Fertility Restorer Gene Research Articles

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.

Insights into cellular crosstalk regulating cytoplasmic male sterility and fertility restoration.

Cytoplasmic male sterility has been a popular genetic tool in development of hybrids. The molecular mechanism behind maternal sterility varies from crop to crop. An understanding of underlying mechanism can help in development of new functional CMS gene in crops which lack effective and stable CMS systems. In crops where seed or fruit is the commercial product, fertility must be recovered in F1 hybrids so that higher yield gains can be realized. This necessitates the presence of fertility restorer gene (Rf) in nucleus of male parent to overcome the effect of sterile cytoplasm. Fertility restoring genes have been identified in crops like wheat, maize, sunflower, rice, pepper, sugar beet, pigeon pea etc. But in crops like eggplant, bell pepper, barley etc. unstable fertility restorers hamper the use of Cytoplasmic genic male sterility (CGMS) system. Stability of CGMS system is influenced by environment, genetic background or interaction of these factors. This review thus aims to understand the genetic mechanisms controlling mitochondrial-nuclear interactions required to design strong and stable restorers without any pleiotropic effects in F1 hybrids.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

BackgroundUnderstanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to several challenges, like inadequate variation of agronomic traits, incomplete sterility, weak resilience of restorer lines, and difficulty in combining strong dominance. Therefore, the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.ResultsTwo F2 segregating populations of cotton were constructed from cytoplasmic male sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants. Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants. However, sterile plants exhibited smaller floral organs, shortened filament lengths, and anther atrophy on the flowering day in comparison with the fertile plants. When performed scanning electron microscopy (SEM), the two F2 populations revealed morphological variations in the anther epidermis. Cellular analysis showed no significant differences in pollen development before pollen maturation. Interestingly, between the pollen maturation and flowering stages, the tapetum layer of sterile plants degenerated prematurely, resulting in abnormal pollen grains and gradual pollen degradation.ConclusionThe results of this study suggest that fertility-restoring genes are controlled by a single dominant gene. Sterile plants exhibit distinctive floral morphology, which is characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between pollen maturity and flowering, indicating that premature tapetum degradation may be the primary cause of pollen abortion. Overall, our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations, mechanisms of sterility, and the role of sterile and 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.

Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines.

BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker-assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.

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 enhancement of reproductive stage drought tolerance in RPHR-1005R and derivative rice hybrids through marker-assisted backcross breeding in rice (Oryza sativa L.).

Drought stress is considered as one of the major production constraints in rice. RPHR-1005R is a restorer line (R-Line) with a popular, medium-slender grain type, and is the male parent of the popular Indian rice hybrid, DRR-H3. However, both the hybrid and its restorer are highly vulnerable to the drought stress, which limits the adoption of the hybrid. Therefore, the selection of the restorer line RPHR-1005R has been made with the objective of enhancing drought tolerance. In this study, we have introgressed a major QTL for grain yield under drought (qDTY 1.1) from Nagina22 through a marker-assisted backcross breeding (MABB) strategy. PCR based SSR markers linked to grain yield under drought (qDTY1.1 - RM431, RM11943), fertility restorer genes (Rf3-DRRM-Rf3-10, Rf4-RM6100) and wide compatibility (S5n allele) were deployed for foreground selection. At BC2F1, a single plant (RPHR6339-4-16-14) with target QTL in heterozygous condition and with the highest recurrent parent genome recovery (85.41%) and phenotypically like RPHR-1005R was identified and selfed to generate BC2F2. Fifty-eight homozygous lines were advanced to BC2F4 and six promising restorer lines and a hybrid combination (APMS6A/RPHR6339-4-16-14-3) were identified. In summary, the six improved restorer lines could be employed for developing heterotic hybrids possessing reproductive stage drought tolerance. The hybrid combination (APMS6A/RPHR6339-4-16-14-3) was estimated to ensure stable yields in drought-prone irrigated lowlands as well as in directly seeded aerobic and upland areas of India.

Genetic basis of the phenomenon of male sterility and fertility restoration in maize (Zea mays L.) – a review

The phenomenon of male sterility in higher plants is, apart from protandry (earlier maturation of stamens), protogyny (earlier maturation of pistils), heterostyly (different stigmas) and self-incompatibility, one of the evolutionarily conditioned mechanisms forcing external pollination. Due to the elimination of the time- and cost-consuming emasculation of maternal lines, male-sterile lines are an object of interest in the seed production of hybrid cultivars of many plant species, including maize. Seed production of hybrid cultivars using male-sterile lines requires the establishment of maternal lines that are male-sterile in different environments and suitable paternal lines with fertility restorer genes. This paper summarizes the findings on the genetics of male sterility and fertility restoration in maize.

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.

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.

Mapping of Rf20(t), a minor fertility restorer gene for rice wild abortive cytoplasmic male sterility in the maintainer line 'Zhenshan97B'.

Three-line hybrid rice has primarily been developed on wild abortive (WA)-type cytoplasmic male sterility (CMS) and has helped increase the yield of rice globally. The development of WA-type CMS lines and hybrids was expedited through the identification and mapping of the fertility restorer gene (Rf) in maintainers. This study observed fertile plants in WA-TianfengA/Zhenshan97B//TianfengB population, indicating that the maintainer line 'Zhenshan97B' should carry Rfs for WA-type CMS. Several advanced backcross populations were generated with the genetic background of the 'WA-TianfengA,' and the pollen fertility levels of the backcrossed individuals in BC3F1, BC4F1 and BC4F2 populations are governed by a new gene, Rf20(t), from 'Zhenshan97B.' Employing bulk segregant analysis of fertile and sterile pools from the BC4F1 population, Rf20(t) was genetically mapped to a candidate region on chromosome 10. Subsequently, Rf20(t) was located between RM24883 and RM24919 through recombination analysis of molecular markers using the BC4F2 population. Implementing a substitution mapping strategy, Rf20(t) was ultimately mapped to a 245-kb region between the molecular markers STS10-122 and STS10-126 and obtained the most likely candidate gene LOC_Os10g02650, which is predicted to encode pentatricopeptide repeat-containing (PPR) protein. These results enhance our understanding of the fertility restoration of WA-type CMS lines, facilitating the development of high-quality pairs of WA-type CMS and maintainer lines.

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.

Fine mapping and candidate gene analysis of a novel fertility restorer gene for C-type cytoplasmic male sterility in maize (Zea mays L.).

A novel strong fertility restorer gene Rf12 for C-type cytoplasmic male sterility of maize was finely mapped on chromosome 2. Its best candidate gene Zm00001d007531 is predicted to encode a p-type PPR protein. The lack of strong restorer gene of maize CMS-C greatly limits its application in hybrid seed production. Therefore, the cloning of maize CMS-C novel strong restorer genes is necessary. In this study, a strong restorer line ZH91 for maize CMS-C was found, and the novel restorer gene named Rf12 in ZH91 had been mapped in a 146kb physical interval on maize chromosome 2. Using the third-generation high-throughput sequencing (ONT), the whole genome sequence of ZH91 was got, and with integrating the annotation information of the reference genome B73_RefGen_v4 and B73_RefGen_v5, four candidate genes were predicted in ZH91 within the mapping region. Then using gene cloning, stranded specific RNA sequencing, qRT-PCR analysis and subcellular localization, Zm00001d007531 was identified as the most likely candidate gene of Rf12. Zm00001d007531 encodes a p-type PPR protein with 19 PPR motifs and targets mitochondria and chloroplast. Stranded specific RNA sequencing and qRT-PCR results both show that the expression of Zm00001d007531 between anthers of near-isogenic lines C478Rf12Rf12 and C478rf12rf12 was significantly difference in pollen mother cell stage. And the result of sequence alignment for Zm00001d007531 gene in 60 materials showed that there are twelve SNPs in CDS region of Zm00001d007531 were tightly linked to the fertility. The finding of a novel strong restorer germplasm resource ZH91 for maize CMS-C can greatly promote the application of maize CMS-C line in maize hybrid seeds production, and the identification of candidate gene Zm00001d007531 can accelerate the backcrossing process of maize CMS-C strong restorer gene Rf12 to some extent.

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.

Validation for the major fertility restorer genes, Rf3 and Rf4 of F4 generation of CBSN 25/ WRM 21-24 and CBSN 25/WRM 93-20 crosses in rice (Oryza sativa L.)

Validation for the major fertility restorer genes, Rf3 and Rf4 of F4 generation of CBSN 25/ WRM 21-24 and CBSN 25/WRM 93-20 crosses in rice (Oryza sativa L.)

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.

Development of new sources of male sterility in sunflower (Helianthus annuus L.) : Limitations and opportunities

Cytopasmic male sterility (CMS) is a reasonably common characteristic of higher plants. The discovery of astable source of CMS was the first step towards the practical use of heterosis in sunflower. The second step was the discovery of fertility restoration gene(s). The first CMS (CMS89) in sunflower was reported in descendants of an interspecific hybrid between Helianthus petiolaris Nutt. and cultivated sunflower (the variety Armavirskiy-9345). Although CMS89 or its derivatives are used to produce all commercial sunflower hybrids, more than 72 additional sources ofsunflower CMS have been described by variousscientists. Male sterility may have multiple causes. It can result from adverse growth conditions, from diseases, or from mutations. In this paper, CMS sources obtained by different methods like hybridization (both intraspecific and interspecific crosses), mutagenesis, chemical and spontaneous occurrence of male sterility as well as limitations and opportunities of diverse CMS sources in sunflower were discussed.

Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.

The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.

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.

Refinement of Rf1-gene localization and development of the new molecular markers for fertility restoration in sunflower.

Ability to restore male fertility is important trait for sunflower breeding. The most commonly used fertility restoration gene in the production of sunflower hybrids is Rf1. The localization of Rf1 on the linkage group 13 has been previously shown, however, its exact position, its sequence and molecular mechanism for fertility restoration remain unknown. Therefore, several markers linked to Rf1 gene, commonly used for MAS, don't always allow to identify the genotype of plants. For this reason, the search for new markers and precise localization of the Rf1 gene is an urgent task. Based on previously identified single nucleotide polymorphisms (SNPs) at LG13, significantly associated with the ability to restore male fertility, two markers have been developed that have performed well after careful evaluation. These markers, together with other Rf1 markers, were applied for genotyping 72 diversity panel accessions and 291 individuals of F2 segregating population, obtained from crossing the cytoplasmic male sterility (CMS) AHO33 and restorer RT085HO lines. The analysis revealed no recombinants between Rf1 gene and SRF833 marker, the distance between Rf1 and SRF122 marker was 1.0cM. Data obtained made it possible to specify the localization of the Rf1 gene and reduce the list of candidate genes to the 3 closely linked PPR-genes spanning a total of 59 Kb. However, it cannot be ruled out that analysis of the candidate region in the genome of fertility restorer lines can reveal new candidate genes in this locus that are absent in the cytoplasmic male sterility maintainer reference sequence.