Disentangling species ID within a genus and verification

21.05.2019 0 Céline Blanc-Jolivet

Beijing

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


Beijing

Timber identification with DNA analysis?

Sample to analyse DNA extraction and analysis

Comparison with reference data

Asia

America

Europe

continent

species

region


Beijing

Building reference data: sampling


Beijing

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


Beijing

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)


Beijing

Bioinformatic work

SNP detection

Select good quality and informative SNP loci

Prescreening of up to 480 SNPs


Beijing

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!


Beijing

Routine analysis in reference laboratories

14-plex Swietenia

SNaPshot Sets: 12-15 “Golden” and “Silver loci”


Beijing

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


Beijing

Species ID and origin of Entandrophragma sp.

Entandrophragma cylindricum


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Genetic reference data Entandrophragma cylindricum: N=1192, 74 loci

Misidendification in the field !


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13 chloroplast loci: species verification of reference samples

Low coverage sequencing of individuals of all genetic groups =>


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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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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)


Beijing


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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Herbarium vouchers were then analyzed…


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Final results with 149 selected loci

Nuclear Plastid


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Cost-effective species ID with diagnostic DNA markers

D. punctata

D. odorata

D. ferrea

D. micrantha

D. alata


Beijing

Conclusions

Controlling species ID and geographic origin: how to proceed?

Genetics

species

Wood anatomy

species genus

origin

Genetics and/or other chemical methods


Beijing

Thanks !

Documents

Disentangling species ID within a genus and verification