The monotypic genus Sinocalycanthus chinensis W. C. Cheng & S. Y. Chang within the family Calycanthaceae is an endemic, endangered plant species in China. Sinocalycanthus chinensis is a diploid (2n = 22; Jin et al., 2010), deciduous shrub characterized by large, individual flowers with a diameter of 4.5–7 cm (Cheng and Chang, 1964). Its high ornamental and medicinal value results in overharvesting and a highly restricted geographic distribution (Li and Jin, 2006). Some studies have focused on the genetic diversity and phylogeny of S. chinensis using random-amplified polymorphic DNA (RAPD) (Li and Jin, 2006), inter-simple sequence repeat (ISSR) (Ye et al., 2006; Jin and Li, 2007), amplified fragment length polymorphism (AFLP) (Zhao et al., 2014), and chloroplast simple sequence repeat (cpSSR) (Li et al., 2012) markers, but with limited resolution, low reproducibility, and/or low stability. In this study, microsatellites, a more powerful and effective marker due to their codominance, were developed for use in genetic investigation of three populations of S. chinensis.

METHODS AND RESULTS

Leaves of S. chinensis were collected from three populations (30 individuals in each population) distributed across three locations in China: Daleishan (DLS) (28.988717°N, 120.811367°E) in Tiantai County, Damingshan (DMS) (30.039817°N, 118.972933°E) in Lin'an city in Zhejiang Province, and Longxushan (LXS) (30.069167°N, 118.700167°E) in Jixi County in Anhui Province (Appendix 1). Leaves of Calycanthus floridus L. were collected from Zhenru Garden (31.253708°N, 121.398147°E) in Shanghai and Hangzhou Botanic Garden (30.255113°N, 121.116163°E) in Zhejiang Province in China (Appendix 1). Total genomic DNA was extracted from silica-dried leaves using the Plant Genomic DNA Kit (Tiangen, Beijing, China). A microsatellite-enriched library of S. chinensis was constructed using the biotin-streptavidin capture method (Zane et al., 2002). Genomic DNA was digested using MseI (New England Biolabs, Beverly, Massachusetts, USA) at 37°C for 3 h, followed by 80°C for 20 min. After visualization by agarose gel electrophoresis, the DNA fragments (200–800 bp after digestion) were ligated to a MseI-adapter pair (F: 5′-TACTCAGGACTCAT-3′, R: 5′-GACGATGAGTCCTGAG-3′) at 37°C for 2 h and then 65°C for 10 min. The ligation products were amplified as follows: 95°C for 3 min, followed by 20 cycles of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min. The PCR products were hybridized with a 5′ biotinylated probe (AG)15 and captured with streptavidin-coated magnetic beads (Promega Corporation, Madison, Wisconsin, USA). The enriched fragments were amplified as follows: 95°C for 3 min; 30 cycles of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min; and 72°C for 8 min. After separation by agarose gel electrophoresis, the PCR products were purified using the Multifunctional DNA Purification Kit (BioTeke, Beijing, China). The purified PCR products were ligated to pMD 19-T vector (TaKaRa Biotechnology Co., Dalian, China) at 72°C for 1 h, and then transformed into strain JM109 of Escherichia coli by transient thermal stimulation (ice bath for 30 min, 42°C water bath for 90 s, followed by ice bath for 2 min).

A total of 716 positive clones were chosen and tested by PCR using primers of (AG)₁₀ and M13F/M13R, respectively. One hundred and twenty-seven screened clones contained potential microsatellite motifs and were sequenced using an ABI 3730 DNA Sequence Analyzer (Applied Biosystems, Foster City, California, USA). A total of 107 (75 in the initial sequencing and 32 in the second sequencing) primer pairs were designed by the program Primer Premier 5 (PREMIER Biosoft International, Palo Alto, California, USA). These primers were tested for polymorphism in 90 S. chinensis individuals within the DLS, DMS, and LXS populations. PCR amplification was performed in a 10-µL reaction: 20 ng of genomic DNA template, 1.0 µL of 10× PCR buffer (with Mg²⁺), 0.15 mM of each dNTPs, 0.05 µM of each primer, and 0.5 units of DNA Taq polymerase (TaKaRa Biotechnology Co.). Microsatellite loci were amplified under the following conditions: 94°C for 3 min; 30 cycles of 94°C for 30 s, 41–60°C (annealing temperature) for 30 s, 72°C for 30 s; and extension at 72°C for 5 min. PCR products were visualized on 1.5% agarose gels and then resolved on a Fragment Analyzer automated capillary electrophoresis system (Advanced Analytical Technologies, Ankeny, Iowa, USA; kit DNF-900-K0500).

Table 1.

Characteristics of 31 microsatellite loci developed from Sinocalycanthus chinensis.

Table 2.

Genetic diversity of 21 polymorphic microsatellite markers in three Sinocalycanthus chinensis populations.^a

The number of alleles, observed heterozygosity, expected heterozygosity, and linkage disequilibrium were estimated with the software FSTAT 2.9.3.2 (Goudet, 2001), and Hardy–Weinberg equilibrium was assessed using GenAlEx 6.3 (Peakall and Smouse, 2006). Of the 31 loci, 21 loci were polymorphic in at least two of the three tested populations, and the remaining 10 loci were monomorphic (Table 1). The number of alleles per locus ranged from one to 20, with an average of 4.677. In the 21 polymorphic markers, the average observed and expected heterozygosity in all three populations were 0.403 ± 0.061 (mean ± SEM [standard error of the mean]) (0.033–1.000 per locus) and 0.510 ± 0.043 (0.032–0.797 per locus), respectively (Table 2). Seven loci (SC056, SC124, SC367, SC375, SC424, SC492, SC537) significantly deviated from Hardy-Weinberg equilibrium in all three tested populations after Bonferroni correction (P < 0.001) (Table 2). Of these 31 loci, 29 were successfully amplified in C. floridus and also revealed high levels of polymorphism (Table 3).

CONCLUSIONS

In this study, 31 microsatellite markers were developed from the Chinese endemic endangered plant species S. chinensis. Twenty-one loci were polymorphic in three tested populations. The high transferability of these markers will provide a more effective method to research the population genetics and phylogeography of S. chinensis and the closely related species C. floridus.

Table 3.

Characterization of 31 microsatellite loci developed from Sinocalycanthus chinensis in two populations of Calycanthus floridus. ^a