Twenty Years of Apple/Grape/Cherry Germplasm Management

Twenty Years of Germplasm Management: Geneva, NY to the Forests of Central Asia, China,

Russia, and Turkey

Philip Forsline April 5, 2004

Plant Genetic Resources Unit (PGRU) located at Cornell University, Geneva, New York

CornellGeneva

PGRU facilitiesexpanding

New officewing andgreenhouses

Greenhouse& screen-house forclonalcollections

History of facilities: Clonal repository at PGRU

Campus Bldg 1985 Campus Bldg 1985

Equipment storage, 1984

Farm preparation, 1984 Tile drainage 1984

Farm development 1985 to present

1985 priorto planting

2002 fromwest toeast

2002 fromeast towest

1990: addition of farm for seedsection ofPGRU

Clonal collection withdeveloping plantings

History of significant events and personnel additions at PGRU, Geneva, NY

1953 – Northeast Regional PI Station (NERPIS) established for vegetables and ornamental germplasm

1982 – CSRS grant to Cornell; 50 acres purchased for Clonal Repository (CR)

1983 – CR Farm Manager hired; 1st propagations of Malus and Vitis

1984 – Curator hired; campus building started

1986 – CR building occupancy and dedication; 1st orchard and vineyard plantings; merger with NERPIS

1987 – S. Kresovich hired as 1st Research Leader for NERPIS and CR – new name: Plant Genetic Resources Unit (PGRU)

1988 – J. McFerson hired as curator of seed group; Cryopreservation project began; 1st

of seven germplasm expeditions initiated

1989 – 40 acres purchased for seed group 1993 – J. McFerson appointed RL for PGRU;

PGRU scientists granted ‘Courtesy/Adjunct’ appointments with H.S. at NYSAES

1995 – Sour cherry collection established at PGRU

1996 – S. Hokanson hired as post doc 1998 – W. Lamboy appointed RL; USDA apple

rootstock breeder hired at PGRU; L. Robertson hired as curator of seed group

1999 – Grape genetics group of PGRU initiated with hiring of grape rootstock breeder; Molecular geneticist hired for seed group

2002 – Grape scion breeder hired 2003 – C. Simon hired as RL after serving 5 years

as RL at Davis, CA Clonal Repository; Grape pathologist and computational biologist hired;

2004 – Grape genomics specialist hired

Vegetatively-propagated crops at PGRU

Apple 3909 accessions Grape 1175 accessions Sour Cherry 87 accessions Total 5171 accessions

Major Seed-Propagated Crops Conserved at Geneva

Larry Robertson: Curator/GeneticistJoanne Labate: Molecular Biologist

CCCrrroooppp NNNuuummmbbbeeerrr ooofff aaacccccceeessssssiiiooonnnsss

TTTooommmaaatttooo 555111444111 BBBrrraaassssssiiicccaaa 222111999333 OOOnnniiiooonnn 111111777999 SSSqqquuuaaassshhh 888333000 RRRaaadddiiissshhh 777111111 CCCeeellleeerrryyy 111999888 BBBuuuccckkkwwwhhheeeaaattt 888777

USDA grapevine genetics -- Geneva 1999 rootstock breeder/geneticist (Peter

Cousins) 2002 molecular genetics/genomics of scion

traits (Chris Owens) 2003 molecular plant-microbe interactions of

fungal pathogens (Lance Cadle-Davidson) 2003 Computational biologist – also working

across Clonal and Seed projects of PGRU (Angela Baldo)

2004 evolutionary genomics/population genetics (Amanda Garris)

The Geneva® Apple Rootstock Breeding ProgramGennaro Fazio, USDA/ARS, PGRU in cooperation

with Cornell University

Staff for clonal collections at PGRU SYs FTE

- P. Forsline1.0

Horticulturist/Curator- C. Simon

0.85Research Geneticist / RL- H. Schwaninger 1.0Molec. Biol. / Support Sci.

Technical FTE

- W. Srmack 1.0Collections Manager /

Supervisor- D. Beckhorn 1.0

Field Assistant III - R. Vaughan 1.0

Biol. Sci. Technician (field) - N. Lepak 1.0 Agri. Res. Sci. Technician

(field)- Vacant 1.0

Biol. Sci. Technician (molecular)

Database- R. Nearpass 0.40Supervisory IT specialist- D. Dellefave 1.0Germplasm Program Assistant

Current CRIS Project:Conservation and Utilization of the Genetic

Resources of Apples, Grapes, and Tart Cherries

Objective 1: Acquire and maintain genetic resources and associated information of apples, cold-hardy grapes, and tart cherries and refine the collection based on morphological and molecular characterization and distribute germplasm to the user community.

Objective 2: Enhance efficiency and effectiveness of germplasm maintenance by applying genomic sequencing and molecular marker techniques to genetically characterize germplasm and determine phylogenies.

Objective 3: Enhance value and utilization of genetic resources of apples, cold-hardy grapes, and tart cherries by systematic characterization and evaluation for important morphological and horticultural traits.

Acquisition and Maintenance of Clonal Collection

Vitis collection at Plant Genetic Resources UnitGeneva, NY, U.S.A.

1045 clones - all maintained as duplicate field plantings- 650 hybrid; 395 (23 Vitis sp)- cryogenic storage (?)- 138 are part of a core collection

130 accessions of wild Vitis sp (seed lots and/or seedling populations) - 420 seedlings under evaluation from 30 of these acc.

A total of 1175 accessions at PGRU** Remainder of Vitis collection at Davis, CA

- 814 hybrids; 1838 (45 Vitis sp)* 3827 accessions of Vitis in NPGS

Vitis collection at PGRU

Vineyard (1045 clones) Early / Late leaf fall

Diversity among clones

DiversityAmongclusters

Digital imaging of Vitis clones

PI 181481‘Vignoles’

PI 588070‘Catawba’

PI 588058‘V. coignetiae’

PI 588054‘V. riparia’’

Prunus (tetraploid cherry) collection at Plant Genetic Resources Unit

Geneva, NY, U.S.A. 87 clones - all maintained as duplicate field

plantings- 57 Prunus cerasus (sour cherry)- 12 P. fruticosa (wild progenitor of sour cherry)- 7 interspecific hybrids- 12 other (misc. Prunus sp)- 52* backed up in cryogenic storage at NCGRP

* 8 also in cryogenic storage on-site at PGRU

Cherry collection at PGRU

Collection (87 acc.) P. cerasus / P. fruticosa

Early / late bloomspreading

upright

Cherry digital imaging

Montmorency Balaton

Danube Schatten Morello

Malus collection at Plant Genetic Resources UnitGeneva, NY, U.S.A.

2376 clones - all maintained as duplicate field plantings- 1362 M.x domestica; 329 hybrid; 685 (54 M. sp)- 2146 backed up in cryogenic storage at NCGRP, Ft Collins, CO

* 436 also in cryogenic storage on-site at PGRU- 195 are part of a core collection (multi-state plantings)

* 60 new additions to core in 2003 1533 accessions of wild M. spp. (stored as seed) from World

centers of origin - 3300 seedlings under evaluation from 340 of these acc.

897 of wild acc. are Malus sieversii from Central Asia A total of 3909 accessions

Diversity in Malus

Bloom

BloomM. baccata

Dormant“Kansas 14”

Early leaf fall

Late leaf fall

Fruit

Malus / seedling collection at PGRU

Early stage after planting 1986 More advanced stage

Apples/seedling

vineyard

Tree removal after repropagation on EMLA 7

Malus / EMLA 9 collection at PGRU

Initial plantings afterestablishment in 1986

Established plantings summer

Established plantings at bloom

Tree removal for conversion to EMLA 7

Digital imaging of Malus clones

GalaBelle de Boskoop

GenevaMalus hupehensis

Malus core collection at PGRU

Planting in 2000

Apples/seedling

Apples/EMLA7 Extension orchard 02

Core collection in bloom: 2001Label systemM. transitoria

Success w/ core collection indiscovering new virus indicator

Core collection fruiting: 2001

clean

infected

infected

Malus / EMLA 7 collection at PGRU

Planting in 2000

Establishment 2001

Apples/seedling

Apples/EMLA 7

Extension orchard, 2002

Fire blight challenges and control in the Malus collection at PGRU

Heavy FB onblossoms; 2000

Severe FB epidemic; 1996 425 trees removed

‘Apogee’ treatment ‘Apogee’

No ‘Apogee’treatment

Codes for fire blight, shootY (natural) of apple

Code Definition No. of Accessions

1 Very resistant - no occurrence 596

2 Moderately resistant - only light rating 127

3 Intermediate - light to medium rating 174

4 Moderately susceptible - medium to heavy rating 363

5 Very susceptible - very heavy rating 1091 2351

YCumulative records 1990 – 2003 for entire ‘clonal collection’

Measures being used to minimize fire blight in the PGRU apple collection

Cryogenic storage of dormant buds; as a result, ~ 90 accessions that have died (fire blight) have been rescued by direct grafting of cryopreserved buds

Repropagation of collection to EMLA 7 rootstock: Replacing collection previously held on seedling and EMLA 9 rootstock

Application of ‘Apogee’ post-bloom to control vegetative growth reducing shoot blight

Removal of all lesions in dormant season; timely annual copper spray; and corrective pruning of infected shoots throughout the growing season

Continued use of antibiotics under defined protocols to minimize blossom blight

Cryopreservation of MalusA back up collection at $1/accession/yr!

Processing 35 mm bud segments

Active collection (436 acc.)at PGRU Geneva, NY

Recovery of accessions by bud grafting

Base collection (2146 acc.)at NCGRP, Ft. Collins, CO

Hundreds of seedlings budded withcryopreserved buds testing viability

Cryopreservation of Prunus

Active collection (8 acc.) at PGRU Geneva, NY

Injury to primary budfollowing LN exposure

Typical recovery scenario from axillary bud

Base collection (52 acc.)at NCGRP, Ft. Collins, CO

Pilot Project: Forsline, P.L., C. Stushnoff, L.E. Towill, J.W. Waddell, W.F. Lamboy and J. R. McFerson. 1998. Recovery and longevity of cryopreserved apple buds.

J. Amer. Soc. Hort. Sci. 123:365-370.

84 accessions were processed periodically over 4-yr period

12/12/1988 – 8 accessions 02/07/1989 – 8 accessions

12/12/1989 – 6 accessions 01/19/1990 – 9 accessions

12/14/1990 – 12 accessions 01/15/1991– 13 accessions

12/18/1991– 13 accessions 01/18/1992– 15 accessions

Pilot project to determine protocol for cryogenic storage of dormant buds: % bud recovery of 84 apple accessions (processed 1989-1992); tested after up to 8 years of storage in liquid nitrogen

Treatment Recovery %Desiccated Control 85.3 aZ

Storage one month 63.0 b

Storage one year 64.2 b

Storage two years 66.5 b

Storage four years 68.6 b

Storage after eightY years 68.3 b

ZSeparation of grand means of 84 accessions at P < 0.01 by test for differences between two proportions (LSD = 7.2)Y Fifteen year test in process 2004 to 2007

Annual cryopreservation of Malus accessions at NCGRP following the 4-yr pilot project 1988-1992

Year M. x domestica Hybrids Other species Total1993 86 8 5 991994 108 7 26 1411995 167 25 50 2421996 169 24 49 2421997 223 71 70 3641998 209 66 64 3391999 120 55 71 2462000 108 46 84 2382003 52 6 56 1142004 22 2 97 121Total 1264 310 572 2146

Successful cryopresevation for > 90% of accessions stored at NCGRP: those with < 30% viability will be reprocessed

0

100

200

300

400

500

600

700

Categories of success based on bud recovery via grafting

0-10 %20-30 %40-50 %60-70 %80-90 %100%

No.

of a

cces

sion

s in

sto

rage

171 or 9% of totalwere unsuccessful

1760 or 91% were successful

0-10 20-30 40-50 60-70 80-90 100

Successful cryopresevation for > 95% of accessions stored at PGRU: those with < 30% viability will be reprocessed

020406080

100120140160180

Categories of success based on bud recovery via grafting

0-10 %20-30 %40-50 %60-70 %80-90 %100%

No.

of a

cces

sion

s in

sto

rage

18 or 4% of totalwere unsuccessful

418 or 96% were successful

0-10 20-30 40-50 60-70 80-90 100

Distribution of Germplasm

Annual distribution of germplasm from 1988 to 2004

0

500

1000

1500

2000

2500

3000

3500

4000

4500

'88 '89 '90 '91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02 '03 '04

Grapes Apples

Apple total no. = 41,111Apple total orders = 1514 (mean:89)Apple orders/yr (range: 30-163Z)

mean = 2418

mean = 692

Grape total no. = 11,765Grape total orders = 782 (mean:46)Grape orders/yr (range = 14-72Z )

Z

Z

Cherry distributions started in 1998: 191 accessions to 21 orders

No.

of a

cces

sion

s an

nual

ly

Characterization and Evaluation

Characterization of collections at Plant Genetic Resources Unit, Geneva, NY

Malus Clones- 1150 acc. with 33 descriptors- 900 digital images

Malus species characterization at origin site- 1088 with 25 descriptors plus other passport records

Malus species grow-out- 465 seedlings with 33 descriptors- 375 digital images

Vitis clones - 850 acc. with 16 descriptors- 414 digital images

Prunus clones- 50 acc. with 8 descriptors & 50 digital images

Collaborative evaluation with Specific Cooperative Agreements (SCA) of clonal

collections in process or recently completed NPGS-funded projects in Malus

evaluation

1) Preservation of alleles from wild collections: Volk, Walters, Richards

2) Antioxidants in Malus collection: Stushnoff

3) Evaluation of wild apple species for disease resistance: Aldwinckle

4) Evaluation of elite M. sieversii and some of its hybrids for apple scab resistance genes: Aldwinckle, Luby, Gardiner and Bus

5) Molecular characterization of seedling populations of 10 Malus species with 90 mapped microsatellite markers in relation to apple rootstocks: Fazio, Baldo

NPGS-funded projects in Vitis and Prunus evaluation

1) Evaluation of Vitis for susceptibility to crown gall: Burr

2) Evaluation of Vitis for resistance to Phomopsis viticola and powdery mildew: Wilcox and Reisch

3) Evaluation of Russian tetraploid cherry selections for cherry leaf spot resistance: Iezzoni

4) Evaluation and rescue of sour cherry germplasm for use as sweet cherry rootstocks: Iezzoni

Molecular studies on clonal germplasm at PGRU: past & present

Apple core collection – Hokanson Wild apple germplasm – Benson and Dickson

(PhD projects) Grape genotyping – Lamboy Grape phylogeny – Schwaninger Overall coordination presently – Simon

(Research leader / molecular geneticist)

New Acquisitions

The origin of the cultivated apple

Dr. B. Juniper: Theory on earlyand recent evolution of thecultivated apple

Ancient Malus species ofChina: bird disseminated to Central Asia

M. sieversiiof CentralAsia

North Americabecame asecondary center of origin: ‘RedDelicious’,‘Golden Delicious’, etc.

Johnny Appleseed

Mammal disseminated

Germplasm collections to add mostly wild Malus species to the PGRU collection

Year Country Species Personnel1987 –1988

Western U.S. & CanadaEastern U.S. & Canada

Malus fusca, M. ioensis, M. coronaria & M. angustifolia

Weeden, Dickson

1989 Uzbekistan, Tajikistan, Kazakhstan

Malus sieversii Aldwinckle, Dickson, Sperling

1993 Kazakhstan, Kyrgyzstan

Malus sieversii, Vitis vinifera, misc.

Forsline, Dickson, Mink, New Zealand scientist

1995 Kazakhstan Malus sieversii, Vitis vinifera, misc.

Forsline, Dickson, Luby, S. African scientists

1996 Kazakhstan Malus sieversii, Vitis vinifera, misc.

Forsline, Hokanson, Unruh, Pellett

1997 China (Sichuan) Malus species (7 endemic to Sichuan)

Forsline, Aldwinckle. Benson

1998 Russia Malus orientalis, Prunus cerasus and misc Prunus spp.

Forsline, Iezzoni, Karle, German scientist

1999 Turkey Malus orientalis, selected local Malus cultivars

Forsline, Aldwinckle

Collection team for 1989 Central Asian expedition to Uzbekistan, Tajikistan and Kazakhstan (site 3)

UzbekistanElizabeth Dickson

Herb Aldwinckle‘The late’ Calvin Sperling

Professor Djangaliev

Geneva:making plans, 1992

Organizing expedition: first morning, Sept. 1993; ready to leave to boardhelicopter

Collection admiration, 1995 One of frequent lectures; 1996

Local Kazak media highlightingAmerican & S. African invasion

1995

1993 collection team: USA & New Zealand

Preparation for departure Transfer in Moscow – hotel and visit to Red Square

Moscow to Alma Ata via ‘Aeroflot’ Team in Alma Ata with Kazakh hosts

Noiton MinkDickson

Significant travel by helicopter

1995 USA and S. African collection team

DicksonBritzForslineLubyHuman

Arrival at site 9

Meals in yurt: site 5

Housing in sanitarium at HQ in Almaty Gala dinner with hosts in Almaty: return to USA

Seed extraction at hotel: site 9

1996 USA collection teamHokansonUnruhForslinePellett

Site 5

Site 9

Site 12

HQ in Almaty

SITE INFORMATION RECORDED

Latitude (GPS) Longitude (GPS) Elevation Slope Aspect Light/Shade

Assoc. Tree spp. Assoc. Shrub spp. Assoc. Herb. spp. Population Abun./Dis. Climate Soil

Site descriptions in Central AsiaCountry/Region Site Lat oN / Long oE Elevation (m) Rainfall (mm)

Tajikistan / --- 1 39 / 68 --- ---

Uzbekistan/ --- 2 41 / 69 --- ---

Kazakstan/Zailisky 3 43 / 77 1170-1690 700

Kazakstan/Djungarsky 4 45 / 80 1170-1760 800 5 46 / 81 1190-1360 850

Kazakstan/Karatau 6 43 / 70 600-910 25011 42 / 70 780-1230 250

Kyrgyzstan/ --- 7 41 / 73 1300-1500 1300

Kazakstan/Tarbagatai 9 48 / 82 870-1120 450

Kazakstan/ Ketmen 10 44 / 80 1600-1700 650

Kazakstan/Talasky 12 42 / 71 1000-1025 320

Kazakhstan site 3: ’89, ’93, ’95, ‘96

Fruit from 30 randomly-collected treesDepletedby dachas

Habitat 1940

Habitatpresent

Studies by ProfessorA. Djangaliev

Kazakhstan site 4, ’93, ’95, ‘96

Scab infected tree

Kazakhstan site 5: ’93, ’95, ‘96Forestry camp at 1200 mheadquarters in ’93, ’95 & ‘96

300 yr oldM. sieversii

Bear scat w/ many apple seeds

Appleforest:1800 m

1100 m

First collection morning (Sept. 1993) starting out at 600 m with hike to 1800 m

Kazakhstan site 6: ’93 & ’95

Camp at 600 m:headquarters

Standing at 900 m

M. sieversiiat 900 m

Fruit from 30randomly-collectedtrees at 900 m

Kyrgyzstan site 7: 1993Apple and walnutforests as seen fromhelicopter

Heavy grazing in M. sieversii areasVillage inKyrgyzstannearcollectionsites

Kazakhstan site 9: ’95 & ‘96Grazed areas – M. sieversii on slopes

Diverse, elite M. sieversii

Super-elite M. sieversii

Heavy grazing of habitatFruit from 30randomly-collectedM. sieversii trees

M. sieversii

Kazakhstan site 10: 1996

Fruit from 20 randomly-collected trees

Breakdowns; common occurrence

Uygur tribes tradition of bread bakingM. niedzwetzkyana

Kazakhstan site 11: 1996

Elite ‘Yellow transparent’-typeM. sieversii

Kazakhstan site 12: 1996Canyon 400 mdeep w/ M. sieversiion the N-facing wall

Fruit from10 randomly-collectedM. sieversiitrees

Bottom ofcanyon;M. sieversiion thisside

Trail to bottom

ACCESSION INFORMATION RECORDED

+ Fruit Over Color/Intensity + Fruit Size + Fruit Ground Color + Fruit Texture+ Fruit Russet + Fruit Flavor+ Fruit Shape + Harvest Season+ Stem Character + Tree Habit

+ Diseases/Insects:Leaves/Fruit

Variability of fruit size of Malus sieversii among sites in Central Asia No. collected Mean size (mm) Size range (mm)

Site / Yr(s) Elite Random Elite Random Elite Random

12 / (‘96) 2 10 65 42 60-74 32-50 * 5 / (‘96) 14 54 58 35 54-65 25-49 9 / (‘95/’96) 57 150 56 43 46-72 28-62 11 / (‘96) 13 40 55 42 44-76 29-63 10 / (‘96) 2 20 49 40 47-51 29-51 * 5 / (‘95) 14 60 46 36 37-56 27-48 3 / (‘95/’96) 11 80 45 34 32-56 26-49 4 / (‘95/’96) 10 87 44 34 32-55 28-44 6 / (‘95) 25 64 42 41 33-46 28-54

Totals 148 565 51 38 32-76 25-63

* Note difference in fruit size observed in 1995 and 1996 at Site 5

Summary of Central Asian M. sieversii collection, distribution and storage

Accessions Seeds Seeds Storage Storage Group Obtained Obtained Distributed at PGRU at NSSL1989 and 1993 179 33,000 9200 13,900 10,900Collections

1995 and 1996 148z 67,000 16,000 29,200 21,800Elites

1995 and 1996 565 30,000 4100 19,100 6800x

Random populations

Totals 892 130,000 28,300Y 62,200 39,500

z 44 of best accessions also obtained as clonesY Distributed to 24 evaluatorsx Stored as 19 bulked populations (each population includes seeds from 10 to 60 trees with an average of 30 trees / population)

Seed storage at -200 C

Evaluation of New Material

PGRU / Cornell cooperative evaluation of Malus sieversii

Lab Evaluation Type No. of Seedlings

H. Aldwinckle Disease resistance 5124Z

PGRU Horticultural / Molecular part of 5124 - P. Forsline - S. Hokanson - W. Lamboy - G. Fazio - L. Benson N. Weeden Isozyme / MolecularS. Mehlenbacher “ / “W. Reissig Insect resistanceAldwinckle / Korea Rosellinia / Helicobasidium 400S. Brown Genetic dwarf 250 I. Merwin Nematode resistance 200

Z 2108 of this group of screened seedlings were sent to OH, NJ, WA, MN and WI for further evaluation

PGRU / SAES cooperative evaluation of M. sieversii

State / Lab Evaluation type No. of seedlings

MN / Luby Hort, Disease, Cold H. 1498Y

NJ / Goffreda Hort, Disease, Molecular, Elite clones 1899Y

CO / Stushnoff Hort, Cold H., Antioxidants 720WA / Barritt Hort, Sunburn, Cold H. 624Y

OHZ / Lynd, Miller Hort, Disease, Late Bloom, Elite clones 950Y

WI / Smith Hort, Disease, Cold H. 655Y

AR / Rom Hort, Disease, Late Bloom ~400AK / McBeath Hort, Disease, Cold H. 346IL / Korban Hort, Disease, Molecular ~200

Z Seedlings planted at Dawes Arboretum, Newark, OH; c/o D. Miller

Y Some seedlings at @ of these sites screened for apple scab, fire blight and cedar-apple rust at Cornell, Geneva by Dr. Aldwinckle

PGRU / International cooperative evaluation of Malus sieversii

No. of Country / Lab Evaluation type seedlings

New Zealand / Bus, Noiton, Gardiner Disease, Hort 4426Germany / Buttner, Geibel, Höfer Disease, Hort 1367Norway / Roen Disease, Hort 692N.S.,Canada / Deslauriers, Embree Disease, Hort 1155Man., Canada / Davidson Disease, Hort, Cold H. 169B.C., Canada / Quamme, Hampson Disease, Hort 325N.B., Canada / Hunter Hort ~120Japan / Bessho Disease, Hort ~100UK, Reading / Farrel Molecular ~300UK, Oxford / Juniper Molecular ~150S. Africa / Human, Britz Dis., Insect, Sunburn, Chilling ?Z

Netherlands / Kemp Elite clones --Italy / Sansivini Elite clones --

Z Parallel collections were made in 1995 expedition in Kazakhstan

M. sieversii seedling grow outs in Germany and New Zealand

Dresden,Germany Vincent Bus; Havelock,

North, New Zealand

Disease Resistance Screening

Screening young M. sieversii seedlings for apple scab (Venturia inaequalis)

Codes used in rating apple scab resistanceH. S. Alwinckle and H. L. Gustafson

1 = Pits, small pin-prick marks (similar to Vm gene) - RESISTANT 2 = Chlorotic lesions (similar to Vf gene) - RESISTANT 3 = Necrotic lesions ( brown) - RESISTANT 4 = Sporulation - SUSCEPTIBLE 5 = Nonsporulating - RESISTANT 6 = Abaxial sporulation - SUSCEPTIBLE 7 = No symptoms - RESISTANT 8 = Cupped or convoluted (similar to Vf gene) - RESISTANT 9 = Stellate (star shaped) necrotic (similar to Vr gene) - RESISTANT

A = Usually has some cupping and or chlorosis but may have little or no symptoms[(Characteristic of Vf gene) ( numbers: 2,5,7,& 8)] - RESISTANT

B = Stellate necrotic [(Characteristic of Vr gene) (number: 9)] - RESISTANT Other resistant reactions

- LN = Large necrotic lesions - RESISTANT- N = Necrotic lesions - RESISTANT- P = Pits ( number 1 ) - RESISTANT

Apple scab resistance of Malus sieversii populations from Central Asia

H. Aldwinckle, P. Forsline, H. Gustafson and S. Hokanson Site No. Seedlings Inoculated % Resistant

4 405 50 6 705 37 5 1224 29 9 1325 28 7 383 25 2 101 24 11 244 23

3 460 17 12 133 14 10 123 6 1 21 5

Totals 5124 28

Wild Malus sieversii seedling plantings

Early stage after planting 1986 More advanced stage1993 collection1995/96 collections (1200 sdgs)

M. sieversii in bloom

1989 collection

M. niedzwetzkyanaform of M. sieversii

M. sieversii fruiting

0

10

20

30

40

50

60

70

Percentage Scab

Resistance

3 6 4 5 9 11 12 10 All

Sites in Kazakhstan

Other-type

B-type resistance (%)

A-type resistance (%)

Summary of scab resistance in grow-out of 1188Malus sieversii seedlings in Geneva, New York

Cedar apple rust resistance in M. sieversii seedlings from 8 different sites in Kazakhstan

0

10

20

30

40

50

60

70

80

Sites in Kazakhstan

Total (1903 sdgs)Site 9 (520 sdgs)Site 6 (207 sdgs)Site 4 (194 sdgsSite 3 (40 sdgs)Site 5 (610 sdgs)Site 11 (162 sdgs)Site 10 (66 sdgs)Site 12 (104 sdgs)To

tal 9 6 4 3 5 11 10 12

% o

f pop

ulat

ion

resi

stan

t

Natural incidence of fire blight on shoots of the Malus sieversii grow-out in Geneva, New York

Fire blight

Natural occurrence of fire blight in M. sieversii seedlings from 7 different sites in Kazakhstan

0

10

20

30

40

50

60

70

0 to light heavy to VH

Total (1150 sdgs)site 9 (374 sdgs)site 5 (167 sdgs)site 11 (131 sdgs)site 12 (98 sdgs)site 4 (28 sdgs)site 6 (205 sdgs)site10 (55 sdgs)

Fire Blight Severity

% o

f tr

ees

in e

ach

cate

gory

Codes 1 & 2 Codes 4 & 5

669

9

6

Horticultural Evaluations

Descriptors used to characterize M. sieversii at: http://www.ars-grin.gov/cgi-bin/npgs/html/crop.pl?115

Date collected / Harvest season Fruit bloom Fruit flesh color Fruit flesh firmness Fruit flesh texture Fruit flesh flavor Fruit flesh oxidation Fruit skin overcolor Overcolor pattern Overcolor intensity Fruit ground color Fruit juiciness (sp. gravity) Fruit size (LxW in mm) Fruit weight (g)

Fruit russet type Fruit russet location Fruit russet intensity Fruit shape Fruit shape uniformity Fruit size uniformity Fruit-top shape Stem cavity Stem thickness Stem length Calyx basin Fruit tenacity to premature abscission Soluble solids Bloom date Budbreak date

Wild Malus sieversii clones and seedlings in grow-out plantings

More advanced stage

vineyard

PI 613972 clone in bloom

PI 613975 clones fruiting

‘Empire’ ‘Gala’

13 different M. sieversii clones

‘Empire’ ‘Gala’

Fruit from half-sib seedlings ofsome of the M. sieversii clones

PI 613978 &

Fruit size, flavor and red color of M. sieversii on-site in Kazakhstan, and as grow-outs in yrs 6 to 8 (2001 to 2003) in Geneva, New York

Site Category No. of

trees

Mean diam (mm /fruit)

Diam range (mm / fruit)

Mean weight

(g / fruit)

Weight range

(g / fruit)

Accept-able flavor

(%)

% of trees w/ red fruit

Red intensity

mean (%)

9 Trees in Kazakhstan

29 51 34-66 NA NA 89 93 45

9 Progeny grow-out

159 46 29-63 42 11-102 60 65 28

6 Trees in Kazakhstan

14 42 30-50 NA NA 72 71 53

6 Progeny grow-out

138 43 29-60 32 15-78 39 54 27

9 Elites in Kazakhstan

11 56 43-64 NA NA 91 100 55

9 Elite grow-out

11 68 58-72 120 74-180 91 100 47

Harvest season and soluble solids of M. sieversii fruit from grow-outs in yrs 5 - 7 (2001 - 2003) in Geneva, New York: in addition the no. of trees to characterize in 2004 (yr 8 or 7)

Site(s) Category No. of trees completed

Harvest season range

Soluble solid range

No. of trees to test in

20049 Elite clones 11 10 Aug-13 Sep 9.5-14.1 109 Seedlings 159 8 Aug-17 Sep 8.0-17.9 786 Seedlings 138 8 Aug-3 Nov 8.9-15.9 415 Seedlings 38 8 Aug-20 Sep 6.8-21.0 98

11 Seedlings 37 8 Aug-30 Sep 9.4-15.4 67

12 Seedlings 29 8 Aug-15 Oct 8.5-14.8 523 Seedlings 5 14 Aug-10 Sep 8.5-10.5 194 Seedlings 7 14 Aug-17 Sep 9.0-12.1 80

10 Seedlings 20 8 Aug-23 Sep 8.7-12.2 324,5,6,11,12 Elite clones 5 22 Aug-10 Oct 9.8-15.3 11

Fruit shape in M. sieversii seedling populations from 6 sites in Kazakhstan

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Globose Flat Conical

Total (421 sdgs )Site 5 (38 sdgs)Site 6 (138 sdgs)Site 9 (159 sdgs)Site 10 (20 sdgs)Site 11 (37 sdgs)Site 12 (29 sdgs)

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Digital imaging of Malus sieversii seedlings

From site 5 in Kazakhstan From site 6 in Kazakhstan

From site 9 in Kazakhstan From site 11 in Kazakhstan

Digital imaging of Malus sieversii clones

From site 5 in KazakhstanFrom site 6 in Kazakhstan

From site 12 in Kazakhstan From site 9 in Kazakhstan

Elite clone (PI 613971) and progeny (3 half-sibs)

CloneSeedling .a

Seedling .b Seedling .h

Elite clone (PI 613976) and progeny (3 half-sibs)

CloneSeedling .a

Seedling .k Seedling .n

Seed bulk-up from flowering M. sieversii seedlings to save additional genetic diversity

In spring 2004 we will make controlled pollinations on seedlings from sites 6 and 9 in order to bulk up seeds for long term storage to supplement original seed collected in 1995

In spring 2005 the same project will be accomplished with seedlings from sites 4, 5, 10, 11, and 12

Other Recent Collections

1997 Sichuan collection teamGeneva, 11/96: Prof. Li; plans Hotel in Chengdu; Prof. Li & wife;

H & B Aldwinckle

Crew collecting minutes before monkey thievery

Crew at 3500 m pass in mountains

Post expedition conference with Yunnan and Guizhou researchers in Chonqing

Laura Benson

monkey

Malus sp. collections in Sichuan, China

M. hupehensis

M. toringoidesM. prattii

Maluskansuensis

1997 collections of Malus spp. in Sichuan, China

Phil Forsline, Laura Benson and Herb AldwinckleSpecies Site(s) No. of accessions No. of seedsMalus prattii 01, 05, 06 22 1475 Malus transitoria 02 9 650Malus hupehensisZ 03, 04, 07 23 1100Malus toringoides 03, 04 16 900Malus kansuensis 03 13 800Malus sieboldiiZ05 7 375Malus zhaojiaoensis 06 11 1900Total -- 101 7200

Z Likely to be apomictic

Chinese Malus spp. screened for fire blight in greenhouse and field: replanted for horticultural evaluation

Greenhousefire blight screen

Temporary field location for gradstudent L. Bensonand field f.b. screen

Digging sdgsfollowing gradstudent projectand f.b. screen

‘Permanent’ fieldlocation for Horticulturalevaluation

Many of these sdgs distributedto 10 arboreta for preservation

Screening 7 Malus species from Sichuan, China for fire blight, apple scab and cedar apple rust

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Speciesscreened

for fireblight

All spcecis (388 sdgs)

M. hupehensis (96 sdgs)

M. sieboldii (53 sdgs)

M. transitoria (18 sdgs)

M. toringoides (900 sdgs)

M. zhaojiaoensis (43sdgs)

M. kansuensis (32 sdgs)

M. yunnanensis (56sdgs)

These seedlings were also screened for: Apple scab – 385 seedlings were screened

and 98% of them were resistant M. hupehensis (03&04), M. toringoides

(04), M. kansuensis, M. zhaojioensis, and M. sieboldii were mostly ‘A-type’ resistance

M. hupehensis (07), M. transitoria, M. toringoides (03) and M. yunnanensis were a mix of ‘A-type and P- type’ resistance

M. sieboldii (91% resistant) was the only species with some susceptible seedlings

Cedar apple rust – 370 sdgs were screened and 93% of them were resistant. M. sieboldii (55% resistant) was the

only species with some susceptible seedlings

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Variability of fire blight resistance in M. hupehensis and M. toringoides collected

from different sites in Sichuan, China

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Sites in Sichuan,China

M.toringoides -site 4 (51sdgs)M.toringoides -site 3 (39sdgs)

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Sites in Sichuan,China

M.hupehensis -site 3(27sdgs)M.hupehensis -site 4 (38sdgs)M.hupehensis -site 7 (30sdgs)

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Apple scab resistance in 5 Malus species collected in Sichuan, China in 2002 by M. Geibel

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Malus species

M. hupehensis (12 sdgs;4 populations)M. kansuensis (11 sdgs;2 populations))M. sieboldii (13 sdgs; 1population)M. toringoides (24 sdgs;4 populations)M. transitoria (133 sdgs;6 populations)

Fire blight and cedar apple rust screen is in process

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Malus orientalis in Russian Caucasus

Vavilov Inst. St. Petersburg

Malus orientalis screening for apple scab, cedar apple rust, and fire blight

Seedlings germinatedSeedlingsscreened for apple scab

Seedlings screened for fire blightSeedlings in high density orchardfor horticultural evaluation

Malus orientalis in Turkey

Local type‘Seker Elmasii’Sugar apple

Q 42413

Forsline, Aldwinckle & 6 Turks

Screening populations of M. orientalis from Russian Caucsus, Turkey and Armenia for fire

blight, apple scab and cedar apple rust

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Russian Caucasus Turkey Armenia

Apple scab Fire blight Cedar apple rust

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No. of sdgs screened Russia Turkey Armenia

Scab 238 786 191

F.B. 206 565 77

C.A.R. 129 191 TBD

27 populations 62 populations 7 populations

Screening 5 populations of European wild crabapple (M. sylvestris) received from

gene bank in Dresden, Germany

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Diseases screened

Apple scab ( 85 sdgs)

Fire blight (107 sdgs)

Cedar apple rust (85sdgs)

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Expedition to Russia to exchange sour cherry and cherry rootstock for crop improvement, July 10-30, 1998

A. Iezzoni, R. Karle, P. Forsline and M. Fischer

Breakdown Orel Michurinsk

Krymsk Prunus nursery

G. & V. Eremin; M. Fischer

Cherry accession in Orel

A. Iezzoni

Cherries in St. Petersburg

Prof. Yushev M. FischerR. Karle

Expedition to Russia to Exchange sour cherry, cherry rootstock, and Malus germplasm for crop improvement

July 10-30, 1998A. Iezzoni, R. Karle, P. Forsline and M. Fischer

Goals of expedition1) tart cherry elite germplasm at two sites (Orel, and Michurinsk)2) broad spectrum Prunus spp (Krymsk)3) Germplasm in 1 & 2 with resistance to ‘cherry leaf spot’ (Blumeriella

jaapii) and ‘twig brown rot’ (Monilinia laxa)4) wild apple collections (Maikop)

Trip participants1) Amy Iezzoni and Renate Karle, Michigan State University2) Philip Forsline, USDA-ARS Plant Genetic Resources Unit3) Manfred Fischer, Genebank OBST, Dresden-Pillnitz, Germany

Material collected1) Orel – 25 accessions2) Michurinsk – 12 accessions3) Krymsk – 24 accessions4) Maikop area – 28 seed lots (6500 seeds) of wild M. orientalis

Vitis collections in Kazakhstan in 1993, 1995 and 1996 in sites 6 and 11

Accessions collected•1993 – 17 populations: 8324 seeds and 10 local cultivars (cuttings)•1995 – 33 populations: 5723 seeds•1996 – 1 population: 318 seeds

Germplasm Utilization and Enhancement

Presence / absence of RAPD markers for 4 scab-resistance genes in elite clones of Malus sieversii

S. Mehlenbacher and N. Weeden

Scab-resistance Genes / RAPD Markers

Vr Vm Vf Vb

Accession No. P415B UBC562 OPB12 CS5 UBC220

GMAL 4326 YES YES NO NO GMAL 4327* YES YES YES YES GMAL 4331* YES YES NO NO GMAL 4333* YES NO NO NO GMAL 4334* YES YES NO NO

* These have been used as pollen parents X ‘Gala” (see next slide)

Germplasm enhancement to studygenetics of apple scab resistance

1. Gala X M. sieversii (GMAL 4335*) – 67% of 230 sdgs resistant2. Gala X M. sieversii (GMAL 4448*) – 57% of 209 sdgs resistant3. Gala X M. sieversii (GMAL 4455*) – 52% of 209 sdgs resistant4. Gala X M. sieversii (GMAL 4331Z) – 29% of 90 sdgs resistant5. Gala X M. sieversii (GMAL 4334Z) – 24% of 206 sdgs resistant6. Gala X M. sieversii (GMAL 4333Z) – 11% of 136 sdgs resistant7. Gala X M. sieversii (GMAL 4327Z) – 9% of 209 sdgs resistant

Total – 38% of 1289 sdgs resistant

*These clones are scab resistant; A-type vs. B-type resistance of sdgs from these crosses in ~ 1:1 ratio

Z >50% of progeny from these clones are scab resistant: clones themselves are not; Sdgs from crosses are all A-type resistance

Selected publications (1 & 2) and media (3 & 4): The origin of apples

Horticultural Reviews, vol. 29. 2003. “Wild Apple and Fruit Trees of Central Asia”. Wiley, New York. J. Janick, P. Forsline, E. Dickson, R. Way and M. Thompson (eds.).

Chapter 1 – “Collection, Maintenance, Characterization and Utilization of Wild Apples of Central Asia”, p. 1-62. P.L. Forsline, H.S. Aldwinckle, E.E. Dickson, J. J. Luby, and S.C. Hokanson. 2003. Chapter 2 - Translation from Russian: “The Wild Apple Tree of Kazakhstan”, p. 63-304. A.D. Djangaliev Chapter 3 – Translation from Russian: “The Wild Fruit and Nut Plants of Kazakhstan”, p. 305-371. A.D. Djangaliev and T.N. Salova

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Conclusions and Future Directions1) Apple

Gene pool greatly increased with collection of wild species with passport data …..Make selections of seedlings w/ unique traits

Planning additional collections of wild species in Southwest China Evaluate 7 populations (1300 seedlings of Gala X M. sieversii crosses)

2) Grape Need to make additional collections in China and North America Grow out and evaluate collections made in Kazakhstan Expand cryopreservation research w/ NCGRP

3) Sour Cherry Gradually building collection based on evaluations of Dr. A. Iezzoni of

Michigan State University4) Activities

Continue morphological characterization on current as well as additional descriptors

Continue digital imaging of fruit samples for all accessions and begin digital imaging of leaves, flower and tree

Molecular characterization – in house and collaboration

ACKNOWLEDGEMENTS Professor Aimak Djangaliev, Kazakstan

Academy of Sciences, Almaty, Kazakstan; Host for Expeditions

USA and international scientists who provided data on evaluation progress

NPGS Plant Exploration Office that provided funding for expeditions: C. Sperling, K. Williams, N. Garvey

NPGS administrative leadership: H. Shands, A. Stoner, P. Bretting

NPGS GRIN program personnel USDA- ARS International Programs: R. Soper, R. Bennnet, E. Rosenquist USDA- ARS National Center for

Germplasm Resources Preservation: S. Eberhart, L. Wiesner, H. Shands, G. Volk, L. Towill, C. Stushnoff, C. Walters

Apple, Grape and Prunus Crop Germplasm Committees (CGC)

All plant exploration team members PGRU administrative leadership:

S. Kresovich, J. McFerson, W. Lamboy, C. Simon

PGRU administrative assistants: D. Emerson, T. Fisk, S. Walburn

Staff assigned to Clonal Repository of PGRU

Former CR technicians who completed cryo & virus indexing: Sheffer & Holleran

Cornell staff responsible for disease screening: H. Aldwinckle, H. Gustafson, T. Momol

Cornell and other SAES scientists with SCAs to evaluate germplasm

PGRU Grape Genetics Staff Horticulture Sciences staff at NYSAES

for early oversight of the CR establishment and continuing activities