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

i

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 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

3. CHAPTER III: MATERIALS AND METHODS 44

3.1 Sampling of plant materials 44

iii

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

4. 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

in A. mangium superbulk

77

iv

4.7 Genetic diversity and correlations with environmental and

physiological factors

83

4.8 Fixation indices for A. mangium superbulk populations

based on 13 loci

88

4.9 Genetic differentiation of A. mangium superbulk populations 90

4.10 Genetic relatedness among A. mangium superbulk

populations

93

4. CHAPTER V: CONCLUSION AND RECOMMENDATION 96

5.1 Conclusion 96

5.2 Recommendations for future studies 98

REFERENCES 99

APPENDICES 128

v

LIST OF TABLES

Table Page

Table 2.1 Height and DBH (cm) of Acacia mangium from different provenance

regions planted at trial sites inSarawak.

11

Table 2.2 Expected heterozygosity and multiplex ratios for several marker systems

in soybeans

40

Table 2.3 Utilization of genic simple sequence repeats (SSR) markers for

estimation of genetic diversity

42

Table 3.1 Site descriptions of the A. mangium superbulk populations surveyed in

this study

46

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

three populations

63

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

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Table 4.4 Allele sizes for all 16 EST-SSR primers for 3 different populations of

Acacia mangium superbulk

81

Table 4.5 Summary of genetic diversity for 3 populations of Acacia mangium

superbulk in this study

84

Table 4.6 Fixation indices (F) of 13 polymorphic loci for three populations of A.

mangium superbulk.

89

Table 4.7 Summary of G-statistics (Nei, 1973, 1977) calculated for each 13

polymorphic loci averaged over 3 populations of A. mangium superbulk

92

Table 4.8 Genetic similarity matrix for A. mangium superbulk populations using 13

EST-SSR primers.

94

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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

Similajau, Sarawak. Source: Umpit (2007).

9

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

from White et al. 2007)

21

Figure 2.7 Tree breeding and seed production processes. (Source: Forest Genetic

Council, 2001)

25

Figure 2.8 Potential applications of biotechnology in tree improvement through

the use of vegetative reproduction, gene insertion and genetic

markers. (Source: Forest Genetic Council, 2001)

27

Figure 2.9 A schematic representation of the development and application of

genic simple sequence repeat (SSR) markers. (Source: NCBI,

National Center for Biotechnology Information, Bethesda, MD, USA)

37

Figure 3.1 Acacia mangium superbulk trial in BTSSSB plantation where the

sampling was conducted

45

Figure 3.2 Acacia mangium superbulk trial at Daiken plantation 45

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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

electrophoresis. 100bp DNA ladder were used in the electrophoresis

70

Figure 4.4 Polymorphic EST-SSR markers on PAGE gel

73

Figure 4.5 EST-SSR genotyping profile using ABI PRISM 3100 Genetic

Analyzer.

(a) Homozygote allele peaks displayed by Genemapper software

(b) Heterozygote allele peaks displayed by Genemapper software

78

Figure 4.6 An example of artefacts consists of stutter peaks and spurious peaks

detection

79

Figure 4.7 The distribution of allele frequency in 3 populations of A. mangium

superbulk on 13 polymorphic loci

85

Figure 4.8 Relationship between expected heterozygosity and latitude.

86

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Figure 4.9 Relationship between expected heterozygosity and longitude.

87

Figure 4.10 Relationship between expected heterozygosity and population mean

annual increment of diameter-breast height.

87

Figure 4.11 Relationship between expected heterozygosity and population mean

annual increment of height.

88

Figure 4.12 Dendogram constructed using UPGMA based on Nei’s distance

method (DA) for thirteen EST-SSR primers

94

x

LIST OF ABBREVIATIONS

AFLP amplified fragment length polymorphism

ATCG nucleotide containing the base adenine, thymine, cytosine

and guanine, respectively oC degree Celsius

DNA deoxyribonucleic acid

EDTA ethylene diaminetetraacetic acid

EST expressed sequence tag

Kb kilobase

Mg milligram

ml millilitre

mM millimolar

M molar

MgCl2 magnesium chloride

ng nanogram

PAGE polyacrylamide gel electrophoresis

PCR polymerase chain reaction

RAPD random amplified polymorphic DNA

SSR simple sequence repeat

µl microliter

µM micro molar

UV ultra violet

λ lambda

xi

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

xii

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.

xiii

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,

xiv

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

1

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

2

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.

3

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

4

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.

5

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.

6

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

7

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 self-

compatible (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.