International Journal of Legal Medicine

, Volume 133, Issue 2, pp 373–384 | Cite as

13-loci STR multiplex system for Brazilian seized samples of marijuana: individualization and origin differentiation

  • Mauro Sander Fett
  • Roberta Fogliatto MariotEmail author
  • Eduardo Avila
  • Clarice Sampaio Alho
  • Valdir Marcos Stefenon
  • Flávio Anastácio de Oliveira Camargo
Original Article


It is known that Cannabis in Brazil could either originate from Paraguay or be cultivated in Brazil. While consumer markets in the North and Northeast regions are maintained by national production, the rest of the country is supplied with Cannabis from Paraguay. However, the Brazilian Federal Police (BFP) has exponentially increased the seizure number of Cannabis seeds sent by mail. For this reason, the aim of the study was to assess the 13-loci short tandem repeat (STR) multiplex system proposed by Houston et al. (2015) to evaluate the power of such markers in individualization and origin differentiation of Cannabis sativa samples seized in Brazil by the BFP. To do so, 72 Cannabis samples seized in Brazil by BFP were analyzed. The principal coordinate analysis (PCoA) and probability identity (PI) analysis were computed. Additionally, the Cannabis samples’ genotypes were subjected to comparison by Kruskal-Wallis H, followed by a multiple discriminant analysis (MDA). All samples analyzed revealed a distinct genetic profile. PCoA clearly discriminated the seizure sets based on their geographic origin. A combination of seven loci was enough to differentiate samples’ genotypes, and the PI for a random sample is approximately one in 50 billion. The Cannabis samples were 100% correct as classified by Kruskal-Wallis H, followed by an MDA. The results of this study demonstrate that the 13-loci STR multiplex system successfully achieved the aim of sample individualization and origin differentiation and suggest that it could be a useful tool to help BFP intelligence in tracing back-trade routes.


Cannabis sativa L. Genotype Short tandem repeat Marker 



We would like to thank Dr. David Gangitano and Dr. Rachel Houston for provide the panel, the bins, the allelic ladder, and two Cannabis-positive control samples. We also want to thank Dr. Claudia Paiva Nunes, Lucas Ribeiro, and Pietra Graebin for their technical assistance.

Funding information

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of Brazil (grant PRO-FORENSE 25/2014) and National Institute of Forensic Science and Technology (CNPq/INCT Forense grant 465450/2014-8).

Compliance with ethical standards


The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    United Nations Office on Drugs and Crime (UNODC) (2016) World drug report 2016. UNODC, New YorkGoogle Scholar
  2. 2.
    United Nations Office on Drugs and Crime (UNODC) (2018) World drug report 2018. UNODC, New YorkCrossRefGoogle Scholar
  3. 3.
    Moreira EM (2005) A despenalização do trabalhador rural preso no cultivo da Cannabis Sativa. Confluência - Rev Interdiscip Sociol e Direito 4(1):7–21CrossRefGoogle Scholar
  4. 4.
    Fraga PCP (2014) Illicit crops in Brazil. The rural workers between income and the exploitation of drug trafficking. Humanit Soc Sci 2:211–222. Google Scholar
  5. 5.
    United Nations Office on Drugs and Crime (UNODC) (2015) World drug report 2015. UNODC, New YorkGoogle Scholar
  6. 6.
    De Backer B, Maebe K, Verstraete AG, Charlier C (2012) Evolution of the content of THC and other major cannabinoids in drug-type Cannabis cuttings and seedlings during growth of plants. J Forensic Sci 57:918–922. CrossRefGoogle Scholar
  7. 7.
    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 10(8):e0133292. CrossRefGoogle Scholar
  8. 8.
    Laursen L (2015) Botany: the cultivation of weed. Nature 525:S4–S5. CrossRefGoogle Scholar
  9. 9.
    Miller Coyle H, Palmbach T, Juliano N, Ladd C, Lee HC (2003) An overview of DNA methods for the identification and individualization of marijuana. Croat Med J 44:315–321Google Scholar
  10. 10.
    Ribeiro A, Dias V, Mello I, Silva R, Sabino B, Garrido R, Seldin L, Moura-Neto RS (2013) O gene rbcL como barcode para identificação forense de Cannabis sativa. Saúde, Ética & Justiça 18 (Ed. Especial): 67–71.
  11. 11.
    Gilmore S, Peakall R, Robertson J (2003) Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations. Forensic Sci Int 131:65–74. CrossRefGoogle Scholar
  12. 12.
    Howard C, Gilmore S, Robertson J, Peakall R (2008) Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis. J Forensic Sci 53:1061–1067. Google Scholar
  13. 13.
    Howard C, Gilmore S, Robertson J, Peakall R (2009) A Cannabis sativa STR genotype database for Australian seizures: forensic applications and limitations. J Forensic Sci 54:556–563. CrossRefGoogle Scholar
  14. 14.
    Köhnemann S, Nedele J, Schwotzer D, Morzfeld J, Pfeiffer H (2012) The validation of a 15 STR multiplex PCR for Cannabis species. Int J Legal Med 126:601–606. CrossRefGoogle Scholar
  15. 15.
    Houston R, Birck M, Hughes-Stamm S, Gangitano D (2015) Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification. Int J Legal Med 130:635–647. CrossRefGoogle Scholar
  16. 16.
    DNeasy. (2013). Plant handbook. Valencia, CA: QiagenGoogle Scholar
  17. 17.
    Dneasy. (2010). mericon food handbook. Valencia, CA: Qiagen.Google Scholar
  18. 18.
    Valverde L, Lischka C, Scheiper S, Nedele J, Challis R, de Pancorbo MM, Pfeiffer H, Köhnemann S (2014) Characterization of 15 STR Cannabis loci: nomenclature proposal and SNPSTR haplotypes. Forensic Sci Int Genet 9:61–65.
  19. 19.
    Gill P, Brinkmann B, D’Aloja E, Andersen J, Bar W, Carracedo A, Dupuy B, Eriksen B, Jangblad M, Johnsson V, Kloosterman AD, Lincoln P, Morling N, Rand S, Sabatier M, Scheithauer R, Schneider P, Vide MC (1997) Considerations from the European DNA profiling group (EDNAP) concerning STR nomenclature. Forensic Sci Int 87:185–192. CrossRefGoogle Scholar
  20. 20.
    Olaisen B, Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Lincoln P, Mayr WR, Rand S (1998) DNA recommendations 1997 of the International Society for Forensic Genetics. Vox Sang 74:61–63CrossRefGoogle Scholar
  21. 21.
    Scientific Working Group on DNA Analysis Methods. (2016) SWGDAM Validation Guidelines for DNA Analysis Methods—Approved 12/05/2016. Available at:
  22. 22.
    Peakall R, Smouse PE (2012) GenALEx 6.5: genetic analysis in excel. Population genetic software for teaching and research-an update. Bioinformatics 28:2537–2539. CrossRefGoogle Scholar
  23. 23.
    Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295. CrossRefGoogle Scholar
  24. 24.
    Nei M (1972) Genetic distance between populations. Amer Nat 106:283–292CrossRefGoogle Scholar
  25. 25.
    Brenner C, Morris J (1990) Paternity index calculations in single locus hypervariable DNA probes: validation and other studies. Proc Int Symp Hum Identif 1989:21–53Google Scholar
  26. 26.
    Akram NA, Farooqi SR (2012) DNASF: a statistical package to analyze the distribution and polymorphism of CODIS STR loci in a heterogeneous population. J Forensic Res 3:1–6. Google Scholar
  27. 27.
    Budowle B, Onorato AJ, Callaghan TF, Della MA, Gross AM, Guerrieri RA, Luttman JC, Mcclure DL (2009) Mixture interpretation: defining the relevant features for guidelines for the assessment of mixed dna profiles in forensic casework. J Forensic Sci 54:810–821. CrossRefGoogle Scholar
  28. 28.
    Pasquale AA De, Tumino G, Pasquale RC De, Pasquale RC De (1974) Micromorphology of the epidermic surfaces of female plants of Cannabis sativa L. Bull Narcotics 26: 27–40.Google Scholar
  29. 29.
    Metz SG, Sharma HC, Armstrong TA, Mascia PN (1988) Chromosome doubling and aneuploidy in anther-derived plants from two winter wheat lines. Genome 30:177–181CrossRefGoogle Scholar
  30. 30.
    Sharma CB (1990) Chemically induced aneuploidy in higher plants. Mutagenesis 5:105–125. CrossRefGoogle Scholar
  31. 31.
    Adams KL, Wendel JF (2005) Polyploidy and genome evolution in plants. Curr Opin Plant Biol 8:135–141. CrossRefGoogle Scholar
  32. 32.
    Bagheri M, Mansouri H (2015) Effect of induced polyploidy on some biochemical parameters in Cannabis sativa L. Appl Biochem Biotechnol 175:2366–2375. CrossRefGoogle Scholar
  33. 33.
    Knight G, Hansen S, Connor M, Poulsen H, McGovern C, Stacey J (2010) The results of an experimental indoor hydroponic Cannabis growing study, using the ‘Screen of Green’ (ScrOG) method—yield, tetrahydrocannabinol (THC) and DNA analysis. Forensic Sci Int 202:36–44. CrossRefGoogle Scholar
  34. 34.
    Hsieh HM, Hou RJ, Tsai LC, Wei CS, Liu SW, Huang LH, Kuo YC, Linacre A, Lee JCI (2003) A highly polymorphic STR locus in Cannabis sativa. Forensic Sci Int 131:53–58. CrossRefGoogle Scholar
  35. 35.
    Shirley N, Allgeier L, Lanier T, Coyle HM (2013) Analysis of the NMI01 marker for a population database of Cannabis seeds. J Forensic Sci 58:176–182. CrossRefGoogle Scholar
  36. 36.
    Shibuya EK, Sarkis JES, Negrini-Neto O, Martinelli LA (2007) Carbon and nitrogen stable isotopes as indicative of geographical origin of marijuana samples seized in the city of São Paulo (Brazil). Forensic Sci Int 167:8–15. CrossRefGoogle Scholar
  37. 37.
    Houston R, Birck M, Hughes-Stamm S, Gangitano D (2017) Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling. Legal Med 26:33–40. CrossRefGoogle Scholar
  38. 38.
    Hair Jr JF, Black WC, Babin BJ, Anderson RE, Tatham RL (2009) Análise multivariada de dados (6th ed.). Porto Alegre: BookGoogle Scholar
  39. 39.
    Szczerbowska-Boruchowska M, Lankosz M, Adamek D (2011) First step toward the “fingerprinting” of brain tumors based on synchrotron radiation X-ray fluorescence and multiple discriminant analysis. J Biol Inorg Chem 16:1217–1226. CrossRefGoogle Scholar
  40. 40.
    Valverde L, Lischka C, Erlemann S, De Meijer E, De Pancorbo MM, Pfeiffer H, Köhnemann S (2014) Nomenclature proposal and SNPSTR haplotypes for 7 new Cannabis sativa L. STR loci. Forensic Sci Int Genet 13:185–186. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Mauro Sander Fett
    • 1
    • 2
  • Roberta Fogliatto Mariot
    • 1
    Email author
  • Eduardo Avila
    • 2
    • 3
  • Clarice Sampaio Alho
    • 3
  • Valdir Marcos Stefenon
    • 4
  • Flávio Anastácio de Oliveira Camargo
    • 1
  1. 1.Soil Science Department, Agronomy FacultyFederal University of Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  2. 2.Setor Técnico-Científico, Superintendência Regional do Departamento de Polícia Federal no Rio Grande do SulPorto AlegreBrazil
  3. 3.Bioscience FacultyPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Porto AlegreBrazil
  4. 4.Interdisciplinary Center of Biotechnology ResearchFederal University of the Pampa (UNIPAMPA)São GabrielBrazil

Personalised recommendations