Transcriptome Analyses of Near Isogenic Lines Reveal Putative Drought Tolerance Controlling Genes in Wheat

Abstract

Drought stress, especially at the grain-filling stage, is a major constraint for wheat production. Drought tolerance is a complex trait controlled by a large array of genes and pathways. This study conducted gene expression profiling on two pairs of near-isogenic lines (NILs) for an important qDSI.4B.1 QTL conferring drought tolerance on the short arm of chromosome 4B in wheat. Analysis showed 1,614 genome-wide differentially expressed genes (DEGs) between the tolerant and susceptible isolines in both NIL pairs. Six common DEGs were found between NIL1 and NIL2 at both 7 and 14 days after stress induction, with two of them having single nucleotide polymorphism (SNP) variants. These six genes that were confirmed by quantitative real-time PCR (qRT-PCR) expression analysis are considered candidate genes for drought tolerance mediated by qDSI.4B.1 QTL with their main contributions to gene regulation, cell elongation, protein quality control, secondary metabolism, and hormone signaling. These six candidate genes and the highest number of DEGs and variants (SNPs/indels) were located between 49 and 137 Mbp of 4BS, making this interval the most probable location for the qDSI.4B.1 locus. Additionally, 765 and 84 DEGs were detected as responsive genes to drought stress in tolerant and susceptible isolines, respectively. According to gene ontology (GO), protein phosphorylation, oxidation reduction, and regulation of transcription were top biological processes involved in the drought response and tolerance. These results provide insights into stress responses regulated by the 4BS locus and have identified candidate genes and genetic markers that can be used for fine mapping of the qDSI.4B.1 locus and, ultimately, in wheat breeding programs for drought tolerance.

Keywords: DEGs; NILs; QTL; RNA-seq; SNP; chromosome 4B; drought stress; qRT-PCR.

FIGURE 1

Representative wheat plants of the tolerant (T) and susceptible (S) near isogenic lines of NIL1 at panels (A) 7 days and (B) 14 days after drought stress initiation at anthesis.

FIGURE 2

(A) Venn diagrams and (B) Smear plots for differentially expressed genes (DEGs) related to each of the isolines (T vs. T and S vs. S) at 7 days after drought stress initiation in comparison to control and 14 days in comparison to 7 days after stress. The X-axis is the average of log counts per million. The Y-axis is the log2 fold change. Symbols are “C” for control; “T” for tolerant isoline; “S” for susceptible isoline; “7d” and “14d” for 7 and 14 days after drought stress initiation at anthesis, respectively. The DEGs were detected with the threshold false discovery rate (FDR) of ≤0.05 and the absolute value of log2 fold change ≥1 or ≤−1.

FIGURE 3

Heatmaps showing the expression of the important (A) upregulated and (B) downregulated genes responsive to drought stress in the tolerant isolines. Color keys represent the log2 of normalized expression values and a histogram of the counts. Each row represents a gene and each column a sample. Symbols are “Con” for control; “T” for tolerant isoline; “7d” and “14d” for 7 and 14 days after drought stress initiation at anthesis, respectively. The DEGs were determined with the threshold false discovery rate (FDR) of ≤0.05 and the absolute value of log2 fold change ≥1 or ≤−1.

FIGURE 4

(A) Venn diagrams and (B) Smear plots for differentially expressed genes (DEGs) between the tolerant and susceptible isolines (T vs. S) at 7 and 14 days after drought stress initiation at anthesis. The X-axis is the average of log counts per million. The Y-axis is the log2 fold change. Symbols are “C” for control; “T” for tolerant isoline; “S” for susceptible isoline; “7d” and “14d” for 7 and 14 days after drought stress initiation at anthesis, respectively. The DEGs were determined with the threshold false discovery rate (FDR) of ≤0.05 and the absolute value of log2 fold change ≥1 or ≤−1.

FIGURE 5

Physical distribution of panel (A) the differentially expressed genes (DEGs) (B) the single nucleotide polymorphisms (SNPs) and/or indels on chromosome 4B. (C) Physical distribution of DEGs and genes containing SNPs and/or indels within a 150-Mb interval of the 4BS. The most probable interval of the qDSI.4B.1 QTL is shown between the arrows. Genes with SNPs and/or indels are shown in gray; The DEGs with higher expression in tolerant (T) and susceptible (S) isolines are shown in blue and red, respectively. Genes with an asterisk (*) were common between NIL1 and NIL2. Rht1 is the reduced plant height gene; MQTL4B.1, MQTL4B.2, and MQTL4B.3 are major meta-QTLs for yield according to Liu et al. (2020).

FIGURE 6

Gene ontology analysis (GO) of the differentially expressed genes (DEGs) identified when comparing tolerant and susceptible (T vs. S) isolines. Top significantly enriched pathways in panels (A) biological processes, (B) cellular components, and (C) molecular function are illustrated with p-value < 0.05. Symbols are “T” for tolerant isoline; “S” for susceptible isoline; “7D” and “14D” for 7 and 14 days after drought stress initiation at anthesis, respectively.

FIGURE 7

Quantitative real time PCR relative expression values of the selected genes in tolerant and susceptible isolines at panels (A) 7 days and (B) 14 days after stress initiation. Mean relative expression is the mean of NIL1 and NIL2. The expression data measured by subtracting the Ct number of the reference gene (Actin) from that of the target gene followed by calculation of 2^{– ΔΔCT}. Values are means ± standard deviation, and the statistical significance was determined by two-sided t-test (*p < 0.05 and **p < 0.01).