Dominant markers

Dominant markers are also known as multi-locus markers because they simultaneously generate data from multiple loci (Figure 2.4). They typically work by using random primers to amplify anonymous regions of the genome, producing a pattern of multiple bands from each individual. Because they use random primers to amplify fragments of DNA, no prior sequence knowledge is required and therefore the development time may be relatively short. Furthermore, because dominant markers each characterize multiple regions of the genome, they often show reasonably high levels of polymorphism that can be useful for inferring close genetic relationships; however, dominant markers are generally unable to resolve more distant evolutionary relationships. Perhaps the biggest drawback to these markers is that their dominant nature means that only one allele can be identified at each locus, and therefore heterozygotes cannot be differentiated from homozygotes. The presence of a band means that an individual is either homozygous (AA, with both alleles producing fragments) or heterozygous (Aa, with only the dominant allele producing a fragment) at that particular locus. Individuals that are homozygous recessive will not produce a band.

The inability to differentiate between homozygotes and heterozygotes makes it difficult to calculate allele frequencies from dominant markers. Methods for doing this do exist but several assumptions must be made, such as the population being in Hardy-Weinberg equilibrium, and as we shall see in the next chapter this is not always the case. The anonymity of dominant markers also can make it difficult to detect contamination and to compare data between studies. Nevertheless, as we will see below, dominant markers have been employed successfully in many studies of molecular ecology.

Random amplified polymorphic DNA

In 1990 a PCR-based technique known as random amplified polymorphic DNA

(RAPDs) was introduced as a method for genotyping individuals at multiple loci (Welsh and McClelland, 1990; Williams et al, 1990). RAPDs are generated using short (usually 10 bp) random primers in a PCR reaction. As there are about one million possible primers that can be made from ten bases, there is plenty of scope for detecting polymorphism by using a different primer in each reaction. A single primer is added to each PCR reaction, and multiple bands arise by chance when the primer happens to anneal to two reasonably proximate sites. The banding pattern for each individual will depend on where suitable primer binding sites are located throughout that individual's genome (Figure 2.8). Although RAPDs can provide a relatively quick and straightforward method for quantifying the genetic

Individual 1 1 2

Individual 2

Figure 2.8 (a) RAPD priming sites (indicated by black boxes) are distributed throughout the genome, although here only two partial chromosomes are represented. The sizes of the products (shaded in grey) that will be amplified during PCR will depend on the locations of these priming sites. (b) Diagrammatic representation of the gel that would follow PCR-RAPD screening of these two individuals. Recall that the rate at which a band migrates through the gel is inversely proportional to its size similarity of individuals, its reproducibility depends on stringent laboratory conditions. The amplification of bands will often vary, depending on a variety of factors including the starting concentration of DNA, the parameters that are used in the PCR cycle, the type of PCR machine used (results often vary between laboratories) and the particular brand of the reagents used. Furthermore, the random nature of RAPDs means that it can be difficult to identify bands that have been amplified from non-target DNA.

If the problems of reproducibility and contamination are overcome, RAPDs can be used to estimate the genetic similarity between two individuals based on the number of bands they share. However, despite their ease of use, a general lack of reproducibility, combined with their dominant nature, has decreased the popularity of RAPDs in recent years. The journal Molecular Ecology actively discourages researchers from submitting manuscripts that report population genetic studies that are based primarily on RAPD data, in part because other more reliable markers now are widely available. That is not to say that there is no role for RAPDs at all. As the Molecular Ecology editorial board points out, RAPDs can be useful in genetic mapping studies. They can also provide markers that are diagnostic of a given species or trait if RAPD screening identifies a band that consistently differs between two groups. This was the case in the toxic dinoflagellate Gymnodinium catenatum, which apparently was introduced to Tasmanian waters in the ballast water of cargo ships (Bolch et al., 1999). Unique RAPD banding patterns from different populations allowed the authors of this study to eliminate Spain, Portugal and Japan as source populations, and led them to conclude that the populations causing algal blooms around Tasmania were restricted largely to local estuaries. They did not, however, have enough data to pinpoint the source of the original Tasmanian introductions.

Amplified fragment length polymorphisms

A more labour intensive, but also more reliable, method than RAPDs for generating PCR-based multi-band profiles is known as amplified fragment length polymorphism (AFLPs) (Vos et al., 1995). AFLP markers are generated by first digesting DNA with two different restriction enzymes that cut the DNA so that one strand overhangs the other strand by one or a few bases, thereby producing overlapping ('sticky') ends. Meanwhile, short DNA linkers are synthesized so that one end of the linker is compatible with the overhanging sequence of DNA. The linkers are ligated to the original DNA fragments, leaving a collection of fragments that have the same DNA sequence at the end. These fragments can then be amplified using primers that anneal to the linker DNA sequence (Figure 2.9). Specificity of primers is usually increased by adding one to three nucleotides at one end of the sequence, because PCR requires a perfect match between the target sequence and the 3' end of the primer. This results in the amplification of multiple

Digest with Eco Rl and Mse I

Adaptor ligation

Preamplification

Selective amplification

-AATTC-

Eco Rl adaptor

5 x -CTCGTAGACTGCGTACCAATTC-3 x - CATCTGACGCATGGTTAAG-

Mse I adaptor

-TTACTCAGGACTCA-3x -AATGAGTCCTGAGTAGCAG-5'

Eco Rl primer

5 x -CTCGTAGACTGCGTACCAATTC— 3 x-CATCTGACGCATGGTTAAG—

-TTACTCAGGACTCA-3 x -AATGAGTCCTGAGTAGCAG-5'

Eco Rl primer

GACTGCGTACCAATTC(+n+3n)

(+n)AATGAGTCCTGAGTAGCAG

Mse I primer

-CTCGTAGACTGCGTACCAATTC-3 x-CATCTGACGCATGGTTAAG-

-TTACTCAGGACTCA-3 x -AATGAGTCCTGAGTAGCAG-5'

(+n+3n)AATGAGTCCTGAGTAGCAG

Mse I primer

Figure 2.9 Schematic diagram showing how AFLP genotypes are generated. Digestion with two restriction enzymes produces sticky ends to which linkers can be ligated. During preamplification, the addition of a single base to the 3' end of each primer will reduce the number of amplified fragments to 1/16 of the number of fragments that otherwise would be amplified. The addition of three more bases to the 3' primer ends during selective amplification further reduces the chance of a perfect match between primers and target sequences, and as a result only 1/65 536 of the original set of fragments will be amplified fragments that appear as a series of different-sized bands when run out on a gel. The pattern of bands will depend on the sequences that are immediately adjacent to the linkers, and also on the distances between the restriction sites. As with RAPDs, the generation of bands is essentially random; in contrast to RAPDs, however, this method has a much higher level of reproducibility and therefore has become a more popular method of dominant genotyping.

The genetic similarity of individuals and populations can be inferred from the numbers of AFLP bands that they have in common. Additional information can be obtained by modifying the standard AFLP method to study gene expression. By ligating linkers to digests of cDNA, researchers can compare the banding patterns of genes that have been expressed, as opposed to the entire genome. This method was used to compare two genetically distinct lines of the endoparasitic wasp Venturia canescens that differ in a number of ways, including oviposition behaviour, numbers of eggs laid and growth rates in the early stages of embryonic development. Researchers used the cDNA-AFLP method to compare gene expression in ovarian tissue between the two groups and found differences in a number of expressed genes, including some that apparently are involved in the regulation of protein degradation during stress responses (Reineke, Schmidt and Zebitz, 2003). This study provided some interesting suggestions about the importance of gene expression during early development.

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