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Cannabis sativa L. - Botany and Biotechnology pp 395–418Cite as

Assessing Genetic Diversity in Cannabis sativa Using Molecular Approaches

Abstract

Cannabis sativa L. represents plants cultivated for their psychoactive and medicinal properties (marijuana) or as a source of fibre, seed and oil (hemp). Breeding and selection efforts have produced marijuana genotypes (strains) with a range of levels of the cannabinoid Δ9-tetrahydrocannabinolic acid (THCA) and other non-psychoactive cannabinoids, e.g. cannabidiolic acid (CBDA). Hemp lines have been bred for high fibre content and seed production and have low/no THCA. There are currently hundreds of marijuana strains which differ in THCA:CBDA ratios, growth characteristics, morphological features, THCA and CBDA contents, disease resistance, as well as overall medicinal value. The extent of genetic diversity among these marijuana strains, as well as between marijuana and hemp, has been studied using a range of molecular approaches. The results from these studies have demonstrated that considerable genetic diversity exists among marijuana as well as hemp strains. Using ISSR markers, we have shown that distinct DNA banding patterns can allow for the initial discrimination between many of the strains tested, and provide an insight into the possible genetic relationships among strains. Some strains, e.g. ‘Jack’, ‘Super Sour Skunk’, ‘Jilly Bean’, exhibited unique patterns that can be used to develop strain-specific DNA fingerprints. In addition, a number of “landraces” and strains originating from remote geographic locations, showed unique and distinct ISSR patterns and morphologies. A very high degree of genetic diversity was exhibited among the strains studied. Additional molecular studies, including DNA sequencing approaches, should provide more insight into the genetic relationships that exist within strains of a complex plant species and could augment future breeding efforts for genetic improvement of C. sativa.

Keywords

  • Amplify Fragment Length Polymorphism
  • Inter Simple Sequence Repeat
  • Female Flower
  • Male Flower
  • Female Plant

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  • Alghanim HJ, Almirall JR (2003) Development of microsatellite markers in Cannabis sativa for DNA typing and genetic relatedness analyses. Anal Bioanal Chem 376:1225–1233

    CAS  CrossRef  PubMed  Google Scholar 

  • Atsmon D, Tabbak C (1979) Comparative effects of gibberellin, silver nitrate and aminoethoxyvinyl glycine on sexual tendency and ethylene evolution in the cucumber plant (Cucumis sativus L). Plant Cell Physiol 20:1547–1555

    CAS  CrossRef  Google Scholar 

  • Bagyawant SS (2016) RAPD-SCAR markers: an interface tool for authentication of traits. J Biosci Med 4:1–9

    Google Scholar 

  • Brady JL, Scott NS, Thomas MR (1996) DNA typing of hops (Humulus lupulus) through application of RAPD and microsatellite marker sequences converted to sequence tag sites (STS). Euphytica 91:277–284

    CAS  CrossRef  Google Scholar 

  • Clarke RC, Merlin MD (2013) Cannabis: evolution and ethnobotany. University of California Press, Berkeley, CA

    Google Scholar 

  • Caliskan M (ed) (2012) Genetic diversity in plants. Published by InTech, 510 p. doi:10.5772/2640

  • Chandra S, Lata H, Khan IA, ElSohly MA (2013) The role of biotechnology in Cannabis sativa propagation for the production of phytocannabinoids. In: Chandra S et al (eds) Biotechnology for medicinal plants. Springer, Berlin Heidelberg

    CrossRef  Google Scholar 

  • Chen S, Rodriguez G, Punja ZK (2015) A PCR method to distinguish between male and female plants of Cannabis sativa. Proceedings of the 10th Canadian plant genomics workshop, Victoria, BC

    Google Scholar 

  • Cheng J, Long Y, Khan MA, Wei C, Fu S, Fu J (2015) Development and significance of RAPD- SCAR markers for the identification of Litchi chinensis Sonn. by improved RAPD amplification and molecular cloning. Electron J Biotech 18:35–39

    CAS  CrossRef  Google Scholar 

  • Datwyler SL, Weiblen GD (2006) Genetic variation in hemp and marijuana (Cannabis sativa) according to amplified fragment length polymorphisms. J Foren Sci 51:371–375

    CAS  CrossRef  Google Scholar 

  • David S (2014) Hydroponics autoflowering marijuana: seed to harvest in 70 days or less. Big Bds. Archived website. https://web.archive.org/web/20140426234644/ http://bigbudsmag.com/grow/article/hydroponics-autoflowering-marijuana-seed-harvest-70-days-or-less-october-2011. Accessed 26 Oct 2016

  • Devaiah K, Balasubramani SP, Venkatasubramanian P (2011) Development of random amplified polymorphic DNA based SCAR marker for identification of Ipomoea mauritiana Jacq (Convolvulaceae). Evid Based Complement Altern Med 868:720–726

    Google Scholar 

  • Dufresnes C, Jan C, Bienert F, Goudet J, Fumagalli L (2017) Broad-scale genetic diversity of cannabis for forensic applications. PLoS ONE 12(1): e0170522

    Google Scholar 

  • Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES et al (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6(5). doi:10.1371/journal.pone.0019379, PMID: 21573248

  • Elsohly MA, Slade D (2005) Chemical constituents of marijuana: the complex mixture of natural cannabinoids. Life Sci 78:539–548

    CAS  CrossRef  PubMed  Google Scholar 

  • Faeti V, Mandolino G, Ranalli P (1996) Genetic diversity of Cannabis sativa germplasm based on RAPD markers. Plant Breed 115:367–370

    Google Scholar 

  • Fernandez M, Figueiras A, Benito C (2002) The use of ISSR and RAPD markers for detecting DNA polymorphism, genotype identification and genetic diversity among barley cultivars with known origin. Theor Appl Gen 104:845–851

    CAS  CrossRef  Google Scholar 

  • Flachowsky H, Schumann E, Weber WE, Peil A (2001) Application of AFLP for the detection of sex-specific markers in hemp. Plant Breed 120:305–309

    CAS  CrossRef  Google Scholar 

  • Forapani S, Carboni A, Paoletti C, Moliterni VMC, Ranalli Mandolino G (2001) Comparison of hemp varieties using random amplified polymorphic DNA markers. Crop Sci 41:1682–1689

    CAS  CrossRef  Google Scholar 

  • Gao C, Xin P, Cheng C, Tang Q, Chen P, Wang C et al. (2014) Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers. PLoS ONE 9:e110638

    Google Scholar 

  • Gigliano GS, Finizio AD, Caputo P, Cozzolino S (1995) Cannabis fingerprints by using random amplified polymorphic DNA (RAPD). Delpinoa 8:35–37

    Google Scholar 

  • Gillan R, Cole MD, Linacre A, Thorpe JW, Watson ND (1995) Comparison of Cannabis sativa by random amplification of polymorphic DNA (RAPD) and HPLC of cannabinoid: a preliminary study. Sci Justice 35:169–177

    CAS  CrossRef  PubMed  Google Scholar 

  • Gilmore S, Peakall R, Robertson J (2003) Short tandem repeats (STR) markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations. Forensic Sci Int 131:65–74

    CAS  CrossRef  PubMed  Google Scholar 

  • Gilmore S, Peakall R, Robertson J (2007) Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa. Forensic Sci Internat 172, 179–190

    Google Scholar 

  • Goodwillie C, Kalisz S, Eckert CG (2005) The evolutionary enigma of mixed mating systems in plants: occurrence, theoretical explanations, and empirical evidence. Annu Rev Ecol Evol Syst 36:47–79

    CrossRef  Google Scholar 

  • Hakki EE, Uz E, Sag A, Atasoy S, Akkaya SM (2003) Genotyping of Cannabis sativa L. accessions from Turkey using RAPD and AFLP markers. Forensic Sci Int 136:31

    Google Scholar 

  • Hakki EE, Kayis SA, Pinarkara E, Sag A (2007) Inter simple sequence repeats separate efficiently hemp from marijuana (Cannabis sativa L.). Electron J Biotech 10:570–581

    CrossRef  Google Scholar 

  • Hamrick JL (1989) Isozymes and the analysis of genetic structure in plant populations. In: Soltis DE, Soltis PS (eds) Isozymes in plant biology. Dioscorides Press, Portland, OR, pp 87–105

    Google Scholar 

  • Hillig K (2005) Genetic evidence for speciation in Cannabis (Cannabaceae). Genet Resour Crop 52:161–180

    CAS  CrossRef  Google Scholar 

  • Hsieh H-M, Hou R-J, Tsai L-C, Wei C-S, Liu S-W, Huang L-H, Kuo Y-C, Linacre Alee J-C (2003) A highly polymorphic STR locus in Cannabis sativa. Forensic Sci Int 131:53–58

    Google Scholar 

  • Hu Z G, Guo H Y, Hu X L, Chen X, Liu X Y, Guo M B et al. (2012) Genetic diversity research of hemp (Cannabis sativa L) cultivar based on AFLP analysis. J. Plant Genet Resource 13: 555–561

    Google Scholar 

  • Igic B, Kohn JR (2006) The distribution of plant mating systems: study bias against obligately outcrossing species. Evolution 60:1098–103

    CrossRef  PubMed  Google Scholar 

  • Jagadish V, Robertson J, Gibbs A (1996) RAPD analysis distinguishes Cannabis sativa samples from different sources. Forensic Sci Int 79:113–121

    CAS  CrossRef  Google Scholar 

  • Jaske J, Bandelji D, Javornik B (2002) Eleven new microsatellites for hop (Humulus lupulus L.). Mol Ecol Notes 2:544–546

    CrossRef  Google Scholar 

  • Kayis SA, Hakki EE, Pinarkara E (2010) Comparison of effectiveness of ISSR and RAPD markers in genetic characterization of seized marijuana (Cannabis sativa L.) in Turkey. Afr J Agric Res 52:2925–2933

    Google Scholar 

  • Khan S, Husaini AM, Kiran U, Ram KM, Abdin MZ (2008) SCAR markers for authentication of herbal drugs. Med Arom Plant Sci Biotech 2:79–85

    Google Scholar 

  • Kojoma M, Iida O, Makino Y, Sekita S, Satake M (2002) DNA fingerprinting of Cannabis sativa using inter-simple sequence repeat (ISSR) amplification. Planta Med 68:60–63

    CAS  CrossRef  PubMed  Google Scholar 

  • Kumar K, Parvatam G, Ravishankar GA (2009) AgNO3—a potential regulator of ethylene activity and plant growth modulator. Electron J Biotechnol 12(2). doi:10.2225/vol.12-issue2-fulltext-1

  • Lata H, Chandra S, Techen N, Khan IA, ElSohly MA (2010) Assessment of the genetic stability of micropropagated plants of Cannabis sativa by ISSR markers. Planta Med 76:97–100

    CAS  CrossRef  PubMed  Google Scholar 

  • Lata H, Chandra S, Techen N, Khan IA, ElSohly MA (2011) Molecular analysis of genetic fidelity in Cannabis sativa L. plants grown from synthetic (encapsulated) seeds following in vitro storage. Biotech. doi:10.1007/s10529-011-0712-7

    Google Scholar 

  • Lynch RC, Vergara D, Tittes S, White K, Schwartz CJ, Gibbs MJ, Ruthenburg TC, deCesare K, Land DP, Kane NC (2017) Genomic and chemical diversity in Cannabis. Crit Rev Plant Sci (in press)

    Google Scholar 

  • Mandolino G, Carboni A, Forapani S, Faeti V, Ranalli P (1999) Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.). Theor Appl Genet 98:86–92

    CAS  CrossRef  Google Scholar 

  • Mechoulam R (2005) Plant cannabinoids: a neglected pharmacological treasure trove. Br J Pharmacol 146:913–915

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  • Mendoza MA, Mills DK, Lata H, Chandra S, ElSohly MA, Almirall JA (2009) Genetic individualization of Cannabis sativa by a short tandem repeat multiplex system. Anal Bioanal Chem 393:719–726

    CAS  CrossRef  PubMed  Google Scholar 

  • Ming R, Bendahmane A, Renner SS (2011) Sex chromosomes in land plants. Ann Rev Plant Biol 62:485–514

    CAS  CrossRef  Google Scholar 

  • Mohan Ram HYM, Jaiswal VS (1970) Induction of female flowers on male plants of Cannabis sativa L. by 2-chloroethanephosphonic acid. Experientia 26:214–216

    CrossRef  Google Scholar 

  • Mohan Ram HYM, Jaiswal VS (1972) Induction of male flowers on female plants of Cannabis sativa by gibberellins and its inhibition by abscisic acid. Planta 105:263–266

    CrossRef  Google Scholar 

  • Mohan Ram HY, Sett R (1981) Modification of growth and sex expression in Cannabis sativa by aminoethoxyvinylglycine and ethephon. Z Pflanzenphysiol 105:165–172

    CrossRef  Google Scholar 

  • Patzak J (2001) Comparison of RAPD, STS, ISSR and AFLP molecular methods used for assessment of genetic diversity in hop (Humulus lupulus L.). Euphytica 121:9–18

    CAS  CrossRef  Google Scholar 

  • Perez de la Torre M, Garcia MN, Heinz R, Escandon A (2012) Analysis of genetic variability by ISSR markers in Calibrachoa caesia. Electron J Biotechnol 15(5)

    Google Scholar 

  • Piluzza G, Sonah H, Bastien M, Iquira E, Tardivel A, Légaré G, Boyle B et al (2013) An improved genotyping by sequencing (GBS) approach offering increased versatility and efficiency of SNP discovery and genotyping. PLoS ONE 8(1):e54603. doi:10.1371/journal.pone.0054603

    CrossRef  Google Scholar 

  • Pinarkara E, Kayis SA, Hakki EE, Sag A (2009) RAPD analysis of seized marijuana (Cannabis sativa L.) in Turkey. Electronic. J Biotech 12(1):1–12

    Google Scholar 

  • Potter DJP (2009) The propagation, characterization and optimization of Cannabis sativa L. as a phytopharmaceutical. PhD Thesis, Department of Pharmaceutical Science Research, King’s College London

    Google Scholar 

  • Punja ZK, Rodriguez G, Chen S (2015) Assessing genetic diversity among Cannabis sativa strains using ISSR markers. Proceedings of the 10th Canadian plant genomics workshop, Victoria, BC

    Google Scholar 

  • Rosenthal E (1991) Marijuana growers handbook—indoor/greenhouse edition. Published by Bookpeople. Available online at: http://www.getfreeride.org/filmy/eBooks/ANG/The%20Marijuana%20Grower’s%20Handbook%20-%20Ed%20Rosenthal%20-%202006.pdf. Accessed 26 Oct 2016

  • Rosenthal E (2000) Marijuana growing tips, 2nd edn. Quick American Archives

    Google Scholar 

  • Sakamoto K, Abe T, Matsuyama T, Yoshida S, Ohmido N, Fukui K, Satoh S (2005) RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L. Genome 48:931–936

    CAS  CrossRef  PubMed  Google Scholar 

  • Sakamoto K, Akiyama Y, Fukui K, Kamada H, Satoh S (1998) Characterization of genome sizes and morphology of sex chromosomes in hemp (Cannabis sativa L.). Cytologia 63:459–464

    CrossRef  Google Scholar 

  • Sakamoto K, Shimomura K, Komeda Y, Kamada H, Satoh S (1995) A male-associated DN sequence in a dioecious plant, Cannabis sativa L. Plant Cell Physiol 36:1549–1554

    CAS  PubMed  Google Scholar 

  • Sawler J, Stout JM, Gardner KM, Hudson D, Vidmar J, Butler L, Page JE, Myles S (2015) The genetic structure of marijuana and hemp. PLoS ONE. doi:10.1371/journal.pone.0133292

    Google Scholar 

  • Schlüter PM, Harris SA (2006) Analysis of multilocus fingerprinting data sets containing missing data. Mol Ecol Notes 6:569–572

    CrossRef  Google Scholar 

  • Schultes RE, Klein WM, Plowman T, Lockwood TE (1974) Cannabis: an example of taxonomic neglect. Bot Mus Leaflet Harvard Univ 23:337–367

    Google Scholar 

  • Shirota O, Watanabe A, Yamazaki M, Saito K, Shibano K, Sekita S, Satake M (1998) Random amplified polymorphic DNA and restriction fragment length polymorphism analysis of Cannabis sativa. Nat Med 52:160–166

    CAS  Google Scholar 

  • Small E (2015) Evolution and classification of Cannabis sativa (marijuana, hemp) in relation to  human utilization. Bot Rev 81: 189–294

    Google Scholar 

  • Small E, Cronquist A (1976) A practical and natural taxonomy for Cannabis. Taxon 25:405–435

    CrossRef  Google Scholar 

  • Srivastava MK, Li C-N, Li Y-R (2012) Development of sequence characterized amplified region (SCAR) marker for identifying drought tolerant sugarcane genotypes. Aust J Crop Sci 6:763–767

    CAS  Google Scholar 

  • Stankovic L, Prodanovic S (2002) Silver nitrate effects on sex expression in cucumber. Acta Hortic 579:203–206

    CAS  CrossRef  Google Scholar 

  • Stajner N, Satovic Z, Cerenak A, Javornik B (2008) Genetic structure and differentiation in hop (Humulus lupulus L.) as inferred from microsatellites. Euphytica 161:301–311

    CAS  CrossRef  Google Scholar 

  • Torjek O, Bucherna N, Kiss E, Homoki H, Finta-Korpelova Z, Bocsa I, Nagy I, Heszky LE (2001) Novel male-specific molecular markers (MADC5, MADC6) in hemp. Euphytica 127:209–218

    CrossRef  Google Scholar 

  • United Nations Office on Drugs and Crime (2009) Recommended methods for the identification and analysis of Cannabis and Cannabis products. Published by Laboratory and Scientific Section, UNODC, United Nations, Vienna. ISBN 978-92-1-148242-3

    Google Scholar 

  • Van Bakel H, Stout JM, Cote AG, Tallon CM, Sharpe AG, Hughes TR, Page JE (2011) The draftgenome and transcriptome of Cannabis sativa. Genome Biol 12:R102. doi:10.1186/gb-2011-12-10-r102

    CrossRef  PubMed  PubMed Central  Google Scholar 

  • Vijayan K (2005) Inter simple sequence repeat (ISSR) polymorphism and its application in mulberry genome analysis. Int J Indust Entom 10:79–86

    Google Scholar 

  • Weising K, Nybom H, Wolff K, Kahl G (2005) DNA fingerprinting in plants. Principles, methods and applications, 2nd edn. Taylor and Francis

    Google Scholar 

  • Welling MT, Shapter T, Rose TJ, Liu L, Stanger R, King GJ (2016) A belated green revolution for Cannabis: virtual genetic resources to fast-track cultivar development. Front Plant Sci 7:1113

    Google Scholar 

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Acknowledgements

Funding for this research was provided through the Natural Sciences and Engineering Research Council of Canada (NSERC), Engage and Engage Plus grants, in addition to funding from the industry partner, Agrima Botanicals, Inc. We thank Philip Campbell and Bianca Rizzo for providing photographic images and the various individuals who provided access to plant materials from which DNA samples could be obtained for the research. We also thank InnoTech Alberta for providing seed of ‘Silesia’ hemp.

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Authors and Affiliations

  1. Department of Biological Sciences, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada

    Zamir K. Punja, Gina Rodriguez & Sarah Chen

  2. Agrima Botanicals, 260-22529 Lougheed Hwy, Maple Ridge, BC, V2X 0T5, Canada

    Gina Rodriguez

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  1. Zamir K. Punja
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  2. Gina Rodriguez
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Correspondence to Zamir K. Punja .

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Editors and Affiliations

  1. University of Mississippi , Oxford, Mississippi, USA

    Suman Chandra

  2. School of Pharmacy, University of Mississippi School of Pharmacy, Oxford, Mississippi, USA

    Hemant Lata

  3. School of Pharmacy, University of Mississippi School of Pharmacy, Oxford, Mississippi, USA

    Mahmoud A. ElSohly

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Punja, Z.K., Rodriguez, G., Chen, S. (2017). Assessing Genetic Diversity in Cannabis sativa Using Molecular Approaches. In: Chandra, S., Lata, H., ElSohly, M. (eds) Cannabis sativa L. - Botany and Biotechnology. Springer, Cham. https://doi.org/10.1007/978-3-319-54564-6_19

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