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sequences. Two synthetic oligonucleotides called primers corresponding to
the known sequences are used to initiate the amplification. PCR can be
modified in numerous, complex ways to achieve special results. The majority
of DNA marker techniques nowadays are based on PCR amplifications.
RAPD (random amplified polymorphic DNA) was a novel DNA
polymorphism assay based on the amplification of random DNA segments
with single primers of arbitrary nucleotide sequence (Welsh and McClelland
1990; Williams et al. 1990). The amplification depends on the correct
orientation and proper distances of the randomly distributed sequences
complementary to the decanucleotide primers in the genome being tested.
The polymorphic markers revealed by RAPD are due to the gain or loss of
priming sites and insertions or deletions between the priming sites. Therefore,
it does not require prior DNA sequence information to run RAPD, since the
primers will bind somewhere in the template and enable the DNA polymerase
to amplify fragments. This was an obvious advantage at that time when
DNA sequence information was scarce for most of the species of economic
importance. In addition, RAPD is easy to perform, requires a low investment
for equipment (a thermal cycler and a horizontal gel electrophoresis system),
and the universal random 10-mer primers are inexpensive. These made the
RAPD assay the most popular in the plant research community. Sunflower
researchers used RAPD for genome mapping and for tagging resistance
genes to rust (Lawson et al. 1998) and broom rape (Lu et al. 2000). In recent
years, RAPD has given way to other marker techniques because it has lower
resolution due to being dominant in nature, because it is anonymous and
not specific to the target sites, and it has a less satisfactory reproducibility
among laboratories where different equipment and reagents are used (Jones
et al. 1997). In addition, Rieseberg (1996) observed that only 91% of 220
pairs of RAPD fragments amplified from related species had homology as
tested by cross-hybridization and restriction digestion.
3.3.3 AFLP
AFLP (amplified fragment length polymorphism) is another novel DNA
fingerprinting technique based on PCR (Vos et al. 1995). It converts the
detection of RFLP from the hybridization-based system to an amplification-
based system. In other words, AFLP methodology employs PCR to detect
RFLP indirectly and collectively. The procedures for AFLP assay are lengthy
and complicated. The first step is the digestion of the DNA samples with
two different restriction enzymes, usually, one six-base cutter and one four-
base cutter. The second step is the ligation of the carefully designed, synthetic
oligonucleotides (adapters) to the ends of the restriction fragments. The
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