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Item Open Access Transcriptome sequencing and digital expression analyses of flower morphogenesis genes in Clivia miniata(University of the Free State, 2023) Naidoo, Palesa; Maleka, M. F.Flower morphology is an important aspect in horticulture as it can be used to create new varieties, it plays an important role in pollination and it influences the visual appeal of a plant. The genus Clivia is important in horticulture due to the plant’s ornamental value, adaptability, low maintenance requirements, easy propagation and showy flowers. The genus comprises plants that exhibit two distinct flower morphologies, namely, the pendulous (drooping) and non-pendulous (upright) forms. The aims of the study were to sequence the transcriptome of the two Clivia flower shapes; identify flower morphogenesis genes and identify differentially expressed morphogenesis genes between the pendulous and non-pendulous Clivia flowers. Illumina sequencing and analyses generated a 200 MB Clivia flower transcriptome. A total of 36 flower development-related genes from the Flower Interactive database (FLOR-ID) were identified, including AGAMOUS, APETALA, SEPALLATA, PISTILATA, TERMINAL FLOWER1 and WUSCHEL. DE analysis resulted in 7,303 DEGs which were annotated using the SwissProt/Uniprot, KEGG, GO, PANTHER, Pfam, TMHMM, SignalP, EggNog and RNAmmer databases. The top three species with sequence homologies to the annotated Clivia transcriptome were Arabidopsis thaliana, Zea mays and Oryza sativa. Several transcription factors were found to be unique to each flower form, including a MADS-box gene likely to be responsible for the different morphologies. Our results contribute towards understanding the molecular mechanisms of flowering, flower development and morphogenesis in Clivia.Item Open Access Skin microbiome diversity of batrachochytrium dendrobatidis-challenged phofung river frogs (amietia hymenopus) in the Drakensberg mountain range(University of the Free State, 2022) Kloppers, George Antonie Ehlers; Gryzenhout, M.; Gryzenhout, C.; Cason, E.; Ghosh, S.Infectious diseases are pressuring amphibian populations globally, especially threatened frog species occurring in restricted areas. Chytridiomycosis, which is caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is arguably the most devastating panzootic disease confronting wildlife today and was determined to be a major driving force of the current amphibian extinction crisis. A healthy amphibian host has a symbiotic relationship with its cutaneous microbial community that benefits the host in many ways including the protection against malicious microbes. The current trend in amphibian skin microbiome studies concentrate predominantly on its bacterial component, while other components of the microbiome such as filamentous fungi and eukaryotes have been neglected. In this study Amietia hymenopus, a Bd-challenged frog population of southern Africa, was sampled from remote locations of the frog?s natural distribution on the Drakensberg mountain range. The cutaneous microbiome diversity was analysed using next generation sequencing and bioinformatics tools, investigating both the bacterial and fungal components of the frogs? skin microbial community. Results indicated high diversity dissimilarities in both alpha and beta diversity when comparing individual specimens, and sampling areas, respectively. This study touched on an important topic regarding the microbial biodiversity found on frogs, and sheds light on the complex interaction between host and microbial communities in amphibian populations. Knowledge on this intricate relationship between host and their microscopic cutaneous symbionts could help inform amphibian conservation efforts against infectious disease.Item Open Access Description of novel species of psychedelic mushrooms from Southern Africa(University of the Free State, 2022) Maloka, Onalerona Orefilempho; Gryzenhout, M.; Ghosh, S.The chemical compound psilocybin, responsible for causing hallucinations, is found in mushroom species of genera such as Gymnopilus, Panaeolus, Pluteus, and Psilocybe. Psilocybin also has a number of psychiatric and medical applications. Psilocybin-producing mushrooms have a wide distribution in South Africa and other parts of the world, but the biodiversity of these fungi is poorly known in South Africa. This study focused on the species identification of two sets of collections of Psilocybe, one from Lesotho and the other from Pretoria, based on morphology and different DNA sequence phylogenetic markers. A multi-locus phylogeny was constructed using the Internal Transcribed Spacer (ITS), RNA Polymerase II (RPB 1), and Translational Elongation Factor 1α (TEF-1α) gene regions. More than one marker was used to confirm identifications, and by combining the sequences, to also obtain better statistical support for groupings. By using the additional genes besides ITS, the usefulness of these additional markers to identify Psilocybe species was also investigated. Results showed that the two collections of samples were unique and different from each other based on all of the genes, except for the RPB 1 region that was found wanting. Although the TEF-1α was found sufficiently variable to also distinguish species similar to the ITS region, a relatively small number of species have been sequenced up to date. The distinct grouping of the two collections was confirmed by a number of macro- and micromorphological characteristics, and described as Psilocybe malotiensis prov. nom. and Psilocybe orontawuli prov. nom. respectively. Results from this study represent an important breakthrough where Southern African samples can now be sequenced and compared with specimens from elsewhere and should illustrate the presence of numerous novel species occurring in this region.Item Open Access Identification of mushrooms from pine plantations within the Tsitsikamma region, South Africa(University of the Free State, 2022) Herselman, Maryke; Gryzenhout, M.Mushrooms have been exploited for ages by mankind for their astoundingly wide application as a sustainable dietary supplement that also carries economical, ecological value and medicinal qualities. Although some mushrooms are considered edible and flavoursome others are deadly. Mushrooms also play ecologically vital roles in nature as decomposers, pathogens and symbionts of plants, animals and humans. Mushrooms have in recent times been heavily explored for new-age biotechnological and medical innovations, but without knowledge of species present in a country, regulation is difficult. In South Africa, knowledge about the biodiversity of macro fungi seems to be lacking. To expand this biodiversity knowledge, this study focused on the coastal Tsitsikamma region in the Eastern Cape province, which represents the largest native forest area of South Africa. However, these forests are interspersed with commercial tree plantations, agriculture and urban development. Specifically, this study focused on mushrooms occurring in plantation areas, to initiate a knowledge base of macro fungi associated with these alien plants, before future studies can determine which are more likely native mushrooms, and if mushrooms from these alien plants can also be found in native vegetation. Therefore, the first aim of the study was to collect and document mushroom diversity and morphology from plantations, and to highlight distinguishable and identifiable characteristics. Morphological studies were aided in the second aim of using rDNA nuclear Internal Transcribed Spacer (ITS) DNA sequence comparisons to confirm specimen identities. A total of 13 species were collected and identified from various plantations in the region. These included species of Amanita, Russula and Lactarius, as well as Panaeolus, Chlorophyllum, Clitopilus, Imleria and Gymnopilus. One specimen identified to be a Chlorophyllum species could not be identified to species level, and may possibly represent a novel species. The study yielded three first reports for South Africa, namely L. quieticolor, P. antillarum and A. morissi, with the latter species having vulnerable red list status and is only known from North America. It was also found that the South African described R. capensis could possibly be conspecific to R. caerulea, which occurs widely in the Northern Hemisphere. A large number of species found were also ectomycorrhizal, having a symbiotic relationship with plant roots, which were pines in this study. The use of DNA sequence comparisons in this study revealed novel associations and reports, in some cases different from the better known morphologically identified species previously known from the region. This study thus shows that careful surveys should be done in future, using both morphological and DNA sequence based identification.Item Open Access Developmental and gene expression changes during intra-puparial development in Chrysomya albiceps (Diptera: Calliphoridae)(University of the Free State, 2021-11) Van der Westhuizen, Lucinda; Wessels, L.; Maleka, M. F.Criminal investigations are dependent on postmortem interval estimations (PMI) to solve crimes by determining when a homicide occurred. Time of death is estimated using several components, including pathology; however, a pathologist can only determine time of death up to 72 hours postmortem. Therefore, during and beyond this period, entomological evidence is very valuable as the age of the eldest immature fly stage available is used to determine the time of death. In forensic investigations, it is important to have validated growth information at hand for entomological samples, involving all developmental stages, to determine the age of the samples collected. In forensic investigations, blowflies (Diptera: Calliphoridae) are important as primary colonizers of cadavers, especially in exposed areas, and current age estimation methods of entomological samples are based on morphological changes of the immature stages. Larvae, often encountered at crime scenes, already have established age estimation methods and preservation protocols. However, pupal developmental and preservation studies are in the minority. The main reason for the lack of data is based on the difficulty of working with pupal samples due to the puparium which conceals morphological changes. Considering the importance of the pupal stage, additional techniques such as molecular analysis are required to augment techniques to improve age estimation of the pupal stage. This dissertation reports on a study that followed a multidisciplinary approach to age estimation of Chrysomya albiceps (blowfly) pupae, which are abundant at crime scenes due to their predatory nature. Investigating the morphological changes occurring within the puparium and the possibility to link this to differential gene expressions changes. Two methods for age estimation were therefore developed: external morphology changes and temporal gene expression changes. In addition, different preservation protocols were tested to determine the most effective preservative dependant on the type of analysis. Validated collection and preservation methods are not readily available for pupal samples and the use of unvalidated collection and preservation methods ultimately hinders age estimation. Laboratory colonies of C. albiceps, reared from wild samples, collected at the University of the Free State, were utilized for pupal sample collection during three trials to obtain optimum number of samples. Three preservation protocols (pierced before hot water kill (HWK) or not pierced before HWK and placing in 70% ethanol or 10% buffered formalin) for morphological examination were tested on 580 – 590 pupae aiming to retain the native morphology. For nucleic acid integrity, to conduct age estimation by gene expression, 78 pupae were subjected to liquid nitrogen as a killing method and samples were preserved at -80˚C. After morphological and molecular examination of pupae the following preservation method is proposed: for morphological examination pupae are pierced prior to HWK and then stored in 70% ethanol, for long- and short-term preservation as ethanol is the best for retaining native morphology; and for gene expression analysis, pupae are immediately subjected to liquid nitrogen and stored at -80˚C. In addition to previously identified markers, we proposed four new landmarks and based on these results, age estimation can be refined up to 96 hours at 26˚C. The temporal gene expression levels based on 78 pupal samples were quantified using qRT-PCR. It emerged that actin, which was previously used as a reference and target gene, cannot be used as a reference gene due to the changes in expression levels during pupal development and that RpS17, 18S and Rp49 are the best reference genes for normalization. This study made a valuable contribution to the forensic science field in criminal investigations by validating current suggested reference genes, analysing previously identified target genes and identifying new morphological markers which can be used as a baseline for future C. albiceps studies.Item Open Access Implications of secondary DNA transfer & public perceptions on the national DNA database of South Africa(University of the Free State, 2017-01) Mokoma, Polo M.; Ehlers, Karin; Wessels, Letecia; Hanckock, CarolynAbstract not availableItem Open Access The role of emotional intelligence and a functional polymorphism in the MAO-A gene on aggression in humans(University of the Free State, 2012-05) Laubscher, Nadia; Spies, J. J.Article 1: Aggression is a complex trait, with both genetic and environmental factors important in its aetiology. It is a universal problem, for which better solutions are needed. This study will focus mainly on the influence of genetic and environmental factors on two subtypes of aggression, namely reactive- and proactive aggression. The moderate heritability values of these subtypes make them ideal candidates for such a study. The genetic components of aggression include the upper-limit heritability estimates for the subtypes by means of correlations between first-degree relatives. Thereafter the role of variants of one specific gene, the MAO-A gene will be examined. The role of emotional intelligence as a specific environmental factor influencing aggression is discussed. Very few studies have been done on the possible influence of emotional intelligence on aggression. Traumatic event exposure will also be studied as a possible secondary influencing factor. Since self-report measures are used, the effect of social desirability bias will be determined. In this chapter each of the variables under study is briefly described, followed by the most salient motivations why these specific variables were seen as the most suitable for this investigation. In addition, the specific aims of the dissertation are briefly outlined. Article 2: Aggression is a highly prevalent and costly problem in societies throughout the world. Treatment options are available, but needs to be improved or adjusted to really be able to curb the problem of aggression. The paper aims to highlight key points in the aggression literature in order to improve researchers’ understanding of the construct of aggression and possible causes underlying aggression, as well as factors that may exacerbate aggression. Therefore, this paper reviews the literature on aggression in humans, and covers aspects relating to the defini-tion of aggression, the various subtypes of aggression, evidence for variable en-vironmental and heritable influences on aggression, as well as looking at specific genes and hormones influencing aggression. Specifically, the influence on ag-gression of the monoamine oxidase A enzyme, the gene that encodes it (MAO-A), and the neurotransmitters that it metabolizes (serotonin and norepinephrine) are looked at. In addition, emotional intelligence as a possible influencing factor on the occurrence of aggression is also covered. This is done to provide a start-ing point for research aiming to develop treatments for aggression, whether they are psychotherapeutic programmes aimed at improving emotional intelligence or psychopharmaceutic drugs aimed at the genetic and hormonal mechanisms un-derlying aggression.Item Open Access Aspects of the genetics of human aggressive behaviour(University of the Free State, 2012-12) Odendaal, Zurika; Spies, J. J.; Spies, P.Item Open Access DNA profiling from the crop content of sarcophagidae spp. larvae(University of the Free State, 2017-01) Barnard, Adri Marlene; Wessels, L.; Ehlers, K.; Brink, S. L.English: Morphological analysis of insect evidence plays a significant role in crime scene investigation. With the influence of DNA analysis in forensic cases, which now also plays a key role in forensic entomology, more emphasis has been placed on a dual preservation goal when collecting insect evidence. Previous studies indicated that it might be possible to identify the last meal of sarcophagid maggots using gut content combined with DNA profiling. For gut content analysis it is imperative to be familiar with the internal morphology as well as maggot gastric emptying. However, insufficient information is available on the morphology of Sarcophaga cruentata maggot alimentary canals as well as the rate of maggot gastric emptying. Also, considering the use of insects for both PMI and DNA analysis, the current preservation methods are not necessarily suitable for maggot preservation for DNA analysis of the crop content. Various preservation methods were examined for optimal preservation of both morphology and gut content. In order to understand gastric emptying, the internal gut structures and movement of food through the gut was examined. Fully fed maggots were removed from the food source and hot water killed at 3 hour intervals for up to 30 hours to investigate gastric emptying. Crop content DNA analyses were performed to attempt identification and DNA profiling of the maggots’ last meal. Due to the inhibiting effect of formalin on DNA analysis and the extensive dehydration of prolonged ethanol storage, -80°C was investigated for sample preservation which generally provided good results. Inconsistent results were obtained using the various preservation and DNA analysis combinations tested. Sarcophagidae gut morphology analysis indicated gross anatomical similarity to Chrysomya megacephala. External tracking of food movement proved difficult. After digestive tract dissection it was found that the mid- and hindgut coiled around each other. Due to the coiled structure of the gut, the exact location of the food bolus could therefore not be determined without dissection. The expected gastric emptying was not consistently observed with pronounced variability in results. Nevertheless, it was observed that the maggot crops were completely empty by 30 hours post removal from food source. DNA profiling of the crop content supported previous findings, although only partial STR profiles were obtained. It is unlikely that full profiles will be obtained when analyzing gut content due to the degraded nature of the food source, as well as the effect of digestive enzymes present in the maggot saliva regurgitated onto the food source. Various recommendations can be made based on the results. At crime scenes maggots should be killed in warm water (± 60°C for 30 seconds), dried on paper towel and stored in 80% ethanol while transported to the laboratory. Samples should be removed from the ethanol within 24 hours after collection, dried and stored individually in microcentrifuge tubes at -80°C (or similar low temperatures) until analysis is performed. During analysis samples should be handled on ice, ensuring the integrity of the sample, as it was found that samples defrosted rapidly after removal from the -80°C storage. It is further recommended to use commercially available kits when analyzing maggot gut samples due to the additional clean-up steps present in the kits. These clean-up steps aid in limiting the addition of fats and lipids from the maggot internal structures that could inhibit downstream DNA analysis of samples. Overall this study reinforced the possibility of using maggot crop content for providing STR profiles of the victim and/or perpetrator. Although only partial STR profiles were obtained, it indicated that, with further investigation and optimization, this is an interesting avenue for future research with many unexplored avenues for aiding in crime scene investigation.Item Open Access The association of specific polymorphisms in the serotonergic system with aggressive, impulsive and suicidal behaviour(University of the Free State, 2016-06) Louw, Susan; Spies, J. J.; Heathfield, L. J.; Schneider, S. R.English: Suicidal behaviour and the consequences thereof are a major global issue and need to be researched in order to promote a better understanding of this maladaptive behaviour. However, understanding the aetiology of suicidal behaviour is important, but difficult, as it is multifactorial and complex. Although there is a growing body of research pertaining to suicidal behaviour, there is a lack of research on the genetic contribution towards an endophenotype for suicidal behaviour in South Africa. Due to the complex nature of suicidal behaviour, it was suggested that the use of an endophenotype would contribute to the possible intervention in this maladaptive behaviour. It has been suggested that some people were genetically predisposed to suicidal behaviour, and more importantly, the tendency to act on suicidal thoughts, and that this genetic vulnerability, underlain by the serotonergic system, might possibly be linked to inherited personality traits such as impulsiveness and aggression. Impulsiveness and aggression have therefore been suggested as possible endophenotypes of suicidal behaviour. Variation in the chosen genetic markers of the serotonergic system may modify the endophenotype of impulsivity and aggression, and in turn, influence the phenotype, suicidal behaviour. The aim of the research was to determine if impulsivity and aggression can act as a potential endophenotype for suicidal behaviour, and therefore, firstly, to determine whether aggression and impulsivity, as personal variables, are associated with suicidal behaviour, and secondly, to investigate the possible association of candidate polymorphisms (HTR1A rs6295, HTR1B rs6296, HTR2A rs6311 and SLC6A4 HTTLPR) of the serotonergic system to impulsivity, aggression and attempted suicide. Genes can thus be studied as a contributing factor and not the only factor that influence suicidal behaviour. A cohort of 25 research participants with a previous suicide attempt were recruited and matched to 25 healthy controls. All participants completed the BIS-11, RPQ, and BPAQ as quantifiable measures. Participants were also genotyped for HTR1A (rs6295), HTR1B (rs6296), HTR2A (rs6311) and SLC6A4 HTTLPR. The results for this study indicated that the suicide attempters scored significantly higher than the control group in all the questionnaires for aggression and impulsivity. This led to the conclusion that impulsivity and aggression is positively associated with suicidal behaviour. However, with regards to the molecular genetic analysis, only the HTR2A gene variant, rs6311, showed a significant difference between the suicide attempters and controls, with the A allele being more frequent in the suicide attempters (p = 0.0066). The suicide attempters were the only group that presented with the X allele for SLC6A4 HTTLPR and further studies are needed to replicate this finding. Interesting trends were observed regarding the other genetic variants, but no significant results were obtained. For HTR1A rs6295, the homozygous GG genotype conferred the highest risk for impulsive and aggressive behaviours in suicide attempters. The G allele for HTR2A rs6311, together with the S allele for HTTLPR, also seemed to increase impulsive and aggressive traits. In the case of HTTLPR, this finding was valid irrespective of the presence of suicidal behaviour. Overall, the results provide support for the use of behavioural measures of impulsivity and aggression as an endophenotype for suicidal behaviour. Some support was also found for the use of impulsive aggression as a single construct with regards to suicidal behaviour. The combination of psychology and genetic results are among the first ever reported for suicide attempters in South Africa. Despite the limited size of the study, perhaps due to the sensitivity of the construct under investigation, this study nevertheless adds significant value to the body of research pertaining to the under-studied topic of suicidal behaviour in South Africa. This study can improve phenotyping that will ultimately benefit South-African individuals.Item Open Access The environmental and genetic aetiology of the severity and presence of attention and hyperactivity related disorders in a population from South Africa(University of the Free State, 2015-07) Mansfield, Jade; Odendaal, Z.; Schneider, S-R.English: Attention-Deficit Hyperactivity Disorder is a neurodevelopmental disorder characterised by symptoms of inattention, hyperactivity, and impulsivity. This disorder has been increasingly diagnosed in children and adults since the early 1990s. Genetics has been largely implicated in the aetiology of ADHD, with environment influencing the severity of the condition. The purpose of this research was to study the influence of polymorphisms in the serotonin system on ADHD, and to investigate the extent to which certain environmental factors affect the severity of the condition. In total, 74 individuals took part in this study by completing an online self-report survey, a semi-structured interview, and/or providing genetic material in the form of saliva (52 individuals in total). Of these, a sub-set of comparative participants comprised of 45 individuals, and a sub-set of participants previously diagnosed with ADHD comprised of 29 individuals. Environmental analysis involved the assessment of medical, psychological, and developmental problems, learning disorders, sleep problems, nicotine dependence, and exposure to oxygen deprived conditions. Impairments in various other aspects were also assessed, including life skills, social concept, work or education, family life, and risky activities. Molecular analysis focussed on three genes in the serotonin system. These genes are responsible for various aspects of the functioning of the system. These genes encode for two serotonin receptors (HTR1B and HTR2A) and the serotonin transporter (SLC6A4). Three single nucleotide polymorphisms (SNPs) were genotyped using restriction enzyme digestion and confirmatory sequencing. A single variable number of tandem repeats (VNTR) in the SLC6A4 gene was also assessed. Results of environmental analysis alone revealed that exposure to hypoxic conditions and/or second-hand nicotine inhalation worsen inattentive and hyperactive symptoms of ADHD. Aspects found to be present alongside ADHD were increased psychological, medical and learning problems, and high nicotine dependence. The most significant impairments in life functioning in ADHD individuals arose in familial, social, and school or work environments, as well as significant impairments in self-concept and an increased affinity for risky behaviours. Exercise was found to have a positive self-reported effect on ADHD symptoms. Molecular analysis showed that the serotonin system plays a significant role in modulating ADHD. The HTR2A (rs6311) SNP in heterozygous form is linked to high levels of hyperactivity. The heterozygous form of the 5-HTT SNP (located in the SLC6A4 gene) was indicative of the presence of ADHD. The GG genotype of the SNP HTR1B (rs6296) showed significant associations with inattention score. The S allele of VNTR in the SLC6A4 gene showed the strongest association to ADHD in terms of type of ADHD (mainly inattentive), presence of ADHD, and severity of inattention and hyperactivity symptoms. Molecular and environmental components taken together reveal an indirect association between the HTR1B (rs6296) GG genotype and increased psychological problems. The HTR2A polymorphism (AG genotype), on the other hand, is associated with poor self-perception. This polymorphism, coupled with the 5-HTTLPR S allele, and medical problems shows significant associations with inattentive type ADHD. The 5-HTTLPR VNTR showed the strongest association to ADHD, even when coupled with environmental influences. The S allele of this VNTR, along with increased medical and psychological problems show significant correlations to ADHD. This study also shows that methods of measurement, while different, produce similar quantifiable information, however, may produce differing resulting diagnoses and should thus be viewed in context for study purposes. In concluding, this study provides a potential profile for the diagnosis of ADHD, in terms of both environmental and genetic components. The research also implicates the importance of the serotonin system as an integral modulator of the presence and severity of ADHD and its symptoms.Item Open Access Aspects of the genetics of human aggressive behaviour(University of the Free State, 2016-02-11) Odendaal, Zurika; Spies, J. J.; Spies, P.English: Abstract indicated in Script chapter by chapterItem Open Access Cross-species microsatellite markers for the detection of hybrids in the genus connochaetes(University of the Free State, 2013-07) Wessels, Letecia; Grobler, J. P.; Kotze, A.; Ehlers, K.Black wildebeest (Connochaetes gnou), a species endemic to South Africa, experienced two bottlenecks in the last century and the number of animals ultimately decreased to approximately 300. These bottlenecks led to a decrease in the genetic diversity of black wildebeest populations across South Africa. An additional threat to the genetic integrity of the black wildebeest was discovered between the 1960s and late 1980s, when researchers noted that hybridization between blue and black wildebeest occurs and that these hybrid animals are fertile. Identification of the hybrid individuals is crucial and various molecular techniques were researched, with microsatellite markers proving to be the most successful. The aim of the current study was to investigate the effectiveness of previously identified cross-species microsatellite markers and statistical approaches for the identification of hybrid herds and individuals on various Nature Reserves in the Free State Province as well as privately owned game farms in and around the Province. Two previously identified diagnostic microsatellite markers (BM1824 and ETH10) were used to screen the populations for putative hybrids. The genetic diversity of the black wildebeest populations studied supported earlier findings showing lower genetic diversity in black wildebeest compared to blue wildebeest. The addition of new reference material in the current study revealed that some of the alleles previously assumed to be unique to a specific species were in fact shared between the two species. This reinforced the need to use more reference populations of adequate size. Nominally blue wildebeest alleles were found in five populations on different game farms and Nature Reserves. The presence of these alleles could be an indication that hybrids are present at these localities or alternatively, support the finding that the number and distribution of reference populations should be increased. Assignment of populations to specific clusters using different software programmes revealed that, due to the large amount of genetic material shared between blue and black wildebeest, no clear assignment of individuals to a specific cluster could be obtained. Molecular analysis of two known hybrid animals did indicate that the two microsatellite markers chosen were able to identify first generation hybrids and possibly even second generation hybrids. The study also investigated the persistence of introgression of blue wildebeest genetic material into black wildebeest populations using simulation software. The simulation tests revealed that introgressed alleles could still be detected after ten generations of backcrossing. This has serious implications for the management of hybrid populations. Various recommendations can be made in terms of the future management and conservation of black wildebeest on Nature Reserves and game farms. The most practical approach for dealing with hybrid animals would first be to develop additional molecular techniques for the accurate identification of populations that contain hybrid animals. Positively identified hybrid populations should be kept separate and no introductions of these animals should be made into pure populations. A more drastic approach would be to cull animals with hybrid ancestry. This would however have serious implications on the already reduced level of genetic diversity in the black wildebeest populations. The most pragmatic approach for dealing with hybrid populations would be to keep pure blue and black wildebeest in protected areas and allow black wildebeest with moderate introgression on game ranches exclusively used for sport hunting.Item Open Access Genetic variation in the most primitive Clivia species(University of the Free State, 2010-11) Van der Westhuizen, Hester Maria; Spies, J. J.; Spies, P.English: The genus Clivia Lindl., which belongs to the family Amaryllidaceae J. St-Hil. (1805), is comprised out of seven different species. Clivia nobilis, C. caulescens, C. miniata, C. gardenii, C. mirabilis, C. robusta and the natural hybrid Clivia xnimbicola all forms part of this genus. Clivia mirabilis is found in the Northern Cape Province and is geographically isolated from the six other species which grows along the Eastern Coast and escarpment of South Africa. Conrad et al. (2003) proved that C. mirabilis and C. nobilis were the two most primitive species in the genus Clivia. During this study sequencing results were used to detect barcodes/SNPs for C. nobilis and C. mirabilis and to reveal genetic variation between the Clivia species. Clivia nobilis and C. mirabilis were tested with cross-species microsatellite makers to reveal intraspecific variation. Seven different gene regions were sequenced. Six were chloroplast regions, namely the atpH-I, matK, rpoB, rpoC1, rpl16, the trnL-F regions and one was a nuclear region, ITS1. The regions used for sequencing were evaluated as potential barcoding/SNP regions for future use. They were also used to infer the evolutionary development of C. nobilis and C. mirabilis. All seven Clivia species were analysed but this study focused mainly on morphologically different specimens of C. nobilis and C. mirabilis. The sequences were aligned and edited with Geneious Pro. A total of forty-seven polymorphic sites were observed between al seven species. Within the rpl16 region eleven parsimony informative sites were observed. The matK and trnL-F regions each had eight parsimony informative sites. ITS1 had three sites and rpoB and rpoC1, one parsimony informative site each. Within the atpHI region no parsimony informative sites were observed. The sequencing data obtained could be used for species identification and, therefore, showed great potential as barcoding regions. We propose that matK, rpl16 and trnL-F are used as a barcode in C. nobilis and C. mirabilis because they had the most parsimony informative sites. The cladogram obtained from the combined data set (atpH-I, rpoB, rpoC1, matK and trnL-F) confirmed that C. nobilis and C. mirabilis are two separate species. Clivia caulescens and C. xnimbicola forms a monophyletic group. Within the rpl16 chloroplast region intraspecific variation in C. mirabilis and interspecific variation between C. nobilis and C. mirabilis were observed. The phylogentic tree representing the sequencing results of the rpl16 region revealed three distinctive groups within the four different C. mirabilis populations. Two plants within one of the Donkerhoek populations showed more variation than the rest of the population. The rpl16 gene region proved to be ideal in order to test intraspecific variation in C. nobilis and C. mirabilis. To evaluate the use of cross-species markers, microsatellite makers designed for Phaedranassa tunguraguae, Hymenocallis coronia and Clivia miniata were tested on C. nobilis and C. mirabilis. Although amplification was obtained, in most cases the results could not be optimized in order to provide reliable analysis. In future species specific primers for C. nobilis and C. mirabilis will be developed. This study undoubtedly identified barcodes/SNPs for C. nobilis and C. mirabilis which can be used to eliminated mistaken identity. Gene regions specific for intra- and interspecific variation were identified and can be used in future for population studies.Item Open Access Identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia(University of the Free State, 2010) Snyman, Marius Christian; Spies, J. J.; Viljoen, C. D.English: Anthocyanins belong to a large group of secondary plant metabolites, the flavonoids, and fulfil a range of biological functions that include the cyanic pigmentation they provide to flowers, fruits, vegetables and leaves. The anthocyanin biosynthetic pathway has been well elucidated and much effort has been made by researchers to modify some of the catalytic steps, thereby changing the colour of some ornamental and cut flower species. The genus, Clivia, is an ornamental monocot indigenous to South Africa and there has been a growing interest among local and international Clivia breeders to introduce novel flower colour varieties into the market. Transgene technology holds new possibilities to ensure modification of Clivia flower colour. However, the genetics and biochemistry of the Clivia anthocyanin biosynthetic pathway must first be investigated before any attempts regarding biotechnology can be made. The current study is the first to deal with the identification and expression analysis of flavonoid biosynthetic genes in the genus Clivia, specifically those involved in anthocyanin biosynthesis, thus identifying future prospects and motivating research in unexplored territory. A previous study concerning an HPLC analysis of Clivia anthocyanin content confirmed the presence of cyanidin and pelargonidin derivatives as the main pigments in the tepals and fruits. This enabled the establishment of a putative Clivia anthocyanin biosynthetic pathway illustrating each enzymatic event. Conventional PCR with degenerate primers and a tepal cDNA template was used to isolate four different target sequences. Consensus cDNA fragments of 586 bp, 326 bp, 510 bp and 225 bp confirmed the existence of Clivia orthologues for Chalcone synthase (CHS), Chalcone isomerase (CHI), Flavanone 3-hydroxylase (F3H), and Dihydroflavonol 4-reductase (DFR), respectively. The deduced amino acid sequences of CHS, DFR and F3H harboured important conserved residues that confirmed the existence of functional enzymes. Furthermore, nucleotide sequence analyses between each new Clivia cDNA fragment and the corresponding fragments of other higher plants, regarding similarity/identity and phylogeny demonstrated closer homologies and evolutionary relatedness to other monocot species. The identification of the Clivia flavonoid biosynthetic genes enabled the expression analyses of CHS and DFR. These structural genes encode enzymes responsible for two important controlling steps necessary to determine the nature of the final end-product(s) of the pathway. Real-time quantitative RT-PCR involving SYBR® Green chemistry was used to investigate the temporal expression of the two genes in the tepal, stamen and carpel tissues during five flower developmental stages of an orange and yellow variety of Clivia miniata. Statistical analyses were used to support any findings where possible. Each respective tissue type revealed its own trend in expression for both CHS (an early biosynthetic gene) and DFR (a late biosynthetic gene) throughout flower development except in the stamens of the yellow flowers. These findings suggested the co-ordinate regulation of the Clivia miniata anthocyanin biosynthetic genes as a single module, a model of transcriptional regulation that is often found in certain monocot species (Dooner et al., 1991; Meldgaard, 1992; Martin and Gerats, 1993). To understand the regulatory system that confers flowers colouration, genes that encode transcription factors should be isolated and their spatial and temporal expression investigated. The „parallelism‟ between anthocyanin biosynthetic gene expression and anthocyanin production in the tepals of the orange and yellow Clivia miniata varieties was also investigated. UV-visible spectrophotometry at A530nm was used to quantify total anthocyanins at each developmental stage after extraction. At full bloom the orange flowers had almost 16 times more anthocyanins, which support orange colour development, than the yellow flowers. It was confirmed by the outcomes of statistical analyses that the trends in expression of CHS and DFR and anthocyanin production were similar. Methods such as HPLC are recommended for more precise qualitative and quantitative determination of total monomeric anthocyanins.Item Open Access A taxonomic review of the genus Microacontias (Scincidae: Acontiinae) based on DNA and morphological data(University of the Free State, 2009) Janse van Vuuren, Lucas Cornelius; Heideman, N. J. L.; Daniels, S. R.The recent taxonomic review of the legless, fossorial skink genus Acontias by Daniels et al. (2006), using DNA sequence data, led to the erecting of the new genus Microacontias for all the small-bodied taxa. These are Microacontias lineatus lineatus, Microacontias lineatus tristis, Microacontias lineatus grayi and Microacontias litoralis. The latter species was, however, nested in the lineatus complex which raised questions about its true taxonomic status. Furthermore, the taxonomic status of the lineatus complex is questioned following the poor relationships between taxonomic diversity and morphological character states in these sub-terrestrial skinks (Daniels et al., 2002, 2005, and 2006). This study therefore addresses the following two questions: (i) should the taxonomic status of the lineatus complex be accepted as it stands and (ii) is Microacontias litoralis an independent species or does it form part of the lineatus complex. It is hypothesized that M. l. lineatus and M. l. tristis do not represent separate taxa (near identical morphology), while M. l. grayi and M. litoralis should be given independent status. The diagnostic methods utilized were (i) Mitochondrial DNA (mtDNA) sequence data (16S rRNA and Cytochrome b), (ii) morphometric data comprising head and body measurements, and (iii) meristic data comprised of various scale counts. The findings suggest that at least in terms of the gene fragments sequenced, the subspecies of the lineatus complex are very closely related. The phylogeny reports no distinct grouping of the taxa, nor could the morphological analyses separate the taxa as independent evolutionary units. The low genetic divergence but extensive color variation can be viewed as an example of morphological diversification despite genetic conservatism.Item Open Access Genetic variation of Clivia caulescens(University of the Free State, 2011-06) Stegmann, Suzanne; Spies, J. J.English: At present, the genus Clivia consists of six species, including Clivia nobilis Lindl., C. miniata (Lindl.) Regel, C. gardenii Hook., C. caulescens RA Dyer, C. mirabilis Rourke and C. robusta Murray, Ran, De Lange, Hemmet, Truter & Swanevelder. Many of the species and cultivars are extensively grown worldwide, making this group of considerable horticultural importance. This study mostly focused on Clivia caulescens with a natural habitat on the escarpment from Limpopo to Swaziland through Mpumalanga. The overlapping distribution between C. miniata and C. caulescens resulted in the formation of a natural hybrid between these species at the Bearded Man Mountain. The occurrences of natural hybrids between the various species are rarely recorded. In an attempt to find out if genetic erosion is currently a threat to the various C. caulescens populations and Bearded Man Mountain clivias, this study was conducted to establish if genetic variation is present. Genetic variation refers to the variation in the genetic material of a population, and includes the nuclear, mitochondrial, ribosomal DNA as well as the DNA of other organelles. The relative genetic diversity among individuals or populations can be determined using morphological and molecular markers. Five chloroplast DNA regions, i.e. atpH-I, matK, rpoB, rpoC and trnL-F, were used in an attempt to study the molecular diversity of C. caulecsens. This study concentrated on Single Nucleotide Polymorphisms (SNPs) from these regions and microsatellites to study genetic variation. The aim of this study was to determine the genetic variation between and within the different populations of C. caulescens, to determine whether gene flow occur between the different populations and to determine which of the DNA regions included in the study can contribute to the identification of plants from a specific geographical area. Regarding the study of Clivias situated at the Bearded Man Mountain, the main objectives were to estimate genetic diversity and determine the genetic relationship among the different species of Clivia (C. miniata, C. caulescens and C. xnimbicola) from this area. Of the initial five regions that were sequenced, trnL-F amplification failed repeatedly, and this region was therefore excluded from all analyses. The other four regions showed variation between the different populations of C. caulescens and for the Bearded Man Mountain clivias, except the rpoC1 region. When the results of the phylogenetics and statistical analysis (genetic distances) were combined, it was detected that most Bearded Man Mountain specimens and God‟s Window specimens clustered together in the cladograms and in the mean distances tables. Intraspecific variation was present in all the regions and combined dataset. All attempts during this study to amplify STRs and test allelic diversity in 13 microsatellite loci for 20 specimens failed. Cross-species amplification was not as effective as hoped. Microsatellites‟ species-specific nature could have a negative effect on obtaining results, although other researchers (as mentioned in the introduction of Chapter 4) could employ cross species markers successfully. Glen & Schabble (2005) reported that a given pair of microsatellite primers rarely works across broad taxonomic groups, so primers are usually developed anew for each species. The next step would therefore be to attempt the designing of specific primers for C. caulescens.Item Open Access Bt expression in maize plant tissues and the impact of gene flow(University of the Free State, 2012) Richardson, Grant Anthony; Viljoen, C. D.In 2007, the first case of field resistance to Cry1Ab was reported in South Africa, which is a concern as it negates the benefit of this technology. It has been suggested that a major contributing factor to the development of resistance in the target insect was the lack of compliance by commercial farmers to plant refugia. However, another possible mechanism of resistance development is the production of sub-lethal doses of Cry1Ab that could have resulted in a high selective pressure for resistance alleles. Although there are studies that have determined levels of Cry1Ab in different tissue in MON810 maize, the available data is not complete, especially for important feeding tissue of B. fusca larvae, such as silk and cob sheath. In this study, a comprehensive analysis of levels of Cry1Ab within and between different tissue over the growing season was conducted, taking the effect of gene flow also into account. Field trials were performed over the 2008/2009 and 2009/2010 growing seasons under conventional farming practice. Gene flow was allowed to occur between IR and non-IR maize in the 2008/2009 growing season, and the F1 seed was planted in the 2009/2010 growing season. The levels of Cry1Ab were monitored over both growing seasons, including the F1 plants in the second season. Notably, this study was the first to determine levels of Cry1Ab in cob sheath, which is considered one of the primary food sources for B. fusca larvae. It was found that there was considerable variation in levels of Cry1Ab within and between different tissue over the growing season. The data for the majority of the sampling points was moderately to highly skewed, indicating the non-parametric range in variation of Cry1Ab levels. There was a significant difference in Cry1Ab production between the two growing seasons, which was attributed to the lower than average rainfall in the 2008/2009 growing season and a higher than average rainfall in the 2009/2010 growing season. The overall trend in Cry1Ab production was congruent with the pattern of target insect larval survival after feeding on different tissue as reported by Van Rensburg (2009). Based on these data we suggest that important insect feeding tissue, namely silk, cob sheath and cob, could be producing sub-lethal doses of Cry1Ab that may result in ineffective control of insect pests. It appears that the decline in Cry1Ab production at late growth stages, in conjunction with variable levels of Cry1Ab between different tissue, may compromise the high dose/refugia strategy, resulting in selective pressure for the evolution of resistance. The gene flow study determined that outcrossing between IR and non-IR maize adversely affects the level of Cry1Ab in F1 plants. The levels of Cry1Ab were significantly lower in F1 maize when compared to a commercial MON810 maize hybrid, possibly as a result of reduced fitness. These data support the observation of increased insect larvae damage to F1 plants, suggesting that F1 maize may produce sub-lethal doses of endotoxin, and consequently will not effectively control insect pests. The considerably lower expression of Cry1Ab in F1 plants is a consideration in respect to subsistence farming practice in Africa, where seed is saved or exchanged among farmers. We postulate that the introduction of IR maize in subsistence farming could promote the development of insect resistance if not managed correctly. In conclusion, the current study has determined that there is a wide range of level of Cry1Ab within and between different tissue over the growing season. Gene flow adversely affects Cry1Ab production, potentially due to reduced fitness of the F1 plants. These data support the observation of differential rates of larvae survival when feeding on different IR maize tissue. Finally, the study provides an important basis for understanding the potential role that variable levels of Cry1Ab may have had on the development of resistance in B. fusca in South Africa.Item Open Access Genetic diversity in the Afrikaner cattle breed(University of the Free State, 2014) Pienaar, Lené; Grobler, J. P.; Neser, F. W. C.; Scholtz, M. M.; Ehlers, K.𝑬𝒏𝒈𝒍𝒊𝒔𝒉 This study was a first attempt to use microsatellite markers to determine the genetic diversity in an indigenous cattle breed, namely the Afrikaner. It was also the first study to combine genetic markers and pedigree analysis to estimate the genetic variability within an indigenous cattle breed. The objectives of the study were to estimate genetic diversity and inbreeding levels within the breed and to utilize the results to preserve and ultimately improve the genetic resources offered by the breed. A total of 1214 stud animals (representing 28 herds) and 166 commercial animals (nine herds) from different geographical areas within and adjoining South Africa were included in this study. Animals were genotyped at the two major animal molecular laboratories in South Africa, with both using the same standardized 11 marker microsatellite set. Estimates of genetic diversity did not support the hypothesis of significant loss of genetic diversity in the Afrikaner breed. Heterozygosity estimates ranged from 0.737-0.456 within individual populations, with an overall heterozygosity estimate of 0.568 for the Afrikaner breed. Assignment methods (based on STRUCTURE software) revealed a real structure consisting of four genetic populations (K=4). No consistent pattern of significant differentiation between stud- and commercial herds could be identified. Pedigree information, based on a total of 244714 recorded animals from 1940 to 2011, were analysed to determine the mean level of inbreeding (F), effective population size (Ne), generation interval, effective number of founders (fe), effective number of ancestors (fa) and average relatedness (AR). The average inbreeding coefficient calculated was 1.83% and the effective population size computed using the increase in the individual rate of inbreeding was estimated at 167.54. A total of 84138 animals (34.4%) were inbred to some degree. The effective numbers of founders and ancestors were 288 and 226 respectively, with an average relatedness of 0.44% and with results confirming a total of six complete generations. The average generation interval for the whole population was calculated as 6.554 ± 3.883 years. It is concluded that a moderate to high degree of variation is still present within the Afrikaner cattle breed, despite the recent decline in numbers of this indigenous breed. Levels of inbreeding appear to be at acceptable and at manageable levels. The current study provided results than can be utilized by farmers and breeders’ society to conserve the Afrikaner and develop the breed to its full potential.Item Open Access Screening of young and/or familial African breast cancer patients for the presence of BRCA mutations(University of the Free State, 2014-01) Peter, Namhla; Van der Merwe, N. C.; Visser, B.English: Screening for mutations within the BRCA1 and BRCA2 genes is a daunting task due to the length of the genes and the absence of mutational hotspots. An additional contributing factor is the genetic diversity within the Black ethnic groups of Africa, including the Sotho/Tswanas of the Free State. As no information was available regarding the prevalence of BRCA mutations within this group, this pilot study was launched in an attempt to determine the genetic component attributable to BRCA mutations in BC development. The selection criteria were not optimal and possible sporadic or single cases were included which according to literature, is not associated with mutations within the two familial BC genes. This resulted in a low percentage of disease-causing mutations being detected. It is proposed that the selection criteria in the future should emphasize the selection of bilateral BC cases with or without a positive family history. This characteristic seems to be more closely associated with familial BC in the Black patients than an early age at onset. The latter could be masking the familial BC cases, as the median age of onset of the disease in Black ethnic groups is 48. Two disease-causing mutations were identified, one within each of the genes. Both mutations were detected with PTT as they are located within exon 11. This indicates that this technique, although based on older technology, is still a valuable screening technique as it is cost-effective and less time consuming than screening the larger exons with for example high resolution melting (HRM). The two mutations are both situated within critical regions of the genes. BRCA1 c.2069_2072delAAAG,p.Lys653SerfsX699 is located within the binding domain of Rad50 whereas BRCA2 c.6455_6455delT,p.Lys2075ArgfsX2078 is located in BRC repeat 8. The presence of both these mutations will result in a truncated protein that would probably not be able to participate in DNA repair in response to DNA damage and cell cycle control (Green and Lin, 2012). This could result in chromosome instability and therefore tumour formation. Both mutations are novel and have not been detected internationally nor in the Black population residing in Gauteng SA. As all mutations detected thus far for the Black SA population seem to be limited to a single family and with no founder mutations found, full screening of both these genes remains the golden standard. Functional studies should be performed for the intronic variant BRCA2 c.517-4C>G (g.32900632C>G, rs81002804) detected in various patients. As this intronic variant is located only four bp from the start of exon 7, it could play a role in creating an alternative splice site. These studies together with the analysis of control individuals from the various Black SA ethnic groups will resolve the question whether this variant has the potential to be disease-causing.