Ploidy Levels and Relative Genome Sizes of Diverse Species, Hybrids, and Cultivars of Rhododendron Jeff R. Jones, Thomas G. Ranney, and polyploidy occurs naturally in some Pryor and Frazier (1970) determined that Nathan P. Lynch rhododendron species, particularly the evergreen azalea hybrids ‘Redwing’ Department of Horticultural Science within the Pentanthera and Rhododendron and ‘Ablaze’ were triploids and also Mountain Horticultural Crops Research subgenera, with ploidy levels ranging documented the existence of mixed ploidy and Extension Center from three to twelve (Ammal, 1950; cytochimeras resulting from colchicine North Carolina State University, Fletcher, Ammal et al., 1950). Sax (1930) completed treatment. Heursel and DeRoo (1981) North Carolina one of the first surveys of chromosome completed chromosome counts on 47 numbers of rhododendron, including cultivars of evergreen azaleas and found Stephen L. Krebs sixteen species, and determined a base they were all diploid with the exception of David G. Leach Research Station chromosome complement of x= 13 the triploid, ‘Euratom’. The Holden Arboretum for the genus and identified both R. The chromosomes in rhododendron Kirtland, Ohio calendulaceum and R. canadense (deciduous are small and can be difficult to view azaleas in subgenus Pentanthera) as natural and count (Eiselein, 1994; Tolstead olyploidy has been an important tetraploids. Nakamura (1931) surveyed and Glencoe, 1991). Light microscopy Ppathway in the evolution of plants and fifteen Japanese species of rhododendron is therefore not a practical method can contribute to reproductive isolation, and found them all to be diploid. Ammal for determining ploidy levels of large increased heterozygosity, novel gene et al. (1950) completed an extensive survey numbers of individual cultivars and combinations, modified gene expression, of chromosome numbers and ploidy clones. However, flow cytometry can enzymatic multiplicity, and ultimately levels in 360 species of rhododendron provide a fast and accurate determination divergence and speciation (Soltis and and found the elepidote rhododendrons of nuclear DNA content (genome size) Soltis, 1993; 2000; Wendel, 2000). The (subgenus Hymenanthes), evergreen azaleas that is related directly to ploidy level origins, adaptive significance, and genetic (subgenus Tsutsusi), and the deciduous among closely related taxa (de Laat et al., implications of polyploidy continue to be azaleas (with the exception of the 1987; Doležel, 1991; Doležel et al., 1998; an active field of research (Bennett, 2004; tetraploid R. calendulaceum and R. canadense) Galbraith et al., 1983). Flow cytometry is Soltis et al., 2003; Chen and Ni, 2006). to be predominantly diploid. Ammal et al. also effective for detecting mixaploidy or For plant breeders, ploidy level is an (1950) further reported a high frequency cytochimeras and individual histogenic important consideration because it can of polyploids in the scaly-leaved species of layers can be analyzed by sampling influence male and female fertility, cross subgenus Rhododendron, with taxa ranging appropriate tissue (DeSchepper et al., fertility, plant vigor, and gene expression from triploids to dodecaploids. In a survey 2001). Flow cytometry has been used (Chahal and Gosal, 2002; Contreras et of fifteen deciduous azaleas from Eastern successfully to determine relative DNA al., 2007; Ranney, 2006; Thomas, 1993). North America, Li (1957) reported that content and ploidy levels of Rhododendron In some cases, polyploid plants, including all of the species were diploid with the spp. (DeSchepper et al., 2001; Eeckhaut rhododendrons, can have desirable exception of the tetraploid R. calendulaceum. et al., 2004; Sakai et al., 2003, 2004a, characteristics including thicker leaves, However, a single triploid R. atlanticum 2004b, 2006; Ureshino and Miyajima, enhanced vigor, and larger flowers with was also identified among the otherwise 1998; Väinölä, 2000). De Schepper et thicker petals that persist longer (Barlup, diploid species. Among lepidotes, chrom- al. (2001), for example, determined the 2002; Hosoda et al., 1953; Kehr, 1996a; osome counts for 27 species in the tropical ploidy level for six species and 88 cultivars Leach, 1961). As a result, there continues subgenus Rhododendron section Vireya within the evergreen azalea subgenus to be interest in identifying naturally indicated that they were uniformly diploid Tsutsusi by using flow cytometry. The vast occurring polyploids and inducing (Atkinson et al., 2000). majority were found to be diploid with the (through mitotic doubling agents) artificial Published information on chromo- exception of three triploids (‘Red Wing’, polyploids as a component of rho- some counts of specific cultivars or clones ‘Euratom’, and ‘Euratom Orange’*) and dodendron breeding programs (Barlup, of rhododendron is less extensive. Hosada one mixaploid (‘Casablanca Tetra’) that 2002; Kehr, 1996b; Paden et al., 1990; et al. (1953) completed chromosome was found to be diploid in the LI and LII Pryor and Frazier, 1970; Leach, 1961). counts on twelve cultivars of Satsuki layers and tetraploid in the LIII. Eeckhaut Most of the more than 800 Rhododen- azaleas (R. lateritium) and identified et al. (2004) studied various Ghent and dron species have been reported to be diploid, triploid (‘Bangaku’), and tetraploid Rustica deciduous azalea hybrids by diploid with 2n = 2x = 26. However, (‘Banka’, ‘Taihei’, and ‘Wako’) plants. using flow cytometry and found them 220 FALL 2007 to be either triploid (‘Mina van Houtte’, replicated, chromosome complement level. Data were subjected to analysis of ‘Daviesii’, ‘Quadricolor’, ‘Gloria Mundi’, irrespective of ploidy level) were deter- variance and means separation by using ‘Van Houtte Flore Pleno’, ‘Norma’, and mined via flow cytometry (de Laat et the Waller procedure (PROC GLM; SAS ‘Phébé’) or tetraploid (‘Nancy Waterer’, al., 1987; Doležel, 1991; Galbraith et al., version 8.02, SAS Institute., Cary, N.C.; ‘Unique’, ‘Narcissiflorum’, ‘Jozef 1983; Greilhuber et al., 2005). Diverse SAS Institute, 1988). Baumann’, ‘Maja’, ‘Rosetta’, ‘Semiramis’, species and cultivars were acquired Chromosome counts. In situations where ‘Souvenir du President Carnot’, ‘Marie from various sources that included taxa cytometric results were not consistent Verschaffelt’, ‘Batholo Lazarri’*, ‘Guelder from the Hymenanthes, Rhododendron, with published research, chromosomes Rose’, ‘Coccineum Major’, ‘Raphael de Tsutsusi, and Pentanthera subgenera along were counted by using standard cyto- Smet’, ‘General Trauff’, ‘Graf von Meran’, with several inter-subgeneric hybrids logical techniques (Contreras et al., ‘Goldlack’, ‘Fénelon’, and ‘Racine’). In (Table 1). Approximately 1 cm2 of newly 2007). Chromosomes were counted in contrast to the survey by Ammal et al. expanded leaf or petal tissue was finely mitotic cells from young root tips of (1950), Eeckhaut et al. (2004) found three chopped with a razor blade in a Petri rhododendron cuttings. Roots were clones of R. luteum to be tetraploid, not dish with 500 mL of nuclei extraction collected before 11 a.m. and root tips d i p l o i d . S a k a i e t a l . ( 2 0 0 6 ) i d e n t i fi e d t w e n t y- buffer (CyStain UV Precise P Nuclei were placed in a pre-fixative solution of three diploid, six triploid (‘Daisetsuzan’, Extraction Buffer, Partec, Münster, 2mM 8-hydroxyquinoline for 4 hours at ‘Goko’, ‘Horiuchikanzaki’*, ‘Issho-no- Germany). The solution was incubated 12 °C in the dark. Root tissue was fixed haru’, ‘Meicho’, and ‘Yuhime’*), nine for 1 to 2 min at approximately 24 °C and in a 1 : 3 solution of propionic acid : 95% tetraploid (‘Ayaka’*, ‘Eiko’, ‘Hoshuku’*, then filtered through Partec CellTrics™ ethanol solution for 24 hours at room ‘Hoshun’, ‘Sachi-no-haru’*, ‘Shunka’*, disposable filters with a pore size of 50 temperature and then hydrolyzed in 1N ‘Taihei’, ‘Taikonotsuki’*, and R. kiusianum mm to remove tissue debris. Nuclei were HCl for 15 minutes at room temperature × R. eriocarpum No. 5) and four mixaploid stained with 1.5 mL 4’, 6-Diamidino- and for 25 minutes at 60 °C, followed by (‘Koyo’, ‘Miharu’*, ‘Shinsen’, and ‘Sulsen’*) 2-phenylindole (DAPI) staining buffer a rinse in distilled water. Root tips were evergreen azaleas and eight diploid and (CyStain UV Precise P Staining Buffer, excised and placed on a glass microscope five tetraploid (‘Golden Flare’, ‘Golden Partec). Stained nuclei were analyzed with slide with a drop of 1% acetocarmine. Sunset’, ‘Klondyke’, ‘Melford Yellow’, and a flow cytometer (Partec PA-I, Partec) to Slides with tissue samples were heated R. japonicum f. flavum No. 6) deciduous determine relative genome size. Counts to approximately 70°C for 10 to 15 s, azaleas. Although flow cytometry can be exceeded a minimum of 3000 cells per squashed with a coverslip, and viewed used to directly compare relative genome sample. Genome sizes were determined under a light microscope (Nikon Eclipse sizes of tissue from related taxa, inclusion by comparing mean relative fluorescence 80i, Nikon, Melville, NY) at 1,500× using of an internal standard with a known of each sample with an internal standard, oil immersion. genome size allows the calculation of the Pisum sativum L. ‘Ctirad’, with a known Results and Discussion sample genome size (Doležel and Bartoš, genome size of 9.09 pg (Bennett and Smith, Flow cytometry was an effective method 2005), which enables comparisons among 1976; Doležel et al., 1998) and calculated for determining genome sizes and ploidy