Genomic characterization of African cultivated and wild Oryza species

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 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.