Analyses of the large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. A detailed analysis of populations in centre of diversity should enable to infer the adaptive capacity of the pathogen and identify potential sources for new invasions. Puccinia striiformis f.sp. tritici (PST) is the causal agent of wheat yellow/stripe rust, and despite a worldwide distribution, this fungus remains a model species for invasion studies, due to its long-distance migration capacity and recurrent local emergence of new strains. Little is known about the ancestral relationship of the worldwide PST population with unknown center of origin. We used multilocus microsatellite genotyping to infer the worldwide population structure of PST and the origin of new invasions, analysing a set of isolates representative of sampling performed over six continents. Bayesian and multivariate clustering methods partitioned the isolates into six distinct genetic groups, corresponding to distinct geographic areas. The assignment analysis confirmed the Middle East-Red Sea Area as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. The existence of strong population subdivision at worldwide level shows that major genetic groups are not markedly affected by recent dispersal events. However, the sources for recent invasions and the migration routes identified emphasize the importance of human activities on the recent long-distance spread of the disease. The analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan (China) regions and a predominant clonal population structure in other regions. To explain the variability in diversity and recombination of worldwide PST populations, we assessed their sex ability in terms of telial production, the sex-specific structures that are obligatory for PST sexual cycle, in a set of 56 isolates representative of these worldwide geographical origins. We confirmed that the variability in genotypic diversity/ recombination was linked with the sex ability, pinpointing the Himalayan region as the possible center of origin of PST, from where it then spread worldwide. The reduced sex ability in clonal populations certainly reflects a loss of sexual function, associated to migration in areas where sexual alternate host is lacking, or not necessary for the completion of epidemic cycle. Approximate Bayesian computation analyses confirmed an out of Himalaya spread of PST, with Pakistan and China being the most ancestral population. A detailed analysis of Pakistani population at regional level revealed the existence of a strong population subdivision, a high genotypic diversity and the existence of recombination signature at each location reflecting the role of sexual recombination in the temporal maintenance at local level. A time spaced sampling of PST in the valley of Tianshui (China) inspired the development of a new estimator, allowing to quantify the relative contribution of sexual reproduction and effective population size on the basis of clonal resampling within and between years. A sexual reproduction rate of 74% (95% confidence interval [CI]: 38-95%) and effective population size of 1735 (95% CI: 675-2800) was quantified in Chinese PST population. The description of the origin and migration routes of PST populations worldwide and at its centre of diversity contributes to our understanding of PST evolutionary potential, and is helpful to build disease management strategies.

It is important to understand the underlying plant defense mechanisms in order to establish best strategies to reduce losses due to diseases in cereals. The current available information is mostly on model organisms and their plant-pathogen interactions. However, this study is focused on the identification of genes involved in the resistance mechanism of one of the most devastating diseases of wheat, yellow rust. The strategy undertaken was to use differential display method (DD) together with microarray technology, on yellow rust differential lines of wheat (Avocet-Yr1 and Avocet-Yr10) infected with the virulent and avirulent Puccinia striiformis f. sp. tritici races (Pst: PST17, PST45, 169E136 and 232E137) together with appropriate control infections. DD primer combinations of ninety allowed the detection of fourteen differentially expressed genes which were also confirmed by real-time QRT-PCR analysis. All of but one were found to be novel sequences in wheat genome. Among those, two very important genes were identified as full ORF including 5&rsquo / and 3&rsquo / end untranslated regions (UTR) / namely cyclophilin like protein (putative antifungal activity) and ubiquitin conjugating enzyme (E2). The sequence homology analysis of the cloned gene fragments reveled that the genes detected have roles in ubiquitinylation, programmed cell death (apoptosis), putative antifungal activities, disease resistance, pathogen related responses, including a few with no known function. In addition to DD analysis, using wheat Affymetrix &ldquo / GeneChip&rdquo / , we identified 93 differentially expressed ESTs of wheat in response to avirulent pathogen attack. We also investigated the differential expression profiles of wheat leaves during the virulent infections and determined 75 differentially regulated ESTs. 1Selected ESTs were further analyzed using QRT-PCR analysis and 15 were confirmed to be differentially regulated. For the further characterization of the identified genes, such as determination of their putative roles in disease response, functional studies have to be performed. For this purpose, BSMV (Barley Stripe Mosaic Virus) mediated virus induced gene silencing (VIGS) method is optimized in this thesis for wheat. We have successfully managed to silence the endogenous PDS gene (Phytoene desaturase) of wheat which can be used as a positive control for the monitoring of silencing of the genes we have identified. Our results show that BSMV mediated VIGS can be used efficiently and effectively to silence wheat genes that we identified through differential display and microarray analysis and can be used to study the functions of those genes

Disease resistance is critical in soft red winter wheat (Triticum aestivum L.) cultivars. Leaf rust caused by Puccinia triticina Eriks and stripe rust caused by Puccinia striiformis Westend. f.sp. tritici Eriks. are destructive pathogens of wheat. From 2014 to 2015 phenotypic data was collected at diverse locations for resistance to leaf rust (North Carolina, Texas, and Virginia) and stripe rust (Arkansas, North Carolina, Georgia, Texas, and Virginia) in a Pioneer ‘25R47’ /‘Jamestown’ (P47/JT) population composed of 186 F5:9 recombinant inbred lines (RILs). Analysis of the P47/JT population identified two quantitative trait loci (QTL) for leaf rust resistance on chromosome 5B and two QTL for stripe rust resistance on chromosomes 3B and 6A. Phenotypic variation (%) explained by the putative leaf rust resistance QTL of Jamestown on 5B was as high as 22.1%. Variation explained by the putative stripe rust resistance QTL of Jamestown on 3B and 6A was as high as 11.1 and 14.3%, respectively. Jamestown is postulated to contain gene Lr18. Seedlings of 186 F5:9 recombinant inbred lines from the P47/JT population and 200 F2 seedlings from eight other crosses including Jamestown and/or the Lr18 host differential line RL6009 (Thatcher*6/Africa 43) were screened with P. triticina race TNRJJ. Genetic analysis of the populations was conducted to validate the presence of Lr18 in Jamestown. Results of linkage analysis identified SNP maker IWB41960 linked within 5 cM of gene Lr18 in all three populations. From 2016 to 2017 phenotypic data was collected at diverse locations for resistance to leaf rust (Illinois, North Carolina, and Virginia) in a ‘2013412’ (PI 667644) / VA10W-21 (PI 676295) population (412/21) composed of 157 doubled haploid (DH) lines. The 412/21 DH lines were genotyped via genotyping by sequence (GBS). Analysis of the 412/21 population identified one quantitative trait loci (QTL) region associated with adult plant resistance to leaf rust on chromosome 1B. Phenotypic variation (%) explained by the putative leaf rust resistance QTL of 2013412 on 1B was as high as 40.1%. Kompetitive allele-specific (KASP) markers KASP_S1B_8414614 and KASP_S1B_8566239 were developed as markers for use in marker assisted selection. / Ph. D. / Disease resistance to leaf rust and stripe rust is important when growing soft red winter wheat. Genetic resistance can have a benefit to cost ratio of up to 27:1, considerably better than that of fungicide treatments. From 2013 to 2017 disease data was collected across multiple locations spanning the eastern United States (Arkansas, Georgia, Illinois, North Carolina, Texas, and Virginia). DNA molecular markers were used to identify specific chromosome regions containing genes associated with leaf and stripe rust resistance. DNA markers associated with genes conferring resistance to leaf rust resistance were identified in three chromosome regions, and genes in two regions were associated with stripe rust resistance. These genes and molecular markers associated with them can be used by scientists to further enhance resistance in wheat cultivars. Another study was conducted to determine if Lr18, a gene for leaf rust resistance that has a large effect, is present in the Virginia Tech soft red winter wheat breeding material. This gene (Lr18) is known to have been introduced from an ancestral species highly related to wheat. Wheat seedlings derived from crosses between lines postulated to carry Lr18 with susceptible lines were tested for resistance to a specific strain of leaf rust lacking virulence to Lr18. Genetic analysis of the ratio of resistant versus susceptible seedlings and association between DNA molecular markers and resistant seedlings were conducted to validate the presence of gene Lr18. A molecular marker linked tightly to gene Lr18 was identified in the study. This gene was found to be widely distributed in soft red winter wheat breeding materials and the molecular marker associated with gene Lr18 will be useful for scientists to further improve resistance in wheat cultivars.

Wheat is one of the most essential food sources in the world. However, there has been serious yield loss of wheat production due to stripe rust disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici. The cost-effective and long-lasting defense to the disease can be achieved by generating genetically resistant crops against the disease forming pathogens. To accomplish this, first step is to acquire knowledge in the plant pathogen interactions of the crop and the pathogen of interests at the cellular and the molecular level. In this thesis research, PstHa2a5 candidate effector gene from Puccinia striiformis f. sp. tritici is investigated to identify its role and interaction between host factors in yellow rust infected Triticum aestivum L. The gene construct was engineered with FLAG-tag fusion at its N-terminus, and synthesized. This construct was cloned into pJL48-TRBO vector for an expression in Nicotiana benthamiana via agrobacterium-mediated gene transformation. The expressed protein structure with FLAG-tag was purified, and immunoprecipitated with one putative N. benthamiana interactor by immunoprecipitation experiments. This candidate interactor protein will be identified with Mass Spectroscopy. In addition to this, subcellular localization of the effector candidate was examined in N. benthamiana plant. This was achieved by cloning PstHa2a5 gene construct in pK7WGF2 gateway destination vector and localization is determined by GFP expression in N. benthamiana after agrobacterium-mediated gene transformation.

Leaf rust and stripe rust of wheat (Triticum aestivum L.) are caused by the fungal pathogens Puccinia triticina, and Puccinia striiformis f.sp. tritici, respectively. In North America, the incorporation of adult-plant resistance (APR) genes into breeding lines has been an important strategy to achieve durable resistance to both diseases. Previously, the spring wheat cultivar AC Domain was reported to express an effective level of adult-plant resistance (APR) to leaf rust under field conditions. Early gene postulation work had suggested AC Domain might carry the APR gene Lr34 due to its phenotypic similarity to other Lr34 carrying lines. However, new gene specific markers have shown that AC Domain is not a carrier of Lr34. The objective of this research was to genetically localize the resistance in AC Domain, which is important because the cultivar has frequently been used as a parent in Canadian breeding programs, primarily for its value as a source of pre-harvest sprouting resistance. A mapping population of 185 doubled haploid (DH) lines derived from the cross ‘RL4452’ by ‘AC Domain’ was used for this study. RL4452 is a known carrier of Lr34. During 2011-2012, the DH population was evaluated in field leaf rust nurseries at Saskatoon, SK and Portage, MB and at a stripe rust nursery at Lethbridge, AB. Field results indicated that rust resistance in the mapping population was variable, with lines ranging from highly resistant, to highly susceptible. DH lines carrying Lr34 showed a high level of resistance to both diseases. Thus, the non-Lr34 carriers were genotyped using select SSR markers, and by an Illumina 9k Infinium iSelect SNP assay for subsequent quantitative trait loci (QTL) analysis. QTL analysis revealed that AC Domain donated a major resistance QTL located on chromosome 2BS, that mapped 46 cM proximal to markers linked to Lr16, and explained a significant portion of the leaf and stripe rust phenotypic variance in all test environments. In addition, this QTL was significantly associated with the expression leaf tip necrosis (LTN), reduction in area under the disease progress curve (AUDPC), and coefficient of infection (CI). In certain environments the interaction between the 2B QTL and Lr34 was additive resulting in a superior level of rust resistance. Indoor rust testing showed AC Domain was susceptible to both diseases at the seedling stage. Taken together these results suggest that the identified resistance in AC Domain is likely due to the presence of an APR gene, on chromosome 2BS.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Microsatellites were used to differentiate Leaf (Puccinia triticina Eriks.) and Yellow rust (Puccinia striiformis Westend. f. sp. tritici Eriks.) pathotypes. There was sufficient diversity in the Leaf rust microsatellite markers to differentiate the pathotypes and create a phylogenetic tree of Leaf rust. Three of the microsatellite markers were sufficient to differentiate all the Leaf rust pathotypes. Sufficient diversity in the Yellow rust microsatellite markers was also observed which made it possible to differentiate the pathotypes. Only three pathotypes were used so no phylogenetic inference was made. Two microsatellite markers were sufficient to differentiate all the yellow rust pathotypes. Microsatellite and Amplified Fragment Length Polymorphisms (AFLP) markers were used to differentiate Stem rust (Puccinia graminis f. sp. tritici Eriks. and Henn.) pathotypes, and the data was combined for phylogenetic analysis. AFLP bands unique to each Stem rust pathotype were converted to Sequence Characterised Amplified Region (SCAR) markers. A single specific SCAR marker was created for UVPgt52. A second SCAR marker amplified four of the eight pathotypes. None of the other SCAR markers were specific. A 270 basepair fragment of the ITS1 region of the rDNA gene of all the Puccinia spp. was also sequenced in order to develop pathotype specific primers that could be used in a Real Time-PCR to determine relative levels of pathogen inoculum in a sample. Unfortunately insufficient diversity in the sequences of the ITS1 region of the rDNA gene did not allow unique primers to be designed for each pathotype making it impossible to proceed with the relative quantification using Real Time-PCR. Following marker development ninety one field isolates were collected from eleven sites in the Overberg and Swartland regions during 2008 and 2009. In the field isolates, four different Leaf rust pathotypes were identifiable. UVPgt13 and UVPgt10 were most prevalent. The most prevalent Stem rust pathotypes were UVPgt50, UVPgt52, UVPgt54 and UVPgt57. Only 6E16A- was identifiable in the Yellow rust isolates. There were no apparent patterns in the distribution of Leaf, Stem or Yellow rust. Leaf and Stem rust were widely distributed, while Yellow rust was confined to three sites in the central South Cape, the only sites where climatic conditions were favourable for its development during the sampling period. The low levels of diversity found in the rust population when compared to international populations are probably due to the relatively small population size, the lack of a host for sexual reproduction, the small sample size, the effective monoculture and the strong selective pressure created by artificial control methods. / AFRIKAANSE OPSOMMING: Mikrosatellietmerkers is gebruik om Blaar- (Puccinia triticina Eriks.) en Geelroes-( Puccinia striiformis Westend. f. sp. tritici Eriks.) patotipes te onderskei. Daar was genoeg diversiteit in die Blaarroesmerkers om verskillende patotipes te kon onderskei en om „n filogenetiese-boom te kon saamstel. Met drie van die mikrosatellietmerkers was dit moontlik om al die Blaarroespatotipes te kon onderskei. Daar was genoeg diversiteit in die Geelroesmerkers om al die patotipes te kon skei en met twee van die mikrosatellietmerkers kon al drie Geelroespatotipes van mekaar onderskei word. Mikrosatelliet- en ge-Amplifiseerde-Fragment-Lengte-Polimorfismes (AFLP) is gebruik om die Stamroes- (Puccinia graminis f. sp. tritici Eriks. and Henn.) patotipes te skei. AFLP-fragmente uniek aan „n spesifieke patotipe is omgeskakel na Volgorde-Spesifieke-ge-Amplifiseerde-Streek (SCAR) merkers. „n Spesifieke SCAR-merker is gemaak vir UVPgt52. „n Tweede SCAR-merker het vier van die patotipes geidentifiseer. Nie een van die ander SCAR-merkers was spesifiek t.o.v. „n spesifieke patotipe nie. Die volgorde van „n 270 basispaar fragment van die ITS1-streek van die rDNS-geen van al die Puccinia spp. is bepaal om patotipe spesifieke inleiers te kon ontwerp. Hierdie inleiers kan gebruik word om „n Intydse-Polimerase-Ketting-Reaksie (RT-PCR) te ontwerp om sodoende die relatiewe vlakke van die patogeen besmetting in „n monster te bepaal. Daar was nie genoeg diversiteit in die bepaalde volgordes om die spes1fieke inleiers te kon identifiseer nie en dus is RT-PCR laat vaar. Na die ontwikkeling van die merkers was een-en-negentig veldmonsters ingesamel afkomstig van elf lokaliteite in die Overberg en Swartland gedurende 2008 en 2009. Vier Blaarroespatotipes was uitkenbaar. Blaarroespatotipes UVPrt10 en UVPrt13 was die mees algemeenste. UVPgt50, UVPgt52, UVPgt54 en UVPgt57 was die mees algemene Stamroespatotipes. Net 6E16A- is geidentifiseer by die Geelroes-isolate. Daar was geen patroon in die verspreiding van Blaar-, Stam- of Geelroes patotipes. Blaar- en Stamroes was die wydste versprei, maar Geelroes het net by drie lokale in die sentrale Suid-Kaap voorgekom. Die lokaliteite is die enigste waar die weersomstandighede gunstig was vir Geelroes ontwikkeling gedurende die periode van monsterneming. Die lae vlakke van diversiteit wat in die roespopulasie gevind was is in teenstelling met internasionale populasies. Dit mag moontlik wees as gevolg van die relatief beperkte populasie grootte, die afwesigheid van „n gasheer vir seksuele voortplanting, die beperkte hoeveelheid monsters wat ingesamel is en die sterk selektiewe druk weens kunsmatige beheer.