Elsevier

Marine Genomics

Volume 4, Issue 2, June 2011, Pages 83-91
Marine Genomics

In silico mining and characterization of simple sequence repeats from gilthead sea bream (Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

https://doi.org/10.1016/j.margen.2011.01.003Get rights and content

Abstract

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project (‘Marine Genomics Europe’ Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream (Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number.

EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.

Research Highlights

► We screened for simple sequence repeats (SSRs) found in EST development project. ► EST-SSRs were used as template for primer design. ► 206 pairs of primers were investigated for cross-species amplification in sixteen teleost fish. ► The characteristics of 63 EST-SSR loci were studied in a wild gilthead sea bream population.

Introduction

Since first described in the 1980s, microsatellites (also known as simple sequence repeats—SSRs, or short tandem repeats—STRs) have become one of the most important molecular genetic markers currently in use (Ellegren, 2004); they have been widely accepted as a common tool employed in population genetics, molecular ecology, systematics, biodiversity and conservation studies and more recently in linkage mapping, traits association studies and comparative genome analysis (Avise, 2004, Hirschhorn and Daly, 2005, Selkoe and Toonen, 2006). Microsatellites refer to specific DNA sequences consisting usually of one to six base pair motifs tandemly repeated and which are abundant, codominant, hypervariable (extra-polymorphic and multi-allelic) and highly reproducible (Schlotterer, 2000). They are present not only within most eukaryotic genomes, mainly in non-coding (intergenic and intronic) regions, but also within coding (exonic) regions (Tóth et al., 2000).

In the last decade, the plethora of expressed sequence tag (EST) databases already available, as well as most importantly, those that may be created for a given species or genus, have proved to be a valuable source to rapidly obtain microsatellite loci (hereafter EST-simple sequence repeats or EST-SSRs), thereby reducing financial constraints and time-consuming library preparation and screening. Indeed, the development of microsatellites from genomic libraries is limited to those motifs for which the initial hybridization or enrichment was performed and in most cases the PCR primers used to amplify SSRs are species-specific, which implies that markers developed in one taxon cannot be easily and readily applied to others. On the other hand, because EST-SSRs are exonic, their flanking regions are expected to be more conserved across closely related species (Slate et al., 2007) and represent a potential source of Type I (coding and functionally-important) markers. Nevertheless, there is a bias of this in silico detection and validation of exonic microsatellites in these databases in favor of economically important plants and to a lesser extent in aquaculture fish and mollusks (Vasemägi et al., 2005, Guyomard et al., 2006, Wang et al., 2007, Wang et al., 2008, Vidal et al., 2009, Yu and Li, 2007, Bouza et al., 2008, Qiu et al., 2009).

The great potential for the use of EST-SSRs not only in population genetic analyses, but also for their transferability to phylogenetically-close species, is highlighted by the fact that in general a small number of markers, less than 20, are genotyped. Although the estimated ratio of EST-SSRs in any in silico analysis is highly dependent on the search parameters (type of the motif, length of the microsatellite, and flanking regions), 1.5–7.3% of all ESTs are thought to harbor SSRs (Ju et al., 2005). Taking into account that many of these EST-SSRs (at least 25% and up to 80–90%) are typically found to be polymorphic, it therefore seems likely that EST databases containing around 1000 sequences could provide enough microsatellite markers to be used in population genetic analyses (Slate et al., 2007). When we compare these rates to the typical SSR isolation and amplification rates, it is expected that even modest EST collections could prove to be of great value to evolutionary biologists (Ellis and Burke, 2007).

The gilthead sea bream Sparus aurata, together with the European sea bass (Dicentrarchus labrax), is one of leading marine species in European and Mediterranean waters from an aquaculture viewpoint; more than 125,000 t were produced by the aquaculture industry in 2007 whereas commercial fishery catches were no higher than 7500 t (FAO, 2009). Genomic resources have grown exponentially over the last few years, from the development of 24 dinucleotide microsatellite loci (Batargias et al., 1999, Launey et al., 2003, Brown et al., 2005) and their use in several population genetics studies (Palma et al., 2001, Alarcón et al., 2004, Miggiano et al., 2005, De Innocentiis et al., 2005a, De Innocentiis et al., 2005b) to the construction of a first-generation genetic linkage map (Franch et al., 2006) and an RH map (Senger et al., 2006). Comparison between the two maps showed that there is good consistency, with the majority of markers in a single linkage group (LG) also located in the same RH group (Sarropoulou et al., 2007). Moreover, there are several ongoing parentage and pilot QTL analyses aiming to identify the genetic loci involved in the determination of economically important traits (Castro et al., 2007, Castro et al., 2008, Navarro et al., 2009). Therefore, although a large set of microsatellite markers already exists for gilthead sea bream, advanced population and linkage mapping studies will greatly benefit from the addition of markers found in expressed regions of the genome.

In this paper, we present the results of collaborative research carried out in the “Fish & Shellfish” node of the Marine Genomics Europe (MGE) Network of Excellence (GOCE CT-2004-505403, http://www.marine-genomics-europe.org). New cDNA libraries and EST collections were produced for gilthead sea bream (S. aurata) to increase the genomic information in this species. More specifically, an EST dataset was produced for gilthead sea bream that currently accounts for little less half of those reported in NCBI entries (Entrez taxonomy browser) with 29,895 sea bream ESTs (Louro, 2010). In order to develop tools and polymorphic markers for population genetics studies, there was a first screening for simple sequence repeats (SSRs) found in the ESTs of the MGE database using bioinformatic pipeline analyses, after which their use in gilthead sea bream (S. aurata) population studies was evaluated and also their potential for cross-species amplification in fifteen teleost fish currently being studied in IMBG investigated. Gilthead sea bream is progressing fast within the group of fish which have advanced genomic tool resources. Since the development of genetic tools in this species has benefited from existing information from other model fish, the increasing number of EST data available for this species will facilitate maximization of the potential for the development of SSR and SNP-based EST maps, and hasten the implementation of more SSRs in genetic studies.

Section snippets

Detection of SSRs and primer design

A total of 18,196 unigenes (5268 contigs and 12,928 singletons) from the MGE EST-dataset was used (Louro et al., 2010) and we searched for the presence of perfect SSR motifs based on the MIcro SAtellite identification Perl tool script (MISA) available at http://pgrc.ipk-gatersleben.de/misa/misa.html. The search was restricted to motifs of at least 18 bp length, except for the hexanucleotides; therefore, sequences with a minimum number of 9 repeats for dinucleotide, 6 for trinucleotide, 5 for

SSR types, distribution and position in the expressed sequences

We finally detected 899 ESTs that harbor 997 SSRs in the 5268 contigs and 12,928 singletons (18,196 unigenes) analyzed. EST-SSRs frequency for gilthead sea bream was 4.94% and is well in the range reported for other fish species which ranged from 1.5% in Xiphophorus to 7.3% in zebrafish (2.2% in Fundulus and 2.6% in medaka, Ju et al., 2005), 2.0 to 2.7% in Atlantic salmon Salmo salar (Vasemägi et al., 2005, Ng et al., 2005), 2.1% in turbot (Bouza et al., 2008), 2.2% in European eel (Pujolar et

Conclusion

The development of type I markers based on EST-SSRs and data mining of the newly-developed Marine Genomics Europe gilthead sea bream's EST database has proved to be efficient leading to 58 polymorphic microsatellite markers ready to be used in population genetics and genomic studies in this species. EST-SSRs in gilthead sea bream are expected to increase genome information and type II marker density on the available genetic map (Franch et al., 2006) and to enable the development of comparative

Acknowledgments

This work was partly financed by the ‘Marine Genomics Europe’ Network of Excellence (COGE-CT-2004-505403) and the European Union research project “AQUAFIRST” (SSP8-CT-513692). We also thank Erika Souche and Filip Volckaert for fruitful discussions and M. Eleftheriou for linguistic assistance.

References (57)

  • K. Belkhir et al.

    GENETIX, logiciel sous Windows TM pour la génétique des populations

  • C. Bouza et al.

    Characterization of EST-derived microsatellites for gene mapping and evolutionary genomics in turbot

    Anim. Genet.

    (2008)
  • R.C. Brown et al.

    Additional microsatellites for Sparus aurata and cross-species amplification within the Sparidae family

    Mol. Ecol. Notes

    (2005)
  • S.L. Chen et al.

    Isolation and characterization of polymorphic microsatellite loci from an EST-library of red sea bream (Chrysophrys major) and cross-species amplification

    Mol. Ecol. Notes

    (2005)
  • S.L. Chen et al.

    Isolation and characterization of polymorphic microsatellite loci from an EST library of turbot (Scophthalmus maximus) and cross-species amplification

    Mol. Ecol. Notes

    (2007)
  • D.A. Chistiakov et al.

    A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L

    Anim. Genet.

    (2008)
  • S. De Innocentiis et al.

    Tracing the geographical origin of individual breeders from gilthead sea bream (Sparus aurata) hatchery broodstocks by multilocus microsatellite profiles

    Aquaculture

    (2005)
  • H. Ellegren

    Microsatellites: simple sequences with complex evolution

    Nat. Rev. Genet.

    (2004)
  • J.R. Ellis et al.

    EST-SSRs as a resource for population genetic analyses

    Heredity

    (2007)
  • FAO 2009. FishStat Plus - Universal software for fishery statistical time series,...
  • R. Franch et al.

    A genetic linkage map of the hermaphrodite teleost fish Sparus aurata L

    Genetics

    (2006)
  • J. Goudet

    Fstat version 1.2: a computer program to calculate F statistics

    J. Hered.

    (1995)
  • S. Gupta et al.

    Development and characterization of genic SSR markers in Medicago truncatula and their transferability in leguminous and non-leguminous species

    Genome

    (2009)
  • R. Guyomard et al.

    A type I and type II microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) with presumptive coverage of all chromosome arms

    BMC Genomics

    (2006)
  • J.N. Hirschhorn et al.

    Genome-wide association studies for common diseases and complex traits

    Nat. Rev. Genet.

    (2005)
  • C. Iseli et al.

    ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences

    Proc Int Conf Intell Syst Mol Biol

    (1999)
  • Z. Ju et al.

    An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus

    In Silico Biol.

    (2005)
  • H. Kuhl et al.

    The European sea bass Dicentrarchus labrax genome puzzle: comparative BAC-mapping and low coverage shotgun sequencing

    BMC Genomics

    (2010)
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