Molecular charaterization (SSRs) of some European cultivarsarchive-ecpgr.cgiar.org/fileadmin/... ·...

Molecular charaterization(SSRs) of some European

cultivars

F. Laurens, L. Feugey

Malus/Pyrus ECP-GR group meeting; Tbilissi 26/10/06

1 –Preliminary SSR Analysis on some French local cultivars

Quantification and characterization of thegenetic diversity in French local apple cultivars

to study the structure of the variability

Identification + distinction between cultivars

Aims :

Markers

Perfect

Perfect

Imperfect

Imperfect

Perfect

Perfect

Perfect

Imperfect

Perfect

SSR repeattype

0.76

nd

nd

0.78

0.77

0.82

0.85

0.79

nd

Expectedheterozygoty

220-240

174-227

155-186

219-239

233-257

210-254

216-254

236-256

136-160

allelesize

range

Liebhard et al (2002)Single locus10COL

Liebhard et al (2002)Multilocus ?7CH04E05

Liebhard et al (2002)Multilocus ?17CH04C06

Liebhard et al (2002)Single locus10CH02C11


Liebhard et al (2002)Single locus11CH02D08


Liebhard et al (2002)Single locus9CH01H02

Liebhard et al (2002)Multi-locus4-12CH01D03

ReferenceLocus typeLinkage groupSSR

Material

⇒142 French local apple cultivarsfrom various geographical origins

⇒ 9 SSRs→ available→ easily to read and to interpretate→ mapped and at least 20 cM far from one to the other

Methods

• DNA isolation QIAGEN DNeasy-kit protocol

• AmplificationSimplex

• AnalysisGel electrophoresis coupled with silverstaining

MUTANTS

SYNONYMS

Smith, 1971 : Belle Fleur de France =

Double Bon pommier = Double Bon Ente

SSR bands :clo ne no m C H 02C 11 C OL C H 01D 03 C H 02C 06 C H 01H 02 C H 04C 06 C H 02C 09 C H 02D 08 C H 04E05

X 4291 D oub le bonne Ent e 5-7-16 1-8-12 3-6 6-24 4-10b 5-6 1-7 4-22?-23? x

X 2326 D oub le bon pommier 7-14-16 1-8-12 3-4-6 6-24 4-10b x-5-6 1-7-12 4-22?-23? x-3

Smith, 1971 : Belle Fleur de France =

Double Bon pommier = Double Bon Ente

SSR bands:clo ne no m C H 02C 11 C OL C H 01D 03 C H 02C 06 C H 01H 02 C H 04C 06 C H 02C 09 C H 02D 08 C H 04E05

X 4291 D oub le bonne Ent e 5-7-16 1-8-12 3-6 6-24 4-10b 5-6 1-7 4-22?-23? x


clo ne no m C H 02C 11 C OL C H 01D 03 C H 02C 06 C H 01H 02 C H 04C 06 C H 02C 09 C H 02D 08 C H 04E05

X 4291 D oub le bo nne Ent e 5-7-16 1-8-12 3-6 6-24 4-10b 5-6 1-7 4-22?-23? x


X 778 F ranc bon pommier 7-16 8-12 3-6 6-24 4-10b 6 1-7 4-23 x

Franc bon pommier x ?

=

Double Bonne Ente et Double bon pommier

Evaluation of SSR potential

0.680.73nd

0.950.830.870.850.78nd

Heterozygotes rate

0.800.70nd

0.880.830.840.850.75nd

Expectedheterozygoty

1214141611

17,18 or 192111

20 or 21

allelenumber

0.0670.95<218-242COL0.1160.88<197-255CH04E05

ndnd<166-195CH04C060.0240.97209->247CH02C110.0490.95<233-257CH02C090.0450.94204-258CH02D080.0350.97<208->262CH02C060.0990.90230-252CH01H02

ndnd134- >162CH01D03

Identityprobability

Discrimination power

Sequencedallele size

(pb)SSR

Evaluation of SSR potential

• CH04C06• CH02C11• CH02C06

• CH02C09

IP < 2 x 10-5

IP < 10 -6

2- Characterization of someEuropean cultivars using 4 SSRs

markersPreliminary results

Material

Laurence Feugey, François Laurens

Tarja HietarantaM. Plekhanova

Anna Lisek

Dag Roen

Thomas Sotiropoulos

BronislovasGelvonauskis

Correspondant

-

07/07/200301/07/2003

01/10/2003

19/05/2003

16/04/2003

03/04/2003Received

INRA Angers

Plant Production Research

Research Institute of Pomology and Floriculture

Planteforsk Njos

Pomology Institute

Lithuanian Institute of Horticulture

Organisation

192France

25 (2)Finland61 (1)Russia

41 (1)Poland

71 (1)Norway

7 (1)Greece

68 (1)LitthuaniaCultivars Country

(1) dry-frozen leaves (2) Whatman FTA cards

Methods• DNA isolation

CTAB method from Tanksley a la Paul

• Amplification multiplex for CH04C06, CH02C11, CH02C09simplex for CH02C06

• Analysispooling of multiplex + simplexanalysis through a capillary sequencer

(ABI 310 - 1 capillary INRA Angers + ABI 3130 - 16 capillary Ouest-Genopôle)

Preliminary results (oct 2006)

193

0

61

41

71

7

68

SSR analysis

INRA Angers

Plant Production Research

Research Institute of Pomology and

Floriculture

Planteforsk Njos

Pomology Institute

Lithuanian Institute of Horticulture

Organisation

162France

0Finland

49Russia

36Poland

52Norway

5Greece

27Litthuany

Results withoutmissing data

Country

331441

Preliminary results (oct 2006)

21

27

19

33

# alleles

155-201CH04C06

190-252CH02C11

222-273CH02C09

240-303CH02C06

Sequenced allele size (pb)SSR

Factorial analysis: Axes 1 / 2

-.5 -.4 -.3 -.2 -.1 .1 .2 .3 .4 .5

.5

.45

.4

.35

.3

.25

.2

.15

.1

.05

-.05

-.1

-.15

-.2

-.25

-.3

-.35

-.4

c

c

c

c

c

c

c

c

c

c

c

c

c

c

GR

GR

GR

GR

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

LI

NO

NO

NO

NO

NO

NO

NONO

NO

NONO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NONO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

PO

POPO

PO

PORU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RURU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RURU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RU

RURU

RU

RU

n

n

n

n

n

n

n

n

n

n

n

n

n

n

n

n

o

o

o

o

o

o

o

o

o o

o

o

o o

o

o

o o

o

o

o

o

o

o

oo

oo

oo

o

o

o

o

o

o

o

o

o

o

oo

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

o

PC 1 (5,8 %)

PC 2 (4.7 %)

Factorial analysis (axes 1 and 2)

DARwin-5.0 software

RussiaPolandLithuaniaNorwayGreeceFrance (old cvars)New cultivarsCider

Factorial analysis: Axes 3 / 4

-.45 -.4 -.35 -.3 -.25 -.2 -.15 -.1 -.05 .05 .1 .15 .2 .25 .3 .35 .4

.4

.35

.3

.25

.2

.15

.1

.05

-.05

-.1

-.15

-.2

-.25

-.3

-.35

-.4

c LI

NO

NO PO

RU

RU

o

o

o

c

LI

NO

NO

PO

RU

RU

o

o

o

c LI

NO

NO

PO

RU

RU

o

o

o

c

LI

NO

NO

PO

RU

RU

oo

o

c

LI

NO

NO

PO RU

RU

o

o

o

c

LI

NO

PO

PO

RU

RU

o

o

o

c

LI

NO

PO

PO

RURU

o

o

o

c

LI

NOPO

PO

RU

n

o

o

o

c

LI

NO

PO PO

RU

n

o

o

o

c

LI

NO

PO

PO

RU

n

o

o

o

cLI

NO

PO

PO

RU

n

o

o

o

c

LI

NO

PO

PO

RUn

o

o

oc

LI

NO

PO

RU

RU

n

o

o

o

c

LI

NO

PO

RU

RU

n

o

oo

GR

LI

NOPO

RU

RU

n

o o

o GR

LI

NO

PO

RU

RU

n

o

o

oGR

NO

NO

PO

RU

RU

n

o

o

o

GR

NO

NO

PO

RU

RU

n

o

o

o

LI

NO

NO

PO

RU

RU

n

o

o

o

LI

NO

NO

PO

RU RU

n

o

o

o LI

NO

NO

PORU

RU

n

o

o

LI

NO

NO

PO

RU

RU

n

o

o

LINO

NO

PO

RU

RU

n

o

oLI

NO

NO

PO

RURU

o

o

o

LI

NO

NO

PO

RURU

o

o

o

PC 3 (3.8 %)

PC 4 (3.4 %)

Factorial analysis (axes 3 and 4)

DARwin-5.0 software

RussiaPolandLithuaniaNorwayGreeceFrance (old cvars)New cultivarsCider

Dissimilarity calcultated from allelic data, dissimilarity indice : simple matching, bootstrap : x 10 000

Tree constructed by the Neighbor-Joining method

Clustering

DARwin-5.0 software

00.2

CX3735 ELI33

ENO36

ENO69

EPO33 ERU25

ERU50

oX1349

oX7190

oX8236

CX3742

ELI39

ENO37

ENO70

EPO35

ERU27

ERU51

oX156

oX7193

oX8238

CX3779

ELI41

ENO38ENO71

EPO36

ERU28

ERU52

oX1641

oX7199

oX8239

CX3799ELI42 ENO39

ENO8

EPO37

ERU29

ERU59

oX1708

oX7200

oX8241

CX3919

ELI44

ENO41

ENO9

EPO38

ERU3

ERU6

oX1846

oX7202

oX8242

CX3945

ELI46

ENO45

EPO10

EPO39

ERU30

ERU7

oX1853

oX7205

oX8244

CX3980ELI5

ENO47

EPO12

EPO4

ERU31

ERU9

oX2315

oX7250

oX8246

CX5056

ELI50

ENO49

EPO13

EPO41

ERU32

nX2740

oX2643

oX7279

oX8252CX5112

ELI52

ENO50

EPO14 EPO5

ERU34

nX2770

oX2919

oX7358

oX8253

CX5114ELI55

ENO51

EPO15

EPO6

ERU35

nX2808

oX352

oX8200

oX8255

CX5118

ELI56

ENO52

EPO16

EPO7

ERU36

nX2836

oX4488

oX8201

oX8377

CX5119

ELI59

ENO54

EPO17

EPO8

ERU37

nX2957

oX468oX8203 oX8379

CX5120

ELI66

ENO56

EPO19

ERU1

ERU38

nX3069

oX4831

oX8207

oX8382

CX5122

ELI7

ENO57

EPO2

ERU10

ERU39

nX4102

oX554

oX8209

oX8383

EGR2

ELI8

ENO58

EPO20

ERU13

ERU4

nX4712

oX585

oX8210oX8384

EGR4

ELI9

ENO59

EPO21

ERU14

ERU40

nX557

oX6171

oX8212

oX8388

EGR6

ENO2

ENO60

EPO22

ERU15

ERU41

nX6138

oX6173

oX8214

oX8392

EGR7ENO22

ENO61

EPO23

ERU16

ERU42 nX6315

oX6194

oX8219

oX8398

ELI2

ENO23

ENO62

EPO24

ERU18 ERU43

nX6553

oX6354

oX8220

oX8414

ELI20

ENO24

ENO63

EPO25

ERU19

ERU44

nX6571oX6394

oX8224

oX843

ELI24

ENO26 ENO64

EPO26

ERU2

ERU45

nX7762

oX661

oX8225

ELI26

ENO27

ENO65

EPO27

ERU20

ERU46

nX922

oX6905

oX8227

ELI29ENO3

ENO66

EPO28

ERU21

ERU47

nX972

oX6918

oX8228

ELI3

ENO32

ENO67

EPO29

ERU22

ERU48 oX1115

oX6920

oX8232

ELI30

ENO33

ENO68

EPO32

ERU24

ERU5

oX1212

oX699

oX8234

30

58

20

32

13

100

14

12

19

13

30

40

18

70

29

32

50

31 23

29

33

2324

13

41

13

18

48

54

37

35

22 31

17

30

12

23

33

62

10

16

27

44

10

63

35

29

12

20

58

27

41 19

49

10

52

32

40

78

27

45

35

36

33

20

20

19

35

12

90

1322

39

12

30

19

28

32

99

27

38

38

75

38

21

16

33

15

46

23

57

24

59

47

34

23

29

37

46

32

40

26

23

29

13

33

30

72

24

44

19

18

28

12 22

44

38

1618

14

19

14

18

P-K

osztelaP

-Polonez

1P

-Skierniew

ice 2

N-B

otunepleN

-Stölene

L-H2

L-H32 R

-Korichnoe

Aananasoe

R-E

rmak

R-B

ukisshkyuA

uksinisR

-Bryanskoe

Mestnoe

L-Ponemunis

BaltosisL-H

110

L-Nr 18010

L-Nr 17977

F-Bondon

F-Gueule de m

outon

P-N

°3P

-N°2

P-B

ankroftP

-Lodel

G-M

egaA

lexandrosF-M

amula

R-B

ukhovkaR

-Borsdorf-K

itaika

Clustering

R-A

nisikiOm

skyR

-Antonovka

Krasnobochka

Results …• Some kind of structure; but not enough

markers …

• Some synomyms

• Some related cultivars : to be precisedwith more markers

Perspectives• Fill in the gaps for the 441 accessions with the 4

markers

• Increase the number of markers (wait for EastMalling meeting in December) at least on theFrench collection

– Which markers ? Neutral, CG, …?

– What to do ?• Identification ?• Structure studies ?• Association genetics ?

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Origin

Not Tested

heterozygous on HS, W, DC

heterozygous on HS, W ; homozygous on DC

Not Tested

0

heterozygous on DC, W ; homozygous on HS

Not Tested

0

heterozygous on DC, W ; homozygous on HS

heterozygous on W, HS ; homozygous on DC

Preliminary results on Pyrus communis (Harrow-Sweet=HS, Williams=W, Doyénné du Comice=DC)

+Yamamoto et al. 2002NH008b

+Gianfranceschi et al. 1998CH02C11

+Gianfranceschi et al. 1998CH02B12



+Gianfranceschi et al. 1998CH02A08

+Gianfranceschi et al. 1998CH01H10

0Gianfranceschi et al. 1998CH01G12

+Gianfranceschi et al. 1998CH01F02

+Gianfranceschi et al. 1998CH01D08

Results on INRA Pyrusspecies collection

Obtained bySSR

First SSR results on Pyrus

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Apple

Origine

0

0

0

0

+ but non informative

non informative ; - on HS

0


heterozygous HS, W ; homozygous on DC

2 bands very close; + for DC

OK when Tm=58°C

+ but non informative except for DC

0

non informative on DC, informative on HS

heterozygous on DC ; homozygous on HS, W


Results on Pyrus communis (Harrow-Sweet=HS, Williams=W, Doyénné du Comice=DC)

NTNZ28F4

NTGianfranceschi et al. 1998col

NTGianfranceschi et al. 1998CH03H03

NTGianfranceschi et al. 1998CH03D01

NTGianfranceschi et al. 1998CH03C02



NTGianfranceschi et al. 1998CH02G01

NTGianfranceschi et al. 1998CH02F06






NTGianfranceschi et al. 1998CH01F03b

NTGianfranceschi et al. 1998CH01F03a

réponse sur collection Pyrus espèces

Obtained bySSR

First SSR results on Pyrus

Thanks to :

Mathieu LascostesLaurent Gouère et Mélanie Collin

Molecular charaterization (SSRs) of some European cultivarsarchive-ecpgr.cgiar.org/fileadmin/... ·...

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