Perkinson et al.: Evaluation of the stock structure of Rachycentron canadum in the southeastern United States 
223 
Figure 1 
Map of zones where cobia (Rachycentron canadum) were tagged and recaptured off the coast of the 
southeastern United States from 1988 through 2017. Designated for the purpose of partitioning 
and analyzing tag-recapture data, zones include both inshore and offshore waters adjacent to each 
location. BR=Brevard County; N-BR=north of Brevard; and S-BR=south of Brevard. 
globe. In all cases, small tissue samples were collected 
from the anal or caudal fin and stored in either 95% 
non-denatured ethanol (EtOH) or a sarkosyl-urea pres¬ 
ervation solution (8 M urea, 1% sarkosyl, 20 mM sodium 
phosphate, 1 mM EDTA) until processed. For the current 
project, sample selection included those collected along 
the U.S. coast in the GOM and western North Atlantic 
Ocean during cobia spawning season defined by each state 
on the basis of temperature-based patterns and gonadoso- 
matic indices (SEDAR, 2013b). Available samples ranged 
from Virginia south along the Atlantic coast around the 
Florida peninsula into the GOM and westward to Texas. 
Spawning season was defined for each state in this way: 
Virginia, June-August; North Carolina, May-July; South 
Carolina and Georgia, April-July; Florida, March-August; 
Mississippi, May (only samples available); and Texas, 
April-August. 
The sarkosyl-urea preservative simultaneously stabi¬ 
lizes sample DNA and serves as a preliminary cell lysis 
solution. The EtOH-stored samples were subjected to a 
proteinase K cell lysis prior to DNA isolation. All DNA iso¬ 
lation, microsatellite amplification, and genotyping meth¬ 
ods followed the method of Darden et al. (2014). Briefly, 
DNA was isolated from all samples by using a magnetic 
bead isolation procedure. Ten polymorphic microsatellite 
loci were then amplified through polymerase chain reac¬ 
tion (PCR) in 3 multiplexed groupings. These loci have 
been optimized and multiplexed previously and were 
used to document both global and local population struc¬ 
ture in cobia. PCR was conducted in 11-pL reactions with 
lx 5PRIME 6 HotMaster buffer kit (5PRIME HotMaster 
Taq DNA Polymerase and lOx 5PRIME HotMaster Buffer, 
5000 U [5 u/pL]; Qiagen Beverly, Inc., Beverly, MA) and 
with 2.5 mM Mg 2+ , 0.2 mM dNTPs, 0.3 units 5PRIME Hot¬ 
Master Taq DNA Polymerase, 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 template. Individual primer concentrations 
differ among loci and are given in Darden et al. (2014). 
Forward primers for all loci were labeled with We 11 RED 
fluorescent dyes (Beckman Coulter, Inc., Fullerton, CA). 
Thermal cycling for PCR used a modified 60°C touchdown 
protocol (Renshaw et al., 2006) consisting of an initial 
denaturation step at 94°C for 2 min, followed by 34 cycles 
of denaturing at 94°C for 30 s, annealing at 60°C, 57°C, 
6 Mention of trade names or commercial companies is for identi¬ 
fication purposes only and does not imply endorsement by the 
National Marine Fisheries Service, NOAA. 
