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

doi:10.1111/j.1365-2052.2008.01786.x

A combined AFLP and microsatellite linkage map and pilotcomparative genomic analysis of European sea bass Dicentrarchuslabrax L.

D. A. Chistiakov*, C. S. Tsigenopoulos†, J. Lagnel†, Y.-M. Guo‡, B. Hellemans*, C. S. Haley§,

F. A. M. Volckaert* and G. Kotoulas†

*Laboratory of Animal Diversity and Systematics, Katholieke Universiteit Leuven, Ch. Deberiotstraat 32, B-3000 Leuven, Belgium.†Hellenic Centre for Marine Research Crete, Institute of Marine Biology and Genetics, Thalassocosmos, PO Box 2214, Gournes Pediados,

715 00 Heraklion, Crete, Greece. ‡Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China,

Jiangxi Agricultural University, Nanchang 330045, China. §Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, UK

Summary European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery

and is commonly farmed in the Mediterranean basin, but has not undergone much long-

term genetic improvement. An updated genetic linkage map of the European sea bass was

constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and

two single nucleotide polymorphisms. From the 45 new microsatellite markers (including

31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were

assembled into 35 linkage groups. Among these markers, 28 represented type I (coding)

markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes

(linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned

1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas

the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate

ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the

European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage

map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed

with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily

conserved regions with the three-spined stickleback.

Keywords amplified fragment length polymorphism, comparative map, Dicentrarchus

labrax, genomics, microsatellite, single nucleotide polymorphism, synteny.

Introduction

Among vertebrates, fishes are evolutionarily and ecologi-

cally a highly diverse and successful group. They offer key

answers to questions on human evolution and are a main

source of animal protein through fisheries and aquaculture

(Boffelli et al. 2004). Although the role of aquaculture is

increasing annually, most species of fish have a very short

domestication history. There is an obvious lack of genetic

knowledge on fishes and their populations, but the steadily

growing access to genomic resources provides great

opportunities for enhanced genetic improvement. Increased

production can be envisaged through selective breeding for

a range of traits (such as growth, oxygen stress, disease

resistance and production traits), breeding of sex-controlled

populations, hybridization, chromosome and gene manip-

ulation (Hulata 2001). A high-density linkage map greatly

benefits the location of molecular markers for traits suitable

for selective breeding. Moreover, high-density genetic maps

were a prerequisite for the location of a growing number of

quantitative trait loci (QTL) in fish (Liu 2003; Rogers &

Bernatchez 2005; Shirak et al. 2006).

The comparison of teleost genomes facilitates the identi-

fication of evolutionarily conserved chromosomal fragments

(Chen et al. 2004). The genomes of the model species

medaka, zebrafish, fugu and spotted green pufferfish are

widely used for such comparisons (Woods et al. 2000;

Address for correspondence

D. A. Chistiakov, Laboratory of Animal Diversity and Systematics,

Katholieke Universiteit Leuven, Ch. Deberiotstraat 32, B-3000 Leuven,

Belgium.

E-mail: [email protected]

Accepted for publication 18 June 2008

� 2008 The Authors, Journal compilation � 2008 International Society for Animal Genetics, Animal Genetics, 39, 623–634 623

Rexroad et al. 2005; Kasahara et al. 2007). Comparisons of

genomes within fish families, such as for the Salmonidae

(Gharbi et al. 2006), have also been made. Major findings

include evidence for an ancestral vertebrate karyotype of 12

chromosomes and high levels of conservation of synteny,

although this decreases with increasing phylogenetic

distance (Chen et al. 2004; Jaillon et al. 2004). Once

conservation of synteny is established, the identification of

heritable traits of interest will be facilitated. This can be

achieved either by a genome scan or by comparative QTL

analyses directed in a targeted way by synteny conservation

and associated gene content information.

Over the past few years, European sea bass Dicentrarchus

labrax L. (Moronidae, Perciformes, Teleostei) has become

one of the most intensively studied fish species because of its

importance as a food source and because of some unique

characteristics such as temperature-dependent sex change

(Pavlidis et al. 2000; Piferrer et al. 2005), adaptive plasticity

to a range of habitats (Nebel et al. 2005) and feeds (Kaushik

et al. 2004) and its compact genome size (Peruzzi et al.

2005). Genomic resources have expanded at a steady pace

over the past 10 years (for a review, see Volckaert et al.

2008). Several kinds of polymorphic genetic markers were

developed, including microsatellites (Garcıa de Leon et al.

1995; Castilho & McAndrew 1998; Ciftci et al. 2002;

Tsigenopoulos et al. 2003; Chistiakov et al. 2004, 2005),

random amplified polymorphic DNA (Caccone et al.

1997), mitochondrial DNA markers (Patarnello et al. 1993;

Cesaroni et al. 1997), amplified fragment length polymor-

phisms (AFLPs, this study) and single nucleotide polymor-

phisms (SNPs) (Chistiakov et al. 2007). Except for AFLPs, all

markers were successfully used in studies of the population

structure of sea bass and particularly microsatellites (Naciri

et al. 1999; Bahri-Sfar et al. 2000; Lemaire et al. 2000,

2005; Castilho & Ciftci 2005).

In sea bass, the first exploratory mapping experiment was

based on 23 microsatellites and resulted in a genetic map of

eight linkage groups (Chistiakov et al. 2004). Then, a first-

generation linkage map comprised 162 microsatellite

markers (including two genes) assembled into 25 linkage

groups; the length of the sex-average map was 815 centi-

morgans (cM); the map based on female meioses covered

906 cM and was 1.6-fold longer than the male map, which

encompassed 567 cM (Chistiakov et al. 2005).

Microsatellites are very useful for building solid map

frameworks, which could be later enriched with other types

of markers, as for example, the case with the linkage maps

of rainbow trout (Nichols et al. 2003), brown trout (Gharbi

et al. 2006) and zebrafish (Woods et al. 2000). A limited

number of AFLP maps were made in animals to provide

quick and high-density genome coverage (Liao et al. 2007).

The dominant nature of the marker complicates the inte-

gration of an AFLP map into any other inter- or intraspecific

genetic map that is based on codominant markers. More

recently, SNPs have become increasingly popular because of

their common occurrence in the genome (on average every

90 bp in sea bass; E. Souche, personal communication),

uniqueness, ease of scoring and, above all, reproducibility

(Liu & Cordes 2004).

Here, an updated linkage map of the European sea bass is

presented. It is significantly denser than a first-generation

map (Chistiakov et al. 2005) and consists of a higher

number of microsatellites and additional types of polymor-

phic markers such as AFLPs and SNPs. The effectiveness of

this map was assessed in a pilot comparative analysis of

microsatellite flanking regions between the European sea

bass and five model ray-finned fish species. A high level of

conservation of synteny in comparison with the three-

spined stickleback was established.

Materials and methods

Isolation of polymorphic markers

Microsatellites were isolated from microsatellite-enriched

genomic libraries as described by Tsigenopoulos et al.

(2003) and Chistiakov et al. (2004). Microsatellites from

genes and mRNA of D. labrax, expression sequence tags

(ESTs) from a liver cDNA library (Chini et al. 2006) avail-

able in the GenBank database and ESTs from a brain cDNA

library were developed as reported by Chistiakov et al.

(2005). Characteristics of new polymorphic microsatellite

markers are shown in Table S1.

AFLP markers were developed using a total of 64 com-

binations of each of EcoRI-specific (EcoRI-ACC and EcoRI-

ACG) primers with each of MseI-specific primers. The AFLP

procedure was performed essentially as described by Vos

et al. (1995). The Venezia Fbis family consisting of both

parents and 50 full-sib progeny (biparental diploids) (Chis-

tiakov et al. 2004) was fingerprinted using the LI-COR 4200

DNA sequencer (LI-COR BioSciences).

SNPs were developed by resequencing several genes and

cDNAs of D. labrax, whose sequences were available in the

GenBank database, in 10 unrelated sea bass individuals,

using the ABI 3130 Genetic Analyzer (Applied Biosystems).

A PCR-RFLP approach was used to develop new SNPs. SNP

characteristics are summarized in Table 1.

Linkage analysis

For linkage analysis, the Venezia Fbis family was genotyped

using the newly developed microsatellite, AFLP and SNP

markers. Using the CRI-MAP program v3.0 (Green et al.

1990), the analysis was performed as described by Chis-

tiakov et al. (2005). The microsatellite-based linkage map of

Chistiakov et al. (2005) was used as a framework to

incorporate new markers mainly represented by AFLPs.

Sex-averaged and sex-specific linkage distances were

estimated for each linkage group assuming the Kosambi

(1944) mapping function.

� 2008 The Authors, Journal compilation � 2008 International Society for Animal Genetics, Animal Genetics, 39, 623–634

Chistiakov et al.624

Comparative genomics

The genomes of teleost fishes, the spotted green pufferfish

Tetraodon nigroviridis (v. 7.40), the tiger pufferfish or fugu

Takifugu rubripes (v. 4.40), medaka Oryzias latipes (v. 1.41),

the three-spined stickleback Gasterosteus aculeatus (v. 1.41)

and zebrafish Danio rerio (v. 6.40), were downloaded from

ftp://ftp.ensembl.org/pub/. Local BLAST searches were per-

formed under default settings using individual sequences of

186 sea bass genomic clones as queries and the different

genomes� repeat-masked sequences as subjects. Hits with

e < 10)5 were considered significant. The aligned region

and the flanking sequence of the subject (±1 kb) were then

extracted from the genomes. Searches for non-repeat-

masked sequences and a Smith–Waterman alignment were

performed with the FASTA v.2.0 package (Pearson & Lipman

1988) with sea bass microsatellite clones as queries.

Microsatellites were identified and characterized with a PERL

script based on the MISA script (http://pgrc.ipk-gatersleben.

de/misa). The parameters were set for the detection of di- to

hexanucleotide motifs with a minimum of 6, 4, 3, 3 and 3

repeats respectively. For the analysis of the structure of the

transcripts, local SQL expressions were developed on the

Ensembl (Hubbard et al. 2007) databases (v. 40 and 41 for

both Ensembl and MART ftp://ftp.ensembl.org/pub). The

MAPCHART software v2.2 (Voorrips 2002) was used for the

graphical visualization of the maps. The above procedure

was automated in a pipeline; Perl scripts are available upon

request to JL.

Results

Development of new microsatellite markers

Fourteen new type II (non-coding) polymorphic dinucleo-

tide microsatellites (DLA0038–DLA0051) were isolated

from two (AC)12 and (AG)12 microsatellite-enriched DNA

libraries of D. labrax (Table S1). The screening of 510 sea

bass liver ESTs and 179 brain ESTs resulted in the selection

of 111 sequences containing putative microsatellites.

Thirty-one type I markers (13 extracted from the liver and

18 from brain cDNA libraries respectively) showed poly-

morphism in the population sample of 21 unrelated sea bass

individuals. The polymorphic microsatellites were desig-

nated DLA0250E–DLA269E and DLA271E–DLA281E

(Table S1). Locus DLA0254PXN1 showed strong homology

with pentraxin-1, a member of the family of pentraxins

representing cytokine-inducible acute phase proteins, which

are implicated in a variety of functions, such as innate

immunity, sperm-egg fusion and neuronal synapses.

Table 1 Characteristics of newly developed SNPs of the European sea bass, including location (gene and SNP position), PCR primer sequences and

conditions (annealing temperature, Mg2+ concentration and number of PCR cycles), restriction enzyme digestion information (name of enzymes,

definitions of alleles and lengths of digestion products) and allelic frequencies.

Gene SNP location PCR primers (5¢–3¢)TA

(�C)

Mg2+

(mM)

No.

PCR

cycles

Restriction

enzyme to

digest PCR

product

Definition of alleles

and lengths of

digestion

products (bp)

Allelic frequency

(no. individuals

tested, n)

ACTB c.*575T>C F: CATGGTTGTAGTATCGCTTGTA

R: GCACCCCATGTTACCCCTTG

60 2.0 40 AatII C: 178+20

T: 198

C/T = 0.675/0.325 (20)

ERB1 c.*147C>T F: GACACACACATTTTTAGAACGGAA

R: GGCACATGAAGTCGTTCATTATC

54 2.0 35 EcoRI C: 222

T: 220+20

C/T = 0.864/0.136 (22)

IL1B c.76+52C>T F: TTCTGGAGCAGATTTTAATCA

R: CATCCGACAGAATAGATCTG

54 2.0 40 PagI T: 129+19

C: 148

C/T = 0.952/0.048 (21)

IL1B c.76+157A>G F: TTCTGGAGCAGATTTTAATCA

R: CATCCGACAGAATAGATCTG

54 2.0 40 NheI A: 128+22

G: 148

A/G = 0.952/0.048 (21)

IL1B c.76+215A>G F: CTATGATATGGCAGCTGTACGC

R: CATTTCCTTTCCTTGAATATAAA

54 2.0 40 PauI A: 143

G: 122+21

A/G = 0.975/0.025 (20)

IL1B c.76+310A>G F: CTATGATATGGCAGCTGTACGC

R: CATTTCCTTTCCTTGAATATAAA

54 2.0 40 SspI A: 119+24

G: 143

A/G = 0.868/0.132 (19)

IL1B c.300+263C>T F: CATGCTGGAGAGCATAGTGG

R: CAGACAACAGGACAATAATAGACAT

58 1.0 35 XceI C: 270+24

T: 294

C/T = 0.929/0.061 (21)

IL1B c.300+329C>T F: GGTGTTTAGGGTTTGTTAAAAGGC

R: TGTGTCTCTTCATGCGCAATCGA

58 2.0 35 Eco147I C: 127+23

T: 150

C/T = 0.976/0.024 (21)

IL1B c.300+431A>C F: GGTGTTTAGGGTTTGTTAAAAGGC

R: TGTGTCTCTTCATGCGCAATCGA

58 2.0 35 Bsu15I A: 128+22

C: 150

A/C = 0.905/0.095 (21)

TCRB c.*241C>T F: GTTCAAAGGGAAAGCAGAACTAC

R: CTCTGAAAAGTTTTTATTTAGATGAAT

58 1.0 35 Eco47III C: 258+49

T: 307

C/T = 0.818/0.182 (22)

SOX10 c.86+148C>A F: AATTATAAGCTCAAATTGTTGATTT

R: AAATAAAGTTTGAGTAAAAATTATAT

55 2.0 40 DraI C: 129

A: 104+25

A/C = 0.773/0.237 (22)


Genomic analysis of D. labrax 625

Analysis of the nucleotide sequence of several D. labrax

genes and cDNAs revealed two sequences containing poly-

morphic type I microsatellites. DLA0261ERA contains a

dinucleotide motif located at intron 3 of the ERA gene

encoding estrogen receptor-a, and DLA0281NROB1 is

situated within the NROB1 gene. DAX-1, a product of the

NROB1 gene, has a DNA-binding domain and functions as

an anti-testis gene by acting antagonistically to the Sry

locus (Zhang et al. 2000). Thus, the addition of 45 new

microsatellites (31 type I) reported here increases the total

number of available microsatellite loci of D. labrax to 246,

including 59 type I markers. Of the 59 coding (type I)

markers, eight microsatellites reside in known genes.

Development of AFLPs

The analysis of 64 combinations of EcoRI/MseI primers in

the Venezia Fbis family revealed a total of 221 AFLPs,

which, on average, corresponded to 3.45 polymorphic

bands per primer combination (Table S2). Various numbers

of markers were produced depending on the primer com-

binations. The combinations of MseI-CAG/EcoRI-ACC and

MseI-ACC/EcoRI-ACC were the most informative primers,

resulting in 14 and 10 polymorphic bands respectively.

Among the 221 AFLPs detected, 44 were heterozygous in

both parents, while the remaining 177 were sex-specific [i.e.

heterozygous in the male (96 bands) or female (81 bands)

parent].

Identification of SNPs

For SNPs, we screened the following sequences: 3¢untranslated regions (UTRs) of mRNA encoding estrogen

receptor b1 (ERB1; AJ489523) and b2 (AJ489524), cyclo-

oxygenase 2 (COX2; AY336080), gonadotropin-releasing

hormone receptor (GRHR; AJ419594), actin b (ACTB;

AJ537421) and T-cell receptor b (TCRB; AJ493441);

intronic sequences of genes encoding sex-determining re-

gion Y-box 10 (COX10; AY247003) and fructose 1,6

diphosphate aldolase (FDA; AJ493441) and the complete

genomic sequence of the interleukin-1b gene (IL1B;

AJ311925). A total of eleven SNPs were developed

(Table 1). These SNPs include ACTB c.*575T>C, ERB1

c.*147C>T and TCRB c.*241C>T and are located at the

3¢ UTRs of the corresponding mRNAs. The SOX10

c.86+148C>A was found in the intronic sequence of the

sex-determining region Y-box 10 gene. Within the IL1B

gene, a total of seven polymorphic nucleotide

substitutions were identified. Four of them, c.76+52C>T,

c.76+157A>G, c.76+215A>G and c.76+310A>G, are

located in intron 1. Three other SNPs, c.300+

263C>T, c.300+329C>T and c.300+431A>C, reside in

intron 2 of the IL1B gene (Fig. S1). All polymorphisms

found in the IL1B gene were in strong linkage disequi-

librium.

Linkage mapping

As mentioned above, the first-generation microsatellite-

based linkage map (Chistiakov et al. 2005) was used as a

framework for incorporating new markers, mainly AFLPs.

Of the 45 newly developed microsatellites, 28 were infor-

mative. Of the 246 polymorphic microsatellites in total, 201

were informative (i.e. heterozygous at least in one of the

parents) in the Venezia Fbis family. Before linkage analysis,

AFLPs were checked to determine whether their frequency

distribution in the F1 progeny followed Mendelian inheri-

tance, either 3:1 or 1:1 (for dominant markers heterozygous

in both parents or in one of the parents respectively), using

a chi-squared test. The screening resulted in the elimination

of 15 AFLPs the observed genotype distribution of which

significantly deviated from Mendelian inheritance. The

remaining 221 AFLP markers were used in linkage analysis.

Linkage mapping resulted in a new version of the linkage

map of D. labrax consisting of 368 genetic markers: 190

microsatellites, 176 AFLPs and two SNPs grouped in 35

linkage groups. Sixty-seven markers (11 microsatellites, 47

AFLPs and nine SNPs) remained unlinked. The current

linkage map includes 24 anonymous type I microsatellites

derived from ESTs and six genes including peptide Y

(DLA0237; LG1), CYP19 (DLA0238; LG6), SOX10 (LG7),

PXN1 (DLA0254, LG16), ERA (DLA0261; LG17) and TCRB

(LG27).

The total length of the sex-averaged map is 1373.1 cM

(Fig. 1). The number of markers ranged from 2 to 39 per

linkage group. Among linkage groups, LG1 is the longest

having a size of 162.4 cM; LG18 is a fusion of the first-

generation LG18 and LG21. In the map, the intermarker

distance varies from 0 to 27.3 cM, with an average of

3.67 cM; 71.5% of the intermarker intervals are in the

range from 0 to 5 cM, 16.7% range from 5 to 10 cM, 10.3%

range from 10 to 20 cM and only 1.5% are larger than

20 cM.

We also built sex-specific linkage maps. The male map

has a length of 1046.9 cM, an average distance between

markers of 3.03 cM and a maximum marker interval of

24.3 cM (Fig. S2). The length of the female map is

1380.0 cM, with an average intermarker distance of

4.05 cM and a maximum marker interval of 37.8 cM

(Fig. S3). The female map is thus 1.32-fold that of the male

map. Most linkage groups are smaller in males than in

females.

Comparative mapping between the sea bass linkagemap and the genomes of five model fish species

Similarity searches of the microsatellite sequences of

D. labrax against the genome sequences of five teleosts

(green spotted pufferfish, fugu, medaka, three-spined stick-

leback and zebrafish) revealed a high percentage of evolu-

tionarily conserved regions (ECRs) between pairs of species.



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The percentage of ECRs decreased from the stickleback,

where we observed the highest similarity (66/

196 = 33.6%), to pufferfish, fugu and medaka (approxi-

mately 20%) and finally to the zebrafish genome (only

6/196 = 3.1%) (Table 2). A similar trend was also observed

with a higher threshold of e < 10)10; there was still great

similarity with the stickleback genome (53 out of 196 se-

quences) and less so with the fugu, medaka, pufferfish and

zebrafish genomes. The mean size of the aligned region

against the species mentioned above ranges from 135 bp

against the zebrafish to 285 bp against the stickleback, with

mean sequence identity ranging from 73% to 78% (Ta-

ble 2).

ECRs, which flank microsatellites associated with ESTs or

genes, seem to be more informative for comparative map-

ping than those within non-coding genomic regions. More

specifically, in the sea bass – stickleback comparison, while

58 out of 66 ECRs refer to stickleback chromosome positions

(Tables 2 and 3), 13 of them are type I markers (46% of the

28 included) and only 45 are non-coding microsatellites

(27% of the 165 used in the analysis). These data are

compatible with the sequence conservation of cis-regulatory

elements and their distribution is close to (i.e. proximal)

and/or inside structural genes (Davidson 2006). A majority

of ECRs (47.0%) are located in genes, mainly in introns.

A comparison between the stickleback genome and the

sea bass linkage map reveals several regions that display

evidence for conservation of synteny as shared putative

homologues (i.e. two or more putative homologues located

in the same region in both species; see Table 3 and Fig. 2).

The large LG1 of sea bass showed homology with LGV of

stickleback, while the sex-determining LGXIX of stickleback

had the highest number of shared sequence fragments with

sea bass at LG6 (Peichel et al. 2001). Overall, for 66

sequences shared between the sea bass and stickleback,

there were 101 pairs of sequences that map to the same sea

bass linkage group. Of these pairs, 74 (73%) were located on

the same chromosome or assembly in stickleback. Similarly,

for the comparison to the spotted green pufferfish, there

were 36 pairs of sequences mapped to the same sea bass

linkage group and 18 of these pairs (50%) were located on

the same chromosome or assembly in pufferfish. For the

comparison to medaka, there were 34 pairs of sequences

mapped to the same sea bass linkage group and 13 of these

pairs (38%) were located on the same chromosome or

assembly in medaka. This indicates substantially greater

conservation of synteny between sea bass and stickleback

than between sea bass and pufferfish or between sea bass

and medaka. The comparison cannot be made for fugu

where no assembly is available.

Likewise, comparative studies may help in resolving the

linkage of non-chromosomal markers and/or regions of

well-studied species, tentatively assigning them to already

described chromosomes (Table 3). For instance, similarity

hits between three markers of sea bass LG1 to corresponding

stickleback non-chromosomal markers could be linked to

G. aculeatus chromosome V, which appears orthologous to

sea bass LG1 on the basis of four markers.

Discussion

The genomic resources of European sea bass now include a

medium-density linkage map, a large number of ESTs based

on 14 tissue libraries (A. Canario, personal communica-

tion), a BAC library (Whitaker et al. 2006), a radiation

hybrid panel (F. Galibert, personal communication) and a

growing inventory of candidate genes. In comparison with

the first version of the linkage map of European sea bass

(Chistiakov et al. 2005), the updated map includes new

types of markers (AFLPs and a few SNPs) in addition to a

larger number of microsatellites.

Compared to microsatellites, AFLPs present several prob-

lems because of the low information content of these mark-

ers, which are generally informative only from one parent or

Table 2 Comparison of 186 microsatellite

sequences of European sea bass with the

three-spined stickleback (Gasterosteus

aculeatus, v. 1.41), fugu (Takifugu rubripes,

v. 4.40), spotted green pufferfish (Tetraodon

nigroviridis, v. 7.40), Japanese medaka

(Oryzias latipes, v. 1.41) and zebrafish

(Danio rerio, v. 6.40).

Species Stickleback Fugu Pufferfish Medaka Zebrafish

Total hits (e < 10)5) 66 40 39 35 6

No. hits with e < 10)10 53 29 24 24 2

Total hits in chromosomes 58 0 29 24 6

Total hits in genes 31 17 18 21 4

Total hits in exons 8 10 10 5 4

Total hits in introns 21 7 8 14 0

Total hits in UTRs 2 0 0 2 0

Mean size alignment (bp) 284.89 278.98 289.54 234.46 135.33

Mean alignment in

Dicentrarchus labrax (%)

71.51 60.74 64.54 56.92 31.36

Minimum alignment (bp) 90 47 90 84 94

Maximum alignment (bp) 835 829 815 605 157

Mean identity (%) 75.46 74.07 73.07 75.37 77.95

Minimum identity (%) 60.3 60.5 62.2 61.2 73.7

Maximum identity (%) 98.9 89.7 89.9 88 85.4



Table 3 Oxford plot comparing the sea bass linkage groups (LG) to the chromosomes of the three-spined stickleback (S), spotted green pufferfish

(T), medaka (M) and zebrafish (Z); Fugu (F) sequence data are assembled into scaffolds, so no chromosomes are available.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 24 25 34

S non-chr 3 1 1 1 1 1

S-I 1

S-II 2 1

S-III 2 1

S-IV 5

S-V 4

S-VI 3

S-VII 2 1 4

S-VIII 3

S-IX 3

S-X 1

S-XI 1 5

S-XIII 2

S-XIV 1

S-XV 2

S-XVI 1

S-XVII

S-XVIII 5

S-XIX 6

S-XX 1 1

F non-chr 2 2 2 2 1 6 2 5 2 1 2 1 1 2 2 2 1 2 1 1

T non-chr 1 2 2 1 1 1 1 1

T-1 1 2

T-2 3 1

T-3 3

T-5 1

T-7 1

T-9 1

T-10 1

T-12 2

T-13 4

T-14 2

T-15 1

T-16 1

T-17 2

T-18 1

T-20 1 1

M non chr 1 2 2 1 1 1 1 1 1

M-1 1

M-2

M-3 1 1

M-4 1

M-5 1

M-6 3

M-7

M-8 3

M-9 2

M-10 1

M-14 1

M-17 1

M-18 2 1

M-19 3

M-22 1

M-24 1

Z-3 1



one sex in our case and exhibit a potentially higher error rate

in genotyping. As a consequence, AFLP markers largely

developed the existing map and in a few cases, we may expect

AFLP markers to link existing maps. Here, this happened

when LG18 and LG21 of the first-generation map fused into a

single linkage group designated LG18 + 21 through the link

between two AFLPs, MSEI-MCAA/ECORI-EACC-188 and

MSEI-MACA/ECORI-EACG-260.

The total sex-averaged length was increased 1.7-fold,

from 815 to 1373.1 cM. The number of linkage groups has

also increased from 25 to 35, including six new linkage

groups that contain only AFLPs. In the current map, 29

linkage groups have three or more markers. The recombi-

nation length is close to the estimated map size of D. labrax

(1769.5 cM; calculations are based on the method of

Postlethwait et al. 1994). We expect that the addition of

new markers will make the direct correspondence of the 24

linkage groups to the 24 chromosomes (Sola et al. 1993) a

biological reality for the European sea bass. This may also

be independently achieved by the development of physical

maps, such as BAC clone paired-end sequencing (H. Kuhl,

personal communication), assembly of a partially or fully

sequenced genome or/and the recombination-indepen-

dent radiation hybrid map (F. Galibert, personal

communication).

With the addition of markers, the new map became

denser, as is reflected by a decrease in the intermarker

spacing from 5.03 to 3.67 cM. Despite the increase in

length of the current sex-specific maps compared to the first

generation maps, their average intermarker interval has

decreased from 3.5 to 3.03 cM (male, M) and from 5.59 to

4.05 cM (female, F). It is consistent with the results from the

first-generation map (Chistiakov et al. 2005). The F:M

recombination ratio in sea bass is typical for most vertebrate

species, whose sex recombination rate was measured. In

higher vertebrates, this ratio varies between 1.0 and 2.0

(Dib et al. 1996; Dietrich et al. 1996; Mellersh et al. 1997).

The F:M ratio in sea bass is the lowest among those reported

in fishes, for which F:M recombination usually exceeds 2.0,

such as in rainbow trout (Sakamoto et al. 2000), zebrafish

(Singer et al. 2002), Arctic char (Woram et al. 2003) and

fugu (Kai et al. 2005). However, in fish such as Japanese

flounder, a higher recombination rate was shown in males

compared to females (Coimbra et al. 2003). The association

of sea bass LG6 with stickleback LGXIX, which hosts the

sex-determining locus at the distal end and the somewhat

reduced recombination rates in the male, make this LG the

target for further analysis. The mechanism of sex determi-

nation in sea bass remains unknown. The DAX1 (NROB1)

gene was shown to be involved in sex differentiation in

several vertebrates, but not in sea bass (Martins et al. 2007).

Sex differentiation is dependent on strain and parents and is

environmentally (temperature) induced (Pavlidis et al.

2000; Piferrer et al. 2005). As the male-specific suppression

in recombination rate (Haldane�s rule) seems common in a

range of fishes and vertebrates, sexual heterogeneity is likely

to be an ancestral feature.

In the updated map, microsatellites represent half of the

mapped markers. Sequenced markers such as microsatel-

lites and ESTs can be easily related to physical maps and

genome sequences. In addition, the current map is

sufficiently dense to be used for the medium-resolution

mapping of economically and physiologically important

QTL. Mapped genes such as SOX10, ERA and CYP19

(with known polymorphic markers inside) are functional

candidates for searching quantitative traits related to sex

differentiation and maturation, whereas TCRB seems sig-

nificant in mapping traits associated with the resistance to

pathogens and immunity. The collection of microsatellites

would be useful for marker-assisted selective breeding to

establish commercially improved strains of farmed sea bass

(Chatziplis et al. 2007). The collection of markers is also

critical for characterizing the genetic background of wild

and cultured sea bass to maintain heterozygosity of cul-

tured stock and for assigning parentage (Garcıa de Leon

et al. 1995).

A growing number of the sea bass microsatellites originate

from coding regions, which may improve the chances for

matches between genomes. A total of 33.6% of the sea bass

microsatellite sequences match with three-spined stickleback

and a lower, but still considerable number with other

Acanthopterygii. Only forty hits (20.4%) were found between

the sea bass and pufferfish. Using the same stringency

(e < 10)5), Stemshorn et al. (2005) and Franch et al. (2006)

reported higher rate of similarity for microsatellite sequences

between the bullhead sea bream and pufferfish (45.0%) and

between the gilthead sea bream and pufferfish (30.4%). Using

a more stringent threshold for BLASTN searches (e < 10)8),

Table 3 Continued.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 24 25 34

Z-5 1 1

Z-6 1

Z-8 1

Z-18 1

Only the sea bass LGs (24 out of 35) and the five species chromosomes (or scaffolds) that show a hit (e < 10)5) are presented. Sea bass sequence hits

against unique chromosomes (excluding non-chromosomal sequences) are shaded.



Figure

2C

om

par

ison

of

the

sex-

aver

aged

linka

ge

map

(lin

kage

gro

ups

1–2

0)

of

the

Euro

pea

nse

abas

sw

ith

the

gen

om

eof

the

thre

e-sp

ined

stic

kleb

ack.

Signifi

cant

BL

AS

Thits

and

thei

rre

lative

posi

tions

on

the

stic

kleb

ack

chro

moso

mes

(I–X

XI)

are

indic

ated

by

lines

bet

wee

nth

ese

abas

slin

kage

gro

up

and

the

stic

kleb

ack

chro

moso

me.



Wang et al. (2007) found high homology in 11% and 22.9%

of the loci for tilapia and barramundi respectively. In sea

bream, a majority of ECRs (72%) were found either in tran-

scribed regions or in introns of the green spotted pufferfish

genome, but a substantial proportion could not be associated

with any known gene (Franch et al. 2006).

Nearly half of the ECRs are found in stickleback genes

(31 out of 66), and most of them are in introns (Table 2).

The average size of highly similar ECRs observed between

the sea bass and the three-spined stickleback (285 bp) is

among the highest values obtained in a synteny analysis

between sea bream and fugu (182 bp; Franch et al. 2006)

and between fugu and human (199 bp; Walter et al.

2005).

As expected, non-Acanthopterygii such as zebrafish

showed the lowest number of matches. Phylogenetic dis-

tance is a key aspect here. The high level of conservation of

synteny between sea bass and stickleback fits with the

paraphyly of the Perciformes and Gasterosteiformes. It

opens very good perspectives for a detailed comparison

between the genome of a model species, such as three-

spined stickleback or fugu and a phylogenetically related

non-model species, such as European sea bass. However, to

have better insight into the evolution of fish chromosome

number, more type I markers (i.e. SNPs and microsatellites

in ESTs and/or genes) are needed for comparative mapping.

In the future, the linkage map of D. labrax will be further

enriched with type I markers, such as genes, ESTs and SNPs.

The high frequency of ECRs or homologous synteny blocks

facilitates the identification of comparative anchor points,

some of them covering a broad phylogenetic range. The

linkage map of sea bass will become even more suitable and

robust for fine-mapping QTL, functional genomics and

comparative analysis with other teleosts. A promising

development is that partial and even full genome

sequencing has become affordable.

Acknowledgements

We thank A. Canario (CCMAR, Faro, Portugal) for kindly

providing the cDNA brain library of sea bass and M. Lopes

dos Santos for excellent project guidance. Research was

funded by EU FP5 project BASSMAP (project no. QSR5-

2001-01701).

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Supporting information

Additional supporting information may be found in the

online version of this article.

Figure S1 Locations of SNPs within the nucleotide

sequences of Dicentrarchus labrax genes.

Figure S2 Male sex-specific linkage maps of European sea

bass. Distances in Kosambi centimorgans (cM) are given to

the left of each ideogram. Microsatellites are indicated as

DLAxxx, AFLP markers as MSEIxxx/ECORIxxx and type I

markers as DLAxxxE.

Figure S3 Female-specific linkage map of European sea

bass. Distances in Kosambi centimorgans (cM) are given to

the left of each ideogram. Microsatellites are indicated as

DLAxxx, AFLP markers as MSEIxxx/ECORIxxx and type I

markers as DLAxxxE.

Table S1 Characteristics of newly developed type I and II

polymorphic microsatellites of the European sea bass.

Table S2 AFLP primer combinations used and number of

markers generated with each primer combination specific

for the male parent, female parent and both parents of

European sea bass.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting information

supplied by the authors.



Figure S1 Locations of SNPs within the nucleotide sequences of D. labrax genes. Coding

sequences are shown in bold. The translation start ATG codon is capitalized. Nucleotide

substitutions (SNPs) are highlighted in red.

The mRNA for actin β (ACTB) (AJ537421):

SNP:

1 cccacccaaa gtttagccAT Ggaagatgaa atcgccgcac tggttgttga caacggatcc

61 ggtatgtgca aagccggatt cgccggagac gacgcccctc gtgctgtctt cccctccatc

121 gtcggtcgcc ccaggcatca gggtgtgatg gtgggtatgg gccagaagga cagctacgtt

181 ggtgatgaag cccagagcaa gagaggtatc ctgaccctga actaccccat cgagcacggt

241 attgtgacca actgggatga catggagaag atctggcatc acaccttcta caacgagctg

301 agagttgccc ctgaggagca cccagtcctg ctcacagagg cccccctgaa ccccaaagcc

361 aacagggaga agatgaccca gatcatgttc gagaccttca acacccccgc catgtacgtt

421 gccatccagg ccgtgctgtc cctgtatgcc tctggtcgta ccactggtat tgtcatggac

481 tccggtgatg gtgtgaccca cacagtgccc atctatgaag gctacgccct gccccacgcc

541 atcctgcgtc tggacttggc tggccgcgac ctcacagact acctcatgaa gatcctgaca

601 gaacgtggct actccttcac caccacagcc gagagggaaa tcgtgcgtga catcaaggag

661 aaactgtgct atgtcgccct ggacttcgag caggaaatgg gcactgctgc ctcctcctcc

721 tccctggaga agagctacga gctgcctgac ggacaggtca tcaccatcgg caatgagagg

781 ttccgttgcc cagaggccct tttccagcct tccttcctcg gtatggantc ctgcggaatc

841 cacgagacca cctacaacag catcatgaag tgcgacgttg acatccgtaa ggacctgtat

901 gccaacactg tgctgtctgg aggtaccacc atgtaccccg gcatcgccga caggatgcag

961 aaggagatca cagccctggc cccatccacc atgaagatca agatcattgc cccacctgag

1021 cgtaaatact ctgtctggat cggaggctcc atcctggcct ctctgtccac cttccagcag

1081 atgtggatca gcaagcagga gtacgatgag tccggcccct ccatcgtcca ccgcaaatgc

1141 ttctaacaga ctgttcctcc tccccctccc caacaaactc tcaacaactt cagctctgtg

1201 caaaacgacc acaccacaca tttctcatac acattcaggc gcagagcatt agatgaccaa

1261 ctcattggca tggcttcagt tatttttggc gcttgactca ggattttaaa aaaactggaa

1321 cgatgaagga gacttaatgt ttttggctag gtttaaaaaa gcaccccagg gttctgcagt

1381 tgcatctggg gacttaaaaa aatgtacatt ttgttttttt ctggattcat tccaaatgtt

1441 tgttaactgc attgttccag acacgagatt ccaaatgtta aatgcattgt tcagacaagg

1501 tattggcctg tgtgaaggct gcccagtggt tggggcatac ttaaacatgg ttgtagtatc

1561 gcttgtaagt aaattatgtg tgggtttttg gtactttcag ccttaaaaat atttaggtcc

1621 agtttaattt tgttgtttta gttatgcgaa acccaatggt gacctttttt tccccctgtt

1681 ggaggtttca ttcctggggt ggtggggcaa ggggttttga T/Cgtgaagggg taacatgggg c.*575T>C

1741 tgccagaccg gtggggccaa cctgtacact gactaaaaat cccaataaag tgcacatgtg

1801 ttccgacatg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a

The SRY (sex determining region Y)-box 10 (SOX10) gene, partial (AY247003):

SNP:

1 gcacaggcgg cgaggaggaa gctggccgat cagtaccccc acctgcacaa cgcggagctc

61 agcaagaccc tgggcaaact gtggaggttt gtatggggtg gttgtctcag aaaagggaac

121 agcgtggcca tacagtggca catttcctgt gtgcgtaaag gatacatggg gatgtagatg

181 ctgaactcaa atgtaaaagt gggcaaatta taagctcaaa ttgttgattg taaC/Acataga c.86+148C>A

241 aaacacacag aagcagaaat caatgtggtg tttctttaat ctcatgtata atgtataatg

301 tgtactatat ataattttta ctcaaacttt atttaaatca gcctaaatgg cgagtttcac

361 cctcttaatt tcctgtaggc atatttacca attacgcacg gggttgaatt cgacattatt

421 gataattttc acatgctaag gacatacttt cacattaact caggcggcgt gtcagactaa

481 aatcgctcct tgtttttctc cccccaggct gctgaacgag aacgacaaga ggccattcat

541 cgaggaggcg gagaggctga ggaag

Partial mRNA for T-cell antigen receptor chain (TCRB) (AJ493441)

SNP:

1 gaaaaagaca aagagattac cggaccatca gtgaaagtgc ttcaaccttc gccaaaggag 61 tgcaaaaatg aaaaagacaa acaaaggaag aagaccttgg tttgtgtggc caaagacttc

121 tacccagacc atgtcagtgt atcctgggag atcaatgggc aaaatgtcac taatggtgtg

181 gcgacggacg aagctgccca gctgatgccg gaaaagaagt tttaccaaat caccagcagg

241 ttgagggtac ctgccaaaga ctgggaaaac tcagataatg aattcaagtg cattgtcaat

301 ttcttcaata aaacccatac tgtcccttac acagattcaa tctatggtga agctgtgaca

361 acagcaaatg tcatgacaag agagaaatat gtgaagatca cacaggctgc caaactcaca

421 tacagtgttt tcatcgccaa gagctgcatt tacggggcct ttgtggtgtt tctggtgtgg

481 aagcttcagg gttcaaaggg aaagcagaac tactgagagc tgaggccaaa tctattctag

541 gagatctaga agaagatgtt ggtttctgta aataaaggct gaaaactttt cacaactttg

601 tatattgaag tgcatttgca tttattactc aattacataa tctaattctg ctaaaataga

661 tgaacagttc taaacatatt atttttttaa tctcaaagct gctttggcat tacatttgat

721 aaatacttag aataataatc ctgtgtttgt agagcgC/Tttg tttgcttaat attcatctaa c.*241C>T

781 ataaaaactt ttcagaggaa aaaaaaaa

Partial mRNA for estrogen receptor β1 (ERB1) (AJ489523): SNP:

1 atcacctctc cgtacaccga cctcaaccac gacttcacca ccataccttt ctacagtccg 61 actctcttca gttatgcggg tcaggctatt tcagactgcc cttccgtcca tcagtcgcta

121 agcccctcct tgttctggcc cagccacggc cacgtggggc cccccatacc cctgcaccac

181 tcccaggctc ggtctcagca cagacagcca atccagagtc catgggtgga actgtcaccg

241 ctggaaaatg taacaaccag taagagtgtg atgaggcgtt ctcagcagag cgaggagggc

301 gtggtgtcat ctggcgggaa ggcggacctc cactactgtg ctgtgtgtca cgactatgcc

361 tcgggttacc actacggcgt ctggtcatgt gaggggtgta aggccttctt caagaggagc

421 atccaaagac acaatgacta catctgtcca gcaaccaatg aatgcactat agacaagaac

481 cgccgcaaga gctgccaggc gtgtcgcctt cgcaaatgct atgaagttgg catgacgaag

541 tgtggtatgc gaaaggaacg tggaaactac aagaaccccc agacgaggcg agtgacccgc

601 ctgtcctcac agagtagaac acacggacca gatgtgttaa ctggatcatc agcaatgggt

661 ttgttaaacg cgccccgtcc tcctgcactg acttcagagc agctgattga gcgaataatg

721 gaggcagagc cgccagagat ctacctcatg aaggacatga ggaggcctct gactgaagcg

781 aacgtcatga tgtcgctcac caacctggca gataaggagt tggttcacat gatcagctgg

841 gccaaaaaga ttccagggtt cattgagctc ggtctcttgg accaggtgca cctgctggag

901 tgctgctggc tggaagtgct gatggtcgga ctgatttgga ggtcagtgga ccatccaggg

961 aaacttatct tctcccctga cctcagcctg agcagagaag aggggagctg tgtccagggc

1021 tttgtggaga tctttgatat gctgatagct gccacgtcca gggtgagaga gctcaagctc

1081 cagagagagg agtacctctg cctcaaggcc atgatcctcc ttaactccaa catgtgcctc

1141 agctcctcag agggcagcga ggagctgcag agtcgctcca agctgctgca tcttctggac

1201 gctgtaacgg acgctctggt gtgggccatc gccaaaaccg gccttagctt ccgccaacag

1261 tacacccgcc tcgcccacct gcttatgctg ctctcacaca tccgtcatgt cagcaacaaa

1321 ggcatggacc acctccactg catgaaaatg aagaacatgg ttcctttgta cgacctgttg

1381 ctggagatgt tggatgccca catcatgcac ggctcccggc tgccccatcg gcccccacag

1441 caggagtcca gggaccagag ggaagctcct gctcagccac agagttctga taacggcccc

1501 tcaaacacct gggctcccag cagcagcact ggaggtggag gtgaaccaca gtagtcggat

1561 cagaattcag atgcaatgaa ttttttttcc gctttgcact agttcgcaag actgatgaga

1621 cgtcgttttg cagagcgctg cattctgtga actctcattg gtgaaactaa agctgcgaca

1681 cacacatttt tagaacgtta C/Ttctgcagac agaccaaact tgcagtattc attgccttac c.*147C>T

1741 caccacgtac tgaaaagcca gagtggaaca ggaatgtgaa gaaattactg tcatactttg

1801 gccagtagca ccaccacaga actgaacaaa acgttcatgt aatatgtgaa tttcatatca

1861 gataatgaac gacttcatgt gccaaggtga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

1921 aaaaaaaaaa aaaa

The interleukin-1β (IL1B) gene (exons 1-5; AJ311925): SNPs: 1 atcaacattg acagagcaac tgttgggatc ttaaccaaac acactaaagc aacttctaat 61 ctactgacct taacaggtaa aatgactttt cttatgtttt ctttttttct ggagcagatt

121 ttaaacaC/Tga tcttttgctt ttttaagtta ctttgagtat agttgtggat tgaatcttgt c.76+52C>T

181 tacaggacag tttctttaac atttacagcc tgttgtgtca ttaaaaactg ctA/Ggaagatc c.76+157A>G

241 tattctgtcg gatgaaaacc tttttactat gatatggcag ctgtacaagc A/Gccttgcagt c.76+215A>G

301 cacatgctga tacagtagtg agtcatgaat cactgcagca gcagacatag atgtagcctt

361 tacaacagag agtaaaagga taaatA/Gatta tattcaagga aaggaaatgt aataacaatg c.76+310A>G

421 gaaacaggct aataaacagg tagagttaaa tttgaaaatc tagctacatt gcctgaagtt

481 gctttctgtc ttcctacaga aacATGgaat ctgagatgaa atgcaacatg agcgagatgt

541 ggagatccaa gatgccccag ggactggact tggagattac ccaccaccca ctgacaatga

601 ggcgcgtggt caacctcatc attgccatgg agagactgaa gggcttcagt tcagagacac

661 tgatgagcac tgagttcaga gatgaaaacc tgctaaacat catgctggag agcatagtgg

721 aaggtaatca cacagacaag acttaaacgc tattgcatta aaaagccctg gactgttttc

781 tctgtaaccc atcctgccgt aagtcctcct aaaggaactg tgggaagttg aagggaagtg

841 aacagttcag ttacagatcc tgttagtcta actttaaagt ttattatttt ctgtaactgg

901 aaaccaattg aactaactgt aacacctgtg agtgaagccc ttatgtccca cactcagtca

961 gactgtatat tgctatatct ctataC/Tatct ctattattgt cctgttgtct gctgtaacta c.300+263C>T

1021 atatgtgggt gtttagggtt tgttaaaagg aC/Tttttactg ttccataaac taaaatattc c.300+329C>T

1081 tgtatatctc cagtggggtg atgaagttgt tgataaacag cagtaaatta ctcagatttt

1141 tggcccttca agaA/Ctcaatt gcgcatgaag agacacagtc tatggctgtt attgcgattc c.300+431A>C

1201 ctgctgcttg ttgataaaag aagaaaacat actctgaaaa acataactca acagatgtca

1261 atttttcttt gcccccatca gagaaaattg tgtttgagcg cggaacaact ccaacagcgc

1321 agtacagcaa gcgacgcgag gtccaatgca gcgtgaccga cagcgaaaag aggagcttag

1381 ttctggtccc gaacagcatg gagctccacg cagtgatgct gcagggaggc tcggacagat

1441 gcaaaggtaa ctaactgcat gtgacagata gctcagatgg ttggaaaatc atcctctggg

1501 tcatggtaga tgtttgcaat aagggttgac aggtttggtt ttagtttatc tgacaattgt

1561 tggcttgaga ttttaagctg aagctgttta atggtgataa tttatttatt tttcaaatgt

1621 aggcctacac ctaaaatgtc cccaaaattc agtgttttca ccactactgc aggatggaaa

1681 acacacagta acactaatac cgagcttgtc ttggctctgt tacatagaca cagatgtgaa

1741 cttttagtga agtgcacaga agacagaggt tgacattgtc tttcagtttt catgaaaatg

1801 aacccttata ctctgcttct ctcctcagtc caactgaaca tgtcaaccta cttggaccgt

1861 acacccagtg ctgaggctca gactgtggct ctgggcatca agggcacaaa ttactacctg

1921 tcctgccaca aggatggaga ggagccaacc ttgcatctgg aggtaaaaac tgtgactcat

1981 tacacaaatc tcacataaag attacacact ttgtagtcct gctgatttga tctacctggt

2041 cgtctgacat gtcatctttt cacacctcag gtggtggaca aagccagtct ggcgaacatc

Figure S2

Figure S3

1

Table S1 Characteristics of newly developed type I and II polymorphic microsatellites of the European sea bass.

Locus Repeat motif PCR primers (5’- 3’)

TA (°C)

Mg2+ (mM)

Allele size range (bp)

No. alleles HO HE PIC

Accession no.

DLA0038

(GT)27 F: AGGTTCTTGGCCTGGGAATC R: AGTGACAGCAGCCTCCAGAG 57 1.5 234-266 16 0.91 0.93 0.86 DQ363861

DLA0039

(CT)27 F: TCCCACCATCCTGTCATC R: CAAATGTATCTCCATTATGTGAGTG 56 1.5 192-214 10 0.81 0.83 0.81 DQ363862

DLA0040

(AG)28 F: CCCCTAACGTAGTTTTATCTCCA R: GCTGCTGAACACCATAACAA 57

1.5 266-284 10 0.86 0.88 0.86 DQ363863

DLA0041

(CT)7(CA)22AA(CA)3AA (CA)3

F: AAAAGGAACAGCCCTCCAC R: CTCAACATTCCTCCCTCTGCA 59

1.5 176-198 12 0.9 0.93 1 DQ363864

DLA0042

(AC)13 F: AGCATTGTTCTTCTGAGTGACC R: TCCATCATTTGGGATCGAATA 53

1.5 152-166 5 0.72 0.73 0.52 DQ363865

DLA0043

(AC)9 TC (AC)10 F: AGGTCATCCAGAGGAACTGC R: ACATTAAGTATCTTAGCATG 51

1.5 150-174 8 0.78 0.8 0.8 DQ363866

DLA0044

(CT)19(T)4(CT)5 F: TCCGCTCCGCACCGAGTGAC R: ACCGCCCAAGGGTTGGACTG 51

1.5 105-129 7 0.79 0.81 0.67 DQ363867

DLA0045

(AC)13 F: AACGAGAGACTGGAGAGATATAA R: ATCCAGTTAGCGACCCAGT 54 3.0 111-119 5 0.71 0.73 0.57 DQ363868

DLA0046

(GT)3 AT (GT)13 F: TGACTGTCAGGTGCCTGGAG R: TGGGTCATTCGACTTGCAGC 58

1.5 187-209 5 0.55 0.56 0.38 DQ363869

DLA0047

(AC)9 AAAC F: GCAAAAGGACGCACCAACTC R: AGCCAAGGTCACACCAAAGG

55

1.5

98-106

4

0.48

0.5

0.62

DQ363870

DLA0048

(GT)19 F: TGCAGGCTGAATGTGCAGTC R: AGGCGCAGAAGAGAGCAGTC 55

1.5 134-156 7 0.65 0.66 0.62 DQ363871

DLA0049

(CT)26 F: CCTAAACTCTTTGAAGTGCGTGG R: CACTGTTGACCATGCTCAGG 58

1.5 86-112 10 0.75 0.77 0.57 DQ363872

DLA0050

(CA)2 ACAA (CA)16 F: TGTTTATGAGCAGCAAAAGGTC R: AATTGCATCGTCCACTGAGG 57

1.5 188-224 7 0.74 0.75 0.6 DQ363873

DLA0051

(GT)17 F: AGGTTCTTGGCCTGGGAATC R: AGTGACAGCAGCCTCCAGAG 50

1.5 149-181 8 0.69 0.71 0.81 DQ363874

DLA0250E

(TTG)7 F: TACTGGACCACAACCCCATT R: TTCAGGTTTAAGCGGACGTT 54 1.0

141-149

4

0.4

0.378

0.351

CV186277

DLA0251E

(GAA)8 F: TGTGATTGATGTCCCAGACTT R: GGCAGAATTAAGACTCCGATAA 54 2.0

155-165

5

0.524

0.546

0.512

CV186260

DLA0252E

(TGA)10CGA(TGA)3 F: GTCCTCCCCTAGCTTTTCCA R: GCCTTGTAGCAAAAGAGAAGC 54 1.0

115-141

8

0.842

0.82

0.799

CV186185

2

DLA0253E

(GAG)5AAG(GAG) F: CTCAGCAGCAGCATGTGAAG R: GCTGCGTGTGATCCTTCTCT 54 1.0

157-165

3

0.381

0.468

0.415

CV186183

DLA0254PXN1

(CAA)2CGG(CAA)3 F: CGAATCAATGGAGCCGAGAT R: GCCATCCATCCCATTACACT 54 2.0

294-310

3

0.143

0.135

0.13

CV186181

DLA0255E

(TCC)5…( TCC)3… (TCC)3(CCT)2(TCC)7

F: TCCCCCTTCTTACTCTCACCT R: TCCTTCCTGTTCTCGCTTGT 54 1.0

153-155

2

0.143

0.133

0.129

CV186155

DLA0256E

(ATC)3GTC(ATC) F: CAGTTGCTGATCCTGCTG R: TTACTGGATGAAGCGTCTGA 54 2.0

109-137

3

0.714

0.564

0.502

CV186144

DLA0257E

(TA)7 F: CTGGGAGAACCAGCCTACAA R: AGGAGGCCAAATCAACCTTT 54 2.0

135-139

3

0.333

0.541

0.453

CV186125

DLA0258E

(GCAGACCAGAACTAGCA)11

F: CAGTTCTCAAGTTCAACTCTCAA R: TCCACCAATCAGAACCTG 54 2.0

184-370

9

0.75

0.836

0.819

CV186114

DLA0259E

(TG)6 F: TGTTTCTTTGACTTGGATGC R: AAATAAACCAGCTCTGACTGC 54 2.0

120-124

2

0.05

0.21

0.188

CV186113

DLA0260E

(AAAA)(TAAA) (TAAAA)3

F: AACAGTCTCATTCCTTTTTAGCC R: CACGGATCCATTGTTGATGA 54 2.0

131-136

2

0.25

0.375

0.3

CB043827

DLA0261ERA

(GT)20 F: GCTGGTGGATGCAGATAAGC R: ACACAGCAGGGGTTTAATGC 58 1.0

297-321

8

0.905

0.799

0.774

AJ505009

DLA0262E

(AATG)7 F: GGAACGTGTAAATTACCATCCTCA R: ACCCACGCTCAAGCTAACTC 54 2.0

122-132

3

0.714

0.602

0.562

DQ020006

DLA0263E

(TAGCC)6(TAGCA) F: TGCAAAGAATATGAAGCTGGAG R: TTTTTAGAGGGAGTTACGTGGTG 54 1.0

105-110

2

0.476

0.472

0.417

DQ020007

DLA0264E

(AAT)3C(AAT)2 F: ACCAGCGTGGAGAAAGAGAA R:TCTGTCAGTTATTGGTTTATTGTTG 54 2.0

121-124

2

0.268

0.289

0.35

CX535581

DLA0265E

(TA)2TG(TA)6 F: TGTGAACTTAAGCCCCGAAT R: CATATTTAGCGCAGGCATCA 54 1.0

131-141

3

0.0

0.177

0.173

DQ020005

DLA0266E

(AC)3AG(AC)4AG(AC)2 GT(AC)3(AC)AT(AC)AT (AC)2

F: TCATCAACCAACAGAGTGCAA R: GCTCAGCCTGTATCCATTTCA

54 1.0

134-148

4

0.238

0.218

0.213

DQ020008

DLA0267E

(TG)19 F: TGTGTTTGACAGAGTATGTGTGAGAG R: GAGCTGCACACTTTCCCATT 54 1.0

109-127

8

0.952

0.827

0.815

DQ020009

DLA0268E

(TG)3(TTG)3(TG)(TTG) F: TGGCACTTAACTCAAGAGCGTA R: TCATGCGCTACAATTGGCTA 54 1.0

165-171

2

0.414

0.439

0.415

DQ020010

DLA0269E

(AT)3(A)2(AT)3(A)2(AT)2 F: GACGAGAATGATCGCCTACC R: GCTGATCCAGAACCGTGAAT

54

1.0

133-137

2

0.191

0.172

0.165

DQ020013

DLA0271E

(CT)9 F: CGTAAGCTCCCTCGCTTT R: CTTTCACCTCAAGATATGAAGATCC 54 1.0

141-145

3

0.572

0.444

0.421

DQ020015

DLA0272E

(TAT)8 F: GTTCAGATAGATCCTATGTTC R: AATTCTTTAA ATTCTCAGGT 54 1.0

229-238 4 0.614 0.456 0.437 DQ020016

DLA0273E

(TG)6AG(TG)8 F: GTGCGTTTCCTACCACTGCT R: GCCACATACAAAGCCCAGAG

54

1.0

99-109

4

0.524

0.423

0.405

DQ020017

3

DLA0274E

(GT)9 F: CATGCGCATACACACAATGA R: AAAACCTCAGCCGTGTTCTG 54 1.0

150-160

4

0.35

0.379

0.367

DQ020018

DLA0275E

(ATCA)2...(ATCA)3 F: GGGGCTCGAAGAAATAACAG R: ATTGAGCGCTTGTGTGTCAG 54 1.0

115-119

2

0.368

0.301

0.278

DQ020011

DLA0276E

(TG)6 F: GCCCCTAGCGTTGTGTTTTA R: CTCAGAGGGGGTTTCATTCA 54 1.0

158-162

3

0.2

0.267

0.264

DQ020012

DLA0277E

(AC)10(GC)2(AC)3 F: CCACTTGTCAGTTGCTTTGC R: CCACTGATGTCATTACTGTACAAAC 54 1.0

188-192

2

0.45

0.469

0.414

DQ020019

DLA0278E

(AC)9 F: GTATTTGACCGTGGGCTGAT R: TCCCCGTTCTGACTTCTGAC 54 1.0

193-198

5

0.5

0.701

0.675

DQ020020

DLA0279E

(TTG)2(GT)3(TTG)3 F: TCCACAGCGTTTGTTACTGG R: TAAGCGGACGTTGTGTTTCA 54 2.0

164-173

4

0.35

0.306

0.297

DQ020021

DLA0280E

(GTT)3(GT)(GTT)2 GTAT(GTT)2

F: GAAGCGTTCAGAGGTCCATC R: CGTACGACGTGACTCATGCT 54 1.0

137-149

6

0.65

0.664

0.64

CX535572

DLA0281NR0B1

(CAA)13 F: AGAGTGACAGCCTTGCGACT R: GGTGGAGGAGGTCTTGTGAA 54 2.0

124-144

6

0.333

0.435

0.425

AJ633646

Table S2 AFLP primer combinations used and number of markers generated with each primer combination specific for the male parent, female parent and both parents of European sea bass.

EcoRI-ACC primer EcoRI-ACG primer

MseI

primers Male parent

Female

parent

Both

parents Male parent

Female

parent

Both

parents Total

AAA 1 1 1 1 2 0 6

AAT 0 0 0 2 2 0 4

AAG 0 0 0 0 2 1 3

AAC 0 2 0 3 3 1 9

ATT 2 1 0 2 1 3 9

ATG 0 0 0 1 1 0 2

ATC 0 0 0 4 2 0 6

ATA 1 0 0 0 0 0 1

ACA 2 2 1 1 1 0 7

ACT 1 0 1 0 1 2 5

ACG 3 2 0 3 2 1 11

ACC 5 4 1 3 4 0 17

AGA 3 2 3 3 0 0 11

AGT 2 0 0 1 4 0 7

AGG 2 0 0 0 0 0 2

AGC 3 1 0 1 0 3 8

CCC 0 0 1 2 0 1 4

CCA 1 0 0 1 0 0 2

CCT 0 1 0 0 0 1 2

CCG 1 0 1 1 1 0 4

CAC 1 2 0 0 2 0 5

CAA 2 3 1 4 2 1 13

CAT 3 0 0 0 2 1 6

CAG 4 5 5 1 0 0 15

CTC 0 2 0 5 3 4 14

CTA 2 2 0 2 4 0 10

CTT 1 2 0 2 0 0 5

CTG 1 2 1 2 2 1 9

CGC 0 2 0 3 3 2 10

CGA 2 0 1 0 1 0 4

CGT 1 1 0 1 1 0 4

CGG 1 0 0 2 1 2 6

Total 45 37 20 51 47 24 221

Documents

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