http://www.paper.edu.cn Aquatic Botany 84 (2006) 301–307 www.elsevier.com/locate/aquabot The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers Jin-Ming Chen a, Wahiti Robert Gituru b, Yu-Hang Wang a, Qing-Feng Wang a,* a Laboratory of Plant Systematics and Evolutionary Biology, College of Life Sciences, Wuhan University, Wuhan, 430072 Hubei, PR China b Botany Department, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya Received 30 April 2005; received in revised form 10 October 2005; accepted 30 November 2005 Abstract Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identification of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson’s diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets. # 2006 Elsevier B.V. All rights reserved. Keywords: Caldesia grandis; Clonal structure; Genetic diversity; ISSR; RAPD; Rare plant 1. Introduction 1998). A genet is composed of all tissue originating from one zygote, whereas a ramet is a potentially independent individual In the angiosperms, vegetative propagation is extremely derived from a single genet (Richards, 1986; Eriksson, 1993). widespread and common (Albert et al., 2003). Most perennial For a clonal plant population, the genetically effective flowering plants combine sexual reproduction with some form population size cannot be determined from counting the of asexual reproduction through vegetative propagation, for number of ramets present; what appears to be a ‘‘large’’ example by rhizomes, bulbils, layering, tillering, or rooting of population may be in fact be ‘‘small’’ in terms of genotypes surface runners (Cook, 1983; Richards, 1986; Eckert et al., (Esselman et al., 1999). Populations of clonal plants consisting 1999). Clonal growth is almost ubiquitous in aquatic or wetland of few genets tend to be subject to similar genetic processes that plants (Sculthorpe, 1967; Cook, 1990). affect any small population, such as genetic drift and inbreeding Clonal plants present special problems for the analysis of (Barrett and Kohn, 1991). In order to obtain information about genetic variation in populations because individuals can be population dynamics and evolution in clonal plants, the recognized at two different organizational levels: genets and effective population size cannot be determined just by counting ramets (Kays and Harp, 1974; Harper, 1977; Escaravage et al., ramets; genets as well as ramets must be studied (Eriksson, 1993). Any study of the conservation biology of clonal plants should be concerned with the number of genetic individuals * Corresponding author. Tel.: +86 27 68752869; fax: +86 27 68752869. (genets) found within the ramets of a population (Sipes and E-mail address: [email protected] (Q.-F. Wang). Wolf, 1997; Esselman et al., 1999). 0304-3770/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.11.008 转载 中国科技论文在线 http://www.paper.edu.cn 302 J.-M. Chen et al. / Aquatic Botany 84 (2006) 301–307 Caldesia grandis Samuelsson is a perennial, erect marsh by scattered patches of Viburnum macrocephalum. C. grandis herb that belongs to the aquatic family Alismataceae. This in BH population is found in four patches in BH marsh species is confined to mountainous bogs and marshes in (2580703000N; 09883304700E) in Yunnan Province. A total of 342 Southeast Asia and has been found in China and the eastern individuals were sampled for RAPD with 134 from LPH Himalayas (Cook, 1996). C. grandis in China has been reported population, 82 from GH, and 126 from BH. A subset from Hubei, Hunan, Guangdong and Yunnan Provinces in comprising 96 of the 342 individuals were examined for ISSR Mainland China as well as from the island of Taiwan (Gituru markers: 36 from LPH, 12 from GH and 48 from BH. Leaves of et al., 2002). The species is rare, occurring as small populations plants from LPH, GH and BH were collected from different in China. In our recent field investigation, only three natural plots and these plots represent almost the full spatial extent of populations including one population in Yunnan Province and the species within the populations (there are very few two populations in Hunan Province were found in Mainland individuals occurred between the sampled plots). We sampled China. C. grandis is a self-compatible species, which can every individual for each plot in the three populations except for reproduce both sexually by selfed and out-crossed seeds and plots 4 in GH population; in plot 4 of GH population sampled vegetatively through bulbils that commonly occur in the plants grew at least 1.5 m apart (the number of the individuals inflorescences (Gituru et al., 2002). C. grandis produces many in this plot is relative large). flowers and seeds, but seedling recruitment is rarely observed (Gituru et al., 2002). 2.2. DNA extraction Identification of different clones in populations of clonal plants have been greatly facilitated by the use of molecular Total genomic DNA was isolated from 0.5 g of silica-dried markers, such as allozymes (Widen et al., 1994) and leaf tissue following the procedure described by Fu et al. polymerase chain reaction (PCR)-based markers like random (2003). amplified polymorphic DNA (RAPD) (Esselman et al., 1999; Persson and Gustavsson, 2001; Hangelbroek et al., 2002; Albert 2.3. RAPD amplification et al., 2003), inter-simple sequence repeat (ISSR) (Esselman et al., 1999; Li and Ge, 2001) and amplified fragment length Reactions were carried out in a volume of 25 ml containing polymorphism (AFLP) (Albert et al., 2003; Escaravage et al., 0.25 mmol/l each of dNTP, 2.5 mlof10Â Taq buffer 1998; Suyama et al., 2000). Although allozymes analysis has [10 mmol/l Tris–Hcl (PH 8.3), 1.5 mM MgCL2 and 50 mM long been used to identify clones and to study population KCL], 1 mmol/l primer, 1 U Taq Polymerase (Tian Yuan genetics of clonal plants, it usually underestimates genetic Biotech) and 40 ng of DNA template. Amplification of polymorphism and has a limited ability to distinguish genetic genomic DNA was made on a PTC-100TM thermocycler (MJ individuals (Esselman et al., 1999; Wang et al., 1999). The Research, Inc.), and commenced with 4 min at 94 8C, followed PCR-based DNA markers evolve rapidly enough to be variable by 45 cycles of 1 min at 94 8C, 1 min annealing at 34 8C, and within a population, thus they are suited for detecting genotypic 2 min extension at 72 8C, and a final extension cycle of 7 min at diversity (Esselman et al., 1999). In the present study we 72 8C. Amplification products were resolved electrophoreti- employ RAPD and ISSR markers (1) to genetically identify C. cally on 1.5% agarose gels run at 100 V in 0.5 Â TBE (Tris– grandis clones as well as to estimate the diversity of these boric acid–EDTA), visualized by staining with ethidium clones; (2) to estimate the genetic diversity in C. grandis bromide, and photographed under ultraviolet light. Sixty populations; (3) to partition the genetic diversity among and RAPD primers from Genbase Co. Ltd. (Shanghai, China) were within populations. screened on six randomly selected individuals. The six samples were amplified twice with the same primer. Seven primers that 2. Materials and methods produced clear and 100% reproducible fragments were selected for further analysis (Table 2). 2.1. Study sites and sampling 2.4. ISSR amplification During July and August 2004, three wild populations of C. grandis occurring in three marshes Lang Pan Hu, Guai Hu, and PCR reactions were conducted in volumes of 25 ml Bei Hai (referred to as LPH, GH and BH populations) in Hunan containing 0.25 mM each of dNTP, 2.5 mlof10Â Taq buffer and Yunnan Provinces in China were sampled. In Hunan [10 mmol/l Tris–Hcl (PH 8.3), 1.5 mM MgCL2 and 50 mM Province, C. grandis is found in two populations (LPH and GH) KCL], 1 mM primer, 1U Taq polymerase (Tian Yuan Biotech) occurring in two marshes close to the center of Mangshan and 60 ng of DNA template. A PTC-100TM thermocycler (MJ Nature Reserve in Hunan Province. The LPH marsh Research) was used with the thermocycle program set at: 94 8C (2485200000N; 11284301900E) is densely covered with a thick for 2 min, followed by 35 cycles of 30 s at 94 8C, 1 min at mat comprised mainly of the peat moss Sphagnum cuspidatum 55 8C, 1.5 min at 72 8C, and ending with 7 min at 72 8C.