03 wp2 progresses&results-20130221
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  • 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