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Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide. EEA Pergamino Fil: Montecchia, Juan Francisco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina Fil: Fass, Mónica I. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina Fil: Cerrudo, Ignacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Quiroz, Facundo José. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Nicosia, Salvador Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnologoía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina Fil: Maringolo, Carla Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Di Rienzo, Julio. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina Fil: Troglia, Carolina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnologoía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina Fil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Escande, Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina Fil: Gonzalez, Julio Horacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Sección Girasol; Argentina Fil: Alvarez, Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina Fil: Heinz, Ruth Amelia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnologoía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina Fil: Lia, Veronica Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnologoía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina Fil: Lia, Veronica Viviana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnologoía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Agrobiotecnología y Biología Molecular (IABIMO); Argentina

We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.

Snakin‐1 (SN1), a cysteine‐rich peptide with broad‐spectrum antimicrobial activity in vitro, was evaluated for its ability to confer resistance to pathogens in transgenic potatoes. Genetic variants of this gene were cloned from wild and cultivated Solanum species. Nucleotide sequences revealed highly evolutionary conservation with 91–98% identity values. Potato plants (S. tuberosum subsp. tuberosum cv. Kennebec) were transformed via Agrobacterium tumefaciens with a construct encoding the S. chacoense SN1 gene under the regulation of the ubiquitous CaMV 35S promoter. Transgenic lines were molecularly characterized and challenged with either Rhizoctonia solani or Erwinia carotovora to analyse whether constitutive in vivo overexpression of the SN1 gene may lead to disease resistance. Only transgenic lines that accumulated high levels of SN1 mRNA exhibited significant symptom reductions of R. solani infection such as stem cankers and damping‐off. Furthermore, these overexpressing lines showed significantly higher survival rates throughout the fungal resistance bioassays. In addition, the same lines showed significant protection against E. carotovora measured as: a reduction of lesion areas (from 46.5 to 88.1% with respect to the wild‐type), number of fallen leaves and thickened or necrotic stems. Enhanced resistance to these two important potato pathogens suggests in vivo antifungal and antibacterial activity of SN1 and thus its possible biotechnological application. Instituto de Biotecnología Fil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Fil: Bazzini, Ariel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Fil: Vazquez Rovere, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Almasia, Natalia Ines. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Área de Biotecnología; Argentina Fil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Área de Biotecnología; Argentina

Tetraploid Paspalum notatum (bahiagrass) is a valuable forage grass with aposporous apomictic reproduction. In a previous study, we showed that apospory in bahiagrass is under the control of a single dominant gene with a distorted segregation ratio. The objective of this work was to identify molecular markers linked to apospory in tetraploid P. notatum and establish a preliminary syntenic relationship with the genomic region associated with apospory in P. simplex. A F1 population of 290 individuals, segregating for apospory, was generated after crossing a completely sexual plant (Q4188) with a natural aposporous apomictic plant (Q4117). The whole progeny was classified as sexual or aposporous by embryo sacs analysis. A bulked segregant analysis was carried out to identify molecular markers co-segregating with apospory. Four hundred RAPD primers, 30 AFLP primers combinations and 85 RFLP clones were screened using DNA from both parental genotypes and aposporous and sexual bulks. Linkage analysis was performed with cytological and genetic information from the complete progeny. Cytoembryological analysis showed 219 sexual and 71 aposporous F1 individuals. Seven different molecular markers (2 RAPD, 4 AFLP and 1 RFLP) were found to be completely linked to apospory. The RFLP probe C1069, mapping to the telomeric region of the long arm of rice chromosome 12, was one of the molecular markers completely linked to apospory in P. notatum. This marker had been previously associated with apospory in P. simplex. A preliminary map of the chromosome region carrying the apospory locus was constructed.

Post-transcriptional gene silencing is an RNA degradation mechanism that can be induced by viruses. Recent evidence indicates that silencing may also be involved in virus synergism, tissue limitation of virus spread, non-host resistance, virus transmission through seeds and in more general mechanisms of defense such as that mediated by salicylic acid. The analysis of Arabidopsis mutants, and of viruses carrying silencing suppressors, has led to a greater understanding of post-transcriptional gene silencing pathways. Much still remains to be discovered, however, not least to allow the successful exploitation of gene silencing in conferring pathogen resistance to transgenic plants.

Fructans are fructose polymers synthesized in a wide range of species such as bacteria, fungi and plants. Fructans are synthesized by fructosyltransferases (FTs) and depolymerized by fructan exohydrolases (FEHs). Bromus pictus is a graminean decaploid species from the Patagonian region of Argentina, which accumulates large amounts of fructans even at temperate temperatures. The first gene isolated from B. pictus fructan metabolism was a putative sucrose:fructan 6-fructosyltransferase (6-SFT). Here, a complete cDNA of the first fructan exohydrolase (FEH) from B. pictus (Bp1-FEHa) was isolated using RT-PCR strategies. The Bp1-FEHa encoding gene is present as a single copy in B. pictus genome. Functional characterization in Pichia pastoris confirmed Bp1-FEHa is a fructan exohydrolase with predominant activity towards beta-(2-1) linkages. Its expression was analyzed in different leaf sections, showing the highest expression levels in the second section of the sheath and the tip of the blade. Bp1-FEHa expression was studied along with FEH and FT activities and fructan accumulation profile in response to chilling conditions during a 7-day time course experiment. Bp1-FEHa expression and FEH activity followed a similar pattern in response to low temperatures, especially in basal sections of the sheaths. In these sections the FEH and FT activities were particularly high and they were significantly correlated to fructan accumulation profile, along with cold treatment.

Clones derived from thirty-one different accessions (nineteen of Argentine origin) belonging to eightSolanum species were screened for resistance to infection by potato virus X strain cp (PVX cp) by mechanical inoculation of plantlets that had been micropropagated in vitro. Estimates of PVX multiplication obtained by enzyme linked immunosorbent assay and slot blot nucleic acid hybridization allowed the identification of resistant clones derived from five accessions belonging toS. commersonii S. oplocense, S. sparsipilum andS. tuberosum andigena. Resistant genotypes supported PVX concentrations 5 to 15 times smaller than did the susceptible control cultivar Spunta. Graft inoculation test confirmed the presence of extreme resistance similar to that conferred by the ‘immunity’ gene X1 (also called RXact).

A library of double stranded cDNA clones was constructed using mRNA isolated from developing barley endosperms as template. The cDNA clones were classified by restriction endonuclease mapping and by hybridization to single stranded cDNA prepared from mRNA of two hordein deficient mutants of barley. This classification and nucleotide sequence analysis identified cDNAs coding for B1 hordein polypeptides. Hybridization of selected cDNA sequences to a B1 hordein cDNA probe at different conditions of stringency demonstrated the presence of a group of related but not identical sequences. Hybridization of the B1 hordein cDNA probe to restriction endonuclease fragments of barley nuclear DNA suggests that the B1 hordein polypeptides are encoded by a multigene family. Abundant mRNA sequences ranging in size from 1,200 to 1,400 nucleotides were detected by hybridization to the B1 hordein cDNA probe. This size is sufficient to code for the B1 hordein polypeptide precursor, which is estimated to have a molecular weight in the range of 29,000 to 35,000. Mutant Riso 56 (hor2ca), which is defective in the synthesis of B hordein polypeptides, lacked B1 hordein mRNAs, thus strongly indicating that the mutation prevents the transcription of these genes. Mutant Riso 1508 (lys3a), which is deficient in the synthesis of all hordein polypeptides, contained detectable amounts of RNA sequences homologous to the B1 hordein cDNA, although the hybridization level was reduced in comparison to the wild type. Failure of these RNA sequences to be translated into B1 hordein polypeptides in a cell free protein synthesizing system hints that thelys3a gene is involved in the post-transcriptional modification of the messenger RNA. Major deletions were not detected in the gene cluster coding for B hordein polypeptides by hybridization of the B1 hordein cDNA to restriction endonuclease fragments of the nuclear DNA from the two mutants Riso 56 and Riso 1508.

The amount of mRNA encoding hordein, protein Z and β-amylase was analyzed in developing endosperms of the barley variety Bomi and the high-lysine mutant Riso 56. Spikes were cultured in liquid media containing different amounts of ammonium nitrate and endosperms harvested between 15 and 25 days after anthesis. Levels of mRNA were determined by in vitro translations. In addition, B hordein and protein Z mRNA species were titrated by molecular hybridization to cDNAs encoding B hordein and protein Z polypeptides, respectively. Increasing amounts of mRNAs were found in response to increasing nitrogen application. The amount of mRNA for C hordein and protein Z increased linearly with increasing nitrogen concentrations in the medium, whereas the rise in β-amylase mRNA was slight. A concentration of 0.5g×l−1 nitrogen stimulated the transcription of B hordein mRNA, but further increases of nitrogen supply up to 2 g×l−1 resulted in little additional B hordein mRNA production. At high nitrogen levels C hordein and protein Z mRNA are preferentially transcribed whereby these proteins serve as major nitrogen sinks. Hordein and protein Z mRNA are effectively augmented by application of nitrogen after the onset of grain filling and storage protein synthesis. Removal of nitrogen in the medium during grain filling led to a short term decrease in the amounts of these mRNAs. Thus it is possible to modulate the mRNA levels by changing the nitrogen concentration in the medium. The absence of mRNA for B hordein polypeptides in the high-lysine mutant Riso 56, (hor 2 ca), does not influence the effect of nitrogen on the expression of the C hordein, protein Z and β-amylase genes.

An 18 residue amino terminal sequence of the barley endosperm protein Z polypeptide is presented. In vitro translation of barley endosperm mRNA and subsequent immunoaffinity isolation using protein Z antibodies identified two major protein Z precursor polypeptides of M.W.44,000 and 46,000 among the translation products. Partial nucleotide sequence analysis identified a cDNA clone to encode the amino terminal portion of protein Z. Messenger RNA sequences 1,800 nucleotides in length, sufficient to code for the protein Z precursor polypeptides, were detected by hybridization to the protein Z cDNA probe. Hybridization of the probe to restriction endonuclease fragments of barley nuclear DNA suggests, that protein Z is encoded by a small multigene family.

Background Association analysis is a powerful tool to identify gene loci that may contribute to phenotypic variation. This includes the estimation of nucleotide diversity, the assessment of linkage disequilibrium structure (LD) and the evaluation of selection processes. Trait mapping by allele association requires a high-density map, which could be obtained by the addition of Single Nucleotide Polymorphisms (SNPs) and short insertion and/or deletions (indels) to SSR and AFLP genetic maps. Nucleotide diversity analysis of randomly selected candidate regions is a promising approach for the success of association analysis and fine mapping in the sunflower genome. Moreover, knowledge of the distance over which LD persists, in agronomically meaningful sunflower accessions, is important to establish the density of markers and the experimental design for association analysis. Results A set of 28 candidate genes related to biotic and abiotic stresses were studied in 19 sunflower inbred lines. A total of 14,348 bp of sequence alignment was analyzed per individual. In average, 1 SNP was found per 69 nucleotides and 38 indels were identified in the complete data set. The mean nucleotide polymorphism was moderate (θ = 0.0056), as expected for inbred materials. The number of haplotypes per region ranged from 1 to 9 (mean = 3.54 ± 1.88). Model-based population structure analysis allowed detection of admixed individuals within the set of accessions examined. Two putative gene pools were identified (G1 and G2), with a large proportion of the inbred lines being assigned to one of them (G1). Consistent with the absence of population sub-structuring, LD for G1 decayed more rapidly (r2 = 0.48 at 643 bp; trend line, pooled data) than the LD trend line for the entire set of 19 individuals (r2 = 0.64 for the same distance). Conclusion Knowledge about the patterns of diversity and the genetic relationships between breeding materials could be an invaluable aid in crop improvement strategies. The relatively high frequency of SNPs within the elite inbred lines studied here, along with the predicted extent of LD over distances of 100 kbp (r2~0.1) suggest that high resolution association mapping in sunflower could be achieved with marker densities lower than those usually reported in the literature.

Background. Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags) were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results. Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion. Eighty genes isolated from organ-specific cDNA libraries were identified as candidate genes for sunflower early response to low temperatures and salinity. Microarray profiling of chilling and NaCl-treated sunflower leaves revealed dynamic changes in transcript abundance, including transcription factors, defense/stress related proteins, and effectors of homeostasis, all of which highlight the complexity of both stress responses. This study not only allowed the identification of common transcriptional changes to both stress conditions but also lead to the detection of stress-specific genes not previously reported in sunflower. This is the first organ-specific cDNA fluorescence microarray study addressing a simultaneous evaluation of concerted transcriptional changes in response to chilling and salinity stress in cultivated sunflower. Fil:Fernandez, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Hopp, H.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Heinz, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.