Diversity of Genome Size and Ty1-copia in Epimedium Species Used for Traditional Chinese Medicines

Epimedium species are traditional Chinese medicinal plants as well as potential groundcover and ornamental plants. In this study, genome size and genome structures of Epimedium species were investigated using flow cytometric and fluorescence in situ hybridization (FISH). The nuclear DNA content of Epimedium species ranged from 8.42 pg/2C (8230.7 Mbp) to 9.97 pg/2C (9752.8 Mbp). The pairwise nucleotide diversity (p) of the fragments of the genes for reverse transcriptase (rt) of Ty1-copia retrotransposon within a species of rt fragments ranged from 0.251 to 0.428 in 10 Epimedium species. Phylogenetic analysis of the sequences revealed four major clades with the largest subclade containing 72 sequences of relatively low nucleotide diversity. FISH indicated that Ty1-copia retrotransposons are distributed unevenly along the pachytene chromosomes of E. wushanense and E. sagittatum, mostly associated with the pericentromeric and terminal heterochromatin. The relatively low sequence heterogeneity of Ty1-copia rt sequences implies that the Epimedium genomes have experienced a few relatively large-scale proliferation events of copia elements, which could be one of the major forces resulting in the large genome size of Epimedium species. Epimedium L. (2n = 2x = 12), referred to as yin yang huo in Chinese, belongs to the basal eudicot plant family, berberidaceae. The genus of Epimedium is composed of more than 50 species (Stearn, 2002), most of which are widely distributed in China and commonly used as traditional Chinese medicinal herbs (Ying, 2002) and as ornamental plants (Stearn, 2002). In particular, Epimedium species are of great interest because of their pharmacological properties in the treatment of impotence, spermatorrhea, infertility, amenorrhea, and in improving menopause symptoms (Wu et al., 2003). In China, Herba Epimedii is usually comacerated in wine with other traditional medicines contributing to prevent disease and strengthen immunity (Ma et al., 2011). Four flavonoids, epimedin A, epimedin B, epimedin C, and icarrin, were believed as the major active components in Epimedium and were regarded as markers for quality control (Chen et al., 2007; Xie and Sun, 2006; Xie et al., 2010). Five species are officially recorded as medicinal plants in the Chinese Pharmacopoeia, including E. brevicornum Maxim, E. sagittatum (Sieb. et Zucc), E. pubescens Maxim, E. wushanense T. S. Ying, and E. koreanum Nakai (Chinese Pharmacopoeia Commission, 2005). Repetitive DNA is the major components of plant genomes (Kubis et al., 1998), which is the primary determinant of genome size and structure and plays an important role in genome evolution. Plant genome size differs as a result of variable amounts of repetitive DNA (Bennett and Leitch, 2011; Flavell et al., 1974). Polyploidization, unequal recombination, and illegitimate recombination leading to plant genome expansion and contraction may be the major driving forces for plant genome size variation (Bennetzen, 2002). Moreover, retrotransposon insertions through a ‘‘copy and paste’’ mechanism also can increase host genome size rapidly (Hawkins et al., 2006; Piegu et al., 2006). Known as the most abundant repetitive DNA, plant transposable elements (TEs) are classified as RNA-mediated TEs (Class 1) and DNA-mediated TEs (Class 2) according to their transposition intermediate (Feschotte et al., 2002). Class 1 TEs are divided into long terminal repeat retrotransposons (LTR) and non-LTR retrotransposons. LTR etrotransposons can be further classified as Ty1-copia and Ty3-gypsy elements based on the order of their coding domains. Ty1-copia group retroransposons have been shown to be present throughout almost all plant genomes with high copy numbers (Flavell et al., 1992a). Sequence analyses of polymerase chain reaction (PCR) fragments of reverse transcriptase conserved domains have revealed very high degrees of sequence heterogeneity in many plants (Flavell et al., 1992b; Kumar et al., 1997). Phylogenetic studies of these LTR retrotransposon families provide information of unknown genomic components and suggest causes of genome size variation. Most recent studies on Epimedium have concentrated on its chemical composition (Jiang et al., 2009; Wu et al., 2011; Zhao et al., 2008), pharmacological properties (Wong et al., 2009), phylogenetic relationship (Sun et al., 2005), karyotype (Sheng et al., 2010; Zhang et al., 2008), and genetic diversity (Xu et al., 2007; Zhou et al., 2007). Despite its great potential value, genomic characteristics, including genome size and genome structure of Epimedium, have received little attention. Genomic analyses of Epimedium will provide basic information on genome duplication, speciation, and its complex metabolism. In this study, we aimed to characterize the Epimedium genome in terms of the nuclear DNA contents, the sequence diversity, and genomic distribution of Ty1-copia retrotransposons. Received for publication 23 Jan. 2012. Accepted for publication 9 May 2012. This paper was part of the colloquium ‘‘Research Highlights and Commercial Application of Medicinal Plants’’ held 27 Sept. 2011 at the ASHS Conference, Waikoloa, HI, and sponsored by the Working Group of Asian Horticulture (WGAH) and the Association of Horticulturists of Indian Origin (AHIO). This work was supported by the National Natural Science Foundation of China (No. 30800624), CAS/SAFEA International Partnership Program for Creative Research Teams Project, Knowledge Innovation Project of The Chinese Academy of Sciences (KSCX2-EW-J-20), and Distinguished Young Scientist Project in Hubei (2009CDA073). We thank Dr. J. Dolezel for providing seeds of Vicia faba cv. Inovec, Dr. Jianfang Gui (Institute of Hydrobiology, Chinese Academy of Sciences) and Dr. Yan Wang (Wuhan University Medicine Division) for sharing flow cytometry, Dr. Yanqin Xu and Ms. Xuejun Zhang for their assistance in material collecting, Dr. Andrew Flavell for helpful advices on designing degenerate primers, and Dr. Alice Hayward and Joao Loureiro for helpful comments on our article. We also gratefully acknowledge the valuable comments by three anonymous reviewers. To whom reprint requests should be addressed; e-mail [email protected]. HORTSCIENCE VOL. 47(8) AUGUST 2012 979 MATERIALS AND METHODS Plant materials. Wild populations of 20 to 50 plants of Epimedium species were collected from various locations (Tables 1 and 2) and grown in soil outside at Wuhan Botanical Garden, Chinese Academy of Sciences. Young leaves grown 2 weeks of seven species were used for flow cytometric analysis (Table 1). The Ty1-copia reverse transcriptase (rt) fragments were cloned from 10 species (Table 2). The species of E. acuminatum, E. dolichostemon, E. pubesens, E. sagittatum, and E. wushanense were included in both studies. Flow cytometric analysis. Relative DNA content was determined using propidium iodide-stained flow cytometry (Dolezel et al., 2007). The analysis was performed by an EPICS ALTRA flow cytometer (BECKMAN COULTER; Brea, CA) equipped with a water-cooled argon ion laser emitting at 488 nm for flow cytometry. Young leaves of the analyzed individuals and a reference standard were cochopped with a razor blade in a glass petri dish containing 0.5 mL of icecold MgSO4 buffer [9.53 mM MgSO4 7H2O, 47.67 mM KCl, 4.77 mM HEPES, 6.48 mM dithiothreitol, 0.25% (w/v) Triton X-100; pH 8.0] (Arumuganathan and Earle, 1991). The crude nuclei suspension was filtered through a 50-mm nylon mesh. Subsequently, 0.5 mL solution containing RNase and propidium iodide (both 50 mg mL) was added. After incubation for 10 min at room temperature, relative fluorescence intensity of nuclei was analyzed. Young leaves of Vicia faba cv. Inovec (2C = 26.9 pg) were used as the internal standards. At least 5000 nuclei were analyzed in each measurement and CV values were below 5.0%. Values obtained were converted to Mbp of nucleotides by the Eq. 1 pg = 978 Mbp (Dolezel et al., 2003). For a given species, 10 individuals were randomly selected from each population for the flow cytometric analysis. Differences in DNA content between species or populations were tested by one-way analysis of variance, and multiple comparison tests were determined by the Tukey’s honestly significant difference test (P # 0.05) with SSPS 13.0 for Windows (IBM, Chicago, IL). Cloning of reverse transcriptases and phylogenetic analysis. Total genomic DNA for PCR amplification was extracted from fresh young leaves using the CTAB method (Doyle and Doyle, 1987). The reverse transcriptase (rt) domain of Ty1-copia retrotransposons was amplified from genomic DNA using degenerate PCR with primers designed against conserved residues from RT domains of retrotransposons. Primer sequences were 5#-ACNGCNTTYYTNCAYGG-3#, encoding the peptide TAFLHG (amino-terminal), and 5#-ARCATRTCRTCNACRTA-3#, encoding YVDDML (carboxy-terminal), where R = A/G, Y = C/T, H = A/T/C, and N = A/G/C/T (Kumar et al., 1997). PCR was carried out in 50 mL containing 100 to 200 ng genomic DNA, 1.5 mM MgCl2, 1 · PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl), 200 mM dNTPs, 3 mM of each degenerate primer, and 2 U Taq DNA polymerase (Fermentas, Burlington, Ontario, Canada) on a Mastercycler gradient PCR cycler (Eppendorf; Hamburg-Nord, Hamburg, Germany). The PCR cycling profile included an initial denaturation step at 94 C for 5 min followed by 35 cycles of 30 s at 94 C, 50 s annealing at 47 C, and 45 s at 70 C with a final extension step at 72 C for 10 min. Purified PCR products (E. Z. N. A. Gel Extraction Kit; Omega Bio-tek, Norcross, GA) were ligated into the pMD18-T plasmid vector (TaKaRa Bio; Dalian, LiaoNing, China) and transformed into competent Escherichia coli (strain DH5a). Clones with positive inserts were detected by PCR using M13 primers. Twenty to 25 positive clones of each species were selected randomly and sequenced using an ABI 3100 (Applied Biosystems) by Shanghai Invitrogen Biotech Co. Ltd. BLASTX searches of the cloned sequences against the National Center for Biotechnology Information plant protein database were performed using default parameters. All sequences were translated into peptides using BioEdit 7.0.1 to find conserved residues (Hall, 1999). Nucleotide diversity Pi (p), between the sets of sequences from each species including the primer regions was calculated using DnaSP 4.10 (Rozas et al., 2003). It is given by the following formula: