Thesis (MSc Natural and Agricultural Sciences (Genetics))--University of Pretoria, 2007. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

Thesis (M.Sc. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2007.

Wheat (Triticum aestivum and T. Durum) is an extremely important agronomic crop produced worldwide. Wheat consumption has doubled in the last 30 years with approximately 600 million tons consumed per annum. According to the International Maize and Wheat Improvement Center, worldwide wheat demand will increase over 40 percent by 2020, while land as well as resources available for the production will decrease significantly if the current trend prevails. The wheat industry is challenged with abiotic and biotic stressors that lead to reduction in crop yields. Increase knowledge of wheat’s biochemical constitution and functional biology is of paramount importance to improve wheat so as to meet with this demand. Pesticides and fungicides are being used to control biotic stress imposed by insect pest and fungi pathogens but these chemicals pose a risk to the environment and human health. To this effect, there is re-evaluation of pesticides currently in use by the Environmental Protection Agency, via mandates of the 1996 Food Quality Protection Act and those with higher perceived risks are banned. Genetic resistance is now a more environmental friendly and effective method of controlling insect pest and rust diseases of wheat than the costly spraying with pesticides and fungicides. Although, resistant cultivars effectively prevent current prevailing pathotypes of leaf rust and biotypes of Russian wheat aphid from attacking wheat, new pathotypes and biotypes of the pathogen/pest may develop and infect resistant cultivars. Therefore, breeders are continually searching for new sources of resistance. Proteomic approaches can be utilised to ascertain target enzymes and proteins from resistant lines that could be utilised to augment the natural tolerance of agronomically favourable varieties of wheat. With this ultimate goal in mind, the aim of this study was to elucidate the mechanism and synchronicity of wheat resistance to leaf rust (Puccinia triticina) and Russian wheat aphid (Duiraphis noxia) SA1. To determine the resistance mechanism of the wheat cultivars to leaf rust infection and Russian wheat aphid infestation, a proteomics approach using two-dimensional gel electrophoresis was used in order to determine the effect of RWA SA1 on the wheat cultivars proteome. Differentially expressed proteins that were up or down regulated (appearing or disappearing) were identified using PDQuestTM Basic 2-DE Gel analysis software. Proteins bands of interest were in-gel trypsin digested as per the protocol described in Schevchenko et al. (2007) and analysed using a Dionex Ultimate 3000 RSLC system coupled to an AB Sciex 6600 TripleTOF mass spectrometer. Protein pilot v5 using Paragon search engine (AB Sciex) was used for comparison of the obtained MS/MS spectra with a custom database containing sequences of Puccinia triticina (Uniprot Swissprot), Triticum aestivum (Uniprot TrEMBL) and Russian wheat aphid (Uniprot TrEMBL) as well as a list of sequences from common contaminating proteins. Proteins with a threshold of ≥99.9 percent confidence were reported. A total of 72 proteins were putatively identified from the 37 protein spots excised originating from either leaf rust or Russian wheat aphid experiments. Sixty-three of these proteins were associated with wheat response to stress imposed by RWA SA1 feeding while 39 were associated with infection by Puccinia triticina. Several enzymes involved in the Calvin cycle, electron transport and ATP synthesis were observed to be differentially regulated suggesting greater metabolic requirements in the wheat plants following aphid infestation and leaf rust infection. Proteins directly associated with photosynthesis were also differentially regulated following RWA SA1 infestation and P.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald Reeck / Pea aphids (Acyrthosiphon pisum) are sap-sucking insects that feed on the phloem sap of some plants of the family Fabaceae (legumes). Aphids feed on host plants by inserting their stylets between plant cells to feed from phloem sap in sieve elements. Their feeding is of major agronomical importance, as aphids cause hundreds of millions of dollars in crop damage worldwide, annually. Salivary gland transcripts from plant-fed and diet-fed pea aphids were studied by RNASeq to analyze their expression. Most transcripts had higher expression in plant-fed pea aphids, likely due to the need for saliva protein in the aphid/plant interaction. Numerous salivary gland transcripts and saliva proteins have been identified in aphids, including a glutathione peroxidase. Glutathione peroxidases are a group of enzymes with the purpose of protecting organisms from oxidative damage. Here, I present a bioinformatic analysis of pea aphid expressed sequence tag libraries that identified four unique glutathione peroxidases in pea aphids. One glutathione peroxidase, ApGPx1 has high expression in the pea aphid salivary gland. Two glutathione peroxidase genes are present in the current annotation of the pea aphid genome. My work indicates that the two genes need to be revised.

M.Sc. (Botany) / Cereals such as oats, rye, rice, barley, maize and wheat are a major source of food worldwide. Wheat (Triticum aestivum L.) is the largest winter cereal crop produced in South Africa (Crop Estimates Committee, 2011; South African Department of Agriculture, Forestry and Fisheries, www.daff.gov.za/crop estimates). Wheat production in South Africa includes both the summer and winter rainfall areas. Like other crops wheat is cultivated under monoculture conditions to increase yield per hectare. This increases the risk to pathogen exposure, as monocultures are genetically very similar or even identical, which makes them particularly vulnerable to both abiotic and biotic stresses (Haile, 2001). Both of these stressors negatively influence crop yield (Peterson and Higley, 2001; Wang et al., 2003).

Masters of Science / Genetic analysis for resistance to Woolly Apple Aphid in apple rootstock breeding populations MC Selala MSc Thesis, Department of Biotechnology, Faculty of Science, University of the WesternCape. The Woolly Apple Aphid (WAA) Eriosoma lanigerum (Hausm.) (Homoptera: Aphididae) is economically one of the most important pests in apple commercial production in the Western Cape province, South Africa. The apple cultivar Northern Spy possesses a single major gene (Er1) responsible for E. lanigerum resistance. This cultivar has been used as a commercial rootstock in apple breeding programmes. There are other genes also implicated in resistance to E. lanigerum from other cultivars. Manipulation and pyramiding of the E. lanigerum resistance genes (Er1, Er2 and Er3) might provide a necessary control for commercial apple production. The aim of this study was to construct a genetic linkage map for apple using microsatellite markers. The use of marker-assisted selection would greatly benefit local apple breeding programmes. Ninety six seedlings from a Northern Spy × Cox Orange Pippin mapping population were used for genetic linkage construction. Phenotypic data collection and analysis were performed to determine the E. lanigerum infestation patterns and the levels of resistance conferred by the Er1 gene from Northern Spy using 52 in vitro propagated seedlings in the greenhouse. Classification and quantification analysis showed association patterns between first assessments (30 days) to second assessment (60 days) in all replicate blocks. Roots and shoots data showed that it could be useful in quantitative trait loci (QTL) analysis, but may be used in different QTLs beingidentified due to the variations between roots and shoots data. A preliminary linkage map was constructed using a mapping population from Northern Spy × Cox Orange Pippin (96 seedlings).Fluorescently labelled published and predicted microsatellite markers were used in map construction. Primers were optimised using single apple cultivar and the detection of polymorphisms using nine apple cultivars. Optimised markers were multiplexed for high throughput data generation using the Polymerase Chain Reaction (PCR) technique. Multiplexed PCR products were pooled and analysed on an ABI 310 PRISM™ Genetic Analyser to determine allele fragment sizes, and the inherited segregation types in the seedlings. Computer software GenoTyper® 2.5.2 and JoinMap® 3.0 was used in data analysis from ABI 310 PRISM™Genetic Analyser and linkage map construction. Seventy two markers were used in linkage map construction, which produced nine linkage groups with some segments from the same linkage group. Twenty-one markers were aligned on the map 20 published and one predicted. Only one linkage group consisted of five markers while other linkage groups had two markers each. This study has proved that th preliminary linkage map could be used as the basis of a complete linkage map of Northern Spy × Cox Orange Pippin.

No description available.

Myzus persicae (Sülzer), commonly known as green peach aphid (GPA), utilizes its slender stylet to penetrate the plant tissues intercellularly and consume copious amounts of photoassimilates present in the phloem sap causing extensive damage to host plants. The compatible interaction between GPA and Arabidopsis thaliana enabled us to characterize plant response to aphid infestation. Upon GPA infestation, Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT4) gene modulates premature leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf. Senescence mechanism is utilized by plants to limit aphid growth. In addition, PAD4 provides antixenosis (deters insect settling and feeding) and antibiosis (impair aphid fecundity) against GPA and adversely impact sieve element availability to GPA. Basal expression of PAD4 contributes to antibiosis, and the GPA-induced expression of PAD4 contributes to antixenosis. Mutation in the Arabidopsis stearoyl-ACP desaturase encoding SSI2 (suppressor of SALICYLIC ACID [SA] insensitivity2) gene that results in an accelerated cell death phenotype and dwarfing, also conferred heightened antibiosis to GPA. Results of this study indicate that PAD4 is required for the ssi2-mediated enhanced antibiosis to GPA. The PAD4 protein contains conserved Ser, Asp and His residues that form the catalytic triad of many α/β fold acyl hydrolases. Arabidopsis plants expressing mutant versions of PAD4 [PAD4(S118A) and PAD4(D178A)] supported higher numbers of GPA as compared to wild type (WT) plants in no-choice tests. Furthermore, Electrical Penetration Graph (EPG) studies revealed that S118 residue in PAD4 is essential to limit GPA feeding from the sieve elements. However, the ability to deter insect settling in choice tests was not impacted by the PAD4(S118A) and PAD4(D178A) mutations, thus suggesting that PAD4s involvement in deterring insect settling and in antibiosis are determined by separate regions of PAD4. The MPL1 (MYZUS PERSICAE INDUCED LIPASE1) gene is another critical component of Arabidopsis defense against GPA. Like PAD4, MPL1 expression is induced in response to GPA infestation. However, MPL1 is required only for antibiosis and is not essential for antixenosis against GPA. EPG analysis suggests that the mpl1 mutant allele does not impact aphid feeding behavior. Since, MPL1 exhibits lipase activity, and ssi2 petiole exudates contain elevated levels of antibiosis, we propose that antibiosis to GPA requires a lipid(s), or a product thereof.

Ces travaux de thèse reposent sur le double objectif de proposer des approches innovantes pour l’étude des relations entre un hôte et son microbiote, et de les appliquer à la description fine de l’holobionte du puceron du pois par des données métagénomiques. Les relations symbiotiques façonnent le fonctionnement et l’évolution de tous les organismes, mais restent décrites de manière imparfaite, notamment à cause de la difficulté à caractériser la diversité génomique des partenaires microbiens constitutifs des holobiontes. L’essor des technologies de séquençage métagénomique révolutionne l’étude de ces systèmes, mais pose également des problèmes méthodologiques pour analyser les jeux de données métagénomiques. La métagénomique est ici appliquée au puceron du pois, un modèle d’étude des relations symbiotiques qui abrite une communauté bactérienne d’une complexité modérée, idéale pour développer de nouvelles approches de caractérisation de la diversité microbienne. Cette thèse vise à mieux décrire la communauté de symbiotes qu’abrite cet holobionte, notamment en distinguant les différents niveaux de variabilité génomique en son sein. Nous présentons une démarche pour l’analyse métagénomique d’holobiontes, qui repose d’abord sur l’assignation taxonomiques des lectures par alignement à des génomes de référence ou préalablement assemblés, puis sur la recherche de variants génomiques. L’étude de génotypes complets de symbiotes permet de retracer l’histoire évolutive des relations hôte-microbiote avec une résolution élevée. Chez le puceron du pois, nous mettons en évidence des niveaux et structures de diversité génomique différents selon les symbiotes, que nous proposons d’expliquer par les modalités de transmission ou l’histoire évolutive propre à chacun des partenaires microbiens. Cette approche repose sur la disponibilité d’un génome de référence adapté, qui est souvent difficile à obtenir en métagénomique. Dans un second temps, nous présentons donc une méthode d’assemblage guidé par référence en contexte métagénomique. Cette méthode se déroule en deux temps : le recrutement et l’assemblage de lectures par alignement sur un génome de référence distant, puis l’assemblage de novo ciblé des régions manquantes, permis par des développements complémentaires apportés au logiciel MindTheGap. Comparativement à un assembleur métagénomique, cette méthode permet l’assemblage d’un seul génome en un temps réduit, et permet de détecter d’éventuels variants structuraux sur le génome ciblé. Appliqué au puceron du pois, MindTheGap a réalisé l’assemblage du symbiote obligatoire Buchnera en un seul contig, et a permis d’identifier différents variants structuraux du bactériophage APSE. Ces travaux ouvrent la voie à la fois à une caractérisation plus précise des relations hôte-microbiote chez le puceron du pois par des approches fonctionnelles et métaboliques, ainsi qu’à l’application des outils présentés à des systèmes plus complexes. / The aim of this PhD thesis is to develop innovative approaches to characterize host-microbiota relationships, and to apply them to finely explore the pea aphid microbiota using metagenomic data. Symbiotic relationships play a major role in the life and evolution of all organisms, but are imperfectly described, essentially because of the difficult characterization of the genomic diversity of the microbial partners. The rise of high throughput metagenomic sequencing is a game changer for the study of those systems, but also raises methodological issues to analyze large metagenomic datasets. Metagenomic is here applied to the pea aphid holobiont, a model system for the study of symbiotic relationships, sheltering a moderately complex microbial community. This level of complexity seems to be ideal to develop new approaches for the strain-resolved characterization of host-microbiota relationships. This thesis aims at a better description of this symbiotic community by distinguishing several scales of metagenomic diversity. In a first part, we present a framework for the metagenomic analysis of holobionts, relying first on the taxonomic assignation of reads by alignment to reference or newly assembled genomes, and then on the detection of genomic variants. Whole genome variant profiles make possible to track the evolutionary history of host-microbiota associations with a high resolution. In the case of the pea aphid, we highlight different scales and structures for the metagenomic diversity of the different symbionts, accounting for different transmission modes or evolutionary histories specific to each microbial partner. This framework is based on the availability of a suitable reference genome, that may be hard to obtain in a metagenomic context. In a second part, we therefore present a novel method for reference guided genome assembly from metagenomic data. This method is based on two steps. First, the recruitment and assembly of reads by mapping metagenomic reads on a distant reference genome, and second, the de novo assembly of the missing regions, allowed by the development of an improved version of the software MindTheGap. Compared to a standard metagenomic assembler, this methods makes possible to assemble a single genome in a reasonable time, and allows to detect eventual structural variations within the targeted genome. When applied to the pea aphid holobiont, MindTheGap yielded single contig assemblie of the obligatory symbiont Buchnera aphidicola, and helped to identify different structural variants of the bacteriophage APSE. This works paves the way to a finer characterization of host-microbiota interactions, and to the application of the presented approaches to more complex systems.

The Russian wheat aphid (Diuraphis noxia, RWA), is a serious pest in most wheat producing countries around the world. Infestation of wheat fields by this pest has a severe economic impact, as a result of heavy losses in crop yield. Because of the importance of wheat as a food source and its ever growing supply demand, the study of wheat-Russian wheat aphid interactions on the molecular level are integral to the development of management strategies. This is highlighted by the fact that new RWA biotypes that overcome resistance in a number of wheat varieties, continually emerge. Therefore, this study aims to contribute to this endeavour, by elucidating the molecular mechanisms by which the RWA resistance gene Dn7 confers resistance to three different RWA biotypes (one from SA, and two from the USA). Firstly, suppression subtractive hybridization (SSH) was applied in order to isolate transcripts differentially expressed in the RWA resistant wheat line, 94M370, carrying the Dn7 gene. There are two main advantages to this technique. One is that the relative representation of rare transcripts is increased in the subsequent cDNA population, and it is these low abundance transcripts that are arguably the ones of particular interest. Secondly, this method allows for the isolation of unknown transcripts, without the need for existing sequence information. Experiments with this method however, failed, leading to an investigation as to probable causes. The various steps involved in the SSH procedure were individually assessed in an attempt to identify and correct the problem. Various adjustments were made to PCR procedures, template enzyme digestions and ligation reactions, without success. After creating a basic cDNA-AFLP fingerprint from the existing cDNA template, in order to confirm that the template is not responsible for experimental difficulties – it was decided to apply a different strategy in order to meet research objectives. Consequently, the study on Dn7 mediated defence responses was continued with cDNA-AFLP. In addition to studying the response by Dn7 to South African biotype RWA infestation, its responses to infestation by two United States RWA biotypes was also explored. This allowed us to gain a greater comprehension of the methods by which Dn7 activates defences against different aphid eliciting agents. Findings suggest that this gene activates responses that are unique to each of the different aphid interactions. Although the interactions between Dn7 and the two US biotypes were very similar, this can possibly be explained by the fact that the differences between these two biotypes on molecular level are minuscule. Dn7 responds to the South African biotype of the RWA in a completely different manner, as judged by the very dissimilar expression patterns obtained during cDNA-AFLP analysis. Reasons for this phenomenon could include molecular differences between the South African and US RWA biotypes, differences in response generating elicitor molecules (which has indeed been shown to be the case between South African and US aphid biotypes), or a combination of both. The sequencing of fragments displaying differential expression patterns during cDNA-AFLP fingerprinting, provides us with additional information as to the exact mechanisms potentially involved. As expected, various compounds related to plant defence were identified, such as a number of Leucine rich repeat (LRR) domain containing proteins, genes related to cell signalling and genes involved in protein processing (proteases, peptidases). Finally, these results are consistent with theories that Dn7 may recognise and interact with its distinct aphid elicitors either directly, by the presence of multiple bindings sites on the same protein, or indirectly. In that case, in accordance with the guard hypothesis, Dn7 may simply monitor interactions between aphid elicitors and other recognition factors- after which a response cascade is activated. Useful potential research would focus on Dn7 itself, including mapping, isolation as well as structural and functional characterization. / Dissertation (MSc)--University of Pretoria, 2007. / Genetics / unrestricted