ORIGINAL ARTICLE Characterization of new chloroplast markers to determine biogeographical origin and crop type of Cannabis sativa Madeline G. Roman 1 & David Gangitano 1 & Rachel Houston 1 Received: 25 June 2019 /Accepted: 6 August 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA. Despite its schedule I classification by the federal government, 33 states and the District of Columbia have legalized its use for medicinal or recreational purposes. This state-specific legalization has created a new problem for law enforcement: preventing and tracking the diversion of legally obtained Cannabis to states where it remains illegal. In addition, trafficking of the drug at the border with Mexico remains an issue for law enforcement agencies. C. sativa crops can be classified as marijuana (a drug containing the psychoactive chemical delta-9-tetrahydrocannabinol) or hemp (the non-drug form of the plant). Differentiation between crop types is important for forensic purposes. In addition, investigation of trafficking routes into and within the USA requires genetic association of samples from different seizures, and determining where the crop originated could provide important leads. This project seeks to exploit sequence variations in C. sativa chloroplast DNA (cpDNA) to allow genetic determination of biogeographic origin, discrimina- tion between marijuana and hemp, and association between cases for C. sativa samples. Due to the limited discriminatory ability of common barcoding markers, the authors sought to discover more informative polymorphic regions. By comparing published whole genome cpDNA sequences, 58 polymorphisms and seven hotspot regions were identified. Hemp samples from the USA and Canada, marijuana samples from Mexico and Chile, and medical marijuana samples from Chile were evaluated using two cpDNA hotspot regions, rpl32-trnL and trnS-trnG. Principal component analysis supported some differences between the groups based on their crop type and biogeographic origin. Keywords Forensic plant science . Chloroplast DNA barcoding . Haplotype . Cannabis sativa Introduction Cannabis sativa crops can be broadly classified as marijuana (a drug containing the psychoactive chemical delta-9-tetrahydro- cannabinol, or THC) or hemp (the non-drug form of the plant, which is cultivated for its use as oil and fiber) [1]. Marijuana is the most commonly used illicit drug in the USA [2], and as a result, it is highly trafficked, with many seizures occurring at the border with Mexico. Despite the federal government ’s classification of marijuana as a schedule I controlled substance, 33 states and the District of Columbia have legalized marijuana for medicinal use, with ten of these states and the District of Columbia also allowing recreational use of the drug. State- specific legalization of the drug poses a new challenge for law enforcement, which must now prevent and investigate the di- version of legal marijuana to states where it remains illegal. Identification of marijuana usually consists of confirming the presence of THC and cystolithic hairs on the leaves of the plant [3, 4]. However, these chemical and microscopic tech- niques can neither give information about the biogeographical origin of the sample nor be used to link seized samples. Since the 1990s, researchers have been interested in developing mo- lecular techniques for identifying and individualizing C. sativa samples in order to demonstrate associations be- tween samples. These techniques include Random Amplified Polymorphic DNA (RAPD) [5, 6], Amplified Fragment Length Polymorphism (AFLPs) [7], Inter Simple Sequence Repeats (ISSRs) [5, 8], barcoding regions in the Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00414-019-02142-w) contains supplementary material, which is available to authorized users. * Rachel Houston rmh034@shsu.edu 1 Department of Forensic Science, College of Criminal Justice, Sam Houston State University, 1003 Bowers Blvd., Huntsville, TX 77340-2525, USA International Journal of Legal Medicine https://doi.org/10.1007/s00414-019-02142-w