ORIGINAL PAPER Comparative analysis of superfamilies of NBS-encoding disease resistance gene analogs in cultivated and wild apple species Received: 16 September 2002 / Accepted: 8 January 2003 / Published online: 4 March 2003 Ó Springer-Verlag 2003 Abstract Eleven distinct families of resistance gene an- alogs (RGAs) with the characteristic nucleotide-binding sequence (NBS) were identified in two wild apple species, Malus prunifolia and M. baccata, and two cultivated apple cultivars, M. domestica cv. Fuji and M. domestica cv. Hong-ok, using PCR approaches with degenerate primers based on two conserved motifs of known NBS-LRR resistance genes. These RGA families were found to be represented in all the apple species tested, including wild and cultivated species. However, their sequences are very divergent from each other. Furthermore, the low level of recombination detected within their RGA families supports the idea that the evolution of NBS-encoding sequences in apple species involves the gradual accumulation of mutations. Despite the high diversity of the RGA families found in all apple species, the apparent lack of differentiation between wild and cultivated forms suggests that other factors, such as the capacity to tolerate pathogens, might play an important role in the survival of wild-type species. Keywords NBS-LRR Æ Apple Æ Disease resistance genes Æ Diversity Introduction Plants utilize a variety of strategies to withstand attack by the huge assortment of pathogens found in their habitats. The existence of resistance genes (R-genes) that are able to detect the presence of specific pathogen races by recog- nizing ligands encoded by the so-called avirulence genes (Avr) of pathogens is one of the most effective and pre- dominant strategies (Richter and Ronald 2000). Recently, more than twenty R -genes that confer resistance to a diverse spectrum of pathogens have been isolated from a wide range of plant species (Boyes et al. 1998; Gassmann et al. 1999; Tai et al. 1999; Cooley et al. 2000; Vinatzer et al. 2001; van der Biezen et al. 2002). These genes share striking structural similarities, a finding which suggests that certain signaling events are common to all or most plant defense systems. So far, five structural classes of cloned R-genes have been identified: the NBS-LRR, protein kinase, eLRR-TM, LRR, and toxin reductase classes. Among these, the NBS-LRR class, the members of which have a nucleotide-binding site (NBS) and a C-terminal leucine-rich repeat (LRR) of variable length, is the most prevalent, and seems to encode receptors that detect the presence of specific pathogens (Bent 1996; Hammond-Kosack and Jones 1997). Phylogenetic analyses have indicated that the NBS- LRR proteins encoded by plant R-genes can be divided into two major types based on their N-terminal sequences (Whitham et al. 1994; Meyers et al. 1999). The TIR group contains an N-terminal domain that plays a role in pathogen recognition and shows similarity to proteins of the Toll/interleukin-1 receptor (IL-1R) family (TIR). The non-TIR group contains a leucine zipper sequence (LZ) at the N terminus, which serves as a pathogen-recognition domain in place of the TIR domain. All plant R proteins of the NBS-LRR-class, both TIR and non-TIR, have a highly conserved backbone of amino acid motifs, which allows one to design degenerate primers for the isolation of disease resistance gene analogs (RGAs) from plant genomes. This approach has been successfully used to isolate NBS-LRR R -genes from various species, which has resulted in the identification of a remarkable number of NBS-encoding genes in plant genomes (Kanazin et al. 1996; Leister et al. 1996; Yu et al. 1996; Shen et al. 1998; Meyers et al. 1999; Noir et al. 2001). The aim of this study was to compare the RGAs that occur in wild and cultivated apple species (Malus sp.). The Mol Gen Genomics (2003) 269: 101–108 DOI 10.1007/s00438-003-0816-4 S.-Y. Lee Æ J.-S. Seo Æ M. Rodriguez-Lanetty D.-H. Lee Communicated by R. Hagemann S.-Y. Lee Æ J.-S. Seo Æ M. Rodriguez-Lanetty Æ D.-H. Lee (&) Department of Life Sciences, Ewha Womans University, 120-750 Seoul, Korea E-mail: lee@ewha.ac.kr Tel.: +82-2-32772376 Fax: +82-2-32772385