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. Introduction 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 - dardent@dnr.sc.gov 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). Methods 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 from expectations. 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 population management. Acknowledgments 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. 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