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The Problem
Kamchatka steelhead (Oncorhynchus mykiss) are included in the Russian Red Book of Rare and Disappearing Species, and in the U.S. Pacific Northwest, anadromous salmonids (Oncorhynchus spp.), bull trout (Salvelinus confluentus), and other char species are of Department of the Interior interest due to declining population status, Endangered Species Act listings, and human use values (e.g., subsistence). The systematics and taxonomy of many species remain unresolved and improved genetic techniques are needed to evaluate faunal diversity and develop greater understanding of population and life history characteristics, evolutionary relationships, and geographic distribution. This information will enhance the scientific foundation for management of these species throughout their ranges. This U.S./Russian collaboration will investigate genetics and life histories of Kamchatka Peninsula rainbow trout and steelhead (O. mykiss), and Dolly Varden, white-spotted, and arctic char (S. malma, S. leucomaensis, and S. alpinus, respectively). Both anadromous and resident forms of these generally occur in Kamchatka rivers that are free from hatchery and watershed development influences, providing research opportunities pertaining to natural patterns in genetic diversity that do not exist in the U.S. Declines in Pacific Northwest salmonids engender numerous genetic questions related to hatchery/wild interactions, effects of transporting genetic material among watersheds, and effects of artificial barriers on gene flow and diversity. Because of the lack of genetically pure wild populations needed as natural
This research was initiated in FY 2000 to determine if microsatellite markers would discriminate Kamchatka steelhead within select Kamchatka watersheds. Contemporary molecular DNA techniques, microsatellite analysis and genomic scanning, are being used to study steelhead and char. The resolving power of these techniques is greater than allozymes and both involve non-lethal sampling methods, and produce analytical results that may be converted into rapid diagnostic systems. Objectives In FY 2003, the existing U.S.-Russian partnership and new relationships will be pursued and expanded to enhance genetic research on Salmonidae of interest. Existing tissue sample inventories in the U.S. and elsewhere will be reviewed and samples acquired as needed to address evolutionary and diversity goals of the research. Specific objectives with respect to Onchorynchus and Salvelinus species are to (1) develop novel molecular markers for genetic stock identification and diversity investigations; (2) develop a diagnostic system to rapidly, economically, and non-lethally screen tissues samples for these genetic markers; (3) assess genetic diversity in relation to life history diversity, stock differentiation, habitat, and geographic distribution; and (4) communicate findings and transfer molecular tools and technologies to Russian and U.S. fisheries managers. Methodology Field and other coordinated sampling activities will place most emphasis on Salvelinus species. This group is geographically widespread, has resident and anadromous forms, and contain species that are listed, or are in need of special protection. Many systematic and taxonomic questions remain in the chars and new methods are needed to quickly evaluate stocks at risk. Sampling in Kamchatka and, more opportunistically, from other portions of the Russian Far East will address conservation objectives of the AREA V international agreement encompassing fisheries research. Similarly, North American and European samples of Salvelinus spp. will be acquired primarily from archived materials and ongoing collection programs. Coordination. During FY 2002, the WFRC contacted several investigators in Alaska, Canada, Russia, and the Pacific Northwest regarding use of archived tissues or with respect to obtaining new genetic material from ongoing programs. During FY 2003, identified sources will be solicited for materials. Field. The WFRC will participate in field work on the Kamchatka Peninsula sampling selected rivers utilizing Russian base camps. Sites
Laboratory. Intra and inter-population genetic differences will be evaluated using PCR-based tools (i.e., apPCR). ApPCR allows for taxonomic/genetic discrimination of organisms lacking sufficient numbers of morphological characters and organisms that express extreme morphological variation but may be closely related. This technique uses short oligonucleotide primers (10 - 20 base pairs) that anneal to complimentary DNA sequences. When sequences, which are complimentary to a specific primer, are located in opposite orientation on separate strands of DNA, a double stranded DNA product may be synthesized by DNA polymerase. The number and size of amplified products is dependent on the frequency and distribution of primer hybridization sites are not possible to predict without extensive characterization of the genome. However, the number of apPCR products generated from individual primers ranges from less than 5 to more than 20 depending on primer composition and the reaction conditions. Therefore, more than one hundred genetic markers may be generated by apPCR from 10-20 oligonucleotide primers. If greater numbers are required, then the number of primers utilized can be increased. Research has demonstrated that by utilizing oligonucleotide primers 15-16 bp in length and composed of simple sequence repeats [eg. (CAG)5, (GACA)4], 80 to 100% of the DNA products generated from apPCR amplification are shared among individuals of the same species for any given primer. Alternatively, individuals from different species share between 0 to 20% of the DNA products generated from these primers. As a result, apPCR band patterns are species specific and may be used for unequivocal taxonomic identification. DNA will be extracted from tissues from 10 samples from each population sampled and compared by apPCR with up to 50 simple sequence repeat (ssn) primers. ApPCR products will be separated by agarose gel electrophoresis and visualized by ethidium bromide staining to identify polymorphic markers within and between populations. A maximum of 75 markers will cloned into the vector pT7blue (Novagen) and subsequently sequenced. Primers will then be constructed based on terminal sequences from each marker and tested for marker specificity by dpPCR. These markers will focus on polymorphic differences between individuals within and between sampled populations. Highlights and Key Findings Planning meetings were held in FY2001 and FY2002 to develop this project. A Russian scientist has visited the Western Fisheries Research Center Seattle Laboratory on two occasions to begin genetic analysis of tissue samples previously collected by the Russians. A Joint U.S.-Russian expidition was undertaken in Fall, 2002 on the Zhypanova River (eastern Kamchatka), in collaboration with the the Russian Academy of Sciences, Moscow State University, and the Wild Salmon Center, (a U.S. non-profit orgnization). The accomplishments include:
Where Are We Headed In 2003 In spring of 2003 a Russian scientist will visit the WFRC Seattle Laboratory, and will bring the genetic material collected during the Fall, 2002 expedition. Russian and U.S. investigators will work together at the Seattle Lab, also in collaboration with the Wild Salmon Center and University of Montana Flathead Lake Biological Station, to continue development of genetic markers to assess genetic diversity and discriminate salmonid life-forms. In collaboration with Russian scientists and the Wild Salmon Center, we will plan for additional field work in 2003 to expand representation of the life stages and behavioral patterns of salmonids in Kamchatka. Project Contact Carl Ostberg Email: carl_ostberg@usgs.gov Publications |
