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RhODIS®
The Rhino DNA Index System:A forensic tool that can help save a flagship speci es
from extinction
Cindy HarperVeterinary Genetics LaboratoryFaculty of Veterinary Science
University of PretoriaOnderstepoort
• 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
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
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
• Early 1900’s 850 000• 1960 ±100 000• 1970 ± 65 000• 1995 ± 2400• 2007 ± 4000• 2013 ± 5055
↓ 96%
Black Rhino
Black Rhino subspeciesD.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
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)MalawiSwaziland – forensic casesBotswana (White)ZambiaKenya – (White and D.b.michaeli) – MOU with Kenya Wildlife Services
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
Rhinos Poached in South Africa
0
200
400
600
800
1000
1200
1400
2007 2008 2009 2010 2011 2012 2013 2014 2015
13
83112
333
448
668
1004
1215
120*
Arrests of poachers in South Africa
0
200
400
600
800
1000
1200
1400
1600
1800
2010 2011 2012 2013 2014
20102011
20122013
2014
165
232267
343 344
Like cocaine, minus the risk……(International Business Times, April 2014)
Why is this happening?
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• 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
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
• 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
Reference Assisted chromosome assembly of white rhino scaffolds with STR’s – domestic horse as referenceLocus Repeat motif Location ChromosomeBlRh1B (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
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
• 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
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)
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
Dedicated to all the amazing people who work tireles sly in the field to investigate rhinoceros poaching and risk their l ives to stop and apprehend the criminals and do all this to save mor e animals from absolute cruelty and a very real risk of complete e xtinction.