Macroarray analysis of gene transcription during sucrose accumulation in sugar beet (Beta vulgaris L.) root: identification of developmental and metabolism related candidate genes

The presented work integrates molecular data on gene expression with anatomical and biochemical data to analyze the development and the sucrose accumulation process in sugar beet (Beta vulgaris L.) roots. Sugar beet is a biennial Chenopodiacean plant, and it is the major crop for sucrose production in temperate regions. A special root morphology and physiology allow the accumulation of sucrose up to 20% of the fresh weight of the mature root. Approaches to study this storage process at the molecular level have so far been limited to known genes involved in pathways related to sugar metabolism which were mapped and tested for their association with QTLs for sugar yield and quality (Schneider et al., 1999, 2002). In the study presented here, transcription levels in sugar beet roots were analyzed to select candidate genes for the sucrose accumulation process. For this purpose macroarrays were generated from two cDNA collections. The first experiment was performed with 3840 redundant sugar beet cDNAs. A procedure for the analysis including control steps was developed. The performance of the macroarrays was evaluated and compared to commercially produced nylon filters. Both systems could detect transcripts present in as little as 10 copies per cell in agreement with reports by Desprez et al. (1998). Their capacity to analyse transcripts of low abundance was demonstrated in a case study using resistance gene analogues (RGAs). Within an interval of two-fold variation in signal intensities, reproducibility between spots on the same filter was determined to be 98.9%, between spots on different filters 89.8%, and reproducibility after hybridization with two probes synthesized from the same poly(A)+RNA sample was 97.6%. Hybridizations with probes synthesized from different field grown samples of the same organ showed reproducibility for 69.7% of the spots on average. Some precautions were introduced to reduce the sampling effects caused by the variability of environmental conditions. Expression profiles from roots, leaves and inflorescences were generated for 2048 unique cDNAs of the first cDNA clone set. Expression values for each organ were determined by stringent statistical analysis based on eight replica for each clone. Differential expression among the three organs was shown for 917 unique cDNAs, and for 76 unique cDNAs, the amount of detected transcript in roots was at least twice as high as in other organs. For 40 of them a map position was identified and linkage to QTLs is discussed. Additionally, possible functions of preferentially root-expressed candidate genes in taproot morphology and physiology are proposed. As a technical validation, macroarray expression data were confirmed by Northern blot analysis and quantitative RT-PCR experiments. The second set of macroarray experiments was performed with 11520 unique cDNA clones to identify candidate genes in sugar beet roots related to sucrose accumulation or development. For this purpose, a time-course experiment was repeated in two different years. Plants were characterized morphologically and metabolically with respect to their sucrose content during the development. Among the genes differentially expressed in the development, 599 clones with highest expression in the early stages of the first vegetation period were identified in both years. For additional 175 clones, a reproducible preferential expression in the last stages of the development was demonstrated. These candidate genes were classified with respect to their function, and their putative role during development and sucrose accumulation is discussed. Additionally, strategies to focus on the validation of candidates related to sucrose accumulation are discussed. In conclusion, the macroarray technology as established here, together with the selection and characterization of appropriate physiological samples, proved to be a valuable tool to identify new candidate genes related to development and to the sucrose accumulation in the sugar beet root. This is of special importance to sugar beet research because the considered processes cannot be analyzed in model systems without a root storage organ for sucrose

The Modest Impact of the Modern Confrontation Clause

The Sixth Amendment\u27s Confrontation Clause grants criminal defendants the right to be confronted with the witnesses against them. A strict reading of this text would transform the criminal justice landscape by prohibiting the prosecution\u27s use of hearsay at trial. But until recently, the Supreme Court\u27s interpretation of the Clause was closer to the opposite. By tying the confrontation right to traditional hearsay exceptions, the Court\u27s longstanding precedents granted prosecutors broad freedom to use out-of-court statements to convict criminal defendants. The Supreme Court\u27s 2004 decision in Crawford v. Washington was supposed to change all that. By severing the link between the Sixth Amendment and the hearsay rules, Crawford ushered in a revolution in the world of evidence and criminal prosecutions. But the excitement did not last. Shifting majorities filled in the details of Crawford\u27s lofty rhetoric, muddying the distinction between the new jurisprudence and what had gone before. This Article takes stock of the Crawford Revolution. First, it explores changes in confrontation doctrine since 2004 and examines, as a theoretical matter, how those changes map onto the state and federal hearsay exceptions that Crawford purportedly rendered irrelevant to constitutional analysis. This interplay between the hearsay rules and the Confrontation Clause is critical. The constitutional right would seem to have little significance if all it does is bar evidence that is already forbidden by nonconstitutional hearsay rules. Second, the Article reports the results of an empirical survey designed to test the theory by carefully cataloguing the hearsay pathways that generated Confrontation Clause challenges in hundreds of federal and state cases. The findings reveal an underappreciated role of the modern confrontation right, and changes to that role after 2004

Genetic approaches to exploit landraces for improvement of Triticum turgidum ssp. durum in the age of climate change

Addressing the challenges of climate change and durum wheat production is becoming an important driver for food and nutrition security in the Mediterranean area, where are located the major producing countries (Italy, Spain, France, Greece, Morocco, Algeria, Tunisia, Turkey, and Syria). One of the emergent strategies, to cope with durum wheat adaptation, is the exploration and exploitation of the existing genetic variability in landrace populations. In this context, this review aims to highlight the important role of durum wheat landraces as a useful genetic resource to improve the sustainability of Mediterranean agroecosystems, with a focus on adaptation to environmental stresses. We described the most recent molecular techniques and statistical approaches suitable for the identification of beneficial genes/alleles related to the most important traits in landraces and the development of molecular markers for marker-assisted selection. Finally, we outline the state of the art about landraces genetic diversity and signature of selection, already identified from these accessions, for adaptability to the environment

Plants expressing murine pro-apoptotic protein Bid do not have enhanced PCD

The purpose of this study was to explore whether plant programmed cell death (PCD) cascade can sense the presence of the animal-only BH3 protein Bid, a BCL-2 family protein known to play a regulatory role in the signaling cascade of animal apoptosis

Selection of candidate genes controlling veraison time in grapevine through integration of meta-QTL and transcriptomic data

Background High temperature during grape berry ripening impairs the quality of fruits and wines. Veraison time, which marks ripening onset, is a key factor for determining climatic conditions during berry ripening. Understanding its genetic control is crucial to successfully breed varieties more adapted to a changing climate. Quantitative trait loci (QTL) studies attempting to elucidate the genetic determinism of developmental stages in grapevine have identified wide genomic regions. Broad scale transcriptomic studies, by identifying sets of genes modulated during berry development and ripening, also highlighted a huge number of putative candidates. Results With the final aim of providing an overview about available information on the genetic control of grapevine veraison time, and prioritizing candidates, we applied a meta-QTL analysis for grapevine phenology-related traits and checked for co-localization of transcriptomic candidates. A consensus genetic map including 3130 markers anchored to the grapevine genome assembly was compiled starting from 39 genetic maps. Two thousand ninety-three QTLs from 47 QTL studies were projected onto the consensus map, providing a comprehensive overview about distribution of available QTLs and revealing extensive co-localization especially across phenology related traits. From 141 phenology related QTLs we generated 4 veraison meta-QTLs located on linkage group (LG) 1 and 2, and 13 additional meta-QTLs connected to the veraison time genetic control, among which the most relevant were located on LG 14, 16 and 18. Functional candidates in these intervals were inspected. Lastly, taking advantage of available transcriptomic datasets, expression data along berry development were integrated, in order to pinpoint among positional candidates, those differentially expressed across the veraison transition. Conclusion Integration of meta-QTLs analysis on available phenology related QTLs and data from transcriptomic dataset allowed to strongly reduce the number of candidate genes for the genetic control of the veraison transition, prioritizing a list of 272 genes, among which 78 involved in regulation of gene expression, signal transduction or development

Combining next-generation pyrosequencing with microarray for large scale expression analysis in non-model species

<p>Abstract</p> <p>Background</p> <p>The next generation sequencing technologies provide new options to characterize the transcriptome and to develop affordable tools for functional genomics. We describe here an innovative approach for this purpose and demonstrate its potential also for non-model species.</p> <p>Results</p> <p>The method we developed is based on 454 sequencing of 3' cDNA fragments from a normalized library constructed from pooled RNAs to generate, through <it>de novo </it>reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence, is not available. This "virtual transcriptome" provides extensive coverage depth, and can be used for the setting up of a comprehensive microarray based expression analysis. We evaluated the potential of this approach by monitoring gene expression during berry maturation in <it>Vitis vinifera </it>as if no other sequence information was available for this species. The microarray designed on the berries' transcriptome derived from half of a 454 run detected the expression of 19,609 genes, and proved to be more informative than one of the most comprehensive grape microarrays available to date, the GrapeArray 1.2 developed by the Italian-French Public Consortium for Grapevine Genome Characterization, which could detect the expression of 15,556 genes in the same samples.</p> <p>Conclusion</p> <p>This approach provides a powerful method to rapidly build up an extensive catalog of unique transcripts that can be successfully used to develop a microarray for large scale analysis of gene expression in any species, without the need for prior sequence knowledge.</p

Genetic approaches to exploit landraces for improvement of Triticum turgidum ssp. durum in the age of climate change

Addressing the challenges of climate change and durum wheat production is becoming an important driver for food and nutrition security in the Mediterranean area, where are located the major producing countries (Italy, Spain, France, Greece, Morocco, Algeria, Tunisia, Turkey, and Syria). One of the emergent strategies, to cope with durum wheat adaptation, is the exploration and exploitation of the existing genetic variability in landrace populations. In this context, this review aims to highlight the important role of durum wheat landraces as a useful genetic resource to improve the sustainability of Mediterranean agroecosystems, with a focus on adaptation to environmental stresses. We described the most recent molecular techniques and statistical approaches suitable for the identification of beneficial genes/alleles related to the most important traits in landraces and the development of molecular markers for marker-assisted selection. Finally, we outline the state of the art about landraces genetic diversity and signature of selection, already identified from these accessions, for adaptability to the environment

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety

<p>Abstract</p> <p>Background</p> <p>Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete <it>Plasmopara viticola</it>, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and <it>P. viticola</it>.</p> <p>Results</p> <p>In this paper we describe the emergence of a <it>P. viticola </it>isolate (isolate SL) that specifically overcomes <it>Rpv3</it>, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two <it>P. viticola </it>isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related.</p> <p>Conclusions</p> <p>Our results are the first description of a resistance-breaking isolate in the grapevine/<it>P. viticola </it>interaction, and show that, despite the reduced genetic variability of <it>P. viticola </it>in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances.</p

Functional genomic analysis of constitutive and inducible defense responses to Fusarium verticillioides infection in maize genotypes with contrasting ear rot resistance.

Background: Fusarium verticillioides causes ear rot in maize (Zea mays L.) and accumulation of mycotoxins, that affect human and animal health. Currently, chemical and agronomic measures to control Fusarium ear rot are not very effective and selection of more resistant genotypes is a desirable strategy to reduce contaminations. A deeper knowledge of molecular events and genetic basis underlying Fusarium ear rot is necessary to speed up progress in breeding for resistance. Results: A next-generation RNA-sequencing approach was used for the first time to study transcriptional changes associated with F. verticillioides inoculation in resistant CO441 and susceptible CO354 maize genotypes at 72 hours post inoculation. More than 100 million sequence reads were generated for inoculated and uninoculated control plants and analyzed to measure gene expression levels. Comparison of expression levels between inoculated vs. uninoculated and resistant vs. susceptible transcriptomes revealed a total number of 6,951 differentially expressed genes. Differences in basal gene expression were observed in the uninoculated samples. CO441 genotype showed a higher level of expression of genes distributed over all functional classes, in particular those related to secondary metabolism category. After F. verticillioides inoculation, a similar response was observed in both genotypes, although the magnitude of induction was much greater in the resistant genotype. This response included higher activation of genes involved in pathogen perception, signaling and defense, including WRKY transcription factors and jasmonate/ ethylene mediated defense responses. Interestingly, strong differences in expression between the two genotypes were observed in secondary metabolism category: pathways related to shikimate, lignin, flavonoid and terpenoid biosynthesis were strongly represented and induced in the CO441 genotype, indicating that selection to enhance these traits is an additional strategy for improving resistance against F. verticillioides infection. Conclusions: The work demonstrates that the global transcriptional analysis provided an exhaustive view of genes involved in pathogen recognition and signaling, and controlling activities of different TFs, phytohormones and secondary metabolites, that contribute to host resistance against F. verticillioides. This work provides an important source of markers for development of disease resistance maize genotypes andmay have relevance to study other pathosystems involving mycotoxin-producing fungi

Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed