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Soybean mosaic virus (SMV; Genus Potyvirus; Family Potyviridae) is one of the most widespread viruses in soybean (Glycine max [L.] Merr.). Hutcheson, a cultivar developed in Virginia, is resistant to the common strains of SMV. However, new resistance-breaking (RB) isolates of SMV have emerged in natural infections to break the resistance of Hutcheson containing the Rsv1y allele. These RB isolates are SMV-G5 and G6-like based on the differential reactions on soybean cultivars with the Rsv1 locus, and are more G6-like based on the amino acid sequence of the coat protein (CP). The CP of the RB isolates is diverse at the amino and carboxy termini and highly conserved in the core region. RB isolates reduce the yield of susceptible cultivars and cause mottling of the seed coat. Dual infection of soybeans with SMV and BPMV increased the severity of symptoms, including plant stunting and SMV titer in comparison to single SMV inoculations. The reactions of Hutcheson and herbicide-tolerant Hutcheson RR were similar with or without herbicide application. Resistance to SMV is controlled by single dominant genes at three distinct loci, Rsv1, Rsv3 and Rsv4. The mechanisms of resistance at the Rsv3 and Rsv4 loci were investigated by tracking virus accumulation and movement over time using leaf immunoprints. The mechanisms of Rsv3 resistance include extreme resistance, hypersensitive response, or restriction to virus replication and movement, which are strain specific. The Rsv4 gene was found to function in a non-strain specific and non-necrotic manner. The mechanisms of Rsv4 resistance involve restricting both cell-to-cell and long distance movement of SMV. The Rsv1, Rsv3 and Rsv4 resistance genes exhibit a continuum of SMV-soybean interactions, and include complete susceptibility, local and systemic necrosis, restriction of virus movement (both cell-to-cell and long distance), reduction in virus accumulation, and extreme resistance with no detectable virus. Cultivars containing two genes for resistance, Rsv1 and Rsv3 or Rsv1 and Rsv4, were resistant to multiple strains of SMV tested and show great potential for gene pyramiding efforts to ensure a wider and more durable resistance to SMV in soybeans. / Ph. D.

No description available.

Student Number : 0413249D - MSc research report - Faculty of Science / Cassava is one of the most important staple crops in the world and is consumed by over 700 million people around the globe and is a profitable product commercially due to the high starch content of its tubers. One of the future aims is to produce cassava that is high yielding, resistant to cassava mosaic geminiviruses (CMGs) and high in starch content. To be able to achieve commercially attractive cassava varieties, research need to be carried out to investigate the virus resistance status of different cassava cultivars, which can later be used in the future breeding programme. In South Africa, cassava is used for commercial starch manufacturing purposes, as a cash crop and a food source by small-scale farmers. Cassava Mosaic Disease (CMD) is having a negative impact on yield of the crop globally and therefore dropping profitability of cassava on a commercial scale. The aims of this research were to propagate thirteen cassava cultivars and then to test them for virus susceptibility or resistance. Eleven cassava cultivars received from the International Institute of Tropical Agriculture (IITA) were tested for resistance or susceptibility against South African cassava mosaic virus (SACMV). Two local, commercial cultivars T200 and T400, were tested for East African cassava mosaic virus (EACMV) and African cassava mosaic virus (ACMV) resistance. Cassava cultivars were successfully propagated in vitro and thereafter transferred into soil and acclimatized to adapt to environmental conditions. When the plantlets were three weeks old, the plantlets were infected with cassava mosaic viruses. Plants were infected with SACMV via Agrobacterium-mediated transfer and infectious EACMV and ACMV monomers were used to biolistically bombard the plantlets. Resistance/susceptibility results of seven of the thirteen cultivars were obtained with SACMV, these cultivars being T200 (susceptible), T400 (susceptible), TME3 (highly resistant), I30572 (susceptible), I420251 (highly susceptible), I60506 (susceptible) and TMS60444 (susceptible). Due to destruction by fungal gnats eating the roots of the plants, acclimatization of the remaining six cultivars was not possible. Also, due to the nature of the biolistic equipment, infection of the cultivars with EACMV and ACMV was not achieved as the plantlets were not robust enough to survive the pressure.

Most plants are capable of mounting resistance responses to various pathogen attacks. For a hypersensitive response (HR) to occur, a dominant or semi-dominant resistance (R) plant gene is required to recognize a dominant avirulence (Avr) factor of the pathogen. Three types of Arabidopsis thaliana, Dijon-17 (Di-17), Dijon-3 (Di-3), and Columbia-0 (Col-0), are significant in understanding the genetics of Turnip crinkle virus (TCV) resistance. It has been shown that three genes are needed for successful resistance to TCV in A. thaliana: the dominant R gene HRT, the recessive gene rrt, and a third gene, TIP. Crosses of Di-17 and Di-3 plants, and crosses of Di-3 and Col-0 plants are being analyzed to determine the genotype of the F1 progeny. Using cleaved amplified polymorphic sequence (CAPS) markers, it is possible to determine the genotype of the progeny compared to the wild-type parents at the HRT and TIP loci. Additionally, protein analysis tools will be employed to compare the Di-3 and Di-17 TIP alleles to determine if there are any significant differences in the protein.

Rift Valley Fever Virus is a plebovirus that causes epidemics and epizootics in sub-Saharan African countries but has expanded to Egypt and the Arabian Peninsula. The laboratory rat (Rattus norvegicus) is susceptible to RVFV and has been shown to manifest the characteristic responses of humans and livestock. The rat has frequently been used as a model to study RVFV pathogenesis. Several strains have been infected and some found to be resistant to hepatic disease while others were not. This resistance was found to be associated with a dominant gene inherited in Mendelian fashion. The congenic rat strain WF.LEW and several substrains of the parental strains were used to try and locate the resistance gene. Microsatellites and single nucleotide polymorphisms were used to characterize the genomes of various rat substrains in an attempt to map the gene. Breeding and viral challenge experiments were used to further characterize the strains and assign a location to the resistance gene. The LEW/SsNHsd rats showed approximately 37% genomic difference as compared with LEW/MolTac rats, and 8% difference as compared with LEW/Crl rats. WF/NHsd rats demonstrated a difference of approximately 8% as compared with WF/CrCrl rats. Genotyping of the congenic WF.LEW revealed Lewis markers on RNO3 and RNO9. Subsequent backcross experiments and viral challenge experiments assigned the resistance gene to the distal end of RNO3.

New technologies are making it possible to engineer organisms with fundamentally new and useful properties. In vivo genome engineering technologies capable of manipulating genomes from the nucleotide to the megabase scale were developed and applied to reassign the genetic code of Escherichia coli. Such genomically recoded organisms show promise for thwarting horizontal gene transfer with natural organisms, resisting viral infection, and expanding the chemical properties of proteins.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, but also have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viral and anti-fungal properties, but the exact mechanism of these proteins still need to be elucidated.The mechanism of resistance however, appears to be independent of the pathogen. For resistance the RIP terminates virus infected plant cells and stops the reproduction and spread of the virus. Transgenic plants containing RIPs should thus be resistant to a wide range of viruses. The ultimate goal of the larger project of which this forms part is the development of virus resistant plants. To monitor the expression of a RIP in a transgenic plant a detection method had to be developed. Antibody detection of the RIP was decided upon as the most cost effective method. The RIP, Dianthin 30 from Dianthus caryophyllus (carnation), was used and expressed in bacterial and insect expression systems. The bacterial expression experiments were done using the pET expression system in BL21(DE3)pLysS cells. The expression in this system yielded recombinant protein at a very low concentration. Expression experiments were also performed in insect tissue culture with the baculovirus vector BAC-TO-BAC™.With this system the expression was also too low to be used for the production of antibodies. A Dianthin 30 specific peptide was then designed and then produced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin 30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if this detection method was effective to monitor the expression in plants, tobacco plants were transformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plant expression vector. The putative transformed plants were analysed with peR and Southern blots. / AFRIKAANSE OPSOMMING: Tans word Ribosomale-inaktiverende proteïene (RIPs) geklassifiseer as rRNA N-glikosidase wat ook polinukleotied: adenosien glikosidase aktiwiteit bevat. Daar word geglo dat RIPs anti-virale en anti-fungus eienskappe bevat, maar die meganisme van beskerming word nog nie ten volle verstaan nie. Dit is wel bewys dat die meganisme van weerstand onafhanklik is van die patogeen. Virus geinfekteerde plantselle word deur die RIP gedood om die voortplanting en verspreiding te bekamp en sodoende word weerstand bewerkstellig. Transgeniese plante wat dan 'n RIP bevat sal dus weerstandbiedend wees teen 'n wye spektrum virusse. Die hoofdoel van die breër projek, waarvan die projek deel uitmaak: is die ontwikkeling van virusbestande plante. Om die uitdrukking van die RIP in die transgeniese plante te kontroleer, moes 'n deteksie metode ontwikkel word. Die mees koste effektiewe deteksie metode is met teenliggame. Die RIP, Dianthin 30 from Dianthus caryophyllus (angelier) was gebruik vir uitdrukking in bakteriele- en insekweefselkultuur. Die bakteriele uitdrukkingseksperimente was gedoen met die pET uitdrukkings sisteem III BL21(DE3)pLysS selle. Die uitdrukking in die sisteem het slegs rekombinante proteïene gelewer in uiters lae konsentrasies. Uitdrukkingseksperimente was ook gedoen in insekweefselkultuur met die baculovirus vektor BAC-To- BACTM. Met die sisteem was die uitdrukking ook veels te laag om bruikbaar te wees vir die produksie van teenliggame. Daar is toe 'n peptied ontwerp wat Dianthin 30 kan verteenwoordig vir die produksie van teenliggame. Die teenliggame is getoets teen Dianthus caryophyllus proteïene. Om vas te stel of die deteksiemetode wel die uitdrukking van Dianthin 30 sal kan monitor, is tabak ook getransformeer met Dianthin 30. Die transformasies is gedoen met die hulp van Agrobacterium tumefaciens en die pART27 plant uitdrukkings vektor. Die plante is getoets met die polimerase ketting reaksie en Southern klad tegnieke.

Le bar est une espèce économique majeure de l’aquaculture méditerranéenne. La nécrose nerveuse virale (VNN), une maladie qui affecte au moins 70 espèces aquatiques, est devenue la menace la plus grave pour l’aquaculture de cette espèce. Bien que de nombreuses études aient été réalisées afin de contrôler cette maladie, aucune procédure simple et efficace n’est disponible. Dans cette thèse, nous évaluons la variabilité génétique de la résistance à cette pathologie et le potentiel d’amélioration génétique pour lutter contre cette menace.Après une introduction générale (premier chapitre) et une revue de la littérature sur la nodavirose en aquaculture (second chapitre), nous explorons dans le troisième chapitre la variabilité génétique de résistance de populations sauvages de bar, Atlantique Nord (NAT), Méditerranée ouest (WEM), Nord-Est Méditerranée (NEM) et Méditerranée Sud-Est (SEM). Pour ce faire, 2011 descendants d’un croisement factoriel complet, où 9 mères WEM ont été croisées avec 60 pères NAT, WEM, NEM et SEM (15 mâles par population), ont été élevés en "common garden". Après 202 jours, 1472 poissons ont été infectés par injection intrapéritonéale nodavirus à 15.8g de poids moyen. Le reste des poissons a été conservé pour collecter les paramètres de performance. Après la récupération du pedigree, nous révélons une forte variabilité de résistance en fonction de l’origine des pères (de 53 à 90%), les descendants de pères Est-Méditerranéens étant les plus résistants (83 à 90% de survie), les descendants WEM étant intermédiaires (62% de survie) et les descendants de père NAT étant les plus sensibles (53% seulement de la survie). Une héritabilité modérée mais significative pour la résistance (0,26 ± 0,11) a été estimée et des corrélations négatives entre la résistance et les traits de production ont été montrées.Dans le quatrième chapitre une recherche de loci à effets fort (QTL) sur la résistance a été effectuée avec une carte de liaison moyenne-densité. Pour cela, 1717 individus appartenant à 397 familles de plein-frères et leurs parents ont été génotypés pour 2722 marqueurs SNP imprimés sur une puce SNPs. À partir de 1274 loci significatifs, une carte de liaison contenant 24 groupes de liaison, ainsi que des cartes sexe-spécifiques et origine-spécifiques ont été construites. Ces résultats révèlent une hétérochiasmie, avec un taux de recombinaison 1,25 fois plus fort chez les femelles par rapport aux mâles. La recherche de QTL a été effectuée à partir de différentes méthodes, mais bien qu’aucun QTL pour le «temps de survie» ou la survie, n’ait été identifié, nous discutons de l’effet du plan expérimental utilisé.Dans le quatrième chapitre, une étude association génomique a été effectuée en deux étapes: non pondérée (GWAS) puis pondérée (wGWAS) à partir de modèles mixtes linéaires utilisant les mêmes SNP que pour la cartographie de QTL, l’objectif étant de détecter des SNPs liés à la résistance au VNN. Un SNP significatif expliquant 3.11% de la résistance appartenant à LG9 a pu être détecté. Le potentiel de prédiction de la génomique pour la résistance au VNN en utilisant différents modèles génomiques a enfin été évalué, mais aucune différence significative n’a été montrée entre les valeurs génétiques estimées à partir des données génomiques ou à partir du pedigree.En conclusion, cette étude montre forte variation génétique de la résistance au VNN des populations sauvages de bar avec des corrélations génétiques négatives avec les traits de production. Ces derniers résultats sont précieux pour aider à définir des stratégies d’amélioration génétique de la résistance au VNN du bar. Enfin, de premières hypothèses sur l’emplacement de QTL putatifs plaident pour une future cartographie fine pour localiser ces QTLs, une valeur ajoutée dans un schéma de sélection assistée par marqueurs pour améliorer la résistance au VNN du bar. / European seabass is one of the most economic species in aquaculture in Mediterranean areas. Viral nervous necrosis (VNN), a disease affecting at least 70 aquatic species, has become the most serious threat to seabass cultured industry. While numerous studies have been performed in order to control this disease, no simple and effective procedures are available. In this thesis, we question genetic variability and the potential of selective breeding as an opportunity to address thwart this threat.After a general introduction (first chapter) and a deep literature review of nodavirus in aquaculture (second chapter), we explore in the third chapter the genetic variability of resistance of different wild populations of European seabass, namely Northern Atlantic (NAT), Western Mediterranean (WEM), Northern-East Mediterranean (NEM) and Southern-East Mediterranean (SEM). To address this question, 2011 fish derived from a full-factorial mating scheme, where 9 WEM dams were crossed with 60 sires originated from NAT, WEM, NEM and SEM (15 sires per population), were reared in “common garden”. At 202 days, 1472 were challenged by nodavirus intraperitoneal injection at a mean body weight of 15.8 g. The rest of fish were kept in a single tank in order to collect performance traits. Strikingly, after pedigree recovery, we reveal a very strong and significant differentiation in VNN resistance among sires’ origin (ranging from 53 to 90%), offspring from East Mediterranean sires being the most resistant (83-90% of survival), offspring from WEM sires being intermediate (62% of survival) and offspring from NAT sires being the most sensitive (53% of survival only). A moderate liability heritability for VNN resistance (0.26±0.11) was estimated and negative correlations between resistance and production traits were shown.In the fourth chapter, a search of Quantitative Trait Loci (QTL) linked to the resistance was performed using a medium linkage map as examined. Therefore, 1717 individuals belonging 397 full-sib families and their parents were genotyped for 2722 SNP markers spotted on a SNPChip. From 1274 significant loci, a 24 linkage groups medium-density linkage map was constructed, as well as sex-specific and Origin-specific linkage maps. From these results, we show a 1.25-fold sex-biased heterochiasmy in favor to female recombination rate. Finally, genome scans for QTLs were performed in different methods, and while no QTLs were identified for both “time to death” or survival, we discuss the effect of the experimental design used.In the fifth chapter, a two-step unweighted then weighted Genome-Wide Association Study (GWAS & wGWAS) was carried out based on linear mixed models using the same SNPs as for QTL mapping. The aim was to determine whether we can detect significant individual SNPs linked to resistance against VNN. After SNPs weight calculation, the wGWAS detected one significant SNP explaining 3.11% of the resistance belonging to LG9. Finally, the potential for genomics prediction for VNN resistance using the different genomic models was performed and extensively presented. However, no significant differences were observed between genomic-based estimated breeding values and pedigree-based estimated breeding values.In conclusion, this study depicts a large genetic variation for VNN resistance in wild seabass populations but with negative genetic correlations with production traits. These latter results are valuable to help to define strategies for genetic improvement of resistance against VNN of European seabass. Moreover, the first assumptions on the location of potential QTLs claim for a fine QTL mapping and an expectable add-value of the use of genomic information in potential marker-assisted selection to VNN resistance in European seabass.

The potential benefits of plant genetic engineering for disease and pest resistance have been widely acknowledged in many studies, and although genetically modified crops are still encountering public wariness, these benefits warrant continued exploration. Because of its intrinsic economic benefits, the development of true potato seed (TPS) cropping systems has been instituted in many regions of the world. The incorporation of transgenic resistance could further the economic gain of farmers who are seeking ways to sustain their livelihood in the most efficient way possible. It is, however, largely unresearched how sexual hybridization of a transgenic crop would affect the behavior of a transgene in the resultant progeny. In the initial part of this study, transgenic lines were developed with a Cry3Aa transgene. These plants were then used in 4x-4x reciprocal crosses and 4x-2x hybridization schemes to determine the stability of the transgene after sexual hybridization. There was no observed parent of origin effect on transgene expression; however, a highly significant, non-mendelian inheritance of the Cry3Aa transgene was seen in the maternally inherited transgene of one set of progeny from a reciprocal cross. Additional transgenic lines of potato were developed with a PVY-O coat protein transgene. These plants were challenged with PVY-O and monitored for symptoms visually and for virus serologically. One transgenic line exhibited complete resistance to PVY-O while two others showed a delay in symptom occurrence. Further examination of the expression levels of the PVY-O coat protein transgene will be necessary to determine the type and usefulness of the observed resistance. / Master of Science