Genomewide nonadditive gene regulation in Arabidopsis allotetraploids

Abstract

Polyploidy has occurred throughout the evolutionary history of all eukaryotes and is extremely common in plants. Reunification of the evolutionarily divergent genomes in allopolyploids creates regulatory incompatibilities that must be reconciled. Here we report genomewide gene expression analysis of Arabidopsis synthetic allotetraploids, using spotted 70-mer oligo-gene microarrays. We detected >15% transcriptome divergence between the progenitors, and 2105 and 1818 genes were highly expressed in Arabidopsis thaliana and A. arenosa, respectively. Approximately 5.2% (1362) and 5.6% (1469) genes displayed expression divergence from the midparent value (MPV) in two independently derived synthetic allotetraploids, suggesting nonadditive gene regulation following interspecific hybridization. Remarkably, the majority of nonadditively expressed genes in the allotetraploids also display expression changes between the parents, indicating that transcriptome divergence is reconciled during allopolyploid formation. Moreover, >65% of the nonadditively expressed genes in the allotetraploids are repressed, and >94% of the repressed genes in the allotetraploids match the genes that are expressed at higher levels in A. thaliana than in A. arenosa, consistent with the silencing of A. thaliana rRNA genes subjected to nucleolar dominance and with overall suppression of the A. thaliana phenotype in the synthetic allotetraploids and natural A. suecica. The nonadditive gene regulation is involved in various biological pathways, and the changes in gene expression are developmentally regulated. In contrast to the small effects of genome doubling on gene regulation in autotetraploids, the combination of two divergent genomes in allotetraploids by interspecific hybridization induces genomewide nonadditive gene regulation, providing a molecular basis for de novo variation and allopolyploid evolution.

Figure 1.

(A) Production of stable synthetic allotetraploids (Allo733 and 738). A self-fertile A. thaliana autotetraploid (Ler, At4) was pollinated with a natural A. arenosa tetraploid (Aa). Multiple independent allotetraploids in S1 were self-pollinated by single-seed descent to the S5 generation. Allo733 and Allo738 resembled A. arenosa and natural A. suecica (Comai et al. 2000). Fluorescence in situ hybridization (FISH) analysis indicates that two sets of centromeres in Allo733 are derived from A. thaliana (At4) and A. arenosa (Aa), respectively. Bar, 5 mm. Allo, allotetraploid.

Figure 2.

Logarithm fold change vs. per-gene standard deviation in a replicated experiment containing four dye swaps. The hybridization probes were cDNAs from Allo733 and two parents, A. thaliana tetraploid and A. arenosa. The data were analyzed using a linear model as previously described (Tian et al. 2005). Green, black, and red dots indicate the pools of significant genes selected by multiple comparison tests (false discovery rate, FDR, α = 0.05) using a per-gene variance, a common variance, and the intersection of the two, respectively. Statistical significance for extremely smallfold changes was detected for two features replicated 6 and 49 times within each microarray slide, indicating the power of replication in microarray experiments.

Figure 3.

Transcriptome divergence and nonadditive gene expression between allotetraploids and their progenitors. (A) The proportion of transcriptome that was highly expressed in A. thaliana (At4), A. arenosa (Aa), or equally expressed (both). (B) Venn diagram showing the number of genes with expression divergence between the progenitors (blue) and between Allo733 (red) or Allo738 (green) and the midparent value (MPV, supplemental Figure 1 at http://www.genetics.org/supplemental/). (C) Chromosomal distribution of the 820 genes displaying nonadditive expression in both allotetraploids (see text).

Figure 4.

Downregulation of A. thaliana genes in the synthetic allotetraploids. (A) Distribution of nonadditively expressed genes detected in each allotetraploid (Allo733 or Allo738) or both allotetraploids (Allos). (B) The nonadditively expressed genes in each allotetraploid matched the genes that were highly expressed in the A. thaliana autotetraploid. (C) The nonadditively expressed genes in each allotetraploid matched the genes that were highly expressed in A. arenosa. (D) The nonadditively expressed genes matched the genes that were equally expressed in both parents. The percentages of downregulated genes are indicated above the columns in each histogram.

Figure 5.

Classification of nonadditively expressed genes detected in synthetic Arabidopsis allotetraploids. (A) The 820 genes detected in both Allo733 and Allo738 lines (Allos) were classified into 15 functional categories using the PEDANT analysis system (http://mips.gsf.de/proj/thal/db/index.html) (Arabidopsis Genome Initiative 2000). (B) The percentages of the genes in each functional category detected in Allo733, Allo738, or both (Allos). The relative ratios in the y-axis were estimated using the percentage of genes detected in each functional category in an Allo line divided by the percentage of all ∼26,000 annotated genes in the Arabidopsis genome (Arabidopsis Genome Initiative 2000). The percentage of the genes detected in the Allo line equal to that of all genes in the whole genome is shown as 100% (dashed line).

Figure 6.

Nonadditive gene regulation occurs in various pathways. (A) Progenitor-dependent repression of the genes involved in the ethylene biosynthesis pathway in Arabidopsis allotetraploids. Each number in parentheses below an enzyme or molecule in the pathways indicates the fold change for the expression of a gene, homolog (h), or putative (p) homolog detected by microarray analysis. Red, green, blue, and purple colors indicate that gene expression differences are detected in both allotetraploids (Allo733 and -738), in Allo738, in Allo733, and between the two parents, respectively. (B) Repression of heat-shock protein (HSP) genes in Arabidopsis allotetraploids. Thirty-one out of 33 HSPs were repressed in each allotetraploid (Allo733 or -738). The length and directions of vertical bars indicate logarithm fold changes in up- (above the line) or downregulation (below the line) of the HSPs relative to the midparent in the allotetraploid lines.

Figure 7.

Developmental and parental contributions to nonadditive gene expression. (A) Venn diagrams of the genes that displayed nonadditive expression in leaves and flower buds in Allo733. Only 175 of 2542 genes showed overlap between leaves and flower buds. (B) Verification of 11 genes detected in microarrays by qRT–PCR. The gene expression levels in the parents were higher in A. thaliana (At > Aa), higher in A. arenosa (Aa > At4), and equal (At4 = Aa). (C) SSCP and CAPS analyses showing parental contributions to nonadditive gene regulation in the allotetraploids. The genes studied in B and C are in the functional classifications of stress (HSP or HSP90 and HSP17.6b); cell cycle, defense, and aging (CYC, CHI, LRR, PDF, and WRKY); metabolism and energy (BCB, PORa, PORb, and SPP); and flower development (COL2 and FLC). The restriction enzymes used in CAPS analysis are indicated in parentheses.