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Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species
Fungal Genetics and Biology 45 (2008) 1582–1590
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Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species Lisa Solieri a,*, Stefano Cassanelli a, Maria Antonietta Croce b, Paolo Giudici a a b
Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42100 Reggio Emilia, Italy Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy
a r t i c l e
i n f o
Article history: Received 25 June 2008 Accepted 2 October 2008 Available online 15 October 2008 Keywords: Zygosaccharomyces rouxii Ploidy Flow cytometry Cell cycle analysis Genome size Yeast Karyotype
a b s t r a c t Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7 ± 0.2 Mb for strain CBS 732T, 21.9 ± 0.2 Mb for ATCC 42981, 28.1 ± 1.3 Mb for ABT 301, and 39.00 ± 0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its ability to grow in environments with high soluble solids concentration. It is involved in the production of high sugary and/or salt fermented food, such as miso and traditional balsamic vinegar, respectively. Furthermore it has been also recognized as one of the main spoilage microorganisms in food industry. According to its incapacity to produce petite mutant, Z. rouxii shows a preferentially respirative metabolism (Merico et al., 2007) and it exhibits a vegetative haploid life style with zygote formation occurring immediately before sporulation (Fowell, 1969; Mori and Onishi, 1967). Phylogenetically Z. rouxii species is closely related to Saccharomyces cerevisiae, but precedes the whole genome duplication which took place in an ancestor of S. cerevisiae (Wong and Wolfe, 2006). Several phenotypical and molecular approaches based on cloning procedure, single-gene PCR amplification and karyotyping highlight a high variability within Z. rouxii species. Two extensively studied Z. rouxii strains, CBS 732T and ATCC 42981, showed different susceptibility to transformation by electroporation, as well as different salt tolerance, glycerol production and karyotype, whereas a conservation of synteny has been observed (Pribylova and Sychrova, 2003; Pribylova et al., 2007a). James et al. (2005) reported that strain CBS 9951 (NCYC 3042) could belong to a novel * Corresponding author. Fax: +39 0522 522027. E-mail address: [email protected] (L. Solieri). 1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.10.001
species (called Z. pseudorouxii) and hybridization events could occur among Z. rouxii and Z. pseudorouxii species [hybrid strain CBS 4837 (NCYC 1682)]. Solieri et al. (2007) proposed a new putative species to describe two strains (namely ABT 301 and ABT 601) isolated from traditional balsamic vinegar, showing a high polymorphism in 5.8S rDNA and two flanking internal transcribed spacers sequences (5.8S-ITS region), as well as two copies of ZSOD genes (ZSOD2-22 and ZSOD22) and HIS3 genes, encoding Na+/H+-antiporter and imidazole–glycerol–phosphate dehydratase, respectively. Recently Gordon and Wolfe (2008) have more extensively investigated the genome structure of ATCC 42981 by random sequencing, showing the presence of T subgenome derived from Z. rouxii (with 97–100% DNA sequence identity to CBS 732T) and P subgenome derived from Z. pseudorouxii (about 80–90% identity). The results suggested that ATCC 42981 is an allodiploid yeast. However, no genome size and ploidy level estimations of this strain as well as of ABT 301 and ABT 601 strains have been attempted. In recent decades, ploidy level and genome size have been regarded as important subjects for inferring genome and sex evolution, even considering the paleopolyploid nature of the S. cerevisiae genome (Wong et al., 2002). Moreover, changes in ploidy introduce potentially significant cytological and physiological effects, without requiring any long-term evolutionary processes (Zeyl, 2004). Indeed Keogh et al. (1998) showed that a correlation between gene order evolution and chromosome number in S. cerevisiae and its close relatives. Several studies have been performed to elucidate the mechanisms of fast evolution of polyploid genomes toward diploidization in S. cerevisiae (Wolfe and Shields,
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1997; Wolfe, 2001; Kellis et al., 2004; Cliften et al., 2006; Scannell et al., 2007). Other studies have focused on how and why haploid and diploid phases can advantage or disadvantage adaptation in a new environment and provide high or low fitness benefits (Perrot et al., 1991; Korona, 1999; Mable and Otto, 1998, 2001; Zeyl et al., 2003; Zeyl, 2004; Anderson et al., 2004). For instance, the ploidy of wine yeasts confers advantages in adapting to variable environments or by increasing the dosage of some genes important for fermentation (Bakalinsky and Snow, 1990; Salmon, 1997). These findings shed a new light on ploidy level as an adaptive evolutionary trait (Katz Ezov et al., 2006). The genome size (C value) of unicellular eukaryote can be estimated by biochemical assays, which determine the phosphate content of DNA isolated from a defined number of cells (Shapiro, 1970); analysis of reassociation kinetics, using hydroxyl apatite chromatography to separate single-stranded and double-stranded DNA (dsDNA) (Britten and Kohne, 1986); pulsed-field gel electrophoresis (PFGE), in which the lengths of whole chromosomes are measured directly (Johnston and Mortimer, 1986; VaughanMartini et al., 1993); more recently by Real-time PCR (Wilhelm et al., 2003). Ploidy level can be obtained comparing the haplotype size generated by PFGE with data obtained by microfluorimetry or by flow cytometry-based methods (FCM). In FCM assay the DNA content of individual nuclei is analyzed by fluorescence measurements after staining with intercalating dyes, such as propidium iodide, ethidium bromide or SYBR Green I, and quantified using reference strains with known genome size (Fortuna et al., 2000). Recently FCM-based methods were successfully employed to determine the genome size and ploidy level of multibudding yeast Paracoccoioides brasiliensis (Almeida et al., 2006, 2007) and of spoilage yeast Z. bailii (Rodrigues et al., 2003). In this study, genome size and ploidy level estimation, as well as ZSOD gene chromosome mapping were used to elucidate the taxonomic relationships among Z. rouxii strains, CBS 732T and three wild strains, such as ATCC 42981 isolated from miso (Kiuchi et al., 1980) and ABT 301 and ABT 601 from traditional balsamic vinegar (Solieri et al., 2006, 2007). 2. Material and methods
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(YNB5%G) at 24 °C with aeration on a mechanical shaker (180 rpm). 2.2. Cell cycle analysis Zygosaccharomyces rouxii and Saccharomyces cerevisiae yeast strains were grown in YPD and YNB5%G medium batch culture to mid-log phase (OD600nm = 0.50.6). Cell samples in YNB5%G medium were inducted to arrest in G1 phase by treatment with 8hydroxyquinoline at a final concentration 100 mg/l for 24 h, as previously described (Johnston and Singer, 1978; Stöver et al., 1998). Cells were harvested by centrifugation (8000 g for 5 min at 4 °C), fixed overnight with 70% ethanol (vol/vol) at 4 °C and subjected to cell cycle analysis as previously described (Fortuna et al., 2000) and modified as follows. After fixation, cells were harvested, washed, and suspended at 5 106 cells/ml in 500 ll of sodium citrate buffer (50 mM; pH 7.0). Cells suspension was treated for 120 min at 50 °C with RNAse A (0.25 mg/ml) and/or proteinase K (2 mg/ml). A cell suspension of 2.5 106 cell/ml was stained overnight at 4 °C with propidium iodide (PI; Sigma) at a final concentration of 4 lg/ml. To remove of clumps and excessive debris, two consecutive ultrasound pulses were applied before FMC analysis at 70% of total output for 20 s with an interval of 1–2 s between the two pulses by using Microson Ultrasonic Cell Disruptor XL (Misonix Inc., New Highway Farmingdale, NY, USA). 2.3. Flow cytometry and estimation of genome size All FCM experiments were performed in triplicates on an EPICs XL (Coulter, Instrumentation Laboratory, Milano, Italia) flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. A minimum of 20,000 cells per sample were acquired at low flow rate and an acquisition protocol was defined to measure forward scatter (FS LOG) and side scatter (SS LOG) on a four-decade logarithmic scale and red Fluorescence (FL3) on a linear scale. For DNA content analysis, doublets and larger aggregates were discarded by electronic analysis of integral- and height-signals from the particles analyzed. Offline data were analyzed with the Windows Multiple Document Interface for Flow Cytometry 2.8 (WinMDI 2.8).
2.1. Strains and culture media 2.4. Chromosomal DNA preparation and pulse-field gel electrophoresis Zygosaccharomyces strains used in this study were listed in Table 1. In addition S. cerevisiae haploid strain BY4742, Euroscarf Acc. No. Y10000 (MATa; his3 D1; leu2 D0; lys2 D0; ura3 D0) and diploid strain BY4743, Euroscarf Acc. No. Y23146 (Mat a/a; his3 D1/his3 D1; leu2 D0/leu2 D0; lys2 D0/LYS2; MET15/met15 D0; ura3D0/ura3 D0; YBR011c::kanMX4/YBR011c) were included. All yeast strains were maintained at 24 °C by periodic subculturing in slanted tubes with YPD solid medium (1% yeast extract, 1% peptone, 2% glucose, 2% agar). For the assays carried out in this study yeast cells were routinely grown in YPD liquid medium and in yeast nitrogen base medium supplemented with 5% (g/v) glucose Table 1 Zygosaccharomyces strains used in this work. Strains
Species
Country of isolation
Source
Citation
CBS 732T
Z. rouxii
Italy
Sacchetti (1932)
ABT 301
Z. pseudorouxii
Italy
ABT 601 ABT 808 ABT 401 ATCC 42981
Z. Z. Z. Z.
‘‘ ‘‘ ‘‘ Japan
Concentrated blackgrape must Traditional balsamic vinegar ‘‘ ‘‘ ‘‘ Miso paste
pseudorouxii rouxii mellis rouxii
Solieri et al. (2006) ‘‘ ‘‘ ‘‘ Kiuchi et al. (1980)
Pulse-field gels were run on a Bio-Rad CHEF (contour-clamped homogeneous electric field) apparatus. Chromosomal DNA was isolated as described by Carle and Olson (1985), with the exception of 5 h incubation with Lyticase (Sigma), in spite of an incubation of 2 h at 37 °C with Zymoliase (Pribylova et al., 2007a). The 1% agarose gels were prepared with Seakem GTG agarose (FMC Bioproducts) and run in 0.5X TBE buffer at 13 °C. The running conditions were: switch time 300 s, run time 100/120 h, angle 106°, voltage 3 V/cm. The chromosomal DNA size marker of S. cerevisiae S288 C (Bio-Rad Laboratories) was used to estimate the chromosome size. Pictures of the gels were digitally captured using the BioDocAnalyze gel imaging and analysis system (Biometra, Göttingen, Germany). The DNA banding patterns were analyzed with BioNumerics version 3.0 (Applied Maths, Belgium). Similarity matrix was calculated using Pearson’s product moment correlation coefficient. Cluster analysis of similarity matrix was performed by the Unweighted Pair Group Method with Arithmetic mean (UPGMA) algorithm. 2.5. Southern blot Chromosomal DNAs separated by PFGE were transferred onto a Hybond-N+ membrane (Amersham) by upward capillary transfer.
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Hybridizations (overnight at 43 °C) were performed with 2 Kb probes generated by PCR using the following primer pairs: kar2f 50 -CAGCCTCATGGATCCTCTTGATGGTT-30 (Kinclova et al., 2001) and SOD1R (James et al., 2005) specific for both ZSOD2-22 and ZSOD2 genes; kar22f 50 -GGTTTGATAGGAGTCGGAAACACTTAC-30 (modified from Kinclova et al., 2001) and SOD2R (James et al., 2005) specific for ZSOD22 gene. All probes used in this study were labeled with digoxigenin-labeled 11-dUTP by using the DIG-DNA labeling kit (Roche). Detection was carried out by chemiluminescence, using an antidigoxigenin antibody and CDP-star (Roche), as recommended by the manufacturer.
size was 12.7 ± 0.21 Mb, while wild strains showed a double or more genome size. According to Rodrigues et al. (2003), we calculated two DNA indexes (DNA In): DNA In1 is in relation to the MFI of the G0/G1 peak of S. cerevisiae haploid strain B4742; DNA In2 is in relation to that of Z. rouxii strain CBS 732T. These DNA In1 values indicated that the strain CBS 732T had 0.97 times as much DNA as S. cerevisiae haploid strain, whereas the wild strains showed a double (ATCC 42981 and ABT 301) or triple (ABT 601) DNA content compared to S. cerevisiae haploid strain. The DNA In2 confirmed the wild strains had a bigger genome size respected to CBS 732T. These differences could be arisen both from ploidy level and chromosome number for haploid set.
3. Results 3.1. Genome size estimation To determine Z. rouxii strains genome size, we optimized a previously described FCM protocol for cell cycle profile analysis of S. cerevisiae and Z. bailii (Fortuna et al., 2000). The FCM analysis of ethanol-fixed Z. rouxii cells grown in YPD medium revealed a low specificity for nuclear PI staining, resulting in a cytometric pattern which did not show the characteristic bimodal profile linked to G0/ G1 and G2/M phases. Pre-treatments with RNase and proteinase K and sonication improved the pattern resolution and allowed to discriminate two major subpopulations of cells with different DNA content corresponding to G0/G1 and G2/M cell cycle phases, respectively (Fig. 1 A1, B1, C1, and D1). All tested strains showed a higher cells percentage in G2/M than in G0/G1 and S. The halfpeak coefficient of variation (HPCV) of G0/G1 peak is lower than 11% (data not shown), indicating an acceptable resolution of dsDNA measurements. According to Rodrigues et al. (2003), a direct correlation can be established between mean fluorescence intensity (MFI) and the amount of dsDNA. In addition treatment with 8-hydroxyquinoline in YNB5%G medium was performed to arrest Z. rouxii cells in G0/G1 and to confirm the peak corresponding to this phase (Fig. 1 A2, B2, C2, and D2). This chemical chelates zinc which is required for the G0/G1 to S transition and has been widely used in cell cycle studies involving S. cerevisiae (Johnston and Singer, 1978). As found for C. albicans (Stöver et al., 1998), 8hydroxyquinoline at a concentration of 100 mg/l is suitable for the cell cycle arrest of Z. rouxii strains, resulting in an increased fraction of the cell population in G0/G1 (Table 2). Saccharomyces cerevisiae haploid and diploid strains, isogenic to the previously sequenced yeast S288C with a haploid genome of 13.1 Mb (Goffeau et al., 1996), were used as references for direct DNA estimation. A concurrent cell cycle analysis of S. cerevisiae haploid and diploid strains revealed three distinct peaks (Fig. 2A), corresponding to 1n, 2n, and 4n DNA contents, where the MFI values of each peak was directly correlated to the amount of DNA (Mb) of its corresponding cell subpopulation (Fig. 2B). A preliminary analysis of mixed cell populations of Z. rouxii strains and S. cerevisiae haploid or diploid strain was used to determine single-cell DNA content (Fig. 1A3–D3). In particular we found three peaks, corresponding to 1n, 2n, and 4n DNA content by mixing CBS 732T and S. cerevisiae diploid cells, suggesting a haploid DNA content for Z. rouxii type strain (Fig. 1 A3). On the contrary we mixed strains ATCC 42981 and ABT 301 cells to S. cerevisiae haploid cells to obtain three peaks, showing a diploid DNA content for these Zygosaccharomyces strains (Fig. 1B3 and C3). Regarding strain ABT 601, we obtained four peaks by mixing it and S. cerevisiae diploid cells, indicating that it could be diploid but with a bigger DNA content per cell compared to S. cerevisiae (Fig. 1 D3). Genome size of each Z. rouxii strain was estimated in accordance with MFI of the G0/G1 subpopulation by using S. cerevisiae standard curve (Fig. 2B). The average amount of DNA per cell varied from 12.7 to 39.0 Mb (Table 3). For strain CBS 732T the genome
3.2. Separation of chromosome-sized DNA molecules by PFGE and ploidy level estimation Ploidy level is generally defined by the number of copies of each chromosome individual set per nucleus (Zeyl, 2004). The ploidy level of our strains was obtained by comparing the DNA content of Z. rouxii cells in G0/G1 phase determined by FCM (Table 2) with the average haploid genome size estimated by PFGE. Z. rouxii ABT 808 and Z. mellis ABT 401strains were used for comparison in PFGE experiment. The chromosomal bands were comparable in size and number to those of yeasts belonging to the genus Zygosaccharomyces, which are in low number ranging from 3 to 8 bands and whose electrophoretic separation requires long pulse times over a period of 5 days (De Jonge et al., 1986; Török et al., 1993; Oda and Tonomura, 1995; Sychrova et al., 2000; Esteve-Zarzoso et al., 2003). Two different migration times (100 and 120 h) were tested to resolve the most similar and large chromosomes (Fig. 3A and B). The karyotyping associated distinct chromosomal migration patterns to each strain, highlighting a high polymorphism in size and number of the bands. In particular the PFGE profiles of Z. rouxii CBS 732T and ABT808 consisted of 6 and 5 bands, respectively, ranging from 2.2 and 1.1 Mb, whereas the wild strains ATCC 42981, ABT 301, and ABT 601 showed more complex PFGE profiles consisting of a higher bands number, 7, 10 and 11, respectively. Also Z. mellis ABT 401 showed a complex chromosomal pattern with 9 chromosomal bands. Karyotype profiles were compared by cluster analysis (Fig. 3 C). ATCC 42981 did not belong to the main cluster including all the other Zygosacchaormyces strains. This cluster was further divided into two subclusters which merged at a similarity level of about 50%: one including only CBS 732, the other one including strains ABT 808, ABT 401, ABT 301, and ABT 601. Differences in genome sizes were also provided by densitometric analysis of PFGE patterns, using S. cerevisiae as reference. The haploid genome size was reported in Table 4. The DNA content of cells in G0/G1 phase determined by FCM and the average haploid genome size estimated by PFGE were used to infer the ploidy ratio. Z. rouxii CBS 732T showed a ploidy level of approximately 1.3, in line with a haploid DNA content. Conversely the ploidy ratio was higher for strains ATCC 42981, ABT 301, and ABT 601 (2.1, 2.0, and 2.3, respectively), indicating a diploid, or at least an aneuploid DNA content. 3.3. Mapping of ZSOD gene by chromo-blotting The ploidy level can also be estimated by analysis of heterologous status of single-copy genes. Strain ATCC 42981 contains two similar ZSOD genes, ZSOD2 and ZSOD22 (85% DNA sequence identity; Iwaki et al., 1998), whereas CBS 732T contains only one copy of the ZSOD gene, namely ZSOD2-22, that is almost identical (99% identity) to ATCC 42981 ZSOD2 but lacks the 45-bp deletion at the 30 end of the gene that is seen in ATCC 42981 (Kinclova et al., 2001). Strains ABT 301 and ABT 601 showed another combination of ZSOD copies, namely ZSOD2-22 and SOD22 (Solieri et al., 2007). We used
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Fig. 1. Cell cycle analysis histograms of Z. rouxii CBS 732T(A), ATCC 42981 (B), ABT 301 (C) and ABT 601 (D) yeast cells in three different conditions: grown in YPD medium to the early exponentially growing phase (A1–D1); arrested with 8-hydroxyquinoline (A2–D2) in YNB5%G medium; mixed to S. cerevisiae haploid or diploid strains grown to the exponential phase in YPD medium (A3–D3).
the ZSOD genes of strains CBS 732T, ATCC 42981, ABT 301 and ABT 601 as targets in a chromoblot analysis to verify whether the ZSOD
genes represent allelic variants of the same gene locus located on the same chromosome or homeologous genes located on different
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Table 2 Percentages of cells treated and non-treated with 8-hydroxyquinoline in G0/G1 and G2/M phases. Strains
CBS 732 ATCC 42981 ABT 301 ABT 601
Conditiona
C HQ C HQ C HQ C HQ
Percentage of cells
Table 3 Genome size (Mb) of Z. rouxii CBS 732T and related strains estimated by flow cytometry (FCM) of PI-stained cells in G0/G1 phases. Strains
G0/G1
G2/M
20.5 64.3 25.1 85.2 21.3 75.52 21.7 78.9
76.6 35.8 72.4 11.9 68.7 19.9 61.7 10.8
S. cerevisiae BY4742 BY4743 Z. rouxii CBS 732T ATCC 42981 ABT 301 ABT 601
DNA In1
DNA In2
Genome size ± SD (Mb)
103.3 ± 3.0 198.0 ± 4.03
1.00 2.00
1.03 2.06
13.1a 26.2b
93.36 ± 2.21 165.74 ± 0.37 201.95 ± 1.44 282.9 ± 2.8
0.97 1.67 2.12 2.98
1.00 1.72 2.21 3.07
12.7 ± 0.2 21.9 ± 0.2 28.1 ± 1.3 39.0 ± 0.3
MFI of G0/G1 cells ± SD
a C, control, cells grown to YNB5%G medium to early growing phase; HQ, exponential-growing culture after 24 h in YNB5%G medium with 8-hydroxyquinoline (100 mg/l).
a Genome size of S. cerevisiae haploid and diploid strains including repeated rDNA sequences (Goffeau et al., 1996). Genomic size was expressed in megabase (Mb) as means among three replicas ± standard deviation (SD).
chromosomes. A preliminary southern blot analysis was carried out to estimate the number of ZSOD2-22 and ZSOD22 copies for strains ABT 301 and ABT 601. Similarly to that found for CBS 732T and ATCC 42981 (Kinclova et al., 2001), hybridization of the probes ZSOD2-22 and ZSOD22 to genomic DNAs of strains ABT 301 and ABT 601 cut with four restriction enzymes showed only one copy of both the ZSOD2-22 and ZSOD22 genes (data not shown). Then chromoblot analysis with two 2 kb long digoxigenine-labeled probes targeted to ZSOD2-22/ZSOD2 and ZSOD22 genes, respectively, allowed to assign these probe to distinct chromosomes. As reported in Fig. 4, Z. rouxii CBS 732T ZSOD2-22 was located on chromosome IV and the ZSOD22 did not give any signal, according to Sychrova et al. (2000) and Kinclova et al. (2001). Strain ATCC 42981 showed two distinctive signals corresponding to chromosome VI for ZSOD2 gene and chromosome VII for ZSOD22, respectively. Similarly, ZSOD2-22 genes were located on chromosomes VII and VIII in strains ABT 301 and ABT 601, respectively, whereas ZSOD22 laid on bigger chromosomes difficult to be resolved on the basis of the migration pattern, namely IX/X for ABT 301 and X/XI for ABT 601. According to that found by Gordon and Wolfe (2008) our results suggested that ZSOD2 and ZSOD2-22 are allele of the same locus, whereas ZSOD22 is the homeologous gene with 85% DNA sequence identity. Therefore ZSOD genes mapping highlighted a similar genome organization among ATCC 42981 and ABT 301 and ABT 601, that is different compared to Z. rouxii CBS 732T.
4. Discussion The genus Zygosaccharomyces underwent quite complex taxonomic changes through the decades and ploidy represented an open question still under discussion. It was introduced in 1901 by Barker to describe osmophilic yeast species that differ from the genus Saccharomyces for their vegetative growth, in which sexual conjugation between cells or a cell and its bud, precedes the sporulation (Barker, 1901). Diploidization through zygote formation means that vegetative life cycle is mainly haploid. The distinction between haploid genus Zygosaccharomyces and diploid genus Saccharomyces has been then considered invalid on the basis of observations that some haploid Z. rouxii yeasts can shift to diploid state and ascosporulation can occur without conjugation (as quoted by Winge and Roberts, 1958). Thus, Lodder and Kreger-Van Rij (1952) considered Zygosaccharomyces as a subdivision within the genus Saccharomyces including species that exist exclusively or partially as haploid cells in vegetative phase. Taxonomically Zygosaccharoymces has been then reinstated as a separate genus by Yarrow (1984). Nowadays it consists of 11 species, which recently have been splitted into three phylogenetically related genera, according to multi-gene sequence analysis (Kurtzman, 2003). However, the topic of ploidy assessment of Zygosaccharomyces species and particularly of Z. rouxii has not been deepened.
Fig. 2. Cell cycle analysis of mixed cell populations of S. cerevisiae haploid and diploid strains (A) and standard curve relating mean fluorescence intensity (MFI) and theoretical amount of DNA per cell (B).
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Fig. 3. Karyotype profiles of Z. rouxii strains obtained by 100 h (A) and 120 h long (B) PFGE and dendrogram tree showing the clustering based on chromosome length polymorphism evaluated by Pearson product moment correlation coefficient (r) and the unweighted pair group method with arithmetic mean (UPGMA) (C). (A) Lanes: 1, S. cerevisiae YNN 295 (chromosome length in Mb; Bio-Rad Laboratories); 2, Z. mellis ABT 401; 3, Z. rouxii ATCC 42981; 4, Z. rouxii CBS 732T; 5, Z. rouxii ABT 808; 6, Z. rouxii ABT 301; 7, Z. rouxii ABT 601. The numbers reported in lane 1 indicate the sizes of the S. cerevisiae chromosomal bands in megabases (Mb). (B) Lanes: 1, Z. rouxii CBS 732T; 2, Z. rouxii ABT 808; 3, Z. rouxii ABT 301; 4, Z. rouxii ABT 601; 5, Z. mellis ABT 401; 6, Z. rouxii ATCC 42981.
Table 4 Comparison of the genome size determined by flow cytometry (FCM) with data regarding the chromosome-sized DNA separated by pulsed-field gel electrophoresis (PFGE). Strains
Genome size ± SD (Mb)
Number of chromosomes
R of PFGE bands (Mb)
Ploidy ratio
CBS 732T ATCC 42981 ABT 301 ABT 601
12.7 ± 0.2 21.9 ± 0.2 28.1 ± 1.3 39.0 ± 0.3
6 7 10 11
9.99 10.07 13.94 16.55
1.3 2.1 2.0 2.3
Genome size and sum of PFGE bands were expressed in megabase (Mb).
Since ploidy is an essential genetic feature that underlies significant cytological and physiological characteristics (Zeyl, 2004), the main goal of this work was to obtain new insights regarding Z. rouxii genome size and ploidy, by including the type strain Z. rouxii
CBS 732T and the wild strains ATCC 42981, ABT 301 and ABT 601. Previously we demonstrated that wild strains ABT 301 and ABT 601 have three divergent 5.8S-ITS rDNA copies and two different copies of both the nuclear genes ZSOD (ZSOD2-22 and ZSOD22, respectively) and HIS3. Their genome composition seems to be similar to that of strain ATCC 42981, which has two subgenomes, including two different copies of 5.8-ITS and 26S rDNA regions in its nucleus (Gordon and Wolfe, 2008). In this work, we set up a PI-based FCM protocol to characterize the DNA content of G0/G1 population. Cell clusters were a major constraint regarding the application of this technique for the analysis of Z. rouxii species. Nonetheless, our results show that the cytofluorimetric analysis of this yeast can give acceptable estimation of genome size, by using pre-treatments consisting in incubations at 50 °C with RNase A (0.25 mg/ml) and proteinase K (2 mg/ml) for 120 min each, followed by ultrasonication. Nevertheless, this technique measures
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Fig. 4. Mapping of ZSOD genes by PFGE and southern hybridization. Chromosomal DNA was isolated from the following strains: 1, CBS 732T; 2, ABT 808; 3, ABT 301; 4, ABT 601;5, ABT 401; 6, ATCC 42981. For Southern hybridization analysis two 2 Kb probes were employed for targeting ZSOD2-22/ZSOD2 (A) and ZSOD22 genes (B), respectively.
extranuclear DNA also (e.g., mitochondrial DNA) and so a slight overestimation would be expected. We found that there is a high variability of DNA content among tested strains, with values ranging from 12.7 ± 0.2 to 39.00 ± 0.3 Mb and that the genome size of wild strains are about 2–3-fold that of type strain CBS 732T, suggesting a variation of ploidy level and/or of chromosome number in the haploid set. Electrophoretic karyotyping provided complementary data to FCM in determining the ploidy level as the haploid genome size may be figured out by PFGE pattern. The species of Saccharomyces sensu stricto group display similar basic karyotypic characters, i.e. the same haploid number of chromosomes (n16) and similar range of chromosomal bands (from 250 to 2200 Kb). Conversely a high variability of chromosomal numbers was found within the genus Zygosaccharomyces, including some species having few chromosomes ranging from 4 to 5 chromosomes and others one with higher chromosome number (8–10 chromosomes). Even though heterogeneity at intraspecific level has also been observed, generally the patterns of strains belonging to the same species are very similar. Thus, the PFGE technique has been proposed as a powerful tool for differentiating Zygosaccharomyces species (Esteve-Zarzoso et al., 2003). However, difficulties related to band size estimation, co-migrating chromosomes, and electrophoretic conditions, can complicate the interpretation of PFGE results, mainly when arisen from different laboratories (Keogh et al., 1998). Noteworthy we analyzed our strains in the same PFGE conditions to improve the karyotypic profiles comparison. On the basis of our results, CBS 732T and ATCC 42981 seemed to have quite similar karyotypes with 6 and 7 chromosomal bands, respectively, corresponding to 9.99 and 10.07 Mb in genome size (Table 4). The chromosome number estimated for CBS 732T agreed to that found by De Jonge et al. (1986), whereas Sychrova et al. (2000) and Pribylova et al. (2007a) reported karyotypes of 7 chromosomes for CBS 732T and of 8 chromosomes for ATCC 42981, supporting a difference of one chromosome between two strains. In particular, comparing our result to that of Pribylova and coworker (2007a), the missing chromosomal bands could be located between chromosomes IV and V (2.2–1.6 Mb; corresponding to chromosome number V in Pribylova et al., 2007a) for CBS 732T and between chromosomes VI and VII for ATCC 42981 (2 Mb; corresponding to chromosome number VII in Pribylova et al., 2007a). Finally strains ABT 301 and ABT 601 showed a very similar PFGE profile (similarity higher than
90%), which did not overlap to those from CBS 732T and ATCC 42981. Combining FCM results and karyotypic data, the ploidy ratio was about 1.3 for type strain CBS 732T and between 2 and 2.3 for wild strains. Differently from that reported by de Montigny et al. (2000), our results suggested a haploid DNA content for CBS 732T. This finding has been also confirmed by isolation of auxotrophic mutants of CBS 732T (Pribylova et al., 2007b). However, a diploid DNA content was found for ATCC 42981, ABT 301, and ABT 601. The change in ploidy could be also confirmed by doubling of genes involved in osmotic adaptation (ZSOD, HOG, GPD, and GCY1) (Iwaki et al.,1999, 2001), as well as by divergent rDNA genes occurring in ATCC 42981 (Gordon and Wolfe, 2008) and in ABT 301 and ABT 601 (Solieri et al., 2007), but not in CBS 732T. In particular ZSOD genes encode Na+/H+-antiporters involved in halotolerance (Iwaki et al., 1998). Gene probe-chromosome hybridization targeted to ZSOD genes showed that strains with a diploid DNA content have the two partially divergent ZSOD copies located on different chromosomes. According to that previously found for ATCC 42981, this result could be explained considering that there are two divergent paralogs ZSOD genes located on non-co-migrating chromosomes arisen from interspecies hybridization in ABT 301 and ABT 601 genomes. Pribylova et al. (2007a) and Gordon and Wolfe (2008) suggested an allodiploidy origin for ATCC 42981. In particular ZSOD2 and ZSOD2-22 are two alleles of a locus in the T-subgenomes, and ZSOD22 is the homeologous locus in the P-subgenomes. However, ZSOD2-22 and ZSOD22 could be two independent genes on two different chromosomes, each existing twice in strains with diploid DNA content. If a recent allodiploidization event determined two nuclear subgenomes coming from two parental strains, we should find this genomic structure only in strains with a diploid DNA content. Conversely Sujaya and coworkers (2003) reported that haploid heterothallic strain CBS 4838 (mating type a) showed two divergent ITS regions, similarly to wild strains with diploid DNA content. The other mating type strain CBS 4837 (NCYC 1682) is haploid (Wickerham and Burton, 1960; Mori and Onishi, 1967) and has with two different copies of ZSOD, HIS3, and ADE2 genes (James et al., 2005). In these cases one haplotype could contain both copies of this gene. Regarding the origin of wild strains with a diploid DNA content, Wickerham and Burton (1960) and Mori and Onishi (1967) reported the occur-
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rence of mating between haploid heterothallic strains to obtain stable cells with diploid DNA content. To conclude, we applied complementary molecular strategies to dissect the complex picture in genome organization within the Z. rouxii clade and highlight that genome size and ploidy of strains classified as this species, are substantially divergent. In particular we demonstrated differences in genome size and copy number between CBS 732T (1n) and the wild strains ATCC 42981, ABT 301, and ABT 601 (2n). The role of diploidization in Z. rouxii has a great interest, mainly considering the ploidy level as an adaptive trait in continue evolutionary modification (Mable and Otto, 1998). In addition, the strong divergence among karyotype patterns found in this work could suggest that Z. rouxii clade could contain more than one species. This hypothesis is also supported by the high number of strains isolated from high sugary environments and very similar to ABT 301 and ABT 601 in terms of karyotype, 5.8SITS and 26S rDNA gene sequences (Solieri et al., 2006). Beside the availability of the CBS 732T whole genome sequence (the Genolevures III project), further analyzes both of MAT locus and cell cycle in different growth conditions could be help to disclose physiological responses and taxonomic status of these diploid strains, as well as to provide new insights on haploid–diploid shift in Z. rouxii strains. Acknowledgment Hana Sychrova and Lenka Pribylova are highly acknowledged for kind help with karyotyping. References Almeida, A.J., Martins, M., Carmona, J.A., Cano, L.E., Restrepo, A., Leao, C., Rodrigues, F., 2006. New insights into the cell cycle profile of Paracoccidioides brasiliensis. Fungal Genet. Biol. 43, 401–409. Almeida, A.J., Matute, D.R., Carmona, J.A., Martins, M., Torres, I., McEwen, J.G., Restrepo, A., Leão, C., Ludovico, P., Rodrigues, F., 2007. Genome size and ploidy of Paracoccidioides brasiliensis reveals a haploid DNA content: Flow cytometry and GP43 sequence analysis. Fungal Genet. Biol. 44, 25–31. Anderson, J.B., Sirjusingh, C., Ricker, N., 2004. Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae. Genetics 168, 1915– 1923. Bakalinsky, A.T., Snow, R., 1990. The chromosomal constitution of wine strains of Saccharomyces cerevisiae. Yeast 6, 367–382. Barker, B.T.P., 1901. A conjugating ‘yeast’. Philos. Trans. R. Soc. Lond. B 194, 467– 485. Britten, R., Kohne, D.E., 1986. Repeated sequences in DNA. Hundreds and thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science 161, 529–540. Carle, G.F., Olson, M.V., 1985. An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA 82, 3756–3760. Cliften, P.F., Fulton, R.S., Wilson, R.K., Johnston, M., 2006. After the duplication: gene loss and adaptation in Saccharomyces genomes. Genetics 172, 863–872. de Montigny, J., Straub, M.L., Potier, S., Tekaia, F., Dujon, B., Wincker, P., Artiguenave, F., Souciet, J.L., 2000. Genomic exploration of the hemiascomycetous yeasts: 8. Zygosaccharomyces rouxii. FEBS Lett. 487, 52–55. De Jonge, P., De Jongh, F.C.M., Meijers, R., Steensma, H.Y., Scheffers, W.A., 1986. Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeast. Yeast 2, 193–204. Esteve-Zarzoso, B., Zorman, T., Belloch, C., Querol, A., 2003. Molecular characterisation of the species of the genus Zygosaccharomyces. Syst. Appl. Microbiol. 26, 404–411. Fortuna, M., Sousa, M.J., Corte-Real, M., Leao, C., Salvador, A., Sansonetty, F., 2000. Cell cycle analysis of yeast. In: Current Protocols in Flow Cytometry. John Wiley & Sons, New York, pp. 11.13.1–11.13.9. Fowell, R.R., 1969. Life cycles in yeasts. In: Rose, A.H., Harrison, J.S. (Eds.), The Yeasts. Academic press, New York, pp. 464–471. Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., et al., 1996. Life with 6000 genes. Science 274, 546.563–546.567. Gordon, J.L., Wolfe, K.H., 2008. Recent allopolyploid origin of Zygosaccharomyces rouxii strain ATCC42981. Yeast 25, 449–456. Iwaki, T., Higashida, Y., Tsuji, H., Tamai, Y., Watanabe, Y., 1998. Characterization of a second gene (ZSOD22) of Na+/H+ antiporter from salt-tolerant yeast Zygosaccharomyces rouxii and functional expression of ZSOD2 and ZSOD22 in Saccharomyces cerevisiae. Yeast 14, 1167–1174.
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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi
Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species Lisa Solieri a,*, Stefano Cassanelli a, Maria Antonietta Croce b, Paolo Giudici a a b
Department of Agricultural and Food Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42100 Reggio Emilia, Italy Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy
a r t i c l e
i n f o
Article history: Received 25 June 2008 Accepted 2 October 2008 Available online 15 October 2008 Keywords: Zygosaccharomyces rouxii Ploidy Flow cytometry Cell cycle analysis Genome size Yeast Karyotype
a b s t r a c t Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7 ± 0.2 Mb for strain CBS 732T, 21.9 ± 0.2 Mb for ATCC 42981, 28.1 ± 1.3 Mb for ABT 301, and 39.00 ± 0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its ability to grow in environments with high soluble solids concentration. It is involved in the production of high sugary and/or salt fermented food, such as miso and traditional balsamic vinegar, respectively. Furthermore it has been also recognized as one of the main spoilage microorganisms in food industry. According to its incapacity to produce petite mutant, Z. rouxii shows a preferentially respirative metabolism (Merico et al., 2007) and it exhibits a vegetative haploid life style with zygote formation occurring immediately before sporulation (Fowell, 1969; Mori and Onishi, 1967). Phylogenetically Z. rouxii species is closely related to Saccharomyces cerevisiae, but precedes the whole genome duplication which took place in an ancestor of S. cerevisiae (Wong and Wolfe, 2006). Several phenotypical and molecular approaches based on cloning procedure, single-gene PCR amplification and karyotyping highlight a high variability within Z. rouxii species. Two extensively studied Z. rouxii strains, CBS 732T and ATCC 42981, showed different susceptibility to transformation by electroporation, as well as different salt tolerance, glycerol production and karyotype, whereas a conservation of synteny has been observed (Pribylova and Sychrova, 2003; Pribylova et al., 2007a). James et al. (2005) reported that strain CBS 9951 (NCYC 3042) could belong to a novel * Corresponding author. Fax: +39 0522 522027. E-mail address: [email protected] (L. Solieri). 1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.10.001
species (called Z. pseudorouxii) and hybridization events could occur among Z. rouxii and Z. pseudorouxii species [hybrid strain CBS 4837 (NCYC 1682)]. Solieri et al. (2007) proposed a new putative species to describe two strains (namely ABT 301 and ABT 601) isolated from traditional balsamic vinegar, showing a high polymorphism in 5.8S rDNA and two flanking internal transcribed spacers sequences (5.8S-ITS region), as well as two copies of ZSOD genes (ZSOD2-22 and ZSOD22) and HIS3 genes, encoding Na+/H+-antiporter and imidazole–glycerol–phosphate dehydratase, respectively. Recently Gordon and Wolfe (2008) have more extensively investigated the genome structure of ATCC 42981 by random sequencing, showing the presence of T subgenome derived from Z. rouxii (with 97–100% DNA sequence identity to CBS 732T) and P subgenome derived from Z. pseudorouxii (about 80–90% identity). The results suggested that ATCC 42981 is an allodiploid yeast. However, no genome size and ploidy level estimations of this strain as well as of ABT 301 and ABT 601 strains have been attempted. In recent decades, ploidy level and genome size have been regarded as important subjects for inferring genome and sex evolution, even considering the paleopolyploid nature of the S. cerevisiae genome (Wong et al., 2002). Moreover, changes in ploidy introduce potentially significant cytological and physiological effects, without requiring any long-term evolutionary processes (Zeyl, 2004). Indeed Keogh et al. (1998) showed that a correlation between gene order evolution and chromosome number in S. cerevisiae and its close relatives. Several studies have been performed to elucidate the mechanisms of fast evolution of polyploid genomes toward diploidization in S. cerevisiae (Wolfe and Shields,
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1997; Wolfe, 2001; Kellis et al., 2004; Cliften et al., 2006; Scannell et al., 2007). Other studies have focused on how and why haploid and diploid phases can advantage or disadvantage adaptation in a new environment and provide high or low fitness benefits (Perrot et al., 1991; Korona, 1999; Mable and Otto, 1998, 2001; Zeyl et al., 2003; Zeyl, 2004; Anderson et al., 2004). For instance, the ploidy of wine yeasts confers advantages in adapting to variable environments or by increasing the dosage of some genes important for fermentation (Bakalinsky and Snow, 1990; Salmon, 1997). These findings shed a new light on ploidy level as an adaptive evolutionary trait (Katz Ezov et al., 2006). The genome size (C value) of unicellular eukaryote can be estimated by biochemical assays, which determine the phosphate content of DNA isolated from a defined number of cells (Shapiro, 1970); analysis of reassociation kinetics, using hydroxyl apatite chromatography to separate single-stranded and double-stranded DNA (dsDNA) (Britten and Kohne, 1986); pulsed-field gel electrophoresis (PFGE), in which the lengths of whole chromosomes are measured directly (Johnston and Mortimer, 1986; VaughanMartini et al., 1993); more recently by Real-time PCR (Wilhelm et al., 2003). Ploidy level can be obtained comparing the haplotype size generated by PFGE with data obtained by microfluorimetry or by flow cytometry-based methods (FCM). In FCM assay the DNA content of individual nuclei is analyzed by fluorescence measurements after staining with intercalating dyes, such as propidium iodide, ethidium bromide or SYBR Green I, and quantified using reference strains with known genome size (Fortuna et al., 2000). Recently FCM-based methods were successfully employed to determine the genome size and ploidy level of multibudding yeast Paracoccoioides brasiliensis (Almeida et al., 2006, 2007) and of spoilage yeast Z. bailii (Rodrigues et al., 2003). In this study, genome size and ploidy level estimation, as well as ZSOD gene chromosome mapping were used to elucidate the taxonomic relationships among Z. rouxii strains, CBS 732T and three wild strains, such as ATCC 42981 isolated from miso (Kiuchi et al., 1980) and ABT 301 and ABT 601 from traditional balsamic vinegar (Solieri et al., 2006, 2007). 2. Material and methods
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(YNB5%G) at 24 °C with aeration on a mechanical shaker (180 rpm). 2.2. Cell cycle analysis Zygosaccharomyces rouxii and Saccharomyces cerevisiae yeast strains were grown in YPD and YNB5%G medium batch culture to mid-log phase (OD600nm = 0.50.6). Cell samples in YNB5%G medium were inducted to arrest in G1 phase by treatment with 8hydroxyquinoline at a final concentration 100 mg/l for 24 h, as previously described (Johnston and Singer, 1978; Stöver et al., 1998). Cells were harvested by centrifugation (8000 g for 5 min at 4 °C), fixed overnight with 70% ethanol (vol/vol) at 4 °C and subjected to cell cycle analysis as previously described (Fortuna et al., 2000) and modified as follows. After fixation, cells were harvested, washed, and suspended at 5 106 cells/ml in 500 ll of sodium citrate buffer (50 mM; pH 7.0). Cells suspension was treated for 120 min at 50 °C with RNAse A (0.25 mg/ml) and/or proteinase K (2 mg/ml). A cell suspension of 2.5 106 cell/ml was stained overnight at 4 °C with propidium iodide (PI; Sigma) at a final concentration of 4 lg/ml. To remove of clumps and excessive debris, two consecutive ultrasound pulses were applied before FMC analysis at 70% of total output for 20 s with an interval of 1–2 s between the two pulses by using Microson Ultrasonic Cell Disruptor XL (Misonix Inc., New Highway Farmingdale, NY, USA). 2.3. Flow cytometry and estimation of genome size All FCM experiments were performed in triplicates on an EPICs XL (Coulter, Instrumentation Laboratory, Milano, Italia) flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. A minimum of 20,000 cells per sample were acquired at low flow rate and an acquisition protocol was defined to measure forward scatter (FS LOG) and side scatter (SS LOG) on a four-decade logarithmic scale and red Fluorescence (FL3) on a linear scale. For DNA content analysis, doublets and larger aggregates were discarded by electronic analysis of integral- and height-signals from the particles analyzed. Offline data were analyzed with the Windows Multiple Document Interface for Flow Cytometry 2.8 (WinMDI 2.8).
2.1. Strains and culture media 2.4. Chromosomal DNA preparation and pulse-field gel electrophoresis Zygosaccharomyces strains used in this study were listed in Table 1. In addition S. cerevisiae haploid strain BY4742, Euroscarf Acc. No. Y10000 (MATa; his3 D1; leu2 D0; lys2 D0; ura3 D0) and diploid strain BY4743, Euroscarf Acc. No. Y23146 (Mat a/a; his3 D1/his3 D1; leu2 D0/leu2 D0; lys2 D0/LYS2; MET15/met15 D0; ura3D0/ura3 D0; YBR011c::kanMX4/YBR011c) were included. All yeast strains were maintained at 24 °C by periodic subculturing in slanted tubes with YPD solid medium (1% yeast extract, 1% peptone, 2% glucose, 2% agar). For the assays carried out in this study yeast cells were routinely grown in YPD liquid medium and in yeast nitrogen base medium supplemented with 5% (g/v) glucose Table 1 Zygosaccharomyces strains used in this work. Strains
Species
Country of isolation
Source
Citation
CBS 732T
Z. rouxii
Italy
Sacchetti (1932)
ABT 301
Z. pseudorouxii
Italy
ABT 601 ABT 808 ABT 401 ATCC 42981
Z. Z. Z. Z.
‘‘ ‘‘ ‘‘ Japan
Concentrated blackgrape must Traditional balsamic vinegar ‘‘ ‘‘ ‘‘ Miso paste
pseudorouxii rouxii mellis rouxii
Solieri et al. (2006) ‘‘ ‘‘ ‘‘ Kiuchi et al. (1980)
Pulse-field gels were run on a Bio-Rad CHEF (contour-clamped homogeneous electric field) apparatus. Chromosomal DNA was isolated as described by Carle and Olson (1985), with the exception of 5 h incubation with Lyticase (Sigma), in spite of an incubation of 2 h at 37 °C with Zymoliase (Pribylova et al., 2007a). The 1% agarose gels were prepared with Seakem GTG agarose (FMC Bioproducts) and run in 0.5X TBE buffer at 13 °C. The running conditions were: switch time 300 s, run time 100/120 h, angle 106°, voltage 3 V/cm. The chromosomal DNA size marker of S. cerevisiae S288 C (Bio-Rad Laboratories) was used to estimate the chromosome size. Pictures of the gels were digitally captured using the BioDocAnalyze gel imaging and analysis system (Biometra, Göttingen, Germany). The DNA banding patterns were analyzed with BioNumerics version 3.0 (Applied Maths, Belgium). Similarity matrix was calculated using Pearson’s product moment correlation coefficient. Cluster analysis of similarity matrix was performed by the Unweighted Pair Group Method with Arithmetic mean (UPGMA) algorithm. 2.5. Southern blot Chromosomal DNAs separated by PFGE were transferred onto a Hybond-N+ membrane (Amersham) by upward capillary transfer.
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Hybridizations (overnight at 43 °C) were performed with 2 Kb probes generated by PCR using the following primer pairs: kar2f 50 -CAGCCTCATGGATCCTCTTGATGGTT-30 (Kinclova et al., 2001) and SOD1R (James et al., 2005) specific for both ZSOD2-22 and ZSOD2 genes; kar22f 50 -GGTTTGATAGGAGTCGGAAACACTTAC-30 (modified from Kinclova et al., 2001) and SOD2R (James et al., 2005) specific for ZSOD22 gene. All probes used in this study were labeled with digoxigenin-labeled 11-dUTP by using the DIG-DNA labeling kit (Roche). Detection was carried out by chemiluminescence, using an antidigoxigenin antibody and CDP-star (Roche), as recommended by the manufacturer.
size was 12.7 ± 0.21 Mb, while wild strains showed a double or more genome size. According to Rodrigues et al. (2003), we calculated two DNA indexes (DNA In): DNA In1 is in relation to the MFI of the G0/G1 peak of S. cerevisiae haploid strain B4742; DNA In2 is in relation to that of Z. rouxii strain CBS 732T. These DNA In1 values indicated that the strain CBS 732T had 0.97 times as much DNA as S. cerevisiae haploid strain, whereas the wild strains showed a double (ATCC 42981 and ABT 301) or triple (ABT 601) DNA content compared to S. cerevisiae haploid strain. The DNA In2 confirmed the wild strains had a bigger genome size respected to CBS 732T. These differences could be arisen both from ploidy level and chromosome number for haploid set.
3. Results 3.1. Genome size estimation To determine Z. rouxii strains genome size, we optimized a previously described FCM protocol for cell cycle profile analysis of S. cerevisiae and Z. bailii (Fortuna et al., 2000). The FCM analysis of ethanol-fixed Z. rouxii cells grown in YPD medium revealed a low specificity for nuclear PI staining, resulting in a cytometric pattern which did not show the characteristic bimodal profile linked to G0/ G1 and G2/M phases. Pre-treatments with RNase and proteinase K and sonication improved the pattern resolution and allowed to discriminate two major subpopulations of cells with different DNA content corresponding to G0/G1 and G2/M cell cycle phases, respectively (Fig. 1 A1, B1, C1, and D1). All tested strains showed a higher cells percentage in G2/M than in G0/G1 and S. The halfpeak coefficient of variation (HPCV) of G0/G1 peak is lower than 11% (data not shown), indicating an acceptable resolution of dsDNA measurements. According to Rodrigues et al. (2003), a direct correlation can be established between mean fluorescence intensity (MFI) and the amount of dsDNA. In addition treatment with 8-hydroxyquinoline in YNB5%G medium was performed to arrest Z. rouxii cells in G0/G1 and to confirm the peak corresponding to this phase (Fig. 1 A2, B2, C2, and D2). This chemical chelates zinc which is required for the G0/G1 to S transition and has been widely used in cell cycle studies involving S. cerevisiae (Johnston and Singer, 1978). As found for C. albicans (Stöver et al., 1998), 8hydroxyquinoline at a concentration of 100 mg/l is suitable for the cell cycle arrest of Z. rouxii strains, resulting in an increased fraction of the cell population in G0/G1 (Table 2). Saccharomyces cerevisiae haploid and diploid strains, isogenic to the previously sequenced yeast S288C with a haploid genome of 13.1 Mb (Goffeau et al., 1996), were used as references for direct DNA estimation. A concurrent cell cycle analysis of S. cerevisiae haploid and diploid strains revealed three distinct peaks (Fig. 2A), corresponding to 1n, 2n, and 4n DNA contents, where the MFI values of each peak was directly correlated to the amount of DNA (Mb) of its corresponding cell subpopulation (Fig. 2B). A preliminary analysis of mixed cell populations of Z. rouxii strains and S. cerevisiae haploid or diploid strain was used to determine single-cell DNA content (Fig. 1A3–D3). In particular we found three peaks, corresponding to 1n, 2n, and 4n DNA content by mixing CBS 732T and S. cerevisiae diploid cells, suggesting a haploid DNA content for Z. rouxii type strain (Fig. 1 A3). On the contrary we mixed strains ATCC 42981 and ABT 301 cells to S. cerevisiae haploid cells to obtain three peaks, showing a diploid DNA content for these Zygosaccharomyces strains (Fig. 1B3 and C3). Regarding strain ABT 601, we obtained four peaks by mixing it and S. cerevisiae diploid cells, indicating that it could be diploid but with a bigger DNA content per cell compared to S. cerevisiae (Fig. 1 D3). Genome size of each Z. rouxii strain was estimated in accordance with MFI of the G0/G1 subpopulation by using S. cerevisiae standard curve (Fig. 2B). The average amount of DNA per cell varied from 12.7 to 39.0 Mb (Table 3). For strain CBS 732T the genome
3.2. Separation of chromosome-sized DNA molecules by PFGE and ploidy level estimation Ploidy level is generally defined by the number of copies of each chromosome individual set per nucleus (Zeyl, 2004). The ploidy level of our strains was obtained by comparing the DNA content of Z. rouxii cells in G0/G1 phase determined by FCM (Table 2) with the average haploid genome size estimated by PFGE. Z. rouxii ABT 808 and Z. mellis ABT 401strains were used for comparison in PFGE experiment. The chromosomal bands were comparable in size and number to those of yeasts belonging to the genus Zygosaccharomyces, which are in low number ranging from 3 to 8 bands and whose electrophoretic separation requires long pulse times over a period of 5 days (De Jonge et al., 1986; Török et al., 1993; Oda and Tonomura, 1995; Sychrova et al., 2000; Esteve-Zarzoso et al., 2003). Two different migration times (100 and 120 h) were tested to resolve the most similar and large chromosomes (Fig. 3A and B). The karyotyping associated distinct chromosomal migration patterns to each strain, highlighting a high polymorphism in size and number of the bands. In particular the PFGE profiles of Z. rouxii CBS 732T and ABT808 consisted of 6 and 5 bands, respectively, ranging from 2.2 and 1.1 Mb, whereas the wild strains ATCC 42981, ABT 301, and ABT 601 showed more complex PFGE profiles consisting of a higher bands number, 7, 10 and 11, respectively. Also Z. mellis ABT 401 showed a complex chromosomal pattern with 9 chromosomal bands. Karyotype profiles were compared by cluster analysis (Fig. 3 C). ATCC 42981 did not belong to the main cluster including all the other Zygosacchaormyces strains. This cluster was further divided into two subclusters which merged at a similarity level of about 50%: one including only CBS 732, the other one including strains ABT 808, ABT 401, ABT 301, and ABT 601. Differences in genome sizes were also provided by densitometric analysis of PFGE patterns, using S. cerevisiae as reference. The haploid genome size was reported in Table 4. The DNA content of cells in G0/G1 phase determined by FCM and the average haploid genome size estimated by PFGE were used to infer the ploidy ratio. Z. rouxii CBS 732T showed a ploidy level of approximately 1.3, in line with a haploid DNA content. Conversely the ploidy ratio was higher for strains ATCC 42981, ABT 301, and ABT 601 (2.1, 2.0, and 2.3, respectively), indicating a diploid, or at least an aneuploid DNA content. 3.3. Mapping of ZSOD gene by chromo-blotting The ploidy level can also be estimated by analysis of heterologous status of single-copy genes. Strain ATCC 42981 contains two similar ZSOD genes, ZSOD2 and ZSOD22 (85% DNA sequence identity; Iwaki et al., 1998), whereas CBS 732T contains only one copy of the ZSOD gene, namely ZSOD2-22, that is almost identical (99% identity) to ATCC 42981 ZSOD2 but lacks the 45-bp deletion at the 30 end of the gene that is seen in ATCC 42981 (Kinclova et al., 2001). Strains ABT 301 and ABT 601 showed another combination of ZSOD copies, namely ZSOD2-22 and SOD22 (Solieri et al., 2007). We used
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Fig. 1. Cell cycle analysis histograms of Z. rouxii CBS 732T(A), ATCC 42981 (B), ABT 301 (C) and ABT 601 (D) yeast cells in three different conditions: grown in YPD medium to the early exponentially growing phase (A1–D1); arrested with 8-hydroxyquinoline (A2–D2) in YNB5%G medium; mixed to S. cerevisiae haploid or diploid strains grown to the exponential phase in YPD medium (A3–D3).
the ZSOD genes of strains CBS 732T, ATCC 42981, ABT 301 and ABT 601 as targets in a chromoblot analysis to verify whether the ZSOD
genes represent allelic variants of the same gene locus located on the same chromosome or homeologous genes located on different
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Table 2 Percentages of cells treated and non-treated with 8-hydroxyquinoline in G0/G1 and G2/M phases. Strains
CBS 732 ATCC 42981 ABT 301 ABT 601
Conditiona
C HQ C HQ C HQ C HQ
Percentage of cells
Table 3 Genome size (Mb) of Z. rouxii CBS 732T and related strains estimated by flow cytometry (FCM) of PI-stained cells in G0/G1 phases. Strains
G0/G1
G2/M
20.5 64.3 25.1 85.2 21.3 75.52 21.7 78.9
76.6 35.8 72.4 11.9 68.7 19.9 61.7 10.8
S. cerevisiae BY4742 BY4743 Z. rouxii CBS 732T ATCC 42981 ABT 301 ABT 601
DNA In1
DNA In2
Genome size ± SD (Mb)
103.3 ± 3.0 198.0 ± 4.03
1.00 2.00
1.03 2.06
13.1a 26.2b
93.36 ± 2.21 165.74 ± 0.37 201.95 ± 1.44 282.9 ± 2.8
0.97 1.67 2.12 2.98
1.00 1.72 2.21 3.07
12.7 ± 0.2 21.9 ± 0.2 28.1 ± 1.3 39.0 ± 0.3
MFI of G0/G1 cells ± SD
a C, control, cells grown to YNB5%G medium to early growing phase; HQ, exponential-growing culture after 24 h in YNB5%G medium with 8-hydroxyquinoline (100 mg/l).
a Genome size of S. cerevisiae haploid and diploid strains including repeated rDNA sequences (Goffeau et al., 1996). Genomic size was expressed in megabase (Mb) as means among three replicas ± standard deviation (SD).
chromosomes. A preliminary southern blot analysis was carried out to estimate the number of ZSOD2-22 and ZSOD22 copies for strains ABT 301 and ABT 601. Similarly to that found for CBS 732T and ATCC 42981 (Kinclova et al., 2001), hybridization of the probes ZSOD2-22 and ZSOD22 to genomic DNAs of strains ABT 301 and ABT 601 cut with four restriction enzymes showed only one copy of both the ZSOD2-22 and ZSOD22 genes (data not shown). Then chromoblot analysis with two 2 kb long digoxigenine-labeled probes targeted to ZSOD2-22/ZSOD2 and ZSOD22 genes, respectively, allowed to assign these probe to distinct chromosomes. As reported in Fig. 4, Z. rouxii CBS 732T ZSOD2-22 was located on chromosome IV and the ZSOD22 did not give any signal, according to Sychrova et al. (2000) and Kinclova et al. (2001). Strain ATCC 42981 showed two distinctive signals corresponding to chromosome VI for ZSOD2 gene and chromosome VII for ZSOD22, respectively. Similarly, ZSOD2-22 genes were located on chromosomes VII and VIII in strains ABT 301 and ABT 601, respectively, whereas ZSOD22 laid on bigger chromosomes difficult to be resolved on the basis of the migration pattern, namely IX/X for ABT 301 and X/XI for ABT 601. According to that found by Gordon and Wolfe (2008) our results suggested that ZSOD2 and ZSOD2-22 are allele of the same locus, whereas ZSOD22 is the homeologous gene with 85% DNA sequence identity. Therefore ZSOD genes mapping highlighted a similar genome organization among ATCC 42981 and ABT 301 and ABT 601, that is different compared to Z. rouxii CBS 732T.
4. Discussion The genus Zygosaccharomyces underwent quite complex taxonomic changes through the decades and ploidy represented an open question still under discussion. It was introduced in 1901 by Barker to describe osmophilic yeast species that differ from the genus Saccharomyces for their vegetative growth, in which sexual conjugation between cells or a cell and its bud, precedes the sporulation (Barker, 1901). Diploidization through zygote formation means that vegetative life cycle is mainly haploid. The distinction between haploid genus Zygosaccharomyces and diploid genus Saccharomyces has been then considered invalid on the basis of observations that some haploid Z. rouxii yeasts can shift to diploid state and ascosporulation can occur without conjugation (as quoted by Winge and Roberts, 1958). Thus, Lodder and Kreger-Van Rij (1952) considered Zygosaccharomyces as a subdivision within the genus Saccharomyces including species that exist exclusively or partially as haploid cells in vegetative phase. Taxonomically Zygosaccharoymces has been then reinstated as a separate genus by Yarrow (1984). Nowadays it consists of 11 species, which recently have been splitted into three phylogenetically related genera, according to multi-gene sequence analysis (Kurtzman, 2003). However, the topic of ploidy assessment of Zygosaccharomyces species and particularly of Z. rouxii has not been deepened.
Fig. 2. Cell cycle analysis of mixed cell populations of S. cerevisiae haploid and diploid strains (A) and standard curve relating mean fluorescence intensity (MFI) and theoretical amount of DNA per cell (B).
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Fig. 3. Karyotype profiles of Z. rouxii strains obtained by 100 h (A) and 120 h long (B) PFGE and dendrogram tree showing the clustering based on chromosome length polymorphism evaluated by Pearson product moment correlation coefficient (r) and the unweighted pair group method with arithmetic mean (UPGMA) (C). (A) Lanes: 1, S. cerevisiae YNN 295 (chromosome length in Mb; Bio-Rad Laboratories); 2, Z. mellis ABT 401; 3, Z. rouxii ATCC 42981; 4, Z. rouxii CBS 732T; 5, Z. rouxii ABT 808; 6, Z. rouxii ABT 301; 7, Z. rouxii ABT 601. The numbers reported in lane 1 indicate the sizes of the S. cerevisiae chromosomal bands in megabases (Mb). (B) Lanes: 1, Z. rouxii CBS 732T; 2, Z. rouxii ABT 808; 3, Z. rouxii ABT 301; 4, Z. rouxii ABT 601; 5, Z. mellis ABT 401; 6, Z. rouxii ATCC 42981.
Table 4 Comparison of the genome size determined by flow cytometry (FCM) with data regarding the chromosome-sized DNA separated by pulsed-field gel electrophoresis (PFGE). Strains
Genome size ± SD (Mb)
Number of chromosomes
R of PFGE bands (Mb)
Ploidy ratio
CBS 732T ATCC 42981 ABT 301 ABT 601
12.7 ± 0.2 21.9 ± 0.2 28.1 ± 1.3 39.0 ± 0.3
6 7 10 11
9.99 10.07 13.94 16.55
1.3 2.1 2.0 2.3
Genome size and sum of PFGE bands were expressed in megabase (Mb).
Since ploidy is an essential genetic feature that underlies significant cytological and physiological characteristics (Zeyl, 2004), the main goal of this work was to obtain new insights regarding Z. rouxii genome size and ploidy, by including the type strain Z. rouxii
CBS 732T and the wild strains ATCC 42981, ABT 301 and ABT 601. Previously we demonstrated that wild strains ABT 301 and ABT 601 have three divergent 5.8S-ITS rDNA copies and two different copies of both the nuclear genes ZSOD (ZSOD2-22 and ZSOD22, respectively) and HIS3. Their genome composition seems to be similar to that of strain ATCC 42981, which has two subgenomes, including two different copies of 5.8-ITS and 26S rDNA regions in its nucleus (Gordon and Wolfe, 2008). In this work, we set up a PI-based FCM protocol to characterize the DNA content of G0/G1 population. Cell clusters were a major constraint regarding the application of this technique for the analysis of Z. rouxii species. Nonetheless, our results show that the cytofluorimetric analysis of this yeast can give acceptable estimation of genome size, by using pre-treatments consisting in incubations at 50 °C with RNase A (0.25 mg/ml) and proteinase K (2 mg/ml) for 120 min each, followed by ultrasonication. Nevertheless, this technique measures
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Fig. 4. Mapping of ZSOD genes by PFGE and southern hybridization. Chromosomal DNA was isolated from the following strains: 1, CBS 732T; 2, ABT 808; 3, ABT 301; 4, ABT 601;5, ABT 401; 6, ATCC 42981. For Southern hybridization analysis two 2 Kb probes were employed for targeting ZSOD2-22/ZSOD2 (A) and ZSOD22 genes (B), respectively.
extranuclear DNA also (e.g., mitochondrial DNA) and so a slight overestimation would be expected. We found that there is a high variability of DNA content among tested strains, with values ranging from 12.7 ± 0.2 to 39.00 ± 0.3 Mb and that the genome size of wild strains are about 2–3-fold that of type strain CBS 732T, suggesting a variation of ploidy level and/or of chromosome number in the haploid set. Electrophoretic karyotyping provided complementary data to FCM in determining the ploidy level as the haploid genome size may be figured out by PFGE pattern. The species of Saccharomyces sensu stricto group display similar basic karyotypic characters, i.e. the same haploid number of chromosomes (n16) and similar range of chromosomal bands (from 250 to 2200 Kb). Conversely a high variability of chromosomal numbers was found within the genus Zygosaccharomyces, including some species having few chromosomes ranging from 4 to 5 chromosomes and others one with higher chromosome number (8–10 chromosomes). Even though heterogeneity at intraspecific level has also been observed, generally the patterns of strains belonging to the same species are very similar. Thus, the PFGE technique has been proposed as a powerful tool for differentiating Zygosaccharomyces species (Esteve-Zarzoso et al., 2003). However, difficulties related to band size estimation, co-migrating chromosomes, and electrophoretic conditions, can complicate the interpretation of PFGE results, mainly when arisen from different laboratories (Keogh et al., 1998). Noteworthy we analyzed our strains in the same PFGE conditions to improve the karyotypic profiles comparison. On the basis of our results, CBS 732T and ATCC 42981 seemed to have quite similar karyotypes with 6 and 7 chromosomal bands, respectively, corresponding to 9.99 and 10.07 Mb in genome size (Table 4). The chromosome number estimated for CBS 732T agreed to that found by De Jonge et al. (1986), whereas Sychrova et al. (2000) and Pribylova et al. (2007a) reported karyotypes of 7 chromosomes for CBS 732T and of 8 chromosomes for ATCC 42981, supporting a difference of one chromosome between two strains. In particular, comparing our result to that of Pribylova and coworker (2007a), the missing chromosomal bands could be located between chromosomes IV and V (2.2–1.6 Mb; corresponding to chromosome number V in Pribylova et al., 2007a) for CBS 732T and between chromosomes VI and VII for ATCC 42981 (2 Mb; corresponding to chromosome number VII in Pribylova et al., 2007a). Finally strains ABT 301 and ABT 601 showed a very similar PFGE profile (similarity higher than
90%), which did not overlap to those from CBS 732T and ATCC 42981. Combining FCM results and karyotypic data, the ploidy ratio was about 1.3 for type strain CBS 732T and between 2 and 2.3 for wild strains. Differently from that reported by de Montigny et al. (2000), our results suggested a haploid DNA content for CBS 732T. This finding has been also confirmed by isolation of auxotrophic mutants of CBS 732T (Pribylova et al., 2007b). However, a diploid DNA content was found for ATCC 42981, ABT 301, and ABT 601. The change in ploidy could be also confirmed by doubling of genes involved in osmotic adaptation (ZSOD, HOG, GPD, and GCY1) (Iwaki et al.,1999, 2001), as well as by divergent rDNA genes occurring in ATCC 42981 (Gordon and Wolfe, 2008) and in ABT 301 and ABT 601 (Solieri et al., 2007), but not in CBS 732T. In particular ZSOD genes encode Na+/H+-antiporters involved in halotolerance (Iwaki et al., 1998). Gene probe-chromosome hybridization targeted to ZSOD genes showed that strains with a diploid DNA content have the two partially divergent ZSOD copies located on different chromosomes. According to that previously found for ATCC 42981, this result could be explained considering that there are two divergent paralogs ZSOD genes located on non-co-migrating chromosomes arisen from interspecies hybridization in ABT 301 and ABT 601 genomes. Pribylova et al. (2007a) and Gordon and Wolfe (2008) suggested an allodiploidy origin for ATCC 42981. In particular ZSOD2 and ZSOD2-22 are two alleles of a locus in the T-subgenomes, and ZSOD22 is the homeologous locus in the P-subgenomes. However, ZSOD2-22 and ZSOD22 could be two independent genes on two different chromosomes, each existing twice in strains with diploid DNA content. If a recent allodiploidization event determined two nuclear subgenomes coming from two parental strains, we should find this genomic structure only in strains with a diploid DNA content. Conversely Sujaya and coworkers (2003) reported that haploid heterothallic strain CBS 4838 (mating type a) showed two divergent ITS regions, similarly to wild strains with diploid DNA content. The other mating type strain CBS 4837 (NCYC 1682) is haploid (Wickerham and Burton, 1960; Mori and Onishi, 1967) and has with two different copies of ZSOD, HIS3, and ADE2 genes (James et al., 2005). In these cases one haplotype could contain both copies of this gene. Regarding the origin of wild strains with a diploid DNA content, Wickerham and Burton (1960) and Mori and Onishi (1967) reported the occur-
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rence of mating between haploid heterothallic strains to obtain stable cells with diploid DNA content. To conclude, we applied complementary molecular strategies to dissect the complex picture in genome organization within the Z. rouxii clade and highlight that genome size and ploidy of strains classified as this species, are substantially divergent. In particular we demonstrated differences in genome size and copy number between CBS 732T (1n) and the wild strains ATCC 42981, ABT 301, and ABT 601 (2n). The role of diploidization in Z. rouxii has a great interest, mainly considering the ploidy level as an adaptive trait in continue evolutionary modification (Mable and Otto, 1998). In addition, the strong divergence among karyotype patterns found in this work could suggest that Z. rouxii clade could contain more than one species. This hypothesis is also supported by the high number of strains isolated from high sugary environments and very similar to ABT 301 and ABT 601 in terms of karyotype, 5.8SITS and 26S rDNA gene sequences (Solieri et al., 2006). Beside the availability of the CBS 732T whole genome sequence (the Genolevures III project), further analyzes both of MAT locus and cell cycle in different growth conditions could be help to disclose physiological responses and taxonomic status of these diploid strains, as well as to provide new insights on haploid–diploid shift in Z. rouxii strains. Acknowledgment Hana Sychrova and Lenka Pribylova are highly acknowledged for kind help with karyotyping. References Almeida, A.J., Martins, M., Carmona, J.A., Cano, L.E., Restrepo, A., Leao, C., Rodrigues, F., 2006. New insights into the cell cycle profile of Paracoccidioides brasiliensis. Fungal Genet. Biol. 43, 401–409. Almeida, A.J., Matute, D.R., Carmona, J.A., Martins, M., Torres, I., McEwen, J.G., Restrepo, A., Leão, C., Ludovico, P., Rodrigues, F., 2007. Genome size and ploidy of Paracoccidioides brasiliensis reveals a haploid DNA content: Flow cytometry and GP43 sequence analysis. Fungal Genet. Biol. 44, 25–31. Anderson, J.B., Sirjusingh, C., Ricker, N., 2004. Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae. Genetics 168, 1915– 1923. Bakalinsky, A.T., Snow, R., 1990. The chromosomal constitution of wine strains of Saccharomyces cerevisiae. Yeast 6, 367–382. Barker, B.T.P., 1901. A conjugating ‘yeast’. Philos. Trans. R. Soc. Lond. B 194, 467– 485. Britten, R., Kohne, D.E., 1986. Repeated sequences in DNA. Hundreds and thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science 161, 529–540. Carle, G.F., Olson, M.V., 1985. An electrophoretic karyotype for yeast. Proc. Natl. Acad. Sci. USA 82, 3756–3760. Cliften, P.F., Fulton, R.S., Wilson, R.K., Johnston, M., 2006. After the duplication: gene loss and adaptation in Saccharomyces genomes. Genetics 172, 863–872. de Montigny, J., Straub, M.L., Potier, S., Tekaia, F., Dujon, B., Wincker, P., Artiguenave, F., Souciet, J.L., 2000. Genomic exploration of the hemiascomycetous yeasts: 8. Zygosaccharomyces rouxii. FEBS Lett. 487, 52–55. De Jonge, P., De Jongh, F.C.M., Meijers, R., Steensma, H.Y., Scheffers, W.A., 1986. Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeast. Yeast 2, 193–204. Esteve-Zarzoso, B., Zorman, T., Belloch, C., Querol, A., 2003. Molecular characterisation of the species of the genus Zygosaccharomyces. Syst. Appl. Microbiol. 26, 404–411. Fortuna, M., Sousa, M.J., Corte-Real, M., Leao, C., Salvador, A., Sansonetty, F., 2000. Cell cycle analysis of yeast. In: Current Protocols in Flow Cytometry. John Wiley & Sons, New York, pp. 11.13.1–11.13.9. Fowell, R.R., 1969. Life cycles in yeasts. In: Rose, A.H., Harrison, J.S. (Eds.), The Yeasts. Academic press, New York, pp. 464–471. Goffeau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon, B., Feldmann, H., et al., 1996. Life with 6000 genes. Science 274, 546.563–546.567. Gordon, J.L., Wolfe, K.H., 2008. Recent allopolyploid origin of Zygosaccharomyces rouxii strain ATCC42981. Yeast 25, 449–456. Iwaki, T., Higashida, Y., Tsuji, H., Tamai, Y., Watanabe, Y., 1998. Characterization of a second gene (ZSOD22) of Na+/H+ antiporter from salt-tolerant yeast Zygosaccharomyces rouxii and functional expression of ZSOD2 and ZSOD22 in Saccharomyces cerevisiae. Yeast 14, 1167–1174.
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