Molecular Genetic Characterization of Photoperiodic genes in Cassava (Manihot esculenta Crantz) and attempts to manipulate their expression to promote floral induction

Flowering and its control is a critical developmental transition in angiosperms. The developmental timing of flowering has been widely studied in the temperate species Arabidopsis, which was well aided by the sequencing of its genome. However, the timing of flowering is not well understood in tropical species. Efforts are being made to extend the understanding of the molecular pathways controlling flowering from those of Arabidopsis to that of Cassava by taking advantage of genomics information from this temperate species. This should be an invaluable tool for understanding and exploiting floral timing in a tropical plant. Cassava is a crop that is critically important for food security in the tropics and sub-tropical regions of the world. Because, it is an orphan crop of limited research interest, almost nothing is known regarding its molecular basis of flowering. Therefore, it remains unclear what molecular pathways are implicated in flowering in Cassava and how this can be used to improve breeding. In this thesis, I provide insight to the discovery of photoperiod genes in Cassava and exploit this pathway to manipulate floral timing. Several genes of interest were sought for in this tropical plant, ranging from clock genes, to photoperiod genes, to the floral integrator, and finally, floral meristem-identity genes. Several genes were found and characterized. From there, transgenic approaches revealed that reproductive timing can be modified in Cassava. In Chapter 3, I identified and characterized three classes of photoperiod genes. The first gene isolated and characterized was MeGI, which revealed sequence similarity and conservation regions when compared with Arabidopsis. The comparison with GIGANTEA- related sequences from other species revealed by phylogeny that Cassava GI clearly clades with dicots and is more closely related in sequence to Castor bean GI. The MeGI expression was measured in Cassava and it was shown to track dusk. I found that MeGI peaks in expression at dusk under both long day or short day growth conditions. This is consistent with similar studies from other species. Another set of genes I found in Cassava was the CO-like genes, which I named MeCOL1, MeCO, and MeCOL2, respectively. The predicted protein encoded by MeCOL2 possesses two adjacent zinc-finger motifs, which is specific for genes in this family. COL2 was shown by phylogeny as the closest to the three MeCO-related genes. The expression of MeCO-like genes in Cassava showed they anticipated dawn. They were found to be acutely induced by light at dawn. Under short day growth conditions, they peaked two hours before dawn and this peak sharply increased at dawn under long days. This expression was decreased over the light period of the day. Thus, MeCOL genes have pre-dawn expression. Finally, I studied in genetic detail a Cassava orthologue of Arabidopsis ELF4. MeELF4 was found to complement elf4 by restoring circadian-rhythm defects of this Arabidopsis mutant. Therefore, I successfully characterized in Cassava the GI, members of the CO-like gene family, and the ELF4 orthologue, all of which are candidate genes for photoperiodic regulators. I bridged the gap between basic and applied research by exploiting the photoperiod- integrator gene FLOWERING LOCUS T (FT) as a tool in the manipulation of floral timing in Cassava. To generate this tool, I placed the Arabidopsis FT gene under the control of an ethanol-inducible promoter. I showed that the construct was functional, as it promoted flowering in a late-flowering genotype of Arabidopsis after ethanol application. This technology was then transferred to Cassava. I transformed a shy flowering Cassava genotype with Arabidopsis FT under the control of an ethanol-inducible promoter. FT expression was found to increase after spraying the Cassava transgenics with ethanol. These plants then flowered. This system is hoped to be applied in conventional breeding programs in order to be able to induce flowering at will, and thus make desired crosses that will lead to the improvement of the genetic basis of the crop