Julio A. Di Rienzo

Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

BackgroundConsidering 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.ResultsAbiotic-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.ConclusionEighty 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.

Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis

The selection and validation of reference genes constitute a key point for gene expression analysis based on qPCR, requiring efficient normalization approaches. In this work, the expression profiles of eight genes were evaluated to identify novel reference genes for transcriptional studies associated to the senescence process in sunflower. Three alternative strategies were applied for the evaluation of gene expression stability in leaves of different ages and exposed to different treatments affecting the senescence process: algorithms implemented in geNorm, BestKeeper software, and the fitting of a statistical linear mixed model (LMModel). The results show that geNorm suggested the use of all combined genes, although identifying α-TUB1 as the most stable expressing gene. BestKeeper revealed α-TUB and β-TUB as stable genes, scoring β-TUB as the most stable one. The statistical LMModel identified α-TUB, actin, PEP, and EF-1α as stable genes in this order. The model-based approximation allows not only the estimation of systematic changes in gene expression, but also the identification of sources of random variation through the estimation of variance components, considering the experimental design applied. Validation of α-TUB and EF-1α as reference genes for expression studies of three sunflower senescence associated genes showed that the first one was more stable for the assayed conditions. We conclude that, when biological replicates are available, LMModel allows a more reliable selection under the assayed conditions. This study represents the first analysis of identification and validation of genuine reference genes for use as internal control in qPCR expression studies in sunflower, experimentally validated throughout six different controlled leaf senescence conditions.

Coexistence and Within-Host Evolution of Diversified Lineages of Hypermutable Pseudomonas aeruginosa in Long-term Cystic Fibrosis Infections

The advent of high-throughput sequencing techniques has made it possible to follow the genomic evolution of pathogenic bacteria by comparing longitudinally collected bacteria sampled from human hosts. Such studies in the context of chronic airway infections by Pseudomonas aeruginosa in cystic fibrosis (CF) patients have indicated high bacterial population diversity. Such diversity may be driven by hypermutability resulting from DNA mismatch repair system (MRS) deficiency, a common trait evolved by P. aeruginosa strains in CF infections. No studies to date have utilized whole-genome sequencing to investigate within-host population diversity or long-term evolution of mutators in CF airways. We sequenced the genomes of 13 and 14 isolates of P. aeruginosa mutator populations from an Argentinian and a Danish CF patient, respectively. Our collection of isolates spanned 6 and 20 years of patient infection history, respectively. We sequenced 11 isolates from a single sample from each patient to allow in-depth analysis of population diversity. Each patient was infected by clonal populations of bacteria that were dominated by mutators. The in vivo mutation rate of the populations was ∼100 SNPs/year–∼40-fold higher than rates in normo-mutable populations. Comparison of the genomes of 11 isolates from the same sample showed extensive within-patient genomic diversification; the populations were composed of different sub-lineages that had coexisted for many years since the initial colonization of the patient. Analysis of the mutations identified genes that underwent convergent evolution across lineages and sub-lineages, suggesting that the genes were targeted by mutation to optimize pathogenic fitness. Parallel evolution was observed in reduction of overall catabolic capacity of the populations. These findings are useful for understanding the evolution of pathogen populations and identifying new targets for control of chronic infections.

Quantifying Functional Biodiversity

1. Introduction 1.1 Functional Diversity Approach To Quantify the Biodiversity 1.2 Functional Diversity Assessment 1.3 Classification of Ecosystem Services 1.4 Selection of Traits According to Ecosystem Service 1.5. Functional Diversity Quantification 2. Functional Groups 2.1 Selecting Trait and Its Relation With Ecosystem Services 2.2 A Guide For Data Arrangement 2.3 Statistical Procedures To Define Functional Groups 2.3.1 The Selection of a Dissimilarity Measure 2.3.2 Standardization 2.3.3 Choosing the Linkage Algorithm Method 2.3.4 Assessing the Number of Functional Groups 2.4 Functional Characterization of Coastal Sandy Plain Vegetation in Southeast Brazil 2.4.1 The Data Set 2.4.2 Plant Functional Types From a Restinga Vegetation 2.5 Functional Groups For Bird Species in Nicaragua 2.5.1 The Data Set 2.5.2 Bird Functional Types From Nicaragua 2.5.3 Characterization of Bird Functional Types of Nicaragua 2.5.4 Relationship of Functional Types With Land Uses 3. Functional Diversity Indices 3.1 About Functional Diversity Indices and Measures 3.2 Species Diversity Indices 3.3 Single-trait Metrics and Indices: Properties and Estimation 3.3.1 Community Weighted Mean 3.3.2 Functional Divergence 3.3.3 Functional Regularity 3.4 Multi-trait Indices: Properties and Estimation 3.4.1 Functional Attribute Diversity 3.4.2 Functional Diversity Based on Dendrograms 3.4.3 Convex Hull Hyper-volume 3.4.4. Quadratic Entropy 3.4.5 Extended FD 3.4.6 Functional Richness, Evenness, Divergence and Dispersion 3.5 Ability of Indices to Detect Some Ecological Processes 4. How To Estimate Functional Diversity Indices 4.1 The FDiversity Software: Capabilities and Data Management 4.1.1 How To Install FDiversity 4.1.2 Data Menu 4.1.3 Statistics and Output Menus 4.2 Case study: Changes in Functional Diversity in an Altitudinal Gradient 4.2.1 Sample Design and Trait Evaluation 4.2.2 The Database 4.2.3 Changes of Plant Functional Types With the Altitude 4.2.4 Changes of Functional Diversity Indices With the Altitude 4.3 Case study: Changes in Functional Diversity in a Chronosequence 4.3.1 Sample Design and Trait Evaluation 4.3.2 The Database 4.3.3 Changes of Plant Functional Types in the Chronosequence 4.4 Multivariate Graphical Projection Methods Index Index

Association mapping in sunflower for sclerotinia head rot resistance

BackgroundSclerotinia Head Rot (SHR) is one of the most damaging diseases of sunflower in Europe, Argentina, and USA, causing average yield reductions of 10 to 20 %, but leading to total production loss under favorable environmental conditions for the pathogen. Association Mapping (AM) is a promising choice for Quantitative Trait Locus (QTL) mapping, as it detects relationships between phenotypic variation and gene polymorphisms in existing germplasm without development of mapping populations. This article reports the identification of QTL for resistance to SHR based on candidate gene AM.ResultsA collection of 94 sunflower inbred lines were tested for SHR under field conditions using assisted inoculation with the fungal pathogen Sclerotinia sclerotiorum. Given that no biological mechanisms or biochemical pathways have been clearly identified for SHR, 43 candidate genes were selected based on previous transcript profiling studies in sunflower and Brassica napus infected with S. sclerotiorum. Associations among SHR incidence and haplotype polymorphisms in 16 candidate genes were tested using Mixed Linear Models (MLM) that account for population structure and kinship relationships. This approach allowed detection of a significant association between the candidate gene HaRIC_B and SHR incidence (P < 0.01), accounting for a SHR incidence reduction of about 20 %.ConclusionsThese results suggest that AM will be useful in dissecting other complex traits in sunflower, thus providing a valuable tool to assist in crop breeding.

Integrating transcriptomic and metabolomic analysis to understand natural leaf senescence in sunflower.

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.

Nitric oxide is required for determining root architecture and lignin composition in sunflower. Supporting evidence from microarray analyses

Nitric oxide (NO) is a signal molecule involved in several physiological processes in plants, including root development. Despite the importance of NO as a root growth regulator, the knowledge about the genes and metabolic pathways modulated by NO in this process is still limited. A constraint to unravel these pathways has been the use of exogenous applications of NO donors that may produce toxic effects. We have analyzed the role of NO in root architecture through the depletion of endogenous NO using the scavenger cPTIO. Sunflower seedlings growing in liquid medium supplemented with cPTIO showed unaltered primary root length while the number of lateral roots was deeply reduced; indicating that endogenous NO participates in determining root branching in sunflower. The transcriptional changes induced by NO depletion have been analyzed using a large-scale approach. A microarray analysis showed 330 genes regulated in the roots (p≤0.001) upon endogenous NO depletion. A general cPTIO-induced up-regulation of genes involved in the lignin biosynthetic pathway was observed. Even if no detectable changes in total lignin content could be detected, cell walls analyses revealed that the ratio G/S lignin increased in roots treated with cPTIO. This means that endogenous NO may control lignin composition in planta. Our results suggest that a fine tuning regulation of NO levels could be used by plants to regulate root architecture and lignin composition. The functional implications of these findings are discussed.

Evaluation of pathogenesis caused in cattle and guinea pig by a Mycobacterium bovis strain isolated from wild boar

BackgroundIn many regions of the world, wild mammals act as reservoir of Mycobacterium bovis, a situation that prevents the eradication of bovine tuberculosis. In order to observe whether a strain isolated from a wild boar, previously tested as highly virulent in a mice model, is also virulent in cattle, we performed cattle experimental inoculation with this strainResultsGroups of Friesian calves were either infected with the wild boar strain M. bovis 04-303 or with the bovine strain NCTC10772 as a control. We found that antigen-specific IFN-γ release in whole blood samples occurred earlier in animals infected with M. bovis 04-303. Both M. bovis strains resulted in a positive skin test, with animals infected with the wild boar isolate showing a stronger response. These results and the presence of more severe organ lesions, with granuloma and pneumonic areas in cattle demonstrate that the wild boar isolate is more virulent than the NCTC10772 strain. Additionally, we tested the infectivity of the M. bovis strains in guinea pigs and found that M. bovis 04-303 had the highest pathogenicity.ConclusionsM. bovis strains isolated from wild boars may be pathogenic for cattle, producing TB lesions.

Basal area growth curves for Pinus patula in two areas of the Calamuchita Valley, Córdoba, Argentina

Abstract This study was carried out in the Calamuchita Valley, in West Central Cordoba, Argentina. Two stands of Pinus patula were chosen, one in each of two different areas: Yacanto de Calamuchita and Rio de los Sauces. The objectives of the study were: 1. to develop basal area (BA) mean annual increment (MAI) and current annual increment (CAI) curves for P. patula in two areas of the Valle de Calamuchita; 2. to estimate the timing of intermediate and final cuts in P. patula plantations; 3. to evaluate P. patula BA growth rate vs. other pine species that grow in the region; and 4. to identify the best growing species for each area. Thirty trees were randomly selected in each stand. An increment core was taken from each sampled tree from bark to bark through the pith. Growth rings were measured and BA-MAI and BA-CAI were calculated. Growth curves were fitted for each stand and intersection points between the MAI and CAI curves were found. Regression analysis was performed for BA-CAI vs. time up to the age of 10 years in both areas. P. patula performance was compared to that of P. elliottii , P. taeda and P. radiata growing in the same areas. It is concluded that: 1. The proper time for the first commercial thinning is about 10–12 years, just before the BA-CAI curve declines. 2. The biological rotation age is expected to occur no sooner than Year 17 in this region. 3. P. patula can perform better than P. elliottii , P. taeda and P. radiata during the first 10 years, depending on site quality and seed genetic quality.

Functional Diversity Indices

Functional diversity may be summarized using indices based on trait values and species importance in the community, like abundance, cover and biomass. This chapter includes taxonomy of the indices based upon the information they used and the output they offer. For each index we have included its definition, the information needed to estimate it, their statistical and ecological properties, and some reference to explore its application to real cases. To facilitate the comprehension of all indices and diversity measures we used homogeneous notation.