Commelina communis L. (Commelinaceae), commonly known as dayflower, is a pseudometallophyte (facultative metallophyte) that is distributed extensively on both cupriferous habitats and surrounding nonmetalliferous sites (Tang et al., 1999, 2001; Ye et al., 2012). It is an annual multibranched herb that exhibits sexual reproduction and clonal propagation (Tang et al., 1999; Ushimaru et al., 2007; Ye et al., 2012). This species can also accumulate extraordinarily high concentrations of copper, with foliar Cu concentration reaching as much as 1000 mg/kg (Tang et al., 1997; Shu et al., 2001). Because of its good reproductive capacity and high biomass production, C. communis has been considered an important pioneer plant for phytoremediation of copper-contaminated soil and restoration of mined land (Tang et al., 1997, 1999).
To effectively use wild metal-tolerant plants for phytoremediation and ecological restoration, an accurate knowledge of their life history traits and population genetics (notably gene flow, breeding system, and genetic diversity organization) is needed (Salt et al., 1998; Escarré et al., 2000). Compared with dominant markers such as random-amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP), microsatellite or simple sequence repeat (SSR) markers are useful in studies of genetic diversity, population genetic structure, and genome mapping because of their high level of polymorphism and codominance (Jarne and Lagoda, 1996; Zhang and Hewitt, 2003). However, microsatellite loci have yet to be developed in C. communis or in congeneric species. In this study, we developed and characterized 12 polymorphic microsatellite loci for C. communis and tested the applicability of these SSR loci to estimate the genetic diversity of C. communis in metallicolous and nonmetallicolous populations.
METHODS AND RESULTS
Two populations of C. communis were sampled in Central China (nonmetalliferous population CS: Changsha, Hunan Province; metalliferous population YP: Jiangxi Province; Appendix 1). Voucher specimens (CS: WH06051793, YP: WH06051794) were deposited at the Wuhan University Herbarium (WH). Soil Cu concentrations at these sampling sites were measured following the method described by Ye et al. (2012) (Appendix 1). Total genomic DNA was extracted from silica gel–dried leaves of one individual of C. communis sampled from the CS population using the QIAGEN DNA Extraction Kit (QIAGEN, Hilden, Germany). Microsatellite loci from an enriched (AG)n library were isolated following the procedure of Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) (Zane et al., 2002). Approximately 250 ng of genomic DNA was completely digested by MseI (Fermentas, Burlington, Ontario, Canada) and then ligated to an MseI adapter pair (F: 5′-TACTCAGGACTCAT-3′; R: 5′-GACGATGAGTCCTGAG-3′) with T4 ligase (Promega Corporation, Madison, Wisconsin, USA). A total of 5 µL of a 10-fold diluted digestion-ligation mixture was directly amplified with 1 µL of the MseI-N primer (5′-GATGAGTCCTGAGTAAN-3′; 25 µM), 1 unit of Taq DNA polymerase (TaKaRa Biotechnology Co., Dalian, China), 2 µL of 10× PCR buffer, 1.6 µL of dNTPs (2.5 mM each), and 1.2 µL of MgCl2 (25 mM) in a total volume of 20 µL using the following thermocycler conditions: 3 min of denaturation at 95°C; followed by 26 cycles of 30 s of denaturation at 94°C, 1 min of annealing at 53°C, and 1 min of extension at 72°C; with a final extension of 72°C for 5 min. Amplified DNA fragments with a range of 200–800 bp were enriched for microsatellite repeats by magnetic bead selection with 5′-biotinylated (AC)15 and (AG)15 probes. Nonspecific DNA fragments were removed by three nonstringency washes with TEN1000 (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl [pH 7.5]) and then three stringency washes using 0.2× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS). After stringent washing, the enriched DNA fragments were eluted in 50 µL of 1× TE and then amplified with MseI-N primers for 26 cycles as described above. The PCR products were purified using a Gel Extraction Kit (TaKaRa Biotechnology Co.) according to the manufacturer's instructions. The purified PCR products with enriched microsatellite repeats were ligated into pGEM-T vector (Promega Corporation) and transformed into DH5α competent cells (TransGen Biotech, Beijing, China). Recombinant clones were screened by blue/white selection, and positive clones were tested by PCR using (AC)10/(AG)10 and T7/Sp6 as primers.
Characteristics of 34 microsatellite loci developed in Commelina communis.
Results of initial primer screening in two populations of Commelina communis.a
The 125 clones with positive inserts were sequenced with an ABI PRISM 3730xl DNA sequencer (Applied Biosystems, Foster City, California, USA). These sequences were analyzed for microsatellite repeat motif regions using the software SSR Hunter (Li and Wang, 2005). Of the 125 sequences analyzed, 90 had microsatellite motifs. After exclusion of redundant sequences, 42 high-quality sequences were selected for microsatellite primer design using OLIGO 7.0 software (Rychlik, 2010) and evaluated in 20 individuals from the CS population. Thirty-four pairs of primers (Table 1) that showed single and clear bands were chosen and labeled with the fluorescent dyes 6-FAM, ROX, or HEX (Invitrogen, Carlsbad, California, USA). Polymorphisms at the 34 SSR loci were assessed using 40 individuals from two populations of C. communis (Appendix 1), each with 20 individuals. Amplifications were performed in a total volume of 20 µL containing 30–50 ng genomic DNA, 0.6 µM of each primer, 7.5 µL of 2× Taq PCR MasterMix (0.1 unit Taq polymerase/µL, 0.5 mM dNTP each, 20 mM Tris-HCl [pH 8.3], 100 mM KCl, 3 mM MgCl2; Tiangen, Beijing, China). The thermocycling conditions were: 95°C for 3 min, 30 cycles of 94°C for 30 s, with the annealing temperature optimized for each specific primer for 30 s (Table 1), 72°C for 60 s, and a final extension step at 72°C for 7 min. The amplified products were separated using an ABI PRISM 3730xl DNA sequencer with GeneScan 600 LIZ (Applied Biosystems) as an internal size standard, and the sizes were determined using GeneMapper version 4.0 (Applied Biosystems).
Out of the 34 primer pairs, 12 primer pairs displayed polymorphism among individuals of the two populations of C. communis (Table 1). For each population, the number of alleles per locus (A), observed and expected heterozygosity (Ho and He), deviation from Hardy–Weinberg equilibrium (HWE), and linkage disequilibrium (LD) between all pairs of polymorphic loci were analyzed using GENEPOP version 4.2 (Rousset, 2008). Across the two populations of C. communis, A ranged from one to 11, with a mean of 4.5 in the YP population and 5.4 in the CS population. The Ho and He per locus ranged from 0.000 to 1.000 and from 0.195 to 0.941, respectively. A relatively high level of genetic diversity was found in the CS population (Ho = 0.338, A = 5.4) compared with the YP population (Ho = 0.288, A = 4.5). Some loci showed significant deviation from HWE (Table 2).
The SSR markers developed here will enable the estimation of genetic diversity in populations of C. communis under different edaphic conditions, and will be helpful for exploring the origin and evolutionary history of metallicolous populations under heavy metal stress in this pseudometallophyte. Their use at larger scales will provide detailed information on the genetic consequences of heavy metal concentration on C. communis that may guide the sustainable management plans for phytoremediation and ecological restoration.
 The authors thank R. L. Guo, Y. Gao, Y. Zhang, L. Fen, and W. Du (College of Life Sciences,Wuhan University) for assistance with the study. This work was supported by the Postdoctoral Science Foundation of Central South University and the National Natural Science Foundation of China (grant no. 31100173, 31000106).