Computational protocol: Expression dynamics and genome distribution of osmoprotectants in soybean: identifying important components to face abiotic stress

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Protocol publication

[…] The selection of seed sequences (Additional file ) was based in a literature search in the PubMed database [] using the key words "Osmoprotectants" AND "Plant Stress". In the selected articles the NCBI [] descriptors for posterior mining were selected and retrieved from the Uniprot SwissProt (cutoff e-10) using BLASTx. In order to confirm their involvement in the biosynthesis of osmoprotectants (proline, trehalose, Myo-inositol and glycine betaine) the sequences were aligned (BLASTx, cutoff e-10) against the soybean peptide database at Phytozome v. 8.0 [], also allowing the identification of the respective transcripts from soybean transcriptome used to associate with the available SuperSAGE tags.Biological material, experimental design and stress application - Soybean HT-SuperSAGE libraries were generated according to the procedures described by Matsumura et al. [] at GenXPro GmbH, with posterior SOLEXA sequencing of the tags. The generated tags are distributed into four libraries (Additional file ) including root tissues subjected to dehydration: two libraries from the drought tolerant cultivar Embrapa 48 [Tolerant after stress (TS) and negative control (TC)] and two libraries from a drought sensible cultivar BR-16 [Sensible after Stress (SS) and negative control (SC)]. The conditions for the generation of the mentioned libraries, time frame experiments, and laboratory protocols used are described in Soares-Cavalcanti et al. []. The generated sequences are available at the GENOSOJA database (Brazilian Soybean Genome Consortium) [].Statistical analysis, tag-gene annotation and the tag fold change estimation - The in silico procedures are illustrated in Figure . Initially 26 bp-tags were analyzed with the DiscoverySpace (v.4.01) software [] aiming to identify unique tags (unitags) and those unitags differentially expressed (p ≤ 0.05) considering a contrast among two libraries. Tags counted only once (singlets) were excluded from the present evaluation. Unitags were annotated by BLASTn [] against nucleotide sequences from the soybean Phytozome database v8.0 (Glyma1 cDNA dataset) [,]. BLASTn alignments (tag-hit) with e-values of 0.0001 or less and tolerating a single mismatch maximum (TSM) were taken into account. Moreover, only plus/plus alignments without mismatches regarding the four first bases CATG were accepted, in order to guarantee the integrity of the SuperSAGE tag. Specific keyword searches on the original glyma annotations were performed looking for the transcripts and tags candidates. Values reflecting expression data (p-value and up- or down-regulation regarding each tag) were associated to the data matrix including the respective tag annotation, the normalized frequencies in the libraries and the fold change values (FC). FC estimative were based on the ratio (R) of the normalized frequencies of the tag in the contrast of the two libraries, where the 'zero' frequency was replaced by 'one'. When R > 1 the FC were directly considered and when R < 1 the FC = - 1/R. Negative FC values indicated repressed tags. [...] Sequence matches for the nine selected osmoprotectant-related genes were aligned against the SoyBase pseudochromosomes aiming to infer about their distribution in the virtual chromosomes available at SoyBase. BLAST algorithm parameters (score, e-value and percentage of identity) were adjusted to allow the anchoring of soybean sequences position along the soybean virtual chromosomes. Afterwards the identified anchoring positions were submitted to the Circos program [] and so edited to generate a picture of higher resolution. This approach allowed the generation of a graph based on a circular organization of the soybean chromosomes (n = 20), allowing the identification of a virtual ideogram with linear distribution of the osmoprotectants identified, the associated SuperSAGE tags, as well as redundant portions. […]

Pipeline specifications

Software tools BLASTX, DiscoverySpace, BLASTN, Circos
Databases SoyBase Phytozome
Applications Miscellaneous, Genome data visualization
Organisms Glycine max, Arabidopsis thaliana
Chemicals Inositol