METAL ANALYSIS AND R4NDOM AMPLIFIED POLYMORPMC DNA CHARACTERIZATION OF JACK PINE (Pinus banksiana)
POPULATIONS FROM THE SUDBURY REGION
by Wayne Stéphane Granon
A thesis submitted in partial hlfiliment of the requirements
for the degree of Master of Science in Biology
School of Graduate Studies Laurentian University
Sudbury, Canada November, 1998
a Copyright by Wayne Stéphane Gratton 1998.
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Abstract
Present day levels of metals in soi1 and jack pine (Pinus banksiana) needles taken near
smelters in Sudbury, Canada and fiom uncontaminated sites were measured by ICP-MS.
Significantly bigher levels of cadmium, copper, nickel and Iead in needles were observed
near Sudbury smelters compared to control sites. In contrast, zinc concentrations were
significantly Iower. Significant negative correlations between copper and zinc (r=-0.63,
ps0.05 ), nickel and zinc (r-0.69, ps0.05) and Iead and zinc (r-0.70, p d . 0 5 ) in jack pine
needles suggest possible antagonistic interactions. Analysis of soil samples indicated higher
accumulations of cadmium, cobalt, copper, nickel and lead in the fust 5 cm of soil
decreasing with soil depth Observation of tree cores collected near Sudbury smelters
reveded distinct sensitive ring patterns for years 196 1 - 1962, i 966- 1967 and 197 1 for most
cores.
Random arnplified polymorphic DNA ( M D ) analysis of jack pine trees fiom Sudbury and
uncontarninated sites indicated low levels of polymorphisms within and among populations.
Six out of eleven primers screened did not produce any amplification, two amplified poorly
and the remaining three primers produced scoreable bands. Species-specific markers were
identified when red and jack pines were compared.
Acknowledpents
1 would like to thank Dr. K. Nkongolo for helping me reaiize that hard work, dedication and
perseverance are essentiai to become a good scientist. Your guidance, patience and
encouragement will aiways be remembered. I am grateful to Dr. P. Beckett and Dr. V.
Clulow for their advice and cntical reviews and to Dr. G. Spiers for his insight on metal
analysis. Technicd assistance of Messrs. Luc Taillefer, Danny Gratton and Normand Graiton
tt-ith smple coIlections is gratefuiiy acknowIedged, Thanks to Dr. B. Bowin, Mr. John
Hechler and Ms. Seanna Hoendermis fiom the Ministry of Northem Development and
Mines for ail their analytical assistance. Thanks are also extended to Pierre Thibodeau, staff
and faculty of Laurentian University BioIogy Department, Mary Taylor tiom the Laurentian
University Audio Visual Centre, Petawawa Nationai Forest Institute and the Ontario Ministry
of Natural Resources in Angus, Ontario for ail their technicd support. Special thanks to the
Robert Spencer Foundation for financiai support.
Table of contents
Abstract .............................................................. iv
....................................................... Acknowledgments v
....................................................... Table of contents vi
... .......................................................... List of tables vin
.......................................................... List of figures ix
..................................................... General introduction 1
Chapter 1 : Literature review .............................................. - 3
Chapter 2: Metal accumulations in soi1 and jack pine needles ..................... 8
2.1. Introduction ............................................... - 8
2.2. Materials and Methods ....................................... - 9
2.2.1. SampIe collection ..................................... - 9 ..................................... 2.2.2. Tree core analysis - 9
2.2.3. Metai analysis ........................................ 12 2.2.4. Statistical analysis ..................................... 13
2.3. Results and Discussion ...................................... 14
2.3.1. Tree core dendrochronology ............................. 14 ........................... 2.3.2. Jack pine needle metal analysis 13
2.3.3. Total soi1 metal analysis ................................. 30 2.3.4.SoilpH ........................................... - 3 8
Chapter 3: RAPD characterization of jack pine populations fiom the Sudbury region and uncontaminated sites ........................................ 40
3.1. Introduction ........................................... - 4 0
3.2. Materials and Methods ................................... 32
...................... 3.2.1. Jack pine seedling germination -42 .................................. 3.2.2. DNA extraction - 4 2
...................... 3 .2.3. Amplification of RAPD markers 43
3.3. Resdts and Discussion ...................................... -45
................. 3.3.1. DNA concentration and purity values - 4 5 ......... . 3.3 .2 RAPD characterization of jack pine populations - 45
..................................................... Generalconclusions 56
............................................................ References 58
vii
List of tables
Table 1. Longitude and latitude coordinates of sampling sites .................. 10
Table 2. Age determination of jack pine populations fkom the Sudbury region ........................................ and uncontaminated sites 1 5
Table 3. Analyticai results of pine needle reference material (NIST 1 575) ......... 1 7
Table 4. Metal concentrations in jack pine needies fiom the Sudbury region ........................................ and uncontaminated sites. 18
Table 5. Metal range concentrations in jack pine needles from the Sudbury region ....................................... and uncontaminated sites. - 2 0
Table 6. Spearman correlation coeficients of total metal concentrations in jack pine needles. .............................................. -22
Table 7: Anaiyticai r e d t s of soi1 reference material (CCRMP-Till 1) ............. 3 1
Tabie 8: Total metal concentrations in soil fiom the Sudbury region and ........................................... uncontaminated sites - 3 2
Table 9: S p e m a n common correlation coefficients of total metal concentrations in soi1 samples ................................................ - 3 5
Table 10. Total metal concentrations in 0-5 cm soil profiles fiom the Sudbury region and uncontaminated sites. ....................................... - 3 6
Table 1 1 : DNA concentration and purity values from jack pine seedling sarnpies. .... 46
Table 12. RAPD markers fiom jack pine populations fiom Sudbury and .......................................... uncontaminated sites. - 4 7
Table 13. RAPD markers and species-specific bands for jack and red pine populations. ........................................... - 4 8
viii
List of figures
Figure 1 . Location of soil aud jack pine population sampling sites within the ............................................. Sudbury region 11
Figure 2 . Tree cores collected near Sudbury smelters revealing distinct sensitive ringpatterns .................................................. 16
Figure 3 . Lead concentrations (mg kg*') in jack pine needles collected fiom Sudbury ................................................ and control sites 23
Figure 4 . Copper concentrations (mg kg-') in jack pine needles collected fiom Sudbury ............................................... and control sites- 25
Figure 5 . Nickel concentrations (mg kg-') in jack pine needles collected nom Sudbury ............................................... and control sites- 26
Figure 6 . Cadmium concentrations (mg kg-') in jack pùie needles collected fiom Sudbury andcontrolsites- ............................................... 27
Figure 7 . Zinc concentrations (mg kg') in jack pine needles collected ftom Sudbury ................................................ and control sites 29
Figure 8 . pH values of soi1 depths fiom Sudbury and control sites . . . . . . . . . . . . . . . . 39
Figure 9 . RAPD markers fiom jack pine populations using primer P.2 . . . . . . . . . . . . 49
Figure 10 . RAPD markers fiom jack pine populations using primer P.8 ............ 50
Figure I 1 . RAPD markers in jack pine populations using primer P.9 . . . . . . . . . . . . . 51
General introduction
The Sudbury region is known for the rnining and smelting of high sulphide ores containing
nickel, copper, iron and precious metals. Early practice of open roast yard rnining, followed
by the construction of smelters, released large quantities of sulphur dioxide and metai
particulates to the atmosphere resulting in elevated metal content in soi1 and vegetation near
Sudbury smelters (Freedman and Hutchinson, 1980; Hutchinson and Whitby, 1977:
Hutchinson and Whitby, 1974). Years of intense Wigation of more than 100 million tonnes
of suIphur dioxide and tens of thousands of tomes of metal particulates created barren and
semi-barren lands near smelters (Amiro and Courtin, 198 1 ; Struik, 1973). Forest
comrnunities with a 15 km radius of smelters were nearly eliminated.
Plant species near Sudbuy smelters have been characterized mainly as birch and maple
transition communities (Amiro and Courtin, 198 1). Communities are composed essentially
of white birch (Berula papyrzyera), although red maple (Acer rubrurn), large tooth aspens
(Popttlus grandidentata), red oak (Quercus borealis), red pine (Pinus resinosa) and jack
pine (Pinus banksiana) do exist. The colonization of the barren landscapes by metal-tolerant
species like Deschampsia caespitosa and Agrostis gigantea have suggested possible genetic
base tolerance (Winterhalder, 1 995). Previous investigations of the Sudbury ecosystem have
provided information on metal levels in soils and their uptake and accumulation by plants,
but knowledge of genetic effects of plants growing in these contaminated areas is limited.
Recently, differences in the genetic structure of plants growing in contaminated areas have
been reported (Müller-Starck, 1989, 1985; Bergmann and Scholz, 1987; Mejnartowicz.
1983).
Although reduction in industrial emissions during the last 30 years has resulted in significant
improvements to the local ecosystem, continued investigation and monitoring of soil and
vegetation is essential. Random amplified polymorphic DNA (RAPD) analyses ofjack pine
trees were conducted to determine if genetic differences exist between populations growing
in Sudbury contaminated soil and uncontaminated sites. Determination of current levels of
metal content in soil and jack pine needies fiom these sites were also investigated.
Chapter 1: Literature review
The community of Sudbury located in Northern Ontario (46O30' N, 8 1°00' W, 259 m above
mean sea level) has a long history of mining. The discovery of a rich ore body in 1883 durhg
the construction of the transcontinental route of the Canadian Pacific Railway caused an
enormous mhing boom. The deposits, extending dong the 150 km rirn ofthe Sudbury Basin,
contained high sulphide ores of nickel, copper, iron and precious metds. Mthough fur
trading and lumbering existed within the region, Sudbury becarne known for its nch sulphide
ores. Several mining companies within the region soon established themselves, however, the
International Nickel Company (Inco Ltd.) and Falconbridge Lirnited became dominant in
Sudbury.
Early metal extraction fiom sulphide ores involved open roast yard rnining. Large quantities
of ore were piled on beds of corewood, ignited and aliowed to bwn for long periods.
Sulphides within the ore oxidized and were released as sulphur dioxide. Smelting then
followed to separate metals. Although dense sulphur dioxide fimes, emitted fiom roast beds,
mostly at ground level, killed plants and acidified soils, widespread contamination of
Sudbury area soils was caused mainly by smelter fiimes fiom stacks containing high levels
of sulphur dioxide, copper and nickel particles. R o m beds produced Iarge amounts of
sulphur dioxide but only localized metal particdate deposition, resulting in fewer permanent
effects to the surrounding soils (Winterhalder, 1 995).
Investigations over the last 40 years have provided critical information. The combinations
of roast beds and mining smelters interacted to produce dramatic changes to the Sudbury
landscape. Fumigation by area smelters caused the elimination of vegetation and ground
cover near smelters- Erosion caused rich nutrient soil horizons to be washed away
(Lautenbach, 1987). Acidification and metal toxicity of soils caused serious breakdowns in
plant-soi1 relationships (Winterhalder, 1984). Ecological degradation soon followed and
large areas of forest communities near smelters disappeared creating barren and semi-barren
landscapes (Stniik, 1973)-
Early investigations by Limon (1 958) and Gorham and Gordon (1960) revealed few plant
species near smelters. Sensitive species such as lichens and white pine were virtudly absent
(Leblanc and Rao, 1966; Gorham and Gordon, 1960). Amiro and Courtin (1 98 1 ) showed that
white birch was the dominant species within the serni-barren iandscapes. Severe surface soil
contaminations were also noted near smelters (Freedman and Hutchinson, 1 980; Hutchinson
and Whitby, 1977). High levels of copper, nickel and sulphur dioxide were found in airborne
pollutants, rainfall and snow sarnples (Freedman and Hutchinson, 1980; Hutchinson and
Whitby, 1 977; Hutchinson and Whitby, 1974). Extensive ecological investigation and long
term precipitation and air monitoring prograrns were then developed. Early reports indicated
that sulphur dioxide concentration had a direct effect on vegetation and soil, However.
permanent and widespread metal contamination in soil and vegetation was attributed to
smelter fumes emitted fiom stacks containing high metal content and suiphur dioxide
(Winterhdder, 1996; Freedman and Hutchinson, 1980; Hutchinson and Whitby, 1974).
Reports of soil and vegetation sampling indicated higher metal concentrations within
fumigation and pollution zones established by Struik (1 973) and Dreisinger and McGovem
(1 969). Soi1 profile analysis indicated higher metai content in surface soils (Hazlett er al.,
1 983 ; Freedman and Hutchinson, 1980; Rutherford, and Bray, 1 979; Hutchinson and Whitby,
1977). Plant metal accumulations were above nomal levels (Freedman and Hutchuison,
1980; Hutchinson and Wbitby, 1974), however, metal levels declined substantially in soil
and vegetation with increasing distance fiom smelters (Freedman and Hutchinson, 1980;
Whitby et al. 1976; Hutchinson and Whitby, 1974). Plant species richness aiso increased
m-i th distance (Hutchinson and Whitby, 1974).
The effects of mining activities have aiso k e n recognized in other parts of the country and
around the world. in Canada, both Trail (BC) and Wawa (ON) exhibited effects of smeiting
(Archibold, 1978; Gordon and Gorham, 1963). Internationally, the devastations caused by
the copper-nickel smelter of Monchegorsk in Russia have been compared to Sudbury
(Kryuchkov, 1993). Contaminations of local biotaand soils near mining complexes have also
been reported elsewhere (Jordan, 1975; Little and Martin, 1972)-
Much attention has focused on the environmental effects of mining pollution. Although
reports provide information of metai levels in soil and their uptake and accumulation by
plants, knowledge of genetic effects on plants growing in contaminated areas is limited.
Several authors have reported differences in the genetic structure of plants growing in
contarninated areas ( Müller-Starck, 1989,1985; Bergmann and Scholz, 1987; Mejnartowicz.
1 983). Enzymatic studies of Norway spruce revealed genetic differences between groups of
sensitive trees in polluted areas (Bergmann and Scholz, 1987; Scholz and Bergmann, 1 984).
Obsemations of higher heterozygosity in tolerant plants of European beech in Germany
(Müller-Starck, 1985), scots pine in Gemany and Great Britain (Geburek et al., 1987; Farrar
et al., 1977) and trembling aspen and red maple in the United States (Berrang et al., 1986)
have been reported. Mejnartowicz (1983) presented evidence of loss of genes and
heterozygosity in tolerant scots pines.
During the last 10 years, DNA technologies have been used to study plant population
structure and species differentiation. However, many of these new techniques are costiy and
laborious for large numbers of individuals (such as restriction fragment length
polyrnorphisms (RFLP)) or limited in their genomic distribution and level of diversity they
reveal (such as isozyrnes) (Sobral and Honeycutt, 1994). The newly developed polymerase
chôin reaction technique (PCR) is simple and cost efficient. It consists of 3 steps: thermal
denaturation of DNA, annealing of oligonucleotide prhers to template DNA and primer
extension by DNA. polyrnerase with nucleotide triphosphates (Saiki er al., 1985). The
discoveries of thermally stable DNA polymerases (Saiki er al., 1988) and the development
of automated thermal cyclers have allowed DNA amplification to become widely used by
scientists interested in comparing organisrns at the molecular level.
Various methods of PCR such as random amplified polymorphic DNA (RAPD) have
evolved recently. RAPD markers, obtained by PCR amplification of random DNA segments
fiom single arbitrary primers, are based on mismatches in primer binding sites andior
insertioddeletion events resulting in the presence or absence fiom a single locus (Welsh et
a' 1 990, Williams er al. 1990). Genetic maps of Arabidopsis sp. (Reiter et al., I 992),
sugarcanes (Al-Janabi et al., 1 993), soybeans (Williams et al., 1990) and pine species
(Carlson el al., 199 1 ) have been produced using RAPD methodology. RAPD has been
successfûl in plant systematics and population genetic studies (Nkongolo, 1998, Furman et
al. 1997 Gunter et al., 1996, Yeh, et al., 1995; Sweeney and Danneberger, 1995; Van
Coppenolle el al., 1993).
Chapter 2: Metal accumulations in soi1 and jack pine needles
2.1. introduction
Environmental conditions within the Sudbury area have improved considerably during the
last 30 years (Dudka et al., 1995; Gunderman and Hutchinson, 1993; Negusanti and
McIlveen, 1990). Improvements to smelting technology, greater emission dispersal by
smelting stacks, closure of the Coniston smelter and stricter governmental emission
guidelines have reduced both sulphur dioxide and metal particle emissions (Gunn et al. 1 996;
Winterhalder, 1996; Negusanti, 1995). Furthemore, reclamation efforts by the Municipality
of Sudbury and local mining companies have resulted in significant improvements of the
Sudbury landscape. Vascular and nonvascular plants such as conifers, birches and lichens
have re-invaded semi-barren landscapes (Gunn, 1996). However, recolonization of bmen
lands is Iimited to very few species, mostly metal-tolerant (Rauser and Winterhalder. 1985).
Lack of reestablishment and/or stunted growth exhibited by some species are presumably in
response to a combination of unfavorable microclimate conditions and continued soil toxicity
(Courtin, 1 994).
Continued investigation and monitoring of soil and vegetation within the Sudbury region are
essential to understand the recovery. Jack pine populations, consisting mainly of small
groups ranging fiom 30 to 50 individuals, were analyzed for metal content. Soi1 profile
analysis for metal content was also conducted- Sites were selected based on population size.
location and direction in relation to Sudbury smelters.
2.2, Materials and Methods
2.2.1. Sample collection
Samples of soil, jack pine needles and tree cores were collected fiom 8 sites within 15 km
of Inco Ltd- and Falconbrïdge Ltd. smelters during September and October 1995 (Table 1,
Figure 1). Two control sites, each located approximately 100 km northwest and northeast
from Sudbury, were aiso sampled (Table 1). Needles were rinsed with deionized distilled
water, oven dried for 16 h and homogenized. Soi1 sarnples, collected in intervals of 5 cm to
a 15 cm depth, were air dried, lightly ground with a ceramic mortar and pestle and sieved to
2 mm. Tree core samples, collected using an Uicrement borer, were air dried and mounted
on wooden backings. Samples were stored until analysis.
2.2.2 Tree core analysis
Annual rings were cross-dated using the narrow ring pattern method from living trees
(Yamaguchi, 199 1). Tree cores were sanded using grades of sandpaper, fiom 60 to 600 grit.
to clariQ ring structures. Rings were counted backwards microscopically in time from the
outermost ring, labeling each decade ring (e.g. 1990, 1980, etc.). Years of rings that were
noticeably narrower than adjacent rings were recorded and Iabeled as marker nngs. Cores
fiom living trees were aged and cross-dated quickly and efficiently by listing the narrow
rings patterns (marker nngs) present in each core and comparing to other cores for shared
narrow rings patierns.
Table 1 . Latitude and longitude coordinates of sampling sites
Sites Location Latitude Longitude
near Falconbridge smelter
near Falconbridge smelter
near Falconbridge smelter
near Inco smelter
near h c o smelter
near h c o smelter
Inco tailing
Faiconbridge property
Temagarni (control)
Low Water Lake (control)
Figure 1. Location of soi1 and jack pine population sampling sites within the Sudbury region.
2.2.3. Metal analysis
Metal analyses were performed as described by Marr (1979) and Carter (1993) with some
modifications. Samples (0.2 g) of homogenized soi1 in Teflon beakers were completely
digested to clear liquid with 15 ml of reagent T4A (400 ml HF, 40 ml HCIO, 40 ml HCI)
and placed on a hotplate at 120°C for 24 h. An additional 15 ml of reagent T4B (30 ml
HCIO,, 70 ml HCI, 380 ml distilled H,O) was added and let to stand for 24 h at 120°C- Eight
drops of HCl, 1 ml of HNO, and 4 drops of HF were added individually, swirled, cooled for
1 min and let to react for approximately 2-3 min. Fifieen millilitres of distilied H,O was then
added. beakers placed on hotplate for 30 min and volumes brought down to approximately
10 ml. SoIutions were diluted to a final volume of 100 ml with distilled H,O.
Samples (0.5 g) of needles were oven dried at 400 "C and digened using a modified HN0,-
30% H,O, - - procedure (Jones et al., 1 99 1 ). Five millilitres of concentrated HNO, were added
to needle samples in 50 ml centrifuge tubes and subjected to cold digestion for 16 h. Samples
were then heated to 1 10 O C for 1 h on digestion blocks, dlowed to cool to 70°C and 2 ml of
30% H201 was added and maintained at 70°C for 45 min. Samples were cooled and diluted
to a final volume of 10 ml with distilled H20. Al1 solutions were analyzed by ICP-MS for
total metal content.
Soi1 and needle sample digests were analyzed for metal concentrations of Cd, Co, Cu, Fe,
Mn, Ni, Pb and Zn at the Geoscience Laboratories of the Ministry of Northem Development
and Mines, Sudbury, Canada. Dataquality was assessed by digestion and analysis ofcertified
reference material for vegetation NIST 1575 (NBS 1575) and soil (Till 1, CANMET) and
analytical and method duplicates. The QNQC analyzed totaled 25%.
Soil pH was measured by adding 20 ml of distilled H,O to 10 g of air dried soil (c 2 mm)
material. Soil suspension was çtirred intermittently for 30 min and let to stand for 1 h
(Carter, 1993). Using a Fisher Scientific Accumet pH meter 910 rnodel, soil pH was
measured three times, 10 min apart, and averaged.
2.2.4. Statistical analysis
Statistical analyses were performed on soil and needle samples using SPSS-X and SPSS 7.5
for Windows (SPSS Inc., 1996)- For al1 statistical tests, differences were significant at
ps0.05. Kolmogorov-Smimov normality test was performed on soil and needle samples to
determine the degree of normality in data distribution. Both Bartiett-Box F and Cochrane's
C were used to evaiuate the degree of homogeneity of variance between data subsets.
Nonparametric analysis of variance Kniskal-Wallis followed by nonparametric Tukey-type
multiple cornparison (Zarr, 1996) were perfonned on soil and needle samples since data
transformations were unsuccessful to satis@ criteria of normality and homogcneity of
variance. Potential reiationships between metal content in needles were evaluated using the
Speamian rank correlation coefficients. individual soil depth correlation coefficients for
metal content were pooled when no significant differences were determined (Zarr, 1996).
Speaman common correlation coefficients were used to evduate any possible relationships.
2.3. Results and discussion
2.3.1. Tree core dendrochronology
Cores analyses revealed that mean age of populations ranged fiom 28.1 to 55.3 years for
sampling sites located near Sudbury smelters (Table 2). Ages fiom transpIanted trees on h c o
Ltd. tailing and Falconbndge Ltd. property were much younger. Control sites, Temagarni and
Low Water Lake, had mean ages of 37.9 and 81 -6 years respectively. Observation of cores
collected near smelters revealed distinct sensitive ring patterns prominent during the 1960's
and earl y 1 970's (Figure 2). Narrow rings for years 196 1 - 1962, 1 966- 1 967 and 1 97 1 were
observed on most of the sampling cores fiom Sudbury. Previous reports indicated tree ring
widths are influenced by age, geometric conshn t s and environmentai interactions (Cook,
1987). High levels of sulphur dioxide have been shown to cause reduced tree ring widths
(Thompson, 198 1 ; Kelly, 1980; Linzon, 197 1)- This was not investigated in the present study
but high levels of sulphur dioxide have been well docurnented in Sudbury during the 1 960's
and ear!y 1 970's (Potvin and Negusanti, 1995, Chan and Luis, 1 985).
2.3.2. Jack pine needle metal analysis
Reliability of the analytical digestion indicated that results were within acceptable ranges
for certified reference material NIST 1575 (Table 3). Only lead was below certified ranges.
Meta1 concentrations in jack pine needles are shown in table 4. High Ievels of cadmium,
copper, nickel and lead were found in needles fiom the Sudbury region. Concentrations
within the Sudbury area were between 3 and 10 times higher compared to control sites.
Table 2. Age determination of jack pine populations fiom the Sudbury region and uncontaminated sites
Sitea Range of age in years Mean age in years
10 85- 1 O 0 90.6 "Sites I,2,3 = sites located near Falconbridge Ltd. smelter; sites 4,5,6 = sites located near lnco Ltd. smelter; sites 7 = lnco Ltd. tailing; site 8 = site located on Falconbridge Ltd. property; site 9 = Temagarni (control site) and site 10 = Low Water Lake (control site).
Figure 2. Tree cores colIected near Sudbury smelters revealing distinct sensitive ring patterns. Figures A and B represent ring patterns fiom a tree cotlected £iom site 2 and 5 respectively.
Table 3. Analfical results of pine needle reference material (NIST 1575); concentrations are in m g kg", dry wt.
Elements Certified mead Reference rangea Study mean % Deviation
WIST, Washington. bElement should be interpreted with caution-
Table 4: Metai concentrations (*SEM) in jack pine needles (n=100) fiom the Sudbury region and uncontaminated sites; concentrations are in mg kg-', dry wt.
Elements
o . i ) ( 0 i ) (*0.05), (11 31, (* 1 51, (*0.25), ( 0 - 1 (*2-0), Means in columns with common subscripts are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric comp&son ( p a ~ . ~ 5 ) .
"Sites 1,2,3 = sites located around Falconbridge Ltd. smelter; sites 4,5,6 = sites located around Inco Ltd. smelter; site 7 = Inco Ltd- tailing, site 8 = site located on Falconbridge Ltd. property; site 9 = Temagarni and site 10 = Low Water Lake.
Reports of high concentrations of nickel aad copper have k e n observed in plant species
within the Sudbury region (Freedman and Hutchinson, 1980; Hutchinson and Whitby,
1974). However, since copper and nickel requirernents in plant tissue tend to be low, a
dramatic increase in either or both element content may be attributed to pollution (Adriano.
1986). Background levels of copper in gyrnnosperms have been quoted at 15 ppm (1 5 mg kg'
') by Bowens (1966) and 5 to 25 mg kg-' of copper are needed for normal physiological
processes in plants (Adriano, 1986) (Table 5). In this study, copper concentrations in jack
pine needles fiom Sudbury ranged fiom 10.3 to 28.6 mg kg-'. Although significantly different
in cornparison to control sites, values in general were within normal levels found in tissue
(Kabata-Pendias and Pendias, 1992; Jones et al., 1 99 1 ) and comparable, or lower than, those
previously detennined in gymnosperms and angiosperms species located near Sudbury
smelters (Negusanti and McIlveen, 1990; Freedman and Hutchinson. 1980; Hutchinson and
Wlitby. 1977). Only site 5, located north of the Copper Cliff smelter, exceeded Ontario
Ministq of Enviromnent and Energy (OMEE) guidelines of 20 mg kg-' of copper in
vegetative tissue (Table 4). Higher copper concentration results fiom the sites close
proximity to the smelter and the effects of prevailing winds fiom the south and south-west.
In contrast, nickel concentrations in jack pine needles tiom Sudbury are elevated.
Concentrations ranged fiom 28.9 to 50.8 mg kg-' exceeding both typicai background levels
and controI site values 7 to 10 fold. Normal concentration of nickel in plants seldom
exceeds 5 mg kg" (Kabata-Pendias and Pendias, L 992; Bowen, 1966). Concentrations
obsewed in Sudbury fa11 within excessive or toxic levels reported in vegetation and exceeded
Table 5. Meîai range concentrations in jack pine needles from the Sudbury region and uncontaminated sites
Eiements Sudbury Uncontaminated Background Excessive or OMEEc region regions levelsa toxic levelsb upper limits (mg kg-') (mg kg-') (mg kg-') 0% kg-') (mg kg-')
Cd 0.2-0.3 0.05-0.1 0-05-0.2 5-30 d a
Fe 145-342 107-222 w'a d a 500
Zn 1 O .4-20 43 -0-66.4 27- 1 50 100-400 d a
'Levels considered normai in vegetation (Kabata-Pendias and Pendias, 1992; Jones er al., 199 1 ; Bowens, 1966).
bLevels considered toxic or excessive in vegetation (Kabata-Pendias and Pendias, 1992). 'OMEE upper Iimits guidelines for individual etements (Negusanti and Mcllveen, 1990). n/a=not available.
the OMEE upper Limit guidelines of 30 mg kg-' of nickel in plant tissue for nearly al1 sites
(Table S)(Kabata-Pendias and Pendias, 1 992; Negusanti and McIlveen, 1 990).
Particulate emissions have decreased since the early 1970's (Negusanti and McIlveen, 1990).
Negusanti ( I 995) reported that nickel concentration decreased significantly in white birch
during the Iast 20 years. However, comparison of total nickel content in jack pine needles
obtained in this study to previous data h m red pine needle analyses (Beckett and
Negusanti. 1990; Freedman and Hutchinson, 1980) indicate continued elevated levels of
nickel in plant species. Interestingly, Beckett et al. (1995) found that metal content in pine
needles increased with age. This higher concentration indicates continued elevated nickel
deposition surrounding smelters and/or higher nickel availability for plant root uptake from
soi1 as a result of historical deposition.
Significant correlation between copper and nickel concentrations were also observed (r=0.82.
ps 0.05) indicating a uniform deposition pattern (Table 6). Emission studies conducted by
the OMEE revealed that copper and nickel are major elements emitted by Sudbury smelters
(Negusanti and McIlveen, 1990). Chan and Luis (1985) demonstrated that copper and nickel
showed the greatest impact of smelting within the Sudbury region, with lead and cadmium
input being slightly less significant. Cornparison of lead content in needles fiom Sudbury
showed significant differences compared to control sites (Figure 3). Elevated metal
content were between 3 and 10 times higher in Sudbury. However, normal concentration
of 30 mg kg-' of lead tissue was not exceeded at any sites (Table 4) (Negusanti and
Table 6. Spearman correlation coefficients of total metai concentrations in jack pine needles (n= 1 00)
Speannan correlation coefficients
Cd Co Cu Fe Mn Ni Pb Zn
Zn -0.31* -0.43* -0.63* -0.33* 0.10 -0.69* -0.704 1.00 'Correlation is significant at the 0.05 level.
Sudbury sites Contml sites
E3 Pb concentration
Figure 3. Lead concentrations (mg kg-') in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric comparison (pz0.05).
McIlveen, 1990). Iron, considered a major element emitted fiom Sudbury smelters, was
within normal levels of 500 mg kg" of iron in tissue. Concentrations in needles from
Sudbury were only significantly different fiom the Low Water Lake control site (Table 4).
Udike copper, nickel and iron, cadmium, cobalt and manganese are present in considerably
iower arnounts in smelter emissions (Negusanti and McIlveen, 1990). Significant differences
for cadmium in needles fiom Sudbury were found in cornparison to contro1 sites (Table 4).
Although slightly higher than normal levels found in vegetation, cadmium concentrations
were well below toxic levels (Kabata-Pendias and Pendias, 1992). In general, cobalt
concentrations in Sudbury jack pine needles were found only to be significantly different
than the control site of Low Water Lake (Table 4). The Temagarni control site showed
sirnilar concentrations to some sites within the Sudbury area Cobalt concentrations observed
\\ithin the Sudbury region were within normal levels found in tissue (Kabata-Pendias and
Pendias. 1 992). Significant positive correlations between copper and cobalt (r=0.77, p r0.05)
and nickel and cobalt (r=0.74, ps0-05) suggest sarne deposition pattern (Table 6). Unlike
cobalt. some sites located in Sudbury showed elevated levels of manganese in needles.
Values observed exceed typical values found in vegetation (Kabata-Pendias and Pendias.
1992; Bowen, 1979). Elevated levels of manganese were also found at the Temagarni site.
Spatial distribution for metd concentrations in needles indicated a variability arnong sites
in Sudbury. Higher copper and nickel levels were found primarily near Falconbridge Ltd.
Sudbury sites Control sites
El Cu concentration
Figure 4. Copper concentrations (mg kg") in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (&SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (pz0.05).
Sudbury sites Control sites
Iii3 Ni concentration
Figure 5. Nickel concentrations (mg kg") in jack pine needles (n=100) collected from Sudbury and control sites. Means (*SEM) with common notations are not sig nificantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (p10.05).
Sudbury sites Control sites
El Cd concentration
Figure 6. Cadmium concentrations (mg kg") in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (*SEM) with common notations are not significantly different as indicated by Kniskal-Wallis test followed by nonparametric cornparison (pz0.05).
smelter (site 1,2 and 8), near Inco Ltd. smelter (site 5) and Lnco Ltd. tailing (site 7)(Figure
4 and 5). Cadmium concentrations were consistent among dl sampling sites in Sudbury
(Figure 6). Cobalt and lead distribution patterns were similar to nickel and copper. High
metal deposition patterns north of the smelters seem to be influenced by the dominant
regional wind vector fiom the south and south-west.
In contrast to hi& nickel, copper, cadmium and fead fevels, a decreased concentration of zinc
was observed in jack pine needles fiom Sudbury. Zinc, an essential trace element necessary
for numerous enzyme activities, had a concentration range fiom 43 to 66 mg kg' fiom the
Low Water Lake and Temagarni sites respectively (Table 4). Significant differences were
found between sites fiom Sudbury and control sites (Figure 7). Jack pine needle
concentrations fiom Sudbury were 3 to 4 times lower than control sites and below normal
ranges found in vegetation (Kabata-Pendis and Pendias, 19%; Jones et al., 199 1 ).
Significant negative correlations between copper and zinc (F-0.63, psO.OS), nickel and
zinc (r-0.69, ps0.05), and Iead and zinc (r-0.70, psO.05) suggest a possible antagonistic
interaction (Table 6). Zinc foliar deficiency in birch leaves observed near a copper-nickel
smelter in Russia was also suggested as possible antagonistic interaction between copper and
zinc (Kozlov er al. 1 995). Antagonistic interactions between anthropogenic elements copper,
nickel and lead with zinc have k e n reported (Kabata-Pendias and Pendias, 1992; Adriano,
1986).
bcd -7
Sudbury rites Control sites
E l Zn concentration
Figure 7. Zinc concentrations (mg kg-') in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (SEM) with cornmon notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (p~0.05).
2.3.3. Total soil metal analysis
Reliability of the analytical method for total soil digest was acceptable for al1 metals (Table
7 ). Results of soil depth analysis indicated that most sites located near Sudbury smelters had
significantly higher metal concentrations in the first 5 cm of soil compared to the lower soil
depths (Table 8). In general, concentrations o f nickel, copper, lead, cobalt and cadmium were
2 to 10 times higher in the first 5 cm of soil. Sites located dong the dominant wind vector
had the highest surface metal concentrations exceeding background levels and ministry
guidelines. No significant differences in metal concentratims between soil depths were
found in control sites.
Previous soil sampling in Sudbury have concentrated near the closed Coniston smelter
(Rutherford and Bray, 1979; Hutchinson and Urhitby, 1977; Hutchinson and Whitby, 1974).
Results indicated that nickel and copper were the main contaminants of soil. Hi&
concentrations of both metals were found in surface horizons nearthe smelter, decreasing
with both depth and distance (Hutchinson and Whitby, 1977; Hutchinson and Whitby. 1974).
Metd analyses of copper and nickel in this study fiom sites located near Inco and
Falconbridge smelters were 3 to 8 times higher in the first 5 cm of soil compared to lower
soil depths,exceeded ministry guideiines of60 mg kg-' ofcopper or nickel in uncontaminated
soil. Concentrations were also higher in cornparison to the first 5 cm of soil surveys fiom the
Sudbury and Garson regions (Negusanti and Mcllveen, 1990). Extensive sampling of topsoil
(first 20 cm) by Dudka et al. (i935) indicated that only 25% and 38% of 73 locations in
Sudbury had concentration of copper and nickel be tow rninisûy guidelines. Strong positive
Table 7: Anaiytical results o f soi1 reference materiai (CCRMP-Till 1); concentrations are in mg kg-', dry wt unless othenvise noted
EIement Certified mean' Study means % R.S.Db % Deviation (n=2)
Cd d a 0.25 6.8 nia
Zn 98 89 5 9 'CANMET, Natural Resources Canada, Ottawa, bReiative standard deviation. n/a=not available.
Table 8: Total mean metal concentrations (*SEM) in soi1 (n=9) from the Sudbury region aiid uncoiitaminated sites; concentrations in mg kg". dry wt. ---
Sampling sites"
Elcm- Dcpth I 2 3 4 5 6 7 8 9 10 ents (cm)
correlation between copper and nickel (r=0.82, p s 0.05) fUrther acknowledges wide spread
copper and nickel deposition near smelters (Table 9).
Spatial distribution indicated that metal concentrations were quite variable in Sudbury and
probably reflected contamination fiom smelters in the past. Significant differences between
soi1 depths for cadmium, cobalt, manganese and lead were found primarily at sites 2, 3
(near Falconbridge Ltd. smelter) and 5,6 (near Inco Ltd- smeiter). Furthemore, both copper
and nickel showed the highest concentrations in these sites. Al1 metal concentrations from
the upper soil profiles for these sarnpling sites exceeded OMEE guidelines and/or
background levels (Negusanti and McIlveen, 1990; Spiers et al., 1989; McKeague et al..
1979) (Table 10). Manganese concentration exceeded background levels o d y at site 5 (near
Inco Ltd. smelter). High metal levels in these sites are probably a result of metal deposition
fiom adjacent smelters? influence ofthe prevailing winds fiom the south and south-west and
sampling sites close proximity to the smelters.
In contrast, zinc and iron concentrations showed no significant differences between soil
depths for nearly al1 sampling sites. Zinc is ernitted only in small quantities fiom Sudbury
smelters (Negusanti and McIlveen, 1990; Chan and Luis, 1985) and is considered by some
a non pol lutant (Freedman and Hutchinson, 1 980; Hutchinson and Whitby, 1 977). Although
iron is considered a major element emitted fiorn smelters, concentrations observed were
comparable to previous studies (Hutchinson and Whitby, 1977; Hutchinson and Whitby.
1974). Most concentrations were within normal guidelines of 3.5 % Fe in soil established
Table 9. Spearman common correlation coefficients of total metal concentrations in soi1 samples (n=30)
- --
Spearman common correlation coefficients
Cd Co Cu Fe' Mn Ni Pb Zn
Cd
Co
Cu
Fe"
M n
Ni
Pb
Zn 'Correlation is significant at the 0.05 level. "iron concentration in mg kg" dry weight was used for soi1 correlation. n/a=not available.
Table 10. Total metal concentrations in 0-5 cm soil profiles fiom the Sudbury region and uncontaminated sites
Metals Sudbury Inco Falconbridge Uncontaminated Background OMEEb reg ion tailing P r O P e q regions levels' UPPer
(mg kg-9 (mg kg-') (mg 43-9 (mg kg-') (mg kg-') limits (mg kg'')
Cd 0.09-1 -21 0.26 0.27 O- 1 8-0.22 0.40- 1.1 d a
Zn 23-60 38 80 22-56 40- 125 nia "Normal levels in soil (Kabata-Pendias and Pendias, 1992; McKeague et al., 1979). b O ~ E ~ upper 1 imits guidel ines for individual elements (Negusanti and Mc1 lveen, 1 990). 'Fe concentrations are expressed in percentage (Fe %).
by the OMEE, aithough variability and naturaily high concentrations of uon in Sudbury area
soils have been shown in the past (Negusanti and McIlveen, 1990; Rutherford and Bray.
1979; Hutchinson and Whitby, 1977).
Metal soil analyses from inco tailing and Faiconbndge property sites indicated high
concentrations of cobalt, copper, lead and nickel. High levels of iron and manganese were
also observed at Faiconbridge Ltd.. Most soil metal concentrations exceeded both OMEE
guidelines and/or background levels. Since h c o tailing is a mixture of ore and sludge
containing iron-copper-nickel sulphides (Bouillon, 1995; Heale, 1993, high levels of these
metals were expected. High metal concentrations observed at the Falconbridge property site
were much lower than previous reports of soi1 metal analyses near the Coniston smelter
(Hutchinson and Whitby, 1977; Hutchinson, Whitby, 1974), probably a reflection of vastly
improved emission management at the new Falconbridge cornplex.
Except nickel and cadmium fiom the Inco tailing, soi1 profile analyses indicated no
significant differences in metal content between soil depths from sites inco tailing and
Falconbridge property (Table 8). Results contradict soil analyses Iiom sites near Sudbury
smelters which showed a decrease in metal content with depth. Lower soil depth analyses
from sites near Sudbury smelters revealed no significant differences for any metals
suggesting that most metal deposition is within the first 5 cm of soil, relatively immobile
within the pedon, and remains locaiized. Since Inco tailing is a combination of waste rock
and sludge mixed and pilled in large disposal areas and the site on Falconbridge property is
located next to the srnelter, consistent high levels of metals distributed throughout the soil
profile would be expected.
2.3.4 Soil pH
Soil pH values in Sudbury ranged fiom 3.8 to 4.6. Soil depth analyses indicated lower pH
values in the first 5 cm of soil increasing with depth. Although soil pH in the past have been
low (Negusanti and Mcnveen, 1990; Freedman and Hutchinson, I980), only site 2 and 3.
each located near Falconbrïdge Ltd. smelter, had a pH lower than 4.0. Lower than normal
pH values (pH Iower than 4.4) were found pnrnarily in the first 5 cm of soil within the
Sudbury region. In contrast, pH values increased with depth within normal ranges suggesting
sulfates are localized primarily withh the upper soil profiles. Significant differences in pH
values were found only at site 2, 3 and 8. (Figure 8). Control sites showed no significant
differences in pH values between soil depths. Values for Temagarni were within normal
range, while Low Water Lake had Iower pH values. inco tailing and Falconbndge property
had higher pH values because of the rehabilitation and liming of sites.
Sudbury rites Control sites
HO-5 cm depth 63 6-10 cm depth Ei11-15 cm depth
Figure 8. pH values of soi1 depths from Sudbury and uncontaminated sites. pH values w lh wmmon notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (pr0.05).
Chapter 3: RAPD characterization of jack pine populations from the Sudbury region and uncontaminated sites
3.1. Introduction
Environmental studies of rnining pollution in Sudbury have focused in the past on the effects
of fùmigation and metal deposition. Reports provide information of landscape degradation,
so il toxicity , acidification and plant metal accumulation. It has been suggested recently that
anthropogenic stresses may affect the genetic structure of plant populations. Several authors
have reported differences in the genetic structure of plants growing in contaminated areas
(Müller-Starck, 1989,1985; Bergrnann and Scholz, 1987; Mejnartowicz, 1983). Different
responses to air pollution exposure have been observed for many forest trees species
indicating genetic differences among individuals (Bergmann and Scholz, 1989;
Mejnartowicz, 1983).
Metal analysis results presented in this study demonstrate that metal accumulation in soi1 and
plants growing in Sudbury is still an important problem. Resuits indicate that jack pine
populations from the Sudbury region continue to growth in elevated metal contaminated
soils. High metal concentrations are toxic to p lam and may impose selective pressure on
plant cornmunities. Since anthropogenic stresses have k e n implicated in large-scale forest
declines around the world, selection pressures may result in considerable changes to plant
community structure living in polluted environrnents. Lost of rare alleles, lower
heterozygosity and directional selection have been concerns of plant populations subjected
to air pollution (Bergmann and Scholz, 1989).
However, studies on the genetic structure of tree populations growing in environments
polluted by metal ernissions are lacking. Previous investigations of the Sudbury ecosystem
have provided information on metal levels in soi1 and their uptake and accumulation by
plants, but knowledge of genetic effects of plants growing in these contarninated areas is
lirnited. Studies of metal-tolerant gras species such as red top (Agrostis gigmtea), tickle
grass (Agrosris scabra) and tufied hair grass (Deschampsia cespifosa) colonizing the barren
and semi-barren landscapes of Sudbury have suggested enhanced metai tolerance
(Archambault and Winterhalder, 1995; Cox and Hutchinson, 1979; Hogan and Rauser,
1979). Plant colonization of metal contaminated soils around Sudbury area mines suggest
possible different genetic makeup (Winterhalder, 1 996). In the present study, jack pine
populations fiom Sudbury and non contarninated areas were analyzed by random arnplified
polymorphic DNA to determine any variation in ailelic frequency. The usefùiness of this
technique to differentiate pine species was also investigated.
3.2. Materiah and Methods
3.2.1. Jack pine seedling germination
Approximately 50 jack pine cones fiom the region of Sudbury were collected randomly fiom
jack pine trees at five sites (1,3,5,6,7) during September and October, 1995. Cones fiom
Temagarni (controi site) were also selected. Seeds were extracted, dewinged and bulked
together by the Ontario Ministry of Naturai Resource in Angus, Ontario as per normal
procedures for commercial lots. Seeds were placed in cIear polycarbonated "Petawawa
germination boxes" containing wet "Kimpak" celluIose paper and kept in a germinator at
25°C. Twelve day old seedlings were collected, roots and seed debris discarded, weighed,
fiozen in liquid nitrogen and stored at -80°C until DNA extractions.
3.2.2. DNA extraction
Jack pine DNA was extracted followïng a procedure described by Nkongolo er a[. (1 998).
Two grarns of eesh pine seedlings were finely ground in liquid nitrogen and transferred to
a 45 ml centri fùge tube. Twenty millilitres of 2X CTAB extraction buffer (2X buffer: 1.4 M
NaCl. 100 mM Tris-HC1 (pH 8.0), 20 m M EDTA, 2% hexadecyltrimethylarnmoniurn
bromide w/v) was added to the tube and the mixture was incubated at 60°C for 1 h.
Following incubation, 1 5 ml of chloroform:octanol(24: 1) was added, mixed and centrifùged
(1 3000 rpm, 15 min, 25°C). The aqueous phase was transferred to a fiesh centrifuge tube and
nucleic acids were precipitated by addition of equal volume of isopropanol. The nucleic acid
pellet was collected by centrifugation (6500 rpm, 3 min, 25°C) and resuspended in 10 ml of
TE buffer (10 m M Tns-HC1 (pH 8.0), ImM EDTA).
A second chloroform:octanol purification was perfomed followed by centrifugation ( 1 3 000
rpm, 5 min, 25°C) and aqueous phase was transferred to fresh tubes. Nucleic acids were
reprecipitated with 1 ml of 7.5 ammonium acetate, followed by 10 ml of cold ethanol
rnixing. The pellet was collected by centrifiigation (6 500 rpm, 3 min, 25°C)- drained, then
redissolved in 10 ml of 200 mM ammonium acetate, and a second ethanol precipitation was
performed. After the final centrifùgation (1 3 000 rpm, 2 min, 25°C) the pe!let was drained,
dried bnefly under vacuum and resuspended in 2 ml of TE buffer. DNA purity and
concentrations were estimated using a spectrophotometer.
3.2.3. Amplification of RAPD markers
Total DNA prepared from d l jack pine samples were used for PCR reactions. in a 25 pl
volume. approximately 200 ng plant DNA, 1.0 pM primers and 200 pM each of dATP.
dCTP, dGTP, dTTP were mixed with 10X PCR reaction buffer (Perkin Elmer) and
0.625unitd25 pl of AmpliTaqa DNA polymerase (Perkin Elmer). Samples were amplified
in a Perkin Elmer DNA thermal cycler model. After an initial denaturation step at 95°C for
5 min and a hot start step at 85°C for 2 min, 42 cycles consisting of 60 s denaturation at 95°C.
120 s annealing at 55°C and 60 s extension at 72°C were perfonned pnor to a final extension
of 5 min at 72°C. DNA amplifications were then cooled to 4°C. Analyses of RAPD
amplifications were preforrned on 1 .O% Gibco agarose gels in TAE buffer using a mixture
of 7 pl of PCR reaction and 5 pl of gel loading b a e r (Maniatis er al. 1989) and
electrophoresis was performed at 44 V for 2.5 h. Gels were stained in 0.5 p g h l ethidiurn
bromide solution and photographed in ultraviolet light. Eleven oligonucleotide primers
synthesized by the University of Calgary, University Core DNA described in table 12 were
used for the study. Primen were selected based on their ability to ampliS. spruce DNA
(Mcongolo, 1 998).
3.3. Results and Discussion
3.3.1. DNA concentration and purity values
Genomic DNA concentrations of twelve day old jack pine seedlings are s h o w in table 1 1.
DNA concentrations ranged fiom 93 to 333 &nl with an average of 205 ~ g / m l . Mean
DNA purity values varied fiom 1.92 to 2.00 within acceptable ranges of 1.8 to 2.0.
3.3.2. RAPD characterizrition of jack pine popuiations
Six of the eleven primers screened (55%) did not produce any amplifications, two primers
(18%) amplified poorly and three primers (27%) consistently produced sharp and
reproducible RAPD bands (Table 12). A total of 18 W D markers ranging from 220 to
137 1 base pairs (bp) were produced by the 3 pnmers Cigure 9, 10 and 1 1). The level of
polymorphisms within and among different populations was low, only 4 loci (22 %) were
poIyrnorphic.
M D analysis using primer P-2 show no variation in patterns among al1 populations (Figure
9). Four RAPD bands ranging fiom 745 to 1265 bp were produced. Jack pine populations
fiom Northern Ontario and New Brunswick show sirnilar RAPD profiles. However, species-
specific RAPD markers were identified when jack and red pine populations were compared
(Table 1 3). Of the 5 bands produced, 4 fragments were present in both pine species while one
band (962 bp) was specific to red pine.
Polyrnorphic bands were also observed when pnmers P-8 and P-9 were used. Six to eight
Table 1 1 : DNA concentration and pwity values from jack pine seedling samples; ranges within parenthesis
Study sitesa Mean DNA concentration Mean DNA purïty Olg/ml)
1 195 (146 -278) 2.00 (1 -99 - 2-06)
9 157 (93 - 214) 1.93 (1 -88 - 1.99) 'Sites 1 and 3= sites located near Falconbridge Ltd. smelter, sites 5 and 6= sites located near Inco
Ltd. smelter; site 7 = Inco Ltd, taiIing, site 9 = Temagami (control site).
Table 1 2. Number of fragments, polymorphic RAPD markers and their size using jack pine DNA fiom Sudbury and uncontaminated populations
-- -
Primer Nucleotide Number of Fragment size Polymorphic identification sequence fragments range bands
(5' to 3') (range) @PI ACGACGTAGG
CCGCGGTTCC
CCGGCTGGAA
GAGGGCGTGA
GCTCCCCCAC
CGATGGCTTT
TAGCCCGCTT
GTAGACGAGC
GTGCGTCCTC
GTTCTCGTGT
AACACACGAG
Table 13. Mean number of RAPD and species-specifk markers for jack and red pine populations
Primer Nucleotide Mean bands amplified Number of species- identification sequence specific bands
(5' to 3') jack pine red pine jack pine red pine
ACGACGTAGG
CCGCGGTTCC
CCGGCTGGAA
GAGGGCGTGA
GCTCCCCCAC
GTAGACGAGC
GTGCGTCCTC
GTTCTCGTGT
AACACACGAG
Figure 9. RAPD markers fiom jack pine popdations using primer P-2. Numbers represent individuals from different sites. Species-specific marker is shown with an arrow. (Kb=l kb ladder; site 1 (near Falconbridge smelter) =1 to 3; site 3 (near Falconbridge smelter) = 4 and 5; site 5 (near Inco smelter) = 6 to 7; site 6 (near inco smelter) =8 and 9; site 7 (Inco tailing) =10 to 12; site 9 (Temagrni) = 13 to 15; New Bninswick=16 to 17; red pine= 18 and 19, blank=20).
Kb 1 2 3 4 5 6 7 8 9 10 Kbll 12 13 14 15 16 17 181920
Figure 10. RAPD markers fiom jack pine popdations using primer P-8. Numbers represent individuals from different sites. Species-specific markers are shown with an arrow. (Kb=l kb [adder; site 1 (near Falconbridge smelter) =1 to 3; site 5 (near Lnco smelter) = 4 and 5; site 7 (hco tailing) = 6 and 7; site 6 (near Inco smelter) =8 to 10; site 7 (Temagami) = 1 1,12,15; site 3 (near Fdconbridge smelter) = 1 3 and 14; New Brunswick=l6 to 17; red pine= 18 and 19; blank=20)-
Figure 1 1 . RAPD markers fÏom jack pine populations using primer P-9. Nunibers represent individuals from different sites. Species-specific markers are s h o w with an arrow. ( K k l kb ladder; site 1 (near Falconbi-idge smelter) =1 to 3; site 3 (near Faiconbridge smelter) = 4 and 5; site 5 (near Inco smelter) = 6 and 7; site 6 (near Inco smelter) =8 and 9; site 7 (hco tailing) =10 to 12; site 7 (Temagarni) = 13 to 15; New Bninswick=l6 to 17; red pine= 18 and 19; blank=20).
RAPD markers ranging fiom 220- 1 12 1 bp were produced with primer P-8 (Table 1 2). Two
trees, each found at site 1, located near the Garson-Nickel City durnp, had a missing band
of 344 bp. Two other trees, located near Skead (site 3) had an additional band of 1 121 bp.
Primer P-9 produced 4 to 6 RAPD hgments ranging fiom 396 to 1371 bp. Faint bands (71 7
and 1 195 bp) in some individuals were interpreted to be an artifact of weak amplification
rather than a polymorphism at that locus (Figure 1 1 ) . Close examination of the samples
showed extremely faint fragments withïn the background (Figure 1 1). However, tree samples
9 and 16 had either missing or additional bands. The two primers, P-8 and P-9 produced
species-specific markers when jack and red pines were compared (Figure 10 and Figure 1 1 ).
In general, amplification of control samples containing no genomic DNA produced no bands.
In isolated cases, such samples did generate some amplified products. However, none of
these bands corresponded to any jack or red pine RAPD markers. This phenornenon has been
attnbuted to primer rnultimer formation and would likely disappear when template DNA is
added to control samples (Yu et al., 1993).
The characterization of jack pine populations in Sudbury indicate low levels of genetic
diversity arnong populations. This corroborates observations made by Mosseler ef al. (1 992)
studying red pine populations in Newfoundland. These authors demonstrated by RAPD
analysis that red pine exhibited low levels of genetic variability. This contrasts results fiom
several allozyme studies which reported existence of genetic variability in ponderosa pine
(Pinus pondesora), Iodgepole pine (Pinus coniorta var Iarifolia) and jack pine (Pinus
banksiana) populations (Danick and Yeh, 1982; OYMal1ey et a', 1979; Yeh and Layton,
1979). Sirnikir analyses reveaied that the levels of genetic variability were lower in jack and
red pines compared to other pine species (Mosseler et al., 199 1 ; Danick and Yeh, 1983).
Although both isozymes and RAPD allow the anaiysis of genetic variability in plant species.
fundamentai differences exist between both methods. Isozyrne analysis reflect alterations
in the DNA sequence through changes in amino acid composition. (Hamrick, 1989). Changes
in amino acid composition will often alter protein charges thereby producing a change in
electrophoretic mobility. These differences in electrophoretic mobility of enzymes provide
an estremely useful method of evaluating levels of variation between individuais and
popuIations on the basis of gene loci coding for specific enzymes (Weeden and Wendel.
1 989). Although isozyme markers have been extremely informative in popuiation genetic
studies, the limited nurnber of isozymes (approximately 30) reflect only a small number of
the genome.
RAPD is a DNA based molecular marker. Genomic DNA is amplified using randomly
constructed oligonucleotides as primers. Unlike isoqmes, RAPD is relatively easy to apply
and the nurnber of loci that can be exarnined is essentiaily unlimited. Since the primers
consist of random sequences and do not discriminate between coding and noncoding regions.
the technique samples the genome more randomly than conventional methods (Lynch and
Milligan, 1994). RAPD results in the amplification of specific portions oftemplate DNA that
binds DNA primers while isozymes involve the differentiation of amino acid sequences
caused by alteration in DNA.
One of the difficulties in studying jack pine populations in Sudbury is the existence of the
small population size (approxùnately 30 to 50 trees). Most of the forest ecosystem within the
Sudbury region have been destroyed by Iogging, erosion and air pollution. Natural ranges of
forest trees are nearly non existent within 1 5 km fiom smelters. Very Little genetic differences
were observed even between populations with 30 year diRerences. Little genetic differences
within different populations studied were expected, but the lack of variation among trees
fiom contarninated and non-contaminated sites was surpnsing. Even jack pine populations
fiom New Brunswick showed similar RAPD patterns in cornparison to those fiom Sudbury.
Further studies using microsatellites (SS) and amplified fragment length polymorphisms
(AFLP) are warranted to confïnn these observations.
Plausible explanation for low genetic variability is a possible bottleneck due to the last
glaciation. The entire area of the present-day distribution ofjack pine is thought to have been
covered by ice during the last glacial stages. Geological and paleobotanical evidences fiom
fossil pollen depositions indicate that jack pine survived glaciation in an extensive refbgiurn
centered on the Appalachian Highlands of eastern North America (Yeatman, 1967). Upon
recession of the Wisconsin icecap, jack pine migration northward is though to have happened
rapidl y (Daubenmire, 1 978).
Evolution of jack pine in North America could have possibly followed the same pattern of
red pine which despite increases in population numbers and mutations, have not produced
much detectable genetic variation (Mosseler et al-, 1992). Fowler and Moms (1977)
attributed lack of genetic diversity in red pine to the occurrence of a genetic bottleneck
during the Holocene giaciation in which red pine migrated southward and survived in an
isolated glacial refugium. Self -p lba t ion may also have contributed to a loss of genetic
variation through inbreeding in smdl populations (Nei et al. 1975). The lack of continuous
forest within the Sudbury ecosystem could possibIy continue to contribute to low levels of
genetic variability between jack pine populations and other pine species since gene flow is
limited to small nurnber of individuds within the same populations.
Despite some limitations, RAPD is an effective tool in studying identity and relatedness
among plant species. Both jack and red pine species were easily differentiated based on their
RAPD fragment profiles. By s c o ~ g the presence and absence of RAPD markers. phenetic
relationships among pine species can easily be accomplished. This could shed light on
problems such as evolution divergences between cIosely related species and possibly help
in the understanding of evolution of species within the genus Pinus. RAPD c m also be useful
in the analysis of gene uitrogession between species. A classical example would be the
characterization of natural hybrids fiom jack and lodgepole pine in Western Canada where
the ranges of the two species overlap.
General conclusions
Aunospheric deposition of metals due to mining has ken, and still is an important
contributor to elevated rnetal levels in soil within the Sudbury region. Although reduction
of atmospheric metal deposition in the last 30 years has shown a decline in metal particdates
in soils fiom the Sudbury area (Ducika et al. 1995; Gundermann and Hutchinson, 1993).
element concentrations observed in this study continue to exceed OMEE upper limit
guidelines for uncontaminated soils. The range of values for these metals indicate a strong
impact of metal deposition fiom Sudbury smelters. Spatial distribution of metal
concentrations indicate that study sites located within dominant wind vector had higher
concentrations of metal contaminants compared to other sites. Sites near Falconbridge and
inco Ltd, smelters show metal increases of 3 to 8 times higher in the first 5 cm of soil. No
significant differences were observed within soil depths for control sites.
With the exception of nickel, reports of metal content above upper limits of normal
concentration for individual elements in needles seem to have decreased relative to recent
historical data. Reduction in particdate emissions have probably contributed to a decrease
in metal deposition on vegetation near smelters. Wind direction seems to have a profound
effect on location of metal depositions within the Sudbury region. Declining industrial
emissions should continue to diminish the anthropogenic effects on the local environment.
Genetic characterization of jack pine populations growing in Sudbury show linle variation
in RAPD profiles. RAPD patterns of jack pine trees fkom the Sudbury area closely resemble
those of control populations fiom Northern Ontario and New Brunswick. niese results
corroborate observations made by Mosseler et al. (1 992) studying red pine populations in
Newfoundland. However, results contrast allozyme studies sho wing genetic variabili ty in
pine species (Danick and Yeh, 1982; O'Malley et al., 1979; Yeh and Layton, 1979). The low
genetic variability in Sudbury is probably not related to pollution but possibly a result of a
bottIeneck occurrence during the last glaciation. Other possibilities include the different
abiiities of the RAPD primers to detect variation or to the limited nwnber of primers used
to characterize pine populations. Further in depth analysis of Sudbury populations is needed
to possibly identiQ variability. The potential of RAPD as a phylogenetic tool for the genus
Pinus was demonstrated. Jack and red pine species were easily differentiated based on their
RAPD profiles.
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Appendices
Appendix 1 : Age determination of jack pine populations -- -
Age determination of tree core samples c r o s y i d
A-5
-approximated age. da= not available. n= narrow ring, vn= very narrow ring.
Age of core
Tree
A- 10
Marker rings shared by most cores: 88,82,80,77,71 vn, 69,66vn, 62.60 Sensitive ring pattern shared by most cores: 77-78,73-74,64-65
93,92,88,84,82,80,77n, Mn, 71vn, 70,69,66vn, 61,62,60vn
Marker rings
Trees identified A represents trees from site 1 (near Falconbridge srnefter). Core samples were collected in October 1995.
93.9 1,88,86,85,83, 82,8 1,78,76vn, 7 1 vn, 69vn, 68,66vn, 63,62,60,59,57vn
1957
Last ring year
1954
Sensitive ring pattern
77-78, 64-65, 62-63
38
74-75, 70-7 1 , 64-65, 6 1 -62
41
Appendix 1 : continue
Age determination of tree core samples
8-8 1 92-90, 88, 82,79,77, 7 1,70,67vn, 65,6 ln, 1 1943 170-71.61-62 60,57,54,5 1 n, 49 1 52
using c r o s s - d a t i n n d
Core samples were collected in October 1995.
Age of core
B-10
n= narrow ring, vn= very narrow ring.
Sensitive ring pattern
Marker rings shared by most cores: 92,88,82,71,67 (vn or n), 6 ln, 57 Sensitive ring pattern shared by rnost cores: 70-7 1.66-67,6 1-62
Last ring Y-
Tree
Trees identified B represents trees fiom site 2 (near Falconbridge smeher).
92,9 1, 89, 85,82,80vn, 78, 77,74,7 1,68, 67-6 1,60,58
Marker rings
1953 6 1-62 42
Appendix 1 : continue
Age determination of tree core samples
Marker rings shared by most cores: 90,87,82n, 71 n, 67,62 (vn or n) Sensitive ring pattern shared by most cores: 70-7 1,66-67,6 1-62
7n, 64vn,63vn, 62n, 6 ln
C-5
C-6
C-7
C-8
C-9
C-10
Trees identified C represents trees fiom site 5 (near inco srnelter). Core samples were collected in October 1995. 'approximated age. n= narrow ring, vn= very narrow ring.
68n ,67,64,62,6 1,60,57
91.87, 86,82,79,74, 7Sn, 67n, 63,62n, 6 1 n, 57, 56n, 54vn,
93,9 In, 90,87,83n, 82n,79,77,76,74,71, 65.63
94,90n, 89n, 87,83, 82,72,71,67n, 65, 6Zvn, 60,58,57vn, 5611,
93n, 9 1,87,82n, 78,76,74,7 1 n. 70,67,62, 57,56n,54n
90,89,87vn, 85,84vn, 82vn,77,75n, 72, 69,67,64,62,58,56,55
91,88,87,83,81,78,75,71,68,67,64,62, 61
1952
1961'
1953
1950
1951
1957
76-77, 66-67, 61-62
?a-7 1
70-7 1, 67-68, 6 1-62
7 1-72, 6 1-62. 56-57
70-7 1, 66-67, 6 1-62
7 1-72, 67-68, 6 1-62
43
34'
42
45
44
3 8
Appendix 1 : continue
Age determination of tree core samples usin~ross-da!
Tree Marker rings
ig method
Last ring Sensitive ring Year pattern
Age of core
D- 1 9 1,86n, 8Svn, 8211-8 1 vn, 79,78vn, 74n, 7 ln, 68n, 66n, 64,60,57,53,5 1 vn, 4 8 ~ 1 , 45,44,43
0-7 1 9 ln, 89,88,86,84,8 1,78,76,74,73,70, 68n,66n
D-IO 1 92n,91n,90un,88.84,81,80n,77.75, 72.70
Core samples were collected in October 1995. n= narrow ring, vn= very nmow ring.
Marker rings shared by most cores: 9 1 (vn or n), 84 (vn or n), 78,76vn, 68,66,48vn Sensitive ring pattern shared by most cores: 70-71.66-67,60-6 1
Appendix 1 : continue
Age determination of tree core samples
94,93,92,88,85,82n, 8On,77,67n, 65n,60 1957 66-6 7 1 38 Trees identified E represents trees fiom site 3 (near Falconbridge smelter).
- -
E-3
4
E-5
E-6
Core samples were collected in October 1995. n= narrow ring, vn= very narrow ring.
-
1955
1965
- - - ---
94,93,9 1,85, 82n780,78n, 77, 69,67,62 6 1 n, 56n
93,9 ln, 90n, 86,82n,77n,76 72,7 1.67n
Marker rings shared by most cores: 82n,80,77,67(vn or n) Sensitive ring panem shared by most cores: 7 1-72,6647
93-9 ln, 87,85,82n, 80n
93n. 89n, 88n, 86,82,77n, 74n, 69n, 68n,67vn, 6 1,60vn, 57n, 5 In, 42n,38,35vn, 3 1.28n
78-79, 66-67,
6 1 -6243
77-78, 7 1 -72
40
30
1978
1920
- 61-62-63,49-
50
17
75
Appendix 1 : continue
Age detemination of tree core samples -
u-oss-dath]
Marker rings
metbod
Last rimg Sensitive ring Age of Ymr pattern core
1932 66-67 63
Tree
93n, 89,87,83vn, 8 1 vn, 79,75,71,69,68vn, 67vn, 63vn,58,56,52,49,44,38vn, 3 4 , 3 2
66-67, 63-64, 54-55
er). Trees iden ied F represents tr& fiorn site 6 (near Inco sm~ Core samples were collected in October 1995. 'approximated age. n= narrow ring, vn= very n m w ring. Marker rings shared by most cores: 88,82,79,77,68,48,44 Sensitive ring pattern shared by most cores: 66-67,63-64
Appendix 1 : continue
Age determination of tree core samples bod 1
u s cross-dating m4 !tl
m
. I
1 997 Trees identified T represents trees fiom Low Water Lake area (control site). Core sarn ples were colIected in October 1995. n= narrow ring, vn= very nanow ring.
Tree Age of core
Marker rings Lln M g 1 Sensitive ring Year pattern
94,92n,86n,83,80n, 78vn. 75th 74n, 66n,65vnT 64 vn, 60vn,58vn, 54n,48vn, 44,40vn 3 9 ~ 3 6 , 28,24, 12n, 8n
- -
T- 7 94,92n, 89,84,83,78,72n, 67n, 63n, 56,55,54, 50,48,47,44, 40,36,34,29,28,26,25,24, Zln, 18, 14, 13vn
T-8 92n,86,85,78,77,74n, 67,65,64m, 63vn,57, 56,54n,53n, 46n,44vn, 40,36n, 33,3 1 vn, 2811, 26,22, 12vn
94,92,90,88n, 86, 84,82,81,79,77,76,74,72, 70, 65,64, don, 58,57,56n, 54,53,51,49,45, 44,42,4Ivn,38,36,31vn,28,27,25n, 17, 13, IO, 894, 1
---
93,91,88,86,84,81,77,76,75,74vn, 72vn, 67vn,65vn, 64,63 vn, 61vn,60vn, 57vn,S6vn, 53, 50n,43n, 38n,31,30, 28,25, 19, 18, 12, 11,
Marker rings shared by most cores: 92n, 86n, 78(vn or n), 64(vn or n), 63n, S4(vn or n), 44vn,40 (vn or n), 13
Sensitive ring pattem shared by most cores: 65-75,3545
Appendix 1 : continue
Age determination of tree core samples using cross-dating method
I 1 1 I Tree 1 Marker rings 1 Last ring 1 Sensitive ring ( Ageof
1 1 ~ e a r 1 pattern 1 core
1 46vn, 44 1 1 1 Trees identified R represents trees fiom Ternagarni area (control site). Core sarnples were collected in October 1995.- 'approxirnated age. n= narrow ring, vn= very narrow ring.
Marker rings shared by most cores: 88n, 82(vn or n ), 66(n or vn), 63,58,47 Sensitive ring pattern shared by most cores: no distinct pattern
Appendix 1 : continue
Age detemination of tree core samples
-
pattern
- --
X-IO 93n, 90,87, 85 1 1984 5ed X represents nees fiom site 7 (Inco tailing). Trees ideni
Core samples were collected in October 1995. 'approsimated age. n= narrow ring, vn= very narrow ring.
Marker rings shared by most cores: 93n,89,87n, 8511 Sensitive ring pattern shared by most cores: no distinct pattern
Appendix 1 : continue
Age determination of tree core samples
Y -9 9 I vn, 89n,86,85 1982 - Y- 1 0 9 1 vn, 89vn,86,85,84 1980 -
Trees identified Y represents trees fiom site 8 (on FaItonbndge property). Core sarnpIes were collected in October 1995. 'approximated age. n= narrow ring, vn= very narrow ring.
- -
Age of core
Marker rings shared by most cores: 9 I(vn or n), 89(vn or n), 85 Sensitive ring pattern shared by most cores: no distinct pattern
f3 Ni concentration Cu concentration
concentration
Sudbury sites Contml sites
Appendix 2. Nickel, copper and zinc concentrations (mg kg-') in jack pine needles (n=lOO) from pooled sampling sites from Sudbury and control sites. Means (&SEM) with wmmon notations are not significantly different as indicated by Kruskal-Wallis test followed by nonpararnetric cornparison (~10.05).
Eà 0-5 cm soif depth Q 6-1 O cm soi1 depth a11 -1 5 cm soi1 depth
Sudbury sites Control sites
Appendix 3. Copper concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury region and control sites. Means (SEM) with common notations are not significantly different as indicated by Kniskal-Wallis test followed by nonparametric cornparison (pz0.05).
l30-5 cm soi1 depth H6-10 cm soit depth 9 1 1-1 5 cm soi1 depth
Sudbury sites Contml sites
Appendix 4. Nickel concentrations (mg kg") in pooled soi1 samples (n=30) from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametnc cornparison (pz0.05).
OO-5 cm soi! depth H6-10 cm soi1 depth Ei 4 1-1 5 cm soi1 depth
Sudbury sites Control sites
Appendix 5. Cadmium concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by non parametric cornparison (pz0 -05).
Sudbury s b Control sites
no-5 cm soi1 depth 6-10 cm soi1 depth
El 11-15 cm soi1 depth
Appendix 6. Lead concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric comparison (pz0.05).
Sudbury sites Control sites
El O S cm soi1 depth 0 6-10 cm soi1 depth IB 11-1 5 cm soi1 depth
Appendix 7. Zinc concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury and control sites. Means (*SEM) with cornmon notations are not significantly different as indicated by Kniskal-Wallis test followed by nonparametric cornparison (pz0.05).
Appendix 8: DNA concentrations (pg ml-') of jack pine samples
SampIes Site Site Site Site Site Site Site Site Site Site 1 2 3 4 5 6 7 8 9 10
Sites 1,2,3 = sites located near Falconbridge Ltd. smelter; sites 4,5,6 = sites located near Inco Ltd. smelter, sites 7= Inco Ltd. tailing; site 8 = site located on Falconbridge Ltd. property: site 9 = Temagami (control site) and site 10 = Low Water Lake (control site).
Appendix 9: DNA degradation tests of jack pine sarnples. (Kb= 1 kb ladder, site 1= 1 to 7, site 3= 8 to 14, site 5= 15 to 21 and site 6=22 to 28).
Appendix 10: Random amplified polymorphic DNA protocol
Components Volume per reaction Final concentration
Sterile deionized distilled H20: 10 X PCR buffer iI: dATP (IOmM): dCTP (1 OmM): dTTP (1 O m M ) : dGTP ( I OmM): Primer: Template DNA: AmpliTaq@ DNA Polymerase: 25 m M MgCI, Solution:
1X 10 rnM Tris-HCI, pH 8.3,50 mM KCl
200 pM 200 pM 200 pM 200 pM 1-0 gM 200 ng
0.625 units/25 pl 4.0 mM
Final volume: 25 DI
Appendix 1 1 : Formulas and products.
Concentration of DNA (pg ml-') = (Absorbance, - Absorbance 320) X 50 ~ ( g ml-' X dilution
DNA Purity = Absorbame!,, / Absorbance,,,
Products: a) Chloroform Molecular Biology Grade (Fisher Scientific) b) l -0ctanol (Sigma) c) Isopropanol anhydrous (Sigma) d) EDTA (United State Biochemicais) e) Hexadecyl trimethyl ammonium bromide (Sigma) f) Sodium Chloride (Fisher Scientific) g) Agarose Ultra Pure (Gibco BRL) h) 1M Tris/HCI pH 8.0 (United State Biochemicals) i) Ammonium Acetate (Sigma) j) 50X TE (United State Biochernicais) k) Polaroid 667 B and W instant film 1) 1 Kb DNA Ladder (Gibco BRL)
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