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A high-resolution linkage map of the Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility in radish (Raphanus sativus L.) produced by a combination of bulked segregant analysis and RNA-Seq

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We utilized a combination of BSA and RNA-Seq to identify SNPs linked to the Rfd1 locus, a restorer-of-fertility gene in radish. A high-density linkage map was constructed using this approach.

Abstract

Male fertility of cytoplasmic male sterility conditioned by the Dongbu cytoplasmic and genic male-sterility cytoplasm can be restored by a restorer-of-fertility locus, Rfd1, in radish. To construct a high-density linkage map and to identify a candidate gene for the Rfd1 locus, bulked segregant analysis and RNA-seq approaches were combined. A total of 26 and 28 million reads produced from male-fertile and male-sterile bulked RNA were mapped to the radish reference unigenes. After stringent screening of SNPs, 327 reliable SNPs of 109 unigenes were selected. Arabidopsis homologs for 101 of the 109 genes were clustered around the 4,000 kb region of Arabidopsis chromosome 3, which was syntenic to the Rfd1 flanking region. Since the reference unigene set was incomplete, the contigs were de novo assembled to identify 134 contigs harboring SNPs. Most of SNP-containing contigs were also clustered on the same syntenic region in Arabidopsis chromosome. A total of 21 molecular markers positioned within a 2.1 cM interval including the Rfd1 locus were developed, based on the selected unigenes and contigs. A segregating population consisting of 10,459 individuals was analyzed to identify recombinants containing crossovers within this interval. A total of 284 identified recombinants were then used to construct a high-density map, which delimited the Rfd1 locus into an 83-kb syntenic interval of Arabidopsis chromosome 3. Since no candidate gene, such as a pentatricopeptide repeat (PPR)-coding gene, was found in this interval, 231 unigenes and 491 contigs containing putative PPR motifs were analyzed further, but no PPR gene in linkage disequilibrium with the Rfd1 locus could be found.

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Acknowledgments

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Development of molecular breeding technology for major traits in radish and Chinese cabbage, Project No. PJ00799201)”, Rural Development Administration and Golden Seed Project (Center for Horticultural Seed Development, No 213003-04-1-SB910). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.

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The authors declare that they have no conflict of interest.

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The authors declare that the experiments comply with the current laws of Republic of Korea.

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  1. Biotech Research Center, Dongbu Advanced Research Institute, Dongbu Hannong Co., Ltd., Daejeon, 305-708, Korea

    Young-Pyo Lee & Youngcho Cho

  2. Department of Plant Biotechnology, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Korea

    Sunggil Kim

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  1. Young-Pyo Lee
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Correspondence to Sunggil Kim.

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Lee, YP., Cho, Y. & Kim, S. A high-resolution linkage map of the Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility in radish (Raphanus sativus L.) produced by a combination of bulked segregant analysis and RNA-Seq. Theor Appl Genet 127, 2243–2252 (2014). https://doi.org/10.1007/s00122-014-2376-x

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