Vandersea et al Identification of larval Centropristis spp using ribosomal DNA-specific molecular assasy 
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Table 2 
Polymerase chain reaction (PCR) reagent concentrations used amplify fish internal transcribed spacer regions and conduct PCR 
and restriction fragment length polymorphism (RFLP) assays for Centropristis striata (black sea bass), C. ocyurus (bank sea 
bass), and C. philadelphica (rock sea bass). 
Reagent 
Fish ITS PCR 
reaction mix 
C. striata 
assay 
C. ocyurus 
assay 
C. philadelphica 
assay 
RFLP PCR 
reaction mix 
Tris-HCl 
100 mM, pH 8.8 
100 mM, pH 8.8 
200 mM, pH 8.4 
100 mM, pH 8.8 
100 mM, pH 8.3 
MgCl 2 
15 mM 
15 mM 
30 mM 
15 mM 
15 mM 
KC1 
750 mM 
750 mM 
500 mM 
750 mM 
750 mM 
Primer I 
25 pmoles 
25 pmoles 
25 pmoles 
25 pmoles 
25 pmoles 
Primer II 
25 pmoles 
25 pmoles 
25 pmoles 
25 pmoles 
25 pmoles 
dNTP mix 
10 mM 
10 mM 
10 mM 
10 mM 
10 mM 
DMSO 
5% 
5% 
5% 
5% 
5% 
Taq DNA polymerase 
0.25 units 
0.25 units 
0.25 units 
0.25 units 
0.25 units 
DNA template 
20-50 ng 
20-50 ng 
20-50 ng 
20-50 ng 
20-50 ng 
Reaction volume 
50 pL 
50 pL 
50 pL 
50 pL 
50 pL 
Table 3 
Primer pairs used to conduct the Centropristis (sea bass) polymerase chain reaction (PCR) and restriction fragment length poly- 
morphism (RFLP) assays. bp=base pairs. 
Species 
Primer 
Primer sequence 5'— 3' 
PCR product size 
C. striata assay 
Cstri-ITSF2 (forward) 
Cstri-ITSR4 (reverse) 
TGGACCGGCTTTCCTCCCG 
CAATGAGGGTTGGAGAAAGGG 
230 bp 
C. ocyurus assay 
Cocy-ITSF5 (forward) 
Cocy-ITSR7 (reverse) 
CTCGTCCTCCTTGCGGTGG 
GGAGGTTTTGGTTTGTGTAGG 
158 bp 
C. philadelphica assay 
Cphil-ITSF5 (forward) 
Cphil-ITSR5 (reverse) 
CACTGCCACTGCCTCCAC 
ACGGAGCCAGCTTTCACC 
238 bp 
Centropristis 
RFLP assay 
CentropFWl (forward) 
CentropREVl (reverse) 
GATCATTACCGGTCGGTTGC 
GTAGTCGAAAAGTGGAGGCAG 
-1200 bp 
Hastings algorithm. The phylogenetic analysis employed 
two concurrent analyses of four chains each. In each 
analysis, one cold and three incrementally heated 
chains were used where the heat of the tth chain is B = 
l/[l+(i-l)T] and T = 0.2. Initial phylogenetic trees for 
each chain were random and used default starting values 
of MrBayes. A single run consisting of 100,000 genera- 
tions was conducted. The run was sampled at every 100 th 
tree. Only trees sampled after a stable burn-in of 1000 
generations were used. The results were plotted as a 
rooted phylogram with Diplectrum formosum as the 
outgroup. An outgroup is any species occurring outside a 
particular branch or clade, i.e., a species that is further 
towards the root of a phylogenetic tree. 
Development of species-specific PCR assays 
for identifying larval fish 
The 3' SSU — 5' LSU sequences for Centropristis striata 
( EF472473-EF472481), C. philadelphica (EF472482- 
EF472490), C. ocyurus (EF472491-EF472499), Diplectrum 
formosum (EF472472), Diplodus holbrooki (EF472471), 
Epinephelus morio (EF472468), Epinephelus adscensionis 
(EF472470), Epinephelus drummondhayi (EF472469), 
Hypoplectrus unicolor (EF472467), Mycteroperca micro- 
lepis (EF472466), Serraniculus pumilio (EF472465), 
Serranus phoebe (EF472464), and Serranus subligarius 
(EF472500) were aligned by using the CLUSTAL-X algo- 
rithm. The alignments were used to identify unique ITS 
sequences and to develop species-specific PCR assays. 
For all three Centropristis assays, we used forward and 
reverse primers located within the ITS1 region (Fig. 2B). 
The PCR primers used for each assay are listed in 
Table 3. The assays were conducted under touchdown 
PCR cycling conditions with an MJ Research PTC-150 
MiniCycler. The PCR reaction profile for the C. striata 
assay was as follows: 2 min. at 95°C, 35 cycles each 
consisting of 30 sec. denaturation at 95°C, 40 sec. ini- 
tial annealing temperature at 66°C which decreased by 
0.5°C per cycle for four cycles and 64°C thereafter, and 
