Figure 1.
Diagram outlining the meanings of terms relating to allopolyploid transcription that are used in this study, and which derive from Yoo et al. [46]. Illustrated is an example of a gene, “A”, that is present as orthologs in the parents E8 and AR5, and as homeologs in the allopolyploid Lp1. The three homeolog expression terms are possible outcomes for relative gene expression in the allopolyploid. Homeolog expression bias: differential expression is not found between the two orthologs, but has arisen in the allopolyploid [43]. Homeolog expression blending: expression differences between the two orthologs have been lost in the allopolyploid. Homeolog expression reversal: the highly expressed ortholog has lower expression than the other homeolog in the allopolyploid.
Figure 2.
Phylogeny and culture morphology of Epichloë and Neotyphodium species.
(A) Phylogeny of Epichloë and Neotyphodium fungi. Haploid species are indicated in black, while some of the many natural allopolyploid species in these genera are shown to the right. Colored lines indicate the parents of these allopolyploids, including the ancestors of the allopolyploid investigated in this study, Lp1 (solid red line). Phylogeny modified from published sources [49], [51], [82]. Culture morphology of (B) N. lolii AR5, (C) allopolyploid N. lolii×E. typhina Lp1 and (D) E. typhina E8. All culture panels 30 mm square.
Table 1.
Read counts from Illumina sequencing of Lp1 and its parents.
Table 2.
SNP counts by diagnostic class.
Figure 3.
Parental and allopolyploid expression patterns from extreme differentially expressed genes.
Plots show the relative expression level between the two homeologs in Lp1 (“Lp1”), between AR5 and E8 orthologs (“Parents”), between the AR5 ortholog and the AR5-like Lp1 homeolog (“AR5 copy”), and between the E8 ortholog and the E8-like Lp1 homeolog (“E8 copy”). (A) Genes with zero expression for one Lp1 homeolog, but non-zero expression for the other; (B) genes with >50-fold difference in expression between the Lp1 homeologs; (C) genes with >50-fold difference in expression between AR5 and E8 orthologs. Heat spectrum (bottom) varies between similar expression (white) and different expression (black). Vertical bars to the right indicate where genes are physically clustered; these are labeled by supercontig number. Genes on supercontig 148, present in (A) and (B), are physically contiguous in the genome. Genes 049470 and 049490, despite their numbering, are not physically adjacent.
Figure 4.
Gene loss in clustered extreme differentially expressed genes in Lp1.
Three clusters of genes with extreme differential expression are shown; the supercontig from which they derive is indicated above. Orange arrows represent genes with extreme differential expression; blue, genes not showing extreme differential expression; and grey, genes culled by our mapping criteria (no expression information). Raw read numbers from AR5 and E8 homeologs, respectively, are shown in ellipses near each gene. Green dotted lines represent AT-rich regions. Below the genomic maps are PCR-RFLP results (reverse images of ethidium bromide-stained polyacrylamide gels) from Lp1, E8, and AR5 genomic DNA. The gene is indicated in bold above, the restriction enzyme to the right, and size marker positions to the left.
Figure 5.
Cumulative distributions of gene expression ratios for Lp1 and parents.
Plotted are the cumulative distributions of genes by normalized gene expression ratio between homeologs in Lp1 (A), and between orthologs from the parents (B). Those with higher expression from the E8-derived copy are shown in orange (left); higher AR5-derived expression in green (right). Grey shading indicates the pool of genes with less than 2-fold expression difference between copies (not differentially expressed). The bias towards more orthologs in E8 having greater relative expression can be seen in (B).
Figure 6.
Fate of gene expression in the allopolyploid compared to the parents.
Relative expression of the AR5-derived and E8-derived gene copies was calculated for Lp1 and its parents; ‘biased’ is 2-fold or greater expression difference. The right indicates numbers of genes in each of the nine possible combinations of biased/non-biased expression [46]. Box-graphs represent relative expression ratios, with the position of the line within the box indicating the E8:AR5 expression ratio. Specifically, red lines high in the box indicate E8 homeolog/ortholog expression bias, black lines mid-way in the box indicate equal expression, and blue lines low in the box indicate AR5 bias. These are grouped into four expression response categories: maintenance of the parental expression pattern in Lp1; homeolog expression blending; homeolog expression bias; and homeolog expression reversal, indicated by the colored boxes on the right. On the left are proportions of genes showing bias towards AR5 or E8 expression or no bias for the parents (top) and Lp1 (bottom). The colored half-circle tabs link the nine expression combinations to the plots on the left.
Table 3.
Similarities in the transcriptional response to allopolyploidy in Lp1 and natural cotton allopolyploids.
Figure 7.
Frequent expression-level dominance from expression changes in the non-dominant homeolog.
Genes showing differential expression in the parents were divided into those where total homeolog expression in Lp1 matches the lowly expressed parental ortholog, the highly expressed parental ortholog, or an intermediate level of expression (left panel; percentages are of all genes showing differential parental expression). Expression-level dominance patterns are indicated by blue boxes. On the right, expression-level dominance genes are divided into those where the non-dominant homeolog changed expression relative to the parents (orange boxes), and those where it did not (percentages are of all expression-level dominant genes). The heights of the yellow and blue dots represent expression of the parental orthologs. The green dot(s) represents expression of homeolog(s): larger green dots on the left represent both homeologs combined; on the right these have been separated into the two individual homeolog expression levels, as indicated below. The ± sign indicates the homeolog may or may not have changed expression. Expression-level dominance of only one parent is illustrated, but the numbers are for both.
Figure 8.
Intrinsic gene regulatory features underlie transcriptional response patterns in the allopolyploid.
Change in expression of a gene (in blue) from the two parents (circles) to the allopolyploid (ellipse) is shown symbolically where a modulon (orange star; see text for details) positively regulates gene expression (green interaction). (A) Expression is high in one parent, with the modulon not active in the other. In the allopolyploid, this modulon only acts on its own homeolog, resulting in simple inheritance of biased gene expression. Failure of the modulon to recognize the other homeolog can result from a cis-acting change (purple cross) in one ortholog (B). The parental situation is the same as in (A), but the modulon acts on both homeologs, yielding homeolog expression blending in the allopolyploid. (C) Expression of the two orthologs is similar in the parents, but the modulon preferentially stimulates transcription of one homeolog in the allopolyploid, generating homeolog expression bias as a result of a cis-acting difference (purple cross) that does not change expression in the parent. Analogous outcomes will be achieved in all cases if the modulon is repressive rather than stimulatory (Figure S4). The promoter is indicated by a transcription arrow, and transcript levels by the number of thin wavy lines.