Browsing by Author "Sandenbergh, Lise"
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- ItemIdentification of SNPs associated with robustness and greater reproductive success in the South African merino sheep using SNP chip technology(Stellenbosch : Stellenbosch University, 2015-04) Sandenbergh, Lise; Cloete, Schalk W. P.; Roodt-Wilding, Rouvay; Van der Merwe, Aletta; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Reproduction and robustness traits are integral in ensuring sustainable, efficient and profitable sheep farming. Increases in genetic gain of reproduction and robustness traits are however, hampered by low heritability coupled with the difficulty in quantification of these traits for traditional selective breeding strategies. The aim of the current study was therefore to identify genomic regions underlying variation in reproduction traits and elucidate quantitative trait loci (QTL) and/or genes associated with reproductive traits. The Elsenburg Merino flock has been divergently selected for the ability to raise multiple offspring and has resulted in a High and a Low line that differ markedly with regard to reproductive output and other robustness traits. The flock thus served as an ideal platform to identify genomic regions subject to selection for reproductive traits. To pinpoint genomic regions subject to selection, a whole-genome genotyping platform, the OvineSNP50 chip, was selected to determine the genotype of more than 50 000 SNPs spread evenly across the ovine genome. The utility of the OvineSNP50 chip was determined for the Elsenburg Merino flock as well as additional South African Merino samples and three other important South African sheep breeds, the Blackheaded Dorper, South African Mutton Merino (SAMM) and the Namaqua Afrikaner. Although genotyping analysis of the Elsenburg Merino flock indicated some signs of poor genotype quality, the overall utility of the genotype data were successfully demonstrated for the South African Merino and the other two commercial breeds, the Dorper and SAMM. Genotyping results of the Namaqua Afrikaner and possibly other indigenous African breeds may be influenced by SNP ascertainment bias due to the limited number of indigenous African breeds used during SNP discovery. Analysis of pedigree, phenotypic records and SNP genotype data of the Elsenburg Merino cohort used in the current study, confirmed that the lines are phenotypically as well as genetically distinct. Numerous putative genomic regions subject to selection were identified by either an FST outlier approach or a genomic scan for regions of homozygosity (ROH) in the High and Low lines. Although annotated genes with putative roles in reproduction were identified, the exact mechanism of involvement with variation in reproduction traits could not be determined for all regions and genes. Putative ROH overlapped with QTL for several reproduction, milk, production and parasite resistance traits, and sheds some light on the possible function of these regions. The overlap between QTL for production and parasite resistance with putative ROH may indicate that several, seemingly unrelated traits add to the net-reproduction and may have been indirectly selected in the Elsenburg Merino flock. A SNP genotyping panel based solely on reproduction traits may therefore be ineffective to capture the variation in all traits influencing reproduction and robustness traits. A holistic selection strategy taking several important traits, such as robustness, reproduction and production into account may as such be a more effective strategy to breed animals with the ability to produce and reproduce more efficiently and thereby ensure profitable and sustainable sheep farming in South Africa.
- ItemOptimization of gene transfer in Haliotis midae by means of polyplex mediation(Stellenbosch : Stellenbosch University, 2010-12) Sandenbergh, Lise; Roodt-Wilding, R.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Haliotis midae is the most important aquaculture species in South Africa, with abalone farming contributing 80% of the Rand value of the aquaculture industry. Although genetic research has benefited the abalone industry, several issues still hinder increases in abalone production. Progress towards an increase in H. midae growth rate by utilizing conventional genetic studies and selective breeding has been relatively slow. Gene transfer has therefore become a plausible option to address this problem. Genes that code for certain desirable traits, such as increased growth rate, could be incorporated into the genome of commercial abalone. The current study undertook the optimization of a chemically-mediated gene transfer technique using Polyethylenimine (PEI) as transfection reagent and fluorescent proteins as reporter genes. Before gene transfer could be undertaken, several complementary studies also needed to be undertaken due to the novel nature of the study. The auto fluorescence of H. midae, the suitability of several H. midae tissues as targets for gene transfer and the cytotoxic effect of transfection reagents and selection antibiotics were assessed before gene transfer optimization could be attempted. Also, genes linked to an increase in growth rate were characterized for differential expression in different abalone age-groups to determine the suitability of these genes for incorporation into a homologous gene construct in future transfection studies. The auto fluorescence of ova, embryos and larvae were found to be comparable to that of the fluorescent reporter genes, EGFP and DsRed. A PCR-based transfection validation method was therefore employed to confirm the presence of internalized transgenes. It was established that sperm, ova, larvae and haemocyte cell culture were the most suitable target tissues for transfection. The transfection reagents, a 25kDa PEI and ExGen 500, were not cytotoxic to sperm, embryos and haemocyte cell cultures. The minimum lethal concentration of the selection antibiotics, neomycin and zeocin, was determined for larvae and haemocytes. After transfection treatment of sperm and fertilization of untreated ova, the presence of internalized transgenes could be verified for larvae. The presence of internalized transgenes could not be detected after transfection treatment of ova and larvae. Fluorescent flow cytometry and microscopy analysis of haemocytes could not detect the expression of the fluorescent reporter genes. Expression of two of the growth related genes was found to differ between age-groups. The perlustrin gene was upiv regulated in older animals, while the insulin related peptide receptor gene was down regulated in older animals. The third gene, a thrombospondin-1 precursor was stably expressed in all age-groups. This study represents the first report of transfection studies carried out on H. midae. Future studies will benefit from the groundwork established in H. midae transfection.