Transcriptome profiling and gene editing for biofortification of cassava

Cassava (Manihot esculenta Crantz) is an important food crop for millions of people in sub- Saharan Africa. Cassava serves as a food security crop and a good source of energy, but it has relatively low nutritional quality, which has nutritional implications for those who rely on the crop as their main source of calorie intake. Vitamin A deficiency (VAD) is a health problem in populations whose diet constitutes mainly starchy crops like cassava. A possible solution is biofortification by conventional breeding or biotechnology to increase β-carotene content. Unfortunately, some studies report a negative correlation between β-carotene and dry matter content in certain genotypes, which could pose a challenge to these types of cassava biofortification measures. Field-grown cassava landraces were analyzed for agronomic traits and carried out specific and global transcript analyses by real-time quantitative RT-PCR and RNA-sequencing (RNA-seq). This was combined with targeted starch and carotenoids analysis by HPLC and non-targeted metabolite analysis by GC/LC-MS to understand the regulation of key enzymes and intermediate metabolites to identify genes influencing β- carotene accumulation in cassava. Also, using the CRISPR/Cas9-mediated gene editing system in the cassava cultivar TMS60444 we tried to introduce knockout mutations into cassava β-carotene hydroxylase (MeChyβ), lycopene-ε-cyclase (MeLcyε), and 9- cisepoxycarotenoid dioxygenase 1 (MeNced1) which are key genes of the carotenoid pathway. In a separate study, the biosafety regulations and policies in Kenya, Nigeria, Uganda, and the EU represented by Sweden were examined by comparing legislative texts and conducting interviews to determine if policy and regulatory frameworks present problems to perform R&D using new breeding technologies. In the cassava landraces analyzed, we found a weak negative correlation between starch and β-carotene content, whereas there was a strong positive correlation between root yield and carotenoids. Also, cassava landraces with reasonably high content of starch and β-carotene were identified that could be candidates for biofortification by further breeding or plant biotechnological means. Global gene expression profiles grouped cassava landraces into white and yellow landraces; however, at the same time there was no general correlation between the expression profiles of individual genes involved in carotenoid synthesis and accumulation with the storage root color. Gene Ontology (GO) enrichment showed over-representation of upregulated genes involved in protein-related metabolic and catabolic processes in yellow landraces while GO related to photosynthesis and light reactions were enriched in white landraces. Interestingly, we identified a previously reported amino acid change from Alanine to Aspartic acid in MePsy1 at position 191 to distinguish the yellow lines from the white lines; however, this change was absent in the paleyellow lines, confirming that the mutation in psy is not solely responsible for carotenoid accumulation in cassava. Non-targeted metabolite analysis revealed higher abundance of several amino acids in white lines, but also higher levels of a few osmolytes indicating differences in stress response. Transformation of cassava FECs with gene targets Lcyε, and Nced1 did not produce transgenic regenerated shoots, whereas MeChyβ produced in vitro plantlets still under investigation. Finally, our study showed that biosafety regulations on GMO approval in Kenya, Nigeria, and Uganda are not a major hurdle for R&D but might rather be influenced by factors outside of the regulatory framework such as perceptual and financial factors including funding opportunities.