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SYSTEMATICS STUDIES OF THREE Aquailarill SPECIES Nur Qisttna Bintt Othman Master of Science 2012
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SYSTEMATICS STUDIES OF THREE Aquailarill SPECIES
Nur Qisttna Bintt Othman
Master of Science 2012
SYSTEMATICS STUDIES OF THREE A QUILARIA SPECIES
Nur Qistina Othman
This thesis is submitted in fulfilment of the requirement of Master of Science
(Botany)
. Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
April 2012
DECLARATION
I, hereby, declare that no portion of the work referred to in this thesis has submitted in
support of an application for another degree of qualification of this to any other university
or institution of higher learning.
(NUR QISTINA BINTI OTHMAN)
Matric No: 07021261
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DEDICATION
My dedication goes to my dearest family members especially my father Encik Othman
Hj. Khalid, my mother Puan Aini Abu Bakar, my step father Encik Mohd. Hisyam
Yahya, brothers and sisters, and Ilham Halabi, for their supports and inspirations given in
completing this thesis successfully.
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ACKNOWLEDGEMENTS
Bismillahirrahmanirrahim. Thanks Allah for His blessing in completing this thesis. This
thesis would not have been possible without the guidance and the help of several
individuals who in one way or another contributed and extended their valuable assistance
in the preparation and completion of this study. First and foremost, I offer my sincerest
gratitude to my supervisors, Professor Dr. Cheksum @ Supiah Tawan, Assoc. Prof. Dr.
Hairul Azman Roslan and Professor Dr. Isa Ipor for their encouragements, assistances,
guidance and supervisions.
I would like to extend my gratitude to the Universiti Malaysia Sarawak for awarding me
the Zamalah UN/MAS scholarship to enable me to pursue my postgraduate and to
Ministry of Higher Education (MOHE) for the financial grant FRGS/O 1 (0 1 )/606/2006(39)
given to enable to conduct this project successfully. My thanks also goes to Sarawak
Forestry Department for the permission to conduct the research and to Forest Research
Institute for providing the seedlings of Aquilaria malaccensis Lam.
My appreciation also goes to all the Faculty of Resource Science and Technology
supporting staff especially to Encik Mohd Mohd Rizan Abdullah, Encik Sekudan Tedong,
Encik Muhd Najib Fardos and Encik Salim Arip for their cooperation and help
throughout my studies. My thanks also goes to Puan Ting Woei and Encik Amin Manggi
who have assisted me in using the scanning electron microscope. Special thanks to my
fellow friends Nur Diana Anuar, Tan Sia Hong, luraidah Salimun, Wee Ching Ching,
Hashimatul Fatma Hashim, Nur Hafizah Azizan and Angeline Simon.
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t
ABSTRACT
A systematic study of Aquilaria beccariana Tiegh., Aquilaria malaccensis Lam. and
Aquilaria microcarpa Baill. based on morphology and molecular characteristics were
conducted. They are important species for producing the aromatic resin known as gaharu
which fetches high market value and have been listed in Appendix II of CITES (Convention
of International Endangered Species). The herbarium specimens were used for the
morphological study while fresh samples of the seedlings grown in the green house at
Universiti Malaysia Sarawak and collection from the field were used for the molecular study.
The objectives of this study were to document the morphological characteristics and to
develop species-specific DNA markers for these three species to be used as a tool to identify
and differentiate the species. This study described in detail the vegetative, floral, and fruits
macro and micromorphology to provide detail taxonomic information for easy identification
of the species. Two molecular techniques were chosen, the RAPD (Random Amplified
Polymorphic Deoxyribonucleic Acid) fingerprinting and RAPD-SCAR (Sequence
Characterized Amplification Region). Based on the RAPD fingerprinting, the genetic profiles
were obtained for A. beccariana and A. microcarpa generated using universal primers M 13
and OPA 10 but for A. malaccensis the profile was generated only through primer OPA 10.
The amplicons obtained were subjected to RAPD-SCAR technique and successfully obtained
the species specific DNA marker. Two primer pairs were developed and designated for each
A. beccariana and A. microcarpa based on primer M 13 and OPA 10 while only one primer
pair was designated for A. malaccensis based on primer OPA 10. All five primer pairs were
considered novel findings. All morphology and molecular information obtained in this study
would be most useful especially in resolving the difficulty in differentiating between species
and important knowledge in implementing any conservation purposes and selection of
seedlings for commercial plantation.
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Kajian Sistematik Terhadap riga Spesies Aquilaria
ABSTRAK
Kajian sistematik terhadap Aguilaria beccariana Teigh., Aguilaria malaccensis Lam. dan
Aguilaria microcarpa Bail!. berdasarkan ciri-ciri moifologi dan molekular telah dilakukan.
Ke/iga-tiga spesies ini penting dalam menghasilkan resin aromatik gaharu yang mempunyai
nilai pasaran yang tinggi. Spesies ini telah disenaraikan dalam Appendik II CITES (Convention
of International Trade of Endangered Species). Spesimen herbarium telah digunakan untuk
kajian moifologi dan sampel segar dari anak pokok di rumah hijau di Universiti Malaysia
Sarawak dan juga dari lapangan digunakan untuk kajian molekular. Objektif kajian ini adalah
untuk mendokumentasi ciri-ciri morfologi dan mendapatkan penanda DNA yang spesifik bagi
memudahkan pengecaman dan membezakan ketiga-tiga spesies berkenaan. Penelitian terhadap
ciri makro dan mikro bahagian vegetatif, bunga, dan buah untuk menghasilkan maklumat
taksonomi yang terperinci telah dilakukan. Teknik molekular cap jari RAPD (Random
Amplified Polymorphic Deoxyribonucleic acid) dan RAPD-SCAR (Sequence-Characterised
Amplified Region) DNA telah gunakan. Hasil daripada cap jari RAPD, profil genetik telah
dihasil bagi d. beccariana and d. microcarpa mengunakan primer universal M 13 dan OP A 10
tetapi untuk d. malaccensis profil hanya diperolehi mengunakan primer OPA 10. Amplikon
yang diperlolehi telah diuji mengunakan teknik RAPD-SCAR dan telah berjaya menghasilkan
penanda DNA yang spesies spesifik. Hasil daripada ini dua pasang primer telah dihasilkan dan
dikhusukan untuk d. beccariana dan d. microcarpa masing-masing berdasarkan primer M 13
dan OPA 10 sementara satu pasang primer dihasilkan untuk d. malaccensis berdasarkan OPA
10. Kelima-lima hasil pasangan primer tersebut merupakan penemuan baru. Kesemua maklumat
moifologi dan molekular yang diperolehi dalam kajian ini penting untuk membantu dalam
menyelesaikan masalah pengecaman spesies dan pengetahuan ini penting untuk tujuan
pemuliharaan dan juga pemilihan spesies untuk tanaman secara komersial.
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Pusat Khidmat MaldulDtAkademik UNIVERSm MALAYSIA SARAWAK
TABLE OF CONTENTS
PAGE
TITLE PAGE
DECLARATION 11
TABLE OF CONTENTS Vll
LIST OF TABLES Xlll
DEDICATION III
ACKNOWLEDGEMENTS IV
ABSTRACT V
ABSTRAK VI
LIST OF FIGURES XIV
LIST OF ABBREVIA nONS XIX
CHAPTER ONE
1.0 INTRODUCTION
1.1 Introduction 1
1.2 Objectives 3
1.3 Benefits obtained from the study 4
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Taxonomy of the Three Aquilaria Species
2.1.1 The Family Thymelaeaceae 6
2.1.2 The Genus Aquilaria Lam. 6
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2.2 Anatomical Characteristics 8
2.3 Distribution and Habitat 10
2.4 Gaharu and its F onnation 13
2.5 Uses of Gaharu 15
2.6 Harvesting, Collection and Trade of Gaharu 18
2.7 Conservation Status and Trade Controls of Gaharu 19
2.8 Cultivation 20
2.9 Molecular Study of Thymelaeaceae 22
2.10 Random Amplified Polymorphic DNA (RAPD) as Genetic Markers 24
2.11 Sequence Characterized Amplified Region (SCAR) Markers 25
CHAPTER THREE
3.0 VEGETATIVE, FLORAL AND FRUIT MORPHOLOGY
3.1 Introduction 27
3.2 Materials and Methods
3.2.1 Observation on Vegetative Characters 29
3.2.2 Leaf Clearing for Venation Studies 29
3.2.3 Preparation of Leaves for Scanning Electron Microscope 30
3.2.4 Floral MacIomorphology 30
3.2.5 Floral Micromorphology 30
3.2.6 Fruit Morphology 31
3.3 Results and Discussion
3.3.1 Bark 31
3.3.2 Leaf Macromorphology 34
3.3.3 Leaf Micromorphology 41
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3.3.4 Floral Morphology
3.3.4.1 Inflorescence
3.3.4.2 Pedicels
3.3.4.3 Flower Type and Size
3.3.4.4 Calyx Tube and Calyx Indumentum
3.3.4.5 Petaloid Appendages
3.3.4.6 Stamens
3.3.4.7 Pistil
3.3.5 Fruits
3.3.5.1 Fruit Shape and Colour
3.3.5.2 Fruit Size
3.3.6 Seeds
3.3.6.1 Seed Size, Shape and Colour
3.3.6.2 Seed Micromorphology
3.3.7 Taxonomic Description
3.3.8 Key to Identification of Species
3.4 Conclusion
CHAPTER FOUR
4.0 RAPD FINGERPRINTING
4.1 Introduction
4.2 Materials and Methods
4.2.1 Plant Materials
4.2.2 Isolation of Plant Total Genomic DNA
4.2.3 Purification of DNA
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4.2.4 Genomic DNA Analysis and Storage 78
4.2.5 Random Amplified Polymorphic DNA-PCR (RAPD-PCR)
4.2.5.1 Fingerprinting Using M13 Universal Primer 78
4.2.5.2 Fingerprinting Using OPA 10 Primer 80
4.2.6 PCR Product Analysis
4.2.6.1 Agarose Gel Electrophoresis 81
4.2.6.2 Data Analysis 81
4.3 Results and Discussion
4.3.1 Isolation of Total DNA 82
4.3.2 Genomic DNA Analysis 85
4.3.3 Fingerprinting Generated by MI3 and OPA 10 Primers 88
4.4 Conclusion 94
CHAPTER FIVE
5.0 RAPD-SCAR MARKER DEVELOPMENT FOR SPECIES IDENTIFICATION
5.l Introduction 95
5.2 Materials and Methods
5.2.1 Identification of Species-Specific RAPD Amplicons
(Diagnostic Bands) 96
5.2.2 Analysis of RAPD Amplicons for Diagnostic Bands 96
5.2.3 Purification of PCR Product 97
5.2.4 Confirmation of Desired Band after Purification 97
5.2.5 Cloning ofRAPD Amplicons (Diagnostic Bands)
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,...
5.2.5.1 Calcium Chloride (CaCh) Bacterial
Competent Cells Preparation 97
5.2.5.2 LAIX Plates Preparation 98
5.2.5.3 Ligation 99
5.2.5.4 Transformation Using the pGEM®-T Easy
Vector Ligation Reactions 99
5.2.5.5 Screening Transformants for Inserts
through Blue/White Colony Selection 100
5.2.5 .6 PCR Amplification for Confirmation 100
5.2.5 .7 Plasmid Extraction 101
5.2.5.8 Restriction Enzyme for Second Confirmation 102
5.2.6 Sequencing 102
5.2.7 SCAR Primer Designing, Primer Synthesis and
Bioinformatics 103
5.2.8 Primer Testing 103
5.3 Results and Discussion
5.3.1 Identification of Diagnostic Bands from RAPD
Amplification 105
5.3.2 Diagnostic Bands for Each Species 108
5.3.3 Cloning of Diagnostic Bands III
5.3.4 Confirmation of Desired Band with PCR and
Restriction Enzyme Analysis 112
5.3.5 Sequencing, Primer Design and Bioinformatics 119
5.3 .5.1 Multiple Sequence Alignment 119
5.3 .5.2 Species-Specific Primers Design 125
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L
5.3.5.2.1
5.3.5.2.2
5.3.5.2.3
Species-Specific Primers for
A. beccariana
Species-Specific Primer for
A. microcarpa
Species-Specific Primers for
A. malaccensis
5.3.6 Primer Testing on Specific Aquilaria Species and
on Other Plant Species
5.4 Conclusion
CHAPTER SIX
6.0 GENERAL DISCUSSION AND CONCLUSION
6.1 Taxonomy of the Genus Aquilaria
6.2 Identification of the Three Aquilaria Species Based
on the Morphology
6.3 Identification of the Three Aquilaria Species Based on
Molecular Studies
6.4 Conclusions and Recommendations
REFERENCES
APPENDIX 1 Specimens of the three Aquilaria species used in the various studies
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151
154
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LIST OF TABLES
PAGE
Table 4.0 Locality of the samples used 76
Table 4.1 MI3-PCR reaction mixture of25 ~l volume reaction 79
Table 4.2 MI3-PCR amplification parameters 79
Table 4.3 OPA IO-PCR reaction mixture of 25 ~l volume reaction 80
Table 4.4 OPA 10-PCR amplification parameter 81
Table 5.0 PCR reaction mixture of 12.5 JlI volume reaction 101
Table 5.1 PCR amplification parameters for confirmation of inserts 101
Table 5.2 PCR reaction mixture of25 JlI volume reaction 104
Table 5.3 PCR amplification parameters 104
Table 5.4 Summary of fragment size of diagnostic bands 131
Table 5.5 Summary of fragment size of SCAR primer 131
Table 5.6 Summary of specific primer designed for specific identification of
A. microcarpa, A. beccariana and A. malaccensis 131
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LIST OF FIGURES PAGE
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.7
Figure 3.8
Figure 3.9
Figure 3.10
Figure 3.11
Figure 3.12
Figure 3.13
Figure 3.14
Figure 3.15
Photographs showing the outer bark surfaces of a mature tree trunk of the
tree species 33
Showing the leaves arrangement and the size ofwhole leaf of
A. beccariana, A. malaccensis and A. microcarpa 37
Light micrographs showing the venation of cleared leaves of the
three species 40
Scanning electron micrographs of A. beccariana showing the
sculpturing of the leaf surfaces 42
Scanning electron micrographs of A. malaccensis showing the
sculpturing of the leaf surfaces 43
Scanning electron micrographs of A. microcarpa showing the
sculpturing of the leaf surfaces 44
Scanning electron micrographs showing the structure of stomata
of the three species 47
Scanning electron micrographs showing floral shape of the
three species 51
Scanning electron micrographs showing the indumentums
of the anthers of the three species 54
Scanning electron micrographs showing the pistil of the three species 56
Scanning electron micrographs showing the shape of the stigmas
of the three species 57
Photographs showing the fruit shape of the three species 60
Fruit shape of some Aquilaria species 61
Light micrographs showing the seed shape and size of the three species 63
Scanning electron micrographs showing the seed surfaces of the three species 65
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Figure 3.16A Herbarium specimen of A. beccariana 67
Figure 3.16B Herbarium specimen ofA. malaccensis 69
Figure 3.1 6C Herbarium specimen of A. microcarpa 70
Figure 4.0 Agarose gel electrophoregram of DNA extracted from A. beccariana 87
Figure 4.1 Agarose gel electrophoregram of DNA extracted from A. malaccensis 87
Figure 4.2 Agarose gel electrophoregram ofDNA extracted from A. microcarpa 88
Figure 4.3 RAPD profiles generated by M13 Universal Primer
(S'- TTATGAAACGACGGCCAG T - 3') separated on 2.S% agarose gel 92
Figure 4.4 RAPD profile generated by OPA 10 primer (S'- GTGATCGCA G- 3')
separated on 2.S% agarose gel 93
Figure 5.0 RAPD profile generated by M 13 Universal Primer
(S'- TTATGAAACGACGGCCAGT - 3') separated on 2.S% agarose gel 106
Figure S.l RAPD profile generated by OPA 10 primer (S'- GTGATCGCAG- 3')
separated on 2.S% agarose gel 107
Figure 5.2 Visualization of purified PCR product ofA. beccariana's diagnostic band
(from RAPD using M13 primer) 109
Figure S.3 Visualization of purified PCR product ofA. microcarpa's diagnostic band
(from RAPD using M13 primer) 109
Figure 5.4 Visualization of purified PCR product ofA. beccariana's diagnostic band
(from RAPD using OPAI0 primer) 110
Figure 5.5 Visualization of purified PCR product of A. malaccensis's diagnostic band
(from RAPD using OPAI0 primer) 110
Figure 5.6 Visualization of purified PCR product ofA. microcarpa's diagnostic band
(from RAPD using OPAl 0 primer) III
Figure 5.7 Visualization ofT7/SP6 PCR product ofA. beccariana (diagnostic band
from RAPD using M13 primer) 114
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Figure 5.8 Restriction enzyme product of A. beccariana (diagnostic band from RAPD
using M 13 primer) 114
Figure 5.9 Visualization ofT7/SP6 PCR product ofA. microcarpa (diagnostic band
from RAPD using M 13 primer) 115
Figure 5.1 0 Restriction enzyme product ofA. microcarpa (diagnostic band from RAPD
using M13 primer) 115
Figure 5.11 Visualization ofT7/SP6 PCR product of A. beccariana (diagnostic band
from RAPD using OPA 10 primer) 116
Figure 5.12 Restriction enzyme product ofA. beccariana (diagnostic band from RAPD
using OPA 10 primer) 116
Figure 5.13 Visualization ofT7/SP6 PCR product ofA. malaccensis (diagnostic band
from RAPD using OPAl 0 primer) 117
Figure 5.14 Restriction enzyme product ofA. malaccensis (diagnostic band from RAPD
using OPA 10 primer) 117
Figure 5.15 Visualization ofT7/SP6 PCR product ofA. microcarpa (diagnostic band
from RAPD using OPA 10 primer) 118
Figure 5.16 Restriction enzyme product ofA. microcarpa (diagnostic band from RAPD
using OPA 10 primer) 118
Figure 5.17 ClustalW multiple alignment ofA. beccariana (RAPD from M13 primer) 120
Figure 5.18 ClustalW multiple alignment of A. microcarpa (RAPD from M13 primer) 121
Figure 5.19 ClustalW multiple alignment ofA. beccariana (RAPD from OPA 10 primer) 122
Figure 5.20 ClustalW multiple alignment ofA. malaccensis (RAPD from OPA 10
primer) 123
Figure 5.21 ClustalW multiple alignment ofA. microcarpa (RAPD from OPA 10
primer) 124
Figure 5.22 Generation of forward and reverse primers for A. beccariana (RAPD
from M 13 primer) by Primer 3 online software 127
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Figure 5.23 Generation of forward and reverse primers for A. beccariana (RAPD
from OPAl 0 primer) by Primer 3 online software 127
Figure 5.24 Generation of forward and reverse primers for A. microcarpa (RAPD
from M 13 primer) by Primer 3 online software 128
Figure 5.25 Generation of reverse and forward primers for A. microcarpa (RAPD
from OPA 10 primer) by Primer 3 online software 129
Figure 5.26 Generation of forward and reverse primers for A. malaccensis (RAPD
from OPAl 0 primer) by Primer 3 online software 130
Figure 5.27 PCR product of specific fragment size amplified by species-specific
primer for each species 132
Figure 5.28 Agarose gel electrophoregram of PCR amplification of A. microcarpa
using the I st set of species-specific primer (MP I) 134
Figure 5.29 Agarose gel electrophoregram of PCR amplification ofA. microcarpa
using the 2nd set of specific primer (MP 2) 135
Figure 5.30 Agarose gel electrophoregram ofPCR amplification ofA. beccariana
using the 1 st set of specific primer (BC 1) 136
Figure 5.31 Agarose gel electrophoregram of PCR amplification ofA. beccariana
using the 2nd set of specific primer (BC 2) 137
Figure 5.32 Agarose gel electrophoregram of PCR amplification ofA. malaccensis
using the specific primer (MC 1) 138
Figure 5.33 Agarose gel electrophoregram of PCR amp1ification of
Cryptocoryne narutoi, C. longcauda, Morinda citrifolia and
Amorphophallus hewitti using MP I primer 139
Figure 5.34 Agarose gel electrophoregram of PCR amplification of
Cryptocoryne narutoi, C. longcauda, Morinda citrifolia and
Amorphophallus hewitti using MP 2 primer 140
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Figure 5.35 Agarose gel electrophoregram of peR amplification of
Figure 5.36
Figure 5.37
Cryptocoryne narutoi, C. /ongcauda, Morinda citrifolia and
Amorphopha//us hewitti using Be 1 primer 141
Agarose gel electrophoregram of peR amplification of
Cryptocoryne narutoi, C. /ongcauda, Morinda citrifolia and
Amorphopha//us hewitti using Be 2 primer 142
Agarose gel electrophoregram of peR amplification of
Cryptocoryne narutoi, C. /ongcauda, Morinda citrifolia and
Amorphopha//us hewitti using Me 1 primer 143
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AP-PCR
CaCl2
CAPS
CIA
CITES
CTAB
DAF
dATP
dCTP
ddH20
dGTP
dTIP
dNTP
DNA
EtBr
FAA
FRIM
GC
GC-MS
HUMS
ITS
IUCN
IPTG
LIST OF ABBREVIATIONS
Arbitrary primed DNA amplification-Polymerase chain reaction
Calcium chloride
Cleaved amplified polymorphic sequence
Chloroform isoamyl alcohol
Convention on International Trade in Endangered Species of Wild
Fauna & Flora
Cetylmethylammonium bromide
DNA amplification fingerprinting
Deoxyadenosine triphosphate
Deoxycytidine triphosphate
Double distilled water
Deoxyguanosine triphosphate
Deoxythymidine triphosphate
Deoxyribonucleotide triphosphate
Deoxyribonucleic acid
Ethidium bromide
Formalin Acetic Alcohol
Forest Research Institute of Malaysia
Gas chromatography
Gas chromatography- mass spectrometry
Herbarium of Universiti Malaysia Sarawak
Internal transcribed spacer
International Union for Conservation ofNature
Isopropyl-~-D-thiogalactopyranoside
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KEP Kepong
Luria Bertani
MgCh Magnesium chloride
MTm The Malaysian Timber Industry Board
PCR Polymerase chain reaction
PVP Polyvinyl pyrrolidone
RAPO Random Amplified Polymorphic DNA
RFLP Restriction fragment length polymorphism
SAN Sandakan
SAR Sarawak
SCAR Sequence-characterized amplification region
SOS Sodium dodecyl sulphate
SEM Scanning Electron Microscope
SOC medium - Super optimal broth with catabolic repressor
STS Sequence tagged site
UNIMAS Universiti Malaysia Sarawak
LB
5-bromo-4-chloro-3-indolyl-~-D-galactopyranosideX-Gal
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CHAPTER ONE
INTRODUCTION
IJ Introduction
The genus Aquilaria Lam. from family Thymelaeceae are well known for the production
ofbigh demand resinous wood which is used in producing medicine, incense and perfume
across Asia and the Middle East. There are many common names for this resinous wood,
including agar, agarwood, aloe(s)wood, eaglewood, gaharu and kalamabak (Barden et al.,
2000).
Fifteen species have been identified in the genus Aquilaria and those speCIes are
distributed all along India, Myanmar, Burma, China, Indochina and the Malesian region.
In Malaysia, fi ve species had been recorded namely Aquilaria beccariana Van Tiegh.,
Aquilaria filaria (Oken) Merr., Aquilaria malaccensis Lamk., Aquilaria microcarpa
Baill. and one incompletely unknown species named as Aquilaria sp. 1. Most of the
Aquilaria species are found in the mixed dipterocarp forest on mineral loam soil of an
a1tirude below 1000 m a.s.l. and rarely inhibiting the swampy forest (Beniwal, 1989).
Identification at the species level among the Aquilaria species is well facilitated by their
vegetative and floral characteristics. However, the harvested wood which impregnated
with the resinous material, the 'gaharu' posed a great difficulty to the enforcement officer
either from The Department of Forestry or custom officer to declare and identify at the
species level in the absent of vegetative and floral samples. In the current practices, they
1
usually assumed and declared that the shipment of any traded gaharu products in form of
chips, powder or oil in Malaysia composed of only A. malaccensis.
Almost all of the Aquilaria species can be found in the wild but uncontrolled harvesting
and trade of the trees for the gaharu will soon decreased its populations in their natural
habitat of the tropical rainforests. Data obtained from International Union for
Conservation of Nature (IUCN) Red List Categories of the world shows that populations
of seven Aquilaria species are now facing a high risk of extinction in the wild and
currently being placed under the vulnerable status in the list. These species are A.
banaensae Phamh., A. beccariana, A. cumingiana (Decne) Ridley, A. hirta Ridl., A.
maiaccensis, A. microcarpa, and A. sinensis (Lour.) Gilg, while A. crassna Pierre is
considered critically endangered from over exploitation for gaharu.
Demand for gaharu is increasing day by day and all species of Aquilaria are significantly
threatened by trade. The problem we are facing now is that, people are still unable to
differentiate all the gaharu producing species. Almost all gaharu producing species of the
genus Aquilaria is harvested using the name of A. malaccensis which is considered
inaccurate. This could result in gaharu being declared as A. malaccensis but is actually
other Aquilaria species, or gaharu not declared as such could yet contain material from
the species.
In the current situation, the government of Malaysia has impose strict regulation III
accordance to the requirement by CITES (Convention on International Trade in
Endangered Species of Wild Fauna and Flora) that exporter of A. malaccensis and other
2
pharu producing species must obtain CITES export permits. All Aquilaria spp. are listed
in Appendix II in CITES which trade must be controlled in order to avoid extinction.
Nevertheless, effort to follow CITES requirements, law and enforcement are slowed
down by difficulties in detecting the wood to species level during trading.
1.2 Objectives
It is very difficult for people to recognize the exact species in the wild during the felling
of the tree and also during import and export activities. Specialist or botanist is needed in
order to determine the correct species and in most cases, both the vegetative and floral
characteristics for species identification are often missing during harvesting process.
Therefore, an additional identification tool is needed here as well. My study demonstrates
that molecular markers can be that additional tool. Therefore, three main objectives of
this study were to:
1) document morphological characteristics of the three selected species, A.
maiaccensis, A. microcarpa and A. beccariana.
2) document anatomical characteristics of the three selected species, A. maiaccensis,
A. microcarpa and A. beccariana.
3) develop species-specific DNA markers for these three species to be used as a tool
to differentiate the species in the wild or during trading.
This study describes the use of RAPD (Rapid Amplified Polymorphic DNA) technique on
the DNA of three Aquilaria species studied in order to detect and formulate genetic
markers unique to each species. By converting one of the RAPD-PCR-derived markers
3
into a sequence-characterized amplification region (SCAR), a simple PCR procedure for
direct detection of the three Aquilaria species can be done.
With all of these new infonnation and compilations of morphology and part of the
anatomical data, it will be most useful especially in resolving the difficulty in
differentiating between species as well as implementing the knowledge in any
conservation purposes. With the design of species-specific primers, it is hoped that these
specific primers could be used in population monitoring and habitat conservation for
species management in the natural habitat.
1.3 Benefits obtained from the study
The fact that many of tree species could not be easily identified to the species level
indicates that molecular markers are promising tools for identification. A. malaccensis, A.
microcarpa and A. beccariana have been logged for the non-timber product since
thousands of years ago and their populations in the natural habitat are greatly decreasing
and have been classified as threatened species. Hence all Aquilaria species are now being
listed under Appendix II of the CITES. This is due to the excessive and indiscriminate
cutting of the tree in the wild for their valuable gaharu. To improve the control of illegal
harvesting by responsible authorities, the development of molecular marker is newly
much needed.
The molecular technique is considered efficient and will be very useful in controlling and
monitoring the international trade of gaharu. Thus, the molecular technique can be
implemented in the Sustainable Forest Management System. Markers developed in this
4
