Cyperus fuscus L. (Cyperaceae) is an annual herb that is native in the Mediterranean region and temperate Eurasia and introduced in North America. It grows on muddy, sandy, or gravelly substrata, on shores of rivers or lakes, and is also found in anthropogenic habitats like gravel pits, wet fields, and traditionally used fish ponds. It has a short life cycle, taking just two to three months from seedling to ripe fruits (von Lampe, 1996).
Cyperus fuscus is anemophilous and self-compatible. With 0.24 pg/1C (or 234.72 Mbp; Doležel et al., 2003), the genome is relatively small (Tremetsberger et al., unpublished data). Plants with 2n = 36 and 72 chromosomes are known (Krahulcová, 2003), most probably corresponding to diploid and tetraploid cytotypes (Roalson, 2008). The large amounts of seeds produced build up a persistent soil seed bank, which can also function as a “genetic memory” by storing the genetic variability in viable seeds (Leck, 1989). We developed 21 microsatellite markers to compare the genetic variation in the seed bank of various natural and manmade habitats.
METHODS AND RESULTS
Plants were grown in the greenhouse from ripe seeds collected in the field (Appendix 1). Genomic DNA of fresh leaves from one plant was extracted with the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions and sent to LGC Genomics (Berlin, Germany) for next-generation sequencing (NGS) on a Genome Sequencer FLX Titanium Instrument (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). In this first run, 143,027 sequence reads with an average length of 238 bp were obtained (Table 1). NGS data are deposited in the GenBank Sequence Read Archive (BioProject no. PRJNA275048). MSATCOMMANDER version 0.8.2 (Faircloth, 2008) was used to detect 520 sequences with simple sequence repeat (SSR) motifs (options: dinucleotide repeats ≥10 repeat units, tri- and tetranucleotide repeats ≥6 repeat units, combine multiple arrays within a sequence if within 50 bp distance). Primers for microsatellite-containing sequences were also designed in MSATCOMMANDER using Primer3 (Rozen and Skaletsky, 1999), with a GTTT PIG-tail (Brownstein et al., 1996) added to the 5′ end of one primer and a CAG or M13R tail (CAG: 5′-CAGTCGGGCGTCATCA-3′; M13R: 5′-GGAAACAGCTATGACCAT-3′) added to the 5′ end of the other primer (Schuelke, 2000). Due to the shortness of the sequences (range = 7–762 bp, mean = 238 bp), only 101 out of the 520 SSR-containing sequences were suitable for primer design. PCR amplifications were performed in a 25-µL final volume of REDTaq ReadyMix PCR Reaction Mix (Sigma-Aldrich, St. Louis, Missouri, USA) with 0.40 µM 5′ FAM-labeled universal CAG or M13R primer, 0.40 µM GTTT-tailed primer, 0.04 µM CAG- or M13R-tailed primer, and 1 µL diluted DNA extract (2–20 ng DNA). Reactions were performed using a touchdown PCR protocol in an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany), with an initial 5 min of denaturation at 95°C; 24 cycles with denaturation at 95°C for 45 s, annealing at 63–48.6°C (0.6°C decrease per cycle) for 90 s, and extension at 72°C for 60 s; 19 cycles with denaturation at 95°C for 45 s, annealing at 50°C for 90 s, and extension at 72°C for 60 s; and a final extension at 72°C for 5 min and 60°C for 30 min. Amplified fragments were analyzed on a 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) and sized using GeneMarker 2.4 (SoftGenetics, State College, Pennsylvania, USA). The markers were tested on seven individuals from different localities (Appendix 1). Seven loci could be unambiguously scored in all seven test individuals. Four of these were applied to a larger number of individuals (primers with the prefix Cf in Table 2; remaining loci are shown in Appendix 2).
A second NGS run of an SSR-enriched library was performed at ecogenics (Balgach, Switzerland), starting from a mix of genomic DNA of two individuals (Appendix 1). Size-selected fragments from genomic DNA were enriched for SSR content by using magnetic streptavidin beads and biotin-labeled CT, GT, AAG, and ATGT repeat oligonucleotides. The SSR-enriched library was analyzed on a Roche 454 platform using the GS FLX Titanium reagents (454 Life Sciences, a Roche Company). In total, 4877 reads with a mean length of 415 bp were obtained and deposited in the GenBank Sequence Read Archive (BioProject no. PRJNA275048), of which 967 contained SSR motifs (MSATCOMMANDER search and primer design settings same as above; Table 1). Four hundred ninety-four reads were suitable for primer design. Ecogenics sent 80 primer pairs also designed with Primer3, containing an M13 tail at the 5′ end of the forward primer (5′-TGTAAAACGACGGCCAGT-3′; Schuelke, 2000) and no PIG-tail. For primer testing, the concentrations and volumes for PCR were the same as above, but we used Jumpstart REDTaq ReadyMix Reaction Mix (Sigma-Aldrich) and a regular PCR protocol, with an initial 5 min of denaturation at 95°C; 38 cycles of denaturation at 95°C for 45 s, annealing at 56°C for 60 s, and extension at 72°C for 1 min; and a final extension at 72°C for 5 min and 60°C for 30 min. Of these 80 markers, 22 showed no PCR product or had a weak signal, failures, or were unspecific. The remaining 58 markers showed clear peaks. Ten of these were monomorphic and 48 polymorphic. Seventeen polymorphic markers were selected for further analysis and combined into four multiplex PCRs with Multiplex Manager version 1.0 (Holleley and Geerts, 2009; PCR multiplex sets 1–4 in Table 2). The remaining loci are shown in Appendix 2. For application of PCR multiplex sets 1–4 to a larger number of individuals, a GTTT PIG-tail was added to the reverse primers (as for primers with the prefix Cf). For multiplex PCR reactions, the forward primers were directly labeled with a fluorescent dye at the 5′ end (Table 2).
Characteristics of the two 454 GS FLX Titanium sequencing runs.a
The 21 newly developed microsatellite markers were applied to 25 individuals from each of two fish pond populations in the Czech Republic (Appendix 1). Interpretation of electropherograms in all loci and all individuals is compatible with a diploid cytotype. The number of alleles, observed (Ho) and expected heterozygosity (He), fixation index, and exact test for Hardy–Weinberg equilibrium (HWE) were calculated with Arlequin version 18.104.22.168 (Excoffier and Lischer, 2010). The mean number of alleles per locus is 2.6 in Zahrádky and 2.9 in Libohošt (Table 3). Ho ranges from 0 to 0.32 (mean = 0.109) in Zahrádky and from 0 to 0.24 (mean = 0.135) in Libohošt. He ranges from 0.078 to 0.706 (mean = 0.405) in Zahrádky and from 0.040 to 0.667 (mean = 0.470) in Libohošt. Deviation from HWE is very high in most loci in both populations, with fixation indices ranging from 0.341 to 1 (mean = 0.756) in Zahrádky and from 0 to 1 (mean = 0.687) in Libohošt.
The 21 polymorphic loci developed in this study will be useful for studying genetic diversity of C. fuscus and the role of the soil seed bank in the life cycle of this ephemeral plant in natural and anthropogenic habitats. The inbreeding coefficients of the two tested populations attest to the very high selfing rate of this species.
Characteristics of 21 SSR loci developed in Cyperus fuscus.
Genetic diversity of 21 newly developed SSR markers in two fish pond populations of Cyperus fuscus.a
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Voucher information for Cyperus fuscus populations used in this study. All vouchers are deposited at the Institute of Botany. University of Natural Resources and Life Sciences, Vienna (WHB). Individuals were grown from seeds in the greenhouse.
 The authors thank K. Bubíková (Brno), Z. Hroudová, P. Kúr, S. Píšová (Prague), and Z. Kącki (Wrocław) for assistance collecting in the field; K. Takayama (Tokyo) and A. Buser (Balgach) for advice in marker development; and K. Fohringer and G. Kohl (Vienna) for help in the laboratory. Financial support was provided by the Austrian Science Fund (FWF, project P 24558-B16), the Czech Science Foundation (14-36079G), and the Institute of Botany of the Czech Academy of Sciences (project RVO 67985939).