T Cgt Tact Caggactcatcgtc


| AFLP primer

Selective bases

Figure 2.6 Outline of the AFLP DNA-fingerprinting method. Top: double-stranded EcoRI-MseI restriction fragment with 'sticky ends'—that is, overhanging single-strand sequences. Centre: the same fragment after ligation of the EcoRI and MseI adapters (boxed). Bottom: both strands with AFLP primers annealed. Each primer has three parts: the 5' part corresponding to the adapter, the restriction site sequence, and three selective bases at the 3' end, which make sure that on average (1/4)3 = 1.6% of the fragments generated are amplified. The number of selective bases can be adjusted to genome size. One of the primers is labelled, with either g-32P or a fluorophore, to allow detection of the bands. After Vos et al. (1995), by permission of Oxford University Press.

around 10. This variant of the technique is called three-enzyme AFLP (Van der Wurff et al. 2000).

Because AFLP generates a large number of bands (50-150) for each genotype, it became the preferred method for mapping ecologically relevant traits in the 1990s. Many traits that determine the fitness of an organism in the environment are measurable in the phenotype as a quantitative score (body size, clutch size, flowering time, longevity, disease resistance, etc.). The genomic segment underlying such quantitative traits is called a quantitative trait locus (QTL). The identification of QTLs in the genome is a major area of research in ecological genetics and plant and animal breeding (Tanksley 1993, Jansen and Stam 1994). QTL mapping uses controlled crosses, preferrably starting with two inbred parents that differ in the trait of interest, to correlate the segregation of bands in AFLP (or other) fingerprints with the segregation of the trait. When the offspring from two inbred parents are sib-mated for several generations, recombination breaks up the linkage between traits in the parental chromosomes and recombinant inbred lines develop, each of which contains a nearly homozygous segment from one of the parental chromosomes. The degree of precision that may be obtained is obviously dependent on the recombination frequency around the locus. In general it proves to be very difficult to pinpoint individual genes in this way; often a region of several thousand to some millions of base pairs remains for molecular analysis, so a QTL is not a genetic locus in the strict sense.

The genomic revolution has opened up new prospects for QTL mapping by using single nucle-otide polymorphisms (SNPs; Borevitz and Nordberg 2003). SNPs are positions in the genome at which at least some individuals of a species have a base pair different from the most common form (see Section 3.1). SNPs are contrasted with other types of genetic variation, such as insertions/deletions and duplications. SNPs and insertions/deletions constitute the predominant source of variation in a population. Depending on the species, there is an SNP every 50 (in Drosophila) to every 1000 (in humans) base pairs in the genome. High-throughput genomics technology for SNP geno-typing has provided a very powerful instrument

Map position

0 0

Post a comment