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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 locus15CH02C09
Liebhard et al (2002)Single locus11CH02D08
Liebhard et al (2002)Single locus2CH02C06
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
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
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 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
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
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. 1998CH02B10
+Gianfranceschi et al. 1998CH02B03
+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
+ but non informative
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
+ but non informative
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. 1998CH03C01
NTGianfranceschi et al. 1998CH02H11
NTGianfranceschi et al. 1998CH02G01
NTGianfranceschi et al. 1998CH02F06
NTGianfranceschi et al. 1998CH02D12
NTGianfranceschi et al. 1998CH02D08
NTGianfranceschi et al. 1998CH02C09
NTGianfranceschi et al. 1998CH02C06
NTGianfranceschi et al. 1998CH01H01
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
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