Download Phylogenetic DNA profiling : a tool for the investigation of poaching

Crime in Australia: International Connections
Australian Institute of Criminology International Conference
Hilton on the Park, Melbourne, Australia
29-30 November 2004
CONFERENCE PAPER:
PHYLOGENETIC DNA PROFILING
A Tool for the Investigation of Poaching
Paul Roffey, Pam Provan, Michelle Duffy, Aisuo Wang, Chris Blanchard and
Lyndall Angel
School of Biomedical Sciences, Charles Sturt University (New South Wales)
http://www.aic.gov.au/conferences/2004/index.html
PHYLOGENETIC DNA PROFILING - A TOOL FOR THE INVESTIGATION OF
POACHING
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Paul Roffey, 2Pam Provan, 3Michelle Duffy,
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Aisuo Wang, 5Chris Blanchard and 6Lyndall Angel
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Lecturer, School of Biomedical Sciences, Charles Sturt University
Honours Graduate, School of Biomedical Sciences, Charles Sturt University
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Honours Graduate, School of Biomedical Sciences, Charles Sturt University
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PhD Candidate, School of Biomedical Sciences, Charles Sturt University
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Senior Lecturer, School of Wine and Food Science, Charles Sturt University
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Head of School of Biomedical Sciences, Charles Sturt University
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Abstract
Poaching of abalone is a multi-million dollar crime and single-handedly is the greatest threat to the
viability of the Australian and New Zealand abalone industries.
Contained within the genome of each organism is a full history of its ancestry, documented as a
series of accumulated mutations. The rate at which mutations accumulate in any particular gene
sequence directly correlates with its importance in life functions where sequences involved with
critical life functions accumulate mutations less rapidly. Hence mutation provides a window
through which evolutionary relationships can be examined. Traditionally this procedure, known as
phylogenetic DNA profiling, has been used to investigate the taxonomy of life forms and to study
the origin and development of species. However, it has powerful potential as a forensic tool to
assist in the investigation of poaching by being able to trace ancestry, and hence geographical
origin, of poached specimens.
In this paper we will show the forensic application of phylogenetic DNA profiling to the
investigation of poaching using abalone as a model.
Introduction
Phylogeny is the term given to the field of science that focuses on the assessment of evolutionary
relationships between organisms. Analyses of this type can be conducted across taxa from
individuals to kingdoms. The analysis relies on the comparison of heritable traits. Pre-“DNA,”
morphological, biochemical and immunological markers were used to determine relationships,
however with the development of sophisticated molecular genetic technology emphasis has shifted
towards the utilisation of genetic markers. Ultimately DNA is a more accurate measure of change
since changes in morphology, biochemistry and immunology all result from changes at the genetic
level. DNA essentially functions as an evolutionary clock, where the changes that occur over time
are recorded as inherited mutations. The rate of mutation is constant, however the inheritance of
mutations is dependant upon the function of the mutated DNA sequence; sequences that are
involved in critical life processes accumulate mutations at a slower rate than those with less
important function.
In many ways the science behind phylogenetics is akin to the basic principles of photography. To
be successful it must be focussed at a particular evolutionary depth. This is achieved through the
careful selection of sequences that will provide the exposure, depth of field and resolution that is
sought. The sequence chosen must be present in all the organisms to be examined (exposure). The
more important the sequence, the more likely it will be maintained in a line of descent (depth of
field). However, the more important the sequence the less likely the variation and therefore ability
to distinguish (resolution). This process is less complicated than it sounds, for in many cases the
examination of a single gene can provide resolution across large evolutionary scales. This is
because different regions within genes are under different selective pressures. This in turn
manifests as variations in sequence flexibility.
In this paper we present phylogenetic analyses that were primarily targeted to Australian and New
Zealand abalone species using a number of genomic and mitochondrial gene markers. The
application of this technology to the abalone industry and the investigation of poaching is
discussed.
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Materials and Methods
Abalone Species: Australian species examined were Haliotis rubra, H.conicopora, H.roei,
H.laevigata and H.scalaris. The New Zealand species examined were H.iris, H.australis and
H.virginea. Also examined was H.asinina which represents a species that inhabits diverse global
geographical locations.
Genomic DNA Extraction: Whole cell DNA was extracted from specimens using either the Chelex
extraction method described by Walsh, et al, (1991) or salt purified (Aljanabi & Martinez, 1997)
followed by standard phenol chloroform extraction (Sambrook, et al, 1989).
Gene Amplification: Where possible, sequences were amplified using a standardised PCR
procedure. Reactions were performed in a total volume of 25 µl using 1-10 ng template DNA. The
final concentration of the components in reaction mix was as follows; 10 mM Tris-HCl (pH 8.3),
50 mM KCl, 2.5 mM MgCl2, 0.25 µM each primer, 200 µM dNTP’s, 1 unit Taq polymerase (Life
Technologies, Australia). After an initial denaturation at 94°C for 2 min, the amplifications were
performed using a “touchdown” amplification procedure consisting of 10 cycles of denaturation at
94°C for 45 sec, annealing for 10 sec at 62°C (decreasing 0.5°C per cycle) and extension at 72°C
for 30 sec, then 35 cycles of denaturation at 94°C for 45 sec, annealing for 10 sec at 55-57°C and
extension at 72°C for 30 sec. A final extension was performed for 10 mins at 72°C. Positive and
negative controls were included in each reaction batch to verify the specificity and efficiency of
amplification and to monitor for contamination.
Genetic Loci: Sequences within the following genetic loci were targeted for amplification; 28s
rRNA (genomic), Cytochrome Oxidase 1 (CO1, mitochondrial), 16s rRNA (mitochondrial), 12s
rRNA (mitochondrial), Cytochrome Oxidase 2 (CO2, mitochondrial) and NAD Dehydrogenase 2
(ND2, mitochondrial). The 28s rRNA gene is a highly conserved genetic sequence whereas the
mitochondrial loci represent more variable sequences. Of the mitochondrial sequences cytochrome
oxidase 1 is the gene with greatest conservation.
Electrophoretic Techniques: Preliminary analysis of amplification products was performed
following separation in a 2% agarose gel. The size of fragments was estimated by comparison with
known molecular weight markers (100 bp ladder, Progen) using the method of Priefer (1984) and
the quantity of specifically amplified product estimated by comparison of fluorescence with bands
of known concentration in the marker.
DNA Sequencing, Sequence Analysis and Phylogenetic Studies: DNA sequencing was performed
on 20-100 pg PCR amplified product using the DyeNamic fluorescent dye terminator sequencing
kit (Amersham Australia) and analysed using an ABI Prism® 377 DNA Sequencer (Applied
Biosystems). Sequences were compared to databases using the FastaA algorithm within the
Australia Nucleic Acid Genome Information Service (ANGIS; http://www.angis.org.au), aligned using
ClustalW and phylogenetic reconstructions performed using MEGA v2.1 (Kumar, et al, 2001)
using the neighbour-joining (NJ) method.
Results
Phylogenetic analysis of the highly conserved 28s rRNA gene sequence.
The H.australis 28s rRNA sequence was compared with sequences within databases containing
DNA sequences of known origin. This analysis identified close similarities with over a thousand
known ribosomal RNA sequences, and amongst those with the highest similarity were 18
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gastropods each with a sequence similarity over 91%. By comparison, primates demonstrated a 8182% sequence identity when compared to H.australis and a 96-99% identity when compared to one
another. Phylogenetic analyses clustered the gastropods into two major subgroups that were linked
at a more distant level (Figure 1). This arrangement is consistent with the current classification of
the Vetigastropoda and Ceanogastropoda respectively (both subgroups of the class Gastropoda). By
comparison the primates grouped separately and demonstrated a very distant link with the
gastropods. Haliotis (abalone) correctly grouped within the Vetigastropoda; this is a well
documented and recognised evolutionary relationship.
Phylogenetic analysis of mitochondrial gene sequences.
The CO1 sequences were retrieved from all species tested. When compared with DNA databases
CO1 sequences from a number of other abalone species were identified, including representatives
from North America, and South Africa. Phylogenetic reconstruction using all CO1 sequences
clearly separated the abalone species according to geographical location (Figure 2). Phylogenetic
reconstructions for the 16s rRNA gene, 12s rRNA gene, ND2 and CO2 showed similar
geographical separations.
Discussion
The abalone industry is a lucrative trade. Australia and New Zealand are amongst the few countries
in the world that still have thriving abalone industries based on natural stock. Not surprisingly these
countries are also prime targets for poaching. The policing of poaching is a difficult task which
largely relies on either catching the poachers “red-handed” or detecting the sale of illegal harvests.
In either case subsequent prosecution and conviction can often hinge on the ability of fisheries
scientists to identify the abalone species and/or subspecies type and therefore the likely country of
origin. Genetic tests that could identify abalone at the species and sub-species level will greatly
improve the detection and prosecution of those involved in the illegal trade and/or poaching of
abalone.
The use of phylogeny in forensic application is not new. Indeed the standard DNA tests for human
discrimination are classic examples of phylogenetic tests. Hence the application of this technology
to poaching does not represent a completely new facet to forensic science. The challenge in
application to non-human samples is to find the suitable sequences that are sufficiently different to
be useful for phylogenetic studies but sufficiently similar to be used across the desired range of
target species. Genes such as the 28s rRNA gene are very highly conserved hence have application
at higher taxonomic levels. Indeed when applied to the gastropods it successfully resolved the order
Vetigastrpoda from the remainder of the gastropods. This follows conventional taxonomic theory
and reinforces a number of other genetic studies (eg Tillier, et al, 1992; Tillier, et al, 1994;
McArthur & Koop, 1999; Colgan, et al, 2000; Ponder & Lindberg, 1996; Ponder & Lindberg,
1997; Salvini-Plawen, 1980; Salvini-Plawen & Haszprunar, 1987). It is clear that application of this
gene to forensic samples would be useful to confirm abalone identification at the genus, family and
order levels.
Species identification requires genetic sequences with greater variation. The mtDNA genes
examined to date appear suited for this level of resolution. In forensic application mtDNA markers
have three great advantages over genomic DNA markers. First, mtDNA genes code for essential
life processes hence remain conserved over many generations. Second, each cell has thousands of
identical copies hence tests targeted to mtDNA provide a significant improvement in sensitivity and
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robustness over the single copy genomic markers. And third, mtDNA shows maternal rather than
Mendelian inheritance. This means the reshuffling of chromosomes that takes place during sexual
reproduction does not influence the inheritance of mtDNA. MtDNA remains essentially over many
generations hence is a more stable marker for family lines and populations.
The mtDNA genes examined to date clearly demonstrate their ability to provide useful information
about gross geographical location. In theory mtDNA should be able to provide a much higher level
of geographical resolution. The level of precision will be determined by the variability of the
fragment chosen where fine detail mapping will require genes with even greater variation. If
genetic sequences can provide high geographical resolution (eg within a 100 kilometre radius) then
the genetic data may be used in conjunction with vessel movements to substantially improve the
usefulness of forensic evidence in poaching investigations. Studies are currently in progress to find
suitable sequences.
Acknowledgements
The authors would like to thank Dr Graeme Bremner (New Zealand Fisheries) for specimens and
for advice on the usefulness of tests to fisheries investigations. This research was supported by
funds from the Charles Sturt University, the Australian Research Council and New Zealand
Fisheries.
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Figure 1 –Phylogram generated from the comparison of 28s rRNA sequences. The family
designations for each gastropod species are shown in parentheses. Major clades are highlighted.
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Figure 2 - Phylograms generated from alignment of CO1 sequences. Geographical locations are
indicated.
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Figure 3 - Phylograms generated from alignment of 16s rRNA, 12s rRNA, ND2 and CO2.
Geographical locations are indicated. Unnamed branches represent non-Haliotis species.
16s rRNA
12s rRNA
ND2
CO2
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