Transcriptome Analysis of Roots from Wheat (Triticum aestivum L.) Varieties in Response to Drought Stress

Abstract

Under climate change, drought is one of the most limiting factors that influences wheat (Triticum aestivum L.) production. Exploring stress-related genes is vital for wheat breeding. To identify genes related to the drought tolerance response, two common wheat cultivars, Zhengmai 366 (ZM366) and Chuanmai 42 (CM42), were selected based on their obvious difference in root length under 15% PEG-6000 treatment. The root length of the ZM366 cultivar was significantly longer than that of CM42. Stress-related genes were identified by RNA-seq in samples treated with 15% PEG-6000 for 7 days. In total, 11,083 differentially expressed genes (DEGs) and numerous single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were identified. GO enrichment analysis revealed that the upregulated genes were mainly related to the response to water, acidic chemicals, oxygen-containing compounds, inorganic substances, and abiotic stimuli. Among the DEGs, the expression levels of 16 genes in ZM366 were higher than those in CM42 after the 15% PEG-6000 treatment based on RT-qPCR. Furthermore, EMS-induced mutants in Kronos (T. turgidum L.) of 4 representative DEGs possessed longer roots than the WT after the 15% PEG-6000 treatment. Altogether, the drought stress genes identified in this study represent useful gene resources for wheat breeding.

Keywords: DEGs; GO; RNA-seq; RT-qPCR; stress treatment; wheat.

Figure 1

Root growth of CM42 and ZM366. (A) CM42 root growth under hydroponic conditions. (B) ZM366 root growth under hydroponic conditions. (C) CM42 root growth under the 15% PEG-6000 treatment. (D) ZM366 root growth under the 15% PEG-6000 treatment. Root growth was measured on days 2, 4, 5, and 7.

Figure 2

Root growth length statistics. (A) Statistics of CM42 and ZM366 under hydroponics and the 15% PEG-6000 treatment. (B) Statistics of Kronos and 4 EMS-induced mutants under the 15% PEG-6000 treatment. Growth was measured on days 2, 4, 5, and 7. The experiment was repeated three times, and the error bar represents the SD of the means (n = 3). ** represents p < 0.01. ns indicates not significant.

Figure 3

Statistics for variant sites, including InDels and SNPs. (A) InDel-impact was plotted for four levels: high, moderate, low, and modifier. (B) InDel-region was counted for the following gene structure regions: downstream, exon, intron, intergenic, splice_site_acceptor, splice_site_donor, and splice_site_region, transcript, upstream, utr_3_prime and utr_5_prime. (C) SNP-function was statistically plotted for three conditions: synonymous mutation, missense mutation, and nonsense mutation. (D) SNP-impact was statistically plotted for four levels: high, moderate, low, and modifier. (E) SNP-region was counted and mapped for the following gene structure regions: downstream, exon, intron, intergenic, splice_site_acceptor, splice_site_donor, and splice_site_region, transcript, upstream, utr_3_prime and utr_5_prime.

Figure 4

Principle Component Analysis (PCA) (A) CM42-hydroponics, CM42-15% PEG, ZM366-hydroponics, and ZM366-15% PEG PCA of a PCA 2D plot. (B) CM42-hydroponics, CM42-15% PEG, ZM366-hydroponics, and ZM366-15% PEG PCA of a PCA 3D plot. PC1 refers to the top contribution rate, which is the factor that has the greatest influence on variation, and PC2 is the second factor.

Figure 5

Transcriptomic analysis of ZM366 and CM42 treated with 15% PEG-6000. (A) Venn diagram of expressed genes in ZM366 and CM42 under 15% PEG-6000 treatment conditions. The yellow represents the genes expressed in ZM366. The purple represents the genes expressed in CM42. (B) Volcano map of differentially expressed genes (DEGs) in ZM366 and CM42 under 15% PEG-6000 treatment conditions. Up, 6239; down, 4844. (C) GO enrichment analysis bubble diagrams. The abscissa represents the ratio of the number of DEGs annotated with a GO term to the total number of DEGs, and the ordinate is the GO term; the size of the point represents the number of genes annotated with a GO term, and the color change from red to purple represents the significance of the enrichment. (D) Expression heatmap of the GO enrichment DEGs between CM42 and ZM366 under 15% PEG-6000 treatment conditions.

Figure 6

Expression patterns revealed by RT-qPCR. The expression patterns of 16 highly expressed genes in ZM366 compared with CM42 (both treated with 15% PEG-6000) as revealed by RT-qPCR. The experiment was repeated three times, and the error bar represents the SD of the means (n = 3). ** represents p < 0.01. * represents p < 0.05.

Figure 7

Root growth of Kronos and 4 EMS-induced mutants under the 15% PEG-6000 treatment. (A) Kronos root growth conditions. (B) Root growth conditions of mutant 3935. (C) Root growth conditions of mutant 3216. (D) Root growth conditions of mutant 3538. (E) Root growth conditions of mutant 3557. Root growth was measured on days 2, 4, 5, and 7.

Figure 8

Expression patterns revealed by RT-qPCR treated with 15% PEG-6000. (A) The expression patterns of 16 highly expressed genes in mutant 3538 compared with Kronos, mutant 3557 (B), in mutant 3216 (C), and in mutant 3935 (D). The experiment was repeated three times, and the error bar represents the SD of the means (n = 3). ** represents p < 0.01. ns indicates not significant.