Metal content in soil and genetic variation in Deschampsia cespitosa

Дата канвертавання19.04.2016
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Metal content in soil and genetic variation in Deschampsia cespitosa populations from Northern Ontario (Canada): application of ISSR markers.

K.K. Nkongolo1, *, M. Mehes1, A. Deck1, and P. Michael1

1Department of Biological Sciences, Laurentian University, Sudbury, Ontario, Canada, P3E 2C6. *Corresponding author:


The Sudbury region located in Northern Ontario has a long history of mining and smelting and high depositions of metals in the ecosystems. Concentrations of cadmium, cobalt, copper, lead, nickel, and zinc in soil samples from nine sites in Northern Ontario were measured in the present study using ICP–MS. Metal concentrations reported continue to exceed Ontario ministry of Environment and Energy (OMEE) upper limit guidelines for uncontaminated soil at some sites. The sites from the Cobalt region have higher levels of metals than the Sudbury region. DNA samples from Deschampsia cespitosa populations growing on these sites were analyzed using Inter-simple sequence repeat (ISSR) assay. The mean levels of polymorphic loci detected in populations from the Cobalt, Sudbury, and Manitoulin Islands regions were 46%, 74% and 69%, respectively. ISSR fingerprinting of DNA samples from sites with high metal content revealed low level of polymorphic markers. The between-populations variance contributed only 13.6 % of the total molecular variance while the within-population variance accounts for 71.2%. The genetic distance data revealed that the D. cespitosa populations from Northern Ontario are different but genetically closely related

Key words: ISSR markers, Deschampsia cesptitosa, D. flexuosa, heavy metal contamination, genetic variations.


The Sudbury region in Ontario Canada is known for the mining and smelting of high sulphide ores containing nickel, copper, iron, and precious metals. Years of intense fumigation of more than 100 million tones of sulphure dioxide and tens of thousands of tones of metal particulates created barren and semi-barren lands near smelters in the Sudbury (Ontario) region (Amiro and Courtin 1981). There are signs of recovery since emissions have been reduced and reforestation efforts were implemented in 1978 (Dudka et al. 1995; Winterhalder, 1995). Continued investigation and monitoring is essential for understanding ecosystem recovery and sustainability. Much attention has focused on the environmental effects of mining and smelter pollution. Although reports provide information of metal levels in soil and their uptake and accumulation by plants, knowledge of genetic effects on plants growing in contaminated areas is limited (Gratton et al., 2000; Hazlett et al., 1983; Hutchinson and Whitby, 1974). Several authors have reported differences in genetic structure of plants growing in contaminated areas (Muller-Stark 1985; Scholz and Bergmann 1984). Enzymatic studies of Norway spruce revealed genetic differences between groups of sensitive trees in polluted areas (Scholz and Bergmann 1984). It has been demonstrated that the evolution of heavy-metal tolerant ecotypes occurs at an unexpectedly rapid rate (Wu et al., 1975), and that despite founder effect and selection, in several cases, the recently established tolerant-populations maintain a high level of variation and appear to be at least as variable as non-tolerant populations. Observations of higher heterozygosity in tolerant plants of European beech in Germany (Muller- Starck 1985), scots pine in Germany and Great Britain (Geburek et al. 1987) and trembling aspen and red maple in the United States (Berrang et al. 1986) have been reported. Several studies, however, have reported the detection of bottleneck effects (Bush and Barnett, 1993; Vekemans and Lefebre, 1997; Nordal et al., 1999). Mejnatowicz (1983) presented evidence of loss of genes and heterozygosity in tolerant Scots pines. The frequent lack of a bottleneck effect has been explained by different hypotheses (Ducousso et al. 1990): successive colonization events, a high number of tolerant plants in the primary populations, pollen flow from the neighboring populations, environmental heterogeneity (Hdrick et al., 1976) and human disturbance (Gouyon et al. 1983).

The colonization of the barren landscapes by metal-tolerant species like Deschampsia caespitosa in the Sudbury region has suggested possible genetic base tolerance (Winterhalder, 1995). The metal tolerance capabilities of D. cespitosa allow this species to survive on the mine tailings in Sudbury and the abandoned mine sites in Cobalt. The soil in these two areas contained high levels of Cu, Ni, Co, Al, and Co, Ni, and As, respectively (Von Frenckell and Hutchinson 1993). Knowledge of genetic characteristics of plant populations growing in metal contaminated areas is still sketchy.
Nkongolo et al. (2001) observed a high level of aneuploidy in Deschampsia cespitosa (tufted hair grass) from Northern Ontario, but made no link between genetic characteristics and heavy metal contamination in soil. Cox and Hutchinson 1980, established a tentative colonization history of D. cespitosa in the Sudbury. They reported that the two most likely areas that could have supplied the colonizers are Cobalt, a small mining community about 150 km NE of Sudbury (near the Quebec border), and Manitoulin Island, which is about 150 km SW of Sudbury (Cox and Hutchinson, 1980).
Dechamspsia flexuosa ( wavy hair-grass) along with Agrostis gigantea (redtop) and Poa compressa (Canada bluegrass) are other grass species that have colonized Sudbury sites to a more modest degree (Winterhalder 1995). D. cespitosa and D. flexuosa can be distinguished by their canopy, but information on their genetic relationships is sketchy.
Among the genetic markers, RAPD markers are the only molecular markers that have been used to study populations of metal-tolerant plants (Nkongolo et al. 2001). The downside of the RAPD system is the repeatability of the amplification results. Since its introduction in 1994, the Inter-Simple Sequence Repeat (ISSR) random marker system has grown in popularity and has superseded RAPD method (Zietkiewicz et al., 1994). The ISSR marker system is based on the use of a small 15-20 bp primers designed to be complimentary to a short tandem repeat (STR) sequence found on a multitude of eukaryotic genomes. Because ISSR primers have longer lengths compared to RAPD primers, the required annealing temperatures are higher and as such, non-specific binding is reduced and banding patterns have higher reproducibility (Qian et al. 2001).
The main objectives of the present study were 1) to monitor the current levels of metal contamination in Northern Ontario and their effects on genetic variation in D. Cespitosa populations that colonized these sites; 2) to determine the level of genetic variation and the degree of relatedness within and among D. cespitosa populations using ISSR markers; Attempts were also made to determine genetic distance among D. cespitosa and D. flexuosa populations and to differentiate these two species using ISSR markers.

Material and Methods
Metal analysis

Soil samples were collected from four sites in the Sudbury area (Coniston, Falconbridge, Coppercliff, and Walden), three abandoned mine sites in the Cobalt area (Cobalt-3, Cobalt-4, and Cobalt-5) and two control sites in the Manitoulin Island area (Little Current, Mississagi Lighthouse)(Fig.1). Four soil samples were taken at each site from the surface to a depth of 15 cm. Samples were air dried, lightly ground with a ceramic mortar and pestle, sieved to 2 mm, and stored prior to further analysis. The soil samples were analyzed for total cadmium, cobalt, copper, lead, nickel, and zinc. A total of 0.5 grams of each sample was digested totally using a digestion reagent that consisted of three parts hydrochloric acid and one part nitric acid. The solution was heated for 30 min, then filtered. Hydrochloric acid (2%) was added to each tube to reach a final volume of 100 mL. All solutions were analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for total metal content. Data quality was assessed by digestion and analysis of certified reference material for soil (Till 1, CANMET) and analytical and method duplicates. The total QA/QC (quality assurance / quality control) analyzed was 25%.

The data for the metal levels in soil samples were analyzed using SPSS 7.5 for Windows. All the data were transformed using a log10 transformation to achieve a normal distribution. ANOVA followed by Tukey HSD multiple comparison analysis were performed to determine significant differences (p < 0.05) among the nine sites.

Figure 1. Map of Northern Ontario showing the location of the three regions where Deschampsia cespitosa and D. flexuosa were collected.

Molecular analysis
Genetic material

Leaves of D. cespitosa were collected from the same sites as soil samples and leaf samples of Deschampsia flexuosa were collected from two sites in Sudbury, Daisy Lake and Garson. For each site, 20 plants (representing 10 to 15% of the D. cespitosa or D. flexuosa population) were collected. Additional D. cespitosa seed samples originating from Poland and Switzerland were obtained from Rutgers University, New Jersey. These populations along with D. flexuosa populations were used as reference and for comparison purposes.

DNA extraction and amplification with ISSR primers

Total DNA was isolated from each sample as described by Nkongolo (1999). Each sample was individually primed with each of the 9 primers (Table 1). The ISSR amplification was carried out in accordance with the method described by Nagaoka and Ogihara (1997), with some modifications. The PCR amplifications were performed in 10 mmol Tris-HCL/L (pH 9.1), 50 mmol/L KCL, 0.1% Triton X-100, 1.5 mmol MgCl2/L, 0.2 mmol deoxynucleoside triphosphate (dNTP)/L, 2% formamide, 1 umol/L of primer, 1 unit of Taq polymerase, and 20 ng of genomic DNA per 25 uL reaction. Initial denaturation was for 7 min at 94oC, followed by a hot start for 2 min at 85oC, and 45 cycles of 1 min at 94oC, 1 min at 52oC, 2 min at 72oC, and a final 7 min extension at 72oC. PCR products were analyzed on 2% agarose gels in 1 x Tris-borate-EDTA (TBE) buffer, then stained with ethidium bromide, and scored for band presence or absence.

ISSR analysis

ISSR assays of each population were performed at least twice. Only reproducible amplified fragments were scored. For each sample, the presence or absence of fragments was recorded as 1 or 0, respectively, and treated as a discrete character. Pairwise comparison of banding patterns was evaluated using RAPDistance version 1.04 (Armstrong et al. 1994). The data were analyzed to generate Jaccard similarity coefficients and genetic distances. These similarity coefficients were used to construct dendrograms, using neighbour-joining analysis (Saitou and Nei 1987). Analysis of molecular variance (AMOVA) was applied, to estimate variance components for ISSR phenotypes. Variations was partitioned among individuals (within regions) and between regions. Levels of significance were determined using nonparametric permutational methods, with the Winamova program (Excoffier et al., 1992).

Heavy metal analysis

Recovery and precision for all elements in reference soil samples were within acceptable range. The estimate levels of metal content in different sites are illustrated in Figure 2. The levels of the metals measured were low in the control sites. Overall, the results indicated that nickel and copper continue to be the main contaminants of soil in Coniston while cadmium, cobalt, copper, lead, zinc and to some extent nickel, were found in high concentration in Cobalt sites.

The Cobalt 4 site is by far the most contaminated of all the sites (Fig. 2). For example, the average mean level of zinc at Cobalt 4 is at least twenty-one fold than that of Sites from Sudbury. Cobalt-4 was also among the sites with the highest accumulation of copper, lead, and nickel. Unlike the Sudbury sites, which are located near smelters, Cobalt-4 site is located in the concrete remains of the foundation of an abandoned mine site. This could explain the extraordinarily high heavy metal accumulation in that area. This particular site also does not appear to have been detoxified or rehabilitated like the Sudbury sites have been. Cobalt 3 showed relatively lower level of heavy metal accumulation than Cobalt 4 and 5 and was typically in the middle of the spectrum of contaminated sites.
The two control sites, Little Current and Mississagi Lighthouse were always among the least contaminated for the metals analysis. Three of the Sudbury sites, Falconbridge, Coppercliff, and Walden were also consistently among the least contaminated. Coniston was found to be on average significantly more contaminated than the other Sudbury sites (Fig. 2).

Figure 2. Cadmium, cobalt, copper, lead, nickel, and zinc concentrations in soil samples collected from the Sudbury, Cobalt, and Manitoulin Island regions. Means with common notations are not significantly different as indicated by Tukey HSD analysis (p > 0.05).

ISSR analysis

Six of the primers screened produced amplifications ranging from 160 bp to 1300 bp. Figures 3 and 4 show ISSR markers generated with the UBC 841 and 17898B primers. The level of polymorphic loci among populations was 63 %. This value was much lower than the polymorphisms level (90%) reported in RAPD analysis of the samples from the same sites (Nkongolo et al 2001). The polymorphism within each population varied between 44% observed in Cobalt-5 and 92% in Falconbridge (Table 2). When the three regions were compared, the highest polymorphism was observed in samples from the Sudbury region (Coniston, Falconbridge, Copper Cliff, Walden), with an average of 74 % of polymorphism, followed by the control sites (Little Current and Mississagi Lighthouse) with 69%. The lowest level of polymorphic loci of 46 % on the average was observed in samples from the Cobalt region (Cobalt-3, Cobalt-4, and Cobalt-5) which also has the highest accumulation of heavy metal. The between- populations variance contributed 13.6 % of the total variance; the within-population variance accounts for 71.2%. Using a nonparametric test, we found that between-population differences were significant (Table 3). No single locus appears to be specific to contaminated sites.

Table 1. Nucleotide sequences of the primers used to produce ISSR profiles by amplification of genomic DNA from sixteen populations of Deschampsia cespitosa and D. flexuosa.

Primer Identification

Nucleotide sequence

(5’→ 3’)


Fragment size range (bp)

ISSR 17898B




UBC 818




UBC 823




UBC 825




UBC 827




UBC 835




UBC 841




UBC 844




UBC 849




Table 2. Levels of polymorphisms within Deschampsia cespitosa populations from Northern Ontario generated with ISSR primers.



Total number

of bands

Number of

polymorphic bands


bands (%)

Sudbury region

















Cobalt region













Manitoulin Island region

Little Current




Mississagi Lighthouse




Table 3. Analysis of molecular variance (AMOVA) for ISSR variation among Deschampsia cespitosa populations from several locations in Northern Ontario.

Sources of variation



Variance component

% Total


Among regions






Populations within region






Individual within populations






Figure 3. ISSR profiles of genomic DNA from Deschampsia cespitosa individuals amplified using primer UBC 841. Lanes 1 and 21 contain 1-Kb+ DNA ladder. Lanes 2 to 10 contain amplified product of Deschampsia cespitosa individuals from Coniston. Lanes 11 to 20 contain amplified product of D. cespitosa individuals from Falconbridge.

In general, the genetic distances among the D. cespitosa populations from Northern Ontario values varied from 0.06 to 0.77 on a scale of 0 (for identical populations) to 1 (for populations that are different for all criteria) (Table 3). The closest genetic distance value (0.06) was observed between the populations from Cobalt 5 and Cobalt 3. The genetic distance data showed that the four D. cespitosa populations from the Sudbury area (Coniston, Falconbridge, Copper Cliff and Walden) were closely related. These data also revealed that the D. cespitosa populations from the Cobalt region (Cobalt-3, Cobalt-4, and Cobalt-5) were closely related to the D. cespitosa population from little Current (Table 4 and Fig. 5). The results were supported by the cluster analysis that illustrated that the four D. cespitosa populations from the Sudbury region clustered together and the three Cobalt populations cluster with the populations from Little Current and Mississagi Lighthouse (Fig. 5). Overall, the molecular analysis using ISSR markers showed that the D. cespitosa populations from Northern Ontario are different but closely related.

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