Population polymorphism of nuclear mitochondrial DNA insertions reveals widespread diploidy associated with loss of heterozygosity in Debaryomyces hansenii

Abstract

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767(T), and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.

Fig. 1.

Separation on agarose gels of PCR products from regions carrying a NUMT in CBS 767^T, amplified on the genomic DNA of various D. hansenii strains. (A) Single NUMT D18. Lane 1, CBS 767^T; lane 2, CBS 5139; lane 3, CLIB 609; lane 4, CLIB 613; lane 5, CLIB 629; lane 6, CBS 1795; lane 7, CLIB 920. (B) NUMT mosaic 10. Lane 1, CBS 767^T; lane 2, CLIB 237; lane 3, CLIB 238; lane 4, CLIB 239; lane 5, CLIB 656; lane 6, CLIB 672. (C) NUMT mosaic 15. Lane 1, CBS 767^T; lane 2, CLIB 380; lane 3, CLIB 542; lane 4, CLIB 657; lane 5, CBS 1795. M, 1-kb⁺ ladder (BioLabs) molecular weight marker.

Fig. 2.

Sequence comparison of the single NUMT D18 region in various D. hansenii isolates. An alignment of the two heterozygous alleles of the NUMT D18 region of CLIB 380 with the same regions of CBS 767^T and CBS 1795 is shown. White capital letters on black background indicate the conserved sequence between CBS 767^T and the various studied strains. Black capital letters on gray background indicate the sequence divergence between CBS 767^T and the various studied strains. Lowercase letters depict the NUMTs.

Fig. 3.

Comparison of the structures of the NUMT mosaic 10 regions in CBS 767^T, CLIB 236, and CBS 1795. The NUMTs are represented by large open arrows according to their orientation. The gray box represents the linear plasmid insertion, hatched boxes indicate a sequence with no BLAST hit, and thick black lines depict the ENO1-like sequences. Deletions are indicated by dashed lines, with double arrows showing their sizes. The structure of this region in CBS 767^T (top) is derived from that of CLIB 236 (middle) by a large deletion (499 bp) encompassing the distal left NUMT, part of the adjacent NUMT, and a fragment of the upstream ENO1-like sequences carrying a linear plasmid insertion. The CBS 1795 region (bottom) does not carry any insertions and contains longer ENO1-like sequences. The two regions that are deleted in CLIB 236 (and in CBS 767^T) are shown.

Fig. 4.

Fluorescence histograms of various D. hansenii isolates after propidium iodide staining. (A) CBS 767^T; (B) CLIB 667; (C) CLIB 380; (D) CLIB 702.

Fig. 5.

PFGE karyotypes of various Debaryomyces isolates. Chromosomes of various D. hansenii strains and of D. tyrocola CLIB 660 were separated by electrophoresis, as described in Materials and Methods. The group for each strain, as defined in Table S1 in the supplemental material, is indicated. The sizes of the seven D. hansenii CBS 767^T chromosomes, as deduced from their sequences, are indicated, except for ribosomal DNA carrying chromosome G, whose size was estimated from a comparison to that of Hansenula wingei chromosomes (not shown).

Fig. 6.

Chronology of NUMT insertions. (A) Four sets of NUMT loci harboring the same presence/absence profile (across the 28 strains analyzed in Table 2) were defined. Each set was placed on a tree according to the presence of the corresponding NUMTs in the different clades of D. hansenii and in D. tyrocola. A single strain represents each D. hansenii clade and the D. tyrocola species. The two alleles of mosaic 10 split the strains of clade 1 into two branches. (B) Histogram showing the average percentages of sequence identity between the NUMTs and their mitochondrial counterparts in each of the four sets of NUMTs defined in panel A. The black bars indicate the standard deviation from the percentage of sequence identity within each set.