Rf Genes Research Articles

The effect of T. aestivum chromosomes 1A and 1D on fertility of alloplasmic recombinant (H. vulgare)-T. aestivum lines depending on cytonuclear compatibility.

The effect of T. aestivum L. chromosomes 1A and 1D on fertility of recombinant bread wheat allolines of the same origin carrying the cytoplasm of barley H. vulgare L. and different levels of cytonuclear compatibility was studied. Alloline L-56 included mainly fully sterile (FS) and partially sterile (PS) plants, alloline L-57 included partially fertile (PF) plants and line L-58 included fertile (F) ones. Analysis of morphobiological traits and pollen painting indicated complete or partial male sterility in plants of allolines L-56 and L-57. To differentiate genotypes with cytonuclear coadaptation and genotypes with cytonuclear incompatibility, PCR analysis of the 18S/5S mitochondrial (mt) repeat was performed. Heteroplasmy (simultaneous presence of barley and wheat mtDNA copies) was found in FS, PS, PF and some F plants, which was associated with a violation of cytonuclear compatibility. Wheat-type homoplasmy (hm) was detected in the majority of the fertile plants, which was associated with cytonuclear coadaptation. The allolines used as maternal genotypes were crossed with wheat-rye substitution lines 1R(1A) and 1R(1D). In F1, all plants of PF×1R(1A) and PF×1R(1D) combinations were fertile, and in F2, a segregation close to 3 (fertile) : 1 (sterile) was observed. These results showed for the first time that chromosomes 1A and 1D carry one dominant Rf gene, which controls the restoration of male fertility of bread wheat carrying the cytoplasm of H. vulgare. All plants of F1 combinations FS×1R(1A), FS×1R(1D), PS×1R(1A), PS×1R(1D) were sterile, which indicates that a single dose of genes localized on wheat chromosomes 1A or 1D is not enough to restore male fertility in FS and PS plants. All plants of hybrid combinations F(hm)×1R(1A) and F(hm)×1R(1D) in both F1 and F2 were fertile, that is, fertility of allolines with cytonuclear coadaptation does not depend on wheat chromosomes 1A and 1D.

Fertility restorer gene CaRf and PepperSNP50K provide a promising breeding system for hybrid pepper.

Cytoplasmic male sterility (CMS) is pivotal in plant breeding and widely employed in various crop hybrids, including pepper. However, the functional validation of the restorer of fertility (Rf) gene in pepper has been lacking until now. This study identifies and characterizes CaRf, a single dominant locus crucial for restoring CMS in the pepper strong recovery inbred line Zhangshugang. The CaRf gene encodes a mitochondria-targeted pentatricopeptide repeat protein, validated through the induction of male sterility upon its silencing in hybrid F1 plants. To enhance pepper breeding efficiency, 176 important pepper breeding parent materials were resequenced, and a PepperSNP50K liquid-phase breeding chip was developed, comprising 51 172 markers. Integration of CaRf functional characterization and PepperSNP50K facilitated the development of a high-quality red pepper hybrid. These findings provide significant insights and practical strategies for advancing molecular-designed breeding in peppers.

Identification and variation of a new restorer of fertility gene that induces cleavage in orf138 mRNA of Ogura male sterility in radish

Key messageA new restorer of fertility gene, Rfs, of Ogura cytoplasmic male sterility (CMS) in radish encodes a pentatricopeptide repeat protein that binds to 15 nucleotides in mRNA of the CMS gene, orf138. Nucleotide substitutions in both Rfs and orf138 determine effectiveness and specificity of restoration.Cytoplasmic male sterility (CMS) in plants caused by the expression of abnormal mitochondrial genes results from impaired pollen production. The manifestation of CMS is suppressed by the restorer of fertility (Rf) genes in the nuclear genome. Thus, the CMS-Rf system is a suitable model for studying the direct interactions of mitochondrial and nuclear genes. At least nine haplotypes, of which Type B is ancestry, have been reported for the Ogura CMS gene, orf138, in radish (Raphanus sativus). We previously observed that Rfo encoding a pentatricopeptide repeat (PPR) protein, ORF687, which inhibits the translation of orf138 is ineffective in one haplotype (i.e., Type H). Here, we carried out map-based cloning of another Rf gene (Rfs) that cleaves the orf138 mRNA of Type H. Rfs produces a PPR protein consisting of 15 PPR motifs that binds to the mRNA, cleaving the mRNA at about 50nt downstream of the binding site. However, Rfs was ineffective for Type A because of a single nucleotide substitution in the binding site. Both Rfo and Rfs suppress orf138 expression in ancestral Type B, but they are rendered ineffective in Type H and Type A, respectively, by a single nucleotide substitution in orf138.

Cytoplasmic male sterility in plants with special emphasis on sugar beet

The phenomenon of cytoplasmic male sterility (CMS) in plants is characterized by a disturbance in the development of functional pollen. The cause of this disorder is the incompatibility of the mitochondrial and nuclear genome. Determinants of CMS are found in the mitochondrial genome and are inherited maternally. Nuclear Rf genes are responsible for restoring male fertility in CMS plants most often by posttranscriptional and/or posttranslational activity on mitochondrial gene expression. Two fertility restoring genes have been identified in sugar beet plants, of which Rf1 is the best characterized. It is concluded that the translation product of this gene acts as a molecular chaperone leading to inactivation of a specific mitochondrial protein that is a marker for the CMS trait in sugar beet. The CMS phenomenon is applied in the commercial breeding of hybrid varieties of many crop species. Exploiting heterosis is easier with the knowledge of cytoplasmic (mitochondrial) determinants of sterility and corresponding restorer genes.

Marker assisted identification of improved restorer lines with yield enhancing genes adaptable for aerobic conditions

To feed the world's growing population, development and introduction of climate resilient rice varieties/hybrids with increased yield ability are the need of the hour. This can be accomplished through novel genetic approaches such as hybrid rice technology, using a diverse set of parental lines with high restoring ability and specific desirable traits. In our study, for perse genetic yield improvement in aerobic restorer line AR 9-18R, hybridization was carried out with yield enhancing donor YPK 198 possessing Gn1a and OsSPL14 during kharif, 2018 and generated F1s were fixed for its hybridity through morphological and molecular analysis. In kharif, 2019 two hundred and five F2 population obtained were screened for two yield-enhancing Gn1a and OsSPL14 genes along with two fertility restorer genes Rf3 and Rf4 at ICAR-IIRR, Hyderabad. c2 analysis revealed that the c2 value of both yield enhancing genes was non-significant as the population was segregated in a ratio of 1:2:1. whereas c2 value for fertility restorer genes was significant as population deviated from 1:2:1. Phenotypic evaluation of F2 population for yield related traits exhibited high GCV and PCV for number of productive tillers, number of grains per panicle and single plant yield. Plant height, number of productive tillers, number of grains per panicle and single plant yield displayed high heritability along with high genetic advance as per cent of mean. Correlation and path co-efficient analysis revealed that plant height, number of productive tillers and number of grains per panicle are very important traits as selection criteria for effective yield improvement.

Analysis of the cytohistological phenotype of anthers in sunflower with ASI-AG3 in the context of the PET1 mytotype and the allelic genotype for the Rf1 gene

Microsporogenesis and male gametogenesis are processes that, although under tight morphogenetic control, can often be affected by deviations of internal or external stimuli causing androsterility. The article examines the cytohistological phenotypes of sunflower anthers with gibberellin induced androsterility in plants with different mitotype and allelic genotype for the Rf1 gene, in comparison with the cytohistological phenotypes of fertile and sterile anthers (ASC-PET1). The treatment with gibberellic acid (AG3, 0.01%) was carried out by spraying the inflorescence at the budding stage. Cytological samples and those with semi-thin anther sections were analysed by light microscopy. It was determined that the gametocidal effects induced by AG3 associated with degenerative and early apoptotic events in tapetum cells were more severe in plants with sterile cytoplasm and nuclear Rf1 genes compared to those with fertile cytoplasm and no fertility-restoring genes. In plants with ASC, the exogenous gibberellin stimulus does not cause any visible compensatory events of the sterile phenotype, on the contrary, the effects of microsporogenesis inhibition are more temporally advanced, with sporogenous and sporophytic tissue cells showing various lesions in the premeiosis. The general common and differentiation characteristics found at the androsteril phenotypes suggest interactions between orfH522 and Rf1 gene expression products of negative feedback type in untreated plants and positive feedback type in AG3-treated ones. The research is relevant in the context of elucidating the physiological regulatory processes of the sensitivity of male gametophyte development to various stress conditions, which prospectively makes possible regulatory intervention in plants with different androsterility/androfertility control systems.

Genetic assessment of diverse rice germplasm for yield-related and stress tolerance traits

AbstractThe development of stress-resistant crops without compromising their yield and nutritional quality is one of the major challenges in plant biology. Plant breeders have made considerable effort in developing cultivars with high yield and tolerance to biotic and abiotic stresses. This research aimed to evaluate pre-breeding resources for favorable genes conferring good yield and resistance to biotic and abiotic stresses for rice varietal improvement. Two hundred forty diverse rice cultivars from top countries with which the United States imports and exports rice were selected from the Germplasm Resource Information Network (GRIN) collection, and these were characterized using molecular markers for various desirable traits. Among the cultivars, 16 were used as checks. The genes of various traits evaluated were grain number (Gn1a), panicle weight (WFP), cold stress tolerance (qSCT-11 and qCTS-12), anaerobic germination (AG1 and AG2), rice blast disease resistance (PiB, Pi-ta, Ptr, and Piz), wide compatibility (S5-n), and restoring ability (Rf4). Out of these 240 accessions, 192 have the Gn1a gene, 168 possess the qSCT-11 cold tolerance gene, 139 carry the Rf4 gene, 152 have the qCTS12 cold tolerance gene, 87 have blast resistance genes, and 44 possess the WC gene. None of the 240 accessions possess the WFP (Wealthy Farmer’s Panicle) gene. The selections with favorable genes can be used by breeders as potential donor parents for varietal improvement programs to produce new and promising cultivars with desired specific traits that may contribute towards alleviating global issues related to food security.

The trait-specific collection of large-seeded sunflower at VIR: ba source for breeding cultivars and hybrids

Background. In recent years, an increase has been observed in the share of sunflower seeds in the confectionery and food industries. There are 30 cultivars and only 3 hybrids for confectionery use among 839 zoned sunflower cultivars and hybrids listed in the State Register for Selection Achievements Admitted for Usage in 2023. Thus, the development of source material for breeding large-seeded cultivars and hybrids remains a relevant task.Materials and methods. The studied material included 90 accessions selected for their large seed size from VIR’s sunflower collection. The following characters were assessed: 1000 seed weight, days from sprouting to maturation, plant height, head diameter, cypsela color and size, and resistance to downy mildew. DNA markers were used to identify accessions with the Rf1 gene and CMS.Results. Accessions k-2818 (Primorsky Territory), k-3633, k-3748, k-3782 (China), and k-3578 (Ukraine) were the best in 1000 seed weight. Russian cvs. ‘Alekseyevsky Krupnoplodny 2’ (k-3552), ‘Gyar-Gyar’ (k-1589), ‘SPK’ (k-3426), and ‘Lakomka’ (k-3526) confirmed their large seed size. The sterile type of cytoplasm was identified in 12 accessions. Fourteen accessions with fertile cytoplasm had diagnostic markers of the nuclear Rf1 gene.Conclusion. The large-seeded cultivars developed at VNIIMK, ‘SPK’, ‘Lakomka’, and k-3782 from China were the best not only in their large seed size but also in downy mildew resistance. As a result of long-term studies, a trait-specific collection was established for the large seed size trait. It included 90 accessions that can be used in the development of confectionery cultivars. Large-seeded sunflower accessions carrying the Rf1 gene were identified. Some of them became the ancestors of donor lines for both pollen fertility restoration genes and large seed size.

Unveiling a half-century mystery of molecular bases for three-line hybrid rice breeding system.

Hybrid rice derived from Wild Abortive (WA) cytoplasmic male sterility type three-line systems is widely used in production. The fertility restoration gene Rf4 can reduce WA352c transcript levels in a dose-dependent manner. A two-copy haplotype of Rf4 (H1) is identified as the most valuable haplotype for future hybrid rice breeding.

Identification of SSR markers linked to new fertility restoration trait in sorghum (Sorghum bicolor (L.) Moench) for A4 (maldandi) male sterile cytoplasm

AbstractIdentification of markers associated with fertility restoration (Rf) genes is essential because they can streamline the breeding of new CMS lines and production of commercial hybrid seeds. Therefore, in the present study, F2 populations (M31‐2A × DSMR 8) was utilized to identify markers linked to Rf loci on maldandi (A4) cytoplasm through bulk segregant analysis (BSA). The F2 population was analysed for seed set percentage. Chi‐square (χ2) analysis showed that the fertility restoration trait followed expected digenic ratio. By BSA, simple sequence repeats (SSRs) markers, namely, Xtxp 34 and Xtxp 69 located on chromosome 3 and SB 3956 and Xtxp 312 located on chromosome 7, showed clear polymorphism between two groups of fertile and sterile bulks. The genomic region harbouring Rf locus on chromosome 3 (2.61 Mbp) predicted to encode five pentatricopeptide repeat (PPR) genes whereas, on chromosome 7, the gene SORBI_3007G047400 predicted to encode MYB (myeloblastosis) domain containing proteins. These predicted genes could be the candidate for restoring fertility on A4 cytoplasm. This finding will be fundamental in the production and rapid selection of novel restorer lines.

Genetic variability studies of yield, its attributing traits and oil content in RHAlines of sunflower (Helianthus annuus L.)

Sunflower inbred lines (RHA) derived from different sources with Rf gene were evaluated during 2018- 2020.Genetic variability, phenotypic and genotypic coefficients of variation, heritability and genetic advance were assessed for seven traits related to seed yield, its attributing traits and oil content in 50 RHA lines of sunflower. The analysis of variance revealed highly significant differences for all the characters among the RHA lines. The magnitude of differences between PCV and GCV was relatively low for all the traits, indicating less environmental influence. The estimates of GCV and PCV were high for seed yield/plant. High heritability in broad sense estimates (>90%) were recorded for all the traits under study indicating, the predominance of additive gene action and selection of RHA lines based on these characters would be more valid and selected RHA lines are likely produce high yielding hybrids. Effective for target plant selection in early generation.

Strong and selective interactions of palmatine with G-rich sequences in TRF2 promoter; experimental and computational studies

G-rich sequences have the potential to fold into G-quadruplexes (GQs). G-quadruplexes, particularly those positioned in the regulatory regions of proto-oncogenes, have recently garnered attention in anti-cancer drug design. A thermal FRET assay was employed to conduct preliminary screening of various alkaloids, aiming to identify stronger interactions with a specific set of G-rich double-labeled oligonucleotides in both K + and Na + buffers. These oligonucleotides were derived from regions associated with Kit, Myc, Ceb, Bcl2, human telomeres, and potential G-quadruplex forming sequences found in the Nrf2 and Trf2 promoters. Palmatine generally increased the stability of different G-rich sequences into their folded GQ structures, more or less in a concentration dependent manner. The thermal stability and interaction of palmatine was further studied using transition FRET (t-FRET), CD and UV-visible spectroscopy and molecular dynamics simulation methods. Palmatine showed the strongest interaction with T RF2 in both K+ and Na+ buffers even at equimolar concentration ratio. T-FRET studies revealed that palmatine has the potential to disrupt double-strand formation by the T RF2 sequence in the presence of its complementary strand. Palmatine exhibits a stronger interaction with G-rich strand DNA, promoting its folding into G-quadruplex structures. It is noteworthy that palmatine exhibits the strongest interaction with T RF2, which is the shortest sequence among the G-rich oligonucleotides studied, featuring only one nucleotide for two of its loops. Palmatine represents a suitable structure for drug design to develop more specific ligands targeting G-quadruplexes. Whether palmatine can also affect the expression of the T RF2 gene requires further studies. Communicated by Ramaswamy H. Sarma

Copy number variation of the restorer Rf4 underlies human selection of three-line hybrid rice breeding

Cytoplasmic male sterility (CMS) lines are important for breeding hybrid crops, and utilization of CMS lines requires strong fertility restorer (Rf) genes. Rf4, a major Rf for Wild-Abortive CMS (CMS-WA), has been cloned in rice. However, the Rf4 evolution and formation of CMS-WA/Rf system remain elusive. Here, we show that the Rf4 locus emerges earlier than the CMS-WA gene WA352 in wild rice, and 69 haplotypes of the Rf4 locus are generated in the Oryza genus through the copy number and sequence variations. Eight of these haplotypes of the Rf4 locus are enriched in modern rice cultivars during natural and human selections, whereas non-functional rf4i is preferentially selected for breeding current CMS-WA lines. We further verify that varieties carrying two-copy Rf4 haplotype have stronger fertility restoration ability and are widely used in three-line hybrid rice breeding. Our findings increase our understanding of CMS/Rf systems and will likely benefit crop breeding.

Heat-responsive microRNAs participate in regulating the pollen fertility stability of CMS-D2 restorer line under high-temperature stress

Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA–mRNA–gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.

The top Bulgarian contributions to sunflower breeding

Abstract The sunflower (Helianthus annuus L.) is a fundamental oil-yielding culture for Bulgaria and the world and thus, special attention is paid to it. Various selection methods have been used over the years and as a result of successful breeding programs new sunflower forms, lines (В/А and R) and hybrids (oil and confectionery type) were created in Bulgaria. Some of these new forms have new plant architectonic, different vegetation periods, seeds with different sizes and coloration, high combining ability, resistance to diseases, the parasite Broomrape and some herbicides, high seed oil and fatty acid content, genes for restoration of fertility (Rf genes), and different types of cytoplasmic male sterility (CMS).

Genetic Analysis of Novel Fertility Restoration Genes (qRf3 and qRf6) in Dongxiang Wild Rice Using GradedPool-Seq Mapping and QTL-Seq Correlation Analysis.

The improvement of grain yield, quality, and resistance can be achieved through the utilization of heterosis. The combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) gene(s) greatly facilitates the commercial development of three-line hybrid rice based on heterosis. The basis for investigating the relationship between CMS and Rf genes lies in the rapid localization of wild rice fertility restoration genes. A set of the BC4F5 population derived from interspecific crosses between Xieqingzao B (XB) and the BC1F9 XB//Dongxiang wild rice (DWR)/XB line L5339 was used to detect quantitative trait loci (QTL) for fertility restoration. The population was then crossed with two male sterile lines, Zhong9A (Z9A) and DongB11A (DB11A), in order to generate a testcrossing population for investigating spikelet fertility. Based on the linkage mapping, seven QTLs were detected on chromosomes 1, 3, 5, 6, 8, and 10, explaining 2.76 to 12.46% of the phenotypic variation. Of them, two novel fertility restoration QTLs, qRf3 and qRf6, can restore fertility of the CMS-DWR line DB11A by 16.56% and 15.12%, respectively. By employing joint QTL-seq and GradedPool-Seq methods, two novel Rf QTLs for DB11A, qRf3 and qRf6, were identified at the physical locations of 10,900,001-11,700,000 bp and 28,016,785-31,247,556 bp, respectively. These findings are useful for exploring the natural variations of Rf genes in rice. Therefore, rice's new genetic resources for the selection and breeding of rice restorer lines provide promising candidates for QTL fine localization and clarification.

Genome-Wide Dissection of Quan 9311A Breeding Process and Application Advantages

Germplasm resource innovation is a crucial factor for cultivar development, particularly within the context of hybrid rice breeding based on the three-line system. Quan 9311A, a cytoplasmic male sterile (CMS) line, has been successfully cultivated using rice restoration materials and extensively empolyed as a female parent in hybrid breeding program in China. This line was developed by crossing the CMS line Zhong 9A with a two-line restorer line 93-11, with the intention of eliminating the restoring ability of 93-11 while retaining the sterility gene WA352c from Zhong 9A. Quan 9311A effectively amalgamates the most favorable agronomic traits from both parental lines. In this study, the relationship between phenotypic characteristics and the known functional genes of Quan 9311A were analyzed using the rice genome navigation analysis technology based on whole-genome sequencing. The findings revealed that Quan 9311A harbors multiple superior alleles from both 93-11 and Zhong 9A, providing exceptional agronomic traits that were unavailable in earlier CMS lines. Despite the removal of the fertility restorer gene Rf3 from 93-11, numerous chromosomal segments from 93-11 persist in the Quan 9311A genome. Furthermore, the hybrid rice Quanyousimiao (QYSM) and the restorer line Wushansimiao (WSSM) were used as examples to illustrate the important role of Quan 9311A as the female parent in heterosis. It was found that QYSM carries a great number of superior alleles, which accounts for its high grain yield and wide adaptability. These insights not only advanced the utilization of hybrid rice pairing groups but also provided guidance for future breeding endeavors. The study introduced innovative concepts to further integrate genomics with traditional breeding techniques. Ultimately, Quan 9311A signifies a significant milestone in rice breeding technology, opening up novel avenues for hybrid rice development.

Dose effects of restorer gene modulate pollen fertility in cotton CMS-D2 restorer lines via auxin signaling and flavonoid biosynthesis.

Dose effects of Rf1 gene regulated retrieval mechanism of pollen fertility for CMS-D2 cotton. Cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) is an economical pollination control system for producing hybrid cotton seeds compared to artificial and chemical emasculation methods. However, the unstable restoring ability of restorer lines is a main barrier in the large-scale application of "three-line" hybrid cotton in China. Our phenotypic investigation determined that the homozygous Rf1Rf1 allelic genotype had a stronger ability to generate fertile pollen than the heterozygous Rf1rf1 allelic genotype. To decipher the genetic mechanisms that control the differential levels of pollen fertility, an integrated metabolomic and transcriptomic analysis was performed at two environments using pollen grains of four cotton genotypes differing in Rf1 alleles or cytoplasm. Totally 5,391 differential metabolite features were detected, and 369 specific differential metabolites (DMs) were identified between homozygous and heterozygous Rf1 allelic genotypes with CMS-D2 cytoplasm. In addition, transcriptome analysis identified 2,490 differentially expressed genes (DEGs) and 96 unique hub DEGs with dynamic regulation in this comparative combination. Further integrated analyses revealed that several key DEGs and DMs involved in indole biosynthesis, flavonoid biosynthesis, and sugar metabolism had strong network linkage with fertility restoration. In vitro application of auxin analogue NAA and inhibitor Auxinole confirmed that over-activated auxin signaling might inhibit pollen development, whereas suppressing auxin signaling partially promoted pollen development in CMS-D2 cotton. Our results provide new insight into how the dosage effects of the Rf1 gene regulate pollen fertility of CMS-D2 cotton.

Identification of Good Restorer Lines through Molecular Confirmation of Rf3 and Rf4 Genes in Rice Hybrids

Pollen fertility can be restored by nuclear-encoded genes known as fertility restorer (Rf) genes. The initial stage in creating high-yielding heterotic hybrids involves the identification of restorers capable of effectively restoring the fertility of CMS lines. Hybrid rice breeding programs have proven effective in achieving these goals by utilizing cytoplasmic male sterility (CMS) systems in combination with restorer genes. Among these, Rf3 and Rf4 genes play critical roles in restoring male fertility and are essential components of successful hybrid rice breeding. In this study, we aimed to investigate the molecular conformation of Rf3 and Rf4 genes in a set of restorer lines and hybrids in rice. DNA was isolated from 10 restorer lines and 30 rice hybrids, two checks and KMR3R were used for comparison for positive alleles. Two functional markers, RM SF21-5 for Rf3 and RMS-PRR-9-1 for Rf4, were employed to identify the allelic status of the fertility restorer genes. Results revealed that among the 42 rice entries screened, five rice hybrids lacked both Rf3 and Rf4 genes and Four rice restorer lines and four hybrids were found to possess both Rf3 and Rf4 genes, making them valuable for hybrid breeding programs. Conversely, five hybrids lacked restorer genes and were unsuitable for such breeding programs. These findings establish Rf3and Rf4 as major fertility restoring genes in rice, consistently contributing to complete fertility restoration. The genotyping results provide valuable insights for selecting appropriate parental lines and restorers in hybrid rice breeding programs. The presence or absence of these fertility-restoring genes plays a crucial role in developing high-yielding and productive rice varieties through effective breeding strategies.

A unique C-terminal domain contributes to the molecular function of Restorer-of-fertility proteins in plant mitochondria.

Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants.