Development of a Molecular Test That Identifies Strains of Cannabis sativa

As the legal cannabis industry continues to mature, it has become increasingly apparent that the need for rapid and simple tests for forensic investigations and industrial quality control has become crucial.  To that end, a group of Japanese investigators developed a polymerase chain reaction (PCR) method called loop-mediated isothermal amplification (LAMP) that can differentiate Cannabis sativa from hemp (1, 2).

The assay is based on gene amplification of highly conserved DNA sequences of the tetrahydrocannabinolic acid (THCA) synthase gene that plays a major role in the Cannabis THC biosynthetic pathway.  Researchers used this test on 21 known Cannabis sativa varieties and were able to detect THCA sequence in each of them within 90 minutes (1).  In additional experiments, the researchers were able to distinguish between multiple strains of Cannabis sativa (which all possessed THCA DNA sequences) and multiple varieties of hemp (which do not possess detectable THCA sequences (2).  Based on these results, the researchers suggested that LAMP represents a rapid, sensitive, highly specific and convenient (in the field) method for detecting Cannabis sativa and differentiating it from non-psychoactive Cannabis varieties.

While this research represents a step in the right direction for forensic analysis and industrial Cannabis quality control, LAMP in its current form does not allow differentiation between different varieties of Cannabis sativa. Additional molecular analyses of different Cannabis genomes and a more in depth evolutionary analysis of genetic divergence in THCA genes from different cannabis varieties will be required to make this test useful in forensic analysis of different cannabis brands and industrial quality control programs.

  References 

  1. Kitamura M, Aragane M, Nakamura K, Watanabe K, Sasaki Y  Development of Loop-mediated isothermal amplification (LAMP) assay for rapid detection of Cannabis sativa Biol Pharm Bull. 2016; 39:1144-1149.
  2. Kitamura M, Aragane M, Nakamura K, Watanabe K, Sasaki Y  Rapid identification of drug-type strain in Cannabis sativa using loop-mediated isothermal amplification assay. J Nat.Med 2017;71:86-95.

Cannabis By Any Other Name

The number of species that compose the genus Cannabis is often debated. Some experts’ claim that the genus is composed of three distinct species designated Cannabis sativa, Cannabis indica and Cannabis ruderalis (1, 2). Others contend that the genus is composed of a single species—C. sativa L—which exhibits high degrees of variations and heterogeneity within different subpopulations.

According to most current scientific conventions, the plant is usually classified as a single species C. sativa.  Historically, most of the groupings or subdivisions in the genus have been made on physical characteristics or uses of the plant. For example, hemp plants are described as tall and skinny do not produce cannabinoids or terpenes (3) are not psychoactive when consumed and are mainly used for the extraction of fiber and oil (4).  Indica plants are typically short with broad leafs and generally are associated with sedative effects after consumption. Sativa plants are tall and skinny with narrow leafs, produce high levels of cannabinoids and terpenes and mediate psychoactive effects after consumption. (4). In addition, a large number of so-called Cannabis hybrids (crosses between the three Cannabis types) also exist.

Recent genomic and molecular biological analyses are beginning to provide new insights into the actual classification of the Cannabis genus. At present, there are six whole-genomes assemblies (two each) of three different strains of C. sativa. (4) These include Purple Kush (5, 6); Chemdawg (4) and LA confidential (4). In addition, there are two sativa transcriptomes (whole genome RNA analyses) [5], four mitochondrial genome assemblies (three sativa and one hemp) [4, 5, 7, 8], 6 chloroplast genome assemblies (5, 7, 9) and over 393 additional genomic resources that are being used to learn more about Cannabis classification (4).

Result of studies using the above mentioned genetic tools suggest that hemp is a distinct group and that two marijuana-type groups may also exist (5, 10, 11). To interpret their results, researchers suggested a naming convention based on leaf phenotypes: narrow-leaf drug type (NLDT) aka sativa, broad-leaf drug type (BLDT) aka indica and hemp (11). While 86% of plants classified as hemp fell into the hemp category, only 19% of popular sativa cultivar/brands fell into the NLDT category (sativa) and 27% of indica strains clustered within the BLDT (indica) group. Interestingly, 36% of so-called hybrid strains fell into the BLDT (indica) group and 62% were placed in the NLDT (sativa) category (4). Further, cultivars/brands that are most popularly reported as 100% sativa can be more closely related to be 100% indica (4).

Thanks to these genomic analyses it appears that the colloquial classifications given to individual Cannabis plants are not accurate. That said, it is important to note that the current genomic classification scheme is based on a small sample size and may not represent the actual genetic variation that exists in the Cannabis genus. To confirm or refute this possibility, more genomic analyses of existing Cannabis cultivars/brands must be performed before the genus can be accurately classified.

While current genomic studies may appear to be a purely academic exercise to some, accurate classification of Cannabis plants will be vitally important as Cannabis growers attempt to validate the identity, quality and properties of the cultivars/brands that they sell for medical or recreational purposes.

References 

  1. Hillig, K. W., and Mahlberg, P. G. A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae) Am. J. 2004; Bot. 91:966–975
  2. Hillig, K. W. Genetic evidence for speciation in Cannabis (Cannabaceae). Genet. Resources Crop Evol 2005; 52: 161–180
  3. Small, E, and Cronquist, A. A practical and natural taxonomy for Cannabis. Taxon. 1976; 25:405–435
  4. Vergara D,  Baker, H, Clancy K,  Keepers KG, Mendieta JP, Pauli CS,  Tittes SB, White KH, Kane,NC  Genetic and Genomic Tools for Cannabis sativa, Critical Reviews in Plant Sciences, 2016; 35:5-6, 364-377, DOI:10.1080/07352689.2016.1267496
  5. van Bakel,H., Stout, J. M., Cote, A. G., Tallon, C. M., Sharpe, A. G., Hughes, T. R., and Page, J. E. The draft genome and transcriptome of Cannabis sativa. Genome Biol. 2011;   12:1241–1250
  6. Li, R., Zhu, H., Ruan, J., Qian, W., Fang, X., Shi, Z., Li, Y., Li, S., Shan, G., and Kristiansen, K. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res. 2010; 20: 265–272
  7. Vergara, D.,White, K. H., Keepers, K. G., and Kane, N. C. The complete chloroplast genomes of Cannabis sativa and Humulus lupulus. Mitochondrial DNA Part A 2015; 27: 3793–3794
  8. White, K. H., Vergara, D., Keepers, K. G., and Kane, N. C. The complete mitochondrial genome for Cannabis sativa. Mitochondrial DNA Part B 2016; 1: 715–716
  9. Oh, H., Seo, B., Lee, S., Ahn, D.-H., Jo, E., Park, J.-K., and Min,G.-S. Two complete chloroplast genome sequences of Cannabis sativa varieties. Mitochondrial DNA Part A. 2015; 27:2835–2837
  10. Sawler, J., Stout, J. M., Gardner, K. M., Hudson, D., Vidmar, J., Butler, L., Page, J. E., and Myles, S. The genetic structure of marijuana and hemp. PloS One 2015; 10: e0133292
  11. Lynch, R. C., Vergara, D., Tittes, S., White, K., Schwartz, C. J., Gibbs, M. J., Ruthenburg, T. C., Land, D. P., and Kane, N.C. Genomic and Chemical Diversity in Cannabis. Crit. Rev. Plant Sci. 2016: 35: 349–363

Cannabis Genomics, Terpenes and the “Entourage Effect”

In addition to pharmacologically active cannabinoids, cannabis resins also contain a variety of terpenes (monoterpenes and sesquiterpenes) that are responsible for the scent of cannabis flowers and contribute to the unique, characteristic flavor qualities of cannabis-derived products. (1)  Over 200 terpenes have been reported in Cannabis sativa (2)

Differences in the medicinal properties of different cannabis strains have been attributed to interactions (or entourage effect) between cannabinoids and various terpenes (2, 3). For example, several cannabis terpenes (most notably, β-Caryophyllene (BCP) have been reported to interact with human cannabinoid receptors (4).  Put simply, terpenes plus cannabinoids—not cannabinoids alone—may be responsible for some of the medicinal benefits attributed to cannabis.  Consequently, it has been proposed that blends of cannabinoids and terpenes could be used in medicinal cannabis preparations to maximize therapeutic benefits via the so-called entourage effect (5). Finally, other research shows that terpenes may contribute to the anxiolytic, antibacterial, anti-inflammatory and sedative effects of Cannabis (2).

While much is known about the phytochemical composition of terpenes for forensic analysis and cannabis breeding, little is know about the molecular biology of terpene biosynthesis in cannabis.  In a recent paper, Booth et al (1) successfully identified nine terpene genes that appear to be involved in all stages of cannabis terpene biosynthesis. The authors suggested that knowledge of the genomics and gene functions of terpene biosynthesis may allow genetic manipulation of cannabis for desirable terpene profiles.  Further, genetic manipulation of terpene biosynthesis may help to scientifically unravel the so-called entourage effect and maximize the medicinal benefits of individual cannabinoids and cannabis-derived pharmaceuticals.

References

  1. Booth JK, Page JE, Bohlmann J. Terpene synthases from Cannabis sativa. PLoSOne 2017; 12:e0173911
  2. Russo EB. Taming THC: potential cannabis synergy and phytocannabinoid‐terpenoid entourage effects. British Journal of Pharmacology. 2011; 163: 1344–64
  3. ElSohly MA, editor. Marijuana and the cannabinoids. Springer Science & Business Media; 2007. November 15.
  4. ElSohly MA, editor. Marijuana and the cannabinoids. Springer Science & Business Media; 2007. November 15.
  5. Wagner H, Ulrich-Merzenich G. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 2009; 16: 97–110

Finding a Science Job in the Cannabis Industry

According to a recent report by the Cannabis website Leafly, America’s legal cannabis industry now supports more than 122,000 full-time jobs in 29 States and Washington DC. I

A recent article by Bruce Barcott entitled “How to Find a Job in the Cannabis Industry” offers some insights on the types of jobs that are available and how to land one.

He offered, like most industries the best way to land a job in the Cannabis industry is to network yourself into one. Also, working with a recruiting firm can be helpful.  Interestingly, recruiting firms and staffing companies that specialize in Cannabis jobs are popping up daily in many states where medical and recreational Cannabis are legal. However, before you take the plunge it is important to educate yourself to determine what is out there and whether or not you are a good fit for a Cannabis career.

So what do we know?  Most of the open jobs are in the Western states, California, Colorado, Oregon, Washington, Nevada and Arizona with a growing presence in Minnesota and Massachusetts. There are a smattering of jobs emerging in New York, Connecticut, Maryland  and Washington DC.  While 40 percent of open positions are specific to the Cannabis industry, roughly 60 are jobs that exist in other industries such as executive assistants, human resources specialists retail operations directors bookkeepers and staff accountants.That said, there are a number of Cannabis business operators who are looking for pharmaceutical sales representatives, or in horticulturalists from large commercial plant growing operations.

So question is: are there are any jobs in the Cannabis for the average Bio Job Blog reader?  The answer is YES!!!!!!  Here are a few examples: Laboratory chemist, operations manager, analytical chemist/production manager, software developer, food productions manager, and my favorite professional joint roller.  Of course there will be many more opportunities as the industry continues to grow (pun intended). That said, relocation is likely required but then again if you are qualified and possess the skills the company may offer a relocation package.  There is a ton of money being made in the industry!