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13. SUPPLEMENTARY MATERIALS
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containing 0.5 µg/ml ethidium bromide, separated in 1 x TBE buffer (Tris-Borate-EDTA;
Sambrook et al. 1989) at 100 V constant power, visualized under UV light (312 nm), photographed
and stored as .jpg files. All bands that could be reliable read were scored either
present (1) or absent (0).
ANNEX 13.11. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)
The power of AFLP is based on genetic variation that exists between closely related
species, varieties or cultivars. AFLP technology is a DNA fingerprinting technique that combines
both classical restriction digestion and PCR-based fingerprinting. It is based on selective
amplification of a subset of genomic restriction fragments using PCR. The steps of AFLP
reaction are as follows (Figure 13.11.1):
• digestion of DNA with restriction endonucleases (Table 13.11.1),
• ligation of double-stranded DNA adapters to the ends of the DNA fragments to generate
template DNA for amplification (Table 13.11.2),
• the sequence of the adapters and the adjacent restriction site serve as a primer binding
sites for subsequent amplification of restriction fragments (Table 13.11.3),
• in the next PCR, selective nucleotides extending into the restriction fragments are added
to the 3’ ends of the PCR primers such that only a subset of the restriction fragments are
recognized (Table 13.11.4),
• the subset of amplified fragments are analyzed by denaturing gel electrophoresis.