Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored. We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp. The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.

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The draft genome and transcriptome of Cannabis sativa

Sex chromosomes in land plants can evolve as a consequence of close linkage between the two sex determination genes with complementary dominance required to establish stable dioecious populations, and they are found in at least 48 species across 20 families. The sex chromosomes in hepatics, mosses, and gymnosperms are morphologically heteromorphic. In angiosperms, heteromorphic sex chromosomes are found in at least 19 species from 4 families, while homomorphic sex chromosomes occur in 20 species from 13 families. The prevalence of the XY system found in 44 out of 48 species may reflect the predominance of the evolutionary pathway from gynodioecy towards dioecy. All dioecious species have the potential to evolve sex chromosomes, and reversions back from dioecy to various forms of monoecy, gynodioecy, or androdioecy have also occurred. Such reversals may occur especially during the early stages of sex chromosome evolution before the lethality of the YY (or WW) genotype is established.

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Sex Chromosomes in Land Plants

Sex determination systems in plants have evolved many times from hermaphroditic ancestors (including monoecious plants with separate male and female flowers on the same individual), and sex chromosome systems have arisen several times in flowering plant evolution. Consistent with theoretical models for the evolutionary transition from hermaphroditism to monoecy, multiple sex determining genes are involved, including male-sterility and female-sterility factors. The requirement that recombination should be rare between these different loci is probably the chief reason for the genetic degeneration of Y chromosomes. Theories for Y chromosome degeneration are reviewed in the light of recent results from genes on plant sex chromosomes.

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Plant sex determination and sex chromosomes

Cannabis (Cannabis sativa, or hemp) and its constituents—in particular the cannabinoids—have been the focus of extensive chemical and biological research for almost half a century since the discovery of the chemical structure of its major active constituent, Δ9-tetrahydrocannabinol (Δ9-THC). The plant’s behavioral and psychotropic effects are attributed to its content of this class of compounds, the cannabinoids, primarily Δ9-THC, which is produced mainly in the leaves and flower buds of the plant. Besides Δ9-THC, there are also non-psychoactive cannabinoids with several medicinal functions, such as cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG), along with other non-cannabinoid constituents belonging to diverse classes of natural products. Today, more than 560 constituents have been identified in cannabis. The recent discoveries of the medicinal properties of cannabis and the cannabinoids in addition to their potential applications in the treatment of a number of serious illnesses, such as glaucoma, depression, neuralgia, multiple sclerosis, Alzheimer’s, and alleviation of symptoms of HIV/AIDS and cancer, have given momentum to the quest for further understanding the chemistry, biology, and medicinal properties of this plant.

Phytochemistry of Cannabis sativa L.

Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure -9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F1's were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F1 plants were self-fertilized, and 10 inbred F2 offspring were collected and analyzed. In all cases, a segregation of the three chemotypes (pure CBD, mixed CBD-THC, and pure THC) fitting a 1:2:1 proportion was observed. The CBD/THC ratio was found to be significantly progeny specific and transmitted from each F1 to the F2's derived from it. A model involving one locus, B, with two alleles, BD and BT, is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype BD/BT at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either BD/BD or BT/BT). It is suggested that such codominance is due to the codification by the two alleles for different isoforms of the same synthase, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The F2 segregating groups were used in a bulk segregant analysis of the pooled DNAs for screening RAPD primers; three chemotype- associated markers are described, one of which has been transformed in a sequence-characterized amplified region (SCAR) marker and shows tight linkage to the chemotype and codominance.

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The inheritance of chemical phenotype in Cannabis sativa L

A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8400, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8400 marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8400 were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp.

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

Random amplified polymorphic DN A (RAPD) markers were generated from 13 cultivars and accessions of Cannabis sativa L. Approximately 200 fragments generated by 10 primers of arbitrary sequence were used to assess the level of DNA variation. Statistical analysis was performed using the Dice coefficient of similarity and principal coordinate analysis. The grouping of the accessions according to the cluster analysis was in good agreement with their origin and lines with common ancestors were grouped together. Principal coordinates 1 and 2 revealed a clear separation of Italian and Hungarian germplasm and a third group, including a mixture of genotypes coming from different places; the third coordinate separated the Korean group which is probably the most divergent germplasm. Variability within the two cultivars ‘Carmagnola’ and ‘Fibranova1’ was also shown, suggesting good possibilities for long–term selection work. RAPD markers provide a powerful tool for the investigation of genetic variation in cultivars/accessions of hemp.

Genetic diversity of Cannabis sativa germplasm based on RAPD markers

The restriction and amplification patterns of potato genomic DNA of eight different cultivars and breeding clones has been studied. The analysis was carried out by polyacrylamide high resolution gels, and subsequent blotting and hybridization to potato DNA probes, and by agarose gel electrophoresis of the amplification products obtained with a variety of random primers. Fingerprinting of the genotypes was possible using two enzyme-probe combinations (RsaI-GP35 and RsaI-CP6) and three random primers (OPA4, OPA19 and OPG12). Based on the same techniques, a number of plants from the cvs. Monalisa and Spunta originated from the sprouting of in vitro-induced tubers (vitrotubers) were analysed to test the reproducibility of RFLP and PCR patterns. No variations were found with RFLP analysis. Some different RAPD patterns were seen, showing putatively vitrotuber-specific variations.

V. Faeti

Papers

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

Genetic diversity of Cannabis sativa germplasm based on RAPD markers

Stability of fingerprints of Solanum tuberosum plants derived from conventional tubers and vitrotubers