Use of AFLP in cereals research

Abstract

Bands of interest can be excised from gels that have been dried onto 3MM filter paper (Whatman) or from dried silver stained gels17xCloning and mapping of variety-specific rice genomic DNA sequences: amplified fragment length polymorphisms (AFLP) from silver stained polyacrylamide gels. Cho, Y.G. et al. Genome. 1996; 39: 373–378Crossref | PubMedSee all References17, and re-hydrated overnight at 4°C. The DNA can then be re-amplified with the original primers and used as a probe, for direct sequencing or for cloning. Also, the AFLP technique can be modified so that one primer is obtained from a known sequence to detect sequence-specific amplification polymorphisms (S-SAP). This approach has been used successfully to map Bare-1 transposable elements throughout the barley genome18xGenetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Waugh, R. et al. Mol. Gen. Genet. 1997; 253: 687–694Crossref | PubMed | Scopus (330)See all References18.Increasingly, AFLP is being used for several specialist applications to assist the rapid isolation and characterization of interesting genes. AFLP-based mRNA fingerprinting has been used to isolate sequences mapping to deleted chromosome segments in cereals or other species19xAFLP-based mRNA fingerprinting. Money, T. et al. Nucleic Acids Res. 1996; 24: 2616–2617Crossref | PubMed | Scopus (68)See all References19 and cDNA-AFLP has been used to monitor the expression of genes20xVisualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber expression. Bachem, C.W.B. et al. Plant J. 1996; 9: 745–753Crossref | PubMedSee all References20. AFLP is also being used to screen superpools of plasmid DNA from 384 clones of bacterial artificial chomosomes (BACs; C. Goldstein and M. Dixon, unpublished), enabling the alignment of contigs and the rapid isolation of genes tightly linked to markers identified through high density mapping. The discriminating power of AFLP has also revealed the cryptic genome changes that could occur in transgenic rice plants generated by particle bombardment or intact cell electroporation21xMolecular analysis of the genome of transgenic rice (Oryza sativa L.). Plants produced via particle bombardment or intact cell electroporation. Arencibia, A. et al. Mol. Breed. 1998; 4: 99–109Crossref | Scopus (37)See all References21. Analysis of complex traits has so far been limited by mapping techniques with a low marker index (MI)22xThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Powell, W. et al. Mol. Breed. 1996; 2: 225–238CrossrefSee all References22. With AFLP the MI is more suitable, and AFLP has recently been applied to the analysis of quantitative traits in barley22xThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Powell, W. et al. Mol. Breed. 1996; 2: 225–238CrossrefSee all References22 and rice23xMapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping. Nandi, S. et al. Mol. Gen. Genet. 1997; 255: 1–8Crossref | PubMed | Scopus (110)See all References23.One of the major limitations of AFLP is the objective difficulty in identifying allelic variants at a specific locus, which has resulted in the technique being used almost exclusively for dominant markers. Recently, however, new software for image analysis of fluorescent PCR products has been developed by several companies including Keygene and Perkin Elmer. This will probably be developed for use with AFLP, which would enable AFLP to be used for scoring semi-dominant markers.As more maps of a given species are produced by various research groups, methods to integrate the different maps are clearly necessary to ensure that the same marker can be investigated. In a limited comparison within a single laboratory, accurately sized bands were found to be sufficient to identify homologous products between six different barley crosses5xHomology of AFLP products in three mapping populations of barley. Waugh, R. et al. Mol. Gen. Genet. 1997; 255: 311–321Crossref | PubMed | Scopus (140)See all References5. The reliability of this approach might be enhanced if more selective nucleotides were included to improve the discrimination of the polymorphic band. Further ambiguity could be eliminated by obtaining the sequence of the band.In conclusion, AFLP is a robust and reliable technique that has already proved its value in mapping and phylogenetic studies with a range of cereals. Increasingly, the technique is proving invaluable for the analysis of complex traits, for variety identification and for the rapid isolation of important genes. AFLP is rapidly becoming the preferred molecular technique for many types of investigation and will undoubtedly continue to find new applications in many areas of cereals research.Note: The AFLP technique is covered by patents and/or patent applications pending owned by Keygene n.v. Use of the technique for purposes other than research requires a license from Keygene n.v.