3 Populations and Pathways
81
The most powerful use of short-read sequence data is comparison to other
genomes that are sequenced at high quality. For instance, Vera et al. (2008) used
the silkworm Bombyx mori to BLAST sequences and to further establish useful
contigs for the Nymphalid butterfly (Melitaea cinxia). The B. mori’s genome has
been estimated to have about 18,000 genes, and assuming that this is representative
for Lepidoptera, Vera et al. (2008) obtained 9,000 non redundant hits in their study.
This may suggest that at least half of all genes in M. cinxia are now at least partially identified using a single pyrosequencing run. As Ellegren (2008) noted about
Vera et al.’s (2008) study, this technology offers support for association mapping,
QTL and population genetics studies, and candidate gene studies (e.g. Saastamoinen
and Hanski 2008). Even though the M. Cinxia genome data is still a draft, such a
study and that of Toth et al. (2007) on the Polistes wasp proved that high-throughput
techniques of cDNA sequencing can be reliably used in species that are of ecological and evolutionary relevance. A further example of the use of such technology is
given in Section 3.3.4 and the analysis of hybrid vigour. As stated earlier, the use of
this technique will increase and, as costs decrease, it becomes a viable alternative to
gene chip expression analyses.
3.1.6 DNA and RNA Studies: Targeted Gene Analyses
Targeted gene analyses or the candidate gene approach (Tabor et al. 2002) are where
a particular gene or a small set of genes is intensively investigated in an organism
or population under different conditions (either natural or artificially induced). It is
difficult to perform targeted gene analyses on non-model organisms where there is
either little or no sequence data available. Gene sequences can be produced via EST
libraries and then specific primers designed for the gene of interest, or a more random approach can be adopted using degenerate PCR. This latter technique involves
identifying the gene of interest in several different species, hopefully including data
from the same taxa as the species of interest and designing primers from the amino
acid sequence incorporating codon usage degeneracy.
In expression work, targeted gene analyses are currently used where there is
some knowledge of which genes may change in expression levels between environmental conditions/treatments and knowledge of the actual gene sequences. These
days, analysis of target genes in expression studies is carried out using Q-PCR.
DNA studies have historically focused on phylogenetic analyses and a specific
set of genes such as cytochrome c oxidase subunit I (COI) and the ribosomal
genes (18s, 16s and 28s) (http://www.barcoding.si.edu/; http://rdp.cme.msu.edu/,
http://bioinformatics.psb.ugent.be/webtools/rRNA/). The COI gene is the gene of
choice for barcoding, a technique that is described in more detail in Chapter 1
However, developments in sequencing technologies have made it possible to resequence a target gene in many individuals from a population in parallel. The
resulting haplotype information is then used to determine either whether specific
alleles can be associated with traits of interest or coalescence-based methods to
tackle selective and hybridizing processes in speciation of closely-related species
81
The most powerful use of short-read sequence data is comparison to other
genomes that are sequenced at high quality. For instance, Vera et al. (2008) used
the silkworm Bombyx mori to BLAST sequences and to further establish useful
contigs for the Nymphalid butterfly (Melitaea cinxia). The B. mori’s genome has
been estimated to have about 18,000 genes, and assuming that this is representative
for Lepidoptera, Vera et al. (2008) obtained 9,000 non redundant hits in their study.
This may suggest that at least half of all genes in M. cinxia are now at least partially identified using a single pyrosequencing run. As Ellegren (2008) noted about
Vera et al.’s (2008) study, this technology offers support for association mapping,
QTL and population genetics studies, and candidate gene studies (e.g. Saastamoinen
and Hanski 2008). Even though the M. Cinxia genome data is still a draft, such a
study and that of Toth et al. (2007) on the Polistes wasp proved that high-throughput
techniques of cDNA sequencing can be reliably used in species that are of ecological and evolutionary relevance. A further example of the use of such technology is
given in Section 3.3.4 and the analysis of hybrid vigour. As stated earlier, the use of
this technique will increase and, as costs decrease, it becomes a viable alternative to
gene chip expression analyses.
3.1.6 DNA and RNA Studies: Targeted Gene Analyses
Targeted gene analyses or the candidate gene approach (Tabor et al. 2002) are where
a particular gene or a small set of genes is intensively investigated in an organism
or population under different conditions (either natural or artificially induced). It is
difficult to perform targeted gene analyses on non-model organisms where there is
either little or no sequence data available. Gene sequences can be produced via EST
libraries and then specific primers designed for the gene of interest, or a more random approach can be adopted using degenerate PCR. This latter technique involves
identifying the gene of interest in several different species, hopefully including data
from the same taxa as the species of interest and designing primers from the amino
acid sequence incorporating codon usage degeneracy.
In expression work, targeted gene analyses are currently used where there is
some knowledge of which genes may change in expression levels between environmental conditions/treatments and knowledge of the actual gene sequences. These
days, analysis of target genes in expression studies is carried out using Q-PCR.
DNA studies have historically focused on phylogenetic analyses and a specific
set of genes such as cytochrome c oxidase subunit I (COI) and the ribosomal
genes (18s, 16s and 28s) (http://www.barcoding.si.edu/; http://rdp.cme.msu.edu/,
http://bioinformatics.psb.ugent.be/webtools/rRNA/). The COI gene is the gene of
choice for barcoding, a technique that is described in more detail in Chapter 1
However, developments in sequencing technologies have made it possible to resequence a target gene in many individuals from a population in parallel. The
resulting haplotype information is then used to determine either whether specific
alleles can be associated with traits of interest or coalescence-based methods to
tackle selective and hybridizing processes in speciation of closely-related species