The document summarizes results from prebreeding activities across multiple organizations. It describes: 1) Evaluation and introduction of disease resistance traits into apple and peach through conventional breeding and pyramiding genes. 2) Use of early flowering transgenic lines to accelerate breeding, including lines with the transgene on different chromosomes and cultivars with an inducible promoter. 3) Introgression of fire blight resistance from Evereste and Malus x robusta 5 into apple. Molecular analysis identified major QTL conferring resistance.

1. WP2 – Establishment of prebreeding material
Results achieved since the beginning
of the project and plans for 2013

2. Reminder of the main objectives of the
WP
• Task T2.1: Evaluation and description of available
material
• Task T2.2: Conventional pre-breeding Introduction of
new traits in to apple and peach
• Task T2.3: Fast pre-breeding
 Main aim is to develop advanced
material with new traits for the breeders

3. Results achieved since the beginning
of the project
• Task 2.1: Description and evaluation of available plant material regarding
the traits powdery mildew, brown rot –peach, scab resistance (Rvi11,
Rvi12), fire blight resistance (Evereste, Mr5) material with pyramided
powdery mildew resistances (Pl1Pl2Pld, Pl1Pl2Plm)

4. Results achieved since the beginning
of the project
• Task 2.2:
– Introduction of traits
• Brown rot, powdery mildew resistance – peach
• Rvi11, Rvi12, Low chilling
– Combination of traits
• Resistance x resistance, resistance x quality
– Pyramiding of resistance genes for a trai
• Scab, powdery mildew

5. Crosses at CRA (peach)
Female parent Traits Male parent Traits Year
No. of
seedlings
CRA
IFF 1147 Resistance to mildew BO 92038173 resistance to brown rot 2011 41
IFF 1148 Resistance to mildew BO 92038173 resistance to brown rot 2011 36
Gialla tardiva tolerant to brown rot IFF 1148 resistance to mildew 2011 77
Percoca di
Romagna 7 tolerant to brown rot
Gilda Rossa
quality 2011
60
IFF 638 tolerant to brown rot IFF 1148 Resistance to mildew 2011 17
Ma 25-01-186 Self 2011 53
Ma 28-06-040 self 85
sum 369
IFF 1147 Resistance to mildew BO 92038173 resistance to brown rot 2012
IFF 1148 Resistance to mildew Contender 2012
IFF 1412 IFF 1148 Resistance to mildew 2012
IFF 1495 IFF 1148 Resistance to mildew 2012
Ma 28-06-040 self 2012
sum
sum total 369

6. Crosses at INRA (peach)
INRA
S7392 gg; yy, Dd; Del Cid (P. dulcis) PPV, green peach aphid 2011 10
S2605 Rm2Rm2; YY; dd; GG BC2-C224 powdery mildew res. QTL 2011 40
S2605 Rm2Rm2; YY; dd; GG BC2-F104 powdery mildew res. QTL 2011 20
S2605 Rm2Rm2; YY; dd; GG BC2-F149 powdery mildew res. QTL 2011 20
S2605 Rm2Rm2; YY; dd; GG BC2-F162 powdery mildew res. QTL 2011 10
Bolinha res. to Monilinia laxa Bolinha brown rot resistance 2011 140
S4577 peach leaf curl res. S8337 gg; Yy; bfbf 2011 15
S6699 Rm1rm1; yy; gg; dd S8572 S*s*; Dd; Gg; Yy; 2011 30
S6699 Rm1rm1; yy; gg; dd S7392 gg; yy; Dd 2011 30
S6699 Rm1rm1; yy; gg; dd S8104 gg; Yy; Dd 2011 10
S6699 Rm1rm1; yy; gg; dd S8363 gg ; Yy ; Dd 2011 52
S6699 Rm1rm1; yy; gg; dd S7135 gg ; Yy; Dd 2011 35
S6699 Rm1rm1; yy; gg; dd S8373 gg; yy; dd; maturity 2011 15
S6699 Rm1rm1; yy; gg; dd R6044 gg; Yy; bfbf; maturity 2011 6
S7392 gg; yy, Dd "pollens mix" … Rm1rm1; yy; gg; S*s* 2011 35
S9390 Vr2vr2; Yy; GG S6699 Vr1vr1; yy; gg; dd 2011 212
S9390 Vr2vr2; Yy; GG; Dd S8872 Yy; DD; ... ; maturity 2011 120
S9390 Vr2vr2; Yy; GG; Dd S8363 gg; Yy; Dd; maturity 2011 46
R4024 gg; Yy; bfbf; dd S9390 Vr2vr2; GG; Yy; Dd 2011 24
R6044 gg; Yy; bfbf S9390 Vr2vr2; GG; Yy; Dd 2011 62
sum 932
S7392 gg; yy, Dd Bolinha brown rot resistance 2012 30
(S7392 x Del Cid) green peach aphid, mildew (S7392 x Del Cid)1 green peach aphid, mildew 2012 30
Boc 79 bloody flesh nectarine R7 brown rot resistance; Rm1/rm1 2012 45
C203 brown rot, powdery mildew R7 brown rot resistance; Rm1/rm1 2012 20
C203 brown rot, powdery mildew Bolinha brown rot resistance 2012 30
S6699 Rm1rm1; yy; gg; dd Bolinha brown rot resistance 2012 30
S6699 Rm1rm1; yy; gg; dd F120 brown rot; powdery mildew QTL 2012 25
S6699 Rm1rm1; yy; gg; dd C203 brown robrown rot; powdery mildew QTL 2012 33
S2678 Rm2Rm2; YY; dd; GG BC2-C224 powdery mildew res. QTL 2012 15
S2678 Rm2Rm2; YY; dd; GG BC2-F104 powdery mildew res. QTL 2012 17
S2678 Rm2Rm2; YY; dd; GG BC2-F149 powdery mildew res. QTL 2012 13
S2678 Rm2Rm2; YY; dd; GG; BC2-F162 powdery mildew res. QTL 2012 20
sum 308
sum total 1240

7. Crosses at ACW
Female parent Traits Male parent Traits Year
Pollinated
flowers Seeds
No. of
seedlings
ACW
ACW16924
Rvi6, Rvi4, Pl2,
PlD ACW16102 Rvi6, Rvi2, Rvi4, Pl1 2011 274 258 258
ACW16956 Rvi6, Rvi2, Pl2 ACW16924 Rvi6, Rvi4, Pl2, PlD 2011 172 187 187
ACW17053 Rvi6, Pl2, AE, GE ACW16102 Rvi6, Rvi2, Rvi4, Pl1 2011 396 324 324
ACW18933 Pl2 Anna low chill 2011 274 2
ACW18921 Maribelle Quality, size, AEGE, Rvi2 2011 229 79
ACW18913 Maribelle Quality, size, AEGE, Rvi2 2011 112 225
sum 1457 1075 769
ACW 18933 Pl2 Maribelle Quality, size, AEGE, Rvi2 2012 574
ACW 18941 AE, GE Maribelle Quality, size, AEGE, Rvi2 2012 344
ACW 18954
Rvi12 (CH01f02),
Pl2 Anna low chill 2012 147
ACW 18971
Pl2, Pl1,
Anna low chill 2012 58
shelf live ( Md-
ACS)
sum 1123 0 0
sum total 2580 1075 769

8. Crosses at JKI
JKI
Ariwa Pl1, Rvi6 ACW 8259 Pl2 Pl2 2011 101 0
Ariwa Pl1, Rvi6 ACW 13597 Pld Pld 2011 73 0
E6 32,138 ACW8099 Pl2, Rvi6 2011 260 140 74
E6 33,126 ACW 13597 Pld 2011 394 40 27
E6 36,164 ACW 8259 Pl2 2011 357 157 78
Gala royal Quality A 722-7 Vbj Rvi11 2011 181 110 74
GD Quality A 722-7 Rvi11 2011 229 139 59
sum 1595 586 312
GD Quality ACW8476 Rvi11 2012 216 116 116
Gala Quality ACW8476 Rvi11 2012 80 120 120
GD Quality Anna low chilling 2012 245 233 50
Gala Quality Anna low chilling 2012 97 85 50
Braeburn Quality A 722-7 Rvi11 2012 245 88 88
Ariwa Pl1, Rvi6 ACW8259 Pl2 2012 19 52 52
Ariwa Pl1, Rvi6 ACW8099 Pl2, Rvi6 2012 18 71 71
GD Quality 06006/... Pl1, Pl2, Plmis 2012 1344 2488 400
GD Quality AA03 2,... Pl1, Pl2, Pld 2012 841 1995 300
06006/... Pl1, Pl2, Plmis E6 33,124 Rvi2, Rvi4, Rvi6, FbF7, Sd1 2012 218 475 475
sum 3323 5723 1722
sum total 4918 6309 2034

9. Results achieved since the beginning
of the project
• Task 2.3:
– Application and improvement of the fast breeding
approach
• Introduction of fire blight resistance in to apple
– Evereste
– Malus xrobusta 5
• Establishment of early flowering lines
– With the transgene on another LG
– Different cultivars with the early flowering gene
– Use of a inducible promoter and a different
flowering gene

10. Fb_E
Major QTL identified in the
ornamental apple Evereste on
the bottom of Linkage Group
(LG) 12 (R2 = 50-70 %).
Durel et al., 2009 – Genome
Parravicini et al., 2011 – Molecular Plant
Pathology
General objective: selecting apple pre-breeding genotypes carrying major
genes/quantitative trait loci (QTL) for disease resistance from wild apples,
with as less as possible „wild“ genome.
 Introgression of a highly efficacious fire blight (FB) resistance locus Fb_E from
Evereste.
Using T1190 at Agroscope ACW
Introduction of fire blight resistance from Evereste

11. X
X
X
X
X
EveresteT1190
F1
3 Fb_E + BpMADS4
Topaz, Diwa® and Maloni Sally ®
Royal Gala
GrannySmith
Braeburn2013
2010
2011
End 2008
beg. 2009
2012
BC‘1
9 Fb_E + BpMADS4
BC‘2
13 Fb_E + BpMADS4
BC‘3
? Fb_E + BpMADS4
BC‘4
? Fb_E without BpMADS4
2014
2018 first flowers of this pre-breeding material
2012/2013
111 seeds were
harvested and
stratified = 25 %
potential BC’3 plants
State of the art

12. Donor for fire blight resistance: Malus xrobusta 5
Idared Malus x robusta 5
M. x robusta 5

13. QTL for resistance to fire blight
QTL-Analysis
QTL explains up to 80% of
phenotypic variance
CH03E030.0
Fem181.5
PAGM48_777.9
CH03G0712.8
PAGM38_37416.2
P12M49_16018.1
HI03D0618.6
E33M32_30421.0
GD12_L123.3
E40M33_37624.9
PAGM42_16627.6
PAGM32_21728.6
PAGM38_35630.2
AU22365736.2
P12M40_27140.4
MS14H0346.6
PAGM32_22847.8
P14M49_28149.5
CH03G12B58.8
LG3

14. Introduction of fire blight resistance from Malus
×robusta 5
T1190 x (Idared x Mr5) BpMADS4 x FbR3 Pl1
Year Flowers
pollinated
Fruits
harvested
Seeds
planted
Plants
obtained
Plants with
BpMADS4,
FbR3, Pl1
2011 1026 77 363 130 37
(T1190 x (Idared x Mr5)) x GD (BpMADS4, FbR3, Pl1)
x Quality
2012 41 11 41 41

15. 15
Fast and dirty isolation of DNA

16. Multiplex DNA with BpMADS4, NPTII, EF

17. Early flowering lines with the transgene on other LGs
• 3 transgenic lines of the cv. ‘Pinova’ carrying the BPMADS4 on
different linkage groups (LG) than LG 4
Methods:
• Isolation of genomic regions flanking T-DNA insertions in the transgenic lines by genome
walking procedure
• Detection of additionally inserted vector backbone by PCR analysis
17
Line Genotype Linkage group Integrated vector backbone
1189 PN 8 None
1236 PN 16 None
1240 PN 14 Ca. 1700 bp after BL (tests outstanding)
Improvement of the fast breeding approach

18. Molecular genetic evaluation of propagated transgenic lines
• Verification of the detected genomic regions flanking T-DNA insertions as well as
the integration point in two transgenic lines by PCR analysis; re-sequencing
necessary for 1240
GM
GM
GM
GM
GM
GM
1189
1189
1189
1236
1236
1236
PN
NK
PN
NK
PN
NK
PN
NK
PN
NK
PN
NK
Detection of integration point with specific
primers binding to the T-DNA flanking contigs
of the apple genome.
Detection of T-DNA flanking genomic regions using specific
primers binding to the T-DNA and its flanking contigs of the
apple genome.
RB - right border, LB – left border.
RB site LB site RB site LB site
500 bp
1000 bp
1000 bp
3000 bp
6000 bp
Improvement of the fast breeding approach

19. Other cultivars carrying the early flowering gene
• Successful transformation with early flowering genes under control of 35S
promotor in different apple cultivars
• Generation of transgenic lines of different apple cultivars overexpressing
BpMADS4 under control of 35S
cv. ‘Pinova’:3, ‘Gala’: 5, ‘Santana’: 1, ‘Gala Mitchgla’: 1
• Transgenic lines flowered during in vitro cultivation
19
GA 35S BpMADS4 SAN 35S BpMADS4 PN 35S BpMADS4
vitro cultivation of transgenic apple lines.
Early flowering apple plants during in vitro
cultivation.
Improvement of the fast breeding approach

20. • Successful silencing of MdTFL1 under control of an inducible promotor in
different apple cultivars: Gala and Pinova
• Generation of currently 1 transgenic line of the apple cv. ‘Pinova’ silenced
in the expression of MdTLF1 under control of Gmhsp17.5-E (HSP)
20
intron T35S MdTFL1T35SGmhsp 17.5-E Gmhsp 17.5-ENpt II BRBL T35SUBQ10
Generation and selection of transgenic
regenerates after silencing MdTFL1.
In vitro cultivation of transgenic apple line of
the cv. ‘Pinova’.
Improvement of the fast breeding approach

21. Publications
Flachowsky H, Le Roux P-M, Peil A, Patocchi A, Richter K, Hanke M-V. (2011)
Applying a high-speed breeding technology to apple (Malus × domestica) based
on transgenic early flowering plants and marker-assisted selection. New
Phytologist 192, 364-377
Le Roux P-M, Flachowsky H, Hanke M-V, Gessler C, Patocchi A (2011) Use of a
transgenic early flowering approach in apple (Malus × domestica Borkh.) to
introgress fire blight resistance from ‘Evereste’. Molecular Breeding, in press.

22. What else?
• Fire blight inoculation tests
– Inoculation by injection of Erwinia amylovora strain CFBP 1430 (108 cfu/ml)
– Scoring of the lesion length in % of the shoot length 21 days after inoculation
High efficacy of the fire blight resistance locus Fb_E is confirmed in the
T1190 × Evereste background
• Background selection
– Estimation of the proportion of left Evereste genome with 79 SSR markers
distributed on all LGs
Assumption of Evereste chromosome eliminated in BC’1 is 78.5 %.
Genome coverage in BC’1 plants was on average 52 to 72 % and the
proportion of Evereste genome left ranged from 13.8 to 38.8 %.

23. Main challenges for 2013
• Identification of new traits to be introduced in
to apple and peach
• Selection of parents for the next crosses
(phenotyping for brown rot)
• Development of early flowering lines with TFL
under the control of a inducible
HeatShockPromoter (HSP)

24. Action Plan for 2013
WHAT WHO HOW DEADLINE
Introducing, combining, pyramiding
traits
ACW, CRA, INRA,
JKI
Crossing
Selection of interesting progenies ACW, CRA, INRA,
JKI
Genotyping,
phenotyping
Application of the fast breeding
approach (enhancing quality of
material resistant to fire blight)
ACW, JKI Crossing,
genotyping,
phenotyping
Establishment of transgenic lines
with TFL1 und HSP
JKI Transformation

25. Interactions between your WP and the
rest of the project
• Interactions planned with other WPs of the project:
– From your WP
• WP7: delivering description of donors to be integrated in to the
database
– To your WP
• WP5: reliable brown rot screening
• Interactions planned with other EU projects or
stakeholders of the project
– A day meeting with peach Italian stakeolders*:
Fruit exhibition and presentation
* To be extended to european?

26. Thanks for you attention