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Browsing Genetics by Author "Dalton, D. L."
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Item Open Access The application of new technologies in conservation genetics(University of the Free State, 2016-01) Labuschagne, Christiaan De Jager; Grobler, P.; Dalton, D. L.; Kotzé, A.English: Over the past decade, the development of high-throughput DNA techniques has expanded the scope of conservation genetics and molecular markers have become indispensable tools for the management of wildlife species and populations. There are several molecular markers available for biodiversity analysis, but their selection depends on the objective of the study, the molecular information sought (and reliability thereof) and the facilities and/or resources available. In order to develop and apply new genetic techniques I have decided on using one bird and one mammal species of interest in South Africa. The bird species chosen is the African Penguin (Spheniscus demersus) which has suffered serious population declines and is listed in the IUCN Red Data Book as an endangered species. Due to world-wide attention to rhinoceros conservation and population decline, the white rhinoceros (Ceratotherium simum) was selected as mammal species. Three different markers and their utility in aid of South African wildlife biodiversity conservation were investigated in these diverse species. The complete mitochondrial genome of the African Penguin was sequenced. The Spheniscus demersus mtDNA genome is very similar, both in composition and length, to both the Eudyptes chrysocome and E. minor genomes. This is the first report of the complete nucleotide sequence for the mitochondrial genome of the African Penguin. These results can be subsequently used to provide information for penguin phylogenetic studies and insights into the evolution of genomes. Furthermore, the study reported eight species specific microsatellite markers as well as 31 SNP markers as new molecular tools for the investigation, management and reintroduction of African penguin. Utilising these new tools, the study generated molecular genetic information to verify/complement studbook-based pedigree data from ex-situ populations of African Penguin. In addition, we compared the relative and combined utility of MS and SNP markers for parentage assignment. We found that a combined subset of these two types of markers attained a > 99% correct cumulative parentage assignment probability. This study further reported on 34 novel SNP markers for the white rhinoceros, identified through sequencing of CATS loci as well as SNP enriched libraries. The utility of 33 Single Nucleotide Polymorphisms and 10 microsatellites in isolation and in combination for assigning parentage in captive white rhinoceros were compared. It was found that a combined dataset of SNPs and microsatellites was most informative and showed the highest confidence level. This study thus provides a useful set of SNP and MS markers for parentage and relatedness testing in white rhinoceros. Furthermore, assessment of the utility of SNP and MS markers over multiple (> three) generations and the incorporation of a larger variety of relationships among individuals (e.g. half-siblings or cousins) is strongly recommended. Developed SNP markers could be used to define the genetic mating system of this species, for forensic applications and to determine population structure and variability when other markers prove problematic.Item Open Access 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.Item Open Access Linking geographic origin and mitogenomes of the ground pangolin in southern Africa(University of the Free State, 2021-11) Du Toit, Zelda; Grobler, J. P.; Kotze, A.; Dalton, D. L.; Jansen, R.The order Pholidota includes the most trafficked mammal species globally. The family Maninae with recognized species are categorized as Endangered and Critically Endangered on the IUCN Red List of Threatened Species. Due to the sharp decline in Asian pangolin numbers, the shift was towards the African pangolin species, to meet the demand in Asia and in Africa. Subsequently, the status of Smutsiinae has been revised by the IUCN and varies between Vulnerable and Endangered. This study contributes to clarify the genetic status of the four African pangolin species using molecular technologies. The first part of the study aimed to assemble mitogenomes of the four African pangolin species using NGS technologies. Whole mitogenomes were assembled and compared with published mitogenomes available in online databases. Mitogenomes were only available for six of the eight pangolin species. Our results showed different genome lengths for the four species: Temminck’s pangolin (16 558 bp), giant pangolin (16 540 bp), white-bellied pangolin (16 565 bp) and black-bellied pangolin (16 649 bp). Three distinctive clusters were observed supporting three genera. The first cluster consisted of the Asian pangolins (Manis), the second cluster was the African tree pangolins (Phataginus) and the third cluster represented the African ground pangolins (Smutsia). There were also two insertions found in the control region of the black-bellied pangolin which could indicate a recent mutation or selection event. The second part of the study focused on the development of species-specific STRs for Temminck’s pangolin and in combination with previous developed mtDNA markers the aim was to assess the population structure across the distribution range in southern Africa. Three mtDNA loci were analysed (Co1, Cytb, control region) with 30 STRs. A total of 62 individuals from six countries were included in the study. The results showed little to no geographical differentiation across the identified sampling locations. This indicates high levels of gene flow between the populations. Three distinctive clusters were observed within the different sampling areas. This could indicate inherited ancestral traits in juveniles with dispersal into new home ranges following a stepping stone model. Temminck’s pangolin has been reported to cover vast distances in search of new home ranges or territories. This study provides the first account of a combination of mtDNA and STR markers to investigate possible geo-referencing of Temminck’s pangolin in southern Africa. It is evident that the mtDNA and STRs markers used in this study, are not robust enough to discriminate between different sampling locations or populations. Further in-depth investigations, including whole genome analysis and SNPs are recommended. A more comprehensive sampling strategy is required to include all areas within the distribution range as well as more individuals for an in-depth genome analyses.Item Open Access Molecular age estimation based on promotor CpG methylation using Methylation Sensitive PCR(2018-06) Le Clercq, L.; Dalton, D. L.; Kotze, A.Chronological age is a key factor in animal ecology, as many biological traits appear and change over time. Such traits include development, age of reproductive maturity, reproductive success, future reproductive potential, and mortality. Several molecular methods have emerged as potential vehicle for biological age determination. The aim of this experiment is to ascertain if a Methylation Sensitive PCR (MSP) could be developed to screen for methylation at a previously identified site in the GRIA2 gene. Primers were designed by eye for both methylated and unmethylated target CpG’s in the GRIA2 promoter, and MSP conducted with the EpiScope® MSP kit. After optimization, the assay was able to successfully amplify the unmethylated control DNA with an efficiency of 97.6% and R²=99% across a range of 0.3–20 ng input DNA. Comparable results were, however, obtained with the methylated control DNA. Thus, the primers designed for the GRIA2 CpG was able to amplify the selected CpG with great efficiency making MSP a promising method of methylation screening, but primer design to assay a specific site faces many problems and selectivity for methylated vs. unmethylated may not be achievable.Item 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.