RESUMO
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.
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Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.
Assuntos
Ascomicetos/patogenicidade , Resistência à Doença/genética , Genoma de Planta , Helianthus/genética , Doenças das Plantas/genética , Argentina , Mapeamento Cromossômico , Helianthus/imunologia , Helianthus/microbiologia , Fenótipo , Melhoramento Vegetal/métodos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Locos de Características QuantitativasRESUMO
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.
Assuntos
Ascomicetos/genética , Helianthus/microbiologia , Doenças das Plantas/microbiologia , Cruzamento/métodos , Parede Celular/microbiologia , Resistência à Doença , Expressão Gênica/genética , Genótipo , Oxirredução , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
Helianthus/genética , Desequilíbrio de Ligação , Polimorfismo Genético , Banco de Sementes , Cromossomos de Plantas/genética , Melhoramento Vegetal/métodosRESUMO
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.
Assuntos
Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Helianthus/genética , Biologia de Sistemas , Transcriptoma , Genômica , Helianthus/fisiologia , Fenótipo , Folhas de Planta/genética , Folhas de Planta/fisiologia , Especificidade da Espécie , Fatores de TempoRESUMO
Leaf senescence is a complex mechanism controlled by multiple genetic and environmental variables. Different crops present a delay in leaf senescence with an important impact on grain yield trough the maintenance of the photosynthetic leaf area during the reproductive stage. Additionally, because of the temporal gap between the onset and phenotypic detection of the senescence process, candidate genes are key tools to enable the early detection of this process. In this sense and given the importance of some transcription factors as hub genes in senescence pathways, we present a comprehensive review on senescence-associated transcription factors, in model plant species and in agronomic relevant crops. This review will contribute to the knowledge of leaf senescence process in crops, thus providing a valuable tool to assist molecular crop breeding.
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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.
Assuntos
Petunia/genética , Fatores de Transcrição/metabolismo , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Petunia/fisiologia , Filogenia , Melhoramento Vegetal , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polinização , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Sclerotinia Head Rot (SHR), a disease caused by Sclerotinia sclerotiorum, is one of the most limiting factors in sunflower production. In this study, we identified genomic loci associated with resistance to SHR to support the development of assisted breeding strategies. We genotyped 114 Recombinant Inbred Lines (RILs) along with their parental lines (PAC2 -partially resistant-and RHA266 -susceptible-) by using a 384 single nucleotide polymorphism (SNP) Illumina Oligo Pool Assay to saturate a sunflower genetic map. Subsequently, we tested these lines for SHR resistance using assisted inoculations with S. sclerotiorum ascospores. We also conducted a randomized complete-block assays with three replicates to visually score disease incidence (DI), disease severity (DS), disease intensity (DInt) and incubation period (IP) through four field trials (2010-2014). We finally assessed main effect quantitative trait loci (M-QTLs) and epistatic QTLs (E-QTLs) by composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively. As a result of this study, the improved map incorporates 61 new SNPs over candidate genes. We detected a broad range of narrow sense heritability (h2) values (1.86-59.9%) as well as 36 M-QTLs and 13 E-QTLs along 14 linkage groups (LGs). On LG1, LG10, and LG15, we repeatedly detected QTLs across field trials; which emphasizes their putative effectiveness against SHR. In all selected variables, most of the identified QTLs showed high determination coefficients, associated with moderate to high heritability values. Using markers shared with previous Sclerotinia resistance studies, we compared the QTL locations in LG1, LG2, LG8, LG10, LG11, LG15 and LG16. This study constitutes the largest report of QTLs for SHR resistance in sunflower. Further studies focusing on the regions in LG1, LG10, and LG15 harboring the detected QTLs are necessary to identify causal alleles and contribute to unraveling the complex genetic basis governing the resistance.
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Ascomicetos/fisiologia , Resistência à Doença/genética , Epistasia Genética , Helianthus/genética , Helianthus/microbiologia , Doenças das Plantas/microbiologia , Locos de Características Quantitativas/genética , Mapeamento Cromossômico , Ligação Genética , Marcadores Genéticos , Genótipo , Endogamia , Fenótipo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
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.
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Regulação da Expressão Gênica de Plantas/fisiologia , Helianthus/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição/metabolismo , Água/metabolismo , Clorofila/metabolismo , Helianthus/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise Serial de Proteínas , RNA de Plantas/genética , RNA de Plantas/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
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Redes Reguladoras de Genes , Genômica/métodos , Helianthus/genética , Metabolômica/métodos , Regulação da Expressão Gênica de Plantas , Helianthus/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Perfilação da Expressão Gênica/métodos , Helianthus/genética , Helianthus/metabolismo , Metabolômica/métodos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genes de Plantas , Íons , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/genética , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
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Etiquetas de Sequências Expressas , Variação Genética , Genética Populacional , Helianthus/genética , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Argentina , Teorema de Bayes , Análise Multivariada , Melhoramento Vegetal , Polimorfismo GenéticoRESUMO
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.
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Perfilação da Expressão Gênica , Helianthus/crescimento & desenvolvimento , Helianthus/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Metabolismo dos Carboidratos/genética , Clorofila/metabolismo , Genômica , Helianthus/metabolismo , Nitrogênio/metabolismo , Fotossíntese/genética , Folhas de Planta/metabolismo , Solubilidade , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.
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Helianthus/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologiaRESUMO
Background: Calibrachoa Cerv. (ex La Llave & Lexarza) is a genus of the Solanaceae family (La Llave and Lexarza, 1825). This genus has a high ornamental and economic value due to its intrinsic variability and multiplicity of flower colours. In Argentina there are eight native species, and one of them is Calibrachoa caesia. The genetic diversity among 35 accessions of C. caesia, from five departments in the province of Misiones, was analyzed using ISSR markers. Results: Thirteen ISSR primers yielded a reproducible banding pattern, with 701 amplified loci and 98 percent of polymorphism. The ISSR primers 5CT, 5CA, 5GA, 5GACA, 3CAC, 3TG and 3TC generated 100% polymorphic patterns. The Rp values ranged from 23.20 to 10.29 for 5GACA and 3AG primers, respectively, while the average values for MI and PIC were 0.367 and 0.231, respectively. The more informative primers were 5GACA and 5GA, and the less informative was 3AC. Simple matching coefficient of similarity varied from 0.8875 to 0.6659, indicating high levels of genetic similarity among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters; one comprised genotypes of the five departments, while the second included individuals from Guaraní and Oberá regions and the third cluster included the San Pedro individuals. The overall grouping pattern is in agreement with principal coordinate analysis (PCoA). Conclusions: The Bayesian cluster analysis revealed structuring of the C. caesia population and two clusters were identified, which correspond to UPGMA major clades. The AMOVA test for all populations showed highest genetic variation within populations (90 percent), meanwhile the Fst coefficient was 0.098, indicating a medium differentiation between populations. These results showed a great intrapopulation genetic diversity but no significant difference was detected among populations...
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Marcadores Genéticos , Variação Genética , Repetições de Microssatélites , Petunia/genética , Solanaceae/genéticaRESUMO
BACKGROUND: Sclerotinia 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. RESULTS: A 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 %. CONCLUSIONS: These results suggest that AM will be useful in dissecting other complex traits in sunflower, thus providing a valuable tool to assist in crop breeding.
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Ascomicetos/patogenicidade , Mapeamento Cromossômico/métodos , Resistência à Doença/genética , Helianthus/genética , Doenças das Plantas/imunologia , Locos de Características Quantitativas/genética , Sequência de Bases , Brassica napus/genética , Produtos Agrícolas , DNA de Plantas/genética , Genes de Plantas/genética , Genótipo , Helianthus/imunologia , Helianthus/microbiologia , Endogamia , Dados de Sequência Molecular , Fenótipo , Doenças das Plantas/microbiologia , Análise de Sequência de DNARESUMO
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.
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Cromossomos Artificiais Bacterianos/genética , Cromossomos de Plantas , Helianthus/genética , Hibridização in Situ Fluorescente/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Marcadores Genéticos , Locos de Características QuantitativasRESUMO
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.
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Análise de Sequência de DNA/métodos , Cromossomos de Plantas , Cromossomos Artificiais Bacterianos/genética , Helianthus/genética , Hibridização in Situ Fluorescente/métodos , Sequência de Bases , Marcadores Genéticos , Locos de Características QuantitativasRESUMO
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.
Assuntos
Adaptação Fisiológica , Bromus/enzimologia , Congelamento , Hexosiltransferases/metabolismo , Nicotiana/genética , Pichia/genética , Sequência de Bases , Cromatografia por Troca Iônica , Primers do DNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.