WP2 – Establishment of prebreeding material

Results achieved since the beginning of the project and plans for 2013

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

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)

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

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

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

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

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

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

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

X

X

X

X

X

Evereste T1190

F1 3 Fb_E + BpMADS4

Topaz, Diwa® and Maloni Sally ®

Royal Gala

GrannySmith

Braeburn 2013

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

Donor for fire blight resistance: Malus xrobusta 5

Idared Malus x robusta 5

M. x robusta 5

QTL for resistance to fire blight

QTL-Analysis

QTL explains up to 80% of phenotypic variance

CH03E03 0.0 Fem18 1.5

PAGM48_77 7.9

CH03G07 12.8

PAGM38_374 16.2 P12M49_160 18.1 HI03D06 18.6 E33M32_304 21.0 GD12_L1 23.3 E40M33_376 24.9 PAGM42_166 27.6 PAGM32_217 28.6 PAGM38_356 30.2

AU223657 36.2

P12M40_271 40.4

MS14H03 46.6 PAGM32_228 47.8 P14M49_281 49.5

CH03G12B 58.8

LG3

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

Fast and dirty isolation of DNA

Multiplex DNA with BpMADS4, NPTII, EF

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

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

11

89

11

89

11

89

12

36

12

36

12

36

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

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

• 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 MdTFL1 T35S Gmhsp 17.5-E Gmhsp 17.5-E Npt II BR BL T35S UBQ10

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

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.

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 %.

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)

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

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?

Thanks for you attention