2.3 DNA based marker techniques
2.3.3 Amplified Fragment Length Polymorphism (AFLP)
AFLPs are based on PCR-based techniques that combine the benefits of RFLPs and RAPDs. The technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The principle of the AFLP technique is the amplification of subsets of genomic restriction fragments using PCR (Vos et al., 1995).
There are four steps in the AFLP technique: DNA digestion, ligation, amplification and gel analysis. Genomic DNA is first digested by two restriction enzymes. Double-stranded oligonucleotide adapters, homologous to either 5'- or 3'-end generated during restriction digestion, are ligated to the DNA fragments. The ligated DNA fragments are amplified by PCR using primers complementary to the adapter and restriction site sequence with additional selective nucleotides at their 3'-end. The use of selective primers reduces the complexity of the mixture. Only those fragments with complementary nucleotides extending beyond the restriction site will be amplified by the selective primers under the stringent conditions. Figure 2.3 shows the steps involved in the AFLP technique.
Polymorphisms are revealed by analysis of amplified fragments on a denaturing polyacrylamide gel, and comparison of the patterns generated for each sample (Blears et al., 1998).
The AFLP technique can be used for DNAs of any origin or complexity. Fingerprints are produced without any prior sequence knowledge using a limited set of generic primers.
The number of fragments detected in a single reaction can be "tuned" by selection of specific primer sets. The AFLP technique is robust and reliable because stringent reaction conditions are used for primer annealing: the reliability of the RFLP technique is combined with the power of the PCR technique (Blears et al., 1998).
The capacity to reveal many polymorphic bands in one reaction is a major advantage of AFLP markers. The numerous bands on a gel are analyzed simultaneously making AFLP an extremely efficient technique. AFLP has the capacity to screen a much greater number of loci for polymorphism than other available PCR-based techniques, such that the number of polymorphisms detected per reaction is much higher. AFLP is superior in terms of the number of sequences amplified per reaction and their reproducibility. The markers produced are reliable and reproducible within and between laboratories, and are relatively easy and inexpensive to generate. A virtually unlimited number of markers can be generated by simply varying the restriction enzymes, and the nature and number of selective nucleotides (Blears et al., 1998).
AFLP is a DNA fingerprinting technique that detects genomic restriction fragments and resembles in that respect the RFLP technique, with the major difference that PCR amplification instead of Southern hybridization is used for detection of the restricted fragments (Vos et al., 1995). Due to the nature of the RFLP technique, only the restriction site is scanned for differences in DNA sequence. The selective nucleotides included in AFLP provide additional possibilities for polymorphisms to be detected beyond the restriction site itself. AFLP has the capacity to detect more point mutations than RFLPs.
In a single hybridization experiment, RFLP can detect, at most, a few genetic loci compared to the 100-200 loci detected by AFLP. In addition to a greater number of polymorphisms per reaction, AFLP is also superior in terms of efficiency, as it does not require template DNA sequencing. Fingerprints are produced without prior knowledge of DNA sequences (Blears et al., 1998).
The resemblance with the RFLP technique was the basis to choose the name AFLP. The name AFLP should not be used as an acronym, because the technique displays the absence or presence of restriction fragments rather than length differences. Other fingerprinting techniques, e.g. RAPDs, DNA amplified fingerprinting (DAF) and Arbitrarily
primed PCR (AP-PCR) are very sensitive to reaction conditions, DNA quality and PCR temperature profiles and this therefore limits their application (Vos et al., 1995).
RAPD is also a PCR-based technique similar to AFLP. However, AFLP uses primers specific to the adapter and restriction site sequence, whereas RAPD uses arbitrary primers. RAPD analysis is easier to perform than AFLP but the RAPD technique is very sensitive to reaction conditions, template DNA concentration and purity, and PCR temperature profiles, limiting its application. AFLP analysis uses stringent annealing conditions, which guarantee a better reproducibility (Folkertsma et al., 1996).
The AFLP technique is not simply a fingerprinting technique but is an enabling technology in genome research, because it can bridge the gap between genetic and physical maps. The AFLP technique is a very effective tool to reveal restriction fragment polymorphisms. These fragment polymorphisms, i.e. AFLP markers can be used to construct high-density genetic maps of genomes or genome segments. In most organisms AFLP will prove to be the most effective way to construct genetic DNA marker maps compared to other existing marker technologies. AFLPs are amendable to high throughput marker generation and have gained popularity in mapping and germplasm comparison studies (Vos et al., 1995).
The AFLP technique is powerful and reliable in identifying markers closely linked to genes of interest, but has some disadvantages for use in MAS and map based cloning.
Limitations to the large scale, locus-specific application of AFLPs includes their dominant type of inheritance, the intensity of labor involved, and the high costs. Hence, conversion of AFLP markers into sequence specific PCR markers (eg. SCAR markers) is required for screening large breeding populations at low costs (Dussle et al., 2002).
The AFLP technique was chosen for this study as it is reliable and is highly reproducible across laboratories. It also detects a higher number of polymorphisms in one reaction compared to the other DNA based techniques. No prior knowledge of sequence of the organism needs to be known before the technique is attempted.