218 



Fishery Bulletin 102(1) 



Chinook 



Coho 



Sockeye 



Chum 



Pink 



Steelhead 



Cutthroat 



Chinook 



Coho 



Sockeye 



Chum 



Pink 



Steelhead 



Cutthroat 



Chinook 



Coho 



Sockeye 



Chum 



Pink 



Steelhead 



Cutthroat 



Chinook 



Coho 



Sockeye 



Chum 



Pink 



Steelhead 



Cutthroat 



20 40 60 

 GGAGCTTTAGACACCAGGCAGATCACGTCAAACAACCTTGAATTAACAAGTAAAAACGCAGT 

 G 



80 100 120 



GACCCCTAGCCCATATGTCTTTGGTTGGGGCGACCGCGGGGGAAAATTAAGCCCCCATGTGG 



140 160 180 

 ATGGGGGCATGCCCCCACAGCCAAGAGCCACAGCTCTAAGCACCAGAATATCTGACCAAAAA 

 T T...A 



200 220 



TGATCCGGCAAACGCCGATCAACGGACCGAGTTACCCTAG. . . 



Figure 2 



Aligned sequences of a variable portion of the 16s gene for seven species of the genus Oncorhynchus. 

 Sequence identity in relation to the chinook salmon "A" sequence is denoted by dots; nucleotide 

 substitutions are indicated. 



Hatchery) and hatchery stocks descended from the Upper 

 Columbia River spring run (Carson Hatchery) had the "A" 

 (uncut) haplotype at a frequency of 83% and 91%, respec- 

 tively, whereas those from the Lower Columbia River ESU 

 were invariant for the "B" (cut) haplotype. The "B" hap- 

 lotype was also invariant in the other lineages examined 

 (Sacramento River, CA; Puget Sound, WA; and the Fraser 

 River, BC). Despite this Dpn II polymorphism, the haplo- 

 type patterns were still chinook-specific. 



Extractions from the trypsin-treated cutthroat trout 

 bones, used as positive controls, were amplified consis- 

 tently, but of the 116 salmonid bones from harbor seal 

 scats, only 78 (67%) were amplified. Failed samples were 

 repeated several times with all possible primer sets. Be- 

 cause each scat contained multiple bones, we were able 

 to amplify bones representing 35 of the 39 scats (90%). 

 The smallest bone we successfully amplified was a O.'2-mg 

 tooth and the largest was a 21.8-mg vertebra. There did 

 not appear to be a relationship between bone size and DNA 

 extraction success; no significant difference in mean bone 

 size was detected between 32 bones that either amplified 

 or failed (P=0.280; unpaired t-test; SYSTAT 8.0 [Chicago, 

 IL| ). The bone samples that failed to amplify repeatedly 

 were also tested by using the evolutionarily conserved 

 16s primers. Some samples were still refractory to PCR, 

 indicating that the overall DNA quality or quantity was 



insufficient for this assay; however, those samples that did 

 amplify were identified by sequencing as salmon. In an un- 

 related study using river otter bones (data not presented), 

 one bone sample morphologically identified as salmonid 

 yielded a sequence with 100% identity to the published 16s 

 sequence available for Northern squawfish {Ptychocheilus 

 oregonensis) (Simons and Mayden, 1998). 



After verifying the specificity of the RFLP analysis for 

 differentiating the Pacific salmon species, the assay was 

 applied to the bone samples. Restriction enzyme digestion 

 required some modification when applied to bone. On occa- 

 sion, the restriction enzyme protocol developed for the fresh 

 tissue resulted in degradation of the amplified bone PCR 

 product. Enzyme amount and digestion times were scaled 

 back for the analysis of the bone samples. The Fok I enzyme 

 proved the most difficult for the bone samples, which was 

 likely due to nonspecific restriction that occurs when the 

 enzyme is present at a high concentration in relation to its 

 target or if the reaction is allowed to digest for more than 

 two hours. In some cases, only very weak amplification was 

 achieved with the bone samples and it was difficult to get 

 digestion without degradation. Although sequencing was 

 the main technique used for bone identification. 23 bones 

 in this study were identified by using the RFLP technique. 

 Fourteen of these 23 bones were additionally confirmed by 

 sequencing and the two techniques gave matching results. 



