presentation - Thermo Fisher Scientific

RhODIS®
The Rhino DNA Index System:
A forensic tool that can help save a flagship species
from extinction
Cindy Harper
Veterinary Genetics Laboratory
Faculty of Veterinary Science
University of Pretoria
Onderstepoort
RhODIS®: Rhinoceros Project
• RhODIS® – Rhino DNA Index System / CODIS –
Combined DNA Index System
• Central standard secure database of rhinoceros DNA
profiles
• Consists of a forensic cases (poaching and related
cases) and routine cases (stockpiles, live and hunted
rhinos)
• Microsatellite based individual ID inventory
History of RhODIS ®
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•
•
•
•
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Wildlife DNA Forensics Course – 2007
Project theme identified
Aim develop individual ID system from rhino horn
SANParks approved project
Late 2009 – presented results
Early 2010 – Attended 1st poaching scene in KNP
History of RhODIS ®
• June 2010 first successful case linking horns to
carcasses using DNA
History of RhODIS ®
• June 2010 – start of database - samples from all
carcasses collected and samples from all live animals
and horns - DNA profiles recorded on the database –
RhODIS named
• April 2012 – legislation published in SA
• March 2013 – CITES report following CoP16 in Bangkok
Black Rhino
•
•
•
•
•
•
Early 1900’s 850 000
1960 ±100 000
1970 ± 65 000
↓ 96%
1995 ± 2400
2007 ± 4000
2013 ± 5055
Black Rhino subspecies
D.b.bicornis (Namibia and
Northern Cape SA)
± 1700
D.b.minor (Zimbabwe and
SA) – reintroduced to
Zambia, Malawi,
Botswana)
± 1900
D.b.michaeli (Kenya and
small introduced
population in SA)
± 700
D.b.longipes (Western
Africa) – declared extinct
2011
White Rhino (Ceratotherium simum)
Southern White – C.s.simum (18 000)
Northern White – C.s.cottoni (5) – practically extinct
History of the Southern White Rhino
• 1895 – 20 to 50 southern white rhinos left in Hluhluwe-Umfolozi
region of KZN : decreased through uncontrolled hunting
• 1897 Hluhluwe and Umfolozi declared as game reserves
• 1960’s Ian Player / Natal Parks Board – re-introduction of white
rhino to previous ranges, private game farms and zoos all over the
world
• 2012 - 18 000 southern white rhino
98% population
in South Africa
RhODIS ® : Where are we now?
Total number of animals (alive and dead) and horns
– 15 000
Received samples from:
South Africa – all 9 provinces and KNP (White and D.b.minor)
State rhinos and Private owners (5000)
Namibia (White and D.b.bicornis)
Zimbabwe (White and D.b.minor)
Malawi
Swaziland – forensic cases
Botswana (White)
Zambia
Kenya – (White and D.b.michaeli) – MOU with Kenya Wildlife Services
Number of forensic cases on RhODIS ®
Sudan 1938
Tool to assist poaching investigations
Match individual DNA profiles from evidence items and horn to individual
rhinoceros – links the criminal to the scene of the crime and provides the
investigator with information
Rhinos Poached in South Africa
1400
1215
1200
1004
1000
800
668
600
448
333
400
200
83
120*
112
13
0
2007
2008
2009
2010
2011
2012
2013
2014
2015
Arrests of poachers in South Africa
343
344
267
232
165
2010
2011
2012
2013
1800
1600
1400
1200
1000
800
600
400
200
0
2010
2011
2012
2013
2014
2014
Why is this happening?
Like cocaine, minus the risk……(International
Business Times, April 2014)
Most valuable illegally traded commodity
Why Rhino Horn?
• Rhino horn and Traditional Asian Medicine (TCM) –
cure fever, poisoning, liver problems
• Cure for cancer – rumour in Vietnam
• Vietnam’s cancer mortality rate = 73% (highest in
the world)
• Rhino horn – the party drug and status symbol
• Used as powder with alcoholic drinks – cleans the
liver
• Used to flaunt new wealth – whole horns and
ornaments carved from the horns
DNA extraction from rhino horn
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•
•
•
•
Horn is not hair
Lacks bony core
Epidermal derivative ~ horse hoof
Consists of keratinized tubules of cells
Intercellular matrix with melanin and
calcium phosphate salts
• Continuous growth 5-6cm/year
• Can be harvested from the live animal
PrepFiler® Forensic DNA Extraction kit and magnetic
particle processor
Sudan 1938
How efficient is DNA extraction from
the horn?
DNA
concentratio
n*
% alleles Nr of Loci
correct³ amplified²
Amount of horn
powder used
0.54
89
3.81
100
23 1mg
4.64
100
23 2mg
7.43
100
23 5 mg
12.88
100
23 10 mg
11.08
100
23 15 mg
13.93
100
23 20 mg
19.31
100
23 25 mg
Sudan 1938
21 0.1mg
Walking stick – 1888
Smallest pieces: 0.1 - 20mg of horn (microscopic)
Best area to sample
Sudan 1938
Taking samples from the horn
Detecting blood on items
Bluestar® Forensic Latent blood Reagent
Field collection program: RhODIS® routine
samples from live animals
RhODIS®: Database
• Genotype data is automatically screened for new
alleles – flagged
• 24 STR’s – improved marker systems under
development
- Applied Biosystems® / LifeTechnologies™
partnership international STR panel for rhinoceros
- NGS: distant future
• Black and white species specific monomorphic
markers – ID black vs white
• Match probability calculation
- low genetic variability of rhinos
- Very large representative database
Reference Assisted chromosome assembly of white rhino
scaffolds with STR’s – domestic horse as reference
Locus
BlRh1B
BlRh1C
BlRh37D
BR6
DB1
DB23
DB44
DB52
DB66
IR10
IR12
IR22
RH12
SR63
SR74
SR262
SR268
SR281
7B
7C
12F
32A
32F
Repeat motif
(gt)13
(TG)6CGCG(TG)11
(tg)6CA(Tg)17(ag)6
(TG)15
(GT)16
(AC)6
(tg)19
(CA)12
(CA)10TA(CA)12
(ca)22
(Ac)22
(Ac)13
(TC)8TT(TC)8
(GT)5AT(GT)8
(aC)19
(AC)11
(AC)13
(AC)17
(tg)16AG(TG)4
(ct)14(at)11
(GT)10AT(GT)7…(Ta)9
(ac)6…(ac)16
(ac)11
Location
Chromosome
JH767724:8,299,703-8,299,802
Chr4
JH767759:3,308,328-3,308,518
Chr5
JH767760:5,803,569-5,803,831
Chr3
JH767793:958,941-959,116
Chr13
JH767726:55,212,707-55,212,874
Chr14
JH767779:4,018,091-4,018,257
Chr15
JH767765:2,229,186-2,229,302
Chr11
JH767728:49,254,161-49,254,310
Chr1
JH767780:644,242-644,385
NF
Unplaced scaffold00031
????
JH767774:6,674,158-6,674,305
ChrX
JH767747:9,369,432-9,369,535
NF
JH767735:34,492,757-34,492,857
Chr20
JH767726:49,561,075-49,561,304
Chr14
JH767831:1,676,337-1,676,424
ChrX
JH767723:55,939,772-55,939,942
NF
JH767757:15,973,899-15,974,168
Chr9
JH767802:6,346,286-6,346,528
Chr13
JH767853:1,478,532-1,478,698
Chr7
JH767727:10,444,144-10,444,335
Chr2
JH767782:5,765,753-5,766,031
Chr15
JH767776:13,563,190-13,563,319
Chr6
JH767732:27,885,389-27,885,530
Chr17
Gender test - Rhinoceros
Zinc Finger protein intron size difference between X and Y
chromosome
• 2 fragments differ by 7bp
• Black and white rhinos
Peppin et al, 2010
RhODIS®: DNA Sample collection Kits
• Forensic sample collection kits developed in collaboration with South
African Police Service forensic laboratory and SANParks
• Ensures chain of custody from sample collection to evidentiary report
and standard method of sample and data collection
eRhODIS™: electronic data collection
• VGL developed app to collect field data with kits
RhODIS® Training
• Training of EMI, Police investigators, field rangers, veterinarians,
prosecutors on use of rhino DNA evidence and to collect DNA
samples (all provinces; Swaziland, Namibia, India – including
Crime Scene training)
Arrested – 16 Jan 2012
Sentenced – 27 Nov 2012
Sudan 1938
International Cases
Singapore:
Vietnamese man jailed for 15 months on 09 January 2014 for having
eight pieces of rhinoceros horn worth while in transit at Changi Airport
in Singapore.
DNA profiling linked a set of the horns to a carcass in KNP poached
approximately 3 – 4 weeks earlier
Dedicated to all the amazing people who work tirelessly in the field
to investigate rhinoceros poaching and risk their lives to stop and
apprehend the criminals and do all this to save more animals from
absolute cruelty and a very real risk of complete extinction.
PrepFiler kit is For Research, Forensic and Paternity Use
Only. Not for use in diagnostic procedures.
Speaker was provided travel and hotel support by Thermo
Fisher Scientific for this presentation, but no remuneration.