1. Pilot studies on Marker
Assisted Breeding: Apple
Markus Kellerhals, Lucie Leumann, Simone Schütz, Isabelle Baumgartner, Andrea Patocchi,
Agroscope
YOUR LOGO

2. Apple breeding at Agroscope: selection
steps
seedlings
Crosses (MAPS)
10 000
2
years
1
Selection in glasshouse (MASS) and
container seedling nursery (MASS)
Grafting on M27 with interstem
4000
4-8
Step 1: 1 tree on M27
600
8-12
Stage A: 3 trees on M9
30
10-17
Stage B: 4 x 4 trees on M9
4
14-20
Stage C: 50 trees, 1 row, on M9
2
3
600
Commercialisation: 1 variety out
of 30 000 to 50 000 seeds after 14 to 20 years
YOUR LOGO

3. Marker application in apple breeding
Fruitbreedomics
WP1: Increasing efficiency of (marker-assisted)
breeding (MAB) of new cultivars
 Pilot studies to check the practical application of
MAB: Agroscope (CH) and University Bologna (I)
 Use of newly developed SNP (Single Nucleotide
Polymorphism) markers instead of SSR (Simple
Sequence Repaeat) markers

4. Early selection towards pyramided disease
resistances and fruit quality
Disease resistance
Scab (V. inaequalis)
 Major resistance genes: Rvi6 (Vf), Rvi2 (Vh2)
Mildew (P. leucotricha)
 Major resistance gene: Pl2
Fruit quality
 Crispness, texture, acidity
 Shelf life, storage (ethylene)

5. Pilot Studies: Crosses at Agroscope
Parents were analysed with 20k SNP chip at Wageningen UR
Mother
Father
Flowers Fruits
Seeds
ACW 11303
(Rvi6=Vf)
ACW 18522
(Rvi6=Vf, Rvi2=Vh2)
2246
352
2227
ACW 13652
(Rvi6=Vf, Pl2)
ACW 11567
(Rvi2=Vh2)
1370
323
2793

6. Steps
 Breeding strategy developed and crosses made (2011)
 New information on false positive reactions with some molecular markers
(SSR marker present but gene not) led to adaptation of the strategy
 Sowing and screening for scab resistance (phenotypic), spring 2013,
susceptible plants removed (except in subpopulations)
 Molecular selection with SNP’s (June 2013)
 Potting of selected plants for container field (July 2013)
 Phenotypic selection of the potted plants (October 2013)
 Grafting for selection field and for second scab screening in the glasshouse
(second screening in 2015)

7. Crosses and glasshouse screening

9. Sampling for molecular analysis
- No lyophilisation necessary
- Deep well block, put the leaf rondelle,
a plastic film and silica gel

10. Markers used
Trait / Locus
Marker
Scab resistance Rvi6=Vf (LG1)
Rvi6_42M10SP6_Y124 (SNP)
Scab resistance Rvi2=Vh2 (LG2) Rvi2_region53_M417 (SNP)
Mildew resistance Pl2 (LG11)
Pl2_3_Y211 (SNP)
Fruit texture Md-PG1 (LG10)
PG_FEM_LC_19
Acidity (LG16)
Acidity_SNP2 (ss475876558; SNP2 from RosBREED)
Crispness (LG16)
Crispness_SNP1 (ss475881704; SNP1 from RosBREED)
Shelf life Md-ACO1 (LG10)
ACO_FEM_cg_4
Shelf life Md-ACS1 (LG15)
ACS_FEM_cg_5

11. MAB experiences
• Plates of 6 x 4 pots allowed plant identification
without labelling
• Missing plants were replaced by living plants to fill
up gaps in the plates (cost), laborious
• Puncturing the leaves and expedition of the plates
was efficient
• Parents had to be checked first for the
polymorphism of the planned SNP markers
• The whole procedure was more time-consuming
than expected
• Costs are relatively low, DNA extraction is the most
expensive, data point to low price
• Close interaction with the company
KASP - SNP Genotyping
(LGC genomics) was required and successful

12. Analyses with LGC Genomics (UK)
• Analysis of 2500 progeny plants (1250 from
each progeny) financed by Fruitbreedomics
• Analysis of remaining progeny plants financed
by Agroscope (useful backup pool)

13. Pilot studies ‘Fruitbreedomics’
Individual plant
Data sheet with
the results of molecular
analysis
Rvi6_42M10SP6_Y124
T = Vf resistance
Cross, plate position
MasterPlate MasterWell Call
1206-5
H06
C:T
1206-5
D09
C:T
1206-5
G09
C:T
1206-5
F10
C:T
1206-5
G10
C:T
1206-5
C12
C:T
1206-6
B01
C:T
1206-6
H01
C:T
1206-6
F02
C:T
1206-6
G02
C:T
1206-6
A03
C:T
1206-6
H04
C:T
1206-6
E05
C:T
1206-6
G05
C:T
1206-6
H05
C:T
Pl2_3_Y211
T = PL2 resistance
Marker
SNPID
MasterPlate MasterWell Call
Rvi6_42M10SP6_Y124 1206-5
H06
C:T
Rvi6_42M10SP6_Y124 1206-5
D09
C:T
Rvi6_42M10SP6_Y124 1206-5
G09
C:T
Rvi6_42M10SP6_Y124 1206-5
F10
C:T
Rvi6_42M10SP6_Y124 1206-5
G10
C:T
Rvi6_42M10SP6_Y124 1206-5
C12
C:T
Rvi6_42M10SP6_Y124 1206-6
B01
C:T
Rvi6_42M10SP6_Y124 1206-6
H01
C:T
Rvi6_42M10SP6_Y124 1206-6
F02
C:T
Rvi6_42M10SP6_Y124 1206-6
G02
C:T
Rvi6_42M10SP6_Y124 1206-6
A03
C:T
Rvi6_42M10SP6_Y124 1206-6
H04
C:T
Rvi6_42M10SP6_Y124 1206-6
E05
C:T
Rvi6_42M10SP6_Y124 1206-6
G05
C:T
Rvi6_42M10SP6_Y124 1206-6
H05
C:T
SNPID
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211
Pl2_3_Y211

14. Strategy and Reality cross 1
cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2): keep 50 random plants, keep 50
scab susceptible plants (class 4), keep all plants with two or more resistances according to
molecular analyses (take out of the resistant part 50 without fruit quality-markers)
Segregation expected and observed (based on the marker analysis)
Rvi6Rvi6Rvi2
observed
% observed
expected from
1250
% expected
Selected for
grafting
Rvi6Rvi6
Rvi6Rvi2
Rvi6
Rvi2
no resistance
total
67
235
298
258
164
75
1097
6.1 %
21.4 %
27.2 %
23.5 %
15.0 %
6.8 %
100 %
156
156
312
312
156
156
1250
12.5 %
12.5 %
25.0 %
25.0 %
12.5 %
12.5 %
100 %
31
5
93
8
10
29
176

15. Strategy and Reality cross 2
cross 2: ACW 13652 (Rvi6, Pl2) x ACW 11567 (Rvi2): keep 50 random plants,
keep 50 scab susceptible plants, keep all plants with three resistances according to
molecular analyses
Rvi2Rvi6Pl2
observed
observed %
expected
from 1250
% expected
Selected
for grafting
Rvi2
Rvi6
Rvi6
Pl2
Rvi2
Pl2
Pl2
Rvi6
Rvi2
no
total
resistance
147
66
1061
13.8 %
6.2 %
133
12.5 %
148
13.9 %
162
15.2 %
150
14.2 %
71
6.7 %
184
17.3 %
156
156
156
156
156
156
156
156
1250
12.50%
47
12.50%
2
12.50%
5
12.50%
6
12.50%
7
12.50%
3
12.50%
11
12.50%
19
100%
100

16. Envisaged further selection
Jan 2014
Feb 2014 or 2015
Winter/Spring 2015
Jun/July 15
Jun/July 16
grafting on rootstock M 27 with intermediate
‘Schneiderapfel’
planting to level 1
second glasshouse inoculation with different scab
strain
first assessment for fruit quality (about 30-40%
fruiting) and scoring of tree characteristics
first major assessment for fruit quality (about
80% fruiting), tree scoring, first comparison of
fruit quality and marker results for fruit quality,
comparison of tree characteristics and marker
set, powdery mildew in cross 2

17. Plant identification

18. Conclusions
 MAB is a useful tool to increase efficiency in fruit
breeding
 Careful parent selection and molecular
characterization is important to avoid
misinterpretation
 Check with the phenotype
 Close interaction with the company which analyses
the samples