A ferrugem da folha é a doença que causa maiores prejuízos à triticultura. A resistência genética é comprovadamente o método mais eficiente e econômico de controle. Em trigo, a resistência à ferrugem da folha pode ser específica à raça e não específica à raça. Atualmente a melhor estratégia para manter uma proteção efetiva contra a ferrugem da folha consiste em empregar combinações de genes, independente do tipo de resistência que conferem. A piramidização de genes é facilitada com o uso de marcadores moleculares. Os mecanismos moleculares de resistência dos genes que conferem resistência específica à raça têm sido amplamente estudados, no entanto da resistência não específica à raça pouco se sabe. Assim, este projeto teve como objetivos: I) validar marcadores moleculares para seleção assistida para a resistência à ferrugem da folha em genótipos brasileiros de trigo; II) identificar marcadores moleculares potencialmente associados a genes de resistência não específica à raça à ferrugem da folha; III) identificar e caracterizar análogos a genes de resistência em trigo;IV) caracterizar mecanismos moleculares da resistência não específica à raça a ferrugem da folha em trigo. Para a validação foram avaliados cinco marcadores PCR-específcos associados aos genes Lr1, Lr9, Lr10 e Lr24. Os marcadores associados aos genes Lr9, Lr10 e Lr24 apresentaram potencial para uso na seleção assistida, pois foram específicos para plantas contendo estes genes. Para a identificação de RGAs foram utilizadas três combinações de primers degenerados. A maioria das seqüências apresentou os motivos comuns a genes de resistência. Uma seqüência apresentou alta homologia com genes de resistência previamente identificados. Os primers derivados das seqüências identificadas apresentaram alto polimorfismo, sendo que um destes mostrou-se associado ao gene Lr26 e a resistência de planta adulta à ferrugem da folha. A combinação da técnica de AFLP com linhagens diferenciais contrastantes para os genes Lr13 e Lr34 permitiu identificar duas seqüências potencialmente associadas ao gene Lr34. O uso da SSH permitiu identificar genes diferencialmente expressos na resistência não específica à raça à ferrugem da folha no cultivar Toropi. Dentre as seqüências identificadas estão genes codificadores de proteínas de membranas potencialmente associados à percepção do patógeno e genes com domínios característicos de sinalizadores secundários para a resistência como NPR1, Rar1, Sgt1 e calmodulina. Além destas, foram identificados também seqüências com homologia a genes codificadores de enzimas chaves no controle de rotas de síntese de substâncias relacionadas à defesa e envolvidas no metabolismo primário, principalmente produção de energia. Os marcadores e seqüências obtidas e mecanismos elucidados neste trabalho representam ferramentas valiosas para a obtenção de cultivares de trigo com resistência ampla e durável à ferrugem da folha. / Leaf rust is one of the most important wheat diseases worldwide. The genetic resistance is the most efficient and economical method to leaf rust control. Host resistance to leaf rust can either be race-specific or no race-specific. The best strategy to maintain an effective protection against leaf rust consists in resistance gene combinations, independent of the resistance type. Marker-assisted selection greatly facilitates gene pyramidization. The molecular mechanisms of race-specific resistance genes have been well studied, however there is little known about the no race-specific resistance gene mechanisms. The objectives of this project were: I) to validate molecular markers for marker-assisted selection to leaf rust resistance genes in Brazilian wheat cultivars; II) to isolate and characterize resistance gene analogues (RGAs) in wheat; III) to identify molecular markers potentially associated to leaf rust no race-specific resistance genes; IV) to characterize molecular mechanisms from no race-specific resistance genes to leaf rust in wheat. Five PCR-specific markers previously identified as associated to the Lr1, Lr9, Lr10 and Lr24 genes were evaluated. The markers associated to the Lr9, Lr10 and Lr24 genes were specific to the cultivars carrying them in the Brazilian wheat cultivars, demonstrating potential for marker-assisted selection. Three degenerate primer combinations were used for the RGAs identification and most of the identified sequences showed the conserved motifs from resistance genes. One sequence showed high homology with previous identified plant resistance genes. The primers from identified sequences showed high polimorphism, and one of these is associated to the Lr26 leaf rust resistance gene and to the adult plant resistance present in cultivar BR35(?). The use of AFLP (amplified fragment length polymorphism) technique with near-isogenic lines carrying Lr13 and Lr34 genes from wheat cv. Thatcher allowed to identify two sequences potentially associated to the Lr34 gene. SSH were used to identify leaf rust induced genes from no race-specific resistance wheat cultivar Toropi. Among the identified sequences there are proteins of membranes for genes potentially associated to the pathogen perception and genes with secondary signals resistance domains as NPR1, Rar1, Sgt1 and calmodulin binding protein. Besides these, we identified sequences with homology to genes for key enzymes in the control of the related defense substances synthesis and involved in the primary metabolisms, mainly energy production. The markers and sequences obtained and the mechanisms elucidated in this work represent a valuable tool for the development of wheat cultivars with more reliable resistance to leaf rust.

Pre-harvest sprouting (PHS) in bread wheat (<i>Triticum aestivum</i> L.) is one of the major abiotic constraints influencing the production of high quality grain. The flour milled from sprouted wheat grains has increased Ñ-amylase activity as compared to non-sprouted grain. PHS negatively affects the properties of flour with deleterious effects on bread and noodle quality. White-grained wheat is generally more susceptible to PHS damage than red-grained wheat. The objectives of this study were to identify a suitable method for phenotyping PHS resistance and to identify PHS resistance genomic regions and markers that could be used for marker-assisted selection in wheat improvement programs. A doubled haploid (DH) mapping population from a cross between two white-grained spring wheat genotypes, Argent (non-dormant) and W98616 (dormant) was used in this study. Forty DH lines (20 dormant and 20 non-dormant) were evaluated for germination frequency, Falling Number, and Ñ-amylase activity in dry and water-imbibed seeds and spikes. The germination test was the most reliable method for measurement of PHS resistance, whereas the Falling Number and Ñ-amylase activity in dry harvested seeds could not be correlated to dormancy levels. However, a positive association (r = 0.60***) was detected between germination frequency and Ñ-amylase activity in imbibed seeds. To identify the genomic regions associated with PHS resistance, a genetic linkage map with a total genome coverage of 2,577 cM was developed. The map was constructed from 913 scored markers (356 SSR, 290 AFLP, 258 DArT and 9 EST) with an average marker density of 3.7 cM/marker. Five genomic regions on chromosomes 1A, 3A, 4A, 7A and 7D were associated with PHS resistance by interval mapping and all regions were contributed by the dormant parent W98616. A total of 60 Canadian wheat cultivars and experimental lines were screened with three SSR markers, DuPw004, barc170 and wmc650, located under the major quantitative trait locus (QTL) on chromosome 4A. The SSR markers explained 60-75% of the total variation in germination frequency among different wheat genotypes. By using the DuPw004 marker in marker-assisted back crossing, the population size in the BC1F1 and BC2F1 generations were reduced by 41% and 59%, respectively. Thus, the 4A QTL markers have been proven useful for marker-assisted selection of PHS resistance for wheat improvement.

Pre-harvest sprouting (PHS) in bread wheat (<i>Triticum aestivum</i> L.) is one of the major abiotic constraints influencing the production of high quality grain. The flour milled from sprouted wheat grains has increased Ñ-amylase activity as compared to non-sprouted grain. PHS negatively affects the properties of flour with deleterious effects on bread and noodle quality. White-grained wheat is generally more susceptible to PHS damage than red-grained wheat. The objectives of this study were to identify a suitable method for phenotyping PHS resistance and to identify PHS resistance genomic regions and markers that could be used for marker-assisted selection in wheat improvement programs. A doubled haploid (DH) mapping population from a cross between two white-grained spring wheat genotypes, Argent (non-dormant) and W98616 (dormant) was used in this study. Forty DH lines (20 dormant and 20 non-dormant) were evaluated for germination frequency, Falling Number, and Ñ-amylase activity in dry and water-imbibed seeds and spikes. The germination test was the most reliable method for measurement of PHS resistance, whereas the Falling Number and Ñ-amylase activity in dry harvested seeds could not be correlated to dormancy levels. However, a positive association (r = 0.60***) was detected between germination frequency and Ñ-amylase activity in imbibed seeds. To identify the genomic regions associated with PHS resistance, a genetic linkage map with a total genome coverage of 2,577 cM was developed. The map was constructed from 913 scored markers (356 SSR, 290 AFLP, 258 DArT and 9 EST) with an average marker density of 3.7 cM/marker. Five genomic regions on chromosomes 1A, 3A, 4A, 7A and 7D were associated with PHS resistance by interval mapping and all regions were contributed by the dormant parent W98616. A total of 60 Canadian wheat cultivars and experimental lines were screened with three SSR markers, DuPw004, barc170 and wmc650, located under the major quantitative trait locus (QTL) on chromosome 4A. The SSR markers explained 60-75% of the total variation in germination frequency among different wheat genotypes. By using the DuPw004 marker in marker-assisted back crossing, the population size in the BC1F1 and BC2F1 generations were reduced by 41% and 59%, respectively. Thus, the 4A QTL markers have been proven useful for marker-assisted selection of PHS resistance for wheat improvement.

Fusarium head blight (FHB) caused by Fusarium graminearum and Septoria tritici blotch (STB) caused by Septoria tritici are economically important wheat diseases in Ontario. Both reduce yield, FHB is associated with mycotoxin accumulation including deoxynivalenol (DON). Different F. graminearum chemotypes produce either DON/15-acetyldeoxynvialenol (ADON) or DON/3-ADON. The majority (97.5%) of F. graminearum isolates collected from commercial fields across Ontario were 15-ADON chemotype, the remaining were 3-ADON. In inoculated field experiments 3-ADON chemotypes were more aggressive and toxic compared with 15-ADON chemotypes as measured by FHB symptoms and DON content. In inoculated field experiments with a population derived from ‘RCATL33’ and ‘RC Strategy’ soft red winter wheat parents, genetic resistance was more effective than fungicide application at controlling FHB. Field trials with the hard red winter wheat population derived from ‘Maxine’ and ‘FTHP Redeemer’ parents revealed that STB and FHB phenotypic resistance had no negative impact on grain yield in the absence of disease.

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Salinity stress limits the growth and productivity of agricultural crops in many regions of the world. Whole plant tolerance to soil salinity involves numerous processes in many different tissues and cell types. For many cereals, sensitivity to salinity is due to the accumulation of sodium (Na⁺) to toxic concentrations in the leaves. This thesis investigates a mechanism of control of Na⁺ accumulation in leaves of wheat. Bread wheat excludes sodium from the leaves better than durum wheat. Bread wheat is hexaploid (AABBDD) whereas durum wheat is tetraploid (AABB). The D-genome in bread wheat carries a major locus for sodium exclusion, Kna1, which may contribute to the differences in sodium exclusion between bread wheat and durum wheat. An unusual durum wheat, Line 149, excludes sodium to a similar degree as bread wheat. Line 149 was derived from a cross between a Triticum monococcum (accession C68-101; AA) and a durum wheat (T. turgidum ssp. durum cv. Marrocos; AABB). Line 149 had been found to contain two major genes for sodium exclusion, named Nax1 and Nax2, which appeared to retrieve sodium from the xylem sap in the roots and so prevent it reaching the leaves. Line 149 had been crossed with the durum wheat cv. Tamaroi, which accumulates high concentrations of Na⁺ in the leaves, and near-isogenic single-gene mapping populations had been developed for Nax1 and Nax2. Nax1 had been located on chromosome 2A. The objective of this thesis was to map Nax2 and identify a candidate gene. Nax2 mapped to chromosome 5AL based on linkage to microsatellite markers. A high-affinity potassium (K⁺) transporter (HKT)-like gene, HKT1;5 was considered as a candidate gene for Nax2, based on similarity of the phenotype to a rice orthologue. Sequence information from a wheat HKT1;5-like expressed sequence tag in the public database was used to develop a probe for use in Southern hybridsation. A HKT1;5-like fragment was identified in Line 149 and T. monococcum C68-101, but was absent in Tamaroi. The HKT1;5-like gene, named TmHKT1;5-A, co-segregated with Nax2 in the Nax2 single-gene mapping population. The HKT1;5 probe identified three putative HKT1;5-like genes on the long arm of chromosome 4B, and one HKT1;5-like gene on the long arm of chromosome 4D, in Langdon (T. turgidum ssp. durum) substitution lines, and in Chinese Spring (T. aestivum) ditelomeric lines. No A-genome HKT1;5 like gene was identified in Langdon or Chinese Spring. The D-genome HKT1;5 gene, named TaHKT1;5-D, was found to co-locate with Kna1, the gene for sodium exclusion in bread wheat, in Chinese Spring chromosome 4D deletion lines. Nax2 (TmHKT1;5-A) was found to be homoeologous with the gene for sodium exclusion in bread wheat, Kna1 (TaHKT1;5-D). TmHKT1;5-A and TaHKT1;5-D, and their promoters, were 94% identical, and both were expressed in the roots of wheat plants. This is consistent with the genes being located in the stele of the roots and retrieving Na⁺ from the xylem sap as it flows towards the shoot, and so excluding Na⁺ from the leaves. A marker for TmHKT1;5-A was developed to track this gene in durum wheat breeding programs. A study of the HKT1;5 gene in diploid ancestors of wheat indicated that this gene is present in most Triticum monococcum accessions, some T. boeoticum accessions, but not present in any T. urartu accessions. T. urartu is the likely A genome ancestor of modern wheat. This may explain the absence of HKT1;5 in the A genome of modern wheat. The protein encoded by TaHKT1;5-D transported sodium when expressed in Xenopus laevis oocytes. The inward currents were specific to Na⁺, but at particular mole fractions of Na⁺ and K⁺ outward currents were observed that were consistent with outward K⁺ transport. These data were consistent with the putative physiological function, of retrieving Na⁺ from the xylem sap as it flows to the leaves, and resulting in a net exchange with K⁺. A construct designed to silence the expression of TaHKT1;5-D was introduced to bread wheat cv. Bob White. Nineteen putative transgenic plants were developed. The leaf Na⁺ concentrations and genotype of the T1 individuals were assayed. The data from two of the transgenic plants indicated that TaHKT1;5-D may have been silenced and that this may have lead to the increase in Na⁺ accumulation in the leaves. However, this data is not conclusive at this time. The information gained from this study will assist the introduction of the Na⁺ exclusion trait into current durum cultivars, which are poor at excluding Na⁺ and are salt sensitive. This information will also contribute to the body of knowledge of ion transport in plants and salinity tolerance in wheat. / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Licentiatavhandling (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 2 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2007. / Härtill 5 uppsatser + 3 appendix.