As keystone species, fig trees (Ficus L., Moraceae) play a significant role in both tropical and subtropical ecosystems by providing food for many vertebrates (Shanahan et al., 2001). Some Ficus species are widely cultivated outside their natural distribution range, providing a valuable opportunity to explore the mechanism of range limits in the genus (Lin et al., 2007). Ficus virens Aiton is a monoecious fig tree species in the subgenus Urostigma (Endl.) Miq. (Bartholomaeus et al., 2009). Its natural distribution is in South and Southeast Asia, Melanesia, and northern Australia (McPherson, 2005). It is also planted widely in southwestern China outside its original distribution regions, extending to the most northern regions for Ficus species in China (Zhang et al., 2014). Therefore, the fine-scale population genetic structure and gene flow among the northern populations of F. virens can offer valuable information for interpreting the mechanism of range limits of Ficus species. In this study, we have developed high-resolution microsatellite loci to provide crucial information for fine-scale genetic studies of this species (Rout and Aparajita, 2009).
METHODS AND RESULTS
Plant material and DNA extraction—Young and healthy leaves of F. virens were collected in Chengdu (30.68271°N, 104.10363°E), Mianyang (31.47661°N, 104.78943°E), and Chongqing (29.8217°N, 106.42913°E) in southwestern China (Appendix 1). The sampled leaves were kept in allochroic silica gel for drying. Genomic DNA was extracted from 0.03 g dry leaves using a Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China).
Development of microsatellite primers—Microsatellite primers were developed using the biotin-streptavidin capture method following the procedure of Kijas et al. (1994) and Zhang et al. (2016). MseI (New England Biolabs, Beverly, Massachusetts, USA) was applied to digest the genomic DNA in a 25-µL reaction volume overnight at 37°C. Then the fragments were ligated to MseI adapters (F: 5′-TACTCAGGACTCAT-3′, R: 5′-GACGATGAGTCCTGAG-3′) and amplified with MseI-N primer (S′-GATGAGTCCTGACTAAN-3′) with the following conditions: 3 min denaturation at 95°C; followed by 20 cycles of 30 s denaturation at 94°C, 1 min annealing at 53°C, and 1 min extension at 72°C; with a final extension of 72°C for 8 min. The PCR products were mixed with 5′-biotinylated probe (AC)15 followed by 5 min denaturation at 95°C, and 2 h hybridization at 48°C. Hybridization products were captured with streptavidin-coated magnetic beads (Promega Corporation, Madison, Wisconsin, USA). The enriched product was amplified with MseI-N as primers for an additional 30 cycles according to the conditions described above. Purification was then performed with a multifunctional DNA Extraction Kit (Sangon Biotech, Shanghai, China), the products were ligated into pGM-T vector (Tiangen Biotech), and then transformed into Escherichia coli strain DH5α (Tiangen Biotech). In total, 108 positive clones were selected and sequenced. These were tested by PCR using (AG)10 and M13+M13- as primers.
The selected positive clones were sequenced with primer M13+/M13- on an AB13730 DNA Sequence Analyzer (Applied Biosystems, Foster City, California, USA) at Sangon Biotech. Eighty-two clones contained simple sequence repeats, 47 of which were discarded because their sequences were too short for primer design. The remaining clones were used to design primers with Primer Premier version 5.0 (PREMIER Biosoft International, Palo Alto, California, USA). The criteria for primer design were as follows: (1) product size from 100 to 300 bp; (2) primer size from 16 to 23 bp with an optimum size of 20 bp; (3) primer melting temperature from 45°C to 65°C with an optimum temperature of 55°C; and (4) GC content of primers from 40% to 60%.
Thirty-five pairs of microsatellite primers were designed and successfully amplified. The polymorphism of each designed primer was tested using 23 randomly selected individuals. PCRs were performed in a 15-µL volume containing 1.5 µL of 10× PCR buffer (with Mg2+), 0.3 µL dNTPs (2 mM each), 0.1 µL each primer (10 µM), 2 µL of 50 ng/µL DNA, 0.2 µL Blend Taq (2.5 units; Toyobo Co. Ltd., Osaka, Japan), and 12.6 µL of DNase-free water. PCR cycling was performed using the program described by Zhang et al. (2016). The products were first checked on 1% agarose gels, then settled on 8% denaturing polyacrylamide gels, and visualized by silver staining with pUC19 DNA/MapI (HpaII) (Fermentas International, Burlington, Ontario, Canada) as the ladder. Of the 35 microsatellite primers, 20 were monomorphic and 15 were polymorphic.
Characteristics of 15 microsatellite loci developed in Ficus virens.
Genetic variation of three populations (Chengdu, Mianyang, and Chongqing) was measured using selected polymorphic primers to further test the amplification stability of each primer. The forward primers of each polymorphic locus were labeled with fluorescent dyes (5′TAMRA, 5′ROX, 5′6-FAM [FITC]; Sangon Biotech) for scoring fragment length on an ABI 3130 automated sequencer (Applied Biosystems), using GeneScan 500 LIZ (Applied Biosystems) as an internal lane standard. The fragment lengths were calculated by GeneMapper 4.0 (Applied Biosystems) software.
Genetic diversity of the 15 polymorphic microsatellite in three Ficus virens populations.a
Data analysis—Linkage disequilibrium and Hardy–Weinberg equilibrium (HWE) were calculated using GENEPOP 4.0 (Rousset, 2008), and occurrence of null allele frequencies was tested with MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004). The genetic variations were estimated for two sampled populations by FSTAT 184.108.40.206 (Goudet, 2001), using the parameters of the number of alleles at each locus, observed heterozygosity, and expected heterozygosity.
Amplification of 15 microsatellite loci developed for Ficus virens in eight other Ficus species.
Linkage disequilibrium was not detected in any loci. The number of alleles per locus ranged from three to 17, with an average of 9.333 (Table 1). The observed heterozygosity and expected heterozygosity of each population ranged from 0.0667 to 0.9286 and 0.0650 to 0.8890, respectively (Table 2). Five loci (V70, V159, V183, V212, V254) in Chengdu, four loci (V98, V159, V212, V254) in Mianyang, and three loci (V42, V212, V254) in Chongqing showed significant departure from HWE. Two loci (V212, V254) showed consistent deviation from HWE. This could be caused by the presence of null alleles (present in six loci: V70, V98, V159, V183, V212, and V254) or by homozygote excess.
Cross-amplification in additional species—Cross-amplification of polymorphic microsatellite primers was performed on eight related species (n = 5 for each species), including F. altissima Blume, F. benjamina L., F. deltoidea Jack, F. hispida L. f., F. microcarpa L. f., F. montana Burm. f., F. rumphii Blume, and F. sarmentosa Buch.-Ham. ex Sm.
All 15 polymorphic primers amplified successfully (amplified a distinct band when the PCR products were checked on 1% agarose gel) in at least one of the eight additional Ficus species tested (Table 3). Two of the markers (V188, V254) were successfully amplified in all eight species, while one locus (V212) was only successfully amplified in F. altissima. Successful amplification across species did not appear to be correlated with the closeness of the genetic relationship of the species tested with F. virens. However, the cross-species sample size was limited and thus it is still likely that these markers will be most useful for closely related species.
The authors thank Yan Chen and Jun-Ying Deng for their suggestions on data analysis, Lu Tan for her assistance in the experiment, and Xiang-Yue Ding and Xi-Qian Yang for their help in sample collections. This study was supported by the Open Item of Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education; XNYB11-2).