Statistics of the Trinity assembly after clustering.

<p>Statistics of the Trinity assembly after clustering.</p

Mapping of high expressed unigenes on MapMan pathways.

<p>The genes exhibiting high expression (≥100 normalized RPKM values) were mapped on functional bins assigned to different pathways in MapMan. Blue squares represent high expressed genes. Grey circles represent genes not exhibiting high expression (≥100 normalized RPKM) in our dataset.</p

Summary of read statistics from RNA sequencing of <i>B</i>. <i>juncea</i> var. CS52.

<p>Summary of read statistics from RNA sequencing of <i>B</i>. <i>juncea</i> var. CS52.</p

Mapping results of predicted unigenes on A) <i>B</i>. <i>rapa</i> transcripts and B) ESTs available for <i>B</i>. <i>rapa</i>, <i>B</i>. <i>nigra</i> and <i>B</i>. <i>juncea</i>.

<p>A) Pie chart showing proportion of total unigenes mapped on <i>B</i>. <i>rapa</i> transcripts and those which could not be mapped (non-rapa). B) Results of EST mapping on total unigenes, <i>B</i>. <i>rapa</i> mapped unigenes and non-rapa unigenes are presented in the form of bar graph.</p

MapMan depiction of gene regulation in functional categories associated with different pathways.

<p>Distribution of unigenes into different pathways, generated in MapMan, are illustrated. Log₂ fold changes are indicated by the color scale, red squares represent up regulated genes and green squares represent down regulated genes. The grey circles represent genes not differentially expressed in our study.</p

<i>De Novo</i> Assembly and Characterization of Stress Transcriptome in a Salinity-Tolerant Variety CS52 of <i>Brassica juncea</i>

<div><p>Oilseed mustard, <i>Brassica juncea</i>, exhibits high levels of genetic variability for salinity tolerance. To obtain the global view of transcriptome and investigate the molecular basis of salinity tolerance in a salt-tolerant variety CS52 of <i>B</i>. <i>juncea</i>, we performed transcriptome sequencing of control and salt-stressed seedlings. <i>De novo</i> assembly of 184 million high-quality paired-end reads yielded 42,327 unique transcripts longer than 300 bp with RPKM ≥1. When compared with non-redundant proteins, we could annotate 67% unigenes obtained in our study. Based on the mapping to expressed sequence tags (ESTs), 52.6% unigenes are novel compared to EST data available for <i>B</i>. <i>juncea</i> and constituent genomes. Differential expression analysis revealed altered expression of 1469 unigenes in response to salinity stress. Of these, 587, mainly associated with ROS detoxification, sulfur assimilation and calcium signaling pathways, are up regulated. Notable of these is <i>RSA1 (SHORT ROOT IN SALT MEDIUM 1) INTERACTING TRANSCRIPTION FACTOR 1 </i>(<i>RITF1</i>) homolog up regulated by >100 folds in response to stress. <i>RITF1</i>, encoding a bHLH transcription factor, is a positive regulator of <i>SOS1</i> and several key genes involved in scavenging of salt stress-induced reactive oxygen species (ROS). Further, we performed comparative expression profiling of key genes implicated in ion homeostasis and sequestration (<i>SOS1</i>, <i>SOS2</i>, <i>SOS3</i>, <i>ENH1</i>, <i>NHX1</i>), calcium sensing pathway (<i>RITF1</i>) and ROS detoxification in contrasting cultivars for salinity tolerance, <i>B</i>. <i>juncea</i> and <i>B</i>. <i>nigra</i>. The results revealed higher transcript accumulation of most of these genes in <i>B</i>. <i>juncea</i> var. CS52 compared to salt-sensitive cultivar even under normal growth conditions. Together, these findings reveal key pathways and signaling components that contribute to salinity tolerance in <i>B</i>. <i>juncea </i>var. CS52.</p></div

Histogram showing GOSlim terms assigned to <i>B</i>. <i>juncea</i> unigenes.

<p>The results are summarized for biological process, cellular component and molecular function categories. The left Y-axis represents the number of genes and the right Y-axis shows the percentage of total unigenes.</p

qRT-PCR validation of RNA-seq results.

<p>Expression of eight genes, showing varied expression patterns in RNA-sequencing experiment, were analyzed in CTRL as well as SS seedlings using qRT-PCR. The error bars show standard error between two biological replicates used for this experiment.</p

Schematic illustration of various cellular pathways mitigating salt stress in <i>B</i>. <i>juncea</i> var. CS52.

<p>A) Schematic overview of key pathways and genes affected in response to salinity stress in <i>B</i>. <i>juncea</i> is presented. Information about highlighted genes was gathered from published articles. Up regulated genes and those exhibiting high expression in absence of stress are highlighted with a red upward arrow and star, respectively. ASC: Ascorbate; GSH: Glutathione; ATPS: ATP sulfurylase; APR: Adenosine 5'-phosphosulfate reductase; P5CS: Delta1-pyrroline-5-carboxylate synthetase; SOD: Superoxide dismutase; GR: Glutathione reductase; GPX: Glutathione peroxidase; DHAR: Dehydroascorbate reductase; APX: Ascorbate peroxidase; MDHAR6: Monodehydroascorbate reductase 6; AtGSTU20: Glutathione S-transferase TAU 20; GLU1: Glutamate synthase 1; CCS: Copper chaperone for superoxide dismutase; RSA1: Short Root In Salt Medium 1; RITF1: RSA1 Interacting Transcription Factor 1; SOS3: Salt Overly Sensitive 3; SOS2: Salt Overly Sensitive 2; SOS1: Salt Overly Sensitive 1; ENH1: Enhancer of sos3-1; NHX1: Sodium/Hydrogen exchanger 1; MAPK1: Mitogen-activated protein kinase. B) Heat map showing comparative expression patterns of candidate genes in <i>B</i>. <i>juncea</i> and <i>B</i>. <i>nigra</i> seedlings under normal growth conditions. Log₂ expression values are indicated by the color scale with red representing high expression, black representing medium expression and green representing low level expression. The colored bars on the right demarcate genes representing different pathways. I: Ion homeostasis and sequestration; II: Sulfate assimilation; III: Gene Regulation and IV: ROS detoxification.</p

Pathway analysis showing relative significance of affected pathways for differentially expressed unigenes in response to salinity stress.

<p>The number of genes is represented on the Y-axis. The pathways enriched among up regulated genes are marked by *.</p