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)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plant-pathogen interactions have been intensively investigated in the last decade. This major drive towards understanding the fundamental aspects involved in plant disease resistance is propelled by the obvious agricultural and economical benefits that are intrinsically linked to disease and stress resistant plants. It is, therefore, not surprising that fundamental research in this area is not just restricted to model organisms, such as Arabidopsis and tobacco, but also extends to more traditional crop plants, such as maize, bean, soybean, apples, grapevine etc. In grapevine for instance, several genes involved in disease resistance have been isolated. One of these genes, encoding for a polygalacturonase inhibiting protein (PGIP), has been studied extensively. PGIPs are cell wall bound, contain leucine rich repeats (LRR) and are found in all dicotyledonous plants so far examined. In most cases, pgip genes occur in small multigene families and expression is often tissue specific and developmentally regulated. Up-regulation of PGIP-encoding genes typically occurs upon pathogen infection, treatment with elicitors, salicylic acid (SA), jasmonic acid (JA), cold treatment and wounding. Differential regulation and specificity have been shown to occur between members of the same multigene family. Differential regulation even extends to the utilization of separate pathways to induce pgip genes from the same family in response to a single stress stimulus. PGIPs interact with cell wall macerating polygalacturonases (PGs) that are secreted by pathogenic fungi during the infection process. The antifungal action of PGIPs is thought to depend on a dual action. The physical interaction of PGIP with PGs has an inhibitionary effect, resulting in (i) a slower fungal infection rate and (ii) the prolonged existence of long chain oligogalacturonides (OGs). These oligosaccharides are able to elicit a general plant defense response, enabling the plant to further retard or curb the spread of infection. The main objective of this study was to investigate the regulatory aspects underlying PGIP expression in grapevine. Unlike most characterized PGIP encoding genes from other dicotyledonous plant species, no evidence to support the existence of a V. vinifera PGIP multigene family could be found from either genetic or biochemical analyses. Recently, a genomic DNA fragment from Vitis vinifera cv Pinotage was pathogen interactions with regards to the fundamental processes underlying defense gene regulation. / AFRIKAANSE OPSOMMING: Die ooglopende voordele wat, vanuit 'n landboukundige én ekonomiese oogpunt, uit siekte- en stresbestande plante spruit, het gedurende die laaste dekade aanleiding gegee tot die ontwikkeling van plantpatogeen-interaksies as "n baie belangrike studieveld. Dit was dus ook te verwagte dat fundamentele navorsing in hierdie area nie net beperk gebly het tot modelorganismes soos Arabidopsis en tabak (ook natuurlik van landboukundige belang) nie, maar ook na meer tradisionele landbougewasse soos mielies, boontjies, sojaboontjies, appels, druiwe, ens. oorgevloei het. Verskeie siekteweerstands-verwante gene is byvoorbeeld al vanuit wingerd geïsoleer. Een só "n geen wat vir "n poligalakturonase-inhiberende proteïen (PGIP) kodeer, vorm deel van hierdie groep gene. Die funksie en regulering van PGIP's is baie goed bestudeer. Hierdie proteïene word normaalweg in die selwande van die meeste dikotiele plante aangetref. Leusienryke herhalings is algemeen in PGIP's en hierdie tipe van herhalings is kenmerkend van proteïene betrokke by proteïen-proteïen-interaksies. Verder word pgip-gene gewoonlik in klein multigeenfamilies aangetref, waar in die meeste gevalle die uitdrukking weefselspesifiek en die regulering spesifiek ten opsigte van die ontwikkelingsfase is. Verskeie faktore kan tot die induksie van pgip-gene lei, soos onder andere patogeen-infeksie, elisitoor-, salisiensuur-, jasmoonsuur- en kouebehandeling, asook verwonding. Differensiële regulering word in baie gevalle tussen lede van dieselfde multigeenfamilie aangetref. Hierdie differensiële regulering kan selfs bemiddel word deur onafhanklike reguleringsweë in reaksie op dieselfde induksiestimulus. PGIP's is in staat om te reageer met poligalakturonases (PGs), wat selwande afbreek en wat gedurende die infeksieproses deur swamme of fungi afgeskei word. Die effek van hierdie interaksie is tweeledig: (i) Die fisiese interaksie tussen PGIP en PG moduleer die aktiwiteit van die PG deur die ensiemaksie te inhibeer, en (ii) PGinhibisie lei tot die verhoogde stabiliteit van langketting-oligogalakturonades, molekules wat daartoe in staat is om die weerstandsrespons van plante te ontlok. Die inhibisie van die patogeen-PG's, tesame met die geïnduseerde weerstandrespons, stel die plant dan in staat om verdere infeksie te vertraag of te verhoed. Die doel van hierdie studie was om die onderliggende aspekte van PGIPregulering in wingerd te bestudeer. In teenstelling met die meeste plantspesies waar pgip-gene in klein multigeenfamilies aangetref word, is daar nie 'n pgip-multigeenfamilie in wingerd nie. Veelvuldige kopieë van In enkele pgip-geen word egter in die wingerdgenoom aangetref. Daar is onlangs in ons laboratorium In genoom-DNAfragment vanaf Vitis vinifera cv Pinotage geïsoleer wat die oopleesraam en 5'-stroomopsekwense van In PGIP-enkoderende geen (Vvpgip1) bevat. In hierdie studie is die uitdrukkingspatroon van Vvpgip1 ten opsigte van weefselspesifisiteit, korrelontwikkelingsfase, asook die effek van verskeie omgewings en patogeenverwante stres-stimuli ontleed. Die regulatoriese meganismes van Vvpgip1 bevat spesifieke in planta-ontwikkelingsfaseseine wat verder deur spesifieke faktore, insluitende omgewings- en patogeenstres, gereguleer word. In lyn hiermee is mRNS-transkripte van Vvpgip1 tot wortel- en korrelweefsels beperk, terwyl die mRNS-vlakke ook tussen verskillende korrelontwikkelingsfases wissel. Kumulatiewe uitdrukking kon waargeneem word in veráison-korrels in reaksie op verwonding en osmotiese stres. Die weefselspesifieke uitdrukkingspatroon tipies van wingerd-PGIP is in blare opgehef in reaksie op Botrytis cinerea-infeksie, verwonding, osmotiese stres, ouksien (indoolasynsuur) en salisiensuur. PGIP-uitdrukking word ook onderdruk deur In staurosporien-sensitiewe proteïenkinase, wat In goeie aanduiding is van die betrokkenheid van proteïenfosforilasie in die seintransduksiekaskade wat tot PGIPuitdrukking aanleiding gee. Die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare kan ook nageboots word in tabak wat met die Vvpgip1-geen en -promotor getransformeer is. PG-inhibisie-eksperimente met membraan-geassosieerde proteïenekstrakte van geïnduseerde wingerdblare het ook dieselfde profiel getoon as dié van PGIP wat deur die Vvpgip1-geen geënkodeer is. Die uitdrukkingsprofiel van PGIP in die transgeniese tabakplante het ook bewys dat die promotor van die Vvpgip1-geen vir die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare verantwoordelik is. In silica-analise van die promotorarea dui op die teenwoordigheid van verskeie cis-werkende elemente. Die kern promotor en transkripsie-aanvangsgedeelte is gevolglik eksperimenteel bepaal. Verder het uitdrukkingseksperimente met promotorfragmente verskeie dele van die promotor geïdentifiseer wat by stimulis-geassosieerde uitdrukking betrokke is. Posisioneel is hierdie fragmente in goeie konteks met die voorspelde cis-werkende elemente en kan dus die basis vorm vir verdere studies oor Vvpgip-regulering. Met hierdie studie word die eerste data verskaf waar die regulering van PGIP deur omgewingsverwante faktore verbind kan word met onwikkelingspesifieke toestande in die plant. Verder verskaf die resultate verdere bewyse vir die rol van PGIP in plant-patogeen-interaksies en lewer spesifieke bydraes tot die onderliggende prosesse wat by die regulering van siekteweerstandverwante gene betrokke is.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / The infectious disease tuberculosis (TB) is one of the leading causes of death worldwide. The idea that infectious diseases are the most important driving force in natural selection and that they sustain frequent polymorphisms in the human genome was formally suggested by Haldane in 1949. This hypothesis implicated the human genetic component in the response to infectious disease. Today the involvement of host genetics in TB has been proven unequivocally and, together with environmental factors (e.g. nutrition and crowding) and the causative bacterium, Mycobacterium tuberculosis (M.tuberculosis), may influence the outcome of disease. As is evident, TB is a complex disease and the implication for studying genetic susceptibility is that a number of genes will be involved. Interferon gamma (IFN-7) is the major macrophage-activating cytokine during infection with M.tuberculosis and its role has been well established in animal models and in humans. This cytokine is produced by activated T helper 1 (Th1) cells. These Th1 responses can best deal with intracellular pathogens such as M.tuberculosis. We selected twelve candidate genes based on the hypothesis that genes which regulate the production of IFN-7 may influence TB susceptibility. We also selected polymorphisms from 27 other candidate genes, which may affect immunological pathways involved in TB, to investigate as susceptibility factors based on the following hypotheses: 1) granulomatous diseases can share susceptibility genes; 2) gene expression studies done by DNA-array analysis experiments may reveal TB susceptibility genes; 3) genomewide linkage studies in TB can determine susceptibility loci and genes in this region are possibly susceptibility factors; and 4) functional susceptibility polymorphisms in genes involved in immune-mediated diseases other than TB may contribute to susceptibility to TB. This research tested the association of 136 genetic polymorphisms in 39 potentially important genes with TB in the South African Coloured population. Well-designed case-control association studies were used and we attempted to replicate these findings in an independent sample set using family-based case-control designs (transmission disequilibrium tests (TDTs)). In addition, haplotypes and linkage disequilibrium (LD) in the candidate genes were also investigated. During the case-control analyses we found significant associations for 6 single nucleotide polymorphisms (SNPs) in the following genes: SH2 domain protein 1A, tolllike receptor 2, class II major histocompatibility complex transactivator, interleukin 1 receptor antagonist, runt-related transcription factor 1 and tumour necrosis factor superfamily, member 1B. Discrepant results were obtained during the TDT analyses. The number of families available was small and for this reason we cannot conclude that the case-control results were spurious. We also tested the association of haplotypes with TB. Haplotypes in the interleukin 12, beta (IL12B) and toll-like receptor 4 genes were nominally associated with TB in both the case-control and TDT analyses. We observed strong LD for the genes in the South African Coloured population. In total 17 novel SNPs were identified and one novel allele was found for a microsatellite in IL12B. This research contributes to the increasing amount of information available on genes involved in TB susceptibility, which in the future may help to predict high risk individuals.

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy