A highly polymorphic STR locus in Cannabis sativa Hsing-Mei Hsieh a , Rur-Jyun Hou a , Li-Chin Tsai a , Chih-Sheng Wei a , Su-Wen Liu a , Li-Hung Huang b , Yi-Chen Kuo b , Adrian Linacre c , James Chun-I Lee a,* a Department of Forensic Science, Central Police University, 56 Shu-Jen Road, Kwei-San, Taoyuan 33334, Taiwan, ROC b Forensic Science Laboratory, Central Police University, 56 Shu-Jen Road, Kwei-San, Taoyuan 33334, Taiwan, ROC c Department of Pure and Applied Chemistry, Forensic Science Unit, University of Strathclyde, Glasgow G1 1XW, UK Received 31 May 2002; accepted 11 October 2002 Abstract We report on the ®rst short tandem repeat (STR) locus to be isolated from the plant Cannabissativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is speci®c to C. sativa and is highly reproducible. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cannabis sativa; Microsatellite DNA; Short tandem repeat 1. Introduction The techniques in molecular analysis of plants usually include random ampli®ed polymorphic DNA (RAPD) [1], ampli®ed fragment length polymorphism (AFLP) [2] and restriction fragment length polymorphism (RFLP) [3], and the speci®c markers analyzed are internal transcribed spacer (ITS) [4], large subunit of ribulose-1, 5-bisphosphate car- boxylase (rbcL) [5], intergenic spacer (IGS) [6] and micro- satellites [7,8]. The plant Cannabis sativa has long been characterized by the observation of cystolithic hairs on the leaves [9]. The legal requirement in most countries is to con®rm the presence of cannabinoids [10±14]. Tests using polymorphic DNA loci have been used to determine not only the presence of cannabis but also their possible geographical origin [15±21]. For the whole genome analysis, RAPD provides fast image of genomic variation at species or population level [15], but due to problems with reproduci- bility RAPD has been superseded by sequence polymorphic tests. The ITS I and II of nuclear ribosomal DNA and intergenic spacer region of chloroplast DNA (trnL±trnF IGS) provides species speci®c identi®cation and some poly- morphic information [16±21]. Identi®cation of the species is possible using such tests but little information on the phylogeny or genetic relationship of individual samples can be gained by these methods and markers. As a means of producing a more informative test, we describe the isolation and characterization of the ®rst microsatellite DNA in cannabis identi®cation. Poly- morphic microsatellite DNA loci are ubiquitous and dis- persed in many genomes [8,22] including plants [7]. They are now widely used in personal identi®cation of forensic applications [23]. This study presents its potential in the individualization of vegetative material from the plant C. sativa and the ®rst real test to link two seizures of vege- tative material. Forensic Science International 131 (2003) 53±58 * Corresponding author. Tel.: 886-3-3275907; fax: 886-3-3275907. E-mail address: jimlee@sun4.cpu.edu.tw (J.C.-I. Lee). 0379-0738/03/$ ± see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. PII:S0379-0738(02)00395-X