- ItemAnalysis of starch metabolism in South African pigeon pea (Cajanus cajan) varieties(Stellenbosch : Stellenbosch University, 2023-03) Kulu, Nokwanda; Lloyd, James R. ; Peters, Shaun Wayne; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is a major storage polyglucan in plants that is composed of two fractions, amylose and amylopectin. The biosynthesis and degradation pathways of starch are well documented, with phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase) catalysing the first two steps in its biosynthesis. This project examined starch in five pigeon pea (Cajanus cajan) varieties: uDhali, SEFA, Nondolo, Lari and India by measuring both total and resistant starches in the seeds and leaves, activities of PGM and AGPase as well as expression of the genes encoding these enzymes. The findings demonstrated that the seeds from these South African pigeon pea varieties are rich in starch, containing an average of 47% starch on a dry weight basis; however, one variety (SEFA) contained only 0.3% starch. The starch in the high- starch varieties contained a minimum of 50% resistant starch, with the India variety reaching 70%. Assessment of soluble sugars in seeds revealed sucrose to be the only sugar present in abundance in all varieties while amounts of galacto-oligosaccharides were low in all seeds. Starch in leaves was observed to be 10 fold less than that found in seeds and the amount of resistant starch in leaves was less than 2 mg/g fresh weight (7.6% of the total). The AGPase gDNA nucleotide sequence from one variety was identical to an already sequenced pigeon pea variety, whereas amplification PGM gDNA was unsuccessful. Amplification of coding sequences (CDSs) for both AGPase and PGM were also identified to be the same as the already sequenced AGPase and PGM genes from the pigeon pea genome resource database. Gene expression for both genes varied throughout a 24 h period and was at its peak during the day (light period). Activities of both AGPase and PGM were determined in seeds from all varieties whereas the AGPase enzyme activity was the same in leaves throughout the day while PGM activity varied between the day (light) and night (dark).
- ItemAnalysis of phosphoglucomutase isoforms from physcomitrium patens(Stellenbosch : Stellenbosch University, 2023-03) De Stadler, Jessica Amy; Lloyd, James R.; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is the main storage polymer found in most plants and plays a significant role in plant fitness. It is a polyglucan and is composed of two separate fractions named amylose and amylopectin. In photosynthetic tissues starch is synthesized in chloroplasts during the day and degraded at night to provide energy which supports metabolism. The pathways of starch synthesis and degradation have been extensively characterised in Angiosperms, however, in non-vascular plants these are less well understood. Physcomitrium patens is a Bryophyte that is an excellent non-vascular model plant due to its fully sequenced genome and the ease at which mutants can be created using homologous recombination. As Bryophytes have been classified as a transitionary species between water-based algae and land plants, research using them sheds light on how biochemical processes (such as starch metabolism) have changed during land colonisation. The first committed step of the starch biosynthetic pathway is the creation of ADP-glucose from glucose 1-phosphate (G1P). The formation of G1P is catalysed by plastidial isoforms of phosphoglucomutase (PGM) and these enzymes are encoded by a highly conserved family of genes. Previous studies in Angiosperms have demonstrated that mutating the plastidial isoform of PGM results in plants that accumulate almost no starch in all organs (Hanson & McHale, 1988; Harrison, et al., 2000; Vriet, et al., 2010). Cytosolic phosphoglucomutase isoforms are also present and the protein sequences of these are similar to those of plastidial isoforms. The first aspect of this project was to identify PGM genes in P. patens that demonstrate high similarity to PGM genes from Arabidopsis thaliana. Four were identified (named PpPGM1-4), and the amino acid sequences of the translated P. patens PGM polypeptides have high similarity to other phosphoglucomutases. The predicted intron-exon boundaries showed that PpPGM1 and PpPGM2 genes contain no introns whereas PpPGM3 and PpPGM4 contain seventeen introns each. Phylogenetic analysis of PGM sequences from red algae, Cyanobacteria and Viridiplantae demonstrated that sequences could be divided into three clades. One contained red algal and cyanobacterial sequences while the other two contained only Viridiplantae PGM’s. One of the Viridiplantae clades contained all isoforms that have been demonstrated experimentally to be plastidial (and PpPGM1 & 2), and the other all isoforms that have been demonstrated experimentally to be localised in the cytosol (and PpPGM3 & 4). The sub-cellular localization of the PGM protein was examined by transiently expressing PGM genes, which had been fused to a gene encoding the green fluorescent protein, in P. patens protoplasts. This demonstrated that PpPGM1 and PpPGM2 were imported into plastids and PpPGM3 and PpPGM4 were localised in the cytosol. All four genes were shown to encode active proteins and their expression restored the wildtype phenotype in an Escherichia coli pgm mutant. Furthermore, analysis of the PGM amino acid sequence showed that PpPGM1 and PpPGM2 both contain a SASHNP active site motif whereas in PpPGM3 and PpPGM4 this is TASHNP. Similarly, a metal binding motif differed between the four polypeptides, being DGDGD in PpPGM1 and PpPGM2 and DGDAD in PpPGM3 and PGM4. The known sugar binding sequence CGEESF was found in all four proteins. These versions of the active sites were conserved across the two Viridiplantae clades. The final aspect of this project was to create knockout mutants of the two plastidial PGM genes in P. patens to identify their effect on phenotype, this was attempted using PEG mediated homologous transformations, several plants survived selection but were demonstrated to be untransformed.
- ItemDetermining the phenotypic and molecular diversity within a collection of wheat wild relatives(Stellenbosch : Stellenbosch University, 2023-03) Bester, Cecile; Botes, Willem; Le Maitre, Nicholas; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Common bread wheat, Triticum aestivum L., is the third most important staple crop, after rice and maize. Wheat provides 20% of the globally consumed calories, and is cultivated on 242 389 108 ha globally. In South Africa, wheat was planted on 523 500 ha with a total production of 2 285 000 tonnes in the 2021/2022 season. The continuous improvement of wheat is important for global and local food security. Crop wild relatives are rich sources of genetic diversity that have been used in 4 157 documented cases of plant improvement by 2022. Wheat wild relatives, like Triticum and Aegilops spp. have been employed 333 times for wheat improvement by 2022. The Stellenbosch University Plant Breeding Laboratory has a collection of 1246 different wheat wild relatives belonging to the genera Triticum and Aegilops. There is limited information on the accessions from this collection, and the diversity present within, restricting the use of these plant genetic resources in wheat improvement. Obtaining more information that can assist when planning introductions, selections, renewal, description, and characterisation of the material is therefore vital. The development of digital phenotyping and characterisation methods can aid in the determination of morphological diversity and species identification. Chloroplast DNA is universal in plants and allows for the assessment of molecular diversity between species with different genomic combinations. From the Stellenbosch University Plant Breeding Laboratory crop wild relative collection, 92 entries were renewed, identified, characterised, and described. The collection showed a high level of morphological diversity in grain yield, flag leaf area and awn types. To improve phenotyping, a digital method of spike length estimation, using ImageJ, was compared to actual physical measurements. There was no significant difference between values obtained by these two methods, verifying the technique. Transfer Learning was applied to train pre-trained MobileNet Convolutional Neural Network models to distinguish between genus, species, subspecies and variety, with accuracies of 0.8750, 0.923, 0.742 and 0.600, respectively. These models can be applied to increase the accuracy of plant identification. Twelve chloroplast simple sequence repeats in four multiplex reactions were used to determine the genetic diversity within the wheat wild relative collection. Polymorphic information content ranged from 0.615 to 0.972 and gene diversity from 0.663 to 0.9724. These values indicate that there is a high level of diversity present within the wheat wild relative collection. Due to the high morphological, and molecular diversity within the ex situ species material collection, the plant genetic resources have a good potential for use in wheat breeding for crop improvement. The information can assist breeders to select the best wild relatives to be used in interspecies crosses to improve common bread wheat.
- ItemWheat pre-breeding towards disease resistance of fungal pathogens(Stellenbosch : Stellenbosch University, 2023-03) White, Michaela-Anne; Botes, Willem C.; Rose, Lindy J.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Bread wheat is an important grain crop cultivated globally, serving as the main source of calories and protein to approximately 4 billion individuals. To sustain increasing consumer demands, the production of wheat is required to increase with 60% by 2050. Wheat production is often threatened by the occurrence of both abiotic and biotic stressors, resulting in reduced yields. The most economically important biotic stressors constraining the production of wheat are fungal diseases. Management strategies have been developed to control disease outbreaks. The integration of disease resistance is highly favoured as it is cost-effective, environmentally friendly and reduces the risk of disease outbreaks. The aim of the study was to identify crossing parents to introduce into the male-sterility mediated marker-assisted recurrent selection pre-breeding programme. The SU-PBL’s 2020 wheat nursery and Fusarium head blight CIMMYT nursery used as male crossing parents, were genotypically and phenotypically assessed for powdery mildew resistance (Pm37, Pm4b, MLAG12, MLUM15) and Fusarium head blight resistance (Qfhs.ifa.5A, 7AQTL, Qfhs.ndsu.3BS), respectively. Two superior genotypes with FHB resistance were identified from the FHB CIMMYT nursery. The two wheat lines displayed low disease severity, low DON content and minimal FDK. Five superior wheat lines with powdery mildew resistance were identified from the SU-PBL’s 2020 wheat nursery. These superior wheat lines displayed promising genotypic and phenotypic expressions for introduction into the MS-MARS pre-breeding scheme. Future studies should include additional molecular and phenotypic characterisation of the nurseries. It should also include active powdery mildew resistance breeding. Due to the expected change in climatic conditions worldwide, an increased risk of powdery mildew outbreaks is expected for South Africa. Extensive breeding for powdery mildew disease resistance through the deployment of several viable genes would reduce this threat.
- ItemImproving water stress tolerance of triticum aestivum by means of male sterility marker assisted recurrent selection(Stellenbosch : Stellenbosch University, 2022-12) Khambule, Mhlengi Sboniso; Botes, Willem; Hess, Lezaan; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Wheat is the most widely grown cereal. More than 90% of the crop is grown for direct human consumption with increasing alternative potential for starch, glutens and biofuels. Wheat agronomic and genetic improvements will increasingly play key roles in meeting current and future food security needs and/or demands, particularly in the face of climate change. With the crop’s genetic complexity, the use and potential of molecular markers in the breeding process cannot be overlooked. The aim of this study was to select crossing parents for wheat vegetative water stress improvement under low water retention. The 4th Wheat Yield Consortium Yield Trial nursery (4-WYCYT) from CIMMYT was used as a prospective donor and evaluated for vegetative water stress tolerance at different vegetative growth stages before selection and crossing with an existing MS-MARS base population. The initial evaluation was undertaken in a growth chamber setting, selection trials were undertaken in in glasshouse conditions while crossing events used a combination of these environments. Seeds resulting from the crossing events were evaluated for early vegetative vigour under glasshouse conditions. Water retention and water availability regimes facilitated various stress combinations in both the evaluation and selection trials. Water availability was a strong factor under growth chamber (GC) conditions. Early vegetative water stress survival ranged from 16-30 plants and varying across the four retention regimes under growth chamber conditions. Survival was comparable under glasshouse conditions despite growth stage of water stress onset. Evaluated entry (EE) 005 (particularly accessions 005A and 005D) performed consistently in both growth chamber trials despite stage of stress onset, and when stress occurred during early vegetative stages despite growth environment. The said entry recorded the highest evaluated average entry yield for early vegetative stress (average of yield or total seed mass performance of available accessions for a given entry) although a single accession from EE - 006 recorded the highest individual yields (significant from two other accessions of the same genotype) when stress occurred during mid-vegetative growth under glasshouse (GH) conditions. All evaluated local checks (5 in GC and 10 in GH) performed significantly below the best performing accession and/or entry for yields in both the respective glasshouse trials, with an above average performance only when stress occurred later in the vegetative growth stages. Mid-vegetative stressed trials however recorded the least yields comparatively. The 4-WYCYT was however robust and displayed varying tolerances to water stress. This informed the selection of entries of this nursery which resulted to a male fertility segregating population produced through male sterility mediated introgression. Low retention evaluated F1 male fertile seedlings were taller on average compared to their infertile counterparts. Future studies should aim to improve wheat vegetative resilience and growth in varied and relevant water stress combinations.