Occurrence of the rust resistance gene Lr37 from Aegilops ventricosa in Argentine cultivars of wheat

Leaf rust of wheat ( Triticum aestivum L.) caused by the fungus Puccinia triticina (formerly P. recondita f. sp. tritici), is one of the most important foliar diseases of this crop. Lr37 rust resistance gene, which confers resistance in wheat against leaf rust, was introgressed into cultivated wheat from Aegilops ventricosa Tausch. Rust races with virulence to Lr37 have been identified in different countries, but it still provides resistance to a wide range of races and is useful in combination with other resistance genes. There are no reports about the presence, frequency and origin of Lr37 in Argentinean wheat cultivars. In this work, we analyzed 88 registered Argentinean wheat cultivars developed by different breeding companies and institutions during the last 15 years by means of a molecular marker which is diagnostic of the 2NS-2AS translocation which carries Lr37. Only 4 cultivars showed the amplification product associated with this chromosome fragment. These four cultivars which carry the translocated 2NS-2AS chromosome were registered by the same breeding company during the last seven years and all of them have European germplasm in their genealogy. To the best of our knowledge this is the first report of the presence of Lr37 in registered South American cultivars

Contribution of non-target-site resistance in imidazolinone-resistant imisun sunflower

The first commercial herbicide-resistant trait in sunflower (Helianthus annuus L.) is known as ‘Imisun’. Imidazolinone resistance in Imisun cultivars has been reported to be genetically controlled by a major gene (known as Imr1 or Ahasl1-1) and modifier genes. Imr1 is an allelic variant of the Ahasl1 locus that codes for the acetohydroxyacid synthase, which is the target site of these herbicides. The mechanism of resistance endowed by modifier genes has not been characterized and it could be related to non-target-site resistance. The objective of this study was to evaluate the role of cytochrome P450 monooxygenases (P450s) in Imisun resistance. The response to imazapyr herbicide in combination with P450s inhibitor malathion was evaluated in 2 Imisun lines, IMI-1 and RHA426. Malathion reduced herbicide efficacy in both lines, but IMI-1 was affected in a greater extent. A significant reduction in plant growth in response to P450s inhibitors 1-aminobenzotriazole and piperonyl butoxide treatment was detected in the Imisun line HA425. The increased susceptibility to imazapyr after P450s-inhibitor treatment indicates that herbicide metabolism by P450s is a mechanism involved in Imisun resistance. These results also suggest the involvement of different P450s isozymes in endowing resistance to imazapyr in Imisun cultivars.Fil: Breccia, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Gil, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Vega, Tatiana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Altieri, Emiliano. Nidera S. A. Departamento de Biotecnología; ArgentinaFil: Bulos, Mariano. Nidera S. A. Departamento de Biotecnología; ArgentinaFil: Picardi, Liliana Amelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Nestares, Graciela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentin

Identification, characterization, mapping and genomic organization of genes responsible for rust resistance in Sunflower

La roya más común en los girasoles cultivados y silvestres se encuentra en todos los países donde se cultiva girasol y ha causado graves pérdidas de rendimiento en Argentina, Canadá, Rusia, E.U.A. y Australia. La información obtenida a partir de monitoreos continuos de las zonas de producción de girasol con la intención de identificar las razas del patógeno presentes en las diferentes regiones y entender la dinámica producida a lo largo de los años, luego del uso de distintas metodologías de control, es una herramienta fundamental para definir las estrategias de mejoramiento. La sustitución de cultivares de polinización abierta susceptibles con híbridos que poseen resistencia a una o más razas ha disminuido considerablemente las pérdidas por roya. Sin embargo, a medida que se producen cambios en la población de roya y las razas capaces de atacar híbridos aumentan en frecuencia, las epidemias de roya se han presentado nuevamente. Existen hoy en día, numerosas fuentes de resistencia a roya identificadas en girasol, de diversos orígenes, pero solo unas pocas han sido extensivamente utilizadas, y sólo algunas de ellas han sido genéticamente caracterizadas. Además de dilucidar el control genético detrás de cada una de las fuentes de resistencia disponibles, y con el objetivo de poder hace un uso eficiente de estos genes, introgresarlos y apilarlos en germoplasma elite, es crítico conocer su ubicación genómica. Esto permitirá detectar marcadores ligados que faciliten su seguimiento a lo largo de cruzas y sus progenies derivadas. Este trabajo tuvo como objetivo general el estudio del control genético de la resistencia a roya en girasol. Se condujo un estudio de la distribución del patógeno en las regiones donde se produce el cultivo y una detallada caracterización racial. Esto permite hacer uso de los aislamientos obtenidos como una herramienta de selección y de estudio de especificidades de los factores implicados en la resistencia. Además, se trabajó para determinar la herencia de la resistencia a roya en fuentes que todavía no han sido caracterizadas genéticamente y determinar su relación con otras fuentes descriptas. Mapear en el genoma de girasol genes previamente descriptos utilizando marcadores moleculares del tipo SSR, SNP y marcadores diseñados de novo para las regiones genómicas de interés fue unos de los objetivos de mayor importancia. Al mismo tiempo, se realizó la piramidización o desagregación de genes de resistencia con el objetivo de determinar así sus especificidades. Se condujeron trabajos de recolección y análisis de aislamientos del patógeno a nivel nacional. Dichos aislamiento fueron posteriormente evaluados con materiales diferenciales, siendo algunos de los mismos seleccionados para nuestros objetivos centrales. Con el fin de entender el control genético subyacente a la resistencia se produjeron poblaciones recombinantes a partir del cruzamiento con diferentes fuentes de resistencia. Utilizando la metodología de Análisis de Segregantes en Masa se pudo establecer de manera rápida y eficiente la localización de los factores de control del carácter en el genoma del cultivo. Para dos de estas fuentes, HAR6 y P386, materiales que mostraban ser de gran utilidad para los programas de mejoramiento a nivel global dado su perfil de resistencia, se continuó con un proceso de mapeo aún más detallado. Este trabajo implicó el diseño de nuevos marcadores que permitieran mejorar la saturación en las regiones de interés, teniendo en cuenta información de secuencia previamente generada. Se realizó también un estudio de alelismo entre el factor que gobierna la resistencia en P386 y el presente en HAR4. La información obtenida permitirá la piramidización de estos factores en una misma línea endocriada de girasol. De este modo, se logró establecer un mapa de las razas fisiológicas de roya prevalentes en nuestro país y realizar comparaciones con información histórica, así como también inferir sobre los posibles motivos de los cambios observados a través del tiempo. Se pudo establecer que el control genético de la resistencia en las fuentes utilizadas estaba conferido por un gen o factor dominante, y que en todos los casos se localizaban en el grupo de ligamiento 13 del mapa público de girasol. En particular para HAR6 y P386, se mapeo de manera más detallada los factores determinantes de sus resistencias, RHAR6 y Pu6, respectivamente, y se logró establecer su posición con respecto a sus marcadores flanqueantes. Esta información permitió, además de validar una estrategia de selección asistida por marcadores para este carácter, establecer herramientas para la eliminación del arrastre por ligamiento de las regiones adyacentes. En el caso de Pu6, se logró establecer su relación alélica con R4, de HAR4. El diseño de nuevos marcadores para lograr la saturación del grupo de ligamiento 13 se llevó a cabo mediante dos estrategias. La primera, basada en el diseño de nuevos marcadores microsatélites a partir de secuencias de una BAC localizada en la región de interés. Esto dió como resultado la obtención de 3 nuevos marcadores, uno de ellos, NidGi1, logró ser ubicado en los mapas de ligamiento desarrollados. La segunda estrategia estuvo basada en el diseño de cebadores degenerados con alta similitud a regiones aminoacídicas y nucleotídicas conservadas presentes en análogos de genes de resistencia. Mediante esta estrategia se logró generar productos de amplificación que, sin embargo, no mostraron polimorfismo para el material analizado. Los resultados de este trabajo permitieron contribuir al conocimiento existente sobre la genética de la resistencia a roya. Esto permitirá el diseño de germoplasma utilizando una estrategia sostenible de control de la enfermedad. Además proporciona herramientas para la asistencia a los programas de mejoramiento en la definición de fuentes de tolerancia, estrategias de introgresión y piramidización de genes en materiales elite. Finalmente, las estrategias, metodologías, herramientas y conocimientos desarrollados en esta tesis contribuyen al desarrollo del cultivo y promueven la adopción de la genómica en las distintas etapas del mejoramiento de girasol.The most common rust in cultivated and wild sunflowers is present in all countries where this oil crop is grown and has caused severe yield losses in Argentina, Canada, Russia, USA and Australia. The information obtained from continuous monitoring of sunflower production areas with the aim of identifying rust races present in different regions and the understanding of the shifts produced over the years, after the utilization of several control methodologies, is a fundamental tool to define improvement strategies. The substitution of susceptible open pollination varieties with hybrids having resistance to one or more races has reduced the yield losses produced by rust attacks in an effective way. However, as there exist shifts in the rust populations and the races having virulence to the existing resistance genes increment their frequencies, rust breakouts were observed again. Up today, there exist several rust resistance sources developed from different origins, however only few of them were extensively deployed and only a few of them were genetically characterized. In addition to the elucidation of the genetic control in each one of the available resistance sources, and with the aim of have an efficient use of these genes, to introgress and piramidized them into elite germplasm, it is a critical to known their genomic position. This knowledge will allow the development of linked molecularmarkers in order to track those genetic factors in different crosses and through their derived progenies. The general objective of this work is to understand the genetic control behind the rust resistance in sunflower. The characterization of the pathogen distribution in the sunflower production areas and their race distribution was determined. The collection of isolates will permits the generation of tools for germplasm selection and the differentiation of the genetic factors involved in the different rust resistance phenotypes under analysis. Additionally, this work covered the genetic governance elucidation in the selected resistance sources and its relationship with other previously characterized ones. The localization of already descripted sunflower genes using molecular markers like SSRs, SNPs or de novo designed ones, based on the sequence of targeted genomic regions was one of the most important goals of this work. At the same time, the piramidization or separation of the linked resistance genes by recombination to understand their specificities was done. Isolates of the pathogen were obtained through the country. Those isolates were analyzed and characterized using a set of reference lines, and some of them were selected for further use in our work. Several recombinant sunflower populations obtained from different rust resistance sources were developed. In order to unravel the genetic control behind the resistance in each population a Bulk Segregant Analysis methodology was applied. This approach let us establish in an efficient way the localization of the genetic factors involved in the control of the resistance. A detailed mapping process was carried out in HAR6 and P386, two lines showing potential for global deployment in sunflower breeding programs because of their resistance pattern. This work implied the design of new molecular markers, using already generated DNA sequence information, to saturate the resistance target genomic regions. An allelic study was carried out among P386 and HAR4 to understand the relationship between the factors controlling their resistance. The information obtained would allow the piramidization these factors into a single sunflower line. The results of this study led to the establishment of a rust physiological race map for Argentina, and the comparison of this result with already available historic records to infer the possible reasons of the evolution patterns observed. The genetic control behind the resistance sources was established. In all the cases, the resistance was conferred by a single dominant gene or genetic factor, which was located in LG 13 of the public consensus sunflower map. In particular, the genetic factors present in HAR6 and P386, RHAR6 and Pu6 respectively, were precisely located between two highly linked markers. The information generated permit us to, in addition to validate marker assisted selection strategy for these traits, develop tools for linkage drag elimination during their introgression. Additionally, it was possible to determine the allelic relationship between Pu6 and R4, the genetic factor controlling the resistance in HAR4. The design of new markers to increase the resolution of the recombination map of LG13, was done following two different strategies. The first one, based on the development of new microsatellite markers through the analysis of the BAC sequence located on the targeted region. This yield three new molecular markers, one of these markers NidGi1, was successfully located in one of the recombination map. The second strategy, was based on the design of degenerated oligo nucleotides having high similarity with amino acidic and nucleotidic conserved regions found in resistance gene analogs. This strategy yielded amplification products that were not polymorphic among the genotypes under analysis. The results of this work contribute to the knowledge of the genetics behind the rust resistance in sunflower. This will permit the development of novel sunflower germplasm using a sustainable strategy for disease control. It will also provide tools for breeding programs in order to define sources of resistance to be used, an introgression strategy and the chance of piramidization of the responsible genes in elite material. Finally, the strategies, methodologies, tools and knowledge developed in this PhD thesis contribute to the improvement of sunflower and promote the adoption of genomics in the different stages of the sunflower breeding process.Fil: Bulos, Mariano. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Occurrence of the rust resistance gene Lr37 from Aegilops ventricosa in Argentine cultivars of wheat

Leaf rust of wheat ( Triticum aestivum L.) caused by the fungus Puccinia triticina (formerly P. recondita f. sp. tritici), is one of the most important foliar diseases of this crop. Lr37 rust resistance gene, which confers resistance in wheat against leaf rust, was introgressed into cultivated wheat from Aegilops ventricosa Tausch. Rust races with virulence to Lr37 have been identified in different countries, but it still provides resistance to a wide range of races and is useful in combination with other resistance genes. There are no reports about the presence, frequency and origin of Lr37 in Argentinean wheat cultivars. In this work, we analyzed 88 registered Argentinean wheat cultivars developed by different breeding companies and institutions during the last 15 years by means of a molecular marker which is diagnostic of the 2NS-2AS translocation which carries Lr37. Only 4 cultivars showed the amplification product associated with this chromosome fragment. These four cultivars which carry the translocated 2NS-2AS chromosome were registered by the same breeding company during the last seven years and all of them have European germplasm in their genealogy. To the best of our knowledge this is the first report of the presence of Lr37 in registered South American cultivars

Effect of Ahasl1-1 and Ahasl1-4 alleles on herbicide resistance and its associated dominance in sunflower

BACKGROUND: Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1-1 and Ahasl1-4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole-plant levels, and to determine the dominance relationships for imazapyr and metsulfuron-methyl herbicides. RESULTS: Resistant near-isogenic lines showed significantly lower specific AHAS activity than susceptible near-isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose–response for six near-isolines carrying different combinations of Ahasl1-1 and Ahasl1-4 alleles and two herbicides (imazapyr and metsulfuron-methyl) were evaluated at whole-plant and enzymatic levels. Ahasl1-1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron-methyl. Conversely, Ahasl1-4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole-plant level showed a semi-dominant behavior among the alleles for both herbicides. CONCLUSION: Ahasl1-4 allele confers higher resistance levels than Ahasl1-1 when evaluated with imazapyr and metsulfuron-methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids.Fil: Breccia, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; ArgentinaFil: Gianotto, Laura Noelia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; ArgentinaFil: Altieri, Emiliano. Nidera S. A. Departamento de Biotecnología; ArgentinaFil: Bulos, Mariano. Nidera S. A. Departamento de Biotecnología; ArgentinaFil: Nestares, Graciela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; Argentin

History of the race structure of Orobanche cumana and the breeding of sunflower for resistance to this parasitic weed: A review

<p class="Default">Broomrape, caused by <em>Orobanche cumana</em>, has affected sunflowers since the early 20^th century in Eastern Europe. Currently, it limits sunflower oil production in Southern and Eastern Europe and in some areas of Asia, causing around 50% seed losses when susceptible hybrids are grown. Covered in this review are aspects such as: biological processes that are common to <em>Orobanche</em> spp. and/or particular to <em>O. cumana</em> in sunflower, genetic resistance and its mechanisms, races of the parasite identified in different countries throughout the time and their increasing virulence, and breeding for resistance to some herbicides as a novel control option. The main purpose is to present an updated and, as far as possible, complete picture of the way both the parasitic weed and its host crop have evolved in time, and how they co-exist in the current agriculture. Additionally, we propose a system for determining the races of the parasite that can be internationally adopted from now. In the context of minimal harmful effects on the environment, changing patterns of land use in farming systems, and global environment changes, the final goal of this work is to provide all those interested in parasites from field crops and their integrated management compiled information on the sunflower – <em>O. cumana</em> system as a case study.</p

History of the race structure of Orobanche cumana and the breeding of sunflower for resistance to this parasitic weed: A review

Broomrape, caused by Orobanche cumana, has affected sunflowers since the early 20th century in Eastern Europe. Currently, it limits sunflower oil production in Southern and Eastern Europe and in some areas of Asia, causing around 50% seed losses when susceptible hybrids are grown. Covered in this review are aspects such as: biological processes that are common to Orobanche spp. and/or particular to O. cumana in sunflower, genetic resistance and its mechanisms, races of the parasite identified in different countries throughout the time and their increasing virulence, and breeding for resistance to some herbicides as a novel control option. The main purpose is to present an updated and, as far as possible, complete picture of the way both the parasitic weed and its host crop have evolved in time, and how they co-exist in the current agriculture. Additionally, we propose a system for determining the races of the parasite that can be internationally adopted from now. In the context of minimal harmful effects on the environment, changing patterns of land use in farming systems, and global environment changes, the final goal of this work is to provide all those interested in parasites from field crops and their integrated management compiled information on the sunflower – O. cumana system as a case study