ORIGINAL PAPER Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.) Received: 29 September 2002 / Accepted: 12 March 2003 / Published online: 24 April 2003 Ó Springer-Verlag 2003 Abstract We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from rad- ish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F 2 plants with adjacent RAPD-STS markers to isolate recombi- nants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification frag- ment length polymorphism (AFLP) markers surround- ing the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high- resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened k and cosmid libraries. The k and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 k and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four k clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants. Keywords Raphanus sativus L. Æ Cytoplasmic male sterility Æ Fertility restorer gene Æ Amplified fragment length polymorphism (AFLP) Æ Lambda contig Introduction Cytoplasmic male sterility (CMS) and fertility restorer (Rf) genes confer agronomic traits that are important for the production of hybrid seed on a commercial scale. CMS-restorer systems have been identified in many plants and are used for hybrid seed production of several crops (Schnable and Wise 1998). We have isolated a homologue of an ogura CMS-associated gene, orf138, in the radish cultivar Kosena, which carries CMS cytoplasm (Iwabuchi et al. 1999). The CMS-associated gene encodes 125 amino acids (orf125), and the protein (ORF125) accumulates as a mitochondrial-membrane protein in various tissues of the radish. Although fertility restoration is always accompanied by a considerable decrease in the levels of ORF125 in floral buds, no obvious effects have been observed at the mRNA level. At least two Rf loci (Rf1 and Rf2) are required for fertility restoration in the CMS radish, and the Rf1 locus (hereinafter termed Rfk1) might regulate the levels of ORF125 in the mitochondria of radish and CMS Brassica napus strains (Koizuka et al. 2000). Markers surrounding the target gene could be obtained by using bulked segregant pools (Michelmore et al. 1991). With the development of bulk segregant analysis and PCR-based methods, such as random polymorphic DNA and amplification fragment length polymorphism (RAPD and AFLP), it has become fea- sible to isolate a number of markers surrounding a given target gene rather easily in a relatively short time (Tanksley et al. 1995). RAPD and AFLP markers that are tightly linked to fertility restorer genes have been isolated in several species (Ma et al. 1995; Delourme et al. 1998; Li et al. 1998; Bentolila et al. 1998; Bentolila and Mol Gen Genomics (2003) 269: 388–394 DOI 10.1007/s00438-003-0847-x R. Imai Æ N. Koizuka Æ H. Fujimoto Æ T. Hayakawa T. Sakai Æ J. Imamura Communicated by R. Hagemann The first three authors contributed equally to this work R. Imai Æ N. Koizuka Æ H. Fujimoto Æ T. Hayakawa T. Sakai Æ J. Imamura (&) Plantech Research Institute, 1000 Kamoshida Aoba-ku, 227-0033 Yokohama, Japan E-mail: pri0006@cc.m-kagaku.co.jp Fax: +81-45-9627492