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Diversity and adaptation of flor yeast : new data for an old question
Jean Luc Legras, Frederic Bigey, Anna Lisa Coi, Virginie Galeote, Claudine Charpentier, Sylvie Dequin
To cite this version:
V
Saccharomyces cerevisiae, our favorite model organism has been used for millennia for the production of all sort of fermented beverages as well as for bread rising. In contrast with these fermentation lifestyles, during flor wine aging Saccharomyces cerevisiae strains are growing aerobically at the surface of wine as a biofilm. As a significant part of the nutriment has been depleted from wine during alcoholic fermentation, and glucose has been replaced by ethanol, flor yeast must have overcome the different stress imposed by this niche.
This work aims at characterizing Flor strains in order to unravel the basis of flor yeast adaptation to sherry like biological aging.
Context and Goals
.
Diversity of flor strains evaluated
with microsatellites markers
Diversity and adaptation of flor yeast : new data for an old
question
Jean-Luc Legras*, Fréderic Bigey, Anna Lisa Coi, Virginie Galeote, Claudine Charpentier et
Sylvie Dequin
Experimental Approaches
Journées des microbiologistes de l’INRA, Avignon, 13-15 Novembre 2012
- Diversity of Flor strain from different countries was evaluated with 12 microsatellite markers according to Legras et al 2007 . 119 flor strains (64 from Jura, 30 from Sardinia, 43 from Spain) were genotyped and compared to 500 strains already
characterized .
- aCGH on Yeast2 Affymetrix arrays was performed for 6 flor strains from: France (Jura 2), Hungary (Tokay 1), Italy (Sardinia 1), Spain (Jerez 2) in comparison to three wine strains (one classical wine U13, one hybrid Eg8 and one Eg25 flor type wine strains).
- Genome sequencing (Hiseq2000, 100bp pair ends reads, 250 x average coverage) of 8 wine strains and 8 flor strains
(France (Jura 2), Hungary (Tokay 2), Italy (Sardinia 1), Spain (Jerez 2, Cordoba 1) ). Assemblies were obtained with SOAP and SNP scored with GATK.
Ø Flor strains from Hungary, Italy, Spain,
and France belong to one main origin
and few Spanish strains are clustered in a second group
Ø Related strains can be encountered in
several vineyards (Romania, Lebanon and France (Alsace)).
Ø FLO11 core size varies according to the
clusters. Some clusters do not carry the deletion deletion in the promoting region described by Fidalgo et al. 2006
Ø Flor strains present a variable ability to
sporulate and produce viable spores . Interestingly strains from (Jura3)
sporulate nicely whereas Spanish flor strains did not produce viable spores
Flor HXT3 allele, has been described from strain Fermichamp: 1 mutation (625 A → G : Ile209 → Val) gives Hxt3p a higher affinity to fructose (Guillaume et al., 2007
AEM, Blondin et al unpublished data)
Figure : 3D structure of HXT3 : V209 is facing Q206 in the hexose channel
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI
Genomic specificities of flor strains evaluated with
aCGH
Infante et al 2003 described large aneuploidies when comparing two flor strains that they associated to velum growth adaptation
CNV were detected with DNAcopy
Venkatraman et al., 2007 Bioinformatics)
Large aneuploidies can be detected for Eg8 hybrid strain or Eg25 given here as a control, but only for flor strain CECT11758 (Chromosome I)
For each flor strain 31 to 55 genes present a lower hybridization
Only two genes YKL222C and YKL221w were amplified in 4 out of 6 flor strains
Flor yeast Genomics preliminary results – HGT introgressions
⇒Region A was not detected in sequenced flor strains (but in some other flor genotypes)
⇒Region B is detected in half of flor strains
⇒Region C is detected in most flor strain (FOT1-2 and FSY1 genes) ⇒Other genes: Ty5-6 of S. paradoxus
also found in QA23
Strain Region A Region B Region C
F25 7.7 P3 Guf54 TA12 TS12 Sarde2D F12 EC1118 ☞
1INRA, UMR1083, Sciences pour l’œnologie – Montpellier, France 2University of Sassari, Italy
Global phylogeny of Flor yeast obtained from Genome
sequencing
E C 11 1 8 /Q A 2 3 K = 8 (10 runs combined with CLUMP)Density of SNP specific to flor yeast reveals divergent regions
(BWA alignment of reads on S288C genome sequence, SNP call made with genotypes were called with the Genome Analysis Toolkit) 7 flor strains were compared to 8 wine strains
P A U 11 F L O 9 F L O 1 H X T 3 F L O 5 H P F 1 T Y 1 Te lo m er e C h r1 2
⇒Detection of different divergent genes (ie PAU11, FZF1, FLO5 -9, HXT3 ….) ⇒Other events: translocations, chimeric genes
Conclusions:
Flor strains are found mainly in one group of S. cerevisiae strains, and represent a specific lineage
CNV is very likely not the main mechanism involved in adaptation to velum growth, but polymorphism (translocation, mutations, …) with specific alleles. Flor strains are more fructophilic than classical wine strains as they carry an improved low affinity transporter for fructose allele of HXT3 and the high
affinity fructose transporter FSY1 of region C of EC1118 (Galeote et al. 2010).
References:
Legras et al. 2007. Mol Ecol 16, 2091–2102 Fidalgo 2006 PNAS 103, 1122811233. Infante 2003, Genetics Evanno 2005, MolEcol
Three HGT caused introgressions (region A BC and) have been
characterized in EC1118 that we tried to detect in the genome of Flor strains:
Fig : Phylogenetic Network obtained with SPLITREE from the matrix of number of SNP per kb estimated from the pairwise genome sequence alignment of 95 strains with MUMmer 3.0
Yeast genomes from Liti et al. 2009, Fay et al. http://www.genetics.wustl.edu/jflab/data4.html
Novo et al 2009, Borneman et al 2011, Argueso et al 2009, Akao et al 2011.
Structure output obtained form a subset of 5000 non
ambiguous positions obtained from genomes alignments . Optimum K =8 evaluated with 10 runs per K according to Evanno’s method (2005) F Z F 1 AWRI796 6320-A7 AWRI1631 D47-6-4A K1-28-1A RM11-1A BC187 F12-3B JAY291 WU_CBS7960 WU__T73 WU_YJM280 WU_CLIB324 Sigma1278b MaxPl_W303 S288C CENPK113_7D WU_FL100 WU_YJM653WU_CLIB382 322134S DBVPG1853 WU_IL01 YJM789 WU_YJM320 WU_YJM428 S2_Y55 WU_PW5 S2_DB6044 NCYC110 S2_SK1 S2UWOPS872421 K11 Kyokai7 WU_UC5 WU_Y9 WU_Y12 S2_Y12 Y9 WU_NC02 W_YPS1009 WU_YPS163 S2_YPS128 YPS606 WU_T7 S2_UWOPS837873 UWOPS05_2272 UWOPS05_2173 WU_Y10 WU_YJM269 WU_YJM451 EC9-8 378604X DBVPG6040 NCYC361 YS2 YS4 ForstersO ForstersB YS9 273614N YIIc17_E5 WU_I14 59A EC1118 TS12-A7 TA12-2 P3-D5 GUF54-A1 7-7 F25 FS2D 20B2 VL3 W_CLIB215 GE7-4A WU_M22 S2_DB1373 22A4 YJM981 YJM978S2_YJM975DBVPG1788 1014-F5 S2_DB6765 L1374 S2_L1528 S2_DB1106 L1414 WU_WE372 Vin13 QA23 WU_YJM326 WU_YJM421 S2_W303 S2_UWOPS034614 0.0010 Wine Flor
African palm wine
Japan sake USA oaks Lab. 0.001 MY138 VI 481-S4 MY91 CECT11756 CECT11757 10.13 CECT11762 II 45.1 CECT12765 F25- c ord 16.29 7.7 11.2 8.21 11.3 9.12 III 17.29 UCD580 X XVIII VIII Jerez 1 TS12CUB CAW22 CAW22B CAW24 CAX22 CAH54 BAD52 BAE52 PII31 PIA64 CAM43 PII33 XRG22 XRG24 XRG25 LR PIN34 PIE31 PIO32 Jura 1 V75 V9 V69 V63 V66 Sarde2D A41 A33 Sard-M25 1043 V5 V16 Sardinia M23 M49 M12 M3 XRA22 XRA23 T19CUB T8CUB V23 V3 FloNero XRC21 V56 V59 M39 M38 M4 M8 MAH53 MAC51 MAD51 MA51jura MAE54 MAF54 SAC53 MAF53 MAI53 SAC56 4.2mjxx MJ104 M14mjxx MJ34 D2mjxx MJ100 Lebanon 480-S4 MJ170 GUF55 GUF54 GUF51 TR05CUB TA12CUB GUE51 GUG55A SAA55 CBA13 SAA52P GUG55B SAA52G CBE54 28640 ARC43 ARC41 ARC44 ARC42 ARC46 CAX21 CAV23 CBB52 CBB01 P5 P3 2Val.Cal Eg25 10402 CBD55 CBC01 CBE51 CBD05 CBA14 MTF1097 CBD04 CBE05 CBB04 CAV21 cheFFA 10418 10419 3238-32 A51 TP32CUB 6038 P7 M66
Eg8 hybrid cluster
Jura 3 10423 6bPenciu VS290 UHA14 COG24 KL21 EC1118 KL1 MJ73 UCD535 322134S DBVPG1853 Fa9623Bdx 1.28.2 1.28.3 VPDN ET7 V IV I CECT11760 11.76.3 CECT11763 CECT11758 CECT11759 Jerez 2 Flor strains Wine COG27 CogFA5 MCN5C TA02 MJ54 Rum strains 1 F12-cord Flor 2 Bread strains 315 397 Bread strains 2 KR645 3Val.Cal Rum strains 2 Beer strains
Fermented milk strains Sake and rice wine strains
lab strains
YPS1000 UW87-2421
CBS7957 CBS7958
USA Oak strains
UW05-227.2 UW05-217.3 African strains 1 YJM326 YJM320 YJM451 DBVPG6040 YJM269 UW83-787.3 NC02-1764 YPS1009 African strains 2 0.1 Hungarian Jura 2 3.7+0.7 3.7+0.4 4.8+0.9 3.7+0.7 2.7+0.5 3.6+0.2 3.0+0.6 2.7+0.7 FLO11 core size
111bp deletion in FLO11 promoter Wild type promoter of FLO11
EC1118 and QA23 are the result of a cross between a wine and a flor strain.
