From yeast diversity to yeast domestication

(1)

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From yeast diversity to yeast domestication

Jean Luc Legras

To cite this version:

Jean Luc Legras. From yeast diversity to yeast domestication. 45. Conference on Yeast, May 2018, Smolenice, Slovakia. 2018. �hal-01837857�

(2)

From yeast diversity to yeast domestication

Jean Luc Legras

SPO Montpellier, France

(3)

Diversity of Saccharomyces cerevisiae habitats

Environments associated to humans

Natural ressources

LEGRAS JL May 15-18th 2017 – Smolenice, Slovakia

2

(4)

3 Remains of the most ancient beverage found in China in a Neolithic tomb in Jiahu village (-7000 BC) ( Mc Govern PNAS 2004)

http://www.penn.museum/research-asian-

section/387-the-earliest-alcoholic-beverage-in-the- world.html

(5)

Origin of Vine culture and wine making

http://www.penn.museum/sites/wine/wineneolithic.html

•

potteries dated from 5400 BC in Iran (Hajji Firuz Tepe) (McGovern 1996 )

•

Identification tartaric acid and resins residuals => wine

4

(6)

Molecular methods for the

characterization of yeast diversity

Pulsed Field Gel electrophoresis

(Schwartz & Cantor, Cell 1984, Blondin et al 1990…)

mDNA RFLP (Aigle et al, J Inst Brew 1984, Querol 1992…)

AFLP (Azumi et Goto-Yamamoto, Yeast 2001)

RAPD (Pafeti et al 1995…)

Interdelta typing (Ness et al. 1993, Legras et al. 2003)

Microsatellite Typing (SSR) (Field & Wills PNAS 1998)

Sequencing

Multi Locus Sequence Typing (Fay & Benavides 2005...)

Restriction site associated sequencing (Rad-Seq) (Cromie et al 2013)

Genome Sequencing (Liti et al 2009 Nature , Peter et al. 2018 Nature)

5

strain 1

strain 2

strain 3

(7)

6

•

200 strains from 30 sites in Italian vineyards (DPCA)

Population structure obtained with:

mDNA RFLP and Microsatellite Typing

ADNm RFLP Microsatellite

(8)

Are strains of various niches different?

7

(9)

Population structure and yeast domestication

- lower diversity for wine and sake yeast

=> wine yeast and sake yeast 2 domestication events

divergence

wine strains 2,700 – 27,000 BP sake strains 3,800 – 38,000 BP

Fay et Benavides, 2005

Wine

Sake

8

(10)

1000 strains genotyped at 12 microsatellite loci, updated from Legras et al. 2007

Bruvo’s distance

Yeast strains clustered according to their origins

Among wine strains:

- Few identical strains from distant vineyards, except starters

- 2 groups of very related strains

(11)

•

Several groups of S. cerevisiae * S. kudriavzevii hybrids

•

isolated in

–

Alsace (France) (1)

–

Geisenheim (Allemagne) (1),

–

Hungary(1)

•

or:

–

Wadenswill (2 and4) (Suisse)

–

Alsace (3) :

•

Hybrides S. cerevisiae*S. uvarum

•

(Alsace 2004, Diois, …)

=> Adaptation to cold climate wine making conditions

Interspecific hybrids are frequent

Erny et al , AEM 2012.

Palm Wine Beer Wine

Sake USA Oak tree Bread

Cheese Asia misc Laborat.

10

(12)

Population differentiation

Fst genetic distance

11

(13)

Fst genetic distance NJ tree between groups;

Modified from Legras et al 2007

F- Rhone Valley F- Burgundy

F- Nantes F- Alsace

Galice USA

F- Bordeaux Austria

Italy

Portugal

F- Montpellier Cognac

Spain Misc Bulgary Romania

South Africa

Hungary Germany

AlsaceCEG

Lebanon

French Indies French Indies

Japan Sake Italy Brd2

Bread

BeerAle Nigeria

fermented milk Distillery China

China

RiceWine 0.02

Relationships between populations

§ Wine yeast separated from other strains

possible Mesopotamia origin .

§

• For other groups

• 26% of genetic distance between groups explained by geography suggests local domestications

Wine

12

(14)

Diversity of S. cerevisiae wine populations

13

(15)

Differentiation of yeast populations in 3 Italian vineyards (Treviso) Coll. V Corrich Padova

220 isolates genotyped with microsatellite markers

(Viel et al. 2017 Frontiers Microbiology)

14

(16)

Diversity of yeast populations from vineyards of the Bordeaux region coll. I Masneuf ISVV

Bordeaux

Borlin et al. In prep Migration pattern

(inferred with Migrate)

15

(17)

Vine - Cellar relationship

16

(18)

Detection of grapes isolates related to yeast starters in

- Oregon (Martiniuk et al. 2016) - Italy (Viel et al. 2017 )

- Bordeaux (Borlin et al. 2018) but not 100 % identical

Up to 10 to 25% of isolates

3mabid3 173maco3 123caco4 33caco3 193caco3 8433maco1 53caco4 53maco3 263caco5 53mabid3 123mabid3 6583caco3 183maco1 11 3maco3

30 25

3maco1 10 3maco1 6 3m

aco3 19 3maco3 27 28

Acti522D

L13 69

2y fco1

202

yfco1 10 F33 2yfco1 9 26

3132

51

3m aco3 24 3cjco2 13

3cjco2 5 3hpco1 24

3hpco1 29 38

39

2in racoG1 82dcco4 18

2dubi3 10 36

26

3fzc3fo4zco4 29 7 55

3fz3fzcoco4 204 5 70

86

3cjco2 12 3cjco

2 8

3hpco3 18 3hpco3 21

3cjco2 15 3hpco1 11 3hpco1 22

46

3hpco1 28 3cjco2 3 3cjco2 7 3hpco13h 27pco1 3 3hpco1 4

28

3hpco1 25 3hpco1 13 3hpco3hp1 20co1 9 40

41 26

3cjco1 19 3mabi1 15 3mabi1 9 84 3fzco1 13

41

3cjco1 2 3cjco1 11 3cjco1 16

64 66

97

38

2cjco5 18

3ribi4 17

2mlbi1 12 3maco3 8 3inra6 2

25

2lhco6 9 3ca3caco2co2 21 22

3hpco2 18

2pacoG1 30 2pacoG1 11

2pacoG1 17

28

36

2pacoG1 14 2pacoG1 21 2pacoG1 18

2pacoG1 20 2pacoG1 27 2pacoG1 1

2pacoG1 16 49 2pacoG1 28

2pacoG1 7 45

42 2pacoG1 22 2pacoG1 24

53 2pacoG1 10 2pacoG1 9

2fz1c2dccoo3 304 16 2inracoG1 5 2dcco4 15

3hpco3 9 2dcco4 255

3hpco3 13 35

3hpco3 16 3hpco3 19

3hpco3 23 38 51 37 69

50 38

BC187

3caco2 10

42

3bebi3 11

3bebi3 14 100

3fzco5 13 2caco4 5 3caco2 16 2cjco5 23 2bibi2 1 2bibi2 4

3caco4 19 3caco3 103lhco2 19 3lhco2 153lhco2 291 71 25

3caco5 15 3caco5 17

99 2cjco2 3

3lhco1 8

3lhco3 303lhco4 24 40 32

3lhco4 19 48 3lhco4 253lhco4 4 383lhco4 3

51 3lhco4 14

63 2cmco4 14

2cmco4 22

50 ICV-GRE2caco3 2

26 2incoA2 12bibi1 5

2lhco1 10 3caco5 13caco5 3 43 3caco4 15

38 2caco4 15

42 2caco3 52caco3 92caco3 28

82 2caco3 6 2caco3 273caco3 1666

73

42 2caco3 172caco3 7

32 2caco4 222caco32caco3 29 14

100

34 3caco2 17 3caco4 2 3caco2 8

3caco3 12 3caco2 23

3caco2 4 2caco4 16

2caco4 30 2caco4 1 2caco4 20

47 59 2caco4 21 2caco4 4

53 2caco4 27

90 2yfco2 22 2yfco2 23 2yfco2 24

51 98 2clico5 172clico2cjco5 65 8 30 2clico5 26 2clico5 9

66

56 2lhco5 11

32 2clico5 7 2clico5 1

2clico5 27

49 2lhco5 3

2lhco5 5

38 2cjco1 29

3caco2 30 3caco5 10

43 2cmco4 23

2cmco4 28 2bibi3 11 2bibi3 14

85 2bibi6 28

29 3caco5 4 2cmco5 10 2caco3 12 2cmco5 202bibi6 93lh co3 15 3lhco3 26 3lhco3 22 FX10

F10 2cmco5 9

3caco3 4 3caco5 19 3bebi3 26 3bebi3 132bibi6 26

3bebi3 13cac0o4 17 3caco5 14

3caco5 737

3mac67o1 43caco5 203caco3 6co3 13caco3 903ca3 1co2 263lh3lhco 8 2iA 22pabiAab762p 62pabiAabiA 32paco4 13c310abiA 32po3 20 163cac3hpco4o4 83hpc78 582pabiA 162pabiA 42pabiA 272pabiA 152pabiA 1969943lhco2 13lhco2 1644423hpco1 233hpco1 193hpco1 26813hpco1 82ribi3 102ribi3 7

88

2lhco3 152fz1co3 10

2lhco3 1

29 2lhco3 112lhco3 82lhco3 16 32 2lhco3 3 2lhco3 6

41

2lhco3 12

2lhco3 2

33 2lhco3 5

2lhco3 13

2lhco3 9

70 2lhco3 7

2lhco3 10 2ggbi6 5 2lhco3 20 2dcco3 2 2lhco3 4 2ggbi6 18

2incoA 2 15 2incoA2 25

67 2incoA2 24 95

2incoA2 21

100 R2

3hpco1 21 3hpco3 2

81

SPorganic VitQuartz

78 lalfermbi LalQA23 89

67 CIVC8130

52 Oe nfermBi

79 3fzco2 11 3fzco2 2

67 3fzco2 1 3fzco2 3 67

99

60

2bibi2 16 2bibi2 9

99

67

LevuliALS

Vin13

54 3fzco2 12

3mabi1 1

3mabi1 8

48

35 56

67

3inra6 17

2bibi3 1 2ribi3 24 38 3inra6 16

3inra6 10

37 2bibi1 10

2bibi3 2

RX60

2ribi3 12 X52bibi7 10

2ribi2 11 2bibi3 9 2ribi2 28 2728 32

29

51

52 CY

3caco3 13

38 3caco5 2

43 3caco3 7 3caco3 11 3caco3 17 56 49 34

3caco3 21 3caco3 5

56

ExcelC1

41

2ribi4 12ribi4 11 70 2ribi4 28 66

VL3

42 3maco3 18

40

2lhco4 5 2lhco4 232lhco4 4 2lhco4 15 2lhco4 9 45 35 65 43

X16 Zyma001

93 2bcbi1 14

2bcbi1 4 3mabi1 10 3mabi1 21 2bcbi1 15

3mabi1 19 2bcbi1 11 27

3mabi1 16 35

3mabi1 4 37

2bcbi1 29 2bcbi1 24

2fzco2 20 2fzco2 21

2bcbi1 12 2bcbi1 18 2bcbi1 23 34

2fzco2 10 2fzco2 12 84

2fzco2 2

39 2fzco2 8

3654 2fzco2 62fzco2 9 62

FermArom Z1 59 2bcbi1 2 2bcbi1 16 2bcbi1 28 VL1 2bcbi1 10 25

DBVPG1788 L-1374 2incoA2 19 YJM981

YJM978

YJM975 Fermivin

45 38 60

B2 K1 50

2bibi2 22 2bibi2 23 94 28

L-1528

2bcbi2 9

YIIc17 E5 2lhco6 4

82

2ma2b 12 2ma2b 19 31

2ma2b 16 43

2yfco3 1 2yfco3 14 38

2ma2b 29 27

2ma2b 17 2yfco3 20 2yfco3 7 2ma2b 20 2yfco3 15 45

2yfco3 22 L2226

2bibi7 16 3fzco4 1

3fzco4 27 80

63

LevuliBRG 2bibi1 9

2bibi1 12incoA2 22 72

2bibi1 21 42

2bibi7 13

2bibi7 1 7 2bibi7 4 2bibi7 14 2bibi7 7 7051 57 33 60 84

DBVPG6765 DBVPG1106

DBVPG1373 BM45

2fzco5 18 2fzco5 6 34

2lhco3 17 3cjco2 17 3cjco2 6 90

3cjco2 10 66

3hpco2 8

3hpco2 20 3hpco2 23 57 54 27

3cjco2 16 3cjco2 4 3cjc63o2 9 71

2cjco1 9 2cjco1 1 2cjco1 19 68 86 50

3bebi3 23

3hpco2 15 56

2ma2b 4

2dubi3 1 2fzco5 19 2fzco5 27 57

2fzco5 11

2fzco5 8 2fzco5 29 38 3caco4 9 2fzco5 24

3hpco2 4F15

56

3rib3ribi5 10i5 3 42 3ribi5 13 74

3ribi5 9 100 2bcbi2 20 2bcbi2 1

2bcbi2 15 2bcbi2 3 2bcb

i2 8 91

2bcbi2 29 42

2bcbi2 28 2bcb i2 25

2bcbi2 24 2bcbi2 19 2bcbi2 4 29

2bcbi2 17 2bcb i2 16 2bcb i2 26

2bcbi2 2

2bcbi2 23

443051

UvaCEG 2mlbi5 18

2pabiB 20

2pabiB 4

100

32 Lalvin71B

YS9YS2YS4

544932 DBVPG1853

273614XD

BVPG 6044

NCYC11097SK174Y5597 UWOPS83

YPS606

4425 UWOPS03UWOPS05.2UWOPS

05.3 78100378604XUWOPS8730S288cW30356X2180 100

DBVPG

6040Y12Y9 72

K113ribi5 172inbiB 182inbiB 62inbiB 142inbiB 12inbiB 19 476497 56 38

0.05

Yeast starters Médoc

Saint Emilion:

Entre-Deux-Mers Pessac-Léognan Bergerac

522D

VL3

Wine - cellar relationship

collaboration ISVV Bordeaux

17

(19)

4 cellars et 4 vineyards from 3 areas (2 x 72 souches) (removal of yeast starters )

1 – small differentiation between cellar and vineyard (Fst= 0.03) 2 – modellisation of gene flow (Bayesian inference with migrate 4.2.14)

Vine – Cellar connection

collaboration ISVV Bordeaux I. Masneuf

Nm = 191 : 166-226

Nm = 55 : 25-83

Two compartments in equilibrium with a higher grapes / vat flow

(Börlin et al. 2018 in prep)

18

(20)

Inter vintage differentiation (Sauternes area, Börlin et al 2016 AEM)

= > Higher differentiation with time (blue)

Vintage Comparison

% of genetic

variation (APWG) Fst P (r<0) 2012 - 2014 5.14 0.113 <0.00000

1 2013-2014 7.17 0.067 <0.00000

1 1992-1993 11.91 0.088 <0.00000

1 1992-2014 15.51 0.294 <0.00000

1 Persistence of yeast populations in cellar

collaboration ISVV Bordeaux I. Masneuf

19

(21)

Yeast vectors

• Birds (Francesca et al. 2012)

• Insects :

–

Drosophila

–

Bees (Goddard et al. 2009)

–

Wasp

(Stefanini et al. 2012)

20

(22)

21 Diversity of S. cerevisiae isolated from wasps’

guts

(Stefanini et al. 2012 PNAS, coll. D. Cavalieri)

(23)

Is there a strain phenotypic diversity associated to strains of different

origins ?

22

(24)

Two examples of yeast phenotypic diversity

Probability of completing alcoholic fermentation in synthetic grape must (20% glucose)

0 100 200 300

Time (h)

400 500

0.00.20.40.60.81.0

Probability of incompletefermentation

Bread/Beer Cheese Flor Grape/Wine MedOak NA Oak Rum/Bioethanol

0 1

0 5 1 0 1 5 2 0 2 5

O.D.660nm

T im e ( h )

0 0 .0 5 0 .1 0 .1 5 0 .2 0 .2 5 0 .3 0 .3 5

R u m / B io e t h a n o l

C h e e se G r a p e / w in e B e e r / B re a d M e d O a k N A O a k F lo r

µ (h

-1

)

A - B -

Growth on a media containing galactose

***

23

(25)

Obtaining sequencing Data

82 strains

§

Grapes (8) Wine (23), 6 haploids,

§

Flor ageing (10), 3 haploids,

§

Bread and Beer (7)

§

Cheese(8) , 1 haploid

§

Rum and bioethanol fermentations (Brazil) (8) 1 haploid,

§

Oak(8)

§

African strains (2 palm wine and NCYC110),

§

Sake (2)

§

Other misc strains ..

v

Illumina HiSeq 2000 sequencing with (20 to -400X 1 )

v

Genotyping with GATK: 367863 biallelic variant positions 20018 indels

24

(26)

Overview of yeast diversity

Bootstrap values >95

Maximum likelihood tree from 320000 snps Legras et al. 2018 Mol Biol Evol

Wine Flor

Rum and Bioethanol

Cheese

Mediterranean oak

Laboratory

Beer and bread

Palm wine

Sake

NA Oak

25

(27)

26 Analysis with FineStructure

(phased data)

(Lawson PloS Genetics 2012)

Legras et al. 2018 Mol Biol Evol

(28)

Can phenotypic diversity be explained by specific genomic content?

27

(29)

28 Distribution of new genomic regions among S. cerevisiae strains

36 transfer events from 1.5 to 42 kb, 28 are prevalent in several populations, and 6 are specific to one or two niches

Region C3 and C4 65kb ( T. microellipsoides, Marsit et al 2015 ):

truncated in wine yeast (FOT1-2)

full length in flor yeast FSY1 : High affinity fructose symporter Region T 6.8 kb

GAL1: Galactokinase

GAL7: Galactose-1-phosphate uridyl transferase

GAL10: UDP-glucose-4-epimerase

GAL7 GAL1 0

GAL 1

Legras et al. 2018 Mol Biol Evol

(30)

Ecological advantage offered by one HGT : region C

1st

fermentation

2nd fermentation

Evolution of frequencies of strains carrying or not FOT genes

- fot

Time (h)

Marsit et al. (2015)

t0

Time (h)

+fot

+ fot

- fot

29

(31)

Ecological advantage offered by Region T in cheese strains

The same region of S. uvarum when transferred in S. cerevisiae improves growth on galactose

improves growth on glucose + galactose media (Roop et al 2016 Nature)

chees e

0 1

0 5 1 0 1 5 2 0 2 5

O.D.660nm

Tim e (h)

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Rum / Bioeth ano l

Ch eese G rape/ w in e Beer/ Bread M e d O ak N A O ak Flo r

µ (h

-1

)

A - B -

30

(32)

May highly differentiated genomic

regions explain phenotypic diversity and adaptation?

31

(33)

May highly differentiated genomic regions explain phenotypic diversity and adaptation?

Flor strain in comparison to wine : Dxy , PCA (Coi et al. 2017 Mol Ecol)

32

(34)

Chromosomal regions differentiating wine and flor yeasts

Z R T 1 S L N 1 IC R 1 Y JL 04 5w S R Y 1 P IK 1 S M F 1

YAR028W YER186C

R G A 2

Genes involved in key functions:

ü

Metal ions homeostasis (ZRT1 SMF1 COT1 ALR1 SKY1 …)

ü

HXT3, HXT4 HXT6

ü

…

ü

Extra cellular cell wall(FLO5 MUC1 DAN4 PIR3 HPF1…)

ü

ICR1, PWR1, FLO11 + IRA1 ….

33

(35)

Impact of allelic variation on flor phenotypes

34 Velum surface

(36)

Searching for highly differentiated regions that may explain phenotypic diversity and adaptation

W

R

OE 1

2

3 4

Whole Genome tree

W

R

OE

Haplotypes Local tree

1

2

3 4

35 hapFLK (Fariello 2013 Genetics): for multiple populations wine, Rum,

Mediterranean Oak

(37)

•

q.value < 0.05 : AUS1, ATG34 –ATG19, THI72, MCH5

•

…

Search for selection from population differentiation (hapFlk, Fariello, 2013 Genetics)

4 populations : Wine Rum, Mediterranean oak, USA oak as a root

•

q.value < 0.05 :

–

AUS1, ATG34 –ATG19: Wine ,

–

YME2- ADH2- FKS3 : Oak and rum

–

THI72, IME1, HAP4 : Oak Med Oak and rum

36

(38)

Searching adaptation to environment

from the signatures of selective sweeps inside a population

The fixation of an advantageous allele is accompanied by a reduction of diversity around the adaptive allele

1 2

Neutral allele Adaptive allele

3

37

(39)

Searching adaptation to environment

from the signatures of selective sweeps inside a population

Wine Cheese

•

Wine : Enrichment for amino acid transport genes (BAP2, TAT1, AVT3, GNP1)

•

Cheese sweep for genes involved in galactose metabolism : Region T (FUR4) and highly divergent galactose transporter GAL2 (SIC1)

38

(40)

39

•

Conclusions

–

The use of new molecular methods has changed our vision of S. cerevisiae diversity :

•

revealed the existence of specific clusters per human associated environments

•

revealed the domestication of some groups

•

offered the possibility to estimate gene flow between regions , cellar and vineyards …

•

enabled the evaluation of demographic/historical scenarii

=> necessity to adopt these molecular methods

especially for ecological studies

(41)

40

•

Conclusions

–

S. cerevisiae populations from anthropic

environments present several clues for adaptation :

•

for wines : amino acid transport and sterol uptake are the two major known constraints : and we found targets for these two constraints

•

for cheeses : adaptation to galactose assimilation (GAL1 GAL7 GAL10 cluster + GAL2 + GAL80) => rewiring of galactose assimilation network

–

mechanisms permitting adaptation : CNV, HGT, mutation accumulation

–

S. cerevisiae: a species complex of specialized

populations with customized genomes

(42)