Journal:Broad-scale genetic diversity of Cannabis for forensic applications

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Full article title Broad-scale genetic diversity of Cannabis for forensic applications
Journal PLOS ONE
Author(s) Dufresnes, Christophe; Jan, Catherine; Bienert, Friederike; Goudet, Jérôme; Fumagalli, Luca
Author affiliation(s) University of Lausanne, Centre Universitaire Romand de Médecine Légale,
Primary contact Email: Luca dot Fumagalli at unil dot ch
Editors Scali, Monica
Year published 2017
Volume and issue 121
Page(s) e0170522
DOI 10.1371/journal.pone.0170522
ISSN 1932-6203
Distribution license Creative Commons Attribution 4.0 International
Website http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170522
Download http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0170522&type=printable (PDF)
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Abstract

Cannabis (hemp and marijuana) is an iconic yet controversial crop. On the one hand, it represents a growing market for pharmaceutical and agricultural sectors. On the other hand, plants synthesizing the psychoactive THC produce the most widespread illicit drug in the world. Yet, the difficulty to reliably distinguish between Cannabis varieties based on morphological or biochemical criteria impedes the development of promising industrial programs and hinders the fight against narcotrafficking. Genetics offers an appropriate alternative to characterize drug vs. non-drug Cannabis. However, forensic applications require rapid and affordable genotyping of informative and reliable molecular markers for which a broad-scale reference database, representing both intra- and inter-variety variation, is available. Here we provide such a resource for Cannabis, by genotyping 13 microsatellite loci (STRs) in 1,324 samples selected specifically for fiber (24 hemp varieties) and drug (15 marijuana varieties) production. We showed that these loci are sufficient to capture most of the genome-wide diversity patterns recently revealed by next-generation sequencing (NGS) data. We recovered strong genetic structure between marijuana and hemp and demonstrated that anonymous samples can be confidently assigned to either plant types. Fibers appear genetically homogeneous whereas drugs show low (often clonal) diversity within varieties, but very high genetic differentiation between them, likely resulting from breeding practices. Based on an additional test dataset that includes samples from 41 local police seizures, we showed that the genetic signature of marijuana cultivars could be used to trace crime scene evidence. To date, our study provides the most comprehensive genetic resource for Cannabis forensics worldwide.

Introduction

Cannabis is one of humanity’s oldest cultivated plant. It is thought to have originated in central Asia and was domesticated as early as 8,000 BP for food, fiber, oil, medicines and as an inebriant. This crop was since distributed across the world during the last two millennia and, due to its recent legalization in several countries, is increasingly exploited by several industrial sectors (hemp) and as a recreational drug (marijuana). The taxonomic status of Cannabis has always been disputed, as it encompasses multiple cultural, geographic, historical, and functional aspects.[1][2][3][4] Whereas most authors now consider it a monotypic panmictic taxon, Cannabis sativa, three species or subspecies (sativa, indica and ruderalis) are often mentioned but without a comprehensive taxonomic grouping so far. The nomenclature may thus differ depending on whether it refers to morphological or chemical variation, geographic distribution, ecotype, as well as crop-use characteristics and intoxicant properties resulting from human selection.[4][5][6][7] Cannabis presumably diversified following selection for traits enhancing fiber and seed production (”hemp”) or psychoactive properties ("drug"). Importantly, Cannabis types differ in their absolute and relative amounts of terpenophenolic cannabinoids, notably Δ1-tetrahydrocannabinol (THC), the well-known psychoactive compound of marijuana, and the non-psychoactive cannabidiol (CBD). In this context, drug-type Cannabis (marijuana) is broadly characterized by a higher overall cannabinoid content than fiber-types. However, the most widely recognized criteria to assign a Cannabis plant to either “drug” or “hemp” type is the THC:CBD ratio, according to which three main chemical phenotype (chemotype) classes are recognized: hemp-type plants with a low ratio (THC:CBD < 1), drug-type plants with a high ratio (THC:CBD > 1), and intermediate-type plants with a ratio close to one.[6][8] The informal designation sativa and indica may have various, controversial meanings. Morphologically, the name sativa designates tall plants with narrow leaves, while indica refers to short plants with wide leaves. Among the marijuana community however, sativa rather refers to equatorial varieties producing stimulating psychoactive effects (THC:CBD ≈ 1), whereas indica-type plants from Central Asia are used for relaxing and sedative drugs (THC:CBD > 1).[8]

The commercial interest for Cannabis declined during the twentieth century due, e.g., to the development of synthetic fibers and the stringent policies regarding its exploitation, but this iconic weed is recently regaining attention in many countries for its high medicinal, industrial, and agricultural potentials.[9] However, its usage is still controversial, in particular from agro-economic, public health, and forensic perspectives. Due to its intoxicant properties, the cultivation and possession of Cannabis is under strict legal regulations. High-THC:CBD varieties are prohibited in many countries but remain the most frequently-used illicit drug worldwide[10] (~180 million consumers in 2013[11]), in the form of marijuana (dried inflorescences) or hashish (resin). In contrast, low-THC:CBD hemp crops can be exploited under licensed control for seed oil, fibers, and pharmaceuticals. For instance, quantitative measures of THC content are currently considered by the European Union (EU) for approval as a licensed hemp cultivar (below 0.2% THC weight per weight in the mature dry inflorescences; http://ec.europa.eu/food/plant_en). Yet hemp and marijuana varieties are hardly distinguishable morphologically, and discrimination of drug vs. non-drug chemotypes by quantitative THC dosage has also proven inadequate due to its dependence on environmental factors, to the strong variation during the plant’s life cycle, as well as between individual plants.[12][13] In addition, the qualitative assessment of THC:CBD ratio is also problematic for an unequivocal discrimination between fiber and drug types due to the presence of a largely variable intermediate chemotype class, the occurrence of several exceptions (e.g., hemp accessions with a THC-predominant chemotype[14][15][16]), and the common practice among drug breeders to produce hybrid varieties.

This issue largely impedes crops’ improvement and full-scale industrial development; it even causes a security risk, as licensed crops may be used as a cover for illegal drug production. Moreover, it significantly limits the ability of law enforcement agencies to trace drug seizures and link illegal producers to organized crime syndicates supplying the black market of Cannabis drugs. In addition, Cannabis can have long-distance dispersal capabilities[17], and fiber crops might face cryptic contamination by pollen from drug varieties.

Genetic tools offer a promising avenue to overcome these issues, especially to distinguish between drug vs. non-drug plants.[18] Importantly, genetics requires small amounts of tissues as a DNA source, whereas chemical analyses necessitate inflorescences. A promising aspect has been to genotype loci directly linked to THC synthesis[8][19] in association with chemotype profiling. However, this association is not ubiquitous[14][15], and genotyping may be compromised by complex gene duplications, pseudogenes[20][21][22], and the fact that only a limited number of varieties among the tremendous Cannabis diversity has been validated[15]; moreover, chemotype seem to greatly vary even among genotypes.[20]

References

  1. ↑ Small, E.; Crongquist, A. (1976). "A practical and natural taxonomy for cannabis". Taxon 25 (4): 405–435. doi:10.2307/1220524. 
  2. ↑ Clarke, R.C.; Merlin, M.D. (2013). Cannabis: Evolution and Ethnobotany. University of California Press. pp. 434. ISBN 9780520270480. 
  3. ↑ Small, E. (2015). "Evolution and Classification of Cannabis sativa (Marijuana, Hemp) in Relation to Human Utilization". The Botanical Review 81 (3): 189–294. doi:10.1007/s12229-015-9157-3. 
  4. ↑ 4.0 4.1 Welling, M.T.; Shapter, T.; Rose, T.J. et al. (2016). "A Belated Green Revolution for Cannabis: Virtual Genetic Resources to Fast-Track Cultivar Development". Frontiers in Plant Science 7: 1113. doi:10.3389/fpls.2016.01113. PMC PMC4965456. PMID 27524992. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965456. 
  5. ↑ de Meijer, E.P.M.; van Soest, L.J.M. (1992). "The CPRO Cannabis germplasm collection". Euphytica 62 (3): 201–11. doi:10.1007/BF00041754. 
  6. ↑ 6.0 6.1 de Meijer, E.P.M. (2014). "The Chemical Phenotypes (Chemotypes) of Cannabis". In Pertwee, R.. Handbook of Cannabis. Oxford University Press. pp. 89–110. ISBN 9780199662685. 
  7. ↑ Hillig, K.W. (2005). "Genetic evidence for speciation in Cannabis (Cannabaceae)". Genetic Resources and Crop Evolution 52 (2): 161–80. doi:10.1007/s10722-003-4452-y. 
  8. ↑ 8.0 8.1 8.2 Hillig, K.W.; Mahlberg, P.G. (2004). "A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae)". American Journal of Botany 91 (6): 966–75. doi:10.3732/ajb.91.6.966. PMID 21653452. 
  9. ↑ Andre, C.M.; Hausman, J.F.; Guerriero, G. (2016). "Cannabis sativa: The Plant of the Thousand and One Molecules". Frontiers in Plant Science 7: 19. doi:10.3389/fpls.2016.00019. PMC PMC4740396. PMID 26870049. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740396. 
  10. ↑ Anderson, P. (2006). "Global use of alcohol, drugs and tobacco". Drug and alcohol review 25 (6): 489–502. doi:10.1080/09595230600944446. PMID 17132569. 
  11. ↑ United Nations Office on Drugs and Crime (2015) (PDF). World Drug Report 2015. United Nations. pp. 162. ISBN 9789211482829. https://www.unodc.org/documents/wdr2015/World_Drug_Report_2015.pdf. 
  12. ↑ Rowan, M.G.; Fairbairn, J.W. (1977). "Cannabinoid patterns in seedlings of Cannabis sativa L. and their use in the determination of chemical race". Journal of Pharmacy and Pharmacology 29 (8): 491–4. PMID 19599. 
  13. ↑ Baker, P.B.; Gough, T.A.; Taylor, B.J. (1982). "The physical and chemical features of Cannabis plants grown in the United Kingdom of Great Britain and Northern Ireland from seeds of known origin". Bulletin of Narcotics 34 (1): 27-36. PMID 6291677. 
  14. ↑ 14.0 14.1 Welling, M.T.; Liu, L.; Shapter, T. et al. (2016). "Characterisation of cannabinoid composition in a diverse Cannabis sativa L. germplasm collection". Euphytica 208 (3): 463–75. doi:10.1007/s10681-015-1585-y. 
  15. ↑ 15.0 15.1 15.2 Staginnus, C.; Zörntlein, S.; de Meijer, E. (2014). "A PCR marker linked to a THCA synthase polymorphism is a reliable tool to discriminate potentially THC-rich plants of Cannabis sativa L.". Journal of Forensic Sciences 59 (4): 919-26. doi:10.1111/1556-4029.12448. PMID 24579739. 
  16. ↑ Tipparat, P.; Natakankitkul, S.; Chamnivikaipong, P.; Chutiwat, S. (2012). "Characteristics of cannabinoids composition of Cannabis plants grown in Northern Thailand and its forensic application". Forensic Science International 215 (1–3): 164-70. doi:10.1016/j.forsciint.2011.05.006. PMID 21636228. 
  17. ↑ Cabezudo, B.; Recio, M.; Sánchez-Laulhé, J. et al. (1997). "Atmospheric transportation of marihuana pollen from North Africa to the Southwest of Europe". Atmospheric Environment 31 (20): 3323-3328. doi:10.1016/S1352-2310(97)00161-1. 
  18. ↑ Miller Coyle, H.; Palmbach, T.; Juliano, N. et al. (2003). "An overview of DNA methods for the identification and individualization of marijuana". Croatian Medical Journal 44 (3): 315–21. PMID 12808725. 
  19. ↑ de Meijer, E.P.; Bagatta, M.; Carboni, A. et al. (2003). "The inheritance of chemical phenotype in Cannabis sativa L.". Genetics 163 (1): 335–46. PMC PMC1462421. PMID 12586720. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462421. 
  20. ↑ 20.0 20.1 Weiblen, G.D.; Wenger, J.P.; Craft, K.J. et al. (2015). "Gene duplication and divergence affecting drug content in Cannabis sativa". The New Phytologist 208 (4): 1241–50. doi:10.1111/nph.13562. PMID 26189495. 
  21. ↑ McKernan, K.J.; Helbert, Y.; Tadigotla, V. et al. (2015). "Single molecule sequencing of THCA synthase reveals copy number variation in modern drug-type Cannabis sativa L.". bioRxiv. doi:10.1101/028654. 
  22. ↑ van Bakel, H.; Stout, J.M.; Cote, A.G. et al. (2011). "The draft genome and transcriptome of Cannabis sativa". Genome Biology 12 (10): R102. doi:10.1186/gb-2011-12-10-r102. PMC PMC3359589. PMID 22014239. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359589. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added.

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