2015 International FruitBreedomics Conference

1. YOUR LOGO
Pilot studies for efficient
early phenotypic and molecular
selection for disease resistance and
fruit quality traits in apple
Markus Kellerhals, Isabelle Baumgartner, Lucie Leumann, Simone Schütz
Andrea Patocchi, Agroscope

2. 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
Repeat) markers
Marker assisted apple breeding

3. 1 variety with commercial potential out
of 30’000 to 50’000 seeds after 14 to 20 years
years
Stage C: 50 trees, 1 row, on M9
Stage B: 4 x 4 trees on M9
Stage A: 4 trees on M9
Step 1: 1 tree on M27
Grafting on M27 with interstem
Selection in glasshouse (MASS) and
container seedling nursery (MASS)
Crosses (MAPS)
14-20
10-17
8-12
4-8
3
2
1
2
4
30
600
600
4000
10 000
seedlings
Pilot studies integrated in the selections steps of
the Agroscope apple breeding program

4. Mother (Resistances) Father (Resistances) 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
Pilot Studies: Crosses at Agroscope (2011)
Progeny 1
Progeny 2

5. 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)
Early selection towards pyramided disease
resistances and fruit quality

6. 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 (SNP)
Shelf life Md-ACO1 (LG10) ACO_FEM_cg_4 (SNP)
Shelf life Md-ACS1 (LG15) ACS_FEM_cg_5 (SNP)
Crispness (LG16) Crispness_SNP1 (ss475881704; SNP1 from RosBREED)
Acidity (LG16) Acidity_SNP2 (ss475876558; SNP2 from RosBREED)
Markers used

7.  Breeding/selection strategy developed and two crosses made (2011)
 New information on false positive reactions with some molecular markers (SSR marker
present but gene not) in the parents led to adaptation of the strategy
 Sowing and screening for phenotypic scab resistance (spring 2013), susceptible plants
removed (except in subpopulations)
 Molecular analysis with SNP’s (June 2013)
 Potting of selected plants based on resistance for container plot (July 2013)
 Phenotypic selection (juvenility, mildew) of the potted plants also considering breeding
strategy and the molecular analysis (October 2013)
 Grafting one tree per genotype for fruit selection in the orchard and one tree per genotype
for the test of the scab resistance pyramid (inoculation with Rvi6-virulent strain in the
glasshouse in 2015)
 Planting to orchard (spring 2015)
Steps

8. Crosses and glasshouse scab screening

9. Progeny 1 Progeny 2

10.  Polymorphism of the SNP alleles checked in the parents
 No lyophilisation necessary
 Deep well block, put the leaf rondelle, a plastic film and silica gel
 Shipment to LGC Genomics for analysis
Sampling leaf material for molecular analysis
(SNP’s)

11. Rvi6_42M10SP6_Y124 Pl2_3_Y211
Cross, plate position T = Vf resistance Marker T = PL2 resistance
MasterPlate MasterWell Call SNPID MasterPlate MasterWell Call SNPID
1206-5 H06 C:T Rvi6_42M10SP6_Y124 1206-5 H06 C:T Pl2_3_Y211
1206-5 D09 C:T Rvi6_42M10SP6_Y124 1206-5 D09 C:T Pl2_3_Y211
1206-5 G09 C:T Rvi6_42M10SP6_Y124 1206-5 G09 C:T Pl2_3_Y211
1206-5 F10 C:T Rvi6_42M10SP6_Y124 1206-5 F10 C:T Pl2_3_Y211
1206-5 G10 C:T Rvi6_42M10SP6_Y124 1206-5 G10 C:T Pl2_3_Y211
1206-5 C12 C:T Rvi6_42M10SP6_Y124 1206-5 C12 C:T Pl2_3_Y211
1206-6 B01 C:T Rvi6_42M10SP6_Y124 1206-6 B01 C:T Pl2_3_Y211
1206-6 H01 C:T Rvi6_42M10SP6_Y124 1206-6 H01 C:T Pl2_3_Y211
1206-6 F02 C:T Rvi6_42M10SP6_Y124 1206-6 F02 C:T Pl2_3_Y211
1206-6 G02 C:T Rvi6_42M10SP6_Y124 1206-6 G02 C:T Pl2_3_Y211
1206-6 A03 C:T Rvi6_42M10SP6_Y124 1206-6 A03 C:T Pl2_3_Y211
1206-6 H04 C:T Rvi6_42M10SP6_Y124 1206-6 H04 C:T Pl2_3_Y211
1206-6 E05 C:T Rvi6_42M10SP6_Y124 1206-6 E05 C:T Pl2_3_Y211
1206-6 G05 C:T Rvi6_42M10SP6_Y124 1206-6 G05 C:T Pl2_3_Y211
1206-6 H05 C:T Rvi6_42M10SP6_Y124 1206-6 H05 C:T Pl2_3_Y211
Data sheet with
the results of molecular
analysis
Individual plant
Seedling selection

12. Rvi2Rvi6Rvi6 Rvi2Rvi6 Rvi2 Rvi6Rvi6 Rvi6 no resistance total
Observed (N) 67 298 164 235 258 75 1097
Observed (%) 6.1 % 27.2 % 15.0 % 21.4 % 23.5 % 6.8 % 100 %
Expected from 1250 (N) 156 312 156 156 312 156 1250
Expected (%) 12.5 % 25.0 % 12.5 % 12.5 % 25.0 % 12.5 % 100 %
Selected for grafting (N) 31 93 10 5 8 29 176
cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2)
 keep 50 random plants
 keep 50 scab susceptible plants (phenotyping class 4)
 keep all plants with two resistances according to molecular analyses
(subset: out of the resistant part 50 with undesired fruit quality-marker combinations)
Segregation expected and observed (based on the molecular analysis)
Strategy and Reality cross 1

13. 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
Strategy and Reality cross 2
Segregation expected and observed (based on the molecular analysis)
Rvi2Rvi6Pl2 Rvi2Rvi6 Rvi2Pl2 Rvi6Pl2 Rvi2 Rvi6 Pl2 no resistance total
Observed (N) 133 148 150 162 147 184 71 66 1061
Observed (%) 12.5 % 13.9 % 14.2 % 15.2 % 13.8 % 17.3 % 6.7 % 6.2 %
Expected from
1250 (N)
156 156 156 156 156 156 156 156 1250
Expected (%) 12.50% 12.50% 12.50% 12.50% 12.50% 12.50% 12.50% 12.50% 100%
Selected for
grafting (N)
47 2 6 5 11 3 7 19 100

14. PG_FEM_LC_19 Crispness_SNP1 Acidity_SNP2
avoid T T:T less crisp than T:C,
C:C best
A:A more acid than C:C,
A:C intermediate
ACW 11303 C:C T:C intermediate A:C intermediate
ACW 18522 T:C C:C best A:A more acid
Prefered
genotypes (%)
in progeny
C:C (46%) C:C best (52%) A:C intermediate (48%)
Segregation of fruit quality markers
PG_FEM_LC_19 ACO_FEM_cg_4 ACS_FEMcg_5 Crispness_SNP1 Acidity_SNP2
avoid T -> not
possible
G = good A = good,
A:A best
T:T less crisp than T:C,
C:C best
A:A more acid than C:C,
A:C intermediate
ACW13652 T:C A:G good A:A best T:T less crisp A:C intermediate
ACW11567 T:T A:A A:G good T:C intermediate A:C intermediate
Prefered
genotypes (%)
in progeny
T:C (50%) A:G good (51%) A:A best (44%) T:C intermediate (54%)A:C intermediate (50%)
cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2)
cross 2: ACW 13652 (Rvi6, Pl2) x ACW 11567 (Rvi2)

15.  (Four) 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 (LGC
genomics) was required and successful KASP - SNP Genotyping
MAB experiences

16. Container plot
From 5020 seeds 350 selections
planted as grafted trees to
selection step 1 orchard (spring
2015)

17.  2 series (1205: March 2015, 1206: April 2015)
 Inoculum: Rvi6 (Vf)-virulent strain, Isolate D42, spray
concentration: 1.35 x 105 sp/ml (series 1), 2.2 x 10
105 sp/ml (series 2) in security glasshouse level 2
 germination rate 24h: 68% (series 1), 75% (series 2)
Germinated conidia
Stellate necrosis: typical for
Rvi2 scab resistancePlants with tent (humidity > 90%)
Test of the scab resistance pyramid (spring 2015)

18. Scab class /
symptom
0 1 2 3a 3b
Stellate
necrosis
Stellate
chlorosis
0 - 3b 4
Scab res. gene
combination
Number of plants
(percent of total plants)
Rvi2, Rvi6 28 86% 0% 14% 0% 0% 7% 4% 100% 0%
Rvi2 9 44% 0% 56% 0% 0% 33% 11% 100% 0%
Rvi6 11 0% 0% 9% 0% 0% 9% 0% 9% 91%
none 14 0% 0% 7% 0% 0% 0% 7% 7% 93%
Scab class /
symptom
0 1 2 3a 3b
Stellate
necrosis
Stellate
chlorosis
0 - 3b 4
Scab res. gene
combination
Number of plants
(percent of total plants)
Rvi2, Rvi6 26 58% 0% 42% 0% 0% 42% 0% 100% 0%
Rvi2 12 25% 0% 67% 8% 0% 75% 8% 100% 0%
Rvi6 4 0% 0% 0% 0% 0% 0% 0% 0% 100%
none 16 0% 0% 0% 0% 0% 0% 0% 0% 100%
Results of scab inoculation with Rvi6-virulent strain
cross 1: ACW 11303 (Rvi6) x ACW 18522 (Rvi6, Rvi2)
cross 2: ACW 13652 (Rvi6, Pl2) x ACW 11567 (Rvi2)

19. autumn 2016
 first assessment for fruit quality
(about 30-40% fruiting)
 scoring of tree characteristics
autumn 2017
 major assessment for fruit quality
(about 80% fruiting)
 scoring of tree characteristics
 comparison of phenotypic fruit quality and
marker results for fruit quality traits
 cross 2: comparison of phenotypic powdery
mildew resistance with Pl2 marker results
Further steps

20.  Plant plate to extraction plate logistics and plant identification
throughout the whole process needs further improvement
 MAB for disease resistance loci is more advanced than for fruit
quality traits
 Careful parent selection and molecular characterization (check for
false positive and polymorphism of the markers) is important to
avoid misinterpretation
 Check with the phenotype (scab pyramid, fruit quality, etc.)
 Close interaction with the company which analyses the samples
 The defined strategy could not be 100% realised
 MAB is a useful tool to increase efficiency in fruit breeding
Conclusions

21. Acknowledgements
The team Financial support
 Fruit-growing team Agroscope
 Cameron Peace, WSU
 FEM
 WUR for the scab isolate