This document discusses the use of marker-assisted selection (MAS) in the apple breeding program at Agroscope Changins-Wädenswil Research Station (ACW) in Switzerland. The goals of the ACW breeding program are to develop apple varieties with resistance to multiple diseases like apple scab, powdery mildew and fire blight, as well as high fruit quality. MAS is used to select seedlings with desired disease resistance genes and phenotypes at various stages of the breeding process, from initial crosses to field trials, to increase selection efficiency. Marker analyses are outsourced to different laboratories. The costs of MAS per seedling or tree are estimated to range from 1 euro for initial scab screening to

1. Federal Department of Economic Affairs FDEA
Agroscope Changins-Wädenswil Research Station ACW
The use of markers in the Wädenswil
apple breeding programme
M. Kellerhals, L. Leumann, I.O Baumgartner, J.E. Frey
and A. Patocchi
Agroscope Changins-Wädenswil, Switzerland

2. Outline
 Marker-assisted breeding (MAB)
 Breeding targets
 Selection process
 Methods
 Results
 Cost calculation
 Conclusions and outlook
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M. Kellerhals

3. Marker-assisted breeding (MAB)
 Selection of parents (MAPS)
 Markers mainly developed and used for disease
resistance selection (apple scab, powdery mildew, fire
blight)
 MAB used with specific seedling progenies (MASS)
 Marker analyses applied at different stages in the
selection process
Aim: increase selection efficiency
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4. ACW apple breeding targets
 Disease resistance
A) combined resistances B) pyramided resistances
against different pathogens against one pathogen
 Fire blight (E. amylovora)
 Apple scab (V. inaequalis)
 Powdery mildew (P. leucotricha)
C) homozygous resistance genes
 High fruit quality
 good tree features (yield, etc.)
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5. Selection process at ACW
1 Crosses (MAPS) 10 000
Nb of seedlings
2+3 Selection in glasshouse (MASS) and 4000
seedling nursery (MASS)
3 Grafting on M27 with interstem 600
years
5-8 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
Commercialisation: 1 variety out of 30 000 to 50 000
seeds after 14 to 20 years. Cost: 2‘000‘000.- €/variety
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6. Seedlings labeling and leaf sampling
for marker analyses
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7. Plant identification
1
2
database
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8. MAB with SSR and SCAR markers
• DNA extraction according to Frey et al. 2004
• PCR with primers for apple scab, powdery mildew and fire
blight according to Patocchi et al. 2009
• Fragment analysis on a 3130xl Genetic Analyzer and
data analysis with GeneMapperTM Software (Applied
Biosystems)
1 2 3
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9. Lab locations and costs
Different labs for analyses:
- ACW (Jürg Frey)
- ETH Zürich (Giovanni Broggini)
- Ecogenics GmbH (www.ecogenics.ch)
Costs:
- Depend on the number of markers and the assembly of the
multiplexes
- For one multiplex with 4-5 SSR-markers costs are
around 10 € per sample
Number of analyses per year:
1‘000 to 3‘000 samples
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10. Sources of resistance against scab
and powdery mildew
Scab (V. inaequalis)
 Major resistance genes: Rvi6 (Vf), Rvi2 (Vh2), Rvi4 (Vh4),
Rvi12 (Vb), Rvi11 (Vbj), etc.
 Partial resistance: VA, ‚traditional‘ and modern
varieties with partial resistance
Mildew (P. leucotricha)
 Major resistance genes: Pl1, Pl2, PlD, etc.
 Partial resistance
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11. Mapped sources of resistance against
fire blight (E. amylovora) in apple
 ‘Fiesta’ FBF7 QTL: 35-40% of phenotypic
variation explained (Calenge et al. 2005, Kahn et al. 2006, 2007)
 ‘Robusta 5’: 67-83% of phenotypic variation explained (Peil et al.
2007, 2008)
 ‘Evereste’: FB_E: 50-70% of phenotypic variation explained (Durel et
al. 2009)
 ‘Florina’: QTL on LG10, 15% of phenotypic variation explained
(Le Roux et al. 2010)
Fruitbreedomics AnnualUniversität Prague, February 7th, 2012
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Isabelle Baumgartner

12. Markers used
Resistance Marker
Rvi6 CHVf1 (SSR)
Rvi2 CH02B10 (SSR)
Rvi4 CH02C02a (SSR)
Pl1 AT20-450 (SCAR)
Pl2 CH04H02 (SSR), N18 (SCAR)
FBF7 AE 10_375 (SCAR), GE-8019 (SCAR)
Use of SSR‘s is most efficient and infomative at the moment
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13. Examples of progenies examined
Crosses from 2009 with MAPS
a. ACW 12143 (Rvi6, Pl1) (Florina (Rvi6) x Ariwa (Rvi6, Pl1)) x
ACW 17358 (Rvi6, Pl2) (ACW 8244 (Rvi6, Pl2) x ACW
11907 (Rvi6) (= Mairac x H23-10))
b. ACW 13434 (Rvi6, Pl2) (ACW 6375 (-) x ACW 8881 (Rvi6,
Pl2)) x ACW 17053 (Rvi6, Pl2, FBF7) (ACW 8099 (Rvi6,
Pl2) x Enterprise (Rvi6, FBF7))
c. ACW 12382 (Rvi6, Rvi4, Pl2, FBF7) (Elise x ACW 8099
(Rvi6, Pl2)) x Enterprise (Rvi6, FBF7)
 Germination and growing in glasshouse
 Marker analysis (Rvi6, Rvi4, Pl1, Pl2, FBF7)
 Scab screening in glasshouse
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14. Results of MASS
Progeny c. ACW 12382 (Rvi6, Rvi4, Pl2, FBF7) (Elise x ACW 8099
(Rvi6, Pl2)) x Enterprise (Rvi6, FBF7)
Rvi6
Rvi6 Rvi6 Rvi4 Pl2 FBF7 Rvi6Rvi6, Rvi4, Pl2, FBF7
N = 286 69 154 160 142 215 13
Observed 24% 54% 56% 50% 75% 4.5%
 Combined resistance against apple scab (Rvi6, Rvi4), powdery mildew
(Pl2) and fire blight (FBF7)
 Pyramided scab resistance (Rvi6 and Rvi4)
 Homozygous scab resistance (Rvi6Rvi6)
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15. MASS of 2250 samples in 2010
Cross Mother (Resistances) Father (Resistances) Number of Plants
0901 ACW 6707 (-) Evereste (Rvi6, Fb_E) 543( after Pre-Selection)
0902 ACW 13434 (Rvi6, Pl2) ACW 17053 (Rvi6, Pl2, FBF7) 69
0903 ACW 13434 (Rvi6, Pl2) ACW 17038 (Rvi6, FBF7) 89
0904 ACW 12143 (Rvi6, Pl1) ACW 17358 (Rvi6, Pl2) 152
0905 ACW 12382 (Rvi4, Rvi6, Pl2, FBF7) Enterprise (Rvi6, FBF7) 309
0906 ACW 12382 (Rvi4, Rvi6, Pl2, FBF7) E10-34-100 (Rvi6) 250
0908 ACW 11907 (Rvi6) KAZ 96 02-01P-11 (FBF7) 234
0909 SIR KEP 93-1 (-) ACW 16960 (Rvi2, Rvi6, Pl2, FBF7) 202 (after Pre-Selection)
0916 ACW 11567 (Rvi2, Rvi4) E-10-34-90 (Rvi6) 346
0820 Enterprise (Rvi6, FBF7) ACW 13434 (Rvi6, Pl2) 44 (after Pre-Selection)
0824 ACW 11582 (Rvi2) ACW 12382 (Rvi4, Rvi6, Pl2, FBF7) 66 (after Pre-Selection)
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16. Results of MASS in 2010
Crosses Number – Outcrosses Expected Genotype Observed Expected
= Total
0901 543 – 23 = 520 Rvi6, Fb_E 126 (24%) - (preselection)
0902 69 – 2 = 67 Rvi6Rvi6, Pl2Pl2, FBF7 4 (6%) 6%
0903 89 – 1 = 88 Rvi6Rvi6, Pl2, FBF7 7 (8%) 6%
0904 152 – 1 = 151 Rvi6Rvi6, Pl1, Pl2 5 (3%) 6%
0905 309 – 23 = 286 Rvi4, Rvi6Rvi6, Pl2, 13 (5%) 2%
FBF7FBF7
0906 250 – 18 = 232 Rvi6Rvi6, Rvi4, Pl2, FBF7 6 (3%) 2%
0908 234 – 4 = 230 Rvi6, FBF7 75 (33%) 25%
0909 202 – 3 = 199 Rvi2, Rvi6, Pl2, FBF7 13 (7%) - (preselection)
0916 346 – 21 = 325 Rvi2, Rvi4, Rvi6 34 (10%) 13%
0820 44 – 1 = 43 Rvi6Rvi6, Pl2, FBF7 10 (23%) - (preselction)
Total 296 (13%)
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17. Cost calculation
Progeny of 2000 seedlings Cost estimation per
seedling/tree/ha
Scab screening 2‘000 € (all) 1€
MASS (1 multiplex) 20‘000 € (all) 10 €
Container trees 20 € x 300 plants = 6‘000 € 20 €
Tree on M27 with 15 € x 200 trees = 3‘000 € 15 €
interstem
Maintenance of 200 x 0.5 m x 3.5 m = 350 m2 8‘000 € per ha and
orchard step 1 = 280 € year
Total for 5 years in 32‘400 €
step 1
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18. Example: Fire blight inoculation test in
ACW 16436 x FlxNEG 3524 progeny plants
100
90
Can molecular analysis
80
70
substitute phenotypic testing?
60
50
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19. MAB allows to focus on outstanding
selections
Future with MASS: select only a few elites
and test them with a higher number of trees
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20. Conclusions and Outlook
 MAB allows more efficient selection
 Progeny plants with pyramided disease resistance identified
with molecular markers
 New markers (SNP’s, Fruitbreedomics)
 More efficient high-throughput methods (Fruitbreedomics)
 MAB for additional traits (fruit quality, yield, etc.)
 Find the most efficient combination of phenotypic and
molecular selection
 Discussion: will MAB allow to develop more varieties, better
varieties (larger progenies to start with) or reduce the number
of breeding programmes?
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21. Acknowlegements
Team Fruit-Growing and Horticulture ACW
Team Cesare Gessler, ETH
Projects ZUEFOS (Züchtung feuerbrandtoleranter Obstsorten) and
BEVOG (Beschreibung von Obstgenressourcen)
BLW, SOV, VariCom
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22. ACW apple varieties
Milwa (Diwa®, Junami®) La Flamboyante (Mairac®)
Galiwa Ladina
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