Chromosomal polymorphism and ploidy divergence in Zygosacharomyces rouxii strains isolated from Traditional Balsamic Vinegars

The genus Zygosaccharomyces traditionally describes osmophilic yeasts that differ from the those of the genus Saccharomyces for their vegetative growth, in which sexual conjugation between cells or a cell and its bud, precedes the sporulation1. Diploidization through zygote formation means that vegetative life cycle is mainly haploid. In the last past decade phenotypical and molecular approaches based on cloning procedure, single-gene PCR amplification and karyotyping highlighted a high variability within Zygosaccharomyces rouxii species, mainly between type strain CBS 732 and some wild strains isolated from miso and traditional balsamic vinegar (ATCC 42981, ABT 301, and ABT 601, respectively)2,3,4. Recently random fragments sequencing from strain ATCC 42981 and allele-specific PCR reaction from strain ABT301 and ABT 601 have revealed that all these wild strains have two copies of several genes, including ITS-5.8S rDNA, 26S D1/D2 regions, ZSOD and HIS34,5. Genome size is one of the most fundamental genetic properties of living organisms. However no study has been attempted to evaluate genome size and ploidy for Z. rouxii strains. In this work genome size and ploidy level estimation, as well as ZSOD gene chromosome mapping were used to elucidate the taxonomic relationships among these wild strains compared to Z. rouxii CBS 732T.In particular, we applied flow cytometry to determine DNA content of cells in G0/G1 phase. The genome size was estimated 11.89±0.21 Mb (12.1±0.22 fg) for strain CBS 732T, 21.08±0.37 Mb (21.56±0.37 fg) for ATCC 42981, 25.68±1.11 Mb (26.27±1.14) for ABT 301, and 36.00±0.37 Mb (36.80±0.38 fg) for ABT 601. Moreover pulsed field gel electrophoresis (PFGE) confirmed the genome size divergence with polymorphic PFGE patterns consisting in different number of chromosomal bands: 6, 7, 10, and 11 for CBS 732T, ATCC 42981, ABT 301, and ABT 601, respectively. By densitometric analysis of PFGE profile, the haploid genome size has been obtained as sum of chromosomal bands size. These data have been combined to DNA content from flow cytometric analysis in order to calculate the ploidy level for each strain. The results showed CBS 732T has a haploid DNA content, whereas wild strains ATCC 42981, ABT 301, and ABT 601 a diploid DNA content. Finally the change in ploidy has been also confirmed by doubling of genes involved in osmotic adaptation (ZSOD, HOG, GPD, and GCY1), as well as by polymorphic rDNA genes occurring in ATCC 429815,6 and in ABT 301 and ABT 6014, but not in CBS 732T 2. For this purpose chromosome mapping of ZSOD genes (encoding Na+/H+-antiporters involved in halotolerance) showed that, differently from haploid CBS 732T, diploid strains ATCC 42981, ABT 301 and ABT 601 have two partially divergent ZSOD copies located on different chromosomes. In conclusion genome size and ploidy are two fundamental and informative traits mainly for yeasts with complex diploid/haploid shift in their life cycle. Our complementary molecular strategies highlighted a naturally occurring variation in genome size and ploidy level within Z. rouxii, suggesting that there is a complex picture in genome organization and the strains now recognized as Z. rouxii could belong to more than one species. References1. Barker, B.T.P., Trans. R. Soc. Lond. B., 194, 467–485 (1901).2. Kinclova, O., et al.,. J. Biotechnol., 88, 151–158. (2001)3. Pribylova, L., et al., Yeast, 24, 171-180 (2007)4. Solieri, L., et al., Yeast, 24, 403–417 (2007)5. Gordon, J., Wolfe, K.H. Yeast, 25, 449-456 (2008) 6. Iwaki T., et al., Microbiol. 145, 241-248 (1999).