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Item Open Access Economically viable 𝘌𝘶𝘤𝘢𝘭𝘺𝘱𝘵𝘶𝘴 species and hybrid clones for commercial afforestation of mined sand dunes in the Richards Bay area of KwaZulu-Natal(University of the Free State, 2023) Komakech, Christopher Otim; Fossey, Annabel; Grobler, Paul𝗕𝗮𝗰𝗸𝗴𝗿𝗼𝘂𝗻𝗱: Richards Bay Minerals (RBM) is South Africa’s largest mineral sands producer with its principal operations located along the coast just north of Richards Bay in KwaZulu-Natal Province. By dredging the coastal sand dunes, the dune sand becomes nutrient poor and does not support economical land use. Casuarina equisetifolia was initially planted for sand dune stabilisation and fuelwood, but its low economic value was of little benefit to the local community. RBM thus undertook a joint venture with the Council for Scientific and Industrial Research (CSIR) to identify genotypes of tree species that were suitable for deployment on the mined sand dunes as alternatives to C. equisetifolia. 𝗠𝗲𝘁𝗵𝗼𝗱𝘀: In this longitudinal study, a multi-method approach which also included Principal Component Analysis (PCA) was followed to gather and analyse quantitative data over a 6-year period of tree growth. Four trials were established in an alpha lattice design on the mined sand dunes to test 70 genotypes of Eucalyptus species and hybrids of related species. Single plot trees were planted in 10 replications per trial, making up 700 trees per trial. The traits of survival, diameter at breast height (DBH), and stem quality were measured over the 6-year period. At 6 years of tree growth, 26 trees belonging to 13 genotypes were felled to measure their wood and Kraft properties which included, kappa number, pulpability factor, screen pulp yield, basic density and fibre yield. Coppicing ability and pests and diseases were recorded on 6-month-old felled tree stumps. Through a stepwise approach, several genotypes with the potential for deployment on the mined sand dunes were identified. Identification of genotypes was achieved by ranking the 70 genotypes according to their relative performance of the traits survival, DBH, height and stem straightness, by calculating a Relative Performance Index (RPI). The 13 genotypes of the felled tree population were also ranked according to Relative Performance Index (RPI) values using the traits kappa number, pulpability factor, screen pulp yield, basic density and fibre yield. All 13 genotypes (interspecific Eucalyptus hybrid clones) of the felled trees were ranked in the top 15 of the 70 genotype ranking. It thus made sense that these 13 genotypes were appropriate for deployment on the mined sand dunes. They were analysed for their appropriateness for use in the pulp and paper industry, as well as for the woodchips export market, by calculating Economic Performance Indices (EPI) for both market sectors. 𝗥𝗲𝘀𝘂𝗹𝘁𝘀: The results clearly showed two categories of genotype suitable for deployment on the mined sand dunes. The EPIs revealed a group of genotypes particularly suited to the pulp and paper industry and they consisted of different interspecific hybrid clones of E. grandis and E. urophylla (GxU). Similarly, the EPIs also revealed a group of genotypes that produced dense wood that would be appropriate for woodchip production for the export market. These genotypes were all different interspecific hybrids of E. grandis and E. camaldulensis (GxC). The relatively high-ranking pure species of E. urophylla (Au7 and Au9) and C. equisetifolia (Cas400 and Cas402) also warrant consideration, because they are deployed as seedlings and are substantially cheaper financially to procure than clonal material. 𝗦𝗶𝗴𝗻𝗶𝗳𝗶𝗰𝗮𝗻𝗰𝗲: The knowledge obtained during this study will facilitate the rehabilitation of the mined sand dunes in the Richards Bay area and will also help local communities in gaining access to markets and becoming economically viable.Item Open Access Linking geographic origin and mitogenomes of the ground pangolin in southern Africa(University of the Free State, 2021-11) Du Toit, Zelda; Grobler, J. P.; Kotze, A.; Dalton, D. L.; Jansen, R.The order Pholidota includes the most trafficked mammal species globally. The family Maninae with recognized species are categorized as Endangered and Critically Endangered on the IUCN Red List of Threatened Species. Due to the sharp decline in Asian pangolin numbers, the shift was towards the African pangolin species, to meet the demand in Asia and in Africa. Subsequently, the status of Smutsiinae has been revised by the IUCN and varies between Vulnerable and Endangered. This study contributes to clarify the genetic status of the four African pangolin species using molecular technologies. The first part of the study aimed to assemble mitogenomes of the four African pangolin species using NGS technologies. Whole mitogenomes were assembled and compared with published mitogenomes available in online databases. Mitogenomes were only available for six of the eight pangolin species. Our results showed different genome lengths for the four species: Temminck’s pangolin (16 558 bp), giant pangolin (16 540 bp), white-bellied pangolin (16 565 bp) and black-bellied pangolin (16 649 bp). Three distinctive clusters were observed supporting three genera. The first cluster consisted of the Asian pangolins (Manis), the second cluster was the African tree pangolins (Phataginus) and the third cluster represented the African ground pangolins (Smutsia). There were also two insertions found in the control region of the black-bellied pangolin which could indicate a recent mutation or selection event. The second part of the study focused on the development of species-specific STRs for Temminck’s pangolin and in combination with previous developed mtDNA markers the aim was to assess the population structure across the distribution range in southern Africa. Three mtDNA loci were analysed (Co1, Cytb, control region) with 30 STRs. A total of 62 individuals from six countries were included in the study. The results showed little to no geographical differentiation across the identified sampling locations. This indicates high levels of gene flow between the populations. Three distinctive clusters were observed within the different sampling areas. This could indicate inherited ancestral traits in juveniles with dispersal into new home ranges following a stepping stone model. Temminck’s pangolin has been reported to cover vast distances in search of new home ranges or territories. This study provides the first account of a combination of mtDNA and STR markers to investigate possible geo-referencing of Temminck’s pangolin in southern Africa. It is evident that the mtDNA and STRs markers used in this study, are not robust enough to discriminate between different sampling locations or populations. Further in-depth investigations, including whole genome analysis and SNPs are recommended. A more comprehensive sampling strategy is required to include all areas within the distribution range as well as more individuals for an in-depth genome analyses.Item Open Access Chemical profiling of the street cocktail drug ‘nyaope’ in South Africa using gas chromatography-mass spectrometry (GC-MS)(University of the Free State, 2020) Mthembi, Pabalala Meshack; Mwenesongole, E. M.; Cole, M. D.; Grobler, J. P.𝑬𝒏𝒈𝒍𝒊𝒔𝒉 The street cocktail drug nyaope, commonly found in South Africa, is a mixture of low-grade heroin, cannabis products, antiretroviral drugs and other materials added as bulking agents. This research led to the development of an analytical method for the identification and profiling of the street cocktail drug nyaope, using gas chromatography – mass spectrometry. This study determined for the first time the most suitable organic solvent in which the common components of nyaope, namely Δ9-tetrahydrocannabinol, heroin, caffeine, dextromethorphan, phenacetin and the antiretrovirals efavirenz and nevirapine, which have different chemical characteristics, are stable prior to an analysis of nyaope samples. The main nyaope components, when extracted into tertiary butyl alcohol, exhibited the greatest autosampler stability of up to 72 hours of storage. From these results it can be determined that tertiary butyl alcohol is a suitable solvent for the identification, comparison and profiling of nyaope samples. With regard to analytical method validation, the method gave acceptable repeatability with the %RSD less than 10% for the 10.0 and 1000.0 mg/L concentration levels for the majority of the components. The linear concentration ranges managed linearity with r2 ≥ 0.997. The detection limits varied between 9.90–39.0 pg on column and the limit of quantitation between 30.0–120 pg on column. The method exhibited acceptable recoveries and ruggedness. The method developed is fit for the purpose of quantitative profiling of the major components of nyaope using gas chromatography-mass spectrometry. The majority of the components in the street cocktail drug nyaope were shown to be stable up to at least 24 hours when stored in a refrigerator. For profiling purposes, samples need to be extracted within 24 hours of seizure in a solvent in which they are stable, such as tertiary butyl alcohol and analysed within 72 hours. At all times the samples need to be protected from light to prevent the photo-decomposition of Δ9-tetrahydrocannabinol and from moisture to prevent the hydrolysis of diamorphine. The chemical components of simulated nyaope samples as well as actual seized street nyaope samples were successfully identified and quantitatively determined using gas chromatography – mass spectrometry. The simulated and actual seized street samples were successfully discriminated into original batches using the identified nyaope components and two unsupervised chemometric methods, namely principal component analysis and hierarchical clustering, as well as chromatographic profiles. Thus, for the first time, a validated analytical method has been developed for the identification, quantitation and profiling of nyaope using gas chromatography – mass spectrometry. The method will assist law enforcement agencies in the identification and comparison of nyaope samples and facilitate the prosecution of illicit drug trafficking offences. ___________________________________________________________________Item Open Access The application of new technologies in conservation genetics(University of the Free State, 2016-01) Labuschagne, Christiaan De Jager; Grobler, P.; Dalton, D. L.; Kotzé, A.English: Over the past decade, the development of high-throughput DNA techniques has expanded the scope of conservation genetics and molecular markers have become indispensable tools for the management of wildlife species and populations. There are several molecular markers available for biodiversity analysis, but their selection depends on the objective of the study, the molecular information sought (and reliability thereof) and the facilities and/or resources available. In order to develop and apply new genetic techniques I have decided on using one bird and one mammal species of interest in South Africa. The bird species chosen is the African Penguin (Spheniscus demersus) which has suffered serious population declines and is listed in the IUCN Red Data Book as an endangered species. Due to world-wide attention to rhinoceros conservation and population decline, the white rhinoceros (Ceratotherium simum) was selected as mammal species. Three different markers and their utility in aid of South African wildlife biodiversity conservation were investigated in these diverse species. The complete mitochondrial genome of the African Penguin was sequenced. The Spheniscus demersus mtDNA genome is very similar, both in composition and length, to both the Eudyptes chrysocome and E. minor genomes. This is the first report of the complete nucleotide sequence for the mitochondrial genome of the African Penguin. These results can be subsequently used to provide information for penguin phylogenetic studies and insights into the evolution of genomes. Furthermore, the study reported eight species specific microsatellite markers as well as 31 SNP markers as new molecular tools for the investigation, management and reintroduction of African penguin. Utilising these new tools, the study generated molecular genetic information to verify/complement studbook-based pedigree data from ex-situ populations of African Penguin. In addition, we compared the relative and combined utility of MS and SNP markers for parentage assignment. We found that a combined subset of these two types of markers attained a > 99% correct cumulative parentage assignment probability. This study further reported on 34 novel SNP markers for the white rhinoceros, identified through sequencing of CATS loci as well as SNP enriched libraries. The utility of 33 Single Nucleotide Polymorphisms and 10 microsatellites in isolation and in combination for assigning parentage in captive white rhinoceros were compared. It was found that a combined dataset of SNPs and microsatellites was most informative and showed the highest confidence level. This study thus provides a useful set of SNP and MS markers for parentage and relatedness testing in white rhinoceros. Furthermore, assessment of the utility of SNP and MS markers over multiple (> three) generations and the incorporation of a larger variety of relationships among individuals (e.g. half-siblings or cousins) is strongly recommended. Developed SNP markers could be used to define the genetic mating system of this species, for forensic applications and to determine population structure and variability when other markers prove problematic.Item Open Access Taxonomy of the Genus Lycium L. (Solanaceae) in Africa(University of the Free State, 2000-06) Venter, Andor Margaretha; Venter, H. J. T.; Verhoeven, R. L.No abstract available.Item Open Access The development of Lachenalia cultivars(University of the Free State, 2013-09) Kleynhans, Riana; Spies, J. J.The floriculture and ornamental industry is constantly looking for new products. South Africa is blessed with an exceptional rich bio-diversity and many South African plants have found their way onto international markets. The local development of products for the international market unfortunately is limited. The genus Lachenalia is one of the exceptions, with local development and production of cultivars for the international pot plant market. This thesis thus aimed to establish the different aspects and requirements needed for the development of new Lachenalia cultivars and to use the basic genetic information generated through research to develop specific breeding strategies for the development of new cultivars. The thesis established the wider requirements of the complete value chain for the development of new floriculture crops and identified the strong need to establish basic research information in order to successfully develop new cultivars in the genus Lachenalia. The diversity amongst the 133 described species of Lachenalia and the breeding and research on production that facilitated the release of cultivars to the international market indicated the suitability of the genus for development. The genetic variation present in the genus includes various different basic chromosome numbers, polyploidy, B-chromosomes, different karyotypes within the same basic chromosome number, different phylogenetic groups and the existence of possible hybrid species. Relationships between specific basic chromosome numbers were shown and possible evolutionary history was proposed, but conclusions in this regard needs further investigation. The development of new cultivars is possible from both conventional and mutation breeding processes, but the availability of basic genetic information is essential for future progress. Inter-specific as well as complex hybrid/hybrid crosses are used for the development of new cultivars. To facilitate future crosses the cross-ability among Lachenalia species was investigated. The cross-ability data supports the phylogenetic relationships identified by various authors and both are strongly linked to basic chromosome numbers. Phenotypic characters cannot be used to predict the success of inter-species crosses, except where clear mechanical isolation (female long style species crossed with male short style species) is present. Clear unilateral cross-ability is present among several species and this is not linked to self-incompatibility. Self-incompatibility seems to be present in specific species, but can be overcome by crossing different accessions of the same species. Clear differences in the level of success of crossing combinations were statistical shown through AHC cluster and principle component analysis. A limited number of crosses showed good cross-ability with the production of many normal seeds. Most of these crosses were between species with the same basic chromosome number with only four exceptions, which were between basic x = 7 and x = 8, confirming the close relationship between these two basic chromosome numbers. Some intermediate success rates between basic x = 11 with both x = 7 and x = 8 was also present possible supporting the basal nature of x = 11. Basic chromosome numbers are currently the best criterion for predicting the success rate of inter-species crossing combinations but it does not guarantee success. The data presented clearly indicated the importance of well characterized (phenotypic and genotypic) germplasm material, including the maintenance of various accessions of a species. Good breeding parents were identified to assist breeders to reach specific goals. The importance of an in-depth investigation on the nature and extent of the crossing barriers and continued research on the genetics and molecular systematic of the genus was determined. This study clearly shows that the availability of basic genetic information and data on the cross-ability among species is essential for the selection of breeding parents to ensure better success rates for inter-species crossing combinations and the future development of new Lachenalia cultivars. Afrikkans:Item Open Access Genetic control of wood properties of Pinus patula in southern Africa(University of the Free State, 2013) Nel, André; Fossey, A.; Grobler, J. P.; Kanzler, A.English: Pinus patula is the most widely planted softwood species in Southern Africa and is utilised for various solid wood and pulp and paper products. Tree improvement programmes for forestry species started in Southern Africa during the 1950’s, with an initial focus on volume improvements. The focus for many advanced tree improvement programmes has moved to the improvement of wood properties. This quantitative genetics study utilized half-rotation age P. patula progeny material from a 5 × 5 full diallel mating design and additional factorial crosses. A radial wood sample at 1.3 m above ground level from each of 300 trees was used to study a range of wood density, tracheid cross-sectional and -dimension characteristics. A large range of family variation was found for all wood properties. The combining ability analysis indicated that general combining ability was the most predominant effect and that specific combining ability effects were absent for nearly all the investigated wood density and tracheid traits. Reciprocal, maternal and non-maternal effects were also not significant for all but a few traits. Some of the wood properties were influenced by the specific site where trees were grown. Heritability estimates for many of the important wood density and tracheid traits were moderate to high, indicating strong additive genetic control of these properties. Wood density traits were under strong genetic control, with a pith-to-bark increase in wood density traits. Latewood proportion and earlywood density had a strong effect on weighted mean wood density. There were also strong positive correlations for density traits between growth rings, indicating that early selection would be possible. Tracheid cross-sectional properties were also strongly inherited, and strong correlations were found between the cross-sectional traits and calculated pulp and paper traits. Tracheid dimension traits such as tracheid length, width and cellwall thickness had lower heritability estimates than those found for wood density and tracheid cross-sectional traits. These were, however, of higher magnitude than growth trait heritabilities. Several strong positive and negative correlations were found between growth, wood density and tracheid property traits. These negative correlations would make multi-trait selections very problematic. Predicted gains for some of the studied wood properties were substantial, but correlated responses between primary and secondary selection traits were often negative. The structuring of genetic material into wood property specific sub-populations would ensure the improvement of selected important wood properties. These different properties can then be combined by means of controlled pollinations, and be deployed via vegetative propagation. This study has provided novel information on the genetic inheritance of physical wood properties of P. patula grown in Southern Africa, and will allow tree breeders to include some of these properties in breeding programmes.Item Open Access Metabolic aspects of the early response of leaf rust-infected wheat(University of the Free State, 2008) Huang, Ju-Chi; Visser, B.; Van der Westhuizen, A. J.English: The aim of this study was to study various aspects of the defence response of wheat infected by Puccinia triticina. SSH was used to isolate differentially expressed cDNA fragments from P. triticina infected wheat. Once sequenced, two cDNA clones were selected for further analysis. One of the cDNA clones, LRW268, showed similarity to an EST derived from de-etiolated wheat. It showed a 16.5 fold induced expression at 9 hpi in the P. triticina infected wheat. In P. striiformis infected wheat, an induced expression was found in the resistant Avoset-Yr1 plants. Application of chemical elicitors showed an induction of LRW268 expression for plants treated with H2O2, MeJA and menadione. To obtain the full length gene, 5’-RACE was attempted, but it was unsuccessful. However, a contig was assembled using ESTs present in the GenBank database. This yielded a 603 bp contig encoding a 96 amino acid sequence that showed good homology to a RLK interacting protein. The presence of a putative MAPK docking motif and a phosphorylation site indicated that LRW268 could play a role in cell signalling. The presence of LRW268 in different wheat cultivars signified that it could form part of the general defence response of wheat. The second cDNA clone, LRW222, which was similar to an EST from wheat infected with powdery mildew, showed a 4.6 fold induction of expression at 15 hpi in wheat infected with P. triticina, while the susceptible Thatcher cultivar showed an induction at an earlier time interval. P. striiformis infected wheat showed a more constant expression of LRW222. Putative induced expression of LRW222 was observed in H2O2, MeJA and menadione treated wheat. Assembly of a contig using published ESTs yielded a 668 bp contig which encoded an 89 amino acid polypeptide showing homology to various wound-induced protease inhibitors. The presence of a putative MAPK docking motif on LRW222 suggested that it could be a general or pathogen specific protease inhibitor. The effect of light on the wheat defence response was also examined. The photosynthetic capacity of all treatments was measured and fluorescence microscopy performed. Infection caused a decrease in the photosynthetic capacity of the susceptible plants with the resistant plants showing less fluctuation. The infected resistant plants recovered faster and better than the infected resistant plants after the dark incubation. Plants that were additionally dark incubated showed a lower photosynthetic capacity compared to the control treatments. This difference in photosynthetic capacity was not observed on molecular level with photosynthesis related genes showing unaltered expression. The putative expression of certain defence related genes did however show a light dependency. An induced defence was observed in the uninfected plants, confirming a putative volatile signalling event that was detected during an earlier study involving this particular plant/pathogen interaction. The importance of light in wheat resistant towards P. triticina could thus be attributed to the ability of plant to photosynthesise optimally. It must however be emphasized that the expression of all tested genes were not quantitatively determined, since end-point analysis using RT-PCR was used. Future research will include the use of techniques that allows quantitative measurement of gene expression.Item Open Access Managing gene flow: a prerequisite for recombinant DNA biotechnology(University of the Free State, 2008) Chetty, Lukeshni; Viljoen, C. D.Over centuries, crop domestication and improvement has led to modern commercial agriculture. Agricultural biotechnology is considered by many a natural step in the course of crop improvement by utilizing genetic engineering. Currently, the global production of biotech crops is approximately 34% of global agriculture. The major biotech crops in terms of production volumes are canola, cotton, maize and soybean. In Africa, South Africa is the only country to accept and commercially produce genetically modified (GM) crop. The 2007, GM traits per crop with environmental release status in South Africa included insect resistant (IR) and herbicide tolerant (HT) cotton (including the stack for both traits) (90% of total cotton production), IR and HT maize (including the stack for both traits) (57% of total production) and HT soybean (80% of total production). There are several factors that impact on the application of this technology in terms of commercial as well as small scale farming. These include: intellectual property rights, socio-economics, regulatory frameworks, agriculture, environment, niche markets and cost benefit. Of all of these aspects, gene flow from GM to non-GM or organic products, land races and wild relatives is a critical consideration. In this study, the impact of potential pollen mediated gene flow (PPMGF) and pollen mediated gene flow (PMGF) was studied in GM soybean and maize, two of the most important GM food crops in terms of production volumes. In this study, GM gene flow was found to have occurred up to 0.9 m from a GM source at two locations over two seasons, despite being considered a selfpollinating crop (Greytown 2005/2006 and Delmas 2006/2007, respectively). However, it was also found that GM soybean pollen was not wind borne and we suggest that the gene flow observed was due to insect-mediation. Future studies of PPMGF in South Africa should include a survey of insects present with the potential to act as a pollen vector in soybean. In the maize component of this study, molecular technology was used to detect GM maize pollen up to 400 m from a GM pollen source. Furthermore, it was found that out-crossing of GM to non-GM maize was possible at a distance of 300 m from the GM field. Based on the statistical analysis of out-crossing data, I have determined that the average theoretical zero (0.0001%) level of out-crossing was between 1.3 km and 2.0 km over different geographic locations. However, what was unexpected is the difference in out-crossing per location for a specific direction. For example, in Bainsvlei (2005/2007) for the ENE direction, the calculated distance to achieve 0.01% out-crossing is 79 km, yet the average is 113 m. Similarly in the second season for the same direction, the calculated distance is 956 m and the average is 135 m. The implication of these data is that it is not possible to establish a one size fits all isolation distance to minimize or prevent gene flow. Different threshold levels of commingling require different isolations distances and should be determined by the acceptable level of tolerance for commingling. For non-GM production in South Africa, based on the 1.0% threshold applied by the Department of Agriculture, I suggest a minimum isolation distance of between 120 m up to 200 m, assuming that the weather patterns are comparable to those of the current study as well as that the non-GM seed being planted contains 0% GM. However, for more stringent thresholds, the isolation distance would need to be extended. For organic crop production, at 0% adventitious GM, as well as field trials of second and third generation GMOs, it is suggested that the isolation distance be set at a minimum of 1.5 km and 2.0 km, respectively. In addition, for non-GM seed production (with a mandatory 0% tolerance so as not to contravene patents) I recommend a 1.5 km isolation distance. These suggested isolation distances are based on the absence of time isolation. It is hoped that this study will help to inform regulatory as well as on farm decision making and that it could be used as a blueprint for other GM crops, especially indigenous African crops such as sorghum and cassava.