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This thesis investigates the effect of Russian wheat aphid (RWA: Diuraphis noxia) infestation on the defence responses of the bread wheat line, PI 137739, on a molecular level. PI 137739 is known to contain the RWA resistance gene, Dn1. The study was conducted by utilising and combining a vast array of molecular biological techniques. Chapter 1 introduces the reader to a summary of the resistance responses observed within infested plants. A detailed description of the Russian wheat aphid follows and the genes responsible for RWA resistance in wheat is discussed. A brief report of research performed on the bread wheat genome is given and the biochemical defence responses of plants against insect infestation are discussed. This is followed by a concise description of resistance (R) genes and resistance gene categories in plants. The last discussion concerns microarray technology, a molecular tool utilised during this study. Chapter 2 aims at identifying genes involved in resistance against RWA infestation: specifically, genes containing the conserved nucleotide binding site leucine rich repeat (NBS-LRR) motif. Genomic, as well as complementary DNA (cDNA), was utilised in order to compare functional gene expression in wheat infested with the RWA. This was executed by employing PCR-based methods, single-pass sequencing and basic local alignment search tool (BLAST) analyses. Chapter 3 introduces suppression subtractive hybridisation (SSH) as a tool to further identify NBS-LRR or other resistance-related sequences in RWA infested wheat plants. SSH allows the comparative analysis of differential gene expression in RWA infested and uninfested wheat in order to identify resistance- related genes expressed in the infested, resistant wheat plants. The effect of RWA infestation on wheat resistance responses was examined further in chapter 4 through microarray analysis. The aim was the introduction and establishment of the microarray technique and to test the feasibility of using microarrays for differential gene expression and regulation studies. Microarray slides were assembled in order to monitor the up- and down regulation of genes at different time intervals - day 2, day 5 and day 8 - of RWA infestation. Clones isolated throughout this study were assembled on microarray slides and probed with control and RWA infested RNA. Differential gene regulation was assessed and further confirmed through Northern blot analyses, as well as quantitative real-time PCR. The thesis concludes with a general summary of the results obtained in chapter 5 and future prospects are outlined.
Diuraphis noxia is an insect pest on barley and wheat in Ethiopia. Yield losses of barley can reach up to 79%. Control is through use of resistant barley cultivars. Biotypes can render control with resistance uncertain. In this book it was indicated that no biotypic variation exist in RWA clones on barley in Ethiopia. Susceptibility of one resistant wheat line (Dn4) to the Ethiopian RWA clones indicated biotypic differences between American and Ethiopian RWA clones. Anholocyclic life cycle of RWA in Ethiopia, dependence on land races and fact that barley land races show tolerance to RWA support the finding of no biotypic variation in the Ethiopian RWA clones. It was also indicated in the same book that molecular markers like mtDNA, two microsatellite loci and dnaN and ptrpE genes of the bacterial endosymbiont Buchnera did not reveal genetic variation among worldwide collections of RWA. Only a single substitution was found in the pseudogene (ptrpE) in the Turkish populations. Amercian populations did not reveal a different genetic pattern compared to Ethiopian ones. The absence of genetic variation could be due to the recent and fast spread 0f the insect from its origin.
Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), is a widely distributed species in western north America, and a major economic pest of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). The objectives of this study were to : 1) determine the inheritance of seedling and adult plant resistances in a wheat line PI 294994 ; 2) establish whether greenhouse and field resistances were related ; 3) determine the effect of RWA infestation on the dry matter, grain yield, and harvest index in the crosses Moro/PI 294994 and Hyak/PI 294994 ; 4) determine whether there was a difference in the three traits between different genotypes due to RWA infestation ; and, 5) assess the degree of protection genetic resistance provided in the two crosses. The club wheat cultivars 'Moro' and 4Hyak' were crossed with PI 294994. Progenies from these two crosses were artificially infested with RWA in the field and the crosses were compared for their dry matter, grain yield, and harvest index. Plant reactions of F2 seedlings, F2 adult plants, and F3 seedlings indicated that both seedling and adult plant resistances are controlled by two genes with dominant and recessive mode of inheritance and that plant reactions in the field were the same as those in the greenhouse. Comparison of mean values of dry matter, grain yield, and harvest index showed that the three traits were affected by RWA infestation and genotype in both crosses. Resistant genotypes and noninfested population were comparable with respect to the three traits in Moro/PI 294994 but they were not in Hyak/PI 294994.
The Russian wheat aphid Diuraphis noxia (Kurdjumov) is a serious pest of world cereal grain crops, primarily barley and wheat. A phenotypic characteristic of D. noxia feeding, leaf rolling, creates a leaf pseudo gall which protects aphids, making it difficult to treat infested plants with insecticides or biological control agents. Therefore, the use of D. noxia-resistant crops is a desirable aphid management tactic. Because of the development of virulent D. noxia biotypes, the identification of new sources of barley and wheat resistance is necessary. Virus-induced gene silencing (VIGS) utilizes the plant defense system to silence viruses in inoculated plants. The accumulation of virus RNA in plants triggers the defense system to silence sequences homologous to the introduced virus and sequences of interest from a plant are inserted into the virus and silenced along with the virus. The VIGS method was tested to determine the ability of barley stripe mosaic virus (BSMV) to serve as a VIGS vector in wheat plants containing the Dnx gene for resistance to D. noxia. Dnx leaves with silenced BSMV virus yielded D. noxia populations that were significantly no different from populations produced on healthy Dnx leaves. Thus, BSMV silencing does not interfere with Dnx resistance. Several different methods were examined to determine how best to confine aphids to the silenced leaf, and a modified plastic straw cage was chosen as the optimum cage type. Microarray and gene expression data were analyzed to select two NBS-LRR type disease resistance protein genes - TaAffx. 104814.1.S1_at and TaAffx. 28897.1.S1 - (NBS-LRR1 and NBSLRR2), in order to assess their role in Dnx resistance. NBS-LRR1 and NBSLRR2 were silenced by inoculating leaves of Dnx plants with barley stripe mosaic virus (BSMV) containing sequences of each gene. Controls included Dnx and Dn0 plants inoculated with BSMV and non-BSMV inoculated plants. Aphids were allowed to feed on control and treatment plants to assess aphid population and mean weight of aphids surviving at the end of the experiment. There were no differences among treatments based on aphid population, but there were significant differences the mean weights of aphids reared on several different treatments.