Precision phenotyping of imidazolinone-induced chlorosis in sunflower

Chlorosis level is a useful parameter to assess imidazolinone resistance in sunflower (Helianthus annuus L.). The aim of this study was to quantify chlorosis through two different methods in sunflower plantlets treated with imazapyr. The genotypes used in this study were two inbred lines reported to be different in their resistance to imidazolinones. Chlorosis was evaluated by spectrophotometrical quantification of photosynthetic leaf pigments and by a bioinformatics-based color analysis. A protocol for pigment extraction was presented which improved pigment stability. Chlorophyll amount decreased significantly when both genotypes were treated with 10 μM of imazapyr. Leaf color was characterized using Tomato Analyzer® color test software. A significant positive correlation between color reduction and chlorophyll concentration was found. It suggests that leaf color measurement could be an accurate method to estimate chlorosis and infer chlorophyll levels in sunflower plants. These results highlight a strong relationship between imidazolinone-induced chlorosis and variations in leaf color and in chlorophyll concentration. Both methods are quantitative, rapid, simple, and reproducible. Thus, they could be useful tools for phenotyping and screening large number of plants when breeding for imidazolinone resistance in this species.Fil: Ochogavía, Ana Claudia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Laboratorio de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Gil, Mercedes. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Picardi, Liliana Amelia. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; Argentina. Universidad Nacional de Rosario. Consejo de Investigaciones de la Universidad de Rosario; ArgentinaFil: Nestares, Graciela María. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentin

Um método rápido para fenotipagem da resistência à imazamox em trigo

The availability of imidazolinone (IMI) resistant cultivars has provided an effective option for weed control in wheat production systems. IMI herbicides control several weeds, including Avena fatua and Lolium multiflorum that are the most frequent grass weeds in wheat crops of Argentine Pampas. The aim of this study was to develop a soil-less method that allows a rapid phenotyping of IMI resistance in wheat. Nine wheat cultivars differing in IMI resistance were evaluated through a between-paper germination method. Herbicide concentrations required to reduce shoot and root length by 50% for resistant cultivars were > 75-fold that of the susceptible cultivars. The response of resistant and susceptible commercial cultivars was assessed in the between-paper and a top-paper methods at 100 µM imazamox as discriminating dose. The Z´-factor was calculated for evaluation of the quality of the screening methods. Both germination methods showed Z´-factors > 0 indicating that the assays were appropriate but the between-paper method allowed to save space in the growth chamber. The germination methods were useful for distinguishing between susceptible and resistant plants carrying at least one resistance gene. The rapid, simple and cost-effective method described in the present study could be a potential tool when selecting for IMI resistance in wheat in breeding programs.A disponibilidade de cultivares resistentes à imidazolinona (IMI) proporcionou uma opção eficaz para o controle de plantas daninhas em sistemas de produção de trigo. Os herbicidas IMI controlam várias plantas daninhas, incluindo Avena fatua e Lolium multiflorum, as gramíneas mais frequentes nas culturas de trigo nos Pampas argentinos. O objetivo deste trabalho foi desenvolver um método sem solo que permita a fenotipagem rápida de resistência ao IMI em trigo. Avaliaram-se nove cultivares de trigo, diferentes na resistência ao IMI, por meio de um método de germinação entre folhas de papel. As concentrações de herbicidas necessárias para reduzir o comprimento da parte aérea e da raiz em 50% para cultivares resistentes foram > 75 vezes que as dos cultivares suscetíveis. A resposta de cultivares comerciais resistentes e suscetíveis, avaliada nos métodos entre folhas de papel e papel, foi superior a 100 mM de imazamox como dose discriminante. O fator Z’ foi calculado para a avaliação da qualidade dos métodos de triagem. Ambos os métodos de germinação mostraram Z’-fatores > 0, indicando que foram apropriados, porém o método entre folhas de papel permitiu economizar espaço na câmara de crescimento. Os testes de germinação foram úteis para distinguir entre plantas suscetíveis e resistentes portadoras de pelo menos um gene de resistência. O método rápido, simples e econômico descrito no presente estudo pode ser uma ferramenta importante para selecionar trigo resistente ao IMI em programas de melhoramento.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: Bisio, María Belén. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Departamento de Biología. Cátedra de Genética; Argentin

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

Fenotipificación de próxima generación en plantas: viejos problemas, nuevas promesas

Current progress in DNA sequencing technology has enabled to gather a large amount of genomic information. Genotyping has become a routine activity with lower costs as new technology emerges. However, genomic information cannot provide much insight into how a given genotype performs. In fact, improvement in understanding phenotypes and the ability to generate good phenotypic data became essential for better utilization of genotypic data and understanding the effect of environment. In this post-genomic era, phenomics emerge as a new discipline that aims the acquisition of high-throughput phenotypic information at all levels of biological organization integrating different “omics” data. New technology in plant phenotyping comprises a wide range of complexity including image analyzing softwares, large-scale platforms and fieldphenotyping systems. The increasingly number of tools for accurate phenotyping as well as the advantages and opportunities for plant sciences are being discussedLos rápidos avances en la tecnología de secuenciación de ADN han permitido obtener una gran cantidad de información a nivel genómico. Actualmente, el principal interés se ha desplazado hacia el estudio del fenotipo con la expectativa de que los progresos en los sistemas de fenotipado puedan aumentar nuestro conocimiento de las relaciones genotipo-fenotipo-ambiente. En esta era post-genómica, la fenómica emerge como una nueva disciplina que integra las demás “ómicas” y busca la adquisición de información en todos los niveles de la organización biológica. En este sentido, las nuevas tecnologías abarcan un amplio rango de complejidad incluyendo programas para el análisis digital de imágenes, plataformas de fenotipado a gran escala y sistemas de fenotipado a campo. En el presente trabajo se discute la importancia de contar con herramientas para la determinación precisa de fenotipos y las oportunidades que las mismas ofrecen para el mejoramiento vegetalFil: Breccia, Gabriela. Universidad Nacional de Rosario. Facultad de Cs.agrarias. Departamento de Biologia. Cat.de Genetica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nestares, Graciela María. Universidad Nacional de Rosario. Facultad de Cs.agrarias. Departamento de Biologia. Cat.de Genetica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Decoding Non-Target-Site Herbicide Resistance in Sunflower: The Beginning of the Story

In the last years, many efforts have been made to develop sunflower cultivars showing important agronomical characteristics such as herbicide resistance. These approaches have been focused mainly on resistance to herbicides with the same mode of action, that is acetohydroxyacid synthase (AHAS) inhibitors. To date, four induced and natural AHAS mutations have been found that confer resistance to these herbicides and many of these alleles are being used for the production of sunflower hybrids resistant to herbicides and to develop different non-transgenic technologies for weed control. However, little is known about the bases of non-target-site-based resistance (NTSR) developing cross-resistance to herbicides with different modes of action in sunflower. These mechanisms diminish the number of active herbicide molecules that reach the target and are generally polygenic. Elucidating the nature of NTSR would allow evaluating maximal efficiency conditions for the herbicide and would enable to establish weed management strategies in sunflower crop. Nowadays, mining of NTSR genes can be more easily accomplished taking advantage of up-to-date omics-based approaches: high-throughput techniques involving genomics, transcriptomics, proteomics and metabolomics. Considering the difficulties in the discovery of new compounds with a broad spectrum of weed control, it results essential to broaden the use of former herbicides which are highly efficient and ecologically desirable. Full understanding of NTSR mechanisms in sunflower would allow detecting specific genes potentially useful as biotechnological tools for the phytoremediation of herbicides and modern plant breeding.Fil: 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: 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

Combining ability evaluation in orange flint lines of maize

In species that show heterosis, information about combining ability with genetically divergent testers is useful to classify the germplasm in heterotic groups. The objective of this study was to evaluate 48 orange flint maize (Zea mays) lines for their combining ability with the testers: sB73 and sMo17 from the Reid x Lancaster pattern and with the flint testers HP3 and P5L2 from the local orange flint pattern during the 1991/92 season at four environments. Analyses of variance were computed for each environment separately and combined across environments for: seed yield, ear height, days to 50% silk, number of ear rows, ear length and diameter, 300 kernels weight, and test weight. Spearman correlation coefficients for grain yield of testcrosses with each tester were computed. Line x tester interactions were highly significant for the majority of the traits and there was no correlation between the merit order of lines based on testcrosses performance. These results suggest the importance of non-additive effects and the ability of the testers to rank the lines.En las especies que exhiben heterosis, el conocimiento de la aptitud combinatoria del germoplasma en cruzamientos con probadores genéticamente divergentes permite su clasificación según grupos heteróticos. El objetivo del presente trabajo fue evaluar 48 líneas de maíz (Zea mays) flint colorado en base a su comportamiento en cruzamientos de prueba con los probadores dentados sB73 y sMo17 del patrón heterótico Reid x Lancaster y con los probadores flint HP3 y P5L2 del patrón heterótico local HP3 x P5L2 en cuatro ambientes durante la campaña 1991/92. Se realizaron los análisis de variancia por ambiente y en forma combinada a través de ambientes para: rendimiento en grano, altura de inserción de espiga, días a floración masculina, número de hileras de grano, largo y diámetro de espiga, peso de 300 granos y peso hectolítrico. Se estimaron los coeficientes de correlación de Spearman para el rendimiento de los cruzamientos de prueba con cada probador. Las interacciones línea por probador fueron altamente significativas para la mayoría de las variables y no hubo correlación entre el ordenamiento de mérito de las líneas basado en su comportamiento en cruzamientos de prueba. Ello refleja la importancia de efectos génicos no aditivos y la capacidad discriminatoria de estos probadores en cruzamientos con las líneas evaluadas.In species that show heterosis, information about combining ability with genetically divergent testers is useful to classify the germplasm in heterotic groups. The objective of this study was to evaluate 48 orange flint maize (Zea mays) lines for their combining ability with the testers: sB73 and sMo17 from the Reid x Lancaster pattern and with the flint testers HP3 and P5L2 from the local orange flint pattern during the 1991/92 season at four environments. Analyses of variance were computed for each environment separately and combined across environments for: seed yield, ear height, days to 50% silk, number of ear rows, ear length and diameter, 300 kernels weight, and test weight. Spearman correlation coefficients for grain yield of testcrosses with each tester were computed. Line x tester interactions were highly significant for the majority of the traits and there was no correlation between the merit order of lines based on testcrosses performance. These results suggest the importance of non-additive effects and the ability of the testers to rank the lines

Evaluación de líneas de maíz flint colorado por aptitud combinatoria

En las especies que exhiben heterosis, el conocimiento de la aptitud combinatoria del germoplasma en cruzamientos con probadores genéticamente divergentes permite su clasificación según grupos heteróticos. El objetivo del presente trabajo fue evaluar 48 líneas de maíz (Zea mays) flint colorado en base a su comportamiento en cruzamientos de prueba con los probadores dentados sB73 y sMo17 del patrón heterótico Reid x Lancaster y con los probadores flint HP3 y P5L2 del patrón heterótico local HP3 x P5L2 en cuatro ambientes durante la campaña 1991/92. Se realizaron los análisis de variancia por ambiente y en forma combinada a través de ambientes para: rendimiento en grano, altura de inserción de espiga, días a floración masculina, número de hileras de grano, largo y diámetro de espiga, peso de 300 granos y peso hectolítrico. Se estimaron los coeficientes de correlación de Spearman para el rendimiento de los cruzamientos de prueba con cada probador. Las interacciones línea por probador fueron altamente significativas para la mayoría de las variables y no hubo correlación entre el ordenamiento de mérito de las líneas basado en su comportamiento en cruzamientos de prueba. Ello refleja la importancia de efectos génicos no aditivos y la capacidad discriminatoria de estos probadores en cruzamientos con las líneas evaluadas

Cultivar variation for imazamox resistance in wheat (Triticum aestivum L.): Insights into enzymatic assays for early selection

Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the target site of several herbicide classes including imidazolinones. Imidazolinone resistance in wheat is conferred by two major genes AhasL-D1 and AhasL-B1. The objective of this work was to evaluate the in vitro and in vivo AHAS activity and plant growth in response to imazamox of nine wheat cultivars. Dose-response curves for two-gene resistant cultivars were significantly different from the single-gene resistant and susceptible cultivars in the in vitro AHAS assay. Resistance levels atthe in vivo AHAS and whole-plant assays for resistant cultivars were >10-fold higher than susceptible cultivars. Moreover, in vivo dose-response curves showed differences among cultivars with the same number of resistance genes. It was concluded that in the in vitro AHAS assay cultivar variability was due to differences in target-site sensitivity while the in vivo AHAS assay reflected the resistance at whole-plant level. Both in vitro and in vivo AHAS dose-response curves could be useful tools when exploring mechanisms involved in imidazolinone resistance in different wheat genetic backgrounds and for the selection of higher resistant genotypes.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: 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; Argentina. Universidad Nacional de Rosario. Consejo de Investigaciones de la Universidad 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 of suitable reference genes by quantitative real-time PCR for gene expression normalization in sunflower

Quantitative real-time PCR (qPCR) is currently the most accurate method for detecting differential gene expression, but depends greatly on normalization to stably expressed housekeeping genes. Transcriptomics analyses and experimental validation in different plant species have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. Thus, reliable validation of reference genes is required to ensure proper normalization. This paper presents a systematic comparison of ten potential reference genes in sunflower: five commonly used genes (Actin, Elongation Factor1, Plastid-encode RNA polymerase, Tubulin, and Ubiquitin, as ACT, EF1, PEP, TUB, and UBQ respectively), as well as five new candidates (Translation initiation factor, MicroRNA precursors 171 and 156, Ask-interacting protein, and Protein of unknown function, as ETIF5, MIR171, MIR156, SKIP, and UNK2 respectively). Reference gene expression stability was examined by qPCR across 20 biological samples, representing different tissues at various developmental stages. Expression of all 10 genes was variable to some extent, but that of ACT, UNK2, and EF1 was overall the most stable. A combination of ETIF5/UNK2/EF1 would be appropriate to use as a reference panel for normalizing gene expression data among vegetative tissues, whereas the combination of ACT/MIR156/UNK2 is most suitable for reproductive tissues. Reference genes selected in this study were further validated by examining relative expression of ahas1, one of three acetohydroxyacid synthase genes of sunflower. Our identification and validation of suitable normalizer genes will be of use to ensure accurate results in future transcriptomics studies in this crop.Fil: Ochogavía, Ana Claudia. 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: Novello, Maria Angelina. 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: 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; Argentina. Consejo de Investigaciones de la Universidad Nacional 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