Development of novel EST‐SSR markers for Ephedra sinica (Ephedraceae) by transcriptome database mining

Premise of the Study Ephedra sinica (Ephedraceae) is a gymnosperm shrub with a wide distribution across Central and Eastern Asia. It is widely cultivated as a medicinal plant, but its wild populations are monitored to determine whether protection is needed. Methods and Results Thirty‐six microsatellite markers, including 11 polymorphic markers, were developed from E. distachya RNA‐Seq data deposited in the National Center for Biotechology Information dbEST database. Among 100 genotyped E. sinica individuals originating from five different population groups, the allele number ranged from three to 22 per locus. Levels of observed and expected heterozygosity ranged from 0 to 0.866 (average 0.176) and 0 to 0.876 (average 0.491), respectively. Allelic polymorphism information content ranged from 0.000 to 0.847 (average 0.333). Cross‐species amplifications were successfully conducted with two related Ephedra species for all 11 di‐ or trinucleotide simple sequence repeats. Conclusions This study provides the first set of microsatellite markers for genetic monitoring and surveying of this medicinal plant.


METHODS AND RESULTS
A total of 4981 ESTs generated from mRNA sequencing of E. distachya were retrieved from the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags database (dbEST) (accessed by searching with "(Ephedra) AND "Ephedra distachya"[porgn:__txid3389]"). Microsatellites with a minimum repeat number of five were detected for 324 ESTs with a minimum length of 200 bp. We obtained 203 unique EST-SSR loci by an allagainst-all BLAST analysis and successfully designed primers for 171 unique EST-SSR loci. All bioinformatic operations were performed using the microsatellite detection and development pipeline QDD version 3.1 (Meglécz et al., 2014). Finally, we selected 88 di-or trinucleotide loci with at least five repeats for further evaluation.
We sampled five populations (100 individuals total) of E. sinica in Datong, Shanxi Province, China (Appendix 1). Voucher specimens were deposited in the Herbarium of Beijing Forestry University (BJFC). In order to test for successful amplification of the 88 EST-SSR loci selected, we conducted PCR analysis using eight individual plants of E. sinica. These eight individuals were collected in the Beijing Botanical Garden, Chinese Academy of Sciences. The genomic DNA was extracted from dried leaves using the cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). An M13 tail (FAM, HEX, TAMRA, ROX) was attached to the forward primer (Meglécz et al., 2014) for visualization. The final PCR volume was 20 μL, containing 10 μL of 2× Taq PCR Mix (Tiangen, Beijing, China), 4 μL of fluorescent dye-labeled M13 primer (4 pM), 4 μL of mixed forward and reverse primers, and 2 μL (20 ng) of DNA. The following PCR conditions were used: 94°C incubation for 5 min; 25 cycles at 94°C for 40 s, 55°C for 40 s, and 72°C for 45 s; 10 cycles at 94°C for 40 s, 53°C for 40 s, and 72°C for 45 s; and a final extension at 72°C for 10 min.
Among the 88 identified di-or trinucleotide loci, 38 displayed the expected size bands. After final capillary electrophoresis analysis on an ABI 3730 sequencer (Applied Biosystems, Waltham, Massachusetts, USA), SSR alleles were called with GeneMarker version 2.20 (SoftGenetics, State College, Pennsylvania, USA). Of these 38 loci, 36 showed clear, single peaks for each allele as essential for confident scoring, and 11 of these loci were polymorphic among the initially screened eight individuals. Characteristics of the 25 pairs of monomorphic microsatellite loci developed for E. sinica are shown in Appendix 2. The 11 polymorphic primer pairs were subsequently used to screen five E. sinica populations (with sample sizes n = 20 per population) and two additional populations originating from E. likiangensis Florin (n = 20) and E. equisetina Bunge (n = 6) (Appendix 1). Table 1 shows the primer sequences, repeat motifs, amplification sizes, GenBank accession number of the target sequences, and functional annotations determined with the protein family database, Pfam (Finn et al., 2014). We employed GenAlEx version 6.5 (Peakall and Smouse, 2012) to calculate genetic diversity parameters. The allelic polymorphism information content (PIC) was calculated using CERVUS 3.0 (Kalinowski et al., 2007). Allele numbers ranged from three to 22, with an average of 11.55 alleles per locus. Levels of observed and expected heterozygosity ranged from 0 to 0.842 (average 0.176) and 0 to 0.883 (average 0.491), respectively. In addition, PIC values ranged from 0 to 0.847 (average 0.333). The genetic parameters calculated for the 11 polymorphic EST-SSR loci are detailed in Table 2. The target sequences for all microsatellite loci are provided in Appendices S1 and S2.
Furthermore, we conducted cross-species amplification of the 11 polymorphic primer pairs on two related species: E. likiangensis from Yulong, Yunnan Province, and E. equisetina from Datong, Shanxi Province, China (Appendix 1). All 11 primer pairs successfully amplified E. likiangensis, except for locus E-20, which produced monomorphic bands in the species (Table 3). For E. equisetina, nine out of the 11 primers tested were polymorphic, and two loci failed to amplify. The interspecific amplification profile may be partially related to the phylogenetic relationships between species, as the relationship between E. equisetina and E. sinica is more distant (Ickert-Bond and Wojciechowski, 2004). In terms of polymorphisms, except for primers at the E-49 locus, the remaining primer pairs showed moderate polymorphism in E. equisetina, possibly due to the small sample size.

CONCLUSIONS
The EST-SSR polymorphic markers developed in this study will be potentially useful for studies of population structure and genetic diversity in E. sinica conservation genetics. These new markers will also be applicable for E. likiangensis and E. equisetina and can enrich the number of DNA markers available for Ephedra.

ACKNOWLEDGMENTS
The authors thank Dr. X.-R. Wang and Dr. X.-Y. Kang for their valuable suggestions. This study was supported by grants from the Fundamental Research Funds for the Central Universities (no. YX2013-412018BLCB08).

DATA ACCESSIBILITY
Expressed sequence tags used for primer development were downloaded from the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags database (dbEST). GenBank accession numbers for target sequences of both polymorphic and monomorphic SSR loci are provided in Table 1 and Appendix 2.

SUPPORTING INFORMATION
Additional Supporting Information may be found online in the Supporting Information section at the end of the article.
APPENDIX S1. Monomorphic microsatellite target sequences from microsatellite marker development in Ephedra sinica.
APPENDIX S2. Polymorphic microsatellite target sequences from microsatellite marker development in Ephedra sinica.