Mating-Type Locus Organization and Mating-Type Chromosome Differentiation in the Bipolar Edible Button Mushroom Agaricus bisporus

Abstract

In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR, encoding pheromone precursors and pheromone receptors, and HD, encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus, and of the related variety burnettii. We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and β-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the β-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between β-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus, constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.

Keywords: Agaricus; automixis; basidiomycete; bipolar; genetic map; heterothallic; homeodomain genes; mating-type chromosome; mating-type genes; pheromone genes; pheromone receptor genes; pseudo-homothallic; recombination suppression; sex chromosomes; tetrapolar.

Figure 1

Mapping the mating-type (MAT) locus on chromosome 1 of A. bisporus var. bisporus strain H97 with genetic data on markers previously published [38,54]. Only markers that made possible to anchor the scaffold 1 sequence are located. PRxxx, ACMxx are CAPS (cleaved amplified polymorphism sequence) markers; AbSSRxx are microsatellites. When available, the GenBank reference number is given in bracket. The position of the BSN locus controlling the presence of two or four spores in basidia is also indicated.

Figure 2

Sequence alignment of (A) HD2 and (B) HD1 homeodomain motifs of A. bisporus mating-type proteins compared to HD1 and HD2 mating type proteins of C. cinerea (GenBank EAU92789, CAA44210, CAA566131, CAA56132). Marked are the positions of the three helixes of the homeodomains and the conserved aa in the DNA-binding motif in Helix III (after [80]). Dashes in the HD2 sequences denote the position of insertion of the three extra amino-acid in the HD1 sequences.

Figure 3

Comparison of the region with the MAT locus on chromosome 1 of A. bisporus var. bisporus H97 and A. bisporus var. burnettii JB137-S8. Shared genes are shown by black boxes, and HD1 and HD2 genes by boxes striped from bottom left to top right and from top left to bottom right, respectively. Closed arrows beneath genes indicate direction of transcription. Genes of DNA similarity or high identity between the two strains are marked with percentages of identity. Insertions are shown by the insets (note the different relative size of the inset in strain JB137-S8). Direct or inversely repeated sequences are marked by arrowheads, and grey boxes indicate DNA sequences that in blastx searches in the NCBI GenBank hit proteins with transposon/retrotransposon activities.

Figure 4

Comparison of (A) the first 74 aa of HD2 protein a2-1 of A. bisporus var. bisporus H97 with a translated sequence from a footprint (fp) of gene a2-2 in A. bisporus var. burnettii JB137-S8 (a2-2 fp) and (B) of 71 aa from the C- terminus of Mip1 of strain JB137-S8 and the translated sequence of a mip1 footprint (Mip1 fp) in the MAT locus of the strain (dashes in the sequences are introduced for optimization of the alignment; * mark positions of no aa due to stop codons, ▲ and ▼, beneath the figure positions of frameshifts by insertion or deletion of a base, respectively).

Figure 5

Phylogenetic analysis of the N-terminal dimerization domains and the homeodomains of (A) HD1 and (B) HD2 proteins from Agaricomycotina. Bars mark paralogous proteins from fungi with more than one HD1-HD2 gene pair. Proteins with N-terminal truncations (A. bisporus var. bisporus H97 b2-1, Tremella mesenterica Sxi2) in the ClustalW alignment were manually adjusted to assign them appropriately in position. Extra aa were added to the short N-terminal sequence of C. cinerea a2-2 from EAU92788 since the non-functional gene contains a preliminary stop codon in the 5′ end of the gene shortly after the normal ATG start codon [85]. Proteins from Tremellomycetes served as outgroup. Accession numbers refer either to NCBI GenBank or to protein IDs at the fungal JGI databases.

Figure 5

Phylogenetic analysis of the N-terminal dimerization domains and the homeodomains of (A) HD1 and (B) HD2 proteins from Agaricomycotina. Bars mark paralogous proteins from fungi with more than one HD1-HD2 gene pair. Proteins with N-terminal truncations (A. bisporus var. bisporus H97 b2-1, Tremella mesenterica Sxi2) in the ClustalW alignment were manually adjusted to assign them appropriately in position. Extra aa were added to the short N-terminal sequence of C. cinerea a2-2 from EAU92788 since the non-functional gene contains a preliminary stop codon in the 5′ end of the gene shortly after the normal ATG start codon [85]. Proteins from Tremellomycetes served as outgroup. Accession numbers refer either to NCBI GenBank or to protein IDs at the fungal JGI databases.

Figure 6

Sequence alignment of pheromone precursors of A. bisporus var. bisporus H97 and A. bisporus var. burnettii JB137-S8. Stretches of charged amino acids for possible N-terminal processing [8,93] and the typical CAAX motif at the C-terminus for farnesylation and carboxymethylation [99] are underlined.

Figure 7

Phylogenetic analysis of the seven-transmembrane regions of putative mating-type-specific and non-mating-type STE3-like pheromone receptors from the Agaricomycetes A. bisporus var. bisporus H97, A. bisporus var. burnettii JB137-S8, C. cinerea [87], L. bicolor [86], R. placenta [89] and S. commune [92]. The α mating type pheromone receptor Ste3p from the ascomycete Saccharomyces cerevisiae served as an outgroup. Black bars mark proteins whose genes are localized within PR mating type loci (in the bipolar P. placenta, the orthologous PR locus with pheromone precursor and pheromone receptor genes seem to have lost mating function [89]. Bα and Bβ locus and B subgroup 1 to 3 indicate paralogous mating type proteins within a species as defined by classical genetics and transformation analyses [8,93]. Grey bars mark genes that are present on the same chromosome than the PR mating-type locus. Accession numbers refer either to NCBI GenBank or to protein IDs at the fungal JGI databases. _m denotes adjustments done to C. cinerea gene models [8].

Figure 8

Synonymous substitution rates between the homokaryotic genomes of A. bisporus var. bisporus H97 and H39 (A) and between A. bisporus var. burnettii H119p1 and H119p4 (B). The Y-axis shows the synonymous divergence levels between the alleles found in the homokaryotic genomes of H97 and H39 of alternative mating types, issued from the U1 cultivar. The X-axis shows the gene order along the H97 genome. Yellow highlights on the background show the putative centromeric regions. Genes found in and around the mating-type loci are highlighted with different colours (blue for HD genes, orange for β-fg1 gene, brown for mip1 gene and magenta for STE3-like putative pheromone receptor genes). Other chromosomes are shown in Supplementary Figure S2.

Figure 9

Identity and organization of mating-type genes in the A. bisporus var. burnettii 119/9 genome. The two constitutive homokaryon genomes (i.e., H119p1 and H119p4) are shown in the top and bottom, respectively. Homeodomain (HD) genes are shown with striped boxes, and flanking β-fg and mip1 genes are shown with solid black boxes. Orthologous gene pairs are linked with grey shaded rectangles. The mating-type genes as well as flanking genes are found in three distinct contigs in the homokaryon H119p1 genome (indicated by black bars, the β-fg2 gene being split onto two contigs), while they are all found (β-fg genes in tandem) in one contig in the H119p4 genome. A ca. 140 kb-long inversion was identified between the two genomes of different mating types, figured by the pair of dark grey triangles. The inversion breakpoints are indicated by light grey symbols within the involved genes and non-coding DNA regions.

Figure 10

Genetic to physical distance plot for adjacent markers for a 1 MB genomic region on chromosome 1 of A. bisporus var. burnettii strain 119/9. The red dot indicates the position of the marker pair flanking the HD genes on chromosome 1.