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Aquaculture xx (2006) xxx xxx www.elsevier.com/locate/aqua-online

Technical paper

Development of a multiplex PCR microsatellite assay in brown trout Salmo trutta, and its potential application for the genus
Estelle Lerceteau-Khler , Steven Weiss
Institut fr Zoologie, Karl-Franzens Universitt, Universittsplatz 2, A-8010 Graz, Austria Received 25 November 2005; received in revised form 21 April 2006; accepted 23 April 2006

Abstract The use of multiplex PCR assays to screen microsatellite variation is a rapid, economically efficient and robust approach to acquire population genetic data. To date, however, few such multiplex assays have been reported in large-scale studies of a fishery. We developed a two reaction, 12 locus microsatellite assay for brown trout, one of the most important aquaculture species in Europe. We describe the steps taken to choose loci that provide unambiguous data in an applied fisheries context. Several parameters were tested for assay optimisation, among which the relative concentration of each primer pair and the PCR elongation time and temperature were the most important. The assay was optimised for the two lineages of brown trout (Atlantic and Danubian) found in Austria. Evaluation on additional lineages of Salmo trutta as well as other Salmo species showed that with few adjustments the assay could be reliably used in all lineages of brown trout as well as S. marmoratus, S. ohridanus, and S. obtusirostris, but probably not for S. salar without major change. 2006 Elsevier B.V. All rights reserved.
Keywords: Salmo trutta; Multiplex PCR; Microsatellites; S. marmoratus; S. ohridanus; S. obtusirostris

1. Introduction The screening of microsatellite variation is the most efficient procedure for assaying genetic variation in applied fisheries, particularly when the maximum level of stock resolution is needed. For large-scale-, or routine assays multiple loading of simplex or du/tri-plex reactions on an automated sequencer increases throughput and reduces costs (e.g. Koskinen and Primmer, 2001; Berthier et al., 2004; Paterson et al., 2004). Yet more efficiency can be achieved with multiplex PCR, but few large multiplex
Corresponding author. Tel.: +43 316 380 55 99; fax: +43 316 380 98 75. E-mail address: Estelle.lerceteau@uni-graz.at (E. Lerceteau-Khler). 0044-8486/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.04.028

SSR assays have been reported for natural fish populations (e.g. a 6-plex assay for Atlantic cod (Gadus morhua) (Delghandi et al., 2003)). Here we report the development of a two reaction, 12 locus microsatellite assay in the valuable aquaculture species brown trout Salmo trutta. We validate the assay on different brown trout lineages, as well as other Salmo species. We emphasize the steps undertaken to achieve an efficient and reliable high-throughput assay, and consider some of the potential pitfalls that may occur in multiplex development and application. 2. Materials and methods A total of 118 samples from the genus Salmo were used for assay development. Genomic DNA was extracted

AQUA-627073; No of Pages 5

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2 E. Lerceteau-Khler, S. Weiss / Aquaculture xx (2006) xxxxxx Table 1 Characteristics of the multiplex assay in other lineages and species Lineage Ind N Dye Development study At/Da (N = 56) SsoSL438 Str60INRA Ssa85 SSsp2216 Str73INRA SsoSL417 Ssa410Uos Ssa408Uos SsaD190 SSsp2213 OMM1064 SsaD71 Ned Hex Fam Ned Fam Hex Fam Hex Fam Hex Ned Fam 89109 93103 101113 129/ 133215 138144 169195 172310 205305 115157 159229 163286 183239 Salmo trutta At (N = 6) 99119 93105 107111 129/ 137151 138146 169/187 216248 235285 123161 167236 199263 215/239 Me (N = 15) 89135 9199 103111 129/ 137199 138168 161209 184316 229245 115144 155236 163219 195255 Ad (N = 15) 99139 9197 107109 129/ 167229 138147 165177 224328 227297 123173 159229 179334 195455 Salmo marmoratus Ma (N = 2) 111/113 93 103107 129/ 211227 148/162 185205 256292 273297 131135 203 191/311 267/365 Salmo ohridanus (N = 5) 9197 91 101107 129/ 163187 166178 171207 250308 227326 131165 139 167/171 195229 Salmo obtusirostris (N = 5) 103121 97 105 129/ 191195 144 171252 239277 139161 155207 247259 191199 Salmo salar (N = 2) 112/132 165173 115131 129/ 139256 202246 177189 218286 277301 123337 187207 144 265331

. Locus not readable in the mentioned population. 129. Single presumably non-target fragment outside the allelic range, absent in some Adriatic drainage populations. For each locus and population, the allelic range in base pairs is given. N is the number of individuals analysed. The abbreviations At, Da, Me, Ad and Ma represent the five major mtDNA lineages of brown trout, whereby Ma is listed as another species S. marmoratus.

from fin clips preserved in 96% ethanol using a high-salt extraction technique (Miller et al., 1988). The assay was optimised on 56 samples of brown trout in Austria, and subsequently tested on other lineages as well as S. ohridanus, S. obtusirostris and S. salar (see Table 1). Forty-three SSRs (Simple Sequence Repeats) were chosen from the literature, including 31 tetra-nucleotide, three tri-nucleotide and nine dinucleotide loci. Each locus was assessed for potential duplication, excessive stutter, imperfect repeats and lack of motif conservation among lineages. Four loci (Str15INRA, Str60INRA, Str73INRA, T3-13) were isolated from S. trutta (Estoup et al., 1993, Estoup et al., 1998), 25 from Salmo salar (SsoSL438Slettan, 1995; SsoSL417Slettan et al., 1995; Ssa85, Ssa171, Ssa197, Ssa202O'Reilly et al., 1996; Ssa407Uos, Ssa408Uos , Ssa410Uos, Ssa413Uos, Ssa418Uos Cairney et al., 2000; SSspG7, SSsp1605, SSsp1606, SSsp2201, SSsp2213, SSsp2215, SSsp2216, SSsp3016 Paterson et al., 2004; SsaD48, SsaD71, SsaD144, SsaD157, SsaD190, SsaD486King et al., 2005), 13 from Onchorhynchus mykiss (OMM1322, OMM1330, OMM1347, OMM1351Palti et al., 2002; OMM1054, OMM1064 , OMM1073 , OMM1082 , OMM1096 , OMM1097, OMM1105, OMM1108, OMM1120Rexroad et al., 2002), and one from O. kisutch (Oki10Smith et al., 1998). For initial testing, single locus PCR was carried out in a total volume of 10 l with 1X GeneAmp PCR buffer II (Applied Biosystems), 0.2 mM of each dNTP,

0.25 M of each primer, 1.5 mM MgCl2, 0.5 U AmpliTaq DNA polymerase (Applied Biosystems) and 2050 ng of DNA using a GeneAmp PCR system 9700 (Applied Biosystems). The reaction consisted of 3 min denaturation at 94 C, 35 cycles of 45 s at 94 C, 20 s at 5264 C, and 30 s at 72 C, and a final extension step of 10 min at 72 C. Unlabelled primers were first used and PCR products visualized on 2.5% agarose gels. To verify microsatellite motif composition, PCR products were cleaned using the ExoSAP-IT kit (Amersham Biosciences), and sequenced using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) following manufacturer's recommendations. Sequences were visualized on an ABI-3100 genotyper. For heterozygous individuals with sufficiently separated fragments, individual alleles were excised from the agarose gel and cleaned via centrifugation for 2 min at 4000 rpm using a home-made spin column composed of cotton and a perforated Eppendorf tube. Subsequently, forward primers were labelled with Fam, Hex, or Ned fluorescent dye. For multiplex development, SSRs were sequentially added to a single locus PCR reaction, and after each addition, the multi-locus profiles were compared to those produced in simplex, to detect the loss or gain of products due to primer interactions. Amplification profiles were further optimised by altering the dye set for better coverage of allelic size ranges, and adjusting the relative concentration of each SSR primer (beginning with equimoler concentrations) to obtain a homogeneous signal across all loci. Due to primer interactions, overlapping

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E. Lerceteau-Khler, S. Weiss / Aquaculture xx (2006) xxxxxx Table 2 List of the microsatellites included in the 8-plex and 4-plex Locus 8-plex Dinucleotide SsoSL438 Str60INRA Ssa85 Str73INRA SsoSL417 Tetranucleotide SSsp2216 Ssa410Uos Ssa408Uos 4-plex Tetranucleotide SsaD190 SSsp2213 OMM1064 SsaD71 Ref Focal species Repeat motif Dye Primer concentration (M) Allele N Allelic size range (bp) 3

4 1 2 1 3 6 5 5

S. S. S. S. S.

salar trutta salar trutta salar

(CA)x (GT)x (CA)x(CG)2(CA)2 (GT)3(CTATTT)(GT)x (GT)x (TAAC)x (CAGA)x (GACA)x

Ned Hex Fam Fam Hex Ned Fam Hex

0.2 0.15 0.1 0.2 0.4 0.05 1 0.75

8 4 6 3 11 13 25 23

89109 93103 101113 138/142/144 169195 129 /133215 172310 205305 115157 159229 163286 183239

S. salar S. salar S. salar

7 6 8 7

S. salar S. salar O. mykiss S. salar

(GATG)x (GGTT)4TGATTA(GTTA)x (ACAG)x(ACAG)2 (ACAAAC)(ACAG)x (TATC)x

Fam Hex Ned Fam

0.0375 0.875 0.375 0.0875

11 17 31 13

1. Estoup et al., 1993. 2. O'Reilly et al., 1996. 3. Slettan et al., 1995. 4. Slettan, 1995. 5. Cairney et al., 2000. 6. Paterson et al., 2004. 7. King et al., 2005. 8. Rexroad et al., 2002. The focal species, repeat motif composition, the dye used in the multiplex, primer concentration, the number of alleles (N), and allelic ranges seen in the 56 screened individuals are shown. Non-target fragment, common to all lineages (see Table 1).

allelic ranges and the restriction of three colour dyes (Fam, Hex and Ned), two sets of SSRs had to be combined into two multiplex reactions (an 8-plex and 4-plex; Table 2). Finally, several parameters (MgCl2/dNTPs/DNA concentrations and different Taq DNA polymerases) were tested in an attempt to improve the resolution of amplification patterns. In general, higher MgCl2/dNTPs concentrations (4 mM MgCl2/400 M dNTPs) gave better resolution of the highest peaks but generated intensity disequilibrium between loci. Consequently, the initial concentrations (1.5 mM MgCl2/200 M dNTPs) were retained. DNA concentrations between 20 and 50 ng were optimal. Among the two different Taq DNA polymerases tested (AmpliTaq; Applied Biosystems and Peqgold; Peqlab Biotechnologie), Peqgold gave better defined peaks but generated a higher background. Therefore AmpliTaq was chosen. Cycling conditions were modified as follows: 3 min denaturation at 94 C, 35 cycles of 45 s at 94 C, 1 min 30 at 57 C and 1 min at 65 C, and a final extension of 30 min at 60 C as suggested by Henegariu et al. (1997) and the Qiagen multiplex PCR handbook (2004). Amplification products were separated on an ABI 3100 automated sequencer (Applied Biosystems). PCR product was dried at 60 C and added to a master mix containing 10 l of deionized formamide and 0.3 l GeneScan-350 Rox size-standard (Applied Biosystems). Samples were heated at 60 C for 2 min and cooled down on ice. SSR

profiles were recorded using the GeneMapper Software v3.7 (Applied Biosystems). 3. Results 3.1. Locus screening and selection Thirty-one loci were discarded due to: poor quality amplification or inappropriate allele size range (Oki-10, OMM1054 , OMM1073 , OMM1082 , OMM1097 , OMM1322, OMM1351, SsaD48, SsaD144, SSsp2215, SSspG7); imperfect or composite repeats or length variation in the flanking region (OMM1105, OMM1120, Ssa413Uos, Ssa418Uos, Ssa197, Ssa171, SSsp1605, SsaD157, Str15INRA, T3-13); duplication (OMM1330 and Ssa202); discrepancies between expected allele sizes and the repeat increment (OMM1108, Ssa407Uos, SSsp2201); or a lack of sufficient variation after sequencing multiple individuals from diverse populations (OMM1096, OMM1347, SsaD486, SSsp1606, SSsp3016). The allelic ranges of the remaining twelve SSRs (seven tetra-and five di-dinucleotide repeats; Table 2) were evaluated by screening 56 samples using the automated genotyper and fluorescent labelled primers. Twenty-five to 100% of the alleles per locus were sequenced to control for abnormalities in the repeat motif or flanking region.

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3.2. Multiplex development The 12 loci were combined into two multiplex reactions (an 8-plex and 4-plex) as described in Table 2. Among the parameters tested, changes in the PCR cycle as described in Materials and methods (elongation time and temperature) proved to be the most important factor for improving amplification patterns, notably in reducing stutter bands. The only locus for which some stutter bands were unavoidable was SsoSL417. The 8-plex and 4-plex cover a range of alleles from 94 to 308 bp and 115 to 286 bp, respectively. The number of alleles per locus ranged from 3 to 31 with an average of 13.8 (Table 2). 3.3. Extension to other brown trout lineages and Salmo species Excluding S. salar, nearly all 12 loci could be analysed without difficulties in other brown trout lineages or species tested (Table 1). Reading problems occurred regionally with SsoSL417, for which either stutter bands or no amplification was observed. Despite larger allele size variation generated at some loci, allele size overlaps were observed only in one Adriatic (locus, Ssa410Uos) and one Mediterranean (loci, SSoSL438 and SSsp2216) S. trutta population. For the latter two loci, an additional overlap was observed in Lake Ohrid brown trout (Ad lineage). For SSsp2216, the overlap was the result of a 129 bp fragment, which is apparently a non-target amplification common to most lineages in the study (see Tables 1 and 2). In contrast, typing in S. salar is impaired by many overlapping allele size ranges and thus a different combination of loci and/or dyes would have to be developed for this species. 4. Discussion

mended by a European concerted action (TroutConcert, 2000), the assay should be of high use for the European community.There is also the potential to add loci to the 4plex in the size range window of 90140 bp. The assay is also applicable to S. obtusirostris and S. ohridanus) but application to S. salar would require the exchange of a few loci. Extensive sequencing of alleles emphasizes general caveats of applying microsatellites to non-focal species or diverse lineages within a species. Amplification in non-focal taxa often shows shifts in allelic ranges, a lack of repeat motif conservation, and for salmonid fishes, previously unobserved duplication. Whether multiplex or simplex PCR is used, allelic series in sample sets containing diverse lineages should be verified with sequencing. If not, the population genetic statistics generated for mixed populations (e.g. where hybridization and introgression has occurred) may be invalid. The success of developing and applying a multiplex microsatellite assay relies as much on a proper choice of loci as on the ability to combine them. We mainly focused on tetranucleotide microsatellites as they exhibit little or no stuttering in comparison to dinucleotides (Edwards et al., 1992; Urquhart et al., 1995) and we additionally restricted our selection to loci with perfect or near-perfect repeat motifs, as these should more closely follow a mutation model such as SMM (Estoup and Cornuet, 1999). Moreover, verification of repeat motif integrity by sequencing a stepwise allelic series at each locus establishes confidence in allele sizing. We emphasize that we found reasons to discard 31 (72%) of the original pool of 43 loci. Despite the required time investment, multiplex microsatellite assays should prove themselves valuable, where large numbers of fish must be screened, or routine stock monitoring. Acknowledgements

This study demonstrates that although attention must be given to locus choice and PCR optimisation, developing high-throughput multiplex microsatellite assays in salmonid fishes is achievable. Our assay was optimised to screen both hatchery and wild populations of brown trout in Austria and was recently applied to a total of 700 individuals (data not shown). This effort required 1496-well plate PCRs and approximately one week of run time on a 16-capillary genotyper, an extraordinary time reduction compared to simplex PCR, even with multiple loading. Our assay is applicable to brown trout lineages outside of Austria. As some loci are already in use (e.g. Carlsson and Carlsson, 2002; Ruzzante et al., 2004) and recom-

The authors would like to thank Guenter Unfer and staff from the Institut fr Hydrobiologie, Fischereiwirtschaft und Aquakultur, Universitt fr Bodenkultur in Vienna for the sampling in Lower Austria, Rui Faria for samples from Portugal, Patrick Berrebi for samples from France, and Craig Primmer for the sample of Salmo salar. We thank Simona Sunik for testing the multiplex PCR on Balkan species of Salmo. The research was supported by the following Austrian entities: Bundesministerium fr Land-und Forstwirtschaft, Umwelt und Wasserwirtschaft; the Amt der Niedersterreichischen Landesregierung (Geschftsstelle des N Landschaftfonds) and the N Landesfischereiverband (St Plten).

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