A fertility-restoring genotype of beet (Beta vulgaris L.) is composed of a weak restorer-of-fertility gene and a modifier gene tightly linked to the Rf1 locus

Abstract

Cytoplasmic male sterility (CMS) is a plant trait that involves interactions between nuclear- and mitochondrial genomes. In CMS, the nuclear restorer-of-fertility gene (Rf), a suppressor of male-sterility inducing mitochondria, is one of the best known genetic factors. Other unidentified genetic factors may exist but have not been well characterized. In sugar beet (Beta vulgaris L.), CMS is used for hybrid seed production, but few male-sterility inducing nuclear genotypes exist. Such genotypes could be introduced from a closely related plant such as leaf beet, but first the fertility restoring genotype of the related plant must be characterized. Here, we report the discovery of a Japanese leaf beet accession 'Fukkoku-ouba' that has both male-sterility inducing and fertility restoring genotypes. We crossed the leaf beet accession with a sugar beet CMS line, developed succeeding generations, and examined the segregation of two DNA markers that are linked to two sugar beet Rfs, Rf1 and Rf2. Only the Rf2 marker co-segregated with fertility restoration in every generation, implying that the Rf1 locus in leaf beet is occupied by a non-restoring allele. Fertility restoration was incomplete without a genetic factor closely linked to Rf1, leading to the assumption that the Rf1 locus encodes a modifier that cannot restore fertility by itself but perhaps strengthens another Rf. We sequenced the apparently non-restoring 'Fukkoku-ouba' rf1 gene-coding region and found that it closely resembles a restoring allele. The protein product demonstrated its potential to suppress CMS in transgenic suspension cells. In contrast, 'Fukkoku-ouba' rf1 transcript abundance was highly reduced compared to that of the restoring Rf1. Consistently, changes in protein complexes containing CMS-associated mitochondrial protein in anthers were very minor. Accordingly, we concluded that 'Fukkoku-ouba' rf1 is a hypomorph that acts as a non-restoring allele but has the potential to support another Rf, i.e. it is a modifier candidate.

Fig 1. Marker analysis of s17 in F1 plants and a ‘Fukkoku-ouba’ DNA gel blot probed with the orf20-3' UTR.

(A) Agarose gel electrophoresis of s17 cleaved amplified fragments derived from representative F1 plants of the cross TA-33BB-CMS x ‘Fukkoku-ouba’ #2 and a TA-33BB-CMS plant. Male fertility of each plant is indicated as S, completely sterile, or N, normal. Size markers are shown on the left (kbp). (B) Agarose gel electrophoresis of s17 markers derived from TA-33BB-CMS and ‘Fukkoku-ouba’ plants. Size markers are shown on the left (kbp). (C) A DNA gel blot containing two genomic DNAs of ‘Fukkoku-ouba’ plants probed with the orf20-3' UTR. Size markers are shown on the left (kbp).

Fig 2. Marker analysis of o7 in F1 plants.

Agarose gel electrophoresis of o7 amplified fragments derived from representative F1 plants of the cross TA-33BB-CMS x ‘Fukkoku-ouba’ #2 and TA-33BB-CMS x ‘Fukkoku-ouba’ #1, and a TA-33BB-CMS plant. Male fertility of each plant is indicated as S, completely sterile, or N, normal. Size markers are shown on the left (kbp).

Fig 3. Comparison of the genomic organization between ‘Fukkoku-ouba’ rf1 and NK-198 Rf1.

‘Fukkoku-ouba’ has a single copy of an orf20-like gene whereas homologs are clustered in NK-198. Boxes indicate genes (introns are not shown). Brackets denote the most similar regions. A single nucleotide substitution in exon 2 of orf20_fukkoku is shown by a lollipop. The scale bar is shown below. The NK-198 Rf1 sequences correspond to DDBJ/EMBL/GenBank accession numbers AB646135 and AB646133.

Fig 4. Immunoblot analysis of transgenic suspension cells expressing FLAG-fused orf20_fukkoku.

(A) Total cellular proteins were separated by SDS-PAGE and probed with αFLAG or αCOXI. Lanes 1 to 6 are transgenic cell lines expressing FLAG-fused orf20_fukkoku. Lane M and lane N are the size marker (that does not react with αFLAG or αCOXI) and non-transgenic cells, respectively. Molecular masses are shown on the right. (B) Mitochondria were electrophoresed by BN-PAGE and probed with αpreSATP6. Lanes 1–3, lanes 4–5, and lane 7 are transgenic cell lines expressing FLAG-fused orf20_fukkoku, FLAG-fused orf20_NK-198, and FLAG-fused orf20L, respectively. Lane N is the non-transgenic cells control. Size markers are shown on the left. An arrow indicates the position of the 200-kDa signal band.

Fig 5. Protein complexes containing preSATP6 in anthers.

(A) Anthers of a plant heterozygous for ‘Fukkoku-ouba’ rf1 (lane 1), TA-33BB-CMS (lane 2), and NK-198 (lane 3) were subjected to BN-PAGE, and the protein complexes were blotted onto membranes and probed with αpreSATP6. Size markers are shown on the left. Exposure time was 1 min. (B) The same blot as panel A but exposed for 2 hr. (C) The same blot as panel A but probed with αCOXI. (D) Anthers of a plant heterozygous for ‘Fukkoku-ouba’ rf1 (lane 1), TA-33BB-CMS (lane 2), and NK-198 (lane 3) were subjected to SDS-PAGE and αpreSATP6 was used for immunodetection.