Genetics

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Open Access
Allelic diversity of selected human neurotransmitter genes in South African ethnic groups
(University of the Free State, 2014-06) Malan, Clement; Schneider, S. R.; Spies, J. J.
English: Inadequate information on the allelic frequency of neurotransmitter genes exist among South African ethnic groups. The analyses of neurotransmitter genes with populations world-wide have benefited behaviour, population and medical fields. Population genetic fields have benefited due to increased information on population differentiation, structure and variation. Neuropsychiatric and behavioural disorders are prevalent among South African populations. In addition, research on neurotransmitter genes is limited for South African populations. Information on South African populations is inadequately based on one or two population groups and a limited number of neurotransmitter genes. This lack of information prompted the analyses of the Khwe, Xhosa, Sotho and Afrikaner ethnic groups with the DRD4, MAO-A and 5-HTT (5-HTTLPR) in the current study. Volunteers from the mentioned South African populations provided saliva samples. In total, 349 individuals were successfully analysed for the DRD4 VNTR, MAO-A-uVNTR and 5-HTTLPR. Significant genetic diversity was determined among the different populations. The Xhosa and Sotho are the closest related populations in the study followed by the Xhosa and Khwe. The Afrikaner population, an African population with mostly European ancestral heritage is the most genetic divergent population. From the analysis of the South African populations a hypothesis was constructed that the DRD4 and 5-HTTLPR allele frequencies may be associated with historic migration distance due to novelty seeking. It was theorised that populations that have migrated the greatest distance from Africa would have the highest levels of novelty seeking alleles. With the use of the South African populations and world-wide populations from revised literature positive correlations were identified for the S allele of the 5-HTTLPR. Positive correlations were also determined for the 7-repeat, >5-repeat and the combined 2- and >5-repeat alleles of the DRD4 VNTR. The 4-repeat allele of the DRD4 VNTR was determined the greatest among the analysed Khwe, Xhosa, Sotho and Afrikaner ethnic groups. The Khwe population had the highest frequency (0.711) for the 4-repeat allele among the analysed populations. The Khwe, Xhosa and Sotho populations DRD4 frequencies were similar to other African populations. The Afrikaner population has similar frequencies to its ancestral populations for the DRD4 VNTR. A remarkable finding of the study is the high frequencies of the long 5-HTTLPR alleles (L, VL and XL) in the Khwe population. The Khwe 5-HTTLPR allele frequencies are similar to that of the pygmy Mbuti population from central Africa. The Khwe is the most genetically diverse population analysed in this study and is possibly associated with the first modern humans. In contrast to the Khwe, the Afrikaner population has reduced L allele frequencies and less genetic diversity. The 4.5- and 3.5-repeat MAO-A-uVNTR alleles were the most common among the analysed populations. A significant finding in this study was that the Khwe, Xhosa and Sotho 3.5-repeat allele frequencies were significantly reduced compared to African Americans. The analysis of the study populations with the MAO-A-uVNTR provided information to an area that is lacking. Few correlations were previously possible due to the MAO-A-VNTR not being analysed in African populations. This study proved that allele frequencies for neurotransmitter genes differ between South African ethnic groups. A high degree of genetic relation was determined among linguistically related groups (Xhosa and Sotho) compared to non-related groups (Afrikaner). Neurotransmitter genes were determined significant factors in past human migrations. Novelty seeking alleles were associated with populations that migrated great distances.
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.
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 chapter
Open Access
Aspects of the genetics of human aggressive behaviour
(University of the Free State, 2012-12) Odendaal, Zurika; Spies, J. J.; Spies, P.
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.
Open Access
Biological clock measures: assessing the association between the circadian and epigenetic clock as predictors of migration phenology and biological aging in wildlife
(University of the Free State, 2024) Le Clercq, Louis Stephanus; Dalton, D. L.; Grobler, J. P.; Kotzé, A.
This thesis explores the use of biological clocks, studied at the molecular level, in understanding and predicting animal traits that change over time. In particular, the thesis focuses on two biological clocks: the circadian clock and the epigenetic clock. The study aimed to shed light on how these clocks, along with specific genes associated with them, influence traits such as migration patterns in birds and age in animals. In the investigation of circadian clock genes, this thesis presents a detailed review of existing literature, presenting both supporting and conflicting results on the association between clock gene polymorphisms and migration patterns. This review summarized the central hypotheses tested in these studies, identified several candidate genes that have been used, revealed distinct patterns in terms of the taxonomy and phylogeny of studied species, and present new insights into why conflicting results happen as well as what future research is needed. Furthermore, the review emphasizes the importance of considering molecular clock differences between lineages when studying multiple species. The two most studied candidate genes were then tested for associations to phenology in an intra-African migrant, the Diederik cuckoo, and provides the first evidence that phenotypic correlates identified in Eurasian passerine species is conserved for these species. The second part of the thesis delves into epigenetic clocks, examining two widely used methods: methylation and telomere length. A systematic review and meta-analysis were performed to compare the performance of these methods across vertebrate classes. Methylation was found to outperform telomere length in predictive power, with both methods showing promise as biomarkers for age determination in animals. To demonstrate the practical application of epigenetic clocks, the study included the development of a new age determination model on the African cheetah. Six candidate genes were identified, and a model using CpG methylation levels was created using machine learning techniques to refine the model, resulting in accurate age predictions. This approach offers a less invasive means of age estimation for population monitoring. The research also introduces two new PYTHON tools, PAReTT, to incorporate phylogenetic and molecular clock data into ecological and evolutionary reviews, and ABCal, to address potential biases in systematic reviews in evolution and ecology. The thesis concludes by highlighting the broader implications of the study, emphasizing the utility of biological clocks in understanding temporal traits, from annual life events in birds to lifelong aging in mammals. The generated datasets and tools contribute to ecological systematic reviews and individual studies, expanding our knowledge of biological clocks and guiding future research endeavours.
Open Access
Biological clocks as age estimation markers in animals: a systematic review and meta-analysis
(Wiley, 2023) Le Clercq, Louis-Stéphane; Kotzé, Antoinette; Grobler, J. Paul; Dalton, Desiré Lee
Various biological attributes associated with individual fitness in animals change predictably over the lifespan of an organism. Therefore, the study of animal ecology and the work of conservationists frequently relies upon the ability to assign animals to functionally relevant age classes to model population fitness. Several approaches have been applied to determining individual age and, while these methods have proved useful, they are not without limitations and often lack standardisation or are only applicable to specific species. For these reasons, scientists have explored the potential use of biological clocks towards creating a universal age-determination method. Two biological clocks, tooth layer annulation and otolith layering have found universal appeal. Both methods are highly invasive and most appropriate for post-mortem age-at-death estimation. More recently, attributes of cellular ageing previously explored in humans have been adapted to studying ageing in animals for the use of less-invasive molecular methods for determining age. Here, we review two such methods, assessment of methylation and telomere length, describing (i) what they are, (ii) how they change with age, and providing (iii) a summary and meta-analysis of studies that have explored their utility in animal age determination. We found that both attributes have been studied across multiple vertebrate classes, however, telomere studies were used before methylation studies and telomere length has been modelled in nearly twice as many studies. Telomere length studies included in the review often related changes to stress responses and illustrated that telomere length is sensitive to environmental and social stressors and, in the absence of repair mechanisms such as telomerase or alternative lengthening modes, lacks the ability to recover. Methylation studies, however, while also detecting sensitivity to stressors and toxins, illustrated the ability to recover from such stresses after a period of accelerated ageing, likely due to constitutive expression or reactivation of repair enzymes such as DNA methyl transferases. We also found that both studied attributes have parentally heritable features, but the mode of inheritance differs among taxa and may relate to heterogamy. Our meta-analysis included more than 40 species in common for methylation and telomere length, although both analyses included at least 60 age-estimation models. We found that methylation outperforms telomere length in terms of predictive power evidenced from effect sizes (more than double that observed for telomeres) and smaller prediction intervals. Both methods produced age correlation models using similar sample sizes and were able to classify individuals into young, middle, or old age classes with high accuracy. Our review and meta-analysis illustrate that both methods are well suited to studying age in animals and do not suffer significantly from variation due to differences in the lifespan of the species, genome size, karyotype, or tissue type but rather that quantitative method, patterns of inheritance, and environmental factors should be the main considerations. Thus, provided that complex factors affecting the measured trait can be accounted for, both methylation and telomere length are promising targets to develop as biomarkers for age determination in animals.
Open Access
Birds of a feather flock together: a dataset for Clock and Adcyap1 genes from migration genetics studies
(Nature Research, 2023) Le Clercq, Louis-Stéphane; Bazzi, Gaia; Obiol, Joan Ferrer; Cecere, Jacopo G.; Gianfranceschi, Luca; Grobler, J. Paul; Kotzé, Antoinette; Riutort León, Marta; González-Solís, Jacob; Rubolini, Diego; Liedvogel, Miriam; Dalton, Desiré Lee
Birds in seasonal habitats rely on intricate strategies for optimal timing of migrations. This is governed by environmental cues, including photoperiod. Genetic factors affecting intrinsic timekeeping mechanisms, such as circadian clock genes, have been explored, yielding inconsistent findings with potential lineage-dependency. To clarify this evidence, a systematic review and phylogenetic reanalysis was done. This descriptor outlines the methodology for sourcing, screening, and processing relevant literature and data. PRISMA guidelines were followed, ultimately including 66 studies, with 34 focusing on candidate genes at the genotype-phenotype interface. Studies were clustered using bibliographic coupling and citation network analysis, alongside scientometric analyses by publication year and location. Data was retrieved for allele data from databases, article supplements, and direct author communications. The dataset, version 1.0.2, encompasses data from 52 species, with 46 species for the Clock gene and 43 for the Adcyap1 gene. This dataset, featuring data from over 8000 birds, constitutes the most extensive cross-species collection for these candidate genes, used in studies investigating gene polymorphisms and seasonal bird migration.
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.
Open Access
Characterization of the endophytic mycobiome in cowpea (Vigna unguiculata) from a single location using illumina sequencing
(MDPI, 2022) Kinge, Tonjock Rosemary; Ghosh, Soumya; Cason, Errol D.; Gryzenhout, Marieka
Cowpea is an important crop for small-scale farmers in poor areas but is also being developed for commercial agriculture as a possible substitute for commercial legumes. Endophytic fungi are omnipresent and play crucial but diverse roles in plants. This study characterized the endophyte component of the cowpea mycobiome from leaves, main and crown stems and roots using Illumina MiSeq of the ITS2 region of the ribosomal operon. Ascomycetes exhibited the highest diversity, with Molecular Operational Taxonomic Units (MOTUs) assigned as Macrophomina, Cladosporium, Phoma, Fusarium and Cryptococcus, among the most dominant genera. Certain MOTUS showed preferential colonization patterns for above or below ground tissues. Several MOTU generic groups known to include phytopathogenic species were found, with relative abundances ranging from high to very low. Phylogenetic analyses of reads for some MOTUs showed that a level of identification could be obtained to species level. It also confirmed the absences of other species, including phytopathogens. This is the first study that adopted a holistic metagenomic typing approach to study the fungal endophytes of cowpea from a single location, a crop that is so integral for low-income households of the world.
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. ___________________________________________________________________
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.
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.
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:
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.
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.
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, Paul
𝗕𝗮𝗰𝗸𝗴𝗿𝗼𝘂𝗻𝗱: 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.
Open Access
The effect of selected polymorphisms in the p53 pathway as potential genetic modifiers of cancer risk and penetrance in female Afrikaner BRCA2 carriers
(University of the Free State, 2007-11) Dajee, Bhavini Kiran; Van der Merwe, N. C.; Visser, B.
Germline mutations in BRCA2 confer a high risk for the development of breast cancer in the Afrikaner population. A great deal of variability in the development of the disease has been observed among mutation positive family members. Evidence suggested that genes affecting breast cancer risk in the general population could potentially also affect breast cancer risk in BRCA mutation carriers. The cell cycle control pathway was selected as a candidate as the functional loss of the tumour suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumourigenesis. The aim of the study was an attempt to identify potential genetic modifiers of breast cancer risk and penetrance in Afrikaner women carrying the South African founder BRCA2 c.8162delG mutation. It involved environmental factors as well as six polymorphisms detected in critical genes of the Tp53 pathway. The investigated polymorphisms included three variants previously detected in Tp53 (intron 3, exon 4 and intron 6), a polymorphism present in the promoter of MDM2 and two SNPs identified in WAF1 (intron 2 and exon 2). The epidemiological study failed to identify any specific characteristic associated with an increased or protective breast cancer risk and did not explain the observed residual variation. Of the six polymorphisms studied, only one proved to be statistically significant, namely the 5’ splice-site variant in intron 2 of WAF1. This polymorphism seemed to explain the variation in penetrance for some of the families, but needs to be confirmed by more extensive studies. A breast cancer recombinant haplotype was compiled using the most informative variants, namely the polymorphism in the MDM2 promoter, the 5’ splice-site variant in intron 2 of WAF1 and the SNP in exon 4 of Tp53, but proved to be uninformative. Association studies including gene to gene and gene to environment interactions could assist researchers in their understanding of the mechanistic basis of the polygenic nature of breast cancer.
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.
Open Access
Filamentous fungi for sustainable remediation of pharmaceutical compounds, heavy metal and oil hydrocarbons
(Frontiers, 2023) Ghosh, Soumya; Rusyn, Iryna; Dmytruk, Olena V.; Dmytruk, Kostyantyn V.; Onyeaka, Helen; Gryzenhout, Marieka; Gafforov, Yusufjon
This review presents a comprehensive summary of the latest research in the field of bioremediation with filamentous fungi. The main focus is on the issue of recent progress in remediation of pharmaceutical compounds, heavy metal treatment and oil hydrocarbons mycoremediation that are usually insufficiently represented in other reviews. It encompasses a variety of cellular mechanisms involved in bioremediation used by filamentous fungi, including bio-adsorption, bio-surfactant production, bio-mineralization, bio-precipitation, as well as extracellular and intracellular enzymatic processes. Processes for wastewater treatment accomplished through physical, biological, and chemical processes are briefly described. The species diversity of filamentous fungi used in pollutant removal, including widely studied species of Aspergillus, Penicillium, Fusarium, Verticillium, Phanerochaete and other species of Basidiomycota and Zygomycota are summarized. The removal efficiency of filamentous fungi and time of elimination of a wide variety of pollutant compounds and their easy handling make them excellent tools for the bioremediation of emerging contaminants. Various types of beneficial byproducts made by filamentous fungi, such as raw material for feed and food production, chitosan, ethanol, lignocellulolytic enzymes, organic acids, as well as nanoparticles, are discussed. Finally, challenges faced, future prospects, and how innovative technologies can be used to further exploit and enhance the abilities of fungi in wastewater remediation, are mentioned.