WO2019234231A1

WO2019234231A1 - Plants comprising wheat g-type cytoplasmic male sterility restorer genes and uses thereof

Info

Publication number: WO2019234231A1
Application number: PCT/EP2019/064983
Authority: WO
Inventors: Mark Davey; John Jacobs; Colin Cavanagh; Antje ROHDE; Arne VERSTICHELE; Michel Van Thournout; Alex WHAN; Jose BARRERO; Aswin SINGARAM; Andrew SPRIGGS; Bill BOVILL
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; BASF SE
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO; BASF SE
Priority date: 2018-06-08
Filing date: 2019-06-07
Publication date: 2019-12-12
Anticipated expiration: 2020-12-08

Abstract

Methods are described for selecting or producing a cereal plant comprising a functional restorer gene Rf3-PPR122 for wheat G-type cytoplasmic male sterility and nucleic acids for use therein.

Description

Plants comprising wheat G-type cytoplasmic male sterility restorer genes, and uses thereof

Field of the invention

[1] The present invention relates generally to the field of plant breeding and molecular biology and concerns a method for selecting or producing a cereal plant comprising a restorer gene for wheat G-type cytoplasmic male sterility, and nucleic acids for use therein.

Background

[2] Cytoplasmic male sterility (CMS) is a major trait of interest in cereals such as wheat in the context of commercial hybrid seed production (Kihara, 1951 , Cytologia 16, 177-193; Wilson and Ross, Wheat Inf Serv.(Kyoto) 14:29-30, 1962; Lucken, 1987 (Hybrid wheat. In Wheat and wheat improvement. Edited by E.G. Heyne. American Society of Agronomy, Madison, Wis.); Sage, 1976, Adv. Agron. 28, 265-298). The cytoplasms of Triticum timopheevii (G-type) and Aegilops kotschyi (K-type) are widely studied as inducers of male sterility in common wheat ( Triticum aestivum), due to few deleterious effects (Kaul, Male sterility in higher plants. Springer Verlag, Berlin ,1988; Lucken, 1987, supra; Mukai and Tsunewaki, Theor. Appl. Genet. 54, 153-60, 1979).

[3] In hybrid seed production system using the G-type cytoplasm, fertility restoration is a critical problem. Most of the hexaploid wheats do not naturally contain fertility restoration (Restorer of fertility or“Rf”) genes (Ahmed et al.2001 , Genes and Genet. Syst. 76, 33-38). In the complicated restoration system of T. timopheevii, eight Rf loci are reported to restore the fertility against T. timopheeviii cytoplasm, and their chromosome locations have been determined, namely, Rf1 (Chr 1A), Rf2 (Chr 7D), Rf3 (Chr 1 B), Rf4 (Chr 6B), Rf5 (Chr 6D), Rf6 (Chr 5D), Rf7 (Chr 7B) and Rf8 (Tahir & Tsunewaki, 1969, Jpn J Genet 44: 1 - 9; Yen et al., 1969, Can. J. Genet. Cytol. 11 , 531-546; Bahl & Maan, 1973, Crop Sci. 13, 317-320; Du et al., 1991 , Crop Sci , 31 : 319-22; Sihna et al., Genetica, 2013, http://dx.doi.org/10.1007/s10709-013-9742-5). Ma et al. (Genome 34:727-732, 1991) transferred an Rf locus from Aegilops umbellulata to wheat; two independent translocation lines with the Rf locus being located on either chromosome 6AS or 6BS were created (from Zhou et al., 2005, Euphytica 141(1-2):33-40, doi: 10.1007/si 0681 -005-5067-5).

[4] Ma and Sorrels (Crop Science 1995, 35, 1137-1143) reported the linkage of Rf3 to RFLP markers Xbcd249 and Xcdo442 on chromosome 1 BS.

[5] Kojima (Genes Genet Syst (1997) 72, p.353-359) localized a fertility Rf locus from wheat variety‘Chinese Spring’ termed Rf3 at a position 1.2 cM and 2.6 cM distant from RFLP markers Xcdo388 and Xabd 56, respectively. It was estimated that that Rf3 could exist within a region of 500 Kbp of the adjacent RFLP markers. [6] Ahmed et al (2001 , supra) determined the close linkage of a major Rf QTL against G-type cytoplasm on chromosome 1B with RFLP marker XksuG9c, close to marker Xabd 56 as reported by Kojima et al (supra).

[7] Zhang et al., (Acta Genetic Sinica (Yi Chuan Xue Bao), 2003, 30(5):459-464) describe an Rf QTL located on 1 BS with a genetic distance of 5.1 cM from microsatellite marker Xgwm550.

[8] Zhou et al (2005, supra) describe results indicating that the Rf 3 locus is located either between SSR markers Xgwm582 and Xbarc207 or between Xbarc207 and Xgwm131 , but very close to Xbarc207. Since the previously identified RFLP markers of Kojima, Ahmed and Ma & Sorrels were not mapped in Zhou et al’s mapping population, a linkage map including these RFLP markers could not be constructed to better estimate the distance between Rf 3 and the identified SSR markers.

[9] More accurate markers to identify and track Rf loci in breeding have been disclosed in WO2017158127 and WO2017158128, as well as by Geyer et al (2016, Molecular Breeding 36, 167). The 2 closest SNP markers, IWB14060 and IWB72107 described by Geyer et al, 2016, supra were flanking the Rf 3 locus on chromosome 1 B at 0.4 and 2.3 cM distance. WO2018015403 and WO2018015404 describe identification of Rf3-PPR genes located within the Rf 3 locus.

[10] There nevertheless remains a need to identify additional and/or alternative PPR genes which can be used to develop improved methods for fertility restoration in wheat with T. timopheevii cytoplasm, including by combination with other identified Rf genes The present invention provides a contribution over the art by disclosing a functional Rf gene from the Rf 3 locus on chromosome 1 B.

Summary of the invention

[11] In one embodiment, the invention provides a(n) (isolated or modified) nucleic acid molecule(s) encoding a functional restorer gene allele for wheat G-type cytoplasmic male sterility, wherein the functional restorer gene allele is a functional allele of a pentacotripeptide (PPR) gene comprised within the nucleotide sequence of SEQ ID NO: 1. The functional restorer gene may comprise a nucleotide sequence selected from a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 4 from the nucleotide at position 51 to the nucleotide at position 2423; a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 4; or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5. The functional restorer gene allele may encode a PPR protein capable of binding alone or in combination with other proteins to the mRNA of orf256, preferably to nt 137-154 of SEQ ID NO: 2, although the PPR protein may also be capable of interacting with the ORF256 protein, or with other mitochondrial transcripts or peptides, and may be obtainable from USDA accession number PI 583676. The nucleotide sequence of SEQ ID NO: 4 may be transcribed at least 2-fold higher, or at least 7-fold higher, or at least 10-fold higher in wheat lines with a functional Rf3 restorer, than in non-Rf3 wheat lines. [12] In another embodiment of the invention, a(n) (isolated or modified) polypeptide is provided encoded by the nucleic acid molecules described herein, or comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, preferably over the entire length of the polypeptide.

[13] In yet another embodiment of the invention, a chimeric gene is provided comprising the following operably linked elements (a) a plant-expressible promoter; (b) a nucleic acid comprising the nucleic acid molecule herein described or encoding the polypeptide herein described; and optionally (c) a transcription termination and polyadenylation region functional in plant cells, wherein at least one of the operably linked elements is heterologous with respect to at least one other element, or contains a modified sequence. The plant expressible promoter may be capable of directing expression of the operably linked nucleic acid at least during (early) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores.

[14] The invention further provides cereal plant cells or cereal plants or seeds thereof, such as wheat plant cells or plant or seed thereof, comprising the nucleic acid molecules or the polypeptides or the chimeric genes herein described, preferably wherein the polypeptide, the nucleic acid, or the chimeric gene in each case is heterologous with respect to the plant cell or plant or seed.

[15] It is yet another embodiment of the invention to provide a method for producing a cereal plant cell or plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising a functional restorer gene for wheat G-type cytoplasmic male sterility, or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant or cell, such as a wheat plant, comprising the step of providing the plant cell or plant with the nucleic acid molecules or the chimeric genes herein described, it being understood that the step of providing comprises providing by transformation, crossing, backcrossing, genome editing or mutagenesis, in such a way that the nucleic acid molecules are transcribed at least 2-fold higher in developing spikes.

[16] The invention further provides a method for producing a cereal plant cell or plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising a functional restorer gene for wheat G-type cytoplasmic male sterility, or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the steps of increasing the expression of a polypeptide as herein described in the plant cell or plant or seed. The Rf-PPR polypeptide may be provided by modifying the genome of the plant to comprise the nucleic acid molecule or the chimeric gene herein described wherein the step of modifying includes by transformation, crossing, backcrossing, genome editing or mutagenesis. Further provided herein is a modified nucleic acid encoding a Rf-PPR-122 protein wherein said nucleic acid is modified by genome editing or mutagenesis (e.g., EMS mutagenesis).

[17] Also provided is a method for converting a non-restoring cereal plant, such as a wheat plant, into a restoring plant for wheat G-type cytoplasmic male sterility (“CMS”), or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the step of modifying the genome of the plant to comprise the nucleic acid molecule or the chimeric gene herein described wherein the step of modifying comprises modifying by transformation, crossing, backcrossing, genome editing or mutagenesis.

[18] In another embodiment, a method is provided for converting a non-restoring cereal plant, such as a wheat plant, into a restoring plant for wheat G-type cytoplasmic male sterility (“CMS”), or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising modifying the genome of the plant to increase the expression of one or more polypeptides, as herein described, in the plant.

[19] The invention further provides cereal plant cells or cereal plants or seeds thereof, such as a wheat plant cells or plants or seeds thereof, obtained according to the methods herein described, preferably wherein the plant has an increased restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”), preferably wherein the PPR polypeptide described is expressed at least during (early) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores. The plant cell, plant or seed may be a hybrid plant cell, plant or seed. In one embodiment, such plant has a modified Rf3-PPR-122 nucleic acid and/or protein that results in improved restoration of G-type CMS in a cereal plant, such as a wheat plant, compared to the restoration obtained with the nucleic acid sequence of SEQ ID NO: 1 or 4 or the protein sequence of SEQ ID NO: 5 in said plant. In one embodiment, such modified Rfl-PPR-122 nucleic acid is that of SEQ ID NO: 1 or 4, or a nucleic acid encoding a modified Rf3-PPR-08 protein as described herein. In one embodiment, a modified Rf3- PPR-122 protein comprises at least one, or one or more, of the following amino acid modifications by reference to the amino acid sequence in SEQ ID No. 4 (or 5): adding an RL or RH dipeptide between the amino acid corresponding to amino acid position 12 and 22 (such as between the amino acid corresponding to amino acid position 21 and 22), change the Arginine amino acid at the amino acid position corresponding to amino acid position 107 to a Histidine amino acid (R107H mutation), change the Alanine amino acid at the amino acid position corresponding to amino acid position 219 to a Valine amino acid (A219V mutation), change the Threonine amino acid at the amino acid position corresponding to amino acid position 591 to a Alanine amino acid (T591A mutation), change the Serine amino acid at the amino acid position corresponding to amino acid position 607 to a Phenylanaline amino acid (S607F mutation), change the Glutamic Acid amino acid at the amino acid position corresponding to amino acid position 771 to an Aspartic Acid amino acid (E771 D mutation), change the Alanine amino acid at the amino acid position corresponding to amino acid position 778 to a Threonine amino acid (A778T mutation), change the Arginine amino acid at the amino acid position corresponding to amino acid position 781 to a Glutamine amino acid (R781Q mutation), change the Arginine amino acid at the amino acid position corresponding to amino acid position 783 to a Histidine amino acid (R783H mutation), change the Valine amino acid at the amino acid position corresponding to amino acid position 789 to a Isoleucine amino acid (V789I mutation). In one embodiment, an otherwise modified Rf3-PPR-122 protein retains at least one, or one or more, of the following amino acids at the following positions by reference to the amino acid sequence in SEQ ID No. 4 (or 5): a Valine at an amino acid position corresponding to amino acid position 73 (V73), a Methionine at an amino acid position corresponding to amino acid position 642 (M642), a Glutamic Acid at an amino acid position corresponding to amino acid position 730 (E730), a Leucine at an amino acid position corresponding to amino acid position 734 (L734), a Methionine at an amino acid position corresponding to amino acid position 741 (M741), a Glutamic Acid at an amino acid position corresponding to amino acid position 754 (E754), and a Valine at an amino acid position corresponding to amino acid position 744 (V744).

[20] In one embodiment, an otherwise modified Rf3-PPR-122 protein retains each of the following amino acids at the following positions by reference to the amino acid sequence in SEQ ID No. 4 (or 5): V73, M642, E730, L734, M741 , E754, V744.

[21] In yet another embodiment of the invention, a method for selecting a cereal plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility or for producing a cereal plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility, is provided, comprising the steps of (a) identifying the expression or transcription, such as by transcription analysis, of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423; and optionally selecting the plant expressing/transcribing the nucleotide sequence.

[22] The invention also provides a method for restoring fertility in a progeny of a G-type cytoplasmic male sterile cereal plant or for producing a fertile progeny plant from a G-type cytoplasmic male sterile cereal parent plant, comprising the steps of (a) providing a population of progeny plants obtained from crossing a female cereal parent plant with a male cereal parent plant, wherein the female parent plant is a G-type cytoplasmic male sterile cereal plant, and wherein the male parent plant comprises a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising and transcribing the nucleotide sequence of SEQ ID NO: 1 (partially) or SEQ ID NO: 4; (b) identifying in the population a fertile progeny plant comprising and expressing or transcribing the nucleotide sequence of SEQ ID NO: 1 (partially) or SEQ ID NO: 4; and optionally (c) selecting the fertile progeny plant; and optionally (d) propagating the fertile progeny plant.

[23] As another embodiment of the invention, a method is provided for identifying and/or selecting a cereal (e.g. wheat) plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising the steps of (a) identifying or detecting in the plant the expression or transcription of a nucleic acid or of the PPR polypeptide or of chimeric genes as herein provided and optionally selecting the plant expressing or transcribing the nucleic acid or polypeptide or chimeric gene.

[24] It is also an objective of the invention to provide a method for producing a cereal plant, such as a wheat plant, comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility, comprising the steps of (a) crossing a first cereal plant as herein described or provided with a second cereal plant; and (b) identifying, and optionally selecting, a progeny plant comprising and expressing or transcribing a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising the nucleotide sequence of SEQ ID NO: 4. [25] It is a further objective of the invention to provide a method for producing hybrid seed, comprising the steps of: (a) providing a male cereal parent plant, such as a wheat plant as herein provided, the plant comprising and expressing the functional restorer gene allele for wheat G-type cytoplasmic male sterility, wherein the functional restorer gene allele is preferably present in homozygous form; (b) providing a female cereal parent plant that is a G-type cytoplasmic male sterile cereal plant, (c) crossing the female cereal parent plant with a the male cereal parent plant; and optionally (d) harvesting seeds.

[26] The invention also provides use of the nucleic acid as herein described to identify one or more further functional restorer gene alleles for wheat G-type cytoplasmic male sterility.

[27] Further provided are uses of nucleic acids, polypeptides or chimeric genes as herein described for the identification of a plant comprising and expressing a functional restorer gene allele for wheat G-type cytoplasmic male sterility.

[28] The plants comprising and expressing the functional restorer gene for wheat G-type cytoplasmic male sterility as herein described may be used for restoring fertility in a progeny of a G-type cytoplasmic male sterile cereal plant, such as a wheat plant and/or for producing hybrid seed or a population of hybrid cereal plants, such as wheat seed or plants.

[29] In one embodiment, also provided herein are plants comprising a modified or mutated (such as a knock-out) Rf3- PPR-122 gene so that the fertility restoration of that gene is decreased or destroyed (e.g., it becomes non-functional), as can be used in making a maintainer line from a CMS female wheat plant. Also included herein is any method to reduce fertility in wheat plants containing an Rf gene, by inactivating the Rf3-PPR-122 gene or protein, or by reducing or blocking expression of an Rf-PPR-122 protein. In one embodiment, all Rf-PPR genes are inactivated in such a plant, or expression of all Rf-PPR proteins is reduced or blocked.

Brief description of the drawings

[30] Figure 1 : (A) - Predicted gene structure for the identified Rf3-PPR-122 gene. @ indicates coding sequences (CDS); # indicates 5’ UTR; and ^* indicates 3’ UTR. (B) amino acid sequence of identified PPR gene indicating the PPR motifs (alternatingly underlined and not underlined) including the 5th and 35th amino acid implied in RNA recognition (bold). (C) Graphical representation of the structure of the PPR protein with PPR motifs.

[31] Figure 2: Mean normalized expression levels of Rf3-PPR-122 in tissues of Rf3 restorer and wild-type (non-restorer) F4 progeny of a cross between‘PI 583676’ and a CMS line. R/3-containing progeny were identified following KASP genotyping with fine-mapping markers and phenotyped to confirm restoration of fertility.

[32] Figure 3: Mean Relative Expression levels of Rb-PPR-122 across 6 contrasting NIL pairs each with/without the Rf3 locus. R/3-containing progeny were identified following KASP genotyping with fine-mapping markers and phenotyped to confirm restoration of fertility. Detailed description

[33] The present invention describes the identification of a functional restorer of fertility {Rf} gene for wheat G-type cytoplasmic male sterility (i.e., T. timopheevii cytoplasm) located on chromosome 1 B (short arm 1 BS), as well as markers associated therewith. Said markers can be used in marker-assisted selection (MAS) of cereal plants, such as wheat, comprising said functional restorer gene located on chromosome 1 B. The identification of the gene and markers are therefore extremely useful in methods for hybrid seed production, as they can be used e.g. in a method for restoring fertility in progeny of a plant possessing G-type cytoplasmic male sterility, thereby producing fertile progeny plants from a G-type cytoplasmic male sterile parent plant. Likewise, the present disclosure also allows identifying plants lacking the desired allele, so that nonrestorer plants can be identified and, e.g., eliminated from subsequent crosses. The identification of a restorer gene underlying the Rf 3 locus on chromosome 1 B further allows targeted engineering to e.g. increase expression thereof, or targeted combination of the gene underlying the Rf 3 locus with other restorer loci or genes., or targeted engineering to e.g. decrease expression thereof, or decreased activity (such as to make a maintainer line).

[34] The Rf 3 locus on chromosome 1 B was mapped to a segment along the chromosome 1 B, in an interval of about 15.8 cM. Further fine-mapping narrowed the 1 B region to an interval of about 1.25 cM (from 6.8 to 8.05 cM) (see published PCT application WO2017/158127- incorporated herein by reference in its entirety).

[35] Male sterility in connection with the present invention refers to the failure or partial failure of plants to produce functional pollen or male gametes. This can be due to natural or artificially introduced genetic predispositions or to human intervention on the plant in the field. Male fertile on the other hand relates to plants capable of producing normal functional pollen and male gametes. Male sterility/fertility can be reflected in fertile seed set upon selfing, e.g. by bagging heads to induce self-fertilization. Likewise, fertility restoration can also be described in terms of seed set upon crossing a male sterile plant with a plant carrying a functional restorer gene, when compared to fertile seed set resulting from crossing (or selfing) fully fertile plants. Partial failure to produce pollen or male gametes preferably refers to plants which produce less than 20%, less than 15% or less than 10% fertile seed upon selfing, or even less than 5%.

[36] A male parent or pollen parent, is a parent plant that provides the male gametes (pollen) for fertilization, while a female parent or seed parent is the plant that provides the female gametes for fertilization, said female plant being the one bearing the seeds.

[37] Cytoplasmic male sterility or“CMS” refers to cytoplasmic-based and maternally-inherited male sterility. CMS is total or partial male sterility in plants as the result of specific nuclear and mitochondrial interactions and is maternally inherited via the cytoplasm. Male sterility is the failure of plants to produce functional anthers, pollen, or male gametes, although CMS plants still produce viable female gametes. Cytoplasmic male sterility is widely used in agriculture to facilitate the production of hybrid seed. [38] “Wheat G-type cytoplasmic male sterility”, as used herein refers to the cytoplasm of T. timopheevii that can confer male sterility when introduced into common wheat (i.e. T. aestivum), thereby resulting in a plant that is male sterile carrying common wheat nuclear genes but cytoplasm from T. timopheevii. The cytoplasm of T. timopheevii (G-type) as inducers of male sterility in common wheat have been extensively studied (Wilson and Ross, 1962, supra; Kaul, Male sterility in higher plants. Springer Verlag, Berlin.1988; Lucken 1987, Hybrid wheat. In Wheat and wheat improvement. Edited by E.G. Heyne. American Society of Agronomy, Madison, Wisconsin; Mukai and Tsunewaki, Theor. Appl. Genet. 54, 153-60, 1979; Tsunewaki, Jpn. Soc. Prom. Sci. . (Tokyo), 49-101 , 1980 ((In: Tsunewaki K. (ed.) Genetic diversity of the cytoplasm in Triticum and Aegilops); Tsunewaki et al., Genes Genet. Syst. 71 , pg. 293-311 ,1996). The origin of the CMS phenotype conferred by T. timopheevii cytoplasm has been reported tp be due to attributed to the presence of a novel mitochondrial chimeric gene termed orf256, which is upstream of cox1 sequences and is co-transcribed with an apparently normal cox1 gene. Antisera prepared against polypeptide sequences predicted from orf256 recognize a 7-kDa protein present in the CMS line, but not in the parental or restored lines (Song and Hedgcoth, Genome 37(2), 203-209, 1994; Hedgcoth et al., Curr. Genet. 41 , 357-365, 2002).

[39] As used herein“a functional restorer gene for wheat G-type cytoplasmic male sterility” indicates a gene that restores, or contributes to the restoration of fertility in the progeny of a cross with a G-type cytoplasmic male sterility (“CMS”) line, i.e., a line carrying common wheat nuclear genes but cytoplasm from T. timopheevii. Restoration against G-type cytoplasm has e.g. been described by Robertson and Curtis (Crop Sci. 7, 493—495, 1967), Yen et al. (Can. J. Genet. Cytol. 11 , 531-546, 1969), Bahl and Maan (1973, supra), Talaat et al. (Egypt. J. Genet. 2, 195-205, 1973) Zhang et al., (2003, supra) Ma and Sorrels (1995, supra), Kojima (1997, supra), Ahmed et al (2001 , supra), Zhou et al (2005, supra). Such restorer genes or alleles are also referred to as Rf genes and Rf alleles. As described at least in the examples, the restorer gene herein described is more highly expressed, particularly in developing spikes, in wheat lines identified to comprise the Rf3 locus (e.g. using the markers described in WO2017/158127) when compared to wheat lines which were identified as not comprising the Rf3 locus or compared to non-restoring wheat lines (even though such lines may comprise a nucleotide sequence for the Rf gene herein described). The mean relative expression level of the R gene in wheat lines identified to comprise the restoring Rf3 locus compared to the mean relative expression level of the restorer gene in wheat lines identified as not comprising the restoring Rf3 locus (particularly mean relative expression level in developing spikes) ranges from about 2 fold to at least about 25 fold higher, such as between 7-fold and 12-fold higher. Usually the ratio is about 10-fold higher. It is expected that protein levels encoded by the Rf3 gene are also increased in wheat lines identified to comprising the restoring Rf3 locus when compared to wheat lines identified as not comprising the restoring Rf3 locus and may equally be at least 2 fold higher, or ranging between about 2 fold to at least about 25 fold higher, such as between 7-fold and 12-fold higher.

[40] The term "maintainer" refers to a plant that when crossed with the CMS plant does not restore fertility, and maintains sterility in the resultant progeny. The maintainer is used to propagate the CMS line, and may also be referred to as a nonrestorer line. Maintainer lines have the same nuclear genes as the sterile line (i.e. do not contain functional Rf genes), but differ in the composition of cytoplasmic factors that cause male sterility in plants i.e. maintainers have "fertile" cytoplasm. When a male sterile line is crossed with its maintainer, the resultant progeny has the male sterile genotype. [41] The term“cereal” relates to members of the monocotyledonous family Poaceae which are cultivated for the edible components of their grain. These grains are composed of endosperm, germ and bran. Maize, wheat and rice together account for more than 80% of the worldwide grain production. Other members of the cereal family comprise rye, oats, barley, triticale, sorghum, wild rice, spelt, einkorn, emmer, durum wheat and kamut.

[42] In one embodiment, a cereal plant according to the invention is a cereal plant that comprises at least a B genome or related genome, such as hexaploid wheat (T. aestivunr, ABD), spelt (T. spelta ABD), durum wheat (T. turgidurrr, AB), barley ( Hordeum vulgare-, H) and rye (Seca/e cerea/e; R) . In a specific embodiment, the cereal plant according to the invention is hexaploid wheat (T. aestivunr, ABD).

[43] A particularly useful assay for detection of SNP markers is for example KBioscience Competitive Allele-Specific PCR (KASP, see www.kpbioscience.co.uk), For developing the KASP-assay 70 base pairs upstream and 70 base pairs downstream of the SNP are selected and two allele-specific forward primers and one allele-specific reverse primer is designed. See e.g. Allen et al. 2011 , Plant Biotechnology J. 9, 1086-1099, especially p1097-1098 for KASP assay method.

[44] The position of the chromosomal segments identified, and the markers thereof, when expressed as recombination frequencies or map units, are provided herein as a matter of general information. The embodiments described herein were obtained using particular wheat populations. Accordingly, the positions of particular segments and markers as map units are expressed with reference to the used populations. It is expected that numbers given for particular segments and markers as map units may vary from cultivar to cultivar and are not part of the essential definition of the DNA segments and markers, which DNA segments and markers are otherwise described, for example, by nucleotide sequence.

[45] A locus (plural loci), as used herein refers to a certain place or position on the genome, e.g. on a chromosome or chromosome arm, where for example a gene or genetic marker is found. A QTL (quantitative trait locus), as used herein, refers to a position on the genome that corresponds to a measurable characteristic, i.e. a trait, such as the Rf loci.

[46] As used herein, the term“allele(s)”, such as of a gene, means any of one or more alternative forms of a gene at a particular locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes or possibly on homeologous chromosomes.

[47] As used herein, the term“homologous chromosomes” means chromosomes that contain information for the same biological features and contain the same genes at the same loci but possibly different alleles of those genes. Homologous chromosomes are chromosomes that pair during meiosis.“Non-homologous chromosomes”, representing all the biological features of an organism, form a set, and the number of sets in a cell is called ploidy. Diploid organisms contain two sets of non-homologous chromosomes, wherein each homologous chromosome is inherited from a different parent. In tetraploid species, two sets of diploid genomes exist, whereby the chromosomes of the two genomes are referred to as“homeologous chromosomes” (and similarly, the loci or genes of the two genomes are referred to as homeologous loci or genes). Likewise, hexaploid species have three sets of diploid genomes, etc. A diploid, tetraploid or hexaploid plant species may comprise a large number of different alleles at a particular locus. The ploidy levels of domesticated wheat species range from diploid (T. monococcum , 2n = 14, AA), tetraploid (T. turgidum , 2n = 28, AABB) to hexaploid (T. aestivum , 2n = 42, AABBDD).

[48] As used herein, the term“heterozygous” means a genetic condition existing when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell. Conversely, as used herein, the term“homozygous” means a genetic condition existing when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell.

[49] An allele of a particular gene or locus can have a particular penetrance, i.e. it can be dominant, partially dominant, co-dominant, partially recessive or recessive. A dominant allele is a variant of a particular locus or gene that when present in heterozygous form in an organism results in the same phenotype as when present in homozygous form. A recessive allele on the other hand is a variant of an allele that in heterozygous form is overruled by the dominant allele thus resulting in the phenotype conferred by the dominant allele, while only in homozygous form leads to the recessive phenotype. Partially dominant, co-dominant or partially recessive refers to the situation where the heterozygote displays a phenotype that is an intermediate between the phenotype of an organism homozygous for the one allele and an organism homozygous for the other allele of a particular locus or gene. This intermediate phenotype is a demonstration of partial or incomplete dominance or penetrance. When partial dominance occurs, a range of phenotypes is usually observed among the offspring. The same applies to partially recessive alleles.

[50] Cytoplasmic male-sterility or“CMS” is caused by one or more mutations in the mitochondrial genome (termed“sterile cytoplasm”) and is inherited as a dominant, maternally transmitted trait. For cytoplasmic male sterility to be used in hybrid seed production, the seed parent must contain a sterile cytoplasm and the pollen parent must contain (nuclear) restorer genes (Rf genes) to restore the fertility of the hybrid plants grown from the hybrid seed. Accordingly, such Rf genes are preferably at least partially dominant, most preferably dominant, in order to have sufficient restoring ability in offspring.

[51] Integration of the fine map with partial genome sequences led to the identification of a genomic region as represented by SEQ ID NO: 1 comprising the functional Rf3 restorer gene or allele. Thus, in any of the herein described embodiments or aspects, the functional restorer gene or allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B may localize to the genome region as represented by SEQ ID NO: 1.

[52] A“contig”, as used herein refers to set of overlapping DNA segments that together represent a consensus region of DNA. In bottom-up sequencing projects, a contig refers to overlapping sequence data (reads); in top-down sequencing projects, contig refers to the overlapping clones that form a physical map of the genome that is used to guide sequencing and assembly. Contigs can thus refer both to overlapping DNA sequence and to overlapping physical segments (fragments) contained in clones depending on the context. [53] In a further embodiment, said functional restorer gene allele is a functional allele of a gene encoding a pentatricopeptide repeat (PPR) protein (i.e. a PPR gene) localising within the genomic region described in WO2017/158127.

[54] PPR proteins are classified based on their domain architecture. P-class PPR proteins possess multiple canonical amino acid motifs, typically consisting of 35 amino acid residues, although the motifs can range between 34 and 36 or even 38 amino acids. P-class PPR proteins may contain a mitochondrial targeting peptide, but normally lack additional domains. Members of this class have functions in most aspects of organelle gene expression. PLS-class PPR proteins have three different types of PPR motifs, which vary in length; P (35 amino acids), L (long, 35-36 amino acids) and S (short, ~31 amino acids), and members of this class are thought to mainly function in RNA editing. Subtypes of the PLS class are categorized based on the additional C-terminal domains they possess (reviewed by Manna et al., 2015, Biochimie 113, p93-99, incorporated herein by reference).

[55] Most fertility restoration (Rf) genes come from a small clade of genes encoding P-class PPR proteins (Fuji et al., 2011 , PNAS 108(4), 1723-1728 - herein incorporated by reference). PPR genes functioning as fertility restoration (Rf) genes are referred to in Fuji (supra) as Rf-PPR genes. Functional PPR proteins are comprised primarily of tandem arrays of 15-20 PPR motifs, each composed of about 35 amino acids.

[56] Most Rf-PPR genes belong to the P-class PPR subfamily, although also PLS-class Rf-PPR genes have been identified. High substitution rates observed for particular amino acids within otherwise highly conserved PPR motifs, indicate diversifying selection, and prompted the conclusion that these residues might be directly involved in binding to RNA targets. This has led to the proposal of a“PPR code” which allows the prediction of RNA targets of naturally occurring PPR proteins as well as the design of synthetic PPR proteins that can bind RNA molecules of interest, whereby sequence specificity is ensured by distinct patterns of hydrogen bonding between each RNA base and the amino acid side chains at positions 5 and 35 in the aligned PPR motif (see Melonek et al., 2016, Nat Sci Report 6:35152, Barkan et al., 2012, PLoS Genet 8(8): e1002910, Barkan and Small 2014, Annu. Rev. Plant Biol. 65:415-442 (https://doi.org/10.1146/annurev-arplant-050213- 040159); Miranda, McDermott, and Barkan 2017, Nucleic Acids Res. 46, 2613-2623 (https://doi.org/10.1093/nar/gkx1288); and Shen et al. 2016, Nat. Commun. 7, 11285 (https://doi.org/10.1038/ncomms11285); all incorporated herein by reference).

[57] Accordingly, a functional allele of a Rf-PPR gene, as used herein, refers to an allele of a Rf-PPR gene that is a functional restorer gene allele for wheat G-type cytoplasmic male sterility as described herein, i.e. that when expressed in a (sexually compatible) cereal plant has the capacity to restore or contribute, directly or indirectly, to the fertility in the progeny of a cross with a G-type cytoplasmic male sterile cereal plant. Such a functional allele of a PPR gene may also be referred to as a PPR-Rf gene (or Rf-PPR gene), which in turn encodes a PPR-Rf (or Rf-PPR) protein.

[58] In one embodiment, a modified Rf3-PPR-122 protein comprises at least one, or one or more, of the following amino acid modifications by reference to the amino acid sequence in SEQ ID No. 4 (or 5): adding an RL or RH dipeptide between the amino acid corresponding to amino acid position 12 and 22 (such as between the amino acid corresponding to amino acid position 21 and 22), change the Arginine amino acid at the amino acid position corresponding to amino acid position 107 to a Histidine amino acid (R107H mutation), change the Alanine amino acid at the amino acid position corresponding to amino acid position 219 to a Valine amino acid (A219V mutation), change the Threonine amino acid at the amino acid position corresponding to amino acid position 591 to a Alanine amino acid (T591A mutation), change the Serine amino acid at the amino acid position corresponding to amino acid position 607 to a Phenylanaline amino acid (S607F mutation), change the Glutamic Acid amino acid at the amino acid position corresponding to amino acid position 771 to an Aspartic Acid amino acid (E771 D mutation), change the Alanine amino acid at the amino acid position corresponding to amino acid position 778 to a Threonine amino acid (A778T mutation), change the Arginine amino acid at the amino acid position corresponding to amino acid position 781 to a Glutamine amino acid (R781Q mutation), change the Arginine amino acid at the amino acid position corresponding to amino acid position 783 to a Histidine amino acid (R783H mutation), change the Valine amino acid at the amino acid position corresponding to amino acid position 789 to a Isoleucine amino acid (V789I mutation). In one embodiment, an otherwise modified Rf3-PPR-122 protein retains at least one, or one or more, of the following amino acids at the following positions by reference to the amino acid sequence in SEQ ID No. 4 (or 5): a Valine at an amino acid position corresponding to amino acid position 73 (V73), a Methionine at an amino acid position corresponding to amino acid position 642 (M642), a Glutamic Acid at an amino acid position corresponding to amino acid position 730 (E730), a Leucine at an amino acid position corresponding to amino acid position 734 (L734), a Methionine at an amino acid position corresponding to amino acid position 741 (M741), a Glutamic Acid at an amino acid position corresponding to amino acid position 754 (E754), and a Valine at an amino acid position corresponding to amino acid position 744 (V744).

[59] In one embodiment, an otherwise modified Rf3-PPR-122 protein retains each of the following amino acids at the following positions by reference to the amino acid sequence in SEQ ID No. 4 (or 5): V73, M642, E730, L734, M741 , E754, V744. Included in this invention is also a modified nucleic acid sequence encoding any of the above modified Rf3-PPR-122 proteins.

[60] In one embodiment, the invention relates to the promoter of Rf3-PPR-122, such as a promoter sequence with at least at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the sequence shown in SEQ ID No. 1 from the nucleotide position corresponding to nucleotide position 1 to the nucleotide position corresponding to nucleotide position 3000, or an active fragment thereof. In one embodiment such promoter is a modified promoter that includes regulatory elements that increase transcription, such as an certain enhancer element, but also by inactivating or removing certain negative regulatory elements, such as repressor elements or target sites for miRNAs or IncRNAs.

[61] Although not intending to limit the invention to a specific mode of action, it is thought that a functional restorer gene allele encodes a polypeptide, such as a PPR protein that has the capacity to (specifically) directly, or in combination with other proteins, bind to the mitochondrial orf256 (SEQ ID NO: 2) transcript responsible for the CMS phenotype. By scavenging the orf256 mRNA, the CMS phenotype can be reversed. As used herein,“bind to” or“specifically bind to” or“(specifically) recognize” means that according to the above described PPR code, the Rf-PPR protein contains a number of PPR motifs with specific residues at positions 5 and 35 and which are ordered in such a way so as to be able to bind to a target mRNA, such as the orf256 mRNA, in a sequence-specific or sequence-preferential manner.

[62] Alternatively, the functional restorer gene allele may encode a polypeptide, such as a PPR protein that has the capacity to (specifically) bind to other mitochondrial mRNAs or chimeric mRNAs responsible for the pollen lethality and the CMS phenotype. The functional restorer gene allele may also encode a polypeptide, such as a PPR protein that has the capacity to (specifically) bind to multiple mitochondrial mRNAs, influencing transcription etc. Via an another alternative mode of action, the functional restorer gene allele may encode a polypeptide, such as a PPR protein that is able to form a complex with additional interacting proteins such as a glycine rich protein (GRP), a hexokinase, or a DUF-WD40, to direct breakdown or cleavage of orf256 and/or other cytotoxic mitochondrial mRNAs, or to inhibit transcription thereof, or to inhibit translation of the cytotoxic, chimeric peptides responsible for the CMS phenotype.

[63] For example, the functional restorer gene allele can encode a PPR protein containing PPR motifs with specific residues at the above indicated positions so as to recognize a target sequence within orf256 mRNA. In one example, the predicted recognition sequence of PPR122 herein described can be NNAUUUNUNNNCNUACGU (SEQ ID NO: 3).

[64] Furthermore, PPR proteins may work in conjunction with other PPR proteins, which may be encoded by a gene in the same Rf locus, and/or by a gene located in any of the other Rf loci, including the Rf1 locus identified on chromosome 1A (described in WO2017/158126). ). In one embodiment, the Rf3-PPR-122 gene is used in cereal plants such as wheat plants in combination with one or more of the Rf loci or Rf genes selected from the group of Rf1 , Rf2, Rf4, Rf5, Rf6, Rf7, and Rf8; such as in combination with Rf1 and Rf6, in combination with Rf1 and Rf7, in combination with Rf1 and Rf4, in combination with Rf4 and Rf6, or in combination with Rf4 and Rf7. In one embodiment, such a combination of Rf loci or Rf genes with the Rfl-PPR-122 gene of the invention occurs at the same locus in the wheat genome (e.g., by translocation, transformation or genome engineering into one locus). In one embodiment, such Rf1-PPR-08 gene is a gene encoding the modified protein as specifically described herein, such as a modified Rf3-PPR-122 protein comprising at least one of the following amino acid modifications by reference to the amino acid sequence in SEQ ID No. 4 (or 5): adding an RL or RH dipeptide between the amino acid corresponding to amino acid position 12 and 22 (such as between the amino acid corresponding to amino acid position 21 and 22), the R107H mutation, the A219V mutation, the T591A mutation, S607F mutation, the E771 D mutation, the A778T mutation, the R781Q mutation, the R783H mutation, or the V789I mutation. In one embodiment, an otherwise modified Rf3-PPR-122 protein retains at least one, or one or more, or all, of the following amino acids at the following positions by reference to the amino acid sequence in SEQ ID No. 4 (or 5): V73, M642, E730, L734, M741 , E754, and/or V744.

[65] A functional restorer gene or allele can for example comprise the nucleotide sequence of SEQ ID NO: 4 or encode a polypeptide having the amino acid sequence of SEQ ID NO: 5.

[66] A functional restorer gene allele can for example also encode a PPR protein, having one or more mutations (insertion, deletion, substitution) that may affect mRNA or protein stability, for example a mutation that increases mRNA or protein stability, thereby resulting in an increased expression of the PPR protein, especially during early pollen development and meisosis, such as in anther or, more specifically, tapetum, or developing microspore.

[67] In one embodiment, the functional restorer gene allele is a functional allele of the Rf-PPR gene comprising the nucleotide sequence of SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, or SEQ ID NO: 4 or encoding the polypeptide sequence of SEQ ID NO: 5. The functional restorer gene allele can also comprise a nucleotide sequence that is substantially identical (as defined herein) to SEQ ID NO: 4, such as having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423. The percent sequence identity is preferably calculated over the entire length of the nucleotide sequence of SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423. The functional restorer gene allele can also comprise a nucleotide sequence that encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. The amino acid sequence is preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 5. The percent sequence identity is preferably calculated over the entire length of the polypeptide of SEQ ID NO: 5.

[68] In a further embodiment, the functional restorer gene allele is a functional restorer gene allele as present in (and as derivable from) at least Accession number PI 583676 (USDA National Small Grains Collection, also known as Dekalb 582M and registered as US PVP 7400045).

[69] The invention further describes a method for producing a cereal (such as a wheat) plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility, comprising the steps of

a. crossing a first cereal plant comprising and expressing or transcribing a functional restorer gene for wheat G- type cytoplasmic male sterility located on chromosome 1 B and having a nucleotide sequence substantially similar to SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially similar to SEQ ID NO: 5 with a second cereal plant;

b. identifying (and optionally selecting) a progeny plant comprising and expressing or transcribing the functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B.

[70] Also provided is a method for producing a cereal (such as a wheat) plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B, comprising the steps of

a. crossing a first cereal plant homozygous for a functional restorer gene for wheat G-type cytoplasmic male sterility located on chromosome 1 B and having a nucleotide sequence substantially similar to SEQ ID NO: 4, or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially similar to SEQ ID NO: 5 with a second cereal plant; b. obtaining a progeny plant, wherein said progeny plant comprises a functional restorer gene allele for wheat G- type cytoplasmic male sterility located on chromosome 1 B.

[71] The second cereal plant can be a cereal plant devoid of a functional restorer gene for wheat G-type cytoplasmic male sterility located on chromosome 1 B, including a cereal plant not transcribing or expressing the identified restorer gene.

[72] In an even further embodiment, the invention provides a method for producing F1 hybrid cereal seeds or F1 hybrid cereal plants, comprising the steps of:

a. providing a male cereal (e.g. wheat) parent plant comprising and expressing or transcribing a functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B and having a nucleotide sequence substantially similar to SEQ ID NO: 4, or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially similar to SEQ ID NO: 5;

b. crossing said male parent plant with a female cereal (e.g. wheat) parent plant, wherein the female parent plant is a G- type cytoplasmic male sterile cereal plant;

c. optionally collecting hybrid seeds from said cross.

[73] The F1 hybrid seeds and plants preferably comprise and express at least one functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B as described herein, and the F1 plants grown from the seeds are therefore fertile. Preferably, the male parent plant is homozygous for the functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B.

[74] In the above method, the male parent plant used for crossing can be selected or identified by analyzing the transcription or expression of a nucleotide sequence substantially similar to SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, or SEQ ID NO: 4, or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially similar to SEQ ID NO: 5. Analysing the transcription or expression can be achieved by (directly) measuring RNA transcribed from the nucleotide sequence substantially similar to SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423 using any available means, including RT-qPCR, transcriptome analysis or the like. Analysing the expression can be achieved by (directly) measuring levels of polypeptides comprising an amino acid sequence substantially similar to SEQ ID NO: 5.

[75] The invention also provides cereal plants, such as wheat plants, obtained by any of the above methods, said cereal plant comprising and transcribing or expressing the functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B, preferably comprising a nucleotide sequence substantially similar to SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, or SEQ ID NO: 4, or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially similar to SEQ ID NO: 5.

[76] Also described is a cereal plant, plant part, plant cell or seed comprising at least one functional restorer gene allele for wheat G-type cytoplasmic male sterility located on chromosome 1 B, said plant comprising and expressing or transcribing a nucleotide sequence substantially similar to SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, or SEQ ID NO: 4, or a nucleotide sequence encoding a polypeptide comprising an amino acid sequence substantially similar to SEQ ID NO: 5. Such plants, plant parts, plant cells or seeds may contain the functional restorer gene allele for wheat G-type cytoplasmic male sterility in a different genomic context, and may e.g. be devoid of the nucleotide sequence of SEQ ID NO: 1 from position 1 to position 3000 and/or of the nucleotide sequence of SEQ ID NO: 1 from position 5799 to position 8810, or being devoid of any of the following nucleotide sequences, or combinations thereof: the nucleotide sequence of SEQ ID NO: 1 from position 1 to position 500, the nucleotide sequence of SEQ ID NO: 1 from position 1 to position 1000, the nucleotide sequence of SEQ ID NO: 1 from position 1 to position 1500, the nucleotide sequence of SEQ ID NO: 1 from position 1 to position 2000, the nucleotide sequence of SEQ ID NO: 1 from position 1 to position 2500, the nucleotide sequence of SEQ ID NO: 1 from position 500 to position 1000, the nucleotide sequence of SEQ ID NO: 1 from position 500 to position 1500, the nucleotide sequence of SEQ ID NO: 1 from position 500 to position 2000, the nucleotide sequence of SEQ ID NO: 1 from position 500 to position 2500, the nucleotide sequence of SEQ ID NO: 1 from position 500 to position 3000, the nucleotide sequence of SEQ ID NO: 1 from position 1000 to position 1500, the nucleotide sequence of SEQ ID NO: 1 from position 1000 to position 2000, the nucleotide sequence of SEQ ID NO: 1 from position 1000 to position 2500, the nucleotide sequence of SEQ ID NO: 1 from position 1000 to position 3000; the nucleotide sequence of SEQ ID NO: 1 from position 1500 to position 2000, the nucleotide sequence of SEQ ID NO: 1 from position 1500 to position 2500, the nucleotide sequence of SEQ ID NO: 1 from position 1500 to position 3000, the nucleotide sequence of SEQ ID NO: 1 from position 2000 to position 2500, the nucleotide sequence of SEQ ID NO: 1 from position 2000 to position 3000, the nucleotide sequence of SEQ ID NO: 1 from position 2500 to position 3000, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 3500, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 4000, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 4500, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 5000, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 5500, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 6000, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 3001 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 4000, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 4500, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 5000, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 5500, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 6000, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 3500 to position 8810, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 4500, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 5000, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 5500, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 6000, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 4000 to position 8810; the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 5000, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 5500, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 6000, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 4500 to position 8810, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 5500, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 6000, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 5000 to position 8810, the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 6000, the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 5500 to position 8810; the nucleotide sequence of SEQ ID NO: 1 from position 6000 to position 6500, the nucleotide sequence of SEQ ID NO: 1 from position 6000 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 6000 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 6000 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 6000 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 6000 to position 8810; the nucleotide sequence of SEQ ID NO: 1 from position 6500 to position 7000, the nucleotide sequence of SEQ ID NO: 1 from position 6500 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 6500 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 6500 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 6500 to position 8810, the nucleotide sequence of SEQ ID NO: 1 from position 7000 to position 7500; the nucleotide sequence of SEQ ID NO: 1 from position 7000 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 7000 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 7000 to position 8810, the nucleotide sequence of SEQ ID NO: 1 from position 7500 to position 8000, the nucleotide sequence of SEQ ID NO: 1 from position 7500 to position 8500, the nucleotide sequence of SEQ ID NO: 1 from position 7500 to position 8810, the nucleotide sequence of SEQ ID NO: 1 from position 8000 to position 8500 or the nucleotide sequence of SEQ ID NO: 1 from position 8000 to position 8810.

[77] Also provided are plant parts, plant cells and seed from the cereal plants according to the invention comprising and expressing the functional restorer gene allele as herein described. The plants, plant parts, plant cells and seeds of the invention may also be hybrid plants, plant parts, plant cells or seeds. [78] Also provided is a method for the identification and/or selection of a cereal (e.g. wheat) plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising the steps of

a. Identifying or detecting in said plant the expression or transcription of the nucleic acid or the polypeptide encoding a functional restorer gene for wheat G-type cytoplasmic male sterility as described herein

b. and optionally selecting said plant comprising said nucleic acid or polypeptide.

[79] Likewise, identifying or detecting can involve obtaining a biological sample (e.g. protein) or genomic DNA and determining the presence of the nucleic acid or polypeptide according to methods well known in the art, such as hybridization, PCR, Rt-PCR, Southern blotting, Southern-by-sequencing, SNP detection methods, western blotting, Elisa etc, e.g. based on the sequences provided herein.

[80] The invention also provides the use of the nucleotide sequence of the functional restorer gene for wheat G-type cytoplasmic male sterility located on chromosome 1 B for the identification of at least one marker comprising an allele linked to said functional restorer gene for wheat G-type cytoplasmic male sterility located on chromosome 1 B. Such markers are also genetically linked or tightly linked to the restorer gene and are also within the scope of the invention. Markers can be identified by any of a variety of genetic or physical mapping techniques. Methods of determining whether markers are genetically linked to a restore gene are known to those of skill in the art and include, for example, interval mapping (Lander and Botstein, (1989) Genetics 121 :185), regression mapping (Haley and Knott, (1992) Heredity 69:315) or MQM mapping (Jansen, (1994) Genetics 138:871), rMQM mapping. In addition, such physical mapping techniques as chromosome walking, contig mapping and assembly, amplicon resequencing, transcriptome sequencing, targeted capture and sequencing, next generation sequencing and the like, can be employed to identify and isolate additional sequences useful as markers.

[81] Also provided is the use of a plant obtained by any of the methods as described herein and comprising and expressing at least a functional restorer gene for wheat G-type cytoplasmic male sterility located on chromosome 1 B as described herein, for restoring fertility in a progeny of a G-type cytoplasmic male sterile cereal plant, such as a wheat plant, or for producing a population of hybrid cereal plants, such as a wheat plants or for producing hybrid seed.

[82] Further provided is a recombinant nucleic acid molecule, especially a recombinant DNA molecule, which comprises a functional restorer gene as described herein. In one embodiment the recombinant DNA molecule comprises a plant expressible promoter, preferably a heterologous plant promoter, operably linked to a nucleotide sequence having substantial identity as herein defined to a nucleotide sequence of SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, or to the nucleotide sequence of SEQ ID NO: 4 or encoding a polypeptide comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 5. The recombinant gene may optionally comprise a transcription termination and polyadenylation, preferably functional in plant cells. Also, a DNA vector is provided comprising the recombinant DNA molecule. The recombinant DNA molecule or DNA vector may be an isolated or modified nucleic acid molecule. The DNA comprising the functional restorer gene may be in a microorganism, such as a bacterium (e.g. Agrobacterium or E.coli). [83] The term "heterologous" refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources. For example, a promoter is heterologous with respect to an operably linked nucleic acid sequence, such as a coding sequence, if such a combination is not normally found in nature. In addition, a particular sequence may be "heterologous" with respect to a cell or organism into which it is inserted (i.e. does not naturally occur in that particular cell or organism). In one embodiment the term“heterologous” as used herein when referring to a nucleic acid or protein occurring in a certain plant species, also includes a nucleic acid or protein whose sequence has been modified or mutated compared to the previously existing nucleic acid or protein sequence occurring in said plant species. Hence, after the deletion, addition or substitution of one or more nucleotides in a nucleic acid or one or more amino acids in a protein sequence occurring in a wheat plant (e.g., modifying a native promoter to include regulatory elements that increase transcription, such as an enhancer element, or modifying a native promoter by inactivating or removing certain negative regulatory elements, such as repressor elements or target sites for miRNAs or IncRNAs), such a modified nucleic acid or protein is also considered heterologous to the wheat plant or to the operably-linked sequence.

[84] The functional restorer gene allele can also encode a PPR protein having a mutation in an a-helical domain of a PPR motif, such as a mutation that affects hairpin formation between two of the a-helices making up a PPR motif.

[85] The functional restorer gene allele can also encode a PPR protein having a mutation that affects dimerization of the PPR protein. It has e.g. been found that‘Thylakoid assembly 8’ (THA8) protein is a pentatricopeptide repeat (PPR) RNA- binding protein required for the splicing of the transcript of ycf3, a gene involved in chloroplast thylakoid-membrane biogenesis. THA8 forms an asymmetric dimer once bound to single stranded RNA, with the bound RNA at the dimer interface. This dimer complex formation is mediated by the N-terminal PPR motifs 1 and 2 and the C-terminal motifs 4 and 5 (Ke et al., 2013, Nature Structural & Molecular Biology, 20,1377-1382).

[86] The functional restorer gene allele can also encode a PPR protein which when expressed is targeted to the mitochondrion. This can e.g. be accomplished by the presence of a (plant-functional) mitochondrial targeting sequence or mitochondrial signal peptide, or mitochondrial transit peptide. A mitochondrial targeting signal is a 10-70 amino acid long peptide that directs a newly synthesized protein to the mitochondria, typically found at the N-terminus. Mitochondrial transit peptides are rich in positively charged amino acids but usually lack negative charges. They have the potential to form amphipathic a-helices in non-aqueous environments, such as membranes. Mitochondrial targeting signals can contain additional signals that subsequently target the protein to different regions of the mitochondria, such as the mitochondrial matrix. Like signal peptides, mitochondrial targeting signals are cleaved once targeting is complete. Mitochondrial transit peptides are e.g. described in Shewry and Gutteridge (1992, Plant Protein Engineering, 143-146, and references therein), Sjoling and Glaser (Trends Plant Sci Volume 3, Issue 4, 1 April 1998, Pages 136-140), Pfanner (2000, Current Biol, Volume 10, Issue 11 , pages R412-R415), Huang et al (2009, Plant Phys 150(3): 1272-1285), Chen et al. (1996, PNAS, Vol. 93, pp. 11763-11768), Fuji et al. (Plant J 2016, 86, 504-513). The amino acid sequence of SEQ ID NO:5 from position 1 to position 33 is an example of such mitochondrial targeting sequence. [87] In a further embodiment, said functional restorer gene allele encoded by said (isolated) nucleic acid molecule is obtainable from USDA accession number PI 583676.

[88] Also provided is a(n) (isolated or modified) polypeptide encoded by the nucleic acid molecule as described above (said polypeptide encoding a functional restorer protein for wheat G-type cytoplasmic male sterility) or comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 5.

[89] The functional restorer gene allele may also be cloned and a chimeric gene may be made, e.g. by operably linking a plant expressible promoter to the functional restorer gene allele and optionally a 3’ end region involved in transcription termination and polyadenylation functional in plants. Such a chimeric gene may be introduced into a plant cell, and the plant cell may be regenerated into a whole plant to produce a transgenic plant. In one aspect the transgenic plant is a cereal plant, such as a wheat plant, according to any method well known in the art.

[90] Thus, in a particular embodiment a chimeric gene is provided comprising a(n) (isolated or modified) nucleic acid molecule encoding the functional restorer gene allele as described above, operably linked to a heterologous plant-expressible promoter and optionally a 3’ termination and polyadenylation region.

[91] The use of such a (isolated or modified or extracted) nucleic acid molecule and/or of such a chimeric gene and/or of such a chromosome fragment for generating plant cells and plants comprising a functional restorer gene allele is encompassed herein. In one aspect it may be used to generate transgenic cereal (e.g. wheat) cells, plants and plant parts or seeds comprising the functional restorer gene allele and the plant having the capacity to restore fertility against wheat G-type cytoplasmic male sterility as described above.

[92] A host or host cell, such as a (cereal plant cell or (cereal) plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising the (isolated or modified) nucleic acid molecule, (isolated or modified) polypeptide, or the chimeric gene as described above is provided, wherein preferably said polypeptide, said nucleic acid, or said chimeric gene in each case is heterologous with respect to said plant cell or plant or seed, or is modified. The host cell can e.g also be a bacterium, such as E.coli or Agrobacterium sp. such as A. tumefaciens.

[93] Thus, also provided is a method for producing a cereal plant cell or plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising a functional restorer gene for wheat G-type cytoplasmic male sterility, or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the steps of providing said plant cell or plant with the (recombinant) chromosome fragment or the (isolated or modified) nucleic acid molecule or the chimeric gene as described herein wherein said providing comprises transformation, crossing, backcrossing, genome editing or mutagenesis. Restoration capacity, as used herein, means the capacity of a plant to restore fertility or to contribute to the restoration of fertility in the progeny of a cross with a G-type cytoplasmic male sterility (“CMS”) line. Preferably, said plant expresses or has an increased expression of the polypeptide according to the invention. Preferably, said (increase in) expression is at least during (the early phases of) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores (where said plant did not express or to a lesser extent expressed the polypeptide prior to the providing step).

[94] Thus, also provided is a method for producing a cereal plant cell or plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising and expressing a functional restorer gene for wheat G-type cytoplasmic male sterility, or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the steps of increasing the expression of the (isolated or modified) polypeptide as described herein in said plant cell or plant or seed. Preferably, said (increase in) expression is at least during (the early phases of) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores. Prior to the increasing step, the initial plant did not express or to a lesser extent expressed the polypeptide and/or did not have or to a lesser extent had restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”)). In one embodiment, the expression of the polypeptide as described herein is increased by engineering the nucleotide sequence encoding the restorer polypeptide, including by deliberate modification of the nucleotide sequence of the gene encoding the restorer polypeptide, such as increasing gene copy number of the gene, inserting modifications that increase stability of the RNA transcribed from the gene or of the polypeptide expressed from the gene, modifications of the upstream region/promoter region, modifications of the transcription termination and polyadenylation region etc.

[95] Increasing the polypeptide expression can be done by providing the plant with the (recombinant) chromosome fragment or the (isolated or modified) nucleic acid molecule or the chimeric gene as described herein, whereby the nucleic acid encoding the functional restorer gene allele is under the control of appropriate regulatory elements such as a promoter driving expression in the desired tissues/cells, but also by providing the plant with transcription factors that e.g. (specifically) recognise the promoter region and promote transcription, such as TALeffectors, dCas (“dead” CAS), dCpfl (Dead Cpf1) etc. coupled to transcriptional enhancers.

[96] Further described is a method for converting a cereal plant, such as a wheat plant, not having the capacity to restore fertility in the progeny of a cross with a G-type cytoplasmic male sterility (“CMS”) line (a non-restorer plant) into a plant having the capacity to restore fertility in the progeny of a cross with a G-type cytoplasmic male sterility (“CMS”) line (a restorer plant), comprising the steps of modifying the genome of said plant to comprise and express the (isolated or modified) nucleic acid molecule or the chimeric gene encoding a functional restorer gene allele for wheat G-type cytoplasmic male sterility as described herein wherein said modifying comprises transformation, crossing, backcrossing, genome editing or mutagenesis, preferably by transformation, genome editing or mutagenesis. Preferably, said plant expresses the polypeptide according to the invention, particularly at least during (the early phases of) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores. Prior to the modification, said the initial plant did not express or to a lesser extent expressed the polypeptide and/or did not have or to a lesser extent had restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”).

[97] Thus, also provided is a method for converting a non-restoring cereal plant, such as a wheat plant, into a restoring plant for wheat G-type cytoplasmic male sterility (“CMS”), or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the steps of modifying the genome of said plant to increase the expression of a polypeptide according to the invention in said plant. Preferably, said (increase in) expression is at least during (the early phases of) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores. Prior to said modification said plant did not express or to a lesser extent expressed the polypeptide and/or did not have or to a lesser extent had restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”).

[98] Modifying the genome to increase expression of the polypeptide can for example be done by modifying the native promoter to include regulatory elements that increase transcription, such as certain enhancer element, but also by inactivating or removing certain negative regulatory elements, such as repressor elements or target sites for miRNAs or IncRNAs. The Rf3 5’ upstream region including the promoter region is included in SEQ ID NO 1 upstream of nucleotide position 3051.

[99] Also described is a plant cell or plant, preferably a cereal plant cell or cereal plant or seed thereof, such as a wheat plant cell or plant or seed thereof, produced according to any of the above methods, preferably wherein said plant has an increased restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) compared to said plant prior to the providing step or the modification step. Use of such a plant obtained according to the above methods to restore fertility in the progeny of a cross with a G-type cytoplasmic male sterility (“CMS”) plant or to produce hybrid plants or hybrid seed is also described. Such a plant cell, plant or seed can be a hybrid plant cell, plant or seed.

[100] Genome editing, as used herein, refers to the targeted modification of genomic DNA using sequence-specific enzymes (such as endonuclease, nickases, base conversion enzymes) and/or donor nucleic acids (e.g. dsDNA, oligo’s) to introduce desired changes in the DNA. Sequence-specific nucleases that can be programmed to recognize specific DNA sequences include meganucleases (MGNs), zinc-finger nucleases (ZFNs), TAL-effector nucleases (TALENs) and RNA-guided or DNA-guided nucleases such as Cas9, Cpfl , CasX, CasY, C2c1 , C2c3, certain Argonaut-based systems (see e.g. Osakabe and Osakabe, Plant Cell Physiol. 2015 Mar;56(3):389-400; Ma et al., Mol Plant. 2016 Jul 6;9(7):961-74; Bortesie et al., Plant Biotech J, 2016, 14; Murovec et al., Plant Biotechnol J. 15:917-926, 2017; Nakade et al., Bioengineered Vol 8, No.3:265-273, 2017; Burstein et al., Nature 542, 37-241 ; Komor et al., Nature 533, 420-424, 2016; all incorporated herein by reference). Donor nucleic acids can be used as a template for repair of the DNA break induced by a sequence specific nuclease, but can also be used as such for gene targeting (without DNA break induction) to introduce a desired change into the genomic DNA.

[101] Accordingly, using these technologies, plants lacking a functional restorer gene for wheat G-type cytoplasmic male sterility (non-restoring plants) can be converted to restoring plants by making the desired changes to existing PPR genes or alternatively to introduce one or more complete sequences encoding functional PPR-Rf proteins, e.g. as described herein, at a specific genomic location.

[102] Mutagenesis as used herein, refers to e.g. EMS mutagenesis or radiation induced mutagenesis and the like.

[103] Transgenic cereal cells, e.g. transgenic wheat cells, comprising in their genome a recombinant chromosome fragment as described or an (isolated or modified) nucleic acid molecule as described or a chimeric gene as described comprising a functional restorer gene allele as described are also an embodiment of the invention. In one aspect, the DNA molecule comprising Rf allele is stably integrated into the cereal (e.g. wheat) genome.

[104] Thus, cereal plants, plant parts, plant cells, or seeds thereof, especially wheat, comprising a chromosome fragment or a nucleic acid molecule according to the invention or a polypeptide according to the invention or a chimeric gene according to the invention encoding a functional restorer gene according to the invention, are provided, said plant having the capacity to restore fertility against wheat G-type cytoplasmic male sterility are provided herein. In one embodiment, the chromosome fragment, nucleic acid molecule, polypeptide or chimeric gene is heterologous to the plant, such as transgenic cereal plants or transgenic wheat plants. This also includes plant cells or cell cultures comprising such a chromosome fragment or nucleic acid molecule, polypeptide or chimeric gene, independent whether introduced by transgenic methods or by breeding methods. The cells are e.g. in vitro and are regenerable into plants comprising the chromosome fragment or nucleic acid molecule, or chimeric gene of the invention. Said plants, plant parts, plant cells and seeds may also be hybrid plants, plant parts, plant cells or seeds.

[105] Such plants may also be used as male parent plant in a method for producing F1 hybrid seeds or F1 hybrid plants, as described above.

[106] A plant-expressible promoter as used herein can be any promoter that drives sufficient expression at least during (early) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspore. This can for example be a constitutive promoter, an inducible promoter, but also a pollen-, anther- or, more specifically tapetum- or microspore-specific/preferential promoter.

[107] A constitutive promoter is a promoter capable of directing high levels of expression in most cell types (in a spatio- temporal independent manner). Examples of plant expressible constitutive promoters include promoters of bacterial origin, such as the octopine synthase (OCS) and nopaline synthase (NOS) promoters from Agrobacterium, but also promoters of viral origin, such as that of the cauliflower mosaic virus (CaMV) 35S transcript (Hapster et al., 1988, Mol. Gen. Genet. 212: 182- 190) or 19S RNAs genes (Odell et al., 1985, Nature. 6;313(6005):810-2; U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al., 1989, EMBO J. 8:2195-2202), the enhanced 2x35S promoter (Kay at al., 1987, Science 236:1299-1302; Datla et al. (1993), Plant Sci 94:139-149) promoters of the cassava vein mosaic virus (CsVMV; WO 97/48819, US 7,053,205), 2xCsVMV (W02004/053135) the circovirus (AU 689 311) promoter, the sugarcane bacilliform badnavirus (ScBV) promoter (Samac et al., 2004, Transgenic Res. 13(4):349-61), the figwort mosaic virus (FMV) promoter (Sanger et al., 1990, Plant Mol Biol. 14(3):433-43), the subterranean clover virus promoter No 4 or No 7 (WO 96/06932) and the enhanced 35S promoter as described in US 5,164,316, US 5,196,525, US 5,322,938, US 5,359,142 and US 5,424,200. Among the promoters of plant origin, mention will be made of the promoters of the plant ribulose-biscarboxylase/oxygenase (Rubisco) small subunit promoter (US 4,962,028; W099/25842) from Zea mays and sunflower, the promoter of the Arabidopsis thaliana histone H4 gene (Chaboute et al., Plant Mol. Biol. 8, 179-191 , 1987), the ubiquitin promoters (Holtorf et al., 1995, Plant Mol. Biol. 29:637-649, US 5,510,474) of Maize, Rice and sugarcane, the Rice actin 1 promoter (Act-1, US 5,641 ,876), the histone promoters as described in EP 0 507 698 A1 , the Maize alcohol dehydrogenase 1 promoter (Adh-1) (from http://www.patentlens.net/daisy/promoters/242.html)). Also the small subunit promoter from Chrysanthemum may be used if that use is combined with the use of the respective terminator (Outchkourov et al., Planta, 216: 1003-1012, 2003).

[108] Examples of inducible promoters include promoters regulated by application of chemical compounds, including alcohol-regulated promoters (see e.g. EP637339), tetracycline regulated promoters (see e.g. US 5464758), steroid-regulated promoters (see e.g. US5512483; US6063985; US6784340; US6379945; W001/62780), metal-regulated promoters (see e.g. US4601978) but also developmental^ regulated promoters.

[109] Pollen/microspore-active promoters include e.g. a maize pollen specific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161 168), PTA29, PTA26 and PTAI 3 (e.g., see U.S. Pat. No. 5,792,929) and as described in e.g. Baerson et al. (1994 Plant Mol. Biol.26: 1947-1959), the NMT19 microspore-specific promoter as e.g. described in W097/30166. Further anther/pollen-specific or anther/pollen-active promoters are described in e.g. Khurana et al., 2012 (Critical Reviews in Plant Sciences, 31 : 359-390), W02005100575, WO 2008037436. Other suitable promoters are e.g. the barley vrn1 promoter, such as described in Alonso-Peral et al. (2011 , PLoS One. 6(12):e29456).

[110] Examples of tissue specific promoters include meristem specific promoters such as the rice OSH1 promoter (Sato et al. (1996) Proc. Natl. Acad. Sci. USA 93:8117-8122) rice metallothein promoter (BAD87835.1) WAK1 and WAK2 promoters (Wagner & Kohorn (2001) Plant Cell 13(2): 303-318, spike tissue specific promoter D5 from barley (US6291666), the lemma/palea specific Lem2 promoter from barley (Abebe et al. (2005) Planta, 221 , 170-183), the early inflorescence specific Pvrnl promoter from barley (Alonse Peral et al. 2011, PLoS ONE 6(12) e29456), the early inflorescence specific Pcrs4/PrA2 promoter from barley (Koppolu et al. 2013, Proc. Natl. Acad. Sci USA, 110(32) 13198-13203), the meristem specific pkn1 promoter with the Act1 intron from rice (Zhang et al., 1998, Planta 204: 542-549, Postma-Haarsma et al. 2002, Plant Molecular Biology 48: 423-441) the SAM/inflorescence specific promoter from Dendrobium sp. Pdomadsl (Yu et al.2002, Plant Molecular Biology 49: 225-237), or an anther or tapetum-specific promoter such as the tapetum-specific Osg6B promoter (Yokoi et al., Plant Cell Rep. 1997, Vol. 16 (6):363-367).

[111] It will be clear that the herein identified nucleic acids and polypeptides encoding functional restorer genes can be used to identify further functional restorer genes for wheat G-type cytoplasmic male sterility. Thus, the invention also provides the use of the (isolated or modified) nucleic acids or polypeptides as disclosed herein, such as SEQ ID NO: 4, to identify one or more further functional restorer genes for wheat G-type cytoplasmic male sterility.

[112] Further, homologous or substantially identical functional restorer genes can be identified using methods known in the art. Flomologous nucleotide sequence may be identified and isolated by hybridization under stringent or high stringent conditions using as probes a nucleic acid comprising e.g. the nucleotide sequence of SEQ ID NO: 4 or part thereof, as described above. Other sequences encoding functional restorer genes may also be obtained by DNA amplification using oligonucleotides specific for genes encoding functional restorer genes as primers, such as but not limited to oligonucleotides comprising or consisting of about 20 to about 50 consecutive nucleotides from SEQ ID NO: 4 or its complement. Flomologous or substantially identical functional restorer genes can be identified in silico using Basic Local Alignment Search Tool (BLAST) homology search with the nucleotide or amino acid sequences as provided herein.

[113] Functionality of restorer genes or alleles thereof, such as identified as above, can be validated for example by providing, e.g. by transformation or crossing, such a restorer gene under control of a plant-expressible promoter in a cereal (wheat) plant that does not have the capacity to restore fertility of offspring of a G-type cytoplasmic male sterile wheat plant, crossing the thus generated cereal plant with a G-type cytoplasmic male sterile wheat plant and evaluating seed set in the progeny. Alternatively, a restorer line can be transformed with an RNAi construct or gene-edited with e.g. CRISPR-Cas technology or any other sequence specific nuclease so to generate a loss of function that renders the plant non-restoring. Similarly, other means for mutating the restorer gene (e.g. EMS, g-radiation) can be used to evaluate the effect of a loss of function mutation on restoring ability.

[114] In any of the herein described embodiments and aspects the plant may comprise or may be selected to comprise or may be provided with a further functional restorer gene or locus for wheat G-type cytoplasmic male sterility (located on or obtainable from the same or another chromosome), such as Rf1 (1A), Rf2 (ID), Rf4 (6B), Rf5 (6D), Rf6 (5D), Rf7 (7B), Rf8 (6AS or 6BS) (Tahir & Tsunewaki, 1969, supra; Yen et al., 1969, supra; Bahl & Maan, 1973, supra; Du et al., 1991 , supra; Sihna et al., 2013, supra; Ma et al., 1991 , supra; Zhou et al., 2005, supra).

[115] Any of the herein described methods, markers and marker alleles, nucleic acids, polypeptides, chimeric genes, plants etc. may also be used to restore fertility against S ype cytoplasm, as e.g. described in Ahmed et al 2001 (supra).

DEFINITIONS

[116] As used herein a“chimeric gene” refers to a nucleic acid construct which is not normally found in a plant species. A chimeric nucleic acid construct can be DNA or RNA.“Chimeric DNA construct” and“chimeric gene” are used interchangeably to denote a gene in which the promoter or one or more other regulatory regions, such as the a transcription termination and polyadenylation region of the gene are not associated in nature with part or all of the transcribed DNA region, or a gene which is present in a locus in the plant genome in which it does not occur naturally or present in a plant in which it does not naturally occur. In other words, the gene and the operably-linked regulatory region or the gene and the genomic locus or the gene and the plant are heterologous with respect to each other, i.e. they do not naturally occur together. This includes the situation wherein one or more of the regulatory elements (such as the promoter or the 3’ end formation and polyadenylation region) or the coding region, of a wheat gene (such as the Rf3-PPR-122 gene of the current invention), is a modified nucleic acid (as that is not normally found in wheat, and is heterologous to the gene elements it is operably-linked to).

[117] A first nucleotide sequence is“operably linked" with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. When recombinantly produced, operably linked nucleic acid sequences are generally contiguous, and, where necessary to join two protein-coding regions, in the same reading frame (e.g. in a polycistronic ORF). However, nucleic acids need not be contiguous to be operably linked.

[118] “Backcrossing” refers to a breeding method by which a (single) trait, such as fertility restoration (Rf), can be transferred from one genetic background (a“donor”) into another genetic background (also referred to as“recurrent parent”), e.g. a plant not comprising such an Rf gene or locus. An offspring of a cross (e.g. an F1 plant obtained by crossing an Rf containing with an Rf lacking plant; or an F2 plant or F3 plant, etc., obtained from selfing the F1) is“backcrossed” to the parent. After repeated backcrossing (BC1 , BC2, etc.) and optionally selfings (BC1S1 , BC2S1 , etc.), the trait of the one genetic background is incorporated into the other genetic background.

[119] “Marker assisted selection” or“MAS” is a process of using the presence of molecular markers, which are genetically linked to a particular locus or to a particular chromosome region (e.g. introgression fragment), to select plants based on the presence of the specific locus or region (introgression fragment). For example, a molecular marker genetically and physically linked to an Rf locus, can be used to detect and/or select plants comprising the Rf locus. The closer the genetic linkage of the molecular marker to the locus, the less likely it is that the marker is dissociated from the locus through meiotic recombination.

[120] A“biological sample” can be a plant or part of a plant such as a plant tissue or a plant cell, or an extract of a plant or part of a plant, including protein.

[121] Wheat, as used herein, refers to any of the following Triticum species: T. aestivum, T. aethiopicum, T. araraticum, T. boeoticum, T. carthlicum, T.com actum, T. dicoccoides, T. dicoccon, T. durum, T. ispahanicum, T. karamyschevii, T. macha, T. militinae, T. monococcum, T. polonicum, T. spelta, T. sphaerococcum, T.timopheevii, T. turanicum, T. turgidum, T. urartu, T. vavilovii, T. zhukovskyi Faegi. Wheat also refers to species of the genera Aegilops and Triticale.

[122] “Providing genomic DNA” as used herein refers to providing a sample comprising genomic DNA from the plant. The sample can refer to a tissue sample which has been obtained from said plant, such as, for example, a leaf sample, comprising genomic DNA from said plant. The sample can further refer to genomic DNA which is obtained from a tissue sample, such as genomic DNA which has been obtained from a tissue, such as a leaf sample. Providing genomic DNA can include, but does not need to include, purification of genomic DNA from the tissue sample. Providing genomic DNA thus also includes obtaining tissue material from a plant or larger piece of tissue and preparing a crude extract or lysate therefrom.

[123] “Isolated DNA” as used herein refers to DNA not occurring in its natural genomic context, irrespective of its length and sequence. Isolated DNA can, for example, refer to DNA which is physically separated from the genomic context, such as a fragment of genomic DNA. Isolated DNA can also be an artificially produced DNA, such as a chemically synthesized DNA, or such as DNA produced via amplification reactions, such as polymerase chain reaction (PCR) well-known in the art. Isolated DNA can further refer to DNA present in a context of DNA in which it does not occur naturally. For example, isolated DNA can refer to a piece of DNA present in a plasmid. Further, the isolated DNA can refer to a piece of DNA present in another chromosomal context than the context in which it occurs naturally, such as for example at another position in the genome than the natural position, in the genome of another species than the species in which it occurs naturally, or in an artificial chromosome. .“Isolated”, as used herein, when referring to a protein (sequence) also includes a protein (sequence) that has been modified by man (e.g., by modifying the nucleic acid encoding that protein) as is done in an effort to obtain some improvement of protein activity (such as restoration activity). “Isolated”, as used herein, when referring to a nucleic acid (sequence) also includes a nucleic acid (sequence) that has been modified by man (e.g., by inserting, deleting or substituting one or more nucleotides in the native nucleic acid) as is done in an effort to obtain some improvement (like improvement in RNA or protein expression, targeting or stability, or improvement in protein activity (such as restoration activity)). A“modified” nucleic acid or protein (sequence), as used herein, refers to a nucleic acid or protein (sequence) that is different to the native nucleic acid or protein, by modifying or mutating the nucleic acid or protein (or the nucleic acid encoding the protein), as is done in an effort to obtain some improvement. Examples of modified nucleic acids are those of SEQ ID NO: 1 or 4 wherein nucleotides are changed so as to encode the modified Rf3-PPR-122 proteins as specifically described herein.

[124] Whenever reference to a“plant” or“plants” according to the invention is made, it is understood that also plant parts (cells, tissues or organs, seed pods, seeds, severed parts such as roots, leaves, flowers, pollen, etc.), progeny of the plants which retain the distinguishing characteristics of the parents (especially the restoring capacity), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived there from are encompassed herein, unless otherwise indicated. In some embodiments, the plant cells of the invention may be non-propagating cells.

[125] The obtained plants according to the invention can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the characteristic of the presence of the restorer gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. The obtained plants can further be used for creating propagating material. Plants according to the invention can further be used to produce gametes, seeds, flour, embryos, either zygotic or somatic, progeny or hybrids of plants obtained by methods of the invention. Seeds obtained from the plants according to the invention are also encompassed by the invention.

[126] “Creating propagating material”, as used herein, relates to any means known in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twinscaling), sexual reproduction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridization).

[127] Transformation, as used herein, means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence. Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium- mediated transformation.

[128] As used herein, the term“homologous” or“substantially identical" or“substantially similar” may refer to nucleotide sequences that are more than 85% identical. For example, a substantially identical nucleotide sequence may be 85.5%; 86%; 87%; 88%; 89%; 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or 99.5% identical to the reference sequence. A probe may also be a nucleic acid molecule that is“specifically hybridizable” or“specifically complementary” to an exact copy of the marker to be detected (“DNA target").“Specifically hybridizable” or“specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and the DNA target. A nucleic acid molecule need not be 100% complementary to its target sequence to be specifically hybridizable. A nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under stringent hybridization conditions, preferably highly stringent conditions. The term“homologous” or“substantially identical" or “substantially similar” may also be used in the context of amino acid sequences that are more than 85% identical. For example, a substantially identical amino acid sequence may be 85.5%; 86%; 87%; 88%; 89%; 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or 99.5% identical to the reference sequence. In one embodiment, such substantially identical/similar or homologous amino acid or DNA sequences refers to: a) DNA sequences different from SEQ ID No. 1 or 4 , but having at least 85 %, or at least 85.5%; 86%; 87%; 88%; 89%; 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or at least 99.5% sequence identity to SEQ ID No. 1 or 4, or b) amino acid sequences different from SEQ ID No. 5, but having at least 85 %, or at least 85.5%; 86%; 87%; 88%; 89%; 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or at least 99.5% sequence identity to SEQ ID No. 5. In one embodiment, an amino acid sequence substantially identical or substantially similar to SEQ ID NO: 5 is more than 85%; 86%; 87%; 88%; 89%; 90%; 91 %; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or more than 99.5% identical to SEQ ID NO:5, and has any one or more of the following amino acid substitutions by reference to the amino acid sequence in SEQ ID No. 5: adding an RL or RH dipeptide between the amino acid corresponding to amino acid position 12 and 22 (such as between the amino acid corresponding to amino acid position 21 and 22), the R107H mutation, the A219V mutation, the T591A mutation, S607F mutation, the E771 D mutation, the A778T mutation, the R781Q mutation, the R783H mutation, or the V789I mutation. In one embodiment, such otherwise modified Rf3-PPR-122 protein retains at least one, or one or more, or all, of the following amino acids at the following positions by reference to the amino acid sequence in SEQ ID No. 4 (or 5): V73, M642, E730, L734, M741 , E754, and/or V744.

[129] “Stringent hybridization conditions” can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequences at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridizations (Northern blots using a probe of e.g. 10Ont) are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.

[130] “High stringency conditions” can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCI, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (100X Denhardt’s contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 m9/ihI denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0.1 * SSC, 0.1% SDS.

[131] “Moderate stringency conditions” refers to conditions equivalent to hybridization in the above described solution but at about 60-62°C. Moderate stringency washing may be done at the hybridization temperature in 1x SSC, 0.1% SDS.

[132] “Low stringency” refers to conditions equivalent to hybridization in the above described solution at about 50-52°C. Low stringency washing may be done at the hybridization temperature in 2x SSC, 0.1 % SDS. See also Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY) and Sambrook and Russell (2001 , Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY).

[133] For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues. The“optimal alignment” of two sequences is found by aligning the two sequences over the entire length according to the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, 1970, J Mol Biol 48(3):443-53) in The European Molecular Biology Open Software Suite (EMBOSS, Rice et al., 2000, Trends in Genetics 16(6): 276—277; see e.g. http://www.ebi.ac.uk/emboss/align/index.html) using default settings (gap opening penalty = 10 (for nucleotides) / 10 (for proteins) and gap extension penalty = 0.5 (for nucleotides) / 0.5 (for proteins)). For nucleotides the default scoring matrix used is EDNAFULL and for proteins the default scoring matrix is EBLOSUM62. It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference to nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application.

[134] As used herein“comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a nucleic acid which is functionally or structurally defined, may comprise additional DNA regions etc.

[135] In certain jurisdictions, plants according to the invention, which however have been obtained exclusively by essentially biological processes, wherein a process for the production of plants is considered essentially biological if it consists entirely of natural phenomena such as crossing or selection, may be excluded from patentability. Plants according to the invention thus also encompass those plants not exclusively obtained by essentially biological processes.

[136] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Cray, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[137] All patents, patent applications, and publications or public disclosures (including publications on internet) referred to or cited herein are incorporated by reference in their entirety.

[138] The sequence listing contained in the file named”BCS18-2015-W01_ST25“, contains 10 sequences SEQ ID NO: 1 through SEQ ID NO: 10, is filed herewith by electronic submission and is incorporated by reference herein.

[139] The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

[140] Throughout the description, reference is made to the following sequences:

SEQ ID NO: 1 : genomic sequence of the region comprising the Rf3-PPR-122 gene

Nt 1-3000: genomic region upstream of cDNA Rf3-PPR-122

Nt 3001-5798: cDNA Rf3-PPR-122 corresponding to SEQ ID NO: 4

Nt 5799-8810: genomic region downstream of cDNA Rf3-PPR-122

SEQ ID NO: 2: ORF256 nucleotide sequence

SEQ ID NO: 3: predicted target sequence within ORF256

SEQ ID NO: 4: cDNA / mRNA1 Rf3-PPR-122

Nt 1-50: 5’UTR Nt 51-2423: CDS

Nt 2424-2798: 3’UTR

SEQ ID NO: 5: amino acid sequence Rf3-PPR-122

SEQ ID NO: 6: Forward primer (Example 5)

SEQ ID NO: 7: Reverse primer (Example 5)

SEQ ID NO: 8: Probe (Example 5)

SEQ ID NO: 9: Forward primer (Example 6)

SEQ ID NO: 10: Reverse primer (Example 6)

Examples

Example 1 Plant materials and genetic mapping

[141] The Rf3 QTL was mapped on Chromosome 1 B as described extensively in Examples 1 to 3 of WO2017158127 (herein incorporated by reference).

Example 2 - Further fine-mapping of Rf region in IB (F4) and in silico analysis

[142] A set of SNP markers that were used for fine-mapping of the Rf 3 locus were aligned to appropriate reference genome(s) to define a physical region representing the Rf3 QTL region on the reference genome. This QTL region was used to identify potential candidate genes and to develop additional markers for BAC-library screening (see below). Structural annotation of the Rf3 QTL region using ah initio gene annotation programs an in-house annotation pipeline, as well as by alignment of wheat EST sequences, wheat FL-cDNA sequences, wheat gene models and known restorer genes from orthologous species available from public databases. Functional annotation of genes in the QTL region was carried out using Blast2GO and PLAZA software programs as well as consultation of published literature. These candidate genes were then prioritized on the basis of their predicted functionality and their homology to known Rf genes (Chen and Liu, 2014, Annu. Rev. Plant Biol. 65, 579-606; Dahan and Mireau, 2013, RNA Biol. 10, 1469-1476).

[143] Mapping fine-mapping genetic markers to the‘Chinese Spring’ reference genome defined a region of ~1.3Mb on chromosome 1 B that represented the Rf 3 QTL region. In the‘Chinese Spring’ reference genome, this region contained the identified pentacotripeptide (PPR) gene (SEQ ID NOs: 4 and 5). PPR proteins are a large family of proteins that are characterized by possession of the canonical, degenerate 35-amino acid repeat motifs and that have been identified in other crops as being involved in restoration of fertility. This is mainly through mechanisms involving modification of the processing and/or transcription of cytotoxic mitochondrial transcripts (Dahan and Mireau, 2013, supra; Gaborieau et al., 2016, Front. Plant Sci. 7, 1816) (Chen and Liu, 2014, supra; Schmitzlinneweber and Small, 2008, Trends Plant Sci. 13, 663-670). Restoration of fertility-type PPRs (Rf-PPRs) are members of the P-class of PPR proteins that typically bind single-stranded RNA in a sequence-specific fashion (Barkan et al., 2012, supra; Binder et al., 2013, RNA Biol. 10, 1511-1519; Chen and Liu, 2014, supra; Gaborieau et al., 2016, supra; Schmitzlinneweber and Small, 2008, supra). Comparison of the sequences of the PPR gene sequences present in the Rf3 QTL region showed that they clustered with known P-class Rf-PPR orthologues from other crop species (data not shown).

Example 3 - BAC libraries of restorer line

[144] In parallel with the insUico analysis (see above), a BAC library was constructed for the above described wheat restorer line PI 583676, by digesting high-molecular weight‘PI 583676’ gDNA with a restriction enzyme, and transforming the resultant fragments (mean insert size ~80 - 130 Kb), into E. coli. The fine-mapping SNP marker sequences, or markers developed from the Rf3 QTL region on the reference genome, were then used to design PCR primers to screen the pooled BAC clones. Once PCR-positive BAC pools had been identified, BACs from the pool were individualized and screened again with the same marker. Individual, PCR-positive BACs were then subjected to BAC-end sequencing to confirm integrity and the presence of the screening marker sequences. Finally verified positive BACs were deep sequenced using PacBio technology and reads assembled to generate a consensus sequence for the BAC insert. Sequenced, positive BACs were then aligned either by de novo assembly, or by assembly to the reference genome or tiled using the screening markers to generate a new‘PI 583676’ reference sequence for the Rf3 QTL region. The‘PI 583676’ Rf 3 QTL reference sequence was then structurally and functionally annotated to identify the gene content.

[145] The‘PI 583676’ BAC library was screened multiple times using PCR markers developed from fine-mapping markers, reference genomes or isolated BAC sequences. Sequenced BACs were then tiled to create a contiguous sequence. These contigs represent the unique‘PI 583676’ genome sequence for the Rf 3 QTL region and were found to capture the Rf3-PPR- 122 candidate gene initially identified.

[146] As shown in fig.1 A, the gene structure for Rf3-PPR-122 is consisting of a single exon. This relatively simple gene structure appears to be typical for Rf-PPRs.

[147] Comparison of the‘PI 583676’ Rf3-PPR-122 candidate gene to the‘Chinese Spring’ orthologue indicated that the sequence is highly conserved and there are no SNPs present neither in the coding sequence nor in the genomic region +/- 2Kb upstream or downstream of the gene. There is a deletion of two consecutive nucleotides (nt 8247-8248) in the restorer line sequence when compared to the Chinese Spring reference sequence.

[148] SEQ ID NO: 1 represents the genomic DNA sequence comprising the Rf3-PPR-122 gene.

Example 4 - Annotation of the PPR122 amino acid sequence

[149] Known Rf-PPRs are members of the P-class of PPR proteins, and contain up to ~30 PPR motifs per protein, with each motif typically comprising 35 amino acids (Gaborieau et al., 2016, supra). Structurally PPR proteins consist of 2 a-helices that form a hairpin and a super-groove, and it is this super groove that interacts with an RNA molecule. The amino acid composition of the individual PPR motifs determines the RNA nucleotide that is recognized, and the number of PPR motifs determines the length of the RNA sequence recognized on the target transcript. Here the Rf3-PPR-122 candidate was annotated to identify PPR motifs and other sequence features and the results summarized in fig.1 B and C.

[150] Rf3-PPR-122 is 790 amino acids long and contains 18 consecutive complete PPR motifs comprising 35 amino acid residues, whereby motifs 2 and 10 contain 36 amino acid residues (SEQ ID NO: 5). This is similar to the Rf-1 A gene cloned from rice, which is 791 amino acids long and contains 16 PPR repeats (Akagi et al., 2004, Theor. Appl. Genet. 108, 1449— 1457; Komori et al., 2004, Plant J. 37, 315-325). Also, Rf3-PPR-122 has a predicted transit peptide that targets the protein to the mitochondria (as predicted by PredSL (Evangelia et al.(2006) Geno. Prot. Biolnfo Vol 4, No.1 , 48-55), with a (strong) mTP (mitochondrial targeting peptide) score of 0,999756 in PredSL).

[151] Each PPR motif consists of 2 antiparallel helices that form a hairpin structure that interacts with a single stranded RNA molecule. Studies have demonstrated the existence of a recognition code linking the identity of specific amino acids within the repeats and the target RNA sequence of the PPR protein studied (Barkan et al., 2012, supra; Yagi et al., 2013, PLoS ONE 8, e57286). In particular the identity of the 5th and the 35th amino acids of each motif have been shown to be important. On the basis of the identity of the amino acids at positions 5 and 35 in the Rf3-PPR-122 motif the target transcript sequence for Rf3-PPR-122 can be predicted. Following the PPR code (as described in Melonek et al., 2016, supra, supplemented with Miranda et al. 2017, RNA 23(4):586-599 and Shen et al. 2016, Nature Communications DOI: 10.1038/ncomms11285), the predicted RNA target sequence is thus 5’- NNAUUUNUNNNCNUACGU -3’ (SEQ ID NO:3, see also Table 3 below). This predicted target sequence can be found in orf256 (position 137- 154 of SEQ ID NO: 2).

Table 1 - PPR motifs and base recognition, see also Figure 1.

Example 5 Expression analysis mRNA

[152] Total RNA was isolated from ~70 - 100 mg fw tissue using the Sigma Spectrum Plant Total RNA Kit (Sigma-Aldrich), and any gDNA contamination removed using the Qiagen RNase-Fee DNase Set (Cat. No. 79254). Nuleic acid concentration and integrity were determined with an Agilent Expert BioAnalyser. Tissue was sampled at five developmental stages (young leaf, spike 2.5-3.5, spike 3.5-4.5, spike 4.5-5.5 cm and anthers), using individuals from an F4 population of progeny derived from‘PI 583676’. These progenies were genotyped using fine-mapping markers, phenotyped for fertility traits, and classified as either non-restoring or homozygous for Rf3. Three individual biological replicates were prepared per tissue type per genotype. qRT-PCR analyses

[153] mRNA from each of the tissue/R/3 genotypes was converted into cDNA using the EcoMix dry kit from Clontech. Gene- specific probes were designed to quantify gene expression levels using the TaqMan assay as summarized in Table 2. Probe specificity and efficiency were tested and optimised and expression analyses carried out on cDNA samples generated as above.

Table 2 - TaqMan primer and probe sequences used for gene expression analyses.

[154] Gene expression was examined in individual plants selected from F4 fine-mapping progeny segregating for the Rf3 locus, in four different tissues. Young leaf, developing spike 2.5 - 3.5 cm, developing spike 3.5-4.5 cm, developing spike 4.5 - 5.5 cm and anthers. Since it is expected that the cytoplasmic male sterile (CMS) phenotype is due to the production of non- viable pollen, Rf genes must at least be expressed during the period of pollen development and meiosis. It is also expected that Rf gene expression will be highest in the early stages of pollen development. [155] As shown in fig. 2, it is clear that mean expression of the Rf3-PPR-122 gene, is exclusively associated with the presence of the Rf 3 locus and is also highest at the 3.5 - 4.5 cm stage of spike development.

Example 6 Confirmation of gene expression and link to fertility restoration in an independent population

[156] Additionally, the link between Rf3-PPR-122 gene expression with fertility was tested in near-isogenic lines developed from a 16-way MAGIC population. This population was developed by intercrossing 16 founder lines, among which there was one cytoplasmic male sterile line derived from T. timopheevii and two potential restorer lines, called R1 and R2. The 16-way MAGIC population was intercrossed for 5 generations and subsequently fixed through single-seed descent to F5. Throughout the line-fixation process, lines were genotyped and phenotyped for fertility. This allowed for the selection of families segregating for restoration of fertility as well as for fine-mapping of the Rf loci. At F5, individuals displaying heterozygosity at the previously mapped Rf3 locus were identified and used to create multiple near-isogenic line (NIL) pairs either with or without the Rf3 locus in their progeny. Six such NIL pairs were selected, grown, and phenotyped. RNAseq and qPCR experiments were performed on developmental spikes at 3 stages from six NIL pairs and also on the respective parental lines. Bioinformatics analysis of the RNAseq data allowed the identification of differentially expressed transcripts between restorer and non-restorer genotypes. The identified candidate transcripts mapped into the QTL regions, were demonstrated to be derived from the correct (restoring) founder line.

Table 3 - Primer sequences used for the gene expression analysis.

[157] As shown in fig. 3 and using similar methods as in example 5, it is clear that the mean expression of the Rf3-PPR- 122 gene is exclusively associated with the presence of the Rf3 locus in developing spikes of 3.5 cm length.

Example 7 - Gene validation

By mutagenesis

[158] A mutagenized population of the restorer line is constructed by EMS mutagenesis. Based on sequencing of the region around the Rf3-PPR-122 gene, mutant plants with an inactivating mutation in the Rf3-PPR-122 gene are identified. The homozygous mutant plants and their wildtype segregants are screened for anther development, pollen development and fertility restoration capacity. The plants that have a mutated Rf3-PPR-122 gene no longer have restoring ability, confirming that the identified R/3-PPR-122 gene is a functional Rf gene. By overexpression

[159] The coding sequence of the Rf3-PPR- 122 gene is cloned under the control of a constitutive UBIQUITIN promoter (e.g. pllbiZm from maize), or under the control of a constitutive cauliflower mosaic virus promoter (p35S), or under the control of a vernalisation-related barley promoter (pvrnl), or a tapetum-specific promoter (e.g., Yokoi et al., supra (or under control of its native promoter), in a T-DNA expression vector comprising a selectable marker, such as the bar gene. The resulting vector is transformed into a wheat line having no restoration capacity such as the transformable variety Fielder (or Chinese spring) according to methods well known in the art for wheat transformation (see e.g.lshida et al Methods Mol Biol. 2015;1223:189- 98). The copy number of the transgene in the transgenic plant is determined by real time PCR on the selectable marker gene. The transformed plants comprising the Rf3-PPR-122 gene cassette, preferably in single copy, are transferred to the greenhouse. Expression of the transgene in leaf tissue and in young developing spikes is tested by qRT-PCR. Transgenic TO plants expressing the Rf3-PPR-122 gene are crossed as male parents to a G-type cytoplasmic male sterile (“CMS”) wheat line. F1 progeny of the crosses contain the G-type cytoplasm and show partial or complete restoration of male fertility due to the presence of the Rf3-PPR-122 gene.

[160] The level of restoration in F1 progeny is tested using four different assays. In the first assay the mitochondrial ORF256 protein is quantified on Western blot using polyclonal antibodies raised against synthetic ORF256 protein. Expression of a functional Rf3-PPR-122 gene leads to reduced accumulation of the ORF256 protein. In the second assay pollen accumulation and pollen viability is quantified using the AmphaZ30 device. Expression of a functional Rf3-PPR-122 gene leads to higher numbers of viable pollen. In the third assay the integrity of anther tissues is inspected microscopically. Expression of a functional Rf3-PPR-122 gene leads to better preservation of functional tapetum layer. In the fourth assay seed set per earfrom self-pollination is quantified. Expression of a functional candidate Rf3-PPR-122 gene leads to higher number of grains per ear. In all tests the F1 progeny from crosses of non-transgenic Fielder plants to the same G-type cytoplasmic male sterile (“CMS”) wheat line serves as a control.

By targeted knock-out

[161] Guide RNAs for CRISPR-mediated gene editing targeting the mRNA coding sequence, preferably the protein coding sequence of the Rf3-PPR-122 gene, or the immediately upstream promoter sequence of the Rf3-PPR-122 gene are designed by using e.g. the CAS-finder tool (e.g., https://omictools.com/casfinder-tool). Preferably four unique or near-unique guide RNAs are designed per target gene. The guide RNAs are tested for targeting efficiency by PEG-mediated transient co-delivery of the gRNA expression vector with an expression vector for the respective nuclease, e.g. Cas9 or Cpf1 , under control of appropriate promoters, to protoplasts of a wheat restorer line containing the Rf3-PPR- 122 gene of interest, preferably the line designated as USDA Accession number PI 583676. Genomic DNA is extracted from the protoplasts after delivery of the guide RNA and nuclease vectors. After PCR amplification, integrity of the targeted Rf3-PPR-122 gene sequence is assessed by sequencing.

[162] The one or two most efficient guide RNAs are used for stable gene editing in same wheat restorer line also containing the G-type CMS cytoplasm. For this purpose, the selected guide RNA expression vector, together with a nuclease expression module and a selectable marker gene, are introduced into embryos isolated from the before mentioned wheat restorer line using e.g. particle gun bombardment. Transgenic plants showing resistance to the selection agent are regenerated using methods known to those skilled in the art. Transgenic TO plants containing gene targeting events, preferably small deletions likely resulting in a non-functional target Rf3-PPR-122 gene are identified by PCR amplification and sequencing.

[163] Transgenic TO plants containing the G-type CMS cytoplasm and likely to contain a functional knock-out of the Rf3- PPR-122 gene, preferably in homozygous state, but alternatively in heterozygous state, are crossed as female parents to a spring wheat line with normal cytoplasm and without PPR-Rf genes. The F1 progeny of the crosses contains the G-type“CMS” cytoplasm and 50% (in case of heterozygous TO) or 100% (in case of homozygous TO) of the F1 progeny will lack a functional version of the target Rf PPR gene. The F1 plants lacking a functional target Rf3-PPR-122 gene are identified using genomic PCR assays. The F1 plants show partial or complete loss of male fertility due to the knock-out of the Rf3-PPR-122 gene.

[164] The level of male fertility in the F1 progeny lacking a functional version of the Rf3-PPR-122 gene is tested using four different assays. In the first assay the mitochondrial ORF256 protein is quantified on Western blot using polyclonal antibodies raised against synthetic ORF256 protein. The knock-out of a functional Rf3-PPR-122 gene leads to increased accumulation of the ORF256 protein. In the second assay pollen accumulation and pollen viability is quantified using the AmphaZ30 device. The knock-out of a functional Rf3-PPR-122 gene leads to lower numbers of viable pollen. In the third assay the integrity of anther tissues is inspected microscopically. The knock-out of a functional Rf3-PPR-122 gene leads to early deterioration of the tapetum layer. In the fourth assay seed set per ear from self-pollination is quantified. The knock-out of a functional Rf3-PPR- 122 gene leads to reduced number of grains per ear. In all tests the F1 progeny from crosses of non-edited Rf plants to the same spring wheat line serve as a control.

[165] Alternatively, guide RNAs for CRISPR-mediated gene editing targeting the promoter region comprised within the nucleotide sequence of SEQ ID NO: 1 from nucleotide position 1 to 3000 are designed and tested in wheat protoplasts of a wheat line of interest in the manner described above. The one or two most efficient guide RNAs are used for stable gene editing in same wheat line as described above, but additionally repair DNA comprising the substation, insertion or deletion of interest (one or more nucleotides) between flanking sequences homologous to the target DNA are also introduced. Plants comprising the edited upstream region are identified by PCR amplification and sequencing and tested for the level of male fertility as described above. Background References

[166] Binder, S., Stoll, K., and Stoll, B. (2013). P-class pentatricopeptide repeat proteins are required for efficient 5' end formation of plant mitochondrial transcripts. RNA Biol. 10, 1511-1519.

[167] Chen, J., Zheng, Y., Qin, L, Wang, Y., Chen, L, He, Y., Fei, Z., and Lu, G. (2016). Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis. BMC Plant Biol. 16, 80.

[168] Ding, J., Lu, Q., Ouyang, Y., Mao, H., Zhang, P., Yao, J., Xu, C., Li, X., Xiao, J., and Zhang, Q. (2012). A long noncoding RNA regulates photoperiod-sensitive male sterility, an essential component of hybrid rice. Proc. Natl. Acad. Sci. 109, 2654-2659.

[169] Fang, Y.-N., Zheng, B.-B., Wang, L, Yang, W„ Wu, X.-M., Xu, Q., and Guo, W.-W. (2016). High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis). BMC Genomics 17, 591.

[170] Wei, M., Wei, H., Wu, M., Song, M., Zhang, J., Yu, J., Fan, S., and Yu, S. (2013). Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton. BMC Plant Biol. 13, 66.

[171] Wei, X., Zhang, X., Yao, Q., Yuan, Y., Li, X., Wei, F„ Zhao, Y., Zhang, Q., Wang, Z„ Jiang, W„ et al. (2015). The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high- throughput sequencing of miRNAs, degradomes, and transcriptomes. Front. Plant Sci. 6.

[172] Xia, R., Meyers, B.C., Liu, Z., Beers, E.P., Ye, S., and Liu, Z. (2013). MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots. Plant Cell Online 25, 1555-1572.

[173] Yagi, Y., Hayashi, S., Kobayashi, K., Hirayama, T., and Nakamura, T. (2013). Elucidation of the RNA Recognition Code for Pentatricopeptide Repeat Proteins Involved in Organelle RNA Editing in Plants. PLoS ONE 8, e57286.

Claims

1. A nucleic acid molecule encoding a functional restorer gene allele for wheat G-type cytoplasmic male sterility, wherein said functional restorer gene allele is a functional allele of a PPR gene comprised within the nucleotide sequence of SEQ ID NO: 1.

2. The nucleic acid molecule of claim 1 , wherein said functional restorer gene allele comprises a nucleotide sequence selected from:

a. a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 4 from the nucleotide at position 51 to the nucleotide at position 2423; preferably over the entire length of SEQ ID NO: 4 from the nucleotide at position 51 to the nucleotide at position 2423.

b. a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 4; preferably over the entire length of SEQ ID NO: 4; or

c. a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5, preferably over the entire length of SEQ ID NO: 5.

3. The nucleic acid molecule of any one of claims 1-2, wherein said functional restorer gene allele encodes a PPR protein capable of binding to the mRNA of ORF256, preferably to nt 137-154 of SEQ ID NO: 2.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein said functional restorer gene allele is obtainable from USDA accession number PI 583676.

5. The nucleic acid molecule of any one of claims 1 to 4, wherein said functional restorer gene allele comprises the nucleotide sequence of SEQ ID NO: 4 or encodes the polypeptide of SEQ ID NO: 5.

6. The nucleic acid molecule of any one of claims 1 to 5, which is an isolated or modified nucleic acid molecule.

7. The nucleic acid molecule of any one of claims 1 to 5, which is an exogenous nucleic acid molecule.

8. The nucleic acid molecule of any one of claims 1 to 5, which is an chimeric or recombinant nucleic acid molecule.

9. A polypeptide encoded by the nucleic acid molecule of any one of claims 1 to 5 or comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5.

10. A chimeric gene comprising the following operably linked elements

a. a plant-expressible promoter;

b. a nucleic acid comprising the nucleic acid molecule of any one of claims 1-5 or encoding the polypeptide of claim 6; and optionally

c. a transcription termination and polyadenylation region functional in plant cells,

wherein at least one of said operably linked elements is heterologous with respect to at least one other element.

11. The chimeric gene of claim 10, wherein said promoter is capable of directing expression of the operably linked nucleic acid at least during (early) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspores.

12. A cereal plant cell or cereal plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising and expressing the nucleic acid molecule of any one of claims 1 to 8, the polypeptide of claim 9, or the chimeric gene of claim 10 or 11 , wherein said polypeptide, said nucleic acid, or said chimeric gene in each case is heterologous with respect to said plant cell or plant or seed.

13. A method for producing a cereal plant cell or plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising a functional restorer gene for wheat G-type cytoplasmic male sterility, or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the step of providing said plant cell or plant with the nucleic acid molecule of any one of claims 1 to 5 or the chimeric gene of claim 10 or 11 , wherein said step of providing comprises providing by transformation, crossing, backcrossing, genome editing or mutagenesis.

14. A method for producing a cereal plant cell or plant or seed thereof, such as a wheat plant cell or plant or seed thereof, comprising a functional restorer gene for wheat G-type cytoplasmic male sterility, or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the steps of increasing the expression of a polypeptide according to claim 9 in said plant cell or plant or seed, and preferably identifying the increase of transcription by measuring increased polypeptide.

15. A method for converting a non-restoring cereal plant, such as a wheat plant, into a restoring plant for wheat G-type cytoplasmic male sterility (“CMS”), or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising the step of modifying the genome of said plant to comprise and express the nucleic acid molecule of any one of claims 1 to 5 or the chimeric gene of claim 10 or 11 , wherein said step of modifying comprises modifying by transformation, crossing, backcrossing, genome editing or mutagenesis.

16. A method for converting a non-restoring cereal plant, such as a wheat plant, into a restoring plant for wheat G-type cytoplasmic male sterility (“CMS”), or for increasing restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”) in a cereal plant, such as a wheat plant, comprising modifying the genome of said plant to increase the expression of a polypeptide according to claim 9 in said plant.

17. A cereal plant cell or cereal plant or seed thereof, such as a wheat plant cell or plant or seed thereof, obtained according to the method of any one of claims 13 to 16, preferably wherein said plant has an increased restoration capacity for wheat G-type cytoplasmic male sterility (“CMS”).

18. The plant cell, plant or seed of claim 12 or 17 wherein the polypeptide of claim 9 is expressed at least during (early) pollen development and meiosis, such as in anther or, more specifically, tapetum development, or developing microspore.

19. The plant cell, plant or seed of claim 12, 17 or 18, which is a hybrid plant cell, plant or seed.

20. A method for selecting a cereal plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility or for producing a cereal plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility, comprising the steps of: (a) identifying the expression or transcription of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423, preferably by measuring level of RNA transcribed from the nucleotide sequence of SEQ ID NO: 4 from nucleotide position 51 to nucleotide position 2423; and optionally

(b) selecting the plant comprising and expressing said at least one marker allele, wherein said plant comprises said functional restorer gene for wheat G-type cytoplasmic male sterility located on chromosome 1 B.

21. A method for restoring fertility in a progeny of a G-type cytoplasmic male sterile cereal plant or for producing a fertile progeny plant from a G-type cytoplasmic male sterile cereal parent plant, comprising the steps of:

(a) providing a population of progeny plants obtained from crossing a female cereal parent plant with a male cereal parent plant, wherein the female parent plant is a G-type cytoplasmic male sterile cereal plant, and wherein the male parent plant comprises and expresses a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4;

(b) identifying in said population a fertile progeny plant comprising and expressing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, preferably by measuring RNA level transcribed from SEQ ID NO: 4; and optionally

(c) selecting said fertile progeny plant; and optionally

(d) propagating the fertile progeny plant.

22. A method for identifying and/or selecting a cereal (e.g. wheat) plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising the steps of

a. identifying or detecting in said plant the expression or transcription, preferably by measuring RNA level transcribed from SEQ ID NO: 4 or level of protein having amino acid sequence of SEQ ID NO: 5, of a nucleic acid of any one of claims 1 to 5 or of the polypeptide according to claim 9, or the chimeric gene of claim 10 or 11.

b. and optionally selecting said plant comprising and expressing or transcribing said nucleic acid or polypeptide or chimeric gene.

23. The method of claim 22, wherein said polypeptide is expressed at least during (early) pollen development and meiosis, such as in anther or, more specifically, tapetum, or developing microspore.

24. A method for producing a cereal plant, such as a wheat plant, comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility, comprising the steps of

a. crossing a first cereal plant, such as a wheat plant of any one of claims 12, 17 or 18 with a second cereal plant;

b. identifying, and optionally selecting, a progeny plant comprising and expressing a functional restorer gene allele for wheat G-type cytoplasmic male sterility comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

25. A method for producing hybrid seed, comprising the steps of:

a. providing a male cereal parent plant, such as a wheat plant according to claim 12, 17 or 18, said plant comprising and expressing said functional restorer gene allele for wheat G-type cytoplasmic male sterility, wherein said functional restorer gene allele is preferably present in homozygous form.

b. providing a female cereal parent plant that is a G-type cytoplasmic male sterile cereal plant.

c. crossing said female cereal parent plant with a said male cereal parent plant; and optionally

d. harvesting seeds.

26. Use of the nucleic acid of any one of claims 1 to 5, to identify one or more further functional restorer gene alleles for wheat G-type cytoplasmic male sterility.

27. Use of the nucleic acid of any one of claims 1 to 5, of the polypeptide according to claim 9 or the chimeric gene of claim 10 or 11 for the identification of a plant comprising a functional restorer gene allele for wheat G-type cytoplasmic male sterility.

28. Use of a plant of any one of claims 12, 17 or 18 or a plant obtained by the method of any one of claims 13 to 16, said plant comprising said functional restorer gene for wheat G-type cytoplasmic male sterility, for restoring fertility in a progeny of a G-type cytoplasmic male sterile cereal plant, such as a wheat plant.

29. Use of a plant of any one of claims 12, 17 or 18 or a plant obtained by the method of any one of claims 13 to 16, said plant comprising said functional restorer gene for wheat G-type cytoplasmic male sterility, for producing hybrid seed or a population of hybrid cereal plants, such as wheat seed or plants.

30. A method for increasing, in a cereal plant, the expression of a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5 by modification of the genome, preferably directed modification or engineering of the genome.

31. The method according to claim 30, wherein the expression is increased at least 2 fold.

32. The method according to claim 30, wherein the expression is increased at least 10 fold.

33. A plant cell comprising a chimeric gene encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5.

34. The plant cell of claim 33, which is a wheat plant cell.