Characterization and expression analysis of WRKY genes during leaf and corolla senescence of Petunia hybrida plants.

Several families of transcription factors (TFs) control the progression of senescence. Many key TFs belonging to the WRKY family have been described to play crucial roles in the regulation of leaf senescence, mainly in Arabidopsis thaliana. However, little is known about senescence-associated WRKY members in floricultural species. Delay of senescence in leaves and petals of Petunia hybrida, a worldwide ornamental crop are highly appreciated traits. In this work, starting from 28 differentially expressed WRKY genes of A. thaliana during the progression of leaf senescence, we identified the orthologous in P. hybrida and explored the expression profiles of 20 PhWRKY genes during the progression of natural (age-related) leaf and corolla senescence as well as in the corollas of flowers undergoing pollination-induced senescence. Simultaneous visualization showed consistent and similar expression profiles of PhWRKYs during natural leaf and corolla senescence, although weak expression changes were observed during pollination-induced senescence. Comparable expression trends between PhWRKYs and the corresponding genes of A. thaliana were observed during leaf senescence, although more divergence was found in petals of pollinated petunia flowers. Integration of expression data with phylogenetics, conserved motif and cis-regulatory element analyses were used to establish a list of candidates that could regulate more than one senescence process. Our results suggest that several members of the WRKY family of TFs are tightly linked to the regulation of senescence in P. hybrida. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01243-y.

On-field phenotypic evaluation of sunflower populations for broad-spectrum resistance to Verticillium leaf mottle and wilt.

Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.

Exploring sunflower responses to Sclerotinia head rot at early stages of infection using RNA-seq analysis.

Sclerotinia head rot (SHR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating sunflower crop diseases. Despite its worldwide occurrence, the genetic determinants of plant resistance are still largely unknown. Here, we investigated the Sclerotinia-sunflower pathosystem by analysing temporal changes in gene expression in one susceptible and two tolerant inbred lines (IL) inoculated with the pathogen under field conditions. Differential expression analysis showed little overlapping among ILs, suggesting genotype-specific control of cell defense responses possibly related to differences in disease resistance strategies. Functional enrichment assessments yielded a similar pattern. However, all three ILs altered the expression of genes involved in the cellular redox state and cell wall remodeling, in agreement with current knowledge about the initiation of plant immune responses. Remarkably, the over-representation of long non-coding RNAs (lncRNA) was another common feature among ILs. Our findings highlight the diversity of transcriptional responses to SHR within sunflower breeding lines and provide evidence of lncRNAs playing a significant role at early stages of defense.

Genetic Diversity, Population Structure and Linkage Disequilibrium Assessment among International Sunflower Breeding Collections.

Sunflower germplasm collections are valuable resources for broadening the genetic base of commercial hybrids and ameliorate the risk of climate events. Nowadays, the most studied worldwide sunflower pre-breeding collections belong to INTA (Argentina), INRA (France), and USDA-UBC (United States of America-Canada). In this work, we assess the amount and distribution of genetic diversity (GD) available within and between these collections to estimate the distribution pattern of global diversity. A mixed genotyping strategy was implemented, by combining proprietary genotyping-by-sequencing data with public whole-genome-sequencing data, to generate an integrative 11,834-common single nucleotide polymorphism matrix including the three breeding collections. In general, the GD estimates obtained were moderate. An analysis of molecular variance provided evidence of population structure between breeding collections. However, the optimal number of subpopulations, studied via discriminant analysis of principal components (K = 12), the bayesian STRUCTURE algorithm (K = 6) and distance-based methods (K = 9) remains unclear, since no single unifying characteristic is apparent for any of the inferred groups. Different overall patterns of linkage disequilibrium (LD) were observed across chromosomes, with Chr10, Chr17, Chr5, and Chr2 showing the highest LD. This work represents the largest and most comprehensive inter-breeding collection analysis of genomic diversity for cultivated sunflower conducted to date.

Exploring gene networks in two sunflower lines with contrasting leaf senescence phenotype using a system biology approach.

BACKGROUND: Leaf senescence is a complex process, controlled by multiple genetic and environmental variables. In sunflower, leaf senescence is triggered abruptly following anthesis thereby limiting the capacity of plants to keep their green leaf area during grain filling, which subsequently has a strong impact on crop yield. Recently, we performed a selection of contrasting sunflower inbred lines for the progress of leaf senescence through a physiological, cytological and molecular approach. Here we present a large scale transcriptomic analysis using RNA-seq and its integration with metabolic profiles for two contrasting sunflower inbred lines, R453 and B481-6 (early and delayed senescence respectively), with the aim of identifying metabolic pathways associated to leaf senescence. RESULTS: Gene expression profiles revealed a higher number of differentially expressed genes, as well as, higher expression levels in R453, providing evidence for early activation of the senescence program in this line. Metabolic pathways associated with sugars and nutrient recycling were differentially regulated between the lines. Additionally, we identified transcription factors acting as hubs in the co-expression networks; some previously reported as senescence-associated genes in model species but many are novel candidate genes. CONCLUSIONS: Understanding the onset and the progress of the senescence process in crops and the identification of these new candidate genes will likely prove highly useful for different management strategies to mitigate the impact of senescence on crop yield. Functional characterization of candidate genes will help to develop molecular tools for biotechnological applications in breeding crop yield.

Identification and expression analysis of NAC transcription factors potentially involved in leaf and petal senescence in Petunia hybrida.

Progression of leaf senescence depends on several families of transcription factors. In Arabidopsis, the NAC family plays crucial roles in the modulation of leaf senescence; however, the mechanisms involved in this NAC-mediated regulation have not been extensively explored in agronomic species. Petunia hybrida is an ornamental plant that is commonly found worldwide. Decreasing the rate of leaf and petal senescence in P. hybrida is essential for maintaining plant quality. In this study, we examined the NAC-mediated networks involved in regulating senescence in this species. From 41 NAC genes, the expression of which changed in Arabidopsis during leaf senescence, we identified 29 putative orthologs in P. hybrida. Analysis using quantitative real-time-PCR indicated that 24 genes in P. hybrida changed their transcript levels during natural leaf senescence. Leaf-expressed genes were subsequently assessed in petals undergoing natural and pollination-induced senescence. Expression data and phylogenetic analysis were used to generate a list of 10-15 candidate genes; 7 of these were considered key regulatory candidates in senescence because of their consistent upregulation in the three senescence processes examined. Altogether, we identified common and distinct patterns of gene expression at different stages of leaf and petal development and during progression of senescence. The results obtained in this study will contribute to the understanding of NAC-mediated regulatory networks in petunia.

Integration of transcriptomic and metabolic data reveals hub transcription factors involved in drought stress response in sunflower (Helianthus annuus L.).

KEY MESSAGE: By integration of transcriptional and metabolic profiles we identified pathways and hubs transcription factors regulated during drought conditions in sunflower, useful for applications in molecular and/or biotechnological breeding. Drought is one of the most important environmental stresses that effects crop productivity in many agricultural regions. Sunflower is tolerant to drought conditions but the mechanisms involved in this tolerance remain unclear at the molecular level. The aim of this study was to characterize and integrate transcriptional and metabolic pathways related to drought stress in sunflower plants, by using a system biology approach. Our results showed a delay in plant senescence with an increase in the expression level of photosynthesis related genes as well as higher levels of sugars, osmoprotectant amino acids and ionic nutrients under drought conditions. In addition, we identified transcription factors that were upregulated during drought conditions and that may act as hubs in the transcriptional network. Many of these transcription factors belong to families implicated in the drought response in model species. The integration of transcriptomic and metabolomic data in this study, together with physiological measurements, has improved our understanding of the biological responses during droughts and contributes to elucidate the molecular mechanisms involved under this environmental condition. These findings will provide useful biotechnological tools to improve stress tolerance while maintaining crop yield under restricted water availability.

Network and biosignature analysis for the integration of transcriptomic and metabolomic data to characterize leaf senescence process in sunflower.

BACKGROUND: In recent years, high throughput technologies have led to an increase of datasets from omics disciplines allowing the understanding of the complex regulatory networks associated with biological processes. Leaf senescence is a complex mechanism controlled by multiple genetic and environmental variables, which has a strong impact on crop yield. Transcription factors (TFs) are key proteins in the regulation of gene expression, regulating different signaling pathways; their function is crucial for triggering and/or regulating different aspects of the leaf senescence process. The study of TF interactions and their integration with metabolic profiles under different developmental conditions, especially for a non-model organism such as sunflower, will open new insights into the details of gene regulation of leaf senescence. RESULTS: Weighted Gene Correlation Network Analysis (WGCNA) and BioSignature Discoverer (BioSD, Gnosis Data Analysis, Heraklion, Greece) were used to integrate transcriptomic and metabolomic data. WGCNA allowed the detection of 10 metabolites and 13 TFs whereas BioSD allowed the detection of 1 metabolite and 6 TFs as potential biomarkers. The comparative analysis demonstrated that three transcription factors were detected through both methodologies, highlighting them as potentially robust biomarkers associated with leaf senescence in sunflower. CONCLUSIONS: The complementary use of network and BioSignature Discoverer analysis of transcriptomic and metabolomic data provided a useful tool for identifying candidate genes and metabolites which may have a role during the triggering and development of the leaf senescence process. The WGCNA tool allowed us to design and test a hypothetical network in order to infer relationships across selected transcription factor and metabolite candidate biomarkers involved in leaf senescence, whereas BioSignature Discoverer selected transcripts and metabolites which discriminate between different ages of sunflower plants. The methodology presented here would help to elucidate and predict novel networks and potential biomarkers of leaf senescence in sunflower.

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.

Population structure and genetic diversity characterization of a sunflower association mapping population using SSR and SNP markers.

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.

Identification of candidate genes associated with leaf senescence in cultivated sunflower (Helianthus annuus L.).

Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha-NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ethylene insensitive 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important roles as potential triggers of leaf senescence and thus can be considered putative candidate genes for senescence in sunflower.

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes.

Bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and cycling-primed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect C-PRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of chromosomes containing key genes and/or QTL associated to agronomic important traits in sunflower.

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and cycling-primed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect C-PRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of chromosomes containing key genes and/or QTL associated to agronomic important traits in sunflower.

Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance.

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

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 ß-TUB as stable genes, scoring ß-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.

Identification of single nucleotide polymorphisms and analysis of linkage disequilibrium in sunflower elite inbred lines using the candidate gene approach.

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

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

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

Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project.

BACKGROUND: Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. RESULTS: Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes. CONCLUSIONS: The application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects.