Scientia Horticulturae 109 (2006) 153–159 www.elsevier.com/locate/scihorti Genetic relationship and differentiation of Paphiopedilum 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 Plant Science, Chung Ang University, 456-756 Ansung, Republic of Korea Received 20 April 2005; received in revised form 16 March 2006; accepted 3 April 2006 Abstract The genetic diversity and relationship between Paphiopedilum and Phragmipedium species and cultivars 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 genus 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 southeast Asia, northern morphological traits to Paphiopedilum (Bechtel et al., 1981). India, southern China, Myanmar, Thailand and New Guinea. 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 plants, exception of Paphiopedilum lowii, 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 cultivar identification and can be differentiated from that of Cypripedium based on tissue genetic diversity studies of many plants (Wolff and Rijn, 1993; color, shape, the existence of variegated leaves, 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 (Orchidaceae) 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 leaf 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 Paphiopedilum malipoense 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 Paphiopedilum stonei 9 Paphiopedilum sukhakulii 2.2. RAPD amplification 10 Paphiopedilum argus 11 Paphiopedilum purpuratum 12 Paphiopedilum callosum RAPD DNA amplification was performed in a volume of 13 Paphiopedilum concolor 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 Paphiopedilum insigne 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 Phragmipedium caudatum warscewizianum performed at 94 8C for 3 min before beginning the cycling 22 Phragmipedium besseae 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 Phragmipedium pearcei 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.