J. AMER.SOC.HORT.SCI. 138(3):198–204. 2013. Interspecific Hybridizations in Ornamental Flowering Cherries Validated by Simple Sequence Repeat Analysis Margaret Pooler1 and Hongmei Ma USDA-ARS, U.S. National Arboretum, 10300 Baltimore Avenue, Building 010A, Beltsville, MD 20705 ADDITIONAL INDEX WORDS. simple sequence repeat, molecular markers, plant breeding, Prunus ABSTRACT. Flowering cherries belong to the genus Prunus, consisting primarily of species native to Asia. Despite the popularity of ornamental cherry trees in the landscape, most ornamental Prunus planted in the United States are derived from a limited genetic base of Japanese flowering cherry taxa. Controlled crosses among flowering cherry species carried out over the past 30 years at the U.S. National Arboretum have resulted in the creation of interspecific hybrids among many of these diverse taxa. We used simple sequence repeat (SSR) markers to verify 73 of 84 putative hybrids created from 43 crosses representing 20 parental taxa. All verified hybrids were within the same section (Cerasus or Laurocerasus in the subgenus Cerasus) with no verified hybrids between sections. Ornamental flowering cherry trees are popular plants for pollen parent bloomed before the seed parent, anthers were street, commercial, and residential landscapes. Grown primar- collected from the pollen donor just before flower opening and ily for their spring bloom, flowering cherries have been in the allowed to dehisce in gelatin capsules which were stored in United States since the mid-1850s (Faust and Suranyi, 1997), paper coin envelopes in the refrigerator before use. In most and they gained in popularity after the historic Tidal Basin cases, the seed parent was emasculated before pollination. cherries were planted in Washington, DC, in 1912. Over 1.2 Generally, at least 50 flowers were pollinated for each cross, million plants are sold wholesale each year at a value of more and some crosses were attempted over several years. As seeds than $32 million [U.S. Department of Agriculture (USDA), developed, they were covered with mesh bags to prevent loss 2010]. Despite the large number of Prunus species with diverse and harvested when ripe. Seeds were cleaned of fruit, sown in origins and ornamental traits, the most widely cultivated flats containing a soilless potting mix (milled sphagnum and flowering cherry trees planted in the United States represent course sand, 1:1), and then moist-stratified for three months in only a few species, primarily P. serrulata, P. subhirtella, and the dark at 4 °C. After stratification, flats were placed in a 21 °C P. yedoensis. The U.S. National Arboretum has an ongoing greenhouse for seed germination. Seedlings were transplanted breeding program aimed at broadening this genetic base by to containers and ultimately to the field. developing new cultivars of flowering cherries with disease and DNA EXTRACTION, SIMPLE SEQUENCE REPEAT PRIMERS, AND pest resistance, tolerance to environmental stresses, and superior POLYMERASE CHAIN REACTIONS. Total genomic DNA was ornamental characteristics. Hundreds of interspecific Prunus extracted using the rapid one-step extraction (ROSE) method hybridizations have been carried out at the U.S. National as described previously (Ma et al., 2009; Steiner et al., 1995), Arboretum over the past 30 years. Verifying whether these except that frozen leaves were ground in 200 mLROSEbuffer resulting plants are true hybrids is complicated by the fact that using lysing matrix tubes in a FastPrep machine (MP Bio- morphological characteristics such as flower, leaf, or bark traits medicals, Santa Ana, CA). Seven SSR primer pairs developed for are quite variable and heterogeneous among the various taxa and other (non-ornamental) Prunus species were used to amplify the are therefore often inadequate for confirming hybridity. The following loci (Table 2): GA59, GA77, CPDCT006, CPDCT033, objective of this study was to verify the pedigree of a number of pchgms3, UDP96001, and UDP97-403. SSR primer synthesis, the putative interspecific hybrids created at the U.S. National reactions, and data collection were as described previously (Ma Arboretum. et al., 2009) with fluorescently labeled polymerase chain reaction (PCR) product sizes determined by analysis using an ABI310 Genetic Analyzer (Applied Biosystems, Foster City, CA) using Materials and Methods GeneScan software Version 3.1.2 (Applied Biosystems). Geno- typer software Version 2.5.2 (Applied Biosystems) was used to PLANT MATERIALS AND HYBRIDIZATIONS. Prunus parent taxa process and view GeneScan-sized peaks. and hybrids used in this study are listed in Table 1. Controlled crosses were performed in the field during the year indicated. Crosses were made by covering clusters of unopened flower Results and Discussion buds with a pollination bag and then hand-pollinating flowers as they opened using a camel hair paintbrush. In cases in which the In this study, 84 plants representing 43 interspecific hybrids using 20 taxa of ornamental flowering cherry were evaluated with SSR markers to verify putative hybrids; 73 of these 84 Received for publication 8 Jan. 2013. Accepted for publication 4 Feb. 2013. plants could be verified as hybrids (Table 1). In some cases, 1Corresponding author. E-mail: [email protected]. these hybrids represent a sampling of a larger population; in 198 J. AMER.SOC.HORT.SCI. 138(3):198–204. 2013. Table 1. Cross, parents, and simple sequence repeat (SSR) profiles of Prunus parents and hybrids tested in this study.z Year Individual of cross Code Parentage Parental SSR alleles hybrid testedy Hybrid SSR alleles 1980 46-80 P. maackii · GA77-(180, 184, 186) · (176, 186); 1# GA77-(176, 180, 186); P. ‘Snow Goose’ CPDCT006-(172,174) · (181, 183) CPDCT006-(172, 174, 183) 1980 64-80 P. padus var. grandiflorus · CPDCT033–125 · (133, 144); 1# CPDCT033-(125, 133); P. virginiana ‘Schubert’ GA77-(170, 172) · 174) GA77-(172, 174) 2# CPDCT033-(125, 133); GA77-(170, 174) 3# CPDCT033-(125, 144); GA77-(172, 174) 1982 02-82 P. ·incam ‘Okame’ · GA59- (163, 187, 207) · (215, 248); 1 GA59-(199, 207); P. campanulata CPDCT033-(131, 141) · (139, 141) CPDCT033-(117, 135) 2# GA59-(187, 248); CPDCT033-(131, 139) 3 GA59- (163, 187, 207); CPDCT033-(139, 141) 1982 09-82 P. yedoensis · CPDCT033-(133, 139) · (139, 141); 1# CPDCT033-(133, 141); P. campanulata GA77-(176, 186) · 180 GA77-(180, 186) 1988 09-88 P. nipponica var. kurilensis · CPDCT033-(139, 141) · (139, 143); 1 CPDCT033-133; P. ‘Oh-Kanzakura’ GA77-178 · (176, 180) GA77-(184, 186) 2 CPDCT033-133; GA77-(182, 184) 3 CPDCT033-133; GA77-(182, 184, 186) 1988 14-88 P. cyclamina · GA77-(182, 188) · 180; 1 GA77-(180, 182); P. campanulata CPDCT033-(135, 141) · 141 CPDCT033-141 1988 15-88 P. cyclamina · GA59-199· 248; 1# GA59-(199, 248); P. ‘Oh-Kanzakura’ seedling UDP97-(129, 146) · (110, 114) UDP97-(114, 129) 1989 08-89 P. cyclamina · GA77-(182, 188) · (180, 186); 1# GA77-(180, 188); (P. ‘Okame’ · UDP96-(127, 139) · (129, 131) UDP96-(129, 139) P. campanulata.) 2# GA77-(180, 182); UDP96-(129, 139) 1991 02-91 P. subhirtella ‘Autumnalis GA77-(184, 186) · (180, 186); 1# GA77-(180, 184); Rosea’ · (P. ‘Autumnalis Rosea’ · pchgms3-(204, 216) · (195, 204); pchgms3-(195, 216) P. campanulata) UDP97-(114, 135) · (101, 110, 136); 2# UDP97-(110, 135); CPDCT033-(131, 133) · (131, 141) CPDCT033-(131, 141) 1992 01-92 (P. ‘Okame’ · UDP96-(129, 131) · 133; 1, 2 UDP96-(129, 133); P. campanulata) · P. campanulata UDP97-146 · 131 UDP97-146 1994 01-94 P. verecunda · CPDCT033-146 · (133, 137); 1# CPDCT033-(137, 146); P. takesimensis GA77-(176, 180) · 178 GA77-(178, 180) 2#, 3# CPDCT033-(137, 146); GA77-(176, 178) 1994 03-94 P. verecunda · GA77-(176, 180) · (178, 182); 1# GA77-(176, 178); P. incisa GA59-199 · (205, 207); GA59-(199, 207) UDP96-120 · (125, 127) 2#, 3# GA77-(176, 182); UDP96-(120, 125) 1994 05-94 P. takesimensis · CPDCT033-133 · 141; 1#, 3# CPDCT033-(133, 141); P. incisa UDP96-120 · (126, 128) UDP96-(120, 126) 2# CPDCT033-(133, 141); UDP96-(120, 128) 1994 7-94 (P. ‘Okame’ open-pollinated GA59-(187, 199) · (203, 248); 5130# GA59-(187, 248); selection) · (P. ‘Autumalis Rosea’ · GA77-(180, 182) · (180, 186) GA77-(182, 186) P. campanulata) Continued next page J. AMER.SOC.HORT.SCI. 138(3):198–204. 2013. 199 Table 1. Continued. Year Individual of cross Code Parentage Parental SSR alleles hybrid testedy Hybrid SSR alleles 1994 08-94 (P. ‘Okame’ · GA59-248 · (187, 199); 1 GA59-(187, 248); P. campanulata) · (P. ‘Okame’ open-pollinated GA77-(180, 186) · (180, 182) GA77-(180, 182) selection) 2# GA59-(199, 248); GA77-(182, 186) 1994 13-94 (P. ‘Umineko’ · P. incisa) · GA77-(176, 186) · (180, 182); 1# GA77-(176, 182); (P. ‘Okame’ open-pollinated pchgms3-(196, 202) · (194, 198) pchgms3-(194, 196) selection) 2# GA77-(182, 186); pchgms3-(196, 198) 3# GA77-(176, 182, 186); pchgms3-(196, 198, 202) 1994 14-94 (P. ‘Umineko’ · P.incisa) · pchgms3-(197, 203) · (195, 204); 5127# pchgms3-(197, 204); (P. ‘Autumnalis Rosea’ · GA59-207 · (203, 248); GA59-(203, 207) P. campanulata) CPDCT033-137 · (131, 141) 5129# pchgms3-(197, 204); GA59-(207, 248) 5131# pchgms3-(197, 204); CPDCT033-(131, 137) 1995 03-95 P. maackii · UDP97-(117, 120) · (110, 136); 1# UDP97-(110, 117, 120); (P. ‘Autumnalis Rosea’ · pchgms3-(197, 205, 214) · (195, 204) pchgms3-(195, 197, 205) P. campanulata) CPDCT033-139 · (131, 141) 2# UDP97-(110, 117); pchgms3-(195, 197, 205) 3# UDP97-(110, 117, 120); CPDCT033-(139, 141) 1995 07-95 P. maximowiczii · CPDCT033-(127, 137) · (131, 141); 1# CPDCP033-(127, 131); (P. ‘Autumnalis Rosea’ · GA77-(176, 186) · (180, 186) GA77-(176, 180) P. campanulata) 1995 09-95 P. maackii · CPDCT033-(129 · 133); 1 CPDCT033-(129, 141); P. yedoensis GA77-(178, 182, 184) · (176, 180) GA77-(178, 180, 186) 2 CPDCT033-139; GA77-(180, 184, 186) 3 CPDCT033-141; GA77-(184, 186) 1995 10-95 P. maackii · CPDCT006-(172, 180) · (163, 187); 1# CPDCT006-(172, 180, 187); (P.