The Effect of Chromosome Arm 1BS on the Fertility of Alloplasmic Recombinant Lines in Bread Wheat with the Hordeum vulgare Cytoplasm

The genetic mechanisms of fertility restoration in alloplasmic bread wheat with the barley cytoplasm are poorly explored. The effect of the 1BS chromosome arm on the fertility of bread wheat with the H. vulgare cytoplasm was studied depending on the incompleteness/completeness of the cytonuclear compatibility. (i) Three self-fertile (SF) lines and one partially fertile (PF) line with an incomplete cytonuclear compatibility and (ii) four self-fertile (SF) lines with a complete cytonuclear compatibility were studied. For the lines in group (i), the heteroplasmy (simultaneous presence of barley and wheat copies) of the 18S/5S mitochondrial (mt) repeat was revealed as well as the barley-type homoplasmy of chloroplast simple sequence repeats (cpSSRs). In the lines in group (ii), the 18S/5S mt repeat and cpSSRs were found in the wheat-type homoplasmic state. In all of the lines, the 1BS chromosome arm was substituted for the 1RS arm. The F1 plants of SF(i)-1BS × 1RS hybrids were fertile. The results of a segregation analysis in the F2 plants of SF(i)-1BS × 1RS showed that 1BS carries a single dominant fertility restorer gene (Rf) of bread wheat with the H. vulgare cytoplasm. All of the F1 plants of PF(i)-1BS × 1RS hybrids were sterile. A single dose of this restorer gene is not sufficient to restore fertility in this alloplasmic PF(i) line. All of the F1 and F2 plants of SF(ii)-1BS × 1RS hybrids were self-fertile.

A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5′ untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

High-generation near-isogenic lines combined with multi-omics to study the mechanism of polima cytoplasmic male sterility

Background Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration. Result In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins. Conclusions We propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS’s potential molecular mechanism and will help breed B. napus hybrids.

Mapping of the New Fertility Restorer Gene Rf-PET2 Close to Rf1 on Linkage Group 13 in Sunflower (Helianthus annuus L.)

The PET2-cytoplasm represents a well characterized new source of cytoplasmic male sterility (CMS) in sunflower. It is distinct from the PET1-cytoplasm, used worldwide for commercial hybrid breeding, although it was, as PET1, derived from an interspecific cross between Helianthus. petiolaris and H. annuus. Fertility restoration is essential for the use of CMS PET2 in sunflower hybrid breeding. Markers closely linked to the fertility restorer gene are needed to build up a pool of restorer lines. Fertility-restored F1-hybrids RHA 265(PET2) × IH-51 showed pollen viability of 98.2% ± 1.2, indicating a sporophytic mode of fertility restoration. Segregation analyses in the F2-population of the cross RHA 265(PET2) × IH-51 revealed that this cross segregated for one major restorer gene Rf-PET2. Bulked-segregant analyses investigating 256 amplified fragment length polymorphism (AFLP) primer combinations revealed a high degree of polymorphism in this cross. Using a subset of 24 AFLP markers, three sequence-tagged site (STS) markers and three microsatellite markers, Rf-PET2 could be mapped to the distal region of linkage group 13 between ORS1030 and ORS630. Three AFLP markers linked to Rf-PET2 were cloned and sequenced. Homology search against the sunflower genome sequence of HanXRQ v1r1 confirmed the physical location of Rf-PET2 close to the restorer gene Rf1 for CMS PET1. STS markers were mapped that can now be used for marker-assisted selection.

Early in vitro selection of winter oilseed rape (Brassica napus L.) plants with the fertility restorer gene for CMS Shaan 2A via non-destructive molecular analysis of microspore-derived embryos

Cotyledons of 201 microspore-derived oilseed rape (Brassica napus) embryos were analysed for the presence of the cytoplasmic male sterility Shaan 2A restorer gene using the primer pair 3F/4R. In total, 172 plants regenerated and were grown to the generative stage. Among phenotypically non-haploid flowering plants, there was a broad spectrum of different flower types from normally developed flowers to flowers with short anthers, stamens, or deformed flowers with atypically developed floral organs, often with traces of pollen. For this reason, only 115 typically fertile or sterile accessions were selected for further phenotype-genotype comparisons. In 14 plants (12.2%) the phenotype did not match the genotype: two plants were marked as fertile, but without the Rf gene. Vice versa, in 12 sterile plants the Rf gene was determined. Nevertheless, the two fertile plants did not produce any pods after self-pollination. In total, 47 doubled haploid (DH) lines with good seed yield, carrying the Rf gene were harvested.

Development and Validation of Markers for the Fertility Restorer Gene Rf1 in Sunflower

Hybrid breeding in sunflowers based on CMS PET1 requires development of restorer lines carrying, in most cases, the restorer gene Rf1. Markers for marker-assisted selection have been developed, but there is still need for closer, more versatile, and co-dominant markers linked to Rf1. Homology searches against the reference sunflower genome using sequences of cloned markers, as well as Bacterial Artificial Chromosome (BAC)-end sequences of clones hybridizing to them, allowed the identification of two genomic regions of 30 and 3.9 Mb, respectively, as possible physical locations of the restorer gene Rf1 on linkage group 13. Nine potential candidate genes, encoding six pentatricopeptide repeat proteins, one tetratricopeptide-like helical domain, a probable aldehyde dehydrogenase 22A1, and a probable poly(A) polymerase 3 (PAPS3), were identified in these two genomic regions. Amplicon targeted next generation sequencing of these nine candidate genes for Rf1 was performed in an association panel consisting of 27 maintainer and 32 restorer lines and revealed the presence of 210 Single Nucleotide Polymorphisms (SNPs) and 67 Insertions/Deletions (INDELs). Association studies showed significant associations of 10 SNPs with fertility restoration (p-value < 10−4), narrowing Rf1 down to three candidate genes. Three new markers, one co-dominant marker 67N04_P and two dominant markers, PPR621.5R for restorer, and PPR621.5M for maintainer lines were developed and verified in the association panel of 59 sunflower lines. The versatility of the three newly developed markers, as well as of three existing markers for the restorer gene Rf1 (HRG01 and HRG02, Cleaved Amplified Polymorphic Sequence (CAPS)-marker H13), was analyzed in a large association panel consisting of 557 accessions.

Association Mapping of Fertility Restorer Gene for CMS PET1 in Sunflower

The phenomenon of cytoplasmic male sterility (CMS), consisting in the inability to produce functional pollen due to mutations in mitochondrial genome, has been described in more than 150 plant species. With the discovery of nuclear fertility restorer (Rf) genes capable of suppressing the CMS phenotype, it became possible to use the CMS-Rf genetic systems as the basis for practical utilization of heterosis effect in various crops. Seed production of sunflower hybrids all over the world is based on the extensive use of the PET1 CMS combined with the Rf1 gene. At the same time, data on Rf1 localization, sequence, and molecular basis for the CMS PET1 type restoration of fertility remain unknown. Searching for candidate genes of the Rf1 gene has great fundamental and practical value. Therefore, in this study, association mapping of fertility restorer gene for CMS PET1 in sunflower was performed. The genome-wide association study (GWAS) results made it possible to isolate a segment 7.72 Mb in length on chromosome 13, in which 21 candidates for Rf1 fertility restorer gene were identified, including 20 pentatricopeptide repeat (PPR)family genes and one Probable aldehyde dehydrogenase gene. The results will serve as a basis for further study of the genetic nature and molecular mechanisms of pollen fertility restoration in sunflower, as well as for further intensification of sunflower breeding.