Dissertationen zum Thema „Grapes Genetics“

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 (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts of sequence data, representing the genomes of multiple organisms of which no prior knowledge is necessarily available. In this study, a metagenomic NGS approach was used to detect multiple novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD) vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were generated from each of the samples and these reads were trimmed and filtered for quality before being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST (Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology Information) database and classified according to database sequences with which they had the highest identity. Twenty-six putative mycovirus species were identified, belonging to the families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be novel mycoviruses not present in the NCBI database. Primers were designed from the de novo assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were performed. These runs generated more and higher quality sequence data than the first sequencing run. Twenty-two of the putative mycoviral sequences initially detected were detected in the subsequent sequence datasets, as well as an additional 29 species not identified in the first HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as many as 19 putative species identified in a single vine. This indicates that the complete virome of diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus (GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates, namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are not known to be naturally associated with two distinctly different fungus genera. Mycoviral sequence data generated in this study can be used to further investigate the diversity and the effect of mycoviruses in grapevine.
AFRIKAANSE OPSOMMING: Metagenomiese studies, wat gebruik maak van volgende-generasie volgordebepalingstegnologie, het die vermoë om die genetiese samestelling van veelvoudige onbekende organismes te bepaal deurdat dit groot hoeveelhede data genereer. Die bogenoemde tegniek was in hierdie studie aangewend om aantal nuwe mikovirusse in die floëem weefsel van wingerd te identifiseer. Dubbelstring-RNS was gesuiwer vanuit twee druiwestokke met rolbladsiekte en drie met shirazsiekte en Illumina HiScanSQ instrument is gebruik om meer as 3.2 miljoen volgorde fragmente te genereer van elk van die monsters. Lae-kwaliteit volgordes was verwyder en die oorblywende kort volgorde fragmente was saamgestel om langer konstrukte te vorm wat met behulp van BLAST soektogte teen die NCBI databasis geïdentifiseer kon word. Ses-en-twintig mikovirus spesies, wat aan die families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae en Totiviridae behoort, was geïdentifiseer. Twee van die geïdentifiseerde mikovirusse, naamlik grapevine-associated chrysovirus (GaCV) en grapevine-associated mycovirus 1 (GaMV-1), was voorheen al in wingerd gekry terwyl die res nuwe mikovirusse is wat tans nie in die NCBI databasis voorkom nie. Inleiers was ontwerp vanaf die saamgestelde mikovirus basisvolgordes en gebruik om wingerd dubbelstring-RNS sowel as swamme wat vanuit die wingerd geïsoleer is te toets vir die teenwoordigheid van hierdie mikovirusse. Slegs twee mikovirusse, wat onderskeidelik verwant is aan sclerotinia sclerotiorum partitivirus S en chalara elegans endornavirus 1 (CeEV-1), kon deur middel van die inleiers in wingerd en swam isolate geïdentifiseer word. Twee addisionele volgordebepalingsreaksies, wat gebruik gemaak het van die Illumina HiScanSQ en ABI SOLiD 5500xl volgordebepalingsplatforms, was gebruik om die teenwoordigheid van mikovirusse in wingerd te bevestig. Groter hoeveelheid volgorde fragmente was geprodusser wat ook van hoër gehalte was as dié van die eerste volgordebepalingsreaksie. Twee-en-twintig mikovirus spesies kon weer geïdentifiseer word, sowel as 29 spesies wat nie in die eerste HiScanSQ basisvolgorde datastelle gevind was nie. Die wingerdstokke wat in hierdie studie ondersoek was, het hoë diversiteit van mikovirusse bevat aangesien daar tot 19 mikovirus spesies in enkele wingerdstok geïdentifiseer was. Dit is aanduiding dat volledige virus profiele van siek wingerdstokke aantal mikovirusse sal insluit. Die vollengte genoomvolgorde van voorheen onbekende endornavirus was saamgestel vanuit een van die tweede HiScanSQ volgorde datastelle. Dit is die eerste mikovirus wat in wingerd gevind word waarvan die volledige genoomvolgorde bepaal is en ons stel die naam grapevine endophyte endornavirus (GEEV) voor vir hierdie virus. Grapevine endophyte endornavirus is die naaste verwant aan CeEV-1 en is dieselfde virus wat voorheen in wingerd dubbelstring-RNS en swam isolate gevind was deur middel van die mikovirus inleiers. Swam isolate waarin GEEV gevind is, was geïdentifiseer as Stemphylium sp. en Aureobasidium pullulans. Dit is van belang dat GEEV in twee swam isolate gevind is wat aan verskillende genusse behoort aangesien hierdie verskynsel nog nie voorheen in die natuur gevind is nie. Mikovirus nukleiensuurvolgordes wat in hierdie studie bepaal was kan gebruik word in toekomstige studies om die verskeidenheid en impak van mikovirusse in wingerd verder te ondersoek.
National Research Foundation (NRF)
Stellenbosch University

Dissertation (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L.
AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species.
AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.

Thesis (MSc)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Grapevine is worldwide an agronomically important crop. Traditionally selective breeding has been used to improve existing cultivars. In the last ten years, however, the advent of biotechnology has shortened these breeding programmes by producing transgenic grapevine. Because this new technology is aimed at the possible genetic manipulation of the ripening process in grape berries, it is important to elucidate all the mechanisms that may be involved in ripening. The aim of the present study was the identification of genes that play an important role during the ripening process in grape berries. This was achieved by investigation of putative differentially expressed genes in ripening Chardonnay berries isolated through subtractive hybridisation. Two subtraction libraries, representing early and late ripening stages were constructed. Four of the ten genes analysed exhibited expression during berry ripening. One of the four genes was expressed in a tissue and stage specific manner. Further characterisation of eight of the DNA and protein sequences revealed that the putative translation products of these clones had homologues that are involved in amongst others cell wall structure in other species. These included UDP-glucose dehydrogenase, which is involved in the synthesis of hemicellulose precursors. The remaining seven clones encoded putative stress response proteins. These included two heat shock proteins, a vacuolar pyrophosphatase and a protein involved in cell division. It is suggested that specific grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion. These processes occur rapidly during the ripening of berries. Accumulation of specific mRNAs can be attributed to part of the normal ripening developmental programme.
AFRIKAANSE OPSOMMING: Druiwe is wêreldwyd 'n belangrike landbougewas en kultivars word tradisioneel deur middel van tydsame selektiewe teling verbeter. Die tyd wat hieraan bestee word, kan verkort word deur die implementering van biotegnologie en die produksie van transgeniese duiwe. Omdat hierdie nuwe tegnologie op die moontlike genetiese manipulering van die rypwordingsproses in druiwe gemik is, is dit belangrik dat alle meganismes betrokke by rypwording ondersoek en verstaan word. Die doel van hierdie studie was om gene wat moontlik tydens die rypwordingsproses in druiwe 'n rol kan speel, te identifiseer. Hierdie doel is bereik deurdat differensieel uitgedrukte gene uit die kultivar Chardonnay geïsoleer is met behulp van verrykingsbiblioteke vanuit jong en volwasse druiwekorrels. Vier van die tien gene wat geanaliseer is, word uitgedruk tydens die rypwordingsproses. Verder het een van die vier gene weefsel- en rypwordingstadium- spesifisiteit getoon. Volledige karakterisering van agt van die DNA- en proteïenvolgordes het aangedui dat die proteïenprodukte van hierdie gene homoloog is aan volgordes wat onder andere by selwandstruktuur betrokke is. Dit sluit UDP-glukose dehidrogenase in, wat betrokke is by die sintese van hemi-sellulose boustene. Die ander sewe gene kodeer vir moontlike spanningsproteïene. Twee hitteskokproteïene, 'n vakuolêre pirofosfatase en 'n proteïen wat betrokke is by selverdeling is geïdentifiseer. Daar word voorgestel dat druiwe mRNA versamel in reaksie op spanningsituasies soos die berging van hoë konsentrasies suikers en selvergroting. Hierdie prosesse vind baie vinnig plaas tydens rypwording. Versameling van spesifieke mRNAs kan toegeskryf word as 'n normale deel van die rypwordingsproses.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast genome. The characterization and sequencing of a number of algal and plant chloroplast genomes has facilitated researchers understanding of cellular functions and metabolism. Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies evolutionary relationships and this organelle offers an alternative means of expressing foreign genes. Although a number of species' chloroplast genomes have been characterized and sequenced, no previous attempts of this kind have been made for a chloroplast genome of the family Vitaceae. In this study, attempts were made to characterize and partially sequence the chloroplast genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1 cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one PstI-digested clone were obtained in this manner. Walking outwards from a previously sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a further -3310 bp region of the Sultana chloroplast genome. BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became available later in the project. It was decided to use these clones for further library construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC clones were obtained. These were compared to sequences found in the NCBI database to find - homologous chloroplast regions and determine the size of each BAC insert. One clone appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region. This clone was selected for further analysis. The BAC clone DNA was isolated and restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies were screened by isolating the plasmid DNA and digesting it with appropriate restriction enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the Atropa belladonna chloroplast genome. A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA contamination. Genomic DNA either originated from the plant nuclear genome or from the bacterial host cells in which the BAC clones were maintained. Many of the clones screened contained genomic DNA, and these could only be identified and removed once the clones had been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the clones screened contained genomic DNA. This kit was specifically designed for the isolation of genomic DNA-free large constructs. The clones obtained from the two strategies provided a good representation of the grapevine chloroplast genome. The only region not represented was the Small Single Copy (SSC) region. Approximately 40% of the grapevine chloroplast genome was covered by these clones. This provides a basis for further genome characterization, physical mapping and sequencing of the grapevine chloroplast genome.
AFRIKAANSE OPSOMMING: Die chloroplasgenoom kodeer VIr 'n hele aantal proteïene wat essensieel is VIr die voortbestaan van 'n plant. Die karakterisering en volgorde bepaling van 'n aantal alg en plant chloroplasgenome het dit. vir navorsers moontlik gemaak om sellulêre funksies en metabolisme van plante te ontrafel. Chloroplas DNA (cpDNA) is ook gebruik om intra- en interspecies evolusionêre verwantskappe vas te stel. Dié organel verskaf ook 'n alternatiewe manier vir die uitdrukking van transgene. Alhoewel die chloroplasgenome van 'n hele aantal species al gekarakteriseer is en die DNA volgorde daarvan bepaal is, is daar nog geen navorsing van bogenoemde aard op die chloroplasgenoom van die Vitaceae familie gedoen rue. In hierdie studie is beoog om die chloroplasgenoom van Vitis vinifera te karakteriseer en gedeeltelike volgordebepaling daarvan te doen. Chloroplas DNA is geïsoleer vanaf Sultana en Sugra 1 kultivars en restriksie-ensiem vertering is gedoen met ensieme wat cpDNA fragmente, met geskikte grootte vir klonering, produseer. Dié fragmente is in 'n plasmiedvektor gekloneer met die haelgeweer-metode en wit kolonies is gesif deur middel van PKR en die kolonieklad metode. Op hierdie manier is drie EcoRI-verteerde klone en een PstI-verteerde kloon verkry. Deur uitwaarts te loop, deur middel van PKR, vanaf 'n druif rrnl6 geenstreek, waarvan die volgorde voorafbepaal is, was dit vir ons moontlik om ook die volgorde te bepaal van 'n verdere ~3310 bp streek van die Sultana chloroplasgenoom. BAC klone wat V. vinifera cv L. Cabernet Sauvignon cpDNA fragmente bevat, het later in die projek beskikbaar geraak. Daar is besluit om hierdie klone, i.p.v. die geïsoleerde cpDNA, te gebruik vir verdere biblioteek konstruksie. Die 5' en 3' entpuntvolgordes van sewe uit die 24 BAC ~lone is verkry. Hierdie volgordes is vergelyk met volgordes in die NCB Idatabasis om homoloë chloroplas streke te identifiseer, en die grootte van elke BAC fragment te bepaal. Die het geblyk dat die hele druif chloroplasgenoom in een van die klone vervat is, behalwe vir 'n 500 bp streek. Die BAC-kloon DNA is geïsoleer en die restriksie-verteerde fragmente is in 'n plasmiedvektor gekloon d.m.V. die haelgeweer-metode. Wit kolonies is gesif deur die isolering van plasmied DNA en die vertering daarvan met geskikte restriksie-ensieme. Die volgorde van die 5' en 3' entpunte van skynbare positiewe klone is bepaal en gekarteer op die Atropa belladonna chloroplasgenoom. In hierdie studie is 'n totaal van 15 klone verkry. Vyf hiervan bevat cpDNA wat vanaf druifblare geïsoleer is, en 10 bevat fragmente wat vanaf die BAC-klone gesubkloneer is. Genorniese DNA kontaminasie was die grootste probleem wat ondervind is tydens beide metodes wat gebruik is vir biblioteek konstruksie. Genomiese DNA was afkomstig vanaf óf die plant nukleêre genoom óf die bakteriële gasheerselle waarin die BAC-klone gehou is. Baie van die klone wat gesif is, het genomiese DNA bevat, en dit kon eers geïdentifiseer en verwyder word nadat die volgorde van die klone bepaal is. Selfs al is 'n kommersiële produk vir BAC-kloon isolasie gebruik, het 31% van die gesifde klone steeds genomiese DNA bevat. Dié kommersiële produk is spesifiek vir die isolasie van groot konstrukte, wat genomiese DNA vry is, ontwerp. Die klone wat deur die twee strategeë verkry is, het 'n goeie verteenwoordiging van die druif chloroplasgenoom gegee. Die enigste streek wat die verteenwoordig is nie, was die Klein Enkelkopie (SSC) streek. Ongeveer 40% van die druif chloroplasgenoom is deur hierdie klone gedek. Dit verskaf 'n basis vir verdere genoomkarakterisering, fisiese kartering en volgordebepaling van die druif chloroplasgenoom.

Dissertation (PhD)--University of Stellenbosch, 2004.
152 Leaves printed single pages, preliminary pages i-xiv and 129 numberd pages. Includes bibliography. List of abbreviations.
ENGLISH ABSTRACT: Despite increased focus on ripening-related gene transcription in grapevine, and the large number of ripening-related cDNAs identified from grapes in recent years, the molecular basis of processes involved in grape berry ripening is still poorly understood. Moreover, little is known about the mechanisms involved in the ripening-related regulation of fruit-specific genes, since the isolation and characterisation of no ripening-related, fruit-specific promoter elements has been reported to date. This study was aimed at the isolation and characterisation of a fruit-specific, ripeningregulated gene from Vitis vinifera L. In the first phase of the work, gene transcription in ripening berries of Cabernet Sauvignon (a good quality wine cultivar) and Clairette blanche (a poor quality wine cultivar) were studied by Amplified Fragment Length Polymorphism analysis of complementary DNA (cDNA-AFLP analysis). Total RNA from immature (14-weeks post flowering, wpf) and mature (18-wpf) berries was used for the analysis. A total of 1 276 cDNA fragments were visualised, of which 175 appeared to be ripening related. Average pairwise difference of the fragments amplified from immature and mature Clairette and Cabernet berries, suggested that ripening-related gene transcription in these two phenotypically different cultivars is remarkably similar. Nevertheless, it was shown that seventy percent of the 175 ripening-related cDNA fragments were cultivar-specific. It was suggested that these differences should be targeted to identify genes related to the phenotypical differences between the two cultivars, but also to identify genes possibly involved berry quality. Moreover, the analysis illustrated the usefulness of cDNA-AFLPs for the analysis of ripening-related gene transcription during grape berry ripening. In the second phase of the work, one of the ripening-related cDNAs identified by the cDNA-AFLP analysis, was selected for further characterisation. This work highlighted the limitation placed on the isolation of a single specific sequence from a cDNA-AFLP gel, indicating the presence of multiple ripening-related genes in a single band excised from a cDNA-AFLP gel. Steps to overcome this limitation of cDNA-AFLP analysis to identify and clone a specific ripening-related gene, were implemented. In short, the band corresponding to the particular ripening-related cDNA was band was excised from the cDNA-AFLP polyacrylamide gel and re-amplified. Northern blot analysis using the re-amplified, uncloned product confirmed the ripening-related transcription demonstrated by cDNA-AFLP analysis. The re-amplified, uncloned product was then cloned. Sequence analysis of two randomly selected candidate clones revealed two distinctly different sequences, of which neither hybridised to messenger RNA from ripening grape berries. Furtheranalysis revealed an additional five cDNAs with terminal sequences corresponding to the selective nucleotides of the primers used for selective amplification, in the re-amplified, uncloned product. Of these, only two were abundantly expressed in ripening grape berries, accounting for the ripeningrelated transcription visualised by cDNA-AFLP analysis. All seven cDNAs identified from the particular excised band were shown to be ripening-regulated during berry development, although most were characterised by low levels of transcription during berry ripening. One of the clones, based on the relative high levels of the transcript and the initiation of gene transcription at the onset of véraison (10- to 12-wpf), was identified for isolation and characterisation of the full length coding sequence. In the third phase of the work, it was shown that this cloned sequence corresponded to a gene encoding a proline-rich protein (PRP) associated with ripening in Merlot and Chardonnay (mrip1, Merlot ripening-induced protein 1). It was shown that the gene is specifically transcribed in the fruit tissue, seed and bunchstems of grapes, from 10-wpf (véraison) to the final stages of berry ripening. The results showed that mrip1 encodes a distinct member of the plant PRP family. Most obvious is the central region of mrip1, which is comprised of eight consecutive repeats of 19 amino acid residues each. In comparison with other grapevine PRPs, mrip1 revealed single amino acid differences and deletion of one of the 19 amino acid residues repeats, all in the central region of mrip1. In situ hybridisation studies showed that accumulation of the mrip1 transcript in the ripening berry is limited to the mesocarp and exocarp cells of the ripening grape berry. No transcript with high sequences similarity to mrip1 could be detected in ripening strawberry or tomato fruit. Based on the properties and proposed function of PRPs, and the results obtained in this study, potential applications for the use of this gene in the control of cell wall architecture in fruits, were proposed. Furthermore, as manipulation of fruit properties in grape berries would be most important in the later stages of ripening, mrip1 was proposed an ideal candidate gene for the isolation of a fruit- and late-ripening-specific promoter to achieve transgene transcription in genetically modified grapevine. The final phase of the work was dedicated to the isolation and characterisation of the mrip1 promoter element. A 5.5 kb sequence corresponding to the mrip1 5’ untranslated (UTR) flanking region was isolated and characterised by sequence analysis. In the 2.8 kb sequence directly upstream of the mrip1 transcription initiation site, several putative cis-acting regulatory elements were identified. These include a spectrum of hormone-, light-, phytochrome-, sugar-and stressresponsive elements, as well as elements implicated in tissue-specific transcription. Analysis of the sequence further upstream (3.6 – 5.5 kb) of the mrip1 transcription initiation site (TIS), revealed the presence of another proline-rich protein directly upstream of mrip1. Sequence identity of this sequence (mprp2) to the mrip1 coding sequence was 88%. This information provided the first insight into the chromosomal organisation of grapevine PRPs. For functional analysis of the mrip1 promoter element, the 2.2 kb sequence directly upstream of the mrip1 TIS, was translationally fused to the sgfpS65T reporter gene. Functionality of the mrip1:sgfpS65T fusion was verified by transient expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacteriummediated transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was developmentally-regulated and specific to the ovary and nectary-tissue of the developing flower. Whilst low in immature flowers, the green fluorescent protein (GFP) rapidly accumulated to the high level of expression visualised in the flower in full-bloom, followed by a decrease in the final stages of ovary development. These observations suggested that the 2.2 kb mrip1 promoter is functional and that this promoter region harbours cis-elements necessary for tissue- and developmental-specific regulation of GFP accumulation. It furthermore suggested that the transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine berries and tobacco flowers. Results presented, suggest that the use of tobacco as heterologous system for the analysis of ripening-related promoters, can be more generally applied. Evidently, characterisation of the mrip1 promoter region contributes towards a better understanding of the regulatory mechanisms involved in non-climacteric fruit ripening, and forms a basis for future experiments defining the cis-acting elements necessary for tissue- and cell-specific gene regulation in fruit, more specifically in grapevine. Moreover, the mrip1 promoter is an ideal candidate for the ripening-related, tissue-specific regulation of transgene transcription in genetically modified grapevine.
AFRIKAANSE OPSOMMING: Ten spyte van toenemende fokus op rypwordings-verwante geentranskripsie in druiwe, en die groot aantal rypwordings-verwante komplimentere DNA (cDNA) fragmente wat gedurende die laaste paar jaar in druiwe geïdentifiseer is, word die molekulêre basis van prosesse betrokke by die rypwording van die druif, steeds swak begryp. Nog te meer, is baie min bekend oor die meganismes betrokke in the rypwordings-verwante regulering van vrugspesifieke gene, aangesien die isolering en karakterisering van nie een rypwordings-verwante, vrugspesifieke promoter tot dusver gerapporteer is nie. Die doel van hierdie studie was die isolering en karakterisering van ‘n vrugspesifieke, rypwordings-verwante geen uit druiwe (Vitis vinifera L). In die eerste fase van die werk, is geentranskripsie in rypwordende druiwekorrels van Cabernet Sauvignon (‘n goeie kwaliteit wyn kultivar) en Clairette blanche (‘n swak kwaliteit wyn kultivar) bestudeer deur middel van cDNA-AFLP vingerafdrukke. Totale RNA van onvolwasse (14-weke na blom vorming) en volwasse (18-weke na blom vorming) druiwekorrels was gebruik vir die analise. ‘n Totaal van 1 276 cDNA fragmente is gevisualiseer, waarvan 175 as rypwordings-verwant voorgekom het. Gemiddelde paarsgewyse verskille van die fragmente wat vanaf onvolwasse en volwasse Clairette en Cabernet druiwekorrels geamplifiseer is, het aangedui dat rypwordingverwante geentranskripsie in die twee kultivars, wat fenotipies baie van mekaar verskil, merkwaardig soortgelyk is. Nieteenstaande, is daar gewys dat sewentig persent van die 175 rypwordings-verwante cDNA fragmente, kultivar-spesifiek is. Daar is voorgestel dat hierdie spesifieke cDNAs verder geanaliseer word om gene betrokke by die fenotipiese verskille tussen die twee kultivars te identifiseer; maar ook om gene te identifiseer wat moontlik by die kwaliteit van die druiwekorrel betrokke is. Voorts, het die analise die bruikbaarheid van die cDNA-AFLP tegniek vir die karakterisering van rypwordings-verwante geentranskripsie in rypwordende druiwekorrels, geïllustreer. In die tweede fase van die werk, is een van die rypwordings-verwante cDNAs wat met die cDNAAFLP analise geïdentifiseer is, geselekteer vir verdere karakterisering. ‘n Aantal rypwordingsverwante cDNAs is in die enkele band wat uit die cDNA-AFLP gel gesny is, geïdentfiseer. Dit het die beperking wat geplaas word op die isolering van ‘n enkel, spesifieke cDNA uit die cDNA-AFLP gel, beklemtoon. Stappe om hierdie beperking te oorkom, en ‘n spesifieke rypwordings-verwante cDNA te identfiseer en te kloneer, is beskryf. In kort, die band oorstemmend met die spesifieke rypwordings-verwante cDNA, is uit die cDNA-AFLP poli-akrielamied gel gesny en gereamplifiseer. Noordelike klad analise waarin die ge-reamplifiseerde, ongekloneerde produk aspeiler gebruik is, het die rypwordings-verwante transkripsie soos deur cDNA-AFLP analise aangedui, bevestig. Die ge-reamplifiseerde, ongekloneerde produk is daarna gekloneer. Nukleotied volgorde bepaling van twee ewekansig geselekteerde kandidaat klone, het twee duidelik verskillende cDNAs aangetoon, waarvan nie een enige hibridisering met boodskapper RNA van rypwordende druiwekorrels getoon het nie. Verder analise het die teenwoordigheid van ‘n verder vyf cDNAs met terminale nukleotied volgordes ooreenstemmend met die selektiewe nukleotiede van die voorlopers wat gebruik is vir selektiewe amplifisering, aangetoon. Van hierdie, het slegs twee hoë vlakke van geentranskripsie in rypwordende druiwekorrels getoon; heel moontlik verteenwoordigend van die rypwordings-verwante geentranskripsie wat met die cDNA-AFLP analise gevisualiseer is. Die studie het gewys dat al sewe cDNAs rypwordings-verwant is, alhoewel die meeste van hierdie cDNAs baie lae vlakke van geentranskripsie tydens duiwekorrel rypwording getoon het. Gebaseer op relatief hoë vlakke van die transkrip, en die inisiering van geen transkripsie met die aanvang van vrugrypwording (véraison, 10- tot 12-weke na blomvorming), is een van die cDNAs geselekteer vir isolering en karakterisering van die vollengte koderings volgorde. In die derde fase van die werk, is dit aangetoon dat hierdie cDNA ooreenstem met ‘n geen wat vir ‘n proline-ryke proteïen (PRP), geassosieerd met vrugrypwording in Merlot en Chardonnay, kodeer. Hierdie geen is genoem Merlot rypwording-geïnduseerde proteïen 1 (mrip1). Die studie het verder aangetoon dat hierdie geen spesifiek in die weefsel van druiwekorrels, saad and stammetjies van die druiwetros getranskribeer word, vanaf 10-weke na blomvorming (véraison) tot 16-weke na blomvorming. Resultate het aangetoon dat mrip1 vir ‘n unieke lid van die plant PRP familie kodeer. Mees opvallend, is die sentrale gedeelte van mrip1, wat uit agt opeenvolgende herhalings van negentien aminosure elk bestaan. In vergelyking met ander druif PRPs, toon mrip1 enkel aminosuur verskille en ‘n delesie van een van die negentien aminosuur herhalings, alles in die sentrale gedeelte van mrip1. In situ hibridisering het getoon dat akkumulering van die mrip1 transkrip net in selle van die mesocarp en eksokarp van die rypwordende druif plaasvind. Geen transkip met hoë nukleotied gelyksoortigheid aan mrip1 kon in rypwordende aarbeie of tamatie vrugte aangetoon word nie. Gebaseer op die eienskappe en funksie van PRPs soos voorgestel in die literatuur, en die bevindinge van hierdie studie, is potensiële toepassings vir die gebruik van die geen in die beheer van selwand argitektuur in vrugte, voorgestel. Verder, aangesien die manipulering van vrugkwaliteit in die druif veral belangrik is vanaf die aanvang van vrugrypwording (véraison), is daar voorgestel dat mrip1 ‘n ideale kandidaat is vir die isolering van ‘n vrugspesifieke en rypwording-verwante promoter vir gebruik in geneties gemodifiseerde druiwe. Die laaste fase van die studie was gewy aan die isolering en karakterisering van die mrip1 promotor element. ‘n 5.5 kb fragment ooreenstemmend met die mrip1 5’ ongetransleerde area is geisoleer en gekarakteriseer deur middel van nukleotied volgorde bepaling. In die 2.8 kb area direk stroomop van die mrip1 transkripsie inisiasie punt (TIS), is verskeie moontlike cis-beherende regulatoriese elemente geïdentifiseer. Hierdie sluit in ‘n spektrum van hormoon-, lig-, fitochroom-, suiker- en stress-reagerende elemente, asook elemente geïmpliseer in weefselspesifieke geentranskripsie. Analise van die area verder stroomop (3.6 – 5.5 kb) van die mrip1 TIS, het die teenwoordigheid van ‘n ander PRP direk stroomop van mrip1 getoon. Nukleotied gelyksoortigheid van hierdie geen (MPRP2) aan die mrip1 koderingsgebied was slegs 88%. Hierdie inligting verskaf die eerste insig in die chromosomale organisasie van druif PRPs. Vir funksionele analise van die mrip1 promotor element, is die 2.2 kb area direk stroomop van die mrip1 TIS transkripsioneel verenig met die sgfpS65T merker geen. Funksionaliteit van die mrip1: sgfpS65T fusie is bevestig deur middel van kortstondige (transient) geenuitdrukking in die perikarp van groenrissie, voordat dit ingevoer is in tabak met Agrobacterium-bemiddelde genetiese transformasie. In transgeniese tabak was transkripsie van die mrip1:sgfpS65T fusie ontwikkelingsstadium-gereguleerd, en spesifiek in die ovarium en heuningsakkie (nektarium) van die ontwikkelende blomme. Terwyl die vlak van geenuitdrukking laag was in die jong blomme, het GFP baie vinnig akkumuleer tot die hoë vlakke wat in die blomme in volle-blom gevisualiseer is. Daarna het dit weer vinnig afgeneem tydens die finale stadiums van ovarium ontwikkeling. Hierdie waarnemings dui daarop dat die 2.2 kb mrip1 promotor element funksioneel is en dit al die nodige cis-beherende regulatoriese element bevat wat nodig is vir weefsel- en ontwikkelingsstadium-spesifieke regulering van GFP akkumulering. Dit dui verder daarop dat transkripsionele aktivering van mrip1 beheer word deur ontwikkelingsstadium seine teenwoordig in beide die druif en tabakblomme. Hierdie resultate stel voor dat tabak meer algemeen gebruik kan word as heteroloë sisteem vir die analise van rypwording-verwante promotors. Duidelik dra die karakterisering van die mrip1 promoter element by tot ‘n beter begrip van die regulatoriese meganismes betrokke by die rypwordingsproses van nie-klimateriese vrugte, en vorm die basis vir toekomstige eksperimente waarin die cis-beherende regulatoriese elemente vir vrug- en sel-spesifieke geen regulering, meer spesifiek die druif, bepaal sal word. Meer nog, is die mrip1 promotor ‘n ideale kandidaat vir weefsel-spefieke en rypwording-verwante regulering van transkripsie van die transgeen in geneties gemodifiseerde druiwe.

Thesis (MSc)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance.
AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3) die beste gekarakteriseerd is. Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié geen was gebruik in die eerste demonstrasie van patogeen-afgeleide weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese en algemene gebruikte metode om virus weerstand in plant gewasse te verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3 geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal. Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer. Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3 CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin (pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor, pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie, Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA 3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die teenwoordigheid van die plant selekteerbare merker geen, bar, op elke konstruksie lui tot dié weerstand. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is deur PKR saam met die bar geen spesifieke inleiers getoets, en geen amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer is nie. Hierdie projek verskaf voorlopige werk vir die daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n poging om virus bestandheid te verskaf.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.

Thesis (MSc (Genetics))--Stellenbosch University, 2008.
The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevine in many grape and wine producing countries around the world. It is renowned not only for the considerable damage it infers to grapevine of its own accord, but in particular for its role in transmitting deleterious viral diseases such as grapevine leafroll disease, Kober stem grooving, Shiraz disease and corky bark. Incidentally, it is an exceptionally tenacious antagonist of grapevine, being resistant to both chemical and biological control mechanisms. As a result, finding an effective strategy for P. ficus control has become a main priority of viticultural industries worldwide. Possible implementation of biotechnological approaches to pest management has resulted in a need for P. ficus genetic data - of which there are currently very little available. The transcribed genes of an organism can be captured in a cDNA library, and the sequences of the various transcripts can then be characterized. In this study altogether five cDNA libraries were constructed from the transcribed sequences of Planococcus ficus (Signoret). Instrumental to their construction was the identification of an RNA extraction protocol that provided large quantities of high quality RNA from mealybugs. The five cDNA libraries were the result of a set of modifications to the Creator™ SMART™ cDNA Library Construction Kit (used for Primary Library construction), and differed mainly with regards to range of insert sizes they contain. Whereas an abundance of short fragments were found in the Primary Library (42% of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries contained inserts of specific size ranges that were more-or-less equally represented. The broadest size range was found in Fractionated Library 4, for which a uniform distribution over the range 0.25 kb - 4 kb was observed. Average insert sizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kb and 2 kb respectively. These results demonstrated the importance of using a protocol designed to circumvent the bias towards incorporation of shorter transcripts in cDNA libraries. Although the libraries were not exhaustively analyzed, the outcome of a pilot investigation indicated that 41% of the submitted sequences had matches in the non-redundant database of the National Center for Biotechnology Information (NCBI, E-value 6 10-5), and that approximately 82% of these were of insect origin. Moreover, two potential targets for an RNAi-mediated approach to P. ficus pest control were identified. With one exception, these sequences seemed to be unique to arthropods. Future research needs to investigate the efficiency by which these sequences are able to constrain P. ficus proliferation, and their suitability for grapevine transformation.

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, 2014.
ENGLISH ABSTRACT: South Africa is ranked eighth in the world as far as international wine production is concerned and in terms of area under bearing vines South Africa is ranked 12th. In 2011 the wine industry contributed R4 204.4 million to the South African economy in state revenue from wine products. The importance of viticulture to the economy of South Africa forces the industry to limit the effect of all disease causing pathogens in order to keep their competitive edge. Aster yellows (AY) phytoplasma 16SrI-B subgroup was reported for the first time in grapevine (Vitis vinifera L. (Vitaceae)) in South Africa in 2006. Worldwide phytoplasma diseases of grapevine cause serious damage ranging from lower yields to the death of vines. The lack of knowledge about the epidemiology of AY disease makes it difficult to determine the impact of the disease on the South African wine industry. The aim of this study was to conduct surveys in disease-affected vineyards in the Vredendal region to determine the incidence and spatial distribution of the disease in a variety of cultivars. The field surveys based on visual symptoms of AY disease were confirmed by polymerase chain reaction (PCR). A survey was also conducted in and around AY-infected vineyards in search of possible alternative host plants of the phytoplasma. Spatial distribution of AY-affected vines were analysed using the PATCHY spatial analysis package. A rapid decline of AY-affected Chardonnay eventually leading to the death of vines was observed, confirming the sensitivity of Chardonnay towards grapevine yellows infections. Symptomless AY infections occurred and AY could not be detected in all symptomatic vines, which indicate uneven distribution of AY in individual vines. Molecular analyses using PCR-RFLP showed that all vines sampled in the Vredendal vicinity contained AY phytoplasma only. No phytoplasmas were present in any weeds or other possible host plants tested. Although the mean yearly disease incidences of Chardonnay (29.95%) and Chenin blanc (16.64%) were higher than Pinotage (5.80%) over the four-year survey period, there was no statistically significant difference between the disease incidences of these three cultivars. The mean yearly disease incidence showed a trend over time and the disease incidence of the first year was significantly lower than that of the other years. Chardonnay showed a cumulative disease incidence of 37.77% at the end of the 4-year study which means that Chardonnay vineyards can be 100% AY infected in ten years’ time. Spatial distribution patterns of AY-infected vines were mostly non-random with clustering of disease affected vines along and across vine rows. With the exception of one vineyard, aggregation of AY-affected vines mostly occurred on the edge of vineyards adjacent to infected vineyards. This epidemiological study gives an indication of the sensitivity of the different cultivars towards AY, the tempo of spreading and the future impact of the disease on the South African wine industry. It also contributes valuable information towards the development of a management strategy for grapevine yellows disease in South African vineyards.
AFRIKAANSE OPSOMMING: Suid- Afrika is op agtste op die wêreld ranglys wat internasionale produksie van wyn aan betref, en in terme van oppervlakte onder wingerd, is Suid-Afrika 12de. In 2011 het die wynbedryf R4 204.4 miljoen tot die Suid-Afrikaanse ekonomie bygedra in staats inkomste uit wyn produkte. Die belangrikheid van wingerd tot die ekonomie van Suid-Afrika dwing die bedryf om die effek van alle siekteveroorsakende patogene te beperk, om sodoende hul kompeterende voordeel te behou. Aster vergeling (AY) fitoplasma 16SrI-B subgroep is vir die eerste keer in 2006 in wingerd (Vitis vinifera L. (Vitaceae)) in Suid-Afrika waargeneem. Fitoplasma siektes van wingerd veroorsaak wêreldwyd ernstige skade wat wissel van laer opbrengste tot die afsterf van wingerdstokke. Die gebrek aan kennis oor die epidemiologie van astervergeling siekte maak dit moeilik om die impak van die siekte op die Suid-Afrikaanse wynbedryf te bepaal. Die doel van hierdie studie was om ‘n opname te maak in siekte geaffekteerde wingerde in die Vredendal omgewing om sodoende siekte voorkoms en verspreidingspatrone van die siekte in 'n verskeidenheid van kultivars te bepaal. Die veld opnames, gebaseer op visuele simptome van aster vergeling siekte, was bevestig deur polimerase kettingreaksie (PKR). ‘n Opname is ook in en om aster vergeling geaffekteerde wingerde uitgevoer, op soek na moontlike alternatiewe gasheer plante van die fitoplasma. Verspreidingspatrone van astervergeling geaffekteerde wingerde is ontleed met behulp van die PATCHY ruimtelike analise pakket. 'n Vinnige agteruitgang van AY geaffekteerde Chardonnay, wat uiteindelik gelei het tot die afsterf van wingerde, is waargeneem, wat die sensitiwiteit van Chardonnay teenoor wingerdvergeling infeksie bevestig. Simptoomlose astervergeling fitoplasma infeksies kom voor en astervergeling fitoplasma kon nie opgespoor word in alle simptomatiese wingerdstokke nie, wat op oneweredige verspreiding van AY fitoplasma in individuele wingerdstokke dui. Molekulêre ontledings met behulp van PKR-RFLP het getoon dat alle wingerdstokke, wat in die Vredendal omgewing getoets is, slegs astervergeling fitoplasma bevat. Geen fitoplasmas was teenwoordig in enige onkruide of ander moontlike gasheer plante. Hoewel die gemiddelde jaarlikse siekte voorkoms van Chardonnay (29,95%) en Chenin Blanc (16,64%) oor die vier-jaar opname periode hoër was as dié van Pinotage (5,80%), was daar geen statisties beduidende verskil tussen die siekte voorkoms van hierdie drie kultivars nie. Die gemiddelde jaarlikse siekte voorkoms het 'n tendens oor tyd getoon, en die siekte voorkoms van die eerste jaar was betekenisvol laer as dié van die ander jare. Chardonnay het ‘n kumulatiewe siekte voorkoms van 37.77% aan die einde van die 4-jaar studie getoon, wat beteken dat Chardonnay wingerde binne 10 jaar 100% besmet kan wees met AY. Verspreidingspatrone van AY geaffekteerde wingerdstokke was meestal nie-ewekansig met bondeling van geaffekteerde wingerdstokke in en oor wingerd rye. Bondeling van AY geaffekteerde wingerdstokke het, met die uitsondering van een wingerd, meestal op die kant van wingerde aanliggend aan besmette wingerde, voorgekom. Die epidemiologiese studie gee 'n aanduiding van die sensitiwiteit van die verskillende kultivars ten opsigte van AY, die tempo van die verspreiding en die toekomstige impak van die siekte op die Suid-Afrikaanse wynbedryf. Dit dra ook waardevolle inligting by tot die ontwikkeling van 'n strategie vir die bestuur van wingerdvergeling siekte in Suid-Afrikaanse wingerde.

Thesis (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole.
AFRIKAANSE OPSOMMING: Plante het nie die vermoë om stres te ontwyk nie en moet dus vinnig op veranderinge in hulomgewingstoestande kan reageer. Indien hulle nie die fotosinteseproses kan kontroleer en reguleer tydens streskondisies nie, sal dit tot die vorming van hoogs reaktiewe suurstofspesies lei, wat beide die pigmente en proteiene van die fotosintetiese apparaat sal beskadig. Lig is die primêre energiebron vir fotosintese en daarom is dit noodsaaklik dat plante deurgaans 'n dinamiese balans tussen fotosintese en fotobeskerming moet handhaaf. Karotenoiëde is voorlopers vir die vorming van absisiensuur, maar meer belangrik vir die plant, ook integrale komponente van die fotosintetiese apparaat. Tydens fotosintese word karotenoiëde vir die opneem van lig benodig, terwyl dit ook die fotosintetiese apparaat beskerm wanneer lig 'n versadigingspunt bereik vir fotosintese. Weens die belang van karotenoiëde vir plant- en menslike gesondheid (as Vitamiene A voorlopers), het hierdie studie beoog om gene te isoleer en karakteriseer wat direk of indirek 'n rol in karoteenbiosintese in Vitis vinifera speel. Elf vollengte- en agt gedeeltelike gene is geïsoleer, gekloneer, en gekarakteriseer. Hierdie gene kan in die volgende biosintetiese paaie gegroepeer word: (i) die 1- deoksi-D-xilulose 5-fosfaat (DOXP)/2-C-metiel-D-eritritol-4-fosfaat (MEP) pad (d.w.s. die plastiediese isopenteniel difosfaat biosintetiese pad); (ii) die mevalonaat pad (d.w.s. the sitosoliese/mitokondriale IPP biosintetiese pad); (iii) die karotenoiëd biosintetiese pad; (iv) die absisiensuur biosintetiese pad (as 'n afbraak produk van karotenoiëde) en die algemene isoprenoïed bisintetiese paaie (as voorlopers van karotenoiëde ). Die vollengte gene (d.w.s. vanaf die geskatte ATG tot die STOP kodon) van DOXP-sintase (DXS), 4-hidroksi-3-metielbut-2-eniel difosfaatreduktase (lytB), IPPisomerase (IPI), 3-hidroksi-3-metielglutariel koensiem A sintase (HMGS), fitoeën sintase (PSY), likopeen p-siklase (LBCY), p-karoteen hidroksilase (BCH), zeaxantien oksidase (ZEP), 9-cis-epoksi karotenoiëd dioksigenase (NCED), farnesiel difosfaat sintase (FPS)en geranielgeraniel difosfaat sintase (GGPS) is met behulp van. RTPKR vanaf eDNA geïsoleer. Die vollengte genomiese kopieë en die verwagte promotors van die DXS, PSY, LBCY, BCH, NCED and ZEP gene is ook geïsoleer d.m.v. die opstel en sifting van subgenomiese biblioteke. Vergelykende analises van die genoom- en eDNA kopieë het insiggewende data oor die genomiese rangskikking van die gene, insluitende die intron-ekson setels in V. vinifera gelewer. Die kopiegetalle van DXS, PSY, LBCY, BCH, NCED en ZEP is bepaal. DXS, PSY, BCH en ZEP is in die Vitis-genoom as enkel kopieë teenwoordig, terwyl LBCYen NCED twee en drie kopieë, repektiewelik, beslaan. Die transkipsionele aktiwiteit van die verwagte promotors van ses van die geïsoleerde gene (naamlik DXS, PSY, LBCY, BCH, ZEP en NCED) is d.m.v. 'n tydelike verklikkergeentoets ondersoek. Geeneen van die promotors het die transkripsie van die verklikkergeen bemiddel nie. Die transkripsie van die gene is egter wel bewys deur van northernhibridisasies en/of RT-PKR gebruik te maak. Die promotors van hierdie gene kan dus as transkipsioneel aktief beskou word. Voorlopige uitdrukkingsprofiele van PSY, LBCY, BCH, en ZEP is in verskillende plantorgane bepaal; die profiele was deurgaans hoër in fotosinteties aktiewe weefsels. Die uitdrukkingsprofiele van die gene is verder ook in reaksie op verskillende induktiewe behandelings (absisiensuur, NaCI en beskadiging) bepaal. Vyf van die vollengte gene (IPI, GGPS, PSY, LBCYen BCH) is funksioneel bewys in 'n bakteriese funksionele kleurkomplementasiesisteem. In silico analises van die afgeleide proteïene van al elf geïsoleerde gene het 'n hoë vlak van homologie met ooreenstemende proteiene van ander plantspesies getoon. Gekonserveerde domeine is ook in die proteïensekwense van die geïsoleerde gene teenwoordig. Hierdie proteïene het deurgaans dieselfde domeinprofiele vertoontoon as homoloë in ander spesies (bakterieë, alge en plante). Die sub-sellulêre teikening van die gene kon voorspel word deur die seinpeptiede in die proteiensekwense te eien. Aangesien hierdie gene betrokke is by biosintetiese paaie wat in diskrete kompartemente plaasvind; is die sub-selluiêre lokalisering van hierdie proteïene voorspelbaar. Die karotenoïed biosintetiese gene (PSY, LBCY, BCH en ZEP), die absisiensuur biosintetiese geen, NCED, sowel as die DOXP/MEP pad se gene (DXS, lytB en IPI) kom almal in die chloroplast voor. Die mevalonaatpadgeen, HMGS, word na beide die sitosol en die mitokondria geteiken, terwyl die algemene isoprenoïed voorlopergene, FPS en GGPS, onderskeidelik na die sitosol en die chloroplast geteiken word. Die verkreë voorspellings stem met die lokalisering van die biosintetiese paaie in die selooreen. Om ons kennis rakende karotenoïed biosintese en veral hulle funksie(s) in plante te verbreed, het ons een van die geïsoleerde gene, BCH, in die model plant, Nicotiana tabacum, konstitutief ooruitgedruk. Plante wat die BCH geen in die "sense" orientasie uitgedruk het, kon normale fotosintetiese aktiwiteit handhaaf onder kondisies wat foto-inhibisie en foto-osidatiewe skade in die ongetransformeerde kontrole plante veroorsaak het. Hierdie resultaat is met chlorofil fluoresensie analises aangetoon terwyl dit met CO2 assimilasie- en huidmondjie geleidingseksperimente bevestig is. Chlorofil fluoresensie metings het aangetoon dat die beskermingsvermoë van die transgeniese plante verhoog is, en dit dan die plante in staat stelom fotosintetese te handhaaf onder streskondisies van hoë lig. Proteïen analises het aangetoon dat 'n integrale fotosintetiese proteien, die 01 proteïen, word veral deur die verhoogde zeaxantien vlakke in die BCH transgeniese plante beskerm. Plante wat verminderde zeaxantien vlakke gehad het, weens die konstitutiewe ooruitdrukking van die BCH geen in die anti-"sense" orientasie, het die teenoorgestelde bewys. Met ander woorde. laer BCH vlakke (en dus laer zeaxantien vlakke) het tot plante wat meer vatbaar was vir hoë lig geïnduseerde stress gelei. Hierdie resultate het die essensiële beskermende rol wat karotenoiede tydens fotosintese speel, uitgelig. Die vermoë om hierdie beskermende meganisme te manipuleer in transgenies plante het aangetoon dat die sisteem in plante, alhoewel effektief, nie optimaal is nie. Enige verbetering in 'n plant se inherente vermoë om streskondisies te weerstaan sal die plant se algemene gesondheid en dus produktiwiteit beïnvloed. As sulks sal hierdie in meeste gewasspesies toepassing vind. Hierdie studie beskryf die isolering en karakterisering van gene wat direk, of indirek, by karotenoïedbiosintese betrokke is. Verdere studies, en veral die manipulering van hierdie gene in model plante, sal die fisiologiese rol van spesifieke karotenoïeede in fotosintese, en die plant as 'n geheel, ontrafel.

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type member of the genus Ampelovirus in the family Closteroviridae and is considered to be the main contributing agent of grapevine leafroll disease (GLD) worldwide. A metagenomic sequencing study of a grapevine leafroll-diseased vineyard led to the discovery of a new variant of GLRaV-3 in South Africa. This new variant was most related to a New Zealand isolate, NZ-1. In this study, we sequenced two isolates, GH11 and GH30, of the new variant group of GLRaV-3. These isolates have less than 70% nucleotide (nt) identity to other known GLRaV-3 variants, indicating that they should be considered variants of a different strain of GLRaV-3. We propose that the GLRaV-3-like virus identified in this study be grouped together with NZ-1 and some Napa Valley isolates as Group VI of GLRaV-3. This study also provided further evidence that next-generation sequencing is an invaluable approach to identify novel viruses and variants, in that the draft sequence generated with bioinformatic tools in this study was 98% identical to the GH11 sequence generated using Sanger sequencing. The study further confirmed that the industry standard ELISA is still an effective GLRaV-3 diagnostic method and that it is able to detect all known variant groups of GLRaV-3. However, this assay is not able to differentiate between GLRaV-3 variant groups. In the current study therefore, a real-time RT-PCR was designed that is able to detect GLRaV-3 variant groups I, II, III and VI, using a single primer pair targeting the Hsp70h gene of GLRaV-3. If high-resolution melting (HRM) curve analysis is added to the real-time RT-PCR, it is possible to differentiate between variant groups based on three melting point intervals. The RT-PCR HRM assay provides a more sensitive and rapid tool to detect and differentiate between different GLRaV-3 variant groups. Finally, a multiplex RT-PCR was designed to differentiate between the variant groups present in South Africa. This multiplex RT-PCR offers a validation method for the RT-PCR HRM and provides an end-point PCR alternative for variant identification. In order to investigate the spread and impact of different GLRaV-3 variants in vineyards, sensitive diagnostic techniques are a necessity. The abovementioned tools will contribute to the understanding of the pathogenesis of GLD and aid epidemiological studies to investigate how these different GLRaV-3 variant groups are spreading, the association of specific GLRaV-3 variants to disease symptoms and the mealybug vector transmission efficiency for each GLRaV-3 variant.
AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ’n lid van die genus Ampelovirus in die familie Closteroviridae en word beskou as die hoof bydraende faktor van wingerd-rolbladsiekte wêreldwyd. ’n Metagenomiese studie het bewys dat daar ’n nuwe variant van GLRaV-3 bestaan wat nog nie voorheen in Suid Afrika opgespoor kon word met die huidige opsporingsmetodes nie. Hierdie nuwe variant was naaste verwant aan ’n Nieu-Seelandse isolaat, NZ-1. In hierdie studie is die genoomvolgorde van twee isolate, GH11 en GH30, van hierdie nuwe GLRaV-3 variant groep bepaal. Hierdie twee isolate was minder as 70% identies aan ander GLRaV-3 variante, wat daarop dui dat hulle as variante van ’n nuwe virus-ras beskou behoort te word. Ons beveel aan dat hierdie GLRaV-3-verwante virus geklassifiseer word saam met die NZ-1 isolaat en ander isolate uit Kalifornië, as groep VI van GLRaV-3. Hierdie studie het ook verdere bewyse verskaf dat volgende-generasie volgordebepalingstegnologie ’n waardevolle benadering is om nuwe virusse en variante te identifiseer, deurdat die huidige studie gewys het dat die voorlopige volgorde, wat gegenereer is deur bioinformatika-instrumente, 98% identies was aan die GH11 volgorde wat met Sanger volgordebepaling verkry was. Hierdie studie het ook gevind dat die industrie-standaard ELISA, nog steeds ’n effektiewe GLRaV-3 diagnostiese metode is en wel infeksies, veroorsaak deur al die variant-groepe, sal kan identifiseer. Die ELISA toets is egter nie in staat om te onderskei tussen GLRaV-3 variant-groepe nie. In hierdie studie is ’n variant-identifiseerbare in-tyd tru-transkripsie polimerase ketting reaksie (PKR) ontwerp wat GLRaV-3 variant-groepe I, II, III en VI kan identifiseer deur middel van ’n enkele inleier-stel wat die GLRaV-3 Hsp70h-geen teiken. As hoë-resolusie smeltingskurwe-analise bygevoeg word by die in-tyd tru-transkripsie PKR, is dit moontlik om te onderskei tussen variant-groepe op grond van drie smeltingspunt intervalle. Die tru-transkripsie hoë-resolusie smeltingskurwe-toets verskaf meer sensitiewe en geoutomatiseerde metodes om GLRaV-3 variant-groepe te identifiseer en te onderskei. ’n Veelvuldige tru-transkripsie PKR is ook ontwerp om tussen variante wat tans in Suid-Afrika aangetref word, te onderskei en te dien as ’n valideringsmetode vir die in-tyd tru-transkripsie hoë-resolusie smeltingskurwe-toets. Sensitiewe en akkurate toetse, soos bogenoemde, is noodsaaklik vir die bestudering van die verspreiding en impak van die verskillende GLRaV-3 variante in wingerd. Hierdie metodes kan gebruik word om kennis ten opsigte van rolblad patogenese te verbreed en om by te dra tot epidemiologiese studies wat ondersoek hoe hierdie variant-groepe versprei, of daar ’n assosiasie bestaan tussen ’n spesifieke variant en siekte-simptome en of daar ’n verskil is in die witluisvektor oordragseffektiwitiet vir elke GLRaV-3 variant.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Thesis (MSc (Genetics))--University of Stellenbosch, 2011.
Includes bibliography
ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development.
AFRIKAANSE OPSOMMING: Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie-spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP-aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus-bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the ever-growing world population. Among the many challenges faced by food producers, crop losses and damage caused by fungal plant pathogens is a major problem. The study of fungal pathogens and the interaction between plants and fungi is therefore essential, and has been carried out for many years. Much has been learned in this time, but the full mechanisms of the various modes of fungal attack and plant defence have still not been elucidated. Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first enzymes to be secreted at the start of fungal attack, and many of them are considered to be essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of fungi, including Botrytis cinerea. An important defence mechanism of plants involves the inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act on fungal ePGs. Many different PGIPs from a number of diverse plant species have been described to date. They are known to have differential inhibition capabilities that often result from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains. Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP activity and a strong resistance response against Botrytis, leading to the characterisation of these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic analysis of these lines it was found that they display differential expression of cell wall metabolism genes and biochemical characteristics that might indicate possible cell wall strengthening compared to wild-type tobacco under uninfecting conditions. The V. vinifera cultivars are all very susceptible to fungal attack, whereas other grapevine species, specifically the North American Vitis species, are known for their strong resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have previously been isolated from these more resistant species. The amino acid sequences of the active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera PGIPs separated into 14 subgroups based on their active domain sequences. For this study, one PGIP from each group was selected for functional analysis in tobacco. The selected PGIP-encoding genes were transformed into tobacco by means of Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco populations consisting of three to twelve individually transformed lines of nine different nonvinifera PGIPs were obtained. A subset of the resultant transgenic lines was infected with B. cinerea in two independent whole plant infections over 11-14 days in order to investigate the disease resistance afforded by the various PGIPs towards this fungus. A line from the previously characterised VvPGIP1 population was included as reference; all the infections were contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs displayed very strong disease reduction in comparison to the WT control: after initial primary lesion formation, the spread of fungal infection was contained and halted in these lines, while wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions, indicating very strong resistance phenotypes. The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1 line and the WT control were also used to further evaluate the previous observation that overexpression might lead to changes in expression of cell wall genes. Analysis of the expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic lines tested. This confirmed previous results and have confirmed in all grapevine PGIP overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell wall. From previous work it is known that downregulation of this gene leads to strengthening of the wall. The results obtained in this study showed that the PGIP-specific resistance phenotype seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance towards B. cinerea provides an interesting angle for further investigation into the structural differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen interactions.
AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie. Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme (SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas. Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage (Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1 plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van infeksie. Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies, spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak. Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op baie sterk weerstandsfenotipes. Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van ‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die plantselwand. Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen interaksies.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.
Includes bibliography.
Title page: Dept. of Genetics, Faculty of Science
ENGLISH ABSTRACT: Next-generation sequencing technologies are increasingly used in metagenomic studies, largely due to the high sequence data throughput capacity and unbiased approach in determining the genetic composition of an unknown environmental sample. This study investigated the applicability of the Illumina next-generation sequencing platform for metagenomic sequencing of grapevine viruses to provide the first complete viral profile, or virome, of a diseased vineyard. Leaf material was harvested from 44 randomly selected vines in a leafroll-diseased vineyard in South Africa. Sample material was pooled and double-stranded RNA extracted. The dsRNA was sequenced as a paired-end sequencing run using the Illumina sequencing-by-synthesis technique, and more than 19 million sequence reads, equivalent to approximately 837 megabases of metagenomic sequence data, were obtained. Of these data, approximately 400 megabases could be assembled into 449 scaffolds, using the de novo assembler Velvet. These scaffolds were subjected to BLAST searches against the NCBI databases and top hit scores were used for virus identification. Based on the BLAST results, suitable sequences were selected from the NCBI database and used as reference sequence in MAQ mapping assemblies. The bioinformatic analyses allowed for the determination of the virus species present, the most prominent variants, and the relative abundance of each. Four known grapevine viral pathogens were identified. Grapevine leafroll-associated virus 3, representing 59% of the analyzed short read sequence data, was identified as the most prominent virus species. Three variants of this virus were detected: GP18 was the most abundant, followed by a minor Cl766/NY1 variant and a potential novel grapevine leafroll-associated ampelovirus. A single Grapevine rupestris stem pitting ]associated virus variant, similar to SG1, and a Grapevine virus A variant, a member of molecular group III, were identified. This study is also the first to report the presence of Grapevine virus E (GVE) in South African vineyards. Grapevine virus E was further genetically characterized and the genome sequence of GVE isolate SA94 determined. The GVE SA94 genome sequence, 7568 nucleotides in length, is the first complete genome sequence for the virus species. The genome organization of GVE SA94 is typical of vitiviruses, but in contrast to other RNA viruses, the AlkB domain is located within the helicase domain in open reading frame 1 (ORF 1). Grapevine virus E SA94 shares nearly 100% nucleotide identity with the Japanese TvP15 isolate and GVE 3404, a de novo scaffold generated from the metagenomic sequence data. Bioinformatic analysis of metagenomic sequence data further revealed the presence of three fungus-infecting viral families, Chrysoviridae, Totiviridae and the unclassified dsRNA virus, Fusarium graminearum dsRNA mycovirus 4. A virus from the family Chrysoviridae, similar to Penicillium chrysogenum virus, was the second most abundant virus detected. We demonstrated the successful application of a short read sequencing technology, such as the Illumina platform, for viral profiling of an infected vineyard. To our knowledge this is the first application of the Illumina technology for this purpose.
AFRIKAANSE OPSOMMING: Volgende-generasie tegnologie om basis volgordes van nukleiensure te bepaal, word al meer gebruik in metagenomiese studies. Dit is veral weens die hoe data-omset kapasiteit en onbevooroordeelde aanslag in die bepaling van die genetiese samestelling van onbekende omgewingsmonsters. Hierdie studie het die aanwending van die Illumina volgende-generasie volgorde-bepalingsplatform in 'n metagenomiese studie van wingerdvirusse, ondersoek. Dit het ten doel gehad om die eerste volledige virus profiel, of viroom, van 'n geinfekteerde wingerd saam te stel. Blaarmateriaal is verkry vanaf 44 lukraak-gekose wingerdstokke in 'n rolblad-geinfekteerde wingerd in Suid-Afrika. Monster materiaal is saamgevoeg en dubbelstring-RNS geekstraheer. Die dubbelstring-RNS is onderwerp aan gepaarde-ent volgorde-bepaling deur gebruik te maak van die Illumina volgorde-bepaling-deur-sintese tegniek. Meer as 19 miljoen volgorde reekse, ekwivalent aan ongeveer 837 megabasisse volgorde data, is verkry. Van hierdie data kon ongeveer 400 megabasisse saamgevoeg word in 449 konstrukte ("scaffolds"), deur gebruik te maak van die de novo samesteller Velvet. Hierdie konstrukte is onderwerp aan BLAST soektogte teen die NCBI databasisse en die hoogste trefslag-telling is gebruik vir virus identifikasie. Op grond van die "BLAST" resultate is geskikte volgordes geselekteer vanaf die NCBI databasis en gebruik as verwysingvolgordes in MAQ kartering-analises. Met die bioinfomatika analises kon die virus spesies teenwoordig, asook die mees prominente variante en relatiewe voorkoms van elk, bepaal word. Vier bekende virus wingerdpatogene is geidentifiseer. Grapevine leafroll-associated virus 3, verteenwoordig deur 59% van die geanaliseerde kort-reeks volgorde data, is identifiseer as die mees prominente virus spesie. Drie variante van die virus is in die wingerdmonster opgespoor: GP18 kom die mees algemeen voor, gevolg deur 'n CL-766/NY1 variant en 'n potensiele nuwe wingerd rolblad-geassosieerde ampelovirus. 'n Enkele Grapevine rupestris stem pitting-associated virus variant, soortgelyk aan SG1, en 'n Grapevine virus A variant, 'n lid van molekulere groep III, is geidentifiseer. Hierdie studie is ook die eerste om die teenwoordigheid van Grapevine virus E (GVE) in Suid-Afrikaanse wingerde te rapporteer. Grapevine virus E is verder geneties gekarakteriseer en die genoomvolgorde van GVE isolaat SA94 is bepaal. Die GVE SA94 genoomvolgorde, 7568 nukleotiede lank, is die eerste volledige genoomvolgorde vir hierdie virus spesie. Die genoomorganisasie is tipies van vitivirusse, maar in kontras met ander RNA virusse is die AlkB domein binne-in die helikase domein van oopleesraam 1 (ORF 1) geleë. Grapevine virus E SA94 deel byna 100% nukleotied identiteit met die Japannese TvP15 isolaat en GVE 3404, 'n de novo konstruk gegenereer vanaf die metagenomiese volgorde data. Bioinformatika analises van die metagenomiese volgorde data het verder die teenwoordigheid van drie swam-infekterende virus families, die Chrysoviridae, Totiviridae en ongeklassifiseerde dubbelstring-RNS virus, Fusarium graminearum dsRNA mycovirus 4, aangetoon. 'n Virus van die Chrysoviridae familie, soortgelyk aan Penicillium chrysogenum virus, het die tweede meeste voorgekom in die wingerd monster. Hierdie studie demonstreer die suksesvolle toepassing van 'n kort reeks volgorde-bepalingstegnologie soos die Illumina platform, vir die opstel van 'n virusprofiel van 'n geinfekteerde wingerd. Sover ons kennis strek is hierdie die eerste aanwending van die Illumina tegnologie vir hierdie doel.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010.
Includes bibliography.
Title page: Dept. of Genetics, Faculty of Science
ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described.
AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010.
Includes bibliography.
ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines using crude plant extracts in both quantitative and conventional reverse transcription polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of differentiating GRSPaV sequence variants. The application of this technique is for the largescale screening of diseased vines to associate sequence variants of GRSPaV with disease symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays (qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant RNA-polymerase and triple gene block movement protein). The qPCR-HRM technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR products and cloned cDNA gave the most consistent amplification plots and dissociation profiles. RT-PCR products generated from total RNA extracts were used as template for qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the three regions of the GRSPaV genome was performed with published GenBank sequences to confirm the HRM data. The dominant sequence variants found in the South African sample set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected samples can in future be subjected to qPCR-HRM assays developed during this study. This can be performed to establish similarity to known genotypes and therefore phylogenetic groups. Mixed infection of sequence variants and quasi-species were a common occurrence. The assay will be useful in establishing correlation of specific genotypes to different phenotypical expression of viral disease. This could provide insight into the etiology of diseases associated with GRSPaV.
AFRIKAANSE OPSOMMING: Die eerste doel van hierdie studie was om die virus wat met Rupestris-stamverpitting (Grapevine rupestris stem pitting-associated virus of “GRSPaV”) in wingerd verbind is, vinnig en betroubaar op te spoor. Dit is bereik deur 94 wingerdstokke vir die teenwoordigheid van die virus te toets met beide kwantitatiewe en konvensionele trutranskripsie polimerase kettingreaksies (RT - PCR) vanaf ongesuiwerde plant-ekstraksies. Die tweede doel was die daarstelling van ’n tegniek om onderskeid te tref tussen variante van GRSPaV met verskillende nukleotiedvolgordes. Hierdie tegniek kan op groot skaal gebruik word om ge-affekteerde wingerdstokke te toets om sodoende siektesimptome met spesifieke variante van GRSPaV te verbind. Ge-neste kwantitatiewe polimerase-kettingreaksies (qPCR) en hoë-resolusie smelt-analises (HRM) is ontwikkel vir drie streke van die GRSPaV-genoom (mantelproteïen, RNS-afhanklike RNS-polimerase en trippelgeenblok bewegingsproteïen). Die tegniek van qPCR-HRM met die hoë-versadingingskleurstof EvaGreen™ en die Rotor- Gene™ 6000 ontleder se geldigheid is bevestig deur vergelyking met ’n paneel van sestien virus-isolate waarvan die volgorde reeds bepaal is. Verdunde RT-PCR-produkte en gekloneerde DNS het die mees konsekwente amplifikasie-uitstipping en dissosiasieprofiele opgelewer. RT-PCR-produkte wat vanuit totale RNS-ekstrakte verkry is, is as templaat vir qPCR-HRM-analises gebruik. Dieselfde produkte is ook gebruik, om die volgorde van sestien monsters in drie streke direk te bepaal. Die gemiddelde amplifikasiedoeltreffendheid van die qPCR was 1.52±0.04. Gebruiker-gedefinieerde genotipes is deur middel van outooproeping teen ’n vertroue-interval van 70% uitgevoer. Filogenetiese analises vir drie streke van die GRSPaV-genoom is uitgevoer met gepubliseerde GenBank-volgordes om die HRMdata te bevestig. Die dominante volgorde-variante in die stel Suid-Afrikaanse monsters het ooreengestem met Groep II, vollengte-verwysingsvariant GRSPaV-SG1. Monsters wat met GRSPaV besmet is kan in die toekoms onderwerp word aan die qPCR-HRM-analises wat in hierdie studie ontwikkel is. Dit kan uitgevoer word om ooreenkomste met bekende genotipes te bepaal, en dus ook met filogenetiese groepe. Die besmetting van plante met meer as een volgorde-variant het algemeen voorgekom. Die kwasi-spesies populasie-struktuur van die virus het ook gedurig na vore gekom. Die toets sal nuttig wees in die bepaling van korrelasies tussen spesifieke genotipes en verskillende fenotipiese voorkomste van virussiektes. Dit kan insig verleen in die etiologie van siektes wat met GRSPaV verbind word.

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

Orientador: Laura Sterian Ward
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A Doença de Graves é um processo imunológico em que a combinação de fatores genéticos e ambientais é fundamental. Vários genes têm sido propostos como envolvidos no desenvolvimento da doença, particularmente os genes do sistema HLA e os polimorfismos do gene CTLA-4. Com relação à Oftalmopatia de Graves, além dos fatores genéticos, o tabagismo é conhecido como um importante fator de seu desenvolvimento. Nós previamente demonstramos que a herança de polimorfismos em genes relacionados ao metabolismo e detoxificação de xenobióticos, além de genes relacionados a apoptose celular, como TP53, tem uma importante ação na suscetibilidade para essas doenças. Nosso objetivo foi determinar as relações entre o polimorfismo do gene CTLA-4 região promotora -318, CYP1A1m1, GSTP1 e 72TP53 e os riscos para Doença de Graves e Oftalmopatia de Graves. Avaliar a relação entre fatores clínicos (idade, sexo, etnia, tabagismo, tamanho do bócio), laboratoriais (TSH, T4livre, T3total, ANTITPO, antiTg, TRAb), de imagem (captação tiroidiana com tecnécio ou iodo131) e de tipo de tratamento (radioiodo, drogas antitiroidianas e cirurgia) . Estudamos um total de 193 pacientes com Doença de Graves comparados com 200 indivíduos-controle, pareados por idade e etnia. A análise genética foi realizada através de PCR-RFLP em DNA extraído de sangue periférico. Confirmando nossos dados anteriores, as variantes dos genes GSTP1 (p = 0,0007) e CYP1A1m1 (p < 0,0001 ) eram mais frequentes em pacientes com Doença de Graves do que em controles, mas isso não ocorreu com o gene CTLA-4, região promotora -318 (p = 0,12) ou nas variantes 72TP53 (p = 0,27). Estudando o mesmo polimorfismo na Oftalmopatia de Graves, observamos que o genótipo CT do CTLA-4 região promotora -318 era mais frequente entre pacientes com oftalmopatia do que aqueles sem esse acometimento (p = 0,005). Constatamos que o gênero masculino (p = 0,01), o tabagismo (p= 0,001) e o bócio (p = 0,01) eram fatores de risco para o desenvolvimento da Oftalmopatia de Graves. A análise de regressão logística identificou o genótipo CT na região promotora posição -318 do gene CTLA-4 (p=0,04; OR=4,13, 95% IC=1,01-16,8) e o sexo masculino (p=0,01; OR=7,59, IC=1,55-37,23) como fatores de risco para o desenvolvimento de oftalmopatia. Concluímos que os polimorfismos dos genes GSTP1 e CYP1A1m1, mas não da região promotora do gene CTLA-4 posição -318, estão relacionados à suscetibilidade a Doença de Graves na população brasileira investigada. Foram fatores importantes no desenvolvimento da Oftalmopatia de Graves: polimorfismo do gene CTLA-4 região promotora posição -318 sexo masculino, tabagismo e tamanho do bócio.
Abstract: Graves' disease is an immunologic process in which the combination of environmental and genetic factors is fundamental. Various genes have been proposed as involved in the development of the disease, particularly HLA system genes and the polymorphisms of CTLA-4 gene. Concerning Graves' ophthalmopathy, besides the genetic factor, smoking is a well accepted factor of its development. We previously demonstrated that the inheritance of polymorphisms in genes related to the metabolism and detoxification of xenobiotics, such as CYP1A1 and GSTP1 genes, besides the genes related to cellular apoptosis, such as TP53, have an important role in the susceptibility to these diseases. Our objective was to determine the relationship among CTLA-4 gene and CYP1A1m1, GSTM1, GSTP1 and 72TP53 genes in the risk for Graves' disease. We studied a total 193 Graves' disease patients compared to 200 control individuals, matched for age and ethnicity. The genetic analysis was done with the use of PCR-RFLP in DNA extracted from peripheral blood. Reinforcing our previous data, GSTP1 (p=0.0007) and CYP1A1m1 (p<0.0001) variants were more frequent among Graves' disease patients than in controls, but this did not happen to CTLA-4 position 318 (p=0.12) or to TP53 variants (p=0.27). Studying the same polymorphisms in Graves' ophthalmopathy, we observed that the CT genotype of CTLA-4 position 318 was more frequent among patients with than in patients without ophthalmopathy (p=0.005). We found male gender (p=0.01), smoking (p=0.001) and goiter (p=0.01) to be factors of risk to the development of Graves' ophthalmopathy. The logistic regression analysis identified CTLA-4 position 318 CT genotype (p=0.04; OR=4.13, 95% IC=1.01-16.8) and male gender (p=0.01; OR=7.59, IC=1.55-37.23) as factors of risk to the development of ophthalmopathy. We concluded that the polymorphisms of GSTP1 and CYP1A1, but not of the promoter region of CTLA4, are related to the susceptibility of Graves' disease in the Brazilian population investigated while CTLA-4, male, smoking and the size of the goiter were important in the development of Graves' ophthalmopathy.
Doutorado
Clinica Medica
Doutor em Clínica Médica

Engineering design is a complex task, which typically involves multiple physical domains. It can benefit from a modeling tool that can represent different domains in a unified manner. For complex designs, optimization by traditional techniques (such as gradient-based methods) may not be appropriate. Evolutionary methodologies may be used in design optimization of complex engineering systems. This research is based on a framework for evolutionary design system consisting of a machine health monitoring system, model-based evolutionary design optimization system, and a design expert system. The design weaknesses and faults of an existing engineering system are identified using a machine health monitoring system. Then under supervision of the design expert system, an optimal design is evolved using genetic programming. This thesis primarily addresses the modeling and evolutionary aspects and their integration. The thesis develops the integrated system consisting of bond-graph modeling and genetic programming. The performance of the developed system is studied using both experimentation and simulation. The drawbacks of the fitness calculation methodologies that are presented in literature are identified and improved fitness functions are developed in the present work. A methodology to automatically obtain the state-space model of a system represented using bond graphs is also developed. While previous researchers have investigated the integration of bond graphs and genetic programming in design, they have not applied the method in a real engineering system. The present work specifically addresses the application of the developed method for design improvement for an industrial machine. For this purpose a linear bond graph model of the industrial fish processing machine is developed and the parameter values are identified using genetic programming. The design of the actual system is modified according to the evolved bond graph model and the results are validated using the data from the actual engineering system. The proposed method is applicable particularly to actual systems, first because the initial model can be tested by comparing its simulated results with the corresponding results from the actual system, and second because the design improvements as suggested by the evolutionary design framework may be implemented and tested against the behaviour of the corresponding model.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Department of Mathematics, 2013.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 149-155).
In this thesis, we present several algorithmic results for problems in spectral graph theory and computational biology. The first part concerns the problem of spectral sparsification. It is known that every dense graph can be approximated in a strong sense by a sparse subgraph, known as a spectral sparsifier of the graph. Furthermore, researchers have recently developed efficient algorithms for computing such approximations. We show how to make these algorithms faster, and also give a substantial improvement in space efficiency. Since sparsification is an important first step in speeding up approximation algorithms for many graph problems, our results have numerous applications. In the second part of the thesis, we consider the problem of inferring human population history from genetic data. We give an efficient and principled algorithm for using single nucleotide polymorphism (SNP) data to infer admixture history of various populations, and apply it to show that Europeans have evidence of mixture with ancient Siberians. Finally, we turn to the problem of RNA secondary structure design. In this problem, we want to find RNA sequences that fold to a given secondary structure. We propose a novel global sampling approach, based on the recently developed RNAmutants algorithm, and show that it has numerous desirable properties when compared to existing solutions. Our method can prove useful for developing the next generation of RNA design algorithms.
by Alex Levin.
Ph.D.

Thesis (MScAgric)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes
AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第11839号
論農博第2593号
新制||農||928(附属図書館)
学位論文||H18||N4151(農学部図書室)
24381
UT51-2006-J536
(主査)教授 米森 敬三, 教授 矢澤 進, 教授 谷坂 隆俊
学位規則第4条第2項該当

The one- and two-sided bipartite graph drawing problem alms to find a layout of a bipartite graph, with vertices of the two parts placed on parallel imaginary lines, that has the minimum number of edge-crossings. Vertices of one part are in fixed positions for the one-sided problem, whereas all vertices are free to move along their lines in the two-sided version. Many different heuristics exist for finding approximations to these problems, which are NP-hard. New sequential and parallel methods for producing drawings with low edgecrossings are investigated and compared to existing algorithms, notably Penalty Minimisation and Sifting, the current leaders. For the one-sided problem, new methods that include those based on simple stochastic hillclimbing, simulated annealing and genet.ic algorithms were tested. The new block-crossover genetic algorithm produced very good results with lower crossings than existing methods, although it tended to be slower. However, time was a secondary aim, the priority being to achieve low numbers of crossings. This algorithm can also be seeded with the output of an existing algorithm to improve results; combining with Penalty Minimisation in this way improved both the speed and number of crossings. Four parallel methods for the one-sided problem have been created, although two were abandoned because they gave bad results for even simple graphs. The other two methods, based on stochastic hill-climbing, produced acceptable results in faster times than similar sequential methods. PVM was used as the parallel communication system. Two new heuristics were studied for the two-sided problem, for which the only known existing method is to apply one-sided algorithms iteratively. The first is based on a heuristic for the linear arrangment problem; the second is a method of performing stochastic hill-climbing on two sides. A way of applying anyone-sided algorithm iteratively was also created. The linear arrangement method based on the Koren-Harel multi-scale algorithm achieved the best results, outperforming iterative Barycentre (previously the best method) and iterative Penalty Minimisation. Another area of this work created three new heuristics for the k-planar drawing problem where k > 1. These are the first known practical algorithms to solve this problem. A sequential genetic algorithm based on TimGA is devised to work on k-planar graphs. Two parallel algorithms, one island model and the other a 'mesh' model, are also given. Comparison of results for k = 2 indicate that the parallel island method is better than the other two methods. MPI was used for the parallel communication. Overall, 14 new methods are introduced, of which 10 were developed into working algorithms. For the one-sided bipartite graph drawing problem the new block-crossover genetic algorithm can produce drawings with lower crossings than the current best available algorithms. The parallel methods do not perform as well as the sequential ones, although they generally achieved the same results faster. All of the new two-sided methods worked well; the weighted two-sided swap stochastic hill-climbing method was comparable to the existing best method, iterative Barycentre, and generally produced drawings with lower crossings, although it suffered with needing a good termination condition. The new methods based on the linear arrangement problem consistently produced drawings with lower crossings than iterative Barycentre, although they were nearly always slower. A new parallel algorithm for the k-planar drawing problem, based on the island model, generally created drawings with the lowest edge-crossings, although no algorithms were known to exist to make comparisons.

Thesis (MSc)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are potent toxins produced by a wide range of evolutionarily diverse plants. These toxins cause cell death by physically dismantling ribosomal RNA and shutting down protein synthesis. They also have a strong antiviral activity. Some believe that the antiviral property of RIPs is a function of ribosomal inactivation, others believe that the two properties are unrelated. RIPs are non-specific in their antiviral activity. Transgenic RIPexpressing plants are resistant to a wide spectrum of viruses. Many different viruses threaten grapevine. It is not practical to design individual remedies for each of these viruses. In this study, we screen the grapevine genome for the presence of a RIP gene using degenerate PCR primers. If a RIP gene does exist in grapevine, it is not being expressed in a useful way. We also clone several well-documented RIP genes from various plants into pGEM-T Easy: dianthin from Dianthus caryophyllus; p-Iuffin from Luffa octandra and mirabilis antiviral protein (MAP) from Mirabilis jalapa. These isolated genes are then subcloned into a selection of expression vectors: dianthin into pKK223-3, a bacterial expression vector; p-Iuffin into pCambia3301, a plant expression vector; and MAP into pFLAG, a yeast expression vector. The constructs prepared in this project may be used for the synthesis of RIP molecules. The exogenous application of RIPs has been shown to protect plants from viruses. Transformation of grapevine with the RIP-containing plant expression vector may result in a variety of vine that is resistant to a wide range viruses. This thesis describes preliminary work in an attempt to impart broad-spectrum antiviral resistance to grapevine.
AFRIKAANSE OPSOMMING: Ribosomale-inaktiverende proteïne (RIPs) is kragtige toksienes wat deur 'n wye verskeidenheid evolusionêr diverse plante verskaf word. Hierdie toksienes veroorsaak die dood van die selle deur fisies die ribosomale RNA af te breek en proteïensintese stop te sit. Hulle toon ook 'n sterk antivirale aktiwiteit. Sommige voel dat die antivirale eienskap van RIPs 'n funksie van ribosomale inaktivering is, terwyl ander glo dat die twee eienskappe onafhanklik optree. RIPs is in hul antivirale aktiwiteit onspesifiek. Transgeniese RIP-weergewende plante toon weerstand teen 'n wye spektrum virusse. Wingerd word deur baie verskillende virusse aangeval. Dit is onprakties om spesifieke teenmiddels vir elk van die virusse te ontwerp. In hierdie studie word die wingerdgenoom vir die voorkoms van 'n RIP-geen ondersoek, deur die gebruik van degeneratiewe PKR primers. As daar wel 'n RIP-geen in wingerd voorkom, word dit nie in 'n nuttige manier uitgedruk nie. Ons het ook 'n groep goedgedokumentêre RIP-gene vanuit verskeie plante in pGEM- T Easy gekloneer: dianthin vanuit Dianthus caryophyllus; p-Iuffin vanuit Luffa octandra; en mirabilis antivirale proteïen (MAP) vanuit Mirabilis jalapa. Hierdie geïsoleerde gene is toe in verskeie uitdrukkingsvektore gesubkloneer: dianthin in pKK223-3, 'n bakterïele uitdrukkingsvektor; p-Iuffin in pCambia3301, 'n plant uitdrukkingsvektor; en MAP in pFLAG, 'n gis uitdrukkingsvektor. Die constructs wat in hierdie projek voorberei is, kan gebruik word vir die sintese van RIP molekules. Dit is gevind dat die eksogeniese toepassing van RIPs plante teen virus-infeksie beskerm. Die transformasie van wingerd met die RIP-bevattende plant ekspressievektor kan 'n wingerd wat teen 'n wye verskeidenheid virusse bestand is tot stand bring. Hierdie tesis beskryf die voorlopige werk in 'n poging om breë-spektrum antivirale weerstand in wingerd deelagtig te maak.

As doenças autoimunes da tireoide (DAIT) representadas, principalmente pela doença de Graves (DG) e tireoidite de Hashimoto (TH), apresentam causas multifatoriais, incluindo fatores genéticos e ambientais. Diversos genes estão envolvidos, entre eles CTLA4, PTPN22 e mais recentemente IFIH1, principalmente quando associados a diabetes mellitus tipo 1 (DM1). OBJETIVOS: Determinar a frequência alélica e genotípica dos polimorfismos: -318C/T, A49G e CT60 do CTLA4, R620W do PTPN22 e A946T do IFIH1 em pacientes pediátricos portadores de DG, TH e DM1 associado a TH e em uma população controle normal, e determinar a associação com características clínicas e laboratoriais. MATERIAL E MÉTODOS: Foram estudados 142 pacientes menores de 21 anos ao diagnóstico. Os dados clínicos e laboratoriais foram obtidos em prontuário. A genotipagem foi realizada por PCR em tempo real de todos os polimorfismos. Dados clínicos e laboratoriais como sexo, idade de início, bócio, anticorpos anti-GAD, IA2 e IAA e níveis de TRAb e anti-TPO foram analisados. RESULTADOS: Sessenta e dois pacientes foram diagnosticados com DG, com idade média ao diagnóstico (IMD) de 10,8 ± 4,4 anos, sendo 43 do sexo feminino; TH esteve presente em 44 pacientes, sendo 37 meninas, com IMD de 10,3 (± 2,9 anos); e 36 pacientes com DM1 associado a TH, sendo 21 meninas, com IMD de 6,2 (± 4,0 anos) no momento do diagnóstico de DM1 e de 11,6 (± 4,6 anos) ao diagnóstico de TH. O grupo controle foi constituído por 81 indivíduos sem diabetes, função tireoidiana normal e ausência de anticorpos antitireoidianos. Todos os polimorfismos estavam em equilíbrio de Hardy-Weinberg. O polimorfismo -318C/T não esteve associado com nenhum dos grupos. O alelo de risco G do polimorfismo A49G foi mais frequente em pacientes com TH (p=0,047) e o genótipo patogênico (AG e GG) foi mais frequente em pacientes com DG (p=0,049). O alelo de risco G do polimorfismo CT60 foi mais frequente apenas em pacientes com DG (p=0,035). O alelo de risco T do polimorfismo R620W foi mais frequente em pacientes com DM1 associado a TH (p=0,043). O alelo de risco T do polimorfismo A946T foi mais frequente em pacientes com DM1 associado a TH (p=0,009), assim como o genótipo patogênico (CT e TT) quando comparado ao grupo controle (p=0,007). Quando agrupamos todas as DAIT, observamos associação com A49G (p=0,024) e R620W (p=0,047). Quando agrupamos apenas pacientes com TH, encontramos diferença no A49G (p=0,018) e no A946T (p=0,041). O polimorfismo CT60 foi associado com menor duração da terapia medicamentosa no grupo DG (p=0,004), mas não com os níveis de TRAb ou presença de bócio. No DM1 com HT, o alelo de risco do A49G foi mais frequentemente encontrado no sexo masculino (p=0,04); R620W foi relacionado com a presença de bócio (p=0,03), enquanto A946T foi associado com níveis de anti-TPO mais baixos (p=0,047). Os níveis de anti-GAD, IA2 e Resumo IAA não foram associados aos polimorfismos. CONCLUSÃO: Encontramos associações genéticas diferentes entre os pacientes com DAIT, sugerindo que as crianças possuem provavelmente padrões genéticos distintos, apesar do menor tempo de exposição a fatores ambientais
Autoimmune thyroid diseases (AITD) represented by Graves\' disease (GD) and Hashimoto\'s thyroiditis (HT), have multifactorial causes, including genetic and environmental factors. Several genes are involved, including CTLA4, PTPN22 and more recently IFIH1, especially when associated with type 1 diabetes (T1D). OBJECTIVES: To determine the allelic and genotypic frequencies of the polymorphisms: -318C/T, A49G and CT60 of CTLA4, R620W of PTPN22 and A946T of IFIH1 in pediatric patients with GD, HT and T1D associated with HT and in a control population and determine association with clinical and laboratory features. MATERIAL AND METHODS: We studied 142 patients under 21 years at diagnosis. Clinical and laboratory data were obtained from medical records. Genotyping was performed by real time PCR for all polymorphisms. Clinical and laboratory data were analyzed, such as gender, age of onset, goiter, anti-GAD, IA2 and IAA levels and TRAb and anti-TPO levels. RESULTS: Sixty-two patients were diagnosed with GD, with mean age at diagnosis (MAD) of 10.8 ± 4.4 years, 43 females; HT was present in 44 patients, 37 girls, MAD 10.3 (± 2.9 years) and type 1 diabetes associated with HT was present in 36 patients, 21 girls, MAD 6.2 (± 4.0 years) at diagnosis of T1D and 11.6 (± 4.6 years) at diagnosis of HT. Control group consisted of 81 subjects without diabetes, normal thyroid function and absence of antithyroid antibodies. All polymorphisms were in Hardy-Weinberg equilibrium. The polymorphism -318C/T was not associated with any of the groups. The risk allele G of A49G polymorphism was more frequent in patients with HT (p=0.047) and the pathogenic genotype (AG and GG) was more frequent in patients with GD (p=0.049). The risk allele G of CT60 polymorphism was more frequent only in patients with GD (p=0.035). The risk allele T of R620W polymorphism was more frequent in patients with T1D associated with HT (p=0.043). The risk allele T of A946T polymorphism was more frequent in patients with T1D associated with HT (p=0.009), as well as the pathogenic genotype (CT and TT) compared to control group (p=0.007). When all AITD is grouped, we observed association with A49G (p=0.024) and R620W (p=0.047). Only when patients with HT are grouped, we found differences in A49G (p=0.018) and A946T (p=0.041). CT60 polymorphism was associated with a shorter duration of drug therapy on GD group (p=0.004), but no association with TRAb levels or presence of goiter were found. In T1D with HT, the risk allele of A49G was more often found in males (p=0.04); R620W was associated with presence of goiter (p=0.03), while A946T was associated with anti-TPO levels (p=0.047). The anti-GAD, IAA and IA2 levels were not associated with any polymorphisms. CONCLUSION: We found different genetic associations among patients with AITD, suggesting that children are likely to have distinct genetic background, despite shorter exposure to environmental factors

Background: Many questions in statistical genomics can be formulated in terms of variable selection of candidate biological factors for modeling a trait or quantity of interest. Often, in these applications, additional covariates describing clinical, demographical or experimental effects must be included a priori as mandatory covariates while allowing the selection of a large number of candidate or optional variables. As genomic studies routinely require mandatory covariates, it is of interest to propose principled methods of variable selection that can incorporate mandatory covariates. Methods: In this article, we propose the ridge-lasso hybrid estimator (ridle), a new penalized regression method that simultaneously estimates coefficients of mandatory covariates while allowing selection for others. The ridle provides a principled approach to mitigate effects of multicollinearity among the mandatory covariates and possible dependency between mandatory and optional variables. We provide detailed empirical and theoretical studies to evaluate our method. In addition, we develop an efficient algorithm for the ridle. Software, based on efficient Fortran code with R-language wrappers, is publicly and freely available at https://sites.google.com/site/zhongyindaye/software. Results: The ridle is useful when mandatory predictors are known to be significant due to prior knowledge or must be kept for additional analysis. Both theoretical and comprehensive simulation studies have shown that the ridle to be advantageous when mandatory covariates are correlated with the irrelevant optional predictors or are highly correlated among themselves. A microarray gene expression analysis of the histologic grades of breast cancer has identified 24 genes, in which 2 genes are selected only by the ridle among current methods and found to be associated with tumor grade. Conclusions: In this article, we proposed the ridle as a principled sparse regression method for the selection of optional variables while incorporating mandatory ones. Results suggest that the ridle is advantageous when mandatory covariates are correlated with the irrelevant optional predictors or are highly correlated among themselves.

Context-based Reasoning (CxBR) and Contextual Graphs (CxGs) involve the modeling of human behavior in autonomous and decision-support situations in which optimal human decision-making is of utmost importance. Both formalisms use the notion of contexts to allow the implementation of intelligent agents equipped with a context sensitive knowledge base. However, CxBR uses a set of discrete contexts, implying that models created using CxBR operate within one context at a given time interval. CxGs use a continuous context-based representation for a given problem-solving scenario for decision-support processes. Both formalisms use contexts dynamically by continuously changing between necessary contexts as needed in appropriate instances. This thesis identifies a synergy between these two formalisms by looking into their similarities and differences. It became clear during the research that each paradigm was designed with a very specific family of problems in mind. Thus, CXBR best implements models of autonomous agents in environment, while CxGs is best implemented in a decision support setting that requires the development of decision-making procedures. Cross applications were implemented on each and the results are discussed.
M.S.Cp.E.
Department of Electrical and Computer Engineering
Engineering and Computer Science
Computer Engineering

A geração de grades horárias de qualidade é um fator crítico em qualquer instituição de ensino, tanto em escolas de ensino fundamental/médio como em universidades. Este problema é considerado complexo, pois devem ser relacionados e otimizados diversos recursos, tais como horários, disciplinas, professores e alunos. Em grande parte das instituições de ensino, a geração de grades horárias é realizada manualmente, o que vem a tornar este processo custoso e sujeito a falhas. Diversas abordagens são também encontradas na literatura para resolução deste problema, nas quais foram aplicados métodos de busca estocástica, devido à sua inerente complexidade. As estratégias de busca formuladas e comparadas no presente trabalho foram baseadas no uso de algoritmos genéticos e de sistemas imunológicos artificiais. Tais técnicas foram capazes de fornecer soluções de qualidade para o problema de geração automática de grades horárias. Neste trabalho foram desenvolvidos dois sistemas de apoio à decisão, nos quais foram combinadas técnicas heurísticas aos algoritmos genéticos e ao algoritmo de seleção clonal. O propósito desta investigação é realizar uma análise comparativa entre as duas técnicas a fim de verificar qual delas apresenta resultados mais promissores para a resolução do problema de geração automática de grades horárias.
The generation of timetables with good quality is a critical factor in any educational institution. This is considered a complex problem because it involves several types of information, such as schedules, course subjects, teachers and students. Several search strategies have been applied to solve timetabling problems, whose constraints may vary from one educational institution to another. Most educational institutions still prepare their timetables manually, which is a highly time-consuming process and subjected to errors. Several approaches to solve this problem are also found in technical studies, which use stochastic search methods due to the problems complexity. The search optimization methods used in this work to solve the timetabling problem are genetic algorithms and the clonal selection algorithm, whose satisfactory results when applied to optimization problems are reported in the literature. Two decision support systems were developed in this work, combining heuristic techniques with the genetic algorithms and the clonal selection algorithm. The purpose of this research is to make a comparative analysis of these two techniques in order to determine which one offers the most promising results for solving the timetabling problem.

Le développement de la baie de raisin est un processus dynamique présentant une courbe de croissance sigmoïde avec deux phases de croissance séparée par une phase de latence. Il se caractérise par une biosynthèse coordonnée de métabolites primaires et secondaires. À la fin de la phase de latence, un phénomène appelé véraison se produit, au cours duquel le fruit commence à prendre de la couleur et le processus de maturation est initié. Les anthocyanes sont responsables de la coloration des baies et la régulation de leur biosynthèse été largement étudiée. Cependant, peu d'études ont porté sur la caractérisation métabolique et génétique de variétés de baies de couleurs fortement contrastées, dans un même fond génétique. En combinant la technologie RNAseq et des analyses métabolomiques, nous avons effectué une caractérisation comparée de deux variétés de raisin de table : Chimenti Globe (CG, rose pâle) et Red Globe (RG, rouge foncé). L'originalité de ce modèle est que CG a été généré à partir d'un événement de mutation spontanée de RG, dans un vignoble de production, permettant ainsi l’étude de la régulation de la biosynthèse des anthocyanes dans un même fond génétique. L'analyse du contenu métabolique des baies a démontré l'importance des stades de développement, de la véraison à la maturation, dans les deux variétés de raisin. En particulier, des différences marquées dans les concentrations de certains métabolites de la voie des phénylpropanoïdes (shikimate, UDP-glucose, phénylalanine) et du tréhalose-6-phosphate ont été mises en évidence en post-véraison. De plus, les différences entre les variétés étaient dues à des changements dans les métabolites liés à la biosynthèse du saccharose et de l'anthocyanine. Les baies de CG ne contenaient que des anthocyanines dihydroxylées (péonidine et cyanidine), et aucune quantité détectable d’anthocyanes trihydroxylées (malvidine, delphinidine et pétunidine), qui sont abondamment présentes dans les pellicules des baies de RG. Ceci explique le phénotype de couleur rose pâle des baies de CG. Une analyse transcriptomique globale par RNAseq montre que 109 gènes sont exprimés de façon différentielle chez CG, en comparaison avec RG, y compris de nombreux gènes liés au métabolisme des flavonoïdes. Notamment, 11 gènes codant pour des 3'5'-hydroxylases flavonoïde, une enzyme-clé pour la biosynthèse des anthocyanes trihydroxylées, sont réprimés dans CG. A partir de cette analyse, un gène candidat pour la régulation de la voie de biosynthèse des anthocyanes a été sélectionné : le gène Cytb5, qui code pour un cytochrome b5, non caractérisé à ce jour chez la Vigne. La surexpression de Cytb5 par transgénèse dans des vignes hybrides V. berlandieri x V. rupestris cv. 110R suggère fortement un rôle clé pour ce gène dans la régulation de la biosynthèse des anthocyanes chez la vigne : les embryons obtenus présentaient une forte coloration rouge, indiquant la présence d’anthocyanes en quantités importantes dans les tissus végétatifs. De plus les embryons transgéniques ont un développement accéléré par rapport aux embryons sauvages. Ces travaux ont permis de mieux comprendre la régulation de l’accumulation des anthocyanes responsables de la coloration des baies de raisin, et plus largement, la régulation du métabolisme des flavonoïdes
El desarrollo de la uva es un proceso dinámico caracterizado por una curva de crecimiento doble sigmoidea, separada por una fase lag, en donde ocurre una biosíntesis coordinada de metabolitos primarios y secundarios. Al final de la fase lag, ocurre un fenómeno llamado pinta correspondiente al comienzo de la coloración de la baya e iniciándose también el proceso de maduración. Las antocianinas son las responsables de la coloración de la piel de las bayas y su regulación ha sido ampliamente estudiada. Sin embargo, pocos estudios se han enfocado en la caracterización genética y metabólica, utilizando variedades contrastantes de color de piel. Utilizando análisis metabólico y la tecnología de RNA-seq, se realizó una nueva caracterización comparativa de dos uvas de mesa, Chimenti Globe (CG) y Red Globe (RG), que poseen un color de piel de la baya contrastante: CG tiene un color rojizo claro y RG posee un color morado. La originalidad de este modelo es que CG fue generada en un evento espontáneo de campo desde una rama de una planta RG. Por lo tanto, el background genético responsable del cambio de color es el mismo. El análisis del contenido metabólico de las pieles de las bayas reveló la importancia de las etapas de desarrollo, pinta y maduración, en ambas variedades en estudio. En particular, la diferencia en la concentración de metabolitos de la ruta fenilpropanoide, tales como shikimato y fenilalanina y otras moléculas como UDP-glucosa y trehalosa-6-fosfato, entre otros. Asimismo, las diferencias entre las variedades estuvieron dadas por cambios relacionados con la biosíntesis de sacarosa y antocianinas. CG solo contenía antocianinas dihidroxiladas, cianidina y peonidina, y no las del tipo trihidroxiladas, malvidina, delfinidina y petunidina, lo cual fue consistente con el fenotipo del color de piel observado. A partir del análisis transcriptómico, generamos un heatmap con 109 genes expresado diferencialmente en CG en comparación con RG, siendo muchos de estos asociados a la ruta biosíntesis de flavonoides. Además, observamos que 11 copias del gen flavonoide 3'5'-hidroxilasa, que codifica una enzima clave para la biosíntesis de antocianinas trihidroxiladas, no estaban inducidas en CG. A partir de este análisis, se seleccionó un gen candidato para contribuir en el estudio de la ruta de biosíntesis de antocianinas: citocromo b5 (Cytb5) que codifica una proteína clave donadora de electrones no caracterizada en vides. La sobreexpresión de Cytb5 en embriones V. berlandieri x V. rupestris cv. 110R sugirió fuertemente la participación en la ruta, ya que los embriones transgénicos exhibieron un color rojizo e incluso, un desarrollo acelerado en comparación con el control. Con estos resultados, hemos sido capaces de proporcionar información sobre la regulación de las antocianinas en vides responsables de la coloración de la piel de las bayas, abriendo nuevos terrenos en la búsqueda de reguladores moleculares de la vía flavonoide

Thesis (PhD (Genetics))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future.
AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V. Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus (GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD), ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5 (Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V. vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors, aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13 (Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as ’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5 en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N. benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer. Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor (35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N. benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5- ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie. Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA- GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare, het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n moontlike belangrike rol kan speel. Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.