Differential Gene Expression between Fungal Mating Types Is Associated with Sequence Degeneration

Abstract

Degenerative mutations in non-recombining regions, such as in sex chromosomes, may lead to differential expression between alleles if mutations occur stochastically in one or the other allele. Reduced allelic expression due to degeneration has indeed been suggested to occur in various sex-chromosome systems. However, whether an association occurs between specific signatures of degeneration and differential expression between alleles has not been extensively tested, and sexual antagonism can also cause differential expression on sex chromosomes. The anther-smut fungus Microbotryum lychnidis-dioicae is ideal for testing associations between specific degenerative signatures and differential expression because 1) there are multiple evolutionary strata on the mating-type chromosomes, reflecting successive recombination suppression linked to mating-type loci; 2) separate haploid cultures of opposite mating types help identify differential expression between alleles; and 3) there is no sexual antagonism as a confounding factor accounting for differential expression. We found that differentially expressed genes were enriched in the four oldest evolutionary strata compared with other genomic compartments, and that, within compartments, several signatures of sequence degeneration were greater for differentially expressed than non-differentially expressed genes. Two particular degenerative signatures were significantly associated with lower expression levels within differentially expressed allele pairs: upstream insertion of transposable elements and mutations truncating the protein length. Other degenerative mutations associated with differential expression included nonsynonymous substitutions and altered intron or GC content. The association between differential expression and allele degeneration is relevant for a broad range of taxa where mating compatibility or sex is determined by genes located in large regions where recombination is suppressed.

Keywords: differential gene expression; mating-type chromosomes; premature stop codon; sequence degeneration; sexual antagonism; transposable elements.

Fig. 1.

—Heatmap and hierarchical clustering of differentially expressed genes (FDR < 0.05) between haploid a₁ and a₂ cultures of Microbotryum lychnidis-dioicae under a low nutrient condition. Each column shows a replicate for each haploid cell culture. The Z-score denotes the relative gene expression level, with blue and red representing high and low expression, respectively. On each node of the clustering tree, bootstrap support values are shown based on 10,000 replicates.

Fig. 2.

—Interaction plots for pairs of explanatory variables in overall GLM of differential gene expression between mating types of Microbotryum lychnidis-dioicae. Y-axes are GLM-predicted response values of differential expression ratio between alleles in a₁ and a₂ haploid genomes, and x-axes are allele differences between alleles in a₁ and a₂ haploid genomes in predicted protein length as the predictor variable, then binned into levels of interacting categorical predictor variables (i.e., panel A, genomic compartment) or other interacting continuous predictor variables (i.e., panels B–D; the lowest bin being no differences between alleles, and low and high bins being split at the median value among genes with non-zero differences between alleles). (A) Interaction plot between protein length differences and genomic compartment. Genomic compartments include autosomes, PARs, youngest and oldest evolutionary strata. (B) Interaction plots between protein length differences and differences in TE insertions. (C) Interaction plots between protein length differences and nonsynonymous substitution (dN) rate differences. (D) Interaction plots between protein length differences and GC content differences.

Fig. 3.—

Comparisons of differentially expressed (DE) versus nondifferentially expressed (non-DE) genes between mating types of Microbotryum lychnidis-dioicae for various degeneration-associated traits within genomic compartments. (A) Nonsynonymous sequence divergence, dN, between alleles of DE and non-DE genes. (B) TE insertion number differences between alleles within 20 kb (upstream and downstream) of DE and non-DE genes. (C) Proportions of differentially expressed (DE) and non-differentially expressed (non-DE) genes with different protein lengths between alleles. (D) Intron content proportional differences between alleles of DE and non-DE genes. (E) Total GC content (GC0) proportional differences between alleles of DE and non-DE genes. Analyzed allele differences represent absolute value comparisons (i.e., unoriented with regard to allelic expression levels). Comparisons in panels (A), (C)–(E) reflect Wilcoxon rank sum tests; panel (B) reflects a two-proportion Z-test. Significance levels shown as ***P < 0.001, *P < 0.05; non-significant test results shown in supplementary tables S4 and S6–S9, Supplementary Material online. Genomic compartments include autosomes, PARs, youngest and oldest evolutionary strata. The notation “a” indicates that the youngest evolutionary strata contained only one DE gene, precluding comparisons to non-DE genes within this compartment. For boxplot, the horizontal bars (from bottom to top) represent the 25% quartile, median, and 75% quartile, respectively.

Fig. 4.

—Significant predictors of the degree of differential expression between mating types of Microbotryum lychnidis-dioicae testing directional effects of degeneration-associated traits. (A) Relationship between expression ratio and oriented TE insertion differences in the region from 10 kb upstream to the gene, where the trait was calculated as the TE number for the allele with lower expression minus the TE number for the allele with higher expression; a positive value thus represented an excess of TEs in the allele with lower expression. (B) Relationship between expression ratio and oriented predicted protein length differences, where the trait was calculated as the ratio of the length for the allele with higher expression divided the length for the allele with lower expression; a larger ratio thus represented a shorter length for the allele with lower expression.

Fig. 5.

—Average indel numbers and proportions of genes with different stop codon positions between alleles of differentially expressed genes of Microbotryum lychnidis-dioicae. Among genes having alleles with different predicted protein lengths, boxplot of average indel numbers for both differentially expressed (DE, in black) and non-DE genes (in gray) across various genomic compartments (A), and barplots for proportions of genes with different stop codon positions for both DE and non-DE genes across genomic compartments (B). **P < 0.01, *P < 0.05, ‘.’: P < 0.1. NS, not significant. Genomic compartments correspond to autosomes, PARs, youngest and oldest evolutionary strata. For boxplot, the horizontal bars (from bottom to top) represent the 25% quartile, median, and 75% quartile, respectively.