Evolution of Suppressed Recombination in Sex Chromosomes of Microbotryum

1
Evolution of Suppressed Recombination in Sex
Chromosomes of Microbotryum Jessica L. Abbate and
Michael Hood Jessie L. Abbate and Michael E. Hood
Dept. of Biology, University of
Virginiajla8k_at_virginia.edu, http//www.people.vir
ginia.edu/jla8k
Figure 1 Microbotryum violaceum strains on
Silene species

• Discussion
• These results suggest that several of the
  sequenced loci have come into the non-recombining
  regions of the fungal sex chromosomes multiple
  times since the divergence of Microbotryum on
  different host genera. Also, subsequent
  differentiation can occur independently in sister
  lineages (i.e. apparent non-recombining of DGS1
  in samples from Dianthus hosts, alongside
  recombination from Saponaria hosts in the same
  gene).
• These results are qualitatively similar to those
  for birds3 and mammals4, and suggest that
  non-recombining regions of sex chromosomes are
  very dynamic and may be subject to similar
  evolutionary forces across the broadest diversity
  of eukaryotes.
• In clades where A1 and A2 sequences are not
  differentiated, it is unclear whether the locus
  is recombining on the fungal sex chromosomes or
  resides on an autosome. To distinguish between
  these possibilities, we are sequencing flanking
  regions and probing karyotypes (i.e. whether the
  patterns of differentiation are determined by an
  expanding and contracting region of
  non-recombination on the sex chromosomes versus
  translocations involving autosomes).
• We hope that this investigation into the
  evolution of sex chromosomes in a haploid
  organism will either support theories present in
  the field, which is dominated by studies in
  diploids, or help to generate a more universal
  perspective.

• Abstract
• The haploid-mating fungus Microbotryum violaceum
  serves as a model for the evolution of dimorphic
  sex chromosomes for comparison with canonical
  patterns of X-Y divergence in diploid-mating
  animals and plants1. In this study, we
  reconstructed the phylogenetic histories of the
  alternate fungal sex chromosomes using multiple
  loci linked to the mating-types (A1 or A2) in
  Microbotyryum from Silene latifolia and
  S.caroliniana. One resulting phylogeny included
  1) separate clades where A1 and A2-linked
  sequences appear to have ceased recombination and
  diverged 2) clades where there is little to no
  difference between the A1 and A2 sequences,
  indicative of continued recombination and 3) a
  paradoxical clade, again with no base pair
  differentiation between the A1 and A2 sequences,
  and with identity among fungal samples expected
  to be highly divergent. As recently found in some
  diploid-mating organisms2, these results suggest
  that locus has come into linkage with the
  non-recombining regions of the fungal sex
  chromosomes more than once, and that subsequent
  differentiation can occur independently in
  related lineages. By identifying and constructing
  phylogenies of other sex chromosome-linked genes,
  we hope to gain further insight into 1) how the
  recruitment of genes into linkage with mating
  type varies across lineages, and 2) whether
  different genes come into linkage with the A1 or
  A2 mating type independently, or if they are
  transferred from one to the other via
  recombination.

• Methods
• Samples of the parasitic fungus Microbotryum
  were isolated from the host genera Silene,
  Saponaria, and Dianthus in Europe and the US
  (Fig. 1).
• Haploid meiotic products of contrasting mating
  types (A1 and A2) were obtained by the isolation
  of linear tetrads using micro-manipulation.
• Baseline relatedness among samples was estimated
  using nuclear ITS and gamma-tubulin sequences
  (Table 1).
• We obtained the first mating-type linked
  sequence (DGS 1) from a genomic shotgun survey
  with sex chromosome-specific DNA. It had a BLAST
  match to a sex-inducing pheromone in Volvox.
  (ca. 400 bp)
• A list of sequences from mating-type associated
  genes in other basidiomycetes on GenBank was
  compiled and blasted against our shotgun library
  databases for S.latifolia and S.caroliniana. Any
  matches that were significant (with an e-value of
  0.01 or less) were investigated for function in
  their organism, and 26 of these genes were fitted
  with primers and amplified.
• PCR product presence or absence, product size
  differences, and DNA sequences were analyzed for
  both mating types of Microbotryum strains from
  the two focal species, and then with isolates
  representing the full phylogenetic spectrum.
• At this time, we have constructed five
  additional phylogenies, for genes Sxi2, Bsp1,
  Znf1, Myo2, and Mip1, using the same set of A1
  and A2 isolates from the 13 different
  Microbotryum taxa listed in Table 1.

• Work In Progress
• Expanding from DGS1 in both directions, we have
  gained approximately 500 additional bases, and
  expect to have another 500 sequenced soon, which
  will hopefully tell us more about the locus, its
  function, and explain the phylogenetic patterns
  we have demonstrated.
• A total of 26 primer sets have been developed
  for this investigation. With a focal group of 13
  taxa (A1 and A2 isolates), the goal is to
  systematically construct full phylogenies for all
  26 putative linked genes. Concordance between
  phylogenies, in addition to possible FISH
  staining or karyotype probing, may help
  demonstrate more clearly how genes come into (and
  out of) linkage with the mating type.
• Once cessation of recombination has been
  established, as with DGS1, we will expand the
  sample size within that clade to determine
  whether the pattern is consistent among
  populations.
• Genes of particular interest can be further
  investigated by walking (as with DGS1) to
  sequence the flanking nucleotide regions.

• References
• 1. Hood, M. E., Antonovics, J., and Koskella, B.
  2004. Shared forces of sex chromosome evolution
  in haploid-mating and diploid-mating organisms.
  Genetics (in press).
• 2. Filatov, DA. Evolutionary History of Silene
  latifolia Sex Chromosomes Revealed by Genetic
  Mapping of Four Genes. Genetics 170975-979.
• 3. Ellegren, H. and Carmichael. A. 2001.
  Multiple and independent cessation of
  recombination between avian sex chromosomes.
  Genetics 158325-331.
• 4. Marais, G. and Galtier, N. 2003. Sex
  chromosomes how X-Y recombination stops. Current
  Biology 13R641-R643.

• Results
• DGS1
• Sex chromosome sequences of Microbotryum from S.
  latifolia and Dianthus spp. were in complete
  linkage disequilibrium with the A1 and A2 mating
  types.
• In contrast, for the other clades the sequences
  had much lower levels of differentiation by
  mating type.
• The amount of variation among populations within
  a clade (Fig.2) reflects that previously
  observed background relatedness (see Table 1).
• There is an unexpected identity for the
  sequences from the North American and some
  European lineages, which are known to be highly
  divergent based upon other studies.
• Sxi2
• Two taxa showed weak evidence for cessation of
  recombination S.ocymoides and D.sylvestris.
• Bsp1
• None of the taxa so far have shown linkage
  between Bsp1 and mating type.
• Znf1
• There is evidence for cessation of recombination
  between mating types of S.latifolia,
  D.sylvestris, and S.paradoxa.
• Myo2
• Only S.acaulis showed any evidence of divergence
  between mating types for this locus.
• Mip1
• Three SNPs between mating types of S.acaulis
  demonstrates good evidence for cessation of
  recombination in this taxon.

Sxi2mating-type specific sexual development
regulator in C.neoformans
Bsp1 Close association with MAT locus in
Cryptococcus spp. Function unknown.
Mip1Mitochondrial intermediate peptidase,
mitochondrial precursor from Saccharomyces
cerevisiae
Znf1 Zn-finger/PHD-finger transcription factor
in Cryptococcus spp.
Myo2 Myosin heavy chain required for the
transport of various organelles and proteins
during segregation in C.neoformans