Forensic Science International 152 (2005) 121–131 www.elsevier.com/locate/forsciint A molecular identification system for grasses: a novel technology for forensic botany J. Warda,*, R. Peakalla, S.R. Gilmorea,b, J. Robertsonc aSchool of Botany and Zoology, Australian National University, Canberra, ACT 0200, Australia bCentre for Forensic Science, Canberra Institute of Technology, GPO Box 826, Canberra, ACT 2601, Australia cNational Manager, Forensic and Technical Services Division, Australian Federal Police, GPO Box 401, Canberra, ACT 2601, Australia Received 29 April 2004; received in revised form 2 July 2004; accepted 7 July 2004 Available online 21 September 2004 Abstract Our present inability to rapidly, accurately and cost-effectively identify trace botanical evidence remains the major impediment to the routine application of forensic botany. Grasses are amongst the most likely plant species encountered as forensic trace evidence and have the potential to provide links between crime scenes and individuals or other vital crime scene information. We are designing a molecular DNA-based identification system for grasses consisting of several PCR assays that, like a traditional morphological taxonomic key, provide criteria that progressively identify an unknown grass sample to a given taxonomic rank. In a prior study of DNA sequences across 20 phylogenetically representative grass species, we identified a series of potentially informative indels in the grass mitochondrial genome. In this study we designed and tested five PCR assays spanning these indels and assessed the feasibility of these assays to aid identification of unknown grass samples. We confirmed that for our control set of 20 samples, on which the design of the PCR assays was based, the five primer combinations produced the expected results. Using these PCR assays in a ‘blind test’, we were able to identify 25 unknown grass samples with some restrictions. Species belonging to genera represented in our control set were all correctly identified to genus with one exception. Similarly, genera belonging to tribes in the control set were correctly identified to the tribal level. Finally, for those samples for which neither the tribal or genus specific PCR assays were designed, we could confidently exclude these samples from belonging to certain tribes and genera. The results confirmed the utility of the PCR assays and the feasibility of developing a robust full- scale usable grass identification system for forensic purposes. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Grasses; Molecular identification system; Forensic botany; Indels; PCR assay; Mitochondrial genome 1. Introduction either awareness or botanical knowledge among evidence- collection teams and prosecutors, and the difficulty in rou- Forensic botany is the study of plants and plant matter as tinely identifying trace material by traditional morphologi- they pertain to criminal investigations [1]. Botanical evi- cal methods, using whole-plant identification or botanical dence remains under-utilised in forensics due to the lack of experts [1]. Grasses are amongst the most likely plant species * Corresponding author. Tel.: +61 2 6125 8161; encountered in trace evidence searches, and have the poten- fax: +61 2 6125 5573. tial to provide links between crime scenes and individuals, E-mail address: [email protected] (J. Ward). relate an item to a suspect, support or disprove alibis or 0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.07.015 122 J. Ward et al. / Forensic Science International 152 (2005) 121–131 provide vital crime scene information. They provide con- sampled set of 20 grass taxa representing the Pooideae siderable potential as contact DNA evidence because of their and the Panicoideae subfamilies [13]. Within each sub- ubiquitous nature in both urban and rural environments, and family, two major tribes were represented and within each their morphological adaptations for seed dispersal [2]. Many tribe, two major genera were represented by each of two grass seeds have hooks, barbs, spines, hairs or sticky cover- species. Within the Poeae tribe, sampling was extended to ings that allow them to be transported easily by attaching to examine variation within species, with two cultivars of the bodies of animals and thus humans [3–5]. each of the four species represented (Table 1). The phy- Molecular technology is now being used routinely in logenetic framework for the sample design was based forensic investigations involving humans [6–8], but applica- on Watson and Dallwitz, Clark et al., GPWG and GPWG tion of these techniques to plant evidence is still novel [14–17]. [9–12]. Our present inability to routinely, rapidly and accu- The 20 samples, which were sequenced and used for rately identify trace botanical evidence remains the major primer design [13], provide our ‘control’ set of samples for impediment to the routine application of forensic botany. testing the PCR assays developed in this study. A further set Our objective is to design a molecular DNA-based of 26 species from within the Pooideae or Panicoideae identification system for grasses consisting of several subfamilies represent our ‘experimental’ set of samples PCR assays that, like a traditional morphological taxonomic (Table 1). All samples were sourced as seeds that were key, provide criteria that progressively identify an unknown obtained from seed, grain and turf companies within grass sample to a given taxonomic rank depending on the Australia and subsequently germinated in sterile soil in outcome of the previous step. Ultimately for those grasses of glasshouse conditions. Shortly after germination, leaf tissue potential widespread forensic interest, such as turf grasses samples were collected for DNA extractions. that are frequently encountered in the urban environment, identification to cultivar level will be essential. This might be 2.2. DNA extraction followed by further population level analyses in an attempt to match samples to a point of origin. Total genomic DNA was isolated from 0.5–1.0 g of As the first step towards the development of this grass seedling tissue that was ground in liquid nitrogen using a molecular identification system, we examined DNA sequence modified CTAB protocol of Doyle and Doyle [18]. The variation among a phylogenetically representative and hier- crude DNA was resuspended in 200 mL of TE Buffer (Tris archically sampled set of 20 grass species at chloroplast, 100 mM, EDTA 1 mM, pH 8.0) and excess polysaccharides mitochondrial and nuclear DNA loci [13]. The sampling removed by adding 1/15th volume of 20% (w/v) SDS and strategy was based on a sound phylogenetic framework to one-third volume of 5 M K-acetate (pH unadjusted) and maximise the chance of finding diagnostic markers, at dif- vortexing. The samples were then incubated at 4 8C for ferent taxonomic levels, that will characterise both known 30 min and the polysaccharides removed by centrifugation and unknown samples. Based on this analysis we identified a followed by a phenol: chloroform (1:1) extraction. The DNA set of putatively informative indels of variable size that were was then washed in 70% ethanol before final re-suspension diagnostic of subfamily, tribe and genus ranks within our set in 50 mL of TE buffer. DNA yield was estimated by elec- of 20 samples. The objective of this study was to develop trophoresis and visual comparison with lambda DNA PCR assays for these putatively diagnostic indels and to test (BioLabs) of known concentration. their utility for identifying unknown grass samples. Our specific objectives were: 2.3. Primer design of taxa-specific molecular 1. to develop PCR assays for five putatively informative markers indels located in the grass mitochondrial genome; 2. to develop a PCR method for the reliable assay of smaller DNA sequences spanning putatively informative indels indels on agarose gels; in the mitochondrial genome were selected as the starting 3. to test the utility of these molecular markers for grass point for primer design in this study. Primers were designed identification of twenty six unknown grass taxa; in conserved flanking regions on either side of the indels 4. to evaluate the future research needs for our continuing using the online primer design program Primer3 [19]. Our development of a robust grass identification system for goal was to obtain primers that would amplify all grasses forensic purposes. under standard PCR conditions without the need for species- specific optimisation, while providing diagnostic informa- tion for target groups. This is an important consideration for 2. Material and methods unknown samples. Primer sequences and PCR product sizes are summarised in Table 2. Below we briefly summarise 2.1. Sampling information about the target indels for these PCR assays and our predictions in terms of their utility for identification in Our initial study of the patterns, extent and location of unknown grass samples based on our knowledge of DNA DNA sequence variation was based on a hierarchically sequence variation [13]. J. Ward et al. / Forensic Science International 152 (2005) 121–131 123 Table 1 The sample design and outcomes of the PCR assays for the 20 control samples and 26 experimental samples Subfamily Tribe Genus Species POO PAN POE TRI PANI AND PAC PAS SOR ZEA ID- ID- ID- SF Tribe Genera Control Pooideae Poeae Lolium perenne Pooideae Poeae Lolium perenne Pooideae Poeae Lolium multiflorum Pooideae Poeae Lolium multiflorum Pooideae Poeae