Thesis (MSc)--Stellenbosch University, 2012. / Triticale is an amphidiploid that consists of wheat (A and B) and rye (R) genomes. This cereal is fast becoming important on a commercial basis and warrants further assessment for the better management and breeding of the hybrid. The assessment of the genetic diversity among the wheat and rye genomes within triticale can be obtained by using molecular markers developed in both donor genomes. Simple sequence repeats markers (SSRs) and amplified fragment length markers (AFLPs) have been previously used to assess the genetic diversity among triticale lines. SSRs are highly polymorphic markers that are abundant and which have been shown to be highly transferable between species in previous studies while AFLP markers are known to generate plenty of data as they cover so many loci. Thus, the aim of this study was to develop a marker system suitable to assess the genetic diversity and relationships of advanced breeding material (and cultivars) of the Stellenbosch University’s Plant Breeding Laboratory (SU-PBL). Therefore, both AFLP and SSR markers were initially analysed using eight triticale cultivars (with known pedigrees) to facilitate cultivar identification. Fourty-two AFLP primer combinations and 86 SSR markers were used to assess the genetic diversity among the Elite triticale cultivars. The AFLP primer combinations generated under average polymorphism information content (PIC) values. Furthermore, these markers generated neighbour-joining (NJ) and unweighted pair group method with arithmetic average (UPGMA) dendograms that displayed relationships that did not correspond with the available pedigree information. Therefore, this marker system was found not to be suitable. A set of 86 SSRs previously identified in both wheat and rye, was used to test the genetic diversity among the eight cultivars. The markers developed in wheat achieved 84% transferability while those developed in rye achieved 79.3% transferability. A subset of SSR markers was able to distinguish the cultivars, and correctly identify them by generating NJ and UPGMA dendograms that exhibited relationships that corroborated the available pedigree data. This panel of markers was therefore chosen as the most suitable for the assessment of the advanced breeding material. The panel of seven SSR markers was optimised for semi-automated analysis and was used to screen and detect the genetic diversity among 306 triticale entries in the F6, Senior and Elite phases of the SU-PBL triticale breeding programme. An average PIC value of 0.65 was detected and moderate genetic variation was observed. NJ and UPGMA dendograms generated showed no clear groupings. However, the panel of markers managed to accurately identify all cultivars within the breeding program. The marker panel developed in this study is being used to routinely distinguish among the advanced breeding material within the SU-PBL triticale breeding programme and as a tool in molecular-assisted backcross.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Apoptosis represents a finely orchestrated and highly conserved natural form of cell death. It exhibits unique morphological and biochemical characteristics which culminate in the controlled dismantling of a cell from within followed by its discreet removal by phagocytic cells. Apoptosis is vital for the preservation of cell and tissue homeostasis but also performs several defensive and protective functions. Owing to its importance, apoptosis is highly regulated and a large number of proteins have been shown to mediate and safeguard the process. Furthermore, deregulated or altered levels of apoptosis can have severe pathological consequences; indeed, apoptosis has been shown to play a central role in several diseases, including neurological and autoimmune diseases as well as a variety of cancers. Consequently, the search for apoptotic-based therapies has received much attention and of vital importance to this quest is the characterisation of the specific mediators of apoptosis and their regulation as well as the identification of novel genes or proteins that can have a regulatory effect on apoptosis. It is thus the aim of this study to assist in this characterisation and also to identify novel candidate genes potentially involved in apoptosis. In a previously performed pilot study, three novel candidate genes potentially involved in apoptosis were identified by performing promoter-trap mutagenesis experiments. These genes were lipoic acid synthetase (LIAS), cyclophilin A (CYPA) and ribosomal protein L9 (RPL9). Since the methodology for this pilot study involved the use of functionally haploid cells, it was aimed in this study to verify these results in a diploid mouse cell line. Candidate gene knockdown was achieved by means of RNA interference and apoptosis assays were performed. A potential role for LIAS and CYPA in apoptosis was successfully verified in this study; however this could not be achieved for RPL9 and the gene was thus excluded from further study. In addition, nucleotide sequences isolated during the promoter-trap mutagenesis experiments in the pilot study were also investigated in order to identify additional novel candidate genes involved in apoptosis. By performing nucleotide BLAST searches, two potential candidate genes were identified, namely AHNAK nucleoprotein (AHNAK) and serum amyloid A-like 1 (SAAL1). Further bioinformatic analyses were performed with the four candidate genes in order to ascertain possible associations with apoptosis or cancer. Lastly, to further characterise the four candidate genes, the relative gene expression was investigated by means of quantitative PCR in two cancer and control cell lines. The results revealed significant differential expression for the majority of genes in the cancer cell lines when compared to the control cell lines. In conclusion, this study identified and characterised four novel genes potentially involved in apoptosis. Results obtained during this study can aid in the complete characterisation and functional annotation of these genes. Potential ties to apoptosis and associations with cancer are discussed for all four candidate genes and the possibilities of therapeutic strategies for anticancer treatments involving these candidate genes are noted. / AFRIKAANSE OPSOMMING: Apoptose verteenwoordig ‘n fyn georganiseerde en hoogs gekonserveerde natuurlike vorm van seldood. Dit vertoon unieke morfologiese and biochemiese eienskappe wat uitloop in die beheerde afbreek van ‘n sel vanuit die binnekant waarna dit onopsigtelik deur fagositiese selle verywder word. Apoptose is uiters belangrik vir die bewaring van sel en weefsel homeostase, maar dit vervul ook menigde afwerende and beskermde funksies. Vanweë sy noodsaaklikheid is apoptose hoogs gereguleer and ‘n groot aantal proteïene is al aangewys as bemiddelaars en beskermers van die proses. Verder, wangereguleerde en veranderde vlakke van apoptose kan ernstige patalogiese nagevolge hê; inderdaad, ‘n sentrale rol vir apoptose in verskeie siektes is al bevestig, insluitend neurologiese en outo-immuun siektes asook ‘n verskeidenheid van kankers. As gevolg hiervan ontvang die soektogte vir apoptose-gebaseerde terapieë vele aandag en uiters noodsaaklik vir hierdie soektogte is die karakterisering van die spesifieke bemiddelaars van apoptose en hul regulering asook die identifisering van nuwe gene of proteïene wat ‘n regulerende effek kan hê op apoptose. Dit is dus die doel van hierdie studie om by te dra tot hierdie karakterisering en ook om nuwe kandidaat gene wat moontlik betrokke kan wees in apoptose te identifiseer. In ‘n vorige loodsprojek is drie gene moontlik betrokke in apoptose geïdentifiseer deur middel van promoter-strik mutagenese eksperimente. Hierdie gene is lipoic acid synthetase (LIAS), cyclophilin A (CYPA) en ribosomal protein L9 (RPL9). Aangesien die metodiek in the loodsprojek gebruik gemaak het van funksionele haploïede selle, was dit die doel van hierdie studie om die resultate te bevestig in ‘n diploïede muis sellyn. Ribonukleïensuur (RNS) steuring is uitgevoer vir die uitklopping van die kandidaat gene en apoptose toetse is ook gedoen. Die bevestiging van ‘n moontlike rol vir LIAS en CYPA in apoptose was suksesvul in hierdie studie; alhoewel dit was nie bereikbaar vir RPL9 nie en hierdie geen is dus uitgesluit in verdere studies. Bykomend is nukleotied volgordes wat geïsoleer is tydens die promoter-strik mutagenese eksperimente in die loodsprojek ook nagesien om moontlike addisionele nuwe kandidaat gene te identifiseer wat moontlik betrokke kan wees by apoptose. Twee potensiële kandidaat gene, naamlik AHNAK nucleoprotein (AHNAK) en serum amyloid A-like 1 (SAAL1), was geïdentifiseer deur middel van nukleotied BLAST soektogte. Addisionele bioinformatiese analises is uitgevoer op die vier kandidaat gene om moontlike redes vir ‘n assosiasie met apoptose of kanker vas te stel. Laastens, om die kandidaat gene verder te karakteriseer, is daar ondersoek ingestel op die relatiewe geen uitdrukking van die kandidaat gene in twee kanker en twee normale sellyne. Die resultate het betekenisvolle differensiële regulering getoon vir meeste van die gene in die kanker sellyne in vergelyking met die normale sellyne. Ten slotte, vier kandidaat gene moontlik betrokke in apoptose is in die huidige studie geïdentifiseer en gekarakteriseer. Die resultate verwerf in hierdie studie kan moontlik bydra tot die volkome karakterisering en funksionele annotering van die kandidaat gene. Moontlike skakels met apoptose en assosiasies met kanker is bepreek vir die vier kandidaat gene en die moontlikheid van terapeutiese strategieë gebaseer rondom die kandidaat gene word ook genoem. / National Research Foundation (NRF)

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also known as Lessertia frutescens, is a leguminous shrub indigenous to southern Africa. Traditionally this plant has been used for the treatment of various ailments; current interest in this plant has escalated after it was announced that extracts could aid in the relief and treatment of HIV/AIDS. These extracts contain an array of metabolites, including sutherlandins, sutherlandiosides L-arginine, L-canavanine, asparagine, gamma-aminobutyric acid (GABA), and various other amino acids, which have been linked to medicinal uses. This study focused on the use of hormones to promote the growth and metabolite production of S. frutescens in vitro cultures. The growth promoting substances used in this study were synthetic analogues of strigolactones, GR24 and Nijmegen-1, and auxins, indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA). The first part of this study focused on the effects strigolactones and auxins, alone and combined, had on the growth of S. frutescens in vitro nodal explants. The S. frutescens nodal explants had the most significant improvement in growth with treatments that contained 1 mg/L NAA. These treatments increased growth via fresh and dry mass and plant length. The metabolite content of these nodal explant cultures was evaluated using liquid chromatography/mass spectrometry (LC/MS) metabolite analysis. The treatments that contained 1 mg/L NAA differed in metabolite composition and showed an increase in metabolite quantity. The SU1 content of the treated plants was also quantified using LC/MS techniques and a combination of 1 mg/L NAA and Nijmegen-1 doubled the amount of SU1. The effect of strigolactones was also studied using hairy root cultures of S. frutescens. Strigolactones alone slightly inhibited the formation of lateral transgenic roots, but when these chemicals were used in combination with auxins, significant reduction in dry mass and lateral root outgrowth resulted. Of the treatments tested in this study, 0.1 mg/L IBA caused noticeable alterations to the metabolite pool, with amino acids such as GABA and arginine accumulating at higher levels than the control explants. The exploitation of hormones to up-regulate the growth and metabolism of the medicinally important plant, Sutherlandia frutescens, proved successful in this study. The use of in vitro nodal explants along with hairy root cultures has assisted in the establishment of a stable system for the up-regulation of metabolites. / AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens, is 'n peulagtige struik inheems tot suider Afrika. Tradisioneel is die plant vir 'n groot verskeidenheid van kwale gebruik; huidige belangstelling in die plant het toegeneem nadat dit bekend gemaak was dat ekstraksies vanaf hierdie plant verligting kan bied vir MIV/VIGS. Hierdie ekstrakte bevat 'n verskeidenheid van metaboliete, insluitend sutherlandins, sutherlandiosiede L-arginien, L-kanavanien, asparagien, gamma-aminobottersuur (GABS), asook verskeie ander aminosure wat medisinale gebruike het. Die studie het gefokus op die gebruik van hormone om die groei en metaboliete van S. frutescens in vitro kulture te vermeerder. Die groei reguleerders wat in hierdie studie gebruik was, was die sintetiese analoë van strigolaktoon, GR24 en Nijmegen-1, asook die ouksiene, indool-3-bottersuur (IBS) en naftaleen asynsuur (NAS). Die eerste deel van die studie het gefokus op die effek van strigolaktoon en ouksien, alleen en in kombinasie, op die groei van S. frutescens in vitro nodale mikrostingels. Die S. frutescens nodale mikrostingels wat behandel was met 1 mg/L NAS het die aansienlikste toename in groei getoon. Hierdie behandeling het groei bevorder deur middel van vars en droë massa en plant lengte. Die metaboliet inhoud van die behandelde mikrostingels was met behulp van vloeistofchromatografie/massa spektrometrie (VC/MS) ondersoek. Al die behandelinge wat 1 mg/L NAS bevat het, het in metaboliet samestelling verskil en het ook 'n toename in metaboliet hoeveelheid getoon. Die SU1 inhoud van die behandelde plante was ook met behulp van VC/MS tegnieke gekwantifiseer en dit was gevind dat 'n kombinasie van 1 mg/L NAS en Nijmegen-1 die hoeveelheid SU1 verdubbel het. Die effek van strigolaktoon op harige wortel kulture van S. frutescens was ook ondersoek. Strigolaktoon alleen het die formasie van laterale transgeniese wortels effens inhibeer, maar wanneer hierdie chemikalieë saam met ouksiene gebruik was, was die aansienlike afname van die massa en inhibisie van die laterale wortel uitgroeisels meer prominent. Van al die behandelinge wat in hierdie studie getoets is, het 0.1 mg/L IBS die mees merkbare veranderinge in metaboliete meegebring en aminosure soos GABS en arginien het teen hoër vlakke versamel. Die uitbuiting van hormone om groei en metaboliet produksie te bevorder in die belangrike medisinale plant, Sutherlandia frutescens, was suksesvol in hierdie studie. Die gebruik van nodale mikrostingels asook harige wortel kulture het bygedra om 'n stabiele sisteem te vestig vir die vermeerdering van metaboliete.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Starch is widely used in industries around the word, some of these are food, oil drilling, paper milling and cosmetics. It is a polymer which has two components, amylose and amylopectin. The production and degradation if starch in plants is fairly well studied and a sizable number of enzymes have been identified which play critical roles in its metabolism. There are still remaining questions, namely if there are more unidentified enzymes that play roles and how the enzymes interact with each another. To study the effect on starch metabolism possible novel starch metabolic genes were studied by analysing Arabidopsis T-DNA insertion mutants for two genes, designated SP1 (At5g39790) and CBD1 (At5g01260). cDNAs for these two was used to produce recombinant protein and investigated potential activities. The cbd1 mutant plants had a starch excess phenotype with iodine staining but this could not be confirmed with quantitative starch measurements. The sp1 mutants did not have a significant difference in all the lines and time points when compared to the Wt plants. No link could be established between the SP1 kinase domain and glucan phosphorylation. From my data a clear involvement of these two genes could not yet be elucidated. To study the interactions of starch metabolic proteins (BEI, BEII, GWD and ISA2) chimeric RNAi constructs was built and transformed into potato. Only StBEI and StBEII lines could be analysed and it was found that the G6P content was increased in both StBEI and StBEII. The BEII leaves and tubers had increased amylose contents. Intriguingly it would appear that starch isolated from both the tubers and leaves of StBEI lines demonstrated a reduction in amylose, with the leaves showing a much bigger decease than the tubers. This needs to be confirmed and the remaining lines need to be analysed. Gaining knowledge about starch metabolism is critical in producing engineered crops that can produce more starch in a smaller agricultural area. With the population growing beyond 8 billion individuals it will be one of the best routes to enhance cop yields through biotechnology. / AFRIKAANSE OPSOMMING: Stysel word reg oor die wereld benut in ‘n verskyndenheid van industiee. Dit is divers en sluit die voedsel, oliebooring, papiermeule en die kosmetiese bedryf in. Dit is ‘n polimeer wat uit twee komponete: amylose en amylopektien bestaan. Stysel metabolisme, wat die vervaardiging en afbreek van dit insluit, is al baie goed bestudeer. Die ensieme wat ‘n kritiese rol speel is al gevind, maar daar bly nogsteeds ‘n paar vrae wat moet beantwoord word. Is daar nog ensieme wat ‘n rol speel wat nog nie geidentifiseer is nie? Wat is die manier hoe die bekende ensieme met mekaar ‘n interaksie het? Om die invloed van twee moonlike nuwe stysel metabolisme gene te bestudeer, is T-DNA insersie mutante ondersoek. Hulle word na verwys in die studie as SP1 (At5g39790) en CBD1 (At5g01260). cDNAs vir hierdie twee was gemaak vir die vervaardeging van rekombonante proteine. Hierdie rekombinante proteine was dan ondersoek vir moonlike aktiwiteite. ‘n Oormaat stysel was wel gevind in die cbd1 mutant plante wanner n jodium vlek tegniek gebruik was. Ongelukkig kon hierdie oormaat die bevestig word wanner n kwantitatiewe metode gebruik was nie. Daar was nie ‘n beduidende verskil in stysel wanner die sp1 mutante plante vergelyk was met die wilde tiepe nie. Daar kon ook geen verbintenis gevind word tussen die kinase area en die fosforilasie van stysel nie. Volgens hierdie data kon daar die n duidelike verbintenis gevind word tussen die twee gene en stysel metabolisme nie. Om die interaksies tussen bekende stysel metabolisme proteine (BEI, BEII, GWD en ISA2) te bestudeer was chimeriese RNAi konstrukte gebou en toe in aartappels in getransformeer. Slegs die StBEI and StBEII kon geanalisser word en daar was bevind dat die G6P hoeveelheid in beide hoër was. Amilose was in groter hoeveelheide teenwoordig in beide BEII blare en knolle. ‘n Onverwagse obserwasie was gemaak toe die BEI lyne ondersoek was. Daar was gevind dat in die blare en knolle daar ‘n laer hoeveelheid amilose was. Die blare het wel baie laer amilose gehad as die knolle. Die obserwasie moet bevestig word met n ander tegniek en die orige RNAi lyne moet nog bestudeer word. Om al die fasette van stysel metabolisme te ken is uiters belangrik vir die vervaardiging van gewasse wat groter opbrengste lewer in n kleiner area. Met die wereld bevolking wat al verby 8 biljoen individue gestyg het is dit moontlik al hoe almal voor gesorg kan word in terme van voeding.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Parkinson's disease (PD) is a severely debilitating neurodegenerative disorder that results in motor circuit dysregulation and ultimately, causes impairment of movement. This condition is due to the selective degradation of the dopaminergic neurons in the substantia nigra pars compacta in the midbrain, which subsequently results in the pathological symptoms namely bradykinesia, resting tremor, postural instability and rigidity. It was initially hypothesized that individuals who develop PD were exposed to an environmental trigger(s) that caused the onset of the disease, but more recently, a significant genetic component, coupled to environmental factors have been implicated in disease pathogenesis. Currently, there are eight genes (Parkin, PINK1, LRRK2, SNCA, DJ-1, ATP13A2, EIF4G1 and VPS35) that have been directly implicated in PD. Worldwide, the prevalence of neurodegenerative disorders is increasing as populations are living longer. In Europe, Canada and USA, it has been projected that the prevalence of PD may increase by a factor of two between 2010 and 2050; approximately a 92% increase. In Tanzania (the only study done in sub-Saharan Africa) an even larger increase of 184% between 2005 and 2025 is predicted, due to the fact that the speed of populations ageing in developing countries, will exceed that of developed countries. Research into the causes and risk factors underlying neurodegenerative disorders such as PD is therefore urgently needed for policy makers and governments in developing nations to take appropriate action to deal with this impending health care problem. The aim of the present study was to investigate the molecular aetiology of a group of South African PD patients. A total of 262 patients from various ethnic backgrounds were recruited for the study, and 35% had a positive family history of PD with the average age at onset (AAO) being 54.3 years of age (SD = 12.5 years). Mutation screening of the known PD genes (Parkin, PINK1, LRRK2, SNCA and DJ-1) was performed using high resolution melt and Sanger sequencing. Genotyping was done using fluorescently-labelled PCR primers followed by electrophoresis on an ABI 3130xl genetic analyser (for CTG repeats in JPH3) and with a KASP™ Genotyping Assay (for a 16bp indel in DJ-1). In order to identify a novel PD-causing gene, whole exome sequencing (WES) was conducted on three Afrikaner probands with an Illumina Genome Hiseq 2000TM and the sequences were aligned using the NCBI Human Reference Genome 37.2. The BORG (Bio-Ontological Relationship Graph) semantic database, which models the relationship of human and model organism genes to functions, pathways and phenotypes, was used to filter and prioritise genetic variants shared between the three PD exomes. It was determined that the known PD genes do not play a significant role in disease pathogenesis in the South African patients as only 15/262 (5.7%) of the patients harboured mutations: seven in Parkin, one in PINK1, six in LRRK2 and one in SNCA. Only one of the patients harboured a 16bp indel variant at the transcription start site of DJ-1. None of the Black PD patients had pathogenic repeat expansions in JPH3 thereby excluding Huntington disease-like 2 as a cause of the disease phenotype. Genealogical analysis revealed that six of the apparently unrelated Afrikaner PD probands were related to a founder couple that immigrated to South Africa in the 1600s which suggests that there is a possible founder effect for the disease. Bioinformatics analysis of WES data on three of the probands identified 21 variants in 12 genes that were present in all three PD exomes and fulfilled various criteria. Sanger sequencing was used for verification of five variants and of these, two (in CDC27 and NEDD4) were found to be artefacts. The remaining three (in HECDT1, TBCC and RNF40) were excluded based on the lack of cosegregation with disease and the high frequency of the allele in controls. Further work is necessary to verify the presence of the remaining sixteen variants and to characterise each of them for their possible pathogenicity. The discovery of novel PD-causing genes is important as this may shed light on the pathways or processes that are involved. A current hypothesis implicates the lysosome-dependent pathway as a unifying biochemical pathway that can account for the phenotypic spectrum within PD. Notably, although Mendelian forms are thought to account for only about 10- 15% of cases, the study of Mendelian inherited variants is likely to provide insight into the pathophysiology of the more common sporadic form of this condition. Dissecting the key molecular mechanisms underlying PD will provide critical information for improved treatment strategies and drug interventions that will ultimately prevent or halt neuronal cell loss in susceptible individuals. / AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is 'n erge neurodegeneratiewe bewegings-siekte, wat motorstroombaan disregulasie veroorsaak. Dit lei uiteindelik tot beperkte bewegings vermoëns. Hierdie toestand word veroorsaak weens die selektiewe agteruitgang van die dopaminergeniese neurone in die substantia nigra pars compacta in die midbrein, wat later lei tot die patologiese simptome naamlik: bradykinesia, rustende spiersametrekkings, posturale onstabiliteit en rigiditeit. Daar is aanvanklik vermoed dat individue wat PS ontwikkel, aan 'n omgewingsfaktor(e) blootgestel is wat die aanvang van die siekte veroorsaak het, terwyl meer onlangs is daar 'n aansienlike genetiese komponent tesame met omgewingsfaktore geïdentifiseer, wat betrokke is by die patogenese van die siekte. Tans is daar agt gene (Parkin, PINK1, LRRK2, SNCA, DJ-1, ATP13A2, EIF4G1 en VPS35) wat direk by PS geïmpliseer is. Wêreldwyd is daar ‗n toenemende voorkoms van neurodegeneratiewe siektes aangesien bevolkings langer leef. In Europa, Kanada en die VSA, is daar geprojekteer dat die voorkoms van PS tussen 2010 en 2050 met 'n faktor van twee verhoog kan word. Dit is ongeveer 'n 92%- verhoging. In Tanzanië (die enigste studie wat tot dusver in sub-Sahara Afrika gedoen is) word daar selfs ‗n groter toename, van 184% tussen 2005 en 2025 voorspel. Dit is te danke aan die feit dat die bevolkings- veroudering in ontwikkelende lande die van ontwikkelde lande sal oorskry. Ondersoeke na die oorsake en risiko-faktore onderliggend aan neurodegeneratiewe siektes, byvoorbeeld PS, word dus dringend benodig deur beleidmakers en regerings in ontwikkelende lande, sodat hulle die nodige stappe kan neem om hierdie dreigende gesondheidsorg-probleem op te los. Die doel van die huidige studie was om ondersoek in te stel na die molekulêre etiologie van 'n groep Suid-Afrikaanse PS pasiënte. 'n Totaal van 262 pasiënte van verskillende etniese agtergronde, is gewerf vir die studie. Hiervan het 35% 'n positiewe familiegeskiedenis van PS en die gemiddelde aanvangs ouderdom (AAO) was 54,3 jaar (SD = 12,5 jaar). Mutasie-analise van die bekende PS gene is uitgevoer met behulp van hoë resolusie smelt en Sanger volgordebepaling. Genotipering is gedoen met behulp van fluoresserend geëtiketteerde PKR inleiers met elektroforese, op 'n ABI 3130xl genetiese analiseerder (CTG herhalings in JPH3), en met 'n KASP ™ Genotipering toets (vir 'n 16bp indel in DJ-1). Ten einde, om 'n nuwe PSveroorsakende geen te identifiseer was heel eksoom volgordebepaling (WES) uitgevoer op drie Afrikaner PS positiewe pasiënte met 'n Illumina Genome Hiseq 2000™ en die volgorders is gerangskik met behulp van die NCBI Menslike Verwysings Genoom 37.2. Die BORG (Bio- Ontologiese Verhoudings Grafiek) semantiese databasis, wat gebaseer is op die verhouding van die mens en model organisme gene funksies, paaie en fenotipes, en is gebruik om genetiese variante, wat gedeel word tussen die drie PS exome te filtreer en te prioritiseer. Daar is vasgestel dat die bekende PS gene nie 'n belangrike rol in die patogenese van die siekte in die Suid-Afrikaanse pasiënte speel nie. Dit is aangesien slegs 15/262 (5.7%) van die pasiënte bekende mutasies dra: sewe in Parkin, een in PINK1, ses in LRRK2 en een in SNCA. Slegs een van die pasiënte het 'n 16bp delesie variant in die transkripsie promotor area van DJ-1 gedra. Geen van die Swart PS pasiënte het patogeniese herhalings in JPH3 vertoon nie. Gevolglik is Huntington siekte-agtige 2 uitgesluit as 'n oorsaak van die siekte fenotipe. Genealogiese analise het getoon dat ses van die skynbaar onverwante Afrikaner PS pasiënte verwant is aan 'n stigter paartjie wat in die 1600's na Suid-Afrika geïmigreer het, wat daarop dui dat daar 'n moontlike stigter effek vir die siekte is. Bioinformatiese analise van WES data vir drie van die pasiënte, het 21 variante in 12 gene geïdentifiseer, wat in al drie PS exome teenwoordig was en verskeie kriteria vervul het. Sanger volgordebepaling is gebruik vir die bevestiging van vyf variante en van hierdie, is twee (in CDC27 en NEDD4) bevind om artefakte te wees. Die oorblywende drie (in HECDT1, TBCC en RNF40) is uitgesluit gebaseer op die gebrek aan gesamentlike-segregasie met die siekte en die hoë frekwensie van die allele in die kontrole groep. Verdere werk is nodig om die teenwoordigheid van die oorblywende variante te verifieer en om elkeen van hulle te karakteriseer vir hulle moontlike patogenisiteit. Die ontdekking van die nuwe PS-veroorsakende gene is belangrik aangesien dit lig kan werp op die stelsels of prosesse wat betrokke is. 'n Huidige hipotese impliseer die lisosoom-afhanklike pad as 'n verenigende biochemiese padweg, wat verantwoordelik is vir die fenotipiese spektrum binne PS. Alhoewel Mendeliese vorms vermoedelik verantwoordelik is vir slegs omgeveer 10-15% van die gevalle, is die studie van Mendelse gene geneig om insig te verkry in die patofisiologie van die meer algemene sporadiese vorm van hierdie toestand. Ontleding van die kern molekulêre meganismes onderliggend aan PS sal kritiese inligting vir beter strategieë vir behandeling en geneesmiddel-intervensies voorsien, wat gevolglik neuronale sel verlies in vatbare individue sal voorkom of beëindig. / Medical Research Council / National Research Foundation / Harry Crossley Foundation

Thesis (PhD (Genetics))—University of Stellenbosch, 2007. / Association analysis was used to improve the efficiency of breeding sugarcane varieties for the negatively correlated traits of resistance to sugarcane smut and the eldana stalk borer. 275 RFLP and 1056 AFLP markers were scored across a population of 77 genotypes representing the genetic variation present within the SASRI breeding programme. Genetic diversity analysis did not detect significant structure within the population. Regression analysis identified 64 markers significantly associated with smut rating and 115 markers associated with eldana rating at r2 > 6.25%. Individual markers with the largest effects explained 15.9% of the phenotypic variation in smut rating and 20.2% of the variation in eldana. Five markers were significantly associated with both smut and eldana. In each case the marker effect was negatively correlated between the two traits, suggesting that they are genetically as well as phenotypically negatively correlated.

Thesis (PhD (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South African abalone, Haliotis midae, has been the subject of major concern regarding its survival and conservation over the last decade or more. Being the only one of five endemic species with commercial value, there is considerable interest and urgency in genetic management and improvement of this species. Limited genetic information and the increasing conservation concern of this species are considered the key motivations for generating information on the micro- and macro-evolutionary processes of H. midae, the overall objective of this study. This study reported the first microsatellite and Single Nucleotide Polymorphism (SNP) markers developed specifically for Haliotis midae. Both these marker types were applied to elucidate the degree of gene flow in nine natural abalone populations whilst testing for two contrasting hypotheses; panmixia versus restricted gene flow. Data was analysed using a series of methodological approaches ranging from traditional summary statistics to more advanced MCMC based Bayesian clustering methods with and without including spatial information. Using only microsatellite data, the historical demography of the species was also examined in terms of effective population size and population size fluctuations. Finally, the evolutionary positioning and origin of Haliotis midae with regards to other Haliotis species was investigated based on mitochondrial and nuclear sequence data. Both microsatellite and SNP data gave evidence for subtle differentiation between West and East coast populations that correlates with a hydrogeographic barrier in the vicinity of Cape Agulhas. Population substructure was supported by AMOVA, FCA and Bayesian clustering analysis. Clustering utilizing spatial information further indicated clinal variation on both sides of the proposed barrier with a region in the middle coinciding with a secondary contact zone, indicating possible historical isolation during glacial periods. Overall, the similar degree of substructure observed with both microsatellites and SNPs supported the existence of contemporary and/or historical factors with genome-wide effect on gene flow. The population expansion measured with the microsatellites was inconsistent with the known recent decline but taking the species’ life cycle and large effective population size into account, a shrinkage in population size will probably only be apparent in a few generations time. On a macro-evolutionary scale, this study presents the first classification of South African abalone as a monophyletic group within the Haliotidae family. The topology based on the combined mitochondrial and nuclear dataset is highly suggestive of a relatively recent radiation of the SA species from the Indo-Pacific basin. The study concludes by describing the most likely factors that could have affected overall population structure and makes suggestions on how the given genetic information should be incorporated into strategies aimed towards the effective management and conservation of Haliotis midae. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is oor die laaste dekade of meer die onderwerp van groot bekommernis betreffende die spesie se oorlewing en bewaring. Aangesien dit die enigste van vyf endemiese SA spesies is met kommersiёle waarde, is daar besonderse belang en erns in die genetiese beheer en verbetering van die spesie. Beperkte genetiese inligting en ‘n toenemende behoefte om die spesie te bewaar is die hoof motivering agter die generering van informasie rakende mikro- en makro-evolusionêre prosesse in Haliotis midae en is die oorhoofse doel van hierdie studie. Hierdie studie beskryf die eerste mikrosatelliete en enkel basispaar polimorfismes wat ontwikkel is spesifiek vir Haliotis midae. Beide tipe merkers is aangewend om die mate van gene vloei in nege wilde perlemoen populasies te ondersoek terwyl twee hipoteses ondersoek is; panmiksie versus beperkte gene vloei. Data is geanaliseer deur gebruik te maak van ‘n reeks metodieke benaderings wat wissel van tradisionele opsommings statistieke tot meer gevorderde MCMC gebasseerde groeperings metodes met of sonder die gebruik van geografiese data. Mikrosatelliet data is ook aangewend om die historiese demografie van die spesie te bepaal in terme van effektiewe populasie grootte asook veranderinge in populasie groottes. Laastens is die evolusionêre posisionering en oorsprong van Haliotis midae teenoor ander Haliotis spesies ondersoek deur gebruik te maak van mitokondriale en nukleêre DNA volgorde data. Beide mikrosatelliet en enkel basispaar polimorfisme data lewer bewys van ‘n subtiele genetiese verskil tussen wes en ooskus populasies wat verband hou met ‘n hidrografiese skeiding in die omgewing van Kaap Agulhas. Populasie struktuur is ondersteun deur die analise van molekulêre variansie (AMOVA), faktoriale komponente analise asook Bayesiese groeperings analise. Groeperings analise wat geografiese informasie insluit dui klinale genetiese variasie aan beide kante van die skeiding aan met ‘n area in die middel wat ooreenstem met ‘n sekondêre kontak gebied. In totaal, ondersteun die soortgelyke mate van struktuur verkry met beide die mikrosatelliete en enkel basispaar polimorfismes die bestaan van hedendaagse en/of historiese faktore met genoom wye invloed op gene vloei. Die toename in populasie grootte vasgestel deur die mikrosatelliet data stem nie ooreen met die onlangse afname waargeneem in die spesie nie, maar met inagneming van Haliotis midae se lewenssiklus en groot effektiewe populasie grootte, sal die afname in populasie grootte moontlik eers oor ‘n paar generasies na vore kom. Op ‘n makro-evolusionêre skaal lewer hierdie studie die eerste klassifikasie van Suid-Afrikaanse perlemoen as ‘n monofiletiese groep binne die Haliotidae familie. Die topologie gebaseer op ‘n gesamentlike mitkondriale en nukleêre datastel is hoogs aanduidend van ‘n relatiewe onlangse verspreiding van die Suid-Afrikaanse spesies uit die Stille-Indiese Oseaan. Die studie sluit af deur die mees algemene faktore te bespreek wat populasie struktuur kon beïnvloed het en maak voorstelle op watter wyse hierdie genetiese inligting aangewend kan word vir die effekiewe beheer en bewaring van Haliotis midae.

Thesis (PhD (Agric)) -- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Chromosome arm 7DL of common wheat carries genes for agronomically important traits such as leaf rust, stem rust, Russian wheat aphid and eye spot resistance. Some of these genes occur on introgressed foreign chromatin, which restricts their utility in breeding. The 7DL genetic maps are poorly resolved, which seriously hampers attempts to manipulate the genes and introgressed regions in breeding. This dissertation represents an attempt to improve our knowledge of the relative map positions of three resistance genes that have significant potential for use in local breeding programmes. The leaf rust resistance gene, Lr19, is located on a Thinopyrum ponticum-derived translocation which occupies a large part of the terminal end of 7DL. The translocation also carries genes for less favourable traits such as yellow flour colour. Attempts have been made to reduce the size of the translocation through allosyndetic pairing induction; the primary aims being to remove deleterious genes and to minimise the amount of foreign chromatin associated with Lr19 so it can be recombined with other useful 7DL genes. Twenty-nine 'Indis'-derived Lr 19 deletion mutants were previously produced by gamma irradiation and a physical map was constructed. In this study, the set of mutant lines were further analysed using 144 Sse8387I/Msei and 32 EcoRI/Msel amplified fragment length polymorphism (AFLP) primer combinations. The previous physical map, which was based on five restriction fragment length polymorphism (RFLP) markers and five structural gene loci, was extended and now includes 95 novel AFLP markers (86 Sse8387I/Msei and 9EcoRI!Msel markers), of which seven map close to Lr 19. Most of the deletions could be ordered according to size and the improved map has already been used to characterise shortened recombinant forms of the Lr 19 translocation. An unsuccessful attempt was made to convert one of the seven markers closest to Lr 19 into a sequence-specific marker. However, an AFLP marker located distally from Lr 19 was successfully converted into a sequence-specific marker in collaboration with other researchers. An attempt was also made to map and tag the Russian wheat aphid (RWA) resistance gene, Dn5. A doubled haploid mapping population consisting of 94 lines was created and typed for Dn5, four microsatellite loci and the endopeptidase locus, Ep-Dl. The Dn5 locus mapped 25.4 cM and 28.6 cM distally from Xg.vm111 and Xg.vm437, respectively, but was not linked to Xgwm428, Xgwm3 7 or Ep-Dl. Tagging of Dn5 was attempted by screening twelve homozygous resistant and seven homozygous susceptible F2 lines from a cross between 'Chinese Spring' and 'PI 294994' with 70 Sse8387IIi\1sei AFLP primer combinations. Only two potentially useful polymorphisms (one in coupling and one in repulsion phase) were identified. Conversion of the coupling phase marker to a sequence-specific marker was not successful. The eyespot resistance gene, Pchl , was derived from Triticum ventricosum and is present in the wheat VPM-1. Close association between Pchl and the endopeptidase Ep-Dlb allele has been reported previously. Pchl/Ep-Dl was tagged by screening ten wheat genotypes (each homozygous for the confirmed presence or absence of Pchl and/or Ep-Dl b) with 36 Sse83 87I/ Msei AFLP primer combinations. Three AFLP markers were closely associated with Pchl I Ep-D 1, one of which was targeted for conversion into a sequence-specific marker. The sequence-specific marker contained a microsatellite core motif and was found to be useful for tagging Pchl!Ep-Dl. A genetic distance of 2 cM was calculated between the novel microsatellite marker and Ep-Dl. The microsatellite marker was also polymorphic for the Lr 19 translocation and it was possible to map it between the Wsp-Dl and Sr25 loci. In this dissertation, mapping and/or tagging of three important resistance genes were achieved. Due to the fact that all markers used in these studies were not polymorphic between all of the targeted regions, it was not possible to fully integrate the data obtained for the three regions. / AFRIKAANSE OPSOMMING: Chromosoom arm 7DL van broodkoring dra gene vir agronomies-belangrike kenrnerke soos blaarroes, stamroes, Russiese koringluis en oogvlek weerstand. Sommige van hierdie gene kom voor in blokke spesie-verhaalde chromatien wat hul bruikbaarheid in teling beperk. Die genetiese kaarte van 7DL is swak ontwikkel en dit maak dit baie moeilik om hierdie gene en spesie-verhaalde streke tydens teling te manipuleer. Hierdie proefskrif verteenwoordig 'n paging om kennis van die relatiewe kaart liggings van drie weerstandsgene, met betekenisvolle potensiaal in plaaslike tee! programme, te verbreed. Die blaarroes weerstandsgeen, Lr 19, kom voor op 'n Thinopyrum ponticum-verhaalde translokasie wat 'n groot terminale gedeelte van 7DL beslaan. Die translokasie dra ook gene vir minder gewensde kenrnerke soos gee! meelkleur. Pogings is aangewend om die translokasie deur homoeoloe parings-induksie te verkort. Die doe! was om nadelige gene te verplaas en die hoeveelheid vreemde chromatien geassosieer met Lr 19 te minimiseer sodat dit met ander nuttige gene op 7DL gerekombineer kan word. Nege-en-twintig 'Indis'-verhaalde Lr 19 delesie mutante is vroeer met gamma bestraling geproduseer en gebruik om 'n fisiese kaart op te stel. Teenswoordig is die stel mutante verder ontleed met behulp van 144 Sse8387I!Msei en 32 EcoRII Msel amplifikasie-fragment-lengte-polimorfisme (AFLP) inleier kombinasies. Die bestaande fisiese kaart, wat gebaseer was op vyf restriksie-fragment-lengte-polimorfisme (RFLP) merkers en vyf strukturele geen loki, is uitgebrei en sluit nou 95 unieke AFLP merkers (86 Sse8387I/Msel en 9EcoRI/Msel merkers) in, waarvan sewe naby aan Lr19 karteer. Die meeste van die delesies kon op grond van hulle grootte gegroepeer word en die verbeterde fisiese kaart is alreeds gebruik om verkorte rekombinante vorms van die Lr 19 translokasie te karakteriseer. 'n Onsuksesvolle paging is aangewend om een van die sewe merkers naaste aan Lr 19 om te skakel na 'n volgorde-spesifieke merker. 'n AFLP merker wat distaal van Lr 19 karteer is egter wel suksesvol in samewerking met ander navorsers omgeskakel na 'n volgordespesifieke merker. 'n Paging is ook aangewend om die Russiese koringluis (RKL) weerstandsgeen, Dn5, te karteer en merkers gekoppel aan die geen te identifiseer. 'n Verdubbelde-haplo!ede karteringspopulasie van 94 lyne is geskep en getipeer vir Dn5, vier mikrosatelliet loki en die endopeptidase lokus, Ep-D1. Die Dn5 lokus karteer 25.4 cM en 28.6 cM distaal van Xgwml11 en Xgwm437, respektiewelik, maar was me gekoppel met Xgwm428, Xgwm37 of Ep-D1 me. Twaalf homosigoties weerstandbiedende en sewe homosigoties vatbare F2 lyne uit die kruising: 'Chinese Spring' I 'PI 294994' is met 70 Sse8387VMsel AFLP inleier kombinasies getoets in 'n poging om merkers vir Dn5 te identifiseer. Slegs twee moontlik bruikbare polimorfismes (een in koppelings- en een in repulsie fase ), is ge'identifiseer. Omskakeling van die koppelingsfase merker na 'n volgorde-spesifieke merker was onsuksesvol. Die oogvlek weerstandsgeen, Pch1, is uit Triticum ventricosum oorgedra en kom voor in die koringlyn, VPM-1. Noue koppeling van Pch1 en die endopeptidase alleel, Ep-D1 b, is vantevore gerapporteer. Merkers is vir P chl I Ep-D 1 gevind deur tien koring genoti pes ( elkeen homosigoties vir die bevestigde teenwoordigheid of afwesigheid van Pch1 en/of Ep-D1 b) te toets met 36 Sse83871/kfsel AFLP inleier kombinasies. Drie AFLP merkers is gevind wat nou koppel met Pchl!Ep-D1 , waarvan een gekies is vir omskakeling na 'n volgorde-spesifieke merker. Die volgorde-spesifieke merker het 'n mikrosatelliet kernmotief bevat en was nuttig as merker vir Pch1/Ep-D1. 'n Genetiese afstand van 2 cM is tussen die unieke mikrosatelliet merker en Ep-D1 bereken. Die mikrosatelliet merker was ook polimorfies vir die Lr 19 translokasie en dit is tussen die Wsp-D1 en Sr25 loki gekarteer. Kartering en/of identifikasie van merkers vir drie belangrike weerstandsgene was suksesvol in hierdie studie. Omdat al die merkers wat gebruik is, nie polimorf was tussen al die streke van belang nie, was dit nie moontlik om die data vir elk van die drie streke ten volle te integreer nie.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The slow growth rate of Haliotis midae impedes the optimal commercial production of this most profitable South African aquaculture species. To date, no comprehensive effort has been made to identify genes associated with growth variation in farmed H. midae. The aim of this study was therefore to investigate growth variation in H. midae and to identify and quantify the expression of selected growth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as a time-efficient and economical way of studying abalone transcriptomics and cell biology. Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were used to elucidate differential gene expression between two sibling groups of abalone demonstrating significant growth variation. Following transcriptome sequencing, genes involved in growth and metabolism, previously unknown in H. midae, were identified. The expression of selected target genes involved in growth was subsequently analyzed by quantitative real-time PCR (qPCR). The feasibility of primary cell cultures for H. midae was furthermore investigated by targeting embryo, larval and haemolymph tissues for the initiation of primary cell culture. Larval cells and haemocytes could be successfully maintained in vitro for limited periods. Primary haemocyte cultures demonstrated to be a suitable in vitro system for studying gene expression and were subsequently used for RNA extraction and qPCR, to evaluate differential growth induced by bovine insulin and epidermal growth factor (EGF). Gene expression was thus quantified in fast and slow growing abalone and in in vitro primary haemocyte cultures treated with different growth stimulating factors. The results obtained from transcriptome analysis and qPCR revealed significant differences in gene expression between large and small abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and in vitro qPCR experiments, the up-regulation of genes involved in the insulin signaling pathway provides evidence for the involvement of insulin in enhanced growth rate for various H. midae tissues. Besides the regulation of target genes, valuable knowledge was also gained in terms of reference genes, during qPCR experimentation. By quantifying the stable expression of two genes (8629, ribosomal protein S9 and 12621, ornithine decarboxylase) in various tissues and under various conditions, suitable reference genes, that can also be used in future H. midae qPCR studies, were identified. By providing evidence at the transcriptional level for the involvement of insulin, insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) in improved growth rate of H. midae, the relevance of investigating ways to stimulate insulin/IGF release in aquaculture species was again emphasized. As nutritional administration remains the most probable route of introducing agents that can stimulate the release of insulin-related peptides, continuous endeavours to stimulate abalone growth through a nutritional approach is encouraged. This is the first time next generation sequencing is used towards the large scale transcriptome sequencing of any haliotid species and also the first time a comprehensive investigation is launched towards the establishment of primary cell cultures for H. midae. A considerable amount of sequence data was furthermore annotated for the first time in H. midae. The results obtained here provide a foundation for future genetic studies exploring ways to optimise the commercial production of H. midae. / AFRIKAANSE OPSOMMING: Die stadige groeitempo van Haliotis midae belemmer die optimale kommersiele produksie van hierdie mees winsgewende Suid-Afrikaanse akwakultuur spesie. Tot op hede is geen omvattende poging aangewend om gene verwant aan groeivariasie in H. midae te identifiseer nie. Die doel van hierdie studie was dus om groeivariasie in H. midae te ondersoek en om spesifieke groei-gekoppelde gene te identifiseer en hul uitdrukking te kwantifiseer. Ter bereiking van hierdie doel is molekulêre metodes en selkulture gekombineer as 'n en tydsbesparende en ekonomiese manier om perlemoen transkriptomika en selbiologie te bestudeer. Moderne Illumina volgordebepaling-deur-sintese tegnologie en daaropvolgende annotasie is gebruik om verskille in geenuitdrukking tussen naby-verwante groepe perlemoen, wat noemenswaardige groeivariasie vertoon, toe te lig. Na afloop van die transkriptoom volgordebepaling is gene betrokke by groei en metabolisme, vantevore onbekend in H. midae, geïdentifiseer. Die uitdrukking van uitgesoekte teikengene betrokke by groei is vervolgens ge-analiseer deur kwantitatiewe "real-time PCR" (qPCR). die lewensvatbaarheid van 'n primêre selkulture vir H. midae is ook ondersoek deur embrio, larwe en hemolimf weefsels te teiken vir die daarstelling van primêre selkulture. Larweselle en hemosiete kon in vitro suksesvol onderhou word vir beperkte periodes. Primêre hemosietkulture het geblyk 'n gepaste in vitro sisteem te wees om geenuitdrukking te bestudeer en dit is vervolgens gebruik vir RNS ekstraksie en qPCR, om differensiële groei, geïnduseer deur insulien en epidermale groeifaktor (EGF), te evalueer. Geenuitdrukking is dus gekwantifiseer in vinnig- en stadiggroeiende perlemoen en in in vitro primêre hemosiet selkulture wat behandel is met verskillende groei stimulante. Die resultate wat verkry is van transkriptoomanalise en qPCR het noemenswaardige verskille in geenuitdrukking tussen groot en klein perlemoen, en tussen behandelde en onbehandelde hemosiet selkulture uitgelig. Die op-regulering van gene betrokke by die insulien sein-padweg, tydens in vivo en in vitro qPCR eksperimente, bied getuienis vir die betrokkenheid van insulien in die verhoogde groeitempo van verskeie H. midae weefsels. Benewens die regulering van teikengene is waardevolle kennis ook ingewin in terme van verwysingsgene tydens qPCR eksperimentering. Deur die stabiele uitdrukking van twee gene (8629, ribosomale proteien S9 en 12621, ornitien dekarboksilase) te kwantifiseer in verskeie weefsels en onder verskeie kondisies is gepaste verwysingsgene, wat ook in toekomstige H. midae qPCR eksperimente aangewend kan word, geïdentifiseer. Deur getuienis vir die betrokkenheid van insulien, insuliensoortige groeifaktor en insuliensoortige groeifaktor-bindingsproteïene by verbeterde groei van H. midae op transkripsievlak te bied, is die toepaslikheid van bestudering van maniere om insulienvrystelling in akwakultuurspesies te stimuleer, beklemtoon. Aangesien voeding die mees waarskynlike roete is om middele wat insuliensoortige peptiedvrystelling stimuleer daar te stel, word vogehoue pogings om perlemoengroei deur die regte voeding te stimuleer, aangemoedig. Hierdie is die eerste studie wat volgende generasie volgordebepaling (“next generation sequencing”) gebruik vir die grootskaalse transkriptoom volgordebepaling van enige haliotied spesie. Dit is ook die eerste keer dat ‘n omvattende ondersoek geloods word na die daarstelling van primêre selkulture vir H.midae. ‘n Aansienlike hoeveelheid volgorde data is ook vir die eerste keer geannoteer in H. midae. Die resultate wat hier verkry is bied ‘n basis vir toekomstige genetiese studies wat maniere ondersoek om die kommersiële produksie van perlemoen te optimiseer.

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is a commercially valuable mollusc and is mostly exported to the Far East. Genetics research on H. midae has increased substantially since a genetic improvement programme was introduced in 2006 by collaboration between Stellenbosch University, government and industry partners. The development of molecular markers, QTL-mapping, gene-expression and genome manipulations are the main focuses of the research currently being conducted. The end goal is to create high quality and fast growing animals for the industry. The present study focused on the development of microsatellite markers and the detection of quantitative trait loci (QTL) affecting growth traits (shell length, shell width, wet weight) in this species. A combination of three methods, namely selective genotyping and bulked segregant analysis (pooling analysis), single marker regression and interval mapping were used to identify putative QTL in two full-sib families from two different farmed locations. Additional methods and protocols were developed that can assist the industry in other molecular research aspects. A total of 125 microsatellite loci were characterised. A total of 82 of these loci were isolated using second generation sequencing, a first for any abalone species. A preliminary, low-density framework linkage map was constructed containing 50 loci that mapped to 18 linkage groups. The observed genome length was 148.72cm with coverage of ±47%. QTL analyses revealed two putative QTL for shell width and wet weight, with 17% and 15% variance explained, that mapped on one linkage group in the first family and three putative QTL, for shell length, shell width and wet weight, with 33%, 28.5% and 31.5% variance explained, that mapped on one linkage group in the second family. Additional methods and protocols developed include an automated high-throughput DNA isolation protocol, a real-time PCR assay for H. midae x H. spadicea hybrid verification, a triploid verification microsatellite assay and a pre- and post-PCR multiplex setup and optimisation protocol. Future studies focussing on QTL and marker assisted selection (MAS) should verify the QTL found in this study and also utilise additional family structures and determine QTL-marker phase within the commercial populations. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is ’n kommersieel waardevolle weekdier en word hoofsaaklik na die Verre-Ooste uitgevoer. Genetiese navorsing op H. midae het aansienlik toegeneem sedert ’n genetiese verbeteringsprogram in 2006 deur samewerking tussen die Universiteit van Stellenbosch, die regering en industrievennote ingebring is. Die ontwikkeling van molekulêre merkers, KEL-kartering, geen-uitdrukking en genoom manipulasies is die hooffokusse van die navorsing wat tans uitgevoer word. Die einddoel is om hoë kwaliteit en snelgroeiende diere vir die industrie te skep. Die huidige studie het op die ontwikkeling van mikrosatelliet merkers en die opsporing van groeiverwante (skulplengte, -breedte en nat gewig) kwantitatiewe eienskap lokusse (KEL) in hierdie spesie gefokus. ’n Kombinasie van drie metodes, naamlik selektiewe genotipering en versamelde segregaat analise (samevoegingsanalise), enkel merker regressie en intervalkartering is gebruik om waarskynlike KEL in twee vol-sibbe families van twee verskillende produksiegebiede te identifiseer. Aanvullende metodes en protokolle is ontwikkel wat die industrie in ander molekulêre navorsingsaspekte kan ondersteun. ’n Totaal van 125 mikrosatelliet lokusse is beskryf. ’n Totaal van 82 van hierdie lokusse is deur die gebruik van derde generasie volgordebepaling geïsoleer, ’n eerste vir enige perlemoen spesie. ’n Voorlopige, laedigtheid raamwerkkoppelingskaart is saamgestel met 50 lokusse wat op 18 koppelingsgroepe gekarteer is. Die waarneembare genoomlengte was 148.72cm met ’n dekking van ±47%. KEL-analises het twee waarskynlike KEL vir skulpbreedte en nat gewig blootgelê wat 17% en 15% variasie verduidelik en is op een koppelingsgroep in die eerste familie gekarteer asook drie waarskynlike KEL, vir skulplengte, -breedte en nat gewig wat 33%, 28.5% en 31.5% variasie verduidelik en is op een koppelingsgroep in die tweede familie gekarteer. Aanvullende metodes en protokolle wat ontwikkel is, sluit ’n geoutomatiseerde hoë-deurgang DNS-isolasieprotokol, ’n intydse PKR-proef vir H. midae x H. spadicea hibried verifikasie, ’n triploïed verifikasie mikrosatellietproef en veelsoortige pre- en post-PKR opstelling en optimaliseringsprotokol in. Toekomstige studies wat fokus op KEL en merker ondersteunde seleksie (MOS) behoort die KEL wat in hierdie studie gevind is te verifieer en ook bykomende familie strukture te benut om KEL-merker fases binne die kommersiële populasie te bepaal.