Identification and characterisation of an altered gene in the novel ABA insensitive Beyma mutant of Lotus japonicus Nur Fatihah Mohd Yusoff BSc. MSc. A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Agriculture and Food Sciences Centre for Integrative Legume Research i Abstract Various approaches can be implemented to identify a gene responsible for a phenotype of interest due to mutagenesis. Current next generation sequencing (NGS) technology allows whole genome sequencing of a mutant and accelerates the identification of mutation-induced polymorphisms in mutagenised organisms. In the model legume Lotus japonicus ecotype Miyakojima (MG-20), an abscisic acid (ABA) insensitive mutant called Beyma was previously isolated by ethyl methanesulphonate (EMS) mutagenesis and originally identified as a heterozygous dominant mutation. Beyma is slow-growing, wilty and incapable of regulating stomatal opening. A wild type segregant of the Beyma mutant (WTS) was also isolated from a self-generation of heterozygous Beyma mutants. ABA plays numerous roles in plant growth and development as well as morphogenetic responses including nodulation in legumes. Yet, there is a lack of ABA studies in legumes. Therefore, the Beyma mutant in L. japonicus allows a wide range of studies that will provide in-depth information of ABA signaling in nodulation as well as stress responses in legumes. This project presents an attempt to identify a causal gene in the ABA insensitive Beyma using the NGS technology. Tissue from a homozygous Beyma mutant, WTS and MG-20 wild type (WT) plants was subjected to the whole genome sequencing, generating about 300 million paired end of short-sequence reads. The Kazusa MG-20 genome was used as reference for read mapping. Single nucleotide polymorphisms (SNPs) were called based on mutations in the Beyma and WTS genomes as compared to the re-sequenced MG-20 genome. As a preliminary study, three procedures of read mapping and variant calling were performed to undertake a genomic comparative analysis and identify the causal gene. Sequencing of single genomes of the three plants showed a mutation occurred in every 208 kb (WTS) and 202 kb (Beyma) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes but each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. A total of 59 SNPs were identified as potential putative causal Beyma mutations, which were located in various annotated genes in the MG-20 genome. Verification of these mutations could not be done due to time constraint but will be performed in future. A candidate gene i approach was also carried out by selecting ABA-related genes based on their roles in ABA biosynthesis to signalling, directly or indirectly. Mutations were found in loci of ABA INSENSITIVE (ABI) 1, ABI2, HAB1, HAB2, ABI3, ABI4, and ABSCISIC ACID 8'- HYDROXYLASE 2 in both mutant genomes or only in the WTS genome. Unique mutations also occurred in EARLY RESPONSIVE TO DEHYDRATION 7 and ABSCISIC ACID 8'- HYDROXYLASE 1/ P450 CYP707A1 genes, which were predicted to be impaired in their downstream regions. Although the candidates were not affected in the essential region of the genes, the candidate gene approach has eliminated all the candidates as the putatively causal Beyma gene. In order to intensify the identification of the causal Beyma gene, re-sequencing of the Beyma and WTS genomes was performed on pooled DNA. In this analysis, the presence of mutations was more frequent in both mutagenised genomes (~18-35% increase), resulting in higher rate of base changes and demonstrated that pooled DNA sequencing increased the mutation frequency. In addition, 69 unique Beyma SNPs were predicted to cause nonsynonymous changes and will be verified in future study. Nevertheless, a mutation (locus named chr3.CM0451.1060.r2.d) was found in both batches of sequencing. It was a C-to-T mutation, which changed glutamic acid to lysine in an F-box family gene. This gene could be the Beyma gene but it requires verification. In conjunction with the genome sequence analysis, other analyses were also done to prepare plant materials for sequencing and future verification. Plants were subjected to ABA treatment on seed germination and root development to select good mutant lines and WTS plants. Outcross between Beyma and L. japoncus ecotype Gifu was also performed for the segregation analysis of the putative causal SNPs in the F2 plants carrying homozygous WT alleles. This project highlighted the overall molecular changes produced in the whole genome of MG-20 mutants due to EMS mutagenesis. In future, the identification of the causal Beyma gene will possibly show a novel gene involved in ABA sensitivity in legume systems. In addition, it should be of great interest for researchers in forward genetics in legume studies. ii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii Publication during candidature Peer-reviewed paper Mohd-Yusoff NF, Ruperao P, Tomoyoshi NE, Edwards D, Gresshoff PM, Biswas B and Batley J (2014). Scanning ethyl methanesulphonate effects on the whole genome of Lotus japonicus using second generation sequencing analysis. G3: GenesIGenomesIGenetics 5: 559-567. Conference abstract Mohd Yusoff NF, Kazakoff S, Jacqueline B, Bandana B and Gresshoff P (Sept 2012). An altered gene in the novel ABA insensitive Beyma mutant of Lotus japonicus. Combio, Adelaide, Australia. Publication included in this thesis Mohd-Yusoff NF, Ruperao P, Tomoyoshi NE, Edwards D, Gresshoff PM, Biswas B and Batley J (2014). Scanning ethyl methanesulphonate effects on the whole genome of Lotus japonicus using second generation sequencing analysis. G3: GenesIGenomesIGenetics (submitted and accepted subject to minor revisions) – incorporated as Chapter 2. Contributor Statement of contribution Designed experiment (30%) Nur Fatihah Mohd-Yusoff Conducted experiment (55%) (Candidate) Wrote manuscript (100%) Pradeep Ruperao Conducted experiment (33%) Nurain Emylia Tomoyoshi Conducted experiment (1%) David Edwards Conducted experiment (1%) Designed experiment (15%) Peter M Gresshoff Edited paper (10%) Designed experiment (25%) Bandana Biswas Conducted experiment (10%) Edited paper (45%) Designed experiment (30%) Jacqueline Batley Edited paper (45%) iv Contributions by others to the thesis Chapter 4 Dr Stephen Kazakoff and Mr. Pradeep Ruperao conducted experiments and contributed to experimental design of read mapping and SNP calling analyses. Chapter 5 Dongxue Li and Dr Satomi Hayashi conducted experiments for re-sequencing. Ms. Jenny Lee conducted read mapping and SNP calling analyses. All Chapters Supervisors, Prof. Peter M Gresshoff, Dr Bandana Biswas and Prof. Jacqueline Batley, assisted in experimental design and editing of the writing. Statement of parts of the thesis submitted to qualify for the award of another degree None v Acknowledgements First, I would like to thank Allah for giving me strength and wisdom to complete this study. It is my pleasure to acknowledge the Ministry of Education, Malaysia and Universiti Putra Malaysia for sponsoring my study at the University of Queensland. I would also like to thank the Centre for Integrative Legume Research (CILR) and School of Agriculture and Food Sciences for giving the opportunity to conduct my PhD research and providing funds for conference attendance and travel. No words will be able to describe my heartfelt gratitude and appreciation to my supervisor Prof. Peter M Gresshoff for his constant guidance, invaluable advice, stimulating discussions and ideas throughout the course of this project. Special thanks are extended to other advisors, Dr Bandana Biswas and Prof. Jacqueline Batley, for their advices, comments and guidance whenever sought. I really appreciate their patience and understanding. I would also like to thank all CILR staff and students, especially former CILR student Dr Stephen Kazakoff, for their technical assistance whenever needed, the brainstorming discussion we had together and creating a good environment working in the laboratory. My appreciation also goes to all of my friends, who studied in the University of Queensland, for their friendships and supports while living in Brisbane. Not forgotten, my special thanks go to Ayah, my siblings and family for giving me support while studying oversea. Last but not least, I would like to express my gratitude to my beloved husband, Uzaeir, and kids, Uthman and Ulfah, for always being there and supporting my PhD. I also appreciate my husband for his technical assistance in my PhD research whenever he visited me in Brisbane. vi Keywords legumes, Lotus japonicus, next generation sequencing, Beyma, EMS mutagenesis, abscisic acid, candidate gene Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060702, Plant Cell and Molecular Biology, 20% ANZSRC code: 060705, Plant Physiology, 20% ANZSRC code: 060102, Bioinformatics, 60% Field of Research (FoR) Classification FoR code: 0604, Genetics (35%) FoR code: 0607, Plant Biology (65%) vii Table of Contents Content Page Abstract i Acknowledgements vi List of figures xi List of tables xii List of abbreviations xiv Chapter 1 General introduction 1 1.1 Abstract 1 1.2 Introduction 2 1.2.1 Lotus japonicus 3 1.2.2 Lotus japonicus genome project 5 1.2.3 Bioinformatics resources available on Lotus japonicus and other 5 legumes 1.2.4 Next generation sequencing 7 1.2.5 Next generation sequencing in the legume genomes 9 1.2.6 ABA perception and signaling 10 1.2.7 ABA roles and genes involved in legumes 12 1.2.8 Description of Beyma 13 1.3 Statement of thesis aims and structures 14 Chapter 2 15 Mohd-Yusoff NF, Ruperao P, Tomoyoshi NE, Edwards D, Gresshoff PM, Biswas B and Batley J (2014). Scanning ethyl methanesulphonate effects on the whole genome of Lotus japonicus using second generation sequencing analysis. G3: GenesIGenomesIGenetics 5: 559-567. Chapter 3 Identification of mutation in an ABA insensitive Beyma mutant 25 using a candidate gene approach 3.1 Abstract 25 3.2 Introduction 26 3.3 Materials and methods 27 3.3.1 Selection of genes involved in ABA perception and signaling 27 viii pathways 3.3.2 Identification of orthologs of candidate genes 28 3.3.3 Identification of unique base changes in the Beyma genome 28 3.3.4 Identification of SNPs in candidate loci 29 3.4 Results 29 3.4.1 Candidate genes in Arabidopsis and other plants 29 3.4.2 Orthologs of candidate genes 29 3.4.3 Mutation in candidate genes 34 3.5 Discussion 36 3.6 Conclusion 38 Chapter 4 Identification of a potentially causative mutation in the Beyma 39 mutant 4.1 Abstract 39 4.2 Introduction 40 4.3 Materials and methods 42 4.3.1 Plant materials 42 4.3.2 Genomic DNA extraction 42 4.3.3 Dehydration screening 43 4.3.4 Genomic sequencing 43 4.3.5 Read mapping and SNP calling 43 4.3.6 SNP analysis 45 4.3.7 PCR sequencing 46 4.4 Results 46 4.4.1 Selection of homozygous Beyma lines 46 4.4.2 Screening of F2 population 47 4.4.3 Putative SNPs in the Beyma genome 48 4.4.4 Putative causative mutation 55 4.4.5 Verification of causative mutation in F2 plants 57 4.4.6 Verification of putative causative SNPs in the mutants 58 4.5 Discussion 58 4.5.1 Phenotyping of F2 plants 58 4.5.2 Identification of putative causative SNPs 59 4.5.3 Background mutation 61 ix