Molecular genetics

Recent advances in molecular biology have resulted in methodologies that can be used to determine the sex of individual birds, paternity and kinship, geographical structuring within species, phylogenetic relationships among species and the timing of speciation events. See Brown (2001) for further information on DNA technologies.

DNA can be obtained from a wide range of sources. From dead birds, any tissue can be used. From living birds, blood is the most practical, and this can be obtained as a blood sample following venepuncture, or from the base of a plucked feather. An advantage of birds over mammals is that their red blood cells are nucleated and so only a small sample is required. DNA can also be obtained directly from shed feathers (Eguchi and Eguchi 2000; Bello et al. 2001). DNA is extracted from the tissue sample using protein-denaturing agents, salt and solvents.

Identifying individuals and relatives. In a major breakthrough that led to the process of DNA fingerprinting or profiling, Jefferies et al. (1985a) discovered that specific nucleotide sequences occurred in a repeated order, called tandem repeats, with high levels of variation meaning that individuals differ in their numbers of repeats. Thus by determining the lengths of these repeated sequences it is possible to discriminate between different individuals. To achieve this, restriction enzymes are added that cut the DNA whenever a given sequence of bases (e.g. GACCAT) occurs, so providing a large numbers of fragments of DNA. When these fragments are added to a gel with an electric current the shorter fragments move more rapidly. Probes are added that attach (hybridize) to the specific repeated sequence and make them visible. The different length fragments thus produce different bands. As chromosomes occur in pairs there will be two bands for each repeat sequence region and as the tandem repeats occur in a number of places in the genome the result is a series of bands (the DNA fingerprint) that is unique to an individual (Jeffreys et al. 1985b; Burke and Bruford 1987). See Burke (1989a,b), Burke et al. (1991), and Krawczak and Schmidtke (1998) for methodological details.

Repetitive sequence elements that occur in tandem are known as minisatellite sequences (typically 10 to 100 bases long) or microsatellite sequences (less than 10 bases). In practice minisatellites are used for DNA fingerprinting as described above. However, this method requires good quality DNA and can be difficult to analyze. As a result there is an increasing use of microsatellites and "single locus probes." These use exactly the same concept, but analyze just a single region of tandem repeats so an individual will have just two bands, one from each chromosome (or a single band if homozygous). This method typically incorporates polymerase chain reaction (PCR), which makes multiple copies of a few stands of DNA, so allowing the analysis of extremely small samples.

Each individual will have two bands at each microsatellite locus, one inherited from each parent. By combining many loci it is possible to estimate relatedness among individuals, for example, Hoglund and Shorey (2003) used microsatellites to determine the frequency of full sib and half sib relationships on a White-bearded Manakin Manacus manacus lek. This method is routinely used to determine the parentage of offspring and the frequency and source of dumped eggs.

Relatedness among species and populations. Because mitochondrial DNA (mt DNA) is passed down the maternal line, it is of no value in establishing paternity. However, sequencing regions of the mitochondrial genome can be used to investigate phylogenetic relationships among populations of the same species and among species. Mitochondrial DNA is thought to have a mutation fixation rate several times greater than nuclear DNA, making it extremely variable and has the further advantage of not being recombined during meiosis so giving a clear line of descent. It is easy to work with because it is single copy gene (one allele per individual) yet has multiple copies in terms of number of molecules per cell. For example, by comparing sequences of yellow wagtails across the Palearctic it has been possible to determine the phylogeny, assess differentiation within and between regions and show evidence for bottlenecks and rapid expansion (Odeen and Bjorklund 2003).

Human genetic studies have increasingly used single nucleotide polymorphisms (SNPs), which determine single base differences at a range of locations across the genome. This technique has recently been applied to birds (Primmer et al. 2002). These have a number of advantages such that they occur at a high frequency across the genome and this multilocus approach probably gives more reliable results than just comparing one sequence. Another advantage is that SNPs can be analyzed using automated processes.

Sex determination. A high proportion of bird species are sexually monomorphic and are therefore difficult or impossible to sex, except by laparotomy to examine the gonads. Nestlings or embryos are obviously difficult to sex. A DNA test that can be used to establish the sex of most species of birds (Griffiths et al. 1998) is based on two conserved chromo-helicase-DNA-binding (CHD) genes that are located on the sex chromosomes. Unlike mammals, in birds the females are heterozygous (ZW) and males are homozygous (ZZ). The CHD-W gene is located on the W chromosome and is therefore unique to females. CHD-Z is on the Z chromosome and therefore occurs in both sexes. The test involves PCR with a single set of primers. It amplifies homologous sections of both genes which incorporate introns whose lengths usually differ. When examined on a gel, there is a single band in males (CHD-Z) but in females there is a distinct second band (CHD-W). Sexing can be done for nestlings and even embryos, but may be unreliable in eggs that have not yet developed a visible embryo (Kalmbach et al. 2001).

Prey species. Another potential use of molecular techniques is to identify prey species in the gut contents, feces or regurgitated pellets of predator species (Symondson 2002). This is carried out by amplification of the prey DNA using PCR and then comparing sequences with online DNA databases of previously studied genes.

Acknowledgements

George Bentley, Tony Williams, Shelley Hinsley, Bengt Silverin, Sara Goodacre, and Brent Emerson provided useful information for this chapter.

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