Allozyme analysis

9.2.1 Allozyme analysis

standing of stock structure, their reconciliation into a unified model can be challenging (McQuinn The first genetic tool to be widely adopted for fish 1997).

population analysis was protein electrophoresis. Genetic analyses are not only useful in helping One of the earliest studies was Sick’s (1961)

Table 9.1 The major current genetic tools used in fisheries science, with some general comments on their properties. The comments are necessarily partly subjective and sometimes oversimplified. For example, the costs of microsatellite analysis are significantly less when primer sequences are already available than when they have to be developed anew. The suitability of these tools in different areas of fisheries science is summarized in Table 9.2.

Expense Genetics

Tissue

Number of

Variability Coding/

Comments

of markers non-coding Allozymes

Still an excellent detectable protein

tool if suitable variation

frozen

tissue available MtDNA-RFLP

Fragment size

Effectively a variation following

single marker restriction enzyme

digestion MtDNA-PCR-

Effectively a RFLP

As above, but using

PCR products

single marker

inherited

preserved

Effectively a Genetics sequencing

MtDNA Nucleotide sequence

single marker

inherited

preserved

Randomly DNA fragment size

Reproducibility a amplified

non-coding concern; highly polymorphic

variation following

frozen,

sensitive to PCR DNA (RAPD)

use of primers of

preserved

random sequence conditions Microsatellite

Variation in number

Non-coding Development time of tandem repeats of

a deterrent; dinucleotide/

(some

frozen,

scoring problems tetranucleotide motifs

recessive null

preserved

not unknown Minisatellite

alleles)

As above but tandem

Non-coding Usually scored repeats longer

with Southern

blots; not popular Amplified

preserved

Still novel for fish; fragment length of fragments after

Presence/absence

non-coding multiple bands can polymorphism

frozen,

hinder cross-gel (AFLP)

selective

preserved

amplification comparisons Exon-primed

Non-coding Still novel, but intron-crossing

Sequence or size

with considerable 201 PCR (EPIC)

variation in introns

frozen,

potential PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; mtDNA, mitochondrial DNA.

preserved

Chapter 9

Table 9.2 Suitability/uses of the current genetic tools in fisheries science. Columns graded * to *****; for example allozymes are very suitable for species identification and somewhat less so for aquaculture genetics. Scorings are overall scores; for example mtDNA sequencing is a very powerful approach to species identification but given its expense is rated below the mtDNA-PCR-RFLP approach. Scores marked with a ? indicate that these approaches have been little explored as yet, but that these are likely scores based on the properties of these markers. Column headings are broad; for example, aquaculture genetics embraces diversity estimation, pedigree analysis and gene mapping studies. Some tools might be better in some subcategories than others; for example AFLPs will likely be of more use in aquaculture in mapping than studies of gene diversity.

Systematics Aquaculture genetics Allozymes

Species identification

Population genetics

** MtDNA sequencing

***? AFLP, amplified fragment length polymorphism; EPIC, exon-primed intron-crossing PCR; mtDNA, mitochondrial

DNA; PCR, polymerase chain reaction; RAPD, randomly amplified polymorphic DNA; RFLP, restriction fragment length polymorphism.

examination of haemoglobin variation in whiting with the limited number of loci that can be as- (Merlangius merlangus) and cod (Gadus morhua). sessed (usually <50), can give little variation for The subsequent advent of enzyme-specific histo- screening purposes. There is also a lingering debate chemical staining made it possible to examine a over whether the variation detected is or is not large range of enzymes, a procedure often referred selectively neutral. Lewontin (1974) gives a classic to as allozyme analysis. The seminal studies of account of this controversy. Population genetic Lewontin and Hubby (1966) and Harris (1966) interpretation is generally based on the null showed that genetically determined allozyme hypothesis of selective neutrality, so that popula- variation was widespread in Drosophila and tion differences are held to reflect reproductive iso- human populations. Fish populations were soon lation and genetic drift. However, it is possible that shown to be no different, and for the first time a some of the amino acid differences responsible for simple, quick and reliable genetic method was allozyme separation affect function in some way; available for examining fish population structure. if this is so, then population differences might Practical details may be found in Richardson et al. reflect differences in selection pressures rather (1986), Whitmore (1990), Manchenko (1994) and than isolation. Hillis et al. (1996).

A modification of protein electrophoresis is

Allozyme analysis remains a highly attractive isoelectric focusing (see Whitmore 1990), where approach, when fresh or frozen tissue is available, proteins are electrophoretically separated on a pH in terms of speed and cost. Many allozymes are gradient gel on the basis of their surface electrical tissue-specific, so the availability of different tis- charge alone, rather than size and charge as in sues including muscle, liver and nerve (eye) is ad- conventional electrophoresis. They are sharply vantageous. Significant disadvantages include low ‘focused’ at their isoelectric point, the pH at which average degrees of variability which, when coupled the protein has a net surface charge of zero.

Genetics

9.2.2 Female DNA analysis

Male

a c The ability to examine DNA variation directly, Nuclear DNA rather than indirectly through protein variants,

b d came with the recognition and isolation of restric- Mitochondrial

X Y tion enzymes. There are many such enzymes, DNA

which cut or ‘restrict’ DNA at enzyme-specific nucleotide sequences, usually of four or six bases. Unfortunately, with the constant development of

Genotypes of offspring new analytical DNA techniques, comprehensive

(male or female) manuals are lacking. Two good sources of in-

formation are Hillis et al. (1996), who provide a a b b an overview of molecular techniques and data

c d c d analysis, and Caetano-Anollés and Gresshoff (1997), who provide details on selected methods of

YY DNA marker analysis and their applications, in-

cluding methods not discussed here. A brief out- Fig. 9.1 Inheritance of nuclear DNA and mitochondrial line of some of the major and more commonly used DNA. The male parent has genotype a/b at a nuclear methods in fisheries population genetics follows; DNA locus, and mitochondrial haplotype X. The female parent has genotype c/d at the nuclear DNA locus, and other approaches are included, briefly, in Tables mitochondrial haplotype Y. The offspring have one of

9.1 and 9.2. four nuclear DNA genotypes (a/c, a/d, b/c and b/d) but all have the mitochondrial DNA haplotype Y.

Mitochondrial DNA

Most initial DNA analyses were of mitochondrial DNA (mtDNA), largely because this genome ing it possible to choose a relatively variable region (usually about 16–18 kb) is much smaller than for population studies or a relatively conserved nuclear DNA (nDNA) and was easier to examine region for taxonomic or systematic studies. with the techniques available some 10–15 years

However, mtDNA also has significant disad- ago. Mitochondrial DNA was extracted and cut vantages. Principal among these is that it is a non- with restriction enzymes; the resulting fragments recombining genome and best treated as a single were separated by electrophoresis and visualized. character, whereas nDNA assessment can be This is a restriction fragment length polymor- based on many independent characters (loci). phism (RFLP) analysis.

Mitochondrial DNA is inherited differently

Polymerase chain reaction

from nDNA (Fig. 9.1): it is only inherited from the maternal parent, with very rare exceptions, and is Analyses of DNA variation, both mtDNA and haploid in nature. The genetically effective popu- nDNA, were considerably enhanced by the lation size of mtDNA is thus, other factors being development of polymerase chain reaction (PCR) equal, only one-quarter that of nDNA, which is techniques. PCR analysis allows the repeated inherited from both parents and is diploid in each amplification of a DNA region lying between parent. This makes it potentially a more sensitive two DNA primers, the primers generally being indicator of genetic drift than nDNA. Further- sequences of around 20 bp that are complementary more, its overall rate of evolution is about an order to sequences in the target DNA. These techniques of magnitude faster than single-copy nDNA. The typically allow the amplification of a DNA region, non-coding control region (d-loop) of mtDNA flanked by the complementary primer sequences, evolves more rapidly than coding segments, mak- that is some hundreds of base pairs long, although

Chapter 9

regions of several thousand base pairs can now be amplified successfully. Once the target fragment has been amplified, it can be examined for size or sequence variation.

This general approach is very flexible and lends itself to a wide variety of adaptations and modifica- tions. Many of these relate to primer design and make it possible to target different classes of mtDNA or nDNA sequence. The amplified fragments can be assessed by a wide variety of methods, including RFLP analysis, sequencing, size analysis, single-strand conformational poly- morphisms and dot plots. Furthermore, very little tissue is needed as the target DNA is amplified many-fold from the small amount initially avail- able. This permits non-invasive and non-lethal sampling. DNA can be extracted and amplified from old fish scales (Nielsen et al. 1999) or museum specimens (Pichler and Baker 2000), permitting historic surveys of changes in genetic variability. Tissue can be stored at room tempera- ture in alcohol or other chemicals such as dimethyl sulfoxide, simplifying the logistics of sampling and freight. PCR analyses have now largely supplanted non-PCR analyses.

Microsatellites

Microsatellites are nDNA regions of repeated sequences where the repeat sequence or motif is short, 2–5 bp. An example is TCTCTCTC, which is a 2-bp or dinucleotide repeat. The total micro- satellite length is usually less than 300 bp (Tautz 1989). Microsatellites are flanked by non- repeat sequences that are conserved within species, making it possible to design PCR primers which will amplify the microsatellite region. Microsatellite variability arises principally from variation in the number of repeats of the motif, leading to size changes in the PCR product that can

be identified on electrophoresis gels. Microsatel- lites are usually in non-coding regions and thus likely to be selectively neutral. The mutation rate of microsatellite loci is very high, and consequently dozens of alleles per microsatellite locus are not uncommon. Allele data are most readily collected using primers with fluorescent

labels and an automated DNA sequencer, al- though non-automated approaches are also popular. Unfortunately, primers tend to be specific to an individual species or species group, as muta- tions that accumulate in the flanking region quickly reduce the binding ability of the primers. Microsatellite primers generally have to be devel- oped anew for each species or species group; this time-consuming development phase can require several months of skilled labour.

Microsatellites provide an abundant supply of hypervariable codominant markers for fish studies (O’Connell and Wright 1997). This is an advantage in pedigree or mapping studies, but too many very low frequency alleles can reduce the power of population structure analyses. Scoring of geno- types can sometimes prove difficult, especially when there are many alleles of similar size and when PCR products of a single allele produce multiple bands on gels, a phenomenon known as band ‘stuttering’ or ‘laddering’. Non-amplifying or ‘null’ alleles have also been recorded; these can arise from mutations in the primer site so that a particular allele is not amplified or from the PCR process not amplifying a long allele as well as a short allele. Null alleles are recessive alleles. In population surveys, an individual heterozygous for an expressed allele and a null allele will usually

be mistakenly scored as homozygous for the ex- pressed allele.

Randomly amplified polymorphic DNA

Another PCR technique, less widely used in fish- eries, is the analysis of randomly amplified poly- morphic DNA (RAPD; Welsh and McClelland 1990; Williams et al. 1990). Primers of random nucleotide sequence, typically around 10–20 bp, are used to amplify anonymous segments of DNA. Variation in primer binding sites leads to variation in PCR products, which are again scored from size variation in electrophoresis gels. This is a quick and relatively simple technique, although amplifi- cation occurs in conditions of low stringency and repeatability of observations requires careful attention to all aspects of the procedure. It is a dominant/recessive system, and DNA fragments

Genetics

of any particular locus in any individual are gener- of distantly related species (e.g. Quattro and Jones ally scored as present (genotypes + + or + -) or ab- 1999).

sent (genotype - -).