US20180343816A1

US20180343816A1 - Methods and Compositions for Obtaining Useful Plant Traits

Info

Publication number: US20180343816A1
Application number: US16/056,024
Authority: US
Inventors: Sally Ann Mackenzie; Ying-Zhi Xu
Original assignee: NuTech Ventures Inc
Current assignee: NuTech Ventures Inc
Priority date: 2013-11-07
Filing date: 2018-08-06
Publication date: 2018-12-06
Also published as: US20150189842A1; US10058044B2

Abstract

The present disclosure provides methods for obtaining plants that exhibit useful traits by perturbation of organellar function in plants. Methods for identifying genetic loci that provide for useful traits in plants and plants produced with those loci are also provided. In addition, plants and grafted plants that exhibit, contain, or harbor the useful traits, parts of the plants including seeds, and products of the plants are provided as well as methods of using the plants. Recombinant DNA vectors and transgenic plants comprising those vectors that provide for organellar perturbation are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. Non-Provisional patent application claims the benefit of U.S. Provisional Patent Application No. 61/901,349, filed Nov. 7, 2013 and incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under a grant from the National Science Foundation (IOS 1126935). The government has certain rights to this invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named “46589_136656_SEQLST.txt”, which is 110,576 bytes in size (measured in operating system MS-Windows), contains 56 sequences, and which was created on Nov. 3, 2014, is contemporaneously filed with this specification by electronic submission (using the United States Patent Office EFS-Web filing system) and is incorporated herein by reference in its entirety.

BACKGROUND

Evidence exists in support of a link between environmental sensing and epigenetic changes in both plants and animals (Bonasio et al., Science 330, 612, 2010). Trans-generational heritability of these changes remains a subject of active investigation (Youngson et al. Annu. Rev. Genom. Human Genet. 9, 233, 2008). Previous studies have shown that altered methylation patterns are highly heritable over multiple generations and can be incorporated into a quantitative analysis of variation (Vaughn et al. 2007; Zhang et al. 2008; Johannes et al. 2009). Earlier studies of methylation changes in Arabidopsis suggest amenability of the epigenome to recurrent selection and also suggest that it is feasible to establish new and stable epigenetic states (F. Johannes et al. PLoS Genet. 5, e1000530 (2009); F. Roux et al. Genetics 188, 1015 (2011). Manipulation of the Arabidopsis met1 and ddmt mutants has allowed the creation of epi-RIL populations that show both heritability of novel methylation patterning and epiallelic segregation, underscoring the likely influence of epigenomic variation in plant adaptation (F. Roux et al. Genetics 188, 1015 (2011)). In natural populations, a large proportion of the epiallelic variation detected in Arabidopsis is found as CpG methylation within gene-rich regions of the genome (C. Becker et al. Nature 480, 245 (2011), R. J. Schmitz et al. Science 334, 369 (2011).
Induction of traits that exhibit cytoplasmic inheritance (Redei Mutat. Res. 18, 149-162, 1973; Sandhu et al. Proc Natl Acad Sci USA. 104:1766-70, 2007) or that exhibit nuclear inheritance by suppression of the MSH1 gene has also been reported (WO 2012/151254; Xu et al. Plant Physiol. Vol. 159:711-720, 2012).

SUMMARY

Methods for producing a plant having a useful trait that exhibits nuclear inheritance comprising the steps of: (a) selfing a first plant wherein said plant or a parent plant thereof is or had been subjected to perturbation of organellar function; (b) screening a population of progeny plants obtained from the selfed plant of step (a) for the useful trait; and, (c) selecting one or more progeny plants having the useful trait that exhibits nuclear inheritance and having recovered organellar function are provided herein. In certain embodiments of the methods, organellar function has been recovered in any of: (i) the selfed first plant in step (a); (ii) at least a portion of the progeny plants of step (b); or (iii) one or more of the selected progeny plants in step (c). In certain embodiments of the methods, the selfed first plant of step (a) exhibits a wild type phenotype or an improvement in a useful trait in comparison to a control plant. In certain embodiments, the selfed first plant of step (a) does not exhibit any MSH1-dr phenotypes. In certain embodiments, the selfed first plant of step (a) exhibits one or more MSH1-dr phenotypes. In certain embodiments, the selfed first plant of step (a) exhibits all MSH1-dr phenotypes. In certain embodiments of the methods, the perturbed organellar function is a plastid function selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function. In certain embodiments of the methods, the photosystem II function and/or sensor function is perturbed by suppressing expression of a gene selected from the group consisting of an MSH1, PPD3, a PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene. In certain embodiments of the methods, selfed plant in step (a) is an inbred plant. In certain embodiments of the methods, about 1% to about 45% of the population of progeny plants in step (b) are selected for the useful trait in step (c). In certain embodiments of any of the aforementioned methods, the method further comprises making a first cross of the selected progeny plant(s) of step (c) to one or more second plant(s). In certain embodiments of the aforementioned method, the selected progeny plant(s) and the second plant(s) used in the first cross are in a single heterotic group. In certain embodiments of the aforementioned methods, the second plant is isogenic to the plant or parent plant of step (a). In certain embodiments of the aforementioned methods, the second plant and the selected plant are in distinct heterotic groups. In certain embodiments of the aforementioned methods, the method further comprises the step of selecting of one or more progeny of the first cross that exhibit an improvement in a useful trait in comparison to progeny of a control cross. In certain embodiments the aforementioned methods, the selected progeny of the first cross comprise about 1% to about 45% of the population of progeny plants obtained from the first cross. In certain embodiments of the aforementioned methods, the plants used in the control cross are of the same heterotic group as the selected progeny plant(s) and the second plant(s), but had not been subjected to organellar perturbation. In certain embodiments of the aforementioned methods, the plants used in the control cross are isogenic to the selected plant and the second plant, but had not been subjected to organellar perturbation. In certain embodiments of the aforementioned methods, the methods further comprise the step of making a second cross of the one or more of the selected progeny plant(s) of the first cross to one or more third plants. In certain embodiments of the aforementioned methods, the methods further comprise the step of selecting of one or more progeny of the second cross that exhibit an improvement in a useful trait in comparison to progeny of a control cross. In certain embodiments of any of the aforementioned methods, the methods further comprise the second plant or a parent plant thereof had been subjected to perturbation of organellar function. In certain embodiments of any of the aforementioned methods, the selfed plant in step (a) is a plant wherein a scion is grafted to rootstock that had been subjected to perturbation of organellar function. In certain embodiments of any of the aforementioned methods, the organellar function is a plastid function or a mitochondrial function. In certain embodiments of any of the aforementioned methods, the useful trait is transmitted by using selected progeny plant(s) or progeny thereof as pollen donors. Also provided are plants or progeny thereof that exhibit a useful trait that are made by the aforementioned methods. Plant parts obtained from the plant or progeny thereof made by the aforementioned methods are also provided. In certain embodiments, the part is selected from the group consisting of a seed, leaf, stem, fruit, and a root. Processed plant products obtained from the plant parts are also provided. Clonal propagates obtained from the plants, the progeny thereof or from the plant parts are also provided.
Also provided herein are methods for producing a plant having a useful trait that exhibits nuclear inheritance comprising the steps of: (a) crossing one or more first plant(s) to one or more second plant(s), wherein at least said first plant(s) or a parent plant thereof is or had been subjected to perturbation of organellar function and wherein either: (i) the first plant or a parent plant thereof does not exhibit any MSH1-dr phenotypes; or (ii) wherein the first plant or a parent plant thereof exhibits one or more MSH1-dr phenotypes and the perturbation of does not comprise direct suppression of MSH 1 gene expression; (b) screening a population of progeny plants obtained from the cross of step (a) for the useful trait; and, (c) selecting one or more progeny plants having the useful trait that exhibits nuclear inheritance and having recovered organellar function, thereby producing a plant exhibiting a useful trait that exhibits nuclear inheritance. In certain embodiments of the methods, organellar function has been recovered in any of: (i) the first plant in step (a); (ii) at least a portion of the population of progeny plants of step (b); or (iii) one or more of the selected progeny plants in step (c). In certain embodiments of the methods, the first plant(s) of step (a) exhibit a wild type phenotype or an improvement in a useful trait in comparison to a control plant. In certain embodiments of the methods, about 1% to about 45% of the population of progeny plants in step (b) are selected for the useful trait in step (c). In certain embodiments of the methods, the perturbed organellar function is a plastid function selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function. In certain embodiments of the methods, the photosystem II function and/or sensor function is perturbed by suppressing expression of a gene selected from the group consisting of an MSH1, PPD3, a PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene, with the proviso that the gene is not MSH1 when first plant or a parent plant thereof exhibits one or more MSH1-dr phenotype(s). In certain embodiments of the methods, the first plant in step (a) is an inbred plant. In certain embodiments of any of the aforementioned methods, the method further comprises making a second cross of the selected progeny plant(s) of step (c) to one or more third plant(s). In certain embodiments of the aforementioned methods, the selected progeny plant(s) and the third plant are in a single heterotic group. In certain embodiments of the aforementioned methods, the third plant is isogenic to the first plant or parent plant of step (a). In certain embodiments of the aforementioned methods, the methods further comprise the step of selecting of one or more progeny of the second cross that exhibit an improvement in a useful trait in comparison to progeny of a control cross. In certain embodiments of the aforementioned methods, the selected progeny of the second cross comprise about 1% to about 45% of the population of progeny plants obtained from the second cross. In certain embodiments of the aforementioned methods, the methods comprise the step of making a third cross of the one or more of the selected progeny plants of the second cross to one or more fourth plants. In certain embodiments of the aforementioned methods, the second plants or a parent plant thereof had been subjected to perturbation of organellar function. In certain embodiments of the aforementioned methods, the third plants or a parent plant thereof had been subjected to perturbation of organellar function. In certain embodiments of any of the aforementioned methods, the first plant(s) or a parent plant thereof in step (a) that is or had been subjected perturbation of organellar function is a plant wherein a scion is grafted to grafted to rootstock that had been subjected to perturbation of organellar function. In certain embodiments of any of the aforementioned methods, the organellar function is a plastid or a mitochondrial function. In certain embodiments of the aforementioned methods, the useful trait is transmitted by using selected progeny plant(s) or progeny thereof as pollen donors. Also provided are plants or progeny thereof that exhibit a useful trait that are made by the aforementioned methods. Plant parts obtained from the plant or progeny thereof made by the aforementioned methods are also provided. In certain embodiments, the part is selected from the group consisting of a seed, leaf, stem, fruit, and a root. Processed plant products obtained from the plant parts are also provided. Clonal propagates obtained from the plants, the progeny thereof, or from the plant parts are also provided.
Also provided herein are methods of identifying a plant harboring a useful trait comprising the steps of: (a) crossing a candidate plant to a second plant, wherein the candidate plant is progeny of: (i) a selfed plant wherein said plant or a parent plant thereof is or had been subjected to perturbation of organellar function; or of (ii) a cross wherein at least one crossed plant or a parent plant thereof is or had been subjected to perturbation of organellar function; and, (b) identifying one or more progeny plants from the cross in step (a) that exhibit a useful trait to a greater extent than the candidate plant, the second plant, or a control plant, thereby identifying the candidate plant as a plant that harbors a useful trait. In certain embodiments of the methods, the control plant is progeny of a cross between; (i) a plant that is not progeny of a selfed plant, a crossed plant, or parent thereof that is or had been subjected to organellar perturbation; and (ii) a plant that is isogenic to the second plant. In certain embodiments of the methods, the selfed plant or a parent plant thereof in (i) or the crossed plant or parent plant thereof in (ii) does not exhibit any MSH1-dr phenotypes. In certain embodiments of the methods, the selfed plant or a parent plant thereof in (i) or the crossed plant or parent plant thereof in (ii) exhibits one or more MSH1-dr phenotypes. In certain embodiments of the methods, the perturbation does not comprise direct suppression of MSH 1 gene expression. In certain embodiments of any of the aforementioned methods, the candidate plant is used as a pollen donor in the crossing step (a). In certain embodiments of any of the aforementioned methods, the selfed plant in (i) or at least one of the plants used in the cross of (ii) that is or had been subjected perturbation of organellar function is a plant wherein a scion is grafted to grafted to rootstock that had been subjected to perturbation of organellar function. In certain embodiments of any of the aforementioned methods, the organellar function is a plastid or a mitochondrial function. In certain embodiments of any of the aforementioned methods, the identifying further comprises showing that the harbored trait exhibits nuclear inheritance. Also provided is a plant, progeny thereof; or seed thereof that harbors a useful trait, wherein said plant, progeny thereof or seed thereof is identified or identifiable by any of the aforementioned the methods.
Also provided herein are methods of identifying a organellar perturbation agent that comprises: (a) assaying one or more candidate agents for inhibition of an interaction of an MSH-1 protein or fragment thereof with at least one assistant protein selected from the group consisting of PPD3, PsbA (D1), a PsbO-1, a PsbO-2, PetC, CAD9, KAB1, GOS12, ELI3-1, STT3B, a fragment thereof and combinations thereof; and, (b) selecting an agent that inhibits the interaction of MSH-1 or fragment thereof with the assistant protein, thereby identifying a organellar perturbation agent. In certain embodiments of the methods, the fragment of MSH-1 comprises a peptide of at least about 10 amino acids located within Domain 2, 3, or 6 of MSH-1. In certain embodiments of the methods, the agent is selected from the group consisting of a compound, a peptide, and a peptidomimetic compound. In certain embodiments of the methods, the peptide is comprises a peptide of a peptide of at least about 10 amino acids located within Domain 2, 3, or 6 of MSH-1.
Plants comprising a scion grafted to rootstock that had been subjected to perturbation of organellar function are provided herewith. Such grafted plants can be used in methods for producing a plant exhibiting useful traits, methods for identifying one or more altered chromosomal loci in a plant that can confer a useful trait, and in methods for obtaining plants comprising modified chromosomal loci that can confer a useful trait. Such grafted plants that exhibit useful traits, progeny of the grafted plants exhibiting the useful traits, parts of the grafted or progeny plants including cells, leafs, stems, flowers and seeds, methods of using the grafted or progeny plants and plant parts, and products of those plants and plant parts, including processed products such as a feed or a meal are also provided herein.
Plants comprising a scion to which a rootstock had been grafted, where the rootstock is obtained from a plant or a parent plant thereof had been subjected to perturbation of organellar function are provided herein. In certain embodiments, the rootstock confers to the grafted plant or to the progeny thereof an improvement in a useful trait in comparison to a control plant which lacks a graft to the rootstock or in comparison to progeny of the control plant. In certain embodiments, the rootstock that is grafted to the scion in step (a) is obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation. In certain embodiments, the plant comprising rootstock obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation exhibits the useful trait. In certain embodiments, the organellar function that is perturbed is a plastid function selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function. In certain embodiments, the perturbation comprises suppression of a sensor gene selected from the group consisting of MSH1 and PPD3. In certain embodiments, the photosystem II function is perturbed by suppressing expression of a gene selected from the group consisting of an PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene. In certain embodiments, the control plant comprises either: (i) a scion grafted to rootstock that had not been subjected to organellar perturbation; or (ii) a whole plant that lacks any root graft and that had not been subjected to organellar perturbation.
Also provided are methods for producing a plant exhibiting a useful trait comprising the steps of: (a) obtaining a population of progeny plants from a grafted plant comprising a scion to which a rootstock had been grafted, wherein the rootstock is obtained from a plant or a parent plant thereof had been subjected to perturbation of organellar function; and, (b) selecting one or more progeny plants from the population, wherein the selected progeny plant exhibit an improvement in the useful trait in comparison to a control plant, thereby producing a plant that exhibits a useful trait. In certain embodiments, the population of progeny plants are obtained from seed of the grafted plant of step (a). In certain embodiments, the population of progeny plants are obtained from clonal propagates of the grafted plant of step (a). In certain embodiments, organellar function has been recovered in the rootstock that is grafted to the scion in step (a). In certain embodiments, the rootstock that is grafted to the scion in step (a) is obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation. In certain embodiments, the grafted plant comprising rootstock obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation exhibits the useful trait. In certain embodiments, the plant comprising rootstock obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation. In certain embodiments, the organellar perturbation is a perturbation of a plastid function is selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function. In certain embodiments, the perturbation comprises suppression of a sensor gene selected from the group consisting of MSH1 and PPD3. In certain embodiments, the photosystem II function was perturbed by suppressing expression of a gene selected from the group consisting of an PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene. In certain embodiments, the control plant comprises either: (i) a scion grafted to rootstock that had not been subjected to organellar perturbation; or (ii) a whole plant that lacks any root graft and that had not been subjected to organellar perturbation. In certain embodiments of any of the aforementioned methods, the 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 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 perturbation of organellar function. In certain embodiments, the epigenetic changes are associated with the improvement in the useful trait. In certain embodiments, the 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 perturbation of organellar function. In certain embodiments, the scion and/or the rootstock exhibit CG hypermethylation of a region encompassing a MSH1 locus in comparison to a control plant that had not been subjected to the organellar perturbation. In certain embodiments, the scion and/or the rootstock exhibit pericentromeric CHG hyper-methylation in comparison to a control plant that had not been subjected to the organellar perturbation. In certain embodiments, the 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 organellar perturbation. 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. 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. In certain embodiments, the plant part is selected from the group consisting of a seed, leaf, stem, fruit, and a root. Also provided are clonal propagates obtained from the plant or progeny thereof that were made by any of the aforementioned methods.
Plants comprising a scion that had been subjected to perturbation of organellar function grafted to rootstock that had not been subjected to plastid perturbation are also provided herewith. Such grafted plants can be used in methods for producing a plant exhibiting useful traits, methods for identifying one or more altered chromosomal loci in a plant that can confer a useful trait, and in methods for obtaining plants comprising modified chromosomal loci that can confer a useful trait. Such grafted plants that exhibit useful traits, progeny of the grafted plants exhibiting the useful traits, parts of the grafted or progeny plants including cells, leafs, stems, flowers and seeds, methods of using the grafted or progeny plants and plant parts, and products of those plants and plant parts, including processed products such as a feed or a meal are also provided herein. In certain embodiments, a tiller, shoot or other clonal propagate from the bottom rootstock of the grafted plant is used to regenerate a plant, progeny thereof, or seed therefrom that exhibit or contain the useful trait.
Plants comprising a scion to which a rootstock had been grafted, where the scion is obtained from a plant or a parent plant thereof had been subjected to perturbation of organellar function are provided herein. In certain embodiments, the scion confers to the grafted plant or to the progeny thereof an improvement in a useful trait in comparison to a control plant which lacks a graft to the rootstock or in comparison to progeny of the control plant. In certain embodiments, the scion that is grafted to the rootstock is obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation. In certain embodiments, the grafted plant comprising the scion obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation exhibits the useful trait. In certain embodiments, the organellar function that is perturbed is a plastid function selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function. In certain embodiments, the perturbation comprises suppression of a sensor gene selected from the group consisting of MSH1 and PPD3. In certain embodiments, the photosystem II function is perturbed by suppressing expression of a gene selected from the group consisting of an PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene. In certain embodiments, the control plant comprises either: (i) a scion that had not been subjected to organellar perturbation grafted to rootstock; or (ii) a whole plant that lacks any scion graft and that had not been subjected to organellar perturbation.
Also provided are methods for producing a plant exhibiting a useful trait comprising the steps of: (a) obtaining a population of progeny plants from a grafted plant comprising a scion to which a rootstock had been grafted, wherein the scion is obtained from a plant or a parent plant thereof had been subjected to perturbation of organellar function; and, (b) selecting one or more progeny plants from the population, wherein the selected progeny plant exhibit an improvement in the useful trait in comparison to a control plant, thereby producing a plant that exhibits a useful trait. In certain embodiments, the population of progeny plants are obtained from seed of the grafted plant of step (a). In certain embodiments, the population of progeny plants are obtained from clonal propagates of the grafted plant of step (a). In certain embodiments, the clonal propagates comprise shoots or tillers from the grafted plant. In certain embodiments, organellar function has been recovered in the scion that is grafted to the rootstock in step (a). In certain embodiments, the scion that is grafted to the rootstock in step (a) is obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation. In certain embodiments, the grafted plant comprising a scion obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation exhibits the useful trait. In certain embodiments, the plant comprises a scion obtained from a plant that was selected for the useful trait and that was derived from a parent plant that had been subjected to organellar perturbation. In certain embodiments, the organellar perturbation is a perturbation of a plastid function is selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function. In certain embodiments, the perturbation comprises suppression of a sensor gene selected from the group consisting of MSH1 and PPD3. In certain embodiments, the photosystem II function was perturbed by suppressing expression of a gene selected from the group consisting of an PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene. In certain embodiments, the control plant comprises either: (i) a scion that had not been subjected to organellar perturbation grafted to rootstock; or (ii) a whole plant that lacks any scion graft and that had not been subjected to organellar perturbation. In certain embodiments of any of the aforementioned methods, the 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 rootstock also 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 rootstock was obtained. In certain embodiments, the epigenetic change(s) are also present in the scion that had been subjected to perturbation of organellar function. In certain embodiments, the epigenetic changes are associated with the improvement in the useful trait. In certain embodiments, the scion contain(s) one or more epigenetic changes in one or more nuclear chromosomes that are absent from nuclear chromosomes of scion obtained from a plant or are absent from nuclear chromosomes of a parent plant thereof had not been subjected to perturbation of organellar function. In certain embodiments, the scion and/or the rootstock exhibit CG hypermethylation of a region encompassing a MSH1 locus in comparison to a control plant that had not been subjected to the organellar perturbation. In certain embodiments, the scion and/or the rootstock exhibit pericentromeric CHG hyper-methylation in comparison to a control plant that had not been subjected to the organellar perturbation. In certain embodiments, the 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 organellar perturbation. 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. 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. In certain embodiments, the plant part is selected from the group consisting of a seed, leaf stem, fruit, and a root. Also provided are clonal propagates obtained from the plant or progeny thereof that were made by any of the aforementioned methods. In certain embodiments, the clonal propagates are shoots or tillers.
Also provided are methods for producing a plant exhibiting a useful trait comprising the steps of: (a) crossing a first plant to a second plant, wherein the first plant is any of the aforementioned plants comprising a scion to which a rootstock had been grafted; and, (b) selecting one or more progeny plants obtained from the cross for an improvement in the useful trait in comparison to a control plant, thereby producing a plant exhibiting a useful trait. In certain embodiments, the control plant is selected from the group consisting of 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, and progeny of a self of the second plant. In certain embodiments, at least the scion of the first plant is from a different heterotic group than the second plant. In certain embodiments, the scion and the rootstock of the first plant are from a different heterotic group than the second plant. In certain embodiments, the scion and the rootstock of the first plant are both from the same heterotic group but are from a different heterotic group than the second plant. In certain embodiments, at least the scion of the first plant is from the same heterotic group as the second plant. In certain embodiments, the scion and the rootstock of the first plant are from the same heterotic group as the second plant. In certain embodiments the second plant and at least the scion of the first plant are isogenic. In certain embodiments, the second plant and the scion and the rootstock of the first plant are isogenic. In certain embodiments of any of the aforementioned methods, the second plant or a parent thereof had also been subjected to perturbation of organellar function. In certain embodiments of any of the aforementioned methods, the 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. Also provided are plants obtained by any of the aforementioned methods. Also provided are plant parts obtained from plants made by any of the aforementioned methods. In certain embodiments, the plant part is selected from the group consisting of a seed, leaf stem, fruit, and a root. Also provided are processed plant products obtained from plants made by any of the aforementioned methods or plant parts obtained from those plants.
Also provided are methods for producing a plant exhibiting a useful trait comprising the steps of: (a) selfing a plant, wherein the plant is any of the aforementioned plants comprising a scion to which a rootstock had been grafted or wherein the plant is a plant made by any of the aforementioned methods; and, (b) selecting one or more progeny plants obtained from the self for an improvement in the useful trait in comparison to a control plant, thereby producing a plant exhibiting a useful trait. In certain embodiments, the control plant is a progeny plant of a self of a plant which lacks a graft to the rootstock. In certain embodiments of any of the aforementioned methods, the 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. Also provided are plants obtained by any of the aforementioned methods. Also provided are plant parts obtained from plants made by any of the aforementioned methods. In certain embodiments, the plant part is selected from the group consisting of a seed, leaf, stem, fruit, and a root. Also provided are processed plant products obtained from plants made by any of the aforementioned methods or plant parts obtained from those plants.
Also provided are methods for producing a seed lot comprising: (i) growing a population of plants, wherein said population comprises two or more of any of the aforementioned plants comprising a scion to which a rootstock had been grafted and/or plants made by any of the aforementioned methods; (ii) selecting a first sub-population of plants exhibiting a useful trait; and, (ii) obtaining a seed lot from the first selected sub-population of step (i) or, optionally, repeating steps (i) and (ii) on a second population of plants grown from the seed obtained from the first selected sub-population of plants. Also provided are seed lots produced by the aforementioned methods, as well as plants, plant parts, and processed plant products obtained from the seed lots.
Also provided are methods for producing a seed lot comprising: (i) growing a population of plants, wherein said population comprises two or more of any of the aforementioned plants comprising a scion to which a rootstock had been grafted and/or plants made by any of the aforementioned methods; and (ii) obtaining a seed lot from the population. Also provided are seed lots produced by the aforementioned method as well as plants, plant parts, and processed plant products obtained from the seed lots.
Also provided are methods for identifying plants harboring a useful trait comprising the steps of: (a) crossing a candidate plant to a second plant, wherein the candidate plant is progeny of: (i) any of the aforementioned 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 had been subjected to perturbation of organellar function and/or plants made by any of the aforementioned methods; or (ii) a plant that had been subjected to perturbation of organellar function or progeny thereof; and, (b) identifying one or more progeny plants from the cross in step (a) that exhibit a useful trait to a greater extent than the candidate plant, the second plant, or a control plant, thereby identifying the candidate plant as a plant that harbors a useful trait. In certain embodiments of the methods, the control plant is progeny of a cross between a plant that is not progeny of a plant or a grafted plant that had been subjected to organellar perturbation and a plant that is isogenic to the second plant. Also provided are plants or progeny thereof that harbor a useful trait, wherein said plant or progeny thereof is identified or identifiable by any of the aforementioned methods.
Also provided are methods of identifying a plant harboring a useful trait comprising the steps of: (a) selfing a candidate plant, wherein the candidate plant is progeny of: (i) any of the aforementioned 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 perturbation of organellar function; or (ii) a plant that had been subjected to perturbation of organellar function or progeny thereof; and, (b) identifying one or more progeny plants from the self in step (a) that exhibit a useful trait to a greater extent than the candidate plant or a control plant, thereby identifying the candidate plant as a plant that harbors a useful trait. In certain embodiments of the methods, the control plant is progeny of a self of plant that is not progeny of a plant or a grafted plant that had been subjected to organellar perturbation but is otherwise isogenic to the candidate plant. Plants or progeny thereof that harbor a useful trait, wherein the plant or progeny thereof is identified or identifiable by the aforementioned methods are also provided.
In certain embodiments, any of the aforementioned plants, parental plants or progeny thereof; plant parts, or processed products thereof produced by the methods provided herein exhibit 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, organellar function has been recovered in the plant. In certain embodiments, the plants, parental plants, progeny thereof; plant parts, or processed products thereof contains one or more epigenetic changes in one or more nuclear chromosomes, wherein the epigenetic changes are absent from the nuclear chromosomes of a control plant, plant part, or processed product thereof. In certain embodiments, the epigenetic changes are associated with the improvement in the useful trait. In certain embodiments, the plants, parental plants, progeny thereof; plant parts, or processed products thereof exhibit CG hypermethylation of a region encompassing a MSH1 locus in comparison to a control plant that had not been subjected to the organellar perturbation. In certain embodiments, plants, parental plants, progeny thereof; plant parts, or processed products thereof exhibit pericentromeric CHG hyper-methylation in comparison to a control plant that had not been subjected to the organellar perturbation. In certain embodiments, plants, parental plants, progeny thereof, plant parts, or processed products thereof 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 organellar perturbation. In certain embodiments, the organellar perturbation comprises perturbation of plastid function or perturbation of mitochondrial function. In certain embodiments, the plant is selected from the group consisting of a crop plant, a tree, a bush, turf grass, pasture grass, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part of the specification, illustrate certain embodiments of the present disclosure. In the drawings:

FIGS. 1A, B, C, D, E, and F illustrate the enhanced growth phenotype of MSH1-epi lines in sorghum. The transgene and crossing procedure used to derive sorghum cpi-populations is indicated. A. The phenotype of the epi-F1 progeny derived from crossing Tx430×MSH1-dr. B. Field grown epi-F2, F3 and F4 sorghum lines show variation in plant architecture and height. C, Panicles from Tx430 (on left, 66 g, 8 mm stem) versus epi-F2 individual (on right, 112 g, 11 mm stem). D, Seed yield from the panicles shown in c. E, The MSH1-dr sorghum phenotype under field conditions. F. Sorghum MSH1-epiF2, epiF3 and epiF4 populations grown in progeny rows in the field in Summer, 2011. Wild-type inbred Tx430 is indicated. Dramatic variation visible in plant height, flowering time and plant architecture is apparently non-genetic; all plants shown are non-transgenic and Tx430 genotype.

FIG. 2A, B illustrate the enhanced phenotypic variation in sorghum MSH1-epiF2 lines. A, Boxplots of within-row field variance for several traits, with values normalized as a proportion of the maximum observed row variance for that trait. Differences in variances between the epi-F2 and wild-type populations were significant for plant height (Brown-Forsythe test, p<0.001) and grain yield (p<0.01). Method of harvesting biomass precluded within-row variance assessment. B, Example of the distribution of values for grain yield when measured per panicle, and plant height in an epi-F2 family (epi-F2 individuals derived from same parental cross) compared to wild-type.

FIG. 3A, B illustrate the phenotypic variation in sorghum MSH1-epiF2, F3 and F4 lines. A. Selection had varying results, with response for yield into the F3 generation, but not into the F4 generation. For each lineage, the mean generation performance is represented as a point. B. Boxplots of epiF2, F3, and F4 line means for various traits, giving a population-wide view of epi-line performance. Dashed lines indicate the 95% confidence interval for wild-type TX430 mean.

FIG. 4 illustrates the epi-type×environment interactions. Joint regression (with Wright modification) indicates differential response between lines to location.

FIG. 5 illustrates that the MSH1-F₄generation shows significant trait differences in multiple lines (ordered by increasing mean value) compared to wild-type Tx430. Means and 95% confidence intervals (error bars) were estimated by mixed model analysis. Only lines with stable within-row height and data for all five traits were used for mean comparison here. Type I error was conservatively controlled by Bonferroni adjustment (m=165, total number of comparisons including all traits).

FIG. 6 illustrates sample SSR marker analysis. Sorghum genomic DNAs were prepared from wildtype Tx430, Tx430 MSH1-DR line (transgene-null, displaying the dwarfed, tillered, delayed flowering phenotype), one epi-F2 and seven epi-F4 lines selected for phenotypic diversity. Sweet sorghum line ‘Wray’ was included as a control. The SSR marker shown is generated with SAM16073 primers. Arrow shows detected DNA polymorphism. M designates marker lane, with fragment sizes (bp) shown at left. The 1500 and 35 bp fragments are internal markers used to calibrate each lane.

FIG. 7 illustrates a sorghum genetic map with markers displaying heterozygous genotype.

FIG. 8 illustrates that no correlation was found between plant height and grain yield (Spearman's rho=0.02, p=0.83). Each point represents a line mean.

FIG. 9A, B illustrate that Epi-line×environment effects were detected from a multiple location experiment. A) Analysis for significant effects using a mixed model indicates that line, location, and line×location are all significant. Sample size N=121. B) Although three epi-F4 lines were similar to wild-type Tx430 in grain yield when grown in one location (Havelock), significant differences emerged when grown another location with a more challenging environment (Mead). Data were collected from a field trial in 2012.

FIG. 10A, B. Evidence of growth variation in millet Tift23BDE following suppression of MSH1 in a T0 plant and recovery of MSH1 function in T3 progeny. (A) Wildtype control plant (left) and transgene-null T3 plant with bagged panicles (right; obtained from selfed progeny of a selfed MSH1-suppressed T0 plant). (B) Sample panicle from wildtype Tift23BDE control line that had not been subjected to MSH1 suppression. (C) Sample panicle from the robust, transgene-null T3 millet line showing markedly larger size.

FIG. 11 A, B, C, D. Drought tolerant and robust 2nd generation msh1 mutants. A) Late flowering 2nd generation plants in flat. Note the large plant in the center (arrow). B) Large 2nd generation plant (arrow) compared to two normal flowering/normal phenotype siblings. C) Four days after drought conditions 2nd generation msh1 plants (left) remain green while Col-0 (yellow stake) and 1st generation msh1 plants (green flags) fail to recover. D) Comparison of the large 2nd generation msh1 plant (left) to Col-0 (right) four days after drought conditions.

FIG. 12 A, B, C, D. Robust growth in a transgene-null line of Rutgers tomato obtained by selfing progeny of a selfed T0 tomato plant subjected to MSH1 suppression with a transgene. (A) Rutgers (left) is wild-type control. Transgene-plus T5 generation self containing MSH1 suppressing transgene is designated “T17-12-15-4-9-4 (+ transgene)” (second from left). Transgene-null T5 generation robust self is designated “T17-12-15-4-6-3 (− transgene)” (third from left). The epiF2 line (far right) was derived by crossing a dwarf msh1-dr type×Rutgers wildtype to generate an F2 population. (B) Single plant fruit production from Rutgers and the transgene negative T6 generation robust inbred T17-12-15-4-6-1-6. (C) Graph of fruit production in T5 generation progeny from the robust selection of T5 generation progeny of T17-12-15-4-6. (D) Graph of total yield from Rutgers versus the T6 generation robust selection of selfed progeny.

DESCRIPTION

As used herein, the phrase “chromosomal modification” refers to any of a) an “altered chromosomal loci” and an “altered chromosomal locus”; b) “mutated chromosomal loci”, a “mutated chromosomal locus”, “chromosomal mutations” and a “chromosomal mutation”; or c) a transgene.
As used herein, the phrases “altered chromosomal loci” (plural) or “altered chromosomal locus (singular) refer to portions of a chromosome that have undergone a heritable and reversible epigenetic change relative to the corresponding parental chromosomal loci. Heritable and reversible genetic changes in altered chromosomal loci include, but are not limited to, methylation of chromosomal DNA, and in particular, methylation of cytosine residues to 5-methylcytosine residues, and/or post-translational modification of histone proteins, and in particular, histone modifications that include, but are not limited to, acetylation, methylation, ubiquitinylation, phosphorylation, and sumoylation (covalent attachment of small ubiquitin-like modifier proteins). As used herein, “chromosomal loci” refer to loci in chromosomes located in the nucleus of a cell.
As used herein, the phrase “clonal propagate” refers to a plant or progeny thereof obtained from a plant cell. Clonal propagates can be obtained by methods including but not limited to regenerating whole plants from plant cells, plant embryos, cuttings, and the like. Various techniques used for such clonal propagation include, but are not limited to, meristem culture, somatic embryogenesis, thin cell layer cultures, adventitious shoot culture, and callus culture.
As used herein, the term “comprising” means “including but not limited to”.
As used herein, the phrase “crop plant” includes, but is not limited to, cereal, seed, grain, fruit, and vegetable crop plants.
As used herein, the phrases “mutated chromosomal loci” (plural) (plural), “mutated chromosomal locus” (singular), “chromosomal mutations” and “chromosomal mutation” refer to portions of a chromosome that have undergone a heritable genetic change in a nucleotide sequence relative to the nucleotide sequence in the corresponding parental chromosomal loci. Mutated chromosomal loci comprise mutations that include, but are not limited to, nucleotide sequence inversions, insertions, deletions, substitutions, or combinations thereof. In certain embodiments, the mutated chromosomal loci can comprise mutations that are reversible. In this context, reversible mutations in the chromosome can include, but are not limited to, insertions of transposable elements, defective transposable elements, and certain inversions. In certain embodiments, the chromosomal loci comprise mutations are irreversible. In this context, irreversible mutations in the chromosome can include, but are not limited to, deletions.
As used herein, the term “discrete variation” or “V_D” refers to distinct, heritable phenotypic variation, that includes one or more but not all MSH1-dr traits of male sterility, dwarfing, variegation, and/or delayed flowering time that can be observed either in any combination or in isolation.
As used herein, the phrase “heterologous sequence”, when used in the context of an operably linked promoter, refers to any sequence or any arrangement of a sequence that is distinct from the sequence or arrangement of the sequence with the promoter as it is found in nature. As such, an MSH1 promoter can be operably linked to a heterologous sequence that includes, but is not limited to, MSH1 sense, MSH1 antisense, combinations of MSH1 antisense and MSH1 sense, and other MSH1 sequences that are distinct from, or arranged differently than, the operably linked sequences of the MSH1 transcription unit as they are found in nature.
As used herein, the phrase “MSH1-dr phenotypes refers to phenotypes that include leaf variegation, cytoplasmic male sterility (CMS), a reduced growth-rate phenotype, delayed or non-flowering phenotype, leaf wrinkling, increased plant tillering, decreased height, decreased internode elongation, plant tillering, and/or stomatal density changes that are observed in plants subjected to suppression of organellar perturbation target genes.
As used herein, the phrase “organellar perturbation target genes” includes plastid perturbation target genes and mitochondrial perturbation target genes. Organellar target genes that can be suppressed to produce an MSH1-dr phenotype include, but not limited to, MSH1 and PPD3.
As used herein, the phrase “organellar perturbation” includes perturbation of plastid function and/or mitochondrial functions. For proteins that function in plastids and mitochondria, organellar perturbation can include perturbation of plastid function, perturbation of mitochondrial function, or perturbation of both functions. Proteins that function in both plastids and mitochondria include, but are not limited to, the MSH1 protein.
As used herein, the term “heterotic group” refers to genetically related germplasm that produce superior hybrids when crossed to genetically distinct germplasm of another heterotic group.
As used herein, the term “progeny” refers to any one of a first, second, third, or subsequent generation obtained from a parent plant or plant cell.
As used herein, the phrase “quantitative variation” or “V_Q” refers to phenotypic variation that is observed in individual progeny lines derived from outcrosses of plants where MSH1 expression was suppressed and that exhibit discrete variation to other plants.
As used herein the terms “microRNA” or “miRNA” refers to both a miRNA that is substantially similar to a native miRNA that occurs in a plant as well as to an artificial miRNA. In certain embodiments, a transgene can be used to produce either a miRNA that is substantially similar to a native miRNA that occurs in a plant or an artificial miRNA.
As used herein, the phrase “obtaining a nucleic acid associated with the altered chromosomal locus” refers to any method that provides for the physical separation or enrichment of the nucleic acid associated with the altered chromosomal locus from covalently linked nucleic that has not been altered. In this context, the nucleic acid does not necessarily comprise the alteration (i.e. such as methylation) but at least comprises one or more of the nucleotide base or bases that are altered. Nucleic acids associated with an altered chromosomal locus can thus be obtained by methods including, but not limited to, molecular cloning, PCR, or direct synthesis based on sequence data.
The phrase “operably linked” as used herein refers to the joining of nucleic acid sequences such that one sequence can provide a required function to a linked sequence. In the context of a promoter, “operably linked” means that the promoter is connected to a sequence of interest such that the transcription of that sequence of interest is controlled and regulated by that promoter. When the sequence of interest encodes a protein and when expression of that protein is desired, “operably linked” means that the promoter is linked to the sequence in such a way that the resulting transcript will be efficiently translated. If the linkage of the promoter to the coding sequence is a transcriptional fusion and expression of the encoded protein is desired, the linkage is made so that the first translational initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made so that the first translational initiation codon contained in the 5′ untranslated sequence associated with the promoter is linked such that the resulting translation product is in frame with the translational open reading frame that encodes the protein desired. Nucleic acid sequences that can be operably linked include, but are not limited to, sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5′ untranslated regions, introns, protein coding regions, 3′ untranslated regions, polyadenylation sites, and/or transcriptional terminators), sequences that provide DNA transfer and/or integration functions (i.e., site specific recombinase recognition sites, integrase recognition sites), sequences that provide for selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker functions (i.e., reporter genes), sequences that facilitate in vitro or in vivo manipulations of the sequences (i.e., polylinker sequences, site specific recombination sequences, homologous recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomous replication sequences, centromeric sequences).
As used herein, the phrases “suppressing expression” of a gene refer to any genetic or environmental manipulation that provides for decreased levels of functional gene activity in a plant or plant cell relative to the levels of functional gene activity that occur in an otherwise isogenic plant or plant cell that had not been subjected to this genetic or environmental manipulation.
As used herein, the term “transgene”, in the context of a chromosomal modification, refers to any DNA from a heterologous source that has been integrated into a chromosome that is stably maintained in a host cell. In this context, heterologous sources for the DNA include, but are not limited to, DNAs from an organism distinct from the host cell organism, species distinct from the host cell species, varieties of the same species that are either distinct varieties or identical varieties, DNA that has been subjected to any in vitro modification, recombinant DNA, and any combination thereof.
As used herein, the term “non-regenerable” refers to a plant part or plant cell that cannot give rise to a whole plant.
To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.
Methods for introducing heritable and epigenetic and/or genetic variation that result in plants that have useful traits exhibiting nuclear inheritance are provided herewith along with plants, plant seeds, plant parts, plant cells, and processed plant products obtainable by these methods. In certain embodiments, methods provided herewith can be 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, methods provided herewith can be used to introduce epigenetic and/or genetic variation into plants that are also amenable to hybridization.
Also provided herein are grafted plants comprising a scion to which a rootstock had been grafted, wherein either the scion or the rootstock is obtained from a plant or a parent plant thereof had been subjected to perturbation of organellar function, as well as progeny plants and clonal propagates obtained from the grafted plant. Such scions or 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.
Scions and rootstocks useful for introducing epigenetic and/or genetic variation into plants can be obtained from a variety of scion or rootstock source plants that had been subjected to organellar perturbation. In certain embodiments, the scion or rootstock source plant is a plant that had itself been subjected to organellar perturbation. In other embodiments, the scion or rootstock source plant is the progeny of a parental plant that had itself been subjected to organellar perturbation. Various methods of making scion or rootstock source plants by organellar perturbation are provided herein. Plants that can serve as scion or rootstock source plants and methods of making such plants are also disclosed in U.S. Patent Application Publication No. 20120284814, U.S. patent application Ser. No. 14/454,518 and U.S. patent application Ser. No. 14/495,498, which are all co-assigned and are each specifically incorporated herein by reference in their entireties. Methods for obtaining grafted plants and the progeny thereof by grafting rootstock subjected to plastid perturbation are also provided in co-assigned U.S. Provisional patent application Ser. No. 14/495,498, which is specifically incorporated herein by reference in its entirety.
In certain embodiments where the plant, a scion source plant, a rootstock source plant, or a parental plant thereof; had been subjected to organellar perturbation, a population of progeny plants obtained from the plant or 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 plant or the grafted plant comprising the scion or rootstock had been subjected to organellar perturbation 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 plants or the grafted plant comprising the scion or 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.
In certain embodiments where the plant, the scion source plant, or the rootstock source plant is the progeny of a parental plant that had been subjected to organellar perturbation, the plant, the scion source plant, or 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. Grafting a scion from a plant that had been selected for a useful trait to rootstock from a plant that does not exhibit the trait can impart the trait to the resultant grafted plant or to progeny thereof. Resultant plants, 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.
In plants, grafted plants or progeny thereof, perturbation of organellar function in the plants, scions, or rootstock can be continuous and ongoing or can be transient. Non-limiting and useful methods for effecting continuous and ongoing perturbation of organellar function in the plants, scions, or rootstock include suppressing expression of a organellar perturbation target gene with mutations in the endogenous gene or with a transgene that yields a product that suppresses expression of the endogenous gene. Alternatively, the perturbation of organellar function in the plants, scions, or rootstock can be transient or have occurred in a parental plant from which the plant or rootstock was obtained but not in the rootstock that was used in the graft. Non-limiting and useful methods for effecting transient suppressing of organellar function in the plants, scions, or rootstock include suppressing expression of a organellar perturbation 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, with a heterozygous transgene insert that is removed from the rootstock by segregation, or by use of a Viral Induced Gene Silencing (VIGS) vector that suppresses expression of a organellar perturbation target gene. Any of the methods described herein for restoring organellar function after perturbation can be used to generate the plants, scions, or rootstock used in certain embodiments.
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^thEdition. 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.
Plants, progeny thereof; scions thereof, or rootstocks thereof subjected to organellar perturbation or obtained from a parental plant that had been subjected to organellar perturbation can exhibit modifications of one or more nuclear chromosomes. In certain embodiments, such plants, progeny thereof scions, or 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 suppression of an MSH1 target gene include, but are not limited to, those patterns disclosed in Example 2 and Example 4 of U.S. patent application Ser. No. 14/454,518, the data and disclosure of which is specifically incorporated herein by reference in its entirety. In certain embodiments, a scion or rootstock of first generation progeny of a plant subjected to suppression of a organellar perturbation target gene will exhibit CG differentially methylated regions (DMR) of various discrete chromosomal regions that include, but are not limited to, regions that encompass the MSH1 locus. In certain embodiments, a CG hypermethylated region that encompasses the MSH1 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 organellar perturbation target gene will also exhibit changes in plant defense and stress response gene expression. In certain embodiments, a plant, progeny thereof, a scion, a rootstock grafted thereto, 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 organellar perturbation target 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. Pat. No. 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 organellar perturbation target 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 organellar perturbation target 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 organellar perturbation. In certain embodiments, such aforementioned changes in the methylation patterns exhibited by plants, progeny thereof, scions that are grafted to the rootstocks, rootstocks that are grafted to the scions, 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 plant or grafted plant.
Also provided herein are various methods for producing a plant exhibiting a useful trait that comprise crossing plants that had been subjected to perturbation of organellar function or grafted plants comprising a scion grafted to rootstock that had been subjected to perturbation of organellar function with another plant or a scion that had been subjected to perturbation of organellar function grafted to rootstock, or crossing progeny plants obtained from the grafted plant with another second 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 perturbation of organellar function, a scion that had been subjected to perturbation of organellar function grafted to rootstock, progeny plants obtained from a grafted plant comprising a scion grafted to rootstock that had been subjected to perturbation of organellar function, any other ungrafted plant that had been subjected to perturbation of organellar function, or any other ungrafted plant obtained from one or more parental plants that had been subjected to perturbation of organellar function. 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 perturbation of organellar function. 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 first plant or 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 than the second plant.
Also provided herein are various methods for producing a plant exhibiting a useful trait that comprise selfing plants that had been subjected to perturbation of organellar function or grafted plants comprising scions or rootstocks that had been subjected to perturbation of organellar function with another plant, or selfing progeny plants obtained from the plant OR 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 the progeny of a parental plant that had been subjected to organellar perturbation. In certain embodiments, the selfed plant is the progeny of a parental plant that had been subjected to organellar perturbation that was selected for and exhibits one or more useful traits. 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 organellar perturbation 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 that was not subjected to organellar perturbation, 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 organellar perturbation, and progeny of a self of a plant that is isogenic to the plant source of the scion of the grafted plant.
In certain embodiments, useful traits provided herein can be exhibited to a greater extent in subsequent generations of plants that are obtained from any of the plants, grafted plants, parental plants, or parental plant cells that had been subjected to organellar perturbation that are provided herein. As such, a given initial plant obtained from a parent plant that was subjected to organellar perturbation can be selfed to obtain first, second, third, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial plant or in comparison to a control plant. An initial plant subjected to organellar perturbation, or an initial grafted plant comprising a scion grafted to rootstock subjected to organellar perturbation or to rootstock obtained from a parent plant that had been subjected to organellar perturbation, can be selfed to obtain first, second, third, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial plant, the initial grafted plant or in comparison to a control plant. In other embodiments, a given initial plant or initial grafted plant obtained from a parent plant that was subjected to organellar perturbation can be outcrossed to obtain F1, F2, F3, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial plant or in comparison to a control plant. In certain embodiments, a useful trait harbored by an initial plant or an initial grafted plant is not exhibited, or is exhibited to a lesser degree extent, in the initial plant or an initial grafted plant. However, the useful trait harbored by such an initial plant or an initial grafted plant is exhibited or is exhibited to a greater extent in progeny obtained by outcrossing the initial plant or the initial grafted plant to another plant. A useful trait harbored by such an initial plant or an initial grafted plant can also be exhibited or is exhibited to a greater extent in progeny obtained by selfing the initial plant or the initial grafted plant. In certain embodiments, plants or grafted plants that are selfed or outcrossed can be inbred lines. In certain embodiments, a useful trait harbored by an inbred line is not exhibited, or is exhibited to a lesser degree extent, in the inbred line. However, the useful trait harbored by such inbred lines is exhibited or is exhibited to a greater extent in progeny obtained by outcrossing the inbred line to another plant. An initial plant or an initial grafted plant comprising a scion grafted to rootstock subjected to organellar perturbation or to rootstock obtained from a parent plant that had been subjected to organellar perturbation can be outcrossed to obtain F1, F2, F3, or later generations of progeny that exhibit a given useful trait to a greater extent in comparison to either the initial plant or the initial grafted plant or in comparison to a control plant. Outcrosses of such initial plants or grafted plants can be to isogenic plants or to genetically distinct plants. In the methods provided herein, initial or subsequent generations of progeny obtained from such selfs or crosses can thus be selected for useful traits. The methods provided herein also permit the identification of plants that harbor, but do not necessarily exhibit to a full extent, various useful traits.
In certain embodiments, methods provided herewith involve suppressing expression of plant organellar perturbation target genes, restoring expression of a functional plant organellar perturbation target gene, and selecting progeny plants that exhibit one or more useful traits. In certain embodiments, these useful traits are associated with either one or more altered chromosomal loci that have undergone a heritable and reversible epigenetic change.
In certain embodiments, methods for selectively suppressing expression of plant organellar perturbation target genes in sub-populations of cells found in plants that contain plastids referred to herein as “sensory plastids” are provided. Sensory plastids are plastids that occur in cells that exhibit preferential expression of at least the MSH1 promoter. In certain embodiments, MSH1 and other promoters active in sensory plastids can thus be operably linked to a heterologous sequence that perturbs plastid function to effect selective suppression of genes in cells containing the sensory plastids. In certain embodiments, MSH1 and other promoters active in sensory plastids can thus be operably linked to a heterologous sequence that perturbs mitochondrial function to effect selective suppression of genes in cells containing the sensory plastids. In addition to the distinguishing characteristic of expressing MSH1, such cells containing sensory plastids can also be readily identified as their plastids are only about 30-40% of the size of the chloroplasts contained within mesophyll cells. Other promoters believed to be active in sensory plastids include, but are not limited to, PPD3 gene promoters. Selective suppression of organellar perturbation target genes in cells containing sensory plastids can trigger epigenetic changes that provide useful plant traits. Suppression of plant plastid perturbation target genes including but not limited to, photosynthetic components, in specific sub-sets of plant cells that contain the sensory plastids is preferred as suppression of those genes in most other plant cell types is detrimental or lethal to the plant due to impairment of its photosynthetic or other capabilities.
Plastid perturbation target genes that can be suppressed by various methods provided herein to trigger epigenetic or other changes that provide useful traits include, but are not limited to, genes that encode components of plant plastid thylakoid membranes and the thylakoid membrane lumen. In certain embodiments, the plastid perturbation target genes are selected from the group consisting of sensor, photosystem I, photosystem II, the NAD(P)H dehydrogenase (NDH) complex of the thylakoid membrane, the Cytochrome b6f complex, and plastocyanin genes. A non-limiting and useful list of plastid pertubation targets is provided in Table 1. The plastid functions of the organellar perturbation target MSH1 and PPD3 genes can be suppressed to impart useful traits. However, embodiments where perturbation of both mitochondrial and plastid functions of the MSH1 and the PPD3 genes, perturbation of only any mitochondrial functions of the MSH1 and PPD3 genes, or perturbation of only any plastid functions of the MSH1 and PPD3 genes are used to impart the useful traits are also provided herein.

TABLE 1

Plastid Perturbation Target Genes

		Target Genes Database
		Accession Numbers and/or
Category	Gene name(s) and/or Activity	SEQ ID NO

Sensor	MSH1	SEQ ID NO: 1, 3-11.
Sensor	PPD3	AT1G76450; SEQ ID NO: 16-40
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAG AT1G55670.1
	G, PSAG
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAD-2 AT1G03130.1
	D-2, PSAD-2
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAO AT1G08380
	O, PSAO
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAK AT1G30380.1
	K, PSAK
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAF AT1G31330.1
	F, PSAF
Photosystem I	Photosystem I PsaN, reaction	PsaN AT1G49975.1
	centre subunit N
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAH-2, PSAH2, PSI-H
	H-2, PHOTOSYSTEM I	AT1G52230.1
	SUBUNIT H2, PSAH-2,
	PSAH2, PSI-H
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAE-2 AT2G20260.1
	E-2, PSAE-2
Photosystem I	PHOTOSYSTEM I P	PSAP AT2G46820.1
	SUBUNIT, PLASTID
	TRANSCRIPTIONALLY
	ACTIVE 8, PSAP, PSI-P,
	PTAC8, THYLAKOID
	MEMBRANE
	PHOSPHOPROTEIN OF 14 KDA,
	TMP14
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAH-1 AT3G16140.1
	H-1, PSAH-1
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAD-1AT4G02770
	D-1, PSAD-1
Photosystem I	PHOTOSYSTEM I SUBUNIT	PSAL AT4G12800
	L, PSAL
Photosystem I	PSAN	PSAN AT5G64040
	LHCA5, PHOTOSYSTEM I	LHCA5 AT1G45474
	LIGHT HARVESTING
	COMPLEX GENE 5
Photosystem II	PsbY	PsbY AT1G67740
Photosystem II	PsbW	PsbW AT2G30570
Photosystem II	PsbW-like	PsbW-like AT4G28660
Photosystem II	PsbX	PsbX AT2G06520
Photosystem II	PsbR	PsbR AT1G79040
Photosystem II	PsbTn	PsbTn AT3G21055
Photosystem II	PsbO-1	PsbO-1 AT5G66570
Photosystem II	PsbO-2	PsbO-2 AT3G50820
Photosystem II	PsbP1	PsbP1 AT1G06680
Photosystem II	PsbP2	PsbP2 At2g30790
Photosystem II	PsbS	PsbS AT1G44575
Photosystem II	PsbQ-1	PsbQ-1, AT4G21280
Photosystem II	PsbQ-2,	PsbQ-2, AT4G05180
Photosystem II	PPL1	PPL1 At3g55330
Photosystem II	PSAE-1	PSAE-1 AT4G28750
Photosystem II	LPA2	LPA2 AT5G51545
Photosystem II	PsbQ-like PQL1	PQL1 AT1G14150
Photosystem II	PsbQ-like PQL2	PQL2 AT3G01440
Photosystem II	PsbQ-like PQL3	PQL3 AT2G01918
NAD(P)H dehydrogenase	PHOTOSYNTHETIC NDH	PPL2 At2g39470
(NDH) Complex	SUBCOMPLEX L 1, PNSL1,
	PPL2, PSBP-LIKE PROTEIN 2
NAD(P)H dehydrogenase	NAD(P)H	NDH48 AT1G15980
(NDH) Complex	DEHYDROGENASE
	SUBUNIT 48, NDF1, NDH-
	DEPENDENT CYCLIC
	ELECTRON FLOW 1,
	NDH48, PHOTOSYNTHETIC
	NDH SUBCOMPLEX B 1,
	PNSB1
NAD(P)H dehydrogenase	NDF6, NDH DEPENDENT	NDF6 AT1G18730
(NDH) Complex	FLOW 6,
	PHOTOSYNTHETIC NDH
	SUBCOMPLEX B 4, PNSB4
NAD(P)H dehydrogenase	NAD(P)H	NDH45 AT1G64770
(NDH) Complex	DEHYDROGENASE
	SUBUNIT 45, NDF2, NDH-
	DEPENDENT CYCLIC
	ELECTRON FLOW 1,
	NDH45, PHOTOSYNTHETIC
	NDH SUBCOMPLEX B 2,
	PNSB2
NAD(P)H dehydrogenase	NDF5, NDH-DEPENDENT	NDF5 AT1G55370
(NDH) Complex	CYCLIC ELECTRON FLOW 5
NAD(P)H dehydrogenase	CHLORORESPIRATORY	NDHL AT1G70760
(NDH) Complex	REDUCTION 23, CRR23,
	NADH DEHYDROGENASE-
	LIKE COMPLEX L, NDHL
NAD(P)H dehydrogenase	NAD(P)H:PLASTOQUINONE	NDHO AT1G74880
(NDH) Complex	DEHYDROGENASE
	COMPLEX SUBUNIT O,
	NADH DEHYDROGENASE-
	LIKE COMPLEX), NDH-O,
	NDHO
NAD(P)H dehydrogenase	PIFI, POST-ILLUMINATION	PIFI AT3G15840
(NDH) Complex	CHLOROPHYLL
	FLUORESCENCE
	INCREASE
NAD(P)H dehydrogenase	NDF4, NDH-DEPENDENT	NDF4AT3G16250
(NDH) Complex	CYCLIC ELECTRON FLOW
	1, PHOTOSYNTHETIC NDH
	SUBCOMPLEX B 3, PNSB3
NAD(P)H dehydrogenase	NADH DEHYDROGENASE-	NDHM AT4G37925
(NDH) Complex	LIKE COMPLEX M, NDH-M,
	NDHM, SUBUNIT NDH-M
	OF
	NAD(P)H:PLASTOQUINONE
	DEHYDROGENASE
	COMPLEX
NAD(P)H dehydrogenase	FK506-BINDING PROTEIN	AT4G39710
(NDH) Complex	16-2, FKBP16-2,
	PHOTOSYNTHETIC NDH
	SUBCOMPLEX L 4, PNSL4
NAD(P)H dehydrogenase	CYCLOPHILIN 20-2,,	PNSL5 AT5G13120
(NDH) Complex	CYCLOPHILIN 20-2, CYP20-
	2, PHOTOSYNTHETIC NDH
	SUBCOMPLEX L 5, PNSL5
NAD(P)H dehydrogenase	CHLORORESPIRATORY	NDHU AT5G21430
(NDH) Complex	REDUCTION L, CRRL,
	NADH DEHYDROGENASE-
	LIKE COMPLEX U, NDHU
NAD(P)H dehydrogenase	CHLORORESPIRATORY	CRR7 AT5G39210
(NDH) Complex	REDUCTION 7, CRR7
NAD(P)H dehydrogenase	NAD(P)H	NDH18 AT5G43750
(NDH) Complex	DEHYDROGENASE 18,
	NDH18, PHOTOSYNTHETIC
	NDH SUBCOMPLEX B 5,
	PNSB5
NAD(P)H dehydrogenase	NADH DEHYDROGENASE-	NDHN AT5G58260
(NDH) Complex	LIKE COMPLEX N, NDHN
Cytochrome b6f complex	Rieske iron-sulfur protein	PetC At4g03280
	containing a [2Fe—2S] cluster,
	OetC
Cytochrome b6f complex	ferredoxin: NADP-reductase	FNR1 AT5G66190
	[FNR1 and FNR2]	FNR2 AT1G20020
plastocyanin	PETE1, PLASTOCYANIN 1	PETE1 AT1G76100
plastocyanin	PETE2, PLASTOCYANIN 2	PETE2 AT1G20340
other	PPD1, PSBP-DOMAIN	PPD1 At4g15510
	PROTEIN1
other	PPD2, PSBP-DOMAIN	PPD2 At2g28605
	PROTEIN2
other	PPD4, PSBP-DOMAIN	PPD4 At1g77090
	PROTEIN4
other	PPD5, PSBP DOMAIN	PPD5 At5g11450
	PROTEIN 5
other	PPD6, PSBP-DOMAIN	PPD6 At3g56650
	PROTEIN 6
other	PPD7, PSBP-DOMAIN	PPD7 At3g05410
	PROTEIN 7
MSH1 interacting proteins	CAD9 (CINNAMYL ALCOHOL	CAD9 AT4G39330
identified by Yeast Two Hybrid	DEHYDROGENASE 9); binding/
	catalytic/oxidoreductase/zinc
	ion binding
MSH1 interacting proteins	KAB1 (POTASSIUM	KAB1 AT1G04690
identified by Yeast Two Hybrid	CHANNEL BETA SUBUNIT);
	oxidoreductase/potassium
	channel
MSH1 interacting proteins	GOS12 (GOLGI SNARE 12);	GOS12 AT2G45200
identified by Yeast Two Hybrid	SNARE binding
MSH1 interacting proteins	ELI3-1 (ELICITOR-	ELI3-1 AT4G37980
identified by Yeast Two Hybrid	ACTIVATED GENE 3-1);
	binding/catalytic/
	oxidoreductase/zinc ion binding
	(CAD7), response to bacterium,
	plant-type hypersensitive response
MSH1 interacting proteins	STT3B (staurosporin and	STT3B AT1G34130
identified by Yeast Two Hybrid	temperature sensitive 3-like b);
	oligosaccharyl transferase
MSH1 interacting proteins	tRNA synthetase beta subunit	AT1G72550
identified by Yeast Two Hybrid	family protein, FUNCTIONS IN:
	phenylalanine-tRNA ligase
	activity, RNA binding,
	magnesium ion binding,
	nucleotide binding, ATP binding
	(unknown to date)
MSH1 interacting proteins	high mobility group (HMG1/2)	AT4G23800
identified by Yeast Two Hybrid	family protein, FUNCTIONS IN:
	sequence-specific DNA binding
	transcription factor activity;
	LOCATED IN: nucleus,
	chloroplast
MSH1 interacting proteins	Protein kinase superfamily	AT3G24190
identified by Yeast Two Hybrid	protein, FUNCTIONS IN: protein
	kinase activity, ATP binding;
	INVOLVED IN: protein amino
	acid phosphorylation; LOCATED
	IN: chloroplast
MSH1 interacting proteins	Protein kinase superfamily	AT1G64460
identified by Yeast Two Hybrid	protein, FUNCTIONS IN: inositol
	or phosphatidylinositol kinase
	activity, phosphotransferase
	activity (interacts with SNARE
	At2G45200)
MSH1 interacting proteins	RNA-binding (RRM/RBD/RNP	AT1G20880
identified by Yeast Two Hybrid	motifs) family protein;
	FUNCTIONS IN: RNA binding,
	nucleotide binding, nucleic acid
	binding; (interactomes map)
MSH1 interacting proteins	unknown protein, LOCATED IN:	AT5G55210
identified by Yeast Two Hybrid	chloroplast
MSH1 interacting proteins	ATPase, F0/V0 complex, subunit	AT4G32530
identified by Yeast Two Hybrid	C protein; FUNCTIONS IN:
	ATPase activity; INVOLVED IN:
	ATP synthesis coupled proton
	transport (vacuole)
MSH1 interacting proteins	RNA binding; FUNCTIONS IN:	AT3G11964
identified by Yeast Two Hybrid	RNA binding; mRNA processing,
	RNA processing

Useful plastid perturbation target genes from Arabidopsis with the accession number for the corresponding sequences in the Arabidopsis genome database (on the world wide web at the address “Arabidopsis.org”) are provided in Table 1. Orthologous genes from many crop species can be obtained through the BLAST comparison of the protein sequences of the Arabidopsis genes above to the genomic databases (NCBI and publically available genomic databases for specific crop species), as well as from the specific names of the subunits. Specifically the genome, cDNA, or EST sequences are available for apples, beans, barley, Brassica napus, rice, Cassava, Coffee, Eggplant, Orange, sorghum, tomato, cotton, grape, lettuce, tobacco, papaya, pine, rye, soybean, sunflower, peach, poplar, scarlet bean, spruce, cocoa, cowpea, maize, onion, pepper, potato, radish, sugarcane, wheat, and other species at the following internet or world wide web addresses: “compbio.dfci.harvard.edu/tgi/plant.html”; “genomevolution.org/wiki/index.php/Sequenced_plant_genomes”; “ncbi.nlm.nih.gov/genomes/PLANTS/PlantList.html”; “plantgdb.org/”; “arabidopsis.org/portals/genAnnotation/other_genomes/”; “gramene.org/resources/”; “genomenewsnetwork.org/resources/sequenced_genomes/genome_guide_p1.shtml”; “jgi.doe.gov/progams/plants/index.jsf”; “chibba.agtec.uga.edu/duplication/”; “mips.helmholtz-muenchen.de/plant/genomes.jsp”; “science.co.il/biomedical/Plant-Genome-Databases.asp”; “jcvi.org/cms/index.php?id=16”; and “phyto5.phytozome.net/Phytozome_resources.php”. The main protein complexes involved in photon capture and electron transport of photosystem II (PSII), NAD(P)H dehydrogenase (NDH), Cytochrome b6f complex, plastocyanin, photosystem I (PSI), and associated plastid proteins that represent certain plastid perturbation targets are also described in Grouneva, I., P. J. Gollan, et al. (2013) Planta 237(2): 399412 Ifuku, K., S. Ishihara, et al. (2010). J Integr Plant Biol 52(8): 723-734.
In general, methods provided herewith for introducing epigenetic and/or genetic variation in plants simply require that organellar perturbation target gene expression be suppressed for a time sufficient to introduce the variation and/or in appropriate subsets of cells (i.e cells containing sensory plastids). As such, a wide variety of organellar perturbation target gene suppression methods can be employed to practice the methods provided herewith and the methods are not limited to a particular suppression technique.
Sequences of organellar perturbation target gene genes or fragments thereof from Arabidopsis and various crop plants are provided herewith. In certain embodiments, such genes may be used directly in either the homologous or a heterologous plant species to provide for suppression of the endogenous organellar perturbation target gene in either the homologous or heterologous plant species. A non-limiting demonstrative example where an MSH1 plastid perturbation target gene from one species was shown to be effective in suppressing the endogenous MSH1 gene in both a homologous and a heterologous species is provided by Sandhu et al. 2007, where a transgene that provides for an MSH1 inhibitory RNA (RNAi) with tomato MSH1 sequences was shown to inhibit the endogenous MSH1 plastid perturbation target gene genes of both tomato and tobacco. A transgene that provides for a organellar perturbation target gene inhibitory RNA (RNAi) with maize organellar perturbation target gene sequences can be used in certain embodiments to inhibit the endogenous organellar perturbation target gene genes of millet, sorghum, and maize. Organellar perturbation target gene genes from other plants including, but not limited to, cotton, canola, wheat, barley, flax, oat, rye, turf grass, sugarcane, alfalfa, banana, broccoli, cabbage, carrot, cassava, cauliflower, celery, citrus, a cucurbit, eucalyptus, garlic, grape, onion, lettuce, pea, peanut, pepper, potato, poplar, pine, sunflower, safflower, soybean, blackberry, blueberry, sugar beet, sweet potato, tobacco, strawberry, sugar beet, sweet potato, Jatropha, Camelina, and Agave can be obtained by a variety of techniques and used to suppress expression of either the corresponding organellar perturbation target gene in those plants or the organellar perturbation target gene in a distinct plant. Methods for obtaining organellar perturbation target genes for various plants include, but are not limited to, techniques such as: i) searching amino acid and/or nucleotide sequence databases comprising sequences from the plant species to identify the organellar perturbation target gene by sequence identity comparisons; ii) cloning the organellar perturbation target gene by either PCR from genomic sequences or RT-PCR from expressed RNA; iii) cloning the organellar perturbation target gene from a genomic or cDNA library using PCR and/or hybridization based techniques; iv) cloning the organellar perturbation target gene from an expression library where an antibody directed to the organellar perturbation target gene protein is used to identify the organellar perturbation target gene containing clone; v) cloning the organellar perturbation target gene by complementation of an organellar perturbation target gene mutant or organellar perturbation target gene deficient plant; or vi) any combination of (i), (ii), (iii), (iv), and/or (v). The DNA sequences of the target genes can be obtained from the promoter regions or transcribed regions of the target genes by PCR isolation from genomic DNA, or PCR of the cDNA for the transcribed regions, or by commercial synthesis of the DNA sequence. RNA sequences can be chemically synthesized or, more preferably, by transcription of suitable DNA templates. Recovery of the organellar perturbation target gene from the plant can be readily determined or confirmed by constructing a plant transformation vector that provides for suppression of the gene, transforming the plants with the vector, and determining if plants transformed with the vector exhibit the characteristic responses that are typically observed in various plant species when MSH1 expression is suppressed that include leaf variegation, cytoplasmic male sterility (CMS), a reduced growth-rate phenotype, and/or delayed or non-flowering phenotype. The characteristic responses of MSH1 suppression have been described previously as developmental reprogramming or “MSH1-dr1” (Xu et al. Plant Physiol. Vol. 159:711-720, 2012).
In certain embodiments, organellar perturbation target genes or fragments thereof used in the methods provided herein will have nucleotide sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% nucleotide sequence identity to one or more of the organellar perturbation target genes or fragments thereof provided herein that include, but are not limited to, genes provided in Table 1 and orthologs thereof found in various crop plants. In certain embodiments, organellar perturbation target genes or fragments thereof used in the methods provided herein encode organellar perturbation target gene proteins or portions thereof will have amino acid sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to one or more of the organellar perturbation target gene proteins provided herein that include, but are not limited to, the organellar perturbation target gene proteins encoded by genes provided in Table 1. In certain embodiments, organellar perturbation target genes or fragments thereof used in the methods provided herein will have nucleotide sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% nucleotide sequence identity to one or more of the PPD3 organellar perturbation target genes fragments thereof orthologs thereof or homologs thereof provided herein that include, but are not limited to, SEQ ID NO:16-40. In certain embodiments, organellar perturbation target gene genes or fragments thereof used in the methods provided herein encode organellar perturbation target gene proteins or portions thereof will have amino acid sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to one or more of the PPD3 organellar perturbation target gene proteins or organellar perturbation target gene homologs provided herein that include, but are not limited to, the proteins encoded by SEQ ID NO:16-40. PPD3 organellar perturbation target gene genes from plants other than those provided herein can also be identified by the encoded regions with homology to the PsbP1 and PsbP2 gene domains that characterize many PPD3 genes. In certain embodiments, organellar perturbation target genes or fragments thereof used in the methods provided herein will have nucleotide sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% nucleotide sequence identity to one or more of the MSH1 organellar perturbation target genes fragments thereof orthologs thereof or homologs thereof provided herein that include, but are not limited to, SEQ ID NO:1, 3-11. In certain embodiments, organellar perturbation target gene genes or fragments thereof used in the methods provided herein encode organellar perturbation target gene proteins or portions thereof will have amino acid sequences with at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity to one or more of the MSH1 organellar perturbation target gene proteins or organellar perturbation target gene homologs provided herein that include, but are not limited to, the proteins encoded by SEQ ID NO:1, 3-11.
It is anticipated that organellar perturbation target gene nucleic acid fragments of 18 to 20 nucleotides, but more preferably 21 nucleotides or more, can be used to effect suppression of the endogenous organellar perturbation target gene. In certain embodiments, organellar perturbation target gene nucleic acid fragments of at least 18, 19, 20, or 21 nucleotides to about 50, 100, 200, 500, or more nucleotides can be used to effect suppression of the endogenous organellar perturbation target gene. Regions of 20, 50, 100, 500, or more nucleotides are suitable for this purpose, with lengths of 100 to 300 bases of the target gene sequences preferable, and lengths of 300 to 500 bp or more being most preferable. In certain embodiments, regions of 20, 50, or 100 to 200, 300, 500, 750, or a 1000 or more nucleotides are used to effect suppression of the organellar target gene. For use in a hairpin or inverted repeat knockdown design, a spacer region with a sequence not related to the sequence of the genome of the target plant can be used. A hairpin construct containing 15, 18, or 20 to 500 bp or more of a target gene sequence in the antisense orientation, followed by a spacer region whose sequence is not critical but can be a intron or non-intron. If the spacer is an intron, the caster bean catalase intron which is effectively spliced in both monocots and dicots (Tanaka, Mita et al. Nucleic Acids Res 18(23): 6767-6770, 1990), is known to those skilled in the art and is useful for the present embodiment. After the spacer the same target gene sequence in the sense orientation is present, such that the antisense and sense strands can form a double stranded RNA after transcription of the transcribed region. The target gene sequences are followed by a polyadenylation region. 3′ polyadenylation regions known to those skilled in the art to function in monocots and dicot plants include but are not limited to the Nopaline Synthase (NOS) 3′ region, the Octapine Synthase (OCS) 3′ region, the Cauliflower Mosaic Virus 35S 3′ region, the Mannopine Synthase (MAS) 3′ region. Additional 3′ polyadenylation regions from monocotyledonous genes such as those from rice, sorghum, wheat, and maize are available to those skilled in the art to provide similar polyadenylation region and function in DNA constructs in the present embodiments. In certain embodiments, a transgene designed to suppress a target gene in dicots is designed to have the following order: promoter/antisense to target gene/catalase intron/sense gene A/polyadenylation region. In embodiments where a gene is designed to suppress a target gene in monocots can have the following order: promoter/intron for monocots/antisense to target gene/catalase intron/sense gene A/polyadenylation region.
Sequences that provide for suppression of a organellar perturbation target gene can include sequences that exhibit complementarity to either strand of the promoter, 5′ or 3′ untranslated region, intron, coding regions, and/or any combination thereof. A target gene promoter region for gene suppression can include the transcription start site, the TATA box, and upstream regions. The promoter region for gene silencing can be about 20, 50, 80, or 100 nucleotides in length, and more preferably is about 100 to 500 nucleotides in length. The promoter region used for such suppression can be from different regions in the upstream promoter, preferably containing at least about 500 nucleotides upstream from the start of transcription, and most preferably containing at least about 500 nucleotides upstream from the start of translation of the native coding region of the native gene. This would include the UTR which may or may not be part of the promoter. A description of various recombinant DNA constructs that target promoter and/or adjoining regions of target genes are described in U.S. Pat. No. 8,293,975, which is incorporated herein by reference in its entirety.
For gene targets with closely related family members, sense, antisense or double hairpin suppression designs can include sequences from more than one family member, following the designs described above. In certain embodiments, a transgene to suppress two genes, target gene A and target gene B, is designed to have the following order: promoter/optional intron/antisense to target gene A/antisense to target gene B/spacer sequence/sense target gene B/sense gene A/polyadenylation region. In certain embodiments, this spacer sequence can be an intron. Useful embodiments include, but are not limited to, the following combinations of gene family members that can each be arranged in a single recombinant DNA construct any order that provides for hairpin formation and suppression of the gene targets:
(a) Construct 1: PsbQ-like PQL1, PsbQ-like, PsbQ-like PQL3, and any combination thereof;

(b) Construct 2: PsbO-1 and PsbO-2;

(c) Construct 3: PsbP1 and PsbP2;

(d) Construct 4: PsbQ-1 and PsbQ-2;

(e) Construct 5: FNR1 and FNR2;

(f) Construct 6: PETE1 and PETE2; and,

(g) Construct 7: PsbW and PsbW-like.

In certain embodiments, suppression of organellar perturbation target gene in a plant is effected with a transgene. Transgenes that can be used to suppress expression of organellar perturbation target gene include, but are not limited to, transgenes that produce dominant-negative mutants of a organellar perturbation target gene, a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNA that provide for inhibition of the endogenous organellar perturbation target gene. U.S. patents incorporated herein by reference in their entireties that describe suppression of endogenous plant genes by transgenes include U.S. Pat. No. 7,109,393, U.S. Pat. No. 5,231,020 and U.S. Pat. No. 5,283,184 (co-suppression methods); and U.S. Pat. No. 5,107,065 and U.S. Pat. No. 5,759,829 (antisense methods). In certain embodiments, transgenes specifically designed to produce double-stranded RNA (dsRNA) molecules with homology to the organellar perturbation target gene can be used to decrease expression of the endogenous organellar perturbation target gene. In such embodiments, the sense strand sequences of the dsRNA can be separated from the antisense sequences by a spacer sequence, preferably one that promotes the formation of a dsRNA (double-stranded RNA) molecule. Examples of such spacer sequences include, but are not limited to, those set forth in Wesley et al., Plant J., 27(6):581-90 (2001), and Hamilton et al., Plant J., 15:737-746 (1998). One useful and non-limiting vector that has been shown to provide for suppression of organellar perturbation target gene in tobacco and tomato has been described by Sandhu et al., 2007 where an intron sequence separates the sense and antisense strands of the organellar perturbation target gene sequence. The design of recombinant DNA constructs for suppression of gene expression are also described in Helliwell, C. and P. Waterhouse (2003). “Constructs and methods for high-throughput gene silencing in plants.” Methods 30(4): 289-295.
In certain embodiments, transgenes that provide for organellar perturbation target gene suppression can comprise regulated promoters that provide for either induction or down-regulation of operably linked organellar perturbation target gene inhibitory sequences. In this context, organellar perturbation target gene inhibitory sequences can include, but are not limited to, dominant-negative mutants of organellar perturbation target gene, a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNA that provide for inhibition of the endogenous organellar perturbation target gene of a plant. Such promoters can provide for suppression of organellar perturbation target gene during controlled time periods by either providing or withholding the inducer or down regulator. Inducible promoters include, but are not limited to, a PR-1a promoter (U.S. Patent Application Publication Number 20020062502) or a GST II promoter (WO 1990/008826 A1). In other embodiments, both a transcription factor that can be induced or repressed as well as a promoter recognized by that transcription factor and operably linked to the organellar perturbation target gene inhibitory sequences are provided. Such transcription factor/promoter systems include, but are not limited to: i) RF2a acidic domain-ecdysone receptor transcription factors/cognate promoters that can be induced by methoxyfenozide, tebufenozide, and other compounds (U.S. Patent Application Publication Number 20070298499); ii) chimeric tetracycline repressor transcription factors/cognate chimeric promoters that can be repressed or de-repressed with tetracycline (Gatz, C., et al. (1992). Plant J. 2, 397-404), and the like.
In certain embodiments, a promoter that provides for selective expression of a hctcrologous sequence that suppresses expression of the target gene in cells containing sensory plastids is used. In certain embodiments, this promoter is an MSH1 or a PPD3 promoter. In certain embodiments, this promoter is an MSH1 or a PPD3 promoter and the operably linked heterologous sequence suppresses expression of a target gene provided in Table 1 (above). MSH1 promoters that can be used to express heterologous sequences in cells containing sensor plastids include, but are not limited to, the Arabidopsis, sorghum, tomato, and maize promoters provided herewith (SEQ ID NO: 11, 12, 13, 14, and 41) as well as functional derivatives thereof that likewise provide for expression in cells that contain sensor plastids. In certain embodiments, deletion derivatives of the MSH1 promoters comprising about 1500 Bp, 1000 Bp, or about 750 Bp of SEQ ID NO:11, 12, 13, 14, and 41 can also be used to express heterologous sequences. PPD3 promoters that can be used to express heterologous sequences in cells containing sensor plastids include, but are not limited to, the Arabidopsis, rice, and tomato promoters provided herewith as SEQ ID NO:52, 53, and 54 as well as functional derivatives thereof that provide for expression in cells that contain sensor plastids. In certain embodiments, deletion derivatives of the MSH1 promoters comprising about 800 Bp, 600 Bp, or about 500 Bp of SEQ ID NO: 52, 53, and 54 can also be used to express heterologous sequences. In certain embodiments, PPD3 promoters comprising SEQ ID NO:52, 53, and 54 and an additional 200, 500, or 1000 base pairs of the endogenous 5′ PPD3 promoter sequences can be used to express heterologous sequences. Additional 200, 500, or 1000 base pairs of the endogenous 5′ PPD3 promoter sequences can be obtained by methods including, but not limited to, retrieval of sequences from databases provided herein and recovery of the adjoining promoter DNA by PCR amplification of genomic template sequences or by direct synthesis. In certain embodiments, recombinant DNA constructs for suppression of dicot target genes can comprise a MSH1 or PPD3 promoter from a dicotyledonous species such as Arabidopsis, soybeans or canola, is attached to a hairpin construct containing 300 to 500 bp or more of a target gene sequence in the antisense orientation, followed by a spacer region whose sequence is not critical but can be a intron or non-intron. The caster bean catalase intron (Tanaka, Mita et al. Nucleic Acids Res 18(23): 6767-6770, 1990), can be used as a spacer in certain embodiments. After the spacer the same target gene sequence in the sense orientation is present, such that the antisense and sense strands can form a double stranded RNA after transcription of the transcribed region. The target gene sequences are followed by a polyadenylation region. Various 3′ polyadenylation regions known to function in monocots and dicot plants include but are not limited to the Nopaline Synthase (NOS) 3′ region, the Octopine Synthase (OCS) 3′ region, the Cauliflower Mosaic Virus 35S 3′ region, the Mannopine Synthase (MAS) 3′ region. In certain embodiments recombinant DNA constructs for suppression of monocot target genes can comprise MSH1 or PPD3 promoter from a monocot species such as rice, maize, sorghum or wheat can either be attached directly to the hairpin region or to a monocot intron before the hairpin region. Monocot introns that are beneficial to gene expression when located between the promoter and coding region are the first intron of the maize ubiquitin (described in U.S. Pat. No. 6,054,574, which is incorporated herein by reference in its entirety) and the first intron of rice actin 1 (McElroy, Zhang et al. Plant Cell 2(2): 163-171, 1990). Additional introns that are beneficial to gene expression when located between the promoter and coding region are the maize hsp70 intron (described in U.S. Pat. No. 5,859,347, which is incorporated herein by reference in its entirety), and the maize alcohol dehydrogenase 1 genes introns 2 and 6 (described in U.S. Pat. No. 6,342,660, which is incorporated herein by reference in its entirety).
In still other embodiments, transgenic plants are provided where the transgene that provides for organellar perturbation target gene suppression is flanked by sequences that provide for removal for the transgene. Such sequences include, but are not limited to, transposable element sequences that are acted on by a cognate transposase. Non-limiting examples of such systems that have been used in transgenic plants include the cre-lox and FLP-FRT systems.
In certain embodiments, organellar perturbation target gene suppression can be effected by Viral Induced Gene Silencing (VIGS) methods. In general, such methods entail insertion of a organellar perturbation target gene sequence into a cloned viral genome that can be introduced directly into a target plant or target plant cell to effect organellar perturbation target gene suppression or that can produce an infectious nucleic acid that is introduced into a target plant or target plant cell to effect organellar perturbation target gene suppression. Various methods and vectors used for suppression of other gene targets by VIGS can also be adapted for use in suppressing organellar perturbation target genes by use of appropriate organellar perturbation target gene sequences disclosed herein. Such VIGS methods and vectors that can be adapted for suppressing organellar perturbation target genes include, but are not limited to, those disclosed in U.S. Pat. Nos. 5,922,602, 6,635,805, 6,369,296, and 7,229,829 that are especially, but not exclusively, useful for performing VIGS in dicot plants. Other VIGS methods and vectors that can be adapted for suppressing organellar perturbation target genes include, but are not limited to, those disclosed in U.S. Pat. No. 6,800,748 that are especially, but not exclusively, useful for performing VIGS in monocot plants. U.S. Pat. Nos. 5,922,602, 6,635,805, 6,369,296, 6,800,748, and 7,229,829 are each incorporated herein by reference in their entireties with respect to their disclosure of VIGS vectors and methods. VIGS vectors and methods based on cloned Hordeivirus (including, but not limited to, barley stripe mosaic virus (“BSMV”), poa semilatent virus (“PSLV”), lychnis ringspot virus (“LRSV”), and anthoxanthum latent blanching virus (“ALBV”)), tobacco mosaic virus (TMV), Cucumber Green Mottle Mosaic virus watermelon strain (CGMMV-W); Brome Mosaic virus (BMV), Potyvirus (including, but not limited to, Rice Necrosis virus, and Potato Virus Y (PVY)), Rice tungro bacilliform virus (RTBV) and Geminivirus genomes all can be used to suppress organellar perturbation target genes. In certain embodiments, cloned viral genomes containing a organellar perturbation target gene sequence can be operably linked to a promoter that is active in cells containing sensor organellars to effect suppression of the organellar perturbation target gene in those cells. Such promoters that can be used to express heterologous sequences in cells containing sensor organellars include MSH1 and PPD3 promoters. In certain embodiments of any of the aforementioned methods, a organellar perturbation target gene sequence is inserted into a Barley stripe mosaic virus (BSMV), a Brome Mosaic Virus (BMV), or a Rice tungro bacilliform virus (RTBV) genome to effect suppression of a organellar perturbation target gene in a monocot plant. In certain embodiments of any of the aforementioned methods, a organellar perturbation target gene sequence is inserted into a viral genome of Table 2 to effect suppression of a organellar perturbation target gene in a dicot plant.

TABLE 2

Viral Genomes Used for VIGS in Dicot plants

	ALSV	Apple latent spherical virus
	ACMV	African cassava mosaic virus
	BPMV	Bean pod mottle virus
	BCTV	Beet curly top virus
	CymMV	Cymbidium mosaic virus
	CbLCV	Cabbage leaf curl virus
	CMV	Cucumber mosaic virus
	CLCrV	Cotton leaf crumple virus
	GVA	Grapevine virus A
	PVX	Potato virus X
	PEBV	Pea early browning virus
	PopMV	Poplar mosaic virus
	SHMV	Sunn-hemp mosaic virus
	TMV	Tobacco mosaic virus
	TRV	Tobacco rattle virus
	TYLCCV	Tomato yellow leaf curl China virus
	TYMV	Turnip yellow mosaic virus
	ToMV	Tomato mosaic virus

Methods for identifying agents that provide for organellar perturbation are also provided herein. Such methods broadly comprise screening and selecting for agents that inhibit or interfere with binding or other productive interactions between an MSH1 protein or a fragment thereof and at least one assistant proteins or fragment thereof that interact with MSH1 and fragments thereof. Assistant proteins that interact with MSH1 include, but are not limited to proteins selected from the group consisting of PPD3, PsbA (D1), a PsbO-1, a PsbO-2, PetC, CAD9, KAB1, GOS12, ELI3-1, STT3B, a fragment thereof; and combinations thereof. In certain embodiments, inhibitors of MSH1 interactions with the assistant proteins can be identified in biological “one-hybrid” or “two-hybrid” assays based on microbial, yeast, or mammalian systems (Velasco-García R, Vargas-Martínez R. The study of protein-protein interactions in bacteria. Can J Microbiol. 2012 November; 58(11):1241-57; Rajagopala S V, et al. Studying protein complexes by the yeast two-hybrid system. Methods. 2012 December; 58(4):392-9; Lievens et al. The use of mammalian two-hybrid technologies for high-throughput drug screening. Methods. 2012 December; 58(4):335-42). A non-limiting example of a yeast two-hybrid assay that can be used to screen and select for such agents is disclosed in co-assigned U.S. patent application Ser. No. 14/454,518, which is specifically incorporated herein by reference in its entirety. In certain embodiments, the assays can comprise biochemical assays for inhibition of binding of MSH1 or fragments thereof to an assistant protein or fragment thereof. In certain embodiments, the fragment of MSH-1 comprises a peptide of at least about 10 amino acids located within Domain 2, 3, or 6 of MSH-1. Various domains of the MSH-1 protein suitable for use in the assays are described in Abdelnoor et al. Proc Natl Acad Sci USA. 2003 May 13; 100(10): 5968-5973; and in US Patent Application Publication 20060248614, which is incorporated herein by reference in its entirety. Sequences of certain domains of MSH1 are as follows:

Combined Domains II III IV

CILVEYAGLNPFGGLRSDSIPKAGCPIMNLRQTLDDLTRNGYSVC

IVEEVQGPTPARSRKGRFISGHAHPGSPYVYGLVGVDHDLDFPDPMPVVG

ISRSARGYCMISIFETMKAYSLDDGLTEEALVTIKRTRRCHHLFLHASLR

HNASGTCRWGEFGEGGLLWGECSSRNFEWFEGDTLSELLSRVKDVYGLDD

EVSFRNVNVPSKNRPRPLHLGTATQIGALPTEGIPCLLKVLLPSTCSGLP

SLYVRDLLLNPPAYDIALKIQETCKLMSTVTCSIPEFTCVSSAKLVKLLE

QREANYIEFCRIKNVLDDVLHMHRHAELVEILKLLMDPTWVATGLKIDFD

TFVNECHWASDTIGEMISLDENESHQNVSKCDNVPNEFFYDMESSWRGRV

KGIHIEEEITQVEKSAEALSLAVAEDFHPIISRIKATTASLGGPKGEIAY

AREHESVWFKGKRFTPSIWAGTAGEDQIKQLKPALDSKGKKVGEEWFTTP

KVEIALVRYHEASENAKARVLELLRELSVKLQTKINVLVFASMLLVISKA

LFSHACEGRRRKWVFPTLVGFS

Domain VI ENDONUCLEASE

MGAENVEGQTKPTWKLTDGVCRESLAFETAKREGVPESVIQRAEALYLSV

YAKDASAEVVKPDQIITSSNNDQQIQKPVSSERSLEKDLAKAIVKICGKK

MIEPEAIECLSIGARELPPPSTVGSSCVYVMRRPDKRLYIGQTDDLEGRI

RAHRAKEGLQGSSFLYLMVQGKSMACQLETLLINQLHEQGYSLANLADGK

HRNFGTSSSLSTSDVVSIL

In certain embodiments, the agents that are screened are selected from the group consisting of compounds, peptides, and peptidomimetic compounds.

Organellar perturbation target gene suppression can be readily identified or monitored by molecular techniques. In certain embodiments where the endogenous organellar perturbation target gene is intact but its expression is inhibited, production or accumulation of the RNA encoding organellar perturbation target gene can be monitored. Molecular methods for monitoring organellar perturbation target gene RNA expression levels include, but are not limited to, use of semi-quantitive or quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) techniques. The use of semi-quantitive PCR techniques to monitor organellar perturbation target gene suppression resulting from RNAi mediated suppression of plastid perturbation target gene has been described (Sandhu et al. 2007). Various quantitative RT-PCR procedures including, but not limited to, TaqMan™ reactions (Applied Biosystems, Foster City, Calif. US), use of SCORPION™ or Molecular Beacon™ probes, or any of the methods disclosed in Bustin, S. A. (Journal of Molecular Endocrinology (2002) 29, 23-39) can be used. It is also possible to use other RNA quantitation techniques such as Quantitative Nucleic Acid Sequence Based Amplification (Q-NASBA™) or the Invader™ technology (Third Wave Technologies, Madison, Wis.).
In certain embodiments where organellar perturbation target gene suppression is achieved by use of a mutation in the endogenous organellar perturbation target gene of a plant, the presence or absence of that mutation in the genomic DNA can be readily determined by a variety of techniques. Certain techniques can also be used that provide for identification of the mutation in a hemizygous state (i.e. where one chromosome carries the mutated msh1 gene and the other chromosome carries the wild type organellar perturbation target gene gene). Mutations in organellar perturbation target DNA sequences that include insertions, deletions, nucleotide substitutions, and combinations thereof can be detected by a variety of effective methods including, but not limited to, those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of which are incorporated herein by reference in their entireties. For example, mutations can be detected by hybridization to allele-specific oligonucleotide (ASO) probes as disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No. 5,210,015 discloses detection of annealed oligonucleotides where a 5′ labelled nucleotide that is not annealed is released by the 5′-3′ exonuclease activity. U.S. Pat. No. 6,004,744 discloses detection of the presence or absence of mutations in DNA through a DNA primer extension reaction. U.S. Pat. No. 5,468,613 discloses allele specific oligonucleotide hybridizations where single or multiple nucleotide variations in nucleic acid sequence can be detected by a process in which the sequence containing the nucleotide variation is amplified, affixed to a support and exposed to a labeled sequence-specific oligonucleotide probe. Mutations can also be detected by probe ligation methods as disclosed in U.S. Pat. No. 5,800,944 where sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe. U.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein provide methods for identifying mutations with mass spectroscopy. These various methods of identifying mutations are provided as examples and are not intended to be limiting as the methods of the present disclosure can be used in conjunction with any polymorphism typing method to identify the presence of absence of mutations in an organellar perturbation target gene in genomic DNA samples. Furthermore, genomic DNA samples used can include, but are not limited to, genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA. The use of mutations in endogenous PPD3 genes is specifically provided herein.
Mutations in endogenous plant organellar perturbation target gene genes can be obtained from a variety of sources and by a variety of techniques. A homologous replacement sequence containing one or more loss of function mutations in the organellar perturbation target gene and homologous sequences at both ends of the double stranded break can provide for homologous recombination and substitution of the resident wild-type organellar perturbation target gene sequence in the chromosome with an msh1 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 organellar perturbation target gene that result in either a complete loss of organellar perturbation target gene function or a loss of organellar perturbation 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 MSH1 organellar perturbation target gene include, but are not limited to, frameshift mutations, 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 msh1 mutation that are engineered into endogenous plastid perturbation target gene plant 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 msh1 sequence (i.e. which provides a loss of function mutation in an organellar perturbation target gene sequence) can also be introduced into a targeted nuclease cleavage site by non-homologous end joining or a combination of non-homologous 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 organellar perturbation target 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 organellar perturbation target gene sequence (WO/06097853A1, WO/06097784A1, WO/04067736A2, U.S. 20070117128A1). It is thus anticipated that one can select or design a nuclease that will cut within a target organellar perturbation target gene sequence. In other embodiments, at least one site specific double stranded break can be introduced in the endogenous organellar perturbation target 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 endogenous organellar perturbation target gene 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 organellar perturbation target gene (Henikoff et al., Plant Physiol. 2004, 135:630-636). The recovery of mutations in endogenous PPD3 genes is specifically provided herein.
Any of the recombinant DNA constructs provided herein can be introduced into the chromosomes of a host plant via methods such as Agrobacterium-mediated transformation, Rhizobium-mediated transformation, Sinorhizobium-mediated transformation, particle-mediated transformation, DNA transfection, DNA electroporation, or “whiskers”-mediated transformation. Aforementioned methods of introducing transgenes are well known to those skilled in the art and are described in U.S. Patent Application No. 20050289673 (Agrobacterium-mediated transformation of corn), U.S. Pat. No. 7,002,058 (Agrobacterium-mediated transformation of soybean), U.S. Pat. No. 6,365,807 (particle mediated transformation of rice), and U.S. Pat. No. 5,004,863 (Agrobacterium-mediated transformation of cotton), each of which are incorporated herein by reference in their entirety. Methods of using bacteria such as Rhizobium or Sinorhizobium to transform plants are described in Broothaerts, et al., Nature. 2005, 10; 433(7026):629-33. It is further understood that the recombinant DNA constructs can comprise cis-acting site-specific recombination sites recognized by site-specific recombinases, including Cre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. Methods of integrating DNA molecules at specific locations in the genomes of transgenic plants through use of site-specific recombinases can then be used (U.S. Pat. No. 7,102,055). Those skilled in the art will further appreciate that any of these gene transfer techniques can be used to introduce the recombinant DNA constructs into the chromosome of a plant cell, a plant tissue or a plant.
Methods of introducing plant minichromosomes comprising plant centromeres that provide for the maintenance of the recombinant minichromosome in a transgenic plant can also be used in practicing certain embodiments of this disclosure (U.S. Pat. No. 6,972,197 and U.S. Patent Application Publication 20120047609). In these embodiments of the present disclosure, the transgenic plants harbor the minichromosomes as extrachromosomal elements that are not integrated into the chromosomes of the host plant. It is anticipated that such mini-chromosomes may be useful in providing for variable transmission of a resident recombinant DNA construct that suppresses expression of a organellar perturbation target gene.
In certain embodiments, it is anticipated that PPD3 suppression can be effected by exposing whole plants, or reproductive structures of plants, to stress conditions that result in suppression of an endogenous PPD3 gene. Such stress conditions include, but are not limited to, high light stress, and heat stress. Useful and non-limiting high light stress conditions include continuous exposure to about 300 to about 1200 μmol photons/m2·s for about 24 to about 120 hours. Useful and non-limiting heat stress conditions include continuous exposure to temperatures of about 32° C. to about 37° C. for about 2 hours to about 24 hours. Useful and non-limiting heat, light, and other environmental stress conditions that can provide for MSH1 suppression are also disclosed for heat (Shedge et al. 2010), high light stress (Xu et al. 2011) and other environmental stress conditions (Hruz et al. 2008) and can also be adapted to effect PPD3 suppression.
Methods where organellar perturbation target gene suppression is effected in plant cells or cultured plant cells are also provided herein. In certain embodiments, organellar perturbation target gene suppression can be effected by culturing plant cells under stress conditions that result in suppression of endogenous organellar perturbation target gene. Such stress conditions include, but are not limited to, high light stress. Useful and non-limiting high light stress conditions include continuous exposure to about 300 to about 1200 μmol photons/m2·s for about 24 to about 120 hours. Useful and non-limiting heat stress conditions include continuous exposure to temperatures of about 32° C. to about 37° C. for about 2 hours to about 24 hours. Useful and non-limiting heat, light, and other environmental stress conditions also that can provide for organellar perturbation target gene suppression are also disclosed for heat (Shedge et al. 2010), high light stress (Xu et al. 2011) and other environmental stress conditions (Hruz et al. 2008). In certain embodiments, organellar perturbation target gene suppression is effected in plant cells or cultured plant cells by introducing a nucleic acid that provides for such suppression into the plant cells. Nucleic acids that can be used to provide for suppression of organellar perturbation target gene in cultured plant cells include, but are not limited to, transgenes that produce a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNA directed to the organellar perturbation target gene. Nucleic acids that can be used to provide for suppression of organellar perturbation target gene in plant cells or cultured plant cells include, but are not limited to, a small inhibitory RNA (siRNA) or a microRNA (miRNA) directed against the endogenous organellar perturbation target gene. RNA molecules that provide for inhibition of organellar perturbation target gene can be introduced by electroporation. Introduction of inhibitory RNAs to cultured plant cells to inhibit target genes can in certain embodiments be accomplished as disclosed in Vanitharani et al. (Proc Natl Acad Sci USA., 2003, 100(16):9632-6), Qi et al. (Nucleic Acids Res. 2004 Dec. 15; 32(22):e179), or J. Cheon et al. (Microbiol. Biotechnol. (2009), 19(8), 781-786). The suppression of endogenous PPD3 genes in cultured plant cells is specifically provided herein.
Methods where organellar perturbation target gene suppression is effected in vegetatively or clonally propagated plant materials are also provided herein. Such vegetatively or clonally propagated plant materials can include, but are not limited to, cuttings, cultured plant materials, and the like. In certain embodiments, recovery of such plant or clonally propagated plant materials that have been subjected to organellar perturbation can be accomplished by methods that allow for transient suppression of the organellar perturbation target gene. In certain non-limiting examples, plant or clonally propagated plant materials that have been subjected to plant organellar perturbation are recovered by placing recombinant DNA constructs that suppress a organellar perturbation target gene in vectors that provide for their excision or segregation. In certain embodiments, such excision can be facilitated by use of transposase-based systems or such segregation can be facilitated by use of mini-chromosomes. In certain embodiments, such excision or segregation can be facilitated by linking a transgene that provides for a “conditional-lethal”counter selection to the transgene that suppresses a organellar perturbation target in the recombinant DNA construct. Vegetatively or clonally propagated plant materials that have been subjected to organellar perturbation and lacking recombinant DNA constructs that suppress a organellar perturbation target gene can then be screened and/or selected for useful traits. Also provided are methods where vegetatively or clonally propagated plant materials are obtained from a plant resulting from a self or outcross or from a cultured plant cell, where either the plant or plant cell had been subjected to suppression of a organellar perturbation target gene. Such vegetatively or clonally propagated plant materials obtained from such plants resulting from a self or outcross or from a plant cell that have been subjected to organellar perturbation can also be screened and/or selected for useful traits. Also provided herein are methods where a sexually reproducing plant or plant population comprising useful traits is vegetatively or clonally propagated, and a plant or a plant population derived therefrom is then used to produce seed or a seed lot. In certain embodiments of any of the aforementioned methods, the organellar perturbation target gene can be a MSH1 or a PPD3 gene.
Organellar perturbation target gene suppression can also be readily identified or monitored by traditional methods where plant phenotypes are observed. For example, organellar perturbation target gene suppression can be identified or monitored by observing organellar effects that include leaf variegation, cytoplasmic male sterility (CMS), a reduced growth-rate phenotype, and/or delayed or non-flowering phenotype. Phenotypes indicative of MSH1 organellar perturbation target gene suppression in various plants are provided in U.S. Patent Application Publication No. US20140157452, which is incorporated herein by reference in its entirety. These phenotypes that are associated with organellar perturbation target gene suppression are referred to herein as “discrete variation” (V_D). Organellar perturbation target gene suppression can also produce changes in plant phenotypes including, but not limited to, male sterility, plant tillering, height, internode elongation and stomatal density (referred to herein as “MSH1-dr phenotypes”) that can be used to identify or monitor organellar perturbation target gene suppression in plants. Other biochemical and molecular traits can also be used to identify or monitor organellar perturbation target gene suppression in plants. Such molecular traits can include, but are not limited to, changes in expression of genes involved in cell cycle regulation, Giberrellic acid catabolism, auxin biosynthesis, auxin receptor expression, flower and vernalization regulators (i.e. increased FLC and decreased SOC1 expression), as well as increased miR156 and decreased miR172 levels. Such biochemical traits can include, but are not limited to, up-regulation of most compounds of the TCA, NAD and carbohydrate metabolic pathways, down-regulation of amino acid biosynthesis, depletion of sucrose in certain plants, increases in sugars or sugar alcohols in certain plants, as well as increases in ascorbate, alphatocopherols, and stress-responsive flavones apigenin, and apigenin-7-o-glucoside, isovitexin, kaempferol 3-O-beta-glucoside, luteolin-7-O-glucoside, and vitexin. In certain embodiments, elevated plastochromanol-8 levels in plant stems can serve as a biochemical marker that can be used to identify or monitor organellar perturbation target gene suppression. In particular, plastochromanol-8 levels in stems of plants subjected to organellar perturbation target gene suppression can be compared to the levels in control plants that have not been subjected to such suppression to identify or monitor organellar perturbation target gene suppression. It is further contemplated that in certain embodiments, a combination of both molecular, biochemical, and traditional methods can be used to identify or monitor organellar perturbation target gene suppression in plants.
Organellar perturbation target gene suppression that results in useful epigenetic changes and useful traits can also be readily identified or monitored by assaying for characteristic DNA methylation and/or gene transcription patterns that occur in plants subject to such perturbations. In certain embodiments, characteristic DNA methylation and/or gene transcription patterns that occur in plants subject suppression of an MSH1 target gene can be monitored in a plant, a plant cell, plants, seeds, and/or processed products obtained therefrom to identify or monitor effects mediated by suppression of other target plant organellar perturbation genes. Such plant organellar perturbation genes that include, but are not limited to, genes provided herewith in the sequence listing and Table 1 are expected to give rise to the characteristic DNA methylation and/or gene transcription patterns that occur in plants subject suppression of an MSH1 target gene. Such characteristic DNA methylation and/or gene transcription patterns that occur in plants or seeds subjected suppression of an MSH1 target gene include, but are not limited to, those patterns disclosed in Examples 2 and 4 of U.S. patent application Ser. No. 14/454,518, which is specifically incorporated herein by reference in its entirety. In certain embodiments, first generation progeny of a plant subjected to suppression of a organellar perturbation target gene will exhibit CG differentially methylated regions (DMR) of various discrete chromosomal regions that include, but are not limited to, regions that encompass the MSH1 locus. In certain embodiments, a CG hypermethylated region that encompasses the MSH1 locus will be about 5 to about 8 MBp (mega base pairs) in length. In certain embodiments, first generation progeny of a plant subjected to suppression of a organellar perturbation target gene will also exhibit changes in plant defense and stress response gene expression. In certain embodiments, a plant, a plant cell, a seed, plant populations, seed populations, and/or processed products obtained therefrom that has been subject to suppression of a organellar perturbation target 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. Pat. No. 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 plants or seed that had been subjected to suppression of a organellar perturbation target 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 organellar perturbation target gene. A variety of methods that provide for suppression of organellar perturbation target gene in a plant followed by recovery of progeny plants where organellar perturbation target gene function is recovered are provided herein. In certain embodiments, such progeny plants can be recovered by downregulating expression of a organellar perturbation target gene-inhibiting transgene or by removing the organellar perturbation target gene-inhibiting transgene with a transposase. In certain embodiments of the methods provided herein, organellar perturbation target gene is suppressed in a target plant or plant cell and progeny plants that express organellar perturbation target gene are recovered by genetic techniques. In one useful and non-limiting embodiment, progeny plants can be obtained by selfing a plant that is heterozygous for the transgene that provides for organellar perturbation target gene segregation. Selfing of such heterozygous plants (or selfing of heterozygous plants regenerated from plant cells) provides for the transgene to segregate out of a subset of the progeny plant population. Where a organellar perturbation target gene is suppressed by use of a recessive mutation in an endogenous organellar perturbation target gene can, in yet another useful and non-limiting embodiment, be crossed to wild-type plants that had not been subjected to organellar perturbation and then selfed to obtain progeny plants that are homozygous for a functional, wild-type organellar perturbation target gene allele. In other embodiments, a organellar perturbation target gene is suppressed in a target plant or plant cell and progeny plants that express the organellar perturbation target gene are recovered by molecular genetic techniques. Non limiting and useful embodiments of such molecular genetic techniques include: i) downregulation of an organellar perturbation target gene suppressing transgene under the control of a regulated promoter by withdrawal of an inducer required for activity of that promoter or introduction of a repressor of that promoter, or, ii) exposure of the an organellar perturbation target gene suppressing transgene flanked by transposase recognition sites to the cognate transposase that provides for removal of that transgene.
Plants or rootstocks subjected to organellar perturbation, and scions grafted to such rootstocks, as well as the progeny thereof; can exhibit a variety of nuclear chromosomal DNA methylation patterns that are absent from control plants, rootstocks, or scions that were not subjected to organellar perturbation. Such methylation patterns can include, but are not limited to, CG hypermethylation, pericentromeric CHG hypermethylation, and/or additional characteristic methylation patterns observed in plants or progeny thereof that had been subjected to suppression of MSH1 gene expression. Such methylation patterns can also include, but are not limited to, changes in 5-hydroxymethylation and in particular, the occurrence of 5-hydroxymethylcytosine (5-hmC). Changes in 5-hmC can be monitored by immunoassays (Quest 5-hmC™ DNA ELISA Kit, Zymo Research Corp., Irvine, Calif., USA; or EpiSeeker™ hydroxymethylated DNA Quantification Kit, Abcam, Inc., Cambridge, Mass.). It is anticipated that plants, plant parts, processed plant products, rootstocks, and scions provided herein or produced by the methods provided herein can be identified by comparing methylation patterns in the genomic DNA of such materials to the methylation patterns of control plants, plant parts, processed plant products, rootstocks, and scions.
In certain embodiments of the methods provided herein, progeny plants derived from plants where organellar perturbation target gene expression was suppressed that exhibit one or more of male sterility, dwarfing, variegation, and/or delayed flowering time (i.e. MSH1-dr phenotypes) and express functional organellar perturbation target gene are obtained and maintained as independent breeding lines or as populations of plants. It has been found that such phenotypes appear to sort, so that it is feasible to select a cytoplasmic male sterile (CMS) plant displaying normal growth rate and no variegation, for example, or a stunted, male fertile plant that is highly variegated. We refer to this phenomenon herein as discrete variation (V_D). A plant that exhibits such discrete variation is a plant that exhibits one or more, but not all, MSH1-dr phenotypes that occur in that plant type when it is subject to MSH1 suppression. MSH1-dr phenotypes that occur in sorghum, millet, soybean, tobacco, and tomato include CMS, leaf variegation, dwarfed growth and reduced internode elongation, enhanced branching, altered leaf morphology, extended juvenility, and delayed flowering. In soybean, the altered leaf morphology can comprise a leaf wrinkling phenotype that resembles viral infection and dramatic delays in flowering. In sorghum, the MSH1-dr phenotypes include enhanced plant tillering, reduced height, reduced internode elongation, and changes in stomatal density. Useful and non-limiting illustrations of the discrete variation (V_D) phenomenon as it occurs in selfed plant populations that have lost an MSH1 organellar perturbation target gene-inhibiting transgene by segregation have been disclosed (U.S. Patent Application Publication No. US20140157452, incorporated herein by reference in its entirety). It is further contemplated that such individual lines that exhibit discrete variation (V_D) can be obtained by any of the aforementioned genetic techniques, molecular genetic techniques, or combinations thereof.
Individual lines or populations of plants obtained from plants where organellar perturbation target gene expression was suppressed that exhibit discrete variation (V_D) or that exhibit all MSH1-dr phenotypes can be crossed to other plants to obtain progeny plants that lack the phenotypes associated with discrete variation (V_D) (i.e. male sterility, dwarfing, variegation, and/or delayed flowering time). In certain embodiments, progeny of such outcrosses can be selfed to obtain individual progeny lines that exhibit significant phenotypic variation. Such phenotypic variation that is observed in these individual progeny lines derived from outcrosses of plants where organellar perturbation target gene expression was suppressed and that exhibit discrete variation to other plants is herein referred to as “quantitative variation” (V_Q). Certain individual progeny plant lines obtained from the outcrosses of plants where organellar perturbation target gene expression was suppressed to other plants can exhibit useful phenotypic variation where one or more traits are improved relative to either parental line and can be selected. Useful phenotypic variation that can be selected in such individual progeny lines includes, but is not limited to, increases in fresh and dry weight biomass relative to either parental line. An useful and non-limiting illustration of this phenomenon as it occurs in F2 progeny of outcrosses of plants that exhibit discrete variation to plants that do not exhibit discrete variation is provided in U.S. Patent Application Publication No. US20140157452, which is incorporated herein by reference in its entirety. Such selected individual lines with the useful trait can either be bred (i.e. crossed or selfed) individually or as a population. In certain embodiments, about 1% to about 45% of the population of progeny plants are selected for the useful trait and subsequently crossed the selected individual plants are crossed or selfed as a collected group of two or more selected plants to obtain populations of progeny plants that are enriched for the trait. In certain embodiments, about 1% to about 45% of the population of progeny plants are selected for the useful trait and subsequently crossed or selfed to obtain progeny plant populations that are enriched for the trait.
Individual lines or populations of plants obtained from plants where organellar perturbation target gene expression was suppressed that exhibit discrete variation (V_D) or that exhibit all MSH1-dr phenotypes can also be selfed to obtain progeny plants that lack the phenotypes associated with discrete variation (V_D) (i.e. male sterility, dwarfing, variegation, and/or delayed flowering time). Recovery of such progeny plants that lack the undesirable phenotypes can in certain embodiments be facilitated by removal of the transgene or endogenous locus that provides for organellar perturbation target gene suppression. In certain embodiments, progeny of such selfs can be used to obtain individual progeny lines or populations that exhibit significant phenotypic variation. Certain individual progeny plant lines or populations obtained from selfing plants where organellar perturbation target gene expression was suppressed can exhibit useful phenotypic variation where one or more traits are improved relative to the parental line that was not subjected to organellar perturbation target gene suppression and can be selected. Useful phenotypic variation that can be selected in such individual progeny lines includes, but is not limited to, increases in fresh and dry weight biomass relative to the parental line. Such selected individual lines with the useful trait can either be bred (i.e. crossed or selfed) individually or as a population. In certain embodiments, the selected individual plants are crossed or selfed as a collected group of two or more selected plants to obtain populations of progeny plants that are enriched for the trait. In certain embodiments, about 1% to about 45% of the population of progeny plants are selected for the useful trait and subsequently crossed or selfed to obtain progeny plant populations that are enriched for the trait.
In certain embodiments of the methods provided herein, progeny plants derived from plants where organellar perturbation target gene expression was suppressed that do not exhibit any MSH1-dr phenotypes and thus do not exhibit discrete variation (V_D) are obtained and maintained as independent breeding lines or as populations of plants that exhibit, contain, or harbor useful traits with nuclear inheritance. Useful traits that exhibit nuclear inheritance can thus occur in progeny plants derived from either selfs or crosses of plants that had been subjected to organellar perturbation but that did not exhibit any of the MSH1-dr phenotypes. More specifically, a plant that is subjected to perturbation of organellar target gene suppression that exhibits a wild type phenotype, a more robust growth habit, or even an improvement in a useful trait in comparison to a control plant that had not been subjected to perturbation of organellar target gene suppression can exhibit, contain, or harbor useful traits with nuclear inheritance. In certain embodiments, the useful traits that are exhibited, contained, or harbored in such plants that do not exhibit any MSH1-dr phenotypes and thus do not exhibit discrete variation (V_D) can be transmitted to progeny plants by selfing or outcrossing. In certain embodiments, the useful traits that are exhibited, contained, or harbored in such plants that do not exhibit any MSH1-dr phenotypes and thus do not exhibit discrete variation (V_D) can be transmitted to a scion from a plant that had not been subjected to organellar perturbation by using the plant that exhibits, contains, or harbors the useful trait as the rootstock in a graft to the scion. It is further contemplated that such individual lines that have the useful traits but that do not exhibit any MSH1-dr phenotypes and thus do not exhibit discrete variation (V_D) can be obtained by any of the aforementioned genetic techniques, molecular genetic techniques, or combinations thereof.
Individual lines or populations of plants obtained from plants where organellar perturbation target gene expression was suppressed that have the useful traits but that do not exhibit any MSH1-dr phenotypes and thus do not exhibit discrete variation (V_D) can be crossed to other plants to obtain progeny plants that exhibit the useful trait. In certain embodiments, the plants where organellar perturbation target gene expression was suppressed that have the useful traits but that do not exhibit any MSH1-dr phenotypes exhibit a wild type phenotype, a more robust growth habit, or even an improvement in a useful trait in comparison to a control plant that had not been subjected to perturbation of organellar target gene suppression can exhibit, contain, or harbor useful traits with nuclear inheritance. In certain embodiments, progeny of such outcrosses can be selfed to obtain individual progeny lines that exhibit significant phenotypic variation. Certain individual progeny plant lines obtained from the outcrosses of plants where organellar perturbation target gene expression was suppressed to other plants can exhibit useful phenotypic variation where one or more traits are improved relative to either parental line and can be selected. Such selected individual lines with the useful trait can either be bred (i.e. crossed or selfed) individually or as a population. Useful phenotypic variation that can be selected in such individual progeny lines includes, but is not limited to, increases in fresh and dry weight biomass relative to either parental line and/or increases in seed yield. Such selected individual lines with the useful trait can either be bred (i.e. crossed or selfed) individually or as a population. In certain embodiments, the selected individual plants are crossed or selfed as a collected group of two or more selected plants to obtain populations of progeny plants that are enriched for the trait. In certain embodiments, about 1% to about 45% of the population of progeny plants are selected for the useful trait and subsequently crossed or selfed to obtain progeny plant populations that are enriched for the trait.
Individual lines or populations of plants obtained from plants where organellar perturbation target gene expression was suppressed that have the useful traits but that do not exhibit any MSH1-dr phenotypes and thus do not exhibit discrete variation (V_D) that can also be selfed to obtain progeny plants that exhibit, contain, or harbor the useful traits. In certain embodiments, the plants where organellar perturbation target gene expression was suppressed that have the useful traits but that do not exhibit any MSH1-dr phenotypes exhibit a wild type phenotype, a more robust growth habit, or even an improvement in a useful trait in comparison to a control plant that had not been subjected to perturbation of organellar target gene suppression can exhibit, contain, or harbor useful traits with nuclear inheritance. Recovery of such progeny plants that lack the undesirable phenotypes can in certain embodiments be facilitated by removal of the transgene or endogenous locus that provides for organellar perturbation target gene suppression. In certain embodiments, progeny of such selfs can be used to obtain individual progeny lines or populations that exhibit significant phenotypic variation. Certain individual progeny plant lines or populations obtained from selfing plants where organellar perturbation target gene expression was suppressed can exhibit useful phenotypic variation where one or more traits are improved relative to the parental line that was not subjected to organellar perturbation target gene suppression and can be selected. Such selected individual lines with the useful trait can either be bred (i.e. crossed or selfed) individually or as a population. Useful phenotypic variation that can be selected in such individual progeny lines includes, but is not limited to, increases in fresh and dry weight biomass relative to the parental line. Such selected individual lines with the useful trait can either be bred (i.e. crossed or selfed) individually or as a population. In certain embodiments, the selected individual plants are crossed or selfed as a collected group of two or more selected plants to obtain populations of progeny plants that are enriched for the trait. In certain embodiments, about 1% to about 45% of the population of progeny plants are selected for the useful trait and subsequently crossed or selfed to obtain progeny plant populations that are enriched for the trait.
In certain embodiments, an outcross of an individual line or lines exhibiting, containing, or harboring the useful traits can be to a plant or plants that have not been subjected to organellar perturbation target gene suppression but are otherwise isogenic to the individual line or lines. In certain embodiments, a line or lines exhibiting, containing, or harboring the useful traits is obtained by suppressing organellar perturbation target gene in a given germplasm and can outcrossed to a plant having that same germplasm that was not subjected to organellar perturbation target gene suppression. In other embodiments, an outcross of an individual line or lines exhibiting, containing, or harboring the useful traits can be to a plant or plants that have not been subjected to organellar perturbation target gene suppression but are not isogenic to the individual line(s). Thus, in certain embodiments, an outcross of an individual line or lines exhibiting, containing, or harboring the useful traits can also be to a plant or plants that comprise one or more chromosomal polymorphisms that do not occur in the individual line(s), to a plant or plants derived from partially or wholly different germplasm, or to a plant or plant of a different heterotic group (in instances where such distinct heterotic groups exist). It is also recognized that such an outcross can be made in either direction. Thus, an individual line exhibiting discrete variability can be used as either a pollen donor or a pollen recipient to a plant that has not been subjected to organellar perturbation target gene suppression in such outcrosses. In certain embodiments, the progeny of the outcross are then selfed to establish individual lines that can be separately screened to identify lines with improved traits relative to parental lines. Such individual lines that exhibit the improved traits are then selected and can be propagated by further selfing. An useful and non-limiting illustration of this procedure where F2 progeny of outcrosses of plants that exhibit discrete variation to plants that do not exhibit discrete variation are obtained is provided in co-assigned U.S. Patent Application Publication No. US20140157452, which is incorporated herein by reference in its entirety. Such F2 progeny lines are screened for desired trait improvements relative to the parental plants and lines exhibiting such improvements are selected.
In certain embodiments, the methods provided herein can comprise selecting one or more progeny plants having the useful trait that exhibit nuclear inheritance. Nuclear inheritance can be established by demonstrating that the trait is pollen transmissible. Nuclear inheritance can also be established by demonstrating that the trait is associated with one or more chromosomal alterations that are present in the plants, grafted plants, or progeny thereof subjected to organellar perturbation but that are absent from control plants that had not been subjected to such organellar perturbation.
In certain embodiments, sub-populations of plants comprising the useful traits and epigenetic changes induced by suppression of the organellar perturbation target gene can be selected and bred as a population. Such populations can then be subjected to one or more additional rounds of selection for the useful traits and/or epigenetic changes to obtain subsequent sub-populations of plants exhibiting the useful trait. Any of these sub-populations can also be used to generate a seed lot. In one embodiment, organellar perturbed plants that do or do not exhibit an MSH1-dr phenotype can be selfed or outcrossed to obtain an F1 generation. A bulk selection at the F1, F2, and/or F3 generation can thus provide a population of plants exhibiting the useful trait and/or epigenetic changes or a seed lot. In certain embodiments, it is also anticipated that populations of progeny plants or progeny seed lots comprising a mixture of inbred and hybrid germplasms can be derived from populations comprising hybrid germplasm (i.e. plants arising from cross of one inbred line to a distinct inbred line). In certain embodiments, such sub-populations can comprise plants that had been subjected to organellar perturbation or grafted plants comprising a scion grafted to rootstock that had been subjected to organellar perturbation. Sub-populations of plants or grafted plants where the plant or the rootstock source plant is the progeny of a parental plant that had been subjected to organellar perturbation and that was selected for one or more useful traits can also be selected and bred as a population. Any of the aforementioned subpopulations can comprise 2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, or 10,000 or more plants. Seed lots thus obtained from these methods or other methods provided herein can comprise seed wherein at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of progeny plants grown from the seed exhibit a useful trait. The selection would provide the most robust and vigorous of the population for seed lot production. Seed lots produced in this manner could be used for either breeding or sale. In certain embodiments, a seed lot comprising seed wherein at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of progeny plants grown from the seed exhibit a useful trait associated with one or more epigenetic changes, wherein the epigenetic changes are associated with CG hyper-methylation and/or CHG hyper-methylation at one or more nuclear chromosomal loci in comparison to a control plant that does not exhibit the useful trait, and wherein the seed or progeny plants grown from said seed that is epigenetically heterogenous are obtained. A seed lot obtainable by these methods can include at least 100, 500, 1000, 5000, or 10,000 seeds.
In certain embodiments, methods for producing a seed lot comprising: (i) growing a population of plants, wherein said population comprises two or more plants that had been subjected to organellar perturbation, two or more grafted plants comprising a scion and rootstock obtained from a plant that had been subjected to organellar perturbation, or two or more plants from a parental plant that had been subjected to organellar perturbation; and (ii) obtaining a seed lot from the population are provided. Populations of grafted plants where the rootstock source plant is the progeny of a parental plant that had been subjected to organellar perturbation and that was selected for one or more useful traits can also be selected and bred as a population. Any of the aforementioned populations can comprise 2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, or 10,000 or more plants. Seed lots thus obtained from these methods or other methods provided herein can comprise seed wherein at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of progeny plants grown from the seed exhibit a useful trait. The selection would provide the most robust and vigorous of the population for seed lot production. Seed lots produced in this manner could be used for either breeding or sale. In certain embodiments, a seed lot comprising seed wherein at least 25%, 50%, 60%, 70%, 80%, 90%, or 95% of progeny plants grown from the seed exhibit a useful trait associated with one or more epigenetic changes, wherein the epigenetic changes are associated with CG hyper-methylation and/or CHG hyper-methylation at one or more nuclear chromosomal loci in comparison to corresponding nuclear chromosomal loci of a control plant that does not exhibit the useful trait, and wherein the seed or progeny plants grown from said seed that is epigenetically heterogenous are obtained. A seed lot obtainable by these methods can include at least 100, 500, 1000, 5000, or 10,000 seeds.
Altered chromosomal loci that can confer useful traits can also be identified and selected by performing appropriate comparative analyses of reference plants that do not exhibit the useful traits and test plants obtained from a parental plant or plant cell that had been subjected to organellar perturbation target gene suppression and obtaining either the altered loci or plants comprising the altered loci. It is anticipated that a variety of reference plants and test plants can be used in such comparisons and selections. In certain embodiments, the reference plants that do not exhibit the useful trait include, but are not limited to, any of a) a wild-type plant; b) a distinct subpopulation of plants within a given F2 population of plants of a given plant line (where the F2 population is any applicable plant type or variety); c) an F1 population exhibiting a wild type phenotype (where the F1 population is any applicable plant type or variety); and/or, d) a plant that is isogenic to the parent plants or parental cells of the test plants prior to suppression of organellar perturbation target gene in those parental plants or plant cells (i.e. the reference plant is isogenic to the plants or plant cells that were later subjected to organellar perturbation target gene suppression to obtain the test plants). In certain embodiments, the test plants that exhibit the useful trait include, but are not limited to, any of a) any non-transgenic segregants that exhibit the useful trait and that were derived from parental plants or plant cells that had been subjected to transgene mediated organellar perturbation target gene suppression, b) a distinct subpopulation of plants within a given F2 population of plants of a given plant line that exhibit the useful trait (where the F2 population is any applicable plant type or variety); (c) any progeny plants obtained from the plants of (a) or (b) that exhibit the useful trait; or d) a plant or plant cell that had been subjected to organellar perturbation target gene suppression that exhibit the useful trait.
In general, an objective of these comparisons is to identify differences in the small RNA profiles and/or methylation of certain chromosomal DNA loci between test plants that exhibit the useful traits and reference plants that do not exhibit the useful traits. Altered loci thus identified can then be isolated or selected in plants to obtain plants exhibiting the useful traits.
In certain embodiments, altered chromosomal loci can be identified by identifying small RNAs that are up or down regulated in the test plants (in comparison to reference plants). This method is based in part on identification of altered chromosomal loci where small interfering RNAs direct the methylation of specific gene targets by RNA-directed DNA methylation (RdDM). The RNA-directed DNA methylation (RdDM) process has been described (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4): 331-343). Any applicable technology platform can be used to compare small RNAs in the test and reference plants, including, but not limited to, microarray-based methods (Franco-Zorilla et al. Plant J. 2009 59(5):840-50), deep sequencing based methods (Wang et al. The Plant Cell 21:1053-1069 (2009)), and the like.
In certain embodiments, altered chromosomal loci can be identified by identifying histone proteins associated with a locus and that are methylated or acylated in the test plants (in comparison to reference plants). The analysis of chromosomal loci associated with methylated or acylated histones can be accomplished by enriching and sequencing those loci using antibodies that recognize methylated or acylated histones. Identification of chromosomal regions associated with methylation or acetylation of specific lysine residues of histone H3 by using antibodies specific for H3K4me3, H3K9ac, H3K27me3, and H3K36me3 has been described (Li et al., Plant Cell 20:259-276, 2008; Wang et al. The Plant Cell 21:1053-1069 (2009).
In certain embodiments, altered chromosomal loci can be identified by identifying chromosomal regions (genomic DNA) that has an altered methylation status in the test plants (in comparison to reference plants). An altered methylation status can comprise either the presence or absence of methylation in one or more chromosomal loci of a test plant comparison to a reference plant. Any applicable technology platform can be used to compare the methylation status of chromosomal loci in the test and reference plants. Applicable technologies for identifying chromosomal loci with changes in their methylation status include, but not limited to, methods based on immunoprecipitation of DNA with antibodies that recognize 5-methylcytidine, methods based on use of methylation dependent restriction endonucleases and PCR such as McrBC-PCR methods (Rabinowicz, et al. Genome Res. 13: 2658-2664 2003; Li et al., Plant Cell 20:259-276, 2008), sequencing of bisulfite-converted DNA (Frommer et al. Proc. Natl. Acad. Sci. U.S.A. 89 (5): 1827-31; Tost et al. BioTechniques 35 (1): 152-156, 2003), methylation-specific PCR analysis of bisulfite treated DNA (Herman et al. Proc. Natl. Acad. Sci. U.S.A. 93 (18): 9821-6, 1996), deep sequencing based methods (Wang et al. The Plant Cell 21:1053-1069 (2009)), methylation sensitive single nucleotide primer extension (MsSnuPE; Gonzalgo and Jones Nucleic Acids Res. 25 (12): 2529-2531, 1997), fluorescence correlation spectroscopy (Umezu et al. Anal Biochem. 415(2):145-50, 2011), single molecule real time sequencing methods (Flusberg et al. Nature Methods 7, 461-465), high resolution melting analysis (Wojdacz and Dobrovic (2007) Nucleic Acids Res. 35 (6): e41), and the like.
Methods for introducing various chromosomal modifications that can confer a useful trait into a plant, as well as the plants, plant parts, and products of those plant parts are also provided herein. Chromosomal alterations and/or chromosomal mutations induced by suppression of organellar perturbation target gene can be identified as described herein. Once identified, chromosomal modifications including, but not limited to, chromosomal alterations, chromosomal mutations, or transgenes that provide for the same genetic effect as the chromosomal alterations and/or chromosomal mutations induced by suppression of organellar perturbation target gene can be introduced into host plants to obtain plants that exhibit the desired trait. In this context, the “same genetic effect” means that the introduced chromosomal modification provides for an increase and/or a reduction in expression of one or more endogenous plant genes that is similar to that observed in a plant that has been subjected to organellar perturbation target gene suppression and exhibits the useful trait. In certain embodiments where an endogenous gene is methylated in a plant subjected to organellar perturbation target gene suppression and exhibits both reduced expression of that gene and a useful trait, chromosomal modifications in other plants that also result in reduced expression of that gene and the useful trait are provided. In certain embodiments where an endogenous gene is methylated in a plant subjected to organellar perturbation target gene suppression and exhibits both increased expression of that gene and a useful trait, chromosomal modifications in other plants that also result in increased expression of that gene and the useful trait are provided. In certain embodiments where an endogenous gene is demethylated in a plant subjected to organellar perturbation target gene suppression and exhibits both increased expression of that gene and a useful trait, chromosomal modifications in other plants that also result in increased expression of that gene and that useful trait are provided. In certain embodiments where an endogenous gene is demethylated in a plant subjected to organellar perturbation target gene suppression and exhibits both decreased expression of that gene and a useful trait, chromosomal modifications in other plants that also result in decreased expression of that gene and that useful trait are provided.
In certain embodiments, the chromosomal modification that is introduced is a chromosomal alteration. Chromosomal alterations including, but not limited to, a difference in a methylation state can be introduced by crossing a plant comprising the chromosomal alteration to a plant that lacks the chromosomal alteration and selecting for the presence of the alteration in F1, F2, or any subsequent generation progeny plants of the cross. In still other embodiments, the chromosomal alterations in specific target genes can be introduced by expression of a siRNA or hairpin RNA targeted to that gene by RNA directed DNA methylation (Chinnusamy V et al. Sci China Ser C-Life Sci. (2009) 52(4): 331-343; Cigan et al. Plant J 43 929-940, 2005; Heilersig et al. (2006) Mol Genet Genomics 275 437-449; Miki and Shimamoto, Plant Journal 56(4):539-49; Okano et al. Plant Journal 53(1):65-77, 2008).
In certain embodiments, the chromosomal modification is a chromosomal mutation. Chromosomal mutations that provide for reductions or increases in expression of an endogenous gene of a chromosomal locus can include, but are not limited to, insertions, deletions, and/or substitutions of nucleotide sequences in a gene. Chromosomal mutations can result in decreased expression of a gene by a variety of mechanisms that include, but are not limited to, introduction of missense codons, frame-shift mutations, premature translational stop codons, promoter deletions, mutations that disrupt mRNA processing, and the like. Chromosomal mutations that result in increased expression of a gene include, but are not limited to, promoter substitutions, removal of negative regulatory elements from the gene, and the like. Chromosomal mutations can be introduced into specific loci of a plant by any applicable method. Applicable methods for introducing chromosomal mutations in endogenous plant chromosomal loci include, but are not limited to, homologous double stranded break repair (Wright et al., Plant J. 44, 693, 2005; D'Halluin, et al., Plant Biotech. J. 6:93, 2008), non-homologous end joining or a combination of non-homologous end joining and homologous recombination (reviewed in Puchta, J. Exp. Bot. 56, 1, 2005; Wright et al., Plant J. 44, 693, 2005), meganuclease-induced, site specific double stranded break repair (WO/06097853A1, WO/06097784A1, WO/04067736A2, U.S. 20070117128A1), and zinc finger nuclease mediated homologous recombination (WO 03/080809, WO 05/014791, WO 07014275, WO 08/021207). In still other embodiments, desired mutations in endogenous plant chromosomal loci can be identified through use of the TILLING technology (Targeting Induced Local Lesions in Genomes) as described (Henikoff et al., Plant Physiol. 2004, 135:630-636).
In other embodiments, chromosomal modifications that provide for the desired genetic effect can comprise a transgene. Transgenes that can result in decreased expression of an gene by a variety of mechanisms that include, but are not limited to, dominant-negative mutants, a small inhibitory RNA (siRNA), a microRNA (miRNA), a co-suppressing sense RNA, and/or an anti-sense RNA and the like. U.S. patents incorporated herein by reference in their entireties that describe suppression of endogenous plant genes by transgenes include U.S. Pat. No. 7,109,393, U.S. Pat. No. 5,231,020 and U.S. Pat. No. 5,283,184 (co-suppression methods); and U.S. Pat. No. 5,107,065 and U.S. Pat. No. 5,759,829 (antisense methods). In certain embodiments, transgenes specifically designed to produce double-stranded RNA (dsRNA) molecules with homology to the endogenous gene of a chromosomal locus can be used to decrease expression of that endogenous gene. In such embodiments, the sense strand sequences of the dsRNA can be separated from the antisense sequences by a spacer sequence, preferably one that promotes the formation of a dsRNA (double-stranded RNA) molecule. Examples of such spacer sequences include, but are not limited to, those set forth in Wesley et al., Plant J., 27(6):581-90 (2001), and Hamilton et al., Plant J., 15:737-746 (1998). Vectors for inhibiting endogenous plant genes with transgene-mediated expression of hairpin RNAs are disclosed in U.S. Patent Application Publication Nos. 20050164394, 20050160490, and 20040231016, each of which is incorporated herein by reference in their entirety.
Transgenes that result in increased expression of a gene of a chromosomal locus include, but are not limited to, a recombinant gene fused to heterologous promoters that are stronger than the native promoter, a recombinant gene comprising elements such as heterologous introns, 5′ untranslated regions, 3′ untranslated regions that provide for increased expression, and combinations thereof. Such promoter, intron, 5′ untranslated, 3′ untranslated regions, and any necessary polyadenylation regions can be operably linked to the DNA of interest in recombinant DNA molecules that comprise parts of transgenes useful for making chromosomal modifications as provided herein.
Promoters useful for expression of transgenes include, but are not limited to, enhanced or duplicate versions of the viral CaMV35S and FMV35S promoters (U.S. Pat. No. 5,378,619, incorporated herein by reference in its entirety), the cauliflower mosaic virus (CaMV) 19S promoters, the rice Act1 promoter and the Figwort Mosaic Virus (FMV) 35S promoter (U.S. Pat. No. 5,463,175; incorporated herein by reference in its entirety). Introns useful for transgene expression include, but are not limited to, the maize hsp70 intron (U.S. Pat. No. 5,424,412; incorporated herein by reference in its entirety), the rice Act1 intron (McElroy et al., 1990, The Plant Cell, Vol. 2, 163-171), the CAT-1 intron (Cazzonnelli and Velten, Plant Molecular Biology Reporter 21: 271-280, September 2003), the pKANNIBAL intron (Wesley et al., Plant J. 2001 27(6):581-90; Collier et al., 2005, Plant J 43: 449-457), the PIV2 intron (Mankin et al. (1997) Plant Mol. Biol. Rep. 15(2): 186-196) and the “Super Ubiquitin” intron (U.S. Pat. No. 6,596,925, incorporated herein by reference in its entirety; Collier et al., 2005, Plant J 43: 449-457). Polyadenylation sequences include, but are not limited to, and Agrobacterium tumor-inducing (Ti) plasmid nopaline synthase (NOS) gene and the pea ssRUBISCO E9 gene polyadenylation sequences.
Plant lines and plant populations obtained by the methods provided herein can be screened and selected for a variety of useful traits by using a wide variety of techniques. In particular embodiments provided herein, individual progeny plant lines or populations of plants obtained from the selfs or outcrosses of plants where organellar perturbation target gene expression was suppressed to other plants are screened and selected for the desired useful traits.
In certain embodiments, the screened and selected trait is improved plant yield. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to one or more parental line(s) under non-stress conditions. Non-stress conditions comprise conditions where water, temperature, nutrients, minerals, and light fall within typical ranges for cultivation of the plant species. Such typical ranges for cultivation comprise amounts or values of water, temperature, nutrients, minerals, and/or light that are neither insufficient nor excessive. In certain embodiments, such yield improvements are improvements in the yield of a plant line relative to parental line(s) under abiotic stress conditions. Such abiotic stress conditions include, but are not limited to, conditions where water, temperature, nutrients, minerals, and/or light that are either insufficient or excessive. Abiotic stress conditions would thus include, but are not limited to, drought stress, osmotic stress, nitrogen stress, phosphorous stress, mineral stress, heat stress, cold stress, and/or light stress. In this context, mineral stress includes, but is not limited to, stress due to insufficient or excessive potassium, calcium, magnesium, iron, manganese, copper, zinc, boron, aluminum, or silicon. In this context, mineral stress includes, but is not limited to, stress due to excessive amounts of heavy metals including, but not limited to, cadmium, copper, nickel, zinc, lead, and chromium.
Improvements in yield in plant lines 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) and can be readily determined by growing plant lines obtained by the methods provided herein in parallel with the parental plants. 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 F G and Gumpertz M L. 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 disclosure) 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, R A. 1986. Biometrics 42: 231-251.).
In certain embodiments, the screened and selected trait is improved resistance to biotic plant stress relative to the parental lines. Biotic plant stress includes, but is not limited to, stress imposed by plant fungal pathogens, plant bacterial pathogens, plant viral pathogens, insects, nematodes, and herbivores. In certain embodiments, screening and selection of plant lines that exhibit resistance to fungal pathogens including, but not limited to, an Alternaria sp., an Ascochyta sp., a Botrytis sp.; a Cercospora sp., a Colletotrichum sp., a Diaporthe sp., a Diplodia sp., an Erysiphe sp., a Fusarium sp., Gaeumanomyces sp., Helminthosporium sp., Macrophomina sp., a Nectria sp., a Peronospora sp., a Phakopsora sp., Phialophora sp., a Phoma sp., a Phyatotrichum sp., a Phytophthora sp., a Plasmopara sp., a Puccinia sp., a Podosphaera sp., a Pyrenophora sp., a Pyricularia sp, a Pythium sp., a Rhizoctonia sp., a Scerotium sp., a Sclerotinia sp., a Septoria sp., a Thielaviopsis sp., an Uncinula sp, a Venturia sp., and a Verticillium sp. is provided. In certain embodiments, screening and selection of plant lines that exhibit resistance to bacterial pathogens including, but not limited to, an Erwinia sp., a Pseudomonas sp., and a Xanthamonas sp. is provided. In certain embodiments, screening and selection of plant lines that exhibit resistance to insects including, but not limited to, aphids and other piercing/sucking insects such as Lygus sp., lepidoteran insects such as Armigera sp., Helicoverpa sp., Heliothis sp., and Pseudoplusia sp., and coleopteran insects such as Diabroticus sp. is provided. In certain embodiments, screening and selection of plant lines that exhibit resistance to nematodes including, but not limited to, Meloidogyne sp., Heterodera sp., Belonolaimus sp., Ditylenchus sp., Globodera sp., Naccobbus sp., and Xiphinema sp. is provided.
Other useful traits that can be obtained by the methods provided herein include various seed quality traits including, but not limited to, improvements in either the compositions or amounts of oil, protein, or starch in the seed. Still other useful traits that can be obtained by methods provided herein include, but are not limited to, increased biomass, non-flowering, male sterility, digestability, seed filling period, maturity (either earlier or later as desired), reduced lodging, and plant height (either increased or decreased as desired). Still other useful traits that can be obtained by methods provided herein include, but are not limited to, delayed leaf 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 addition to any of the aforementioned traits, particularly useful traits for sorghum that can be obtained by the methods provided herein also include, but are not limited to: i) agronomic traits (flowering time, days to flower, days to flower-post rainy, days to flower-rainy; ii) fungal disease resistance (sorghum downy mildew resistance—glasshouse, sorghum downy mildew resistance-field, sorghum grain mold, sorghum leaf blight resistance, sorghum rust resistance; iii) grain related trait: (Grain dry weight, grain number, grain number per square meter, Grain weight over panicle. seed color, seed luster, seed size); iv) growth and development stage related traits (basal tillers number, days to harvest, days to maturity, nodal tillering, plant height, plant height-postrainy); v) infloresence anatomy and morphology trait (threshability); vi) Insect damage resistance (sorghum shoot fly resistance-post-rainy, sorghum shoot fly resistance-rainy, sorghum stem borer resistance); vii) leaf related traits (leaf color, leaf midrib color, leaf vein color, flag leaf weight, leaf weight, rest of leaves weight); viii) mineral and ion content related traits (shoot potassium content, shoot sodium content); ix) panicle related traits (number of panicles, panicle compactness and shape, panicle exertion, panicle harvest index, panicle length, panicle weight, panicle weight without grain, panicle width); x) phytochemical compound content (plant pigmentation); xii) spikelet anatomy and morphology traits (glume color, glume covering); xiii) stem related trait (stem over leaf weight, stem weight); and xiv) miscellaneous traits (stover related traits, metabolised energy, nitrogen digestibility, organic matter digestibility, stover dry weight).

EXAMPLES

The following examples are included to demonstrate certain embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques determined by the inventors to function well in the practice of the present disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Example 1. Implementing Selection of MSH1-Induced Non-Genetic Variation in Sorghum bicolor as a Model for Epigenetic Breeding

Here we investigate the implications of MSH1 modulation in a crop species. We found that MSH1-mediated phenotypic variation in Sorghum bicolor is heritable, responsive to selection, and potentially valuable for crop breeding. We observed phenotypic variation for grain yield, plant height, flowering time, panicle architecture, and above-ground biomass. Focusing on grain yield and plant height, we selected these traits for three cycles with progress each cycle. Based on amenability of this system to implementation in a range of crops, and the scope of phenotypic variation that is derived, our results suggest that MSH1 suppression provides a novel model for epigenetic breeding in crops.
Here we investigate the consequences of incorporating the MSH1-dr condition to plant selection, using sorghum as a model. We show that crossing with a transgene-null MSH1-dr line produces an unexpected range of phenotypic variation that is both heritable and responsive to selection. This variation appears to be stable over at least four generations. We also show evidence of epi-type×environment interactions. Finally, we demonstrate gains in grain yield over only two generations of selection, suggesting that this non-genetic variation may prove valuable for agricultural production as a novel crop breeding strategy.
Plant Materials and Growth Conditions.
Sorghum MSH1-dr plants used in these experiments were derived as described in Xu et al (2012). Six T₃individuals displaying the MSH1-dr phenotype but null for the MSH1-RNAi transgene were used as females in crosses to wild-type inbred Tx430 to derive F, seed. Another three T₃individuals were used as males in the reciprocal crosses to Tx430. The number of F₁plants derived from each cross ranged from 5 to 19 individuals. Parents and F₁progeny 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 F₁plants was harvested individually to generate corresponding F₂families.
Field Experiments and Phenotyping.
Plants were thinned to a final density of 15 plants/m². The 2011 F₂, F₃, and F₄combined field experiment comprised seven blocks of 28 entries each (28×7 alpha lattice design), with two replicates grown under fertilized soil conditions. In total, the 2011 field experiment comprised 48 F₂entries, 77 F₃entries, and 39 F₄, with additional wild-type Tx430 rows as a control.
For estimating grain yield, threshed panicles from three plants were pooled and converted to grams/m², with 2-3 such measurements taken per row. The histogram figure for yield variation used individual panicle grain yield (prior to pooling). For flowering time, plant height, and rachis length, measurements were taken on individual plants. For each dry biomass measurement, three fully dried plants were pooled together then converted to grams/plant.
The 2012 multi-location experiment included Lincoln, Nebr. (40° 51′ N, 96° 35′ W) and Mead, Nebr. (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 replications. All sites were fertilized identically according to standard growing practices. For this experiment, grain yield was estimated by taking threshed panicles from a meter-length area of each row and converting to grams/m².
Statistical Analysis.
For evaluations in a single environment, mean phenotypic values and standard errors for each line were estimated using the linear mixed model
y_ijk=μ+α_i+r_k+(b/r)_jk+ε_ijkwhere y_ijkis the trait response, μ is the population mean, α_iis the effect of line i, r_kis the effect of replicate k, (b/r)_jkis the effect of block j nested within replicate k, and ε_ijkis the residual error. For evaluations over multiple environments, mean phenotypic values and standard errors for each line were estimated using the linear mixed model y_ijkm=μ+α_i+e_m+(r/e)_km+(b/r/e)_jkm+(αe)_im+ε_ijkmwhere y_ijkmis the trait response, μ is the population mean, α_iis the effect of line i, e_mis the effect of environment m, (r/e)_kmis the effect of replicate nested within environment, (b/r/e)_jkmis the effect of block j nested within replicate k of environment m, (αe)_imis the interaction between line i and environment m, and ε_ijkmis the residual. Line, environment, and line×environment effects were treated as fixed while block and replicate effects were treated as random. Models were fit by restricted maximum likelihood using the R package “nlme” (Pinheiro et al. 2013). Residuals were graphically examined for anomalies. When deemed appropriate, Box-cox transformations were performed. In some cases, heteroscedasticity was modeled by specifying separate variance estimates stratified by generation/type (e.g. epi-F₂, wild-type Tx430, etc.) or broad height class (short, tall, mixed). F₄mean comparisons excluded lines exhibiting mixed heights or lines with inadequate data for one or more traits of interest. Contrasts for differences in trait means between lines were performed using the R package “multcomp” (Hothorn et al. 2008).
PCR Assay for RNAi Transgene and SSR Marker Analysis.
PCR assay for MSH1-RNAi transgene presence in sorghum materials used primers RNAi-F 5′GTGTACT CATCTGGATCTGTATTG-3′ (SEQ ID NO:55) and RNAi-R 5′GGTTGAGGAGCCTGAATCTCTGAAC3′ (SEQ ID NO:56) Positive and negative controls were included from a confirmed transgenic line and wildtype Tx430, respectively.
SSR marker analysis used SSR primers that were developed and mapped previously (Schloss et al. 2002; Li et al. 2009). Fragments were assayed by capillary electrophoresis on an Advanced Analytical Fragment Analyzer™ (Advanced Analytical Technologies, Inc. Ames, Iowa) using the dsDNA Reagent kit, 35-1,500 bp 500S that separates DNA in the size range of 35-1,500 bp. Of 136 primers that were tested, 43 produced unambiguous polymorphisms between Tx430 and the sweet sorghum control line ‘Wray’ and were used for testing the epi-lines.
Sorghum SNP Survey.
Leaf tissue sample was collected from plants grown under controlled greenhouse conditions three weeks after germination. Genomic DNA was extracted from freeze-dried leaf tissue and processed following manufacturer's recommendations prior to Infinium™ beadchip hybridization (Illumina, San Diego, Calif.). The genotyping of five MSH1-epiF₄lines and wild type Tx430 was carried out at the Monsanto Applied Genotyping Labs (Chesterfield, Mo.). The platform used was an exclusive custom-designed Sorghum bicolor Infinium™ high-density beadchip containing 1,885 internally validated SNP markers.
For the six samples, 107 of the 1,885 SNP markers, ca 5.68%, provided invalid data due to one of the following: low marker signal intensity, marker failed data QC, or unscorable allele calls. The remaining 1,778 SNP markers were used for the analysis. These 1,778 SNP markers are distributed across all 10 sorghum chromosomes with genome coverage approximating 90%. The number of heterozygotes (#Het) and percentage of heterozygotes (% Het) were calculated based on the 1778 SNP markers.

Results

MSH1-Altered Lines and Reciprocal Crosses.
Previously, we described MSH1-RNAi lines displaying numerous physiological changes, a condition of developmental reprogramming that was termed MSH1-dr (Xu et al. 2012). Segregation of the MSH1-RNAi transgene gave rise to some MSH1+/+ individuals that retained the characteristic msh1 phenotype despite having normal MSH1 transcript levels (Xu et al. 2012). These plants maintain the altered MSH1-dr growth phenotype through multiple (at least nine, to date) generations of self-pollination.
To investigate the mechanism of inheritance, we performed reciprocal crosses in sorghum of MSH1-dr individuals to their wild-type counterpart. FIG. 1 illustrates the transgene and crossing process used in this study, with all sorghum materials generated from the inbred line Tx430 (Miller 1984). When crossed to the wild-type inbred Tx430 line, the transgene-null MSH1-dr individuals produced progeny that were restored to normal phenotype (FIG. 1a ). The derived F₁progeny no longer showed the dwarfed, tillering, and late flowering phenotype; instead, many of the plants grew taller and produced more seed than the wild-type. This was repeatedly observed in F₁populations derived from nine separate crosses, three of which used an MSH1-dr plant as the pollen donor (Xu et al. 2012).
Lack of the MSH1-dr phenotype in the F₁generation from either direct or reciprocal crosses argues against the observed phenotypes being inherited via cytoplasmic organellar genomes. Analogously generated crosses in Arabidopsis with msh1 point or T-DNA insertion mutations also display enhanced vigor, and whole-genome bisulfite sequencing of those materials revealed significant changes in pericentromeric CHH methylation and in CG methylation of particular genomic regions (Xu et al. 2013). In other species, including tomato, soybean and tobacco, heritable MSH1-dr phenotypes also persist despite restored MSH1 expression following RNAi silencing (Xu et al. 2012 and unpublished), and crosses in those species to their respective wild-type counterparts similarly produce progeny with enhanced growth phenotypes (unpublished). Taken together, the evidence suggests that the MSH1-dr and F₁observations involve a conserved, programmed epigenetic pathway, and we therefore designated the F₁progeny as MSH1-epiF₁.
MSH1-epiF₂Populations Show Enhanced Variation.
Self-pollination of the MSH1-cpiF₁plants produced an F₂population (MSH1-cpiF₂) variable in plant phenotype (FIG. 1 b, c, d, f, FIG. 2, Table 3), with a minority exhibiting the MSH1-dr phenotype (FIG. 1e ). Notably increased variation in the F₂was detected for grain yield and plant height (FIG. 2, Table S1). Although we did not detect a very significant increase in variance for flowering time or panicle length in the cpi-F₂, by the F₄we were able to detect lines different from wild-type Tx430 for those traits (FIG. 5), indicating modest but heritable variation for flowering time and panicle length.

TABLE 3

Sorghum epi-lines families showed overall increased variation compared
to wild-type Tx430.

			Mean		Brown-
			within-		Forsythe
		Population	row		test
Trait	Population	Size	variance	F-value	p-value

Grain Yield	Tx430		55	38.02	—	—
(grams/m²)
	epiF2	318	64.41	9.6194	<0.01
	epiF3	348	87.04	19.582	<0.001
	epiF4	235	45.75	6.9213	<0.01
Plant Height	Tx430	192	38.03	—	—
(cm)
	epiF2	1493	789.94	301.04	<0.001
	epiF3	1587	668.49	306.61	<0.001
	epiF4	947	356.42	79.396	<0.001
Flowering	Tx430	134	1.04	—	—
Time (DAS)
	epiF2	908	1.15	0.2843	>0.1
	epiF3	635	0.86	16.819	<0.001
	epiF4	524	0.76	3.0162	<0.1
Rachis	Tx430	288	3.31	—	—
Length (cm)
	epiF2	1980	4.20	0.1348	>0.1
	epiF3	2412	4.75	3.3373	>0.1
	epiF4	1404	3.86	0.328	>0.1
Dry Biomass	Tx430	23	86.48	—	—
(grams/plant)
	epiF2	163	183.05	17.137	<0.001
	epiF3	188	199.31	18.313	<0.001
	epiF4	116	174.46	7.2468	<0.01

Data for Table 3 were acquired from a 2011 field experiment. Brown-Forsythe tests for homogeneous variances were performed between epi-line generation and wild-type (e.g. all epiF2 vs Tx430, all epiF3 vs Tx430).
A small proportion of greenhouse-grown MSH1-epiF₃families also showed the MSH1-dr phenotype, with an overall frequency of ca. 8% (Table 4). By the F₄generation, we estimate that the overall frequency drops to below 2%. Although the progeny from these sporadic MSH1-dr types in advanced generations have not been thoroughly investigated, some families appear more likely than others to produce this phenotype. When MSH1-dr frequencies were compared between parental and progeny generations, each derived from a single individual, the phenotype was only observed in progeny generations whose parental generation had some incidence of the phenotype (Table 5). Currently, we cannot rule out that the overall rarity of the MSH1-dr phenotype by the F₄generation may be the consequence of inadvertent selection rather than a natural tendency to gradually stabilize away from the phenotype.

TABLE 4

Frequency of MSH1-dr phenotype (8.4%) in epi-F3 families derived from
sorghum MSH1-dr × Tx430, and grown in the greenhouse.

			# Individuals	# Individuals
F3	Sample	Mean plant	with wild-type	with
family	size	height (cm)	height or higher	dwarf height

	1	10	160	10	0
	2	9	208	9	0
	3	10	167	10	0
	4	10	189	10	0
	5	8	186	7	1
	6	10	114	10	0
	7	9	203	9	0
	8	7	102	6	1
	9	2	107	2	0
	10	9	116	9	0
	11	4	89	3	1
	12	6	118	6	0
	13	10	187	10	0
	14	8	150	6	2
	15	7	81	3	4
	16	10	143	7	3
	17	5	122	5	0
	18	10	137	9	1
	19	10	98	10	0
Total	19	154	—	141	13

TABLE 5

From each of ten epi-lines, a single individual that did not display the
MSH1-dr phenotype was grown along with its parental generation. Parental and progeny
generation frequencies were then counted with N ≥105 in each generation.

Frequency of MSH1-dr phenotype

Line	Parental Frequency	%	Progeny Frequency	%

Msh1 . . . #11 × Tx430 #2-6-10-8	0/118	0%	0/114	0%
Msh1 . . . #11 × Tx430 #2-6-3-23	0/116	0%	0/119	0%
Msh1 . . . #15 × Tx430 #7-1-1-5	0/118	0%	0/117	0%
Msh1 . . . #15 × Tx430 #7-1-3-1	3/105	2.86%	2/122	1.64%
Msh1 . . . #15 × Tx430 #7-1-9-10	0/115	0%	0/111	0%
Msh1 . . . #22 × Tx430 #4-3-1-3	9/119	7.56%	1/112	0.89%
Msh1 . . . #22 × Tx430 #4-4-10-28	0/126	0%	0/125	0%
Msh1 . . . #24 × Tx430 #13-8-6	0/118	0%	0/128	0%
Msh1 . . . #24 × Tx430 #19-11-7	5/116	4.31%	7/122	5.74%
Msh1 . . . #28 × Tx430 #13-3-1	0/119	0%	0/120	0%

To ensure that the observed variation was not the consequence of inadvertent seed contamination or outcrossing, 50 SSR markers were used to test a number of derived lines, which produced no evidence of polymorphism (FIG. 6; Table 6). This analysis was extended with 1778 SNP markers that, when assayed across five different MSH1-epiF2 individuals and the wildtype Tx430, detected less than 0.8% variation (Tables 7, 8; FIG. 7). In Arabidopsis, the msh1 mutant genome was DNA sequenced, with genome alignment and de novo assembly producing no evidence of unexplained genome rearrangement or unusual mutation frequency (Xu et al. 2013). These data, together with reproducibility of the phenomenon, argue against the developmental reprogramming phenotype as a consequence of genome hypermutability.

TABLE 6

SSR marker polymorphism data for 43 markers. Markers were scored as
+ or − relative the pattern of Tx430 wildtype. SSR markers were selected based on their
polymorphic behavior in comparisons of Tx430 and ‘Wray’. Assays included a transgene-
null Tx-430 line displaying the developmental reprogramming phenotype (DR), one epi-F2,
two epi-F3 and seven epi-F4 lines.

Tx430-

F2-

F3-

F4-

SSR Marker

Tx430

DR

Wray

22.9

53

68

22.9

2b.3

2b.10

5a.3

12.3

14.1

17.2

XCUP 1

−

+

−

XCUP 5

−

+

−

XCUP 26

−

+

−

XCUP 28

−

+

−

XCUP 32

−

+

−

XCUP 48

−

+

−

XCUP 50

−

+

−

XCUP 61

−

+

−

XCUP 69

−

+

−

SAM 03605

−

+

−

SAM 06337

−

+

−

SAM 16073

−

+

−

SAM 19028

−

+

−

SAM 18581

−

+

−

SAM 21112

−

+

−

SAM 01312

−

+

−

SAM 51414

−

+

−

NA

−

SAM 56359

−

+

−

NA

−

SAM 55010

−

+

−

NA

−

SAM 56942

−

+

−

NA

−

SAM 62005

−

+

−

NA

−

SAM 62186b

−

+

−

NA

−

SAM 59974

−

+

−

NA

−

SAM 61376

−

+

−

NA

−

SAM 65125

−

+

−

NA

−

SAM 66160

−

+

−

NA

−

SAM 26858a

−

+

−

SAM 27170

−

+

−

SAM 33545

−

+

−

NA

−

SAM 36890

−

+

−

NA

−

SAM 47801

−

+

−

NA

−

SAM 48589

−

+

−

NA

−

SAM 48870

−

+

−

NA

−

SAM 49411b

−

+

−

NA

−

SAM 64056

−

+

−

NA

−

SAM 62693

−

+

−

NA

−

SAM 67633

−

+

−

NA

−

SAM 63126c

−

+

−

NA

−

SAM 64809

−

+

−

NA

−

SAM 01952

−

+

−

NA

−

−−

−

SAM 40439

−

+

−

NA

−

SAM 42610

−

+

−

NA

−

SAM 39963

−

+

−

NA

−

TABLE 7

SNP marker analysis: Summary of Het %

Pedigree	Total #markers	#Het	% Het

(Msh1epi11/Tx430):0002.0006.0010.	1778	13	0.73%
(Msh1epi15/Tx430):0007.0001.0001.	1778	13	0.73%
(Msh1epi15/Tx430):0008.0001.0002.	1778	13	0.73%
(Msh1epi22/Tx430):0004.0004.0010.	1776	14	0.79%
(Msh1epi24/Tx430):0019.0004.	1774	13	0.73%
RTx430WT	1773	14	0.79%

TABLE 8

SNP marker analysis: A list of all the markers with heterozygous
genotype.

		Genetic	Physical			(MSH1epi11/
		Distance	Distance			Tx430):	(MSH1epi15/
Marker	Chr	(cM)	(bp)	a1	a2	0002.0006.0010.	Tx430): 0007.0001.0001.

SNP1	1	18.8	19772012	C	G	CG	CG
SNP2
	1	27.7	31762071	A	G	AG	AG
SNP3
	3	62.9	33270511	C	T	CT	CT
SNP4
	3	66.6	42615046	C	G	GG	GG
SNP5
	3	66.9	35965148	A	G	AG	AG
SNP6	4	53.6	13022975	C	T	CT	CT
SNP7	4	58.4	23297070	A	G	AG	AG
SNP8	4	66.9	41492135	C	T	CT	CT
SNP9	4	67.3	42325806	C	T	CT	CT
SNP10
	5	62.6	16905084	A	G	AG	AG
SNP11	8	55.7	12142806	C	G	CC	CC
SNP12	9	73.1	43494421	C	T	CT	CT
SNP13	9	73.6	43961814	C	T	CT	CT
SNP14	9	77.8	46574813	A	G	AG	AG
SNP15
	10	54.2	10653756	G	T	GT	GT

		(MSHh1epi22/	(MSH1epi24/
	(MSH1epi15/	Tx430):	Tx430):
Marker	Tx430): 0008.0001.0002.	0004.0004.0010	0019.0004.	RTx430WT

SNP1	CG	CG	CG	CG
SNP2	AG	AG	AG	AG
SNP3	CT	CT	CT	CT
SNP4	GG	CG	GG	GG
SNP5	AG	AG	AG	AG
SNP6	CT	CT	CT	CT
SNP7	AG	AG	AG	AG
SNP8	CT	CT	CT	CT
SNP9	CT	CT	CT	CT
SNP10	AG	AG	AG	AG
SNP11	CC	CC	CC	CG
SNP12	CT	CT	CT	CT
SNP13	CT	CT	CT	CT
SNP14	AG	AG	AG	AG
SNP15	GT	GT	GT	GT

In Table 8, markers with heterozygous genotypes are ordered by chromosome and genetic distance. The remainder (not shown) had homozygous genotypes. Marker genotypes of the six lines are similar but for the two markers highlighted in yellow. The markers showing a heterozygous genotype represent the true heterozygous genotype, not heterogeneity at the markers since only a single plant was sampled for DNA.
Significant Increases in Trait Values Persist for Multiple Generations.
From the MSH1-epiF₂families, individuals were self-pollinated and selected for grain yield and plant height to the F₃and F₄generations. F₄lines, along with F₃and F₂lines from remnant seed, were evaluated together in the same 2011 field experiment. Despite weak selection intensity (33% and 38% of phenotyped plants were propagated to F₃and F₄, respectively), derived F₃and F₄lines showed significant differences in grain yield and plant height, as well as differences in dry biomass and panicle length (FIGS. 3, 5, Table 9). Differences were detectable even when F3 and F4 lines were analyzed separately or when a model term for generation was included, indicating that the variation did not simply come from maternal effects. While some traits appeared to be correlated, such as flowering time and grain yield, no correlation was detected between plant height and grain yield, indicating that height was not pleiotropically affecting grain yield (FIG. 8).

TABLE 9

Data for each trait listed below were fit to a linear mixed model (see
methods), with results indicating differences between lines. Line
was treated as a fixed effect while block and replicate were treated
as random effects. Separately analyzing lines by generation or
general height class, or adding a model term for generation and
height class, did not affect conclusions. The models were used to
estimate trait means and confidence intervals (FIGS. 3B, 5).

	Response variable		Sample	F-statistic of	p-value of
Population	of model	df	size	Line effect	Line effect

All lines	Grain Yield	160	956	4.985	<0.001
	(g/m²)^a
	Plant height (cm)^b	167	3537	159.6	<0.001
	Flowering time	136	1874	6.13	<0.001
	(DAS)^a,b
	Rachis Length	165	3495	5.06	<0.001
	(cm)^a,b
	Dry Biomass	167	490	5.1	<0.001
	(g/plant)^a

^aBox-cox transformed for model fit.
^bSeparate variances estimated by generation or height strata.

Although the F₃generation showed higher variance for some traits compared to the F₂generation, for all measured traits the F₄generation showed lower variance compared to the F₂generation (FIG. 2A). Furthermore, in contrast to the F₂generation, we did not find significant heterogeneity for variance in grain yield among wild-type, F₃and F₄lines (p>0.1, Brown-Forsythe test; p<0.01 in F₂with wild-type).
Analysis of direct lineages from F₂to F₄showed high response to selection for plant height but variable response for grain yield; however, in the most extreme examples, we observed gains of up to 87% over wild-type for above-ground dry biomass and 61% for grain yield (FIG. 3A). Overall, gains in the F₄were more modest compared to the F₃, implying progress may taper off by F₄in self-pollinated lineages. Indeed, there is evidence that the epi-F₃generation may be the most vigorous. As a population, it appears to have slightly higher overall grain yield than the epi-F2 or epi-F4. Nevertheless, the population mean for grain yield in the epi-F4 remains higher than that of wild-type Tx430 (FIG. 3B).
Line×Environment Interactions Suggest an Epigenetic Component to G×E.
We evaluated the yield performance of three MSH1-epiF₅families alongside wild-type Tx430 at two different locations. The two locations displayed a large difference in environmental means. Surprisingly, the lines showed little between-line difference at the site of the earlier experiments; however, they showed large differences at the second site, which was more drought-stressed, demonstrating a line×environment effect (FIGS. 4, 9A,B). Results at the first site also suggest that, depending on conditions, epigenetic variation in these materials could begin to dissipate at around the F₅generation. The outcomes of these experiments indicate that plant materials with little to no genetic variation have the potential to exhibit substantial variation in response to environmental influence, which may reflect epigenetic×environmental interactions.
The substantial range of sorghum phenotypic variation observed in this study appears to be non-genetic, and is induced by crossing to a MSH1-dr line, altered through MSH1 suppression in a previous generation. The MSH1-dr lines used in this study were maintained as transgene-nulls seven generations following segregation of the transgene, suggesting that the epigenetic properties of the MSH1-dr line are stable through multiple rounds of self-pollination (Xu et al. 2012). We do not presume that all of the variation observed is non-genetic; the observed bimodal distribution for plant height could support an alternative hypothesis of markedly enhanced reversion frequency for the dwarfing gene, dw3, in line Tx430 (Multani et al. 2003). If this is the case, the unusually high reversion rate may be the consequence of increased local recombination, one side-effect of cytosine methylation pattern redistribution (Colomé-Tatché et al. 2012; Migicovsky and Kovalchuk 2013). We are investigating this possibility presently. Nevertheless, we see additional height variation within short and tall plants, indicating variation beyond a single-locus.
The range of phenotypic variation observed is surprising. While we were not able to take measurements of all parameters for this initial study, the F₃and the F₄generations showed highly significant increases in above-ground biomass and grain yield over Tx430 wild type. One interpretation of these increases would be that dw3 reversion could cause pleiotropic changes in plant architecture. However, the greater range of plant height, panicle architecture and yield variation observed in this study appears to exclude that possibility (Brown et al. 2008).
The observation of epi-type×environment interaction in test plots suggests that at least some portion of the genotype×environment interaction that is commonly observed in varietal studies may be non-genetic, which is supported by other recent studies (Zhang et al. 2013). The MSH1 system may be useful in understanding this type of environmental influence and selecting for enhanced stability of crop performance.
To date, relatively few plant systems have permitted resolution of epigenetic variation as it influences plant phenotype. In Arabidopsis, mutation of genes that comprise the DNA methylation machinery, followed by crossing to wild-type for development of recombinant inbred lines, has provided valuable information on the phenotypic consequences of epigenomic perturbation, as well as heritability and stability of epigenetic changes (Johannes et al. 2009; Reinders et al. 2009). It has been suggested that doubled haploids, subjected to recursive selection for mitochondrial behavior, can produce epigenetic variation that may be amenable to selection (Hauben et al. 2009). Somaclonal variation derived from plant tissue culture has also been associated with epigenetic changes (Stroud et al. 2013).
MSH1-dr transgene null lines developed on elite inbred genetic backgrounds would permit direct incorporation of the MSH1-enhanced growth phenomenon to hybrid production. However, studies to date have not observed the greatest gain in growth to occur in the derived F₁populations, suggesting that the effects we observe in this system may be distinct from heterosis. It is possible that self- or open-pollination breeding will prove more effective at capturing maximal growth gain derived from MSH1 manipulation. The transgene-null MSH1-dr line crossed to its wild-type counterpart produces maximum variation in the epiF₂population, at which point selection appears to be most effective. Large-scale seed increase in F₃and F₄generations permits rapid capture of the growth enhancement as variation tapers off Extrapolation of this approach from sorghum data in this study would predict at least 25% overall enhancement of yield. In our experience with this system, variation observed in the epi-F₂population tends to produce above-wildtype performance more often than below (FIG. 3B, FIG. 8). Consequently, development of MSH1-dr in an elite line followed by selection in the epi-F₂, appears to result in, by the epiF₄, a population that is uniform genetically, variable epigenetically, and significantly enhanced in growth vigor and productivity.
The progress, response to selection, and final phenotypic outcomes observed in this study were of sufficient magnitude to suggest that untapped epigenetic potential may reside within crops. Whether crop enhancement using MSH1 manipulation will produce crop vulnerabilities not yet considered is under investigation. However, the performance of these plant materials under low rainfall conditions suggests that this methodology holds significant promise.

LITERATURE CITED

Abdelnoor R V, Yule R, Elo A, Christensen A C, Meyer-Gauen G, et al. 2003 Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS. Proc. Natl. Acad. Sci. USA 100:5968-5973.
Brown, P J, Rooney W L, Franks C, Kresovich S. 2008. Efficient mapping of plant height quantitative trait loci in a sorghum association population with introgressed dwarfing genes. Genetics 180: 629-637.
Cheptou P O, Donohue K. 2013 Epigenetics as a new avenue for the role of inbreeding depression in evolutionary ecology. Heredity (Edinb). 110:205-6.
Colomé-Tatché M, Cortijo S, Wardenaar R, Morgado L, Lahouze B, et al. 2012. Features of the Arabidopsis recombination landscape resulting from the combined loss of sequence variation and DNA methylation. Proc Natl Acad Sci USA. 109:16240-5.
Cowling, W. 2013. Sustainable plant breeding. Plant Breeding 132:1-9.
Dooner H K, Weil D F 2007. Give-and-take: interactions between DNA transposons and their host plant genomes. Curr Opin Genet Dev 17:486-492.
Gressel, J. 2008 Genetic Glass Ceilings: Transgenics for Crop Biodiversity. John Hopkins Univ. Press
Groszmann M, Greaves I K, Albert N, Fujimoto R, Helliwell C A, Dennis E S, Peacock W J. 2011 Epigenetics in plants-vernalisation and hybrid vigour. Biochim Biophys Acta. 1809:427-37.
Hauben M, Haesendonckx B, Standacrt E, Van Der Kelen K, Azmi A, Akpo H, Van Breusegem F, Guisez Y, Bots M, Lambert B, Laga B, De Block M. 2009. Energy use efficiency is characterized by an epigenetic component that can be directed through artificial selection to increase yield. Proc Natl Acad Sci USA. 106:20109-14.
Heo J B, Sung S. 2011 Encoding memory of winter by noncoding RNAs. Epigenetics. 6:544-7
Hothorn T, Bretz F, and Westfal P. 2008. Simultaneous Inference in General Parametric Models. Biometrical Journal 50: 346-363.
Johannes F, Porcher E, Teixeira F K, Saliba-Colombani V, Simon M, et al. 2009. Assessing the impact of transgenerational epigenetic variation on complex traits. PLoS Genet. 5(6):e1000530.
Li, M. Yuyama, N., Le Luo, Mariko Hirata, Cai, H. 2009. In silico mapping of 1758 new SSR markers developed from public genomic sequences for sorghum. Mol. Breed. 24: 41-47.
Lu, P. et al. 2012 Analysis of Arabidopsis genome-wide variations before and after meiosis and meiotic recombination by resequencing Landsberg erecta and all four products of a single meiosis. Genome Res. 22: 508-518.
Mcaburn E L, Schalkwyk L C, Mill J. 2010. Allele-specific methylation in the human genome: implications for genetic studies of complex disease. Epigenetics. 5:578-82.
Migicovsky Z, Kovalchuk I. 2013. Changes to DNA methylation and homologous recombination frequency in the progeny of stressed plants. Biochem Cell Biol. 91:1-5
Miller F R 1984. Registration of RTx430 sorghum parental line. Crop Sci. 24:1224.
Multani D S, Briggs S P, Chamberlin M A, Blakeslee J J, Murphy A S, Johal G S. 2003. Loss of an MDR transporter in compact stalks of maize br2 and sorghum dw3 mutants. Science 302:81-4
Pinheiro J, Bates D, DebRoy S, Sarkar D and the R Development Core Team. 2013. Nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1-109.
Reinders J, Wulff B B, Mirouze M, Mari-Ordóñez A, Dapp M, et al. 2009. Compromised stability of DNA methylation and transposon immobilization in mosaic Arabidopsis epigenomes. Genes Dev. 23:939-50.
Schloss, S. J. et al. 2002. Characterization of RFLP probe sequence for gene discovery and SSR development in Sorghum bicolor (L.) Moench. Theor. Appl. Genet 105, 912-920.
Schmitz R J, Amasino R M. (2007) Vernalization: a model for investigating epigenetics and eukaryotic gene regulation in plants. Biochim Biophys Acta. 1769:269-75.
Shedge V, Davila J, Arrieta-Montiel M P, Mohammed S, Mackenzie S A 2010. Extensive rearrangement of the Arabidopsis mitochondrial genome elicits cellular conditions for thermotolerance. Plant Physiol. 152:1960-1970.
Shivaprasad P V, Dunn R M, Santos B A, Bassett A, Baulcombe D C. 2012 Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs. EMBO J. 31:257-66.
Smith A M, Hansey C N, Kaeppler S M 2012. TCUP: A novel hAT transposon active in maize tissue culture. Front Plant Sci 3:6.
Stokes, T. L., Kunkel, B. N. & Richards, E J 2002. Epigenetic variation in Arabidopsis disease resistance. Genes Dev 16:171-182.
Stroud H, Ding B, Simon S A, Feng S, Bellizzi M, Pellegrini M, Wang G L, Meyers B C, Jacobsen S E. 2013. Plants regenerated from tissue culture contain stable epigenome changes in rice. Elife 2:e00354. doi: 10.7554/eLife.00354.
Tsaftaris, A. S., Polidoros, A. N., Kapazoglou, A., Tani, E. and Kovačević, N. M. 2008 Epigenetics and Plant Breeding, in Plant Breeding Reviews, Volume 30 (ed J. Janick), John Wiley & Sons, Inc., Hoboken, N.J., USA. doi: 10.1002/97804703801 30.ch2
Xu Y Z, Arrieta-Montiel M P, Virdi K, De Paula W B M, Widhalm J R, et al. 2011. MSH1 is a nuclcoid protein that alters mitochondrial and plastid properties and plant response to high light. Plant Cell 239:3428-41
Xu, Y-Z, Santamaria, R., Virdi K S, Arrieta-Montiel M P, Razvi F, et al. 2012. The chloroplast triggers developmental reprogramming when MUTS HOMOLOG1 is suppressed in plants. Plant Physiol. 159:710-20.
Xu Y-Z, Laurie J D, Wang D, Virdi K S, Feng S, Yu J, Wamboldt Y, Chen M, Riethoven J J M, Arrieta-Montiel M P, Kundariya H, Mackenzie S A. 2013. MSH1 mutation alters the epigenome to produce heritable changes in plant growth. Submitted.
Zhang Y Y, Fischer M, Colot V, Bossdorf O. 2013. Epigenetic variation creates potential for evolution of plant phenotypic plasticity. New Phytol. 197:314-22.

Example 2. Use of Msh1-Perturbed Plants that do not Exhibit an MSH1-Dr Phenotype to Obtain Progeny Plants with Useful Traits

Transformation of tomato and millet with a transgene that provides for RNAi suppression of the MSH-1 gene of the MSH1 gene results in suppression of MSH1 expression (as described in US Patent Application Publication No. 20120284814, which is incorporated herein by reference in its entirety) and the emergence of variant plant phenotypes. Progeny obtained by selfing the progeny of selfed T0 transformants with MSH1 suppression were grown, and displayed segregation for the MSH1 suppressing transgene and a wide range of variant phenotypes. Selection and self-pollination of the dwarf types produced predominantly dwarf types. However, selection and self-pollination of the larger or normal growth types produced progeny displaying a wide range of phenotypes. This is illustrated in FIGS. 10A and 10B for millet, where a robust plant progeny line lacking the transgene was obtained from a subsequent generation of selfed progeny of a selfed MSH1 suppressed transgenic parent. In tomato, it has been possible to identify a robust transgene-null T5 and T6 line that was obtained by selfing five (T5) or six (T6) generations of progeny from selfed T0 plants that had been subjected to MSH1 suppression (FIG. 12A). This robust tomato line obtained by selfing progeny of a selfed selfed MSH1 suppressed transgenic parent out-performed the isogenic wildtype control plants in plant growth rate and fruit number (FIG. 12B, C, D. To date, tomato has been shown to breed true for this enhanced growth capacity for two additional generations. Data comparing the robust inbred tomato plant lines obtained by selfing progeny of a selfed selfed MSH1 suppressed transgenic parent (i.e. progeny from selfs of line T17-12-15-4-6; shown in boldface) are provided in the following Tables.

TABLE 10

Height Data.

Height data	6		10	12	14
(cm)	weeks	8 weeks	weeks	weeks	weeks

Rutgers-1	28.6	53.6	103.6	131.2	151.2
Rutgers-3	26.2	58.9	119.4	141.8	158.8
Rutgers-4	26.4	63.8	120.6	154.2	169.4
Rutgers-5	25.8	61.0	101.9	134.8	147.4
AVG	26.8	59.3	111.4	140.5	156.7
SD	1.3	4.3	10.0	10.1	9.7
(TXR7)-1 F2	39.2	64.5	102.5	113.9	125.5
(TXR7)-2 F2	40.2	88.5	112.5	118.3	141.4
(TXR7)-3 F2	44.8	80.5	107.8	120.4	145.7
(TXR7)-4 F2	38.2	82.5	124.9	156.2	174.6
(TXR7)-5 F2	39.0	72.8	108.2	115.4
AVG	40.3	77.8	111.2	124.8	146.8
SD	2.6	9.3	8.5	17.7	20.5
(TXR8)-1 F2	41.4	83.1	117.2	135.2	136.9
(TXR8)-2 F2	37.5	86.4	127.5	146.4	161.8
(TXR8)-3 F2	39.5	77.4	116.8	128.7	141.8
(TXR8)-4 F2	34.4	78.9	127.2	157.3	183.6
(TXR8)-5 F2	44.7	85.4	115.4	123.7	138.6
AVG	39.5	82.2	120.8	138.3	152.5
SD	3.9	4.0	6.0	13.6	20.0
(TXR3)-1 F2	21.0	65.2	99.2	133.4	139.5
(TXR3)-2 F2	36.1	64.5	107.9	123.8	135.2
(TXR3)-3 F2	33.8	68.6	104.5	118.8	134.3
(TXR3)-4 F2	38.2	76.6	112.6	142.3	162.1
AVG	32.3	68.7	106.1	129.6	142.8
SD	7.7	5.5	5.6	10.4	13.1
(TXR5)-1 F2	41.5	80.4	108.8	127.2	131.4
(TXR5)-2 F2	30.1	69.8	104.5	145.9	165.8
(TXR5)-3 F2	56.2	105.6	128.8	146.8	150.3
(TXR5)-4 F2	39.6	76.8	110.5	139.4	155.2
(TXR5)-5 F2	36.2	76.8	102.1	122.0	140.6
AVG	40.7	81.9	110.9	136.3	148.7
SD	9.7	13.8	10.5	11.2	13.2
(T17-12-15-4-	39.5	69.8	131.5	174.2	204.8
6)-1
(T17-12-15-4-	16.2	55.8	121.6	179.6	222.2
6)-2
(T17-12-15-4-	35.2	79.6	138.5	188.8	194.2
6)-3
(T17-12-15-4-	27.2	62.8	119.7	168.5	205.2
6)-4
(T17-12-15-4-	29.8	75.4	134.2	181.9	180.2
6)-5
AVG	29.6	68.7	129.1	178.6	201.3
SD	8.9	9.6	8.1	7.7	15.5
(T17-12-15-4-	18.6	40.5	63.2	98.4	116.8
8)-1
(T17-12-15-4-	12.5	21.5	40.8	61.4	77.1
8)-2
(T17-12-15-4-	6.5	8.7	24.2	44.9	48.2
8)-3
(T17-12-15-4-	16.2	22.6	40.9	65.5	96.5
8)-4
(T17-12-15-4-	12.0	23.3	43.6	63.5	89.6
8)-5
AVG	13.2	23.3	42.5	66.7	85.6
SD	4.6	11.3	13.9	19.5	25.4
(T17-12-15-4-	12.8	28.6	58.5	85.4	94.2
9)-1
(T17-12-15-4-	9.8	19.8	40.5	60.8	68.8
9)-2
(T17-12-15-4-	13.2	23.8	42.8	65.2	87.5
9)-4
(T17-12-15-4-	10.5	20.7	35.8	58.4	70.8
9)-5
AVG	11.6	23.2	44.4	67.5	80.3
SD	1.7	4.0	9.8	12.3	12.5
(T17-12-15-4-	33.2	63.8	104.5	129.8	148.8
14)-1
(T17-12-15-4-	22.2	58.3	92.8	117.4	145.9
14)-2
(T17-12-15-4-	27.2	57.2	76.8	109.8	141.6
14)-3
(T17-12-15-4-	26.8	60.5	90.1	129.6	163.9
14)-4
(T17-12-15-4-	28.2	57.8	92.5	116.8	149.8
14)-5
AVG	27.5	59.5	91.3	120.7	150.0
SD	3.9	2.7	9.9	8.8	8.4

Data for robust inbred in bold.

TABLE 11

Total Fruit Number

Total fruit	12		16		20
number	weeks	14 weeks	weeks	18 weeks	weeks

Rutgers-1	9.0	10.0	10.0	7.0	12.0
Rutgers-3	12.0	13.0	13.0	13.0	13.0
Rutgers-4	12.0	14.0	15.0	15.0	16.0
Rutgers-5	15.0	14.0	13.0	14.0	17.0
AVG	12.0	12.8	12.8	12.3	14.5
SD	2.4	1.9	2.1	3.6	2.4
(T X R 7)-1 F2	18.0	27.0	28.0	26.0	36.0
(TXR7)-2 F2	12.0	17.0	29.0	26.0	27.0
(TXR7)-3 F2	17.0	24.0	30.0	30.0	35.0
(TXR7)-4 F2	16.0	20.0	23.0	26.0	34.0
AVG	15.8	22.0	27.5	27.0	33.0
SD	2.6	4.4	3.1	2.0	4.1
(TXR8)-1 F2	19.0	26.0	38.0	26.0	32.0
(TXR8)-2 F2	15.0	29.0	31.0	33.0	38.0
(TXR8)-3 F2	16.0	21.0	27.0	28.0	32.0
(TXR8)-4 F2	13.0	16.0	23.0	19.0	24.0
(TXR8)-5 F2	15.0	24.0	20.0	30.0	34.0
AVG	14.8	22.5	25.3	27.5	32.0
SD	1.3	5.4	4.8	6.0	5.9
(TXR3)-1 F2	11.0	14.0	15.0	24.0	25.0
(TXR3)-2 F2	17.0	23.0	30.0	32.0	32.0
(TXR3)-3 F2	8.0	20.0	22.0	31.0	32.0
(TXR3)-4 F2	10.0	17.0	17.0	17.0	20.0
AVG	11.5	18.5	21.0	26.0	27.3
SD	3.4	3.4	5.8	6.0	5.1
(TXR5)-1 F2	16.0	21.0	22.0	24.0	28.0
(TXR5)-2 F2	14.0	20.0	23.0	30.0	31.0
(TXR5)-3 F2	18.0	31.0	32.0	31.0	38.0
(TXR5)-4 F2	13.0	23.0	26.0	29.0	39.0
(TXR5)-5 F2	14.0	17.0	19.0	17.0	26.0
AVG	15.0	22.4	24.4	26.2	32.4
SD	2.0	5.3	4.9	5.8	5.9
(T17-12-15-4-6)-1	26.0	37.0	51.0	59.0	64.0
(T17-12-15-4-6)-2	14.0	28.0	24.0	29.0	34.0
(T17-12-15-4-6)-3	26.0	33.0	46.0	45.0	49.0
(T17-12-15-4-6)-4	24.0	32.0	47.0	51.0	51.0
(T17-12-15-4-6)-5	23.0	31.0	45.0	51.0	52.0
AVG	22.6	32.2	42.6	47.0	50.0
SD	5.0	3.3	10.6	11.2	10.7
(T17-12-15-4-8)-1	3.0	4.0	5.0	6.0	6.0
(T17-12-15-4-8)-2	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-3	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-4	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-5	0.0	0.0	0.0	0.0	0.0
AVG	0.6	0.8	1.0	1.2	1.2
SD	1.3	1.8	2.2	2.7	2.7
(T17-12-15-4-9)-1	2.0	7.0	7.0	8.0	7.0
(T17-12-15-4-9)-2	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-9)-4	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-9)-5	0.0	0.0	0.0	0.0	0.0
AVG	0.5	1.8	1.8	2.0	1.8
SD	1.0	3.5	3.5	4.0	3.5
(T17-12-15-4-	8.0	10.0	17.0	17.0	20.0
14)-1
(T17-12-15-4-	8.0	10.0	10.0	22.0	25.0
14)-2
(T17-12-15-4-	8.0	8.0	14.0	27.0	17.0
14)-3
(T17-12-15-4-	5.0	10.0	14.0	24.0	25.0
14)-4
(T17-12-15-4-	5.0	8.0	11.0	14.0	20.0
14)-5
AVG	6.8	9.2	13.2	20.8	21.4
SD	1.6	1.1	2.8	5.3	3.5

Data for robust inbred in bold.

TABLE 12

Red Fruit Number

	14	15	16	17	18	19	20
red fruit number	weeks	weeks	weeks	weeks	weeks	weeks	weeks

Rutgers-1	0.0	2.0	5.0	5.0	5.0	7.0	7.0
Rutgers-3	0.0	0.0	1.0	5.0	13.0	13.0	13.0
Rutgers-4	1.0	2.0	4.0	5.0	12.0	15.0	15.0
Rutgers-5	1.0	3.0	3.0	3.0	7.0	10.0	11.0
AVG	0.5	1.8	3.3	4.5	9.3	11.3	11.5
SD	0.6	1.3	1.7	1.0	3.9	3.5	3.4
(TXR7)-1 F2	4.0	9.0	14.0	16.0	20.0	22.0	24.0
(TXR7)-2 F2	2.0	5.0	7.0	7.0	9.0	9.0	11.0
(TXR7)-3 F2	5.0	10.0	15.0	16.0	19.0	24.0	24.0
(TXR7)-4 F2	0.0	4.0	7.0	16.0	15.0	16.0	24.0
(TXR7)-5 F2
AVG	2.8	7.0	10.8	13.8	15.8	17.8	20.8
SD	2.2	2.9	4.3	4.5	5.0	6.8	6.5
(TXR8)-1 F2	3.0	16.0	16.0	6.0	10.0	12.0	20.0
(TXR8)-2 F2	3.0	7.0	12.0	15.0	16.0	21.0	30.0
(TXR8)-3 F2	1.0	4.0	5.0	17.0	17.0	20.0	25.0
(TXR8)-4 F2	1.0	2.0	6.0	12.0	11.0	13.0	16.0
(TXR8)-5 F2	3.0	7.0	9.0	16.0	22.0	25.0	27.0
AVG	2.0	5.0	8.0	15.0	16.5	19.8	24.5
SD	1.2	2.4	3.2	2.2	4.5	5.0	6.0
(TXR3)-1 F2	0.0	3.0	7.0	14.0	17.0	18.0	19.0
(TXR3)-2 F2	1.0	13.0	15.0	17.0	20.0	24.0	29.0
(TXR3)-3 F2	1.0	7.0	8.0	10.0	18.0	24.0	26.0
(TXR3)-4 F2	1.0	6.0	7.0	12.0	12.0	14.0	16.0
AVG	0.8	7.3	9.3	13.3	16.8	20.0	22.5
SD	0.4	3.6	3.3	2.6	2.9	4.2	5.2
(TXR5)-1 F2	0.0	6.0	12.0	17.0	20.0	20.0	21.0
(TXR5)-2 F2	1.0	5.0	6.0	14.0	16.0	17.0	19.0
(TXR5)-3 F2	4.0	15.0	15.0	12.0	15.0	20.0	24.0
(TXR5)-4 F2	1.0	6.0	9.0	12.0	17.0	20.0	24.0
(TXR5)-5 F2	1.0	6.0	7.0	10.0	12.0	13.0	15.0
AVG	1.4	7.6	9.8	13.0	16.0	18.0	20.6
SD	1.5	4.2	3.7	2.6	2.9	3.1	3.8
(T17-12-15-4-6)-1	2.0	13.0	17.0	19.0	27.0	35.0	40.0
(T17-12-15-4-6)-2	0.0	3.0	5.0	8.0	18.0	23.0	26.0
(T17-12-15-4-6)-3	3.0	5.0	6.0	12.0	20.0	24.0	36.0
(T17-12-15-4-6)-4	2.0	7.0	14.0	15.0	27.0	28.0	39.0
(T17-12-15-4-6)-5	1.0	6.0	16.0	19.0	25.0	31.0	34.0
AVG	1.6	6.8	11.6	14.6	23.4	28.2	35.0
SD	1.1	3.8	5.7	4.7	4.2	5.0	5.6
(T17-12-15-4-8)-1	0.0	0.0	1.0	1.0	5.0	5.0	5.0
(T17-12-15-4-8)-2	0.0	0.0	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-3	0.0	0.0	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-4	0.0	0.0	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-5	0.0	0.0	0.0	0.0	0.0	0.0	0.0
AVG	0.0	0.0	0.2	0.2	1.0	1.0	1.0
SD	0.0	0.0	0.4	0.4	2.2	2.2	2.2
(T17-12-15-4-9)-1	0.0	0.0	0.0	2.0	1.0	6.0	6.0
(T17-12-15-4-9)-2	0.0	0.0	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-9)-4	0.0	0.0	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-9)-5	0.0	0.0	0.0	0.0	0.0	0.0	0.0
AVG	0.0	0.0	0.0	0.5	0.3	1.5	1.5
SD	0.0	0.0	0.0	1.0	0.5	3.0	3.0
(T17-12-15-4-14)-1	0.0	2.0	3.0	7.0	10.0	11.0	15.0
(T17-12-15-4-14)-2	0.0	1.0	2.0	8.0	11.0	11.0	10.0
(T17-12-15-4-14)-3	0.0	0.0	1.0	7.0	7.0	8.0	11.0
(T17-12-15-4-14)-4	0.0	0.0	2.0	6.0	9.0	10.0	12.0
(T17-12-15-4-14)-5	0.0	2.0	5.0	2.0	4.0	4.0	6.0
AVG	0.0	1.0	2.6	6.0	8.2	8.8	10.8
SD	0.0	1.0	1.5	2.3	2.8	2.9	3.3

Data for robust inbred in bold.

TABLE 13

Inflorescence Number

	8	10	12	14	16
Inflorescence Number	weeks	weeks	weeks	weeks	weeks

Rutgers-1	1.0	2.0	3.0	10.0	9.0	8.0
Rutgers-3	1.0	2.0	3.0	9.0	7.0	10.0
Rutgers-4	1.0	2.0	8.0	12.0	11.0	15.0
Rutgers-5	1.0	2.0	7.0	10.0	6.0	10.0
AVG	1.0	2.0	5.3	10.3	8.3	10.8
SD	0.0	0.0	2.6	1.3	2.2	3.0
(T17-12-5-11 X Rutgers 7)-1	3.0	6.0	16.0	19.0	21.0	19.0
F2
(T17-12-5-11 X Rutgers 7)-2	2.0	4.0	13.0	20.0	17.0	22.0
F2
(T17-12-5-11 X Rutgers 7)-3	4.0	6.0	16.0	20.0	20.0	21.0
F2
(T17-12-5-11 X Rutgers 7)-4	2.0	3.0	14.0	21.0	20.0	27.0
F2
(T17-12-5-11 X Rutgers 7)-5	2.0	4.0	13.0	19.0
F2
AVG	2.6	4.6	14.4	19.8	19.5	22.3
SD	0.9	1.3	1.5	0.8	1.7	3.4
(T17-12-5-11 X Rutgers 8)-1	2.0	4.0	16.0	18.0	20.0	23.0
F2
(T17-12-5-11 X Rutgers 8)-2	2.0	3.0	14.0	22.0	28.0	24.0
F2
(T17-12-5-11 X Rutgers 8)-3	2.0	4.0	13.0	17.0	16.0	16.0
F2
(T17-12-5-11 X Rutgers 8)-4	1.0	3.0	12.0	17.0	21.0	19.0
F2
(T17-12-5-11 X Rutgers 8)-5	2.0	4.0	13.0	21.0	18.0	22.0
F2
AVG	1.8	3.6	13.6	19.0	20.6	20.8
SD	0.4	0.5	1.5	2.3	4.6	3.3
(T17-12-5-11 X Rutgers 3)-1	1.0	1.0	7.0	13.0	15.0	15.0
F2
(T17-12-5-11 X Rutgers 3)-2	2.0	4.0	15.0	16.0	17.0	19.0
F2
(T17-12-5-11 X Rutgers 3)-3	2.0	3.0	10.0	20.0	21.0	21.0
F2
(T17-12-5-11 X Rutgers 3)-4	2.0	4.0	17.0	22.0	21.0	23.0
F2
AVG	1.8	3.0	12.3	17.8	18.5	19.5
SD	0.5	1.4	4.6	4.0	3.0	3.4
(T17-12-5-11 X Rutgers 5)-1	2.0	3.0	16.0	20.0	18.0	21.0
F2
(T17-12-5-11 X Rutgers 5)-2	2.0	4.0	12.0	20.0	25.0	27.0
F2
(T17-12-5-11 X Rutgers 5)-3	2.0	6.0	16.0	20.0	16.0	17.0
F2
(T17-12-5-11 X Rutgers 5)-4	2.0	5.0	15.0	17.0	24.0	29.0
F2
(T17-12-5-11 X Rutgers 5)-5	2.0	3.0	16.0	21.0	18.0	22.0
F2
AVG	2.0	4.2	15.0	19.6	20.2	23.2
SD	0.0	1.3	1.7	1.5	4.0	4.8
(T17-12-15-4-6)-1	2.0	4.0	10.0	14.0	17.0	24.0
(T17-12-15-4-6)-2	1.0	1.0	4.0	11.0	15.0	29.0
(T17-12-15-4-6)-3	1.0	2.0	5.0	16.0	14.0	20.0
(T17-12-15-4-6)-4	1.0	2.0	8.0	16.0	16.0	20.0
(T17-12-15-4-6)-5	1.0	4.0	8.0	14.0	12.0	18.0
AVG	1.2	2.6	7.0	14.2	14.8	22.2
SD	0.4	1.3	2.4	2.0	1.9	4.4
(T17-12-15-4-8)-1	0.0	0.0	4.0	10.0	21.0	24.0
(T17-12-15-4-8)-2	0.0	0.0	1.0	2.0	7.0	10.0
(T17-12-15-4-8)-3	0.0	0.0	0.0	0.0	0.0	0.0
(T17-12-15-4-8)-4	0.0	0.0	0.0	0.0	4.0	8.0
(T17-12-15-4-8)-5	0.0	0.0	2.0	7.0	8.0	6.0
AVG	0.0	0.0	1.4	3.8	8.0	9.6
SD	0.0	0.0	1.7	4.5	7.9	8.9
(T17-12-15-4-9)-1	0.0	0.0	3.0	10.0	13.0	17.0
(T17-12-15-4-9)-2	0.0	0.0	1.0	2.0	2.0	0.0
(T17-12-15-4-9)-4	0.0	0.0	2.0	9.0	10.0	14.0
(T17-12-15-4-9)-5	0.0	0.0	1.0	3.0	6.0	1.0
AVG	0.0	0.0	1.8	6.0	7.8	8.0
SD	0.0	0.0	2.7	10.1	12.5	16.8
(T17-12-15-4-14)-1	1.0	2.0	11.0	21.0	25.0	30.0
(T17-12-15-4-14)-2	1.0	1.0	9.0	13.0	18.0	16.0
(T17-12-15-4-14)-3	1.0	2.0	9.0	15.0	18.0	17.0
(T17-12-15-4-14)-4	1.0	2.0	7.0	13.0	20.0	25.0
(T17-12-15-4-14)-5	1.0	2.0	7.0	12.0	12.0	18.0
AVG	1.0	1.8	8.6	14.8	18.6	21.2
SD	0.0	0.4	1.7	3.6	4.7	6.1

Data for robust inbred in bold.

A similar range of plant phenotypes arise in Arabidopsis msh1 T-DNA insertion mutants that are selfed. In brief, an Arabidopsis strain that was heterozygous for an T-DNA insertional mutation in the endogenous Msh1 gene and obtained from the Arabidopsis Stock Center (Arabidopsis Biological Resource Center, Columbus, Ohio, USA) was selfed to obtain msh1 plants homozygous for this T-DNA insertion that lack a functional MSH1 gene. These homozygous msh1 null plants are subsequently selfed to obtain subsequent generations of msh1 null plants that exhibit phenotypic variability that includes certain useful traits. Useful traits observed in the selfed msh1 lines include increased above-ground biomass and drought tolerance (FIG. 11A, B, C, D).
At low frequency, it is feasible in all of these cases to identify robust variants that out-perform isogenic wildtype in above-ground biomass (Arabidopsis), fruit number (tomato) or panicle size (millet) by selecting for robust progeny of plants subjected to MSH1 suppression. For tomato and millet, this result was obtained by selfing and did not entail outcrossing. This is in contrast to previously reported results described in U.S. patent application Ser. No. 13/155,505 where variants that out-perform isogenic wildtype were obtained by outcrossing plants subjected to MSH1 suppression and selecting for progeny that exhibited one or more MSH1-dr phenotypes (CMS, leaf variegation, dwarfed growth and reduced internode elongation, enhanced branching, altered leaf morphology, extended juvenility, and delayed flowering).

Example 3. Summary Tables of Nucleic Add Sequences and SEQ ID NO

TABLE 14

Nucleotide Sequences provided in the Sequence Listing

Internet Accession	SEQ ID
Information	NO	Comments

The Arabidopsis Information Resource	1	Arabidopsis
(TAIR)		MSH1
1009043787		Full length cDNA (DNA
on the internet (world wide web) at		sequence)
arabidopsis.org
The Arabidopsis Information Resource	2	Arabidopsis
(TAIR)		MSH1 Protein (amino acid
1009118392		sequence)
on the internet (world wide web) at
arabidopsis.org
NCBI AY856369	3	Soybean MSH1
on the world wide web at		>gi\|61696668\|gb\|AY856369.1\|
ncbi.nlm.nih.gov/nuccore		Glycine max DNA mismatch
		repair protein (MSH1)
		complete cds; (DNA sequence)
NCBI Accession	4	Zea mays MSH1
AY856370		gi\|61696670\|gb\|AY856370.1\|
on the world wide web at		Zea mays DNA mismatch
ncbi.nlm.nih.gov/nuccore		repair protein (MSH1),
		complete cds; (DNA sequence)
NCBI Accession	5	Tomato MSH1
AY866434.1		>gi\|61696672\|gb\|AY866434.1\|
on the world wide web at		Lycopersicon esculentum DNA
ncbi.nlm.nih.gov/nuccore		mismatch repair protein
		(MSH1), partial cds; (DNA
		sequence)
NCBI	6	Sorghum MSH1
XM002448093.1		>gi\|242076403: 1-3180
on the world wide web at		Sorghum bicolor hypothetical
ncbi.nlm.nih.gov/nuccore		protein; (DNA sequence)
Os04g42784.1	7	Rice (Oryza sativa) MSH1
Rice Genome Annotation Project - MSU		coding sequence (DNA
Rice Genome Annotation (Osa1)		sequence)
Release 6.1
Internet address
rice.plantbiology.msu.edu/index.shtml
Brachypodium	8	Brachypodium
Bradi5g15120.1		MSH1 coding region (DNA
On the world wide web at		sequence)
gramene.org/Brachypodium_distachyon/Gene/
Summary?db=core;g=BRADI5G15120;
r=5:18500245-18518223;t=BRADI5G15120.1
GSVIVT01027931001	9	Vitis Vinifera
On the world wide web at		MSH1 cDNA (DNA sequence)
genoscope.cns.fr/spip/Vitis-viniferae.html
Cucsa.255860.1	10	Cucumber (Cucumis sativa)
On the internet (world wide web) at		MSH1 coding sequence; (DNA
phytozome.net/		sequence)
GenBank Accession	11	Cotton (Gossypium hirsutum)
ES831813.1		MSH1 partial cDNA sequence
on the world wide web at		(EST); (DNA sequence)
ncbi.nlm.nih.gov/nucest
Oryza_sativa_msh1_2000up	12	Oryza_sativa_msh1_Promoter
>Rice-LOC_Os04g42784		and 5′ UTR
Solanum_lycopersicum_2000up	13	Solanum_lycopersicum msh1
>Tomato-Solyc09g090870.2		promoter and 5′ UTR
Sorghum_bicolor_MSH1_2000up_Phytozome	14	Sorghum bicolor msh1
>Sb06g021950		promoter and 5′ UTR
Arabidopsis-Col0-MSH1	15	Arabidopsis-Col0-MSH1
		promoter and 5′ UTR
>gi\|145337631\|ref\|NM_106295.3\|	16	Arabidopsis PPD3 coding
Arabidopsis thaliana photosystem II		region
reaction center PsbP family protein
cDNA, complete cds
>gi\|297839518\|ref\|XM_002887595.1\|	17	Arabidopsis PPD3 coding
Arabidopsis lyrata subsp. lyrata		region
hypothetical protein, cDNA
>gi\|449522158\|ref\|XM_004168047.1\|	18	Cucumis sativus PPD3 coding
PREDICTED: Cucumis sativus psbP domain-		region
containing protein
3, chloroplastic-like
(LOC101211525), cDNA
>gi\|255539323\|ref\|XM_002510681.1\|	19	Ricinus communis PPD3
Ricinus communis conserved		coding region
hypothetical protein cDNA
>gi\|359491869\|ref\|XM_002273296.2\|	20	Vitis vinifera PPD3 coding
PREDICTED: Vitis vinifera psbP domain-		region
containing protein
3, chloroplastic-like
(LOC100263326), cDNA
>gi\|357467178\|ref\|XM_003603826.1\|Medicago	21	Medicago truncatula PPD3 coding
truncatula PsbP domain-containing protein		region
(MTR_3g116110) cDNA, complete cds
>gi\|224083365\|ref\|XM_002306962.1\| Populus	22	Populus trichocarpa PPD3 coding
trichocarpa predicted protein, cDNA		region
>gi\|388521576\|gb\|BT149056.1\|Lotus	23	Lotus japonicus PPD3 coding
japonicus clone JCVI-FLLj-8L12		region
unknown cDNA
gi\|470131466\|ref\|XM_004301567.1\|	24	Fragaria vesca PPD3 coding
PREDICTED: Fragaria vesca subsp.		region
vesca psbP domain-containing protein 3,
chloroplastic-like (LOC101302662),
mRNA
>gi\|356517169\|ref\|XM_003527214.1\|	25	Glycine max PPD3 coding
PREDICTED: Glycine max psbP		region
domain-containing protein 3,
chloroplastic-like (LOC100805637),
mRNA
Solanum lycopersicum psbP domain-	26	Solanum lycopersicum PPD3
containing protein
3, chloroplastic-like		coding region
(LOC101247415), mRNA
>gi\|502130964\|ref\|XM_004500773.1\|	27	Cicer arietinum PPD3 coding
PREDICTED: Cicer arietinum psbP domain-		region
containing protein
3, chloroplastic-like
(LOC101499898), transcript variant X2, mRNA
>gi\|241989846\|dbj\|AK330387.1\| Triticum	28	Triticum aestivum PPD3
aestivum cDNA, clone: SET4_F09, cultivar:		coding region
Chinese Spring
>gi\|115477245\|ref\|NM_001068754.1\|	29	Oryza sativa PPD3 coding
Oryza saliva Japonica Group		region
Os08g0512500 (Os08g0512500)
mRNA, complete cds
>gi\|357141873\|ref\|XM_003572329.1\|	30	Brachypodium distachyon
PREDICTED: Brachypodium		PPD3 coding region
distachyon psbP domain-containing
protein 3, chloroplastic-like
(LOC100840022), mRNA
>gi\|242383886\|emb\|FP097685.1\|	31	Phyllostachys edulis PPD3
Phyllostachys edulis cDNA clone:		coding region
bphylf043n24, full insert sequence
>gi\|326512571\|dbj\|AK368438.1\|	32	Hordeum vulgare PPD3 coding
Hordeum vulgare subsp. vulgare mRNA		region
for predicted protein, partial cds, clone:
N1ASHv2073K06
>gi\|195613363\|gb\|EU956394.1\|Zea	33	Zea mays PPD3 coding region
mays clone 1562032 thylakoid lumen
protein mRNA, complete cds
>gi\|242082240\|ref\|XM_002445844.1\|	34	Sorghum bicolor PPD3 coding
Sorghum bicolor hypothetical protein,		region
mRNA
>gi\|514797822\|ref\|XM_004973837.1\|	35	Setaria italica PPD3 coding
PREDICTED: Setaria italica psbP		region
domain-containing protein 3,
chloroplastic-like (LOC101754517),
mRNA
>gi\|270145042\|gb\|BT111994.1\|Picea glauca	36	Picea glauca PPD3 coding
clone GQ03308_J01 mRNA sequence		region
>gi\|215274040\|gb\|EU935214.1\|Arachis diogoi	37	Arachis diogoi PPD3 coding
clone AF1U3 unknown mRNA		region
>gi\|168003548\|ref\|XM_001754423.1\|	38	Physcomitrella patens PPD3
Physcomitrella patens subsp. patens		coding region
predicted protein
(PHYPADRAFT_175716) mRNA,
complete cds
>gi\|302809907\|ref\|XM_002986600.1\|	39	Selaginella moellendorffii
Selaginella moellendorffii hypothetical		PPD3 coding region
protein, mRNA
>gi\|330318510\|gb\|HM003344.1\|	40	Camellia sinensis PPD3 coding
Camellia sinensis clone U10BcDNA		region
3162
Zea_mays_2000up_phytozome	41	Zea mays MSH1 promoter and
>GRMZM2G360873		5′ UTR
AT5G67120RING-F	42	primer
AT5G67120RING-R	43	primer
AT1G20690SWI-F	44	primer
AT1G20690SWI-R	45	primer
AT3g271501stMir2-F	46	primer
AT3g271501stMir2-R	47	primer
AT3g271502ndMir2-F	48	primer
AT3g271502ndMir2-R	49	primer
RNAi-F	50	primer
RNAi-R	51	primer
upstream_1kb\|photosystem II	52	Arabidopsis thaliana PPD3
reaction center PsbP family protein		promoter
mRNA
upstream_1kb\|Oryza sativa Japonica	53	Oryza sativa PPD3 promoter
Group Os08g0512500
(Os08g0512500) mRNA
upstream_1kb\|PREDICTED:	54	Solanum lycopersicum
Solanum lycopersicum psbP domain-		PPD3 promoter
containing protein
3, chloroplastic-
like

Sequence Listing is provided herewith as a computer readable form (CRF) named “46589_126309_SEQ_LST.txt” and is incorporated herein by reference in its entirety. This sequence listing contains SEQ ID NO:1-56 that are referred to herein.
The embodiments were chosen and described in order to best explain the principles of the present disclosure and its practical application to thereby enable others skilled in the art to best utilize the present disclosure in various embodiments and with various modifications as are suited to the particular use contemplated.
As various modifications could be made in the constructions and methods herein described and illustrated without departing from the scope of the present disclosure, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative rather than limiting. Thus, the breadth and scope of the present present disclosure should not be limited by any of the above-described embodiments, but should be defined only in accordance with the following claims appended hereto and their equivalents.

Claims

What is claimed is:

1. A method for producing a plant having a useful trait that exhibits nuclear inheritance comprising the steps of:

(a) selfing a first plant wherein said plant or a parent plant thereof is or had been subjected to perturbation of organellar function;

(b) screening a population of progeny plants obtained from the selfed plant of step (a) for the useful trait; and,

(c) selecting one or more progeny plants having the useful trait that exhibits nuclear inheritance and having recovered organellar function, thereby producing a plant having a useful trait that exhibits nuclear inheritance.

2. The method of claim 1, wherein organellar function has been recovered in any of: (i) the selfed first plant in step (a); (ii) at least a portion of the progeny plants of step (b); or (iii) one or more of the selected progeny plants in step (c).

3. The method of claim 1, wherein the selfed first plant of step (a) exhibits a wild type phenotype or an improvement in a useful trait in comparison to a control plant.

4. The method of claim 1, wherein the selfed first plant of step (a) exhibits one or more MSH1-dr phenotypes.

5. The method of claim 1 wherein the perturbed organellar function is a plastid function selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function.

6. The method of claim 5, wherein the photosystem II function and/or sensor function is perturbed by suppressing expression of a gene selected from the group consisting of an MSH1, PPD3, a PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene.

7. The method of claim 1, wherein said selfed plant in step (a) is an inbred plant.

8. The method of claim 1, wherein about 1% to about 45% of the population of progeny plants in step (b) are selected for the useful trait in step (c).

9. The method of any one of claims 1-8, wherein the method further comprises making a first cross of the selected progeny plant(s) of step (c) to one or more second plant(s).

10. The method of claim 9, wherein the selected progeny plant(s) and the second plant(s) are in a single heterotic group.

11. The method of claim 9, wherein the second plant is isogenic to the plant or parent plant of step (a).

12. The method of claim 9, wherein the second plant and the selected plant are in distinct heterotic groups.

13. The method of claim 9, further comprising the step of selecting of one or more progeny of the first cross that exhibit an improvement in a useful trait in comparison to progeny of a control cross.

14. The method of claim 13, wherein the selected progeny of the first cross comprise about 1.0% to about 45% of the population of progeny plants obtained from the first cross.

15. The method of claim 13, wherein plants used in the control cross are of the same heterotic group as the selected progeny plant(s) and the second plant(s), but had not been subjected to organellar perturbation.

16. The method of claim 13, wherein plants used in the control cross are isogenic to the selected plant and the second plant, but had not been subjected to organellar perturbation.

17. The method of claim 13, further comprising the step of making a second cross of the one or more of the selected progeny plant(s) of the first cross to one or more third plants.

18. The method of claim 17, further comprising the step of selecting of one or more progeny of the second cross that exhibit an improvement in a useful trait in comparison to progeny of a control cross.

19. The method of claim 9, wherein the second plant or a parent plant thereof had been subjected to perturbation of organellar function.

20. The method of any one of claims 1-19, wherein the organellar function is a plastid function or a mitochondrial function.

21. The method of any one of claims 1-20, wherein the useful trait is transmitted by using selected progeny plant(s) or progeny thereof as pollen donors.

22. A plant or progeny thereof that exhibits a useful trait that is made by the method of any one of claims 1-21.

23. A plant part obtained from the plant or progeny thereof of claim 22.

24. The plant part of claim 23, wherein said part is selected from the group consisting of a seed, leaf, stem, fruit, and a root.

25. A processed plant product obtained from the plant part of claim 23.

26. A clonal propagate obtained from the plant of claim 22 or from the plant part of claim 23.

27. A method for producing a plant having a useful trait that exhibits nuclear inheritance comprising the steps of:

(a) crossing one or more first plant(s) to one or more second plant(s), wherein at least said first plant(s) or a parent plant thereof is or had been subjected to perturbation of organellar function and wherein either: (i) the first plant or a parent plant thereof does not exhibit any MSH1-dr phenotypes; or (ii) wherein the first plant or a parent plant thereof exhibits one or more MSH1-dr phenotype(s) and the perturbation of does not comprise direct suppression of MSH 1 gene expression;

(b) screening a population of progeny plants obtained from the cross of step (a) for the useful trait; and,

(c) selecting one or more progeny plants having the useful trait that exhibits nuclear inheritance and having recovered organellar function, thereby producing a plant exhibiting a useful trait that exhibits nuclear inheritance.

28. The method of claim 27, wherein organellar function has been recovered in any of: (i) the first plant in step (a); (ii) at least a portion of the population of progeny plants of step (b); or (iii) one or more of the selected progeny plants in step (c).

29. The method of claim 27, wherein the first plant(s) of step (a) exhibit a wild type phenotype or an improvement in a useful trait in comparison to a control plant.

30. The method of claim 27, wherein about 1% to about 45% of the population of progeny plants in step (b) are selected for the useful trait in step (c).

31. The method of claim 27 wherein the perturbed organellar function is a plastid function selected from the group consisting of a sensor, photosystem I, photosystem II, NAD(P)H dehydrogenase (NDH) complex, cytochrome b6f complex, and plastocyanin function.

32. The method of claim 31, wherein the photosystem II function and/or sensor function is perturbed by suppressing expression of a gene selected from the group consisting of an MSH1, PPD3, a PsbO-1, a PsbO-2, PsbY, PsbW, PsbX, PsbR, PsbTn, PsbP1, PsbP2, PsbS, PsbQ-1, PsbQ-2, PPL1, PSAE-1, LPA2, PQL1, PQL2, and a PQL3 gene, with the proviso that the gene is not MSH1 when first plant or a parent plant thereof exhibits an MSH1-dr phenotype.

33. The method of claim 27, wherein said first plant in step (a) is an inbred plant.

34. The method of any one of claims 27-33, wherein the method further comprises making a second cross of the selected progeny plant(s) of step (c) to one or more third plant(s).

35. The method of claim 34, wherein the selected progeny plant(s) and the third plant are in a single heterotic group.

36. The method of claim 34, wherein the third plant is isogenic to the first plant or parent plant of step (a).

37. The method of any one of claims 34-36, further comprising the step of selecting of one or more progeny of the second cross that exhibit an improvement in a useful trait in comparison to progeny of a control cross.

38. The method of claim 37, wherein the selected progeny of the second cross comprise about 1% to about 45% of the population of progeny plants obtained from the second cross.

39. The method of claim 38, further comprising the step of making a third cross of the one or more of the selected progeny plants of the second cross to one or more fourth plants.

40. The method of claim 27, wherein the second plants or a parent plant thereof had been subjected to perturbation of organellar function.

41. The method of claim 34, wherein the third plants or a parent plant thereof had been subjected to perturbation of organellar function.

42. The method of any one of claims 27-41, wherein the organellar function is a plastid or a mitochondrial function.

43. The method of any one of claims 27-42, wherein the useful trait is transmitted by using selected progeny plant(s) or progeny thereof as pollen donors.

44. A plant or progeny thereof that exhibits a useful trait that is made by the method of any one of claims 27-43.

45. A plant part obtained from the plant or progeny thereof of claim 44.

46. The plant part of claim 45, wherein said part is selected from the group consisting of a seed, leaf, stem, fruit, and a root.

47. A processed plant product obtained from the plant part of claim 45.

48. A clonal propagate obtained from the plant of claim 44 or from the plant part of claim 45.

49. A method of identifying a organellar perturbation agent that comprises: (a) assaying one or more candidate agents for inhibition of an interaction of an MSH-1 protein or fragment thereof with at least one assistant protein selected from the group consisting of PPD3, PsbA (D1), a PsbO-1, a PsbO-2, PetC, CAD9, KAB1, GOS12, ELI3-1, STT3B, a fragment thereof and combinations thereof; and, (b) selecting an agent that inhibits the interaction of MSH-1 or fragment thereof with the assistant protein, thereby identifying a organellar perturbation agent.

50. The method of claim 49, wherein the fragment of MSH-1 comprises a peptide of at least about 10 amino acids located within Domain 2, 3, or 6 of MSH-1.

51. The method of claim 50, wherein the agent is selected from the group consisting of a compound, a peptide, and a peptidomimetic compound.

52. The method of claim 51, wherein said peptide is comprises a peptide of a peptide of at least about 10 amino acids located within Domain 2, 3, or 6 of MSH-1.

53. A method of identifying a plant harboring a useful trait comprising the steps of:

(a) crossing a candidate plant to a second plant, wherein the candidate plant is progeny of: (i) a selfed plant wherein said plant or a parent plant thereof is or had been subjected to perturbation of organellar function; or of (ii) a cross wherein at least one crossed plant or a parent plant thereof is or had been subjected to perturbation of organellar function; and,

(b) identifying one or more progeny plants from the cross in step (a) that exhibit a useful trait to a greater extent than the candidate plant, the second plant, or a control plant, thereby identifying the candidate plant as a plant that harbors a useful trait.

54. The method of claim 53, wherein the control plant is progeny of a cross between; (i) a plant that is not progeny of a selfed plant, a crossed plant, or parent thereof that is or had been subjected to organellar perturbation; and (ii) a plant that is isogenic to the second plant.

55. The method of claim 53, wherein the selfed plant or a parent plant thereof in (i) or the crossed plant or parent plant thereof in (ii) does not exhibit any MSH1-dr phenotypes.

56. The method of claim 55, wherein the selfed plant or a parent plant thereof in (i) or the crossed plant or parent plant thereof in (ii) exhibits one or more MSH1-dr phenotypes.

57. The method of claim 56, wherein the perturbation does not comprise direct suppression of MSH 1 gene expression.

58. The method of any one of claims 53-57, wherein the candidate plant is used as a pollen donor in the crossing step (a).

59. The method of any one of claims 53-58, wherein the identifying further comprises showing that the harbored trait exhibits nuclear inheritance.

60. A plant, progeny thereof or seed thereof that harbors a useful trait, wherein said plant, progeny thereof, or seed thereof is identified or identifiable by the method of any one of claims 53-59.

61. A method for producing a seed lot comprising:

(i) growing a population of plants, wherein said population comprises two or more of the plants or progeny thereof of claim 22, 44, or 60;

(ii) selecting a first sub-population of plants exhibiting a useful trait; and,

(ii) obtaining a seed lot from the first selected sub-population of step (i) or, optionally, repeating steps (i) and (ii) on a second population of plants grown from the seed obtained from the first selected sub-population of plants.

62. A seed lot produced by the method of claim 61.

63. A method for producing a seed lot comprising:

(i) growing a population of plants, wherein said population comprises two or more of the plants or progeny thereof of claim 22, 44, or 60; and,

(ii) obtaining a seed lot from the population.

64. A seed lot produced by the method of claim 63.