New wheat CENH3 allele

Abstract

Provided are wheat plants with mutations causing amino acid sequence changes in centromere histone H3 (CENH3), which have the biological activity of a haploid inducer. In addition, provided are methods for producing the wheat plants and obtaining haploid and doubled haploid wheat plants by crossing the wheat plants with wild-type wheat plants.

Description

Technical Field

[0001] The present disclosure relates to the field of agriculture. In particular, the present disclosure relates to CenH3 proteins and polynucleotides encoding them, methods for producing haploid and subsequently doubling haploid plants, and plants and seeds derived therefrom, particularly in wheat species.

[0002] Claiming priority

[0003] Under the Paris Convention, this application claims priority to PCT / CN 2018 / 110063, filed on October 12, 2018, the entire text of which is incorporated herein.

[0004] Sequence Listing

[0005] The present application is accompanied by a sequence listing, which is designated as 81696WOPCT_ST25.txt, created on September 30, 2019, and is approximately 112 kilobytes in size. The sequence listing is incorporated herein by reference in its entirety. The sequence listing is submitted with this application via EFS-Web and complies with 37 CFR §1.824(a)(2)-(6) and (b). Background Art

[0006] High levels of heterozygosity in breeding materials can make plant breeding and selection of beneficial traits very time-consuming. Even with the latest molecular breeding tools, extensive population screening is laborious and expensive. Creating haploid plants followed by chemical or spontaneous genome doubling has proven to be an effective way to address the problem of high heterozygosity and accelerate the breeding process. Such techniques are also known as double haploid production systems. The use of the double haploid production system allows breeders to achieve homozygosity at all loci in a single generation through whole genome duplication. This effectively eliminates the need for selfing or backcrossing, where at least 7 generations of selfing or backcrossing are usually required to reduce heterozygosity to acceptable levels.

[0007] Haploid plants can be produced according to different methods. For example, haploid plants can be produced in some crops by using a method called microspore culture. However, this method is expensive, time-consuming and not suitable for all crops. In some crop species, (double) haploid plants can be obtained by parthenogenesis of egg cells or by eliminating one of the parental genomes. However, such methods are not optimal because they are only applicable to a few selected crop species and the yield of (double) haploid plants is quite low.

[0008] WO 2011 / 044132 discloses a method for producing haploid plants, the method consisting of inactivating or altering or knocking out centromere-specific H3 (CenH3) proteins in plants. In the first step, the method comprises eliminating or knocking out the endogenous CenH3 gene in the plant. In the second step, an expression cassette encoding a mutated or altered CenH3 protein is introduced into the plant. The mutated or altered CenH3 protein is produced by fusing the H3.3 N-terminal domain, optionally with a GFP tag, to the endogenous CenH3 histone fold domain. This method is also referred to as "GFP-tail swap" or "tail swap" (also reviewed in Britt and Kuppu, Front Plant Sci. [Plant Science Frontier] 2016; 7: 357). Hybridization of plants carrying this tail swap with wild-type plants (i.e., having functional endogenous CenH3 proteins without tail swaps) results in elimination of the uniparental genome, which in turn results in the production of haploid plants. By adding GFP to the N-terminus of endogenous CenH3 (without tail swapping), some haploid induction was also found, albeit at a lower frequency. However, this approach is not ideal because it is laborious, time-consuming and requires the generation of transgenic plants.

[0009] WO 2014 / 110274 describes a method for producing haploid plants, the method comprising crossing a first plant expressing an endogenous CenH3 gene with a second plant, called a haploid induced plant, the second plant having genomes from at least two species, wherein the majority of the genome is from the first species, and the genome comprises a heterologous genomic region from the second species, wherein the heterologous genomic region encodes a CenH3 polypeptide that is different from the CenH3 of the first species (also described in Maheshwari et al., PLoS Genet. 2015 Jan 26; 11(1):e1004970). However, this method is not optimal because it encounters the same pitfalls as above - laborious, time-consuming and requires the production of transgenic plants. In addition, the method is associated with low yields of haploid plants.

[0010] Other methods include introducing one or more point mutations that result in a single amino acid change in the C-terminal histone fold domain of the CenH3 protein or the CenH3 gene encoding the CenH3 protein. Examples of such mutations in the C-terminal histone fold domain of the CenH3 protein are reported in Karimi-Ashtiyani et al. (2015) Proc Natl Acad Sci [Proceedings of the National Academy of Sciences of the United States] USA. 2015 September 8; 112(36): 11211-16; Kuppu, et al. (2015) PLoS Genet. [PLoS Genetics] 2015 September 9; 11(9): e1005494. However, the success of such methods is mitigated for some reasons, as it was found that not all of these mutations were sufficient to induce uniparental genome elimination after hybridization with wild-type plants to produce haploid plants.

[0011] Common wheat (Triticum aestivum) is a particularly complex organism to edit or mutate genes in because it is a hexaploid organism. After thousands of years of evolution and several cross-breedings with ancestral wheat species, common wheat contains three genomes: A (probably from T. monoccum or Einkorn wheat), B (probably from T. searsii), and D (probably from T. tauschii). Each genome has 7 chromosomes. Common wheat has two copies of each genome, namely AA BB DD; therefore, it has a total of 42 chromosomes (6 complete genomes, each with 7 chromosomes). See generally www.cerealsdb.uk.net / cerealgenomics / WheatBP / Documents / DOC_Ev olution.php The Evolution of Wheat [ The Evolution of Wheat , last accessed July 10, 2019. In addition, an edit or mutation in one copy of a gene might not show an observable effect in common wheat because the other five copies might compensate for the mutant copy. To truly see the effect of a knockout mutation, all six copies would have to be mutated.

[0012] Therefore, it remains elusive which mutations or modifications in the CenH3 protein or the CenH3 gene encoding the CenH3 protein are capable or sufficient to induce uniparental genome elimination to produce haploid plants. Therefore, there remains a need in the art for alternative or improved methods that allow efficient production of haploid plants (e.g., less labor intensive, less time consuming, less costly and / or not necessarily requiring the preparation of transgenic plants), which can then be doubled to produce doubled haploid plants. Using a doubled haploid production system, homozygosity can be achieved in one generation. Summary of the invention

[0013] To meet this need, one embodiment of the present invention is a wheat plant comprising at least an A genome, a B genome and a D genome, wherein the B genome comprises a knockout mutation in the CENH3 gene, and optionally, wherein the D genome comprises a knockout mutation in the CENH3 gene, and further, wherein the A genome comprises a mutated CENH3 gene, and the mutated CENH3 gene comprises at least one knockdown mutation at the 5' splice site of the intron. On the one hand, the knockdown mutation is a repaired frame shift mutation or a large deletion mutation. In another embodiment, the wheat plant is homozygous for the CENH3 gene knockout mutation in the B genome. In an alternative embodiment, the wheat plant is biallelic for the CENH3 gene knockout mutation in the B genome. In another embodiment, the wheat plant is homozygous for the CENH3 gene knockout mutation in the D genome. In an alternative embodiment, the wheat plant is biallelic for the CENH3 gene knockout mutation in the D genome. In yet another embodiment, the wheat plant is homozygous, biallelic, or a combination thereof for the CENH3 knockout mutation in the B genome and the D genome. In another embodiment, the wheat plant is homozygous for the repaired frame-shift CENH3 mutation; or it is heterozygous for the repaired frame-shift CENH3 mutation; or it is biallelic for the repaired frame-shift CENH3 mutation.

[0014] Another aspect of the present invention is a method for producing a haploid induced wheat plant, the method comprising: (a) obtaining at least a wheat plant cell comprising at least three genomes; (b) mutating two of the three genomes to obtain a homozygous knockout mutation of the CENH3 gene; (c) mutating the third genome to obtain a homozygous knockdown mutation of the CENH3 gene; and (d) producing therefrom a wheat plant comprising a homozygous knockout mutation in the CENH3 gene of two of the three genomes and further comprising a homozygous knockdown mutation in the CENH3 gene of the third genome; whereby when hybridized with a wild-type wheat plant, the wheat plant produced by step (d) produces a haploid offspring. In one embodiment, the three genomes comprise an A genome, a B genome, and a D genome. In another embodiment, the CENH3 gene knockout mutation occurs in the B and D genomes. In yet another embodiment, the CENH3 gene knockdown mutation occurs in the A genome. On the one hand, the CENH3 gene knockdown mutation in the A genome is a repaired reading frame shift mutation. On the other hand, the repaired frame shift mutation is selected from the group consisting of: SEQ ID NO:56, a nucleic acid sequence with 70% identity to SEQ ID NO:56, SEQ ID NO:63, a nucleic acid sequence with 70% identity to SEQ ID NO:63, SEQ ID NO:69, and a nucleic acid sequence with 70% identity to SEQ ID NO:69.

[0015] Another aspect of the invention is a wheat plant comprising a mutated CENH3 gene comprising at least one deletion mutation in the N-terminal domain, resulting in a frame shift, a repaired frame shift, or a large deletion. Yet another aspect is a wheat plant comprising a mutated CENH3 gene comprising at least one insertion mutation in the N-terminal domain, resulting in a frame shift, a repaired frame shift, or a large deletion.

[0016] Another aspect of the invention is a method of producing an engineered repaired reading frame shift in a gene of a cell, comprising: (a) contacting the genome with a site-directed nuclease ("SDN") and at least two guide nucleic acids, wherein the at least two guide nucleic acids target at least two target sequences within the gene; (b) allowing the SDN to cut the gene at the at least two target sequences, thereby losing the intervening sequence between the at least two target sequences; and allowing endogenous DNA repair to occur; whereby the endogenous DNA repair results in a gene having an engineered repaired reading frame shift. In one embodiment, the lost intervening sequence of step (b) comprises (N) base pairs, wherein (N) is a multiple of 3.

[0017] Yet another aspect of the present invention is a method for producing a haploid wheat plant, comprising: (a) obtaining a wheat plant; (b) hybridizing the wheat plant with the wheat plant comprising a mutated CENH3 gene; and (c) selecting an offspring produced from the hybridization step; wherein the offspring is a haploid wheat plant. In one embodiment, the wheat plant of step (a) is the male parent. In another embodiment, the wheat plant of step (a) is the female parent. In another embodiment, the method comprises the further step of converting the offspring wheat plant into a doubled haploid wheat plant.

[0018] Another aspect of the present invention is to provide a wheat plant comprising a mutant CENH3 allele, the mutant CENH3 allele comprising a nucleic acid sequence having at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs: 53-73, wherein the mutation is a repaired frame shift mutation, and wherein the wheat plant produces haploid offspring when crossed with a wild-type diploid wheat plant. In one embodiment, the wheat plant comprises at least one copy of the mutant CENH3 allele; in another embodiment, the wheat plant comprises at least two copies of the mutant CENH3 allele; in yet another embodiment, the wheat plant comprises at least three copies of the mutant CENH3 allele. In one embodiment, the mutant CENH3 allele comprises a nucleic acid sequence having 80%, 90%, 95% or 100% identity to SEQ ID NOs: 53-73.

[0019]

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The TaCenH3α gene structure and relative gRNA positions are shown. Exons are numbered and represented by thick bars. Introns are represented by thin lines. The length of both is represented by width.

[0022] definition

[0023] The present invention is not limited to the specific methods, schemes, cell lines, plant species or genera, constructs and reagents described herein. The terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, and the present invention will be limited only by the appended claims. It should be noted that, as used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms "one / kind" and "the / said" include plural references. Therefore, for example, referring to "a plant" refers to one or more plants and includes its equivalents known to those skilled in the art, etc. As used herein, the word "or" means any one member of a specific list and also includes any combination of members of the list (that is, also including "and").

[0024] The term "about" is used herein to mean approximately, roughly, about, or around. When the term "about" is used in conjunction with a numerical range, it defines the range by extending the boundaries above and below the stated values. Typically, the term "about" is used herein to define a numerical value to a variation of 20%, preferably 10% above and below (higher or lower) above and below the specified value. With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., no "about") is preferred.

[0025] As used herein, the term "amplification" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleotide molecule or multiple copies complementary to the nucleic acid molecule. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS), and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., ed., American Society for Microbiology, Washington, DC, (1993). The amplified product is referred to as an "amplicon".

[0026] The term "biallelic gene" refers to a gene pair that is neither homozygous (AA or aa) nor heterozygous (Aa). More specifically, both genes in the pair have been edited, but are not identical. For example, the CenH3 gene pair on chromosome A in the present invention can include an RFS mutation in one allele, resulting in knockout of the gene when expressed, while the other allele includes a gene knockout mutation. This can be symbolically represented as "A*a" and indicates a biallelic mutation.

[0027] The term "specific DNA sequence" refers to a polynucleotide sequence having a nucleotide sequence homology greater than 80%, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, still more preferably greater than 97%, and most preferably greater than 99% to another specified sequence.

[0028] "cDNA" refers to a single-stranded or double-stranded DNA that is complementary to and derived from mRNA. As used herein, the term "centromere-specific variant of histone H3 protein" ("CenH3 protein" or "CENH3" for short) refers to a protein that is a member of the kinetochore complex. CenH3 protein is also known as CENP-A protein. The kinetochore complex is located on the chromatids, where the spindle fibers attach during cell division, thereby pulling the sister chromatids apart. CenH3 protein belongs to a class of well-characterized proteins that are variants of H3 histones. These proteins are essential for proper kinetochore formation and function and help the kinetochore to associate with DNA. Cells lacking CenH3 are unable to localize kinetochore proteins on chromatids and show strong chromosome segregation defects (i.e., all chromosomes expressing the defective CenH3 protein from the plant are eliminated or lost, resulting in changes in the ploidy of somatic cells (e.g., reduction in the number of chromosome sets (such as diploid to haploid)). Therefore, the potential use of CenH3 proteins in double haploid production systems has been widely studied. CenH3 proteins are characterized by a variable tail domain composed of three α-helical regions connected by a hook portion (also called "N-terminal domain" or "N-terminal tail domain") and a conserved histone fold domain (also called "C-terminal domain"). The CenH3 histone fold domain is relatively conserved between CenH3 proteins of different species. The histone fold domain is located at the carboxyl terminus of endogenous CenH3 proteins. In contrast to the histone fold domain, the N-terminal tail domain of CenH3 is highly variable even between closely related species.

[0029] "A polynucleotide encoding CenH3 having one or more active mutations" refers to a polynucleotide encoding CenH3 that has been mutated non-endogenously or endogenously, encoding a CenH3 protein having one or more active mutations, which, when present in a plant without the presence of its polynucleotide encoding endogenous CenH3 and / or endogenous CenH3 protein, renders the plant viable and allows the production of haploid offspring or offspring with abnormal ploidy when the plant is crossed with a wild-type plant. Plants comprising a polynucleotide encoding CenH3 having one or more active mutations may be referred to as "modified plants". The percentage of haploid offspring or offspring with abnormal ploidy produced after crossing with a wild-type plant may be, for example, at least 0.1%, 0.5%, 1%, 5%, 10%, 20% or more. A mutation that causes a conversion from an endogenous polynucleotide encoding CenH3 to a polynucleotide encoding CenH3 having one or more active mutations is referred to herein as an active mutation. Active mutations in the context of the CenH3 protein may lead to, among other factors, reduced centromere loading during cell division, reduced function of the CenH3 protein, and / or reduced function of chromosome segregation. Introduction of one or more active mutations into a polynucleotide encoding CenH3 may be accomplished by any of several methods well known to those skilled in the art, for example, by random mutagenesis (such as induced by treating seeds or plant cells with chemicals or radiation), by directed mutagenesis, by the use of endonucleases, by generating partial or complete protein domain deletions, or by fusion with heterologous sequences.

[0030] Plants can be made lacking polynucleotides encoding endogenous CenH3 by knocking out or inactivating the polynucleotides encoding endogenous CenH3. Alternatively, the polynucleotides encoding endogenous CenH3 can be modified to encode an inactive or non-functional CenH3 protein.

[0031] A modified plant comprising a polynucleotide encoding CenH3 having one or more active mutations as taught herein can be hybridized with a wild-type plant as a pollen parent or an ovule parent. In one embodiment, the CenH3 protein having one or more active mutations may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more amino acid changes relative to the endogenous CenH3 protein. In one embodiment, the polynucleotide encoding CenH3 having one or more active mutations has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity with the polynucleotide encoding endogenous CenH3 (preferably over the full length). A skilled person will be able to easily determine whether a modified plant as taught herein comprises one or more active mutations. For example, a technician can use a prediction tool such as SIFT (Kumar P, Henikoff S, Ng PC. (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc; 4 (7): 1073-81. doi: 10.1038 / nprot.2009.86) to propose such an active mutation. The one or more active mutations can then be made in the plant, and the expression of the endogenous CenH3 protein in the plant should be knocked out. When the percentage of haploid offspring or offspring with abnormal ploidy produced after hybridization with wild-type plants is at least 0.1%, 0.5%, 1%, 5%, 10%, 20% or more, the plant can be considered to contain one or more active mutations.

[0032] A plant lacking a polynucleotide encoding an endogenous CenH3 or lacking endogenous CenH3 protein expression and expressing a CenH3 protein with one or more active mutations is hybridized with a wild-type plant (i.e., expressing an endogenous CenH3 protein) as a pollen or ovule parent, resulting in offspring being haploid or showing abnormal ploidy. Such a plant contains only the chromosomes of the parent expressing the endogenous CenH3 protein, and does not have the chromosomes of the plant expressing the CenH3 protein with one or more active mutations.

[0033] As used herein, the term "abnormal ploidy" refers to a situation where a cell contains an abnormal or anti-constant set of chromosomes of interest. For example, a cell having one or three sets of chromosomes per cell when the usual number is two is a cell with abnormal ploidy. In the present invention, active mutant CenH3 proteins and methods of use thereof can be used to produce mutant plants with abnormal ploidy, for example, to produce haploid plants, while non-mutant plants are diploid. The haploid plants can be used to accelerate breeding programs to create homozygous lines and eliminate the need for inbreeding.

[0034] As used herein, the terms "chimeric construct," "chimeric gene," "chimeric polynucleotide," or "chimeric nucleic acid" (and similar terms) refer to a construct or molecule comprising two or more polynucleotides from different sources assembled into a single nucleic acid molecule. The terms "chimeric construct," "chimeric gene," "chimeric polynucleotide," or "chimeric nucleic acid" refer to any construct or molecule that contains (1) a polynucleotide (e.g., DNA) including a regulatory polynucleotide and a coding polynucleotide that are not found together in nature (i.e., at least one polynucleotide is heterologous with respect to at least one of its other polynucleotides), or (2) a polynucleotide that encodes a portion of a protein that is not naturally contiguous, or (3) a portion of a promoter that is not naturally contiguous. In addition, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid may comprise a regulatory polynucleotide and a coding polynucleotide derived from different sources, or comprise a regulatory polynucleotide and a coding polynucleotide derived from the same source but arranged in a manner different from that found in nature. In a preferred aspect of the invention, the chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid comprises an expression cassette comprising a polynucleotide of the invention under the control of a regulatory polynucleotide, particularly a regulatory polynucleotide functional in plants.

[0035] The term "chromosome" as used herein is as recognized in the art and refers to a self-replicating genetic structure in the nucleus of a cell that contains the cell's DNA and has a linear array of genes.

[0036] "Coding polynucleotide" is a polynucleotide that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). Preferably, the RNA is then translated in an organism to produce a protein. It may consist of an "uninterrupted coding polynucleotide" as in cDNA, i.e. lacking introns, or it may include one or more introns bounded by suitable splice junctions. "Introns" are poly(ribo)nucleotides that are contained in the original transcript but are removed by cleavage and reconnection of the RNA in the cell to create a mature mRNA that can be translated into a protein.

[0037] As used herein, the term "double haploid plant" refers to the genotype formed when a haploid cell undergoes chromosome doubling. The artificial production of double haploids is very important in plant breeding. Double haploids can be produced in vivo or in vitro. Haploid embryos are produced in vivo by parthenogenesis, pseudogamy or chromosome elimination. Various in vitro methods for producing double haploid organisms from haploid organisms are known. Non-limiting examples of methods for producing double haploids in vitro include treating somatic haploid cells, haploid embryos, haploid seeds or haploid plants produced by haploid seeds with chromosome multipliers such as colchicine. In the present invention, homozygous double haploid plants can be regenerated from haploid cells by contacting haploid cells with chromosome multipliers (such as colchicine), anti-microtubule herbicides or nitrous oxide to produce homozygous double haploid cells. Methods for chromosome doubling are disclosed in, for example, U.S. Pat. Nos. 5,770,788; 7,135,615 and U.S. Patent Publication Nos. 2004 / 0210959 and 2005 / 0289673; Antoine-Michard, S. et al., Plant Cell, Tissue Organ Cult. [Plant Cell, Tissue Organ Culture], Cordrecht, The Netherlands, Kluwer Academic Publishers 48(3):203-207 (1997); Kato, A., Maize Genetics Cooperation Newsletter [Maize Genetics Cooperation Newsletter] 1997, 36-37; and Wan, Y. et al., Trends Genetics [Genetics Trend] 77:889-892 (1989). Wan, Y. et al., Trends Genetics [Genetics Trend] 81:205-21 1 (1991), the disclosures of which are incorporated herein by reference. Doubled haploid plants can be further crossed with other plants to produce F1, F2 or subsequent generations of plants with desired traits. Conventional inbreeding processes require seven generations to reach approximately complete homozygosity, whereas haploid doubling requires only one generation.

[0038] The term "E0" first refers to an edited plant. That is, a plant cell edited by, for example, CRISPR and then matured into a plant has become an E0 plant. E1 plants are the edited offspring of E0 (usually but not necessarily self-fertilized). Similarly, E2 plants are the edited offspring of E1 plants (usually but not necessarily self-fertilized). E3, E4, E5, etc. plants are likewise separated from E0 plants generation by generation.

[0039] The terms "gene editing", "editing", "genome editing", "GE", etc. refer to site-specific mutations performed at a target sequence. This may also be referred to as "directed mutagenesis". As used herein, the term "directed mutagenesis" or "mutagenesis strategy" refers to any mutagenesis method that results in the intentional mutagenesis of a selected gene. Directed mutagenesis includes methods CRISPR, TILLING, TALEN, and other methods that have not yet been discovered but can be used to achieve the same results. Mutagenesis can be performed according to any technique known in the art, such as, but not limited to, synthesizing oligonucleotides having one or more mutations within the sequence of a specific regulatory sequence. Specifically, site-specific mutagenesis is a technique that can be used to prepare promoter mutants by specific mutagenesis of the underlying DNA. RNA-guided endonucleases ("RGEN", such as CRISPR / Cas9) can also be used. For example, in combination with one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into DNA, the technology further provides the ready-made ability to prepare and test sequence variants. Site-specific mutagenesis allows the use of specific oligonucleotide sequences encoding the desired mutant DNA sequence and a sufficient number of adjacent nucleotides to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion connection fragment being studied in detail to produce a mutant. Typically, a primer of about 17 to about 75 nucleotides or more nucleotides in length is preferred, wherein about 10 to about 25 or more residues on both sides of the sequence connection fragment are changed. Generally referring to U.S. Patent number 10,285,348, which is incorporated herein by reference in its entirety.

[0040] The terms "edited N-terminal tail" or "edited N-terminal domain" are used interchangeably throughout this document.

[0041] The term "endogenous" used in combination with a protein or gene in the context of the present invention means that the protein or gene originates from the plant that still contains it. Typically, endogenous genes will be present in the plant in its normal genetic background. In another context, the term "endogenous" can refer to the normal function of the cell. For example, but not limited to, "endogenous DNA repair" refers to the normal DNA repair mechanisms, enzymes and processes of the cell.

[0042] When used with reference to polynucleotides (such as genes, ORFs or parts thereof, or transgenics of plants), the term "expression" refers to the process of converting the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by the "transcription" of a gene (i.e., via the enzymatic action of an RNA polymerase), and into a protein by the "translation" of mRNA in applicable cases (e.g., if the gene encodes a protein). Gene expression can be regulated at many stages of the process. For example, in the case of an antisense construct or a dsRNA construct, expression may refer to only the antisense RNA or only the transcription of the dsRNA, respectively. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" may also refer to the production of a protein.

[0043] The terms "homology", "sequence similarity" or "sequence identity" of nucleotide or amino acid sequences mean the degree of identity or similarity of two or more sequences and can be routinely determined by using known software or computer programs such as Best-Fit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis., 53711). BestFit uses Smith and Waterman, Advances in The local homology algorithm of Applied Mathematics [Progress in Applied Mathematics] 2:482-489 (1981) is used to find the best segment of identity or similarity between two sequences. Sequence comparisons between two or more polynucleotides or polypeptides are usually performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is usually from about 20 to 200 consecutive nucleotides. Gap uses the method of Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453 (1970) Perform a global alignment of all one sequence with all other similar sequences. When using a sequence alignment program (such as BestFit) to determine the degree of DNA sequence homology, similarity or identity, the default settings can be used, or an appropriate scoring matrix can be selected to optimize the identity, similarity or homology scores. Similarly, when using a program (such as BestFit) to determine the sequence identity, similarity or homology between two different amino acid sequences, the default settings can be used, or an appropriate scoring matrix (such as blosum45 or blosum80) can be selected to optimize the identity, similarity or homology scores.

[0044] The term "locus" refers to a position on a chromosome in a given species (eg, the position of a gene, genetic marker, etc.).

[0045] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target when placed under conditions that induce the synthesis of a primer extension product (e.g., in the presence of nucleotides and reagents such as DNA polymerase for polymerization and at a suitable temperature and pH), and the amplification target allows DNA polymerase to attach, thereby serving as a starting point for DNA synthesis. (Amplification) primers are preferably single-stranded, to obtain maximum amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers are generally long enough to initiate the synthesis of extension products in the presence of reagents for polymerization. The exact length of a primer will depend on many factors, including the temperature and composition (A / T and G / C content) of the primer. A pair of bidirectional primers is composed of a forward primer and a reverse primer commonly used in the field of DNA amplification (such as in PCR amplification). It should be understood that, as used herein, "primer" may refer to more than one primer, particularly when there is some ambiguity in the information about one or more terminal sequences of a target region to be amplified. Therefore, "primer" includes a set of primer oligonucleotides containing sequences representing possible variations in the sequence, or includes nucleotides that allow typical base pairing. Oligonucleotide primers can be prepared by any suitable method. Methods for preparing oligonucleotides of specific sequences are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, phosphodiester or triester methods, diethylaminophosphoryl ester methods, and solid phase support methods disclosed in, for example, U.S. 4,458,066. If necessary, primers can be labeled by incorporating means that can be detected by, for example, spectral methods, fluorescent methods, photochemical methods, biochemical methods, immunochemical methods, or chemical methods. The template-dependent extension of one or more oligonucleotide primers is catalyzed by a polymerization agent in the presence of an appropriate amount of four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, and dTTP; i.e., dNTP) or analogs, in a reaction medium (comprising appropriate salts, metal cations, and pH buffer systems). Suitable polymerization agents are enzymes known to catalyze primers and template-dependent DNA synthesis. Known DNA polymerases include, for example, Escherichia coli (E.coli) DNA polymerase or its Klenow fragment, T4 DNA polymerase and Taq DNA polymerase. The reaction conditions for catalyzing DNA synthesis with these DNA polymerases are known in the art. The synthesized product is a double-stranded molecule consisting of a template strand and a primer extension strand, which includes a target sequence. These products in turn serve as templates for another round of replication. In the second round of replication, the primer extension strand of the first round of circulation is annealed with its complementary primer; synthesis produces a "short" product, which is combined at both the 5'-end and the 3'-end by the primer sequence or its complement. Repeated cycles of denaturation, primer annealing and extension result in an exponential accumulation of the target region defined by the primer. Sufficient cycles are performed to achieve a desired amount of polynucleotides containing a nucleic acid target region.The required amount can vary and is determined by the function of the product polynucleotide. The PCR method is well described in the manual and is known to those skilled in the art. After amplification by PCR, the target polynucleotide can be detected by hybridization with a probe polynucleotide, which forms a stable hybrid with the polynucleotide of the target sequence under low, moderate or even highly stringent hybridization and washing conditions. If the probe is expected to be substantially fully complementary to the target sequence (i.e., about 99% or more), highly stringent conditions can be used. If it is expected that there are some mismatches, such as if the variant variety is expected to cause the probe to be incompletely complementary, the stringency of hybridization can be reduced. However, typically the conditions for excluding non-specific / accidental binding are selected. Conditions that affect hybridization and are selected for non-specific binding are known in the art and are described in, for example, Sambrook and Russell, 2001. Typically, lower salt concentrations and higher temperatures increase the stringency of hybridization conditions. "PCR primers" are preferably understood within the scope of the present invention to refer to relatively short fragments of single-stranded DNA used in the PCR amplification of a specific region of DNA.

[0046] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0047] The term "promoter" refers to a polynucleotide, usually upstream (5') of its coding polynucleotide, which controls the expression of the coding polynucleotide by providing the recognition of RNA polymerase and other factors required for proper transcription.

[0048] The term "site-directed nuclease" refers to any enzyme directed to a target sequence within a DNA chain by a nucleotide sequence. Site-directed nucleases are preferably based on CRISPR, but can also be meganucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases. Site-directed nucleases can be referred to as "SDNs". SDNs include but are not limited to meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Cas9 nucleases, Cpf1 (Cas12a) nucleases, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytosine deaminases, chimeric Cas9-adenine deaminases, chimeric FEN1-FokI, and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nucleases, and dCpf1 non-FokI nucleases; and further the guide nucleic acid is a guide RNA.

[0049] The term "stringent conditions" or "stringent hybridization conditions" includes conditions that refer to conditions where the degree of hybridization of a polynucleotide to its target sequence will be detectably higher (e.g., at least 2 times the background) than the degree of hybridization to other sequences. Stringent conditions are sequence-dependent and will be different in different situations. By controlling the stringency of the hybridization and / or washing conditions, a target polynucleotide that is 100% complementary to the probe (homologous detection) can be identified. Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequence so that a lower degree of similarity is detected (heterologous detection). Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ions (or other salts), the pH is 7.0 to 8.3, and the temperature is at least 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37°C using a buffer solution of 30% to 35% formamide, 1M NaCl, 1% SDS (w / v; sodium dodecyl sulfate), and washing in 1× to 2× SSC (20×SSC=3.0M NaCl / 0.3M trisodium citrate) at 50°C to 55°C. Exemplary moderate stringency conditions include hybridization at 37°C in 40% to 45% formamide, 1M NaCl, 1% SDS, and washing in 0.5× to 1× SSC at 55°C to 60°C. Exemplary high stringency conditions include hybridization at 37°C in 50% formamide, 1M NaCl, 1% SDS, and washing in 0.1× SSC at 60°C to 65°C. Specificity typically depends on post-hybridization washing, with the critical factors being the ionic strength of the final wash solution and the temperature. For DNA-DNA hybrids, Tm can be estimated from the equation of Meinkoth and Wahl (Anal. Biochem., 138:267-284, (1984)): Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L; where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. For every 1% mismatch, Tm decreases by about 1°C; therefore, Tm, hybridization and / or washing conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, Tm can be decreased by 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.However, extremely stringent conditions can utilize hybridization and / or washing at 1°C, 2°C, 3°C, or 4°C below the thermal melting point (Tm); moderately stringent conditions can utilize hybridization and / or washing at 6°C, 7°C, 8°C, 9°C, or 10°C below the thermal melting point (Tm); and low stringency conditions can utilize hybridization and / or washing at 11°C, 12°C, 13°C, 14°C, 15°C, or 20°C below the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and the desired Tm, one of ordinary skill in the art will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. If the desired degree of mismatching results in a Tm below 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York (1995). Methods for stringent hybridization are known in the art, and these conditions can be calculated by methods known in the art. This is disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, Cold Spring Harbor, NY, and in Current Protocols in Molecular Biology, Ausebel et al., eds., John Wiley and Sons, Inc., 2000.Methods for determining percent sequence identity are known in the art, an example of which is the GCG computer sequence analysis software (GCG, Inc, Madison Wis.).

[0050] As used herein, the term "repaired reading frame shift" ("RFS") refers to a mutation or a series of mutations in a gene that interrupts the coding sequence of a gene, either alone or in combination, but does not change the reading frame of the coding sequence. This may also be referred to as "repairing reading frame synchronization". For example, a DNA coding sequence comprises a series of codons. Each codon comprises three nucleotides, and each codon (when transcribed into RNA) encodes an amino acid during translation. An insertion / deletion mutation ("insertion gap") of one or two nucleotides in the coding sequence will result in an offset ("reading frame shift") of the coding frame. However, insertions or deletions, whether alone or in combination, occur cumulatively in multiples of three, repairing the codon to its original reading frame even if the coding sequence itself has changed. See, for example, BN Ames and HJ Whitfield, Jr., Frameshift Mutagenesis in Salmonella [Salmonella reading frame shift mutation], Cold Spring Harb. Symp. Quant. Biol. [Cold Spring Harb. Symp. Quant. Biol. 31: 221-225 (1966). For example, and within the scope of this definition, a sequence comprising at least two indel deletions (whether or not consecutive, and the indels result in the repair of the reading frame to its original reading frame) is a sequence comprising a repaired reading frame shift mutation. The term "engineered repaired reading frame shift" can also be used to describe RFS mutations that have been generated by genome editing or genome modification.

[0051] As used herein, the term "large deletion" ("LD") refers to a mutation that causes the loss of several consecutive nucleotides. In particular, a large deletion refers to the loss of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 or more nucleotides. In some embodiments, the sequence lost in the LD will be a multiple of 3 (i.e., 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, etc.). In other embodiments, the LD mutation may also occur together with an insertion mutation elsewhere in the same sequence, thereby causing a repaired frame shift mutation.

[0052] In the context of the present invention, the term "wild type" or "wild type plant" is used to refer to plants that do not carry mutant CenH3 proteins or genes (i.e., do not contain one or more active mutations taught herein) and that endogenously express or produce functional CenH3 genes and proteins. DETAILED DESCRIPTION

[0053] Here, we induced alternative splicing in wheat (common histones) by applying CRISPR-Cas9 to edit cis-splicing sequences containing the 5' and 3' splice sites. We chose wheat as a target model organism because wheat is hexaploid, thus conferring functional genomic redundancy to wheat. As a target gene, we selected the centromere protein-encoding gene CENTROMERIC HISTONE 3 ("CenH3") because modification of this gene should produce plants with crop breeding value. CenH3 is responsible for the faithful segregation of chromosomes during cell division. Unlike H3 and other conventional histones, CENH3 has a long, hypervariable N-terminal tail. See J. Monen et al., Separase Cleaves the N-Tail of the CENP-A Related Protein CPAR-1at the Meiosis I Metaphase-Anaphase Transition in C. elegans, PLoS One 10:e0125382 (2015). Direct or natural modification of the tail triggers compensatory changes in the kinetochore, which may allow CENH3 to drive speciation through meiosis or inhibit zygotic chromosome segregation. See I. Lermontova et al., Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation, Plant J 68:40-50 (2011) and M. Ravi and R. Bondada, Genome Elimination by TailswapCenH3: In Vivo Haploid Production in Arabidopsis thaliana, Methods Mol Biol 1469:77-99 (2016).Swapping the N-terminal tail for the H3 tail results in haploids in Arabidopsis (M. Ravi and S. Chan, Haploid plants produced by centromere-mediated genome elimination, Nature 464:615-618 (2010)) and maize (T. Kelliher et al., Maternal Haploids are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize, Front. Plant Sci., doi.org / 10.3389 / fpls.2016.00414). 31(7):414(2016). Haploid induction is an abnormal reproductive process that results in a decrease in ploidy from one generation to the next. Haploids can double to produce inbred lines, saving the six generations of self-pollination normally required to produce new purebred populations. Delivering tail-swapping methods to crops requires multiple generations to assemble natural allelic knockouts and stable transgene insertions. We were able to induce AS by directly editing the N-terminal sequence of wheat CenH3. These new CenH3 sequences were investigated to determine whether and in what combinations mutant CENH3 proteins could cause haploid induction in wheat. In the case we describe, it indeed could.

[0054] Therefore, one embodiment of the present invention is a wheat plant comprising at least an A genome, a B genome and a D genome, wherein the B genome comprises a knockout mutation in a CENH3 gene, and optionally, wherein the D genome comprises a knockout mutation in a CENH3 gene, and further, wherein the A genome comprises a mutated CENH3 gene, and the mutated CENH3 gene comprises at least one knockdown mutation at the 5' splice site of an intron. On the one hand, the knockdown mutation is a repaired frame shift mutation or a large deletion mutation. In another embodiment, the wheat plant is homozygous for a CENH3 gene knockout mutation in the B genome. In an alternative embodiment, the wheat plant is biallelic for a CENH3 gene knockout mutation in the B genome. In another embodiment, the wheat plant is homozygous for a CENH3 gene knockout mutation in the D genome. In an alternative embodiment, the wheat plant is biallelic for a CENH3 gene knockout mutation in the D genome. In yet another embodiment, the wheat plant is homozygous, biallelic, or a combination thereof for a CENH3 gene knockout mutation in the B genome and the D genome. In another embodiment, the wheat plant is homozygous for the repaired frame-shift CENH3 mutation; or it is heterozygous for the repaired frame-shift CENH3 mutation; or it is biallelic for the repaired frame-shift CENH3 mutation.

[0055] Another aspect of the present invention is a method for producing a haploid induced wheat plant, the method comprising: (a) obtaining at least a wheat plant cell comprising at least three genomes; (b) mutating two of the three genomes to obtain a homozygous knockout mutation of the CENH3 gene; (c) mutating the third genome to obtain a homozygous knockdown mutation of the CENH3 gene; and (d) producing therefrom a wheat plant comprising a homozygous knockout mutation in the CENH3 gene of two of the three genomes and further comprising a homozygous knockdown mutation in the CENH3 gene of the third genome; whereby when hybridized with a wild-type wheat plant, the wheat plant produced by step (d) produces a haploid offspring. In one embodiment, the three genomes comprise an A genome, a B genome, and a D genome. In another embodiment, the CENH3 gene knockout mutation occurs in the B and D genomes. In yet another embodiment, the CENH3 gene knockdown mutation occurs in the A genome. On the one hand, the CENH3 gene knockdown mutation in the A genome is a repaired reading frame shift mutation. On the other hand, the repaired frame shift mutation is selected from the group consisting of: SEQ ID NO:56, a nucleic acid sequence with 70% identity to SEQ ID NO:56, SEQ ID NO:63, a nucleic acid sequence with 70% identity to SEQ ID NO:63, SEQ ID NO:69, and a nucleic acid sequence with 70% identity to SEQ ID NO:69.

[0056] Another aspect of the invention is a wheat plant comprising a mutated CENH3 gene comprising at least one deletion mutation in the N-terminal domain, resulting in a frame shift, a repaired frame shift, or a large deletion. Yet another aspect is a wheat plant comprising a mutated CENH3 gene comprising at least one insertion mutation in the N-terminal domain, resulting in a frame shift, a repaired frame shift, or a large deletion.

[0057] Another aspect of the invention is a method of producing an engineered repaired reading frame shift in a gene of a cell, comprising: (a) contacting the genome with a site-directed nuclease ("SDN") and at least two guide nucleic acids, wherein the at least two guide nucleic acids target at least two target sequences within the gene; (b) allowing the SDN to cut the gene at the at least two target sequences, thereby losing the intervening sequence between the at least two target sequences; and allowing endogenous DNA repair to occur; whereby the endogenous DNA repair results in a gene having an engineered repaired reading frame shift. In one embodiment, the lost intervening sequence of step (b) comprises (N) base pairs, wherein (N) is a multiple of 3.

[0058] Yet another aspect of the present invention is a method for producing a haploid wheat plant, comprising: (a) obtaining a wheat plant; (b) hybridizing the wheat plant with the wheat plant comprising a mutated CENH3 gene; and (c) selecting an offspring produced from the hybridization step; wherein the offspring is a haploid wheat plant. In one embodiment, the wheat plant of step (a) is the male parent. In another embodiment, the wheat plant of step (a) is the female parent. In another embodiment, the method comprises the further step of converting the offspring wheat plant into a doubled haploid wheat plant.

[0059] Another aspect of the present invention is to provide a wheat plant comprising a mutant CENH3 allele, the mutant CENH3 allele comprising a nucleic acid sequence having at least 70% identity to a sequence selected from the group consisting of SEQ ID NOs: 56-73, wherein the mutation is a repaired frame shift mutation, and wherein the wheat plant produces haploid offspring when crossed with a wild-type diploid wheat plant. In one embodiment, the wheat plant comprises at least one copy of the mutant CENH3 allele; in another embodiment, the wheat plant comprises at least two copies of the mutant CENH3 allele; in yet another embodiment, the wheat plant comprises at least three copies of the mutant CENH3 allele. In one embodiment, the mutant CENH3 allele comprises a nucleic acid sequence having 80%, 90%, 95% or 100% identity to SEQ ID NOs: 56-73.

[0060] Examples

[0061] Example 1: Theory of using two N-terminal guide RNAs

[0062] The CENH3-tail exchange transgene, when heterologously expressed in a line in which the natural CENH3 gene is knocked out, results in haploid induction. See, e.g., U.S. Patent Application Publication No. 2019 / 0136250, which is incorporated herein by reference. This is referred to as the tail exchange method. Importantly, there is no wild-type allele in the tail exchange haploid induction line. It is presumed that the transgene is partially functional and is capable of producing sufficiently stable centromeres, which, when homozygous, are sufficient to obtain normally developed plants. However, when the tail exchange transgene in the cell is heterologous to the wild-type CENH3, the tail exchange transgene is unstable and results in successful haploid induction during outcrossing. In the design of these tail exchange plants, it is crucial to knock out the natural CENH3 gene, and the N-terminal domain of the tail exchange transgene undergoes significant changes, while the C-terminal domain undergoes only minor changes, or preferably zero. Even if the mutant CENH3 gene encodes a CENH3 protein that retains normal or near-normal function, haploid induction will not occur.

[0063] To achieve haploid induction in wheat, we directly edited the six CENH3α genes to knock out multiple copies and modify the N-terminal domain (leaving the C-terminal domain intact) in the other copies. Based on our experiments measuring CENH3α gene expression from the A, B, and D genomes, we were specifically focused on creating N-terminal modifications in the A genome and knocking out in the B and D genomes. If our editing was successful, we would leave zero copies of CENH3α normal (intact): all genes would be edited, but the outcome of the editing would be different. Importantly, our editing design did not include any CENH3 transgenes - we simply wanted to create a partially functional, N-terminally modified version of the A genome CENH3α by direct editing.

[0064] Typically, large changes can be made to protein-coding regions through CRISPR SDN II genome editing, also known as allelic replacement ("AR") or homologous recombination ("HR"), but the efficiency of this technology is very low in plants and has only been rarely achieved in wheat. Therefore, we designed a strategy for editing using two guide RNAs ("gRNAs") that could modify CENH3α to produce partial loss-of-function alleles with large changes (more than 5 amino acid changes) in the native (unchanged) sequence of the N-terminal domain and the C-terminal domain. This would require specific cleavage at both guide RNA sites. We knew that the selected guide RNAs would also edit the B and D genomic copies of CENH3α, which was intentional. In fact, due to the unpredictability of the outcome of each specific editing by the two guide RNAs, we expected that most of the edited alleles would be complete loss-of-function alleles (in the A, B, and D genomic copies) due to frame shifts in the coding sequence that resulted in truncated CENH3α protein products and thus complete knockout of premature stop codons of the native gene. However, we also knew that if we knocked out all copies of the CENH3α gene, the plants would die, since plant development requires a partial function of CENH3α. It would help us if the same guides that created the modified haploid inducing allele in the A genome could simultaneously (in the same plant) knock out the alleles in the B and D genomes: the result would be the perfectly designed haploid inducing line. Therefore, we aimed to mimic the tail-swap transgenic system using two gRNAs by direct editing, but our key creative step was not to perform any allelic replacement or CENH3α transgenesis, but to generate new modified variants by small insertions after nuclease cleavage at the two guide RNAs in the N-terminal domain.

[0065] However, not every pair of guides could potentially combine to produce an altered N-terminal domain paired with a functional C-terminal domain: not every pair of guides would produce copies with those altered “edited N-terminal tails” that were partially functional and haploid inducing potential. After examining the modifications that would result from the edits in terms of amino acid sequence, many guides resulted in premature stop codons. In other words, we realized that we had to specifically select guide RNAs that we predicted would produce combinatorial edits at the target site that would produce an amino acid sequence in the mature CENH3α protein product that contained a significantly altered N-terminal domain, but left the C-terminal domain unaffected. In particular, we planned to screen plants and identify those that contained such high-yielding, N-terminally modified alleles of the A genome copy of CENH3α, as well as plants with knockout alleles of the B and D genome copies of CENH3α. Knowing that site-directed nuclease-mediated editing does not always occur immediately during transformation, we reasoned that in the E0 generation we might generate knockout (complete loss of function) alleles for some copies of the CENH3α gene, and partial loss of function (haploid induction) alleles for other copies, and that these materials might be able to generate haploids by self-pollination—assuming that the male and female gametes (sperm and egg cells) have different editing patterns or different combinations of alleles and therefore different underlying centromeres. In other words, we thought we might find some haploids in the E1 generation plants. We hoped to let these observations guide us in selecting certain E1 plants for genotyping (i.e., genotyping diploid siblings in a population that would generate some haploids upon self-pollination) and identifying E1 plants that are homozygous for the partial loss of function allele. If we were able to do this, we would then use those specific plants to test haploid induction rates by outcrossing. In summary, we thought that once stable mutant lines were generated, we would be able to test whether they were indeed haploid inducers, but we knew that we could not do this in the first generation because the editing might not be ‘complete’ by that point in time – and we would need to test and retest haploid induction in different genotypes in the E1 and E2 generations.

[0066] We can choose from many guide RNAs with appropriate PAM sites, but only a few selection pairs can produce the haploid induced alleles we need. Regarding the desired allele, one way a pair of guide RNAs can produce such alleles is to edit the first gRNA target site to produce a frame shift, and then repair the frame shift by a compensatory mutation at the second target site, which returns the transcript to the correct (natural) reading frame. This will result in a change in the amino acid sequence of the N-terminal domain of the intervening amino acid string between the two guide RNA target sites, and will repair the "natural" sequence of the C-terminal domain. We decided to call such alleles repaired frame shift ("RFS") alleles. Alternatively, simultaneous or near-simultaneous cutting at two guide RNA target sites may result in the deletion of intervening nucleic acid sequences. In many cases, the deletion will produce a frame shift in the downstream sequence, but in some cases, this deletion may just leave the 3' sequence of the transcript in the normal reading frame, so that a large part of the N-terminal domain amino acid sequence does not exist in the resulting protein product, but the C-terminal domain remains intact. We decided to call these large deletion (“LD”) alleles. Finally, in some cases, we designed gRNAs to target splice site junctions, and editing at these target sites could generate alternative splicing patterns (e.g., could result in intron retention or exon skipping). These alternative splicing (“AS”) alleles would result in premature stop codons and gene knockout in most cases, but we also found a guide RNA that, if edited correctly and spliced ​​correctly, would not result in any premature stop codons in the reading frame. Instead, it might result in a large insertion in the mature transcript that would cause a significant change in the N-terminal domain by inserting a longer stretch of amino acids, but then a specific edit at a second target site might bring the sequence back into the reading frame of the C-terminal domain. In other words, using intelligent design of gRNAs, splice site mutations could be predicted to produce mature mRNA transcript variants that would change the amino acid code of the N-terminal domain but repair the normal reading frame and sequence of the C-terminal domain.

[0067] In the following examples, we describe in detail the specific guide RNAs and edits we recovered, as well as the combination of edits in specific plants. We will show in detail how to generate haploid induction lines in wheat by directly editing the native CENH3 gene using two guide RNAs targeting the hypervariable N-terminal domain. By design, plants with mutations at both target sites in some cases produce protein products containing significant changes to the amino acid sequence of the N-terminal domain without affecting the amino acid sequence of the C-terminal domain. We selected and maintained edited lines of alleles of CENH3 alleles with N-terminal tail changes of these types of edits, and ensured that the other copies of the CenH3 gene (from the A, B, and D genomes) were knocked out by mutations generated by those same guide RNAs. We recovered and tested haploid induction in lines with the desired mutations, including a combination of the A genome RFS allele with knockouts of the B and D genomes. These same lines, plus the correct combination of edits that we expected to produce haploids, did lead to haploid induction.

[0068] Example 2: Determination of gRNA sequence to edit the CENH3α gene of the Fielder genome.

[0069] There are two CenH3 genes in hexaploid wheat, TaCen3α and TaCenH3β. Using primers designed for the genome sequence of the "Chinese Spring_v2" variety, the A, B and D genomes of the two genes were cloned into the "Fielder" wheat variety. The sequence (SEQ ID NO:1-12) is given. Previous studies have shown that virus-induced gene silencing ("VIGS") of TaCenH3α leads to dwarfing and reduced root fecundity, while TaCenH3β silencing reduces seed setting (Yuan et al., New Phytol. [New Plant Scientist] 206 (2): 839-51.2015). Like most wheat genes, the specific expression pattern and function of each homolog of the A, B and D genomes have not been well studied. In order to perform genome editing, we chose to modify the TaCenH3α of the Fielder spring wheat variety, on the grounds that the mutation of this gene should not affect seed setting as much as the mutation of TaCenH3β. The expression levels of TaCENH3α-A, TaCENH3α-B and TaCENH3α-D (SEQ ID NO: 13-21) in reproductive tissues (pollen, ovary and anther) and young leaf tissues were queried using homolog-specific Taqman qPCR assays. TaCenH3α-A and TaCenH3α-B were highly expressed in anthers, pollen and ovaries, while TaCenH3α-D expression transcripts were almost absent (Table 2). In leaves, TaCenH3α-A was the major transcript, which may indicate that the loss of function of this gene leads to the dwarf phenotype after TaCenH3α silencing.

[0070] Table 2. Relative expression of TaCenH3α-A, TaCenH3α-B and TaCenH3α-D.

[0071] Relative expression (mean ± SD)

[0072]

[0073] Guide RNAs were selected using NGG PAM sites and based on our predictions of the types of amino acid sequences that would result in CENH3 protein products if certain edits were made at those target sites. Importantly, most of the guide RNAs we considered failed to generate edit combinations that produced either RFS or AS alleles. We focused on selecting a few gRNA pairs that could conceivably produce the same results. Guide RNA1 (ACGTCGGCGACACCG GT GCG; SEQ ID NO: 25) (underlined is the approximate site of double-strand break induced by CRISPR-Cas9 complex) is located at the exon 2-intron 2 junction. The gRNA1 is driven by the TaU6 promoter. Guide RNA2 (CTTGTGGGAGCAGGG GC AAC; SEQ ID NO:26) is targeted just after the intron 3-exon 4 junction driven by TaU6. In most cases, guide RNA2 will not edit the 3' splice acceptor site of intron 3. The choice of using two guide RNAs is to allow us to produce significant changes in the N-terminal domain, such as RFS, LD or AS alleles, while still retaining the C-terminal domain in the reading frame. For example, in some plants and edited alleles, both gRNAs will cut at the same time, resulting in the loss of intervening sequences. In some cases, the repair effect produced will produce a frame shift, thereby knocking out the protein. In other cases, it will produce a shortened LD transcript lacking intron 2, all exon 3, and part of exon 4, which approximately removes the amino acid sequence RAGRAAAPGGAQGA (SEQ ID NO:76) from the protein product, constituting a significant change in the N-terminal domain.

[0074] Alternatively, non-simultaneous cleavage of the two sites may produce a frame shift at gRNA1 (e.g., any indel that hits the coding sequence and is not a multiple of 3), which is repaired by a complementary indel at the gRNA2 site, placing the coding sequence back into its normal reading frame. For example, a deletion of 1 nucleotide ("nt") at gRNA1 and an insertion of 1 nucleotide at gRNA2 would repair the coding frame, resulting in an RFS allele. Assuming there are no stop codons in the sequence intervening the frame shift, the allele is likely not a loss-of-function allele. From our evaluation of the potential changes, we can see that at least one editing combination will produce an RFS between gRNA1 and gRNA2 without any stop codons in the intervening sequence. Therefore, we can predict which pair of small indels on gRNA1 and gRNA2 will combine to give us a functional RFS allele.

[0075] These two guide RNAs can generate alternative splicing ("AS") alleles that also have the ability to act as RFS or large insertion alleles. Guide RNA1 will cut between GT (SEQ ID NO:25; underlined above). That is the 5' splice donor at the end of exon 2. If GT is modified so that intron 2 is not properly spliced, an AS allele may be generated, resulting in intron 2 remaining in the coding sequence. After translation, the ribosome will read this intron and generate a new insertion of 44-47 amino acids, depending on the nature of the indel on gRNA1 and gRNA2. This new insertion can be predicted by reading the new coding frame after considering the indel and the translation of the new mature mRNA. For example, if gRNA1 and gRNA2 both produce insertions of a single A nucleotide at two sites of TaCENH3α-A, the transcript can be alternatively spliced, thereby inserting the amino acid sequence "VARDLPGSLPFRFVLFSVFWSDLLVTCSTECRGEPGGRRPQGGLKGQ" (SEQ ID NO: 77) before the normal sequence is repaired by the gRNA mutation, removing the WT sequence "RRAGRAAAPGGAQGA" (SEQ ID NO: 76) of exon 3. Similarly, it can be similarly predicted that different mutations of gRNA1 (e.g., deletion of GTG) combined with gRNA2 (deletion of C) will result in alternative splicing, resulting in a new "GTFPGRFLFVSSCFLFFGLTCSSPVRRNAEASRAGGGPRGGSRG" (SEQ ID NO: 78) insertion, removing the native sequence "RAGRAAAPGGAQGA" (SEQ ID NO: 76). We can also predict that other mutation combinations at gRNA1 and gRNA2 will produce frame shifts that are not put back into the reading frame, resulting in loss of function alleles. Similarly, alternative splicing alleles induced at other sites in the N-terminal region of CenH3 will not be able to generate a modified mRNA sequence with the C-terminal domain repaired to a normal amino acid sequence because a stop codon will be generated in the retained intron or after skipping one or more exons. Therefore, gRNAs can be selected based on their ability to produce large changes in the N-terminal domain while leaving the C-terminal domain intact and translated normally.

[0076] Example 3: Construct design and plant transformation.

[0077] The specific sequences of TaCENH3α-A, TaCENH3α-B, and TaCENH3α-D of Fielder variety were cloned using primers designed for Chinese Spring genome sequence, and then gRNA was selected according to the PAM site. gRNA1 (ACGTCGGCGACACCG GTGCG; SEQ ID NO: 25) is located in the exon 2-intron 2 junction ( Figure 1 ). gRNA2 (CTTGTGGGAGCAGGGGCAAC; SEQ ID NO: 26) targets after the intron 3-exon 4 junction. The SpCas9 gene was wheat codon optimized with two NLSs at both ends and driven by the sugarcane Ubi promoter with two enhancers. The gRNA cassette containing the wheat U6 promoter and gRNA scaffold was synthesized by GenScript (www.genscript.com) and cloned into a binary vector, construct 24194 (SEQ ID NO: 74).

[0078] Fielder was used for transformation, which is a spring wheat inbred line. Immature embryos with a diameter of about 2.0-2.5 mm were harvested, sterilized with 70% ethanol for 1 min, and sterilized with 1% sodium hypochlorite for 10 min. After sterilization, the immature embryos were separated into a small test tube with a scalpel and a scraper, and centrifuged for 10 min in an inoculation medium at 20,000 × g, 4°C. The separated embryos were infected with Agrobacterium for 5 min, and then transferred to a co-cultivation medium at 23°C in the dark for 2 days. The embryonic axis was cut from the immature embryo, then transferred to a dormancy medium, cultured in the dark at 25°C for 5 days, and then transferred to a selection medium containing 15g / L mannose. See Y. Ishida et al., Wheat (Triticum aestivum L.) Transformation Using Immature Embryos, Methods in Molecular Biology 1223: 189-198 (2015). After 4 weeks, the vigorously growing callus was transferred to regeneration medium to produce green plants. The surviving plants were subjected to Taqman examination, which analyzed the presence or absence of DNA fragments in the transgenic DNA insertion; only plants that were positive for 35S and PMI Taqman assays were sent to the greenhouse.

[0079] Example 4: Sequencing of E0-edited plants.

[0080] Genomic DNA was isolated from young leaves of Taqman positive E0 plants. Sequencing was performed using a high-fidelity DNA polymerase, i.e., KOD-Plus-Neo (source: TOYOBO Life Science). TaCenH3α-A allele-specific primers (FA, SEQ ID NO: 50; R3, SEQ ID NO: 51) were used. PCR was performed as follows: 95°C for 5 min; 35 cycles of 95°C for 30 sec, 65°C for 30 sec, 68°C for 1 min; 68°C for 10 min. The PCR reaction mixture contained 11.5 μl of distilled water, 2.5 μl of 10X PCR buffer for KOD-Plus-Neo, 1 μl of 2 mM dNTPs, 1 μl of 25 mM MgSO4, 1 μl of KOD-Plus-Neo DNA polymerase, 1 μl of forward primer FA (10 μM), 1 μl of reverse primer R3 (10 μM) and 1 μl of genomic DNA. PCR products were sequenced directly by SQ-1 primer (SEQ ID NO: 79) or cloned into pEASY-Blunt Zero cloning vector (Transgen Biotech, Beijing). M13R (SEQ ID NO: 52) and M13F (SEQ ID NO: 53) were used for colony sequencing.

[0081] Example 5: Wheat Event A004A

[0082] The wheat event A004A shows haploid induction. The event number A004A is one of hundreds of transgenic plants produced by transformation construct 24194. Direct sequencing after Taqman determination shows that the genotype of TaCENH3α gene is AA*BBdd at E0 seedling stage. Here, capital letters indicate unedited wild-type TaCENH3α alleles, lowercase letters indicate alleles with loss of function, and capital letters with asterisks (*) indicate repaired reading frame shift (RFS), large deletions (LD) or alternative splicing (AS) alleles, which represent putative haploid induction alleles. The A004A plant A* allele includes an adenine insertion at the target site of gRNA 1, and includes another adenine insertion at the target site of gRNA2 (SEQ ID NO:56). The adenine insertion at gRNA1 is actually located in an intron, 3bp downstream of the end of exon 2, immediately after the 5' splice junction. It does not disrupt the coding sequence itself, but may change the splicing pattern in some cases. The adenine on gRNA2 is inserted in exon 3 and shifts the reading frame of the coding sequence. Predictions for the splicing pattern induced by the insertion of adenine at the gRNA1 target site suggest that this may be an AS allele that shows intron retention (IR) of intron 2, because having an extra adenine after the 5' splice donor site can alter the initiation of intron removal, triggering alternative splicing. In this case, the alternative splicing would be an insertion of many amino acids into exon 3. If alternative splicing is present, analysis of the results suggests that the sequence in exon 3 will be out of frame until gRNA2 editing (another insertion of adenine) repairs the normal reading frame and amino acid code of the C-terminal domain.

[0083] To verify that the A004A A* allele is alternatively spliced ​​and generates a putative haploid induced allele, we examined the mature mRNA sequence of the CENH3α-A gene in the E0 leaves of A004A juvenile stage. Total RNA of young leaves was extracted using INVITROGEN TRIzol according to the manufacturer's instructions. cDNA was synthesized from 1 mg of total RNA using oligo-dT primers by Superscript III First Chain Synthesis System (Invitrogen). TaCenH3α-A transcripts were amplified using KOD-Plus-Neo (TOYOBO) with primers F1 (SEQ ID NO: 54) and R1 (SEQ ID NO: 55). PCR was performed according to the manufacturer's instructions as follows: 95°C for 5 min; 35 cycles of 95°C for 30 seconds, 62°C for 20 seconds, 68°C for 20 seconds, 35 cycles; 68°C for 10 min. The PCR reaction mixture comprises 11.5 μl of distilled water, 2.5 μl of 10X PCR buffer for KOD-Plus-Neo, 1 μl of 2mM dNTP, 1 μl of 25mM MgSO4, 1 μl of KOD-Plus-Neo, 1 μl of F1 primer (10 μM), 1 μl of R1 primer (10 μM) and 1 μl of cDNA. The PCR product was purified by GeneJET PCR purification kit (Thermo Scientific) and cloned into pEASY-Blunt Zero cloning vector (Beijing Quanshijin Biology). Primers M13R and M13F were used for colony sequencing. Several clones of each PCR product were sequenced and analyzed by Vector NTI software (Invitrogen). The relative expression of splicing variants was calculated by the number of clones. PCR sequencing analysis of colonies showed that TaCENH3α-A mRNA in A004A had two transcripts, indicating variable splicing. One of the transcripts found in 8 (44%) of the 18 clones was spliced ​​using the canonical 5' splice site (SEQ ID NO: 58). For these transcripts, normal splicing of intron 2 means that gRNA1 editing will not affect the amino acid translation of mature mRNA; however, gRNA2 editing causes a frame shift. Therefore, in this case, the constitutively spliced ​​mature mRNA is actually a knockout transcript. On the other hand, 10 (56%) of the 18 colonies have mature mRNA transcripts (SEQ ID NO: 57) that retain intron 2, resulting in the N-terminal RFS allele containing 47 new amino acids, so 32 amino acids are inserted by intron 2, together with 15 amino acids changed by frame shift in exon 3. This 32aa insertion and 47aa overall change severely alter the N-terminal domain of the protein produced by RFS mRNA translation.Importantly, the mature mRNA sequence we obtained for the AS allele A004A-TaCENH3α-A* was the exact mature mRNA sequence result we predicted would occur. The A004A E0 plants were maintained through flowering. We did not observe any abnormal phenotypes, and E1 seeds were harvested from the panicles produced from this plant (following self-pollination).

[0084] The ploidy levels of E1 progeny seeds produced by self-pollination of A004A plants were examined. The seeds were planted, the germinated seedlings were sampled, and their DNA content was analyzed by flow cytometry. Haploids were obtained in the first batch of progeny plants sown: the haploid induction rate ("HIR") was 3.8%. In the second batch of seedlings, haploids were observed again, and the HIR was 4.2%. Wheat haploids are smaller than their diploids, similar to haploids in rice and corn. Importantly, these plants either did not have both copies of the "B" allele knocked out or did not have a repaired reading frame shift caused by a mutation at the gRNA2 target site. This suggests that only the correct editing combination of the two target sites of gRNA1 and gRNA2 in the CENH3α-A gene is sufficient to trigger haploid induction when paired with the knockout of both copies of the "B" allele.

[0085] In the first batch, we observed two plants with twin seedlings; both seedlings were haploid according to flow cytometry examination. This indicates that there are two haploid embryos in one seed. Twin embryos may be caused by the disruption of ovule development, which may be partially triggered by the editing of CENH3α, although more experimental work is needed to confirm this.

[0086] Although we were somewhat surprised to observe haploidy after self-pollination (since haploidy is usually induced only in CENH3 modifications during outcrossing), the continuous ability to edit E0 plants implies that male and female sex cells may inherit different sequences (edits) and, therefore, differ in centromere binding and kinetochore architecture from each other, leading to haploidy after selfing.

[0087] Example 6: Wheat Event C003A.

[0088] Plant C003A was edited so that the TaCenH3α genotype was A*abbdd at the E0 plant stage. A* was introduced by deleting guanine in gRNA1 and inserting adenine in gRNA2. At the protein level, there were 11 amino acid differences in the N-terminal domain compared to the wild-type sequence. SEQ ID NOs: 63-65 show the A* genomic CenH3 sequence, A* CDS sequence, and A* protein sequence of C003A, respectively.

[0089] E1 seeds are produced by selfing C003A E0 plants. In greenhouse, E1 plants with A*A*bbdd genotype are grown to determine their ability to induce haploids after outcrossing. Wild-type plants (test 03S0352-22) are selected as pollen donors. Manually remove E1 C003A and pollinate with wild-type pollen. Haploids are detected by SNP markers (SEQ ID#29-43), and the differences between Fielder and 03S0352-22 can be distinguished, and then confirmed by flow cytometry. In 208 strain F1 plants, we obtained a haploid. This shows only the male genotype for four markers, but shows the female genotype (KW11091) for one marker.

[0090] Example 7: Wheat Event A073A.

[0091] At the E0 seedling stage, plant A073A had the genotype AA*B*bdd of TaCENH3α. The A073A A* allele had an adenine insertion caused by gRNA 3 and a guanine deletion caused by gRNA4 in the genomic DNA. This triggers a repaired reading frame shift at the protein level, with a 31 amino acid difference in the N-terminal domain between the wild type and the edited type. The plant was highlighted as a potential ancestor of the haploid induction line because of its ability to produce offspring A*A*bbdd (an ideal genetic combination for triggering haploid induction). SEQ ID NO:69-71 shows the A* genomic CenH3 sequence, A*CDS sequence, and A* protein sequence of A073A, respectively.

[0092] E1 seeds are produced by allowing A073A E0 plants to self-pollinate. E1 plants are sequenced, and plants with genotype combination AA*bbdd are selected to be further grown in a greenhouse to measure the haploid induction potential after outcrossing. Use the test line 03S0352-22 selected as a pollen donor, manually remove the plant edited by E1, and manually pollinate. Haploid is detected by SNP markers, which can distinguish Fielder and 03S0352-22 on genotype. The total DNA content is then checked by flow cytometry, confirming the haploid as determined by the inferred homozygosity of these markers. In 57 strains of F1 plants, 53 strains are mainly heterozygous for SNP markers, and are diploids by ploidy inspection, indicating that they are hybrids. In contrast, only four strains have paternal genome SNP markers, and are haploids by flow cytometry detection, and the outcrossing haploid induction rate reaches 7%.

[0093] E1 plants with AA*bbdd also produced E2 haploids during seedling formation (Table 3). We observed one haploid from 13 E2 plants (7.7% haploid induction rate). At the same time, we observed several plants with partial chromosome elimination.

[0094] Table 3: Ploidy levels of selfed E2 plants derived from A073A.

[0095]

[0096] Sequence Listing <110> Syngenta Participations AG Syngenta Biotechnology China Co., Ltd. Lv, Jian Yu, Kun Wei, Juan Liu, Chunxia Zhou, Hongju Kelliher, Timothy <120> New wheat CENH3 allele <130> 81696-WO-REG-ORG-P-1 <150> PCT / CN2018 / 110063 <151> 2018-10-12 <160> 79 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Common wheat <400> 1 agcgaatgaa acaacacacg 20 <210> 2 <211> 20 <212> DNA <213> Common wheat <400> 2 gaagtcggtg acctccttga 20 <210> 3 <211> 20 <212> DNA <213> regular wheat <400> 3 caaggaggtc accgacttct 20 <210> 4 <211> 20 <212> DNA <213> regular wheat <400> 4 gccttgcaag ctgtatgtcc 20 <210> 5 <211> 20 <212> DNA <213> regular wheat <400> 5 actagacgag tcgggacgaa 20 <210> 6 <211> 20 <212> DNA <213> regular wheat <400> 6 gaagtcggtg acctccttga 20 <210> 7 <211> 20 <212> DNA <213> regular wheat <400> 7 caaggaggtc accgacttct 20 <210> 8 <211> 18 <212> DNA <213> regular wheat <400> 8 acgcctcgcg agctgtat 18 <210> 9 <211> 20 <212> DNA <213> regular wheat <400> 9 gggtccaaga aagacacacg 20 <210> 10 <211> 20 <212> DNA <213> Common wheat <400> 10 gaagtcggtg acctccttga 20 <210> 11 <211> 20 <212> DNA <213> Common wheat <400> 11 caaggaggtc accgacttct 20 <210> 12 <211> 18 <212> DNA <213> Common wheat <400> 12 acgcctcgcg agctgtat 18 <210> 13 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 13 ggttcaggcc aggcacg 17 <210> 14 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 14 caaatggtgc aaacgggat 19 <210> 15 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 15 aggtatcaga agtcg 15 <210> 16 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 16 gtcgttagaa agtattgtag gtgtatcatt 30 <210> 17 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 17 ctgaatgcaa aagtcgaatg atc 23 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 18 tagatgtgtc ttcaaagtt 19 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 19 cgttcgactc gctggagtag t 21 <210> 20 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> 20 cctgccgatt gtttgtttta ctg 23 <210> twenty one <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> twenty one tagtagtaac cgcgcctccc gcac 24 <210> twenty two <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> twenty two tcgatgccgc tggtaagac 19 <210> twenty three <211> 18 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> twenty three tccgatggtt gggatggt 18 <210> twenty four <211> 29 <212> DNA <213> Artificial sequence <220> <223> Primer / probe <400> twenty four ctacaagctc aagctcggag agatcgtca 29 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> sgRNA targeting the exon 2-intron 2 junction <400> 25 acgtcggcga caccggtgcg 20 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <220> <223> sgRNA targeting the intron 3-exon 4 junction <400> 26 cttgtgggag caggggcaac 20 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <220> <223> sgRNA targeting exon 1 <400> 27 gccttggtct tcctgacggc 20 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> sgRNA targeting the intron 2-exon 3 junction <400> 28 gacggaatgc agaggcgagc 20 <210> 29 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 29 ggatcttgct ctgcctgcct gtt 23 <210> 30 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 30 gcagctcatt tcagttacaa acttag 26 <210> 31 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 31 gcagctcatt tcagttacaa acttac 26 <210> 32 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 32 tcggagttca ctattgcctc aggaa 25 <210> 33 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 33 gcccatcact tggctgatct ctt 23 <210> 34 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 34 cccatcactt ggctgatctc tc 22 <210> 35 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 35 cggaccagtt aatgtgattc gacagat 27 <210> 36 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 36 aaatcagcaa acccaaaatg gcatgat 27 <210> 37 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 37 atcagcaaac ccaaaatggc atgac 25 <210> 38 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 38 gtggttgttc tgctcggcca ct 22 <210> 39 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 39 cagactgaat caagtgcatc cct 23 <210> 40 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 40 cagactgaat caagtgcatc ccc 23 <210> 41 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 41 tgttttcctt ggcgctggcg atttt 25 <210> 42 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 42 cagagacaag aatcaggagc a 21 <210> 43 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 43 ctcagagaca agaatcagga gcg 23 <210> 44 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Primers <400> 44 gacagtggtc ccaaagatgg a 21 <210> 45 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <400> 45 cgtggttgga acgtcttctt tt 22 <210> 46 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Probe <400> 46 ccccacccac gaggagcatc g 21 <210> 47 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 47 ccctatcccc gttgacgac 19 <210> 48 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 48 ctgatagtgg tctccttgtc gct 23 <210> 49 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Probe <400> 49 ttcgccttca gcctgcacga cct 23 <210> 50 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 50 caaaacagat aaaacaaaat gtagggaatg t 31 <210> 51 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 51 ggtgcaaacg ggatgagaaa gtc 23 <210> 52 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 52 gtaaaacgac ggccagt 17 <210> 53 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 53 caggaaacag ctatgac 17 <210> 54 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 54 tggcccgcac caagc 15 <210> 55 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Primers <400> 55 gctgtatgtc cttttgcatg acg 23 <210> 56 <211> 4401 <212> DNA <213> Common wheat <400> 56 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgcc ccgcgcagcg gcggcaggag acaggtcagc ccttccctcc ttcctccccc 120 cgtctcccgc ctctcgagcg aacgccgtcg ccatttcgtc gcgagagatg gacggacgga 180 cgccccacgc atgacgctaa tgtgctcttc ctctcccttt gcagatggcg cgggcacgtc 240 ggcgacaccg gtagcgcggg accttcccgg gtcgcttcct tttcgtttcg tcttgttttc 300 tgttttttgg tctgacttgc tcgtcacctg ttcgacggaa tgcagaggcg agccgggcgg 360 gcggcggccc cagggggggc tcaaggtgcg gccttctttg cgcttttcgg ttttccgccg 420 cgtgtttagg gccatttccg tcttgtttgg gggtgcgcgg ggcgggggct tgtttttttt 480 cctcccccct tcgttgttgc gcacattgct cgggaatgct gccaggagcg gttgcggttc 540 ttctttgacc cttcgggagg gctggatcgg cagtttcttc gcttccttgc tccagatttt 600 agttcatctt gtaccagtac agtagcaaga tgatggatgg gggcttgttt tttctttctc 660 cttccttgtt ccggacattt ctctggagcg acttttatgc atttcccagt attgtccttt 720 gtccttagag ggtagtggat cggcagtttt cttcgcctca ttggtccaga ttttagttta 780 tcttgtacgg taccaagatg atggatgtgg cacaaagttc tcagtttggg ggttgcgctc 840 ttccgggcag ttgttatttt ggtctgtgat gactaactcg tatctattct tgtgggagca 900 ggggcaaact gggcaaccca agcagaggaa accacaccgg ttcaggccag gcacggtggc 960 actgcgggag atcaggaggt atcagaagtc ggtcgacttt ctcatcccgt ttgcaccatt 1020 tgtccgtctg gtgggtacct ctgtctgtca tatcctctcg ctctctctac aaacgatctg 1080 cagtgcagag tgtaattgga atattttgtt cctgacaaat ttgcagatca aggaggtcac 1140 cgacttcttc tgtcctgaaa tcagccgctg gactccccaa gcgctcgtcg cgattcaaga 1200 ggtcagtgct aaaacctggc atgtactatt agatctgatg gtttgattag agtactacaa 1260 tgcagatgaa ttcaatatcc gaaaaccatg aactgtgggg tagatacatg tatcgcctta 1320 attcatggtt tctgaatgct ctgctattaa ttcagtttga tatatttatt tagcagcatg 1380 gtattgtttt ggtggctggt aaatcaaaac tgaaatgtga ttacgagcaa aacggtatcg 1440 attgtcgatc ctgtgtgttt ttgtgcacat ctggttgttt ggtcaagatg tgtttgtgca 1500 catcttgcaa catgatcctg cccacacact caaaactgac tattggttag gttccatttg 1560 tcttatggaa tttagggtgt aactgagagg tgagcaagtg gtagtaacgt tcaattttga 1620 ttcaggatga ggatatgtg atccagaaaa ttgcatgtta tggttatgtg tccaaacgcc 1680 aaatgattat gtctatatcc agtactttag aaccagtaca acaacaaaaa gtactttaga 1740 accagtcaag tttatgtgc atttatacaa gagtgttgtt tgcacaatag acttgcttta 1800 gtcgtctctt gccagaaatg ccttcttctg cacaacgagc aaaaataaca taaagttgac 1860 tatactcagt gtggcctaag aaatgagttg tactttttag tcactggcct gtgtatttgc 1920 tttgaattga cacataattc ccttttcctt tccctctgca ggctgcagag tatcacctcg 1980 tcgacgtatt tgaaagggca aatcactgtg ccatccatgc aaagcgtgtt accgtcagta 2040 agttctcact gaatgaaaac tccctttctt ttacaatatt gcgcagaagg aaacatgcca 2100 gttatgaaag agtttcaatt acaggatcac ctttgctttc atttgatgtg atatctagtt 2160 ttgatgttgt ttcaaggttc aagaattcta atgataaatg ataggatcca caattgttat 2220 atctctgcag ctcctcgtat ctgttgtcca cgaacaaaca tatcaaacaa ttcattaaaa 2280 agatgaagaa gtcaaacaaa ftatatgt gcactgcata ttagatatca aacctggtta 2340 ctatacgact gatctggcct gacccccgcg tcgcctctcc ctggcggcac ggggggaacc 2400 actccggcgc cgccaccttc cctcctccct ccacccccca cctcgccgcc gcctgaggag 2460 ttcgccggcg aagcccggtc tggctccagg gagggtggcg gcggggcatc tctctgcgag 2520 gcgtgagggc gcatctcgtg cgcgggcgcg gcgagcttgg gcgggatcgc gggcgcggct 2580 ctggctcggg ctggtgaggc tccggcggtc cgaggggtgt cgcggcgcgg cgggggtctc 2640 gctccggcgc gggcggtccg agggcgcgcg ggatctggcg ctccagcagg ggcgccgtca 2700 aggggagccg ggcagggaag ctcgtcaggc gggctcgtca ggcaggcgtg acgcggggcg 2760 gcggccgcgg gctggaagcc atggcgtttt ggccatggtg tcgtgtgcgc tcgcttctca 2820 ttcgcagctt gaggtgctgc tgctgagggt ggtgcgcggt gaagcttggt ggtcggcgtt 2880 ggagagtgca aggcgcaaca ggaatggctc caatgatctc cacgcttctg ggtggatcag 2940 atctgcggcc ctccataggg gtgtgttccg ggcgaaagcc ttgacccgac tttgtcggtg 3000 ccgtcgacgg cggcgctttc gggcgtcgtt tccctccttg gaggcgtcgt tgtggaactc 3060 atcttcttct atgtggggct cgggctctcc gggtgaaaac ctaagctcca gattttccgg 3120 agcgggcgat ggcggcgtct tcgtcgtttc cctcttgggg gcgttgcttg ggagagttag 3180 cttgtgcttg gtgcgtttgg ttttctccta cgtcgggttt ggtggatgcc ggggcagcgg 3240 ccccggacgg ctgatgaacg ccgaggcggc ggcctcggaa agtgatgcgc ggtgcggctc 3300 catggggcgg ggcggtggct cggccttcac ttgggtggca atcttgggcc acttgggtgg 3360 caggcttgtc ggtccggtcg acgcgttcca gagggggcgg tctgactttg cgtcggggcg 3420 gcggccccgg atgtggtgcg acgttcgtgg tctgcgagcg gttgccttga gcagcgtggg 3480 ctgcgggcag ctgggtcgcg cggcgttgct gctcgagcgg agcggtggta cgtcggggcg 3540 gcggccccgg aaggtgatgc cggttgattg cgctgggcgt gacggagcgg tggatgtcgg 3600 ggcggcggcc ccgagaaaat cactgtggcg tccagatttc tgtggcaacg atgatggtgg 3660 gagcgatgtc ggcgacgcgg caatggttgc gatagtcggc tcttctccgg cgtgtccacg 3720 atattgcctc ggtttgtttg ttgctgtgga gtcgaagctg cggcggcgag gccctgtggt 3780 atacgatgac tggttccagg tgtcctttcg tcgatcttcc gtggcgccag ccgtgcctgg 3840 tttcgttctt ccgagttctc cgtcagaatc ggagctgcgt tgtctgtccg caggtcgaca 3900 tgttgtcgag aagggtgggc tttgccctgt gtgtttcagt ctatgcgagt gggctcggcc 3960 cttgttgttc tggtttttgc ccggttttcc gtaattaact gggcaattct cttctgctta 4020 attaatagat gaggcaatct ttgcctccct ttcaaaaaaa aacctggtta ctatagcagg 4080 aaattcaggg ttgattactt tatttcttat ctgaaggata aacattgtat caaatcagaa 4140 ttttatttgt aagttacatt tttttttact tataaaactt ggaaactgtt ttactgtgac 4200 aaatagatgc cactagaatc atgatcacat cgtggctgtt gctattctaa caaataaatg 4260 ctcctgaaca aatgggaact atatatgaag atgtatggac cagcatgttc ctgttaacct 4320 gacctttttc ctttttttgc tgctgcagtg caaaaggaca tacagcttgc aaggcgtatc 4380 ggcgggagga ggctttggtg a 4401 <210> 57 <211> 588 <212> DNA <213> Common wheat <400> 57 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgcc ccgcgcagcg gcggcaggag acagatggcg cgggcacgtc ggcgacaccg 120 gtagcgcggg accttcccgg gtcgcttcct tttcgtttcg tcttgttttc tgttttttgg 180 tctgacttgc tcgtcacctg ttcgacggaa tgcagaggcg agccgggcgg gcggcggccc 240 cagggggggc tcaaggggca aactgggcaa cccaagcaga ggaaaccaca ccggttcagg 300 ccaggcacgg tggcactgcg ggagatcagg aggtatcaga agtcggtcga ctttctcatc 360 ccgtttgcac catttgtccg tctgatcaag gaggtcaccg acttcttctg tcctgaaatc 420 agccgctgga ctccccaagc gctcgtcgcg attcaagagg ctgcagagta tcacctcgtc 480 gacgtatttg aaagggcaaa tcactgtgcc atccatgcaa agcgtgttac cgtcatgcaa 540 aaggacatac agcttgcaag gcgtatcggc gggaggaggc tttggtga 588 <210> 58 <211> 493 <212> DNA <213> Common wheat <400> 58 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgcc ccgcgcagcg gcggcaggag acagatggcg cgggcacgtc ggcgacaccg 120 aggcgagccg ggcgggcggc ggccccaggg ggggctcaag gggcaaactg ggcaacccaa 180 gcagaggaaa ccacaccggt tcaggccagg cacggtggca ctgcgggaga tcaggaggta 240 tcagaagtcg gtcgactttc tcatcccgtt tgcaccattt gtccgtctga tcaaggaggt 300 caccgacttc ttctgtcctg aaatcagccg ctggactccc caagcgctcg tcgcgattca 360 agaggctgca gagtatcacc tcgtcgacgt atttgaaagg gcaaatcact gtgccatcca 420 tgcaaagcgt gttaccgtca tgcaaaagga catacagctt gcaaggcgta tcggcgggag 480 gaggctttgg tga 493 <210> 59 <211> 195 <212> PRT <213> Common wheat <400> 59 Met Ala Arg Thr Lys His Pro Ala Val Arg Lys Thr Lys Ala Pro Pro 1 5 10 15 Lys Lys Gln Leu Gly Pro Arg Pro Ala Gln Arg Arg Gln Glu Thr Asp 20 25 30 Gly Ala Gly Thr Ser Ala Thr Pro Val Ala Arg Asp Leu Pro Gly Ser 35 40 45 Leu Pro Phe Arg Phe Val Leu Phe Ser Val Phe Trp Ser Asp Leu Leu 50 55 60 Val Thr Cys Ser Thr Glu Cys Arg Gly Glu Pro Gly Gly Arg Arg Pro 65 70 75 80 Gln Gly Gly Leu Lys Gly Gln Thr Gly Gln Pro Lys Gln Arg Lys Pro 85 90 95 His Arg Phe Arg Pro Gly Thr Val Ala Leu Arg Glu Ile Arg Arg Tyr 100 105 110 Gln Lys Ser Val Asp Phe Leu Ile Pro Phe Ala Pro Phe Val Arg Leu 115 120 125 Ile Lys Glu Val Thr Asp Phe Phe Cys Pro Glu Ile Ser Arg Trp Thr 130 135 140 Pro Gln Ala Leu Val Ala Ile Gln Glu Ala Ala Glu Tyr His Leu Val 145 150 155 160 Asp Val Phe Glu Arg Ala Asn His Cys Ala Ile His Ala Lys Arg Val 165 170 175 Thr Val Met Gln Lys Asp Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg 180 185 190 Arg Leu Trp 195 <210> 60 <211> 106 <212> PRT <213> Common wheat <400> 60 Met Ala Arg Thr Lys His Pro Ala Val Arg Lys Thr Lys Ala Pro Pro 1 5 10 15 Lys Lys Gln Leu Gly Pro Arg Pro Ala Gln Arg Arg Gln Glu Thr Asp 20 25 30 Gly Ala Gly Thr Ser Ala Thr Pro Arg Arg Ala Gly Arg Ala Ala Ala 35 40 45 Pro Gly Gly Ala Gln Gly Ala Asn Trp Ala Thr Gln Ala Glu Glu Thr 50 55 60 Thr Pro Val Gln Ala Arg His Gly Gly Thr Ala Gly Asp Gln Glu Val 65 70 75 80 Ser Glu Val Gly Arg Leu Ser His Pro Val Cys Thr Ile Cys Pro Ser 85 90 95 Asp Gln Gly Gly His Arg Leu Leu Leu Ser 100 105 <210> 61 <211> 2618 <212> DNA <213> Common wheat <400> 61 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgctgcccaa gaagcagctc 60 gggacgcgcc cctcggccgg gacgccgcgg cggcaggaga caggtgagcc cgcccctcct 120 tccttcctcc cccccatctc gagcgaacgc cgtcgcctcg tcgcgagaga tggacgcccc 180 acgcgtgatg ctaatgtgtt cttcctctcc ctttgcagat ggcgcgggca cgtcggcgac 240 tccggtgcgc gggccttcct gggtcacttc cttttgtttc gtcttgtttt ctgtttttgg 300 tctgacttgc tcgtctcctg ttcgacggaa tgcagaggcg agccgggcgg gcggcggccc 360 caggggcggc tgaaggtgcg cccttctttg tgcttttcgg ttttccgccg cgtgtttagg 420 gctatttccg tcttgtttgg ggcggggact tgtatcttct ccccccttcg ttgttgcgca 480 catttctcgg gaatgctacc aggagcagtt gcggttcttt ggcccttagg gagggctgca 540 ccggcaattt cttcgcttcc ttgctccaga ttttagttta tcttctacag tagcaagatg 600 atggatgggg acttgcgttt ttctttcccc ttccttgttc cgggcatttc tctggagcaa 660 tgtttatgca tttcccagta ttgtcctttg ccctcagagg ggaggggatc ggcagttttc 720 ttcgcttcat cggtccagat tttagtttat cttgtacagt agcaagatga tggatgtgac 780 acaaagttct cagtttgggg gttgcgctct tccgggtagt tgttaatttc gtctgtgact 840 gactcgtatc tattcttgtg ggagcagggg caactgggca acccaagcag aggaagccac 900 accggttcag gccaggcacg gtggcactgc gggagatcag gaagtaccag aaatcggtcg 960 actttctcat cccgtttgca ccatttgttc gtctggtggg tacctctgtc atatcctctc 1020 gctctctcta ccaacgatct ggagtgcgga gtgtaaattg gaatcttttg ttcctgacaa 1080 atttgtagat caaggaggtc accgacttct tctgtcctga aatcagccgc tggactcccc 1140 aagcgctcgt tgcaattcaa gaggtcagtg ctcaaacctg gcatgtacta ttagatctga 1200 tggtttgatt agagtactac aatgcagatg aattcaagat ctgaaaacca tgaactgtgg 1260 ggtagatgca tgtatcatag tgtctgaatg ctctggtatt aattcagttt gatatattta 1320 tttagcagca tggtattgtt ttggtggctg gtaacccaaa actgaaacgt gattacgagc 1380 aaaatggtat caattgtctt ttctgtgtgt ttgtgcaaat ctggttgttt ggtcaagata 1440 tacagtcttg caacatgaat acatgatcct gcccacacac tcaaaactga actattggtt 1500 agatgccata tgtcttatgg aatttagggt gtaactgaga ggtgagcaag tggtagtgac 1560 gttcaatttt gattcaggat gaggatattg tgatccagaa aattgcatgt tatggttatg 1620 tgtccgaacg ccaaatgatt atgtctatat ccactacatg cttatgtgtc ccaaccccca 1680 aatgattatc tctgtttcca gtacaacaac aaaaagtact ttagaaccag tcaagtttat 1740 tgtgcattta tacgagattg ttgtttgcac aatagactta ctttagtcgc ctcttgccag 1800 aaatgccttc ttctgcacaa cgagcaaaaa taacataaag ttgactatac tcagtgtggc 1860 ctaagaaatg agttagtact tttagtcact ggcctgtgta tttgctttga attgacgcat 1920 aattcccttt tcctttccct ctgcaggctg cagagtatca cctcgtcgac gtatttgaaa 1980 gggcaaatca ctgtgccatc catgcaaagc gtgttaccgt cagtaagttc tcactgaatg 2040 aaaacttcct tccatttaca atattatgca gaaggaaaca tgccagttat gaaagagttt 2100 caattacagg atcacctttg ctttcatttg atgtgatatc tagttttgat gttgtttcaa 2160 ggttcaagaa ttctaacgat taatgatagg atccacaatt gttatatctc tgcagctcct 2220 cgtatctgtt gtcaacgaac aaacatgtca aacaattcat taaaaaaaac gaagatgtca 2280 aaccgggtta ctatagcagg aaattcaggg ttgattacct atttattcag ggttgattac 2340 ctatttattt cttatctgaa ggataaacat tatataaaat cagaatttta tttgtaggtt 2400 acattttttg tttccttata aaacttggaa actgttgaga atcatcatca tcatggctgt 2460 tgctattcta acaaataaaa cggatgctcc tgaataaatg gaactatata tgaagatgta 2520 tttactagca tgttcctgtt aacctgactt tttttgctgc tacagtgcaa aaggacatac 2580 agctcgcgag gcgtatcggc gggaggaggc tttggtga 2618 <210> 62 <211> 2676 <212> DNA <213> Common wheat <400> 62 atggcccgta ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgtc ccgcgcagcg gcggcaggag acaggtcagc ccgcccgccc gtcgctcctc 120 ccttccttcc cccccccgtc tcccgcctct cgaccgaacg ccgccgcctt gtttgttgcg 180 gtacagaggt gcccgtccgt gatgctaaca ggttcttcct ctcctttgca gatggcgcgg 240 gcacgtcggc gacaccggtg cgcgggtctt cccgggtcgc ttccttttgt ttcgtcttgt 300 tttctgtttt ttggtctgac ttgctcgtca cctgttcgac ggaatgcaga ggcgagccgg 360 gcgggcggcg gccccagggg gcgctgaagg tgcggccttc tttgcgcttt tcggttttcc 420 gccgcgtgtt tagggccatt tccgtcttgt ttgggggtgg gtggggcggg ggcttgattt 480 ttttcctcct cccttcgttg ttgcgcacat ttctcgggaa tgcttccagg agcggttgca 540 gttcttcttt ggccttaggg agggctggat cggcagtttc ttcgcttcct tgctccagat 600 tttagtttat cttgtagtag tacagtagca agatgatgga tggggacttg ttttttcttt 660 ccccttcctt gttccggaca tttctttgga gcaactttta tgcatttccc ggtattgtcc 720 tttgccctta gagtggagtg gatcggcagt tttcttcgct tccctggtcc agattttagt 780 ttatctttac agtagcaaga tgatgtatgt gacacaaagt tctcagtttg ggggttgcgc 840 tcttccaggt gtagttgtta tttttcgtct gtgactgact cgtatctatt cttgtgggag 900 caggggcaaa ctgggcaacc caagcagagg aagccacacc ggttcaggcc aggcacggtg 960 gcactgcggg agatcaggag gtaccagaag tcggtcgact ttctcatccc gtttgcacca 1020 tttgtccgtc tggtgggtac ctctgtctgt catatcctct cgctctctct accaacaatc 1080 tggagtgcgg ggtgtaaact ggaatctttt gttcctgaca aatttgcaga tcaaggaggt 1140 caccgacttc ttctgtcctg aaatcagccg ctggactccc caagcgctcg tcgcgattca 1200 agaggtcagt gctcaaacct ggcatgtact attagatctg atggtttgat tagagtacta 1260 caatgcagat gaattcaaga tctgaaaacc atgaactgtg gggtagatgc atgtatcgcc 1320 ttaattcata gtttctgaat gctctggtat taattcagtt tgatatattt atttagctgc 1380 atggtattgt tttggtggct ggtaattcaa aactgaaatg tgattacgag caaaatggta 1440 tcaattgtct tttctgtgca catctggttg tttggtcaag atatacagtc ttgcaacatg 1500 atcctgccca cacactcaaa actgaactat tggttagatt ccattttgct tatggaattt 1560 agggtgtaac tgagaggtga gcaggtggca gtgaccagag ctacgttcaa ttttgattca 1620 ggaagaggat cttgcgatcc agaaaattgc atgttatggt tatgtgtccg aaggccaaat 1680 gattatctct atatccagta catgcttatg tgtccgaacc cccaaatgat tatctctata 1740 tccagtacat gcttatgtgt ccgaaccatt caagttattg tgcatttatt caagattgtt 1800 gttgcacaa tagacttact ttagtcgcct cttgccagaa atgcctcctt ctgcacaatg 1860 agcaaaaata acataaagtt gattatgctc agtgtggcct aattgtactt tcagttactg 1920 gcctgtgtat ttgctttgaa ttgacacata attcccttt cctttccctc tgcaggctgc 1980 agagtatcac ctcgtcgacg tatttgaaag ggcaaatcac tgtgccatcc atgcaaagcg 2040 tgttaccgtc agtaagttct cactgaatga aaacttcctt tcttttacaa tattatgcag 2100 aaggaaacat gccagttatg aaagagtttc aattacaaga tcacctttgc tttcatttga 2160 tgtggtatct agttttgatg ttgtttcaag gttcaagaat tctaatgatt aatgatagga 2220 tccacaattg ttatatctct gcagctcctc gtatctgacg aacaaacatg tcaaacaatt 2280 cattaaaaaa tgaagatgtc aaacaaacag tatatgcact gcatattgga tatcaaacct 2340 ggttactaca gcaggaagtt cagggttgat tactttattt cttatctgaa ggataaacat 2400 tgtatcaaat cagaatttta tttgtaagtt gcattttttg tttactcata aaacttggaa 2460 actgttttac tgtgagaaat agatgcccac tagaatcatg atcatcatca tggctgttgc 2520 tattctaaca aataaaacgg atgctcctga acaaatggaa ctatgtatga agatgtatgg 2580 actagcatgt tcctgttaac ctgacttttt tttgctgcta cagtgcaaaa ggacatacag 2640 ctcgcgaggc gtatcggtgg gaggaggctt tggtga 2676 <210> 63 <211> 4399 <212> DNA <213> Common wheat <400> 63 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgcc ccgcgcagcg gcggcaggag acaggtcagc ccttccctcc ttcctccccc 120 cgtctcccgc ctctcgagcg aacgccgtcg ccatttcgtc gcgagagatg gacggacgga 180 cgccccacgc atgacgctaa tgtgctcttc ctctcccttt gcagatggcg cgggcacgtc 240 ggcgacaccg tgcgcgggac cttcccgggt cgcttccttt tcgtttcgtc ttgttttctg 300 ttttttggtc tgacttgctc gtcacctgtt cgacggaatg cagaggcgag ccgggcgggc 360 ggcggcccca gggggggctc aaggtgcggc cttctttgcg cttttcggtt ttccgccgcg 420 tgtttagggc catttccgtc ttgtttgggg gtgcgcgggg cgggggcttg ttttttttcc 480 tccccccttc gttgttgcgc acattgctcg ggaatgctgc caggagcggt tgcggttctt 540 ctttgaccct tcgggagggc tggatcggca gtttcttcgc ttccttgctc cagattttag 600 ttcatcttgt accagtacag tagcaagatg atggatgggg gcttgttttt tctttctcct 660 tccttgttcc ggacatttct ctggagcgac ttttatgcat ttcccagtat tgtcctttgt 720 ccttagaggg tagtggatcg gcagttttct tcgcctcatt ggtccagatt ttagtttatc 780 ttgtacggta ccaagatgat ggatgtggca caaagttctc agtttggggg ttgcgctctt 840 ccgggcagtt gttattttgg tctgtgatga ctaactcgta tctattcttg tgggagcagg 900 ggcaaactgg gcaacccaag cagaggaaac cacaccggtt caggccaggc acggtggcac 960 tgcgggagat caggaggtat cagaagtcgg tcgactttct catcccgttt gcaccatttg 1020 tccgtctggt gggtacctct gtctgtcata tcctctcgct ctctctacaa acgatctgca 1080 gtgcagagtg taattggaat attttgttcc tgacaaattt gcagatcaag gaggtcaccg 1140 acttcttctg tcctgaaatc agccgctgga ctccccaagc gctcgtcgcg attcaagagg tcagtgctaa aacctggcat gtactattag atctgatggt ttgattagag tactacaatg 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320 tcatggtttc tgaatgctct gctattaatt cagtttgata tatttattta gcagcatggt attgttttgg tggctggtaa atcaaaactg aaatgtgatt acgagcaaaa cggtatcgat tgtcgatcct gtgtgttttt gtgcacatct ggttgtttgg tcaagatgtg tttgtgcaca 1560. tcttgcaaca tgatcctgcc cacacactca aaactgacta ttggttaggt tccatttgtc ttatggaatt tagggtgtaa ctgagaggtg agcaagtggt agtaacgttc aattttgatt caggatgagg atattgtgat ccagaaaatt gcatgttatg gttatgtgtc caaacgccaa atgattatgt ctatatccag tactttagaa ccagtacaac aacaaaaagt actttagaac cagtcaagtt tattgtgcat ttatacaaga gtgttgtttg cacaatagac ttgctttagt cgtctcttgc cagaaatgcc ttcttctgca caacgagca aaataacata aagttgacta tactcagtgt ggcctaagaa atgagttgta ctttttagtc actggcctgt gtatttgctt 1920 tgaattgaca cataattccc ttttcctttc cctctgcagg ctgcagagta tcacctcgtc 1980 gacgtatttg aaagggcaaa tcactgtgcc atccatgcaa agcgtgttac cgtcagtaag 2040 ttctcactga atgaaaactc cctttctttt acaatattgc gcagaaggaa acatgccagt 2100 tatgaaagag tttcaattac aggatcacct ttgctttcat ttgatgtgat atctagtttt 2160 gatgttgttt caaggttcaa gaattctaat gataaatgat aggatccaca attgttatat 2220 ctctgcagct cctcgtatct gttgtccacg aacaaacata tcaaacaatt cattaaaaag 2280 atgaagaagt caaacaaaca gtatatgtgc actgcatatt agatatcaaa cctggttact 2340 atacgactga tctggcctga cccccgcgtc gcctctccct ggcggcacgg ggggaaccac 2400 tccggcgccg ccaccttccc tcctccctcc accccccacc tcgccgccgc ctgaggagtt 2460 cgccggcgaa gcccggtctg gctccaggga gggtggcggc ggggcatctc tctgcgaggc 2520 gtgagggcgc atctcgtgcg cgggcgcggc gagcttgggc gggatcgcgg gcgcggctct 2580 ggctcgggct ggtgaggctc cggcggtccg aggggtgtcg cggcgcggcg ggggtctcgc 2640 tccggcgcgg gcggtccgag ggcgcgcggg atctggcgct ccagcagggg cgccgtcaag 2700 gggagccggg cagggaagct cgtcaggcgg gctcgtcagg caggcgtgac gcggggcggc 2760 ggccgcgggc tggaagccat ggcgttttgg ccatggtgtc gtgtgcgctc gcttctcatt 2820 cgcagcttga ggtgctgctg ctgagggtgg tgcgcggtga agcttggtgg tcggcgttgg 2880 agagtgcaag gcgcaacagg aatggctcca atgatctcca cgcttctggg tggatcagat 2940 ctgcggccct ccataggggt gtgttccggg cgaaagcctt gacccgactt tgtcggtgcc 3000 gtcgacggcg gcgctttcgg gcgtcgtttc cctccttgga ggcgtcgttg tggaactcat 3060 cttcttctat gtggggctcg ggctctccgg gtgaaaacct aagctccaga ttttccggag 3120 cgggcgatgg cggcgtcttc gtcgtttccc tcttgggggc gttgcttggg agagttagct 3180 tgtgcttggt gcgtttggtt ttctcctacg tcgggtttgg tggatgccgg ggcagcggcc 3240 ccggacggct gatgaacgcc gaggcggcgg cctcggaaag tgatgcgcgg tgcggctcca 3300 tggggcgggg cggtggctcg gccttcactt gggtggcaat cttgggccac ttgggtggca 3360 ggcttgcgg tccggtcgac gcgttccaga gggggcggtc tgactttgcg tcggggcggc 3420 ggccccggat gtggtgcgac gttcgtggtc tgcgagcggt tgccttgagc agcgtgggct 3480 gcgggcagct gggtcgcg gcgttgctgc tcgagcggag cggtggtacg tcggggcggc 3540 ggccccggaa ggtgatgccg gttgattgcg ctgggcgtga cggagcggtg gatgtcgggg 3600 cggcggcccc gagaaaatca ctgtggcgtc cagatttctg tggcaacgat gatggtggga 3660 gcgatgtcgg cgacgcggca atggttgcga tagcggctc ttctccggcg tgtccacgat 3720 attgcctcgg tttgtttgtt gctgtggagt cgaagctgcg gcggcgaggc cctgtggtat 3780 acgatgactg gttccaggtg tccttcgtc gatcttccgt ggcgccagcc gtgcctggtt 3840 tcgttcttcc gagttctccg tcagaatcgg agctgcgttg tctgtccgca ggtcgacatg 3900 ttgtcgagaa gggtgggctt tgccctgtgt gtttcagtct atgcgagtgg gctcggccct 3960 tgttgttctg gttttgccc ggttttccgt attaactgg gcaattctct tctgcttaat 4020 taatagatga ggcaatcttt gcctcccttt caaaaaaaaa cctggttact atagcaggaa 4080 attcagggtt gattacttta tttcttatct gaaggataaa cattgtatca aatcagaatt 4140 ttatttgtaa gttacatttt tttttactta taaaacttgg aaactgtttt actgtgacaa 4200 atagatgcca ctagaatcat gatcacatcg tggctgttgc tattctaaca aataaatgct 4260 cctgaacaaa tgggaactat atatgaagat gtatggacca gcatgttcct gttaacctga 4320 cctttttcct ttttttgctg ctgcagtgca aaaggacata cagcttgcaa ggcgtatcgg 4380 cgggaggagg ctttggtga 4399 <210> 64 <211> 492 <212> DNA <213> Common wheat <400> 64 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgcc ccgcgcagcg gcggcaggag acagatggcg cgggcacgtc ggcgacacca 120 ggcgagccgg gcgggcggcg gccccagggg gggctcaagg ggcaaactgg gcaacccaag 180 cagaggaaac cacaccggtt caggccaggc acggtggcac tgcgggagat caggaggtat 240 cagaagtcgg tcgactttct catcccgttt gcaccatttg tccgtctgat caaggaggtc 300 accgacttct tctgtcctga aatcagccgc tggactcccc aagcgctcgt cgcgattcaa 360 gaggctgcag agtatcacct cgtcgacgta tttgaaaggg caaatcactg tgccatccat 420 gcaaagcgtg ttaccgtcat gcaaaaggac atacagcttg caaggcgtat cggcgggagg 480 aggctttggt ga 492 <210> 65 <211> 163 <212> PRT <213> Common wheat <400> 65 Met Ala Arg Thr Lys His Pro Ala Val Arg Lys Thr Lys Ala Pro Pro 1 5 10 15 Lys Lys Gln Leu Gly Pro Arg Pro Ala Gln Arg Arg Gln Glu Thr Asp 20 25 30 Gly Ala Gly Thr Ser Ala Thr Pro Gly Glu Pro Gly Gly Arg Arg Pro 35 40 45 Gln Gly Gly Leu Lys Gly Gln Thr Gly Gln Pro Lys Gln Arg Lys Pro 50 55 60 His Arg Phe Arg Pro Gly Thr Val Ala Leu Arg Glu Ile Arg Arg Tyr 65 70 75 80 Gln Lys Ser Val Asp Phe Leu Ile Pro Phe Ala Pro Phe Val Arg Leu 85 90 95 Ile Lys Glu Val Thr Asp Phe Phe Cys Pro Glu Ile Ser Arg Trp Thr 100 105 110 Pro Gln Ala Leu Val Ala Ile Gln Glu Ala Ala Glu Tyr His Leu Val 115 120 125 Asp Val Phe Glu Arg Ala Asn His Cys Ala Ile His Ala Lys Arg Val 130 135 140 Thr Val Met Gln Lys Asp Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg 145 150 155 160 Arg Leu Trp <210> 66 <211> 2619 <212> DNA <213> Common wheat <400> 66 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgctgcccaa gaagcagctc 60 gggacgcgcc cctcggccgg gacgccgcgg cggcaggaga caggtgagcc cgcccctcct 120 tccttcctcc cccccatctc gagcgaacgc cgtcgcctcg tcgcgagaga tggacgcccc 180 acgcgtgatg ctaatgtgtt cttcctctcc ctttgcagat ggcgcgggca cgtcggcgac 240 tccggtgcgc gggccttcct gggtcacttc cttttgtttc gtcttgtttt ctgtttttgg 300 tctgacttgc tcgtctcctg ttcgacggaa tgcagaggcg agccgggcgg gcggcggccc 360 caggggcggc tgaaggtgcg cccttctttg tgcttttcgg ttttccgccg cgtgtttagg 420 gctatttccg tcttgtttgg ggcggggact tgtatcttct ccccccttcg ttgttgcgca 480 catttctcgg gaatgctacc aggagcagtt gcggttcttt ggcccttagg gagggctgca 540 ccggcaattt cttcgcttcc ttgctccaga ttttagttta tcttctacag tagcaagatg 600 atggatgggg acttgcgttt ttctttcccc ttccttgttc cgggcatttc tctggagcaa 660 tgtttatgca tttcccagta ttgtcctttg ccctcagagg ggaggggatc ggcagttttc 720 ttcgcttcat cggtccagat tttagtttat cttgtacagt agcaagatga tggatgtgac 780 acaaagttct cagtttgggg gttgcgctct tccgggtagt tgttaatttc gtctgtgact 840 gactcgtatc tattcttgtg ggagcagggg caaactgggc aacccaagca gaggaagcca 900 caccggttca ggccaggcac ggtggcactg cgggagatca ggaagtacca gaaatcggtc 960 gactttctca tcccgtttgc accatttgtt cgtctggtgg gtacctctgt catatcctct 1020 cgctctctct accaacgatc tggagtgcgg agtgtaaatt ggaatctttt gttcctgaca 1080 aatttgtaga tcaaggaggt caccgacttc ttctgtcctg aaatcagccg ctggactccc 1140 caagcgctcg ttgcaattca agaggtcagt gctcaaacct ggcatgtact attagatctg 1200 atggtttgat tagagtacta caatgcagat gaattcaaga tctgaaaacc atgaactgtg 1260 gggtagatgc atgtatcata gtgtctgaat gctctggtat taattcagtt tgatatattt 1320 atttagcagc atggtattgt tttggtggct ggtaacccaa aactgaaacg tgattacgag 1380 caaaatggta tcaattgtct tttctgtgtg tttgtgcaaa tctggttgtt tggtcaagat 1440 atacagtctt gcaacatgaa tacatgatcc tgcccacaca ctcaaaactg aactattggt 1500 tagatgccat atgtcttatg gaatttaggg tgtaactgag aggtgagcaa gtggtagtga 1560 cgttcaattt tgattcagga tgaggatatt gtgatccaga aaattgcatg ttatggttat 1620 gtgtccgaac gccaaatgat tatgtctata tccactacat gcttatgtgt cccaaccccc 1680 aaatgattat ctctgtttcc agtacaacaa caaaagtac tttagaacca gtcaagttta ttgtgcattt atacgagatt gttgtttgca caatagactt actttagtcg cctcttgcca gaaatgcctt cttctgcaca acgagcaaaa attack gttgactata ctcagtgtgg cctaagaaat gagttagtac ttttagtcac tggcctgtgt atttgctttg aattgacgca taattccctt ttccttttccc tctgcaggct gcagagtatc acctcgtcga cgtatttgaa agggcaaatc actgtgccat ccatgcaaag cgtgttaccg tcagtaagtt ctcactgaat gaaaacttcc ttccatttac aatattatgc agaaggaac atgccagtta tgaaagagtt tcaattacag gatcaccttt gctttcattt gatgtgatat ctagttttga tgttgtttca aggttcaaga attctaacga ttaatgatag gatccacaat tgttatatct ctgcagctcc tcgtatctgt tgtcaacgaa caaacatgtc aaacaattca ttaaaaaaaa cgaagatgtc aaaccgggtt actatagcag gaaattcagg gttgattacc tatttattca gggttgatta cctatttatt tcttatctga aggataaaca ttattaaaa tcagaatttt atttgtaggt tacatttttt gtttccttat aaaacttgga aactgttgag aatcatcatc atcatggctg 2460 ttgctattct aacaaataaa acggatgctc ctgaataaat ggaactatat atgaagatgt 2520 atttactagc atgttcctgt taacctgact ttttttgctg ctacagtgca aaaggacata 2580 cagctcgcga ggcgtatcgg cgggaggagg ctttggtga 2619 <210> 67 <211> 502 <212> DNA <213> Common wheat <400> 67 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgctgcccaa gaagcagctc 60 gggacgcgcc cctcggccgg gacgccgcgg cggcaggaga cagatggcgc gggcacgtcg 120 gcgactccga ggcgagccgg gcgggcggcg gccccagggg cggctgaagg ggcaaactgg 180 gcaacccaag cagaggaagc cacaccggtt caggccaggc acggtggcac tgcgggagat 240 caggaagtac cagaaatcgg tcgactttct catcccgttt gcaccatttg ttcgtctgat 300 caaggaggtc accgacttct tctgtcctga aatcagccgc tggactcccc aagcgctcgt 360 tgcaattcaa gaggctgcag agtatcacct cgtcgacgta tttgaaaggg caaatcactg 420 tgccatccat gcaaagcgtg ttaccgtcat gcaaaaggac atacagctcg cgaggcgtat 480 cggcgggagg aggctttggt ga 502 <210> 68 <211> 2676 <212> DNA <213> Common wheat <400> 68 atggcccgta ccaagcaccc ggccgtcagg aagaccaagg cgccgcccaa gaagcagctc 60 gggccccgtc ccgcgcagcg gcggcaggag acaggtcagc ccgcccgccc gtcgctcctc 120 ccttccttcc cccccccgtc tcccgcctct cgaccgaacg ccgccgcctt gtttgttgcg 180 gtacagaggt gcccgtccgt gatgctaaca ggttcttcct ctcctttgca gatggcgcgg 240 gcacgtcggc gacaccggtg cgcgggtctt cccgggtcgc ttccttttgt ttcgtcttgt 300 tttctgtttt ttggtctgac ttgctcgtca cctgttcgac ggaatgcaga ggcgagccgg 360 gcgggcggcg gccccagggg gcgctgaagg tgcggccttc tttgcgcttt tcggttttcc 420 gccgcgtgtt tagggccatt tccgtcttgt ttgggggtgg gtggggcggg ggcttgattt 480 ttttcctcct cccttcgttg ttgcgcacat ttctcgggaa tgcttccagg agcggttgca 540 gttcttcttt ggccttaggg agggctggat cggcagtttc ttcgcttcct tgctccagat 600 tttagtttat cttgtagtag tacagtagca agatgatgga tggggacttg ttttttcttt 660 ccccttcctt gttccggaca tttctttgga gcaactttta tgcatttccc ggtattgtcc 720 tttgccctta gagtggagtg gatcggcagt tttcttcgct tccctggtcc agattttagt 780 ttatctttac agtagcaaga tgatgtatgt gacacaaagt tctcagtttg ggggttgcgc 840 tcttccaggt gtagttgtta ttttcgtct gtgactgact cgtatctatt cttgtgggag 900 caggggcaaa ctgggcaacc caagcagagg aagccacacc ggttcaggcc aggcacggtg 960 gcactgcggg agatcaggag gtaccagaag tcggtcgact ttctcatccc gtttgcacca 1020 tttgtccgtc tggtgggtac ctctgtctgt catatcctct cgctctctct accaacaatc 1080 tggagtgcgg ggtgtaaact ggaatcttttt gttcctgaca aatttgcaga tcaaggaggt 1140 caccgacttc ttctgtcctg aaatcagccg ctggactccc caagcgctcg tcgcgattca 1200 agaggtcagt gctcaaacct ggcatgtact attagatctg atggtttgat tagagtacta 1260 caatgcagat gaattcaaga tctgaaaacc atgaactgtg gggtagatgc atgtatcgcc 1320 ttaattcata gtttctgaat gctctggtat taattcagtt tgatatattt atttagctgc 1380 atggtattgt tttggtggct ggtaattcaa aactgaaatg tgattacgag caaaatggta 1440 tcaattgtct tttctgtgca catctggttg tttggtcaag atatacagtc ttgcaacatg 1500 atcctgccca cacactcaaa actgaactat tggttagatt ccattttgct tatggaattt 1560 agggtgtaac tgagaggtga gcaggtggca gtgaccagag ctacgttcaa ttttgattca 1620 ggaagaggat cttgcgatcc agaaaattgc atgttatggt tatgtgtccg aaggccaaat 1680 gattatctct atatccagta catgcttatg tgtccgaacc cccaaatgat tatctctata 1740 tccagtacat gcttatgtgt ccgaaccatt caagttattg tgcatttatt caagattgtt 1800 gtttgcacaa tagacttact ttagtcgcct cttgccagaa atgcctcctt ctgcacaatg 1860 agcaaaaata acataaagtt gattatgctc agtgtggcct aattgtactt tcagttactg 1920 gcctgtgtat ttgctttgaa ttgacacata attccctttt cctttccctc tgcaggctgc 1980 agagtatcac ctcgtcgacg tatttgaaag ggcaaatcac tgtgccatcc atgcaaagcg 2040 tgttaccgtc agtaagttct cactgaatga aaacttcctt tcttttacaa tattatgcag 2100 aaggaaacat gccagttatg aaagagtttc aattacaaga tcacctttgc tttcatttga 2160 tgtggtatct agttttgatg ttgtttcaag gttcaagaat tctaatgatt aatgatagga 2220 tccacaattg ttatatctct gcagctcctc gtatctgacg aacaaacatg tcaaacaatt 2280 cattaaaaaa tgaagatgtc aaacaaacag tatatgcact gcatattgga tatcaaacct 2340 ggttactaca gcaggaagtt cagggttgat tactttattt cttatctgaa ggataaacat 2400 tgtatcaaat cagaatttta tttgtaagtt gcattttttg tttactcata aaacttggaa 2460 actgttttac tgtgagaaat agatgcccac tagaatcatg atcatcatca tggctgttgc 2520 tattctaaca aataaaacgg atgctcctga acaaatggaa ctatgtatga agatgtatgg 2580 actagcatgt tcctgttaac ctgacttttt tttgctgcta cagtgcaaaa ggacatacag 2640 ctcgcgaggc gtatcggtgg gaggaggctt tggtga 2676 <210> 69 <211> 4399 <212> DNA <213> Common wheat <400> 69 atggcccgca ccaagcaccc ggccagtcag gaagaccaag gcgccgccca agaagcagct 60 cgggccccgc cccgcgcagc ggcggcagga gacaggtcag cccttccctc cttcctcccc 120 ccgtctcccg cctctcgagc gaacgccgtc gccatttcgt cgcgagagat ggacggacgg 180 acgccccacg catgacgcta atgtgctctt cctctccctt tgcagatggc gcgggcacgt 240 cggcgacacc ggtgcgcggg accttcccgg gtcgcttcct tttcgtttcg tcttgttttc 300 tgttttttgg tctgacttgc tcgtcacctg ttcgacggaa tgcagaggca gccgggcggg 360 cggcggcccc agggggggct caaggtgcgg ccttctttgc gcttttcggt tttccgccgc 420 gtgtttaggg ccatttccgt cttgtttggg ggtgcgcggg gcgggggctt gttttttttc 480 ctcccccctt cgttgttgcg cacattgctc gggaatgctg ccaggagcgg ttgcggttct 540 tctttgaccc ttcgggaggg ctggatcggc agtttcttcg cttccttgct ccagatttta 600 gttcatcttg taccagtaca gtagcaagat gatggatggg ggcttgtttt ttctttctcc 660 ttccttgttc cggacatttc tctggagcga cttttatgca tttcccagta ttgtcctttg 720 tccttagagg gtagtggatc ggcagttttc ttcgcctcat tggtccagat tttagtttat 780 cttgtacggt accaagatga tggatgtggc acaaagttct cagtttgggg gttgcgctct 840 tccgggcagt tgttattttg gtctgtgatg actaactcgt atctattctt gtgggagcag 900 gggcaactgg gcaacccaag cagaggaac cacaccggtt caggccaggc acggtggcac tgcgggagat caggaggtat cagaagtcgg tcgactttct catcccgttt gcaccatttg tccgtctggt gggtacctct gtctgtcata tcctctcgct ctctctacaa acgatctgca 1080 gtgcagagtg taattggaat attttgttcc tgacaaattt gcagatcaag gaggtcaccg acttcttctg tcctgaaatc agccgctgga ctccccaagc gctcgtcgcg attcaagagg tcagtgctaa aacctggcat gtactattag atctgatggt ttgattagag tactacaatg 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320. 1320 tcatggtttc tgaatgctct gctattaatt cagtttgata tatttattta gcagcatggt attgttttgg tggctggtaa atcaaaactg aaatgtgatt acgagcaaaa cggtatcgat 1440 tgtcgatcct gtgtgttttt gtgcacatct ggttgtttgg tcaagatgtg tttgtgcaca 1500 tcttgcaaca tgatcctgcc cacacactca aaactgacta ttggttaggt tccatttgtc 1560 ttatggaatt tagggtgtaa ctgagaggtg agcaagtggt agtaacgttc aattttgatt 1620 caggatgagg atattgtgat ccagaaaatt gcatgttatg gttatgtgtc caaacgccaa 1680 atgattatgt ctatatccag tactttagaa ccagtacaac aacaaaaagt actttagaac 1740 cagtcaagtt tattgtgcat ttatacaaga gtgttgtttg cacaatagac ttgctttagt 1800 cgtctcttgc cagaaatgcc ttcttctgca caacgagcaa aaataacata aagttgacta 1860 tactcagtgt ggcctaagaa atgagttgta ctttttagtc actggcctgt gtatttgctt 1920 tgaattgaca cataattccc ttttcctttc cctctgcagg ctgcagagta tcacctcgtc 1980 gacgtatttg aaagggcaaa tcactgtgcc atccatgcaa agcgtgttac cgtcagtaag 2040 ttctcactga atgaaaactc cctttctttt acaatattgc gcagaaggaa acatgccagt 2100 tatgaaagag tttcaattac aggatcacct ttgctttcat ttgatgtgat atctagtttt 2160 gatgttgttt caaggttcaa gaattctaat gataaatgat aggatccaca attgttatat 2220 ctctgcagct cctcgtatct gttgtccacg aacaaacata tcaaacaatt cattaaaaag 2280 atgaagaagt caaacaaaca gtatatgtgc actgcatatt agatatcaaa cctggttact 2340 atacgactga tctggcctga cccccgcgtc gcctctccct ggcggcacgg ggggaaccac 2400 tccggcgccg ccaccttccc tcctccctcc accccccacc tcgccgccgc ctgaggagtt 2460 cgccggcgaa gcccggtctg gctccaggga gggtggcggc ggggcatctc tctgcgaggc 2520 gtgagggcgc atctcgtgcg cgggcgcggc gagcttgggc gggatcgcgg gcgcggctct 2580 ggctcgggct ggtgaggctc cggcggtccg aggggtgtcg cggcgcggcg ggggtctcgc 2640 tccggcgcgg gcggtccgag ggcgcgcggg atctggcgct ccagcagggg cgccgtcaag 2700 gggagccggg cagggaagct cgtcaggcgg gctcgtcagg caggcgtgac gcggggcggc 2760 ggccgcgggc tggaagccat ggcgttttgg ccatggtgtc gtgtgcgctc gcttctcatt 2820 cgcagcttga ggtgctgctg ctgagggtgg tgcgcggtga agcttggtgg tcggcgttgg 2880 agagtgcaag gcgcaacagg aatggctcca atgatctcca cgcttctggg tggatcagat 2940 ctgcggccct ccataggggt gtgttccggg cgaaagcctt gacccgactt tgtcggtgcc 3000 gtcgacggcg gcgctttcgg gcgtcgtttc cctccttgga ggcgtcgttg tggaactcat 3060 cttcttctat gtggggctcg ggctctccgg gtgaaaacct aagctccaga ttttccggag 3120 cgggcgatgg cggcgtcttc gtcgtttccc tcttgggggc gttgcttggg agagttagct 3180 tgtgcttggt gcgtttggtt ttctcctacg tcgggtttgg tggatgccgg ggcagcggcc 3240 ccggacggct gatgaacgcc gaggcggcgg cctcggaaag tgatgcgcgg tgcggctcca 3300 tggggcgggg cggtggctcg gccttcactt gggtggcaat cttgggccac ttgggtggca 3360 ggcttgtcgg tccggtcgac gcgttccaga gggggcggtc tgactttgcg tcggggcggc 3420 ggccccggat gtggtgcgac gttcgtggtc tgcgagcggt tgccttgagc agcgtgggct 3480 gcgggcagct gggtcgcgcg gcgttgctgc tcgagcggag cggtggtacg tcggggcggc 3540 ggccccggaa ggtgatgccg gttgattgcg ctgggcgtga cggagcggtg gatgtcgggg 3600 cggcggcccc gagaaaatca ctgtggcgtc cagatttctg tggcaacgat gatggtggga 3660 gcgatgtcgg cgacgcggca atggttgcga tagcggctc ttctccggcg tgtccacgat 3720 attgcctcgg tttgtttgtt gctgtggagt cgaagctgcg gcggcgaggc cctgtggtat 3780 acgatgactg gttccaggtg tccttcgtc gatcttccgt ggcgccagcc gtgcctggtt 3840 tcgttcttcc gagttctccg tcagaatcgg agctgcgttg tctgtccgca ggtcgacatg 3900 ttgtcgagaa gggtgggctt tgccctgtgt gtttcagtct atgcgagtgg gctcggccct 3960 tgttgttctg gttttgccc gttttccgt attaactgg gcaattctct tctgcttaat 4020 tatagatga ggcaatcttt gcctcccttt caaaaaaaaa cctggttact atagcaggaa 4080 attcagggtt gattacttta tttcttatct gaaggataaa cattgtatca aatcagaatt 4140 ttatttgtaa gttacattttt ttttactta taaaacttgg aaactgtttt actgtgacaa 4200 atagatgcca ctagaatcat gatcacatcg tggctgttgc tattctaaca aataaatgct 4260 cctgaacaaa tgggaactat atatgaagat gtatggacca gcatgttcct gttaacctga 4320 cctttttcct ttttttgctg ctgcagtgca aaaggacata cagcttgcaa ggcgtatcgg 4380 cgggaggagg ctttggtga 4399 <210> 70 <211> 492 <212> DNA <213> Common wheat <400> 70 atggcccgca ccaagcaccc ggccagtcag gaagaccaag gcgccgccca agaagcagct 60 cgggccccgc cccgcgcagc ggcggcagga gacagatggc gcgggcacgt cggcgacacc 120 gaggcagccg ggcgggcggc ggccccaggg ggggctcaag gggcaactgg gcaacccaag 180 cagaggaaac cacaccggtt caggccaggc acggtggcac tgcgggagat caggaggtat 240 cagaagtcgg tcgactttct catcccgttt gcaccatttg tccgtctgat caaggaggtc 300 accgacttct tctgtcctga aatcagccgc tggactcccc aagcgctcgt cgcgattcaa 360 gaggctgcag agtatcacct cgtcgacgta tttgaaaggg caaatcactg tgccatccat 420 gcaaagcgtg ttaccgtcat gcaaaaggac atacagcttg caaggcgtat cggcgggagg 480 aggctttggt ga 492 <210> 71 <211> 163 <212> PRT <213> Common wheat <400> 71 Met Ala Arg Thr Lys His Pro Ala Ser Gln Glu Asp Gln Gly Ala Ala 1 5 10 15 Gln Glu Ala Ala Arg Ala Pro Pro Arg Ala Ala Ala Ala Gly Asp Arg 20 25 30 Trp Arg Gly His Val Gly Asp Thr Glu Ala Ala Gly Arg Ala Ala Ala 35 40 45 Pro Gly Gly Ala Gln Gly Ala Thr Gly Gln Pro Lys Gln Arg Lys Pro 50 55 60 His Arg Phe Arg Pro Gly Thr Val Ala Leu Arg Glu Ile Arg Arg Tyr 65 70 75 80 Gln Lys Ser Val Asp Phe Leu Ile Pro Phe Ala Pro Phe Val Arg Leu 85 90 95 Ile Lys Glu Val Thr Asp Phe Phe Cys Pro Glu Ile Ser Arg Trp Thr 100 105 110 Pro Gln Ala Leu Val Ala Ile Gln Glu Ala Ala Glu Tyr His Leu Val 115 120 125 Asp Val Phe Glu Arg Ala Asn His Cys Ala Ile His Ala Lys Arg Val 130 135 140 Thr Val Met Gln Lys Asp Ile Gln Leu Ala Arg Arg Ile Gly Gly Arg 145 150 155 160 Arg Leu Trp <210> 72 <211> 2619 <212> DNA <213> Common wheat <400> 72 atggcccgca ccaagcaccc ggccgtcagg aagaccaagg cgctgcccaa gaagcagctc 60 gggacgcgcc cctcggccgg gacgccgcgg cggcaggaga caggtgagcc cgcccctcct 120 tccttcctcc cccccatctc gagcgaacgc cgtcgcctcg tcgcgagaga tggacgcccc 180 acgcgtgatg ctaatgtgtt cttcctctcc ctttgcagat ggcgcgggca cgtcggcgac 240 tccggtgcgc gggccttcct gggtcacttc cttttgtttc gtcttgtttt ctgtttttgg 300 tctgacttgc tcgtctcctg ttcgacggaa tgcagaggcg aagccgggcg ggcggcggcc 360 ccaggggcgg ctgaaggtgc gcccttcttt gtgcttttcg gttttccgcc gcgtgtttag 420 ggctatttcc gtcttgtttg gggcggggac ttgtatcttc tccccccttc gttgttgcgc 480 acatttctcg ggaatgctac caggagcagt tgcggttctt tggcccttag ggagggctgc 540 accggcaatt tcttcgcttc cttgctccag attttagttt atcttctaca gtagcaagat 600 gatggatggg gacttgcgtt tttctttccc cttccttgtt ccgggcattt ctctggagca 660 atgtttatgc atttcccagt attgtccttt gccctcagag gggaggggat cggcagtttt 720 cttcgcttca tcggtccaga ttttagttta tcttgtacag tagcaagatg atggatgtga 780 cacaaagttc tcagtttggg ggttgcgctc ttccgggtag ttgttaattt cgtctgtgac 840 tgactcgtat ctattcttgt gggagcaggg gcaactgggc aacccaagca gaggaagcca 900 caccggttca ggccaggcac ggtggcactg cgggagatca ggaagtacca gaaatcggtc 960 gactttctca tcccgtttgc accatttgtt cgtctggtgg gtacctctgt catatcctct 1020 cgctctctct accaacgatc tggagtgcgg agtgtaaatt ggaatctttt gttcctgaca 1080 aatttgtaga tcaaggaggt caccgacttc ttctgtcctg aaatcagccg ctggactccc 1140 caagcgctcg ttgcaattca agaggtcagt gctcaaacct ggcatgtact attagatctg 1200 atggtttgat tagagtacta caatgcagat gaattcaaga tctgaaaacc atgaactgtg 1260 gggtagatgc atgtatcata gtgtctgaat gctctggtat taattcagtt tgatatattt 1320 atttagcagc atggtattgt tttggtggct ggtaacccaa aactgaaacg tgattacgag 1380 caaaatggta tcaattgtct tttctgtgtg tttgtgcaaa tctggttgtt tggtcaagat 1440 atacagtctt gcaacatgaa tacatgatcc tgcccacaca ctcaaaactg aactattggt 1500 tagatgccat atgtcttatg gaatttaggg tgtaactgag aggtgagcaa gtggtagtga 1560 cgttcaattt tgattcagga tgaggatatt gtgatccaga aaattgcatg ttatggttat 1620 gtgtccgaac gccaaatgat tatgtctata tccactacat gcttatgtgt cccaaccccc 1680 aaatgattat ctctgtttcc agtacaacaa caaaaagtac tttagaacca gtcaagttta 1740 ttgtgcattt atacgagatt gttgtttgca caatagactt actttagtcg cctcttgcca 1800 gaaatgcctt cttctgcaca acgagcaaaa ataacataaa gttgactata ctcagtgtgg 1860 cctaagaaat gagttagtac ttttagtcac tggcctgtgt atttgctttg aattgacgca 1920 taattccctt ttcctttccc tctgcaggct gcagagtatc acctcgtcga cgtatttgaa 1980 agggcaaatc actgtgccat ccatgcaaag cgtgttaccg tcagtaagtt ctcactgaat 2040 gaaaacttcc ttccatttac aatattatgc agaaggaaac atgccagtta tgaaagagtt 2100 tcaattacag gatcaccttt gctttcattt gatgtgatat ctagttttga tgttgtttca 2160 aggttcaaga attctaacga ttaatgatag gatccacaat tgttatatct ctgcagctcc 2220 tcgtatctgt tgtcaacgaa caaacatgtc aaacaattca ttaaaaaaaa cgaagatgtc 2280 aaaccgggtt actatagcag gaaattcagg gttgattacc tatttattca gggttgatta 2340 cctatttatt tcttatctga aggataaaca ttatataaaa tcagaatttt atttgtaggt 2400 tacatttttt gtttccttat aaaacttgga aactgttgag aatcatcatc atcatggctg 2460 ttgctattct aacaaataaa acggatgctc ctgaataaat ggaactatat atgaagatgt 2520 atttactagc atgttcctgt taacctgact ttttttgctg ctacagtgca aaaggacata 2580 cagctcgcga ggcgtatcgg cgggaggagg ctttggtga 2619 <210> 73 <211> 2673 <212> DNA <213> Common wheat <400> 73 atggcccgta ccaagcaccc ggccaggaag accaaggcgc cgcccaagaa gcagctcggg 60 ccccgtcccg cgcagcggcg gcaggagaca ggtcagcccg cccgcccgtc gctcctccct 120 tccttccccc ccccgtctcc cgcctctcga ccgaacgccg ccgccttgtt tgttgcggta 180 cagaggtgcc cgtccgtgat gctaacaggt tcttcctctc ctttgcagat ggcgcgggca 240 cgtcggcgac accggtgcgc gggtcttccc gggtcgcttc cttttgtttc gtcttgtttt 300 ctgttttttg gtctgacttg ctcgtcacct gttcgacgga atgcagaggc gaagccgggc 360 gggcggcggc cccagggggc gctgaaggtg cggccttctt tgcgcttttc ggttttccgc 420 cgcgtgttta gggccatttc cgtcttgttt gggggtgggt ggggcggggg cttgattttt 480 ttcctcctcc cttcgttgtt gcgcacattt ctcgggaatg cttccaggag cggttgcagt 540 tcttctttgg ccttagggag ggctggatcg gcagtttctt cgcttccttg ctccagattt 600 tagtttatct tgtagtagta cagtagcaag atgatggatg gggacttgtt ttttctttcc 660 ccttccttgt tccggacatt tctttggagc aacttttatg catttcccgg tattgtcctt 720 tgcccttaga gtggagtgga tcggcagttt tcttcgcttc cctggtccag attttagttt 780 atctttacag tagcaagatg atgtatgtga cacaaagttc tcagtttggg ggttgcgctc 840 ttccaggtgt agttgttatt tttcgtctgt gactgactcg tatctattct tgtgggagca 900 ggggcaactg ggcaacccaa gcagaggaag ccacaccggt tcaggccagg cacggtggca 960 ctgcgggaga tcaggaggta ccagaagtcg gtcgactttc tcatcccgtt tgcaccattt 1020 gtccgtctgg tgggtacctc tgtctgtcat atcctctcgc tctctctacc aacaatctgg 1080 agtgcggggt gtaaactgga atcttttgtt cctgacaaat ttgcagatca aggaggtcac 1140 cgacttcttc tgtcctgaaa tcagccgctg gactccccaa gcgctcgtcg cgattcaaga 1200 ggtcagtgct caaacctggc atgtactatt agatctgatg gtttgattag agtactacaa 1260 tgcagatgaa ttcaagatct gaaaaccatg aactgtgggg tagatgcatg tatcgcctta 1320 attcatagtt tctgaatgct ctggtattaa ttcagtttga tatatttatt tagctgcatg 1380 gtattgtttt ggtggctggt aattcaaaac tgaaatgtga ttacgagcaa aatggtatca 1440 attgtctttt ctgtgcacat ctggttgttt ggtcaagata tacagtcttg caacatgatc 1500 ctgcccacac actcaaaact gaactattgg ttagattcca ttttgcttat ggaatttagg 1560 gtgtaactga gaggtgagca ggtggcagtg accagagcta cgttcaattt tgattcagga 1620 agaggatctt gcgatccaga aaattgcatg ttatggttat gtgtccgaag gccaaatgat 1680 tatctctata tccagtacat gcttatgtgt ccgaaccccc aaatgattat ctctatatcc 1740 agtacatgct tatgtgtccg aaccattcaa gttattgtgc atttattcaa gattgttgtt 1800 tgcacaatag acttacttta gtcgcctctt gccagaaatg cctccttctg cacaatgagc 1860 aaaaataaca taaagttgat tatgctcagt gtggcctaat tgtactttca gttactggcc 1920 tgtgtatttg ctttgaattg acacataatt cccttttcct ttccctctgc aggctgcaga 1980 gtatcacctc gtcgacgtat ttgaaagggc aaatcactgt gccatccatg caaagcgtgt 2040 taccgtcagt aagttctcac tgaatgaaaa cttcctttct tttacaatat tatgcagaag 2100 gaaacatgcc agttatgaaa gagtttcaat tacaagatca cctttgcttt catttgatgt 2160 ggtatctagt tttgatgttg tttcaaggtt caagaattct aatgattaat gataggatcc 2220 acaattgtta tatctctgca gctcctcgta tctgacgaac aaacatgtca aacaattcat 2280 taaaaaatga agatgtcaaa caaacagtat atgcactgca tattggatat caaacctggt 2340 tactacagca ggaagttcag ggttgattac tttatttctt atctgaagga taaacattgt 2400 atcaaatcag aattttattt gtaagttgca ttttttgttt actcataaaa cttggaaact 2460 gttttactgt gagaaataga tgcccactag aatcatgatc atcatcatgg ctgttgctat 2520 tctaacaaat aaaacggatg ctcctgaaca aatggaacta tgtatgaaga tgtatggact 2580 agcatgttcc tgttaacctg actttttttt gctgctacag tgcaaaagga catacagctc 2640 gcgaggcgta tcggtgggag gaggctttgg tga 2673 <210> 74 <211> 18400 <212> DNA <213> Artificial sequence <220> <223> Construct 24195 <220> <221> Features not yet classified <222> (4)..(259) <223> bNRB-05 <220> <221> Features not yet classified <222> (101)..(125) <223> bNRB-01-01 <220> <221> Enhancer <222> (168)..(259) <223> eNOS-01 <220> <221> Enhancer <222> (292)..(485) <223> eFMV-06 <220> <221> Enhancer <222> (492)..(784) <223> e35S-11 <220> <221> Promoter <222> (791)..(2592) <223> prSoUbi4-02 <220> <221> Features not yet classified <222> (1170)..(1234) <223> u5SoUbi4-02 <220> <221> Intron <222> (1235)..(2592) <223> iSoUbi4-02 <220> <221> Features not yet classified <222> (2607)..(2657) <223> NLS <220> <221> Gene <222> (2607)..(6827) <223> CRISPR-related 9 <220> <221> Features not yet classified <222> (2610)..(2610) <223> +4G <220> <221> Features not yet classified <222> (2611)..(2611) <223> +5C <220> <221> mutation <222> (6147)..(6149) <223> L to V mutation <220> <221> mutation <222> (6192)..(6194) <223> I to V mutation <220> <221> Features not yet classified <222> (6762)..(6824) <223> NLS <220> <221> Terminator <222> (6834)..(7835) <223> tZmMTL-01 <220> <221> Features not yet classified <222> (7836)..(7847) <223> xSTOPS-01 <220> <221> Promoter <222> (7855)..(8216) <223> prTau6-01 <220> <221> Features not yet classified <222> (8217)..(8236) <223> xTaCenh3 Target-03 <220> <221> Features not yet classified <222> (8217)..(8321) <223> gRNA TaCenH3-03 <220> <221> Features not yet classified <222> (8237)..(8248) <223> crRNA-01 <220> <221> Features not yet classified <222> (8253)..(8321) <223> tracrRNA-01 <220> <221> Promoter <222> (8322)..(8683) <223> prTaU6-01 <220> <221> Features not yet classified <222> (8684)..(8703) <223> xTaCenH3 Target-04 <220> <221> Features not yet classified <222> (8684)..(8788) <223> Guide RNA TaCenH3-04 <220> <221> Features not yet classified <222> (8704)..(8715) <223> crRNA-01 <220> <221> Features not yet classified <222> (8720)..(8788) <223> tracrRNA-01 <220> <221> Features not yet classified <222> (8795)..(8806) <223> xSTOPS-01 <220> <221> Promoter <222> (8807)..(10799) <223> prUbi1-10 <220> <221> TATA_Signal <222> (9677)..(9685) <223> TATA box <220> <221> Intron <222> (9790)..(10799) <223> iUbi1-02-01 <220> <221> Gene <222> (10811)..(11989) <223> cPMI-12 <220> <221> Terminator <222> (11994)..(13028) <223> tUbi1-01 <220> <221> Features not yet classified <222> (13072)..(13083) <223> xSTOPS-01 <220> <221> Features not yet classified <222> (13084)..(13123) <223> xTAG-02 <220> <221> Features not yet classified <222> (13132)..(13261) <223> bNLB-05 <220> <221> Features not yet classified <222> (13167)..(13191) <223> bNLB-01-01 <220> <221> Gene <222> (13541)..(14329) <223> cSpec-03 <220> <221> Promoter <222> (14424)..(14554) <223> prVirG-01 <220> <221> Gene <222> (14629)..(15354) <223> cVirG-01 <220> <221> Gene <222> (15384)..(16457) <223> cRepA-03 <220> <221> Unclassified feature <222> (16500)..(16904) <223> oVS1-02 <220> <221> Unclassified feature <222> (17582)..(18388) <223> oCOLE-06 <400> 74 attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 60 taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 120 tgtcaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 180 attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 240 aactgacaga accgcaacgc tgcaggaatt ggccgcagcg gccatttaaa tagctgcttg 300 tggggaccag acaaaaaagg aatggtgcag aattgttagg cgcacctacc aaaagcaact 360 ttgcctttat tgcaaagata aagcagattc ctctagtaca agtggggaac aaaataacgt 420 ggaaaagagc tgtcctgaca gcccactcac tattgcgttt gacgaacgca gtgacgacca 480 caaaactcga gacttttcaa caaagggtat tatccggaaa cctctcgga ttccattgcc 540 cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc 600 atcattgcga taaaggaaag gctatcgttg aagatgcctc tgccgacagt ggtcccaaag 660 atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 720 agcaagtgga ttgatgtgat atctccactg acgtaagggt tgacgaacaa tcccactatc 780 cttcggaccc gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaat 840 gttctaaaaa aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta 900 acctgatcta gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcg 960 tccccctcct cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggg 1020 gacggattcc acgagaccgc gacgcaaccg cctctcgccg ctgggcccca caccgctcgg 1080 tgccgtagcc tcacgggact ctttctccct cctccccgt tataaattgg cttcatcccc 1140 tccttgcctc atccatccaa atcccagtcc ccaatcccat cccttcgtag gagaaattca 1200 tcgaagctaa gcgaatcctc gcgatcctct caaggtactg cgagttttcg atccccctct 1260 cgacccctcg tatgtttgtg tttgtcgtag cgtttgatta ggtatgcttt ccctgtttgt 1320 gttcgtcgta gcgtttgatt aggtatgctt tccctgttcg tgttcatcgt agtgtttgat 1380 taggtcgtgt gaggcgatgg cctgctcgcg tccttcgatc tgtagtcgat ttgcgggtcg 1440 tggtgtagat ctgcgggctg tgatgaagtt atttggtgtg atctgctcgc ctgattctgc 1500 gggttggctc gagtagatat gatggttgga ccggttggtt cgtttaccgc gctagggttg 1560 ggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact tgcgtttgat 1620 tgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg tagcttctgc 1680 ttatgtgcca gatgcgccta ctgctcatat gcctgatgat aatcataaat ggctgtggaa 1740 ctaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag ctacaggtgt 1800 agggacttgc gtctaattgt ttggtccttt actcatgttg caattatgca atttagttta 1860 gattgtttgt tccactcatc taggctgtaa aagggacact gcttagattg ctgtttaatc 1920 tttttagtag attattattat attggtaact tattacccct attacatgcc atacgtgact 1980 tctgctcatg cctgatgata atcatagatc actgtggaat taattagttg attgttgaat 2040 catgtttcat gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttac 2100 tgatccatgt atgatttgcg tggttctcta atgtgaaata ctatagctac ttgttagtaa 2160 gaatcaggtt cgtatgctta atgctgtatg tgccttctgc tcatgcctga tgataatcat 2220 atatcactgg aattaattag ttgatcgttt aatcatatat caagtacata ccatgccaca 2280 atttttagtc acttaaccca tgcagattga actggtccct gcatgttttg ctaaattgtt 2340 ctattctgat tagaccatat atcatgtatt ttttttggt aatggttctc ttattttaaa 2400 tgctatatag ttctggtact tgttagaaag atctgcttca tagtttagtt gcctatccct 2460 cgaattagga tgctgagcag ctgatcctat agctttgttt catgtatcaa ttctttgtg 2520 ttcaacagtc agttttgtt agattcattg taacttatgg tcgcttactc ttctggtcct 2580 caatgcttgc agcctagtaa gccgccatgg ccccaaagaa gaagaggaag gtcggcatcc 2640 acggcgtccc agctgcgatg gatagaagt actccatcgg cctcgacatc ggcaccaca 2700 gcgtgggctg ggccgtcatc accgacgagt acaggtgcc atccagaag ttcaggtcc 2760 tgggcacac cgaccgccac agcatcaaga agaacctcat cggcgctctc ctgttcgact 2820 ccggcgagac ggctgaggct accaggctca agcgcaccgc caggaggagg caccagga 2880 ggaagaacag gatctgctac ctccaagaga tctctccaa cgagatggcc aaggtggacg 2940 actccttctt ccaccgcctg gaggagagct tcctcgtcga ggaggacaag aagcacgaga 3000 ggcaccaat cttcggcaac atcgtggacg aggtcgccta ccacgagaag tacccaacca 3060 tctaccacct gaggagaag ctcgtggact ccaccgacaa ggccgacctc cgcctgatct 3120 acctcgccct ggcccacatg atcaagttca ggggccactt cctgatcgag ggcgacctca 3180 acccagacaa cagcgacgtg ggaagctgt tcatccaact cgtccagacc tacaaccacc 3240 tcttcgagga gaacccgatc aacgcttccg gcgtggacgc taaggctc ctgagcgcta 3300 ggctctccaa gagcaggagg ctcgagaacc tgatcccca gctcccaggc gagagagaga 3360 acggcctgtt cggcaacctc atcgctctct ccctgggcct caccccaaac ttcaagagca 3420 acttcgacct cgccgaggac gccaagctgc aactctccaa ggacacctac gacgacgacc 3480 tggacaacct cctggcccag atcggcgacc aatacgccga cctgttcctc gccgccaaga 3540 acctgtccga cgccatcctc ctgagcgaca tcctccgcgt gaacaccgag atcaccaagg 3600 ccccactctc cgccagcatg atcaagcgct acgacgagca ccaccaggac ctgaccctcc 3660 tgaaggccct ggtcaggcaa cagctcccag agaagtacaa ggagatcttc ttcgaccaga 3720 gcaagaacgg ctacgctggc tacatcgacg gcggcgcctc ccaagaggag ttctacaagt 3780 tcatcaagcc aatcctggag aagatggacg gcaccgagga gctcctggtg aagctcaaca 3840 gggaggacct cctgaggaag cagcgcacct tcgacaacgg cagcatccca caccaaatcc 3900 acctcggcga gctgcacgct atcctgagga ggcaagagga cttctaccca ttcctcaagg 3960 acaacaggga gaagatcgag aagatcctga ccttccgcat cccctactac gtcggcccac 4020 tcgctagggg caactccagg ttcgcttgga tgacccgcaa gagcgaggag acgatcaccc 4080 cgtggaactt cgaggaggtc gtcgacaagg gcgcttccgc tcagagcttc atcgagagga 4140 tgaccaactt cgacaagaac ctgccaaacg agaaggtgct cccaaagcac tccctcctgt acgagtactt caccgtctac aacgagctca ccaaggtgaa gtatgtgacc gagggcatgc gcaagccagc cttcctgagc ggcgagcaga agaaggccat cgtggacctc ctgttcaaga 4320 4380. ccacagga ggtgaccgtc aagcaactca aggaggacta cttcaagaag atcgagtgct tcgactccgt ggagatcagc ggcgtcgagg accgcttcaa cgcctccctc ggcacctacc 4440 acgacctcct gaagatcatc aaggacaagg acttcctgga caacgaggag aacgaggaca tcctcgagga catcgtgctg accctcaccc tgttcgagga cagggagatg atcgaggagc 4560 gcctgaagac ctacgcccac ctcttcgacg acaaggtcat gaagcaactc aagaggagga 4620 ggtacaccgg ctggggcagg ctgagccgca agctcatcaa cggcatccgc gacaagcagt 4740. ccggcaagac catcctcgac ttcctgaaga gcgacggctt cgccaacagg aacttcatgc aactgatcca cgacgactcc ctcaccttca aggaggacat ccaaaaggct caggtgtccg gccaggggcga cagcctgcac gagcacatcg ctaacctcgc tggcagccca gccatcaaga 4860 agggcatcct gcagaccgtg aaggtcgtcg acgagctcgt gaagttcatg ggcaggcaca 4920 agccagagaa catcgtcatc gagatggccc gcgagaacca gaccaccag aagggccaaa 4980 agaactccag ggagcgcatg aagcgcatcg aggaggcat ggcagctg ggcagccaaa 5040 tcctcagga gcaccagtg gagacaccc aactgcagaa cgagaagctc tacctgtact 5100 acctccagaa cggcagggac atgtatgtgg accagagct ggacatcac cgccctccg 5160 actacgacgt ggaccacatc gtcccacagt ccttcctgaa ggacgacagc atcgacaaca 5220 aggtgctcac caggagcgac aagaccgcg gcaagtccga caacgtccca agcgaggagg 5280 tggtcaagaa gatgaaaaac tactggaggc agctcctgaa cgccaagctg atcacccaa 5340 ggaagttcga aacctcacc aaggctgaga ggggcggcct ctccgagctg ggaaggccg 5400 gcttcattaa aaggcagctg gtggagacgc gccaaatcac caagcacgtc gcccaatcc 5460 tcgacagccg catgacacc aagtacgacg agaacgacaa gctgatcagg gaggtgagg 5520 tcatcaccct gaagtccaag ctcgtgagcg acttcaggaa ggacttccag ttctacaagg 5580 tccgcgagat caataattac caccacgccc acgacgctta cctcaacgct gtggtcggca 5640 ccgccctgat taaaaagtac ccaaagctcg agtccgagtt cgtgtacggc gactacaagg 5700 tgtacgacgt ccgcaagatg atcgccaagt ccgagcaaga gatcggcaag gccaccgcca 5760 agtacttctt ctacagcaac atcatgaact tcttcaagac cgagatcacc ctggccaacg 5820 gcgagatcag gaagcgccca ctcatcgaga cgaacggcga gacgggcgag atcgtgtggg 5880 acaagggcag ggacttcgcc accgtgcgca aggtcctctc catgccacag gtgaacatcg 5940 tcaagaagac cgaggtccaa accggcggct tctccaagga gagcatcctg ccaaagagga 6000 acagcgacaa gctcatcgcc cgcaagaagg actgggatcc aaagaagtac ggcggcttcg 6060 actccccaac cgtggcctac agcgtcctcg tggtcgccaa ggtggagaag ggcaagtcca 6120 agaagctgaa gagcgtgaag gagctcgtcg gcatcaccat catggagagg tccagcttcg 6180 agaagaaccc agtggacttc ctcgaggcca agggctacaa ggaggtcaag aaggacctga 6240 tcattaaact cccaagtac agcccttcg agctggagaa cggcaggaag cgcatgctgg 6300 cttccgctgg cgagctccaa agggcaacg agctcgccct gccatccaag ttgtgact 6360 tcctctacct ggcctcccac tcgagaagc tcaagggcag cccagaggac acgagcaaa 6420 agcagctgtt cgtcgagcag cacaaccact acctcgacga gatcatcgag caatctccg 6480 agttcagcaa gcgcgtgatc ctcgccgacg ccaacctgga caagctcctc tccgcctaca 6540 acaagcacag ggacaagcca atccgcgagc aggccgagaa catcatccac ctctcaccc 6600 tgaccaacct cggcgctcca gctgccttca agtacttcga caccaccatc gaggaagc 6660 gctacaccctc caccaggag gtgctggacg ccacccat ccaccagtcc atcaccggcc 6720 tctacgagac gaggatcgac ctgagccac tcggcggcga ctccagccca ccaaagaga 6780 agaggaaggt cagctggaag gacgcttccg gctggagccg catgtgaggt acctcacatc 6840 gatcgacgac caggatatg attattatct atctagcttg tggtggtggt tgaacaataa 6900 taagcgaggc cgagctggct gccatacata ggtattgtgt ggtgtgtgtg agagagagag 6960 aaacagagtt cttcagtttg ctatctctct ctgcatgttt ggcgtcagtc tttgtgctca 7020 tgtacgtacg tgtgtctacc tgcatgttgg ttgatccgat tgcatctgct gtaaccatat 7080 attaattggt ccacgatgat atgatttgat actatatata tactaaaacc ggacttctta 7140 ttataatact tgtagtatat aagtttctta cgcccgcaat tgatcgattc agaaggagtt 7200 ctagctagct aaaacatgca gattcagaat atcagatttt taggactact ggagatccag 7260 aaccttcgtg tccttgtacc cgtgattttg gatccccttt ctccccacta caatcgttgg 7320 cgcaatcttg ttctgctcgc ctagaatggt acgccctcac cgaccatgtc ctcgccgatg 7380 cctaccccgc cacgccatct ggatggtgca tgaatgtcgt cgttttgtcc tggatgctag 7440 gcacactctc ccccgagttg atggagcctc ctcgcacctc tggtggcact gcgccgcgcc 7500 tggcttgcca tcgaggagca attcctcggc aatcgcgaag ctcgcacact tcgtctcgac 7560 gccgagttcc atgtcttcat gtaggcgatc tcttcgtcag cgactattgt ctattgacgc 7620 aagatgaagg ggatggacga ggcccttggt gatcttggtg aggtcatcca tgaccgtacc 7680 cttgtcctaa acgtgttgtg tggtctgaat gagaggtttg cccacataaa ggtccacttc 7740 aagcactcga atccgttccc ctccttcacc gacgtttgta atgatctcat ccttgaggag 7800 atcgactcca gcgcgcctcc tccgcctccg accacctaat tagctaaggg acccgaccaa 7860 gcccgttatt ctgacagttc tggtgctcaa cacatttata tttatcaagg agcacattgt 7920 tactcactgc taggagggaa tcgaactagg aatattgatc agaggaacta cgagagagct 7980 gaagataact gccctctagc tctcactgat ctgggtcgca tagtgagatg cagcccacgt 8040 gagttcagca acggtctagc gctgggcttt taggcccgca tgatcgggct tttgtcgggt 8100 ggtcgacgtg ttcacgattg gggagagcaa cgcagcagtt cctcttagtt tagtcccacc 8160 tcgcctgtcc agcagagttc tgaccggttt ataaactcgc ttgctgcatc agacttgcct 8220 tggtcttcct gacggcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 8280 atcaacttga aaaagtggca ccgagtcggt gctttttttt tgaccaagcc cgttattctg 8340 acagttctgg tgctcaacac atttatattt atcaaggagc acattgttac tcactgctag 8400 gaggaatcg aactaggaat attgatcaga ggaactacga gagagctgaa gataactgcc 8460 ctctagctct cactgatctg ggtcgcatag tgagatgcag cccacgtgag ttcagcaacg 8520 gtctagcgct gggcttttag gcccgcatga tcgggcttt gtcgggtggt cgacgtgttc 8580 acgattgggg agagcaacgc agcagttcct cttagtttag tcccacctcg cctgtccagc 8640 agagttctga ccggtttata aactcgcttg ctgcatcaga cttgacggaa tgcagaggcg 8700 agcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 8760 agtggcaccg agtcggtgct ttttttttcc taggctaatt agctaactgc agtgcagcgt 8820 gacccggtcg tgcccctctc tagagataat gagcattgca tgtctaagtt ataaaaatt 8880 accacatatt ttttttgtca cacttgtttg aagtgcagtt tatctatctt tatacatata 8940 tttaaacttt actctacgaa taatataatc tatagtacta caataatatc agtgttttag 9000 agaatcatat aaatgaacag ttagacatgg tctaaaggac aattgagtat tttgacaaca 9060 ggactctaca gtttatctt tttagtgtgc atgtgttctc ctttttttt gcaaatagct 9120 tcacctatat aatacttcat ccattttatt agtacatcca tttagggttt agggttaatg 9180 gtttttatag actaattttt ttagtacatc tattttattc tattttagcc tctaaattaa 9240 gaaaactaaa actctatttt agttttttta tttaataatt tagatataaa atagaataaa 9300 ataaagtgac taaaaattaa acaaataccc tttaagaaat taaaaaaact aaggaaacat 9360 ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc cgtcgacgag tctaacggac 9420 accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga agcagacggc acggcatctc 9480 tgtcgctgcc tctggacccc tctcgagagt tccgctccac cgttggactt gctccgctgt 9540 cggcatccag aaattgcgtg gcggagcggc agacgtgagc cggcacggca ggcggcctcc 9600 tcctcctctc acggcaccgg cagctacggg ggattccttt cccaccgctc cttcgctttc 9660 ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc ccaacctcgt 9720 gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg tcggcacctc 9780 cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct ctagatcggc 9840 gttccggtcc atggttaggg cccggtagtt ctacttctgt tcatgtttgt gttagatccg 9900 tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca 9960 cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg gctctagccg 10020 ttccgcagac gggatcgatt tcatgattt ttttgtttcg ttgcataggg tttggttgc 10080 ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct tttcatgctt 10140 ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga tcggagtaga 10200 attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg tgtgccatac 10260 atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga taggtataca 10320 tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct tggttgtgat 10380 gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat actgtttcaa 10440 actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat cttcatagtt 10500 acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg atgtgggttt 10560 tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta accttgagta 10620 cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat atacttggat 10680 gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata cgctatttat 10740 ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta cttctgcagt 10800 gactaatag atgcagaagc tgatcaacag cgtgcagaac tacgcctggg gcagcaagac 10860 cgccctgacc gagctgtacg gcatggagaa ccccagcagc cagcccatgg ccgagctgtg 10920 gatgggcgcc caccccaaga gctcaagccg cgtgcagaac gccgccggg atatcgttag 10980 cctgcgcgac gtgatcgaga gcgacaagag caccctgctg ggcgaggcg tggccaagcg 11040 cttcggcgag ctgcccttc tgttcaaggt gctgtgcgcc gctcagcccc tgagcatcca 11100 ggtgcaccct aacaagcaca acagcgagat cggcttcgcc aaggagaacg ccgccggcat 11160 ccccatggac gccgccgagc gcaactacaa ggaccccaac cacaagcccg agctggtgtt 11220 cgccctgacc cccttcctgg ccatgaacgc cttccgcgag ttcagcgaga tcgttagcct 11280 gctgcagccc gtggccggcg cccaccccgc tatcgcccac ttccttcagc agcccgacgc 11340 cgagcgcctg agcgagctgt tcgccagcct gctgaacatg cagggtgagg agaagtcacg 11400 cgccctggcc your atcct gcgccctgga cagccagcag ggcgagccct ggcagacaat 11460 ccgcctgatc agcgagttct accccgagga tagcggcctg ttcagcccccc tgctgctgaa 11520 cgtggtgaag ctgaaccccg gcgaggccat gttcctgttc gccgagaccc cccacgccta 11580 cctgcagggc gtggccctgg aggtgatggc caacagcgac aacgtgctgc gcgccggcct 11640 gacccccaag tacatcgaca tccccgagct ggtggccaac gtgaagttcg aggctaagcc 11700 cgccaaccag ctgctgaccc agcccgtgaa gcagggcgcc gagctggact tccctatccc 11760 cgttgacgacgac ttcgccttca gcctgcacga cctgagcgac aaggacca ctatcagcca 11820 gcagagcgcc gcgatcctgt tctgcgtgga gggcgacgcc accctgtgga agggcagcca 11880 gcagctgcag ctgaagcccg gcgagagcgc ctttatcgcc gccaacgaga gccccgtgac 11940 cgtgaagggc cacggccgcc tggcccgcgt gtacaacaag ctgtgatagc tacgtcatgg 12000 gtcgtttaag ctgccgatgt gcctgcgtcg tctggtgccc tctctccata tggaggttgt 12060 caaagtatct gctgttcgtg tcatgagtcg tgtcagtgtt ggtttaataa tggaccggtt 12120 gtgttgtgtg tgcgtactac ccagaactat gacaaatcat gaataagttt gatgtttgaa 12180 attaaagcct gtgctcatta tgttctgtct ttcagttgtc tcctaatatt tgcctgcagg 12240 tactggctat ctaccgtttc ttacttagga ggtgtttgaa tgcactaaaa ctaatagtta 12300 gtggctaaaa ttagttaaaa catccaaaca ccatagctaa tagttgaact attagctatt 12360 tttggaaaat tagttaatag tgaggtagtt atttgttagc tagctaattc aactaacaat 12420 ttttagccaa ctaacaatta gtttcagtgc attcaaacac ccccttaatg ttaacgtggt 12480 tctatctacc gtctcctaat atatggttga ttgttcggtt tgttgctatg ctattgggtt 12540 ctgattgctg ctagttcttg ctgaatccag aagttctcgt agtatagctc agattcatat 12600 tatttatttg agtgataagt gatccaggtt attackatgt tagctaggtt ttttttacaa 12660 ggataaatta tctgtgatca taatcttat gaagcttta tgtttcctgg aggcagtggc 12720 atgcaatgca tgacagcaac ttgatcacac cagctgaggt agatacggta acaaggttct 12780 taaatctgtt caccaaatca ttggaaca cacatacaca ttctgccag tctggttag 12840 agaaatttca tgacaaatg ccaagctgt cttgactctt cactttggc catgagtcgt 12900 gacttagttt gttttaatgg accggttctc ctagcttgtt ctactcaaaa ctgttgttga 12960 tgcgaataag tgtgatggt tgatctctgg attttgttt gctctcaata gtggacgaga 13020 ttagatagct taagcctgca ggcggaccgc ctgcaggccc gggggcgcc cctattagc 13080 taacggccag gatcgccgcg tgagcctta gcaactagct agattatta acgcaatctg 13140 ttattaagtt gtctaagcgt caatttgttt acaccacaat atatcctgcc accagccagc 13200 caacagctcc ccgaccggca gctcggcaca aaatcaccac tcgatacagg cagcccatca 13260 gattaattc tcatgtttga cagcttatca tcgactgcac ggtgcaccaa tgcttctggc 13320 gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc 13380 gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt 13440 tctggcaaat attctgaaat gagctgttga caattaatca tccggctcgt ataatgtgtg 13500 gaattgtgag cggataacaa tttcacacag gaaacagacc atgagggaag cgttgatcgc 13560 cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac 13620 gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga 13680 tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat 13740 caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga 13800 agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact 13860 gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat 13920 cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg 13980 tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa 14040 tgaaactta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt 14100 gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt 14160 cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc 14220 taggcaggct tatcttggac aagaagatcg cttggcctcg cgcgcagatc agttggaaga 14280 atttgttcac tacgtgaaag gcgagatcac caaagtagtc ggcaaataaa gctctagtgg 14340 atctccgtac ccagggatct ggctcgcggc ggacgcacga cgccggggcg agaccatagg 14400 cgatctccta aatcaatagt agctgtaacc tcgaagcgtt tcacttgtaa caacgattga 14460 gaatttttgt cataaaattg aaatacttgg ttcgcattttt tgtcatccgc ggtcagccgc 14520 aattctgacg aactgcccat ttagctggag atgattgtac atccttcacg tgaaaatttc 14580 tcaagcgctg tgaacaaggg ttcagatttt agattgaaag gtgagccgtt gaaacacgtt 14640 cttcttgtcg atgacgacgt cgctatgcgg catcttatta ttgaatacct tacgatccac 14700 gccttcaaag tgaccgcggt agccgacagc acccagttca caagagtact ctcttccgcg acggtcgatg tcgtggttgt tgatctagat ttaggtcgtg aagatgggct cgagatcgtt 14880. cgtaatctgg cggcaaagtc tgatattcca atcataatta tcagtggcga ccgccttgag gagacggata aagttgttgc actcgagcta ggagcaagtg attttatcgc tagccgttc agtatcagag agtttctag acgcattcgg gttgccttgc gcgtgcgccc caacgttgtc cgctccaaag accgacggtc tttttgtttt actgactgga cacttaatct caggcaacgt cgcttgatgt ccgaagctgg cggtgaggtg aaacttacgg caggtgagtt caatcttctc ctcgcgtttt tagagaacc ccgcgacgtt ctatcgcgcg agcaacttct cattgccagt cgagtacgcg acgaggaggt ttatgacagg agtatagattg ttctcatttt gaggctgcgc 15240 cgcaaacttg aggcagatcc gtcaagccct caactgataa aaacagcaag aggtgccggt tatttctttg acgcggacgt gcaggtttcg cacgggggga cgatggcagc ctgagccaat tcccagatcc ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg gtacaaatcg 15420 gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 15480 aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 15540 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 15600 gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 15660 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 15720 tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 15780 ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 15840 accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 15900 acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 15960 cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 16020 gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 16080 gcgaaaccgg gcggccggag tacatcgaga tcgagctggc tgattggatg taccgcgaga 16140 tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 16200 ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 16260 gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 16320 gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 16380 aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 16440 catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 16500 aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 16560 ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 16620 tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 16680 ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 16740 aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 16800 16860. gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgctgaggt ctgcctcgtg 16920 aagaaggtgt tgctgactca taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca gttggtgatt ttgaactttt 17040 gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac cattacca attctgatta gaaaaactca tcgagcatca aatgaaactg 17280. 17280. 17280. 17280. 17280. 17280. 17280. 17280. 17280 aggagaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa tagttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gctctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 17520 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 17580 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 17640 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 17700 tccgcccccc tgacgagcat cacaaaaaatc gacgctcaag tcagaggtgg cgaaacccga 17760 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 17820 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 17880 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctggggct 17940 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 18000 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 18060 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 18120 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 18180 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 18240 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 18300 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 18360 caaaaaggat cttcacctag atccttttga tccggaatta 18400 <210> 75 <211> 18402 <212> DNA <213> Artificial sequence <220> <223> Construct 24194 <220> <221> Features not yet classified <222> (4)..(259) <223> bNRB-05 <220> <221> Features not yet classified <222> (101)..(125) <223> bNRB-01-01 <220> <221> Enhancer <222> (168)..(259) <223> eNOS-01 <220> <221> Enhancer <222> (292)..(485) <223> eFMV-06 <220> <221> Enhancer <222> (492)..(784) <223> e35S-11 <220> <221> Promoter <222> (791)..(2592) <223> prSoUbi4-02 <220> <221> Features not yet classified <222> (1170)..(1234) <223> u5SoUbi4-02 <220> <221> Intron <222> (1235)..(2592) <223> iSoUbi4-02 <220> <221> Features not yet classified <222> (2607)..(2657) <223> NLS <220> <221> Gene <222> (2607)..(6827) <223> CRISPR associated 9 <220> <221> Features not yet classified <222> (2610)..(2610) <223> +4G <220> <221> Features not yet classified <222> (2611)..(2611) <223> +5C <220> <221> mutation <222> (6147)..(6149) <223> L to V mutation <220> <221> mutation <222> (6192)..(6194) <223> I to V mutation <220> <221> Features not yet classified <222> (6762)..(6824) <223> NLS <220> <221> Terminator <222> (6834)..(7835) <223> tZmMTL-01 <220> <221> Features not yet classified <222> (7836)..(7847) <223> xSTOPS-01 <220> <221> Promoter <222> (7855)..(8216) <223> prTaU6-01 <220> <221> Features not yet classified <222> (8217)..(8322) <223> Guide RNA TaCenH3-01 <220> <221> Features not yet classified <222> (8218)..(8237) <223> xTaCenH3 Target-01 <220> <221> Features not yet classified <222> (8238)..(8249) <223> crRNA-01 <220> <221> Features not yet classified <222> (8254)..(8322) <223> tracrRNA-01 <220> <221> Promoter <222> (8323)..(8684) <223> prTaU6-01 <220> <221> Features not yet classified <222> (8685)..(8790) <223> Guide RNA TaCenH3-02 <220> <221> Features not yet classified <222> (8686)..(8705) <223> xTaCenH3 Target-02 <220> <221> Features not yet classified <222> (8706)..(8717) <223> crRNA-01 <220> <221> Features not yet classified <222> (8722)..(8790) <223> tracrRNA-01 <220> <221> Features not yet classified <222> (8797)..(8808) <223> xSTOPS-01 <220> <221> Promoter <222> (8809)..(10801) <223> prUbi1-10 <220> <221> TATA_Signal <222> (9679)..(9687) <223> TATA box <220> <221> Intron <222> (9792)..(10801) <223> iUbi1-02-01 <220> <221> Gene <222> (10813)..(11991) <223> cPMI-12 <220> <221> Terminator <222> (11996)..(13030) <223> tUbi1-01 <220> <221> Features not yet classified <222> (13074)..(13085) <223> xSTOPS-01 <220> <221> Features not yet classified <222> (13086)..(13125) <223> xTAG-02 <220> <221> Features not yet classified <222> (13134)..(13263) <223> bNLB-05 <220> <221> Features not yet classified <222> (13169)..(13193) <223> bNLB-01-01 <220> <221> Gene <222> (13543)..(14331) <223> cSpec-03 <220> <221> Promoter <222> (14426)..(14556) <223> prVirG-01 <220> <221> Gene <222> (14631)..(15356) <223> cVirG-01 <220> <221> Gene <222> (15386)..(16459) <223> cRepA-03 <220> <221> Features not yet classified <222> (16502)..(16906) <223> oVS1-02 <220> <221> Features not yet classified <222> (17584)..(18390) <223> oCOLE-06 <400> 75 attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 60 taaatccg attattctaa taaacgctct tttctcttag gttacccgc caatatatcc 120 tgtcaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 180 attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 240 aactgacaga accgcaacgc tgcaggaatt ggccgcagcg gccattaaa tagctgcttg 300 tggggaccag acaaaaaagg aatggtgcag aattgttagg cgcacctacc aaaagcaact 360 ttgcctttat tgcaaagata aagcagattc ctctagtaca agtggggaac aaaataacgt 420 ggaaaagagc tgtcctgaca gcccactcac tattgcgttt gacgaacgca gtgacgacca 480 caaaactcga gactttcaa caaagggtat tatccggaaa cctcctcgga ttccattgcc 540 cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc 600 atcattgcga taaaggaaag gctatcgttg aagatgcctc tgccgacagt ggtcccaaag 660 atggacccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 720 agcaagtgga ttgatgtgat atctccactg acgtaagggt tgacgaacaa tcccactatc 780 cttcggaccc gaattcatta tgtggtctag gtaggttcta tatataagaa aacttgaaat 840 gttctaaaaa aaaattcaag cccatgcatg attgaagcaa acggtatagc aacggtgtta 900 acctgatcta gtgatctctt gcaatcctta acggccacct accgcaggta gcaaacggcg 960 tccccctcct cgatatctcc gcggcgacct ctggcttttt ccgcggaatt gcgcggtggg 1020 gacggattcc acgagaccgc gacgcaaccg cctctcgccg ctgggcccca caccgctcgg 1080 tgccgtagcc tcacgggact ctttctccct cctccccgt tataaattgg cttcatcccc 1140 tccttgcctc atccatccaa atcccagtcc ccaatcccat cccttcgtag gagaaattca 1200 tcgaagctaa gcgaatcctc gcgatcctct caaggtactg cgagttttcg atccccctct 1260 cgacccctcg tatgtttgtg tttgtcgtag cgtttgatta ggtatgcttt ccctgtttgt 1320 gttcgtcgta gcgtttgatt aggtatgctt tccctgttcg tgttcatcgt agtgtttgat 1380 taggtcgtgt gaggcgatgg cctgctcgcg tccttcgatc tgtagtcgat ttgcgggtcg 1440 tggtgtagat ctgcgggctg tgatgaagtt atttggtgtg atctgctcgc ctgattctgc 1500 gggttggctc gagtagatat gatggttgga ccggttggtt cgtttaccgc gctagggttg 1560 ggctgggatg atgttgcatg cgccgttgcg cgtgatcccg cagcaggact tgcgtttgat 1620 tgccagatct cgttacgatt atgtgatttg gtttggactt tttagatctg tagcttctgc 1680 ttatgtgcca gatgcgccta ctgctcatat gcctgatgat aatcataaat ggctgtggaa 1740 ctaactagtt gattgcggag tcatgtatca gctacaggtg tagggactag ctacaggtgt 1800 agggacttgc gtctaattgt ttggtccttt actcatgttg caattatgca atttagttta 1860 gattgtttgt tccactcatc taggctgtaa aagggacact gcttagattg ctgtttaatc 1920 tttttagtag attatattat attggtaact tattacccct attacatgcc atacgtgact 1980 tctgctcatg cctgatgata atcatagatc actgtggaat taattagttg attgttgaat 2040 catgtttcat gtacatacca cggcacaatt gcttagttcc ttaacaaatg caaattttac 2100 tgatccatgt atgatttgcg tggttctcta atgtgaaata ctatagctac ttgttagtaa 2160 gaatcaggtt cgtatgctta atgctgtatg tgccttctgc tcatgcctga tgataatcat 2220 atatcactgg aattaattag ttgatcgttt aatcatatat caagtacata ccatgccaca 2280 atttttagtc acttaaccca tgcagattga actggtccct gcatgttttg ctaaattgtt 2340 ctattctgat tagaccatat atcatgtatt ttttttggt aatggttctc ttattttaaa 2400 tgctatatag ttctggtact tgttagaaag atctgcttca tagtttagtt gcctatccct 2460 cgaattagga tgctgagcag ctgatcctat agctttgttt catgtatcaa ttctttgtg 2520 ttcaacagtc agttttgtt agattcattg taacttatgg tcgcttactc ttctggtcct 2580 caatgcttgc agcctagtaa gccgccatgg ccccaaagaa gaagaggaag gtcggcatcc 2640 acggcgtccc agctgcgatg gacaagaagt actccatcgg cctcgacatc ggcaccaaca 2700 gcgtgggctg ggccgtcatc accgacgagt acaaggtgcc atccaagaag ttcaaggtcc 2760 tgggcaacac cgaccgccac agcatcaaga agaacctcat cggcgctctc ctgttcgact 2820 ccggcgagac ggctgaggct accaggctca agcgcaccgc caggaggagg tacaccagga 2880 ggaagaacag gatctgctac ctccaagaga tcttctccaa cgagatggcc aaggtggacg 2940 actccttctt ccaccgcctg gaggagagct tcctcgtcga ggaggacaag aagcacgaga 3000 ggcacccaat cttcggcaac atcgtggacg aggtcgccta ccacgagaag tacccaacca 3060 tctaccacct gaggaagaag ctcgtggact ccaccgacaa ggccgacctc cgcctgatct 3120 acctcgccct ggcccacatg atcaagttca ggggccactt cctgatcgag ggcgacctca 3180 acccagacaa cagcgacgtg gacaagctgt tcatccaact cgtccagacc tacaaccagc 3240 tcttcgagga gaacccgatc aacgcttccg gcgtggacgc taaggctatc ctgagcgcta 3300 ggctctccaa gagcaggagg ctcgagaacc tgatcgccca gctcccaggc gagaagaaga 3360 acggcctgtt cggcaacctc atcgctctct ccctgggcct caccccaaac ttcaagagca 3420 acttcgacct cgccgaggac gccaagctgc aactctccaa ggacacctac gacgacgacc 3480 tggacaacct cctggcccag atcggcgacc aatacgccga cctgttcctc gccgccaaga 3540 acctgtccga cgccatcctc ctgagcgaca tcctccgcgt gaacaccgag atcaccaagg 3600 ccccactctc cgccagcatg atcaagcgct acgacgagca ccaccaggac ctgaccctcc 3660 tgaaggccct ggtcaggcaa cagctcccag agaagtacaa ggagatcttc ttcgaccaga 3720 gcaagaacgg ctacgctggc tacatcgacg gcggcgcctc ccaagaggag ttctacaagt 3780 tcatcaagcc aatcctggag aagatggacg gcaccgagga gctcctggtg aagctcaaca 3840 gggaggacct cctgaggaag cagcgcacct tcgacaacgg cagcatccca caccaaatcc 3900 acctcggcga gctgcacgct atcctgagga ggcaagagga cttctaccca ttcctcaagg 3960 acaacaggga gaagatcgag aagatcctga ccttccgcat cccctactac gtcggcccac 4020 tcgctagggg caactccagg ttcgcttgga tgacccgcaa gagcgaggag acgatcaccc 4080 cgtggaactt cgaggaggtc gtcgacaagg gcgcttccgc tcagagcttc atcgagagga 4140 tgaccaactt cgacaagaac ctgccaaacg agaaggtgct cccaaagcac tccctcctgt 4200 acgagtactt caccgtctac aacgagctca ccaaggtgaa gtatgtgacc gagggcatgc 4260 gcaagccagc cttcctgagc ggcgagcaga agaaggccat cgtggacctc ctgttcaaga 4320 4380. ccacagga ggtgaccgtc aagcaactca aggaggacta cttcaagaag atcgagtgct tcgactccgt ggagatcagc ggcgtcgagg accgcttcaa cgcctccctc ggcacctacc 4440 acgacctcct gaagatcatc aaggacaagg acttcctgga caacgaggag aacgaggaca tcctcgagga catcgtgctg accctcaccc tgttcgagga cagggagatg atcgaggagc 4560 gcctgaagac ctacgcccac ctcttcgacg acaaggtcat gaagcaactc aagaggagga 4620 ggtacaccgg ctggggcagg ctgagccgca agctcatcaa cggcatccgc gacaagcagt 4740. ccggcaagac catcctcgac ttcctgaaga gcgacggctt cgccaacagg aacttcatgc aactgatcca cgacgactcc ctcaccttca aggaggacat ccaaaaggct caggtgtccg gccagggcga cagcctgcac gagcacatcg ctaacctcgc tggcagccca gccatcaaga agggcatcct gcagaccgtg aaggtcgtcg acgagctcgt gaaggtcatg ggcaggcaca agccagaga catcgtcatc gagatggccc gcgagacca gaccacccag aagggccaaa 5040. ggcagccag ggcagccaaa 5040. ggcagccaaa tcctcaagga gcacccagtg gagaacaccc aactgcagaa cgagaagctc tacctgtact 5100 acctccagaa cggcagggac atgtatgtgg accaagagct ggacatcaac cgcctctccg 5160 actacgacgt ggaccacatc gtcccacagt ccttcctgaa ggacgacagc atcgacaaca 5220 aggtgctcac caggagcgac aagaaccgcg gcaagtccga caacgtccca agcgaggagg 5280 tggtcaagaa gatgaaaaac tactggaggc agctcctgaa cgccaagctg atcacccaaa 5340 ggaagttcga caacctcacc aaggctgaga ggggcggcct ctccgagctg gacaaggccg 5400 gcttcattaa aaggcagctg gtggagacgc gccaaatcac caagcacgtc gcccaaatcc 5460 tcgacagccg catgaacacc aagtacgacg agaacgacaa gctgatcagg gaggtgaagg 5520 tcatcaccct gaagtccaag ctcgtgagcg acttcaggaa ggacttccag ttctacaagg 5580 tccgcgagat caataattac caccacgccc acgacgctta cctcaacgct gtggtcggca 5640 ccgccctgat taaaaagtac ccaaagctcg agtccgagtt cgtgtacggc gactacaagg 5700 tgtacgacgt ccgcaagatg atcgccaagt ccgagcaaga gatcggcaag gccaccgcca 5760 agtacttctt ctacagcaac atcatgaact tcttcaagac cgagatcacc ctggccaacg 5820 gcgagatcag gaagcgccca ctcatcgaga cgaacggcga gacgggcgag atcgtgtggg 5880 acaagggcag ggacttcgcc accgtgcgca aggtcctctc catgccacag gtgaacatcg 5940 tcaagaagac cgaggtccaa accggcggct tctccaagga gagcatcctg ccaaagagga 6000 acagcgacaa gctcatcgcc cgcaagaagg actgggatcc aaagaagtac ggcggcttcg 6060 actccccaac cgtggcctac agcgtcctcg tggtcgccaa ggtggagaag ggcaagtcca 6120 agaagctgaa gagcgtgaag gagctcgtcg gcatcaccat catggagagg tccagcttcg 6180 agaagaaccc agtggacttc ctcgaggcca agggctacaa ggaggtcaag aaggacctga 6240 tcattaaact cccaaagtac agcctcttcg agctggagaa cggcaggaag cgcatgctgg 6300 cttccgctgg cgagctccaa aagggcaacg agctcgccct gccatccaag tatgtgaact 6360 tcctctacct ggcctcccac tacgagaagc tcaagggcag cccagaggac aacgagcaaa 6420 agcagctgtt cgtcgagcag cacaagcact acctcgacga gatcatcgag caaatctccg 6480 agttcagcaa gcgcgtgatc ctcgccgacg ccaacctgga caagctcctc tccgcctaca 6540 acaagcacag ggacaagcca atccgcgagc aggccgagaa catcatccac ctctcaccc 6600 tgaccaacct cggcgctcca gctgccttca agtacttcga caccaccatc gaggaagc 6660 gctacaccctc caccaggag gtgctggacg ccacccat ccaccagtcc atcaccggcc 6720 tctacgagac gaggatcgac ctgagccac tcggcggcga ctccagccca ccaaagaga 6780 agaggaaggt cagctggaag gacgcttccg gctggagccg catgtgaggt acctcacatc 6840 gatcgacgac caggatatg attattatct atctagcttg tggtggtggt tgaacaataa 6900 taagcgaggc cgagctggct gccatacata ggtattgtgt ggtgtgtgtg agagagagag 6960 aaacagagtt cttcagtttg ctatctctct ctgcatgttt ggcgtcagtc ttgtgctca 7020 tgtacgtacg tgtgtctacc tgcatgttgg tgatccgat tgcatctgct gtaaccatat 7080 attaattggt ccacgatgat atgatttgat actatatata tactaaaacc ggactctta 7140 ttataatact tgtagtatat aagtttctta cgcccgcaat tgatcgattc agaaggtt 7200 ctagctagct aaaacatgca gattcagaat atcagatttt taggactact ggagatccag 7260 aaccttcgtg tccttgtacc cgtgattttg gatccccttt ctccccacta caatcgttgg 7320 cgcaatcttg ttctgctcgc ctagaatggt acgccctcac cgaccatgtc ctcgccgatg 7380 cctaccccgc cacgccatct ggatggtgca tgaatgtcgt cgttttgtcc tggatgctag 7440 gcacactctc ccccgagttg atggagcctc ctcgcacctc tggtggcact gcgccgcgcc 7500 tggcttgcca tcgaggagca attcctcggc aatcgcgaag ctcgcacact tcgtctcgac 7560 gccgagttcc atgtcttcat gtaggcgatc tcttcgtcag cgactattgt ctattgacgc 7620 aagatgaagg ggatggacga ggcccttggt gatcttggtg aggtcatcca tgaccgtacc 7680 cttgtcctaa acgtgttgtg tggtctgaat gagaggtttg cccacataaa ggtccacttc 7740 aagcactcga atccgttccc ctccttcacc gacgtttgta atgatctcat ccttgaggag 7800 atcgactcca gcgcgcctcc tccgcctccg accacctaat tagctaaggg acccgaccaa 7860 gcccgttatt ctgacagttc tggtgctcaa cacatttata tttatcaagg agcacattgt 7920 tactcactgc taggagggaa tcgaactagg aatattgatc agaggaacta cgagagagct 7980 gaagataact gccctctagc tctcactgat ctgggtcgca tagtgagatg cagcccacgt 8040 gagttcagca acggtctagc gctgggcttt taggcccgca tgatcgggct tttgtcgggt 8100 ggtcgacgtg ttcacgattg gggagagcaa cgcagcagtt cctcttagtt tagtcccacc 8160 tcgcctgtcc agcagagttc tgaccggttt ataaactcgc ttgctgcatc agacttgacg 8220 tcggcgacac cggtgcggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt 8280 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ttgaccaagc ccgttattct 8340 gacagttctg gtgctcaaca catttatatt tatcaaggag cacattgtta ctcactgcta 8400 ggagggaatc gaactaggaa tattgatcag aggaactacg agagagctga agataactgc 8460 cctctagctc tcactgatct gggtcgcata gtgagatgca gcccacgtga gttcagcaac 8520 ggtctagcgc tgggctttta ggcccgcatg atcgggcttt tgtcgggtgg tcgacgtgtt 8580 cacgattggg gagagcaacg cagcagttcc tcttagttta gtcccacctc gcctgtccag 8640 cagagttctg accggtttat aaactcgctt gctgcatcag acttgcttgt gggagcaggg 8700 gcaacgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 8760 aaagtggcac cgagtcggtg ctttttttt cctaggctaa ttagctaact gcagtgcagc 8820 gtgacccggt cgtgcccctc tctagagata atgagcattg catgtctaag ttataaaaaa 8880 ttaccacata ttttttgtgt cacacttgtt tgaagtgcag tttatctatc tttatacata 8940 tatttaaact ttactctacg aataatataa tctatagtac tacaataata tcagtgtttt 9000 agagaatcat ataaatgaac agttagacat ggtctaaagg acaattgagt atttgacaa 9060 caggactcta cagttttatc ttttagtgt gcatgtgttc tccttttttt ttgcaaatag 9120 cttcacctat ataatacttc atccatttta ttagtacatc catttagggt ttagggttaa 9180 tggtttttat agactaattt ttttagtaca tctattttat tctattttag cctctaaatt 9240 aagaaaacta aaactctatt ttagtttttt tatttaataa tttagatata aatagaata 9300 aaataaagtg actaaaaatt aaacaaatac cctttaagaa attaaaaaaa ctaaggaaac 9360 atttttcttg tttcgagtag ataatgccag cctgttaaac gccgtcgacg agtctaacgg 9420 acaccaacca gcgaaccagc agcgtcgcgt cgggccaagc gaagcagacg gcacggcatc 9480 tctgtcgctg cctctggacc cctctcgaga gttccgctcc accgttggac ttgctccgct 9540 gtcggcatcc agaaattgcg tggcggagcg gcagacgtga gccggcacgg caggcggcct 9600 cctcctcctc tcacggcacc ggcagctacg ggggattcct ttcccaccgc tccttcgctt 9660 tcccttcctc gcccgccgta ataaatagac accccctcca caccctcttt ccccaacctc 9720 gtgttgttcg gagcgcacac acacacaacc agatctcccc caaatccacc cgtcggcacc 9780 tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc tctctacctt ctctagatcg 9840 gcgttccggt ccatggttag ggcccggtag ttctacttct gttcatgttt gtgttagatc 9900 cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga 9960 cacgttctga ttgctaactt gccagtgttt ctctttgggg aatcctggga tggctctagc 10020 cgttccgcag acgggatcga tttcatgatt ttttttgttt cgttgcatag ggtttggttt 10080 gcccttttcc tttatttcaa tatatgccgt gcacttgttt gtcgggtcat cttttcatgc 10140 ttttttttgt cttggttgtg atgatgtggt ctggttgggc ggtcgttcta gatcggagta 10200 gaattctgtt tcaaactacc tggtggattt attaattttg gatctgtatg tgtgtgccat 10260 acatattcat agttacgaat tgaagatgat ggatggaaat atcgatctag gataggtata 10320 catgttgatg cgggttttac tgatgcatat acagagatgc tttttgttcg cttggttgtg 10380 atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga atactgtttc 10440 aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac atcttcatag 10500 ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt tgatgtgggt 10560 tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc taaccttgag 10620 tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg atatacttgg 10680 atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca tacgctattt 10740 atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt tacttctgca 10800 gtgactaaat agatgcagaa gctgatcaac agcgtgcaga actacgcctg gggcagcaag 10860 accgccctga ccgagctgta cggcatggag aaccccagca gccagcccat ggccgagctg 10920 tggatgggcg cccaccccaa gagctcaagc cgcgtgcaga acgccgccgg cgatatcgtt 10980 agcctgcgcg acgtgatcga gagcgacaag agcaccctgc tgggcgaggc cgtggccaag 11040 cgcttcggcg agctgccctt cctgttcaag gtgctgtgcg ccgctcagcc cctgagcatc 11100 caggtgcacc ctaacaagca caacagcgag atcggcttcg ccaaggagaa cgccgccggc 11160 atccccatgg acgccgccga gcgcaactac aaggacccca accacaagcc cgagctggtg 11220 ttcgccctga cccccttcct ggccatgaac gccttccgcg agttcagcga gatcgttagc 11280 ctgctgcagc ccgtggccgg cgcccacccc gctatcgccc acttccttca gcagcccgac 11340 gccgagcgcc tgagcgagct gttcgccagc ctgctgaaca tgcagggtga ggagaagtca 11400 cgcgccctgg ccatcctgaa gagcgccctg gacagccagc agggcgagcc ctggcagaca 11460 atccgcctga tcagcgagtt ctaccccgag gatagcggcc tgttcagccc cctgctgctg 11520 aacgtggtga agctgaaccc cggcgaggcc atgttcctgt tcgccgagac cccccacgcc 11580 tacctgcagg gcgtggccct ggaggtgatg gccaacagcg acaacgtgct gcgcgccggc 11640 ctgaccccca agtacatcga catccccgag ctggtggcca acgtgaagtt cgaggctaag 11700 cccgccaacc agctgctgac ccagcccgtg aagcagggcg ccgagctgga cttccctatc 11760 cccgttgacg acttcgcctt cagcctgcac gacctgagcg acaaggagac cactatcagc 11820 cagcagagcg ccgcgatcct gttctgcgtg gagggcgacg ccaccctgtg gaagggcagc 11880 cagcagctgc agctgaagcc cggcgagagc gcctttatcg ccgccaacga gagccccgtg 11940 accgtgaagg gccacggccg cctggcccgc gtgtacaaca agctgtgata gctacgtcat 12000 gggtcgttta agctgccgat gtgcctgcgt cgtctggtgc cctctctcca tatggaggtt 12060 gtcaaagtat ctgctgttcg tgtcatgagt cgtgtcagtg ttggtttaat aatggaccgg 12120 ttgtgttgtg tgtgcgtact acccagaact atgacaaatc atgaataagt ttgatgtttg 12180 aaattaaagc ctgtgctcat tatgttctgt ctttcagttg tctcctaata tttgcctgca 12240 ggtactggct atctaccgtt tcttacttag gaggtgtttg aatgcactaa aactaatagt 12300 tagtggctaa aattagttaa aacatccaaa caccatagct aatagttgaa ctattagcta 12360 tttttggaaa attagttaat agtgaggtag ttatttgtta gctagctaat tcaactaaca 12420 atttttagcc aactaacaat tagtttcagt gcattcaaac acccccttaa tgttaacgtg 12480 gttctatcta ccgtctccta atatatggtt gattgttcgg tttgttgcta tgctattggg 12540 ttctgattgc tgctagttct tgctgaatcc agaagttctc gtagtatagc tcagattcat 12600 attatttatt tgagtgataa gtgatccagg ttattactat gttagctagg ttttttttac 12660 aaggataaat tatctgtgat cataattctt atgaaagctt tatgtttcct ggaggcagtg 12720 gcatgcaatg catgacagca acttgatcac accagctgag gtagatacgg taacaaggtt 12780 cttaaatctg ttcaccaaat cattggagaa cacacataca cattcttgcc agtcttggtt 12840 agagaaattt catgacaaaa tgccaaagct gtcttgactc ttcactttg gccatgagtc 12900 gtgacttagt ttggtttaat ggaccggttc tcctagcttg ttctactcaa aactgttgtt 12960 gatgcgaata agttgtgatg gttgatctct ggattttgtt ttgctctcaa tagtggacga 13020 gattatag cttaagcctg caggcggacc gcctgcaggc ccggggcgc gccctaatta 13080 gctaacggcc aggatcgccg cgtgagcctt tagcaactag ctagattaat taacgcaatc 13140 tgttattaag ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccaccagcca 13200 gccaacagct ccccgaccgg cagctcggca caaaatcacc actcgataca ggcagcccat 13260 cagaattaat tctcatgttt gacagcttat catcgactgc acggtgcacc aatgcttctg 13320 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat 13380 13440 gttctggcaa atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg 13500 13560 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg 13620 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt 13680 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg 13740 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta 13800 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa 13860 ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg 13920 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta 13980 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta 14040 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta 14100 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat 14160 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa 14220 gctaggcagg cttatcttgg acaagagat cgcttggcct cgcgcgcaga tcagttgga 14280 gaatttgttc actacgtgaa aggcgagatc accaaagtag tcggcaaata aagctctagt ggatctccgt acccagggat ctggctcgcg gcggacgcac gacgccgggg cgagaccata 14400 ggcgatctcc taatcaata gtagctgtaa cctcgaagcg tttcacttgt aacaacgatt gagaattttt gtcataaaat tgaaatactt ggttcgcatt tttgtcatcc gcggtcagcc gcaattctga cgaactgccc atttagctgg agatgattgt acatccttca cgtgaaaatt tctcaagcgc tgtgaacaag ggttcagatt ttagttgaa aggtgagccg ttgaaacacg ttcttcttgt cgatgacgac gtcgctatgc ggcatcttat tattgaatac cttacgatcc acgccttcaa agtgaccgcg gtagccgaca gcacccagtt cacaagagta ctctcttccg cgacggtcga tgtcgtggtt gttgatctag atttaggtcg tgaagatggg ctcgagatcg ttcgtaatct ggcggcaaag tctgatattc caatcataat tatcagtggc gaccgccttg aggagacgga taaagttgtt gcactcgagc taggagcaag tgattttatc gctaagccgt 14940. tcagtatcag agagtttcta gcacgcattc gggttgcctt gcgcgtgcgc cccaacgttg 15000 tccgctccaa agaccgacgg tctttttgtt ttactgactg gacacttaat ctcaggcaac 15060 gtcgcttgat gtccgaagct ggcggtgagg tgaaacttac ggcaggtgag ttcaatcttc 15120 tcctcgcgtt tttagagaaa ccccgcgacg ttctatcgcg cgagcaactt ctcattgcca 15180 gtcgagtacg cgacgaggag gtttatgaca ggagtataga tgttctcatt ttgaggctgc 15240 gccgcaaact tgaggcagat ccgtcaagcc ctcaactgat aaaaacagca agaggtgccg 15300 gttatttctt tgacgcggac gtgcaggttt cgcacggggg gacgatggca gcctgagcca 15360 attcccagat ccccgaggaa tcggcgtgag cggtcgcaaa ccatccggcc cggtacaaat 15420 cggcgcggcg ctgggtgatg acctggtgga gaagttgaag gccgcgcagg ccgcccagcg 15480 gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg caagcggccg ctgatcgaat 15540 ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg tcgattagga agccgcccaa 15600 gggcgacgag caaccagatt ttttcgttcc gatgctctat gacgtgggca cccgcgatag 15660 tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag cgtgaccgac gagctggcga 15720 ggtgatccgc tacgagcttc cagacgggca cgtagaggtt tccgcagggc cggccggcat 15780 ggccagtgtg tgggattacg acctggtact gatggcggtt tcccatctaa ccgaatccat 15840 gaaccgatac cgggaaggga agggagacaa gcccggccgc gtgttccgtc cacacgttgc 15900 ggacgtactc aagttctgcc ggcgagccga tggcggaaag cagaaagacg acctggtaga 15960 aacctgcatt cggttaaaca ccacgcacgt tgccatgcag cgtacgaaga aggccaagaa 16020 cggccgcctg gtgacggtat ccgagggtga agccttgatt agccgctaca agatcgtaaa 16080 gagcgaaacc gggcggccgg agtacatcga gatcgagctg gctgattgga tgtaccgcga 16140 gatcacagaa ggcaagaacc cggacgtgct gacggttcac cccgattact ttttgatcga 16200 tcccggcatc ggccgttttc tctaccgcct ggcacgccgc gccgcaggca aggcagaagc 16260 cagatggttg ttcaagacga tctacgaacg cagtggcagc gccggagagt tcaagaagtt 16320 ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg ccggagtacg atttgaagga 16380 ggagggcgggg caggctggcc cgatcctagt catgcgctac cgcaacctga tcgagggcga 16440 agcatccgcc ggttcctaat gtacggagca gatgctaggg caaattgccc tagcagggga 16500 aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac attgggaacc caaagccgta 16560 cattgggaac cggaacccgt acattgggaa cccaaagccg tacattggga accggtcaca 16620 catgtaagtg actgatataa aagagaaaaa aggcgatttt tccgcctaaa actctttaaa 16680 acttattaaa actcttaaaa cccgcctggc ctgtgcataa ctgtctggcc agcgcacagc 16740 cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc tccctacgcc ccgccgcttc 16800 gcgtcggcct atcgcggccg ctggccgctc aaaaatggct ggcctacggc caggcaatct 16860 accagggcgc ggacaagccg cgccgtcgcc actcgaccgc cggcgctgag gtctgcctcg 16920 tgaagaaggt gttgctgact cataccaggc ctgaatcgcc ccatcatcca gccagaaagt 16980 gagggagcca cggttgatga gagctttgtt gtaggtggac cagttggtga ttttgaactt 17040 ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc 17100 agcaaaagtt cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc 17160 cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac 17220 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat 17280 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg 17340 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta 17400 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagctctgca 17460 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 17520 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 17580 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 17640 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 17700 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 17760 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 17820 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 17880 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 17940 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 18000 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 18060 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 18120 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 18180 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 18240 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 18300 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 18360 atcaaaaagg atcttcacct agatcctttt gatccggaat ta 18402 <210> 76 <211> 14 <212> PRT <213> Common wheat <400> 76 Arg Ala Gly Arg Ala Ala Ala Pro Gly Gly Ala Gln Gly Ala 1 5 10 <210> 77 <211> 47 <212> PRT <213> Common wheat <400> 77 Val Ala Arg Asp Leu Pro Gly Ser Leu Pro Phe Arg Phe Val Leu Phe 1 5 10 15 Ser Val Phe Trp Ser Asp Leu Leu Val Thr Cys Ser Thr Glu Cys Arg 20 25 30 Gly Glu Pro Gly Gly Arg Arg Pro Gln Gly Gly Leu Lys Gly Gln 35 40 45 <210> 78 <211> 44 <212> PRT <213> Common wheat <400> 78 Gly Thr Phe Pro Gly Arg Phe Leu Phe Val Ser Ser Cys Phe Leu Phe 1 5 10 15 Phe Gly Leu Thr Cys Ser Ser Pro Val Arg Arg Asn Ala Glu Ala Ser 20 25 30 Arg Ala Gly Gly Gly Pro Arg Gly Gly Ser Arg Gly 35 40 <210> 79 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 79 cgactcgcct cgcgctagta 20

Claims

1. A method for producing haploid induced wheat plants, the method comprising: a. obtaining at least a wheat plant cell comprising at least three genomes, wherein the three genomes include an A genome, a B genome, and a D genome; b. mutating two of the three genomes to obtain a homozygous knockout mutation of the CENH3α gene, wherein the CENH3α gene knockout mutation occurs in the B and D genomes; c. mutating a third genome to obtain a homozygous knockdown mutation of the CENH3α gene at the 5' splice site of the intron, wherein the CENH3α gene knockdown mutation occurs in the A genome, and wherein the knockdown mutation results in an altered N-terminal domain paired with a functional C-terminal domain; and d. producing therefrom a wheat plant comprising a homozygous knockout mutation in the CENH3α gene of two of the three genomes and further comprising a homozygous knockdown mutation in the CENH3α gene of the third genome; The wheat plant produced by step (d) thereby produces haploid offspring when crossed with a wild-type wheat plant.

2. The method of claim 1, wherein the CENH3α gene knockdown mutation is a repaired frame shift mutation.

3. The method of claim 2, wherein the repaired frame shift mutation is selected from the group consisting of: SEQ ID NO:56, SEQ ID NO:63 and SEQ ID NO:

69.

4. A method for producing a haploid wheat plant, comprising: a. obtaining wheat plants; b. crossing the wheat plant with a second wheat plant comprising a homozygous knockout mutation of the CENH3α gene in the B and D genomes and a knockdown mutation of the CENH3α gene at the 5' splice site of the intron in the A genome, wherein the A genome comprises an altered N-terminal domain paired with a functional C-terminal domain; and c. selecting offspring produced from said hybridization step; wherein the progeny are haploid wheat plants.

5. The method of claim 4, wherein the wheat plant of step (a) is the male parent.

6. The method of claim 4, wherein the wheat plant of step (a) is a female parent.

7. The method of claim 4, further comprising converting the progeny wheat plant into a doubled haploid wheat plant.

8. The method of claim 4, wherein the mutated CENH3α allele comprises a sequence selected from the group consisting of SEQ ID NO:56, SEQ ID NO:63, and SEQ ID NO:69.

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