Genome-wide association, RNA-seq and iTRAQ analyses identify candidate genes controlling radicle length of wheat

Abstract

The radicle, present in the embryo of a seed, is the first root to emerge at germination, and its rapid growth is essential for establishment and survival of the seedling. However, there are few studies on the critical mechanisms underlying radicle and then radicle length in wheat seedlings, despite its importance as a food crop throughout the world. In the present study, 196 wheat accessions from the Huanghuai Wheat Region were screened to measure radicle length under 4 hydroponic culture environments over 3 years. Different expression genes and proteins (DEGs/DEPs) between accessions with extremely long [Yunong 949 (WRL1), Zhongyu 9,302 (WRL2)] and short roots [Yunong 201 (WRS1), Beijing 841 (WRS2)] were identified in 12 sets of root tissue samples by RNA-seq and iTRAQ (Isobaric tags for relative and absolute quantification). Phenotypic results showed that the elongation zone was significantly longer in root accessions with long roots compared to the short-rooted accessions. A genome-wide association study (GWAS) identified four stable chromosomal regions significantly associated with radicle length, among which 1A, 4A, and 7A chromosomes regions explained 7.17% to12.93% of the phenotypic variation. The omics studies identified the expression patterns of 24 DEGs/DEPs changed at both the transcriptional and protein levels. These DEGs/DEPs were mainly involved in carbon fixation in photosynthetic organisms, photosynthesis and phenylpropanoid biosynthesis pathways. TraesCS1A02G104100 and TraesCS2B02G519100 were involved in the biosynthesis of tricin-lignins in cell walls and may affect the extension of cell walls in the radicle elongation zone. A combination of GWAS and RNA-seq analyses revealed 19 DEGs with expression changes in the four accessions, among which, TraesCS1A02G422700 (a cysteine-rich receptor-like protein kinase 6, CRK6) also showed upregulation in the comparison group by RNA-seq, iTRAQ, and qRT-PCR. BSMV-mediated gene silencing also showed that TaCRK6 improves root development in wheat. Our data suggest that TaCRK6 is a candidate gene regulating radicle length in wheat.

Keywords: GWAS analysis; RNA-seq analysis; iTRAQ analysis; radicle length; wheat (Triticum aestivum L.).

Figure 1

Radicle length after 10 days of hydroponic growth in different accessions. (A) Images of the radicle length. Red arrows indicate radicle; (B) Average radicle length difference; (C) Frequency distribution of the radicle length for 196 accessions in the four hydroponic environments; (D) Image of radicle tips in bright field microscopy with stain. Red frames indicate elongation zones; (E) Elongation zone in radicle tip. Significant differences are indicated as p < 0.05 (*).

Figure 2

Quantile-quantile plots and Manhattan plots for radicle length across 196 accessions and SNP chromosome locations. (A,B) Wheat hydroponically grown in greenhouse; (C,D) Wheat hydroponically grown outdoor in 2017; (E,F) Wheat hydroponically grown outdoor in 2018; (G,H) Wheat hydroponically grown outdoor in 2019.

Figure 3

Transcriptome data of wheat accessions with long and short radicles. (A,B) Quality control of all samples; (C) Number of transcripts per accession; (D) Number of DEGs in pair-wise comparisons.

Figure 4

Statistics and annotation of the DEPs in short and long radicles of wheat. (A) Venn diagram of the number of proteins identified in 3 replicates; (B) Number of DEPs in pair-wise comparisons; (C) DEP-enriched GO annotations; (D) DEP-enriched KEGG annotations.

Figure 5

Statistics and annotation of the DEGs in candidate segments of radical length. (A) A heat map of 19 candidate genes, (B) Candidate genes-enriched GO annotations, (C) Candidate genes-enriched KEGG annotations, (D) Candidate genes-enriched KEGG annotations.

Figure 6

Phenylpropanoid biosynthesis pathways including DEPs and DEGs for wheat radicle length. Red frames are annotated metabolic pathway targets with DEPs, and blue frames are annotated metabolic pathway targets with DEGs.

Figure 7

Silencing of TaCRK6 in wheat using the BSMV-VIGS system. (A) BSMV and photobleaching were evident in plants infected by BSMV:PDS after 20 days post-inoculation, (B) Radicle length at the third-fourth leaf stage with BSMV:PDS, BSMV:TaCRK6, or BSMV:WT at 20 days post-inoculation. Red arrows indicate radicle, (C) Average radicle length of different treatments after sowing 30-days, (D) Relative transcript levels of TaCRK6 in knock-down plants and control wheat roots. The values of root are the mean ± SD from four samples, and significant differences are indicated as p < 0.05 (*) and p < 0.01 (**).

Figure 8

A molecular regulatory model controlling wheat radicle length.

Figure 9

Protein–protein interaction (PPI) network analysis of the DEPs between long and short wheat radicles. Red circles represent upregulated proteins and blue circles downregulated proteins. Edges with purple represent classes of KEGG pathway.