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Fishery Bulletin 102(1) 



appropriate target for our assay. We chose to explore 

 smaller regions of the mitochondrial genome, including the 

 d-loop (Shedlock et al., 1992), a portion of the 16s ribosomal 

 gene (Parker and Kornfield, 1996), and a region spanning 

 the cytochrome oxidase III, t-RNA glycine, and ND3 genes 

 (hereafter, referred to as COIII/ND3) (Domanico and Phil- 

 lips, 1995 ). Significant interspecific variation but not intra- 

 specific variation was observed in the COIII/ND3 region 

 among salmonid species in previous studies, making it a 

 particularly good candidate region for the development of 

 diagnostic markers (Domanico and Phillips, 1995). 



In the first phase of the study, we developed and vali- 

 dated the genetic tools for species identification by using 

 frozen or ethanol-preserved tissues collected from known 

 species and populations. In the second phase, we applied 

 these tools to the identification of bone remains from har- 

 bor seal scats collected at the Umpqua River (Oregon). 

 A number of Pacific salmonid species are present in the 

 Umpqua River but of particular concern were the sea- 

 run cutthroat (Oncorhynchus clarki) that were listed as 

 endangered under the ESA during 1996 (Johnson et al., 

 1999). Here we report the method associated with these 

 two phases of the project. The salmonid bones that were 

 identified genetically were incorporated into a larger study 

 of the harbor seal diet and are reported in a companion 

 paper (Orr et al., 2004). 



Materials and methods 



Salmonid tissue samples of known species have been 

 collected over the past decade by geneticists from the 

 Conservation Biology Molecular Genetics Laboratory 

 (NOAA/NMFS/NWFSC) or generously donated by others 

 (see "Acknowledgments" section) and maintained either 

 frozen at -80°C or preserved in 95% ethanol. Reference 

 populations were chosen to represent the geographic 

 range of chinook salmon (O. tshawytscha), coho salmon 

 (O. kisutch), sockeye salmon (O. nerka). pink salmon (O. 

 gorbuscha), chum salmon (O. keta), steelhead (O. mykiss), 

 coastal cutthroat trout (O. clarki clarki), and Yellowstone 

 cutthroat trout ( O. clarki bouvieri ) ( collection information is 

 listed in Table 1 ). Tissues were extracted with either a stan- 

 dard phenol and chloroform extraction (Sambrook et al., 

 1989) or by using the DNAeasy 96-well tissue kit (Qiagen, 

 Valencia, CA), following the manufacturer's instruction 

 for tissue preparations. PCR primers were either taken 

 directly from the published studies or designed from the 

 reported sequences (Table 2). All primers were cycled with 

 2.5 mM MgCl 2 , 0.8 mM dNTPs, 0.04 ,«M primers, 0.25 units 

 of Taq DNA polymerase (Promega, Madison, WI), 20-40 ng 

 of DNA, and cresol red loading buffer (final concentration 

 2' < sucrose and 0.005% cresol red) for 35-45 cycles of 

 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 

 1 minute. 



A single individual of each salmonid species listed in 

 Table 1 was sequenced for both the 16s rRNA and COIII/ 

 ND3 regions. For DNA sequencing, the PCR products were 

 purified with an Ultrafree MC column (Millipore, Beverly, 

 MA i and resuspended in 20 ,uL of sterile water. The puri- 



fied product (1-10 uL depending on band intensity) was 

 manually sequenced by using the USB ThermoSeque- 

 nase cycle sequencing kit (Cleveland. OH), following the 

 manufacturer's instructions. MACDNASIS (Miraibio Inc., 

 Alameda. CA) and SEQUENCHER (Gene Codes Corp., Ann 

 Arbor. MI) were used for sequence alignment and identifi- 

 cation of diagnostic restriction enzyme cut sites. 



RFLP analysis of the unpurified COIII/ND3 PCR product 

 was performed in the presence of a cresol red loading buf- 

 fer. Restriction digests were incubated for 6 to 12 hours at 

 37°C for Dpn II, Sau 961, Fok I, Ase I, at 50° for Apo I, and 

 at 60°C for Bst NI with the supplied buffers (NEB, Beverly, 

 MA) and 1-5 units of enzyme. Restricted products were 

 electrophoresed in a 47c 3:1 high-resolution and medium- 

 resolution agarose gel (Continental Laboratory Products, 

 San Diego, CA). DNA bands on the agarose gels were 

 visualized with SYBR Gold, following the manufacturer's 

 instructions (Molecular Probes, Eugene, OR). 



Personnel from the National Marine Mammal Laboratory 

 (NMML) collected and processed harbor seal scat samples 

 from the Umpqua River (Orr et al., 2004). NMML research- 

 ers identified bone remains to either family or species level 

 by using morphological characteristics of skeletal remains 

 (Orr et al., 2004). From 39 harbor seal scats, 116 bones were 

 identified morphologically to the genus Oncorhynchus and 

 subjected to DNA analysis for species identification. For a 

 positive DNA extraction control, we simulated digestion 

 by treating coastal cutthroat bones (collected from Cowlitz 

 Trout Hatchery, Winlock, WAi in a mixture of laboratory- 

 grade trypsin (a digestive enzyme), baking soda, and water 

 for 1 to 2 days. These trypsin-treated bones from a coastal 

 cutthroat trout were used as positive DNA extraction and 

 amplification control. 



To prepare samples for DNA extraction, bones were 

 soaked in 107c sodium hypochlorite for 10 minutes to 

 destroy any contaminating DNA that may have adhered 

 to the outside of the bone and were rinsed twice in sterile 

 water. Bones ranged in weight from 0.1 to 105.6 mg and 

 included teeth, vertebrae, gillrakers, radials, and bone 

 fragments (hereafter, all bony parts and teeth will be re- 

 ferred to as "bone"). The bones were decalcified overnight 

 in 0.5M EDTA solution (Hochmeister et al., 1991); fragile 

 or small fragments were not decalcified. The EDTA was 

 removed and the decalcified samples were extracted with 

 the QIAamp tissue extraction kit (Qiagen. Valencia. CA) 

 according to the manufacturer's instructions with the 

 following modifications: 1) samples were proteinase K 

 digested overnight or until completely digested; 2) 10 

 mg/«L yeast t-RNA carrier was added to the extractant 

 before placement on the QIAQuick column; and 3) DNA 

 was eluted in a reduced volume (50-100 «L) of buffer AE. 

 Negative controls containing no tissue were simultane- 

 ously processed to verify that the extraction was free of 

 contaminating DNA. The trypsin-treated coastal cutthroat 

 bones were used as positive extraction and PCR controls. 



Five to ten microliters of the extracted DNA were used 

 in each amplification reaction. Amplification success was 

 determined by electrophoresis through a 27c agarose gel 

 followed by staining with ethidium bromide or the more 

 sensitive SYBR Gold i Molecular Probes). Species identifi- 



