Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9854-8) contains supplementary material, which is available to authorized users.

Restorer-of-fertility (Rf) is a suppressor of cytoplasmic male sterility (CMS), a mitochondrion-encoded trait that has been reported in many plant species. The occurrence of CMS is considered to be independent in each lineage; hence, the question of how Rf evolved was raised. Sugar beet Rf resembles Oma1, a gene for quality control of the mitochondrial inner membrane. Oma1 homologues comprise a small gene family in the sugar beet genome, unlike Arabidopsis and other eukaryotes. The sugar beet sequence that best matched Arabidopsis atOma1 was named bvOma1; sugar beet Rf (RF1-Oma1) was another member. During anther development, atOma1 mRNA was detected from the tetrad to the microspore stages, whereas bvOma1 mRNA was detected at the microspore stage and RF1-Oma1 mRNA was detected during the meiosis and tetrad stages. A transgenic study revealed that, whereas RF1-Oma1 can bind to a CMS-specific protein and alter the higher-order structure of the CMS-specific protein complex, neither bvOma1 nor atOma1 show such activity. We favour the hypothesis that an ancestral Oma1 gene duplicated to form a small gene family, and that one of the copies evolved and acquired a novel expression pattern and protein function as an Rf, i.e. RF1-Oma1 evolved via neofunctionalization.

Cytoplasmic male sterility in plants is caused by male sterility-inducing mitochondria, which have emerged frequently during plant evolution. Nuclear Restorer-of-fertility (Rf) genes can suppress their cognate male sterility-inducing mitochondria. Whereas many Rfs encode a class of RNA binding protein, the sugar beet (Caryophyllales) Rf encodes a protein resembling Oma1, which is involved in the quality control of mitochondria. In this study we investigated the molecular evolution of Oma1 homologues in plants. We analyzed 37 plant genomes and concluded that a single copy is the ancestral state in Caryophyllales. Among the sugar beet Oma1 homologues, the orthologous copy is located in a syntenic region that is preserved in Arabidopsis thaliana. The sugar beet Rf is a complex locus consisting of a small Oma1 homologue family (RF-Oma1 family) unique to sugar beet. The gene arrangement in the vicinity of the locus is seen in some but not all Caryophyllalean plants and is absent from A. thaliana. This suggests a segmental duplication rather than a whole genome duplication as the mechanism of RF-Oma1 evolution. Among the positively selected codons in RF-Oma1, many are located in predicted transmembrane helices. Phylogenetic network analysis indicated that homologous recombination among the RF-Oma1 members played an important role to generate protein activity related to suppression. Together, our data illustrate how an evolutionarily young Rf has emerged from a lineage-specific paralogue. Interestingly, several evolutionary features are shared with the RNA binding protein type Rfs. Hence, the evolution of the sugar beet Rf is representative of Rf evolution in general.

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