UNIVERSITI PUTRA MALAYSIA

GENETIC DIVERSITY ANALYSIS OF SWEET POTATO (IPOMOEA BATATAS L.) GERMPLASM FROM MALAYSIA AND

INDONESIA USING RAPD MARKERS

RAMISAH BTE MOHD SHAH

IB 2001 2

GENETIC DIVERSITY ANALYSIS OF SWEETPOTATO (IPOMOEA BATATAS L.) GERMPLASM FROM MALAYSIA AND

INDONESIA USING RAPD MARKERS

By

RAMISAH BTE MOHO SHAH

Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Science in the Institute of Bioscience

Universiti Putra Malaysia

September 2001

Success

This success begins with our own will ...

It's all in the state

of MIND.

Life battles are not always won.

By those who are stronger or faster,

Sooner or later

The person who wins

Is the person who thinks he can

May ALLAH a/ways

Give the STRENGTH to all of us

and keep us in his BLESSING al/ over a year.

AMIN ...

1\

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

GENETIC DIVERSITY ANALYSIS OF SWEETPOT ATO (IPOMOEA BATATAS L.) GERMPLASM FROM MALAYSIA AND

INDONESIA USING RAPD MARKERS

By

RAMISAH BTE MOHO SHAH

September 2001

Chairman: Associate Professor Dr. Mohd Said b. Saad

Faculty: I nstitute of Bioscience

Genetic variation among sweetpotato accession (Ipomoea batatas l.)

from Malaysia and Indonesia has not been extensively examined with

molecular markers. The objectives of this study are to use random

amplified polymorphic DNA (RAPD) marker to determine the degree of

polymorphism in the sweetpotato germ plasm and to study the genetiC

diversity and relationships among sweetpotato accessions from two

different regions, Malaysia and Indonesia.

A total of 92 accessions of sweet potato germplasm from the two

countries maintained at Universiti Putra Malaysia were characterized

using five RAPD· markers. Thirty-nine accessions were collected from

seven different states of Malaysia and 53 accessions came from two

different sub-regions of Indonesia (Irian Jaya and Java).

The results of this study indicated that the levels of polymorphism

among all sweetpotato were extremely high. From five random primers

iii

used (OPB 07, ope 1 0, OPO 01, OPO 06 and OPG 1 4), 1 94 fragments

were amplified, of which 1 92 (98. 97%) were polymorphic. Only two

fragments were monomorphic. The fragment size ranged from 1 1 7bp -

3 240bp.

An NTSYS-pc computer program was further employed for data analysis

using Jaccard's coefficient of simi larity as a base for dendrogram

construction via the UPGMA method. The Jaccard's similarity values

ranged from 0.08 to 0.6 9 showing high levels of genetic variability

among sweetpotato accessions. The cluster analysis separated

Malaysian and Indonesian accessions i nto a d ifferent group with a

number of additional clusters. Some of the Malaysian and Indonesian

accessions were clustered based on their geographic source. The

analysis indicates that very large genetic variation exists among

sweetpotato accessions used in this study and the sweetpotato

collection is a valuable as a genetic resource. This could be done by

selecting cultivars from different groups delineated by cluster analysis for

hybridization programs.

Genetic diversity analysis within the sweetpotato germplasm collection

had provided useful information for managing this collection. RAPO

appears to be useful for discerning variation within crop germplasm and

to assess the genetic relationships among sweetpotato germ plasm from

Malaysia and Indonesia.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai me menu hi keperluan untuk ijazah Master Sains

ANALISIS DIVERSITI GENETIK 01 DALAM GERMPLASMA KELEDEK (IPOMOEA BATATAS L.) DARI MALAYSIA DAN

INDONESIA MENGGUNAKAN PENANDA RAPD

Oleh

RAMISAH BTE MOHO SHAH

September 2001

Pengerusi: Profesor Madya Dr. Mohd Said bin Saad

Fakulti: Institut Biosains

Variasi genetik dalam germplasma tanaman keledek (Ipomoea batatas

L.) yang berasal dari Malaysia dan Indonesia belum dikaji dengan

mendalam menggunakan teknik penanda molekul. Objektif utama kajian

ini adalah untuk menggunakan penanda RAPD bagi menilai darjah

polimorfisma di dalam germ plasma keledek dan mengkaji diversiti dan

pertalian genetik di antara aksesi keledek dari dua kawasan yang

berbeza, Malaysia dan Indonesia.

Sejumlah 92 aksesi germplasma keledek dari dua negara di pelihara di

Universiti Putra Malaysia telah di cirikan dengan menggunakan lima

penanda RAPD. 39 aksesi diperolehi dari tujuh negeri di Malaysia and

53 aksesi lagi dari dua sub-kawasan di Indonesia (Irian Jaya dan Jawa).

Keputusan daripada kajian ini menunjukan paras polimorfisma di antara

tanaman keledek ini adalah sangat tinggi. Dari lima primer rawak yang

v

digunakan (OPB 07, ope 1 0, OPO 0 1 , OPO 06 dan OPG 14), 1 94

frakmen telah diamplifikasikan di mana 1 92 (98.97%) daripadanya

adalah polimorfik. Hanya dua frakmen adalah monomorfik. Saiz frakmen

adalah di antara 1 1 7bp - 3240bp.

Program komputer NTSYS-pc digunakan untuk mengalisis data

menggunakan koeffisien persamaan Jaccard sebagai asas untuk

membina dendrogram berdasarkan teknik UPGMA. Nilai anggaran

persamaan adalah di antara 0.08 - 0.69 menunjukkan paras

kepelbagaian genetik yang tinggi di antara aksesi keledek. Analisis

kelompok yang dihasilkan telah mengasingkan aksesi Malaysia dan

Indonesia dengan beberapa kelompok tambahan. Ada aksesi dari

Malaysia dan Indonesia dikelompokkan berdasarkan kepada sumber

geografi aksesi tersebut. Keputusan ini menunjukkan terdapat variasi

genetik yang luas di antara aksesi keledek dan koleksi keledek ini

berguna sebagai sumber genetik. lanya dapat dilakukan dengan memilih

kultivar dari kumpulan yang berbeza hasil daripada anal isis kelompok di

dalam program hibridisasi.

Analisis variasi di dalam koleksi germ plasma keledek ini dapat

memberikan pengetahuan yang berguna bagi tujuan pengurusan. RAPO

didapati berguna untuk tujuan pengecaman variasi di dalam

germplasma tanaman dan untuk menentukan pertal ian genetik di antara

germplasma keledek dari Malaysia dan Indonesia.

vi

ACKNOWLEDGMENTS

PERPUSTAKAAN

JNIV�lt�ITI PUTKA MALAYSIA

I am very thankful to ALLAH S.W.t. for giving me the guidance, peace

and patience in completing this study.

I would like to express my sincere appreciation to my supervisor Assoc.

Prof. Dr. Mohd Said Saad for his suggestion, constructive criticism and

guidance throughout the course of my research. Thanks are also due to

Dr. Abdul Ghani Yunus and Assoc. Prof. Dr. Nor Aini Abdul Shukor for

their help and co-operation for allowing me to use their equipment during

the work of this project.

Special thanks to my dear friends, Khairul Hasni Mat Isa and Rogayah

Sekeli for their help and moral support to make this project possible. Not

forgetting, thanks to all the supporting staffs at PSGT, UPM especially to

Auntie Marie and Julia Abdul Aziz for their concerns.

My sincere thanks also goes to my beloved husband, Zukhairi b. Harun

@ Halim for his love, sacrifice, continuous support and patience during

the process of completing my study. To my lovely son, Muhammad

Za'im Akmal, for giving me the happiness. To my parents, brothers and

sister, Kak G and all my family members, thank you for giving me the

encouragement and understanding.

The research grant IPGRI (LOA-AP097/009) supported by IPGRI

(International Plant Genetic Resource Institute) was gratefully

acknowledged. In addition, I am also indebted to UPM PASCA

scholarship for the financial support.

vii

I certify that an Examination Committee met on 10th September 2001 to conduct the final examination of Ramisah bte Mohd Shah on her Master of Science thesis entitled "Genetic Diversity Analysis of Sweetpotato (Ipomoea batatas L) Germplasm from Malaysia and Indonesia using RAPD Markers" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

MIHDZAR ABDUL KADlR, Ph.D.

Faculty of Agriculture Universiti Putra Malaysia (Chairman)

MOHO SAID SAAD, Ph.D. Associate Profesor Institute Bioscience Universiti Putra Malaysia (Member)

ABDUL GHANI YUNUS, Ph.D. Faculty of Agriculture Universiti Putra Malaysia (Member)

NOR AINI ABDUL SHUKOR, Ph.D. Faculty of Forestry Universiti Putra Malaysia (Member)

ProfessorlDeputy Dean of Graduate School Universiti Putra Malaysia

Date: 1 0 NOV t001

viii

This thesis was submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Master of Science.

ix

AINI IDERIS, PhD. Professor, Dean of Graduate School, Universiti Putra Malaysia.

Date:

11 0 JAN 2002

I hereby declare that the 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 UPM or other institutions.

RAMISAH BTE MOHO SHAH

Date: 1 6 NOV 2001

x

TABLE OF CONTENTS

Page

DEDiCATION .. .... . ........... . ........ ... .. . ....... . . .. ....... . . , . . . ... . .. .. .. i i ABSTRACT ......................... .. ... ....... .. . . .............. ... . ......... . i i i ABSTRAK .......... . ............. ..... ......... ........ ...... ...... ...... . .... .. v ACKNOWLEDGEMENTS . . .... .... .... .. . ... .... ... . ..................... .. vii APPROVAL SHEET .............. ..... .... ..... .... .. ........ .... . .......... . viii DECLARATION FORM ... .. . . . ..... ..... . .. . . . .. . ..... . . ......... ... . . ..... . x LIST OF TABLES ........ ... .... ... ... .... . . . . . . . .. . . . .. . . ..... . ........ . ..... xiii LIST OF FIGURES ............... . .... ... ...... . . .. .. .. .. . .. . . ............. .. xiv LIST OF PLATES ........ ... . ... ... . ... ..... . . . . . .. .. ..... .... .... ..... .. . .. .. xv LIST OF ABBREVIATIONS AND GLOSSARy. ... ... . .. . . . .. .... ... .. . xvi

CHAPTER

INTRODUCTION 1

II LITERATURE REVIEW ... .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . .. 5 Sweet potato (Ipomoea batatas L. ) - Background .. . . . . . . . . . . . 5 Origin and Distribution . . . . . . . . .. . . . . . . . . . . . .. .. .. . . . . .. . . . . . . . . . . , . . . . 8 Sweet potato Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 1 0 Sweetpotato Util ization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 Variabil ity in Sweetpotato . . . . . . . . . . .. . . .. . . .. . . . . . .. .. .. .. . . . . . . , . . . . 1 3 Molecular Genetic Studies in Sweetpotato . . . . . . . . . . . . . . . . . .. . . . 1 6 Molecular Markers in Plant Studies .. . . . . . . . . . . . . . ... . . . . . . . . . . . . . 1 9 Types of DNA Markers .. .. . .. . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . .. . . . . . . 26

Polymerase Chain Reaction (PCR) . .. . .. . . . . . . . . . . . . . . . . . . . . 29 Random Amplified Polymorphic DNA (RAPD) Marker . . . . . . . 3 2

Principles of RAPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Optimization of RAPD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 3 6 The Advantages and Limitation of RAPD Technique . . . . 38

Simple Sequence Repeat (SSR) Marker. . .... .. . ... . . ... ... . .. . . . 4 1 Amplified Length Polymorphism (AFLP) Marker... ... . . . . . . . . . . 43 The Measurement of Similarity Coefficient for RAPD Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

III MATERIALS AND METHODS .. . . . . . . . . . . . . . ... . . . . . ... . . . . . . . . . . . . 49 Plant Material . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 49 DNA Extraction . . . .. . . . . . . . . . . . ... . . . . . . . . . . . . . . .. . . . .. . . . ... . . . .. . .. ... .. 53

Sarkosyll Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 53 CTAB Method... . . . . . . . . . . .. . . .. .. . . .. ... ... . .. . . . . .. . . . . . . .. . . . . . .. 54 Determination of DNA Purity . . . . .. . . .. ... . .. . . . . . . . . . . . . . . . .... . 56

Screening of RAPD Primer . . . . . . .. . . . .. " ... . ... . . . . . . . . . . . ... . . . . . . . 57 RAPD-PCR Analysis . . .. . .. . . , . . ..... .... , . .. . .. . . . . . , . . .... . '" .. .. . . . 57 Detection of Amplification Product . . . . . . . . . . .. .. . . .. ... . .. . . . .. .. ... 58

xi

Gel Scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Data Analysis . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

IV RESULTS AND DiSCUSSiON ................ , . . . ' " . . . . . . . . . . . . . . .

DNA Extraction . . .. . . . . . . . . . ' " . . . . . . . . . . , . . . . . . . . . . . . . . . . ' " . , . . . . . . . . . .

Identification of Primer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

RAPD Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Genetic Relationships Among Sweetpotato Accessions . . . . .

Genetic Relationships Among Malaysia Sweetpotato Accessions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Genetic Relationships Among Indonesia Sweetpotato Accessions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V CONCLUSIONS AND RECOMMENDATIONS ............... .

Conclusions . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62 62 66 68 78

84

88

92 92 94

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

APPENDICES

A.O List of N inety two Accessions of Sweetpotato (Ipomoea batatas L.) Used in The Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 6

B.O Binary Data of RAPD Character for 92 Malaysian and Indonesian sweetpotato accessions using 5 Primers (O=Absent band, 1 =Present band) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 9

B. 1 Binary Data of RAPD Character for 39 Malaysian sweet potato accessions using 5 Primers (O=Absent band, 1 =Present band) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 25

B.2 Binary Data of RAPD Character for 53 Indonesian sweetpotato accessions using 5 Primers (O=Absent band, 1 =Present band) . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 28

C.O Similarity matrix for 92 Malaysian and Indonesian Sweetpotato Accessions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 32

C. 1 Similarity matrix for 39 Malaysian Sweetpotato Accessions . . 1 39 C.2 Similarity matrix for 53 Indonesian Sweetpotato Accessions 1 4 1 0.0 Preparation of Phenol Chloroform . . . . . . . . . . . . . '" . . . . . .. . . . . . '" . . . 1 44 0. 1 Preparation of Stock solution (1 l itre) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 45 0.2 Preparation of Agarose gels . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . '" . . . 1 46

BIODATA OF THE AUTHOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 47

xii

LIST OF TABLES

Table Page

1 Country of origin and number of sweetpotato (Ipomoea batatas L.) accessions used in the study . . . . . . . . . . .. . . . . . . . 50

2 The time period, quality and amount of DNA extracted from sweetpotato (Ipomoea batatas L. ) using Sarkosyll and CTAS method .. . ... . . . . .... . . . . . . . . ... . . . .. . . . . . . . . . . . . . . . ... 62

3 Values of UV spectrophotometer readings for 1 0 sweetpotato (Ipomoea batatas L.) accessions from Irian Jaya, Indonesia . .. . . . . . . . . . . . . . . . .. . . . . . . . ... . . . . . . ... . . . . .... . .. . . 65

4 List of the primer tested and selected on the screening of sweet potato (Ipomoea batatas L.) accessions . . . . . . . . . 66

5 The five arbitrary primers that were found suitable for RAPD-PCR analysis on sweetpotato (Ipomoea batatas L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

6 RAPD analysis on 92 accessions of sweetpotato (Ipomoea batatas L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

7 The number of unique fragments generated using five primers in the RAPD analysis on the ninety-two accessions of sweet potato (Ipomoea batatas L . ) . . . .. . . . . . 74

8 Number of amplified fragments generated from RAPD analysis using five primers on sweetpotato (Ipomoea batatas L. ) from three different regions .. . ... .. . . . . . . . . . . . . . . 77

9 The d istribution of sweetpotato (Ipomoea batatas L.) accessions into groups revealed by cluster analysis according to reg ion . . . . . . . . . . . . ... . . . . . . . . . . . . . . ... , . . . . .. . . . . . . . . . 8 1

xiii

LIST OF FIGURES

Figure Page

1 Schematic diagram of polymerase chain reaction process by using a pair of primer . . . . . . . . . . . .. . , . . . . . . . ... . . . . 30

2 Schematic representation of the ampification of DNA using two primers in the standard peR versus a single primer in RAPD assay . . . . . .. . . , . . . . . . . . . .. . . .. . . , . . . . . . . . . . . . ,., 34

3 Map of Malaysia showing the seven sub-regions where the thirty-nine accessions of sweetpotato (Ipomoea batatas L.) were collected . .... . . . ... . . .... . ...... ... . . . ... . ..... 51

4 Map of Indonesia showing the two sub-regions where the fifty-three accessions of sweetpotato (Ipomoea batatas L.) were collected . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . 52

5 Schematic diagram of P late 4 showed clear banding pattern of monomorphic (220 bp) and polymorphic (2000 bp) fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . 72

6 Dendrogram from U PGMA clustering analysis using Jaccard's coefficient of similarity on 92 sweet potato accessions from Malaysia and Indonesia .. . . . . . . . . . . . . . . . . . 80

7 Dendrogram from U PGMA clustering analysis using Jaccard's coefficient of similarity on 39 sweetpotato accessions from Malaysia ... . .. ...... . .... .... . . . . . . .. . ......... 85

8 Dendrogram from U PGMA clustering analysis using Jaccard's coefficient of Similarity on 53 sweet potato accessions from Indonesia ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . 89

xiv

LIST OF PLATES

Plate Page

1 Quality of sweetpotato (Ipomoea batatas L.) DNA extracted using CT AB and 8arkosyll method . . . .. ... . . . 63

2 Poor quality sweetpotato (Ipomoea batatas L.) DNA especially in lanes 1 ,2,6 ,8,9, 1 1 and 1 2 indicating RNA contamination .. ..... .. .... . . . . .. ... . . . . . . .... . . ....... .. . 64

3 Fragments of sweetpotato (Ipomoea batatas L.) DNA without smear indicating good qual ity such purified DNA which is suitable for PCR analysis . . . .. . . . . . . . . .... . 64

4 DNA ampl ification profiles of ten accessions from Java and ten accessions from Irian Jaya using primer OPG 1 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

5 The present of two unique fragments from primer OPG 14 for accessions 4 1 2T (240 bp) and 388T (2077 bp), both from Terengganu, Malaysia . . . . . . . . .. . . 75

6 The present of one unique fragment from primer OPS 07 for accession 801 94 ( 1 60 bp) from Java, Indonesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

xv

A260nm

A280nm

AFLP

AP-PCR

bp

CIP

OAF

dATP

dCTP

dGTP

DNA

dNTP

dTTP

EDTA

FAO

IPGRI

Kb

M

MgCI2

Min

mM

I')g

NaCI

LIST OF ABBREVIATIONS AND GLOSSARY

Absorbance at 260nm

Absorbance at 280nm

Amplified fragment length polymorphism

Arbitrary primed polymerase chair reaction

base pair

International Potato Center

DNA amplification fingerprinting

deoxyadenine-S' -triphosphate

deoxycytidine-S'-triphosphate

deoxyguanocine-S' -triphosphate

Deoxyribonucleic acid

deoxynucleotide-S' -triphosphate

deoxythymidine-S' -triphosphate

Ethylenediamine tetra-acetic acid

Food and Agriculture Organization

International Plant Genetic Resource Institute

Kilobase

Molar

Magnesium chloride

minute (s)

milimolar

nanogram

Sodium chloride

xvi

00 Optical density

peR Polymerase chain reaction

RAPD Random amplified polymorphic DNA

RFLP Restriction fragment length polymorphism

rpm revolution per minute

SSR Simple sequence repeat

Taq Thermus aquaticus

TBE Tris-borate EDTA

TE Tris-EDTA

I-Ig microgram

1-11 microliter

UV Ultraviolet

xvii

GLOSSARY

Amplification The production of many DNA copies from one master region of DNA.

Anneal The spontaneous pairing of complementary DNA or RNA sequences by hydrogen bonding to form a double-stranded polynucleotide.

Arbitrary primer A short oligonucleotide primer used in certain PCR methods to initiate DNA synthesis at random locations on the target DNA.

Base The chemical unit which characterises a nucleotide. In DNA the bases found are adenine, guanine, thymine and cytosine.

Base pair Two nucleotide bases on different strands of a nucleic acid molecule that are held together by hydrogen bonds. Bases can pair in only one way -adenine with thymine and guanine with cytosine in DNA.

Cross-pollination The fertilization of one plant with pollen from another. This outcrossing ends to enhance genetiC diversity in plant populations because more diverse genetic mixtures occur.

Cultivar A contraction of cultivated variety.

Dendrogram A graphical representation of the results of a clustering procedure in which the vertical axis consists of the objects or individuals and the horizontal axis represents the number of clusters formed at each step of the clustering procedure.

DNA Deoxyribonucleic acid - genetic material found in all living organisms. Every characteristic of every living organism can be traced to the code of its DNA.

Electrophoresis A technique for separating molecules in a matrix (such as agarose or starch gels) according to their electrical charge and size.

Enzyme

Gene

A specialized protein catalyses biochemical reactions.

The chemical units of heredity that, when expressed, determine an organism's traits.

xviii

Genetic diversity The range of genetic differences among individuals or groups of organisms.

Genetic resources Germplasm characteristics organisms.

containing of plants,

potentially animals and

useful other

Genotype The genetic constitution of an individual or group that may be either expressed or unexpressed , depending on the environmental effects of a given location.

Germplasm The collection hereditary materials within a species.

Heterosis The intensified expression of desirable genetic traits that makes a hybrid superior to its parents.

Hybridization A procedures that involves the deliberate act of applying male pollen onto the female stigma to effect fertilization.

Hybrid vigor The intensified expression of desirable genetic traits that makes a hybrids superior to its parents.

Isozymes Variations of an enzyme

Loci Plural of locus.

Locus A specific site on a chromosome, usually of a gene or other marker.

Marker An identifiable physical location on a chromosome whose inheritance can be monitored .

Monomorphic The situations in which all the individuals in a population are the same genetic type or have the same allele.

Mutation Alterations in the genetic code due to environmental influences or errors in replication.

Natural selection Nature's selection of organism that adapt best to environmental and/or hereditary changes.

Polymorphism A detectable difference at a particular marker occurring among individuals.

xix

Primer

Selection

Species

A short DNA fragment annealed to a single­stranded DNA, to which further nucleotides can be added by DNA polymerase.

To discriminate deliberately among individuals in the number of offspring contributed to the next generation.

In taxonomy, the next step below genus. Individuals within a species often look alike and can interbreed.

xx

CHAPTER 1

INTRODUCTION

Sweetpotato (Ipomoea batatas L.) is an important food crop which is

grown in a wide cl imatic conditions located between 1 5°S and 45°N

(MacKay, 1 989). It currently ranks seventh among the most important

food crops after rice, wheat, maize, potato, barley and cassava (FAO,

1 997). World sweetpotato production exceeds 1 40 mill ion tons in an

area of about 9.4 mill ion ha (FAO, 200 1 ) .

Sweetpotato has been regarded as the 'potato' of the warm tropics due

to its abil ity to grow under h igh temperatures and low inputs of water and

ferti lizer (Bohac et a/., 1 995). Sweetpotato has and will play an important

role in solving g lobal issues associating food, natural resources and the

environment of the 2 1 st century (Kozai et a/., 1 997).

Sweetpotato exhibits great diversity in morphological and phenotypic

traits, such as growth habits, leaf shape and storage root flesh and skin

color (Woolfe, 1 992; Saad, 1 993). There are thousands of different

sweetpotato genotypes cultivated around the world. In Papua New

Guinea, it is estimated that there are about 5000 cultivars of

sweet potatoes. The island of New Guinea has been considered as the

secondary center of diversity for sweetpotato because of its range of

2

isolated ecological niches and the large number of cultivars found within

a small area (Zhang et al., 1998).

Saad (1993) reported that some Malaysian farmers grow more than 10

cultivars at one time. Most sweetpotato farmers grow their own cultivars.

Many of these cultivars were not selected, but some farmers selected

their own cultivars from several varieties obtained from other farmers or

their friends. Nevertheless, some advanced farmers have brought in

varieties from other countries such as China and Indonesia (Saad and

Anang, 1994).

Many of these cultivars have arisen through systematic breeding efforts,

but an appreciable number of them haves also arisen through natural

hybridization and mutations. Sweetpotato is hexaploid and cross­

pollinated. Continuous outcrossing between different genotypes leads to

formation of many segregants. The crop's outcrossing nature, combined

with vegetative propagation capabilities has created a vast number of

cultivated genotypes around the world.

Sweet potato germplasm collection, characterization and conservation

are important prerequisites for the utilization in crop improvement

programmes. To facilitate efficieflt germplasm collection and

management practices, there is a continual need for a greater

understanding of the extent of genetic variation within the germ plasm

3

collections and the nature of genetic relationships among the

accessions.

Genetic variation within and between populations of crop is a major

interest to plant breeders and populations geneticists. Knowledge of the

distribution of genetic diversity is essential for rational germplasm

conservation. Information on genetic identity and relationships of

genotypes is crucial to the development of source materials or core

collection (Frankel, 1984). A core collection is essential for rationalizing

the management and enhancing the utilization of the genetic diversity

available in the entire sweet potato germ plasm collection. A core

collection contains a subset of accessions from entire collection that

captures most of the available genetic diversity of the species (Brown,

1989).

Genetic variation assessments of agricultural species traditionally are

based on differences in morphological and agronomic characteristics.

These types of data are often influenced by environmental factors. In

recent years a variety of molecular techniques have been developed for

measuring genetic variability in plant genetic collections.

Molecular markers can afford many benefits for identifying variation and

estimating biological diversity (Virk et a/., 1995). These techniques allow

researchers to identify accessions at the taxonomic level, assess the

4

relative diversity within and among species and locate diverse

accessions for breeding purpose.

Random amplified polymorphic DNA (RAPD) has been well established

in the past few years as a cost-effective means of assessing genetic

variation at the DNA sequence level without requiring a prior knowledge

of species DNA sequences (Williams et aI., 1990; Hadrys et aI., 1992;

and Huff et al. , 1993). These techniques are widely accepted and have

been used successfully for different purposes, such as to investigate the

genetic relationships between different cultivars (Moeller and Schaal,

1999), to construct genetic maps (Eujayl et al., 1998), to identify

molecular markers linked to genes of interest (Nair et aI., 1996) and to

detect genetic diversity (Wachira et al. , 1995).

The objective of this study were i) to determine the degree of RAPD

polymorphism in the sweetpotato germplasm and ii) to study the genetic

relationship between the Malaysian and Indonesian sweet potato

accessions.