Genetic analyses of a hybrid cross between serotypes A and D strains of the human pathogenic fungus Cryptococcus neoformans

Abstract

Cryptococcus neoformans has two varieties, var. grubii and var. neoformans, that correspond to serotypes A and D, respectively. Molecular phylogenetic analyses suggest that these two varieties have diverged from each other for approximately 18 million years. The discovery of pathogenic serotype AD hybrid strains in nature indicates that intervariety mating in C. neoformans occurs in the natural environment. However, little is known about the genetic consequences of hybridization in C. neoformans. Here, we analyzed a hybrid population of 163 progeny from a cross between strains of serotypes A (CDC15) and D (JEC20), using 114 codominant nuclear PCR-RFLP markers and 1 direct PCR marker. These markers were distributed on all 14 chromosomes of the sequenced strain JEC21 that was isogenic to one of the parents (JEC20) in our cross. Our analyses identified that of the 163 progeny, 5 were heterozygous at all 115 loci, 1 was completely homozygous and identical to one of the parents (CDC15), and the remaining 157 each contained at least 1 heterozygous locus. Because all 163 progeny inherited mitochondria from the MATa parent JEC20, none of the progeny had a genotype identical to either of the two parents or to a composite of the two parents. All 115 nuclear loci showed three different genotypes in the progeny population, consistent with Mendelian segregation during meiosis. While the linkage analysis showed independent reassortment among loci on different linkage groups, there were significant differences in recombination frequencies among chromosomes and among regions within certain chromosomes. Overall, the linkage-map length from this hybrid cross was much shorter and the recombination frequency much lower than those constructed using serotype D strains, consistent with suppressed recombination in the intervariety cross between strains of serotypes A and D. We discuss the implications of our results in our understanding of the speciation and evolution of the C. neoformans species complex.

Figure 1.—

Distribution of markers along the 14 chromosomes used in this study. The lengths of the chromosomes as well as the positions of the markers analyzed here on each chromosome are shown along the top bar.

Figure 2.—

The UPGMA phenogram showing the overall genetic similarity among the 163 progeny as well as their relationships to the two parental strains CDC15 and JEC20 and their composite genotype P1P2. Genotypes represented by >3 progeny are marked to the right.

Figure 3.—

Segregation ratios of the molecular markers along chromosomes. Each colored curve represents a chromosome. The x-axis shows the physical locations of the markers on the chromosome. The y-axis represents the frequency of CDC15 alleles. Dotted lines represent the boundaries for the 95% confidence interval around the null hypothesis of random segregation (1:1 ratio). Markers showing significantly skewed segregations are indicated by black arrows: all markers on chromosome 2 and two markers, CNN01880 and CNN02060, on chromosome 12.

Figure 4.—

A representative linkage group and the confirmation of a translocation breakpoint in this linkage group constructed from an intervariety cross in C. neoformans. (A) LG 3 of the hybrid genetic linkage map of C. neoformans. Marker names are indicated on the right of the linkage group. Numbers on the left are genetic distances in centimorgans. The diagram on the right of the linkage group is the distributions of heterozygosity at each marker locus located on LG 3, with the marker orders following their physical locations in chromosome 3 of JEC21. The x-axis shows the level of heterozygosity and the y-axis shows the physical distance from one end of chromosome 3. Arrows indicate markers with positions in the linkage groups different from their physical locations in chromosomes 3 of JEC21 (for putative inversions and translocations, see results). Dotted lines indicate the approximate locations of the candidate centromeric region in chromosome 3 of JEC21 as identified by Loftus et al. (2005). (B) The locations of primers used to confirm the putative translocation of marker CNC07180 in LG 3. Arrows indicate the positions and directions of primers in chromosome 3 of strain JEC21 and contig 32.1 of strain H99. (C) Gel electrophoresis of PCR products amplified from different strains using different primer pairs to detect translocation. The strain names are indicated at the top of each lane. The primer pairs used for PCR are indicated at the bottom. Primer sequences are listed in Table 2.

Figure 4.—

A representative linkage group and the confirmation of a translocation breakpoint in this linkage group constructed from an intervariety cross in C. neoformans. (A) LG 3 of the hybrid genetic linkage map of C. neoformans. Marker names are indicated on the right of the linkage group. Numbers on the left are genetic distances in centimorgans. The diagram on the right of the linkage group is the distributions of heterozygosity at each marker locus located on LG 3, with the marker orders following their physical locations in chromosome 3 of JEC21. The x-axis shows the level of heterozygosity and the y-axis shows the physical distance from one end of chromosome 3. Arrows indicate markers with positions in the linkage groups different from their physical locations in chromosomes 3 of JEC21 (for putative inversions and translocations, see results). Dotted lines indicate the approximate locations of the candidate centromeric region in chromosome 3 of JEC21 as identified by Loftus et al. (2005). (B) The locations of primers used to confirm the putative translocation of marker CNC07180 in LG 3. Arrows indicate the positions and directions of primers in chromosome 3 of strain JEC21 and contig 32.1 of strain H99. (C) Gel electrophoresis of PCR products amplified from different strains using different primer pairs to detect translocation. The strain names are indicated at the top of each lane. The primer pairs used for PCR are indicated at the bottom. Primer sequences are listed in Table 2.