1. Concepts of Apple Rootstock Breeding and Selection: A Journey Through the Development of New Apple Rootstocks G. Fazio, H. Aldwinckle, T. Robinson

2. Introduction Breeding work for the Geneva rootstocks was initiated by Drs. Cummins and Aldwinckle in 1967. The USDA/Cornell program is actively breeding and selecting new rootstocks (about 2,500 in the pipeline) Dr. Aldwinckle and Dr. Robinson represent Cornell University in the program. The program, has always focused on developing yield efficient, disease resistant rootstocks (fire blight, etc). It is now focusing on characterization of other important traits such as replant disease resistance, drought tolerance, cold tolerance, etc. 3. Apple Harvest Doud family farm (1916, Miami Co. Indiana) 4. Auvil Fruit Farm (Vantage, WA 2005 next to Columbia River) 5. Benefits from the implementation of dwarfing rootstocksLess sprays Less ladder accidents Increased productivity 6. Improving Rootstocks for Superior Tree Performance Fruit Color and Quality Fruit Size Disease ResistancePlant Architecture Dwarfing Molecular mapping and selection tools GenomicsYield and productivity (Nutrition) Precocity Abiotic Stress Resistance (Cold) Disease Resistance Fire blight ($40M 2000 epidemic, MI) Replant disease complexTRANSGENIC ROOTSTOCKS for a plethora of traits 7. Active Apple Rootstock Breeding Programs 1970s and 80s QuebecMallingSweden Norway RussiaVineland Poland JapanMichigan Geneva GermanyCzech 8. Active Apple Rootstock Breeding Programs 2005Russia S. Korea Japan ChinaGeneva New Zealand 9. New and Experimental Apple Rootstocks in the U.S. PolishCzhecMallingRussiaVinelandQuebecJapanGermanyGenevaP.14JTE-BAR-86-1-20Bud 57-195V.1SJP84-5218JM1Supp. 1G.11P.22JTE-CAR-86-1-25Bud 60-160V.2SJP84-5217JM2Supp. 2G.16JTE-DAR-295-6Bud 61-31V.3SJP84-5198JM3Supp. 3G.41AR-931-15Bud 62-396V.4SJP84-5162JM7Supp. 4G.65AR-440-1Bud 64-194V.7SJP84-5231MarubakaidoPiAu 56-83G.935AR-680-2Bud 65-838SJP84-5174G.30AR-486-1Bud 67-5(32)SJP84-5189CG.2001AR-628-2Bud 70-8-8SJP84-5180CG.2003AR-69-7Bud 70-20-21CG.2006AR-360-19Bud 71-3-150CG.2022M.20Bud 71-7-22CG.2034 CG.2406CG.3142CG.3736CG.3902CG.4001CG.4002CG.4003CG.4004CG.4005CG.3001CG.4011CG.4013CG.4018CG.4019CG.4021CG.4038CG.4049CG.4088CG.3007CG.4094CG.4113CG.4172CG.4210CG.4213CG.4214CG.4247CG.4288CG.3029 10. Geneva Rootstock Selection Traits TRAITFire Blight resistance Phytopthora resistanceReplant Disease Complex Wholly apple aphid res.EVALUATION YEARS 1 or 7 1 1 or 7 1LOCATION Greenhouse/Field Greenhouse Greenhouse/field GreenhouseJuvenility - Spines3-4Field/StoolbedStoolbed rooting3-4Field/StoolbedGrowth habit - Brittleness3-4Field/StoolbedDwarfing8-12OrchardPrecocity8OrchardSuckering8OrchardYield Biennial bearing12OrchardCold hardiness Drought tolerance Graft union compatibility15Orchard4Orchard5Orchard 11. Insects and diseases of apple rootstocks Fire blight (Erwinia amylovora) Crown rot, root rot (Phytophthora spp.) Woolly Apple Aphid (Eriosoma lanigerum) Southern Blight (Sclerotium rolfsii) White root rot (Rosellinia necatrix) Texas root rot (Phymatotrichum omnivora) 12. Apple Rootstock Breeding: Resources and Activities Orchard Production Lab & Greenhouse -Seedling inoculations -Molecular markers -Tissue culture -Plant pathology -ETC.-Individual yield and growth -Disease incidence -Scion compatibility -ETC.Nursery -Liner production -Tree Production -Stoolbed evaluation -Transplant Evaluation -ETC.Apple Rootstock Breeding is a very resource intensive endeavor. 13. Apple Rootstock Breeding and Selection Protocols Stage 1Stage 2Stage 3Stage 4Stage 5Stage 6Stage 7Stage 8Stage 9Stage 10Years 1-2Objective 1.1Years 3-4Years 5-6Years 7-12Years 10-15Years 16-18Years 19-21Years 22-24Years 25-27Years 27-301. Select elite parentsCritical Juncture: Molecular information about allelic constitution at disease resistance loci and about other important traits can be used to bypass first test orchard.2. Generate F1 populations 3. Stratify and plant populations 4. MAS for dwarfing, precocity, disease resistance, etc. 5. Disease screening 6. Plant selected stoolsRepeated inoculations with Fireblight, Wooly Apply Aphid, Phytophtora, Powdery Mildew and comparison to known standards7. Stool selection 8. Propagation and grafting 9. First test orchard 10. First test evaluation and selection 11. Elite stoolbed establishment 12. Stress tolerance tests - drought, cold hardiness, graft union strength 13. Elite liner and tree production 14. Elite stoolbed selection and distribution to nurseries 15. Intermediate replicated orchard 16. Intermediate orchard evaluation 17. Commercial stoolbed evaluation 18. NC-140 and cooperator trials (national and international) 19. Commercial production ramp up Patent and UPOV protection 20. Comercial sale Contingent on goodness of MAS Highly replicated first test orchard at multiple sites Test orchard Evaluation and Selection Elite stoolbed establishment Stress tolerance tests - drought, cold hardiness, graft union strength Elite stoolbed selection and distribution to nurseries Commercial stoolbed evaluation and distribution of trees to trial sites NC140 and cooperator trials (national and internationa) Commercial production ramp up Commercial saleFeedback from Horticultural Traits evaluations, disease resistance evaluations, stress tolerance evaluationswill be combined with molecular data and used in subsequent cycles of selection. Elite material from these evaluations are used as parents in subsequent cycles.These Stress Tolerance Tests willl have to be highly replicated at each location - therefore only a few selections at a time will be tested. Grower cooperators in cold or drought prone areas in the US will be selected for cold hardiness and drought stress. For graft union compatibility and strength a set of rootstocks will be grafted with multiple scions and graft unions tested after 3 years. 14. Breeding and Selection of Apple Rootstocks - Simplified 10,000Number of genotypes observedNumber of replications per genotypeNumber of plants1000Transgenic value-added traits100 10 1 12345 6 7 8 Time (3 year stages)910 B. Johnson 15. Criteria for Parent Selection Phenotype and Molecular Markers Dwarfing Precocity Disease Resistance Fire Blight AD13 SCAR scab marker (Boudichevskaia et al. 2006) Phytopthora Powdery Mildew Apple ScabYield and Field Performance New Gene Pools EM M01 SCAR powdery mildew marker (Evans et al. 2003) 16. New Gene Pools at the Plant Genetic Resources Unit (PGRU) Geneva, New York Malus - Apple - 3995 accessions 2430 clones (grafted) and 1565 seedlots from wild2808 wild Malus seedlings from 310 populations from Kazakhstan, Russia, China & Turkey 17. Malus sieversii from Kazakhstan 1989 - 1996Site 9 w/ depletion by grazing animals Excellent apple-scab resistance from some sitesSite 5Site 6 18. Gene Pool Identification Combining SSR, SCAR Markers MM.111 PiAu5683M.2 MM.106M.13 P.14 M.1 M.7P.18PiAu5111 J-TE-C V.1 M.27 M.11 G.65 M.10 M? M.30.81G.41 0.14G.202J.9 Pi80 M.9T337 B.118 PiAu514 M.9 M.26 J-TE-G M.8 M.20M.25 V.7 V.2G.11 G.30 G3007 G.935 G5179R.5 III IIIAlnarp M.4O.3B.9P.1 B.491 JM.10-0.52 -0.52 1.00 0.47 0.47 -1.19II -0.06G.16 Novole Maruba NAGA-0.59 -1.11 -1.86 -1.86 -1.03 -0.59 -0.15I0.29 0.29G3007 G.935 O.3 G5179 G.202 G.30 G.11 B.9 M.8 M.20 J.9 JM.10 J-TE-G Pi80 M.9 M.9T337 V.2 M? P.1 P.14 G.16 V.1 G.41 M.27 G.65 PiAu5111MW M.10 M.13 M.26 Alnarp PiAu514 M.1 M.2 M.4 M.11 M.3 PiAu5683 M.7 PiAu5111 P.18 R.5 V.7 J-TE-C M.25 MM.106 MM.111 B.491 B.118 Maruba NAGA Novole 0.000.720.210.42Coefficient0.630.83 19. Crossing Parents Stage 1 20. Crossing Parents Stage 1 21. Seed Harvest Stage 1 2,000-10,000 seeds per cross 22. Disease Screens Stage 1 3,000 to 10,000 seedlings/year 23. Disease Screens Stage 1 3,000 to 10,000 seedlings/year 24. Fire blight - Erwinia amylovora Major disease for apple rootstocks in North America Bacterial disease with strain differentiation Resistance sources available Rootstock infection routes: suckers injuries systemic movement of bacteria from scion 25. Fire Blight Screening Stage 1 500 to 2,000 seedlings 26. Integration of Marker Assisted Selection Stage 2 High throughput PCR markers SCARs, SSRs Target traits: Dwarfing Powdery mildew resistance Scab resistance Wooly apple aphid resistanceUse published and in house markers 27. Propagation and Evaluation of Layering Stool-Bed Properties 28. Harvest of Rootstock Liners Evaluation of Rooting 29. Rootstock Liners in Tree Nursery for Budding/Grafting 30. First Test Orchard Stage 3 3-10 replicates per rootstock genotype 50-100 different genotype selections every year All grafted with same scion Evaluated for 8-12 years 31. Early field selection of precocious genotypes Stage 4 32. Expansion of Layering Beds to Increase Replications Stage 4 33. Evaluation of Layering Stool Beds Stage 5 34. Rootstock Liner Evaluation Stage 5 35. Second Test for Resistance to Biotic Stresses Stage 5 Inoculation with Wooly Apple Aphid (Eriosoma lanigerum)Water Logging test with Phytophthora InoculationFire Blight Inoculations with Multiple Strains 36. Replicated Orchard Trials in Multiple Locations Stage 6 Precocity Yield Fruit Size Dwarfing Tree Survival Disease Incidence Tree Architecture Burr Knots 37. Cumulative Yield Efficiency Measurements Descriptive Statistics M.9Variable: CUM-YEFF Anderson-Darling Normality Test A-Squared: P-Value:0.52.03.55.017.106 0.0008.095% Confidence Interval for Mu2.59040 1.19212 1.42114 1.75248 3.70960 317Minimum 1st Quartile Median 3rd Quartile Maximum6.5Mean StDev Variance Skewness Kurtosis N0.24800 1.87200 2.20500 2.91550 8.1850095% Confidence Interval for Mu 2.45867 2.082.182.282.382.482.582.682.782.7221495% Confidence Interval for Sigma 1.105981.2929295% Confidence Interval for Median95% Confidence Interval for Median2.138062.34268 38. Replicated Orchard Trials in Multiple Locations Stage 6 Descriptive Statistics Variable: Suckers Anderson-Darling Normality Test A-Squared: P-Value:10407010013016095% Confidence Interval for MuMean StDev Variance Skewness Kurtosis N21.1430 29.4424 866.858 2.55072 8.75868 317Minimum 1st Quartile Median 3rd Quartile Maximum19027.087 0.0000.000 2.000 9.500 29.166 204.00095% Confidence Interval for Mu 17.889 5152524.39795% Confidence Interval for Sigma 27.31531.93295% Confidence Interval for Median95% Confidence Interval for Median6.71913.403 39. Timing of fire blight screens Number of genotypes observedNumber of replications per genotype 9Rootstock blight orchard trialsNumber of plants1Transgene mediated resistanceStrain specific direct inoculation 5 screens 12 Time 40. Screening for Resistance to Fire Blight (E. amylovora)FIELD INOCULATIONS ON FINISHED TREESGREENHOUSE INOCULATIONS ON LINERS 41. Commercial Stool Bed Trials Stage 7 On site trials of elite rootstocks at commercial nursery locations Evaluate liner productivity and quality under commercial conditions Generates nursery stock for major orchard trials (NC-140, large grower trials) 42. Program ProtocolTrials with NC-140 Cooperators Stage 8 43. Drought Tolerance TestsWork of Dr. PARRA 44. Graft Union Strength TestsPictures Courtesy of Mike Parker (NC State University) 45. On Farm and Nursery Trials in Several U.S. Locations Stage 9 Large scale trials planted in WA, PA, MI, NY Trials include 20-45 different genotypes 46. Nursery Tree Measurements on 10-15 Trees per Rootstocks Branch LengthTree HeightBranch Angle 0=Flat 90=UprightTotal Number of Branches Branch Height 47. Branch Angles of Brookfield Gala Trees on Several Dwarfing Rootstocks for 7 W hirlsBranch Angle (0=Flat 90=Upright)60 2034 2406 304150 404210 4213 4214 4814 5935 B9 G1130 20 10 0 1234 Whirl (3 branches)567G16 G? M9 48. Stages of Micro-Propagation Prior to Release Stage 10 49. Commercial Release and Continued Testing Stage 10 Program has released 6 new rootstock genotypes to date. G.16 and G.30 G.202, G.41, G.935, G.11 are commercially available in U.S. Release decision for six more elite rootstock genotypes expected in 2008. 50. Large Scale Production of Rootstock Liners 51. Production of High Quality Nursery Trees and Adoption By GrowersNursery trees on Geneva 202 rootstocks and planted in high density orchard. 52. QTL Mapping of Apple Rootstock Yield & Disease Resistance TraitsF20.0 1.5 2.6 3.7 4.8 7.7 12.0CH02f06 E32M41-0208 E31M41-0089 G15-1250 CH02c02a E35M41-3500 E35M37-031815.4 17.0E35M41-0193 E35M32-027222.5 23.5E35M42-0282 E33M36-01921.20Sieversii596280 Sieversii596283 Orientalis594101 PI594103 Sieversii596282 6589 Pi-Au56-83 NAGA Kansuenesis59409 AR486-1 Maruba M8 Asiatica594009 WijcikMacP18 Novole AR295-6 ReinetteSim Seiboldii594094 B9 YellowTrans J9 AR628-2 P1 Fusca589975 Pi-Au51-4 M26 Fusca589941EsopusSpitzenJM7 5046 NSpy Pi-Au51-11 J-TE-G B118 J-TE-CM27CrimsonBeauty P14 Tschonoskii58939 GoldenDel M7 JM4 V1 MM111 Supp4 Orientalis590015 M9 P22 NAKB337 B491 MM106 V2 G65 G16 Toringoides58939 Transitoria58942 6006G30 Transitoria58938 7707 5257179 4213 5030 54214 4013 4210 5935 6143 Ioensis590015 6210 50126874 5757 5087 3041 6969 6879 5890 Angustifolia5897 4814 G11 6253 3007 GH589882 4202 0.85 II 0.210.55III -0.1044.8 46.4 47.4 48.1 48.7 49.5 50.3 52.3 53.5 63.3 65.7 66.8 67.7 75.3AD04-450 CH02c06 AA12-2200 E35M35-0178 E35M32-0460 E36M41-0590 CH03d10 E35M41-1000 E35M35-1500 E32M41-0650 CH02b10 C10-750 CH02a04y K13-350 E37M32-0113 E33M37-0172 E33M36-0194 CH05e03 E37M36-0195 CH03d01 NZ03c01z P14-750 E32M42-0340 F05-1500-0.75-1.05-1.69 -1.40 -1.53-0.42Coronaria589976 Nertchinsk R5-0.87I -0.21 0.45 1.11Dr. Wan Yizhen Construct a molecular map of Apple Rootstock using microsatellites, SNP, SCAR. Map and develop markers for plant architecture and disease resistance traits. Develop basic knowledge on Chinese apomictic species for seed propagated rootstocks. Transgenic approaches for improving rootstock performance. 53. Research Work on Apple Rootstocks Requires Many Collaborators and Institutions Cornell University: T. Robinson (Orchard Systems) I. Merwin (Horticulture - Replant) H. Aldwinckle (Plant Pathology) L. Cheng (Physiology) S. Brown (Scion Breeding)Michigan State University: R. Perry (Rootstocks) S. VanNocker (Genomics)Washington State University: B. Barrit (Scion Breeding) D. Main (BioInformatics)USDA ARS PGRU: A. Baldo (BioInformatics) P. Forsline (Apple Collection)USDA ARS AFRS Kearneysville: J. Norelli (Transgenics) C. Bassett (Stress Physiology)USDA ARS Wenatchee: M. Mazzola (Plant Pathology) Y. Zhu (Genomics)PENN State University: T. McNellis (Genomics) J. Schupp (Horticulture)Over 40 scientists as NC-140 collaborators Washington Tree Fruit Research Commission 54. BASIC SCIENCE Genomics, Proteomics Gene Discovery, Expression PhysiologyAPPLIED SCIENCE Plant Breeding Genetic Transformation VERY APPLIED SCIENCE Horticultural Trait Evaluation Widespread Field Performance Field Recommendations NC-140INDUSTRY, GROWERS, PROCESSORS, CONSUMERS 55. Genomic Revolution We know that M.7 rootstock is less precocious than M.9. Do we know why? We know that M.9 dwarfs more than M.26? Do we know why? Through Genomics much is being discovered about how rootstocks do all that they do. NSF funded project that aims to discover what genes are turned on and off in the apple scion by different rootstocks. (Dr. McNellis, Penn State)Tree architecture modified by apple rootstocks. Wealth of new genetic material 56. The Geneva Apple Rootstock Breeding ProgramTHANK YOUTodd Holleran, Sarah Bauer, Yizhen Wan Funding from IDFTA, WTFRC, USDA, Cornell 57. The Road Ahead (2003 NC-140 Mtgs. Door County, Wisconsin)