Faculty of Resource Science and Technology
GENETIC DIVERSITY OF Acacia mangium PLUS TREE (SUPERBULK)
USING EST-SSR MOLECULAR MARKER
Angela Tida ak Henry Ganie
Master of Science
(Botany)
2013
GENETIC DIVERSITY OF Acacia mangium PLUS TREE (SUPERBULK) USING
EST-SSR MOLECULAR MARKER
ANGELA TIDA AK HENRY GANIE
A thesis submitted
In fulfilment of the requirements for the degree of Masters in Forest Biotechnology
Faculty of Resource Science and Technology
UNIVERSITI MALAYSIA SARAWAK
2013
DECLARATION
I hereby declare that this thesis is based on my original work except for quotations and
citations which have been duly acknowledged. I also declare that it has not been previously
or concurrently submitted for any other degree at UNIMAS and any other institutions.
____________________________
ANGELA TIDA HENRY GANIE
Date: 21 August 2013
ACKNOWLEDGEMENTS
I would like to express my gratitude to the Lord for the strength and courage He bestowed
upon me throughout completing this master project to success. This project was made
possible with all the guidance and support by a lot of people and agencies, to whom I owed so
much and would like to express to them my deepest gratitude and appreciation.
First of all, to my supervisor Dr. Ismail Jusoh for his constant guidance and encouragement,
constructive critism, brilliant suggestions and motivation throughout the path of completing
this study. You have given me the opportunity, freedom, and resources to fully pursue a
graduate education. I also warmly thank my co-supervisors Dr. Ho Wei Seng and Prof
Wickineswari Ratnam from Universiti Kebangsaan Malaysia, for their willingness to provide
professional guidance and expertise in forest genetics. Also to postgraduate students in UKM
Forest Genetics Laboratory especially Ngu Mee Siing for her assistance and guidance during
my 6-month attachment in their laboratory. My appreciation also goes to UKM itself and
MNA (Malaysian Nuclear Agency), for allowing me to use their equipment and materials in
order to carry out this project.
My genuine appreciation also goes to my husband, Bartholomew Alvin, my family, friends
and loved ones for their encouragement, devotion, and understanding which always been the
ultimate source of my inspiration. Lastly, I dedicate this humble effort, the fruit of my study
to my late father, Henry Ganie Chundie who inspired me through lives thick and thins.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
i
TABLE OF CONTENTS
ii
LIST OF TABLES
v
LIST OF FIGURES
vii
LIST OF ABBREVIATIONS
x
ABSTRACT
xi
ABSTRAK
xiii
1.
CHAPTER I : INTRODUCTION
1
2.
CHAPTER II : LITERATURE REVIEW
6
2.1
3.
Species review
6
2.1.1
Acacia mangium
9
2.1.2
Acacia mangium superbulk
8
2.2
Adaptive genetic variation
13
2.3
Genetic diversity
16
2.4
Tree Improvement
19
2.5
Potential applications of biotechnology in tree improvement
22
2.6
Molecular markers
28
CHAPTER III: MATERIALS AND METHODS
44
3.1
44
Sampling of plant materials
ii
4.
3.2
Growth rate
47
3.3
DNA isolation and purification
47
3.3.1
Chemicals and reagents
47
3.3.2
DNA isolation
47
3.3.3
DNA purification
48
3.4
DNA quantification
49
3.5
Polymerase Chain Reaction (PCR)
50
3.5.1
Polymerase Chain Reaction
50
3.5.2
Primer screening
51
3.5.3
Agarose gel electrophoresis
51
3.6
Polyacrylamide gel electrophoresis
52
3.7
Genotyping
55
3.8
Genotype Scoring
57
3.9
Sequencing
57
4.0
Statistical Analysis
58
CHAPTER IV: RESULTS AND DISCUSSION
63
4.1
Growth rate
63
4.2
A. mangium superbulk phenotype assessment
64
4.3
Acacia mangium superbulk DNA isolation and purification
66
4.4
EST-SSR primers screening for polymorphism detection
68
4.5
Polymorphism of A.mangium superbulk DNA
72
4.6
Fragment analysis and distribution of genotypes
77
in A. mangium superbulk
iii
4.7
Genetic diversity and correlations with environmental and
83
physiological factors
4.8
Fixation indices for A. mangium superbulk populations
88
based on 13 loci
4.
4.9
Genetic differentiation of A. mangium superbulk populations
90
4.10
Genetic relatedness among A. mangium superbulk
populations
93
CHAPTER V: CONCLUSION AND RECOMMENDATION
96
5.1
Conclusion
96
5.2
Recommendations for future studies
98
REFERENCES
99
APPENDICES
128
iv
LIST OF TABLES
Table
Table 2.1
Page
Height and DBH (cm) of Acacia mangium from different provenance
11
regions planted at trial sites inSarawak.
Table 2.2
Expected heterozygosity and multiplex ratios for several marker systems
40
in soybeans
Table 2.3
Utilization of genic simple sequence repeats (SSR) markers for
42
estimation of genetic diversity
Table 3.1
Site descriptions of the A. mangium superbulk populations surveyed in
46
this study
Table 3.2
PCR condition for primer screening
50
Table 3.3
Thermal Cycling Profile for primer screening
51
Table 3.4
Components for 7% polyacrylamide gel preparation
53
Table 3.5
Panels Arrangement for EST-SSR markers used for fragment analysis
56
Table 4.1
Mean Annual Increment for diameter-breast height and height for all
63
three populations
Table 4.2
Mean diameter and height of 12 months old trial of Acacia species
64
Table 4.3
Polymorphic EST-SSR markers for Acacia mangium superbulk
75
v
Table 4.4
Allele sizes for all 16 EST-SSR primers for 3 different populations of
81
Acacia mangium superbulk
Table 4.5
Summary of genetic diversity for 3 populations of Acacia mangium
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superbulk in this study
Table 4.6
Fixation indices (F) of 13 polymorphic loci for three populations of A.
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mangium superbulk.
Table 4.7
Summary of G-statistics (Nei, 1973, 1977) calculated for each 13
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polymorphic loci averaged over 3 populations of A. mangium superbulk
Table 4.8
Genetic similarity matrix for A. mangium superbulk populations using 13
EST-SSR primers.
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94
LIST OF FIGURES
Figure
Page
Figure 2.1 A. mangium superbulk inflorescence
7
Figure 2.2 A four year-old A. mangium superbulk after 50% thinning in
9
Similajau, Sarawak. Source: Umpit (2007).
Figure 2.3 A. mangium superbulk tree
12
Figure 2.4 A. mangium superbulk bark
12
Figure 2.5 A. mangium superbulk seed
13
Figure 2.6 The breeding cycles in forest tree improvement programs. Adapted
21
from White et al. 2007)
Figure 2.7 Tree breeding and seed production processes. (Source: Forest Genetic
25
Council, 2001)
Figure 2.8 Potential applications of biotechnology in tree improvement through
27
the use of vegetative reproduction, gene insertion and genetic
markers. (Source: Forest Genetic Council, 2001)
Figure 2.9 A schematic representation of the development and application of
37
genic simple sequence repeat (SSR) markers. (Source: NCBI,
National Center for Biotechnology Information, Bethesda, MD, USA)
Figure 3.1 Acacia mangium superbulk trial in BTSSSB plantation where the
45
sampling was conducted
Figure 3.2 Acacia mangium superbulk trial at Daiken plantation
vii
45
Figure 3.3 Three different populations of A. mangium superbulk
46
Figure 3.4 Flow chart of methods used to conduct this study in this study.
62
Figure 4.1 Unpurified DNA samples from BTSSSB and Daiken populations
66
Figure 4.2 Purified DNA samples from BTSSSB and Daiken populations.
67
Figure 4.3 DNA bands after running the products on 1.2% agarose gel
70
electrophoresis. 100bp DNA ladder were used in the electrophoresis
Figure 4.4 Polymorphic EST-SSR markers on PAGE gel
73
Figure 4.5 EST-SSR genotyping profile using ABI PRISM 3100 Genetic
78
Analyzer.
(a) Homozygote allele peaks displayed by Genemapper software
(b) Heterozygote allele peaks displayed by Genemapper software
Figure 4.6 An example of artefacts consists of stutter peaks and spurious peaks
79
detection
Figure 4.7 The distribution of allele frequency in 3 populations of A. mangium
85
superbulk on 13 polymorphic loci
Figure 4.8 Relationship between expected heterozygosity and latitude.
viii
86
Figure 4.9
Relationship between expected heterozygosity and longitude.
87
Figure 4.10
Relationship between expected heterozygosity and population mean
87
annual increment of diameter-breast height.
Figure 4.11
Relationship between expected heterozygosity and population mean
88
annual increment of height.
Figure 4.12
Dendogram constructed using UPGMA based on Nei’s distance
method (DA) for thirteen EST-SSR primers
ix
94
LIST OF ABBREVIATIONS
AFLP
amplified fragment length polymorphism
ATCG
nucleotide containing the base adenine, thymine, cytosine
and guanine, respectively
degree Celsius
o
C
DNA
deoxyribonucleic acid
EDTA
ethylene diaminetetraacetic acid
EST
expressed sequence tag
Kb
kilobase
Mg
milligram
ml
millilitre
mM
M
MgCl2
ng
PAGE
PCR
RAPD
SSR
µl
millimolar
molar
magnesium chloride
nanogram
polyacrylamide gel electrophoresis
polymerase chain reaction
random amplified polymorphic DNA
simple sequence repeat
microliter
µM
micro molar
UV
ultra violet
λ
lambda
x
ABSTRACT
The application of biotechnology in forest plantation industry is getting more popular over the
last decades and many researches have been conducted to develop marker-assisted selection
for tree improvement programme in Malaysia. However, to date, marker assisted selection has
not yet widely implemented in Sarawak and conventional breeding would take years to select
the best characteristics in trees. Thus, this study was conducted to look into the effectiveness
in using Expressed Sequenced Tag derived Simple Sequence Repeats (EST-SSRs) marker to
estimate the distribution of genotypes and allele polymorphism of Acacia mangium superbulk
populations in Sarawak;
to determine the relationship or correlation between genetic
variation of these populations to environmental and physiological factors and to determine the
genetic differentiation of the Acacia mangium superbulk populations in comparison with
other Acacia species. In this study, 93 fresh young leaf samples were collected from three
different areas, namely Similajau (Borneo Tree Seeds Seedlings Supply Sdn Bhd. (BTSSSB),
Bintulu (DAIKEN) and Kota Samarahan (UNIMAS arboretum) and total genomic DNA of
the samples were extracted using modified CTAB method. Thirteen EST-SSR markers were
chosen to determine the genetic diversity of A. mangium superbulk and fragment analysis was
conducted by using ABI PRISM® 3100 Genetic Analyzer and Genemapper™ Version 4.0
software (Applied Biosystems, USA). Statistical analysis performed using PowerMarker
software over 13 polymorphic loci revealed that the mean expected heterozygosity or He for
the three populations was 0.432 and polymorphic information content (PIC) value of 0.398,
respectively. Borneo Tree Seeds Seedlings Supply Sdn. Bhd. population showed the highest
level of genetic diversity (N = 40; He = 0.474; PIC = 0.432) compared to Daiken (N = 40; He
= 0.458; PIC = 0.429) and UNIMAS Arboretum N = 13; He = 0.364; PIC = 0.333). Mean FST
xi
ranged from 0.411 to 0.533 which indicates that the surplus of homozygotes generally occur
within these populations. This might be due to inbreeding factor or partial selfing in A.
mangium superbulk populations and Mean GST value of 0.038 further revealed that a low
gene differentiation between all populations (at 3.8%), which signifies that these populations
are genetically similar to each other. UPGMA dendogram was also constructed on the A.
mangium superbulk populations and it revealed that the three populations belong to the same
cluster, with BTSSSB and Daiken populations have the highest similarity coefficient (0.890).
Future studies should look into thebreeding patterns in Acacia plus tree populations; the effect
of silvicultural regimes in forest plantations to the genetic structure of Acacia species; and the
effect of interaction between temporal variations in outcrossing rates with temporal and
spatial variations in seedling selection towards genetic structure of Acacia species.
Keywords: Acacia mangium superbulk, EST-SSR, UPGMA, genetic diversity.
xii
KEPELBAGAIAN GENETIK POKOK Acacia mangium TERBAIK (SUPERBULK)
MENGGUNAKAN KAEDAH PENANDA JUJUKAN TERUNGKAP TERBITAN
MIKROSATELIT (EST-SSR)
ABSTRAK
Penggunaan bioteknologi di dalam industri perladangan hutan menjadi lebih popular pada
10 tahun kebelakangan ini dan banyak penyelidikan telah dijalankan untuk membangunkan
kaedah pemilihan pokok menggunakan penanda genetik di Malaysia. Walaubagaimanapun,
sehingga kini pemilihan berasaskan penanda genetik belum diamalkan dengan meluas di
Sarawak dan pembiakan secara konvensional memerlukan beberapa tahun untuk memilih
pokok yang berkarakter baik. Maka, projek ini bertujuan untuk mengkaji keberkesanan
penanda jujukan terungkap terbitan mikrosatelit (EST-SSR) bagi menganggarkan taburan
genotip dan alel polimorfik pada populasi Acacia mangium superbulk di Sarawak; untuk
menentukan perhubungan dan korelasi di antara variasi genetik pada populasi Acacia
tersebut dengan faktor persekitaran dan fisiologi dan menentukan perbezaan genetik populasi
A. mangium superbulk berbanding spesis pokok Acacia yang lain. Dalam kajian ini, 93 daun
muda diambil dari tiga kawasan berbeza, iaitu Similajau (Borneo Tree Seeds Seedlings
Supply Sdn Bhd atau BTSSSB), Bintulu (DAIKEN) dan Kota Samarahan (Aboretum Unimas)
dan jumlah genomik DNA telah diekstrak menggunakan kaedah CTAB yang telah diubahsuai.
Tiga belas penanda-penanda EST-SSR ialah dipilih untuk menentukan kepelbagaian genetik
A. mangium superbulk dan analisis cebisan dijalankan dengan menggunakan ABI PRISM®
3100 Genetic Analyzer and Genemapper™ Version 4.0 perisian (Applied Biosystems, USA).
Analisis statistic menggunakan perisian PowerMarker ke atas 13 tempat sebenar polimorf
menunjukkan yang purata heterozigot yang dijangka atau He untuk tiga populasi ialah 0.432,
xiii
dengan nilai kandungan maklumat polimorfik atau PIC ialah 0.398. Populasi
BTSSSB
menunjukkan kepelbagaian paras genetic tertinggi (N = 40; He = 0.474; PIC = 0.432)
berbanding Daiken ((N = 40; He = 0.458; PIC = 0.429) dan UNIMAS Arboretum N = 13; He
= 0.364; PIC = 0.333). Nilai min FST adalah di antara 0.411 dan 0.533 di mana ia
menunjukkan lebihan homozigot berlaku dalam populasi ini. Ini mungkin disebabkan oleh
pembiakbakaan dalaman atau separa kacukan sendiri di dalam populasi A. mangium
superbulk dan purata GST pada 0.038 seterusnya menunjukkan perbezaan gen yang rendah
antara semua populasi (pada 3.8%), ini bermakna populasi-populasi ini mengandungi
genetik yang sama. Dendogram UPGMA telah yang dibina berdasarkan analisis terhadap
persamaan di antara ketiga-tiga populasi A. mangium superbulk menunjukkan bahawa
ketiga-tiga populasi tersebut terdiri daripada kluster yang sama, di mana populasi BTSSSB
dan Daiken mempunyai koefisien keserupaan yang tertinggi iaitu (0.89). Cadangan untuk
kajian di masa hadapan adalah untuk mengkaji corak pembiakan pokok yang terbaik untuk
Acacia mangium superbulk; kesan amalan silvikultur di ladang hutan terhadap struktur
genetik spesis Acacia; dan kesan interaksi di antara variasi waktu untuk kadar kacukan
dengan variasi waktu dan ruang di dalam pemilihan anak benih terhadap struktur genetik
spesis Acacia.
Kata kunci: Acacia mangium superbulk, EST-SSR, UPGMA, kepelbagaian genetik
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CHAPTER I
INTRODUCTION
The timber resource of Malaysia has undergone a change in character from colonial times to
the present. Originally, vast virgin forests in most regions of the country permit harvesting of
trees for species and quality in a manner that is considered wasteful by today’s standard.
Traditionally, the management of forest resources in Malaysia has always emphasized on the
selective felling of matured tress and leaving sufficient trees behind for future crop. The
prescription of conventional felling regime worked very well in the 1950's through the 1970's
when harvesting was confined mainly to lowland dipterocarp forests, and the demand from
timber industries was rather limited. Enrichment of indigenous timber species was carried out
only in areas where natural regeneration was found to be inadequate. Then followed a period
of sharp increased in the number of species utilized and an increased in the use of smaller and
lower quality logs. This trend is continued to the present day. As a result, the supply of
today’s log is limited due to the diminishing forest areas. Thus, the wood-based industry is
experiencing shortages in log supply to cope with the ever increasing demand for wood
products. To cope with the demand, the industry now accepts additional species and lower
quality trees that come from a group of trees called Lesser Known Species or Lesser Used
Species.
At the present day, the industry is beginning to see harvest from manmade forest, forest
plantation. Commercial forest plantation started in Malaysia in 1957 with the planting of teak
(Tectona grandis) in the northern states of Peninsular Malaysia (Sahri and Bokhari, 2003).
Several attempts were made to establish commercial forest plantation mainly by the Forestry
1
Departments in Peninsular Malaysia and by government corporations in Sabah through the
1960's and 1970's (FAO, 1994). During this period, plantation development in Peninsular
Malaysia has been shifted toward establishment of fast growing tropical pines (Sahri et al.
2003). However, the planting of these species were stopped in the late 1970’s due to
difficulties in obtaining good quality seeds.
In the early years, the planting of forest species was limited to line planting of some selected
indigenous species, particularly heavy hardwood species such as Neobalanocarpus heimii
(chengal), Intsia palembanica (merbau) and Shorea spp (balau), as well as Palaquium spp.
(nyatoh) (FAO, 1994). Trial planting of indigenous species carried out produced variable
results, with little success in the open planting as compared to line planting under certain
degree of shades. According to FAO (1994), the search for fast growing hardwood species
began in the late 1970's when severe timber shortage was envisaged to occur by the year 1990
for the Peninsula. Several exotics including Acacia mangium, Paraserienthes falcataria and
Gmelina arborea were short-listed as the potential for the establishment of short-rotation crop
to produce general utility timber (FAO, 1994). A. mangium was first planted in Sabah in 1966
as firebreak (Ding et al., 2003). Due to its fast growth, satisfactory wood quality and good site
adaptability, it was trial planted by Sabah Softwoods Bhd (SSB) in 1976. Later, A. mangium
was pioneered to other parts of the country, accounting for about 84% and 65% of the total
timber plantation areas established in Peninsular Malaysia and Sabah, respectively. Eight
species of Acacia: A. aulocarpa, A. auriculiformis, A. crassicarpa, A. farneciana, A.
holosericea, A. mangium, A. podalyriaefolia, and A. richii are currently being planted for
various purposes throughout Malaysia (Sahri et al. 2003). Acacia mangium and A.
auriculiformis are the most widely planted among the eight species.
2
Acacia species are normally diffuse porous species. Acacia species do not have distinctive
growth ring, and the sapwood can be differentiated from the hardwood (Sahri and Bokhari,
2003). The parenchyma is of paratracheal type and partially surrounded the vessel or other
vessel groups (Khairudin, 1994). Some of their parenchyma consists of calcium compounds
crystal (Wu et al., 1988). The fibre of the Acacia species is normally imperforated, axially
elongated and tapered to a painted tip. The fibre length and length width ratio are shorter and
lower than other hardwoods. The variability of the fibre properties might be influenced by the
factors such as species, geographic variations such as provenances and genetics.
This study is focusing on Acacia mangium superbulk, which is the improved generation of
Acacia mangium after going through many years of selected planting in tree improvement
program. The name ‘superbulk’ was given by the Borneo Tree Seed and Seedlings Supply
Sdn, Bhd (BTSSSB) in Bintulu, Sarawak due to the superior characteristics of the tree
compared to the original Acacia mangium. Thus, it is also known as the Acacia mangium plus
tree. According to Juing (2007), visual inspection in BTSSSB plantation confirmed that A.
mangium superbulk continue to display the fastest growth rate and also much better at
confining the site due to its big canopy which provides deeper shading compare to other
species planted at their plantation in Similajau.
Expressed Sequenced Tag derived Simple Sequence Repeats (EST-SSRs) molecular marker
has become the marker of choice for population genetic analyses due to the potential for
analysis of functional diversity (Ayres et al. 1997; Saha et al. 2004) and a higher
transferability across taxa than SSR markers generated from genomic DNA libraries (Ellis
3
and Burke, 2007). They are also relatively easy and inexpensive to develop using publicly
available EST databases and genomic softwares, plus easier to analyse compared to their
anonymous counterparts (Pashley et al. 2006).
To date, marker-assisted selection has not yet widely implemented in Acacia mangium
plantations in Sarawak. As a result, conventional breeding practice in tree improvement
programmes would take years to select the best characteristics in A. mangium trees.
Therefore, this study looked into the effectiveness of EST-SSR molecular marker in assessing
the genetic diversity of Acacia mangium superbulk populations in Sarawak and providing
valuable information that can contribute to the tree improvement and breeding programs of
Acacia plus trees. The specific objectives of this study were:
1. To estimate the distribution of genotypes and allele polymorphism of Acacia
mangium superbulk populations in Sarawak by using EST-SSR molecular
marker.
2. To determine the relationships between genetic variation of these populations
to environmental and physiological factors.
3. To determine the genetic differentiation of the Acacia mangium superbulk
populations in comparison with other Acacia species.
4
The result of this study will provide information for forestry program to establish A. mangium
superbulk plantation through marker assisted selection in the future. It will also help in the
management and conservation of genetic resource of Acacia mangium plus tree (superbulk)
by providing information on the genetic diversity of species and population level.
5
CHAPTER II
LITERATURE REVIEW
2.1 Species review
2.1.1 Acacia mangium.
Acacias are pioneer species that renowned for their robustness and adaptability, which makes
them good plantation species. They demand full light for good development. The generic
names Acacia comes from the Greek word ‘akis’, meaning a point or a barb. The Acacia
mangium was originally described as Mangium montanum Rumph. in Herbarium
Amboinense (1750) but changed to an Acacia in 1806. The specific name is an allusion that
this tree resembled ‘mangge’ (mangroves in Indonesia). Acacia mangium occurs in the Aru
Islands, Irian Jaya, Seram and the Sula Islands of Indonesia, Western Province of Papua New
Guinea and north-eastern Queensland, Australia. It comes from family Fabaceae and is
commonly known as black wattle, brown salwood, mangium in English, maber in Filipino,
mangge hutan in Indonesian, mangium in Malay, krathin-thepha in Thai and zamorano in
Spanish.
Acacia mangium is single-stemmed evergreen tree that can grow up to 25-35 m in height. It
grows in the humid, tropical lowland zones and tolerates pH levels between 4.5 and 6.5. The
reproductive biology of this species involves precocious flowering which produces seed that
can be harvested 24 months after planting. The flowers of A. mangium occur as inflorescences
consisting of many flowers borne on loose, pendulus spikes.
The flower is regular in
symmetry and consists of five sepals, five petals, numerous stamens and gynoecium. In
6
general, the flowers are hermaphroditic, with some occurrence of staminate flowers. Pollen
occurs in polyads of 16 pollen grains. A. mangium generally outcrosses and pollinators are
commonly insects which Trigona and Apis spp. being the active pollen vectors. However, it
tends to self-pollinated due to the fact that its flowers are monogamus and partially selfcompatible (Awang and Taylor, 1993). These characteristics are shown in Figure 2.1 below.
Figure 2.1. A. mangium superbulk inflorescence.
Acacia mangium is versatile in its growth and also tolerant to various site conditions. Its
ability to adapt to different planting objectives also makes it the most popular species for
plantation in Malaysia. This species can grow reasonably well in difficult sites with pH as low
as 4.5, even on degraded forest sites and denuded catchments and grasslands dominated by
Imperata cylindrica, as long as the rainfall and temperature are favourable.
7