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Conservation Genetics Resources

, Volume 11, Issue 3, pp 253–257 | Cite as

SNP discovery in European lobster (Homarus gammarus) using RAD sequencing

  • Tom L. JenkinsEmail author
  • Charlie D. Ellis
  • Jamie R. Stevens
Open Access
Technical Note

Abstract

The European lobster (Homarus gammarus) is a decapod crustacean with a high market value and therefore their fisheries are of major importance to the economies they support. However, over-exploitation has led to profound stock declines in some regions such as Scandinavia and the Mediterranean. To manage this resource sustainably, knowledge of population structure and connectivity is crucial to inform management about dispersal, recruitment, stock identification and food traceability. We used restriction-site associated DNA sequencing to develop novel SNP markers from 55 individuals encompassing much of the species range; SNPs were quality filtered, ranked using F-statistics and the top 96 SNPs adequate for primer design were retained. SNP markers were developed with the aim of maximising the power to detect genetic differentiation between: (i) Atlantic and Mediterranean lobsters and (ii) Atlantic lobsters. This panel of SNPs provides a useful resource for future studies of population genetic structure and assignment in H. gammarus.

Keywords

Conservation genetics Fisheries management Homarus gammarus Population assignment RAD-seq Single nucleotide polymorphism 

The European lobster (Homarus gammarus) is a decapod crustacean belonging to the family Nephropidae. They are found on hard substrates hiding in crevices or on compressed muds, typically at depths from the low tide mark to 50 m, but they can occur at depths up to 150 m. Homarus gammarus is widely distributed, ranging from Morocco to Arctic Norway, including Skagerrak, and also in the Mediterranean where they are generally found more sparsely. The species’ high market value makes it a highly-prized seafood product, so its fisheries are of great importance to the local and regional economies they support. However, current and historical over-exploitation has led to stock declines, some of which have been quite profound in several regions (e.g. Scandinavia, Mediterranean) and from which recovery has been slow or stagnant (Kleiven et al. 2012). This has led to the rearing of H. gammarus larvae in lobster hatcheries to produce juveniles which are released into the wild to supplement productive stocks where the risk of over-exploitation is high (Ellis et al. 2015).

Over the last decade, genetic diversity and population structure has been investigated in H. gammarus using traditional molecular markers including random amplification of polymorphic DNA (RAPDs) (Ulrich et al. 2001), allozymes (Jorstad et al. 2005), mtDNA restriction fragment length polymorphisms (RFLPs) (Triantafyllidis et al. 2005) and microsatellites (Huserbraten et al. 2013; Watson et al. 2016; Ellis et al. 2017). However, single nucleotide polymorphisms (SNPs) are becoming the marker of choice in molecular ecology studies, particularly for non-model organisms without a well-annotated genome, because they are (i) abundant and generally widespread in the genome, (ii) eligible for high-throughput screening and automation, and (iii) reproducible across labs (Seeb et al. 2011). Moreover, genomics now enables thousands to tens of thousands of SNPs to be discovered in non-model marine organisms, meaning we have greater power over previous genetic markers to resolve spatial patterns of genetic differentiation, which is thought to be particularly beneficial when studying highly dispersive marine species that exhibit typically weak genetic differentiation (e.g. American lobster, Benestan et al. 2015). These advances have also led to the development of small panels of informative SNPs (e.g. Nielsen et al. 2012; Villacorta-Rath et al. 2016) that are likely to be useful for assessments of genetic structure, population assignment and connectivity.

In this study, we used restriction-site associated DNA (RAD) sequencing to isolate and characterise 96 novel SNP markers in H. gammarus. Genomic DNA was extracted from v-notch or pleopod tissue using a modified salting-out protocol (Li et al. 2011) (S1 Supplementary Material). The RAD library was prepared in-house using Illumina Nextera XT barcodes and comprised 55 individuals from 27 geographically separate sampling locations, ranging from the Mediterranean to the British Isles and Skagerrak (S2 Supplementary Material). The library was sequenced on an Illumina HiSeq 100 bp paired-end rapid run platform. Raw reads (available from Dryad,  https://doi.org/10.5061/dryad.2pc6v) were cleaned and truncated to 97 bp using the process_radtags program in Stacks v1.45 (Catchen et al. 2013) and RAD loci were built using the denovo_map.pl wrapper script in Stacks using optimised parameters of m = 3, M = 3 and n = 3 following the methods of Paris et al. (2017). The populations program was run using all 55 individuals and initial results indicated genetic differentiation between Mediterranean, Skagerrak and the remaining Atlantic samples (S3 Supplementary Material). Therefore, the program was also re-run using only samples from the Atlantic (excluding Mediterranean and Skagerrak samples). This approach maximised the potential to find SNPs that are most informative for detecting hierarchical genetic differentiation between Atlantic lobsters. Full details of the bioinformatics and parameters used are available in S3 Supplementary Information.

In total, 276 million reads were generated and a mean average of 97.9% across all samples were retained after quality control. After initial filtering in Stacks, 7022 biallelic SNPs were identified using all samples and 4377 biallelic SNPs were identified using only Atlantic samples. These SNPs were then ranked by highest \(G_{{{\text{ST}}}}^{{\prime \prime }}\) (Meirmans and Hedrick 2011), sorted by the number of SNPs per RAD locus, and filtered for primer design adequacy and suitability for high-throughput genotyping on a Fluidigm EP1 system. The SNP panel was composed of the highest-ranked remaining SNPs; 21 SNPs were chosen from the dataset composed of all samples (aiming to capture differentiation between Atlantic and Mediterranean lobsters) and 78 SNPs were chosen from the dataset composed of only Atlantic samples (aiming to capture any potential hierarchical differentiation in the Atlantic).

Using these 96 SNP markers and all of our samples, we calculated several population genetic statistics for each locus (Table 1). The observed and expected heterozygosity ranged from 0.049 to 0.630 and 0.179 to 0.504, respectively. The minor allele frequency and the inbreeding coefficient ranged from 0.100 to 0.504 and − 0.457 to 0.470, respectively. After false discovery rate correction, six SNPs deviated significantly from Hardy–Weinberg equilibrium (P < 0.05). To our knowledge, this is the first development of SNP markers in H. gammarus, and therefore these novel markers offer a valuable tool for future studies of spatial genetic structure and population assignment in this species.

Table 1

Summary information for the 96 SNP markers developed for the European lobster (Homarus gammarus)

Locus ID

Sequence length (bp)

SNP

H o

H e

MAF

F IS

P HWE

H_gam_03441

442

G/A

0.407

0.460

0.355

0.045

0.640

H_gam_04173

496

C/T

0.599

0.492

0.409

− 0.457

0.273

H_gam_06157

264

G/C

0.383

0.425

0.282

− 0.094

0.701

H_gam_07502

97

C/T

0.568

0.499

0.445

− 0.184

0.574

H_gam_07892

97

A/T

0.204

0.268

0.155

0.089

0.273

H_gam_08953

496

G/T

0.222

0.423

0.308

0.470

0.018

H_gam_09441

496

A/G

0.414

0.378

0.264

− 0.080

0.691

H_gam_11071

400

G/A

0.179

0.251

0.145

0.304

0.239

H_gam_11183

130

A/G

0.537

0.496

0.445

− 0.072

0.716

H_gam_11291

270

T/G

0.167

0.213

0.120

− 0.274

0.306

H_gam_12971

496

A/G

0.395

0.426

0.309

0.056

0.702

H_gam_14047

496

C/T

0.401

0.417

0.300

− 0.252

0.759

H_gam_14742

496

G/A

0.216

0.331

0.222

0.217

0.097

H_gam_15109

496

T/A

0.265

0.423

0.300

0.215

0.087

H_gam_15128

142

C/T

0.383

0.425

0.291

− 0.094

0.532

H_gam_15435

496

C/T

0.173

0.190

0.109

0.029

0.611

H_gam_15531

122

G/A

0.284

0.476

0.391

0.074

0.029

H_gam_15581

107

A/G

0.290

0.365

0.236

0.013

0.298

H_gam_18512

496

G/T

0.290

0.337

0.218

− 0.179

0.426

H_gam_18652

201

A/G

0.451

0.473

0.364

0.037

0.712

H_gam_19266

175

C/T

0.284

0.296

0.182

− 0.125

0.759

H_gam_19460

247

C/T

0.432

0.477

0.382

0.016

0.646

H_gam_20354

142

C/T

0.469

0.430

0.309

− 0.194

0.626

H_gam_21197

163

C/T

0.525

0.498

0.436

− 0.197

0.759

H_gam_21880

496

A/C

0.481

0.503

0.463

0.054

0.706

H_gam_22323

439

G/A

0.586

0.491

0.418

− 0.358

0.465

H_gam_22365

176

A/T

0.340

0.449

0.318

− 0.038

0.291

H_gam_22740

138

T/C

0.370

0.386

0.245

− 0.328

0.689

H_gam_23146

174

T/C

0.315

0.442

0.300

0.156

0.206

H_gam_23447

114

T/C

0.358

0.472

0.382

0.229

0.275

H_gam_23481

137

T/A

0.296

0.369

0.236

0.159

0.307

H_gam_23677

228

A/G

0.370

0.488

0.418

0.202

0.229

H_gam_23787

496

T/G

0.185

0.246

0.155

0.078

0.267

H_gam_24020

496

C/G

0.216

0.223

0.127

− 0.076

0.759

H_gam_25229

230

C/G

0.259

0.227

0.118

− 0.395

0.759

H_gam_25580

101

C/T

0.630

0.497

0.436

− 0.302

0.276

H_gam_25608

97

C/T

0.185

0.264

0.164

0.268

0.161

H_gam_27329

97

T/C

0.407

0.504

0.464

0.122

0.462

H_gam_28357

496

G/A

0.444

0.497

0.436

0.175

0.587

H_gam_29410

97

T/C

0.420

0.476

0.391

0.001

0.553

H_gam_29801

496

A/G

0.179

0.267

0.164

0.100

0.113

H_gam_29889

496

A/G

0.383

0.483

0.400

0.172

0.451

H_gam_30339

496

G/A

0.228

0.231

0.136

− 0.285

0.759

H_gam_31462

140

C/A

0.327

0.455

0.345

0.211

0.198

H_gam_31618

496

A/G

0.333

0.369

0.236

0.059

0.595

H_gam_31967

195

A/C

0.302

0.429

0.318

0.203

0.180

H_gam_31979

182

G/T

0.259

0.380

0.245

− 0.080

0.223

H_gam_32358

496

G/A

0.074

0.198

0.109

− 0.169

0.036

H_gam_32362

213

C/T

0.630

0.497

0.436

− 0.302

0.276

H_gam_32435

496

T/C

0.210

0.246

0.145

0.070

0.489

H_gam_33066

218

C/A

0.370

0.386

0.245

− 0.328

0.685

H_gam_33784

136

A/G

0.463

0.504

0.491

0.002

0.715

H_gam_34443

302

G/A

0.346

0.453

0.327

0.186

0.215

H_gam_34818

192

A/C

0.259

0.281

0.173

0.066

0.671

H_gam_35584

97

A/T

0.346

0.445

0.336

0.149

0.306

H_gam_36910

97

A/G

0.395

0.482

0.400

0.096

0.458

H_gam_39107

127

C/T

0.216

0.223

0.127

0.016

0.759

H_gam_39876

134

C/T

0.296

0.312

0.200

− 0.155

0.574

H_gam_41521

97

A/T

0.438

0.451

0.355

0.051

0.759

H_gam_42395

496

T/C

0.314

0.472

0.380

0.107

0.119

H_gam_42529

496

A/C

0.364

0.365

0.227

− 0.166

0.759

H_gam_42821

190

G/A

0.167

0.185

0.100

0.006

0.581

H_gam_44670

251

T/C

0.204

0.398

0.255

0.402

0.000

H_gam_45154

496

G/A

0.377

0.470

0.373

0.207

0.472

H_gam_45217

496

G/A

0.265

0.259

0.145

− 0.136

0.759

H_gam_51159

97

T/G

0.432

0.398

0.273

− 0.283

0.692

H_gam_51507

97

G/A

0.308

0.357

0.224

− 0.250

0.443

H_gam_53052

496

A/T

0.407

0.368

0.227

− 0.288

0.684

H_gam_53263

496

T/A

0.383

0.392

0.255

− 0.304

0.691

H_gam_53314

496

T/C

0.327

0.345

0.218

− 0.114

0.691

H_gam_53720

96

C/T

0.568

0.495

0.435

− 0.335

0.483

H_gam_53889

496

G/C

0.191

0.194

0.118

− 0.016

0.631

H_gam_53935

468

C/T

0.284

0.476

0.391

0.074

0.018

H_gam_54240

97

A/C

0.444

0.420

0.287

− 0.182

0.759

H_gam_54762

496

C/T

0.531

0.491

0.436

− 0.345

0.651

H_gam_55111

146

C/T

0.488

0.503

0.500

− 0.222

0.759

H_gam_55142

178

T/G

0.327

0.490

0.426

0.270

0.164

H_gam_55564

496

G/A

0.370

0.503

0.482

0.128

0.264

H_gam_56423

182

C/T

0.420

0.427

0.291

0.127

0.705

H_gam_56785

99

T/C

0.444

0.497

0.436

0.175

0.575

H_gam_57131

97

T/G

0.377

0.407

0.282

− 0.090

0.698

H_gam_57989

408

A/T

0.451

0.450

0.336

− 0.027

0.759

H_gam_58053

97

A/G

0.049

0.179

0.100

0.046

0.000

H_gam_59503

97

T/A

0.593

0.492

0.427

− 0.274

0.335

H_gam_59586

201

G/T

0.296

0.487

0.394

0.261

0.062

H_gam_59967

178

C/T

0.358

0.382

0.255

0.090

0.693

H_gam_60546

167

C/A

0.333

0.494

0.427

0.252

0.166

H_gam_63140

496

C/T

0.321

0.341

0.209

− 0.070

0.683

H_gam_63267

97

G/C

0.395

0.381

0.255

− 0.085

0.759

H_gam_63581

139

T/C

0.426

0.437

0.318

− 0.101

0.705

H_gam_63605

132

T/C

0.451

0.487

0.409

− 0.147

0.716

H_gam_63771

97

A/G

0.346

0.454

0.343

0.227

0.287

H_gam_63798

188

G/A

0.568

0.486

0.418

− 0.237

0.443

H_gam_65064

496

C/A

0.370

0.386

0.245

− 0.328

0.685

H_gam_65376

496

C/A

0.364

0.429

0.309

0.134

0.511

H_gam_65576

173

A/C

0.352

0.376

0.236

− 0.457

0.592

Sequences and additional SNP information can be found in S4 Supplementary Material

SNP single nucleotide polymorphism, Ho observed heterozygosity, He expected heterozygosity, MAF minor allele frequency, FIS inbreeding coefficient, PHWEP-values for Hardy–Weinberg equilibrium corrected for multiple comparisons using the false discovery rate

Notes

Acknowledgements

We thank the many people who provided lobster tissue samples for this research. We also thank Karen Moore and staff at the Exeter Sequencing Service (Exeter, UK) for constructing the RAD libraries. This research was funded by a Natural Environment Research Council UK GW4 + DTP studentship (Grant No. NE/L002434/1), Natural England (Ref. PO 904130) and the University of Exeter, and forms part of the PhD of TLJ.

Supplementary material

12686_2018_1001_MOESM1_ESM.docx (24 kb)
Supplementary material 1 (DOCX 24 KB)
12686_2018_1001_MOESM2_ESM.docx (381 kb)
Supplementary material 2 (DOCX 380 KB)
12686_2018_1001_MOESM3_ESM.docx (227 kb)
Supplementary material 3 (DOCX 227 KB)
12686_2018_1001_MOESM4_ESM.xlsx (31 kb)
Supplementary material 4 (XLSX 32 KB)

References

  1. Benestan L, Gosselin T, Perrier C et al (2015) RAD-genotyping reveals fine-scale genetic structuring and provides powerful population assignment in a widely distributed marine species; the American lobster (Homarus americanus). Mol Ecol 24:3299–3315.  https://doi.org/10.1111/mec.13245 CrossRefGoogle Scholar
  2. Catchen J, Hohenlohe PA, Bassham S et al (2013) Stacks: an analysis tool set for population genomics. Mol Ecol 22:3124–3140.  https://doi.org/10.1111/mec.12354 CrossRefGoogle Scholar
  3. Ellis CD, Hodgson DJ, Daniels CL et al (2015) European lobster stocking requires comprehensive impact assessment to determine fishery benefits. ICES J Mar Sci 72:i35–i48.  https://doi.org/10.1093/icesjms/fsu196 CrossRefGoogle Scholar
  4. Ellis CD, Hodgson DJ, Daniels CL et al (2017) Population genetic structure in European lobsters: implications for connectivity, diversity and hatchery stocking. Mar Ecol Prog Ser 563:123–137.  https://doi.org/10.3354/meps11957 CrossRefGoogle Scholar
  5. Huserbraten MBO, Moland E, Knutsen H et al (2013) Conservation, spillover and gene flow within a network of northern European Marine Protected Areas. PLoS ONE 8:e73388.  https://doi.org/10.1371/journal.pone.0073388 CrossRefGoogle Scholar
  6. Jorstad KE, Faresteit E, Kelly E, Triantaphyllids C (2005) Allozyme variation in European lobster (Homarus gammarus) throughout its distribution range. N Z J Mar Freshw Res 39:515–526.  https://doi.org/10.1080/00288330.2005.9517330 CrossRefGoogle Scholar
  7. Kleiven AR, Olsen EM, Vølstad JH (2012) Total catch of a red-listed marine species is an order of magnitude higher than official data. PLoS ONE 7:1–7.  https://doi.org/10.1371/journal.pone.0031216 CrossRefGoogle Scholar
  8. Li Y, Wang W, Liu X et al (2011) DNA extraction from crayfish exoskeleton. Indian J Exp Biol 49:953–957Google Scholar
  9. Meirmans PG, Hedrick PW (2011) Assessing population structure: F ST and related measures. Mol Ecol Resour 11:5–18.  https://doi.org/10.1111/j.1755-0998.2010.02927.x CrossRefGoogle Scholar
  10. Nielsen EE, Cariani A, Aoidh EM et al (2012) Gene-associated markers provide tools for tackling illegal fishing and false eco-certification. Nat Commun 3:851.  https://doi.org/10.1038/ncomms1845 CrossRefGoogle Scholar
  11. Paris JR, Stevens JR, Catchen JM (2017) Lost in parameter space: a road map for stacks. Methods Ecol Evol 8:1360–1373.  https://doi.org/10.1111/2041-210X.12775 CrossRefGoogle Scholar
  12. Seeb JE, Carvalho G, Hauser L et al (2011) Single-nucleotide polymorphism (SNP) discovery and applications of SNP genotyping in nonmodel organisms. Mol Ecol Resour 11:1–8.  https://doi.org/10.1111/j.1755-0998.2010.02979.x CrossRefGoogle Scholar
  13. Triantafyllidis A, Apostolidis AP, Katsares V et al (2005) Mitochondrial DNA variation in the European lobster (Homarus gammarus) throughout the range. Mar Biol 146:223–235.  https://doi.org/10.1007/s00227-004-1435-2 CrossRefGoogle Scholar
  14. Ulrich I, Muller J, Schutt C, Buchholz F (2001) A study of population genetics in the European lobster, Homarus gammarus (Decapoda, Nephropidae). Crustaceana 74:825–837.  https://doi.org/10.1163/15685400152682593 CrossRefGoogle Scholar
  15. Villacorta-Rath C, Ilyushkina I, Strugnell JM et al (2016) Outlier SNPs enable food traceability of the southern rock lobster, Jasus edwardsii. Mar Biol 163:163:223.  https://doi.org/10.1007/s00227-016-3000-1 CrossRefGoogle Scholar
  16. Watson HV, McKeown NJ, Coscia I et al (2016) Population genetic structure of the European lobster (Homarus gammarus) in the Irish Sea and implications for the effectiveness of the first British marine protected area. Fish Res 183:287–293.  https://doi.org/10.1016/j.fishres.2016.06.015 CrossRefGoogle Scholar

Copyright information

© The Author(s) 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Tom L. Jenkins
    • 1
    Email author
  • Charlie D. Ellis
    • 2
  • Jamie R. Stevens
    • 1
  1. 1.Department of Biosciences, College of Life and Environmental SciencesUniversity of ExeterExeterUK
  2. 2.National Lobster HatcheryPadstowUK

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