A Journey Through the Development of New Apple Rootstocks G

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Concepts of Apple Rootstock Breeding and Selection: A Journey Through the Development of New Apple Rootstocks G. Fazio, H. Aldwinckle, T. Robinson Introduction z Breeding work for the Geneva® rootstocks was initiated by Drs. Cummins and Aldwinckle in 1967. z 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. z The program, has always focused on developing yield efficient, disease resistant rootstocks (fire blight, etc). z It is now focusing on characterization of other important traits such as replant disease resistance, drought tolerance, cold tolerance, etc. Apple Harvest Doud family farm (1916, Miami Co. Indiana) Auvil Fruit Farm (Vantage, WA 2005 – next to Columbia River) Benefits from the implementation of dwarfing rootstocks

Less sprays

Less ladder accidents

Increased productivity Improving Rootstocks for Superior Tree Performance

z Fruit Color and Quality z Fruit Size z Disease Resistance

z Plant Architecture – Dwarfing z Molecular mapping and selection tools z Genomics z Yield and productivity (Nutrition) z Precocity z Abiotic Stress Resistance (Cold) z Disease Resistance z Fire blight ($40M 2000 epidemic, MI) z Replant disease complex z TRANSGENIC ROOTSTOCKS for a plethora of traits Active Apple Rootstock Breeding Programs 1970s and 80s

Quebec Malling Sweden Norway Russia Vineland

Poland Michigan Japan Geneva Germany Czech Active Apple Rootstock Breeding Programs 2005

Russia S. Korea

Japan

Geneva China New Zealand New and Experimental Apple Rootstocks in the U.S.

Polish Czhec Malling Russia Vineland Quebec Japan Germany Geneva

P.14 JTE-B AR-86-1-20 Bud 57-195 V.1 SJP84-5218 JM1 Supp. 1 G.11

P.22 JTE-C AR-86-1-25 Bud 60-160 V.2 SJP84-5217 JM2 Supp. 2 G.16

JTE-D AR-295-6 Bud 61-31 V.3 SJP84-5198 JM3 Supp. 3 G.41

AR-931-15 Bud 62-396 V.4 SJP84-5162 JM7 Supp. 4 G.65

AR-440-1 Bud 64-194 V.7 SJP84-5231 Marubakaido PiAu 56-83 G.935

AR-680-2 Bud 65-838 SJP84-5174 G.30

AR-486-1 Bud 67-5(32) SJP84-5189 CG.2001

AR-628-2 Bud 70-8-8 SJP84-5180 CG.2003

AR-69-7 Bud 70-20-21 CG.2006

AR-360-19 Bud 71-3-150 CG.2022

M.20 Bud 71-7-22 CG.2034

CG.2406

CG.3142 CG.3736 CG.3902 CG.4001 CG.4002 CG.4003 CG.4004 CG.4005 CG.3001

CG.4011 CG.4013 CG.4018 CG.4019 CG.4021 CG.4038 CG.4049 CG.4088 CG.3007

CG.4094 CG.4113 CG.4172 CG.4210 CG.4213 CG.4214 CG.4247 CG.4288 CG.3029 Geneva Rootstock Selection Traits

TRAIT EVALUATION YEARS LOCATION Fire Blight resistance 1 or 7 Greenhouse/Field Phytopthora resistance 1 Greenhouse Replant Disease Complex 1 or 7 Greenhouse/field Wholly apple aphid res. 1 Greenhouse Juvenility - Spines 3-4 Field/Stoolbed Stoolbed rooting 3-4 Field/Stoolbed Growth habit - Brittleness 3-4 Field/Stoolbed Dwarfing 8-12 Orchard Precocity 8 Orchard Suckering 8 Orchard Yield – Biennial bearing 12 Orchard Cold hardiness 15 Orchard Drought tolerance 4 Orchard Graft union compatibility 5 Orchard Insects and diseases of apple rootstocks z Fire blight (Erwinia amylovora) z Crown rot, root rot (Phytophthora spp.) z Woolly Apple Aphid (Eriosoma lanigerum) z Southern Blight (Sclerotium rolfsii) z White root rot (Rosellinia necatrix) z Texas root rot (Phymatotrichum omnivora) Apple Rootstock Breeding: Resources and Activities

Orchard Production -Individual yield and Lab & Greenhouse growth -Seedling inoculations -Disease incidence -Molecular markers -Scion compatibility -Tissue culture -ETC. -Plant pathology -ETC. Nursery -Liner production -Tree Production -Stoolbed evaluation -Transplant Evaluation -ETC.

Apple Rootstock Breeding is a very resource intensive endeavor. Apple Rootstock Breeding and Selection Protocols Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 Stage 7 Stage 8 Stage 9 Stage 10 Objective 1.1 Years 1-2 Years 3-4 Years 5-6 Years 7-12 Years 10-15 Years 16-18 Years 19-21 Years 22-24 Years 25-27 Years 27-30

1. Select elite parents Critical Juncture: Molecular 2. Generate F1 populations information about allelic constitution at disease resistance 3. Stratify and plant populations loci and about other important traits can be used to bypass first 4. MAS for dwarfing, precocity, test orchard. disease resistance, etc. These Stress Tolerance Tests willl have to be highly replicated 5. Disease screening at each location - therefore only a few selections at a time will 6. Plant selected stools Repeated inoculations with be tested. Grower cooperators Fireblight, Wooly Apply Aphid, in cold or drought prone areas 7. Stool selection Phytophtora, Powdery Mildew and in the US will be selected for comparison to known standards 8. Propagation and grafting cold hardiness and drought stress. For graft union 9. First test orchard compatibility and strength a set of rootstocks will be grafted 10. First test evaluation and selection with multiple scions and graft unions tested after 3 years. 11. Elite stoolbed establishment Feedback from 12. Stress tolerance tests - drought, Horticultural Traits cold hardiness, graft union strength evaluations, disease resistance evaluations, 13. Elite liner and tree production stress tolerance 14. Elite stoolbed selection and evaluationswill be distribution to nurseries combined with molecular 15. Intermediate replicated orchard data and used in subsequent cycles of 16. Intermediate orchard evaluation selection. Elite material from these evaluations 17. Commercial stoolbed evaluation are used as parents in subsequent cycles. 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 sale Breeding and Selection of Apple Rootstocks - Simplified

Number of Number of 10,000 Transgenic genotypes replications “value-added” observed per genotype 1000 traits

a l 100

e b 10

N 1

152 34 678910 Time (3 year stages) B. Johnson Criteria for Parent Selection – Phenotype and Molecular Markers z Dwarfing z Precocity z Disease Resistance z Fire Blight z Phytopthora AD13 SCAR scab marker (Boudichevskaia et al. 2006) z Powdery Mildew z Apple Scab z Yield and Field Performance z “New” Gene Pools

EM M01 SCAR powdery mildew marker (Evans et al. 2003) New Gene Pools at the Plant Genetic Resources Unit (PGRU) Geneva, New York

Malus - Apple - 3995 accessions 2430 clones (grafted) and 1565 seedlots from wild

2808 wild Malus seedlings from 310 populations from Kazakhstan, Russia, China & Turkey Malus sieversii from Kazakhstan 1989 - 1996

Site 9 w/ depletion by grazing animals Site 6

Excellent apple-scab resistance from some sites

Site 5 Gene Pool Identification – Combining SSR, SCAR Markers

G3007 G.935 O.3 MM.111 G5179 G.202 M.2 PiAu5683 G.30 MM.106 G.11 AlnarpM.4 B.9 M.8 M.20 M.1M.13 M.7P.14P.18PiAu5111 J.9 J-TE-C M.27 JM.10 0.81 G. 6 5 V. 1 M.11 J-TE-G Pi80 M.3M.10 M? M.9 J.9 M.9T337 Pi80 V. 2 G. 4 1 M.9T337B.118M.9 PiAu514M.26 M? M.25 M.8J-TE-G P.1 V. 7 M.20 P.14 0.14 V. 2 G.16 G. 2 0 2 B.9 V. 1 O.3 P.1B.491 G. 1 1 G. 3 0 JM.10 G.41 G3007 M.27 R.5 G.935 PiAu5111MW G.65 G5179 M.10 M.13 IIIIII M.26 -0.52-0.52 Alnarp 1.00 PiAu514 M.1 M.2 0.470.47 M.4 G. 1 6 M.11 M.3 -1.19-1.19 IIII Novole PiAu5683 -0.06-0.06 M.7 PiAu5111 NAGAMaruba P.18 -0.59-0.59 R.5 V. 7 J-TE-C M.25 -1.11-1.11-1.86-1.86 MM.106 -1.03-1.03 MM.111 -0.59 B.491 -0.59 B.118 -0.15-0.15 Maruba 0.290.29 NAGA II 0.00 0.21 0.42 0.63 0.83 0.72 Novole Coefficient Crossing Parents – Stage 1 Crossing Parents – Stage 1 Seed Harvest – Stage 1 – 2,000-10,000 seeds per cross Disease Screens – Stage 1 – 3,000 to 10,000 seedlings/year Disease Screens – Stage 1 – 3,000 to 10,000 seedlings/year Fire blight - Erwinia amylovora

z Major disease for apple rootstocks in North America z Bacterial disease with strain differentiation z Resistance sources available z Rootstock infection routes: z suckers z injuries z systemic movement of bacteria from scion Fire Blight Screening – Stage 1 500 to 2,000 seedlings Integration of Marker Assisted Selection – Stage 2 z High throughput PCR markers – SCARs, SSRs z Target traits: z Dwarfing z Powdery mildew resistance z Scab resistance z Wooly apple aphid resistance z Use published and “in house” markers Propagation and Evaluation of Layering Stool-Bed Properties Harvest of Rootstock Liners – Evaluation of Rooting Rootstock Liners in Tree Nursery for Budding/Grafting First Test Orchard – Stage 3 z 3-10 replicates per rootstock genotype z 50-100 different genotype selections every year z All grafted with same scion z Evaluated for 8-12 years Early field selection of precocious genotypes – Stage 4 Expansion of Layering Beds to Increase Replications – Stage 4 Evaluation of Layering Stool Beds – Stage 5 Rootstock Liner Evaluation – Stage 5 Second Test for Resistance to Biotic Stresses – Stage 5

Inoculation with Wooly Apple Aphid (Eriosoma lanigerum)

Water Logging test with Phytophthora Inoculation

Fire Blight Inoculations with Multiple Strains Replicated Orchard Trials in Multiple Locations – Stage 6 z Precocity z Yield z Fruit Size z Dwarfing z Tree Survival z Disease Incidence z Tree Architecture z Burr Knots Cumulative Yield Efficiency Measurements Descriptive Statistics Anderson-Darling Normality Test 17.106 M.9 Variable: CUM-YEFF 0.000 A-Squared: 2.59040 P-Value: 1.19212 1.42114 Mean 1.75248 StDev 3.70960 Variance 317 Skewness Kurtosis 0.24800 N 1.87200 2.20500 Minimum 2.91550 0.5 2.0 3.5 5.0 6.5 8.0 95%1st Confidence Quartile Interval for8.18500 Mu Median 3rd Quartile 95% ConfidenceMaximum Interval for 2.72214Sigma 95% Confidence Interval for Mu 95% Confidence Interval for Median 2.45867 1.29292

2.08 2.18 2.28 2.38 2.48 2.58 2.68 2.78 1.10598 2.34268

95% Confidence Interval for Median 2.13806 Replicated Orchard Trials in Multiple Locations – Stage 6

Descriptive Statistics

Variable: Suckers

A-Squared: Anderson-DarlingP-Value: Normality Test

Mean 27.087 StDev 0.000 10 40 70 100 130 160 190 Variance Skewness Kurtosis 21.1430 N 29.4424 866.858 Minimum 2.55072 1st Quartile 8.75868 95% Confidence Interval for Mu Median 317 3rd Quartile Maximum 0.000 2.000 9.500 5 15 25 17.889 29.166 204.000 27.315

95% Confidence Interval for Median 95% 6.719 Confidence Interval 24.397 for Mu

31.932 95% Confidence Interval for Sigma

13.403 95% Confidence Interval for Median Timing of fire blight screens

Number of Number of Transgene genotypes replications mediated observed per genotype resistance 9

t Rootstock blight

a orchard trials

r Strain specific

b direct inoculation

m 5 u screens

N 1 1 2

Time Screening for Resistance to Fire Blight (E. amylovora)

FIELD INOCULATIONS ON FINISHED TREES

GREENHOUSE INOCULATIONS ON LINERS Commercial Stool Bed Trials – Stage 7 z On site trials of elite rootstocks at commercial nursery locations z Evaluate liner productivity and quality under commercial conditions z Generates nursery stock for major orchard trials (NC-140, large grower trials)

o r P m a r g r

P o c o t o l Trials with NC-140 Cooperators – Stage 8 Drought Tolerance Tests

Work of Dr. PARRA Graft Union Strength Tests

Pictures Courtesy of Mike Parker (NC State University) On Farm and Nursery Trials in Several U.S. Locations – Stage 9 z Large scale trials planted in WA, PA, MI, NY z Trials include 20-45 different genotypes Nursery Tree Measurements on 10-15 Trees per Rootstocks

Branch Length

Tree Height

Branch Angle Total Number of 0=Flat Branches 90=Upright

Branch Height Branch Angles of Brookfield Gala Trees on Several Dwarfing Rootstocks for 7 Whirls

60 2034 2406 50 3041 40 4210 4213 30 4214 4814 20 5935 B9 10 G11

Branch Angle (0=Flat 90=Upright) G16 0 G? M9 1234567 Whirl (3 branches) Stages of Micro-Propagation Prior to Release – Stage 10 Commercial Release and Continued Testing – Stage 10 z Program has released 6 new rootstock genotypes to date. z G.16 and G.30 G.202, G.41, G.935, G.11 are commercially available in U.S. z Release decision for six more elite rootstock genotypes expected in 2008. Large Scale Production of Rootstock Liners Production of High Quality Nursery Trees and Adoption By Growers

Nursery trees on Geneva 202 rootstocks and planted in high density orchard. QTL Mapping of Apple Rootstock Yield & Disease Resistance Traits

z Dr. Wan Yizhen z Construct a molecular map of Apple Rootstock using microsatellites, SNP, SCAR. z Map and develop markers for F2 plant architecture and disease 0.0 CH02f06 1.5 E32M41-0208 2.6 E31M41-0089 3.7 G15-1250 CH02c02a 1.20 resistance traits. 4.8 7.7 E35M41-3500 12.0 E35M37-0318 Sieversii596280Sieversii596283 Orientalis594101 PI594103 15.4 E35M41-0193 Sieversii596282 17.0 E35M32-0272 6589 Pi-Au56-83NAGA 0.55 Kansuenesis59409MarubaAR486-1 z M8 Develop basic knowledge on P18 22.5 E35M42-0282 Asiatica594009WijcikMac 23.5 E33M36-0192 NovoleAR295-6 Reinet t eSim Seiboldii594094 B9 YellowTransJ9 Fusca589975AR628-2Pi-Au51-4P1 III JM7 J-TE-GM26 Fusca589941NSpyEsopusSpitzenPi-Au51-11M275046 B118J-TE-CP14CrimsonBeautyV1 -0.10 Tschonoskii58939GoMM111 lden DelM7 Chinese apomictic species for Orientalis590015B491Supp4JM4M9V2NAKB337P22 MM106 G6 5 G1 6 Toringoides58939 44.8 AD04-450 Transitoria58942 6006G30 46.4 CH02c06 AA12-2200 Transitoria58938 525777075179 50304213 40134214 E35M35-0178 E35M32-0460 Ioensis590015 421061436210 501268745935 47.4 575750873041 seed propagated rootstocks. E36M41-0590 -0.75 696968795890 Angustifolia5897 4814 CH03d10 E35M41-1000 G1 1 6253 3007 48.1 GH589882 E35M35-1500 E32M41-0650 4202 CH02b10 C10-750 II 0.85 48.7 0.21 CH02a04y 0.21 -0.42 49.5 K13-350 -1.05 Coronaria589976 50.3 E37M32-0113 E33M37-0172 -1.69-1.40 z Transgenic approaches for R5 52.3 E33M36-0194 -1.53 Nertchinsk 53.5 CH05e03 E37M36-0195 -0.87 63.3 CH03d01 NZ03c01z -0.21 65.7 I 66.8 P14-750 improving rootstock performance. 67.7 E32M42-0340 0.45

75.3 F05-1500 1.11 Research Work on Apple Rootstocks Requires Many Collaborators and Institutions z Cornell University: z USDA ARS AFRS z T. Robinson (Orchard Systems) Kearneysville: z I. Merwin (Horticulture - Replant) z J. Norelli (Transgenics) z H. Aldwinckle (Plant Pathology) z C. Bassett (Stress Physiology) z L. Cheng (Physiology) z USDA ARS Wenatchee: z S. Brown (Scion Breeding) z M. Mazzola (Plant Pathology) z Michigan State University: z Y. Zhu (Genomics) z R. Perry (Rootstocks) z PENN State University: z S. VanNocker (Genomics) z T. McNellis (Genomics) z Washington State University: z J. Schupp (Horticulture) z B. Barrit (Scion Breeding) z Over 40 scientists as NC-140 z D. Main (BioInformatics) collaborators z USDA ARS PGRU: z Washington Tree Fruit z A. Baldo (BioInformatics) Research Commission z P. Forsline (Apple Collection) BASIC SCIENCE Genomics, Proteomics Gene Discovery, Expression Physiology

APPLIED SCIENCE Plant Breeding Genetic Transformation VERY APPLIED SCIENCE Horticultural Trait Evaluation Widespread Field Performance Field Recommendations NC-140

INDUSTRY, GROWERS, PROCESSORS, CONSUMERS Genomic Revolution z We know that M.7 rootstock is less precocious than M.9. Do we know why? z We know that M.9 dwarfs more than M.26? Do we know why? z Through Genomics much is being discovered about how rootstocks do all that they do. z 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) z Tree architecture modified by apple rootstocks…. z Wealth of new genetic material The Geneva® Apple Rootstock Breeding Program THANK YOU

Todd Holleran, Sarah Bauer, Yizhen Wan

Funding from IDFTA, WTFRC, USDA, Cornell The Road Ahead (2003 NC-140 Mtgs. Door County, Wisconsin)