Biochemical Systematics and Ecology 39 (2011) 425–433 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Genetic diversity of the traditional Chinese medicinal plant Ypsilandra thibetica (Melanthiaceae): Applications for conservation Hong-Tao Li a,b, Hong Wang a, Jun-Bo Yang a,b, De-Zhu Li a,b,* a Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Yunnan 650204, China b The Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China article info abstract Article history: Twelve microsatellite markers were developed to determine the genetic diversity and Received 31 December 2010 genetic structure of Ypsilandra thibetica, represented by a total of 90 individuals from six Accepted 3 June 2011 natural populations. All twelve microsatellite loci were polymorphic, and the results Available online 28 June 2011 indicated that a high genetic diversity was present within populations (mean RS ¼ 4.996; mean HE ¼ 0.615), with high levels of genetic structure (mean FST ¼ 0.165; mean Keywords: FIS ¼ 0.692) among populations. This pattern is likely attributable to consanguineous Conservation mating, and this hypothesis is supported by a low relatedness coefficient. Our study Genetic diversity fl Gene flow suggested that environment factors might restrict gene ow among populations. In Microsatellite markers addition, physical distances between populations were not related to genetic distances, Ypsilandra thibetica implying that ancestral populations might have been distributed over a wider area. These results suggest that Y. thibetica should be a high priority for conservation managers. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The genus Ypsilandra Franchet (Melanthiaceae) comprises six herbaceous plant species and is mainly distributed in China (Chen and Minoru, 2000; Chen et al., 2003). Species of Ypsilandra are perennial with thick rhizomes, and are associated with moist hillsides, shady slopes and forested habitats. Ypsilandra thibetica Franchet is endemic to southwestern China. It is distinguished from related species by the length of its pedicels and tepals (6–10 mm), and by the 10–18 mm long stamens that extend beyond its tepals at anthesis (Chen and Minoru, 2000). Y. thibetica has been used in traditional Chinese medicine especially in southwestern China (Xie et al., 2009). The pharmacological effects of Y. thibetica materials include hemostatic (Jiangsu, 1977; Zhou et al., 2003) and anticancer (Li et al., 1995) properties. Traditional medical treatments involving phytotherapy have played important roles for people throughout history, and modern medicine owes some if its success to these practices. More than 80% of the world’s population use medical treatments based on plant remedies, and more than 20% of the world’s pharmaceutical medicines are derived from plants (Rai et al., 2000). Unfortunately, overharvesting has resulted in a severe decline of many plant species and even the extinction of some (Mills, 2006). Preliminary investigations in China indicated that Y. thibetica habitats have shrunk and population numbers have declined over the last two decades. In our survey of its habitat, no wild populations of Y. thibetica harbored more than 30 individuals, and most contained fewer than 10 individuals. Their habitats have become highly fragmentized by anthropogenic disruptions. As such, successful conservation of this species will depend upon judicious interventions based on sound genetic diversity data of these populations. In this study, twelve polymorphic microsatellite markers for Y. thibetica * Corresponding author. Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Yunnan 650204, China. Tel.: þ86 871 5223503; fax: þ86 871 5217791. E-mail addresses: [email protected] (H.-T. Li), [email protected] (H. Wang), [email protected] (J.-B. Yang), [email protected] (D.-Z. Li). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.06.004 426 H.-T. Li et al. / Biochemical Systematics and Ecology 39 (2011) 425–433 were developed and evaluated their diversity among 6 natural populations. The research objectives were to describe the genetic diversity of Y. thibetica and to propose conservation measures for this important medicinal herb. 2. Materials and methods 2.1. Materials More than 20 wild populations of Y. thibetica were surveyed, of which most contained fewer than 10 individuals and only six had more than 20 individuals. Fourteen to 16 individuals from each of six natural populations of Y. thibetica were sampled from locations across its range, for a total of 90 individuals (Fig.1, Table 1). A 10 m minimum distance between individuals was implemented to reduce the potential of sampling ramets from the same genetic. Healthy, clean leaves were collected from each individual, then quickly desiccated and dried in silica gel. A portion of each sample was separated and deposited as a herbarium specimen at the Herbarium of the Kunming Institute of Botany, the Chinese Academy of Sciences (KUN). 2.2. DNA extraction, microsatellite marker development For each sample, total genomic DNA was isolated from 0.2 g silica gel-dried leaf material ground in liquid nitrogen following a modified CTAB method (Doyle and Doyle, 1987), using 4% CTAB instead of 2%, and the addition of approximately 1% polyvinyl polypyrrolidone (PVP) and 2% b-mercaptoethanol. The isolation of microsatellite loci was performed according to the FIASCO method (fast isolation by AFLP of sequences containing repeats) (Zane et al., 2002). Total genomic DNA (about 500 ng) was completely digested with MseI and then ligated to an MseI AFLP adapter. A diluted digestion-ligation mixture (1:10) was amplified with adapter-specific primers (50-GAT- GAGTCCTGAGTAAN-30). Amplified DNA fragments of sizes ranging from 200 to 800 bp were enriched for repeats by magnetic 0 bead selection with 5 -biotinylated (AC) 15,(AG)15, and (AAG) 10 probes. Subsequently, enriched fragments were amplified again with adapter-specific primers. Polymerase chain reaction (PCR) products were purified using an EZNA Gel Extraction Kit (Omega Bio-Tek). Purified DNA fragments were ligated into the pGEM-T vector (Promega), and transformed into DH5a cells. Positive clones were tested by PCR using (AC) 10/(AG) 10/(AAG) 7 and T7/Sp6 primers. In total, 320 clones with positive inserts were sequenced with an ABI PRISM 3730XL DNA sequencer. A total of 240 (75%) sequences were found to contain Fig. 1. Distribution and sampling locations for Ypsilandra thibetica in southwestern China. H.-T. Li et al. / Biochemical Systematics and Ecology 39 (2011) 425–433 427 Table 1 Localities and sample sizes for the six Ypsilandra thibetica populations. Provinces Populations Codes Coordinates na Chongqing Nanchuan NC 29.10N, 107.05E 15 Guangxi Longsheng LS 25.78N, 110.02E 15 Guangxi Maoershan ME 25.81N, 110.66E 16 Hunan Xinning XN 26.44N, 110.84E 15 Sichuan Luding LD 29.92N, 102.25E 14 Yunnan Zhaotong ZT 26.90N, 102.92E 15 a The sample sizes (n) represent number of individuals analyzed. microsatellite repeats, and 60 of them were suitable for designing locus-specific primers, using the primer 5.0 program (Clarke and Gorley, 2001). All 60 microsatellite loci were screened for polymorphisms using genomic DNA of 30 samples of Y. thibetica from six natural populations in southwest China. The PCR reactions were performed in 15 ml of reaction containing 30–50 ng genomic DNA, 0.6 mM of each primer, 7.5 ml2Â Taq PCR MasterMix (Tiangen), 0.1 U Taq Polymerase/ml, 0.5 mM dNTP each, 20 mM Tris– HCl (PH8.3), 100 mM KCl, 3 mM MgCl2). PCR amplifications were conducted under the following conditions: 97 C for 3 min; 32 cycles at 94 C for 40 s, specific annealing temperature (Table 2) for 40 s, and 72 C for 1 min; followed by a final extension step at 72 C for 7 min. PCR products were separated and visualized using QIAxcel gel electrophoresis system (QIAGEN, Irvine, USA). Genotypes of 90 individuals were determined using 12 nuclear simple sequence repeat (nSSR) markers developed in this study (Y182, Y536, Y371, Y420, Y406, Y407, Y448, YL2, Y501, YL4, Y58, Y463). 2.3. Data analysis The numbers of observed alleles (Na), numbers of alleles per population (Nap) and number of effective alleles (Ne)were calculated for each of the 12 loci, and overall Na and Ne were calculated for each of the six populations using GenALEX 6 (Peakall and Smouse, 2006). Summary statistics for allelic richness (El Mousadik and Petit, 1996), observed heterozygosity (HO), expected heterozygosity (HE; Nei, 1987), and fixation index (FIS ¼ 1ÀHO/HE) were calculated over all loci and populations using FSTAT version 2.9.3.2 (Goudet, 1995), GENETIX 4.0.5 (Belkhir et al., 2001) and GENEPOP version 4.0 (Raymond and Rousset, 1995). FSTAT version 2.9.3.2 was used to test departures from Hardy–Weinberg equilibrium at each locus, as well as linkage disequilibrium between loci (alleles were randomized 1000 times over all samples). Table 2 Specific primer sequences and allelic characteristics for fifteen microsatellite loci isolated from Ypsilandra thibetica. 0 0 Locus Repeat motif Primer sequences (5 –3 )Ta(C) Expect size(bp) Allele size (bp) A HO HE GenBank Accession no. Y182 (AG)6AA(AG)7 F:TACTTAGGTGGGGTGGGC 55 228 224–248 8 0.242 0.680 GQ856940 R:AGGAACAAAAGGTGGTGA