Isolation and Characterization of Eleven Polymorphic Microsatellite Loci for the Valuable Medicinal Plant Dendrobium huoshanense and Cross-Species Amplification

Dendrobium huoshanense (Orchidaceae) is a perennial herb and a widely used medicinal plant in Traditional Chinese medicine (TCM) endemic to Huoshan County town in Anhui province in Southeast China. A microsatellite-enriched genomic DNA library of D. huoshanense was developed and screened to identify marker loci. Eleven polymorphic loci were isolated and analyzed by screening 25 individuals collected from a natural population. The number of alleles per locus ranged from 2 to 5. The observed and expected heterozygosities ranged from 0.227 to 0.818 and from 0.317 to 0.757, respectively. Two loci showed significant deviations from Hardy-Weinberg equilibrium and four of the pairwise comparisons of loci revealed linkage disequilibrium (p < 0.05). These microsatellite loci were cross-amplified for five congeneric species and seven loci can be amplified in all species. These simple sequence repeats (SSR) markers are useful in genetic studies of D. huoshanense and other related species and in conservation decision-making.


Introduction
Dendrobium huoshanense, a perennial epiphytic orchid herb, is endemic to Huoshan County town in Anhui province in southeast China. It is a widely used medicinal plant in traditional Chinese medicine (TCM), whose stems have been used for centuries in China and Southeast Asia as a therapeutic agent for treatment of cataract, throat inflammation, fever, chronic superficial gastritis, and as a tonic to improve health, mentality and to delay aging [1]. Recent researches revealed that the pharmacological activity of D. huoshanense was due to several active components, such as alkaloids, stilbenoid, glycosides and polysaccharides [2][3][4]. Moreover, D. huoshanense is of great horticultural interest due to the delicate lime flowers that bloom from May [4]. However, human activities have brought this species to the brink of extinction in the last few decades. Extensive deforestation and increased utilization in TCM have caused a dramatic decline in the natural populations. Although the morphology and pharmacology of D. huoshanense has been extensively studied, information on molecular phylogeny and population genetics is largely unknown.
During recent decades, SSRs (simple sequence repeats), also known as microsatellites, have become the most popular source of genetic markers owing to their high reproducibility, multi-allelic nature, codominant inheritance, high abundance, and wide genome coverage [5,6]. They are an ideal tool for genome mapping, population genetics, conservation biology, marker-assisted selection and other studies [7][8][9][10]. Previous work by Lu et al. showed the development and characterization of SSR markers for Dendrobium officinale [11]. In addition, Ding et al. reported that 13 of the 15 tested D. officinale SSR primer pairs were successful in amplifying corresponding products in D. huoshanense [12]. Here, we report the isolation and characterization of eleven polymorphic microsatellite loci from D. huoshanense using a modified biotin-capture method for the first time [13].

Results and Discussion
Eleven polymorphic microsatellite loci were isolated and deposited in GenBank (JQ400190-JQ400200; Table 1). Polymorphism at each locus was determined using 25 D. huoshanense individuals from a natural population. The number of alleles per locus ranged from two to five. The largest number of alleles (five) was found at Hs48 locus ( Table 1). The observed heterozygosity (H o ) ranged from 0.227 to 0.818 and the expected heterozygosity (H e ) ranged from 0.317 to 0.757 (Table 1).
Deviations from Hardy-Weinberg expectation (HWE) were detected for each locus, nine loci conformed to HWE and two loci (Hs39, Hs50) significantly deviated from HWE in the sampled population after Bonferroni's correction (p < 0.05). Linkage disequilibrium (LD) is important for evolutionary and population genetics and so LD between pairs of loci was calculated. Four of the pairwise comparisons (Hs40 and Hs47, Hs41 and Hs54, Hs50 and Hs57, Hs48 and Hs54) exhibited significant linkage disequilibrium (p < 0.05). The cross-species amplification was carried out. The results of the screen are summarized in Table 2. Notably, seven loci (Hs39, Hs41, Hs44, Hs47, Hs48, Hs56, Hs57) can be amplified in all five species, while three loci (Hs40, Hs50, Hs54) can be amplified in four species and one loci (Hs42) can be amplified in two species. These results suggest that the identified microsatellite markers are useful in genetic studies of D. huoshanense and other Dendrobium species. The meaning of the provided values is the size range of PCR products and minus sign (−) denotes no visible PCR product.

Plant Materials and DNA Extraction
The leaves of D. huoshanense from a natural population in Huoshan (31°0'35''N, 115°0'53''E, Anhui, China) were collected, dried in silica gel in sealed polyethylene bags and stored at room temperature until use. The total genomic DNA of 25 individuals was extracted using the standard cetyltrimethyl ammonium bromide (CTAB) method [14].

Isolation of Microsatellite Markers
About 500 ng genomic DNA from a single individual were digested by Sau3AI (TaKaRa), and the fragments ranging from 400 to 900 bp were purified and ligated to the Sau3AI adaptors oligoA (5'-GGCCAGAGACCCCAAGCTTCG-3') and oligoB (5'-PO4-GATCCGAAGCTTGGGGTCTCTGG CC-3') [13]. The products were then hybridized to the single-stranded 3'-biotinylated (CT) 15 oligonucleotide probes and captured by streptavidin-coated beads (Promega). Captured fragments were amplified and ligated to pMD18-T vector (TaKaRa). The recombinant plasmid was transformed into Escherichia coli DH5α and the transformants were distinguished by blue-white screening. The insert-positive clones were identified by PCR using oligo(A) and (CT) 15 oligonucleotides as the primers. The clones that yielded two or more bands contained microsatellite fractions.
In total, fifty-one positive colonies were sequenced on the ABI-PRISM 3730 automated sequencer, and thirty-six contained microsatellites. Thirty-six pairs of primers were then designed using PRIMER PREMIER 5.0 software [15] and synthesized. Eleven sets of primers that gave consistent and specific PCR products were tested for allelic polymorphism in 25 individuals from a natural population. PCR amplifications (15 μL) contained 6.25 μL GoTaq ® Green Master Mix (Promega, Nanjing, China), 0.5-1.0 μmol of each primer and 10-20 ng DNA, and were performed in an iCycler thermocycler (Bio-Rad). PCR conditions were 4 min at 94 °C followed by 35 cycles of 45 s at 94 °C, 35 s at optimized annealing temperature (Table 1) and 30 s at 72 °C, with a final extension time of 8 min at 72 °C. PCR products were separated on an 8% denaturing polyacrylamide gel using a LI-COR 4200 automated DNA sequencer and analyzed using LI-COR SAGA GT software.

Data Analysis
The number of alleles (N a ) per locus, observed heterozygosity (H o ) and expected heterozygosity (H e ), Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were tested using GENEPOP version 4.0 [16]. MICRO-CHECKER version 2.2.3 was used to determine the presence of null alleles [17]. All results for multiple tests were corrected using Bonferroni's correction [18].

Cross-Species Transferability
Cross-species amplification of microsatellite markers was performed for five congeneric species, D. aduncum, D. henanense, D. moniliforme, D. nobile and D. officinale. DNA extraction and PCR amplification were performed as described for D. huoshanense except that the annealing temperature was re-optimized for each locus. Five individuals from each species were screened at 11 microsatellite loci.

Conclusions
We report the isolation and characterization of eleven polymorphic microsatellite loci for D. huoshanense and cross-amplification in five congeneric species. The low level of polymorphism here was possibly caused by heterozygote deficiency resulting from inbreeding or sampling related individuals. These eleven microsatellite markers are considered useful for population genetic studies and conservation decision-making for this medicinal plant.