1. Testing for a signal of selection at olfactory receptor gene-linked markers in coho salmon (Oncorhynchus kisutch) Marc A. Johnson & Michael A. Banks Coastal Oregon Marine Experiment Station, Hatfield Marine Science Center Department of Fisheries and Wildlife, Oregon State University 2030 SE Marine Science Drive Newport, OR 97365 U.S.A. Marc.Johnson@oregonstate.edu Introduction Classical population genetic studies have often focused on estimating demographic parameters such as genetic effective population size (Ne), and migration rates (m). Most models used to estimate these parameters assume neutrality at the loci under consideration, and researchers have accordingly made efforts to employ neutral genetic markers. Within the context of fisheries biology, neutral genetic markers can provide valuable information for the management and conservation of populations. However, as neutral loci, free from selection, they reveal little about the adaptive potential of populations. Natural populations of coho salmon (Oncorhyncus kisutch) spawn in coastal rivers throughout the northern Pacific rim. During spawning migrations, these semelparous, anadromous fish utilize olfactory cues to accurately navigate to and spawn in their natal streams. It is believed that this behavior isolates populations, generates genetic structure and promotes the evolution of locally adaptive traits. Despite this, recent microsatellite studies have shown that only weak structure exists among natural populations of coho salmon along the Oregon coast (Johnson & Banks 2007, Ford et al. 2004), possibly due to straying at time of spawning migration. Under such a scenario, markers linked to genes under selection may prove to carry greater discriminatory power among populations, and provide an assay for adaptive genetic diversity. In this study, we characterize four hatchery coho populations from the state of Oregon, USA, at twelve putatively neutral microsatellite loci and four olfactory receptor gene-linked markers. These populations were originally founded by wild Oregon coho stock, between 20 and 40 years ago. Since that time, managers at these hatcheries have either largely (Cole Rivers, Coos and Cow Creek hatcheries) or exclusively (Nehalem) spawned marked, hatchery coho that have accurately homed back to their hatchery of origin. We test for a signal of this ongoing selection by examining patterns of F ST associated with olfactory receptor gene-linked loci, and comparing them with the overall F ST pattern generated from a panel of putatively neutral microsatellites. This effort forms the first step toward disentangling the genome-wide effects of drift from the locus-specific effects of selection over a suite of genes, presumably responsible for accurate olfactory mediated homing. Results We have characterized the full length coding sequence (BLAST predicted) of seven main olfactory receptor genes from coho salmon We have identified one or more length polymorphisms (micro- and minisatellites) in the upstream sequence flanking each of these genes, and genotyped four hatchery populations of coho salmon at four of these variable markers: - OR2a, OR2d, OR3a and OR4a (Number of alleles = 2, 3, 2, 15, respectively) For all pairwise tests, at least two OR gene-linked markers appear as “outliers” - In all but one test, one or two “neutral” µsat markers also appear as outliers Methods Olfactory receptor (OR) genes amplified from coho salmon genomic DNA with degenerate primers designed with program CODEHOP (Rose et al. 2003) Entire coding and variable lengths of flanking sequences characterized via “forward-then-back-again” primer walking, using BD Biosciences Genome Walker kit and ABI 3730XL DNA Analyzer Fluorescent labeled primers designed to amplify novel length polymorphisms in sequences flanking olfactory receptor genes OR gene-linked markers used to genotype coho from four hatchery populations: Nehalem (n= 43), South Umpqua (n= 83), Coos (n=95) and Rogue (n=87) All populations genotyped at 12 putatively neutral microsatellite loci - Ots206, Ots215, Ots505, Ots519, Ots520, One13, One111, Ots2, Ots3, p53, Ocl8, One1011 Program DETSEL used to detect “outlier” loci, potentially under selection Cole Rivers Hatchery on the Rogue River Nehalem River Hatchery Adult male coho salmon Pacific Ocean Bandon Hatchery raises Coos River stock coho and releases these smolts into that system Rock Creek Hatchery raises South Umpqua River stock coho and releases these smolts into that system Organism and systemGenes and associated markers Evidence for selection At left: Neighbor joining tree of protein sequences for 136 Danio rerio olfactory receptor genes from Alioto & Ngai (2005) and six predicted coho main olfactory receptor genes characterized through this study. Below: Schematic representations of coho olfactory receptor genes and polymorphic sites in flanking sequence. The following markers have been used thus far, and are included in our results: OR4a minisatellite, OR3a microsatellite, OR2a microsatellite, OR2d microsatellite. Tissue samples of adult coho salmon were collected from each of the four hatcheries described below: NehalemCole RiversCoos Cow Creek OR2a, OR2d, OR3aOR2a, OR3aOR2d, OR4a Coos OR2a, OR2d, OR3a Cole Rivers OR2d, OR2a An example of DETSEL (Vitalis et al. 2003) results: Coos x Nehalem California Oregon Washington Nevada Idaho Utah Montana Arizona Pacific Ocean 50 km Envelope defining the 95% high probability region derived through simulation of microsatellite data under a neutral model of divergence Potential “outlier loci” Acknowledgements This research has been funded by the Oregon Watershed Enhancement Board, as well as a Mamie Markham graduate research scholarship awarded to MAJ. Coho salmon tissue samples were graciously provided by the Oregon Department of Fish and Wildlife (ODFW). Many helpful suggestions were provided by members of the MFGL. Literature Cited Alioto T. S., Ngai, J. 2005. The odorant receptor repertoire of teleost fish. BMC Genomics 6, 173 Ford M. J., Teel D., Van Doornik D. M., Kuligowski D., Lawson P. W. 2004. Genetic population structure of central Oregon Coast coho salmon (Oncorhynchus kisutch). Conservation Genetics 5, 797-812. Johnson M. J., Banks M. A. 2007. Genetic structure, migration and patterns of allelic richness among coho salmon (Oncorhynchus kisutch) populations of the Oregon Coast. Canadian Journal of Fisheries and Aquatic Sciences. In press. Rose T., Henikoff J., Henikoff S. 2003. CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Research 31, 3763-3766. Vitalis R., Dawson K., Boursot P., Belkhir K. 2003. DetSel 1.0: A Computer Program to Detect Markers Responding to Selection. Journal of Heredity 94, 429-431. At left: An example output of the program DETSEL (Vitalis et al. 2003), indicating markers OR2a, OR3a and OR2d as potential “outliers”, under selection, for the Coos x Nehalem hatchery populations comparison. Inset at left (shaded table): OR gene-linked loci identified as potential “outliers” for all pairwise population comparisons. Below: Maximum-likelihood tree inferred from putatively neutral microsatellite data for wild and hatchery coho populations of the Rogue, Nehalem, South Umpqua and Coos Rivers. Data for OR gene linked loci not yet available for wild populations. Question of interest: Will a tree inferred from only OR gene-linked loci, potentially under selection, re- structure to form separate hatchery and wild clades? Future Directions Expand panel of “putatively neutral” microsatellites to obtain more representative sample of neutral variation Genotype wild populations, at OR gene-linked markers for comparison with hatchery populations - Wild populations have likely not experienced the same selection pressures as hatchery populations Explore additional methods of neutrality testing