Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Oral Communication Abstract - S3b

THE CONTROL OF THE CHEMICAL PHENOTYPE IN CANNABIS SATIVA L.: GENETIC ANALYSIS AND MOLECULAR MARKERS

G. MANDOLINO*, A. CARBONI*, D. PACIFICO*, P. RANALLI*, E. DE MEIJER**

*) Istituto Sperimentale per le Colture Industriali, Via di Corticella 133, 40128 Bologna (I)

**) GW Pharmaceuticals plc., Porton Down Science Park, Salisbury, Wiltshire SP4 0JQ (U.K.)

Cannabis, cannabinoids, THC-synthase, CBD-synthase, molecular markers

Cannabinoids are terpenophenolic compounds unique to the genus Cannabis. These secondary products are important for breeding and legal reasons; in fact, UE subsidies are issued only if the amount of the psychoactive cannabinoid delta-9-tetrahydrocannabinol (THC) in the fibre crops is certified to be lower than 0.2 % of the inflorescence dry matter. Besides, the plants containing high levels of this cannabinoid are considered illegal, and are strongly repressed by police actions. On the other hand, high percent of specific cannabinoids (and especially of the psychoactive ones) are desirable during the breeding for the production of pure lines for pharmaceutical purposes. Therefore, the type and amount of cannabinoids produced by the Cannabis plants, and the mechanisms of their inheritance are of major interest for the different and sometimes contrasting purposes of breeding for this ancient no food plant.

The two most common cannabinoids found in Cannabis plants are delta-9-tetrahydrocannabinol and cannabidiol (CBD, non psychoactive). These two compounds are synthetized from the common precursor cannabigerol (CBG, non psychoactive) by the two enzymes THC- and CBD-synthase. Inbred lines were obtained, endowed with only one of the two main cannabinoids (THC or CBD). Hybrid F₁ progenies were obtained, and several individual F₁ plants were selfed to obtain F₂ generations. All the F₁ and F₂ plants were analysed by gas-chromatography to determine their cannabinoids profile. The results obtained support a model of determination of the chemotype by one single locus (B) with two codominant alleles, B_D and B_T, each probably coding for one of the two synthases involved in cannabinoid biosynthesis; the chemotype distribution in all the F₂ examined fitted well the expected 1.2:1 ratio. A null allele, B₀, is also postulated, explaining the occurrence of CBG-accumulating plants. Sequence-characterized markers were developed using the two F₂ pure-chemotypes segregating groups in a Bulk Segregant Analysis. These markers show tight linkage with the chemotype in the progenies studied. Besides, sequence-specific markers for each of the two alleles involved in chemotype determination were developed; a multiplex assay was set up for the simultaneous recognition, in one single PCR reaction, of the genotype at the B locus (B_T/B_T, B_D/B_D or B_T/B_D). Such assay allows the identification at very early stages of the type of cannabinoid each plant will produce at maturity. It has been noted that mixed chemotype plants (either the F₁ hybrids or the 50% of each F₂ progeny) fall into two different classes, depending on their CBD/THC content ratio. These different ratios are stably inherited from F₁ hybrids to F₂ heterozygous they generate, and suggest the existence of different isoforms of THC- or CBD-synthases, with different efficiencies in competing for the precursor CBG, and presumably encoded by different B_T and B_D alleles. This hypothesis is presently being tested by sequencing the THC- and CBD-synthase genes of F₂ plants showing such different ratios of the two main cannabinoids.

Within each chemotype (i.e. within each genotype at the B locus), the cannabinoid content is a typically quantitative trait, probably related with the density of the glandular trichomes, the sites of the cannabinoid synthesis. Therefore, the overall chemotype is the net result of both a mendelian determination at one single locus (B) and of quantitative and environmental factors affecting the amount of each cannabinoid synthetized by the plant. Both these factors must be taken into consideration for the practical exploitation of these class of secondary products in breeding programs.