Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Both qualitative and quantitative resistance mechanisms are important contributors to disease resistance in rice. To identify useful sources of durable resistance for Korean breeding programs, the distribution of rice blast isolates compatible to widely used resistance (R) genes was analyzed. Interactions of 3,747 Korean rice Magnaporthe oryzae isolates with eight monogenic lines, each harboring a major blast R gene, were tested. Lines with R gene Pi-9 and Pi-5 were susceptible to the fewest M. oryzae isolates, and therefore, this gene might be applied for blast resistance in breeding programs in Korea. Six major blast resistance genes were susceptible to more than 60 % of the population, suggesting limited utility of these genes in breeding programs. Quantitative trait loci (QTL)-based resistance is predicted to provide durable and broad spectrum resistance to rice diseases. A candidate gene approach was applied to a population of 164 recombinant inbred lines to identify sources of quantitative resistance. Resistance gene analogs and defense response genes were mapped on the rice chromosomes, and analyzed for their association with blast and bacterial blight resistance QTL. A total of 21 putative QTL for blast resistance were identified on chromosomes 1, 4, 5, 6, 8, 9 and 12. Four putative QTL for bacterial blight resistance were identified on chromosome 3, 5 and 10. Thirteen RGA markers were associated with 11 different QTL on chromosome 1, 5, 8, and 9. The role of one disease resistance QTL associated gene, Os02g39330, encoding a chitinase was investigated for contributing to basal defense responses. RNAi silencing was used to evaluate contributions of the gene for the resistance to Rhizoctonia solani and M. oryzae. Five transgenic lines harboring the silencing construct and which differed in the level of expression of Os02g39330 were screened for responses to R. solani and M. oryzae. The chitinase gene expression levels were inversely correlated with sheath blight disease severity, suggesting a role for this defense gene in resistance to R. solani. Rice blast disease was not affected by silencing Os02g39330. Both qualitative and QTL-based resistances provide valuable sources of disease resistance, and a combination of R gene Pi-9 and QTL harboring the Os02g39330 chitinase may help to stabilize resistance.

Wheat is infected by leaf rust disease (Puccinia triticina Eriks.) almost everywhere it is cultivated. The dynamic nature of P. triticina populations affects the effective life span of genes conferring leaf rust resistance (Lr genes). Genetic diversity and combinations of Lr genes should be used to achieve durable resistance. Toropi, a Brazilian wheat cultivar, has maintained leaf rust resistance since its release in 1965, suggesting that it is a good candidate for durable resistance. Two recessive complementary adult plant genes were previously described in Toropi. The objective of this study was to characterize and map the sources of resistance present in Toropi. Double haploid (DH) populations developed by crossing the susceptible leaf rust cultivar Thatcher with Toropi were analysed in Canada (Glenlea – 2010 and Portage La Prairie – 2011), New Zealand (Lincoln – 2010 and 2011) and in Brazil (Parana – 2011), and in greenhouse. The leaf rust reactions indicated the presence of at least four leaf rust resistance genes in Toropi: one seedling gene and three adult plant genes. The seedling resistance gene Trp-Se, responsible for immune response in New Zealand, was mapped on chromosome 3D. QTL analyses identified a QTL associated with leaf rust resistance (QLr.crc-5AL.1) on chromosome 5AL, which overlapped with a QTL for stripe rust (QStr.crc-5AL.1) in the same population. This gene, designated Trp1, is believed to be one of the two adult plant complementary partial resistance genes. The position of the Trp-2 is not confirmed yet. One minor race specific adult plant gene, temporarily designated Trp-3, was mapped on 4BL chromosome. The Lr genes in Toropi confer minor effects against leaf rust, except for Trp-Se, which conditioned immunity in New Zealand. However, when the Toropi Lr genes were combined an almost immune response resulted. Toropi had a very good leaf rust resistance in South and North America, and in New Zealand. The molecular markers identified during this project could facilitate the incorporation of the Toropi genes in new cultivars, helping to achieve more diverse and durable wheat.

Wheat is infected by leaf rust disease (Puccinia triticina Eriks.) almost everywhere it is cultivated. The dynamic nature of P. triticina populations affects the effective life span of genes conferring leaf rust resistance (Lr genes). Genetic diversity and combinations of Lr genes should be used to achieve durable resistance. Toropi, a Brazilian wheat cultivar, has maintained leaf rust resistance since its release in 1965, suggesting that it is a good candidate for durable resistance. Two recessive complementary adult plant genes were previously described in Toropi. The objective of this study was to characterize and map the sources of resistance present in Toropi. Double haploid (DH) populations developed by crossing the susceptible leaf rust cultivar Thatcher with Toropi were analysed in Canada (Glenlea – 2010 and Portage La Prairie – 2011), New Zealand (Lincoln – 2010 and 2011) and in Brazil (Parana – 2011), and in greenhouse. The leaf rust reactions indicated the presence of at least four leaf rust resistance genes in Toropi: one seedling gene and three adult plant genes. The seedling resistance gene Trp-Se, responsible for immune response in New Zealand, was mapped on chromosome 3D. QTL analyses identified a QTL associated with leaf rust resistance (QLr.crc-5AL.1) on chromosome 5AL, which overlapped with a QTL for stripe rust (QStr.crc-5AL.1) in the same population. This gene, designated Trp1, is believed to be one of the two adult plant complementary partial resistance genes. The position of the Trp-2 is not confirmed yet. One minor race specific adult plant gene, temporarily designated Trp-3, was mapped on 4BL chromosome. The Lr genes in Toropi confer minor effects against leaf rust, except for Trp-Se, which conditioned immunity in New Zealand. However, when the Toropi Lr genes were combined an almost immune response resulted. Toropi had a very good leaf rust resistance in South and North America, and in New Zealand. The molecular markers identified during this project could facilitate the incorporation of the Toropi genes in new cultivars, helping to achieve more diverse and durable wheat.

Wheat diseases such as rusts and powdery mildews are among the most important and ancient diseases that affect wheat cultivation worldwide. The pathogen race specific resistance genes cannot maintain long lasting resistance. On the other hand, the presence of genes confers the non-race specific resistance last much longer. The durable resistance phenotypes in wheat against various rust and powdery mildew diseases were reported as Lr34, Yr18, and Pm38 separately / nevertheless, they were known to locate very close to each other based on linkage analysis. Recently, it was shown that all of these resistance phenotypes are indeed conferred by the same gene, encoding Adenosine triphosphate&ndash / binding cassette transporter (ABC-transporter) type protein. The way with which this transporter is functioning to maintain the durable resistance against different types of pathogens is still unknown. v The presence of LR34 (or ABC-transporter) gene resistant allele can be detected by specially designed markers. In spite of the few DNA sequence differences between the resistant and the susceptible alleles, the easily applicable PCR based markers allow the detection of the presence of this durable resistant allele on any given variety or cultivar. In our study, 62 different Turkish bread wheat varieties were screened by the gene specific molecular markers, developed from those LR34 gene mutation sites. The 11 cultivars determined to contain the resistant allele. This study is the first screening of Turkish cultivars for the presence of LR34 gene.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant diseases are among the major causes of food insecurity. In South Africa the wheat fungal diseases including stem rust caused by Puccinia graminis f. sp. tritici, leaf rust caused by P. triticina and stripe rust caused by P. striiformis f. sp. tritici are the most important. Genetic resistance is a viable way of protecting wheat crops against the wheat rusts, especially cultivars carrying multiple genes that confer durable resistance. In order to breed for multi-gene resistance an effective breeding strategy that allows for selecting multiple resistance genes and other desirable traits needs to be devised. The aim of this study was to identify a number of genotypes with combinations of different rust resistance genes, good grain yield and end-use quality out of an existing pre-breeding population and thereby identify superior parents. In order to achieve the stated aim the following objectives have been identified: identify wheat lines through marker-assisted selection (MAS) carrying the gene complexes, Sr31/Lr26/Yr9, Lr24/Sr24, Lr37/Sr38/Yr17, Lr34/Yr18 and Sr2; to develop inbred lines to evaluate selected lines under field trials. From the initial subset of 64 lines, 60 were chosen and advanced to the doubled haploid (DH) phase and seed multiplication. The 60 lines either carried one or more of the three rust resistance gene complexes. The genes that were the most prominent were Sr31/Lr26/Yr9 and Lr24/Sr24. The selected lines were incorporated into a DH seed multiplication phase. After 4 cycles of seed increases and preliminary field evaluation during multiplication, 15 lines were chosen and subjected to multi-location field trails. The extensive multi-location field trails carried out in this study aided in identifying genotypes from the 15 MS-MARS lines with good adaptability and stability in regards to yield and baking quality. An important observation was that the molecular markers employed to indentify quality loci correlated well with the genes encoding the HMW-GS 5, 10 and 12 as observed with the Agilent© 2100 Bioanalyzer. In future studies the lines which performed the best could be re-introduced into the existing MSMARS pre-breeding programme of the Stellenbosch University’s Plant Breeding Laboratory (SUPBL). The frequencies of desired alleles could be increased in this manner. Since the majority of these characteristics are influenced by quantitatively inherited alleles, using these lines as recurrent parents will increase the frequencies of these alleles in the existing SU-PBL pre-breeding population. / AFRIKAANSE OPSOMMING: Plantsiektes is van die belangrikste oorsake van voedselonsekerheid ter wêreld. In Suid-Afrika is die roesswamme van die belangrikste plantsiektes wat koring produksie beïnvloed. Hierdie siektes sluit in, stamroes wat veroorsaak word deur Puccinia graminis f. sp. tritici, blaarroes wat veroorsaak word deur P. triticina en streeproes wat veroorsaak word deur P. striiformis f. sp. tritici. Genetiese weerstand is ‘n uitstekende manier om koring te beskerm teen hierdie swamsiektes. Weerstand wat gebasseer is op veelvuldige weerstandsgene is veral ‘n goeie middel om genetieseweerstand op ‘n volhoubare basis in koringteling toe te pas. Om veelvuldige weerstandsgene in koringkultivars in te teel word ‘n effektiewe telingstrategie benodig. Die doel van die studie was om genotipes te identifiseer met kombinasies van veelvuldige weerstandsgene vir roes, sowel as goeie eienskappe belangrik vir graanopbrengs en bakkwaliteit. Lyne is geïdentifiseer uit ‘n bestaande voortelingspopulasie van Stellenbosch Universiteit se Planteteelt Laboratorium (SU-PTL) wat geteel was met spesifiek weerstand en opbrengs potensiaal in gedagte. Om die doel van die studie te bereik is sekere doelwitte daar gestel. Hierdie doelwitte sluit in om lyne uit die populasie te selekteer deur middel van merker bemiddelde seleksie (MBS) vir gene naamlik Sr31/Lr26/Yr9, Lr24/Sr24, Lr37/Sr38/Yr17, Lr34/Yr18 en Sr2; om die geselekteerde lyne suiwertelend te maak; sowel as om die suiwertelende lyne in veld proewe in te sluit. Van die oorspronklike stel van 64 lyne, is 60 gekies vir verdere studie. Deur middel van die verdubbelde haploïed (VH) tegniek is die lyne suiwertelend gemaak. Die 60 lyne het een of meer van die geselekteerde gene bevat. Die mees prominente gene was die twee geen komplekse Sr31/Lr26/Yr9 en Lr24/Sr24. Na vier siklusse van saadvermeerdering en voorloppige seleksies is 15 lyne ingesluit by ‘n multi-omgewing veldproef. Hierdie uitgebreide multi-omgewing veldproewe het gehelp om individue uit die 15 lyne te identifiseer wat oor goeie aanpasbaarheid en stabiliteit beskik met betrekking tot opbrengs en bak kwaliteit. Die molekulêre merkers gebruik om die gene verantwoordelik vir die kodering van HMGGS 5, 10 en 12 op te spoor het goed gekorreleer met die HMG-GS bande bepaal met behulp van die Agilent© 2100 Bioanalyzer. Toekomstige studies kan moontlik insluit die gebruik van die lyne wat geïdentifiseer was met goeie kenmerke in die bestaande MS-MARS teelprogram van die SU-PTL. Die frekwensies van die verlangde allele kan op hierdie manier in die populasie verhoog word.

Doctor of Philosophy / Department of Plant Pathology / Scot H. Hulbert / The rice cultivar Oryzica Llanos 5 (OL5) possesses a high level of resistance to the fungus Magnaporthe grisea. The number and chromosomal location of quantitative trait loci (QTL) conferring resistance against eight isolates of the blast fungus were tested in two different populations of recombinant inbred lines from the cross Fanny x OL5. Twenty one QTL were detected and associated with the resistance traits, disease leaf area and lesion type, on 9 rice chromosomes. Eight of these 21 resistance loci had significant resistance effects in both experiments, while the others had effects that were only statistically significant in one experiment. Most, but not all, of the QTL occurred in the same genomic regions as either genes with major race-specific effects or other resistance QTL that had been described in previous experiments. Most of the QTL appeared to be race-specific in their effects but it is possible some of the QTL with smaller effects were nonspecific. One of the blast isolates used was FL440, which causes limited disease on OL5 and was probably virulent on most or all of the major genes from OL5. Three QTL affected resistance to FL440 in both experiments, one of which mapped to a region on chromosome 9 where no blast resistance genes have yet been mapped. An advanced backcross strategy with marker-assisted selection for OL5 alleles in QTL regions was used to generate five BC2F3 populations carrying five different target regions associated with partial resistance to rice blast disease. Three of five of these populations were analyzed for segregation for resistance to the M. grisea isolate FL440. One QTL designated qrbr-11.3 near the bottom of rice chromosome 11 was found to be significantly associated with partial blast resistance in 120 lines of a BC2F3 population (P< 0.01). This QTL accounted for 12.4% and 8.0% of the phenotypic variation in diseased leaf area and lesion type observed under greenhouse inoculation. Examination of the genomic sequence at the qrbr-11.3 locus showed that twenty-nine candidate resistance genes are present at that locus (~1.8 Mb), twenty-seven of which are predicted NBS-LRR genes. Ultimately, the information from this study can be integrated into the development of improved lines with OL5-derived QTL for resistance.

Phytophthora sojae, the causal organism of stem and root rot, and <i>Soybean mosaic virus</i> (SMV) cause two of the most highly destructive diseases of soybean (<i>Glycine max</i> L. Merr). <i>P. sojae</i> can be managed either through deployment of race-specific resistance or through quantitative resistance termed partial resistance. In the current study, partial resistance to <i>P. sojae</i> was mapped in an interspecific recombinant inbred line (RIL) population of <i>Glycine max</i> by <i>Glycine soja</i>. One major quantitative trait loci (QTL) on molecular linkage group (MLG)-J (chromosome 16) and two minor QTL on MLG-I (chromosome 20) and -G (chromosome 18) were mapped using conventional molecular markers. Additionally, partial resistance to <i>P. sojae</i> was mapped in the same RIL population using single feature polymorphism (SFP) markers that further fine mapped the <i>P. sojae</i> QTL and identified potential candidate genes contributing to resistance. In a separate study, race-specific resistance was characterized in PI96983 discovering a potentially new allele of <i>Rps4</i> on MLG-G. Finally, using the newly available whole-genome shotgun sequence of soybean, <i>Rsv4</i> conferring resistance to strains of SMV known in the US, was localized to an approximately 100 kb region of sequence on chromosome 2 (MLG-D1B). Newly designed PCR-based markers permit for efficient selection of <i>Rsv4</i> by breeding programs. Identified candidate genes for <i>Rsv4</i> are discussed. Genomic resources developed in all of these studies provide breeders the tools necessary for developing durable resistance to both SMV and <i>P. sojae</i>. / Ph. D.

Le mildiou de la vigne est causé par l’oomycète Plasmopara viticola, qui s’attaque aux parties aériennes non-lignifiées affectant la production viticole. Une alternative à l’utilisation de pesticides est l’utilisation de variétés de vigne à la résistance durable, et un programme pour leur création par croisement entre espèces résistantes et la vigne cultivée, Vitis vinifera, est en cours. Ce programme nécessite l’identification de nouveaux gènes de résistance, ce que le projet vise à faire par (1) le criblage de vignes résistantes avec des effecteurs conservés chez P. viticola, (2) l’étude fonctionnelle d’effecteurs candidats. Le criblage de plantes résistantes n’a conduit à l’identification d’aucun nouveau facteur de résistance majeur. L’étude fonctionnelle d’effecteurs a permis la mise en évidence d’une nouvelle famille d’effecteurs chez P. viticola et a conduit à l’identification de deux effecteurs Pv33, nucléaire, et Pv47, associé au réticulum endoplasmique, qui induisent des défenses végétales. / Grapevine downy mildew is caused by the oomycete Plasmopara viticola, which attacks the aerial non-lignified tissues affecting wine production. An alternative to the use of pesticides is the use of vine varieties with sustainable resistances. A programme aiming to create such varieties by crossing resistant species with the cultivated grapevine, Vitis vinifera, is ongoing. Within this program requiring the indentification of new resistance genes, which the project aims to do by (1) screening resistant vines with effectors stored in P. viticola, (2) performing a functional study of candidate effectors. The screening of resistant plants did not lead to the identification of any new major resistance factors. The functional study of effectors revealed a new family of effectors in P. viticola and led to the identification of two effectors Pv33, nuclear, and Pv47, associated with the endoplasmic reticulum, which induce plant defences.

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.