Development, characterization, and cross‐amplification of microsatellite markers for Psammosilene tunicoides (Caryophyllaceae)

Premise of the Study Psammosilene tunicoides (Caryophyllaceae) is a narrowly distributed and endemic plant species in southwestern China. The overexploitation of natural P. tunicoides has led to the destruction of many populations. Population and genetic studies will provide crucial data for the protection and management of P. tunicoides. In this study, we develop simple sequence repeat markers of P. tunicoides to analyze population diversity. Methods and Results Microsatellite loci of P. tunicoides were isolated with FIASCO. Eleven polymorphic and 10 monomorphic primers were developed. The 11 polymorphic primers were tested in three P. tunicoides populations, yielding two to nine alleles per locus. Levels of observed heterozygosity varied from 0.000 to 1.000, and levels of expected heterozygosity ranged from 0.000 to 0.615. In addition, three of these loci were successfully amplified, and showed polymorphism, in three Silene species. Conclusions These microsatellite markers can be valuable tools to investigate the genetic diversity and population structure of P. tunicoides.


METHODS AND RESULTS
Total genomic DNA was extracted from silica gel-dried leaf tissue from seven samples of P. tunicoides from different populations (Appendix 1) using the DNeasy Plant Mini Kit (Tiangen Biotech, Beijing, China). These microsatellite markers were developed using the Fast Isolation by AFLP of Sequences COntaining repeats protocol (FIASCO) with modifications (Zane et al., 2002). Approximately 500 ng of genomic DNA was digested with MseI restriction enzyme (New England Biolabs, Beverly, Massachusetts, USA). Then, using the universal adapter pair (F: TACTCAGGACTCAT, R: GACGATGAGTCCTGAG) combined with its fragment, the digested product was placed at 37°C. This mixture was amplified by PCR using a reaction program containing an initial denaturation of 94°C for 3 min; followed by 20 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 60 s; with a final extension of 72°C for 8 min.
PCR product hybridization was performed with 5′-biotinylated (AC) 15 /(AG) 15 probes, and hybridization products were enriched using magnetic beads. The collected enriched products were used as a template to conduct a PCR reaction according to the above program. After the amplified product was purified, the purified product was ligated into a pGM-T vector (Tiangen Biotech) and transformed using Trans1-T1 Phage Resistant Chemically Competent Cells (TransGen Biotech, Beijing, China) to carry out amplification and expression. Positive strains were selected using Luria-Bertani medium containing ampicillin. A total of 359 positive clones were selected and sequenced using the ABI PRISM 3730XL DNA sequencer (Applied Biosystems, Foster City, California, USA), including 217 sequences containing an SSR. Out of these 217 sequences, 46 successfully yielded clear bands; the others showed multi-banding patterns or no amplification.
Polymorphisms were validated using the 46 designed primers in 30 samples of P. tunicoides that were collected from three locations in China: Yunnan, Sichuan, and Guizhou (Appendix 1). PCR was performed in a 25-μL reaction volume, containing 1 μL of template DNA (5 ng/μL), 0.5 μL of reference primer, 1 unit of Taq polymerase (TaKaRa Biotechnology Co., Dalian, China), 2.5 μL of 10× PCR buffer, 2.0 μL of MgCl 2 (25 mM), 0.5 μL of dNTP mixture (10 mM each), and 17.8 μL ddH 2 O. The amplification reaction was conducted using the following protocol: initial denaturation at 95°C for 3 min; followed by 30 cycles of 95°C for 30 s, 45-56°C for 1 min (Table 1), and 72°C for 30 s; and a final extension at 72°C for 10 min. After PCR amplification, PCR products were separated and visualized using an ABI 3730 automated sequencer (ABI 3730XL, Applied Biosystems), and the size of the alleles at each locus was scored by GeneMapper version 3.2 (Applied Biosystems). All sequences were deposited in GenBank (Table 1).
GENEPOP version 3.4 (Rousset, 2008). Only 11 primer pairs displayed polymorphism among these three populations (Table 1), and the amplification products were within the expected size range. The number of alleles per locus ranged from two to nine, with an average of 3.81 alleles. The average levels of H o and H e in all three populations were 0.31 ± 0.06 and 0.29 ± 0.04, respectively (Table 2). Five loci (E10, Z6, Z11, Z14, Z16) in the Lijiang population, three loci (Z5, Z11, Z14) in the Yanyuan population, and four loci (E7, E10, Z11, Z12) in the Weining population showed significant deviations from Hardy-Weinberg equilibrium, indicating heterozygote deficiencies. In addition to the 11 polymorphic loci, 10 monomorphic microsatellite loci were obtained and the sequence information was deposited to NCBI (Appendix 2).
We also tested 11 primer pairs in three species of the related genus Silene: S. gracilicaulis, S. huguettiae, and S. gonosperma. The results revealed that only three primers (E5, Z3, and Z14) show amplified bands, with lower H o and H e in these loci (Table 3).

CONCLUSIONS
This is the first study to characterize microsatellite markers specifically for P. tunicoides. The 11 polymorphic markers developed here will enable further studies investigating the population genetic structure, the development of conservation strategies, and markerassisted selection breeding of this species. However, the crossspecies amplification of these markers indicates that they may be less useful in related genera, such as Silene, because of the distant phylogenetic relationships between P. tunicoides and other species in Caryophyllaceae.