Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, commonly known as perlemoen, is an economically important gastropod mollusc. Historically, this species maintained a lucrative fisheries sector; however with increasingly lower landings there has now been a shift to aquaculture. Efforts to conserve natural populations and to improve abalone aquaculture production are thus running in parallel. Previous studies reported significant disparities in parental contributions in aquaculture populations that could explain the rapid divergence of commercial stocks from wild populations. Furthermore, subtle, but significant, population differentiation has also been reported for wild populations on the west-, south-, and east coast of the South African coastline. This study therefore aimed to investigate the evolutionary forces, in particularly selection, facilitating population divergence in wild and cultured H. midae populations using a population genomics approach. By using both microsatellite- and single nucleotide polymorphism (SNP) markers it was found that approximately 10% to 27% of the H. midae genome may be influenced by selection. When incorporating these loci into analyses of population differentiation (e.g. AMOVA, factorial correspondence analysis and estimates of genetic distance) there was a marked increase in genetic divergence between wild and cultured populations (especially when using microsatellite loci) and amongst populations from different geographic regions (particularly supported by the SNP loci). The differences in population clustering as highlighted by microsatellite- and SNP markers can most likely be attributed to the genomic distribution of the respective loci: The SNP markers were developed from EST sequences and therefore mostly represents protein structural variation; whereas the microsatellite markers, found to be putatively under selection, were mainly located in regulatory motifs. The results of this study therefore confirmed previous observations of divergence amongst wild- and cultured populations, but more importantly demonstrated that selection is an important factor driving this divergence. In wild populations selection probably facilitates adaptation to local environmental conditions, whilst amongst aquaculture population adaptation to captivity, husbandry practices and artificial selection may be important determinants. There is evidence for population bottlenecks in wild- and cultured populations; nonetheless long-term effective population sizes seem to be large. Amongst the wild populations, however, short-term population sizes appear to be small most likely due to differential spawning rates amongst reproductively active animals leading to temporal fluctuation in genetic diversity. The results indicate that contact between wild and cultured abalone should be minimised to prevent any adverse effects due to outbreeding depression. With regards to conservation, an emphasis on maintaining adaptive diversity of the wild stocks might be warranted. Continued genetic monitoring is advisable for both wild and cultured abalone populations as to optimally manage the abalone resource for both conservation and commercial viability and sustainability. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is 'n ekonomies belangrike buikpotige weekdier. Histories het hierdie spesie 'n winsgewende vissery gehandhaaf, maar met steeds dalende vangste is daar nou 'n verskuiwing na akwakultuur. Pogings om natuurlike populasies te bewaar en perlemoen te verbeter vir verhoogde akwakultuur produksie loop dus in parallel. Vorige studies het bevind dat beduidende verskille in ouerlike bydraes tot die nageslag, in akwakultuur populasies, kan verduidelik hoekom die populasies so vinnig divergeer van die wilde voorouers. Verder, is subtiele, maar betekenisvolle genetiese differensiasie tussen wilde populasies aan die wes-, suid-en ooskus van die land gevind. Hierdie studie is dus daarop gemik om ondersoek in te stel na die mate waartoe verskeie evolusionêre prosesse, in besonder seleksie, die populasie divergensie in beide wilde en gekweekte H. midae teweegbring deur gebruik te maak van ‘n populasie genomika benadering. Deur gebruik te maak van beide mikrosatelliet- en enkel nukleotied polimorfisme (ENP) merkers is dit bevind dat ongeveer 10% tot 27% van die H. midae genoom moontlik beïnvloed word deur seleksie. Met die gebruik van loki onder seleksie tydens die ontleding van populasie differensiasie (bv. AMOVA, faktoriaal korrespondensie analise en genetiese afstand ramings) was daar 'n merkbare toename in genetiese divergensie tussen wilde- en gekweekte populasies (veral wanneer mikrosatelliet loki gebruik is) en onder die populasies vanuit verskillende geografiese gebiede (veral ondersteun deur die ENP loki). Die verskille in die populasie groeperings soos uitgelig deur die mikrosatelliet- en ENP-merkers kan waarskynlik toegeskryf word aan die genomiese verspreiding van die onderskeie loki: Die ENP-merkers is ontwikkel vanaf uitgedrukte volgorde merker (UVM) volgordes en daarom verteenwoordig dit meestal proteïen strukturele veranderinge, terwyl mikrosatelliet merkers eerder in regulatoriese motiewe geleë is. Die resultate van hierdie studie steun dus vorige waarnemings, maar meer belangrik, het dit getoon dat seleksie ‘n betekenisvolle faktor in populasie divergensie in beide wilde en gekweekte populasies is. In wilde populasies fasiliteer seleksie waarskynlik die aanpassing tot plaaslike omgewingstoestande terwyl seleksie onder die gekweekte populasies teweeggebring kan word as gevolg van aanpassing tot aanhouding, boerdery praktyke en kunsmatige seleksie. Daar is bewyse vir populasie bottelnekke in wilde- en gekweekte populasies; tog blyk langtermyn effektiewe populasiegroottes om redelik groot te wees. Onder die wilde populasies is egter gevind dat kort-termyn populasiegroottes klein kan wees, waarskynlik as gevolg van differensiële broeikoerse onder reproduktiewe diere. Dit het tot gevolg dat daar beduidende fluktuasies is in temporale genetiese diversiteit. Die resultate dui daarop dat kontak tussen wilde en gekweekte perlemoen tot 'n minimum beperk moet word om enige nadelige effekte weens uitteling depressie te voorkom. Verder, met betrekking tot bewaring, is ‘n klem op die handhawing van aangepaste genetiese diversitiet dalk geregverdig. Voortgesette genetiese monitering word aanbeveel vir beide wilde- en gekweekte perlemoen populasies ter wille van die optimale bestuur van die perlemoen hulpbron vir beide bewaring en kommersiële lewensvatbaarheid en volhoubaarheid. / International Foundation for Science / National Research Foundation of South Africa / Stellenbosch University

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine producing regions in SA. To date, only three GFLV isolates have been completely sequenced internationally, and limited sequence information is available for SA GFLV isolates. In this study, the first full-length GFLV genome sequence from a South African isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for phylogenetic analysis and revealed that the SA isolates are separate from other sequenced GFLV isolates. Full-length sequences were also used to investigate putative intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2 between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different recombination analysis software packages, the most notable of the putative recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2 5’ UTR might have evolved from an interspecies recombination event between GFLVF13- type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA) associated with South African GFLV isolates was also investigated. In a collaborative study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants were screened for satRNAs. SatRNAs were present in only two plants, containing isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were determined. No significant sequence variation could be detected between the GFLV isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44 RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3, which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen from a common ancestor, which may have originated from an interspecies recombination event. The GFLV-SACH44 satRNA was found to be more closely related to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA clone was however abolished when co-inoculated with an ArMV helper virus, even though it is phylogenetically more closely related to ArMV satRNAs. The full-length satRNA clones were modified to be used as vectors for expression and/or silencing of foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial sequences downstream of the open reading frame of the satRNA. These constructs were cloned into a binary vector to allow for agro-infiltration into plants. Full-length cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA expression and silencing constructs had to be aborted. Attempts to understand this failure revealed that, among other point mutations, four frameshifts had occurred in the RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious. Strategies to correct the mutations are discussed. Once these mutations have been corrected this study can continue in evaluating the use of the satRNA component for expression and silencing analysis. / AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is. Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3, bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3 RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse, met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’ ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44 RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens ‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44 satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44 satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2 klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise gebruik kan word.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sulfated galactans (agarans and carrageenans) are accumulated in the cell wall of various red algae (Rhodophyta) species. These polysaccharides are of commercial importance in the food, pharmaceutical and biotechnology industries due to their unique physicochemical properties. Although having received significant research attention over the last 20 years, events regarding their biosynthesis have not been elucidated. Aiming for the identification of galactosyltransferase (GalT) genes involved in sulfated galactan biosynthesis, cDNA expression libraries were constructed from the prolific agar-producing South African red seaweed Gelidium pristoides (Turner) Kützing and screened by functional complementation of UDP-galactose 4-epimerase deficient mutants (E. coli and S. cerevisiae). Regretfully, no GalTs were identified. The study however yielded the first UGE enzyme described for a red seaweed. Southern hybridization indicated the presence of two UGE copies and confirmed the gene originated from G. pristoides. Bioinformatic analysis of G. pristoides UGE shows amino acid sequence homology to known UGEs from various organisms. The enzyme was shown to be functional in E. coli crude extracts and showed affinity for UDP-D-galactose, similar to other UDP-galactose 4-epimerases. Further, the isolated G. pristoides UGE (GpUGE) was biochemically characterized and its kinetic parameters determined. We found that there was no kinetic difference between this enzyme and previously described UGE enzymes except enhanced activity in the presence of exogenously added NAD+. The UDP-galactose 4-epimerase (UDP-glucose 4-epimerase, UGE, EC 5.1.3.2) is an essential Leloir pathway enzyme facilitating the catalytic inter-conversion between UDP-D-glucose and UDP-D-galactose. UDP-D-galactose is the nucleotide sugar required by galactosyltransferases for the production of red algae sulfated galactans. UGE is suspected as being responsible for supplying UDP-D-galactose for the synthesis of sulfated galactans. In planta monitoring of GpUGE transcript levels with respect to dark and light cycling indicated high expression of the enzyme at night, while expression diminished during the day. The occurrence of increased nocturnal UGE expression correlates with floridean starch breakdown at night. Evidence for hydrolysis of floridean starch is also reflected in obtained G. pristoides transcriptome sequence data. In red algae, floridean starch degradation coincides with sulfated galactan production. The detection of starch hydrolysis enzyme transcripts alongside increased expression of GpUGE suggests the enzyme plays a role in supplying UDP-Dgalactose for sulfated galactan production. As far as we know, this the first report of sequencing and biochemical characterization of a UGE from red seaweed.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Increased demand of petroleum, declining fossil fuel reserves, geopolitical instability and the environmentally detrimental effects of fossil fuels have stimulated research to search for alternative sources of energy such as plant derived biofuels. The main feedstocks for production of first generation biofuels (bioethanol) are currently sucrose and starch, produced by crops such as sugarcane, sugarbeet, maize, and cassava. The use of food crop carbohydrates to produce biofuels is viewed as competing for limited agronomic resources and jeopardizing food security. Plants are also capable of storing sugars in their cell walls in the form of polysaccharides such as cellulose, hemicelluloses and pectin, however those are usually cross-linked with lignin, making their fermentation problematic, and are consequently referred to as lignocellulosics. Current technologies are not sufficient to degrade these cell wall sugars without large energy inputs, therefore making lignocellulosic biomass commercially unviable as a source of sugars for biofuel production. In the present study genes encoding for enzymes for cellulosic, hemicellulosic and starch-like polysaccharides biosynthesis were heterologously expressed to increase the amount of fermentable sugars in sugarcane. Transgenic lines heterologously expressing CsCesA, encoding a cellulose synthase from the marine invertebrate Ciona savignyi showed significant increases in their total cellulose synthase enzyme activity as well as the total cellulose content in internodal tissues. Elevation in cellulose contents was accompanied by a rise in hemicellulosic glucose content and uronic acid amounts, while total lignin was reduced in internodal tissues. Enzymatic saccharification of untreated lignocellulosic biomass of transgenic sugarcane lines had improved glucose release when exposed to cellulose hydrolyzing enzymes. Calli derived from transgenic sugarcane lines ectopically expressing galactomannan biosynthetic sequences ManS and GMGT from the cluster bean (Cyamopsis tetragonoloba) were observed to be capable of producing a galactomannan polysaccharide. However, after regeneration, transgenic sugarcane plants derived from those calli were unable to produce the polymer although the inserted genes were transcribed at the mRNA level. While the ectopic expression of Deinococcus radiodurans amylosucrase protein in the cytosol had a detrimental effect on the growth of transgenic lines (plants showed stunted growth through the 18 months growth period in greenhouse), contrastingly targeting the amylosucrase protein into the vacuole resulted in 3 months old transgenic lines which were having high maltooligosaccharide and soluble sugar (sucrose, glucose and fructose) levels in leaves. After 18 months growing in the greenhouse, the mature transgenic lines were morphologically similar to the untransformed lines and also contained comparable maltooligosaccharide and soluble sugar and starch amounts. The non-biosynthesis of galactomannan and amylose polysaccharides in the matured transgenic plants may be due to post-transcriptional protein processing and or protein instability, possibly explainable by other epigenetic mechanisms taking place to regulate gene expression in the at least allo-octaploid species of sugarcane under investigation in this study.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-­‐virus interaction. This study made use of next-­‐generation sequencing to investigate the effect of virus-­‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐derived sRNAs and vsiRNAs. Broad and narrow size-­‐range datasets were generated using sRNA libraries prepared from total and size-­‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-­‐ and trans-­‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-­‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-­‐halves. Reads associated with tRNA-­‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-­‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple. / AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-­‐virus interaksie is beskikbaar. Hierdie studie het van volgende-­‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl. miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐afkomstige sRNAs en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-­‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte. Deur middel van bioinformatiese data-­‐ontleding is 130 genomiese loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-­‐beskikbare degradoom datastel. Cis-­‐ en trans-­‐natuurlike antisense transkripte (NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook geïdentifiseer. Vir 25 van hierdie loci is 'n in-­‐fase miRNA teiken geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs het ooreengestem met tRFs en tRNA-­‐helftes. Volgordes geassosieer met tRNA-­‐helftes was prominent in die breë grootte-­‐verspreiding datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek voorbereiding op die interpretasie van resultate. Ontleding van die differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies het geen verskil tussen gesonde en geïnfekteerde plante getoon nie, behalwe vir die tRNA-­‐afkomstige sRNAs, wat wel verandering die vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat in hierdie studie geïdentifiseer is, het die bestaande kennis van appel sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige funksionele studies in appel. Hierdie studie bied ook die eerste, en mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in appel.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: see item for full text / AFRIKAANSE OPSOMMING: sien item vir volteks

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines. / AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie. / The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work

Thesis (MSC)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: South Africa is shouldering the highest burden of HIV-infection. Inter-individual differences in response to antiretroviral treatment (ART) and the limited availability of second and third-line ART regimens call for optimising first-line ART in South African populations. Measuring antiretroviral drug levels in patients may be of clinical value as an intermediate indicator of treatment response and may moreover serve to assess the genetic variation underlying differential drug exposure. This study aimed to determine the effect of SNPs in the CYP2B6 gene and efavirenz (EFV) levels measured in hair on ART outcomes in females of two South African populations. Female Xhosa (XH) (n = 81) and Mixed Ancestry (MA) (n = 53) patients receiving the first-line regimen component EFV for at least three months donated saliva for genomic DNA extraction and 20 strands of hair for determination of EFV concentrations by high performance liquid chromatography. Regulatory and exonic regions in the CYP2B6 gene, which codes for the major metabolising enzyme of EFV, were subjected to bi-directional sequence analysis in 15 XH and 15 MA individuals to assess common genetic variation in these populations. Out of 45 single nucleotide polymorphisms (SNPs) identified, 17 SNPs of known or predicted functional importance in EFV metabolism, including four novel SNPs, were genotyped in the entire patient cohort by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. All SNPs were tested for Hardy-Weinberg equilibrium (HWE) and maximum likelihood haplotypes and assessed for an association with EFV levels measured in hair, likelihood of developing adverse drug reactions (ADRs) and virological response to EFV-based treatment. After correcting for age and ethnicity, homozygous carriers of c.516G>T (CYP2B6*6) had significantly increased EFV levels (p = 0.0021; mean: 12.0 ng/mg; IQR: 3.95 – 6.99 ng/mg; n = 12), as did heterozygotes of c.983T>C (CYP2B6*18) (p = 0.0005; mean: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). No CYP2B6*18 homozygotes were detected. No association between EFV levels and virological response was evident (p = 0.8467), but CYP2B6*6 predicted increased odds of virological failure (VL > 80 copies/ml) after correcting for adherence, race, age, weight, time on treatment, baseline CD4, smoking, alcohol and WHO disease stage (p = 0.0328). Carriers of the CYP2B6*1 allele had increased odds (OR = 5) of favourable treatment outcome (VL < 80 copies/ml). In accordance with other studies, this study provides evidence that genetically predisposed poor metabolisers of EFV may be at increased risk of virological failure, possibly following non-adherence. Concurrently, these patients may be more vulnerable to adverse drug reactions and are more frequent in the XH (13%) than MA (4%). These results should be verified in larger patient cohorts, but contribute to a better understanding of the effect of genetic factors on EFV exposure and ART outcome in two South African populations. The outcomes of this study may thus provide recommendations for prospective studies and impact future clinical decisions. / AFRIKAANSE OPSOMMING: Suid-Afrika dra die grootste las van MIV-infeksies. Inter-individuele verskille in reaksie op anti-retrovirale terapie (ART) en die beperkte beskikbaarheid van tweede- en derde-linie ART-reekse regverdig die optimisering van eerste-linie ART in Suid-Afrikaanse bevolkings. Meting van antiretrovirale middel-vlakke in pasiënte, as ‘n intermediêre aanduiding van reaksie op behandeling, kan van kliniese belang wees en kan ook die waarde van die bepaling van genetiese variasie, onderliggend aan differensiële blootstelling aan middels, bepaal. Die doel van hierdie studie is om die effek van enkel-nukleotied polimorfismes (SNPs) in die CYP2B6-geen en efavirenz (EFV)-vlakke in hare op ART-uitkoms te bepaal in vroue van twee Suid-Afrikaanse bevolkingsgroepe. Vroulike Xhosa (XH) (n = 81) en Gemengde Herkoms (GH) (n = 53) pasiënte wat EFV as deel van eerste-linie ART vir ten minste drie maande ontvang het, het speekselmonsters vir genomiese DNA-ekstraksie en 20 hare vir die bepaling van EFV-konsentrasies deur hoë werkverrigting vloeistofchromatografie (“HPLC”) geskenk. Regulatoriese en eksoniese areas in die CYP2B6-geen, wat vir die vernaamste metaboliserende ensiem van EFV kodeer, is deur middel van tweerigting-volgordebepalings-analise in 15 XH en 15 GH individue ondersoek om gemeenskaplike genetiese variasie in hierdie bevolkings te bepaal. Uit ‘n totaal van 45 SNPs wat geïdentifiseer is, is 17 SNPs wat bekende of voorspelde belangrike rolle in EFV-metabolisme speel, insluitend vier nuwe SNPs, ondersoek. Hierdie SNPs is in die volledige pasiënt-kohort gegenotipeer deur polimerase-ketting reaksie gebaseerde restriksie fragment lengte-polimorfisme (PKR-RFLP) analise. Alle SNPs is getoets vir Hardy-Weinberg-ewewig (HWE) en maksimum waarskynlikheidshaplotipes en is geassesseer vir assosiasie met EFV-vlakke gemeet in hare, die waarskynlikheid om ongunstige reaksies tot die middel te ontwikkel en virologiese reaksie op EFV-gebaseerde behandeling. Nadat vir ouderdom en herkoms gekorrigeer is, het homosigotiese draers van c.516G>T (CYP2B6*6) beduidend verhoogde EFV-vlakke (p = 0.0021; gemiddeld: 12.0 ng/mg; IQR: 3.95 – 6.99; n=12) getoon, so ook heterosigote vir c.983T>C (CYP2B6*18) (p = 0.0005; gemiddeld: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). Geen CYP2B6*18 homosigote is gevind nie. Daarbenewens is geen duidelike assosiasie tussen EFV-vlakke en virologiese reaksie gevind nie (p = 0.8467), maar CYP2B6*6 het verhoogde waarskynlikheid op virologiese mislukking (VL > 80 kopieë/ml) getoon nadat daar vir mddel-getrouheid, ras, ouderdom, gewig, tydsduur van behandeling, basis-CD4, rook, alkohol en Wêreld Gesondheids Organisasie siekte-fase gekorrigeer is (p = 0.0328). Draers van die CYP2B6*1-alleel het verhoogde waarskynlikheid (OR = 5) op gunstige behandelingsuitkomste getoon (VL < 80 kopieë/ml). In ooreenstemming met ander studies verskaf hierdie studie bewyse dat pasiënte wat geneties geneig is tot stadige metabolisme van EFV ‘n hoër risiko kan hê vir virologiese mislukking, wat moontlik ‘n gevolg is van middel-ontrouheid. Hierdie pasiënte kan ook meer geneig wees tot vatbaarheid vir ongunstige middel-reaksie en kom meer voor in die XH (13%) as in die MA (4%). Hierdie resultate moet in groter pasiënt-kohorte gestaaf word, maar dra by tot ‘n beter begrip van die effek van genetiese faktore op blootstelling aan EFV en ART-uitkoms in twee Suid-Afrikaanse bevolkings. Die uitkomste van hierdie studie kan dus as aanbevelings gebruik word vir voornemende studies en ook toekomstige kliniese besluite beïnvloed. / The Medical Research Fund (MRC) for funding this project. The University Centre for Studies in Namibia (TUCSIN) and Deutscher Akademischer Austausch-Dienst (DAAD) for financial support

Resistances to bean common mosaic virus, halo, common and Ascochyta blight, angular leaf spot, anthracnose and rust pathogens of beans in South Africa were combined by reverse dichotomous crossing. Full resistance to Uromyces appendiculatus from Carioca 80 was conditioned by a single dominant gene. Partially dominant resistance to Phaeoisariopsis griseola was conditioned by a single gene in Carioca 80 and two genes in PAl 127. Differences in aggressiveness of isolates of Phoma exigua var. exigua were found. Different levels of Ascochyta blight resistance were found in the glasshouse, but field testing showed little difference after flowering. Inoculations of differential cultivars indicated the presence of at least eight races of U. appendiculatus and the a-Brazil race of Colletotrichum lindemuthianum . Inoculations of the old set of halo blight differential cultivars identified races 1 and 2. Forty-five lines with partial resistance to rust were obtained by recurrent selection. Very highly significant differences were noted between ratings of percentage leaf area affected by rust and yield of 23 cultivars planted in field trials. Significant genotype x environment interaction was noted for rust ratings. Ratings at different dates within a trial were correlated with one another, showing few ratings are required per trial, and a correlation of -0.678 between yield and rust rating was found. Inheritance of partial resistance and improved yield of eight cultivars crossed in a full diallel was mostly due to additive effects but non-additive effects were also very highly significant. Reciprocal effects were not significant for yield and rust ratings. Genotype x environment interactions were significant for rust ratings and yield. High estimates of narrow-sense heritability for rust resistance were obtained. No relationship between resistance and time to flowering, pustule size, leaf hairs and stomata was found. Latent periods in unifoliate leaves did not correlate with resistance but a closer match was found in the fourth trifoliate leaves. Inoculations with three additional single-pustule isolates of the 23 parent cultivars indicated the cultivars had similar levels of resistance. Ring necrosis was found in nine cultivars or crosses with them. The ring reaction was conditioned by a single dominant gene and possibly by the epistatic interaction of two dominant genes in Carioca 80. Differences in symptom severity in plants derived from Epicure indicated the possibility of additional gene interaction. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

No abstract available. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.