Genomic and evolutionary evidence for drought adaptation of allopolyploid Brachypodium hybridum

Abstract

Climate change is increasing the frequency and severity of drought worldwide, threatening the environmental resilience of cultivated grasses. However, the genetic diversity in many wild grasses could contribute to the development of climate-adapted varieties. Here, we elucidated the impact of polyploidy on drought responses using allotetraploid Brachypodium hybridum (B. hybridum) and its progenitor diploid species Brachypodium stacei (B. stacei). Our findings suggest that progenitor species' genomic legacies resulting from hybridization and whole-genome duplications conferred greater ecological adaptive advantages to B. hybridum compared with B. stacei. Genes related to stomatal regulation and the immune response from S-subgenomes were under positive selection during speciation, underscoring their evolutionary importance in adapting to environmental stresses. Biased expression in polyploid subgenomes (B. stacei-type and B. distachyon-type) significantly influenced differential gene expression, with the dominant subgenome exhibiting more differential expression. B. hybridum adapted a drought escape strategy characterized by higher photosynthetic capacity and lower intrinsic water-use efficiency than B. stacei, driven by a highly correlated coexpression network involving genes in the circadian rhythm pathway. In summary, our study shows the influence of polyploidy on ecological and environmental adaptation and resilience in model Brachypodium grasses. These insights hold promise for informing the breeding of climate-resilient cereal crops and pasture grasses.

Keywords: Brachypodium hybridum; Brachypodium stacei; drought response strategy; genomics; polyploidy; stomatal regulation; transcriptomics.

Fig. 1.

Genomic evolution analysis of Brachypodium species. (A) Density of synonymous substitution rates (Ks) of Brachypodium hybridum (Bh), Brachypodium stacei (Bs), Brachypodium distachyon (Bd), Bhd versus Bs (Bhd_Bs), and Bhs versus Bd (Bhs_Bd). Ks values >1 were removed to eliminate saturated synonymous sites. The three peaks represent the divergence time between the subgenomes of the allotetraploid and each diploid genome and whole-genome duplication events of the allotetraploid. (B) Circos diagram depicting the genomic relationships of Bh and Bs. The chromosomes are shown along with their relative position. The outer track shows the distribution of long terminal repeat retrotransposons over each chromosome as stacked histograms; blue bars and orange bars represent Copia-domain distribution and Gypsy-domain distribution, respectively. The text in the inner ring demonstrates homeolog genes with the highest Ka/Ks values between the two species. The inner arcs designate intergenomic rearrangements.

Fig. 2.

Stomatal structure of Brachypodium stacei and Brachypodium hybridum in response to drought. (A) Stomatal aperture length and width, (B) subsidiary cell length and width, (C) guard cell length and width; (D–I) comparison of stomatal parameters between B. stacei and B. hybridum. In (A–C), the data are visualized using scatter density plots, where the color gradient represents the density of neighboring data points (n_neighbors), with yellow indicating higher density and purple indicating lower density. The upper panels show measurements made under drought conditions and the lower panels show measurements made under well-watered conditions. The results highlight differences in stomatal and subsidiary cell structure between the two species and the impact of drought on these parameters. In (D–I), differences between groups were determined using one-way ANOVA with a post-hoc Tukey HSD test. Significant differences (P<0.05) are indicated with different letters above the plots.

Fig. 3.

Global transcriptome analyses of the drought response in Brachypodium hybridum and Brachypodium stacei. (A) Venn plot of differentially expressed genes (DEGs) in subgenomes of B. hybridum and in B. stacei; genes that are differentially expressed in more than 30% of the accessions within each species were considered DEGs. (B) Gene Ontology enrichment analysis for the intersections of DEGs in the Venn plot; ‘inner’ represents the 136 genes differentially expressed in Bhd, Bhs, and Bs. (C) Expression differentiation of paired homeolog genes between B. hybridum subgenomes. Homeolog gene pairs were identified by collinear blocks using the Bhs subgenome as the reference for each pair. The black horizontal bars on the left of the figure show the number of paired homeolog genes dominated by the Bhs/Bhd subgenome under well-watered (control; C) or drought (treatment; T) conditions. The lines with dots indicate genes sharing the same homeolog expression bias. The gene numbers of intersections are represented by the vertical bars.

Fig. 4.

Proportion of non-differentially expressed genes (A) and differentially expressed genes (DEGs) (B) balanced or predominantly expressed in each subgenome of Brachypodium hybridum and their homologous genes in Brachypodium stacei. Different colors distinguish the subgenomes that dominate gene expression under control (well-watered) or treatment (drought) conditions. Each bar represents a specific category of DEGs, with ‘n’ showing the number of genes in each category. Bhd-DEG and Bhs-DEG refer to DEGs specific to the Bhs or Bhd subgenome, respectively; Bhd-DEG & Bs_DEG and Bhs-DEG & Bs_DEG represent DEGs shared between the Bhd/Bhs subgenome and their corresponding homologous genes in B. stacei, respectively. The P-values above each bar denote the statistical significance among different expression patterns in each category.

Fig. 5.

Significant associations between gene clusters and physiological traits in (A) Brachypodium hybridum and (B) Brachypodium stacei. A triangular correlation plot was created to display the relationships between physiological and stomatal parameters. Red indicates positive correlations, blue represents negative correlations, and asterisks denote the significance of the correlations (*P<0.05, **P<0.01, ***P<0.001). The lines connected to each parameter represent coexpressed gene modules, with only those showing significant correlations retained. Red lines indicate positive correlations, green lines indicate negative correlations, and the thickness of the lines represents the Spearman’s r value. A, net CO₂ assimilation; Ci, intercellular CO₂ concentration; gs, stomatal conductance; Tleaf, leaf temperature; VPD, vapor pressure deficit; WUE, water use efficiency.

Fig. 6.

Gene Ontology terms of gene modules significantly positively (A) and negatively (B) correlated with physiological traits across different subgenomes.

Fig. 7.

Genome resequencing and the relationship between genes under selective pressure and expression patterns in Brachypodium hybridum and Brachypodium stacei. (A) Circos plot representing, from the outer to the inner circle, the number of variants with high impact (gain/lost stop_codon) of each gene of Bh (blue) and Bs (orange), single nucleotide polymorphism (SNP) density (red), and indel density (green). The inner arcs designate interchromosomal rearrangements; red and green lines represent enriched high-impact variants of Bh or Bs, respectively. (B) Gene Ontology terms for genes with high-impact SNPs. piN/piS indicates the ratio of non-synonymous to synonymous SNPs. (C) Proportion of genes under selective pressure with balanced or predominant expression in each subgenome of B. hybridum. Different colors distinguish the subgenomes that show dominant gene expression under control (well-watered) or treatment (drought) conditions. Each bar represents a specific category of genes, with ‘n’ showing the number of genes in each category. The P-values above each bar denote the statistical significance among different expression patterns in each category.