DNA Genotyping of Duabanga moluccana (sawih) using ISSR Markers

Supervisor: Dr Ho Wei Seng Prepared by: Cinderella Sio (23332)

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Declaration by Candidate

I hereby declare that this thesis is my own work and effort and that it has not been submitted

anywhere for any reward. Where other sources of information have been used, they have been

acknowledged.

______________________________________

Cinderella anak Sio (23332)

Date: June 18, 2012

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

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Acknowledgement

I would like to extend my greatest gratitude to my supervisor, Dr. Ho Wei Seng for his guidance

and dedication throughout the project. Besides, I would like to thank our postgraduate students,

Ms. Tiong Sing Yiing and Ms. Lai Pei Sing for their advice and guidance. I also want to thank

our lab assistant, Mdm. Kamaliawati for her assistance. Thanks also to my labmates Natalie Gali,

Flora Lapik, Valeria S. Kabalon, Nurul Haniza and Nurfarah Natasha. Last but not least, thanks

to my family for their supports.

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Table of Contents

Declaration i

Acknowledgement ii

Table of Contents iii

List of Abbreviations v

List of Tables vii

List of Figures viii

Abstract ix

1.0 Introduction 1

2.0 Literature Review

2.1 Duabanga moluccana 3

2.2 DNA Genotyping 4

2.3 Inter Simple Sequence Repeats (ISSR) Marker 5

2.4 Polymerase Chain Reaction (PCR) 6

2.5 POPGENE Version 1.31 7

3.0 Materials and Methods

3.1 Plant Material 8

3.2 DNA Extraction 8

3.3 PCR Optimization 9

3.3 ISSR-PCR 9

3.5 ISSR Analysis 10

4.0 Result 12

4.1 Data Scoring 12

4.2 ISSR-PCR Analysis 13

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4.2.1 Mukah 14

4.2.2 Niah 16

4.2.3 Tatau 18

4.3 Estimation of sizes of ISSR Fragment 20

4.4 Data Analysis 21

4.4.1 Polymorphisms detected by ISSR 21

4.4.2 Genetic diversity in three populations of D. moluccana 21

4.4.3 Genetic structure in three populations of D. moluccana 22

4.4.4 Dendrograms obtained with ISSR markers 23

5.0 Result 27

5.1 Application of ISSR in genetic diversity study 27

5.2 Genetic diversity of D. moluccana 28

5.3 Genetic structure of D. moluccana 29

6.0 Conclusion 31

References 32

Appendix

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List of Abbreviations

μl microliter

μM micromolar

mM millimolar

m meter

mm/year millimetre per year

mm millimetre

ng nanogram

cm centimetre

min minute

DNA deoxyribonucleic acid

ISSR inter simple sequence repeats

oC degree Celcius

dpi dots per inch

PCR polymerase chain reaction

UPGMA unweighted pair group method with the arithmetic averaging algorithm

UV ultraviolet

PPL percentage polymorphic loci

H Nei’s gene diversity

I Shannon’s Information Index

Nm Gene flow estimation

Gst Genetic differentiation coefficient

Na observed number of alleles

Ne effective number of alleles

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Ht total gene diversity

Hs gene diversity within populations

NTSYS-pc Numerical Taxonomy Multivariate Analysis System

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List of Tables

1 Location and sampling size of the three cultivation populations of

D. moluccana

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2 Tm and range of Ta for selected ISSR primers 8

3 PCR ingredients for 25 μl of reaction mixture 9

4 Thermal cycling profile for PCR reaction 9

5 List of selected ISSR primers and their Ta (oC) 10

6 ISSR primers and their respective loci 12

7 DNA fragment size at each locus of two ISSR primers 20

8 Genetic variation in three populations of D. moluccana 22

9 Nei's genetic distance below diagonal and genetic identity above

diagonal

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viii

List of Figures

1 Pictures of D. moluccana 4

2 Three populations of D. moluccana (Mukah, Niah and Tatau) 8

3 ISSR fingerprints obtained on 2% agarose gel for 30 D.

moluccana samples from Mukah with primer CAG(CA)8.

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4 ISSR fingerprints obtained on 2% agarose gel for 30 D.

moluccana samples from Mukah with primer (ACC)6G.

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5 ISSR fingerprints obtained on 2% agarose gel for 30 D.

moluccana samples from Niah with primer CAG(CA)8.

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6 ISSR fingerprints obtained on 2% agarose gel for 30 D.

moluccana samples from Niah with primer (ACC)6G.

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7 ISSR fingerprints obtained on 2% agarose gel for 30 D.

moluccana samples from Tatau with primer CAG(CA)8.

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8 ISSR fingerprints obtained on 2% agarose gel for 30 D.

moluccana samples from Tatau with primer (ACC)6G.

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9 UPGMA dendogram showing relationship among thirty D.

moluccana samples within Mukah population

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10 UPGMA dendogram showing relationship among thirty D.

moluccana samples within Niah population

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11 UPGMA dendogram showing relationship among thirty D.

moluccana samples within Tatau population

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12 UPGMA dendogram showing relationship among ninety D.

moluccana samples among Mukah, Niah and Tatau

populations

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DNA Genotyping of D. moluccana Using ISSR Marker

Cinderella anak Sio

Program of Resource Biotechnology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

The genetic diversity of Duabanga moluccana in 3 populations was characterized using inter-simple sequence

repeats (ISSR) technique. Two ISSR primers: CAG(CA)8 and (ACC)6G were screened, of which both are

polymorphic and provide informative patterns to determine genetic relationships. ISSR amplification was conducted

on 90 samples from 3 populations, and all 40 loci detected were polymorphic. The percentage of PPL at Mukah,

Niah and Tatau were 82.5%, 90.00% and 62.50% respectively, with an average of 78.33%. Nei’s gene diversity (h)

and Shannon’s Information Index (I) of D. moluccana at the species level were 0.2206 and 0.3569, respectively. The

genetic differentiation coefficient (Gst) among populations was 0.1234. The gene flow (Nm) among populations was

1.7759, indicating that gene flow was high among populations of D. moluccana.

Keywords D. moluccana, ISSR, Nei’s gene diversity, Shannon’s Information Index, polymorphic, gene flow.

ABSTRAK

Kepelbagaian genetik Duabanga moluccana yang terdapat di 3 populasi: Mukah, Niah dan Tatau dikategorikan

menggunakan teknik ISSR. Dua primer ISSR iaitu CAG(CA)8 dan (ACC)6G telah disaring. Kedua-dua primer

adalah polimorfik dan memberikan corak yang informative bagi mengenalpasti perkaitan genetik. Amplifikasi ISSR

telah dijalankan ke atas 90 sampel dari 3 populasi tersebut. Kesemua 40 lokus yang telah dikenalpasti adalah

polimorfik. Peratusan polimorfik pada populasi Mukah, Niah dan Tatau adalah masing-masing 82.5%, 90.0% dan

62.5%. Purata bagi peratusan tersebut adalah 78.33%. Kepelbagaian gen Nei (h) dan Indeks Informasi Shannon (I)

pada peringkat spesis adalah masing-masing 0.2206 dan 0.3569. Coefficient perbezaan genetik (Gst) antara

populasi adalah 0.1234. Aliran gen (Nm) dalam kalangan populasi adalah 1.7759, menunjukkan bahawa aliran gen

adalah tinggi antara populasi.

Kata kunci D. moluccana, ISSR, kepelbagaian gen Nei, Indeks Informasi Shannon, polimorfik, aliran gen.

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1.0 Introduction

Sarawak is blessed with rainforest which has become the habitat of various species including the

indigenous plant species such as sawih (Duabanga moluccana), kelampayan (Neolamarckia

cadamba), meranti sarang punai (Shorea parvifolia), and kapur bukit (Dryobalanops spp.).

These trees species have high commercial and medicinal values. Hence, there is an urgent need

for us to study their genetic variability. Wickneswari and Ho (2003) stated that a complete

understanding in the genetic information of certain plant species is fundamental for the

advancement in its breeding program.

Since less study of D. moluccana had been done, a further and deeper study on its genetic

diversity is crucial to improve its documentation and subsequently aids in the improvement of its

future varieties for commercialization purposes. Furthermore, the study is tremendously

important for varietal identification, classification, proper purity maintenance, conservation and

plant breeding advancement. D. moluccana from Sonneratiaceae family can be found in Java,

Lesser Sunda Islands, Borneo, Philippines, Celebes, Moluccas, and New Guinea. It is selected

for this study due to its fast growth rate and good tree form which is ideal for plywood and

furniture manufacturing.

Researchers in various fields of plant improvement has utilized DNA markers for various

functions such as cultivar identification (Kantety et al., 1995; Pejic et al., 1998; Fang et al.,

1997), parentage analysis, evaluation of genetic diversity (Al-Huqail and Al-Saad, 2010), and the

construction of genetic linkage maps (Godwin et al., 1997, Yamamoto et al., 2006) and complete

genome maps (Simon and Muehlbauer, 1997). According to Carson et al. (1996), managing tree

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advancement programs is more efficient once marker system is available and at the same time

can help to minimize the mislabeling errors in forest tree species.

Inter-simple sequence repeat (ISSR) marker was utilized in this study. ISSR is a simple,

cost-efficient, robust, multilocus marker method which is extremely useful in determining

genetic variability (Gupta et al., 1994, Zietkiewicz et al., 1994, Reddy et al., 2002). ISSR has

been successfully applied for the determination of genetic diversity, cultivar identification and

phylogenetic studies (Kantety et al., 1995; Pejic et al., 1998; Prevost and Wilkinson, 1999; Fang

et al., 1997; Nagaoka and Ogihara, 1997; Metais et al., 2000; Martin and Sanchez-Yelamo,

2000; Bhagyawant and Srivastava, 2008; Al-Huqail and Al-Saad, 2010).

The objective of this study was to determine the genetic diversity of D. moluccana within

and among three populations (Mukah, Niah and Tatau) using ISSR marker.

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2.0 Literature Review

2.1 Duabanga moluccana

Duabanga moluccana is a member of the family Sonneratiaceae and genus Duabanga.

Sometimes it is included in the family Lythraceae. It is distributed in Java, Lesser Sunda Islands,

Borneo, Philippines, Celebes, Moluccas, and New Guinea. The taxonomical classification of D.

moluccana is shown as below:

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Myrtales

Family : Sonneratiaceae

Genus : Duabanga

Species : Duabanga moluccana

D. moluccana is the indigenous species in Sarawak. It is a fast-growing plant and has a

short life cycle of 15 years. The vernacular names of D. moluccana are sawih, benung kasung,

binuang, binuang laki, magas, mas, sawak, sawi and sawik. It grows in open forests at an altitude

of 300-1200 m dpi, with an average rainfall 2000-3500 mm/year. The optimum temperature for

the growth of D. moluccana is at the range of 27-32 oC during the day and 15-24

oC at night. The

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tree can grow up to 45 m, with trunk diameters of 70-100 cm. The trunk which is rod straight and

round is ideal for plywood and furniture manufacturing.

Figure 1 Pictures of D. moluccana

2.2 DNA Genotyping

DNA genotyping which is also known as DNA fingerprinting or DNA profiling can distinguish

plants from different families, genera, species, cultivars and even siblings’ plants with the aid of

DNA markers (Hong, 2007). DNA genotyping reflects plant variability directly at genetic level

with reliable and enormous data set for reproducible estimation of genetic diversity for

investigation of phylogenetic relationship of plant species compared to morphological traits

(Wang et. al, 2009).

The commonly used DNA markers are inter-simple sequence repeats (ISSR), simple

sequence repeats (SSR), fluorescent inter-simple sequence repeats (FISSR), restriction fragment

length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified

fragment length polymorphism (AFLP), single nucleotide polymorphism (SNP), and sequence-

related amplified polymorphisms (SRAP).

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2.3 Inter-Simple Sequence Repeat (ISSR) Marker

Inter-simple sequence repeats (ISSR) marker is a useful tool for the analysis of genetic diversity

in various species (Gupta and Varshney, 2000). It has been established as a reliable, rapid,

simple, cost effective, easy to generate, and versatile set of marker that does not oblige previous

knowledge of the genome sequence to generate DNA markers (Zietkiewicz et al., 1994, Gupta et

al., 1994, Bornet and Branchard, 2001 and Bornet et al., 2002). According to Sheppard and

Smith (2000), the marker is easily generated by using minimal equipment and are hypervariable,

thus yielding a reasonable cost to the researcher.

Inter-simple sequence repeats (ISSRs) technique involves polymerase chain reaction

(PCR) to amplify DNA fragments between two simple sequence repeats (SSRs) with inverse

orientations using primers with a single SSR motifs anchored at the 30- or 50-end by a few

nucleotides (Zietkiewicz et al., 1994). Peng et al. (2006) stated that ISSR markers are usually

highly polymorphic in plant populations, providing a genotyping system with features of

consistency, reliability and codominancy. Furthermore, ISSR markers are present in both nuclear

and organellar genomes (Peng et al., 2006).

The study done by Nagaraju et al. (2002) has proved that ISSR method is an efficient

molecular marker to reveal genetic relationship in traditional and evolved Basmati and semi

dwarf non-Basmati rice varieties. The genetic diversity of Cicer arietinum L. (Bhagyawant and

Srivastava, 2008), barley (Matus and Hayes, 2002; Hou et al., 2005) and Nigella sativa L. (Al-

Huqail and Al-Saad, 2010) also has been successfully determined using ISSR marker. Besides, it

has been used for cultivar identification in maize (Kantety et al., 1995; Pejic et al., 1998),

potatoes (Prevost and Wilkinson, 1999), trifoliate orange (Fang et al., 1997), wheat (Nagaoka

and Ogihara, 1997), bean (Metais et al., 2000), and Diplotaxis (Martin and Sanchez-Yelamo,

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2000). In recent study, ISSR polymorphism was determined in orchid Cymbidium goeringii

cultivars (Wang et al., 2009).

2.4 Polymerase Chain Reaction (PCR)

Campbell and Reece (2002) defined PCR as an in vitro technique to amplify DNA quickly by

incubation with special primers, DNA polymerase molecules, and other PCR ingredients. PCR is

an important technique in DNA studies which enzymatically amplified DNA fragments and

allows the specific DNA fragment to be exponentially amplified (Karp et al., 2002; Elliot &

Elliot, 2005). PCR which is also known as molecular photocopying is a fast and inexpensive

method to amplify small fragments of DNA. It consists of three major steps which are

denaturation, annealing and extension, which are carried out in thermocycler or automated

cycler.

Denaturation step requires temperature between 92oC to 99

oC. During this step, the

hydrogen bonding between complementary bases is disrupted by heat. Hence, single-stranded

DNA is formed. This step is important to ensure the DNA template is accessible for the binding

of primers. The required temperature for annealing step is depending on the length and

nucleotide sequence of the primers. During this step, the primers will recognize and bind to the

complementary sequences on DNA template. The extension step is done at 72oC, which is

suitable for DNA polymerase. This is the step where the dNTPs or nucleotides are sequentially

added.

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2.5 POPGENE Version 1.31

POPGENE is a user-friendly Microsoft® Window-based computer package for the analysis of

genetic variation among and within natural populations using co-dominant and dominant

markers and quantitative traits (Yeh et al., 1999). It is designed specifically for the analysis of

co-dominant and dominant markers using haploid and diploid data. It performs most types of

data analysis encountered in population genetics and related fields.

It can be used to compute summary statistics (e.g., allele frequency, gene diversity,

genetic distance, F-statistics, multilocus structure, etc.) for (1) single-locus, single populations;

(2) single-locus, multiple populations; (3) multilocus, single populations and (4) multilocus,

multiple populations (Yeh et al., 1999). POPGENE has been used in the study of genetic

diversity of the mangrove Kandelia obovata in China (Shao-Bo Chen et al., 2010), Centaurea

nivea in Turkey (Sozen and Ozaydin, 2009) and Sarotherodon galilaeus (Saad, 2009).

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3.0 Materials and Methods

3.1 Plant materials

In this study, a total of 90 samples were obtained from three cultivated population. The details on

sample collection are given in Table 1.

Table 1 Location and sampling size of the three cultivation populations of D. moluccana

Population Location No. of samples

Mukah Mukah, Sarawak 30

Niah Niah, Miri, Sarawak 30

Tatau Tatau, Bintulu, Sarawak 30

Figure 2 Three populations of D. moluccana (Mukah, Niah and Tatau)

3.2 DNA extraction

The DNA was extracted using cetyltrimethylammonium bromide (CTAB) method (Doyle and

Doyle, 1990). The DNA was quantified using spectrophotometer and stored at -20 oC until used.

The samples used in this study were isolated by previous student in 2010.

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3.3 PCR Optimization

PCR optimization was performed for each primer using Mastercycler®

Gradient PCR

(Eppendorf, Germany) to determine the optimum annealing temperature (Ta). The range of Ta

was estimated based on the melting temperature (Tm) of each primer.

Table 2 Tm and range of Ta for selected ISSR primers

ISSR Primer Tm (oC) Ta (

oC)

(ACC)6G 60.0 55.0, 56.5, 58.0, 59.5, 61.0, 62.5

CAG(CA)8 58.0 51.0, 52.5, 54.0, 55.5, 57.0, 58.5

3.3 ISSR-PCR

Two selected ISSR primers were used for PCR [CAG(CA)8 and (ACC)6G]. Each 25 μl

amplification reaction consisted of 10 X PCR buffer, 0.2 mM of each dNTPs (Promega), 2.0 mM

of MgCl2, 0.5 units Taq polymerase (Invitrogen, USA), template DNA, and 10.0 pmol of

primers. PCR amplification was performed under the following conditions: initial denaturation of

2.0 min at 95oC, followed by 40 cycles of 0.5 min denaturation at 94

oC, 40 cycles of 0.5 min of

annealing at 44-61 oC, 40 cycles of 1.0 min of extension at 72

oC, and a final extension at 72

oC

for 10.0 min. From the 25 μl obtained after each amplification, aliquots of 8 μl were separated in

2% agarose (Promega) gel, in 1X TAE buffer at 70 V for 2 hours, and stained with ethidium

bromide (0.5 μg/ml). The gels were visualized and photographed under UV light. A 100 bp DNA

ladder (Invitrogen, USA) was used to determine the molecular size of the fragments.

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Table 3 PCR ingredients for 25 μl of reaction mixture

Reagent Concentration Volume (μl)

10 X PCR buffer 1 X 2.50

dNTPs 0.2 mM 2.50

MgCl2 2.0 mM 1.25

Primer 2.5 pmol 4.00

Taq polymerase 0.5 unit 1.00

Template DNA - 1.00

Distilled water - 12.75

Total volume 25.0

Table 4 Thermal cycling profile for PCR reaction

Parameter Temperature (oC) Time (min) No. of cycles

Initial denaturation 95 2.0

Denaturation 94 0.5

Annealing 44-65 0.5 40

Extension 72 1.0

Final extension 72 10.0

Table 5 List of selected ISSR primers and their Ta (oC)

Primer Sequence Ta (oC)

CAG(CA)8 5’-CAGCACACACACACACACA-3’ 58.0

(ACC)6G 5’-ACCACCACCACCACCACCG-3’ 52.0

3.5 ISSR Analysis

ISSR was the dominant marker, and all bands amplified by the same primer pair with identical

electrophoretic mobility were homologous. ISSR bands were used to assign loci for each primer

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and scored for presence (1) or absence (0). The binary data was done using Microsoft Excel 2007

before transferred into NTedit 1.07c program. Assuming Hardy–Weinberg equilibrium, the

binary data matrix was input into POPGENE (Yeh et al., 1999).

The following indices were used to quantify the amount of genetic diversity within and

among the populations examined: The percentage of polymorphic loci (PPL and Shannon’s

Information index (Lewontin, 1972). Genetic differentiation among the populations was

estimated by Nei’s gene diversity statistics (Nei, 1973). The total gene diversity (Ht), the gene

diversity within populations (Hs) and the genetic differentiation coefficient (Gst= (Ht-Hs)/ Ht)

were also calculated. The level of gene flow among these populations was estimated as Nm=

(1/Gst -1)/4 (Slatkin and Barton, 1989).

The binary data produced was also used to calculate the genetic similarity matrices of

each sample. The numerical and taxonomical analyses were conducted using Numerical

Taxonomy Multivariate Analysis System (NTSYS-pc) software version 2.02 (Exeter Software,

Setauket, N.Y.). Dendrograms showing the phylogenetic relationships of D. moluccana within

each population and between the three populations were constructed with unweighted pair group

method based on the arithmetic averaging algorithm (UPGMA) using the SAHN subroutine and

Tree plot of NTSYS-pc.

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4.0 Results

4.1 Data scoring

The presence of band was scored as (1) and absence of band as (0). In this study, 40 loci were

determined from two ISSR primers: CAG(CA)8 and (ACC)6G. 25 and 15 loci were identified

from CAG(CA)8 and (ACC)6G respectively. Each locus was named based on the primer’s name.

Table 6 ISSR primers and their respective loci

Primer Locus name

CAG01

CAG02

CAG03

CAG04

CAG05

CAG06

CAG07

CAG08

CAG09

CAG(CA)8 CAG10

CAG11

CAG12

CAG13

CAG14

CAG15

CAG16

CAG17

CAG18

CAG19

CAG20

CAG21

CAG22

CAG23

CAG24

CAG25

ACC01

13

ACC02

ACC03

ACC04

ACC05

ACC06

ACC07

(ACC)6G ACC08

ACC09

ACC10

ACC11

ACC12

ACC13

ACC14

ACC15

4.2 ISSR-PCR Analysis

ISSR-PCR was carried out by screening 90 samples of D. moluccana from Mukah, Niah and

Tatau using two ISSR primers: CAG(CA)8 and (ACC)6G. The results of the ISSR-PCR are

shown in Figures 4-9. Multiple bands were successfully obtained for all samples except Mukah’s

sample M15. Meanwhile, Tatau’s sample T14 and Niah’s sample N22 failed to produce any band

when amplified using CAG(CA)8 and (ACC)6G primers respectively. At least two bands had

been amplified using the selected primers. Both primers showed different DNA profiling where

CAG(CA)8 primer produced most bands.

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4.2.1 Mukah

Figure 3: ISSR fingerprints obtained on 2% agarose gel for 30 D. moluccana samples from Mukah with primer

CAG(CA)8. Lane M: 100 bp DNA Ladder (Invitrogen®), Lane 1-10: M1 to M10, Lane 11-20: M11 to M20 and Lane

21-30: M21 to M30.

L 1 2 3 4 5 6 7 8 9 10

L 11 12 13 14 15 16 17 18 19 20

L 21 22 23 24 25 26 27 28 29 30

100

1500

2702

600

100

600

1500

2702

100

2702

600

1500