Effect of Metal Contamination on the Genetic Diversity of Deschampsia cespitosa Populations from Northern Ontario: An Application of ISSR and Microsatellite Markers

Sudbury, Ontario (Canada) has been subjected to intense sulphur dioxide fumigation, soil contamination by aerial metal fallout and acid precipitation since the discovery of nickel and copper deposits in the late 1800s (Cox and Hutchinson, 1979; Bush and Barrett, 1993). The discovery of silver ore in Cobalt, Ontario (Canada) in the early 1900s, has been associated with arsenide and sulfarsenide mineral contamination of the soil in the region (Dumaresq, 1993). Although both these sites have been able to recover to some extent over the past 30 years due to emission reductions and remediation efforts (Dudka et al., 1995; Nkongolo et al., 2008), fact remains that such highly contaminated mine tailings often have metal concentrations that are increased to a level that are toxic for the majority of plants (JiménezAmbriz et al., 2007). The toxic metal pollutants, accompanied by the detrimental physical disturbances in the environment can influence plant survivorship, recruitment, reproductive success, mutation rates and migration, all of which affect the genetic diversity of the exposed populations (Deng et al., 2007). To date, hundreds of metal-tolerant genotypes have been identified from a wide variety of plant species surviving on such metal contaminated soils with many different life stories, pollinating systems and life-spans at an unexpectedly high rate (Mengoni et al., 2000; Wu et al., 1975). This type of rapid and widespread adaptation to metal pollution suggests that the evolution of metal tolerance is one of the major strategies for plant colonization of mining spoils (Deng et al., 2007). Several investigations on the genetic diversity among metal tolerant populations relative to their non-metal tolerant counterparts have been carried out. Despite founder effect and selection, studies on Silene paradoxa (Mengoni et al., 2000) and Agrostis stolonifera (Wu et al., 1975) demonstrated that the genetic diversity of the contaminated population was the same as that of the uncontaminated populations. Other studies reported a high heterozygosity in tolerant plants such as European beech (Muller-Starck, 1985), Scots pine (Geburek et al., 1987), trembling aspen and red maple (Berrang et al., 1986). Contrary to these results, a reduction of genetic diversity has been found in other tolerant populations such as Armeria maritime (Vekemans and Lefèbvre, 1997).


Introduction
Sudbury, Ontario (Canada) has been subjected to intense sulphur dioxide fumigation, soil contamination by aerial metal fallout and acid precipitation since the discovery of nickel and copper deposits in the late 1800s (Cox and Hutchinson, 1979;Bush and Barrett, 1993).The discovery of silver ore in Cobalt, Ontario (Canada) in the early 1900s, has been associated with arsenide and sulfarsenide mineral contamination of the soil in the region (Dumaresq, 1993).Although both these sites have been able to recover to some extent over the past 30 years due to emission reductions and remediation efforts (Dudka et al., 1995;Nkongolo et al., 2008), fact remains that such highly contaminated mine tailings often have metal concentrations that are increased to a level that are toxic for the majority of plants (Jiménez-Ambriz et al., 2007).The toxic metal pollutants, accompanied by the detrimental physical disturbances in the environment can influence plant survivorship, recruitment, reproductive success, mutation rates and migration, all of which affect the genetic diversity of the exposed populations (Deng et al., 2007).To date, hundreds of metal-tolerant genotypes have been identified from a wide variety of plant species surviving on such metal contaminated soils with many different life stories, pollinating systems and life-spans at an unexpectedly high rate (Mengoni et al., 2000;Wu et al., 1975).This type of rapid and widespread adaptation to metal pollution suggests that the evolution of metal tolerance is one of the major strategies for plant colonization of mining spoils (Deng et al., 2007).Several investigations on the genetic diversity among metal tolerant populations relative to their non-metal tolerant counterparts have been carried out.Despite founder effect and selection, studies on Silene paradoxa (Mengoni et al., 2000) and Agrostis stolonifera (Wu et al., 1975) demonstrated that the genetic diversity of the contaminated population was the same as that of the uncontaminated populations.Other studies reported a high heterozygosity in tolerant plants such as European beech (Muller-Starck, 1985), Scots pine (Geburek et al., 1987), trembling aspen and red maple (Berrang et al., 1986).Contrary to these results, a reduction of genetic diversity has been found in other tolerant populations such as Armeria maritime (Vekemans and Lefèbvre, 1997).

DNA extraction
The total cellular DNA from individual samples was extracted from seedling tissue using the method described by Nkongolo (1999), with some modifications.The modification involved addition of PVP (polyvinylpyrrolidone) and β-mercaptoethanol to the CTAB extraction buffer.The DNA concentration was determined using the fluorochrome Hoechst 33258 (bisbensimide) fluorescent DNA quantitation kit from Bio-Rad (cat. # 170-2480) and the purity was determined using a spectrophotometer (Varian Cary 100 UV-VIS spectrophotometer).

ISSR analysis
All DNA samples were primed with each of the nine primers (ISSR 17898B, UBC 818, UBC 823, UBC 825, UBC 827, UBC 835, UBC 841, UBC 844, and UBC 849) (Mehes et al., 2007).The ISSR amplification was carried out in accordance with the method described by Nagaoka and Ogihara (1997), with some modifications described by Mehes et al. (2007).All PCR products were loaded into 2% agarose gel in 1X Tris-Borate-EDTA (TBE) buffer.Gels were pre-stained with 4μl of ethidium bromide and run at 3.14V/cm for approximately 120 minutes.These agarose gels were visualized under UV light source, documented with the Bio-Rad ChemiDoc XRS system and analyzed for band presence or absence with the Discovery Series Quantity One 1D Analysis Software.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's 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 were 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).

Microsatellite analysis
A total of 31 microsatellite primers, developed in several members of the Poaceae family (Table 2), were screened for cross-species consrvation in Deschampsia cespitosa.Of these, 5 were from Elymus caninus (Sun et al., 1999), 7 were from Avena sativa (Li et al., 2000), 3 were from Triticum aestivum (Röder et al., 1995) and 17 were developed in Hordeum vulgare (Liu et al., 1996;MacRitchie and Sun, 2004;Struss et al., 1998).The microsatellites primers used in this study were selected based on the phylogenetic relationship between the species of origin and D. cespitosa, the polymorphic index of the alleles within their respective species and, in some cases, previous reports of cross-species conservation of the microsatellite locus.DNA amplification was performed following the procedure described by Mehes et al. (2010).Of the 31 microsatellite primer pairs screened, only those that successfully amplified a clear, reproducible, distinguishable band, demonstrated microsatellite characteristics and showed a certain degree of polymorphism were used in the study.The agarose gels were documented using the Bio-Rad ChemiDoc XRS system and analyzed with the Discovery Series Quantity One 1 D Analysis Software.Nine microliters of 3X loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol) was added to the Effect of Metal Contamination on the Genetic Diversity of Deschampsia cespitosa Populations from Northern Ontario: An Application of ISSR and Microsatellite Markers 119 remainder of the 18 μl of the amplified products and 2.5 μl of 3X loading buffer was added to the mixture of 5 μl of water and 1.5 μl of 10 bp ladder (Invitrogen).The samples and the 10 bp ladder (Invitrogen) were denatured at 99 o C for 10 min and snap cooled for 2 min on ice prior to loading on denaturing gels.The PCR products were electrophoresed on a 0.4 mm denaturing 6% polyacrylamide gels containing 8 M Urea and 1X TBE buffer at constant power of 73 W, 2 100 V and 90 mA and were equilibrated to 55 o C (DNA Sequencing System, FisherBiotech, Fisher Scientific).The amplifications products were visualized with the Silver Staining Sequence DNA Sequencing System according to the manufacturers protocol (Promega Corporation).Resolved fragments were sized by The Quantity One and Genescan softwares.The presence and absence of alleles yielded by the microsatellite primer pairs were scored as 1 or 0, respectively in order to determine the polymorphism of each locus.Such designations were carried out with the Quantity One software by establishing the alleles of interest and comparing them to the 10 bp ladder which served as a marker system.The data was computed into the Popgene software, version 1.32 (Yeh et al., 1997) and used to determine the intra-and interpopulation genetic diversity parameters such as number of alleles per locus, mean number of alleles across loci, percentage of polymorphic loci and Shannon's information index.In order to determine whether the observed allelic proportions met Hardy-Weinberg expectations, the populations were tested using an exact test (Guo and Thompson, 1992) by the computer program GENEPOP version 1.2 (Raymond and Rousset, 1995).Hardy-Weinberg Equilibrium deviations were further tested with heterozygote deficiency and excess alternative hypotheses.Wright's F statistics including inbreeding coefficients and fixation index (Weir and Cockerham, 1984) as well as gene flow were determined using Popgene.The PowerMarker software, version 3.25 (Liu and Muse, 2005) was used to calculate the genetic distances between the populations based on the Cavalli-Sforza and Edwards Chord's Distance (1967).Finally, the relationship between the matrices based on microsatellite data and geographical location was calculated using the Pearson's correlation coefficient and the significance of the correlation was determined by the Mantel test (Mantel, 1967) (Liu et al., 1996) Table 2. List of the microsatellite primer pairs screened for transferability in D. cespitosa.

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Relevant Perspectives in Global Environmental Change 122

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).

ISSR analysis
Five of the nine primers screened (Table 3) produced good amplification products ranging from 160 bp to 1300 bp.They include 17898B, UBC 818, UBC 827, UBC835 and UBC 841 had good amplifications products (Figure 8).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 to 92% in Falconbridge (Table 4).The overall values for the three regions were compared.The highest level of polymorphism was observed in samples from the Sudbury region (Coniston, Falconbridge, Copper Cliff, Walden) with an average of 74 %, followed by the Manitoulin Island region (Little Current and Mississagi Lighthouse) with an average of 69%.The lowest level of polymorphic loci was observed in samples originating from the Cobalt region (Cobalt-3, Cobalt-4, and Cobalt-5) with a mean value of 46%.Interestingly, the Cobalt region was also the site which exhibited the highest accumulation of metals in the soil.The between-populations variance contributed 13.6 % of the total variance while the withinpopulation variance accounted for 71.2%.Using a nonparametric test, we found that between-population differences were significant (Table 5).No single locus appears to be specific to contaminated sites.In general, the genetic distances among the nine D. cespitosa populations from Northern Ontario values varied from 0.059 to 0.488.The scale utilized denotes a 0 for identical populations to a 1 for populations that are different for all criteria (Table 6).The closest genetic distance value (0.06) was observed between the populations from Cobalt-5 and Cobalt-3.The two most genetically distant populations were Cobalt-5 and Falconbridge (0.49).The genetic distance data also 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 6, Fig. 3).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 clustered with the populations from Little Current and Mississagi Lighthouse (Fig. 3).Overall, the molecular analysis using ISSR markers showed that the D. cespitosa populations from Northern Ontario are different but closely related.

Nuclear microsatellite analysis
Only primer pairs HVM3, HVM5, HVM20, HVM65 and WMS6 successfully amplified a clear, reproducible, distinguishable band within an acceptable range of the expected size while demonstrating polymorphism.This represents only 14% of the microsatellite primer pairs screened for transferability within D. cespitosa.All alleles detected were scored according to the guidelines previously outlined.Every possible microsatellite loci pair, in every population, was surveyed for association following the null hypothesis that genotypes at one locus are independent from genotypes at the other locus.Based on the results, no evidence of linkage disequilibrium between the microsatellite loci used in this study was observed.Only data related to genetic diversity are discussed in the present report.

Genetic diversity
Genetic diversity within each individual, population, region and locus was assessed using standard descriptive statistics.The five polymorphic microsatellite markers detected a total of 40 alleles.The mean number of alleles per locus across populations was 2.1, 2, 7.3, 4.6 and 2.6 for locus HVM 3, HVM 5, HVM 20, HVM 65 and WMS 6 respectively.The mean number of alleles per populations across loci was 3 for Little Current, 3.2 for Mississagi Lighthouse and Walden, 3.6 for Falconbridge and Copper Cliff, 3.8 for Coniston and Cobalt-5, 4 for Cobalt-4 and 5.2 for Cobalt-3.At the population level, the observed mean heterozygosity (H O ) and the expected mean heterozygosity (H E ) ranged from 0.413 and 0.48 in the Mississagi Lighthouse population to 0.65 and 0.76 in the Cobalt-3 population.At the regional level, the H O and the H E ranged from 0.40 and 0.46 in the Cobalt region to 0.34 and 0.42 in the Manitoulin Island region.Finally, the H O and the H E observed by individual loci ranged from 0.28 and 0.44 for HVM3 to 0.85 and 0.99 for HVM20.Shannon's Information Index (i) was also calculated as an additional measure of genetic diversity.Values varied from 0.54 at the HVM5 locus to 2.58 at the HVM20 locus with a mean of 1.417 (Table 7).
Inbreeding is defined as the non-random uniting of gametes which results in a decrease of heterozygotes.The level of inbreeding within a population is determined by the inbreeding coefficient, F IS , where -1 (all individuals heterozygous) ≤ F IS ≤ 1 (no observed heterozygotes).The inbreeding coefficients (F IS ) were determined for each population per loci based on the null hypothesis of no inbreeding represented as F IS = -1.All nine populations exhibited a negative F IS value at loci HVM3, HVM5 and HVM20 indicating an excess of heterozygotes.Only Walden and Cobalt-3 populations presented negative F IS values at locus HVM65.Finally, six of the nine populations exhibited negative F IS values at the WMS6 locus.As a result, the overall inbreeding coefficient for D. cespitosa populations were -0.18, -0.08, -0.07, -0.35, -0.19, -0.07, -0.12, -0.21 and -0.17 for Coniston, Falconbridge, Copper Cliff, Walden, Cobalt-3, Cobalt-4, Cobalt-5, Little Current and Mississagi Lighthouse, respectively.The fixation index (F ST) is a measure of the extent of genetic differentiation among populations due to genetic drift.Values can range from 0.0, indicating no differentiation, to 1.0, indicating complete differentiation.However, because the observed maximum is usually much less than 1.0, a value between 0.0 and 0.05 is considered as little genetic differentiation, 0.05 and 0.15 as moderate genetic differentiation, 0.15 and 0.25 as great  8).Finally, HVM3 locus represented a very great deal of genetic differentiation with an F ST value of 0.311 (Table 8).
The F ST values were subsequently used to estimate the level of gene flow (N m) for each locus according to Nei (1987), where N m = 0.25(1-F ST )/F ST .The mean level of gene flow was 0.933 with individual N m values ranging from 0.5424 for the HVM65 locus to 2.347 for the HVM5 locus (Table 8).Genetic distance coefficients were calculated according to the Cavalli-Sforza and Edwards chord distance (D C ).This particular scale ranges from 0, indicating no genetic difference to 1, indicating differences at all conditions criteria.The genetic distance coefficients varied between 0.57 between the Coniston and Cobalt-3 populations and 0.18 between the Falconbridge and Copper Cliff populations (Table 9).Based on these values, an un-rooted Neighbour-Joining phylogenetic tree was constructed with 101-bootstrap.The resulting tree illustrates three major clades (Fig. 4).The first is composed of the Little Current population, the second comprises all four Sudbury region populations and the Mississagi Lighthouse population and the third includes all three Cobalt region populations.Within the second clade, the Mississagi Lighthouse population is the most distantly related, while the Falconbridge and Copper Cliff populations and the Walden and Coniston populations form clusters.Within the third clade, the Cobalt-5 population appears to be the most distantly related.The Mantel test results reveal a significant correlation between the two distance matrices (r = 0.514, p = 0.01) suggesting a congruence between the genetic distance generated from microsatellite data and the geographical distance between populations.

ISSR analysis
In general, the efficiency of a molecular marker technique depends on the amount of polymorphism it can detect among the set of accessions investigated.In the present study, the level of polymorphism detected with the ISSR system was lower than that observed with the RAPD method.Similar results were obtained by Fang and Roose (1997) who showed that RAPD PCR had identified a higher level of variation in Citrus spp.than ISSRs.Other studies conducted by Nagoaka and Ogihara (1997), Nkongolo et al. (2005), Raina et al. (2001) and Qian et al. (2001) have shown that ISSRs reveal a higher level of variation than RAPD markers in other plant species.Technically, RAPD and ISSR markers are different systems targeting different areas of the genome.RAPD markers cover the entire genome, revealing length polymorphism in coding or non-coding regions as well as repeated or single-copy sequences (Williams et al. 1993).Unlike RAPD products, the origin of the amplification products in ISSR-PCR is known to be from the sequences between two simple-sequence repeat primer sites where length variation does not necessarily reflect simple-sequence length polymorphism (Zietkiewicz et al. 1994).The level of variation detected with each system greatly depends on the primers used, thus making comparisons regarding levels of polymorphisms generated with RAPD and ISSR primers rather inconsistent.
The ISSR analysis revealed great variation in regards to the genetic relatedness of the samples analyzed.In general, the genetic distance values revealed that D. cespitosa from the nine Northern Ontario sites are genetically close.The genetic distance values between Cobalt-3 and Cobalt-5 indicate that these two populations are quite genetically similar.This suggests that these sites were likely seeded with the same genetic materials.The relative small genetic distance values among Sudbury populations and their clustering on the dendrogram are consistent with the previous RAPD data (Nkongolo et al., 2001).These findings also corroborate with the speculations from several ecologists that these populations might be the result of a single colonization event (Winterhalder, 2002;Personal communication).In general, the genetic similarity between the nine D. cespitosa populations from Northern Ontario may suggest that these populations could have originated from a common source.Furthermore, based on the genetic distance data, the theory of Cobalt and Little Current populations as the source of D. cespitosa which colonized the Sudbury area around 1972 can not be rejected.Previous genetic analysis of these populations aiming at establishing relationships among these nine sites using RAPD markers was inconclusive (Nkongolo et al., 2001).Also, the study conducted by Bush and Barrett (1993) using isozyme markers indicated that the Sudbury and Cobalt samples showed enough variation to reject the theory of Cobalt D. cespitosa colonizing Sudbury.Although isozymes and ISSRs allow the analysis of genetic variability in plant species, fundamental differences exist between these two methods.Isozyme analysis reflects alterations in the DNA sequence of coding regions in the genome leading to changes in amino acid composition which can go undetected (Hamrick 1989).
ISSRs target microsatellites sequences located throughout the entire eukaryotic genome, most of which are selectively neutral areas.These areas are known to evolve rapidly and as such, have been deemed good tools for any study in genetic diversity in many organisms (Blair et al., 1999).Attempts were made in the present study to use environmental conditions for appropriately interpreting genetic information.The effects of novel and toxic environments have been examined in considerable detail in the study of life history evolution.There are theoretical reasons for expecting the genetic variance of a life history character to increase when the population is challenged with a novel environment, an expectation that has been upheld empirically by numerous studies (Service and Rose, 1985;Holloway et al., 1990).If metal tolerance is controlled by many genes as suggested by Von Frenkell-Insam and Hutchinson (1993) and McNair (1993), it is very likely that allelic frequency in an out-crossing and perennial species like D. cespitosa will be maintained over time resulting in a neutral genetic variation.The high level of genetic variability within D. cespitosa populations from the Sudbury region could be ascribed in part to these conditions.If the toxic stress continues at a sub-lethal level for many generations, resistance could develop, resulting in a decrease in genetic variation through selection.This might be the case of D. cespitosa populations from Cobalt where the high accumulation of heavy metal for several years appears to have significant impacts on the genetic structure of the D. www.intechopen.com

Effect of Metal Contamination on the Genetic Diversity of Deschampsia cespitosa
Populations from Northern Ontario: An Application of ISSR and Microsatellite Markers 131 cespitosa populations in that region by processes which are assumed to have selective effects.Metals impose severe stress on plants, especially in the rooting zone, which has led to the evolution of metal-resistant ecotypes in several herbaceous species like D. cespitosa (Cox and Hutchinson, 1979).Plants possess homeostatic cellular mechanisms to regulate the concentration of metal ions inside the cell to minimize the potential damage that could result from the exposure to nonessential metal ions.These mechanisms serve to control the uptake, accumulation and detoxification of metals (Foy et al., 1978).Selection of metal-resistant genotypes has been demonstrated to occur rapidly, within one or two generations, in populations that contain the necessary genetic information (Wu et al. 1975).These authors identified two factors that may affect the plant's ability to tolerate metals; the intensity of the contamination and the amount of time the population has been exposed to the toxic levels.The populations of D. cespitosa in Cobalt have been there for a much greater amount of time than the populations in Sudbury and the Cobalt soil is more contaminated than the Sudbury soil.This has resulted in a decrease and possibly a loss of alleles at some loci and many rare alleles that has lead ultimately to a lower genetic diversity in D. cespitosa populations from the Cobalt region.Evidence of a loss of genetic diversity at the population level caused by pollution has been demonstrated in other species (Lopes et al., 2004;Van Straalen and Timmermans, 2002).The low level of genetic variation in samples from D. cespitosa population from Cobalt-3 could be explained by the fact that this abandoned mine waste site that was likely as contaminated as Cobalt-4 and Cobalt-5 sites has been covered with a clay cap.This fresh clay that was brought in was not as contaminated as the surrounding area.
There is also a slight possibility that the small level of polymorphic loci detected in Cobalt samples could be caused by a founder effect.This is a form of genetic bottleneck occurring where new populations are established by a small number of individuals, or by a group of individuals whose genetic variation is not representative of the parent population.However, the possibility of a founder effect occurring only in Cobalt populations and not in Little Current where the D. cespitosa population has been isolated for several generations is quite small (Peter Beckett, 2006, personal communication).It should also be pointed out that D. cespitosa is an out-crossing species which produces a lot of seeds every year.These characteristics alone usually negate possible founder effects in many species (Hedrick et al., 1976).Muller et al. (2004) also indicated that high frequency of heavy metal tolerance in natural populations can reverse the effects of an initial genetic bottleneck.

Microsatellite analysis
The various application of microsatellite markers are the direct result of their hypervariability, co-dominant nature, abundance throughout the genome and reproducibility.Their primary disadvantage lies is their time consuming and expensive de novo development and, as such, has been restricted to a few agriculturally important crops.In light of this, a growing number of studies have examined the ability of microsatellite primer pairs to amplify across closely related species.In this study, we were able to identify several polymorphic microsatellite loci in Deschampsia cespitosa via SSR primer pairs developed in Hordeum vulgare (barley) and Triticum aetivum (wheat).The five microsatellite primer pairs, namely HVM3, HVM5, HVM20, HVM65 and WMS6 were originally selected because of their high polymorphic index and documented conservation across species.Primers HVM3, HVM20 and HVM65, originally developed in barley showed conservation in Avena species with polymorphic information content (PIC) of 0.44, 0.77 and 0.72, respectively.In addition, a PIC of 0.15 for HVM3 and 0.50 for HVM20 was reported in oat cultivars (Li et al., 2000).The HVM5 primer originally developed from barley, showed conservation while displaying polymorphism in Elymus and Pseudoroegneria species (MacRitchie and Sun, 2004).WMS6, originally developed in wheat, had been documented as conserved and polymorphic in both barley and rye (Röder et al., 1995).In Deschampsia cespitosa, these primers ranged from having 2 alleles at the HVM5 locus with a PIC of 0.29 to 17 alleles at the HVM20 with PIC values of 0.91.Our findings are in accordance with other studies conducted by Gaitán-Solís et al. (2002), andSaha et al. (2004), which all report the occurrence of cross-species microsatellite primer pair transferability at an elevated rate.Such success depends heavily on the conservation of priming sites within the flanking regions and as such the evolutionary relatedness of the species sampled.Particular to the Poaceae family, believed to have radiated about 60 million years ago, genetic mapping has revealed remarkable conservation of gene content and gene order.Studies have shown that the linear organization of genes in some nine different genomes varying in chromosome number, from 5 to 12 and nuclear DNA amount, from 400 to 6000Mb, can be described in terms of only 25 rice linkage blocks (Gale and Devos, 1998).We also report shorter SSRs at HVM3, HVM5 and HVM 20 loci in D. cespitosa than in the species of origin with a difference in size of 36bp, 33bp and 5bp respectively.HVM65 has an allelic range of 196-220 in D.cespitosa whereas its size in barley is noted as 129 bp, a difference of 63 bp.Previous studies have shown that mutations at microsatellite loci are not solely restricted to the hypervariable region and can occur in the flanking regions at nonnegligeable rate, both of which can contribute to variations or lack thereof in allele size (Chapuis and Estoup, 2006).As such, inferring complete sequence homology based solely on the presence of amplification product is premature.

Genetic diversity
Mine tailings are typically a difficult medium for plant establishment and growth as these sites often contain elevated levels of metals, low nutrients and organic matter as well as being subjected to wind and water erosion.Some plant species and/or adapted populations have successfully colonized these toxic environments, however such inhospitable conditions often leave these areas with only scattered patches of vegetation (Mining in the Yukon).Deschampsia cespitosa has shown a remarkable ability to colonize and dominate such plots of land with great success having naturally colonized over 1000s of hectares of barren lands around Sudbury, following the constructions of the Super Stack in 1972.As a direct result of the mining activity in both regions, Cobalt and Sudbury present extremely hostile environments that are believed to have imposed strong selection pressures on colonizers resulting in reductions in genetic diversity.Detailed analysis of our nine populations with microsatellite markers reveals the observed mean heterozygosity (Ho) and the expected mean heterozygosity (He) ranged from 0.413 and 0.48 in the Mississagi Lighthouse population to 0.645 and 0.76 in the Cobalt-3 region.In addition, genetic diversity measures based on Nei's and Shannon's index demonstrated a similar patter with values ranging from 0.39 and 0.84 in Mississagi Lighthouse to 0.61 and 1.21 in Cobalt-3.As far as soil analysis, the Cobalt-4 site was shown to be the most contaminated of all the sites with significantly higher levels of arsenic, lead, zinc, cadmium and cobalt whereas the Mississagi Lighthouse and Little Current consistently www.intechopen.com

Effect of Metal Contamination on the Genetic Diversity of Deschampsia cespitosa
Populations from Northern Ontario: An Application of ISSR and Microsatellite Markers 133 grouped as the sites with the significantly least amount of metals.Cobalt-3 typically placed in the middle of the spectrum among contaminated sites.Therefore, based on our microsatellite data, the level of genetic diversity at the population level does not decrease in terms of increased metal contamination.These findings are in conjunction with the results reported by Bush and Barrett (1993) on isozyme diversity that indicate that mine populations were no less polymorphic than uncontaminated populations.The retention of such elevated levels of genetic diversity within these mining populations can be attributed to number of selective, reproductive and demographic factors.As described by Bourret et al. (2007) if tolerance to the adverse environmental condition increases as a function of individual heterozygosity and/or if the contaminant is a mutagen, genetic variation within the affected population will remain elevated and may increase.Also, this species is a wind-pollinated outbreeder and, as a result, founders from such outbreeding populations are likely to heterozygous at many loci.In turn, this enhances the gene pool of small, founding populations by increasing the probability that at least the common alleles in the source population are represented in the new population (Bush and Barrett, 1993).Examination of the Hardy-Weinberg equilibrium across the nine populations revealed only two deviating populations, Walden and Cobalt-3 which were identified as having a significant heterozygote excess with values of 0.0021 and 0.0060 (p<0.05),respectively.Further analysis revealed inbreeding coefficients (F IS ) ranging from -0.346 in Walden to -0.19 in Cobalt-3 to -0.066 in Cobalt-4.These presences of these negative values across all nine populations imply a substantial amount of outbreeding, which as discussed earlier is in agreement with the reproductive pattern of the species.These findings also explain the occurrence of such highly heterozygote saturated populations because as stated above, outbreeders are more likely to be heterozygous at many loci (Bush and Barrett, 1993).Finally, the degree of differentiation among population (F ST ) was measured and was found to vary between moderate genetic differentiations with a value of 0.096 at locus HVM5 to very high with a value of 0.298 at locus HVM3.The mean degree of population differentiation was 0.194 in the Deschampsia population analyzed, indicating that 19.4% of the total genetic diversity is attributed to differences among populations.

Gene flow
Gene flow was examined to give an estimate of the average migration between all the populations studied per generation.The mean level of gene flow (N M ) in Deschampsia cespitosa based on our microsatellite analysis was 1.04 which is interpreted as the absolute number of individuals exchanged between populations.The level of genetic differentiation of 0.19 is regarded as high genetic differentiation between populations.It is also inversely proportional to Nm because as gene flow between populations increase, the genetic differentiation between these populations would decrease as a direct result.The low level of gene flow can be explained by the geographic distance between the nine populations, as the two closest sites are 2.1km away from each other and the two most distant sites are 319 km, despite the wind-pollinating reproductive strategy of the species.

Phylogenetic relationship
Studies by Bush and Barrett (1993) support the hypothesis that the metal-tolerant populations of D. cespitosa evolved at least twice in recent evolutionary history based on isozyme analysis.Secondary to their work, RAPD analysis (Nkongolo et al., 2001) of these same populations reveal a relatively small genetic distance between the four Sudbury populations which suggest that they are the results of a single colonization event.The Cavalli-Sforza and Edward's (1967) chord distance, D C , was used to estimate the genetic distance among our nine D. cespitosa populations.This particular algorithm is relatively unaffected by the presence of null alleles with low to moderate frequency and it relies on allele frequencies in order to determine the geometric placement of populations in a multidimensional sphere, rather than a mutational model (Chapuis and Estoup, 2007;Khasa et al., 2006).The distance matrix based on our microsatellite data revealed that the Walden and Coniston populations were the most genetically closely related populations even though their geographic locations were not the closest (18.5km), whereas the populations from Coniston and Cobalt-3 exhibited the greatest genetic distance despite the fact that these two populations were not the furthest geographically (133.7km).The four Deschampsia populations from the Sudbury region clustered together along with the Mississagi Lighthouse population.These findings are partly in accordance with the findings of Nkongolo et al. (2000) based on RAPD analysis, which also identified the four Sudbury population as a single cluster along with Little Current as well as the ISSR data which also clustered the four Sudbury populations.As such, this lends support to the theory that these four populations are the result of a single colonization event.The dendrogram also clustered the three Cobalt populations, which is not similar to the groupings of Nkongolo et al. (2001).In fact, the analysis of microsatellite and ISSR data suggests a very close genetic relationship between Cobalt-3 and Cobalt-4, followed by Cobalt-5.This is in disagreement with the proposed hypotheses that Cobalt-3 population arose from an unspecified seed mix (Nkongolo et al. 2001).The data described in the present study tend to lend additional supports to the allozyme findings of Bush and Barrett (1993) which suggest that Cobalt and Sudbury have independent evolutionary histories.Finally, the Little Current population appears as very genetically distantly related from the Sudbury grouping.As such, it does not lend support to the Hutchinson theory which describes the possible colonization of Sudbury region through the railway (Nkongolo et al., 2001).Finally, based on the work of isozymes (Bush and Barrett, 1993), RAPDs (Nkongolo et al., 2001), ISSRs and microsatellites, the Mississagi Lighthouse and Little Current populations never cluster together, despite both being located on the island.The Mantel test did show a correlation between the genetic matrix and the geographic distance matrix, although this relationship does not seem to be based on the concentration of metal contaminants in the soil.
In conclusion, monitoring the genetic diversity of D. cespitosa populations has been useful in detecting trends that should alert ecologists to potential problems.The high genetic variability detected in the Deschampsia populations from Sudbury and Cobalt suggests that these are healthy populations with the evolutionary potential to respond favourably and adapt to changes or disturbances in the environment.

Acknowledgement
We express our appreciation to the Natural Sciences and Engineering Council of Canada (NSERC), Vale Limited (Sudbury) and Xstrata Limited for financial among populations.

Fig. 1 .
Fig. 1.Map of Northern Ontario illustrating the three regions, Manitoulin Islands, Sudbury and Cobalt, where samples of Deschampsia cespitosa were collected for this study.

Fig. 2 .
Fig. 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).

Fig. 3 .
Fig. 3. Dendrogram of the genetic relationships between the nine D. cespitosa populations from Northern Ontario using data generated from the Jaccard similarity matrix from ISSR profiles.The values below the branches indicate the patristic distances based on the neighbor-joining analysis.The D. cespitosa populations from Europe and the D. flexuosa populations (Northern Ontario) were used as outgroups.

Fig. 4 .
Fig. 4. Dendrogram of the genetic relationship between the nine populations of Deschampsia cespitosa from Northern Ontario using the data generated from Chord's distance (Cavalli-Sforza and Edwards, 1967) based on the microsatellite profiles.This is an un-rooted tree based on neighbor-joining (NJ) analysis constructed with 101-bootstrap.

Table 1 .
using XLSTAT version 7.5 software package (www.xlstat.com).Geographic coordinates of the nine D. cespitosa sampling sites located throughout Northern Ontario.

Table 3 .
Nucleotide sequences of the primers used to produce ISSR profiles by amplification of genomic DNA from nine populations of Deschampsia cespitosa.

Table 4 .
Levels of polymorphisms within Deschampsia cespitosa populations from Northern Ontario generated with ISSR primers.
Effect of Metal Contamination on the Genetic Diversity of Deschampsia cespitosaPopulations from Northern Ontario: An Application of ISSR and Microsatellite Markers 127 genetic differentiation and values above 0.25 as very great genetic differentiation.The F ST values were 0.079 for HVM5 and 0.088 for HVM20, indicating moderate genetic differentiation.Locus HVM65 and locus WMS6 demonstrated great genetic differentiation with 0.22 and 0.22 F ST values, respectively (Table

Table 8 .
Genetic diversity parameters for the five microsatellite primer pairs of Deschampsia cespitosa.

Table 9 .
Cavalli-Sforza and Edward's chord's distance matrix (1967)generated from microsatellite data used in neighbor-joining analysis of Deschampsia cespitosa populations.