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1.
Int J Cancer ; 146(2): 521-530, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31403184

RESUMO

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , MicroRNAs/metabolismo , Neoplasias da Glândula Tireoide/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Processamento Alternativo/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Intervalo Livre de Doença , Feminino , Seguimentos , Perfilação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais/genética , Taxa de Sobrevida , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/mortalidade , Neoplasias da Glândula Tireoide/patologia
2.
Theranostics ; 9(17): 4946-4958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410193

RESUMO

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.

3.
Bioengineering (Basel) ; 6(3)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349743

RESUMO

Computational tools are essential in the process of designing a CRISPR/Cas experiment for the targeted modification of an organism's genome. Among other functionalities, these tools facilitate the design of a guide-RNA (gRNA) for a given nuclease that maximizes its binding to the intended genomic site, while avoiding binding to undesired sites with similar sequences in the genome of the organism of interest (off-targets). Due to the popularity of this methodology and the rapid pace at which it evolves and changes, new computational tools show up constantly. This rapid turnover, together with the intrinsic high death-rate of bioinformatics tools, mean that many of the published tools become unavailable at some point. Consequently, the traditional ways to inform the community about the landscape of available tools, i.e., reviews in the scientific literature, are not adequate for this fast-moving field. To overcome these limitations, we have developed "WeReview: CRISPR Tools," a live, on-line, user-updatable repository of computational tools to assist researchers in designing CRISPR/Cas experiments. In its web site users can find an updated comprehensive list of tools and search for those fulfilling their specific needs, as well as proposing modifications to the data associated with the tools or the incorporation of new ones.

5.
Am J Hum Genet ; 104(4): 651-664, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929736

RESUMO

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.


Assuntos
Aciltransferases/genética , Neoplasias das Glândulas Suprarrenais/genética , Mutação em Linhagem Germinativa , Paraganglioma/genética , Feocromocitoma/genética , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese , Domínio Catalítico , Ciclo do Ácido Cítrico , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade
6.
Plant Physiol ; 177(3): 1234-1253, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29853599

RESUMO

Seedlessness is greatly prized by consumers of fresh grapes. While stenospermocarpic seed abortion determined by the SEED DEVELOPMENT INHIBITOR (SDI) locus is the usual source of seedlessness in commercial grapevine (Vitis vinifera) cultivars, the underlying sdi mutation remains unknown. Here, we undertook an integrative approach to identify the causal mutation. Quantitative genetics and fine-mapping in two 'Crimson Seedless'-derived F1 mapping populations confirmed the major effect of the SDI locus and delimited the sdi mutation to a 323-kb region on chromosome 18. RNA-sequencing comparing seed traces of seedless and seeds of seeded F1 individuals identified processes triggered during sdi-determined seed abortion, including the activation of salicylic acid-dependent autoimmunity. The RNA-sequencing data set was investigated for candidate genes, and while no evidence for causal cis-acting regulatory mutations was detected, deleterious nucleotide changes in coding sequences of the seedless haplotype were predicted in two genes within the sdi fine-mapping interval. Targeted resequencing of the two genes in a collection of 124 grapevine cultivars showed that only the point variation causing the arginine-197-to-leucine substitution in the seed morphogenesis regulator gene AGAMOUS-LIKE11 (VviAGL11) was fully linked with stenospermocarpy. The concurrent postzygotic variation identified for this missense polymorphism and seedlessness phenotype in seeded somatic variants of the original stenospermocarpic cultivar supports a causal effect. We postulate that seed abortion caused by this amino acid substitution in VviAGL11 is the major cause of seedlessness in cultivated grapevine. This information can be exploited to boost seedless grape breeding.


Assuntos
Proteínas de Domínio MADS/genética , Mutação de Sentido Incorreto , Proteínas de Plantas/genética , Sementes/genética , Vitis/fisiologia , Substituição de Aminoácidos , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Locos de Características Quantitativas , Ácido Salicílico/metabolismo , Sementes/crescimento & desenvolvimento , Vitis/genética
7.
Genet Med ; 20(12): 1644-1651, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29740169

RESUMO

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , DNA (Citosina-5-)-Metiltransferases/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Sistemas CRISPR-Cas/genética , Metilação de DNA , Feminino , Mutação com Ganho de Função , Predisposição Genética para Doença , Genótipo , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Paraganglioma/patologia , Feocromocitoma/patologia , Sequenciamento Completo do Exoma
8.
Plant Physiol ; 175(2): 786-801, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28811336

RESUMO

Grape (Vitis vinifera) color somatic variants that can be used to develop new grapevine cultivars occasionally appear associated with deletion events of uncertain origin. To understand the mutational mechanisms generating somatic structural variation in grapevine, we compared the Tempranillo Blanco (TB) white berry somatic variant with its black berry ancestor, Tempranillo Tinto. Whole-genome sequencing uncovered a catastrophic genome rearrangement in TB that caused the hemizygous deletion of 313 genes, including the loss of the functional copy for the MYB transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species.


Assuntos
Antocianinas/metabolismo , Rearranjo Gênico , Genoma de Planta/genética , Perda de Heterozigosidade/genética , Vitis/genética , Cor , Frutas/genética , Frutas/fisiologia , Ligação Genética , Mutação , Fenótipo , Pigmentação , Vitis/fisiologia
9.
Clin Cancer Res ; 23(20): 6315-6324, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28720665

RESUMO

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR.


Assuntos
Ciclo do Ácido Cítrico/genética , Exoma , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Paraganglioma/genética , Feocromocitoma/genética , Análise por Conglomerados , Metilação de DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Humanos , Metaboloma , Metabolômica/métodos , Paraganglioma/metabolismo , Feocromocitoma/metabolismo
10.
J Mol Diagn ; 19(4): 575-588, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28552549

RESUMO

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/diagnóstico , Adulto , Análise Mutacional de DNA/métodos , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico
11.
Int J Food Microbiol ; 241: 60-68, 2017 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-27756034

RESUMO

Advances in microbial wine biotechnology have led to the recent commercialization of several non-Saccharomyces starter cultures. These are intended to be used in either simultaneous or sequential inoculation with Saccharomyces cerevisiae. The different types of microbial interactions that can be stablished during wine fermentation acquire an increased relevance in the context of these mixed-starter fermentations. We analysed the transcriptional response to co-cultivation of S. cerevisiae and Torulaspora delbrueckii. The study focused in the initial stages of wine fermentation, before S. cerevisiae completely dominates the mixed cultures. Both species showed a clear response to the presence of each other, even though the portion of the genome showing altered transcription levels was relatively small. Changes in the transcription pattern suggested a stimulation of metabolic activity and growth, as a consequence of the presence of competitors in the same medium. The response of S. cerevisiae seems to take place earlier, as compared to T. delbrueckii. Enhanced glycolytic activity of the mixed culture was confirmed by the CO2 production profile during these early stages of fermentation. Interestingly, HSP12 expression appeared induced by co-cultivation for both of S. cerevisiae and Torulaspora delbrueckii in the two time points studied. This might be related with a recently described role of Hsp12 in intercellular communication in yeast. Expression of S. cerevisiae PAU genes was also stimulated in mixed cultures.


Assuntos
Fermentação , Microbiologia de Alimentos , Saccharomyces cerevisiae/metabolismo , Torulaspora/metabolismo , Vinho/análise , Vinho/microbiologia , Dióxido de Carbono/química , Técnicas de Cocultura , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Glicólise , Proteínas de Choque Térmico/química , Proteínas de Saccharomyces cerevisiae/química , Análise de Sequência de RNA , Especificidade da Espécie , Transcrição Genética , Transcriptoma , Fermento Seco/metabolismo
12.
Front Microbiol ; 7: 1545, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27733850

RESUMO

Adaptation to changes in osmolarity is fundamental for the survival of living cells, and has implications in food and industrial biotechnology. It has been extensively studied in the yeast Saccharomyces cerevisiae, where the Hog1 stress activated protein kinase was discovered about 20 years ago. Hog1 is the core of the intracellular signaling pathway that governs the adaptive response to osmotic stress in this species. The main endpoint of this program is synthesis and intracellular retention of glycerol, as a compatible osmolyte. Despite many details of the signaling pathways and yeast responses to osmotic challenges have already been described, genome-wide approaches are contributing to refine our knowledge of yeast adaptation to hypertonic media. In this work, we used a quantitative fitness analysis approach in order to deepen our understanding of the interplay between yeast cells and the osmotic environment. Genetic requirements for proper growth under osmotic stress showed both common and specific features when hypertonic conditions were induced by either glucose or sorbitol. Tolerance to high-glucose content requires mitochondrial function, while defective protein targeting to peroxisome, GID-complex function (involved in negative regulation of gluconeogenesis), or chromatin dynamics, result in poor survival to sorbitol-induced osmotic stress. On the other side, the competitive disadvantage of yeast strains defective in the endomembrane system is relieved by hypertonic conditions. This finding points to the Golgi-endosome system as one of the main cell components negatively affected by hyperosmolarity. Most of the biological processes highlighted in this analysis had not been previously related to osmotic stress but are probably relevant in an ecological and evolutionary context.

13.
BMC Genomics ; 17: 74, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26801623

RESUMO

BACKGROUND: The two-spotted spider mite, Tetranychus urticae, is an extreme generalist plant pest. Even though mites can feed on many plant species, local mite populations form host races that do not perform equally well on all potential hosts. An acquisition of the ability to evade plant defenses is fundamental for mite's ability to use a particular plant as a host. Thus, understanding the interactions between the plant and mites with different host adaptation status allows the identification of functional plant defenses and ways mites can evolve to avoid them. RESULTS: The grapevine genome-wide transcriptional responses to spider mite strains that are non-adapted and adapted to grapevine as a host were examined. Comparative transcriptome analysis of grapevine responses to these mite strains identified the existence of weak responses induced by the feeding of the non-adapted strain. In contrast, strong but ineffective induced defenses were triggered upon feeding of the adapted strain. A comparative meta-analysis of Arabidopsis, tomato and grapevine responses to mite feeding identified a core of 36 highly conserved genes involved in the perception, regulation and metabolism that were commonly induced in all three species by mite herbivory. CONCLUSIONS: This study describes the genome-wide grapevine transcriptional responses to herbivory of mite strains that differ in their ability to use grapevine as a host. It raises hypotheses whose testing will lead to our understanding of grapevine defenses and mite adaptations to them.


Assuntos
Regulação da Expressão Gênica de Plantas , Tetranychidae/fisiologia , Transcriptoma/genética , Vitis/genética , Vitis/parasitologia , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/parasitologia
14.
J Exp Bot ; 67(1): 259-73, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26454283

RESUMO

Seedlessness is a relevant trait in grapevine cultivars intended for fresh consumption or raisin production. Previous DNA marker analysis indicated that Corinto bianco (CB) is a parthenocarpic somatic variant of the seeded cultivar Pedro Ximenes (PX). This study compared both variant lines to determine the basis of this parthenocarpic phenotype. At maturity, CB seedless berries were 6-fold smaller than PX berries. The macrogametophyte was absent from CB ovules, and CB was also pollen sterile. Occasionally, one seed developed in 1.6% of CB berries. Microsatellite genotyping and flow cytometry analyses of seedlings generated from these seeds showed that most CB viable seeds were formed by fertilization of unreduced gametes generated by meiotic diplospory, a process that has not been described previously in grapevine. Microarray and RNA-sequencing analyses identified 1958 genes that were differentially expressed between CB and PX developing flowers. Genes downregulated in CB were enriched in gametophyte-preferentially expressed transcripts, indicating the absence of regular post-meiotic germline development in CB. RNA-sequencing was also used for genetic variant calling and 14 single-nucleotide polymorphisms distinguishing the CB and PX variant lines were detected. Among these, CB-specific polymorphisms were considered as candidate parthenocarpy-responsible mutations, including a putative deleterious substitution in a HAL2-like protein. Collectively, these results revealed that the absence of a mature macrogametophyte, probably due to meiosis arrest, coupled with a process of fertilization-independent fruit growth, caused parthenocarpy in CB. This study provides a number of grapevine parthenocarpy-responsible candidate genes and shows how genomic approaches can shed light on the genetic origin of woody crop somatic variants.


Assuntos
Flores/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Proteínas de Plantas/genética , Transcriptoma , Vitis/genética , Flores/genética , Frutas/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Análise de Sequência de RNA , Vitis/crescimento & desenvolvimento , Vitis/metabolismo
15.
Theor Appl Genet ; 129(2): 227-42, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26536891

RESUMO

KEY MESSAGE: A set of SNP markers associated to bunch compactness and related traits were identified in grapevine. ABSTRACT: Bunch compactness plays an important role in the sanitary status and perceived quality of table and wine grapes, being influenced by cultural practices and by environmental and genetic factors, which are mostly unknown. In this work, we took advantage of genetic, genomic and bioinformatic advances to analyze part of its molecular basis through a combination of transcriptomic and association analyses. Results from different transcriptomic comparisons between loose and compact grapevine clones were analyzed to select a set of candidate genes likely involved in the observed variation for bunch compactness. Up to 183 genes were sequenced in a grapevine collection, and 7032 single nucleotide polymorphisms (SNPs) were detected in more than 100 varieties with a frequency of the minor allele over 5%. They were used to test their association in three consecutive seasons with bunch compactness and two of its most influencing factors: total berry number and length of the first ramification of the rachis. Only one SNP was associated with berry number in two seasons, suggesting the high sensitiveness of this trait to seasonal environmental changes. On the other hand, we found a set of SNPs associated with both the first ramification length and bunch compactness in various seasons, in several genes which had not previously related to bunch compactness or bunch compactness-related traits. They are proposed as interesting candidates for further functional analyses aimed to verify the results obtained in this work, as a previous step to their inclusion in marker-assisted selection strategies.


Assuntos
Frutas/crescimento & desenvolvimento , Genes de Plantas , Polimorfismo de Nucleotídeo Único , Vitis/genética , Alelos , DNA de Plantas/genética , Modelos Genéticos , Fenótipo , Característica Quantitativa Herdável , Análise de Sequência de DNA , Transcriptoma
16.
BMC Plant Biol ; 15: 253, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26499326

RESUMO

BACKGROUND: Domestication and selection of Vitis vinifera L. for table and wine grapes has led to a large level of berry size diversity in current grapevine cultivars. Identifying the genetic basis for this natural variation is paramount both for breeding programs and for elucidating which genes contributed to crop evolution during domestication and selection processes. The gene VvNAC26, which encodes a NAC domain-containing transcription factor, has been related to the early development of grapevine flowers and berries. It was selected as candidate gene for an association study to elucidate its possible participation in the natural variation of reproductive traits in cultivated grapevine. METHODS: A grapevine collection of 114 varieties was characterized during three consecutive seasons for different berry and bunch traits. The promoter and coding regions of VvNAC26 gene (VIT_01s0026g02710) were sequenced in all the varieties of the collection, and the existing polymorphisms (SNP and INDEL) were detected. The corresponding haplotypes were inferred and used for a phylogenetic analysis. The possible associations between genotypic and phenotypic data were analyzed independently for each season data, using different models and significance thresholds. RESULTS: A total of 30 non-rare polymorphisms were detected in the VvNAC26 sequence, and 26 different haplotypes were inferred. Phylogenetic analysis revealed their clustering in two major haplogroups with marked phenotypic differences in berry size between varieties harboring haplogroup-specific alleles. After correcting the statistical models for the effect of the population genetic stratification, we found a set of polymorphisms associated with berry size explaining between 8.4 and 21.7% (R(2)) of trait variance, including those generating the differentiation between both haplogroups. Haplotypes built from only three polymorphisms (minihaplotypes) were also associated with this trait (R(2): 17.5 - 26.6%), supporting the involvement of this gene in the natural variation for berry size. CONCLUSIONS: Our results suggest the participation of VvNAC26 in the determination of the grape berry final size. Different VvNAC26 polymorphisms and their combination showed to be associated with different features of the fruit. The phylogenetic relationships between the VvNAC26 haplotypes and the association results indicate that this nucleotide variation may have contributed to the differentiation between table and wine grapes.


Assuntos
Frutas/anatomia & histologia , Frutas/genética , Genes de Plantas , Estudos de Associação Genética , Haplótipos/genética , Polimorfismo de Nucleotídeo Único/genética , Vitis/genética , Sequência de Bases , Cloroplastos/genética , Marcadores Genéticos , Genética Populacional , Dados de Sequência Molecular , Tamanho do Órgão , Fenótipo , Filogenia
17.
Bioessays ; 37(3): 237-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25545039

RESUMO

Nowadays, in the Internet databases era, certain knowledge is being progressively lost. This knowledge, which we feel is essential and should be acquired through education, is the understanding of how the pioneer researchers faced major questions in their field and made their discoveries.


Assuntos
Biologia do Desenvolvimento/educação , Animais , Bibliometria , Caenorhabditis elegans/citologia , Linhagem da Célula , Biologia do Desenvolvimento/história , História do Século XX , História do Século XXI , Humanos
18.
Plant Cell Physiol ; 54(7): 1200-16, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23659918

RESUMO

Berry organoleptic properties are highly influenced by ripening environmental conditions. In this study, we used grapevine fruiting cuttings to follow berry ripening under different controlled conditions of temperature and irradiation intensity. Berries ripened at higher temperatures showed reduced anthocyanin accumulation and hastened ripening, leading to a characteristic drop in malic acid and total acidity. The GrapeGen GeneChip® combined with a newly developed GrapeGen 12Xv1 MapMan version were utilized for the functional analysis of berry transcriptomic differences after 2 week treatments from veraison onset. These analyses revealed the establishment of a thermotolerance response in berries under high temperatures marked by the induction of heat shock protein (HSP) chaperones and the repression of transmembrane transporter-encoding transcripts. The thermotolerance response was coincident with up-regulation of ERF subfamily transcription factors and increased ABA levels, suggesting their participation in the maintenance of the acclimation response. Lower expression of amino acid transporter-encoding transcripts at high temperature correlated with balanced amino acid content, suggesting a transcriptional compensation of temperature effects on protein and membrane stability to allow for completion of berry ripening. In contrast, the lower accumulation of anthocyanins and higher malate metabolization measured under high temperature might partly result from imbalance in the expression and function of their specific transmembrane transporters and expression changes in genes involved in their metabolic pathways. These results open up new views to improve our understanding of berry ripening under high temperatures.


Assuntos
Adaptação Fisiológica/genética , Frutas/genética , Temperatura Ambiente , Vitis/genética , Ácido Abscísico/metabolismo , Aminoácidos/metabolismo , Antocianinas/metabolismo , Proteínas de Ligação a DNA/genética , Frutas/metabolismo , Frutas/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Luz , Malatos/metabolismo , Redes e Vias Metabólicas/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transcriptoma , Vitis/metabolismo , Vitis/fisiologia
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