disentangling species id within a genus and verification
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21.05.2019 0 Céline Blanc-Jolivet
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Disentangling species ID within a genus and verification of timber origin with DNA methods Céline Blanc-Jolivet, Euridice Honorio, Yulai Yanbaev, Malte Mader, Maike Paulini, Bernd Degen Thünen Institute of Forest Genetics
Beijing
21.05.2019
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Timber identification with DNA analysis?
Sample to analyse DNA extraction and analysis
Comparison with reference data
Asia
America
Europe
continent
species
region
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Building reference data: sampling
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Building reference data: Wokflow
RAD sequencing
Selection of loci (480)
MassARRAY
Pre-Screening: 94 samples
Best loci (120)
MassARRAY
Screening: all samples
Reference data for
geographic origin
MiSeq/ Skimming
10 High quality samples
Sampling
DNA extraction All samples
Bioinformatics Population genetics
Laboratory work
Sampling, data collection (Local Teams)
MassARRAY
DataBase entry
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Building reference data: NGS
RAD sequencing
N
Cell
Nucleus
Mt CP
Mapping of Mt reads
Mapping of CP reads
Mapping of nucleus reads
CP = Chloroplast Mt = Mitochondrium
Genome Skimming (plastid genome)
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Bioinformatic work
SNP detection
Select good quality and informative SNP loci
Prescreening of up to 480 SNPs
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Genotyping work: MassARRAY
Mass spectrometry spectra
C variant A variant DNA amplification C A
G
T
Primer extension
C
A
Primer C variant A variant
C variant A variant C variant A variant
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Genotyping work: selection of loci
Analysis with different grouping of data: selection of different signals
look for “diagnostic markers”
Then new MassARRAY design…
Diagnostic markers have priority!
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Routine analysis in reference laboratories
14-plex Swietenia
SNaPshot Sets: 12-15 “Golden” and “Silver loci”
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Species ID and origin of Larix sp.
Genetic reference data: 253 loci, 1885 individuals
Wood anatomy: Larix sp.
Siberian Larch?
Genetics: Larix sibirica West und East
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Species ID and origin of Entandrophragma sp.
Entandrophragma cylindricum
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Species ID and origin of Entandrophragma sp.
Genetic reference data Entandrophragma cylindricum: N=1192, 74 loci
Misidendification in the field !
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Species ID and origin of Entandrophragma sp.
13 chloroplast loci: species verification of reference samples
Low coverage sequencing of individuals of all genetic groups =>
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Species ID and origin of Entandrophragma sp.
Genetic reference data Entandrophragma utile: N=541, 105 loci
Total samples N= 648
- 32 genus probably wrong
- 39 E. angolense (DRC)
- 11 E. candolei
- 25 E. cylindricum
Also 38 blind samples with wrong identification!
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Species ID and origin of Entandrophragma sp.
Similar genetic patterns in African timber species
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Species ID and origin of Dipteryx sp.
Field collection D. odorata (FG, Brazil, Bolivia), D. micrantha (Peru), D. alata (North Peru)
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Species ID and origin of Dipteryx sp.
Prescreening of 73 individuals, 292 loci
French Guiana Brazil Brazil Peru Bolivia
K1+K2+K5: D. odorata… K3+K4: D. micrantha…
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Species ID and origin of Dipteryx sp.
Herbarium vouchers were then analyzed…
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Species ID and origin of Dipteryx sp.
Final results with 149 selected loci
Nuclear Plastid
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Species ID and origin of Dipteryx sp.
Cost-effective species ID with diagnostic DNA markers
D. punctata
D. odorata
D. ferrea
D. micrantha
D. alata
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Conclusions
Controlling species ID and geographic origin: how to proceed?
Genetics
species
Wood anatomy
species genus
origin
Genetics and/or other chemical methods
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Thanks !