Wheat comprising male fertility restorer allele
阅读说明：本技术 包含雄性育性恢复等位基因的小麦 (Wheat comprising male fertility restorer allele ) 是由 P·瓦雷纳 J·科玛德兰 S·斯皮瑟尔 A·穆里格纽 J·梅洛内克 I·斯玛尔 P·佩雷 于 2018-10-31 设计创作，主要内容包括：本发明属于植物遗传学和植物育种领域。本发明更具体地涉及携带对提莫菲维小麦CMS细胞质具有特异性的育性基因的恢复子的小麦转基因植物。(The present invention is in the field of plant genetics and plant breeding. The invention more specifically relates to wheat transgenic plants carrying a restorer of a fertility gene specific to the CMS cytoplasm of the tiffany wheat.)
1. An isolated Rf1 nucleic acid encoding a fertility Rf1 protein restorer of the CMS cytoplasm of tiffany wheat, 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.
2. The isolated nucleic acid of claim 1, comprising SEQ ID NO 3119.
3. A transgenic wheat plant comprising the Rf1 nucleic acid of claim 1 or 2, and optionally one or more nucleic acids comprising an Rf3, Rf4, and/or Rf7 restorer allele as a transgenic element.
4. A genetically engineered wheat plant comprising as a genetically engineered element an Rf1 nucleic acid of claim 1 or 2, and optionally one or more nucleic acids comprising an Rf3, Rf4 and/or Rf7 restorer allele.
5. The wheat plant of claim 3 or 4, wherein said transgenic or genetically engineered element expresses a polypeptide that restores or improves male fertility of said plant as compared to a parent plant that does not contain such transgenic or genetically engineered element.
6. A wheat plant restorer of fertility to the CMS cytoplasm of Triticum aestivum wheat comprising the Rf1 restorer allele of claim 1 or 2, and at least two fertility restorer alleles selected from Rf3, Rf4, and Rf7 within a restorer locus, wherein,
the Rf3 locus is located at most 10cM from the marker cfn1249269 of SEQ ID NO. 3205 or the marker BS00090770 of SEQ ID NO. 3228,
the Rf7 locus is located at most 10cM away from the marker cfn0919993 of SEQ ID NO:3231, and
the Rf4 locus is located at most 10cM from the marker cfn0393953 of SEQ ID NO: 3233.
7. The wheat plant of any one of claims 3-6, wherein the plant comprises Rf1, Rf3, and Rf7 restorer alleles.
8. The wheat plant of any one of claims 3-7 characterized in that it comprises at least one Rf3 restorer allele within the Rf3 locus which Rf3 restorer allele is located within the chromosomal fragment between the SNP markers cfn1249269 and BS 00090770.
9. The wheat plant of claim 8, wherein the corresponding amino acid sequence of the Rf3 restorer allele has at least 95% identity, preferably at least 96%, 97%, 98%, 99% or 100% identity, with an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO 158, SEQ ID NO 676 and SEQ ID NO 684.
10. The wheat plant of claim 8 or 9, wherein the Rf3 locus comprises SEQ ID NO 1712, SEQ ID NO 3147 or SEQ ID NO 2230, SEQ ID NO 3148 or SEQ ID NO 2238.
11. The wheat plant of any one of claims 3-10 comprising at least one Rf7 restorer allele within the Rf7 locus, said Rf7 locus being characterized by the presence of one or more of the following restorer SNP alleles:
12. the wheat plant of any one of claims 3-11, characterized in that it comprises at least one Rf4 restorer allele of the Rf4 protein restorer for fertility encoding the CMS cytoplasm of the tiffany wheat, wherein the corresponding amino acid sequence has at least 95% identity, preferably at least 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of seq id no:477 and 3135-3138 SEQ ID NOs.
13. The wheat plant of any one of claims 3-5 and 7-12 comprising Rf1, Rf3, and Rf7 restorer alleles at the same locus.
14. A method of producing the transgenic wheat plant of any one of claims 3 and 5-13, wherein the method comprises the steps of: transforming a parent wheat plant with one or more nucleic acids encoding a protein restorer of the CMS cytoplasm of tiffany wheat of claim 1 or 2, selecting a plant comprising said one or more nucleic acids as a transgene, regenerating and growing said wheat transgenic plant.
15. A method of producing the genetically modified wheat plant of any one of claims 4-13, wherein the method comprises the steps of: preferably, a parent wheat plant is genetically modified by genome editing to obtain in its genome one or more nucleotide sequences encoding a protein restorer of the CMS cytoplasm of tiffany wheat as defined in claim 1 or 2, selecting a plant comprising said one or more nucleotide sequences as a genetic engineering element, regenerating and growing said genetically engineered wheat plant.
16. A method of producing the wheat plant of claim 6, comprising the steps of:
a. providing a first wheat plant comprising one or two restorer alleles selected from the group consisting of Rf1, Rf3, and Rf7 restorer alleles,
b. Crossing said first wheat plant with a second wheat plant comprising one or two restorer alleles selected from the group consisting of Rf1, Rf3 and Rf7 restorer alleles, wherein Rf1, Rf3 and Rf7 restorer alleles occur at least once in a set of restorer alleles provided by said first plant and said second plant,
c. collecting the seeds of the F1 hybrid seeds,
d. obtaining a homozygous plant from the F1 plant,
e. optionally detecting the presence of the restored alleles of Rf1, Rf3, and Rf7 in the hybrid seeds and/or in each generation.
17. The method of claim 14, 15 or 16, wherein the fertility score of the obtained wheat plant is higher than the fertility score of the parent wheat plant.
18. A method of producing a transgenic or genetically engineered wheat plant, wherein the level of fertility of said plant is altered, comprising the step of knocking down the expression of a Rf1 restorer allele, wherein the Rf1 restorer allele comprises the nucleic acid of claim 1 or 2.
19. A method for altering fertility levels in a wheat plant by genome editing comprising providing genome editing means capable of modulating the expression of a Rf1 restorer allele, wherein the Rf1 restorer allele comprises a nucleotide sequence as defined in claim 1 or 2.
20. A method of producing a hybrid wheat plant comprising the steps of:
a. crossing a sterile female comprising the cytoplasm of a tiffany wheat with a fertile male wheat plant of any one of claims 3-13;
b. collecting hybrid seeds;
c. optionally detecting the level of hybrid seed hybridization.
21. The method of claim 20, further comprising the step of: detecting the presence of the cytoplasm of the Trimopivirus wheat and/or at least three Rf loci selected from Rf1, Rf3, Rf4, and Rf7 in the hybrid seed.
22. A wheat hybrid plant obtained by the method of claim 20 or 21.
23. A method of identifying a wheat plant according to any one of claims 3-13 wherein said wheat plant is identified by detecting the presence of at least one restorer allele Rf1 and optionally one or more other restorer alleles selected from Rf3, Rf4 and Rf 7.
24. Nucleic acid probes or primers for the specific detection of the restorer allele Rf1 and optionally one or more of Rf3, Rf4 and Rf7 restorer alleles in wheat plants.
25. A recombinant nucleic acid comprising the nucleic acid encoding the protein restorer of the CMS cytoplasm of tiffany wheat of claim 1 or 2 operably linked to regulatory elements.
26. A vector for transforming a wheat plant comprising the recombinant nucleic acid of claim 25.
Technical Field
The present invention is in the field of plant genetics and plant breeding. More specifically, the invention relates to wheat plants carrying a restorer for a fertility gene specific for the CMS cytoplasm of the wheat (t.timopheevii) tiffany.
Background
Hybrid production is based on crossing two parental lines to increase hybrid vigor and in fact increase genetic variability, resulting in new varieties or genotypes with higher yields and better adaptation to environmental stresses. Even in the major self-fertilized species (e.g., wheat), studies have shown that hybrid lines exhibit improved quality and greater tolerance to environmental and biotic stresses.
In order to promote a commercially viable rate of hybrid production, self-flower fertilization, i.e., fertilization of the female organ by pollen from the same plant, must be avoided. It is desirable that the female organ of the female parent is fertilized only with pollen of the male parent. In order to obtain a reliable and efficient system for producing seeds for hybrid production, three basic elements are generally required: means for inducing male sterility, means for breeding sterility and means for restoring fertility. For example, a completely genetic-based system consists of a male sterile line (the female parent), a fertile maintainer line (the male parent that allows the male sterile line to propagate), and a fertility restorer line (the male parent for hybrid production).
Male sterility can be achieved in three different ways. Manual detasseling is the simplest and is still used in some species (e.g. corn) that separate male and female flowers. However, in species that contain both female and male organs in the flower (such as wheat), this is impractical. Male sterility can also be induced by Chemical Hybridizing Agents (CHA) which have gametocidal effects. Currently, only a few commercial hybrid wheat varieties are based on this technology because it can bear significant financial risks.
Finally, male sterility can also be induced by genetic means. There are many examples of crossing systems in maize or sorghum based on male sterility induced by genetic means, indicating that this technique dominates over the two previously mentioned techniques. However, hybrid production remains a challenge in other species that are primarily self-pollinating (e.g., wheat) (Longin et al, 2012).
The first case of male sterility was observed in wheat in 1951 (Kihara,1951), where sterility was observed to be caused by incompatibility between the cytoplasm of Aegilops caudate (Aegilops caudata L.) and the nucleus of the erythrospermum variety of common wheat. Subsequent studies of the Tomopivirus wheat cytoplasm revealed that this cytoplasm was able to induce sterility in T.aestivum (Wilson and Ross,1961, Crop Sci,1: 191-193). Orf256 was previously identified as a gene specific for the mitochondrial genome of Triticum tikitamurensis wheat (Rathburn and Hedgcoth, 1991; Song and Hedgcoth,1994), however, Orf256 has yet to be demonstrated to be a genetic determinant of Triticum tikitamurensis wheat CMS. It is expected that this cytoplasm can be used in a hybrid production system. However, the main limitation is the difficulty in finding a stable fertility restorer gene that is completely dominant and free of negative side effects (especially on yield).
Fertility restoration of male sterile plants carrying the tiffany wheat CMS cytoplasm (T-CMS cytoplasm) has been reported, and eight major restorer loci (designated Rf1-Rf8) have been identified and are located approximately in the wheat genome. One of the most effective restoration loci is Rf3(Ma and Sorrells, 1995; Kojima et al, 1997; Ahmed et al 2001; Geyer et al 2016). Two SNP markers allow the locus of Rf3 to be located within the 2cM fragment on
Although it is understood that restoring normal pollen fertility may require two or more Rf loci, it is well known that, depending on environmental conditions, there are modifier loci that have little effect with lower penetrance (Zhou et al 2005, Stojalowski et al 2013), or have inhibitory effect on fertility (Wilson, 1984). It is not clear which combination of genes or loci is required to fully restore T-CMS under different genetic background and environmental conditions.
In this case, it is important to develop a technique capable of completely restoring pollen fertility to wheat. Therefore, it is an object of the present invention to propose a suitable fertility restorer gene in wheat to develop a hybrid production system which can be used in seed industry.
Summary of The Invention
A first object of the present disclosure relates to an isolated Rf1 nucleic acid encoding a Rf1 protein restorer for fertility of the CMS cytoplasm of diaphraphytine wheat, 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 an Rf1 nucleic acid comprises SEQ ID NO: 3119.
The present disclosure also relates to transgenic wheat plants comprising as a transgenic element such an Rf1 nucleic acid and optionally one or more nucleic acids comprising an Rf3, Rf4 and/or Rf7 restorer allele.
Another aspect relates to genetically engineered wheat plants comprising as a genetically engineered element such an Rf1 nucleic acid and optionally one or more nucleic acids comprising an Rf3, Rf4 and/or Rf7 restorer allele.
In particular embodiments, the transgenic or genetically engineered element expresses a polypeptide that restores or improves male fertility of the plant as compared to a parent plant that does not contain such transgenic or genetically engineered element.
Another aspect relates to a wheat plant restorer of fertility to the CMS cytoplasm of Triticum aestivum wheat comprising such a Rf1 restorer allele, and at least two fertility restorer alleles selected from Rf3, Rf4, and Rf7 at the restorer locus, wherein,
The locus of Rf3 is located at most 10cM from the marker cfn1249269 of SEQ ID NO. 3205 or the marker BS00090770 of SEQ ID NO. 3228,
the Rf7 locus is located at most 10cM away from the marker cfn0919993 of SEQ ID NO:3231, and,
the locus of Rf4 is located at most 10cM from the marker cfn0393953 of SEQ ID NO: 3233.
The present disclosure also provides a method of 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 a protein restorer of the CMS cytoplasm of the tiffany wheat, selecting a plant comprising said one or more nucleic acids as a transgene, regenerating and growing said wheat transgenic plant.
Also part of the present disclosure is a method of producing a genetically modified wheat plant as described above, wherein the method comprises the steps of: preferably, the parent wheat plant is genetically modified by genome editing to obtain in its genome one or more nucleotide sequences encoding the Rf1 protein restorer of the CMS cytoplasm of the tiffany wheat, selecting a plant comprising said one or more nucleotide sequences as a genetic engineering element, regenerating and growing said genetically engineered wheat plant.
The present invention also relates to a method for producing a wheat plant by crossing, said method comprising the following:
providing a first wheat plant comprising one or two restorer alleles selected from the group consisting of Rf1, Rf3 and Rf7 restorer alleles,
crossing said first wheat plant with a second wheat plant comprising one or two restorer alleles selected from the group consisting of Rf1, Rf3 and Rf7 restorer alleles, wherein Rf1, Rf3 and Rf7 restorer alleles occur at least once in a set of restorer alleles provided by said first plant and said second plant,
collecting the seeds of the F1 hybrid,
obtaining a homozygous plant from the F1 plant,
optionally detecting the presence of the restored alleles of Rf1, Rf3 and Rf7 in said hybrid seeds and/or in each generation.
Preferably, in this method, the fertility score of the obtained wheat plant is higher than the fertility score of the parent wheat plant.
The present disclosure also relates to methods for producing a transgenic or genetically engineered wheat plant, wherein the fertility level of the plant is altered, comprising the step of knocking down the expression of a Rf1 restorer allele, wherein the Rf1 restorer allele comprises a Rf1 nucleic acid.
The present disclosure also relates to a method for producing a wheat hybrid plant, comprising the steps of:
crossing a sterile female comprising the cytoplasm of the temofibrinovir wheat with a fertile male wheat plant as described above;
collecting the hybrid seeds;
optionally detecting the level of hybrid seed hybridization.
Wheat hybrid plants 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 other restorer alleles selected from Rf3, Rf4 and
Thus, also disclosed herein are nucleic acid probes or primers for specifically detecting the restorer allele Rf1 and optionally one or more of Rf3, Rf4 and Rf7 restorer alleles in wheat plants.
Another aspect of the present disclosure relates to recombinant nucleic acids comprising an Rf1 nucleic acid encoding an Rf1 protein restorer of the CMS cytoplasm of the wheat of tiffany wheat operably linked to regulatory elements, and vectors useful for transforming wheat plants comprising such recombinant nucleic acids.
Detailed Description
Nucleic acids of the disclosure
One aspect of the present disclosure relates to the cloning and characterization of genes encoding restorer of fertility proteins (hereinafter Rf gene or nucleic acid) acting on the CMS cytoplasm of the tiffany wheat in wheat plants, and the use of the corresponding Rf nucleic acids for the production of transgenic wheat plants, for the modification of wheat plants by genome editing, and/or for the detection of such Rf genes in wheat plants.
Whenever reference is made to "plant", it is understood that plant parts (cells, tissues or organs, seed pods, seeds, segregating parts, e.g., roots, leaves, flowers, pollen, etc.), plant progeny that retain the distinguishing characteristics of the parents (particularly the male fertility associated with the claimed Rf nucleic acid), e.g., seed obtained by selfing or crossing, e.g., unless otherwise indicated, hybrid seed (obtained by crossing two inbred parent plants), hybrid plants, and plant parts derived therefrom, are encompassed herein.
As used herein, the term "wheat plant" refers to species of the genus triticum, such as common wheat (t.aestivum), russian wheat (t.aethiopicum), alabaster wheat (t.ararticum), wild monocot wheat (t.boeoticum), persian wheat (t.carthricum), triticale wheat (t.compactum), wild monocot wheat (t.dicocoides), durum wheat (t.dicococon), durum wheat (t.rudum), iprodium wheat (t.ispahanicum), kohlii (t.karamycchevii), momum wheat (t.maccha), milritan wheat (t.niticiene), monocot wheat (t), polum wheat (t.monicum), polum wheat (t.niru), myrtle (t.sphaericum), cottum wheat (t.sphaericum), fargeum (t.ovatus), fargefitum (t.ovatus). Wheat plants also refer to species of the genera agjol and triticale.
As used herein, the term "fertility restorer of the CMS cytoplasm of the tiffany wheat" refers to a protein whose expression in a wheat plant comprising the CMS cytoplasm of the tiffany wheat contributes to restoration of pollen production in the CMS system of the tiffany wheat.
As used herein, the term "allele" refers to any of one or more alternative forms of a gene at a particular locus. In diploids, alleles of a given gene are located at specific locations or loci on the chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. The same definition is used for plants carrying higher ploidy levels, like in for example the wheat gender where common wheat is a hexaploid plant.
As used herein, the term "restorer allele of the CMS cytoplasm of tiffany wheat" refers to an allele that contributes to restoration of pollen production in the CMS tiffany wheat system.
Restoration of pollen fertility may be partial or complete. Pollen fertility can be assessed by the pollen fertility test described in the examples below. In particular, the fertility score of F1 wheat plants with CMS-T tiffany wheat cytoplasm (from test restorer lines with CMS hybrids) can be calculated by dividing the total number of seeds threshed from the ear by the number of spikelets counted and can be compared to the fertility score of a group of control fertile plants (e.g., elite inbred lines carrying normal wheat cytoplasm) grown in the same area and under the same agricultural environmental conditions. Preferably, the set of lines includes a set of at least 5 elite inbred lines, wherein the lines represent areas where fertility testing is accomplished. Furthermore, it is preferred that for a given experiment at least 10 ears from different individual F1 plants are evaluated.
If the fertility score is not zero, the plant has acquired partial or complete fertility restoration. For each fertility score, a statistical test was calculated to obtain a p-value. Examples of statistical tests are analysis of variance or mean comparison tests. A p-value below the 5% threshold would indicate that the two distributions are statistically different. Thus, a significant reduction in the fertility score of the tested wheat plants compared to the fertility score of the fully fertile control plants indicates that the F1 plants did not obtain complete fertility restoration (i.e., partial restoration). A fertility score of similar or higher indicates that the F1 plant has obtained complete fertility restoration. In a preferred embodiment, a wheat plant according to the present disclosure, such as a transgenic or genetically engineered wheat plant, has obtained complete fertility restoration.
The loci of the restorer alleles of the CMS cytoplasm of the tiffany wheat have been mapped within Rf1, Rf3, Rf4 and
In particular embodiments, representative alleles of the Rf1, Rf3, Rf4, and Rf7 restorer alleles are provided by a seed sample selected from the group consisting of: 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 equals 1% of the probability that a marker at one genetic locus will segregate from a marker at a second locus by a single generation of hybridization.
As used herein, the term "chromosomal interval" refers to a continuous linear range of genomic DNA located on a single chromosome of a plant. Genetic elements or genes located in a single chromosomal interval are physically linked. The size of the chromosomal interval is not particularly limited. In some aspects, genetic elements located within a single chromosomal interval are genetically linked, e.g., the genetic recombination distance is typically less than or equal to 20cM, or less than or equal to 10 cM. That is, two genetic elements located within a single chromosomal interval recombine with a frequency of less than or equal to 20% or 10%.
The present disclosure provides nucleic acids and recombinant forms thereof comprising a coding sequence for Rf1, Rf3, Rf4, Rf7, or Rf-rye restorer of fertility proteins active in the CMS cytoplasm of the tiffany wheat.
As used herein, a "recombinant nucleic acid" is a nucleic acid molecule, preferably a DNA molecule comprising a combination of nucleic acid molecules that do not occur together in nature and are the result of human intervention, e.g., a DNA molecule consisting of a combination of at least two DNA molecules that are heterologous to each other and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide sequence that is different from a polynucleotide sequence normally found in nature.
As described in the examples below, such nucleic acids encoding candidate restorers of fertility proteins of the CMS cytoplasm of the mitriphery wheat have been isolated. Accordingly, a first aspect of the present disclosure is a nucleic acid encoding a protein restorer for fertility of mithramycin wheat, having an amino acid sequence which is at least 95% identical, typically at least 96% identical to any one of the amino acid sequences selected from SEQ ID NO:1 to SEQ ID NO: 1554.
Percentage of sequence identity, as used herein, is determined by calculating the number of matching positions in the aligned amino acid sequences, dividing the number of aligned positions by the total number of aligned amino acids, and multiplying by 100. A matched position is a position in which the same amino acid occurs at the same position in the aligned amino acid sequences. For example, the amino acid sequence can be aligned using CD hits (setting-c 0.96-n 5-G0-d 0-AS 60-A105-G1, see http://weizhongli- lab.org/cd-hit/)。
The above candidate nucleic acids encoding any of polypeptides SEQ ID NOs 1-1554 may be further evaluated for their ability to restore fertility to a sterile wheat plant as described below.
Thus, disclosed herein is a method of assessing the ability of a nucleic acid to restore fertility, wherein the method comprises the steps of:
a. introducing one or more candidate Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids encoding a putative amino acid sequence of at least 95% identity to any one of SEQ ID NO1-SEQ ID NO1554 into the CMS cytoplasm of a parent wheat sterile plant and Triticum mefenovorum wheat,
b. selecting a transgenic plant carrying one or more candidate nucleic acids as a transgene, and
c. evaluating fertility of the transgenic plant compared to the parent wheat sterile plant based on fertility restoration analysis,
wherein an improvement in fertility restoration indicates that the nucleic acid has the ability to restore fertility.
In a particular embodiment, the parent wheat sterile plant is the Fielder line carrying the CMS cytoplasm of the tiffany wheat.
In another specific embodiment of the above method, the Rf1, Rf3, Rf4, Rf7 and/or Rf-rye candidate nucleic acid sequences are selected from those encoding amino acid sequences having at least 95% identity, or at least 96% identity, e.g. 100% identity, to any one of SEQ ID NO1-SEQ ID NO 1554.
Typically, the Rf1, Rf3, Rf4, Rf7, and/or Rf-rye candidate nucleic acids are selected from the following nucleic acids: 1555 to 3107 and 3133.
In further embodiments, the nucleic acid sequence may be optimized to increase expression in transformed plants, as appropriate. By replacing one codon with another codon encoding the same amino acid, conservative codon exchanges, many optimizations can be performed at the DNA level without changing the protein sequence. In addition, the nucleic acid sequence may be modified for cloning purposes. Like optimization, this modification can be achieved without altering the protein sequence.
Rf1 nucleic acid
In certain embodiments, the nucleic acid of the present disclosure is an Rf1 nucleic acid.
As used herein, the term "
In particular, and as shown in example 6, the inventors have identified that the RFL79 sequence of SEQ ID NO:361 (as shown in Table 7) can restore male fertility to CMS-Fielder plants. Thus, in preferred embodiments, examples of Rf1 nucleic acids include the disclosed Rf1 nucleic acid sequence SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1915, SEQ ID NO:1916 or SEQ ID NO:3119, preferably the Rf1 nucleic acid comprises SEQ ID NO: 3119.
Rf3 nucleic acid
In certain embodiments, the nucleic acid of the present disclosure is an Rf3 nucleic acid.
As used herein, the term "
Rf4 nucleic acid
In certain embodiments, the nucleic acid of the present disclosure is an Rf4 nucleic acid.
As used herein, the term "
Rf7 nucleic acid
In certain embodiments, the nucleic acid of the present disclosure is an Rf7 nucleic acid.
As used herein, the term "
Rf-rye nucleic acids
In particular embodiments, the nucleic acids of the present disclosure are Rf-rye nucleic acids.
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 to the CMS cytoplasm of the tiffany wheat, 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, 378 and 859. Examples of corresponding specific Rf-rye nucleic acids are mentioned in table 7.
Rf nucleic acids as transgenes
The present disclosure more specifically relates to DNA molecules comprising one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids. In particular, the present disclosure relates to any DNA molecule resulting from the insertion of a transgene comprising one or more of the above Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids into a wheat plant, and the insertion results in the expression of the corresponding RNA and/or protein in the wheat plant.
Also part of the present disclosure is a nucleic acid extracted from a cell or tissue or from a plant or seed or plant tissue; or can be produced as amplicons of DNA or RNA extracted from cells or tissues or from homogenates of plants or seeds or plant tissues, any of which is derived from such materials from plants comprising the above-described nucleic acids.
As used herein, the term "transgene" or "transgenic element" refers to a nucleic acid (e.g., a DNA molecule) that is incorporated into the genome of a host cell. The term "transgene" or "transgenic element" particularly refers to a sequence that is not normally present in a given host genome in the genetic environment in which the sequence is currently found. In this regard, the sequences may be native with respect to the host genome, but rearranged with respect to other genetic sequences within the host genome sequence. For example, transgenes rearrange at different loci compared to the native gene.
The one or more transgenic elements are capable of expressing a polypeptide that restores or improves male fertility in a plant having the CMS cytoplasm of tiffany wheat as compared to a parent plant that does not comprise the transgenic element.
Particular transgenic elements are recombinant nucleic acids as defined above, for example Rf1 nucleic acids as defined above. In particular embodiments, the transgenic element comprises an Rf nucleic acid under the control of a constitutive promoter (e.g., a ZmUbi promoter).
Recombinant nucleic acids for transforming wheat plants
Such Rf nucleic acids as defined above may also be used for transforming or genetically modifying wheat plants, in particular wheat plants not having one or more fertility restorer alleles Rf1, Rf3, Rf4, Rf7 and Rf-rye.
Another aspect of the present disclosure relates to a vector for transforming a wheat plant comprising one or more of the Rf1, Rf3, Rf4, Rf7 and Rf-rye nucleic acids as described above.
The vector used to transform a wheat plant includes at least the coding sequence (naturally occurring coding sequence or a modified sequence, e.g., a codon optimized sequence) of the corresponding restorer of fertility protein, operably linked to regulatory elements (e.g., a promoter).
As used herein, the term "promoter" refers to a region of DNA upstream of a coding sequence (upstream of the initiation codon) and includes regions of DNA that recognize and bind RNA polymerase and other proteins to initiate transcription of the initiation codon. Examples of constitutive promoters 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
Promoters may be "tissue-preferred", i.e., initiate transcription in certain tissues, or "tissue-specific", i.e., initiate transcription only in certain tissues. Examples of such promoters are embryo-specific DHN12, LTR1, LTP1, phloem-specific SS1, tapetum-specific OSG6B (Gotz et al 2011 and Jones 2015).
Other suitable promoters may be used. It may be an inducible promoter, a developmentally regulated promoter. An "inducible" promoter initiates transcription under certain environmental controls or induction by any stress, such as abiotic stress-induced RD29, COR14b (Gotz et al, 2011).
Constitutive promoters can be used, such as the ZmUbi promoter, typically the ZmUbi promoter of SEQ ID NO: 3134. Finally, the promoters corresponding to SEQ ID NO:3114, SEQ ID NO:3123 and SEQ ID NO:3113 of pTaRFL46, 79 and 104 can also be used.
In particular embodiments, the Rf1, Rf3, Rf4, Rf7, or Rf-rye nucleic acids of the present disclosure are operably linked to a heterologous promoter (i.e., a promoter that is not a native promoter of the corresponding Rf1, Rf3, Rf4, Rf7, or Rf-rye nucleic acids found in wheat). Typical recombinant constructs of Rf3 nucleic acids having heterologous promoters include any of SEQ ID NO 3150 and 3153, and any of SEQ ID NO 3156-SEQ ID NO 3159. A typical recombinant construct of Rf1 nucleic acid having a heterologous promoter includes SEQ ID NO 3122, or the nucleic acid SEQ ID NO 3119 under the control of promoter SEQ ID NO 2123.
The vector may further comprise additional elements, including a selectable gene marker, operably linked to the regulatory elements, which allow for the selection of transformed plant cells comprising the vector, which comprise the nucleic acid of the present disclosure as a transgene.
The vector may further comprise additional elements including a counter-selectable gene marker operably linked to the regulatory elements that allow for counter-selection of transformed plant cells that do not retain the counter-selectable gene marker in their genome.
In a specific embodiment, the vector according to the invention may be a vector suitable for Agrobacterium-mediated transformation, in particular Agrobacterium tumefaciens (Agrobacterium tumefaciens) or Agrobacterium rhizogenes (Agrobacterium rhizogenes) -mediated transformation, as described in the next section.
Method for producing wheat transgenic plant
Another aspect of the disclosure relates to the use of the above-described nucleic acids in the production of wheat transgenic plants expressing a restorer of fertility protein.
The term "transgenic plant" refers to a plant comprising such a transgene. "transgenic plant" includes plants, plant parts, plant cells or seeds whose genome has been altered by stable integration of recombinant DNA. Transgenic plants include plants regenerated from the originally transformed plant cells and progeny of the transgenic plants from progeny or hybrids of the transformed plants. Due to this genomic change, the transgenic plants differ significantly from the relevant wild type plants. Examples of transgenic plants are plants described herein, which comprise one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids, typically as transgenic elements. For example, transgenic plants comprise one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids as transgenes, which are inserted at a locus different from the native locus of the corresponding Rf gene. Thus, disclosed herein is a method of producing a transgenic plant of wheat, wherein the method comprises the steps of:
(i) Transforming parent wheat plants with Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids,
(ii) selecting a plant comprising said one or more nucleic acids as a transgene,
(iii) regeneration and
(iv) growing said wheat transgenic plant.
For transformation methods in plant cells, direct gene transfer methods can be exemplified, such as direct microinjection into plant embryos, vacuum infiltration or electroporation, direct precipitation by the PEG method, or bombardment of particles covered with the target plasmid DNA with a gun.
Preferably, the plant cells are transformed with a bacterial strain, in particular Agrobacterium tumefaciens. In particular, the method described by Ishida et al (Nature Biotechnology,14,745-750,1996) can be used to transform monocots.
A description of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer is provided by Moloney et al, Plant Cell Reports 8:238 (1989). See also U.S. Pat. No.5,591,616, approved on
Alternatively, direct gene transfer may be used. One commonly used method of plant transformation is particle-mediated transformation, in which DNA is carried on the surface of particles of 1-4 microns in size. The expression vector is introduced into plant tissue by a particle gun device that accelerates the particles to a velocity of 300-600m/s sufficient to penetrate plant cell walls and membranes. Sanford et al, part.Sci.Techol.5: 27(1987), Sanford, J.C., Trends Biotechnology.6: 299(1988), Kleinet al, Biotechnology 6: 559-. Several target tissues can be bombarded with DNA-coated microparticles to produce transgenic plants, including, for example, callus (type I or type II), immature embryos, and meristems.
Following transformation of the wheat target tissue, expression of the selectable marker gene allows for preferential selection of transformed cells, tissues and/or plants using regeneration and selection methods currently well known in the art.
The aforementioned transformation methods will typically be used to produce transgenic plants comprising one or more Rf1, Rf3, Rf4, Rf7 or Rf rye nucleic acids as transgenic elements.
The transgenic plant can then be crossed with another (untransformed or transformed) inbred line to produce a new transgenic line. Alternatively, a genetic trait engineered into a particular line using the transformation techniques described above can be transferred into another line using conventional backcrossing techniques well known in the art of plant breeding. For example, the backcross approach can be used to transfer engineered traits from a public non-elite inbred line into an elite inbred line, or from an inbred line that includes a foreign gene in its genome into one or more inbred lines that do not include the gene. As used herein, "hybridization" may refer to the process of simple X hybridization or backcrossing by Y, depending on the context.
When the term transgenic wheat plant is used in the context of the present disclosure, it also includes any wheat plant comprising one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids as transgenic element, and wherein the one or more desired traits are further introduced by a backcross method, whether such a trait is a naturally occurring trait or a transgenic trait. Backcrossing methods can be used with the present invention to improve one or more properties or to introduce one or more properties into the inbreds. As used herein, the term backcross refers to the complex re-crossing of progeny of a cross back to one of the parent wheat plants. A parent wheat plant that contributes one or more genes to a desired trait is referred to as a non-recurrent or donor parent. The term refers to the fact that the non-recurrent parent is used once in the backcrossing scheme and is therefore not recurrent. The parent wheat plant to which one or more genes from the non-recurrent parent are transferred is called the recurrent parent, since it uses several rounds in the backcrossing scheme (Fehr et al, 1987).
In a typical backcrossing scheme, a recurrent parent is crossed with a second non-recurrent parent carrying one or more genes of interest to be transferred. The progeny resulting from this cross are then crossed again with the recurrent parent and the process is repeated until a wheat plant is obtained in which all the desired morphological and physiological properties of the recurrent parent are restored in the transformed plant, except for the gene or genes transferred from the non-recurrent parent. It should be noted that some, one, two, three or more self-pollination and population growth may be included between two consecutive backcrosses.
Method for producing wheat gene engineering plant
One aspect of the present disclosure relates to DNA fragments (naturally occurring coding sequences or improved sequences, e.g., codon optimized sequences) of a respective fertility protein restorer in combination with a genome editing tool (e.g., TALEN, CRISPR-Cas, Cpf1, or zinc finger nuclease tool) to target a respective Rf restorer allele within a wheat plant genome by insertion at any locus in the genome or by partial or full allele replacement at the respective locus.
In particular, the present 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 its genome one or more nucleotide sequences encoding a protein restorer Rf1, Rf3, Rf4 or Rf7 of the CMS cytoplasm of the tiffany wheat as disclosed herein, preferably by genome editing, selecting a plant comprising said one or more nucleotide sequences as a genetic engineering element, regenerating and growing said wheat genetically engineered plant.
As used herein, the term "genetically engineered element" refers to a nucleic acid sequence that is present in the genome of a plant and that has been modified by mutagenesis or by a genome editing tool, preferably by a genome editing tool. In particular embodiments, a genetically engineered element refers to a nucleic acid sequence that is not normally present in a given host genome in the genetic background in which the sequence is currently found, but is introduced into the genome of a plant through the use of genome editing tools. In this regard, the sequence may be native with respect to the host genome, but rearranged with respect to other genetic sequences within the host genome sequence. For example, a genetically engineered element is a gene that is rearranged at a different locus than the 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 below.
The term "genetically engineered plant" or "genetically modified plant" refers to a plant that comprises such genetically engineered elements. "genetically engineered plant" includes plants, plant parts, plant cells or seeds whose genome has been altered by stable integration of recombinant DNA. As used herein, the term "genetically engineered plant" also includes plants, plant parts, plant cells, or seeds whose genome has been altered by genome editing techniques. Genetically engineered plants include plants regenerated from the originally engineered plant cells and progeny of the genetically engineered plants or progeny of genetically engineered plants of hybrids. As a result of this genomic change, genetically engineered plants differ significantly from the relevant wild type plants. Examples of genetically engineered plants are plants described herein, which comprise one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids. For example, a genetically engineered plant comprises as a genetically engineered element one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids, which are inserted at a different locus than the native locus of the corresponding Rf gene.
In particular embodiments, the genetically engineered plant does not include plants that can be obtained by only basic biological processes.
The one or more genetically engineered elements are capable of expressing a polypeptide that restores or improves male fertility in a plant having the CMS cytoplasm of the tiffany wheat as compared to a parent plant that does not comprise the genetically engineered element.
A particular genetic engineering element is an Rf nucleic acid as defined above, for example an Rf1 nucleic acid as defined above. In particular embodiments, the genetically engineered elements include Rf nucleic acids as promoters and/or terminators under the control of the expression elements. Suitable promoters may be constitutive promoters, such as the ZmUbi promoter or the native or modified endogenous Rf promoter.
Another aspect of the present disclosure relates to genetically engineered wheat plants comprising modifications by any genome editing tool (including the base editing tools described in WO 2015089406) or by mutagenesis by insertion or deletion of one or several nucleotide (as genetic engineering element) point mutations of the allele sequence Rf or Rf into the respective Rf or Rf allele.
The present disclosure further includes methods for altering fertility levels in a plant by genome editing comprising providing a genome editing tool capable of partially or completely replacing an Rf1, Rf3, Rf4, Rf7, or Rf-rye non-restorer allele sequence or form thereof in a wheat plant with its corresponding Rf1, Rf3, Rf4, Rf7, or Rf-rye restorer allele sequence disclosed herein.
The term "Rf non-restorer allele sequence or form" can be related to the presence of an Rf non-restorer allele in the genome or to the absence of any Rf or Rf allele in the genome. For example, in the Rf3/Rf3 system, one wheat non-restorer line can be characterized by the presence of an Rf3 allele sequence, while another non-restorer line can be characterized by the absence of any Rf3 or Rf3 allele 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 is characterized by the presence of the Rf4 gene sequence in the genome.
In a particular embodiment, a method for altering fertility level of a plant by genome editing comprises providing a genome editing tool capable of replacing or modifying an Rf3 non-restorer allele to obtain an Rf3 restorer allele comprising SEQ ID NO:3146(RFL29 a). In other particular embodiments, the rf3 non-restorer allele may comprise RFL29c characterized by a frameshift compared to the RFL29a nucleotide sequence shown in example 22 and FIG. 12 and SEQ ID NO: 3457.
The present disclosure further includes methods of altering fertility levels in plants by introducing an endogenous promoter of a restorer Rf gene through genome editing or mutagenesis to increase expression of the corresponding endogenous Rf gene.
In particular embodiments, the present disclosure includes methods of altering fertility levels in plants by genome editing a weakly fertile plant by modifying the 5'UTR sequence of the Rf3 "weak" RFL29b allele, the 5' UTR region including an insertion of 163bp to be deleted as shown in example 15.
In a further specific embodiment, the present invention includes a method of altering fertility levels in a plant by increasing expression of an endogenous Rf gene by mutagenesis or by restoring the endogenous promoter of the daughter Rf gene by genome editing. For example, the sequence of protarl 79 shown in SEQ ID N ° 3123 may be mutated or edited to increase the RFL79 protein level.
In another specific embodiment, whenever a stronger promoter is located upstream of the promoter of the Rf restoring gene, deletion of the promoter and upstream region can be achieved so as to juxtapose the stronger promoter to the Rf gene.
In particular embodiments, one Rf non-restorer allele is partially or completely replaced with any one of Rf1, Rf3, Rf4, Rf7, or Rf-rye restorer alleles. In this disclosure, for example, the non-restorer Rf1 allele may be replaced with an Rf3 or Rf4 or Rf7 or Rf-rye allele.
In another aspect of the present disclosure, at least one restorer allele of Rf1, Rf3, Rf4, Rf7 and Rf-rye can be integrated at one or more target sites of the wheat plant genome to typically obtain expression of said restorer allele. Such expression can be achieved by exploiting the presence of a promoter (more specifically a strong promoter) and/or a terminator at the target locus, and by targeting the expression element with the Rf allele. In particular embodiments, the endogenous Rf allele is deleted from one first locus and further integrated downstream of a suitable promoter at a second locus in the genome of the same plant.
In particular embodiments of the invention, the target site may be located in the Rf1, Rf3, Rf4 and/or Rf7 loci as defined in example 17. In more particular embodiments, the target site may be an Rf1, Rf3, Rf4, Rf7, or Rf-rye endogenous gene sequence or any other target site different from Rf1, Rf3, Rf4, Rf7, or Rf-rye endogenous gene sequence.
In a preferred aspect of the present disclosure, the wheat plant may comprise Rf1, Rf3 and Rf7 restorer alleles at only one locus in its genome.
Such genome editing tools include, but are not limited to, targeted sequence modification provided by double-strand break technology, double-strand break technology based on the use of engineered nucleases, such as, but not limited to, meganucleases, ZFNs, TALENs (WO2011072246) or CRISPR CAS systems (including CRISPR Cas9, WO2013181440), Cpf1 or next generation thereof.
A method for reducing fertility levels in wheat plants:
alternatively, the disclosure further includes methods for altering the fertility level of a plant by restoring a restorer line comprising an Rf allele to a maintenance line comprising an Rf allele. The method may be used for any plant comprising an Rf allele (including Rf 1). Of particular interest is the case in a hybrid production system based on the Rf3 restorer allele, wherein for example the Rf3 sequence is RFL29a and the Rf3 sequence is RFL29 c.
Fertility can be reduced by knocking down the Rf gene or allele as described below.
More specifically, the method may first correspond to attenuating the promoter function of the Rf allele to suppress expression by RNAi against the target Rf allele or by mutagenesis or by genome editing.
On the other hand, a reduction in fertility level is obtained by mutagenesis, classically induced with mutagens, or by weakening the protein function by genome editing techniques, by deleting the gene in whole or in part, or by modifying the reading frame of the gene sequence to weaken the translation of the protein.
In another aspect, the present disclosure relates to transgenic or genetically engineered wheat plants having reduced fertility levels obtained by the above methods.
Transgenic or genetically engineered wheat plants of the disclosure
Another aspect of the present disclosure relates to transgenic or genetically engineered wheat plants comprising one or more Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acids as described in the previous section as transgenic or genetically engineered elements, respectively.
The transgenic or genetically engineered plants can be obtained by the methods described in the previous section.
The transgenic or genetically engineered plants of the present disclosure can be advantageously used as parent plants to produce fertile wheat transgenic or genetically engineered plant restorers of the CMS cytoplasm of the tiffany wheat. In particular, in particular embodiments, the wheat transgenic or genetically engineered plant is a fertile wheat transgenic or genetically engineered plant restorer to the CMS cytoplasm of the tiffany wheat. Typically, transgenic or genetically engineered wheat plants according to the present disclosure comprise 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.
Transgenic or genetically engineered wheat plants disclosed herein may express such protein restorers of fertility Rf1, Rf3, Rf4, Rf7 and/or Rf-rye together due to transgene expression or expression of genetically engineered elements, and may also express other fertility protein restorers due to naturally occurring alleles.
In one embodiment, the combination of at least two, three or four Rf1, Rf3, Rf4, Rf7 and Rf-rye nucleic acids is found in the same locus in the genome of a transgenic or genetically engineered plant. In other embodiments, the corresponding nucleic acids of the combination are located in different loci. In a particular embodiment, the combination may be obtained by crossing transgenic plants of the present disclosure, each of which carries one nucleic acid of the combination as a transgene at a different locus.
Typically, the transgenic or genetically engineered plant comprises as transgenic or genetically engineered elements the following combinations of nucleic acids:
an Rf1 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430 (preferably SEQ ID NO:361), typically an Rf1 nucleic acid comprising SEQ ID NO:3119 and an Rf3 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of 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, SEQ ID NO:676 and SEQ ID NO:684,
An Rf1 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430 (preferably SEQ ID NO:361), typically an Rf1 nucleic acid comprising SEQ ID NO:3119, and an Rf7 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NO:363, SEQ ID NO:516 and SEQ ID NO:768,
an Rf1 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430 (preferably SEQ ID NO:361), typically an Rf1 nucleic acid comprising SEQ ID NO:3119, and an Rf-rye nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NO:227, SEQ ID NO:378 and SEQ ID NO:859,
rf3 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of 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, SEQ ID NO:676 and SEQ ID NO:684, and Rf7 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of SEQ ID NO:363, SEQ ID NO:516 and SEQ ID NO:768,
An Rf3 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of 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, SEQ ID NO:676 and SEQ ID NO:684, and an Rf-rye nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NO:227, SEQ ID NO:378 and SEQ ID NO:859,
an Rf7 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NO 363, SEQ ID NO 516 and SEQ ID NO 768, and an Rf-rye nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NO 227, SEQ ID NO 378 and SEQ ID NO 859,
rf1 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430 (preferably SEQ ID NO:361), typically Rf1 nucleic acids comprise SEQ ID NO:3119 and Rf3 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of 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, SEQ ID NO 676 and SEQ ID NO 684 and Rf7 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of SEQ ID NO 363, SEQ ID NO:516 and SEQ ID NO 768;
An Rf1 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430 (preferably SEQ ID NO:361), typically an Rf1 nucleic acid comprising SEQ ID NO:3119 and an Rf3 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of 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, SEQ ID NO 676 and 378, and an Rf-rye nucleic acid, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any one of SEQ ID NO:227, SEQ ID NO: 859;
an Rf1 nucleic acid, preferably encoding an amino acid sequence having at least 95% identity, e.g. at least 96% identity, to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430 (preferably SEQ ID NO:361), typically an Rf1 nucleic acid comprising SEQ ID NO:3119, and an Rf7 nucleic acid of
Rf3 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of 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, SEQ ID NO:676 and SEQ ID NO:684, and Rf7 nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of SEQ ID NO:363, SEQ ID NO:516 and SEQ ID NO:768, and Rf-rye nucleic acids, preferably encoding an amino acid sequence having at least 95% identity or at least 96% identity to any of SEQ ID NO:227, SEQ ID NO:378 and SEQ ID NO: 859.
In other specific embodiments, the transgenic or genetically engineered plant comprises, in addition to any one of the above defined combinations of Rf1, Rf3, Rf7 and/or Rf-rye nucleic acids as transgenic element, as defined in the preceding paragraphs, 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 NOs 3135-3138.
The present disclosure also relates to a hybrid wheat plant that can be produced by crossing a transgenic or genetically engineered fertility wheat plant restorer according to the present disclosure as described above with a second plant.
In certain embodiments, the wheat plant according to the invention is heterogeneous and comprises the tiffany wheat cytoplasm.
For example, a hybrid wheat plant may be obtained by crossing a wheat plant restorer (preferably comprising a Rf1, Rf3, Rf4, Rf7 or Rf-rye nucleic acid) according to the present disclosure as described above with a wheat plant which does not express the corresponding fertility Rf1, Rf3, Rf4, Rf7 or Rf-rye protein restorer.
Also disclosed herein is a method of producing a wheat hybrid transgenic or genetically engineered plant comprising the steps of:
a. crossing a sterile female wheat plant comprising a tiffany wheat cytoplasm with a fertility restorer of a fertile male transgenic or genetically engineered wheat plant of the present disclosure as described above;
b. collecting hybrid seeds;
c. optionally detecting the presence of the cytoplasm of Trimopivy wheat and/or at least one or more Rf nucleic acids selected from Rf1, Rf3, Rf4, Rf7, and Rf-rye in the hybrid seed as a transgenic element or a genetically engineered element; and the combination of (a) and (b),
d. optionally, the hybrid seed is tested for hybridization levels.
Thus, also disclosed herein are transgenic or genetically engineered plants or lines of wheat according to the present disclosure that are developed to obtain such hybrid plants. Such transgenic or genetically engineered plants or lines typically contain cytoplasmic elements required to implement the corresponding crossing system. Preferably, the transgenic or genetically engineered plant or line comprises a combination of at least two, three or four Rf1, Rf3, Rf4, Rf7 and Rf-rye nucleic acids, and the cytoplasm of the tiffany wheat.
Alternatively, the presence of the cytoplasm of the Tomopivia wheat and at least one or more Rf nucleic acids selected from Rf1, Rf3, Rf4, Rf7, and Rf-rye can be detected on the parental line (step "c" of the above method) to check the genotype before starting the cross (step "a").
In a certain embodiment of the present disclosure, the male wheat plant is taller than the female wheat plant. This can be achieved by using male plants carrying the Rht allele that allow size differences to be obtained. Optionally, the present disclosure further includes the step of applying the herbicide to fertile plants standing above the height of the shorter female plants, and optionally, further includes the step of harvesting the seeds and selecting the seeds to remove unwanted self-fertilized male seeds, using morphological and/or phenotypic characteristics such as size, shape, color, etc. … …. An example of such a method for hybrid production is described in WO 2015135940.
T-CMS cytoplasm can be detected either phenotypically (where plants carrying the rf gene and T-CMS cytoplasm will be sterile) or by molecular means capable of detecting the orf256 gene (as described by Rathburn and Hedgcoth,1991 and Song and Hedgcoth, 1994).
The present disclosure also relates to a method for improving fertility restoration levels of a parent wheat plant having fertility levels below full restoration levels, comprising the step 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 acid in said wheat plant. The method further comprises the steps of selecting a transgenic or genetically engineered wheat plant comprising said Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acid (preferably Rf1, Rf3 and Rf4) as transgenic or genetically engineered element, regenerating and growing said transgenic or genetically engineered wheat plant, wherein said transgenic or genetically engineered wheat plant has an improved level of fertility restoration compared to the parent plant.
The present disclosure also provides a method for restoring fertility to a sterile wheat plant carrying the tiffany wheat CMS cytoplasm, comprising the steps of: transforming parental sterile wheat plants carrying the CMS cytoplasm of the tiffany wheat with Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids as described above, or genetically engineering said sterile plants to express Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids.
Use of the nucleic acids of the present disclosure in the identification of 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 of 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 for, or breeding wheat plants comprise obtaining one or more wheat plants and evaluating their DNA to determine the presence or absence of Rf1, Rf3, Rf4, Rf7, and/or Rf-rye fertility restorer alleles and/or corresponding Rf1, Rf3, Rf4, Rf7, and/or Rf-rye nucleic acids.
For example, the method can be used to determine which progeny produced by a cross have a desired fertility restorer allele or Rf nucleic acid (or combination thereof), and accordingly, whether other desired traits are present to direct the production of plants having the desired fertility restorer allele or Rf nucleic acid.
The method will comprise identifying the presence of Rf1, Rf3, Rf4, Rf7 and/or Rf-rye alleles or nucleic acids in fertile plants, which plants are fertile transgenic plants or non-transgenic plants. Optionally, the method further comprises identifying the absence of Rf1, Rf3, Rf4, Rf7 and/or Rf-rye alleles or nucleic acids in the non-restorer plant and/or sterile plant.
Thus, disclosed herein are means for specifically detecting Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids in wheat plants.
For example, such means include a pair of primers for specifically amplifying fragment nucleotide sequences of Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids from plant wheat genomic DNA.
As used herein, primers encompass any nucleic acid capable of priming synthesis of a nascent nucleic acid in a template-dependent process (e.g., PCR). Typically, the primer is an oligonucleotide of 10 to 30 nucleotides, but longer sequences may be employed. The primer may be provided in a double stranded form, although a single stranded form is preferred.
Alternatively, the nucleic acid probe may be used to specifically detect any of Rf1, Rf3, Rf4, Rf7 and/or Rf-rye nucleic acids.
As used herein, a nucleic acid probe encompasses any nucleic acid of at least 30 nucleotides, and which can specifically hybridize to a defined nucleic acid under standard stringency conditions. As used herein, standard stringency conditions refer to hybridization conditions, such as those described in Sambrook et al 1989, which can comprise 1) immobilizing a plant genomic DNA fragment or library DNA on a filter; 2) filters were prehybridized in 6 XSSC 5 XDenhardt reagent, 0.5% SDS and 20mg/ml denatured carrier DNA for 1-2 hours at 65 ℃; 3) addition of probe (labeled); 4) incubating for 16-24 hours; 5) wash the filter once in 6x SSC, 0.1% SDS for 30 minutes at 68 ℃; 6) filters were washed three times (twice for 30 min in 30ml and once for 10 min in 500 ml) in 2x SSC 0.1% SDS at 68 ℃. The nucleic acid probe may further comprise a labeling agent, such as a fluorescent agent covalently linked to the nucleic acid portion of the probe.
Method for producing wheat plants carrying a modified Rf3 restorer for fertility
The inventors have also identified two types of Rf3 restorers of fertility, one with strong fertility restoration ability, e.g., capable of providing plants with a fertility score above 1.0 (e.g., 1.0-2.0), and the other with weak fertility restoration ability, e.g., capable of providing plants with a fertility score below 0.1 (e.g., 0.5-1.0).
Surprisingly, strong fertility restoration was associated with the absence in the genome of wheat plants carrying Rf3 of SEQ ID NO:3174 about a 163bp fragment.
Accordingly, the present disclosure relates to a method of producing a wheat plant carrying a fertility Rf3 restorer, the method comprising (i) providing a parent wheat plant comprising in its genome a fragment of at least 163bp of SEQ ID NO:3174, and (ii) deleting in the genome of the wheat plant a fragment of at least 10bp of said SEQ ID NO:3174, such as at least 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp or the entire fragment of SEQ ID NO:3174, thereby obtaining the wheat plant carrying a fertility Rf3 restorer.
Advantageously, the fertility score of the obtained wheat plant is higher than that of the parent wheat plant due to the deletion of the fragment depicted in SEQ ID NO 3174. One skilled in the art can select deletions in order to obtain an increase in fertility restoration compared to the parent wheat plant having in its genome the complete fragment of SEQ ID NO: 3174.
In a particular embodiment, the fertility score of the parent wheat plant is below 1, such as 0.5-1.0, and the fertility score of the obtained wheat plant is above 1.0, such as 1.0-2.0.
Fertility can also be restored in plants with an Rf3 non-restorer allele that is frame-shifted due to nucleotide deletions or insertions compared to the Rf3 restorer allele RLF29a sequence of seq id NO:3146 (see also example 22).
Correction of the deletion or frameshift of the genomic fragment can be obtained by any suitable method known to those skilled in the art, including genome editing tools such as, but not limited to, meganucleases, ZFNs, TALENs (WO2011072246) or CRISPR CAS systems (including CRISPR Cas9, WO2013181440) or their next generation double strand break based techniques employing engineered nucleases. Examples of these methods are also described in example 15 and example 22.
Wheat plants obtained by the above methods are also part of the present disclosure. Typically, such wheat plants obtained or obtainable by the above method carry a Rf3 fertility restorer in which only a part, but not all, of the genomic fragment of SEQ ID NO:3174 is deleted in the genome of said wheat plant. Typically, a 10bp-162bp fragment of SEQ ID NO 3174 is deleted in the genome of said wheat plant. The wheat plant with the genome deletion obtained is expected to have a fertility score higher than that measured in a parent wheat plant having the same genome except for the complete sequence of SEQ ID NO:3174 in its genome. Alternatively, such wheat plants obtained or obtainable by the above method carry a fertility Rf3 restorer in which nucleotides of RFL29c have been deleted or added to restore in frame translation.
Method for evaluating fertility restoration of wheat plants
The present disclosure also includes methods for assessing fertility restoration of a wheat plant, the method comprising determining whether a segment of SEQ ID NO:3174 is present in the genome of the plant, wherein the presence of an intact segment indicates weak fertility restoration and the absence of at least a portion of such a segment indicates strong fertility restoration. Typically, the method is performed in wheat plants that are susceptible to carrying a restorer of Rf3 for fertility.
Also disclosed herein is a nucleic acid probe for use in the above method for assessing fertility restoration of a wheat plant, wherein the nucleic acid probe consists of a nucleic acid of at least 10 nucleotides within SEQ ID NO: 3174.
Typically, the nucleic acid probe is a fragment of at least 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp or the complete fragment of SEQ ID NO 3174.
Wheat plant restorer of fertility to the CMS cytoplasm of the tiffany wheat having 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 enables plants to be obtained which fully restore fertility to the CMS cytoplasm of the diaphenanthra wheat.
Accordingly, a first aspect of the present disclosure relates to a wheat plant restorer of fertility to the CMS cytoplasm of diaphraphenanthrene wheat, wherein the plant comprises at least three fertility restorer alleles within a restorer locus selected from the group consisting of Rf1, Rf3, Rf4 and
In a particular embodiment, the plant comprises at least three fertility restorer alleles Rf1, Rf3,
In a particular embodiment, the plant comprises at least three fertility restorer alleles Rf1, Rf4,
In a particular embodiment, the plant comprises at least three fertility restorer alleles Rf1, Rf3,
In a particular embodiment, the plant comprises at least three fertility restorer alleles Rf3, Rf4,
As used herein, the Rf1 locus refers to the locus of the Rf1 restorer allele which is located at a distance of at most 10cM, preferably at most 7cM, more preferably at most 2cM from marker cfn0522096 of SEQ ID No. 4 and/or marker cfn05277067 of SEQ ID No. 10. In a particular embodiment, the wheat plant restorer for fertility according to the present disclosure comprises at least one Rf1 restorer allele which is located within the chromosomal interval between the SNP marker cfn0522096 of SEQ ID NO:3190 and cfn05277067 of SEQ ID NO: 3196. In particular embodiments, the wheat plant restorer for fertility comprises one Rf1 restorer allele at the Rf1 locus, which Rf1 locus is characterized by the presence of one or more SNP alleles as set forth in table 1.
Table 1: SNP marker for locating Rf1 locus
Preferably, the wheat plant restorer for fertility according to the present disclosure comprises one Rf1 restorer allele at the Rf1 locus, said Rf1 locus being characterized by the presence of a SNP3 and/or SNP7 restorer allele as described in table 1. More preferably, the fertile wheat plant restorer is characterized by the haplotype of SNP3 and SNP7 restorer alleles "C" and "a". In certain embodiments, a wheat plant restorer with fertility of the Rf1 restorer allele comprises a Rf1 nucleic acid of the present disclosure as described above. Examples of Rf1 nucleic acids include the disclosed Rf1 nucleic acid sequence SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1915, SEQ ID NO:1916 or SEQ ID NO:3119, preferably, the Rf1 nucleic acid comprises SEQ ID NO: 3119.
As used herein, the Rf3 locus refers to a locus of Rf3 restorer allele which is located at a distance of at most 10cM, preferably at most 7cM, more preferably at most 2cM from marker cfn1249269 of SEQ ID NO:3205 and/or from marker BS00090770 of SEQ ID NO: 3228. In a particular embodiment, the wheat plant restorer for fertility comprises at least one Rf3 restorer allele within the Rf3 locus, which Rf3 restorer allele is located within the chromosomal fragment between the SNP markers cfn1249269 and BS 00090770. In particular embodiments, the wheat plant restorer for fertility comprises one Rf3 restorer allele at the Rf3 locus, which Rf3 locus is characterized by the presence of one or more SNP alleles as set forth in table 2.
Table 2: SNP marker for locating Rf3 locus
Preferably, the wheat plant restorer for fertility according to the present disclosure comprises one Rf3 restorer allele at the Rf3 locus, said Rf3 locus being characterized by the presence of a SNP29 and/or SNP31 restorer allele as described in table 2. More preferably, the fertile wheat plant restorer is characterized by a haplotype of the restoration alleles "T" and "a" at SNP29 and SNP31, respectively.
In another specific embodiment that may be combined with previous embodiments, the wheat plant restorer for fertility according to the present disclosure includes one Rf3 restorer allele at the Rf3 locus, which Rf3 locus is characterized by the presence of SNP38 and SNP41 restorer alleles "a" and "a", respectively.
Preferably, a wheat plant restorer for fertility according to the present disclosure comprises 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 a distance of up to 10cM from the marker cfn0919993 of SEQ ID NO: 3231. In particular embodiments, the wheat plant restorer for fertility comprises one Rf7 restorer allele at the Rf7 locus, which Rf7 locus is characterized by the presence of one or more SNP alleles as set forth in table 3.
Table 3: SNP marker of Rf7 locus
Preferably, the wheat plant restorer for fertility according to the present disclosure comprises one Rf7 restorer allele at the Rf7 locus, said Rf7 locus being characterized by the presence of nine SNP restorer alleles SNP44-SNP46 and SNP49-54 as described in table 3 for the "restorer allele" haplotype.
As used herein, the locus of Rf4 is located at a distance of up to 10cM from the marker cfn0393953 of SEQ ID NO: 3233. In particular embodiments, the wheat plant restorer for fertility comprises one Rf4 restorer allele at the Rf4 locus, which Rf4 locus is characterized by the presence of one or more SNP alleles as set forth in table 4.
Table 4: SNP marker of Rf4 locus
The wheat plant restorer for fertility according to the present disclosure includes one Rf4 restorer allele at the Rf4 locus, which Rf4 locus is characterized by the presence of two SNP restorer alleles SNP47 and SNP48 of haplotypes "C" and "G", respectively, as described in table 4.
In certain embodiments, a wheat plant restorer with fertility of the Rf4 restorer allele comprises a Rf4 nucleic acid of the present disclosure as described above. Examples of Rf4 nucleic acids include the published Rf4 nucleic acid sequence, SEQ ID NO:2031, SEQ ID NO: 3140-3142.
In particular embodiments, the wheat plant restorer for fertility of the CMS cytoplasm of diaphenanthryl wheat comprises one Rf3 restorer allele and two other fertility restorer alleles selected from the group consisting of Rf1, Rf4 and Rf7 restorer alleles. Preferably, the wheat plant restorer for fertility of the temofibrivirus wheat CMSCMS cytoplasm according to the present disclosure comprises Rf1, Rf3 and Rf7 restorer alleles.
Specifically, included herein are wheat plants comprising restored alleles of Rf1, Rf3, and Rf7 provided from seed samples deposited at the NCIMB collection at 2017 on 25.9 under accession numbers NCIMB 42811, NCIMB 42812, NCIMB 42813, NCIMB 42814, NCIMB 42815, NCIMB 42816, and NCIMB 42817.
The present invention also relates to a hybrid wheat plant which can be produced by crossing a fertile wheat plant restorer according to the present disclosure as described above with a second plant.
In certain embodiments, the wheat plant according to the present disclosure is heterogeneous and comprises a tiffany wheat cytoplasm.
For example, a hybrid wheat plant may be obtained by crossing a wheat plant restorer (preferably comprising the Rf1, Rf3, and Rf7 restorer alleles) according to the present disclosure as described above with a wheat plant which does not have the fertility restorer allele.
Also disclosed herein is a method of producing a hybrid plant of wheat, comprising the steps of:
a. crossing a sterile female wheat plant comprising a tiffany wheat cytoplasm with a fertile male wheat plant of the present disclosure as described above;
b. collecting hybrid seeds;
c. optionally detecting the presence of the cytoplasm of the Tomopivirus wheat and/or at least three Rf loci selected from Rf1, Rf3, Rf4, and Rf7 in the hybrid seed; and the combination of (a) and (b),
d. optionally, the hybrid seed is tested for hybridization levels.
Thus, also disclosed herein are wheat plants or lines according to the present disclosure that are developed to obtain such hybrid plants. Such plants or lines typically comprise cytoplasmic elements required for implementing the corresponding crossing system. Preferably, the plant or line comprises fertility restorer alleles Rf1, Rf3 and Rf7 and the tiffany wheat cytoplasm. In particular embodiments, such plants or lines comprise a fertility restorer allele Rf1, comprising a Rf1 nucleic acid of the present disclosure as described above.
Alternatively, the presence of the cytoplasm of the Tomopivia wheat and at least three Rf loci selected from Rf1, Rf3, Rf4, and Rf7 can be tested on the parental lines (step "c" of the above method) to check the genotype before starting the cross (step "a").
T-CMS cytoplasm can be detected either phenotypically (where plants carrying the rf gene and T-CMS cytoplasm will be sterile) or by molecular means capable of detecting the orf256 gene (as described by Rathburn and Hedgcoth,1991 and Song and Hedgcoth, 1994).
Methods of producing and selecting wheat plants of the disclosure
The present disclosure also relates to methods of producing wheat plants having fertility restorer alleles as described in the previous section.
In one embodiment, the method of producing a wheat plant comprises the steps of:
a. providing a first wheat plant comprising one or two restorer alleles selected from the group consisting of Rf1, Rf3, and Rf7 restorer alleles,
b. crossing said first wheat plant with a second wheat plant comprising one or two restorer alleles selected from the group consisting of Rf1, Rf3 and Rf7 restorer alleles, wherein Rf1, Rf3 and Rf7 restorer alleles occur at least once in a set of restorer alleles provided by said first plant and said second plant,
c. collecting the seeds of the F1 hybrid seeds,
d. obtaining a homozygous plant from the F1 plant,
e. optionally detecting the presence of the restored alleles of Rf1, Rf3, and Rf7 in the hybrid seeds and/or in each generation.
Preferably, the female plants in step b) carry T-CMS cytoplasm. In this case, the presence of the restorer allele is assessed in each generation of steps b) to d) by using markers and optionally by further assessing fertility levels.
Methods for producing homozygous plants are generally well known to those skilled in the art. This can be done by repeated backcrossing or by doubled haploid development or by single seed progeny (SSD) methods.
The applicant has deposited, according to budapest treaty on 25.9.2017, seed samples of the wheat plants disclosed as having said Rf1, Rf3 and Rf7 restorer alleles at the NCIMB collection under accession numbers NCIMB42811, NCIMB 42812, NCIMB 42813, NCIMB 42814, NCIMB 42815, NCIMB 42816 and NCIMB 42817.
The present disclosure further includes and provides methods of identifying each of the Rf1, Rf3, Rf4, and/or Rf7 restorer alleles disclosed in the previous sections, and more generally, methods of selecting or breeding wheat plants for the presence or absence of Rf1, Rf3, Rf4, and/or Rf7 fertility restorer alleles. Such methods of identifying, selecting, or breeding wheat plants include obtaining one or more wheat plants and evaluating their DNA to determine the presence or absence of fertility restorer alleles for Rf1, Rf3, Rf4, and/or Rf7 contained in the respective loci.
For example, this method can be used to determine which progeny resulting from a cross have a desired combination of fertility restorer alleles, and to direct the preparation of plants having the desired combination in combination with the presence or absence of other desired traits, accordingly.
Thus, plants can be identified or selected by assessing the presence in the plants of one or more of the individual SNPs appearing in tables 1, 2, 3 and 4 above, as well as the SNP in table 19, to assess the presence of the restorer allele Rf1, Rf3, Rf7 or Rf4, respectively.
More generally, specific means for detecting restorer alleles, more specifically Rf1, Rf3, Rf4 and Rf7 restorer alleles and combinations thereof in wheat plants are disclosed herein.
Thus, the means comprise a marker adapted to mark one or more of: any means of detecting the following SNP markers in SEQ ID NOs 3187-3235.
Any method known in the art can be used 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 Sequencing (GBS), or a combination thereof.
Different PCR-based methods are available to the skilled person. RT-PCR methods or Kaspar method from KBioscience (LGC Group, Teddington, Middlesex, UK) can be used.
KASPTMThe genotyping system uses three target-specific primers: two primers, each of which is specific for each allelic form of an SNP (single nucleotide polymorphism), and the other primer can achieve reverse amplification, which is shared by both allelic forms. Each target-specific primer also has a tail sequence corresponding to one of the two FRET probes: withA label and a band of dyesAnother label for the dye.
Successive PCR reactions were performed. The nature of the emitted fluorescence is used to identify one or more allelic forms present in the mixture from the DNA under study.
The primers identified in Table 5 are particularly suitable for use in KASPTMA genotyping system. Of course, the skilled person may use variant primers or nucleic acid probes of the primers identified in table 5, which have at least 90%, preferably 95% sequence identity to any one of the primers identified in table 5 or to a DNA genome fragment amplified by the corresponding set of primers identified in table 5.
The percentage of sequence identity as used herein is determined by counting the number of matching positions in the aligned nucleic acid sequences, dividing the number of matching positions by the total number of aligned nucleotides, and multiplying by 100. A matched position is a position in which identical nucleotides occur at the same position in the aligned nucleic acid sequences. For example, nucleic acid sequences can be aligned using the
As used herein, primers encompass any nucleic acid capable of priming synthesis of a nascent nucleic acid in a template-dependent process (e.g., PCR). Typically, the primer is an oligonucleotide of 10 to 30 nucleotides, but longer sequences may be employed. The primer may be provided in a double stranded form, although a single stranded form is preferred. Alternatively, nucleic acid probes may be used. Nucleic acid probes encompass any nucleic acid of at least 30 nucleotides, and which can specifically hybridize to a defined nucleic acid under standard stringency conditions. As used herein, standard stringency conditions refer to hybridization conditions, such as those described in Sambrook et al 1989, which can comprise 1) immobilizing a plant genomic DNA fragment or library DNA on a filter; 2) filters were prehybridized in 6 XSSC 5 XDenhardt reagent, 0.5% SDS and 20mg/ml denatured carrier DNA for 1-2 hours at 65 ℃; 3) addition of probe (labeled); 4) incubating for 16-24 hours; 5) wash the filter once in 6x SSC, 0.1% SDS for 30 minutes at 68 ℃; 6) filters were washed three times (twice for 30 min in 30ml and once for 10 min in 500 ml) in 2x SSC 0.1% SDS at 68 ℃.
In particular embodiments, the primers (specific for each allele "X" or "Y" or collectively) used to detect the SNP markers of the disclosure are shown in table 5 below:
Table 5: primers for detecting the SNP marker of fertility restorer of the present invention (as shown by the primer name)
Use of the wheat plants of the present disclosure
Plants according to the present disclosure can be crossed with any other inbred line to produce a new line comprising an increased or decreased level of fertility. Alternatively, a genetic trait engineered into a particular line using the transformation techniques described above can be transferred into another line using conventional backcrossing techniques well known in the art of plant breeding. For example, the backcross approach can be used to transfer engineered traits from a public non-elite inbred line into an elite inbred line, or from an inbred line that includes a foreign gene in its genome into one or more inbred lines that do not include the gene. As used herein, "hybridization" may refer to the process of simple X hybridization or backcrossing by Y, depending on the context.
The wheat plants of the present disclosure are also wheat plants in which one or more desired traits are further introduced by a backcrossing method, whether or not such traits are naturally occurring.
The invention also relates to the use of a wheat plant as described above or a seed thereof in food applications, preferably in flour production and feed applications, or in breeding applications, for example as a parent plant in breeding to improve the agronomic value of a wheat plant, line, hybrid or variety.
As used herein, breeding applications encompass pedigree breeding to improve the agronomic value of a plant, line, hybrid or variety.
For example, the wheat plants disclosed herein may be further used for the production of flour or for feed applications.
Seeds harvested from the plants described herein can be used to prepare flour by any technique available in the art. The wheat plant or flour thereof may also be used as a food composition for humans or animals.
The following examples are provided for illustrative purposes only.
Description of the preferred embodiments
1. An isolated nucleic acid encoding a protein restorer of fertility to Triticum aestivum, wherein the corresponding amino acid sequence has at least 95% identity to an amino acid sequence selected from any one of SEQ ID No. 1 to SEQ ID No. 1554.
2. The nucleic acid of
3. The nucleic acid of
4. The nucleic acid of
5. The nucleic acid of
6. The nucleic acid of
7. The nucleic acid of
8. The nucleic acid of
9. A recombinant nucleic acid comprising a nucleic acid encoding the protein restorer of the CMS cytoplasm of tiffany wheat of any one of embodiments 1-8 operably linked to regulatory elements.
10. Use of a vector to transform a wheat plant comprising the recombinant nucleic acid of
11. A wheat transgenic plant comprising as a transgenic element one or more nucleic acids of any one of embodiments 1-9.
12. The wheat transgenic plant of embodiment 11 which is a fertile wheat plant restorer of the CMS cytoplasm of tiffany wheat and comprises a combination of at least two different transgenic elements selected from the group consisting of Rf1, Rf3, Rf7 and Rf-rye nucleic acids of any one of
13. The transgenic wheat plant of
an Rf1 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430, and an Rf3 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of 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, SEQ ID NO 676 and SEQ ID NO 684,
an Rf1 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430, and an Rf7 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO:363, SEQ ID NO:516 and SEQ ID NO:768,
An Rf1 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430, and an Rf-rye nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 227, SEQ ID NO 378 and SEQ ID NO 859,
an Rf3 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of 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, SEQ ID NO 676 and SEQ ID NO 684, and an Rf7 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 363, SEQ ID NO 516 and SEQ ID NO 768,
an Rf3 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of 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, SEQ ID NO 676 and SEQ ID NO 684, and an Rf-rye nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 227, SEQ ID NO 378 and SEQ ID NO 859,
an Rf7 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 363, SEQ ID NO 516 and SEQ ID NO 768, and an Rf-rye nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 227, SEQ ID NO 378 and SEQ ID NO 859,
Rf1 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430, and Rf3 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of 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, SEQ ID NO 676 and SEQ ID NO 684, and Rf7 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs 363, SEQ ID NO 516 and SEQ ID NO 768;
an Rf1 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430, and an Rf3 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of 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, SEQ ID NO 676 and SEQ ID NO 684, and an Rf-rye nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 227, SEQ ID NO 378 and SEQ ID NO 859;
an Rf1 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NOs359, 361, 362 or SEQ ID NOs428-430, and an Rf7 nucleic acid of
Rf3 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of 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, SEQ ID NO 676 and SEQ ID NO 684, and Rf7 nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 363, SEQ ID NO 516 and SEQ ID NO 768, and Rf-rye nucleic acid encoding an amino acid sequence having at least 95% identity to any one of SEQ ID NO 227, SEQ ID NO 378 and SEQ ID NO 859.
14. The transgenic wheat plant of
15. The transgenic wheat plant of any one of embodiments 11-14, wherein the one or more transgenic elements express a polypeptide that restores or improves male fertility of the plant as compared to a parent plant that does not contain such transgenic element.
16. A method of producing the wheat transgenic plant of any one of embodiments 11-15, wherein said method comprises the steps of: transforming a parent wheat plant with one or more nucleic acids encoding a protein restorer of the CMS cytoplasm of tiffany wheat according to any of embodiments 1-9, selecting a plant comprising said one or more nucleic acids as a transgene, regenerating and growing said wheat transgenic plant.
17. A method of producing a wheat plant carrying a Rf3 restorer of fertility, the method comprising (i) providing a parent wheat plant comprising in its genome a fragment of at least 163bp of SEQ ID NO:3174, and (ii) deleting in the genome of the wheat plant a region of at least 10bp, such as at least 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp or the entire fragment of SEQ ID NO:3174 of said fragment of SEQ ID NO:3174, thereby obtaining the wheat plant carrying a Rf3 restorer of fertility.
18. The method of
19. The method of
20. A wheat plant carrying a fertility Rf3 restorer obtainable by the method of any one of embodiments 17-19, wherein only a part, but not all, of the genomic fragment of SEQ ID NO:3174 is deleted in the genome of said wheat plant.
21. A method for assessing fertility restoration of a wheat plant, the method comprising determining whether a fragment of SEQ ID NO:3174 is present in the genome of the plant, wherein the presence of an intact fragment indicates weak fertility restoration and the absence of at least a portion of such or an intact fragment of SEQ ID NO:3174 indicates strong fertility restoration.
22. A nucleic acid probe for use in the method of any one of
23. A wheat plant restorer of fertility to the CMS cytoplasm of diaphenanthraceae wheat, wherein said plant comprises at least three fertility restorer alleles within a restorer locus selected from the group consisting of Rf1, Rf3, Rf4 and Rf7, wherein:
the Rf1 locus is located at most 10cM from the marker cfn0522096 of SEQ ID NO 3190 or the marker cfn05277067 of SEQ ID NO 3196,
the Rf3 locus is located at most 10cM from the marker cfn1249269 of SEQ ID NO. 3205 or the marker BS00090770 of SEQ ID NO. 3228,
The Rf7 locus is located at most 10cM from the marker cfn0919993 of SEQ ID NO:3231, and
the Rf4 locus is located at most 10cM from the marker cfn0393953 of SEQ ID NO: 3233.
24. The wheat plant of
25. Wheat plant according to any one of
26. The wheat plant of
27. the wheat plant of embodiment 26, wherein the Rf1 locus is characterized by the haplotypes "C" and "a" of the SNP3 and SNP7 recovery alleles as described in the table of embodiment 5.
28. The wheat plant of any one of embodiments 23-27, characterized in that it comprises at least one Rf3 restorer allele within the Rf3 locus, which Rf3 restorer allele is located within the chromosomal fragment between the SNP markers cfn1249269 and BS 00090770.
29. The wheat plant of embodiment 28, wherein the Rf3 locus is characterized by the presence of one or more of the following SNP alleles:
30. The wheat plant of embodiment 29, wherein the Rf3 locus is characterized by the haplotypes "T" and "a" of the SNP29 and SNP31 recovery alleles as described in the table of
31. The wheat plant of any one of embodiments 23-30, wherein the Rf7 locus is characterized by the presence of one or more of the following restorer SNP alleles:
32. the wheat plant of any one of embodiments 23-31, wherein the Rf4 locus is characterized by the presence of one or more of the following SNP alleles, preferably with the haplotypes "C" and "G" restored by SNP47 and SNP 48:
33. the wheat plant of any one of embodiments 23-32, wherein the representative alleles of the Rf1, Rf3, Rf4, and Rf7 restorer alleles are provided by a seed sample selected from the group consisting of: NCIMB 42811, NCIMB 42812, NCIMB 42813, NCIMB 42814, NCIMB 42815, NCIMB 42816 and NCIMB 42817.
34. The wheat plant of any one of embodiments 23-33, wherein the wheat plant is heterogeneous and comprises a tiffany wheat cytoplasm.
35. A method of identifying a wheat plant of any one of embodiments 23-34, wherein said wheat plant is identified by detecting the presence of at least one restorer allele that is genetically linked to a restorer locus selected from the group consisting of the Rf1, Rf3, Rf4 and Rf7 loci.
36. Means for detecting one or more SNPs of SEQ ID NOs 3187-3235.
37. The means of embodiment 36 consisting of one or more primers comprising any one of: SEQID NOs 3253-3444.
38. A method of producing a hybrid wheat plant comprising the steps of:
a. crossing a sterile female wheat plant comprising the cytoplasm of a tiffany wheat with a fertile male wheat plant according to any one of embodiments 23-34;
b. collecting hybrid seeds;
c. optionally detecting the presence of the cytoplasm of the Tomopivirus wheat and/or at least three Rf loci selected from Rf1, Rf3, Rf4, and Rf7 in the hybrid seed; and the combination of (a) and (b),
d. optionally, the hybrid seed is tested for hybridization levels.
Drawings
FIGS. 1A and 1B are tables showing summaries of the plant genomes used and the number of RFLs identified in the study. Collectively, these analyses cover 16 genomes from wheat and 13 genomes from the rice family, respectively, as well as from Brachypodium distachyon (Brachypodium distachyon), tef (eragerotics tef), rye (secaleale), millet (Setaria italica), Sorghum (Sorghum bicolor) and maize (Zea mays). Ryegrass (Lolium perenne) and Triticum turgidum (Triticum turgidum) transcriptome datasets were also used.
FIG. 2: processing of orf256 in T-CMS wheat mitochondria (a) the structure of orf256 identified in the tiffany wheat mitochondrial genome. The binding sites of WORF256 probe (Song and Hedgoth 1994) for Northern blot analysis are shown. (B) Differential treatment of orf256 in wheat lines with different recovery capacity. Orf256 transcripts were not detected in the common wheat, primipii, Anapurna and wheat-rye-6R (WR _6R) lines. Another third band detected in the R197 and R0934F accession numbers is indicated by an asterisk. As a control for gel loading, pictures of ethidium bromide (EtBr) -stained agarose gels are shown.
FIG. 3: FIG. 3 shows a list of RFL groups potentially corresponding to the Rf4 gene.
Fig. 4a and 4 b: FIGS. 4A and 4B show the alignment between nucleotide and amino acid sequences of RFL 120-spring (subject) and RFL 120-time (query), respectively.
FIG. 5A: FIG. 5A shows the protein sequence alignment of RFL29a, RFL29b, RFL29c _1 and RFL29c _ 2.
Fig. 5B and 5C: FIGS. 5B and 5C show protein sequence alignments of RFL164a and 164 RFL164B and RFL166a and 166B, respectively.
FIG. 6: FIG. 6 shows an alignment of the 5' UTR regions identified in the RFL29a and RFL29b genes.
FIG. 7: FIG. 7 shows the positions of the different target sequences around and within the 163bp region identified for the different endonucleases.
FIG. 8: figure 8 shows the relative positions of the Rf1 localization intervals identified on our internal consensus genetic map.
FIG. 9: figure 9 shows the relative positions of the Rf3 localization intervals identified on our internal consensus genetic map.
FIG. 10: FIG. 10A shows the location of the marker within the chromosomal interval of the
FIGS. 10B, 10C, 10D show a subset of the diversity groups showing the restored lines (R197, R204, R0932E), the derived lines LGWR16-0016 and LGWR16-0026 for genetic mapping and the haplotype at the Rf1 locus of the maintained line set. "-": corresponding to a dominant marker that was not amplified in several maintained lines. "H": refers to a heterozygote state in which both alleles are detected.
FIG. 11: FIG. 11A shows the location of the marker within the chromosomal interval of the
Fig. 11B, 11C, 11D show a subset of the diversity group showing the haplotype at the Rf3 locus for the recovery lines LGWR16-0016 and LGWR16-0026, the TJB155 line used as the recovery parent line and a series of maintenance lines in the Rf3 QTL localization.
"-" corresponds to a dominant marker that is not amplified in several maintained lines. "H" refers to a heterozygote state in which both alleles are detected.
FIG. 12: FIG. 12 shows the nucleotide sequence alignment between RFL29a and RFL29c fragment sequences. The PAM motif and target sequence of the CRISPR version are bold and underlined, respectively.
Detailed description of the preferred embodiments
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