Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing

Abstract

Most eukaryotes have genomes that exhibit high levels of gene redundancy, much of which seems to have arisen from one or more cycles of genome doubling. Polyploidy has been particularly prominent during flowering plant evolution, yielding duplicated genes (homoeologs) whose expression may be retained or lost either as an immediate consequence of polyploidization or on an evolutionary timescale. Expression of 40 homoeologous gene pairs was assayed by cDNA-single-stranded conformation polymorphism in natural (1- to 2-million-yr-old) and synthetic tetraploid cotton (Gossypium) to determine whether homoeologous gene pairs are expressed at equal levels after polyploid formation. Silencing or unequal expression of one homoeolog was documented for 10 of 40 genes examined in ovules of Gossypium hirsutum. Assays of homoeolog expression in 10 organs revealed variable expression levels and silencing, depending on the gene and organ examined. Remarkably, silencing and biased expression of some gene pairs are reciprocal and developmentally regulated, with one homoeolog showing silencing in some organs and the other being silenced in other organs, suggesting rapid subfunctionalization. Duplicate gene expression was examined in additional natural polyploids to characterize the pace at which expression alteration evolves. Analysis of a synthetic tetraploid revealed homoeolog expression and silencing patterns that sometimes mirrored those of the natural tetraploid. Both long-term and immediate responses to polyploidization were implicated. Data suggest that some silencing events are epigenetically induced during the allopolyploidization process.

Figure 1

Schematic representation of the phylogenetic history of diploid and allopolyploid Gossypium. Polyploid formation occurred ≈1.5 million years ago after hybridization between A genome and D genome diploids (19). After formation, the ancestral polyploid radiated into lineages represented now by the five species shown.

Figure 2

Homoeologous gene pair expression in cotton ovules. “A” and “D” indicate diploid A- and D-genome species G. herbaceum and G. raimondii, respectively; “AD” indicates allotetraploid G. hirsutum; and “g” indicates genomic DNA control from G. hirsutum. (A and B) Examples of RT-PCR results. Reactions were performed with (+) or without (−) RT. Genes include adhA (A) and gene F12 (B); (C–G) cDNA-SSCP analysis. Genes include (C) COP-1 interacting protein (F12); (D) syntaxin (C3); (E) sulfate transporter (G11); (F) alcohol dehydrogenase A; and (G) flavonoid 3′ hydroxylase (G8). Note equal expression of homoeologs in C–E, but biased expression (unequal intensities of the two bands in the AD lane) in F and G. In some cases, the tetraploid sequences migrate differently from the corresponding diploid sequences because of mutations that have occurred postpolyploidization or because the diploid species are the best living models rather than the actual progenitors of the natural allopolyploid.

Figure 3

Transcript levels from duplicated gene pairs in multiple organs of G. hirsutum, as determined by cDNA-SSCP analysis. “Dt” and “At” denote homoeologous genes from the A or D genome of the natural allopolyploid G. hirsutum, for which the numbers indicate transcript percentages as determined by PhosphorImager. (Note that for some genes At ran further on the gels and for other genes Dt ran further.) Genes include (A) adhA alcohol dehydrogenase A; (B) adhD alcohol dehydrogenase D; (C) putative protein A1520; and (D) oxalate oxidase (gene B5). One strand is shown for RT-PCR products that were labeled on both strands.

Figure 4

Transcript levels of homoeologous gene pairs in synthetic allopolyploid cotton. “Dt” and “At” indicate homoeologous genes from the A or D genome. Numbers indicate transcript percentages as determined by PhosphorImager. Dashes indicate lanes that were not scored on the gel shown. (A) adhA, alcohol dehydrogenase; (B) flavonoid 3′ hydroxylase (gene G8).

Figure 5

Expression of homoeologous gene pairs in three species of allopolyploid cotton. Phylogenetic relationships among the species used are shown in Fig. 1. (A–C) adhA. (D–F) adhD.