WO2018125749A1

WO2018125749A1 - Methods of producing hybrid-like seed lots

Info

Publication number: WO2018125749A1
Application number: PCT/US2017/067907
Authority: WO
Inventors: Sally Ann MACKENZIE; Michael Fromm
Original assignee: Epicrop Technologies Inc; University of Nebraska Lincoln; University of Nebraska System
Current assignee: Epicrop Technologies Inc; University of Nebraska Lincoln; University of Nebraska System
Priority date: 2016-12-30
Filing date: 2017-12-21
Publication date: 2018-07-05
Anticipated expiration: 2019-06-30

Abstract

A method of producing hybrid-like yields in normally self-pollinated crops, wherein the yields are highest in a targeted number of generations, and wherein later generations of plants have lower relative yields is provided herein.

Description

INTERNATIONAL PATENT APPLICATION

FOR

METHODS OF PRODUCING HYBRID-LIKE SEED LOTS

Cross-Reference to Related Applications

[0001] This PCT patent application claims the benefit of U.S. Patent Application No.

62/440,845, filed December 30, 2016 and incorporated herein by reference in its entirety.

Incorporation of Sequences

[0002] The sequence listing that is contained in the file named "46589-172131 ST25", which is 54926 bytes (measured in operating system MS-Windows), created on 12/21/2017, is filed herewith by electronic submission and incorporated herein by reference in its entirety.

Statement Regarding Federally Sponsored Research or Development

[0003] This invention was made with Government Support under IOS1126935 awarded by the National Science Foundation. The government has certain rights in the invention.

Background

[0004] Self-pollinated crops such as rice, wheat, soybeans, cotton, oats, rye, and barley generally suffer from a lack of investment by private industry due to a lack of recurring annual sales. Growers can buy the seeds, grow the crop, and then save some of the seeds from the crop for planting in the subsequent year(s). This is in contrast to hybrid crops where the better yields of hybrid seed relative to subsequent generations provides an economic incentive for the grower to buy seeds every year. A biological system that provides the advantages of hybrids in a self-pollinated crop is needed to encourage investment in improving crop yields in these crops.

[0005] Plant genomes contain relatively large amounts of 5-methylcytosine (5meC; Kumar et al. 2013 J Genet 92(3): 629-666). Other than silencing transposable elements and repeated sequences, the biological roles of 5meC and its transgenerational stability are still emerging. Intercrossing a low methylation mutant plant with a normally methylated plant resulted in heritable changes in DNA methylation in the plant genome that affected some plant phenotypic traits in Arabidopsis (Cortijo et al. 2014 Science. 2014 Mar 7;343(6175): 1 145-8; Kooke et al. Plant Cell. 2015 Feb;27(2):337-48).

[0006] Over expression of Arabidopsis MET1, a DNA methyltransferase predominantly responsible for CG maintenance methylation, in Arabidopsis resulted in plants that flower earlier (U. S. Patents # 6,01 1,200 and 6,444,469). Gene mutations affecting DNA

methylation include those described (Stroud et al., Cell 2013 152(1-2)352-64) that can alter DNA methylation patterns in progeny.

Summary

[0007] The present disclosure provides methods of producing hybrid-like yields in self- pollinated crops wherein the yields are highest in a targeted number of generations, wherein later generations of plants have lower relative yields. The methods identify parent plants that can produce these hybrid-like yields that are highest (e.g., attain a peak yield) in a targeted number of generations with subsequent generations exhibiting lower yields.

[0008] Non-limiting embodiments methods of identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations, are provided herein.

[0009] Non-limiting embodiments of methods of identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications by Mshl suppression- restoration to produce said candidate inbred plants; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and, c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self- pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations, are provided herein.

[0010] Non-limiting embodiments of methods of producing hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations; and, d) producing hybrid-like seeds with peak yields in a targeted number of generations from the seeds of the inbred plant identified in step (c), are provided herein.

[0011] Non-limiting embodiments of methods of producing hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications by Mshl suppression-restoration to produce said candidate inbred plants; b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and, c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for a hybrid-like plant line for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations; and, d) producing hybrid-like seeds with peak yields in a targeted number of generations from the seeds of the inbred plant identified in step (c),are provided herein.

[0012] In certain embodiments of any of the aforementioned methods, the targeted number of generations is selected from the group consisting of 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, 5 to 6, and 6 generations. In certain embodiments of any of the aforementioned methods, the targeted number of generations is selected from the group consisting of 3, 4, 5, and 6. In certain embodiments of any of the aforementioned methods, a single plant harvester is used to measure yields in single plants in one or more generations of step (b).

[0013] In certain embodiments of any of the aforementioned methods, the candidate inbred plants of step (a) are produced from an inbred parent which was suppressed for Mshl function or were derived from an ancestral plant suppressed for Mshl function.

[0014] In certain embodiments of any of the aforementioned methods, the candidate inbred plants are produced from two genetically diverse parental plants at least one of which was suppressed for Mshl function or were derived from an ancestral plant suppressed for Mshl function.

[0015] In certain embodiments of any of the aforementioned methods, seeds of said candidate inbred plants of step (a) are stored at about -18 to -20 C.

[0016] In certain embodiments of any of the aforementioned methods, the hybrid-like plant lines of step (b) are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant, wherein said second parental plant is isogenic to said candidate inbred plant prior to any epigenetic modification steps.

[0017] In certain embodiments of any of the aforementioned methods, the hybrid-like plant lines of step (b) are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant, wherein said second parental plant is isogenic to said candidate inbred plant and wherein said second parental plant is derived from an ancestral plant suppressed for Mshl .

[0018] In certain embodiments of any of the aforementioned methods, the hybrid-like seeds are rice, wheat, soybeans, cotton, oats, rye, or barley seeds. In certain embodiments of any of the aforementioned or other methods provided herein wherein soybean plants and/or soybean rootstock are used to obtain hybrid-like soybean plants or soybean seed, a soybean Mshl gene is subjected to suppression-restoration. In certain embodiments, the soybean Mshl that is subjected to suppression-restoration encodes the polynucleotide of SEQ ID NO:6, SEQ ID NO: 8, or a variant of SEQ ID NO: 6 or SEQ ID NO: 8 having at least 80%, 90%, 95%, 98%, or 99% sequence identity thereto or having one, two, three , four, five or more nucleotide substitutions, deletions, and/or insertions. In certain embodiments, the soybean Mshl gene that is subjected to suppression-restoration comprises a polynucleotide that encodes the polypeptide of SEQ ID NO: 7, SEQ ID NO: 9, or a variant of SEQ ID NO: 7 or SEQ ID NO: 9 having at least 80%, 90%, 95%, 98%, or 99% sequence identity thereto or having one, two, three , four, five or more amino acid residue substitutions, deletions, and/or insertions.

[0019] In certain embodiments of any of the aforementioned or other methods provided herein, the candidate inbred plants are produced in step (a) by suppressing expression of Mshl in a parent plant and recovering a candidate inbred plant wherein Mshl expression has been restored. In certain embodiments of any of the aforementioned or other methods provided herein, the candidate inbred plants are produced in step (a) by introducing a loss-of- function mutation into an endogenous Mshl gene of a parent plant, obtaining a first progeny plant of the parent plant wherein Mshl gene expression is suppressed by the mutation, and recovering from the first progeny plant the candidate inbred plant, wherein Mshl gene expression is restored in the candidate inbred plant.

Description

Definitions

[0020] As used herein, the phrase "producing candidate inbred plants with new epigenetic modifications by suppressing and then restoring Mshl" refers to a multi -generation process of suppressing Mshl in a plant for one or more plant generations and then restoring Mshl function in a later generation to create epigenetic modifications suitable for producing 'hybrid-like plants' .

[0021] As used herein, the phrase "reference plant line" refers to a plant line lacking new epigenetic modifications but otherwise genetically similar or identical to a candidate plant. A reference plant line facilitates comparison to lines with epigenetic contributions to yields and phenotypes.

[0022] As used herein, the phrase "new epigenetic modifications" refers to epigenetic modifications introduced into a plant or plant line by a specific procedure as opposed to epigenetic modifications that occur by traditional breeding procedures. Exemplary non- limiting examples of specific procedures for introducing new epigenetic modifications are altering DNA methyltransferase levels, mutants in DNA methylation pathways, or Mshl suppression-restoration systems.

[0023] As used herein, the phrase "Mshl suppression-restoration" refers to a multi- generation process of suppressing Mshl in a plant for one or more plant generations and then restoring Mshl function in a later generation to create epigenetic modifications in progeny plants and lines.

[0024] As used herein, the phrase "peak yields" refers to yields obtained from a given generation of plants that exceed yields obtained from a parental plant line and that exceed yields obtained from a subsequent generation of plants.

[0025] As used herein, the phrase "plant line" refers to one or more generations of a plant lineage, including ancestor plants, current plants, and multiple generations of progeny plants. A plant line can be an inbred line or a line early in the process of becoming an inbred or can be more genetically heterogeneous than an inbred. A plant line can be derived from a single ancestor or two common ancestors.

[0026] As used herein, the phrase "inbred" refers to a plant or plant line sufficiently homozygous to have progeny with similar phenotypes. Typically an inbred plant has been self-pollinated for at least three generations or has been generated from a double haploid method to have a high degree of genetic and/or epigenetic homozygosity.

[0027] As used herein, the phrase "seeds of the candidate inbred plant" refers to seeds from the candidate inbred plant or plant line, said seeds from earlier, current, or subsequent generations of said candidate inbred plant or plant line.

[0028] As used herein, the phrases "candidate inbred plant" (singular) or "candidate inbred plants" (plural) refer to an inbred plant or plants, and/or an inbred plant line and includes seeds from the candidate inbred plant or plant line, said seeds from earlier, current, or subsequent generations of said candidate inbred plant or plant line. It is understood that the process of screening and/or identifying a candidate inbred plant or candidate inbred plants includes storing the seeds of these plants for later recovery from storage and propagation of plants from these seeds.

[0029] As used herein, the phrase "epigenetic trait" refers to a trait dependent in part on epigenetic modifications. For example, a yield increase that is dependent in part upon DNA methylation modifications.

[0030] As used herein, the term "generation" (singular) or "generations" (plural) refers to one or several complete life cycle (an initial seed is grown to a mature plant that produces seeds).

[0031] As used herein, the term "progeny" refers to a first, second, third, or later generations of plants derived from a parent or ancestor plant.

[0032] As used herein, the term "Fl" refers to seeds produced from a cross of two parents or plants grown from said seeds. If only self-pollination is performed for a plant line, then Fl can refer to the first generation of plant grown. [0033] As used herein, the term "F2" refers to seeds produced from self-pollination of plants grown from Fl seeds or plants grown from F2 seeds.

[0034] As used herein, the term "F3" refers to seeds produced from self-pollination of plants grown from F2 seeds or plants grown from F3 seeds.

[0035] As used herein, the term "Fn" refers to seeds produced from self-pollination of plants grown from F(n-l) seeds or plants grown from Fn seeds.

[0036] As used herein, the phrase "hybrid-like" is used to refer to plants or seeds that are epigenetically modified and that exhibit, or give rise to plants that exhibit, an increase in yield in comparison to a parental plant that lacks the epigenetic modifications. In certain embodiments, the yield increase is diminished or lost in subsequent generations of plants obtained from selfs of certain hybrid like plants or from selfs of a plant obtained from certain hybrid-like seed.

[0037] Various methods for providing plants with hybrid-like yields are provided herein. The methods provided herein can be applied to most crops that contain new epigenetics modifications that increase yields in hybrid-like plants. The Mshl suppression-restoration system is one such suitable system. Other systems for introducing epigenetic modifications include, but are not limited to, using recombinant DNA methyltransferases. The methods of producing the candidate inbred plants as well as the two parental plants in the initial cross can be varied. One variation is to produce candidate inbred plants comprising genetics from two genetically different plants and concurrently creating new epigenetic modifications in said candidate inbred plants. The two parental plants used to initiate the production of hybrid-like plants in the initial cross can have one or both parents comprising new epigenetic

modifications. These two parents can be genetically identical, similar, or genetically more distant from each other. Altering the genetic and epigenetic range in the candidate inbred plants as well as the two parental plants in the initial cross that starts the production of the hybrid-like plants will provide a range of generations in which peak yields occur and these can be identified and selected using the methods or method similar to those disclosed or exemplified herein. Epigenetic modifications, including those induced by Mshl suppression- restoration, are feasible in most crops, making most crop plants suitable for producing hybrid-like plants with peak yields in a targeted number of generations.

[0038] In certain embodiments, the methods use Mshl suppression-restoration techniques. Mshl suppression-restoration is a process that can occur over several generations and can lead to unusual plant phenotypes (Xu YZ, Santamaria Rde L, Virdi KS, Arrieta-Montiel MP, Razvi F, Li S, Ren G, Yu B, Alexander D, Guo L, Feng X, Dweikat FM, Clemente TE, Mackenzie SA. Plant Physiol. 2012 Jun; 159(2):710-20; Virdi KS, Wamboldt Y,

Kundariya H, Laurie JD, Keren I, Kumar KR, Block A, Basset G, Luebker S, Elowsky C, Day PM, Roose JL, Bricker TM, Elthon T, Mackenzie SA. Mol Plant. 2016 Feb 1 ;9(2):245- 60). Mshl suppression-restoration can also lead to subsequently higher yields in progeny after crossing followed by self-pollination in tomatoes (Yang X, Kundariya H, Xu YZ, Sandhu A, Yu J, Hutton SF, Zhang M, Mackenzie SA. Plant Physiol. 2015 May; 168(l):222- 32), sorghum (de la Rosa Santamaria R, Shao MR, Wang G, Nino-Liu DO, Kundariya H, Wamboldt Y, Dweikat I, Mackenzie SA. PLoS One. 2014 Oct 27;9(10):el08407), and Arabidopsis (Virdi KS, Laurie JD, Xu YZ, Yu J, Shao MR, Sanchez R, Kundariya H, Wang D, Riethoven JJ, Wamboldt Y, Arri eta-Monti el MP, Shedge V, Mackenzie SA. Nat Commun. 2015 Feb 27;6:6386).

[0039] Methods and various Mshl nucleic acid sequences for producing epigenetically- modified plants using Mshl suppression in an ancestral plant or plants and restoration include, but are not limited to, those described in U. S. Patent Application Publication No. 20120284814, U.S. Patent Application Publication No. 20150052630, U.S. Patent

Application Publication No. 201501 13679, and U. S. Patent Application No. 20170188512, which are each incorporated herein by reference in their entireties. Such Mshl suppression can be effected by methods including, but not limited to, transgene-mediated suppression (e.g., by RNAi), VIGS-based suppression, introduction of inhibitory polynucleotides (e.g., encoding or comprising RNAi-inducing polynucleotides), or by introduction of mutations in the endogenous Mshl gene (e.g., in the Mshl promoter, transcribed region, splice donor and/or acceptor sites, polyadenylation/terminator region, or any combination thereof). Types of mutations that provide for such Mshl suppression can include, but are not limited to, substitutions, deletions, and/or insertions into the endogenous Mshl gene. Useful mutations in the endogenous Mshl gene include, but are not limited to, frameshift, nonsense, and missense mutations that cause a loss-of function. Mutations in endogenous Mshl genes can be obtained from a variety of sources and by a variety of techniques. In certain embodiments, mutations an endogenous Mshl gene can introduced by physical mutagenesis methods, including but not limited to, chemical (e.g., EMS) or radiation mutagenic treatments,

TILLING, gene editing techniques (e.g., CRISPR-CAS9, meganucleases, TALENS) and the like. Methods for introducing mutations in endogenous genes disclosed in Guha et al.

Comput Struct Biotechnol J. 2017; 15: 146-160 can be adapted for use in introducing mutations in an endogenous Mshl gene. In certain embodiments, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease (e.g., Cas9, Cpfl, S. aureus Cas9 variants, eSpCas9) and a guide RNA are used to introduce the mutation in the endogenous Mshl gene. In certain embodiments, a homologous replacement sequence containing one or more loss of function mutations in the Mshl gene and homologous sequences at both ends of the double stranded break can provide for homologous

recombination and substitution of the resident wild-type Mshl target gene sequence in the chromosome with a mshl replacement sequence with the loss of function mutation(s). Such loss of function mutations include, but are not limited to, insertions, deletions, and

substitutions of sequences within an Mshl gene that result in either a complete loss of Mshl target gene function or a loss of Mshl target gene function sufficient to elicit alterations (i.e. heritable and reversible epigenetic changes) in other chromosomal loci or mutations in other chromosomal loci. Loss-of-function mutations in an Mshl gene also include, but are not limited to, pre-mature translational stop codon insertions, deletions of one or more functional domains that include, but are not limited to, a DNA binding (Domain I), an ATPase (Domain V) domain, and/or a carboxy-terminal GIY-YIG type endonuclease domain, and the like. Also provided herein are mutations analogous the Arabidopsis mshl mutation that are engineered into endogenous Mshl gene to obtain similar effects. Methods for substituting endogenous chromosomal sequences by homologous double stranded break repair have been reported in tobacco and maize (Wright et al., Plant J. 44, 693, 2005; D'Halluin, et al., Plant Biotech. J. 6:93, 2008). A homologous replacement mshl sequence (i.e. which provides a loss of function mutation in an Mshl target gene sequence) can also be introduced into a targeted nuclease cleavage site by non-homologous end joining or a combination of nonhomologous end joining and homologous recombination (reviewed in Puchta, J. Exp. Bot. 56, 1, 2005; Wright et al., Plant J. 44, 693, 2005). In certain embodiments, at least one site specific double stranded break can be introduced into the endogenous Mshl gene by a meganuclease. Genetic modification of meganucleases can provide for meganucleases that cut within a recognition sequence that exactly matches or is closely related to specific endogenous Mshl gene sequence (WO/06097853A1, WO/06097784A1, WO/04067736 A2, U.S. 20070117128A1). It is thus anticipated that one can select or design a nuclease that will cut within a target Mshl gene sequence. In other embodiments, at least one site specific double stranded break can be introduced in the endogenous Mshl gene target sequence with a zinc finger nuclease. The use of engineered zinc finger nuclease to provide homologous recombination in plants has also been disclosed (WO 03/080809, WO 05/014791, WO 07014275, WO 08/021207). In still other embodiments, mutations in Mshl genes can be identified through use of the TILLING technology (Targeting Induced Local Lesions in Genomes) as described by Henikoff et al. where traditional chemical mutagenesis would be followed by high-throughput screening to identify plants comprising point mutations or other mutations in the endogenous Mshl gene (Henikoff et al., Plant Physiol. 2004, 135:630-636). Restoration of Mshl function can be provided by crossing the plants that are homozygous for recessive mutations that provide for such Mshl suppression with plants having wildtype Mshl gene(s) and harvesting progeny plants that are heterozygous for the mshl mutation.

[0040] Methods and various nucleic acid sequences for producing epigenetically-modified plants using recombinant DNA methyltransferases include, but are not limited to, those described in U.S. Patent Application Publication No. 20160032310 and U.S. Patent

Application Publication No. 20170016017, which are each incorporated herein by reference in their entireties.

[0041] Methods of producing hybrid-like plants include the use of freezing seeds as a method of stably storing epigenetically modified seeds (U.S. Patent Application Publication No. 20170223914, which is incorporated herein by reference in its entirety).

[0042] Methods of producing hybrid-like plants include single plant harvesters and row harvesters for selecting individual plants or plant families (see U.S. Patent Application Publication No. 20170188512, which is incorporated herein by reference in its entirety.

[0043] Examples of suitable plants for the present disclosure both as self-pollinated plants as well as plants often produced as hybrids include but are not limited to, those from: barley (Hordeum vulgare), corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower

(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco

(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucijra), sugar beets (Beta vulgaris), sugarcane (Saccharum spp, tomatoes (Lycopersicon esculentum), lettuce (for example, Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo.).

[0044] Improvements in yield in plant lines that include, but are not limited to, peak yields obtained by the methods provided herein, can be identified by direct measurements of wet or dry biomass including, but not limited to, grain, lint, leaves, stems, or seed. Improvements in yield can also be assessed by measuring yield related traits that include, but are not limited to, 100 seed weight, a harvest index, and seed weight. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to one or more parental line(s), subsequent generations, reference lines, controls, or checks and can be readily determined by growing plant lines obtained by the methods provided herein in parallel with the parental plants, subsequent generations, reference lines, controls, or checks. In certain embodiments, field trials to determine differences in yield whereby plots of test and control plants are replicated, randomized, and controlled for variation can be employed (Giesbrecht FG and Gumpertz ML. 2004. Planning, Construction, and Statistical Analysis of Comparative Experiments. Wiley. New York; Mead, R. 1997. Design of plant breeding trials. In Statistical Methods for Plant Variety Evaluation, eds. Kempton and Fox. Chapman and Hall. London.). Methods for spacing of the test plants (i.e. plants obtained with the methods of this invention) with check plants (parental or other controls) to obtain yield data suitable for comparisons are provided in references that include, but are not limited to, any of Cullis, B. et al. J. Agric. Biol. Env. Stat.11 :381-393; and Besag, J. and Kempton, RA. 1986. Biometrics 42: 231-251.).

[0045] In certain embodiments, peak yields obtained from targeted generation(s) of plants can be identified by comparing yields obtained from the targeted generation(s) to yields obtained from a parental line, from a subsequent generation of plants, from parental lines and subsequent generations, or from any other reference, check, or control plant line. In certain embodiments, peak yields obtained from the targeted generation of plants will exceed yields obtained from any of the other aforementioned plants or plant lines by at least about 1%, 2%, 5%, 7%, 8%, 10%, 12%, 14%, or 15%. In certain embodiments, peak yields obtained from the targeted generation of plants will exceed yields obtained from any of the other

aforementioned plants or plant lines by at least about 1%, 2%, or 3% to about 5%, 7%, 8%, 10%, 12%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a 100%. In certain embodiments, peak yields obtained from the targeted generation of plants will exceed yields obtained from any of the other aforementioned plants or plant lines by at least about 25%, 50%, or 100%.

[0046] In certain embodiments, the methods for introducing heritable epigenetic or genetic variation in a plant or progeny thereof can comprise the step of grafting rootstock obtained from a plant or a parent plant thereof had been subjected to suppression of a MSH1 gene. In certain embodiments of any of the aforementioned methods, the heritable epigenetic variation provides a useful trait is selected from the group consisting of improved yield, delayed flowering, non-flowering, increased biotic stress resistance, increased abiotic stress resistance, enhanced lodging resistance, enhanced growth rate, enhanced biomass, enhanced tillering, enhanced branching, delayed flowering time, delayed senescence, increased flower number, improved architecture for high density planting, improved photosynthesis, increased root mass, increased cell number, improved seedling vigor, improved seedling size, increased rate of cell division, improved metabolic efficiency, and increased meristem size in comparison to the control plant. In certain embodiments, the plant, progeny of the plant, or scion contain(s) one or more epigenetic changes in one or more nuclear chromosomes, wherein the epigenetic changes are absent from nuclear chromosomes of the control plant or are absent from nuclear chromosomes of a plant from which the scion was obtained. In certain embodiments, the epigenetic change(s) are also present in the rootstock that had been subjected to MSH1 gene suppression. In certain embodiments, the epigenetic changes in the plant, progeny of the plant, scion, or rootstock are associated with the improvement in the useful trait. In certain embodiments, the epigenetic changes in the plant, progeny of the plant, scion, or rootstock induced by suppression of a MSH1 gene. In certain embodiments, the plant, progeny of the plant, scion, or rootstock contain(s) one or more epigenetic changes in one or more nuclear chromosomes that are absent from nuclear chromosomes of rootstock obtained from a plant or are absent from nuclear chromosomes of a parent plant thereof had not been subjected to MSH1 gene suppression. In certain embodiments, the plant, progeny of the plant, scion and/or the rootstock exhibit CG hypermethylation of a region encompassing a MSHl locus in comparison to a control plant that had not been subjected to the MSH1 gene suppression. In certain embodiments, the plant, progeny of the plant, scion and/or the rootstock exhibit pericentromeric CHG hyper-methylation in comparison to a control plant that had not been subjected to the MSHl gene suppression. In certain embodiments, the plant, progeny of the plant, scion and/or the rootstock exhibit CG hypermethylation and/or CHG hypermethylation at one or more nuclear chromosomal loci in comparison to corresponding nuclear chromosomal loci of a control plant that had not been subjected to the MSHl gene suppression. In certain embodiments, the plant is selected from the group consisting of a crop plant, a tree, a bush, and a vine. In certain embodiments, the crop plant is selected from the group consisting of corn, soybean, cotton, canola, wheat, rice, tomato, tobacco, millet, potato, sugarbeet, cassava, alfalfa, barley, oats, sugarcane, sunflower, strawberry, and sorghum. In certain embodiments, the tree is selected from the group consisting of an apple, apricot, grapefruit, orange, peach, pear, plum, lemon, coconut, poplar, eucalyptus, date palm, palm oil, pine, and an olive tree. In certain embodiments, the bush is selected from the group consisting of a blueberry, raspberry, and blackberry bush. In certain embodiments, the vine is a grape vine. Also provided are plants or progeny thereof obtained by any of the aforementioned methods. Also provided are plant parts obtained from the plant or progeny thereof that were made by any of the aforementioned methods.

[0047] Also provided herein are grafted plants comprising a scion to which a rootstock had been grafted, wherein the rootstock is obtained from a plant or a parent plant thereof that had been subjected to MSH1 gene suppression, as well as progeny plants and clonal propagates obtained from the grafted plant. Such rootstocks can be also used to introduce epigenetic and/or genetic variation into varietal or non-hybrid plants that result in useful traits as well as useful plants, plant parts including, but not limited to, seeds, plant cells, and processed plant products that exhibit, carry, or otherwise reflect benefits conferred by the useful traits. In other embodiments, such rootstocks can also be used to introduce epigenetic and/or genetic variation into plants that are also amenable to hybridization.

[0048] Rootstocks useful for introducing epigenetic and/or genetic variation into plants can be obtained from a variety of rootstock source plants that had been subjected to MSH1 gene suppression. In certain embodiments, the rootstock source plant is a plant that had itself been subjected to MSH1 gene suppression. In other embodiments, the rootstock source plant is the progeny of a parental plant that had itself been subjected to MSH1 gene suppression. Various methods of making rootstock source plants by MSH1 gene suppression are provided herein. Plants that can serve as rootstock source plants and methods of making such plants are also disclosed in US Patent Application Publication No. 20120284814, which is specifically incorporated herein by reference in its entirety. The use of plants with useful traits and methods of making such plants disclosed in para. [0072], [0085], and [0089] in US Patent Application Publication No. 20120284814 as rootstock sources is specifically provided, and each of those paragraphs is specifically incorporated herein by reference in their entireties.

[0049] In certain embodiments where the rootstock source plant, or a parental plant thereof, had been subjected to MSH1 suppression, a population of progeny plants obtained from the grafted plant are screened and individual progeny plants are selected for one or more useful traits. Such populations of progeny plants can be obtained by methods including, but not limited to, selfing or outcrossing the grafted plant comprising the rootstock to obtain seed that give rise to the population. Such populations of progeny plants can also be obtained by methods including, but not limited to, growing a population of plants that are derived from independent clonal propagates obtained from the grafted plant comprising the rootstock. Such selected individual progeny plants that exhibit the useful trait can then be sexually or asexually propagated to yield populations of plants that exhibit the useful trait or seed lots that exhibit or harbor the useful trait. Such sexual propagation can be accomplished by selfing or outcrossing the selected individual progeny plants that exhibit the useful trait.

[0050] In certain embodiments where the rootstock source plant is the progeny of a parental plant that had been subjected to MSHl suppression, the rootstock source plant itself can be a plant that was selected for one or more useful traits. Grafting rootstock from a plant that had been selected for a useful trait to a scion that does not exhibit the trait can impart the trait to the resultant grafted plant or to progeny thereof. Resultant grafted plants or progeny thereof that exhibit the useful trait can then be sexually or asexually propagated to yield populations of plants that exhibit the useful trait or seed lots that exhibit or harbor the useful trait.

[0051] In grafted plants or progeny thereof, MSHl gene suppression in the rootstock can be continuous and ongoing or can be transient. Non-limiting and exemplary methods for effecting continuous and ongoing MSHl gene suppression in the rootstock include suppressing expression of a MSHl gene with mutations in the endogenous MSHl gene or with a transgene that yields a product that suppresses expression of the MSHl endogenous gene. Alternatively, the MSHl gene suppression in the rootstock can be transient or have occurred in a parental plant from which the rootstock was obtained but not in the rootstock that was used in the graft. Non-limiting and exemplary methods for effecting transient suppressing of MSHl function in the rootstock include suppressing expression of a MSHl target gene with a transgene that provides for inducible or repressible expression of a product that suppresses expression of the endogenous gene, with a transgene that can be excised, or with a heterozygous transgene insert that is removed from the rootstock by segregation. Any of the methods described herein for restoring MSHl function after MSHl suppression can be used to generate rootstock used in certain embodiments.

[0052] Grafting can be effected by any method that provides for establishment of a vascular connection between the rootstock and the scion. Methods of grafting that can be used to effect the connection between the scion and the rootstock include, but are not limited to, apical graftage, side graftage, bark graftage, and root graftage. Such methods for effecting grafts of scions to rootstock are disclosed in "Plant Propagation: Principles and Practices; Chapter 12: Techniques of Grafting" Ed. Hartman, Kester, Davies, and Geneve, 7^th Edition. Methods for effecting grafts of monocot plant scions to rootstocks that can be used with the scions and rootstocks provided herein are disclosed in Muzik and La Rue, The Grafting of Large Monocotyledonous Plants, Science 116, No. 3022: 589-591, 1952. [0053] Rootstocks subjected to MSH1 gene suppression or obtained from a parental plant that had been subjected to MSH1 gene suppression can exhibit modifications of one or more nuclear chromosomes. In certain embodiments, such rootstocks can exhibit characteristic DNA methylation and/or gene transcription patterns that occur in plants subjected to suppression of an MSH1 target gene. Such characteristic DNA methylation and/or gene transcription patterns that occur in plants or seeds subjected to suppression of an MSH1 target gene can include, but are not limited to, those patterns disclosed in Example 5. In certain embodiments, rootstock of first generation progeny of a plant subjected to suppression of a MSHl gene will exhibit CG differentially methylated regions (DMR) of various discrete chromosomal regions that include, but are not limited to, regions that encompass the MSHl locus. In certain embodiments, a CG hypermethylated region that encompasses the MSHl locus will be about 5 to about 8 MBp (mega base pairs) in length. In certain embodiments, rootstock of first generation progeny of a plant subjected to suppression of a MSHl gene will also exhibit changes in plant defense and stress response gene expression. In certain embodiments, a rootstock, a scion grafted thereto, and/or a plant cell, a seed, a progeny plant, plant populations, seed populations, and/or processed products obtained therefrom that has been subject to suppression of a MSHl gene will exhibit pericentromeric CHG

hypermethylation and CG hypermethlation of various discrete or localized chromosomal regions. Such discrete or localized hypermethylation is distinct from generalized

hypermethylation across chromosomes that have been previously observed (U.S. Patent 6,444,469). Such CHG hypermethylation is understood to be methylation at the sequence "CHG" where H=A, T, or C. Such CG and CHG hypermethylation can be assessed by comparing the methylation status of a sample from rootstocks, scions of plants grafted to root stocks, plants or seed that had been subjected to suppression of a MSHl gene, or a sample from progeny plants or seed derived therefrom, to a sample from control plants or seed that had not been subjected to suppression of a MSHl gene. In this and certain other contexts, such control plants include, but are not limited to, plants, grafted plants, scions thereof and rootstocks thereof that had not been subjected to MSHl gene suppression. In certain embodiments, such aforementioned changes in the methylation patterns exhibited by scions that are grafted to the rootstocks, or exhibited by a plant cell, a seed, a progeny plant, plant populations, seed populations, and/or processed products obtained from the grafted plant, be used to monitor the effectiveness of the graft in transmitting desirable epigenetic changes or to identify a plant cell, a seed, a progeny plant, plant populations, seed populations, and/or processed products obtained from the grafted plant. [0054] Also provided herein are various methods for producing a plant exhibiting a useful trait that comprise crossing grafted plants comprising a scion grafted to rootstock that had been subjected to MSH1 gene suppression with another plant, or crossing progeny plants obtained from the grafted plant with another plant, and selecting one or more progeny plants obtained from the cross for an improvement in the useful trait in comparison to a control plant. In certain embodiments, the second plant can also be a grafted plant comprising a scion grafted to rootstock that had been subjected to MSH1 gene suppression, a progeny plants obtained from a grafted plant comprising a scion grafted to rootstock that had been subjected to MSH1 gene suppression, any other ungrafted plant that had been subjected to MSH1 gene suppression, or any other ungrafted plant obtained from one or more parental plants that had been subjected to MSH1 gene suppression. Such second plants can be plants that were selected for a useful trait and that were progeny of any plant or grafted plant that had subjected to MSH1 gene suppression. Control plants used as comparators to identify progeny of the cross that exhibit an improvement in the useful trait include, but are not limited to: progeny of a cross between a plant which lacks a graft to the rootstock and a plant that is isogenic to the second plant, progeny of a self of a plant that lacks a graft to the rootstock, progeny of a self of the second plant; progeny of a cross between a plant that is isogenic to the plant source of the scion of the grafted plant and a plant that is isogenic to the second plant; and, progeny of a cross between a plant that is isogenic to the plant source of the scion of the grafted plant and that is isogenic to the plant source of a scion of the second plant when the second plant is a grafted plant. Also provided are methods where at least the scion of the first plant is from a different heterotic group than the second plant or where at least the scion of the first plant is from the same heterotic group as the second plant.

[0055] Also provided herein are various methods for producing a plant exhibiting a useful trait that comprise selfing grafted plants comprising a scion grafted to rootstock that had been subjected to MSH1 gene suppression with another plant, or selfing progeny plants obtained from the grafted plant, and selecting one or more progeny plants obtained from the self for an improvement in the useful trait in comparison to a control plant to produce a plant exhibiting a useful trait. In certain embodiments, the selfed plant is a grafted plant where the rootstock source plant is the progeny of a parental plant that had been subjected to MSH1 gene suppression and the rootstock source plant itself was selected for and exhibits one or more useful traits. Control plants used as comparators to identify progeny of the self that exhibit an improvement in the useful trait include, but are not limited to: progeny of a self of a plant which lacks a graft to the rootstock, progeny of a self of a plant that has a graft to rootstock that had not been subjected to MSH1 gene suppression, and progeny of a self of a plant that is isogenic to the plant source of the scion of the grafted plant.

[0056] The following examples are included to demonstrate certain embodiments. It should be appreciated by those of skill in the art that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Examples

Example 1. Enhanced yields in Sorghum occurs in the F3 and F4 hybrid-like

generations but not the F5 generation.

[0057] Sorghum MSHl-dr plants used in these experiments were derived as described (Xu YZl, Arri eta-Monti el MP, Virdi KS, de Paula WB, Widhalm JR, Basset GJ, Davila JI, Elthon TE, Elowsky CG, Sato SJ, Clemente TE, Mackenzie SA. Plant Cell. 2011 Sep;23(9):3428- 41).

[0058] For development of RNAi transgene constructions, segments encoding 157 amino acids from the MSH1 C terminus were amplified from total cDNA of sorghum using primers zm-msf8 (SEQ ID NO: 1) and zm-msr8 (SEQ ID NO: 2). PCR products were cloned in forward and reverse orientation, separated by an intron sequence in a base vector:

pUCRNAi- intron, which harbors the second intron of the Arabidopsis small nuclear riboprotein (At4g02840), was graciously provided by H. Cerutti (University of Nebraska, Lincoln, NE). The vector pPTN290, a derivative of binary plasmid pPZP212, was used to construct the MSH1 -RNAi cassettes under the control of the maize (Zea mays)

Ubiquitinl promoter coupled with its first intron, with transcription terminated by the CaMV 35S terminator. Agrobacterium tumefaciens strain NTL4 was used for inoculating embryos from sorghum Tx430 lines. Six T₃ individuals displaying the MSHl-dr phenotype but null for the MSHl-RNAi transgene were used as females in crosses to wild type inbred Tx430 to derive F₁ seed. Another three T₃ individuals were used as males in the reciprocal crosses to Tx430. The number of F₁ plants derived from each cross ranged from 5 to 19 individuals. Parents and F₁ progeny were grown under greenhouse conditions on a 14 hr/10 hr day-night cycle with 28°C/22°C day-night temperatures. Self-pollinated seed of F₁ plants was harvested individually to generate corresponding F₂ families.

[0059] In all field plots, plants were thinned to a final density of 15 plants/m² and fertilized according to standard growing practices. The 2010 field experiment was used to propagate F₂ lines, and contained F₂ and wild type Tx430. The 2011 field experiment contained F₂, F₃, and F₄ lines randomized across seven blocks with 28 rows per block (alpha lattice design) and two field replicates. Replicates were augmented with wild type Tx430 (16 rows total).

[0060] For estimating grain yield, threshed panicles from three plants were pooled and converted to grams/m² based on final plant density, with 2-3 such measurements taken per row. Plants showing the DR phenotype were not included in phenotypic variation analysis.

[0061] The 2012 multi -location experiment included Lincoln, NE (40° 51 'N, 96° 35W) and Mead, NE (41° 9'N, 96° 24 W) sites, which received 178 mm and 158 mm of precipitation over the growing season, respectively. Within each location, lines were grown in two-row plots arranged in a randomized complete block design with two replicates. For this experiment, grain yield was estimated by taking threshed panicles from a meter-length area of each row and converting to grams/m².

[0062] Analysis of several direct lineages from F₂ to F₄ showed high response to selection for plant height but variable response for grain yield. Overall, gains in the F₄ were more modest compared to the F₃, implying progress can taper off by F₄ in self-pollinated lineages. Indeed, there is evidence that the F₃ generation can be the most vigorous. As a population, it appears to have slightly higher overall grain yield than the F₂ or F₄. Nevertheless, the population mean for grain yield in the F₄ remains higher than that of wild type Tx430 (see de la Rosa Santamaria R, Shao MR1, Wang G, Nino-Liu DO, Kundariya H, Wamboldt Y, Dweikat I, Mackenzie SA. PLoS One. 2014 Oct 27;9(10):el08407).

[0063] Field tests of the next F5 generation in Nebraska in the summer of 2013 indicated sorghum yields of the hybrid-like F5 generation was similar to that of the control Tx430 parent. The enhanced yield apparent in the F3 and F4 generations was dissipated in the F5 generation.

[0064] Having established that the candidate inbred T3 lines produced the highest yields in the hybrid-like the F3 and F4 generations, stored seeds of the T₃ inbred lines (displaying the MSHl-dr phenotype but null for the MSHl-RNAi transgene) can be used to repeatedly produce hybrid-like yield increases in F3 and F4 targeted generations, but not in the F5 generation. Thereby producing hybrid-like seeds with peak yields in a targeted number of generations from the seeds of the candidate inbred plant. Example 2. Enhanced yield in Soybeans occurs in the F4 hybrid-like generation but not the F6 generation.

[0065] Soybean variety Thorne was transformed with an RNAi construct containing a 556 bp cDNA segment (SEQ ID NO: 3) encoding amino acid 945 to 1131 of Soybean MShl, which represents the region after domain V to the end of GIY-YIG homing endonuclease domain, was PCR amplified from cDNA using primers Soy-MSF4 (SEQ ID NO: 4) and Soy-3Rbam (SEQ ID NO: 5). The PCR amplified fragment was cloned in forward and reverse orientation flanking the second intron of the Arabidopsis small nuclear riboprotein (At4g02840) in the pUCRNAi vector provided by Dr. H. Cerutti (University of Nebraska-Lincoln). The CaMV 35S promoter and transcription terminator regulate expression of the construction and the neomycin phosphotransferase II (npt II) reporter gene, and the insert is flanked by right border (RB) and left border (LB) integration sequences. Agrobacterium tumefaciens stain C58Cl/pMP90 was used for soybean transformation of the variety Thorne.

[0066] Two dr-inbred lines T8 and T9, displaying the MSHl-dr phenotype and null for the MSHl-RNAi transgene were used in reciprocal crosses. Self-pollinated seed of F₁ plants was harvested individually to generate corresponding F₂ families. Three individuals of each F2 family were chosen by their appearance and seed yields to use their seeds for F3 plant production. Seeds from F3 plants were either harvested in total (BULK) or from the top 50% of the highest yielding F3 plants (TOP 50%). These F4 seeds were planted in a replicated random plot field design in 2014. These results are shown in Table 1. The next generation of seeds from 2014 (Now F5 generations) were planted at four locations, each with three replica blocks in a random plot design, in the field in the summer of 2015. Although multiple lines had high hybrid-like yields in 2014 (Table 1), only one of these had high yields (7% above Thorne controls) in the F5 test in 2015, and none of the lines were above Thorne controls in the F6 generation in the field test in 2015.

[0067] These results demonstrate the enhanced yield apparent in the F4 generation was dissipated in the F5 generation, with the exception of one line whose yield advantage was dissipated in the F6 generation. These results demonstrate that the candidate inbred T8 lines produced high yields in the F4 but not the F5. The candidate inbred T9 lines produced high yields in the F4 but not the F6 generation. Thereby producing hybrid-like seeds from a selected parent line will produce peak yields in a targeted number of generations from the seeds of the candidate inbred plant, with the increased yields dissipating in later generations. The generation number of the peak yield and the generation number for which the increased yield dissipates are chosen by screening larger numbers of lines that have the desired number of generations for peak yield and dissipation of the higher yield.

Table 1. Field trial yields of Summer of 2014.

planted in 2014. T8 and T9 refer to epigenetically modified dr-inbred parental lines crossed to Thorne to produce Fl seeds. Fl plants and later generations were then self-pollinated for 3 generations to produce F4 seeds for the field trial. Symbols: WT or Wt is the Thorne parent line; Bulk means no selection in previous F3 generation while Top 50% means the top 50% of F3 plants were selected to produce F4 seeds; Individual lineages end with R-# or P-#. * p-value < 0.05; *** p-value < 0.001.

[0068] As various modifications could be made in the methods described herein without departing from the scope of the disclosure or claims, it is intended that all matter contained in the foregoing description shall be interpreted as illustrative rather than limiting. Thus, the breadth and scope of the present disclosure should not be limited by any of the above- described embodiments.

Claims

CLAIMS What is Claimed is:

1. A method of identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations comprising:

a) producing candidate inbred plants with new epigenetic modifications;

b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and

c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations.

2. A method of identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations comprising:

a) producing candidate inbred plants with new epigenetic modifications by Mshl suppression-restoration to produce said candidate inbred plants;

b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations; and,

3. A method to produce hybrid-like seeds with peak yields in a targeted number of generations comprising: a) producing candidate inbred plants with new epigenetic modifications;

b) screening said candidate inbred plants of step (a) for inbred plants that display higher yields than a reference plant line in a targeted number of generations of hybrid-like plants, wherein said hybrid-like plant lines are produced by crossing grafting a rootstock of a candidate inbred plant to a second parental plant and then self-pollinating the progeny for a targeted number of generations;

c) screening and selecting hybrid-like plant lines of step (b) with higher yields than a reference plant line in a targeted number of generations, for hybrid-like plant lines for which subsequent self-pollination for at least one generation produces plant populations with lower yields than the yield in the targeted number of generations, thereby identifying an inbred plant that produces hybrid-like seeds with peak yields in a targeted number of generations; and,

d) producing hybrid-like seeds with peak yields in a targeted number of generations from the seeds of the inbred plant identified in step (c).

4. A method to produce hybrid-like seeds with peak yields in a targeted number of generations comprising:

5. The method of any one of claims 1 to 4, wherein the targeted number of generations is selected from the group consisting of 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, 5 to 6, and 6 generations.

6. The method of any one of claims 1 to 4, wherein a single plant harvester is used to measure yields in single plants in one or more generations of step (b).

7. The method of any one of claims 1 to 4, wherein said candidate inbred plants of step

(a) are produced from an inbred parent which was suppressed for Mshl function or were derived from an ancestral plant suppressed for Mshl function.

8. The method of claim 7, wherein said candidate inbred plants are produced from two genetically diverse parental plants at least one of which was suppressed for Mshl function or were derived from an ancestral plant suppressed for Mshl function.

9. The method of claims 1 to 4, wherein seeds of said candidate inbred plants of step (a) are stored at about -18 to -20 C.

10. The method of any one of claims 1 to 4, wherein said hybrid-like plant lines of step

(b) are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant, wherein said second parental plant is isogenic to said candidate inbred plant prior to any epigenetic modification steps.

11. The method of any one of claims 1 to 4, wherein said hybrid-like plant lines of step (b) are produced by crossing or grafting a rootstock of a candidate inbred plant to a second parental plant, wherein said second parental plant is isogenic to said candidate inbred plant and wherein said second parental plant is derived from an ancestral plant suppressed for Mshl .

12. The method of any one of claims 1 to 4, wherein the hybrid-like seeds are rice, wheat, soybeans, cotton, oats, rye, or barley seeds.

13. The method of any one of claims 1 to 4, wherein the candidate inbred plants are produced in step (a) by suppressing expression of Mshl in a parent plant and recovering a candidate inbred plant wherein Mshl expression has been restored.

14. The method of any one of claims 1 to 4, wherein the candidate inbred plants are produced in step (a) by introducing a loss-of-function mutation into an endogenous Mshl gene of a parent plant, obtaining a first progeny plant of the parent plant wherein Mshl gene expression is suppressed by the mutation, and recovering from the first progeny plant the candidate inbred plant, wherein Mshl gene expression is restored in the candidate inbred plant.