134. The Mixed Mating System of Cryphonectria parasitica.
R. E. Marra and M. G. Milgroom. Department of Plant Pathology, Comell University, Ithaca NY.
Cryphonectria parasitica, the ascomycete that causes chestnut blight, has been shown in
laboratory crosses to conform to a heterothallic mating system. When perithecia were sampled
from North American populations, a significant portion (~25%) appeared to be the result of self-fertilization, suggesting a mixed mating system. Ascospores from a single perithecium were
considered the result of self-fertilization if there was no segregation at 5 to 7 vegetative
compatibility (vc) loci, 6 unlinked RFLP loci, and 8-15 (or more) unlinked fingerprinting loci.
However, progeny arrays from most putatively selfed perethecia segregate approx. 1:1 for mating
type, even though the maternl parents of these perithecia usually express only one or the other
mating type in the lab. Results from lab matings correlate 100% with southern hybridizations
using a C parasitica Mat-2-specific probe, a 280-bp conserved HMG domain. Although selfing in
the lab is rare, 5 individuals have selfed, and the progeny of lab selfs segregate for mating type,
based on both mating phenotype and HMG hybridizations. Most of our laboratory experiments
begin with single conidia, which are single-celled. We tested the hypothesis that the conidia of
individuals that can self are multinucleate by studying the conidia from three selfing strains. Two
thousand conidia from each of these three strains were examined using epifluorescent microscopy,
and we could not reject a null hypothesis that conidia are uninucleate. Additionally, no
segregation was observed for DNA fingerprints or the HMG domain in ten conidia from each of
these three strains, suggesting that mating type segregation does not occur somatically.
135. Isolation of compatible strains of Podospora anserina with the same mitochondrial rearrangement: analysis of the effects of sexual reproduction on mitochondrial inheritance and life span.
Margaret E. Silliker and Eric M. Nelson, DePaul University, Chicago, IL.
This lab has previously described a stop/start longevity mutant, Mn19, of Podospora
anserina. It is unusual in that it has a stable mitochondrial genome which is a rearrangement of
the wild type, juvenile, race A mitochondrial genome. The Mn19 genome consists of two non-overlapping circular molecules which most likely arose by recombination between short sequence
repeats. Attempts to transmit the Mn19 rearrangement to progeny for genetic analysis have
resulted in preferential inheritance of wild type genomes, even when wild type genomes are rare in
the mate (detected only at the level of PCR). In order test whether the process of sexual
reproduction was contributing to the restoration of the Mn19 mitochondrial genome we wanted
to cross the Mn19 mutant with itself. However, Mn19 was isolated as a monokaryotic strain of
the (+) mating type. Here we describe a confrontation cross where perithecia were produced
exclusively on the Mn19 side of the cross. Spores were isolated and the progeny were screened
by PCR in order to detect isolates with only the Mn19 rearrangement and the (-) mating type.
One such strain was identified and crossed with the original Mn19 strain. Wild type (un-rearranged) sequences have been amplified from the majority of the progeny, though the
rearrangement was transmitted to some strains. We are currently measuring the life span
phenotypes of these progeny.
136. Phylogeneties and evolution of mating type genes.
B. Gillian Turgeon, Cornell Univ. Ithaca, NY. and Mary Berbee, Univ. of British Columbia, Vancouver, BC.
We are exploring the evolution of reproductive strategies in sexual and asexual ascomycetes by comparing mating type (MAT) gene sequences. Although MAT sequences are powerful tools for molecular evolution studies, their usefulness has been limited by difficulties in cloning them from a wide array of fungi. We have developed an efficient PCR-based procedure for cloning MAT genes across genus lines from both sexual and asexual species. With MAT, ITS, and GPD sequence data we are inferring phylogenetic histories. Preliminary comparisons of distances among species pairs suggest that phylogenies from the three regions will be congruent. Trees from ITS sequences divide Cochliobolus species into two groups; one contains serious cereal pathogens like C. heterostrophus and C. carbonum and tends to have Bipolaris asexual states. In the second group, serious pathogens are lacking and Curvularia asexual states are common. Using the phylogeny, we will ask whether MAT genes have co-evolved with genes for primary metabolism. The fungi chosen for this study (Pleosporaceae) have different reproductive strategies, ie., sexual vs. asexual or homothallic (self fertile) vs. heterothallic (self sterile). As sequence data are accumulated we will ask if asexual species arise from sexual, if homothallics arise from heterothallics, and if MAT genes in sexual and asexual fungi are evolving at the same rate. Finally, because MAT-1 and MAT-2 genes never recombine (they are dissimilar sequences occupying the same genetic locus), each gene has a completely clonal phylogenetic history. Conflicting phylogenetic histories may suggest interspecific mating.