Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Biopolymers exhibit the required material properties to replace conventional, non-biodegradable, petroleum-based polymer products. They have a closed carbon cycle, making them carbon neutral and environmentally friendly. Biopolymers are produced from non-toxic substrates during in vivo enzymatic reactions. Biosynthesis of the most commercially important biopolymers is too complex to be reproduced in in vitro reactions. Identification of the genes responsible for their biosynthesis has been under investigation, with some pathways already elucidated. The genes involved in the biosynthesis of these polymers have been targeted for genetic manipulation to increase productivity, as well as create tailor-made polymers. Novel biopolymers and the genes responsible for their synthesis are of interest for their potential commercial applications. Bacteria produce a wide range of biopolymers and are being implemented as the bio-factories for biopolymer production. They are capable of utilising easily accessible and renewable carbon sources such as sucrose for polymer biosynthesis. Bacteria thus allow for economical production of these environmentally beneficial polymers. In this study, the gene responsible for the production of an unknown biopolymer from an unknown bacterium was identified. The biopolymer producing bacteria were grown on media enriched with sucrose as carbon source, during an expression library screening in a previous study. Expression library technology was used to search for the gene and it was identified as a 424 amino acid levansucrase which had a 100% homology to Leuconostoc mesenteroides M1FT levansucrase (AAT81165.1). Biopolymer analysis revealed that the biopolymer was a levan, a polysaccharide consisting of only fructose molecules with a molecular weight of ± 5 kDa. Analysis of a 516 bp fragment of the 16S rRNA determined that the unknown bacteria were a Pseudomonas species. / AFRIKAANSE OPSOMMING: Bio-polimere besit noodsaaklike materiële eienskappe wat toelaat dat dit konvensionele, nie bio-afbreekbare, petroleum-gebasseerde polimeer produkte kan vervang. Hulle het n geslote koolstof kringloop en is dus koolstof neutraal en omgewingsvriendelik. Bio-polimere word vervaardig van nie-toksiese substrate, gedurende ensiematiese reaksies in vivo. Die belangrikste kommersiële bio-polimere se ensiematiese produksie is te kompleks om in ʼn in vitro reaksie te herproduseer. Ondersoeke tot die identifikasie van die gene wat verantwoordelik is vir die produksie van die polimere is onderweg, en sommige produksie paaie is reeds bekend. Die bekende gene word geteiken vir genetiese manipulasie om hulle produktiwiteit te vermeerder en om unieke polimere te produseer. Unieke bio-polimere en die gene wat vir hul produksie verantwoordelik is, is van belang vir hulle potentiële implimentering in komersiële toepassings. Bakteria produseer ʼn verskeidenheid bio-polimere en word as die bio-fabrieke vir polimeerproduksie geimplimenteer. Hulle kan maklik bekombare koolstofbronne, soos sukrose, gebruik om bio-polimere te produseer. Bakteria laat dus die ekonomiese produksie van hierdie omgewingsvriendelike polimere toe. In hierdie studie word die geen wat verantwoordelik is vir die produksie van ʼn onbekende bio-polimeer van ʼn onbekende bakteria, geidentifiseer. Die bakteria was gevind op media, wat verryk was met sukrose as koolstofbron, tydens ʼn vorige studie, waartydens ʼn uitdrukkingsbiblioteek gesif was op hierdie media. Uitdrukkingsbiblioteek tegnologie was gebruik om die geen te vind. Die geen was geidentifiseer as ʼn 424 aminosuur, homo-fruktose-polimeer produseerende geen, ʼn “levansucrase”. Die geen het ʼn 100% homologie met die M1FT “levansucrase” geen (AAT81165.1) van Leuconostoc mesenteroides gehad. Analise van die bio-polimeer het bepaal dat die polimeer ʼn polisakkaried was, wat slegs uit fruktose molekules bestaan het. Die molekulêre gewig van die polimeer was ± 5 kDa. Analise van ʼn 516 bp fragment van die 16S rRNS het bepaal dat die bakteria van die Pseudomonas spesie afkomstig was.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae (commonly referred to as perlemoen) is the only one of five endemic species in South Africa that is commercially valued both locally and internationally. Unfortunately, natural perlemoen populations have become a dwindling resource due to commercial exploitation, poaching and the influx of natural threats, such as the West Coast rock lobster, Jasus lalandii. To preserve the natural diversity and sustainability of natural populations as well as commercial stocks, genetic management and improvement of perlemoen is critical. Genetic management requires the utilisation of molecular markers, which aid in the construction of linkage maps and the identification of quantitative trait loci (QTL) associated with economically significant traits. This will allow improvement of commercial stock management in terms of broodstock selection as well as provide valuable insight into natural population dynamics. Single Nucleotide Polymorphisms (SNPs) were selected as the marker of choice due to their successful employment as molecular markers and their wide distribution and abundance within the genomes of various marine species. This study focuses on the characterisation of novel SNPs from transcript sequences generated by Next Generation Sequencing technology. Approximately 40% of the transcripts facilitated the isolation of 105 putative markers, indicating a SNP frequency of ~1% within the H. midae genome. A subset of 24 markers, in addition to 24 previously developed markers, was characterised using the Illumina GoldenGate genotyping assay with the VeraCode technology, a medium to high-throughput genotyping technology. This is the first reported medium- to highthroughput characterisation of SNPs in H. midae. The selected markers were used to determine the efficiency and overall success rate of the GoldenGate platform. Marker characterisation was completed in both natural and commercial populations to determine the utility of these markers for genetic diversity and population structure inference. An 85% genotyping success rate was achieved with the platform. Statistical analysis indicated that the markers developed in this study are suitable for applications including population genetic structure inference, genetic diversity estimation and possibly other downstream applications such as linkage mapping. These markers are considered to be invaluable for future work regarding the genetic management and conservation of H. midae. / AFRIKAANSE OPSOMMING: Haliotis midae (ook bekend as perlemoen) is die enigste van vyf inheemse spesies in Suid-Afrika wat noemenswaardige kommersiële waarde toon plaaslik sowel as internasionaal. Ongelukkig het kommersiële uitbuiting, wildstropery en natuurlike bedreiging (bv. die Weskus kreef Jasus lalandii), wilde perlemoen populasies noemenswaardig verminder. Dus, om natuurlike diversiteit en die voortbestaan van beide wilde en kommersiële populasies te beskerm, is genetiese bestuur en verbetering absoluut noodsaaklik. Genetiese bestuur vereis die gebruik van molekulêre merkers as ’n hulpmiddel in die opstellingvan koppelingskaarte, en die identifisering van die relevante kwantitatiewe eienskap loki (QTL) tipies geassosieer met ekonomies belangrike eienskappe. Die laasgenoemde beoog om kommersiële voorraad bestuur te verbeter, kragtens deur broeidier seleksie sowel as om insig te verskaf m.b.t. wilde bevolking dinamika. Enkel Nukleotied Polimorfismes (SNPs) is gekies as die toepaslike merker vanweë die omvattende toepaslikheid van hierdie merkers binne die genome van verskeie mariene spesies. Hierdie studie fokus op die karakterisering van nuwe SNPs vanuit transkript volgordes ontwikkel deur middel van Volgende Generasie Volgordebepaling (“Next Generation Sequencing”). ’n Beraamde 40% van transkripte het gelei tot die ontwikkeling van 105 potensiëlemerkers, aanduidend van ’n SNP frekwensie van ~1% binne die H. midae genoom. ’n Sub-versameling van 24 merkers, tesame met 24 bestaande merkers, is gekarakteriseer deur die Illumina GoldenGate genotiperings toets met die VeraCode tegnologie, ’n medium tot hoë deurvloei genotiperingstegnologie. Hierdie is die eerste berig van medium tot hoë deurvloei karakterisering van SNPs in H. midae. Die geselekteerde merkers is gebruik om die doeltreffendheid van die GoldenGate platform te bepaal. Merker karakterisering is uitgevoer in beide wilde en kommersiële bevolkings om die effektiewe bruikbaarheid van hierdie merkers m.b.t. genetiese diversiteit, en bevolking struktuur bepaling, te ondersoek. Die platform het ’n 85% genotiperingsukses syfer getoon. Statistiese analise dui daarop dat merkers ontwikkel tydens hierdie studie toepaslik is vir bevolking genetiese struktuur bepaling, genetiese diversiteitberaming en moontlik ook genetiese koppelingskartering. Hierdie merkers word bestempel as onmisbaar vir toekomstige navorsing in genetiese bestuur en bewaring van H. midae.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The decline of Haliotis midae (perlemoen) populations together with the ensuing collapse of commercial abalone fisheries in South Africa have shifted the responsibility to abalone farms to meet the demand for perlemoen. Attention has recently turned to the genetic enhancement of cultured abalone in order for the farms to remain competitive in the international aquaculture market. To develop a successful breeding programme it is imperative to draw on a good foundation of high levels of genetic diversity and to successfully maintain these levels in order to create an enhanced strain of cultured abalone. A Performance Recording Scheme (PRS) was established as the first breeding programme for Haliotis midae to utilise molecular tools. This programme was aimed at enhancing the growth rate of abalone in order to shorten the production times on farms. The current study made use of 12 species-specific microsatellite markers to assign parentage to a group of faster-growing PRS animals, as selected by the abalone farms, in order to select a diverse on-farm generation of broodstock. Additionally, the influence of standard selection practises on the genetic diversity of a population compared to genotypic selection was investigated. This data was also used to study the differentiation and levels of genetic diversities within and between cultured and wild populations. Selection based on genotypic traits successfully retained genetic diversity while some diversity was lost in phenotypically selected populations. These phenotypic populations differed significantly from each other and wild populations, while the genotypic populations were similar in genetic composition to each other and wild populations of the West coast. The broodstock populations used in the PRS spawning event were representative of the wild populations from where they were sourced, with no significant differentiation between the broodstock and West coast population. When these broodstock populations were compared to their corresponding offspring populations, only two populations displayed a significant loss in diversity; although all of the offspring populations showed significant differentiation with their corresponding broodstock populations. This was attributed to the differential contribution of broodstock and the effect of artificial selection. It was established that the cultured populations of the participating abalone farms should be used with caution in ranching and reseeding programmes. These populations differed significantly from both the East and West coast wild populations. This study concluded that it is possible to retain genetic diversity by selecting breeding animals based on genotypic traits. The loss of diversity in some cultured populations and significant differentiation from the wild populations indicate that animals are exposed to different selection pressures in the cultured environment. The results found in this study highlight the need for the effective management of hatchery practices and the genetic monitoring of the breeding animals. / AFRIKAANSE OPSOMMING: Die afname in Haliotis midae (perlemoen) populasies en die daaropvolgende ineenstorting van die kommersiële perlemoen bedryf in Suid-Afrika het die verantwoordelikheid om in die aanvraag na perlemoen te voorsien, na perlemoen plase verskuif. Die genetiese verbetering van verboude perlemoen geniet tans aandag in ‘n poging om kompeterend te bly in die internasionale mark. Dit is noodsaaklik vir die sukses van ‘n broeiprogram om gebruik te maak van ‘n goeie genetiese basis met hoë vlakke van genetiese diversiteit en die suksesvolle behoud van die vlakke om so ‘n verbeterde lyn te skep. ‘n Groeiprestasie aanteken stelsel [Performance Recording Scheme (PRS)] is gestig as die eerste broeiprogram vir Haliotis midae wat gebruik maak van molekulêre tegnieke. Die doel van hierdie program was om die groeitempo van verboude perlemoen te verbeter om produksie tye te verkort. Die huidige studie het gebruik gemaak van 12 spesie-spesifieke mikrosatelliet merkers om ouerskap toe te ken aan ‘n groep vinnig-groeiende PRS-diere, soos geselekteer deur die perlemoen plase, om ‘n diverse generasie gekultiveerde diere te selekteer wat as broeidiere kan dien. Die invloed van standaard seleksie metodes op die genetiese diversitiet van ‘n populasie in vergelyking met genotipiese seleksie is ook ondersoek. Die ouerskap data is ook gebruik om differensiasie en vlakke van genetiese diversiteit tussen verboude perlemoene en wilde populasies vas te stel. Seleksie gebasseer op genetiese eienskappe het daarin geslaag om genetiese diversiteit te behou, terwyl diversiteit verlore gegaan het in die fenotipies geselekteerde populasies. Hierdie fenotipiese populasies het ook beduidend met mekaar sowel as met die wilde populasies verskil, terwyl genotipiese populasies soortgelyk was in hul genetiese samestelling en nie van die wilde populasies van die Weskus verskil het nie. Die broeidiere wat in die PRS broeiprogram gebruik is, was verteenwoordigend van die wilde populasies vanwaar hulle oorspronlik gekom het, met geen beduidende differensiasie tussen die broeidiere en die Wes kus populasies nie. Met die vergelyking van die broeidiere en hul ooreenstemmende nageslag, het dit geblyk dat slegs twee populasies ‘n beduidende verlies aan genetiese diversiteit getoon het, alhoewel al die nageslag beduidende populasie differensiasie met hul ouers getoon het. Hierdie bevindinge is toegeskryf aan oneweredige bydraes van die broeidiere tydens gameetvrystelling en die invloed van kunsmatige seleksie. Hierdie studie het ook vasgestel dat die verboude perlemoen populasies met sorg gebruik moet word om wilde populasies te herstel, aangesien hierdie populasies beduidend verskil het van wilde populasies van beide die Oos en Wes-kus. Hierdie studie het gevind dat dit moontlik is om genetiese diversiteit te behou deur diere te selekteer op grond van genotipiese eienskappe. Die verlies van diversiteit in sommige van die verboude perlemoen populasies en die beduidende verskil met die wilde populasies dui daarop dat diere in die gekultiveerde omgewing blootgestel word aan verskillende tipes seleksiedruk. Hierdie bevindinge beklemtoon die belang vir effektiewe bestuur van broeiery praktyke en genetiese monitering van broeidiere.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae, or more commonly known as Perlemoen, is an abalone species found along the coast of South Africa. It is the only cultured abalone species in South Africa and has a high demand abroad. Due to its popularity as a seafood delicacy, illegal harvesting has taken its toll on Perlemoen numbers. This increases the need for sustainable farming efforts and efficient implementation of law enforcement practices against poachers. Abalone farms make use of a limited number of broodstock for breeding, so it is necessary to ensure that genetic effects such as inbreeding and bottlenecks do not interfere with the viability of the offspring. Research that focuses on the genetics of Perlemoen will greatly aid the farms to continue sustainable production of this species as well as enhance their breeding efficiency. This study focuses on the construction of a linkage map for H. midae that will allow the future identification of markers associated with genes important to production, such as growth and disease resistance. Identification of these genes will allow breeders to select genetically superior abalone that will be used for breeding programmes in which the phenotype of the offspring will be enhanced. For the construction of a linkage map it is necessary to have enough informative markers for mapping. In this study, gene-linked microsatellite markers were developed by screening a contig assembly of H. midae’s transcriptome. Ninety-eight primer pairs could be developed from the contigs and 60 loci produced amplification products. Twenty-six microsatellites were found to be polymorphic (27% success rate). In addition to these markers, 239 previously developed microsatellites and 48 gene-linked SNPs were used to develop sex-average and sex-specific linkage maps in four full-sib families consisting of approximately 100 offspring each. Of these markers 99 were informative in family DS1 (31% success rate), 81 in family DS2 (26%), 77 in family DS5 (24%) and 71 in family DS6 (23%). These markers were used for linkage analysis (LOD>3). The average number of linkage groups for the sex-average maps ranged from 17-19. The average genome length for these maps ranged from 700cM to 1100cM with an average marker spacing of 8cM. The sex-specific maps’ linkage groups ranged from 13-17 with an average genome length of 600cM to 1500cM. The average marker spacing was approximately 16cM. The integrated map was constructed by merging the sex-average maps. This map contained 25 linkage groups with an average genome length calculation of 1700cM and an average marker spacing of 9.3cM. The linkage maps created in this study are the first to utilize SNPs in H. midae. Further incorporation of SNPs into linkage maps will enhance the density. The maps created in this study are of medium-density (65%) and provide a link to the development of high-density linkage maps to facilitate associations of phenotypic traits to certain markers, to so that QTL mapping can be performed. This information can be used for marker-assisted selection to produce genetically superior abalone. / AFRIKAANSE OPSOMMING: Haliotis midae, of meer algemeen bekend as Perlemoen, is 'n klipkous spesie wat langs die kus van Suid-Afrika voorkom. Dit is die enigste gekweekte klipkous spesie in Suid-Afrika en het 'n hoë aanvraag in die buiteland. As gevolg van sy gewildheid as 'n seekos lekkerny, het onwettige stropery sy tol geneem op Perlemoen getalle. Hierdie verhoog die behoefte vir volhoubare boerdery pogings en doeltreffende implementering van wetstoepassing teen stropers. Perlemoenplase maak gebruik van 'n beperkte aantal broeidiere vir teling, dus is dit nodig om te verseker dat genetiese effekte soos inteling en genetiese bottelnekke nie inmeng met die lewensvatbaarheid van die nageslag nie. Navorsing wat fokus op die genetika van Perlemoen sal grootliks die plase steun om die volhoubare produksie van hierdie spesie voort te sit, sowel as hul teling doeltreffendheid te verbeter. Hierdie studie fokus op die ontwikkeling van 'n genetiese koppelingskaart vir H. midae, wat die toekomstige identifisering van die merkers wat verband hou met die gene wat belangrik is vir die produksie, soos groei en weerstand teen siektes sal verbeter. Identifisering van hierdie gene sal toelaat dat telers genetiese voortreflike Perlemoen kan kies vir teelprogramme waartydens die fenotipe van die nageslag sal verbeter word. Vir die ontwikkeling van 'n genetiese koppelingskaart is dit nodig om genoeg informatiewe merkers vir die kartering te hê. In hierdie studie, is geen-gekoppelde mikrosatelliet-merkers ontwikkel deur ‘contig’ data van H. midae se transkriptoom te ondersoek. Agt en negentig inleier pare kon ontwikkel word uit die ‘contigs’ en 60 loki kon ‘n amplifiseringsproduk lewer. Ses-en-twintig mikrosatelliete was polimorfies (27% suksessyfer). Bykomend tot hierdie ontwikkelde merkers is 239 voorheen ontwikkelde mikrosatelliete en 48 geen-gekoppelde SNPs gebruik om geslagsgemiddelde en geslagspesifieke koppelingskaarte in vier volsib families, wat uit ongeveer 100 nageslag elk bestaan, te ontwikkel. Van hierdie merkers was 99 informatief in familie DS1 (31%), 81 in die familie DS2 (26%), 77 in die familie DS5 (24%) en 71 in die familie DS6 (23%). Hierdie merkers is gebruik vir 'n koppelingsanalise (LOD>3). Die gemiddelde aantal koppelingsgroepe vir die geslagsgemiddelde kaarte het gewissel van 17-19. Die gemiddelde genoom lengte vir hierdie kaarte het gewissel van 700cM tot 1100cM met 'n gemiddelde merker spasiëring van 8cm. Die koppelingsgroepe van die geslagspesifieke kaarte het gewissel van 13-17 met 'n gemiddelde genoom lengte van 600cM tot 1500cM. Die gemiddelde merker spasiëring was ongeveer 16cm. Die geïntegreerde kaart is saamgestel deur die samesmelting van die geslagsgemiddelde kaarte. Die kaart toon 25 koppelingsgroepe met 'n gemiddelde berekende genoom lengte van 1700cM en' n gemiddelde merker spasiëring van 9.3cM. Die genetiese koppelingskaarte wat in hierdie studie ontwikkel is, is die eerste om SNPs te gebruik in H. midae. Verdere insluiting van SNPs in koppelingskaarte sal die digtheid verhoog. Die kaarte wat in hierdie studie ontwikkel is, is van medium digtheid (65%) en bied 'n stap nader aan die ontwikkeling van hoë digtheid koppelingskaarte om fenotipiese eienskappe met sekere merkers te assosieer, vir kwantitatiewe kenmerk lokus kartering. Hierdie inligting kan gebruik word vir merker bemiddelde seleksie om geneties verbeterde Perlemoen te produseer.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Iron metabolism disorders comprise the most common disorders in humans. Hereditary haemochromatosis (HH) is a common condition resulting from inappropriate iron absorption. The most common form of the disease (Type 1) is associated with mutations in the HFE gene. The C282Y homozygous genotype accounts for approximately 80% of all reported cases of HH within the Caucasian population. A second HFE mutation, H63D, is associated with less severe disease expression. The C282Y mutation is extremely rare in Asian and African populations. The H63D mutation is more prevalent and has been observed in almost all populations. Iron overload resulting from haemochromatosis is predicted to be rare in Asian Indian populations and is not associated with common HFE mutations that are responsible for HH in the Caucasian population. The aberrant genes associated with HH in India have not yet been identified. The present study attempted to identify variants in six iron regulatory genes that were resulting in the Type 1 HH phenotype observed in two Asian Indian probands from a highly consanguineous family. The promoter and coding regions of the HMOX1, HFE, HAMP, SLC40A1, CYBRD1 and HJV genes were subjected to mutation analysis. Gene fragments were amplified employing the polymerase chain reaction (PCR) and subsequently subjected to heteroduplex single-strand conformational polymorphism (HEX-SSCP) analysis. Samples displaying aberrations were then analysed using bi-directional semi-automated DNA sequencing analysis to identify any known or novel variants within the six genes. Variants disrupting restriction enzyme recognition sites were genotyped employing restriction fragment length polymorphism (RFLP) analysis. Mutation analysis of the six genes revealed 24 previously identified variants, five novel variants (HFE: 5’UTR-840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR- 1272T→C; HJV: 5’UTR-534G→T, 5’UTR-530G→T), one previously described microsatellite and two novel repeats. Variants identified within the SLC40A1, CYBRD1 and HJV genes do not seem to be associated with the iron overload phenotype. A previously described HAMP variant (5’UTR-335G→T) was observed in the homozygous state in both probands. This variant seems to be the genetic aberration responsible for iron overload in this Indian family. The severe juvenile haemochromatosis phenotype usually associated with HAMP mutations, was not exhibited by the two Indian probands. Their symptoms resembled those observed in classic Type 1 HH. It is suggested that variants identified in the HMOX1 and HFE genes are modifying the effect of the HAMP variant and resulting in the less severe disease phenotype. Although this variant has only been identified in one Indian family, it could shed some light in the hunt for the iron-loading gene in India. / AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is ‘n algemene siektetoestand wat ontstaan as gevolg van oneffektiewe opname van yster in die liggaam. Die mees algemene vorm van die siekte (Tipe 1) word geassosieer met mutasies in die HFE-geen. Die C282Y homosigotiese genotipe is verantwoordelik vir ongeveer 80% van alle gerapporteerde gevalle van OH binne die Kaukasiese bevolking. ‘n Tweede HFE mutasie, H63D, word geassosieer met minder ernstige siekte simptome. Die C282Y mutasie is besonder skaars in Asiese en Afrika bevolkings. Daar word bespiegel dat oorerflike ysteroorlading as gevolg van hemochromatose skaars is in Asiese Indiër bevolkings en word nie geassosieer met algemene HFE mutasies wat verantwoordelik is vir OH in Kaukasiese bevolkings nie. Die abnormale gene wat wél geassosieer word met OH in Indië is tot dusver nog nie identifiseer nie. Die doel van hierdie studie was om die variante in ses yster-regulerende gene te identifiseer wat die Tipe 1 OH fenotipe in hierdie familie veroorsaak. Hierdie fenotipe is waargeneem in twee Asies Indiese familielede afkomstig van ‘n bloedverwante familie. Die promotor en koderingsareas van die HMOX1, HFE, HAMP, SLC40A1, CYBRD1 en HJV gene is gesif vir mutasies. Geen fragmente is geamplifiseer met behulp van die polimerase kettingsreaksie (PKR) en daarna aan heterodupleks enkelstring konformasie polimorfisme (HEX-SSCP) analise blootgestel. PKR produkte wat variasies getoon het, is daarna geanaliseer deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise om enige bekende of nuwe variante binne die ses gene te identifiseer. Variante waar restriksie ensiem herkenningsetels teenwoordig is, is verder analiseer met behulp van die restriksie fragment lengte polimorfisme (RFLP) analise sisteem. Mutasie analise van die ses gene het 24 bekende variante, vyf nuwe variante (HFE: 5’UTR- 840T→G, CYBRD1: 5’UTR-1813C→T, 5’UTR-1452T→C, 5’UTR-1272T→C, HJV: 5’UTR-534G→T, 5’UTR-530G→T), een bekende herhaling en twee nuwe herhalings gewys. Variante wat binne die SLC4041, CYBRD1 en HJV gene geïdentifiseer is, blyk nie om by te dra tot die ysteroorladings-fenotipe nie. Die bekende HAMP variant (5’UTR-335G→T) is waargeneem in die homosigotiese toestand in beide van die aangetaste individue. Hierdie variant blyk om die genetiese fout te wees wat verantwoordelik is vir die ysteroorlading in die betrokke Indiese familie. Die erge juvenielehemochromatose fenotipe wat meestal geassosieer word met HAMP-mutasies, is nie waargeneem in hierdie familie nie. Hul simptome kom ooreen met die simptome van die klassieke Tipe 1 OH. Dit blyk moontlik te wees dat die variante identifiseer in die HMOX1 en HFE gene die impak van die HAMP variant modifiseer en die matiger siekte-fenotipe tot gevolg het. Alhoewel hierdie variant slegs in een Indiese familie geïdentifiseer is, kan dit lig werp op die soektog na die veroorsakende ysterladingsgeen in Indië.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The indigenous abalone Haliotis midae is one of the most remarkable and highly exploited species of marine molluscs in South Africa. It is the only species of southern African Haliotidae to be commercially reared and has been successfully cultured for almost two decades. Its short history of domestication along with market demands and the need to develop efficiency in the production process has resulted in an increased interest in the possible genetic improvement of this species. The unhurried growth rate associated with H. midae is a cause of particular concern to the industry, predominantly with regards to profitability and competitiveness in the market place. A modest amount of work has so far been directed at establishing a means of enhancement for selective breeding on the commercial level. Genetics plays a key role in the establishment of successful improvement programmes in various aquaculture species. The aim of this study was to develop species-specific microsatellite markers for the abalone and subsequently perform parentage assignment on farm produced animals entered into a growth performance recording scheme. Animals were obtained from the hatcheries of three commercial abalone farms situated in the Walker Bay region in the Western Cape. Microsatellites were isolated using the enrichment-based FIASCO method, and characterised into perfect, imperfect and compound repeats according to the structural nature of their repetitive units. From the partial gDNA libraries obtained and 365 screened colonies, a total of 54 loci were located. PCR primers were designed for 36 markers and the 15 primer pairs that displayed loci with the highest level of polymorphism were subsequently chosen for fluorescent labelling. The markers were tested on a subset of 32 wild H. midae individuals to determine their usefulness and efficiency in genotyping. Five markers, along with five others that were previously designed, were chosen for assigning parentage to the animals submitted to the performance recording scheme. Three thousand offspring from each of the three participating farms were equally divided and reared at five different locations. From each location 20 fast growing and 20 slow growing juveniles, as well as the broodstocks, were sampled and genotyped using the ten chosen microsatellite loci. Two farms had 60% of offspring unambiguously assigned to a single parental couple. Assignments showed patterns of dominant male and female brooders, but no trend in brooders specifically contributing to fast or slow growing offspring. Parentage assignment for the third farm was, however, unsuccessful due to lack of broodstock data. In future, screening of all available broodstock will ensure acquisition of relevant pedigree information. The results obtained in this study are an initial step in the development of a genetic improvement programme for commercial Haliotis midae. / AFRIKAANSE OPSOMMING: Die inheemse skulpvis Haliotis midae is een van die mees merkwaardige en hoogs oorbenutte mariene slakspesies in Suid-Afrika. Dit is die enigste suidelike Afrika Haliotidae spesie wat kommersieel benut word en dit word al meer as twee dekades suksesvol geteel. Die spesie se kort domestiseringsgeskiedenis, toenemende mark aanvraag en die behoefte om meer effektiewe produksie daar te stel, het gelei tot toenemende belangstelling in die moontlike genetiese verbetering van die spesie. Die stadige groeitempo geassosieer met H. midae is veral ‘n punt van kommer vir die industrie, veral in terme van winsgewendheid en kompetering in die markplek. Minimale werk is sover gedoen in die daarstelling van verbetering deur selektiewe teling op ‘n kommersiële skaal. Genetika speel ’n sleutelrol in die daarstelling van suksesvolle verbeteringsprogramme van verskeie akwakultuur spesies. Die doel van hierdie studie was om spesie-spesifieke mikrosatelliet merkers vir perlemoen te ontwikkel en vervolgens ouerskapsbepaling van kommersiële diere, wat deelneem aan ‘n groeiprestasie aantekenstelsel, uit te voer. Diere is voorsien deur die teelstasies van drie kommersiële perlemoenplase geleë in die Walker Bay omgewing in die Wes-Kaap. Mikrosatelliete is geïsoleer deur die verrykings-gebaseerde FIASCO metode, en gekarakteriseer as perfekte, onderbroke of saamgestelde herhalings gebaseer op die strukturele aard van die herhalings eenhede. Vanaf die gedeeltelik gDNA biblioteke wat bekom is en 365 gesifte kolonies, is ‘n totaal van 54 loki opgespoor. PKR inleiers is ontwerp vir 36 merkers en die 15 inleierpare, wat loki met die hoogste polimorfisme geamplifiseer het, is vervolgens geselekteer vir fluoreserende merking. Die merkers is getoets op ’n kleiner groep van 32 natuurlike H. midae individue om hulle bruikbaarheid en genotiperingseffektiwiteit te bepaal. Vyf merkers is saam met vyf reeds ontwikkelde merkers gekies vir ouerskapsbepaling van die diere in die prestasie aantekenstelsel. Drieduisend nageslag diere vanaf elkeen van die drie deelnemde plase is gelykop verdeel en grootgemaak op die vyf verskillende lokaliteite. ‘n Monster van 20 vinnig groeiende en 20 stadig groeiende jong perlemoen, sowel as broeidiere, is vanaf elke lokaliteit geneem en gegenotipeer deur middel van die 10 geselekteerde mikrosatelliet loki. Sestig persent van twee van die plase se nageslag is onteenseglik toegesê aan ‘n enkele ouerpaar. Ouerskapstoekenning het patrone van dominante vroulike en manlike broeidiere getoon, maar geen tendens in terme van bydrae tot vinnig en stadig groeiende nageslag kon gevind word nie. Ouerskapstoekenning vir die derde plaas was onsuksesvol as gevolg van ’n gebrek aan data vir die broeidiere. In die toekoms sal genotipering van alle beskikbare broeidiere die daarstelling van relevante stamboominligting verseker. Die resultate verkry in hierdie studie verteenwoordig ‘n eerste stap in die ontwikkeling van ’n genetiese verbeteringsprogram vir kommersiële Haliotis midae.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / Hereditary haemochromatosis (HH), a common autosomal recessive disease, is characterized by increased iron absorption leading to progressive iron accumulation in organs such as the liver, heart and pancreas. In the South African population the disease is prevalent in individuals of Caucasian origin, with a carrier frequency of one in six for the C282Y mutation in the HFE gene. We investigated the role of genes implicated in iron metabolism, including the high-iron gene (HFE), haem oxgenase-1 gene (HMOX1), solute carrier family 40 (iron-regulated transporter) member 1 gene (SLC40A1), cytochrome b reductase gene (CYBRD1), hepcidin antimicrobial peptide gene (HAMP) and the hemojuvelin gene (HJV) in a patient cohort with non-HFE iron overload. DNA analysis was performed on samples from 36 unrelated South African Caucasian patients presenting with primary iron overload, who tested either negative or heterozygous for C282Y. In this study, mutation screening was performed by PCR amplification and HEX-SSCP analysis. Sixteen previously described and two novel variants were identified by semi-automated DNA sequencing. Common variants identified in the HFE gene included C282Y, H63D, IVS2+4T→C, IVS4-44T→C, IVS4+48G→A and IVS5-47G→A. The Q127H mutation in exon 3 of the HFE gene was identified in one patient, who tested negative for both C282Y and H63D. Mutation S65C was identified only in the population-matched controls and was absent in the patient group. Other previously described polymorphisms identified included the IVS5+51delTGGCTGTCTGACT deletion in HMOX1, I109 and V221 in SLC40A1, IVS1-4C→G, IVS2+8T→C and S266N, in the CYBRD1 gene and, S264 and A310G in the HJV gene. The novel variants, -89C→T, in the promoter region of the CYBRD1 gene, was detected in only one patient, while S333 in exon 4 of the HJV gene was present in three patients. These variants were not identified in any of the population-matched controls screened and could explain the non-HFE iron overload presented by these patients. This study clearly demonstrates the importance of modifier genes in patients with iron overload that cannot be explained by the common C282Y mutation. Studies on iron-related genes and the identification of mutations in these genes in non-HFE patients could lead to improved diagnosis and counselling of South African patients presenting with primary iron overload.

Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / Oesophageal cancer is a disease characterised by a disproportionate presentation in certain ethnic groups, with squamous cell carcinoma (SCC) occuring more often in Blacks and adenocarcinoma (ADC) being more prevalent in Caucasians. Several factors have been attributed to the development of OC, including an excess of iron (leading to enhanced tumour growth), oesophageal injury and chronic inflammation. The main aim of this study was to establish the mutation spectrum of six genes (including HFE, HMOX1, SLC40A1, HAMP, CYBRD1 and HJV) involved in iron metabolism, in the Black South African OC population. The patient cohort comprised of 50 (25 male and 25 female) unrelated patients presenting with SCC of the oesophagus, with the control group consisting of 50 unrelated, healthy population-matched individuals. The mutation detection techniques employed included polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformational polymorphism (HEX-SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis and bi-directional semi-automated DNA sequencing analysis of variants identified.

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / Thinopyrum distichum (2n = 4x = 28; J1dJ1dJ2dJ2d) is a hardy, salt-tolerant maritime wheatgrass indigenous to southern Africa. In order to transfer its salt-tolerance to cultivated cereals, the Thinopyrum chromosomes involved must first be characterized with molecular markers. Thinopyrum distichum chromosomes 2J1d, 3J1d, 4J1d and 5J1d have previously been found to be major determinants of salt-tolerance. A genotype panel consisting of two triticale/Th. distichum allopolyploids, two Th. distichum/2*triticale doubled-haploids, eight triticale addition-lines (for chromosomes 2J1d; 2J1dβ; 3J1d; 3J1dL; 4J1d; 4J2d; 5J1d and 7J2d, respectively) and two triticale translocation-lines (involving chromosome arms 3J1dS and 3J1dL, respectively) were used for fluorescence-based, semi-automated AFLP-analyses and to a lesser extent for EST-SSR microsatellite marker-development, to identify molecular markers specific to the critical Th. distichum chromosomes. Thirteen EST-SSR primer pairs produced four putative Th. distichum-specific microsatellite-markers, one of which was specific for critical chromosome 5J1d. AFLP-analysis with 60 selective EcoRI/MseI and 18 Sse8387I/MseI primer combinations produced 159 AFLP-fragments specific for Th. distichum. These included seven putative markers for chromosome 2J1d, 15 for 3J1d, one marker for 4J1d and two for 5J1d. A salt-tolerance experiment was done to determine which chromosome 2J1d and 3J1d regions may carry genes for salt-tolerance. Plants were selected that had a monosomic addition of a chromosome 2J1d variant (either the complete chromosome or a modified version referred to as 2J1dβ) in addition to one of four chromosome 3J1d variants (the complete 3J1d chromosome; a 3J1dL-telosome; a 3J1dS-translocation or a 3J1dL-translocation). The results suggested that Th. distichum chromosome-arms 2J1dL and 3J1dS are probably involved in salt-tolerance. A group of 93 trigeneric (Triticum/Secale/Thinopyrum) F2 secondary hybrids were then analyzed in order to: (i) Evaluate some (ten) of the newly developed putative AFLP-markers; and (ii) attempt to find translocations, telosomes or substitutions involving the critical Thinopyrum chromosomes. Five (50 %) of the ten putative AFLP-markers could be reproduced, but only four proved to be chromosome-specific. It was also possible to assign hese four markers to chromosome arms: E32M49.118 (2J1dS); E41M49.103 (2J1dS); E35M49.137 (3J1d); and E41M49.188 (3J1dL). The selective primer combination that produced marker E41M49.103 (2J1dS), also amplified a fragment of the same size on chromosome 4J1d. These markers will be useful for further mapping and selection of the salt-tolerance genes. The fact that only four of the ten putative AFLP-markers evaluated proved to be repeatable implies that the remaining untested markers need to be confirmed against larger genotype panels as well. Probable reasons for the relatively low frequency of markers that turned out to be reliable are discussed. The marker-association study also revealed that visual examination of all electropherograms produced by AFLP-fragment analysis is necessary to correctly identify all AFLP-fragments. Use of the AFLP- and STS-/SCAR-markers in conjunction with the group of 93 F2 secondary hybrids showed that 18 of these probably carried a 3J1dL-translocation. Several hybrids possibly had translocations involving the 4J1d and 5J1d chromosomes. However, these results need to be confirmed. Various hybrids also appeared to have critical Th. distichum substitutions, although this still requires further confirmation. The identified plant material could prove useful for further characterization of salt-tolerance in Thinopyrum, and its eventual utilization in cereal crops.

Thesis (MSc (Genetics))--Stellenbosch University, 2008.