Chow et al PCR-RFLP analysis on thirteen western Atlantic snappers 



621 



1.5% KC1, pH 7.5) for 10 minutes at room tempera- 

 ture. The muscle fibrils were finely minced and ho- 

 mogenized in 5-10 mL of ice-cold TEK buffer by means 

 of a motor-driven, glass-teflon homogenizer. Five or 

 more strokes of the homogenizer were neccessary to 

 grind the toughest muscle tissue. The homogenate was 

 transferred to a plastic centrifuge tube (50-mL capac- 

 ity), and SDS and proteinase K were added as de- 

 scribed in Chapman and Powers (1984). The sample 

 was then incubated at 65°C for 2 hours or more and 

 vigorously shaken at intervals. This crude sample was 

 phenol-chloroform extracted and ethanol precipitated 

 as described in Chapman and Powers (1984). The 

 amount of DNA was usually too small to be seen as a 

 pellet, but was rehydrated in 500 pi of TE buffer 

 (ImM EDTA, 10 mM Tris-HCl, pH 8.0) and transferred 

 to a 1.5 mL microcentrifuge tube. To concentrate this 

 DNA sample, ethanol precipitation was repeated and 

 the pellet was rehydrated in 10 ul of TE buffer. 



Embryos and larvae Each specimen was transferred 

 to a 1.5-mL microcentrifuge tube. TEK buffer was 

 added and decanted several times to rinse specimens 

 and to remove ethanol. The specimen was then ho- 

 mogenized in 500 pi of TEK buffer and total DNA was 

 isolated as described above. The pellet was rehydrated 

 in 10 pi of TE buffer. 



Amplification of mitochondrial cytochrome b 

 and 12S rRNA genes 



The two pairs of primers used that targeted cytochrome 

 b and 12S rRNA genes were abbreviated forms of those 

 described by Kocher et al. (1989). The nucleotide se- 

 quences of each set of primers were as follows: cyto- 

 chrome 6, 5-GCTTCCATCCAACATCTCAGCATGATG- 

 3' and 5-GCAGCCCCTCAGAATGATATTTGTCCTC-3'; 

 12S rRNA, 5-TCAAACTGGGATTAGATACCCCACTAT- 

 3' and 5-TGACTGCAGAGGGTGACGGGCGGTGTGT- 

 3'. These primers were synthesized t>y R. K. Werner in 

 the Department of Biochemistry and Molecular Biol- 

 ogy at the University of Miami. 



Polymerase chain reaction was carried out in a final 

 volume of 50 pi in a reaction mixture described by 

 Kocher et al. (1989). This reaction mixture was pre- 

 heated at 94° C for 3 minutes followed by 30-35 cycles 

 of amplification (93°C for 1 min, 44°C for 1 min, and 

 72°C for lmin). The same cycle was applied to am- 

 plify both cytochrome b and 12S rRNA genes. After 

 amplification, the reaction mixture was separated from 

 covering oil and transferred to a 1.5-mL microcentrifuge 

 tube. TE buffer (0.5 mL) was added to the reaction 

 mixture, followed by chloroform: isoamylalcohol (24:1) 

 extraction and ethanol precipitation. After centrifuga- 



tion at 12,000 x g for 10 minutes, the pellet was dried 

 under reduced vacuum, rehydrated with 10 to 50 pi of 

 TE, and stored at 4°C. 



Endonuclease digestion for amplified DNA 

 fragments 



Nine restriction endonucleases used were Alu I, Cfo I, 

 Dde I, Hae III, Hin fl, Mbo I, Msp I, Rsa I, and Taq I, 

 all recognizing symmetric 4-base pair sequences. One 

 unit of each enzyme was applied to 1 to 2 pi of ampli- 

 fied PCR product in a final reaction volume of 5 pi. 

 The digested samples were electrophoresed through 

 27c NuSieve (3:1) (FMC BioProducts, Rockland, ME) 

 agarose gels in TBE buffer (90 mM Tris-boric acid, and 

 2mM EDTA). DNA bands were visualized and photo- 

 graphed following electrophoresis and staining with 

 ethidium bromide. 



Data analysis The size of fragment amplified and re- 

 stricted was estimated in comparison with a size stan- 

 dard ( 1 kb ladder, BRL). Nucleotide sequence divegence 

 (p) (Upholt, 1977) was calculated by using the propor- 

 tion of shared restriction fragments between specimens. 

 Restriction patterns by each endonuclease were desig- 

 nated A, B or C for composite haplotype analysis, and 

 the number of haplotype was used to estimate nucleon 

 diversity (/; ) (Nei and Tajima, 1981) within species. 



Results 



Gene amplification 



Each pair of primers successfully amplified 355±5 bp 

 and 450±5bp fragments of the cytochrome b and 12S 

 rRNA genes, respectively. There were no apparent dif- 

 ferences in the fragment size among species. Both DNA 

 fragments in all of the fresh and frozen specimens and 

 in ethanol-preserved embryos and larvae were well 

 amplified. In contrast, the amount of DNA was usu- 

 ally much less in museum specimens, to the extent 

 that PCR did not always amplify one or both of the 

 gene fragments. 



Restriction fragment analysis 



Figure 1, A and B, shows representative restriction 

 patterns with diagnostic enzymes for amplified frag- 

 ments of the cytochrome 6 and 12S rRNA genes. The 

 restricted fragment distributions for nine enzymes of 

 72 individuals in cytochrome b and 59 individuals in 

 12S rRNA genes are shown in Tables 2 and 3. 



