EXPRESSION PROFILING OF HUANGLONGBING DISEASE IN CITRUS THROUGH MOLECULAR TECHNIQUES By Rozina Aslam M. Phil. Biochemistry (UAF) A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biochemistry Department of Biochemistry Faculty of Sciences University of Agriculture Faisalabad, Pakistan 2016 Declaration I, Rozina Aslam, Regd. No. 92-ag-1367, hereby declare that the contents of the thesis “EXPRESSION PROFILING OF HUANGLONGBING DISEASE IN CITRUS THROUGH MOLECULAR TECHNIQUES” are product of my own research and no part has been copied from any published source (except the references, standard mathematical and genetic models/equations/formulas/protocols etc.). I further declare that this work has not been submitted for award of any other diploma / degree. The university may take action if the information provided is found inaccurate at any stage. In case of any default, the scholar will be proceeded against as per HEC plagiarism policy. ROZINA ASLAM 92-ag-1367 To The Controller of Examination University of Agriculture, Faisalabad. "We, the Supervisory Committee, certify that the contents and form of the thesis submitted by Rozina Aslam have been found satisfactory and recommend that it be processed for evaluation by the external Examiner(s) for the award of degree." SUPERVISORY COMMITTEE: CHAIRMAN ------------------------------------- (Prof. Dr. Khalil-ur-Rahman) MEMBER ------------------------------------- (Prof. Dr. Muhammad Asghar Bajwa) MEMBER ------------------------------------- (Prof. Dr. Iqrar Ahmad Khan) Dedicated to Late Father and Mother May Allah rest their souls in peace ACKNOWLEDGEMENTS First of all, immeasurable thanks to Almighty Allah for his mercy and guidance in giving me full strength to complete this task. May Allah’s peace and blessing be upon our beloved prophet Muhammad (PBUH). I am highly grateful to my supervisor, Prof. Dr. Khalil-ur-Rahman whose expertise, generous guidance and support made it possible for me to complete this task. Without his guidance and persistent help this dissertation would not have been possible. I would like to thank my committee member Prof. Dr. Muhammad Asghar for his precious guidance and support which was extremely valuable for my study both theoretically and practically. My deepest gratitude, special thanks and best regards goes to my most respectable committee member, Prof. Dr. Iqrar Ahmad Khan (Vice Chancellor, University of Agriculture, Faisalabad, Pakistan) for his precious supervision and kind guidance, keen interest, prompt inspiration, timely suggestion with kindness, arrangement of all research facilities, scholarly advice and scientific approach which have helped me and made it possible to complete my research project. I would like to express my gratitude to all my teachers for their advices, sharing knowledge and who put their faith in me and urged me to do better. I thank profusely to all the faculty and staff, Department of Biochemistry, Centre for Agricultural Biochemistry and Biotechnology (CABB) and Greenhouse, Institute of Horticultural Sciences for their support and cooperation. I would like to thank my parents and all other family members especially my brother Sher Afgan, sisters and in laws, for their kind help, support, motivation and prayers, all the time to achieve this difficult task. It is my privilege to thank especially my husband for his encouragement, support and help in the lab and field throughout my research period. I express my deepest thanks to Prof. Dr. Mikeal L. Roose (Chairman, Department of Botany and Plant Sciences, University of California, Riverside (UCR), USA.) and Dr. Claire Thomas Federici (UCR) for their kind support and help for completing molecular studies of my research. I also appreciate the kind support and help of Dr. Glen Hicks, Neerja katiar, John weiger and Clay Clark (IIGB, UCR). I also appreciate the kind cooperation and support of Makbule koksal and Kimbely Gentile (UCR). I acknowledge the financial support of HEC (Higher Education Commission), Government of Pakistan, and Pak-US project “Management of citrus greening by producing healthy plants, monitoring vectors and identification of tolerance” due to which this study became possible. I also appreciate and acknowledge Dr. Richard F. Lee, Dr. Robert Kruger and all staff members of USDA National Clonal Germplasm Repository for Citrus and Dates, Riverside, USA for their help and providing citrus germplasm seed. Rozina Aslam TABLE OF CONTENTS Chapter No. TITLE Page No. ACKNOWLEDGEMENTS i LIST OF ABBREVIATIONS iv TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES xii ABSTRACT xiv 1 INTRODUCTION 1 1.1 NEED FOR THE PROJECT 3 1.2 OBJECTIVES 5 2 REVIEW OF LITERATURE 6 2.1 Huanglongbing 6 2.2 Huanglongbing occurance in Pakistan 7 2.3 Liberibacter transmission by Asian citrus psyllid 7 2.4 8 Huanglongbing diagnosis 2.4.1 9 Huanglongbing diagnosis by the presence of natural vector 2.4.2 10 Huanglongbing diagnosis on the basis of symptoms 2.4.3 10 Huanglongbing diagnosis by biochemical test 2.4.4 11 Huanglongbing diagnosis by microscopy 2.4.5 11 Huanglongbing diagnosis by molecular techniques 2.5 13 Expression profiling of huanglongbing 3 MATERIALS AND METHODS 17 3.1 EXPERIMENT-1. Rearing of citrus germplasm in greenhouse 17 3.2 EXPERIMENT-2. Asian citrus psyllid rearing 22 3.3 EXPERIMENT-3. Huanglongbing diagnosis in ACP infested 25 plants and inoculation of citrus germplasm 3.3.1 Huanglongbing diagnosis 25 3.3.1.1 DNA extraction 25 3.3.1.2 DNA quatification by agarose gel electrophoresis 26 3.3.1.3 Polymerase chain reactions for HLB diagnosis 27 3.3.1.4 PCR product analysis 28 3.3.2 Inoculation of citrus germplasm 29 3.4 EXPERIMENT-4 Detection of Candidatus Liberibacter 33 asiaticus in citrus germplasm 3.4.1 Sample collection 33 3.4.2 Shipment of DNA to USA and Zymopurification 34 3.4.3 Citrus germplasm DNA quantification by spectrophotometry 35 3.4.4 Real-time PCR for Candidatus Liberibacter asiaticus detection 36 in citrus germplasm 3.4.4.1 Taqman based qPCR protocol for Las detection in citrus 37 germplasm 3.5 EXPERIMENT-5 Expression profiling of huanglongbing 39 disease in citrus 3.5.1 RNA extraction and shipment to USA 39 3.5.2 Synthesis of cDNA using NEB MMLV enzyme 41 3.5.3 Conventional PCR for the detection of internal control gene 42 malate dehydrogenase 3.5.4 Treatment of RNA samples with DNase 43 3.5.5 Qubit quantification of RNA samples 44 3.5.6 Qubit assay for RNA 45 3.5.7 Qubit assay for DNA 46 3.5.8 Purification of citrus germplasm RNA 48 3.5.9 Expression profiling of huanglongbing disease in citrus by real 53 time quantitative PCR 3.5.9.1 SYBR green based quantitative real time PCR 53 3.5.10 RNA-Seq library preparation for Illumina 59 4 RESULTS AND DISCUSSION 61 4.1 EXPERIMENT-1 Rearing of citrus germplasm in greenhouse 61 4.2 EXPERIMENT- 2 Asian citrus psyllid rearing 65 4.3 EXPERIMENT- 3 Huanglongbing diagnosis in ACP infested 68 plants and inoculation of citrus germplasm 4.3.1 Huanglongbing diagnosis 68 4.3.2 Inoculation of citrus germplasm 68 4.4 Experiment 4. Detection of Candidatus Liberibacter asiaticus 74 in citrus germplasm 4.4.1 Correlation between HLB symptoms and Ct values 75 4.5 Experiment 5. Expression Profiling of Huanglongbing Disease 81 in Citrus Germplasm by real time quantitative PCR 4.6 Assessment of RNA-Seq library quality on bioanalyzer 96 5 SUMMARY 98 6 RECOMMENDATIONS 100 REFERENCES 101 LIST OF TABLES Table No. Title Page No. 3.1 Citrus germplasm sown for the detection and expression 18 profiling of huanglongbing. 3.2 Nutrients composition applied to the germplasm 22 3.3 Composition of CTAB buffer for DNA extraction 26 3.4 The primers sequence for HLB diagnosis by conventional 27 PCR studies 3.5 Composition of reaction mixture for conventional PCR 28 3.6 Thermal profile for conventional PCR 28 3.7 Composition of 5 X TBE buffer for one liter solution 28 3.8 Composition of 50X TAE buffer for one liter solution 29 3.9 DNA clean and concentrator contents 34 3.10 The primer/probe sequence for Las detection by qPCR studies 37 3.11 Reaction mixture for Taqman based qPCR 38 3.12 Composition of RNA extraction buffer 40 3.13 Nucleic acid concentrations for initial evaluation of RNA by 41 nanodrop 3.14 Primers and dNTPs cocktail for cDNA 41 3.15 Reverse transcription (RT) reaction cocktail 42 3.16 Composition of PCR reaction mix for MDH gene 42 3.17 PCR Conditions for MDH gene 43 3.18 Specification of Qubit assay for RNA and DNA 45 3.19 Qubit assay values for RNA and DNA 46 3.20 Comparison of RNA concentrations checked by nanodrop and 48 bioanalyzer along with RIN values 3.21 RNA clean and concentrator contents 48 3.22 Nanodrop concentrations of RNA after purification 49 3.23 Composition of TE buffer for primer dissolving 54 3.24 Thermal conditions for SYBR green based PCR reactions 55 3.25 List of primers used for expression profiling of HLB in citrus 55 by qPCR. 3.26 Citrus germplasm tested for expression profiling of HLB 57 4.1 Survived citrus germplasm from 97sown genotypes of citrus 62 4.2 Citrus genotypes expressing huanglongbing symptoms 70 4.3 Mean cycle threshold values in citrus germplasm DNA for the 79 detection of Candidatus Liberibacter asiaticus 4.4 Comparison of expression of 7 genes in 45 genotypes of HLB 82 infected citrus using qRT PCR analysis 4.5 Sample information for cDNA library concentrations by 96 nanodrop and index primer sequences LIST OF FIGURES Fig. No. Title Page No. 3.1 Greyish black polythene bags having a size of 15x9 inch 18 used for citrus germplasm seed