Transcriptomic profiling of wheat near-isogenic lines reveals candidate genes on chromosome 3A for pre-harvest sprouting resistance

Abstract

Background: Pre-harvest sprouting (PHS) in wheat can cause severe damage to both grain yield and quality. Resistance to PHS is a quantitative trait controlled by many genes located across all 21 wheat chromosomes. The study targeted a large-effect quantitative trait locus (QTL) QPhs.ccsu-3A.1 for PHS resistance using several sets previously developed near-isogenic lines (NILs). Two pairs of NILs with highly significant phenotypic differences between the isolines were examined by RNA sequencing for their transcriptomic profiles on developing seeds at 15, 25 and 35 days after pollination (DAP) to identify candidate genes underlying the QTL and elucidate gene effects on PHS resistance. At each DAP, differentially expressed genes (DEGs) between the isolines were investigated.

Results: Gene ontology and KEGG pathway enrichment analyses of key DEGs suggested that six candidate genes underlie QPhs.ccsu-3A.1 responsible for PHS resistance in wheat. Candidate gene expression was further validated by quantitative RT-PCR. Within the targeted QTL interval, 16 genetic variants including five single nucleotide polymorphisms (SNPs) and 11 indels showed consistent polymorphism between resistant and susceptible isolines.

Conclusions: The targeted QTL is confirmed to harbor core genes related to hormone signaling pathways that can be exploited as a key genomic region for marker-assisted selection. The candidate genes and SNP/indel markers detected in this study are valuable resources for understanding the mechanism of PHS resistance and for marker-assisted breeding of the trait in wheat.

Keywords: Marker-assisted selection; Near-isogenic lines; Pre-harvest sprouting; RNA sequencing; Wheat.

Fig. 1

Diagram showing the physical locations of cloned genes and flanking markers of major QTL on chromosome 3A for seed dormancy and preharvest sprouting resistance. QTL in bold is the targeted QTL in this study. Bar represents 100 Mb physical distance

Fig. 2

Comparison of NIL pairs 1 and 2. A. Phenotypic differences between resistant (R) and susceptible (S) isolines in the two NIL pairs. (a) and (b) were the spike sprouting test of NIL pair 1 (left) and NIL pair 2 (right), respectively, at day 7 of the test; (c) and (d) were seed germination test at day 2 of the test. B. Venn diagrams showing the number of differentially expressed genes (DEGs) that were commonly (a) up-regulated and (b) down-regulated in the resistant isolines compared with those in the susceptible isolines. Numerals inside the parentheses indicate the number of genes expressed at each time point. The total number of DEGs is noted at the bottom of each Venn diagram. C. Volcano plot showing DEGs within each NIL pair at different time-points. X axis represents log2 transformed fold change. Y axis represents -log10 transformed p value significance. Blue points represent up-regulated DEGs. Red points represent down-regulated DEGs. Gray points represent non-DEGs. DPA = days post anthesis

Fig. 3

Gene ontology assignment of differentially expressed genes (DEGs) in the near-isogenic lines. The unigenes were mapped to three main categories: a cellular component, b molecular function, and c biological process. The x-axis indicates the number of annotated DEGs. DPA = days post anthesis

Fig. 4

Pathway enrichment of differentially expressed genes (DEGs) in the near-isogenic lines at a 15 DPA, b 25 DPA and c 35 DPA. The x-axis indicates the rich factor. DPA = days post anthesis

Fig. 5

Distribution of differentially expressed genes (DEGs) in different transcription factor (TF) families in the whole transcriptome