Pollen Fertility Restoration Research Articles

DNA methylation analysis of sterile and fertile CMS-C hybrids and their parents in maize

C-type cytoplasmic male sterile (CMS-C) line plays an important role in hybrid seed production in maize. However, mechanisms of pollen abortion and fertility restoration remain unclear. This study aimed to investigate the mechanisms of CMS-C pollen abortion and fertility restoration, particularly based on epigenetics, and to understand the relationship between male fertility performance and DNA methylation status. Methylation-sensitive amplification polymorphism (MSAP) technique was conducted to analyze DNA methylation levels and patterns of tassels at pollen-mother cell, tetrad, mononuclear, and binuclear stages among four half-sib hybrids and their parents with different fertility. Results showed that DNA methylation levels in fertility restored hybrids were higher than those in sterility maintained hybrids. Nine CCGG sites, which displayed methylation polymorphism between male-fertile and male-sterile hybrids, were screened. To validate MSAP results, we performed methylation-sensitive PCR (MS-PCR) and found consistent observations in cloned methylation sites. Interestingly, a specific site named 16–1 was discovered in the region of Rf5 gene, which is one of the restorer genes of CMS-C. Thus, DNA methylation may participate in regulating the expression of fertility restorer genes of maize CMS-C.

Fertility of CMS wheat is restored by two Rf loci located on a recombined acrocentric chromosome.

Cytoplasmic male sterility (CMS) results from incompatibility between nuclear and cytoplasmic genomes, and is characterized by the inability to produce viable pollen. The restoration of male fertility generally involves the introgression of nuclear genes, termed restorers of fertility (Rf). CMS has been widely used for hybrid seed production in many crops but not in wheat, partly owing to the complex genetics of fertility restoration. In this study, an acrocentric chromosome that restores pollen fertility of CMS wheat in Hordeum chilense cytoplasm (msH1 system) is studied. The results show that this chromosome, of H. chilense origin and named H(ch)ac, originated from a complex reorganization of the short arm of chromosomes 1H(ch) (1H(ch)S) and 6H(ch) (6H(ch)S). Diversity arrays technology (DArT) markers and cytological analysis indicate that H(ch)ac is a kind of `zebra-like' chromosome composed of chromosome 1H(ch)S and alternate fragments of interstitial and distal regions of chromosome 6H(ch)S. PCR-based markers together with FISH, GISH, and meiotic pairing analysis support this result. A restorer of fertility gene, named Rf6H(ch)S, has been identified on the short arm of chromosome 6H(ch)S. Moreover, restoration by the addition of chromosome 1H(ch)S has been observed at a very low frequency and under certain environmental conditions. Therefore, the results indicate the presence of two Rf genes on the acrocentric chromosome: Rf6H(ch)S and Rf1H(ch)S, the restoration potential of Rf6H(ch)S being greater. The stable and high restoration of pollen fertility in the msH1 system is therefore the result of the interaction between these two restorer genes.

Polymorphism among RFL-PPR homologs in sunflower (Helianthus annuus L.) lines with varying ability for the suppression of the cytoplasmic male sterility phenotype

A complex comparative genetic approach was used for the investigation of the structural and functional diversity of genes for the restoration of sunflower pollen fertility. It includes (i) hybridological analysis; (ii) analysis of polymorphism among EST fragments.homologous to the known Rf genes that contain repeated motives of 35 amino acids (RFL-PPR); (iii) the development of molecular markers. Monogenic segregation in three interline cross combinations and the results of molecular marker analysis confirmed the allelic differences of parental lines in the Mendelian locus for CMS PET1 pollen fertility restoration. Introns were found in two RFL-PPR fragments. Two allelic variants of the QHL12D20 fragment were detected among the sixty lines of the sunflower genetic collection. An intron of QHL12D20 fragment was homologous to an intron of the AHBP-1B gene; the product of this gene-has a similarity with the transcription factor of the bZIP-family of Arabidopsis. A relationship between the QHL12D20 polymorphism and the functional state of the Rfl locus was revealed.

Gene action and stability parameters for yield and yield components, maturity duration and protein content of CGMS lines, pollen fertility restorers and their hybrids in pigeonpea [Cajanus cajan (L.) Millsp.

Cytoplasmic genic male sterility system based hybrids were synthesized by line × tester design and evaluated with check GTH 1 in RBD at Sardarkrushinagar, Jagudan and Khedbrahma during kharif 2007. Analysis of variance in individual and across environments revealed significant differences among genotypes and existence of overall heterosis for seed yield per plant and other thirteen traits. The top ranking on the basis of standard heterosis were CMSGT087A × GTR0525 (116.40 %) followed by CMSGT087A × AGTR0534 (108.93 %), CMSGT0307A × AGTR0538 (99.21 %) and CMSGT 0301A × AGTR 0534 (95.51 %) for seed yield per plant and for one or two of its contributing traits. Combining ability analysis revealed presence of both additive and non-additive gene effects. The specific combining ability variance was found more compaired to general combining ability variance for all the characters under studied. This favored a hybrid breeding programme. In stability analysis, linear portion was considerably high for all the traits except for days to flower and pod length. The best stable hybrids for seed yield per plant over environments were CMSGT 308A × AGTR 0534, CMSGT 307A × AGTR 0543, CMSGT 0308A × AGTR 0536 and CMSGT 0311A × GTR 0525 and also recorded stability in any one or more of its contributing traits viz. pods per plant, branches per plant, seed per pod, pod length and test weight. Top ranking hybrids had high mean performance, high heterosis and desirable sca effects and stability for seed yield per plant. Therefore, these hybrids could be valuable for commercial exploitation.

Evaluation of Restorability of Two Fertility Restorer Genes in the Rice Chromosome Single Segment Substitution Lines (SSSLs)

Rice chromosome single segment substitution line (SSSL) S15, a strong restorer line for WA-, and DA-CMS, was recently isolated at Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South China Agricultural University. The present study was carried out with the objective to investigate the genetic mode of fertility restorer (Rf) genes and the genetic relationship between WA- and DA-CMS systems. The SSSL S15 was used to pollinate WA-CMS line of Bobai A and DA-CMS line of Xieqingzao A for generation of two BC3F2 populations by using marker-assisted selection (MAS) and traditional backcrossing. The results were as follows: (1) The genetic effect showed a trend of WA-CMS DA-CMS in the genetic background of SSSL S15, and the effect of Rf4 was slightly larger than that of Rf3 for the two CMS systems. (2) Two pairs of dominant genes governed restoration of pollen fertility restoration, and some modifying or minor genes were involved in the inheritance of restorer ability besides Rf3 or Rf4 in SSSL S15, indicating that the genetic mode of Rf genes showed a qualitative-quantitative characteristic for WA-, and DA-CMS system. (3) When 202 SSR markers were used to analyze the genetic background and average length of substituted chromosome segments of the two BC3F2 individuals, carrying the genotypes Rf3Rf3/rf4rf4 or rf3rf3/Rf4Rf4, the mean of segments of inherited background of them was 1.0, while the average lengths of the substituted chromosome segments, corresponding to Rf3 and Rf4 loci, of them were 12.9 cM and 18.4 cM, respectively.

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.

Genetic diversity of sources of sunflower pollen fertility restorer genes

The genetic diversity of the sunflower collection with respect to components of the CMS-Rf system is studied with the use of molecular markers specific for mitochondrial genes atp9 and orfH522 and well as SCAR markers closely linked with nuclear gene Rf1 (fertility restorer for CMS PET1).

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 A(1) and A(2) 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 A(1) 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 A(1) and A(2) cytoplasms, and modified the extent of restoration conditioned by the major restorer Rf(5) in A(1) 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).

N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N2O) gas to obtain 2n male gametes. N2O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N2O treatment restores fertility in sterile hybrids. To establish optimal N2O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N2O gas at different anther developmental stages from fertile and sterile hybrid lilies. N2O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N2O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC1 plants. Thus N2O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.

Molecular and cytological characterization of an extra acrocentric chromosome that restores male fertility of wheat in the msH1 CMS system

A new CMS system designated as 'msH1' has been reported in bread wheat using the cytoplasm of H. chilense. While testing this system in different wheat backgrounds, a highly fertile line with chromosome number 42 plus an extra acrocentric chromosome was obtained. The extra chromosome did not pair with any wheat chromosome at meiosis, and progeny from this line which lack the acrocentric chromosome showed pollen abortion and male sterility. In order to establish the origin of this chromosome, FISH using H. chilense genomic DNA as probe was used and showed that it had originated from H. chilense chromosome(s). The novel chromosome did not possess sequences similar to wheat rDNA; however, the probe pSc119.2 from S. cereale containing the 120bp family was found to occur at the end of its long arm. Data obtained from FISH and EST molecular markers confirm that the long arm of the acrocentric chromosome is indeed, the short arm of chromosome 1H(ch) from H. chilense. We suggest that the novel chromosome originated from a deletion of the distal part of the long arm of chromosome 1H(ch). Neither the 1H(ch)S short arm, nor the whole chromosome 1H(ch) restores pollen fertility of the alloplasmic wheat. Therefore, the restorer gene on the acrocentric chromosome must be located on the retained segment from the hypothetical 1H(ch)L, while some pollen fertility inhibitor could be present on the deleted 1H(ch)L distal segment. Disomic addition of the acrocentric chromosome was obtained and this line resulted fully stable and fertile.

Genetic Analysis of Fertility Revertants Identified in Rice CMS Populations

The contamination of cytoplasmic male sterile (CMS) populations by fertile plants (FPs), which reduces purity of hybrid seeds, is a common problem in hybrid seed production, but genetic characterization of FPs has not been undertaken in rice (Oryza sativa L.). In this study, two types of FPs were identified among 64,530 plants of three CMS lines of Dian type 1 (CMS‐D1), in which the CMS seeds were produced under strict isolation. One type of FP had a genetic background identical to that of the CMS maintainers, which was revealed by 169 simple sequence repeat (SSR) markers and nine morphological traits. This type of FP was found at a frequency of 0.0418% in the CMS populations. These FPs could have resulted from maintainer contamination. Another type was a fertility revertant (FR), occurring at a frequency of 0.0155% in the CMS population. Although the FRs did not show differences from the corresponding CMS lines in morphological traits and the SSR markers, they possessed a fertility restoring gene of heterozygous type, which acted in gametophytic mode on pollen fertility restoration of the hybrids. The gene was designated temporarily as Rfr(d1). Rfr(d1) was allelic to the fertility restoring gene carried by CMS‐D1 restorers, and it was located at the Rf‐1 locus, which was revealed by allelic testing and cleaved amplified polymorphism sequence markers located within the Rf‐1 locus. Fertility revertants could be generated spontaneously through reversion of CMS lines.

The nearness of you: the effect of population structure on siring success in a gynodioecious species

Theoretically, both balancing selection and genetic drift can contribute to the maintenance of gender polymorphism within and/or among populations. However, if strong differences exist among genotypes in the quantity of viable gametes they produce, then it is expected that these differences will play an important role in determining the relative frequency of the genotypes and contribute to whether or not such polymorphism is maintained. In this issue, De Cauwer et al. (2010) describe an investigation of gynodioecious wild sea beet, which in addition to containing females, contain two types of hermaphrodites: restored hermaphrodites carrying a cytoplasm that causes pollen sterility and a nuclear gene that restores pollen fertility, and hermaphrodites without the sterilizing cytoplasm. The results show that restored hermaphrodites, who have relatively low pollen viability, achieve disproportionately high siring success simply because of where they are located in a patchy population (Fig. 1). Notably, these individuals tend to be close to females because of the genetics of sex determination. These results indicate that population structure caused by drift processes can have an unexpectedly large effect on the fitness of these low quality hermaphrodites, thereby contributing in the short term to the maintenance of gynodioecy in this population. While these results indicate that population structure caused by drift processes can have a large effect on the relative fitness of genetic variants, whether these effects promote or discourage the maintenance of polymorphism in the long term is still up for debate.

Molecular mapping and candidate gene identification of the Rf2 gene for pollen fertility restoration in sorghum [Sorghum bicolor (L.) Moench

The A1 cytoplasmic-nuclear male sterility system in sorghum is used almost exclusively for the production of commercial hybrid seed and thus, the dominant genes that restore male fertility in F(1) hybrids are of critical importance to commercial seed production. The genetics of fertility restoration in sorghum can appear complex, being controlled by at least two major genes with additional modifiers and additional gene-environment interaction. To elucidate the molecular processes controlling fertility restoration and to develop a marker screening system for this important trait, two sorghum recombinant inbred line populations were created by crossing a restorer and a non-restoring inbred line, with fertility phenotypes evaluated in hybrid combination with three unique cytoplasmic male sterile lines. In both populations, a single major gene segregated for restoration which was localized to chromosome SBI-02 at approximately 0.5 cM from microsatellite marker, Xtxp304. In the two populations we observed that approximately 85 and 87% of the phenotypic variation in seed set was associated with the major Rf gene on SBI-02. Some evidence for modifier genes was also observed since a continuum of partial restored fertility was exhibited by lines in both RIL populations. With the prior report (Klein et al. in Theor Appl Genet 111:994-1012, 2005) of the cloning of the major fertility restoration gene Rf1 in sorghum, the major fertility restorer locus identified in this study was designated Rf2. A fine-mapping population was used to resolve the Rf2 locus to a 236,219-bp region of chromosome SBI-02, which spanned ~31 predicted open reading frames including a pentatricopeptide repeat (PPR) gene family member. The PPR gene displayed high homology with rice Rf1. Progress towards the development of a marker-assisted screen for fertility restoration is discussed.

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line (‘E-38') was transformed with Cre gene and the inbred/pure line (‘E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T0 -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T 0 -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

Molecular markers in identification of sunflower pollen fertility restorer genes

The presence of the Rf1 gene (pollen fertility restorer of CMS PET1) in genotypes of most restorer lines is confirmed by molecular and hybridological analysis methods. Molecular markers of the Rf1 locus are identified for the first time in genotypes of autofertile lines of the collection of the Vavilov All-Russian Research Institute of Plant Industry (VIR) as well as in genomes of perennial and annual wild species of the genus Helianthus. The possibility of introgression of the fertility restorer gene into the genome of the cultivated sunflower during distant hybridization is shown.

Chromosome engineering in wheat to restore male fertility in the msH1 CMS system

Pollen fertility restoration of the CMS phenotype caused by H. chilense cytoplasm in wheat was associated with the addition of chromosome 6HchS from H. chilense accession H1. In order to develop an euploid restored line, different genomic combinations substituting the 6HchS arm for another homoeologous chromosome in wheat were evaluated, with the conclusion that the optimal combination was the translocation T6HchS·6DL. The double translocation T6HchS·6DL in H. chilense cytoplasm was obtained. This line is fertile and stable under different environmental conditions. However, a single dose of the T6HchS·6DL translocation is insufficient for fertility restoration when chromosome 6D is also present. Restoration in the msH1 system is promoted by interaction between two or more genes, and in addition to the restorer of fertility (Rf) located on chromosome 6HchS, one or more inhibitor of fertility (Fi) genes may be present in chromosome 6DL.

A study of the inheritance of morphological characters in sunflower. 1. Genetic control of coloration of pseudo-ligulate flowers, branchiness, and restoration of pollen fertility

The inheritance of coloration in pseudo-ligulate flowers in sunflower in the collection of the Yur’ev Institute of Plant Growing is studied. Only the yellow color, which is the most widespread of all the different types of flower coloring, is found in the first generation. Segregation into phenotypic classes in the ratios 3 : 1 and 9 : 3 : 4 is observed in different cross combinations in the second generation, attesting to a difference in the form with respect to one or two genes, respectively. The study of the inheritance of branchiness and the restoration of the fertility of pollen in the inbred sunflower lines studied revealed monogenic control of differences with respect to these characters. It is established that all the genes that control these characters are inherited independently.

Restricted distribution of orf687 as the pollen fertility restorer gene for Ogura male sterility in Japanese wild radish

In radish, the mitochondrial gene orf138 found in Ogura cytoplasm is responsible for male sterility, whereas the nuclear restorer gene (Rf gene) inhibits the expression of orf138. To investigate the distribution of the Rf gene for Ogura male sterile cytoplasm in Japanese wild radish, 226 plants collected from 15 regions in Japan and two additional sites of Korea were used as materials. PCR analysis indicated that 42% of the wild radishes possessed orf138. On the other hand, 207 plants (91.6%) were judged to have an Rf gene by the observation of pollen fertility in wild radishes themselves and in hybrids obtained by crosses with a male sterile variety having Ogura cytoplasm. For plants having restorer function, we analyzed the nucleotide sequence of the Rf gene, orf687, identified to date. Among the 207 plants having the Rf gene, 199 amplified the orf687 fragment of the expected size by PCR. PCR products were further analyzed by mismatch-specific endonuclease digestion and PCR-RFLP analysis. As a result, plants that showed an identical RFLP pattern to the known Rf type of orf687 were restricted to 30 plants (14.5% of the 207 plants) collected mainly in populations of southern region of Japan. It was also found that most of the plants (148 plants; 71.5% of 207 plants) possessed the sequence of the orf687 corresponding to the rf genotype lacking the restorer function. We conclude that the Rf gene widely distributed in Japanese wild radish is not orf687.

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA, Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F1 plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F2 populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.

Cytoplasmic male sterility and restoration of pollen fertility in higher plants

The review deals with cytoplasmic male sterility (CMS) in higher plants: impairment of the pollen formation resulting from interaction of the nuclear and mitochondrial genomes. The information on the known nuclear restorer-of-fertility genes and their effects on the expression of CMS-associated mitochondrial loci are considered. Heteroplasmy of mtDNA in plants and its potential association with CMS inheritance, as well as possible mechanisms of the observed direct and reverse association between altered expression of the CMS-inducing mitochondrial genome, metabolic defects, and pollen sterility are discussed.