338 



Fishery Bulletin 98(2) 



denaturation step of 2 min at 94°C followed by 35 

 cycles of 1 min at 95*^0, 1 min at 50 ^C, and 1 min at 

 72°C, with a final step of 5 min at 72''C. Reaction vol- 

 umes of 100 ]iL contained 1 jiL of DNA extract, 2 mM 

 MgCl,,, 200 pM each dNTP, 2.5 units Tag polymerase 

 (Promega), and 0.17 pM of each primer. An approxi- 

 mately 380-bp fragment of the cyt b gene was amplified 

 with universal primers CB2-H and CBl-L (Palumbi et 

 al., 1991) by using the same protocol, except that the 

 final primer concentrations were 0.2 pM. 



An approximately 680-bp fragment of the ATPase 

 6 gene was amplified with primers ATPase6-F and 

 ATPase6-R by using an initial denaturation step of 2 

 min at 94"C followed by 30 cycles of 45 sec at 94°C, 1 

 min at 52°C, and 2 min at 72°C, with a final step of 

 5 min at 72°C. Reagent and template concentrations 

 were identical to those used to amplify the cvt b frag- 

 ment. This PCR reaction protocol was also used to 

 amplify an approximately 1270-bp fragment of ND2 

 with universal primers t-Met and c-Trp (Park et al., 

 1993) and an approximately 1455-bp fragment con- 

 taining the entire control region, tRNAf*^*^, as well 

 as part of the 12S rRNA gene with universal prim- 

 ers L15995 (L-Pro; Meyer et al., 1994) and 12SAR-H 

 (Palumbi etal., 1991). 



An approximately 240-bp fragment of the LDH 

 intron 6 was amplified with primers LDHA6F1 and 

 LDHA6R (Quattro and Jones, 1999) by using an ini- 

 tial denaturation step of 2 min at 94°C followed by 

 35 cycles of 1 min at 95"C, 1 min at 52°C, and 1 min 

 at 72°C, and a final step of 2 min at 72°C. Reaction 

 volumes of 100 pL contained 3 pL of DNA extract, 2 

 mM MgClg, 200 pM each dNTP, 2.5 units Taq poly- 

 merase, and 0.2 pM of each primer. 



Restriction endonuclease digestion of PCR products 



Restriction enzyme digestions were performed as 

 specified by the manufacturer (New England Bio- 

 Labs, Inc., Beverly, MA) in 20 pi reactions contain- 

 ing 5 units of enzyme per reaction. Digestions were 

 incubated for a minimum of 5 hours before being 

 stopped with loading dye (207. Ficoll 400, 0.1 M 

 Na^EDTA pH 8, m SDS, 0.257r bromophenol blue, 

 0.25% orange G). The digests were run for electro- 

 phoresis on 2% agarose gels for at least 2 hours at 

 100 volts. Gels were stained with ethidium bromide 

 and photographed under UV light. Fragment sizes 

 were determined from migration distances in rela- 

 tion to known standards [BstN I digest of pBR322 

 (New England Biolabs) and Hae III digest of pUC18 

 (Sigma, St. Louis, MO)] with the computer package 

 Anagel (Mrazek and Spanova, 1992). 



A subset of 24 fish, eight from each geographical 

 region, was screened for polymorphisms in the five 



amplified mitochondrial DNA segments with the fol- 

 lowing 16 restriction enzymes: Alu I, Aci I, BsmA I, 

 BstV I, Dde I, Dpn II, Hae III, Hha I, Hiiif I, Mid I, 

 Mse I, Msp I, Ma III, Rsa I, oTaq I, and rsp509 I. 

 Restriction enzyme and mtDNA region combinations 

 that revealed variation in the initial screening, ND2- 

 Hinf I and control region-Hae III, were repeated for 

 the entire sample of 72 fish, 24 from each geographic 

 region. 



Denaturing gradient gel electrophoresis 



Perpendicular gradient denaturing gels were run 

 to determine the approximate denaturing points of 

 the COI, cyt b, and LDH intron PCR products. For 

 each fragment, PCR product was mixed with an 

 equal volume of neutral loading dye (207r sucrose; 10 

 mM Tris-HCl, pH 7.8; 1 mM ethylenediaminetetra- 

 acidic acid [EDTA]; 0.1% bromophenol blue) and run 

 on 6.5^^ acrylamide gels (14 cm x 19 cm, 0.75 mm 

 thick) containing a perpendicular gradient of to 

 80% denaturant [100% denaturant was defined as 

 7 M urea/ 40% (v/v) formamide]. Gels were electro- 

 phoresed at 150 volts for a minimum of 5 hours in a 

 recirculating 1 x TAE buffer bath at 60°C (CBS Sci- 

 entific, Inc., Del Mar, CA). Gels were visualized with 

 ethidium bromide staining and photographed under 

 UV light. 



The entire sample of 72 fish was screened for poly- 

 morphisms in COI, cyt 6, and the LDH intron by 

 means of parallel DGGE. The parallel gradient gels 

 spanned a range of 10% denaturant on either side 

 of the experimentally determined melting point of 

 each region: 40% to 60% gels were used to screen 

 COI and cyt 6, and 20% to 40% gels to screen the 

 LDH intron. Parallel denaturing gels were run at 

 150 volts under conditions identical to those used for 

 the initial perpendicular DGGE; the running times 

 were optimized for resolution of each region: 4 hours 

 for the LDH intron, 5 hours for cyt b, and 6 hours for 

 COI. 



In addition, heteroduplex analysis was performed 

 on each individual from each of the three regions. 

 Heteroduplexes were formed by heating samples 

 containing equal volumes of PCR product from two 

 individuals to 95°C for 5 min and then incubating 

 them at -20'C for at least 30 min. Each heteroduplex 

 sample was allowed to thaw slowly at room temper- 

 ature and was run on the appropriate gradient. To 

 create a chain of comparison linking each individual 

 to all of the others, the samples were sequentially 

 combined: the first was mixed with the second, the 

 second with the third, etc., including the last sample 

 which was combined with the first. Pairs of samples 

 that exhibited a single homoduplex band and no het- 



