Masters Degrees (Genetics)

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Genetic diversity and mating systems in a mass-reared black soldier fly (Hermetia illucens) population
(Stellenbosch : Stellenbosch University, 2021-03) Hoffmann, Lelanie; Rhode, Clint; Bester-van der Merwe, Aletta Elizabeth; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Black soldier flies are gaining popularity as an alternative source of protein in animal feed. They have a high feed conversion ratio and can be reared on biowaste, reducing the energy input required for mass-rearing. As the number of mass-reared colonies is increasing worldwide, the importance of genetic management in commercial populations is becoming clear. This study aimed to determine the effects of domestication and mating behaviour on the genetic diversity of a mass-reared black soldier fly population. Eight microsatellite markers were used to estimate genetic diversity in two temporally separated samples of a wild black soldier fly colony (Wild2015 and Wild2018) and three distinct generations of a mass-reared black soldier fly colony (F28, F48 and F52). Diversity estimates decreased significantly in the mass-reared colony over time, when compared to the two wild samples. The mass-reared colony also saw an increase in relatedness over time, with a relatedness coefficient as high as 0.430 in generation F48. These results indicate severe inbreeding in the mass-reared colony. Effective population sizes of between 22.6 and 59.0 in the mass-reared colony are also a cause for concern, as populations with low effective population sizes are more vulnerable to inbreeding depression and extinction. The high levels of genetic diversity observed in the two wild samples provide the potential for the wild colony to become a donor population, providing immigrants to introduce genetic diversity into the mass-reared colony. However, based on FST estimates, the two populations appear to be diverging from each other over time. Moderate differentiation was observed between Wild2015 and F28 (FST=0.062; p=0.000), while great differentiation was observed between Wild2018 and F52 (FST=0.161; p=0.000). To minimise the risk of outbreeding depression, the compatibility of wild individuals with the artificial environment would therefore need to be tested before immigrants are introduced into the mass-reared population. To study the mating behaviour of the black soldier fly, five mating pairs were randomly sampled in copula from generation F48 of the mass-reared colony. All candidate parents and 25 offspring from each clutch were genotyped and subjected to parentage analysis. Multiple paternity was detected in two of the five families, providing evidence for polyandry. This was a novel finding, as observation had previously led to the hypothesis that this species is monogamous. The occurrence of polyandrous mating provides evidence that adult flies can mate multiply despite being unable to replenish energy through feeding, thereby creating the potential for polygynous mating. Additionally, polyandrous mating has positive implications for the genetic management of commercial black soldier fly populations. However, these results are limited to mass-reared colonies, as the higher population densities found in captive populations increase the probability of remating. Finally, diversity estimates and inbreeding estimates were calculated for the candidate parents, offspring, and the population the parents were sourced from. Individual parent pairs showed increased levels of relatedness when compared to the source population, indicating positive assortative mating. As markers from random genomic regions were used for this study, the observed increase in relatedness may provide additional evidence for inbreeding in the mass-reared population. However, inbred populations show greater genome wide linkage disequilibrium, meaning that mate selection for desirable traits could potentially be detected in markers not directly related to traits of interest.
Genetic diversity and population genetic structure in the South African commercially important shark species, the common smoothhound (Mustelus mustelus)
(Stellenbosch : Stellenbosch University, 2014-12) Maduna, Simo Njabulo; Bester-van der Merwe, Aletta Elizabeth; Roodt-Wilding, Rouvay; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Deciphering patterns of intraspecies population genetic structuring in commercially important shark species is essential for an integrated fisheries management approach to conservation of regional biodiversity. The common smoothhound shark, Mustelus mustelus, is an overexploited, commercially and recreationally important shark species in South Africa. Considering the vulnerable status of the common smoothhound shark and due to very limited available genetic information, this study aimed to develop molecular markers, assess patterns of genetic diversity and population connectivity along the South African coast using multilocus data generated from 12 microsatellite markers and the mitochondrial gene, NADH dehydrogenase subunit 4 (ND4). The cross-species amplification of microsatellites proved useful for genetic diversity and population genetic analysis of the common smoothhound shark. These microsatellites could aid in the molecular characterisation of other endemic and cosmopolitan species and provide valuable tools for the conservation of potentially threatened or exploited shark species. For the microsatellite data, moderate levels of genetic diversity based on the heterozygosity, allelic richness and haplotype diversity were found in a total of 144 individuals sampled across eight study populations. Estimates for pairwise population differentiation, F-statistics, AMOVA and factorial correspondence analysis (FCA) indicated significant genetic structure within and between west- and east coast populations. Additionally, Bayesian clustering analyses detected two putative ancestral gene pools, supporting the presence of a biogeographic barrier at the Cape Agulhas region and therefore genetic discontinuity between the Indian and Atlantic Ocean samples. On the contrary, mitochondrial data indicated that common smoothhound shark is genetically homogenous with substantial interoceanic gene flow. Such conflicting signals found between nuclear and mitochondrial DNA (mitonuclear discordance) can be attributed to a number of factors and could simply be due to the inherent differences in marker properties or an indication of sex biased dispersal. Despite an indication of an expanding common smoothhound shark population based on both marker types, a contemporary genetic bottleneck may have gone undetected as genetic divergence was very low in some of the study populations. Nonetheless, contemporary restriction to gene flow and historical demographics such as range expansion are proposed as the most likely forces explaining genetic structure in present-day common smoothhound sharks in South Africa. For future sustainable exploitation of common smoothhound shark, the possible existence of two genetically differentiated populations and observed asymmetric gene flow along the South African coast should be taken into consideration. It is also recommended that in the future further evaluations of finescale genetic structure and seasonal migration patterns in this commercially important species are conducted in order to allow integration of this knowledge into existing fisheries management practices.
Genetic diversity in a commercial black soldier fly, Hermetia illucens (Diptera: Stratiomydiae), population
(Stellenbosch : Stellenbosch University, 2017-03) Badenhorst, Rozane; Rhode, Clint; Bester-van der Merwe, Aletta Elizabeth; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: The applications of black soldier fly (BSF), Hermetia illucens, as a first-generation biotechnology address two global problems - the pressure on food security arising from unsustainable food production practices which exhausting fisheries resources by fishmeal production; and the vast accumulation of organic waste. Understanding the phenotypic and genetic changes experienced when establishing colonies of H. illucens is important for successfully establishing new colonies and managing already existing mass-rearing facilities. The aim of this study was to assess the changes in genetic diversity coupled with the phenotypic changes associated with colony establishment. Commercially-relevant phenotypic parameters were measured; including pupae weight, percentage eclosion, post-mating longevity, hatchability and egg clutch sizes over six successive generations (F0-F5). Hatchability, clutch size and pupae weight increased from F1 to F4, but a decline was apparent from F3 to F4. Post-mating longevity dramatically decreased from F1 (100%) to F4 (33%) and the F5 generation did not develop beyond the pre-pupae stage resulting in colony-collapse. The phenotypic changes can be explained by a combination of environmental, physiological and genetic effects. The positive trends observed during earlier generations (F0-F1) may reflect the population reacting to relaxed natural selection. While, at the end of the study period (F3-F5), natural selection in captivity resulted in a decrease in phenotypic variables. The colony collapse in the final generation, despite constant environmental conditions, possibly reflects the deleterious effects of random genetic drift and inbreeding depression. For the assessment of the genetic processes which may have resulted in the observed phenotypic changes, ten microsatellite markers were developed and characterised. Marker utility was tested on 37 wild individuals using three multiplex-PCR reactions. The number of alleles for each locus ranged from 4 to 21. Polymorphism information content ranged from 0.52 to 0.90, while observed and expected heterozygosity ranged from 0.30-0.65 and 0.55-0.91, respectively. Furthermore, the markers are useful in individual identification (PID = 1.2x10-11and PIDsib = 1.1x10-4) and parentage analysis (P1=1; P2 = 1). The markers proved useful in the assessment of genetic diversity and were used to understand the genetic mechanisms underlying phenotypic trends previously described. The assessment of genetic diversity revealed significant population differentiation as described by FST estimates across the experimental generations (P < 0.01), accompanied by a significant loss of genetic diversity. The final generation indicated a significantly higher number of private alleles and significantly lower observed heterozygosity in comparison to preceding generations (P < 0.05). Population bottlenecks and inbreeding were evident in the estimation of effective population size (Ne) and relatedness. Phenotypic parameters which significantly correlated (Pearson’s r) to changes in genetic diversity were pupation (%), clutch size, pupae weight and female post-mating longevity (P < 0.05). Results indicate the effects of inbreeding depression and genetic drift were amplified by a decrease in effective population size and increased relatedness amongst individuals. In summation, the assessment of changes in phenotypic measurements and genetic diversity during the establishment of an H. illucens colony, gave valuable insight into the genetic processes which occur during colony establishment; and are applicable to colony establishment and the management of already existing colonies.
Marine forensics : a molecular tool for trade monitoring and compliance in southern African fisheries, with focus on commercially exploited elasmobranch species
(Stellenbosch : Stellenbosch University, 2019-12) Asbury, Tamaryn Anne; Bester-van der Merwe, Aletta Elizabeth; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Marine forensic science can be described as protecting fisheries resources, marine mammals and endangered species based on enforcement of the nation's laws. Species identification becomes challenging when morphological features (such as fins, scales and heads) are removed, or if confiscated samples are already in a processed state. The harvesting of elasmobranchs (sharks, rays and skates) is driven by the international shark meat and shark fin trade. In recent decades, a combination of increasing demand and economic globalisation has created a global market for elasmobranch products, especially the highly prized shark fins for Asian markets. In this study, marine forensics was assessed as a tool for complementing traditional identification methods – through the development of a mini-barcoding assay as well as investigating High Resolution Melting (HRM) analysis – for species identification, with focus on elasmobranch species occurring in the southern African region. Firstly, this involved the testing and optimisation of the standard barcoding region of the cytochrome oxidase c subunit I (COI) gene, and then using traditional barcoding primers as well as nested polymerase chain reaction (PCR) primers in a multiplex assay. Preliminary results (only a 22 % species identification success rate) indicated the limitations of using only the traditional COI primers and warranted the inclusion of alternate COI gene fragments for species identification in future related forensic cases. A mini-barcoding multiplex assay, comprising three primer sets, was optimised and applied to a wide range of forensic case studies involving confiscated shark fins, possibly for illegal trade. A significant number of CITES-listed and endangered species were identified when confiscated specimens from various regions in southern Africa were tested. Secondly, PCR amplification of a 16S ribosomal RNA (16S rRNA) gene fragment was optimised based on six southern African houndshark species and seven other commercially exploited species, including: hammerhead sharks Sphyrna lewini and Sphyrna zygaena, copper shark Carcharhinus brachyurus, dusky shark Carcharhinus obscurus, bull shark Carcharhinus leucas, blacktip shark Carcharhinus limbatus and blue shark Prionace glauca. High Resolution Melting analysis using the 16S rRNA gene region was investigated as a species identification method for these species. The HRM assay was successfully applied for the identification of seven commercially exploited shark species, including some of the top commercially important sharks and one endemic houndshark Scylliogaleus quecketti. Although further optimisation is required, this relatively fast and cost-effective approach will be a valuable tool for the initial screening of detained shipments, for possible illicit trade. Accordingly, this research presents species identification assays suitable for various shark related forensic case studies, and in future could be applied to identify most, if not all, elasmobranch species involved in trade regionally.
Medium-throughput SNP genotyping and linkage mapping in Haliotis midae
(Stellenbosch : Stellenbosch University, 2012-12) Du Plessis, Jana; Roodt-Wilding, Rouvay; Bester-van der Merwe, Aletta Elizabeth; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: Haliotis midae (locally also known as perlemoen) is the largest of five endemic species found along the coast of South Africa. It is the only species with commercial value contributing to the exploitation of these animals. Due to declines of natural stocks, farming practices were established during the early 1990s in order to supply the international demand. To facilitate efficient breeding methods and ensure the sustainability of these commercial populations, genetic management, which can be accomplished with the use of molecular markers such as single nucleotide polymorphisms (SNPs), is necessary. Single nucleotide polymorphisms have become the markers of choice in various applications in aquaculture genetics due to their abundance in genomes, reduction in developmental costs and increased throughput of genotyping assays. Identification of SNPs in non-model species such as H. midae can be achieved by in silico approaches. In silico methods are suitable for de novo SNP identification and are both cost- and time-efficient. It is based on the analysis of multiple alignments where mismatches may be reported as candidate SNPs. Various medium-throughput genotyping methods are available to confirm putative SNPs, but the ideal method depends on factors such as cost, accuracy and multiplexing capacity. Although SNP markers can have various applications within the aquaculture environment the focus for this current study was saturating the linkage map of H. midae with additional markers. This would assist in the identification of quantitative trait loci associated with economically important traits, which in turn could ultimately be employed for marker-assisted selection and improved molecular breeding programs. In order to identify in silico SNPs, sequenced transcriptome data from a previous study was used and subjected to a series of criteria: minor allele frequency 10%, minimum coverage 80, 60 bp flanking regions. Selected loci were genotyped using a 192-plex assay with the Illumina GoldenGate genotyping assay with the VeraCode technology on the BeadXpress platform, in individuals from six mapping families. A conversion rate of 69.35% and global success rate of 76.34% was achieved. Polymorphic loci were subjected to linkage analysis using JoinMap® v.4.1 to create sex-average and sex-specific maps and to saturate the current linkage map for H. midae. Along with previously developed markers, 54% of the newly developed SNPs could be successfully incorporated into the linkage map of H. midae. A total of 18 linkage groups were observed with an average marker spacing of 6.9 cM and genome coverage of 79.1%. Bioinformatic analyses and setting stringent criteria to identify SNPs from sequenced transcriptomic data proved to be an efficient way for SNP discovery in the current study. Genotyping of the identified loci with the GoldenGate genotyping assay demonstrated a high success rate; providing a genotyping assay adequate for species with little genomic information. The linkage map created in this study illustrated the utility of SNP markers in conjunction with microsatellite markers for linkage map construction and the adequate marker spacing obtained provides a step closer to quantitative trait loci mapping in this species.
Molecular aspects of exploited sharks in South Africa : multiple paternity and identification of novel molecular markers
(Stellenbosch : Stellenbosch University, 2015-12) Rossouw, Charne; Bester-van der Merwe, Aletta Elizabeth; Stellenbosch University. Faculty of Science. Dept. of Genetics.
ENGLISH ABSTRACT: Sharks have existed for the past 400 million years and play an important role in the oceanic ecosystem as they occupy the upper categories of the food web. Since the 1920s they have been fished on a commercial scale as the demand for shark related products increased. This placed considerable pressure on shark populations, resulting in a global decline of many populations and an increased risk of population extinction. As the genetic diversity of a population determines it’s resilience to changing environmental factors, including such information has become paramount for short and long-term management and conservation of individual species. This study therefore aimed to add to the growing body of biological and genetic data by investigating mating strategy through assessing the presence of multiple paternity (MP) in three commercially important shark species: the common smoothhound Mustelus mustelus, dusky shark Carcharhinus obscurus and the scalloped hammerhead shark Sphyrna lewini; and by identifying potential microsatellite and single nucleotide polymorphism (SNP) markers in two of the species, M. mustelus and C. obscurus, through the use of Next-Generation Sequencing (NGS) platforms. The first aim of this study was achieved by cross-amplifying microsatellite markers developed in closely related species to the study species. A total of 22 microsatellite markers were initially tested on four litters of M. mustelus and C. obscurus in order to determine the most informative markers for parentage analysis. Reduced marker panels of five to six microsatellites were selected and parentage analysis in GERUD and COLONY revealed the presence of MP in all three species. Mustelus mustelus had the highest frequency of MP (67%), followed by S. lewini (46%) and C. obscurus (35%). The second aim of this study entailed reduced genome sequencing of one M. mustelus and one C. obscurus individual using the HiSeq Illumina and Ion Proton platforms, respectively. For M. mustelus, 51,5 million reads with an average read length of 250bp were obtained, whereas C. obscurus yielded 27,6 million reads with an average read length of 213bp. Contigs were constructed for both species in order to search for perfect repeat motifs. In total, 2 700 and 1 255 microsatellite-containing regions were identified for M. mustelus and C. obscurus respectively. In order to search for SNP-containing regions, both sample species were aligned to previously assembled scaffolds of the ghost shark Callorhinchus milii, which served as a reference genome. After quality filtering, only 767 SNP-containing regions were identified for M. mustelus, whereas the identification of potential SNPs for C. obscurus was not successful. The insights gained into the mating strategies of M. mustelus, C. obscurus and S. lewini as well as the identification of potential species-specific molecular markers add to the growing body of information and genetic resources available for exploited species. In future, this information could be used for further molecular assessment of shark populations and a more intergrated approach to conservation and management of these already vulnerable sharks.
Molecular species identification and spatio-temporal assessment of genetic diversity in the smooth hammerhead shark Sphyrna zygaena in South Africa
(Stellenbosch : Stellenbosch University, 2017-03) Kuguru, Gibbs; Bester-van der Merwe, Aletta Elizabeth; Rhode, Clint; Gennari, E.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
ENGLISH ABSTRACT: The South African coast hosts a unique oceanographic regime with an enriched habitat able to support a diverse biota of chondrichthyans (sharks, skates, rays and chimaeras). Investigating these species and populations on a molecular level could aid in conserving this rich chondrichthyan biodiversity. As a precursor, a case study regarding the composition of species in three different South African fisheries was evaluated to determine the utility of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene in delimiting species identity. Through this, a number of issues surrounding misidentification and cryptic speciation were recognized, and the efficacy of CO1 was tested and proved to be useful in identifying chondrichthyans affected in South African fisheries. One of these species, the smooth hammerhead shark (Sphyrna zygaena) displays a high degree of site fidelity to Mossel Bay as evidenced by the rise in the number of neonate and juvenile hammerheads during the summer season. This species is vulnerable as they are in danger of overfishing and the destruction of their natural habitat. With a low fecundity and a long generational time, recovery of near-depleted populations is prolonged. In this study mitochondrial sequence data and microsatellite markers were used to assess genetic diversity within and between S. zygaena sampling cohorts collected from Mossel Bay to the KwaZulu Natal coast. Additionally, kinship between the juvenile individuals was determined and parental genotypes were reconstructed from the neonate and juvenile smooth hammerhead sharks sampled in the Mossel Bay area. Significant population subdivision was evident between individuals sampled in the warm temperate south coast (Mosselbay) and the subtropical east coast (Algoa Bay and KwaZulu Natal), with asymmetric gene flow mainly from the south to the east coast. Highly significant population differentiation was seen between sampling years, indicative of differential temporal stocks utilizing Mossel Bay each year. Analysis of kinship revealed a high degree of sibling relationships within and between seasons, which is likely due to an overlap of some parental genotypes across seasons. The results obtained here can assist with decisions regarding the conservation of chondrichthyan biodiversity in South Africa while it is also recommended that genetic structure and temporal variation of S. zygaena populations be evaluated on a finer scale in the future.
Population dynamics of two endemic catshark species inferred from molecular and tag-recapture data
(Stellenbosch : Stellenbosch University, 2023-03) Hubble, Emma Rebecca; Klein, Juliana Dunia; Bester-van der Merwe, Aletta Elizabeth; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
ENGLISH ABSTRACT: The endemic catshark genus, Poroderma, consists of two closely related species, the pyjama catshark Poroderma africanum and the leopard catshark Poroderma pantherinum. They are found within the southern African oceans and are threatened by overexploitation due to being a bycatch product of commercial fisheries. In recent years, this previously understudied genus has gained more attention for their role as important meso-predator species within the South African marine environment. There is a lack of information regarding the movement patterns and population dynamics of these catsharks and the effective management and conservation of these sharks remains a huge challenge. This study aimed at addressing these knowledge gaps using a multifaceted approach; 1) molecular markers to infer the genetic variation within and between species, and 2) a long-term tag-recapture dataset from 1984 to 2020, to investigate the habitat usage, movement patterns and site fidelity of both species. Successful cross-amplification of microsatellite markers from the small-spotted catshark Scyliorhinus canicula was done to complement the de novo-developed Poroderma microsatellite markers. Traditional population genetics analyses (F-statistics and AMOVA) of nuclear data did not reveal any significant intraspecific genetic differentiation (P > 0.05) while multivariate (Discriminant Analysis of Principal Components) and Bayesian clustering analyses also indicated a high level of gene flow between populations i.e. very weak intraspecific genetic differentiation for both species. Nonetheless, all analyses indicated clear genetic differentiation between species, with a slightly higher level of genetic diversity and clinal structure observed in Poroderma pantherinum. Each species was assigned to distinct genetic clusters based on the nuclear data and there were no mitochondrial (NADH-2) haplotypes shared between the species. The presence of only two mutation events between the major haplotypes for each species highlighted the low sequence divergence that has previously been noted for these catsharks. Regarding their movement patterns, a high overall recapture rate was observed (10.36%) for both species and high site fidelity was confirmed for this genus since 72.52% of all tag-recaptures occurred in the same location as the tagging. The tag- recapture data indicated that several individuals from both species appeared to travel further distances (>30 km) than other endemic catsharks, which may result in the notable lack of genetic differentiation and population structure. Investigation into the utility of seasonal or no-take marine protected areas for these catsharks could be worthwhile as it would reduce the vulnerability of these species to the fishing trade and allow their unique habitats to recover from previous fishing pressure. Despite the lack of genetic differentiation, the high site fidelity means that there are specific locations that play a crucial role in the ecology and reproduction of these catsharks. Accordingly, the protection and management of these critical habitats could help prevent these species from becoming even more threatened.

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