26 
Fishery Bulletin 1 12(1) 
Figure 1 
Map of sampling locations for Cobias (Rachycentron canadum) from 
2008 to 2009 along the Atlantic coast of the southeastern United 
States. Locations included inshore Virginia (VA), offshore North 
Carolina (NCoff), inshore South Carolina (SC), and offshore South 
Carolina (SCoff)- 
gear until 2-3 years of age, thus creating a potential 
lag in recruitment and subsequent genetic identifica- 
tion of up to 3 years. Collection locations were provided 
for each specimen by participating anglers. Our 2008 
collection comprised a broader geographic scope than 
that of 2009. In 2008, we obtained 488 samples from 
Virginia (VA), NC, and SC; whereas, our 2009 collection 
comprised 290 samples from only NC and SC. Although 
our SC-collected sample sizes were high in both years 
(349 and 234), specific capture location details were 
missing from the associated collection data for many 
samples. As described later, samples were included in 
each analysis only if appropriate associated data were 
available (Table 2). Collections during both years were 
confined to limited areas; the southern portion of the 
state of SC, the offshore area of the Albemarle Sound 
in NC, and the vicinity of Chesapeake Bay in VA (Fig. 
1). Collected fin tissue was stored at room tempera- 
ture in a solution for the stabilization of DNA and cell 
lysis — a solution made of 8 M urea, 1% sarkosyl, 
20 mM sodium phosphate, and 1 mM EDTA. Ex- 
ternally tagged Cobia were recaptured during the 
previously described sample collections as well as 
through additional tag reporting by recreational 
anglers during both years. 
Molecular protocols 
Total DNA was isolated from the sarkosyl-urea 
solution and tissue lysate by using the Agencourt 
SprintPrep plasmid purification system (Beckman 
Coulter, Inc., Brea, MA) according to the manufac- 
turer’s instructions. Ten microsatellite loci were 
amplified in 3 multiplexed polymerase chain re- 
actions. Each reaction contained 0.2 mM dNTPs, 
IxHotMaster buffer with 2.5 mM Mg 2+ , 0.025 
units Hotmaster Tag DNA Polymerase (5 Prime, 
Inc., Gaithersburg, MD), and 0.5 mM MgCl 2 , 0.20 
mg/mL BSA, 0.3 pM forward and reverse primers, 
and 1 pL of 1:10 diluted DNA isolate. All forward 
primers were labeled with WellRED fiorescent 
dyes (Beckman Coulter, Inc.); individual primer 
concentrations differed for each locus (Table 3). 
All amplifications were performed in 11-pL reac- 
tion volumes in iCycler systems (Bio-Rad Labora- 
tories, Hercules, CA) by using a 60°C touchdown 
protocol (modified from Renshaw et al., 2006) that 
consisted of 3 steps after initial denaturation at 
94°C for 2 min. Step 1 included 7 cycles of 94°C 
for 30 s, 60°C for 1 min and 64°C for 2 min. Step 
2 included 7 cycles of 94°C for 30 s, 57°C for 1 
min and 64°C for 2 min. Step 3 included twenty 
cycles of 94°C for 30 s, 54°C for 1 min and 64°C 
for 2 min, followed by a final extension at 64°C 
for 60 min. The protocol includes substantial de- 
creases in extension times from that of Renshaw 
et al. (2006) to shorten the overall length of the 
protocol. All amplifications were run with 2 nega- 
tive controls. Reaction products and size stan- 
dards (GenomeLab DNA Size standard Kit 400; Beck- 
man Coulter, Inc.) were separated with a CEQ8000 
automated DNA sequencer (Beckman Coulter, Inc.), 
and fragment size analysis was performed with the 
CEQ8000 software package. All chromatograms were 
scored manually, and genotypes were verified indepen- 
dently by a second reader. Approximately 10% of the 
samples were regenotyped to provide validation. 
Marker statistics and parentage analysis 
The sample data pooled over collection years were used 
to test all loci for adherence to Hardy-Weinberg equi- 
librium (HWE), linkage disequilibrium, and the pres- 
ence of genotyping artifacts at each collection locality 
separately (i.e., for SC samples, only those with known 
collection information were included). Examinations for 
departures from HWE and for linkage disequilibrium 
between loci pairs were performed in the program Ar- 
