Abstract

   The cannabis plant and its active ingredients (i.e., cannabinoids and
   terpenoids) have been socially stigmatized for half a century. Luckily,
   with more than 430,000 published scientific papers and about 600
   ongoing and completed clinical trials, nowadays cannabis is employed
   for the treatment of many different medical conditions. Nevertheless,
   even if a large amount of high-throughput functional genomic data
   exists, most researchers feature a strong background in molecular
   biology but lack advanced bioinformatics skills. In this work, publicly
   available gene expression datasets have been analyzed giving rise to a
   total of 40,224 gene expression profiles taken from cannabis plant
   tissue at different developmental stages. The resource presented here
   will provide researchers with a starting point for future
   investigations with Cannabis sativa.

   Keywords: Cannabis sativa, gene expression, cannabinoid pathway

Introduction

   The cannabis plant has been used for medical purposes for centuries,
   before being socially stigmatized for the last half century ^[23]1.
   Nevertheless, more than 430,000 published scientific papers exist, with
   about 25,600 works published in 2016 (
   [24]https://scholar.google.com/). In addition, there are about 600
   ongoing and completed clinical trials involving cannabis (
   [25]https://www.clinicaltrials.gov/).

   The endocannabinoid system is involved in virtually every biological
   function ^[26]2, so it is not surprising that cannabis is being used to
   treat neurological ^[27]3, psychiatric ^[28]4, immunological ^[29]5,
   cardiovascular ^[30]6, gastrointestinal ^[31]7, and oncological ^[32]8
   conditions.

   Today, a large amount of high-throughput functional genomic data
   exists. Nonetheless, even in the era of ‘omics, the great majority of
   researchers feature a strong background in molecular biology but lack
   advanced bioinformatics skills ^[33]9.

   In the present work, publicly available gene expression data taken from
   cannabis plant tissue at different developmental stages (shoot, root,
   stem, young and mature leaf, early-, mid- and mature-stage flower) have
   been analyzed, giving rise to 40,224 gene expression profiles.
   Moreover, the expression patterns of 23 cannabinoid pathway related
   genes are described. The data note provided here will aid future
   studies by providing researchers with a powerful resource for future
   investigations.

Material and methods

Gene expression analysis

   Gene expression datasets were downloaded from the NCBI SRA directory
   ^[34]10 ( [35]https://www.ncbi.nlm.nih.gov/sra/) with accession numbers
   [36]SRP006678 and [37]SRP008673. Raw sequences were mapped to the
   canSat3 reference genome ^[38]11 with TopHat2 v2.1.0 ^[39]12. Gene
   counts and relative transcript levels were obtained with Cufflinks
   v2.2.1.0 ^[40]13, and submitted to NCBI GEO (
   [41]https://www.ncbi.nlm.nih.gov/geo/) with accession number
   [42]GSE93201. Cannabinoid related genes were found within the canSat3
   transcripts with the Cannabis genome browser BLAT web tool ^[43]11 (
   [44]http://genome.ccbr.utoronto.ca/cgi-bin/hgBlat?command=start). Gene
   expression heatmaps and unsupervised hierarchical clustering were
   carried out with GENE-E ^[45]14.

Results

   The Cannabis sativa reference genome and transcriptome have been
   published, although data analysis is still at the preliminary stages
   ^[46]11. In other words, we know what the presumptive genes are, but we
   do not know the chromosomes they are located in, nor their molecular
   functions. Given that this high-throughput gene expression data is
   publicly available, expression analysis of these yet unidentified genes
   can be performed. To this end, public repositories have been surveyed
   for transcriptional profiling datasets derived from Cannabis sativa. In
   total, 31 RNA-seq datasets derived from one hemp and two different
   psychoactive strains (NCBI SRA accession numbers: [47]SRP006678 and
   [48]SRP008673) of Cannabis sativa shoot, root, stem, young and mature
   leaf, early-, mid- and mature-stage flower have been analyzed.
   Unsupervised hierarchical clustering of gene expression values revealed
   six clusters of genes with specific tissue/stage expression (
   [49]Figure 1). Cluster 1 genes display high expression levels in
   shoots, mature leaves, and flowers; cluster 2 genes in leaves and
   flowers; cluster 3 genes in roots and stems; cluster 4 genes in roots,
   stems, and flowers; cluster 5 genes in hemp flowers and cluster 6 genes
   in shoots, roots, stems, and flowers.

Figure 1. Gene expression profiles taken from cannabis plant tissue at
different developmental stages.

   [50]Figure 1.
   [51]Open in a new tab

   Heatmap showing relative expression values (log2 RPKM) of the highest
   expressed genes. Six gene clusters were defined in accordance with the
   unsupervised hierarchical clustering.

   Genes involved in the biosynthesis of cannabinoids and their precursors
   have been shown to be overexpressed in flowers ^[52]15. To validate
   gene expression profiling, cannabinoid, hexanoate,
   2-C-methyl-D-erythritol 4-phosphate ( MEP) and geranyl diphosphate (
   GPP) pathway genes ^[53]11, [54]16, together with the olivetol synthase
   ( OLS) gene ^[55]17, [56]18, the (-)-limonene terpene synthase ( TPS)
   gene ^[57]19 and the polyketide synthase ( PKS) gene ^[58]20, have been
   analyzed. As expected, most of these genes were overexpressed in
   flowers, although many of the genes also displayed high expression in
   other tissues ( [59]Figure 2; [60]Supplementary table 1).
   Interestingly, virtually all of them were highly expressed in the
   shoot.

Figure 2. Gene expression analysis of the cannabinoid pathway.

   [61]Figure 2.
   [62]Open in a new tab

   Heatmap showing relative expression values (log2 RPKM) of genes
   belonging to cannabinoid and precursor (hexanoate, GPP, MEP, olivetolic
   acid) pathways, together with terpene synthase (TPS) and polyketide
   synthase (PKS).

Discussion

   Today, cannabis and its derivatives are successfully employed for
   treatment of a large number of different pathological conditions
   ^[63]3, [64]5– [65]8. Each year, more articles related to cannabis are
   published, with about 25,600 studies published in 2016 (
   [66]https://scholar.google.com/). Remarkably, only 3% of these papers
   (13,300 out of 432,000) also take genomics into consideration, with
   very few of them directly relating to the genomics of cannabis. This
   could be due to the fact that, for obvious reasons, most researchers
   still lack advanced bioinformatics skills and are therefore limited in
   their research ^[67]9.

   To this end, a total of 40,224 gene expression profiles taken from
   cannabis plant tissue at different developmental stages were obtained
   by exploiting common bioinformatics pipelines ^[68]13. Moreover,
   expression profiles of the genes belonging to the cannabinoid pathway
   ^[69]11, [70]16– [71]20 are provided.

   Even if these data are preliminary, some observations can already be
   made. For instance, virtually all genes found to be highly expressed in
   flowers ( [72]Figure 1, cluster 1 and [73]Figure 2) also displayed high
   expression in the shoot. Having had only one sample at this specific
   developmental stage, these results could be derived from technical
   issues rather than differences in gene expression. However, not all
   transcripts (57%) were found to be overexpressed in the shoot, thus
   pointing toward the possible specificity of these changes. If this is
   confirmed, it may provide researchers with the possibility to study the
   molecular function of flower specific genes directly in sprouting
   plants, without having to wait for the plant to fully bloom.

   Cannabis sativa is a versatile plant - it is being used for medical as
   well as for industrial purposes ^[74]21, [75]22. For this reason,
   cutting-edge genomics technology is currently being applied either to
   ameliorate specific phenotypes, or for breeding purposes ^[76]22–
   [77]27. Cluster 5 genes ( [78]Figure 1) seem of great interest in this
   regard, as they are visibly overexpressed specifically in
   non-psychoactive cannabis flowers. These genes could be downregulated
   in hemp in order to create new strains high in cannabidiol (CBD), but
   with the proper entourage effect commonly found in the psychoactive
   counterparts ^[79]28. On the other hand, hemp specific genes could be
   upregulated in marijuana to produce high fiber/oil containing crops
   harboring therapeutically valuable active principles within their
   flowers. One potential candidate is the Csfad2a gene which was recently
   found to be highly expressed only in some hemp strains. Here, high
   Csfad2a expression was correlated with both higher oil content and
   lower oxidation tendency, eventually leading to the production of a
   significantly better commercial product ^[80]26.

   Perhaps the major pitfall of this kind of analysis comes from the fact
   that although the current cannabis reference genome and transcriptome
   have been published, data analysis is still at the preliminary stages
   ^[81]11. Like in other plants, the cannabis genome is highly redundant
   and difficult to resolve ^[82]29. It is very likely that false
   negatives have caused important transcripts to still be missing.
   Nevertheless, these 40,224 gene expression profiles will provide
   researchers with a valuable resource and important genomic insights for
   future investigations with Cannabis sativa.

Data availability

   The data referenced by this article are under copyright with the
   following copyright statement: Copyright: © 2017 Massimino L

   Raw expression data can be found in the NCBI SRA directory (
   [83]https://www.ncbi.nlm.nih.gov/sra/) with accession numbers
   [84]SRP006678 and [85]SRP008673.

   Processed data can be found in the NCBI GEO repository (
   [86]https://www.ncbi.nlm.nih.gov/geo/) with accession number
   [87]GSE93201.

Funding Statement

   The author(s) declared that no grants were involved in supporting this
   work.

   [version 1; referees: 2 approved]

Supplementary material

   Supplementary table 1. Cannabinoid metabolism related gene profiling in
   different tissues and developmental stages. Gene expression matrix of
   cannabinoid pathway genes. Expression values are expressed in RPKM.
   [88]Click here for additional data file.^ (17KB, tgz)

   .

References