The authors have declared that no competing interests exists.
Conceived and designed the experiments: JLL CE CC. Performed the experiments: JLL CC. Analyzed the data: JLL CC. Contributed reagents/materials/analysis tools: JLL CC. Contributed to the writing of the manuscript: JLL CE CC.
Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including
Numerous fermented beverages have been developed all over the world during history. In addition to alcoholic fermentation, some beverages are obtained through a specific aging process called flor wine aging. During this process, which takes place only after the completion of alcoholic fermentation, a biofilm called velum is formed by yeast at the surface of the wine leading to the progressive oxidation of alcohol and remaining carbohydrates. This yeast oxidative metabolism generates many aromatic compounds (ethanal, sotolon, solerone…)
Flor aging (or biological aging) is performed traditionally in several vineyards in Europe, including Hungary (Tokaj Hegyalja) to produce Szamorodni, Italy (Sardinia) to produce Vernaccia di Oristano, Spain (Jerez area) to produce Xeres, and France (Jura) to produce Vin Jaune. The apparition of the velum is generally spontaneous
Yeast strains adopt a specific lifestyle during flor aging, and adaptation to this ecological niche has long remained the focus of many investigations. Aneuploidies have been described
Flor aging is encountered in highly distant vineyards, which raises the question of the relatedness and origin of these strains. The similar conditions faced by various strains in European vineyards implies that these strains share a similar genomic makeup and features of aneuploidy. In this paper, we compared flor yeast populations from Hungary (Tokaj), France (Jura), Italy (Sardinia) and Spain (Jerez). We used various molecular genetic techniques to investigate the genetic composition of these strains. The polymorphism of microsatellite markers allowed us to infer the structure of the flor yeast population. We measured the ploidy of strains and compared the genomes of several flor strains by CGH on array, which enabled us to detect aneuploidies specific to flor strains. Finally, we also examined polymorphisms within the promoter and protein central core region of
The strains of this study originated from several laboratories in Spain, Hungary, Italy and France. They are described in detail in
Yeast cells were cultivated in 10 ml of YPD medium (36 h, 28°C, 160 rpm). Velum growth was verified on Fornachon medium
The polymorphism of the length of Flo11p was measured from the amplification of
The presence of the 111 bp deletion in
For 5 strains (CAV21, LRJura, CECT11758, TR05CUB, T8CUB), the amplified fragment was sequenced with the same primers. These five sequences are available in GenBank under the accession number (HG965200–HG965204).
Cell hydrophobicity was evaluated following the procedure of Ishigami et al.
Genomic DNA was labeled and hybridized against GeneChip Yeast Genome 2.0 Array from Affymetrix (Santa Clara, CA), which covers all
The full data set has been deposited at the NCBI Gene Expression Omnibus (GEO) with GEO accession number (GSE55925)
For the analysis of cell DNA content, yeast cells were prepared in 96 well plates as described previously
We collected 142 flor stains from various countries. The 64 French strains from Jura were characterized previously by pulsed field gel electrophoresis and inter delta typing
The tree was built from the Dc chord distance and drawn with MEGA5.22. The wine cluster has been condensed due to its large size. Red dots indicate the presence of a 111 bp deletion in the
To confirm the global structure observed from microsatellite typing, we used the software InStruct to detect population structure and assign the various flor strains to a particular origin. InStruct
Each color corresponds to one inferred ancestral group. The proportion of each colors gives the proportion of the corresponding ancestral genome in the genome of each strain. The name of the isolated population is shown at the top of each cluster.
The possible relationship between the different groups of flor strains can also be evaluated from the Fst genetic distance between each population. The neighbor-net network obtained with Splittree
Flor strains have been described as aneuploid
Strain | Ploidy | CV % | Strain | Ploidy | CV % |
FINO 7.7 | 1.9 | 8.0 | ARC42 | 2.0 | 4.3 |
FINO 11.3 | 2.1 | 7.8 | ARC44 | 2.0 | 5.0 |
FINO 1.282 | 2.9 | 4.6 | ARC46 | 2.0 | 6.3 |
Manzanilla-II | 1.9 | 6.1 | BAE52 | 2.1 | 8.0 |
Manzanilla-III | 2.0 | 7.9 | CAW24 | 2.1 | 5.0 |
Manzanilla-VI | 2.0 | 9.0 | CBA13 | 2.1 | 12.6 |
Manzanilla-VIII | 2.1 | 9.2 | CBB01 | 2.0 | 4.4 |
Manzanilla-X | 2.6 | 4.2 | CBB52 | 2.0 | 4.3 |
My138 | 1.9 | 6.6 | CBD05 | 2.1 | 6.9 |
My91 | 1.9 | 6.9 | CBD55 | 2.1 | 4.5 |
F25 | 2.9 | 5.0 | GUF54 | 2.0 | 9.5 |
1682-S4 | 2.0 | 4.2 | LRJura | 2.0 | 5.4 |
CECT11761 | 2.0 | 4.7 | MAC51 | 1.9 | 8.7 |
CECT11764 | 2.0 | 4.8 | MAD51 | 2.1 | 5.7 |
G1 | 2.0 | 5.2 | MAE53 | 2.1 | 5.2 |
MAE54 | 2.1 | 5.0 | |||
2D | 2.0 | 8.3 | MAF53 | 2.0 | 5.7 |
FloraNero | 2.1 | 6.1 | MAF54 | 2.0 | 5.0 |
A33 | 1.9 | 7.7 | P3 | 2.0 | 7.1 |
A41 | 2.0 | 6.3 | PIA64 | 2.1 | 5.2 |
A51 | 2.1 | 7.7 | PII31 | 2.0 | 4.8 |
A9 | 2.0 | 8.4 | PII33 | 2.0 | 6.5 |
M23 | 2.1 | 4.8 | PIN34 | 2.0 | 6.5 |
M3 | 1.9 | 13.0 | PIO32 | 2.0 | 5.5 |
M38 | 2.1 | 7.9 | SAA52 g | 2.0 | 7.2 |
M39 | 2.1 | 7.7 | SAA55 | 2.0 | 6.1 |
M4 | 2.1 | 4.6 | SAC56 | 2.1 | 6.7 |
M49 | 2.1 | 4.6 | XRG25 | 2.1 | 5.5 |
M66 | 2.1 | 5.4 | |||
M8 | 2.1 | 6.4 | |||
V23 | 2.2 | 7.7 | T19CUB | 2.0 | 6.3 |
V5sard | 2.1 | 8.5 | T8CUB | 2.0 | 5.5 |
V63 | 2.0 | 5.6 | TA12CUB | 2.1 | 8.9 |
V75 | 2.0 | 4.9 | TR5CUB | 2.0 | 5.1 |
V80 | 2.0 | 4.9 | TS12CUB | 2.2 | 7.4 |
V9 | 2.0 | 5.0 |
Aneuploidy and gene amplification have been hypothesized as major sources of variation explaining adaptation to flor media
A first analysis carried out with different normalization methods dedicated to Affymetrix arrays (RMA, GCRMA, MAS5) indicated that 1606, 834, and 218 probe sets, respectively varied significantly between strains after correction for multiple tests (adj.
Chromosomes are colored in blue (uneven numbers) or dark blue (even numbers). Mean segment level estimated by DNAcopy is shown as a red line. The red arrow indicates the
In contrast with these aneuploid strains, we did not find substantial aneuploidy in the six flor strains tested. A low hybridization signal for chromosome I suggested the presence of only one copy in the CECT11758 strain, making it the only flor strain with a typical aneuploidy. Interestingly, a low hybridization signal at each subtelomeric region leading to an inverted U hybridization profile occurred in three of the six flor strains tested (TA12CUB, P3, and FloraNero), suggesting divergent alleles or missing genes in these regions.
For all strains, the hybridization signal of several genes was lower than that of the reference strain S288C. This suggests either the existence of divergent genes or genes with a low number of copies. We defined three thresholds to differentiate regions with zero, one, two or three copies: −1, −0.38 and +0.3, taking into account the average values observed for aneuploidies of CECT1158, Eg25 and Eg8 strains (Chromosome I of CECT11758, chromosome III and XVI of Eg25 and chromosomes IV, V, VIII and XVI of Eg8) and the dispersions around this average ratio. Accordingly, we divided regions with a low hybridization signal into two categories according to their log ratio: log ratio between −0.38 and −1, indicating one copy, and regions with hybridization signal lower than −1, indicating no copies. The gene lists corresponding to these thresholds are shown in
A. Genes with a low hybridization signal for most strains. B. Cluster of genes with a low hybridization signal specifically for flor strains. C. Clusters of genes potentially amplified (Log ratio>0.3) in comparison with S288C.
Cluster A (
In addition to the set of genes showing low hybridization, some genes showed moderately low hybridization, as exemplified by two subtelomeric clusters. The first cluster includes
Cluster B (
The presence of several clusters with low hybridization signals in subtelomeric regions is puzzling. These clusters explain the typical “inverted U” observed in
Few functional categories were associated with these genes. Genes involved in maltose metabolism were significantly affected (GO:0000023: maltose metabolic process,
In addition to gene loss, gene amplification may also drive adaptation in response to a selective constraint
The ability to develop a velum is an essential trait of flor yeast and requires high hydrophobicity at the surface of yeast cells. This trait has been related previously to polymorphisms of the
The first sequences were obtained from Genbank and correspond to Spanish and an Italian flor strains
Cluster | Strain | FLO11 diversity | Hydrophobicity | replicates | |||||||
Promoter | Flo11p length | mean per strain | mean per Cluster | ||||||||
Jura 1 | BAE52 | del | 3.2 | 3.2 | 3.7 |
||||||
CAH54 | del | 3.2 | 3.2 | ||||||||
CAW22 | del | 4.2 | 4.2 | ||||||||
CAW24 | del | 4.2 | 4.2 | ||||||||
CAX22 | del | 3.5 | 3.5 | ||||||||
LRJura | del | 3.8 | 3.8 | 91.3 |
3 | ||||||
PIA64 | del | 3.2 | 3.2 | 93.5 |
2 | ||||||
PIN34 | del | 3.6 | 3.6 | 88.4 |
3 | ||||||
SAC56 | del | 3.5; 4.2 | 3.9 | ||||||||
XRG22 | del | 3.8 | 3.8 | 90.0 |
3 | ||||||
Jura 2 | MAC51 | del | 3.6; 4 | 3.8 | 3.6 |
94.7 |
2 | ||||
MAD51 | del | 3.5 | 3.5 | ||||||||
MAI53 | del | 3.5 | 3.5 | ||||||||
XRA22 | del | 3.7 | 3.7 | 92.8 |
3 | ||||||
XRC21 | del | 3; 3.7 | 3.35 | ||||||||
SAC53 | del | ||||||||||
Jura 3 | GUF55 | WT | 4.7 | 4.7 | 4.7 |
90.5 |
3 | ||||
GUF51 | WT | 5.0 | 5 | 88.9 |
3 | ||||||
CBB52 | WT | 4.5 | 4.5 | ||||||||
CAV23 | WT | 4.7 | 4.7 | 90.7 |
2 | ||||||
P5 | WT | 4.7 | 4.7 | 89.9 |
2 | ||||||
CBD04 | WT | 4.8 | 4.8 | ||||||||
GUE51 | WT | 4.8 | 4.8 | ||||||||
GUG55A | WT | 4.9 | 4.9 | ||||||||
CBA13 | WT | 5.0 | 5.0 | ||||||||
SAA52G | WT | 6.0 | 6.0 | 94.7 |
2 | ||||||
CBD05 | WT | 4.5; 5 | 4.8 | ||||||||
CBE05 | WT | 4.5; 5 | 4.8 | ||||||||
CAV21 | WT | 3.7; 4.5 | 4.1 | ||||||||
ARC41 | WT | 4.5; 5 | 4.8 | 76.8 |
3 | ||||||
SAA55 | WT | ||||||||||
Jerez 2 | CECT11758 | del | 4.5; 3.8 | 4.15 | 3.7 |
95.3 |
2 | ||||
CECT11759 | del | 2.7 | 2.7 | 92.4 |
3 | ||||||
CECT11760 | del | 4.5; 3.8 | 4.15 | ||||||||
CECT11763 | del | 94.1 |
3 | ||||||||
ET7 | del | 3.7 | 3.7 | 88.8 |
3 | ||||||
Jerez 1 | 480-SL | del | 3.5 | 3.5 | 3.8 |
92.1 |
3 | ||||
481-SL | del | 4.8; 2.7 | 3.75 | 94.0 |
3 | ||||||
CECT11756 | del | ||||||||||
CECT11757 | del | ||||||||||
CECT11762 | del | ||||||||||
CECT12765 | del | 4.2 | 4.2 | ||||||||
CECT1882 | del | 5; 4 | 4.5 | ||||||||
My138 | del | 3.3 | 3.3 | 95.3 |
2 | ||||||
My91 | del | 3.3 | 3.3 | ||||||||
Sardinia | 1043 | del | 2.5 | 2.8 |
88.4 |
3 | |||||
FloraNero | del | 3.4 | 91.8 |
3 | |||||||
M25 | del | 2.5 | 35.4 |
3 | |||||||
Hungary | T19CUB | del | 2.0 | 3.0 |
|||||||
T8CUB | del | 3.3 | |||||||||
TS12CUB | del | 2.4 | 93.9 |
3 | |||||||
TA12CUB | WT del | 3.5 | 94.8 |
3 | |||||||
TP32CUB | WT del | 3.0 | 95.0 |
3 | |||||||
TR05CUB | WT del | 3.5 | |||||||||
Spanish Flor 2 | G1 | WT | 2.2 | 2.2 | 2.2 | 10.0 |
3 | ||||
Wine Cluster | MAA52 | WT | 2.4 | 2.4 | 2.9 |
||||||
MTF2-K1 | WT | 2.4 | 2.4 | 8.7 |
3 | ||||||
RM11 | WT | 3.8 | 3.8 | ||||||||
Lab | S288C | WT | 3.2 | 16.7 |
3 |
del: presence of the deletion in ICR1, WT: Wild type allele. The size of the core region of Flo11p alleles is given, as well as the mean size per strain. The mean size of Flo11p per cluster is given with standard variation. Hydrophobicity was measured according to Ishigami et al.
*ND: could not be amplified.
We amplified the core region of Flo11p for 59 strains and obtained DNA fragments for 53 strains, with sizes varying from 2.5 to 6 kb. We did not obtain amplification for four Spanish strains and two Jura strains. The mean size for wine strains was 2.9 kb, similar to Hungarian flor strains at 3.0 kb. The core region of Flo11p was longer in other flor groups, including Jura 1 and 2 at 3.6 kb and Jerez 1 and 2 at 3.7 kb.Jura 3 cluster strains had the longest Flo11p core region (4.8 kb). We obtained a mean value of 2.8 kb for three Sardinian strains. The size of the variable central core of
We measured cell hydrophobicity and velum formation to examine the effect of
We assessed the ability of 29 strains to produce a velum by cultivating them on Fornachon’s media. All strains of clusters Jura 1, Jura 2 and Jerez 2 produced a velum (
Microsatellite | Duration | Incubation (days) | ||||
Strain | 2 | 4 | 6 | 8 | 10 | |
BAE52 | 2 | 4 | 4 | 4 | ||
LR | 1 | 4 | 4 | 4 | ||
PIN34 | 2 | 3 | 3 | fell | ||
MAC51 | 4 | 4 | 4 | 4 | ||
GUF55 | 1 | 1 | 1 | 0 | ||
MAD 51 | 4 | 4 | 4 | fell | ||
MAI53 | 4 | 4 | 4 | 4 | ||
ARC41 | 0 | 0 | 0 | 0 | 0 | |
CAV21 | 0 | 0 | 0 | 0 | 0 | |
CBD04 | 0 | 0 | 0 | 0 | ||
GUG55 | 0 | 4 | 4 | 4 | 4 | |
GUE 51 | 1 | 0 | 0 | 1 | ||
P5 | 0 | 0 | 1 | 1 | 0 | |
480 SL | 0 | 0 | 0 | 0 | ||
481 SL | 4 | 4 | 4 | 0 | ||
MY138 | 0 | 3 | 3 | 2 | ||
CECT11758 | 0 | 0 | 1 | 1 | ||
CECT11763 | 0 | 4 | 4 | 4 | ||
ET7 | 0 | 0 | 1 | 1 | ||
1043 | 0 | 0 | 0 | 0 | 0 | |
Flora Nero | 0 | 1 | 3 | 3 | 3 | |
M25 | 0 | 0 | 0 | 0 | 0 | |
T19CUB | 0 | 3 | 3 | 3 | 4 | |
T8CUB | 2 | 4 | 4 | 4 | 4 | |
TR05CUB | 0 | 0 | 0 | 0 | 0 | |
TP32CUB | 0 | 1 | 0 | 1 | 0 | |
TA12CUB | 0 | 0 | 0 | 1 | 1 | |
TS12CUB | 0 | 0 | 0 | 0 | 0 | |
G1 | 0 | 0 | 0 | 0 | 0 | |
S288C | 0 | 0 | 0 | 0 | 0 |
Flor strains are found in several countries in Europe; however, until now no global approaches had been undertaken to compare strains from various vineyards. We showed previously that Jura flor strains carry a specific allele of
In this study, we used microsatellite typing, InStruct clustering and population analysis to reveal for the first time that most flor strains share the same unique origin. Lebanese and Spanish strains showed the most basal position within the population structure; therefore, it is difficult to infer the origin of flor yeast. Interestingly, a flor yeast population was recently characterized in Georgian aged wines produced by the “Kakhetian” method
Wine is a much harsher environment than must for yeast cells during flor aging. During alcoholic fermentation, yeast cells metabolize almost all fermentable sugars and assimilate most nitrogen sources (except proline) and vitamins. As a result, wine contains a high concentration of alcohol (starting from 13% v/v in Jura, and 14–15% in Sardinia and Spain) and a low nitrogen and vitamin content. In addition, yeast cells have an aerobic biofilm lifestyle, and use glycerol and ethanol as carbon sources. Many experiments have shown how yeast are able to adapt to particular environmental conditions
Aneuploidy is a mechanism that fuels adaptation to environmental changes
Several genomic regions showed a low hybridization signal indicating that these regions are missing or contain variations hampering hybridization. One of the most puzzling aspects was the location of most of these events in subtelomeric regions, which was observed previously by other groups
Polymorphism of
In conclusion, our results reveal that flor yeast are a unique family. Flor strains are mainly diploids, with some polyploid Spanish strains. We detected a shared pattern of amplification for two genes in four out of six flor strains (
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The authors are extremely grateful to Prof M. Budroni, University of Sassari, Italy, Prof A. Maráz, Corvinus University of Budapest, Hungary, Dr. Maria Esther Rodríguez Jiménez, Universidad de Cádiz, Spain, Prof J. Jimenez Universidad Pablo de Olavide, Sevilla, Prof JC Garcia Mauricio, Universidad de Cordoba Spain, Prof JA Suárez-Lepe Universidad Politécnica de Madrid, Madrid, Spain, for sending us flor strains from the different vineyards. We also acknowledge two reviewers for their constructive critics that substancially improved this manuscript.
The authors would like also to thank Anne Alais for her helpful technical assistance with microsatellite typing, Pierre Delobel for the analysis of flow cytometry data, and Dr. Frédéric Bigey for mapping affymetrix array probes on S288C reference genome.