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 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

• 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

Sudan 1938

Number of forensic cases on RhODIS ®

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

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PrepFiler® Forensic DNA Extraction kit and magneticparticle processor

How efficient is DNA extraction from the horn?

Smallest pieces: 0.1 - 20mg of horn (microscopic)

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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

Best area to sample

Sudan 1938

Taking samples from the horn

Detecting blood on itemsBluestar® Forensic Latent blood Reagent

Field collection program: RhODIS® routine samples from live animals

• 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

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)

Sudan 1938

Arrested – 16 Jan 2012

Sentenced – 27 Nov 2012

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.

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.