RAD Sequencing for
Applied Conservation
Genetics
RAD Sequencing for
Applied Conservation Genetics
Rob Ogden
TRACE Wildlife Forensics Network
&
The Royal Zoological Society of Scotland
Applied Conservation Genetics
RAD Sequencing for
Applied Conservation
Genetics
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Utilizing classical and applied theory to bridge the gap
between academics and end-users
Cons Gen
Theory
In situ
management
Ex situ
management
Population Genetics
Evolutionary Biology
Authenticity
Traceability
Wildlife Crime
Human Crime
Beavers - Background
RAD Sequencing for
Applied Conservation
Genetics
Castor fiber – European beaver
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Evidence of beaver in Britain from
2M years ago to 400 years ago
Scottish Beaver Trial aims to
evaluate feasibility and impact of
reintroduction
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European history suggests
postglacial recolonisation
complicated by translocations
Two principal lineages
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Very few microsatellite markers
Population genetic studies limited
Probable distribution of
European beaver in
Europe (outwith Russia)
after the last Ice Age
RAD Sequencing for
Applied Conservation
Genetics
(Halley, D & Rosell, F. - 2002)
Beaver distribution by late
19th Century
1. Telemark, Norway. c.100
2. Elbe, Germany. <200
RAD Sequencing for
Applied Conservation
Genetics
3. Rhône, France. c. 30
4. Pripet Marshes, Belarus. <400
Approx population 1200 animals
Largely due to re-introductions
Beaver distribution by
2002
Approx. population
RAD Sequencing for
Applied Conservation
Genetics
639,000 – 742,000 animals
Beavers - Background
RAD Sequencing for
Applied Conservation
Genetics
Genetic Research
Applications:
Norwegian populations
• Pheromone / genotype correspondence
• Population structure / genetic diversity
• Molecular ecology / behaviour
Reintroduction projects
• Founder selection
• Breeding success
• Genetic management
Veterinary research
Beavers – SNP discovery
RAD Sequencing for
Applied Conservation
Genetics
Project design:
Norway:
1 family of 6 – individual barcodes
1 northern pop x 3 animals – individual barcodes
1 southern pop x 3 animals – individual barcodes
Germany
1 Bavarian pop x 3 animals – individual barcodes
Beavers – SNP discovery
RAD Sequencing for
Applied Conservation
Genetics
Project design:
Assume a 3Gb genome with 50% GC
Predict: ~45,000 SbfI rest. sites = 90,000 tags per haploid genome
= 180,000 tags per individual
Target: 30x coverage of each tag,
requires 5.4m reads per individual.
2-3 individuals per lane for complete coverage
Adults:
2 individuals in one lane
Offspring:
4 individuals in one lane
N. Norway/Bavaria
6 individuals in one lane
(reduced coverage – 15x)
Wildlife DNA Forensics
RAD Sequencing for
Applied Conservation
Genetics
Points to prove:
What is it?
Where’s it from?
Who is it? Relatedness?
Species ID
Population ID
Individual ID
Applications:
TCM ID
Food ID
Stock ID
Horn to Carcass Product to Farm
DNA sequencing – Diagnostic SNP genotyping
Allele frequency variation – SNPs or microsatellites
DNA profiling – SNPs or microsatellites
SturSNiP
RAD Sequencing for
Applied Conservation
Genetics
Sturgeon:
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The genus Acipenser contains 17 of 25 caviar producing species
All Acipenser species are listed on CITES I or II
Combination of habitat loss and over fishing
Adriatic sturgeon is practically extinct in the wild
Caspian Sea species under huge pressure
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Taxonomic uncertainty remains for several key species
Relatively little understanding of genetic diversity within and
among species
Large scale hatchery release programmes developing without
sufficient resources for genetic management
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SturSNiP
RAD Sequencing for
Applied Conservation
Genetics
Caviar:
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Bans on sturgeon fishing undermined by illegal caviar trade
EU is largest importer of caviar and is becoming largest
producer of farmed caviar
Protection of species, protection of consumer, crime prevention
Identification and traceability systems for caviar required
Markers required for:
1. Species ID and definition
2. Molecular ecology
3. Hatchery management
4. Source traceability
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Project funded by EC JRC
Collaboration with Italy, Russia, Iran
and Edinburgh University (GenePool)
SturSNiP
RAD Sequencing for
Applied Conservation
Genetics
Project design:
Adriatic:
Siberian:
Russian:
Iranian:
8 fish (2 parents, 6 progeny) individual barcodes
3 pops x 16 fish – population barcodes
3 pops x 16 fish – population barcodes
2 pops x 16 fish – population barcodes
Sturgeon species
Adriatic
Siberian
Russian
Iranian
SturSNiP
RAD Sequencing for
Applied Conservation
Genetics
Design issues:
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Sturgeon genomes are tetraploid (or octaploid?!)
Sturgeon genomes are large (~ 4Gb)
Predicted results:
Estimated output:
5 lanes x 20M paired end reads x 100 bp
Sufficient for 30x coverage of each tag for 16 barcodes
Currently preparing first barcoded libraries
Beavers & SturSNiP
RAD Sequencing for
Applied Conservation
Genetics
SNP discovery:
Utilize paired end sequencing to generate sufficient flanking
sequence for genotyping assays
100 bp forward contig
~500 bp paired end contig
Assemble against existing 454-derived sequence backbone for
related species (Beavers = rat/mouse, Acipenser = Acipenser)
SNPs for Applied Conservation Genetics
Validation:
RAD Sequencing for
Applied Conservation
Genetics
Is it worth validating candidate SNPs individually in the lab?
Genotyping:
Assay design
intron-exon boundaries (cDNA)
flanking region (e.g. RAD-Tag)
chemistry limitations (Golden Gate)
Sample numbers
Massive economies of scale
Not economically viable at low n
Lack of suitable options
Greatest issue for applied work
Outlook / Future Work
RAD Sequencing for
Applied Conservation
Genetics
Techniques
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genome sequence everything? 10k genome project
new platforms promise 15 minute genomes
how to assemble? annotate?
probably be using SNPs and SNP discovery methods for
next five years
Applications
Forensics
Fisheries / timber / foodstuffs
Industry self-certification – compliance
Cons Gen
Limited by discovery rate
Dependent on added value over neutral msats
Requires genotyping solutions for modest sample numbers
Acknowledgements
RAD Sequencing for
Applied Conservation
Genetics
Staff at RZSS WildGenes laboratory and TRACE:
Ross McEwing, Janne Timm & Leonie Grenfell
Collaborators
Beavers:
Telemark University, Norway
Funders
RZSS & Telemark
Sturgeon:
Edinburgh Uni Gene Pool
EC Joint Research Centre
University of Padova
VNIRO, Russia
Iranian Fisheries Research Organization