Subtropical China includes a host of taxa that are presumed to be phylogenetically primitive, with many occurring as monotypic taxa. Cyclocarya paliurus (Batalin) Iljinsk. is a medium-sized (up to 30 m) deciduous tree growing in montane forests (ca. 400–2500 m a.s.l.) (Lu et al., 1999). This species has a scattered distribution in subtropical China and is the only representative in the genus Cyclocarya Iljinsk., often known as a “living fossil” (Ying et al., 1993; Wu et al., 2003). Therefore, it is an ideal candidate for inferring the evolutionary histories of ancient monotypic genera in subtropical China, e.g., inferring refugial locations and the predominant pattern of migration that has led to their present geographical range. Moreover, C. paliurus has multiple commercial uses and is widely exploited. The leaves taste sweet and are used as an ingredient in functional foods or beverages in China. Cyclocarya paliurus has preventive effects against hypolipidemia and diabetes mellitus and improves mental efficiency, antihypertensive action, and immunomodulation (Kurihara et al., 2003; Jiang et al., 2006; Xie et al., 2006). The increasing demand for C. paliurus in tea production and medical uses has already resulted in a rapid decline of population size and local extinctions at many natural places (D.-M. Fan, personal observation). Consequently, it is necessary to quantify patterns of genetic diversity and gene flow to develop adequate management strategies for the long-term conservation of this species and to ensure the rational use of wild genetic resources. In this study, we isolate and characterize 28 novel microsatellite loci for C. paliurus, which is the first step toward investigating the genetic diversity and spatial genetic structure of this species.
METHODS AND RESULTS
We sampled 24 C. paliurus trees in two natural populations (Jinggangshan, Jiangxi: 26.51707°N, 114.09920°E, n = 12; Yuyao, Fujian: 29.75192°N, 121.08393°E, n = 12). Voucher specimens for each population were deposited in the Jiangxi Agricultural University herbarium (accession no.: JXAU35129 and JXAU35158). Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987), and microsatellites were isolated using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol (Zane et al., 2002). A single individual from the Jinggangshan population was used to prepare the microsatellite-enriched library. Total genomic DNA (ca. 250–500 ng) was completely digested with 2.5 U of Mse I restriction enzyme and then ligated to an Mse I AFLP adapter (5′-TACTCAGGACTCAT-3′/5′-GACGATGAGTCCTGAG-3′) using T4 DNA ligase (MBI, Fermentas, Vilnius, Lithuania). The digested-ligated fragments were diluted in a ratio of 1:10, and 5 µL of them were used for amplification reactions with adapter-specific primers (5′-GATGAGTCCTGAGTAAN-3′/5′-TTACTCAGGACTCATCN-3′). The amplified DNA fragments (200–800 bp) were enriched for simple sequence repeats by magnetic bead selection with a 5′-biotinylated probe [(AG)15 or (AC)15, respectively]. Enriched DNA fragments were reamplified with Mse I-N primers. The PCR products were purified using SanPrep Column DNA Gel Extraction Kit (Sangon Bio-Tech, Shanghai, China). Purified DNA fragments were ligated into pGEM-T Easy Vector (Promega Corporation, Madison, Wisconsin, USA), and then transformed into DH5α competent cells (Tiangen Biotech, Beijing, China). The positive clones were tested by PCR using vector primers T7/Sp6 and primers (AC)10/(AG)10. In total, 337 clones with positive inserts were sequenced with an ABI PRISM 3730xl DNA sequencer (Applied Biosystems, Carlsbad, California, USA). A total of 153 sequences contained microsatellite repeats, and 137 with relatively long flanking regions were used to design primers using OLIGO 7.0 software (Rychlik, 2007).
Characteristics of 28 microsatellite primers developed in Cyclocarya paliurus.
Polymorphism of all loci with newly designed primer pairs was assessed with all 24 individuals sampled. The PCR reactions were performed in a 20-µL reaction volume containing 50–100 ng of genomic DNA, 0.5 µM of each primer, and 10 µL 2× Taq PCR MasterMix (0.1 U Taq polymerase/µL, 0.5 mM dNTP each, 20 mM Tris-HCl [pH 8.3], 100 mM KCl, and 3 mM MgCl2; Tiangen Biotech, Beijing, China). PCR amplifications were conducted under the following conditions: 95°C for 3 min followed by 32–35 cycles at 94°C for 45 s, at the annealing temperature for each specific primer (optimized for each locus, Table 1) for 45 s, 72°C for 45 s; and a final extension step at 72°C for 5 min. PCR products were separated by 8% nondenaturing PAGE gel and stained with a silver-staining method. A portion of PCR products were checked using QIAxcel for capillary gel electrophoresis (QIAGEN, Düsseldorf, Germany).
Results of initial primer screening in populations of Cyclocarya paliurus.
Among the 137 primer pairs, 28 successfully amplified in all samples. All 28 primer pairs displayed polymorphism. Standard genetic diversity parameters, e.g., the number of alleles per locus, expected heterozygosity, and observed heterozygosity, were calculated using the package GENEPOP (version 4.0; Raymond and Rousset, 1995). The number of alleles per locus ranged from two to eight, with a mean of 3.3. The expected and observed heterozygosities ranged from 0.153 to 0.802 and from 0 to 0.750, respectively (Table 2). The marker transferability of the polymorphic primer pairs was tested on three closely related species, Juglans regia L., Pterocarya stenoptera C. DC., and Platycarya strobilacea Siebold & Zucc. (three individuals for each species), using the same PCR conditions as previously described. Leaf samples of these three species were collected from cultivated trees in Jiangxi Agricultural University, Nanchang, China (28.76073°N, 115.82740°E; voucher no. JXAU35160–JXAU35162). Twenty-one markers (75.0%) were successfully amplified in J. regia, 22 (78.6%) in P. stenoptera, and 15 (53.6%) in P. strobilacea (Table 1).
The 28 microsatellite markers developed in this study will be useful for detection of genetic diversity in C. paliurus populations and elucidation of population dynamics. These markers will also help to develop viable strategies for the conservation and management of this monotypic genus. In addition, more than half have been successfully amplified in three closely related species; thus, these markers may represent a set of useful molecular tools for population genetic studies in other species of Juglandaceae.
 The authors thank S. Q. Lei for help with the field survey and leaf collection. We are grateful to Z. R. Zhang for assistance with the molecular techniques, and to H. L. Hu, Y. Yang, W. X. Chen, and R. Wu for help with laboratory work. This study was supported by grants from the National Natural Science Foundation of China (grant no. 31160043 and 41061006) and the National Science and Technology Support Program (2012BAC11B02).