Twenty Years of Germplasm Management Geneva NY to

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 Cornell Geneva New office wing and greenhouses PGRU facilities expanding Greenhouse & screenhouse for clonal collections

History of facilities: Clonal repository at PGRU Campus Bldg 1985 Equipment storage, 1984 Farm preparation, 1984 Campus Bldg 1985 Tile drainage 1984

Farm development 1985 to present Clonal collection with developing plantings 1990: addition of farm for seed section of PGRU 1985 prior to planting 2002 from west to east 2002 from east to west

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; 1 st propagations of Malus and Vitis 1984 – Curator hired; campus building started 1986 – CR building occupancy and dedication; 1 st orchard and vineyard plantings; merger with NERPIS 1987 – S. Kresovich hired as 1 st Research Leader for NERPIS and CR – new name: Plant Genetic Resources Unit (PGRU) 1988 – J. Mc. Ferson hired as curator of seed group; Cryopreservation project began; 1 st of seven germplasm expeditions initiated § § § § § 1989 – 40 acres purchased for seed group 1993 – J. Mc. Ferson 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/Geneticist Joanne Labate: Molecular Biologist

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 Program Gennaro Fazio, USDA/ARS, PGRU in cooperation with Cornell University

Staff for clonal collections at PGRU § SYs - P. Forsline FTE 1. 0 Horticulturist/Curator - C. Simon 0. 85 Research Geneticist / RL - H. Schwaninger 1. 0 Molec. Biol. / Support Sci. § Database - R. Nearpass 0. 40 Supervisory IT specialist - D. Dellefave 1. 0 Germplasm Program Assistant § Technical FTE - W. Srmack 1. 0 Collections 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)

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 Unit Geneva, 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 clusters Diversity among clones

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. ) Early / late bloom P. cerasus / P. fruticosa spreading upright

Cherry digital imaging Montmorency Balaton Danube Schatten Morello

Malus collection at Plant Genetic Resources Unit Geneva, 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 Fruit Bloom Late leaf fall Bloom M. baccata Dormant “Kansas 14” Early leaf fall

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 after establishment in 1986 Established plantings at bloom Established plantings summer Tree removal for conversion to EMLA 7

Digital imaging of Malus clones Gala Belle de Boskoop Geneva Malus hupehensis

Malus core collection at PGRU Label system M. transitoria Planting in 2000 Core collection in bloom: 2001 Apples/seedling clean infected Apples/EMLA 7 Core collection fruiting: 2001 Extension orchard 02 Success w/ core collection in discovering new virus indicator

Malus / EMLA 7 collection at PGRU Planting in 2000 Apples/seedling Apples/EMLA 7 Establishment 2001 Extension orchard, 2002

Fire blight challenges and control in the Malus collection at PGRU Heavy FB on blossoms; 2000 No ‘Apogee’ treatment Severe FB epidemic; 1996 425 trees removed ‘Apogee’

Codes for fire blight, shoot. Y (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 YCumulative records 1990 – 2003 for entire ‘clonal collection’ 1091 2351

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 Malus A back up collection at $1/accession/yr! Base collection (2146 acc. ) at NCGRP, Ft. Collins, CO Processing 35 mm bud segments Recovery of accessions by bud grafting Active collection (436 acc. ) at PGRU Geneva, NY Hundreds of seedlings budded with cryopreserved buds testing viability

Cryopreservation of Prunus Base collection (52 acc. ) at NCGRP, Ft. Collins, CO Injury to primary bud following LN exposure Active collection (8 acc. ) at PGRU Geneva, NY Typical recovery scenario from axillary bud

Pilot Project: Forsline, P. L. , C. Stushnoff, L. E. Towill, J. W. Waddell, W. F. Lamboy and J. R. Mc. Ferson. 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 § 12/14/1990 – 12 accessions § 01/15/1991– 13 accessions 12/12/1989 – 6 accessions 01/19/1990 – 9 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 a. Z 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 eight. Y 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 Total 1993 86 8 5 99 1994 108 7 26 141 1995 167 25 50 242 1996 169 24 49 242 1997 223 71 70 364 1998 209 66 64 339 1999 120 55 71 246 2000 108 46 84 238 2003 52 6 56 114 2004 22 2 97 121 Total 1264 310 572 2146

No. of accessions in storage Successful cryopresevation for > 90% of accessions stored at NCGRP: those with < 30% viability will be reprocessed 171 or 9% of total were unsuccessful 0 -10 20 -30 1760 or 91% were successful 40 -50 60 -70 80 -90 100

No. of accessions in storage Successful cryopresevation for > 95% of accessions stored at PGRU: those with < 30% viability will be reprocessed 18 or 4% of total were unsuccessful 0 -10 20 -30 418 or 96% were successful 40 -50 60 -70 80 -90 100

Distribution of Germplasm

Annual distribution of germplasm from 1988 to 2004 No. of accessions annually Z Apple total no. = 41, 111 Apple total orders = 1514 (mean: 89) Apple orders/yr (range: 30 -163 Z) mean = 2418 mean = 692 Z Grape total no. = 11, 765 Cherry distributions started Grape total orders = 782 (mean: 46) in 1998: 191 accessions to 21 orders Grape orders/yr (range = 14 -72 Z )

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) 2) 3) 4) 5) Preservation of alleles from wild collections: Volk, Walters, Richards Antioxidants in Malus collection: Stushnoff Evaluation of wild apple species for disease resistance: Aldwinckle Evaluation of elite M. sieversii and some of its hybrids for apple scab resistance genes: Aldwinckle, Luby, Gardiner and Bus 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) 2) 3) 4) Evaluation of Vitis for susceptibility to crown gall: Burr Evaluation of Vitis for resistance to Phomopsis viticola and powdery mildew: Wilcox and Reisch Evaluation of Russian tetraploid cherry selections for cherry leaf spot resistance: Iezzoni 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 (Ph. D projects) § Grape genotyping – Lamboy § Grape phylogeny – Schwaninger § Overall coordination presently – Simon (Research leader / molecular geneticist)

New Acquisitions

The origin of the cultivated apple Ancient Malus species of China: bird disseminated to Central Asia M. sieversii of Central Asia Mammal disseminated North America became a secondary center of origin: ‘Red Delicious’, ‘Golden Delicious’, etc. Johnny Appleseed Dr. B. Juniper: Theory on early and recent evolution of the cultivated apple

Germplasm collections to add mostly wild Malus species to the PGRU collection Year Country Species Personnel 1987 – 1988 Western U. S. & Canada Eastern 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) ‘The late’ Calvin Sperling Elizabeth Dickson Uzbekistan Herb Aldwinckle

Professor Djangaliev Geneva: making plans, 1992 Collection admiration, 1995 Organizing expedition: first morning, Sept. 1993; ready to leave to board helicopter One of frequent lectures; 1996 Local Kazak media highlighting 1995 American & S. African invasion

1993 collection team: USA & New Zealand Noiton Preparation for departure Mink Dickson Transfer in Moscow – hotel and visit to Red Square Significant travel by helicopter Moscow to Alma Ata via ‘Aeroflot’ Team in Alma Ata with Kazakh hosts

1995 USA and S. African collection team Dickson Britz Forsline Luby Human Arrival at site 9 Meals in yurt: site 5 Seed extraction at hotel: site 9 Housing in sanitarium at HQ in Almaty Gala dinner with hosts in Almaty: return to USA

1996 USA collection team Hokanson Unruh Forsline Pellett HQ in Almaty Site 5 Site 9 Site 12

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 Asia Country/Region Site Lat o. N / Long o. E Elevation (m) Rainfall (mm) Tajikistan / --- 1 39 / 68 --- Uzbekistan/ --- 2 41 / 69 --- Kazakstan/Zailisky 3 43 / 77 1170 -1690 700 Kazakstan/Djungarsky 4 5 45 46 / 80 / 81 1170 -1760 1190 -1360 800 850 6 11 43 42 / 70 600 -910 780 -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 Kazakstan/Karatau

Kazakhstan site 3: ’ 89, ’ 93, ’ 95, ‘ 96 Habitat 1940 Studies by Professor A. Djangaliev Habitat present Depleted by dachas Fruit from 30 randomly-collected trees

Kazakhstan site 4, ’ 93, ’ 95, ‘ 96 Scab infected tree

Kazakhstan site 5: ’ 93, ’ 95, ‘ 96 Forestry camp at 1200 m headquarters in ’ 93, ’ 95 & ‘ 96 Apple forest: 1800 m 1100 m Bear scat w/ many apple seeds First collection morning (Sept. 1993) starting out at 600 m with hike to 1800 m 300 yr old M. sieversii

Kazakhstan site 6: ’ 93 & ’ 95 Camp at 600 m: headquarters Standing at 900 m Fruit from 30 randomly-collected trees at 900 m M. sieversii at 900 m

Kyrgyzstan site 7: 1993 Apple and walnut forests as seen from helicopter Village in Kyrgyzstan near collection sites Heavy grazing in M. sieversii areas

Kazakhstan site 9: ’ 95 & ‘ 96 Grazed areas – M. sieversii on slopes Diverse, elite M. sieversii Super-elite M. sieversii Fruit from 30 randomly-collected M. sieversii trees Heavy grazing of habitat

Kazakhstan site 10: 1996 Breakdowns; common occurrence M. niedzwetzkyana Uygur tribes tradition of bread baking Fruit from 20 randomly-collected trees

Kazakhstan site 11: 1996 Elite ‘Yellow transparent’-type M. sieversii

Kazakhstan site 12: 1996 Canyon 400 m deep w/ M. sieversii on the N-facing wall Bottom of canyon; M. sieversii on this side Trail to bottom Fruit from 10 randomly collected M. sieversii trees

ACCESSION INFORMATION RECORDED + Fruit Over Color/Intensity + Fruit Ground Color + Fruit Russet + Fruit Shape + Stem Character + Fruit Size + Fruit Texture + Fruit Flavor + Harvest Season + Tree Habit + Diseases/Insects: Leaves/Fruit

Variability of fruit size of Malus sieversii among sites in Central Asia No. collected Site / Yr(s) 12 / (‘ 96) * 5 / (‘ 96) 9 / (‘ 95/’ 96) 11 / (‘ 96) 10 / (‘ 96) * 5 / (‘ 95) 3 / (‘ 95/’ 96) 4 / (‘ 95/’ 96) 6 / (‘ 95) Totals Mean size (mm) Elite Random 2 14 57 13 2 14 11 10 Size range (mm) Elite Random 25 10 54 150 40 20 60 80 87 64 65 58 56 55 49 46 45 44 42 42 35 43 42 40 36 34 34 41 60 -74 54 -65 46 -72 44 -76 47 -51 37 -56 32 -55 33 -46 32 -50 25 -49 28 -62 29 -63 29 -51 27 -48 26 -49 28 -44 28 -54 148 565 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 Obtained Seeds Obtained 1989 and 1993 Collections 179 33, 000 1995 and 1996 Elites 148 z Group 1995 and 1996 565 Random populations Totals 892 Seeds Distributed Storage at PGRU Storage at NSSL 9200 13, 900 10, 900 67, 000 16, 000 29, 200 21, 800 30, 000 4100 19, 100 130, 000 28, 300 Y 62, 200 6800 x 39, 500 z 44 of best accessions also obtained as clones Y Distributed to 24 evaluators x 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 H. Aldwinckle PGRU - P. Forsline - S. Hokanson - W. Lamboy - G. Fazio - L. Benson N. Weeden S. Mehlenbacher W. Reissig Aldwinckle / Korea S. Brown I. Merwin Disease resistance Horticultural / Molecular Isozyme / Molecular “ / “ Insect resistance Rosellinia / Helicobasidium Genetic dwarf Nematode resistance No. of Seedlings 5124 Z part of 5124 400 250 200 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 NJ / Goffreda CO / Stushnoff WA / Barritt OHZ / Lynd, Miller WI / Smith AR / Rom AK / Mc. Beath IL / Korban Hort, Disease, Cold H. Hort, Disease, Molecular, Elite clones Hort, Cold H. , Antioxidants Hort, Sunburn, Cold H. Hort, Disease, Late Bloom, Elite clones Hort, Disease, Cold H. Hort, Disease, Late Bloom Hort, Disease, Cold H. Hort, Disease, Molecular 1498 Y 1899 Y 720 624 Y 950 Y 655 Y ~400 346 ~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 Country / Lab New Zealand / Bus, Noiton, Gardiner Germany / Buttner, Geibel, Höfer Norway / Roen N. S. , Canada / Deslauriers, Embree Man. , Canada / Davidson B. C. , Canada / Quamme, Hampson N. B. , Canada / Hunter Japan / Bessho UK, Reading / Farrel UK, Oxford / Juniper S. Africa / Human, Britz Netherlands / Kemp Italy / Sansivini Evaluation type Disease, Hort Disease, Hort, Cold H. Disease, Hort Molecular Dis. , Insect, Sunburn, Chilling Elite clones Z Parallel collections were made in 1995 expedition in Kazakhstan No. of seedlings 4426 1367 692 1155 169 325 ~120 ~100 ~300 ~150 ? Z ---

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 resistance H. 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 6 5 9 7 2 11 3 12 10 1 405 705 1224 1325 383 101 244 460 133 123 21 50 37 29 28 25 24 23 17 14 6 5 Totals 5124 28

Wild Malus sieversii seedling plantings 1989 collection Early stage after planting 1986 1993 collection M. sieversii in bloom 1995/96 collections More advanced stage (1200 sdgs) M. sieversii fruiting M. niedzwetzkyana form of M. sieversii

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

Total % of population resistant Cedar apple rust resistance in M. sieversii seedlings from 8 different sites in Kazakhstan 9 6 4 3 5 11 10 12

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

% of trees in each category Natural occurrence of fire blight in M. sieversii seedlings from 7 different sites in Kazakhstan 9 9 6 6 Codes 1 & 2 Codes 4 & 5 Fire Blight Severity

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 (Lx. W in mm) Fruit weight (g) § § § § Fruit russet type Fruit russet location Fruit russet intensity 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 PI 613972 clone in bloom PI 613978 & PI 613975 clones fruiting More advanced stage 13 different M. sieversii clones Fruit from half-sib seedlings of some of the M. sieversii clones vineyard ‘Empire’ ‘Gala’

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 % of Red -able trees w/ intensity flavor red fruit 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 2004 9 Elite clones 11 10 Aug-13 Sep 9. 5 -14. 1 10 9 Seedlings 159 8 Aug-17 Sep 8. 0 -17. 9 78 6 Seedlings 138 8 Aug-3 Nov 8. 9 -15. 9 41 5 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 52 3 Seedlings 5 14 Aug-10 Sep 8. 5 -10. 5 19 4 Seedlings 7 14 Aug-17 Sep 9. 0 -12. 1 80 10 Seedlings 20 8 Aug-23 Sep 8. 7 -12. 2 32 4, 5, 6, 11, 12 Elite clones 5 22 Aug-10 Oct 9. 8 -15. 3 11

% of seedlings in each category Fruit shape in M. sieversii seedling populations from 6 sites in Kazakhstan

Digital imaging of Malus sieversii seedlings From site 5 in Kazakhstan From site 9 in Kazakhstan From site 6 in Kazakhstan From site 11 in Kazakhstan

Digital imaging of Malus sieversii clones From site 5 in Kazakhstan From site 12 in Kazakhstan From site 6 in Kazakhstan From site 9 in Kazakhstan

Elite clone (PI 613971) and progeny (3 half-sibs) Clone Seedling. a Seedling. b Seedling. h

Elite clone (PI 613976) and progeny (3 half-sibs) Clone Seedling. k Seedling. a 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 team Geneva, 11/96: Prof. Li; plans Crew collecting minutes before monkey thievery Hotel in Chengdu; Prof. Li & wife; H & B Aldwinckle monkey Crew at 3500 m pass in mountains Laura Benson Post expedition conference with Yunnan and Guizhou researchers in Chonqing

Malus sp. collections in Sichuan, China Malus kansuensis M. hupehensis M. toringoides M. prattii

1997 collections of Malus spp. in Sichuan, China Phil Forsline, Laura Benson and Herb Aldwinckle Species Malus prattii Malus transitoria Malus hupehensis. Z Malus toringoides Malus kansuensis Malus sieboldii. Z 05 Malus zhaojiaoensis Total Z Likely to be apomictic Site(s) No. of accessions 01, 05, 06 22 02 9 03, 04, 07 23 03, 04 16 03 13 7 06 11 -101 No. of seeds 1475 650 1100 900 800 375 1900 7200

Chinese Malus spp. screened for fire blight in greenhouse and field: replanted for horticultural evaluation Greenhouse fire blight screen Digging sdgs following grad student project and f. b. screen Many of these sdgs distributed to 10 arboreta for preservation Temporary field location for grad student L. Benson and field f. b. screen ‘Permanent’ field location for Horticultural evaluation

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

3 4 7 M. hupehensis % of sdgs fire blight resistant Variability of fire blight resistance in M. hupehensis and M. toringoides collected from different sites in Sichuan, China 4 3 M. toringoides

transitoria toringoides sieboldii kansuensis hupehensis % of seedlings scab resistant Apple scab resistance in 5 Malus species collected in Sichuan, China in 2002 by M. Geibel Fire blight and cedar apple rust screen is in process

Malus orientalis in Russian Caucasus Vavilov Inst. St. Petersburg

Malus orientalis screening for apple scab, cedar apple rust, and fire blight Seedlings germinated Seedlings screened for apple scab Seedlings screened for fire blight Seedlings in high density orchard for horticultural evaluation

Malus orientalis in Turkey Forsline, Aldwinckle & 6 Turks Local type ‘Seker Elmasii’ Sugar apple Q 42413

Screening populations of M. orientalis from Russian Caucsus, Turkey and Armenia for fire blight, apple scab and cedar apple rust No. of sdgs screened 27 populations 62 populations 7 populations 238 786 191 F. B. Mostly A-type Scab A/B 2: 1 A, B, P-type; 3: 1: 2 ratio % of seedlings resistant Russia Turkey Armenia 206 565 77 C. A. R. 129 191 TBD

% of seedlings resistant Screening 5 populations of European wild crabapple (M. sylvestris) received from gene bank in Dresden, Germany

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 Cherries in St. Petersburg Prof. Yushev Breakdown Orel Cherry accession in Orel M. Fischer R. Karle Michurinsk A. Iezzoni Krymsk Prunus nursery G. & V. Eremin; M. Fischer

Expedition to Russia to Exchange sour cherry, cherry rootstock, and Malus germplasm for crop improvement July 10 -30, 1998 A. Iezzoni, R. Karle, P. Forsline and M. Fischer § Goals of expedition 1) 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 participants 1) Amy Iezzoni and Renate Karle, Michigan State University 2) Philip Forsline, USDA-ARS Plant Genetic Resources Unit 3) Manfred Fischer, Genebank OBST, Dresden-Pillnitz, Germany § Material collected 1) 2) 3) 4) Orel – 25 accessions Michurinsk – 12 accessions Krymsk – 24 accessions 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 scabresistance genes in elite clones of Malus sieversii S. Mehlenbacher and N. Weeden Scab-resistance Genes / RAPD Markers Accession No. GMAL 4326 GMAL 4327* GMAL 4331* GMAL 4333* GMAL 4334* Vr P 415 B UBC 562 YES YES YES Vm Vf Vb OPB 12 CS 5 UBC 220 YES YES NO NO YES NO NO NO * These have been used as pollen parents X ‘Gala” (see next slide)

Germplasm enhancement to study genetics of apple scab resistance 1. 2. 3. 4. 5. 6. 7. Gala X M. sieversii (GMAL 4335*) – 67% of 230 sdgs resistant Gala X M. sieversii (GMAL 4448*) – 57% of 209 sdgs resistant Gala X M. sieversii (GMAL 4455*) – 52% of 209 sdgs resistant Gala X M. sieversii (GMAL 4331 Z) – 29% of 90 sdgs resistant Gala X M. sieversii (GMAL 4334 Z) – 24% of 206 sdgs resistant Gala X M. sieversii (GMAL 4333 Z) – 11% of 136 sdgs resistant Gala X M. sieversii (GMAL 4327 Z) – 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 >50% of progeny from these clones are scab resistant: clones themselves are not; Sdgs from crosses are all A-type resistance Z

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

Conclusions and Future Directions 1) Apple § § § 2) 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) Grape § § § 3) Need to make additional collections in China and North America Grow out and evaluate collections made in Kazakhstan Expand cryopreservation research w/ NCGRP Sour Cherry § 4) Gradually building collection based on evaluations of Dr. A. Iezzoni of Michigan State University 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. Mc. Ferson, 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