Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / Multiple sclerosis (MS) is a disease that causes neurological dysfunction. Studies attempting to elucidate the role of genes in MS development may aid efforts to control the damage caused by the disease that affects two million people worldwide, e.g. improved diagnosis and treatment. Although the association of MS and genes has not been fully characterized the proposed genetic etiology has been supported by the observed association of MS with the Major Histocompatibility Complex (MHC), haplotype HLA-DRB1*1501, DRB5*0101, DQA1*0102, DQB1*0602. Iron, or rather the dysregulation thereof, has also been implicated as a precipitating factor in MS development. Considering the factors of iron dysregulation and the genes involved in iron regulation, this study aims to identify variation within genes involved in iron metabolism namely the high iron gene (HFE), solute-carrier family 40 (iron regulated transporter) member 1 gene (SLC40A1), hepcidin anti-microbial peptide (HAMP), cytochrome b reductase 1 (CYBRD1) and hemojuvelin (HJV). Screening of 40 patients (33 female, seven male; 33 Caucasian, seven Coloured) for each of the five genes was achieved by the Heteroduplex Single-Stranded Conformation Polymorphism (HEX-SSCP) technique. Semi-automated DNA sequencing allowed for verification and characterization of the variants detected. Results included identification of four novel variants present in only the Caucasian patient group, characterized as IVS4-53G→A (HFE) (one of 33 patients; 3%), IVS2-65delA (CYBRD1) (two of 32 patients; 6.3%), 3’UTR+26delACGTCACGTTTCAAAACTA (CYBRD1) (one of 31 patients; 3.2%) and 219delG (HJV) (two of 33 patients; 6%). In addition, a total of 15 previously described variants were identified (seven intronic and eight exonic) of which three were also prevalent in only the Caucasian patient group. This study aimed to investigate the differences ...

Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Rapidly expanding knowledge of the human genome allows new insight into the interaction between drugs and DNA. The heterogeneic nature of schizophrenia is known to cause different patients to display dissimilar drug responses, reflecting distinct genetic profiles. Resulting adverse side effects include tardive dyskinesia (TD), a movement disorder associated with the long-term use of antipsychotic drugs. The identification of a pharmacogenetic basis of TD may have significant clinical implications in the treatment of schizophrenia, allowing individualised prescription of antipsychotic drugs and eventual elimination of undesirable side effects. The current study focussed on a number of South African Xhosa schizophrenia patients, some of whom have been diagnosed with TD. The investigation sought to establish whether the underlying mechanism causing the disorder to manifest only in some individuals, might be attributed to differences in DNA sequences, i.e. genomic susceptibility. A number of candidate polymorphisms in the CYP and MDR1 genes were evaluated in three separate analyses. (The same approach was followed in each investigation, and only known polymorphisms were selected.) The incidences of the various variants were compared between TD and non-TD patients. In addition, potential predisposing factors, i.e. tobacco and cannabis smoking and anhedonia, were taken into consideration. These were analysed concurrently with DNA data and TD status.

Thesis (MSc (Genetics))--University of Stellenbosch, 2006. / Pre-eclampsia is a multisystemic disorder with an incidence of ~6-8% in non-Caucasian women in the Western Cape. Trophoblast invasion is vital for adequate anchorage of the placenta to the uterine wall as well as for the optimisation of utero-placental blood flow in uncomplicated pregnancies. This process is facilitated by the fetal trophoblast cells that digest the extracellular matrix of the uterus by secreting various molecules, including the metalloproteinases (MMP), of which MMP-9 has an increased production during the first trimester. Leptin, an autocrine regulator of MMP-9 secretion, functions via the leptin receptor to prevent over-invasion of maternal tissues. The aim of this study was to investigate the role of the leptin (ob) and leptin receptor (obR) genes in predisposition to pre-eclampsia and involved screening the genes in South African non-Caucasian cohorts and performing statistical analysis to determine whether any variants contributed to the disease profile.

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / Withering syndrome is a severe disease of abalone, Haliotis species that has been associated with mortality ranging from 99% in black, H. cracherodii Leach and 30% in red abalone, H. rufescens Swainson. The disease was first observed in California, along the west coast of North America and is an economically important disease that has led to the closure of the black abalone fishery throughout the southern California State. The causative agent of withering syndrome is a gram-negative intracellular Rickettsiales-like prokaryote designated Candidatus xenohaliotis californiensis. The geographical range of C. xenohaliotis californiensis is broad, besides red and black abalone it has also been reported in yellow, H. corrugate and blue abalone, H. fulgens all caught in Baja California, Mexico. In 2000 a Rickettsiales-like prokaryote resembling the disease-causing agent was observed in the digestive gland of the South African abalone H. midae. In this study we aimed to determine the relationship of the bacterium observed in the local abalone species, H. midae to the disease-causing agent of withering syndrome. A specific PCR and in situ hybridization test using primers and probes specific for the C. xenohaliotis californiesis 16S rDNA gene were used to screen H. midae digestive gland tissues showing classical features of the Rickettsiales-like prokaryote. Both analyses indicated that C. xenohaliotis californiensis is not present in the local abalone species. We therefore aimed the identification of the organism parasiting the local abalone species by DNA sequence analysis of the 16S rDNA gene. The 16S rDNA gene was amplified by PCR, cloned and sequenced. Phylogenetic trees, constructed by maximum parsimony analysis revealed a diverse community comprised of α - Proteobacteria, Mollicutes and Spirochaetes. In the class α - Proteobacteria a novel group of sequences showing phylogenetic affinities to the order Rickettsiales was identified as likely candidate for forming the Rickettsiales-like inclusions in the digestive gland of H. midae. Oligonucleotide probes that bind to four variable regions of the novel group were used to confirm their presence in infected H. midae digestive gland tissue by in situ hybridization. Although these probes did not recognize the inclusions formed by the Rickettsiales-like organisms, they revealed the presence of a group of free-living bacteria abundant in the host tissue. We therefore conclude that (1) C.xenohaliotis californiensis is not present in the South- African abalone, H. midae; (2) the organisms isolated from the digestive gland of H. midae are part of the normal microflora and (3) the group of sequences showing phylogenetic affinities to the order Rickettsiales is not responsible for the Rickettsiales-like inclusions in infected digestive gland tissues but represent a novel group of organisms that are abundant in the host tissue.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Pre-eclampsia is one of the most common hypertensive disorders of pregnancy in South Africa. Presently, the only cure for pre-eclampsia is delivery, which brings with it, additional complications. As an alternative, clinical management of this disorder relies on timely diagnosis. The predictive biomarker, Placental Protein 13 (PP13), is currently used for the early diagnosis of pre-eclampsia, in an ELISA-based diagnostic kit, developed by Diagnostic Technologies Limited (DTL)1. A decrease in serum PP13 levels has been reported during the first trimester of pregnancy in women who later develop pre-eclampsia. The function of PP13 has not been fully elucidated and it is also not known whether the reduction in PP13 levels is a cause or an effect of the disease. The use of PP13 as a predictive biomarker for pre-eclampsia therefore warrants a comprehensive study of this peptide and the encoding gene, LGALS13. The aim of this study was firstly to characterise LGALS13 using a range of in silico tools. PP13 was found to be most homologous to the predicted protein product of a neighbouring “putative” gene, LOC148003. A gene conversion event between these two genes most likely underlies the so-called “hotspot mutation” in LGALS13. Data also demonstrates that the DelT mutation disrupts functionally and structurally important features of the gene and peptide sequences. Through the analysis of the putative promoter region of LGALS13, the presence of a Stimulatory protein-1 (Sp1) binding sequence element was predicted, which has implications for regulation of LGALS13. Secondly, the study aimed to establish a study cohort for the investigation of the effect that the LGALS13 genotype has on the expression of its mRNA and protein products. Serum, plasma and whole blood samples were collected and prepared from 316 pregnant women. Placental tissue samples were obtained from a selected group of these subjects for RNA extraction. Once the sampling on the two remaining targeted deliveries has occurred, the collection of samples will be batched and sent to DTL in Israel, for PP13 measurement. DNA was extracted from the whole blood samples obtained, and all study participants were genotyped for seven sequence variants within the LGALS13 gene using (i) Multiphor Single Stranded Conformational Polymorphism and Heteroduplex (SSCP/HD) analysis, (ii) restriction enzyme analysis and (iii) DNA sequencing. The genotype data sets will be compared with PP13 levels when they become available, and also with clinical parameters, once the deliveries have all occurred and the database is complete. This study demonstrated the power of an in silico approach to direct the focus of future experimental work. The newly established study cohort will be used for prospective studies aiming at a better understanding of the role which LGALS13 and PP13 play in the early prediction of preeclampsia.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is the only endemic species of commercial value. Aquaculture remains the only avenue for expanding the industry, since the closure of the fishery. The current focus is on implementing a molecular breeding programme; thus the development of molecular markers for linkage mapping and QTL analysis is a priority. Various markers, mainly anonymous, have been developed for H. midae; however emphasis is being placed on the development of gene-linked type I molecular markers. The present study investigates and demonstrates the use of public sequence collections to develop type I markers for a species with limited genomic resources, via three strategies: Surveying anonymous H. midae microsatellite markers’ flanking regions to find homology to gene sequences in public databases, cross-species marker transfer of anonymous markers from H. rubra and H. discus hannai demonstrating putative gene associations and lastly EST marker mining (SNP and microsatellites) from various Haliotids and testing transfer to the target species. Approximately 17% of H. midae anonymous markers showed significant similarity to genes. The current study also reports higher cross-species transferability from both H. rubra and H. discus hannai to H. midae (39% and 20.5%, respectively) than previously demonstrated and 15 EST-microsatellites and 16 EST-SNPs were successfully mined. Furthermore, the non-random distribution of microsatellites and high nucleotide diversity in the H. midae genome was confirmed. This is a low cost and time effective method for marker development and presents a continuous and dynamic resource that could be used for future marker development and characterisation as sequence information in public databases grow exponentially. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is die enigste van vyf inheemse spesies van kommersiële waarde. Na die noodgedwonge sluiting van die vissery, is akwakultuur die mees praktiese oplossing om die perlemoen industrie uit te brei. Die huidige fokus is gerig op die implementering van ‘n molekulêre teel-program en dus is die ontwikkeling van molekulêre merkers vir genetiese kartering en kwantitatiewe kenmerk lokus analise, van uiterste belang. Tipe II merkers is voorheen vir die perlemoen ontwikkel, maar huidige tendense lê klem op die ontwikkeling van geen-gekoppelde tipe I merkers. Die huidige studie ondersoek die gebruik van publieke databasisse vir die ontwikkeling van tipe I molekulêre merkers vir ‘n spesie met beperkte genomiese bronne. Drie strategieë is geïmplementeer: Eerstens is ‘n opname gemaak van die homologie van perlemoen tipe II merker-vleuelende volgordes met geen volgordes in databasisse. Verder is die oordraagbaarheid van tipe II merkers vanaf H. rubra en H. discus hannai wat assosiasie met gene toon ondersoek. Laastens is ‘n Uitgedrukte Volgorde Merk (UVM) (Expressed Sequence Tag, EST) merker-ontginnings metode vanaf verskeie Haliotis spesies en toetsing van oordraagbaarheid na die teiken spesie uitgevoer. Ongeveer 17% van die tipe II H. midae merkers het geniese assosiasie getoon. ‘n Hoër tussen-spesie oordraagbaarheid vanaf beide H. rubra en H. discus hannai na H. midae (39% en 20.5%, onderskeidelik) word gerapporteer in vergelyking met vorige studies en 15 UVM-mikrosatelliete en 16 UVM-enkel nukleotied polimorfismes (single nucleotide polimorphism, SNP) is ontwikkel. Verder bevestig die studie die nie-lukrake verspreiding van mikrosatelliete en hoë nukleotied diversiteit in die perlemoen genoom. Die gebruik van publieke databasise vir die ontwikkeling en karakterisering van tipe I molekulêre merkers is tyd- en koste-besparend en bied ‘n volgehoue en dinamiese bron vir toekomstige gebruik.

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.