Progress 08/01/08 to 07/31/11
Outputs
Target Audience: There have been many people affected by this research. An undergraduate student worked on the flowering rush project, created a poster and oral presentations for scientific meetings and presented her research. She won awards for her presentations. Also other Native American students were able to see her presentations and her research and realize that this type of work was also available to them. Her work provided actual examples of the concepts that were discussed in molecular biology courses. Collaborations were developed with Dr. Brenda Grewell at University of California, Davis. Also the water milfoil assay has reached the Flathead Lake Biological Station, and is being utilized by Dr. Gordon Luikart for monitoring the lake. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This grant allowed Karoline Lambert to finish graduate school, having completed her analysis and assay development for the water milfoil. It also allowed an undergraduate student at Salish Kootenai College, Amy Stiffarm, to perform high level research. Amy Stiffarm has since graduated with her Bachelor's of Science in Life Sciences from Salish Kootenai College, and she has nearly completed her Master's Degree in Public Health at University of Montana, Missoula, MT. How have the results been disseminated to communities of interest? The results of the water milfoil test were seen by Dr. Gordon Luikart at the University of Montana Biological Station on Flathead Lake. He requested the reagents and information from Dr. Elizabeth Rutledge at Salish Kootenai College, and successfully performed the test in his laboratory. He is currently monitoring for the types of milfoil in Flathead Lake and plans to publish his findings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
With regard to water milfoil, Karoline Lambert, a graduate student in the laboratory of Dr. Cort Anderson at the University of Idaho, Moscow, ID, successfully developed a Single Nucleotide Polymorphism (SNP) allele discrimination assay to distinguish between three common types of water milfoil: Eurasian, Native, and a hybrid between the two. This assay was the subject of Karoline Lambert's dissertation, and she has now graduated with her degree. The assay utilizes two fluorescently labeled probes (TaqManTM probes) and has been also tested and validated in the laboratory of the Cellular and Molecular Biology laboratory at Salish Kootenai College. In addition, this test for the types of water milfoil is being utilized by Dr. Gordon Luikart at the University of Montana Biological Station on Flathead Lake. He is utilizing this test to monitor Flathead Lake for the presence of types of water milfoil as he also monitors for zebra mussels. To determine the ploidy of the flowering rush, the first technique used was a chromosome squash method to spread the chromosomes and to be able to count them. The squash method did work well to prepare the chromosomes from actively dividing root tips, however, it was too difficult to be able to count the chromosomes with our current microscopy. A sample slide of squashed and preserved root tips was sent to the laboratory of Dr. Brenda Grewell at University of California, Davis. There, the technician, Joy Futrell, was able to very precisely take photographs at various depths through cells and to obtain a series of images that represented the entire depth of a cell. Using these photographs she was able to carefully follow each chromosome from end to end, and thus count all of the chromosomes. These results confirmed that the flowering rush sample from Flathead Lake was triploid, containing 39 chromosomes. While the method for chromosome counting was successful, it was extremely cumbersome, requiring very fresh actively dividing root tips, and requiring specialized microscopic equipment not available at Salish Kootenai College. Obtaining the fresh actively dividing root tips was only possible in the early spring, and was not possible by growing flowering rush plants in the laboratory or the greenhouse. Several attempts were made to induce rhizomes to create fresh root tips in the laboratory, but all failed. Because of the difficulty in obtaining fresh root tip tissue, it was decided to focus efforts on developing an allele discrimination assay, similar to that developed for water milfoil by Karoline Lambert. However, since there was no DNA sequence information in the GenBank database, it was necessary for us to sequence portions of the genome of flowering rush to look for a Single Nucleotide Polymorphism (SNP). A fragment of sequence was obtained by an undergraduate student in the laboratory, and it appeared to have an ambiguous sequence at one nucleotide, which could be a SNP. The sequence was confirmed in a repeat experiment to be ambiguous. More efforts were focused with this sequence to verify and clone this region around the SNP. Many attempts were made to clone this fragment into a plasmid in E. coli, and to test clones for each allele. However, the only clones that were obtained were all of one allele. Also a TaqMan allele discrimination assay was designed and tested, but the assay did not work properly, and lacking the proper controls for this assay (there were no other known SNPs for this plant) it was not possible to troubleshoot within the timeline of the grant period.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2010
Citation:
DNA diagnostics for the identification of the native aquatic plant Myriophyllum sibiricum, its invasive congener Myriophyllum spicatum, and their hybrids in the Pacific Northwest, U. S. A. A Thesis
Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science
with a Major in Environmental Science in the College of Graduate Studies University of Idaho By Karoline Lambert June 2010
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Progress 08/01/08 to 07/31/09
Outputs
Dr. Cort Anderson and his student, Karoline Lambert, designed molecular assays to distinguish water milfoil species by DNA sequence variations in the ITS (internal transcribed spacer) region of Myriophyllum nuclear DNA that differ between M. sibiricum (Northern watermilfoil) and the invasive M. spicatum (Eurasian watermilfoil). They designed PCR primers that specifically amplify this DNA fragment, and made unambiguous species identification. To date they have processed ca. 300 watermilfoil samples. These are primarily from the Pacific Northwest (Idaho, Montana, Oregon, Washington), with smaller numbers of samples from Colorado, Minnesota, Wisconsin, Texas, Utah, and Europe. They designed and tested two independent probes for the allelic discrimination assay, targeting two different, species-diagnostic single nucleotide polymorphisms, and they both performed well. If hybridization between species gives rise to fertile hybrids, subsequent introgression of diagnostic alleles would confound this assay. We are currently performing controlled fertilization studies. Flowering Rush (Butomus umbellatus) samples were collected from several locations in the United States, and have been maintained in a large aquarium tank for the last year. To test whether there could be diagnostic variations in the internal transcribed spacer region, DNA was isolated from a triploid plant from Flathead Lake by Dr. Elizabeth Rutledge, and conditions for PCR amplification of the ITS region were optimized. These PCR products have been sent to Dr. Cort Anderson, University of Idaho, co-investigator, for DNA sequencing to look for nucleotide variations in the ITS of the triploid form. The presence of variations will allow creation of a PCR assay to determine ploidy. Ploidy determination by chromosome squash has been attemped by two students and is currently not optimized to provide results. Work is still in progress. Pat Hurley studied the growth of the flowering rush in Flathead Lake. Six points in each of at least three transects were measured five times from 4/11/08 to 7/30/08, and they included leaf height, soil or water temperature, and water depth. Throughout the 109 days of measurement, the leaf height increased with water depth. It is unknown whether the leaf height would have increased at the same rate if the water depth had not increased. Test plots showed that herbicides were not effective for controlling flowering rush. Virgil Dupuis organized and participated in several meetings: Two public education/scoping meetings on the control and management of flowering rush, participation with the development of the State Aquatic Presentations to the Montana Department of Fish Wildlife and Parks on flowering rush in the Flathead Basin, presentation to the Northwest Vertebrate Society on the impacts of flowering rush on the Columbia River Basin, Nuisance Species Plan for Eurasian watermilfoil, Flowering Rush, and curley leaf pond weed. He is coordinating with the other six reservations in Montana to gain their participatioin and input. Participation with the development of the Flathead Basin Aquatic Nuisance Species Plan, that includes milfoil and rush. PRODUCTS: The first year of this grant has produced one graduate student stipend at University of Idaho, Moscow (Karoline Lambert) mentored by Dr. Cort Anderson, and one undergraduate internship (Montina Threefingers) at Salish Kootenai College. A database of DNA sequences was created that includes the ribosomal internal transcribed spacer region from many samples of water milfoil collected from the United States and Europe. A collection of 11 flowering rush samples from Idaho, South Dakota, Washington, and Montana has been maintained over the winter in an aquarium. Virgil Dupuis has created and attended meetings that created awareness of the problems associated with flowering rush and water milfoil, and the problems associated with taking no action. OUTCOMES: Outcomes are the experience of the students, both undergraduate and graduate, in being exposed to both field and laboratory techniques, following a protocol, troubleshooting a protocol, analyzing their results. They are more able to focus on a question, think critically about what they are doing, think about how to do it better, and think about what the data means. They acquired basic laboratory skills that are useful in any laboratory. DISSEMINATION ACTIVITIES: A data packet was assembled and distributed to attendees of a two day meeting concerning Flathead Lake management. Those present were CSKT tribal members, University of Montana scientists, biologists, land managers, and instructors. FUTURE INITIATIVES: With standard molecular laboratory technology, water milfoil species can be distinguished and the ploidy of flowering rush can be determined. This will inform how and what treatments will be applied. There will be continual efforts to educate local residents, managers, tribal members, and recreational boaters about practices to reduce the spread of flowering rush and water milfoil.
Impacts
Hopefully the impacts will be to prevent water milfoil from becoming established in Flathead Lake, and to reduce and control the spread of flowering rush. There will be more informed management of Flathead Lake, especially with regard to controlling the water levels, to reduce flowering rush spread. The impacts will also be greater awareness in the public of the dangers of spreading aquatic invasive weeds through human activites such as boating.
Publications
- No publications reported this period
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