Mapping and characterization of Rf 5: a new gene conditioning pollen fertility restoration in A1 and A2 cytoplasm in sorghum (Sorghum bicolor (L.) Moench)

With an aim to further characterize the cytoplasmic male sterility–fertility restoration system in sorghum, a major fertility restoration gene was mapped along with a second locus capable of partial restoration of pollen fertility. The major fertility restoration gene, Rf ₅ , was located on sorghum chromosome SBI-05, and was capable of restoring pollen fertility in both A₁ and A₂ male sterile cytoplasms. Depending on the restorer parent, mapping populations exhibited fertility restoration phenotypes that ranged from nearly bimodal distribution due to the action of Rf ₅ , to a more normalized distribution reflecting the action of Rf ₅ and additional modifier/partial restoration genes. A second fertility restoration locus capable of partially restoring pollen fertility in A₁ cytoplasm was localized to chromosome SBI-04. Unlike Rf ₅ , this modifier/partial restorer gene acting alone resulted in less than 10% seed set in both A₁ and A₂ cytoplasms, and modified the extent of restoration conditioned by the major restorer Rf ₅ in A₁ cytoplasm. In examining the genomic regions spanning the Rf ₅ locus, a cluster of pentatricopeptide gene family members with high homology to rice Rf ₁ and sorghum Rf ₂ were identified as potential candidates encoding Rf ₅ .     

Fertility restorer locus Rf1 of sorghum (Sorghum bicolor L.) encodes a pentatricopeptide repeat protein not present in the colinear region of rice chromosome 12

With an aim to clone the sorghum fertility restorer gene Rf1, a high-resolution genetic and physical map of the locus was constructed. The Rf1 locus was resolved to a 32-kb region spanning four open reading frames: a plasma membrane Ca²⁺-ATPase, a cyclin D-1, an unknown protein, and a pentatricopeptide repeat (PPR13) gene family member. An ~19-kb region spanning the cyclin D-1 and unknown protein genes was completely conserved between sterile and fertile plants as was the sequence spanning the coding region of the Ca²⁺-ATPase. In contrast, 19 sequence polymorphisms were located in an ~7-kb region spanning PPR13, and all markers cosegregated with the fertility restoration phenotype. PPR13 was predicted to encode a mitochondrial-targeted protein containing a single exon with 14 PPR repeats, and the protein is classified as an E-type PPR subfamily member. To permit sequence-based comparison of the sorghum and rice genomes in the Rf1 region, 0.53 Mb of sorghum chromosome 8 was sequenced and compared to the colinear region of rice chromosome 12. Genome comparison revealed a mosaic pattern of colinearity with an ~275-kb gene-poor region with little gene conservation and an adjacent, ~245-kb gene-rice region that is more highly conserved between rice and sorghum. Despite being located in a region of high gene conservation, sorghum PPR13 was not located in a colinear position on rice chromosome 12. The present results suggest that sorghum PPR13 represents a potential candidate for the sorghum Rf1 gene, and its presence in the sorghum genome indicates a single gene transposition event subsequent to the divergence of rice and sorghum ancestors.An erratum to this article can be found at     

Isolation and Analysis of Rice Rf1-Orthologus PPR Genes Co-segregating with <Emphasis Type="Italic">Rf3</Emphasis> in Maize

Using an in silico cloning approach, five putative maize pentatricopeptide repeat (PPR)-containing protein genes (PPR-814a, PPR-814b, PPR-814c, PPR-816, PPR-817) with complete open reading frames were identified in the inbred line S-Mo17^Rf3Rf3. The amino acid sequence indicated that these genes encoded mitochondrially targeted proteins containing repeats of a 35-aa PPR motif. The genes were mapped into the interval umc1525–bnlg1520 on chromosome 2. In a non-restoring genotype, we identified three homologous genes that contained deletions or nucleotide substitutions in the coding region. Sequence analysis revealed that one of the three genes (PPR-814a, PPR-814b, PPR-814c) could be considered a candidate restorer gene for S male sterility cytoplasm, and linkage analysis demonstrated that the genes co-segregated with the fertility restorer gene Rf3.     

Molecular markers linked to the Rf2 fertility restorer gene in cotton.

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.     

Development and validation of candidate gene-specific markers for the major fertility restorer genes, <Emphasis Type="Italic">Rf4</Emphasis> and <Emphasis Type="Italic">Rf3</Emphasis> 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.     

Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice

Commercial exploitation of heterosis is essential for enhancing productivity of rice. The use of cytoplasmic male sterility (CMS) and fertility restoration system greatly facilitates large scale production of hybrid seed. The wild abortive (WA) cytoplasm is most widely used for hybrid seed production in rice. The present study was undertaken to develop molecular markers for both WA cytoplasm based male sterility and its fertility restoration for use in efficient hybrid breeding. High degree of genetic differentiation of WA-cytoplasm from its normal fertile counterpart was observed due to DNA rearrangements involving five (coxI, coxIII, cob, atp6 and rps3) mitochondrial genes. Cleaved amplified polymorphic sequence (CAPS) markers based on five mitochondrial genes namely, coxIII, cob, atp9, rps3 and 18SrRNA polymorphic between CMS and maintainer line were developed. The utility of these informative markers was demonstrated in purity testing of the CMS line Pusa6A being used in commercial hybrid seed production. Fertility restoration was found to be controlled by a major locus in the Basmati restorer line PRR78, which was mapped to a short marker interval of 0.8 cM and a physical interval of 163.6 kb on rice chromosome 10. A total of 13 pentatricopeptide repeat (PPR) motif containing genes were predicted in a 1.66 Mb region on the long-arm of this chromosome of which, four were present in the marker interval containing the fertility restorer gene. High degree of conservation of gene order was observed between japonica and indica for the predicted PPR genes. A sequence tagged site (STS) and a genic non-coding microsatellite (GNMS) marker were designed based on one of the candidate PPR motif containing genes present in the marker interval, which were validated using F₂ population and other known restorer lines. The candidate gene based marker identified in the present study would be useful in marker assisted selection (MAS) for fertility restorer gene in hybrid breeding programme based on WA-CMS of rice.     

Genetic architecture of male fertility restoration of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> cytoplasm and fine-mapping of the major restorer locus <Emphasis Type="Italic">Rf3</Emphasis> on chromosome 1B

Key message

Restoration of fertility in the cytoplasmic male sterility-inducing Triticum timopheevii cytoplasm can be achieved with the major restorer locus Rf3 located on chromosome 1B, but is also dependent on modifier loci.

Abstract

Hybrid breeding relies on a hybrid mechanism enabling a cost-efficient hybrid seed production. In wheat and triticale, cytoplasmic male sterility based on the T. timopheevii cytoplasm is commonly used, and the aim of this study was to dissect the genetic architecture underlying fertility restoration. Our study was based on two segregating F₂ triticale populations with 313 and 188 individuals that share a common female parent and have two different lines with high fertility restoration ability as male parents. The plants were cloned to enable replicated assessments of their phenotype and fertility restoration was evaluated based on seed set or staining for pollen fertility. The traits showed high heritabilities but their distributions differed between the two populations. In one population, a quarter of the lines were sterile, conforming to a 3:1 segregation ratio. QTL mapping identified two and three QTL in these populations, with the major QTL being detected on chromosome 1B. This QTL was collinear in both populations and likely corresponds to Rf3. We found that Rf3 explained approximately 30 and 50% of the genotypic variance, has a dominant mode of inheritance, and that the female parent lacks this locus, probably due to a 1B.1R translocation. Taken together, Rf3 is a major restorer locus that enables fertility restoration of the T. timopheevii cytoplasm, but additional modifier loci are needed for full restoration of male fertility. Consequently, Rf3 holds great potential for hybrid wheat and triticale breeding, but other loci must also be considered, either through marker-assisted or phenotypic selection.

     

Identification and mapping of Rf-I an inhibitor of the Rf5 restorer gene for Cms-C in maize (Zea mays L.)

The restoration of the C-type cytoplasmic male sterility (Cms) has been a common agriculture practice in the production of hybrid seed for many years. In this study, a series of crosses between select sterile and restorer lines, as well as a backcross population of (Cms-C77 × 6233) × 6233, were used to investigate the restoration of C-type Cms. Our results demonstrated that there was an inhibitor of the Rf5 restorer gene. This inhibitor gene, Rf-I, maps to chromosome 7 and is tightly linked with SSR markers, umc2326 and umc2327, at a genetic distance 4.7 and 3.4 cM, respectively. After analyzing our data combined with previous studies, we propose that the restoration of C-type Cms has two dominant genes, Rf4 and Rf5. Rf4 has the ability to restore all genotypes of Cms-C lines; however, there exists an inhibitor for the other restorer gene, Rf5; thus, it can restore only those genotypes of Cms-C lines lacking the Rf-I inhibitor.     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

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 will also 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 the gene(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.     

Genetic analysis and molecular mapping of a new fertility restorer gene Rf8 for Triticum timopheevi cytoplasm in wheat (Triticum aestivum L.) using SSR markers

A study on mode of inheritance and mapping of fertility restorer (Rf) gene(s) using simple sequence repeat (SSR) markers was conducted in a cross of male sterile line 2041A having Triticum timopheevi cytoplasm and a restorer line PWR4099 of common wheat (Triticum aestivum L.). The F₁ hybrid was completely fertile indicating that fertility restoration is a dominant trait. Based on the pollen fertility and seed set of bagged spikes in F₂ generation, the individual plants were classified into fertile and sterile groups. Out of 120 F₂ plants, 97 were fertile and 23 sterile (based on pollen fertility) while 98 plants set ≥5 seeds/spike and 22 produced ≤4 or no seed. The observed frequency fits well into Mendelian ratio of 3 fertile: 1 sterile with χ² value of 2.84 for pollen fertility and 2.17 for seed setting indicating that the fertility restoration is governed by a single dominant gene in PWR4099. The three linked SSR markers, Xwmc503, Xgwm296 and Xwmc112 located on the chromosome 2DS were placed at a distance of 3.3, 5.8 and 6.7 cM, respectively, from the Rf gene. Since, no known Rf gene is located on the chromosome arm 2DS, the Rf gene in PWR4099 is a new gene and proposed as Rf8. The closest SSR marker, Xwmc503, linked to the Rf8 was validated in a set of Rf, maintainer and cytoplasmic male sterile lines. The closely linked SSR marker Xwmc503 may be used in marker-assisted backcross breeding facilitating the transfer of fertility restoration gene Rf8 into elite backgrounds with ease.     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.     

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

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

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

Molecular mapping of the fertility restorer gene for ms-CW-type cytoplasmic male sterility of rice

Cytoplasmic male sterility (CMS) of rice (Oryza sativa L.) was first reported using the cytoplasm of a Chinese wild rice, Oryza rufipogon Griff. strain W1. However, it was not possible to characterize this ms-CW-type CMS in more detail until a restorer line had been developed due to the lack of restorer genes among cultivars thus far tested. The breeding of a restorer line (W1-R) was eventually achieved by transferring the restorer gene(s) of W1 to a cultivar. We report here the characterization of the ms-CW pollen grains and mapping of the restorer gene for ms-CW-type CMS. Pollen grains of the male-sterile plants appeared to be normal and viable based on the fluorochromatic reaction test, but they did not germinate on normal stigmas. The 1:1 segregation of fertile and sterile plants in a BC₁F₁ population from a cross between W1-R and a maintainer line demonstrated that fertility restoration is controlled by a single gene. The fertile seed set of all the F₂ plants examined indicated that the fertility restoration functions gametophytically. We designated the fertility restorer gene Rfcw. Using cleaved amplified polymorphic sequence (CAPS) and simple sequence repeat (SSR) markers, we localized Rfcw to chromosome 4 with a genetic distance of 0.6 cM from the nearest SSR marker.     

Mapping of genes for male-fertility restoration in ’Pampa’ CMS winter rye (Secale cereale L.)

Hybrid rye breeding and seed production is based on the cytoplasmic male sterility (CMS)-inducing Pampa (P)-cytoplasm. For restoring male fertility in the hybrids, dominant, nuclear restorer genes are necessary. However, current pollinator lines are only partial restorers. Effective restorers were recently detected in the German inbred line L18 and in materials originating from the Argentinian rye cultivar Pico Gentario and an Iranian primitive rye accession called IRAN IX. F₂ populations were developed for each of these three restorer sources to map the responsible genes by means of RFLP (restriction fragment length polymorphism) markers. For this purpose, homo- and heterologous DNA probes were used leading to 101 polymorphic marker loci in total. For phenotypic evaluation, 100 to 134 randomly chosen plants from each of the populations were cloned and grown at two or three locations with two plants each. Segregation ratios of pollen fertility in the F₂ populations with L18 and IRAN IX were in accordance with a monogenic dominant inheritance. The segregation pattern for Pico Gentario indicated complementary gene action. Major dominant restorer genes were detected on chromosomes 1RS (L18) and 4RL (Pico Gentario, IRAN IX). The gene on 1RS explained 54% of the phenotypic variation and that on 4 RL 59% and 68% in the Pico Gentario and IRAN IX populations, respectively. Additionally, three minor genes from L18 were identified on chromosomes 3RL, 4RL and 5R. In the Pico Gentario population, a dominant modifier gene contributed by the female parent was found on chromosome 6R. This gene significantly enhanced the expression of the major restorer gene but on its own was not able to restore any degree of fertility. The map-distances between the major restorer loci and at least one flanking marker were small in all three F₂ populations (5–6 cM). In Pico Gentario an unfavorable linkage exists between the major restorer gene and a QTL for plant height. Since highly effective restorers are scarce in actual breeding populations, the major restorer genes detected on chromosomes 1 RS and 4RL should be introgressed into actual restorer lines. This is facilitated by using the closely linked molecular markers described. Received: 10 February 2000 / Accepted: 31 March 2000<@head-com-p1a.lf>Communicated by G. Wenzel