Molecular characterization of Als1, an acetohydroxyacid synthase mutation conferring resistance to sulfonylurea herbicides in soybean.

KEY MESSAGE: The AHAS gene family in soybean was characterized. The locus Als1 for sulfonylurea resistance was mapped and the resistant allele was characterized at the molecular level. Sulfonylurea (SU) resistance in soybean is controlled by Als1, a semi-dominant allele obtained by EMS mutagenesis over the cultivar Williams 82. The overall objective of this research was to map Als1 in the soybean genome and to determine the nucleotidic changes conferring resistance to SU. Four nucleotide sequences (GmAhas1-4) showing high homology with the Arabidopsis thaliana acetohydroxyacid synthase (AHAS, EC 4.1.3.18) gene sequence were identified by in silico analysis, PCR-amplified from the SU-resistant line BTK323STS and sequenced. Expression analysis showed that GmAhas1, located on chromosome 4 by in silico analysis, is the most expressed sequence in true leaves. F2:3 families derived from the cross between susceptible and resistant lines were evaluated for SU resistance. Mapping results indicate that the locus als1 is located on chromosome 4. Sequence comparison of GmAhas1 between BTK323STS and Williams 82 showed a single nucleotide change from cytosine to thymine at position 532. This transversion generates an amino acid change from proline to serine at position 197 (A. thaliana nomenclature) of the AHAS catalytic subunit. An allele-specific marker developed for the GmAhas1 mutant sequence cosegregated with SU resistance in the F2 population. Taking together, the mapping, expression and sequencing results indicate that the GmAhas1 sequence corresponds to the Als1 gene sequence controlling SU resistance in soybean. The molecular breeding tools described herein create the basis to speed up the identification of new mutations in soybean AHAS leading to enhanced levels of resistance to SU or to other families of AHAS inhibitor herbicides.

Molecular mapping of a sunflower rust resistance gene from HAR6.

Sunflower rust, caused by Puccinia helianthi Schw., can result in significant yield losses in cultivated sunflower (Helianthus annuus L. var. macrocarpus Ckll.). HAR6 is a germplasm population resistant to most predominant rust races. The objectives of this study were to map the resistance factor present in HAR6 (R HAR6 ), and to provide and validate molecular tools for the identification of this gene for marker assisted selection purposes. Virulence reaction of seedlings for the F2 population and F2:3 families suggested that a single dominant gene confers rust resistance in HAR6-1, a selected rust resistance line from the original population. Genetic mapping with eight markers covered 97.4 cM of genetic distance on linkage group 13 of the sunflower consensus map. A co-dominant marker ZVG61 is the closest marker distal to R HAR6 at a genetic distance of 0.7 cM, while ORS581, a dominant marker linked in the coupling phase, is proximal to R HAR6 at a genetic distance of 1.5 cM. Validation of these markers was assessed by converting a susceptible line into a rust resistant isoline by means of marker assisted backcrossing. The application of these results to assist the breeding process and to design new strategies for rust control in sunflower is discussed.

Phenotypic characterization, genetic mapping and candidate gene analysis of a source conferring reduced plant height in sunflower.

Reduced height germplasm has the potential to increase stem strength, standability, and also yields potential of the sunflower crop (Helianthus annuus L. var. macrocarpus Ckll.). In this study, we report on the inheritance, mapping, phenotypic and molecular characterization of a reduced plant height trait in inbred lines derived from the source DDR. This trait is controlled by a semidominant allele, Rht1, which maps on linkage group 12 of the sunflower public consensus map. Phenotypic effects of this allele include shorter height and internode length, insensibility to exogenous gibberellin application, normal skotomorphogenetic response, and reduced seed set under self-pollination conditions. This later effect presumably is related to the reduced pollen viability observed in all DDR-derived lines studied. Rht1 completely cosegregated with a haplotype of the HaDella1 gene sequence. This haplotype consists of a point mutation converting a leucine residue in a proline within the conserved DELLA domain. Taken together, the phenotypic, genetic, and molecular results reported here indicate that Rht1 in sunflower likely encodes an altered DELLA protein. If the DELPA motif of the HaDELLA1 sequence in the Rht1-encoded protein determines by itself the observed reduction in height is a matter that remains to be investigated.

Root biomass response to foliar application of imazapyr for two imidazolinone tolerant alleles of sunflower (Helianthus annuus L.).

Imisun and CLPlus are two imidazolinone tolerance traits in sunflower (Helianthus annuus L.) determined by the expression of two alleles at the locus Ahasl1. Both traits differed in their tolerance level to imazapyr -a type of imidazolinone herbicide- when aboveground biomass is considered, but the concomitant herbicide effect over the root system has not been reported. The objective of this work was to quantify the root biomass response to increased doses of imazapyr in susceptible (ahasl1/ahasl1), Imisun (Ahasl1-1/Ahasl1-1) and CLPlus (Ahasl1-3/Ahasl1-3) homozygous sunflower genotypes. These materials were sprayed at the V2-V4 stage with increased doses of imazapyr (from 0 to 480 g active ingredient ha(-1)) and 14 days after treatment root biomass of each plant was assessed. Genotype at the Ahasl1 locus, dose of imazapyr and their interaction significantly contributed (P < 0.001) to explain the reduction in root biomass accumulation after herbicide application. Estimated dose of imazapyr required to reduce root biomass accumulation by fifty percent (GR(50)) differed statistically for the three genotypes under study (P < 0.001). CLPlus genotypes showed the highest values of GR(50), 300 times higher on average than the susceptible genotypes, and almost 8 times higher than Imisun materials, demonstrating that both alleles differ in their root biomass response to foliar application of increased doses of imazapyr.

Inheritance and molecular characterization of broad range tolerance to herbicides targeting acetohydroxyacid synthase in sunflower.

Ahasl1 is a multilallelic locus where all the induced and natural mutations for herbicide tolerance were described thus far in sunflower (Helianthus annuus L.). The allele Ahasl1-1 confers moderate tolerance to imidazolinone (IMI), Ahasl1-2, and Ahasl1-3 provides high levels of tolerance solely to sulfonylurea (SU) and IMI, respectively. An Argentinean wild sunflower population showing plants with high level of tolerance to either an IMI and a SU herbicide was discovered and used to develop an inbred line designated RW-B. The objectives of this work were to determine the relative level and pattern of cross-tolerance to different AHAS-inhibiting herbicides, the mode of inheritance, and the molecular basis of herbicide tolerance in this line. Slight or no symptoms observed after application of different herbicides indicated that RW-B possesses a completely new pattern of tolerance to AHAS-inhibiting herbicides in sunflower. Biomass response to increasing doses of metsulfuron or imazapyr demonstrated a higher level of tolerance in RW-B with respect to Ahasl1-1/Ahasl1-1 and Ahasl1-2/Ahasl1-2 lines. On the basis of genetic analyses and cosegregation test, it was concluded that tolerance to imazapyr in the original population is inherited as a single, partially dominant nuclear gene and that this gene is controlling the tolerance to four different AHAS-inhibiting herbicides. Pseudo-allelism test permitted us to conclude that the tolerant allele present in RW-B is an allelic variant of Ahasl1-1 and was designated as Ahasl1-4. Nucleotide and deduced amino acid sequence indicated that the Ahasl1-4 allele sequence of RW-B has a leucine codon (TTG) at position 574 (relative to the Arabidopsis thaliana AHAS sequence), whereas the enzyme from susceptible lines has a tryptophan residue (TGG) at this position. The utilization of this new allele in the framework of weed control and crop rotation is discussed.

Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the Ahasl1 locus of sunflower.

Imisun and CLPlus are two imidazolinone (IMI) tolerance traits in sunflower (Helianthus annuus L.) determined by the expression of different alleles at the same locus, Ahasl1-1 and Ahasl1-3, respectively. This paper reports the level of tolerance expressed by plants containing both alleles in a homozygous, heterozygous and in a heterozygous stacked state to increasing doses of IMI at the enzyme and whole plant levels. Six genotypes of the Ahasl1 gene were compared with each other in three different genetic backgrounds. These materials were treated at the V2-V4 stage with increasing doses of imazapyr (from 0 to 480 g a.i. ha(-1)) followed by an assessment of the aboveground biomass and herbicide phytotoxicity. The estimated dose of imazapyr required to reduce biomass accumulation by 50% (GR(50)) differed statistically for the six genotypes of the Ahasl1 gene. Homozygous CLPlus (Ahasl1-3/Ahasl1-3) genotypes and materials containing a combination of both tolerant alleles (Imisun/CLPlus heterozygous stack, Ahasl1-1/Ahasl1-3) showed the highest values of GR(50), 300 times higher than the susceptible genotypes and more than 2.5 times higher than homozygous Imisun materials (Ahasl1-1/Ahasl1-1). In vitro AHAS enzyme activity assays using increasing doses of herbicide (from 0 to 100 µM) showed similar trends, where homozygous CLPlus materials and those containing heterozygous stacks of Imisun/CLPlus were statistically similar and showed the least level of inhibition of enzyme activity to increasing doses of herbicide. The degree of dominance for the accumulation of biomass after herbicide application calculated for the Ahasl1-1 allele indicated that it is co-dominant to recessive depending on the imazapyr dose used. By the contrary, the Ahasl1-3 allele showed dominance to semi dominance according to the applied dose. This last allele is dominant over Ahasl1-1 over the entire range of herbicide rates tested. At the level of enzymatic activity, however, both alleles showed recessivity to semi-recessivity with respect to the wild-type allele, even though the Ahasl1-3 allele is dominant over Ahasl1-1 at all the herbicides rates used.

Molecular and biochemical characterization of an induced mutation conferring imidazolinone resistance in sunflower.

A partially dominant nuclear gene conferring resistance to the imidazolinone herbicides was previously identified in the cultivated sunflower (Helianthus annuus L.) line CLHA-Plus developed by seed mutagenesis. The objective of this study was to characterize this resistant gene at the phenotypic, biochemical and molecular levels. CLHA-Plus showed a complete susceptibility to sulfonylureas (metsulfuron, tribenuron and chlorsulfuron) but, on the other hand, it showed a complete resistance to imidazolinones (imazamox, imazapyr and imazapic) at two rates of herbicide application. This pattern was in close association with the AHAS-inhibition kinetics of protein extracts of CLHA-Plus challenged with different doses of imazamox and chlorsulfuron. Nucleotide and deduced amino acid sequence comparisons between resistant and susceptible lines indicated that the imidazolinone-resistant AHAS of CLHA-Plus has a threonine codon (ACG) at position 122 (relative to the Arabidopsis thaliana AHAS sequence), whereas the herbicide-susceptible enzyme from BTK47 has an alanine residue (GCG) at this position. Since the resistance genes to AHAS-inhibiting herbicides so far characterized in sunflower code for the catalytic (large) subunit of AHAS, we propose to redesignate the wild type allele as ahasl1 and the incomplete dominant resistant alleles as Ahasl1-1 (previously Imr1 or Ar ( pur )), Ahasl1-2 (previously Ar ( kan )) and Ahasl1-3 (for the allele present in CLHA-Plus). The higher tolerance level to imidazolinones and the lack of cross-resistance to other AHAS-inhibiting herbicides of Ahasl1-3 indicate that this induced mutation can be used to develop commercial hybrids with superior levels of tolerance and, at the same time, to assist weed management where control of weedy common sunflower is necessary.

Experiencias de cuantificación del proceso de enseñanza-aprendizaje / Evaluation tests to quantify the teaching-learning process

El objetivo de este estudio es presentar una experiencia de utilización de una prueba de evaluación del tipo "progreso alcanzado" del área cognoscitiva que pretende cuantificar el proceso de enseñanza aprendizaje, con 35 alumnos de la carrera de medicina en sus trabajos prácticoa (TP) correspondientes al área de cirugía vascular. Al comenzar el TP se mostraron a los alumnos una serie de imágenes arteriográficas y tomográficas computadas en las cuales debían mencionar y señalar en forma individual las diferentes estructuras vasculares normales y patológicas a solicitud del docente. En base al porcentaje de respuestas correctas se confeccionó una tabla de resultados basales de referencia. Posteriormente se realizó el TP donde el objetivo fue "aprender a reconocer las imágenes vasculares normales y patológicas". Finalizado el mismo se procedió a una nueva evaluación individual como se había efectuado al inicio. Con este método se demostró una diferencia significativa en la comparación pre y post-TP. La cuantificación del proceso de enseñanza-aprendizaje se presenta como una herramienta práctica para medir el progreso del aprendizaje en los alumnos, para evaluar la conducción de dicho proceso por parte del docente y en especial para supervisar los tipos de estrategias de enseñanza por él empleados (AU)

Experiencias de cuantificación del proceso de enseñanza-aprendizaje / Evaluation tests to quantify the teaching-learning process

El objetivo de este estudio es presentar una experiencia de utilización de una prueba de evaluación del tipo "progreso alcanzado" del área cognoscitiva que pretende cuantificar el proceso de enseñanza aprendizaje, con 35 alumnos de la carrera de medicina en sus trabajos prácticoa (TP) correspondientes al área de cirugía vascular. Al comenzar el TP se mostraron a los alumnos una serie de imágenes arteriográficas y tomográficas computadas en las cuales debían mencionar y señalar en forma individual las diferentes estructuras vasculares normales y patológicas a solicitud del docente. En base al porcentaje de respuestas correctas se confeccionó una tabla de resultados basales de referencia. Posteriormente se realizó el TP donde el objetivo fue "aprender a reconocer las imágenes vasculares normales y patológicas". Finalizado el mismo se procedió a una nueva evaluación individual como se había efectuado al inicio. Con este método se demostró una diferencia significativa en la comparación pre y post-TP. La cuantificación del proceso de enseñanza-aprendizaje se presenta como una herramienta práctica para medir el progreso del aprendizaje en los alumnos, para evaluar la conducción de dicho proceso por parte del docente y en especial para supervisar los tipos de estrategias de enseñanza por él empleados

Cirugía ambulatoria en patología venosa de los miembros inferiores / Ambulatory surgery in vein pathology of inferior legs

Desde enero de 1990 se efectuaron 60 operaciones por várices de miembros inferiores, 48 se realizaron en forma ambulatoria sin registrarse complicaciones. Se analizan el tipo de operaciones practicadas y la técnica anestésica utilizada. Debido a los resultados logrados durante 1990 la tendencia actual se inclina hacia una selección adecuada para cirugía ambulatoria llegando a un 100%de casos ambulatorios en el primer semestre de 1991

Cirugía ambulatoria en patología venosa de los miembros inferiores / Ambulatory surgery in vein pathology of inferior legs

Desde enero de 1990 se efectuaron 60 operaciones por várices de miembros inferiores, 48 se realizaron en forma ambulatoria sin registrarse complicaciones. Se analizan el tipo de operaciones practicadas y la técnica anestésica utilizada. Debido a los resultados logrados durante 1990 la tendencia actual se inclina hacia una selección adecuada para cirugía ambulatoria llegando a un 100%de casos ambulatorios en el primer semestre de 1991