A Rapid and Efficient Method for Purifying High Quality Total RNA from Peaches (Prunus persica) for Functional Genomics Analyses

http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602005000100010&lng=es&nrm=isoPrunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries

JUICE: a data management system that facilitates the analysis of large volumes of information in an EST project workflow

BACKGROUND: Expressed sequence tag (EST) analyses provide a rapid and economical means to identify candidate genes that may be involved in a particular biological process. These ESTs are useful in many Functional Genomics studies. However, the large quantity and complexity of the data generated during an EST sequencing project can make the analysis of this information a daunting task. RESULTS: In an attempt to make this task friendlier, we have developed JUICE, an open source data management system (Apache + PHP + MySQL on Linux), which enables the user to easily upload, organize, visualize and search the different types of data generated in an EST project pipeline. In contrast to other systems, the JUICE data management system allows a branched pipeline to be established, modified and expanded, during the course of an EST project. The web interfaces and tools in JUICE enable the users to visualize the information in a graphical, user-friendly manner. The user may browse or search for sequences and/or sequence information within all the branches of the pipeline. The user can search using terms associated with the sequence name, annotation or other characteristics stored in JUICE and associated with sequences or sequence groups. Groups of sequences can be created by the user, stored in a clipboard and/or downloaded for further analyses. Different user profiles restrict the access of each user depending upon their role in the project. The user may have access exclusively to visualize sequence information, access to annotate sequences and sequence information, or administrative access. CONCLUSION: JUICE is an open source data management system that has been developed to aid users in organizing and analyzing the large amount of data generated in an EST Project workflow. JUICE has been used in one of the first functional genomics projects in Chile, entitled "Functional Genomics in nectarines: Platform to potentiate the competitiveness of Chile in fruit exportation". However, due to its ability to organize and visualize data from external pipelines, JUICE is a flexible data management system that should be useful for other EST/Genome projects. The JUICE data management system is released under the Open Source GNU Lesser General Public License (LGPL). JUICE may be downloaded from or

Comparative EST transcript profiling of peach fruits under different post-harvest conditions reveals candidate genes associated with peach fruit quality

<p>Abstract</p> <p>Background</p> <p>Cold storage is used to inhibit peach fruit ripening during shipment to distant markets. However, this cold storage can negatively affect the quality of the fruit when it is ripened, resulting in disorders such as wooliness, browning or leathering. In order to understand the individual and combined biological effects that factors such as cold storage and ripening have on the fruit and fruit quality, we have taken a comparative EST transcript profiling approach to identify genes that are differentially expressed in response to these factors.</p> <p>Results</p> <p>We sequenced 50,625 Expressed Sequence Tags (ESTs) from peach mesocarp <it>(Prunus persica </it>O'Henry variety) stored at four different postharvest conditions. A total of 10,830 Unigenes (4,169 contigs and 6,661 singletons) were formed by assembling these ESTs. Additionally, a collection of 614 full-length and 1,109 putative full-length cDNA clones within flanking loxP recombination sites was created.</p> <p>Statistically analyzing the EST population, we have identified genes that are differentially expressed during ripening, in response to cold storage or the combined effects of cold storage and ripening. Pair-wise comparisons revealed 197 contigs with at least one significant difference in transcript abundance between at least two conditions. Gene expression profile analyses revealed that the contigs may be classified into 13 different clusters of gene expression patterns. These clusters include groups of contigs that increase or decrease transcript abundance during ripening, in response to cold or ripening plus cold.</p> <p>Conclusion</p> <p>These analyses have enabled us to statistically identify novel genes and gene clusters that are differentially expressed in response to post-harvest factors such as long-term cold storage, ripening or a combination of these two factors. These differentially expressed genes reveal the complex biological processes that are associated with these factors, as well as a large number of putative gene families that may participate differentially in these processes. In particular, these analyzes suggest that woolly fruits lack the increased boost of metabolic processes necessary for ripening. Additionally, these results suggest that the mitochondria and plastids play a major role in these processes. The EST sequences and full-length cDNA clones developed in this work, combined with the large population of differentially expressed genes may serve as useful tools and markers that will enable the scientific community to better define the molecular processes that affect fruit quality in response to post-harvest conditions and the organelles that participate in these processes.</p

Analysis of Piscirickettsia salmonis Metabolism Using Genome-Scale Reconstruction, Modeling, and Testing

International audiencePiscirickettsia salmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with highly adverse impact in the Chilean salmon farming industry. The development of effective treatment and control methods for piscireckttsiosis is still a challenge. To meet it the number of studies on P. salmonis has grown in the last couple of years but many aspects of the pathogen's biology are still poorly understood. Studies on its metabolism are scarce and only recently a metabolic model for reference strain LF-89 was developed. We present a new genome-scale model for P. salmonis LF-89 with more than twice as many genes as in the previous model and incorporating specific elements of the fish pathogen metabolism. Comparative analysis with models of different bacterial pathogens revealed a lower flexibility in P. salmonis metabolic network. Through constraint-based analysis, we determined essential metabolites required for its growth and showed that it can benefit from different carbon sources tested experimentally in new defined media. We also built an additional model for strain A1-15972, and together with an analysis of P. salmonis pangenome, we identified metabolic features that differentiate two main species clades. Both models constitute a knowledge-base for P. salmonis metabolism and can be used to guide the efficient culture of the pathogen and the identification of specific drug targets

Bacterial communities associated to Chilean altiplanic native plants from the Andean grasslands soils

The rhizosphere is considered the primary place for soil microbiome differentiation and plays a key role in plant survival, especially for those subjected to environmental stress. Using high-throughput sequencing of the 16S rRNA gene, we analyzed and compared soil bacterial communities associated to four of the most abundant high altitude native plant species of the Chilean Andean grasslands. We examined three soil compartments: the rhizosphere (bacteria firmly attached to the roots), the rhizosphere-surrounding soil (bacteria loosely attached to the roots) and the bulk soil (plant-free soil). The rhizosphere microbiome was in all cases the least diverse, exposing that the bulk soil was a more complex environment. Taxonomic analysis revealed an abrupt change between the rhizosphere and the rest of the non-rhizospheric soils. Thus, while rhizobacterial communities were enriched in Proteobacteria (mainly Alphaproteobacteria), Actinobacteria (mostly Blastocatellia

Genome-scale metabolic models of Microbacterium species isolated from a high altitude desert environment

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Structure and co-occurrence patterns in microbial communities under acute environmental stress reveal ecological factors fostering resilience.

International audienceUnderstanding the factors that modulate bacterial community assembly in natural soils is a longstanding challenge in microbial community ecology. In this work, we compared two microbial co-occurrence networks representing bacterial soil communities from two different sections of a pH, temperature and humidity gradient occurring along a western slope of the Andes in the Atacama Desert. In doing so, a topological graph alignment of co-occurrence networks was used to determine the impact of a shift in environmental variables on OTUs taxonomic composition and their relationships. We observed that a fraction of association patterns identified in the co-occurrence networks are persistent despite large environmental variation. This apparent resilience seems to be due to: (1) a proportion of OTUs that persist across the gradient and maintain similar association patterns within the community and (2) bacterial community ecological rearrangements, where an important fraction of the OTUs come to fill the ecological roles of other OTUs in the other network. Actually, potential functional features suggest a fundamental role of persistent OTUs along the soil gradient involving nitrogen fixation. Our results allow identifying factors that induce changes in microbial assemblage configuration, altering specific bacterial soil functions and interactions within the microbial communities in natural environments