1 Journal Section: Evolutionary and Genomic Microbiology
2 The chimerical genomes of natural hybrids between
3 Saccharomyces cerevisiae and Saccharomyces
4 kudriavzevii
5 Carmela Belloch1, Roberto Pérez-Torrado1, Sara S. González1, José E.
6 Pérez-Ortín2, José García-Martínez2, Amparo Querol1 and Eladio Barrio3,*
7
8 1Instituto de Agroquímica y Tecnología de los Alimentos, IATA-CSIC. P.O.
9 Box, 73. E-46100 Burjassot, Spain
10 2Department de Bioquímica i Biologia Molecular, Facultat de Ciències
11 Biològiques, Universitat de València, C/ Dr. Moliner, 50. E-46100 Burjassot,
12 Spain
13 3Institut ‘Cavanilles’ de Biodiversitat i Biologia Evolutiva, Universitat de
14 València. P.O. Box 22185, E-46071 València, Spain
15
16 *Corresponding author:
17 Institut ‘Cavanilles’ de Biodiversitat i Biologia Evolutiva, Universitat de
18 València. P.O. Box 22185, E-46071 València, Spain. Phone +34 963 543 667.
19 Fax +34 963 543 670. E-mail: [email protected]
20
21 Running Title: Chimerical genomes in Saccharomyces hybrids
22
- 1 - 1 ABSTRACT
2 Recently, a new type of hybrids resulting from the hybridization between S.
3 cerevisiae and S. kudriavzevii were described. These strains exhibit
4 physiological properties of potential biotechnological interest. A preliminary
5 characterization of these hybrids showed a trend to reduce the S. kudriavzevii
6 fraction of the hybrid genome.
7 We characterized the genomic constitution of several wine S. cerevisiae x S.
8 kudriavzevii strains by using a combined approach based on the RFLP
9 analysis of gene regions, comparative genome hybridizations with S.
10 cerevisiae DNA arrays, ploidy analysis and gene dose determination by
11 quantitative real-time PCR.
12 The high similarity in the genome structure of the S. cerevisiae x S.
13 kudriavzevii hybrids under study indicates they originated from a single
14 hybridization event. After hybridization, the hybrid genome underwent
15 extensive chromosomal rearrangements, including chromosome losses and
16 the generation of chimerical chromosomes by non-reciprocal recombination
17 between homeologous chromosomes. These non-reciprocal recombinations
18 between homeologous chromosomes occurred in highly conserved regions,
19 such as Ty LTRs, rRNA regions, and conserved protein-coding genes.
20 This study supports the hypothesis that chimerical chromosomes may have
21 been generated by a similar mechanism to the recombination-mediated
22 chromosome loss acting during the meiosis in Saccharomyces hybrids. As a
23 result of the selective processes acting during fermentation, hybrid genomes
24 maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.
- 2 - 1 Keywords: natural hybrids, Saccharomyces cerevisiae, Saccharomyces
2 kudriavzevii, genome hybridization, aneuploidy, aCGH, qRT-PCR,
3 recombination points
4 Accession numbers: PMT1 sequences from this study have been submitted
5 to the EMBL database under accession numbers FM211146 to FM211153,
6 and aCGH data to Gene Expression Omnibus under accession no.
7 GSE12774.
8 INTRODUCTION
9 The genus Saccharomyces consist of seven biological species S. arboricolus,
10 S. bayanus, S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S.
11 paradoxus (29,59) and the partially allotetraploid species S. pastorianus
12 (46,58).
13 The hybrid species S. pastorianus, restricted to lager brewing environments,
14 arose from two or more natural hybridization events between S. cerevisiae
15 and a S. bayanus-like yeast (7,16,28,46). Recent studies on S. bayanus have
16 also revealed the hybrid nature of certain strains of this species, which has
17 subsequently been subdivided into two groups, the S. bayanus var. bayanus,
18 containing a variety of hybrid strains and the S. bayanus var. uvarum, also
19 referred to as S. uvarum, that contains non-hybrid strains (45,46).
20 New hybrids between other species from the genus Saccharomyces have
21 recently been described. Hybrid yeasts between S. cerevisiae and S.
22 kudriavzevii have been characterized among wine (6,20,33) and brewing
23 yeasts (21), even triple hybrids between S. cerevisiae, S. bayanus and S.
24 kudriavzevii have been identified (20,41).
- 3 - 1 The first natural Saccharomyces interspecific hybrid identified, the lager
2 brewing yeast S. pastorianus (S. carlsbergensis) (42,57) has become one of
3 most investigated type of yeast hybrids. The genome structure of these
4 hybrids has been examined by competitive array comparative genome
5 hybridization (aCGH) (5,16,28), complete genome sequencing (28) and PCR-
6 restriction fragment length polymorphism analysis of 48 genes and partial
7 sequences of 16 genes (46). The aCGH analyses of several S. pastorianus
8 strains with S. cerevisiae-only DNA arrays (5,28) revealed the presence of
9 aneuploidies due to deletions of entire regions of the S. cerevisiae fraction of
10 the hybrid genomes. A recent aCGH analysis of S. pastorianus strains with S.
11 cerevisiae and S. bayanus DNA arrays (16) showed two groups of strains
12 according to their genome structure and composition. These groups arose
13 from two independent hybridization events, and each one is characterized by
14 a reduction and an amplification of the S. cerevisiae genome fraction,
15 respectively.
16 The genetic characterization of the wine S. cerevisiae and S. kudriavzevii
17 hybrids by restriction analysis of 5 nuclear genes located in different
18 chromosomes, 5.8S-ITS rDNA region and the mitochondrial COX2 gene
19 revealed the presence of three types of hybrids in Swiss wines, thus indicating
20 the presence of different hybrid genomes (20). In a recent study (21), we
21 identified six new types of S. cerevisiae and S. kudriavzevii hybrids among
22 brewing strains, which were compared to wine hybrids by a genetic
23 characterization based on RFLP analysis of 35 protein-encoding genes. This
24 analysis confirmed the presence of three different genome types among wine
25 hybrids that contain putative chimerical chromosomes, probably generated by
- 4 - 1 a recombination between homeologous chromosomes of different parental
2 origins.
3 The aim of the present study is to investigate the genome composition and
4 structure of wine hybrids between S. cerevisiae and S. kudriavzevii. This has
5 been achieved by a combined approach based on the RFLP analysis of 35
6 gene regions from our previous study, comparative genome hybridizations
7 using S. cerevisiae DNA macroarrays, a ploidy analysis by flow cytometry,
8 and gene dose determinations by quantitative real-time PCR. This multiple
9 approach allowed us to confirm the presence of chimerical chromosomes and
10 define the mechanisms involved in their origins.
11 MATERIALS AND METHODS
12 Strains: The yeast strains used in this study are four S. cerevisiae x S.
13 kudriavzevii hybrids. W27 and W46 were isolated from Pinot noir must
14 fermentations in Jenins and Stäfa, Switzerland, respectively. They correspond
15 to commercial wine yeasts (Lallemand Inc., Montreal, Canada). SPG16-91
16 and 441 were isolated from Muller-Thurgau must fermentations in Wädenswil,
17 Switzerland. The homoploid S. cerevisiae lab strain FY-1679, the haploid S.
18 cerevisiae lab strain S288c and the S. kudriavzevii type strain IFO 1802T were
19 also used in different experiments performed in the present study.
20 DNA labeling and competitive genome hybridization: DNA was extracted
21 from yeast strains grown from single colonies in GPY (glucose 2%, peptone
22 0.5%, yeast extract 0.5%) following the procedure described by Querol et al.
23 (44). Before the labeling reaction, 10 µg of DNA were digested with HinfI
24 (Takara, Japan), according to the manufacturer’s instructions, to an average
25 length of 250bp to 8 Kb. The fragmented sample was purified using a QIA-
- 5 - 1 quick gel extraction kit (Qiagen, Germany), then was heat-denatured for 5
2 minutes at 100ºC and then cooled on ice. DNA was labeled in 50 µl reactions
3 by random priming using GE-Healthcare Ready-To-Go DNA Labeling Beads
4 (GE Healthcare Ltd. Amersham Biosciences, USA). Each Reaction Mix bead
5 contains buffer, FPLCpureTM Klenow fragment (7-12 units), random
6 oligonucleotides (primarily 9-mers) and dATP, dGTP, dTTP. The room-
7 temperature-stable bead is reconstituted with a solution of denatured template
8 (1 µg) and [c-33P]dCTP (40 µC) to total of 50 µl. The mixture is then incubated
9 at 37ºC for 1-2 hours. Unincorporated nucleotides are removed by filtration
10 through a MicroSpinTM S-200 HR column (GE Healthcare Ltd., Amersham
11 Biosciences, Germany). Equal amounts of labeled DNA (5x106 dpm/ml) were
12 used as probes to hybridize to PCR-amplified ORFs of homoploid S.
13 cerevisiae FY 1679 DNA spotted onto membrane macroarrays (described in
14 1; see also http://scsie.uv.es/chipsdna/index.html). New macroarrays are pre-
15 treated for 30 minutes at 80ºC with 0.5% SDS. Filters are pre-hybridized in a
16 rotator oven with 5 ml of pre-hybridization solution (5x SSC, 5x Denhart’s
17 solution, 0.5% SDS and 100 µg herring sperm DNA/ml), at 65ºC for 2 hours.
18 The pre-hybridization solution is replaced with 5 ml of the same solution
19 containing the desired amount of radioactive sample and hybridized for 40
20 hours at 65ºC. After hybridization membranes were washed once at 65ºC for
21 20 minutes in 2x SSC, 0.1% SDS, and twice at 65ºC for 30 minutes at 0.2x
22 SSC, 0.1% SDS. After the washing step, membranes were exposed to an
23 imaging plate (BAS-MP, Fuji Film, Japan) for 3 days.
24 Macroarray scanning and data normalization: Images were acquired using
25 a Fujifilm FLA3000 Phosphoimager. The raw data were processed by using
- 6 - 1 EXCEL spreadsheets. Spot intensities from 3 replicate hybridizations were
2 measured as ARM density (artifact-removed density), background and sARM
3 Density (background-corrected ARM density) by using the Array Vision 7.0
4 software (Imaging Research Inc., Canada). Poor or inconsistent signals were
5 not considered for further analysis. Only signal intensities higher than 1.5
6 times the background were considered as valid data and normalized. The
7 normalization process and the measure of the significance level for each ORF
8 were done by using ArrayStat software (Imaging Research Inc., Canada),
9 considering the data as independent and allowing the program to take a
10 minimum number of two valid replicates in order to calculate the mean and
11 standard deviation values for every gene (only one of the three replicates was
12 allowed to be a removable outlier). Genes with missing values of more than
13 80% were removed.
14 Background-corrected DNA macroarray data intensities were normalized to a
15 common reference in another channel by following a global LOWESS
16 procedure. The common reference used in normalization was generated with
17 respect to the sum of intensities of all genes. Logarithms were calculated and
18 M-A plots represented. The hybridization signal of each ORF from the hybrid
19 and parental strains was normalized to that of the homoploid strain FY1679.
20 Hybridization signals were depicted as log2 of the hybridization signal ratio
21 (hybrid/FY1679) with respect to the S. cerevisiae gene order using the
22 program ChARM v. 1.6 (36).
23 Flow cytometry: Hybrid yeasts and the reference homoploid strain were
24 grown in 10 ml of liquid medium under agitation. Early stationary-phase cells
25 (c. 106 cells mL-1) were harvested by centrifuging, washed and fixed in 70%
- 7 - 1 ethanol at 4ºC for 5 minutes. Fixed cells were harvested by centrifuging and
2 resuspended in 0.01M PBS buffer (pH 7.2) containing 400 µL of RNase (10
3 mg ml-1). After incubation at 37ºC for 30 minutes, cells were harvested by
4 centrifuging and resuspended in 950 µl of 0.01M PBS buffer (pH 7.2)
5 containing 50 µl of propidium iodide (0.005%). Samples were analyzed using
6 a flow cytometer FACScan analyzer (Becton Dickinson, USA). Ploidy
7 determination of the hybrid strains was done by comparison against the
8 haploid S288c and diploid FY1679 strains.
9 Gene copy estimation by qRT-PCR: General and species-specific
10 oligonucleotide PCR primers for genes located in different chromosome
11 regions were designed (see Table S1 in supplementary materials). These
12 general and species-specific primers were designed according to the
13 available genome sequences of the laboratory strain S. cerevisiae S288c and
14 the Japanese type strain of S. kudriavzevii IFO1802T, representatives of the
15 parental species. Specificity, efficiency and accuracy of the primers were
16 tested and optimized by standard PCR reactions, using DNA from parental
17 and hybrid strains. Primers showing amplification were used in the
18 subsequent quantitative real-time polymerase chain reaction (qRT-PCR)
19 analysis. Amplification of gene fragments from different yeast strains was
20 determined by qRT-PCR using a standard curve method (61). DNA from
21 overnight stationed pre-cultures was extracted in triplicate with PrepMan Kit
22 (PE Applied Biosystems, USA) as described (34). qRT-PCR was performed
23 with gene-specific primers (200 nM) in a 20 µl reaction, using the Light Cycler
24 FastStart DNA MasterPLUS SYBR green (Roche Applied Science, Germany) in
25 a LightCycler® 2.0 System (Roche Applied Science, Germany) device. All
- 8 - 1 samples were processed for melting curve analysis, amplification efficiency
2 and DNA concentration determination using LightCycler® 2.0 System. For
3 every strain, DNA extracted from 106 cfu and serial dilutions (10-1 to 10-5)
4 were used for a standard curve. The copy number for each gene was
5 estimated by comparing DNA concentration for S288c (haploid S. cerevisiae
6 strain) or IFO 1802 (diploid S. kudriavzevii strain) with W27 hybrid strain.
7
8 Recombination sites in the chimerical chromosomes: The approximate
9 location of the recombination points in the mosaic chromosomes IV and V
10 was determined from the up and down “jump” locations in the ORFs mapping
11 by macroarray analysis of the hybrid yeast genomes.
12 The recombination site in chromosome IV was approximately placed in the
13 PMT1 gene by amplification with general primers (direct: 5’- -3’, and reverse:
14 5’- -3’) and subsequent sequencing. Once the recombination region was
15 identified, S. cerevisiae (ScePMT1up: 5’-
16 GACAAACAGGTGCATTGTTAAAGCGAGGT-3’, and ScePMT1down: 5’-
17 CTGTCAGGCCATGCCAAGCCAT-3’) and S. kudriavzevii (SkuPMT1up: 5’-
18 GAGGTGCATTGTGCAATACTTAGGCGAAAC-3’, and SkuPMT1down: 5’-
19 GTCTGGCCAGGCGAGGCTGC-3’) specific oligonucleotide primers were
20 designed. PCR amplification with different combinations of the species-
21 specific primers allowed us to corroborate the presence of chimerical
22 chromosomes IV, and the subsequent sequencing revealed the recombination
23 site located in the 5’ end of PMT1 gene.
24 The PCR reaction was prepared with rTaq, buffer and dNTPs mix (Takara,
25 Japan) according to the manufacturer instructions, 4 µl of DNA (10-100 ng)
- 9 - 1 and 1µl of each oligonucleotide to a final volume of 50 µl. PCR reactions were
2 performed in a G-STORM thermocycler (Gene Technologies Ltd., UK).
3 PCR amplification of DNA upstream and downstream of PMT1 gene was as
4 follows: 45 cycles of 94ºC for 45 seconds, 62ºC for 35 seconds and 72ºC for 1
5 minute, with a final extension of 10 minutes at 72ºC. The PCR products were
6 purified using the PerfectPrep kit (Eppendorf, Germany) and directly
7 sequenced by using the BigDyeTM Terminator V3.1 Cycle Sequencing Kit (PE
8 Applied Biosystems, USA), following the manufacturer’s instructions, in an
9 automatic DNA sequencer, Model ABI 3730 (PE Applied Biosystems, USA).
10 RESULTS
11 Comparative genome analysis of hybrid strains based on DNA
12 macroarrays
13 To determine the genome composition and structure of wine hybrids between
14 S. cerevisiae and S. kudriavzevii, we performed a comparative genome
15 hybridization analysis with S. cerevisiae DNA macroarrays. Of the seven wine
16 S. cerevisiae x S. kudriavzevii hybrids described (20), we selected four strains
17 (W27, W46, SPG16-91, and 441), representative of the three genome types
18 characterized by González et al. (21).
19 The analysis of the presence of chromosomal rearrangements among
20 Saccharomyces species (17), as well as the complete genome sequencing of
21 S. kudriavzevii (9,10) showed that this species contains a co-linear (syntenic)
22 genome with respect to that of S. cerevisiae, and hence, a similar position of
23 the genes under analysis is expected in the hybrid’s chromosomes coming
24 from the S. kudriavzevii parent.
- 10 - 1 Relative gene copy numbers in the hybrid strains were determined by
2 comparative genome hybridization with respect to the S. cerevisiae homoploid
3 strain FY1679. This strain contains the same genetic background as the
4 haploid strain S288C, whose complete genome sequence is known (19) and
5 is the basis of the DNA macrochip synthesis (1).
6 The hybridization signal of each ORF from the hybrid strains was normalized
7 to that of FY1679, and shown as log2 of the Signal Ratio (hybrid/FY1679) with
8 respect to the S. cerevisiae gene order in each chromosome (data in
9 supplementary material). Nucleotide divergences among the genomes of the
10 parental species S. cerevisiae and S. kudriavzevii are on average ~30%. Due
11 to the DNA hybridization stringency conditions used (<10% nucleotide
12 divergence), most genes from the S. kudriavzevii genome fraction of hybrids
13 did not hybridize to the S. cerevisiae macroarray and, hence, hybridization
14 differences mainly correspond to the hybrid genome fraction coming from the
15 S. cerevisiae parent.
16 As an example, Figure 1 shows the hybridization ratios vs. chromosomal gene
17 order for the wine hybrid strain W27. When the log2 of the hybridization ratios
18 for the hybrid yeast genes were arranged according the S. cerevisiae gene
19 order, we observed up and down “jumps”. Up “jumps” correspond to those
20 genes that are over-represented, i.e. more copies than average, in the hybrid
21 genome fraction coming from the S. cerevisiae, and down “jumps” to genes
22 either absent or under-represented, with less copies than average. “Jumps”
23 involving large chromosomal regions were observed in chromosomes IV, V,
24 IX, XIV and XV for all four hybrids, and also in chromosome XII for hybrid
- 11 - 1 SPG16-91 (Figure 2a). Other “jumps” involved shorter subtelomeric regions in
2 several chromosomes, or inter-dispersed regions of a few genes.
3 Large over- or under-represented chromosomal regions can be interpreted as
4 indicative of regional duplications and deletions, respectively. But, in the case
5 of hybrid strains, the simplest explanation of the ratios of hybridization data is
6 that the ‘jump’ locations represent regions where the ‘homeologous’
7 chromosomes (i.e. homologous from different parental species) have
8 undergone inter-chromosomal non-reciprocal recombination (4). This way, the
9 different points of recombination between homeologous chromosomes appear
10 as abrupt changes in the hybridization ratios because the extra copies of
11 some of the hybrid genes coming from the S. cerevisiae parent are in the
12 resulting chimerical chromosomes. This is the case of chromosomes IV, V, IX,
13 XIV and XV in all hybrids (Figure 1), as well as chromosome XII in strain
14 SPG16-91 (Figure 2a). As an example, SPG16-91 genes, coming from S.
15 cerevisiae, that are located in the left arm and the first third of the right arm of
16 chromosome XII show log2 hybridization ratios between -0.5 and 0.5, but
17 those located in the rest of the right arm exhibit log2 ratios around 1. These
18 results indicate that the hybrid contain two copies of the genes in the second
19 region per each copy of the genes in the first, which is compatible with the
20 presence in SPG16-91 hybrid genome of a complete and a partial or
21 chimerical forms of S. cerevisiae chromosome XII as indicated in Figure 2a.
22 In a previous study (21), we characterized the genome structure of hybrids
23 according to the presence/absence of the parental alleles for 36 gene regions
24 located in the different chromosomal arms. This analysis, summarized in
25 Figures 1 and 2, demonstrated the absence of the S. kudriavzevii alleles for
- 12 - 1 some genes and their presence for other genes of the same chromosome.
2 These results were already interpreted as evidence of the presence of
3 chimerical chromosomes in the hybrid genomes. As can be seen, these
4 results are congruent with the aCGH analysis, indicating the presence of
5 chimerical forms for chromosomes IV, IX and XV according to the
6 chromosome structures depicted in Figure 1, as well as for chromosome XII in
7 the case of hybrid SPG16-91 (Figure 2a). Both analyses are also congruent
8 for the short subtelomeric rearranged region located in the left arm of
9 chromosome VII, according to the position of gene MNT2 (Figure 1).
10 Nonetheless, it seems to be there a discrepancy between both studies with
11 respect to chromosomes V and XIV. On one hand, the aCGH analysis of all
12 hybrids shows up and down “jumps” in the log2 hybridization ratio of genes
13 located in the left arm of chromosome V and in the right arm of chromosome
14 XIV, respectively, which is indicative of either a regional duplication in
15 chromosome V and a deletion in chromosome XIV, or the presence of
16 chimerical chromosomes. On the other hand, the RFLP analysis of genes
17 located in these chromosomal regions showed the presence of the two
18 parental alleles. However, both kinds of results are compatible with the
19 presence in the hybrid genome of three different structural forms of
20 chromosomes V and XIV, an intact S. cerevisiae chromosome, an entire S.
21 kudriavzevii chromosome and a rearranged form, chimerical chromosome, as
22 depicted in Figure 1.
23 The remaining genes of the S. cerevisiae genome fraction of hybrids, located
24 in chromosomes II, III, VI, VII, VIII, X, XI, XIII, XVI, as well as XII for all hybrids
25 except SPG16-91, in general showed relative hybridization ratios oscillating
- 13 - 1 between -0.5 and 0.5. These results indicate that they are in similar relative
2 copy numbers. In our previous analysis of hybrid genome composition, we
3 could observe that genes located on these chromosomes contain two different
4 alleles, each one coming from a different parent. The only exceptions are
5 genes located in chromosome I for strain 441 (Figure 2b), that exhibit
6 hybridization ratios between 0.5 and 1, which is indicative of the presence of
7 an extra copy of the S. cerevisiae chromosome I in this hybrid genome.
8 Moreover, the RFLP analyses of genes located in this chromosome (21)
9 indicated the absence of S. kudriavzevii chromosome I in the hybrid genome
10 of this strain.
11 Hybrid ploidy and chromosome copy numbers
12 Due to the normalization procedure, the hybridization ratios derived from
13 macroarray analysis shows the relative proportions of each gene respect to
14 the average in the reference strain. Although this method allows to identify
15 those over- and under-represented regions, it does not permit to determine
16 the absolute copy numbers of each S. cerevisiae chromosomal region present
17 in the hybrid genome. The RFLP analysis of gene regions located in the
18 different chromosomes complements the aCGH analysis, providing only
19 qualitative information about the presence/absence of S. cerevisiae and S.
20 kudriavzevii chromosomal regions.
21 Therefore, we used two additional approaches to decipher the precise
22 genome structure and composition of wine hybrids. First, we used flow
23 cytometry to estimate hybrid ploidies. The comparison of the distribution of
24 DNA content in hybrid cells (depicted in Figure 3) to those obtained for the
25 reference haploid (S288C) and diploid (FY1679) strains rendered the
- 14 - 1 approximate ploidies of the hybrid strains. Wine hybrids between S. cerevisiae
2 and S. kudriavzevii displayed an estimated ploidy slightly higher than diploidy
3 (n = 2.2-2.3), which is indicative of a low level of aneuploidy. This estimated
4 ploidy is compatible with a hybrid genome composition of two copies for most
5 chromosomes and three for chromosomes V and XIV, corresponding to an
6 expected ploidy of 2.11.
7 To estimate the copy numbers of the different chromosomes present in the
8 hybrid genomes, we performed a quantitative RT-PCR analysis of several
9 genes, located in the different chromosome regions defined by the aCGH
10 analysis. Since hybrids exhibited the same ploidy and very similar genome
11 composition, this analysis was only done for all gene regions of hybrid strain
12 W27. Figure 4 summarizes the copy numbers estimated for 19 gene regions
13 amplified with general primers (designed and tested for S. cerevisiae and S.
14 kudriavzevii) and species-specific primers, either for S. cerevisiae or for S.
15 kudriavzevii. These primers were designed according to the available genome
16 sequences of the strains S. cerevisiae S288c and S. kudriavzevii IFO1802T,
17 also used for qPCR amplification testing and the generation of the copy
18 number standard curve by the serial DNA dilution method. However, these
19 sequences may differ from the allele sequences present in hybrids, especially
20 in the case of the S. kudriavzevii, alleles according to the sequences available
21 for some other genes (20, 21, 51). This putative sequence divergence
22 explains differences in the amplification efficiency with DNA from hybrids that
23 are responsible of the biases observed in the gene copy number estimates.
24 However, gene copies should correspond to natural numbers and values
25 smaller or higher than expected can be rounded to the closest integer. For
- 15 - 1 example, a value of 1.75 ± 0.07 obtained for CYC3 with general primers is not
2 indicative of a non-sense value of 1 ¾ copies of the gene present in the
3 hybrid, but two copies amplified with a lower efficiency that in the case of the
4 reference strains due to nucleotide divergence.
5 Taking this into consideration, these results supports the hypothesis that the
6 hybrid genome of wine strain W27 contains three homeologous forms of
7 chromosomes V and XIV, two of them corresponding to complete S.
8 cerevisiae and S. kudriavzevii chromosomes and the third to a chimerical
9 chromosome. Chromosomes IV, IX, and XIV are present in two forms, one
10 corresponds to a complete S. cerevisiae chromosome and the other to a
11 chimerical chromosome, with one end coming from S. cerevisiae and the
12 other from S. kudriavzevii. Finally, the remaining chromosomes (I-III, VI-VIII,
13 X-XIII and XVI) are also present in two versions, one coming from S.
14 cerevisiae and the other from S. kudriavzevii, with the exception of some
15 rearranged subtelomeric regions.
16 With respect to the genome composition of the other hybrids, W46 exhibits
17 the same ploidy and identical chromosome structure than W27. Hybrid 441
18 only differs from W27 in the composition of chromosome I. The qRT-PCR
19 quantification of the copy numbers of gene CYC3, located in chromosome I,
20 indicates that strain 441 contains two copies of the gene (total copies per cell
21 2,07 ‒ 0.11, estimated using general primers), but both coming from S.
22 cerevisiae (cerevisiae copies per cell 1.95 ‒ 0.10, estimated with species-
23 specific primers). Finally, hybrid SPG16-91 only differs in the structure of
24 chromosome XII, which contains two copies (total copies of gene PDC5 1.80
- 16 - 1 ‒ 0.15), but one corresponds to a complete S. cerevisiae chromosome and
2 the other to a chimerical chromosome.
3 Recombination sites in chimerical chromosomes
4 In the aCGH analysis of hybrid genomes, “jumps” in the log2 hybridization
5 ratios of genes, located in large chromosomal regions, are due to the
6 presence of chimerical chromosomes generated by non-reciprocal
7 recombination between homeologous chromosomes. This abrupt changes are
8 observed between the genes flanking (or located at the vicinities of) the
9 recombination points where crossing-over occurred. In this way, we could
10 identify in the chimerical chromosomes those regions where the putative
11 recombination sites are located. The conformation of these regions in the
12 hybrid genomes are described in Figure 5.
13 The recombination sites in the chimerical chromosomes XIV and XV are
14 located between genes flanking large regions containing Ty1 delta, Ty3
15 sigma, Ty4 tau elements, and tRNA genes, according to the genome
16 sequences of S. cerevisiae, S. kudriavzevii, and other Saccharomyces
17 species (available at the Saccharomyces Genome Database,
18 http://db.yeastgenome.org/).
19 The recombination site in the chimerical chromosome XII, only present in the
20 hybrid genome of strain SPG16-91, is located within the large cluster of 100-
21 200 tandem repeats containing the highly conserved rRNA (RDN) genes.
22 Finally, recombination sites in chimerical chromosomes IV, V and IX, appear
23 as located within protein-coding genes. The recombination site in
24 chromosome IV is located in the region between genes RPN6 and PMT1,
25 coding respectively for a regulatory subunit of the 26S proteasome required
- 17 - 1 for its assembly and activity and a O-mannosyltransferase involved in
2 glycosylation, respectively. The recombination site in chromosome IX is
3 located in the vicinity of the highly conserved gene RPL34B, encoding a
4 ribosomal protein of the large ribosomal subunit (60S). This region has been
5 identified as a meiotic recombination hotspot in the S. cerevisiae genome
6 (18). Finally, the site in chromosome V is apparently located in the region
7 around NUG1, which encodes a GTPase required for export of 60S ribosomal
8 subunits from the nucleus.
9 The definite demonstration of the presence of chimerical chromosomes in the
10 hybrid genome would require the sequencing of at least one of the
11 recombinant regions. Thus, we decided to amplify by PCR and sequence one
12 of these putative regions. Recombinant sites located in the chimerical
13 chromosomes XII, XIV and XV were discarded because they are located
14 within large potential regions. Among those hypothetical recombinant sites
15 placed in the vicinities of protein-coding sequences, the one located in the
16 chimerical chromosome IV was the more feasible. DNAs from the different
17 hybrid strains were used for PCR amplifications with general primers
18 (described in the supplemental material) of a DNA fragment spanning the
19 RPN6 and PMT1 genes and the region between them. The direct sequencing
20 of the DNA fragments with an automatic sequencer allowed us to determine
21 the location of the recombination region. This region was identified, in the
22 resulting chromatogram, as the nucleotide positions where a transition from
23 double peaks, due to the overlapping S. cerevisiae and S. kudriavzevii
24 sequences, to single peaks, corresponding to the S. cerevisiae sequence, is
25 observed. From these sequences, species-specific primers were designed.
- 18 - 1 The PCR amplification of a 297-bp DNA fragment using the S. cerevisiae–
2 specific primers (described in Methods) pinpointed the presence of a non-
3 recombinant S. cerevisiae chromosome IV. As expected, no amplifications
4 were obtained with both S. kudriavzevii-specific primers (see also Methods),
5 indicative of the absence of a non-recombinant S. kudriavzevii chromosome
6 IV. But the PCR amplification of a 303-bp DNA fragment, using the suitable
7 combination of S. cerevisiae- and S. kudriavzevii-specific primers
8 (ScePMT1up+SkuPMT1down, see Methods), unequivocally demonstrated the
9 presence of a chimerical chromosome IV in the hybrid genomes.
10 The subsequent sequencing of the recombination region of the chimerical
11 chromosome IV from the different hybrid strains showed that they share the
12 same recombination event, which occurred in a short region of
13 ‘microhomology’ located within the PMT1 gene, at codons 11-12 (see Figure
14 6). The coding regions of the two PMT1 alleles from hybrids only differ in two
15 synonymous substitutions, and hence, hybrids contain two alleles encoding
16 the same protein but under the control of divergent promoters of different
17 parent origin.
18
19 DISCUSSION
20 Natural hybridization among Saccharomyces yeasts seems to be more
21 common than previously suspected (53). The best known example of
22 Saccharomyces hybrids are the lager yeasts, hybrids between S. cerevisiae
23 and S. bayanus (28,46). S. cerevisiae x S. bayanus var. uvarum hybrid strains
24 have also been found in wines (2,30,35). A new type of hybrids resulting from
25 the hybridization between S. cerevisiae and S. kudriavzevii, the subject of the
- 19 - 1 present study, were firstly described among wine strains (6,20,33), and
2 recently among brewing yeasts (21).
3 The presence of S. cerevisiae x S. bayanus hybrids is easily explainable,
4 since both species coexist in the same fermentation processes such as
5 brewing (46), cider production (11,40,55) and winemaking (2,13,49,56).
6 Moreover, Le Jeune et al. (30) have recently found S. cerevisiae x S. uvarum
7 hybrids and their putative parents in the same winery.
8 However, in the case of S. cerevisiae x S. kudriavzevii hybrids, it is not so
9 easy to understand how this hybridization occurred and how these hybrids
10 colonized Central European wine fermentations (21,53). Although the parental
11 species S. cerevisiae is the dominant yeast species in most wine
12 fermentations, S. kudriavzevii has never been found so far in fermentative
13 environments, which suggest that hybridizations likely took place in wild
14 environments. The first strains of S. kudriavzevii were isolated from decayed
15 leaves and soil in Japan (38) and, recently, new strains were found on the
16 bark of oak trees in several locations of Portugal (51). Phylogenetic analyses
17 based on some gene sequences showed that hybrids are closer to the
18 Portuguese S. kudriavzevii strains than the Japanese (20,51), which is
19 indicative that hybridization probably occurred in Europe.
20 The high similarity in the genome structure of the S. cerevisiae x S.
21 kudriavzevii hybrids under study clearly indicates they share a common
22 ancestor. This is supported by the geographical origin of the strains (Eastern
23 Switzerland), and the similar fermentation conditions from which they were
24 isolated. Besides, these hybrids have shown similar tolerance to several
25 stress conditions that are important in wine fermentation. (3,22). In a previous
- 20 - 1 study, we observed that some S. cerevisiae x S. kudriavzevii hybrids isolated
2 in Belgium share a similar genome constitution with Swiss wine strains (21).
3 All these observations support that S. cerevisiae x S. kudriavzevii hybrids
4 were originated by a limited number of hybridization events.
5 Hybridization of different Saccharomyces species, either by conjugation of
6 haploid spores or cells, probably mediated by insects (43,50), by rare mating
7 of a vegetative diploid cell and a haploid spore or cell, or by rare mating of
8 vegetative diploid cells (12), produces a hybrid genome consisting of different
9 copies of the parental genomes. Hybrid diploids are sterile (37), and can only
10 be maintained by asexual reproduction. Only allotetraploid (or amphidiploid)
11 hybrids are fertile, producing diploid hybrid spores (39), but S. cerevisiae x S.
12 kudriavzevii wine hybrids are almost diploid and sterile.
13 Meiotic segregation in diploid hybrids is known to be ineffective, and the
14 resulting spores are non-viable (< 1%) due to the high frequency of
15 aneuploidies (25). Previous studies (24,32) have shown that reproductive
16 isolation between the Saccharomyces species is due primarily to sequence
17 divergence acted upon by the mismatch repair system (MRS), which
18 contributes to sterility in an interspecific hybrid by preventing successful
19 meiotic crossing-over leading to aneuploid (25).
20 Chambers et al. (8) demonstrated that MRS reduces meiotic homeologous
21 recombination in artificial S. cerevisiae x S. paradoxus partial hybrids. When
22 recombination between divergent regions is aborted by the MRS, the absence
23 of functional interchromosomal crossovers may result in aberrant
24 chromosome segregation (meiosis II nondisjunction). However, when
25 recombination is initiated in a region with higher homology, the MRS
- 21 - 1 stimulates the loss of one partner of the recombination event in the hybrids
2 and the fixation of the other, a chimerical recombinant chromosome. This
3 mechanism, called ‘recombinant-dependent chromosome loss’ (RDCL), was
4 demonstrated during meiosis and explains the sterility of hybrids, and the
5 correlation between genome divergence and postzigotic reproductive isolation
6 observed in Saccharomyces (32). In the case a similar mechanism is acting
7 during the less-frequent mitotic recombination, it could explain the
8 chromosome rearrangements observed in the S. cerevisiae x S. kudriavzevii
9 hybrids in the present study, as well as those described in different S.
10 cerevisiae x S. bayanus hybrids (5,16,28).
11 The MRS-mediated chromosome instability observed in hybrids could also
12 explain differences in the frequency of hybridization among Saccharomyces
13 species. In the case of a ‘RDCL’ mechanism acting during mitosis, a direct
14 correlation between hybrid genome stability and divergence would be
15 expected. This way, the frequency of homeologous recombination will be
16 lower in a hybrid containing divergent homeologous chromosomes than in a
17 hybrid from closely related species, and hence, the genome stability will be
18 higher in the former than in the latter. Natural hybrids among Saccharomyces
19 species have only been described among the three more distantly related
20 species of this genus, i.e. S. cerevisiae, S. bayanus and S. kudriavzevii (29);
21 but not among closely related species, such as S. cerevisiae and S.
22 paradoxus, which coexist in the same habitats (48,54). Although Liti et al. (31)
23 postulated putative S. cerevisie x S. paradoxus hybrids from the distribution of
24 telomeric repetitive sequences and transposable elements, these strains do
25 not exhibit a hybrid genome (unpublished results from our laboratory), they
- 22 - 1 instead correspond to S. pardoxus strains introgressed with some S.
2 cerevisiae subtelomeric genome regions (32). S. cerevisiae strains
3 introgressed with several S. paradoxus genes have also been described
4 recently (14,60). All these introgressions are clear indications of ancient
5 instable hybridization events between these species.
6 The RDCL model predicts that chimerical chromosomes are generated when
7 the homeologous recombination is initiated in a region of high homology. In
8 the case of S. cerevisiae x S. kudriavzevii hybrids, we have demonstrated that
9 the homeologous recombinations, involved in the generation of chimerical
10 chromosomes, occurred in highly conserved regions, such as defective Ty
11 elements (LTR regions), rRNA regions, and conserved coding genes.
12 Similarly, lager S. cerevisiae x S. bayanus hybrids contain chromosomal
13 rearrangements generated by recombination between homeologous
14 chromosomes in regions positioned at or close to tRNAs, transposon-related
15 sequences, and origins of replication, well-known sites associated with
16 meiosis-induced double-strand breaks (5,16). Even though there seems to be
17 no clear association between homologous recombination hotspots and
18 repetitive DNA elements (18), the comparative analysis of Saccharomyces
19 chromosomes and genomes (15,17,26,27) showed that conserved
20 paralogous genes, transposons, and tRNAs are located at the rearrangement
21 breakpoints generated by ectopic recombination initiated in these repetitive
22 and highly conserved regions.
23 After hybridization, the hybrid genome suffers random genomic
24 rearrangements that, under the strong selective conditions prevailing during
25 wine fermentation (nutrient depletion, osmotic stress, fermenting temperature,
- 23 - 1 increasing levels of ethanol, etc.) are selected. Natural Saccharomyces
2 hybrids have been found so far associated to fermentation processes in
3 temperate areas of Europe, regions of continental climate located in the
4 Northern limit of grapevine distribution such as Northern Spain, France,
5 Germany, Switzerland, Austria, Hungary, etc., where they can also
6 predominate (2,20,30,33). Under such circumstances, however, hybrids may
7 have clear advantages over the parental species (3,52), because although
8 they are generally less suited than the parents to specific environmental
9 conditions, hybrids can be better adapted to intermediate or fluctuating
10 conditions that can be present in these peripheral areas. This is due to the
11 acquisition of physiological properties from both parents, which provide a
12 mechanism for the natural selection of hybrids (22,23,35,62). For example, S.
13 cerevisiae x S. kudriavzevii hybrids, they acquired the physiological properties
14 of both parents, a good alcohol and glucose tolerance and a fast fermentation
15 performance from S. cerevisiae and a better adaptation to low and
16 intermediate temperatures, as well as a higher production of glycerol and
17 aroma compounds from S. kudriavzevii (3,22).
18 As a result of these selective processes, a trend to maintain the S. cerevisiae
19 genome and to reduce the S. kudriavzevii fraction is observed in S. cerevisiae
20 x S. kudriavzevii hybrids (21). On the contrary, a group of lager S. cerevisiae x
21 S. bayanus hybrids exhibit an opposite tendency to preserve the S. bayanus-
22 like genome and to reduce the S. cerevisiae fraction (16,28), while another
23 maintains both genomes (16). However, both types of natural hybrids contain
24 the non-cerevisiae mitochondrial genomes, kudriavzevii-like in S. cerevisiae x
25 S. kudriavzevii hybrids (21) and bayanus-like in S. cerevisiae x S. bayanus
- 24 - 1 hybrids (47), which may impose restrictions to the loss of some non-
2 cerevisiae genes from the nuclear hybrid genome involved in mitochondrial
3 functions.
4 ACKNOWLEDGEMENTS
5 This work was supported by CICYT grants AGL2006-12703-CO2-01/ALI and -
6 02/ALI from the Spanish Government to AQ and EB, respectively. CB
7 gratefully acknowledges an I3P postdoctoral fellowship from the Spanish
8 Research Council (CSIC). Hybrid strains were kindly provided by Dr. Antonio
9 Palacios, Lallemand Inc., and Dr. Jürg Gafner, Swiss Federal Research
10 Station, Wädenswil, Switzerland.
11
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18
19
- 34 - 1 Figure legends
2 Figure 1. Chromosome structures in the S. cerevisiae x S. kudriavzevii hybrid strain
3 W27 as deduced from the DNA array analysis. These putative structures were
4 deduced from the plots of the log2 of the hybridization ratios (W27 hybrid gene
5 signals divided by those of the homoploid S. cerevisiae FY1679 genes) with respect
6 to the S. cerevisiae gene order of each chromosome. Abrupt changes in the
7 hybridization ratios of some chromosome regions are due to the presence of
8 chimerical recombinant chromosomes generated by non-reciprocal recombination
9 between homeologous chromosomes. Chromosomes and chromosome regions
10 coming from the S. cerevisiae parent are represented as black bars and those
11 coming from the S. kudriavzevii parent as white bars, vertical gray bars correspond to
12 centromeres. These chromosome structures are congruent with the results of a
13 previous study on the presence/absence of parental genes based on the RFLP
14 analysis of 35 gene regions (21). The locations of these gene regions are indicated
15 above each chromosome, and the results of presence/absence of the parental alleles
16 are summarized as follows: when both parental genes are present the gene name is
17 depicted in black, but in red when only the S. cerevisiae gene is present. The only
18 presence of a S. kudriavzevii gene was not observed.
19
20 Figure 2. Variable chromosome structures in S. cerevisiae x S. kudriavzevii hybrids.
21 Putative structures, as deduced from the results of macroarray analysis, of those
22 chromosomes of S. cerevisiae x S. kudriavzevii hybrids differing from the structures
23 of strain W27 chromosomes: a) structure differences in chromosome XII of hybrid
- 35 - 1 SPG16-91 with respect to that of hybrid W27; b) structure differences in chromosome
2 I of hybrid 441 with respect to that of hybrid W27. The representation of
3 chromosomes, plots and presence/absence of parental genes is the same than in
4 Figure 1.
5
6 Figure 3. Flow cytometry analysis of the DNA content per cell in the S. cerevisiae x
7 S. kudriavzevii hybrid strains. The DNA content per cell was measured by flow
8 cytometry for the S. cerevisiae x S. kudriavzevii hybrid strains: a) W27, b) SPG 16-
9 91, and c) 441. a) W27, b) SPG 16-91, and c) 441. The signal of the haploid
10 reference strain S288c is depicted in light green, that of the reference diploid strain
11 FY1679 in black, and those of the hybrids under study in purple.
12
13 Figure 4. Chromosome copy numbers in the S. cerevisiae x S. kudriavzevii hybrids.
14 Copy numbers of chromosomes from hybrid W27 were deduced from the gene dose
15 analysis by quantitative RT-PCR of 19 gene regions. The gene copy estimations
16 were performed, as indicated, with general primers designed and tested for S.
17 cerevisiae and S. kudriavzevii, and/or with species-specific primers, either for S.
18 cerevisiae or for S. kudriavzevii. The genes under analysis are located, as depicted,
19 in the different chromosome regions defined by the aCGH analysis. Chromosomes
20 and chromosome regions coming from the S. cerevisiae parent are represented as
21 black bars and those coming from the S. kudriavzevii parent as white bars, the
22 position of the centromeres are shown as vertical gray bars.
- 36 - 1
2 Figure 5. Location of the putative recombination sites in the chimeric chromosomes
3 of S. cerevisiae x S. kudriavzevii hybrids. The location of these recombinant regions
4 was deduced from the abrupt changes in the hybridization ratios depicted in Figures
5 1 and 2. In the case of chromosome XII, the recombinant form is only present in
6 hybrid SPG16-91 but not in the other, as depicted. The hybrid genes coming from the
7 S. cerevisiae parent are indicated as black arrows and those coming from the S.
8 kudriavzevii parent as white arrows, and those genes where the putative
9 recombination site is located are indicated as gray arrows. A gray box represents the
10 large cluster of 100-200 tandem repeats containing the highly conserved rRNA genes
11 (RDN genes), where the putative recombination site in the chimerical chromosome
12 XII of strain SPG16-91 is located. A vertical black bar denotes the position of the
13 centromere in chromosome XIV. Dotted and striped arrows represent tRNA and
14 LTRs (either delta, sigma or tau) from Ty retrotransposones, respectively, which
15 could be involved in the recombination events. The recombination site located within
16 PMT1 was confirmed by sequencing (see Figure 6).
17
18 Figure 6. Alignment of the partial sequences of the nonrecombinant (cer, S.
19 cerevisiae origin) and recombinant (rec) PMT1 alleles found in S. cerevisiae x S.
20 kudriavzevii hybrid strains, and those of the reference strains of the parent species,
21 S. cerevisiae (Sce) S288c and S. kudriavzevii (Sku) type strain IFO1802. A dot
22 indicates nucleotides identical to that from the reference PMT1 sequence of S.
23 cerevisiae S288c. Regions in the hybrid alleles that exhibit a higher similarity to S.
24 cerevisiae and S. kudriavzevii sequences are indicated in positive and negative,
- 37 - 1 respectively. A continuous rectangle highlights the 5’ end of the PMT1 coding region.
2 The black box indicates the crossing-over site involved in the non-reciprocal
3 recombination between homeologous chromosomes V of the common ancestor of
4 the hybrids.
- 38 - I IX
II X
III XI
IV XII
V XIII
XIV
VII XV
VIII XVI a b
XII I
W27 W27
PPR1 MAG2 CYC3 BUD14
SPG16-91 441
PPR1 MAG2 CYC3 BUD14 a
b
c I CYC3 II TIP1 III ARE1
IV UGA3 RPN4 HXT7 V ALD5 VI DAK2 VII KSS1 VIII ERG7 IX GUT2 X ILV3 XI GPM1 XII PDC5 XIII DAK1 XIV EGT2 BRE5 XV RRI2 XVI ALD6 IV SNU23 RPN6 PMT1 PMT5 V
FMP52 YND1NUG1 PAC2 TMA20 SNR14
IX YIL055c YIL054w RHR2 RPL34BMMF1 PCL7 DFG10
Cluster of W27 W46 XII RDN genes ACS2 RNH203 MAS1 YLR164w PUS5 441
Cluster of RDN genes SPG16-91 ACS2 RNH203 MAS1 YLR164w PUS5
XIV
DOM34CEN14 CIT1 YNR001w-A FUN34 RPC34
XV GPM3 THI20 PSH1 AIM39 DDR2 1 kbp Figure 6
PMT1 * 20 * 40 * 60 * Sce S288C AGTATCA------GAAGAGCCTATCAAGAAACAGCTAACAGCTACAAGCACGGTC ATG TCG GAA GAG AAA ACG TAC AAA W27-cer ...... ------...... W46-cer ...... ------...... 441-cer ...... ------...... SPG16-91-cer ...... ------...... W27-rec ..CG.T.AAAAGA...... A...CT...... G...... T...... G W46-rec ..CG.T.AAAAGA...... A...CT...... G...... T...... G SPG16-91-rec ..CG.T.AAAAGA...... A...CT...... G...... T...... G 441-rec ..CG.T.AAAAGA...... A...CT...... G...... T...... G Sku IFO1802 ..CG.T.AAAAGA...... A...CT...... G...... T...... G
80 * 100 * 120 * 140 Sce S288C CGT GTA GAG CAG GAT GAT CCC GTG CCC GAA CTG GAT ATC AAG CAG GGC CCC GTA AGA CCC TTT ATT W27-cer ...... W46-cer ...... 441-cer ...... SPG16-91-cer ...... W27-rec ... ..G ...... W46-rec ... ..G ...... SPG16-91-rec ... ..G ...... 441-rec ... ..G ...... Sku IFO1802 ... ..G ...... A.C ... ..T ...... C ... ..A ...... T.. .A. ..C