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UNIVERSITI PUTRA MALAYSIA

MOLECULAR DISSECTION AND QTL MAPPING OF RICE BLAST DISEASE RESISTANCE USING SIMPLE SEQUENCE REPEAT MARKERS

SADEGH ASHKANI

FP 2011 25

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MOLECULAR DISSECTION AND QTL MAPPING OF RICE BLAST DISEASE RESISTANCE USING SIMPLE SEQUENCE REPEAT MARKERS

By

SADEGH ASHKANI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Doctor of Philosophy

December 2011

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DEDICATIONS

To My Dear Wife, Fatemeh Azad with Innermost and Everlasting Affection and Love To My Dear Children, Hasti and Mobin, the Little Angels Bringing Us Joy and Happiness

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

MOLECULAR DISSECTION AND QTL MAPPING OF RICE BLAST DISEASE RESISTANCE USING SIMPLE SEQUENCE REPEAT MARKERS

By

SADEGH ASHKANI

December 2011

Chairman: Associate Professor Mohd Rafii binYusop, PhD Faculty: Agriculture DNA marker technology and the mapping of the major genes have played important

roles in rice (Oryza sativa L.) disease improvement. Blast caused by Magnaporthe

oryzae is an important disease of rice in Malaysia and all over the world. Its frequent

appearance during all the stages of plant growth greatly decreases yield and grain

quality. Quantitative trait loci (QTL)-based resistance for rice blast disease control is

becoming increasingly important in breeding rice programs. This study used

molecular marker approaches in order to analyse molecular genetics of resistance in

segregating populations and to identify QTL conferring resistance against two

different pathotypes of M. oryzae, namely, P7.2 and P5.0, in F3 families derived from

the cross of Pongsu Seribu 2 (resistant) variety and Mahsuri (susceptible) cultivar.

One hundred and twenty five micro-satellite markers closely linked to the blast

resistant genes (Pi-genes) distributed over 12 chromosomes of the rice genome were

chosen and used in the study to determine polymorphism and potential association

with blast resistance. Twenty three of polymorphic markers were used to identify

blast resistant segregation ratios in 320 individuals of F2 population. Eleven markers

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showed a good fit to the expected segregation ratio (1:2:1) for single gene model

(df = 1.0, p ≤ 0.05). The rest of the markers did not fit the expected segregating

Mendelian ratios. The F3 families were grown in a greenhouse and challenged with

two blast pathotypes, P7.2 and P5.0. In analysis of Chi-square tests for single gene

model, two independent genes and possible different gene models of epistasis effect

different segregation ratio (3R:1S) and (15R:1S) were observed for pathotypes P7.2

and P5.0 in blast lesion trait respectively. Pongsu Seribu 2 was resistant against both

blast pathotypes tested. Pathotype P7.2 was found to be a high virulence blast

pathotype. The plants resistant to blast pathotype P7.2 from F3 population were good

linked with genotypes of four SSR markers, RM413, RM1233, RM8225 and

RM5961, with observed segregation ratio of (1:2:1) for single dominant gene model.

These markers were found as suitable SSR markers for use in marker assisted

selection of blast resistant genes conferring resistance to Magnaporthe oryzae

pathotype, P7.2. A total of 188 F3 families derived from the cross between, Pongsu

Seribu 2 and Mahsuri were used in this experiment to identify QTLs for resistance to

local Magnaporthe oryzae pathotypes, P7.2 and P5.0. There was a positive

correlation for the three traits (BLD, BLT and % DLA) for each isolate. A trait

distribution analysis did not show continuous variation with normal distribution. For

both pathotypes, the distributions of disease severity (DS) were skewed towards

resistance. Sixty three polymorphic SSR markers were used to construct a linkage

map in 188 F3 families. The 63 SSR markers covered 3989.9 cM of the rice genome.

Single marker analysis (SMA), interval mapping (IM) and composite interval

mapping with permutation analysis was used for Quantitative Trait Loci (QTL)

analysis. Twenty eight independent QTLs were detected to be associated with blast

resistance on chromosomes 1, 2, 3, 5, 6, 8, 10, 11 and 12. Six putative QTL (qRBr-

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1.2, qRBr-2.1, qRBr-5.1, qRBr-6.1, qRBr-11.1 and qRBr-11.2) with Logarithmic of

Odds (LOD) > 3.0.and 7 suggestive QTLs (qRBr-1.1, qRBr-3.1, qRBr-6.2, qRBr-

10.1, qRBr-10.2, qRBr-11.3 and qRBr-12.1, LOD < 3.0) were detected for pathotype

P7.2. Meanwhile, three putative QTLs (qRBr-6.1, qRBr-11.4, and qRBr-12.1) with

Logarithmic of Odds (LOD) > 3.0 and 12 suggestive QTLs (qRBr-1.2, qRBr-2.1,

qRBr-4.1, qRBr-5.1, qRBr-6.2 qRBr-6.3, qRBr-8.1, qRBr-10.1, qRBr-10.2, qRBr-

11.1, qRBr-11.2 and qRBr-11.3, LOD < 3.0) were detected for pathotype P5.0.

Likelihood Ratio Statistics (LRS) for the association of the traits with locus at p ≤

0.05 ranged from 4.0 to 32.4. However, only 9 putative QTLs were found with a

single marker analysis and either interval mapping or composite interval mapping (at

LRS up to15) on chromosomes1, 2, 5, 6, 11 and 12. The individual locus found in the

population F3 for traits studied, explained 2-16% of the total phenotypic variance in

resistance against blast pathotypes. QTLs that had effects on both of the two blast

isolates, such QTLs may be commonly involved in the defense response against a

broad range of pathogens infections and others may only be involved in limited

defense responses, thus showing degrees of race specificity. Some of our identified

QTL were mapped to region where previously some Pi genes for blast resistance

have been reported. Such quantitative resistance genes might be defeated major R-

genes. In conclusion, from this research it was found that resistance to blast in

Pongsu Seribu 2 is very complex and is composed of a combination of Pi genes as

well as some unknown genes, and major and minor effects of multiple loci that

appear to contribute to partial resistance to local blast isolates.

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Abstrak tesis dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan ijazah Doktor Falsafah

DISEKSI MOLEKUL DAN PEMETAAN QTL KERINTANGAN PENYAKIT KARAH PADI MENGGUNA PENANDA ULANGAN JUJUKAN RINGKAS

Oleh

SADEGH ASHKANI

Disember 2011

Pengerusi: Profesor Madya Mohd Rafii bin Yusop, PhD

Fakulti: Pertanian

Teknologi penanda DNA dan pemetaan gen memainkan peranan penting dalam

pembiakbakaan penyakit tanaman padi (Oryza sativa L.). Karah yang disebabkan

oleh Magnaporthe oryzae adalah penyakit padi yang penting di Malaysia dan seluruh

dunia. Kekerapan kemuculannya di semua peringkat tumbesaran pokok

mengakibatkan penurunan ketara hasil dan kualiti bijian padi. Pencirian lokus

kuantitatif (QTL) kerintangan yang mengawal penyakit karah padi telah meningkat

kepentingannya di dalam program pembiakbakaan padi.. Kajian ini menggunakan

pendekatan penanda molekul bagi menganalisa genetik molekul yang rintang di

dalam populasi yang bersegregasi dan untuk mengenalpasti QTL yang rintang

terhadap dua patotip M oryzae yang berbeza, P7.2 dan P5.0 dalam famili F3 hasil dari

kacukan antara varieti Pongsu Seribu 2 (rintang) dan Mahsuri (rentan). Satu ratus dua

puluh lima penanda mikrosatelit yang berkait rapat dengan gen rintang penyakit

karah (Pi gen) meliputi kesemua 12 kromosom genom padi dipilih dan digunakan

dalam kajian menentukan polimorfisme dan potensi hubungannya dengan

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kerintangan kepada penyakit karah. Dua puluh tiga penanda polimorfik digunakan

bagi mengenalpasti nisbah kerintangan karah dalam 320 individu dalam populasi F2.

Sebelas penanda menunjukkan kecocokan yang baik dengan kadar segregasi yang

dijangka (1:2:1) untuk model gen tunggal (df=1.0, p≤0.05). Sementara penanda yang

lain segregasi tidak mengikut nisbah jangkaan segregasi Mendel. Famili F3 telah di

tanam di rumah hijau dan dilakukan rawatan saringan dengan patotip karah, P7.2 dan

P5.0. Dalam analisa ujian khi kuasadua untuk model satu gen, model dua gen bebas

dan kemungkinan model gen yang berbeza kesan epistasis, beberapa nisbah segregasi

(3R: 1S) dan (15R:1S) telah diperolehi masing-masing bagi patotip P7.2 dan P5.0.

Pongsu Seribu 2 di dapati ringtang terhapap kedua-dua patotip. Patotip P7.2 didapati

patotip karah yang paling virulen. Pokok yang rintang kepada patotip P7.2 daripada

populasi F3 didapati mempunyai kaitan baik dengan empat genotip penanda SSR,

RM413, RM1233, RM8225 dan RM5961, dimana nisbah segregasi dicerap adalah

(1:2:1) untuk model satu gen dominan. Penanda-penanda SSR ini didapati sesuai

digunakan di dalam pemilihan bantuan penanda gen rintang penyakit karah terhadap

Magnaporthe oryzae patotip P7.2. Sejumlah 188 famili F3 hasil kacukan Pongsu

Seribu 2 dan Mahsuri telah digunakan dalam kajian mengenalpasti QTL rintang

kepada Magnaporthe oryzae tempatan patotip P7.2 dan P5.0. Perhubungan yang

positif diperolehi untuk ketiga-tiga ciri (BLD, BLT and %DLA) bagi setiap patotip.

Analisa taburan sesuatu ciri didapati tidak memberikan variasi beterusan dan

taburannya tidak secara normal. Ujian distribusi keterukan penyakit (DS) didapati,

kedua-dua patotip condong ke arah rintang. Enam puluh tiga penanda SSR yang

polimorfik digunakan untuk membina peta tautan dalam kromosom bagi 188 famili

F3. Enam puluh tiga penanda SSR tersebut meliputi 3989.9 cM genom padi. Analisa

penanda tunggal (SMA), pemetaan interval (IM) dan pemetaan komposit interval

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(CIM) dengan analisa permutasi digunakan dalam analisa QTL. Dua puluh lapan

QTL berasingan telah dikesan mempunyai hubungan dengan kerintangan karah pada

kromosom 1, 2, 3, 5, 6, 8, 10, 11 and 12. Enam putatif QTL (qRBr-1.2, qRBr-2.1,

qRBr-5.1, qRBr-6.1, qRBr-11.1 and qRBr-11.2) dengan Logarithmic of Odds (LOD)

> 3.0 dan 7. sugestif QTL (qRBr-1.1, qRBr-3.1, qRBr-6.2, qRBr-10.1, qRBr-10.2,

qRBr-11.3 dan qRBr-12.1, LOD < 3.0) dikesan bagi patotip P7.2. Manakala, 3

putatif QTL (qRBr-6.1, qRBr-11.4, and qRBr-12.1) dengan Logarithmic of Odds

(LOD) > 3.0 and 12 jangkaan QTLs (qRBr-1.2, qRBr-2.1, qRBr-4.1, qRBr-5.1,

qRBr-6.2 qRBr-6.3, qRBr-8.1, qRBr-10.1, qRBr-10.2, qRBr-11.1, qRBr-11.2 and

qRBr-11.3 LOD < 3.0) dikesan bagi patotip P5.0. Statistik nisbah kebarangkalian

(LRS) untuk perhubungan ciri dengan lokus pada p ≤ 0.05 adalah dari julat 4.0

hingga 32.4. Walaubagaimanapun, hanya 9 putative QTL diperolehi dengan Analisa

penanda tunggal dan samada pemetaan interval dan pemetaan komposit interval

(pada LRS lebih daripada 15). Lokus individu yang dijumpai dalam populasi F3 bagi

ciri-ciri ynag dikaji, menerangkan 2-16% daripada jumlah variasi fenotip kerintangan

terhadap patotip karah. QTL yang mempunyai kesan ke atas kedua patotip,

kemungkinan terlibat dalam gerakbalas pertahanan terhadap jangkitan pathogen dan

yang lain mungkin terlibat pada gerakbalas pertahanan yang terhad, menunjukkan

terdapat spesifisiti terhadap ras patotip. Sebahgian QTL yang ditemui dipeta dalam

rantau Pi-gen yang telah dilaporkan oleh penyelidik sebelum ini. Gen rintang

kuantitatif tersebut mungkin disebabkan oleh R-gen utama. Kesimpulannya, dari

penyelidikan ini didapati kerintangan terhadap karah dalam Pongsu Seribu 2 adalah

sangat komplek, dan ianya dibentuk dari satu kombinasi beberapa Pi gen serta gen

yang tidak diketahui, dan kesan major dan minor pelbagai lokus yang menyumbang

kepada separa rintang terhadap isolasi karah tempatan.

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ACKNOWLEDGEMENTS

My outmost praise goes to The Greatest and Almighty Allah who enabled me to

undertake and complete this dissertation successfully. I hope in some way this work

will glorify His name. It is a pleasure to thank those who helped me to complete this

work.

First and foremost, I would like to express my deep and sincere appreciation to the

chair of my Supervisory Committee, Assoc. Prof. Dr. Mohd Rafii Bin Yusop, for his

thoughtful guidance, friendship and consistent support during the entire period of my

study.

Special thanks are directed to other members of my supervisory committee, Prof. Dr.

Sariah Meon, Assoc. Prof. Datin Dr. Siti Nor Akmar and Dr. Rusli Ibrahim, for their

invaluable advice and guidance, and have continuously provided support and

motivation throughout all the phases of my work.

I am indebted to Dr. Norimah Yusof, Director of Agrotechnology and Biosciences

Division, Malaysian Nuclear Agency (Nuclear Malaysia), for providing me with the

facilities and support to complete the study.

I highly appreciate Dr. Abdul Rahim Harun, from Nuclear Malaysia, for his

significant involvement in my research, whom has continuously provided support,

kind consultations, friendship and helped me in various ways.

I wish to thank Dr. Azhar Mohamad from Nuclear Malaysia, for his invaluable

advice and sharing of experiences on laboratory works. My special thanks go to other

staff of Nuclear Malaysia for their kindness and assistance.

I wish to express my deep appreciation to Tn. Hj. Saad Abdullah from the Malaysian

Agricultural Research and Development Institute (MARDI), Seberang Perai, for his

guidance in blast screening and providing me with starting materials and greenhouse

facilities. I would like to thank all the glasshouse and laboratory staff of MARDI

Seberang Perai for their kind assistance.

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I am grateful to Dr. M. A. Latif from Bangladesh Rice Research Institute, for his

constructive technical advices and help during the publishing of the manuscript of

this dissertation.

I thank the Department of Crop Science, Faculty of Agriculture, Universiti Putra

Malaysia (UPM), for providing me with the facilities and support to complete the

study.

Thanks are due to my colleagues, to my lab mates for sharing knowledge with me

and providing a stimulating and enjoyable environment in which to learn and

develop. I am indebted to my many of friends who helped me in various ways. They

shared many important and memorable moments with me during my stay in Malaysia.

I wish to thank all my brothers, sisters and other family members; and to my

relatives, who are always providing me with moral support until the completion of

my studies, I am extremely thankful. You all love me unconditionally, I couldn’t ask

for more. I love you all. May God bless you mightily.

I would like to thank my mother-in-law, Hajar Nazarzadeh, and brothers and sister-

in-law, for their love, support and encouragement.

I am deeply grateful to my dearest mother Fatemeh Aghaloo and my late father

Golam Reza Ashkani. They bore me, raised me, supported me, taught me, and loved

me, and whose continuous prayers and blessings I seek all the time.

My gratitude goes to my dear children, to my daughter Hasti Ashkani and my son

Mobin Ashkani, the little angels bringing us joy and happiness, who have kept my

feet on the ground.

Most of all, I am deeply indebted to my dear wife, Fatemeh Azad, for her love,

support, continual encouragement, believing in me and patience throughout the

duration of my entire work.

Sincerely

SADEGH ASHKANI

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I certify that a Thesis Examination Committee has met on 15 December 2011 to conduct the final examination of Sadegh Ashkani on his thesis entitled " MOLECULAR DISSECTION AND QTL MAPPING OF RICE BLAST DISEASE RESISTANCE USING SIMPLE SEQUENCE REPEAT MARKERS " in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Thesis Examination Committee were as follows: Mihdzar Bin Abdul Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Maziah binti Mahmood, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Maheran Abd. Aziz, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Muhammad Ashraf, PhD Professor Faculty of Sciences University of Agriculture, Faisalabad Pakistan (External Examiner)

SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirements for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows: Mohd Rafii Bin Yusop, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Sariah Meon, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Member) Siti Nor Akmar Abdullah, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

Rusli Ibrahim, PhD Principal Researches Agrotechnology & Bioscience Division Agensi Nuklear Malaysia (Member)

BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institutions.

SADEGH ASHKANI Date: 15 December 2011

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TABLE OF CONTENTS

Page ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xviii LIST OF FIGURES xxi LIST OF ABBREVIATIONS xxvii CHAPTER

1 INTRODUCTION 1 1.1 General Introduction 1 1.2 Significance of Study 3 1.3 Problem Statement 4 1.4 Research Objectives 5

2 LITERATURE REVIEW 6

2.1 Rice (Oryza sativa) 6 2.1.1 Rice Taxonomy and Biology Description 6 2.1.2 Rice Production and Nutrition Importance 8 2.1.3 The Rice Genome 11 2.1.4 Rice Molecular Genetics Strategy 11 2.2 Rice Blast Disease 12 2.2.1 Magnaporthe oryzae: Infection and Life Cycle 13 2.2.2 Blast Symptoms 15 2.2.3 Pathogenic Variation of Magnaporthe oryzae 16 2.3 Blast Disease Management 17 2.4 Disease Resistance in Plant 17 2.5 Classification of Blast Resistance 19 2.6 Blast Disease Resistance Genes in Rice 20 2.7 Molecular Marker Techniques for Genome Analysis 21 2.7.1 Non- PCR Based Techniques 22 2.7.2 PCR-Based Techniques 23 2.8 Molecular Markers and Blast Disease Improvement 28

2.9 Marker Assisted Selection (MAS) 28 2.10 Genetic Linkage Maps Construction 29 2.11 QTL Mapping 31 2.11.1 Population Structures and Experimental Designs 33

2.11.2 Data for QTL Mapping 34 2.11.3 QTL Mapping Methods 35

3 SELECTION AND USE OF MICROSATELLITES MARKERS

FOR GENETIC ANALYSIS OF RICE BLAST DISEASE

38 3.1 Introduction 38

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3.2 Materials and Methods 40 3.2.1 Plant Materials 40 3.2.2 DNA Extraction 40 3.2.3 SSR Marker Selection 42 3.2.4 Optimisation of Annealing Temperature for SSRs

Primer Pairs

42 3.2.5 PCR Amplification 48 3.2.6 Genotyping for Marker Segregation 49 3.2.7 Disease Evaluation on F2 Populations 49 3.2.8 Statistical Analysis 51

3.3 Results and Discussion 51 3.3.1 DNA Quality and Quantity Identification 51 3.3.2 Optimization of PCR Conditions to Amplify

Microsatellite Loci 54

3.3.3 Marker Polymorphism Between Parents 55 3.3.4 SSR Markers Survey on Parents and F2 Populations 56 3.3.5 Markers Segregation Data Analysis 59 3.3.6 Inheritance of Blast Resistance in F2 Population 61

3.4 Conclusion 65

4 BLAST SCREENING FOR VIRULENCE OF LOCAL M. oryzae PATHOTYPES AND INHERITANCE STUDY IN F3 FAMILIES

66

4.1 Introduction 66 4.2 Materials and Methods 67

4.2.1 Plant Materials 67 4.2.2 Magnaporthe oryzae Pathotypes 68 4.2.3 Media Preparation 69

4.2.4 Single Spore Isolation 70 4.2.5 Induction of M. oryzae Sporulation 70 4.2.6 Preparation of Inoculums 70 4.2.7 Seedling Preparation and Experimental Design 71 4.2.8 Pathogen Inoculation and Disease Scoring 73

4.2.9 Statistical Analysis 76 4.3 Results and Discussion 76 4.3.1 Trait Distribution 76

4.3.2 Trait Correlations 80 4.3.3 Pathotypes Virulence through Disease Reaction

Pattern

81 4.3.4 Evaluation of Blast Resistance in F3 Plants 82 4.3.5 Resistance Traits Segregation in F3 Population 83 4.4 Conclusion 89

5 SSR MARKERS SUITABLE FOR MARKER ASSISTED

SELECTION FOR BLAST RESISTANCE GENES TO IMPROVE RICE (Oryza sativa L.) CULTIVAR

91

5.1 Introduction 91 5.2 Materials and Methods 93 5.2.1 Plant Materials 93

5.2.2 Inoculation Methods and Disease Assessment 94 5.2.3 DNA Extraction 95

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5.2.4 Microsatellite Analysis 96 5.2.5 Marker Genotyping 99 5.2.6 SSR Marker-Assisted Selection 99 5.2.7 Statistical Analysis 99

5.3

Results and Discussion 100 5.3.1 Phenotypic Evaluation 100 5.3.2 Molecular Marker Assays 101 5.3.3 Effects of Markers on Disease Severity 104 5.3.4 Marker-Assisted Selection 104

5.4 Conclusions 107

6 IDENTIFICATION OF QTLs FOR BLAST RESISTANCE USING F3 POPULATIONS DERIVED FROM CROSS BETWEEN PONGSU SERIBU 2 AND MAHSURI

108 6.1 Introduction 108 6.2 Materials and Methods 110

6.2.1 Mapping Population 110 6.2.2 Blast Infection and Assessment 111 6.2.3 DNA Isolation 111 6.2.4 PCR Amplification 112 6.2.5 Genotyping F3 Plants Using Fluorescently-

Labelled Microsatellite Panels

114 6.2.6 Fragment Size Determination 119 6.2.7 Chromatograph 120 6.2.8 Linkage Mapping 120 6.2.9 QTL Analysis 121 6.2.10 Nomenclature 123

6.3

Result and Discussion 123 6.3.1 Trait Evaluation and Distribution in The F3

Populations

124 6.3.2 Genotyping and Fragment Size Determination 128 6.3.3 Linkage Map Construction 131 6.3.4 QTLs Identification 133 6.3.5 QTL Analysis for Resistance to Pathotype P7.2 145 6.3.6 QTL Analysis for Resistance to Pathotype P5.0 147 6.3.7 Confirmation of Significant QTLs Identified in F3

Population

150 6.3.8 Loci Associated with Blast Resistance 160

6.4 Conclusion 169

7 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE RESEARCH

172

7.1 Summary 172 7.2 Conclusion 178 7.3 Recommendations for Future Work 182 REFRENCES/BIBLIOGRAPHY 184 APPENDICES 208 BIODATA OF STUDENT 225