4 Interactions of Wild and Reared Fish and Invertebrates
121
Box 4.1 Molecular genetic methods
The development of molecular techniques has been likened to the stocking of
a toolbox, with ever more powerful tools. The molecular methods used in
interaction studies began to be developed in the 1960s, when the allozyme
technique was first used. This method focuses on enzymes and other specific
proteins, which are the products of functional genes, and as such, is an
indirect genetic technique. It involves protein electrophoresis, usually on
starch gels, where products are separated on the basis of charge, and has
generally been superseded by techniques such as microsatellite analysis (see
below), which concentrate directly on genomic DNA. The latter techniques
show much more genetic variability (alleles per locus) both because of
intrinsic differences such as high mutation rate, but also because proteins are
affected by code redundancy and similarity in charge between products of
different genetic composition. In certain cases, however, the allozyme
technique is still useful, as with brown trout Salmo trutta in Spain where most
reared strains used in stocking are effectively fixed for a LDH-C
* allele (or
the now more commonly used underlying nucleotide sequence (McMeel
et al. 2001)) which is almost absent in wild populations, since the former
originate from a northern European population grouping. Other allozyme loci
such as MEP-2
* in Atlantic salmon Salmo salar are influenced by natural
selection and thus may have an important role in local adaptation, therefore
further investigation of either these enzymes or the genes that code for them,
should prove fruitful.
In the 1970s mitochondrial DNA, studied using restriction (specific- cutting)
enzymes, was added to the suite of methods. Restriction enzymes could not in
general be used with the nuclear genome, because its much greater size
resulted in so many fragments that the results were usually ambiguous or
uninterpretable. MtDNA, because of its haploid nature and greater propensity
to accumulate mutations, offered certain advantages, but it was not exploited in
the context of interactions until the polymerase chain reaction (PCR) was
developed in the 1980s. The PCR allows the localisation and amplification of
a specific segment of DNA, using primers (short segments of DNA which
define either end of the target fragment) and a DNA polymerase, to produce
millions of new duplicate copies in vitro. The PCR can be applied to nuclear or
mitochondrial DNA and has enabled sequencing of specific DNA fragments
and investigation of microsatellite and SNP variability (see below).
Microsatellites have become the “marker of choice” for interaction studies
because of their high variability and relatively high frequency throughout the
genome (one locus every 10,000 bp). Box 4.1 Figure 1 shows a typical microsatellite locus from Atlantic salmon. Microsatellites, the loci currently used in
human forensics, consist of tandemly repeated arrays of two, three or four bases
(di-, tri- and tetranucleotides) of largely unknown function (most do not code for
(continued)
121
Box 4.1 Molecular genetic methods
The development of molecular techniques has been likened to the stocking of
a toolbox, with ever more powerful tools. The molecular methods used in
interaction studies began to be developed in the 1960s, when the allozyme
technique was first used. This method focuses on enzymes and other specific
proteins, which are the products of functional genes, and as such, is an
indirect genetic technique. It involves protein electrophoresis, usually on
starch gels, where products are separated on the basis of charge, and has
generally been superseded by techniques such as microsatellite analysis (see
below), which concentrate directly on genomic DNA. The latter techniques
show much more genetic variability (alleles per locus) both because of
intrinsic differences such as high mutation rate, but also because proteins are
affected by code redundancy and similarity in charge between products of
different genetic composition. In certain cases, however, the allozyme
technique is still useful, as with brown trout Salmo trutta in Spain where most
reared strains used in stocking are effectively fixed for a LDH-C
* allele (or
the now more commonly used underlying nucleotide sequence (McMeel
et al. 2001)) which is almost absent in wild populations, since the former
originate from a northern European population grouping. Other allozyme loci
such as MEP-2
* in Atlantic salmon Salmo salar are influenced by natural
selection and thus may have an important role in local adaptation, therefore
further investigation of either these enzymes or the genes that code for them,
should prove fruitful.
In the 1970s mitochondrial DNA, studied using restriction (specific- cutting)
enzymes, was added to the suite of methods. Restriction enzymes could not in
general be used with the nuclear genome, because its much greater size
resulted in so many fragments that the results were usually ambiguous or
uninterpretable. MtDNA, because of its haploid nature and greater propensity
to accumulate mutations, offered certain advantages, but it was not exploited in
the context of interactions until the polymerase chain reaction (PCR) was
developed in the 1980s. The PCR allows the localisation and amplification of
a specific segment of DNA, using primers (short segments of DNA which
define either end of the target fragment) and a DNA polymerase, to produce
millions of new duplicate copies in vitro. The PCR can be applied to nuclear or
mitochondrial DNA and has enabled sequencing of specific DNA fragments
and investigation of microsatellite and SNP variability (see below).
Microsatellites have become the “marker of choice” for interaction studies
because of their high variability and relatively high frequency throughout the
genome (one locus every 10,000 bp). Box 4.1 Figure 1 shows a typical microsatellite locus from Atlantic salmon. Microsatellites, the loci currently used in
human forensics, consist of tandemly repeated arrays of two, three or four bases
(di-, tri- and tetranucleotides) of largely unknown function (most do not code for
(continued)