Process for the modification of genome structure and gene expression in wheat

Abstract

A method of modifying the genome structure and / or gene expression of wheat by crossing a parental wheat (P♀) with a transgenic barley (X XH+H) containing at least one transgenic barley chromosome (XH+), wherein the following steps are carried out: (i) barley genome is transformed with a pre-designed gene construct that can affect the wheat genome and / or its expression, (ii) the resulting wheat (F1) × wheat × at least one transgenic barley chromosome containing transitional hybrids (F1♀) are selected and bred, (iii) the wheat × at least one transgenic barley chromosome -containing transitional hybrids (F1♀) are backcrossed with a wheat (♂), (iv) and a new generation is produced by selfing the selected backcrosses (BC1), (v) from this new generation, a wheat (BC1S1) is selected which is the product of the process and is free from any chromosomal aberrations and altered in genome structure and / or gene expression.

Description

[0001] Process for the modification of genome structure and gene expression in wheat

[0002] The invention relates to a process that modifies genome structure and / or gene expression in wheat without resulting in transgenesis in the wheat target genome, said process being more efficient than similar processes used in wheat so far, which do not result in transgenesis. The process is a genetic technique that involves a change in the genetic material and / or gene expression of wheat that would not occur in nature without human intervention.

[0003] To modify the genetic material and / or gene expression of wheat, barley genome is transformed with a pre- designed gene construct that can manipulate the wheat genome and / or its expression, and then fertilization of wheat egg cell with the transgenic barley pollen is combined with a demethylation chemical treatment to produce a wheat × barley transitional hybrid plant in which proteins and / or RNAs encoded by the transgene modify the wheat genome. The change in the genetic material and / or in the gene expression of the wheat may be temporary or may be passed on to subsequent generations, depending on the transgcnc carried by the barley genome. The new trait of the wheat plant is created by the transgcnc present in the barley; in the absence of the transgene, the beneficial trait would not develop.

[0004] State of the art

[0005] Modification of genome structure and / or gene expression is usually achieved by the intracellular expression of an executive protein and / or RNA molecule, or an artificial delivery thereof. In many cases, such a modification of genetic material confers an economic advantage to the given organism. This approach includes various gene editing techniques (CRISPR / Cas, TALEN, ZFN, meganucleases) that can be used to impart food, health, or other economically important traits to plants. Unlike previous transgenic techniques, these traits do not necessarily require the insertion of foreign gene sequences; they create new phenotypes through induced gene mutations, similar to other freely available but random mutation techniques (irradiation, chemical treatment, etc.). A major advantage of gene editing over previous mutation techniques is the high degree of specificity of creating the desired gene mutation, provided by a programmed RNA molecule and / or protein. Based on the state of the art, the most efficient method for implementing gene editing mutations is the temporary insertion of the gene of the gene editing protein and, if applicable, an RNA molecule into the genome and then removing them in subsequent generations after the gene mutations have been created. However, temporary transgenesis involving the target genome may raise concerns on the user side due to the currently controversial perception of GMOs (Genetically Modified Organisms), including plants. At the same time, transgenesis has technological difficulties, as the transformability of most plant species / cultivars is limited. Given the above, it is feasible to produce transgenic wheat by gene editing and then by transgene segregation. The problem is that few wheat cultivars can be well transformed, and there is a risk of persistence of the transgene or small gene fragments that are difficult to trace and that have been incorporated during transgenesis.

[0006] For these reasons, procedures have been developed that do not require transgenesis of the target genome. Examples include the biolistic (gene gun) or chemically induced delivery of gene editing protein and / or RNA into embryogenic cells and subsequent plant regeneration from these cells. However, in the absence of selection, these techniques are labour-intensive, and due to the limited lifetime of the gene editing protein in the cells, their efficiency is very low, and the process is not well-suited for industrial application.

[0007] International Publication Document No. WO / 2018 / 102816, which is the closest technical solution to the present invention, describes a new method of gene editing. It does not involve the introduction of a transgene into the target genome, does not require good transfonnability of the target genome, and at the same time, a genome with a transgene and the target genome only temporarily coexist in the cell, so that both gene editing and transgene removal can be perfonned without any doubt.

[0008] The process involves pollinating a plant to be modified with pollen from a known haploid inducer line containing a transgene suitable for plant gene editing. The genome of the resulting haploid progeny is modified by the function of the transgene transferred through crossing, while the gene-modifying transgene is eliminated along with the chromosomes of the haploid inducer line. During the method, there is practically only time to alter the genetic material until the first few cell divisions, as the gene sequence producing gene editing proteins present on the chromosomes of the haploid inducer line is continuously eliminated during cell divisions. Preferred embodiments of the method include modification of the wheat genome. However, the method is actually of little use or can only be used with low efficiency for modifying the genome of hexapioid wheat, since there are three pairs of homeologous subgenomes (A, B, and D) in wheat, and usually all three homeologous gene sequences need to be modified to achieve a particular trait, which is very inefficient in a process where the transgene is only present for a few cell divisions. In many cases, moreover, the gene to be modified is located in a heterochromatic region during embryonic development, which, due to its methylation, is not accessible to the proteins encoded by the transgene during the period until elimination. Other wheat genes (e.g. gliadin storage protein genes) are present in the wheat genome in very many (>100) copies, which would also require very high efficiency or longer time to modify. WO / 2018 / 102816 mentions the wide crossing of wheat with transgenic barley as a preferred embodiment. After crossing wheat and transgenic barley, the F1 progeny plants can be divided into two sets: A) a group of plants without a barley chromosome (early chromosome elimination), which are considered haploid (In) compared to the original wheat with 2n genome, since they contain only the maternal genome; B) a group of plants comprising a barley chromosome, which are partial or complete wheat × barley hybrid plants. The solution according to WO / 2018 / 102816 focuses only on set A) and aims to achieve the transgene-free status by the early elimination of paternal chromosomes, which, however, is not conducive to the modification of wheat genes due to the relatively short presence of the transgene. This embodiment of the cited patent application is not well feasible for all wheat cultivars, since the proportion of transgene-free haploid F1 individuals in wheat × barley crosses is, with few exceptions, relatively low. Thus, compared to other wide crosses (e.g., wheat × maize), wheat × barley crosses can be considered hybridization rather than haploid induction.

[0009] Given the shortcomings identified, we aimed to develop a method that is more efficient than the methods known in the art for modifying the genome structure and / or gene expression in wheat, while ensuring a transgene-free outcome.

[0010] The discovery underlying the invention

[0011] It is known from the state of the art that hybridization occurs in wide crosses of wheat x barley to a technologically useful extent. It has been recognized that the presence of transgene -containing barley chromosomes in hybrids provides the opportunity over longer periods and at different stages of the life cycle to modify the genome structure and / or gene expression in wheat compared to haploid induction techniques, which is favorable to the use of genetic engineering tools that modify wheat genome structure and / or gene expression. We consider genetic engineering tools to be protein families and RNA classes that allow the modification of genome structure and / or gene expression. Without limitation, such tools are CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-Associated Protein), TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), meganucleases, RNA interference constructs (artificial microRNA, inverted repeat), enzymes affecting DNA methylation (mcthylascs, dcmcthylascs). Wc consider genetic engineering to be those techniques that facilitate the use of the above tools. Without limitation, such methods arc: biolistic or agrobactcrial transgenesis; direct cellular delivery of RNAs, RNPs (RiboNucleoProtein), or proteins; and viral delivery (VIGE, Virus Induced Genome Editing).

[0012] In the present specification, the term "crossing" is used in a sense different from the essentially biological process of creating conventionally bred plant cultivars: in the solution according to the present invention, "crossing" includes, inter alia, pollination between distant (genetically and reproductively unrelated) species and a chemical treatment for demethylation. Cross-fertilization between distant species is characterized by the partial or total loss of genetic material from one (often the paternal) parent during the development of the offspring. This process is fundamentally different from directed crossing used in cultivar development, which, on the contrary, aims at combining the genetic material of both parents and passing it on in a stable manner to the progeny. On the other hand, chemical demethylation treatment can compensate with great efficiency for the phenomenon, which often occurs when pollinating distant species, that the genetic material from the parents of the opposite species is incompatible, which prevents the methylation pattern of the genes from being passed on to the progeny cells: as a result, the development of the endosperm does not start, and the lack of which inhibits the growth and development of the embryo. This defect is improved by the demethylation chemical treatment by resetting the methylation pattern, resulting in larger and more developed hybrid embryos. Accordingly, in wheat × barley “crossing”, the efficient production of hybrid embryos is also ensured by chemical treatment with a demethylation agent. (Note that, unlike in the crossing for cultivar development, the progeny grains that are produced after pollination between distant species are non-viable on their own because they lack endosperm, i.e. nutritive tissue, to provide the nutrients necessary for natural germination. Therefore, embryos that are undeveloped and doomed to die must always be dissected out of the grains at an early stage and then grown in vitro on artificial media to provide these missing nutrients.) It is also known from the state of the art that wheat × barley hybrids can be backcrossed with wheat to produce wheat with a 2n genome and no barley chromosomes. Related to this is our further finding that, with the appropriate sequence of steps known from the art in our process, after modifying the genome structure and / or gene expression in the hybrids, the progeny plants can be made transgene-free with a 2n genome by backcrossing and self-fertilization. Thus, the product of the process meets all the state-of-the- art requirements imposed on gene editing technologies in terms of the assessment of GMOs

[0013] In International Publication Document No. WO / 2018 / 102816, barley is used as a haploid-inducing crop for wheat, but we use the hybridization phenomenon that typically occurs with a higher probability. In our method, we, therefore, retain for further crossing those hybrids that still contain transgene-containing barley chromosomes and are more likely to have the desired changes in genome structure and / or gene expression. In the F1 progeny resulting from a wheat × transgenic barley cross, the barley chromosomes may segregate during meiosis, so that after backcrossing the F1 progeny with wheat, some individuals of the BC1 (Back Cross 1) generation no longer contain the barley chromosomes. The individuals without the barley chromosomes can be selected using methods known in the art. The members of the BC1 generation generated by backcrossing, selected using known methods, are heterozygous for a given induced gene mutation. However, members of the BC1S1 (BC1 Self 1) generation generated by selfing BC1 plants, which are homozygous for the desired gene altered in genome structure and / or gene expression and no longer contain the barley chromosome, may be selected.

[0014] It is also important to note that the genetic makeup of barley and wheat is so different from each other that the possibility of gene transfer by recombination in the transitional hybrids, and thus the appearance of the gene-modifying transgene from the barley chromosome on the wheat chromosome, is virtually excluded.

[0015] The objective of the invention

[0016] With the objective in mind, our invention is therefore a method to modify the genome structure and / or gene expression with high efficiency using a transgenic barley genome containing at least one transgenic barley chromosome.

[0017] The objectives of the invention can be achieved by the process described in claim 1, the preferred embodiments of which are set out in the dependent claims.

[0018] Brief description of the invention

[0019] 1. A process for modifying the genome structure and / or gene expression of wheat by crossing a parental wheat with a transgenic barley containing at least one transgenic barley chromosome wherein the following steps are carried out:

[0020] (i) transforming the barley genome with a pre-designed gene construct that can manipulate the wheat genome and / or its expression,

[0021] (ii) fertilizing wheat egg cells with the transgenic barley pollen, then subjecting the wheat to a demethylation treatment to induce partial development of the endosperm and more efficient development of the embryos, and then growing the embryos on artificial media under in vitro conditions;

[0022] (iii) selecting and growing the resulting cross-generation (F1) wheat × barley transitional hybrids containing at least one transgenic barley chromosome,

[0023] (iv) then, backcrossing the wheat × barley transitional hybrids containing at least one transgenic barley chromosome with a wheat

[0024] (v) and then producing a new generation by self-fertilization from the selected individuals (BC1) resulting from backcrossing,

[0025] (vi) selecting from said new generation (BC1S1) a wheat that is free of the barley chromosomes and is modified in its genome structure and / or gene expression, which is the product of the process.

[0026] 2. The process according to Point 1, wherein the wheat used for backcrossing and the parental wheat are of the same cultivar.

[0027] 3. The process according to Point 1, wherein the wheat used for backcrossing and the parental wheat arc of different cultivars.

[0028] 4. The process according to any one of Points 1 to 3, wherein a chemical demethylation treatment is applied during the crossing.

[0029] 5. The process according to any one of Points 1 to 4, wherein the transgene of the transgenic barley chromosome used is a gene editing tool.

[0030] 6. The process according to Point 5, wherein said gene editing tool is one of the genetic engineering tools of CRISPR / Cas, TALENs, ZFNs, RNA interference constructs, or a derivative thereof, or a combination thereof.

[0031] 7. The process according to any one of Points 1 to 3, wherein the transgene of the transgenic barley chromosome used modifies the methylation pattern of the wheat.

[0032] 8. The process according to Point 7, wherein the transgene that modifies the methylation pattern is an RNA interference construct that silences the Met1 and / or Cmt3 genes.

[0033] 9. The process according to any one of Points 1 to 8, wherein one of the transgenes of the transgenic barley chromosome used is a reporter gene.

[0034] 10. The process according to Point 9, wherein one of GFP, YFP, DsRed, Ruby, or a combination thereof is used as the reporter gene. 11. The process according to any one of Points 1 to 10, wherein the growing is carried out by in vitro micropropagation .

[0035] 12. The process of Point 11, wherein the explant in the in vitro micropropagation is an immature embryo or immature inflorescence.

[0036] Description of the figures

[0037] Our invention is described in detail with reference to the attached drawings, wherein

[0038] - Figure 1 shows the steps of our process.

[0039] - Figure 2 shows the application of our process for gene editing.

[0040] - Figure 3 shows the application of our process for transiently modifying gene expression.

[0041] - Figure 4 shows an application of our process to modify the genome structure and / or gene expression of a wheat that hybridizes poorly with barley.

[0042] - Figure 5 shows a process in which the process of meiotic recombination between two wheat cultivars is modified.

[0043] - Figure 6: Analysis of the chromosome composition of the F1 (wheat × barley) generation by multiplex PCR (MPCR). (A) A-genome-specific MPCR products, (B) B-genome-specific MPCR products, (C) D-genome-specific MPCR products, (D) H-genome -specific MPCR products (red asterisk, Cas9-positive plants; red arrow, 445 bp Cas9 amplicon), (E) Template DNA used to identify individual chromosome-specific MPCR product amplicons. M - 100 bp Plus DNA ladder; 1-37 - F1 hybrid plants; M1 - M1 wheat; GP - 'Golden Promise' barley; dw - distilled water.

[0044] - Figure 7: Analysis of wheat mlo gene mutations in the F1 generation by PCR / RE (238 bp Tamlo PCR product digested with Cac8I restriction enzyme). M - 100 bp Plus DNA ladder; 1-37 - F1 hybrid plants (red arrow, mutant plants); w - wild type M1 wheat, digested PCR product; - wild type M1 wheat, undigested PCR product; dw - distilled water.

[0045] - Figure 8: Analysis of chromosome composition of subgenomes of BC1 generation by multiplex PCR. M - 100 bp Plus DNA ladder; 1-17 - BC1 plants (red asterisk, Cas9-positive plants; red arrow, 445 bp Cas9 amplicon); GP - 'Golden Promise' barley; M1 - M1 wheat.

[0046] - Figure 9: Analysis of wheat mlo gene mutations in the BC1 generation by PCR / RE (238 bp Tamlo PCR products digested with Cac81 restriction enzyme). M --- 100 bp Plus DNA ladder; 1-17 -- BC1 plants (red arrow, mutant plant); w - wild type M1 wheat, digested PCR. product; - wild type M1 wheat, undigested PCR product.

[0047] - Figure 10: Analysis of wheat mlo gene mutations (A) and the chromosome composition of the barley genome (B) in in vitro plants derived from an F1 embryo. M - 100 bp Plus DNA ladder; 1-20 - in vitro propagated plants (red arrow, mutant plant); w0 - wild type M1 wheat, undigested PCR product; w - wild type M1 wheat, digested PCR product; dw - distilled water; M1 - M1 wheat; GP - 'Golden Promise' barley.

[0048] - Figure 1 1 : Analysis of wheat mlo gene mutations by PCR / RE in progeny propagated in vitro from immature inflorescence of an F1 plant (7 of Figure 10). M - 100 bp Plus DNA ladder; 1-24 - in vitro propagated plants; w - wild type M1 wheat, digested PCR product; - wild type M1 wheat, undigested

[0049] PCR product.

[0050] - Figure 12 shows a schematic drawing of the pHUER_Tamlosg2 vector (SEQ ID NO: 01) used for the Agrobacterium-mediated transformation of immature barley embryos to create targeted mutations in wheat Tamlo genes.

[0051] The meaning of the reference symbols used in the description and figures is as follows:

[0052] P - Parental generation

[0053] F1 - First (filial) generation of progeny

[0054] BC1 - First backcrossed generation

[0055] BC1 S1 - Generation produced by selfing from the first backcrossed generation

[0056] Xx- At least one chromosome from any subgenome of hexapioid wheat

[0057] - At least one chromosome from any subgenome of hexapioid wheat with altered genome structure

[0058] - Barley transgenic chromosome (in text: XH+) - Barley non-transgenic chromosome (in text: XH)

[0059] - At least one chromosome from any subgenome of hexapioid common wheat

[0060] - One chromosome derived from the recombination of homologous chromosomes from any subgenome of hexapioid common wheat and another wheat

[0061] - The other chromosome derived from recombination of homologous chromosomes from any subgenome of hexapioid common wheat and another wheat

[0062] - Parental wheat

[0063] - Wheat × barley transitional hybrid containing at least one transgenic barley chromosome

[0064] - Wheat used for backcrossing

[0065] Figure 1 shows the steps of our process. After crossing wheat with transgenic barley (step 2), modification of genome structure and / or gene expression can already be achieved. However, changes in the genome structure and / or gene expression of wheat with a 2n genome, homozygous for the desired gene, generated during the process, can also occur in any of the subsequent steps of the process, in which the wheat genome and transgenic barley chromosomes are present in cells together, which is of particular importance in influencing the recombination process, where the effect of the transgene is particularly relevant during the formation of gametes of the BC1 generation. The process steps are as follows:

[0066] Step 1 : Transforming barley with a system that modifies the genome structure and / or expression of the wheat.

[0067] In the process, any transgene that modifies the genome structure and / or gene expression of wheat can be used in the transgenic barley. The transgene is always selected according to the genomic structure and / or gene expression modification objective from among the procedures known in the state of the art. Hereinafter, the transgene selected according to the purpose will be abbreviated as transgene GOI, or Gene of Interest, for better differentiation. Genome structure -modifying transgene GOIs may be, but are not limited to, the following types: CRISPR / Cas, TALEN, meganucleases or ZFN. Gene-expression-altering transgenes may be, but are not limited to, RNA interference constructs (artificial microRNA, inverted repeat), methylases, demethylases, and modifying factors. A transgenic barley carrying a GOI transgene can be produced by methods known in the art. In the process, in addition to the GOI transgene, selected according to the purpose, it is advantageous to use a linked reporter gene (e.g. GFP, YFP, DsRed, Ruby, etc.) to identify transgenic barley chromosomes. Without limitation, transgenic barley may be produced by biolistic or Agrobacterium-mediated gene transfer.

[0068] Step 2: Crossing of parental wheat with transgenic barley containing at least one transgenic barley chromosome

[0069] In this step, barley (Hordeum vulgare L.) is crossed with wheat (Triticum aestivum L.) using barley as a pollen donor. Following crossing, a chemical demethylation treatment is applied to tire wheat plant to promote more efficient endosperm and embryo development following wide crossing.

[0070] Step 3: Selection and growing of wheat x barley transitional hybrids containing at least one transgenic barley chromosome

[0071] Selection can be performed, for example, by using a reporter gene, which was linked to the appropriately selected GOI transgcnc, using a method known in the art, when generating the transgenic barley, or by any other method aimed at determining the karyotype or genotype. The individuals of the F1 generation are raised by embryo rescue, in vitro, on artificial media. Since the efficiency of egg cell fertilization with a distant species is lower than with natural intraspecific pollination, and among the low number of embryos that develop, the number of wheats comprising barley chromosome carrying the GOI transgene, or with modified genome structure and / or gene expression, is also variable; the system can be made more efficient by micropropagating in in vitro culture the generated F1 hybrid embryo or the immature embryo or immature inflorescence of the raised F1 hybrid plant carrying wheat chromosomes comprising the desired modification and barley chromosome(s) comprising the GOI transgene. Through in vitro culturing, more plants can be backcrossed with wheat, which increases the yield of BC1 plants in the subsequent step.

[0072] Step 4: Backcrossing of wheat × barley hybrids containing at least one transgenic barley chromosome with wheat

[0073] To produce a wheat plant containing again 2n wheat genome from the F1 hybrid plant selected in step 3, the selected wheat x transgenic barley hybrids of the F1 hybrids are crossed with wheat using any method known in the art.

[0074] One preferred embodiment of our method is backcrossing with the parental wheat, wherein the hybrid selected in the previous step is crossed with the starting wheat plant.

[0075] Another preferred embodiment of our method is crossing the hybrid selected in the previous step with a common wheat, which is different from the parental wheat and does not hybridize well with barley, but is otherwise more important from an economic point of view. In this case, the resulting BC1 hybrid will either contain the transgenic barley chromosome or not. In a hybrid containing the transgenic barley chromosome, the genome and / or gene expression of the economically more important wheat may be altered.

[0076] According to another preferred embodiment of our method, the GOI transgene comprises a methylase / demethylase enzyme or amethylase / demethylase gene silencing construct, so that recombination can be extended to naturally rarely recombinable or non-recombinable regions of the genome by modifying methylation in two different wheat cultivars during backcrossing.

[0077] Step 5: Self-fertilization of the backcrossed generation

[0078] Self-fertilization occurs naturally on its own. To prevent fertilization by foreign pollen, the ear is insulated, so the only possibility for fertilization is self-fertilization. Self-fertilization, including insulation, can be achieved by any method or means known in the art.

[0079] Step 6: Selection of wheat free of transgenic barley chromosomes and modified in its genome structure and / or gene expression as a product

[0080] From the generation generated by self-fertilization in the previous step, the karyotype of the plants is analyzed, i.e., which chromosomes of which genomes are present, using any method known in the art. Such a method may be genomic in situ hybridization (GISH), or a PCR-bascd chromosome marker system, or any other method known in the art. Furthermore, we examine the occurrence of modifications in genome structure and / or gene expression. In the case of a change in genome structure and / or gene expression, the change affects at least one gene. The change can be verified by PCR / RE (PCR / restriction enzyme), T7 endonuclease assay, possibly Cas9 / guide RNA (RNP) in vitro cleavage, qPCR, RNAseq procedures, or sequencing the target sequence. This can be used to determine whether a mutant sequence different from the original genome sequence is present at the locus of the target sequence and whether it is present in heterozygous or homozygous form.

[0081] The products of the process can become the starting materials for plant breeding processes. Processes that involve editing a gene or genes and / or modifying the gene expression result in a stable product that is homozygous for the given gene or genes, which in turn can be used for plant breeding. However, hybrids containing recombination induced in rarely recombining regions, where the aim is to increase genetic variability and to create new combinations, will in most cases necessarily become products in a form that is mixed homozygous and heterozygous for the given gene and, as such, will be suitable starting materials for a plant breeding process.

[0082] Figure 2 shows the application of our method to modify genome structure. In the figure, only the at least one affected chromosome from both the wheat genome and barley genome is shown. In the parental generation P, we cross a wheat of genotype XXXXwith a transgenic barley of genotype that contains at least one transgenic chromosome (hereinafter: denotes the transgenic barley chromosome), which transgene is capable of modifying the structure and / or gene expression of the target genome on chromosome XX, wherein XXis at least one chromosome of subgenomes A, B, D.

[0083] From the transitional hybrids of the first progeny generation F1 resulting from the crossing, we select, using methods known in the art, those hybrids of genotype X'XXH+in which at least one chromosome of the wheat A, B, and D subgenomes has been modified as a result of hybridization and transgene expression on the XHbarley chromosome, and at least one X'Xgene-edited wheat chromosome has been created. The hybrid thus created contains at least one X'Xgene-edited chromosome.

[0084] Subsequently, the individuals of the F1 generation containing at least one X'Xchromosome are backcrossed with wheat of genotype XXXXto produce the first backcrossed generation BC1, which will again contain hexapioid (2n) wheat genomes and be heterozygous for the gene-edited gene.

[0085] From the members of the BC1S1 self-fertilized generation, which can be generated by self-fertilization from X'XXXand X'XXXXH+individuals of the BC1 generation, those X'XX'Xindividuals that are homozygous for at least one gene-edited gene and no longer contain a barley chromosome can be selected.

[0086] Figure 3 shows the application of our method to transiently modify gene expression. The GOI transgene of the XH+XH transgenic barley containing at least one transgenic barley chromosome used in the parental generation P allows for the temporary modification of gene expression in P wheat of genotype XXXX. From the transitional hybrids of genotype XXXH+of the first progeny generation F1 resulting from the crossing, those in which the expression of the desired gene is modified due to the hybridization and the transgcnc expressed on the XH+transgenic barley chromosome, resulting in the appearance of the N protein and / or RNA in the cell, are selected using known methods. The selection method can be implemented based on phenotypic traits due to temporary changes in gene expression.

[0087] Subsequently, the selected XXXH+transitional hybrid formed in the F1 generation is backcrossed with the wheat of genotype XXXXto produce the first backcrossed generation BC1, which will again contain hexapioid (2n) wheat genomes, within which they will either contain the XH+barley chromosome, i.e. they are XXXXXH+wheat × barley hybrids, or if the XH+barley chromosome has already segregated during meiosis, the individual will be at most a partial wheat × barley hybrid, or if it does not contain a barley chromosome, it will not be a hybrid, i.e. it will be an XXXXwheat.

[0088] From the members of the BC1S1 self-fertilized generation, which can be generated by self-fertilization from any individual of the BC1 generation, those XXXXwheat individuals that no longer contain a barley chromosome can be selected.

[0089] Figure 4 shows an application of our process to modify the genome structure and / or gene expression of a common wheat of genotype XX*XX*that is intrinsically poorly hybridizable with barley. In the figure, only the at least one affected chromosome from both the wheat genome and the barley genome is shown. Figure 4 is a sub-case of Figure 2, in which the genome structure and / or gene expression of common wheat of genotype XX*XX*, which wheat hybridizes poorly with transgenic barley containing at least one transgenic barley chromosome but is important for economic reasons, is modified by BC1 backcrossing, wherein, similarly to Figure 2, transitional hybrids of genotype XXXH+, containing at least one transgenic barley chromosome, are selected from the F1 generation generated from parental wheat of genotype XXXXand XH+XHtransgenic barley containing at least one transgenic barley chromosome, and then hybrids are generated by backcrossing the transitional hybrid of genotype XXXH+containing at least transgenic barley chromosome with common wheat of genotype XX*XX*. Of the resulting BC1 hybrids, the XXXX*XH+hybrid containing the transgenic XH+chromosome is important to us because the transgene can modify the structure and / or gene expression of the chromosomes of the common wheat, thus after the self-fertilization step, the modified X'X*X'X*common wheat is produced in homozygous form, which can be selected from the BC1S1 generation using methods known in the art.

[0090] Figure 5 shows a process in which the process of meiotic recombination between two wheat cultivars is modified. Figure 5 is a sub-case of Figure 3, wherein the gene modified in expression affects the methylation of DNA. In the hybrids containing a transgenic barley chromosome of the F1 generation, enzymes affecting methylation M already appear due to the transgene function. Affecting methylation in the BC 1 generation will be important during gamete formation, since in this phase recombination can occur between the Xx chromosome of the transitional hybrid of genotype XXXH+from the F1 generation and the XX*chromosome of the common wheat of genotype XX*XX*, if the presence of the transgenic XH+ barley chromosome ensures the production of enzymes affecting methylation M and thus the modification of the methylation pattern of the chromosomes. After self-fertilization, XX*XXXX*recombined wheat × wheat hybrids that do not contain barley chromosomes arc selected.

[0091] Description of the sequences used in the invention

[0092] SEQ ID:01

[0093] Sequence

[0094] <212> Type : DNA

[0095] <211> Length : 18380

[0096] ScqucnccNamc : pHUER_Tamlosg2

[0097] SequenceDescription :

[0098] Feature

[0099] Sequence: pHUER_Tamlosg2:

[0100] <221> FeatureKey : misc_feature

[0101] <222> LocationFrom : 21

[0102] <222> LocationTo : 45

[0103] Other Information : RB T-DNA repeat

[0104] CDSJoin : No

[0105] Feature

[0106] Sequence: pHUER_Tamlosg2:

[0107] <221> FeatureKey : gene

[0108] <222> LocationFrom : 16472

[0109] <222> LocationTo : 17101

[0110] Other Information : pVSl StaA

[0111] CDSJoin : No

[0112] Feature

[0113] Sequence: pHUER_Tamlosg2: <221> FeatureKey : gene

[0114] <222> LocationFrom : 14970

[0115] <222> LocationTo : 16043

[0116] Other Information : pVSl RepA

[0117] CDSJoin : No

[0118] Feature

[0119] Sequence: pHUER_Tamlosg2:

[0120] <221> FeatureKey : rep_origin

[0121] <222> LocationFrom : 13452

[0122] <222> LocationTo : 14040

[0123] Other Information : ori

[0124] CDSJoin : No

[0125] Feature

[0126] Sequence: pHUER_Tamlosg2:

[0127] <221> FeatureKey : gene

[0128] <222> LocationFrom : 12571

[0129] <222> LocationTo : 13365

[0130] Other Information : KanR

[0131] CDSJoin : No

[0132] Feature

[0133] Sequence: pHUER_Tamlosg2:

[0134] <221> FeatureKey : misc_feature

[0135] <222> LocationFrom : 12122

[0136] <222> LocationTo : 12146

[0137] Other Information : LB T-DNA repeat

[0138] CDSJoin : No

[0139] Feature

[0140] Sequence: pHUER_Tamlosg2:

[0141] <221> FeatureKey : terminator

[0142] <222> LocationFrom : 11870 <222> LocationTo : 12044

[0143] Other Information : CaMV poly (A) signal

[0144] CDSJoin : No

[0145] Feature

[0146] Sequence: pHUER_Tamlosg2:

[0147] <221> FeatureKey : gene

[0148] <222> LocationFrom : 10805

[0149] <222> LocationTo : 11830

[0150] Other Information : Hptll

[0151] CDSJoin : No

[0152] Feature

[0153] Sequence: pHUER_Tamlosg2:

[0154] <221> FeatureKey : promoter

[0155] <222> LocationFrom : 10060

[0156] <222> LocationTo : 10737

[0157] Other Information : CaMV 35 S promoter (enhanced)

[0158] CDSJoin : No

[0159] Feature

[0160] Sequence: pHUER_Tamlosg2:

[0161] <221> FeatureKey : gene

[0162] <222> LocationFrom : 4833

[0163] <222> LocationTo : 9098

[0164] Other Information : Cas9 with 3x FLAG

[0165] CDSJoin : No

[0166] Feature

[0167] Sequence: pHUER_Tamlosg2:

[0168] <221> FeatureKey : promoter

[0169] <222> LocationFrom : 2823

[0170] <222> LocationTo : 4814

[0171] Other Information : ZmUbi CDSJoin : No

[0172] Feature

[0173] Sequence: pHUER_Tamlosg2:

[0174] <221> FeatureKey : terminator

[0175] <222> LocationFrom : 2520

[0176] <222> LocationTo : 2810

[0177] Other Information : OsU3t

[0178] CDSJoin : No

[0179] Feature

[0180] Sequence: pHUER_Tamlosg2:

[0181] <221> FeatureKey : misc_feature

[0182] <222> LocationFrom : 2444

[0183] <222> LocationTo : 2519

[0184] Other Information : gRNA scaffold

[0185] CDSJoin : No

[0186] Feature

[0187] Sequence: pHUER_Tamlosg2:

[0188] <221> FeatureKey : promoter

[0189] <222> LocationFrom : 1987

[0190] <222> LocationTo : 2423

[0191] Other Information : OsU3p

[0192] CDSJoin : No

[0193] Feature

[0194] Sequence: pHUER_Tamlosg2:

[0195] <221> FeatureKey : promoter

[0196] <222> LocationFrom : 1015

[0197] <222> LocationTo : 1691

[0198] Other Information : CaMV 35 S promoter (enhanced)

[0199] CDSJoin : No Feature

[0200] Sequence: pHUER_Tamlosg2: <221> FeatureKey : terminator <222> LocationFrom : 68

[0201] <222> LocationTo : 242

[0202] Other Information : CaMV poly(A) signal

[0203] CDSJoin : No

[0204] Feature

[0205] Sequence: pHUER_Tamlosg2:

[0206] <221> FeatureKey : gene

[0207] <222> LocationFrom : 277

[0208] <222> LocationTo : 957

[0209] Other Information : dsRed

[0210] CDSJoin : No

[0211] Feature

[0212] Sequence: pHUER_Tamlosg2: <221> FeatureKey : misc_feature <222> LocationFrom : 2424

[0213] <222> LocationTo : 2443

[0214] Other Information : Tamlo_sg2 protospacer sequence

[0215] CDSJoin : No

[0216] SEQ ID: 02

[0217] Sequence

[0218] <213> OrganismName : Artificial Sequence

[0219] <400> PreSequenceString : ggcgcggcac aagaacgcgc tgg 23

[0220] <212> Type : DNA

[0221] <211> Length : 23

[0222] SequenceName : Tamlo_sg2_guide_F SequenceDescription :

[0223] SEQ ID:03

[0224] Sequence

[0225] <213> OrganismName : Artificial Sequence

[0226] <400> PreSequenceString : aaacccagcg cgttcttgtg ccg 23

[0227] <212> Type : DNA

[0228] <211> Length : 23

[0229] SequenceName : Tamlo_sg2_guide_R

[0230] SequenceDescription :

[0231] SEQ ID: 04

[0232] Sequence

[0233] <213> OrganismName : Artificial Sequence <400> PreSequenceString : ctccgtcctc ctggagcacg eg 22

[0234] <212> Type : DNA

[0235] <211> Length : 22

[0236] SequenceName : Tamlo_sg2_seq_F

[0237] SequenceDescription :

[0238] SEQ ID:05

[0239] Sequence

[0240] <213> OrganismName : Artificial Sequence

[0241] <400> PreSequenceString : gtgacggcga gcagcagcga g 21

[0242] <212> Type : DNA

[0243] <211> Length : 21

[0244] SequenceName : Tamlo_sg2_seq_R

[0245] SequenceDescription : SEQ ID:06

[0246] Sequence

[0247] <213> OrganismName : Artificial Sequence

[0248] <400> PreSequenceString : gcctaccacg agaagtaccc tac 23

[0249] <212> Type : DNA

[0250] <211> Length : 23

[0251] SequenceName : Cas9_det_F

[0252] SequenceDescription :

[0253] SEQ ID:07

[0254] Sequence

[0255] <213> OrganismName : Artificial Sequence

[0256] <400> PreSequenceString : ggteategte gtatgtgtcc ttg 23

[0257] <212> Type : DNA

[0258] <211> Length : 23

[0259] SequenceName : Cas9_det_R

[0260] SequenceDescription :

[0261] SEQ ID:08

[0262] Sequence

[0263] <213> OrganismName : Triticum aestivum

[0264] <400> PreSequenceString : gcggcacaag aacgcgcttg gegg 24

[0265] <212> Type : DNA

[0266] <211> Length : 24

[0267] SequenceName : mutation in Tamlo allel of A subgenome

[0268] SequenceDescription : Examples

[0269] Example 1: In the process, a barley plant was transformed using the pHUERTamlosg2 CRISPR / Cas9 vector (SEQ ID NO:01, Figure 12), in which the transfer DNA (T-DNA) contained a wheat gene (Tamlo) specific guide RNA, a constitutively driven (maize ubiquitin promoter) Cas9 gene, a constitutively driven (CaMV 35S promoter) DsRed reporter gene, and a hygromycin phosphotransferase (hptll) gene. We used a guide RNA (target sequence: ) with an efficiency of 17%, i.e. very low, according to the guide RNA designer CRISPR-Cereal (http: / / crispr.hzau.edu.cn / CRISPR- Cereal / ). The target sequence also contains a restriction enzyme (Cac8I) recognition site, which facilitates the detection of mutations using the PCR / restriction enzyme (PCR / RE) method. The construct was generated based on a previously described protocol (Xing, Hui-Li, et al., BMC Plant Biology, 14: 1-12, 2014) using Golden Gate Assembly with Tamlo_sg2_guide_F (SEQ ID NO:02) and Tamlo_sg2_guide_R (SEQ ID NO:03) oligonucleotides. The barley cultivar transgenically modified with the construct is Hordeum vulgare cv. 'Golden Promise'. The barley transformation was carried out using a previously described method (Bartlett, Joanne G., et al., Plant Methods 4: 1-12, 2008).

[0270] Wheat (Triticum aestivum L. 'M1') was crossed with T3 individuals of transgenic barley containing at least one transgenic barley chromosome (which did not show segregation for the presence of the transgene) using a method known from the literature (Polgari, David et al., Plant Cell Reports 38:767-775, 2019), where the pollen donor was the transgenic barley containing at least one transgenic barley chromosome. A total of 37 plants were regenerated in vitro by embryo rescue. To reactivate sex-specifically epigenetically inactivated genes, plants were treated with a cytosine analog (5-azacytidine, Sigma-Aldrich) that carries a nitrogen substitution at the carbon atom 5 (Vieira etal., Genome, 33.5: 707-712, 1990; Haaf, Thomas, Pharmacology & Therapeutics, 65.1: 19-46, 1995). 5-azacytidine dissolved in DMSO (at a concentration of 5 μM - 5 mM) was injected into the last internode cavity below the ear. Treated ears were injected with 1 mL of 100 mg / L 2,4-D solution 1 day after pollination to induce elongation of maternal tissues. Developing seeds were removed from the pollinated inflorescences 14 days after fertilization, and the developing hybrid embryos were rescued after surface sterilization. A multiplex PCR-based chromosome-specific marker system (Ali et al., Plant Methods, 20.1 (2024): 37) was used to determine the karyotype of the hybrids (Figure 6). In five cases, the hybrid plants did not contain any barley chromosomes, 15 plants contained the entire set of barley chromosomes, and 17 plants contained 1-6 barley chromosomes in a random distribution. A universal primer pair (SEQ ID NO:04, 05) designed for all three Tamlo homeoalleles were used to amplify the guide RNA target sequence from DNA extracted from the leaves of the 37 F1 plants, and then the resulting amplicons were subjected to Cac8I restriction enzyme digestion (Figure 7), during which amplicons containing mutations cannot be cleaved by the Cac8I restriction enzyme because the mutations generated by CRISPR / Cas9 are located in the restriction enzyme recognition site. In mutant individuals, we therefore expected to see a product of the same size as the uncleaved amplicon after cleavage. None of the individuals lacking barley chromosomes had a Tamlo gene mutation in the CRISPR / Cas9 guide RNA target sequence detectable by PCR / RE method. Only ~54% of the hybrid individuals containing the CRISPR / Cas9 transgene had targeted gene mutations as determined by PCR / RE. F1 hybrids were pollinated with wheat (Triticum aestivum L. 'M1'). In this experiment, a total of 17 BC1 plants were grown, 12% of which did not contain any barley chromosomes according to the multiplex PCR- based marker system (Figure 8), and the remaining 88% contained barley chromosomes in a random distribution. Individuals containing the CRISPR / Cas9 transgene were present in 18% of tire BC1 generation based on Cas9-specific PCR (Figure 8, H-plex) using the Cas9_det_F and Cas9_det_R oligos (SEQ ID NO:06, 07). In the PCR / RE experiment analysing the Tamlo mutation (Figure 9), no mutation was found in the Tamlo gene, the CRISPR / Cas9 target sequence, among BC1 individuals lacking barley chromosomes, only among individuals containing barley chromosomes. By self-fertilizing the mutant individuals, a mutant individual homozygous for the desired gene can be created using methods known in the art.

[0271] Example 2: In the above example, 18% of the BC1 generation still had the CRISPR / Cas9 transgene present on a barley chromosome, and 33% of them also contained a mutation. Therefore, in these plants, the CRISPR / Cas9 system, still expressed from the transgcnc carried by the barley chromosomes, induces additional mutations in the BC1 generation on the paternal wheat genome. Then, as in Example 1, among the individuals of the BC1S1 self-fertilized generation, those individuals in which the Tamlo gene mutations are present in homozygous form can be identified by the PCR / RE method.

[0272] Example 3 : During the production of the F1 plants of Example 1, an embryo expressing the DsRed reporter gene was not grown in vitro by embryo rescue, but was subjected to callus induction using a method known from the literature (Bartlett, Joanne G., et al., Plant Methods 4.1: 1-12, 2008). Mutation analysis of the 20 in vitro propagated F1 plant clones obtained by the method, using PCR / RE (Figure 10 / A) revealed that one of the plants was highly mutated, which was also confirmed by DNA sequencing data. No mutation in Tamlo in subgenomes B and D, but a T nucleotide insertion in the guide RNA target sequence (SEQ ID NO:8) in that of subgenome A was found. We also examined the presence of barley chromosomes in the plant (Figure 10 / B), where we found that barley chromosome 6 was missing. The immature inflorescence (~1-1.5 cm in length) of this plant was placed on a callus induction medium in vitro. Mutation analysis of the 24 in vitro propagated F1 plant clones thus produced, using PCR / RE (Figure 11) showed that they exhibited the same PCR / RE pattern as the plant subjected to propagation. Then, as in Example 1, these plants can be backcrossed with wild-type wheat, and in the BC 1 generation, individuals that contain Tamlo mutations in the maternal genome and that do not contain barley chromosomes and the transgene- can be selected, which can be self-fertilized to produce homozygous Tamlo mutant wheat plants, or as in example 2, in the BC1 generation, individuals containing CRISPR / Cas9 transgenic barley chromosomes can be selected, which can induce further mutations in the paternal Tamlo gene. Then, as in Example 1, among individuals of the BC1 S1 self-fertilized generation, those individuals in which the Tamlo gene mutations are present in homozygous form can be identified by PCR / RE. Example 4: Modification of the process of meiotic recombination between two wheat cultivars

[0273] During breeding a new cereal cultivar, the diversity of combinations of preferred parental traits determines the success of selecting the desired offspring. Parts of the parental genomes are exchanged during meiotic recombination that occurs early in meiosis. The process of meiotic recombination is highly regulated in time, space, and number. Meiotic recombination starts with a large number of DNA double-strand breaks, which can be physically observed on the chromosomes, in the form of so-called chiasmata, before the first cell division of meiosis. In cereals, however, only a fraction of the DNA double-strand breaks lead to recombination; the remaining breaks are eliminated by the repair mechanism. Another limiting factor is that the resulting chromosomal crossing-overs occur mainly in regions of the chromosome that are further away from the centromere, thus, new combinations are rarely formed in genomic regions close to the centromere. By modifying the processes that regulate the formation of chromosome crossing -overs, plant materials can be created in which recombination can be extended to previously inaccessible genomic regions. In the model plant Arabidopsis thaliana, non-functional mutations of the enzymes DNA mcthyltransfcrasc (MET1) and DNA chromomcthylasc (CMT3), which arc involved in silencing gene function, were used to demonstrate that recombination hotspots in the genome can be relocated. In plants mutant for the met1 gene, the number of chromosomal crossing-overs close to the centromere increased, while the number of those further away decreased. Similarly, the number of recombination events near the centromeres increased in cmt3 gene mutants (Yelina, N.E. et al., Genes & Development 29:2183-2202, 2015; Yelina, N.E., et al., PLoS Genetics 8.8:e1002844, 2012; Underwood, C.J. et al., Genome Research 28:519-531, 2018).

[0274] In wheat, the recombination process can also be modified by silencing analogous genes, allowing the development of genetic materials in which beneficial agronomic traits appear in previously unattainable combinations.

[0275] Our method can also be used to implement the mechanism described above in wheat. In the process, the barley GOI transgene is an inducible, either tissue-specific or constitutive promoter-driven gene silencing construct (e.g., artificial miRNA, inverted-repeat, Cas13 nuclease, dCas9-SunTag) and a constitutively driven reporter gene construct linked to it. The gene silencing construct is created to silence wheat met1 or cmt3 or another gene with a similar function, and then the transgenic barley containing at least one transgenic barley chromosome is pollinated onto a wheat mother plant. The F1 embryos containing the GOI transgene are raised and backcrossed with another wheat. In the resulting BC1 generation, plants containing the transgenic chromosomes are selected using the reporter gene. By the functioning of the gene silencing construct, in the meiosis that occurs before the self-fertilization of the BC1 generation, chromosome crossing-overs are induced in regions where they would occur naturally with a low probability. In the next generation (BC1S1), wheat progeny that no longer contain the reporter gene and the barley chromosomes are selected using the reporter gene and a PCR-based marker system.

[0276] Our method is therefore suitable for modifying the genome structure and / or gene expression of wheat with a hexapioid genome in a temporary or heritable manner, and then, following the modification, producing individuals homozygous for given genes of the target genome, without leaving either the transgenic or other barley chromosomes in the plant. The method is also suitable for the use of gene editing techniques in wheat that were not feasible or were difficult to implement due to the characteristics of wheat. The method can also be used to make changes in a given gene or genes, the product of which is an individual homozygous for the given gene or genes. The process can also be used to recombine two different wheats, the product of which is a heterozygous individual that contains the characteristics of the two wheats mixed within the chromosomes. The products of the process generally provide starting materials for wheat plant breeding processes.

Claims

What is claimed is1. A method for modifying the genome structure and / or gene expression of wheat by crossing a parental wheat with a transgenic barley (X XH+H) containing at least one transgenic barley chromosome (XH+), characterized by the following steps:(i) transforming the barley genome with a pre-designed gene construct that can manipulate the wheat genome and / or its expression,(ii) fertilizing wheat egg cells with the transgenic barley pollen, the wheat is then subjected to a demethylation treatment to induce partial development of the endosperm and more efficient development of the embryos, and growing the embryos on artificial media under in vitro conditions;(iii) selecting and breeding the resulting cross-generation (F1) wheat × transitional hybridscontaining at least one transgenic barley chromosome,(iv) backcrossing transitional hybrids containing at least one transgenic wheat × barley chromosomewith a wheat(v) and then producing a new generation by self-fertilization from the selected individuals (BC1) resulting from backcrossing,(vi) from which a new generation of wheat (BC1S1) is selected, which is the product of the process, free of the barley chromosomes and modified in genome structure and / or gene expression.

2. The process according to Claim 1, characterized in that the backcrossed wheat and the parental wheatare of the same cultivar.

3. The process according to any one of Claims 1 and 2, characterized in that a wheat cultivar differentfrom the parental wheat is used for backcrossing.

4. The process according to any one of Claims 1 to 3, characterized in that the crossing involves a chemical demethylation treatment.

5. The process according to any one of Claims 1 to 4, characterized in that the transgene of the transgenic barley chromosome (XH+) used is a gene-editing tool.

6. The process according to Claim 5, characterized in that said gene-editing tool is a genetic engineering tool, a derivative or combination thereof selected from the group of CRISPR / Cas, TALENs, ZFNs, RNA interference constructs.

7. The process according to any one of Claims 1-3, characterized in that the transgene of the transgenic barley chromosome (XH+) used alters the methylation pattern of the wheat.

8. The process according to Claim 7, characterized in that the methylation pattern modifying transgene is an RNA interference construct that silences the Met1 and / or Cmt3 genes.

9. The process according to any one of Claims 1 to 8, characterized in that one of the transgenic barley chromosomes (XH+) used is a reporter gene.

10. The process according to Claim 9, characterized in that one of GFP, YFP, DsRed, Ruby, or a combination thereof is used as the reporter gene.

11. The process according to any one of Claims 1 to 10, characterized in that the growing is carried out by in vitro micropropagation.

12. The process according to Claim 11, characterized in that in the in vitro micropropagation the explant is an immature embryo or immature ear.

Images