# Multi-Omics Approaches to Study Molecular Mechanisms in _Camanabis sativa_
## 4 Multi-Omics Approaches in Cannabis  
NGS technologies have revolutionized plant biology and elucidated molecular processes in crops of therapeutic importance [82]. Similarly, combined methods, based on the relationship among genomic, transcriptomic and metabolomic approaches, have been developed for a wide set of crop species, finding synergies among these heterogeneous data. Indeed, multi-omics approaches have been employed in the study of cannabis, such as a combined metabolomic and transcriptomic approach used to examine mature infloresances [83]. In this study, the plant chemotype was assessed, and qRT-PCR used to quantify transcript levels of cannabinoid synthesis genes, specifically looking into CBDA and THCA synthesis. These sequences were also compared with other existing sequences obtained by RNA-Seq and led to the identification of several SNPs that correlated to the cannabinoid composition of the inflorescence. The results suggested that these variations can have a functional significance, as well as at least partially explain the existence of different cannabis chemotypes [83]. In a subsequent investigation [15], in order to better understand the diversity and the major cannabis ancestry, the first cannabis genome study [23] was further expanded, by re-mapping sequence reads through whole genome shotgun (WGS) sequencing and genotype-by-sequencing (GBS) approaches. Concurrently, a chemotype analysis was performed by HPLC, and over 350 cannabinoids and terpenids were found. Overall findings showed that, despite several hybridizations, significant ancestral insights related to modern cannabis varieties were found in the genomic results. Particularly, among the studied \(\sim\)350 Cannabis varieties, the existence of at least three principal diversity groups with European hemp varieties more closely related to narrow leaflet drug-types than to broad leaflet drug-types was demonstrated [15]. Trichomes are a crucial aspect of medicinal cannabis varieties, being the primary storage organ for cannabinoids. Investigations have focused on the changes occurring during flower development, the main phase of metabolite accumulation, using metabolomics and transcriptomics in parallel [33]. SPME, GC-MS, and LC-MS techniques were applied for the quantitative analysis of terpen and cannabinoids. RNA-Seq gene co-expression networks were developed, and the Pearson's correlation coefficient was used to quantify the similarity. Results showed how medicinal cannabis trichomes undergo changes to their morphology and metabolite profile during flower development. Furthermore, they demonstrated a high expression of cannabinoid and terpene related genes, as well as the presence of numerous uncharacterized highly co-expressed genes involved in CBDA synthase, suggesting that the glandular trichomes on cannabis flowers are strongly dedicated to cannabinoid and terpene production. In the same year, nine cannabis cultivars were selected according to several characteristics, such as colour or smell, the glandular trichomes were isolated from each of the cultivars, and their metabolome and transcriptome were analysed and compared by WGCNA [43]. The analysis revealed genes involved in the biosynthesis of both cannabinoids and terpenoids. Furthermore, in addition to the previously characterized terpene synthase genes, e.g., _CsTPS14 CT_ [(-)-limonene synthase] and _CsTPS15 CT_ (\(\beta\)-myrrene synthase), other genes, including _CsTPS18 VF_ and _CsTPS19 BL_ (nerolidol/linalool synthases), _CsTPS16 CC_ (germacrene B synthase), and _CsTPS20 CT_ (hedycaryol synthase), were investigated from a functional point of view, by identifying the synthase pathway in which they were involved. The cannabis terpenene synthase gene family is complex, containing at least 55 members. Results demonstrated that subsets of this family are expressed in all plant tissues, including a set of root specific monoterpene synthases [72].