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2009 SOUTHEASTERN NATURALIST 8(4):671–676
Three Multiplexed Microsatellite Panels For Striped Bass
Jennifer Fountain1,2,3, Tanya Darden1,*, Wallace Jenkins1,
and Michael Denson1
Abstract - Microsatellite multiplexing is a useful technique that minimizes the time,
reagents, and cost associated with genetic studies in fisheries biology. Striped Bass is
an important sport and aquaculture species commonly stocked throughout the United
States. We have developed three multiplexed panels that collectively incorporate
twelve different established microsatellite loci. All loci were tested for Hardy-
Weinberg equilibrium, linkage disequilibrium, Mendelian inheritance, and null
alleles in two populations. Loci were comparably polymorphic in two river systems
with similar allele size ranges observed; therefore, these multiplexed panels should
be useful for genetic population studies of Striped Bass both within and between
disparate geographic distributions.
Morone saxatilis Walbaum (Striped Bass), is a long-lived species
that natively inhabits coastal estuaries and rivers along the east coast of
North America and the Gulf of Mexico. Although this is an anadromous
species, Striped Bass can complete their life cycle in freshwater (Scruggs
1957). Striped Bass have been stocked in both freshwater reservoirs and
coastal estuaries of North America in efforts to support a vibrant recreational
fishery. With prevalent stocking of this species, the need arises for management
programs to adhere to a “responsible approach” to stock enhancement.
A basic tenet of responsible stocking is that all stocked fish be marked and
identifiable from their wild cohorts (Blankenship and Leber 1995).
The emerging use of molecular markers for stock identification is advantageous
as it alleviates the stress associated with conventional tagging
methods and identification recovery is non-lethal. While a variety of genetic
markers exist for fish identification, microsatellites are often the preferred
method due to their polymorphic nature and versatile use in applications
including measures of genetic diversity, parentage analysis, and identification
of population structure (Liu and Cordes 2004). Currently, hundreds of
microsatellite primers are available for Striped Bass (Couch et al. 2006,
Rexroad et al. 2006). Developing protocols to combine known primers and
polymerase chain reaction (PCR) amplifications into multiplexed panels reduces
costs compared to single locus reactions as it conserves reagents and
decreases the time needed to prepare reactions.
1South Carolina Department of Natural Resources (SCDNR), 215 Ft. Johnson Road,
Charleston, SC 29412. 2Grice Marine Laboratory, 205 Ft. Johnson Road, Charleston,
SC 29412. 3Current address - Hollings Marine Laboratory, SCDNR, 331 Ft. Johnson
Road, Charleston, SC 29412. *Corresponding author - firstname.lastname@example.org
672 Southeastern Naturalist Vol. 8, No. 4
In this paper, we describe the optimization of three multiplexed panels,
each containing four microsatellite loci. Striped Bass samples collected during
2006 from the Santee-Cooper system, (n = 61) were used to evaluate the
possibility of multiplexing and perform descriptive locus statistics. Samples
from the same year in the Savannah River (n = 40) and a larger dataset of
Santee-Cooper River (n = 140) samples were used to discern potential interbasin
polymorphism at distinct loci, as these systems are believed to have
low levels of gene fl ow (Bulak et al. 2004).
The multiplexed panels were developed using 20 μL PCR amplifications
containing 50–100 ng genomic DNA performed on an iCycler®
(Bio-Rad Laboratories, Hercules, CA) thermal cycler platform. Each multiplexed
panel was optimized to include 0.2 mM dNTPs, 1x HotMaster
buffer with 2.5mM Mg2+, 0.03 units HotMaster Taq (5 Prime, Inc., Gaithersburg,
MD), and either 1.0 mM Mg2+ (total rxn [Mg2+]: 3.5 mM for panels
1 and 2) or 1.5 mM Mg2+ (total rxn [Mg2+]: 4.0 mM for panel 3). Total reaction
and individual primer concentrations for all multiplexed panels are
provided in Table 1.
All multiplexed panels were successfully amplified using the following
60 °C touchdown protocol: initial denaturation at 94 °C for 3 minutes,
followed by 10 repetitions of a second cycle (94 °C for 30 seconds, 60 °C
for 30 seconds, and 62.2 °C for 30 seconds). After the first repetition of the
second cycle, the annealing temperature was decreased by 0.5 °C with each
subsequent repetition. The third cycle—94 °C for 30 seconds, 50 °C for 30
Table 1. Loci sets for multiplexed PCR panels (MP). Fluorescent dye, allele size range, number
of allelic variants found, GenBank accession number, original source, total primer concentration
(μmol) for multiplexed panel, and individual primer concentration (nmol) are provided.
The forward primer of all sets were fl ourescently labelled with Beckman-Coulter dyes as indicated.
Total and individual unlabeled reverse primer concentrations were the same as reported
for the forward primers.
Allele Total Individual
WellRED size # of [primer] [primer]
MP Locus Accession # dye range alleles Source (μmol) (nmol)
1 MSM1144 BV678214 D4 118–156 15 Couch et al. 2006 0.6 37.50
MSM1095 BV678178 D2 168–198 10 Couch et al. 2006 337.50
MSM1096 BV678179 D3 179–199 8 Couch et al. 2006 168.75
MSM1243 BV678663 D4 239–247 5 Couch et al. 2006 56.25
2 MSM1094 BV678177 D4 127–161 9 Couch et al. 2006 0.3 18.80
MSM1526 BV678552 D2 139–161 10 Rexroad et al. 2006 131.20
MSM1208 BV678286 D3 184–198 7 Couch et al. 2006 75.00
MSM1067 BV678238 D4 193–211 5 Couch et al. 2006 75.00
3 MSM1168 BV678235 D4 140–156 5 Couch et al. 2006 0.6 50.00
MSM1139 BV678210 D2 161–213 10 Couch et al. 2006 250.00
MSM1592 BV678609 D3 155–211 18 Rexroad et al. 2006 200.00
MSM1357 BV678321 D4 217–273 16 Rexroad et al. 2006 100.00
2009 J. Fountain, T. Darden, W. Jenkins, and M. Denson 673
seconds, and 62.2 °C for 30 seconds—was repeated 25 times with a final
extension of 62.2 °C for 60 minutes. Amplified fragments were separated on
a CEQ™ 8000 (Beckman Coulter, Inc., Fullerton, CA) automated sequencer
and scored using the CEQ™ 8000 Fragment Analysis Software.
Deviations from Hardy-Weinberg equilibrium (HWE) were evaluated
using a Markov chain randomization method (1000 dememorizations, 100
batches, and 5000 iterations per batch) with an associated FIS statistic following
Weir and Cockerham (1984). Linkage disequilibrium among all
loci and samples was also determined using a Markov chain randomization
method (same parameters). Analyses of HWE and linkage disequilibrium
were performed in Genepop 3.4 (Raymond and Rousset 1995). Microchecker
(Van Oosterhout et al. 2004) was implemented to test for null
alleles and large-allele dropout for each locus. A χ2 test of Mendelian inheritance
for all loci was conducted using offspring (n = 30) from a known
parental cross within the Santee-Cooper River system. Spatial geographic
population structuring among the Santee-Cooper and Savannah Rivers was
assessed by testing the null hypothesis of genetic homogeneity of allelic
distributions using exact tests as implemented in Genepop 3.4. All statistical
results of multiple simultaneous tests were adjusted using a sequential
Bonferroni approach (Rice 1989).
Results and Discussion
Genotypes of all Striped Bass samples were obtained using the three
multiplexed panels. Utilizing the Santee-Cooper samples, HWE and
linkage disequilibrium were verified for all loci (Table 2), with only locus
MSM1357 indicating linkage disequilibrium with MSM1208 and
MSM1592. The χ2 tests confirmed that all loci exhibit Mendelian inheritance
(Table 3). In addition, neither null alleles nor large-allele dropout
were detected for any locus.
Table 2. Locus information for Santee River Striped Bass microsatellite loci based on samples
of 61 fish. Included are Hardy-Weinberg equilibrium probability values, associated standard
error (S.E.), and the inbreeding coefficient (FIS).
Locus P-value S.E. FIS
MSM1144 0.3843 0.011 -0.079
MSM1095 0.7624 0.009 +0.014
MSM1096 0.1447 0.006 +0.105
MSM1243 0.2159 0.004 -0.025
MSM1094 0.3606 0.005 -0.097
MSM1526 0.1457 0.011 +0.033
MSM1208 0.0235 0.002 -0.069
MSM1067 0.7205 0.004 -0.133
MSM1168 0.0714 0.001 +0.085
MSM1139 0.7316 0.007 +0.075
MSM1592 0.2109 0.011 -0.006
MSM1357 0.5721 0.015 +0.017
674 Southeastern Naturalist Vol. 8, No. 4
Based on allele size range, allele frequencies and number of allelic variants
(Table 4), all loci are comparably polymorphic among river systems,
with similar allele size ranges occurring in each river system. Interestingly,
private alleles were found at multiple loci in both populations. Although additional
samples should be evaluated to confirm the true uniqueness of these
alleles, these results indicate that these loci should be useful for a wide range
of studies in Striped Bass populations, including the evaluation of population
structure. Even with low sample sizes, the fixation index (FST = 0.058)
suggests the Santee-Cooper and Savannah Rivers are moderately differentiated,
agreeing with Bulak et al. (2004). Likewise the populations show
significant genic and genotypic differentiation (χ2 = ∞, P = 0.0000 for both
Table 3. Statistical results of Mendelian inheritance analysis for each locus. The x2 value, degrees
of freedom (d.f.), and P-value are reported. Following sequential Bonferroni correction
(total analysis α = 0.05; individual comparison α = 0.004), no loci showed significant deviation
Locus χ2 d.f. P-value
MSM1144 14.10 6 0.0290
MSM1095 5.42 6 0.4912
MSM1096 2.11 5 0.8337
MSM1243 10.85 4 0.0283
MSM1094 22.26 9 0.0081
MSM1526 5.40 1 0.0201
MSM1208 6.31 6 0.3894
MSM1067 6.00 1 0.0143
MSM1168 2.07 3 0.5580
MSM1139 7.20 3 0.0658
MSM1592 8.11 6 0.2302
MSM1357 9.85 4 0.0430
Table 4. Comparison of Striped Bass microsatellite loci across drainage systems. Allele size
range (bp), number of alleles present, number of private alleles, and range of allele frequencies
observed per population for Santee-Cooper River (n = 140) and Savannah River (n = 40)
systems are reported.
Santee River Savannah River
Size Allele Private Allele Size Allele Private Allele
Locus range count alleles frequency range count alleles frequency
MSM1144 122-156 13 4 0.004-0.349 118-154 11 2 0.013-0.325
MSM1095 168-198 9 4 0.004-0.442 170-194 6 1 0.025-0.375
MSM1096 179-199 7 1 0.011-0.356 179-199 7 1 0.025-0.413
MSM1243 239-247 5 0 0.026-0.522 239-247 5 0 0.013-0.600
MSM1094 127-157 6 0 0.075-0.325 127-161 9 3 0.013-0.250
MSM1526 139-161 9 2 0.014-0.604 139-161 8 1 0.013-0.188
MSM1208 184-198 7 2 0.004-0.309 184-192 5 0 0.013-0.388
MSM1067 193-211 5 0 0.004-0.750 193-211 5 0 0.038-0.688
MSM1168 142-152 3 0 0.361-0.375 140-156 5 2 0.013-0.475
MSM1139 161-213 9 4 0.004-0.514 161-197 6 1 0.013-0.475
MSM1592 159-207 14 4 0.004-0.361 155-211 14 4 0.013-0.475
MSM1357 217-269 14 1 0.004-0.300 217-273 15 2 0.013-0.363
2009 J. Fountain, T. Darden, W. Jenkins, and M. Denson 675
tests), which further supports population differentiation between the two
river systems. In addition, the inbreeding coefficient (FIS = 0.035) indicates
that there is not significant inbreeding occurring within these populations.
In summary, we optimized three multiplexed panels for Striped Bass
from previously developed markers in order to cost-effectively evaluate
their potential use in various population genetic applications in two river
systems in South Carolina. We were able to illustrate the effectiveness of the
tool by showing that there is moderate population structuring of the species
between the two river basins. Additionally, these panels have wide applicability
to other Striped Bass populations because of the documented locus
polymorphism among populations. Optimizing primers for multiplexing microsatellites,
as we have done in this study, represents an important technical
application that will facilitate the use of genetic markers as tags for testing
multiple stocking treatments simultaneously, allowing for the implementation
of more complex experimental designs as well as responsible genetic
This work was funded by the National Fish and Wildlife Foundation and was
conducted in collaboration with the Hollings Marine Laboratory, Charleston, SC. We
thank Robert Chapman who has greatly contributed to the development of population
genetic research in South Carolina that led to the application of this technique
along with Stacey Robbins and Laura Borecki for technical assistance. Ana Zimmerman
provided valuable comments on the manuscript. This is publication number
650 from the Marine Resources Division, South Carolina Department of Natural
Resources and publication number 335 from the Grice Marine Laboratory, College
of Charleston, SC.
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