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.

KEY MESSAGE: 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. 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.

Construction of a high-resolution linkage map of Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility conferred by DCGMS cytoplasm in radish (Raphanus sativus L.) using synteny between radish and Arabidopsis genomes.

Cytoplasmic male sterility caused by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome-3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group-2 co-segregated with male fertility in an F(2) population. Using radish ESTs positioned in linkage group-2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome-3. Synteny between the radish and Arabidopsis genomes in the 985-kb interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome-3.