Special Issue "Genetics and Functional Genomics in Cannabis sativa, L."

A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Genetics, Genomics and Biotechnology".

Deadline for manuscript submissions: 31 August 2022 | Viewed by 15576

Special Issue Editor

Dr. Roberta Paris
E-Mail Website
Guest Editor
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria – Centro di Ricerca Cerealicoltura e Colture Industriali (CREA – CI), Via di Corticella 133, 40128 Bologna, Italy
Interests: Cannabis sativa secondary metabolism: identification and isolation of the structural and regulatory genes involved in cannabinoid, terpens, and flavonoids biosynthesis and accumulation. Valorisation of hemp waste as a source for the production of new biochemicals of pharmaceutical, cosmeceutical, and manufacturing interests. In vitro culture and micropropopagation of medical Cannabis. Influence of photoperiod on flowering time and seed set in Cannabis sativa

Special Issue Information

Dear Colleagues,

Cannabis sativa L. is a fascinating and, at the same time, controversial plant widely cultivated for diverse uses, spanning from the production of paper and textiles to pharmaceutical applications. Moreover, it is a low input, high sustainable crop, matching with bioeconomy principles and perfectly fitting in the current scenario of the climate crisis.

The recent interest in Cannabis cultivation, for a long time a neglected plant, derives from the fact that, in addition to being a good source of both cellulosic and woody fibers, it is particularly rich in secondary metabolites, conferring a high added-value to this plant. Female inflorescences are sources of peculiar bioactive compounds, but there are also phytocomplexes in which the beneficial activities of single molecules are potentiated by a synergic effect of the blend made of cannabinoids, terpens, and flavonoids. The molecular comprehension of their biosynthesis and regulation in Cannabis will allow for manipulating their content and to develop markers for breeding. Female inflorescences also produce seeds characterized by flavors and oil composition valuable for human consumption. The increase in grain yield and seed quality are important goals for hemp breeding, and require a deeper knowledge of the basic plant and floral biology, also at the molecular level.

In recent years, a great deal of Cannabis genomic and transcriptomic data have became available, providing the hemp research community with fundamental tools to identify target genes of interest, and with databases to interrogate in the search of candidate genes for a number of traits. Despite significant progress, however, much remains unknown regarding Cannabis genetics and functional genomics, still leaving a lot of room for study in different hemp research areas. A few genes have been identified, especially those involved in the biosynthesis of cannabinoids, but a functional characterization still lags behind for most of them.

This Special Issues, titled “Genetics and Functional Genomics in Cannabis sativa, L.” intends to collect papers ranging from genome-wide to target gene analyses on different hemp research topics. The identification of new genes involved in the synthesis of specialized metabolites or in the expression of other important traits for Cannabis breeding (fiber traits, flowering time, grain yield and quality, sex plasticity, disease resistance, etc.) are welcome, together with papers reporting on the development of methods for reverse genetics in Cannabis, and the characterization of functional target genes. Genome-wide bioinformatic analyses are also welcome, especially when they bring new knowledge about hemp research.

Dr. Roberta Paris
Guest Editor

Manuscript Submission Information

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Keywords

  • Cannabis
  • metabolites
  • seed
  • oil
  • fibers
  • hemp
  • phytocannabinoids
  • flavonoids
  • terpens
  • flavor
  • gene function
  • genetics
  • genomics
  • transcriptomes
  • resistance genes
  • transcription factors
  • tissue culture
  • reverse genetics
  • genome assemblies
  • genomic resources
  • VIGS
  • flowering time
  • sex expression
  • genetic maps
  • bioinformatic
  • molecular biology
  • gene knockout
  • genetic transformation
  • cisgenesis
  • genetic diversity

Published Papers (5 papers)

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Research

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Article
Tobacco Rattle Virus as a Tool for Rapid Reverse-Genetics Screens and Analysis of Gene Function in Cannabis sativa L.
Plants 2022, 11(3), 327; https://doi.org/10.3390/plants11030327 - 26 Jan 2022
Viewed by 1219
Abstract
Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the [...] Read more.
Medical cannabis (Cannabis sativa L.) is quickly becoming a central agricultural crop as its production has continued to increase globally. The recent release of the cannabis reference genomes provides key genetic information for the functional analysis of cannabis genes. Currently, however, the established tools for in vivo gene functional analysis in cannabis are very limited. In this study, we investigated the use of the tobacco rattle virus (TRV) as a possible tool for virus-induced gene silencing (VIGS) and virus-aided gene expression (VAGE). Using leaf photobleaching as a visual marker of PHYTOENE DESATURASE (PDS) silencing, we found that VIGS was largely restricted to the agro-infiltrated leaves. However, when agro-infiltration was performed under vacuum, VIGS increased dramatically, which resulted in intense PDS silencing and an increased photobleaching phenotype. The suitability of TRV as a vector for virus-aided gene expression (VAGE) was demonstrated by an analysis of DsRed fluorescence protein. Interestingly, a DsRed signal was also observed in glandular trichomes in TRV2-DsRed-infected plants, which suggests the possibility of trichome-related gene function analysis. These results indicate that TRV, despite its limited spread, is an attractive vector for rapid reverse-genetics screens and for the analysis of gene function in cannabis. Full article
(This article belongs to the Special Issue Genetics and Functional Genomics in Cannabis sativa, L.)
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Article
Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments
Plants 2021, 10(10), 2174; https://doi.org/10.3390/plants10102174 - 14 Oct 2021
Viewed by 1097
Abstract
Cannabis sativa (2n = 2x = 20) is a popular species belonging to the Cannabaceae family. Despite its use for medical, recreational, and industrial purposes as well as its long history, the genetic research on this species is in its infancy [...] Read more.
Cannabis sativa (2n = 2x = 20) is a popular species belonging to the Cannabaceae family. Despite its use for medical, recreational, and industrial purposes as well as its long history, the genetic research on this species is in its infancy due to the legal implications and the prohibition campaigns. The recent legalization of Cannabis in many countries along with the use of genomics boosted the approaches aimed at marker-assisted selection, germplasm management, genetic discrimination, and authentication of cultivars. Nonetheless, the exploitation of molecular markers for the development of commercial varieties through marker-assisted breeding schemes is still rare. The present study aimed to develop an informative panel of simple sequence repeat markers to be used for the genotyping of high breeding value C. sativa lines. Starting from 41 nuclear SSR designated by in silico analyses, we selected 20 highly polymorphic and discriminant loci that were tested in 104 individuals belonging to 11 distinct hemp varieties. The selected markers were successful in accessing homozygosity, genetic uniformity, and genetic variation within and among varieties. Population structure analysis identified eight genetic groups, clustering individuals based on sexual behaviors (dioecious and monoecious) and geographical origins. Overall, this study provides important tools for the genetic characterization, authentication, conservation of biodiversity, genetic improvement and traceability of this increasingly important plant species. Full article
(This article belongs to the Special Issue Genetics and Functional Genomics in Cannabis sativa, L.)
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Article
Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase
Plants 2021, 10(9), 1857; https://doi.org/10.3390/plants10091857 - 08 Sep 2021
Cited by 1 | Viewed by 1484
Abstract
Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties [...] Read more.
Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids. Full article
(This article belongs to the Special Issue Genetics and Functional Genomics in Cannabis sativa, L.)
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Article
In Silico Identification of MYB and bHLH Families Reveals Candidate Transcription Factors for Secondary Metabolic Pathways in Cannabis sativa L.
Plants 2020, 9(11), 1540; https://doi.org/10.3390/plants9111540 - 11 Nov 2020
Cited by 6 | Viewed by 2091
Abstract
Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up [...] Read more.
Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids’ metabolic pathways, very little is known about their regulation, while the literature on flavonoids’ metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs. Full article
(This article belongs to the Special Issue Genetics and Functional Genomics in Cannabis sativa, L.)
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Review

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Review
The Past, Present and Future of Cannabis sativa Tissue Culture
Plants 2021, 10(1), 185; https://doi.org/10.3390/plants10010185 - 19 Jan 2021
Cited by 17 | Viewed by 8510
Abstract
The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and forms the basis for other [...] Read more.
The recent legalization of Cannabis sativa L. in many regions has revealed a need for effective propagation and biotechnologies for the species. Micropropagation affords researchers and producers methods to rapidly propagate insect-/disease-/virus-free clonal plants and store germplasm and forms the basis for other biotechnologies. Despite this need, research in the area is limited due to the long history of prohibitions and restrictions. Existing literature has multiple limitations: many publications use hemp as a proxy for drug-type Cannabis when it is well established that there is significant genotype specificity; studies using drug-type cultivars are predominantly optimized using a single cultivar; most protocols have not been replicated by independent groups, and some attempts demonstrate a lack of reproducibility across genotypes. Due to culture decline and other problems, the multiplication phase of micropropagation (Stage 2) has not been fully developed in many reports. This review will provide a brief background on the history and botany of Cannabis as well as a comprehensive and critical summary of Cannabis tissue culture. Special attention will be paid to current challenges faced by researchers, the limitations of existing Cannabis micropropagation studies, and recent developments and future directions of Cannabis tissue culture technologies. Full article
(This article belongs to the Special Issue Genetics and Functional Genomics in Cannabis sativa, L.)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: In silico identification of MYBs and bHLH reveal candidate regulatory TFs for key secondary metabolic pathways in Cannabis sativa L.
Authors: Laura Bassolino1*, Matteo Buti2, Flavia Fulvio1, Alessandro Pennesi1, Giuseppe Mandolino1, Justyna Milc3, Enrico Francia3 and Roberta Paris1,*
Affiliation: 1 CREA-Research Centre for Cereal and Industrial Crops Bologna, Italy 2 Department of Agriculture, Food, Environment and Forestry (Dagri), University of Florence, Florence, Italy 3 Department of Life Sciences, Centre BIOGEST-SITEIA, University of Modena and Reggio Emilia
Abstract: Plant secondary metabolic pathways are finely tuned and regulated by the activity of transcription factors like members of bHLH and MYB subfamilies. Cannabis sativa L. is a unique officinal plant species with approximately 600 synthesized metabolites having diverse scale-up industrial and pharmaceutical usage. Despite a comprehensive knowledge of cannabinoids metabolic pathway very little is known about their regulation while literature on flavonoids is scarce. The assembled genome of the CBDRx variety is a genetic resource of great value for bioinformatics studies. In this study, we provide the first genome-wide analysis of bHLH and MYB families in Cannabis which led to the identification of 93 and 89 genes, respectively. Comparative phylogenetic analysis with A. thaliana protein members distributed CsbHLH and CsMYBs into distinct functional subfamilies. Gene structure analysis and motif distribution confirmed that members belonging to the same evolutionary clade share common features at both gene and amino acidic level. Interestingly, such findings resulted in candidate regulatory genes for key metabolic pathways leading to flavonoids and cannabinoids synthesis in Cannabis. Furthermore, a candidate gene approach was used to identify in silico candidate structural enzymes encoding genes for flavonoids and cannabinoids synthesis which are randomly distributed on chromosomes. Taken as a whole, our results represent a valuable resource of candidate genes for further investigating the C. sativa genome for genomic studies and genetic improvements strategies.

Title: Transcriptome Response of Cannabis (Cannabis sativa L.) to the Pathogenic fungus, Golovinomyces cichoracearum
Authors: Dinesh Adhikary
Affiliation: University of Alberta
Abstract: Powdery mildew (PM), caused by the obligate biotrophic fungus Golovinomyces cichoracearum, is an economically important fungal disease of hemp - and marijuana – type cannabis. While the PM disease can be managed effectively by cultivating resistant hosts and maintaining proper sanitation in the growth facilities, there are not known PM resistant genetic variants representing a wide variety of chemotypes. This study represents the first transcriptomic report on hemp cultivar ‘X59’ in response to G. cichoracearum. We have quantified transcript dynamics at 5-, 8-, and 11-days post inoculation (DPI) of C. sativa genotypes and compared the differential expression between the control and inoculated seedlings. Symptoms of pathogen infection was observed 8 days post inoculation (DPI). Reference genome guided transcriptomic analysis revealed 1898 genes that were significantly (q < 0.05) differentially expressed (DE) during Golovinomyces infection. Of these, 241, 315, and 910 were upregulated in the treated samples at 5, 8, and 11 DPI respectively, while 263, 266, and 968 transcripts were downregulated in the treated samples at 5, 8, 11 DPI, respectively. Genes related to phytohormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), were abundantly regulated. Metabolic pathways related to flavonoids, terpenes, phytoalexins, and thaumatin were upregulated indicating their possible roles in defense against the pathogen. The expression of glycosyl hydrolases including chitinases and glucanases gradually increased by 11 DPI. Among the DE genes, there were 45 putative gene transcripts that were potentially involved in the plant response against PM. Our study has contributed to an enhanced understanding of the potential mechanisms that are involved in mediating response to PM, particularly during early stages of PM establishment and colonization.

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