Genetic Analysis of Pacific Salmonids in the Northeast Pacific and the Russian Far East
The Problem
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Aerial
view of the Zhypanova River, about 53 degrees north latitude,
southeastern Kamchatka peninsula, Siberia. This wilderness river
supports large populations of Pacific salmon, resident rainbow
trout, and anadromous char. During a joint U.S./Russian expedition
in Fall, 2002, genetic material was collected from rainbows
and char. |
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
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This
project emphasizes genetic analysis of two anadromous char species.
Shown are an adult Siberian white spotted char, or kundza (Salvelinus
leucomaensis, top), and a pre-spawn Dolly Varden (S.
malma, bottom). |
experimental controls, some crucial
experiments cannot be conducted in the U.S. (outside of Alaska). Kamchatka
populations have much promise in this regard; for example, sympatric resident
rainbow and anadromous steelhead populations unaffected by human barriers
and genetic introgression from fish plants or transport would allow investigation
of the genetic basis of anadromy. The results are expected to produce
a research model applicable to many North American situations where management
suffers from a lack of knowledge regarding genetic relationships.
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
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One
object of the study is to determine if significant genetic variation
exists among different populations of Dolly Varden char that
spawn in different tributaries of the same river system. We
therefore collected juvenile fish from different widely separated
rearing habitats, before they migrated and mingled with other
populations. Shown above is a typical off-channel habitat sampled
by dip net. Below is a juvenile char approximately 10 cm in
length. |
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are selected based on the presence of resident and anadromous forms
of the species of interest, and because they include geographically
representative and distinctive habitat types. Samples will be collected
by gill nets, dip nets (juveniles), and hook and line to obtain material
for genetic analyses. Tissues will be collected from at least 10 individuals
per species (ideally 20) at each location identified for sampling.
Fin clip tissues will be stored in vials of ethanol for shipment to
the WFRC. Standard length/weight measurement, scale collection, otolith
collection, internal organ collection, and reproductive condition
assessment will be undertaken.
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:
- Establishment of an approximately 50 km long river reach as a study
area, with geo-referenced collection locations.
- Collection and preservation of 81 adult S. malma tissue samples
(for genetic investigations) and
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An
adult resident rainbow (pre-spawn female) dissected for
tissue sampling, internal measurements, and spawning condition
assessment. |
otolith sets (for later aging and possible stable isotope analysis),
along with body measurements, sex, and reproductive condition determined
by internal examination.
- Collection and preservation of some 300 additional adult S. malma
tissue samples (for genetic investigations) via non-lethal fin clips
from fish from throughout the mainstem river reach.
- Collection and preservation of 20 juvenile S. malma tissue
samples from each of three distinct juvenile rearing habitats separated
by at least 5 km. These represent fish that have not yet migrated to
sea.
- Collection and preservation of 25 adult S. leucomaensis tissue
samples (for genetic investigations) and otolith sets (for later aging
and possible stable isotope analysis), along with body measurements,
sex and reproductive condition determined by internal examination.
- Collection and preservation of 46 adult O. mykiss tissue
samples (for genetic investigations) and otolith sets and scales (for
later aging and possible stable isotope analysis), along with detailed
meristic body measurement data, samples of four internal tissues for
electrophoresis, sex and reproductive condition determined by internal
examination.
- Collection of non-lethal fin clips, from about 45 O. mykiss
adults from throughout the study reach, for genetic investigations.
- All tissue samples packaged in preservation buffer for transport to
the WFRC Seattle Laboratory by a Russian investigator in Spring, 2003.
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
U.S. Geological Survey
Western Fisheries Research Center
6505 NE 65th St.
Seattle, WA 98115
Email: carl_ostberg@usgs.gov
Phone: 206-526-6282
Fax: 206-526-6654
Publications
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