Abstract
We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. 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 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding 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 λ and cosmid libraries. The λ 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 λ 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 λ 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.
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Acknowledgements
We thank Chie Tamura for her expert technical assistance in the construction of the λ and cosmid contigs, and Yusuke Kimura for his valuable comments on the experiments. This work has been carried out in compliance with the current laws governing genetic experimentation in Japan
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Communicated by R. Hagemann
The first three authors contributed equally to this work
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Imai, R., Koizuka, N., Fujimoto, H. et al. Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.). Mol Gen Genomics 269, 388–394 (2003). https://doi.org/10.1007/s00438-003-0847-x
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DOI: https://doi.org/10.1007/s00438-003-0847-x