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Species identification of botanical trace evidence using molecular markers
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Forensic Science International: Genetics Supplement Series 1 (2008) 630–632 www.elsevier.com/locate/FSIGSS
Research article
Species identification of botanical trace evidence using molecular markers Monique Wesselink *, Irene Kuiper Netherlands Forensic Institute, Laan van Ypenburg 6; 2497 GB The Hague, The Netherlands Received 17 August 2007; accepted 1 October 2007
Abstract Species identification of botanical trace evidence may provide links between crime scenes and individuals, or help verify alibis. Historically, identification has been performed using morphological techniques, preventing small or damaged fragments to be identified at the species level. DNA based techniques can aid in identification of this type of evidence, and are a first step in individual identification, generating high value evidence. Several DNA regions were evaluated for their use as plant-identification marker in a forensic setting. Characteristics include robustness when using bad quality material, reference sequence availability and ‘universal’ use throughout the plant kingdom. Following a theoretical examination of markers described in literature, three chosen DNA regions were amplified and sequenced from several Dutch plants. Comparison of these sequences to each other and to public databases enabled us to determine advantages and drawbacks of each marker. All markers had several positive and negative features, but when combined the correct species could be identified for most samples. Several factors were selected to combine the different markers and determine the level of identification (i.e. species, genus or family) and eventually identify the species of origin. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic botany; Species identification
1. Introduction In the forensic field, botanical trace evidence is used for several purposes. The three main groups are to: (1) establish links between suspects, victims, crime scenes and objects; (2) verification of scenarios by estimating a time frame through vegetation analysis or stomach content; (3) ascertain the possession or trade in forbidden or endangered species. Historically, botanical evidence was classified using morphological or histological characteristics, often preventing small or damaged fragments to be identified to the species level. This has limited the interest for botanical trace evidence, although small amounts of botanical evidence are easily transferred when a crime is committed. Especially abundantly present organisms like grasses, shrubs, weeds or seed pods can often be found on other evidence types, as bodies, vehicles and tools. Identification of these traces may link individuals to crime
* Corresponding author. Tel.: +31 708886244; fax: +31 708886556. E-mail address: [email protected] (M. Wesselink). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.211
scenes, or verify or disprove alibis [1]. Recently the value of botanical trace evidence is being more widely recognized, and new techniques are explored for identifying such fragments. Identification of the species determines if further investigation is necessary, which investigations for individual identification are possible, and what the evidential value of such investigations could be. Botanical evidence is also encountered in wildlife cases where endangered or forbidden plants, or products derived from these plants, are collected or traded [2]. Again, whole plants can easily be identified at the species level through morphological or histological characteristics. However, once plants have been processed to art works, medicines or other objects it can become impossible to identify the species of origin using traditional methods. In these cases molecular markers may be used to identify the species. By doing so the possession or trade in a forbidden species can easily be determined. Phylogenetic studies in plants have evaluated several nuclear and chloroplast sequences, including the internal transcribed spacer, several parts of the rbcL gene, the trnL intron, and intergenic spacers between trnL and trnF and between trnH and
M. Wesselink, I. Kuiper / Forensic Science International: Genetics Supplement Series 1 (2008) 630–632
psbA genes. In these evaluations, not all properties that are important in forensic case work, have been addressed. These include: (1) the ease of amplification in all (or at least most) plant species using universal primers; (2) the ease of amplification in bad quality or microbially contaminated material; (3) sequencing ease in all (or at least most) plant species. Moreover the sequences obtained from a marker useful in forensics should; (4) display many sequence differences between species; (5) display few sequence differences within a species; (6) have many reference sequences available. Ideally a single marker would be applicable in all plants and have enough resolution to separate all species. However as plants are a diverse group of organisms, this truly universal marker for species identification has not yet been found. We have investigated the forensic utility of several markers used in phylogenetics, and the feasibility of combining several markers to create a multi-marker system for species identification of plants, using both publicly available databases as GenBank and newly generated sequences for comparisons. 2. Materials studied Leaf material was collected from 50 plant species occurring naturally throughout The Netherlands, roughly representing all land plants with an emphasis on the grass family. After the species was determined morphologically, approximately 1 cm2 of leaf material was ground under liquid nitrogen with pestle and mortar. The powder was transferred to a 1.5 ml reaction tube and total DNA was extracted using the DNeasy Plant Mini kit (Qiagen GmbH, Germany) following the manufacturers protocol. The internal transcribed spacer region ITS (ITS), and the intergenic spacers between trnL and trnF (trnLF) and between trnH and psbA (psbAS) were amplified using primers ITS4-R and ITS5-F [3,4], A50272 and B49873 [5], and trnH [6] and psbAF [7]. A small volume of each PCR product was subjected to gel electroforesis, ethidium bromide staining and UV detection. Products displaying more than one band were loaded on 1.8% agarose gel in larger quantities separated by electroforesis and visualized as described above. Separate bands were excised from gel, and following purification, the PCR product was re-amplified. Single PCR products were sequenced commercially by BaseClear B.V. (Leiden, The Netherlands) using the forward and/or reverse PCR primers. Chromas were used for sequence analysis, after which sequences were compared with GenBank using the blastn algorithm. To compare individual sequences either ClustalW or the align-two-sequences function in blast were used. 3. Results None of the tested molecular markers could be amplified and sequenced in all plant species. However, in all tested species at least two markers could be amplified and sequenced. ITS was difficult to sequence in bad quality plant material, as the fungal ITS was amplified along with the plant ITS, and sequencing resulted in two sequences. Amplification of marker trnLF
631
resulted in two PCR products with different sizes in some Poaceae. Sequencing both products revealed that one was the expected chloroplast sequence, whereas the other showed homology with mitochondrial sequences. These have been described as being transferred between chloroplast and mitochondria [8]. Marker psbAS was troublesome to amplify in Poaceae. Sequence analysis revealed an inverted repeat sequence that may form secondary structures and thereby inhibit the PCR reaction. The presence of reference sequences in GenBank was determined for the different markers. Markers ITS and trnLF were present in GenBank for all investigated plant families. For most families several genera and species could be found with several entries, enabling inter- and intraspecies sequence comparisons. In the investigated species, both markers displayed little intraspecies sequence variability. The interspecies sequence variability was moderate in general, but differed between families; in some families ITS was more variable than trnLF whereas in other families trnLF was more variable than ITS. Marker psbAS was present with many entries in some families, but hardly any in other families. The total number of sequences was also less than for trnLF and ITS. This limited the inter- and intraspecies comparison possibilities. The comparisons that were performed showed that marker psbAS was quite stable within species, and very variable between species. All sequences obtained from Dutch plants were compared with GenBank to assess the usefulness of public databases as GenBank for species identification. If the species under investigation was present in the database, the highest similarity (97–100%) was found with this species, or with species of the same genus if no interspecies variation was present for that specific marker genus combination (all 100%). If only sequences of species of the same genus were present in the database, the highest similarity was found with these species (80–100%). If only sequences of members of the same family were present, the highest similarity was found with these species, although the level of similarity decreased (70–90%). In some cases no sequences were found in the database with a high similarity to the search sequence. 4. Discussion and conclusions Species identification using molecular markers greatly relies on the availability of high quality reference sequences. Correct determination of starting material and avoidance of contamination are of great importance, but cannot be verified in public databases as GenBank. To incorporate this drawback in decision making, several factors have been selected to weigh when comparing an unknown sequence to a public database. These are: (1) sequence specificity: the percentage of similarity to the best and other matching species; (2) consistency between markers: do all markers yield the same best matching species; (3) completeness of reference data: the number of sequences of that species and genus present in the database and the presence of (all) domestic species; (4) verification: have the sequences been published.
632
M. Wesselink, I. Kuiper / Forensic Science International: Genetics Supplement Series 1 (2008) 630–632
The usage of several markers greatly improves identification ability and reliability, as one single marker may easily result in a misidentification. The combination of markers ITS, trnLF and psbAS is a good combination for most groups of plants. To entirely avoid the drawbacks attached to public databases, reference material or sequences of the suspected species and other species within that genus should be obtained from trustworthy sources. Conflict of interest None. References [1] H. Miller Coyle, C.-L. Lee, W.-Y. Lin, H.C. Lee, T.M. Palmbach, Forensic botany: using plant evidence to aid in forensic death investigation, Croat. Med. J. 46 (2005) 606–612.
[2] A. Linacre, J. Thorpe, Detection and identification of cannabis by DNA, Forensic Sci. Int. 91 (1998) 71–76. [3] B.G. Baldwin, Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an example from the compositae, Mol. Phylogen. Evol. 1 (1992) 3–16. [4] T.J. White, T. Bruns, S. Lee, J. Taylor, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, in: PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., 1990, pp. 315– 322. [5] P. Taberlet, L. Gielly, G. Pautou, J. Bouvet, Universal primers for amplification of three non-coding regions of chloroplast DNA, Plant Mol. Biol. 17 (1991) 1105–1109. [6] M.B. Hamilton, Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation, Mol. Ecol. 8 (1999) 521– 523. [7] T. Sang, D.J. Crawford, T.F. Stuessy, Chloroplast DNA phylogeny, reticulate evolution and biogeography of Paeonia (Paeoniaceae), Am. J. Bot. 84 (1997) 1120–1136. [8] P.B.M. Joyce, M.W. Gray, Chloroplast-like transfer RNA genes expressed in wheat mitochondria, Nucleic Acids Res. 17 (1989) 5461– 5476.
Forensic Science International: Genetics Supplement Series 1 (2008) 630–632 www.elsevier.com/locate/FSIGSS
Research article
Species identification of botanical trace evidence using molecular markers Monique Wesselink *, Irene Kuiper Netherlands Forensic Institute, Laan van Ypenburg 6; 2497 GB The Hague, The Netherlands Received 17 August 2007; accepted 1 October 2007
Abstract Species identification of botanical trace evidence may provide links between crime scenes and individuals, or help verify alibis. Historically, identification has been performed using morphological techniques, preventing small or damaged fragments to be identified at the species level. DNA based techniques can aid in identification of this type of evidence, and are a first step in individual identification, generating high value evidence. Several DNA regions were evaluated for their use as plant-identification marker in a forensic setting. Characteristics include robustness when using bad quality material, reference sequence availability and ‘universal’ use throughout the plant kingdom. Following a theoretical examination of markers described in literature, three chosen DNA regions were amplified and sequenced from several Dutch plants. Comparison of these sequences to each other and to public databases enabled us to determine advantages and drawbacks of each marker. All markers had several positive and negative features, but when combined the correct species could be identified for most samples. Several factors were selected to combine the different markers and determine the level of identification (i.e. species, genus or family) and eventually identify the species of origin. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic botany; Species identification
1. Introduction In the forensic field, botanical trace evidence is used for several purposes. The three main groups are to: (1) establish links between suspects, victims, crime scenes and objects; (2) verification of scenarios by estimating a time frame through vegetation analysis or stomach content; (3) ascertain the possession or trade in forbidden or endangered species. Historically, botanical evidence was classified using morphological or histological characteristics, often preventing small or damaged fragments to be identified to the species level. This has limited the interest for botanical trace evidence, although small amounts of botanical evidence are easily transferred when a crime is committed. Especially abundantly present organisms like grasses, shrubs, weeds or seed pods can often be found on other evidence types, as bodies, vehicles and tools. Identification of these traces may link individuals to crime
* Corresponding author. Tel.: +31 708886244; fax: +31 708886556. E-mail address: [email protected] (M. Wesselink). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.211
scenes, or verify or disprove alibis [1]. Recently the value of botanical trace evidence is being more widely recognized, and new techniques are explored for identifying such fragments. Identification of the species determines if further investigation is necessary, which investigations for individual identification are possible, and what the evidential value of such investigations could be. Botanical evidence is also encountered in wildlife cases where endangered or forbidden plants, or products derived from these plants, are collected or traded [2]. Again, whole plants can easily be identified at the species level through morphological or histological characteristics. However, once plants have been processed to art works, medicines or other objects it can become impossible to identify the species of origin using traditional methods. In these cases molecular markers may be used to identify the species. By doing so the possession or trade in a forbidden species can easily be determined. Phylogenetic studies in plants have evaluated several nuclear and chloroplast sequences, including the internal transcribed spacer, several parts of the rbcL gene, the trnL intron, and intergenic spacers between trnL and trnF and between trnH and
M. Wesselink, I. Kuiper / Forensic Science International: Genetics Supplement Series 1 (2008) 630–632
psbA genes. In these evaluations, not all properties that are important in forensic case work, have been addressed. These include: (1) the ease of amplification in all (or at least most) plant species using universal primers; (2) the ease of amplification in bad quality or microbially contaminated material; (3) sequencing ease in all (or at least most) plant species. Moreover the sequences obtained from a marker useful in forensics should; (4) display many sequence differences between species; (5) display few sequence differences within a species; (6) have many reference sequences available. Ideally a single marker would be applicable in all plants and have enough resolution to separate all species. However as plants are a diverse group of organisms, this truly universal marker for species identification has not yet been found. We have investigated the forensic utility of several markers used in phylogenetics, and the feasibility of combining several markers to create a multi-marker system for species identification of plants, using both publicly available databases as GenBank and newly generated sequences for comparisons. 2. Materials studied Leaf material was collected from 50 plant species occurring naturally throughout The Netherlands, roughly representing all land plants with an emphasis on the grass family. After the species was determined morphologically, approximately 1 cm2 of leaf material was ground under liquid nitrogen with pestle and mortar. The powder was transferred to a 1.5 ml reaction tube and total DNA was extracted using the DNeasy Plant Mini kit (Qiagen GmbH, Germany) following the manufacturers protocol. The internal transcribed spacer region ITS (ITS), and the intergenic spacers between trnL and trnF (trnLF) and between trnH and psbA (psbAS) were amplified using primers ITS4-R and ITS5-F [3,4], A50272 and B49873 [5], and trnH [6] and psbAF [7]. A small volume of each PCR product was subjected to gel electroforesis, ethidium bromide staining and UV detection. Products displaying more than one band were loaded on 1.8% agarose gel in larger quantities separated by electroforesis and visualized as described above. Separate bands were excised from gel, and following purification, the PCR product was re-amplified. Single PCR products were sequenced commercially by BaseClear B.V. (Leiden, The Netherlands) using the forward and/or reverse PCR primers. Chromas were used for sequence analysis, after which sequences were compared with GenBank using the blastn algorithm. To compare individual sequences either ClustalW or the align-two-sequences function in blast were used. 3. Results None of the tested molecular markers could be amplified and sequenced in all plant species. However, in all tested species at least two markers could be amplified and sequenced. ITS was difficult to sequence in bad quality plant material, as the fungal ITS was amplified along with the plant ITS, and sequencing resulted in two sequences. Amplification of marker trnLF
631
resulted in two PCR products with different sizes in some Poaceae. Sequencing both products revealed that one was the expected chloroplast sequence, whereas the other showed homology with mitochondrial sequences. These have been described as being transferred between chloroplast and mitochondria [8]. Marker psbAS was troublesome to amplify in Poaceae. Sequence analysis revealed an inverted repeat sequence that may form secondary structures and thereby inhibit the PCR reaction. The presence of reference sequences in GenBank was determined for the different markers. Markers ITS and trnLF were present in GenBank for all investigated plant families. For most families several genera and species could be found with several entries, enabling inter- and intraspecies sequence comparisons. In the investigated species, both markers displayed little intraspecies sequence variability. The interspecies sequence variability was moderate in general, but differed between families; in some families ITS was more variable than trnLF whereas in other families trnLF was more variable than ITS. Marker psbAS was present with many entries in some families, but hardly any in other families. The total number of sequences was also less than for trnLF and ITS. This limited the inter- and intraspecies comparison possibilities. The comparisons that were performed showed that marker psbAS was quite stable within species, and very variable between species. All sequences obtained from Dutch plants were compared with GenBank to assess the usefulness of public databases as GenBank for species identification. If the species under investigation was present in the database, the highest similarity (97–100%) was found with this species, or with species of the same genus if no interspecies variation was present for that specific marker genus combination (all 100%). If only sequences of species of the same genus were present in the database, the highest similarity was found with these species (80–100%). If only sequences of members of the same family were present, the highest similarity was found with these species, although the level of similarity decreased (70–90%). In some cases no sequences were found in the database with a high similarity to the search sequence. 4. Discussion and conclusions Species identification using molecular markers greatly relies on the availability of high quality reference sequences. Correct determination of starting material and avoidance of contamination are of great importance, but cannot be verified in public databases as GenBank. To incorporate this drawback in decision making, several factors have been selected to weigh when comparing an unknown sequence to a public database. These are: (1) sequence specificity: the percentage of similarity to the best and other matching species; (2) consistency between markers: do all markers yield the same best matching species; (3) completeness of reference data: the number of sequences of that species and genus present in the database and the presence of (all) domestic species; (4) verification: have the sequences been published.
632
M. Wesselink, I. Kuiper / Forensic Science International: Genetics Supplement Series 1 (2008) 630–632
The usage of several markers greatly improves identification ability and reliability, as one single marker may easily result in a misidentification. The combination of markers ITS, trnLF and psbAS is a good combination for most groups of plants. To entirely avoid the drawbacks attached to public databases, reference material or sequences of the suspected species and other species within that genus should be obtained from trustworthy sources. Conflict of interest None. References [1] H. Miller Coyle, C.-L. Lee, W.-Y. Lin, H.C. Lee, T.M. Palmbach, Forensic botany: using plant evidence to aid in forensic death investigation, Croat. Med. J. 46 (2005) 606–612.
[2] A. Linacre, J. Thorpe, Detection and identification of cannabis by DNA, Forensic Sci. Int. 91 (1998) 71–76. [3] B.G. Baldwin, Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an example from the compositae, Mol. Phylogen. Evol. 1 (1992) 3–16. [4] T.J. White, T. Bruns, S. Lee, J. Taylor, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, in: PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., 1990, pp. 315– 322. [5] P. Taberlet, L. Gielly, G. Pautou, J. Bouvet, Universal primers for amplification of three non-coding regions of chloroplast DNA, Plant Mol. Biol. 17 (1991) 1105–1109. [6] M.B. Hamilton, Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation, Mol. Ecol. 8 (1999) 521– 523. [7] T. Sang, D.J. Crawford, T.F. Stuessy, Chloroplast DNA phylogeny, reticulate evolution and biogeography of Paeonia (Paeoniaceae), Am. J. Bot. 84 (1997) 1120–1136. [8] P.B.M. Joyce, M.W. Gray, Chloroplast-like transfer RNA genes expressed in wheat mitochondria, Nucleic Acids Res. 17 (1989) 5461– 5476.