Forensic Research & Criminology International Journal
Genetic Based Species Identification and Tracking of
the Geographic Origin of a Fully Tanned Animal Skin in
Wildlife Forensics
Case Report
Abstract
Species identification and assign the geographic origin from processed products
based on morphological traits is a challenging task in wildlife forensics due to
lack of the reference specimens. Here, we report species identification and assign
the geographical origin of a fully tanned animal skin using complete mtDNA
cytochrome b gene. A nucleotide sequence of 1140 bp cytochrome b was generated
from the DNA extracted from the small piece of skin from the inner ear (IEP-01).
GenBank BLAST search of the unknown Cyt b (1140bp) sequence against the full
range of published Rangifer tarandus has facilitated in identification of species
and ascertaining the species of origin with high meta probability (100%). We
determined that the seized unknown skin is wild reindeer (R.t. groenlandicus)
and has been originated from Canada, where this Least Concern species under
the IUCN red list. We propose to establish genetic database across the range of the
species threatened due to illegal trade to determine hotspots of poaching.
Keywords: Reindeer(Rangifer tarandus); Forensic genetics; Tanned skin; Species
identification; Geographic origin; Cytochrome b gene
Volume 2 Issue 3 - 2016
1
Wildlife Institute of India, India
Survey of India, India
3
Amity Institute of Wildlife Sciences, Amity University, India
4
Veer Kunwar Singh Universities, India
2
*Corresponding author: Surendra Prakash Goyal, Wildlife
Forensic and Conservation Genetic Cell, Wildlife Institute
of India, Post Box# 18, Chandrabani, Dehradun 248001,
Uttarakhand, India, Tel: +91-135-2640112-117; Ext. 235;
Fax: +91-135-2640115; Email:
Received: June 07, 2016 | Published: June 16, 2016
Introduction
Case History
Illegal trading of wild animals, their body parts and derived
products is a major concern in wildlife conservation. Species
identification based on DNA analysis is a critical tool in
wildlife forensics, especially when the specific morphological
characteristics are lost due to transformation or processing [1].
Mitochondrial markers, such as cytochrome b, 16S rRNA, 12S
rRNA and Cytochrome Oxidase showing interspecific variation,
are commonly used to perform species identification and
phylogenetic studies in wildlife forensics [2-5]. Cyt b is highly
informative in mammals and a large database for this genetic
marker is already available for different species and subspecies
in different geographical ranges, making it especially useful in
wildlife forensics [6]. Assignment of samples, we need to amplify
large fragment of mtDNA gene that show more number of SNPs
over a geographic range. In most of the wildlife forensic cases,
the samples received are in highly degraded condition or highly
processed, viz., burn bones/bone piece, cooked meat, tanned skin,
claws, canines, etc. Hence, the genomic DNA will be in low quantity
and quality as well. Therefore, extraction of a good quality DNA
from such type of degraded and tanned sample is a challenging
task for the wildlife forensic experts.
A highly tanned animal skin was seized by the Custom Officer
at Indira Gandhi International Airport, New Delhi, India, during
December, 2012 and sent to the Wildlife Institute of India,
Dehradun for species identification (Figure 1). The skin at visual
inspection appeared to be of a deer species but the morphological
hair patterns did not show definite species assignment due
to the lack of an extensive collection of reference samples for
comparison. A DNA based species identification strategy was
devised to overcome this problem.
In the present study, we aimed to select proper part to high
yield DNA from highly tanned animal skin and amplify the large
fragment of mitochondrial gene to identification of species and
to ascertain its geographical origin that was seized by the Custom
Authority at Indira Gandhi International Airport, New Delhi India.
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Materials and Methods
DNA extraction
Since the sample was highly tanned, we attempted DNA
extraction with commercially available Qiagen DN easy Tissue Kit
(QIAGEN, Germany) following manufacturer instructions. Three
samples from different parts of unknown tanned skin (IEP-1,
OSP-2 and OEP-3) were taken for DNA extraction. The tanning of
the skin potentially caused DNA degradation, therefore a section
of the inner part of the animal’s ear (IEP-1) that seemed to be
relatively less affected by the chemical process was selected for
DNA extraction and we processed remaining samples i.e. the
outer part of skin and ear (OSP-2 and OEP-3) in order to compare
the efficiency of DNA extraction from the different parts of the
tanned skin.
Forensic Res Criminol Int J 2016, 2(3): 00058
Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics
Copyright:
©2016 Kumar et al.
2/7
Results and Discussion
Of the total samples (n = 3), DNA extracted from, two
samples (OSP-2 and OEP-3) did not yield good DNA (Fig 2.A)
and amplification was not observed after PCR (Figures 2A & 2B).
Sample IEP-1 yielded detectable product of all three different
size (200bp, 350bp and 1140bp) Cyt b gene on agarose gel
electrophoresis (Figure 2B).
Figure 1: Animal skin seized by the customs authorities at Indira
Gandhi International Airport, New Delhi, India. A, dorsal view; B,
ventral view.
To determine the quality and concentration of DNA obtained,
the samples were subjected to gel electrophoresis on a 0.8%
agarose gel in 1X TAE buffer and DNA quantified with a UV
spectrophotometer (Amersham Pharmacia) (Figure 2A).
PCR Amplification and Sequencing
In case of non amplified samples (OSP-2 and OEP-3) we
suspected the presence of PCR inhibitors within the DNA, or
inhibitors may be due to chemical used in tanning process or from
the environment. Therefore, we tested the DNA for PCR inhibitors,
by using known positive PCR reactions. The results indicated that
two DNA samples (OSP-2 and OEP-3) contained PCR inhibitors. To
solved this problem, we check various dilution (1x-100x) of DNA
sample, we found that 50x diluted DNA give PCR amplification in
short fragment of Cyt b gene (200 bp) but remaining fragments of
Cyt b gene were not amplified (Figure 2C).
DNA template were subjected to polymerase chain reaction
using universal of Cyt b gene of different size 200bp, 350bp and
1140bp [7-9]. All PCR reactions were carried out on an Applied
Biosystems® 2720 Thermal Cycler (ABI) in a total reaction
volume of 25 µl containing 10 µl 2X PCR mix buffer (Amresco);
10 µM of each primer, and 4 µl of total DNA. The Thermal cycling
consisted of conditions of denaturation step at 94°C for 3 min,
35 cycles of denaturation(94°C for 30 s),annealing (53°C for 45
s) and primer extension (72°C for 40 s) and a final extension
step of 10 min for 72°C 10 min. A small volume of PCR products
(5 μl) were subjected to electrophoresis on 2% agarose gel and
visualized over an UV transilluminator. Extraction and PCR blanks
were incorporated into the analysis. One extraction blank was
incorporated with three samples extracted, and one PCR blank
was subsequently incorporated with every three extracts that
were amplified.
In this study, we used only (IEP-1) PCR amplicon for DNA
sequencing. Amplified 1140 bp PCR product was purified using
Exo-SAP to remove residual oligonucleotides and dNTPs prior to
sequencing reaction. The forward and reverse primers were used
independently for the sequencing reactions using the Big Dye®
Terminator v3.1 Cycle Sequencing kit to generate sequence from
both ends. The products were purified using a standard ethanol
precipitation method and sequenced on an ABI 3130 Genetic
Analyzer (Applied Biosystems, USA).
Data analysis
Cyt b sequence (IEP-1) was cleaned and validated using
SEQUENCHER 4.8 (Gene Codes Corporation, Ann Arbor, MI).
Multiple sequence alignments were performed using the
CLUSTAL W algorithm implemented in BIOEDIT version 7.0.5.3 \
9 [10]. The sequence obtained from the unknown skin specimen
was compared with the sequences publicly available at GenBank
using BLAST search tool of NCBI (http://blast.ncbi.nlm.nih.gov/).
All the sequences that showed similarity with the unknown
sequence (IEP-1) were downloaded (Tables 1 & 2) and used for
phylogenetic analysis using Kimura 2 parameter distance matrix
with the neighbor-joining method as implemented in Mega v5.0
software [11].
Figure 2: Agarose gel electrophoresis of unknown animal skin sample.
(A). Quality of Genomic DNA, Lane 1,2,3 and 4 represent IEP-1, OSP-2,
OEP-3 and –Ve control respectively. (B) Amplified PCR product, Lane
2, 3, 4 and 5 indicate IEP-1, OSP-2, OEP-3 and –Ve control respectively.
Only Unknown skin sample IEP-1 show amplification in all three
different sized primer (200bp, 350bp and 1140bp). (C) Amplification
after 50x dilution Lane 2 to 3 are of OSP-2 and OEP-3 respectively.
Indicate amplification in 200bp only and remaining primer did not
amplified. Lane 4 and 5 are showed +Ve and -Ve control. Lane 1 in (B)
and (C) show 100 bp DNA ladder.
Tanning is an aggressive chemical process that potentially
damage DNA and this was the probable cause for low yield of
DNA in extraction process from the outer parts of the skin sample,
which were visibly fully tanned. UV radiation and overexposing to
heat are also known to cause DNA damages [12,13] and may have
contributed to the negative amplification. The inner ear fragment
was probably less damaged by tanning chemicals, UV radiation
and heat and resulted in yielding good quality PCR product.
Citation: Kumar VP, Rajpoot A, Mukesh, Kumar D, Goyal SP (2016) Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics. Forensic Res Criminol Int J 2(3): 00058. DOI: 10.15406/frcij.2016.02.00058
Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics
Copyright:
©2016 Kumar et al.
Table 1: List of GenBank accession numbers used in this study and their respective names with geographic origin.
Species/Subspecies
Common Name
Origin
Accessions Number
Cervus albirostris
Cervus nippon
Thorold’s deer
China, Qinghai
AY044863.1
Cervus elaphus
Red deer
Cervus elaphus hippelaphus
Middle European red deer
Germany, Encloser
Cervus elaphus brauneri
Sika deer
Krim red deer
China
AB021093.1
Yugoslavia
AY070225.1
Ukraine
Cervus elaphus scoticus
Middle European red deer
Scottish red deer
Scotland
Cervus elaphus atlanticus
Red deer
Cervus elaphus xanthopygus
Isubra
Norway, Hitra
Cervus elaphus canadensis
American wapiti
Cervus elaphus hippelaphus
Cervus elaphus sibericus
Cervus elaphus kansuensis
Siberian wapiti
Kansu red deer
Russia, Anjui
AY070226.1
AY070224.1
AY070223.1
Cervus elaphus corsicanus
Sardinian deer
Tunisia, Tunis
Cervus elaphus maral
Maral
Iran
Tien Shan wapiti
AY244491.1
AB021099.1
China, Dong Da Shan
Shou
Cervus elaphus songaricus
AY148966.1
AF423198.1
Barbary red deer
Cervus elaphus wallichi
Cervus elaphus barbarus
AY118198
North America
China, Mongolia
China, Qinghai
Cervus elaphus macneilli
MacNeill’s deer
France
AY044862.1
AY035875.1
China, Tibet
AY044861.1
Sardinia
AY244489.1
China, Tien Shan-
AY070222.1
AF489280.1
AY035871.1
Spain, La Gaganta
AF489281.1
Cervus elaphus yarkandensis
Bactrian red deer
Tadzikistan
AY142327.1
Cervus unicolor
Sambar
India
Cervus elaphus hispanicus
Spanish red deer
Cervus elaphus hippelaphus
Middle European deer
Cervus elaphus bactrianus
Axis axis
Cervus duvaucelii
Yarkand red deer
Chital
Bulgaria
AF423195.1
China
AY142326.1
India
JN596156.1
JN861032.1
EF079830.1
Swamp deer
India
Axis porcinus
Hog deer
Cervus eldi thamin
Thamin deer
Germany
AY035874.1
Norway
DQ673123.1
Rangifer tarandus tarandus
Reindeer
BLAST analysis of Cyt b gene indicated that the samples of
unknown tanned animal skin (IEP-1) showed 100% similarity
Rangifer tarandus .The neighbor-joining phylogenetic tree with
other deer subspecies also showed that the unknown skin (IEP-1)
is belonged to the Rangifer tarandus species, commonly known
as reindeer, with a strong bootstrap value of 100% (Figure 3).
However, five subspecies of wild reindeer are currently recognized:
Barren-ground caribou (R. t. groenlandicus), woodland caribou
(R. t. caribou), Grant’s caribou (R. t. granti), Peary caribou (R. t.
pearyi), Dawson’s caribou (R. t. dawsoni, extinct), and are still
widely distributed across northern Eurasia and North America
(caribou) [13,14]. Today, almost 50% of the approximate
3,00,0000 reindeer in the Old World are wild animals, and some
of this population are semi-domestic (R. t. tarandus) which are
managed in close coexistence in many areas [15,16].
Thailand
3/7
EF079829.1
We compared complete sequence of cytochrome b (1140
bp) gene of unknown tanned skin sample (IEP-1) with three
subspecies of wild reindeer and semi-domestic reindeer sequence
taken from Cronin et al. [16,17] available in GenBank (Table 2).
The Multiple Sequence Alignments displayed 100% similarity
with wild reindeer Barren-ground caribou (Table 3).
In order to assess the geographic origin of the unknown
sample (IEP-1), we retrieved the n=76 sequences of cytochrome
b gene (from NCBI Gene Bank) and found 70 haplotypes in these
sequences that represent 56 haplotypes from 3 subspecies of wild
reindeer; Grant’s caribou, Canadian barren-ground caribou and
woodland caribou and 14 haplotypes from different geographic
origins of semi-domestic reindeer that were found in 13 herds
from 3 regions: Alaska, Russia, and Scandinavia (Table 2).
Citation: Kumar VP, Rajpoot A, Mukesh, Kumar D, Goyal SP (2016) Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics. Forensic Res Criminol Int J 2(3): 00058. DOI: 10.15406/frcij.2016.02.00058
Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics
Copyright:
©2016 Kumar et al.
4/7
Table 2: List of Haplotypes and GenBank accession numbers used in this study and their respective names with geographic origin. These all Cyt b
sequences taken from Cronin et al. [16,17].
Haplotype
Species Name
Wild/ Semi
domestic
Hap-01
R. tarandus
Semi domestic
R. tarandus
Semi domestic
R. tarandus
Semi domestic
Svalbard Is, Sweden
Hap-08,09, 10,11
R. tarandus
Semi domestic
Russia
Hap-12,13,14
R. tarandus
Semi domestic
Alaska
Hap-15,16,17,18, 19,20
R.t.caribou
Wild
Canada
Hap-21,22,23,24,25,26
R.t.groenlandicus
Wild
Canada
Hap-02,03, 04,05
Hap-06,07
Hap-27,28,29,30,31, 32, 33,34,35
,36,37,38,39,40,41,42,43,44,45,4
6,47,48,49,50,51,52,53,54,55,56
,57,58,59,60,61,62,63,64,65,66,6
7,68,69,70
R.t.granti
Wild
The Alaskan herds represent the geographic range from Siberia,
Russia and extended to the Seward Peninsula, Alaska. While the
reindeer from the Russian herds represent geographical range
restricted to Magadan, district in Siberia and the Scandinavian
herds reindeer are represent the Norway population. The
sequence of unknown sample (IEP-1) matched with the Hap-21, 22
wild reindeer that represents the Canadian population with 57%
bootstrap value and displayed 100% similarity. The remaining
reindeer haplotypes have sequences similarity and bootstrap
Geographical Origin
Norway
Alaska/Russia,
USA
Accessions number
DQ673123.1
DQ673122.1,DQ673127.1,
DQ673130.1,DQ673131.1
DQ673124.1,DQ673125.1
DQ673126.1,DQ673132.1,
DQ673133.1,DQ673135.1
DQ673128.1,DQ673129.1,
DQ673134.1
AY726672.1,AY726673.1,
AY726674.1,AY726677.1,
AY726675.1,AY726676.1
AY726679.1,AY726691.1,
AY726705.1,AY726706.1,
AY726720.1,AY726729.1
AY726680.1,AY726682.1,
AY726685.1,AY726690.1,
AY726697.1,AY726701.1,
AY726724.1,AY726730.1,
AY726703.1,AY726711.1,
AY726712.1,AY726714.1,
AY726715.1,AY726718.1,
AY726721.1,AY726723.1,
AY726728.1,AY726683.1,
AY726686.1, AY72689.1,
AY726700.1,AY726702.1,
AY726684.1,AY726704.1,
AY726707.1,AY726719.1
AY726720.1,AY726726.1,
AY726687.1,AY726688.1,
AY726693.1,AY726696.1,
AY726699.1,AY726692.1,
AY726695.1,AY726713.1,
AY726722.1,AY726694.1,
AY726698.1,AY726709.1,
AY726717.1
values which were lower, when compared with unknown sample
(IEP-1) (Table 3 & Figure 4). The unknown query sequence
showed 100% similarity with the haplotype H-21, 22 that has
been originated from Canada. Therefore, based on the data of
Cronin et al. [16,17] we concluded that the animal skin seized by
Indian Customs is of the wild reindeer (R. t. groenlandicus) and
has been originated from Canada. Wild population of reindeer is
Least Concern species in IUCN, listed on Appendix III of the Bern
Convention.
Citation: Kumar VP, Rajpoot A, Mukesh, Kumar D, Goyal SP (2016) Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics. Forensic Res Criminol Int J 2(3): 00058. DOI: 10.15406/frcij.2016.02.00058
Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics
Copyright:
©2016 Kumar et al.
5/7
Table 3: Similarities in the Cyt b locus between unknown sample (IEP-1) and sequences of reindeer subspecies available in GenBank.
Unknown
Sample
Subspecies with the Highest Similarity (Genbank
Accession)
IEP-1
Rangifer tarandus tarandus
IEP-1
IEP-1
IEP-1
IEP-1
IEP-1
Rangifer tarandus tarandus
Rangifer tarandus tarandus
Rangifer tarandus tarandus
Rangifer tarandus tarandus
Rangifer tarandus tarandus
IEP-1
Rangifer tarandus groenlandicus
IEP-1
Rangifer tarandus caribou
IEP-1
Rangifer tarandus granti
Query Coverage (%)
Similarity (%)
Hap/Geographic Origin
100
99
Hap01/Norway
100
100
100
100
100
99
Hap-02,03.04,05 / All
from Alaska/Russia
99
Hap-07 / Swden
99
99
99
100
100
99
98
100
99
Hap-06 / Svalbard Is
Hap-08,09,10,11 All from
Russia
Hap-12,13,14 / All from
Alaska
Hap-21,22,23,24,25,26/
Canada
Hap-22, 24,27/ USA
Hap-15,16,17/ all from
Canada
Figure 3: Neighbor-joining phylogenetic tree showing the relationships of unknown skin sample with the other deer species of the world.
Citation: Kumar VP, Rajpoot A, Mukesh, Kumar D, Goyal SP (2016) Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics. Forensic Res Criminol Int J 2(3): 00058. DOI: 10.15406/frcij.2016.02.00058
Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics
Copyright:
©2016 Kumar et al.
6/7
Figure 4: Haplotype Neighbor-joining phylogenetic tree with 70 different geographically originated showing the relationships of unknown skin
sample (IEP-1) with the semi domestic (Hap-01 to Hap-14) and three wild subspecies (Hap-15 to Hap-70) of Reindeer. Details of haplotype given
in Table 2.
Conclusion
In conclusion, we describe the utility of large fragment of
cytochrome b (1140 bp) mtDNA gene sequence in the species
identification and ascertaining the geographic origin of a fully
tanned animal skin. In order to assign the geographical origin
of species, it is necessary to use Cyt b gene, which cover the
entire range of probable haplotypes. In many study mtDNA Cyt
b gene sequences were used to determine and investigate the
geographical origin of species [18,19]. This result shows the
importance of genetic analysis in wildlife forensics and the utility
of DNA based analysis in the implementation CITES (Convention
of International Trade in Endangered Species). We suggest that
DNA analysis of tanned skins should target samples from inner
Citation: Kumar VP, Rajpoot A, Mukesh, Kumar D, Goyal SP (2016) Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics. Forensic Res Criminol Int J 2(3): 00058. DOI: 10.15406/frcij.2016.02.00058
Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics
parts of the skins. Furthermore, we defended that database of Cyt
b gene sequences across the range of species threatened due to
illegal poaching and classified in Appendix I should be established
to allow for confident genetic identifications.
Wild reindeer is the least concern species under the IUCN
red list. This means that we have a special responsibility to take
care of and manage the reindeer in a way that will allow future
generations to experience viable population of reindeer. This
database would be of great aid in DNA-based investigations of
illegal trade, the implementation CITES and the detection of
geographic poaching hotspots.
Acknowledgment
The authors are thankful to the Director, Dean and Research
Coordinator, Wildlife Institute of India (WII), Dehradun, for their
strong support. We would like to thank Nodal Officer of Wildlife
Forensic and Conservation Genetic Cell (WFCGC). We would like
to thank all the researchers and staff of the Wildlife Forensic and
Conservation Genetic Cell (WFCGC), for providing scientific and
technical assistance while undertaking the work.
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Citation: Kumar VP, Rajpoot A, Mukesh, Kumar D, Goyal SP (2016) Genetic Based Species Identification and Tracking of the Geographic Origin of a Fully
Tanned Animal Skin in Wildlife Forensics. Forensic Res Criminol Int J 2(3): 00058. DOI: 10.15406/frcij.2016.02.00058