A
Brief History of Alternative Genetic Markers

The first wave of molecular genetic data on sea turtles included a variety of techniques, during a period when DNA sequence data were still expensive and laborious to obtain. For the purposes of sea turtle population studies, those techniques have largely been replaced. However, it is notable that the conclusions based on them have been confirmed (for the most part) with newer technologies based on the polymerase chain reaction (PCR), a genetic-analysis technique that is used to amplify pieces of DNA and generates millions of copies of a particular sequence. As explained in Chapter 2, mitochondrial DNA control-region sequences and hypervariable microsatellites1 are the genetic-analysis techniques of choice for sea-turtle population assessment and are likely to remain the primary ones for the next decade (Bowen and Karl, 2007). One promising genetic-analysis technique that has not yet been applied to sea turtles is single-nucleotide polymorphisms (a DNA-sequence variation that can occur among members of the same species; Vignal et al., 2002), which require extensive nuclear DNA sequence information to identify variable sites throughout the genome (Lee, 2008).


Restriction Fragment Length Polymorphisms (Bowen et al., 1992)—This technique takes advantage of a suite of restriction enzymes,2 which can

1

Also known as DNA fingerprints, these are highly variable DNA sequences that occur in short repeats, such as GAGAGAGAGA. The number of repeats can vary from a few to over 30 so it is possible to have many variants at this hypervariable region.

2

These are useful for cutting genomes into fragments that are small enough to manipulate in cloning or DNA sequencing.



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A Brief History of Alternative Genetic Markers The first wave of molecular genetic data on sea turtles included a variety of techniques, during a period when DNA sequence data were still expensive and laborious to obtain. For the purposes of sea turtle population studies, those techniques have largely been replaced. However, it is notable that the conclusions based on them have been confirmed (for the most part) with newer technologies based on the polymerase chain reaction (PCR), a genetic­ analysis technique that is used to amplify pieces of DNA and generates millions of copies of a particular sequence. As explained in Chapter 2, mito­ chondrial DNA control­region sequences and hypervariable microsatellites1 are the genetic­analysis techniques of choice for sea­turtle population assess­ ment and are likely to remain the primary ones for the next decade (Bowen and Karl, 2007). One promising genetic­analysis technique that has not yet been applied to sea turtles is single­nucleotide polymorphisms (a DNA­ sequence variation that can occur among members of the same species; Vignal et al., 2002), which require extensive nuclear DNA sequence informa­ tion to identify variable sites throughout the genome (Lee, 2008). Restriction Fragment Length Polymorphisms (Bowen et al., 1992)—This technique takes advantage of a suite of restriction enzymes,2 which can 1 Also known as DNA fingerprints, these are highly variable DNA sequences that occur in short repeats, such as GAGAGAGAGA. The number of repeats can vary from a few to over 30 so it is possible to have many variants at this hypervariable region. 2 These are useful for cutting genomes into fragments that are small enough to manipulate in cloning or DNA sequencing. 

OCR for page 155
 APPENDIX A cut DNA at specific four–, five–, or six–base­pair sequences. For exam­ ple, the enzyme EcoR (a restriction enzyme derived from the bacterium Escherichia coli) cuts DNA at sites that contain the nucleotide sequence GAATTC. This is a quick and inexpensive way to get sequence informa­ tion and was widely used in population­genetics studies before the advent of PCR­based sequencing technology. The technique is highly repeatable and robust but has largely been replaced by direct DNA sequencing. Anonymous Single-Copy Nuclear DNA (Karl et al., 1992)—This tech­ nique requires cloning and sequencing fragments of DNA. On the basis of the clones, variation in the nuclear genome can be resolved and char­ acterized. The requirement of cloning (like microsatellites; see Chapter 2) makes this an expensive and labor­intensive approach to initialize, but it is robust and repeatable (Karl and Avise, 1993). In population genetic studies, it is largely replaced by microsatellite methods but has broad applications in phylogeography and phylogenetic studies.3 Minisatellites (Peare and Parker, 1996)—These are the first generation of “DNA fingerprints” and consist of short repeat sequences4 of about 10–60 base pairs that occur in variable copy number, in hundreds of locations in the genome. They are detected with a fluorescent or radioactive probe and can be variable enough to distinguish individuals (Jeffreys, 2005). However, they can be difficult to interpret and have largely been replaced by microsatellites in population­genetics studies. Random Amplification of Polymorphic DNA (Schroth et al., 1996)—This technique uses PCR primers to amplify short segments of the genome ran­ domly; they are then separated and visualized with gel electrophoresis.5 It has the advantage of not requiring prior knowledge of the genome (sequence data) to design primers. However, it is not widely used in population­genetics studies because of problems with interpretation and repeatability. 3 Phylogeography focuses on the geographic distribution of genetic variation, usually at the level of species and genera. Phylogeographic studies often reveal molecular evolutionary separations below the species level, as is the case with green turtles ( Chelonia mydas; Bowen et al., 1992). Phylogenetics is the study of evolutionary history, usually describing relation ­ ships among species, genera, and higher taxonomic categories in the format of trees. 4 These are DNA segments that repeat the same sequence multiple times. They are prone to duplication during cell replication and therefore can produce highly variable genetic markers. 5 This is a method of separating DNA fragments by size. The DNA or protein is inserted into a gelatin slab, and an electric current is run through the gelatin to move fragments toward either the positive or negative end.