# Multi-Omics Approaches to Study Molecular Mechanisms in _Camanabis sativa_
## 4 Multi-Omics Approaches in Cannabis
In the light of the role played by volatile terpen in plant interaction with the soil biome, findings related to the root genes group belonging to CsTPS family, could be the subject of further investigations being particularly interesting for their agronomic impact. For instance, in maize, \(\beta\)-caryophyllene is released from roots under attack of worms [84]. Terpene profiles of cannabis were recently characterized in detail as part of a multimic study also focusing on the transcriptome of trichomes [85]. Sequence analysis was performed on a set of cultivars assembled onto the reference genome of 'PK' and this revealed about 30 different CsTPS genes, as well as expression variations of genes involved in terpenoid and cannabinoid pathways between the different cultivars. This study, using a new annotation of the 'PK' genome, was able to identify \(\sim\)20 CsTPS gene models, as well as additional genes involved in isoprenoid and cannabinoid biosynthesis. Despite these recent discoveries, the relationship between terpene and cannabinoid biosynthesis to date has not been fully described. As the relations between cannabinoid synthase CNVs and cannabinoid content are still not clear [19], some recent studies focussed efforts on this issue. A multi-omics approach, based on metabolomics, genomics and transcriptomics, was employed to study the relationship between cannabinoid synthase CNVs and cannabinoid profile [86]. Cannabinoid profiles of 69 Cannabis cultivars of different lineages were assessed using HPLC. Two de novo cannabis genome assemblies and an additional whole genome shotgun data set from a diversity of cultivars were used, and an RNA-Seq method was applied for the transcriptome analysis. Results confirmed that genes involved in the cannabinoids pathway showed several CNVs and were differentially expressed between the cultivars. Furthermore, new insights about a positive correlation between the accumulation of specific cannabinoids, including THCA, and the copy number of certain synthase paralogs were provided. The sex chromosome evolution in cannabis was also investigated using multi-omics approaches [67]. An RNA-sequencing pipeline was developed using Complete Genomic (CG) sequencing which, unlike other DNA sequencing techniques, allowed sequences with a gapped read structure to be aligned. There, a further genotyping analysis was performed using the cannabis genome developed in 2011 [23] and only SNPs supported by at least three reads were considered. Results identified over 500 sex-linked genes and showed that old plant sex chromosomes can have large non-recombining regions. Particularly, the X-specific cannabis region was found larger compared to other plant systems. Interestingly, the age estimated for the sex chromosomes ranged from \(\sim\)12 myr to \(\sim\)29 myr old, which according to the literature, is among the oldest in plants in which the age of sex chromosomes was investigated by sequencing data [87]. Moreover, it was found that the Y gene loss is about 70%, which is much higher than other species, where this loss has been estimated to be \(\sim\)40% [88]. Cannabis sex-linked genes were also investigated in a subsequent study [25]. Male and female cannabis genomes (cv 'JL') were sequenced with Pac Bio, utilizing long-read single molecule sequencing. These assemblies were further annotated with an Isoform sequencing (Iso-Seq) approach, providing further information about isoform complexity. Genes of interest on the Y chromosome, playing a significant role in sex determination and trichome development, were discussed, among them: _FT_ (Flowering Locus T), _FY_ (Flowering Time control protein), _PIN2_ (Auxin efflux carrier component 2), _CRL5_ (AP2-like ethylene responsive transcription factor), and _TBL6_ (Protein trichome birefringence-like 6).