Grantee Research Project Results
2001 Progress Report: Ecosystem Monitoring via Genetic Diversity Surveys of Dandelions using VNTR multi-locus DNA probes
EPA Grant Number: R826602Title: Ecosystem Monitoring via Genetic Diversity Surveys of Dandelions using VNTR multi-locus DNA probes
Investigators: Rogstad, Steven , Keane, Brian
Institution: University of Cincinnati
EPA Project Officer: Hahn, Intaek
Project Period: July 14, 1998 through July 13, 2001 (Extended to July 13, 2003)
Project Period Covered by this Report: July 14, 2001 through July 13, 2002
Project Amount: $291,045
RFA: Ecological Indicators (1998) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Ecological Indicators/Assessment/Restoration
Objective:
New methodologies in molecular genetics may provide novel types of ecological indicators for monitoring the integrity of natural ecosystems and the sustainability of ecosystems that are affected by anthropogenic influences. One type of an ideal ecological indicator would be an organism that grows in a wide variety of habitats that could easily be used to monitor for the presence of mutagens or anthropogenic factors that alter normal population genetics processes. We are investigating dandelions (Taraxacum officinale Weber; Asteraceae) as a potential model ecological indicator organism because: (1) dandelions have an extremely wide ecological amplitude, growing almost worldwide from sea-level to alpine biomes, and from the tropics to north-temperate habitats; (2) dandelions grow rapidly, and populations can be easily manipulated and monitored; (3) it has been documented that a variety of pollutants can be sequestered in dandelion tissues; (4) dandelion seeds are produced asexually rendering the detection of mutations easy; and (5) we have generated data using variable-number-tandem-repeat (VNTR) deoxyribonucleic acid (DNA) probes (used in DNA fingerprinting), which suggest that mutation rates and population genetic diversity parameters can be analyzed successfully with dandelions. The specific hypothesis to be tested is that dandelions can be used as a sensitive ecological indicator species through comparative analyses of VNTR genetic markers since differences in: (1) mutation rates; and/or (2) population genetic structure can be detected between pollution impacted versus non-impacted populations.
Progress Summary:
Twenty-nine study sites in the central United States were selected to span a range of pollution exposures as determined by analyses of particulate matter (PM10), the amount of airborne particulate matter <10 micrometers, or as determined by a known history of intense exposure (four sites). Soils and dandelion tissues from these sites have been analyzed for eight metals (cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn)). When we examined the data across sites, a positive, statistically significant correlation existed between the mean annual PM10 and soil concentration of each metal. This implies that airborne particulate matter is an effective indicator of soil metal contamination. However, only four of the metals (Cr, Mn, Pb, and Zn) exhibited positive, statistically significant correlations between soil and fall leaf concentrations. Furthermore, sampling dandelion tissues collected from eight sites in both spring versus fall revealed that leaf tissue metal concentrations increased for five of the metals over the growing season. These results indicate that the amount of a metal at a site does not always predict the amount sequestered in the dandelion tissues; other factors such as site seasonal climate history, substrate characteristics, and/or dandelion genotypes present at a site possibly interacting with uptake.
We now have analyzed the transmission of 82,715 VNTR genetic markers in over 120 parent-to-clonal offspring arrays (1,258 offspring) from 16 of these sites (from CO to PA) that span a range of metal exposures. Six VNTR probes were used for each individual, resulting in an average of 65.8 markers scored per offspring. Dandelions provide a rapid system for VNTR genetic marker mutation because the detection of mutations is facilitated by the clonal seed production of dandelions (agamospermy), in which all markers in the seed producing plant should also occur in the offspring. Genetic markers that differ in one offspring are therefore easily recognized and scored. Across sites, the average, single-event, parent-offspring, mini-satellite marker transmission rate is 0.0067 (1 mutant marker per 149 markers transmitted), ranging from 0.002 to 0.015, a 7.5-fold difference. The average rate is among the highest rates of mutation found for genetic markers found for plants, and eukaryotes in general.
We did not find a significant correlation between the single-event mutation rate at sites and either PM10 or soil concentrations of any of the metals. However, single-event mutation rates were significantly (P <0.05) and positively correlated to increasing leaf tissue concentrations of four metals (Cr, Fe, Mn, and Ni) across sites (the other metals exhibited non-significant relationships). Multiple regression analysis suggests that a model including three metals (in order of importance to the model: Cr (P = 0.002), Fe (P = 0.02), and Ni (P = 0.005); overall P for model = 0.001) may improve the ability to predict mutation rate relative to leaf metal concentrations in dandelions. Mutations at mini-satellite loci may provide convenient biomarkers by which to assess the mutagen stressor risk present in environments. The global distribution of the common dandelion, along with the taxon's ability to tolerate a wide range of environmental conditions, make this "species" a particularly attractive candidate to evaluate for its value as a biological monitor of environmental mutation stress, using parent-offspring clonal arrays to rapidly survey for differences in population. Minisatellite mutation rates may be feasible for most sexually apomictic organisms. Manuscripts describing this research have been submitted to journals for publication.
Analysis of VNTR marker variation in populations (three large transects, and sampling at two polluted versus two relatively pristine sites) indicates that some dandelion clones are distributed very widely, although clones with narrow distributions (e.g., restricted to a site) also have been found (Rogstad, et al., in press). In related research, we have determined that unique dandelion clones (identified with VNTR DNA fingerprinting) growing at relatively contaminated sites versus at relatively pristine sites differ in productivity characteristics when grown on contaminated versus pristine media. Specifically, when grown on unpolluted growth media, dandelion plants from unpolluted sites are more productive than dandelions from polluted sites. This productivity difference is reversed when both types of plants are grown on unpolluted media. These results demonstrate that dandelion populations do differ at sites due to selection imposed by metal presence or absence (or correlated factors), and that there is a cost to being either metal pollution tolerant or sensitive. These results suggest that, in the search for plants to be used for phytoremediation, not all populations of a species will interact with metals in the same manner. Populations on contaminated soils, or soils with high metal content, should be the best populations from which to sample lines to be used in phytoremediation studies. If one particular species has been reported to be a good metal accumulator, this does not mean that all populations will be similar in this characteristic. A manuscript has been submitted concerning this research.
Furthermore, we are conducting research to determine whether dandelions from polluted sites are metal tolerant because they are metal excluders, whether they sequester and tolerate metals in their tissues, and whether these characteristics differ among metal contamination sensitive versus tolerant individuals. Preliminary results suggest that plants from polluted sites take up larger amounts of metals into their roots and shoots than plants from non-polluted sites, when both are grown on the same growth media polluted with a defined level of metals. These results indicate that metal-tolerant dandelions are not metal excluders, but rather, are metal-sequestering tolerators, with plants from polluted sites sequestering and tolerating higher levels of metals than dandelions from non-polluted sites.
In the last year, we also have completed the probing and scoring for VNTR markers for dandelions from three separate unpolluted and three separate polluted populations. We will soon analyze these data to determine whether the two types of populations differ in population genetics architecture characteristics (degree of clonality, genetic diversity, level of similarity, etc.).
If all of the earlier mentioned findings hold, we will conclude the following: (1) PM10 is a good indicator of the degree of soil contamination for the metals examined; (2) dandelions are not effective biomonitors in the sense that their tissues sequester metals in proportion to the concentration of metals in the soil; (3) dandelions may be good ecosystem biomonitors that they reflect metal bioavailability (i.e., how the plant population at a site is interacting with the metals to sequester them); (4) dandelions appear to be good mutation stressor biomonitors, indicating that as the tissue exposure to four metals, or possibly other related mutagen stressors increase, mutation rates at a site increase; (5) the metals at a site (or related factors) can select for dandelions that differ in response to metal presence; and (6) dandelions from polluted versus unpolluted sites differ in metal uptake to shoot and root characteristics. Over the next year, we also will determine whether dandelion population genetic architecture differs between polluted and unpolluted dandelion populations.
At the conclusion of this project, we expect to make final determinations as to whether VNTR markers in dandelions can be used as sensitive indicators of anthropogenic changes in population genetic diversity, due to either altered mutation rates or stressor-induced selection. If mutation rates or genetic diversity vary in correlation with increasing pollution, dandelions provide an easily utilized biomonitor to survey the ecological integrity and sustainability of a wide range of habitats across multiple spatial scales throughout the world.
Future Activities:
We intend to complete the submission-publication process for the manuscripts that are in preparation (listed above). In the last year, we have completed the probing and scoring for VNTR markers for dandelions from three separate unpolluted and three separate polluted populations. We will analyze these data to determine whether the two types of populations differ in population genetics architecture characteristics (degree of clonality; genetic diversity; level of similarity; etc.). Grant proposals will be submitted to extend this research. A large amount of the variation in our data may be due to the variation in environmental conditions and differing local genotypes in our study area (from the Colorado mountains to the Pennsylvania coast). We are interested in whether increases in mutation rates can be more clearly detected by using the same genotypes (collected from non-polluted sites) transplanted back to the non-polluted site and to local related polluted sites. In addition, preliminary trials will be attempted with dandelion DNA already in hand to investigate the practicality of utilizing allele chemical cleavage mutation analysis and capillary electrophoresis to analyze for mutation rates.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 27 publications | 15 publications in selected types | All 13 journal articles |
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Keane B, Pelikan S, Toth GP, Smith MK, Rogstad SH. Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes. American Journal of Botany 1999;86(9):1226-1238. |
R826602 (1998) R826602 (1999) R826602 (2000) R826602 (2001) R826602 (Final) |
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Keane B, Lim HW, Rogstad SH. Re-probing DNA blots: wet is better than dry storage of uncharged nylon membranes after removing probes. Plant Molecular Biology Reporter 2000;18(1):17-21. |
R826602 (2001) R826602 (Final) |
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Keane B, Collier MH, Shann JR, Rogstad SH. Metal content of dandelion (Taraxacum officinale) leaves in relation to soil contamination and airborne particulate matter. Science of The Total Environment 2001;281(1-3):63-78. |
R826602 (2001) R826602 (Final) |
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Lim HW, Pelikan S, Rogstad SH. Genetic diversity among populations and size classes of buckeyes (Aesculus: Hippocastanaceae) examined with multilocus VNTR probes. Plant Systematics and Evolution 2002;230(3-4):125-141. |
R826602 (1998) R826602 (1999) R826602 (2000) R826602 (2001) R826602 (Final) |
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Rogstad SH, Keane B, Beresh J. Genetic variation across VNTR loci in central North American Taraxacum surveyed at different spatial scales. Plant Ecology 2002;161(1):111-121. |
R826602 (1999) R826602 (2000) R826602 (2001) R826602 (Final) |
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Supplemental Keywords:
air, soil, risk assessment, ecological effects, genetic polymorphisms, population responses, bioavailability, sensitive populations, mutagens, cumulative effects, particulates, integrated assessment, conservation, phytoremediation, bioremediation, environmental chemistry, monitoring, ecology, genetics, measurement methods, midwest, Colorado, CO, Indiana, IN, Kentucky, KY, Ohio, OH, Pennsylvania, PA., RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Ecological Effects - Environmental Exposure & Risk, Environmental Monitoring, Ecology and Ecosystems, Ecological Risk Assessment, Ecological Indicators, ecological exposure, risk assessment, anthropogenic stresses, dandelions, molecular genetics, genetic polymorphism, multiple spatial scales, survey, ecosystem indicators, assessment methods, DNA, genetic diversityRelevant Websites:
http//www.biology.uc.edu/faculty/rog/steve.htm Exit
http://www.enn.com/enn-news-archive/1999/10/102899/cattail_6793.asp Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.