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Sugar beet BAC library construction and assembly of a contig spanning Rf1, a restorer-of-fertility gene for Owen cytoplasmic male sterility

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Abstract

Rf1 is a nuclear gene that controls fertility restoration in cases of cytoplasmic male sterility caused by the Owen cytoplasm in sugar beet. In order to isolate the gene by positional cloning, a BAC library was constructed from a restorer line, NK198, with the genotype Rf1Rf1. The library contained 32,180 clones with an average insert size of 97.8 kb, providing 3.4 genome equivalents. Five AFLP markers closely linked to Rf1 were used to screen the library. As a result, we identified eight different BAC clones that were clustered into two contigs. The gap between the two contigs was filled by chromosome walking. To map the Rf1 region in more detail, we developed five cleaved amplified polymorphic sequence (CAPS) markers from the BAC DNAs identified, and carried out genotyping of 509 plants in the mapping population with the Rf1-flanking AFLP and CAPS markers. Thirteen plants in which recombination events had occurred in the vicinity of the Rf1 locus were identified and used to map the molecular markers relative to each other and to Rf1. In this way, we were able to restrict the possible location of the Rf1 gene to a minimum of six BAC clones spanning an interval of approximately 250 kb.

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Acknowledgments

The authors wish to thank Drs. M. Watanabe, M. Itoh and S. Nakamura for valuable suggestions and technical advice about BAC library construction and handling. This work was done in part at the Research Center for Molecular Genetics, Hokkaido University, and was supported in part by Grants in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and in part by a grant from the Ministry of Agriculture, Forestry and Fisheries, Japan, (Research Project for Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries No. 1423). E.H. was, and H.M. is, a recipient of the JSPS Fellowship for Japanese Junior Scientists.

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Authors and Affiliations

  1. Graduate School of Agriculture, Hokkaido University, N-9, W-9, Kita-ku, Sapporo, 060-8589, Japan

    Eiki Hagihara, Hiroaki Matsuhira, Minoru Ueda, Tetsuo Mikami & Tomohiko Kubo

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  1. Eiki Hagihara
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  2. Hiroaki Matsuhira
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  3. Minoru Ueda
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  4. Tetsuo Mikami
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  5. Tomohiko Kubo
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Communicated by R. Hagemann

The first two authors contributed equally to this work.

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Hagihara, E., Matsuhira, H., Ueda, M. et al. Sugar beet BAC library construction and assembly of a contig spanning Rf1, a restorer-of-fertility gene for Owen cytoplasmic male sterility. Mol Genet Genomics 274, 316–323 (2005). https://doi.org/10.1007/s00438-005-0024-5

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  • DOI: https://doi.org/10.1007/s00438-005-0024-5

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