Molecular Breeding 7: 203–209, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 203 DNA fingerprinting based on microsatellite-anchored fragment length polymorphisms, and isolation of sequence-specific PCR markers in lupin (Lupinus angustifolius L.) Huaan Yang 1,∗ , Mark W. Sweetingham 1,4 , Wallace A. Cowling 1,2 and Penelope M.C. Smith 3 1 Centre for Legumes in Mediterranean Agriculture, 2 Plant Sciences, and 3 Department of Botany, The University of Western Australia, Nedlands, WA 6907, Australia; 4 Agriculture Western Australia, Private Bag No. 4, Bentley Delivery Centre, WA 6983, Australia; ∗ Author for correspondence (e-mail: hyang@agric.wa.gov.au; fax: 61-8- 93682165) Received 13 June 2000; accepted in revised form 4 December 2000 Key words: DNA fingerprinting, Molecular marker, Microsatellite, Simple sequence repeat Abstract We report a method of microsatellite-anchored fragment length polymorphisms for DNA fingerprinting. The method combines the concept of AFLP and the microsatellite-anchor primer technique. Genomic DNA was digested by one restriction enzyme MseI. One AFLP adaptor (MseI adaptor) was ligated onto the restriction fragments. DNA fingerprints were produced by PCR using one microsatellite-anchor primer in combination with one MseI-primer. The method allows co-amplification of over 100 DNA fragments containing microsatellite motifs per PCR. Polymorphisms detected from lupin by this method included those arising from variation in the number of microsatellite repeat units targeted by the microsatellite-anchor primers, from variation on the annealing sites for the SSR-anchor primers, from insertions/deletions outside the SSR region, and from variation in restriction sites. The first three types of polymorphisms were readily converted into sequence-specific PCR markers suitable for marker-assisted breeding. Introduction Microsatellites, also called simple sequence repeats (SSR), are abundant and highly polymorphic in eu- karyotic genomes (Litt and Luty 1989; Chin et al. 1996). SSR-anchor primers, consisting of a simple sequence repeat component and an anchor sequence component, have been used in various PCR based methods to amplify and detect SSR-related DNA polymorphisms, including inter-simple sequence re- peats (ISSR) (Zietkiewicz et al. 1994) and random- amplified microsatellite polymorphisms (RAMP) (Wu et al. 1994). In recent years, amplified fragment length polymorphism (AFLP) (Vos et al. 1995) has been widely used for DNA fingerprinting. The power of AFLP lies in the fact that it requires no prior se- quence knowledge, it amplifies a large number of restriction fragments in one PCR, and it is highly re- producible (Folkertsma et al. 1996). Witsenboer et al. (1997) described the selectively amplified microsatel- lite polymorphic loci (SAMPL) method, which de- tects compound microsatellite related polymorphic loci. The aim of this study was to develop a method to amplify and detect simple microsatellite sequence- related polymorphisms by combining the concept of AFLP with the SSR-anchor primer technique. Since the protocol of the method resembles AFLP, and each detected fragment contains a microsatel- lite motif sequence, we refer to this technique as MFLP (microsatellite-anchored fragment length poly- morphisms). The requirements of DNA markers for marker- assisted selection (MAS) and for construction of mole- cular genetic maps in plant breeding are different from those for fingerprinting. It is preferable that the mark- ers developed for MAS and for mapping are sequence-