Bembo et al.: Stock discrimination among Engrauhs encrasicolus by means of DNA analysis 



33 



Figure 1 



Map of the northern Mediterranean showing sample locations for Euro- 

 pean anchovy, Engraulis encrasicolus. Samples referred to in the text 

 were Trieste (1), Ancona (2), Vieste, (3), Ionian Sea (4), Sicilian Channel 

 (5), Tyrrhenian Sea (6), and Aegean Sea (7). 



Kb region containing the ND5 and ND6 genes of the 

 NADH dehydrogenase complex (Cronin et al., 1993). 



ND5/6 



5- AAT AGT TTA TCC AGT TGG TCT TAG -3' 24 mer 

 5'- TTA CAA CGA TGG TTT TTC ATA GTC A -3' 25 mer 



Other mtDNA primers assayed amplified a 2.3 Kb 

 region coding for the ND3/ND4 genes (Cronin et al., 

 1993) and a 2.0 Kb fragment coding for ND1 and 16s 

 RNAlHall, 1992 3 ). 



ND3/4 



5'- TAA (C/T)TA GTA CAG (C/T)TG ACT TCC AA -3' 23 mer 



5- TTT TGG TTC CTA AGA CCA A(C/T)G GAT -3' 24 mer 



NDl/16s 



5- ACC CCG CCT GTT TAC CAA AAA CAT -3' 24 mer 

 5- GGT ATG AGC CCG ATA GCT TA -3' 20 mer 



The amount of template DNA for the PCR reac- 

 tion was usually 1 /iL of the 100 fjh volume extrac- 

 tion described above (approximately 50 ng), but oc- 

 casionally a 1/10 dilution was required for efficient 

 amplification. The reaction cocktail (per 50 fuL reac- 



' Hall, H. 1992. Zoological Society of London, Regent's Park, 

 London, U.K. Personal commun. 



tion) contained 5 nh lOx PCR buffer (0.5 M KC1, 0.1 

 M Tris, 2.5 mM MgCl 2 , pH 8.3), 5/iL dNTP mix (at 2 

 mM with respect to each dNTP), 1 jj.h of each primer 

 (approximately 25 pmol), 1 u Boehringer Taq poly- 

 merase, 1 fjh template DNA and 37 jih sterile fil- 

 tered dH.,0. This was overlaid with two drops of ster- 

 ile mineral oil. Amplification cycle conditions for the 

 ND5/6 primers, with the use of a Hybaid Omni-Gene 

 thermal cycler, were 1) 95°C, 5 min; 1 cycle; 2) 49°C, 

 1 min 30 s; 72°C, 1 min 30 s; 94°C, 30 s; 25 cycles; 3) 

 49°C, 1 min 30 s; 72°C, 10 min; 1 cycle. Conditions for 

 amplifying NDl/16s and ND3/4 were identical, except 

 that, for NDl/16s, annealing took place at 51°C. 



DNA restriction and data collection 



From 3 to 5 jiL of PCR product were restricted with 

 a range of endonucleases recognizing four, five, and 

 six nucleotide sequences: Aat II, Alu I, Ava I, Ava II, 

 Cfo I, Eco RI, Hae III (Pal I), Hind III, Hint I, Msp I, 

 Nci I, Pvu I, Rsa I, Sau 3AI, Sau 961, and Taq I. 

 Restriction products were resolved on agarose and 

 polyacrylamide gels by using a TBE buffer system 

 (Sambrook et al., 1989) and visualized by ethidium 

 bromide and silver nitrate staining, respectively. 

 Restriction fragment data were recorded from all 

 gels, and fragment sizes estimated from their mo- 

 bilities relative to lambda-phage DNA-marker frag- 



