Mycol. Res. 100 (9): 1107-1111 (1996) Printed in Great Britain 1107 The use of AFLP fingerprinting for the detection of genetic variation in fungi DOROTHEA MAJER~, RICHARD MITHEN*, BRIAN G. LEWISZ, PIETER vos3 AND RICHARD P. OLIVERZ Department of Brassica and Oilseed Research, ]ohn Innes Centre, Colney Lane, Nonuich NR4 7UH, U.K. "niversity of East Anglia, Nonuirh NR4 3NW, U.K. Keygene N . V., P.O. Box 216, Wageningen, The Netherlands A new PCR-based technique for the detection of inter- and intraspecific genetic variation has been tested on isolates of the fungal phytopathogens Cladosporium fulvurn and Pyrenopeziuz brassicae. The method is based on the selective PCR amplification of restriction fragments from digests of genomic DNA. We show that the technique is very efficient at detecting polymorphisms, even in species where very little variation could previously be found by RFLP analysis. 21 primer combinations were used on four isolates of P. brassicae, detecting a total of 162 polyrnorphisms (mean = 4.1 polymorphisms per primer combination per pair of isolates). Four primer combinations were used on eight isolates of C. fulvum, detecting a total of 32 polymorphisms (mean = 3.3 polymorphisms per primer combination per pair of isolates). Primer combinations varied in their ability to detect variation, ranging from 0 to 24 polymorphisms between P. brassicae isolates and 0 to 10 polymorphisms between C. fulvum isolates. AFLP fingerprints were highly reproducible and have great potential as a tool for evaluating genetic diversity of fungal pathogens. Molecular markers are being increasingly used to characterize fungal plant pathogen populations. Markers can be used to evaluate levels of genetic diversity and phylogenetic relation- ships within and between species, and to identify particular races and pathotypes. Additionally, markers which are closely linked to avirulence genes are being increasingly sought to assist map-based gene cloning. Several different types of markers have been developed. Isozyme markers are relatively cheap and easy to use but tend to reveal low levels of polymorphisms in pathogenic fungi (Burdon & Roelfs, 1985a, b; Tooley, Fry & Villarreal Gonzalez, 1985; Newton, 1987). RFLP markers may be highly informative if appropriate DNA probes are available (Garber & Yoder, 1984; Hulbert & Michelmore, 1988), but in several studies, including those of Cladosporiumfulvurn Cooke (syn. Fulvia fulva (Cooke) Cif.) and Pyrenopeziza brassicae B. Sutton & Rawl. little variation has been revealed with the use of heterologous probes. RAPD markers are being used widely (Chen, Line & Leung Hei, 1993; Assigbetse et al., 1994; Manulis et al., 1994) with different levels of success, and in some laboratories suffer from lack of reproducibility (Devos & Gale, 1992; Hamilton & Aquadro, 1992; Ellesworth, Rittenhouse & Honeycutt, 1993 ; Muralidharan & Wakeland, 1993; Penner et al., 1993; Micheli et a/., 1994). In this paper, we describe the use of a new technique for detecting polymorphisms among fungal isolates. The method, referred to as AFLP fingerprinting, has been used for genetic mapping in plants (Vos ef al., 1995). It has many of the characteristics of an ideal system for detecting genetic variation. For example, variability is assessed at a large number of independent loci, AFLP markers are 'neutral' (i.e. not subject to natural selection), variation is revealed in any part of the genome, data are obtained very quickly, and are extremely reproducible. In this method, genomic DNA is digested simultaneously with two enzymes, a six-base cutter (e.g. EcoR I) and a four- base cutter (e.g. Mse I). A typical fungal genome of about 40000 kb is thus cut into over 150000 fragments, the majority being less than 500 bp long. To visualize a manageable number of fragments, two methods of selection are used. Firstly, Mse I- and biotinylated EcoR I- adaptors are ligated to the fragments. Fragments with ligated EcoR I- adapter (ca 20000 in a fungal genome) are selected by binding to streptavidin beads. The far more numerous Mse-Mse fragments are discarded. Secondly, primers based on the adapter sequences are used to amplify the ligation mix (Fig. 1). To select a manageable fraction of all possible fragments that could be amplified, the primers have arbitrary one, two or three base extensions at their 3' end (the primers are referred to as + 1, +2, and +3, respectively). The number of fragments amplified thus depends on the primer combinations used and can be optimized by altering the length of the 3' extension. Usually 50-70 fragments can be resolved on denaturing polyacrylamide gels. The whole procedure can be completed within 2 d. In this paper we describe the application of this technique to measure intraspecific variation among