficiency of correct identification of the chemotypes was 88% for pure THC plants, 95% for mixed chemotype plants, and 98% for pure CBD plants (de Meijer et al., 2003). However, these markers, very useful within the pedigrees created from the starting inbred lines, were not equally effective in unrelated materials, like the dioecious fiber varieties Carmagnola, Fibranova or Eletta Campana. Besides, despite the very good degree of association with the chemotype shown by marker B190/B200 , it cannot be taken into consideration for the marker-assisted identification of illicit crops and for legal purposes (P. Cantin, personal communication). In this case, in fact, a marker must be 100% linked to the chemotype, for its exploita- tion as an effective and reliable drug repression tool. The only marker with these characteristics is of course the gene itself. In the NCBI database, there are the sequences corresponding to the genes for the THC- and CBD-synthases (entry numbers AB057805, E55107, E55108, E55090 and E55091); these sequences have been patented by a research group of the Taisho Pharmaceuticals Company, Japan. The sequences of the genes coding for THC- and CBD-synthase show very high similarities; the identity along the 1635 bp coding sequence is 89.3%. The major difference is apparently a missing nucleotide triplet in the positions 757–759 of the THC-synthase sequence. The translated protein sequence is 545 and 544 aminoacids, for CBD- and THC-synthase, respectively. The THC-synthase has a missing aminoacid (SER) in position 253 of the sequence. Out of the 545 aminoacids stretch, only 87 (16%) are different between the two enzymes (including the missing one); about half of these variations, however, are between aminoacids of the same type. The aminoacid changes are quite evenly distributed throughout the sequence, the longest variant stretch consisting of six aminoacids in positions 491– 496. These differences are large enough to allow the construction of specific primers, able to identify in the different chemotypes the allelic complement of each plant. In our laboratory, we devised a three-primers system able to amplify, in a single PCR reaction of leaf tissue fragments, the DNA sequences identifying the allelic status at the B locus (A. Carboni, unpublished). Genetic analysis of heterozygous ( B D B T ) plants from different crosses, revealed that the THC:CBD ratio may vary slightly but consistently and heritably around the value of 1 (de Meijer et al., 2003). This sug- gests the possibility that several isoenzymatic forms of THC- and CBD-synthases exist in different germplasm. Confirmation of this hypothesis, presently in progress through the sequencing in our laboratory of these possible variants, could lead to the identification of further alleles of potential interest at the B locus. Besides, the identification, either by progeny analysis or by direct sequencing, of the alleles responsible for the synthesis of the several cannabinoids described in C. sativa , would open the possibility of assisted selection in Cannabis, bred not only as a fiber crop, but also for its pharmaceutical applications (see G. Guy and R. Pertwee contributions in this special issue). Chemotype IV and V, having CBG or no cannabinoids, are of remarkable interest for both fiber and pharmaceutical purposes; the identification of the alleles at the B locus responsible for the accumulation of CBG or for the absence of cannabinoids (Figure 4), would open the way to the development of molecular markers for these chemotypes. The knowledge of the genes and alleles responsible for the different chemotypes could also lead to the manipulation of the pathway in both plants and cell cultures; the availability of chemotype-specific cell cultures in which the cannabinoid biosynthesis is made active by manipulations of the key enzymes of their pathway, could lead to the development of bioreactors useful for the in vitro large-scale production of specific cannabinoids for the pharmaceutical industry. However, C. sativa remains primarily a fiber crop, and a great deal of work is being done for ...
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... presently at GW Pharmaceuticals (UK). Different segregating F2s were obtained from initial crosses between inbred lines with contrasting chemotypes (I and III, i.e. almost pure THC and almost pure CBD); the genetic analysis of the gas-chromatographic data demonstrated that the F1 offspring was completely hybrid (chemotype II), while all three chemotypes were again present in the F2 generations, in a 1:2:1 proportion (pure THC:mixed THC + CBD:pure CBD) within each progeny; this finding was in agreement with the hypothesis of one gene and two codominant alleles ( B D and B T ) for chemotype determination. This hypothesis is not the only possible, but it is the simplest explaining of the presently available data. The F2 segregating groups were screened by RAPD markers using the bulk segregant approach, and several CBD- or THC-associated markers were identified. All these markers behaved as dominant, except one (named B190/B200 ; Figure 4 in- sert), deriving from a CBD-associated RAPD fragment, that once transformed into a SCAR marker, turned out to be codominant, and therefore able to genotype completely at the B locus the plants. The efficiency of correct identification of the chemotypes was 88% for pure THC plants, 95% for mixed chemotype plants, and 98% for pure CBD plants (de Meijer et al., 2003). However, these markers, very useful within the pedigrees created from the starting inbred lines, were not equally effective in unrelated materials, like the dioecious fiber varieties Carmagnola, Fibranova or Eletta Campana. Besides, despite the very good degree of association with the chemotype shown by marker B190/B200 , it cannot be taken into consideration for the marker-assisted identification of illicit crops and for legal purposes (P. Cantin, personal communication). In this case, in fact, a marker must be 100% linked to the chemotype, for its exploita- tion as an effective and reliable drug repression tool. The only marker with these characteristics is of course the gene itself. In the NCBI database, there are the sequences corresponding to the genes for the THC- and CBD-synthases (entry numbers AB057805, E55107, E55108, E55090 and E55091); these sequences have been patented by a research group of the Taisho Pharmaceuticals Company, Japan. The sequences of the genes coding for THC- and CBD-synthase show very high similarities; the identity along the 1635 bp coding sequence is 89.3%. The major difference is apparently a missing nucleotide triplet in the positions 757–759 of the THC-synthase sequence. The translated protein sequence is 545 and 544 aminoacids, for CBD- and THC-synthase, respectively. The THC-synthase has a missing aminoacid (SER) in position 253 of the sequence. Out of the 545 aminoacids stretch, only 87 (16%) are different between the two enzymes (including the missing one); about half of these variations, however, are between aminoacids of the same type. The aminoacid changes are quite evenly distributed throughout the sequence, the longest variant stretch consisting of six aminoacids in positions 491– 496. These differences are large enough to allow the construction of specific primers, able to identify in the different chemotypes the allelic complement of each plant. In our laboratory, we devised a three-primers system able to amplify, in a single PCR reaction of leaf tissue fragments, the DNA sequences identifying the allelic status at the B locus (A. Carboni, unpublished). Genetic analysis of heterozygous ( B D B T ) plants from different crosses, revealed that the THC:CBD ratio may vary slightly but consistently and heritably around the value of 1 (de Meijer et al., 2003). This sug- gests the possibility that several isoenzymatic forms of THC- and CBD-synthases exist in different germplasm. Confirmation of this hypothesis, presently in progress through the sequencing in our laboratory of these possible variants, could lead to the identification of further alleles of potential interest at the B locus. Besides, the identification, either by progeny analysis or by direct sequencing, of the alleles responsible for the synthesis of the several cannabinoids described in C. sativa , would open the possibility of assisted selection in Cannabis, bred not only as a fiber crop, but also for its pharmaceutical applications (see G. Guy and R. Pertwee contributions in this special issue). Chemotype IV and V, having CBG or no cannabinoids, are of remarkable interest for both fiber and pharmaceutical purposes; the identification of the alleles at the B locus responsible for the accumulation of CBG or for the absence of cannabinoids (Figure 4), would open the way to the development of molecular markers for these chemotypes. The knowledge of the genes and alleles responsible for the different chemotypes could also lead to the manipulation of the pathway in both plants and cell cultures; the availability of chemotype-specific cell cultures in which the cannabinoid biosynthesis is made active by manipulations of the key enzymes of their pathway, could lead to the development of bioreactors useful for the in vitro large-scale production of specific cannabinoids for the pharmaceutical industry. However, C. sativa remains primarily a fiber crop, and a great deal of work is being done for ...
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... model illustrating the enzymes involved and the alleles reputed responsible for the different steps of cannabinoid biosynthesis, is shown in Figure 4. The condensation of geranygeraniol diphos- phate with olivetolic acid (catalyzed by geranylgeran- iol:olivetolate transferase, GOT; Fellermeier and Zenk, 1998) is the step leading to the first Cannabis’ exclu- sive product, cannabigerol (CBG); this particular com- pound was also described as the prevalent cannabinoid in some plants (Fournier et al., 1987). These “mutants” could not therefore be considered belonging to any of the three formerly known chemotypes, and were assigned to a new chemotype (prevalent CBG, or chemotype IV; Figure 5). CBG is today widely accepted as the common precursor for the synthesis of both THC and CBD (Fellermeier et al., 2001). A further chemotype was found, with an undetectable amount of cannabinoids. This “zero cannabinoid” type, (we propose for it the creation of a chemotype V; see Figure 5 for its gas-chromatogram) has been described by some authors in different germplasm (G. Grassi and I. Virovets, personal communication), though it is not yet clear whether this absence was due to a metabolic block at the level of GOT, or rather to a very limited for- mation of glandular trichomes, the site of synthesis and accumulation of cannabinoids (Kim and Mahlberg, 2003). Usually, CBG is detected in very small amounts in Cannabis’ extracts, probably because it is almost completely utilized as substrate by the downstream synthases (THC- and CBD-synthases), transforming it into THC, CBD or other less common end products (such as cannabichromene, CBC; Figure 4). The two synthases are respectively coded by B D and B T , the two alleles at the B locus, and have very similar K m and V max (Taura et al., 1995, 1996). This peculiarity explains the fact that, when both enzymes are present (i.e. when the genotype at the B locus is B D B T ), the almost equal efficiency of oxidocycliza- tion of CBG into the respective end products, leads to a ratio close to the unity in the THC:CBD ratio. This produces the distribution along the median line of the THC vs. CDB scatter plots observed for chemotype II plants, and for all the F1 progenies of pure chemotype parentals. One of the main targets for fiber hemp has been for a long time the eradication of hemp plants bearing the B T allele, and consequently synthesizing more or less “illegal” amounts of THC. There have been, in ...
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... model illustrating the enzymes involved and the alleles reputed responsible for the different steps of cannabinoid biosynthesis, is shown in Figure 4. The condensation of geranygeraniol diphos- phate with olivetolic acid (catalyzed by geranylgeran- iol:olivetolate transferase, GOT; Fellermeier and Zenk, 1998) is the step leading to the first Cannabis’ exclu- sive product, cannabigerol (CBG); this particular com- pound was also described as the prevalent cannabinoid in some plants (Fournier et al., 1987). These “mutants” could not therefore be considered belonging to any of the three formerly known chemotypes, and were assigned to a new chemotype (prevalent CBG, or chemotype IV; Figure 5). CBG is today widely accepted as the common precursor for the synthesis of both THC and CBD (Fellermeier et al., 2001). A further chemotype was found, with an undetectable amount of cannabinoids. This “zero cannabinoid” type, (we propose for it the creation of a chemotype V; see Figure 5 for its gas-chromatogram) has been described by some authors in different germplasm (G. Grassi and I. Virovets, personal communication), though it is not yet clear whether this absence was due to a metabolic block at the level of GOT, or rather to a very limited for- mation of glandular trichomes, the site of synthesis and accumulation of cannabinoids (Kim and Mahlberg, 2003). Usually, CBG is detected in very small amounts in Cannabis’ extracts, probably because it is almost completely utilized as substrate by the downstream synthases (THC- and CBD-synthases), transforming it into THC, CBD or other less common end products (such as cannabichromene, CBC; Figure 4). The two synthases are respectively coded by B D and B T , the two alleles at the B locus, and have very similar K m and V max (Taura et al., 1995, 1996). This peculiarity explains the fact that, when both enzymes are present (i.e. when the genotype at the B locus is B D B T ), the almost equal efficiency of oxidocycliza- tion of CBG into the respective end products, leads to a ratio close to the unity in the THC:CBD ratio. This produces the distribution along the median line of the THC vs. CDB scatter plots observed for chemotype II plants, and for all the F1 progenies of pure chemotype parentals. One of the main targets for fiber hemp has been for a long time the eradication of hemp plants bearing the B T allele, and consequently synthesizing more or less “illegal” amounts of THC. There have been, in ...
Citations
... 'Felina 32' is a monoecious cultivar. Monoecious cultivars are reported to be more uniform than dioecious cultivars in plant height and seed yield 45,67 , fibre quality 68 , and flowering time 45,[67][68][69] . This presents an advantage for mechanical harvesting and processing 67,70 . ...
... 'Felina 32' is a monoecious cultivar. Monoecious cultivars are reported to be more uniform than dioecious cultivars in plant height and seed yield 45,67 , fibre quality 68 , and flowering time 45,[67][68][69] . This presents an advantage for mechanical harvesting and processing 67,70 . ...
... Monoecious cultivars are reported to be more uniform than dioecious cultivars in plant height and seed yield 45,67 , fibre quality 68 , and flowering time 45,[67][68][69] . This presents an advantage for mechanical harvesting and processing 67,70 . However, the pollen of monoecious cultivars is reported to be less uniform than the pollen of dioecious cultivars 71 . ...
Hemp (Cannabis sativa L.) is a versatile crop with substantial potential for creating productive, sustainable, and resilient agricultural systems. However, in contrast to other crops such as cereals, hemp is highly heterozygous, resulting in both challenges and opportunities for agriculture, breeding, and research. Here, we utilise the heterozygosity of hemp to explore the genetic basis of phenotypic variability in a population generated from a single self-pollinated hemp plant. The S1 population shows extensive variability in plant growth, development, and reproductive patterns. Using reduced representation sequencing, selection of alleles heterozygous in the parent plant, and a model originally developed for genome-wide association studies (GWAS), we were able to identify statistically significant single nucleotide variants (SNVs) and haplotypes associated with phenotypic traits of interest, such as flowering time or biomass yield. This new approach, which we term genome-specific association study (GSAS), enables the mapping of traits in a single generation without the need for a large number of diverse cultivars or samples. GSAS might be applicable to other highly heterozygous vegetable and fruit crops, informing the breeding of new cultivars with enhanced uniformity and improved performance in traits relevant to various applications.
... Sex chromosomes are classified as either XX or XY. Chemotype is classified as CBD-dominant (III) or CBG-dominant (IV) as defined by de Mandolino and Carboni [40]. Entity or institution from which genotype was provided. ...
Genomic characterization of Cannabis sativa has accelerated rapidly in the last decade as sequencing costs have decreased and public and private interest in the species has increased. Here, we present seven new chromosome-level haplotype-phased genomes of C. sativa. All of these genotypes were alive at the time of publication, and several have numerous years of associated phenotype data. We performed a k-mer-based pangenome analysis to contextualize these assemblies within over 200 existing assemblies. This allowed us to identify unique haplotypes and genomic diversity among Cannabis sativa genotypes. We leveraged linkage maps constructed from F2 progeny of two of the assembled genotypes to characterize the recombination rate across the genome showing strong periphery-biased recombination. Lastly, we re-aligned a bulk segregant analysis dataset for the major-effect flowering locus Early1 to several of the new assemblies to evaluate the impact of reference bias on the mapping results and narrow the locus to a smaller region of the chromosome. These new assemblies, combined with the continued propagation of the genotypes, will contribute to the growing body of genomic resources for C. sativa to accelerate future research efforts.
... Type I cultivars possess a high concentration of THC and low to no CBD content, whereas Type II cultivars possess similar concentrations of THC and CBD and Type III groups cultivars with high CBD and low THC. Additional Types IV and V group plants with cannabigerol as the dominant cannabinoid, and plants with undetectable amounts of cannabinoids, respectively [6,7]. ...
Background
Due to its previously illicit nature, Cannabis sativa had not fully reaped the benefits of recent innovations in genomics and plant sciences. However, Canada’s legalization of C. sativa and products derived from its flower in 2018 triggered significant new demand for robust genotyping tools to assist breeders in meeting consumer demands. Early molecular marker-based research on C. sativa focused on screening for plant sex and chemotype, and more recent research has sought to use molecular markers to target traits of agronomic interest, to study populations and to differentiate between C. sativa cultivars.
Results
In this study, we have conducted whole genome sequencing of 32 cultivars, mined the sequencing data for SNPs, developed a reduced SNP genotyping panel to discriminate between sequenced cultivars, then validated the 20-SNP panel using DNA from the sequenced cultivars and tested the assays on commercially available dried flower. The assay conversion rate was higher in DNA extracted from fresh plant material than in DNA extracted from dried flower samples. However, called genotypes were internally consistent, highlighting discrepancies between genotypes detected using sequencing data and observed using genotyping assays. The primary contributions of this work are to clearly document the process used to develop minimal SNP genotyping panels, the feasibility of using such panels to differentiate between C. sativa cultivars, and outline improvements and goals for future iterations of PCR-based, minimal SNP panels to enable efficient development genotyping tools to identify and screen C. sativa cultivars.
Conclusions
Our key recommendations are to increase sampling density to account for intra-cultivar variability; leverage higher read length paired-end short-read technology; conduct in-depth pre- and post-processing of reads, mapping, and variant calling data; integrate trait-associated loci to develop multi-purpose panels; and use iterative approaches for in vitro validation to ensure that only the most discriminant and performant SNPs are retained.
... Sex chromosomes are classified as either XX or XY. Chemotype is classified as CBD-dominant (III) or CBG-dominant (IV) as defined by de Mandolino and Carboni [40]. Entity or institution from which genotype was provided. ...
Genomic characterization of Cannabis sativa has accelerated rapidly in the last decade as sequencing costs have decreased and public and private interest in the species has increased. Two emerging challenges for the C. sativa research community are to 1) comprehensively capture the genomic diversity of the species and 2) reconcile and leverage the large amounts of genomic data to make meaningful contributions to fundamental research and commercial production systems. Here, we present seven new chromosome-level haplotype-phased genomes of C. sativa. All of these genotypes were alive at the time of publication, and several have numerous years of associated phenotype data. We used a kmer-based pangenome analysis to place these new assemblies in the context of more than 200 existing assemblies. We leveraged linkage maps constructed from F2 progeny of two of the assembled genotypes to characterize the recombination rate across the genome. Lastly, we re-aligned a bulk segregant analysis dataset for the major-effect flowering locus Early1 to several of the new assemblies to evaluate the impact of reference bias on the mapping results. These new assemblies, combined with the continued propagation of the genotypes, will contribute to the growing body of genomic resources for C. sativa to accelerate future research efforts.
... Monoecious cultivars of hemp bears hermaphrodite flowers or bisexual inflorescence is very popular due to several agronomical advantages such as higher seed yields, higher crop homogeneity and easier mechanical harvest (Mandolino and Carboni 2004;Mandolino et al., 2002;Moliterni et al. 2004) [9,10,14] . Genetically, monoecious cultivars of hemp consists of 18 autosomes and two sex chromosomes i.e. ...
... Monoecious cultivars of hemp bears hermaphrodite flowers or bisexual inflorescence is very popular due to several agronomical advantages such as higher seed yields, higher crop homogeneity and easier mechanical harvest (Mandolino and Carboni 2004;Mandolino et al., 2002;Moliterni et al. 2004) [9,10,14] . Genetically, monoecious cultivars of hemp consists of 18 autosomes and two sex chromosomes i.e. ...
... It is a naturally dioecious plant with distinct male and female individuals, but monoecious cultivars have been obtained as a result of long breeding efforts (Salentijn et al. 2019). Five different chemotypes can be distinguished on the basis of the concentration of the psychoactive cannabinoid D 9 -tetrahydrocannabinol (THC) and the two non-psychoactive cannabinoids cannabidiol (CBD) and cannabigerol (CBG): the chemotype I or drug-type (also known as cannabis or marijuana) with high THC ([ 0.3%) and low CBD (\ 0.5%), the chemotype II or intermediate-type with high THC ([ 0.3%) and high CBD ([ 0.5%), and three hemp chemotypes characterized by low THC (\ 0.3%), which include the chemotype III with high CBD ([ 0.5%), the chemotype IV with high CBG ([ 0.3%) and low CBD (\ 0.5%), and the chemotype V, which is almost completely devoid of cannabinoids (Mandolino and Carboni 2004). ...
Cannabis sativa L. is an annual dioecious species native from Central Asia, which has mainly been used for medical purposes by many ancient cultures and is currently used for the treatment of several diseases. The pharmacological properties of C. sativa are related to cannabinoids, a class of secondary metabolites entirely unique to this crop that are produced and stored at high levels in the inflorescences and leaves. In addition to cannabinoids, C. sativa plants also produce a large number of non-cannabinoid secondary metabolites including terpenes, phenolic compounds and others, which have also been associated with health-promoting activities. In recent decades, the interest in secondary metabolites from C. sativa has been increasing due to their potential applications not only as pharmaceuticals, but also as nutraceuticals, food additives, drugs, fragrances, and biopesticides. This has generated a significant increase in the development of effective strategies for improving the production of such bioactive compounds. In this context, elicitation has emerged as an effective tool based on the application of abiotic or biotic factors that induce physiological changes and stimulate defense or stress-related responses in plants, including the biosynthesis of secondary metabolites. The current review gives a comprehensive overview of the available studies on the different elicitation approaches used to enhance the accumulation of the major bioactive compounds in C. sativa, and highlights challenges and opportunities related to the use of external elicitors for improving the added value of this crop.
... Chemotype V (industrial fibre hemp with almost no cannabinoids) has been also recently recognised. [3][4][5][6][7][8] The interest for CBG (and, as consequence, for chemotype IV) has increased in the last decade, since the wide range of biological activities exhibited that, in contrast with what described for Δ 9 -THC and CBD, which act on CB1 and CB2 receptors, [9] are mainly due to the interactions with noncannabinoids receptors such as 5-HT 1A and α-2 receptors. [10][11] The effects include, among the others, anticonvulsant, [12] antibacterial [13] and antinflammatory [14] properties, as well neuroprotective effects, preconized in Huntington disease, amyotrophic lateral sclerosis, Parkinson disease, epilepsy, and multiple sclerosis. ...
The reactivity of Cannabigerol (CBG, 1) in organic solvents under photochemical and acid catalyzed conditions has been investigated in detail. The obtained results pointed out the presence of different reaction paths in the photochemical and the acid‐catalyzed conditions, with a remarkable selectivity toward the formation of a 2,2‐disubstituted‐chromane derivative in the first case.
... For practical and legal reasons prohibiting the cultivation of high ∆ 9 -THC hemp, the cultivars were classified as three main chemotypes: drug-type with higher (>0.3%) levels of ∆ 9 -THC and CBD content lower than 0.5%, an intermediate type (chemotype II) with similar levels of each and fiber-type (chemotype III) with higher CBD content (>0.5%), and THC content lower than 0.3% (Grassi and McPartland 2017). Other chemotypes IV and V are also classified: chemotype IV has a low ∆ 9 -THC level but a relatively high content of CBG (cannabigerol) and chemotype V -synthesize trace amounts of cannabinoids (Mandolino and Carboni 2004;Meijer and Hammond 2005). Whereas the ratio of ∆ 9 -THC/CBD is constant during the plant's life (Meijer et al. 2003;Pacifico et al. 2008), the content of phytocannabinoids may be very variable. ...
Cannabis sativa L. is an extremely variable species. Even within the same cultivar plants can significantly differ in the content and profile of cannabinoids. Therefore, the best method for production of uniform plants and standardized raw material is vegetative propagation using clones. The aim of this study was to determine the content of cannabidiolic acid (CBDA), cannabidiol (CBD), Δ⁹-tetrahydrocannabinolic acid (Δ⁹-THCA), Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabichromene (CBC), cannabigerol (CBG), and cannabinol (CBN) in clonally propagated plants of industrial hemp. One hundred and thirty-nine plants representing 17 different hemp genotypes were regenerated in vitro, hardened, and grown in a vegetation hall until harvest. Single plants of each accession were analyzed using high-performance liquid chromatography with UV/diode-array detection (HPLC-DAD/UV). The results revealed significant variability in the total cannabinoid content (0.55–5.18% in dry weight) among tested genotypes and within the Epsilon 68 cultivar. The highest content of total CBD (4.410%) was recorded for EPS/40 genotype, while the level of total Δ⁹-THC was below the allowed threshold (0.3%). Therefore, we can conclude that some clonally propagated plants provided reproducible hemp material as a potential source of cannabidiol. The results of this study will be useful for breeding and early selection of hemp genotypes.
... Historically, C. sativa plants have been classified into three chemotypes (sometimes also referred to as chemovars) based on the relative amounts of Δ 9 -THC and CBD: chemotype I (Δ 9 -THC > CBD), chemotype II (Δ 9 -THC and CBD at similar concentrations, Δ 9 -THC >0.3%), also indicated with the vernacular name "drug-type", and chemotype III (Δ 9 -THC < CBD, Δ 9 -THC <0.3%), also called "fiber-type" based on their main utilization [9]. Later, other chemotypes have been described, including a cannabigerol (CBG) predominant one (chemotype IV) [10] and chemotype V with no cannabinoids [11]. ...
... Based on both CBDA/THCA ratio and amount of the main cannabinoids, C. sativa plants can be classified in five chemical phenotypes (chemotypes): THCA is prevalent in chemotype I, chemotype II has both CBDA and THCA at similar concentrations, III is CBDA-dominant, IV is CBGA-dominant and V has only traces of cannabinoids (Mandolino and Carboni 2004). Chemotype I, II and III can be easily and rapidly distinguished by using molecular markers soon after seed germination. ...
In Cannabis sativa L. the presence of delta 9-tetrahydrocannabinolic acid (THCA) above legal limit is a challenging issue that still restricts the industrial exploitation of this promising crop. In recent years, the interest of entrepreneurs and growers who see hemp as a dynamic and profitable crop was joined by the growing knowledge on C. sativa genetics and genomics, accelerated by the application of high throughput tools. Despite the renewed interest in the species, much remains to be clarified, especially about the long-standing problem of THCA in hemp inflorescences, which could even result in the seizure of the whole harvest. Although several hypotheses have been formulated on the accumulation of this metabolite in industrial varieties, none is conclusive yet. In this work, individuals of a population of the hemp cultivar 'FINOLA' obtained from commercial seeds were investigated for total THC level and examined at molecular level. A marker linked to THCA synthase was found at a high incidence in both male and female plants, suggesting a considerable genetic variability within the seed batch. Full-length sequences encoding for putatively functional THCA synthases were isolated for the first time from the genome of both female and male plants of an industrial hemp variety and, using transcriptional analysis, the THCA synthase expression was quantified in mature inflorescences of individuals identified by the marker. Biochemical analyses finally demonstrated for these plants a 100% association between the predicted and actual chemotype.
