76
M.S. Clark et al.
technique, a greater number of different sequences are obtained in each library
(gene discovery is more efficient), but no estimate of relative gene copy number is
possible. This technique is more complicated than non-subtracted methodologies.
It can often require larger amounts of RNA or if amplification techniques are
used, sequence bias can result during the amplification process.
Both techniques of library production have their own advantages and disadvantages, the main point is to be aware of these and conduct the in silico analyses
accordingly. The ESTs can then be used either as a data source for DNA markers, or
the clones physically used for the production of gene chips or targeted gene analyses
and to a large extent, it is the former where EST libraries have been put to best use to
date (see Sections 3.1.2 and 3.1.3). Whilst there are numerous papers documenting
the production of EST libraries (e.g. Douglas et al. 2007, Govoroun et al. 2006), they
are invariably gene lists or catalogues for future studies. EST libraries per se cannot
provide accurate information on environmental adaptation and any inferred findings
need to be validated with in-depth functional analyses and experimental manipulation. Also non-model species suffer from lack of functional information associated
with EST data, i.e. many sequences are designated as “unknown” or “putative protein” and this impacts on their usefulness for both functional and population studies
(see Section 3.2.1.1).
3.1.2 DNA Studies: Microsatellites
Microsatellites or SSRs (simple sequence repeats) are small DNA stretches of a
repeated core sequence of few base pairs (e.g. GT repeat units). Because they are
highly polymorphic in length, they have been extensively used since the 1990s
to produce genetic linkage maps and have been used in population assignments,
paternity analyses and fine-scale dispersal analyses. The major drawback of this
technique has long been the tremendous effort needed to generate a statistically
relevant number of such polymorphic loci in non-model organisms (Zane et al.
2002).
The availability of genome databases for model organisms has considerably
enhanced the possibility of finding microsatellites in non-model organisms by defining primers in regions conserved across a large set of organisms, but there is also the
increasing resource of EST libraries where such markers are present in 4% of the
bivalve cDNA sequences in GenBank (Saavedra and Bachere 2006). The frequency
of SSR EST-based data is highly variable across species. However, microsatellites
have been found in numerous ESTs libraries constructed for a wide range of ecologically and/or economically important marine species including invertebrates (e.g. the
European clam Ruditapes decussatus, the blue mussel Mytilus edulis, the Japanese
oyster Crassostrea gigas and the deep-sea vent mussel, Bathymodiolus azoricus,
(Tanguy et al. 2008), the bay scallop Argopecten irradians (Roberts et al. 2005)),
fishes (e.g. cod, Gadus morhua; halibut, Hippoglossus hippoglossus, Douglas et al.
M.S. Clark et al.
technique, a greater number of different sequences are obtained in each library
(gene discovery is more efficient), but no estimate of relative gene copy number is
possible. This technique is more complicated than non-subtracted methodologies.
It can often require larger amounts of RNA or if amplification techniques are
used, sequence bias can result during the amplification process.
Both techniques of library production have their own advantages and disadvantages, the main point is to be aware of these and conduct the in silico analyses
accordingly. The ESTs can then be used either as a data source for DNA markers, or
the clones physically used for the production of gene chips or targeted gene analyses
and to a large extent, it is the former where EST libraries have been put to best use to
date (see Sections 3.1.2 and 3.1.3). Whilst there are numerous papers documenting
the production of EST libraries (e.g. Douglas et al. 2007, Govoroun et al. 2006), they
are invariably gene lists or catalogues for future studies. EST libraries per se cannot
provide accurate information on environmental adaptation and any inferred findings
need to be validated with in-depth functional analyses and experimental manipulation. Also non-model species suffer from lack of functional information associated
with EST data, i.e. many sequences are designated as “unknown” or “putative protein” and this impacts on their usefulness for both functional and population studies
(see Section 3.2.1.1).
3.1.2 DNA Studies: Microsatellites
Microsatellites or SSRs (simple sequence repeats) are small DNA stretches of a
repeated core sequence of few base pairs (e.g. GT repeat units). Because they are
highly polymorphic in length, they have been extensively used since the 1990s
to produce genetic linkage maps and have been used in population assignments,
paternity analyses and fine-scale dispersal analyses. The major drawback of this
technique has long been the tremendous effort needed to generate a statistically
relevant number of such polymorphic loci in non-model organisms (Zane et al.
2002).
The availability of genome databases for model organisms has considerably
enhanced the possibility of finding microsatellites in non-model organisms by defining primers in regions conserved across a large set of organisms, but there is also the
increasing resource of EST libraries where such markers are present in 4% of the
bivalve cDNA sequences in GenBank (Saavedra and Bachere 2006). The frequency
of SSR EST-based data is highly variable across species. However, microsatellites
have been found in numerous ESTs libraries constructed for a wide range of ecologically and/or economically important marine species including invertebrates (e.g. the
European clam Ruditapes decussatus, the blue mussel Mytilus edulis, the Japanese
oyster Crassostrea gigas and the deep-sea vent mussel, Bathymodiolus azoricus,
(Tanguy et al. 2008), the bay scallop Argopecten irradians (Roberts et al. 2005)),
fishes (e.g. cod, Gadus morhua; halibut, Hippoglossus hippoglossus, Douglas et al.