Cereal plants and methods of producing same

Abstract

A cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of a wild-cultivar of a cereal, the plasmotype imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to the domesticated cultivar devoid of the plasmotype, the at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor. Also provided are such plants which are obtained by chloroplast editing and not plasmotype transfer.

Description

[0001] CEREAL PLANTS AND METHODS OF PRODUCING SAME

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Patent Application No. 63 / 753,519 filed February 4, 2025, the contents of which are incorporated herein by reference in their entirety.

[0004]

[0005] The XML file, entitled 105349. xml, created on 3 February 2026, comprising 40,960 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to cereal plants and methods of producing same.

[0008] Breeding for new traits to improve crops requires an initial event of diversity burst followed by rounds of selection for phenotype of interest, where grain yield and related attributes such as size and quality that we consider to be more challenging to improve due to tradeoffs between yield components, e.g. number and size. Wild species are considered a significant source for crop improvement, yet the focus in incorporating such wild alleles is almost wholly relying on variation from the nuclear genome, and the contribution of the plasmotype (chloroplast and mitochondria) in controlling plant performance has rarely been elucidated or utilized for developing new varieties. The inheritance and effects of the cytoplasm on plant fitness and agronomic traits have been known since the beginning of the 20th century, after discoveries by the German botanists Carl Correns and Edwin Baur (Hagemann, 2000). Probably the most prominent example is cytoplasmic male sterility (CMS), which is heavily used for hybrid seed production. Disease resistance has also been shown to be influenced by the genotype of the cytoplasm, the plasmotype. Moreover, especially in grasses the contribution of the plasmotype to yield and grain quality has been demonstrated (Frei et al., 2003; Sanetomo and Gebhardt, 2015). These traits are likely the result of a local adaptation of the original wild alleles, since, for example, in bread wheat (Triticum aestivum), cytoplasmic influence on fruit quality is influenced by genotype-by-environment interactions (Ekiz et al., 1998). Nevertheless, many of these examinations of alloplasmic lines, which contained cytoplasm from distantly related wild relatives, showed no frequent effects on agronomic traits besides grain quality (Frei et al., 2010). One proposed explanation for this is the need for proper cytonuclear interactions and homogeneity of the cytoplasm. For example, in somatic potato hybrids evaluations of the correlation between the cytoplasmic types showed that an interaction between starch content and mt-pt combinations exists(Edssl et al., 2000). In general, the highest field performances were associated with those cytoplasmic configurations that appeared at a high frequency, i.e. better homogeneity of the organelle genomes.

[0009] Additional Related Background Art:

[0010] Hubner et al. 2009 [Mol Ecol 18(7): 1523-36) teaches SSR markers for genetic make -p of nuclear genomes and the correlation between genetic distance with aridity and temperature gradients in wild barley (Hordeum vulgare ssp. spontaneum)}

[0011] Fridman et al. (Authorea, February 17, 2023) teach the effect of variation in the RpoCl gene (Bdolach et al., 2019) on photosynthetic rhythm plasticity by heterologous expression of two barley alleles in the model plant Arabidopsis.

[0012] Tiwari et al. Physiologia Plantarum. 2024;176:el4192.

[0013] Bodenheimer, 2025 www(dot)doi.org / 10.1101 / 2025.04.08.647843

[0014] SUMMARY OF THE INVENTION

[0015] According to an aspect of some embodiments of the present invention there is provided a cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of a wild-cultivar of a cereal, the plasmotype imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to the domesticated cultivar devoid of the plasmotype, the at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor.

[0016] According to an aspect of some embodiments of the present invention there is provided a cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of the domesticated cultivar, the plasmotype comprising a sequence variation in RpoCl as compared to that of the domesticated cultivar, the sequence variation imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to the domesticated cultivar devoid of the sequence variation, the at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor.

[0017] According to some embodiments of the invention, the grain yield is manifested by thousand grain weight (TGW) or kernel weight per growth area.

[0018] According to some embodiments of the invention, the improvement in at least one agriculturally desired trait is stable across different abiotic conditions including drought and heat and exhibits reduced variance (value distribution) in the trait as compared to the variance in domesticated cultivar of cereal devoid of the plasmotype or sequence variation.

[0019] According to some embodiments of the invention, the plant is barley.According to some embodiments of the invention, the plasmotype comprises a sequence variation in RpoCl as compared to that of the domesticated cultivar.

[0020] According to some embodiments of the invention, the sequence variation comprises an N to K mutation at a position corresponding to position 571 of SEQ ID NO: 2.

[0021] According to some embodiments of the invention, the domesticated genome and the plasmotype are of the same species.

[0022] According to some embodiments of the invention, the domesticated genome and the plasmotype are of different species.

[0023] According to some embodiments of the invention, the plant is transgenic.

[0024] According to some embodiments of the invention, the plant is non-transgenic.

[0025] According to some embodiments of the invention, the plasmotype is of BK1-50-04.

[0026] According to an aspect of some embodiments of the present invention there is provided a part of the plant as described herein.

[0027] According to an aspect of some embodiments of the present invention there is provided a method of producing the plant as described herein, the method comprising:

[0028] (a) crossing a domesticated male cereal plant with a wild female cereal plant to obtain a hybrid, the female cereal plant comprising a plasmotype which comprises a sequence variation capable of increasing at least one agriculturally desired trait as compared to the domesticated male cereal plant devoid of the same plasmotype, the at least one agriculturally desired trait being selected from the group consisting of grain yield and / or germination vigor; and

[0029] (b) back-crossing the hybrid to the male domesticated cereal plant to obtain a back-cross; and optionally repeating step (b) where each time a resultant back-cross is crossed with the male domesticated cereal plant.

[0030] According to some embodiments of the invention, the method further comprises selecting a progeny plant of the back-crossing by identifying a sequence variation in RpoCl as compared to that of the domesticated cultivar, wherein the sequence variation is indicative that the progeny is characterized by the improvement of the at least one agriculturally desired trait.

[0031] According to an aspect of some embodiments of the present invention there is provided a method of producing the plant as described herein comprising subjecting a chloroplast of the cereal plant to a gene editing agent to introduce the sequence variation in RpoCl, thereby producing the plant.

[0032] According to some embodiments of the invention, the genome editing agent is selected from the group consisting of CRISPR-Cas, TALEN, and a Zinc Finger Nuclease (ZFN).According to an aspect of some embodiments of the present invention there is provided a food, feed or beverage or any other article of manufacture comprising the plant or part thereof as described herein.

[0033] According to some embodiments of the invention, the food, feed, beverage or article of manufacture comprises a genome of the plasmotype and / or the nuclear genome.

[0034] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0035] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0036] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0037] In the drawings:

[0038] FIGs. 1A-C show build and field trials of the barley cytonuclear multiparent population (CMPP): A) shows the breeding scheme leading to 10 biparental BC2DH segregating for plasmotype and nucleotype genomes. B) shows locations for the field trails during 2022 (Rehovot) and 2023 (Mevo Hama, Yizram, Yotvata). C) shows the means and distribution of the days to flowering (DTF) for the whole CMPP in the three locations, exemplifying the major effects of the environment on plant phenology.

[0039] FIGs. 2A-D show that the wild plasmotype of B1K-50-04 causes plethora of effects related to grain yield and qualities. A) Comparison between carriers of the wild (W) vs cultivated (C) plasmotypes at the CMP33 and CMP50 biparental populations, showing no effects in the former and an +8.5% increase in the latter. B) Effect of the B 1K50 wild plasmotype on stability [index based on (Shukla, 1972)] where lower level refers to increased stability across the different environments. C) Effect of the B1K50 wild plasmotype on kernel weight (KW), with a significant increase of +14.5% for carriers of the wild vs cultivated cytoplasm. D) Effect of the B 1K50 wild plasmotype on reducing the noise in germination, i.e., -57% in the CV between germination vigor of carriers for the wild vs cultivated plasmotype.FIG. 3 shows advanced reciprocal backcrosses between carriers of B 1K50 and elite cultivated (cv. Magal) plasmotypes, grown at different locations. The graphs shows that B1K plasmotypic background (left) mitigates environment-related decrease (right) in average TGW.

[0040] FIG. 4 shows multiple sequence alignment of the RpoCl gene with a highlight in the N to K 571 variation which is present in B1K-50 but not at the corresponding position of B1K-09 or other cereals shown.

[0041] Blk09_rpoCl (SEQ ID NO: 3).

[0042] B1K-50 (SEQ ID NO: 1)

[0043] AKK66_gp090 (SEQ ID NO: 5)

[0044] ZemaCpO14 (SEQ ID NO: 7)

[0045] TraeCpO13 (SEQ ID NO: 9)

[0046] RS19_pO77 (SEQ ID NO: 11)

[0047] SpboiCp013 (SEQ ID NO: 13)

[0048] BrdiC_pO16 (SEQ ID NO: 15)

[0049] FIG. 5 is a photograph depicting the juxtaposition of 10 grains from parents used for the Magal Reciprocal Validation Panel (MRVP). Scale bar equals 1 cm.

[0050] FIGs. 6A-B are graphs showing how variance is explained by Cytoplasm and Cytoplasm x Environment, derived from two-way ANOVA. (Figure 6A) Significant terms within CMPP Validation panel (CMPPV) populations, and (Figure 6B) MRVP populations. Blue bars show the percent change between the Wild cytoplasm and its cultivated counterpart for each trait.

[0051] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0052] The present invention, in some embodiments thereof, relates to cereal plants and methods of producing same.

[0053] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0054] The present invention relates to the introduction of a cytoplasm responsible for high thousand grain weight and total grains from wild barley into cultivated barley plants. More particularly, the present invention relates to cereal plant lines in which the introduction of a specific variation at the chloroplastic RPOC1 gene into a cultivated genetic background resulted in a general cultivated phenotype having a stable grain yield phenotype over diverse environments.The cultivated barley plant according to the present invention is characterized by grains having a weight higher than that of a nearly isogenic barley plant carrying the cultivated cytoplasm, and across different environments, while at the same time having a general phenotype of said nearly isogenic barley plant carrying the cultivated cytoplasm.

[0055] In addition, the cultivated barley plant according to the present invention is characterized by grains having a more uniform level of germination than that of a nearly isogenic barley plant carrying the cultivated cytoplasm.

[0056] The cultivated barley plant according to the present invention is characterized by grains having a more uniform level of germination than that of a nearly isogenic barley plant carrying the cultivated cytoplasm.

[0057] The cultivated barley or wheat plants carrying the RPOC1571Kaccording to the present invention is characterized by higher grain weight across different environments and with grains having a more uniform level of germination than that of a nearly isogenic plant carrying the RPOC1571N

[0058] Specifically, as shown in the Examples section which follows, present inventors proved that introducing the wild Mt. Hermon (B1K-50-04) plasmotype to an elite background increases the stability (phenotype across different environments, ie Rehovot, Mevo Hama, Negev, and Yotveta selected to identify the different environments) and the grain weight in a stand of 2 rows of 20 seeds per plot. This stand is equivalent to the recommended commercial stand in Israel (150 seeds / m2). Based on integrating molecular work on RpoCl with the field trials of the cytonuclear multiparent population (CMPP) (Figures 1A-C and 2A-D), it is conceivable that variation at the RpoCl underlie increases in grain weights and its stability across different commercial fields.

[0059] Thus, according to an aspect of the invention, there is provided a cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of a wild-cultivar of a cereal, the plasmotype imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to the domesticated cultivar devoid of the plasmotype, the at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor.

[0060] According to an alternative aspect there is provided a cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of the domesticated cultivar, the plasmotype comprising a sequence variation in RpoCl as compared to that of the domesticated cultivar, the sequence variation imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to the domesticated cultivar devoid of the sequence variation,the at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor.

[0061] As used herein, the term "plant" refers to an entire plant, its organs (i.e., leaves, stems, roots, flowers etc.), seeds, plant cells, and progeny of the same. The term "plant cell" includes without limitation cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores. According to a specific embodiment, the domesticated plant is a plant line.

[0062] According to a specific embodiment, the domesticated plant is an elite line.

[0063] The phrase "plant part" refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calli, and the like. According to a specific embodiment, the plant part comprises the nucleic acid variation in RpoCl as described below. According to a specific embodiment, the plant part is a seed.

[0064] “Wild” may be considered also as a “donor plant”.

[0065] “Domesticated” or “cultivated” or “line” may be considered also as a “recipient plant”.

[0066] According to a specific embodiment, the donor plant and the recipient plant are from the same species (e.g., barley / barley).

[0067] According to a specific embodiment, the donor plant and the recipient plant are from different species (e.g., barley / corn).

[0068] According to a specific embodiment, the donor plant and the recipient plant are from the nonidentical cultivars (e.g., domesticated / wild).

[0069] As used herein, the phrases "progeny plant" refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof.

[0070] As used herein the term “Gramineae plant” or “Cereal” refers to the cereal grass family, which cultivated species include but are not limited to maize (corn), wheat, rice, barley, rye, sorghum or millet.

[0071] According to a specific embodiment, the cereal plant is barley.

[0072] As used herein the term “barley” refers to a cultivated Hordeum plant.

[0073] As used herein the term “cultivated Hordeum plant” refers to a cultivated grass barley species having a diploid genome, 2n = 14 (HH genome). Examples of domesticated Hordeum species include, but are not limited to, Hordeum vulgare (cultivated barley). The term may be interchanged with the term barley.Domesticated Hordeum varieties contemplated herein according to exemplary embodiments refer to hulled or hull-less (naked) barley, as well as two-row and six-row barley types, and may include feed, malting, and food barley.

[0074] There are two main spike types of Hordeum vulgar e

[0075] Two-row barley (H. vulgare var. vulgare): Produces a single fertile spikelet at each node, commonly used for malting due to more uniform grain size.

[0076] Six-row barley (H. vulgare var. vulgare): Produces three fertile spikelets at each node, typically higher yielding and commonly used for animal feed and certain industrial uses.

[0077] As used herein the term “wheat” is also interchangeably referred to as “Triticum L.” or “Triticum subsp.”.

[0078] As used herein the term “common wheat” is also interchangeably referred to as “Bread wheat” or “Triticum aestivum”.

[0079] As used herein the term “durum wheat” is also interchangeably referred to as “Macaroni wheat” or “Triticum durum Desf.” or “Triticum turgidum subsp. durum”.

[0080] Wheat is conventionally grown for human or animal food or beverages or as a source of raw materials, food supplements, chemicals or fuel. The common wheat plant is allohexaploid (6N=42) in nature, whereas the durum wheat is a tetrapioid (4N=28).

[0081] As used herein the term “rice” refers to a cultivated Oryza plant.

[0082] As used herein the term “cultivated Oryza plant” refers to a cultivated grass rice species having a diploid genome, 2n = 24 (AA genome). Examples of domesticated Oryza species include but are not limited to, Oryza sativa (Asian rice) or Oryza glaberrima (African rice). The term may be interchanged with the term rice.

[0083] Domesticated Oryza varieties contemplated herein according to exemplary embodiments refer to long grain, short grain, white, brown, red and black.

[0084] There are three main varieties of Oryza sativa'.

[0085] Indica: The indica variety is long-grained.

[0086] Japonica: Japonica rice is short-grained and high in amylopectin (thus becoming "sticky" when cooked), and is grown mainly in more temperate or colder regions such as Japan.

[0087] Javanica: Javanica rice is broad-grained and grown in tropical climates.

[0088] Other major varieties include Aromatic and Glutinos.

[0089] As used herein, the term "genome" refers to the complete set of genetic material of an organism or organelle. The genome may be nuclear or extranuclear, including organelle genomes such as the chloroplast genome (plastome) and the mitochondrial genome (mitogenome).In some embodiments, the nuclear genome refers to the chromosomal DNA contained within the nucleus of a cell, inherited biparentally in sexually reproducing plants.

[0090] The plasmotype may include the chloroplast genome, the mitochondrial genome, or both, which are generally maternally inherited in most cereal species.

[0091] As used herein, the term “plasmotype” refers to at least chloroplasts and may include also mitochondria.

[0092] As used herein, the term “improvement” or “increase” refers to at least 5 %, 7 %, 10 %, 12 %, 15 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 1.5 fold, 2 fold or more as compared to a control of the same genetic background without the plasmotype sequence variation (i.e., control plant e.g., the recipient plant).

[0093] As used herein, the term “grain yield” refers to the measure of the yield of grains per unit area of land cultivation.

[0094] Grain yield can be measured in pounds per acre, typically at 10-12% for proper germination and storage. Grain yield of barley can be determined by the number of spikes per unit area, the number of kernels per spike, the number of kernels per unit area, kernel weight per spike and / or the thousand grain weight (TGW), which is the weight of 1,000 grains.

[0095] As used herein “germination vigor” refers to the population or batch of seeds inherent ability to initiate and complete the process of germination in a uniform manner, under optimal or suboptimal environmental conditions, as can be determined by the skilled artisan. For barley seeds, according to some embodiments, of the invention, it is a measure of the seed's physiological potential to undergo early-stage metabolic activation, resulting in the timely emergence of healthy seedlings that exhibit strong, uniform growth. Germination vigor is typically quantified through parameters such as germination rate, seedling growth rate, and the percentage of seeds that successfully develop into viable plants within a specified time under controlled conditions.

[0096] As used herein “RpoCl” refers to a subunit of the RNA polymerase enzyme, which is responsible for synthesizing RNA from a DNA template in the process of transcription. Specifically, RpoCl is part of the core RNA polymerase enzyme in plants, including barley. It codes for the beta prime (P') subunit of the RNA polymerase complex, which plays a crucial role in the elongation process during transcription. Figure 4 shows exemplary RpoCl genes in different cereals. The genes are conserved and the position of the N571K variation is easily identified in the other homologs.

[0097] According to a specific embodiment, the RpoCl is that of Mt. Hermon B1K-50-04.

[0098] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognizedthat residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a nonconservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9],

[0099] Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

[0100] Hence according to a specific embodiment, the variation can arise from different cultivars of the species (e.g., indica to japonica) or different species.

[0101] According to some embodiments of the invention, the identity is a global identity,

[0102]

[0103] an identity over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof.

[0104] As mentioned, this variation that may be of a single base to several hundreds or thousands of nucleotides e.g., 1-200 nucleotides confers the enhanced grain yield.

[0105] As used herein “haplotype” relates to a set of SNP alleles that tend to always occur together (i.e., that are associated statistically).

[0106] Also provided is a method of producing the plant as described herein, the method comprising: (a) crossing a domesticated male cereal plant with a wild female cereal plant to obtain a hybrid, said female cereal plant comprising a plasmotype which comprises a sequence variation capable of increasing at least one agriculturally desired trait as compared to the domesticated male cereal plant devoid of said plasmotype, said at least one agriculturally desired trait being selected from the group consisting of grain yield and / or germination vigor; and(b) back-crossing said hybrid to said male domesticated cereal plant to obtain a back-cross; and optionally repeating step (b) where each time a resultant back-cross is crossed with said male domesticated cereal plant.

[0107] According to a specific embodiment, the method further comprises selecting a progeny plant of said back-crossing by identifying a sequence variation in RpoCl as compared to that of said domesticated line, wherein said sequence variation is indicative that said progeny is characterized by said improvement of said at least one agriculturally desired trait.

[0108] As mentioned, once progeny plants are selected for the trait, the progeny may be crossed to the recurrent parent (it is 'crossed back' to the recurrent parent, hence the term backcross). The progeny of this cross is selected for the trait of interest and then crossed back to the recurrent parent. This process is repeated for as many backcrosses as are needed to create a line that is the recurrent parent with the sequence variation from the donor parent. The goal of backcrossing is to obtain a line as identical as possible to the recurrent parent with the addition of the sequence variation that has been added through breeding.

[0109] In some embodiments, the selection of progeny following each backcross generation includes molecular genotyping for polymorphisms in the RpoCl gene, which encodes a subunit of the plastid-encoded RNA polymerase complex. The identified variation in RpoCl, particularly when located within a coding or regulatory region, may serve as a reliable cytoplasmic marker to confirm the presence of donor-derived plastid genomes. As plastid genes are maternally inherited in most Gramineae, the RpoCl variant provides a stable and specific genetic signal that facilitates cytoplasmic tracking during successive rounds of backcrossing. In addition to serving as a marker for cytoplasmic inheritance, such sequence variation may be functionally linked to phenotypic traits of interest, such as increased grain weight, improved yield stability, or enhanced germination vigor under abiotic stress.

[0110] Numerous ways are known in the art for crossing Gramineae plants or species thereof.

[0111] The term “crossed” or “cross” in the context of this invention means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule are from the same plant or from genetically identical plants).

[0112] “Backcrossing” is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, crossing a first generation hybrid Fi with one of the parental genotypes of the Fi hybrid. The parent to which the hybrid is backcrossed is the “recurrent parent.”.

[0113] Specific primers for identification of sequence variations in the RpoCl gene include the forward primer 10A3 (5’- tttctgttggtgctgatattgcATGCTTATCGGGCTTTAT -3’- SEQ ID NO: 17)as well as the reverse primer 10A4 (5’- acttgcctgtcgctctatcttcCCTGTTAGTGTTCTAAGT -3’ - SEQ ID NO: 18), with the upper-case nucleotides representing the target- specific, and the lower case nucleotides representing the adapter- specific sequence used for barcoding and amplicon sequencing as described in Tiwari et al. 2024.

[0114] The nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence (i.e. the RpoCl gene). Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of a variation in the RpoCl gene. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are capable of specifically hybridizing to one another if the two molecules are capable of forming an antiparallel, double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit "complete complementarity" when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be "minimally complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency" conditions. Similarly, the molecules are said to be "complementary" if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high- stringency" conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985), Departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular solvent and salt concentrations employed.

[0115] Regarding the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, "stringent conditions" are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wildtype sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.

[0116] For example, to determine whether a barley plant resulting from a sexual cross contains the RpoCl variation, DNA extracted from the plasmotype of the barley sample may be subjected to nucleic acid amplification method using a primer pair that includes a primer derived from flanking sequence in the genome of the plant adjacent to the insertion site of inserted heterologous DNA, anda second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the RpoCl variation. The amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair, preferably plus about 5 nucleotide base pairs, more preferably plus about 10 nucleotide base pairs, and even more preferably plus about 20 nucleotide base pairs. Alternatively, a primer pair can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucleotide sequence. A member of a primer pair derived from the plant genomic sequence may be located a distance from the inserted DNA molecule, this distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.

[0117] Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the present invention.

[0118] The amplicon produced by these methods may be detected by a plurality of techniques. One such method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate. Following PCR of the region of interest (using one primer in the inserted sequence and one in the adjacent flanking genomic sequence), a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert / flanking sequence due to successful amplification, hybridization, and single base extension.

[0119] Another method is the pyro sequencing technique as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotide is designed that overlaps the adjacent genomic DNA and insert DNA junction. The oligonucleotide is hybridized to single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin. dNTP's are added individually and theincorporation results in a light signal which is measured. A light signal indicates the presence of the RpoCl variation due to successful amplification, hybridization, and single or multi-base extension.

[0120] Fluorescence polarization as described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect the amplicon of the present invention. Using this method an oligonucleotide is designed which overlaps the genomic flanking and inserted DNA junction. The oligonucleotide is hybridized to single-stranded PCR product from the region of interest (one primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change in polarization using a fluorimeter. A change in polarization indicates the presence of the RpoCl variation due to successful amplification, hybridization, and single base extension.

[0121] Taqman®. (PE Applied Biosystems, Foster City, Calif.) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is designed which overlaps the genomic flanking and insert DNA junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the RpoCl variation due to successful amplification and hybridization.

[0122] Molecular Beacons have been described for use in sequence detection as described in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties that results in the production of a fluorescent signal. The fluorescent signal indicates the presence of the RpoCl variation due to successful amplification and hybridization.

[0123] Other described methods, such as, microfluidics (US Patent pub. 2006068398, U.S. Pat. No.

[0124] 6,544,734) provide methods and devices to separate and amplify DNA samples. Optical dyes used to detect and quantitate specific DNA molecules (WO / 05017181). Nanotube devices (WO / 06024023) that comprise an electronic sensor for the detection of DNA molecules or nanobeads that bind specific DNA molecules and can then be detected.As mentioned, the plasmotype of the donor cultivar typically comprises a sequence variation in RpoCl which imparts the cereal plant with an improvement in at least one agriculturally desired trait.

[0125] The sequence variation in the donor plasmotype may be naturally present (e.g. in a wildcultivar).

[0126] The present invention further contemplates genetically modified plants in which sequence variations are artificially introduced into the plasmotype of plants - e.g. by genome editing. Plants generated accordingly are typically transgenic plants.

[0127] Methods of performing gene editing in chloroplast (and not in the nuclear genome) are well known in the art, see e.g., US20240218384, which is hereby incorporated by reference in its entirety.

[0128] Following is a non-limiting description of genome editing technologies which can be used to upregulate expression according to some embodiments of the invention.

[0129] Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double-stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDS) and non-homologous end-joining (NHEJF). NHEJF directly joins the DNA ends in a double-stranded break, while HDR utilizes a homologous donor sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a donor DNA repair template containing the desired sequence must be present during HDR.

[0130] Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and these sequences often will be found in many locations across the genome resulting in multiple cuts which are not limited to a desired location. To overcome this challenge and create site-specific single- or double-stranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TAEENs) and CRISPR / Cas system.

[0131] Meganucleases - Meganucleases are commonly grouped into four families: the LAGEIDADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGEIDADG family are characterized by having either one or two copies of the conserved EAGLIDADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly inmicrobial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location.

[0132] This can be exploited to make site-specific double-stranded breaks in genome editing. One of skill in the art can use these naturally occurring meganucleases, however the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize a new sequence.

[0133] Alternatively, DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., US Patent 8,021,867). Meganucleases can be designed using the methods described in e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent Nos. 8,304,222; 8,021,867; 8, 119,381; 8, 124,369; 8, 129,134; 8,133,697; 8,143,015; 8,143,016; 8, 148,098; or 8, 163,514, the contents of each are incorporated herein by reference in their entirety. Alternatively, meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0134] ZFNs and TALENs - Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven to be effective at producing targeted double-stranded breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

[0135] Basically, ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the double- stranded break.

[0136] Thus, for example to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. Upon transient expression in cells, the nucleases bind to their target sites and the Fokl domainsheterodimerize to create a double- stranded break. Repair of these double-stranded breaks through the non-homologous end-joining (NHEJ) pathway often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site.

[0137] The deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have been successfully generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). In addition, when a fragment of DNA with homology to the targeted region is introduced in conjunction with the nuclease pair, the double-stranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011; Miller et al., 2010; Umov et al., 2005).

[0138] Although the nuclease portions of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers are typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Approaches for making site-specific zinc finger endonucleases include, e.g., modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low- stringency selection of peptide domains vs. triplet nucleotides followed by high- stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

[0139] Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53. A recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org). TALEN can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

[0140] T-GEE system (TargetGene's Genome Editing Engine) - A programmable nucleoprotein molecular complex containing a polypeptide moiety and a specificity conferring nucleic acid (SCNA) which assembles in-vivo, in a target cell, and is capable of interacting with the predetermined targetnucleic acid sequence is provided. The programmable nucleoprotein molecular complex is capable of specifically modifying and / or editing a target site within the target nucleic acid sequence and / or modifying the function of the target nucleic acid sequence. Nucleoprotein composition comprises (a) polynucleotide molecule encoding a chimeric polypeptide and comprising (i) a functional domain capable of modifying the target site, and (ii) a linking domain that is capable of interacting with a specificity conferring nucleic acid, and (b) specificity conferring nucleic acid (SCNA) comprising (i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site, and (ii) a recognition region capable of specifically attaching to the linking domain of the polypeptide. The composition enables modifying a predetermined nucleic acid sequence target precisely, reliably and cost-effectively with high specificity and binding capabilities of molecular complex to the target nucleic acid through base-pairing of specificity-conferring nucleic acid and a target nucleic acid. The composition is less genotoxic, modular in their assembly, utilize single platform without customization, practical for independent use outside of specialized core-facilities, and has shorter development time frame and reduced costs.

[0141] CRISPR-Cas system (also referred to herein as “CRISPR”)- Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) nucleotide sequences that produce RNA components and CRISPR associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to the DNA of specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen. Studies of the type II CRISPR / Cas system of Streptococcus pyogenes have shown that three components form an RNA / protein complex and together are sufficient for sequence-specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821.).

[0142] It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro. It was also demonstrated that transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double-stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a, b; Jinek et al., 2013; Mali et al., 2013).

[0143] The CRIPSR / Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas9.The gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA / Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA / Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break. Just as with ZFNs and TALENs, the doublestranded breaks produced by CRISPR / Cas can undergo homologous recombination or NHEJ and are susceptible to specific sequence modification during DNA repair.

[0144] The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.

[0145] A significant advantage of CRISPR / Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs. This creates a system that can be readily modified to target modifications at different genomic sites and / or to target different modifications at the same site. Additionally, protocols have been established which enable simultaneous targeting of multiple genes. The majority of cells carrying the mutation present biallelic mutations in the targeted genes.

[0146] However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9.

[0147] Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system. A double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. Thus, if specificity and reduced off-target effects are crucial, using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off-target effect as either gRNA alone will result in nicks that will not change the genomic DNA.

[0148] Modified versions of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9, or dCas9) have no nuclease activity while still able to bind to DNA based on gRNA specificity. ThedCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

[0149] There are a number of publically available tools available to help choose and / or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species such as the Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.

[0150] In order to use the CRISPR system, both gRNA and Cas9 should be expressed in a target cell. The insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available such as the px33O plasmid from Addgene. Use of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology and a Cas endonuclease for modifying plant genomes are also at least disclosed by Svitashev etal., 2015, Plant Physiology, 169 (2): 931-945; Kumar and Jain, 2015, J Exp Bot 66: 47-57; and in U.S. Patent Application Publication No. 20150082478, which is specifically incorporated herein by reference in its entirety.

[0151] “Hit and run” or “in-out” - involves a two-step recombination procedure. In the first step, an insertion-type vector containing a dual positive / negative selectable marker cassette is used to introduce the desired sequence alteration. The insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, electroporated into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.

[0152] The “double-replacement” or “tag and exchange” strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive / negative selectable cassette near the location where the mutation is to be introduced. After electroporation and positive selection, homologously targeted clones are identified. Next, asecond targeting vector that contains a region of homology with the desired mutation is electroporated into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

[0153] Site-Specific Recombinases - The Cre recombinase derived from the Pl bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.

[0154] Basically, the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue- specific manner. Of note, the Cre and Flp recombinases leave behind a Lox or FRT “scar” of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3' UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function.

[0155] Thus, Cre / Lox and Flp / FRT recombination involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.

[0156] According to a specific embodiment, the DNA editing agent is CRISPR-Cas9.

[0157] Also provided are processed products which are produced from the plants described herein and preferably contain the nucleic acid sequence conferring the improved out-crossing rate described herein. Also provided are methods of processing the cereals (e.g., to produce meal) or other processed products.

[0158] DNA detection in the processed products can be performed using methods which are wellknown in the art and are described in some detail hereinabove.

[0159] Thus provided are processed products derived from the cereal plants described herein, which preferably contain the nucleic acid sequence conferring the improved trait described herein. Methods of processing the barley (e.g., to produce meal) or other processed products are also included.

[0160] For instance, barley flour, barley malt, breakfast cereals, soups, bread, animal feed, beer, whisky, barley straw, barley starch, vegan protein, glucose syrups, and paper, textile, and adhesive products

[0161] As mentioned, germination vigor of the harvested grains plays a critical role in the utilization of barley to the beverage industry, i.e. source for producing malt (product of steeping, germination and kiln of the grains). Also, upcycling of malt by-products such as spent grains or malting rootlets for the vegan protein industry. Hence, the disclosed trait is pertinent to the use / product.

[0162] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0163] The term “consisting of’ means “including and limited to”.

[0164] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0165] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0166] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0167] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0168] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0169] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0170] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0171] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0172] EXAMPLES

[0173] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0174] EXAMPLE 1

[0175] METHODS

[0176] Field trials involving the CMPP population were conducted at four locations: Rehovot, Yotveta, Yizream, and Mevo Hama. The trials in Rehovot and Yotveta were irrigated. The Rehovot trial occurred during the 2021 / 2022 season, while the trials at Yotveta, Yizream, and Mevo Hama were conducted during the 2022 / 2023 season. Trials were conducted without replication and the bestlinear unbiased prediction (BLUP) value was calculated for each of the lines to allow comparison of the means BLUP for carriers of the wild vs. cultivated cytoplasm in each family. In Rehovot, six plants were tray-germinated and grown in a greenhouse with 30 cm spacing between plots. In the remaining sites, fifty seeds from each line were divided evenly between two chambers in twelvechamber magazines (Winter Steiger, Austria) and sown mechanically into two rows of 1 meter each, with 25 plants per row. Each line had two empty rows between them and a l m gap between plots. At full maturity, ten spikes were harvested from each plot.

[0177] Generally, the different sites represent different environments:

[0178] Rehovot:

[0179] Heat: Low around 18°C (winter), High around 35°C (summer).

[0180] Humidity: Moderate, around 50-60% in summer.Mevo Hama:

[0181] Heat: Low around 20°C (winter), High around 40°C (summer).

[0182] Humidity: Moderate, slightly higher in summer, around 50-65%.

[0183] Negev:

[0184] Heat: Low around 10°C (winter nights), High often above 40°C (summer).

[0185] Humidity: Very low, around 20-30%.

[0186] Yotveta:

[0187] Heat: Low around 15°C (winter nights), High often above 40°C (summer).

[0188] Humidity: Very low, around 10-20%.

[0189] Field trials of the reciprocal F2 population were carried out by selecting individual doubled haploids carrying the B1K50 plasmotype, and reciprocally backcrossing those lines to the Magal cultivar, which carries the plasmotype belonging to the Australian Hindmarsh cultivar. The selfed reciprocal backcross populations were grown both in Mibhor and in Yotveta using a randomized complete block design, with manual sowing and plots containing seven plants each.

[0190] Grain analysis was performed using a Marvin ProLine II seed analyzer (MARViTECH GmbH, Wittenburg).

[0191] Shukla stability-analysis was performed using the function as implemented in the metan package.

[0192] Germination for 10 seeds was conducted for three days at 20 °C on vertical petri dishes filled with Agar media and with embryos facing down. Germination vigor (GV) was determined by summing the division of the number of emerging rootlets at each time point by time.

[0193] RESULTSWild barley B1K-50-04 was used by the present inventors as a female donor to obtain an agriculturally beneficial line. Ten wild cytoplasms were directionally crossed as females to a cultivated background (cv Noga) and followed with a series of crosses, backcrosses and doubled haploidy process (Figure 1A). This breeding scheme ended with 960 homozygous lines segregating at the plasmotype and the nuclear genomes. Moreover, since the whole CMPP infrastructure include homozygous doubled haploid lines, it was possible to test the very same genotypes in multiple environments during 2022 and 2023 and collect flowering and grain yield measurements (Figures IB, 1C), including size and weight (thousand-grain weight; TGW). For each line, best linear unbiased predictor (BLUP) values across the four environments were calculated. The mean values between carriers of the cultivated (C) vs wild (W) plasmotypes were compared in each of the ten biparental populations. Figure 2A depicts the significant differences for TGW between W and C types, with the wild plasmotype originating from Hermon showing an average increase of 8.5 %, compared to similar TGW values for the Mt Harif (CMP33 family) population. These unique and significant effects of the Mt. Hermon (B1K-50-04) were even more significant while considering stability indices, i.e. the wild B1K-50-04 plasmotype is the one most stabilizing among the ten tested C vs W bi-parental families (Figure 2B).

[0194] In another experiment, conducted by Hazera39 seed company, the CMPP population was examined in a commercial field mini-plot experiment for total kernel yield and biomass, followed by analysis of the grain attributes. Like the results obtained for TGW across different environments, the B1K-50-04 cytoplasm was associated with a significant increase of the kernel weight (KW; +14.5 %; Figure 2C). Finally, germination vigor of the harvested grains plays a critical role in the utilization of barley to the beverage industry, i.e. source for producing malt (product of steeping, germination and kiln of the grains). The present inventors therefore wished to examine whether the wild B IK-50-04 cytoplasm provide any advantage in the germination vigor or its stability (variation between individual germinating grains, measured by coefficience of variance, CV). Figure 2D depicts a significant difference in the CV of the germination vigor between carriers of the wild vs cultivated cytoplasm, with a similar mean value for the germination. Figure 3 presents the results of a reciprocal crossing experiment in which two distinct doubled haploids carrying the B 1K-50-04 plasmotype were crossed with the Magal cultivar as both male and female parents. The resulting F2 populations were grown in two locations in Israel — Mibhor (central) and Yotveta (southern) — which differ in climate and soil conditions. As shown, the TGW of Magal plasmotype carriers was significantly more influenced by the environment than that of B1K-50-04 plasmotype carriers. Specifically, Magal plasmotype carriers exhibited a reduced TGW at the Mibhor site, whereas B1K-50-04 plasmotype carriers maintained a stable TGW across both locations in both reciprocal populations.EXAMPLE 2

[0195] To validate and examine the effects of specific wild cytoplasm, from the original barley CMPP population, in different environments and genetic backgrounds, two targeted validation panels were utilized: the CMPP Validation panel (CMPPV), and the Magal Reciprocal Validation Panel (MRVP) (Table 1), which were evaluated in two contrasting environments (Mibhor and Yotveta). While CMPPV represents 40 % of the original CMPP (4 out of 10 donors), the MRVP introduces reciprocal crosses between wild-cytoplasm-carrying CMPP lines and the smaller-grain Magal cultivar (Figure 5).

[0196] Table 1

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[0207] Table 1: Composition and experimental design of the CMPP Validation Panel (CMPPV) and the Magal Reciprocal Validation Panel (MRVP). Each panel is defined by the populations tested, reciprocal parental combinations, generation type and segregation status, sample sizes in Mibhor (M) and Yotveta (Y), environments and experimental designs, plot structure and harvest unit, and recordedfield traits (GA, Grain Area; GL, Grain Length; GPS, Grains per Spike; LWR, Length- Width-Ratio; TGW, Thousand Grain Weight; BM, Biomass; GPP, Grains per Plant.

[0208] For each population of both the CMPPV and MRPV panels, a two-way ANOVA was fitted to assess the main and interaction effects of cytoplasm and environment for the measured field traits (Table 1). To quantify the proportion of phenotypic variance attributable to each factor, the eta squared (q2) was calculated, which represents the percentage of total variance explained by a given effect. As can be observed from Figure 6A, cytoplasm significantly contributes to TGW variation in the CMPPV panel, explaining 3.24% of variance within the CMP04 population (q2= 3.24%), and 5.89% and 5.92% within CMP29 and CMP50, respectively (q2= 5.89% and q2= 5.92%). These values are in accordance with the 4.8 % of variance previously observed to be explained by cytoplasm in the CMPPforTGW (Bodenheimer et al. 2025, https: / / doi.org / 10.1101 / 2025.04.08.647843). Importantly, the directionality of the cytoplasm effect is also consistent with that described in Example 1, with the CMP04 and CMP50 wild cytoplasms both causing a relative increase in TGW (A = 3.14 % and A = 5.02 %, respectively), whereas the CMP29 wild cytoplasm yielded a relative decrease of TGW by A = -3.62 % (Figure 6A). TGW in the CMPPV panel is strongly driven by changes in GW (R2= 0.72,), which helps explain why the same consistency with that described in Example 1 can be observed for GW, with the wild cytoplasms of CMP29 and CMP50 modifying GW by A = -1.29% and A = 2.23%, respectively. Interestingly, while increasing TGW, the wild CMP50 cytoplasm also positively influenced GPS (q2= 3.47%, A = 12.7 %).

[0209] Having established that the CMP04, CMP29, and CMP50 cytoplasmic effects replicate in the CMPPV, the present inventors next evaluated whether these effects persist when transferred into a distinct nuclear background. The MRVP panel comprises four reciprocal F2 populations: RecipPopi50w 13, RecipPop250w 38, RecipPop304w 06, and RecipPop4Noga, which together introduce the CMP50 wild cytoplasm, the CMP04 wild cytoplasm, and the cultivated Noga cytoplasm into the Magal background (Table 1), a modem Israeli two-row variety carrying the cytoplasm of the Australian Hindmarsh cultivar. Beyond segregating cytoplasms, these populations also carry a complex nucleotype composed of 50 % Magal, and 50 % of either Noga (in the case of RecipPop4Noga), or Noga with residual wild introgressions (in the case of RecipPopi50w 13, RecipPop250w 38, and Reci pP ops04 w 06). Notably, the significant effects observed within the MRVP panel were all localized to the CMP50-derived groups (Figure 6B). In RecipPopi50w 13and RecipPop250w 38, wild cytoplasm significantly explained TGW variation (q2= 8.45% and q2= 5.89%, respectively), accompanied by substantial increases in average TGW under the CMP50 wild cytoplasm (A = 10.74 % and A = 8.67 %). Cytoplasmic contributions were also detected for grain size components. GA showed 4.25% explained variance in RecipPopi50w 13(q2= 4.25%, A = 4.61%), and GW showed 8.16% and 4.58% explained variance in RecipPopi50w l3and RecipPop250w 38q2= 8.16%and r|2= 4.58%, A = 3.30% and A = 2.48%, respectively). A single Cytoplasm x Environment term was detected for LWR in RecipPop250w 38(r|2= 5.07%, A = -0.08%), indicating an environmentdependent modulation of grain shape on the cytoplasm-level (Figure 6B). In contrast, RecipPop304w 06and RecipPop4Nogashowed no significant cytoplasmic main effects, suggesting limited contribution of the CMP04 wild and Noga cytoplasms to trait variation in the given setting.

[0210] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0211] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.REFERENCES

[0212] (OTHER ARE LISTED IN THE DOCUMENT)

[0213] Bdolach, E., Prusty, M.R., Faigenboim-Doron, A., Filichkin, T., Helgerson, L., Schmid, K.J., Greiner, S., and Fridman, E. (2019). Thermal plasticity of the circadian clock is under nuclear and cytoplasmic control in wild barley. Plant Cell and Environment.

[0214] Ekiz, H., Kiral, A.S., Akgin, A., and Simsek, L. (1998). Cytoplasmic effects on quality traits of bread wheat (Triticum aestivum L.). Euphytica 100: 189-196.

[0215] Frei, U., Peiretti, E.G., and Wenzel, G. (2003). Significance of cytoplasmic DNA in plant breeding. In Plant Breeding Reviews, J. Janick, ed (John Wiley & Sons, Inc.: Hoboken), pp. 175— 210.

[0216] Frei, U., Peiretti, E.G., and Wenzel, G. (2010). Significance of Cytoplasmic DNA in Plant Breeding.

[0217] Fridman, E., Tiwari, L., Bdolach, e., Prusty, M., Beery, A., Bodenheimer, S., Doron-Faigenboim, A., Yamamoto, E., and Kashkush, K. (2022). Cytonuclear diversity underlying clock and growth adaptation to warming environments in wild barley ( Hordeum vulgare ssp. spontaneum ) (Preprints).

[0218] Hagemann, R. (2000). Erwin Baur or Carl Correns: Who really created the theory of plastid inheritance? Journal of Heredity.

[0219] Hubner, S., HOffken, M., Oren, E., Haseneyer, G., Stein, N., Graner, A., Schmid, K., and Fridman, E. (2009). Strong correlation of wild barley Hordeum spontaneum) population structure with temperature and precipitation variation. Molecular Ecology 18: 1523-1536.

[0220] Lbssl, A., Gbtz, M., Braun, A., and Wenzel, G. (2000). Molecular markers for cytoplasm in potato: Male sterility and contribution of different plastid-mitochondrial configurations to starch production. Euphytica.

[0221] Sanetomo, R. and Gebhardt, C. (2015). Cytoplasmic genome types of European potatoes and their effects on complex agronomic traits. BMC Plant Biology 15: 162.

[0222] Shukla, G.K. (1972). Some statistical aspects of partitioning genotype-environmental components of variability. Heredity (Edinb) 29: 237-245.

Claims

WHAT IS CLAIMED IS:

1. A cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of a wild-cultivar of a cereal, said plasmotype imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to said domesticated cultivar devoid of said plasmotype, said at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor.

2. A cereal plant comprising a nuclear genome being at least 90 % of a domesticated cultivar and a plasmotype of said domesticated cultivar, said plasmotype comprising a sequence variation in RpoCl as compared to that of said domesticated cultivar, said sequence variation imparting the cereal plant with an improvement in at least one agriculturally desired trait as compared to said domesticated cultivar devoid of said sequence variation, said at least one agriculturally desired trait being selected from the group consisting of grain yield and germination vigor.

3. The plant of claim 1 or 2, wherein said grain yield is manifested by thousand grain weight (TGW) or kernel weight per growth area.

4. The plant of any one of claims 1-3, wherein said improvement in at least one agriculturally desired trait is stable across different abiotic conditions including drought and heat and exhibits reduced variance (value distribution) in said trait as compared to said variance in domesticated cultivar of cereal devoid of said plasmotype or sequence variation.

5. The plant of any one of claims 1-4, being barley.

6. The plant of any one of claims 1, 3-5, wherein said plasmotype comprises a sequence variation in RpoCl as compared to that of the domesticated cultivar.

7. The plant of any one of claims 2, 4 and 6, wherein said sequence variation comprises an N to K mutation at a position corresponding to position 571 of SEQ ID NO: 2 (encoded by SEQ ID NO: 1 for instance).

8. The plant of any one of claims 1, 3-7, wherein said domesticated genome and said plasmotype are of the same species.

9. The plant of any one of claims 1, 3-7, wherein said domesticated genome and said plasmotype are of different species.

10. The plant of any one of claims 1-9, being transgenic.

11. The plant of any one of claims 1-9, being non-transgenic.

12. The plant of any one of claims 1, 3-11, wherein said plasmotype is of BK1-50-04.

13. A part of the plant of any one of claims 1, 3-12.

14. A method of producing the plant of any one of claims 1, 3-12, the method comprising: (a) crossing a domesticated male cereal plant with a wild female cereal plant to obtain a hybrid, said female cereal plant comprising a plasmotype which comprises a sequence variation capable of increasing at least one agriculturally desired trait as compared to the domesticated male cereal plant devoid of said plasmotype, said at least one agriculturally desired trait being selected from the group consisting of grain yield and / or germination vigor; and(b) back-crossing said hybrid to said male domesticated cereal plant to obtain a back-cross; and optionally repeating step (b) where each time a resultant back-cross is crossed with said male domesticated cereal plant.

15. The method of claim 14 further comprising selecting a progeny plant of said back-crossing by identifying a sequence variation in RpoCl as compared to that of said domesticated cultivar, wherein said sequence variation is indicative that said progeny is characterized by said improvement of said at least one agriculturally desired trait.

16. A method of producing the plant of any one of claims 2-5, 7, 10-11 and 13 comprising subjecting a chloroplast of the cereal plant to a gene editing agent to introduce said sequence variation in RpoCl, thereby producing the plant.

17. The method of claim 16, wherein said genome editing agent is selected from the group consisting of CRISPR-Cas, TALEN, and a Zinc Finger Nuclease (ZFN).

18. A food, feed or beverage or any other article of manufacture comprising the plant or part thereof of any one of claims 1-13.

19. The food, feed, beverage or article of manufacture of claim 18, comprising a genome of said plasmotype and / or said nuclear genome.

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