Restorer Locus Research Articles

New Observations of the Effects of the Cytoplasm of Aegilops kotschyi Boiss. in Bread Wheat Triticum aestivum L.

The cytoplasm of Aegilops kotschyi is known for the induction of male sterility and haploidy in wheat. Both systems originally appeared rather simple, but manipulation of the standard chromosome constitution of the nuclear genome revealed additional interactions. This study shows that while there is little or no allelic variation at the main fertility restorer locus Rfmulti on chromosome arm 1BS, additional genes may also be involved in the nuclear-mitochondrial genome interactions, affecting not only male fertility but also the growth rate, from pollen competition for fertilization and early endosperm divisions all the way to seed size and plant maturity. Some of these effects appear to be of a sporophytic nature; others are gametophytic. Induction of parthenogenesis by a rye inducer in conjunction with the Ae. kotschyi cytoplasm is well known. However, here we show that the cytoplasmic-nuclear interactions affect all aspects of double fertilization: producing maternal haploids from unfertilized eggs, diploids from fertilized eggs or synergids, embryo-less kernels, and fertilized eggs without fertilization of the double nucleus in the embryo sack. It is unclear how frequent the inducers of parthenogenesis are, as variation, if any, is obscured by suppressors present in the wheat genome. Genetic dissection of a single wheat accession revealed five distinct loci affecting the rate of maternal haploid production: four acting as suppressors and one as an enhancer. Only when the suppressing haplotypes are confirmed may it be possible to the identify genetic variation of haploidy inducers, map their position(s), and determine their nature and the mode of action.

A Single Nucleotide Polymorphism Unique to the Galanthum-CMS of Onion

Different sources of cytoplasmic male sterility (CMS) are used to produce hybrid onion seed. The most commonly used source of CMS in onion is S cytoplasm (S-CMS), and male fertility is restored by a dominant allele at the nuclear male-fertility restoration locus (Ms). Male-sterile plants possess S cytoplasm and have the homozygous recessive genotype at Ms; seed propagation of male-sterile plants is possible by crossing with a male-fertile maintainer plant or inbred possessing normal (N) male-fertile cytoplasm and the homozygous recessive at the Ms locus (N msms). Some commercially important onion populations possess S-CMS and high frequencies of the dominant Ms allele, eliminating the possibility to develop maintainer lines. An alloplasmic source of CMS (Gal-CMS) was developed by backcrossing the cytoplasm of Allium galanthum into the nuclear background of onion. The advantage of Gal-CMS is that the dominant allele at Ms does not restore male fertility, making this source of CMS useful for the development of male-sterile lines from populations possessing S cytoplasm and dominant allele(s) at Ms. In this research, a single nucleotide polymorphism unique to the cytoplasms of A. galanthum and Gal-CMS was identified, useful to distinguish Gal-CMS from other onion cytoplasms.

Fine mapping of Rf2, a minor Restorer-of-fertility (Rf) gene for cytoplasmic male sterility in chili pepper G164 (Capsicum annuum L.).

Genome re-sequencing and recombination analyses identified Capana06g000193 as a strong candidate for the minor male fertility restoration locus Rf2 in chili pepper G164 harboring two dominant male fertility restoration genes. Male fertility restoration genes of chili pepper restorer line G164 (Capsicum annuum L.) were studied using molecular marker genotypes of an F2 population (7G) of G164 crossed with the cytoplasmic male sterility line 77013A. The ratio of sterile to fertile single plants in the F2 population was 1:15. This result indicates that chili pepper G164 has two dominant restoration genes, which we designated as Rf1 and Rf2. An individual plant recessive for Rf1 and heterozygous for Rf2, 7G-112 (rf1rf1Rf2rf2), was identified by molecular marker selection and genetic analysis, and a single Rf2 gene-segregating population with a 3:1 ratio of fertile to sterile plants was developed from the self-pollination of male fertile individuals of 77013A and 7G-112 hybrid progeny. Bulk segregant analysis of fertile and sterile pools from the segregating populations was used to genetically map Rf2 to a 3.1-Mb region on chromosome 6. Rf2 was further narrowed to a 179.3-kb interval through recombination analysis of molecular markers and obtained the most likely candidate gene, Capana06g000193.

KASP Markers Specific for the Fertility Restorer Locus Rf1 and Application for Genetic Purity Testing in Sunflowers (Helianthus annuus L.).

Single nucleotide polymorphisms (SNPs) were significantly associated with fertility restoration of cytoplasmic male sterility (CMS) PET1 by the restorer gene Rf1. For these SNPs, four Kompetitive allele-specific PCR (KASP) markers were successfully designed. The KASP markers cover the fertility restorer locus Rf1, spanning about 3 Mb, and clearly differentiate restorer and maintainer lines. For genetic purity testing in sunflower hybrid production, the efficiency for detecting contaminations in samples was simulated using mixtures of hypocotyls or leaves. Contaminations of restorer lines with 1%, 3%, 5%, 10%, and 50% of maintainer lines were screened with all four KASP markers. Contaminations of 10% could be clearly detected in pools of 100 plants. Contaminations below this level require detection on a single plant level. For single plant detections, ethyl methanesulfonate-treated sunflower F1 hybrids, which had been phenotypically evaluated for male sterility (potential mutation in the Rf1 gene) were screened. Nine identified either partially male-sterile or male-sterile plants were analyzed with all four KASP markers and only one proved to be a hybrid with a mutation, seven were male-sterile contaminants in the F1 seeds used (1.6%) and one a recombinant plant. The four KASP markers should be valuable tools for marker-assisted selection (MAS) in sunflower breeding regarding the restorer locus Rf1.

Migration restores hybrid incompatibility driven by mitochondrial-nuclear sexual conflict.

In the mitochondrial genome, sexual asymmetry in transmission allows the accumulation of male-harming mutations since selection acts only on the effect of the mutation in females. Called the 'Mother's Curse', this phenomenon induces a selective pressure for nuclear variants that compensate for this reduction in male fitness. Previous work has demonstrated the existence of these interactions and their potential to act as Dobzhansky-Muller incompatibilities, contributing to reproductive isolation between populations. However, it is not clear how readily they would give rise to and sustain hybrid incompatibilities. Here, we use computer simulations in SLiM 3 to investigate the consequences of sexually antagonistic mitochondrial-nuclear interactions in a subdivided population. We consider distinct migration schemes and vary the chromosomal location, and consequently the transmission pattern, of nuclear restorers. Disrupting these co-evolved interactions results in less-fit males, skewing the sex ratio toward females. Restoration of male fitness depends on both the chromosomal location of nuclear restorer loci and the migration scheme. Our results show that these interactions may act as Dobzhansky-Muller incompatibilities, but their strength is not enough to drive population isolation. Overall, this model shows the varied ways in which populations can respond to migration's disruption of co-evolved mitochondrial-nuclear interactions.

Perspective and application of molecular markers linked to the cytoplasm types and male‐fertility restorer locus in onion (Allium cepa)

AbstractThe male sterility trait is an economically essential feature for the exploitation of hybrid vigour in onions; hence, scientists have looked into the molecular component of male sterility in onions. As a result, numerous molecular markers associated with male‐sterile (CMS‐S and CMS‐T) cytoplasms, male‐fertile normal (N) cytoplasm and nuclear‐male‐fertility restorers (Ms locus) were developed. The discovered markers have proven to be helpful in the effective selection and isolation of male‐sterile lines, their maintainer lines and restorer‐of‐fertility lines. These lines are required for breeding F1 hybrid to harness heterosis through marker‐assisted selection (MAS) of cytoplasmic genotypes with Ms or ms loci. Thus, the study came up with a compilation of molecular markers, by revealing their molecular perspective, practical utility and functional effectiveness in the MAS of male‐sterile cytoplasm types with nuclear‐fertility‐restorer locus, to speed up onion breeding, development of F1 hybrids and production of pure hybrid seeds.

Identification and validation of genetic locus Rfk1 for wheat fertility restoration in the presence of Aegilops kotschyi cytoplasm.

Major fertility restorer locus for Aegilops kotschyi cytoplasm in wheat, Rfk1,was mapped to chromosome arm 1BS. Most likely candidate gene is TraesCS1B02G197400LC, which is predicted to encode a pectinesterase/pectinesterase inhibitor. Cytoplasmic male sterility (CMS) is widely used for heterosis and hybrid seed production in wheat. Genes related to male fertility restoration in the presence of Aegilops kotschyi cytoplasm have been reported, but the fertility restoration-associated gene loci have not been investigated systematically. In this study, a BC1F1 population derived from a backcross between KTP116A, its maintainer line TP116B, and its restorer line LK783 was employed to map fertility restoration by bulked segregant RNA-Seq (BSR-Seq). A major fertility allele restorer locus for Ae. kotschyi cytoplasm in wheat, Rfk1, was mapped to chromosome arm1BS, and it was contributed by LK783. Morphological and cytological studies showed that male fertility restoration occurred mainly after the late uninucleate stage. Based on simple sequence repeat and single-nucleotide polymorphism genotyping, the gene locus was located between Xnwafu_6 and Xbarc137 on chromosome arm 1BS. To further isolate the specific region, six Kompetitive allele-specific polymerase chain reaction markers derived from BSR-Seq were developed to delimit Rfk1 within physical intervals of 26.0Mb. After searching for differentially expressed genes within the candidate interval in the anthers and sequencing analysis, TraesCS1B02G197400LC was identified as a candidate gene for Rfk1 and it was predicted to encode a pectinesterase/pectinesterase inhibitor. Expression analysis also confirmed that it was specifically expressed in the anthers, and its expression level was higher in fertile lines compared with sterile lines. Thus, TraesCS1B02G197400LC was identified as the most likely candidate gene for Rfk1, thereby providing insights into the fertility restoration mechanism for K-type CMS in wheat.

Fine mapping of the male fertility restoration gene CaRf032 in Capsicum annuum L.

A novel strong candidate gene CA00g82510 for the male fertility restoration locus CaRf032 in Capsicum annuum was identified by genome re-sequencing and recombination analysis. A single dominant locus (CaRf032) for fertility restoration of cytoplasmic male sterility was identified in the strong restorer inbred line IVF2014032 of chili pepper (Capsicum annuum L.). CaRf032 was localized within an 8.81-Mb candidate intervals on chromosome 6 using bulked segregant analysis based on high-throughput sequencing data. Subsequently, the candidate interval was genetically mapped and defined to a 249.41-kb region using an F2 population of 441 individuals generated by crossing the male-sterile line 77013A and the restorer line IVF2014032. To fine map CaRf032, eight newly developed KASP markers were used to genotype 23 recombinants screened from a larger F2 population of 2877 individuals. The CaRf032 locus was localized to a 148.05-kb region between the KASP markers S1402 and S1354, which was predicted to contain 22 open reading frames (ORFs). One ORF with an incomplete sequence was predicted to contain a PPR motif, and its physical position overlapped with the Rf candidate gene CaPPR6_46. The PPR ORF sequence before the gap showed 100% identity with the CA00g82510 locus of the CM334 reference genome. CA00g82510 encodes a protein of 583 amino acids, containing 14 PPR motifs, and shows significantly differential expression between the flower buds of the maintainer line 77013 and the restorer line IVF2014032. These results indicated that CA00g82510 is a strong candidate gene for CaRf032. Five KASP markers, which detected single-nucleotide polymorphisms in CA00g82510 of 77013 and IVF2014032, co-segregated with CaRf032 and showed 64.4% successful genotyping of 38 maintainer and 63 restorer lines.

Development of a genotyping method for onion (Allium cepa L.) male-fertility based on multiplex PCR

ABSTRACTThe most widely used source of onion cytoplasmic male-sterility (CMS) is controlled by a single nuclear gene and a cytoplasmic factor, and the sterility trait can be recovered by the dominant nuclear gene (Ms). Marker-assisted selection (MAS) has a great potential in the selection of male sterility lines and maintainer lines. In this study, two markers at the Ms loci and one cytoplasm marker were used to a develop multiplexed polymerase chain reaction (PCR) system for MAS of fertility traits in onion. Seven pairs of PCR primers were designed on the basis of the flanking sequences (F1890 and S1887) of the male-fertility restoration (Ms) locus and a mitochondrial sequence (mt472) in onion. One set of compatible primers were selected to develop a multiplex-PCR molecular marker (AcCN), which was established in forty-eight lines. The fertility types of cytoplasm and nuclear were distinguished by one-time PCR detection. The accuracy of the AcCN marker was verified in practical backcross breeding population. The AcCN marker types perfectly matched with their genotypes and phenotypes. It can be concluded that this marker is of great practical significance for MAS in the selection of onion maintainer lines.

Maternal factors have stronger effect on family sex ratio than nuclear factors in a trans-Atlantic cross of Silene vulgaris

ABSTRACTBackground: Sex determination in cyto-nuclear gynodioecious plants (those with females and hermaphrodites) is complex, with maternally inherited mitochondrial genes (CMS factors) causing male sterility and female phenotypes, and bi-parentally inherited nuclear genes (Rf) restoring male fertility, resulting in hermaphrodite phenotypes. Many gynodioecious species harbour multiple CMS and restoration factors.Aims: We tested the hypothesis that these sex determination factors were matched between geographically proximal populations and mismatched between distant populations, with the prediction that crosses from populations from the same continents would produce relatively more hermaphrodites, and crosses across continents would produce relatively more females.Methods: We measured the progeny sex ratios from reciprocal crosses among 10 families of Silene vulgaris from North America and Europe and sequenced the mitochondrial atp1 gene for each family.Results: Our results were inconsistent with the prediction of a mismatch between geographically distant populations. Progeny sex ratios suggested that frequencies of male fertility restorer alleles had low variability worldwide.Conclusions: Progeny sex ratios suggested at least four different matching CMS and restorer loci segregating within the cross and alluded to a cost of restoration. Our results support previous research indicating that both CMS and restorer types are maintained over long time periods.

Undesired fertility restoration in msm1 barley associates with two mTERF genes

The novel Rfm3 locus causing undesired fertility restoration in the msm1 cytoplasm of winter barley is located on the short arm of chromosome 6H. Undesired fertility restoration of cytoplasmic male sterile (CMS) mother lines in absence of the functional Rfm1 restorer gene is a significant problem for hybrid breeding in winter barley. Here, we describe that a novel restorer locus on the short arm of chromosome 6H, designated Rfm3, is closely linked to two mitochondrial transcription termination factor family (mTERF) protein coding genes. Genome-wide association studies in a multiparental mapping population revealed that two of the most significantly associated markers are located very close to these genes, with one marker lying directly within one mTERF gene sequence. Sequences of the candidate genes in the parental lines, segregating individuals and an independent set of breeding lines clearly revealed haplotypes discriminating completely sterile, partially fertile and Rfm1-restorer lines. The haplotypes segregate for several single nucleotide polymorphisms, a 6bp insertion-deletion (InDel) polymorphism and another 2bp InDel. CMS-unstable genotypes carrying haplotypes associated with undesired fertility restoration showed significantly higher grain setting on bagged spikes when plants were subjected to elevated temperatures during anthesis, indicating a temperature influence on pollen fertility. SNPs associated with desirable Rfm3 haplotypes can be implemented in marker-assisted selection of stable CMS mother lines.

QTL mapping and validation of fertility restoration in West African sorghum A1 cytoplasm and identification of a potential causative mutation for Rf2

Key messageMajor A1cytoplasm fertility restoration loci,Rf2andRf5, were found in the West African sorghum. A potential causative mutation forRf2was identified. KASP markers were validated on independent material.To accelerate the identification and development of hybrid parental lines in West African (WA) sorghum, this study aimed to understand the genetics underlying the fertility restoration (Rf) in WA A1 cytoplasmic male sterility system and to develop markers for a routine use in WA breeding programs. We genotyped by sequencing three F2 populations to map the Rf quantitative trait loci (QTL), validated the molecular KASP markers developed from those QTL in two F2:3 populations, and assessed the most promising markers on a set of 95 R- and B-lines from WA breeding programs. Seven QTL were found across the three F2 populations. On chromosome SBI-05, we found a major fertility restorer locus (Rf5) for two populations with the same male parent, explaining 19 and 14% of the phenotypic variation in either population. Minor QTL were detected in these two populations on chromosomes SBI-02, SBI-03, SBI-04 and SBI-10. In the third population, we identified one major fertility restorer locus on chromosome SBI-02, Rf2, explaining 31% of the phenotypic variation. Pentatricopeptide repeat genes in the Rf2 QTL region were sequenced, and we detected in Sobic.002G057050 a missense mutation in the first exon, explaining 81% of the phenotypic variation in a F2:3 population and clearly separating B- from R-lines. The KASP marker developed from this mutation stands as a promising tool for routine use in WA breeding programs.

Mapping a male-fertility restoration locus for the A4 cytoplasmic-genic male-sterility system in pearl millet using a genotyping-by-sequencing-based linkage map

BackgroundPearl millet (Pennisetum glaucum (L.) R. Br., syn. Cenchrus americanus (L.) R. Br) is an important cereal and fodder crop in hot and arid environments. There is great potential to improve pearl millet production through hybrid breeding. Cytoplasmic male sterility (CMS) and the corresponding nuclear fertility restoration / sterility maintenance genes (Rfs) are essential tools for economic hybrid seed production in pearl millet. Mapping the Rf genes of the A4 CMS system in pearl millet would enable more efficient introgression of both dominant male-fertility restoration alleles (Rf) and their recessive male-sterility maintenance counterparts (rf).ResultsA high density linkage map based on single nucleotide polymorphism (SNP) markers was generated using an F2 mapping population and genotyping-by-sequencing (GBS). The parents of this cross were ‘ICMA 02777’ and ‘ICMR 08888’, which segregate for the A4Rf locus. The linkage map consists of 460 SNP markers distributed mostly evenly and has a total length of 462 cM. The segregation ratio of male-fertile and male-sterile plants (3:1) based on pollen production (presence/absence) indicated monogenic dominant inheritance of male-fertility restoration. Correspondingly, a major quantitative trait locus (QTL) for pollen production was found on linkage group 2, with cross-validation showing a very high QTL occurrence (97%). The major QTL was confirmed using selfed seed set as phenotypic trait, though with a lower precision. However, these QTL explained only 14.5% and 9.9% of the phenotypic variance of pollen production and selfed seed set, respectively, which was below expectation. Two functional KASP markers were developed for the identified locus.ConclusionThis study identified a major QTL for male-fertility restoration using a GBS-based linkage map and developed KASP markers which support high-throughput screening of the haploblock. This is a first step toward marker-assisted selection of A4 male-fertility restoration and male-sterility maintenance in pearl millet.

Fine mapping of Rf2, a major locus controlling pollen fertility restoration in sorghum A1 cytoplasm, encodes a PPR gene and its validation through expression analysis

AbstractSorghum is one of the pioneering cereal crops where cytoplasmic male sterility (CMS) was successfully exploited for mass production of F1 hybrid seed. Mapping genes for fertility restoration (Rf) is an important aspect of understanding the molecular basis of fertility restoration in crop plants. In this study, we fine‐mapped a fertility restoration locus, Rf2 of sorghum reported earlier (Jordan, Mace, Henzell, Klein, & Klein, ), involving two F2 populations (296A × RS29 and 296A × DSV1) and newly developed SSR markers delimited Rf2 locus to 10.32‐kb region on chromosome 2. The Rf2 locus was tightly linked with two new SSRs, MS‐SB02‐3460 (0.14 cM) and MS‐SB02‐3466 (0.75 cM) on both sides, and hosted only one gene (Sobic.002G057050) of PPR gene family. Another new SSR marker developed in the study, MS‐SB02‐37912, forms the part of PPR gene and could act as a perfect marker in marker‐assisted breeding for fertility restoration involving Rf2 in sorghum breeding. The strong involvement of Sobic.002G057050 gene in fertility restoration was supported through RNA expression analysis.

Exploring the genetics of fertility restoration controlled by Rf1 in common wheat (Triticum aestivum L.) using high-density linkage maps.

Hybrid wheat breeding has the potential to significantly increase wheat productivity compared to line breeding. The induction of male sterility by the cytoplasm of Triticum timopheevii Zhuk. is a widely discussed approach to ensure cross-pollination between parental inbred lines in hybrid wheat seed production. As fertility restoration in hybrids with this cytoplasm is often incomplete, understanding the underlying genetics is a prerequisite to apply this technology. A promising component for fertility restoration is the restorer locus Rf1, which was first detected on chromosome 1A of the restorer accession R3. In the present study, we performed quantitative trait locus (QTL) analyses to locate Rf1 and estimate its effect in populations involving the restorer lines R3, R113 and L19. Molecular markers linked to Rf1 in these populations were used to analyse the genomic target region in T. timopheevii accessions and common wheat breeding lines. The QTL analyses revealed that Rf1 interacted with a modifier locus on chromosome 1BS and the restorer locus Rf4 on chromosome 6B. The modifier locus significantly influenced both the penetrance and expressivity of Rf1. Whereas Rf1 exhibited expressivity higher than that of Rf4, the effects of these loci were not additive. Evaluating the marker haplotype for the Rf1 region, we propose that the restoring Rf1 allele may be derived exclusively from T. timopheevii. The present study demonstrates that interactions between restorer and modifier loci play a critical role in fertility restoration of common wheat with the cytoplasm of T. timopheevii.

Map-based cloning of the fertility restoration locus Rfm1 in cultivated barley (Hordeum vulgare)

Hybridization technology has proven valuable in enhancing yields in many crops, but was only recently adopted in the small grain cereals. Hybrid varieties in barley (Hordeum vulgare) rely on the cytoplasmic male sterility (CMS) system msm1 derived from Hordeum vulgare ssp. spontaneum. The major restorer gene described for the msm1 system is known as Rfm1 and maps to the top of chromosome 6H. To gain further insight into mechanisms underlying male fertility restoration in barley, we used a map-based cloning approach to identify the nuclear gene involved in the restoration mechanism of this hybridization system. Taking advantage of the available genomic resources in barley in combination with a custom-made non-gridded BAC library developed from a restorer line, we cloned and sequenced the Rfm1 restorer locus. The characterization and annotation of the nucleotide sequence for the Rfm1 restorer allele allowed for the identification of the candidate gene for Rfm1. The Rfm1 locus carries a tandem repeat of a gene encoding a pentatricopeptide repeat (PPR) protein. Surprisingly, Rfm1 belongs to the PLS-DYW subfamily of PPR genes known for their involvement in RNA editing in plants organelles, but that to date have not been identified as restorer genes.

Identification of promoter exchange at a male fertility restorer locus for cytoplasmic male sterility in radish (Raphanus sativus L.)

The cytoplasmic male sterility (CMS)/nucleus-controlled fertility restoration (Rf) system is a major component that exploits hybrid vigor in crops. Here, we have identified a new pentatricopeptide repeat (PPR)-encoding allele at the RsRf3 locus linked to the fertility restoration to CMS in radish. This allele was named as RsRf3-5 and encodes a putative protein with 18 PPRs and a predicted mitochondrial targeting signal. Another allele RsRf3-6 encoding the same protein with RsRf3-5 possesses a different promoter region and has failed to restore fertility to CMS. The non-restoring allele RsRf3-7 encoding a predicted protein with 15 PPRs has an identical promoter region with RsRf3-6, suggesting that RsRf3-6 could result from a recent crossover between RsRf3-5 and RsRf3-7. Interestingly, the RsRf3-5 allele shows higher RNA expression levels in the CMS cytoplasmic background compared with the RsRf3-6 allele.

No evidence for strong cytonuclear conflict over sex allocation in a simultaneously hermaphroditic flatworm

BackgroundCytoplasmic sex allocation distorters, which arise from cytonuclear conflict over the optimal investment into male versus female reproductive function, are some of the best-researched examples for genomic conflict. Among hermaphrodites, many such distorters have been found in plants, while, to our knowledge, none have been clearly documented in animals.MethodsHere we provide a quantitative test for cytonuclear conflict over sex allocation in the simultaneously hermaphroditic flatworm Macrostomum lignano. We used a quantitative genetic breeding design, employing pair-wise crosses of 2 × 15 independent inbred lines, to partition the phenotypic variance in several traits (including sex allocation) into its nuclear and cytoplasmic components.ResultsAlthough the nuclear genetic background had a significant effect on all traits analyzed, we found significant cytoplasmic genetic variation only for ovary size, there explaining just 4.1% of the variance. A subsequent statistical power analysis showed that the experimental design had considerable power to detect cytonuclear interactions.ConclusionWe conclude that there were no strong effects of cytonuclear conflict in the studied populations, possibly because the usually compact mitochondrial genomes in animals have a lower evolvability than the large mitochondrial genomes in plants or because the sampled populations currently do not harbor variation at putative distorter and/or the restorer loci.

Genetic architecture of male fertility restoration of Triticum timopheevii cytoplasm and fine-mapping of the major restorer locus Rf3 on chromosome 1B.

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. 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.

Fertility restoration locus and cytoplasm types in onion.

The objective of this study was the identification of the cytoplasmic types and the genotyping for the fertility restoration nuclear locus (Ms) in 59 onion accessions, aiming at the selection of 'A' and 'B' lines essential for the obtainment of hybrids. Three markers were used to identify the cytoplasm 5' cob, orfA501, and orf725, and two were used for the Ms locus (AcSKP1 and AcPMS1). The two types of male-sterile cytoplasm ('S' and 'T'), as well as fertile cytoplasm ('N'), and the Ms and ms alleles in both homozygosity and heterozygosity were detected in the 59 genotypes evaluated in the experiment. The frequencies of the 5' cob/orfA501 and orf725 markers, as well as of the markers AcSKP1 and AcPMS1, were close in the onion accessions evaluated in this study. In the Brazilian germplasm, the frequencies of the 'N', 'S', and 'T' cytoplasm were approximately 0.47, 0.28, and 0.25, respectively, whereas the allele frequencies of Ms and ms were 0.52 and 0.48, respectively. The accessions Régia, EHCEB 20146, EHCEB 201427, Alvorada, Serrana, Crioula Mercosul, EHCEB 20142, BRS 367, Rainha, Juporanga, and Alfa SF C-XI have potential for the identification of 'A' and 'B' lines, since they presented mixtures of cytoplasm and different allele frequencies for Ms. All the plants of the accessions EHCEB 20142040/EHCEB 20141040, EHCEB 20142028/EHCEB 20141028, and EHCEB 20112006/EHCEB 20111006 were in the Nmsms and Smsms conditions, and have the potential for 'B' and 'A' lines, respectively, for the CMS-S system. All the plants of the accessions EHCEB 20142027/EHCEB 20141027, EHCEB 20102019/EHCEB 20101019, and Alfa SF 'B'/Alfa SF 'A' were in Nmsms and Tmsms conditions, and have the potential for 'B' and 'A' lines, respectively, for the CMS-T system.