Fertility Restorer Lines Research Articles

Genetic Resources for Fertility Restoration Lines and Maintainers of Capsicum annuum L.

Male fertility reactions of one hundred and forty-one accessions of chilies were classified. Three groups were found. Some accessions maintained male sterility and were determined to carry a non-sterile cytoplasm and to lack fertility restoration genes or N rfrf genotype. Some accessions segregated for the ability to restore male sterile cytoplasm and were determined to be heterozygous in restorer genes with genotype N/SRfrf. Some accessions restored fertility of CMS and had the genotype N/SRfRf. A few maintainers with good horticultural characteristics were selected. They were selfed and selected for a few generations and then their progeny were evaluated. There were differences in the genetic stability of cytoplasmic male-sterility among the selected lines. Some lines were good maintainers, but a few lines were discarded. The stable maintainers were distributed to 10 seed companies and the government of China. Some F1 hybrid chilies produced using these lines have been commercially sold both in China and Thailand. Fruit physio-chemical qualities of maintainer accessions, restorers and heterozygous accessions were also recorded. The level of capsaicin of the accessions varied from 3,250 to 8,850 Scoville units. The level of vitamin C showed a range of 4.43 to 103.16 mg./100g.fw. Horticultural characteristics of the accessions were recorded and the fruit physio-chemical qualities of the accessions were reported.

Transcriptome profiling of differentially expressed genes in cytoplasmic male-sterile line and its fertility restorer line in pigeon pea (Cajanus cajan L.)

BackgroundPigeon pea (Cajanus cajan L.) is the sixth major legume crop widely cultivated in the Indian sub-continent, Africa, and South-east Asia. Cytoplasmic male-sterility (CMS) is the incompetence of flowering plants to produce viable pollens during anther development. CMS has been extensively utilized for commercial hybrid seeds production in pigeon pea. However, the molecular basis governing CMS in pigeon pea remains unclear and undetermined. In this study transcriptome analysis for exploring differentially expressed genes (DEGs) between cytoplasmic male-sterile line (AKCMS11) and its fertility restorer line (AKPR303) was performed using Illumina paired-end sequencing.ResultsA total of 3167 DEGs were identified, of which 1432 were up-regulated and 1390 were down-regulated in AKCMS11 in comparison to AKPR303. By querying, all the 3167 DEGs against TAIR database, 34 pigeon pea homologous genes were identified, few involved in pollen development (EMS1, MS1, ARF17) and encoding MYB and bHLH transcription factors with lower expression in the sterile buds, implying their possible role in pollen sterility. Many of these DEGs implicated in carbon metabolism, tricarboxylic acid cycle (TCA), oxidative phosphorylation and elimination of reactive oxygen species (ROS) showed reduced expression in the AKCMS11 (sterile) buds.ConclusionThe comparative transcriptome findings suggest the potential role of these DEGs in pollen development or abortion, pointing towards their involvement in cytoplasmic male-sterility in pigeon pea. The candidate DEGs identified in this investigation will be highly significant for further research, as they could lend a comprehensive basis in unravelling the molecular mechanism governing CMS in pigeon pea.

BREEDING OF WINTER RAPESEED INBRED LINES FOR THE DEVELOPMENT OF PARENT FORMS FOR HYBRIDS

Introduction. The V.S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK) performs the breeding of interline rapeseed hybrids using the Ogura CMS. The program of interline winter rapeseed hybrid development was aimed at studying the efficiency of self-pollinated line breeding from population cultivars, assessing the impact of inbreeding depression on economically important traits, analyzing the changes in the lines’ traits after their transition to the sterile basis, and obtaining fertility restorer lines with an acceptable level of glucosinolate content in seeds.Materials and methods. Intraspecific hybrids, cultivars developed at the Pustovoit Institute and those of foreign breeding were employed as parent material for the development of self-pollinated lines. Self-pollination of rapeseed plants was performed using spunbond isolators. Over 1000 plants were self-pollinated every year. Generations of self-pollinated lines S1 –S4 were planted on 2.25 m2 plots, and during the growth season their visual screening was undertaken to assess their morphobiological traits. Starting with S5, the lines were evaluated for their yield on 7.5 m2 plots.Results. The inbreeding depression was found to not exceed 3–15% of the seed yield. The transition of the lines to sterile cytoplasm did not have any negative consequences. A series of experimental hybrids were developed, exceeding the reference cultivar ‘Loris’ in seed yield by 23–48%. New hybrid combinations also exceeded the reference in seed yield by 21–54%.

Создание линий-восстановителей фертильности пыльцы подсолнечника, устойчивых к наиболее распространенным расам ложной мучнистой росы в Краснодарском крае

Создание линий-восстановителей фертильности пыльцы подсолнечника, устойчивых к наиболее распространенным расам ложной мучнистой росы в Краснодарском крае

Создание линий-восстановителей фертильности пыльцы подсолнечника, устойчивых к наиболее распространенным расам ложной мучнистой росы в Краснодарском крае

Создание линий-восстановителей фертильности пыльцы подсолнечника, устойчивых к наиболее распространенным расам ложной мучнистой росы в Краснодарском крае

Intrapopulation recurrent selection in sweet sorghum for improving sugar yield

Sweet sorghum [Sorghum bicolor (L.) Moench] accumulates fermentable sugars in the stalk making this crop an important bioenergy feedstock. The objectives of this study were to implement an intrapopulation recurrent selection program to increase the sugar yield in sweet sorghum [Sorghum bicolor (L.) Moench] and to estimate the genetic progress after a recurrent selection cycle for the main agro-industrial traits of interest. The base population for cycle 0 (C0) was developed from 15 fertility restorer lines using a genetic male-sterile source to promote recombination. A total of 196 half-sib progeny from C0 were evaluated in experiments with a 14 × 14 lattice design in the 2016–2017 season. The following traits were evaluated: days to flowering (DF), plant height (PH), fresh biomass yield (FBY), total soluble solids content (TSS) and tons of Brix per hectare (TBH). Data were analyzed using the mixed model approach. Genetic variation was observed among the C0 half-sib progeny for all of the agro-industrial traits studied. The 40 best progeny were selected based on the best linear unbiased predictions (BLUPs) for TBH, which were then recombined to form the base population for cycle 1 (C1). Subsequently, 40 half-sib progeny from each cycle (C0 and C1) were randomly selected and evaluated in experiments with a 9 × 9 lattice design to estimate the genetic progress for DF, PH, FBY, TSS and TBH traits. The genetic progress was significant, with increases of 3.73% for PH, 8.04% for FBY, and 8.65% for TBH, demonstrating the efficiency of the intrapopulation recurrent selection program used.

Accelerating hybrid rice breeding through the adoption of doubled haploid technology for R-line development

Hybrid rice holds significant yield advantage over inbred lines, but its adoption rate is very slow in India. Development of cytoplasmic male sterile line (CMS) and fertility restorer line with superior grain quality is the prerequisite in three line hybrid rice breeding programme. Minimum of three to four years are required for development of useful R-line which severely delays the pace of hybrid breeding. Application of doubled haploid (DH) technology will be very useful to develop elite R-lines within short period of time. An elite inbred line LAXMI was crossed with the R-line RPR158 and anthers from the resulting F1 population was used for development of DH plants. Eight hormonal combinations and four different cold pre-treatment conditions were tried in tissue culture. Maximum callusing frequency was observed in N6 medium supplemented with 2.0 mg L−1 2, 4-diclorophenoxyacetic acid (2,4-D), 0.5 mg L−1 napthalene acetic acid (NAA), 0.5 mg L−1 benzyl amino purine(BAP) and 6% maltose with 3 days cold pre-treatment. Highest green plant regeneration frequency was found with Murashige and Skoog medium supplemented with 0.5 mg L−1 NAA, 0.5 mg L−1 kinetin, 2 mg L−1 BAP. A total of 280 DH plants were generated and evaluated under field conditions for agronomic characters and fertility restoration capacity in CMS system. Two distinct DH lines were identified as improved R-lines based on high restoration ability (94%), superior grain quality and medium slender grain type. This study successfully demonstrated that superior R-lines can be developed within two crop seasons through the application of DH technology.

General and specific combining ability in sweet sorghum

In breeding of sweet sorghum hybrids, non-additive genetic effects are important in phenotypic expression of the traits of interest. The aim of this study was to evaluate the general combining ability (GCA) of sweet sorghum lines and the specific combining ability (SCA) of the hybrids for agronomic and technological traits. Five fertility restorer lines, four male-sterile lines, and their hybrids from partial diallel crosses were evaluated in experiments laid out in a 5 x 6 triple rectangular lattice design in the municipalities of Lavras, MG and Sete Lagoas, MG, Brazil. Diallel analysis was performed using the Griffing model adapted to partial diallel crosses. There was a significant effect of GCA and SCA for most of the traits evaluated, indicating the participation of additive or dominant genes in inheritance. The restorer lines CMSX508, BRS 511, CMSXS643, and CMSXS646 show potential for use as parents in sorghum breeding programs.

Identification of fertility restorer and sterility maintainer lines for diversified CMS lines in sunflower (Helianthus annuus L.)

Six diversifiedCMS lines ARG-6-3-1-4, PKUZ, ARG-3, ARG-2-1-2, PRUN-29 and MUT2-8-3-2 were crossed with ninety six inbred lines in a line x tester fashion to study their sterility maintenance or fertility restoration reaction. Out of 96 inbreds tested, 48 inbreds restored fertility in the diversified CMS lines viz., ARG-6-3-1-4, PKUZ, ARG-3, ARG-2-1-2, while 47 and 45 inbreds restored fertility in MUT2-8-3-2 and PRUN-29, respectively. On the other hand, 39, 46, 45, 43, 44 and 46 inbreds maintained sterility in the diversified CMS lines ARG-6-3-1-4, MUT-2-8-2, PUKZ, ARG-3, PRUN-29 and ARG-2-1-2, respectively. Among 96 inbreds, as many as 38 inbreds were identified as common maintainers for all the six diverse CMS lines. Similarly, 39 inbreds were identified as common restorers for all the six diverse CMS lines. Effective restorer lines identified for alternate sources of cytoplasm could be exploited in developing hybrids with better heterosis and it can pave way for broadening the CMS sources used in commercial hybrid development. Identified promising maintainer lines could be used in development of new CMS lines through conversion.

Development of CGMS system in ridge gourd [Luffa acutangula (Roxb.) L.] for production of F1 hybrids

Cytoplasmic male sterile system in ridge gourd has been converted to cytoplasmic genetic male sterile (CGMS) system through the development of analogues of male sterile (MS) line, maintainer line and fertility restorer line. These lines were developed by crossing the MS mutant, regenerated through in vitro culture, with monoecious pollen parents Deepthi, Haritham, LA 101, CO 2, IC 92761 and IC 92685. All hybrids and the BC1 generation developed by crossing with the recurring pollen parents Deepthi, Haritham and LA 101 were male sterile. Male sterile BC1 plants have been advanced to BC6 generation and the parental line LA 101 was proved to be a successful maintainer line, producing male sterile progeny in successive back cross generations. Analogue of cytoplasmic male sterile line, MS LA 101, was developed through back crossing and on crossing with fertility restorer lines Arka Sumeet and LA 102, this line excelled as female parent, resulting heterotic combinations. Mitochondrial marker rpS14 and SCAR Tm-53 were identified to yield male sterility specific markers whereas SSR marker 18956 has generated the male fertility specific marker. These primers are recommended for marker assisted selection of ridge gourd, for utilizing male sterility for hybrid seed production and for developing A, B and C lines in CGMS system.

Effect of Sclerotinia sclerotiorum on Sunflower Seeds Quality

AbstractThe investigation was carried out at Dobrudzha Agricultural Institute – General Toshevo, Bulgaria, and encompassed the period 2009–2010. The experiment involved four double haploid fertility restorer lines (DH-R-2, DH-R-7, DH-R-116 and DH-R-128), which have shown in our previous studies various degree of resistance to Sclerotinia sclerotiorum de Bary under artificial infection conditions. Ten plants from each investigated line were inoculated by the Straw-method at stage 5-6th pair of leaves. The plants were self-pollinated and the seeds obtained from them, as well as the seeds from the check plants (not infected), were analyzed for the traits 1000 seed weight, % of kernel, oil in kernel and protein content in kernel. Variations in the quality characteristics of the sunflower seeds were found in all investigated lines, the degree of quality “deterioration” having different expression according to the tolerance of the line to the pathogen. Lines DH-R-2 and DH-R-7 formed seeds with lower seed weight and percent of kernel. These seeds had lower oil and protein content. The established variations in the quality of the seeds between lines DH-R-116 and DH-R-128 and the check variants were not statistically significant.

Heterosis in sweet sorghum

Abstract: The objective of this work was to evaluate the potential per se of male-sterile and fertility-restorer lines of sweet sorghum (Sorghum bicolor), as well as to detail the heterosis manifested for some traits directly or indirectly related to ethanol production, accumulation rate, and predictability. Evaluations were performed for 20 genotypes, of which 4 are fertility-restorer lines (R), 3 are male-sterile lines (A), and 12 are experimental hybrids (H) resulting from the partial diallel cross between lines A and R, besides a commercial hybrid CV198 used as a check, in four harvest seasons. The experiments were carried out in the municipalities of Lavras and Sete Lagoas, in the state of Minas Gerais, Brazil. The measured traits were plant height, green mass production, juice extraction, total soluble solids content, and megagrams of Brix per hectare. The male-sterile A1 and the fertility-restorer R1 and R3 lines show the best potential per se, considering all traits and their accumulation rate and predictability over harvest times. Heterosis is significant for all traits. The H11, H13, H14, H21, H22, and H33 hybrids are promising because of their better performance per se and higher heterosis.

Molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea (Cajanus cajan (L.) Millsp.)

Key messageWe report molecular mapping and inheritance of restoration of fertility (Rf) in A4 hybrid system in pigeonpea. We have also developed PCR-based markers amenable to low-cost genotyping to identify fertility restorer lines.Commercial hybrids in pigeonpea are based on A4 cytoplasmic male sterility (CMS) system, and their fertility restoration is one of the key prerequisites for breeding. In this context, an effort has been made to understand the genetics and identify quantitative trait loci (QTL) associated with restoration of fertility (Rf). One F2 population was developed by crossing CMS line (ICPA 2039) with fertility restorer line (ICPL 87119). Genetic analysis has shown involvement of two dominant genes in regulation of restoration of fertility. In parallel, the genotyping-by-sequencing (GBS) approach has generated ~ 33 Gb data on the F2 population. GBS data have provided 2457 single nucleotide polymorphism (SNPs) segregating across the mapping population. Based on these genotyping data, a genetic map has been developed with 306 SNPs covering a total length 981.9 cM. Further QTL analysis has provided the region flanked by S8_7664779 and S8_6474381 on CcLG08 harboured major QTL explained up to 28.5% phenotypic variation. Subsequently, sequence information within the major QTLs was compared between the maintainer and the restorer lines. From this sequence information, we have developed two PCR-based markers for identification of restorer lines from non-restorer lines and validated them on parental lines of hybrids as well as on another F2 mapping population. The results obtained in this study are expected to enhance the efficiency of selection for the identification of restorer lines in hybrid breeding and may reduce traditional time-consuming phenotyping activities.

Transcript levels of orf288 are associated with the hau cytoplasmic male sterility system and altered nuclear gene expression in Brassica juncea.

Cytoplasmic male sterility (CMS) is primarily caused by chimeric genes located in the mitochondrial genomes. In Brassica juncea, orf288 has been identified as a CMS-associated gene in the hau CMS line; however, neither the specific abortive stage nor the molecular function of the gene have been determined. We therefore characterized the hau CMS line, and found that defective mitochondria affect the development of archesporial cells during the L2 stage, leading to male sterility. The expression level of the orf288 transcript was higher in the male-sterility line than in the fertility-restorer line, although no significant differences were apparent at the protein level. The toxicity region of ORF288 was found to be located near the N-terminus and repressed growth of Escherichia coli. However, transgenic expression of different portions of ORF288 indicated that the region that causes male sterility resides between amino acids 73 and 288, the expression of which in E. coli did not result in growth inhibition. Transcriptome analysis revealed a wide range of genes involved in anther development and mitochondrial function that were differentially expressed in the hau CMS line. This study provides new insights into the hau CMS mechanism by which orf288 affects the fertility of Brassica juncea.

New Sunflower Genotypes with Resistance to Drought, Main Pathogens and Broomrape (Orobanche cumana), Created at NARDI Fundulea

The development of human society in general depends and is conditioned by the presence of fresh water resources. Certainly, the main cause, which has led to the ascending curve of current needs and consumption, is determined by the demographic explosion and the rapid urbanization process. Therefore, industry and agriculture are among the biggest consumers of water. The real problem is not lack of resources but effective distribution and overall management it is. In the context of the aforementioned, at NARDI Fundulea was developed in 2010 a program to improve drought resistance and obtain new genotypes through introgression of resistance genes from Helianthus argophyllus species in cultivated specie (Helianthus annuus L.) (Project MAKIS, Financed by the World Bank). Helianthus argopyllus is the most interesting species with very pubescent leaves, moderate transpiration and good photosynthetic activity. These characters provide superior water use efficiency compared to cultivated species. For these reasons, it has been used in our research to achieve the above-mentioned goal. In this project, at NARDI Fundulea has successfully used interspecific immature embryo culture to obtain drought-resistant genotypes and shortening the breeding process by getting two to three generations/year. Starting with the BC6 generation of self-pollination, the resulted fertile lines (B, maintainer lines) were cross polinated with an androsterility source in order to produce the sterile analogues of these lines (all being B, maintenance, fertile) were performed. After 4-5 generations of sterile plant selection, sterile analogue lines for each of the new creations were obtained. After this, new sunflower hybrids were obtained using certain fertility restoration lines from the NARDI Fundulea collection. The new hybrids were tested in three very different locations and years in terms of rainfall and temperatures during sunflower vegetation (2014-2015-2016). Based on the results we can conclude that interspecific hybridization may be an additional technique in the breeding of the sunflower and can be successfully used to create new sources of genetic variability to improve germplasm for drought and pathogens resistance. The newly created genotypes have responded positively to testing under different climatic and soil conditions and can be included in the approval list for both classical and ecological culture conditions.

Identification of fertility restorer and maintainer lines of A2 cytoplasm based cms lines in pigeonpea [Cajanus cajan (L) millsp.

Identification of fertility restorer and maintainer lines of A₂ cytoplasm based cms lines in pigeonpea [<i>Cajanus cajan</i> (L) millsp.

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments.

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4. Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but willalso be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of thegene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F1s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F1s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363 and PRR 396 possessing both Rf4 and Rf3 genes and good fertility restoration have been identified which could be used further in hybrid rice breeding.

Expression profiles of a cytoplasmic male sterile line of Gossypium harknessii and its fertility restorer and maintainer lines revealed by RNA-Seq

The Gossypium harknessii background cytoplasmic male sterility (CMS) system has been used in cotton hybrid breeding in China. However, the mechanism underlying pollen abortion and fertility restoration in CMS remains to be determined. In this study, we used RNA-seq to identify critical genes and pathways associated with CMS in G. harknessii based CMS lines (588A), the near isogenic restorer lines (588R), and maintainer lines (588B). We performed an assembly of 80,811,676 raw reads into 89,939 high-quality unigenes with an average length of 698 bp. Among these, 72.62% unigenes were annotated in public protein databases and were classified into functional clusters. In addition, we investigated the changes in expression of genes between 588A and 588B (588R); the RNA-seq data showed 742 differentially expressed genes (DEGs) between 588A and 588B and 748 DEGs between 588A and 588R. They were mainly down-regulated in 588A and most of them distributed in metabolic and biosynthesis of secondary metabolites pathways. Further analysis revealed 23 pollen development related genes were differentially expressed between 588A and 588B. Numerous genes associated with tapetum development were down-regulated in 588A, implicating tapetum dysplasia may be a key reason for pollen abortion in CMS lines. Also, among DEGs between 588A and 588R, we identified two PPR genes which were highly up-regulated in restorer line. This study may provide assistance for detailed molecular analysis and a better understanding of harknessii based CMS in cotton.

Fifty years of hybrid rice research in China

It has been over a half century on the hybrid rice research in China since the first time discovery of male sterile rice by Yuan LongPing in the indica cultivar Dong-Ting-Zao-Xian in 1964. In the last 50 years, hybrid rice contributes substantially on the security for food supply in China, and which makes China take a leading role on hybrid rice around the world. Here, in this context, the trajectory of hybrid rice development in China was reviewed, and strategies for the production and development of hybrid rice in the industrilization era were discussed. In the 1970, the discovery of natural male sterile line named “Wild Abortive” by Li Bihu, assistant of Yuan LongPing, in wild rice Oryza rufipogon in Sanya, Hainan Island, marked a great breakthrough to the hybrid rice development. Two years latter, the first commercial cytoplasmic male sterile line Zhenshan 97A was developed by consecutive backcross of Zhenshan 97 as male parent to the “Wild Abortive” male sterile line, which established a foundation for the three-line hybrid rice. In 1976, the hybrid rice combinations were released for commercial production after a series of elite fertility restorer lines including IR6 and IR64 etc. being discovered. The successful breeding of WA-type hybrid rice inspired people to create new CMS by wide crossing, then, a great many of CMS types including Honglian, Dian I/II, Gang and D-types, and their corresponding restorer lines were developed from a variety of wild rice or landraces by different research groups in 1970s–1980s. These researches greatly enriched the diversity of hybrid rice. As the development of three line hybrid rice, a new genic male sterile line which showed reversible transition between male sterility and fertility under different temperature and photoperiod environments was discovered by Shi MingSong in 1973. This discovery brought the dream to breed two-line hybrid rice into truth in 1990s after tremendous efforts. In order to resolve the problems for hybrid rice development in breeding practice, some questions such as ideal plant architecture, heterosis and combining ability, CMS and fertility restoring genes etc. were raised. These questions drive us to deepen understanding the essentiality of heterosis and rice yield, and lead to a trend that a great many agronomic important genes were cloned in the last two decades, which greatly promotes the application of molecular technologies in hybrid rice development. After half-a-century development, hybrid rice has been spread over forty countries in Southeastern Asia, Africa and Southern American apart from China. It also has been recommended by Food and Agriculture Organization of the United Nations (FAO) as one of the most efficient way to safeguard food security of the world.

Development and validation of candidate gene-specific markers for the major fertility restorer genes, Rf4 and Rf3 in rice

Two major nuclear genes, Rf3 and Rf4, are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9-782-(M,I) and PPR762 (for Rf4) and SF21 (for Rf3), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9-782-(M,I), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer (n = 120) and maintainer lines (n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762, displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9-782-(M,I) is indeed the candidate gene for Rf4. With respect to Rf3, a codominant marker, named RMS-SF21-5 developed targeting SF21, displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4-specific markers. Validation of these markers in a F2 mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3, together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.