1. WP4: Exploring the phenotypic and genetic diversity in European
apple and peach germplasm collections and its exploitation by
association genetics approach
Results achieved since the beginning
of the project and plans for 2013
Maria José Aranzana + all WP4 partners

2. Main objectives of WP 4
 Improve the knowledge of genetic and phenotypic variability in
apple and peach European collections
 Identify genomic regions contributing to the genetic control of
major horticultural traits through genome-wide association
genetics (LD mapping)
 In combination with WP3, supply WP1 with tightly linked
molecular markers for implementation into the MAB pipeline

3. Results achieved since the beginning
of the project
 Description of phenotypic variability in apple
and peach germplasm collections
• Peach genetic variability, population
structure and LD analysis
• Apple genetic variability and population
structure

4. UK; 2237
FR; 1054
BE; 1314
CZ; 256
IT; 421 SWE;195
Selection of accessions in
germplasm collections
UK
310
FR
274
BE
296
CZ
253
IT
232
SWE
195
5,477 accessions in
germplasm collections
1,560
accessions with
phenotypic
observations
APPLE
Removing synonyms and triploids
IT-RO; 943
IT-MI; 575
FR; 764
ISP; 303
CH; 300
2,885 accessions in
germplasm collections
1,296 accessions
with phenotypic
observations
Removing synonyms and selecting
diversity
PEACH
IT-RO
178
IT-MI
140
FR
343
SP
353
CH
282

5. GROUP LABEL DESCRIPTOR PRIORITY
DI1Canker
Tree-twigs - Susceptibility to NECTRIA
CANKER (1-9)
Partial
DI1Mildew
Leave & twigs - Global Susceptibility to
POWDERY MILDEW (1-9)
Partial
DI1ScabFruit Fruit – Global Susceptibility to SCAB (1-9) Partial
DI1 = FUNGI
DISEASE
DI1ScabLeaf Leaf – Global Susceptibility to SCAB (1-9) Partial
FL1Class Flowering Period (1-9) Common
FL1Intensity Flowering Intensity (1-9) PartialFL1 = FLOWERING
PHENOLOGY
FL1Regularity
Relative regularity of flowering - Biennal
Habit (1-9)
Add
FR1OverColorAmount Fruit Global amount of over colour (1-9) Common
FR1Size Fruit Size (1-9) Common
FR1RibApex Fruit Crowning apex (1-9) Partial
FR1RussetAmount Fruit Global Amount russet (1-9) New
FR1Shape Fruit Basic Global shape (1-4) Common
FR1ShapeRatio Fruit Global shape (RATIO Heigt / Width) Partial
FR1 = FRUIT
CHARACTERISATION
FR1StalkLength Fruit Stalk Length (1-9) Partial
FR2Acid Fruit Sensory Acidity (1-9) Partial
FR2AcidSugarRatio Fruit Sensory Flesh Balance Sweet/acid New
FR2Bitter Fruit Sensory Flesh Bitterness (1#9) Partial
FR2Crunch Fruit Crispness (1-9) New
FR2Firm Fruit Sensory flesh Firmness (1-9) New
FR2Granular Fruit Sensory flesh Texture (1-9) Common
FR2Juice Fruit Sensory Juiciness (1-9) New
FR2Mealy Flesh mealiness Add
FR2Sugar Fruit Sensory Sweetness (1-9) Partial
FR2TasteGlobal Fruit – Sensory Global eating quality (1-9) Common
FR2 = FRUIT TASTE
FR2TasteMaturity
Assessment of the optimal ripening stage of
the fruits when picked and/or tasted
New
FR3PeriodKeeping Global Period of Fruit Keeping ability (1-9) PartialFR3 = FRUIT
KEEPING & PICKING
PERIODS
FR3PeriodPicking Fruit Harvest Maturity (1-9) New
FR4Size Fruit Size (Diameter mm) CommonFR4 = FRUIT
INSTRUMENTAL
CHARACTERISATION
FR4Weight Fruit Weight Average (g) Common
TR1Architecture Tree – Global Architecture PartialTR1 = TREE
CHARACTERISATION TR1BearingHabit Tree Type of fruiting (1-4) (Lespinasse) Partial
TR2 = FRUIT
CROPPING
TR2Prod1year Relative Fruit setting (1-9) – Year by year Add
Results: phenotypic variability in apple
and peach germplasm collections
0
5000
10000
15000
20000
25000
READING INRA CRA-W RBIPH UNIBO
Fruit & Tree
Period of flowering
Disease susceptibility
APPLE
• 1,264 accessions with observations
• 81,206 informative data entries in the
phenotypic database

6. Results: phenotypic variability in apple
and peach germplasm collections
APPLE
Data entries per partners and years

7. Results: phenotypic variability in apple
and peach germplasm collections
• 998 accessions with observations
• 37,818 informative data entries in the
phenotypic database
PEACH
CRA-Rome UMIL IRTA INRA ZJU
vigor x x - - x
tree habit x x - - x
gland type x x - - x
flower type x x - - x
male sterility - x - x x
beginning of flowering date x - x x x
beginning of flowering description - - - - x
full blossom date - x x x x
full blossom description - - x - x
flower density x x x x x
beginning of ripening date x x x - x
beginning of ripening description - - x - x
yield x x - - x
fruit size x x - x x
fruit size weight x - x x -
fruit pubescence x x x x x
fruit shape x x x x x
fruit flesh color x x x x x
fruit hue of overcolor x x - - x
fruit extent of overcolor x x - x x
sugar content brix - - x x x
sugar content taste data - x - - -
acidity - - x x -
acidity taste description - x - - x
acidity taste scale - x - - -
flesh firmness x x - x x
texture x x - - x
stony hard - x - - x
adherence of stone to flesh x x - x x
PRODUCTION
FRUIT QUALITY
VEGETATIVE TRAITS
FLOWER TRAITS
FRUIT MORPHOLOGY
FLOWERING
MATURITY
0
1000
2000
3000
4000
5000
6000
CRA-Rome UNIMI INRA IRTA ZJU
Vegetative traits
Flower Traits
Flowering period
Maturity
Production
Fruit Morphology
Fruit Quality
.

8. Results: phenotypic variability in apple
and peach germplasm collections
PEACH
Data entries per partner
0
500
1000
1500
2000
2500
3000
3500
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
Years with phenotypic observations
Numberofdataentries
CRA-Rome
UNIMI
INRA
IRTA
ZJU

9. • Analysis of the distribution of traits variability per collection
Results: phenotypic variability in apple
and peach germplasm collections
Days to full blossom Days to ripen
Sugar content Acidity

10. Results achieved since the beginning
of the project
 Description of phenotypic variability in apple
and peach germplasm collections
 Peach genetic variability, population
structure and LD analysis
• Apple genetic variability and population
structure

11. Results: peach variability, population
structure and LD analysis
1,296 peach
accessions
CRA-Rome
178
UNIMI
140
INRA
343
IRTA
353
ZJU
282
Genotyped with the 9K IPSC SNPs chip:
8,144 SNPs identified through the re-
sequencing of ca. 50 prunus
accessions
SNP genotyping
Good
4379
54%
Nullallele
242
3%
Lack of one
homozygous
827
10%
To check
20
0%
Monomorphic
778
10%
Failed
1898
23%

12. Results: peach variability, population
structure and LD analysis
- ~20% of accessions were clones (98% of identical genotype)
- Average observed heterozygosity 30% (Min 0.3% and Max 68%)
- Average inbreeding coefficient (F): 0.27 (first cousin)

13. Results: peach variability, population
structure and LD analysis
Most representative K is 3:
• Occidental breeding material (352 accessions)
• Chinese / oriental material (58 accessions)
• Occidental old / non breeding material (165 accessions)
• Admixed accessions [Q < 0.8] (665 accessions)

14. Results: peach variability, population
structure and LD analysis
Occidental breeding material Occidental old / non breeding material
Chinese / oriental material Admixed accessions [Q < 0.8]
LD 1Mbp

15. Results achieved since the beginning
of the project
 Description of phenotypic variability in apple
and peach germplasm collections
 Peach genetic variability, population
structure and LD analysis
 Apple genetic variability and population
structure

16. Results: apple genetic variability and
population structure
CH03g12z4.0
Hi02c0725.0
HB11AG38.0
CHVf156.0
CH05g0877.0
LG01
CH02f061.0
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CH02c0642.0
CH05e0351.0
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CH03g12z4.0
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CH03g12z4.0
Hi02c0725.0
HB11AG38.0
CHVf156.0
CH05g0877.0
LG01
CH02f061.0
CN49313924.0
CH02c0642.0
CH05e0351.0
CH03d0167.0
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CH02f061.0
CN49313924.0
CH02c0642.0
CH05e0351.0
CH03d0167.0
LG02
CH03e033.0
CH03g0724.0
Hi03d0643.0
GD1255.0
AU22365776.0
Ms14h0394.0
CH03g12y111.0
LG03
CH03e033.0
CH03g0724.0
Hi03d0643.0
GD1255.0
AU22365776.0
Ms14h0394.0
CH03g12y111.0
LG03
NZ05g080.0
CH04e022.0
Hi23g0218.0
CH05d0232.0
Hi07b0255.0
CH02c02b73.0
LG04
NZ05g080.0
CH04e022.0
Hi23g0218.0
CH05d0232.0
Hi07b0255.0
CH02c02b73.0
LG04
Hi09b045.0
Hi04a0820.0
CH03a0934.0
CH05e0645.0
Hi04d0269.0
CH05f0681.0
CH02a0890.0
CH04e03108.0
LG05
Hi09b045.0
Hi04a0820.0
CH03a0934.0
CH05e0645.0
Hi04d0269.0
CH05f0681.0
CH02a0890.0
CH04e03108.0
LG05
HB09TC9.0
CH03d0723.0
CH03d1240.0
CH05a0578.0
LG06
HB09TC9.0
CH03d0723.0
CH03d1240.0
CH05a0578.0
LG06
CN4447945.0
Hi03a1026.0
CH04e0535.0
Ms06c0954.0
Hi05b0974.0
LG07
CN4447945.0
Hi03a1026.0
CH04e0535.0
Ms06c0954.0
Hi05b0974.0
LG07
CH01c0618.0
CH01f0933.0
CH01h1052.0
Hi23g1262.0
LG08
CH01c0618.0
CH01f0933.0
CH01h1052.0
Hi23g1262.0
LG08
NH0029a12.0
CH01f03b24.0
Hi05e0742.0
CH01h0259.0
CN44454269.0
CH05d0873.0
LG09
NH0029a12.0
CH01f03b24.0
Hi05e0742.0
CH01h0259.0
CN44454269.0
CH05d0873.0
LG09
CH04c06z13.0
CH02b0714.0
CH02c1140.0
CH03d1150.0
CH04g09y64.0
CH02b03b81.0
Ms06g03104.0
LG10
CH04h020.0
Hi07d11x10.0
CH02d0831.0
CH04a1246.0
Hi16d0255.0
CH04g0773.0
LG11
CH05d0413.0
NZ28f0429.0
CH01g1245.0
CH04d0251.0
CH01f0255.0
CH03c0261.0
Hi07f0176.0
CH01d03z89.0
LG12
CH05h054.0
Hi04g0518.0
GD14740.0
NH009b70.0
LG13
CH05g07-10.0
CH01g0510.0
CH04c0719.0
U7894842.0
LG14
NZ02b018.0
CH03b0611.0
Hi03g0623.0
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CH02d1177.0
CH02c09110.0
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CH02a038.0
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CH04c06y0.0
AT00017412.0
CH01h0125.0
Hi03c0535.0
Hi02f1255.0
Hi05c0674.0
CH05d0891.0
Hi07h02100.0
LG17
CH04c06z13.0
CH02b0714.0
CH02c1140.0
CH03d1150.0
CH04g09y64.0
CH02b03b81.0
Ms06g03104.0
LG10
CH04c06z13.0
CH02b0714.0
CH02c1140.0
CH03d1150.0
CH04g09y64.0
CH02b03b81.0
Ms06g03104.0
LG10
CH04h020.0
Hi07d11x10.0
CH02d0831.0
CH04a1246.0
Hi16d0255.0
CH04g0773.0
LG11
CH04h020.0
Hi07d11x10.0
CH02d0831.0
CH04a1246.0
Hi16d0255.0
CH04g0773.0
LG11
CH05d0413.0
NZ28f0429.0
CH01g1245.0
CH04d0251.0
CH01f0255.0
CH03c0261.0
Hi07f0176.0
CH01d03z89.0
LG12
CH05d0413.0
NZ28f0429.0
CH01g1245.0
CH04d0251.0
CH01f0255.0
CH03c0261.0
Hi07f0176.0
CH01d03z89.0
LG12
CH05h054.0
Hi04g0518.0
GD14740.0
NH009b70.0
LG13
CH05h054.0
Hi04g0518.0
GD14740.0
NH009b70.0
LG13
CH05g07-10.0
CH01g0510.0
CH04c0719.0
U7894842.0
LG14
CH05g07-10.0
CH01g0510.0
CH04c0719.0
U7894842.0
LG14
NZ02b018.0
CH03b0611.0
Hi03g0623.0
CH01d0837.0
Hi04c0555.0
CH02d1177.0
CH02c09110.0
LG15
NZ02b018.0
CH03b0611.0
Hi03g0623.0
CH01d0837.0
Hi04c0555.0
CH02d1177.0
CH02c09110.0
LG15
CH02a038.0
CH05c0614.0
Hi04e0430.0
CH05a0447.0
CH04f1076.0
LG16
CH02a038.0
CH05c0614.0
Hi04e0430.0
CH05a0447.0
CH04f1076.0
LG16
CH04c06y0.0
AT00017412.0
CH01h0125.0
Hi03c0535.0
Hi02f1255.0
Hi05c0674.0
CH05d0891.0
Hi07h02100.0
LG17
In red : 8 common SSR
In purple : 4 additional SSR genotyped on the 1000
acc.
SSR Genotyping
SSR data already available on Collections :
UNIBO : 11 SSR
INRA : 24 SSR (29)
Reading/Brogdale : 12 SSR
SSR data newly acquired on Collections :
CRA-W : 12 SSR
RBIPH : 12 SSR
USL-Baslgard : 12 SSR
+
adjusting SSR allele sizes between
INRA/UNIBO/Reading
READING
310
INRA
274
CRA-W
296
RBIPH
253
UNIBO
232
Balsgard
195

17. Results: apple genetic variability and
population structure
Nb of
genotypes
Nb of
accessions
Belgium 190 209
CZ 120 132
France 225 225
Italy 167 184
Sweden 158 160
UK 271 271
2 countries 78 171
3 countries 20 72
4 countries 12 58
1241 1482
0
1
2
3
4
5
6
7
8
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100103106109
Duplicates - diploids Belgium Czech_Republic France Italy Sweden United_Kingdom
Diploids

18. Results: apple genetic variability and
population structure Diversity –
Geographic origin

19. Results: apple genetic variability and
population structure Diversity –
Partner’s collection

20. Position of WP3 parents in the overall diversity
WP3 parents cover well the most frequent variability
WP4 based on large genetic diversity while WP3 in breeding material

21. Population structure
K = 3
K = 2

22. Diversity – Tree distribution
(1230 genotypes)

23. Exemple of European Core Collection
(384 accessions)

24. Publications
• Meeting presentations
– Application of high throughput genotyping techniques in peach germplasm and breeding lines
within the FruitBreedomics project (Micheletti D., Arús P., Barreneche T., Bassi D., Dirlewanger E.,
Gao Z., Lambert P., Laurens F., Pascal T., Quilot B., Rossini L., Troggio M., Van de Weg E., Verde I. and
Aranzana MJ). Poster. PAG 2012
– Genetic variability in a wide germplasm of domesticated peach through high throughput
genotyping. Diego Micheletti, Valeria Aramini, Pere Arus, Elisa Banchi, Teresa Barreneche, Daniele
Bassi , Elisabeth Dirlewanger, Zhongshan Gao, Laura Gazza, Patrick Lambert, François Laurens,
Xiongwei, Thierry Pascal, Bénédicte Quilot-Turion, Laura Rossini, Michela Troggio, Ignazio Verde,
Maria Jose Aranzana. Oral presentation. PAG 2013
– Genetic variability description in a wide germplasm of Peach through High Throughput
Genotyping within Fruitbreedomics. Diego Micheletti,Valeria Aramini, Pere Arús, Elisa Banchi, Teresa
Barreneche,Daniele Bassi, Elisabeth Dirlewanger, Zhongshan Gao, Laura Gazza , Patrick Lambert,
François Laurens, Xiongwei Li, Thierry Pascal, Bénédicte Quilot-TurionLaura Rossini, Michela Troggio,
Ignazio Verde, Maria José Aranzana . Oral presentation. RGC6 2012

25. Publications plan for 2013
Genetic variability in a wide germplasm of
domesticated peach through high throughput
genotyping. (D.4.2).

26. Main challenges for 2013
Task 4.1.- Description of phenotypic variability
 Phenotypic data already in a web database
Search queries and statistical analysis tools in
the database to be defined (jointly with WP1 and
WP3 and done by WP7)

27. Main challenges for 2013
Task 4.2.- apple and peach core collections
peach cc set and analyzed with 9K SNPs
apple cc set and analyzed with SSRs
Apple SNP genotyping:
• LD decays fast in apple  thousands of NSPs needed
• Which genotyping method???
•SNP chip
•Genotyping by sequence

28. Main challenges for 2013: chose
genotyping strategy in apple
• Test of SNP genotyping, 48 old apple accessions
with Illumina SNP Chip
Good
10363
57%
Null allele
110
1%
Lack of one
homozygous;
low frequency;
missing data
5870
33%
Monomorphic
1491
8%
Failed
185
1%
 20K apple SNP Chip
 14 apple accessions re-sequenced
 2.6 million SNPs found  750 K valid
 18,019 SNPs selected (16.3K new and
3.7K from previous chip)
Genetic interval 1cM
LD decay at 55 Kb!!

29. Task 4.3.- Acquiring new phenotypic data
Collect second year of phenotypic data
Main challenges for 2013: chose
genotyping strategy in apple

30. Action Plan for 2013
WHAT WHO HOW DEADLINE

31. Deliverables and milestones for the
next 12 months
D4.2.- Database uploaded with already available phenotypic data (M24)
D4.3.- Submit a manuscript of: (M30)
• Allelic diversity, population structure and LD in peach
• Construction of apple CC
MS15.- SNP SSR data available in the database for apple cultivars (M18)
MS16.- Peach SNP data available in the database (M24)
MS17.- Apple SNP data available in the database (M33)
MS18.- One year of phenotypic and genome-wide data in the database (M36)

32. Interactions between your WP and the
rest of the project
• Interactions planned with other WPs of the project:
– From your WP:
• WP1: provide SNPs linked to monogenic traits
• WP3: share QTLs information for comparison
• WP7: genetic information needs to be included in the
database; search toolbox and statistics of phenotypic
information
– To your WP:
• WP3: share QTLs information for comparison
• WP7: release of

33. Agenda for WP4 session: EDIFICI
POLIVALENT 2.03 room
• Acquiring new phenotypic data (2013) (20´)
– Feedback of 2012 phenotyping
– Problems to solve?
• Apple core collection. Prospects for the paper (15´)
• Draft of variability and LD paper on peach (15´)
• Discussion on the best strategy for apple SNP genotyping
(30´)
• Action plan (10´)