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, 26 (5), 1878-1900

mRNA and Small RNA Transcriptomes Reveal Insights Into Dynamic Homoeolog Regulation of Allopolyploid Heterosis in Nascent Hexaploid Wheat

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mRNA and Small RNA Transcriptomes Reveal Insights Into Dynamic Homoeolog Regulation of Allopolyploid Heterosis in Nascent Hexaploid Wheat

Aili Li et al. Plant Cell.

Abstract

Nascent allohexaploid wheat may represent the initial genetic state of common wheat (Triticum aestivum), which arose as a hybrid between Triticum turgidum (AABB) and Aegilops tauschii (DD) and by chromosome doubling and outcompeted its parents in growth vigor and adaptability. To better understand the molecular basis for this success, we performed mRNA and small RNA transcriptome analyses in nascent allohexaploid wheat and its following generations, their progenitors, and the natural allohexaploid cultivar Chinese Spring, with the assistance of recently published A and D genome sequences. We found that nonadditively expressed protein-coding genes were rare but relevant to growth vigor. Moreover, a high proportion of protein-coding genes exhibited parental expression level dominance, with genes for which the total homoeolog expression level in the progeny was similar to that in T. turgidum potentially participating in development and those with similar expression to that in Ae. tauschii involved in adaptation. In addition, a high proportion of microRNAs showed nonadditive expression upon polyploidization, potentially leading to differential expression of important target genes. Furthermore, increased small interfering RNA density was observed for transposable element-associated D homoeologs in the allohexaploid progeny, which may account for biased repression of D homoeologs. Together, our data provide insights into small RNA-mediated dynamic homoeolog regulation mechanisms that may contribute to heterosis in nascent hexaploid wheat.

Figures

Figure 1.
Figure 1.
Characterization of Nascent Allohexaploid Wheat. (A) Chromosomes in root tip cells. Green arrows indicate the 4A/7B chromosome translocations in synthetic hexaploid and its T. turgidum parent PI 94655 (4x). (B) Seven-day-old seedlings of T. turgidum (4x), synthetic wheat (6x), and Ae. tauschii (2x). (C) Heading stage spikes of the third generation of self-pollinated allohexaploid wheat (S3, 6x) and its parents. (D) Developing seeds of S3 allohexaploid plants and their progenitors. 4x, T. turgidum; 2x, Ae. tauschii; 6x, newly synthesized allohexaploid wheat. (E) Sampling schema. Samples in oval circles have biological replicates. AABB, T. turgidum; DD, Ae. tauschii; S1 to S4, consecutive generation of selfed allohexaploid wheat.
Figure 2.
Figure 2.
Global Characterization of Gene Expression Patterns among Three Tissues in Nascent Hexaploid Wheat and Its Progenitors. AABB, T. turgidum; DD, Ae. tauschii; S3, the third generation of self-pollinated allohexaploid wheat. (A) Correlation coefficients between gene expression data sets from two biological duplicates. (B) Cluster dendrogram showing global relationships of gene expression in different tissues and species. The branch length indicates the degree of variance. (C) to (E) Venn diagram analyses of tissue specific genes in T. turgidum (C), Ae. tauschii (D), and S3 allohexaploid wheat (E). (F) to (H) Functional categories of genes showing tissue-specific expression in seedlings (F), young spikes (G), and immature seed (H) of S3 plants. For each tissue, only genes whose transcripts were detected in both allohexaploid progeny and its progenitor were considered. FDR-adjusted P values, *P < 0.05 and **P < 0.01, respectively. Observed, numbers of genes observed in this study; Expected, numbers of genes in this same category in the GO enrichment analysis program.
Figure 3.
Figure 3.
Nonadditively Expressed Genes in Young Spikes of Nascent Allohexaploid Wheat. (A) Genes differentially expressed in S3 progeny and their tetraploid (AABB) and diploid (DD) progenitors. Numbers close to the species (colored) represent upregulated genes compared with the neighboring species. Percentages indicate those among all expressed genes in young spikes. The total number of genes differentially expressed between two species is given (black). (B) GO enrichment analysis of nonadditively expressed genes. Shown are significantly enriched GO terms (Fisher test FDR < 0.05). BP, biological process; MF, molecular function; CC, cellular component.
Figure 4.
Figure 4.
ELD of Genes in Seedlings and Young Spikes of Nascent Allohexaploid Wheat. ELD-ab, genes with expression level similar to that in T. turgidum; ELD-d, genes with expression level similar to that in Ae. tauschii. (A) Twelve bins of differentially expressed genes. AB, T. turgidum; D, Ae. tauschii; S3, the third generation of self-pollinated allohexaploid progeny. (B) Numbers of genes showing four types of parental ELD in seedlings and young spikes. Bins II, IV, IX, and XI correspond to those in (A). (C) Enriched GO terms of genes showing parental ELD in seedlings and young spikes. Fisher test, *FDR < 0.05 and **FDR < 0.01.
Figure 5.
Figure 5.
Transgressive Inheritance of Genes with Parental ELD in Young Spikes of Nascent Synthetic Wheat. AABB, T. turgidum; DD, Ae. tauschii; S1 to S4, consecutive generations of self-pollinated allohexaploid wheat. ELD-ab, genes with expression level similar to that in T. turgidum; ELD-d, genes with expression level similar to that in Ae. tauschii. (A) Venn diagram of ELD-ab genes among S1, S3, and S4 nascent allohexaploid wheat and their preservation in CS. (B) Venn diagram of ELD-d genes among S1, S3, and S4 nascent allohexaploid wheat and their preservation in CS. (C) Enriched GO terms among genes displaying parental ELD and shared by S1, S3, and S4 generations of nascent allohexaploid wheat. (D) Top five MapMan bins of ELD-ab and ELD-d genes in (C).
Figure 6.
Figure 6.
Biased Homoeolog Expression as Measured by SNP Mapping Depths for Young Spikes of S3 Progeny. (A) and (B) Distribution of the fraction of mapped reads that carry the AB homoeolog. (A) Reads mapped against the T. urartu (A) genome. Dotted line indicates the median number, which is greater than 0.5. (B) Reads mapped against the Ae. tauschii (D) genome. (C) Homoeolog expression regulation during allohexaploidization. T. turgidum homoeologs, AB; Ae. tauschii homoeolog, D; >, =, and < indicate relative expression levels between homologs in parental lines (pa) or homoeologs in progeny (pr). (D) Dynamic regulation of AB or D homoeolog expression in nascent allohexaploid wheat and parental genome ELD. Categories II, IV, IX, and XI correspond to those in Figure 4A. up, upregulated in the progeny relative to the parental expression level; down, downregulated in the progeny relative to the parental expression level. For details, see Supplemental Table 7.
Figure 7.
Figure 7.
Effect of Allohexaploidization on Expression of miRNAs and Their Targets. AABB, T. turgidum; DD, Ae. tauschii; S1 to S4, consecutive generation of selfed allohexaploid wheat. ELD-ab, miRNAs with ELD for T. turgidum; ELD-d, miRNAs with ELD for Ae. tauschii. (A) Left: Hierarchical clustering of miRNAs displaying nonadditive expression. Blue letters, nonadditively repressed miRNAs; black letters, nonadditively activated miRNAs. Right: Hierarchical clustering analysis of miRNAs displaying parental ELD in young spikes. Blue dotted lines connect ELD-ab miRNAs with those nonadditively expressed miRNAs; yellow dotted lines connect ELD-d miRNAs with those nonadditively expressed miRNAs. Blue letters, ELD-d miRNAs; black letters, ELD-d miRNAs. (B) RNA gel blot analysis of miRNAs displaying nonadditive expression in young spikes. U6 RNA is used as a loading control. (C) Inverse correlations between log2 fold changes of a subset of differentially expressed miRNAs and those of differentially expressed targets. Multiple targets of a single miRNA are indicated by a vertical line. r, Pearson correlation efficient; P values are derived from Wilcoxon paired rank sum test.
Figure 8.
Figure 8.
siRNA Expression Patterns during Wheat Allohexaploidization. (A) Venn diagram showing tissue-specific expression of siRNA clusters in S3 plants using the D genome as reference. (B) Clustered siRNA loci (on top of the figure) in young spikes identified using the D genome as a reference. The percentages of S1, S3, S4, and CS siRNA clusters were derived against the number of siRNA clusters from Ae. tauschii (DD, 76,413). (C) Clustered siRNA loci (on top of the figure) in young spikes identified using the A genome as a reference. The percentages of S1, S3, S4, and CS siRNA clusters were derived against the number of siRNA clusters from T. turgidum (AABB, 53,072). (D) Proportions of TAGs and locations of TEs (triangles) in transcribed region (gray box) and their 2-kb upstream and downstream regions (extended lines) of Ae. tauschii (D) and T. urartu (A) gene models. (E) and (F) Small RNA densities (100-bp sliding window) in upstream (2 kb), transcribed, and downstream (2 kb) regions of TAGs and non-TAGs in young spikes (E) and immature seed (F) on the D genome. (G) and (H) Small RNA densities (100-bp sliding window) in upstream (2 kb), transcribed, and downstream (2 kb) regions of TAGs and non-TAGs in young spikes (G) and immature seed (H) on the A genome.
Figure 9.
Figure 9.
The Effect of Differential Expression of siRNAs on Homoeolog Expression Levels. (A) and (B) Numbers of D homoeologs with siRNAs differentially expressed between S3 and Ae. tauschii (DD) or A homoeologs between S3 and T. turgidum (AABB) in young spikes (A) and immature seed (B). (C) GO enrichment analysis of D homoeologs in young spikes with differentially expressed siRNAs between S3 and Ae. tauschii. (D) MapMan pathway analysis in young spikes of A homoeologs between S3 and T. turgidum or D homoeologs between S3 and Ae. tauschii with differentially expressed siRNAs. (E) and (F) Two examples of downregulation of siRNA expression and concerted upregulation of neighboring D homoeolog AEGTA32405 (E) and AEGTA00388 (F) in young spikes of S3 plants compared with their expression in the diploid progenitor Ae. tauschii (DD).
Figure 10.
Figure 10.
A Small RNA–Dominated Model for Homoeolog Expression Regulation during Wheat Allohexaploidization. AABB, T. turgidum; DD, Ae. tauschii; AABBDD, nascent allohexaploid progeny; ELD-ab, genes with expression level similar to that in T. turgidum; ELD-d, genes with expression level similar to that in Ae. tauschii. (A) miRNA regulation module. Nonadditively expressed miRNAs target genes involved in growth, stress, and flowering. (B) siRNA regulation module. Note increased siRNA expression on the D subgenome and reduced transcript production (wavy lines). Rectangles represent genes; yellow lines represent repeat sequences; blue triangles, siRNAs. Question marks indicate unknown conditions in the B genome. (C) Expression patterns of protein-coding genes, their distinct functions (development, growth vigor, and adaptation), and key representative genes in nascent allohexaploid wheat.

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