Genome-Wide Characterization of WRKY Transcription Factors Revealed Gene Duplication and Diversification in Populations of Wild to Domesticated Barley

Abstract

The WRKY transcription factors (WRKYs) are known for their crucial roles in biotic and abiotic stress responses, and developmental and physiological processes. In barley, early studies revealed their importance, whereas their diversity at the population scale remains hardly estimated. In this study, 98 HsWRKYs and 103 HvWRKYs have been identified from the reference genome of wild and cultivated barley, respectively. The tandem duplication and segmental duplication events from the cultivated barley were observed. By taking advantage of early released exome-captured sequencing datasets in 90 wild barley accessions and 137 landraces, the diversity analysis uncovered synonymous and non-synonymous variants instead of loss-of-function mutations that had occurred at all WRKYs. For majority of WRKYs, the haplotype and nucleotide diversity both decreased in cultivated barley relative to the wild population. Five WRKYs were detected to have undergone selection, among which haplotypes of WRKY9 were enriched, correlating with the geographic collection sites. Collectively, profiting from the state-of-the-art barley genomic resources, this work represented the characterization and diversity of barley WRKY transcription factors, shedding light on future deciphering of their roles in barley domestication and adaptation.

Keywords: WRKY transcription factors; adaptation; barley; diversity; domestication.

Figure 1

The phylogenetic tree of barley WRKYs. The WRKYs derived from wild and cultivated barley are highlighted with black and red font respectively, while A. thaliana WRKYs is represented with bold black. The values of the nodes are the bootstraps. The groups and sub-groups are indicated (I, II a–e and III).

Figure 2

Transcriptional profiles of HsWRKY and HvWRKY genes. The expressional profile of WRKYs in different tissues from the wild (A) and cultivated (B) barley. SE, whole seedlings of 15 days after planting (dap); LE, leaves from seedlings at 25 dap; RT, roots collected at 30 dap; ST, first stems dissected at 42 dap; SP, spikelets obtained at anthesis; LN, developing kernels collected at 15 days post-anthesis. EMB, 4-day embryos; ETI, etiolated seedling at 10 dap, dark condition; ROO1, roots from seedlings (10 cm shoot stage); LEA, shoots from seedlings (10 cm shoot stage); NOD, developing tillers, 3rd internode at 42 dap; INF2, developing inflorescences (1–1.5 cm); CAR5, developing grain at 5 days post-pollination (dap); CAR15, developing grain (15 dap); EPI, epidermal strips (28 dap); LOD, inflorescences, lodicule (42 dap); LEM, inflorescences, lemma (42 dap); PAL, dissected inflorescences, palea (42 dap); RAC, inflorescences, rachis (35 dap); ROO2, roots (28 dap); SEN, senescing leaves (56 dap). Heatmap was generated based on logarithm values of FPKM and the colored scale for the expression levels is shown.

Figure 3

Haplotype analysis of nine WRKYs in wild and cultivated barley population. The nucleotide substitutions in the coding region and the haplotype network of WRKY9 (A), WRKY13 (B), WRKY73 (C), WRKY89 (D) and WRKY95 (E). The rectangles represent the coding region of each gene, while the grey box indicates the part without qualified sequences. The size of each circle briefly refers to the number of the accessions containing a particular haplotype. Each short solid line represents a single mutation among haplotypes. The red and black lines indicate the positions of non-synonymous and synonymous mutations in the coding sequence, respectively. ^* Stop codon removed, Hs, Hordeum vulgare ssp. spontaneum; Hv, Hordeum vulgare ssp. vulgare.

Figure 4

Haplotypes of WRKY9 and their geographical distribution. (A) Graphical representation of full-length coding region in each haplotype. The non-synonymous and synonymous substitutions if compared to Hap I (HvWRKY9) were indicated using red and blue lines. Hap VII showed a sequence identical to HsWRKY9 identified from the reference assembly of wild barley ‘AWCS276’. The bold-highlighted haplotypes were detected in cultivated barley. The red and blue lines indicated non-synonymous and synonymous, respectively. The rectangles represent the coding region of each haplotype, while the grey box indicates the part without qualified sequences. (B) Geographical distribution of barley accessions harboring respective haplotypes. Each accession is allocated according to latitude and longitude coordinates of its collection site. The red and blue circles indicate the wild and cultivated barley accessions, respectively.

Figure 5

The subcellular localization and transactivation activity of WRKY9 haplotypes. (A) Graphical representation of the full-length coding sequence of selected haplotypes. Only non-synonymous substitutions are indicated. The blue rectangle represents the WRKY domain. (B) Subcellular localization of WRKY9 haplotypes in N. benthamiana. Scale bar = 20 μm. (C) Transactivation activity of WRKY9 haplotypes in yeast. BD-HvTCP24, which contains a transactivation activity in yeast, serves as a positive control. SD, synthetic dextrose medium; Trp, tryptophan; His, histidine; Ade, Adenine. 0.1, 0.01, 0.001 and 0.0001 denoted the different dilution series.