Genetic variation of dipterocarps: from molecular...

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Genetic variation of dipterocarps: from molecular phylogenies to the identification of the origin of timber

Reiner Finkeldey, Oliver Gailing, Iskandar Siregar, Ulfah Siregar, Randy Villarin, Cuiping Cao, Nga Phi Nguyen, Hani Nuroniah, Yanti Rachmayanti

Büsgen Institute Forest Genetics and Forest Tree Breeding

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Structure of presentation

l  Background –  Dipterocarpaceae

l  Interspecific variation: molecular phylogeny of Dipterocarpaceae

l  Intraspecific variation of selected Shorea spp.

l  Application: Molecular methods to trace the origin of wood

l  Conclusions and outlook l  Literature

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The pantropical distribution of Dipterocarpaceae

Dipterocarpoideae

13 Genera; 470 Species

Pakaraimoideae

1 Genus; 1 Species

Monotoideae

3 Genera; 40 Species

Background: Dipterocarpaceae

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Phylogenie der Dipterocarpaceae

Dipterocarpoideae in insular Southeast-Asia


Number of endemic species Number of non-endemic species

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Dipterocarpoideae in Southeast-Asia

l  Widely distributed –  Mainland and insular SE-Asia

l  Dominating lowland rainforests and monsoon forests in SE-Asia

–  50-80% of wood volume

–  Keystone group

l  Important timber species –  Trade name: Meranti, Balau,

Keruing, Kapur

l  Many endangered taxa

Dipterocarp forest on Borneo


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Dipterocarp: growth habit

l  Medium-sized – emergent trees

–  Mainly climax species

–  Often long, straight boles

–  Slow to fast growth

Dipterocarpus condoriensis

Shorea selanica (<60 years)


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Dipterocarpaceae – Reproductive biology

l  Hermaphrodites

l  Zoogamous (mainly insect- pollinated) –  Bees, butterflies

–  Thrips

l  Self compatible, mixed mating system –  t typical 60-90%


Dipterocarpus condoriensis

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Dipterocarp fruits

l  Typical 2 (0-5) winged diaspores

–  Medium-sized to very large

l  recalcitrant

l  No efficient means of seed dispersal


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Exploitation of dipterocarps

Local International


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Use of dipterocarps

By-products Wood

resin

charcoal

window frames

furniture

plywood

Hintergrund: Dipterocarpaceae

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Order: Malvales; Family Dipterocarpaceae

l  Blume (1825): related to Tiliaceae

l  Cronquist (1968) & Takhtajan (1969): Theales

l  Dahlgren (1975): Malvales

l  Maguire et al. (1977), Dayanandan et al. (1999): Malvales (Sarcolaenaceae)

l  Chase et al. (1993); Alverson et al. (1998); Bayer et al., (1999): Malvales (Bombacaceae, Tiliaceae, Sterculiaceae and Malvaceae)

Phylogeny of Dipterocarpaceae

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Malvales; family Dipterocarpaceae

Phylogeny of dipterocarps

(nach CHASE et al., 1993)

rbcL sequences

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Material for studies on inter- and intraspecific variation

l  Over 3000 samples from dipterocarps in SE-Asia

–  Dried leaves l  Silica gel l  Long-term storage at -60°C

–  Extracted DNA –  Herbarium material maintained

l  Mainly with partners

l  Total of 116 species –  All genera of Dipterocarpoideae –  From six countries

l  More than 40 locations l  Focus on Indonesia (>2500

samples) –  Mainly natural forests (>80%), few

plantations, arboreta, botanical gardens

Material

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Methods: cpDNA polymorphisms

–  PCR-RFLP ofcpDNA regions : trnLF, rbcL, petB, psbA, psaA (Tsumura et al., 1996)

–  cpSSRs (Microsatellites): ccmp2, ccmp6, ccmp10 (Weising et al., 1999)

–  Sequences: trnL; trnLF

PCR RFLPs Sequences


trnL region

cpSSRs

(Indrioko et al., 2006)

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Monotes kerstingiiCotylelobium lanceolatumUpuna borneensisAnisoptera costataAnisoptera marginataAnisoptera reticulataVatica bantamensisVatica bellaVatica granulataVatica paucifloraVatica rassakVatica venulosaDipterocarpus grandiflorusDipterocarpus oblongifoliusDipterocarpus retususDipterocarpus rigidusDipterocarpus tempehesDryobalanops aromaticaDryobalanops lanceolataHopea bancanaHopea celebicaHopea odorataHopea sangalHopea dryobalanoidesHopea nigraHopea griffithiiHopea mengarawanParashorea lucidaParashorea globosaShorea blumutensisShorea guisoShorea montigenaShorea scaberrimaShorea seminisShorea acuminataShorea andulensisShorea javanicaShorea johorensisShorea leprosulaShorea macropteraShorea mecistopteryxShorea ovalisShorea palembanicaShorea parvifoliaShorea platycladosShorea xanthophyllaShorea balangeranShorea selanicaShorea splendidaShorea macrophyllaShorea pinangaShorea stenopteraShorea acuminatissimaShorea dasyphyllaShorea faguetianaShorea multifloraShorea fallaxShorea materialisShorea virescens

99 68

83

99

94

100

99

71

85

66 100

69

59

67

55

77

60 98

<50

<50

<50

<50

95

63

Upuna borneensisCotylelobium lanceolatumVatica bantamensisVatica bellaVatica granulataVatica paucifloraVatica rassakVatica venulosaAnisoptera costataAnisoptera marginataAnisoptera reticulataDipterocarpus grandiflorusDipterocarpus oblongifoliusDipterocarpus retususDipterocarpus rigidusDipterocarpus tempehesDryobalanops aromaticaDryobalanops lanceolataHopea bancanaHopea celebicaHopea odorataHopea sangalHopea dryobalanoidesHopea nigraHopea griffithiiHopea mengarawanParashorea lucidaParashorea globosaShorea blumutensisShorea guisoShorea montigenaShorea scaberrimaShorea seminisShorea acuminataShorea andulensisShorea javanicaShorea johorensisShorea leprosulaShorea macropteraShorea mecistopteryxShorea ovalisShorea palembanicaShorea parvifoliaShorea platycladosShorea xanthophyllaShorea balangeranShorea selanicaShorea splendidaShorea macrophyllaShorea pinangaShorea stenopteraShorea acuminatissimaShorea dasyphyllaShorea faguetianaShorea multifloraShorea fallaxShorea materialisShorea virescens

99

95

99

100

99

80

92

67

100

98 69

50

63

58

58

82

61 98

<50

<50

67

50

94

Outgroup: Upuna Outgroup: Monotes

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Diagnostic characters of endemic species


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Upuna borneensis

Anisoptera reticulata

Shorea fallax


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Phylogeny of tribe Dipterocarpae

l  Based on sequence variation of two cpDNA regions

–  trnL Intron

–  trnL-F intergenic spacer


(Nga, 2009)

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Molecular phylogeny based on AFLPs

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Dipterocarpus retususDipterocarpus oblongifolius

Dipterocarpus rigidusDipterocarpus tempehes

Dipterocarpus glandiflorus 1Dipterocarpus glandiflorus 2

Anisoptera reticulataAnisoptera marginata

Anisoptera costata 1Anisoptera costata 2

Cotylelobium lanceolatumUpuna borneensis

Vatica bantamensis 1Vatica bantamensis 2

Vatica venulosaVatica bellaVatica granulata

Vatica rassakVatica pauciflora 1

Vatica pauciflora 2Hopea celebicaHopea bancana 1Hopea bancana 2

Hopea sangal 1Hopea sangal 2

Hopea griffithiiHopea nigra

Hopea dryobalanoides 1Hopea dryobalanoides 2

Hopea mengarawan 1Hopea mengarawan 2

Parashorea lucidaParashorea globosa

Hopea odorata 1Hopea odorata 2

Shorea seminisShorea macroptera

Shorea montigenaShorea andulensis

Shorea balangeranShorea selanica 1Shorea selanica 2

Shorea xanthophylla 1Shorea xanthophylla 2

Shorea materialisShorea macrophylla 1

Shorea macrophylla 2Shorea splendida 1Shorea splendida 2

Shorea mecistopteryx 1Shorea mecistopteryx 2Shorea mecistopteryx 3

Shorea scaberrimaShorea palembanica 1

Shorea palembanica 2Shorea leprosula 1Shorea leprosula 2

Shorea parvifolia 1Shorea parvifolia 2

Shorea guiso 1Shorea guiso 2

Shorea platyclados 1Shorea platyclados 2

Shorea ovalis 1Shorea ovalis 2

Shorea faguetianaShorea acuminatissima

Shorea javanicaShorea johorensis 1Shorea johorensis 2

Shorea blumutensis 1Shorea blumutensis 2

Shorea acuminata 3Shorea acuminata 1

Shorea acuminata 2Dryobalanops aromatica

Dryobalanops lanceolata 1Dryobalanops lanceolata 2

Shorea virescens 1Shorea virescens 2

The phylogeny based on morphology and cpDNA variation is also reflected at AFLPs, indicating genome-wide differentiation pattern congruent with variation at cpDNA

(from CAO et al., 2006a)


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Intraspecific diversity in Indonesian Shorea spp.

Indonesia

Sumatra

BorneoNS (1, 2, 3, 4, 5, 6)

SLB (7, 8, 9, 10, 11)

Java

Sampling locations

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Diversity within Shorea species

Genetic diversity within populations of nine Shorea species in Indonesia at AFLP loci Pop. ID Distribution Sample size Polymorphic loci PPL na ne He I 1. Spar_NS common 26 38 44.71% 1.447 1.176 0.110 0.174 2. Sacu_NS common 32 42 49.41% 1.494 1.159 0.100 0.162 3. Sdas_NS scattered 20 47 55.29% 1.553 1.273 0.164 0.251 4. Sblu_NS rare 21 53 62.35% 1.624 1.266 0.165 0.257 5. Slep_NS common 16 36 42.35% 1.424 1.224 0.134 0.204 6. Smac_NS common 26 45 52.94% 1.529 1.259 0.155 0.238 Mean 24 44 51.18% 1.512 1.226 0.138 0.214 7. Spar_SLB common 31 44 51.76% 1.518 1.199 0.122 0.193 8. Slep_SLB common 26 39 45.88% 1.459 1.192 0.115 0.178 9. Spal_SLB common 25 52 61.18% 1.612 1.245 0.149 0.232 10. Splat_SLB common 27 56 65.88% 1.659 1.235 0.144 0.230 11. Sjoh_SLB common 24 47 55.29% 1.553 1.183 0.115 0.186 Mean 27 48 56.00% 1.560 1.211 0.129 0.204

Notes: Information of species distribution on islands was obtained from Newman et al. (1996a; 1996b). PPL, percentage of phenotypically polymorphic loci; na, observed number of alleles per locus; ne, effective number of alleles per locus;

He, Nei’s (1973) gene diversity; I, Shannon's information index [Lewontin (1972)]. (from Cao et al., 2009)

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Intraspecific diversity – common, widespread species

Intraspecific variation

NS

TS

HJ, DJ

SmB BaB

SB

TB PS

AS

IB

BkB

CJ

KB

MtB

TJS

BB

WkB

Shorea leprosula and S. parvifolia

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Species N PPL (%)

He Ι Ht Hs Gst

S. parvifolia

117 75 0,1973 0,3062 0,1973 0,1362 0,3121

S. leprosula

152 94,64 0,2404 0,3788 0,2404 0,1814 0,2355

Variation within species

N: sample size PPL. Percentage of polymorphic loci He: Nei's (1973) gene diversity I: Shannon's Information index Ht: Total variation HS : Variation within populations GST: Differentiation among populations


(Cao et al, 2006b)

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G st for AFLP markes in Shorea leprosula

0 0,2 0,4 0,6 0,8

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55

G st for AFLP markers in Shorea parvifolia

0 0,2 0,4 0,6 0,8

1

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55

Genetic differentiation among populations


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Conversion of AFLP bands to SCAR markers 10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SUMATRA 427 GATACAGGGCAGCAATCACATGGATAGACTCAGCAGCTAATGGATAATTTTAGGCTTAAAGGGGGCTATCACATACCAAAAGTTCAGCAAGTAAGGCCGG BORNEO 421 GATACAGGGCAGCAATCACATGGATAGACTCAGCAGCTAATGGATAATTTTAGGCTTAAAGGGGGCTATCACATACCAAAAGTTCAGCAAGTAAGGCCGG BORNEO 418 GATACAGGGCAGCAATCACATGGATAGACTCAGCAGCTAATGGATAATTTTAGGCTTAAAGGGGGCTATCACATACCAAAAGTTCAGCAAGTAAGGCCGG BORNEO 409 GATACAGGGCAGCAATCACATGGATAGACTCAGCAGCTAATGGATAATTTTAGGCTTAAAGGGGGCTATCACATACCAAAAGTTCAGCAAGTAAGGCCGG 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SUMATRA 427 AGCTGGCAGGTTTGGGGGAGAGCCGGCGGGAAGTGCTGGGAAATTTAGGGGAATCTACCGAAAATTGGGWGGAACCAAGAGAAATTAGAGTAAACCCAAG BORNEO 421 AGCTGGCAGGTTTGGGGGAGAGCCGGCGGGAAGTGCTGGGAAATTTAGGGGAATCTACCGAAAATTGGGAGGAACCAAGAGAAATTAGAGTAAACCCAAG BORNEO 418 AGCTGGCAGGTTTGGGGGAGAGCCGGCGGGAAGTGCTGGGAAATTTAGGGGAATCTACCGAAAATTGGGAGGAACCAAGAGAAATTAGAGTAAACCCAAG BORNEO 409 AGCTGGCAGGTTTGGGGGAGAGCCGGCGGGAAGTGCTGGGAAATTTAGGGGAATCTACCGAAAATTGGGAGGAACCAAGAGA---------AAACCCAAG 210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SUMATRA 427 CTAATTCAAGGAAGAACAAAGGAATTAGTGGCTGTTTCTTACCCAAAAATTTGAAGAATTCACACCCACACAATGAAGAGCAGTRGATAGAAATAGAAGT BORNEO 421 CTAATTCAAGGAAGAACAAAGGAATTAGTGGCTGTTTCTTACCCAAAAATTTGAAGAATTCACACCCACACAATGAAGAGCAGTAGATAGAA------GT BORNEO 418 CTAATTCAAGGAAGAACAAAGGAATTAGTGGCTGTTTCTTACCCAAAAAT---AAGAATTCACACCCACACAATGAAGAGCAGTAGATAGAA------GT BORNEO 409 CTAATTCAAGGAAGAACAAAGGAATTAGTGGCTGTTTCTTACCCAAAAAT---AAGAATTCACACCCACACAATGAAGAGCAGTAGATAGAA------GT EcoRI 310 320 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| SUMATRA 427 AACAGAGCAGAGCAGATCCTGAATTTCTCTGCAGGGGGGAGGTTTCAAGGCTCCAAATCTTGTTCTTGAACAAGAAAAGAAAGGAAAAATCAAGCTCTCT BORNEO 421 AACAGAGCAGAGCAGATCCTGAATTTCTCTGCAGGGGGGAGGTTTCAAGGCTCCAAATCTTGTTCTTGAACAAGAAAAGAAAGGAAAAATCAAGCTCTCT BORNEO 418 AACAGAGCAGAGCAGATCCTGAATTTCTCTGCAGGGGGGAGGTTTCAAGGCTCCAAATCTTGTTCTTGAACAAGAAAAGAAAGGAAAAATCAAGCTCTCT BORNEO 409 AACAGAGCAGAGCAGATCCTGAATTTCTCTGCAGGGGGGAGGTTTCAAGGCTCCAAATCTTGTTCTTGAACAAGAAAAGAAAGGAAAAATCAAGCTCTCT 410 420 430 ....|....|....|....|....|....|. SUMATRA 427 TACCGTTTCTTGAGGGTGGGTGCTGTGTCGA BORNEO 421 TACCGTTTCTTAAGGGTGGGTGCTGTGTCGA BORNEO 418 TACCGTTTCTTAAGGGTGGGTGCTGTGTCGA BORNEO 409 TACCGTTTCTTAAGGGTGGGTGCTGTGTCGA MseI

(Nuroniah et al in prep.)


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Shorea leprosula: Allele frequency at a co-dominant SCAR marker

1

2

3

1

2

3

1

2

3

TjS TS

PS AS NS

KB

BB

SmB

TB SB

IB

BkB

WkB MtB

BORNEO

JAVA

1

2

3

427 418

421

418/ 421

1

2

3

1

2

3

1

2

3

1

2

3

4

5

409/ 418

409/ 421

1

2

3

1

2

3

4

5

6

7

1

2

3

4

5

6

7

8

421/ 427 418/ 427

1

2

3

1

2

3


(Nuroniah et al., 2010; Finkeldey et al., 2010)

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Genetic fingerprints to trace the origin of tropical timber

l  Wood from tropical forests is (and will continue to be) traded on international markets

–  origin l  illegal logging l  sustainably managed forests

–  certified forests

l  How to trace the origin of wood and wood products?

–  Confirm or reject the origin from particular enterprises, regions, countries,...

Only the best is good enough!

Application: Timber identification

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The chain of custody

Harvest

Processing

Export & Import

Further processing

Transport

End user


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Material for DNA extraction from wood: Dipterocarps

l  N = 382 –  181 from countries of

origin l  Indonesia

l  Thailand

l  Philippines

l  Vietnam

–  151 processed wood l  Germany

l  Indonesia


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DNA Extraction method

l  Based on DNeasy plant mini kit (Qiagen) with modifications

–  50-100 mg of wood shavings

–  Addition of PVP

l  RACHMAYANTI et al. (2006, 2009)


27-a. first eluat 27-a. second eluat

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Success of DNA extraction and amplification

l  Main impact –  Species

–  Age and processing of wood

–  Inhibitory substances

–  Location on stem disk (outer – inner)

–  Size and genomic origin of PCR fragment

Results: Extraction

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Impact of fragment size and position on disk

Ccmp2 (150 bp)

trnL (0.6 kbp)

trnLF (1.1 kbp)

a m i

0.15 0.6 1

first eluat of a.second eluat of a.

first eluat of m.second eluat of m.

first eluat of i.

second eluat of i.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

PCR success

fragment length (kb)

Results: Extraction

(Rachmayanti et al., unpublished)

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Inhibitory tests: PVP and position on disk

High-quality DNA (from leaves) and diluted or undiluted wood extracts as PCR template

DNA extraction : 1. without PVP addition

2. with 2.6% PVP

3. with 5.0% PVP

2. 1. 3.

Wood extract dilution (x10 time) first eluat

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 1 2 4 8 16 32

wood extract ( x10 times dilution)

inhibitory rate

inner heartwood (i) rings transition wood (m) outer sapwood (a)

Results: Extraction


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Summary: Success of extraction and amplification

Results: Extraction

PCR analysis of dipterocarp wood DNA

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Vietnam (40)

Indonesia (38)

Thailand (53)

Philippine (50)

Enterprises(151)

Tectona grandis (12)

Eusideroxylonzwageri (2)

Prunusarborea (1)

Strombosiaceylanica (1)

Triplochiton scleroxylon

(7)

Populus sp.(25)

Costa Ricawood (3)

Taxus baccata (2)

Salix spp. (1)

Pinus sylvestris (12)

Picea abiesL (8)

Dipterocarp wood Non-dipterocarp tropic wood non-tropic wood

Am

plifi

catio

n su

cces

s ra

te

short fragment (ccmp2, 150 bp; PS,100-200 bp; G3,119-133 bp) middle length fragment (trnL, ca. 600 bp) long fragment (trnLF, ca. 1.1 kb)


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Conclusions and outlook I

l  Molecular genetic tools are in principle suitable to infer the origin of tropical wood

–  DNA extraction from wood is feasible

l  but: limitations (age, inhibitors, processing,...)

–  Development of informative markers is demanding

l  Problems are taxa specific l  Different marker types often

necessary –  Development of sufficient

information (data bases) is costly, time consuming

Conclusion and outlook


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27-a. first eluat 27-a. second eluat

trnL region

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Conclusions and outlook II

Conclusion and outlook

l  The dipterocarps are a suitable model group to further develop and to apply molecular methods to infer the origin of wood

–  Ecologically relevant keystone species

–  Conservation need (endangered species)

–  Important timber species (national, international)

–  Some knowledge on molecular genetic variation

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Selected relevant literature from the group

Tsumura, Y., Kado, T., Yoshida, K., Abe, H., Ohtani, M., Taguchi, M., Fukue, Y., Tani, N., Ueno, S., Yoshimura, K., Kamiya, K., Harada, K., Takeuchi, Y., Diway, B., Finkeldey, R., Na’iem, M., Indrioko, S., Ng, K.K.S., Muhammad, N., Lee, S.L. (2011). Molecular database for classifying Shorea species (Dipterocarpaceae) and techniques for checking the legitimacy of timber and wood products. Journal of Plant Research 124: 35-48

Nuroniah, H.S., Gailing, O, Finkeldey, R. (2010). Development of SCAR markers for species identification in the genus Shorea (Dipterocarpaceae). Silvae Genetica 59: 249-256

Finkeldey, R., Leinemann, L., Gailing, O. (2010). Molecular genetic tools to infer the origin of forest plants and wood. Applied Microbiology and Biotechnology 85: 1251-1258 Open Access review: http://www.springerlink.com/content/u65730127511k770/

Cao, C.P., Gailing, O., Siregar, I.Z., Siregar, U., Finkeldey, R. (2009) Genetic variation in nine Shorea species revealed by AFLPs. Tree Genetics and Genomes 5: 407-420

Rachmayanti, Y., Leinemann, L., Gailing, O., Finkeldey, R. (2009) DNA from processed and unprocessed wood: factors influencing the isolation success. Forensic Science International, Genetics 3: 185-192

Cao, C.P., Finkeldey, R., Siregar, I.Z., Siregar U.J., Gailing, O. (2006). Genetic diversity within and among populations of Shorea leprosula Miq. and Shorea parvifolia Dyer (Dipterocarpaceae) in Indonesia detected by AFLPs. Tree Genetics and Genomes 2: 225-239

Cao, C.P., Gailing, O., Siregar, I., Indrioko, S., Finkeldey, R. (2006). Genetic variation at AFLPs for the Dipterocarpaceae and its relation to molecular phylogenies and taxonomic subdivisions. Journal of Plant Research 119: 553-558

Indrioko, S., Gailing, O., Finkeldey, R. (2006). Molecular phylogeny of Dipterocarpaceae in Indonesia based on chloroplast DNA. Plant Systematics and Evolution 261: 99-115

Rachmayanti, Y., Leinemann, L., Gailing, O., Finkeldey R. (2006). Extraction, amplification and characterization of wood DNA from Dipterocarpaceae. Plant Molecular Biology Reporter 24: 45-55

See also: http://www.uni-goettingen.de/en/82738.html http://aunp.forst.uni-goettingen.de/outputs/teaching_material.shtml

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Acknowledgements

l  The main sponsors

l  The main partners –  Dr. Paciencia Milan, Dr. Tony Quimio,

Leyte State University, Philippines –  Prof. Le-Cong Kiet, Vietnam Natl.

University –  Dr, Suchitra Changtragoon, RFD

Thailand –  Dr. Nyan Htun, UoF, Burma

Thank You for Your attention!

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Intraspecific Variation at cpDNA (cpSSRs and PCR-RFLPs)

Results: Marker development

(Indrioko, 2005) Haplotype distribution in Shorea parvifolia

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Poor geographic structure at cpDNA markers – Why?

Results: Marker development

(SATHIAMURTY and VORIS, 2006)

Sea level in SE-Asia approx. 14580 years ago

Genetic variation of dipterocarps: from molecular...

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