Diagnostic value of Rf1 gene molecular markers in sunflower

Background. Modern production of sunflower seeds is currently based on the cultivation of high-yielding heterotic F1 hybrids from cross-breeding of lines with cytoplasmic male sterility (CMS) of PET1-type and fertility restorer lines. The paternal parent serves as a donor of the nuclear Rf1 gene functional allele, which is responsible for pollen fertility restoration in F1 plants. The detection of carriers of the Rf1 locus recessive and dominant alleles using diagnostic molecular markers accelerates breeding of female and male parental lines for creating hybrids. Materials and methods. The material for the study included 75 lines of various origins from the VIR sunflower genetic collection as well as hybrids from crosses of VIR 116A sterile line with fertile lines differing in the type of cytoplasm (fertile or sterile) and the presence of molecular markers, most of which were linked to the Rf1 locus. For marker validation, two different approaches were used: either by analyzing associations between the ability of a line to restore pollen fertility and the presence of molecular markers in its genotype, or by estimating recombination frequency between the Rf1 locus and marker loci in four segregating hybrid populations. Results. According to the obtained results, no markers demonstrated 100% efficiency in the analysis of the sample of genotypes. The ORS511 marker was most frequently observed among the lines presumably carrying the dominant allele Rf1. Pollen fertility of F1 hybrids from interline crossings was 89-99%. The segregation for fertility/sterility in F2 fitted the theoretical ratio of 3:1 expected in case of the monogenic control of the trait. The markers HRG01, HRG02 and ORS511 were linked to the fertility restoration trait, with recombination rates between Rf1 locus and markers varying in different cross combinations. The analysis of VIR 116А × VIR 740 and VIR 116А × RIL 130 hybrids showed that among the marker loci studied, the ORS511 was closest to the Rf1 locus Rf1 (recombination frequency of 2.2 and 3.3%, respectively). The recombination rate between the Rf1 and ORS511 loci equaled 7.5% in the cross VIR 116А × VIR 210 and 8.9% in VIR 116 × VIR 195. Conclusion. The markers ORS511, HRG01 and HRG02 are the most efficient for the identification of alleles of the Rf1 gene and for the marker assisted selection in hybrid populations produced involving sunflower lines from the VIR collection.

The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

The control of messenger RNA (mRNA) translation has been increasingly recognized as a key regulatory step for gene control, but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf). In this study, we focused on the Ogura CMS system in rapeseed and showed that reversion to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific translation inhibition of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.

Identification of Rf Genes in Hexaploid Wheat (Triticumaestivum L.) by RNA-Seq and Paralog Analyses

Among the natural mechanisms used for wheat hybrid breeding, the most desirable is the system combining the cytoplasmic male sterility (cms) of the female parent with the fertility-restoring genes (Rf) of the male parent. The objective of this study was to identify Rf candidate genes in the wheat genome on the basis of transcriptome sequencing (RNA-seq) and paralog analysis data. Total RNA was isolated from the anthers of two fertility-restorer (Primépi and Patras) and two non-restorer (Astoria and Grana) varieties at the tetrad and late uninucleate microspore stages. Of 36,912 differentially expressed genes (DEGs), 21 encoding domains in known fertility-restoring proteins were selected. To enrich the pool of Rf candidates, 52 paralogs (PAGs) of the 21 selected DEGs were included in the analyses. The expression profiles of most of the DEGs and PAGs determined bioinformatically were as expected (i.e., they were overexpressed in at least one fertility-restorer variety). However, these results were only partially consistent with the quantitative real-time PCR data. The DEG and PAG promoters included cis-regulatory elements common among PPR-encoding genes. On the basis of the obtained results, we designated seven genes as Rf candidate genes, six of which were identified for the first time in this study.

The Effect of Chromosome Arm 1BS on the Fertility of Alloplasmic Recombinant Lines in Bread Wheat with the Hordeum vulgare Cytoplasm

The genetic mechanisms of fertility restoration in alloplasmic bread wheat with the barley cytoplasm are poorly explored. The effect of the 1BS chromosome arm on the fertility of bread wheat with the H. vulgare cytoplasm was studied depending on the incompleteness/completeness of the cytonuclear compatibility. (i) Three self-fertile (SF) lines and one partially fertile (PF) line with an incomplete cytonuclear compatibility and (ii) four self-fertile (SF) lines with a complete cytonuclear compatibility were studied. For the lines in group (i), the heteroplasmy (simultaneous presence of barley and wheat copies) of the 18S/5S mitochondrial (mt) repeat was revealed as well as the barley-type homoplasmy of chloroplast simple sequence repeats (cpSSRs). In the lines in group (ii), the 18S/5S mt repeat and cpSSRs were found in the wheat-type homoplasmic state. In all of the lines, the 1BS chromosome arm was substituted for the 1RS arm. The F1 plants of SF(i)-1BS × 1RS hybrids were fertile. The results of a segregation analysis in the F2 plants of SF(i)-1BS × 1RS showed that 1BS carries a single dominant fertility restorer gene (Rf) of bread wheat with the H. vulgare cytoplasm. All of the F1 plants of PF(i)-1BS × 1RS hybrids were sterile. A single dose of this restorer gene is not sufficient to restore fertility in this alloplasmic PF(i) line. All of the F1 and F2 plants of SF(ii)-1BS × 1RS hybrids were self-fertile.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5′ untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

The effects of Rf5 and Rf6 on fertility restoration in Honglian-type cytoplasmic male sterile (CMS) lines of japonica rice (Oryza sativa L. ssp. japonica)

The Honglian (HL)-type cytoplasmic male sterility (CMS) has only been used in the development of three-line indica rice hybrids, and the fertility of HL-type indica CMS lines can be restored by two nonallelic fertility-restorer (Rf) genes, Rf5 and Rf6. For the development of HL-type japonica hybrid combinations, it is therefore necessary to determine whether Rf5 and Rf6 can restore the fertility of HL-type japonica CMS lines. Here, we genetically characterized HL-type japonica CMS lines and the ability of Rf5 and Rf6 to restore fertility for breeding HL-type japonica hybrids. I2-KI pollen staining revealed that HL-type japonica CMS lines and their derived testcross F1 hybrids had stained abortive pollen grains, unlike HL-type indica CMS lines. Crossing experiments showed that Rf5 and Rf6 partially restored the fertility of HL-type japonica CMS lines, and Rf6 showed higher restorability than Rf5. Furthermore, we found that there were additive and dosage effects of Rf5 and Rf6 with respect to fertility restoration in HL-type japonica CMS lines. These results give critical insight into the breeding of HL-type japonica CMS lines and restorers, which will be helpful for the development of commercial HL-type japonica hybrids.

The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

The control of mRNA translation has been increasingly recognized as a key regulatory step for gene control but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf) genes. In the study, we focused on the Ogura CMS system in rapeseed and showed that the suppression to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific inhibition of the translation of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.