title: Towards genetic mapping of nutritional quality traits in faba bean (Vicia faba L.)
Creators: name: Warsame, Ahmed Omar
Publisher: name: University of Reading
Year: 2021

description: Faba bean (Vicia faba) is a high-yielding and protein-rich legume crop which is grown globally for human and animal nutrition. Given the global importance of its protein content, the aim of this thesis was to address major gaps in our understanding of faba bean seed protein composition by investigating seed protein composition and its diversity, identifying genetic loci associated with total protein content and the abundance of major seed proteins, to understand the accumulation patterns of these proteins during seed development, and finally to fine-map the genetic loci responsible for pale seed hilum colour in faba bean. Diversity in seed protein composition was assessed in 35 diverse faba bean genotypes using one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (1D SDS– PAGE) and size exclusion high performance liquid chromatography (SE-HPLC). On 1D SDS– PAGE gels, 25 protein bands were accurately identified by mass spectrometry and genotypes carrying rare legumin subunits were found. Also, SE-HPLC analysis of fractionated seed proteins showed that the main globulin seed storage proteins (legumin, vicilin and convicilin) consist of heterogeneous subunits with varying solubility in aqueous and salt solutions. In addition, SE-HPLC based quantification of the proportions of legumin and vicilin/convicilin fractions showed significant variation among genotypes with the ratio of legumin: vicilin/convicilin ranging from 1:1 to 1:3. To reveal genetic loci associated with protein quality traits, a genome-wide association study was conducted using a multi-parent structured population that was grown in the field for two seasons (n=149 and 168 in 2019 and 2020, respectively). For the first time in faba bean, Marker Trait Associations (MTAs) were detected for total protein content (3 MTAs) and the abundance of 24 seed protein subunits (59 MTAs). Among protein subunits with novel MTAs were 4 legumins, 2 vicilins and several convicilin subunits. As revealed by examination of gene content of the associated regions using synteny with the Medicago truncatula genome, structural and regulatory seed storage protein candidate causative genes were found. On the other hand, proteomic analysis of developing seeds of the inbred line Hedin/2 at 12 growth stages showed diverse accumulation patterns within and between major protein classes of legumin, vicilin and convicilin. For instance, certain legumin B-type and a vicilin protein were accumulated in the seed early from 45 days after pollination (DAP) while two proteins annotated as legumin J had biphasic pattern during seed development. Finally, through combined bulk segregant analysis and linkage mapping, dihydroflavonol 4-reductase-like (DFR) was identified as the likely candidate gene responsible for the pale hilum colour in faba bean seeds. However, the nature of the mutation associated with the pale colour could not be elucidated further due to the presence of near-identical DFR copies in the candidate interval.