Patent Application
20200347104
The invention is in the field of plant genetics and plant breeding. The invention more specifically relates to wheat plants carrying restorer of fertility genes specific to T. timopheevii CMS cytoplasm.
Hybrid production is based on crossing two parental lines to increase heterosis and de facto, increase genetic variability to create new varieties or genotypes with higher yield and better adapted to environmental stresses. Even in a predominantly autogamous species like wheat, research studies have shown that hybrid lines exhibit improved quality and greater tolerance to environmental and biotic stresses.
In order to promote commercially viable rates of hybrid production, self-fertilization must be avoided, i.e. fertilization of the female organ by the pollen of the same plant. It is desired that the female organ of the female parent is exclusively fertilized with the pollen of the male parent. In order to obtain a reliable and efficient system for producing seeds needed for hybrid production, one generally needs three essential elements: a means to induce male sterility, a means to propagate the sterility, and a means to restore fertility. For example a fully genetically based system is composed of a male-sterile line (female parent), a fertile maintainer line (male parent allowing propagation of the male-sterile line), and a fertility restorer line (male parent for hybrid production).
Male sterility can be achieved by three different ways. Manual emasculation is the simplest one and is still used in some species where male and female flowers are separated, e.g. corn. However, it is impractical in species like wheat where flowers contain both female and male organs. Male sterility can also be induced by chemical hybridization agents (CHAs) with gametocidic effects. Currently, only a few commercial hybrid wheat cultivars are based on this technology as it can bear substantial financial risks.
Finally, male sterility can also be induced by genetic means. There are many examples of hybrid systems in corn or sorghum based on male sterility induced by genetic means showing the preponderance of this technology compared to the two mentioned previously. However, in other species which are predominantly self-pollinated like wheat, hybrid production is still a challenge (Longin et al, 2012).
The first case of male sterility in wheat was observed in 1951 (Kihara, 1951), where it was observed that sterility was caused by incompatibility between the cytoplasm of Aegilops caudata L. and the nucleus of T. aestivum var. erythrospermum. Subsequently research on T. timopheevii cytoplasm showed that this cytoplasm is able to induce sterility in bread wheat (T. aestivum) (Wilson and Ross, 1961, Crop Sci, 1: 191-193). Orf256 was previously identified as a gene specific to the T. timopheevii mitochondrial genome (Rathburn and Hedgcoth, 1991; Song and Hedgcoth, 1994), however, it remains to be shown that orf256 is the genetic determinant of T. timopheevii CMS. It was expected that such a cytoplasm could be used in a hybrid production system. However, major limitations arose from the difficulty in finding a completely dominant and stable fertility restorer gene with no negative side effects (notably on yield).
Fertility restoration of male sterile plants harboring T. timopheevii CMS cytoplasm (T-CMS cytoplasm) has been reported and eight major restorer loci (designated as Rf1 to Rf8) have been identified and located approximate within the wheat genome. One of the most effective restorer loci is Rf3 (Ma and Sorrells, 1995; Kojima et al, 1997; Ahmed et al 2001; Geyer et al 2016). Two SNP markers allowed the location of the Rf3 locus within a 2 cM fragment on chromosome 1B (Geyer et al, 2016). The author notes that these markers are not diagnostic markers.
While it is understood that restoration to normal pollen fertility could require two or more Rf loci, it is also well known that modifier loci exist that have either minor effects with low penetrance (Zhou et al 2005, Stojalowski et al 2013) or inhibitory effects on fertility, depending on environmental conditions (Wilson, 1984). It is not yet understood which combination of genes or loci is needed to complete a full restoration of T-CMS in different genetic backgrounds and environmental conditions.
In this context, the development of technologies that enable a full restoration of pollen fertility is of major importance in wheat. It is therefore the object of this invention to propose suitable fertility restorer genes in wheat for the development of a hybrid production system useful for the seed industry.
A first object of the present disclosure relates to an isolated Rf1 nucleic acid encoding a Rf1 protein restorer of fertility of T. timopheevii CMS cytoplasm, wherein the corresponding amino acid sequence has at least 95% identity, preferably at least 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:361. An example of Rf1 nucleic acid comprises SEQ ID NO:3119.
The disclosure also relates to a transgenic wheat plant comprising such Rf1 nucleic acid, and, optionally, one or more nucleic acids comprising Rf3, Rf4 and/or Rf7 restorer allele(s), as transgenic element(s).
Another aspect relates to a genetically engineered wheat plant comprising such Rf1 nucleic acid, and, optionally, one or more nucleic acids comprising Rf3, Rf4 and/or Rf7 restorer allele(s), as genetically engineered element(s).
In specific embodiments, said transgenic element(s) or genetically engineered element(s) express polypeptides which restore or improve male fertility to the plant as compared to the parent plant without such transgenic element(s) or genetically engineered element(s).
Yet another aspect relates to a wheat plant restorer of fertility of T. timopheevii CMS cytoplasm comprising such Rf1 restorer allele, and at least two fertility restorer alleles within the restorer loci chosen amongst Rf3, Rf4 and Rf7, wherein,
The disclosure also provides methods for producing a transgenic wheat plant as described above, wherein the method comprises the steps of transforming a parent wheat plant with one or more Rf1 nucleic acids encoding protein restorer of T. timopheevii CMS cytoplasm, selecting a plant comprising said one or more nucleic acid(s) as transgene(s), regenerating and growing said wheat transgenic plant.
Also part of the present disclosure is a method for producing a genetically modified wheat plant as described above, wherein the method comprises the steps of genetically modifying a parent wheat plant to obtain in their genome one or more nucleotide sequence encoding Rf1 protein restorer of T. timopheevii CMS cytoplasm, preferably by genome-editing, selecting a plant comprising said one or more nucleotide sequences as genetically engineered elements, regenerating and growing said wheat genetically engineered plant.
The disclosure further relates to a method for producing a wheat plant by crossing, said method includes the following:
Preferably in such methods, the fertility score of the obtained wheat plant has a fertility score higher than the parent wheat plant.
The disclosure also relates to a method for producing a transgenic or genetically engineered wheat plant, wherein the fertility level of said plant is modified, comprising the step of knocking-down Rf1 restorer allele expression, wherein said Rf1 restorer allele comprises a Rf1 nucleic acid.
The disclosure also relates to the method for producing a wheat hybrid plant comprising the steps of:
The wheat hybrid plant as obtained by the above methods are also part of the present disclosure.
The present disclosure also relates to a method of identifying a wheat plant as described above, wherein said wheat plant is identified by detecting the presence of at least one restorer allele Rf1 and, optionally, one or more further restorer alleles selected from the group consisting of Rf3, Rf4 and Rf7.
Accordingly, nucleic acid probes or primers for the specific detection of the restorer allele Rf1 in a wheat plant, and, optionally, one or more of the Rf3, Rf4, and Rf7 restorer alleles, are also disclosed herein.
Another aspect of the disclosure relates to a recombinant nucleic acid comprising a Rf1 nucleic acid encoding a Rf1 protein restorer of T. timopheevii CMS cytoplasm, operably linked to regulatory elements and the vectors for use in transformation of a wheat plant, comprising such recombinant nucleic acids.
Nucleic Acids of the Present Disclosure
An aspect of the present disclosure relates to the cloning and characterization of genes encoding restorer of fertility proteins that act on T. timopheevi CMS cytoplasm (hereafter referred as Rf genes or nucleic acids) in wheat plants and the use of the corresponding Rf nucleic acids for producing transgenic wheat plants, for modifying wheat plants by genome editing, and/or for detecting such Rf genes in wheat plants.
Whenever reference to a “plant” or “plants” 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, male fertility associated with the claimed Rf nucleic acids), such as seed obtained by selfing or crossing, e.g. hybrid seeds (obtained by crossing two inbred parent plants), hybrid plants and plant parts derived therefrom are encompassed herein, unless otherwise indicated.
As used herein, the term “wheat plant” refers to species of the genus Triticum as for example, T. aestivum, T. aethiopicum, T. araraticum, T. boeoticum, T. carthlicum, T. cornpactum, 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 plant also refers to species of the genera Aegilops and Triticale.
As used herein, the term “restorer of fertility of T. timopheevi CMS cytoplasm” refers to a protein whose expression in a wheat plant comprising T. timopheevi CMS cytoplasm contributes to the restoration of the production of pollen in the Triticum timopheevii CMS system.
As used herein, the term “allele(s)” means any of one or more alternative forms of a gene at a particular locus. In a diploid, alleles of a given gene are located at a specific location or locus on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. The same definition is used for plants bearing a higher level of ploidy like in Triticum gender wherein, for example, T. aestivum is an hexaploid plant.
As used herein, the term “restorer allele of T. timopheevi CMS cytoplasm” refers to an allele which contributes to the restoration of the production of pollen in the CMS Triticum timopheevii system.
The restoration of pollen fertility may be partial or complete. The pollen fertility can be evaluated by the pollen fertility tests as described in the Examples below. In particular, the fertility score of F1 wheat plants having CMS-T timopheevii cytoplasm (from test restorer line with CMS hybrids) may be calculated by dividing the total number of seeds threshed from a spike by the number of counted spikelets and may be compared with the fertility scores of a panel of control fertile plants, for example elite inbred lines bearing a normal wheat cytoplasm, grown in the same area and under the same agro-environmental conditions. It is preferred that such panels of lines comprise a set of at least 5 elite inbred lines wherein these lines are representative of the area where the fertility test is achieved. Besides, it is preferred that at least 10 spikes from different individual F1 plants be assessed for a given experiment.
If the fertility score is not null, then the plant has acquired partial or full restoration of fertility. For each fertility score, a statistical test is calculated to obtain a p-value. Examples of statistical tests are the Anova or mean comparison tests. A p-value below a 5% threshold will indicate that the two distributions are statistically different. Therefore, a significant decrease of the fertility score of the tested wheat plant as compared to the fertility score of the fully fertile control plant is indicative that the F1 plant has not acquired full restoration of fertility (i.e. partial restoration). A similar or higher fertility score is indicative that the F1 plant has acquired full restoration of fertility. In a preferred embodiment, the wheat plant, such as transgenic or genetically engineered wheat plant, according to the present disclosure, has acquired full restoration of fertility.
The loci of the restorer alleles of T. timopheevi CMS cytoplasm within Rf1, Rf3, Rf4 and Rf7 have been mapped in the present disclosure. The corresponding restorer alleles are designated Rf1, Rf3, Rf4 and Rf7 restorer alleles and have been described in the art. In particular, a wheat plant source of the Rf3 restorer allele includes the commercial following lines: Allezy, Altigo, Altamira, see table 15. A wheat plant source of the Rf4 restorer allele includes the following lines: R113 or L13.
In specific embodiments, representative alleles of Rf1, Rf3, Rf4 and Rf7 restorer alleles are provided by the seed sample chosen amongst: NCIMB 42811, NCIMB 42812, NCIMB 42813, NCIMB 42814, NCIMB 42815, NCIMB 42816, and NCIMB 42817.
As used herein, the term “centimorgan” (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.
The present disclosure provides nucleic acids and their recombinant forms comprising the coding sequence of either Rf1, Rf3, Rf4, Rf7 or Rf-rye restorer of fertility proteins active in T. timopheevii CMS cytoplasm.
As used herein, a “recombinant nucleic acid” is a nucleic acid molecule, preferably a DNA molecule, comprising a combination of nucleic acid molecules that would not naturally occur together and is the result of human intervention, e.g., a DNA molecule that is comprised of a combination of at least two DNA molecules heterologous to each other, and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide sequence that deviates from the polynucleotide sequence that would normally exist in nature.
Such nucleic acids encoding candidate restorer of fertility proteins of T. timopheevii CMS cytoplasm have been isolated as described in the Examples below. Accordingly, a first aspect of the disclosure are nucleic acids encoding a protein restorer of fertility of T. timopheevii having an amino acid sequence at least 95% identical, typically at least 96% identical, to an amino acid sequence chosen amongst any one of SEQ ID NO:1 to SEQ ID NO:1554.
Percentage of sequence identity as used herein is determined by calculating the number of matched positions in aligned amino acid sequences, dividing the number of matched positions by the total number of aligned amino acids, and multiplying by 100. A matched position refers to a position in which identical amino acids occur at the same position in aligned amino acid sequences. For example, amino acid sequences may be aligned using the CD-hit (settings -c 0.96 -n 5 -G 0 -d 0 -AS 60 -A 105 -g 1, see http://weizhongli-lab.org/cd-hit/).
The above candidate nucleic acids encoding any one of polypeptides SEQ ID NO: 1 to 1554, can further be assessed for their capacity to restore fertility of sterile wheat plant as described below.
It is therefore disclosed herein a method for assessing the capacity of a nucleic acid to restore fertility, wherein the method comprises the steps of:
In a specific embodiment, the parent wheat sterile plant is the Fielder line bearing the T. timopheevii CMS cytoplasm.
In another specific embodiment of the above method, the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye candidate nucleic acid sequence is selected from those encoding an amino acid sequence having at least 95% identity, or at least 96% identity, for example 100% identity, to any one of SEQ ID NO1 to SEQ ID NO1554.
Typically, the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye candidate nucleic acids are selected among the following nucleic acids of SEQ ID NO:1555 to SEQ ID NO:3107 and 3133.
In a further specific embodiment, where appropriate, the nucleic acid sequence may be optimized for increased expression in the transformed plant. There are a number of optimizations that can be performed at the DNA level, without changing the protein sequence, by conservative codon exchanges which replace one codon by another codon encoding the same amino acid. Besides, the nucleic acid sequence can be modified for cloning purpose. Like for optimization, such modification is achieved without changing the protein sequence.
Rf1 nucleic acids
In specific embodiment, the nucleic acid of the present disclosure is a Rf1 nucleic acid.
As used herein, the term “Rf1 nucleic acid” refers to a nucleic acid comprising a gene encoding a Rf1 protein restorer of fertility of T. timopheevii CMS cytoplasm, wherein the corresponding amino acid sequence has at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs1-2, SEQ ID NOs288-290, SEQ ID NOs293-296, SEQ ID NOs343-346, SEQ ID NOs349-354, SEQ ID NOs359, 361 and 362, SEQ ID NOs 396 and 397, SEQ ID NOs428-430, SEQ ID NO517 and 519, SEQ ID NOs752-754, SEQ ID NOs1092, 1093 and 1095, typically, SEQ ID NOs359, 361 and 362 and SEQ ID NO428-430. In a particularly preferred embodiment, the Rf1 nucleic acid encodes an amino acid sequence having at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:361. Examples of corresponding specific Rf1 nucleic acids are referred to in Table 7.
In particular, and as shown in Example 6, the inventors have identified that RFL79 sequence of SEQ ID NO:361 (as depicted in Table 7) can restore male fertility of CMS-Fielder plants. Accordingly, in a preferred embodiment, examples of Rf1 nucleic acids comprises the disclosed Rf1 nucleic acid sequences of SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1915, SEQ ID NO:1916 or SEQ ID NO:3119, preferably a Rf1 nucleic acid comprises SEQ ID NO:3119.
Rf3 Nucleic Acids
In specific embodiment, the nucleic acid of the present disclosure is a Rf3 nucleic acid.
As used herein, the term “Rf3 nucleic acid” refers to a nucleic acid comprising a gene encoding a Rf3 protein restorer of fertility of T. timopheevii CMS cytoplasm, wherein the corresponding amino acid sequence has at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:124 and 125, SEQ ID NO:147, SEQ ID NO:150, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NOs:315-321, SEQ ID NOs:379-381, SEQ ID NOs:553 and 554, SEQ ID NOs:557 and 558, SEQ ID NOs:676 and 677, SEQ ID NOs:684 and 685, SEQ ID NOs:696 and 697, SEQ ID NOs:938 and 939 and SEQ ID NOs:1038 and 1039, typically, SEQ ID NOs:315-321, SEQ ID NOs:379-381, SEQ ID NOs:147 and 150, SEQ ID NOs:156 and 158, SEQ ID NOs297 and 299. Preferred Rf3 nucleic acids encode a Rf3 protein restorer of fertility of T. timopheevii CMS cytoplasm, with the corresponding amino acid sequence having at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:676 and SEQ ID NO:684. Examples of corresponding specific Rf3 nucleic acids are referred to in Table 7 or further described in Example 12. Typically, examples of specific Rf3 nucleic acids comprises SEQ ID NO:1712, SEQ ID NO:2230, SEQ ID NO:2238, SEQ ID NO:3146, SEQ ID NO:3147 or SEQ ID NO:3148.
Rf4 Nucleic Acids
In specific embodiment, the nucleic acid of the present disclosure is a Rf4 nucleic acid.
As used herein, the term “Rf4 nucleic acid” refers to a nucleic acid comprising a gene encoding a Rf4 protein restorer of fertility of T. timopheevii CMS cytoplasm, wherein the corresponding amino acid sequence has at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:477, and SEQ ID NOs3135-3138, typically SEQ ID NO:477 and SEQ ID NOs3136-3138. Examples of corresponding specific Rf4 nucleic acids are listed in Table 7 and further include any of SEQ ID NO: 2031, and SEQ ID NO:3140-3142.
Rf7 Nucleic Acids
In specific embodiment, the nucleic acid of the present disclosure is a Rf7 nucleic acid.
As used herein, the term “Rf7 nucleic acid” refers to a nucleic acid comprising a gene encoding a Rf7 protein restorer of fertility of T. timopheevii CMS cytoplasm, wherein the corresponding amino acid sequence has at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:240-243, SEQ ID NOs303-305, SEQ ID NO:363, SEQ ID NOs375-377, SEQ ID NOs497-499, SEQ ID NO:516, SEQ ID NOs709-711, SEQ ID NO:768, typically SEQ ID NO:363, SEQ ID NO:516 and SEQ ID NO:768. Examples of corresponding specific Rf7 nucleic acids are referred to in Table 7.
Rf-Rye Nucleic Acids
In specific embodiment, the nucleic acid of the present disclosure is a Rf-rye nucleic acid.
As used herein, the term “Rf-rye nucleic acid” refers to a nucleic acid comprising a gene encoding a Rf-rye protein restorer of fertility of T. timopheevii CMS cytoplasm, wherein the corresponding amino acid sequence has at least 95% identity, preferably, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:227, SEQ ID NO:378 and SEQ ID NO:859. Examples of corresponding specific Rf-rye nucleic acids are referred to in Table 7.
Rf Nucleic Acids as Transgene
The present disclosure more specifically relates to DNA molecules including one or more of the Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids. In particular, the disclosure relates to any DNA molecule resulting from the insertion of a transgene in the wheat plant, said transgene including one or more of the above described Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids, and which insertion results in the expression of corresponding RNA and/or protein in the wheat plant.
Also part of the present disclosure is a nucleic acid that has been extracted from cells, or tissues, or homogenate from a plant or seed or plant tissue; or can be produced as an amplicon from extracted DNA or RNA from cells, or tissues, or homogenate from a plant or seed or plant tissue, any of which is derived from such materials derived from a plant comprising such nucleic acid as disclosed above.
As used herein, the term “transgene” or “transgenic element” refers to the nucleic acid (e.g. DNA molecule) incorporated into a host cell's genome. The term “transgene” or ‘transgenic element” refers in particular to a sequence that is not normally present in a given host genome in the genetic context in which the sequence is currently found. In this respect, the sequence may be native to the host genome, but be rearranged with respect to other genetic sequences within the host genomic sequence. For example, the transgene is rearranged at a different locus as compared to the native gene.
Said one or more transgenic element(s) enables the expression of polypeptides which restore or improve male fertility to the plant having T. timopheevii CMS cytoplasm, as compared to the parent plant which do not comprise the transgenic element.
A particular transgenic element is the recombinant nucleic acid as defined above, for example Rf1 nucleic acids as defined above. In specific embodiments, a transgenic element includes an Rf nucleic acid under the control of a constitutive promoter, such as the ZmUbi promoter.
Recombinant Nucleic Acids for Use in Transforming Wheat Plants
Such Rf nucleic acids as defined above are also useful to transform or genetically modify wheat plant, in particular wheat plant which does not have one or more of the fertility restoration alleles Rf1, Rf3, Rf4, Rf7 and Rf-rye.
Another aspect of the present disclosure relates to a vector for use in transforming wheat plant, comprising one or more of Rf1, Rf3, Rf4, Rf7 and Rf-rye nucleic acids as described above.
Vectors for use in transforming wheat plant includes at least the coding sequence of the corresponding protein restorer of fertility (either naturally occurring coding sequence, or improved sequence, such as codon optimized sequence), such coding sequence being operably linked to a regulatory element such as a promoter.
The term “promoter” as used herein refers to a region of DNA upstream of the coding sequence (upstream of start codon) and including DNA regions for recognition and binding of RNA polymerase and other proteins to initiate transcription at the start codon. Examples of constitutive promoters useful for expression include the 35S promoter or the 19S promoter (Kay et al, 1987), the rice actin promoter (McElroy et al, 1990), the pCRV promoter (Depigny-This et al, 1992), the CsVMV promoter (Verdaguer et al. 1996), the ubiquitin 1 promoter of maize (Christensen and Quail, 1996), the regulatory sequences of the T-DNA of Agrobacterium tumefaciens, including mannopine synthase, nopaline synthase, octopine synthase.
Promoters may be «tissue-preferred», i.e. initiating transcription in certain tissues or “tissue-specific”, i.e. initiating transcription only in certain tissues. Examples of such promoters are DHN12, LTR1, LTP1 specific of the embryo, SS1 specific of the phloem, OSG6B specific of the tapetum (Gotz et al 2011 and Jones 2015).
Other suitable promoters could be used. It could be an inducible promoter, a developmentally regulated promoter. An “inducible” promoter initiates transcription under some environmental control or any stress-induced like for example the abiotic stress-induced RD29, COR14b (Gotz et al, 2011).
Constitutive promoters may be used, such as the ZmUbi promoter, typically the ZmUbi promoter of SEQ ID NO:3134. Finally, promoter of SEQ ID NO:3114, SEQ ID NO:3123 and SEQ ID NO:3113 corresponding to pTaRFL46, 79 and 104 can also be used.
In specific embodiments, the Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids of the present disclosure are operably linked to heterologous promoters, i.e. a promoter which is not the natural promoter of the corresponding Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids as found in wheat. Typical recombinant constructs of Rf3 nucleic acids with heterologous promoters include any one of SEQ ID NO:3150-3153, and any one of SEQ ID NO:3156-SEQ ID NO:3159. Typical recombinant constructs of Rf1 nucleic acids with heterologous promoters includes SEQ ID NO:3122, or a nucleic acid of SEQ ID NO:3119 under the regulation of the promoter of SEQ ID NO:2123.
The vector may further comprise additional elements including selection gene marker, operably linked to regulatory element, that allows to select the transformed plant cells containing the vector, comprising the nucleic acids of the present disclosure as transgene.
The vector may further comprise additional elements including counter-selection gene marker, operably linked to regulatory element that allows to counter-select the transformed plant cells which do not have maintained the counter-selection gene marker in its genome.
In a specific embodiment, the vector according to the present disclosure may be vector suitable for Agrobacterium-mediated transformation, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes mediated transformation, as described in the next section.
Methods for Producing a Wheat Transgenic Plant
Another aspect of the present disclosure relates to the use of the above-described nucleic acids for producing wheat transgenic plant expressing protein restorer of fertility.
The term “transgenic plant” refers to a plant comprising such a transgene. A “transgenic plant” includes a plant, plant part, a plant cell or seed whose genome has been altered by the stable integration of recombinant DNA. A transgenic plant includes a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant. As a result of such genomic alteration, the transgenic plant is distinctly different from the related wild type plant. An example of a transgenic plant is a plant described herein as comprising one or more of the Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids, typically as transgenic elements. For example, the transgenic plant includes one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids as transgene, inserted at loci different from the native locus of the corresponding Rf gene(s). Accordingly, it is herein disclosed a method for producing a wheat transgenic plant, wherein the method comprises the steps of
For transformation methods within a plant cell, one can cite methods of direct transfer of genes such as direct micro-injection into plant embryos, vacuum infiltration or electroporation, direct precipitation by means of PEG or the bombardment by gun of particles covered with the plasmidic DNA of interest.
It is preferred to transform the plant cell with a bacterial strain, in particular Agrobacterium, in particular Agrobacterium tumefaciens. In particular, it is possible to use the method described by Ishida et al. (Nature Biotechnology, 14, 745-750, 1996) for the transformation of monocotyledons.
Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Moloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.
Alternatively, direct gene transfer may be used. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 micron. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al., BioTechnology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206 (1990), Klein et al., BioTechnology 10:268 (1992). Several target tissues can be bombarded with DNA-coated microprojectiles in order to produce transgenic plants, including, for example, callus (Type I or Type II), immature embryos, and meristematic tissue.
Following transformation of wheat target tissues, expression of the selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.
The foregoing methods for transformation would typically be used for producing a transgenic plant including one or more of Rf1, Rf3, Rf4, Rf7 or Rf Rye nucleic acids as transgenic element(s).
The transgenic plant could then be crossed, with another (non-transformed or transformed) inbred line, in order to produce a new transgenic line. Alternatively, a genetic trait which has been engineered into a particular line using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred line into an elite inbred line, or from an inbred line containing a foreign gene in its genome into an inbred line or lines which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
When the term transgenic wheat plant is used in the context of the present disclosure, this also includes any wheat plant including, as a transgenic element one or more of Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids and wherein one or more desired traits have further been introduced through backcrossing methods, whether such trait is a naturally occurring one or a transgenic one. Backcrossing methods can be used with the present invention to improve or introduce one or more characteristic into the inbred. The term backcrossing as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental wheat plants. The parental wheat plant which contributes the gene or the genes for the desired characteristic is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental wheat plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Fehr et al, 1987).
In a typical backcross protocol, the recurrent parent is crossed to a second nonrecurrent parent that carries the gene or genes of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a wheat plant is obtained wherein all the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant in addition to the gene or genes transferred from the nonrecurrent parent. It should be noted that some, one, two, three or more, self-pollination and growing of a population might be included between two successive backcrosses.
Methods for Producing a Wheat Genetically Engineered Plant
An aspect of the present disclosure relates to a DNA fragment of the corresponding protein restorer of fertility (either naturally occurring coding sequence, or improved sequence, such as codon optimized sequence) combined with genome editing tools (such TALENs, CRISPR-Cas, Cpf1 or zing finger nuclease tools) to target the corresponding Rf restorer alleles within the wheat plant genome by insertion at any locus in the genome or by partial or total allele replacement at the corresponding locus.
In particular, the disclosure relates to a genetically modified (or engineered) wheat plant, wherein the method comprises the steps of genetically modifying a parent wheat plant to obtain in their genome one or more nucleotide sequence encoding protein restorer of T. timopheevii CMS cytoplasm Rf1, Rf3, Rf4 or Rf7 as disclosed herein, preferably by genome-editing, selecting a plant comprising said one or more nucleotide sequences as genetically engineered elements, regenerating and growing said wheat genetically engineered plant.
As used herein, the term “genetically engineered element” refers to a nucleic acid sequence present in the genome of a plant and that has been modified by mutagenesis or by genome-editing tools, preferentially by genome-editing tools. In specific embodiments, a genetically engineered element refers to a nucleic acid sequence that is not normally present in a given host genome in the genetic context in which the sequence is currently found but is incorporated in the genome of plant by use of genome-editing tools. In this respect, the sequence may be native to the host genome, but be rearranged with respect to other genetic sequences within the host genomic sequence. For example, the genetically engineered element is a gene that is rearranged at a different locus as compared to a native gene. Alternatively, the sequence is a native coding sequence that has been placed under the control of heterologous regulatory sequences. Other specific examples are described hereafter. The term “genetically engineered plant” or “genetically modified plant” refers to a plant comprising such genetically engineered element. A “genetically engineered plant” includes a plant, plant part, a plant cell or seed whose genome has been altered by the stable integration of recombinant DNA. As used herein, the term “genetically engineered plant” further includes a plant, plant part, a plant cell or seed whose genome has been altered by genome editing techniques. A genetically engineered plant includes a plant regenerated from an originally-engineered plant cell and progeny of genetically engineered plants from later generations or crosses of a genetically engineered plant. As a result of such genomic alteration, the genetically engineered plant is distinctly different from the related wild type plant. An example of a genetically engineered plant is a plant described herein as comprising one or more of the Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids. For example, the genetically engineered plant includes one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids as genetically engineered elements, inserted at loci different from the native locus of the corresponding Rf gene(s).
In specific embodiments, said genetically engineered plants do not include plants which could be obtained exclusively by means of an essentially biological process. Said one or more genetically engineered element(s) enables the expression of polypeptides which restore or improve male fertility to the plant having T. timopheevii CMS cytoplasm, as compared to the parent plant which do not comprise the genetically engineered element(s).
A particular genetically engineered element is the Rf nucleic acid as defined above, for example Rf1 nucleic acids as defined above. In specific embodiments, a genetically engineered element includes an Rf nucleic acid under the control of expression elements as promoter and/or terminator. Suitable promoter can be a constitutive promoter, such as the ZmUbi promoter or an endogenous Rf promoter native or modified.
Another aspect of the disclosure relates to a genetically engineered wheat plant, which comprises the modification by point mutation insertion or deletion of one or few nucleotides of an allele sequence rf or Rf, as genetically engineered element, into the respectively Rf or rf allele, by any of the genome editing tools including base-editing tool as described in WO2015089406 or by mutagenesis.
The present disclosure further includes methods for modifying fertility level in a plant by genome editing, comprising providing a genome editing tool capable of replacing partially or totally a rf1, rf3, rf4, rf7 or rf-rye non-restorer allele sequence or form in a wheat plant by its corresponding Rf1, Rf3, Rf4, Rf7 or Rf-rye restorer allele sequence as disclosed herein.
The term “rf non-restorer allele sequence or form” can be related to the presence in the genome of a rf non-restorer allele or to the absence in the genome of any rf or Rf allele. For example, in the rf3/Rf3 system, one wheat non-restorer line can be characterized by the presence of a rf3 allele sequence while another non-restorer line is characterized by the absence of any Rf3 or rf3 allelic sequence. The Rf3 restorer plant will be characterized by the presence of the Rf3 allele sequence in the genome. In the case of the rf4/Rf4 system, the non-restorer plant is characterized by the absence of any rf4 or Rf4 allelic forms while the Rf4 restorer plant will be characterized by the presence in the genome of the Rf4 gene sequence.
In specific embodiments, methods for modifying fertility level in a plant by genome editing comprises providing a genome editing tool capable of replacing or modifying a rf3 non-restorer allele to obtain a Rf3 restorer allele comprising SEQ ID NO:3146 (RFL29a). In other specific embodiments, rf3 non-restorer allele may comprise RFL29c characterized by a frameshift compared to RFL29a nucleotide sequence as shown in Example 22 and
The disclosure further includes methods for modifying fertility level in a plant introducing the endogenous promoter of a restorer Rf gene in order to increase the expression of the corresponding endogenous Rf gene either by genome-editing or by mutagenesis.
In a specific embodiment, the disclosure includes methods for modifying fertility level in a plant by genome editing of a weak fertile plant by modifying the 5′UTR sequence of the Rf3 “weak” RFL29b allele which 5′UTR region includes a 163 bp insertion to be deleted as shown in example 15.
In a further specific embodiment, the invention includes a method for modifying fertility level in a plant by mutagenesis or by genome editing the endogenous promoter of a restorer Rf gene in order to increase the expression of the endogenous Rf gene. As an example, the sequence of proTaRFL79 depicted in SEQ ID No 3123 could be mutated or edited to increase RFL79 protein level.
In another specific embodiment, whenever a stronger promoter is located upstream to the promoter of the Rf restorer gene, a deletion of that promoter and the region upstream can be achieved in order to juxtapose the stronger promoter to the Rf gene.
In a specific embodiment, one rf non-restorer allele is replaced partially or totally by anyone of the Rf1, Rf3, Rf4, Rf7 or Rf-rye restorer allele. In such disclosure, a non-restorer rf1 allele could be replaced, for example, by a Rf3, or a Rf4, or a Rf7, or Rf-rye allele.
In another aspect of the disclosure, at least one restorer allele from among Rf1, Rf3, Rf4, Rf7 and Rf-Rye can be integrated at one or more target sites in the wheat plant genome, typically in order to get an expression of said restorer alleles. Said expression can be achieved either by taking advantage of the presence at the targeting locus of a promoter, more specifically a strong promoter, and/or a terminator and by targeting with the Rf allele said expression elements. In a specific embodiment, the endogenous Rf allele is deleted from one first locus and further integrated downstream a suitable promoter at a second locus in the same plant genome.
In a specific embodiment of the invention, the target sites can be located in the Rf1, Rf3, Rf4 and/or Rf7 locus as defined in example 17. In a more specific embodiment, the target sites can either be the Rf1, Rf3, Rf4, Rf7 or Rf-Rye endogenous gene sequence or any other target sites different from the Rf1, Rf3, Rf4, Rf7 or Rf-Rye endogenous gene sequences.
In a preferred aspect of the disclosure, a wheat plant can comprise in its genome, at only one locus, the Rf1, Rf3 and Rf7 restorer alleles.
Such genome editing tool includes without limitation targeted sequence modification provided by double-strand break technologies such as, but not limited to, meganucleases, ZFNs, TALENs (WO2011072246) or CRISPR CAS system (including CRISPR Cas9, WO2013181440), Cpf1 or their next generations based on double-strand break technologies using engineered nucleases.
Method for Decreasing the Fertility Level in Wheat Plant:
Alternatively, the present disclosure further includes methods for modifying fertility level in a plant by reverting a restorer line comprising a Rf allele to a maintainer line comprising a rf allele. Such method could be used for any plant comprising a Rf allele, including Rf1. It is of particular interest in the case of a hybrid production system based on Rf3 restorer allele, wherein for example, the Rf3 sequence is RFL29a and the rf3 sequence is RFL29c.
The decrease in fertility could be obtained by knocking-down Rf gene or allele as described below.
More specifically, the method can correspond first to an inhibition of the expression by RNAi directed against the Rf allele of interest or by impairing the promoter function of the Rf allele either by mutagenesis or by genome editing.
In another aspect, the fertility level decrease is obtained by mutagenesis, classically induced with mutagenic agents, or by genome editing technologies to impair the protein function, by deleting totally or partially the gene, or by modifying the gene sequence reading frame to impair protein translation.
In a further aspect, the disclosure relates to the transgenic or genetically engineered wheat plant with decreased fertility level obtained by the methods described above.
The Transgenic or Genetically Engineered Wheat Plant of the Present Disclosure
Another aspect of the present disclosure relates to a transgenic or genetically engineered wheat plant comprising one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acid(s) as described in the previous sections, as transgenic or genetically engineered elements respectively.
Said transgenic or genetically engineered plant may be obtained by the methods described in the previous section.
The transgenic or genetically engineered plants of the present disclosure may advantageously be used as parent plant in order to produce fertile wheat transgenic or genetically engineered plant restorer of T. timopheevii CMS cytoplasm. In particular, in specific embodiment, the wheat transgenic or genetically engineered plant is a fertile wheat transgenic or genetically engineered plant restorer of T. timopheevii CMS cytoplasm. Typically, a transgenic or genetically engineered wheat plant according to the present disclosure comprises a combination of at least two different transgenic or genetically engineered elements selected from the group consisting of Rf1, Rf3, Rf4, Rf7 and Rf-rye encoding nucleic acids.
The transgenic or genetically engineered wheat plant as disclosed herein may express such protein restorer of fertility Rf1, Rf3, Rf4, Rf7 and/or Rf-Rye together as the result of the transgene's expression or expression of the genetically engineered element and may additionally express other protein restorer of fertility, as the result of naturally occurring alleles.
In one embodiment, said combination of at least two, three or four of Rf1, Rf3, Rf4, Rf7 and Rf-rye nucleic is found in the same locus in the genome of the transgenic or genetically engineered plant. In other embodiments, the corresponding nucleic acids of the combination are located in distinct loci. In one specific embodiment, said combination may be obtained by crossing transgenic plants of the present disclosure each bearing one nucleic acid of the combination as a transgene at a distinct locus.
Typically, said transgenic or genetically engineered plant includes the following combination of nucleic acids as transgenic or genetically engineered elements:
In other specific embodiments, said transgenic or genetically engineered plant further includes a Rf4 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs:477, and SEQ ID NOs3135-3138 in addition to any one of the above defined combination of Rf1, Rf3, Rf7 and/or Rf-Rye nucleic acids as defined in the previous paragraph, as transgenic elements.
The disclosure also relates to hybrid wheat plants which can be produced by crossing a transgenic or genetically engineered wheat plant restorer of fertility according to the present disclosure as described above with a second plant.
In certain embodiments, the wheat plant according to the disclosure is alloplasmic and comprises the T. timopheevii cytoplasm.
For example, a hybrid wheat plant may be obtained by crossing a wheat plant restorer of fertility according to the present disclosure as described above, preferably comprising Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids, and a wheat plant which does not express corresponding Rf1, Rf3, Rf4, Rf7 or Rf-rye protein restorer of fertility.
It is also disclosed herein a method for producing a wheat hybrid transgenic or genetically engineered plant comprising the steps of:
Therefore, it is also disclosed herein the wheat transgenic or genetically engineered plants or lines according to the present disclosure developed to obtain such hybrid plants. Such transgenic or genetically engineered plants or lines typically comprise the cytoplasmic elements necessary for the implementation of the corresponding hybrid system. Preferably, the transgenic or genetically engineered plants or lines comprise a combination of at least two, three or four of Rf1, Rf3, Rf4, Rf7 and Rf-rye nucleic acids, and T. timopheevii cytoplasm.
Alternatively, the detection of the presence of T. timopheevii cytoplasm and of at least one or more of the Rf nucleic acids chosen amongst Rf1, Rf3, Rf4, Rf7 and Rf-rye (step “c” of the method described above) can be performed on the parent lines in order to check their genotype before to start the cross (step “a”).
In a certain embodiment of the disclosure, the male wheat plant is taller than the female wheat plant. This can be achieved by using male plant bearing Rht alleles that allows to obtain the size differences. Optionally, the disclosure further comprises the step of to applying an herbicide to the fertile plants standing above the height of the shorter female plants, and further optionally, comprises the step of harvesting the seeds and selecting the seeds to remove undesirable self-fertilized male seeds, using a morphological character and/or a phenotypic character like size, shape, color etc . . . . An example of such method for hybrid production is described in WO2015135940.
The T-CMS cytoplasm can be detected either phenotypically wherein a plant bearing rf genes and a T-CMS cytoplasm will be sterile or by molecular means able to detect the orf256 gene as described in Rathburn and Hedgcoth, 1991 and Song and Hedgcoth, 1994.
The disclosure also relates to a method for improving the level of fertility restoration of a parent wheat plant bearing a fertility level lower than a full restoration level, comprising the steps of transforming said parent wheat plant with a vector comprising a Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acid as described above, or genetically engineering said Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids in said wheat plant. The method further comprises the step of selecting a transgenic or genetically engineered wheat plant comprising said Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acid(s) as a transgene or genetically engineered element, preferably Rf1, Rf3 and Rf4, regenerating and growing said transgenic or genetically engineered wheat plant, wherein said transgenic or genetically engineered wheat plant has an improved fertility restoration level as compared to the parent plant.
The disclosure further provides a method for restoring fertility of a sterile wheat plant bearing the T. timopheevii CMS cytoplasm, comprising the step of transforming a parent sterile wheat plant bearing the T. timopheevii CMS cytoplasm, with a Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acid as described above or genetically engineering said sterile plant to express a Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids.
Use of the Nucleic Acids of the Present Disclosure for Identifying Rf1, Rf3, Rf4, Rf7 and Rf-Rye Restorer Alleles or Transgenic Elements
The present disclosure further provides methods of identifying the respective Rf1, Rf3, Rf4, Rf7 and/or Rf-rye restorer alleles and/or Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids as disclosed in the previous sections, and more generally methods of selecting or breeding wheat plants for the presence or absence of the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye fertility restorer alleles and/or corresponding Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids.
Such methods of identifying, selecting or breeding wheat plants comprise obtaining one or more wheat plants and assessing their DNA to determine the presence or absence of the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye fertility restorer alleles and/or corresponding Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids.
Such methods may be used, for example, to determine which progeny resulting from a cross have the required fertility restorer allele or Rf nucleic acids (or combination thereof) and accordingly to guide the preparation of plants having the required fertility restorer allele or Rf nucleic acids in combination with the presence or absence of other desirable traits.
The method will consist of identifying the presence of the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye allele or nucleic acids in the fertile plant, said plant being either a fertile transgenic plant or a non-transgenic plant. Optionally, the method further consists of identifying the absence of the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye allele or nucleic acids in non-restorer plants and/or sterile plants.
Accordingly, it is disclosed herein the means for specifically detecting the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids in a wheat plant.
Such means include for example a pair of primers for the specific amplification of a fragment nucleotide sequence of Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids from plant wheat genomic DNA.
As used herein, a primer encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form though single-stranded form is preferred.
Alternatively, nucleic acid probe can be used for the specific detection of any one of Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids.
As used herein, a nucleic acid probe encompass any nucleic acid of at least 30 nucleotides and which can specifically hybridizes under standard stringent conditions with a defined nucleic acid. Standard stringent conditions as used herein refers to conditions for hybridization described for example in Sambrook et al 1989 which can comprise 1) immobilizing plant genomic DNA fragments or library DNA on a filter 2) prehybridizing the filter for 1 to 2 hours at 65° C. in 6×SSC 5×Denhardt's reagent, 0.5% SDS and 20 mg/ml denatured carrier DNA 3) adding the probe (labeled) 4) incubating for 16 to 24 hours 5) washing the filter once for 30 min at 68° C. in 6×SSC, 0.1% SDS 6) washing the filter three times (two times for 30 min in 30 ml and once for 10 min in 500 ml) at 68° C. in 2×SSC 0.1% SDS. The nucleic acid probe may further comprise labeling agent, such as fluorescent agents covalently attached to the nucleic acid part of the probe.
Methods of Producing a Wheat Plant Carrying a Modified Rf3 Restorer of Fertility
The inventors have also identified two types of Rf3 restorer of fertility, one with a strong fertility restoration, for example, capable of providing plants with a fertility score above 1.0, for example comprised between 1.0 and 2.0 and another with a weak fertility restoration, for example, capable of providing plants having a fertility score below 0.1, for example comprised between 0.5 and 1.0.
Surprisingly, the strong fertility restoration correlates with the absence in the genome of the wheat plant carrying Rf3 of a 163 bp fragment of SEQ ID NO:3174, located in the 5′UTR of Rf3 coding sequence.
Accordingly, the disclosure relates to a method for producing a wheat plant carrying a Rf3 restorer of fertility, said method comprising (i) providing a parent wheat plant comprising in its genome at least a 163 bp fragment of SEQ ID NO:3174, and (ii) deleting a fragment of at least 10 bp of said fragment of SEQ ID NO:3174, for example at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp or the whole fragment of SEQ ID NO:3174 in the genome of said wheat plant, thereby obtaining said wheat plant carrying a Rf3 restorer of fertility.
Advantageously, the fertility score of the obtained wheat plant has a fertility score higher than the parent wheat plant, due to the deletion of said fragment depicted in SEQ ID NO:3174. The skilled person may select the deletion such as to obtain an increase in fertility restoration as compared to the parent wheat plant with the full fragment of SEQ ID NO:3174 in its genome.
In specific embodiments, the parent wheat plant has a fertility score below 1, for example comprised between 0.5 and 1.0 and the obtained wheat plant has a fertility score above 1.0, for example comprised between 1.0 and 2.0.
It is also possible to restore fertility from plants having rf3 non-restorer allele presenting a frameshift due to nucleotides deletion or insertion as compared to the Rf3 restorer allele RLF29a sequence of SEQ ID NO:3146 (see also Example 22).
Such deletion of genomic fragment or correction of frameshift may be obtained by any suitable methods known by the skilled person in the art, including genome editing tools such as, but not limited to, meganucleases, ZFNs, TALENs (WO2011072246) or CRISPR CAS system (including CRISPR Cas9, WO2013181440) or their next generations based on double-strand break technologies using engineered nucleases. Examples of such methods are also described in Example 15 and Example 22.
The wheat plants as obtained by the method described above are also part of the disclosure. Typically, such wheat plant as obtained by the above method, or obtainable by such method, carries a Rf3 restorer of fertility, wherein only a part but not the whole genomic fragment of SEQ ID NO:3174 is deleted in the genome of said wheat plant. Typically, a fragment between 10 bp and 162 bp of SEQ ID NO:3174 is deleted in the genome of said wheat plant. It is expected that the obtained wheat plant with the genome deletion has a fertility score higher than the fertility score as measured in a parent wheat plant with identical genome except for the full sequence of SEQ ID NO:3174 in its genome. Alternatively, such wheat plant as obtained by the above method, or obtainable by such method, carries a Rf3 restorer of fertility, wherein nucleotides of RFL29c has been deleted or added to restore in frame translation.
Methods for Assessing Fertility Restoration in a Wheat Plant
The disclosure also includes a method for assessing fertility restoration in a wheat plant, said method comprising determining the presence or absence of a fragment of SEQ ID NO:3174 in the genome of said plant, wherein the presence of the whole fragment is indicative of a weak restoration of fertility and a deletion of at least a part of such fragment is indicative of a strong restoration of fertility. Typically said method is performed in a wheat plant which is susceptible to carry a Rf3 restorer of fertility.
It is also disclosed herein nucleic acid probes for use in the above methods for assessing fertility restoration in wheat plant, wherein said nucleic acid probe consists of a nucleic acid of at least 10 nucleotides within SEQ ID NO:3174.
Typically, said nucleic acid probe is a fragment of at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp or the whole fragment of SEQ ID NO:3174.
The Wheat Plant Restorer of Fertility of T. timopheevii CMS Cytoplasm with at Least Three Specific Fertility Restorer Alleles
The inventors have shown that a combination of at least 3 specific fertility restorer alleles within the restorer loci Rf1, Rf3, Rf4 and Rf7 enable the obtention of plants with full restoration of fertility of T. timopheevii CMS cytoplasm.
Therefore, a first aspect of the present disclosure relates to a wheat plant restorer of fertility of T. timopheevii CMS cytoplasm, wherein the plant comprises at least three fertility restorer alleles within the restorer loci chosen amongst Rf1, Rf3, Rf4 and Rf7.
In specific embodiments, the plant comprises at least the three fertility restorer alleles Rf1, Rf3, Rf4.
In specific embodiments, the plant comprises at least the three fertility restorer alleles Rf1, Rf4, Rf7.
In specific embodiments, the plant comprises at least the three fertility restorer alleles Rf1, Rf3, Rf7.
In specific embodiments, the plant comprises at least the three fertility restorer alleles Rf3, Rf4, Rf7.
As used herein, the Rf1 locus refers to the locus of the Rf1 restorer allele, which locus is located at most 10 cM, preferably at most 7 cM, more preferably at most 2 cM, from marker cfn0522096 of SEQ ID NO:4 and/or from marker cfn05277067 of SEQ ID NO:10. In a specific embodiment, the wheat plant restorer of fertility according to the present disclosure includes at least one Rf1 restorer allele, said Rf1 restorer allele being located within the chromosomal interval between SNP markers cfn0522096 of SEQ ID NO:3190 and cfn05277067 of SEQ ID NO:3196. In specific embodiments, the wheat plant restorer of fertility includes one Rf1 restorer allele at the Rf1 locus characterized by the presence of one or more of the SNP allele(s) as identified by Table 1.
Preferably, the wheat plant restorer of fertility according to the present disclosure includes one Rf1 restorer allele at the Rf1 locus characterized by the presence of the SNP3 and/or SNP7 restorer alleles(s) as described in Table 1. More preferably, the wheat plant restorer of fertility is characterized by the haplotypes of the SNP3 and SNP7 restorer alleles “C” and “A”. In specific embodiments, the wheat plant restorer of fertility with Rf1 restorer allele comprises a Rf1 nucleic acid of the present disclosure as described above. Examples of Rf1 nucleic acids comprises the disclosed Rf1 nucleic acid sequences of SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1915, SEQ ID NO:1916 or SEQ ID NO:3119, preferably a Rf1 nucleic acid comprises SEQ ID NO:3119.
As used herein, the Rf3 locus refers to the locus of the Rf3 restorer allele, which locus is at most 10 cM, preferably at most 7 cM, more preferably at most 2 cM, from marker cfn1249269 of SEQ ID NO:3205 and/or from marker BS00090770 of SEQ ID NO:3228. In a specific embodiment, the wheat plant restorer of fertility includes at least one Rf3 restorer allele within the Rf3 locus, said Rf3 restorer allele being located within the chromosomal fragment between SNP markers cfn1249269 and BS00090770. In specific embodiment, the wheat plant restorer of fertility includes one Rf3 restorer allele at the Rf3 locus characterized by the presence of one or more of the SNP alleles(s) as identified by Table 2.
Preferably, the wheat plant restorer of fertility according to the present disclosure includes one Rf3 restorer allele at the Rf3 locus characterized by the presence of the SNP29 and/or SNP31 restorer alleles(s) as described in Table 2. More preferably, the wheat plant restorer of fertility is characterized by the haplotype of the SNP29 and SNP31 restorer alleles “T” and “A” respectively.
In another particular embodiment, that may be combined with the previous embodiments, the wheat plant restorer of fertility according to the present disclosure includes one Rf3 restorer allele at the Rf3 locus characterized by the presence of the SNP38 and SNP41 restorer alleles “A” and “A” respectively.
Preferably, the wheat plant restorer of fertility according to the present disclosure includes a Rf3 nucleic acid comprising SEQ ID NO:1712, SEQ ID NO:2230, SEQ ID NO:2238, SEQ ID NO:3146, SEQ ID NO:3147 or SEQ ID NO:3148, preferably SEQ ID NO:3146.
As used herein, the Rf7 locus is located at most 10 cM from marker cfn0919993 of SEQ ID NO:3231. In specific embodiment, the wheat plant restorer of fertility includes one Rf7 restorer allele at the Rf7 locus characterized by the presence of one or more of the SNP alleles(s) as identified by Table 3:
Preferably, the wheat plant restorer of fertility according to the present disclosure includes one Rf7 restorer allele at the Rf7 locus characterized by the presence of the nine SNP restorer alleles SNP44-SNP46 and SNP49-54 of “restorer allele” haplotype, as described in Table 3.
As used herein, the Rf4 locus is located at most 10 cM from marker cfn0393953 of SEQ ID NO:3233. In specific embodiment, the wheat plant restorer of fertility includes one Rf4 restorer allele at the Rf4 locus characterized by the presence of one or more of the SNP alleles(s) as identified by Table 4.
Preferably, the wheat plant restorer of fertility according to the present disclosure includes one Rf4 restorer allele at the Rf4 locus characterized by the presence of the two SNP restorer alleles, SNP47, and SNP48, of the haplotype “C” and “G” respectively, as described in Table 4.
In specific embodiments, the wheat plant restorer of fertility with Rf4 restorer allele comprises a Rf4 nucleic acid of the present disclosure as described above. Examples of Rf4 nucleic acids comprises the disclosed Rf4 nucleic acid sequences of SEQ ID NO:2031, SEQ ID NO:3140 to 3142.
In a particular embodiment, the wheat plant restorer of fertility of T. timopheevii CMS cytoplasm comprises one Rf3 restorer allele and two other fertility restorer alleles selected amongst Rf1, Rf4 and Rf7 restorer alleles. Preferably, the wheat plant restorer of fertility of T. timopheevii CMS cytoplasm according to the present disclosure comprises the Rf1, Rf3, and Rf7 restorer alleles.
In particular, it is hereby included a wheat plant comprising Rf1, Rf3 and Rf7 restorer alleles as provided by the seed samples as deposited on 25 Sep. 2017 under deposit number NCIMB 42811, NCIMB 42812, NCIMB 42813, NCIMB 42814, NCIMB 42815, NCIMB 42816, and NCIMB 42817 at the NCIMB collection.
The disclosure also relates to hybrid wheat plants which can be produced by crossing a wheat plant restorer of fertility according to the present disclosure as described above with a second plant.
In certain embodiments, the wheat plant according to the disclosure is alloplasmic and comprises the T. timopheevii cytoplasm.
For example, a hybrid wheat plant may be obtained by crossing a wheat plant restorer of fertility according to the present disclosure as described above, preferably comprising Rf1, Rf3 and Rf7 restorer alleles, and a wheat plant which does not have said fertility restorer alleles.
It is also disclosed herein a method for producing a wheat hybrid plant comprising the steps of:
Therefore, it is also disclosed herein the wheat plants or lines according to the present disclosure developed to obtain such hybrid plants. Such plants or lines typically comprise the cytoplasmic elements necessary for the implementation of the corresponding hybrid system. Preferably, the plants or lines comprise the fertility restorer alleles Rf1, Rf3 and Rf7 and T. timopheevii cytoplasm. In specific embodiments, such plants or lines comprise a fertility restorer allele Rf1 comprising a Rf1 nucleic acid of the present disclosure as disclosed above.
Alternatively, the detection of the presence of T. timopheevii cytoplasm and of at least three of the Rf locus chosen amongst Rf1, Rf3, Rf4 and Rf7 (step “c” of the method described above) can be performed on the parent lines in order to check their genotype before to start the cross (step “a”).
The T-CMS cytoplasm can be detected either phenotypically wherein a plant bearing rf genes and a T-CMS cytoplasm will be sterile or by molecular means able to detect the orf256 gene as described in Rathburn and Hedgcoth, 1991 and Song and Hedgcoth, 1994.
Method of Producing and Selecting a Wheat Plant of the Disclosure
The present disclosure also relates to the methods to produce the wheat plant with the fertility restorer alleles as described in the previous section.
In one embodiment, said method for producing the wheat plant includes the following step:
Preferentially, the female plant in step b) is bearing the T-CMS cytoplasm. In this case, the presence of the restorer alleles is assessed at every generation from step b) to step d) by using the markers and optionally by further by assessing the fertility level.
Method to generate homozygous plants are generally well known from skilled person of the art. This could be either by repetitive backcross or by double haploid development or by Single Seeds Descent (SSD) methods.
The applicant has deposited a sample of seeds of the disclosed wheat plant with said Rf1, Rf3 and Rf7 restorer alleles, on 25 Sep. 2017 under the Budapest treaty, at NCIMB collection under the number NCIMB 42811, NCIMB 42812, NCIMB 42813, NCIMB 42814, NCIMB 42815, NCIMB 42816, and NCIMB 42817.
The present disclosure further includes and provides methods of identifying the respective Rf1, Rf3, Rf4 and/or Rf7 restorer alleles as disclosed in the previous sections, and more generally methods of selecting or breeding wheat plants for the presence or absence of the Rf1, Rf3, Rf4 and/or Rf7 fertility restorer alleles. Such methods of identifying, selecting or breeding wheat plants comprise obtaining one or more wheat plants and assessing their DNA to determine the presence or absence of the Rf1, Rf3, Rf4 and/or Rf7 fertility restorer alleles contained in the respective locus.
Such methods may be used, for example, to determine which progeny resulting from a cross have the required combination of fertility restorer alleles and accordingly to guide the preparation of plants having the required combination in combination with the presence or absence of other desirable traits.
Accordingly, plants can be identified or selected by assessing them for the presence of one or more individual SNPs appearing in the above Tables 1, 2, 3 and 4, as well as the SNPs in Table 19, for assessing the presence of restorer alleles Rf1, Rf3, Rf7 or Rf4 respectively.
More generally, it is disclosed herein the specific means for detecting the restorer alleles in a wheat plant, more specifically Rf1, Rf3, Rf4 and Rf7 restorer alleles and their combinations.
Said means thus include any means suitable for detecting the following SNP markers within one or more of the following markers: SEQ ID NOs 3187-3235.
Any method known in the art may be used in the art to assess the presence or absence of a SNP. Some suitable methods include, but are not limited to, sequencing, hybridization assays, polymerase chain reaction (PCR), ligase chain reaction (LCR), and genotyping-by-sequence (GBS), or combinations thereof.
Different PCR based methods are available to the person skilled of the art. One can use the RT-PCR method or the Kaspar method from KBioscience (LGC Group, Teddington, Middlesex, UK).
The KASP™ genotyping system uses three target specific primers: two primers, each of them being specific of each allelic form of the SNP (Single Nucleotide Polymorphism) and one other primer to achieve reverse amplification, which is shared by both allelic form. Each target specific primer also presents a tail sequence that corresponds with one of two FRET probes: one label with FAM® dye and the other with HEX® dye.
Successive PCR reactions are performed. The nature of the emitted fluorescence is used to identify the allelic form or forms present in the mix from the studied DNA.
The primers identified in Table 5 are particularly suitable for use with the KASP™ genotyping system. Of course, the skilled person may use variant primers or nucleic acid probes of the primers as identified in Table 5, said variant primers or nucleic acid probes having at least 90%, and preferably 95% sequence identity with any one of the primers as identified in Table 5, or with the DNA genomic fragment amplified by the corresponding set of primers as identified in Table 5.
Percentage of sequence identity as used herein is determined by calculating the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. For example, nucleic acid sequences may be aligned using the BLAST 2 sequences (BI2seq) using BLASTN algorithms (www.ncbi.nlm.nih.gov).
As used herein, a primer encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form though single-stranded form is preferred. Alternatively, nucleic acid probe can be used. Nucleic acid probe encompass any nucleic acid of at least 30 nucleotides and which can specifically hybridizes under standard stringent conditions with a defined nucleic acid. Standard stringent conditions as used herein refers to conditions for hybridization described for example in Sambrook et al 1989 which can comprise 1) immobilizing plant genomic DNA fragments or library DNA on a filter 2) prehybridizing the filter for 1 to 2 hours at 65° C. in 6×SSC 5×Denhardt's reagent, 0.5% SDS and 20 mg/ml denatured carrier DNA 3) adding the probe (labeled) 4) incubating for 16 to 24 hours 5) washing the filter once for 30 min at 68° C. in 6×SSC, 0.1% SDS 6) washing the filter three times (two times for 30 min in 30 ml and once for 10 min in 500 ml) at 68° C. in 2×SSC 0.1% SDS.
In specific embodiments, said primers for detecting the SNP markers of the present disclosure (specific for each allele “X” or “Y” or common) are as listed in the following table 5:
Use of the Wheat Plants of the Disclosure
The plant according to the disclosure can be crossed, with any another inbred line, in order to produce a new line comprising either an increase or a decrease in the fertility level. Alternatively, a genetic trait which has been engineered into a particular line using the foregoing techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred line into an elite inbred line, or from an inbred line containing a foreign gene in its genome into an inbred line or lines which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
The wheat plant of the disclosure is a also a wheat plant wherein one or more desired traits have further been introduced through backcrossing methods, whether such trait is a naturally occurring one or not.
The disclosure also relates to the use of the wheat plant as described above or its seeds, for food applications, preferably for flour production and for feed applications, or for breeding applications, for example for use as a parent plant in breeding for improving agronomical value of a wheat plant, line, hybrid or variety.
As used herein, breeding applications encompass pedigree breeding to improve the agronomical value of a plant, line, hybrid, or variety.
The wheat plants disclosed herein are further useful, for example, for producing flour or for feed applications.
Seeds harvested from plants described herein can be used to make flour by any available techniques in the art. The wheat plants or their flour are also useful as food compositions, for human or animal.
The Examples below are given for illustration purposes only.
The
The
“-” corresponds to dominant markers with no amplification in several maintainer lines. “H” means heterozygote status wherein two alleles are detected.
32 genomic and two transcriptome data sets from 27 cereal plant species and their wild relatives were downloaded from the public sequence depositories and analysed. A complete list of files and databases from which they were downloaded is presented in
The DNA sequences were screened for open reading frames (ORFs) in six-frame translations with the getorf program of the EMBOSS 6.6.0 package (Rice et al., 2000). Predicted ORFs longer than 92 codons were screened for the presence of P- and PLS-class pentatricopeptide repeat (PPR) motifs using hmmsearch from the HMMER 3.1b package (hmmer.org) and hidden Markov models defined by hmmbuild (Cheng et al., 2016). The post-processing of hmmsearch results was carried out according to rules described previously (Cheng et al., 2016). Sequences containing 10 or more P-class PPR motifs were retained for further analysis, as a previous study has shown that Restorer-to-Fertility-Like (RFL) genes are primarily comprised of tandem arrays of 15 to 20 PPR motifs (Fujii et al., 2011).
For identification of RFL sequences among the P-class PPRs, the OrthoMCL algorithm (Li et al., 2003) was used via the OrthoMCL-DB website to cluster P-class PPR proteins from each data set (http://www.orthomcl.org/orthomcl/). The resulting output files were screened for groups containing reference RFLs (Fujii et al., 2011).
In total, 633 RFLs were identified in the 34 cereal data sets by OrthoMCl analysis (see Table of
A. Selection of Germplasm Accessions:
Six wheat accessions were identified as potential restorer lines of Timopheevii-type CMS (T-CMS) derived from the interspecific cross between Triticum timopheevi and Triticum aestivum.
The first accession is a wheat-rye addition line, “Wheat-Rye-6R”, wherein the Rf gene was mapped on the additional long arm of 6R chromosome of rye Secale cereale (Curtis and Lukaszewski, 1993). Four other wheat accessions are characterized by the presence of at least one of the mapped restorer genes in wheat: Rf1, Rf3 and Rf7. The commercial variety, Primepii, carries the Rf3 gene, and three Limagrain lines R197, R0934F and R0932E respectively carry both Rf1 and Rf7 genes, Rf3 or Rf1.
The sixth accession, named Anapurna, is a maintainer line not able to restore T-CMS and carries no known Rf genes. Anapurna is considered as the negative control in this experiment. In addition, a T. timopheevii line was included in the study as it is a fertile line expected to harbor more than one Rf gene able to restore T-type CMS (Wilson and Ross, 1962). To some extent, this line is considered as a positive control in this experiment.
All six accessions were verified in regard to their restorer status by genetic analysis.
Northern blot analysis was performed with restorer and sterile accessions using an orf256-specific probe (
B. Bait Design and RFL-Capture from Different Wheat Genotypes:
The 1188 RFL PPR sequences identified by our bioinformatics analysis underwent a pre-treatment process that included masking of the target sequences against wheat mitochondrial and chloroplastic genome sequences (accessions NC_007579.1 and AB042240) as well as repeated elements of wheat genome. The masked target sequences were used for capture probe design. Briefly, probes were designed to cover the target sequences with a frequency masking algorithm intended to rule out probes that match with high copy number sequences in the targeted genome(s). The final probes were synthetized as a probe pool.
Seeds of each accession were sown and plantlets were grown in etiolated conditions. After DNA extraction, Illumina libraries (referred to as NGS libraries) were prepared from DNA fragments around 600 bp with the KAPA Biosystems chemistry according to the manufacturer's recommendations.
The NGS libraries were then specifically enriched in RFL sequences using the probe pool and a capture protocol. The efficiency of the capture was confirmed by a specific qPCR assay and ultimately libraries were pooled and sequenced in paired-end mode with 300 nt read length on a MiSeq platform.
C. Assembly of Full-Length Gene Sequences Encoding RFL Proteins and Identification of Putative Orthologous Groups:
Sequence reads from the RFL capture experiment were assembled into full-length contigs spanning one or more sequences encoding RFL proteins as described below. Overlapping paired reads were merged into a single sequence using bbmerge from the bbmap package (https://sourceforga.net/projects/bbmap/) with the parameters qtrim2=t trimq=10, 15, 20 minq=12 mininsert=150. Read pairs that could not be merged were discarded. The merged reads were downsampled to 300,000 reads using reformat.sh in the bbmap package (samplereadstarget=300000). The merged and downsampled reads were assembled with Geneious 8 (set to Medium Sensitivity/Fast) (http://www.geneious.com/). Finally, contigs composed of more than 100 merged reads were retained for further analysis, with most of these composed of over 1000 reads. In this way, a total of 1457 contigs were generated (Table 6).
Approximately 220 contigs were obtained from each accession, except for Triticum timopheevii for which only 138 contigs were assembled. This is consistent with the tetraploid nature of the Triticum timopheevii genome. The consistency of the results indicates that the RFL-capture experiment was, a priori, comprehensive.
Triticum
timopheevi
Sequences encoding RFL proteins were identified within these contigs as follows.
Open reading frames (ORFs) within the contigs were identified with getorf from the EMBOSS package (Rice et al. 2000) using the parameters -minsize 630 -find 0 -reverse true. Thus only ORFs longer than 210 amino acids were used for further analysis. In total, 1554 ORFs were identified across the seven accessions (Table 6). The number of ORFs per accession ranged from 143 ORFs in T. timopheevii to 245 in R0934F (Table 6). The ORFs were further analyzed using hmmsearch (with parameters -E 0.1 --domE 100) from the HMMER package (v3.1b1) to detect PPR motifs using the hidden Markov models developed by Cheng et al. (2016) and the post-processing steps described in the same paper. Finally, to identify putatively orthologous RFL sequences across all seven accessions, the 1554 RFL ORFs were clustered using CD-hit (settings -c 0.96 -n 5 -G 0 -d 0 -AS 60 -A 105 -g 1). Across all accessions, 397 non-redundant RFL clusters representing putatively orthologous groups were obtained (Table 6). We define an orthologous RFL group as a set of at least one RFL ORF from at least one accession and wherein, if at least two sequences are present, these sequences share at least 96% sequence identity over the alignment length. Some highly conserved RFL genes are present in all seven accessions, others are found in only a subset of the accessions, or in a single accession. In most cases, each orthologous RFL group contains only one RFL protein from each accession.
Table 7 below is showing the different RFL groups, the corresponding ORF names and the corresponding protein sequence number and DNA encoding protein sequence number.
However, we found that genes encoding RFL-PPR proteins are often inactivated by indels creating frameshifts that break the contiguity of the ORFs, resulting in two shorter ORFs corresponding to a single longer ORF in another accession. In these cases, both shorter ORFs could be within the same orthologous group. In our analysis, only ORF encoding more than 350 amino acids were considered as possibly functional. This threshold was used as it corresponds to 10 PPR motifs (each of 35 amino acids), and all known active Rf proteins contain at least this number of motifs (usually 15-20).
Finally, a set of 397 orthologous RFL groups were identified and numbered from 1 to 397 (see Table 7 below).
A. Fine-Mapping of the Genomic Region Containing Rf1 Genetic Determinants
Three F2 mapping populations segregating for Rf1 (R197xKalahari, R204xAlixan and R0932ExAltigo) encompassing 210, 218 and 212 individuals respectively were phenotyped and genotyped with 18100 SNP markers using Limagrain's internal genotyping platform.
Fertility tests were conducted indoors under controlled growth conditions, either in growth chambers or in greenhouses, enabling normal fertility of the tested wheat plants. The fertility scores indicated have been calculated by dividing the total number of seeds threshed from a spike by the number of counted spikelets. The t-tests conducted were done by comparing the fertility scores of F1s made with a restorer and the fertility scores of a panel of elite inbred lines grown under the same conditions.
Rf1 was first mapped on the short arm of the chromosome 1A between 4 cM and 10.9 cM on Limagrain's internal consensus map and physically delimited by SNP markers cfn1087371 and cfn0530841. These two SNP markers delimit the largest possible interval defined by the three mapping populations.
Subsequently, joint analysis of the three mapping populations and phenotyping of the individual F2 recombinant plants on derived F3 families validated the QTL position and delimited the Rf1 interval between 7 cM and 8.9 cM on Limagrain's internal consensus map and physically delimited by SNP markers cfn1082074 and cfn0523990. We used the genomic resources of the IWGSC Whole genome assembly, ‘IWGSC WGA’ (available from June 2016 from the URGI IWGSC repository) to anchor the locus to the wheat genome reference physical map. The left border (cfn1082074) was anchored on the IWGSCWGAVO2_1AS_scaffold44309 scaffold and the right border (cfn0523990) was anchored on the IWGSCWGAV02_1AS_scaffold47238 scaffold.
Next, the locus was fine-mapped by screening 2976 and 3072 F3 lines from R197xKalahari and R204xAlixan derived from F2 plants heterozygous at the locus. Phenotyping and analysis of recombinant plant progenies within the interval redefined a smaller mapping interval between 7.5 and 8.8 cM delimited by cfn0522096 and cfn0527067 SNP markers on the IWGSCWGAV02_1AS_scaffold44309 scaffold and the IWGSCWGAV02_1AS_scaffold47238 scaffold, respectively.
B. Fine-Mapping of the Genomic Region Containing Rf3 Genetic Determinants
Three F2 mapping populations (TJB155xAnapurna, 2852xAltamira, and AH46xR0946E) encompassing 217, 135, and 246 individuals respectively and a doubled-haploid (DH) population (H46xR934F) consisting of 140 individual plants segregating for Rf3 were phenotyped as described in example 1, and genotyped with 18100 SNP markers using Limagrain's internal genotyping platform. Rf3 was first mapped on the short arm of the chromosome 1B between 18.9 cM and 24.2 cM on Limagrain's internal consensus map and physically delimited by SNP markers cfn0554333 and cfn0560679. These two SNP markers delimit the largest possible interval defined by the four mapping populations.
Subsequently, joint analysis of the four mapping populations and validation of the phenotype of the individual F2/DH recombinant plants on derived F3 families validated the QTL, genetically delimited the locus between 22.2 cM and 22.7 cM on Limagrain's internal consensus map, and physically delimited the Rf3 interval between SNP markers cfn0436720 and cfn0238384. We used the genomic resources of the IWGSC Whole genome assembly, ‘IWGSC WGA’ (available from June 2016 from the URGI IWGSC repository) to anchor the locus to the physical map. The left border (cfn0436720) was anchored on the IWGSCWGAV02_1BS_scaffold35219 scaffold and the right border (cfn0238384) was anchored on the IWGSCWGAV02_1BS_scaffold5117 scaffold.
Next, the locus was fine-mapped by screening 2496 and 672 plants from TJB155xAnapurna and AH46xR0946E F2 plants heterozygous at the locus. Analysis of recombinant F3 plant progenies within the interval redefined a smaller mapping interval between 22.5 and 22.7 cM delimited by cfn1249269 and BS00090770 SNP markers on the IWGSCWGAV02_1BS_scaffold35219 scaffold and the IWGSCWGAV02_1BS_scaffold5117 scaffold, respectively.
C. Mapping of the Genomic Region Containing Rf7 Genetic Determinants
We crossed R197 and Primepii and then derived a population of 176 plants from individuals that were rf1 and rf3, i.e. not carrying the restorer alleles at the loci Rf1 and Rf3. The plants were genotyped with 18100 SNP markers using Limagrain's internal genotyping platform and phenotyped as described in example 1. We mapped the Rf7 locus on chromosome 7BL. Moreover, internal genotyping data showing a strong genetic divergence suggests the presence of an exotic chromosomal fragment which is stably transmitted through generations. We identified a large QTL ranging from 45 cM to 88 cM on chromosome 7B on Limagrain's internal consensus map with a peak on 46.7 cM (cfn0919993 with LOD score of 3.37E-40). Initial analysis of the recombinant plants suggests the Rf7 gene could be located between cfn3407185 and W90K_RAC875_c33564_120 markers delimiting a mapping interval of 0.3 cM between 46.7 cM and 47 cM on Limagrain's internal consensus map.
For each of the 282 RFL groups identified in example 2, the captured RFL ORFs (in the following referred to as protein sequences) were identified and the total number of RFL protein sequences is reported for each accession (Table 7, Table 8).
Only the following RFL clusters will be taken into consideration:
Tables 8A, 9A, 10 and 11 show the lists of the orthologous RFL groups correlating respectively with Rf1, Rf3, Rf7 and Rf-Rye-6R genes after the first screen. T. timopheevii is known to be fertile and, as a consequence, to restore T-CMS. This line is added here as it could contain any of the target Rf1, Rf3, Rf7 or Rf-rye genes.
Finally, only the orthologous RFL groups mapping in the chromosomal interval in wheat Triticum aestivum Chinese Spring reference genome are considered as candidates for further analyses. These orthologous RFL groups will be selected as “candidate Rf groups”.
Mapping was achieved using the tool tblastn from the BLAST+ Suite (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download). Specific parameters (-evalue 1e-25 -best_hit_score_edge 0.05 -best_hit_overhang 0.25) were used in order to keep all best hits.
A. Results for Rf1 Accessions:
Table 8A shows the orthologous RFL groups comprising at least one sequence captured from an accession characterized as bearing the Rf1 gene (R197, R0932E and T. timopheevii). The mapping described in example 3 allows us to discard orthologous RFL groups mapped outside of the chromosomal interval genetically associated with Rf1 fertility on the short arm of chromosome 1A. In this way, four RFLs clusters (79, 104, 185 and 268) were identified as potentially corresponding to the Rf protein encoded by the Rf1 gene.
All protein sequences from group RFL185 contain ˜500 amino acids and only 8.5 PPR motifs. Typically, full-length functional RFL proteins are expected to contain 15-20 PPR motifs. In addition, the last PPR motif of RFL185 is composed of only 15 amino acids. This indicates that RFL185 is truncated. RFL268 is also truncated (382 amino acids). Detailed sequence analysis has shown that RFL185 and RFL268 are remnants of the same gene that was split by a frameshift. Thus, both proteins are unlikely to be functional.
Hence RFL79 and RFL104 are considered as being the best candidate Rf groups for Rf1.
Triticum
timopheevii
It can be noted from Table 8A that the only accession lacking Rf1 containing sequences in these candidate Rf groups is the accession R0934F.
The DNA sequences derived from the contigs identified in example 2 and encoding RFL proteins from candidate Rf groups 79 and 104 were aligned with BWA-MEM software (Li H. and Durbin R., 2010). It was observed that these sequences differ in the 5′ UTR region in R0934F compared to R0932E and R197. One hypothesis is that the DNA sequences in R0934F were generated by a recombination event between the DNA sequences from candidate Rf groups 79 and 104. This recombined sequence may not be functional in R0934F as this line is only known to carry Rf3.
B. Results for Rf3 Accessions:
The same rationale as for the Rf1 accessions was applied to the accessions (Primepii and R0934F) characterized as carrying the Rf3 gene. Table 9A shows that orthologous RFL groups 67, 89, 140, 166 and 252 are candidate Rf groups for the Rf protein encoded by Rf3 as they contain proteins identified in Primepii and R0934, the two accessions characterized as carrying Rf3 restorer gene and are located in the mapped genetic interval on the short arm on chromosome 1B.
Triticum
timopheevii
In order to achieve an exhaustive analysis for selection of Rf3 candidates, all 282 RFL clusters were carefully analyzed with regard to number of RFLs, their length and their origin in relation to Rf3 genotype information. RFL clusters composed of RFL sequences from multiple accessions were screened for full-length protein sequences originating only from Primepii and R0934F genotypes and partial/shorter sequences from the non-Rf3-carrying genotypes. This analysis allowed the identification of four additional Rf3-candidate RFL clusters: RFL28, RFL29, RFL60 and RFL170.
Table 9B shows that:
In candidate group RFL 28, the sequences from Rf3 genotypes (Primepii and R0934F) are 857 amino acids long whereas sequences from all other germplasms (non-Rf3) are truncated (sizes ranging from 323-479 amino acids) and thus are most probably nonfunctional.
In candidate group RFL 29, the sequences from Rf3 accessions (Primepii and R0934F) are 828 amino acids long whereas sequences from all other (non-Rf3) germplasms are truncated and thus most probably nonfunctional.
In candidate group RFL 60, the sequences in non-Rf3 genotypes (R197 and R0932E) are either absent (R0932E) or deemed nonfunctional due to their amino acid length (287 amino acids). The sequences from Rf3 genotypes Primepii and R0934F based on their sequence length appear to be full length and functional. In addition, our mapping analysis positioned RFL60 cluster within the Rf3 interval.
In cluster RFL 170, the sequences from Rf3 genotypes (Primepii and R0934F) are significantly larger (560 amino acids) than sequences from non Rf3 genotypes that appear truncated (below 370 amino acids) and are considered as being nonfunctional. Our detailed sequence analysis has shown that RFL170 is actually a second ORF, in addition to RFL 288, encoded by the same contig. Both ORFs originate from the same RFL gene in which contiguity was disrupted by a frameshift.
C. Rf7 and Rf-Rye Accessions:
The same rationale as for the analysis of Rf1 and Rf3 carrying accessions was applied to the accessions carrying the Rf7 restorer gene (R197 and T. timopheevii). The Rf7 gene was mapped on chromosome 7BL.
Table 10 shows that RFL 80, 128 and 191 are candidate Rf groups for the Rf protein encoded by the Rf7 gene as the proteins assigned to those clusters were found only in either R197 or T. timopheevii.
In regard to a restorer gene that originates from the introgression of rye chromosome 6R into wheat genome, clusters composed of single proteins originating from the Wheat-Rye-6R restorer line are considered as good candidates for a Rye-6R-specific restorer gene. Those criteria are true for the RFL 46, 87 and 208 orthologous groups listed in Table 6. Due to their high sequence divergence compared to Triticum sequences these genes are great candidates for restorer genes originating from rye.
Triticum
timopheevii
Triticum
timopheevii
The nucleic acid sequence encoding any of the RFL proteins from a candidate Rf group could be used for cloning and transformation. However, in the present experiment for cloning and transformation purposes, a DNA sequence encoding the longest RFL protein which was characterized as having a start codon, mitochondrial targeting sequence and number of PPR motifs between 15 and 20 was preferentially used. If at least two longest RFL proteins happen to have the same length, the nucleic acid encoding such RFL protein, and presenting the longest 5′-UTR sequence will be preferentially chosen to perform the cloning and transformation steps.
Wheat-Rye-6R RFL46 sequence derived from Wheat-Rye-6R.300k_Assembly_Contig_35_1 was optimized to provide SEQ ID No3115. This sequence was cloned via a Golden Gate reaction into the destination binary plasmid pBIOS10746, between the constitutive Zea mays ubiquitin promoter (proZmUbi depicted in SEQ ID No 3134) with the Zea mays ubiquitin intron (intZmUbi, depicted in SEQ ID No 3109, Christensen et al 1992) and a 3′ termination sequence of the gene encoding a sorghum heat shock protein (accession number: Sb03g006880); The termination sequence, named terSbHSP, is depicted in SEQ ID No 3110. The sequence of the recombinant construct is depicted in SEQ ID No 3125.
Similarly as above, TaRFL104 sequence (derived from R0932E.300k_Assembly_Contig_82_1), TaRFL67 sequence (derived from Primepii.300k_Assembly_Contig_2_1), TaRFL79 sequence (derived from R197.300k_Assembly_Contig_120_1) and TaRFL89 sequence (derived from R0934F.300k_Assembly_Contig_99_1) were adapted for cloning purpose as depicted respectively in SEQ ID No 3117 to 3120. These sequences were cloned via a Golden Gate reaction into the destination binary plasmid pBIOS10746. The sequences of the recombinant constructs are respectively depicted in SEQ ID No 3131, 3128, 3129 and 3130.
TaRFL104 sequence (depicted in SEQ ID No 3117) was cloned via restriction enzyme reaction, between the native Triticum aestivum promoter (proTaRFL104, SEQ ID No 3113) and the 3′ termination sequence of Triticum aestivum RFL104 encoding gene (terTaRFL104, depicted in SEQ ID No 3112), into the destination binary plasmid pBIOS10747. The sequence of the recombinant construct is depicted in SEQ ID No 3126.
TaRFL79 sequence (depicted in SEQ ID No 3119) was cloned via restriction enzyme reaction, between the native Triticum aestivum promoter (proTaRFL79, SEQ ID No3123) and the 3′ termination sequence of T. aestivum RFL79 encoding gene (terTaRFL79, depicted in SEQ ID No 3124), into the destination binary plasmid pBIOS10747. The sequence of the recombinant construct is depicted in SEQ ID No3122.
Wheat-Rye RFL46 sequence was also cloned via restriction enzyme reaction, between the native Triticum aestivum promoter (proTaRFL46, SEQ ID No 3114) and the 3′ termination sequence of Triticum aestivum RFL46 encoding gene (terTaRFL46, depicted in SEQ ID No 3111), into the destination binary plasmid pBIOS10747. For this construct, Wheat-Rye RFL46 sequence is derived from Wheat-Rye-6R.300k_Assembly_Contig_35_1 coding sequence and modified for cloning purpose without any optimization steps. The coding sequence is depicted in SEQ ID No3116. The sequence of the recombinant construct is depicted in SEQ ID No3127.
The binary destination vectors pBIOS10746 and pBIOS10747 are a derivative of the binary vector pMRT (WO2001018192A3).
All the binary plasmids described above were transformed into Agrobacterium EHA105.
In order to screen for candidate genes involved in fertility restoration, the BGA_Fielder_CMS wheat cultivar harboring both cytoplasmic male sterility and strong transformability and regeneration potential was developed. BGA_Fielder_CMS wheat cultivars were transformed with Agrobacterium strains obtained in example 5 essentially as described by WO 2000/063398. Wheat transgenic events were generated for each construct described above.
For construct comprising RFL46, transformation was also performed with the cultivar Fielder.
All wheat transgenic plants generated in example 6 and control fertile plants were grown in a glasshouse under standard wheat growth conditions (16 h of light period at 20° C. and 8 h of dark period at 15° C. with constant 60% humidity) until control grains of the wild type Fielder cultivar reached maturity stage.
Fertility of the transgenic plants was evaluated by counting the number of seeds and empty glumes per spikes on each plant and comparing with the wild type Fielder and BGA_Fielder_CMS control plants. Plants are also evaluated by observing anther extrusion.
16 transformed CMS-Fielder plants overexpressing the RFL79 sequence recited in SEQ ID No361 (as listed in table 7) under the ZmUbi promoter derived from 11 independent transformation events were analyzed.
All the plants present restoration of male fertility while 100% of untransformed CMS-Fielder plants grown in parallel are fully sterile with no anther extrusion and no seed produced, and 100% of WT-Fielder plants are fertile.
These results confirm that RFL79 can restore fertility of a CMS-T plant and that genetic transformation of CMS-Fielder is an efficient system to test the function of restorer-of-fertility genes.
The E. coli beta-glucuronidase (EcGUS) sequence was optimized (as depicted in SEQ ID No3121) and cloned via restriction enzyme reaction, between the native Triticum aestivum promoter (proTaRFL46, SEQ ID No 3114) and the 3′ termination sequence of T. aestivum gene encoding RFL46 (terTaRFL46, depicted in SEQ ID No 3111), into the destination binary plasmid pBIOS10743 forming pBIOS11468.
Fielder wheat cultivars were transformed with these Agrobacterium strains essentially as described by WO 2000/063398. Wheat transgenic events were generated for each construct described above.
After booting stage until anthesis, heads and floral organs were dissected and incubated in X-Gluc solution (Jefferson, 1987) at 37° C. for 16 hours, to assess GUS expression.
A second capture was achieved using a set of different accessions compared to the capture performed in example 2. The following accessions were used:
GSTR435 is derived from an introgression of Aegilops speltoïdes into Triticum aestivum and is available at USDA (https://npgsweb.ars-grin.gov/gringlobal/search.aspx). The three other accessions, R113 (available via Australian Grains Genebank: 90819), L13 (available via Australian Grains Genebank: 90821) and 17F3R-0377 (derived from R113) are all derived from Triticum timopheevi introgressions into Triticum aestivum.
The bait design and hybridization with DNA fragments from the accessions were performed as in example 2. Then, a subset of 100K read pairs from each accession were mapped to the RFL groups identified in table 7 using Novoalign (version 3.04.06, http://www.novocraft.com/products/novoalign/) with settings allowing multiple hits with approximately 97% of identity (options: -r all -t 240). The average coverage per RFL was then calculated using Bedtools utilities (version 2.26.0, http://github.com/arq5x/bedtools2) coverageBed (option: -d) and groupBy (options: -o mean).
The relative coverage of all RFLs with the reads from each accession was assessed. A first ranking of the RFLs according to their coverage with reads from accessions derived from T. timopheevii was assessed. Only RFL groups showing no coverage (value from 0 to 10) with reads from “non-Rf” (maintainer) or non-Rf4 accessions but showing significant coverage (value >30) with reads from Rf4 accessions were considered.
The coverage with accession GSTR435 was also assessed.
In order to investigate further the sequences related to the RFL120 group, the reads mapping to RFL120 for each Rf4 accession were then assembled in two steps. The first step consisted of merging overlapping read pairs with the utility bbmerge.sh from the BBMAP package (version 36.59 https://sourceforge.net/projects/bbmap/) and assembling them with the utility tadwrapper.sh from the same package (options: k=150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450 bisect=t). The contigs from the first step were deduplicated with the tool dedupe.sh also from the same package and given to another assembler, SPADES (version 3.10.1 http://bioinf.spbau.ru/spades), as “trusted contigs” along with all read pairs from the same accession (options: --coy-cutoff 5 -careful) to generate the final assembly of the accession. Then for each accession the protein sequence RFL120 (SEQ ID No 477 as listed in table 7) best hit was searched using the tblastn utility from the BLAST+ package (version 2.2.30 https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download) using default settings.
The following sequences were finally identified to be included in the RFL120 group:
RFL120-R113 which is depicted in SEQ ID No3138 and is encoded by SEQ ID No3142, RFL120-L13 which is depicted in SEQ ID No3137 and is encoded by SEQ ID No3141, RFL120-17F3R-0377 is depicted in SEQ ID No3135 and is encoded by SEQ ID No3139 and finally RFL120-GSTR 435 (called here RFL120-spelt) is depicted in SEQ ID No3136 and is encoded by SEQ ID No3140.
Alignment between the above amino acid or nucleotide sequences with the corresponding sequences from RFL120, called here “RFL120_timo”, and recited in SEQ ID No477 and SEQ ID No2031 (see table 7) shows that RFL120-17F3R-0377 is truncated. Regarding RFL120-R113 and RFL120-L13, the nucleotide sequences are both identical to RFL120-timo except that they are respectively 357 and 349 nucleotides longer on the 5′UTR region.
In conclusion, the results confirm that the RFL120 group is the strongest candidate for Rf4.
Following the same methods as described in Examples 5 and 6, the nucleotide sequences of RFL120-timo, RFL120-spelt, RFL120-R113 and RFLK120-L13 are, when appropriate, optimized for ensuring proper expression in wheat and adapted for cloning purposes.
These sequences are cloned via a Golden Gate reaction into the destination binary plasmid pBIOS10746, between the constitutive Zea mays ubiquitin promoter (proZmUbi depicted in SEQ ID No 3134) with the Zea mays ubiquitin intron (intZmUbi, depicted in SEQ ID No 3109, Christensen et al 1992) and a 3′ termination sequence of the gene encoding a sorghum heat shock protein (accession number: Sb03g006880); The termination sequence, named terSbHSP, is depicted in SEQ ID No 3110.
Transformation and fertility assays are performed as in Example 6.
Eleven wheat elite lines classified as carrying Rf3 restorer gene known to be involved in restoration of the T-type cytoplasmic male sterility in Triticum timopheevii (T-CMS) were assessed for their capacity to restore the T-CMS cytoplasm and characterized for their fertility genotype. Hybrids resulting from crosses between a sterile CMS wheat line, used as a female parent, and a given wheat restorer line, used as a pollen donor, were studied regarding their ability to produce grain. The elite lines included both commercially available lines such as Altigo, Aristote, Cellule, Altamira, Rubisko, Primepii or Premio as well as Limagrain proprietary lines (Table 12). In addition, Chinese Springlines were included in the study (Table 12).
Fertility tests have been conducted indoors, either in growth chamber or in greenhouse, under controlled growth conditions enabling a normal expression of the fertility phenotype of the tested wheat plants. The plants were grown under 16 hours light period and temperature between 20 and 25° C. and 8 hours dark period at a temperature between 15° C. and 20° C., with humidity between 50 and 70%. The observed restoration of pollen fertility may be partial or complete.
The fertility score of F1 wheat plants carrying T-CMS cytoplasm may be calculated by dividing the total number of seeds threshed from a spike by the number of counted spikelets and may be compared with the fertility scores of a panel of control fertile plants which in this study consists of elite inbred lines bearing a normal wheat cytoplasm, grown in the same area and under the same agro-environmental conditions. It is preferred that such panel of lines comprises a set of at least 5 elite inbred lines. Besides, it is preferred that at least 10 spikes from different F1 individual plant will be assessed for a given experiment.
Fertility score i higher than zero (>0) indicates that the plant has acquired partial or full fertility restoration. For each fertility score, a statistical test is achieved to obtain a p-value. Examples of statistical tests are the Anova or mean comparison tests. A p-value below a 5% threshold will indicate that the two distributions are statistically different. Therefore, a significantly lower fertility score of the tested wheat plant as compared to the fertility score of the fertile control plant is indicative that the F1 plant has not acquired full restoration fertility (i.e. partial restoration). A significantly similar or higher fertility score is indicative that the F1 plant has acquired full restoration of fertility. The fertility scores indicated were calculated by dividing the total number of seeds threshed from a spike by the number of counted spikelets.
The t-tests were conducted by comparing the fertility scores of F1 plants carrying Rf3 restorer gene and the fertility scores of a panel of elite inbred lines grown under the same conditions (633 spikes from 37 winter and spring elite lines; p=2.36, a=0.59).
The results presented in Table 12 show that there are two types of partial Rf3 restorer of fertility in the CMS hybrids. One that can be referred to as “Rf3” and which fertility scores are comprised between 1 and 1.8, and a second type referred to as “Rf3 weak” which fertility scores are less than 1. For example, the CMS-hybrids made with Primepii produced an average fertility score of 1.7 grains/spikelet over 10 individual F1 spikes (“Rf3” phenotype) while hybrids made with Altigo produced an average fertility score of 0.7 grains/spikelet over 47 individual F1 spikes (“Rf3 weak” phenotype) (Table 12). Different Chinese Spring lines were shown by genetic mapping and marker assisted selection to harbor an Rf3 restorer locus (data not shown). CMS-hybrids made with Chinese Spring lines were evaluated for fertility score. They show a mean estimated fertility score of 0.6 grains/spikelet and a “Rf3 weak” phenotype.
The RFL gene capture was achieved with accessions listed in Table 12 as described in Example 2. For each RFL identified in Example 4B, the corresponding protein sequences from each accession were aligned for comparison.
The results show that for RFL29, RFL164 and RFL166, strong association between the phenotype and the genotype exist. For RFL29, three different alleles referred to as “a”, “b” and “c” were identified while two different alleles “a” and “b” are identified either for RFL164 or RFL166. All accessions with an “Rf3” phenotype carry RFL29a, RFL164a and RFL166a alleles while all the “Rf3 weak” accessions carry RFL29b, RFL166b and RFL164b alleles in their genotype
For RFL29, the maintainer line Atomo is characterized by the presence of two truncated ORFs, probably due to a frameshift mutation, RFL29c_1 and RFL29c_2, encoding proteins consisting of 258 and 535 amino acids, respectively. This genotype form is only present in maintainer lines (data not shown).
Following the same methods as described in Examples 5 and 6, the nucleotide sequences of RFL29a (depicted in SEQ ID No3146 or SEQ ID No1712 and encoding a sequence identical to SEQ ID No158), RFL29b (depicted in SEQ ID No 3149 and encoding a sequence identical to SEQ ID No3143), RFL164a (depicted in SEQ ID No 3147 or SEQ ID NO 2230 and encoding a sequence identical to SEQ ID No676), and RFL166a (depicted in SEQ ID No 3148 or SEQ ID NO:2238 and encoding a sequence identical to SEQ ID No684), were cloned via a Golden Gate reaction into destination binary plasmid pBIOS10746, between the constitutive Zea mays ubiquitin promoter (proZmUbi depicted in SEQ ID No 3134) with the Zea mays ubiquitin intron (intZmUbi, depicted in SEQ ID No 3109, Christensen et al 1992) and a 3′ termination sequence of the gene encoding a sorghum heat shock protein (accession number: Sb03g006880 terSbHSP depicted in SEQ ID No 3110). The sequences of each of the recombinant constructs are respectively depicted in SEQ ID No3150, SEQ ID No3151, 3152 and 3153.
Similarly, the RFL29a and RFL29b sequences were cloned downstream of their endogenous promoter pRFL29a (depicted in SEQ ID No3154) and pRFL29b (depicted in SEQ ID No3155) and the terminator sequence terSbHSP. The sequences of each of the recombinant construct are respectively depicted in SEQ ID No3156 and SEQ ID No3157.
Finally, two other cassettes were made identically as described previously with the only exception that the corresponding endogenous terminator sequences terRFL29a (depicted in SEQ ID No3160) and terRFL29b (depicted in SEQ ID No3161) are used. The corresponding expression cassettes are respectively depicted in SEQ ID No3158 and SEQ ID No3159. Transformation and fertility assays are performed as in Example 6.
In order to analyze whether the variation of the level of fertility could be explained by a variation in the gene expression level, the 5′UTR sequence of RFL29a gene was isolated from BACs generated from TJB155 line (Table 12) classified as “Rf3” and the 5′UTR sequence of RFL29b was identified from the Chinese Spring classified as “Rf3weak” line (IWGSC RefSeq v1.0 assembly).
The alignment of the 5′UTR regions identified in the RFL29a and RFL29b genes is shown in
Sequence comparison shows that the 5′UTR sequence of the RFL29a gene comprises a deletion of the 163 bp-long region identified in the 5′UTR of RFL29b corresponding sequence (SEQ ID NO: 3174). Part of this region has been identified in the patent application WO2018015403 as being putatively involved in miRNA-mediated repression of the expression of the PPR gene identified downstream.
Sequence comparison between the different accessions listed in Table 12 shows that all “Rf3weak” accessions harbor the 163 bp and all the “Rf3” accessions harbor the 163 bp deletion.
Because of the 163 bp deletion in the 5′UTR sequence of RFL29a gene, it is expected that the 163 bp region impairs the expression of RFL29b gene such that the fertility level is weak in lines harboring the RFL29b allele compared to lines harboring the RFL29a allele.
Following the same methods as described in Example 5, the nucleotide sequence of RFL29b was expressed under the modified promoter pRFL29bdel (depicted in SEQ ID No3162) which is bearing a deletion of the 163 bp region, from the nucleotide 1876 to nucleotide 2038 of the RFL29b promoter sequence depicted in SEQ ID No3155. The termination sequence, terSbHSP, depicted in SEQ ID No3110 is used. The recombinant construct is depicted in SEQ ID No3163.
Transformation of CMS*Fielder wheat line (which is either not “Rf3” or “Rf3 weak”) is performed as in Example 6 except that the following controls are added: all of the “Rf3” phenotype lines as listed in Table 12, all of the “Rf3 weak” lines as listed in Table 12 and finally the transformed lines with the cassette harboring the pTaRFL29b promoter upstream to RFL29b as described in example 12.
In order to increase the expression of RL29b, different deletions of the 163 bp are performed in the promoter of RFL29b using endonuclease for site-directed mutagenesis.
Table 13 provides the different endonucleases with the associated PAM motif and the corresponding target sequences.
The nucleotide sequence encoding for LbCpf1 endonuclease is optimized (as depicted in SEQ ID No3164) and cloned via a Golden Gate reaction into the destination binary plasmid pBIOS10746, between the constitutive Zea mays ubiquitin promoter (proZmUbi depicted in SEQ ID No 3134) with the Zea mays ubiquitin intron (intZmUbi, depicted in SEQ ID No 3109, Christensen et al 1992) and in the 3′ region, the mouse nuclear import NLS sequence (as depicted in SEQ ID No 3172) and the 3′ termination sequence terZmHSP depicted in SEQ ID No 3170.
The nucleotide sequence encoding for SpCas9 endonuclease is optimized (as depicted in SEQ ID No 3165) and cloned via a Golden Gate reaction into the destination binary plasmid pBIOS10746, downstream to the constitutive Zea mays ubiquitin promoter (proZmUbi depicted in SEQ ID No 3134) with the Zea mays ubiquitin intron (intZmUbi, depicted in SEQ ID No 3109, Christensen et al 1992) and the SV40NLS sequence (as depicted in SEQ ID No 3173) and upstream of the mouse nuclear import NLS sequence and the 3′ termination sequence terAtNos depicted in SEQ ID No 3171.
Each guide sequence is cloned between the pTaU6 promoter (depicted in SEQ ID No 3168) and the termination sequence TerRNApolIII (depicted in SEQ ID No3169).
Each cassette expressing the endonuclease is cloned consecutively to the cassette expressing the corresponding guide sequence. A recombinant cassette expressing LbCpf1 and guide sequences directed to target -23 and target -71 is depicted in SEQ ID No3167. A recombinant cassette expressing SpCas9 and guide sequences directed to target −58 and target −54 is depicted in SEQ ID No3166.
Transformation is performed as in Example 6 except that all “Rf3 weak” lines as listed in Table 12 are transformed. Fertility assays to select lines with an “Rf3” phenotype are performed as in example 14.
Some wheat elite lines possess an ability to partially restore the fertility of the cytoplasmic male sterility Triticum timopheevii (T-CMS). The hybrid formed between a sterile CMS wheat line taken as female and the wheat restorer line taken as male can produce grain. Commercial lines as Allezy, Altamira, Altigo, Aristote, Osado, Cellule or Premio and Limagrain proprietary lines have been tested for their capacity to restore T-CMS. All lines have also been characterized for their fertility genotype.
Fertility tests have been conducted indoor, either in growth chamber or in greenhouse, under controlled growth conditions enabling a normal expression of fertility of the tested wheat plants. The fertility scores indicated have been calculated by dividing the total number of seeds threshed from a spike by the number of counted spikelets. The t-tests conducted were done by comparing the fertility scores of F1s made with a restorer and the fertility scores of a panel of elite inbred lines grown under the same conditions (633 spikes from 37 winter and spring elite lines; p=2.36, a=0.59).
The results in Table 14 show that these lines act as partial restorer of fertility in a CMS hybrid. For example, the hybrids made with Allezy produce an average fertility of 0.99 grains/spikelets over 33 individual F1 spikes. This native ability to partially restore the fertility of the CMS T. timopheevii was previously identified in wheat accessions as Primepii or Maris Hunstmann (Bahl and Maan, 1973), and it is confirmed by our own internal data (See Table 14). Beside this supposedly wheat intrisic source of restoration, the hybridizations between Triticum timopheevii and Triticum aestivum conducted by Wilson (Wilson and Ross 1962) contributed to the introduction into wheat of further restorer genes which chromosome arm localization was identified through monosomic analysis (Bahl and Maan, 1973; Maan and al, 1984).
Following these initial works many breeding programs implemented worldwide aimed at creating restorer lines for the T-CMS such as TJB155 (BBSRC Small Grain Cereals Collection: 2072 to 2075), which ability to restore is also confirmed by our own internal data (Table 14).
L13 (available via Australian Grains Genebank: 90821), is a wheat restorer line selected from R113 (available via Australian Grains Genebank: 90819) which carries the restorer allele Rf4. It produces a low level of fertility restoration when crossed with CMS lines (0.85 seeds/spikelet on average). When compared with the fertility scores of a panel of elite lines grown under the same conditions it appears that none of the tested putative restorer make possible a full restoration of fertility of the hybrid (p-values <0.05. table 14).
Table 14 also shows that restorer lines bearing two different Rf loci, are not able to fully restore the sterility induced by T-CMS. Therefore, all restorer lines tested, with single or two Rf loci are only partial T-CMS restorer lines.
A. Fine-Mapping of the Genomic Region Containing Rf1 Genetic Determinants
Three F2 mapping populations segregating for Rf1 (R197xKalahari, R204xAlixan and R0932ExAltigo) encompassing 210, 218 and 212 individuals respectively were phenotyped as described in example 1 and genotyped with 18100 SNP markers using Limagrain's internal genotyping platform. Fertility of R204 and R197 lines is genetically associated to Rf1 and Rf7 locus. Fertility in R0932E line is associated to Rf1 locus.
Rf1 was first mapped on the short arm of the chromosome 1A between 4 cM and 10.9 cM on Limagrain's internal consensus map and physically delimited by SNP markers cfn1087371 and cfn0530841. These two SNP markers delimit the largest possible interval defined by the three mapping populations (see
Following, joint analysis of the three mapping populations and phenotyping of the individual F2 recombinant plants on derived F3 families validated the QTL position and delimited Rf1 interval between 7 cM and 8.9 cM on Limagrain's internal consensus map and physically delimited by SNP markers cfn1082074 and cfn0523990. We used the genomic resources of the IWGSC Whole genome assembly, ‘IWGSC WGA’ (available from June 2016 from the URGI IWGSC repository) to anchor the locus to the wheat genome reference physical map. The left border (cfn1082074) was anchored on the IWGSCWGAV02_1AS_scaffold44309 scaffold and the right border (cfn0523990) was anchored on the IWGSCWGAV02_1AS_scaffold47238 scaffold.
Next, we decided to enlarge the population sizes to fine-map the locus and screened 2976 and 3072 F3 lines from R197xKalahari and R204xAlixan derived from F2 plants heterozygote at the locus respectively. Phenotyping and analysis of recombinant plant progenies within the interval redefined a smaller mapping interval between 7.5 and 8.8 cM delimited by cfn0522096 and cfn0527067 SNP markers on the M/GSCWGAV02_1AS_scaffold44309 scaffold and the M/GSCWGAV02_1AS_scaffold47238 scaffold respectively.
B. Fine-Mapping of the Genomic Region Containing Rf3 Genetic Determinants
Three F2 mapping populations (TJB155xAnapurna, 2852xAltamira, and AH46xR0946E) encompassing 217, 135, and 246 individuals respectively and a doubled-haploid (DH) population (H46xR934F) consisting of 140 individual plants segregating for Rf3 were phenotyped as described in example 1 and genotyped with 18100 SNP markers using Limagrain's internal genotyping platform. Sources of Rf3 locus are TJB155, Altamira and R0946E.
Rf3 was first mapped on the short arm of the chromosome 1B between 18.9 cM and 24.2 cM on Limagrain's internal consensus map and physically delimited by SNP markers cfn0554333 and cfn0560679. These two SNP markers delimit the largest possible interval defined by the four mapping populations (see
Following, joint analysis of the four mapping populations and validation of the phenotype of the individual F2/DH recombinant plants on derived F3 families validated the QTL, genetically delimited the locus between 22.2 cM and 22.7 cM on Limagrain's internal consensus map and physically delimited the Rf3 interval between SNP markers cfn0436720 and cfn0238384. We used the genomic resources of the IWGSC Whole genome assembly, ‘IWGSC WGA’ (available from June 2016 from the URGI IWGSC repository) to anchor the locus to the physical map. The left border (cfn0436720) was anchored on the IWGSCWGAV02_1BS_scaffold35219 scaffold and the right border (cfn0238384) was anchored on the IWGSCWGAV02_1BS_scaffold5117 scaffold.
Next, we decided to enlarge the population sizes to fine-map the locus and screened 2496 and 672 plants from TJB155xAnapurna and AH46xR0946E derived F2 plants heterozygote at the locus. Analysis of recombinant F3 plant progenies within the interval redefined a smaller mapping interval between 22.5 and 22.7 cM delimited by cfn1249269 and BS00090770 SNP markers on the IWGSCWGAV02_1BS_scaffold35219 scaffold and the IWGSCWGAV02_1BS_scaffold5117 scaffold respectively.
C. Mapping of the Genomic Region Containing Rf7 Genetic Determinants
We crossed R197 (harboring Rf1 and Rf7 locus) and Primepii (harboring Rf3 locus) and then derived a population of 176 plants from individuals that were rf1 and rf3, which means not carrying the restorer alleles at the loci Rf1 and Rf3. The plants were genotyped with 18100 SNP markers using Limagrain's internal genotyping platform and phenotyped as described in example 1. We mapped the Rf7 locus on chromosome 7BL. Moreover, internal genotyping data showing a strong genetic divergence suggests the presence of an exotic chromosomal fragment which is stably transmitted through generation. We identified a large QTL ranging from 45 cM to 88 cM on chromosome 7B on Limagrain's internal consensus map with a peak on 46.7 cM (cfn0919993 with LOD score of 3.37E-40). First expertise of the recombinant plants suggests the Rf7 gene could be located between cfn3407185 and W90K_RAC875_c33564_120 markers delimiting a mapping interval of 0.3 cM between 46.7 cM and 47 cM on Limagrain's internal consensus map.
D. Mapping of the Genomic Region Containing Rf4 Genetic Determinant
A mapping population from the cross between AH46 and L13 (harboring Rf4 locus) consisting of 124 individual plants segregating for Rf4 was genotyped with 18100 SNP markers using Limagrain's internal genotyping platform and phenotyped as described in example 1. A QTL that we named Rf4 was identified on chromosome 6B between 0 cM to 65 cM on Limagrain's internal consensus map with a peak on 43.3 cM (cfn0393953 with LOD score of 1.08E-13). Moreover, internal genotyping data showing a strong genetic divergence suggests the presence of an exotic chromosomal fragment which is stably transmitted through generation. It is expected that the rate of recombination be very low within this chromosomal region and, consequently, any marker in linkage with cfn0393953 is considered to be associated with Rf4 locus.
We constructed a BAC library with a DH line comprising Rf1, Rf3 and Rf7 alleles. This library was used for the identification of BAC clones within the Rf1 and Rf3 QTL intervals. More specifically, we identified and sequenced and genetically validated 3 BAC clones within the Rf1 region and 3 BAC clones within the Rf3 region. The BAC sequences were compared to Chinese Spring reference genome for SNP discovery. We then saturated Rf1 and Rf3 mapping intervals by mining available SNPs from public genomic resources and the newly discovered SNPs from sequenced BAC clones.
Screening for polymorphic and informative SNP markers on a diversity panel consisting of 83 wheat elite plants and known restorers for Rf1, Rf3, Rf4 and Rf7 identified a set of tightly linked markers localized within or in the immediate flanking regions of the Rf1, Rf3, Rf4 and Rf7 mapping intervals. These SNP markers can be used alone and/or in haplotype to select for Rf or rf plants in MAS breeding schemes (
As an example, the identification of the presence of the Rf1 locus in the genome of a plant is achieved by using either the marker 276113_96622_97797 or 104A4_105588 (
Similarly, the identification of the presence of the Rf3 locus in the genome of a plant is achieved by using either the marker 136H5_3 M5_7601 or 136H5_3 M5_89176 (
The identification of the presence of the Rf7 locus in the genome of a plant is achieved by using the markers cfn0917304, cfn0919993 and cfn0920459 (Table 17). In this case the 3 markers might be used in haplotype to distinguish restorer plants from maintainer plants. Thus the haplotype TGC would identify the restorer plants.
Finally, the identification of the presence of the Rf4 locus in the genome of a plant is achieved by using the markers cfn0393953 and cfn0856945 (Table 18).
To test the hypothesis of the cumulative effect of the restorer alleles and to identify the combination(s) of Rf genes that can produce a full fertility in the CMS hybrid, the SNPs linked to the 4 major loci mapped are used to create restorer lines combining the different restoring alleles. The 2 Rf alleles, 3 Rf alleles and 4 Rf alleles combinations (Rf1+Rf3, Rf1+Rf4, Rf1+Rf7, Rf3+Rf4, Rf3+Rf7, Rf4+Rf7, Rf1+Rf3+Rf4, Rf1+Rf3+Rf7, Rf3+Rf4+Rf7 and Rf1+Rf3+Rf4+Rf7) can be created employing different breeding techniques such as pedigree breeding, backcross, single-seed descent or double haploid.
In the example exposed 2 double-haploid lines from the cross TJB155/R204 were created: LGWR16-0016 and LGWR16-0026. Those two lines carry the restorer alleles Rf1, Rf7 (donor R204) and Rf3 (donor TJB155) and are alloplasmic for the cytoplasm from T. timopheevii (donor TJB155).
LGWR16-0016 and LGWR16-0026 have winter growth habits and show a normal fertility (LGWR16-0016: 2.54 seeds/spikelet, average over 29 individual spikes. LGWR16-0026: 2.33 seeds/spikelet, average over 40 individual spikes). LGWR16-0016 and LGWR16-0026 were used as pollinators in a series of crosses onto a series of A-line with elite background (14 A-lines for LGWR16-0016 and 16 A-lines for LGWR16-0026). The F1 plants originating from those crosses were assessed indoor for their fertility.
Fertility Assessment with the Main Tillers
The spikes from the hybrids produced with the restorer lines LGWR16-0016 yielded on average 45.4 grains and did show an average fertility of 2.44 grains/spikelet. The spikes from the hybrids produced with the restorer lines LGWR16-0026 yielded on average 44.7 grains and did show an average fertility of 2.37 grains/spikelet. Neither of these two groups differ significantly in their fertility distribution from the group formed by the elite lines (Table 20. T-test, P-values <0.05). The distribution of the fertility scores are, as a consequence, relatively similar between groups (see Table 20)
Fertility Assessment with all Tillers from F1 Plants
To be considered as complete, the restoration of fertility has to be observed in all the spikes formed by the hybrid plant. For that purpose, the integrality of the spikes of 56 F1 plants produced with LRWG16-0016, of 61 F1 plants produced with LGWR16-0026 and of 52 elite lines were assessed for their fertility and represent, per group, 294, 262 and respectively 288 individual spikes (Table 21). The 56 F1 plants produced with LGWR16-0016 produced on average 41.4 grains/spikes and did show an average fertility of 2.21 grains per spikelet (standard deviation 0.32). The 61 F1 plants produced with LGWR16-0026 produced on average 42.8 grains/spikes and did show an average fertility of 2.27 grains per spikelet (Table 5. Standard deviation 0.32). The fertility distribution of these two groups differ significantly from the fertility distribution of the groups formed by the elite lines (average number of seeds per spike: 38.4. Average fertility: 2.01. Standard deviation 0.44. see Table 21).
With the help of the markers developed in the previous examples, new restorer lines comprising Rf1, Rf3 and Rf7 loci were obtained using different sources of restoration locus. Table 22 shows the list of these restorer lines and their characteristics.
LGWR17-0015 is a winter wheat double haploid line produced from the F1 plants from a complex cross: R0934F was first pollinated by Altigo, the created F1 R0934F/Altigo was then crossed with the F1 R197/Apache taken as male and the resulting four ways F1 “R0934F/ALTIGO/R197/APACHE” was then pollinated with the line Altamira. LGWR17-0015 inherited the T-CMS cytoplasm from the restorer line R0934F. LGWR17-0015 has been selected under local environment and is a fully fertile wheat line.
LGWR17-0022 and LGWR17-0157 are winter wheat double haploid lines arising from a complex cross: the F1 formed from the cross between the restorer lines R204 and R213 was pollinated with Aristote, the resulting 3-ways cross “R204/R213//Aristote” was then pollinated with the line NIC07-5520. LGWR17-0022 and LGWR17-0157 inherited the T-CMS cytoplasm from the restorer line R204. They have both been selected under local environment and are fully fertile wheat lines.
LGWR17-0096 and LGWR17-0154 are winter wheat lines developed by conventional pedigree breeding technique from the 3-ways cross R204/R213//ARISTOTE. LGWR17-0096 and LGWR17-0154 inherited the T-CMS cytoplasm from the restorer line R204. They have both been selected under local environment and are fully fertile wheat lines.
All lines have been genotyped with the markers as described above to check for the presence of Rf1, Rf3 and Rf7 haplotypes.
Representative seed samples for each line have been deposited before NCIMB collection.
Fertility from 4 hybrids produced with the restorer lines LGWR17-0022, LGWR17-0153, LGWR17-0154 and LGWR17-0157 was assessed in field as described in example 19 for the fertility assessment of the main tillers. The restorer lines have the pedigree and haplotypes as described in table 23.
The assays are made with agronomically adapted hybrids in three different countries France, Germany and United Kingdom. In total 28 hybrids are tested and compared to 26 control Elite inbreds. The result show that, in the field, the hybrids comprising the combination of the three alleles Rf1, Rf3 and Rf7 restorers performed as well as the elite inbreds (table 24). It is also worth to be noted that, in this combination, the Rf3 weak or Rf3 do not have any impact on the fertility level.
As shown in example 11,
Such modification could be achieved in Fielder, which comprises a RFL29c as depicted in SEQ ID NO 3457, by designing, as described in example 15, a suitable guide sequence targeting the frameshift and used it in combination with a base-editing technology such as described in WO2015089406.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17306500.4 | Oct 2017 | EP | regional |
| 17306501.2 | Oct 2017 | EP | regional |
| 18305027.7 | Jan 2018 | EP | regional |
| 18306114.2 | Aug 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/079816 | 10/31/2018 | WO | 00 |
