1. Genetic structure of
European apple
germplasm Molecular markers as tools to manage
practical issues in germplasm collections
C.E. DUREL
INRA, Angers

2. Major questions when curating a
germplasm collection :
• Is my accession corresponding to the true genotype ?
(TTT = « True To Type »)
• Is my accession unique or redundant within my
collection or with other collections ?
• Are these 2 accessions related ?
• Are these 2 accessions genetically close or distant ?
• How representative of the genetic diversity is my
collection ?
• Is my collection structured in subgroups ?

3. Markers available in apple
(isozymes, RAPD, AFLP)
SSR :
(Simple Sequence Repeats)
> 660 published
http://www.hidras.unimi.it/
SNP :
(Single Nucleotide Polymorphism)
8k / 18k / … 420k
GBS :
(Genotyping by Sequencing)
(Elshire et al., 2011)

4. Fingerprinting
SSR
Cvrs
SSR1 SSR2 SSR3 SSR4 …
Cvr A 90-100 120-126 164-168 205-209
Cvr B 96-100 126-132 164-170 205-211
Cvr C 98-102 122-130 162-170 207-213
Cvr D 90-100 120-126 164-168 205-209
…
Genetic fingerprinting
SNP
vrs
SNP1 SNP2 SNP3 SNP4 …
vr A AC AG CC GT
vr B AA AG CT GG
vr C CC AA TT TT
vr D AC AG CC GT
The more markers, the better …
i.e., the more specific the fingerprint is.

5. Genetic distances
SSR
Cvrs
SSR1 SSR2 SSR3 SSR4
Cvr A 90-100 120-126 164-168 205-209
Cvr B 96-100 126-132 164-170 205-211
Cvr C 98-102 122-130 162-170 207-213
Cvr D 90-100 120-126 164-168 205-209
 Genetic distances (dissimilarities) according to common/distinct marker profiles
Cvrs
Cvrs
Cvr A Cvr B Cvr C Cvr D
Cvr A 0
Cvr B 0.5 0
Cvr C 1 0.87 0
Cvr D 0 0.5 1 0

6. Is my accession True To Type ?
SSR
Cvrs
SSR1 SSR2 SSR3 SSR4
Cvr A 90-100 120-126 164-168 205-209
Cvr B 96-100 126-132 164-170 205-211
Cvr C 98-102 122-130 162-170 207-213
Cvr D 90-100 120-126 164-168 205-209
A and D : Clones
(+/- mutations)
Same
fingerprints
dij = 0

7. Disctinctness
SSR
Cvrs
SSR1 SSR2 SSR3 SSR4
Cvr A 90-100 120-126 164-168 205-209
Cvr B 96-100 126-132 164-170 205-211
Cvr C 98-102 122-130 162-170 207-213
Cvr D 90-100 120-126 164-168 205-209
A (or B) and C ~ Unrelated
~ No
matching
alleles
dij ~ 0.9 - 1

8. Triploidy checking
N°
echantillon
Diploid /
Triploid
CH01f03b CH01h01 CH02c06 CH02d08 CH04e05 CH05f06 NZ05g08
0001
Bédange de
Nantes
Diploid 174/186 121/127 254/256 215/258 205/205 171/185 127/163
0002 Belle de fumée Triploid 141/174 119/123/127 232/242/254 215/229 178/213 179/189 127/127
0003
Belle fille du
Penthièvre
Triploid 141/162/186 119/121/127 - 229/258 178/205/213 179/187/189 133/149
0004 Blanc d'été Diploid 141/141 125/127 254/254 211/231 205/205 185/189 127/142
0005 Bercelien Triploid 162/174/186 125/137 232/254 215/258 178/213/219 171/181/187 127/129
0006 Boblin Diploid 141/174 105/137 232/262 217/260 178/219 177/189 121/121
0009 Charles pitrel Diploid 174/186 133/137 254/254 258/258 178/219 179/185 129/142
0010 Coaquin Triploid 141/174 125/133 234/238/252 215/229/258 178/205/219 171/183/185 144/151/163
0011 Cul d'oie Diploid 141/141 127/137 232/246 209/229 178/205 177/181 127/127
0012 Cul na Diploid 141/174 119/133 220/232 215/256 178/222 179/181 127/127
0013 Cyriac Diploid 141/186 137/137 232/232 215/229 178/219 181/187 127/127
…
Accessions with 3 alleles per marker  triploid apple varieties

9. Is my accession unique or redundant within my
collection or with other collections ?
0
1
2
3
4
5
6
7
8
1
6
11
16
21
26
31
36
41
46
51
56
61
66
71
76
81
86
91
96
101
106
Duplicates - Belgium
Belgium Czech_Republic France Italy Sweden United_Kingdom
unique 2n
190 genotypes
(209 accessions)
duplicate 2n
69 genotypes
(88 accessions)
unique 3n
59 genotypes
(79 accessions)
duplicate 3n
10 genotypes
(16 accessions)
Collection 1
(408 DNA samples received)

10. Are these 2 accessions related ?
Large data set
 Relatedness coefficient (ML-Relate software ; Kalinowski et al., 2006)
Parent-Offspring (0.5) / Full-sibs (0.25) / Half-sibs (0.125) / …
http://en.wikipedia.org/wiki/
File:Pedigree_marker_information.jpg

11. Parentage analysis
Parentage inference conditionally to the available marker data:
 Software CERVUS (Kalinowsky et al., 2005)
Known variety
Female parent ? Male parent ?
Correct marker allele inheritance ?

12. Parentage analysis
Rose de Berne Rose d’Ajoie Blaser
Pomme raisin Calville rouge d’hiver
Inference of unknown parents:
Full-Sibs:
Lassois et al. (in prep.)
Requirements:
- very large data set
- >= 20 SSR
Caution !:
- Are the accessions TTT ??
- The true cross could derive from mutants
of the identified parents
(2 Swiss cvrs)

13. Global relatedness
Putative relatedness tree in grape:
IBD computed on >5300 SNPs
(Myles et al., 2010)

14. Is my collection structured in
subgroups ?
Dessert - New
Dessert - Old
Cider
Lassois et al. (in prep.)
FRB-funded project

15. Is my collection structured in
subgroups ?
Wild apples
Domesticated apple

16. Is my collection structured in
subgroups ?
FruitBreedomics – WP4 :
2750 accessions fingerprinted
with 16 SSR

17. Is my collection structured in
subgroups ?
Faint structure in 3 European regions
North-East
West
South
(1550 diploid genotypes
with known geographic origin)

18. How representative of the genetic
diversity is my collection ?
Collection 1

19. Collection 2
How representative of the genetic
diversity is my collection ?

20. Can I get a subset of cvrs representative of
my (neutral) genetic diversity ?
0
20
40
60
80
100
0 100 200 300 400 500 600
N° of accessions of the core
Capturedallelicrichness(%)
97%
Lassois et al. (in prep.)
Core collection

21. Technical issue for SSR : ajusting allele sizes
 choice of genotypes covering almost all SSR alleles
96 accessions covering almost
all SSR alleles
 Facilitating the alignment of SSR alleles

22. Limits -
Marker versus Phenotype mutation
Phenotypic mutations :
Colour  Gala, Red Delicious
Architecture  McIntosh vs Wijick
Acidity  Usterapfel LA vs HA
Vigor  M9 rootstock
…
Genetic mutation
Regulation modification
Cell layers L1/L2/L3
Marker mutation rate = 10-4 for SSR
10-6 for SNP

23. SSR marker mutation
1 single
allelic difference
over 24 markers
Allelic mutation
Marker CH03d12
Des moissons
Pomme Livre
Belle Louronnaise
Demie sûre

24. Markers can help germplasm curators
• Quality insurance :
- Grafting/labelling error
- Erroneous pomological identification
• Information about duplicates within and between collections (but not mutants !)
• Possible inferences of parentage (when enough markers and large data set)
 looking backwards how selection has been done empirically
by farmers and gardeners
• Representativeness/specificity of the collection
 To be combined with phenotypic data
• Optimal choice for core collection
• Genome-Wide Association Studies

25. Technical and financial issues
Multiplexing Analysis Cost Information
SSR : Low Long Low High (multi-allelic)
SNP : High Short High Low / marker
8k ~ 50 € but numerous SNP
18k ~ 65 €
420k ~ 165 €
GBS : High Long Low Missing data
(bioinformatic) (imputation)
 Major goal : ~500-1000 SNP at 5-10 € ???

26. Thank you
• M. Lateur, P. Houben (CRA-W)
• S. Tartarini, L. Dondini (UNIBO)
• F. Paprstein, J. Sedlak (RBIPH)
• M. Ordidge (Reading Univ.)
• F. Fernandez, K.M. Evans (EMR)
• H. Nybom, L. Garkava-Gustavsson (SLU)
• C. Miranda, J. Urrestarazu (Un. Navarra)
• J. Gassmann (Agroscope)
• K. Antonius (MTT)
• I. Suprun (SKZNIISIV, Krasnodar)
• A. Pikunova (VNIISPK, Orel)
• C. Denancé, E. Ravon, L. Feugey, A. Guyader, R. Guisnel, L. Lassois (INRA)

27. Thank you for your attention!