Cindy Harper, Veterinary Genetics Laboratory, Faculty of Veterinary Science, University of Pretoria, Onderstepoort

1. 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

2. • 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
RhODIS®: Rhinoceros Project

3. History of RhODIS ®
• 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

4. History of RhODIS ®
• June 2010 first successful case linking horns to
carcasses using DNA

5. 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

6. • Early 1900’s 850 000
• 1960 ±100 000
• 1970 ± 65 000
• 1995 ± 2400
• 2007 ± 4000
• 2013 ± 5055
↓ 96%
Black Rhino

7. 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

8. White Rhino (Ceratotherium simum)
Southern White – C.s.simum (18 000)
Northern White – C.s.cottoni (5) – practically extinct

9. 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

10. Total number of animals (alive and dead) and horns
– 15 000
RhODIS ® : Where are we now?
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

11. Sudan 1938
Number of forensic cases on RhODIS ®

12. 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
Tool to assist poaching investigations

13. Rhinos Poached in South Africa
0
200
400
600
800
1000
1200
1400
2007 2008 2009 2010 2011 2012 2013 2014 2015
13
83
112
333
448
668
1004
1215
120*

14. Arrests of poachers in South Africa
0
200
400
600
800
1000
1200
1400
1600
1800
2010 2011 2012 2013 2014
2010
2011
2012
2013
2014
165
232
267
343 344

15. Like cocaine, minus the risk……(International
Business Times, April 2014)
Why is this happening?
Most valuable illegally traded commodity

16. 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

17. DNA extraction from rhino horn
• 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

18. Sudan 1938
PrepFiler® Forensic DNA Extraction kit and magnetic
particle processor

19. How efficient is DNA extraction from
the horn?
Smallest pieces: 0.1 - 20mg of horn (microscopic)
Sudan 1938
Walking stick – 1888
DNA
concentratio
n*
% alleles
correct³
Nr of Loci
amplified²
Amount of horn
powder used
0.54 89 21 0.1mg
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

20. Best area to sample
Sudan 1938

21. Taking samples from the horn

22. Detecting blood on items
Bluestar® Forensic Latent blood Reagent

23. Field collection program: RhODIS® routine
samples from live animals

24. • 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
RhODIS®: Database

25. Reference Assisted chromosome assembly of white rhino
scaffolds with STR’s – domestic horse as reference
Locus Repeat motif Location Chromosome
BlRh1B (gt)13 JH767724:8,299,703-8,299,802 Chr4
BlRh1C (TG)6CGCG(TG)11 JH767759:3,308,328-3,308,518 Chr5
BlRh37D (tg)6CA(Tg)17(ag)6 JH767760:5,803,569-5,803,831 Chr3
BR6 (TG)15 JH767793:958,941-959,116 Chr13
DB1 (GT)16 JH767726:55,212,707-55,212,874 Chr14
DB23 (AC)6 JH767779:4,018,091-4,018,257 Chr15
DB44 (tg)19 JH767765:2,229,186-2,229,302 Chr11
DB52 (CA)12 JH767728:49,254,161-49,254,310 Chr1
DB66 (CA)10TA(CA)12 JH767780:644,242-644,385 NF
IR10 (ca)22 Unplaced scaffold00031 ????
IR12 (Ac)22 JH767774:6,674,158-6,674,305 ChrX
IR22 (Ac)13 JH767747:9,369,432-9,369,535 NF
RH12 (TC)8TT(TC)8 JH767735:34,492,757-34,492,857 Chr20
SR63 (GT)5AT(GT)8 JH767726:49,561,075-49,561,304 Chr14
SR74 (aC)19 JH767831:1,676,337-1,676,424 ChrX
SR262 (AC)11 JH767723:55,939,772-55,939,942 NF
SR268 (AC)13 JH767757:15,973,899-15,974,168 Chr9
SR281 (AC)17 JH767802:6,346,286-6,346,528 Chr13
7B (tg)16AG(TG)4 JH767853:1,478,532-1,478,698 Chr7
7C (ct)14(at)11 JH767727:10,444,144-10,444,335 Chr2
12F (GT)10AT(GT)7…(Ta)9 JH767782:5,765,753-5,766,031 Chr15
32A (ac)6…(ac)16 JH767776:13,563,190-13,563,319 Chr6
32F (ac)11 JH767732:27,885,389-27,885,530 Chr17

26. 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

27. • 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
RhODIS®: DNA Sample collection Kits

28. eRhODIS™: electronic data collection
• VGL developed app to collect field data with kits

29. 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)

30. Sudan 1938
Arrested – 16 Jan 2012
Sentenced – 27 Nov 2012

31. 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
International Cases

32. 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.

33. 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.