Recent and Massive Expansion of the Mating-Type-Specific Region in the Smut Fungus Microbotryum

The presence of large genomic regions with suppressed recombination (SR) is a key shared property of some sex- and mating-type determining (mat) chromosomes identified to date in animals, plants, and fungi. Why such regions form and how they evolve remain central questions in evolutionary genetics. The smut fungus Microbotryum lychnis-dioicae is a basidiomycete fungus in which dimorphic mat chromosomes have been reported, but the size, age, and evolutionary dynamics of the SR region remains unresolved. To identify the SR region in M. lychnis-dioicae and to study its evolution, we sequenced 12 genomes (6 per mating type) of this species and identified the genomic contigs that show fixed sequence differences between the mating types. We report that the SR region spans more than half of the mat chromosome (>2.3 Mbp) and that it is of very recent origin (∼2 × 10⁶ years) as the average sequence divergence between mating types was only 2% in the SR region. This contrasts with a much higher divergence in and around the mating-type determining pheromone receptor locus in the SR, suggesting a recent and massive expansion of the SR region. Our results comprise the first reported case of recent massive SR expansion documented in a basidiomycete fungus.     

Chaos of Rearrangements in the Mating-Type Chromosomes of the Anther-Smut Fungus Microbotryum lychnidis-dioicae

Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures.     

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.     

The mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma represents a model for early evolution of sex chromosomes

We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains), derived from one N. tetrasperma heterokaryon (mat A+mat a), was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first "evolutionary stratum", genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers.     

Testing for the Footprint of Sexually Antagonistic Polymorphisms in the Pseudoautosomal Region of a Plant Sex Chromosome Pair

The existence of sexually antagonistic (SA) polymorphism is widely considered the most likely explanation for the evolution of suppressed recombination of sex chromosome pairs. This explanation is largely untested empirically, and no such polymorphisms have been identified, other than in fish, where no evidence directly implicates these genes in events causing loss of recombination. We tested for the presence of loci with SA polymorphism in the plant Silene latifolia, which is dioecious (with separate male and female individuals) and has a pair of highly heteromorphic sex chromosomes, with XY males. Suppressed recombination between much of the Y and X sex chromosomes evolved in several steps, and the results in Bergero et al. (2013) show that it is still ongoing in the recombining or pseudoautosomal, regions (PARs) of these chromosomes. We used molecular evolutionary approaches to test for the footprints of SA polymorphisms, based on sequence diversity levels in S. latifolia PAR genes identified by genetic mapping. Nucleotide diversity is high for at least four of six PAR genes identified, and our data suggest the existence of polymorphisms maintained by balancing selection in this genome region, since molecular evolutionary (HKA) tests exclude an elevated mutation rate, and other tests also suggest balancing selection. The presence of sexually antagonistic alleles at a locus or loci in the PAR is suggested by the very different X and Y chromosome allele frequencies for at least one PAR gene.     

A remarkable autosomal heteromorphism in Pseudoryzomys simplex 2n = 56; FN = 54–55 (Rodentia,Sigmodontinae)

Pseudoryzomys simplex, the false rice rat, is a monotypic genus of the Oryzomyini tribe (Sigmodontinae) distributed in part of Bolivia, Paraguay, Argentina and Brazil. Its diploid number has been described as 56 acrocentric chromosomes decreasing in size and no karyotype figure has been depicted. Herein, we present karyotypic data on P. simplex, including chromosome banding and molecular fluorescent in situ hybridization using telomeric sequences and the whole X-chromosome of its sister clade Holochilus brasiliensis (HBR) as probes. A case of remarkable autosomal heteromorphism due to the presence of a whole heterochromatic arm leading to the variability of FN is reported, as well as the occurrence of regions of homology between the X and Y chromosomes (pseudoautosomal regions) after chromosome painting with the HBR X probe on P. simplex metaphases.     

Evidence of the accumulation of allele-specific non-synonymous substitutions in the young region of recombination suppression within the mating-type chromosomes of Neurospora tetrasperma

Currently, little is known about the origin and early evolution of sex chromosomes. This is largely due to the fact that ancient non-recombining sex chromosomes are highly degenerated, and thus provide little information about the early genomic events in their evolution. The Neurospora tetrasperma mating-type (mat) chromosomes contain a young (<6 Mya) and large region (>6.6 Mb) of suppressed recombination, thereby providing a model system to study early stages of sex chromosome evolution. Here, we examined alleles of 207 genes located on the N. tetrasperma mat a and mat A chromosomes to test for signs of genomic alterations at the protein level in the young region of recombination suppression. We report that the N. tetrasperma mat a and mat A chromosomes have each independently accumulated allele-specific non-synonymous codon substitutions in a time-dependent, and gene-specific manner in the recombinationally suppressed region. In addition, examination of the ratio (ω) of non-synonymous substitutions (dN) to synonymous substitutions (dS) using maximum likelihood analyses, indicates that such changes are associated with relaxed purifying selection, a finding consistent with genomic degeneration. We also reveal that sex specific biases in mutation rates or selection pressures are not necessary for genomic alterations in sex chromosomes, and that recombination suppression in itself is sufficient to explain these results. The present findings extend our current understanding of genomic events associated within the young region of recombination suppression in these fungal sex-regulating chromosomes.     

Accumulation of interspersed and sex-specific repeats in the non-recombining region of papaya sex chromosomes

Background

The papaya Y chromosome has undergone a degenerative expansion from its ancestral autosome, as a consequence of recombination suppression in the sex determining region of the sex chromosomes. The non-recombining feature led to the accumulation of repetitive sequences in the male- or hermaphrodite-specific regions of the Y or the Y^h chromosome (MSY or HSY). Therefore, repeat composition and distribution in the sex determining region of papaya sex chromosomes would be informative to understand how these repetitive sequences might be involved in the early stages of sex chromosome evolution.

Results

Detailed composition of interspersed, sex-specific, and tandem repeats was analyzed from 8.1 megabases (Mb) HSY and 5.3 Mb corresponding X chromosomal regions. Approximately 77% of the HSY and 64% of the corresponding X region were occupied by repetitive sequences. Ty3-gypsy retrotransposons were the most abundant interspersed repeats in both regions. Comparative analysis of repetitive sequences between the sex determining region of papaya X chromosome and orthologous autosomal sequences of Vasconcellea monoica, a close relative of papaya lacking sex chromosomes, revealed distinctive differences in the accumulation of Ty3-Gypsy, suggesting that the evolution of the papaya sex determining region may accompany Ty3-Gypsy element accumulation. In total, 21 sex-specific repeats were identified from the sex determining region; 20 from the HSY and one from the X. Interestingly, most HSY-specific repeats were detected in two regions where the HSY expansion occurred, suggesting that the HSY expansion may result in the accumulation of sex-specific repeats or that HSY-specific repeats might play an important role in the HSY expansion. The analysis of simple sequence repeats (SSRs) revealed that longer SSRs were less abundant in the papaya sex determining region than the other chromosomal regions.

Conclusion

Major repetitive elements were Ty3-gypsy retrotransposons in both the HSY and the corresponding X. Accumulation of Ty3-Gypsy retrotransposons in the sex determining region of papaya X chromosome was significantly higher than that in the corresponding region of V. monoica, suggesting that Ty3-Gypsy could be crucial for the expansion and evolution of the sex determining region in papaya. Most sex-specific repeats were located in the two HSY expansion regions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-335) contains supplementary material, which is available to authorized users.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.     

Species and Population Level Molecular Profiling Reveals Cryptic Recombination and Emergent Asymmetry in the Dimorphic Mating Locus of C. reinhardtii

Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT−. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT—gene conversion in the rearranged domains, and crossover exchanges in flanking domains—both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+×MT+ crosses, it was still suppressed in MT−×MT− crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.     

Evolution of the pseudoautosomal boundary in Old World monkeys and great apes

Mammalian sex chromosomes are divided into sex-specific and pseudoautosomal regions. Sequences in the pseudoautosomal region recombine between the sex chromosomes; the sex-specific sequences normally do not. The interface between sex-specific and pseudoautosomal sequences is the pseudoautosomal boundary. The boundary is the centromeric limit to recombination in the pseudoautosomal region. In man, an Alu repeat element is found inserted at the boundary on the Y chromosome. In the evolutionary comparison conducted here, the Alu repeat element is found at the Y boundary in great apes, but it is not found there in two Old World monkeys. During the evolution of the Old World monkey and great ape lineages, homology between the sex chromosomes was maintained by recombination in the sequences telomeric to the Alu insertion site. The Alu repeat element did not create the present-day boundary; instead, it inserted at the preexisting boundary after the Old World monkey and great ape lineages diverged.     

Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes, and, as a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex-determining regions of the genome. Here, we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100-kilobase mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be ~20% between MAT and a flanking marker at 5, 10, 50, or 100 kilobases from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted ~10- to 50-fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. MAT heterozygosity was not required for enhanced recombination, implying that this process is not due to a physical distortion from the two non-paired alleles and could also occur during same-sex mating. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and pathogenic yeasts.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

Gene Conversion between the X Chromosome and the Male-Specific Region of the Y Chromosome at a Translocation Hotspot

Outside the pseudoautosomal regions, the mammalian sex chromosomes are thought to have been genetically isolated for up to 350 million years. However, in humans pathogenic XY translocations occur in XY-homologous (gametologous) regions, causing sex-reversal and infertility. Gene conversion might accompany recombination intermediates that resolve without translocation and persist in the population. We resequenced X and Y copies of a translocation hotspot adjacent to the PRKX and PRKY genes and found evidence of historical exchange between the male-specific region of the human Y and the X in patchy flanking gene-conversion tracts on both chromosomes. The rate of X-to-Y conversion (per base per generation) is four to five orders of magnitude more rapid than the rate of Y-chromosomal base-substitution mutation, and given assumptions about the recombination history of the X locus, tract lengths have an overall average length of ∼100 bp. Sequence exchange outside the pseudoautosomal regions could play a role in protecting the Y-linked copies of gametologous genes from degeneration.     

Gene conversion occurs within the mating-type locus of Cryptococcus neoformans during sexual reproduction

Meiotic recombination of sex chromosomes is thought to be repressed in organisms with heterogametic sex determination (e.g. mammalian X/Y chromosomes), due to extensive divergence and chromosomal rearrangements between the two chromosomes. However, proper segregation of sex chromosomes during meiosis requires crossing-over occurring within the pseudoautosomal regions (PAR). Recent studies reveal that recombination, in the form of gene conversion, is widely distributed within and may have played important roles in the evolution of some chromosomal regions within which recombination was thought to be repressed, such as the centromere cores of maize. Cryptococcus neoformans, a major human pathogenic fungus, has an unusually large mating-type locus (MAT, >100 kb), and the MAT alleles from the two opposite mating-types show extensive nucleotide sequence divergence and chromosomal rearrangements, mirroring characteristics of sex chromosomes. Meiotic recombination was assumed to be repressed within the C. neoformans MAT locus. A previous study identified recombination hot spots flanking the C. neoformans MAT, and these hot spots are associated with high GC content. Here, we investigated a GC-rich intergenic region located within the MAT locus of C. neoformans to establish if this region also exhibits unique recombination behavior during meiosis. Population genetics analysis of natural C. neoformans isolates revealed signals of homogenization spanning this GC-rich intergenic region within different C. neoformans lineages, consistent with a model in which gene conversion of this region during meiosis prevents it from diversifying within each lineage. By analyzing meiotic progeny from laboratory crosses, we found that meiotic recombination (gene conversion) occurs around the GC-rich intergenic region at a frequency equal to or greater than the meiotic recombination frequency observed in other genomic regions. We discuss the implications of these findings with regards to the possible functional and evolutionary importance of gene conversion within the C. neoformans MAT locus and, more generally, in fungi.     

Crossover Heterogeneity in the Absence of Hotspots in Caenorhabditis elegans

Crossovers play mechanical roles in meiotic chromosome segregation, generate genetic diversity by producing new allelic combinations, and facilitate evolution by decoupling linked alleles. In almost every species studied to date, crossover distributions are dramatically nonuniform, differing among sexes and across genomes, with spatial variation in crossover rates on scales from whole chromosomes to subkilobase hotspots. To understand the regulatory forces dictating these heterogeneous distributions a crucial first step is the fine-scale characterization of crossover distributions. Here we define the wild-type distribution of crossovers along a region of the C. elegans chromosome II at unprecedented resolution, using recombinant chromosomes of 243 hermaphrodites and 226 males. We find that well-characterized large-scale domains, with little fine-scale rate heterogeneity, dominate this region’s crossover landscape. Using the Gini coefficient as a summary statistic, we find that this region of the C. elegans genome has the least heterogeneous fine-scale crossover distribution yet observed among model organisms, and we show by simulation that the data are incompatible with a mammalian-type hotspot-rich landscape. The large-scale structural domains—the low-recombination center and the high-recombination arm—have a discrete boundary that we localize to a small region. This boundary coincides with the arm-center boundary defined both by nuclear-envelope attachment of DNA in somatic cells and GC content, consistent with proposals that these features of chromosome organization may be mechanical causes and evolutionary consequences of crossover recombination.