Genetic Linkage Maps: Strategies, Resources and Achievements - Genetics, Genomics and Breeding of Crop Plants - page 92

third step is the pre-amplification of the fragment with primers with
sequences complementary to that of the adapters. The fourth step is the
selective amplification of sets of restriction fragments, which is achieved by
adding one to three selective nucleotides to the 3' ends of the primers in the
pre-amplification step. Only those fragments in which the sequence variation
matches the nucleotides flanking the restriction sites are amplified in this
step. The final step is analysis of the amplified fragments by electrophoresis
with a denaturing polyacrylamide gel and visualizing the fragment with
silver-staining or autoradiography techniques. Typically, each primer
combination can amplify 50-100 fragments that produce a multi-banded
profile on the sequencing gel. AFLP has proven to be a powerful genotyping
technique for almost all organisms with different levels of genome
complexity. Sunflower researchers adapted AFLP for fingerprinting
sunflower germplasm (Hongtrakul et al. 1997) and genome mapping
(Peerbolte and Peleman 1996; Gedil et al. 2001) and for quantitative trait loci
(QTL) analysis of various important traits such as in vitro organogenesis
(Flores Berrios et al. 2000a, b), partial resistance to downy mildew (Al-
Chaarani et al. 2002) and leaf chlorophyll concentration and stomatal
conductance (Hervé et al. 2001). Like the RAPD markers, most of the AFLP
markers are dominant in nature and not specific to the target sites. However,
AFLP can reveal a much greater number of polymorphic loci and has a
better reproducibility than RAPD. As a high-throughput genotyping
technique, it is inevitable that artifacts will be generated. As a result, there
are always 10 to 15% of the AFLP markers that cannot be assigned to linkage
groups in map construction. This is well compensated for by a large number
of markers amplified in each assay.
3.3.4 DALP
DALP (direct amplification of length polymorphism) was proposed by
Desmarais et al. (1998). In this technique, several selective primers are made
by extending the 3' end of the M13-40 universal sequencing primer with
two or four nucleotides. These selective primers can be used with another
universal sequencing primer M13 to generate multi-banded DNA
fingerprints resolved with a sequencing gel. A different fingerprint can be
obtained when a different selective primer is used. This technique is very
easy to perform and offers the possibility to sequence each of the amplified
fragments with the universal sequencing primers. Since the procedure
requires the use of radioisotope to label one of the primers, this technique
was not widely used. However, Langer et al. (2003) used it in combination
with AFLP and specific PCR and produced a sunflower linkage map.