Fig 1.
Phenotypic distribution of the chlorosis and necrosis traits in the OWB population grown under Cd treatment.
The unit of x-axis represents the percentage of leaves in each line showing stress symptoms. The unit of y-axis represents the number of DH lines. The data are represented in three replicates.
Fig 2.
QTL scanning curves for the chlorosis and necrosis in the OWB population.
QTL curves on chromosomes (A) 2H and (B) 6H of the OWB population. The horizontal axis represents the position of markers. The vertical axis represents the LOD score. R: Replicate.
Table 1.
QTL peaks on 2H and 6H for Cd-tolerance in the OWB mapping population.
Fig 3.
General workflow used in the barley RNA-Seq analysis.
After quality control of raw reads by FastQC, we used Trimmomatic to trim low-quality bases. The Bowtie v2.3.4.1 software was used to make a barley genome index. The clean reads obtained from the RNA-Seq data were mapped to the barley genome assembly using Tophat v2.1.1 and the alignments were sorted by SAMTools v1.8. HTSeq-counts v0.9.1 was used to generate read counts of each gene, and DESeq2 v3.7 was used to identify DEGs between treatments and genotypes. Detection of variants was performed using SAMTools v1.8, BCFTools and snpEff pipeline.
Fig 4.
MA plots produced using DESeq2 for differential expression analysis in four comparisons.
The x-axis shows the mean normalized read counts and the y-axis shows log2 fold changes. Points in red show significant DEGs (Padj ≤ 0.05). The positive area shows upregulated genes and the negative area shows downregulated genes. (A) In Rec genotype, between Cd stress and CK treatment (Rec.Cd vs Rec.CK), (B) in Dom genotype, between Cd stress and CK treatment (Dom.Cd vs Dom.CK), (C) between the two genotypes and under Cd treatment (Rec.Cd vs Dom.Cd), and (D) between the two genotypes and under CK treatment (Rec.CK vs Dom.CK). CK: Control.
Table 2.
Summary of variant calling between Rec and Dom.
Fig 5.
Validation of expression profile for three candidate genes by qRT-PCR.
The expression profile of the three candidate genes detected by (A) qRT-PCR and (B) RNA-Seq techniques, which were co-located with the major QTL on 6H chromosome. The relative expression levels of the selected genes were compared with Dom genotype and normalized using an internal control (Actin) and calculated based on the 2−ΔΔCt method. For the qRT-PCR data, the mean ± standard error of three technical replicates are represented. Asterisks indicate levels of significance of differential expression tested by the Student’s t-test (* p ≤ 0.05, ** p ≤ 0.01). Nd: No data.
Table 3.
Candidate genes on detected QTLs related to Cd tolerance.