STUDY OF MOLECULAR AND GENETIC DIVERSITY

OF JAVA TEA (Orthosiphon stamineus Benth.) AS A

BASIS FOR PLANT IMPROVEMENT

NURUL AIN BINTI MAZNI

MASTER OF SCIENCE

2015

Study of Molecular and Genetic Diversity

of Java Tea (Orthosiphon stamineus Benth.) as A

Basis for Plant Improvement

By

Nurul Ain binti Mazni

A thesis submitted in fulfillment of the requirements for the degree of

Master of Science

Faculty of Agro Based Industry

UNIVERSITI MALAYSIA KELANTAN

2015

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original

research and has not been submitted for a higher degree to any other University or

Institution.

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Dated from until

CONFIDENTIAL (Contains confidential information under the Official

Secret Act 1972)*

RESTRICTED (Contains restricted information as specified by the

organization where research was done)*

I acknowledge that University Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of University Malaysia Kelantan.

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IC/PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

ACKNOWLEDGEMENT

Foremost, I would like to express my sincere gratitude to my supervisor Dr. Dwi

Susanto for the continuous support of my Master study and research, for his patience,

motivation, enthusiasm, and immense knowledge. His guidance helped me in all the

time of research and writing of this thesis. I could not have imagined having a better

advisor and mentor for my Master study in Plant Biotechnology at UMK.

Besides my supervisor, I would like to thank the rest of my field supervisor, Dr. Siti

Salwana for guiding me in my research and let me do my research in Laboratory

Genetics in Forest Research Institute Malaysia (FRIM), Dr. Arifullah and Dr. Fatimah

for their encouragement, insightful comments, and hard questions and guiding me in my

research.

I thank my fellow lab mates in University Malaysia Kelantan: Norzielawati, Aishatul

Izzah, Zeti Ermiena, Nurul’Ain, Azwani, Siti Hajar, Qayyum Nadia, Zalina, for the

stimulating discussions, for the sleepless nights we were working together before

deadlines, and for all the fun we have had in the last two years.

Last but not the least; I would like to thank my family: my parents Mazni Che Yusoff

and Liley Abdullah, for giving birth to me at the first place and supporting me

spiritually throughout my life.

TABLE OF CONTENT

NO.

PAGE

THESIS DECLARATION i

ACKNOWLEDGMENTS ii

TABLE OF CONTENTS iii

LIST OF FIGURES vi

LIST OF TABLES viii

ABSTRAK ix

ABSTRACT x

CHAPTER 1

INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 4

2.1. Botany and Taxonomy of Orthosiphon stamineus Benth. 4

2.2. The Use of Orthosiphon 6

2.3. Genetic Diversity 7

2.3.1. Cluster Analysis 8

2.4. Polymerase Chain Reaction (PCR) 9

2.4.1. Random Amplified Polymorphic DNA (RAPD) 12

2.4.2. Restriction Fragment Length Polymorphism (RFLP) 13

2.4.3. Amplified Fragments Length Polymorphism (AFLP) 15

2.4.4. Real-Time PCR (RT-PCR) 16

2.5. Plant Improvement 17

2.5.1 Molecular Breeding 19

2.5.1.1 Genetic Markers 19

CHAPTER 3 MATERIALS AND METHODS 21

3.1. Plant Materials 21

3.2. Morphological Attributes (Variables) 22

3.3. DNA Extraction 28

3.3.1. CTAB Method 28

3.4. DNA Quality and Quantity Confirmation 30

3.4.1. Agarose Gel Electrophoresis 30

3.4.2. Reading Optimal Density (OD) 30

3.5. DNA Dilution 31

3.6. Optimization of PCR Reaction 31

3.7. Primer Screening 33

3.8. Phylogenetic Trees 34

CHAPTER 4 RESULT AND DISCUSSION 35

4.1. Taxonomic Characteristics 35

4.1.1. Plant Height 35

4.1.2.

Leaf Width

38

4.1.3.

Leaf Length

39

4.1.4.

Leaf Area

40

4.1.5.

Petiole Length

42

4.1.6.

Internode Length

43

4.2. Morphological Characteristics 45

4.2.1. Leaves Morphological Characteristics 45

4.2.2. Stem Morphological Characteristics 46

4.2.3. Root Characteristics 46

4.2.4. Flower Morphological Characteristics 49

4.3. Phylogenetic Tree Based on Morphological Characteristic 50

4.4. DNA Extraction 53

4.5. Optimization of PCR Conditions 57

4.6. Optimization of primer annealing temperature, Ta. 60

4.7. Primer Screening 64

4.8. RAPD Profile Analyses 67

4.9. Phylogenetic Tree Based on Molecular Data 71

CHAPTER 5 CONCLUSION 72

5.1 Research Findings 73

5.2 Future Work 76

REFERENCES 78

APPENDIX A 84

APPENDIX B 85

APPENDIX C 86

APPENDIX D 87

APPENDIX E 88

APPENDIX F 89

APPENDIX G 90

APPENDIX H 91

APPENDIX I 93

LIST OF FIGURES

NO

PAGE

2.1. White O. stamineus (Left) and purple O. stamineus (Right) 5

2.2. Leaf morphology of purple variety (leaf), ovate shape with light yellowish

spots on the surface; white variety (right), rombhoid shape and green

veination. Picture label with A is the front leaf and B is the rear leaf

(Chan and Loo, 2006). 5

2.3. Polymerase Chain Reaction 11

3.1. Collection of Orthosiphon stamineus Benth. in Agropark, Jeli. 21

3.2. Leaf Arrangement on Stem (Whiting et al., 2012) 23

3.3. Leaf Arrangement on Petiole (Whiting et al., 2012) 24

3.4. Overall Leaf Shapes (Whiting et al., 2012) 24

3.5. Shapes of Leaf Tip (Whiting et al., 2012) 25

3.6. Shapes of Leaf Base (Whiting et al., 2012) 25

3.7. Types of Leaf Margins (Whiting et al., 2012) 26

3.8. Types of Leaf Venation (Whiting et al., 2012) 27

3.9. Types of Inflorescence (Whiting et al., 2012) 27

3.10. Flowchart of the DNA extraction 29

4.1. Plant Height of 28 accessions of O. stamineus. Lane 1-11 are purple

variety while Lane 12-28 are from the white variety. Samples were taken

from various location as stated in Table 4.1. 37

4.2. Means of Leaf Width of 28 accessions of O. stamineus. Lane 1-11 was

purple variety and Lane 12-28 was white variety. Samples were taken from various location as stated in Table 4.1. 38

4.3. Means of Leaf Length of 28 accessions of O. stamineus. Lane 1-11 was

Purple variety and Lane 12-28 was white variety. Samples were taken

from various location as stated in Table 4.1. 40

4.4. Means of Leaf Area of 28 accessions of O. stamineus. Lane 1-11 was

purple variety and Lane 12-28 was white variety. Samples were taken from

various location as stated in Table 4.1. 41

4.5. Means of Petiole Length of 28 accessions of O. stamineus. Lane 1-11

was purple variety and Lane 12-28 was white variety. Samples were

taken from various location as stated in Table 4.1. 42

4.6. Means of Internode Length of 28 accessions of O. stamineus. Lane 1-11

was purple variety and Lane 12-28 was white variety. Samples were taken

from various location as stated in Table 4.1. 44

4.7 The Inflorescence of java tea. 49

4.8. Phylogenetic tree based on qualitative data of morphological

characteristics. 52

4.9. Product of DNA extraction for 28 accessions of O. stamineus.

M1= 5 µg of DNA concentration, M2 = 10 µg of DNA concentration,

M3 = 25 µg of DNA concentration, M4 = 50 µg of DNA concentration,

Samples were taken from various location as stated in Table 4.1.

Agarose gel electrophoresis was run at 90V for 1 hour. 54

4.10. Purification for 28 samples extracted DNA of O. stamineus.

M1= 5 µg of DNA concentration, M2 = 10 µg of DNA concentration,

M3 = 25 µg of DNA concentration, M4 = 50 µg of DNA concentration,

Lane 1-11 = White variety and Lane 12-28 = Purple variety. Samples were

taken from various location as stated in Table 4.1 56

4.11. Gel electrophoresis of diluted DNA. Lane 1-11 = White variety and

Lane 12-28 = Purple variety. Samples were taken from various location

as stated in Table 4.1. 57

4.12. PCR products with 2.5µl of MgCl2 (lane 1-6), 3.0µl MgCl2

(lane 7-12) and 5.0µl (lane 13-18) using primer J-01 with Ta, 37.2°C on

1% agarose gel run under 90V for 1 hour. 58

4.13. PCR products by 1.5µl of MgCl2 (lane 1-8) using primer J-01

with Ta, 37.2°C on 1% agarose gel run under 90V for 1 hour. 59

4.14. Optimization of annealing temperature (Ta) for primer O-01 at the

range of 33.3°C to 40.2°C on 1.0 % agarose gel under 90V for 1 hour.

Annealing temperature of 38.1 (indicate in the arrow) was chosen for

further analysis. 61

4.15. Banding pattern of Optimization of annealing temperature, Ta using

primer O-02 on 1.0% agarose gel under 90V for 1 hour. 62

4.16. DNA amplification of 28 O. stamineus accessions (as stated in Table 4.1)

with the size ranging from 250bp-1500bp using primer L-02 on

1.0% agarose gel electrophoresis under 90V for 1 hour. 65

4.17. Banding pattern of DNA amplification of 28 O. stamineus accessions

(as stated in Table 4.1) with the size ranging from 250bp-900bp using primer

U-01 on 1.0% agarose gel electrophoresis under 90V for 1 hour. 65

4.18. DNA amplification of 28 O. stamineus accessions (as stated in Table 4.1)

with size ranging from 260bp – 1400bp using primer V-01 on 1.0% agarose

gel under 90V for 1 hour. 66

4.19. DNA amplification of 28 O. stamineus accessions (as stated in Table 4.1)

with size ranging from 450bp- 1400bp using primer H-01 on 1.0%

agarose gel under 90V for 1 hour. 66

4.20. DNA amplification products of 28 samples of O. stamineus using

W-02 primer. Lane M: 1kb DNA ladder. Lane 1-28: DNA amplification

of different O. stamineus accession on 1% agarose gel under

90V for 1 hour. 69

4.21. DNA amplification products of 28 samples of O. stamineus using

O-02 primer. Lane M: 1kb DNA ladder. Lane 1-28: DNA amplification

of different O. stamineus accession on 1% agarose gel under

90V for 1 hour. 69

4.22. Dendogram of O. stamineus from 28 accessions based on

molecular data generated by 20 primers. 70

5.1 Classification of O. stamineus based on the phylogenetic tree produced 75

LIST OF TABLES

NO PAGE

3.1. The quantitative variables studied for morphological traits

of O. stamineus. 22

3.2 Optimization of PCR Mixture 32

3.3. Optimization of PCR Reaction 33

4.1 Locations of O. stamineus from Peninsular Malaysia and Indonesia 36

4.2. Analysis of variance of plant height within 28 O. stamineus accessions. 37

4.3. Analysis of variance of leaf width within 28 O. stamineus accessions. 39

4.4. Analysis of variance of leaf length within 28 O. stamineus accessions. 40

4.5. Analysis of variance of leaf area within 28 O. stamineus accessions. 41

4.6. Analysis of variance of petiole length within 28 O. stamineus

accessions. 43

4.7. Analysis of variance of internode length within 28 O. stamineus

accessions. 44

4.8. The morphological characteristics of 28 accessions java tea from

Malaysia and Indonesia. 47

4.9. Nucleic Acid concentration, ng, purity (A260/A280) from

stock high pure and Concentration of diluted samples (ng). 55

4.10. PCR master mix with different volume of reagents. 58

4.11. Optimal annealing temperature range for 40 primers 63

4.12. List of primers, their sequences, total numbers of amplified

fragments and number of polymorphic and monomorphic bands

generated by PCR using 20 primers. 68

Kajian terhadap Molekular dan Kepelbagaian Genetik Pada Teh Java

(Orthosiphon stamineus Benth.) Sebagai Asas Membaikpulih Tumbuhan

ABSTRAK

Orthosiphon stamineus Benth. dikenali sebagai misai kucing atau Teh Java

merupakan tumbuhan perubatan berasal dari negara-negara Asia Tenggara seperti

Thailand, Indonesia , Filipina , Malaysia dan Brunei. Di Malaysia, dua varieti telah

dikenal pasti berdasarkan warna bunga . Satu varieti mempunyai bunga berwarna putih

manakala satu lagi varieti mempunyai bunga berwarna ungu cerah. Ia telah terbukti

bahawa herba ini berkesan merawat pelbagai penyakit terutamanya yang berkaitan

dengan buah pinggang. Tumbuhan ini juga dipercayai mempunyai anti-alergi, anti-

hipertensi, anti-imflammatory dan sifat diuretik. Maklumat mengenai kepelbagaian

genetik spesies adalah sangat terhad. Dalam kajian ini, dua puluh lapan genotip O.

stamineus daripada pelbagai lokasi dalam Malaysia dan Indonesia telah disaring dengan

menggunakan empat puluh primer RAPD (10 mer). Keputusan menunjukkan bahawa

dua puluh primer menjana corak jalur yang stabil , sejumlah 159 jalur polimorfik yang

konsisten menghasilkan saiz jalur antara 100 bp kepada 1.5 kb. Daripada produk

amplifikasi direkodkan, 35% adalah monomorphic, iaitu kebiasaan kepada semua

spesies . Manakala , 65 % adalah jalur polimorfik yang diturunkan hubungan antara

spesies ini. Data matrik telah digunakan untuk menghasilkan dendogram (UPGMA )

bagi jarak genetik antara O. stamineus . Data matrik digunakan untuk menghasilkan

dendogram perbezaan jarak genetik antara O. stamineus. Keputusan saringan “cap jari”

berdasarkan data molekul menunjukkan 28 kumpulan O. stamineus dibahagikan kepada

2 kluster. Kluster I dikategorikan sebagai varieti putih O. stamineus manakala kluster II

ialah varieti ungu O. stamineus. Pokok filogenetik yang dihasilkan berdasarkan

persamaan matrik membuktikan 74%-96% kesamaan genetik dikalangan 28 kumpulan

O. stamineus. Pokok filogenetik menunjukkan sepsis putih dan ungu O. stamineus

terbahagi kepada beberapa sub-spesis.

Study of Molecular and Genetic Diversity of Java Tea (Orthosiphon stamineus

Benth.) As a Basis for Plant Improvement

ABSTRACT

Orthosiphon stamineus Benth. commonly known as misai kucing or Java Tea is

a medical plant originated from Southeast Asia countries such as Thailand, Indonesia,

Philippines, Malaysia and Brunei. In Malaysia, two varieties had been identified based

on the color of the flowers. One variety has white flower while the other has light purple

flowers. It had been proven that the herb can effectively treat various ailments

especially related to the kidney. The plant was also believed to have anti-allergic,

antihypertensive, anti-inflammatory and diuretic properties. Information on genetic

diversity of the species was very limited. In the presence study, twenty eight genotypes

of O. stamineus from various accessions within Malaysia and Indonesia were screened

by using forty RAPD primers (10 mer). Results showed that twenty primers generated

stable band patterns, a total of 159 consistent and ambiguous polymorphic bands were

produced ranging size from 100 bp to 1.5 kb. From the amplification products recorded,

35% were monomorphic, common to all species. Whereas, another 65% perform

polymorphic this revealed the relationship between these species. Matrix data was used

to generate a dendogram (UPGMA) of genetic distance between O. stamineus. The

result of fingerprinting based on molecular data showed that, the 28 accessions of O.

stamineus are divided by two major clusters. Cluster I categorized as white variety of O.

stamineus while Cluster II is a purple variety of O. stamineus. The phylogenetic tree

based on the similarity matrix revealed 74%-96% genetic relatedness among 28

genotypes. The phylogenetic tree showed that the two species of white and purple O.

stamineus were divided into several subspecies.

CHAPTER 1

INTRODUCTION

In the early 20th century, Orthosiphon stamineus Benth. was introduced to

Europe where it became a popular herbal health tea. Today there are quite a number of

commercial products derived from this plant. In Malaysia, O. staminues commonly

known as misai kucing is a medicinal herb belonging to the family Lamiaceae. It is

widely grown in Southeast Asia and the tropical countries such as Malaysia and

Indonesia. O. stamineus gets its common name misai kucing or cat’s whiskers from its

pale purple flowers with long whispy stamens shaped like cats whiskers (Indubala et al.,

2000). O. stamineus is a herbaceous shrub which grows to a height of 1.5 m. In

Malaysia, O. stamineus was believed to consist of two varieties based on the color of

the flower purple and white (Keng et al., 2006). Leaves of this plant are used commonly

in many countries for herbal tea, well known as “java tea” (Basheer, 2010). Java tea is

believed to have anti-allergic, anti-hypertensive, anti-inflammatory, antioxidant and

diuretic properties (Beaux, 1999; Adam, 2009). It can treat various ailements such as

problem of kidney and bladder due to its mild diuretic action. O. stamineus is also used

for treating gout, diabetes, hypertension and rheumatism (Siddig et al., 2011).

There are studies about O. stamineus mainly related to morphology and

chemical properties (Indubala et al., 2000; Basheer et al., 2010). However, studies on

DNA technology and genetic diversity of O. stamineus is limited. Diversity may relate

to environmental influences or genetic differences. A species may contain two or more

subspecies that typically have some genetic differences compared to each other. Plant

that are geographically close will be interbreed with each other compared to those

which are more distant. This population may have genetically differences from one

another (USDA, 2006). In Malaysia, it is believed that there are only 2 varieties of O.

stamineus, a study must be made because Malaysia is one of the centre of origin of O.

stamineus and there must be diversity of O. stamineus population (Keng et al., 2006). In

the current research, there are 28 O. stamineus accession that was collected within

Malaysia and Indonesia to study genetic diversity of the plant. All collection of O.

stamineus was planted at the Agropark in Universiti Malaysia Kelantan, Kampus Jeli.

There are techniques available for assessing genetic diversity at the molecular

level (Matasyoh et al., 2008). Traditional methods for testing genetic variation is based

on morphological (Pankaj et al., 2007). The application of DNA technology such as

DNA fingerprinting in agriculture research has been made over the last twenty five

years (Nybom, 1990), especially to study genetic diversity in many plant species (Lee et

al., 1996, Scheliro et al., 2001, Gaudeul et al., 2000). However, this method not really

anymore to apply as a result of DNA extraction give poor DNA concentration to be

proceed to amplification process. This is because the presence of secondary metabolites

(alkaloid, flavanoids and phenols), polysaccharides, polyphenols and tannins in

medicinal plants that interfere with DNA extraction procedures which is can inhibited

PCR reaction (Padmalatha et al., 2005).

The aims of this research were as below:

1. To identify and fingerprint the genotypes of O. stamineus accession.

2. To identify genetic variability and genetic distance of Orthosiphon species

from the centre of diversity using molecular markers.

3. To create a base population of Orthosiphon stamineus for further research in

agro industry.

CHAPTER 2

LITERATURE REVIEW

2.1 Botany and Taxonomy of Orthosiphon stamineus Benth.

Java tea locally is a medical plant that is widely grown in tropical areas

such as Malaysia, Thailand and Indonesia. Common names in Southeast Asia

are misai kucing (Malaysia), kumis kucing (Indonesia), Yaa Nuat Maeo

(Thailand) and balbas-pusa (Philippines). The genus name Orthosiphon came

from Latin’s word, “Iorthos” and “siphon”. The word “Iorthos” mean

“straight” while the word “siphon” mean “tube-like structure”(Keng et al.,

2006). Therefore, this word mean “straight tube-like flower” that is produce by

the species, one of the characteristics for Lamiaceae family. The plant is

commonly grown in open areas such as along the roadsides and wastelands in

either the lowlands or highlands.

In Malaysia, there are two varieties of O. stamineus; it is classified based

on the colour of flowers which is white and purple varieties (Keng et al., 2006)

(Figure 2.1). The purple variety has yellowish spots that is scattered unevenly on

the surface of the leaves and it is produced light purple flowers. The white

variety has green leaves without yellowish spots on the surface of the leaves and

it is produced white flowers (Keng et al., 2006) (Figure 2.2). The other

structures of leaves, stems, stigma and pollen grains were same for both

varieties. O. stamineus is an herbaceous shrub, which grows to a height up to 1.5

meter.

Figure 2.1: White O. stamineus (Left) and purple O. stamineus (Right).

The leaves are organized in opposite pairs. They are simple, green, and glabrous

with a lanceolate leaf blade and a notched margin. The leaf apice is acuminate

with an acute leaf base. The petiole is relatively short, about 0.3 cm in length

and reddish purple in color. The stem is quadrangle, reddish in color, erect

with a lot of branching (Chan & Loo, 2006).

1cm 1cm

Figure 2.2: Leaf morphology of purple variety (leaf), ovate shape with light yellowish

spots on the surface; white variety (right), rombhoid shape and green

veination. Picture label with A is the front leaf and B is the rear leaf (Chan

and Loo, 2006).

2.2 The Use of Orthosiphon

Orthosiphon stamineus (Java or Cat’s Whiskers) is one of the most

important and widely used traditional herbal medicines for many centuries in

Southeast Asian countries (Indubala et al., 2000). It is appreciated for treating

ailments of the bladder and kidney. It appears to be a large source of both

rosmarinic acid and methylated flavonoids. Plant extracts, especially herbs are

rich in phenolic secondary metabolites and some have antimicrobial activity (Ho

et al., 2010). Ho et al. (2010) obtained extracts of the Orthosiphon stamineus

plant and tested it for antimicrobial and antioxidant activities against selected

food-borne Vibrio parahaemolyticusin vitro. They found that the extract of O.

stamineus produce high concentration of rosmarinic acid that is act as

antibacterial and free radical scavenging activities for food preservation. O.

stamineus also is a medicinal plant with diuretic properties that can treat various

diseases especially related to kidney, rheumatism, abdominal pain, edema and

gout. Adam et al., (2009) proved that O. stamineus extracts exhibit diuretic

activity and the extracts slightly increased the blood glucose level. Mohana et

al., (2012) stated that more than twenty phenolic compounds were isolated from

this plant including lipophilic flavones, flavonol glycosides and caffeic acid

derivatives such as rosmarinic acid. Other than that, there are various

commercial products related to this plant with antioxidant claims on it (Khamsah

et al., 2006). However, Prior et al., (1998) found that considerable variability of

antioxidant activity depend on several factors including geographical origin.

2.3 Genetic Diversity

Genetic diversity is the level of biodiversity referring to the total number

of genetic characteristics in the genetic makeup of a species and it

describes the tendency of genetic characteristics to vary. Genetic diversity is

very important because the genotypes itself can determine organisms’ physical

form and function, it helps organism cope with current environmental

variability, diversity within populations can reduces potentially deleterious

effects of breeding among close relatives and it is the primary basis for

adaptation to future environmental uncertainty (Donald et al., 2001). Genetic

diversity is shown in the process of inheritance, where nucleotides are shuffled

and recombined to form new combinations that are different from the parents.

The different can be due to the mutations that making changes in one or more

nucleotides in the DNA sequences (Fulton et al., 2011). Because of change in

the DNA sequence affects the encoded protein that is can lead to the damaging

of a cell or organism. Mutations can be caused by external (exogenous) factors

such as sunlight, radiation, and smoking or endogenous (native) factors such as

errors during DNA replication or they may be caused by errors in the

cellular machinery. Physical or chemical agents that induce mutations in DNA

are called mutagens and are said to be mutagenic. There are two general types

of mutations that is point mutation and chromosomal mutations. Point

mutations affect only one or a few nucleotides within a gene. Chromosomal

mutations change the number of chromosomes, the number, or the arrangement

of genes in the chromosomes.

There are many different ways to measure genetic diversity. Traditional

methods for testing genetic variability in plant improvement are based on

morphological or time-consuming physiological assays (Scheliro et al., 2001).

Presently biochemical and molecular technique is more advance for plant

improvement (Williams et al., 1993; Smith et al., 1994; Welsh et al., 1995;

Rafalski et al., 1996). Molecular markers of Amplified Fragment Length

Polymorphism (AFLP), selective amplification of microsatellite polymorphic

loci (SAMPL), inter-simple sequence repeat (ISSR) and random amplified

polymorphic DNA (RAPD) markers were used for the detection of genetic

polymorphism. These methods for nucleotides differences can be effectively

used to run individual or combined data sets of morphological, biochemical or

DNA based data (Aremu, 2012). RAPD (Williams et al. 1990; Welsh and

McClelland 1990) is the least initially expensive route chosen for this project.

Previous studies have indicated that RAPDs can provide valuable tools for

genotype identification, population and pedigree analysis, phylogenetic studies,

the screening of segregating population for linked markers and genetic mapping

(Lee et al. 1996).

2.3.1 Cluster Analysis

The genetic distance and genetic similarities between species can be

calculated from the amplification products. The data must first translate into

binary matrix for statistical analysis. For RAPD markers, the results of DNA

fingerprinting bands were score as (1) or absent (0). All results from each

primer are scored and translated as binary matrix for statistical analysis. The

data source from morphological, biochemical and molecular marker data are

pooled together for cluster analysis. It presents patterns of relationship between

genotypes and hierarchical mutually exclusive grouping such that similar

descriptions are mathematically gathered into same cluster (Hair et al. 1995).

Cluster analysis may use several methods; Unweight Pair Group Method with

Arithmetic Mean (UPGMA), Single Linkages (SLCA), Complete Linkage

(CLCA) and Median Linkage (MLCA). UPGMA provides more accurate

grouping information on breeding materials used in accordance with pedigrees

and calculated results found most consistent with known heterotic groups than

the other clusters (Aremu et al., 2007). In this research, the clustering method

used to create phylogenetic trees (dendrogram) is by using the programme

NTSYSpc based on method UPGMA (Backeljau et al., 1996).

2.4 Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) developed by Kary Mullis (1983) is a

biochemical technology in molecular biology to amplify a single or a few copies

of DNA pieces to generate thousands to millions of copies of a particular DNA

sequence. PCR is a common technique used in various amplifications such as

DNA cloning for sequencing, DNA-based phylogeny, functional analysis of

genes; the diagnosis of hereditary diseases including identification of genetic

fingerprints (used in forensic sciences and paternity testing) and detection and

diagnosis of infectious diseases.

A basic PCR set up requires several components and reagents in a

reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes). PCR

components and reagents include DNA template that contains the DNA region

(target) to be amplified, two primers that are complementary to the DNA target,

taq polymerase, deoxynucleoside triphosphates (dNTPs) for the building-blocks

from which the DNA polymerase synthesizes a new DNA strand, buffer solution

that provide a suitable chemical environment for optimum activity and stability

of the DNA polymerase and magnesium chloride, MgCl2 (Ernő, 2011). The

reaction is set up in a thin walled PCR tube to allow for rapid thermal

equilibration in a thermal cycler.

PCR procedure is divided into several steps. The first step is the DNA

denaturation step that weakens the entire DNA in the reaction into single strand.

This step is accomplished at 94 oC or 95

oC for 30 seconds. The second step is

the primer annealing step which the PCR primers find their complementary

targets and attach themselves to those sequences. The melting point of the

primer determines the temperature of the annealing step. Finally, the last step in

a PCR cycle is the polymerase extension step which the DNA polymerase is

producing a complimentary copy of the target DNA strand. The usual

temperature of this step is 72 oC, considered a good optimum temperature for

thermal-stable polymerases. The cycle repeated for about 30-40 cycles. In

addition to these cycling conditions, it is often to place a single denaturation step

of three to five minutes at 94oC or 95

oC at the beginning of the reaction and a

final extension step of a few minutes at 72 oC (Figure 2.4).

Figure 2.3: Process of Polymerase Chain Reaction.

Nowadays, there are various of PCR techniques in molecular biology

have been developed from the basic PCR method to improve performance and

specificity, and to achieve the amplification of other molecules of interest in a

particular research. Some of these various types of PCR are i. Random

Amplified Polymorphic DNA (RAPD) PCR that randomly amplified several

DNA sequences; ii. Restriction Fragment Length Polymorphism (RFLP)-PCR,

which is using restriction enzymes to detect differences in homologous DNA; iii.

Amplified Fragments Length Polymorphism (AFLP) PCR that has the capability

to detect various polymorphisms in different genomic regions simultaneously;

iv. RT-PCR which generates amplification of RNA by synthesis of cDNA (DNA

complementary to RNA) that is then amplified by PCR; and, v. Real time

PCR which performs absolute or relative quantification of nucleic acid

copies obtained by PCR.