This article is an Advance Online Publication of the authors’ corrected proof. Note that minor changes may be made before final version publication. The Horticulture Journal Preview e Japanese Society for doi: 10.2503/hortj.OKD-087 JSHS Horticultural Science http://www.jshs.jp/ Estimation of Phylogeny of Nineteen Sedoideae Species Cultivated in Korea Inferred from Chloroplast DNA Analysis Mi Sang Lim and Sun Hee Choi* Department of Horticulture, Biotechnology and Landscape Architecture, Seoul Women’s University, Seoul 01797, Korea The genetic diversity and relationships among plants belonging to the subfamily Sedoideae (Crassulaceae), some of which are indigenous to Korea or introduced from other countries, were determined using chloroplast (cp) nucleotide sequence analysis. To analyze genetic diversity and variation among 19 plants including species belonging to Sedum, Hylotelephium, and Phedimus, the tRNA-Leucine gene (trnL [UAA]) and adjoining spacer in chloroplast DNA (cpDNA) were sequenced and compared across species. Species were divided into two main groups based on the cpDNA sequence comparison. The generated phylogeny indicated that many native Sedum species had diverged from S. album. Members of the Phedimus and Hylotelephium species, and several Sedum species analyzed here, clustered distinctively in different groups. Using cpDNA sequence analysis, we successfully discriminated Sedoideae plants cultivated in Korea from each other, even at the intraspecific level, and the results were reflective of the morphological and biogeographical characteristics. These findings could be useful for classifying samples for proper naming, choosing breeding materials for new cultivars, or identifying species for conservation of horticultural crop resources. Key Words: classification, Crassulaceae, Hylotelephium, Phedimus, Sedum. countries, Mexico, and Far Eastern countries like Introduction China, Japan, and Korea (Stephenson, 1994). Sedum is The Crassulaceae family includes 35 genera in six known to contain about 470 species (Mayuzumi and subfamilies that are divided into two lineages, a Ohba, 2004), 22 of which are indigenous to Korea Crassula lineage and a Sedum lineage. The latter line- (http://www.nature.go.kr). The taxonomy of the sub- age is composed of three subfamilies (Echeverioideae, families in Crassulaceae is based on the number and ar- Sedoideae, and Sempervivoideae) (Berger et al., 1930). rangement of floral parts, the degree of sympetaly, and Despite the general consensus that a substantial part of the phyllotaxis, with these factors featuring in each of it is highly artificial, Berger’s classification for the larger genera (Van Ham and ’T Hart, 1998). Plants Crassulaceae has been widely used because of its com- in the genus Sedum have succulent leaves, and the flow- prehensiveness and great practical value (Van Ham ers usually have five petals, and only occasionally four and ’T Hart, 1998). Sedum, the largest genus of or six. These features, along with their chromosome Crassulaceae, is a polyphyletic genus that encompasses counts, define the genus Sedum (Ohba, 1977). While much of the morphological diversity present in the fam- the basic genome chromosome number in Sedum spe- ily. The Sedum genus is, however, still poorly character- cies is eight (x = 8), chromosome doubling has oc- ized and requires further investigation (Mort et al., curred repeatedly, and some Sedum species contain x = 2001). Plants of the genus Sedum occur across the 16, 32, and 64. Polyploidy appears to be common Northern Hemisphere, including in the Mediterranean throughout the Sedum genus, and some members also show euploidy with chromosome counts of x = 7 or 31 Received; February 7, 2017. Accepted; June 5, 2017. (Stephenson, 1994). Especially in Korea, Sedum taxon- First Published Online in J-STAGE on July 12, 2017. omy is mainly based on morphological characteristics, This work was supported by a research grant from Seoul Women’s and there is currently no accurate method of classifying University (2015), and also carried out with the support of Sedum species by genetic variation and divergence “Cooperative Research Program for Agriculture Science & from the closest relative. Hylotelephium and Phedimus Technology Development (Project No. PJ010878042016)” Rural Development Administration, Republic of Korea. species have been previously classified as Sedum spe- * Corresponding author (E-mail: [email protected]). cies (e.g., Sedum telephium) (Ohba, 1977). However, © 2017 The Japanese Society for Horticultural Science (JSHS), All rights reserved. 2 M. S. Lim and S. H. Choi there is still confusion in the common or public naming examine the relationships between them. Sedoideae system in Korea. Sedum species, such as species used in this study either grow naturally or have S. sarmentosum, which is known as ‘Dolnamul’ in been recently introduced to Korea and are cultivated. Korean, have been used traditionally as food. Recently, To discriminate native species from imported species Sedum species were recognized as useful plant re- and to develop hybrid cultivars showing useful new sources for landscape architecture or materials for traits in breeding programs, the genetic characteristics ground cover in Korea. Despite these uses, a classifica- and relationships need to be assessed. Therefore, we tion system for Sedum and related species based on ge- aimed to estimate the genetic relationships between netic identification has not been properly established in Sedoideae plants from various origins by examining Korea. To avoid naming confusion in the commercial cpDNA sequences. The information obtained in this market and improper identification, we aimed to identi- study could be useful for discriminating the native fy indigenous Sedum and related species and those pre- plants from each other for identification during resource viously imported and now cultivated in Korea using conservation programs. The evaluation of molecular molecular genetics data from chloroplast DNA cpDNA markers would allow the development of prop- (cpDNA) sequences to facilitate the conservation of er discrimination methods among Sedoideae plants cul- horticultural resources and breeding materials. tivated in Korea and their application for various Since DNA sequence data has led to reconstructions practical uses, including in breeding programs. of classifications that often conflict with traditional Materials and Methods taxonomy, cpDNA sequences have proven to be a valu- able tool for phylogeny construction in plants because Plant materials and genomic DNA extraction of their small size, high copy number, and maternally Nineteen sample plants were obtained from the inherited nature (Chase and Palmer, 1989; Dong et al., National Institute of Horticultural and Herbal Science 2013; Wu et al., 2010). The simultaneous alignment of located in Gyeonggi province, Korea, in May, 2014. many nucleotide sequences is an essential tool in mo- Samples comprised eleven Sedum species, five lecular biology to detect homology among species, and Phedimus species, and three Hylotelephium species the use of cpDNA in multiple sequence alignment to (Table 1). The closely related genera Aeonium and examine relationships and evolutionary changes is ad- Greenovia were used as outgroups for the analysis. The vantageous. To resolve phylogenetic relationships at species used and their overall characteristics are shown several taxonomic levels, the maturase K (matK) gene in Table 1. As the imported Sedoideae plants have been of cpDNA was used (Soltis et al., 1996). Recently, the cultivated in Korea over several years, they were used phylogeny of the Dendrobium species in Thailand was in addition to the indigenous plants. Genomic DNA was reconstructed using data from a partial matK gene and extracted using an DNeasy Plant Mini Kit (Qiagen, rDNA internal transcribed spacer (ITS) sequences USA). For each sample, 100 mg FW of plant leaves (Srikulnath et al., 2015). Using sequences in a noncod- was used. The DNA quantity and quality were mea- ing region in cpDNA, Crassulaceae species have been sured by absorbance at 260 nm with an ND 2000 spec- analyzed by several research groups for their molecular trophotometer (Nanodrop Technologies, USA), and evolution or discrimination within the genus (Van Ham isolated DNA was checked by visualization on a 1% and ’T Hart, 1998; Van Ham et al., 1994). In particular, agarose gel with ethidium bromide (EtBr). Mayuzumi and Ohba (2004) evaluated the phylogenetic positions of Eastern Asian Sedoideae plants using trnL- Amplification of cpDNA and sequencing F (spacers between tRNA-Leucine and Phenylalanine) To compare cpDNA sequence variation, regions data. Recently, trnL-F regions were applied to estimate consisting of a tRNA-UAA gene (trnL) and trnL-trnF the relationships and track the origins among 31 hostas intergenic spacer (trnL-F) located in the large single- (Lee and Maki, 2015). Random amplified polymorphic copy region of cpDNA were amplified. Samples were DNA (RAPD) has also been used previously to set con- analyzed using primers encompassing trnL and flanking servation priorities and design management strategies two intergenic spacers, trnL-F and trnT-L, as previously for taxa in Sedum species (Olfelt et al., 2001). Korean described by Taberlet et al. (1991). PCR amplification Sedum plants, in particular, are thought to have diverse reactions contained 10–20 ng of genomic DNA, 20 pM phenotypes at both the species and interspecies levels of each primer (Taberlet et