The First Generation Linkage Map of the European Sea Bass

1
The First Generation Linkage Map of the European
Sea Bass
BASSMAP Consortium D. A. Chistiakov, B.
Hellemans, F. A. M. Volckaert (coordinator)
Katholieke Universiteit Leuven (KULeuven),
Belgium, C. S. Tsigenopoulos, G.
Kotoulas Institute of Molecular Biology and
Biotechnology (IMBB), Greece H. Whitaker,
J. B. Taggart, B. McAndrew Institute of
Aquaculture (IoA), UK A. S. Low, N.
Bartley, C.S. Haley Roslin Institute (RI),
UK) D. Bertotto, A. Libertini CNR
Institute of Marine Biology (IBM),
Italy C. Batargias Nireus Chios
Aquaculture S.A. (Nireus), Greece D.
Chatziplis, A. Georgoudis Aristotle University
of Thessaloniki (AUTh), Greece
Source http//www.FishBase.org
EC Fifth Framework Programme
http//www.bassmap.org

• AIMS and STRUCTURE of the EU BASSMAP
  PROJECT
• (2001-2005)
• production of a medium density linkage and
  synteny map of the European sea bass,
  Dicentrarchus labrax.
• construction of a Bacterial Artificial
  Chromosome (BAC) library
• development of informative Expressed Sequence
  Tags (ESTs) and 600
• polymorphic markers (microsatellites and
  amplified fragment length
• polymorphisms (AFLPs))
• breeding of a test panel of sea bass
• genotyping with polymorphic markers for
  mapping purposes
• mapping of 100 functional genes
• application of the linkage map in a pilot
  study (identification of quantitative
• trait loci (QTLs) or candidate genes of
  interest to breeding programmes.

• TEST PANEL (IBM)
• different types of F1 progeny (outbred,
  meiotic gynogenetic, single and double haploid)
  have been produced (Francescon et al., 2004)
• a test panel (two parents and 50
  offspring of a single outbred cross) for further
  genetic mapping studies was selected

• NEW TOOLS (IoA)
• BAC library
• contains 69, 000 clones with an average size of
  genomic DNA insert of 150kb
• 6x coverage of the haploid sea bass
  genome (900 megabases)
• spotted onto high density macroarrays
  (25,000/filter) to facilitate
• facilitate further analyses

Example of 10 Not I digested sea bass BAC clones
separated by PFGE. The clones are flanked on
each side by a ? ladder and a ? Hind III marker.
White lines delineate the 100 - 200 kb gel region
while the black line marks 150 kb. The origin is
at the top of the picture. The lowest band in
each BAC lane represents the linearised vector
(8.1kb kb).
TEST PANEL breeding of test panel
GENOTYPING OF TEST PANEL ESTs microsatellites
AFLPs
PILOT STUDY preliminary analysis of QTLs
NEW TOOLS BAC library ESTs microsatellites AFLP
s

• DATA HANDLING (RI)
• ResSpecies database
• To date, the RESBASS database which is part of
  the ResSpecies database (http//www.resspecies.or
  g) hosted by the Roslin Instutute, contains
• 127 sea bass microsatellites
• one microsatellite from sea
  bream,Sparus aurata (Batargias et al., 1999)
• 226 AFLP markers

MAP BUILDING data base mapping

• NEW TOOLS (IoA, IMBB)
• ESTs
• three types of cDNA libraries (larvae,
  and adult brain and liver) were constructed
• 720 ESTs (520 from the larvae and 200
  from the brain) have been isolated, sequenced
  and screened for similarity with the existing
  gene sequences from nucleotide databases
• among these, the 100 most important ESTs
  will be chosen and screened for polymorphic
  markers (microsatellites and SNPs) to be
  subsequently mapped

• LINKAGE ANALYSIS (RI)
• Map building
• The current version of the sex-averaged
  linkage map of sea bass
• comprises 20 linkage groups of the
  expected n24 (haploid karyotype)
• consists of 111 microsatellite loci
• covers 569.6 cM of the expected 900 cM

• GENOTYPING OF TEST PANEL (KULeuven, IMBB)
• Microsatellites and AFLPs
• five types of microsatellite-enriched
  libraries have been developed using selective
  hybridization with di-, tetra- and
  pentanucleotide probes
• 141 new microsatellites have been isolated
  from these libraries (Tsigeno-
• poulos et al., 2003 Chistiakov et al.,
  2004) and genotyped in the test panel
• 13 of the 14 previously published
  microsatellites of sea bass (Garcia de Leon et
  al., 1995 Castilho and McAndrew, 1998 Ciftci et
  al., 2002) have been reevaluated.
• 31 putative EST-derived microsatellites as the
  result of the screening of 22
• known genes and 154 ESTs from sea bass cDNA
  libraries
• 226 polymorphic AFLPs have been produced by
  genotyping of the test panel (64 combinations
  of AFLP primers have been tested)

HAPLOID KARYOTYPE OF SEA BASS
Example of AFLP genotyping of the test panel. PCR
products of selective amplification, using
genomic DNA from both parents father (F) and
mother (M) and fifty F1 progeny and MseI-ACC
unlabelled and EcoRI-ACG labelled primers, have
been separated in a 7 polyacrylamide gel.
QTL MAPPING (IMBB, AUTh, NIREUS) 2500 F1 progeny
produced by random cross experiments at the
commercial partner NIREUS, will be screened in
two triplex PCRs to select a testing population
of 500 individuals. These 500 fish will be
genotyped at IMBB using polymorphic markers
located across two selected linkage groups to map
QTLs linked to growth and sex determination.
Methodology for the detection of multiple QTLs
using sib pair analysis has been developed at
AUTh.
F M progeny-------------------------------------
--------------------------------------------------
--------------------------------------------------
-----------------------------? F M
REFERENCES Batargias et al. (1999) Mol. Ecol. 8
897-898 Castilho McAndrew (1998) Anim. Genet.
29 151-152 Ciftci et al. (2002) Mol. Ecol. Notes
2 574-576 Chistiakov et al. (2004) Anim. Genet.
35 53-57 Garcia de Leon et al. (1995) Mol. Mar.
Biol. Biotechnol. 4 62-68 Francescon et al.
(2004) Aquaculture 236 231-239 Tsigenopoulos et
al. (2003) Mol. Ecol. Notes 3 352-354
For information Filip Volckaert, Laboratory of
Aquatic Ecology, Katholieke Univeriteit Leuven,
B-3000, Leuven, Belgium. E-mail
filip.volckaert_at_bio.kuleuven.ac.be Poster
prepared by Dimitry Chistiakov (KULeuven)
ACKNOWLEDGEMENTS BASSMAP is funded by the
European Commission. Contract No. Q5RS-2001-01701