Genome sequencing of Sitopsis species provides insights into their contribution to the B subgenome of bread wheat

Abstract

Wheat (Triticum aestivum, BBAADD) is an allohexaploid species that originated from two polyploidization events. The progenitors of the A and D subgenomes have been identified as Triticum urartu and Aegilops tauschii, respectively. Current research suggests that Aegilops speltoides is the closest but not the direct ancestor of the B subgenome. However, whether Ae. speltoides has contributed genomically to the wheat B subgenome and which chromosome regions are conserved between Ae. speltoides and the B subgenome remain unclear. Here, we assembled a high-quality reference genome for Ae. speltoides, resequenced 53 accessions from seven species (Aegilops bicornis, Aegilops longissima, Aegilops searsii, Aegilops sharonensis, Ae. speltoides, Aegilops mutica [syn. Amblyopyrum muticum], and Triticum dicoccoides) and revealed their genomic contributions to the wheat B subgenome. Our results showed that centromeric regions were particularly conserved between Aegilops and Triticum and revealed 0.17 Gb of conserved blocks between Ae. speltoides and the B subgenome. We classified five groups of conserved and non-conserved genes between Aegilops and Triticum, revealing their biological characteristics, differentiation in gene expression patterns, and collinear relationships between Ae. speltoides and the wheat B subgenome. We also identified gene families that expanded in Ae. speltoides during its evolution and 789 genes specific to Ae. speltoides. These genes can serve as genetic resources for improvement of adaptability to biotic and abiotic stress. The newly constructed reference genome and large-scale resequencing data for Sitopsis species will provide a valuable genomic resource for wheat genetic improvement and genomic studies.

Keywords: Aegilops; B subgenome; Sitopsis; conserved blocks; polyploid wheat.

Figure 1

Chromosome-level assembly and annotation of Ae. speltoides. (A) Geographic distribution of the 12 species used in this research: Ae. bicornis, Ae. longissima, Ae. mutica, Ae. searsii, Ae. sharonensis, Ae. speltoides, Ae. tauschii, T. aestivum, T. dicoccoides, T. dicoccum, T. durum, and T. urartu. (B) Circos plot of genomic features of the assembled Ae. speltoides genome: (i) density of HC genes, (ii) distribution of GC content, (iii) TE density along each chromosome, (iv) density of Copia TEs, (v) density of Gypsy elements, and (vi) links between syntenic genes. (C) FISH results with the probes (left) Oligo-pSc119.2 (green) + Oligo-pTa71 (red) and the probe (right) Oligo-(GAA)₇ (red) for the chromosomes of Ae. speltoides (S^sp), Ae. searsii (S^s), Ae. bicornis (S^b), Ae. longissima (S^l), Ae. sharonensis (S^sh), and the wheat B subgenome (B). The numbers 1–7 represent homoeologous chromosome groups 1–7, respectively. (D) Phylogenetic tree constructed with data from 13 species using the neighbor-joining method; barley was used as the outgroup species.

Figure 2

Conserved blocks between the Triticum B genome and Aegilops species are enriched in centromeric regions. (A) Chromosomal distribution of conserved genomic blocks with the T. aestivum B subgenome as the background. (B) Boxplots of conserved genomic block lengths with the T. aestivum B, A, or D subgenome as the background. (C) Evolutionary patterns of orthologous gene pairs estimated using a Ks distribution density plot. (D) Ks distribution in the IWGSC RefSeq v1.0 B subgenome. The figure represents the Ks distribution of an average of 50 genes. The red dotted lines indicate chromosome boundaries. (E) Boxplots of the variation frequency in the gene-coding regions of 12 species. The red dotted line represents the average variation frequency of Ae. speltoides. The top figure shows SNP variation frequency in centromeric regions, and the bottom figure shows SNP variation frequency in non-centromeric regions. (F) SNP variation frequency of different TEs indicated by SNP density values, which ranged from 0 to 0.08. Red represents higher variation frequency, and blue represents lower frequency.

Figure 3

Phylogenetic relationships of genes from Aegilops/Triticum species. (A) Comparative evolutionary relationships of Ae. speltoides and other Aegilops and Triticum genomes. The number of gene families that were expanded (purple) or contracted (orange) in each species was estimated using CAFÉ. (B) Statistical analysis of heat shock protein 20 (HSP20) genes in five representative species. OG, orthologous groups. (C) Neighbor-joining phylogeny of HSP20 genes in Ae. speltoides. CI and CP represent HSP20 subfamilies. (D) Relative expression level of three HSP20 genes under heat stress treatments of different durations measured by RT-qPCR.

Figure 4

Identifying conserved and non-conserved genes between Aegilops and Triticum. (A) Gene classification between Aegilops and Triticum species on the basis of SNP variation frequency. Red and blue represent high and low SNP variation frequency between Aegilops and Triticum, respectively. The horizontal axis shows groups of genes defined by SNP variation frequency, including seven groups (G1–7) and five types of genes. G1 and G6 had similar patterns of SNP variation frequency, as did G2 and G3. The vertical axis shows the different species. (B) Distribution of five types of genes in the wheat B subgenome: (1) B lineage:Sitopsis (B:S) conserved genes, (2) B:S non-conserved genes, (3) B:S^sp conserved genes, (4) B:S^sp non-conserved genes, and (5) B lineage conserved genes. (C) Heatmap of gene expression levels of the non-conserved and conserved gene groups. The vertical axis represents different RNA-seq tissues, and the horizontal axis shows the different types of genes: B:S conserved genes, B:S non-conserved genes, B:S^sp conserved genes, B:S^sp non-conserved genes, and B conserved genes. (D) Distribution proportion of the five types of genes in conserved blocks. (E) Distribution of collinear genes in the conserved and non-conserved gene groups. (F) Distribution of differentially expressed genes and non-differentially expressed genes in the conserved and non-conserved gene groups.

Figure 5

Sitopsis species provide resources for wheat improvement. (A) Identification of Ae. speltoides-specific genes. The right column shows the different species, and the horizontal axis represents the gene coverage rate. The region between the black dotted lines indicates the 789 Ae. speltoides-specific genes, and the remaining regions represent shared genes. (B) GO enrichment analysis of the 789 Ae. speltoides-specific genes. The horizontal axis indicates the enrichment factor values, and the vertical axis represents the GO terms. The circle size represents the gene numbers, and the circle colors indicate the significance of the regulatory pathway, which is indicated by −log₁₀(P value). (C) Expression levels of the 789 Ae. speltoides-specific genes. The histogram above the heatmap shows the numbers of highly expressed genes, which are marked with red rectangles in the heatmap. (D) Co-expression networks of wheat TraesCS1B01G225000 and TraesCS7B01G091900; each node represents a gene. Red circles represent hub genes, red triangles represent Ae. speltoides-specific genes, and darkcyan circles represent T. aestivum BB genes.