49. Cloning and mapping of amplified fragment length polymorphisms (AFLPs) in rice from silver-stained polyacrylamide gels

Yong Gu CHO¹, Matthew W. BLAOR², Olivier PANAUD³ and Susan R. McCOUCH²

1) Division of Cytogenetics, Dept. of Bio-resources, Agricultural Science and Technology Institute, RDA, Suweon 441-707, Korea
2) Dept. of Plant Breeding, Cornell University, Ithaca NY 14850, USA
3) Genetique et evolution des plantes cultivees, Laboratoire devolution et systematique vegetale, Batiment 362, Universite Paris XI, 91405 Orsay, Cedex, France

A number of PCR-based marker technologies have been developed that expedite the construction of high density linkage maps and facilitate genetic analysis, map based cloning, and marker-assisted selection in breeding. These techniques include randomly amplified polymorphic DNA (RAPD) (Williams et al. 1990), digital amplification fingerprinting (DAF) (Caetano-Anolles et al. 1991), multiple arbitarary amplicon profiling (MAAP) (Caetano-Anolles et al. 1993), inter-repeat amplification (IRA) (Zietkiewicz et al. 1994), microsatellite, or simple sequence repeat polymorphism (SSLP) analysis (Tautz, 1989; Panaud et al. 1996), and amplified fragment length polymorphism (AFLP) analysis (Vos et al. 1995). AFLP complements other marker technologies in that it makes it possible to inspect a large number of loci for polymorphism simultaneously within a single lane of a polyacrylamide gel.

Previous demonstrations of the AFLP technique utilized ³²P or ³³P for band detection, but our results using silver staining demonstrate that this visualization procedure has many advantages over the use of radioisotopes. The resolution of the silver stained gels was greater than that of the ³²P labelled gels and a larger number of better-defined bands could be scored (Fig. 1). The difference between silver staining and the use of radioisotope labelling was most apparent in the region of the gel containing bands greater than 300 bp.

An efficient cloning technique is indispensable for harnessing the potential of AFLP or other multi-locus scanning techniques to produce usable polymorphic markers. The conversion of markers developed with random amplification techniques into specific, diagnostic PCR-based markers, and the further characterization of interesting polymorphisms rely on cloning and sequencing of purified DNA fragments. We show that individual AFLP bands can be amplified directly from silver-stained polyacrylamide gels without contamination by alternate fragments. These bands can be reliably cloned after a single round of PCR amplification rather than requiring various amplification steps to obtain a pure product. Among individuals of a mapping population, normal Mendelian segregation for the restriction fragment length polymorphisms of the two AFLP clones was observed, confirming that the AFLPs that we isolated contained rice nuclear DNA (Fig. 2).

Both of the AFLP fragments cloned in this study could be mapped on the molecular map of rice (Causse et al. 1994). The two probes mapped to internal regions of independent chromosomes (Fig. 2). Despite the problems associated with the use of random anonymous sequences as markers, such as the frequent isolation of repeated sequences which are difficult to map by Southern analysis, we were able to obtain AFLPs from rice that were single copy sequences and mappable. The extraction of purified nuclear DNA for our AFLP reaction was important for reducing the risk so contamination by organellar DNA, which would have resulted in unmappable AFLP clones. In cases where AFLP bands contain repeated sequences, the ability to clone and sequence specific bands provides the opportunity to convert them into sequency tagged sites (STS) for further genetic analysis. In a species with a small genome such as rice, the probability of isolating single copy sequences using this method may be greater than in species with large fractions of repeated DNA. We do not have data to suggest the frequency with which multiple copy sequences would be cloned from AFLP profiles in rice. The specific enzyme combination used in template preparation for AFLP may also be an important element in increasing the likelihood of obtaining mappable, nuclear sequences from cloned AFLPs. Our use of Pst 1 and Taq I, both methylation sensitive enzymes, may have enriched for single copy regions of the rice genome.

The frequency of polymorphism in the AFLP patterns of two genotypes are likely to reflect their genetic relatedness. We detected no polymorphisms in the two nearly isogenic lines in Taichung 65 background, while many more polymorphisms were detected in the varieties IR24 (indica) and Taichung 65 (Japonica), which belong to different sub-species. These results are in agreement with studies obtained from RFLP analysis with random Pst 1genomic clones and reassure us that cloned AFLPs will complement the well-characterized set of RFLP markers available for the rice genome (Causse et al. 1994). The large number of bands obtained from the AFLP analysis, combined with the ability to clone out of silver-stained gels, offers a powerful strategy for targetted mapping and detailed analyses of plant genomes.

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