IDENTIFICATION OF POLYMORPHIC MICROSATELLITE LOCI IN NORTH AMERICAN GRASS CARP CTENOPHARYNGODON IDELLA AND APPLICATIONS TO METAPOPULATION DYNAMICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0216767
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007

Performing Department
WILDLIFE & FISHERIES SCIENCES

Non Technical Summary
Grass carp Ctenopharyngodon idella were introduced into the U.S. in 1963 as a biological control for overabundant aquatic plants. Intentional and accidental releases of grass carp, along with dispersals through major river systems, have since created a broad U.S. distribution that now includes 45 states. At moderately high densities, these plant-eating fish are known to affect aquatic ecosystems by reducing vegetation density, altering nutrient cycles, and reducing native plankon-eater abundance. These effects can disrupt entire aquatic environments including changes in the presence or absence of certain fish species and changes in water quality. Thus, the potential for grass carp to reproduce and establish wild populations persists. Grass carp should be evaluated for their potential to escape their designated stocking locations, and invade and colonize new environments. Genetic research into grass carp populations in the Missouri and Mississippi River basins will provide insight into their ability to migrate, interbreed and invade new areas. The presence of grass carp in the Missouri and Mississippi river basins provides an ideal situation for investigating the genetics of invasion biology because of known stocking dates, locations, and genetic origins. Differences in the genetic code at particular regions of the DNA between populations will indicate isolation, whereas similarities between populations will identify potentially interbreeding populations and the extent of migration and range expansion potential. The rate of range expansion from their point of origin as well as the rate of genetic change can be inferred from comparative studies of the genetics and age structure of colonial populations and their distance from source populations. This research will serve as a model for the integration of molecular biology and ecology that is necessary to characterize and address the problems associated with aquatic invasive species. Invasive species, such as grass carp, can cause millions of dollars in damages to aquatic environments and water infrastruture (municipal water facilities, dams, etc.). Understanding their biology with studies such as this one will help managers prevent and mitigate these damages.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
135	0819	1070	25%
135	0819	1080	25%
135	7299	1080	50%

Knowledge Area
135 - Aquatic and Terrestrial Wildlife;

Subject Of Investigation
0819 - Natural fisheries, other; 7299 - Research equipment and methods, general/other;

Field Of Science
1070 - Ecology; 1080 - Genetics;

Keywords

grass carp

ctenopharyngodon idella

microsatellites

metapopulation dynamics

invasive species

missouri river

mississippi river

Goals / Objectives
The three objectives of this project are 1) to demonstrate our ability to accurately detect polymorphic microsatellite loci in North American grass carp, 2) to quantify the number of microsatellites and alleles at each of those microsatellites that can be applied to test the hypotheses in our broader proposal, and 3) quantify the potential divergence within the grass carp metapopulation of the Missouri and Mississippi River basins. This project will generate preliminary data and peer-reviewed publications that will improve our chances of obtaining outside funding for a broader proposal that we intend to submit to the Population and Evolutionary Processes Program in the National Science Foundation's Division of Environmental Biology. The broader proposal will address objectives related to the invasion risk posed by grass carp including 1) their capacity for adaptation (e.g., cold tolerance) to new environmental conditions, 2) their capacity for escape and dispersal, and 3) their capacity for hybridization and gene transfer with other Asian carp. Ultimately, we anticipate that the larger study will enable development of a dynamic demographic model that will provide a predictive framework for the invasion biology of Asian carp.

Project Methods
State and federal agency collaborators throughout the Missouri and Mississippi River basins are currently collecting samples for this project. These samples have or will be obtained from wild individuals basin-wide so that we will generate a more comprehensive analysis of the potential for polymorphism at each microsatellite locus. Additionally, we will request reference samples from long-standing fish producers having access to some of the original grass carp families. Microsatellite analyses will be conducted in the Functional Genomics Core Facility (FGCF) and associated labs in the Northern Plains Biostress Building (SNP) at SDSU. DNA extractions, PCR amplifications, and agarose gel electrophoresis will be conducted in SNP 243, a standard molecular biology lab, in collaboration with D. Auger (Department of Biology and Microbiology, SDSU). Multiplex analysis of the combined amplification products will be accomplished using the ABI 3130xl Genetic Analyzer in the FGCF in SNP 224. For this preliminary work, subsamples derived from selected geographic strata will be genotyped using a microsatellite set comprising the 17 polymorphic SSR markers in grass carp described by Li et al (7). In particular, we will assess the TRAP-derived markers developed for grass carp by Zhang et al (8), the 12 SSR markers found to be highly polymorphic among and within grass carp and other carps by David et al (9), the highly polymorphic orthologous cyprinid microsatellite locus described by Tong et al (10), and other markers identified in related species (11, 12). Following preliminary screening by conventional PCR and fragment separation by agarose gel electrophoresis, a smaller set of polymorphic markers will be analyzed in a larger number of samples representing greater geographic diversity, using the automated multiplex fragment analysis capabilities of the ABI 3130xl. For these analyses, forward primers will be labeled with fluorescent dyes (e.g., the FAM, HEX, NED, ROX set (labeled primers will be synthesized by ABI)) to discriminate among multiplexed amplification products by size and label color. The labeled PCR products will be separated by microcapillary electrophoresis on the ABI 3130xl Genetic Analyzer, using POP7 polymer. Fragment size will be determined in relation to a size standard (600 LIZ, ABI) and analyzed by GeneMapper software (ABI). Population parameters (e.g., deviation from Hardy-Weinberg equilibrium, linkage, allelic variability) will be determined using Genepop and FSTAT. Statistical analyses of geographic distance and genetic divergence will follow procedures outlined by Viard et al. (15).

Progress 10/01/08 to 09/30/10

Outputs
OUTPUTS: We met the 3 objectives of our project: 1) to demonstrate our ability to accurately detect polymorphic microsatellite loci in North American grass carp (Ctenopharyngodon idella), 2) to quantify the number of microsatellites and alleles at each of those microsatellites that can be applied to test the hypotheses in our broader proposal, and 3) to quantify the potential divergence within the grass carp metapopulation of the Missouri and Mississippi River basins. Activities to address these objectives included experiments to detect forty-one published microsatellite markers in grass carp, and we quantified the number and size of alleles for five of those microsatellite markers. Products of this research included formation of a successful collaboration among Drs. Bertrand, Brown, and Auger and a broader network of collaborators in the United States, Hungary, Pakistan, and China to both acquire samples to meet the objectives of this In-house grant and to develop a pre-proposal that was submitted to the NSF PIRE (Partnerships for International Research and Education) program on February 26, 2009. We received positive reviews, but we were not selected for funding. Currently, we are revising our proposal based on reviewer feedback and plan to resubmit our proposal to the NSF PIRE program solicitation in 2011 (the program is only offered biennially). In the interim, our collaborative network is pursuing partial funding through state and federal agencies, and we meet monthly via web conferences and share documents via our wiki (http://pireasiancarp.pbworks.com). Our collaborative work at SDSU included the formal mentorship of an undergraduate, Bobbi Adams, through the Joseph F. Nelson Mentorship Program. Events related to this mentorship included multiple professional society presentations and awards. In February 2009, Bobbi gave a platform presentation on the preliminary results of our study to the Dakota Chapter of the American Fisheries Society in Bismarck, ND, and presented her conclusions to the same society in February 2010 in Spearfish, South Dakota. Ms. Adams traveled to the parent society meeting of the American Fisheries Society in August of 2009 (Nashville, TN) and September of 2010 (Pittsburgh, PA) to present an invited poster on this research during the carp symposia and was also honored to receive a travel award from the Equal Opportunities Section of the American Fisheries Society in 2009. She disseminated her results locally at the Eastern South Dakota Water Conference on November 2, 2009 and SDSU's Undergraduate Research, Scholarship, and Creative Activities Day, April 28, 2010. Dissemination related to this collaboration and mentorship included a summer workshop and a fisheries presentation and research facility tour for visiting students from St. Joseph's Indian School in June of 2009 and 2010. PARTICIPANTS: Dr. Katie Bertrand, Assistant Professor, Department of Wildlife and Fisheries Sciences, South Dakota State University, is the lead principal investigator on the project and administered the daily tasks required to meet project objectives, including mentorship of an undergraduate researcher, Bobbi Adams. Dr. Michael Brown, Professor, Department of Wildlife and Fisheries Sciences, South Dakota State University, is a co-Principal Investigator on the project and provided support to the lead principal investigator. Dr. Donald Auger, Associate Professor, Department of Biology and Microbiology, South Dakota State University, is a collaborator on the project, and provided technical expertise and education in genetic analyses to Bobbi Adams. Bobbi Adams is an undergraduate researcher employed by Dr. Katie Bertrand to perform the genetic analyses for this project. Her mentorship as an undergraduate researcher specifically created a successful training and professional development opportunity on the project. Ms. Adams is currently pursuing a master's degree in the field of fisheries genetics at Washington State University Pullman. Collaborators and partner organizations providing samples for the research included Duane Chapman (United States Geological Survey), Sam Stukel (South Dakota Game, Fish and Parks), Kirk Steffensen (Nebraska Game and Parks Commission), Brandon Eder (Nebraska Game and Parks Commission), Marty Hamel (Nebraska Game and Parks Commission), Van Sterner (Iowa Department of Natural Resources), Bernie Schonhoff (Iowa Department of Natural Resources), Dave Bierman (Iowa Department of Natural Resources), Kirk Hansen (Iowa Department of Natural Resources), Mark Drobish (United States Army Corps of Engineers), Craig Paukert (Kansas State Univeristy Cooperative Research Unit), and Kevin Irons (Illinois Natural History Survey). TARGET AUDIENCES: Bobbi Adams' mentorship as an undergraduate researcher specifically created a successful training and professional development opportunity on the project. Ms. Adams is currently pursuing a master's degree in the field of fisheries genetics at Washington State University Pullman. Outeach related to this collaboration and mentorship included a summer workshop and a fisheries presentation and research facility tour for visiting students from St. Joseph's Indian School in June of 2009 and 2010. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Grass carp are successful invaders outside their native range in China, but little information exists on their biology or ecology in the United States. Knowledge of this species is primarily based on studies conducted within their native range. Our research provides new information about grass carp in the United States and leads to a change in knowledge about species invasions. Our specific objectives for this project were 1) to demonstrate our ability to accurately detect polymorphic microsatellite loci in North American grass carp (Ctenopharyngodon idella), 2) to quantify the number of microsatellites and alleles at each of those microsatellites that can be applied to test the hypotheses in our broader proposal, and 3) quantify the potential divergence within the grass carp metapopulation of the Missouri and Mississippi River basins. We found that the microsatellites characterized in grass carp from their native range also work well in grass carp introduced into North American waters. Additionally, we tested microsatellites developed for closely related species in the family Cyprinidae: common carp (Cyprinus carpio), koi (Cyprinus carpio carpio), and silver carp (Hypophthalmichthys molitrix) for a total of over 31 microsatellites that can be used to assess population structure of grass carps in North America. None of the koi markers were evident in grass carp, but all three of the common carp markers were evident in grass carp. Fourteen of sixteen silver carp markers were evident in grass carp. In the second year of funding, we tested nearly 60 fish at the 17 microsatellites developed specifically for grass carp using the ABI-3130XL capillary sequencer in the Functional Genomics Core Facility at SDSU and found that the North American population is in the early stages of invasion. Among the many positive outcomes of our mentorship of Bobbi Adams (an undergraduate at South Dakota State University), she experienced a change in knowledge and her research produced a change in actions. Ms. Adams learned the skills required to successfully conduct experiments in a genetics laboratory. She applied these skills when she began her graduate study at Washington State University Pullman in Fall 2010. She also helped a new graduate research assistant, Jacob Davis, to learn genetics analysis techniques and develop his skill related to genetics research. Through experimentation, we arrived at an effective method for extracting and purifying DNA from frozen fish fins. We found that traditional phenol-chloroform extraction techniques were not well-suited to this tissue type, and we eventually refined methods from commercially-produced DNA extraction and purification kits to isolate sufficient levels of DNA. This led to a change in actions: we modified our proposed methods for extracting and purifying DNA.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: During the first year of the project we met the first 2 of our 3 objectives: 1) to demonstrate our ability to accurately detect polymorphic microsatellite loci in North American grass carp (Ctenopharyngodon idella) and 2) to quantify the number of microsatellites and alleles at each of those microsatellites that can be applied to test the hypotheses in our broader proposal. Activities to address these objectives included experiments to detect forty-one published microsatellite markers in grass carp, and we quantified the number and size of alleles for 5 of those microsatellite markers. Products of this research included formation of a successful collaboration among Drs. Bertrand, Brown, and Auger and a broader network of collaborators in the United States, Hungary, Pakistan, and China to both acquire samples to meet the objectives of this In-house grant and to develop a pre-proposal that was submitted to the NSF PIRE (Partnerships for International Research and Education) program on February 26, 2009. We received positive reviews, but we were not selected for funding. Currently, we are revising our proposal based on reviewer feedback and plan to resubmit our proposal to the NSF PIRE program solicitation in February of 2011 (the program is only offered biennially). In the interim, our collaborative network, is pursuing partial funding through state and federal agencies, and we meet via web conferences and share documents via our wiki (http://pireasiancarp.pbworks.com). Dr. Bertrand will include preliminary data obtained through this In-House Grant in her NSF CAREER proposal, which will focus on ecological genetics of invasion by Asian carps and be submitted in July 2010. Our collaborative work at SDSU included the formal mentorship of an undergraduate, Bobbi Adams, through the Joseph F. Nelson Mentorship Program. Events related to this mentorship included multiple professional society presentations and awards. In February 2009, Bobbi gave a platform presentation on the preliminary results of our study. Ms. Adams traveled to the parent society meeting of the American Fisheries Society in August of 2009 to present an invited poster on this research during the carp symposium and was also honored to receive a travel award from the Equal Opportunities Section of the American Fisheries Society. She disseminated her results locally at the Eastern South Dakota Water Conference on November 2, 2009, and will present her research again at the annual meeting of the Dakota Chapter of the American Fisheries Society in Spearfish South Dakota in February 2010. Dissemination related to this collaboration and mentorship included a summer workshop and a fisheries presentation and research facility tour for visiting students from St. Joseph's Indian School in June of 2009. PARTICIPANTS: Dr. Katie Bertrand, Assistant Professor, Department of Wildlife and Fisheries Sciences, South Dakota State University, is the lead Principal Investigator on the project and administered the daily tasks required to meet project objectives, including mentorship of an undergraduate researcher, Bobbi Adams. Dr. Michael Brown, Professor, Department of Wildlife and Fisheries Sciences, South Dakota State University, is a co-Principal Investigator on the project and provided support to the lead principal investigator. Dr. Donald Auger, Associate Professor, Department of Biology and Microbiology, South Dakota State University, is a collaborator on the project, and provided technical expertise and education in genetic analyses to Bobbi Adams. Bobbi Adams is an undergraduate researcher employed by Dr. Katie Bertrand to perform the genetic analyses for this project. Her mentorship as an undergraduate researcher specifically created a successful training and professional development opportunity on the project. Ms. Adams plans to pursue a master's degree in the field of fisheries genetics in fall 2010. Collaborators and partner organizations providing samples for the research included Duane Chapman (United States Geological Survey), Sam Stukel (South Dakota Game, Fish and Parks), Kirk Steffensen (Nebraska Game and Parks Commission), Brandon Eder (Nebraska Game and Parks Commission), Marty Hamel (Nebraska Game and Parks Commission), Van Sterner (Iowa Department of Natural Resources), Bernie Schonhoff (Iowa Department of Natural Resources), Dave Bierman (Iowa Department of Natural Resources), Kirk Hansen (Iowa Department of Natural Resources), Mark Drobish (United States Army Corps of Engineers), Craig Paukert (Kansas State Univeristy Cooperative Research Unit), and Kevin Irons (Illinois Natural History Survey). TARGET AUDIENCES: One target audience benefitting from an effort of this project was a group of American Indian high school students, visiting from St. Joseph's High School during June 2009. They participated in a hands-on genetics workshop and a presentation and tour of the fisheries research and education facilities. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Grass carp are successful invaders outside their native range in China, but little information exists on their biology or ecology in the United States. Knowledge of this species is primarily based on studies conducted within their native range. Our research provides new information about grass carp in the United States and leads to a change in knowledge about species invasions. Our specific objectives for this project were 1) to demonstrate our ability to accurately detect polymorphic microsatellite loci in North American grass carp (Ctenopharyngodon idella), 2) to quantify the number of microsatellites and alleles at each of those microsatellites that can be applied to test the hypotheses in our broader proposal, and 3) quantify the potential divergence within the grass carp metapopulation of the Missouri and Mississippi River basins. We were able to detect 31 out of 41 tested microsatellite markers in US grass carp. Among 17 markers developed specifically for grass carp, we were able to detect 14 in fishes collected from US waters. We subsequently tested markers developed for closely related species in the family Cyprinidae: common carp (Cyprinus carpio), koi (Cyprinus carpio carpio), and silver carp (Hypophthalmichthys molitrix). None of the koi markers were evident in grass carp, but all three of the common carp markers were evident in grass carp. Fourteen of sixteen silver carp markers were evident in grass carp. We performed detailed analyses on 5 grass carp-specific markers to explore allelic variation among fish collected from four general geographic regions across the US and from the native range in China. The allele sizes measured in our experiments were within or near the published expected range for the microsatellite marker. We observed allelic variation at the markers Ci1, Ci2, Ci3, Ci4, and Ci5, and these could be useful polymorphic markers for quantifying differences among populations. Among the many positive outcomes of our mentorship of Bobbi Adams (an undergraduate at South Dakota State University), she experienced a change in knowledge and her research produced a change in actions. Ms. Adams learned the skills required to successfully conduct experiments in a genetics laboratory. She will apply these skills when she continues her education as a graduate student in fall 2010. She also helped a new graduate research assistant, Jacob Davis, to learn genetics analysis techniques and develop his skill related to genetics research. Through experimentation, we arrived at an effective method for extracting and purifying DNA from frozen fish fins. We found that traditional phenol-chloroform extraction techniques were not well-suited to this tissue type, and we eventually refined methods from commercially-produced DNA extraction and purification kits to isolate sufficient levels of DNA. This led to a change in actions: we modified our proposed methods for extracting and purifying DNA.

Publications

• No publications reported this period