More microsatellite markers in the sea bass Dicentrarchus labrax L'

1
More microsatellite markers in the sea bass
(Dicentrarchus labrax L.)
D. A. Chistiakov1, B. Hellemans1, C. S.
Tsigenopoulos2, A. Libertini3, G. Kotoulas2, F.
Volckaert1
1 Laboratory of Aquatic Ecology, Katholieke
Universiteit Leuven, Ch. de Bériotstraat 32,
B-3000 Leuven, Belgium 2 Department of Genetics
and Molecular Biotechnology, Institute of Marine
Biology of Crete (IMBC), P.O. Box 2214, Port of
Heraklion, Heraklion, Greece 3 Istituto di
Biologia del Mare di Venezia, Consiglio Nazionale
delle Ricerche, Castello 1364/A, I-30122 Venezia,
Italy
1
MICROSATELLITE ISOLATION AND DEVELOPMENT
INTRODUCTION
Sea bass (Dicentrarchus labrax) aquaculture
maintains its position as one of the
fastest-growing production activities in the
world. From 1991 to 1997 sea bream and sea bass
production in the Mediterranean region grew 6.5
times and yielded almost 65,000 tons in 1997.
However, 20 years of enlarged production of sea
bass have not yet generated a single domesticated
stock. A lack of genetic information strongly
hampers breeding and farming of this species
towards the selective development of economically
important features. Constructing a first
generation linkage and synteny map of the sea
bass genome should support the fastest way to map
to characterize genes responsible for
commercially significant traits. The genetic
management of this species therefore requires
further expansion of the number of polymorphic
genetic markers and microsatellites in
particular. A few dinucleotide microsatellites
of sea bass have been developed starting from
1995. Seven loci have been described by Garcia de
Leon et al. (1995) and two markers were isolated
by Castilho McAndrew (1998). Recently five more
microsatellites were developed by Ciftci et al.
(2002). Here we describe the isolation of eleven
new microsatellites of D. labrax that can be
useful as genetic markers for the development of
a linkage map of sea bass and the genetic
management of this species.
GENOMIC DNA
DIGESTION WITH RSAI
ISOLATION OF SIZE-SELECTED DNA FRAGMENTS (0.2-1
kb) FROM AGAROSE GEL
LIGATION WITH RSAI AFLP ADAPTORS
HYBRIDISATION WITH BIOTINYLATED (AC)12 PROBE
CAPTURE OF DNA BY STREPTAVIDIN PARAMAGNETIC BEADS
THE EU BASSMAP RESEARCH PROJECT
The BASSMAP project (http//www.bassmap.org)
intends to build a first generation medium
density linkage and synteny map of sea bass
consisting of several hundreds of highly
informative polymorphic markers. About one
hundred genes that are responsible for
economically important traits will be mapped
with these polymorphic markers. This will be a
first for a marine fish. It will result in a very
useful tool for breeding sea bass, and it also
sets a standard for other farmed fish and
shellfish on the verge of genetic improvement.
ELUTION OF DNA FROM THE BEADS
PCR AMPLIFICATION WITH RSAI AFLP
ADAPTOR-SPECIFIC PRIMER
TA-CLONING IN pGEM-T VECTOR
STEPS OF THE BASSMAP PROJECT
TRANSFORMATION OF DH-5a E.COLI CELLS
TEST PANEL breeding of test panel
GENOTYPING OF TEST PANEL ESTs Microsatellites A
FLPs
MAP BUILDING data base mapping
PILOT STUDY preliminary analysis of QTLs
BLUE-WHITE SELECTION OF RECOMBINANT CLONES
NEW TOOLS BAC library ESTs microsatellites AFLPs

DNA LIBRARY ENRICHED BY (AC)n AND (TG)n TANDEM
REPEATS
SCREENING FOR REPEAT-CONTAINING CLONES USING
TRIPLE PCR AMPLIFICATION WITH T7, SP6 AND
NON-BIOTINYLATED (AC)12 PRIMERS
RESULTS
Characteristics of the 11 new sea bass
microsatellites
FLUORESCENCE-BASED SEQUENCING OF POSITIVE CLONES
DESIGNING PCR PRIMERS
TESTING PCR PRIMERS
FLUORESCENCE-BASED GENOTYPING USING A TEST PANEL
OF 21 UNRELATED SEA BASS INDIVIDUALS
NEW SEA BASS MICROSATELLITE MARKERS
REFERENCES
Castilho R, McAndrew BJ (1998) Two polymorphic
microsatellite markers in the European seabass,
Dicentrarchus labrax (L.). Animal Genetics, 29,
151-152.
Ciftci Y, Castilho R, McAndrew BJ (2002) More
polymorphic markers in the European sea bass
(Dicentrarchus labrax L.). Molecular Ecology
Notes, 2, 575-576.
CONCLUSIONS
The microsatellite loci showed different
variation patterns in 21 unrelated sea bass
individuals with mean number of alleles 8.6 and
mean observed heterozygosity 0.68. Nine of
these new microsatellites have a number of
alleles (8-13) and observed heterozygosity value
range (0.571-0.857) that are sufficient to be
useful as genetic marker in a constructing sea
bass genome map.
Garcia de Leon FJ, Dallas DJ, Chatain B et al.
(1995) Development and use of microsatellite
markers in seabass, Dicentrarchus labrax
(Linnaeus, 1758) (Perciformes Serranidae).
Molecular Marine Biology and Biotechnology, 4,
62-68.