Abstract Several cloned disease resistance genes from a wide range of plant species are known to share con- served regions with similar structural motifs. Degenerate primers based on conserved sequences of the nucleotide binding site of the genes RPS2, N and L6 were used for polymerase chain reaction (PCR) amplification from ge- nomic DNA of two doubled haploid lines of Brassica ol- eracea. Sequences of amplified products were highly variable, but most of them showed similarity to known disease resistance genes, including RPS5, RPS2 and N, and to disease resistance gene-like sequences (RGLs) from different species. Primers based on B. oleracea se- quences amplified five groups of RGLs. Products were mapped through cleaved amplified polymorphic se- quence assays onto four different linkage groups of B. oleracea. PCR amplification from cDNA and allele anal- ysis indicated that four locus-specific RGL fragments are expressed in cauliflower. Screening of a B. oleracea bac- terial artificial chromosome library (BAC) with four B. oleracea RGL probes identified a small number of clones, suggesting that the four RGLs may not be highly copied. Screening of a BAC library of A. thaliana with the same probes identified clones that mapped onto four different chromosomes. These map positions correspond to known disease resistance loci of A. thaliana. Keywords Brassica · Candidate genes · Genetic mapping · Nucleotide binding site-leucine rich repeat (NBS-LRR) · Degenerate oligonucleotides Introduction In recent years, several genes involved in plant disease resistance have been cloned from a wide range of plant species. The R genes were grouped into different classes based on structural similarities of their predicted protein products (reviewed by Staskawicz et al. 1995; Hammond-Kosack and Jones 1997). The largest class in- cludes gene products that contain a putative nucleotide binding site (NBS) and leucine-rich repeats (LRR) of various lengths. Genes from the NBS-LRR class are widely distributed in dicotyledon and monocotyledon plant species and confer resistance to a variety of plant pathogens including bacteria, fungi, viruses, nematodes and insects. Some of these genes, such as N (Whitham et al. 1994) and L6 (Lawrence et al. 1995), have N-terminal homology to the Toll protein of Drosophila and the inter- leukin-1 receptor of mammals (TIR), while others, such as RPS2 (Bent et al. 1994; Mindrinos et al. 1994) and RPS5 (Warren et al. 1998), contain leucine zipper (LZ) motifs in the N-terminal region. Meyers et al. (1999) de- scribed a number of motifs present on the NBS region of TIR and non-TIR genes. Due to the low overall sequence similarity observed between R genes, heterologous hybridisation, using cloned genes as probes, is unlikely to be a successful strategy for identifying R gene candidates (Leister et al. 1996; Shen et al. 1998). Polymerase chain reaction (PCR) amplification using degenerate primers based on conserved motifs of NBS-LRR genes is a more sensitive and efficient method to identify and characterise resis- tance gene-like sequences (RGLs) (Yu et al. 1996). This approach has been used successfully in the monocotyle- don species maize, wheat, barley and rice (Collins et al. 1998; Seah et al. 1998; Leister et al. 1999; Mago et al. 1999) and in dicotyledon species such as potato (Leister et al. 1996), soybean (Kanazin et al. 1996; Yu et al. 1996), lettuce (Shen et al. 1998), Arabidopsis thaliana (Botella et al. 1997; Aarts et al. 1998; Speulman et al. 1998) and Brassica napus (Joyeux et al. 1999). The primers used in most of these studies were based on mo- Communicated by J.W. Snape The sequence data reported will appear in EMBL GenBank and DDBJ databases under the accession numbers AJ250321 to AJ250326 J.G. Vicente ( ✉ ) · G.J. King Horticulture Research International, Wellesbourne, Warwickshire CV35 9EF, UK e-mail: joana.vicente@hri.ac.uk Theor Appl Genet (2001) 102:555–563 © Springer-Verlag 2001 ORIGINAL ARTICLE J.G. Vicente · G.J. King Characterisation of disease resistance gene-like sequences in Brassica oleracea L. Received: 12 November 1999 / Accepted: 19 June 2000