Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Wheat production is globally affected by several different wheat rust diseases. The rust diseases can effectively be controlled by the deployment of multiple resistance genes that confer durable resistance. One of the most effective strategies to incorporate resistance genes is by the implementation of recurrent mass selection as it maximizes opportunities for gene pyramiding. The implementation of a recurrent mass selection program in wheat can effectively be enhanced with the use of genetic male sterility and the incorporation of maker assisted-selection (MAS). The aim of the study was to pyramid wheat rust resistance genes in wheat lines by utilizing a male sterile mediated marker-assisted recurrent selection breeding (MS-MARS) scheme. An existing segregating MS-MARS base population and resistance donor lines carrying genes of interest (Sr26, Sr35 and Sr45) were used as female and male crossing parents. Potential markers for the genes of interest were first identified and validated on the male population. PCR based markers tested for Sr26 and Sr45 easily distinguished between resistant and non-resistant plants in the study, while markers tested for the detection of Sr35 and Sr45 in most instances failed to do so. The identified Sr26 marker (Sr26#43) was successfully added to the SU-PBL’s standardized marker set in a multiplex reaction. The standardized marker set and the co-dominant PCR marker for Sr45 were used to screen male and female populations before and after cross-pollination. Several wheat rust resistance genes were present in various frequencies in both male and female populations prior to the first crossing cycle, except Sr26 and Sr45. Increases in gene frequencies and combinations were obtained after the first crossing cycle, highlighting the effectiveness of the MS-MARS breeding strategy to improve gene frequencies of desirable genes. Two MS-MARS crossing cycles were successfully completed and large numbers of hybrid seeds were produced in a short period of time by selecting male sterile plants based on distinct characteristics induced by the dominant male sterility gene. Future studies will include the wide deployment of Sr26 and Sr45 in the MS-MARS breeding program as markers are now available and can be included in the SU-PBL’s standardized marker set for the effective detection of these genes, the development of gene-specific markers for Sr35 to ascertain the presence of the gene in the MS-MARS population and the specific selection of male sterile plants with wide open glumes to maximize outcrossing rates. / AFRIKAANSE OPSOMMING: Koring produksie word wêreldwyd aansienlik deur koringroes siektes geaffekteer. Die siektes kan doeltreffend beheer word deur die ontplooing van veelvuldige weerstandsgene, wat langdurige weerstand tot gevolg het. Een van die mees doeltreffendste strategieë om weerstandsgene in n koring plant te inkorporeer is deur die implementering van herhalende massa seleksie (HMS), siende dat dit geleenthede vir geen stapeling maksimaliseer. Die implementering van 'n HMS program in koring kan effektief aangewend word met behulp van genetiese manlike steriliteit en merker bemiddelde seleksie (MBS). Die doelwit van hierdie studie was om veelvuldige koringroes weerstandsgene in koring lyne te stapel met behulp van die manlik steriliteits merker bemiddelde herhalende seleksie (MS-MBHS)-telingsskema. ‘n Gevestigde segregerende MS-MARS basis populasie en donor lyne, wat die gene (Sr26, Sr35 en Sr45) van belang dra, was onderskeidelik as vroulike en manlike kruisingsouers gebruik. Potensiële molekulêre merkers vir die gene van belang was eers geidentifiseer in literatuur en op die donor lyne getoets, voordat dit vir die opsporing van die gene in die nageslag gebruik was. Polimerase ketting reaksie (PKR)-gebaseerde merkers wat getoets was vir Sr26 en Sr45, kon maklik tussen weerstand en nie-weerstandbiedende plante in die studie onderskei, terwyl ander merkers vir die opsporing van Sr35 en Sr45 nie so doeltreffend was nie. Die geidentifiseerde Sr26 merker was suksesvol bygevoeg tot die SU-PBL se gestandardiseerde merkerpaneel, in ‘n multipleks reaksie. Die gestandardiseerde merkerpaneel en die ko-dominante PKR merker vir Sr45 was gebruik om die manlike en vroulike populasie te analiseer vir die teenwoordigheid van verskeie weerstandsgene voor en na kruisbestuiwing. Merker analise het die teenwoordigheid van verskeie koringroes weerstandsgene in verskillende frekwensies in beide die manlike en vroulike populasie voor die eerste kruising siklus aangedui. Sr26 en Sr45 was egter afwesig in beide populasies. ‘n Toename in geen frekwensies en kombinasies was waargeneem na die eerste kruising siklus. Dit het gevolglik die doeltreffendheid van die MS-MARS teling strategie beklemtoon. Twee herhalende kruising siklusse was suksesvol voltooi en groot hoeveelhede bastersaad was verkry vanaf steriele plante wat geselekteer was op grond van unieke eienskappe wat hulle vertoon as gevolg van die manlike steriliteits geen. Toekomstige studies sluit in, die groot skaalse gebruik van Sr26 en Sr45 in die MS-MARS teelprogram aangesien merkers nou beskikbaar is en gebruik kan word in die MS-MARS teelprogram vir die doeltreffende opsporing van hierdie gene, die ontwikkeling van ‘n geen-spesifieke merker vir Sr35 om die teenwoordigheid van die geen in die MS-MARS populasie vas te stel, en die selektering van manlike steriele plante met wyd oop kaffies om kruisbestuiwing te verhoog.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The aim of this study on grapevine was to genetically characterise, validate and map the reported fungal disease resistance genes of Pölöskei Muskotály (PM), Kishmish Vatkana (KV) and Villard Blanc (VB) in South Africa using QTL analysis. These fungal resistant parents were crossed with other varieties that have desirable fruit qualities in an effort to combine fungal disease resistance with desirable fruit qualities in a single variety. The genetic basis of PM’s resistance to downy and powdery mildew has not been investigated before. It does however have VB in its pedigree so the assumption was made that the same QTL/genes present in VB contribute to this resistance. KV’s resistance to powdery mildew reportedly originates from the REN1 gene located on chromosome 13. VB’s powdery and downy mildew resistance is conferred by QTL present on chromosome 15 and chromosome 18 respectively and has been reported in numerous studies. The study populations comprised of 124 F1 PM x Regal Seedless plants, 16 F1 PM x G4-3418 plants, 14 F1 PM x Sunred Seedless plants, 158 F1 Sunred Seedless x KV plants and 250 F1 VB x G1-6604 plants. DNA was extracted from the leaves and all plants were screened using microsatellite markers. Phenotypic evaluations of downy and/or powdery mildew resistance were performed on the appropriate populations. The molecular data was used to generate linkage maps and combined with phenotypic data to perform QTL analysis. From the molecular data generated for the three PM populations it was determined that the F1 progeny inherited almost exclusively maternal alleles, and could not be used in a mapping study. These populations were eliminated from the study and PM will be used as a pollen donor in future. Molecular data from the Sunred Seedless x KV cross was used to generate a linkage map for chromosome 13 comprising eight markers and spanning 45.6 cM. When combined with the data from two powdery mildew phenotypic screens a QTL peak spanning the REN1 gene on chromosome 13 of KV was identified. This locus explains between 44.8% and 57.7% of the phenotypic variance observed. The molecular data from the VB x G1-6604 cross was used to generate partial linkage maps for chromosome 15 and 18. Eleven markers were mapped on chromosome 15 spanning 56.4 cM, and ten markers were mapped on chromosome 18 spanning 101.8 cM. When the chromosome 15 linkage map was combined with the data from two powdery mildew phenotypic screens a QTL associated with powdery mildew resistance was identified on chromosome 15 that explains between 18.9% and 23.9% of the phenotypic variance observed. Likewise a QTL associated with downy mildew resistance was identified on chromosome 18 when the chromosome 18 linkage map was combined with data from two downy mildew phenotypic screens. This QTL explains between 19.1% and 21.2% of the phenotypic variance observed. This study succeeded in genetically characterising the fungal disease resistance genes of two different sources of grapevine and provided exclusionary information on a third resistance source for future breeding applications. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie in wingerd was om die genetiese komponent van die swamweerstandsgene van Pölöskei Muskotály (PM), Kishmish Vatkana (KV) and Villard Blanc (VB) in Suid-Afrika te karakteriseer en die teenwoordigheid daarvan te bevestig deur ʼn Kwantitatiewe Eienskap Lokus (KEL) benadering te volg. In ʼn poging om swamweerstand en goeie vrugeienskappe te kombineer in ʼn enkel variëteit is die weerstandige variëteite met vatbare variëteite gekruis wat goeie vrugeienskappe besit. Die genetiese basis van PM se weerstand teen donsskimmel en witroes is nog nie vantevore bestudeer nie. VB is een van sy voorgeslagte en daar is aangeneem dat dieselfde KEL/gene waarskynlik verantwoordelik is vir die weerstand. Dit is gerapporteer dat KV se witroesweerstand afkomstig is van die REN1 geen op chromosoom 13. Vele publikasies rapporteer VB se weerstand teen witroes en donsskimmel Beide die witroes- en donsskimmelweerstand word oorgedra deur KEL teenwoordig op chromosome 15 en 18 onderskeidelik. Die populasies gebruik in hierdie studie het bestaan uit 124 F1 PM x Regal Seedless plante, 16 F1 PM x G4-3418 plante, 14 F1 PM x Sunred Seedless, 158 F1 Sunred Seedless x KV plante en 250 F1 VB x G1-6604 plante onderskeidelik. Blare is versamel vir DNS isolasie en genotipering met mikrosatellietmerkers. Al drie populasies se weerstand teen donsskimmel en/of witroes is fenotipies geëvalueer. Die molekulêre data is gebruik om genetiese koppelingskaarte op te stel en gekombineer met die fenotipiese data om KEL analise uit te voer. Die molekulêre data van die drie PM populasies het daarop gedui dat die F1 nageslag amper uitsluitlik moederlike allele geërf het en kon gevolglik nie gebruik word in die studie nie. Die PM populasies is uitgesluit uit hierdie studie en PM sal voortaan as stuifmeelskenker gebruik word. Molekulêre data van die Sunred Seedless x KV kruising is gebruik om ʼn koppelingskaart vir chromosoom 13 op te stel wat 45.6 cM lank is en agt merkers bevat. Die KEL analise van die koppelingskaart en twee fenotipiese datastelle vir witroes het ʼn KEL piek geïdentifiseer wat oor die lengte van die REN1 geen-interval strek. Hierdie lokus is verantwoordelik vir 44.8% tot 57.7% van die fenotipiese variasie wat waargeneem word. Molekulêre data van die VB x G1-6604 kruising is gebruik om gedeeltelike koppelingskaarte vir chromosome 15 en 18 op te stel. Elf merkers karteer op die chromosoom 15 kaart van 56.4 cM en tien merkers karteer op die chromosoom 18 kaart van 101.8 cM. KEL analise van chromosoom 15 se koppelingskaart en twee witroes fenotipiese datastelle het ʼn KEL geïdentifiseer wat 18.9% tot 23.9% van die fenotipiese variasie verduidelik. ʼn KEL is ook op chromosoom 18 geïdentifiseer wat 19.1% tot 21.2% van die fenotipiese variasie verduidelik met die gekombineerde analise van chromosoom 18 se koppelingskaart en twee donsskimmel fenotipiese datastelle. Hierdie studie het die genetiese komponent van die swamweerstandsgene van twee Vitis variëteite suksesvol gekarakteriseer en bevestig. Waardevolle telingsinligting oor die derde variëteit is ook onthul.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: With knowledge of the human genome increasing constantly we are continually faced with new and potentially groundbreaking methods for managing, treating and/or identifying diseases and predisposition to diseases and conditions at a genetic level. The human cytochrome P450 (CYP) super-family of genes code for enzymes that can participate in metabolism of drugs and foreign chemicals and in steroid synthesis and metabolism. Mutations in these genes may contribute to clinically relevant diseases. In this study, the effects of mutations within four CYP genes were evaluated in two South African disease groups - variegate porphyria and breast cancer. Variegate porphyria (VP) has an unusually high incidence in South Africa due to the R59W founder mutation in the protoporphyrinogen oxidase (PPOX) gene that causes a disruption in the haem biosynthetic pathway. VP presents with variable clinical symptoms and has a relatively low penetrance. It is expected that environmental factors and modifier genes play a role in the clinical expression of VP. CYP genes are implicated as candidate modifier genes for the expression of VP due to the function they have in metabolising many drugs contraindicated in porphyria patients, and the necessity of haem binding to the apoprotein to produce a functional CYP enzyme. This is the first study to investigate CYPs as possible modifier genes for VP clinical expression. Six CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 - 734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4), associated with four CYP loci, were genotyped in a VP population and a suitable control population. The results observed are suggestive of CYPIAlml and CYPIBI playing a role as modifiers for the clinical expression of VP as they were significantly associated (P<O.05) with the presence ofVP related symptoms. Breast cancer is one of the leading causes of death in women due to cancer. It is believed that breast cancer may be the result of co-participation between common DNA alterations and environmental exposures. Polymorphisms in enzymes involved with the metabolism of environmental exposures, such as the cytochrome P450 enzymes, are therefore expected to play an aetiological role in breast cancer. Two groups of South African breast cancer patients (Caucasian and of mixed ancestry) and population-matched controls were genotyped for four CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A and CYPIBI 8372 A>C). This represents the first investigation of the potential role of CYPs as breast cancer risk modifiers in the two South African populations. Significant differences were observed (P<O.003) in genotype and allele frequencies between the breast cancer patients and controls for the CYPIBI 8372 A>C polymorphism in the population of mixed ancestry. Vast differences in allele frequencies were also observed between the two groups of breast cancer populations. These results emphasize the importance of population-based risk assessment when genetic testing and counselling for complex disease susceptibility is offered. The results of this study provide the first evidence suggesting a role for CYPs in modifying the clinical expression of VP and in acting as risk factors for developing breast cancer in a South African population. / AFRIKAANSE OPSOMMING: Met die konstante toename van kennis oor die mensgenoom kom ons voortdurend te staan voor nuwe metodes vir die beheer, behandeling en/of identifikasie van siektes en vatbaarheid vir siektes op 'n genetiese vlak. Die mens sitochroom P450 geensuperfamilie kodeer vir ensieme betrokke in die metabolisme van medisyne en ander chemiese stowwe en steroïed-sintese en -metabolisme. Mutasies in hierdie gene kan 'n bydrae lewer tot kliniese relevante siektes. In hierdie studie is die effek van mutasies in vier sitochroom gene bestudeer in twee Suid-Afrikaanse siekte groepe, variegate porfirie en borskanker. Variegate porfirie (VP) het 'n besonderse hoë frekwensie in Suid-Afrika as gevolg van die R59W stigter-mutasie in die protoporfirinogeen oksidase (PPOX) geen. Hierdie mutasie lei tot 'n versteuring in die heem biosintese padweg. VP presenteer met variërende kliniese simptome en het 'n betreklike lae penetrasie. Daar word vermoed dat omgewingsfaktore en kandidaat modifiserende gene 'n rol speel in die kliniese beeld van VP. Sitochroom P450 gene is geïdentifiseer as kandidaat modifiserende gene as gevolg van hulle rol in die metabolisme van verbode medikasie vir porfirie pasiënte, asook die binding van heem aan die apoproteïen wat noodsaaklik is vir die produksie van funksionele sitochroom P450 ensiem. Hierdie is die eerste studie wat sitochroom P450 gene as moontlike modifiserende gene vir die kliniese uitdrukking van VP ondersoek. Ses sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4) is ondersoek in beide 'n VP populasie en 'n geskikte kontrole populasie. Die resultate suggereer 'n rol vir CYPIAlml en CYPIBI in die modifisering van die kliniese uitdrukking van VP aangesien hulle betekenisvolle assosiasie (P<O.05) met die voorkoms van VP-verwante simptome getoon het. Borskanker IS een van die vernaamste oorsake van kankersterftes onder vroue. Borskanker IS waarskynlik die resultaat van interaksie tussen algemene DNSveranderinge en omgewings-blootstelling. Daar word dus verwag dat polimorfismes in ensieme betrokke by die metabolisme van omgewingselemente, soos byvoorbeeld die sitochroom P450 ensieme, 'n etiolgiese rol in borskanker sal speel. Die genotipes van twee groepe Suid-Afrikaanse borskanker lyers (Kaukasiërs en van gemengde herkoms) en populasie-passende kontroles is bepaal vir vier sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C). Hierdie studie verteenwordig die eerste ondersoek na die potensiële rol van sitochroom P450s as risiko-modifiserende faktore vir borskanker in die twee populasies. Betekenisvolle verskille (P<O.003) in genotipe en allele frekwensies is waargeneem tussen die borskanker lyers en kontroles vir die CYPIBI 8372 A>C polimorfisme in die gemengde herkoms populasie. Beduidende verskille in alleel frekwensies is ook waargeneem tussen die twee borskanker populasies. Hierdie resultate beklemtoon die belangrikheid van populasie gebaseerde risiko-beraming wanneer genetiese toetse en voorligting vir komplekse siekte-vatbaarheid aangebied word. Die resultate van hierdie studie bied die eerste getuienis dat sitochroom P450s 'n rol kan speel in die modifisering van die kliniese beeld van VP en ook kan optree as as risiko faktore vir die ontwikkeling van borskanker in 'n Suid-Afrikaanse populasie.

Dissertation (PhD (Agric))--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: A breeding programme aimed at the genetic improvement of growth rate of rainbow trout was initiated in 1988 by the Department of Genetics, University of Stellenbosch, in collaboration with the local trout producer's organisations. The first phase of the breeding programme included the collection, evaluation and selection of the best available genetic material from 13 different genetic groups (nine local and four overseas) to make up two separate base populations as odd and even year-groups. This was done to establishment a base population with high genetic merit and variation at the onset of the breeding programme. Statistically significant and commercially valuable genetic differences in terms of weight and length gain were detected between the various hatchery groups. The next two generations of the breeding program included a series of single and double crosses in order to increase the levels of genetic variation in the base populations, and to investigate possible heterosis and specific and general combining ability among the crosses. Significant levels of heterosis (6.7% to 9.6%) and general combining ability was found for weight and length gain during consecutive growth stages. No evidence was found for specific combining ability among the crosses. The crossing of selected offspring from the original genetic groups followed by the application of intensive multi-stage selection for growth rate within progeny groups has led to the establishment of second and third generation parental populations with higher levels of genetic variation and improved individual genetic merit with regard to growth rate. The exploitation of non-additive genetic variation within the base populations through crossbreeding and heterosis during the early stages of the selection programme was delayed in favour of the utilization of additive genetic variance through a procedure of multi-stage selection that incorporated high intensities of selection within and between family groups. The estimation of genetic parameters during the fourth generation on the basis of a hierarchical half-sib family structure confirmed the presence of high levels of additive genetic variation within the respective populations/year-groups. High heritability values in the range of 0.40 to 0.53 were recorded for body weight and length at 150 days. Genetic correlations between the traits were also high, in the range of 0.74 to 0.82. The cumulative realized response of 50% in body length for the EVEN year-group after six generations of selection (8.3% per generation), and the 33% for the ODD year-group after five generations of selection (6.6% per generation) confirms the efficiency of the multi-stage selection procedure to exploit the available additive genetic variation for growth rate within the respective populations. The programme is still ongoing, entering its 7th generation in 2004 and is supplying about 50-60% of commercial material through direct supplies of broodstock, ova and fingerlings and indirect supplies via multiplier stations (commercial hatcheries). The programme was the first of its kind in relation to aquaculture species in the Southern African region, and has since initiated the introduction of programmes of genetic improvement in three other indigenous species, namely tilapia (Oreochromis mossambicus), African catfish (Clarias gariepinus) and abalone (Haliotis midae). / AFRIKAANSE OPSOMMING: ‘n Teelprogram gerig op die verbetering van groeitempo in reënboogforel is in 1988 ingestel onder toesig van die Departement Genetika aan die Universiteit van Stellenbosch, in sameweking met die plaaslike forelprodusenteverenigings. Die eerste fase van die teelprogram behels die versameling, evalasie en seleksie van die beste beskikbare genetiese materiaal vanuit, 13 verskillende genetiese groepe (nege plaaslike en vier van oorsee) om twee basispopulasies te ontwikkel in elk van die gelyke en ongelyke jaargange. Die doel daarvan was om ’n basispopulasie met hoë genetiese meriete en variasie te ontwikkel met die aanvang van die teelprogram gerig op genetiese verbetering, deur middel van seleksie. Statisties betekenisvolle en ekonomies belangrike genetiese verskille in massa- en lengtetoename is aangetref, tussen die onderskeie genetiese groepe. Die daaropvolgende twee generasies binne die teelprogram behels die uitvoering van ’n reeks enkel- en dubbelkruisings ten einde ’n verdere toename in genetiese variasie in die basispopulasies te bewerkstellig, sowel as om die voorkoms van heterose en algemene, sowel as spesifieke kombinerings-vermoë tussen die kruisings te bepaal. Betekenisvolle vlakke van heterose (6.7% tot 9.6%) sowel as algemene kombineringsvermoë, is aangetref ten opsigte van massa- en lengtetoename in opeenvolgende groeifases. Daar kon geen aanduiding van betekenisvolle, spesifieke kombineringsvermoë gevind word nie. Die kruising van geselekteerde nageslag vanuit die oorspronklike genetiese groepe, gevolg deur ‘n multi-fase seleksiemetode vir groeitempo binne nageslaggroepe, het bygedra tot die ontwikkeling van ‘n tweede en derde generasie broeipopulasie wat beskik oor hoër vlakke van genetiese variasie en verbeterde individuele meriete ten opsigte van groeitempo. Die benutting van nie-additatiewe genetiese variasie binne die basispopulasies deur middel van kruisteling en heterose tydens die vroee stadium van die teelprogram is uitgestel ten gunste van die benutting van additatiewe genetiese variasie deur middel van ‘n multi-fase seleksiemetode, wat berus het op die toepassing van hoë vlakke van seleksie-intensteit binne en tussen familiegroepe. Die beraming van genetiese parameters tydens die vierde generasie het die voorkoms van hoe vlakke van additatiewe variasie binne die onderskeie jaargroepe bevestig. Hoë oorerflikhede van 0.40 tot 0.53 is beraam vir ligaamsmassa en -lengte op die ouderdom van 150 dae. Genetiese korrelasies tussen die kenmerke was ook hoog met waardes van 0.74 tot 0.82. Die saamgestelde gerealiseerde seleksierespons van 50% vir liggaamslengte vir die “EVEN”-jaargroep na afloop van ses generasies van seleksie (8.3% per generasie) en die 33% van die “ODD”-jaargroep na afloop van vyf generasies van seleksie (6.6% per generasie) het die doeltreffendheid van die multi-fase seleksiemetode bevestig ten opsigte van die benutting van die additatiewe variasie vir groeitempo binne die onderskeie basispopulasies/jaargroepe. Die teelprogram duur steeds voort en sal die 7de generasie in 2004 bereik. Die program voorsien nagenoeg 50-60% van die kommersiele materiaal vanuit direkte voorsiening van teelmaterial, eiers en vingerlinge asook die indirekte voorsiening via kommersiële teelstasies. Die teelprogram was die eerste van sy soort met betrekking tot akwakultuurspesies in Suider Afrika en het bygedra tot die implimentering van programme van genetiese verbetering in drie inheemse spesies, naamlik die tilapia (Oreochromis mossambicus), die baber (Clarias gariepinus) en die perlemoen (Haliotis midae).

Thesis (PhD (Agric)) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Stripe (yellow) rust of wheat, caused by Puccinia striiformis f.sp. tritici, was first detected as a single introduction into South Africa in 1996. Two additional pathotypes have since been identified. Control of the disease may be achieved by use of genetic adult plant resistance (APR) as is present in the local cultivar 'Kariega'. The aim of this project was to understand the genetic basis of the APR in 'Kariega' to facilitate breeding of new varieties with genetic resistance to stripe rust. A partial linkage map of a 'Kariega X Avocet S' doubled haploid population covering all 21 wheat chromosomes was generated using 208 DNA markers, viz, 62 SSR, 133 AFLP, 3 RGA and 10 SRAP markers, and 4 alternative loci. The different marker techniques detected varying polymorphism, viz, overall SSR: 46%, AFLP: 7%, SRAP: 6% and RGA: 9%, and the markers produced low levels of missing data (4%) and segregation distortion (5%). A significant feature of the linkage map was the low polymorphism found in the D genome, viz, 19% of all mapped DNA markers, 11% of all AFLP markers and 30% of the total genome map distance. A region exhibiting significant segregation distortion was mapped to chromosome 4A and a seedling resistance gene for stem rust (Puccinia graminis f.sp . tritici), Sr26, mapped to chromosome 6A close to three SSR markers. The leaf tip necrosis gene, Ltn, which was also segregating in the population, mapped to chromosome 7D. Protocols for SRAP and RGA were optimised, and SRAP marker use in wheat genetic linkage studies is reported for the first time. The linkage map was used together with growth chamber and replicated field disease scores for QTL mapping. Chromosomes showing statistically significant QTL effects were then targeted with supplementary SSR markers for higher resolution mapping. The quality of disease resistance phenotypic data was confirmed by correlation analysis between the different scorers for reaction type (0.799±0.023) and for transformed percentage leaf area infected (0.942±0.007). Major QTL were consistently identified on chromosome 7D (explaining some 25-48% of the variation) and on chromosome 2B (21-46%) using transformed percentage leaf area infected and transformed reaction type scores (early and final) with interval mapping and modified interval mapping techniques. Both chromosomal regions have previously been identified in other studies and the 7D QTL is thought likely to be the previously mapped APR gene Yr 18. Minor QTL were identified on chromosomes lA and 4A with the QTL on 4A being more prominent at the early field scoring for both score types. A QTL evidently originating from 'Avocet S' was detected under growth chamber conditions but was not detected in the field, suggesting genotype-environment interaction and highlighting the need for modifications of growth chamber conditions to better simulate conditions in the field. The genetic basis of the APR to stripe rust exhibited by 'Kariega' was established by mapping of QTL controlling this trait. The linkage map constructed will be a valuable resource for future genetic studies and provides a facility for mapping other polymorphic traits in the parents of this population with a considerable saving in costs. / AFRIKAANSE OPSOMMING: Streep of geelroes van koring word veroorsaak deur Puccinia striiformis f. sp tritici, en is die eerste keer in 1996 in Suid-Afrika na introduksie van 'n enkele patotipe waargeneem. Twee verdere patotipes is sedertdien in Suid-Afrika gei"dentifiseer. Beheer van die siekte word veral moontlik gemaak deur die gebruik van genetiese volwasseplantweerstand soos gei"dentifiseer in die plaaslike kultivar 'Kariega'. Die doel van hierdie studie was om die genetiese grondslag van die streeproesweerstand te ontrafel ten einde die teling van nuwe bestande kultivars moontlik te maak. 'n Verdubbelde haplo1ede populasie uit die kruising 'Kariega X Avocet S' is aangewend om 'n gedeeltelike koppelingskaart vir die volle stel van 21 koring chromosome saam te stel. Die kaart het uit 208 DNA merkers, nl., 62 SSR, 133 AFLP, 3 RGA, 10 SRAP merkers en 4 ander lokusse bestaan. Totale polimorfisme wat deur die verskillende merkersisteme opgespoor is, was as volg: SSR: 46%, RGA: 9%, AFLP: 7% en SRAP: 6%. Die mate van ontbrekende data was gering (4%) asook die mate van segregasie distorsie (5%) van 'n enkele geval wat op chromosoom 4A gekarteer is. 'n Prominente kenmerk van die koppelingskaart is die relatiewe gebrek aan polimorfiese merkers op die D-genoom, nl., slegs 19% van alle DNA merkers en 11% van alle AFLP merkers wat slegs 30% van die totale genoom kaartafstand bestaan het. Die stamroes (Puccinia graminis f. sp. tritici) saailingweerstandsgeen, Sr26, karteer op chromosoom 6A naby drie SSR merkers. Die geen vir blaartipnekrose, Ltn, karteer op chromosoom 7D. Protokolle vir SRAP en RGA merkers is ge-optimiseer en gebruik van SRAP merkers in koppelings-analise word vir die eerste keer in koring gerapporteer. Die koppelingskaart is in kombinasie met groeikamerdata en gerepliseerde veldproefdata gebruik om die gene (QTL) vir volwasseplant streeproesweerstand te karteer. Chromosome met statisties betekenisvolle QTL is met aanvullende SSR merkers geteiken om die resolusie van kartering verder te verhoog. Die kwaliteit van fenotipiese data, soos in die proewe aangeteken, is bevestig deur korrelasies te bereken tussen lesings geneem deur onafhanklike plantpataloe (0.799 ± 0.023 vir reaksietipe en 0.942 ± 0.007 vir getransformeerde persentasie blaaroppervlakte besmet). Hoofeffek QTL vir die twee maatstawwe van weerstand is deur middel van die metodes van interval QTL kartering en gemodifiseerde interval QTL kartering konsekwent op chromosome 7D (25-48% van variasie verklaar) en 2B (21-46% van variasie verklaar) ge"identifiseer. In vorige studies is aangetoon dat beide chromosome 7D en 2B QTL vir volwasseplant streeproesweerstand dra. Die 7D QTL is waarskynlik die weerstandsgeen, Yr 18. QTL met klein effekte op weerstand is op chromosome lA en 4A ge"identifiseer. Die effek van laasgenoemde geen was meer prominent in die velddata in die vroee datum van weerstandsbeoordeling. Een QTL, afkomstig van 'Avocet S', is slegs onder groeikamertoestande identifiseerbaar. Dit dui op moontlike genotipe-omgewing wisselwerking en beklemtoon die noodsaaklikheid om aanpassings te maak in groeikamertoestande vir beter simulasie van veldproeftoestande. Die genetiese grondslag van volwasseplantweerstand teen streeproes in die kultivar 'Kariega' is deur QTL kartering bepaal. Die 'Kariega X Avocet S' koppelingskaart kan as 'n waardevolle basis dien vir toekomstige genetiese ontledings van ander polimorfiese kenmerke in die populasie.

Thesis (PhD (Genetics))--University of Stellenbosch, 2011. / Includes bibliography / ENGLISH ABSTRACT: Grapevine diseases, in particular virus and virus-like diseases, are threatening grapevine industries worldwide; also in South Africa. Grapevine leafroll (GLR) is one of the most important diseases of grapevines, occurring in all grape-producing countries worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is known to be closely associated with GLR disease and occurs commonly in South African vineyards. In this study three genetic variants of GLRaV-3 were identified in vineyards of the Western Cape, South Africaby single strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. A specific SSCP profile could be assigned to each variant group and these wereconfirmed by sequencing of the ORF5 regions.These results demonstrated that SSCP analysis on this region in ORF5 provides a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5’ ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5’UTRs indicated significant sequence and length variation in this region, between the three South African variant groups. Nucleotide sequence alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three GLRaV-3 variants in South Africa, and that two or three additional variant groups occurs elsewhere in the world. We further investigated the prevalence of these three GLRaV-3 variants in mother blocksof different cultivars and from different vine growing regions, using SSCP analysis. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of the three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows. Some plants in the cluster were also infected with variants not present in the original plant. These infections could have been caused by mealybug vectors feeding on plants from surrounding areas and then infecting these plants. The scientific information generated on GLRaV-3 variants in this project contributed to the advancement of our knowledge of genetic variability and provides a basis of further epidemiology and vector-virus studies. The study showed for the first time that different GLRaV-3 variants were transmitted to adjacent plants in a row and across rows in a GLR disease cluster. The diversity detected in the 5’UTR between variants from the three genetic groups provides a platform for the further study of the biological characteristics of GLRaV-3 variants. / AFRIKAANSE OPSOMMING: Wingerdsiektes, veral virus siektes, bedreig wingerd industrieë wêreldwyd, asook die Suid Afrikaanse wingerdbedryf. Rolbladsiekte is een van die belangrikste siektes op wingerd en kom wêreldwyd voor. Die virus, grapevine leafroll-associated virus 3 (GLRaV-3), word sterk geassosieer met Rolbladsiekte en kom wydverspreid voor in Suid Afrikaanse wingerde. Tydens hierdie studie is drie genetiese variante van GLRaV-3 geïdentifiseer in wingerd moederblokke in die Wes-Kaap. Die GLRaV-3 variante is geïdentifiseer met ‘n tegniek wat ‘single-strand conformation polymorphism (SSCP)’ genoem word. Die SSCP profiele was gegenereer vanaf PKR produkte van die ORF5 area op die genoom van GLRaV-3. Die geamplifiseerde produk van die ORF5 gebied is gebruik om die SSCP profiele te verkry en DNA-volgorde data in die gebied het die drie SSCP profiele gestaaf. Hierdie metode om virus variasie te bestudeer in plante is vinnig en betroubare resultate is verkry. Gemengde infeksies, wat gereeld in wingerd voorkom, kon ook met die tegniek opgespoor word. Die volledige nukleotied-volgorde van elkeen van die drie GLRaV-3 genome is volledig bepaal. Die isolate wat die drie variant groepe verteenwoordig is isolaat 621 (groep I), 623 (groep II) en PL-20 (groep III). Die nukleotiedvolgorde in die 5’UTR is bepaal met die RLM-RACE tegniek. Wanneer die 5’UTRs van die drie variante vergelyk is, het dit getoon dat daar verskille is in die volgordes en lengtes voorgekom het. Ander dele van die genoom, o.a. die dopproteïen (CP) en Hsp70 areas, is filogeneties vergelyk met isolate van regoor die wêreld. In die filogenetiese analise is bevind dat die drie GLRaV-3 variante saamgegroepeer het met ander isolate in die wêreld en dat daar elders ook twee to drie addisionele variant groepe van GLRaV-3 voorkom. Die verspreiding van die drie GLRaV-3 variante in wingerde is bestudeer in verskillende kultivars en in verskillende verbouingsgebiede. Die meerderheid van die plante in die studie was geïnfekteer met die groep II variant wat dieselfde is as isolate 623 en GP18. Die voorkoms van die drie variante in ‘n siekte cluster is bestudeer d.m.v SSCP. Die studie het gewys dat verskillende GLRaV-3 variante versprei word na aangrensende plante in ‘n ry en tussen rye. In sommige gevalle is die variant wat in die oorspronklik geïnfekteerde plant voorkom, oorgedra na naasliggende plante. Sommige van die plante in the infeksie area was ook met ander GLRaV-3 variante geïnfekteer wat moontlik deur wolluise oorgedra is vanaf naburige geïnfekteerde plante. Die wetenskaplike inligting wat tydens hierdie studie beskryf word aangaande die identifikasie van GLRaV-3 variante, dra by tot die molekulêre kennis van GLRaV-3 en verskaf ‘n basis vir verdure epidemiologiese -en insek oordragingstudies. Die studie het vir die eerste keer bewys dat verskillende GLRaV-3 variante na aanliggende plante in ‘n ry asook oor rye oorgedra word. Die diversiteit tussen die GLRaV-3 variant groepe in die 5’UTR moet verder ondersoek word en die deel van die genoom kan ‘n belangrike rol speel in die biologiese eienskappe van die variante.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance. / AFRIKAANSE OPSOMMING: Die Russiese-koringluis (RWA) is ‘n pes wat ‘n belangrike ekonomiese invloed op koring opbrengste het en infestasie kan tot grootskaalse skade en oes verlies lei. Verskeie studies, onder andere komplimentêre DNA amplifiseerde fragment polimorfismes (cDNA-AFLPs), onderdrukkende onderskeidende hibridisaie (SSH) en mikro-reekse wat voorheen op transkriptomiese vlak gedoen is, het moontlike gene wat by RWA weerstand betrokke is, geïdentifiseer. Alhoewel hierdie gene reeds geidentifiseer was, hulle rol is nogtans onbekend. Dié gene moet nog getoets word, duur funksionele genetiese benaderingste maak. In hierdie studie is ‘n gars streep mosaïek virus vektor (BSMV) gebruik om kandidaat-gene van belang in ‘n Dn1-weerstandige geen-bevattende kultivar (TugelaDN) te onderdruk. Ondrukking van gene het deur middel van virus geïnduseerde geen onderdrukking (VIGS) plaasgevind. In hierdie studie is die hipotese gestel dat die gene betrokke by die hipersensitiewe reaksie (HR) ‘n invloed op plantweerstand kan hê en is dus geteiken vir geen-onderdrukking-studies. Hierdie gene het die volgende ingesluit: glutatioon-S-transferase (GST), superoksied dismutase Cu/Zn (SOD) en askorbien peroksidase (APX). Egter, omdat luisinfestasie ook tot verwonding aanleiding gee, is die onderdrukte gene ook onder alleenlik verwondingstoestande getoets. Luis vrugbaarheid is gebruik as indikator van betrokkenheid omdat meer vatbare plante ‘n hoër luis vrugbaarheid tot gevolg het. In die studie is gevind dat onder alleenlik verwondingkondisies, plante wat Dn1 bevat, ‘n groter hipersensitiewe respons vertoon, as vatbare kontroles. Daar is verder gevind dat askorbien peroksidase ‘n belangrike rol tydens verwondings-geïnduseerde weerstand in Dn1-plante speel. Daar is verder bevind dat die onderdrukking van superoksied dismutase Cu/Zn (SOD) en ‘n tilakoïed-geassosïeerde askorbien peroksidase (tAPX). Onder luis-infestasie kondisies, geen effek op luisvrugbaarheid gehad het nie en dus nie direk by die weerstandsrespons betrokke is nie. Die onderdrukking van ‘n phi-klas glutatioon-S-transferase F6 (TaGSTF6) het egter ‘n groot invloed op luis-vrugbaarheid gehad en kan dus ‘n rol in Dn1-weerstand speel. Die moontlike weerstands gene, geïdentifiseer as nukleotied bindings proteïen (NBP) en weestandsgeen anoloog 2 (T10rga2-1D), is getoets onder luis-infestasie kondisies. Die analise van NBP het getoon dat dit ‘n integrale deel van die yster homeostase meganisme in die sitosol, wat vir Fe-kluster samestelling verantwoordelik is, vorm. Onderdrukking van beide die NBP en T10rga2-1D het tot die uitdrukking van ‘n vatbare fenotipe aanleiding gegee. T10rga2-1A is ‘n NBS-LRR proteïen wat bekend is om noodsaaklik te wees tydens roes weerstandigheid in teenwoordigheid van die weerstandsgeen Lr10. T10rga2-1D-onderdrukte behandelings het tot vatbaarheid aangeiding gegee en daartoe gelei dat plante na luis-infestasies doodgaan. Hierdie resultate dui dus ‘n rol vir T10rga2-1D in Dn1-weerstandigheid aan, en suggereer verder dat hierdie geen ‘n goeie stroom-op kandidaat in Dn1-weerstandigheid is.

Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”. / AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / The Russian wheat aphid, Diuraphis noxia (Mordvilko), is a serious insect pest of wheat and barley. It affects the quality and yield of grain by sucking plant sap from the newest growth whilst toxic substances are injected that destroy plant tissue. The Russian wheat aphid also acts as a vector of plant viruses. The cultivation of aphid resistant cultivars is the preferred control strategy and nine resistance genes, designated Dn1 to Dn9, have been identified. Another undesignated gene, Dnx, was found in the wheat accession PI220127. Mapping of the resistance genes relative to known markers will improve their use in breeding programs. The dominant RWA resistance gene, Dn5, was identified in the accession PI294994 and mapped to chromosome arm 7DL. However, recent reports have placed Dn5 on ...

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The indigenous abalone, Haliotis midae has been a successfully cultured aquaculture species in South Africa since 1990. It has a slow growth rate and takes from two to five years to reach market size. Like for most other commercially important abalone species, the slow growth rate of H. midae is a cause of concern with regard to the profitability of farming and global competitiveness of the species. Ploidy manipulation of the maternal genome, a universally growing practice in shellfish culture, is considered a promising method to improve the growth rate of abalone - a desirable trait in aquaculture organisms from a commercial perspective. This manipulation technique is employed to achieve sterility, which results in limited gonad development. The consequent re-allocation of resources to somatic growth results in improved growth. The purpose of this study was to establish a viable method for the induction and validation of triploidy, on a commercial scale, in the South African abalone, H. midae. The focus was on hydrostatic pressure as a method of induction and flow cytometry as the method of validation. The results obtained confirm hydrostatic pressure as an effective method for the induction of triploidy in H. midae, delivering high percentages of triploidy (>80%) over a wide range of pressures and times, in 48 hour-old larvae. Hydrostatic pressure had a negative effect on survival in 20 hour-old larvae. Flow cytometry was validated as a reliable, fast and accurate, though expensive, method for identification of triploidy in H. midae. As an outcome of this study a manual of “Procedures for the Induction and Validation of Triploidy in the abalone” is presented (Appendix 1) together with recommendations for further studies on triploidy in the South African abalone, H. midae. / AFRIKAANSE OPSOMMING: Die inheemse perlemoen, Haliotis midae, is sedert 1990 ‘n suksesvol gekweekte akwakultuur spesie in Suid-Afrika. ‘n Kenmerk van die spesie is die stadige groeitempo van tussen twee en vyf jaar ten einde bemarkbare grootte te bereik. Soos vir die meerderheid perlemoen van kommersiële belang, is hierdie stadige groeitempo rede tot kommer met betrekking tot die winsgewende kweek en wêreldwye mededingendheid van die spesie. Die manipulasie van ploïdie van die moederlike genoom is ‘n toenemende praktyk in skulpvisboerdery en word gereken as ‘n belowende metode om die groeitempo van perlemoen te verbeter. Hierdie manipulasietegniek word gebruik om steriliteit te verkry wat manifesteer as onderdrukte ontwikkeling van die geslagsklier. Die gevolg is die herkanalisering van bronne na somatiese groei. Die doel van hierdie studie was om ‘n lewensvatbare metode vir die induksie van triploïdie op ‘n kommersiële skaal in die Suid-Afrikaanse perlemoen, H. midae, te vestig. Daar is op hidrostatiese druk as metode vir die induksie en vloei-sitometrie as metode vir die geldigverklaring van triploïdie gefokus. Die resultate van hierdie studie bevestig dat hidrostatiese druk ‘n effektiewe metode vir die induksie van triploïdie in H. midae is. Hoë persentasies van triploïdie (>80%) is oor ‘n wye reeks van drukke en tye in 48 uur oue larwes verkry. Daar is gevind dat hidrostatiese drukbehandeling ‘n negatiewe effek op die oorlewing van 20 uur oue larwes het. Vloei-sitometrie is bevestig as ‘n betroubare, vinnig en akkurate, maar duur metode vir die identifikasie van triploïdie in H. midae. As ‘n uitvloeisel van die studie word ‘n handleiding “Procedures for the Induction and Validation of Triploidy in the abalone” (Appendix 1) aangebied tesame met aanbevelings vir verdere studies rakende triploïdie in die Suid-Afrikaanse perlemoen, H. midae.