Hop i Sill yii 4400 2009. tamed culttvars introduced to or selected in North America. Identification of Historic Apple Trees in Modern commercial apple production requires consistency of ripening time, quality retention during processing and shipping, and the Southwestern United States and long storage life, and not all varieties can meet these criteria (Golanij and Bauer. 2004). Implications for Conservation Market pressures have reduced the diversity Kanin J. Routson' of fnut trees once grown in small fiumily orchards- -where diversity of ripening time, Arid Lands Resource Sciences, Unii'ei'sit-t' ofA rizona, 1955 East Si.itli Street, sizes, textures, and flavors were celebra- P.O. Box 210184, Tucson, AZ 85719 ted—to only a few handfuls of commonly planted commercial . Curi'enil y. I Ann A. Reillev, Adam D. Henk, and Gayle M. Volk apple cultivars account for over 90% of the National ('enic,' fin' Genetic Resources Preservation, U. S. Depai'/rnenr of sold in the United States, with 'Red Agriculiui'e, Fort Collins, CO 80521 Delicious' constituting 41% of this hgure Dennis, 2008). In The Fruit. Bern' and A'te! Ai/i/ioonal iiidrv words. .tioliv. simple sequence repeat. genetic diversity J;ii anton- ( Whcalv. 2001) Kent Whealy lists 1500 apple varieties cun'entiv available Abstract. lany apple varieties commonl y planted in the United States a century ago can through U.S. nurseries, many of which have no longer be found in toda y 's orchards and nurseries. Abandoned farmsteads and been developed through modern fruit breed- historic orchards harbor considerable agrobiodiversity , but the extent and location of ing. This suggests a substantial decrease in that (li%ersitv is poorl y understood. We assessed the genetic diversit y of 280 apple (Ma/xis the number of apple cultivars offored through xdo,,iestiea Borkh.) trees growing in 43 historic f'ai'nisktad and orchard sites in Arizona, U.S. nurseries over the past century (779/a by Utah, and New Mexico using seven microsatellite markers. We compared the samples to Ragan's calculations and 89% by Bussey's), 109 cultivars likel y introduced into the southwest in the late 1901 and earl y 20th although we do not know to what extent this centuries. Genetic anal y sis revealed 144 genoty pes represented in the 280 field samples. naming actuall y represented genetically dis- \\e identified 34 of these 144 genot y pes as cultivars brought to the region bN , Stark tinct cultivars. Brothers Nursery and b y USDA agricultural experiment stations. One hundred twenl y of Although tue loss of'on-farni diversity can the total samples (43%) had DNA fingerprints that suggested the y were representative of be lasting, fruit trees have all over these 34 ctiltivars. The remaining 160 samples—representing 110 genotspes—had annual crops because these trees can live to unique fingerprints that did not match all the fingerprinted cultivars. The results remarkably old ages, surviving some fads in of this study confirm for the first time that a high diversit y of historic apple genotypes consumer demand. Sin gle apple trees have remain in homestead orchards in the U.S. southwest. Future efforts targeting orchards in been known to live 150 years or longer, in the southwest should focus on conservation for all unique genot y pes as a means to sustain triany areas, it is still possible to find trees of both cultural heritage and biological genetic diversity. "IlCirloom" cultivars once abundant at the beginning of the 20th century. Remnant orchards planted before the "modern era" The late 19th century is often referred to desirable . and those that were amena- of fruit production (Jackson. 2003) hang on as the golden years of apple growing in the ble to cooking and baking. This period of tenaciousl y around abandoned I'll rtii stead s United States (Calhoun. 1995: Hensle y, 2005). American horticultural history was preceded and historic orchards. Although farmstead Farmstead and kitchen orchards were planted by an era of fruit diversification, which trees often persist without their original with a wide variety of fruit trees to suit spanned much of the I 700s and into the early names being retained, they represent a snap- diverse family needs. Historically. rural live- I 800s. Thousands of trees emerged horn shot of the diversity of fruit varieties avail- lihoods were maintained b growing apples y seedling orchards, planted for and for able over a century ago during the peak of and pears that ripened in summer, would animal feed, that bore high-quality fruits fi'uit tree diversification. "keep" all winter in the cold cellar, produce worthy of naming. conserving, and distribut- Morphological and taxonomic traits typ- ing. These trees were clonally propagated ically used to differentiate between apple from cuttings and traded and sold to become eultivars call ambiguous as a result of Received for publication 16 Dec. 2008. Accepted elements of the diverse orchards of the 19th the broad phenotypic variation under differ- for publication 6 Feb. 2009. century (Beach. 1905: Hedrick, 1950). The ent environmental influences. Furthermore, This project was pat of Kanin Routson's Master's last remnants of 19th century plantings may many 19th century apple cultivars are mor- research at Northern Arizona Universit y. Thank still he alive in landscapes and tire phologically similar to one another and accu- you to Gary Nahhan, Thomas Sisk-, David Osterg- the subject of this regional southwestern rate descriptions are often lacking. making ren, Gery Allan, and other staff at NAU who helped survey. with this study. We also express our gratitude to all conventional identification methods difficult, who have provided funding for this project: the USDA pomologist W.H. Ragan under- if not impossible, for these centur y-old trees. National Park Service and the Margaret T. Morris took the task of recording the names and Genetic fitigerprinti ng. including micro- Foundation. [it we are grateful to Lee characteristics of every apple grown satellites, have become powerful and accu- Calhoun, Ram Fishman, and Philip Forsline for in the United States during the 19th century. rate tools for analyzing genetic diversity sending leaf samples of named varieties used in this In his book, The Nonzenc/ai,o-e q/ the Apple (Hammer et al., 2003). Microsatel lite loci, study and to Christopher Richards for use of (Ragan. 1905). Ragan lists 6654 unique or simple sequence repeats, are short nlicle- facilities at USDA-ARS-NCGRP. Lastly, we thank named apple varieties that lie found refer- otide sequences of up to six hasepairs Gerard Geenen of the Plant C yiornetry Services enced in U.S. literature between the years repeated in Schipnde I. The Netherlands) for determining ploidy tandem, head to tail, without 1804 and 1904. In 1980, [)an l3usse y began for the samples and reference culiivars. interruption. They are highly polymorphic. expanding on Ragan 's register to update Mention of trade nanles or coillmc'rcj at products in codominant markers that have been detected this article is solely for the purpose of providing descriptions of known cultivars and include in every organism thus far studied (I lancock, specific information and does not imply reeom- additional apple cultivars referenced in the 1999). Gianfranceschi et al. (1998), l-Iokanson niendation or endorsement by the U.S. Department U.S. literature up to the year 1980. Over a et al. (1998), and 1-Jemmat et al. (2003) were of Agriculture. decade after beginning this project, Bussey instrumental in developing Ma/us-specific To whom reprint requests should be addressed: (in press) is close to releasing The App/c' in microsatellites in the United States. In e-nlai I kjr53aemail .arizona.edu. North America, which lists over 14,000 Europe. Si lfverberg-Di lworth ci al. (2006)

H0RTSClrNcE Vor. 44(3) JUN! 2009 589 and the II igli-Qual ity Disease Resistant Apples for Sustainable Agriculture have been forefront in developing microsatellites for Ma/us. In a similar study in Spain. Pereira- Lorenzo et al. (2007) evaluated the genetic diversity of 114 Spanish apple cultivars comparing the local Spanish landrace geno- types with 26 commercial apple cultivars found in the region. Similar to this study. Pereira-Lorenzo et al. (2007) found high Utah Colorado levels of genetic diversity in apple trees in I northern Spain based on observed heterozy- 42-43 • 41 gosity. Capitol Reef •" In this study, we sampled tissues from 280 apple trees from 43 historic sites within Arizona. Utah. and New Mexico to assess their diversity. Genotypes identified using seven highly variable markers were com- Taos 38-40 pared with reference genotypes of known 4 eultivars. The term "historic" is used in this text to refer to farmstead and orchard sites 26-33 Santa Fe planted during the late 19th and early 20th 25 Albuquerque centuries, and the term "heirloom" refers to Flagstaff cultivars introduced during the 19th century 21-24 .. and before as opposed to recent introductions Prescott 34-37 . developed through modern fruit breeding programs. 1-19 • 20 Materials and Methods Arizona I New Mexico Field co//eciwns. We collected leaf sam- ples from 280 apple trees located in 43 historic orchard sites on public and private (hut not tribal) lands in Arizona, New Mexico, and Utah (Fig. 1). Sampling took place from June through September of 2007. We targeted Fi g 1. I lisioric apple collection sites (numbered and noted h (tiamonds ) in Utah, Arizona. and Ncs places with presumed historic orchards and Mexico were sampled. Cities are provided for reference purposes (star designation). trees Of Visually differing morphologies. Local experts provided site location information. Sampling permission was obtained and sam- pies were used only for this study, not for gene num tags were nailed to the trees with a Polymerase chain reactions (P(.'Rs) were banking or crop improvement. numerical identifier. For each sample. 50 carried out in 15 p L total volume. Each 15 iL We focused sampling eiThrts primarily on mg of fresh leaf tissue was placed into a 96- reaction contained: 0.3 pL GoTaq Flcxi Taq historic farmstead and orchard sites dating well plate and frozen at —20 °C until extrac- Polymerase (Promega, Madison, WI; 5 units! back to the 1930s and earlier with priority tion. Known cultivars were obtained from the iL). 3 pL Promcga 5x Colorless GoTaq Flex given to trees planted before 1920. For a U SDA-ARS-Plant Genetic Resources Unit, Buffer (10 mu Tris-HCI, 50 mn KCI, and 0.5°/o handful of the orchard sites such as Capitol Geneva, NY (PGRU). Varieties that were Triton X- 100), 1.5 p of 0.25 mu MgCl 2, and ReelNational Park, UT, and Slide Rock State not available through the Geneva facility were 1.5 p of 0.25 inn dNTPs. Forward and reverse Park. AZ, orchard planting dates could be obtained from Lee Calhoiui of Calhoun's primers were added to a final concentration of found in historical documentation. Most of Nursery in Pittshoro, NC: Ram Fishman of 0.25 pM/reaction except for GD 12 at 0.3 pM! the orchard sites lacked written documenta- Greenmantle Nursery in Garberville. CA: and reaction and GD] 00 at 0.5 pM/reaction. tion, however, and we had to rely on oral Gordon 'Tooley of Tooley's Trees in Truchas, Genomic DNA, isolated as described pre- history or visual determination of tree size NM. Leaf samples were processed in the same viously, was added at 0.5 ng to 5 ng/reactlon. and locality for approximate ages. We avoided manner as the unknown samples. Reaction volumes were adjusted to IS gL sampling from seedling trees and rootstock Micrusatel/ile ana!i:is. The genetic anal- using sterile distilled 1-1 20. PCR reactions trees where it appeared the original grafted ysis of the 280 samples and 109 cLiltivars was were multiplexed with the followin g primer top had died. The dry southwest climate performed following procedures described in sets: GDI2. GD100: GD142, GDI47, limits establishment of naturalized seedling Volk et al. (2005). We extracted genomic GD162; and GD IS. GD96 nin together. apple trees and many farmstead orchards in DNA from the leaf samples using Qiagen PCR was carried out using Mi Research the southwest date to the late 19th to early DNeasy 96 plant kits (Qiagen. Valencia, PTC 200 Thermocycicr (Reno, NV). Ampli- 20th centuries, a time when few seedling CA). Two separate sets of DNA were extracted fications were done using touchdown PCR, in orchards were planted in the United States. from each sample and run independently. which the thermocycler reduced the anneal- Leaf samples were collected only from trees Ma/us-specific microsatellites were amplified ing temperature 1° every cycle, starting at that were visibly different from other trees at using unlinked primers (GD 12, GDI5, GD96, 63 °C and ending at 54 °C, followed by an the same site to avoid repeat sampling of GDIOO, GD142, GD147, and GD162) as annealing temperature of 55°C for 18 cycles cultivars. However, this was not always described by 1-lokanson et al. (1998) and by and ending with a 2 min 72 °C extension. possible for trees without fruit. I lemniat et al. (2003). Forward primers were PCR products were diluted 1:1 with a Global positioning system locations for the labeled with either IRD70() or IRDSOO infra- loading buffer of' formamide bromophenol sample trees (Gannin eTrex-Vista handheld red florescent dyes (MWG-Biotech, High blue loading buffer and were denatured at unit: Garmin, Olathe, KS) in UTM (Nad1983) Point, NC). Unlabeled reverse primers were 95 °C for 5 mm. Denatured products were coordinates were recorded, and small alumi- obtained from IDT (Coralville, IA). diluted 1:10 with additional loading buffer.

590 II )RTSc1ENCE VOL. 44(3) JUNF/ 2009 Table 1. Ploidy, origin, and probable date of release are provided for 109 apple cultivars introduced to the southwestern United Stales.' Cultivar name Ploidv Origin Date Southwest introduction' Reflrence' Akin 2x Illinois 186$ Stark 1912 Beach Albemarle Pippin 2x Long Island 1759 Stark 1912 Beach American Summer Peamiain 2x USA 1817 Stark 1912 Smith Antanovka 2x Russia 1826 Texas Smith 2x Arkansas 1870 Arizona, New Mexico, Texas Beach 3x Massachusetts 1784 Arizona. Texas Smith Bank's (iravenstein 2x Nova Scotia 1880 Stark 1912 Whealy Bedfordshire Foundling 2x England 1800 unk.' Smith 2x Southern U.S. Early 1800s Arizona. New Mexico. Texas Smith Benoni 2x Massachusetts 1830 Arizona, Ness Mexico Smith Black Amish 2x USA, Pennsylvania(?( unk. unk. Calhoun Black Ben Davis ((.iano) 2x Missouri 1880 TX Smith Black Oxford 2x Maine 1790 unk. Smith Boiken 2x Germany 182$ Stark 1912 Smith Bonum (Magnum Bonum ) 2x North Carolina 1954 Stark 1912 Smith Buckingham 3x Southern U.S. <1817 New Mexico, Texas Smith Camplield 2x New Jerse y <1817 unk. Smith Canada Red 2x Ness England 1822 Stark 1912 Beach Carter's Blue 2x Alabama 1869 New Mexico. Texas Smith Charlamoff (Duchess of Oldenburg) 2x Russia 1700s New Mexico, Texas Smith Chenartgo Strawberry 2x New York or Connecticut 1851 New Mexico Smith Chestnut Crab 2x Minnesota 1946 unk. Whealy ('ox's Orange 2x England 1850 Stark 1912 Smith Delicious 2x Iowa 1895 Stark 1912 Smith Dol go Crab 2x Russia <1897 unk. Whealy Duchess 2x Scotland? <1883 Stark 1912 Smith Earl y Harvest 2x USA ISOOs Arizona, New Mexico. Texas Smith Early Strawberry 2x New York 183$ Arizona Smith Esopus Spitzenhurg 2x New York <1790 Arizona Smith Fall Cheese 2x Virginia <1905 Stark 1912 Ragan Fall Pippin 3x USA 1806 Arizona. Texas Smith Fameuse 2x Canada 1730 Arizona. New Mexico Smith 2x West Virginia 1890 unk. Smith 2x England Mid- l700s Arizona Smith 2x Australia 1868 sink. Smith Cravenstein Washington Red 3x Italy or German y 1600s Arizona. lexas Smith Crimes Golden 2x Vest Virginia 1804 Arizona, New Mexico Smith Ilcury ('lay 2x USA 1905 Stark 1912 Ragan I luhardston Nonsuch 2x Massachusetts 1832 Stark 1912 Smith Ingram 2x Missouri 1844 Stark 1912 Smith Irish ['each 2x Ireland <1820 unk. Smith Jet Uris 2x Pennsylvania 1830 Texas Smith 2x New York I. Arizona, New Mexico. Texas Smith Keswick Codlin 2x England 1793 Arizona Smith Kinnaird's Choice 2x Tennessee Mid-I 800s Texas Whealy Knobbed Russet 2x England 1820 unk. Smith Lady (Api) 2x France <162$ Stark 1912 Smith Laver 2x Kansas 1868 New Mexico, Texas Smith I,iveland (Lowland) Rasberry 2x Russia <1870 Stark 1912 Smith LongfIeld (Glogerovka) 2x Lithuania or Russia <1903 Texas Smith Lowry 2x Virginia 1850 Stark 1912 Whealy Ly man's (I.ymans Large Summer) 3x Connecticut 1867 unk. Smith Maiden Blush 2x New Jersey <1817 New Mexico, Texas Smith Martha 2x Wisconsin <1905 Arizona. New Mexico Ragan Mcintosh Sunimerland Red 2x Canada 1796 Stark 1912 Smith Mother 2x Massachusetts 1844 Stark 1912 Smith Northern 5p 2x New York 1800 Arizona, New Mexico Smith Northwest Greening 2x Wisconsin 1972 Stark 1912 Whealy Ohio 2x Ohio 1848 Stark 1912 Smith Opalescent 2x Ohio 1899 Stark 1912 Smith Orticy 2x New Jersey <1817 Arizona. New Mexico, Texas Smith Paragon 3x Tennessee 1830 Arizona Beach Peck Pleasant 2x Rhode Island 1832 Arizona Smith Pcwaukee 2x Wisconsin 1870 New Mexico Smith Pi tnlaston Pineapple 2x England 1785 unk. Smithih Porter 2x Massachusetts 1800 New Mexico Smith Primate 2x New York 1840 Stark 1912 Smith 2x Russia <1816 New Mexico Smith Red Bieti gheimer (Rater Stettincr( 3x Germany 1598 Arizona. New Mexico. Texas Smith Red June (Carolina Red June) 2x North Carolina 184$ New Mexico. Texas Smith Red Rails (Rails Janet) 2x Virginia 1800 Arizona. New Mexico Smith 3x Rhode Island 1650 Arizona, New Mexico Smith Ribston 3x England 1707 Arizona Smith Roman Stem 2x New Jersey 1817 Stark 1912 Smith

(Continued on next page)

I-IOWrSCJENCL VOL. 44(3) JuNL 2009 591 Table I ( Coniinus'iI) Pioidy, origin. and probable date of release are provided for 109 apple cultivars ii trodUeed to the soutlisvesterii Looted States/ ('ultivar name Ploidy Origin Date Southss est introduction' Reference' Rome Beauty law 2x Ohio 1848 Arizona, New Mexico. Texas Smith Rosemary Russet 2x England 1831 unk. Smith 3x Massachusetts 1649 Arizona Beach Shockley 2x Georgia 1854 New Mexico. Texas Smith Smiths Cider 2x Pennsylvania <1817 Arizona. Texas Smith Sops of Wine 2x England 1831 New Mexico Smith Stark 3x Ohio 1867 Arizona Smith Starman 3x Kansas 1875 Stark 1912 Smith Summer 2x Arkansas 1897? Stark 1912 Calhoun Swaar 2x New York 1804 Arizona, New Mexico Smith Sweet Bough (Bough) 2x USA 1817 Arizona, Texas Smith Sweet Dixon 2x North Carolina <1905 New Mexico, Texas Ragan Tolman Sweet 2x USA 1822 Arizona. New Mexico Smith Tompkin's King 3x New Jersey 1804 Stark 1912 Smith Transcendent Crab 3x unk. <1844 New Mexico Bussey Twenty Ounce Pippin 2x Connecticut or New York 1844 Texas Smith Virginia Crab (I lewes) 2x Virginia <1803 unk. Busscv Virginia Beauty 2x Virginia 1826 Stark 1912 Whealy Vixin Crab 3x unk. unk. tink. unk. Wagner 2x New York 1791 Stark 1912 Smith 2x minnesota 1860 New Mexico, Texas Smith Westfield Seek No Further 2's Massachusetts 1796 unk. Smith Arizona Smith White Astrachan 3x or Russia 11748 849 White Winter Peannain 2x USA Stark 1912 Smith Wickson 2x California 1944 unk. Whealy 2x New Jersey < 1817 Arizona, New Mexico, Texas Smith Winter Banana 2x Indiana 1876 unk. Smith Winthrop Greening 2x Maine 180)) unk. Smith 2x Wisconsin 1875 New Mexico Smith Yarl ingtnn Mill 2x England Link. unk. W hea ly Yates 2x Georgia 1813 Stark 1912 Smith \elloss Bellflower 2x New .lersey <1817 Arizona, Texas Smith Yellow Newtown Pippin" 2x Long Island 1759 Link. Beach Yellow (White) Transparent 2x Russia or Baltic States 1800s Texas Smith 2x Pennsylvania 1830 Arizona, New Mexico Smith 'Cultivars identified in bold were identified in historic farmsteads.

Diluted products were loaded on gels (6.5% pared with 109 known cultivars introduced locations. A number of trees matched geno- KB Plus actylamide; LI-COR, Lincoln, NE) into the southwest in the late 19lh century by types ofnarned culttvars for all but one or two and run in lx TBE buffer (89 mu Tris, 89 mu USDA agricultural experiment stations and alleles at the locus GD 100 (Table 2). It is not boric acid, and 20 mm EDTA) for 1 h. 45 mm by Stark Brother's Nursery, the largest mail known if these I- or 2-hp shifts represent at 1500 V. 40 W, 40 mA, and 45

592 HotsrSctrNcE VOL. 44(3) JUNE 2009 Table 2. Identification of cultivars identified iii Iiisiiiric farmsteads in the southwestern United States. Trees Present Nursery Cultivar identified no. L and on nership' Orchard typei condition' sources" (no.) Lse Arkansas Black 3 liSPS, private Commercial and farm Maintained 35 Cider, fresh eating, and cooking Ben Davis 16 NFS and private Commercial and farm Abandoned 10 Long storage life l3enoni 2 Private Commercial Abandoned 6 Dessert Delicious 12 NFS and private Commercial and faint Maintained 36 Fresh eating and dessert Duchess I Private Farm Maintained 17 Cooking, poor storage life Early harsest' I Private Farm Abandoned 18 Cooking i'anieuse 2 Private Farm Abandoned 19 ('ider, fresh eating, and cooking Gravenstien. WA Red I Private Farm Abandoned 31 Cooking and cider Grimes Golden 6 NPS and private Commercial and flirni Maintained 35 Cider and dessert Jonat han 17 N ES and private Coin mercial and farm Maintained 44 Cooking and fresh eating Lawyer' I Private Commercial Abandoned I Fresh eating Liveland Rasberry I NFS Fawn Abandoned II Fresh eating Maiden Blush 2 Private Commercial Abandoned 16 Cider, fresh eating, cooking McIntosh Summerland I Private Farm Abandoned 46 Cider, fresh eating. cooking I Private Commercial Abandoned 39 Cider, fresh eating. and cooking Northwest Greening 2 Private Farm Maintained 15 Winter cookin g apple Paragon 2 AZSPS and private Farm Maintained I Winter Red Astrachan' 2 N ES and private Commercial and (arm Abandoned IS Fresh eating and cooking Red June I NFS Farm Abandoned Ii) Cider, fresh eating, and cooking Red RaIls 3 Private Farm Maintained I I Fresh eating Rhode Island Greening' 3 NI'S and private Commercial and farm Maintained 12 Winter cooking apple Rome Beautv Law 2 Private Farm Abandoned II Cooking and cider Stayntan 3 Private Farm Maintained 24 Winter cooking and Tolman Sn cet' 2 Private Commercial and farm Abandoned 1 7 Cider, fresh eating, and cooking Transcendent Crab I NFS Farm Abandoned 2 Cooking Wagner 2 NFS Farm Abandoned 8 Cider, flesh eating, and cooking Wealthy' I NFS Farm Abandoned 24 Cider, fresh eating, and cooking Westfield Seek No Further' 4 NFS Farm Abandoned 16 Dessert White Astrachan' 3 NFS and private Commercial and firm Abandoned 2 Fresh eating and dessert 'A inesap 13 Pro ate Commercial and fanii Abandoned 23 Cider, fresh eating. and cooking 1 NFS Farm Abandoned 27 Cider Wolf River 4 AZSPS and private Commercial and farm Maintained 31 Cooking Yellow Bellflower 3 NFS Farm Abandoned lb Winter cooking apple Yellow Transparent I Private Farm Abandoned 36 Fresh eating and cooking 'Tree(s) were located on private property, National Forest Service land (NFS), National Park Service land (NPS), or Arizona State Park land (ALE'S). Identifies if tree is from a commercial orchard or farmstead orchard. 'Indication if current tree locations are maintained orchards or abandoned. he number of U.S. nurseries that still offered the cultivar in 2000 of 277 surve yed I Whealy, 2001). 'Cultis ars identical to the reference cultis ar except at marker GD 100 where there was a i-or 2-bp difference.

High levels of observed heterozygosity Table 3. Sample size, number of alleles, observed heterotygosity, and expected lieterozygosiiy were (0.92 for GD 142, 0.90 for GD 162, and 0.88 calculated for samples with unique genotypes. for GD 147 and GD96) in the samples suggest Mierosaiellite Sample Number of Observed Expected that relatively high levels ot'genetic diversity marker size number alleles heterozygosity heterozygosity are represented (Table 3). Heterozygosity (iDI2 144 II 0.833 0.751 was calculated frotit a sample size of 144 GD1S 144 2 0.021 0.021 individuals, representin g each of the 144 GD96 144 14 0.875 0.853 distinct genotypes found growing in the GDIOI) 134 9 0.485 0.814 GD142 southwest. Observed heterozygositv was cal- 144 13 0.924 0.864 culated by dividing the number of hetero- GD147 144 12 0.868 0.817 GD162 144 zygotes at a locus by the nttrnberof'individuals 13 0.896 0.827 surveyed. Expected heterozygosity assumes Hardy-Weinberg cqUilibriLlIn but is ineluded as a reference. The multiplicative probability result of the presence of two larger alleles at (Durmmire. 2004) as did the Archbishop Lamy of a niultilocus genot y pe determines the marker GD 12 (182 and 190 bps) instead of of Santa Fe in the 19th century (I [organ, power of diseritiitnalion the high hetero- the more common 148- to 162-bp length 1975). Geographic differentiation could also zsygositv of the sampled loci therefore makes alleles. Such clustering could he indicative imply different apple preferences associated the probability of distinctly different geno- Of' a shared genetic heritage between geno- with different regions. However, there was no types being identical at all seven microsalel- types in the upper left cluster, although there apparent genetic separation byby geographic lite loci very slight. Microsatellite GDIOO does not appear to be any morphological origitt among samples. The historic trees in had 6% missing data as a result of poor association with the groups. the southwest might all share the same recent amplification during PCR for several sam- Unknown gctlotypes were labeled in the parents. or the parent diversity could have ples. We chose not to score this allele in these PC'A with a state prefix attached to the end of beet) obscured by small sample size. Efforts cases to avoid potential scoring error. Het- the sample number to show possible associa- were made to avoid seedling apple trees erozygosity and allele frequencies should be tions of geographic origin to genotype. Geo- durin g sampling. As mentioned in the "Mate- interpreted with dubiety for this allele. graphic separatioti of samples would indicate rials and Methods" of this article, we focused PCA was performed oil samples to that different sources of apple trees were sampling effbt'ts on grafted as opposed to visualize the genetic difference between grown in the various regions. Spanish priests, seedling ti'ees^ however, a small percentage apple genotypes (Fig. 2), In the PCA ordina- explorers, and settlers introduced apple trees of the samples are likely to be of seedling lion, the two distinct clusters appear to he the to New Mexico as early as the I 7th century origin arid not natitecl cultivars. Seedling trees

HOR)S(ii-xcE Vol- 44(3) Jux g 2009 593 .Ar./FLE sequence repc:its Ii tie 3cnetic in: lvsi el apple. Theor. App!. Genet. 96:1096 1076. Goland. C. and S. Bauer. 2004. When the apple [ills close to the tree: Local food systems and the preservation of diversity. Renewable Agri- U55Z N1t8dSU)kr, cultural Food Systems 19:228-236. I lanimer, K., N. Arrowsmiih, and T. Gladis. 2003. Agrobiodiversity with emphasis on plant genetic

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81NM *ete_Ast,chan Maiden_OMeN and C. Schlhttcrer (ei.ls.). Reprinted in 2003. 4751dM Microsatellites: [volution and applications. Wnier B scans Oxford University Press, Oxford, UK. 2C M1 032AZ Hedrick, U.P. 19511.4 hisiory of horticulture in • Mchtesh_Summerland America to 186)). Oxford University Press. BOOM Oxford, UK. U) I Iensmat, M.S.K.B. and N.F. Weeden. 2003. Map- >< ping and evaluation of Ma/u.s xdoniesiIca MS NM mierosatellites in apple and pear. J. Amer. 457051 Soc. Ilort. Sci. t28:515 -520. 522AZ 2T2NZ sSUllM Hensley, T. 2005. A curious tale: The apple in 2651dM 23511,M 038)SZ 207AZ Paragon North America. 5-15. In: Hanson, B. (ed.). I$ 5025AZ , 1)5 Lrr p. 41 fi54U1 ran The best apples to bu y and grow. Brooklyn 1df47ONM 0551 N#1hotGreenng M Benow •'1)25Z Botanic Garden, Inc. Brooklyn. NY. USLZ 0120fr45M 47800 2041dM onoz 025AZ Hokanson, S.C.. A.K. S,cwc-McFaddcn, W.F. .I28NM 04515Z 278NM UUOZ2OAZ - 275814 2, .' • 2071dM 0446Z 2 1285NM Laniboy. and J.R. MeFerson. 1998. Microsa- 4651dM uJ3.Z tini 250W O4NMO^A^ ^ B NM nnao,Oisck tellite (SSR) markers reveal genetic identities, 00344 .U,CPZOSIJT A genetic diversity and relationships in Ma/us 2840 224AZ 4551dM 1501dM 0024Z 41dr,I 1420M BOOM 4711dM xeIanis',vtica Borkh. core subset collection. 277 NM Transcendnnl_CrOSM 84NM245NM 0354 Theor. App!. Genet. 97:671-683. • fiOAZ 065AZ 272NM R,d_RaIlN llorgan. P. 1975. Lamy of Santa Fe: His life and 2801dM OtSA- siOgner 1201dM FanneunR 251dM 8488eZ M O3OAZ 033AZ times, a biography. Farrar, Straus & Giroux. 1551dM New York, NY. 0 23AZ Jackson. J.E. 2003. Biology of apples and pears. Axis 1 Cambridge University Press, Cambridge. UK. Peakall. K. and P.E. Smouse. 2006. GENALEX 6: state origin Fig. 2. Principal component analysis of unique apple genotypes. Sample numbers and of are Genetic analysis in Excel. Population genetic provided for unknown varieties and names are provided for known varieties. software for teaching and research. Mol. Ecol. Notes 6: 288-295. 14 Nov. 2007. . would not share the same "heirloom" status as PGRU will potentially enable us to identify the Pereira-Lorcnzo, S., A.M. Ramos-Cabrer. and named 19th century cultivars but may still unknowns in this survey. Until such work is M.D. Diaz-l-ternándei.. 2007. Evaluation of Possess useful traits or local adaptations. undertaken, these unknown genotypes should genetic identit y and variation of local apple For this study, we fingerprinted cultivars be conserved and analyzed for useful traits. eul ii vars (Mains xdo,nesijca Borkh.) from from the USDA National Germplasm Col- Spain using microsatel!itc markers. Genet. lection and from private nurseries to compare Literature Cited Resources Crop [vol. 54:405-420. Ragan, W.H. 1905. Nomenclature of the apple: A with the unknown samples. We restricted the Beach, S.A. 1905. The apples of New York. catalogue of the known varieties referred to in number of reference cultivars to 109 likely Volumes I & II. Report of the New York American publications from 1804 to 1904. introduced into the southwest. The existence Agriculture Experiment Station for the year USDA-DPI Bulletin #56. Government Printing of a DNA fingerprint database for correctly 1903. J.B. Lyon Company, Albany, NY. Office, Washington, DC. at Bussey, D. The apple in North America: A register identified fruit tree cultivars such as those Sill\erberg-Dilevorth. E., C.L. Matasci, W.E. Van of apple varieties listed in North American the PGRU would allow studies such as this de Weg. M.P. Van Kaauwen. M. Walser, L.P. publications from 1802-1980, based on nomen- one to answer more questions about the Kodde, V. Sog!io, L. (iianfranceschi, C.E. clature of the apple. U.S. Department of Agri- identities of the many unknown apple trees Durel, F. Costa, T. Yamamoto, B. Koller, C. culture Division of Plant Industry Bulletin #56. growing on abandoned farms and in parks Compiled by W.H. Ragan, 1905 tin press). (jessler. and A. Patocehi. 2006. Microsatellite s Xe/aflie'.Su!Ca and forests across the country. Calhoun, C.L. 1995. Old southern apples. McDonald markers spanning the apple (Ma/i Apple plantings in a number of orchards and Woodward Publishing Company, Blacks- Borkh.) genome. Tree Genetics & Genomes and farmsteads cultivated in the 19th and early burg, VA. 2:202-224. Smith, M.W.G. 1971. National apple register of the 20th centuries in the U.S. southwest still Dennis, F.G...Jr. 2008. l.laius xdo,nestica: Apple. United Kingdom. London Ministry of Agricul- survive, although many have been abandoned. p. 661-674. In: Janick, i. and R.E. Paull (eds.). ture, Fisheries and Food. 2002 reprint by From 43 historic sites, 280 apple trees were The encyclopedia of fruit and nuts. CAB] Castlepoint Press, Colvend, UK. sampled and compared with 109 cultivars at Publishing. Cambridge, MA. Dunmire, C.C. 2004. Gardens of new Spain: 110w Volk, G.M., C.M. Richards, A.A. Reilley. A.D. the PGRU and in private nurseries using Mediterranean plants and foods changed Amer- Henk, P.L. Forsline. and [IS. Aldwinekle. niicrosatellite analysis. The 280 samples ica. University of Texas Press, Austin, TX. 2005. Ex situ conservation of vegetatively yielded 34 named cultivars and 110 unique Ferree, D.C. and I.J. Warrington. 2003. Apples: propagated species: Development of a seed- genotypes. These results suggest that the his- Botany, production and uses..(ABI Publishing. based core collection for Ma/u.s ,vie'verxii. I. toric orchards in the southwest had a high Cambridge, MA. Amer. Hort. Sci. 130:203-210. diversity of genotypes. Additional genetic Giafranccschi, L., N. Seglias, R. Tarchini, M. Vhealv, K. 2001. Fruit. berry, and nut inventory. fingerprinting of apple cultivars in the USDA Komjane. and C. Gessler. 1998. Simple 3rd Ed. Seed Savers Exchange. Decorah, IA.

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