Comprehensive Analysis of Differentially Expressed Unigenes under NaCl Stress in Flax (Linum usitatissimum L.) Using RNA-Seq

Abstract

Flax (Linum usitatissimum L.) is an important industrial crop that is often cultivated on marginal lands, where salt stress negatively affects yield and quality. High-throughput RNA sequencing (RNA-seq) using the powerful Illumina platform was employed for transcript analysis and gene discovery to reveal flax response mechanisms to salt stress. After cDNA libraries were constructed from flax exposed to water (negative control) or salt (100 mM NaCl) for 12 h, 24 h or 48 h, transcription expression profiles and cDNA sequences representing expressed mRNA were obtained. A total of 431,808,502 clean reads were assembled to form 75,961 unigenes. After ruling out short-length and low-quality sequences, 33,774 differentially expressed unigenes (DEUs) were identified between salt-stressed and unstressed control (C) flax. Of these DEUs, 3669, 8882 and 21,223 unigenes were obtained from flax exposed to salt for 12 h (N1), 24 h (N2) and 48 h (N4), respectively. Gene function classification and pathway assignments of 2842 DEUs were obtained by comparing unigene sequences to information within public data repositories. qRT-PCR of selected DEUs was used to validate flax cDNA libraries generated for various durations of salt exposure. Based on transcriptome sequences, 1777 EST-SSRs were identified of which trinucleotide and dinucleotide repeat microsatellite motifs were most abundant. The flax DEUs and EST-SSRs identified here will serve as a powerful resource to better understand flax response mechanisms to salt exposure for development of more salt-tolerant varieties of flax.

Keywords: DEUs; EST-SSR; NaCl stress; RNA-seq; flax.

Figure 1

The comparison of clean reads with reference genome in different regions.

Figure 2

The box plot of FPKM in gene expression levels. Abscissa for sample name, ordinate for Log10.FPKM, box plot for each region for five statistics (From top to bottom: maximum, upper quartile, median, lower quartile and minimum), the outlier is shown in black dots.

Figure 3

Comparison of up and down-regulation of DEGs. C-vs-N1, C-vs-N2 and C-vs-N4 representing the DEUs under the exposure time of 12 h, 24 h and 48 h in NaCl solution, respectively.

Figure 4

The Venn diagram of DEUs. Venn diagram representing the distribution of NaCl-responsive genes. The numbers in the Venn diagram indicated total numbers of regulated genes in the unique treatment.

Figure 5

Cluster diagram of DEUs (FDR ≤ 0.05, fold change ≥ 2). The darker color represents the higher of the gene expression level. Each color block on the left represents a cluster of genes with similar expression levels. The Log2.FPKM value was used for clustering, with red for high expression gene and green for low expression gene. The color ranges from green to red, indicating higher gene expression.

Figure 6

Gene enrichment of non-redundant unigenes. The longitudinal axis represents the different pathway and horizontal axis represents the Rich factor. The size of dots indicates the number of differentially expressed genes in this pathway and the color of the point corresponds to a different Qvalue range.

Figure 7

Validation of the RNA-seq data expression profile by qRT-PCR. The relative expression levels of 6 DEUs were calculated according to the 2^−ΔΔCt method using the Actin gene as an internal reference gene. The x-axis indicates the different exposure of 100 mM NaCl solution with 12 h (C-N1), 24 h (C-N2) and 48 h (C-N4). LOG2FC and LOG2QRT represent for the binary logarithm of fold changes of differentially expressed genes in RNA-seq and qRT-PCR, respectively.