PREMISE: The distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species since they create and define ecological communities, contributing disproportionately to ecosystem function.

METHODS: We examined the distribution of genetic diversity and clones, which we defined first as unique multilocus genotypes (MLG), and then by grouping similar MLGs into multilocus lineages (MLL). We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch-size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species.

RESULTS: Every ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single MLL. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three.

CONCLUSIONS: We found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.

Sampling design

We collected tissue samples in August 2010, when the plants were vigorously growing. Thirteen L. chinensis patches were selected haphazardly within an area of 2000m². Patches were defined naturally (by the space occupied by L. chinensis locally), and all patches were separated from others by at least 200m. Patches ranged in area from 2.8m² to 166.8m² We collected vegetative tillers of L. chinensis at distances of 0, 0.5, 1, 2, 4, 8, and 16m from the center of each patch (or to the edge of the patch, whichever came first) in each of eight directions (N, S, E, W, NE, NW, SE, SW). Consequently, we sampled 17-35 ramets from each patch (Table 2). The vegetative ramets of L. chinensis were harvested at the ground surface and each ramet was put into a zip-lock plastic bag with silica gel to preserve the tissue. We transported all samples to the laboratory and stored them at -20℃.

DNA extraction

For each sample, we performed extractions of total genomic DNA using a modified CTAB method (Doyle and Doyle, 1987). We ground tissue of dried young leaves (0.025 to 0.030g) to a fine powder in liquid nitrogen with pure quartz sand in a 2 ml centrifuge tube. Then, we added 400 μl of extraction buffer [2% cetyl trimethyl ammonium bromide (CTAB); 100 mM Tris (pH=8.0); 20 mM ethylenediamine tetra-acetic acid (EDTA); 0.5% NaHSO₃; 1.4 M NaCl; 1% polyvinylpyrrolidone (PVP); 2% β-mercaptoethanol] to the ground sample and incubated at 65℃ for 30 minutes. We performed two rounds of extraction with 400 μl of tris-saturated phenol. After centrifugation, we performed two more rounds of extraction with chloroform: isoamyl alcohol (24:1) and precipitated the DNA with 200 μl of 5 M potassium acetate and 1ml of 100% ice-cold ethanol. The precipitate was washed with 70% ethanol twice, dried, and resuspended in 100ul of 1×Tris-EDTA (TE) buffer (Shimizu et al., 2002). We estimated the quantity of DNA by comparing band intensities with known amounts of lambda DNA on 0.8% agarose gel. DNA quality was checked by spectrophotometry at 260 nm and 280 nm. All the DNA samples were stored at -20℃ for ISSR analysis.

ISSR amplification

Twenty-one primers from the Biotechnology Laboratory, University of British Columbia (UBC set no.9) were synthesized by Sangon (Shanghai Sangon Biological Engineering Technology and Services Co. Ltd, Shanghai, China) for initial screening. Eventually, we selected 11 primers that produced clear bands with good reproducibility and high polymorphism (Table 1).

We performed ISSR amplification in a volume of 10 μl containing 10ng template DNA, 1μl 10×PCR Buffer, 20 mM MgCl₂, 10 mM dNTPs, 1.5 μM primer, and 0.4U of Taq DNA polymerase. All amplifications were carried out in a Veriti 96 Well Thermal Cycler (Applied Biosystems, USA). For primers 6, 10, 13, 807, 818, 827, 834, 842, 848 and 855, the PCR reaction procedure was as followed: an initial denaturation step of 2 min at 94℃, 35 cycles of 1 min at 94℃, 1 min at respective T_m values (Table 1), and 1 min at 72℃, and a final extension step of 10 min at 72℃. For primer 811, the amplifications began with initial denaturation for 5 min at 94℃, followed by 45 cycles of 30 s at 94℃, 45 s at 56℃, and 2 min at 72℃, and a final extension at 72℃ for 5 min. The amplification products obtained were separated on 2% agarose gels containing 0.1% ethidium bromide at 120 V for 2h, and photographed under UV light. The molecular weight of every amplified band was evaluated by using the DL 2000 Marker (Beijing Dingguo Changsheng Biotechnology Co. Ltd, Beijing, China). Each reaction was repeated twice for each sample with each primer. Only those primers which generated reproducible bands in both reactions for each sample were used for the data analysis.

Methods are completely described in the paper. R code is provided.