Search

Citrus canker is a major disease caused by Xanthomonas citri pv. citri. Snakin-1 is an antimicrobial peptide, which was previously shown to be effective against different bacterial and fungal diseases in potato, wheat and lettuce when expressed in transgenic plants. We generated transgenic Citrange Troyer citrus rootstocks constitutively expressing this peptide and 5 different transgenic lines were challenged against virulent X. citri isolates. Challenge assays conducted in vitro using detached leaves and in planta by infiltration revealed a significant reduction of the number and size of canker lesions in some of the transgenic lines.

Background: Sclerotinia sclerotiorum is a necrotrophic fungus that causes Sclerotinia head rot (SHR) in sunflower, with epidemics leading to severe yield losses. In this work, we present an association mapping (AM) approach to investigate the genetic basis of natural resistance to SHR in cultivated sunflower, the fourth most widely grown oilseed crop in the world. Results: Our association mapping population (AMP), which comprises 135 inbred breeding lines (ILs), was genotyped using 27 candidate genes, a panel of 9 Simple Sequence Repeat (SSR) markers previously associated with SHR resistance via bi-parental mapping, and a set of 384 SNPs located in genes with molecular functions related to stress responses. Moreover, given the complexity of the trait, we evaluated four disease descriptors (i.e, disease incidence, disease severity, area under the disease progress curve for disease incidence, and incubation period). As a result, this work constitutes the most exhaustive AM study of disease resistance in sunflower performed to date. Mixed linear models accounting for population structure and kinship relatedness were used for the statistical analysis of phenotype-genotype associations, allowing the identification of 13 markers associated with disease reduction. The number of favourable alleles was negatively correlated to disease incidence, disease severity and area under the disease progress curve for disease incidence, whereas it was positevily correlated to the incubation period. Conclusions: Four of the markers identified here as associated with SHR resistance (HA1848, HaCOI_1, G33 and G34) validate previous research, while other four novel markers (SNP117, SNP136, SNP44, SNP128) were consistently associated with SHR resistance, emerging as promising candidates for marker-assisted breeding. From the germplasm point of view, the five ILs carrying the largest combination of resistance alleles provide a valuable resource for sunflower breeding programs worldwide. Fil: Filippi, Carla Valeria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Zubrzycki, Jeremías Enrique. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Di Rienzo, Julio Alejandro. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina Fil: Quiroz, Facundo José. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Puebla, Andrea Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Alvarez, D.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Maringolo, C. A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Escande, Alberto Raul. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Heinz, Ruth Amelia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Lia, Veronica Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina

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 (IL) 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.

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes., Instituto de Biotecnología y Biología Molecular

33 páginas, 2 tablas, 4 figuras, Background and aims: The number of plastome sequences has increased exponentially during the last decade. However, there is still little knowledge of the levels and distribution of intraspecific variation. The aims of this study were to estimate plastome diversity within Zea mays and analyse the distribution of haplotypes in connection with the landrace groups previously delimited for South American maize based on nuclear markers. Methods: We obtained the complete plastomes of 30 South American maize landraces and three teosintes by means of next-generation sequencing (NGS) and used them in combination with data from public repositories. After quality filtering, the curated data were employed to search for single-nucleotide polymorphisms, indels and chloroplast simple sequence repeats. Exact permutational contingency tests were performed to assess associations between plastome and nuclear variation. Network and Bayesian phylogenetic analyses were used to infer evolutionary relationships among haplotypes. Key results: Our analyses identified a total of 124 polymorphic plastome loci, with the intergenic regions psbE-rps18, petN-rpoB, trnL_UAG-ndhF and rpoC2-atpI exhibiting the highest marker densities. Although restricted in number, these markers allowed the discrimination of 27 haplotypes in a total of 51 Zea mays individuals. Andean and lowland South American landraces differed significantly in haplotype distribution. However, overall differentiation patterns were not informative with respect to subspecies diversification, as evidenced by the scattered distribution of maize and teosinte plastomes in both the network and Bayesian phylogenetic reconstructions. Conclusions: Knowledge of intraspecific plastome variation provides the framework for a more comprehensive understanding of evolutionary processes at low taxonomic levels and may become increasingly important for future plant barcoding efforts. Whole-plastome sequencing provided useful variability to contribute to maize phylogeographic studies. The structuring of haplotype diversity in the maize landraces examined here clearly reflects the distinction between the Andean and South American lowland gene pools previously inferred based on nuclear markers., This work was supported by the Agencia Nacional de Promoción Científica y Técnica (PICT 2012 0325, PICT 2016 1101), the Consejo Nacional de Investigaciones Científicas y Tecnológicas (PIP 11220120100416CO 2013–2015), the Instituto Nacional de Tecnología Agropecuaria (PNBIO 1131044) and the DEANN Project.

Specific mutations in the conserved domains of the acetolactate synthase (ALS) gene conduct to different key amino acid substitutions that can confer herbicide resistance in different plant species. This outcome has been widely exploited to produce herbicide-resistant agronomic crops as well as to direct many genome editing studies. Therefore, the ALS gene has become a model sequence target to improve our technological skills for more precise CRISPR/Cas nucleotide base substitution in plants, which is essential for modulation/modification of gene function as opposed to the more general gene knock out obtained by indels in conventional genome editing studies. This review summarizes the main knowledge and experiences attained from the use of the ALS gene as a target in CRISPR/Cas studies. Instituto de Biotecnología Fil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Lopez Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Sclerotinia head rot (SHR) is one of the most serious constraints to sunflower (Helianthus annuus L. var. macrocarpus) production worldwide. Here, we evaluated the response to SHR in a sunflower inbred panel from a large INTA germplasm collection, consisting of 137 inbred lines (ILs). Field trials were performed over five consecutive seasons using a twice-replicated randomized complete-block design. Disease incidence, disease severity, incubation period and area under disease progress curve for disease incidence and severity were determined after controlled inoculation with the pathogen. Statistical analysis using mixed-effect models detected significant differences among ILs for all variables (P

Aim of study: To validate and characterize new microsatellites or Simple Sequence Repeats (SSR) markers, located within genomic transcribed sequences related to growth and plant developmental traits, in Eucalyptus species. Area of study: Eucalyptus species from different Australian origins planted in Argentina. Material and methods: In total, 134 SSR in 129 candidate genes (CG-SSR) involved in plant development were selected and physically mapped to the E. grandis reference genome by bioinformatic tools. Experimental validation and polymorphism analysis were performed on 48 individuals from E. grandis and interspecific hybrids (E. grandis x E. camaldulensis; E. grandis x E. tereticornis), E. globulus, E. maidenii, E. dunnii and E. benthamii. Main results: 131 out of 134 CG-SSR were mapped on the 11 chromosomes of E. grandis reference genome. Most of the 134 analyzed SSR (> 75%) were positively amplified and 39 were polymorphic in at least one species. A search of annotated genes within a 25 kbp up and downstream region of each SSR location retrieved 773 genes of interest. Research highlights: The new validated and characterized CG-SSR are potentially suitable for comparative QTL mapping, molecular marker-assisted breeding (MAB) and population genetic studies across different species within Symphyomyrtus subgenus. Fil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Rivas, Juan Gabriel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Aguirre, Natalia Cristina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Villalba, Pamela Victoria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Martinez, Maria Carolina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Garcia, Martín Nahuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Marcucci Poltri, Susana Noemí. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina

Restriction site-associated DNA sequencing (RADseq) and its derived protocols, such as double digest RADseq (ddRADseq), offer a flexible and highly cost-effective strategy for efficient plant genome sampling. This has become one of the most popular genotyping approaches for breeding, conservation, and evolution studies in model and non-model plant species. However, universal protocols do not always adapt well to non-model species. Herein, this study reports the development of an optimized and detailed ddRADseq protocol in Eucalyptus dunnii, a non-model species, which combines different aspects of published methodologies. The initial protocol was established using only two samples by selecting the best combination of enzymes and through optimal size selection and simplifying lab procedures. Both single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) were determined with high accuracy after applying stringent bioinformatics settings and quality filters, with and without a reference genome. To scale it up to 24 samples, we added barcoded adapters. We also applied automatic size selection, and therefore obtained an optimal number of loci, the expected SNP locus density, and genome-wide distribution. Reliability and cross-sequencing platform compatibility were verified through dissimilarity coefficients of 0.05 between replicates. To our knowledge, this optimized ddRADseq protocol will allow users to go from the DNA sample to genotyping data in a highly accessible and reproducible way. Instituto de Biotecnología Fil: Aguirre, Natalia Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Filippi, Carla Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zaina, Giusi. University of Udine. Department of Agricultural, Food, Environmental and Animal Sciences; Italia Fil: Rivas, Juan Gabriel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Villalba, Pamela Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Garcia, Martin Nahuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martinez, Maria Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Puebla, Andrea Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morgante, Michele. University of Udine. Department of Agricultural, Food, Environmental and Animal Sciences; Italia Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marcucci Poltri, Susana Noemi. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Snakin/GASA proteins have been involved in different aspects of plant growth and development although their exact role is still intriguing. All of them maintain 12 cysteines of the C-terminus in highly conserved positions that are responsible for their structure and are essential for their biochemical activity as antioxidants. Two members were isolated from Solanum tuberosum to date (Snakin-1 and Snakin-2) and were shown to have antimicrobial activity. We have recently demonstrated that Snakin-1 has additional roles in plant growth and development. We carried out a genome-wide search for new Snakin/GASA family members in potato. 16 Snakin/GASA genes were isolated, sequenced and characterized. Interestingly, we found in Solanum tuberosum subsp. tuberosum cv. Kennebec that Snakin-1, Snakin-2 and Snakin-3 expression is affected by bacterial and/or fungal inoculation. These results strengthen the participation of Snakin-1 and Snakin-2 genes in biotic stress tolerance and suggest that Snakin-3 is also involved in plant defense. The data presented here could be a good starting point for more focused and deep investigations regarding the biological functions of potato Snakin/GASA genes during plant development and in response to environmental stress. Fil: Nahirñak, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Rivarola, Maximo Lisandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: González de Urreta, Martín Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Paniego, Norma Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Hopp, Horacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Almasia, Natalia Ines. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina Fil: Vazquez Rovere, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina

Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant–virus interactions using a reverse genetic approach. Fil: Delfosse, Verónica Cecilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Casse, María F.. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Agrofoglio, Yamila Carla. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bonacic Kresic , Iván. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Ziegler Graff, Véronique. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; Francia Fil: Distéfano, Ana Julia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The discovery of commercial transgenic varieties of orange petunias sold in Europe and the United States although they had never reached the approved status, and the consequent recommendation to destroy them, was the trigger to discuss about biotechnological improvement of ornamental plants. Inside the restricted world of 26 vegetal transgenic species, according to the ISAAA’s reports (http://www.isaaa.org), there are three ornamental species: carnation, rose and the Beijing University developed petunia; all of them with the same trait, a change in their colour. On the other hand, in 2014, the whole-genome sequence of carnation appeared which was the first and until now the only one among ornamental species. In this context, we review the publications from the last five years in petunia, rose, chrysanthemum and carnation. In these papers there are detailed descriptions of modification of the cascade of genes and transcription factors involved in stress situations, in different developmental stages and their regulation through different plant hormones. This knowledge will allow breeding for better and new varieties with changes in their abiotic or biotic stress tolerance, altered growth or yield and modified product quality as colour or fragrance. A descoberta de variedades transgênicas de petúnias laranja vendidas na Europa e nos Estados Unidos que nunca alcançaram o status aprovado e a consequente recomendação de destruí-las foi o fator desencadeador para a discussão sobre a melhoria biotecnológica de plantas ornamentais. Dentro do mundo estrito de 26 espécies transgênicas vegetais, de acordo com os relatórios do ISAAA (http://www.isaaa.org), existem três espécies ornamentais: cravo, rosa e as petúnias desenvolvidas pela Universidade de Pequim que tem como característica mudança na cor. Por outro lado, em 2014 foi realizado pela primeira vez o sequenciamento completo do genoma do cravo que é o único sequenciado entre as espécies ornamentais. Neste contexto, revisamos as publicações dos últimos cinco anos em petúnia, rosa, crisântemo e cravo. Nestes trabalhos, há descrições detalhadas da modificação da cascata de genes e fatores de transcrição envolvidos em situações de estresse, diferentes estágios do crescimento e sua regulação através de diferentes hormônios vegetais. Este conhecimento contribuirá diretamente no melhoramento vegetal, o qual permitirá o desenvolvimento de novas variedades que sejam resistentes a diferentes situações de estresse abiótico ou biótico, alterações nos fatores que contribuem para o crescimento ou produtividade e modificações nos parâmetros de qualidade (como cor ou fragrância). Instituto de Biotecnología Fil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Radonic, Laura Mabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: López Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

An outbreak of a new disease occurred in cotton (Gossypium hirsutum) fields in northwest Argentina starting in the 2009-10 growing season and is still spreading steadily. The characteristic symptoms of the disease included slight leaf rolling and a bushy phenotype in the upper part of the plant. In this study, we determined the complete nucleotide sequences of two independent virus genomes isolated from cotton blue disease (CBD)-resistant and -susceptible cotton varieties. This virus genome comprised 5,866 nucleotides with an organization similar to that of the genus Polerovirus and was closely related to cotton leafroll dwarf virus, with protein identity ranging from 88 to 98%. The virus was subsequently transmitted to a CBD-resistant cotton variety using Aphis gossypii and symptoms were successfully reproduced. To study the persistence of the virus, we analyzed symptomatic plants from CBD-resistant varieties from different cotton-growing fields between 2013 and 2015 and showed the presence of the same virus strain. In addition, a constructed full-length infectious cDNA clone from the virus caused disease symptoms in systemic leaves of CBD-resistant cotton plants. Altogether, the new leafroll disease in CBD-resistant cotton plants is caused by an atypical cotton leafroll dwarf virus. Fil: Agrofoglio, Yamila Carla. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Delfosse, Verónica Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina Fil: Casset, María Andrea. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Kresic, Iván Bonacic. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Distéfano, Ana Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

In the present work we report new tools for the characterization of the complete chromosome complement of sunflower ( Helianthus annuus L.), using a bacterial artificial chromosome (BAC) clone containing repetitive sequences with similarity to retrotransposons and a homologous rDNA sequence isolated from the sunflower genome as probes for FISH. The rDNA signal was found in 3 pairs of chromosomes, coinciding with the location of satellites. The BAC clone containing highly represented retroelements hybridized with all the chromosome complement in FISH, and used together with the rDNA probe allowed the discrimination of all chromosome pairs of sunflower. Their distinctive distribution pattern suggests that these probes could be useful for karyotype characterization and for chromosome identification. The karyotype could be subdivided into 3 clear-cut groups of 12 metacentric pairs, 1 submetacentric pair, and 4 subtelocentric pairs, thus resolving previously described karyotype controversies. The use of BAC clones containing single sequences of specific markers and (or) genes associated with important agricultural traits represents an important tool for future locus-specific identification and physical mapping.

Fructans are fructose polymers synthesized from sucrose in the plant vacuole. They represent short- and long-term carbohydrate reserves and have been associated with abiotic stress tolerance in graminean species. We report the isolation and characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) gene from a Patagonian grass species, Bromus pictus, tolerant to drought and cold temperatures. Structural and functional analyses of this gene were performed by Southern and Northern blot. Sugar content, quality and fructosyltransferase activity were studied using HPAEC-PAD (high-pH anion-exchange chromatography with pulsed amperometric detection), enzymatic and colorimetric assays. The putative 6-SFT gene had all the conserved motifs of fructosyl-transferase and showed 90% identity at the amino acid level with other 6-SFTs from winter cereals. Expression studies, and determination of sugar content and fructosyl-transferase activity were performed on five sections of the leaf. Bp6-SFT was expressed predominantly in leaf bases, where fructosyltransferase activity and fructan content are higher. Bp6-SFT expression and accumulation of fructans showed different patterns in the evaluated leaf sections during a 7 d time course experiment under chilling treatment. The transcriptional pattern suggests that the B. pictus 6-SFT gene is highly expressed in basal leaf sections even under control temperate conditions, in contrast to previous reports in other graminean species. Low temperatures caused an increase in Bp6-SFT expression and fructan accumulation in leaf bases. This is the first study of the isolation and molecular characterization of a fructosyltransferase in a native species from the Patagonian region. Expression in heterologous systems will confirm the functionality, allowing future developments in generation of functional markers for assisted breeding or biotechnological applications.

Leaf senescence is a complex process, which has dramatic consequences on crop yield. In sunflower, gap between potential and actual yields reveals the economic impact of senescence. Indeed, sunflower plants are incapable of maintaining their green leaf area over sustained periods. This study characterizes the leaf senescence process in sunflower through a systems biology approach integrating transcriptomic and metabolomic analyses: plants being grown under both glasshouse and field conditions. Our results revealed a correspondence between profile changes detected at the molecular, biochemical and physiological level throughout the progression of leaf senescence measured at different plant developmental stages. Early metabolic changes were detected prior to anthesis and before the onset of the first senescence symptoms, with more pronounced changes observed when physiological and molecular variables were assessed under field conditions. During leaf development, photosynthetic activity and cell growth processes decreased, whereas sucrose, fatty acid, nucleotide and amino acid metabolisms increased. Pathways related to nutrient recycling processes were also up-regulated. Members of the NAC, AP2-EREBP, HB, bZIP and MYB transcription factor families showed high expression levels, and their expression level was highly correlated, suggesting their involvement in sunflower senescence. The results of this study thus contribute to the elucidation of the molecular mechanisms involved in the onset and progression of leaf senescence in sunflower leaves as well as to the identification of candidate genes involved in this process. Inst. de Biotecnología Fil: Moschen, Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina Fil: Heinz, Ruth Amelia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bengoa Luoni, Sofía. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina Fil: Di Rienzo, Julio A. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina Fil: Caro, María del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán; Argentina Fil: Tohge, Takayuki. Max-Planck-Institut fur Molekulare Pflanzenphysiologie; Alemania Fil: Watanabe, Mutsumi Max-Planck-Institut fur Molekulare Pflanzenphysiologie; Alemania Fil: Hollmann, Julien. University of Kiel. Institute of Botany, Christian-Albrechts; Alemania Fil: García-García, Francisco. Centro de Investigación Príncipe Felipe. Department of Bioinformatics and Genomics; España. Centro de Investigación Príncipe Felipe . National Institute of Bioinformatics. Functional Genomics Node; España Fil: Dopazo, Joaquín. Centro de Investigación Príncipe Felipe. Department of Bioinformatics and Genomics; España. Centro de Investigación Príncipe Felipe . National Institute of Bioinformatics. Functional Genomics Node; España Fil: Hoefgen, Rainer Max-Planck-Institut fur Molekulare Pflanzenphysiologie; Alemania Fil: Fernie, Alisdair R. Max-Planck-Institut fur Molekulare Pflanzenphysiologie; Alemania

Infections by plant virus generally cause disease symptoms by interfering with cellular processes. Here we demonstrated that infection of Nicotiana tabacum ( N.t ) by plant viruses representative of the Tobamoviridae , Potyviridae , and Potexviridae families altered accumulation of certain microRNAs (miRNAs). A correlation was observed between symptom severity and alteration in levels of miRNAs 156, 160, 164,166, 169, and 171 that is independent of viral posttranscriptional gene silencing suppressor activity. Hybrid transgenic plants that produced tobacco mosaic virus (TMV) movement protein (MP) plus coat protein (CP) T42W (a variant of CP) exhibited disease-like phenotypes, including abnormal plant development. Grafting studies with a plant line in which both transgenes are silenced confirmed that the disease-like phenotypes are due to the coexpression of CP and MP. In hybrid MPxCP T42W plants and TMV-infected plants, miRNAs 156, 164, 165, and 167 accumulated to higher levels compared with nontransgenic and noninfected tissues. Bimolecular fluorescence complementation assays revealed that MP interacts with CP T42W in vivo and leads to the hypothesis that complexes formed between MP and CP caused increases in miRNAs that result in disease symptoms. This work presents evidence that virus infection and viral proteins influence miRNA balance without affecting posttranscriptional gene silencing and contributes to the hypothesis that viruses exploit miRNA pathways during pathogenesis.

The expression of a gene that encodes coat protein (CP) of Potato virus X (PVX) in transgenic tobacco plants confers a high level of CP-mediated rresistance (CP-MR) against PVX infection. To determine if posttranscriptional gene silencing (PTGS) plays a role in resistance, transgenic plants expressing PVX CP were challenged against PVX under conditions in which PTGS was suppressed by low temperatures or using viruses carrying PTGS suppressors. The data demonstrate that PTGS does not play a significant role in PVX CP-MR.

Replication of Potato virus X (PVX) was reduced in transgenic protoplasts that accumulated wild-type coat protein (CPWT) of Tobacco mosaic virus (TMV) or a mutant CP, CPT42W, that produced highly ordered states of aggregation, including pseudovirions. This reaction is referred to as heterologous CP-mediated resistance. However, protoplasts expressing a CP mutant that abolished aggregation and did not produce pseudovirions, CPT28W, did not reduce PVX replication. Similarly, in transgenic tobacco plants producing TMV CPWT or CPT42W, there was a delay in local cell-to-cell spread of PVX infection that was not observed in CPT28W plants or in non-transgenic plants. The results suggest that the quaternary structure of the TMV CP regulates the mechanism(s) of heterologous CP-mediated resistance. Similarly, transgenic protoplasts that produced PVX CP conferred transient protection against infection by TMV RNA. Transgenic plants that accumulated PVX CP reduced the cell-to-cell spread of infection and resulted in a delay in systemic infection following inoculation with TMV or TMV RNA. Heterologous CP-mediated resistance was characterized by a brief delay in systemic infection, whilst homologous CP-mediated resistance conferred reduced or no systemic infection.

Molecular genetic analysis was applied to 162 individuals of 37 half-sib selected families belonging to six provenances of Eucalyptus dunnii Maiden in a provenance/family trial. The individuals were selected by a trait selection index and genetic diversity criteria were later applied for designing seedling seed orchards. Genetic diversity and its distribution, as well as relationships among individuals, were assessed on the basis of nine microsatellite loci and 243 amplified fragment length polymorphism markers. High diversity was found with both kinds of markers. Clear-cut genomic patterns of identification (fingerprinting) were obtained for each individual. Genetic differentiation estimates consistently showed low differentiation among provenances (R(ST1) = 0.069, theta(P) = 0.026 and F(CT) = 0.035) and great differentiation among families (R(ST2) = 0.223, theta(S) = 0.174 and F(SC) = 0.164). A high proportion of the total variation was observed within families (around 80% by both marker analyses), suggesting that orchard design should be based on individual or family selection rather than on provenance selection, and that individual ranking by both trait selection index and molecular genetic diversity criteria should be considered. A selection procedure for a seedling seed orchard design is proposed.

A Eucalyptus dunnii Maiden breeding population of 46 accessions originated in Australia and selected for fitness to subtropical and cold environments was screened by Amplified Fragment Length Polymorphism (AFLP) and microsatellite markers to obtain quantitative estimates of genetic diversity. A randomly chosen group of AFLP primers generated 205 AFLP bands that were used to fingerprint the genotypes and to evaluate genetic relationships among accessions. Sixty-eight percent (140) of the bands were polymorphic markers. The mean diversity index (DI) was 0.33 and about 52% of the loci had values greater than 0.4. Cluster analysis derived from similarity indices (SI) revealed no particular grouping among accessions suggesting the absence of closely related genotypes, except for five pairs of genotypes. Bootstrap analysis results confirmed the suitability of AFLP to describe genetic relationships in this breeding population. In addition, four highly informative microsatellites were used to construct an identification matrix that discriminated nearly all of the genotypes. Mean values for the number of alleles per locus, DI and SI among accessions were 13, 0.78 and 0.19, respectively, indicating that the breeding population has high genetic diversity. However, several genotypes showed the presence of single microsatellite bands suggesting a putatively important degree of homozygosity. Molecular data were used to design a clonal seed orchard. To achieve this aim, the nine most divergent pairs of genotypes were chosen, thereby retaining 95.2% of the total number of alleles from the 140 polymorphic AFLP loci and the four microsatellite loci analyzed. Mean DI and SI for AFLP and microsatellites showed no significant differences between the original breeding population and the selected seed orchard, confirming that a seed orchard can be designed with a limited number of individuals, which allows similar accessions to be discarded and avoids inbreeding.

Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of four MRCV genome segments was determined. MRCV S1, S2, S3 and S6 were predicted to encode proteins of 168.4, 134.4, 141.7 and 90 kDa, respectively. MRCV S1 encodes a basic protein that contains conserved RNA-dependent RNA polymerase motifs, and is homologous to Rice black streaked dwarf virus (RBSDV), Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV) polymerases as well as to corresponding proteins of members of other genera of the Reoviridae. MRCV S2 codes for a protein with intermediate homology to the ones coded by RBSDV S4 and FDV S3 'B' spike, which is presumably the B-spike protein. MRCV S3 most probably encodes the major core protein and is highly homologous to corresponding proteins of RBSDV S2 and FDV S3. MRCV S6-encoded protein has low homology to the proteins of unknown function coded by RBSDV S6 and FDV S6. The identity levels between all analyzed MRCV coded proteins and their RBSDV counterparts varied between 84.5 and 44.8%. The analysis of the reported sequences allowed a phylogenetic comparison of MRCV with other reovirus and supported its taxonomic status within the genus.

This is the first sequence-based characterization of Mal de Río Cuarto virus (MRCV), currently classified as a variant of Maize rough dwarf virus (MRDV) and exclusively found in South America. We sequenced and analyzed genome segments S4 and S8. MRCV S4 coded for a putative 131.67 kDa protein while MRCV S8 coded for a putative 68.26 kDa protein containing an ATP/GTP-binding motif. The 5' and 3' ends of MRCV segments, were 5'AAGUUUUU3' and 5'CAGCUnnnGUC3', respectively. Prediction of secondary structure of both segments coding strands showed that terminal regions were able to form structures that are proposed to be replication and packaging signals. MRCV S4 showed identity to members of Fijivirus as well as to two other genera of the Reoviridae family. MRCV S8 revealed identity with Rice black streaked dwarf virus (RBSDV) S8, MRDV S7, Oat sterile dwarf virus (OSDV) S9 and Nilaparvata lugens reovirus (NLRV) S7. While MRDV and RBSDV segments are highly homologous between each other, MRCV identity levels with them was considerably lower. We discussed the evolutionary relationships of MRCV to other Reoviridae, and based on phylogenetic analysis we proposed that although MRCV is related to MRDV, it could be regarded as a new species of the Fijivirus genus.

Use of molecular markers such as Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) and Simple Sequence Repeats (SSR) as descriptors to characterize and differentiate a set of 100 soybean varieties of commercial use in Argentina was taken as a leading case study for plant variety protection (PVP) purposes. Sixteen morphological traits were recorded to compare pedigree relationships among varieties with information derived from conventional descriptors and molecular markers. Analysis of 109 polymorphic loci confirmed the rather low genetic variability of commercial soybean germplasm. Still, genetic fingerprinting of the 100 varieties could be established. Calculated similarity indexes were dependent on the technique, ranging from 0.262 (SSR), 0.407 (RAPD), 0.400 (AFLP) and 0.574 (morphological traits).Dendrograms generated from morphological data matrix showed low value correlation with kinship coefficient matrix (r = 0.216). Still, they were suitable to identify and differentiate each of the 100 varieties analyzed; which was not possible with RAPD or AFLP markers using comparable numbers of polymorphic loci. SSR data showed the best fit to pedigree information (r = 0.353), while maintaining an association to morphologically based separation. Results suggest that the four techniques describe genetic variability in different and specific ways. A combination of SSR and morphological descriptors show the best compromise of regarding genetic relationships and the needs of clear classification for PVP and may help to establish minimum genetic distances for distinctness within PVP Office definition.

Background: Argentina has a long tradition of sunflower breeding, and its germplasm is a valuable genetic resource worldwide. However, knowledge of the genetic constitution and variability levels of the Argentinean germplasm is still scarce, rendering the global map of cultivated sunflower diversity incomplete. In this study, 42 microsatellite loci and 384 single nucleotide polymorphisms (SNPs) were used to characterize the first association mapping population used for quantitative trait loci mapping in sunflower, along with a selection of allied open-pollinated and composite populations from the germplasm bank of the National Institute of Agricultural Technology of Argentina. The ability of different kinds of markers to assess genetic diversity and population structure was also evaluated. Results: The analysis of polymorphism in the set of sunflower accessions studied here showed that both the microsatellites and SNP markers were informative for germplasm characterization, although to different extents. In general, the estimates of genetic variability were moderate. The average genetic diversity, as quantified by the expected heterozygosity, was 0.52 for SSR loci and 0.29 for SNPs. Within SSR markers, those derived from non-coding regions were able to capture higher levels of diversity than EST-SSR. A significant correlation was found between SSR and SNP- based genetic distances among accessions. Bayesian and multivariate methods were used to infer population structure. Evidence for the existence of three different genetic groups was found consistently across data sets (i.e., SSR, SNP and SSR + SNP), with the maintainer/restorer status being the most prevalent characteristic associated with group delimitation. Conclusion: The present study constitutes the first report comparing the performance of SSR and SNP markers for population genetics analysis in cultivated sunflower. We show that the SSR and SNP panels examined here, either used separately or in conjunction, allowed consistent estimations of genetic diversity and population structure in sunflower breeding materials. The generated knowledge about the levels of diversity and population structure of sunflower germplasm is an important contribution to this crop breeding and conservation. Instituto de Biotecnología Fil: Filippi, Carla Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Aguirre, Natalia Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Rivas, Juan Gabriel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Zubrzycki, Jeremias Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Puebla, Andrea Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Cordes, Diego Darío. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina Fil: Moreno, Maria Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina Fil: Fusari, Corina Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Max Planck Institute of Molecular Plant Physiology; Alemania 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 Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lia, Veronica Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Open-pollinated (OPs) and composite populations (CPs) represent a valuable resource for sunflower breeding programmes. However, little is known about the levels and distribution of genetic variation within each of these populations. In this study, quantitative and qualitative traits along with molecular markers were used to evaluate 14 populations from the Instituto Nacional de Tecnología Agropecuaria (INTA) sunflower germplasm collection. These populations were chosen to represent historically important accessions that still play a central role within the INTA sunflower breeding programme due to their extensive variation in diverse agronomically important traits. Nine quantitative and eight qualitative agro-morphological descriptors were recorded and compared with those of a larger set of accessions representative of the phenotypic diversity of the sunflower collection. Molecular characterization was conducted on a total of 311 individuals using 16 microsatellite markers. Overall, the average gene diversity was 0.56 and the average number of alleles per locus was 6.25. No statistically significant differences in genetic diversity were detected between the OPs and CPs. Global estimates of F ST revealed very high levels of differentiation among accessions (F ST= 0.413, P< 0.05). Population structure analyses were consistent with the observed levels of differentiation and identified two major groups. The results of this work show that high global diversity is preserved within the accessions analysed here. Additionally, this study provides a set of reliable and discriminant markers for the cost-effective molecular characterization of sunflower accessions, along with the guidelines for the delineation of sampling strategies for OPs and CPs, thus aiding the efficient management and exploitation of sunflower germplasm collections. Fil: Moreno, M. V.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Nishinakamasu, V.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Loray, M. A.. Instituto Nacional de Semillas; Argentina Fil: Alvarez, D.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Gieco, J.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; Argentina Fil: Vicario, A.. Instituto Nacional de Semillas; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Heinz, Ruth Amelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lia, Verónica Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The PVX coat protein (CP) is involved in many aspects of plant-virus interaction (virion morphology, plant symptoms, viral pathogenesis and virulence, and genomic RNA accumulation). Different virus strains have been distinguished according to their compatibility with the host resistance genes Nx, Nb, and Rx. Substitution of the Thr 122 on the CP with a Lys in PVX strain HB has been shown to affect the response of potato cultivars with the Rx resistance gene. In PVX DX the avirulence determinant for the Nx gene has been localized in the Gln 78 of the coat. PVX strain MS, like PVX HB, is able to overcome the Rx, Nx, and Nb genes. Sequencing of the CP gene of PVX MS (EMBL accession number Z34261) shows that it has a Thr in codon 122 and a Gln in codon 78. These results suggest that, in addition to the coat protein gene, other regions of the viral genome are involved in the pathogenicity.

Snakin/GASA proteins are widely distributed among plant species. They are expressed in different plant organs with high tissue and temporal specificity, and their subcellular localization varies among the different members. Interestingly, all of them maintain 12 cysteines of the C-terminus in highly conserved positions of the aminoacid sequences that are essential for their biochemical activity and probably responsible for their protein structure. Despite their common features, their functions are not completely elucidated and little is known about their mode of action. This review focuses on the current knowledge about this intriguing family of peptides and advances comprising gene regulation analyses, expression pattern studies and phenotypic characterization of mutants and transgenic plants. Furthermore, we discuss the roles of Snakin/GASA proteins in several aspects of plant development, plant responses to biotic or abiotic stress and their participation in hormone crosstalk and redox homeostasis. Instituto de Biotecnología Fil: Nahirñak, Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fil: Almasia, Natalia Ines. 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. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.

A new resistance-breaking isolate of potato virus X (PVX MS; also called PVX fcaOl) that multiplies in genotypes carrying the Rx gene was detected in Argentina. Enzyme-linked immunosorbent assay (ELISA) assessment of PVX multiplication in mechanically inoculated micropropagated plantlets revealed that PVX MS is able to replicate in both resistant (Rx) and suceptible plantlets whereas strain PVX cp is able to replicate only in the latter. Immunoblot detection with specific monoclonal an, tibodies showed that PVX MS belongs to the serotype PVXo (common or European strain), whereas PVX HB is included in the serotype PVXA (Andean). SDS polyacrylamide gel electrophoresis followed by immunoblotting of the coat proteins of the PVXst, rains cp, cp4, HB, and MS showed that they differed in mobility with apparent mol. wts of 27.9, 28.8, 29.9, and 29.4 kDa, respectively.

In order to saturate a sunflower genetic map and facilitate marker-assisted selection (MAS) breeding for stress response, it is necessary to enhance map saturation with molecular markers localized in linkage groups associated to genomic regions involved in these traits. This work describes the identification and characterization of 1,134 simple sequence repeat (SSR) containing expressed sequence tags (EST) from unigenes available databases. Twelve of these functional markers as well as 41 public SSR markers were successfully localized in linkage groups, thus contributing to the saturation of specific regions on a reference genetic-linkage-map derived from recombinant inbred lines (RIL) mapping population from the cross between PAC2 x RHA266 lines. The enriched map includes 547 markers (231 SSR, 9 EST-SSR, 3 insertions/deletions (InDel) and 304 amplified fragment length polymorphisms (AFLP) distributed in 17 linkage groups (LG), spanning genetic size to 1,942.3 cM and improving its mean density to 3.6 cM per locus. As consequence, no gaps longer than 13.2 cM remain uncovered throughout the entire map, which increases the feasibility of detecting genes or traits of agronomic importance in sunflower.

cDNA clones of potato virus X (PVXcp strain), potato virus Y (PVYo strain), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTV) were used separately or combined for the detection of the corresponding RNAs in extracts of infected plants. A general method for the rapid preparation of RNA extracts without use of organic solvents (i.e. phenol) was developed for this purpose. Plant extracts from a range of field, artificially inoculated germplasm genotypes, micro-propagated and protoplast samples, as well as vector insect extracts, were dot-blotted onto nylon or nitrocellulose membranes, subjected to sandwich nucleic acid hybridization with non-labelled specific single-stranded DNA probes followed by a biotin-labelled second step hybridization probe. Each probe was virus-specific but not strain-specific. Healthy or non-related plant extracts developed very faint or no signals. Sensitivity was tested by slot-blot hybridization. Detection levels were between 1.5 to 6 pg of viral nucleic acids and between 20 to 50 times more sensitive than standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The assay developed was tested with material that was prepared for processing in the field (combination of fresh sap with extraction solution) and tested under simple laboratory conditions for detection. It was also successfully employed for screening of germplasm for virus resistance, detection of pathogens in vector insects, plantlets grown in vitro and in more sophisticated quantitative determinations of viral replication in artificially inoculated plants and protoplasts.

Mal de Rio Cuarto virus (MRCV) was recently described as a new species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of two MRCV genome segments was determined. MRCV S5 and S10 were predicted to encode proteins of 106.9 and 63.5 kDa respectively. The protein coded by MRCV S5 had 62.8% and 35.7% identity to fijiviruses RBSDV S5 and FDV S5 coded proteins, and contained a rarely reported type-1 C-terminal peroxisomal targeting signal. The protein coded by MRCV S10 had identity levels of 72.4% and 21.7% to the major outer capsid proteins of fijiviruses RBSDV S10 and NLRV S8.

Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

Background: Prosopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus. Results: Next generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads. Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic. Conclusions: This study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data. The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera. Fil: Torales, Susana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pomponio, María Florencia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina Fil: González, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernández, Paula del Carmen. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: López Lauenstein, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina Fil: Verga, Aníbal Ramón. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Geneticos Vegetales; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marcucci Poltri, Susana Noemí. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina

Nothofagus nervosa is one of the most emblematic native tree species of Patagonian temperate forests. Here, the shotgun RNA-sequencing (RNA-Seq) of the transcriptome of N. nervosa, including de novo assembly, functional annotation, and in silico discovery of potential molecular markers to support population and associations genetic studies, are described. Fil: Torales, Susana Leonor. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; Argentina Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pomponio, Maria Florencia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; Argentina Fil: Fernandez, Paula Del Carmen. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Marchelli, Paula Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Argentina Fil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Azpilicueta, María M. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Gallo, Leonardo A. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marcucci Poltri, Susana Noemi. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

DNA amplification was studied in the two closely related species, Oxalis glaucifolia and O. rhombeo-ovata . Three tissue types of different degrees of differentiation were used for each species: leaf, friable and nodular callus. The relative abundance in the nuclear genome of five genes coding for functional proteins was determined in each tissue. The utilized probes detected the genes for: Histones H3 and H4, ribosomal 25S RNA, ribulose-1,5-bisphosphate carboxylase (small subunit) and ubiquitin. While all callus tissues showed significant amplification of these genes relative to the differentiated leaf tissue, the pattern of amplification was different for the two species. Furthermore, each gene was amplified to a different degree, suggesting an independent control mechanism.

cDNA libraries, representative of potato viruses X (PVXc strain) and Y (PVY degrees strain) genomes were obtained. A PVX cDNA cloned fragment was sequenced and biotinylated to be used as hybridization probe for the detection of purified virus or nucleic acid extracts of infected plants. Dot hybridization assay was sensitive to detect 4 ng of viral particles, corresponding to about 200 pg of viral RNA. The level of detection in infected plant extracts was as effective as that obtained with the ELISA. The presence of biotinylated PVY cDNA in the hybridization mixture did not affect sensitivity of the PVX detection assay, suggesting that a single diagnostic assay for several potato viruses and virus-related pathogens could be developed.