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Development of 14 polymorphic microsatellite loci for Ficus tikoua (Moraceae)1

Premise of the study: Polymorphic microsatellite markers were developed to facilitate studies on the fine-scale population genetic structure of Ficus tikoua (Moraceae), a prostrate shrub known to have highly restricted gene flow. Methods and Results: Microsatellite primers were developed using the biotin-streptavidin capture method and scanned for polymorphism within 76 individuals sampled from three natural F. tikoua populations. Fourteen loci were shown to be polymorphic, with allele numbers ranging from three to 16. The observed and expected heterozygosity in the three populations ranged from 0 to 1 and from 0 to 0.87, respectively. Substantial divergence was found among the populations at some loci. All loci can be successfully amplified in at least eight other Ficus species, indicating good transferability within the genus. Conclusions: The 14 microsatellite loci will be a helpful tool for assessing the fine-scale genetic structure of F. tikoua.

Fig trees (Ficus L., Moraceae) are keystone species in many tropical and subtropical ecosystems, providing food for more frugivorous animal species than other plants (Shanahan et al., 2001). Fig tree species rely on highly species-specific pollinating fig wasps (Agaonidae) for pollen dispersal and seed production, and in turn provide food for fig wasp larvae. With more than 750 species, a diverse range of life forms (trees, shrubs, stranglers, and vines), breeding systems (monoecy and dioecy), and pollination modes (active and passive) among Ficus species (Herre et al., 2008), the interaction between Ficus species and their pollinators continues to stimulate and inform evolutionary and conservation questions (Cook and Rasplus, 2003;Herre et al., 2008).
Ficus tikoua Bureau was previously attributed to subgenus Ficus (Corner, 1965), but was transferred into subgenus Sycomorus (Gasp.) Miq. in a recent phylogenetic study (Cruaud et al., 2012). It is a functionally dioecious shrub with an unusual prostrate life form, so that the figs (syconia) in which the seeds and pollinating fig wasps develop are close to, or partially buried in, the soil. The position of the figs of F. tikoua makes them unusually hidden from its pollinator, potentially restricting the gene flow of both host and pollinator populations. Significant genetic differentiation was detected between F. tikoua populations separated by only 31 km using microsatellite primers developed for other Ficus species (Chen et al., 2011). However, these transferred primers showed low resolution in F. tikoua, with no more than four alleles per locus (see table 1 in Chen et al., 2011). High-resolution microsatellite markers for this species are therefore needed to assess its fine-scale genetic structure and degree of inbreeding, given its highly restricted gene flow.
Microsatellite primers were developed using the biotin-streptavidin capture method following the procedure of Kijas et al. (1994) and Tong et al. (2012). Approximately 250 ng of genomic DNA from each individual was digested with the restriction enzyme MseI (New England Biolabs, Beverly, Massachusetts, USA). The digested fragments were linked to an MseI-adapter pair (F: 5′-TACTCAGGACTCAT-3′, R: 5′-GACGATGAGTCCTGAG-3′) and then amplified with an MseI-N primer (5′-GATGAGTCCTGAGTAAN-3′) using a protocol of 95°C for 3 min; followed by 20 cycles of 94°C for 30 s, 54°C for 1 min, and 72°C for 1 min; with a final extension at 72°C for 5 min. PCR products were hybridized with a 5′-biotinylated probe (AG) 15 at 48°C for 2 h, and microsatellite motifs were then captured with streptavidin-coated magnetic beads (Promega Corporation, Madison, Wisconsin, USA). The enriched motifs were again amplified with an MseI-N primer and purified with a multifunctional DNA Extraction Kit (Sangon Biotech, Shanghai, China). The purified products were ligated into a pMD 19-T vector (TaKaRa Biotechnology Co., Dalian, China) and transformed into Escherichia coli strain JM109. Positive clones were detected by PCR using (AG) 10 and M13 + /M13 − primers.
A total of 106 positive clones were selected and sequenced with M13 + /M13 − primers on an ABI 3730 DNA Sequence Analyzer (Applied Biosystems, Foster City, California, USA) at Sangon Biotech. Ninety-five clones were found to contain simple sequence repeats, of which 51 primer pairs were designed using Primer Premier version 5.0 (PREMIER Biosoft International, Palo Alto, California, USA).
The amplification stability and polymorphism of each primer pair were tested using randomly selected individuals. The 10-μL PCR reaction volumes included 50 ng of genomic DNA, 0.2 mM of each dNTP, 0.1 μM of each primer, 1× PCR buffer (Mg 2+ free), 2.5 mM Mg 2+ , and 1 unit Taq DNA polymerase 1 Manuscript received 29 August 2015; revision accepted 3 November 2015.
The authors thank Jun-Yin Deng and Rong-Hua Fu for laboratory assistance. This study was supported by the National Natural Science Foundation of China (31270387 to Y.C. • Premise of the study: Polymorphic microsatellite markers were developed to facilitate studies on the fine-scale population genetic structure of Ficus tikoua (Moraceae), a prostrate shrub known to have highly restricted gene flow. • Methods and Results: Microsatellite primers were developed using the biotin-streptavidin capture method and scanned for polymorphism within 76 individuals sampled from three natural F. tikoua populations. Fourteen loci were shown to be polymorphic, with allele numbers ranging from three to 16. The observed and expected heterozygosity in the three populations ranged from 0 to 1 and from 0 to 0.87, respectively. Substantial divergence was found among the populations at some loci. All loci can be successfully amplified in at least eight other Ficus species, indicating good transferability within the genus. In total, 14 loci proved to be polymorphic, with allele numbers ranging from three to 16 in total (Table 1) and from one to 11 within populations. No significant LD was found between any pair of loci, and no loci were found to deviate (Sangon Biotech), which was performed under the following conditions: 94°C for 5 min; 30 cycles with each cycle lasting 30 s at 94°C, 30 s at a primerspecific annealing temperature (Table 1), and 30 s at 72°C; and a final extension of 72°C for 8 min. PCR products were first checked on 1.2% agarose gels, resolved on 8% polyacrylamide denaturing gel, and then visualized by silver staining, with pUC19 DNA/MapI (HpaII) (Fermentas International, Burlington, Ontario, Canada) as the ladder.
To rate the polymorphism of each locus, genomic DNA templates of all 76 F. tikoua individuals from the three natural populations were used ( Table 2). The forward primers of each polymorphic locus were labeled with fluorescent dyes (5′TAMRA, 5′ROX, 5′FAM; Sangon Biotech) for scoring fragment length on an ABI 3130 automated sequencer (Applied Biosystems), using  (Table 2). H o and H e ranged from 0 to 1 and from 0 to 0.87, respectively, and substantial between-population differences were found at loci FT02, FT05, and FT07 ( Table 2). The genetic diversity values of developed loci were comparable with those of microsatellite loci developed for other Ficus species, such as for F. hirta Vahl (Zheng et al., 2015). In addition, relatively low genetic variation was found in Mianyang, the northernmost population, despite its large sample size, which further verified the validity of these loci. Cross-species amplification of the 14 developed primers was tested in 12 other Ficus species, using the same procedures described above. The involved species covered most Ficus subgenera distributed in the Asian-Australasian region (four out of five subgenera). All primers successfully amplified in at least eight additional species (Table 3), indicating good transferability of these primers.

CONCLUSIONS
The 14 microsatellite loci developed for F. tikoua showed high genetic diversity and substantial differences among populations. They will be useful for further studies of fine-scale genetic structure and gene flow in F. tikoua.