Translator Disclaimer
24 July 2017 Development and Characterization of Microsatellite Primers for Zanthoxylum schinifolium (Rutaceae)
Author Affiliations +

The genus Zanthoxylum L. (Rutaceae) includes approximately 200 or more species around the world (Wu et al., 2008), but only about six species occur in Korea. Two of these species, Z. schinifolium Siebold & Zucc. and Z. piperitum (L.) DC., are the most representative common species in this genus in Korea. Zanthoxylum schinifolium is a deciduous shrub distributed in China, Japan, Taiwan, and Korea (Hassler, 2016). This species has a unique aromatic flavor on its fruits and leaves and has therefore been traditionally used as an edible or medicinal plant. Recently, significant effectiveness in the antibacterial and anticancer activity of some of the compounds of this species has been scientifically confirmed (Choi et al., 2008; Li et al., 2013). Consequently, this species is attracting attention as a promising medicinal plant.

In many cases, the increasing economic value of a plant species can lead to drastic reductions in wild populations because of extensive use; thus, conservation efforts of genetic resources as breeding materials should be increased (Shippmann et al., 2003). Zanthoxylum schinifolium, a medicinal species that breeders have recently started to cultivate, still has ample natural populations. Therefore, it can be used as a model species to identify the impact of harvest pressure on the genetic diversity patterns of wild populations of medicinal plants. However, most studies on this species have focused on the identification of medicinal compounds. In our review of the literature, the only genetic studies found for the species were studies of molecular identification based on ribosomal DNA sequence information (Sun et al., 2010) and phylogenetic relationships with cpDNA markers (Feng et al., 2016). However, these markers are unsuitable for analyzing the genetic variation of the species for conservation and management (Wan et al., 2004). Microsatellite markers are preferred in studies of genetic variation of individuals or populations due to their high level of polymorphism, codominance, biparental inheritance, and reproducibility of results (Varshney et al., 2005).

We have developed and evaluated microsatellite markers for further genetic studies of Z. schinifolium, and tested their cross-amplification in the related species Z. piperitum.


Sample collection and DNA extraction—A total of 102 samples of Z. schinifolium from three natural populations were collected to develop and validate new microsatellite primers for this species. To identify cross-amplification of the markers to other related species in the same genus, 30 samples of Z. piperitum were collected from one population. Detailed information on all of the samples collected for this study is provided in Appendix 1. Total genomic DNA (gDNA) was isolated from fresh leaves using Biomedic Plant gDNA Extraction Kit (Biomedic, Bucheon, Gyeonggi, South Korea). The DNA and leaf samples collected for this study were stored in the Gene Bank of the National Forest Seed and Variety Center (NFSV, Chungju, South Korea).

Construction of a microsatellite enrichment library—A microsatellite enrichment library was constructed according to the magnetic bead hybridization method of Glenn and Schable (2005) using one sample of Z. schinifolium collected from Chungju-si, Chungcheongbuk-do, South Korea. To obtain DNA fragments ranging from roughly 0.3 to 1 kbp, total gDNA was digested using the restriction enzyme RsaI and then ligated with SuperSNX linkers containing a GTTT PIG-tail. DNA molecules in the ligation products were hybridized with 3′-biotinylated microsatellite probes and then subsequently isolated using streptavidin-coupled (M-280) Dynabeads (Invitrogen, Carlsbad, California, USA). PCR amplification was performed on the collected DNA molecules with Super-SNX-24 primers. This enrichment step was repeated once. The DNA fragments highly enriched with microsatellites were cloned into pGEM-T vectors (Promega Corporation, Madison, Wisconsin, USA) using Escherichia coli DH5α-competent cells. Recombinant clones were identified by colony PCR using M13 forward and reverse primers. The PCR products were purified, and then directly sequenced using the ABI 3730 DNA Analyzer (Applied Biosystems, Waltham, Massachusetts, USA). After the trimming of vector and linker sequences, 182 nonredundant contig sequences (GenBank accession no. KU884701–KU884883) were obtained from the assembly process using Lasergene SeqMan (version 7.0.0; DNASTAR, Madison, Wisconsin, USA).

Microsatellite primer design and validation—Putative microsatellites were mined using MISA software (Thiel et al., 2003) based on the following criteria: more than three repeats for dinucleotides to hexanucleotides and a gap within 100 bp in composite types. Amplicon size (85–350 bp) and annealing temperature (57–60°C) were the main consideration in primer design. A total of 104 primer sets were synthesized by Biomedic Co. Ltd. (; Bucheon, South Korea) and used for preliminary screening. The preliminary screening of markers was performed by conventional PCR using the gDNA from eight samples of Z. schinifolium as templates to identify putative loci. Then, these PCR products were separated on a 2% agarose gel.

Finally, 20 microsatellite marker candidates, which produced amplicons at putative single loci, were selected to validate polymorphism and crossamplification. PCR was performed using an ABI2720 Thermal Cycler (Applied Biosystems) in a 11-µL reaction volume containing 3 µL of template DNA (3 ng/µL), 1.1 µL of 2 mM dNTPs, 0.22 µL of 10 µM 6-FAM fluorescent dye–labeled forward primer and reverse primer, 0.15 µL of NeoTherm Taq DNA polymerase (5 U/µL; GeneCraft, Köln, Germany), 1.1 µL of 10× reaction buffer (containing 25 mM MgCl2; GeneCraft), and 5.21 µL of distilled water. The PCR was performed with an initial denaturation at 94°C for 5 min; followed by 34 cycles of 94°C for 30 s, 57–64°C for 1 min (Table 1), and 72°C for 1 min; and a final extension at 72°C for 10 min. After PCR amplification, 0.2 µL of the fluorescent PCR products were mixed with 9.8 µL of Hi-Di Formamide (Applied Biosystems) and 0.2 µL of GeneScan 500 ROX Size Standard (Applied Biosystems). The mixture was denatured at 95°C for 5 min and placed on ice. The amplified fragments were separated by capillary electrophoresis on an ABI 3730 Genetic Analyzer (Applied Biosystems). Each of the individual genotypes was scored using GeneMapper 4.1 software (Applied Biosystems).

Table 1.

Characteristics of 15 polymorphic and three monomorphic loci developed for Zanthoxylum schinifolium and cross-amplified in Z. piperitum.


Of the 20 candidate primers, 18 (90%) were successfully amplified for Z. schinifolium. Of these 18, 15 produced polymorphic DNA fragments, and the remaining three primers produced monomorphic amplicons (Table 1). The percentage of amplification was 61.1% (11/18) for Z. piperitum. Out of these 11, six primers showed polymorphism with two or more alleles, but the remaining five primers had only one allele in the 30 samples analyzed. In most primers, the size range of alleles overlapped between the two species, but three primers (Zs3027, Zs3038, and Zsm3029) showed completely different size ranges between the two species, so these markers could be used to distinguish between the two species.

Evaluation of genetic properties for use as polymorphic markers—The genetic properties of the 15 polymorphic primers for Z. schinifolium were evaluated using 102 samples from three populations (Table 2). Population genetic diversity parameters (i.e., number of alleles [A], number of effective alleles [Ae], and observed [Ho] and expected [He] heterozygosities) were estimated using GenAlEx version 6.41 software (Peakall and Smouse, 2006). The Hardy– Weinberg equilibrium (HWE) at each locus for each population was tested based on χ2 tests using GenAlEx version 6.41 software (Peakall and Smouse, 2006). Polymorphic information content (PIC) and nonexclusion probability (NEI; identity) were calculated by CERVUS version 3.0.3 (Kalinowski et al., 2007). The test for null allele presence was performed using MICRO-CHECKER (van Oosterhout et al., 2004). Over all samples, A ranged from two to eight, Ae ranged from 1.1 to 3.2, and PIC values were calculated as 0.087 to 0.650. Ho and He ranged from 0.070 to 0.677 and 0.093 to 0.688, respectively. The near-zero NEI value (0.0000005) indicated that the developed markers in this study are useful for individual identification. A ranged from one to four for Z. piperitum, and Ho and He at six polymorphic loci were in the respective ranges 0.077–1.000 and 0.211–0.536. Significant deviations (P < 0.05) from HWE were detected for some primers within each population. Because agreement with HWE depends on certain assumptions including an infinite population size, simple Mendelian inheritance in a diploid organism, discrete generations, and random mating, the test results could not be interpreted without information such as the mating system in the tested populations. Unexpected genotype patterns in the microsatellite data set were reported, of which null allele presence has been frequently mentioned as an explanatory cause (Dakin and Avise, 2004). The null test results indicated a significant possibility of the presence of null alleles at some loci in some populations (Table 2). In particular, Zs2011 showed a significant possibility of the presence of null alleles in all three tested populations; this locus should be used carefully in further genetic studies. Additional testing of known parent– offspring relationships should be used to confirm these results, as they might be affected by the presence of null alleles.

Table 2.

Genetic properties of 15 polymorphic microsatellite loci of Zanthoxylum schinifolium and Z. piperituma



In this study, 15 polymorphic and three monomorphic microsatellite markers were developed for Z. schinifolium. In the cross-amplification test of the developed markers for Z. piperitum, a related species in the same genus, 61% (11/18) were successfully amplified. Three of the 11 cross-amplified primers could be useful for distinguishing between the two species because the amplified fragments have completely different size ranges. These developed markers can be useful for individual identification within species as well as for conservation and management of the genetic resources of Z. schinifolium.


This work was supported by the Research Project of the National Forest Seed and Variety Center, Chungju, South Korea.



Choi, S. I., K. M. Chang, W. S. Lee, and G. H. Kim. 2008. Antibacterial activity of essential oils from Zanthoxylum piperitum A.P. DC. and Zanthoxylum schinifolium. Food Science and Biotechnology 17: 195–198. Google Scholar


Dakin, E. E., and J. C. Avise. 2004. Microsatellite null alleles in parentage analysis. Heredity 93: 504–509. Google Scholar


Feng, S., Z. Liu, L. Chen, N. Hou, T. Yang, and A. Wei. 2016. Phylogenetic relationships among cultivated Zanthoxylum species in China based on cpDNA markers. Tree Genetics & Genomes 12: 45. Google Scholar


Glenn, T. C., and N. A. Schable. 2005. Isolating microsatellite DNA loci. In E. A. Zimmer and E. H. Roalson [eds.], Methods in enzymology, vol. 224: Molecular evolution: Producing the biochemical data, Part B, 202–222. Academic Press, San Diego, California, USA. Google Scholar


Hassler, M. 2016. World plants: Synonymic checklists of the vascular plants of the world (version Feb 2016). In Y. Roskov, L. Abucay, T. Orrell, D. Nicolson, T. Kunze, C. Flann, N. Bailly, et al. [eds.], Species 2000 & ITIS Catalogue of Life, 27 June 2016. Naturalis, Leiden, The Netherlands. Website [accessed 19 July 2016]. Google Scholar


Kalinowski, S. T., M. L. Taper, and T. C. Marshall. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16: 1099–1106. Google Scholar


Li, W., Y. N. Sun, W. T. Yan, S. Y. Yang, E. J. Kim, H. K. Kang, and Y. H. Kim. 2013. Coumarins and lignans from Zanthoxylum schinifolium and their anticancer activities. Journal of Agricultural and Food Chemistry 61: 10730–10740. Google Scholar


Peakall, R., and P. E. Smouse. 2006. GenAlEx 6.41: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6: 288–295. Google Scholar


Shippmann, U., D. J. Leaman, and A. B. Cunningham. 2003. Impact of cultivation and gathering of medicinal plant on biodiversity: Global trends and issues. FAO, vol. 312: Biodiversity and the ecosystem approach in agriculture, forestry and fisheries, 142–167. Inter-departmental Working Group on Biological Diversity for Food and Agriculture, Rome, Italy. Google Scholar


Sun, Y. L., W. G. Park, O. W. Kwon, and S. K. Hong. 2010. Ribosomal DNA internal transcribed spacer 1 and internal transcribed spacer 2 regions as targets for molecular identification of medically important Zanthoxylum schinifolium. African Journal of Biotechnology 9: 4661–4673. Google Scholar


Thiel, T., W. Michalek, R. K. Varshney, and A. Graner. 2003. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theoretical and Applied Genetics 106: 411–422. Google Scholar


van Oosterhout, C., W. F. Hutchinson, D. P. M. Wills, and P. Shipley. 2004. MICRO-CHECKER: Software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4: 535–538. Google Scholar


Varshney, R. K., A. Graner, and M. E. Sorrells. 2005. Genic microsatellite markers: Features and applications. Trends in Biotechnology 23: 48–55. Google Scholar


Wan, Q. H., H. Wu. T. Fujihara, and S. G. Fang. 2004. Which genetic marker for which conservation genetics issue? Electrophoresis 25: 2165–2176. Google Scholar


Wu, Z. Y., P. H. Raven, and D. Y. Hong. 2008. Flora of China, vol. 11. Science Press, Beijing, China, and Missouri Botanical Garden Press, St. Louis, Missouri, USA. Google Scholar


Appendix 1.

Locality information and accession numbers of Zanthoxylum schinifolium and Z. piperitum samples used in this study.a

Young Mi Kim, Aruna Jo, Ji Hee Jeong, Yong Rak Kwon, and Ho Bang Kim "Development and Characterization of Microsatellite Primers for Zanthoxylum schinifolium (Rutaceae)," Applications in Plant Sciences 5(7), (24 July 2017).
Received: 19 November 2016; Accepted: 1 January 2017; Published: 24 July 2017

Back to Top