Use of AFLP in cereals research

Amplified fragment length polymorphism (AFLP) is a PCR-based technique that involves restriction of genomic DNA, followed by ligation of adapters to the fragments generated and selective PCR amplification of a subset of these fragments 1 Zabeau, M. and Vos, P. (1993) Selective restriction fragment amplification: a general method for DNA fingerprinting, European Patent Application number: 92402629.7, Publication number 0 534 858 A1 Google Scholar , 2 Vos P. et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995; 23: 4407-4414 Crossref PubMed Scopus (10564) Google Scholar . The amplified fragments are separated on a sequencing gel and visualized, usually by autoradiography or fluorescent sequencing equipment. Several different restriction enzymes and primers are available, which gives a high degree of flexibility, enabling the complex final fingerprint to be manipulated for particular applications and efficient scanning of the genome for polymorphisms. The choice of enzymes and primer length is crucial for optimizing results in different applications and the rationale for choosing restriction enzymes, adapters and primers is described both here and in the original publications 1 Zabeau, M. and Vos, P. (1993) Selective restriction fragment amplification: a general method for DNA fingerprinting, European Patent Application number: 92402629.7, Publication number 0 534 858 A1 Google Scholar , 2 Vos P. et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995; 23: 4407-4414 Crossref PubMed Scopus (10564) Google Scholar . A summary of the technique is shown in Fig. 1. Other marker techniques commonly used in cereals research are summarized in Box 1. Box 1. Other marker techniques commonly used in cereals research RAPD (Randomly amplified polymorphic DNA) A PCR-based technique relying on the use of single, short (usually 10 bp) arbitrary oligomers used as primers for PCR-mediated amplification of genomic DNA between closely spaced inverted sequences. RFLP (Restriction fragment length polymorphism) A hybridization-based technique involving restriction of genomic DNA followed by electrophoretic separation of the fragments in a gel matrix; the fragments are then transferred on a membrane by Southern blotting and hybridized with a labelled probe from an appropriate cloned sequence. SSR (Simple sequence repeat) or STEMS (sequence-tagged microsatellite site) analysis A PCR-based technique where polymorphisms in the number of repeats in stretches of tandemly repeated short nucleotide motifs are detected by PCR amplification using primers specific for sequences flanking the repeats. A PCR-based technique relying on the use of single, short (usually 10 bp) arbitrary oligomers used as primers for PCR-mediated amplification of genomic DNA between closely spaced inverted sequences. A hybridization-based technique involving restriction of genomic DNA followed by electrophoretic separation of the fragments in a gel matrix; the fragments are then transferred on a membrane by Southern blotting and hybridized with a labelled probe from an appropriate cloned sequence. A PCR-based technique where polymorphisms in the number of repeats in stretches of tandemly repeated short nucleotide motifs are detected by PCR amplification using primers specific for sequences flanking the repeats.