Concepts of Apple Rootstock Breeding and Selection:A Journey Through the

Development of New Apple Rootstocks

G. Fazio, H. Aldwinckle, T. Robinson

IntroductionBreeding 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.

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

Fruit Color and QualityFruit SizeDisease Resistance

Plant Architecture – DwarfingMolecular mapping and selection toolsGenomics

Yield and productivity (Nutrition)PrecocityAbiotic Stress Resistance (Cold)Disease Resistance

Fire blight ($40M 2000 epidemic, MI)Replant disease complex

TRANSGENIC ROOTSTOCKS for a plethora of traits

Active Apple Rootstock Breeding Programs 1970s and 80s

Sweden Norway

Russia

Poland

CzechGermany

Malling

Japan

Quebec

Vineland

Geneva

Michigan

Active Apple Rootstock Breeding Programs 2005

Russia

Japan

Geneva

S. Korea

New Zealand

China

New and Experimental Apple Rootstocks in the U.S.

Polish Czhec Malling Russia Vineland Quebec Japan Germany GenevaP.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

Replant Disease Complex 1 or 7 Greenhouse/field

1

3-4

3-4

3-4

8-12

8

8

12

15

4

5

Greenhouse

Wholly apple aphid res. Greenhouse

Juvenility - Spines Field/Stoolbed

Stoolbed rooting Field/Stoolbed

Growth habit - Brittleness Field/Stoolbed

Dwarfing Orchard

Precocity Orchard

Suckering Orchard

Yield – Biennial bearing Orchard

Cold hardiness Orchard

Drought tolerance Orchard

Graft union compatibility Orchard

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)

Apple Rootstock Breeding: Resources and Activities

Orchard Production-Individual yield and growth-Disease incidence-Scion compatibility-ETC.

Lab & Greenhouse-Seedling inoculations-Molecular markers-Tissue culture-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

Objective 1.1Years 1-2

Stage 1

Years 3-4

Stage 2

Years 5-6

Stage 3

Years 7-12

Stage 4

Years 10-15

Stage 5

Years 16-18

Stage 6

Years 19-21

Stage 7

Years 22-24

Stage 8

Years 25-27

Stage 9

Years 27-30

Stage 10

1. Select elite parents

2. Generate F1 populations

3. Stratify and plant populations4. MAS for dwarfing, precocity,disease resistance, etc.5. Disease screening

6. Plant selected stools

7. Stool selection

8. Propagation and grafting

9. First test orchard

10. First test evaluation and selection

11. Elite stoolbed establishment

13. Elite liner and tree production14. Elite stoolbed selection anddistribution to nurseries15. Intermediate replicated orchard

16. Intermediate orchard evaluation

17. Commercial stoolbed evaluation18. NC-140 and cooperator trials(national and international)19. Commercial production ramp upPatent and UPOV protection20. Comercial sale

12. Stress tolerance tests - drought,cold hardiness, graft union strength

Highly replicated first test orchardat multiple sitesTest orchard Evaluation and Selection

Elite stoolbed establishmentStress tolerance tests - drought,cold hardiness, graft union strengthElite stoolbed selection anddistribution to nurseriesCommercial stoolbed evaluation anddistribution of trees to trial sitesNC140 and cooperator trials(national and internationa)

Commercial production ramp up

Commercial sale

Contingent on goodness of MAS

Critical Juncture: Molecularinformation about allelic

constitution at disease resistanceloci and about other important

traits can be used to bypass firsttest orchard.

Repeated inoculations withFireblight, Wooly Apply Aphid,

Phytophtora, Powdery Mildew andcomparison to known standards

These Stress Tolerance Testswilll have to be highly replicatedat each location - therefore only

a few selections at a time willbe tested. Grower cooperatorsin cold or drought prone areasin the US will be selected forcold hardiness and drought

stress. For graft unioncompatibility and strength a set

of rootstocks will be graftedwith multiple scions and graftunions tested after 3 years.

Feedback fromHorticultural Traits

evaluations, diseaseresistance evaluations,

stress toleranceevaluationswill be

combined with moleculardata and used in

subsequent cycles ofselection. Elite materialfrom these evaluationsare used as parents in

subsequent cycles.

Breeding and Selection of Apple Rootstocks - Simplified

Nu m

b er o

f pla

nts

Time (3 year stages)

1

Number of replications

per genotype

Number of genotypesobserved

Transgenic“value-added”

traits

10,000

10

100

1000

21 53 4 6 7 8 9 10

B. Johnson

Criteria for Parent Selection –Phenotype and Molecular Markers

DwarfingPrecocityDisease Resistance

Fire BlightPhytopthoraPowdery MildewApple Scab

Yield and Field Performance“New” Gene Pools

AD13 SCAR scab marker (Boudichevskaia et al. 2006)

EM M01 SCAR powdery mildew marker (Evans et al. 2003)

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

2808 wild Malusseedlings from 310 populations from Kazakhstan, Russia, China & Turkey

New Gene Pools at the Plant Genetic Resources Unit (PGRU) Geneva, New York

Malus sieversii from Kazakhstan 1989 - 1996

Site 6

Site 5

Site 9 w/ depletion by grazing animals

Excellent apple-scab resistance from some sites

Gene Pool Identification –Combining SSR, SCAR Markers

Coefficient0.00 0.21 0.42 0.63 0.83

PiAu5111MW

G3007 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

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

V.7V.2

V.1

R.5

PiAu5683

PiAu514

PiAu5111

Pi80

P.18P.14

P.1O.3

Novole

M.9T337

NAGA

MM.111

MM.106

Maruba

M.27

M.26M.25 M.20

M.13

M.11M.10 M?

M.9M.8

M.7

M.4

M.3

M.2

M.1

J-TE-G

J-TE-C

JM.10

J.9

G.65

G.30

G.16

G.11B.9

B.118

B.491

Alnarp

G.935G5179

G.202

G.41

G3007

1.001.00

0.470.47

IIII -0.06-0.06

-0.59-0.59

-1.86-1.86-1.11-1.11

-1.03-1.03-0.59-0.59

-1.19-1.19

-0.15-0.15

II 0.290.29

IIIIII-0.52-0.52

0.720.72

0.140.14

0.810.81

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 amylovoraMajor disease for apple rootstocks in North AmericaBacterial disease with strain differentiationResistance sources availableRootstock infection routes:

suckersinjuriessystemic movement of bacteria from scion

Fire Blight Screening – Stage 1 500 to 2,000 seedlings

Integration of Marker Assisted Selection – Stage 2

High throughput PCR markers –SCARs, SSRsTarget traits:

DwarfingPowdery mildew resistanceScab resistanceWooly apple aphid resistance

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 33-10 replicates per rootstock genotype50-100 different genotype selections every yearAll grafted with same scionEvaluated 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

Fire Blight Inoculations with Multiple Strains

Water Logging test with Phytophthora Inoculation

Inoculation with Wooly Apple Aphid (Eriosoma lanigerum)

Replicated Orchard Trials in Multiple Locations – Stage 6

PrecocityYieldFruit SizeDwarfingTree SurvivalDisease IncidenceTree ArchitectureBurr Knots

8.06.55.03.52.00.5

95% Confidence Interval for Mu

2.782.682.582.482.382.282.182.08

95% Confidence Interval for Median

Variable: CUM-YEFF

2.13806

1.10598

2.45867

Maximum3rd QuartileMedian1st QuartileMinimum

NKurtosisSkewnessVarianceStDevMean

P-Value:A-Squared:

2.34268

1.29292

2.72214

8.185002.915502.205001.872000.24800

3173.709601.752481.421141.192122.59040

0.00017.106

95% Confidence Interval for Median

95% Confidence Interval for Sigma

95% Confidence Interval for Mu

Anderson-Darling Normality Test

Descriptive Statistics

Cumulative Yield Efficiency Measurements

M.9

190160130100704010

95% Confidence Interval for Mu

25155

95% Confidence Interval for Median

Variable: Suckers

6.719

27.315

17.889

Maximum3rd QuartileMedian1st QuartileMinimum

NKurtosisSkewnessVarianceStDevMean

P-Value:A-Squared:

13.403

31.932

24.397

204.000 29.166 9.500 2.000 0.000

3178.758682.55072866.85829.442421.1430

0.00027.087

95% Confidence Interval for Median

95% Confidence Interval for Sigma

95% Confidence Interval for Mu

Anderson-Darling Normality Test

Descriptive Statistics

Replicated Orchard Trials in Multiple Locations – Stage 6

Timing of fire blight screensN

u mb e

r of p

lant

s

Time

1

Number of replications

per genotype

Number of genotypesobserved

1 2

5

9

Transgenemediated

resistance

Strain specificdirect inoculation

screens

Rootstock blightorchard trials

Screening for Resistance to Fire Blight (E. amylovora)

GREENHOUSEINOCULATIONS

ON LINERS

FIELD INOCULATIONSON FINISHED TREES

On site trials of elite rootstocks at commercial nursery locationsEvaluate liner productivity and quality under commercial conditionsGenerates nursery stock for major orchard trials (NC-140, large grower trials)

Commercial Stool Bed Trials –Stage 7

Prog

ram

Pro

toco

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)

Large scale trials planted in WA, PA, MI, NYTrials include 20-45 different genotypes

On Farm and Nursery Trials in Several U.S. Locations – Stage 9

Nursery Tree Measurements on 10-15 Trees per Rootstocks

Tree Height

Branch Height

Branch Angle0=Flat

90=Upright

Branch Length

Total Number of Branches

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

0

10

20

30

40

50

60

1 2 3 4 5 6 7Whirl (3 branches)

Bra

nch

Ang

le (0

=Fla

t 90=

Upr

ight

) 20342406304142104213421448145935B9G11G16G?M9

Stages of Micro-Propagation Prior to Release – Stage 10

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.

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

Dr. Wan YizhenConstruct 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.

CH02f060.0E32M41-02081.5E31M41-00892.6G15-12503.7CH02c02a4.8E35M41-35007.7E35M37-031812.0E35M41-019315.4E35M32-027217.0

E35M42-028222.5E33M36-019223.5

AD04-45044.8CH02c06 AA12-220046.4E35M35-0178 E35M32-0460E36M41-059047.4CH03d10 E35M41-1000E35M35-1500 E32M41-065048.1

CH02b10 C10-750CH02a04y48.7K13-35049.5E37M32-0113 E33M37-017250.3E33M36-019452.3CH05e03 E37M36-019553.5CH03d0163.3NZ03c01z65.7P14-75066.8E32M42-034067.7F05-150075.3

F2

R5

M8

YellowTrans

WijcikMac

V2V1

ReinetteSim

Pi-Au56-83

Pi-Au51-4Pi-Au51-11Supp4

PI594103

P22

P1

P18

P14

Novole

Nertchinsk

NAKB337

NAGA

NSpyMM111

MM106

Maruba

M7 M9M27 M26JM7

JM4

J-TE-GJ-TE-C

J9

GH589882

G65G30

G16

G11

EsopusSpitzen CrimsonBeautyB118B9

B491

AR628-2

AR486-1AR295-6

7707

696968796874

GoldenDel

6589

625362106143

6006

Sieversii596283Sieversii596282

Sieversii596280Orientalis594101

Kansuenesis59409

Orientalis590015

Seiboldii594094

Asiatica594009

5935Ioensis590015

Coronaria589976

Fusca589975Fusca589941

Angustifolia5897

Transitoria58942

Tschonoskii58939

Toringoides58939Transitoria58938

58905757

52575179

5087

5046

50305012

4814

421442134210

4202

40133041

3007

0.850.850.210.21

-0.42-0.42IIII

-1.05-1.05-1.40-1.40-1.69-1.69

-1.53-1.53

-0.75-0.75

IIIIII-0.10-0.10

0.550.55

-0.87-0.87

1.201.20

II -0.21-0.21

0.450.45

1.111.11

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 collaboratorsWashington Tree Fruit Research Commission

INDUSTRY, GROWERS, PROCESSORS, CONSUMERS

APPLIED SCIENCEPlant Breeding

Genetic Transformation

BASIC SCIENCEGenomics, Proteomics

Gene Discovery, ExpressionPhysiology

VERY APPLIED SCIENCEHorticultural Trait Evaluation

Widespread Field PerformanceField Recommendations

NC-140

Genomic RevolutionWe 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

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)