Skip to main content

Polymorphic microsatellite markers in the brown seaweed Fucus vesiculosus



Fucus vesiculosus is a brown seaweed dominant on temperate rocky shores of the northern hemisphere and, is typically distributed in the mid-upper intertidal zone. It is an external fertilizer that reproduces sexually, providing an excellent model to address conflicting theories related to mating systems and sexual selection. Microsatellite markers have been reported for several Fucus species, however the genomic libraries from where these markers have been isolated, have originated from two or more species pooled together (F. vesiculosus and F. serratus in one library; F. vesiculosus, F. serratus and Ascophyllum nodosum in a second library), or when the genomic DNA originated from only one species it was from Fucus spiralis. Although these markers cross-amplify F. vesiculosus individuals, the level of polymorphism has been low for relatedness studies.


The microsatellite markers described here were obtained from an enriched genomic library, followed by 454 pyrosequencing. A total of 9 microsatellite markers were tested across 44 individuals from the North of Portugal. The mean number of alleles across loci was 8.7 and the gene diversity 0.67.


The high variability displayed by these microsatellite loci should be useful for paternity analysis, assessing variance of reproductive success and in estimations of genetic variation within and between populations.


The seaweed genus Fucus (Fucaceae, Phaeophyta) dominates the intertidal biomass of the northern hemisphere’s shores, where several species co-occur and in some cases hybridize [1]. Fucus vesiculosus is a dominant producer on temperate rocky shores of the northern hemisphere, typically distributed in the mid-upper intertidal zone. It is dioecious, reproduces sexually, and has external fertilization, thus providing an excellent model to address conflicting theories related to mating systems and sexual selection. Variance in reproductive success can lead to sexual selection on reproductive traits and even to sexual conflict between the sexes [2]. Currently, nothing is known about the consequences of external fertilization and the role it plays in the evolution of reproductive traits in this species. Appropriate genetic markers, such as variable microsatellite loci, would allow for more detailed studies related to this topic as well as how mating systems influence patterns of variation within and between populations.

To date, four studies have reported microsatellite markers for different species of the Fucus genera [3-6]. However, none of the previously described markers have been isolated from the species Fucus vesiculosus alone, rather previous studies have been mainly focused on hybridization and speciation processes that affect several species of this genus. Therefore, genomic libraries with a mixture of two species or more have been developed to obtain common markers between the target species (F. vesiculosus and F. serratus in one library [6]; F. vesiculosus, F. serratus and Ascophyllum nodosum in a second library [3]). In the studies where the genomic DNA originated from only one species it has been from Fucus spiralis [4] and Fucus guiryi ([5], published as F. spiralis before this new species was described). Although these markers cross-amplify for F. vesiculosus individuals, their level of polymorphism has proven low for relatedness studies (unpublished observations, Teixeira S.).

Here we report the development and characterization of polymorphic microsatellite loci for Fucus vesiculosus. These markers will be useful for relatedness and population genetic studies of this species and hence to assess within and between population genetic diversity.

Whole genomic DNA was isolated from sperm cells of three F. vesiculosus males using the CTAB method [7]. We used sperm cells as starting material to avoid cross contamination from the abundant microbial communities commonly found in marine organisms. To isolate the microsatellite sequences, a combination of an SSR-enrichment protocol (standard CT/GT) with 454 pyrosequencing was performed by a commercial company (Ecogenics GmbH, Zürich, Switzerland). The same company designed 48 primers that we tested for polymorphisms across a panel of seven individuals obtained from locations in France (Roscoff [48°42′47.53″N; 4°02′32.95″W]) and Portugal (Viana do Castelo [41°41′32.57″N; 8°50′57.57″W]). An M13-tail (TGTAAAACGACGGCCAGT) was added at the 5′ end of all forward primers to enable fluorescent-dye labelling [8]. We identified nine polymorphic loci, and these results are presented in Table 1.

Table 1 Characterization of the polymorphic microsatellite loci identified in Fucus vesiculosus

PCR amplification was performed in 10 μL reaction volumes containing 10 ng of genomic DNA, 1x Qiagen HotStart Taq buffer, 200 μM of dNTP’s, 0.04 μM of forward primer, 0.16 μM of reverse primer and fluorescently-labeled M13 primer, and 0.5 U of HotStart Taq polymerase (Qiagen). All amplifications were conducted in a Perkin-Elmer GeneAmp7200 (Waltham, MA, USA) with the following program: 15 min at 95°C; 30 cycles composed of 30 s denaturation at 95°C, 45 s at the annealing temperature (Table 1) and 45 s elongation at 72°C, followed by an additional 8 cycles composed of 30 s of denaturation at 95°C, 45 s at 53°C, 45 s elongation at 72°C, and a final extra elongation step for 30 min at 72°C. The forward primer for each set was fluorescently labelled with either FAM, ATT550, or HEX, and PCR products were multiplexed. Fragment analysis was conducted on an ABI 3130XL automated sequencer (Applied Biosystems, Foster City, CA, USA) with Rox350 size standard. Alleles were scored using Peak Scanner 1.0 (Applied Biosystems).

Genetic variation for all markers was tested across 44 individuals sampled in the North of Portugal (Viana do Castelo). The number of alleles per locus (n), expected (H E), observed heterozygosity (H O) and heterozygote deficiency (F IS; Table 1) were calculated using the software GENETIX 4.05 [9]. The polymorphic information content, PIC was calculated using PICcalc [10]. The majority of the optimized markers (9) were highly polymorphic, PIC values ranged from 0.41 (Fves11) to 0.8 (Fves1), the number of alleles found for the 9 loci ranged from 5 (Fves11) to 18 (Fves12) alleles; H E varied from 0.45 (Fves11) to 0.83 (Fves1) and H O from 0.2 (Fves10) to 0.85 (Fves12). Significant heterozygote deficiency was observed for 5 markers (Fves1, 7, 8, 10, and 14), as shown by the high and significant F IS values (Table 1). Null alleles might occur at these loci, as confirmed by the determination of the frequency of null alleles in the dataset using ML- NULLFREQ [11] (Table 1). We tested for linkage disequilibrium between all pairs of loci using the software GENETIX 4.05 [9]. No linkage disequilibrium was found after the correction for multiple tests using the false discovery rate (FDR) approach [12] in QVALUE [13].

Previous studies found for the population of Viana do Castelo and across loci, gene diversities of 0.59 [5] and 0.58 [14], while the mean number of alleles found were 4.3 [5] and 5.6 [14]. The overall gene diversity found for the same population across loci, with our newly developed set of markers was of 0.67 and the mean number of alleles 8.7. The higher variability displayed by these microsatellite loci may be useful for paternity analysis and population genetic studies of this species.

Availability of supporting data

The microsatellite sequences are available through the National Center for Biotechnology Information (see The accession numbers on the repository are the following: GenBank accession no. KP765803 through KP765811.


  1. 1.

    Lüning K. Seaweeds: Their Environment, Biogeography and Ecophysiology. New York: John Wiley & Sons; 1990.

    Google Scholar 

  2. 2.

    Arnold SJ. Is there a unifying concept of sexual selection that applies to both plants and animals? Am Nat. 1994;144:S1–S12.

    Article  Google Scholar 

  3. 3.

    Engel CR, Brawley S, Edwards KJ, Serrão E. Isolation and cross-species amplification of microsatellite loci from the fucoid seaweeds Fucus vesiculosus, F. serratus, and Ascophyllum nodosum (Heterokontophyta, Fucaceae). Mol Ecol Notes. 2003;3:180–2.

    Article  CAS  Google Scholar 

  4. 4.

    Wallace AR, Klein AS, Mathieson AC. Determining the affinities of salt marsh fucoids using microsatellite markers: evidence of hybridization and introgression between two species of Fucus (Phaeophyceae) in a Maine estuary. J Phycol. 2004;40:1013–27.

    Article  CAS  Google Scholar 

  5. 5.

    Perrin C, Daguin C, Van De Vliet M, Engel CR, Pearson GA, Serrão EA. Implications of mating system for genetic diversity of sister algal species: Fucus spiralis and Fucus vesiculosus (Heterokontophyta, Phaeophyceae). Eur J Phycol. 2007;42:219–30.

    Article  CAS  Google Scholar 

  6. 6.

    Coyer JA, Hoarau G, Beszerti B, Pearson G, Olsen JL. Expressed sequence tag derived polymorphic SSR markers for Fucus serratus and amplification in other species of Fucus. Mol Ecol Res. 2009;9:168–70.

    Article  CAS  Google Scholar 

  7. 7.

    Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus. 1990;12:13–5.

    Google Scholar 

  8. 8.

    Schuelke M. An economic method for the fluorescent labelling of PCR fragments. Nat Biotech. 2000;18:233–4.

    Article  CAS  Google Scholar 

  9. 9.

    Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F. GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Montpellier (France): Laboratoire Génome, Populations, Interactions, CNRS UMR 5000, Université de Montpellier II; 1996.

    Google Scholar 

  10. 10.

    Nagy S, Poczai P, Cernák I, Gorji AM, Hegedűs G, Taller J. PICcalc: An online program to calculate polymorphic information content for molecular genetic studies. Biochem Genet. 2012;50:670–2.

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Kalinowski ST, Taper ML. Maximum likelihood estimation of the frequency of null alleles at microsatellite loci. Conserv Genet. 2006;7:991–5.

    Article  CAS  Google Scholar 

  12. 12.

    Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc. 1995;57:289–300.

    Google Scholar 

  13. 13.

    Storey JD. A direct approach to false discovery rates. J R Stat Soc. 2002;64:479–98.

    Article  Google Scholar 

  14. 14.

    Nicastro K, Zardi G, Teixeira S, Neiva J, Serrão E, Pearson GA. Shift happens: trailing edge contraction associated with recent warming trends threatens a distinct genetic lineage in the marine macroalga Fucus vesiculosus. BMC Biol. 2013;11:6.

    Article  PubMed Central  PubMed  Google Scholar 

Download references


This study was supported by the Portuguese Science Foundation (FCT) through project PTCD/MAR/104477/2008 (ST), EXCL/AAG-GLO/0661/2012, and postdoctoral fellowship SFRH/BPD/39097/2007 (ST).

Author information



Corresponding author

Correspondence to Sara Teixeira.

Additional information

Competing interests

The authors declare they have no competing interests.

Authors’ contributions

ST, GAP and EAS were responsible for the design and implementation of the study, supervision of the work and processing interpretation of the results. RC, PCA and ST participated in data analysis and microsatellite marker validation, RC and ST drafted the manuscript. All authors read and approved the final manuscript.

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Candeias, R., Casado-Amezúa, P., Pearson, G.A. et al. Polymorphic microsatellite markers in the brown seaweed Fucus vesiculosus . BMC Res Notes 8, 73 (2015).

Download citation


  • Intertidal
  • Genetic diversity
  • Fucus vesiculosus
  • Brown macroalga
  • Microsatellites