Domestication In Marama Bean (Tylosema Esculentum): Agronomy, Phenotypic And Molecular Characterization For Its Improvement

Abstract

Marama bean [Tylosema esculentum (Burchell) Schreiber] belongs to the family Fabaceae and is a candidate for domestication in arid zones. It is indigenous to the Kalahari regions of Southern Africa thriving in low nutrient and low moisture soils. Marama bean seeds exhibit high oil (up to 48%) and protein (up to 42%) content comparable to peanut and soybean respectively. The main purpose of this study was to determine the usefulness of previously developed microsatellite markers in distinguishing phenotypically characterized marama plants and plants treated with gamma irradiation for improvement. The chromosome number was determined to lay a foundation for molecular mapping. Further to this, the study sought to establish the effect of improved nutrients, moisture and hormone treatments on vegetative growth of marama bean. Grafting was explored as a propagation method to side step juvenility and molecular identification of potential fungal pathogens of leaf tissue was achieved. SSR primers were screened using DNA isolated from phenotypically characterized individuals representing 13 marama bean ecotypes. Two microsatellite markers MARA 039 and MARA077 were found to be candidates for use in detecting differences in internode length as well as distinguishing seeds treated with gamma radiation from untreated seeds. The chromosome number in T. esculentum was found to be n=22 (2n=44) and this will be useful in future mapping efforts. A completely randomized block design was used in assessing the possibilities of enhancing vegetative growth with fertilizer, hormones and water: Lucky plant fertilizer (LS004990-00-00) (1g/L), 100μg/L of hormone Gibberellin (GA3), 200mL water for

iii

the control, 400mL water for the high water treatments were applied. The results obtained were analysed by one way ANOVA and showed no significant difference in internode length (p=0.362>0.05), stem length (p=0.256>0.05) and number of leaves (p=0.466>0.05) suggesting the treatments had no effect on the vegetative growth of T. esculentum. Marama bean was observed to be non-responsive to grafting in the trials carried out prompting the exploration of tissue culture methods for future studies. The overall inoculations of PDA and PDB with leaf tissue showing signs of necrosis and DNA isolation together with the internal transcribed spacer (ITS) region amplification of the total 8 single spore cultures plus sequencing followed by a comparison of the DNA sequences with GenBank revealed the presence of a complex with 8 known species: Penicillium brevicompactum, Epicoccum sorghi, Rhizopus stolonifer, Alternari solani, Fusarium equiseti, Penicillium olsonii, Fusarium chlamydosporum and Fusarium incarnatum. This study has made several contributions to knowledge and current understanding of plant sciences. To our knowledge, this is the first report describing the presence of these fungi on marama bean seedling leaf tissue. This study has made a major contribution to mapping efforts as it identified regions of the genome that can be used in these studies. This study has also confirmed the marama bean chromosome number. Molecular markers linked to internode length or germination rate or any other trait that may be of agronomic importance had not been reported previously. The results from the work on grafting and plant growth regulators will prevent farmers from using high cost agricultural practices in cultivation of marama bean. Based on the findings of this study, the recommended next steps would be to explore Next Generation Sequence data with genome walking to identify genes in marama, particularly genes

iv

controlling flower formation, as well as establishing F1 populations for mapping