AFLP markers in crop improvement
Author: Smita Narendra Shingane
Amplified Fragment Length Polymorphism (AFLP) markers are done by restriction endonuclease digestion of total genomic DNA, followed by selective PCR amplification and electrophoresis of a subset of the fragments resulting in a unique, reproducible fingerprint (or profile) for each individual. The Amplified Fragment Length Polymorphism (AFLP) technique has come a long way since its publication in 1995 (Vos et al.1995) including many technological advances in generating and analyzing AFLP data. AFLP has become the method of choice for many studies on plants and, more recently, for animals, fungi and bacteria, spanning numerous disciplines in genetics, evolution and ecology. Amplified Fragment Length Polymorphism (AFLP) DNA fingerprinting is a firmly established molecular marker technique, with broad applications in population genetics, phylogenetics, linkage mapping.
Comparison of AFLP with other dominant marker systems
Most common techniques for multilocus genomic fingerprinting are AFLP, Random Amplified Polymorphic DNAs (RAPDs), and inter simple sequence repeats (ISSRs). They are PCR-based techniques that use primers to amplify DNA fragments and can be used on organisms for which there is no prior sequence information. These three techniques vary regard to data quality and discriminatory power among individuals. In many studies AFLPs outperform ISSRs and RAPDs in their high reproducibility, robustness, informativeness, and fewer reported reaction artifacts. AFLPs, which like RAPDs and ISSRs are dominant marker systems, can serve as an effective marker system in orphan crops where very little or no genomic information is available.
The AFLP technique can be ideal in the following situations
• When there is no a priori sequence information.
• When genetic variability is low (i.e. when it is necessary to amplify many loci to locate the few that are polymorphic, e.g. crop species).
• When high quality DNA is available.
• Where there are no suitable established markers.
• Where there is access to the appropriate facilities (preferably including a capillary electrophoresis instrument).
Steps in AFLP
The AFLP technique is based on the selective PCR of restriction fragments from a total digest of genomic DNA. The technique involves the following steps.
(i) Restriction of the DNA and ligation of oligonucleotide adapters;
(ii) Selective amplification of sets of restriction fragments and
(iii) PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction site.
(iv) Electrophoretic separation and visualization of the AFLP bands
Important applications of Amplified fragment length polymorphism markers
Linkage mapping and construction of genetic linkage maps. AFLP markers are used for construction of quick genetic maps. In most cases it is used for fine saturation of molecular genetic linkage maps.
Parentage analysis: AFLP markers are used for multilocus finger printing studies especially used in varietal identification studies.
Measuring genetic diversity: Since AFLP markers are mainly used in multilocus fingerprinting studies it gives a quick scan of whole genome for polymorphism. AFLP markers gives more appropriate picture of diversity available in a species.
Since AFLP markers give whole scan of the genome for polymorphism, these are the best markers for estimation of various population parameters such as Analysis of Molecular Variation and Fixation indices.
Development of single locus markers from AFLP for application in MAS
The reproducible amplicons obtained from AFLP analysis with clear distinction between target genotypes can be converted to CAPS/SCAR markers by eluting the bands and thereafter sequencing them for redesigning primer for its used in MAS.
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Ac- ids Res 23: 4407-4414
About Author / Additional Info:
Dr. Smita - Ph.D. in (Genetics and Plant Breeding)