Duplicate genes increase expression diversity in closely related species and allopolyploids

Abstract

Polyploidy or whole genome duplication (WGD) provides raw genetic materials for sequence and expression evolution of duplicate genes. However, the mode and tempo of expression divergence between WGD duplicate genes in closely related species and recurrent allopolyploids are poorly understood. Arabidopsis is a suitable system for testing the hypothesis that duplicate genes increase expression diversity and regulatory networks. In Arabidopsis, WGD occurred more than once before the split between Arabidopsis thaliana and Arabidopsis arenosa, and both natural and human-made allotetraploids are available. Comparative genomic hybridization analysis indicated that single-copy and duplicate genes after WGD were well preserved in A. thaliana and A. arenosa. Analysis of gene expression microarrays showed that duplicate genes generally had higher levels of expression divergence between two closely related species than single-copy genes. The proportion of the progenitors' duplicate genes that were nonadditively expressed in the resynthesized and natural allotetraploids was significantly higher than that of single-copy genes. Duplicate genes related to environmental stresses tended to be differentially expressed, and multicopy duplicate genes were likely to diverge expression between progenitors and in the allotetraploids. Compared with single-copy genes, duplicate genes tended to contain TATA boxes and less DNA methylation in the promoter regions, facilitating transcriptional regulation by binding transcription factors and/or cis- and trans-acting proteins. The data suggest an important role of WGD duplicate genes in modulating diverse and novel gene expression changes in response to external environmental cues and internal genetic turmoil such as recurrent polyploidy events.

Fig. 1.

Sequence comparison between A. thaliana and A. arenosa FLC-containing orthologous regions on chromosome 5. BAC sequence from A. arenosa was aligned to A. thaliana genomic sequence using genome VISTA. Purple, blue, and pink represent protein coding exons, introns, and intergenic regions, respectively. The percentage of nucleotide sequence identity between A. thaliana and A. arenosa is shown at the right. The orientation and names of loci are shown at the top, and the genomic coordinates are shown at the bottom. FLC locus designates At5g10140.

Fig. 2.

An experimental model for testing the effects of polyploidy on expression evolution of single-copy and duplicate genes. (A) A whole genome duplication (WGD) occurred 20–40 Mya in Arabidopsis. (B) WGD is accompanied by mutations, deletions, and chromosomal rearrangements, a process called a diploidization, resulting in loss of genes (marked with an X). (C) Retention of duplicate genes (connected by gray lines) and persistence of single-copy genes (green and blue boxes) in the ancestoral Arabidopsis species. (D) Speciation of A. thaliana and A. arenosa (≈6 Mya) and artificial tetraploidization. Allotetraploids were formed by pollinating A. thaliana tetraploid with A. arenosa outcrossing tetraploid (13). The resynthesized allotetraploids were self-pollinated for 6 generations, and the gene expression changes were examined (13). Two allotetraploids plants (Allo733 and Allo738) shown were derived from independent lineages in the 7th generation of selfing. Two parental loci become homoeologous in allotetraploids.

Fig. 3.

Expression divergence of single-copy and duplicate genes in resynthesized and natural allotetraploids and their progenitors. Proportions of single-copy and duplicate genes are shown to be differentially expressed between A. thaliana (At4) and A. arenosa (Aa) (A) and nonadditively expressed in allotetraploids Allo733 (B), Allo738 (C), and A. suecica (D) relative to the midparent value (MPV). Pearson's χ² tests with Yates' continuity correction are as follows: χ² = 13.8, degrees of freedom (df) = 1, P = 0.0002 (A); χ² = 23.5, df = 1, P = 1.3 × 10⁻⁶ (B); and χ² = 16.5, d.f. = 1, P = 4.8 × 10⁻⁵ (C). (D) Fisher's Exact test, P = 0.049. Unless noted otherwise, the standard errors were estimated using 10,000 replications of bootstrapping.

Fig. 4.

Differential expression of duplicate genes in GOSlim biological process classifications in allotetraploids and their progenitors. (A) Duplicate genes in external biological processes are differentially expressed between A. thaliana and A. arenosa (RS, χ² = 15.3, df = 1, P = 9.4 × 10⁻⁵ and RA, χ² = 10.0, df = 1, P = 0.002). (B) Homoeologous duplicate genes in “response to stress and external stimuli” are nonadditively expressed in the allotetraploid Allo733 relative to the MPV (RS, χ² = 21.6, df = 1, P = 3.4 × 10⁻⁴ and RA, χ² = 15.5, df = 1, P = 8.0 × 10⁻⁵). (C) Homoeologous duplicate genes in “response to stress and external stimuli” are nonadditively expressed in the allotetraploid Allo738 relative to the MPV (RS, χ² = 42.5, df = 1, P = 7.2 × 10⁻¹¹ and RA, χ² = 39.0, df = 1, P = 4.3 × 10⁻¹⁰). (D) Homoeologous duplicate genes in “response to stress” are nonadditively expressed in the natural allotetraploid A. suecica relative to the MPV (randomization test, P = 0.02). Dashed lines indicate the percentage of total differentially expressed duplicate genes. RS: response to stress; RA: response to abiotic or biotic stimulus; EP: energy pathways; DP: developmental processes; CB: cell organization and biogenesis; TP: transport; TC: transcription; ST: signal transduction; PM: protein metabolism; NM: nucleotide metabolism; OB: other biological processes; OC: other cellular processes; and OM: other metabolic processes.

Fig. 5.

High levels of expression divergence among the genes with multiple paralogs and low levels of DNA methylation in duplicate genes. (A) Differentially expressed genes with the number of paralogs between A. thaliana and A. arenosa (open triangle) and nonadditively expressed genes in the allotetraploids Allo733 (open circle), Allo738 (open square), and A. suecica (filled square). (Pearson's χ² test with Yates' continuity correction, degree of freedom = 3, At4 vs. Aa, χ² = 80.0 and P = 0; Allo733, χ² = 69.6 and P = 5.3 × 10⁻¹⁵; Allo738, χ² = 69.5 and P = 5.4 × 10⁻¹⁵; A. suecoca, χ² = 125.4114 and P = 2.2 × 10⁻¹⁶). The P values of logistic regression were 1.2 × 10⁻⁵ (A. thaliana and A. arenosa), 4.7 × 10⁻⁶ (Allo733), 6.6 × 10⁻⁵ (Allo738) and 6.9 × 10⁻¹⁶ (A. suecica). (B) Upstream regions of duplicate genes are less methylated DNA than that of single-copy genes (Pearson's χ² test with Yates' continuity correction, P < 0.001).