Thesis (MSc.)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The detrimental effect of soil salinity on crop production is a growmg problem worldwide (Tanji, 1990b). The degree to which plants can tolerate high concentrations of salt in their rooting medium is under genetic control with different genetic and physiological mechanisms contributing to salt tolerance at different developmental stages (Epstein & Rains, 1987). Only limited variation exists for salt tolerance in the cultivated cereals. This has prompted attempts to select tolerant progeny following hybridisation of cultivated species and wild, salt-tolerant species. Thinopyrum distichum, an indigenous wheatgrass that is naturally adapted to saline environments (McGuire & Dvorak, 1981), was crossed with triticale (x Triticosecale) in an attempt to transfer its salt tolerance and other hardiness characteristics (Marais & Marais, 1998). The aims of this study were to (i) identify Thinopyrum chromosomes carrying genes for salt tolerance and to identify molecular markers for these chromosomes, (ii) identify a number of diverse monosomic and disomie addition plants. Bulked segregant analysis (BSA), in combination with AFLP, RAPD and DAF marker analysis was implemented to screen for polymorphisms associated with salt tolerance. Five putative AFLP markers and two RAPD markers were detected using bulks composed of salt tolerant plants and bulks composed of salt sensitive plants. The distribution of the markers in these bulks suggests that more than one Thinopyrum chromosome carry genes for salt tolerance. Salt tolerant monosomic and disomie addition plants were characterised for AFLP, RAPD and DAF polymorphisms in an attempt to find markers associated with the chromosome(s) conditioning salt tolerance. One salt tolerant monosomic and one disomie addition plant was identified. One AFLP and two RAPD markers were identified for the Thinopyrum chromosome( s) present in the monosomic addition plant, while three AFLP and three RAPD markers were identified for the disomie addition plant. An attempt was also made to identify diverse chromosome addition plants having complete or near complete triticale genomes plus an additional random Thinopyrum chromosome. Plants with 2n = 43 /44 were identified and characterised for molecular markers (AFLP and RAPD). Cluster analysis was used to group the putative monosomic or disomie addition plants according to the specific Thinopyrum chromosomes they retained. Seventeen AFLP and RAPD markers could be used to group the 24 putative addition plants into six broadly similar groups with different additional Thinopyrum chromosomes. While the members of each group are likely to carry the same additional Thinopyrum chromosomes, this may not necessarily be the case as the interpretation of the marker results is complicated by heterogeneity among plants with regard to the triticale background chromosomes they possess. It is also likely that chromosome translocations occurred during backerossing which may further complicate data. Nonetheless, it is now possible to select disomie addition plants from each group that are likely to represent different Thinopyrum chromosomes. The data will also be useful in future attempts to find further addition plants carrying the remaining Thinopyrum chromosomes. / AFRIKAANSE OPSOMMING: Die skadelike effek van grond versouting op gewasproduksie neem wêreldwyd toe (Tanji, 1990b). Die mate waartoe plante hoë konsentrasies sout in die wortelstelsel kan hanteer is onder genetiese beheer en verskillende genetiese en fisiologiese meganismes dra by tot die soutverdraagsaamheid tydens verskillende ontwikkelingstadia (Epstein & Rains, 1987). Slegs beperkte variasie bestaan vir soutverdraagsaamheid in verboude grane. Dit het aanleiding gegee tot pogings om soutverdraagsame nageslag te selekteer na hibridisasie van verboude spesies en wilde, soutverdraagsame spesies. Thinopyrum distichum, 'n inheemse koringgras, wat aangepas is by brak omgewings (McGuire & Dvorak, 1981), is met korog (x Triticosecale) gekruis in 'n poging om die gene vir soutverdraagsaamheid en ander gehardheidseienskappe oor te dra (Marais & Marais, 1998). Die oogmerke van hierdie studie was om (i) Thinopyrum chromosome te identifiseer wat gene bevat vir soutverdraagsaamheid en molekulêre merkers te vind vir hierdie chromosome, (ii) 'n aantal diverse monosomiese en disomiese addisieplante te identifiseer. Bulksegregaatanalise (BSA), gekombineer met AFLP-, RAPD- en DAF-merkeranalise, is gebruik om polimorfismes geassosieerd met soutverdraagsaamheid op te spoor. Vyf moontlike AFLPmerkers en twee RAPD-merkers is geïdentifiseer met gebruik van bulks bestaande uit soutverdraagsame plante en bulks bestaande uit soutgevoelige plante. Die verspreiding van die merkers in soutverdraagsame bulks dui daarop dat meer as een Thinopyrum chromosoom bydra tot soutverdraagsaamheid. Soutverdraagsame, monosomiese en disomiese addisieplante is gekarakteriseer vir AFLP- en RAPD-polimorfismes in 'n verdere poging om merkers te vind vir chromosome betrokke by soutverdraagsaamheid. Een soutverdraagsame monosomiese en een disomiese addisieplant is geïdentifiseer. Een AFLP- en twee RAPD-merkers is geïdentifiseer vir die Thinopyrum chromosoom(e) teenwoordig in die monosomiese addisieplant, terwyl drie AFLP- en drie RAPDmerkers geïdentifiseer is vir die disomiese addisieplant. 'n Poging is ook gemaak om diverse addisieplante te identifiseer met 'n volledige koroggenoom plus 'n addisionele Thinopyrum chromosoom. Plante met 2n = 43 / 44 is geïdentifiseer en gekarakteriseer met molekulêre merkers (AFLP en RAPD). Tros-analise is gebruik om die vermoedelik monosomiese of disomiese addisieplante te groepeer volgens die spesifieke Thinopyrum chromosome wat hulle behou het. Sewentien AFLP- en RAPD-merkers is gebruik om die 24 vermoedelike addisieplante in 6 groepe met verskillende Thinopyrum chromosome te groepeer. Alhoewel dit voorkom of die verskillende plante in 'n groep dieselfde addisionele Thinopyrum chromosoom het, is dit nie noodwendig die geval nie aangesien die interpretasie van die merkers bemoeilik word deur die heterogeniteit tussen die plante wat betref die agtergrond korogchromosome wat hulle besit. Dit is ook moontlik dat chromosoom herrangskikkings plaasgevind het gedurende die terugkruisings, wat die data verder kan bemoeilik. Nietemin, dit is nou moontlik om disomiese addisies te selekteer uit elke groep wat moontlik verskillende Thinopyrum chromosome bevat. Die data kan ook gebruik word om in die toekoms verdere addisieplante te identifiseer wat die oorblywende Thinopyrum chromosome bevat.

Thesis (MScAgric.)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: In cross pollinated crops, recurrent selection is used to increase the frequency of desirable alleles by breaking up existing linkage blocks and forming new gene combinations. Despite promising results from numerous feasibility studies, recurrent selection is seldom routinely used in wheat. A major obstacle has been the inability to readily achieve random interbreeding of large numbers of selected plants. In China the Taigu genetic male sterility gene, Ms2, has however been used to establish a recurrent selection programme in which field grown male sterile plants were pollinated by selected male fertile plants (Huang et al., 1988). Another dominant gene for male sterility, Ms3, was found after EMS treatment of the seeds of an alloplasmie common wheat with Triticum tauschii cytoplasm (Maan et al., 1984) and is located at 3 map units from the centromere on chromosome arm SAS (Maan et al., 1987). In a study done during 1999 at Welgevallen to determine the frequency of natural intererossing under field conditions, Ms3 showed incomplete penetrance and only about two thirds of the seed set on male sterile plants could be attributed to intercrossing. Ms3 has stable expression in plants grown within the normal range of greenhouse temperatures for wheat, 16 - 2SoC. Under warmer field conditions, 21 - 3SoC, its penetrance is, however, incomplete (Maan et al., 1984). The utility of Ms3 under field conditions is therefore unsatisfactory. An attempt to determine the location and origin of an unknown male sterility gene, found in cross 9SK3 of a routine breeding programme, showed that a single locus was not the cause of the male sterility. Chromosome abnormalities and gene imbalances were probably to blame. The male sterility probably relates to a T.urartu addition chromosome in the pedigree of cross 9SK3. To facilitate the production of large numbers of hybrid progeny, a simple hydroponic system was developed in which male sterile tillers cut at the flowering stage can be pollinated and maintained for about 8 weeks, long enough to produce viable seeds. For pollination, florets on male tillers are cut open and placed in a container with a similar number of pollen shedding male tillers. It was found that cut tillers could be maintained in the hydroponic system as long as certain precautions were met: (a) The tillers must be handled with care so as not to damage the flag leaf which must be maintained for as long as period possible. (b) The tillers have a nutrient requirement and a 20% solution showed the best results of the nutrient solutions tested. (c) The sterilizing effect of Jik at O.OS%gave excellent fungal control en helped to sustain the nutrient solution. (d) Although the treatment of tillers with hormones improved seed quality, it was not justified by the additional inputs required. Different selection strategies were used for male and female plants. At the onset of the recurrent selection programme in 1998, a total of 1881 plants were tested for seedling resistance and 597 plants were selected for use as parents and source material for 1999. In total 158 male sterile and 188 male fertile ears were used in the hydroponic pollination and a 63.47% seed set was obtained, resulting in 3410 seeds, forming the 1999 female component. One hundred and fifty seven F2:96K109plants were selected from a field grown population in 1998. These, together with 44 selections from a pedigree programme, formed the male component for 1999. In total 9564 plants were tested for seedling resistance during 1999. A total of 3230 resistant seedling were selected and planted. Again male fertile plants from the previous season were field planted and selected. The selected plants were subjected to mixograph testing. A total of 448 male sterile and 1020 male fertile ears were used for hydroponic pollination. Approximately 12000 seeds were harvested, the seed set being around 75%. The 157 F2:96K109 field selected plants (1999) and 64 selections from a pedigree programme formed the male component for 2000. Seedling resistance testing during 2000 included a total of 6465 plants and 2832 were selected and planted. The hydroponic system was improved during 2000 with new, larger capacity containers being used which improved cross pollination. In total 878 male sterile tillers and 1016 male fertile tillers were cut and intercrossed. In total 25380 seeds were harvested, the seed set being 81.7%. In an attempt to determine the amount of variation within the 157 F2-families selected during 1999, mixograph testing was performed. The data showed variation among families. Seedling resistance testing for leaf and stem rust was performed on the 1999 and 2000 FIs to determine the variation for resistance within the populations. Both populations showed high level of stem rust resistance but lower levels of leaf rust resistance (± 50%). Ms3 can thus be used in combination with hydroponic tiller culture to facilitate recurrent selection. Integration with an excisting pedigree selection programme is viable and requires little additional input. Some of the these results have already been published (Addendum D). / AFRIKAANSE OPSOMMING: Herhalende seleksie word by kruisbestuiwers aangewend om die frekwensie voordelige allele te verhoog deur die opbreek van bestaande koppelingsblokke en vorming van nuwe geen-kombinasies. Hoewel uitstekende resultate m.b.V.herhalende seleksie reeds by koring verkry is, is die roetine aanwending egter beperk weens die gebrek aan effektiewe kruisbestuiwing van groot getalle plante. In China is "Taigu" genetiese manlike steriliteit, Ms2, egter met sukses aangewend vir die vestiging van 'n herhalende seleksieprogram vir landverboude koring. Die manlik-vrugbare plante word vir die bestuiwing van geselekteerde manlik-steriele plante aangewend (Huang et al., 1988). Nog 'n dominante manlike steriliteitsgeen, Ms3, is ontdek na EMS behandeling van sade afkomstig vanaf 'n alloplasmiese gewone koring met 'n Triticum tauschii sitoplasma (Maan et al., 1984) en is gesetelop chromosoom 5AS, 3 kaarteenhede vanaf die sentromeer (Maan et al., 1987). 'n Ondersoek na die frekwensie natuurlike kruisbestuiwing onder landtoestande (Welgevallen, 1999) het getoon dat onvolledige penetrasie van Ms3 lei tot ongeveer 5% selfbestuiwing en dat slegs twee-derdes van die saadset aan kruisbestuiwing toegeskryf kon word. Ms3 word wel stabiel uitgedruk onder normale glashuistemperature tydens blom nl. 16 - 25°C, maar onder warmer landtoestande, 21 - 35°C, is uitdrukking onstabiel met laer penetrasie van die geen (Maan et al., 1984). Die benutbaarheid van Ms3 onder landtoestande was dus onbevredigend. Die ondersoek na die oorsprong en ligging van 'n onbekende, manlike steriliteitsgeen (95K3) wat ontdek is in 'n roetine teelprogram het daarop gedui dat 'n enkellokus waarskynlik me ter sprake is nie, maar eerder chromosoom-abnormaliteite en geenwanbalanse. Die manlike steriliteit kan verband hou met 'n T urartu addisie chromosoom in die stamboom van hierdie bron. Ten einde kruisbestuiwing van 'n groot aantal plante te bewerkstellig, is 'n eenvoudige bestuiwersisteem ontwikkel gegrond op waterkultuurkweking van afgeknipte manlik-steriele (Ms3ms3), are. Manlik-steriele en manlik-vrugbare are is tydens blom geknip. Die manliksteriele are se blommetjies is oopgeknip en toegelaat om deur die manlik-vrugbare are bestuif te word. Die bestuifde manlik-steriele are (Ms3ms3) is hierna vir ongeveer 8 weke gelaat vir saadvorming. Afgeknipte are kan baie suksesvol in voedingsmedium onderhou word mits sekere eenvoudige voorsorgmaatreëls getref word, naamlik: (a) Die are moet met sorg hanteer word en die vlagblaar moet so lank as moontlik behou word. Are moet weekliks teruggeknip word ten einde verstopping en agteruitgang van vaatweefsel teen te werk. Die oorspronklik- afgeknipte halm is dus belangrik. (b) Die are toon 'n definitiewe voedingsbehoefte en 'n 20% voedingsoplossing was die beste van die oplossings wat getoets is. Die voedingsoplossing moet verkieslik weekliks vervang word wanneer are teruggeknip word. Op die tydstip behoort die houers met 'n steriliseringsmiddel gewas te word vir die verwydering van enige moontlike swamgroei aan die houers se wande. (c) Jik was die beter steriliseringsmiddel en het teen 0.05% toediening goeie swaminhibering bewerkstellig. (d) Hormone is nie in die roetinetoepassing gebruik nie aangesien die voordeel hiervan nie die ekstra insette regverdig nie. Verskillende strategieë is aangewend vir die seleksie van manlike en vroulike plante. Met die aanvang van die herhalende seleksieprogram in 1998 is 'n totaal van 1881 plante getoets vir roesweerstand en 597 geselekteer as bronmateriaal vir 1999. In totaal is 158 manliksteriele en 188 manlik-vrugbare are gebruik in die bestuiwersisteem vir die verkryging van die 1999 vroulike komponent. 'n Totaal van 3410 sade is verkry met 'n 63.47% saadset. Tesame met 157 F2:96KI09 landgeselekteerde plante is 44 seleksies vanuit 'n stamboom seleksieprogram gebruik as manlike komponent in 1999. Gedurende 1999 is 9564 plante getoets vir roesweerstand en 3230 geselekteer en geplant. Weereens het landseleksie plaasgevind. Die 157 seleksies is onderwerp aan miksograaf-toetsing. Vierhonderd agt- en - veertig manlik-steriele en 1020 manlik-vrugbare are is gebruik in die bestuiwersisteem. Ongeveer 12138 sade is geoes, teen 'n 75% saadset. Gedurende 2000 is die sade asook 64 seleksies uit 'n stamboom seleksieprogram aangewend as die manlike komponent. Roestoetsing is weereens in 2000 uitgevoer en 6465 plante is geïnokuleer waaruit 2832 plante geselekteer en geplant is. Die bestuiwersisteem is aangepas vir die hantering van groter aantalle are tydens 2000 en in totaal is 878 manlik-steriele are en 'n 1016 manlik-vrugbare are gebruik vir kruisbestuiwing. Die saadset is verhoog na 81.7% en 25380 sade is verkry. Om die hoeveelheid variasie binne die populasie te bepaal, is miksograaftoetsing op die 1999 F2-populasie uitgevoer. Die data het aangetoon dat groot hoeveelhede genetiese variasie beskikbaar is binne die populasie. Roestoetsing van die 1999- en 2000-bestuiwerpopulasies is ook uitgevoer om 'n indikasie te verkry van die verspreiding van weerstand teen blaar- en starnroes. Die blaamoes het 'n relatief lae vlak van weerstand getoon (± 50%) terwyl die stamroesweerstand baie hoë vlakke gehandhaaf het. Ms3 kan dus gebruik word om in kombinasie met waterkultuurkweking van gesnyde halms, 'n herhalende seleksieprogram van stapel te stuur. Integrasie met 'n bestaande stamboom seleksieprogram is ook moontlik en sal relatief min addisionele insette vereis. 'n Gedeelte van die werk is reeds gepubliseer en word hierbyaangeheg as Aanhangsel D.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science. / ENGLISH ABSTRACT: Undeniably, changes in the environment and dwindling traditional energy resources have resulted in the search for viable, renewable energy alternatives such as biofuels. Cellulose is one of the most abundant polymers on earth and can be converted to simple sugars and fermented to ethanol biofuel fairly easily. Cellulose rich biomass that can serve to supply ethanol biofuel production can be sourced from unexploited agricultural waste. The main drawback to using vegetative tissue as opposed to harvested food stocks from crops results from the structural properties of plant cell walls. Although cellulose is abundant, the contaminating hemicellulose and lignin fibres within the cell wall matrix have a negative impact on the digestibility of the cellulose present. Thus, an important step in creating an effective biofuel production system from agricultural excess is developing crops with improved cell wall polymer characteristics that can be converted to ethanol more efficiently. This project consisted of two parts. Firstly, the aim was to assess lignin production in transgenic sugarcane transformed with a construct aimed at down-regulating the 4- (hydroxyl) cinnamoyl CoA ligase (4CL) gene in the lignin biosynthesis pathway. The second part of the project revolved around discovering the mechanism of impared cell growth caused by expressing the gene encoding cellulose synthase from a marine invertebrate, Ciona savignyi, in the yeast Saccharomyces cerevisiae. Several sugarcane lines that had been previously transformed with a hairpin RNAi construct aimed at down-regulating the 4CL gene in the monolignol biosynthesis pathway were subjected to analysis to determine if lignification had been reduced. Although the presence of the hairpin construct in the genomic DNA had been confirmed for all of the transgenic lines, there was no significant decrease in the lignin levels in any of the transgenic lines. PCR analysis of the mRNA and enzyme assays also confirmed that the 4CL gene was still being expressed. Ongoing work will determine the cause of the unsuccessful down-regulation. Previously, it had been proven that the cellulose synthase gene from C. savignyi could be functionally expressed in S. cerevisiae. However, cellulose production resulted in extremely retarded growth of colonies and cultures, to the point of the apparent death of the cultures. The aim of this part of the project was to determine the mechanism (either metabolic or physical) that causes this effect. To generate enough cell mass to perform metabolic analysis, several strategies to impede cellulose production in transgenic yeast were explored. Attempts to stop cellulose production and induce better growth by introducing Isoxaben (a traditional weed killer that targets cellulose synthases) into the growth medium used for the transgenic yeast proved unsuccessful. To control the expression of the transgene, it was attempted to clone the cellulose synthase gene into an expression system containing an inducible promoter. The cloning exercise proved extremely difficult and multiple attempts with several strategies proved unsuccessful. This process is still ongoing as the growth retarding process induced by cellulose production in yeast remains to be identified. / AFRIKAAANSE OPSOMMING: Dit is onontkenbaar dat veranderinge in die omgewing en minderwordende tradisionele energiebronne veroorsaak dat lewensvatbare en hernubare energiebronne soos biobrandstof gevind moet word. Sellulose is een van die mees volop polimere op aarde en kan redelik maklik omgeskakel word na eenvoudige suikers en gefermenteer word tot etanol-biobrandstof. Sellulose-ryk biomassa wat etanol-biobrandstof kan verskaf, kan herwin word van tot op hede ongebruikte landbou-afval. Die komplekse struktuur van plantselwande is die hoofstruikelblok in die omskakeling van vegetatiewe weefsel tot biobrandstof. Hoewel sellulose volop is, het die kontaminerende hemisellulose- en lignienvesels binne die selwand-matriks ’n negatiewe impak op die verteerbaarheid van die sellulose teenwoordig in die selwand. Daarom is ’n belangrike stap in die ontwikkeling van effektiewe biobrandstof-produksiesisteme vanaf landbou-afval om gewasse te ontwikkel met verbeterde selwandpolimeer-eienskappe wat etanol-produksie kan vergemakilik. Hierdie projek het bestaan uit twee dele. Eerstens was die doel om vas te stel of die lignienproduksie geaffekteer is in transgeniese suikerriet getransformeer met ’n konstruk wat mik om die 4-(hidroksie)-cinnamoyl CoA ligase (4CL) geen te af-reguleer in die lignienbiosintese- padweg. Die tweede deel van die projek het daarop gefokus om die meganisme te ondek wat die belemmerde selgroei veroorsaak, as gevolg van die uitdrukking van die geen wat kodeer vir sellulose-sintase in ’n mariene ongewerwelde, Ciona savignyi, in Saccharomyces cerevisiae. Verskeie suikerriet-lyne, wat voorheen getransformeer is met ’n haarnaald-RNAi-konstruk om die 4CL-geen te af-reguleer in die monolignol-biosintese-padweg, is onderwerp aan analise om vas te stel of lignifikasie verminder is. Hoewel die teenwoordigheid van die haarnaald-konstruk in die genomiese DNA bevestig is vir al die transgeniese lyne, was daar geen beduidende vermindering in die lignienvlakke in die transgeniese lyne nie. PKRanalise van die mRNA en ensiem-aktiwiteitstoetse het ook bevestig dat die 4CL-geen steeds uitgedruk word. Verdere ondersoek sal kan vasstel wat die oorsaak van die onsuksesvolle af-regulering is. Voorheen is bewys dat die sellulose-sintase-geen van C. savignyi funksioneel uitgedruk kon word in Saccharomyces cerevisiae. Egter, selluloseproduksie het die gevolg gehad dat groei in die transgeniese kolonies en kulture erg gestrem is, tot die punt dat die kulture dood voorgekom het. Die doel van hierdie deel van die projek was om vas te stel wat die meganisme (òf metabolies òf fisies) is wat hierdie verskynsel veroorsaak het. Om genoeg selmassa te genereer om metaboliese analise uit te voer, is verskeie strategieë om selluloseproduksie in transgeniese gis te verhinder, ondersoek. Pogings om selluloseproduksie te stop en om groei te verbeter deur Isoxaben by te voeg in die groeimedium gebruik vir transgeniese gis, was onsuksesvol. Isoxaben is ’n tradisionele onkruiddoder wat sellulose-sintases teiken en inhibeer. Om die uitdrukking van die transgeen te beheer, is ’n poging aangewend om dié sellulose-sintase-geen in ’n uitdrukking-sisteem te kloon met ’n induseerbare promotor. Die kloneringsoefening was uiters moeilik en veelvoudige pogings met verskeie strategieë was onsuksesvol. Hierdie proses moet verder gevoer word aangesien die groeistremmingsmeganisme veroorsaak deur selluloseproduksie in gis nog geïdentifiseer moet word.

Thesis (MScAgric)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Grapevine supports one of the oldest industries in South Africa today, and is also of significant international importance. With increasing international trade and the transport of fruit and other grapevine-derived products between borders, it has become increasingly important for South African farmers and viticulturalists to ensure their products conform to strict international market requirements if they are to remain competitive. Such requirements include the correct and accurate identification of berries and wines according to cultivar. In light of this, 26 different wine, table grape and rootstock cultivars, as well as a number of clones from KWV's core germplasm collection were characterised at 16 microsatellite marker loci. Microsatellite markers are known for their high level of informativeness, reliability and reproducibility, and are widely used in the identification and characterisation of plant varieties, population analyses and forensic applications. Unique allelic profiles were obtained for all but two plants, which proved to be identical at all loci considered, and thus 'clones'. These profiles were collated to form a database, containing the DNA fingerprints of each sample at each locus. The relative levels of informativeness of each marker used were also determined, and compared with those found in the literature. Six markers proved to be highly informative, and are promising in the potential application of this technology to other cultivars. The applicability of microsatellite markers to such studies is confirmed; this approach could easily be extended to include any number of cultivars of national and international interest. The results of such an investigation would have important implications for both the farming and commercial industries alike. / AFRIKAANSE OPSOMMING: Wingerd ondersteun een van die oudste industriee in Suid-Afrika vandag, en is ook van groat intemasionale belang. Met die toenemende intemasionale ruilhandel en die vervoer van vrugte en ander wingerd produkte tussen grense, het dit toenemend belangrik geword vir SuidAfrikaanse wingerdboere om te. verseker dat hulle produkte voldoen aan die streng vereistes van die intemasional mark, indien hulle kompeterend wil bly. Hierdie vereistes sluit in die korrekte en akkurate identifisering van druiwe en wyn volgens kultivar. Met hierdie vereistes in ag geneem, is 26 verskillende wyn, tafeldruif en wortelstok kultivars, asook 'n aantal klone van die KWV se kern kiemplasma versameling, gekarakteriseer by 16 mikrosatelliet merker loki. Mikrosatelliet merkers word gekenmerk deur 'n hoe vlak van informatiwiteit, betroubaarheid en herhaalbaarheid en word wydverspreid gebruik in die identifisering en karakterisering van plant varieteite, populasie analises en forensiese toepassings. Unieke alleliese profiele is vir a1 die plante verkry, behalwe vir twee plante wat identiese resultate by alle loki opgelewer het en dus as "klone" beskou kan word. Hierdie profiele is bymekaar gevoeg om 'n databasis te vorm wat die DNA vingerafdrukke van elke monster by elke lokus bevat. Die relatiewe vlak van informatiwiteit van al die merkers is ook bepaal en vergelyk met merkers in die literatuur. Ses van die merkers blyk om hoogs informatief te wees en lyk belowend in die potensiele toepassing van hierdie tegnologie op ander kultivars. Die toepaslikheid van mikrosatelliet merkers op sulke studies is bevestig; hierdie benadering kan maklik aangepas word om enige aantal kultivars van nasionale en intemasionale belang in te sluit. Die resultate van s6 'n ondersoek sal belangrike implikasies inhou vir beide die boerdery en kommersiele industriee.

Thesis (MScAgric (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: This study aimed to investigate the suitability of spring wheat in the Western Cape as a potential feedstock for a future bio-ethanol industry as well as initiate a pre-breeding effort to develop bioethanol -directed improved lines. Determined primarily on grain yield, disease resistance and, direct as well as indirect assaying of important parameters, material was selected from a base-population for use as male parents. These were crossed with female parents sourced from the Stellenbosch University Plant Breeding Laboratory (SU-PBL) male sterility -mediated marker-assisted recurrent selection (MSMARS) programe. This programe is constituted by an agronomically and disease-resistance - improved population, containing a dominant male sterility gene (Ms3). The progeny of these crosses was used to initiate the production of doubled haploids in order to ultimately derive higher ethanol yielding lines. Multi-location field trial (MLFT) data revealed that 00K60-16-3-3 was the best adapted and highest yielding (2160.95 litres ethanol per hectare) advanced breeding line (ABL). Its performance was not statistically significantly less than first-ranked 03H86-8-2 (2184.62 litres per hectare) and both ABLs significantly (P≤0.05) out-performed six controls in the study. ABL 00K60-16-3-3 was also the most adapted in terms of potential yield in litres per ton of grain. ABL 03H86-8-1 was second recommended for the Western Cape, performing above the expected mean for yield in litres per hectare. Further adaptation of specific ABLs to the two major sub-regions of the Western Cape i.e. the Swartland and Southern Cape including the Rûens was also elucidated. Napier was significantly the highest yielding trial site although none of the considered sites were both stable and high yielding. It was also determined that entry X locality interaction (GxE) was indeed significant across the whole production area regarding litres per hectare as well as its two subregions. This is expected considering the environmentally diverse nature of the region as a whole. Using several entries as examples, relationships between starch, ethanol production in litres ethanol per hectare and litres per ton where grain yield is not taken into consideration were illustrated. Overall applicable relationships other than clear grouped entry differences could not be established. What was clearly demonstrated however, is that the maximization of grain yield is paramount. Highlighted thus, is the individuality of a specific genotype where MLFTs will always be required to quantify genotype potential. / AFRIKAANSE OPSOMMING: Die studie het ten doel gehad om die geskiktheid van lentekoring vir die produksie van bio-etanol in die Wes Kaap te evalueer. Ook het dit ‘n voortelingsprogram geinisieer vir die teel van lyne met verhoogde bio-etanol opbrengs. Materiaal vir gebruik as manlike ouers in ‘n basis-populasie is geselekteer gegrond grootliks op graanopbrengs, siekteweerstand en direkte sowel as indirekte etanolopbrengs kenmerke. Die gekose materiaal is gekruis met vroulike ouers verkry vanaf Stellenbosch Universiteit se Planteteeltlaboratorium (SU-PTL) se manlike steriliteits gedrewe merker bemiddelde herhalende seleksieprogram. Die program is saamgestel uit ‘n verbeterde populasie ten opsigte van siekteweerstand en agronomiese eienskappe. Dit bevat ook ‘n dominante steriliteitsgeen. Die nageslag van die kruisings is aangewend vir die inisiasie van die produksie van verdubbelde haploied lyne vir die verkryging van lyne met verhoogde etanol opbrangs. Die ontleding van data ten opsigte van die multi-lokaliteitsproewe (MLP) het aangetoon dat gevorderde teellyn (GTL) 00K60-16-3-3 die beste aangepas was en ook die hoogste opbrengs (2160.95 liters etanol per hektaar) gegee het. 00K60-16-3-3 was ook nie statisties betekenisvol swakker as die eerste geplaaste 03H86-8-2 (2184.62 liters etanol per hektaar) en beide GTLs was statisties betekenisvol beter (P≤0.05) as die ses kontroles in die studie. GTL 00K60-16-3-3 was ook die beste aangepaste in terme van etanol opbrengs in liters per ton graan. GTL 03H86-8-1 was tweede aanbevole vir die Wes-Kaap met ‘n prestasie bo die verwagte gemiddelde opbrengs in liters per hektaar. Verdere aanpassing van spesifieke GTLs vir die twee mega-omgewings in Wes- Kaap nl. Swartland en Suid-Kaap insluitend die Rûens was ook afgelei. Napier was betekenisvol beter, maar nie enige van die lokaliteite was beide stabiel en hoë opbrengs lokaliteite nie. Dit was ook bepaal dat die inskrywing by lokaliteits interaksie (GXE) betekenisvol was oor die hele produksiegebied ten opsigte van liters per hektaar asook in die twee mega-omgewings afsonderlik. Dit was egter te verwagte gegewe die diverse aard van die omgewings in die streek as geheel. Deur gebruik te maak van verskeie inskrywings as voorbeelde is die verwantskap tussen stysel, etanol produksie in liters etanol per hektaar en liters etanol per ton graan geillustreer sonder om graanopbrengs in ag te neem. Oorhoofs toepaslike verwantskappe anders as duidelike gegroepeerde inskrywings verskille kon nie afgelei word nie. Wat wel duidelik gedemonstreer kon word is dat maksimum graanopbrengs uiters belangrik was. Dit is dus duidelik dat weens die wisselende aard van spesifieke genotipes MLPs altyd van kardinale belang sal wees vir die kwantifisering van ‘n genotipe se potensiaal.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science. / ENGLISH ABSTRACT: Haliotis midae, or Perlemoen, is the only cultured species of abalone in South Africa and is under great international demand. This species is considered endangered, making sustainable farming practises and law enforcement against poaching essential for maintaining wild stocks. A limited amount of broodstock animals are provided to each farm from which thousands of offspring are grown and exported. The prevention of inbreeding and preservation of genetic diversity within farmed stocks is necessary for future sustainable farming and production of genetically stable offspring. Further research into the genetic dynamics of Perlemoen will provide the knowledge for advanced management programs for optimal farming practises and essentially sustainable production. This study focuses on genetic linkage map development with the intention of future identification of markers associated with genes of economic importance, such as growth rate. Identification of markers linked to genes responsible for such phenotypic traits will ultimately allow farming practises to select naturally genetically superior animals for breeding, thereby enhancing production. For the construction of a genetic linkage map of H. midae, microsatellite markers were developed using two strategies: FIASCO and screening of next generation sequence-bysynthesis contig data. The FIASCO-derived markers were characterised by genotype screening in 32 individuals from a full-sib family and analysed using Mendelian segregation expectations. The Illumina-derived markers were characterised by genotype screening in 32 individuals from wild populations and analysed against Hardy-Weinberg expectations. Forty four microsatellite-family combinations were obtained from FIASCO of which 28 provided informative genotype results (32% success). Twenty two markers were developed from sequence-by-synthesis screening. Fourteen provided reliable genotypes (37%) and six conformed to Hardy-Weinberg expectations. These markers were used, in addition to 156 previously developed markers, to develop sex-specific and sex-average linkage maps in two full-sib families consisting of approximately 100 offspring each. One hundred and six polymorphic loci were used for linkage analysis (LOD>3) in both families. The number of linkage groups obtained from sex-specific maps ranged from 13-16. The average genome length ranged from 500 cM to 800 cM with an average marker spacing of 10 cM. The sex-average linkage map provided 18 linkage groups with an average genome length calculation of 1800 cM and average marker spacing of approximately 13 cM. The linkage maps created in this study are preliminary but provide a stepping stone towards a high density map incorporating high throughput markers. This also provides a base for QTL mapping studies, in which phenotypic traits of interest can be identified and associated to specific locations in the H. midae genome for marker-assisted selection. / AFRIKAANSE OPSOMMING: Haliotis midae, ook bekend as Perlemoen, is in groot internasionale aanvraag en is ook die enigste klipkous spesie waarmee in Suid Afrika geboer word. Hierdie spesie word as bedreig beskou en daarom is volhoubare boerdery bedrywe en wetstoepassing teen stroping noodsaaklik om wilde populasies te beskerm. Elke perlemoenplaas word met ‘n beperkte aantal broeidiere verskaf, waarvan die nageslag dan gekweek en uitgevoer word. Voorkoming van inteling en handhawing van genetiese diversiteit binne gekweekte populasies is noodsaakllik vir toekomstige volhoubare kweking en produksie van ń geneties stabiele nageslag. Verdere ondersoeke na die genetiese dinamika van Perlemoen sal die nodige kennis verskaf om sodoende gevorderde bestuursprogramme te ontwikkel, wat tot optimale kweek praktyke en effektiewe volhoubare produksie sal lei. Hierdie studie fokus op die ontwikkeling van ‘n genetiese koppelingskaart met die voorneme om toekomstige merkers te identifiseer wat met gene van ekonomiese belang, soos byvoorbeeld groei tempo geassosieerd is. Identifisering van merkers wat vir sulke fenotipiese eienskappe verantwoordelik is sal sodoende toelaat dat boerdery praktyke kan selekteer vir diere vir verbeterde teling en produksie. Mikrosatelliet merkers is ontwikkel om die genetiese koppelingskaart saam te stel. Die volgende twee strategieë is benut: FIASCO en sifting van volgende generasie volgordebepaling-deur-sintese “contig” data. Die FIASCO-afgeleide merkers is gekarakteriseer deur genotipiese sifting in 32 individue van ‘n volsib familie en is deur Mendeliese segregasie verwagtinge ge-analiseer. Die Illumina-afgeleide merkers is gekarakteriseer deur genotipiese sifting in 32 individue van wilde populasies en is met Hardy-Weinberg ewewig ge-analiseer. Vier en veertig mikrosatelliet-familie kombinasies is deur FIASCO verky, waarvan 28 informatiewe genotipiese resultate gelewer het (32% sukses). Twee en twintig merkers is vanaf volgordebepaling-deur-sintese sifting ontwikkel. Veertien van hierdie merkers het betroubare genotipes (37%) verskaf en ses het aan Hardy-Weinberg verwagtinge voldoen. Hierbenewens is 156 voorheen ontwikkelde merkers gebruik om geslagspesifieke en geslagsgemiddelde koppelingskaarte in twee volsib families saam te stel. Hierdie volsib families het uit ń naslag van 100 elk bestaan. Een honderd en ses polimorfiese lokusse is vir koppelingsanalise gebruik, waar ‘n LOD waarde groter as drie statisties betekenisvol geag was. Die aantal koppelingsgroepe verkry van geslagspesifieke kaarte het tussen 13 en 16 gewissel. Die gemiddelde genoom lengte het van 500 cM tot 800 cM met ‘n gemiddelde merker spasiëring van 10 cM. Die geslagsgemiddelde koppelingskaart het 18 koppelingsgroepe gehad met ‘n gemiddelde genoom lengte berekening van 1800 cM en ‘n gemiddelde merker spasiëring van ongeveer 13 cM. Die koppelingskaarte wat in hierdie studie geskep is, is voorlopig en verskaf ‘n grondslag vir die ontwikkeling van ‘n hoër digtheidskaart, wat hoë deurset merkers inkorporeer. Dit verskaf ook ‘n basis vir kwantitatiewe kenmerk lokus karteringstudies. Hierdie karteringstudies kan fenotipiese eienskappe van belang identifiseer en assosieer met spesifieke posisies binne die H. midae genoom vir merker bemiddelde seleksie.

Thesis (MSc (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Haliotis midae is the most important aquaculture species in South Africa, with abalone farming contributing 80% of the Rand value of the aquaculture industry. Although genetic research has benefited the abalone industry, several issues still hinder increases in abalone production. Progress towards an increase in H. midae growth rate by utilizing conventional genetic studies and selective breeding has been relatively slow. Gene transfer has therefore become a plausible option to address this problem. Genes that code for certain desirable traits, such as increased growth rate, could be incorporated into the genome of commercial abalone. The current study undertook the optimization of a chemically-mediated gene transfer technique using Polyethylenimine (PEI) as transfection reagent and fluorescent proteins as reporter genes. Before gene transfer could be undertaken, several complementary studies also needed to be undertaken due to the novel nature of the study. The auto fluorescence of H. midae, the suitability of several H. midae tissues as targets for gene transfer and the cytotoxic effect of transfection reagents and selection antibiotics were assessed before gene transfer optimization could be attempted. Also, genes linked to an increase in growth rate were characterized for differential expression in different abalone age-groups to determine the suitability of these genes for incorporation into a homologous gene construct in future transfection studies. The auto fluorescence of ova, embryos and larvae were found to be comparable to that of the fluorescent reporter genes, EGFP and DsRed. A PCR-based transfection validation method was therefore employed to confirm the presence of internalized transgenes. It was established that sperm, ova, larvae and haemocyte cell culture were the most suitable target tissues for transfection. The transfection reagents, a 25kDa PEI and ExGen 500, were not cytotoxic to sperm, embryos and haemocyte cell cultures. The minimum lethal concentration of the selection antibiotics, neomycin and zeocin, was determined for larvae and haemocytes. After transfection treatment of sperm and fertilization of untreated ova, the presence of internalized transgenes could be verified for larvae. The presence of internalized transgenes could not be detected after transfection treatment of ova and larvae. Fluorescent flow cytometry and microscopy analysis of haemocytes could not detect the expression of the fluorescent reporter genes. Expression of two of the growth related genes was found to differ between age-groups. The perlustrin gene was upiv regulated in older animals, while the insulin related peptide receptor gene was down regulated in older animals. The third gene, a thrombospondin-1 precursor was stably expressed in all age-groups. This study represents the first report of transfection studies carried out on H. midae. Future studies will benefit from the groundwork established in H. midae transfection. / AFRIKAANSE OPSOMMING: Haliotis midae is die belangrikste akwakultuur spesie in Suid-Afrika met perlemoen boerdery wat 80% van die Rand waarde van die akwakultuur industrie bydrae. Alhoewel genetiese studies die perlemoen industrie ‘n hupstoot gegee het, is daar steeds sekere struikelblokke wat verdere toename in produksie verhoed. Vooruitgang ten opsigte van ‘n toename in H. midae se groei tempo deur gebruik te maak van konvensionele genetiese studies en selektiewe teling was tot dusver relatief stadig. Genetiese transformasie het daarom ‘n wesenlike alternatief geword wat moontlik hierdie probleem kan oplos. Gene wat kodeer vir sekere eienskappe, soos ‘n toename in groeitempo, kan in die genoom van kommersiële perlemoen inkorporeer word. Die huidige studie het onderneem om ‘n chemies-gemedieerde genetiese transfeksie tegniek te optimiseer en van Polyethylenimine (PEI) as transfeksie reagens en fluoresserende proteine as verklikkers gebruik te maak. As gevolg van die oorspronklikheid van die studie moes verskeie bykomende ondersoeke ook aangepak word voordat genetiese transfeksie uitgevoer kon word. Die outofluoressensie van H. midae, die geskiktheid van verskeie H. midae teiken weefsels en die sitotoksiese effek van die transfeksie reagense en seleksie antibiotika is ondersoek voordat transfeksie uitgevoer is. Gene gekoppel aan ‘n toename in groeitempo is ook gekarakteriseer vir verskille in uitdrukking in verskillende perlemoen ouderdoms-groepe om te bepaal of hierdie gene moontlik in ‘n homoloë geen konstruk ingesluit kan word vir toekomstige transfeksie studies. Dit is gevind dat die outofluoressensie van ova, embrios en larwes vergelykbaar is met die fluoressensie van die verklikker proteïene, EGFP en DsRed. ‘n PKR-baseerde metode om die internalisering van die transgeen te kontroleer is daarom gebruik. Dit is vasgestel dat sperm, ova, larwes en haemosiete die mees geskikte teiken vir transfeksie sou wees. Die transfeksie reagense, ‘n 25kDa PEI en Exgen 500, is nie sitotoksies vir sperm, embrios of haemosiete nie. Die minimum dodelike konsentrasie van die seleksie antibiotika, neomycin en zeocin, is bepaal. Na transfeksie behandeling van sperm en bevrugting van onbehandelde ova, kon die teenwoordigheid van internaliseerde transgene bevestig word vir larwes. Die teenwoordigheid van internaliseerde transgene kon nie bevestig word na transfeksie behandeling van ova en larwes nie. Fluoressente vloei sitometrie en mikroskopiese analise kon nie die uitdrukking van die fluoressente verklikker gene bevestig in haemosiete nie. Die uitdrukking van twee van die gene gekoppel aan groei het verskil tussen ouderdomsgroepe. Die perlustrin geen is meer uitgedruk in ouer diere terwyl die insulien geassosieerde peptied reseptor geen minder uitgedruk is in ouer diere. Die thrombospondin-1 voorloper geen is stabiel uitgedruk in al die ouderdomsgroepe. Hierdie studie verteenwoordig die eerste verslag van transfeksie studies uitgevoer op H. midae. Toekomstige studies sal baat vind by die grondslag wat deur hierdie projek gelê is.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae (locally also known as perlemoen) is the largest of five endemic species found along the coast of South Africa. It is the only species with commercial value contributing to the exploitation of these animals. Due to declines of natural stocks, farming practices were established during the early 1990s in order to supply the international demand. To facilitate efficient breeding methods and ensure the sustainability of these commercial populations, genetic management, which can be accomplished with the use of molecular markers such as single nucleotide polymorphisms (SNPs), is necessary. Single nucleotide polymorphisms have become the markers of choice in various applications in aquaculture genetics due to their abundance in genomes, reduction in developmental costs and increased throughput of genotyping assays. Identification of SNPs in non-model species such as H. midae can be achieved by in silico approaches. In silico methods are suitable for de novo SNP identification and are both cost- and time-efficient. It is based on the analysis of multiple alignments where mismatches may be reported as candidate SNPs. Various medium-throughput genotyping methods are available to confirm putative SNPs, but the ideal method depends on factors such as cost, accuracy and multiplexing capacity. Although SNP markers can have various applications within the aquaculture environment the focus for this current study was saturating the linkage map of H. midae with additional markers. This would assist in the identification of quantitative trait loci associated with economically important traits, which in turn could ultimately be employed for marker-assisted selection and improved molecular breeding programs. In order to identify in silico SNPs, sequenced transcriptome data from a previous study was used and subjected to a series of criteria: minor allele frequency 10%, minimum coverage 80, 60 bp flanking regions. Selected loci were genotyped using a 192-plex assay with the Illumina GoldenGate genotyping assay with the VeraCode technology on the BeadXpress platform, in individuals from six mapping families. A conversion rate of 69.35% and global success rate of 76.34% was achieved. Polymorphic loci were subjected to linkage analysis using JoinMap® v.4.1 to create sex-average and sex-specific maps and to saturate the current linkage map for H. midae. Along with previously developed markers, 54% of the newly developed SNPs could be successfully incorporated into the linkage map of H. midae. A total of 18 linkage groups were observed with an average marker spacing of 6.9 cM and genome coverage of 79.1%. Bioinformatic analyses and setting stringent criteria to identify SNPs from sequenced transcriptomic data proved to be an efficient way for SNP discovery in the current study. Genotyping of the identified loci with the GoldenGate genotyping assay demonstrated a high success rate; providing a genotyping assay adequate for species with little genomic information. The linkage map created in this study illustrated the utility of SNP markers in conjunction with microsatellite markers for linkage map construction and the adequate marker spacing obtained provides a step closer to quantitative trait loci mapping in this species. / AFRIKAANSE OPSOMMING: Haliotis midae (plaaslik ook bekend as perlemoen) is die grootste van vyf inheemse spesies wat langs die kus van Suid-Afrika aangetref word. Dit is die enigste spesie van kommersiële waarde wat bydraend is tot die uitbuiting van hierdie diere. As gevolg van die afname in hierdie natuurlike hulpbron het boerdery praktyke gedurende die vroeë 1990's ontstaan om in die internasionale aanvraag te voorsien. Ten einde doeltreffende teelmetodes te beoefen en die volhoubaarheid van hierdie kommersiële populasies te verseker is genetiese bestuur, wat bewerkstellig kan word deur die gebruik van molekulêre merkers soos enkel nukleotied polimorfismes (ENPs), baie belangrik. Enkel nukleotied polimorfismes is gewilde merkers in verskeie toepassings in akwakultuur genetika as gevolg van hul oorvloed in genome, verlaagde ontwikkelingskoste en verhoogde deurset van ENP-genotiperingstoetse. Identifisering van ENPs in nie-model spesies soos H. midae kan uitgevoer word deur in siliko benaderings te gebruik wat geskik is vir de novo ENP identifisering en ook tyd- en koste-effektief is. Dit word gebaseer op die analise van veelvuldige inlynstellings waar nukleotiedes wat nie ooreenstem nie as kandidaat ENPs gerapporteer kan word. Om kandidaat ENPs te bevestig, kan verskeie medium-deurset genotiperingsmetodes uitgevoer word, maar die ideale metode word bepaal deur faktore soos koste, akkuraatheid en multipleks kapasiteit. Alhoewel ENP merkers in verskeie toepassing binne die akwakultuur omgewing gebruik kan word was die fokus van die huidige studie om die koppelingskaart van H. midae te versadig. Dit sal bydrae tot die identifisering van kwantitatiewe eienskap lokusse wat gekoppel kan word aan ekonomies belangrike eienskappe wat dan op die beurt weer vir merkerbemiddelde seleksie gebruik kan word en uiteindelik ten opsigte van die verbetering van molekulêre teelprogramme aangewend kan word. Ten einde in siliko ENPs te identifiseer is transkriptoomdata van 'n vorige studie gebruik en onderwerp aan 'n reeks kriteria: geringste alleelfrekwensie 10%, minimum dekking 80, 60 bp gebiede weerskante van polimorfisme. Geïdentifiseerde lokus-genotipering is met behulp van 'n 192-pleks toets uitgevoer met die Illumina GoldenGate genotiperingstoets met die VeraCode tegnologie op die BeadXpress-platform, in individue afkomsitg vanaf ses karteringsfamilies. 'n Omskakelingskoers van 69.35% en 'n algehele sukseskoers van 76.34% is bereik. Polimorfiese lokusse is onderwerp aan koppelings-analise met behulp van JoinMap® v.4.1 om geslags-gemiddelde en geslags-spesifieke kaarte te skep asook om die kaart wat beskikbaar is vir H. midae te versadig. Saam met voorheen ontwikkelde merkers is 54% van die nuut ontwikkelde ENPs suksesvol opgeneem in die kaart van H. midae. 'n Totaal van 18 koppelingsgroepe is verkry met 'n gemiddelde merker-spasiëring van 6.9 cM en 'n genoomdekking van 79.1%. Die gebruik van bioinformatiese analises en streng kriteria om ENPs vanaf transkriptoomdata te identifiseer blyk doeltreffend te wees in hierdie studie. Genotipering van die geïdentifiseerde lokusse met die GoldenGate genotiperingstoets dui op 'n hoë suksessyfer en verskaf 'n voldoende genotiperingstoets aan spesies met min genomiese inligting. Die koppelingskaart in hierdie studie het geïllustreer dat die ENP merkers suksesvol saam met mikrosatelliet merkers gebruik kan word vir koppelingskaart konstruksie en dat die voldoende merker-spasiëring verkry 'n stap nader aan kwantitatiewe eienskap lokus kartering in hierdie spesie bied.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Grapevine virus E (GVE) is a newly identified virus that has been detected in an established vineyard in South Africa. This virus is a member of the genus Vitivirus, family Flexiviridae. Members of this genus are known to infecte grapevine and are associated with various disease complexes, such as the Rugose wood complex (RWC) and Shiraz disease (SD). However, the role and impact of GVE in South African vineyards are still unknown. It is important to study these viruses to determine how they infect and the possible impact they may have on vine health. The accurate and early detection of grapevine viruses is the first important step in disease management. In this study, reverse transcription-polymerase chain reaction (RT-PCR), double antibody sandwich enzyme linked immunesorbent assay (DAS-ELISA) and quantitative (q)RT-PCR were used for the detection of GVE in the vineyard (Vitis vinifera cv Merlot) where GVE was first identified in South Africa. Reverse transcription-PCR was used for detection and determining the incidence of GVE. The incidence was as low as 3% in the vineyard surveyed. All the GVE positive plants were co-infected with GLRaV-3 and no disease association could therefore be made. Evaluation of the Bioreba Grapevine virus A (GVA) DAS-ELISA kit showed that it did not detect GVE. No cross-reactivity occurred with epitopes of GVE, confirming this kit to be a valid and specific assay for GVA infection. The relative virus titer of GVE was calculated over the growing season of 2010/2011, using qRT-PCR. No fluctuation in virus titer was observed during that growing season. Transmission experiments were performed in an attempt to transfer GVE from grapevine to an alternative host. Three different transmission buffers as well as nine different herbaceous plant species, that have shown to be susceptible to several plant viruses in previous studies, were evaluated. In these experiments, GVE could not be transmitted to any of the herbaceous species. To further characterize GVE, chimeric clones were constructed with GVA. The ORF2 and ORF5 of GVE were cloned into previously constructed GVA ORF2 and ORF5 deletion mutants. Construction of the chimeric clones, 35S-GVA-GR5-ΔORF2-GVE-ORF2 and 35S-GVA-118-ΔORF5-GVE-ORF5 were successful and they were evaluated for their infectivity in N. benthamiana. The 35S-GVA-GR5-ΔORF2-GVE-ORF2 chimera was able to infect and replicate in these plants and disease symptoms such as yellowing of veins and leaf curling were observed. Virus, derived from this vector, was detected by TPIA, RT-PCR and DAS-ELISA. The 35S-GVA-118-ΔORF5-GVE-ORF5 chimeric vector was not able to infect N. benthamiana as no disease symptoms were observed in any of the infiltrated plants and virus was not detected with serological analysis and RT-PCR. This study was aimed at further characterizing the recently identified virus GVE. Here, insight is given into the prevalence of this virus in the vineyard where it was first identified and attempts to biologically characterize GVE were made. / AFRIKAANSE OPSOMMING: Grapevine virus E (GVE) is „n nuut geïndetifiseerde virus wat onlangs in „n gevestigde wingerd in Suid Afrika opgespoor is. Hierdie virus vorm deel van die genus Vitivirus, familie Betaflexiviridae. Spesies in hierdie genus is bekend vir wingerdinfeksies en word met „n verskeidenheid wingerd siektes geassosieer, soos bv. Rugose wood complex (RWC) en Shiraz siekte (SD). Die rol en impak van GVE is nog onbekend. Dit is dus belangrik om die virus te bestudeer om te bepaal hoe dit infekteer en of dit enige impak het op wingerd gesondheid. Akkurate en vroeë opsporing van virusse is die eerste belangrike stap vir virussiekte beheer. In hierdie studie word tru-transkripsie (TT) – polimerase ketting reaksie (PKR), dubbel teenliggaam (DAS) -ensiem gekoppelde immuno-absorberende analise (ELISA) en qTT-PKR gebruik vir die opsporing van GVE in die wingerd (Vitis vinifera cv Merlot) waar dit vroeër in Suid Afrika geïdentifiseer was. Vir opsporing en bepaling van verspreiding is TT-PKR gebruik. Daar is bepaal dat 3% van die wingerd met GVE geïnfekteer is. Al die GVE-positiewe stokke het ook positief getoets vir GLRaV-3 en geen assosiasie met siekte simptome kon gemaak word nie. Evaluering van die Bioreba GVA DAS-ELISA met GVE positiewe stokke het nie GVE opgespoor nie. Geen kruisreaktiwiteit het plaasgevind met epitope van GVE nie en dus is die DAS-ELISA ʼn betroubare toets vir GVA infeksie. Die relatiewe virus titer van GVE was ook bepaal oor die groeiseisoen van 2010/2011 deur qTT-PKR te gebruik. Geen fluktuasie in virus titer gedurende die groeiseisoen is waargeneem nie. Transmissie eksperimente is gedoen om GVE vanaf wingerd na ʼn alternatiewe gasheer oor te dra. Drie verskillende transmissie buffers en tien verskillende sagteplant spesies, wat voorheen vatbaarheid vir plantvirusse getoon het, is gebruik. In die transmissie eksperimente kon GVE nie na enige van die sagteplante oorgedra word nie. Om GVE verder te karakteriseer is hibried-virusse met GVA gemaak. Die leesraam (ORF) 2 en ORF5 van GVE gekloneer in GVA ORF2 en -ORF5 delesie konstrukte, 35S-GVA-GR5-ΔORF2 en 35S-GVA-118-ΔORF5, onderskeidelik (Blignaut, 2009; Du Preez, 2010). Klonering van die hibried konstrukte, 35S-GVA-GR5-ΔORF2-GVE-ORF2 en 35S-GVA-118-ΔORF5-GVE-ORF5, was suksesvol en is in N. benthamiana geëvalueer. Virus afkomstig van die 35S-GVA-GR5-ΔORF2-GVE-ORF2 hibried konstruk, kon plante suksesvol infekteer en kon repliseer binne hierdie plante. Siektesimptome soos vergeling van die are en rolblaar is ook waargeneem in plante geïnfekteer met hierdie hibried konstruk. Plante is getoets met weefsel afdruk immuno analise (TPIA), TT-PKR en DAS-ELISA en is positief gevind vir virus afkomstig van hierdie konstruk. Die 35S-GVA-118-ΔORF5-GVE-ORF5 hibried kon nie N. benthamiana infekteer nie en geen siektesimptome is waargeneem in enige van die plante geïnfiltreer met hierdie konstruk. Serologiese analise en TT-PKR het ook nie virus in die N. benthamiana plante opgespoor nie. Die doel van hierdie studie was om GVE te karakteriseer. In hierdie studie word insig gegee oor die verspreiding van hierdie virus in Suid Afrika en pogings is gemaak om GVE biologies te karakteriseer.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant diseases are among the major causes of food insecurity. In South Africa the wheat fungal diseases including stem rust caused by Puccinia graminis f. sp. tritici, leaf rust caused by P. triticina and stripe rust caused by P. striiformis f. sp. tritici are the most important. Genetic resistance is a viable way of protecting wheat crops against the wheat rusts, especially cultivars carrying multiple genes that confer durable resistance. In order to breed for multi-gene resistance an effective breeding strategy that allows for selecting multiple resistance genes and other desirable traits needs to be devised. The aim of this study was to identify a number of genotypes with combinations of different rust resistance genes, good grain yield and end-use quality out of an existing pre-breeding population and thereby identify superior parents. In order to achieve the stated aim the following objectives have been identified: identify wheat lines through marker-assisted selection (MAS) carrying the gene complexes, Sr31/Lr26/Yr9, Lr24/Sr24, Lr37/Sr38/Yr17, Lr34/Yr18 and Sr2; to develop inbred lines to evaluate selected lines under field trials. From the initial subset of 64 lines, 60 were chosen and advanced to the doubled haploid (DH) phase and seed multiplication. The 60 lines either carried one or more of the three rust resistance gene complexes. The genes that were the most prominent were Sr31/Lr26/Yr9 and Lr24/Sr24. The selected lines were incorporated into a DH seed multiplication phase. After 4 cycles of seed increases and preliminary field evaluation during multiplication, 15 lines were chosen and subjected to multi-location field trails. The extensive multi-location field trails carried out in this study aided in identifying genotypes from the 15 MS-MARS lines with good adaptability and stability in regards to yield and baking quality. An important observation was that the molecular markers employed to indentify quality loci correlated well with the genes encoding the HMW-GS 5, 10 and 12 as observed with the Agilent© 2100 Bioanalyzer. In future studies the lines which performed the best could be re-introduced into the existing MSMARS pre-breeding programme of the Stellenbosch University’s Plant Breeding Laboratory (SUPBL). The frequencies of desired alleles could be increased in this manner. Since the majority of these characteristics are influenced by quantitatively inherited alleles, using these lines as recurrent parents will increase the frequencies of these alleles in the existing SU-PBL pre-breeding population. / AFRIKAANSE OPSOMMING: Plantsiektes is van die belangrikste oorsake van voedselonsekerheid ter wêreld. In Suid-Afrika is die roesswamme van die belangrikste plantsiektes wat koring produksie beïnvloed. Hierdie siektes sluit in, stamroes wat veroorsaak word deur Puccinia graminis f. sp. tritici, blaarroes wat veroorsaak word deur P. triticina en streeproes wat veroorsaak word deur P. striiformis f. sp. tritici. Genetiese weerstand is ‘n uitstekende manier om koring te beskerm teen hierdie swamsiektes. Weerstand wat gebasseer is op veelvuldige weerstandsgene is veral ‘n goeie middel om genetieseweerstand op ‘n volhoubare basis in koringteling toe te pas. Om veelvuldige weerstandsgene in koringkultivars in te teel word ‘n effektiewe telingstrategie benodig. Die doel van die studie was om genotipes te identifiseer met kombinasies van veelvuldige weerstandsgene vir roes, sowel as goeie eienskappe belangrik vir graanopbrengs en bakkwaliteit. Lyne is geïdentifiseer uit ‘n bestaande voortelingspopulasie van Stellenbosch Universiteit se Planteteelt Laboratorium (SU-PTL) wat geteel was met spesifiek weerstand en opbrengs potensiaal in gedagte. Om die doel van die studie te bereik is sekere doelwitte daar gestel. Hierdie doelwitte sluit in om lyne uit die populasie te selekteer deur middel van merker bemiddelde seleksie (MBS) vir gene naamlik Sr31/Lr26/Yr9, Lr24/Sr24, Lr37/Sr38/Yr17, Lr34/Yr18 en Sr2; om die geselekteerde lyne suiwertelend te maak; sowel as om die suiwertelende lyne in veld proewe in te sluit. Van die oorspronklike stel van 64 lyne, is 60 gekies vir verdere studie. Deur middel van die verdubbelde haploïed (VH) tegniek is die lyne suiwertelend gemaak. Die 60 lyne het een of meer van die geselekteerde gene bevat. Die mees prominente gene was die twee geen komplekse Sr31/Lr26/Yr9 en Lr24/Sr24. Na vier siklusse van saadvermeerdering en voorloppige seleksies is 15 lyne ingesluit by ‘n multi-omgewing veldproef. Hierdie uitgebreide multi-omgewing veldproewe het gehelp om individue uit die 15 lyne te identifiseer wat oor goeie aanpasbaarheid en stabiliteit beskik met betrekking tot opbrengs en bak kwaliteit. Die molekulêre merkers gebruik om die gene verantwoordelik vir die kodering van HMGGS 5, 10 en 12 op te spoor het goed gekorreleer met die HMG-GS bande bepaal met behulp van die Agilent© 2100 Bioanalyzer. Toekomstige studies kan moontlik insluit die gebruik van die lyne wat geïdentifiseer was met goeie kenmerke in die bestaande MS-MARS teelprogram van die SU-PTL. Die frekwensies van die verlangde allele kan op hierdie manier in die populasie verhoog word.