Genomic characterization of African cultivated and wild Oryza species Peterson Weru Wambugu Msc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Queensland Alliance for Agriculture and Food Innovation Abstract Rice is the most important crop in the world, acting as the staple food for over half of the world’s population. It belongs to the Oryza genus and has a wide genetic diversity of both wild and cultivated species, with a substantial proportion of this diversity being found in Africa. In this thesis, African cultivated and wild Oryza species have been characterized using genomic tools with the goal of promoting their conservation and utilization. A desk based study aimed at documenting the current state of conservation and utilization of African Oryza genetic resources revealed that they remain under collected and hence underrepresented in germplasm collections. Moreover, they are under-characterized and therefore grossly underutilized. In order to obtain maximum benefits from these resources, it is imperative that they are collected, efficiently conserved and optimally utilized. Efficient conservation and utilization of the Oryza gene pool is however hindered by lack of conclusive, consistent and well resolved phylogenetic and evolutionary relationships between the various cultivated and wild species. This study therefore sought to define the genetic and evolutionary relationships between the various wild and cultivated African species that form part of the AA genome group. This was done by reconstructing the phylogeny of Oryza AA genome species by massive parallel sequencing of whole chloroplast genomes. The analysis resulted in a well resolved and strongly supported phylogeny of the AA genome species showing strong genetic differentiation. The clearly defined relationships between the various taxa provide a robust platform for supporting the conservation and utility of these species. In an effort at promoting utility of these species, their starch physicochemical properties and the functional diversity relating to these starch related traits were analyzed. African rice was found to have some unique starch structure and properties, among these being high amylose content which is higher than in Asian rice. Amylose content is a key starch trait in rice. The health benefits of high amylose foods are increasingly getting recognized. African rice therefore seems to be a valuable genetic resource for breeding premium-value genotypes with high amylose content. The deployment of African rice genetic resources in rice improvement is however constrained by the inadequate understanding of the genetic mechanism underlying their unique starch traits, specifically amylose content. First, this study analysed key starch biosynthesis genes in the genomes of various cultivated and wild Oryza species. The analysis revealed that the pullulanase gene has undergone gene duplication in African rice. This apparent gene duplication was characterized with the aim of understanding whether it ii could be the molecular mechanism underlying high amylose in African rice. It was hypothesized that this gene duplication may have led to increased pullulanase activity which may have subsequently led to unique starch structure and properties due to protein dosage effects. However, pullulanase activity between O. glaberrima and O. sativa was not significantly different as would have been expected. The results therefore did not support the study hypothesis and it therefore appears that the high amylose might be due to other genetic mechanisms and not due to pullulanase gene duplication. Further analysis was therefore required in order to unravel these genetic mechanisms underlying amylose content. This was conducted by genetic mapping of an interspecific cross between O. sativa and O. glaberrima segregating for amylose content. The aim was to identify genetic variants and candidate genes associated with amylose biosynthesis. Phenotyping for amylose content resulted in a normal distribution of amylose content showing transgressive segregation. Sequencing based bulk segregant analysis enabled the identification of markers putatively linked with amylose content. In particular, a G/A SNP located on chromosome 12 of granule bound starch synthase (GBSS1) that causes an amino acid change from Asp to Asn was identified. This variation may influence the stability of GBSS1 and hence its activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. A total of 106 candidate SNPs clustered with this polymorphism in a genomic region of about 2.1Mbp, most likely due to linkage drag. Finally, an amylose candidate gene analysis was conducted using an integrative genetics approach. This involved a combination of several strategies namely genomic based bulk segregant analysis, gene co-expression analysis and analysis of cis regulatory motifs. Two candidate transcription factors belonging to NAC and CCAAT-HAP5 families were found to be co- expressed with key starch genes and also to share conserved regulatory motifs with known amylose biosynthesis genes. Results of this study suggest that these transcription factors are involved in the transcriptional regulation of the GBSS1 structural gene. These candidate genes provide novel targets for manipulating amylose content. iii 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. iv Publications during candidature Peer reviewed papers Wambugu, P., Furtado, A., Waters, D., Nyamongo, D. & Henry, R. 2013. Conservation and utilization of African Oryza genetic resources. Rice, 6, 29-29. Wambugu, P.W., Brozynska, M., Furtado, A.., Waters, D.L. and Henry, R.J. 2015. Relationships of wild and domesticated rices (Oryza AA genome species) based upon whole chloroplast genome sequences. Scientific Reports 5, 13957 Wang, K., Wambugu, P.W., Zhang, B., Wu, A.C., Henry, R.J., and Gilbert, R.J. 2015. The biosynthesis, structure and gelatinization properties of starches from wild and cultivated African rice species (Oryza barthii and Oryza glaberrima). Carbohydrate Polymers 129, 92- 100 Conference abstracts Wambugu, P.W. A. Furtado and R. Henry. 2015. Molecular and genetic basis of unique starch properties in African rice (Oryza glaberrima). Paper presented at the 65th Australasian grain science conference. 16th-18th September 2015. Publications included in this thesis Wambugu, P.W, Furtado, A., Waters, D., Nyamongo, D. & Henry, R. 2013. Conservation and utilization of African Oryza genetic resources. Rice, 6, 29-29 – incorporated as chapter 2 Contributor Statement of contribution Peterson Wambugu (Candidate) Wrote paper (80%) Designed study (40%) Agnelo Furtado Edited paper (10%) Daniel Waters Edited paper (10%) Desterio Nyamongo Edited paper (10%) Robert Henry Designed study (60%) Wrote paper (20%) Edited paper (70%) v Wambugu, P.W., Brozynska, M., Furtado, A.., Waters, D.L. and Henry, R.J. 2015. Relationships of wild and domesticated rices (Oryza AA genome species) based upon whole chloroplast genome sequences. Scientific Reports 5, 13957 - incorporated as chapter 3 Contributor Statement of contribution Peterson Wambugu (Candidate) Designed experiments (30%) Performed the experiments (100%) Data analysis (90%) Wrote paper (80%) Agnelo Furtado Designed experiments (20%) Edited paper (10%) Daniel Waters Edited paper (10%) Marta Brozynska Statistical analysis of data in Table 14 and Figure 15 (10%) Robert Henry Designed experiments (50%) Edited paper (80%) Wrote paper (20%) vi Contributions by others to the thesis Principal advisor, Prof. Henry Robert contributed in the conception and design of the project. He also edited and critically revised sections of this thesis. Dr. Marie-Noelle Ndjiondjop of Africa Rice Center provided the mapping population that was analysed in this thesis. Statement of parts of the thesis submitted to qualify for the award of another degree None vii Acknowledgements This thesis is the culmination of 4years full time research study conducted at the