Читать книгу Genotyping by Sequencing for Crop Improvement - Группа авторов - Страница 22

This marker technique was developed by Vos et al. (1995) and is patented by Keygene (www.keygene.com). In this technique, DNA is cut into fragments by a combination of restriction enzymes which are frequent (four bases) and rare (six bases) cutters that generate restriction overhangs on both sides of fragments. This is followed by the annealing of double‐stranded oligonucleotide adapters of a few oligonucleotide bases with respective restriction overhangs. The oligonucleotide adapters are designed in such a way that the original restriction sites are not reinstated and also provide the PCR amplification sites. The fragments are PCR amplified and visualized on agarose gel. This method produces many restriction fragments enabling the polymorphism detection. The number of amplified DNA fragments can be controlled by selecting different number or composition of bases in the adapters. The stringent reaction conditions used for primer annealing make this technique more reliable. This method is a combination of both RFLP and PCR techniques and is extremely useful in the detection of polymorphism between closely related genotypes. Like RAPD, AFLP is a dominant marker and is not preferred for genetic mapping studies and MAS. AFLP maps have been constructed in several species and integrated into already existing RFLP maps e.g. tomato (Haanstra et al. 1999), rice (Cho et al. 1997), and wheat (Lotti et al. 2000).