Masters Degrees (Genetics)

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Open Access
Fluorescence in situ hybridization as a diagnostic tool for the detection of the FANCA delE12-31 and delE11-17 mutations
(University of the Free State, 2005-11) Nogabe, Sibongile Joy; Pearson, T.; Theron, M.; Jansen, S.
English: Fanconi anaemia (FA) is a rare autosomal recessive and X-linked disorder characterized by a very high frequency of bone marrow failure and many other manifestations. These include, but are not restricted to, severe birth defects and marked predisposition to malignancies, especially acute myeloid leukaemia and, to a lesser extent, solid tumours (Rodriguez et al, 2005). Cells from FA patients are hypersensitive to agents that produce DNA cross-links, and after in vitro treatment with these agents, display marked chromosome breakage and other cytogenetic abnormalities. FA shows genetic heterogeneity with mutations in any of twelve genes resulting in a similar phenotype. Current diagnostic criteria for FA relies mainly on cytogenetic quantification of chromosomal breakage in response to DEB and/or MMC. The diagnostic value of induced chromosome instability does not appear to be feasible for differentiating between FA carriers and non-carriers, since overlapping in quantitative values between the two groups is common place. In this investigation a population based screening strategy was followed. The method based on fluorescent in situ hybridization (FISH) was applied to allow a rapid and unequivocal identification of two founder Afrikaner FAA gene deletions, in both homozygous and carrier states. Direct labeling by both nick translation and thermal cycling amplification, using dUTP-labeled fluorochromes, resulted in no visible signals after hybridization, even though labeling proved to be successful. This restriction may be ascribed to the relatively small size (1.8kb and 2.3kb, respectively) of the DNA probes. Efficiency of hybridization detection decreases with decreasing probe size and a more sensitive detection method may solve this problem. Indirect labeling by polymerase chain reaction (PCR) amplification using digoxigenin-dUTP (DIG-dUTP) and antibodies (anti-DIG fluorochromes), provides an extremely sensitive method of detection, albeit more time consuming and costly. Bright, clearly defined signals were visualized after hybridization, using fluorescent microscopy. Stringent hybridization conditions, such as formamide contents of the hybridization buffer (70%) and optimal probe concentration (150ng), enhanced target-specificity and reduced background interference to almost none. Predominantly (>70% of interphase nuclei) the number of signals were in agreement with the ploidy of the specific DNA sequence, but the remaining cells revealed a mixture of either one, two or three signals. Target specificity tends to be a problem, especially with the smaller probe. Probes that are too small tend to bind non-specifically and re-hybridize, leaving smaller amounts of probe available for hybridization to the specific target. Even though, after hybridization, both probes resulted in easily visible fluorescent signals, the smaller delE11-17 probe (1.8kb) tended to be more prone to background interference with the signal, and, in addition, less target-specific. Probe hybridization efficiency and background are both influenced by the size of the labeled probe. The length of the probe molecule is critical for probe diffusion and hybridization to the specific target sequence. Probe size should be improved in order to provide a reliable and unequivocal diagnostic tool in the diagnosis of both FA patients and carriers. Longer probes will improve target-specificity and reduce the possibility of hybridization to other complementary regions in the genome. In conclusion, making use of this unique application of FISH offers an effective population directed screening for FA carriers and affected.
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
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.
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.
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.
Open Access
Genetic connectivity, population dynamics and habitat selection of the southern ground hornbill (Bucorvus leadbeateri) in the Limpopo province
(University of the Free State, 2011-03) Theron, Nicholas Terence; Kotze, A.; Grobler, J. P.; Jansen, R.
Southern ground hornbills (Bucorvus leadbeateri) (SGH) are co-operative breeders that occur in groups of 2-9 individuals. Long life spans, large territory sizes (100km²), and low reproductive rates render these birds vulnerable to threats such as loss of habitat, persecution for their habit of breaking windows through territorial aggression, poisoning and loss of suitable nesting sites. As a result, SGH are listed as vulnerable in the red data book of South Africa as well as globally. The main objective of this study was to contribute to our overall understanding of the ecology and biology of the SGH for conservation planning. Data collection was completed in the nonprotected, semi-arid landscape of the Limpopo Valley from June 2008 - September 2009. The seasonal habitat use by a group of SGH, seasonal abundance (numbers) and biomass (volume) of invertebrates using pitfall and sweep net methods was investigated. Furthermore, a total of eight groups and 23 birds were captured in the Limpopo Valley and different statistical analysis were performed to investigate levels of inbreeding, relatedness, sex-biased dispersal and the effects the recent re-colonisation has had on the genetic structure of SGH in the Limpopo Valley. Finally the genetic variation of the species in the rest of Africa was determined using samples from Kenya, Tanzania and three populations in South Africa namely the Limpopo Valley, Kruger National Park (KNP) and Kwa Zulu-Natal (KZN). Genetic analysis revealed SGH have retained comparatively high levels of genetic diversity, even though there are indications of genetic bottlenecks in the Limpopo, KNP and Kenyan populations. The SGH populations studied were grouped into two clusters corresponding to the geographic origin of samples. The birds from Tanzania and Kenya clustered together while the KNP and KZN birds clustered together with the Limpopo population grouping more or less equally between the Kenyan/Tanzanian and South African populations. A large percentage of genetic variation was found within populations while among population variation was low, indicating there is little molecular evidence for the presence of SGH subspecies. The overall home range of one group was approximately 20 000 ha while seasonal home ranges varied between 5000 ha in winter to 13 500 ha in summer. The response of organisms to environmental variables in this extremely seasonal habitat was further revealed by the positive correlations found between the number of invertebrates with mean monthly maximum and minimum temperatures, and the volume of invertebrates with mean monthly rainfall. No significant differences were found between numbers and volume of invertebrates per order, between sites, which was expected in this homogenous vegetation type dominated by mopani shrub and trees (Colophospermum mopane). The re-colonisation of the Limpopo Valley was shown to have occurred by a number of unrelated individuals. This was demonstrable by very low levels of inbreeding and average relatedness of the population, as well as the favourable levels of heterozygosity across age and sex categories. Within group relatedness was high with juveniles related to at least one parent from their natal group. Insights were also gained into the breeding behaviour of SGH, providing evidence for the first time that SGH are not as monogamous as previously thought, with two instances of extra pair copulations recorded between four groups. This study shows that a holistic approach combining genetic techniques, radio telemetry studies and ecological principles has great potential to further investigate SGH, thereby contributing to the preservation of this enigmatic species of the savannah biome.
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.
Open Access
Influence of selected polymorphisms on the expression of breast cancer in Afrikaner BRCA2 carriers
(University of the Free State, 2011-12) Schneider, Sue-Rica; Van der Merwe, N .C.; Visser, B.
English: The aim of the study was to elucidate the variation in phenotypic expression observed within BRCA2 c.8162delG mutation positive families. The study attempted to identify possible genetic factors that contribute to the residual risk conferred by the BRCA2 founder mutation. As BC is a polygenetic disorder, polymorphisms within various low penetrance genes may contribute to the expression of the disease. The selection of the SNPs were based on the results of the CIMBA consortium and have been proven to be associated with an increased BC risk in the general population (Easton et al., 2007) and in BRCA2 mutation carriers specifically (Antoniou et al., 2008). Two SNPs (rs2234693 [PvuII] and rs9340799 [XbaI]) present within ESR1 as well as SNPs present in TNRC9 (rs3803662), LSP1 (rs3817198), MAP3K1 (rs889312) and FGFR2 (rs2981582) identified by GWAS have been implicated in BC risk. These six polymorphisms have been selected to evaluate the risk within the Afrikaner BRCA2 8162delG (c.7934del, p.Arg2645AsnfsX3) mutation carriers specifically. Genotyping of rs2234693 (PvuII) and rs9340799 (XbaI) was done by PCR-RFLP analysis whereas Taqman® assays were used for genotyping rs3803662 (TNRC9), rs3817198 (LSP1), rs889312 (MAP3K1) and rs2981582 (FGFR2). Automated allelic discrimination using the BioRad CFX Manager v1.1.308.1111 software were compared to manual discrimination methods to ensure robust genotyping. Cohen’s kappa analysis suggested a combination of automated (Method 1) and manual (Method 3) genotyping was best suited for accurate allelic discrimination except for LSP1. Due to an putative SNP detected within LSP1, the validity of the LSP1 results should be treated cautiously as no information on the frequency of the second putative SNP in white European individuals is available. Of the six polymorphisms analyzed, only rs2234693 (PvuII), indicated a possible association with BC (P-value = 0.0896), which should be explored within a larger study group. For FGFR2, the HWE results indicated that the deviation observed in the BRCA2 mutation carrier group could possibly be associated with BC. Haplotypes compiled for rs2234693 (PvuII) and rs9340799 (XbaI) as well as the remaining four SNPs were uninformative as it revealed no differences between the BC patients and the Cases. These results may have been due to the high allelic heterogeneity observed within the Afrikaner population, as well as the small test group used.. Although the results of this study did not deliver significant results, it did provide insight into allelic distributions of the SNPs in the Afrikaner BRCA2 8162delG (c.7934del, p.Arg2645AsnfsX3) mutation carriers specifically. Larger scale genotyping could lead to more significant findings to help elucidate the polygenetic nature of BC with the Afrikaner.
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
Genetic origins of the introduced pea weevil (Bruchus pisorum) population in Ethiopia
(University of the Free State, 2012) Scheepers, Loraine Cornelia; Grobler, J. P.
English: This study aimed to determine the origin of pea weevils (Bruchus pisorum) in Ethiopia and to determine the current population structure across that country. The pea weevil is presently a widely distributed pest of peas in Ethiopia, causing huge financial losses. Conflicting hypotheses exist on the origin of B. pisorum in Ethiopia. It was possibly introduced to Ethiopia sometime in the 1970s, or it might have occurred historically in the area in very low numbers. The methodology of this study consisted of finding populations of pea weevils across the globe and then comparing these populations with the population currently found in Ethiopia. Specimens were obtained from Ethiopia, the USA, Germany and Australia. Gene sequences of pea weevils from China and Japan were also downloaded from GenBank to serve as reference material. DNA was extracted, amplified and sequenced using standard protocols, with the exception of the USA sample which was composed of museum specimens that demanded a non-destructive DNA extraction method. Three gene regions were used in this study: the Elongation Factor 1alpha (EF-1α), Cytochrome oxidase subunit one (COX1) and Cytochrome b (Cytb). The COX1 and Cytb sequence data provided insight into a possible source population of pea weevils in Ethiopia, whereas results from EF-1α were uninformative. Pea weevils from the USA were identified as a possible direct source, but it should be noted that these pea weevils are not endemic to the USA. The possibility of an endemic population of pea weevils in Ethiopia is also discussed. Tests for differentiation indicated that there was some differentiation between the Ethiopian subpopulations. This variation is discussed with reference to possible multiple sources of introduction for the current population in Ethiopia, genetic drift since introduction, and the possibility of a mixture of endemic and introduced genetic material in B. pisorum in Ethiopia.
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.
Open Access
Patterns of genetic diversity in vervet monkeys (Chlorocebus aethiops) from the south eastern regions of South Africa
(University of the Free State, 2012-07) Coetzer, W. G.; Grobler, J. P.; Turner, T. R.
Vervet monkeys (Chlorocebus aethiops) are one of the most widely distributed primate species in Africa. The aim of this study was to determine the level of genetic differentiation among conspecific vervet monkey populations in the south-eastern regions of South Africa, as part of a bigger project to determine levels of differentiation across South Africa. For this purpose, samples were taken from four localities in the Free State Province (Soetdoring Nature Reserve (NR), Gariep Dam NR, Sandveld NR and the Parys area), four Eastern Cape locations (Tsolwana NR, Baviaanskloof NR, Shamwari Private Game Reserve (PGR) and the Nelson Mandela Metropolitan University (NMMU) campus, Port Elizabeth), three Kwa-Zulu Natal location (St. Lucia area) and one Limpopo Province locality. Genetic differentiation was quantified using sequence data from a portion of the mtDNA control region. Twelve Haplotypes were identified within the total sample group. The nucleotide diversity for each grouping was calculated over all loci. Nucleotide diversity ranged from 0 to 0.038% ±0.02. Haplotype frequencies distribution among samples was calculated. An analysis of Molecular Variance (AMOVA) test was conducted and population pairwise FST values were estimated. The AMOVA test revealed that the majority of the genetic diversity occurred among the different groups (52.5%), with only 4.9% of the variation found within populations. The populations were assigned to groups according to geographic origins. The pairwise analysis identified significant levels of genetic variation among populations, with an average FST value of 0.851. These haplotypes were found to coincide with the geographical borders of Provinces. A ML tree was constructed using the haplotype data, and results showed clustering corresponding to geographical borders. A phylogenetic network was constructed, and this showed clustering similar to that found with the ML tree analysis. According to these results it is clear that there is genetic structuring among vervet monkey populations in South Africa. This clustering of populations can be potentially explained by female philopatry and geographical barriers. Female philopatry is a well known occurrence amongst Cercopithecine primates. The occurrence of geographical barriers, such as rivers and mountains had influence on migration rates and genetic structuring. This clustering pattern observed with mtDNA analysis contradicts results from previous studies working with nuclear DNA markers. This can be caused by various factors. Except for female philopatry having an effect on mtDNA differentiation patterns, it should be noted that the faster evolutionary rate of mtDNA vs. nuclear DNA can also cause different genetic patterns. The effective population size of mtDNA is also four-fold smaller than that of nuclear genes, and will also cause skewed results when comparing mtDNA data with nuclear DNA data. No reliable recommendations can be made toward the release of rehabilitated vervet monkeys, as further analysis is needed. It is thus suggested to use both genetic markers in follow-up studies. An increase in sample size from a broader geographical range is also recommended. In addition to further work on patterns of genetic variation, the adaptive significance of observed genetic differences should also be investigated.
Open Access
Genetic management of the baboon population in the Suikerbosrand Nature Reserve
(University of the Free State, 2012-10-12) Bubb, Annesca; Ehlers, K.; Kotze, A.; Grobler, J. P.
Genetic management has become a critical part of the overall management of nonhuman primate populations. This dissertation describes a genetic analysis of the chacma baboon population at the Suikerbosrand Nature Reserve. The aim of this study was to apply genetic data as a credible tool to contribute to the conservation and management of chacma baboons at Suikerbosrand Nature Reserve. The specific objectives included individual identification, determining genetic relationships and levels of gene flow within- and among the fourteen troops, and to construct a genetic database with individual genotypes of the whole population. A secondary objective of this study was to determine whether it would be feasible to extract DNA from fecal samples collected from a sleeping site and then use the genetic profiles to determine the number of individuals in that specific troop. The current population is estimated to be between 611 and 764 animals. The sleeping site of the Diepkloof troop was used for this part of the study. A panel of eleven human microsatellite markers was used for DNA analysis. DNA profiles from all the blood samples were successfully constructed and could be used to estimate genetic relationships. The level of genetic diversity in the Suikerbosrand baboon population did not differ significantly from that in the outgroup. Thus, the reintroduction of new individuals into the population to maintain acceptable levels of diversity is not an immediate priority. High levels of gene flow were observed between the troops, especially the troops located in the central part of the reserve. In order to ensure high DNA quality from fecal samples collected at the sleeping site, the collection method for fecal samples were optimized (A manuscript based on the work in this section has been accepted for publication in the European Journal of Wildlife Research). The profiles obtained from the fecal samples that were collected at the Diepkloof site corresponded with two of the thirteen profiles from the reference database. The estimated size of the Diepkloof troop is thirty seven individuals. The results show that non-invasive sampling could be a promising alternative for future research on the reserve, as the samples can be used to determine individual profiles. The genetic data collected can be combined with ecological and behavioral information collected form future research to further understand the population structure of the Suikerbosrand chacma baboons and changes that might occur in the population.
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
Insights into the genetics of the Ground Pangolin (Smutsia temminckii)
(University of the Free State, 2013-02) De Beer, Christle; Kotzé, A.; Dalton, D.; Ehlers, K.; Jansen, R.
English: Little is known about the molecular genetic variation of Ground Pangolin populations in South Africa. In this study it was attempted to assess the genetic diversity of the populations, but this could not be achieved due to insufficient cross‐species markers amplification. It should, however, be emphasized that the molecular work done in this study is novel, and that the results found during this research are key foundations for future studies. During sample collection it was found that three main populations of Ground Pangolins exist in South Africa in the Eastern, Western and Central parts of the country. Isolation protocols have been optimized, and it has been shown that noninvasive samples yield good quality and quantity DNA that is usable for down‐stream applications and perform as well as invasive samples. The PCR protocol was optimized, and the results from the optimization chapter will be of assistance when species‐specific markers are optimized. This study has also shown the need for the development of species‐specific markers, and the use of said markers will give a better indication of the genetic diversity of the Ground Pangolin populations in South Africa. From the statistical analysis it would seem that there are some correlations between the three sampling localities which may indicate a population divergence at some point. Based on the genetic diversity results, it appears that the diversity within Ground Pangolin populations is much lower based on the markers tested than in Malayan Pangolin populations. This will however have to be confirmed with species‐specific markers.
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
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.
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.
Open Access
Population genetic structure of the ground pangolin based on mitochondrial genomes
(University of the Free State, 2014-03) Du Toit, Zelda; Grobler, J. P.; Kotzé, A.; Dalton, D. L.
English: Temminck’s ground pangolin, S. temminckii, is currently listed as Vulnerable on the IUCN Red Data List. However, their numbers are decreasing due to illegal hunting for bush meat and over-harvesting for traditional use in Africa. Pangolins are also exported to Asia as a delicacy and for use in traditional medicine. Currently, the greatest threat to ground pangolins in southern Africa is electrocution by electric fences on game farms. This project consisted of two parts. The first was to sequence the whole mtDNA genome of Temminck’s ground pangolin to identify gene regions and to determine the evolutionary relationship of the order Pholidota. Results generated using the primer walking method, indicated that the whole mtDNA of Temminck’s ground pangolin is 16,559 bp in length. The phylogenetic analysis shows that the order Pholidota form a sister grouping with the order Carnivora rather than with the order Xenarthra as would be expected. Data suggested a Laurasian origin approximately 87 mya and possible migration into Africa during the Paleocene era around 55 mya. The second part of the study was conducted in order to determine the phylogeography of Temminck’s ground pangolins in southern Africa. Twenty five samples were collected from four countries, namely Namibia, Zimbabwe, Mozambique and South Africa (Mpumalanga and the Northern Cape Provinces). The results obtained indicated a high level of genetic variation within populations and only a few individuals displayed private haplotypes, which resulted in an increase in haplotype diversity. Samples from Zimbabwe and Mozambique (Group 1) clustered together while samples from the Northern Cape and Mpumalanga Provinces of South Africa grouped with samples from Namibia (Group 2), suggesting either an ancestral or recent split between Groups 1 and 2. The BEAST analysis indicated that the two groups shared a recent common ancestor between 2.94 and 1.27 mya across the three gene regions. In addition, it was estimated that the Zimbabwe/Mozambique split occurred between 920 and 710 kya and the Kalahari/Namibia/Mpumalanga split between 1.16 mya and 790 kya. This pattern corresponds to the Mega Kalahari Sand Sea forming a barrier between individuals and populations around that time. This study is the first molecular analysis based on the mitochondrial DNA genome of Temminck’s ground pangolin in southern Africa and it provides an insight into the species’ population genetics across its range in southern Africa. However, additional research into the order Pholidota throughout Africa can assist in better understanding of genetic variation within African pangolin species and populations. Furthermore, such studies will also support the conservation of genetic variation within species and contribute to identifying evolutionary distinct populations to assist in developing effective conservation management plans for the different species of the order Pholidota.
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.