Male-sterile Sugar Beet Research Articles

The Effects of DNA Methylation on Cytoplasmic Male Sterility in Sugar Beet.

DNA methylation is widely found in higher plants and can control gene expression by regulation without changing the DNA sequence. In this study, the whole-genome methylation map of sugar beet was constructed by WGBS (whole-genome bisulfite sequencing) technology, and the results of WGBS were verified by bisulfite transformation, indicating that the results of WGBS technology were reliable. In addition, 12 differential methylation genes (DMGs) were identified, which were related to carbohydrate and energy metabolism, pollen wall development, and endogenous hormone regulation. Quantitative real-time PCR (qRT-PCR) showed that 75% of DMG expression levels showed negative feedback with methylation level, indicating that DNA methylation can affect gene expression to a certain extent. In addition, we found hypermethylation inhibited gene expression, which laid a foundation for further study on the molecular mechanism of DNA methylation at the epigenetic level in sugar beet male sterility.

Selection of nuclear genotypes associated with the thermo-sensitivity of Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.).

Cytoplasmic male sterility in sugar beet becomes thermo-sensitive when combined with specific genotypes, potentially offering a means to environmentally control pollination by this trait. The stability of cytoplasmic male sterility expression in several genetic backgrounds was investigated in sugar beet (Beta vulgaris L.). Nine genetically heterogenous plants from open-pollinated varieties were crossed with a cytoplasmic male sterile line to obtain 266 F1 plants. Based on marker analysis using a multiallelic DNA marker linked to restorer-of-fertility 1 (Rf1), we divided the F1 plants into 15 genotypes. We evaluated the phenotypes of the F1 plants under two environmental conditions: greenhouse rooms with or without daytime heating during the flowering season. Three phenotypic groups appeared: those consistently expressing male sterility, those consistently having restored pollen fertility, and those expressing male sterility in a thermo-sensitive manner. All plants in the consistently malesterile group inherited a specific Rf1 marker type named p4. We tested the potential for thermo-sensitive male sterile plants to serve as seed parents for hybrid seed production, and three genotypes were selected. Open pollination by a pollen parental line with a dominant trait of red-pigmented hypocotyls and leaf veins resulted in seed setting on thermo-sensitive male sterile plants, indicating that their female organs were functional. More than 99.9% of the progeny expressed the red pigmentation trait; hence, highly pure hybrids were obtained. We determined the nucleotide sequences of Rf1 from the three genotypes: One had a novel allele and two had known alleles, of which one was reported to have been selected previously as a non-restoring allele at a single U.S. breeding station but not at other stations in the U.S., or in Europe or Japan, suggesting environmental sensitivity.

Post‐translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris)

Genetic conflict between cytoplasmically inherited elements and nuclear genes arising from their different transmission patterns can be seen in cytoplasmic male sterility (CMS), the mitochondrion-encoded inability to shed functional pollen. CMS is associated with a mitochondrial open reading frame (ORF) that is absent from non-sterility inducing mitochondria (S-orf). Nuclear genes that suppress CMS are called restorer-of-fertility (Rf) genes. Post-transcriptional and translational repression of S-orf mediates the molecular action of Rf that encodes a class of RNA-binding proteins with pentatricopeptide repeat (PPR) motifs. Besides the PPR-type of Rfs, there are also non-PPR Rfs, but the molecular interactions between non-PPR Rf and S-orf have not been described. In this study, we investigated the interaction of bvORF20, a non-PPR Rf from sugar beet (Beta vulgaris), with preSatp6, the S-orf from sugar beet. Anthers expressing bvORF20 contained a protein that interacted with preSATP6 protein. Analysis of anthers and transgenic calli expressing a FLAG-tagged bvORF20 suggested the binding of preSATP6 to bvORF20. To see the effect of bvORF20 on preSATP6, which exists as a 250-kDa protein complex in CMS plants, signal bands of preSATP6 in bvORF20-expressing and non-expressing anthers were compared by immunoblotting combined with Blue Native polyacrylamide gel electrophoresis. The signal intensity of the 250-kDa band decreased significantly, and 200- and 150-kDa bands appeared in bvORF20-expressing anthers. Transgenic callus expressing bvORF20 also generated the 200- and 150-kDa bands. The 200-kDa complex is likely to include both preSATP6 and bvORF20. Post-translational interaction between preSATP6 and bvORF20 appears to alter the higher order structure of preSATP6 that may lead to fertility restoration in sugar beet.

Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele.

Key message By genetically eliminating the major restorer - of - fertility gene ( Rf ), a weak Rf gene was unveiled. It is an allele of Z , long known as an elusive Rf gene in sugar beet. In the hybrid breeding of sugar beet, maintainer-genotype selection is a laborious process because of the dependence on test crossing, despite the very low occurrence of this genotype. Marker-assisted selection (MAS) of the maintainer genotype is highly desired by sugar beet breeders. The major restorer-of-fertility gene (Rf) was identified as Rf1, and its non-restoring allele (rf1) was discriminated at the DNA level; however, some of the rf1rf1 selections retained an as yet unidentified Rf, another target locus for MAS. The objective of this study was to identify this Rf. An rfrf1 plant was crossed to a cytoplasmic male-sterile sugar beet and then backcrossed to obtain progeny segregating the unidentified Rf. The progeny exhibited partial male-fertility restoration that was unstable in single plants. The segregation ratio of restored vs. non-restored plants suggested the involvement of a single Rf in this male-fertility restoration, designated as Rf2. We confirmed the feasibility of molecular tagging of Rf2 by identifying four shared amplified fragment length polymorphism (AFLP) fragments specific to 17 restored plants. Bulked segregant analysis also was performed to screen the Rf2-linked AFLP markers, which were subsequently converted into 17 sequence-tagged site markers. All the markers, as well two additional chromosome-IV-assigned markers, were linked to each other to form a single linkage map, on which Rf2 was located. Our data suggested that Rf2 is likely an allele of Z, long known as an elusive Rf gene in sugar beet. We also discuss the importance of Rf2 for sugar beet breeding.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-014-2398-4) contains supplementary material, which is available to authorized users.

Hybrid Breeding Skewed the Allelic Frequencies of Molecular Variants Derived from the Restorer of fertility 1 Locus for Cytoplasmic Male Sterility in Sugar Beet (Beta vulgaris L.)

ABSTRACTHybrid breeding of crops may involve the selection of reproductive traits, such as cytoplasmic male sterility (CMS), whose expression is controlled by cytoplasm and nuclear gene(s). Intense selection of a single cytoplasm and the consequent lack of cytoplasmic divergence is a potential danger, the so‐called genetic vulnerability. However, little is known about the relationship between hybrid breeding and the diversity of nuclear genes that suppress the expression of CMS, termed restorer of fertility (Rf). Despite the multi‐allelic nature of Rf at the molecular level, a common Rf variant was previously found to predominate in sugar beet maintainer lines that were selected for a specific genotype for propagating the CMS line. The question was raised as to the frequency of the common Rf variant before the hybrid breeding era. As the origin of Japanese maintainer lines can be genealogically traced back to seven nonhybrid cultivars, we investigated the allelic diversity of Rf in the seven cultivars using molecular markers. Our results indicated that Rf diversity differs among the cultivars but exceeds that of the maintainers in total, and the common variant in the maintainers is infrequent in all of the cultivars. Therefore, maintainer selection has involved selecting a small number of Rf variants in the founder population in Japan.

Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype

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.

Molecular mapping of a fertility restorer gene for Owen cytoplasmic male sterility in sugar beet

We report here the molecular mapping of a fertility restorer gene (named Rf1) for Owen cytoplasmic male sterility in sugar beet. Eight AFLP and two RAPD markers, tightly linked to the Rf1 locus, were identified using bulked segregant analysis. Three AFLP markers, mAFEM972, mAFEM976 and mAFEM985, were found to co-segregate with the Rf1 allele in our mapping populations. With the help of RFLP markers, previously mapped on the sugar beet genome, we showed that Rf1 is positioned in the terminal region of linkage group Kiel III/Koeln IV. This map location agrees well with that found for the restorer gene X, which suggests that the Rf1 locus corresponds to the X locus. The availability of the molecular markers will facilitate the selection of maintainer-pollinator lines in breeding program and provide the foundation for map-based cloning of the Rf1 gene.

Male Sterility in Sugar Beet Induced by Cooling Treatment and its Application to Cross-pollination for Breeding.

Male sterility was induced in sugar beet plants by cooling treatment at the early bud stage. The percentage of male sterile flowers was higher the longer the cooling treatment and lower the temperature from 5°C to 3°C. Complete male sterility was induced by the treatment at 3°C for 50 days or 5°C for 70 days. Microscopic observations confirmed that the microspores and tapetum cells were more sensitive to cool temperature than somatic cells within a sugar beet anther. Female organs in the male sterile plants under cooling were not fatally injured and they could produce hybrid seeds by crossing with normal pollen. These results indicated that cooling treatment, which induced male sterility in sugar beet plants, was applicable as a method of castration in the breeding system.

Alterations in organization and transcription of the mitochondrial genome of cytoplasmic male sterile sugar beet (Beta vulgaris L.).

We have constructed a physical map of the mitochondrial DNA of a cytoplasmic male sterile (CMS) sugar beet line, TK81-MS, and compared it with that published for normal fertile sugar beet (cv. TK81-O) to clarify the differences between the CMS and normal mitochondrial genomes. The TK81-MS genome is present as a single circular molecule of 481.8 kb, or as two molecules of 184.9 and 296.9 kb. The CMS genome was found to be highly rearranged relative to the normal mitochondrial genome, with at least fifteen rearrangement and/or inversion events being required to align the two DNAs. Analysis of transcription patterns of known mitochondrial genes and rearranged regions revealed six genes, coxI, coxII, atpA, atp6, rps3, and orf324, whose expression is altered in the CMS line relative to the normal line. Of these six, only the coxI transcript pattern differs between male-sterile and fertility-restored genotypes, making it likely that the coxI locus is involved in mediating CMS in sugar beet.

Mapping of a chloroplast RFLP marker associated with the CMS cytoplasm of sugar beet (Beta vulgaris)

The Owen cytoplasm of male-sterile sugar beet is associated with several alterations of mitochondrial DNA and one additional HindIII site of chloroplast DNA. The region of this HindIII site has been cloned and sequenced. The site maps in a small reading frame (orf32) close to the ycf7 (orf31) gene in the petG-psbE region of chloroplast DNA. Possible functional implications of the results are discussed. The chloroplast RFLP marker described could be useful for studies on chloroplast-mitochondrial interactions, CMS of sugar beet, and the origin of the Owen cytoplasm.

Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.

Variation in mitochondrial translation products in fertile and cytoplasmic male-sterile sugar beets

Intact and functional mitochondria were isolated from sugar beet plants (Beta vulgaris L.) containing normal fertile (F) or cytoplasmic male-sterile (S1-S4) cytoplasms. Incorporation of (35)S-methionine by mitochondria isolated from both roots and leaves showed approximately 20 major and ten minor translation products. Comparison of the polypeptide synthesis patterns produced by leaf mitochondria from fertile plants of three different species within the genus Beta revealed several taxonomically related differences. Contrary to this, the patterns of polypeptides synthesized by mitochondria from roots and leaves of sugar beet plants containing the F and S1-S4 cytoplasms were very similar; in the S1 and S2 cytoplasms no qualitative, and only a few quantitative, differences from the F cytoplasm were observed. Thus, in these cases, cytoplasmic male sterility in sugar beet is not correlated with the constitutive expression of variant polypeptides. In the S3 cytoplasm, however, an additional 6 kDa polypeptide was synthesized and in the S4 cytoplasm an additional 10 kDa polypeptide was observed when compared with the F cytoplasm. The expression of cytoplasmic male sterility in sugar beet may be associated with these variant polypeptides. The mitochondrial polypeptides synthesized were identical in plants with different nuclear backgrounds but with identical S1 cytoplasms. Mitochondria from plants with variants of the S4 cytoplasm in the same nuclear genotype also showed identical patterns of polypeptide synthesis, including the synthesis of the 10 kDa S4-specific polypeptide. Pulse-chase experiments did not affect the synthesis of this polypeptide.

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.

High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned α-subunit of F1-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.

Sugarbeet mitochondria contain an open reading frame showing extensive sequence homology to the subunit 2 gene of the NADH: Ubiquinone reductase complex

Sequence analysis of a transcribed region of mitochondrial DNA (mtDNA) from male fertile sugarbeet (Beta vulgaris L.) revealed an open reading frame showing extensive sequence homology to the subunit 2 gene of the NADH: ubiquinone reductase complex (nad2). Sugarbeet nad2 in common with its proposed counterpart in animal mitochondria has no intron and thereby differs from the corresponding chloroplast gene. Northern RNA analysis of sugarbeet nad2 suggested that transcription of this locus gives rise to at least three transcripts. No differences in transcript profile were detected between male fertile and cytoplasmic male sterile sugarbeet. This constitutes the first report of a mitochondrial nad2 gene in higher plants.

Comparison of the organization and expression of mtDNA of fertile and male-sterile sugar beet varieties (Beta vulgaris L.)

Analysis of minicircle occurrence in different samples of sugar beet mitochondrial (mt) DNA invalidates the postulated relationship between cytoplasmic male sterility (CMS) phenotype and the absence of minicircle c and d. In high molecular weight mt DNA, two types of restriction patterns are found for fertile genomes and only one type for the CMS; in spite of the multiplicity of crosses carried out by plant breeders, all the CMS varieties analyzed seem to have derived from the original cytoplasm discovered by Owen in 1945. Southern hybridizations with mitochondrial genes coding for cytochrome oxidase subunits II and III, ATPase subunits α, 6 and 9 and 26S ribosomal RNA indicate that gene organization is different between fertile and sterile genomes but similar in all fertile genomes. Transcription analysis with the same genes indicate several differences between fertile and sterile varieties but also within some fertile varieties. These results suggest that the mt genome found in male-sterile sugar beet may originate not from modifications of the fertile mitochondrial genome but from a particular source of cytoplasm, of which a possible origin is discussed.

In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugarbeet (Beta vulgaris L.)

Haploid plantlets from male fertile and male sterile sugarbeet plants could be induced at frequencies up to 2.2% using ovule culture. Ovary culture on media without charcoal resulted in a similar induction frequency. Plant development was inhibited by callus development originating from the mother tissue. When the callus parts were removed and the ovule transferred to a new medium without 2,4 D, callus formation could be inhibited by adding 0.5% charcoal to the medium. Up to 6.1% haploids were induced. Chromosome counts in leaf tips, chloroplast counts and isozyme patterns revealed that all plants were haploid and originated from the haploid cells of the embryo sac. Root tips showed spontaneous polyploidisation.

Molecular heterogeneity in mitochondrial and chloroplast DNAs from normal and male sterile cytoplasms in sugar beets

Mitochondrial (mt) and chloroplast (ct) DNAs were prepared from normal (N) and male sterile (S) cytoplasmic lines of sugar beet. The DNAs were cleaved with BamHI, EcoRI, HindIII and SalI enzymes, and the resultant fragments were fractionated by agarose gel electrophoresis. The results showed that N and S cytoplasms contained distinct mtDNA. Although most of the DNA fragments were common to these two cytoplasms, each cytoplasm was readily characterized by bands specific to that cytoplasm. In addition, these distinctive cleavage patterns were invariant in different nuclear backgrounds. In contrast to the marked variation in mtDNA, restriction fragment analyses of ctDNA demonstrated little difference between both cytoplasms. Only HindIII digestion showed one band missing in the S genome. The data presented here provides circumstantial evidence for mitochondrial involvement in the inheritance of cytoplasmic male sterility in sugar beet.

Species of small DNA molecules found in mitochondria from sugarbeet with normal and male sterile cytoplasms

Mitochondrial DNA and RNA were isolated from a range of normal and cytoplasmically male sterile sugarbeet varieties and breeding lines. When these nucleic acids were analysed by electrophoresis on agarose gels it was found that mitochondria from normal sugarbeet contain DNA species of sizes 1.3 kilobase pairs (kbp), 1.4 kbp and one of two other species of sizes 1.45 kbp and 1.5 kbp, in addition to their DNA of relatively much higher molecular weight. In contrast mitochondria from cytoplasmically male sterile sugarbeet contained only one of these DNA species, that of 1.5 kbp. Treatment with DNaseI, RNase and nuclease S1 showed that these species consisted of supercoiled circular DNA. It is not known whether the lack of the two smaller DNA species causes cytoplasmic male sterility, or whether the two traits are associated by chance. In addition it was found that mitochondria from some individual sugarbeets contained one or more types of high molecular weight RNA molecules which were probably double stranded. Also mitochondria from some sugarbeet lines and varieties contained a series of DNA molecules with molecular weights in the range 2 to 10 kbp. Neither these DNA molecules nor the RNA molecules were apparently correlated with cytoplasmic male sterility.