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Home , Paphiopedilum, Phragmipedium

Scientia Horticulturae 109 (2006) 153–159 www.elsevier.com/locate/scihorti

Genetic relationship and differentiation of and Phragmepedium based on RAPD analysis S.Y. Chung a, S.H. Choi a, M.J. Kim a, K.E. Yoon a, G.P. Lee b, J.S. Lee a, K.H. Ryu a,* a Division of and Environmental and Life Sciences, Seoul Women’s University, 139-774 Seoul, Republic of Korea b Department of Applied Science, Chung Ang University, 456-756 Ansung, Republic of Korea Received 20 April 2005; received in revised 16 March 2006; accepted 3 April 2006

Abstract The genetic diversity and relationship between Paphiopedilum and and was determined by randomly amplified polymorphic DNA (RAPD) analysis. Twenty-one species and 13 varieties of these two orchids genera were analyzed and the similarity values ranged from 0.629, between P. kolosand and Paphiopedilum koloparkngii, to 0.882, between P. koloparkingii and Phragmipedium ‘Hanne Popow’. The analysis included both total and polymorphic band scores. The orchids examined could be separated into two major subgroups. The first major subgroup, subgroup I, included 28 species, which was composed of all Paphiopedilum species and 8 Phragmipedium species. Subgroup II was comprised of six species. These two subgroups could be further divided into two minor clusters. In this study Paphiopedilum and Phragmipedium were successfully differentiated by RAPD and the results were in good agreement with morphological based classifications. Therefore, our results suggest that the phylogenetic information could be obtained using molecular markers to address the interspecies genetic relationships of Paphiopedilum and Phragmipedium. # 2006 Elsevier B.V. All rights reserved.

Keywords: Paphiopedilum; Phragmipedium; RAPD; Relationship; Genetic diversity; Classification

1. Introduction matopetalum and Cochilopetalum (Bechtel et al., 1981; Braem et al., 1999; Liu et al., 2002). The Phragmipedium is native Paphiopedilum is a subtropical orchid also known as the to southern Mexico, Brazil and Peru, and has similar ‘‘lady’s slipper’’ orchid. It is native to , northern morphological traits to Paphiopedilum (Bechtel et al., 1981). India, southern , Myanmar, Thailand and . Morphological character and isozyme marker analysis have Most Paphiopedilums have a terrestrial character with the been used to classify cultivars or hybrids of a number of , exception of , Paph. parishii, Paph. supardii but both markers have limitations to use due to environment and Paph. stonei, which are epiphytic (Bechtel et al., 1981). The effect and available marker numbers. Therefore, a new molecular genus Paphiopedilum belongs to the Magnoliophyta and has over marker test is required to identify and protect the development of 70 original species and several thousand hybrids (Braem et al., new cultivars and randomly amplified polymorphic DNA 1998, 1999). It is estimated that the introduction of Paphiope- (RAPD) has been widely used for this purpose. Recently, the dilum to Europe occurred some time in the mid-1750s and RAPD technique and DNA sequencing analysis of specific hybrids have been developed since 1869 (Birk, 1983). This genus regions have been used for routine identification and can be differentiated from that of based on tissue genetic diversity studies of many plants (Wolff and Rijn, 1993; color, shape, the existence of variegated , petal size, sepals, Deng et al., 1995; Garcia et al., 1995; Debener and Mattiesch, pollen shape, pollinia viscosity and labellum shape. The genus 1998). Results of population genetic study of Goodyera procera Paphiopedilum can be classified into six subgenera that include () with allozyme and RAPD markers supported that Parvisepalum, Brachypetalum, Polyantha, Paphiopedilum, Sig- RAPD can detect higher levels of genetic variations than allozyme in same populations (Wong and Sun, 1999). Knowl- edge of the level of variation among these species and cultivars * Corresponding author. Tel.: +82 2 9705618; fax: +82 2 9705610. would be of great value to breeders because many species and E-mail address: [email protected] (K.H. Ryu). cultivars are still being used in crosses aimed at producing new

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.04.005 154 S.Y. Chung et al. / Scientia Horticulturae 109 (2006) 153–159 hybrids. Thus, we aimed to evaluate the genetic diversity and orchid genomic DNA was extracted from leaves by a relationships between Paphiopedilum and Phragmipedium modification of the cetyltrimethylammonium bromide (CTAB) species and cultivars by RAPD analysis. method (Knapp and Chandlee, 1996). One hundred milligrams fresh tissue was placed in a mortar and ground to a powder in 2. Materials and methods liquid nitrogen. Six hundred microlitres of cold extraction buffer (3% CTAB, 1.42 M NaCl, 20 mM EDTA, 100 mM Tris–Cl pH 2.1. Plant materials and DNA extraction 8.0, 2% polyvinylpyrrolidone, 5 mM ascorbic acid) was added and the tissue was further homogenized for 2 min. Ground Thirty-four species and cultivars, 20 Paphiopedilums and 14 samples were treated at 65 8C for 15 min and then extracted once Phragmipedium, used in this study are listed in Table 1. The with chloroform–isoamyl alcohol (24:1,v/v) to obtain a clear supernatant. Supernatant containing the plant genomic DNAwas Table 1 transferred to a fresh tube after centrifugation at 12,000 rpm for List of 20 Paphiopedilum species and 14 Phragmipedium species 5 min. A one-fifth volume of 5% CTAB solution in 0.7 M NaCl No. Plant name was added to the aqueous phase, the samples were treated at 65 8C for 15 min, and extracted again with chloroform–isoamyl 1 Paphiopedilum ‘Hamana Wave’ alcohol. The DNA was precipitated from the supernatant by the (‘Dark Roller’  Paph. rothschildianum) 2 Paphiopedilum emersonii addition of two volumes of cold absolute ethanol, incubated at 3 Paphiopedilum ‘Mt. Toro’ À80 8C for 15 min, and the DNA centrifuged at 12,000 rpm for (Paph. stonei  Paph. philippines) 20 min at 4 8C. After rinsing the DNA pellet in cold 70% ethanol, 4 the DNA was dried under a vacuum. The dried DNA was 5 Paphiopedilum kolosand (Paph. resuspended in 100 ml of distilled water. DNA was concentra- koloparkingii  Paph. sanderianum) 6 Paphiopedilum koloparkingii tions were determined by measuring the absorbance at 260 nm. 7 Paphiopedilum chamberlainianum The DNA quality was assessed by examination on a 1% agarose var. chamberlainianum gel stained with ethidium bromide. 8 9 2.2. RAPD amplification 10 11 12 RAPD DNA amplification was performed in a volume of 13 20 ml that contained: 10 ng of template DNA, 0.2 mM each of 14 Paphiopedilum fairrianum Red AM/APS dATP, dGTP, dCTP and dTTP, 50 pM of UBC primer 15 Paphiopedilum hirsutissimum (University of British Columbia, Canada) (Table 2), 20 mM 16 17 Paphiopedilum rothschildianum Tris–Cl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 18 Paphiopedilum wilhelminae ‘Fox valley’ 0.5% Tween 20, 0.5% Nonidet P-40, 50% glycerol, 1 unit of 19 Paphiopedilum ‘Joyce Hasegawa’ Taq DNA polymerase (Takara, Japan), 5 mM MgCl2 and (Paph. delenatii  Paph. emersonii) sterilized water. Amplification was performed in a Model 480 20 Paphiopedilum ‘St.Swithin’ (Paph. thermal cycler (Perkin-Elmer, USA). Denaturation was rothschildianum  Paph. Philippines) 21 warscewizianum performed at 94 8C for 3 min before beginning the cycling 22 flavum protocol. An amplification cycle consisted of 40 s at 94 8C, (‘Inca Gold’  ‘Wings of Gold’) 1 min at 37 8C and 1 min at 72 8C. A total of 40 cycles were 23 Phragmipedium besseae performed. The cycling was terminated with a final extension at (‘Ozon’  ‘Eat My Dust’) 72 8C for 10 min. 24 Phragmipedium longiflorum 25 26 Phragmipedium sargentianum Table 2 27 Phragmipedium schilimi Primers used for identification of Paphiopedilum and Pragmipedium by RAPD 28 Phragmipedium ‘Ardean Five’ (Phrag. and the number of bands produced lindleyanum  Phrag. besseae) Primer Nucleotide sequence GC% No. of No. of Total 29 Phragmipedium ‘Belle Hogue Point’ polymorphic bands (‘Erick Young’  Phrag. caudatum sanderae) bands 30 Phragmipedium ‘Desormers’ (‘Sorcerer’s Apprentice’  Hanne Popow ‘DayGlo’) UBC 241 GCCCGACGCG 90 8 10 180 31 Phragmipedium ‘Don Wimber’ (Phrag. Erick UBC 248 GAGTAAGCGG 60 10 11 Young ‘Rocket Fire’  Phrag. besseae flavum) UBC 249 GCATCTACCG 60 25 27 32 Phragmipedium ‘Bakara LeAnn’ (Phrag. UBC 703 CCCACAACCC 70 9 10 besseae  Phrag. fischeri) UBC 707 GGGAGAAGGG 70 20 23 33 Phragmipedium ‘Mem. Dick Clements’(Phrag. UBC 719 GGTGGTTGGG 70 26 29 besseae flavum ‘4th of July’  Phrag. UBC 764 CACACCACCC 70 17 21 Sargentianum ‘Westwood’) UBC 771 CCCTCCTCCC 80 9 10 34 Phragmipedium ‘Hanne Popow’ (Phrag. UBC 772 CCCACCACCC 80 16 19 besseae flavum ‘El Dorado’  Phrag. Schilimii ‘Golden Halo’) UBC 778 GGAGAGGAGA 60 18 20 S.Y. Chung et al. / Scientia Horticulturae 109 (2006) 153–159 155

2.3. Preliminary screening of primers 3. Results

Two primer sets, UBC #2 and #7, supplied with 100 primers 3.1. RAPD detection of polymorphism each were tested. RAPD was performed with these 200 10-mer length primers to determine he polymorphism extent and to We used RAPD analysis to examine the relationship select primers that detected these polymorphisms within between the genera Paphiopedilum and Phragmipedium. The Paphiopedilum and Phragmipedium. Only those primers that results presented describe the use of this phylogenetically produce identical polymorphic bands two or more times were informative DNA based method to address the interspecific selected for analysis. Primers that were problematic during the genetic relationships. Additionally, a phylogenetic tree was amplification of all samples were not used. constructed using the RAPD results and used to compare the Paphiopedilum and Phragmipedium lineages. All 34 Paphio- pedilum and Phragmipedium exhibited unique polymorphisms 2.4. RAPD data analysis in this study. Ten primers of the prescreened primers with a 60– 90% GC content successfully amplified polymorphic DNA The presence of amplified bands with different intensities bands (Table 2). Amplified DNA bands were detected using the and locations were detected and analyzed with the Quantity Quantity One 4.1 software (BioRad, USA). The plants One 4.1 (BioRad, Hercules, CA, USA) software using the examined produced a total of 180 bands and all were following values: noise filter: 4; lane width: 4.063 mm; size polymorphic. Most of the primers reproducibly produced clear scale: 5 and sensitivity: 4.665. Bands were scored for their differences in banding patterns for all samples (Fig. 1). presence (1) or absence (0) for numerical analysis. Genetic Amplified bands were characterized based on their size, which distances were calculated between all pairs of entries using ranged from approximately 100 to 4000 bp, and the number of Nei’s coefficient of genetic distance (Nei and Li, 1979): bands produced, which ranged from 10 using UBC 241 to 29 S =(2Nxy)/Nx + Ny, D = Àlog 10S; where S is the pair-wise using UBC 719 (Table 2). similarity coefficient, Nx and Ny are total number of bands The UBC 241 primers clearly distinguished Paphiopedilum produced by plant X and Y, respectively, Nxy the number of rothschildianum from the other Paphiopedilums and Phragmi- bands shared by plant X and Y and D is the genetic distance pedium sargentianum, Phrag. pearcei, Phrag. longifolium, between plant X and Y. The relationship between Paphiope- Phrag. ‘Belle Hogue Point’, Phrag. ‘Bakara LeAnn’, Phrag. dilum and Phragmipedium was displayed as a dendrogram, ‘Mem. Dick Clements’, Phrag. ‘Don Wimber’ and Phrag. which was derived from the genetic distance matrix by ‘Hanne Popow’ (Fig. 1). The bands produced from two unweighted pair group method using arithmetic average cultivars (Fig. 1, lanes 22 and 23) of Phrag. besseae had a (UPGMA) (Sneath and Sokal, 1973) and the NTSYS-pc unique band that differentiated them from other orchids clustering method (Rohlf, 1990). (primers UBC 241, UBC 248 and UBC 772).

Fig. 1. RAPD amplified from the genomic DNA of 34 Paphiopedilum and Phragmipedium species using random 10-mer primers (UBC 241, 248). Arrows (UBC 241) in lanes 1 and 17 indicated specific bands for differentiation of samples. Arrow (UBC 248) indicate band specific for lanes 22 and 23. Lane M, DNA size marker; lane 1, Paph. ‘Hamana Wave’; lane 2, Paph. emersonii; lane 3, Paph. ‘Mt. Toro’; lane 4, Paph. malipoense; lane 5, Paph. kolosand; lane 6, Paph. koloparkingii; lane 7, Paph. chamberianum; lane 8, Paph. stonei; lane 9, Paph. sukhakulii; lane 10, Paph. argus; lane 11, Paph. purpuratum; lane 12, Paph. callosum; lane 13, Paph. concolor; lane 14, Paph. fairrianum Red AM/APS; lane 15, Paph. hirsutissimum; lane 16, Paph. insigne; lane 17, Paph. rothschildianum; lane 18, Paph. wilhelminae ‘Fox valley’; lane 19, Paph. ‘Joyce Hasegawa’; lane 20, Paph. ‘St. Swithin’; lane 21, Phrag. caudatum warscewizianum; lane 22, Phrag. besseae flavum; lane 23, Phrag. besseae; lane 24, Phrag. longiflorum; lane 25, Phrag. pearcei; lane 26, Phrag. sargentianum; lane 27, Phrag. schilimii; lane 28, Phrag. ‘Ardean five’; lane 29, Phrag. ‘Belle Hogue Point’; lane 30, Phrag. ‘Desormers’; lane 31, Phrag. ‘Don Wimber’; lane 32, Phrag. ‘Bakara LeAnn’; lane 33, Phrag. ‘Mem. Dick Clements’ and lane 34, Phrag. ‘Hanne Popow’. 156 S.Y. Chung et al. / Scientia Horticulturae 109 (2006) 153–159 1.000 0.719 1.000 0.787 0.787 1.000 0.753 0.697 0.764 1.000 0.753 0.742 0.798 0.753 1.000 0.691 0.747 0.736 0.758 0.747 1.000 0.736 0.725 0.758 0.702 0.725 0.7640.713 1.000 0.770 0.747 0.725 0.758 0.7530.685 0.809 0.708 1.000 0.708 0.719 0.730 0.815 0.736 0.770 1.000 species and cultivars generated from Nei’s estimate of similarity Phragmipedium and 14 Paphiopedilums 12345678910111213141516171819202122232425262728293031323334 1 1.000 2 0.815 1.000 3 0.798 0.758 1.000 4 0.781 0.787 0.792 1.000 5 0.809 0.781 0.865 0.781 1.000 6 0.792 0.775 0.803 0.742 0.8827 1.000 0.803 0.753 0.792 0.742 0.8038 0.787 0.803 1.000 0.775 0.826 0.775 0.8609 0.854 0.792 0.854 0.742 1.000 0.770 0.764 0.792 0.764 0.764 0.775 1.000 27 0.713 0.742 0.736 0.775 0.74728 0.730 0.725 0.753 0.719 0.730 0.725 0.742 0.697 0.730 0.73629 0.758 0.719 0.702 0.719 0.730 0.708 0.719 0.730 0.669 0.758 0.764 0.685 0.742 0.775 0.657 0.76430 0.747 0.663 0.747 0.713 0.742 0.685 0.747 0.730 0.753 0.719 0.747 0.702 0.758 0.697 0.747 0.685 0.764 0.697 0.792 0.725 0.77531 0.691 0.747 0.742 0.736 0.697 0.697 0.792 0.697 0.758 0.680 0.697 0.708 0.764 0.758 0.685 0.691 0.758 0.730 0.747 0.702 0.674 0.719 0.792 0.697 0.71932 0.758 0.792 0.669 0.713 0.730 0.697 0.826 0.691 0.725 0.669 0.730 0.764 0.725 0.691 0.685 0.713 0.770 0.725 0.669 0.691 0.730 0.725 0.713 0.697 0.76433 0.742 0.781 0.680 0.725 0.730 0.702 0.713 0.725 0.736 0.736 0.702 0.719 0.691 0.736 0.697 0.708 0.713 0.792 0.758 0.785 0.713 0.736 0.747 0.742 0.72534 0.708 0.781 0.725 0.697 0.669 0.736 0.815 0.725 0.730 0.685 0.713 0.730 0.736 0.708 0.680 0.674 0.770 0.747 0.742 0.697 0.725 0.747 0.753 0.669 0.713 0.730 0.764 0.629 0.697 0.725 0.753 0.652 0.719 0.702 0.736 0.674 0.719 0.719 0.685 0.708 0.742 0.747 0.674 0.753 0.747 0.680 0.753 0.719 0.652 0.775 0.753 0.685 0.702 0.742 0.702 0.708 0.753 0.685 0.753 0.697 0.730 0.691 0.775 0.691 0.736 0.691 0.753 0.725 0.691 0.691 0.736 0.697 0.680 Table 3 Percent of similarity matrix from 20 10 0.770 0.787 0.781 0.775 0.77011 0.719 0.764 0.742 0.747 0.753 0.764 0.809 0.713 1.000 0.77512 0.758 0.792 0.781 0.753 0.803 0.792 0.825 0.730 0.770 0.81513 1.000 0.798 0.770 0.809 0.753 0.798 0.758 0.809 0.730 0.787 0.78114 0.815 0.775 0.730 1.000 0.742 0.736 0.787 0.798 0.809 0.747 0.775 0.77515 0.781 0.736 0.770 0.809 0.758 0.775 1.000 0.747 0.781 0.770 0.753 0.758 0.73616 0.764 0.730 0.792 0.758 0.753 0.787 0.747 0.775 0.826 1.000 0.787 0.764 0.798 0.86017 0.803 0.831 0.742 0.798 0.831 0.747 0.787 0.876 0.753 0.803 0.798 0.713 1.000 0.764 0.76418 0.770 0.781 0.775 0.820 0.747 0.770 0.787 0.758 0.753 0.770 0.770 0.792 0.787 0.758 0.798 1.000 19 0.730 0.770 0.787 0.781 0.792 0.803 0.781 0.815 0.764 0.775 0.770 0.770 0.725 0.792 0.775 0.78120 0.742 0.792 1.000 0.753 0.792 0.770 0.725 0.792 0.770 0.775 0.775 0.758 0.747 0.781 0.770 0.820 0.82621 0.764 0.792 0.753 0.730 0.826 0.736 1.000 0.691 0.826 0.803 0.708 0.764 0.792 0.736 0.781 0.781 0.719 0.79222 0.753 0.725 0.775 0.742 0.770 0.725 0.809 0.725 0.792 0.736 1.000 0.697 0.753 0.770 0.691 0.747 0.747 0.719 0.78123 0.742 0.725 0.742 0.753 0.747 0.725 0.798 0.725 0.758 0.747 0.798 0.730 0.764 0.792 1.000 0.702 0.803 0.758 0.708 0.75824 0.775 0.713 0.753 0.725 0.747 0.736 0.787 0.652 0.736 0.725 0.764 0.713 0.753 0.747 0.787 0.697 0.758 0.725 1.000 0.713 0.77025 0.742 0.697 0.753 0.719 0.736 0.697 0.753 0.713 0.736 0.719 0.787 0.730 0.753 0.742 0.775 0.725 0.792 0.708 0.809 0.730 0.73626 0.713 1.000 0.736 0.753 0.713 0.719 0.747 0.742 0.719 0.719 0.781 0.775 0.691 0.680 0.758 0.742 0.652 0.685 0.747 0.820 0.713 0.719 0.730 0.831 0.719 0.702 0.747 1.000 0.730 0.713 0.736 0.719 0.702 0.764 0.742 0.725 0.747 0.708 0.770 0.758 0.747 0.725 0.775 0.753 0.736 0.787 0.730 1.000 0.764 0.725 0.787 0.719 0.798 0.719 0.798 0.758 0.775 0.713 0.770 0.736 1.000 0.713 0.702 0.803 0.725 0.685 0.758 S.Y. Chung et al. / Scientia Horticulturae 109 (2006) 153–159 157

Fig. 2. Dendrogram of Paphiopedilums and Phragmipediums based on UPGMA cluster analysis and similarity index. Scale (bottom) and value above the branch are the UPGMA coefficient.

3.2. Analysis of phylogeny according to RAPD sargentianum to form there own subcluster, subcluster I-2-1. Therefore, cluster I-1 was composed of two subclusters, I-1-1 Nei’s method for determining genetic similarity was used to and I-1-2. Cluster I-2 also consisted of two subclusters, I-2-1 construct a similarity matrix (Table 3). The similarity values and I-2-2. Subcluster I-2-2 included only the Phragmipedium were calculated by scoring the total number and the species. Subgroup II was comprised of Phrag. longiflorum, polymorphic number of bands obtained with each primer. Phragmipedium varieties ‘Belle Hogue point’, ‘Bakara Similarity values ranged from 0.629, between Paph. kolosand LeAnn’, ‘Mem. Dick Clements’, ‘Don Wimber’ and ‘Hanne and Paph. koloparkingii,to0.882,betweenPaph. kolopar- Popow’. kingii and Phrag. ‘Hanne Popow’. The results from the UPGMA cluster analysis are shown as a dendrogram in Fig. 2. 4. Discussion As seen in this dendrogram, the UPGMA results segregated the orchids into two major subgroups. Subgroup I included 28 Previous population diversity studies of Goodyera procera species and subgroup II was comprised of 6 species. Subgroup (Orchidaceae)(Wong and Sun, 1999) and Paph. micranthum I included all Paphiopedilum species and eight Phragmipe- (Li et al., 2002) with allozyme and RAPD concluded that there dium species. This subgroup could be further separated into were low genetic diversities in both orchid populations and two minor clusters, we designated as I-1 and I-2. Cluster I-1 possible reasons might be inbreeding, and/or habitat fragmen- was comprised of almost all the Paphiopedilum species except tation. These results suggested that there might be low genetic Paph. rothschildianum, which was clustered with Phrag. diversities exist in some orchid species. 158 S.Y. Chung et al. / Scientia Horticulturae 109 (2006) 153–159

Cultivar developments of Paphiopedilum genus have long closely related to the ‘St. Swithin’, crossed that history and results of breeder’s effort, well diverged originated from parents in Polyantha. Paph. fairrieanum and Paphiopedilum varieties and complex hybrids with complicated Paph. hirsutissium of the Paphiopedilum subgenus are closely pedigree exist (Birk, 1983). Paphiopedilum genus could be related to each other. Paph. sukakuhlii and Paph. purpuratum of grouped as three according to the leaf morphology. These leaf the Paphiopedilum subgenus formed the small subcluster I-1-2. morphological groupings were generally matched with RAPD However, Paph. rothschildianum of the Polyantha subgenus, groupings of this experiment. In subcluster I-2-1, Paph. Mastigiopetalum section was related to Phrag. sargentianum, ‘Hamana Wave’, Paph. emersonii and Paph. malipoense which were previously classified into different genera (Braem shared similar leaf morphologies and Paph. ‘Mt. Toro’, Paph. et al., 1998, 1999; Cribb, 1998). kolosand, Paph. koloparkingii shared similar leaf morphologies In contrast with Paphiopedilum, variety development history and in subcluster I-2-1, Paph. sukhakulii, Paph. purpuratum, of Phragmipedium was not that long and pedigree of Paph. argus, Paph. fairrianum and Paph. hirsutissimum shared Phragmipedium varieties were relatively simpler than similar leaf morphologies (Fig. 2). As contrast, RAPD Paphiopedilum. There was a high degree of homology among groupings were not matched with flower color characteristics. the original species Phrag. caudatum Phrag. warscewizianum, Our results showed that Paph. emersonii and Paph. Phrag. besseae, Phrag. pearci, Phrag. schilimii, and the cross malipoense belong to the subgenus Parvisepalum and were species Phrag. ‘Ardean Five’ (Phrag. lindleyanum crossed with closely related. However, Paph. ‘Joyce Hasegawa’, which was Phrag. besseae), and Phrag. ‘desormers’ (‘Sorcerer’s Appren- known as interbred from Paph. delenatii and Paph. emersonii, tice’ crossed with Hanne Popow ‘DayGlo’). Major subgroup II was determined to be closer to the Sigmatopetalum subgenus was made up of the related species Phrag. longiflorum, Phrag. Paph. callosum. It is unfortunate that Paph. delenatii, which is ‘Belle Hogue Point’, Phrag. ‘Barbara LeAnn’ (Phrag. besseae one of the parents in the pedigree of Paph. ‘Joyce Hasegawa’, crossed with Phrag. fischeri), Phrag. ‘Mem. Dick Clements’ could not be obtained to analyze phylogenetic relationship to (Phrag. besseae ‘4th of July’ crossed with Phrag. sargentianum clarify the reason that Paph. ‘Joyce Hasegawa’ was determined ‘Westwood’), Phrag. ‘Don Wimber (3N)’ (Phrag. Eric Young to be closer to Paph. callosum. Maude type Paphiopedilum ‘Rocket Fire’ 4N crossed with Phrag. besseae flavum) and ‘Hamana Wave’ was more closely related to Paph. emersonii Phrag. ‘Hanne Popow’ (Phrag. besseae flavum ‘El Dorado’ than with Paph. callosum, which was speculated to be a close crossed with Phrag. schilimi ‘Golden Halo’). relative of ‘Hamana Wave’. The genetic relationship between 34 orchids belonging to The Polyantha subgenus Mastigiopetalum was found to two genera was estimated from RAPD results. Disagreement consist of Paph. koloparkingii, Paph. kolosand (interbred from with previous phenotypic classifications (Birk, 1983; Braem Paph. koloparkingii), Paph. stonei, Paph. ‘Mt. Toro’ (interbred et al., 1998, 1999; Cash, 1991; Cribb, 1998; Hunt, 1978) was from Paph. stonei), Paph. rothschildianum and Paphiopedilum noted. This was believed to reflect the quality of the ‘St. Swithin’ (interbred from Paph. rothschildianum and Paph. characteristics rather than the number of the characteristics philippines)(Birk, 1983; Braem et al., 1998, 1999; Cribb, because the target sequences for analysis were random 1998). Paph. koloparkingii and Paph. kolosand were closely according to RAPD method (Clark and Lanigan, 1993; Tingey related phylogenetically based on the RAPD analysis. Cultivar and Tufo, 1993). ‘Mt. Toro’ was closely related to Paph. stonei and Paph. In conclusion, our RAPD results indicated that: (1) insigne. Interestingly, Paph. stonei and Paph. insigne were Paphiopedilum and Phragmipedium were differed from each more closely related than Mastigiopetalum subgenus. other, (2) cultivars or species of were different from each other The RAPD results showed that almost all of the original and (3) classification using RAPD agreed well with some of species and their inbred counterparts belonging to Mastigio- previous morphology based classifications. This study showed petalum were grouped together. This agreed well with the that RAPD markers based on the genomic DNA of phenotype classification. However, these species were closely Paphiopedilum and Phragmipedium provided phylogenetic related to Paph. insigne, which is a standard type of information that addresses the genetic relationship of inter- Paphiopedilum. Paph. chamberlainianum var. chamberlainia- species. The discriminatory band patterns and phylogenetic tree num, including the Cochlopetalum subgenus, was closely created from the results of this study were successfully used to related to the Matigiopetalum genus and Paphiopedilum determine the lineages of Paphiopedilum and Phragmipedium subgenus in this analysis. The species classified into the species. Sigmatopetalum genus, Planipetalum section, which are Paph. sukhakulii and Paph. purpuratum (Birk, 1983; Braem et al., Acknowledgement 1998, 1999), exhibited a close relationship with each other and Paphiopedilum argus, which belongs to Sigmapetalum barbata This study was supported in part by a grant from the section. However, Paph. callosum, which also belongs S. Agricultural Research Center of Namyangjoo City in Korea. barbata section, was genetically distant. Paph. concolor, of the Brachipetalum genus, was closely References related to Paph. ‘Joyce Hasegawa’ and Paph. callosum. Paph. wilhelminae, thought to be mountain type of Paph. prestans in Bechtel, H., Cribb, P., Launert, E., 1981. The Manual of Cultivated Orchid the Mastigiopetalum section of the Polyantha genus, was also Species. MIT Press, p. 443. S.Y. Chung et al. / Scientia Horticulturae 109 (2006) 153–159 159

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