Alpinia oxyphylla Miq. belongs to the genus Alpinia Roxb. (Zingiberaceae), which comprises 250 species and is widely distributed in tropical and subtropical areas. Approximately 51 species are found in southern China (Wu and Larsen, 2000). Alpinia oxyphylla is endemic to southwestern China, and its fruits are used in traditional Chinese medicine for the treatment of intestinal disorders, urosis, and diuresis. Alpinia oxyphylla is also coded in the Chinese Pharmacopeia as an aromatic stomachic. Evidence suggests that the extract from the fruits of A. oxyphylla has significant neuroprotective activity (Yu et al., 2003; Wong et al., 2004).
A first step to better utilizing this medicinal plant is to understand more about the genetic diversity and population structure of the species. The limited research on characterization at the DNA level has hindered the improvement of A. oxyphylla, and little DNA variation has been detected by inter-simple sequence repeat (ISSR) markers (Wang et al., 2012). Here we present a set of polymorphic microsatellite markers that can provide an important tool for understanding genetic diversity and genetic structure of A. oxyphylla. Moreover, such information can serve as a guide to help characterize the chemical composition of this medicinal plant.
METHODS AND RESULTS
Genomic DNA was extracted from dried leaves of A. oxyphylla individuals using the cetyltrimethylammonium bromide (CTAB) method (Doyle, 1990), with slight modifications for using sufficient β-mercaptoethanol as an antioxidant followed by purification using phenol-chloroform. Microsatellites were developed following the methods of Glenn and Schable (2005). Approximately 5 µg of DNA were digested with the restriction enzyme MseI (New England BioLabs, Beverly, Massachusetts, USA). The restricted fragments were ligated with MseI adapters (5′-TACTCAGGACTCAT-3′ and 5′-GACGATGAGTCCTGAG-3′) with T4 ligase (New England BioLabs) overnight at 16°C and then amplified using the MseI-N primer (5′-GATGAGTCCTGAGTAAN-3′). PCR products were hybridized to a 5′ biotin-labeled oligonucleotide probe: (GA)15, (AC)15. Magnetic beads coated with streptavidin (Promega Corporation, Madison, Wisconsin, USA) were used to capture GA and AC repeats. The enriched fragments were amplified by PCR using MseI-N as the primer, ligated into plasmids, cloned into the plasmid vector pGEM-T Easy Vector (TaKaRa Biotechnology Co., Dalian, Liaoning, China), and then transformed into Escherichia coli DH5α competent cells (TaKaRa Biotechnology Co.). The recombinant positive clones were identified by colony PCR using the universal M13R or M13F as primers (TaKaRa Biotechnology Co.). Approximately 200 positive colonies were sequenced (Beijing Genomics Institute, Beijing, China). Sequencing output showed that 85 sequences contained perfect microsatellites with sufficient flanking regions, and these were selected to design primers with the software Primer3 (Rozen and Skaletsky, 2000). Eight individuals were selected for assessment of polymorphisms and the optimal annealing temperature of microsatellite loci. PCR amplification was performed in 10-µL reaction mixtures, consisting of approximately 5 ng of template DNA, 1.5 µL 10× buffer, 5 pmol of each primer, 0.2 mmol of each dNTP, and 1 U of Taq DNA polymerase (TaKaRa Biotechnology Co.). The PCR program included 4 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at annealing temperature (48–64°C), 1 min at 72°C, and a final extension at 72°C for 10 min. Amplified products were subjected to electrophoresis in 6% denatured polyacrylamide gels using silver staining.
Of the 85 primer sets, 58 were capable of amplifying products from A. oxyphylla genomic DNA, of which 35 were monomorphic and 23 were polymorphic (Table 1 and Table 2). Only polymorphic primer pairs were further characterized. Specifically, the forward primer of each polymorphic primer pair was labeled with a fluorescent dye (6-FAM, HEX, or TAMRA). Thirty-two individuals in two geographically distinct populations from Guangdong and Hainan provinces in China (Appendix 1) were chosen to more thoroughly assay polymorphism levels for the 23 primer pairs. PCR amplifications were carried out as described above. Equal volumes of PCR products amplified with two or three different dye-labeled primers were mixed together and used for genotyping. The PCR products were genotyped using an ABI Prism 3730 DNA Sequencer (Applied Biosystems).
Characteristics of 23 polymorphic microsatellite primers developed for Alpinia oxyphylla.
Our results (Tables 1 and Table 2) indicate that these microsatellite markers are highly polymorphic. The number of alleles per locus ranged from one to 13, and observed heterozygosity and expected heterozygosity varied from 0 to 1 (mean: 0.6441) and 0 to 0.887 (mean: 0.6241; values obtained using POPGENE version 1.32; Yeh et al., 2000).
We developed and evaluated 23 polymorphic microsatellite markers from A. oxyphylla. These polymorphic microsatellite markers will be useful for investigating the genetic diversity and population structure of A. oxyphylla. The results should be helpful in efforts to exploit the medicinal value of the species.
Results of polymorphic primer screening in populations of Alpinia oxyphylla. a
 The authors thank Dr. Zheng Feng Wang (South China Botanical Garden, Chinese Academy of Sciences) for his help in the experiments, and Dr. Kyle Dexter and Dr. Markus Ruhsam (Royal Botanic Garden Edinburgh) for their help in revising the manuscript. This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (grant KSCX2-YW-Z-0919), the National Natural Science Foundation of China (grants 30900089, 31200246, 31200176, 30870173), and the South China Botanical Garden–Shanghai Institute of Plant Physiology and Ecology Joint Fund.