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Scientists are using DNA sequencers in efforts to conserve species of Pacific salmon
Sarah E. DeWeerdt

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When the American writer Thomas Wolfe visited the Pacific Northwest in 1938, he wrote that one could "throw a hook into some ordinary-looking creek and pull out a twelve pound salmon." Since then Pacific salmon populations have dwindled due to decades of over-fishing and the destruction of their habitat, although new evidence suggests that some populations may be coming back. Researchers are working hard to save the region's iconic fish, and some are using DNA sequencers to reach that goal.

Top: Chinook salmon

Bottom: Detail of screen capture during DNA analysis. View larger

"Technology and particularly high-throughput genotyping machines have had a major impact on the field of salmon genetics," says Paul Bentzen, of the University of Washington, Seattle. His research team at the Marine Molecular Biotechnology Laboratory (MMBL) uses DNA sequencers to identify genomic differences among both salmon populations and individual fish. Purple, yellow, and orange squares—representing DNA profiles of individual fish—move across computer screens connected to the lab's MegaBACE capillary sequencing machine.

‘Salmon geneticists have been quick to adopt methods developed for human studies.’

"In the world of fish genetics, salmon genetics is at the forefront," Bentzen says. His laboratory is among dozens using DNA-based technologies to develop better captive breeding programs and other strategies for conserving salmon populations at risk of extinction. Five populations of Pacific salmon are listed as endangered, 21 are classified as threatened, and several others may soon be listed.

Research on salmon genetics began in the 1960s, and most studies focused on allozymes, or different forms of the same enzyme. Geneticists assembled large databases detailing allozyme frequencies in different salmon populations. Initially this information was used to help manage salmon fisheries.

Photo by Sarah E. DeWeerdt
Five species of Pacific salmon inhabit the rivers along the West Coast of North America: chinook, pink, coho, sockeye, and chum salmon, as well as the closely related steelhead and cutthroat trout.

But with the decline of salmon populations, these databases would be used to help researchers identify the genetic diversity within fish species. Although whole species of salmon were not being eliminated, explains Robin Waples, of the National Marine Fisheries Service (NMFS) in Seattle, the genetic diversity within species was being lost.

Waples helped define the concept of the Evolutionarily Significant Unit (ESU), which is a genetically and ecologically distinct portion of a species. During the early 1990s, Waples and other NMFS scientists used the allozyme data to divide Pacific salmon species into ESUs and evaluate the status of each. They also used information about the behavior and ecology of different salmon populations. The chinook salmon in Washington's Dungeness River, for example, are part of the Puget Sound Chinook ESU, which is listed as threatened.

Sampling pink salmon on the Dungeness River for captive breeding. Left: a tag being inserted. Right: a fin clip being placed in a vial.

Next, biologists turned their attention to protecting the remaining salmon and restoring depleted populations, a task that requires more detailed information than allozymes could provide. "The power of DNA markers to detect differentiation is typically greater than that exhibited by allozymes," says Jim Shaklee, of the Washington Department of Fish and Wildlife (WDFW). "But for a long time this [DNA research] was impeded by the high cost and low throughput of the available DNA technologies. That has changed in the last four or five years."

Another advantage of DNA studies is that, with the advent of polymerase chain reaction (PCR), they are non-lethal. The development of PCR allowed biologists to amplify sufficient DNA for studies from a snip of a fin; they no longer had to extract allozymes from heart, liver, muscle, and eye tissues. "We've spent the last 10 years developing methods for DNA analysis in salmon," says Paul Moran, a geneticist with NMFS in Seattle. "Now we're able to use that technology to ask the biological questions. So this is an exciting time."

"Salmon geneticists have been quick to adopt methods developed for human studies," says Paul Moran. Much of the work on salmon genetics today involves microsatellites, the short DNA sequences used in fingerprinting techniques in humans. These sequences are repeated at different lengths throughout the genome, and salmon microsatellites might come in dozens of forms. Paul Bentzen and his colleagues have used DNA markers in projects that typify current research on the West Coast.

Photo by Sarah E. DeWeerdt
Incubation room at Dungeness Hatchery. Each tray holds 7,500 coho or chinook salmon fry. The total capacity of the room is 11 million fish.

In one project, Bentzen and graduate student Jeff Olsen teamed up with WDFW biologists to design a pink salmon captive breeding program on the Dungeness River, home to two distinct populations of pink salmon—summer-run and fall-run. The WDFW biologists used captive breeding to boost the numbers of the fall-run population, estimated at fewer than 400 fish when the program began. The two populations look the same to the human eye and 'overlap' in the river for a few weeks in mid-August.

Bentzen and Olsen analyzed seven microsatellites in 171 wild fish that were captured from the river. Using computational tools, the researchers assigned about three-quarters of the fish to one of the two populations. This meant they could select only fish from the fall-run population for the captive breeding program, preserving the unique genetic character of the population.

In a second study, Bentzen, Olsen, and others used microsatellites to produce genetic 'fingerprints' of individual chinook salmon, also from the Dungeness River. The team analyzed 14 microsatellites in 147 fish to determine familial relationships. The researchers also reconstructed DNA profiles of the parents—wild chinook that had spawned the previous fall and that the biologists had never seen—and determined the pedigrees of each family. The technique is similar to human paternity testing, which also makes use of microsatellites.

The fish in this study were collected in the wild as eggs or just-hatched fry for a WDFW captive-breeding program initiated in 1992. At the time, the Dungeness chinook population was estimated at fewer than 150 fish. The WDFW biologists used the genetic information to keep better track of the pedigrees of the fish in their program and minimize inbreeding. "Once you start breeding fish in captivity, you have to manage them genetically," says Bentzen.

Photo by Sarah E. DeWeerdt
Dungeness Hatchery. This tank contains 25,000 young chinook salmon. All the fish are siblings, the offspring of two wild salmon reared in captivity.

Last year, the WDFW biologists found 87 chinook nests on the Dungeness River. This was the largest number in a decade, but not as large as they had hoped for. Even the most ardent hatchery biologist or fish geneticist concedes that genetic management in hatcheries alone won't save wild salmon. What the fish really need is habitat—rivers with plenty of cool, clean water, with a minimum of dams and dikes. "Until the other problems have been fixed, we are trying to keep the fish safe and as genetically wild as possible" says Dick Rogers of the Dungeness Hatchery.

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Bentzen, P. et al. Kinship analysis of Pacific salmon: insights into mating, homing, and timing of reproduction. J Hered (in Press, 2001).

Wakefield, J. Record salmon populations disguise uncertain future. Nature 411, 226 (May 17, 2001).

Olsen, J.B. et al. Microsatellites reveal population identity of individual pink salmon to allow supportive breeding of a population at risk of extinction. Transactions of the American Fisheries Society 129, 232-242 (2000).

Waples. R.S. Evolutionarily significant units and the conservation of biological diversity under the Endangered Species Act. American Fisheries Society Symposium 17, 8-27 (1995).

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