Patent application title: Methods and Compositions for Obtaining Useful Plant Traits
Inventors:
IPC8 Class: AA01H104FI
USPC Class:
1 1
Class name:
Publication date: 2018-12-06
Patent application number: 20180343816
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.Claims:
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.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] 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.
INCORPORATION OF SEQUENCE LISTING
[0003] 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
[0004] 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).
[0005] 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
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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
[0023] The accompanying drawings, which are incorporated in and form a part of the specification, illustrate certain embodiments of the present disclosure. In the drawings:
[0024] 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.times.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.
[0025] 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.
[0026] 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.
[0027] FIG. 4 illustrates the epi-type.times.environment interactions. Joint regression (with Wright modification) indicates differential response between lines to location.
[0028] FIG. 5 illustrates that the MSH1-F.sub.4 generation 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).
[0029] 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.
[0030] FIG. 7 illustrates a sorghum genetic map with markers displaying heterozygous genotype.
[0031] 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.
[0032] FIG. 9A, B illustrate that Epi-line.times.environment effects were detected from a multiple location experiment. A) Analysis for significant effects using a mixed model indicates that line, location, and line.times.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.
[0033] 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.
[0034] 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.
[0035] 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.times.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
[0036] 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.
[0037] 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.
[0038] 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.
[0039] As used herein, the term "comprising" means "including but not limited to".
[0040] As used herein, the phrase "crop plant" includes, but is not limited to, cereal, seed, grain, fruit, and vegetable crop plants.
[0041] 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.
[0042] As used herein, the term "discrete variation" or "V.sub.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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] As used herein, the phrase "quantitative variation" or "V.sub.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.
[0050] 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.
[0051] 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.
[0052] 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).
[0053] 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.
[0054] 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.
[0055] As used herein, the term "non-regenerable" refers to a plant part or plant cell that cannot give rise to a whole plant.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.sup.th Edition. Methods for effecting grafts of monocot plant scions to rootstocks that can be used with the scions and rootstocks provided herein are disclosed in Muzik and La Rue, The Grafting of Large Monocotyledonous Plants, Science 116, No. 3022: 589-591, 1952.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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-US-00001 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
[0071] 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.
[0072] 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.
[0073] 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).
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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;
[0078] (f) Construct 6: PETE1 and PETE2; and,
(g) Construct 7: PsbW and PsbW-like.
[0079] 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.
[0080] 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.
[0081] 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).
[0082] 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.
[0083] 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-US-00002 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
[0084] 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-Garcia R, Vargas-Martinez 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:
TABLE-US-00003 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.
[0085] 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.TM. reactions (Applied Biosystems, Foster City, Calif. US), use of SCORPION.TM. or Molecular Beacon.TM. 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.TM.) or the Invader.TM. technology (Third Wave Technologies, Madison, Wis.).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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 .mu.mol photons/m2s for about 24 to about 120 hours. Useful and non-limiting heat stress conditions include continuous exposure to temperatures of about 32.degree. C. to about 37.degree. 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.
[0091] 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 .mu.mol photons/m2s for about 24 to about 120 hours. Useful and non-limiting heat stress conditions include continuous exposure to temperatures of about 32.degree. C. to about 37.degree. 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.
[0092] 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.
[0093] 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.sub.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.
[0094] 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.
[0095] 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.TM. DNA ELISA Kit, Zymo Research Corp., Irvine, Calif., USA; or EpiSeeker.TM. 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.
[0096] 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.sub.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.sub.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.sub.D) can be obtained by any of the aforementioned genetic techniques, molecular genetic techniques, or combinations thereof.
[0097] Individual lines or populations of plants obtained from plants where organellar perturbation target gene expression was suppressed that exhibit discrete variation (V.sub.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.sub.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.sub.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.
[0098] Individual lines or populations of plants obtained from plants where organellar perturbation target gene expression was suppressed that exhibit discrete variation (V.sub.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.sub.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.
[0099] 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.sub.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.sub.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.sub.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.sub.D) can be obtained by any of the aforementioned genetic techniques, molecular genetic techniques, or combinations thereof.
[0100] 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.sub.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.
[0101] 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.sub.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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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.
[0112] 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).
[0113] 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).
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.).
[0120] 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.
[0121] 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.
[0122] 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
[0123] 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
[0124] 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.
[0125] 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.times.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.
[0126] Plant Materials and Growth Conditions.
[0127] Sorghum MSH1-dr plants used in these experiments were derived as described in Xu et al (2012). Six T.sub.3 individuals 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 T.sub.3 individuals were used as males in the reciprocal crosses to Tx430. The number of F.sub.1 plants derived from each cross ranged from 5 to 19 individuals. Parents and F.sub.1 progeny were grown under greenhouse conditions on a 14 hr/10 hr day-night cycle with 28.degree. C./22.degree. C. day-night temperatures. Self-pollinated seed of F.sub.1 plants was harvested individually to generate corresponding F.sub.2 families.
[0128] Field Experiments and Phenotyping.
[0129] Plants were thinned to a final density of 15 plants/m.sup.2. The 2011 F.sub.2, F.sub.3, and F.sub.4 combined field experiment comprised seven blocks of 28 entries each (28.times.7 alpha lattice design), with two replicates grown under fertilized soil conditions. In total, the 2011 field experiment comprised 48 F.sub.2 entries, 77 F.sub.3 entries, and 39 F.sub.4, with additional wild-type Tx430 rows as a control.
[0130] For estimating grain yield, threshed panicles from three plants were pooled and converted to grams/m.sup.2, 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.
[0131] The 2012 multi-location experiment included Lincoln, Nebr. (40.degree. 51' N, 96.degree. 35' W) and Mead, Nebr. (41.degree. 9' N, 96.degree. 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/m.sup.2.
[0132] Statistical Analysis.
[0133] For evaluations in a single environment, mean phenotypic values and standard errors for each line were estimated using the linear mixed model
[0134] y.sub.ijk=.mu.+.alpha..sub.i+r.sub.k+(b/r).sub.jk+.epsilon..sub.ijk where y.sub.ijk is the trait response, .mu. is the population mean, .alpha..sub.i is the effect of line i, r.sub.k is the effect of replicate k, (b/r).sub.jk is the effect of block j nested within replicate k, and .epsilon..sub.ijk is 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.sub.ijkm=.mu.+.alpha..sub.i+e.sub.m+(r/e).sub.km+(b/r/e).sub.jkm+(.alph- a.e).sub.im+.epsilon..sub.ijkm where y.sub.ijkm is the trait response, .mu. is the population mean, .alpha..sub.i is the effect of line i, e.sub.m is the effect of environment m, (r/e).sub.km is the effect of replicate nested within environment, (b/r/e).sub.jkm is the effect of block j nested within replicate k of environment m, (.alpha.e).sub.im is the interaction between line i and environment m, and .epsilon..sub.ijkm is the residual. Line, environment, and line.times.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-F.sub.2, wild-type Tx430, etc.) or broad height class (short, tall, mixed). F.sub.4 mean 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).
[0135] PCR Assay for RNAi Transgene and SSR Marker Analysis.
[0136] 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.
[0137] 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.TM. (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.
[0138] Sorghum SNP Survey.
[0139] 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.TM. beadchip hybridization (Illumina, San Diego, Calif.). The genotyping of five MSH1-epiF.sub.4 lines 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.TM. high-density beadchip containing 1,885 internally validated SNP markers.
[0140] 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
[0141] MSH1-Altered Lines and Reciprocal Crosses.
[0142] 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.
[0143] 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 F.sub.1 progeny 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 F.sub.1 populations derived from nine separate crosses, three of which used an MSH1-dr plant as the pollen donor (Xu et al. 2012).
[0144] Lack of the MSH1-dr phenotype in the F.sub.1 generation 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 F.sub.1 observations involve a conserved, programmed epigenetic pathway, and we therefore designated the F.sub.1 progeny as MSH1-epiF.sub.1.
[0145] MSH1-epiF.sub.2 Populations Show Enhanced Variation.
[0146] Self-pollination of the MSH1-cpiF.sub.1 plants produced an F.sub.2 population (MSH1-cpiF.sub.2) variable in plant phenotype (FIG. 1b, c, d, f, FIG. 2, Table 3), with a minority exhibiting the MSH1-dr phenotype (FIG. 1e). Notably increased variation in the F.sub.2 was 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-F.sub.2, by the F.sub.4 we 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-US-00004 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/m.sup.2) 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
[0147] 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).
[0148] A small proportion of greenhouse-grown MSH1-epiF.sub.3 families also showed the MSH1-dr phenotype, with an overall frequency of ca. 8% (Table 4). By the F.sub.4 generation, 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 F.sub.4 generation may be the consequence of inadvertent selection rather than a natural tendency to gradually stabilize away from the phenotype.
TABLE-US-00005 TABLE 4 Frequency of MSH1-dr phenotype (8.4%) in epi-F3 families derived from sorghum MSH1-dr .times. 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-US-00006 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 .gtoreq.105 in each generation. Frequency of MSH1-dr phenotype Line Parental Frequency % Progeny Frequency % Msh1 . . . #11 .times. Tx430 #2-6-10-8 0/118 0% 0/114 0% Msh1 . . . #11 .times. Tx430 #2-6-3-23 0/116 0% 0/119 0% Msh1 . . . #15 .times. Tx430 #7-1-1-5 0/118 0% 0/117 0% Msh1 . . . #15 .times. Tx430 #7-1-3-1 3/105 2.86% 2/122 1.64% Msh1 . . . #15 .times. Tx430 #7-1-9-10 0/115 0% 0/111 0% Msh1 . . . #22 .times. Tx430 #4-3-1-3 9/119 7.56% 1/112 0.89% Msh1 . . . #22 .times. Tx430 #4-4-10-28 0/126 0% 0/125 0% Msh1 . . . #24 .times. Tx430 #13-8-6 0/118 0% 0/128 0% Msh1 . . . #24 .times. Tx430 #19-11-7 5/116 4.31% 7/122 5.74% Msh1 . . . #28 .times. Tx430 #13-3-1 0/119 0% 0/120 0%
[0149] 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-US-00007 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- F3- F4- F4- F4- F4- F4- F4- 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 NA - - - - - - - SAM 56359 - - + - NA NA - - - - - - - SAM 55010 - - + - NA NA - - - - - - - SAM 56942 - - + - NA NA - - - - - - - SAM 62005 - - + - NA NA - - - - - - - SAM 62186b - - + - NA NA - - - - - - - SAM 59974 - - + - NA NA - - - - - - - SAM 61376 - - + - NA NA - - - - - - - SAM 65125 - - + - NA NA - - - - - - - SAM 66160 - - + - NA NA - - - - - - - SAM 26858a - - + - - - - - - - - - - SAM 27170 - - + - - - - - - - - - - SAM 33545 - - + - NA NA - - - - - - - SAM 36890 - - + - NA NA - - - - - - - SAM 47801 - - + - NA NA - - - - - - - SAM 48589 - - + - NA NA - - - - - - - SAM 48870 - - + - NA NA - - - - - - - SAM 49411b - - + - NA NA - - - - - - - SAM 64056 - - + - NA NA - - - - - - - SAM 62693 - - + - NA NA - - - - - - - SAM 67633 - - + - NA NA - - - - - - - SAM 63126c - - + - NA NA - - - - - - - SAM 64809 - - + - NA NA - - - - - - - SAM 01952 - - + - NA NA - - - - -- - - SAM 40439 - - + - NA NA - - - - - - - SAM 42610 - - + - NA NA - - - - - - - SAM 39963 - - + - NA NA - - - - - - -
TABLE-US-00008 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-US-00009 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
[0150] 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.
[0151] Significant Increases in Trait Values Persist for Multiple Generations.
[0152] From the MSH1-epiF.sub.2 families, individuals were self-pollinated and selected for grain yield and plant height to the F.sub.3 and F.sub.4 generations. F.sub.4 lines, along with F.sub.3 and F.sub.2 lines 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 F.sub.3 and F.sub.4, respectively), derived F.sub.3 and F.sub.4 lines 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-US-00010 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/m.sup.2).sup.a Plant height (cm).sup.b 167 3537 159.6 <0.001 Flowering time 136 1874 6.13 <0.001 (DAS).sup.a,b Rachis Length 165 3495 5.06 <0.001 (cm).sup.a,b Dry Biomass 167 490 5.1 <0.001 (g/plant).sup.a .sup.aBox-cox transformed for model fit. .sup.bSeparate variances estimated by generation or height strata.
[0153] Although the F.sub.3 generation showed higher variance for some traits compared to the F.sub.2 generation, for all measured traits the F.sub.4 generation showed lower variance compared to the F.sub.2 generation (FIG. 2A). Furthermore, in contrast to the F.sub.2 generation, we did not find significant heterogeneity for variance in grain yield among wild-type, F.sub.3 and F.sub.4 lines (p>0.1, Brown-Forsythe test; p<0.01 in F.sub.2 with wild-type).
[0154] Analysis of direct lineages from F.sub.2 to F.sub.4 showed 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 F.sub.4 were more modest compared to the F.sub.3, implying progress may taper off by F.sub.4 in self-pollinated lineages. Indeed, there is evidence that the epi-F.sub.3 generation 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).
[0155] Line.times.Environment Interactions Suggest an Epigenetic Component to G.times.E.
[0156] We evaluated the yield performance of three MSH1-epiF.sub.5 families 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.times.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 F.sub.5 generation. 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.times.environmental interactions.
[0157] 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 (Colome-Tatche 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.
[0158] The range of phenotypic variation observed is surprising. While we were not able to take measurements of all parameters for this initial study, the F.sub.3 and the F.sub.4 generations 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).
[0159] The observation of epi-type.times.environment interaction in test plots suggests that at least some portion of the genotype.times.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.
[0160] 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).
[0161] 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 F.sub.1 populations, 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 epiF.sub.2 population, at which point selection appears to be most effective. Large-scale seed increase in F.sub.3 and F.sub.4 generations 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-F.sub.2 population 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-F.sub.2, appears to result in, by the epiF.sub.4, a population that is uniform genetically, variable epigenetically, and significantly enhanced in growth vigor and productivity.
[0162] 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.
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[0189] 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.
[0190] Tsaftaris, A. S., Polidoros, A. N., Kapazoglou, A., Tani, E. and Kova evi , 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
[0191] 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
[0192] 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.
[0193] 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.
[0194] 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
[0195] 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-US-00011 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-US-00012 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-US-00013 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-US-00014 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.
[0196] 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).
[0197] 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-US-00015
[0198] 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
[0199] 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.
[0200] 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.
[0201] 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.
Sequence CWU
1
1
5613730DNAArabidopsis thaliana 1agaggactgt gagattgtga attgcatagt
cgtcgtcttc tggcgggaaa agaagcccta 60gaaaaagggt gaaaggtgaa aactctactt
cttcttcttc ttcttcttca gagtgtgaga 120gagatgcatt ggattgctac cagaaacgcc
gtcgtttcat tcccaaaatg gcggttcttc 180ttccgctcct catatcgcac ttactcttcc
ctcaaaccct cctccccaat tctacttaat 240agaaggtact ctgaggggat atcttgtctc
agagatggaa agtctttgaa aagaatcaca 300acggcttcta agaaagtgaa gacgtcaagt
gatgttctca ctgacaaaga tctctctcat 360ttggtttggt ggaaggagag attgcagaca
tgtaagaaac catctactct tcagcttatt 420gaaaggctta tgtacaccaa tttacttggt
ttggacccta gcttgaggaa tggaagttta 480aaagatggaa acctcaactg ggagatgttg
cagtttaagt caaggtttcc acgcgaagtt 540ttgctctgca gagtaggaga attttatgag
gctattggaa tagatgcttg tatacttgtt 600gaatatgctg gtctcaatcc ttttggtggt
cttcgatcag atagtattcc aaaggctggc 660tgcccaatta tgaatcttcg acagactttg
gatgacctga cacgcaatgg ttattcagtg 720tgtattgtgg aggaagttca ggggccaaca
ccagcacgct cccgtaaagg tcgatttatt 780tcagggcatg cacatccagg aagtccttat
gtatatgggc ttgtcggtgt tgaccatgat 840cttgactttc ctgatcctat gcctgttgtt
gggatatctc gttcagcaag ggggtattgt 900atgatatcta ttttcgagac tatgaaagca
tattcgctag atgatggtct aacagaagaa 960gccttagtta ccaagctccg cactcgtcgc
tgtcatcatc ttttcttaca tgcatcgttg 1020aggcacaatg catcagggac gtgccgctgg
ggagagtttg gggaaggggg tctactctgg 1080ggagaatgca gtagcaggaa ttttgaatgg
tttgaaggag atactctttc cgagctctta 1140tcaagggtca aagatgttta tggtcttgat
gatgaagttt cctttagaaa tgtcaatgta 1200ccttcaaaaa atcggccacg tccgttgcat
cttggaacgg ctacacaaat tggtgcctta 1260cctactgaag gaataccttg tttgttgaag
gtgttacttc catctacgtg cagtggtctg 1320ccttctttgt atgttaggga tcttcttctg
aaccctcctg cttacgatat tgctctgaaa 1380attcaagaaa cgtgcaagct catgagcaca
gtaacatgtt caattccaga gtttacctgc 1440gtctcttctg ctaagcttgt gaagcttctt
gagcaacggg aagccaacta cattgagttc 1500tgtcgaataa aaaatgtgct tgatgatgta
ttacatatgc atagacatgc tgagcttgtg 1560gaaatcctga aattattgat ggatcctacc
tgggtggcta ctggtttgaa aattgacttt 1620gacacttttg tcaacgaatg tcattgggcg
tctgatacaa ttggtgaaat gatctcttta 1680gatgagaatg aaagtcatca gaatgtaagt
aaatgtgaca atgtcccgaa cgaattcttt 1740tatgatatgg agtcttcatg gcgaggtcgc
gttaagggaa ttcatataga ggaagaaatc 1800actcaagtag aaaaatcagc tgaggcttta
tctttagcag tagctgagga ttttcaccct 1860attatatcaa gaattaaggc caccactgct
tcacttggtg gcccgaaagg cgaaatcgca 1920tatgcaagag agcatgagtc tgtttggttc
aaggggaaac ggtttacgcc atctatctgg 1980gctggtactg caggggaaga ccaaataaaa
cagctgaaac ctgccttaga ctcgaaagga 2040aaaaaggttg gagaagaatg gtttacgacc
ccaaaggtgg aaattgcttt agtcagatac 2100catgaagcta gtgagaatgc aaaagctcgg
gtgttggaac tgttgcgcga gttatccgtt 2160aaattgcaaa caaaaataaa tgttcttgtc
tttgcatcta tgcttctggt catttcaaaa 2220gcattatttt cccatgcttg tgaagggaga
aggcgaaagt gggtttttcc aacgcttgtc 2280ggattcagtt tagatgaggg cgcaaaacca
ttagatggtg ccagtcgaat gaagctgaca 2340ggcctgtcac cttattggtt tgatgtatct
tctggaaccg ctgttcacaa taccgttgac 2400atgcaatcac tgtttcttct aactggacct
aacggtggtg gtaaatcgag tttgctcaga 2460tcaatatgcg cagctgctct acttggaatt
tccggtttaa tggttccagc tgaatcagct 2520tgtattcctc actttgattc catcatgctt
cacatgaaat catatgacag ccctgtagac 2580ggaaaaagtt ctttccaggt agaaatgtcg
gaaatacgat ctattgtaag ccaggctact 2640tcgagaagcc tagtgcttat agatgagata
tgccgaggga cagagacagc aaaaggcacc 2700tgtatcgctg gtagtgtggt agagagtctt
gacacaagtg gttgtttggg tattgtatct 2760actcatctcc atggaatctt cagtttacct
cttacagcga aaaacatcac atataaagca 2820atgggagccg aaaatgtcga agggcaaacc
aagccaactt ggaaattgac agatggagtc 2880tgcagagaga gtcttgcgtt tgaaacagct
aagagggaag gtgttcccga gtcagttatc 2940caaagagctg aagctcttta cctctcggtc
tatgcaaaag acgcatcagc tgaagttgtc 3000aaacccgacc aaatcataac ttcatccaac
aatgaccagc agatccaaaa accagtcagc 3060tctgagagaa gtttggagaa ggacttagca
aaagctatcg tcaaaatctg tgggaaaaag 3120atgattgagc ctgaagcaat agaatgtctt
tcaattggtg ctcgtgagct tccacctcca 3180tctacagttg gttcttcatg cgtgtatgtg
atgcggagac ccgataagag attgtacatt 3240ggacagaccg atgatcttga aggacgaata
cgtgcgcatc gagcaaagga aggactgcaa 3300gggtcaagtt ttctatacct tatggttcaa
ggtaagagca tggcttgtca gttagagact 3360ctattgatta atcaactcca tgaacaaggc
tactctctgg ctaacctagc cgatggaaag 3420caccgtaatt tcggaacgtc ctcaagcttg
agtacatcag acgtagtcag catcttatag 3480tttgaaacat tagctgtgtt tgtagttgat
catctctatg tgcaattgaa caagtcagtt 3540tgctagaact agagtagatt actaagaaac
catgccgttt ttcattttga gattttgcaa 3600aacggcatgc agttcgggta agtcggatgc
cgcaattacc aattttgggt cagtctgtgt 3660aattgtcgtt tcataaatcc gattaacgtg
tactttgaac aaaactcagc agtaaacttc 3720tttattcatc
373021118PRTArabidopsis thaliana 2Met
His Trp Ile Ala Thr Arg Asn Ala Val Val Ser Phe Pro Lys Trp 1
5 10 15 Arg Phe Phe Phe Arg Ser
Ser Tyr Arg Thr Tyr Ser Ser Leu Lys Pro 20
25 30 Ser Ser Pro Ile Leu Leu Asn Arg Arg Tyr
Ser Glu Gly Ile Ser Cys 35 40
45 Leu Arg Asp Gly Lys Ser Leu Lys Arg Ile Thr Thr Ala Ser
Lys Lys 50 55 60
Val Lys Thr Ser Ser Asp Val Leu Thr Asp Lys Asp Leu Ser His Leu 65
70 75 80 Val Trp Trp Lys Glu
Arg Leu Gln Thr Cys Lys Lys Pro Ser Thr Leu 85
90 95 Gln Leu Ile Glu Arg Leu Met Tyr Thr Asn
Leu Leu Gly Leu Asp Pro 100 105
110 Ser Leu Arg Asn Gly Ser Leu Lys Asp Gly Asn Leu Asn Trp Glu
Met 115 120 125 Leu
Gln Phe Lys Ser Arg Phe Pro Arg Glu Val Leu Leu Cys Arg Val 130
135 140 Gly Glu Phe Tyr Glu Ala
Ile Gly Ile Asp Ala Cys Ile Leu Val Glu 145 150
155 160 Tyr Ala Gly Leu Asn Pro Phe Gly Gly Leu Arg
Ser Asp Ser Ile Pro 165 170
175 Lys Ala Gly Cys Pro Ile Met Asn Leu Arg Gln Thr Leu Asp Asp Leu
180 185 190 Thr Arg
Asn Gly Tyr Ser Val Cys Ile Val Glu Glu Val Gln Gly Pro 195
200 205 Thr Pro Ala Arg Ser Arg Lys
Gly Arg Phe Ile Ser Gly His Ala His 210 215
220 Pro Gly Ser Pro Tyr Val Tyr Gly Leu Val Gly Val
Asp His Asp Leu 225 230 235
240 Asp Phe Pro Asp Pro Met Pro Val Val Gly Ile Ser Arg Ser Ala Arg
245 250 255 Gly Tyr Cys
Met Ile Ser Ile Phe Glu Thr Met Lys Ala Tyr Ser Leu 260
265 270 Asp Asp Gly Leu Thr Glu Glu Ala
Leu Val Thr Lys Leu Arg Thr Arg 275 280
285 Arg Cys His His Leu Phe Leu His Ala Ser Leu Arg His
Asn Ala Ser 290 295 300
Gly Thr Cys Arg Trp Gly Glu Phe Gly Glu Gly Gly Leu Leu Trp Gly 305
310 315 320 Glu Cys Ser Ser
Arg Asn Phe Glu Trp Phe Glu Gly Asp Thr Leu Ser 325
330 335 Glu Leu Leu Ser Arg Val Lys Asp Val
Tyr Gly Leu Asp Asp Glu Val 340 345
350 Ser Phe Arg Asn Val Asn Val Pro Ser Lys Asn Arg Pro Arg
Pro Leu 355 360 365
His Leu Gly Thr Ala Thr Gln Ile Gly Ala Leu Pro Thr Glu Gly Ile 370
375 380 Pro Cys Leu Leu Lys
Val Leu Leu Pro Ser Thr Cys Ser Gly Leu Pro 385 390
395 400 Ser Leu Tyr Val Arg Asp Leu Leu Leu Asn
Pro Pro Ala Tyr Asp Ile 405 410
415 Ala Leu Lys Ile Gln Glu Thr Cys Lys Leu Met Ser Thr Val Thr
Cys 420 425 430 Ser
Ile Pro Glu Phe Thr Cys Val Ser Ser Ala Lys Leu Val Lys Leu 435
440 445 Leu Glu Gln Arg Glu Ala
Asn Tyr Ile Glu Phe Cys Arg Ile Lys Asn 450 455
460 Val Leu Asp Asp Val Leu His Met His Arg His
Ala Glu Leu Val Glu 465 470 475
480 Ile Leu Lys Leu Leu Met Asp Pro Thr Trp Val Ala Thr Gly Leu Lys
485 490 495 Ile Asp
Phe Asp Thr Phe Val Asn Glu Cys His Trp Ala Ser Asp Thr 500
505 510 Ile Gly Glu Met Ile Ser Leu
Asp Glu Asn Glu Ser His Gln Asn Val 515 520
525 Ser Lys Cys Asp Asn Val Pro Asn Glu Phe Phe Tyr
Asp Met Glu Ser 530 535 540
Ser Trp Arg Gly Arg Val Lys Gly Ile His Ile Glu Glu Glu Ile Thr 545
550 555 560 Gln Val Glu
Lys Ser Ala Glu Ala Leu Ser Leu Ala Val Ala Glu Asp 565
570 575 Phe His Pro Ile Ile Ser Arg Ile
Lys Ala Thr Thr Ala Ser Leu Gly 580 585
590 Gly Pro Lys Gly Glu Ile Ala Tyr Ala Arg Glu His Glu
Ser Val Trp 595 600 605
Phe Lys Gly Lys Arg Phe Thr Pro Ser Ile Trp Ala Gly Thr Ala Gly 610
615 620 Glu Asp Gln Ile
Lys Gln Leu Lys Pro Ala Leu Asp Ser Lys Gly Lys 625 630
635 640 Lys Val Gly Glu Glu Trp Phe Thr Thr
Pro Lys Val Glu Ile Ala Leu 645 650
655 Val Arg Tyr His Glu Ala Ser Glu Asn Ala Lys Ala Arg Val
Leu Glu 660 665 670
Leu Leu Arg Glu Leu Ser Val Lys Leu Gln Thr Lys Ile Asn Val Leu
675 680 685 Val Phe Ala Ser
Met Leu Leu Val Ile Ser Lys Ala Leu Phe Ser His 690
695 700 Ala Cys Glu Gly Arg Arg Arg Lys
Trp Val Phe Pro Thr Leu Val Gly 705 710
715 720 Phe Ser Leu Asp Glu Gly Ala Lys Pro Leu Asp Gly
Ala Ser Arg Met 725 730
735 Lys Leu Thr Gly Leu Ser Pro Tyr Trp Phe Asp Val Ser Ser Gly Thr
740 745 750 Ala Val His
Asn Thr Val Asp Met Gln Ser Leu Phe Leu Leu Thr Gly 755
760 765 Pro Asn Gly Gly Gly Lys Ser Ser
Leu Leu Arg Ser Ile Cys Ala Ala 770 775
780 Ala Leu Leu Gly Ile Ser Gly Leu Met Val Pro Ala Glu
Ser Ala Cys 785 790 795
800 Ile Pro His Phe Asp Ser Ile Met Leu His Met Lys Ser Tyr Asp Ser
805 810 815 Pro Val Asp Gly
Lys Ser Ser Phe Gln Val Glu Met Ser Glu Ile Arg 820
825 830 Ser Ile Val Ser Gln Ala Thr Ser Arg
Ser Leu Val Leu Ile Asp Glu 835 840
845 Ile Cys Arg Gly Thr Glu Thr Ala Lys Gly Thr Cys Ile Ala
Gly Ser 850 855 860
Val Val Glu Ser Leu Asp Thr Ser Gly Cys Leu Gly Ile Val Ser Thr 865
870 875 880 His Leu His Gly Ile
Phe Ser Leu Pro Leu Thr Ala Lys Asn Ile Thr 885
890 895 Tyr Lys Ala Met Gly Ala Glu Asn Val Glu
Gly Gln Thr Lys Pro Thr 900 905
910 Trp Lys Leu Thr Asp Gly Val Cys Arg Glu Ser Leu Ala Phe Glu
Thr 915 920 925 Ala
Lys Arg Glu Gly Val Pro Glu Ser Val Ile Gln Arg Ala Glu Ala 930
935 940 Leu Tyr Leu Ser Val Tyr
Ala Lys Asp Ala Ser Ala Glu Val Val Lys 945 950
955 960 Pro Asp Gln Ile Ile Thr Ser Ser Asn Asn Asp
Gln Gln Ile Gln Lys 965 970
975 Pro Val Ser Ser Glu Arg Ser Leu Glu Lys Asp Leu Ala Lys Ala Ile
980 985 990 Val Lys
Ile Cys Gly Lys Lys Met Ile Glu Pro Glu Ala Ile Glu Cys 995
1000 1005 Leu Ser Ile Gly Ala
Arg Glu Leu Pro Pro Pro Ser Thr Val Gly 1010 1015
1020 Ser Ser Cys Val Tyr Val Met Arg Arg Pro
Asp Lys Arg Leu Tyr 1025 1030 1035
Ile Gly Gln Thr Asp Asp Leu Glu Gly Arg Ile Arg Ala His Arg
1040 1045 1050 Ala Lys
Glu Gly Leu Gln Gly Ser Ser Phe Leu Tyr Leu Met Val 1055
1060 1065 Gln Gly Lys Ser Met Ala Cys
Gln Leu Glu Thr Leu Leu Ile Asn 1070 1075
1080 Gln Leu His Glu Gln Gly Tyr Ser Leu Ala Asn Leu
Ala Asp Gly 1085 1090 1095
Lys His Arg Asn Phe Gly Thr Ser Ser Ser Leu Ser Thr Ser Asp 1100
1105 1110 Val Val Ser Ile Leu
1115 33765DNAGlycine max 3gtcagataca gagtccttcc
ctcctcgtgt gtggactgtg gcgggaactc attttgctag 60tttgcttcct ctctctctct
cgttcccatt caacgcaatg tacagggtag ccacaagaaa 120cgtcgccgtt ttcttccctc
gttgctgttc cctcgcgcac tacactcctt ctctatttcc 180cattttcact tcattcgctc
cctctcgttt ccttagaata aatggatgtg taaagaatgt 240gtcgagttat acggataaga
aggtttcaag ggggagtagt agggccacca agaagcccaa 300aataccaaat aacgttttag
atgataaaga ccttcctcac atactgtggt ggaaggagag 360gttgcaaatg tgcagaaagt
tttcaactgt ccagttaatt gaaagacttg aattttctaa 420tttgcttggc ctgaattcca
acttgaaaaa tggaagtctg aaggaaggaa cactcaactg 480ggaaatgttg caattcaagt
caaaatttcc acgtcaagta ttgctttgca gagttgggga 540attctatgaa gcttggggaa
tagatgcttg tattcttgtt gaatatgtgg gtttaaatcc 600cattggtggt ctgcgatcag
atagtatccc aagagctagt tgtcctgtcg tgaatcttcg 660gcagacttta gatgatctga
caacaaatgg ttattcagtg tgcattgtgg aggaggctca 720gggcccaagt caagctcgat
ccaggaaacg tcgctttata tctgggcatg ctcatcctgg 780aaatccctat gtatatggac
ttgctacagt tgatcatgat cttaactttc cagaaccaat 840gcctgtagta ggaatatctc
attctgcgag gggttattgc attaatatgg tactagagac 900catgaagaca tattcttctg
aagattgctt gacagaagaa gcagttgtta cgaagcttcg 960tacttgccaa tatcattact
tatttttgca tacatccttg aggcggaatt cttgtggaac 1020ctgcaactgg ggagaatttg
gtgagggagg gctattatgg ggagaatgta gttctagaca 1080ttttgattgg tttgatggca
accctgtctc cgatcttttg gccaaggtaa aggaacttta 1140tagtattgat gatgaggtta
cctttcggaa cacaactgtg tcttcaggac atagggctcg 1200accattaact cttggaacat
ctactcaaat tggtgccatt ccaacagaag gaataccttc 1260tttgttgaag gttttacttc
catcaaattg caatggatta ccagtattgt acataaggga 1320acttcttttg aatcctcctt
catatgagat tgcatccaaa attcaagcaa catgcaaact 1380tatgagcagt gtaacgtgtt
caattccaga atttacatgt gtttcgtcag caaagcttgt 1440aaagctactt gaatggaggg
aggtcaatca tatggaattt tgtagaataa agaatgtact 1500ggatgaaatt ttgcagatgt
atagtacctc tgagctcaat gaaatattga aacatttaat 1560cgagcccaca tgggtggcaa
ctgggttaga aattgacttt gaaaccttgg ttgcaggatg 1620tgagatcgca tctagtaaga
ttggtgaaat agtatctctg gatgatgaga atgatcagaa 1680aatcaactcg ttctctttta
ttcctcacga attttttgag gatatggagt ctaaatggaa 1740aggtcgaata aaaagaatcc
acatagatga tgtattcact gcagtggaaa aagcagctga 1800ggccttacat atagcagtca
ctgaagattt tgttcctgtt gtttctagaa taaaggctat 1860tgtagcccct ctcggaggtc
ctaagggaga aatatcttat gctcgggagc aagaagcagt 1920ttggttcaaa ggcaaacgct
ttacaccgaa tttgtgggct ggtagccctg gagaggaaca 1980aattaaacag cttaggcatg
ctttagattc taaaggtaga aaggtagggg aggaatggtt 2040taccacacca aaggtcgagg
ctgcattaac aaggtaccat gaagcaaatg ccaaggcaaa 2100agaaagagtt ttggaaattt
taaggggact cgctgctgag ttgcaataca gtataaacat 2160tcttgtcttt tcttccatgt
tgcttgttat tgccaaagct ttatttgctc atgcaagtga 2220agggagaaga aggagatggg
tctttcccac gcttgtagaa tcccatgggt ttgaggatgt 2280gaagtcattg gacaaaaccc
atgggatgaa gataagtggt ttattgccat attggttcca 2340catagcagaa ggtgttgtgc
gtaatgatgt tgatatgcaa tcattatttc tgttgacagg 2400accgaatggt ggtgggaaat
caagttttct taggtcaatt tgtgctgctg cactacttgg 2460gatatgtgga ctcatggttc
ctgcagaatc agccctaatt ccttattttg actccatcac 2520gcttcatatg aagtcatatg
atagtccagc tgataaaaag agttcctttc aggttgaaat 2580gtcagaactt cgatccatca
ttggcggaac aaccaacagg agccttgtac ttgttgatga 2640aatatgccga ggaacagaaa
ctgcaaaagg gacttgcatt gctggtagca tcattgaaac 2700ccttgatgga attgggtgtc
tgggtattgt atccactcac ttgcatggaa tatttacttt 2760gcccctaaac aaaaaaaaca
ctgtgcacaa agcaatgggc acaacatcca ttgatggaca 2820aataatgcct acatggaagt
tgacagatgg agtttgtaaa gaaagtcttg cttttgaaac 2880ggctaagagg gaaggaattc
ctgagcatat tgttagaaga gctgaatatc tttatcagtt 2940ggtttatgct aaggaaatgc
tttttgcaga aaatttccca aatgaagaaa agttttctac 3000ctgcatcaat gttaataatt
tgaatggaac acatcttcat tcaaaaaggt tcctatcagg 3060agctaatcaa atggaagttt
tacgcgagga agttgagaga gctgtcactg tgatttgcca 3120ggatcatata aaggacctaa
aatgcaaaaa gattgcattg gagcttactg agataaaatg 3180tctcataatt ggtacaaggg
agctaccacc tccatcggtt gtaggttctt caagcgtcta 3240tgtgatgttc agaccagata
agaaactcta tgtaggagag actgatgatc tcgagggacg 3300ggtccgaaga catcgattaa
aggaaggaat gcatgatgca tcattccttt attttcttgt 3360cccaggtaaa agcttggcat
gccaatttga atctctgctc atcaaccaac tttctggtca 3420aggcttccaa ctgagcaata
tagctgatgg taaacatagg aattttggca cttccaacct 3480gtatacataa ctagtctata
gacattgata ttatctacct caatcgcgta tttttgcctc 3540ttttaaatgg ctcaaagact
tcaatcatcg atgttaagtt taggaaacaa tgtctgcagc 3600atttttgtta gaattagttg
ctgcagctgc atttatgtcc acatcttcaa gtgtggaaat 3660tcttgttcat tagcttgtaa
gtacaaaagt gtttgtgtac gtttggagtc ccgagagaat 3720atacaagtac aaatgaacaa
atatattagt aatgaatgca ctaga 376543642DNAZea mays
4gcgcactacc ccgagaaacg tgcgacggga acctccgcgg ttccccaagt tcgcctcctt
60cactactctc gcgccccggc acgcctgaaa aaccccaccc ctcctgccgc tccgcctctc
120ccatcacttc ccacgcccct cgccgcctcc cattccagcg tggacacgac gccactcgcc
180agcacggaga cgcgcgcctc gaagcactac tgcactagcc agccgtcgtt cttccgcgcc
240ggcgccatgc accgggtgct cgtgagctcg cttgtggccg ccacgccgcg atggctgccc
300ctcgccgact ccatcctccg gcgccgccgg ccgcgctgct cccctcttcc cgtgctgatg
360ttcgatcgga gggcttggtc caagccaagg aaggtctcac gaggcatttc agtggcgtcc
420aggaaagcta acaaacaggg agaatactgt gatgaaagta tgctgtcgca tatcatgtgg
480tggaaagaga aaatggagag gtgcagaaaa ccatcatcca tacaattgac tcagaggctt
540gtgtattcaa atatattagg gttggatccg aatttaagaa acggaagctt gaaagatgga
600accctgaaca tggagatttt ggtatttaaa tcaaaatttc ctcgtgaggt tctactttgc
660agagtaggag atttctatga agctatcggt tttgatgcct gtattctcgt agagcatgca
720ggcttaaatc cttttggagg tttgcgttcc gacagtattc ctaaagctgg gtgtccagtc
780gtgaatttac ggcagacatt ggatgatttg actcgatgtg gttattccgt gtgcatagtc
840gaggaaattc aaggcccaac tcaagcccgt gctcggaaaa gtcgatttat ttctgggcat
900gcccatcctg gtagtcctta tgtatttggt cttgctgaag tagaccatga tgtagagttc
960cctgatccga tgcctgttgt tgggatttca cattctgcaa aaggttattg cttgatatct
1020gtgctagaga caatgaaaac ttattcagct gaggagggct taacagagga ggctattgtt
1080actaagctcc gcatatgtcg ttatcaccat ctataccttc acaattcttt gaagaataat
1140tcttcaggga catcacgctg gggtgaattc ggtgaaggtg ggctcttgtg gggagagtgc
1200agtgggaagt cctttgagtg gtttgacggt tcacctattc aagaactttt atgcaaggta
1260cgggaaatat atggccttga tgagaaaacg gtttttcgcg atgtcaccgt ctcattggaa
1320ggcaggcccc aacctcttca tcttgggact gctactcaaa ttggagtcat accaactgag
1380ggaataccga gtttgttaag aatggtgctt ccttcaaatt gtggcgggct tccatcaatg
1440tatattagag atcttcttct taatcctcca tcatttgagg ttgcagcagc gatccaagag
1500gcttgcaggc ttatgggcaa cataacctgc tccattcctg aatttacatg catatcagca
1560gcaaagcttg tgaaactact tgagtcgaaa ggggtcaatc acattgaatt ttgtagaata
1620aaaaatgtcc ttgatgagat tatgctcatg aacagggatg ctgagctttc tgcaatcctg
1680catgaattac tggtacctgc ttctgtggct actggtttca aagttgaagc tgatatgcta
1740atgaacggat gtagcattat ttcacaacga atagctgaag tgatttcttt aggtgttgaa
1800agtgatcagg caataacttc attggaatat attccaaagg agttcttcaa tgatatggag
1860tcatcttgga aggggcgcgt gaaaaggatc catgctgaag aagagtttgc aaatgttgat
1920agggctgctg aggcattatc aattgcggtc attgaagatt ttatgccaat tatttcgagg
1980gtgaaatctg tagtgtcctc gaatggaggt ttgaaaggag aaatcggtta tgcaaaagaa
2040catgaagctg tttggtttaa aggaaagaga ttcataccaa atgtatgggc taacacacct
2100ggtgagcagc aaataaaaca actgaagcct gcaattgatt caaaaggcag aaaggttggg
2160gaggaatggt ttacaacaag caaagttgag aatgctttag ccaggtacca tgaagcttgt
2220gataatgcaa gaaataaagt tcttgagctg ttgagaggcc tttctagtga attgcaggac
2280aaaattaaca tacttgtctt ttgctcaaca ctgctcatca ttgcaaaagc actttttggt
2340catgttagtg aggctcgaag aagaggttgg atgcttccta ctatatctcc cttatcaaag
2400gactgtgttg tggaggaaag ttcaagtgca atggatttag taggactatt tccttactgg
2460cttgatgtta atcaaggaaa tgcaatattg aatgatgtcc acatgcactc tttatttgtt
2520cttactggcc caaatggtgg tggtaaatct agcatgttgc gatcagtctg tgcagctgtg
2580cttcttggaa tatgtggcct gatggtacct tcaacttcag ctgtaatccc acattttgat
2640tccattatgc tgcatatgaa agcctatgat agcccagcag atgggaaaag ttcatttcag
2700attgaaatgt cggagatacg tgctttagtc agccgagcta ctgctaggag tcttgttctg
2760attgatgaaa tatgtagagg cacagaaact gcaaaaggaa catgtatagc tggtagcatc
2820attgaaagac ttgataatgt tggctgccta ggcatcatat caactcacct gcatgggatt
2880ttcgacctgc ctctctcact tagcaacact gatttcaaag ctatgggaac tgaagtggtc
2940gatggatgca ttcatccaac atggaaactg attgatggca tatgtagaga aagccttgct
3000tttcaaacag caaggaggga aggcatgcct gacttgataa tcaccagggc tgaggagcta
3060tatttgagta tgagtacaaa taacaagcag ggagcatcag tggcgcacaa tgagcctcct
3120aatggcagcc ccagtgtaaa tggcttggtt gaggagcctg aatctctgaa gaacagacta
3180gaaatgctgc ctggtacctt tgagccgctg cggaaggaag ttgagagtgc tgttactacg
3240atgtgtaaga aaatactgtc ggacctttac aacaaaagta gcatcccaga actggtcgag
3300gtggtctgcg ttgctgtagg tgctagagag caaccaccgc cttccactgt tggcagatct
3360agcatctacg tgattatcag aagcgacaac aggctctatg ttggacagac ggacgatctt
3420ctggggcgct tgaacgccca cagatcgaag gaaggcatgc gggacgctac ggtattatac
3480gtcttggtcc ctggcaagag cgttgcctgc cagctggaaa cccttctcat aaaccagctc
3540ccttcgaggg gcttcaagct catcaacaag gcagacggga agcacaggaa cttcggtata
3600tctcgaatct ctggcgaggc agttgctact ggacggaact ag
364253373DNALycopersicon esculentum 5atgtattggg ttacggcaaa aaacgtcgtc
gtttcagttc cccgttggcg ttcactgtcc 60cttttcctcc gtccaccact tcgccggcgt
ttcttatctt tctctccaca tactctgtgc 120cgagagcaga tacgttgcgt gaaggagcgg
aagttttttg ccacaacggc aaaaaaactc 180aaacaaccaa aaagtattcc agaggaaaaa
gactatgtta atattatgtg gtggaaagag 240agaatggaat tcttgagaaa gccttcttcc
gctcttctgg ctaagaggct tacatattgt 300aacttgctgg gtgtggatcc gagtttgaga
aatggaagtc ttaaagaggg aacacttaac 360tcggagatgt tgcagttcaa gtcaaaattt
ccacgtgaag ttttgctctg tagagtaggt 420gatttttatg aagctattgg attcgatgct
tgtattcttg tggaatatgc tggtttaaat 480ccatttggtg gcctgcactc agatagtata
ccaaaagctg gttgtccagt tgtgaatcta 540agacagacgc ttgatgatct cacacgtaat
ggtttctctg tgtgcgtcgt ggaggaagtt 600cagggtccaa ctcaagctcg tgctcgtaag
agtcgattta tatcagggca tgcacatcca 660ggcagtccct atgtttttgg ccttgttgga
gatgatcaag atcttgattt tccagaacca 720atgcctgttg ttggaatatc ccgttcagcg
aaggggtatt gcattatctc tgtttacgag 780actatgaaga cttactctgt ggaagatggc
ctaactgaag aagccgtagt caccaaactt 840cgtacttgtc gatgccatca tttttttttg
cataattcat tgaagaacaa ttcctcagga 900acatcgcgtt ggggagagtt tggtgaaggt
ggacttttgt ggggagaatg taatgctaga 960cagcaggaat ggttggatgg caatcctatc
gatgagcttt tgttcaaggt aaaagagctt 1020tatggtctca atgatgacat tccattcaga
aatgtcactg ttgtttcaga aaataggccc 1080cgtcctttac accttggaac tgccacacaa
attggtgcta ttccaaccga agggattcca 1140tgtttgttaa aggtgttgct tcctcctcat
tgcagtggtc taccagtcct gtatattagg 1200gatcttcttt taaatccacc agcctatgag
atttcttcag acattcaaga ggcatgcaga 1260cttatgatga gtgtcacatg ttcaattcct
gattttacct gtatttcatc tgcaaagctg 1320gtcaagctgc ttgagttgag ggaggcaaat
cacgttgagt tctgcaaaat aaagagcatg 1380gtcgaagaga tactgcagtt gtatagaaat
tcagagcttc gtgctattgt agagttactg 1440atggatccta cttgggtggc aactgggttg
aaagttgatt ttgatacact agtaaatgaa 1500tgtggaaaga tttcttgtag aatcagtgaa
ataatatccg tacatggtga aaatgatcaa 1560aagattagtt cctatcctat catcccaaat
gatttctttg aagatatgga gttgttgtgg 1620aaaggccgtg tcaagaggat ccatttggag
gaagcatatg cagaagtaga aaaggctgcg 1680gatgctttat ctttagccat aacagaagat
ttcctaccta ttatttcaag aataagggcc 1740acgatggccc cacttggagg aactaaaggg
gagattttgt atgcccgtga gcatggagct 1800gtatggttta agggaaagag atttgtacca
actgtttggg ctggaaccgc tggagaagaa 1860caaattaagc aactcagacc tgctctagat
tcaaagggga agaaggttgg agaagaatgg 1920ttcactacaa tgagggtgga agatgcaata
gctaggtatc acgaggcaag tgctaaggca 1980aagtcaaggg tcttggaatt gctaagggga
ctttcttctg aattactatc taagatcaat 2040atccttatct ttgcatctgt cttgaatgtg
atagcaaaat cattattttc tcatgtgagt 2100gaaggaagaa gaagaaattg gattttccca
acaatcacac aatttaacaa atgtcaggac 2160acagaggcac ttaatggaac tgatggaatg
aagataattg gtctatctcc ttattggttt 2220gatgcagcac gagggactgg tgtacagaat
acagtagata tgcagtccat gtttctttta 2280acaggtccaa atggtggggg caaatcaagc
ttgctgcgtt cgttgtgtgc agctgcattg 2340ctaggaatgt gtgggttcat ggttccagct
gaatcagctg tcattcctca ttttgactca 2400attatgctgc atatgaaatc atatgatagt
cctgttgatg gaaaaagttc atttcagatt 2460gaaatgtctg aaattcggtc tctgattact
ggtgccactt caagaagtct tgtacttata 2520gatgaaatat gtcgaggaac agaaacagca
aaagggacat gtattgctgg aagtgtcata 2580gaaaccctgg acgaaattgg ctgtttggga
attgtatcaa cccacttgca tggaatattt 2640gatttacccc tgaaaatcaa gaagaccgtg
tataaagcaa tgggagctga atatgttgac 2700ggtcaaccaa taccaacttg gaaactcatt
gatgggatct gtaaagagag tctagcattt 2760gaaacagctc agagagaagg aattccagaa
atattaatcc aaagagcaga agaattgtat 2820aattcagctt acgggaatca gataccaagg
aagatagacc aaataagacc tctttgttca 2880gatattgacc tcaatagcac agataacagt
tctgaccaat taaatggtac aagacaaata 2940gctttggatt ctagcacaaa gttaatgcat
cgaatgggaa tttcaagcaa gaaacttgaa 3000gatgctatct gtcttatctg tgagaagaag
ttaattgagc tgtataaaat gaaaaatccg 3060tcagaaatgc caatggtgaa ttgcgttctt
attgctgcca gggaacagcc ggctccatca 3120acaattggtg cttcaagtgt ctatataatg
ctaagacctg acaaaaagtt gtatgttgga 3180cagactgatg atcttgaggg cagagtacgt
gctcatcgct tgaaggaggg aatggaaaac 3240gcgtcattcc tatatttctt agtctctggc
aagagcatcg cctgccaatt ggaaactctt 3300ctaataaatc aacttcctaa tcatggtttt
cagctaacaa acgttgctga tggtaagcat 3360cgtaattttg gca
337363180DNASorghum bicolor 6atgcaccggg
tgctcgtgag ctcgctcgtg gccgccacgc cgcggtggct ccccctcgcc 60gactccatcc
tccggcgccg ccgcccgcgc tgctctcctc ttcccatgct gctattcgac 120cggagggctt
ggtccaagcc aaggaaggtc tcacgaggca tctcagtggc gtctaggaaa 180gctaacaaac
agggagaata ttgtgatgaa agcatgctat cgcatatcat gtggtggaaa 240gagaaaatgg
agaagtgcag aaaaccatca tccgtacaat tgactcagag gcttgtgtat 300tcaaatatat
tagggttgga tccaaatcta agaaatggaa gcttgaaaga tggaaccctg 360aacatggaga
ttttgctatt taaatcaaaa tttcctcgtg aggttctact ttgcagagta 420ggagacttct
atgaagctat tggttttgat gcctgtattc tcgtagagca tgcaggctta 480aatccttttg
gaggtttgcg ttctgacagt atccctaaag ctgggtgtcc agtcgtgaat 540ttacggcaga
cattggatga tttgactcga tgtggttatt ctgtgtgcat agttgaggaa 600attcaaggcc
caacacaagc ccgttcccgg aaaagtcgat ttatttctgg gcatgcccat 660cctggtagtc
cttatgtatt tggtcttgct gaagtagacc atgatgtaga gttccctgat 720ccgatgcctg
ttgttgggat ttcacattct gcaaaaggtt attgcttgat atctgtgcta 780gagacaatga
aaacttattc agctgaggag ggcttaacag aagaggctat tgttactaag 840ctccgcatat
gtcgttatca tcatctatac cttcacaatt ctttgaagaa taattcttca 900gggacatcac
gctggggtga attcggtgaa ggagggctct tgtggggaga gtgcagtggg 960aagtcctttg
agtggtttga tggtttacct attgaagaac ttttatgcaa ggtacgggaa 1020atatatggcc
ttgatgagaa aactgttttt cgcaatgtca ccgtctcatt ggaaggcagg 1080ccccaacctc
tttatcttgg aactgctact caaattggag tcataccaac tgagggaata 1140ccgagtttgc
taaaaatggc actcccttca agttgtggcg ggcttccatc aatgtatatt 1200agagatcttc
ttcttaatcc tccatcattt gatgttgcgg cagcggtcca agaggcttgc 1260aggcttatgg
ggagcataac ttgttctgtt cctgaattta cttgcatatc acttgtgaag 1320ctacttgagt
ctaaagaggt caatcacatt gaattttgta gaataaaaaa tgtccttgat 1380gagattatgc
tcatgaacag gaatgctgag ctttctgcaa tcctgaacaa attgctggta 1440cctggttctg
tggctactgg tttgaaagtt gaagctgata tgctagtcat tgaagatttt 1500atgccaatta
tttcaagggt gaaatctgta gtgtcctcaa atggaggttc gaaaggagaa 1560atctgttatg
caaaagaaca tgaagctgtt tggtttaaag gaaagcgatt cacaccaact 1620gtatgggcta
acacacctgg tgagcagcaa ataaaacaac tgaagcctgc aattgattcg 1680aaaggcagaa
aggttgggga ggaatggttt acaacaagca aagttgagaa tgctttagcc 1740aggtaccatg
aagcttgtga taatgcaaga aataaagttg ttgagctgtt gagagggctt 1800tcaagtgaat
tgcaggacaa aattaacata cttgtctttt gctcaacact gctcatcatt 1860gcaaaagcac
tttttggtca tgttagtgag gctcggagaa gaggctggat gcttcctact 1920atatttccct
tgtcaaagga ctgtgttgca gaggaaagtt caaatgcaat ggatttagta 1980ggactctttc
cttactggct tgatgttaat caaggaaatg caatattgaa tgatgtccac 2040atgcactctt
tatttgttct tactggtcca aatggtggtg gtaaatctag tatgttgcga 2100tcagtctgtg
cagctgcgct gcttggaata tgtggcctga tggtaccttc aacttcagct 2160gtaatcccgc
attttgattc cattatgctg catatgaaag cctacgatag cccagccgat 2220gggaaaagtt
catttcagat tgaaatgtcg gagatacgtg ctttagtcag ccgagctact 2280gctaggagtc
ttgtcctgat tgatgaaata tgtaggggca cagaaactgc aaaaggaacc 2340tgtattgctg
gtagcatcat cgaaaggctg gataatgttg gctgcctagg catcatatca 2400actcacctgc
atgggatttt tgacttgcct ctctcactca gcactactga tttcaaagct 2460atgggaactg
aagtggtcga cgggtgcatt catccaacat ggaaactgat ggatggcatc 2520tgtagagaaa
gccttgcttt tcaaacagcc aggagggaag gcatgcctga gttcataatc 2580agaagggctg
aggagctata tttgactatg agtacaaata acaagcagac cgcatcaatg 2640gtccacaatg
agcctcgtaa tgacagcccc agtgtaaatg gcttggttga gaagcctgaa 2700tatctgaaat
acagactaga aattctgcct ggtacctttg agccgttgcg gagggaagtt 2760gagagtgctg
ttactatgat atgcaagaaa aaactgttgg atctttacaa taaaagtagc 2820atcccagaac
tggttgaggt ggtctgtgtt gctgtaggtg ctagagagca accaccacct 2880tccactgttg
gcaggtctag catctatgtg attatcagaa gcgacaacaa gctttatgtt 2940ggacagacgg
atgatcttct ggggcgcctt cacgcccaca gatcgaagga aggcatgcag 3000gatgctacga
tattatacat cttggttcct ggcaagagcg ttgcctgcca gctggaaacc 3060cttctcataa
atcagcttcc ttcgaggggc ttcaagctca tcaacaaggc agacggaaag 3120cataggaact
tcggtatatc tcgaatctct ggagaggcaa tcgccaccca gctaaactaa
318073399DNAOryza sativa 7atggccattc agcggctgct cgcgagctcg ctcgtggccg
ccacgccgcg gtggcttccc 60gtcgccgccg actcgtttct ccggcgccgc caccgccctc
gctgctcccc gctccccgcg 120ctgctattta acaggaggtc ctggtctaaa ccaaggaaag
tctcacgaag catttccatt 180gtgtctagga agatgaacaa acaaggagat ctctgtaatg
aaggcatgct gccacatatt 240ctgtggtgga aagagaaaat ggagaggtgc aggaaaccat
catcaatgca attgactcag 300agacttgtgt attcaaatat tttaggattg gatccaactt
taagaaatgg aagcttgaag 360gatggaagcc tgaacacgga aatgttgcaa ttcaaatcga
agtttcctcg tgaagttcta 420ctttgcagag tgggagattt ctacgaggct gttgggtttg
atgcatgtat ccttgtggag 480catgcaggct taaatccttt tggaggcttg cgttctgata
gtattccaaa agctggatgt 540ccagtcatga atttgcggca gacattggat gatttgactc
gatgtggtta ctctgtgtgc 600atagttgaag aaattcaagg cccaacccaa gctcgtgcta
ggaaaggccg atttatttct 660ggccatgcac atcctggtag tccttatgta tttggtcttg
ctgaagtaga ccatgatgtt 720gagttccctg atccaatgcc tgtagttggg atttcacgat
ctgcaaaagg ctattgcctg 780atttctgtgc tagagacaat gaaaacatat tcagctgagg
agggcttaac agaggaagca 840gttgttacta agcttcgcat atgccgttat catcatctat
accttcatag ttctttgagg 900aacaattctt caggcacatc acgctgggga gaatttggcg
aaggtgggct attgtgggga 960gagtgcagtg gaaaatcttt tgagtggttt gatggtaatc
ctattgaaga actgttatgc 1020aaggtaaggg aaatatatgg gcttgaagag aagactgttt
tccgtaatgt cagtgtctca 1080ttggaaggga ggcctcaacc cttgtatctt ggaacagcta
ctcaaattgg ggtgatacca 1140actgagggaa tacccagttt gctaaaaatt gttctccctc
caaactttgg tggccttcca 1200tcattgtata ttagagatct tcttcttaac cctccatctt
ttgatgttgc atcatcagtt 1260caagaggctt gcaggcttat gggtagcata acttgctcga
ttcctgaatt tacatgcata 1320ccggcagcaa agcttgtgaa attactcgag tcaaaagagg
ttaatcacat cgaattttgt 1380agaataaaga atgtcctcga tgaggtgttg ttcatgggta
gcaatgctga gctttctgct 1440atcctgaata aattgcttga tcctgccgcc atagttactg
ggttcaaagt tgaagccgat 1500atactagtga atgaatgtag ctttatttca caacgtatag
ctgaagtaat ctctttaggt 1560ggtgaaagtg accaggcaat aacttcatct gaatatattc
cgaaagagtt cttcaatgat 1620atggagtcat cttggaaggg acgtgtaaaa agggtgcatg
ctgaagagga gttctcaaat 1680gttgatatag ctgctgaggc actgtcaaca gcggtcattg
aagattttct gccaattatt 1740tcaagagtaa aatctgtgat gtcctcaaat ggaagttcga
agggagaaat cagttatgca 1800aaagagcatg aatctgtttg gtttaaaggg aggcgattca
caccaaatgt gtgggccaac 1860actcctggtg aactacagat aaagcaattg aagcctgcaa
ttgactcaaa aggtagaaag 1920gtcggagaag aatggttcac cactatcaaa gttgagaatg
ctttaaccag gtaccatgaa 1980gcttgtgata atgcaaaacg taaagttctt gagttgttga
gaggactttc aagtgaattg 2040caggacaaga ttaatgtcct tgtcttttgc tcaacgatgc
tcatcataac aaaagcactt 2100tttggtcatg ttagtgaagg acgaagaagg ggttgggtgc
ttcctactat atctcccttg 2160tgtaaggata atgttacaga ggaaatctca agtgaaatgg
aattgtcagg aacttttcct 2220tactggcttg atactaacca agggaatgca atactgaatg
atgtccatat gcactctttg 2280tttattctta ctggtccaaa cggtggtggt aaatccagta
tgctgagatc agtctgtgct 2340gctgcattac ttggaatatg tggcctgatg gtgccagctg
cttcagctgt catcccacat 2400ttcgattcca tcatgctgca tatgaaagca tatgatagcc
cagctgatgg taaaagttcg 2460tttcagattg aaatgtcaga gatacgatct ttagtctgcc
gagctacagc taggagtctt 2520gttctaattg atgaaatatg taggggcaca gaaacagcaa
aaggaacatg tatagctggt 2580agcatcattg aaagactcga taatgttggc tgcataggca
tcatatcaac tcatttgcat 2640ggcatttttg accttccact gtcactccac aatactgatt
tcaaagctat gggaaccgaa 2700atcatcgata ggtgcattca gccaacatgg aaattaatgg
atggcatctg tagagagagt 2760cttgcttttc aaacagccag gaaagaaggt atgcctgact
tgataattag aagagctgag 2820gaactatatt tggctatgag cacaaacagc aagcagacat
catcagctgt ccaccatgaa 2880atatccatag ccaactctac tgtaaatagc ttggttgaga
agcctaatta cctgagaaat 2940ggactagagc ttcaatctgg ttccttcgga ttactaagaa
aagaaattga gagtgttgtt 3000accacaatat gcaagaagaa actgttggat ctctacaaca
aaaggagcat ctcagaactg 3060attgaggtgg tctgtgttgc tgtgggtgct agggagcaac
ccccaccttc aactgttggc 3120aggtccagca tttatgtaat tatcagacgt gacagcaagc
tctatattgg acagacggat 3180gatcttgtgg gtcgacttag tgctcacaga tcgaaggaag
gtatgcagga tgccacgata 3240ttatatattt tggtacctgg gaagagcatt gcatgccaac
tggaaactct tctcataaat 3300cagctacctt tgaaaggttt caagctcatc aacaaggcag
atggcaagca tcgaaatttc 3360ggtatatctc ttgtcccagg agaggcaatt gccgcatag
339983381DNABrachypodium distachyon 8atgcagcggc
ttctggcgag cacgatcgtg gccgccacgc cgcgttggct ccccctcgcc 60gactctatcg
tccggcgccg ccgcccgcgc cgttccccgc tccccgtcct gctattccac 120agatcattgt
acaaaccaag gaaggtttca cgaggcatta caatggtgtc taataaggtg 180aacaaacagg
gagatctctg caatgaaggc atgctgtcac atattatgtg gtggaaagag 240aaaatggaga
gctgcaggaa accatcatct gtgcagttga ctcagagact tgtgtactct 300aatatattag
ggttggatcc aactttaagg aatggaagct taaaagatgg aaccctgaac 360atggagatgt
tacaatttaa atcaaagttt ccacgtgagg tcctactttg cagagtagga 420gatttctatg
aagccattgg gtttgatgcc tgcattcttg tagagcatgc aggcctaaat 480ccttttgggg
gcttgcgttc tgacagtatt ccaaaagctg gatgtccaat catgaatttg 540cggcaaacat
tggatgattt gactcggtct ggttattctg tgtgcatagt tgaggaaatt 600caaggcccaa
ctcaagcccg tgctcggaaa ggtcgattta tctctggcca tgcgcatcct 660ggcagtcctt
atgtatttgg tcttgctgaa gtagatcatg atcttgagtt tcctgaccca 720atgcctgtag
ttgggatttc acgctctgca aaaggctatt gcttgatttc tgtgctagag 780acgatgaaaa
cttattcagc tgaggagggc ctaacagaag aagctgtagt gactaagctg 840cgcatatgcc
gttatcatca tctatacctt cacagttctt tgaggaataa ttcttcaggg 900acatcacgct
ggggggaatt cggagaggga ggactcttgt ggggagagtg cagtggaaag 960tgttttgaat
ggtttgatgg ttctcctatt gaggaacttt tatgcaaggt aagggagata 1020tatgggctgg
atgagaaaac taatttccgc aatgtcactg tctcattgga agggaggcct 1080caacctttat
atcttggaac tgctactcaa attggagtga tacaaacgga gggaattccc 1140agtttactaa
aaatgctact ccctccaaac tatggcgggc ttccatcaat gtatatcaga 1200gatcttcttc
ttaatcctcc atcttttgat gtcgcgtctg caattcagga ggcttgcagg 1260cttatgggca
gcataacttg ttcgattcct gaatttactt gcataccatc agcgaagctt 1320gtgaaattac
tcgagtcaaa agaggttaat cacattgaat tttgtagaat aaagaatgtc 1380cttgatgaca
ttatattaat gaatggaaac actgagcttt ctgctatcat ggacaaattg 1440ctcgaacctg
cttcggtggt tactggtttg aaagttgatg ctgatatact aattagagaa 1500tgtagcctta
tctcacaacg tataggtgaa gtcatctctt taggtgggga aagcgatcag 1560gcaataactt
catcggaata tattcccaag gagttcttta atgatatgga gtcatcttgg 1620aaggggcgtg
tgaaaagggt tcatgctgaa gaagagttca caaatgtcga tgtagctgct 1680gaagcattat
caaccgcggt aactgaagat tttctgccaa ttattgtaag agttaaatct 1740gtgatatctt
cacatggagg ttctaaaggg gaaatctctt atgcaaaaga acacgaagct 1800gtttggttta
aagggaagcg attcacacca aatgtctggg cgaacacacc tggtgaacaa 1860cagataaaac
aactaaagcc tgcgattgat tcaaaaggta gaaaagttgg ggaggaatgg 1920tttacaacaa
tcaaagttga gaatgcttta gccaggtatc atgaagcttg tgatagtgca 1980aaaggcaaag
ttcttgagct gttgagaggt ctttcaagtg aattgcagga caagattaat 2040atacttgtct
tctgctcgac gctgctcatc atagcaaaag cactttttgg tcatgttagc 2100gagggtctta
gaaggggttg ggtgcttcct gccatatctc ccctatctaa ggactatagt 2160actgaagaag
gctcaagtga aatggattta ttgagactct ttccttactg gcttgacagt 2220aatcaaggga
atgcaatact gaatgatgtc aatatgcact ctttgtttat tctgactggc 2280ccaaatggtg
gaggtaaatc cagtatgttg cgatcagtct gtgcagctgc attgcttgga 2340atatgtggtc
tgatggtgcc agctgcttca gctgtcatcc cacactttga ttccatcatg 2400ctgcatatga
aggcctatga tagcccagct gatgggaaaa gttcgtttca gattgaaatg 2460tcagagatcc
gatctttagt cagccgtgct actggtagga gtcttgttct cattgatgaa 2520atatgtaggg
gcacagaaac tgcaaaagga acttgtatag ctggtagcat catcgaaagg 2580ctcgacgatg
ttggctgcct aggcatcata tcaacccatt tgcatggcat ttttgacttg 2640cctctgtcac
tcggcaatac tgatttcaaa gctatgggaa cagaagttgt caatgggtgc 2700attcagccaa
catggagatt aatggatggt atctgtagag aaagccttgc ttttcaaaca 2760gcaaggaagg
aaggtatgcc tgacttgata attaaaagag cagaggagct atacagtact 2820atgggcagaa
gcaagacgtc atcaacagtc caccatggtc catccgttgc taagtctaaa 2880gcaagtggat
tggttgatat gcctgatggt ctgggaaatg gattagaact tccatctggt 2940gcttttgcac
tgctgcgaaa ggatgtcgaa ataattgtga ccgcaatatg caaggataaa 3000ttgttggatc
tctacaacaa aagaagcatc tcagagctgg ttgaggtggt ttgtgttact 3060gtaggtgcta
gggagcaacc gccaccttca actgttggca ggtccagcat ctacatagtt 3120atcaggcgtg
acaacaagct ctatgttgga cagacggatg atcttgttgg ccgtcttgct 3180gttcatagat
ccaaggaagg tatgcagggt gccacaatat tatatatcgt ggttcctggc 3240aagagcgttg
cgtgccagct ggagacactt ctcataaacc agcttccctc gaaaggtttt 3300aagctcacga
acaaggcaga tggcaagcat cggaacttcg gcatgtctgt tatctctgga 3360gaagccattg
ctgcacactg a
338193520DNAVitis vinifera 9atgtactggc tgtcaaccaa aaacgtcgtc gtttcattcc
ctcgattcta ctctctcgct 60cttcttctcc gttcccctgc ctgcaaatac acttcatttc
gttcttctac acttctactc 120caacagtttg agaagagccg atgtctcaac gaaaggaggg
ttttgaaagg agctggaaga 180atgacaaaaa atgttatagg attgcaaaat gagctagatg
aaaaggatct ttctcacata 240atgtggtgga aggagaggat gcaaatgtgt aaaaagccgt
ccactgtcca ccttgttaaa 300aggcttatat attccaattt gctaggagtg gatcctaact
tgaaaaatgg gaatctaaaa 360gaaggaacgc tgaactggga gatgttgcag ttcaagtcaa
agtttcctcg tgaagtttta 420ctctgcagag taggggattt ttatgaagcc atcggaattg
atgcttgtat tcttgttgaa 480tatgctggtt tgaatccttt tggtggtttg cgctcagaca
gtataccaag agctggctgc 540ccagtcatga atctacgaca aactttggat gacctgacac
gtagcgggta ttcagtttgc 600atagtggagg aagttcaggg tccaactcaa gctcgttctc
gtaaaggtcg ttttatctct 660gggcatgcgc atccgggtag tccttatgta tttggacttg
ttggggttga tcatgatctt 720gattttccag aaccaatgcc tgtagttgga atttctcgtt
ctgcgaaggg ttattctata 780attttagtcc ttgagactat gaagacgttt tcagtagagg
atggtctgac agaagaggct 840ttagttacca agcttcgcac ttgtcactac catcatttat
tgctgcatac atctctgaga 900cgcaactcct caggtacttg tcgttgggga gaatttggtg
agggaggact attatgggga 960gaatgtagtg ctagacactt tgaatggttt gaaggggatc
ctgtatctca acttttgttt 1020aaggtgaagg agctctatgg ttttgatgat caagttacat
ttagaaatgt cactgtgtct 1080tcagagaaaa gaccccgttc tttacacctt ggcacagcta
cacaaattgg tgccatacca 1140acagagggca taccgtgttt gttaaaggtg ttgcttccat
caaattgcac tggtctacct 1200cttttgtatg ttagagatct tcttctcaac cctcctgctt
atgagattgc atccataatt 1260caagcaacat gcagactcat gaacaatgta acgtgctcga
ttcctgagtt tacttgtgtt 1320tcccctgcaa agcttgtgaa gctacttgag cttagggagg
ctaatcatat tgagttctgc 1380agaataaaaa gtgtacttga tgaaatattg cagatgcata
gaaactctga tcttaacaaa 1440atccttaaat tattgatgga tcctacctgg gtggcaactg
gattgaagat tgactttgac 1500acattggtga acgaatgtga atggatttca gctagaattg
gtaaaatgat ctttcttgat 1560ggtgaaaatg atcaaaagat aagttaccat cctatcattc
caaatgactt ttttgaggac 1620atggaatctc cttggaaggg tcgtgtgaag aggatccatg
tagaagaagc atttgctgaa 1680gtggaaagag cagctgaggc attatcttta gctatctccg
aagattttct acctattatt 1740tcaagaataa aagctaccac agccccactt ggaggtccaa
aaggagaagt tgtatatgct 1800cgagagcatg aagctgtttg gttcaaggga aaacgttttg
caccagttgc atgggcaggt 1860actccagggg aagaacaaat taagcagctt agacctgcta
tagattcaaa aggtagaaag 1920gttggattgg aatggtttac cacagtgaag gtggaggatg
cactaacaag gtaccatgag 1980gctggggaca aggcaaaagc aagggtcttg gaattgttga
ggggactttc tgcggagtta 2040caaactaaaa ttaacatcct tatctttgct tccatgttgc
ttgtcattgc aaaggcatta 2100tttgctcatg tgagtgaagg gagaagaagg aaatgggttt
tcccctctct tgtagagttg 2160cataggtcta aggacatgga acctctggat ggagctaatt
ggatgaagat aactggttta 2220tcaccatatt ggttggacgt ggcacaaggc agtgctgtgc
ataatacagt tgatatgaaa 2280tcattgtttc ttttgacagg acctaatggg ggtggtaaat
caagtttgct tcgatcaatt 2340tgtgcagccg cattacttgg aatatgtgga tttatggtgc
ctgcagaatc ggccttgatt 2400cctcattttg attctattat gcttcacatg aaatcttatg
atagcccagc tgatggaaaa 2460agttcatttc agattgaaat gtcagagatg cgatccataa
tcactggagc cacttcaaga 2520agcctggtgc tgatagatga aatctgccga ggaacagaaa
cagcaaaggg gacatgtatt 2580gctggtagca tagttgaaac tcttgataag attggttgtc
tgggtattgt atccactcac 2640ttgcatggta tatttacctt gggactgaat actaagaatg
ctatttgtaa agcaatggga 2700actgaatatg ttgatggcaa aacaaaaccg acctggaagt
tgatagatgg aatctgtaga 2760gaaagccttg cctttgaaac agctcagaag gagggaattc
ctgaaacaat tatccgaaga 2820gcagaagagc tgtatctttc aatccattca aaagacttaa
ttacaggggg aactatttgt 2880cctaaaattg agtcaacaaa tgaaatggaa gtcttacata
agaaagttga gagtgcagtc 2940accattgttt gccaaaagaa gctgaaggag ctctataagc
agaaaaacac gtcaaaactt 3000ccagagataa actgtgtggc cattttgcca ggggaacagc
cgccgccatc aacaattggt 3060gcttcaagtg tgtatgtgtt gtttagcact gataagaaac
tttatgttgg agagacagat 3120gatcttgaag gcagagtccg tgcgcatcga tcaaaggaag
gaatgcagaa ggcctcattc 3180ctttattttg tggtcccagg gaagagcttg gcatgccaac
tcgaaacgct tctcatcaac 3240cagctccctg tccaggggtt ccaactggtc aatagagctg
atggtaaaca tcgaaatttt 3300ggcacattgg atcactccgt ggaagttgtg accttgcatc
aatgagcctg cgctccttgc 3360cacccatttt gtagaatggt tccatctttg aaatatgtac
ttgaatgaca aaaaccagat 3420gaaagtggct gcagcaattt tggttttttg atgtacgttg
ctccacttgc attagtatta 3480tctacctgat gaaatatgca ttgatattgc ttgctctaca
3520103615DNACucumis sativus 10atggaaatat
ccatctatgt cgatgtggca ttgtggcggg aagtatcgga aaccaagggt 60tttctgttcc
ggcgacgacg agttacaaac accctcctca tttcaaacca aaacgcttta 120aaacttccaa
tcacaacaag attgaagctc acaaaccatc catttttatc caccgccatg 180tactgggcgg
caacacgaac cgttgtttct gcttcccggt ggcgttttct ggctcttttg 240attcgcttcc
ctccgcgtaa cttcacctca gttactcatt cgccggcatt tatagaaagg 300caacagcttg
aaaagttgca ctgttggaaa agcagaaaag gttcaagagg aagcatcaaa 360gctgctaaga
agtttaagga taataatatt ctccaagaca ataagtttct ttctcacatt 420ttatggtgga
aagagacggt ggaatcatgc aagaagccgt catctgtcca gctggttaag 480aggcttgact
tttccaactt gctaggttta gatacaaacc tgaaaaatgg gagtcttaaa 540gaaggaactc
ttaactgtga gattctacag ttcaaggcaa agtttcctcg agaagttttg 600ctctgtagag
ttggagattt ttatgaagca attggaatag atgcttgcat acttgtggaa 660tatgctggtt
taaatccttt tggaggtcag cgtatggata gtattccaaa agctggttgc 720cccgttgtga
atcttcgtca aactttggat gatctgacac gcaatgggtt ctcagtgtgc 780atagtggaag
aagttcaggg cccaattcaa gctcgttctc gcaaaggacg ttttatatct 840gggcatgcac
acccaggcag tccctatgtt tttgggcttg tcggggttga tcacgatctt 900gactttccag
aaccgatgcc tgtgattgga atatctcgat ccgcaagggg ctattgcatg 960agccttgtca
tagagaccat gaagacatat tcatcagagg atggtttgac agaagaggcc 1020ttagttacta
aactgcgcac ttgtcaatac catcatttat ttcttcacac gtcattaagg 1080aacaactcct
caggcacttg ccgctggggt gaatttggtg agggtggccg gctatggggg 1140gaatgtaatc
ccagacattt tgagtggttc gatggaaagc ctcttgataa tcttatttct 1200aaggttaaag
agctttatgg tcttgatgat gaagttacat ttagaaatgt tacaatatcg 1260tcagaaaata
ggccacatcc gttaactcta ggaactgcaa cacagattgg tgccatacca 1320acagagggaa
taccttgttt gctgaaggtt ttgcttccat ccaattgtgc tggccttcct 1380gcattgtata
tgagggatct tcttctcaat cctcctgctt atgagactgc atcgactatt 1440caagctatat
gcaggcttat gagcaatgtc acatgtgcaa ttccagactt cacttgcttt 1500cccccagcca
agcttgtgaa gttattggaa acgagggagg cgaatcatat tgaattctgt 1560agaatgaaga
atgtacttga cgaaatatta caaatgcaca aaaattgcaa gctaaacaat 1620atcctgaaat
tgctgatgga tcctgcatct gtggcaactg ggttgaaaat tgactatgat 1680acatttgtca
acgaatgtga atgggcttcc agtagagttg atgaaatgat ttttcttggt 1740agtgaaagtg
aaagtgatca gaaaatcagt tcttatccta ttattcctaa tggttttttc 1800gaggacatgg
aattttcttg gaaaggtcgt gtgaagagga ttcacattga agaatcttgt 1860acagaagttg
aacgggcagc tgaagcactc tcccttgcag ttactgaaga ttttgtccca 1920atcatttcta
gaatcagggc tactaatgca ccactaggag gtccaaaggg agaaatatta 1980tatgctcggg
accatcaatc tgtctggttc aaaggaaaac ggtttgcacc atctgtatgg 2040gctggaagcc
ctggagaagc agaaattaaa caactgaaac ctgctcttga ttcaaaggga 2100aaaaaagttg
gggaggagtg gtttaccacg aagaaggtgg aggattcttt aacaaggtac 2160caagaggcca
ataccaaagc aaaagcaaaa gtagtagatc tgctgaggga actttcttct 2220gaattgttag
ctaaaattaa cgtcctaata tttgcttcca tgctactcat aattgccaag 2280gcgttatttg
ctcatgtgag tgaagggagg aggaggaaat gggtttttcc cacccttgct 2340gcacccagtg
ataggtccaa ggggaaagtt gcgatgaagc tggttggtct atctccctat 2400tggtttgatg
ttgtcgaagg caatgctgtg cagaatacta ttgagatgga atcattattt 2460cttttgactg
gtccaaatgg gggtggaaaa tctagtttgc ttcgatcgat ttgtgctgct 2520actttgcttg
ggatatgtgg atttatggta ccggcagagt ccgccctgat tccccacttc 2580gactcaatta
tgcttcatat gaaatctttt gatagtcctg ctgatggaaa aagttctttt 2640caggtggaaa
tgtcagagat gagatccatt gtcaatagag taacggagag aagtcttgta 2700cttatcgatg
aaatctgtcg tggaacagaa acagcaaaag gaacttgtat tgccgggagc 2760attattgaag
ctcttgataa agcaggttgt cttggcattg tctccactca cttgcatgga 2820atatttgatt
tgcctttaga tacccaaaac attgtgtaca aagcaatggg aactgtttct 2880gcggaaggac
gcacggttcc cacttggaag ttgattagtg gaatatgtcg agagagcctt 2940gcctttgaaa
cagcaaagaa tgaaggaatc tctgaagcta taattcaaag ggctgaagat 3000ttgtatctct
caaattatgc taaagaaggg atttcaggaa aagagacgac agatctgaac 3060ttttttgttt
cttctcatcc aagccttaat ggtaatggca ctggaaaatc caatctcaag 3120tcaaacggtg
tgattgtaaa ggctgatcag ccaaaaacag agacaactag caaaacaggt 3180gtcttgtgga
agaaacttga gagggctatc acaaagatat gccaaaagaa gttgatagag 3240tttcatagag
ataaaaacac attgacacct gctgaaattc aatgtgttct aattgatgca 3300agagagaagc
cacctccatc aacaataggt gcttcgagcg tatatgtgat tcttagaccg 3360gatggcaaat
tctatgttgg acagactgat gatctggatg gtagggtcca atcacatcgt 3420ttaaaggaag
gaatgcggga tgctgcattc ctttatctta tggtgcctgg gaagagctta 3480gcttgccaac
ttgaaactct tctcatcaat cgacttcctg atcacgggtt ccagctaact 3540aacgttgctg
atggaaagca tcggaatttt ggcacagcca atctcttatc cgacaatgtg 3600actgtttgct
catga
361511660DNAGossypium hirsutum 11ggcacgaggt tgctattgct gcaagggaac
agccacctcc atcaactatc ggtgcttctt 60gcgtatatgt catgttcaga cctgataaga
aactatacat tggagagacg gatgatcttg 120atggtcgaat tcgttcgcat cgttcaaagg
acgggatgga aaatgcttct ttcctatatt 180tcacagttcc agggaagagt attgctcgcc
aactcgaaac tcttctaatc aaccaactct 240taagtcaagg cttcccgatc gccaacttgg
ctgacggtaa gcatcagaat tttggcacat 300ccagtctctc atttgacggc ataaccgtag
cctaacgagt taaaatgtat atcaatacgt 360aatttatatc gaaattgaca tagaagtggc
ggcagcaatt ttgcctttga tctcggttgc 420tccacttgct ttgtacatgc atcacccttt
taaccaaggg taaagttttc tagtcataat 480ttaatagcat gtatctatta agtccatttt
gaggtttata tgaatcaggt tttcatcatt 540aattggttaa attctgttat tagctcctct
actttactaa agttgtagat ttagttctta 600tactttaatt agattatttt tactctatac
ttttcgaatg ataaaatttt agtcttcatt 660122225DNAOryza sativa 12agaaaagcta
aaatggatga aaaaaacaga gaaaaagaaa tcctaccctc catagcgagc 60agtggtggtc
tgcactctgc aggcaggcag gcacccaggc ctgcagctcg atggcgccgc 120cgccgccgtc
gccgccgcga tctctcacct ccgtctccct ccggactcct ctgagccctc 180tcctcttcct
acggccagct tcttgcaatc catcggcggt ctccggctcc tgcagcagcg 240gagcatgccg
cggcgtgcgc tgctcggcgg cgaacaagcc ttctccttcc accgctccgg 300gcaccgaggt
aaggtagccg gctagccgcc ccccatattc ttgtttctgt gttgatcgga 360gctcgatggc
tggggtgctc tgggctcgtc gtcgtcggtc gatcgtcatg gcttgcttcg 420tttcttgcag
ctcgacctcc atggcaaaga taaggagtga ggtgctgtcc ccgttccgct 480ccgtgcggat
gttcttctac ctcgcgttca tggccagcgc cgggctcggg gccctcatcg 540cgctcacgca
gctcatcccg gcgctgtcca gcccggcgag ggcggccgcc gcgggggaga 600cgctcaaggg
cctgggcatc gacgtcgcgg cggtctccgt cttcgcgttc ctctactggc 660gcgagagcaa
ggccaaggac gcgcaggtgg cgaagctcac gcgggaggag aacctgtcca 720ggctcaggat
ccgcgccggc gagggccgcc cgcccgtccc gctcggcgag ctgaggggca 780ccgcgcggct
cgtcatcgtc gccggccccg cggcgttcgt caccgagtcg ttccgccgga 840gcaagccgtt
cttgaaggac ctcatggagc gcggcgtgct tgtcgtgccc ttctcgacgg 900acggcaacgc
gccggacctg cagttcgacg aggccgacga ggaggaggag gaggcggcgg 960cggcggctgg
gaagatgaag cggaggctct ggcagctcac tccggtttac acttctgaat 1020gggccaagta
cgcgcaaagc cgggatccca tgaatttagc tgcttaaatt tcttcttcat 1080gtcaatcgaa
attcaaatgc aaattagtat ctcattttca aatcgattgc tgcttcttgc 1140agatggctag
atgagcagaa gaagctagcc aacgtgtcac ctgattcccc cgtgtgagta 1200tcaaaaacta
ctctgaattt gtctgaaaat ataactgaag tttctgcagc tgctgaactg 1260aaaccgcatc
actcttgcag gtatctctcg ctccggctgg acggccgcgt ccgtggcagc 1320ggcgtcgggt
acccgccgtg gcaagcgttc gtggcgcagc tgccgccggt gaaggggatg 1380tggtccggcc
tccttgatgg gatggacggg agggtgcttt gaatatttga ctgatacaga 1440ccgtgaaaac
attagttgat tggagaaaaa aaaggacggc cgggttcgat ctatagctta 1500tactagaaca
agaacaggaa gagtttgatg attgctttaa cttctgtggg gttgattttg 1560cttcctgcat
cccagcgaca tcgcccaagt gaatgtgata tgccatgtgc ccatgtacat 1620gttgttttgc
agcctacgtg acttgattat taacgagaat cctgtgtcaa agatcgcttt 1680ttccgtggta
ggcttctcca ttttatttta tttttgaata tatatacgaa ccgtgacaaa 1740tctgatggaa
cactggacca tgggggtaat gatactgtag tcgcctggtc tttttatcag 1800gcgctaaatg
caaacaatca gacagcttaa acaacctgag gttgttcagc cagcccagaa 1860tacaaaaagc
ccatggaccg tgagcccgtg aaaccatggc ccacccatca gtcacgtcac 1920gtcgggacgt
gtgcgcacta ccccgagaag cgcgcggccg taagccacaa ccacaacccc 1980acaccgcctc
ttggctgctc gcgccctgac acttcccaaa accccaaacc gcccatatct 2040ctctcctctt
ctccgctcct cgcttccccc aaaaccctcg cggtggcggt gggccgccgg 2100tctcattccg
ccggcgtcca cggaggcggc cggagccagg cgctccgcgg gccggggagc 2160caccggaagg
ctggaaccct agcggcggcg ggtctcctcc ccctccccgc gcgccgggcg 2220gcgcc
2225132000DNASolanum lycopersicum 13cacgcatcaa catgtactag ctaactttgt
ccaccaagga atatctatta ccttcaacca 60gcaacaagta tcagctaact ttgtccattt
aggttaaaac cttgactaaa tccattgaaa 120acctattacc tcccaccagc aacttatgcc
aactaacatt gtctaccaag ggatatccgt 180tgcctcaaca ttgtccatcg agggatatcc
attgcctccc agcagcaaca agtaccagct 240aacattgtcc aatcgaggga catccatacc
aaataatttt tgagaaagta attatttagg 300gtgatgttta tttgtgcttg gaaaacaaat
agccattgaa ttagtaaaat ttgcaattaa 360atcacattac aaaaatgtct aaggtaattt
atcctaaata caatagtgat atcaataata 420atatatttga gtgtaatgtc ataatcaaga
atttataaaa ataatatgtc agtaggttta 480attactgata tattaaaatt gtttcgataa
attcttgatt taaagagtac aatcaacaaa 540taaaagaaca aatagtaaaa tataagatac
gtatattttt aaatagtagt gtacagtagg 600actagatgac aaaataaaat tgtaagaaga
agaggatagt ctttaccatt ctcacacaaa 660atcatatctc gtcacaaatg aatttacagt
tactcataaa tatatttaat aaaatgatag 720tattagcaaa acataattga tgtgttagtt
catcagttat tatgcactta aagttattta 780taaaaaaaaa aggtcaaatg ccccccctaa
tctgttgtcc gatttctaag tacgtacgta 840aactttagag gggtcatatc acccctgaac
tgtttaaaat tgaaattttt gtactcctaa 900aaagccataa ccatatttat gttgatgagt
gaaactcacg cgttttgaca catgaaaaat 960ccattagaaa aagtttttct tcaatttttt
cattattctt tattcttttc ttctattttt 1020tcaccatctc tccaacacaa aaatattttc
actattttct ccattatcat aaacaccaaa 1080ggttcactaa tcactacaat cttcaaaacg
aaaaggtcgg cagtgatttt gattcttatt 1140tgtcatttcg cccactactt agttggtgct
aagatcattt tcatgaatac atgtgtcaaa 1200tttctcaaat tagagtaaat gaataacaat
ggaggaaagt cgaatgtggg ttttattaaa 1260aatttaaaat tcttcgtttg tttgaattaa
aattacaaat ttggagcttt gaagaaccaa 1320aaaaagaatt tgccgaccta attgacctta
atttgtcgtt tgaccatgta caaggatgat 1380taacaactcc aataatatcg ataaagagtt
gattttcaaa aactcttaat tccatacaca 1440aattaaaatc aagaaaaatg cgaaaatttt
atgaacaaat ctaataacaa agaacgaaaa 1500aatttctacg aagaacaaag aaaagaataa
ggaagaagat aaaatatttg ttttggtgct 1560cactctatca gggagtgaaa tacattcact
atggagatga taatttcaat tttaaacagt 1620tcaggaaggt gatatgcgta aagtttaagt
taatagataa tcaaagatat ctgttgacgt 1680attgattcaa tgttaaatat acagttgagt
tacttataac ttataaatac aataagtaat 1740ttttttcctt ctaccatttg aaaaaaaata
acgcgtacgt cattgtcttc aatcatcaac 1800gatttattat tttcaatgtg attatattat
taagtaatta attcgtccca aataccaata 1860tctactaaca ttctttgcct aatgtttaat
tgtaattcct acagatttta tttttttgaa 1920aataatataa agtacaccat ttctctgccg
ggaagaacaa atacacagag agagagtgta 1980ttgtgcactg atatcgagca
2000142000DNASorghum bicolor
14tggcacataa gtaaaattgc tgacttggta gggtcattta atgaaggaat ttcatgagat
60gagagagaag tttcatcccc atgaaactca tatggctcgg ttacttggta actgtgtcat
120caaactatgc attgagactg gcctgcatgt ttcatgagag tgtcatgcac attaaataag
180atatcacata agcaaaattg ctgacttagc tgggtcatta aatgaaggag tttcatcaga
240tgagagagga gtttcatctt cataaaactc ttgtggctcg gttacctagt ttttagtctc
300ggtaactgtg tcatgaaact ataatacatg ttttatggaa gtgtcacgca tattataggg
360tgccacataa gtaaaattgt taacttgaca gagtcattaa atgaagaagt ttcatcagat
420gagagaggag tttcatcccc ataaaattca tgtggctcag ttacctagtt tatagtcttg
480gtaactgtgc catgaaacta tgcattgaga ctagcctaac aaggtgataa ggccagtctc
540aatgcatgtt tcataagagt gtcatgcaca ttaaataaga tgccacataa gcaaaattgc
600tgacttgaca gggtcattaa atgaaggagt ttcgttagat gagagaggag tttcatcccc
660atgaaactct tatggctcga ttacctagtt tatagtcttg ataactgtgt catgaaacta
720tgtattaaga ctggagactc aaatacaaat tgtatatagc ctatggctct atttgtttcc
780gcgaccggcc aacccgtgac cacgattaag caaacacgac tcgagatcgt gtatgtataa
840atttggatct tcggtggttt aattttagtt tttttagcta agagaattta tatacatttt
900ccattattaa accatgtttc aaggtttcta attccatgct ctaaaataat atattatttt
960atggaaaata gatatttttg aaattaaatg tatgtataaa ttttcaagac tatataaaat
1020aataaaagct ctaatctatt gatgtagttc aatttttatc tccctagagc tgatgtggca
1080ccacatacgg tgccacctag gttaaaacta acctcaaaac ccggctaggt ttgtgatttg
1140cctcgtattt gatagtttag ggtgtactcc gtataagatt atctgtatat tcagagatta
1200gaaaatcaaa cacatctact aaattttaga acaagcatca ccctgtttgg cggctagtcg
1260taaaagtaac tgatgttaat ttattgtgag agtaaaatac tgttgtttga taataaaact
1320tttacttata atttcaagct aatagagccc ataaagacaa acctgtctca caagttagaa
1380ttcttacaac ttttcgtcta atcacatcga atatttgaac acatgtatat agtattaaat
1440ataataaaaa tatttaattg tacagtttac ctatatttac aagacgaata ttttaaatat
1500aattagttta ttattaaaca ctaattacta taattataaa tatactacaa tataaaaaaa
1560aaactttcgt ttttgcgagc taaacaccgg gacacaaaac gctcctgtct cctgggcggc
1620ttgggcctgc cagagcccgg gaaccgtgag cccgtgaccc cagcgggccc aaccagtcag
1680acgagagtcg aaaggcgtgc gcactacccc gagaaacgtg cgacaggaac ctcccccttc
1740ccggcctggg agcggcctgg cgagtggcga ctggcgtctt ccgcggttcc ccccagttcg
1800cctccttcac tgccgcccgc gcgccccgac acgcctgaaa aaccccacct ctcccctccg
1860ctccgcctct ctcgccctcc acttcccacg ccccacgccg cctgccattc cagttccagc
1920gtggactcga cgccagcgcg gagacgcgcg tctcgaagca ctagcccccc gttgttctgc
1980cgcgccggcg cgccggcgcc
2000152049DNAArabidopsis thaliana 15catatgaaac atttcctgta gcagtgagat
ggttacaaga ggatccatac ccgagtgctg 60tgcaatcaga caaactacaa gcatagtcga
tattatcagg caagtcatcg aggttatatg 120cattagggtc aagaatacac caagttttgg
gcagatactt cacatcttcc acaggaacca 180aaggcttgtc atttccttta cctgacaagt
caagctcata tttcggtctc ccatcaaact 240caaagatccc ccagtgcctc tcaaaagtcc
ccggtgctat actcttggcg tcctcatcaa 300caaggctgaa aaggtaaaca tccataatca
ctcctttcct cgcaggcgtt ccatttccag 360acatagcatg ctttaccatt ccctgattga
atctctttgc actctttaca ttagcgttct 420tgtctccatc cgtaggccac ccgacctctc
ctacaatgat cttcatcccc aagaagctat 480atctctccat agcacaaatc aaagtgtcga
gattcgcatc aaacacattg gtgtagacca 540aatttccatc tctcaaagac ttgttagtcc
catcaaagaa ggcaaaatcc aaaggaaagt 600aagcatttcc atagagacta agaaaagggt
atatgttaac cgtgaaaggc gaatcatgtg 660aatacaagaa attgattatc tcaatcgttg
catcccttag ctcaggtcta aagtctccag 720ctgatggaac agggttcgct tcaggggaaa
aatagatgtc tgcgttgaaa ggaacagtga 780ctttcacatt tttcagatca gcttcctcta
gtgctcgttg gatgttgata agagctggta 840atgtgaactc aacgtaagtt ccattatatg
tctgaaggaa aggctcgttt ccaacagcta 900tgtacttgat gttgactcca ccgttgtaag
aataagcagt aacgttttct tcaacccatg 960aagctgctac agatgtatct tgagccattt
ctttaagaaa ccggtttggt attcctatca 1020tgacttcaat gtctgagcca attagagcgt
ctaagatgtt ttggtcggct tcaaatagtt 1080tcagcttagt gaaactattg tccattagca
tcttcacaac cttttctggt ggaagctggt 1140gactcgccat tattccccag ttaactccta
cattactcgt gttgcttgag gcaatggaaa 1200cttgtgagat gataagaaag taacaaagaa
taatctgatg attataaaag tggtttgttg 1260ttaactttga tctctctcct gccatttttt
tctctgttta tgagtctttt cttctctttt 1320ctttatggag tctttgttaa gggagaagat
gaaatgtgat tggatatttg tgatttgtac 1380ttagttcagt taaagaagca gacacaacat
gcaaaatagc cattggtgaa acactttgtg 1440catgcctatc tgataaatcc attgactcac
cacaaattct tatgtaattc tagatgtttc 1500gtatttgttg tgccaaacaa acacacacac
tcacacactg cactgagtct agacatttag 1560tggttttgtt ttcttattat taatactcat
tagagtatta agtttgtata gaattcagaa 1620acaactgata gtcattttaa gatttctaat
tacaaaactt ttgatcctct ttgaaaagca 1680gagaaattac aatctttaca aacaaaactg
agagattaga gatgtgttca tagagatggg 1740ttctttgtta gacattccaa aaagatacaa
aactagccga tgattaattt tggtaaatta 1800atgaacaaga atgtaatttg aaacattata
gggagcaaat gagaaattac tctttttaaa 1860aggctaaaat cctaattacc tttaaactaa
gaagacaaga agagaagaga aaacatgttt 1920tccattagag gactgtgaga ttgtgaattg
catagtcgtc gtcttctggc gggaaaagaa 1980gccctagaaa aagggtgaaa ggtgaaaact
ctacttcttc ttcttcttct tcttcagagt 2040gtgagagag
204916946DNAArabidopsis thaliana
16aaaagttgaa acagcaaagt agcgatagat ttcgtgaaaa cagagaagcg gacatatctt
60gaaacacatg gcagcgattt ctccatggtt atcttctcct cagagctttt cgaatccccg
120cgttaccatt acagattcca gaagatgttc atcaatttct gcggcaatct ctgttcttga
180cagctccaac gaggaacaac atcgaatttc gtctagagat catgttggga tgaagagaag
240agacgtcatg ttacagatag cttcctctgt tttcttcctt ccattggcca tttcacctgc
300atttgcagag acaaatgcat cagaagcttt ccgtgtgtac acagatgaaa cgaacaaatt
360cgagatatca atcccacaag attggcaagt cgggcaagca gaacctaatg gattcaagtc
420aatcacagct ttttacccac aagaaacttc aacttccaat gtgagtatag cgatcactgg
480actaggtcca gacttcacca ggatggaatc attcggaaag gtcgaagctt tcgccgaaac
540attggtcagt ggattggata gaagctggca aaaaccagta ggagtgactg caaagctaat
600cgatagcaga gcttctaagg gattctatta catcgagtac accttacaaa accctggaga
660agctcgcaag catttgtact ctgcaattgg aatggcaaca aacggatggt acaaccgttt
720atacactgtc acaggacagt ttacagatga agaatctgct gaacaaagct ctaagatcca
780gaagacagtc aagtctttca gattcatctg agaatgtcat tcatatctat cagcggaact
840aaattataga attgatcaaa caatttgttt actgaacaat tacttttttg caatgaaatt
900ctgagaaaag agcctactcc atactttgaa gtaagcttca gtaaac
94617807DNAArabidopsis lyrata 17tccagaagat gttcatcaat tcctgtagca
atctcagctc tagacagctc caacgaggaa 60caacatcgaa tttcgtctag agatcatgtg
gggattaaaa gaagagaagc catgttacag 120atagcttcct ctgttttctt ccttccattg
gccgtttcac ctgcatttgc agagacaaat 180gcatcagaag ctttccgtgt gtacacagat
gaagcgaaca aattcgagat atcaatccca 240caagaagatt ggcaagtcgg gcaagcagaa
cctaatggat tcaagtcaat cacagccttt 300taccctcagg aaacttcaac ttccaacgtg
agcatagcga tcactggact aggtccggac 360ttcaccagga tggaatcttt tggaaaggtc
gaagctttcg ctgaaacact ggtcagtgga 420ttggatagaa gttggcaaaa accagcagga
gtgactgcaa agctaatcga tagcagatct 480tccaagggat tctattacat cgagtacacc
ttacaaaacc ctggagaagc tcgcaagcat 540ctgtactctg caattggaat ggcaacaaac
ggttggtaca accgcttata cactgtcaca 600ggacagttta cagatgaaga atctgctgaa
caaagctcca agatccaaaa gacagtcaag 660tctttcagat tcatctgaga atgttattca
tatctatcag cggaactata ttattgaatt 720gatcaagcaa tttgtttact gaacaatcac
ttttttcaat gaaattctga gaaaagagcc 780aactccatac tttgaagtaa gcttcag
807181054DNACucumis sativus
18ggagcgcatt gtacaaagaa aatccatctc taatctttga gtggactaca agcatggcga
60tggcgtccct tctttcaccc agcgctgtaa tcctacgccc tcactcattc cgcttctcac
120aatcatcact ctccaatgga ttctccatta ttcctatccg ctcaacactt cgtgttttct
180gctctgccaa tggcaacagc atccacactt ctaacaaaaa cccagttatt tggcgagcgg
240ggtcaacaga cgagaaatta tgctagggat tggattcact gcattttcat ttcaagaagt
300tgtttctaat gccctagctg agagtgttgt ggttgctgaa gattatcgga cgtatacaga
360cgaagcgaat aagttcagct tggtgattcc tcaagattgg caagtgggta atggtgaacc
420gaatggattc aagtcggtta cggcattttt tcctcaagaa acttcaactt ccaatgtcag
480tgttgtaatc tcggggcttg gtcctgatta cacgaggatg gaatcctttg gcaaggttga
540ggaatttgct gatacattgg tgagtggact ggacagaagc tggaaaaggc caccaggtgt
600ggcggcgaaa cttatcgact gtagatcatc taaagggata tattacatag agtacacact
660gcagaatcca ggtgaaagcc gcaaacattt atactcagca attgggatgt catccaatgg
720ctggtacaat agactttaca ccataacagg acagtatgca gatgaagaat cggagagcta
780tagctccaaa atcgagaagg ttgtcaattc cttcgctttc atttgatgat tgccacagaa
840ttggcctcca ccacactatc ataatggtta aatgttttcc acatctctct ctaattatag
900ttctcttttg ttattattat tattattatt ttttgtaatg agttctaaac ataatattga
960attgtctttg atgcatctat atttttacat tttcacgagg aatgaattca catttctatt
1020aattcataaa agaatccaca aaacagaaaa aaaa
105419795DNARicinus communis 19atggcttcaa tttctttact ctgttgcaat
tgctacttca catctttctc caacaagaca 60cctctccatc ttttgaaacc taacttaaac
ttcctctctg cttcaccttc ttttcgattt 120aacagttgca gaaagcaaca tcttccatgt
tgcaccaact ctttcccaga cgaagaccaa 180caccaaccat tattctgtcg ttttaggctt
caagaaccat atggaagaag agaagctttg 240ttcagcgtgg catttaccac tgggtttact
tttccagggc ttatttctaa tgcatttgca 300gagattgatg acttccgcct ttatactgat
gatgccaaca agttccaaat atcgattccc 360caagactgga gagtaggtgc tggagaacct
aatgggttca aatcagtgac cgctttctac 420ccagaagaag cttcaggctc tagtgtcagt
gtagtgatca caggactcgg tccggatttt 480actagaatgg agtcttttgg caaagtggaa
gccttcgccg aaactctggt tagtggattg 540gacagaagct ggcaaaggcc cccaggcgtt
gcagcaaaac ttatcgactg taaagcgact 600aaagggattt actacattga gtacacatta
caaaacccag gcgaaggtcg caaacatctg 660ttttctgctc ttgggatggc tttcaatggt
tggtataaca gactgtatac agtgacaggg 720cagtttgtgg aagaggagtc agagaattat
ggatcaaagg ttcagaaggt tgtttcatca 780ttcaagttca tctga
795201028DNAVitis vinifera 20caatcaaaaa
aagcatggct ctgtattttc cacttcctct ccgttctggg tcctgcgact 60tctcagctta
ttcgagtaaa aaaggttatg ggtcaagaac cgggaaatgt ggaaaaaagc 120aacgtgttgt
cttctgcaag aatgagaaca aggaagaaga aaaaacaagt tttgggatta 180aagaacaaca
tggaggtgga agaagggagg ttgtgctaca gatggtgttc agtacaattt 240cccttcaggc
aattgttcct aacgcactgg ccgatactga ggtgccagag gatttcaagg 300tttactcaga
tgaggtcaac aagttcaaaa tacagattcc ccaagattgg caggtgggtt 360caggagaacc
aagtggattt aaatcagtga cagcattcta cccagaagaa gcttctggtt 420caaatgtcag
cgtagttatc actgggcttg gcgcggattt taccagactc gagtcttttg 480gcaaagttga
tgcttttgca gagaatctgg taaatggatt ggatagaagc tggcaaaggc 540cccctggtat
tgctgcaaaa ctcattgact gcagagctgc taatgggttt tattacattg 600agtattggct
tcagaatcct ggggaaagtc gtagacattt attttcagct gttgggatgg 660caaacaacgg
ttggtacaac aggctttata ctgtgaccgg acagtatttg gaagaagaat 720cagaaaaatt
cagttctaaa attgagaagg ttgttgcatc cttcaggttt atttgaagaa 780aaatttgcat
gttcaggata taaactgagg ctgaagatta ctggttcagc aactctgtgg 840atttcacaat
gcacacgaat tggcattgtg caaaaagatg agatgattta tatactcaga 900ttgcatcagg
tgtcttttgt tgtaaaattg taaggaaggg gaagggaaat tatctctatg 960ctaccattga
aaattttttc cacacctttg cagttgcttc acattcattt gcagaattga 1020tggatgag
102821726DNAMedicago truncatula 21atggcatcca tttcatggtt cagctgtcta
cacatccgac caacagccac tgccggcgac 60aaaggtttat catctcccat aaccgtggaa
catcataaaa caagaccaca aaatttactc 120tcatcctcgg aagaaggact tgcgattaat
agaagacaac taattcttta cacatccact 180gcagcaattg cagcttcatc tactgactca
aatgcattgg cactcaatga tgtatctgag 240gattttagta tctacactga tgatgagaac
aagttcaaga tagatattcc acaagagtgg 300caaattggaa caggagagtc tgcagggttc
aaatcattaa ctgctttcta cccaaaagag 360caatctaatt ccaatgtgag cgttgtgatc
acaggagtgg gtccagattt cactaagatg 420gaatcattcg gcaaagttga agaatttgct
gacactctgg ttagtgggtt ggatagaagc 480tggaaaaaac cacctggtgt ggctgctaaa
ctcatagatt gtaaatcatc taaaggattt 540tatttcattg agtatacgct gcaaagtcct
ggtgagggtc gcaaacatct atattcagct 600attgggatgt taacaaatgg ctggtataac
agactgtata cagtgacagg acagtatggg 660gaagaggaaa cagacaagta tgcttccaaa
attcagaagg cagttcgatc gtttaagttc 720atataa
72622579DNAPopulus trichocarpa
22ggaactaaaa gaagagaagc tttattcaat atggtattta ctgcttttac tttccctgca
60attgcctcta ctgcattggc agccacaggc gtggcagagg attcacgtgt ttataccgat
120gatgcgaaca agtttaagat atctattccc caaggctggc aagtaggtgc aggagaacca
180agtggataca aatccgtcac tgctttctat ccagaagaag cttctaattc aagtgtcagc
240gttgtgatca ccgggcttgg tccagatttt actagattgg aatcatttgg caaagttgat
300gcctttgctg agactctggt gggtggattg gacaggagct ggcagaggcc cccgggcgtg
360gcagcaaaac ttatagactc taaagctgct aatgggcttt actacatcga gtatacgctg
420caaaatccag gcgaaagtcg cagacatttg ctttcagcac ttggagttac attcaatggt
480tggtacaaca gactatatac ggtgacaggg cagtttgtcg atgaagaatc agagaaattc
540ggcaccgaga tcaggaaggt atatcagaac tcttcattt
57923951DNALotus japonicus 23ggaatggcgt ccatttcctg gtcttgttgt ctgcgttggc
gaccaacaat atccgaccgc 60acagcctctg cggccgacaa aggtttctca cctcccataa
cattggagca tcataaaaaa 120acaccatgtt tactatcagc acgcaattcc tccattgaag
aaggacatgc ggttaacaga 180agacaacttg ttttctacac gtcactagct gcatttgcag
ctgccccatc tactgtcctg 240aaggcattgg cactcaatga tgtggttgag gatgttcgta
tctacattga tgatgagaac 300aagttcaaga tagagattcc ccaagattgg gaagtaggaa
caggagactc tagtgggttc 360aaatcattaa ctgcattcta ccccaaagag gcatctagtt
ccaatgtgag tgttgctatc 420acagggttgg ggccggattt cactaagatg gagtcgtttg
gcaaggttga tgagtttgct 480gagactctgg ttagtgggct ggacagaagc tggagaaaac
cgcctggtgt agctgctaaa 540ctcataaata gtaaaccatc taaaggaatt tattatatcg
agtactcgtt gcaaaatcct 600ggtgagagtc gcagacatct atattcagct atagggatgg
caacaaatgg ttggtataac 660agactgtata ctgtgacagg acagtatgtg gaagaggaaa
cagacaagta tgcttccgaa 720attcagaagg cggtcacatc atttaagttc atataaagaa
atgctcatga tgaaggagaa 780atttccccac agccatcttt cctatataaa tacagatttg
tgccttccta cagtgtagga 840ttcttatgag caagagagga ttcttatatt tgtctttatg
agcaaaatgg aatacttcat 900tatttcattc ctctcttatg tctcttgctc ctcagattat
gtatattgta t 95124866DNAFragaria vesca 24atggcttctg
ttgcgtcttg gcatcctctg ttccttcgac ctcgcacatc ccatttcacc 60acgacctcct
acaacacagg caccgccata tgtagaaaga gctatctgca atgttgcaac 120aacaaagaac
aagaaccaca accagaacaa gaagaaaaat cggtttttgg gatgcaatgc 180caagccaaga
gaagacaagt tttgcttggg actacttttg ctgcattttc ttttccggaa 240atttattcca
acattgcatt ggccgagaat gacgattttc gtgttttcac cgatgatgtc 300aacaagttcc
agatatcaat tcccctagac tggcaagtag gcgcagggga accaagtggg 360ttcaagtcag
ttactgcttt ttacccggaa gagggatcta gctcaattag tgtcgtaatc 420acggggcttg
gtccggattt tacgaagatg gaatcctttg gcaaagttga cgaattcgct 480gagactctgg
tcagtggact agataggagc tggcaaagga cagcaggagt tgcagcaaaa 540ctcatagatt
gcaaatcatc taaagggatt tactacattg agtattcgct acagaaacct 600ggtgaaagta
tcaagcacct ctattcagct cttgggatgg caaacaacgg ctggtacaac 660agactatata
ccgtcactgg ccagtttgga gaggaggaag cggataaata cagatccaaa 720attgagaagg
ctgtaaaatc cttcaagttc atatgataaa caacctccag aggggcagag 780tttgaattgt
gaactacggt ttaccaattt tgattgggtc agttgtacac aaatttttca 840tcgtaatcta
atgtaataca tttgaa
866251015DNAGlycine max 25gtgaataata atcagaacaa aaccaaccat ttaaaaaaaa
aaaaaaaaga aatggtggca 60gagaagctga tctgaaagga atggcgttca tttggcggtt
ctgtggtgtg tctctatgca 120acttcacagc ctctaatgcc cagaaaggtc cttctccttc
tctgcccata accttggact 180tggagcatca tataacaacc ccatctttac tttcttccat
cgaagaagaa gaaggacgcg 240cggttaatag gagacaactt attcttcaca cgccagtagc
tgcagcagct gcatttgcag 300tcccaaatgc attggcactc aatgatgtgt ctgaggatgt
tcgtgtctac actgacgatg 360agaacaagtt caagattgag attcccgaag agtggcaagt
gggaacagga gacggagaat 420ctagtgggtt taaatccata actgctttct acccaacaca
ggcatccaat tccaatgtga 480gcgttgtgat cacagggctg ggaccggatt tcaccaggat
ggaatccttt ggcaaagttg 540acgagtttgc tcagactcta gttagtgggc ttgacagaag
ctggcgaaaa cccccgggtg 600tggctgctaa actcatagat tgtaaatcat ctaatgggat
ttattacatc gagtatttgc 660tgcaaaatcc tggtgagagt cgcaggtatt tgtattcagc
tattgggatg gcatcaaatg 720gttggtataa cagactgtat accgtgacag gacagtatgt
ggaagaggac acagacaagt 780atgcttcaaa agttcagaag gtagttgcat catttaggtt
catatgaaga aaatggtcat 840gacgaggaag aatttttatc acagcacttc atctattcta
tttcattatg gattttcctg 900gcattgttct ttaagctaga tatggcattc tagatcggac
tggtatgata aaaaccatga 960catttccttc gagattgttg aatgaaagta atatacttag
tggccataat tgaca 1015261159DNASolanum lycopersicum 26ggctgtgaat
tggatacacc aatatctctg cttcttcaaa gaaaacaaaa aaaataaaaa 60cagaaatggc
gactctttca tcttcatctt catcttcatc ttcatcttca tctccatgtt 120tgaaccagta
ccagtatcaa gctattcttc gcttgccacg tgtcccttta atttcctctc 180atcttcttaa
agttcccaag aaaaatcgaa actcacttat tttctgctgc aacaacactg 240tgcctgattc
aagaacaggt gagcaagtta aaggagaatg cttaaccaag agaagagagc 300tcctgctaca
ggcaggctct gttgcatttt ctctgtccgc ctttacatcg attgcattgg 360cagagaagga
tgtcccggag gagtttcgtg tttattcaga tgatgtcaac aagtttaaga 420tcatgatacc
tagtgattgg caaataggcg cgggagaagg tgatggagta aggtcactct 480tagctttcta
tcctccagaa gcttctaact caaatgtcag catagtaatc acaagccttg 540gtgctgattt
caccaagttg gaatctttcg ggaaagttga tgcttttgct gagaatctgg 600tcagcggatt
tgatagaagc tggcaaaggc ctccgggagt gaaagcaaaa ctcatagata 660gcaaagcttc
taaagggttg tattacatcg agtacactct ccaaaatccc ggtgaaagtc 720tcagacatct
attttcagtg cttgggatag caaacaatgg gatttacaac agactgtata 780ctctcactgg
acagtttgta gacgaggagg cagagaaata tggtgccaaa atacagaagg 840ctgtttcttc
tttcagatta atatgatgac atgaacagag agcgcgatat cgcaaatttt 900ggcttgagct
tctggttttt ctcgtttggt gaatggtaaa cataattgag agcgcgatat 960cacagattca
agttctggtt aaggtatatt atgacgactc gagaaaaaac tggagttgta 1020agtatgaact
agcaacttga tcaatgttag agttagtatt tgcatatatc gttatatacc 1080aaaactgtat
cgattttttg ataaaaatat gaccttagtg caaataattt gatgctcaag 1140ttttgattat
atatttgta
1159271680DNACicer arietinum 27tctcgtaaaa gaatgagatt cagttgaagt
tatcgaagaa gaagaatgca gcagaggaaa 60aggcaaagta tggatgggcc tagcatctat
gcgtggttgt tttgtgtcat tggaatgaca 120tcacttttcg gtgctgcttt cctaccacca
gatataggga ttgtgttttt tctcagacaa 180atatgtctgt ttgtgtttcg atctatgtgg
atgacaaggt tggtgttttt tctagctgca 240gctgcacatg tcattgaggc tatctatgct
tggtgcttgg ctagaagact ggatccttcc 300aattcaaggg cttggttttg gcaaacattg
gctctaggct ttttttcatt gcgttttcta 360ttgaaattga aaagatcaaa ggaataactt
tgaaataatc acttcttgta ccattgttat 420tttcagaaat caattatctt tcccatcaat
tgtatgcctt ttttttatgt aaaattaata 480tttttacaag ttgttattag ttatcactgt
cgaataaatt taccatgaca gtacatatct 540tttaattttg aaaattgtcc attttgtttt
cttatctata atggttttct tctctattta 600attttggatt taattaaatt agaaatagtg
tcttctctgt ggaggataaa agtgtaatta 660aatagataat aataataaag gaaatgaagg
gtgagaaaga gaagctgatc taaaaaggaa 720tggcatcaat ttcatggttc agctgtttac
acattccacc aacatcctct gctgccgata 780aaggtttatc atcatctccc ataaccgtgg
aacatcataa aacaacaaca cgtttaatct 840cttcctttga aggacaacaa catgttgtta
atagaagaca actgattctt tatacatcca 900cagcagcaat tgcagcacta tctactgtcc
caaatgcatt ggcactaaat gatgtgtctg 960aggatgttag tatctacact gatgatgaga
acaagttcaa gatagaaatt cctcaagagt 1020ggcagatagg aacaggagag tctgcagggt
ttaaatcctt aactgctttc tacccaaaag 1080atgaatctaa ttccaacgtg agcgttgtga
tcacaggggt cggaccggat ttcactaaga 1140tggaatcatt cggcaaagtt gaagaatttg
ctgacactct ggtaagtggg cttgacagaa 1200gctggaaaag accccctggt gtggctgcta
aactcataaa ttgtaaatca tctaaaggat 1260tttattacat tgagtatacg ctgcaaaatc
ccggcgagag tcgcaagcat ctatattcag 1320ttattgggat gtcaacagtt ggctggtata
acagactgta tactgtgaca ggacagtttg 1380tggaagagga aacagaaaag tatgcttcca
aaattttgaa ggcggttgca tcgtttaagt 1440tcatataaag aaatgcttgt gacgggagag
aaatgttctc attggtttct ttcatgggct 1500gccgtttaat gttttcatga cattttttgt
aagctagaaa tggcgtctaa atgttataaa 1560tatgatattt gctatggtac ttccttcaaa
aactgataaa ccagagtagg ctaaatgaat 1620ggcacaaatt gatgtaatgg ataagatatt
ttgcagtgaa tatcagcacc ttcagttaaa 1680281068DNATriticum aestivum
28gaagcacagc agcgtcgtcg accaccatcg agccgtactc catggctgcc gtgaccaccg
60cctctctctg tcccggcctc ggcaagcccc gccgagacca cgcgaagcca ccgagaacca
120cggtctgcca ttgcctccct gctcggagga cggaggaggg ggtgaagcgg cgggacgccc
180tgctcggcgt cctcctctcc gctaccgccg cgtcgtcggc gccgctgctc gtccccgccg
240aggctttcgc cgaggtcgcc gatgcgcagg aggggttcac cgcgtacgag gacgaggcca
300acaagttcac cctcgtgatt ccacaaggct ggcaggtcgg cgcaggtgaa cgcagcggct
360tcaagaacgt gacagcgttc ttccccgagc aaaaccccaa ctccagcgtc agcgtcgtga
420tcaccgggat cgggccggac ttcaccagcc tcaagtcctt cggtaacgtc gacgagttcg
480ccgaaaacct ggtgaccggc ctggacagga gctggcagag gccggcgggg ctcgcagcga
540agctcatcga ctccaaggca tcaaacggct tgtactacat cgagtacacg ctgcagaacc
600ccggcgagaa gcgccgccac atcgtctccg ccatcgggat ggcattcaac ggctggtaca
660atcggctgta cacggtgaca gggcagtaca tcgacgacga cgaggagtca gccatataca
720aacctgagat agagaagtct gtcaagtcgt tcaagttcac atgaaatgcc cccaaaaagg
780aagttcaggt gagaacaagt atagagtgac agagaagaga gagtatacaa agctagtagc
840tcctgatgtc aagttcaatt agtgagtatg catatgtttg tcgaatttac cggaaagaaa
900agatgaacac cagatgttcg aagacttcga tggcgtagct tggctgagaa cagcattggc
960agcatgagtg tgagatagag catgagtgtc gttggttcta agaaaattgc tagaactctg
1020ttacaaggaa actaaaattg ctctgatgta aaaaaaaaaa aaaaacga
1068291037DNAOryza sativa 29gcaaggcagg cagcagcgac cagacgaagc agagagagcg
cccgcgccgc gccggccatg 60gctgccgccg tgaccaccac caccaccgcc acaaccaccc
atctctgccg tggcctctcc 120tcctcctccg ccgccgccgc caagccgcgg cgagcgacga
cgctcagatg cggcgccgct 180gctcgggtgg aagggctggg gcggagggag gcgttgctcg
gcgtgctcct ctccacggcg 240acggcggcgt ccgcgcccgt cgccgccgtg gccgcgaccg
ccgagttgca ggaggggttc 300cgcacgtacg aggatgaggc caacaagttc agcatcgcca
ttccacaaga ctggctgatc 360ggcgccggcg aggtcagcgg cttcaagtcc gtcacggcgt
tctaccctga ccaagtcgcc 420gactccaatg tcagcgtagc catcaccgga atcggccccg
atttcaccag cctcaagtcg 480ttcggcgacg tcgacgcctt cgcagagacc ctggtgaacg
gcctggacag gagctggaaa 540cggccgccgg gggtcgccgc gaagctcatc aactccaggg
cagccaacgg gttttactac 600atcgagtaca cgctgcagaa ccccggcgag cagcgccggc
acattgtctc ggccatcggg 660atggcgttca acggctggta caaccggctc tacacggtga
caggccagta catcgatgag 720gacggggatg tagacaagta cagggctcag atagagaagt
gtgttcagtc attcaggttc 780acatgaaaga ggagcatcct acacaacatc caacaaggcg
aggacgaaaa acattttgta 840aaccaacgta tttcgttata attgtaaatc aatcagtata
ttcatgtcat cagttcaacc 900aactaaatgt acaccaattg ttccgagatt ttgacgatgc
ggccttgccg aggccaacat 960gagctaatta tgttgtggca agtcataagc attgttttct
atgcattttt aagggagaaa 1020aaacaggtgt atttgtt
1037301269DNABrachypodium distachyon 30atggcccggc
tcggcccggc ccacgtgttt tacagccgtt gggctgggcc gtactgccaa 60aacgtgtcca
aatggcccac gtcgaagacg aactctcacg ggccgacgtc cttggcgcgc 120ggccatggcc
catgggtaag taagatacga gggggccgaa gcaatgtgcg gcggaagttc 180ccggacgacg
acacgacggc cggccaccgt acaaagcttc tcagcaaaat atcctccccc 240tcgaaagcca
ccagcgcagc agcagcgcag agccattcgc tcgccatggc tgccgtgacc 300accgcctcct
ccgccatctg ccccggcttc agcagcaacc cccgccgagg ccacgcgaag 360ccgcggagat
ccacggcctg ggcctgccat tgccgccgct cccctgccct gcgcgagcaa 420caacctacgg
cggccgttgc cgggacggcg gaggaggggc tcaggcggag ggatgccctg 480ctcggcgtcg
tcttctcggc cggcacggcg acgctgctcg ctagtcccgc cggtgctctc 540gccgaggccg
ctgccgaggt gcaggagggg ttcagcgagt accaggacga ggccaacaag 600ttcagcatcg
tggttccgca aggatggcag atgggcgctg gtgagggcag cggcttcaag 660aacgtcacgg
cgttcttccc ggacaaggcc gccgactcga gcgttagcgt ggtcatcacc 720gggatcgggc
cggacttcac cagcctcaag tccttcggcg acgtcgacgc cttcgccgag 780aacctggtga
ccgggctgga caggagctgg cagcggcctg cgggggtcac cgcgaagctc 840atcgactcca
gggcgtccaa cggcatgtac tacatcgagt acacactgca gaaccccggc 900gataagcgcc
ggcacatcgt ctccgccatc ggcatggcgt tcaacggctg gtacaaccgg 960ctctacacgg
tgacagggca gtacatcgaa gatgacgagg agtccgtcaa gttcaagcct 1020cagattgaga
agtctgtcaa atcattcaag ttcacatgaa atgccttcaa aacaaaggtc 1080acatgaaaat
aagtactgct actacttttg aatgaagtac tatatctaag cagagaagag 1140aaggtatata
aaggcagctt ccggtaatgt gtgcagaacg aaatgaacta aacctttgtg 1200aatgtaaggg
ttgtgagctt tgagaatata tatgtttgtc aattttactg aatacatagc 1260tctagactt
1269311024DNAPhyllostachys edulis 31atagtgccat acgccatggc cgccgtaact
accgcctccc tctttcctgg cctctcctcc 60tgcagcccca agcccagaag ccacaggaag
ctgcagagaa cgacggtctg ccaatgccgc 120cctgctcgga tggaggggat gaaacggagg
gaggccttgc ttagcatcct cctctccact 180gccgcttcgg cgccgccgct tgctcctgcc
gaagctttgg ccgagaccac cgagttgcag 240gagggcttcc gtacgtacga ggacgaggct
aacaagttta gcattgcggt tccacaagac 300tggatggtcg gcgcaggcga gggcagcggc
ttcaagtccg tcacggcgtt ctaccctgaa 360ggcgccgact cgagcgtcag cgtcgtgatc
accggaatcg gaccggattt caccagcctc 420aagtccttcg gcgacgtcga cgccttcgcc
gagagcctgg tgaacggcct ggacaggagc 480tggcagaggc cgccggggct cgccgcgaag
ctcatcgact ccagggcagc gaacggtctg 540tactacgtcg agtacacgct gcagaacccc
ggcgaaaagc ggcggcatat cgtctcggcc 600gtcgggatgg cgttcaacgg ctggtacaac
aggctctaca cggtgacagg gcagtacatc 660gatgacgacg acgagccagg caagtacaag
cctcagatag agaagtctgt cctatcgttc 720aggttcacat gaaagaacta aactacagtc
tacccagagt gcaacaatat gcagagaaga 780taaagtagat aaaagccctt ccgcagataa
gttcagaacg gaagatacgt tgtgattttt 840gtcaatcagt gagcatatgt ttgtcgattt
gaccaaataa aatatgtact ccacatgttc 900gacgacttgc tgtgcccagc atgagttaat
tgtaagagaa gttaccatgc gccggacctg 960tcattctgaa actgtgatga gtgacattct
gaaactgtaa catagtaaac gtatgttcag 1020tttt
1024321053DNAHordeum vulgare
32agagccgtac tccatggccg ccgtgaccac cgcctccctc tgcccgggcc tcggcaagac
60ccaacgaggc cacgcgaagc cgccgagaac aacggtctgc cattgcctcc ctgctcggag
120gacggaggag ggggtgaagc ggcgggacgc cctgctcggc gtcctcctct ccgctaccgc
180cgcgtcgtcg gcgccactgc tcgtccccgc cgaggctttc gccgaggccg ccgaggcgca
240ggaggggttc accgcgtacg aggatgaggc caacaagttc accctcgcga ttccacaagg
300ctggcaggtc ggcgcaggtg aacgcagcgg cttcaagaac gtgacggcgt tcttccccga
360gcaaaacccc aactcgagtg tcagcgtcgt gatcaccgga atcgggccag acttcaccag
420cctcaagtcc ttcggtaatg tcgacgagtt cgccgagaac ctggtgacag gcctggacag
480gagctggcag cggccggcgg ggctcaccgc gaagctcatc gactccaagg cagcaaacgg
540tctctactac atcgagtaca cgctgcagaa ccccggcgag aagcgccgcc acatcgtgtc
600cgccatcggg atggcgttca acggctggta caaccggctc tacacggtga caggacagta
660catcgatgac gacgaggatt cagccatata caagcctgag atagagaagt ctgtcaagtc
720tttcaagttc acatgaaatg cctccaaaaa ggaagttcag gtgagaacaa gtatagagga
780acagagaaga gaaagtatac aaaactggta gctcttcatg ttaagttcaa ttagtgagtg
840tgtatatgtt tgtcgaattt accggaagaa aatatgaaca ccaaatgttc aaagacttcg
900atggcgttgc ttggctgagg acagcaatgg cagcatgagg gtatgagata gagcatgaga
960atgtcgtttg ttctgagaac attgctagaa ctccttataa gaaactaaaa ttgctccgat
1020gtaaacttct tcctagcatc tatttttggg ctc
1053331138DNAZea mays 33cccgttgcca cacatacgga tccaacaaaa tctcccgcaa
agacaacggc gagccaacca 60ccaccagcgt cccgcgctag ctgcggcacc gcatggctgc
cgtgaccagc accgcctcca 120tctgcccggc cgcagccggc gccctctctt cgctgccgtc
cttcatcacg cgcaagccca 180ccagcggcag caggaggttg cagcaggcag cagcgacgac
agtctgccac tgccgctctg 240ctcgggtaga ggaggggctg ctgggccgga gggacgcctt
attgctcggc atcgtcttct 300ccgccgcgac gccgccgctg ctcgcccctg ccggcgctct
ggcggacgag gccaccgccg 360agtcgcagga gggcttcact acgtacgagg atgaggccaa
caagttcagc attcaagttc 420cgcaaggctg gctggtcggc gccggcgagg ccagcggcat
caagtctgtc acggcgttct 480accccgagca ggccgccacc gactccaatg tcagcgtcgc
catcaccggg atcgggccgg 540acttcaccag cctcaagtcc ttcggcgacg tcgatgcctt
cgccgagggt ctggtgaacg 600gcctggacag gagctggcag aggccgccgg ggctcgccgc
caagctcatc gactccaggg 660cggcaaacgg cctgtactac ctggagtaca cgctgcagaa
ccccggcgag cgacggcgcc 720acatcgtctc ggccatcggg atggccttca acggctggta
caaccgcctc tacactgtga 780cgggccagta catcgacgac gatgactcgg agaagtacag
gcctcagata gagaaggctg 840ttggatcgtt caggctgaca tgaaagatgc gatgtcatcc
agcaccagca gcagcagccg 900cccacggtac ataaacccta aatatgtatg cggagaggtc
cagcaacatg ttgtgcccga 960aaattgacac cttgccattt cgatgagaca agacaaggca
tgtgcctatt gccctattcc 1020aattcttgag cactgtaaca ctgccaatat gcagagtata
tgttttctgc ctgttgaggt 1080ggatacaaat gcatgctttt ttttttatta ataactcatg
tgtaacactg ctgccttt 1138341162DNASorghum bicolor 34agcgcgagcc
tgccagccaa ccaccgccac cggctttcat cccgcgcgtg cgtcctgcgc 60tagctgcgcc
catggctgcc gtgaccagca ccgcctccct ctgcccggcc gcagccggcg 120gcctctctgc
ctcgtcgtcg tcgccgttca cgcgcaagcc cagcagcagc aggaggctgc 180aggcagcgtc
cacggcctgc cactgccgcc ctgctcgggt agtagagggg ctggaccgga 240gggacgcctt
gctcggcatc gtcctctccg ccgcggtggc gccgctgctc gcccctgccg 300gtgctctagc
ggacgagccc accaccgagt cgcaggaggg cttcactacg tacgaagatg 360aggccaacaa
gttcagcatt caagttccac agggctggct ggtcggcgcc ggcgaggcca 420gcggcatcaa
gtcggtcacg gcgttctacc cggagcaggc agccgccgat tccaacgtca 480gcgtcgccat
caccgggatc gggccggact tcaccagcct caagtccttc ggcgacgtcg 540actccttcgc
cgagggcctt gtgaacggcc tggacaggag ctggcagagg ccgccggggc 600tcgccgccaa
gctcatcgac tccagggcgg caaacggttt gtactacctg gagtacacgc 660tgcagaaccc
cggcgagcgg cggcggcaca tcgtctcggc catcgggatg gcgttcaacg 720gctggtacaa
ccgcctctac acggtgacag gccagtacat cgacgacgat gacgattccg 780aaaagtacag
gcctcagata gagaaggctg ttcgatcgtt caggctgaca tgaaagatgc 840catgtcattc
agcagaggtc ttgtgcctga aaattgacac cttgccattt ccatgagatg 900agacaagaca
agacatgtat gccaattctt gagcactgta acactgcaag tatgcgaata 960tattttctcc
tttttgaggt ggatataaat atgttttttg taactcttgt gtaacgttgc 1020tgcggtgttt
ttttggttgt gtatatgtaa tgtttagagg gtcgggctga aggagcaact 1080atgtgacctt
tattctcttt ttaaggcaaa gttcgtgtca cttcttttca aaacaagcaa 1140atggttttgt
ttcttgagct gg
1162351062DNASetaria italica 35cggtgcctac caaaacacac ggctggaaca
aaatatcccc cacgaaaaca aacggcgagc 60caaccgcaac caccactcgg caccggctgg
cctgcggcgc gcgccatggc tgccgtgacc 120agcaccgcct tcctctgccc ggccgccggc
ggcctctccc cctcgccgcc cttcaggcgc 180aatcccggca gcagcagcag ccgcaggagg
ctgcagctgc aggtctgcca ctgccgccct 240gctcgggtag aggggctgga ccggagggag
gccttgctcg gcgtcgccct ctccgccgcc 300gcgccggcgc tcttcgcccc cgcggctgct
ctggcggccg aggccaccgg tactttgcag 360gagggcttca ccacgtacga ggatgaggcc
aacaagttca gcattgtggt tccacaaggc 420tggctgatcg gcgccggcga gtccagcggc
atcaagtccg tcacggcgtt ctaccccgag 480caggccgccg actccaacgt cagcgtcgcg
atcaccggca tcgggccgga cttcaccagc 540ctcaagtcct tcggcgatgt cgacgccttc
gccgagggcc tggtgaacgg cctggacagg 600agctggcaga ggccgccggg gctcgccgcg
aagctcatcg attccaaggc ggcaaacggt 660ctgtactacg tggagtacac gctgcagaac
cccggcgagc ggcggcggca catcctctct 720gcaatcggga tggcgttcaa cggctggtac
aaccgcctct acacggtgac aggccagtac 780atcgacgacg aggagtcgga gaagttcagg
cctcagattg agaaggcggt tcgatcgttc 840aggctgacat gagagtgctt cgcactgtgt
agcattcaga gatgcacggt atgcaggtgg 900acgcctgtaa attgaccaac tcgtcttcca
cattattaag ttttttttta agcaagtctc 960acggtatgtt ggaaagtaca ttgctacacc
tcaacaatcc catagatcgt ctcatgtaat 1020gccacatata atttttgctg gtgtggagga
aggagggtga tg 1062361091DNAPicea glauca 36acttcaaaat
cccaaaccca tcagaggagg gagttccaac agatgactat cgtggtgaag 60atgggcatgc
ttggcctggt gtcatcacca tcaattccca ctactcacaa tctatgctct 120ccagtccaac
cattcagaag gaatttcaag aatttcaagc aggggaaaaa gcaagtcaca 180ggctgtttcg
aaatccagaa caatctgtcc tcccatgaat tgtcaagaaa tggaagaagg 240caggccatat
gtcagattgc tgctttgttc tcagcgattc cttgtactgt ttcagcggca 300agggcagcag
aaactgagct tcaagaagat tacgagttgt ataaggacga gacagacaaa 360ttttcactac
tagttcctcg agactggata aagggtgaag gaaaaacaga tggacagaga 420gcagtgactg
ccttctaccc tgaaagcggc atagtttcta atgtgaatgt aataataaca 480ggactttctg
ctgactatac aaaaatggaa tcatttggca ctgttgatgc atttgctgag 540accctggtta
attctctaga tagaagctgg aaaagaccgc cagggcaagc agcaaagctg 600cttaatgcaa
aatccaaaaa cggcttgtat tatatagagt attcattgca aaagcctggg 660gagagtaaga
tccatcttct ctctgcgatc ggaatggcaa tgaatggttg gtacaacagg 720ctttacactg
taacggggca gtatctagaa gacgatgctg gcaaatatgg ctcaaagatt 780gaaaagtcca
tttcatcttt cagattagtt tgaaagatta attaccttcc atgtgaggca 840tcaagtatgt
tgggaaaaga cttataatat acaagagcat aaaggtgata aatattaaat 900aattaaaaat
cccccatttt attcatcttc aattatgtct ggaataaact tgatttacct 960tgtaatatat
aatgtatgac ctaatatctt atttggaact aagtgtgaaa ccactcatag 1020ttattccaac
taaattttag tttagacaag ggaataaaat acattcaatg tccttattgt 1080ttactaaaaa a
109137355DNAArachis diogoi 37ggggtgggtc cggattttac taagatggaa tcgtttggta
aagtggaaga atttgccgag 60actctgattg gtggattgga cagaagctgg caaagaccac
cgggtgtggc tgccaaactc 120atagattgta aatcatccaa ggggttttat tacatagagt
attcactgca aaatccgggt 180gagagtcgca gaaccttata ttctgctatt ggaatggcat
caaatggttg gtataacaga 240ctctacacgg tgacaggaca gtttgtagaa gaggaaactg
acaagtatgc ttccaaagtg 300aaaaaggctg ttgcatcatt taggttcata tgaacaaaga
gttcatgagg gagat 355381089DNAPhyscomitrella patens 38gcaggagcaa
tggacgctgt cgttggtcgc acctcatgcc ccttgtctct gtcttcctcg 60tatcaatgga
ttgctgggtc gccatctgct tctcgtgcta cagtcgttgt tagaggtaca 120agccggcgtg
acggtaaaca caaagcagtg cgttgcgagc aggttccaga atgcagcacc 180agcaattgtc
aaacaatgca gagacgagag gttatcggtc aagctctatt agccatgtcc 240atgagctttg
ctcctccagc tcgttcggcc acagacacag atgctgctac tgaatttacg 300acttacgagg
atgcagccga taaattcaca ttgctcgtgc cacaagcctg gaacagaggc 360gaagggaaaa
cgtccgggca aaggaaagtc acggctttct atcctgcgga tggcggtctt 420accaatgtaa
atatagtcat aacaggactc ggagcagatt tcacgagttt aggatccttt 480ggcacggccg
acaatttcgc ggagaatttg gtgaacagtt tggacaggag ttggcagaaa 540cccccgggcc
agaaagcaag gcttgtggat tgtaaatcaa gagcagataa atactatgta 600gaatacacta
tacagagact cggagagcag cagcggcact tagtctcagt tgttgggatt 660ggaaacaatg
gatgggtcaa cagattatac accgtcacgg gccagtactt tgaggaagac 720tcagccaaat
ataaacaaga cattaacaag atcatctcct ctttcaaaat actgtagttc 780atagatcgaa
gactcggggc acagactgca gaccatggag tttatgactg accagcattg 840tggtaaaacc
tgggattttc gttcctcatg cttcgtgtca cagagaagct cattgagttg 900ggagaactga
agtggttgtg taaacacgct ggggttgttt tgcatgtggt gagagcagat 960gtccttcgag
cagccattca taaatctcaa gatgagttta ttctgttctg cagaaactgc 1020cgaacctgga
ttcttgtaat agaaccattc aataatttcc aaggtcacca ttgggcctga 1080gatatcatc
1089392425DNASelaginella moellendorffii 39atgggggagg agaagcgcga
gaatctcatg gagtcgctct tcggggagga gtcggatgac 60gacgccgatg ccgctgcctc
ctactctgag gaggagggcg aaggcgaagg cgaggcagag 120ggcggtggtg ccgagagcgg
tggcgacagg gaagagagcg agggcgagcg ggaagccagt 180gagggcgaca aggaggagag
cgagcatgag agagtgtcca ggaggagcag tgatgacgga 240ggtggtgaag aagggagcgc
cgccagcggc tctgagaacg atggacaagt ccaggaaagg 300gaagcaactc gcagcgagga
ggtggaggag cagcgtggtg ctagaattcg tgcagcattg 360ggtgattctg atgacgacga
tcaggaggaa ggaggagttg acggccctcg caagccatcg 420agtccagagg gggagggctc
ggacttggag ggaagctacc acaaagatag taagcggggt 480gcagaggatg acgaggagca
gtattactct gatgaagagc gcgcagaaat caagaaaccc 540aagggtgaac ctcttacagt
agaagttcca ctcaggcaac ctccaactca ggctcacaac 600gcaagtagtt tggcacttgt
ttctttttca ttatctcgtg agcttacggg cctttgcttt 660cagatgaatc tcgtcaggat
ctccaacatt atggatatag agcaaaggcc ttttgatcca 720aagacatacg tcgaagagga
tggatttgtt gatgaaactg gccgacgccg tatacgtata 780gaggagaacg tggtgcgctg
gagatacgtc aggaatcggg atggctcgcg ctcggccgag 840agcaacgccc ggtttgtgaa
gtggtccgat ggtagcatgc aacttcttct cggcaatgaa 900gttctggatc ttgctgttca
agatgggcaa caagacgaat cacacttgtt tatccgtcag 960cccaagggat tattacaagc
ccaagggagg ctggcacgta agatgagatt catgccttcg 1020tcgttgagat cgaagtcgca
ccgtctctta actgctctcg tggattctac gcacaagaag 1080gtgttcaaag tgaagaatgt
gatcacggac ttcgatcccg agaaggacaa agaacaaaaa 1140gagaaggcag cagaacagag
gattaaaagc aaagaagatc tccaaaaaaa gcaggagaag 1200acgatgcgta aatacccccc
tacacgggag agagaacctc agctttctcc tggatacctt 1260gaaggggcgc ttgaagagga
ggatgaagat tatgacgacg aggggcgtag caaccgacgg 1320taccaagacc agcttgatgc
cgaaagcagg gcggagagac gaatcaacca ggttaagcgg 1380caaccaccaa aggcaatgga
gcgacgccct tcttcgcgga gcaggaggga tattttggaa 1440gacgaggatt cagacgagag
cgaccgcaat agaggtgcgc gaggtgatga aggtgaggaa 1500gagcaggaag atgacgaggg
ggaggaagaa gaggaggaag tggaggcaag ggatagccgc 1560cgcaagagga aagacaggga
cagggaacag ctgcagcagc agcaaaattc gcctcccaga 1620aagcagcaaa cgcacaggcg
gagagcagta gtttggtttc tcgccacagg ttgcaagagt 1680agaatagata ggggcagcca
gggaagtgtg gcgtcatggc aagtgtgcat acaaactgct 1740atgttcgtcc agcagttcca
gccagttcaa gtcgaccgcc attcaatgct cctgctactg 1800ctgcctgtcc accgctgagc
aagagaaatc tcttattgct ggtgcctctg ttggcgatgc 1860cggcgacgcc tgtgtttgct
gctggtattg tacttgtcaa tcgttcgtta aactttgatg 1920caagacagga gcctcagatg
aatatcaagt atacgaggaa caagacaagt tctccctcac 1980tgtacccaaa gactggataa
aaggcgaggg gaaagtcgga tccagaagag tggtggcatt 2040ccatccatcc aaggctactt
tcccaaacgt gaacgtgatc atcacgaacc tgggtgccga 2100tttcaccggg atcggctcac
tgggatccgt ggactcgttc gccgcgagcg tggttggcag 2160catggataga agctacaagc
ggccgcctgg aacagcggct cggctggtga atgctgtgtc 2220gagaaacggc atgtattatc
tcgactacac cgtccagacg cccggggaag cccagcgcca 2280tttcttctcg gtggccggtg
ttggcgagac acagttttac aagcagctct acacggcaac 2340cggccagtac tgggaggctg
atggagacag ggacaggaaa gcattgcaag aggcgataga 2400gtcattccgg attgttcaca
agtga 242540832DNACamellia
sinensismisc_feature(90)..(90)n is a, c, g, or t 40atacccaaag ttgccaagtg
gggccggggg aaataaagga gatcaagtca gtaacggagt 60tgtacccgat gggacctctg
atgaaagtcn cgtggtaatc gcggcctggt gcgggtttta 120ccagaataga aatcttttgg
taaagatgat gcttttgcag agaatctgct acgggctggg 180acaagaagct ggcaaaggcc
accggggggt aagagcaaaa ctcatagact ctaaaactgc 240taatggtttg tattacattg
aatatacact gcaaaatcct ggccaaagtt gcagacattt 300atttcagtgc ttgggatccg
aacaatggtt ggtatccaga ctatataccg tcactggaca 360gtttgttgat gaggattcag
aaaaatatgg ctccaaaatt gagaaggctg tttcatcgtt 420cagattaaac tgagattttg
aggatccttt ccatttttgc tttcaacatc ggctctcatc 480gctgcaacat gtccaattga
agtcaagttt actaaaggaa gcaaagcatt gaatgatgtt 540tgcatcctgg ccgaggattt
ccatgcacct ggagccagct gtttggaaga caccaagagg 600cggctagatt gtggcatatt
tactctctgt tttacttttg ttattcctag cctttcttgc 660aacttttctt gaagtatgct
ggaaccttta ttatttattt gactaggaaa tttattctta 720ataccaccag aaagagatag
acaaaaacta ccttggtgat tgcattgaat taacataaag 780tgcccaaaaa tattttgcta
agaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 832412246DNAZea mays
41tttggtgttt ctcgcagtgc gcagagggct tcagtcatta tcgtctcgtg ctctctattg
60atggtagttt tttgcttggg aagtataggg gcacattgtt ggttgccatt tcatgtgacg
120tggacaacgc attggttctt ttggcatttg ctttggtggt gagggagaac agagatagtt
180cgttttggtt cttgcgactt gttcggatcc atgtcgtggg ccctggtcgg gagattggtg
240tcatatatga cagaaaccag ggtattctta atgtagtgca agagcagatt cctggctaca
300cacccatgca ccacagatgg agcactcgac acctagtaga aaatcttttt cgaaagggtt
360gtaccaagaa taattccccc ctttttgagg aggtctgtcg acagttggag gtttcgttat
420tcgaggataa gttgaaggaa ttaaaagata caacaaatga tgaaggtgaa aaatggattg
480ctagatttcc taactgtatt tcctcgtctc ctacacaaaa aacagtaagt acgaatgcaa
540cattacgact gatgctatca aaaagcaata tcaacctcat cgtaatctac cacatgtcca
600aaaataaccc acaccaagat cagaaggaac taatccatac ttagcttcat taaaaccgcc
660actcattttt tctttttctt tgcctctggc tcaggccaat gggacctagg accatacaac
720cactctatga aactacactt acgcccaccg tatggccgta ggggaataaa ttagtaaatt
780tgtgaaaaaa taacaaaatt ttggacattt gttatgcact cacataatta ctgttgggac
840acacgaagta cttcacagtg tccttacata agacaacacg atctccgcaa tcacaccgag
900gcggagactt cagtcgtctt tctgcgacta agttcttctt ctcatccgtc attggtgtcg
960tgtcggggga ggaggcaccc aacgcttaaa acactgtata ttccttctgg acaaccaatt
1020agcgaaaagg agatatcgat ggtcaaattt atgaggaccg tcgatccatt ggaaaacaca
1080tcaagtgatc ctacagaaat ggaatgaagc attagtacac atgtagtaaa catcaataac
1140acacaagtca taagctacat atgttaaaaa cgccacaagt gtagaagcag cgagcagccg
1200tgtctagata tttgaattga catacccaag gcggtctgcc acagtcacaa ttaggtacag
1260gaagatcagg aggaacatga gcatccttac tagatacatc cagatacaac tcccgaggac
1320gatctctctt ctgccacaac gactcctaat acatgtcttt tcatctaaca aacgattata
1380tattaacaca actatataga aataacagaa atgattttaa cttataaata catgtatttc
1440ataaattatc taacaaactg tagtcattat atgaaccata tatcatagga ttcataacta
1500atagcaatat gagcaacaat caaaactaat taatcgaaaa catacaacgc aatatacgta
1560acaaatgaat cagtgagaca ctataccttg gtcttcaaag ttttccaact cgaatacgaa
1620gattggagca cctctgggtg gtatggaaga agatacaaat ggttgggtgc agctctcggc
1680taggaaacaa ccgatttata ggccttccta gctcggcgcc acagatccat ggcgccgagc
1740tagtgccatg tcggtgctgc atcagccctc gacgctagcg gccttgtgtt accttggtgc
1800tacaacttat gatattgagc aacgagtcca aatgcagaaa taagttatct aggggtctaa
1860acattaaaca cgattttttt cttaaaaagg accaacataa aaaaaactcg ggctggcggc
1920ctgccagcgc ccggggaccg tgagcccgtg accccagcgg gcccaaccag tcaaaaccgc
1980aagagagagt cgtgaggcgt gcgcactacc ccgagaaacg tgcgacggga acctccgcgg
2040ttccccaagt tcgcctcctt cactactctc gcgccccggc acgcctgaaa aaccccaccc
2100ctcctgccgc tccgcctctc ccatcacttc ccacgcccct cgccgcctcc cattccagcg
2160tggacacgac gccactcgcc agcacggaga cgcgcgcctc gaagcactac tgcactagcc
2220agccgtcgtt cttccgcgcc ggcgcc
22464227DNAArtificialsynthetic 42tttttaggaa ttattgagta ttattga
274324DNAArtificialsynthetic 43aaataaaaat
catacccaca tccc
244426DNAArtificialsynthetic 44tgttgaatta ttaagatatt taagat
264525DNAArtificialsynthetic 45tcaaccaata
aaaattacca tctac
254630DNAArtificialsynthetic 46taagtttttt ttaagagttt gtatttgtat
304725DNAArtificialsynthetic 47taaaaataat
caaaacctaa cttac
254825DNAArtificialsynthetic 48attgtttatt aaatgttttt tagtt
254924DNAArtificialsynthetic 49ctaacaattc
ccaaaaccct tatc
245024DNAArtificialsynthetic 50gtgtactcat ctggatctgt attg
245125DNAArtificialsynthetic 51ggttgaggag
cctgaatctc tgaac
25521000DNAArabidopsis thaliana 52acagaaaaaa aaaaactaat ttcgattcca
caatgccgaa acccagaaaa attctcaaaa 60cgaagtcact caagaagata atcaacaaag
cgaaaacgga gaagggtttg ggtttaaggt 120accgtaacga actccggtaa gttccgatga
tggtgttgac agcgaagctt cgtggaacag 180cgtgaccgtc gttactcact ggatttgccg
gtgaatttga tgagtcaaaa atataactag 240cagctggtca aagtaaccac tctgaataag
acacgtgtca gaaactttgt tgaccattct 300gactaaagaa accttgtaac caaaaacatc
cacattttta catgctctgt catatttcaa 360ctgtatacta aagttgaaat atgactatgg
taacttatgt ttttttagtt gaaaactaag 420gtaaactatt ctatgccagc aggtaaaaga
tgtggatgac caactactgc tggcataatt 480tattctatga ctaaggtaaa actaaaagtt
aatatcactc gaaatattta caaaagtatt 540agataaagta ctaaaaattt taagttagaa
atgatgaaat taaaagatat atgtggctta 600tttaggaaaa tagatacttt taatatgatg
gttgggtagt tttattattg tttgggattg 660aaaaaaacta tgaaagtgtc tccaagtaca
gtaatattta acaactaact aggaaggtgt 720acattgttca gtattcatca ttctaattaa
tacaaataat tttaaaaatt tggggtcgtc 780aattgctcaa agatattcat tattttataa
ttttctacat tttatcaaaa acagaattgt 840gtaaatgttg tgtcattatg tgtcgacttg
gatgtatgca ttacataatt ttattttata 900tatgttttta attgttagag catttattcg
aaaattataa atattgaatt ttaatcctca 960gtcacttaat aaaaacatta ttttaggact
aaaaaataat 1000531000DNAOryza sativa 53caacgacggc
ttgcaggaat ttttaaaaaa ctataaaaaa aatcatatca ataatacgga 60tcttaccggt
gtagtgagat gacaaagaag agaagcaatg ggcaggtgag cgtgcgcaaa 120caaatgagat
aggaggcagt tgccacagga gggcatgccg agggtggtaa agtaaataaa 180aaaataatta
taagagttca agtagaatta caatttataa atgaaagaaa tatcacaata 240agaaatattt
atttttgaaa aaaatagtcc atacagacca caacatgaca aattataata 300aaaaaacaag
agaatagacc cataaaaaat tagagtggtg taggtagatg ggacatctaa 360aaactataaa
caaaacctta aatagaatat caatgataat taagaggagg gacaacgagc 420cggtcgttaa
gcagcacgat ctcgacgatt ggagggattt ctagaaagta aaaaaaaact 480ctcaattata
atcatgttcg attttcaaat ctcaacgata ataaaaatgg gaagcaatgg 540acgggccaaa
gaggagtata gtggcggcgt ttagcgagac ttataaaaat tataaaaatg 600aaacccaaca
atacaatgaa ctctaaaacc acaaggtcaa atttgtagag gttccaggaa 660ggatgaatga
aagcattggt agatcgagca agtaaataaa gcgatgataa agaaataatg 720cgtagtgatt
agcatgacta ttaaaagcac cgggatgata aagtcggttt ctaacatata 780gttatttaac
aaattttaag taaaatcata tgtaaaaaat agataatttt gtatgggtgc 840tagcccgcgc
aattgcgcgg gccacctagc tagttttttt aaaaatgtaa atataaaaaa 900ttcgaccacc
acgcatcgcc tgctctgccg cggcgacgcg agccacaaag ccgtcgacca 960ccgcaacaac
aaaatatcct cctcccgacg caaaccggtt
1000541000DNASolanum lycopersicum 54atgtttaatt tatgggtaat gtagaagtgt
atttaaaaaa aaaaaattga taaatatgtt 60tatattatat gtgtaatgtg tgttaatatt
ttcggtgatg gaataatata tgataaatca 120tttagactca tgaagctctt cttaaatata
atgaaaagac ttccaatgta aaaaattata 180atcggaagcg gagccaatat atatttaggg
gttcatttga attctcttta gcgaaaaata 240tagtactatt tatatatgat cagttatttt
tttttatgca tatgtagtag atattaaatc 300tccttcgatt atttcgtgtg tttacttctt
gagattttaa attccctatt caaaaatcct 360agcctcgcca ttgtccgtaa caaagagata
ctaaatcgta tcttgtactc cctgtgtccc 420aatttatgcg acttacgttg ctttttagtc
agtccaaaaa taatgacaca attctatatt 480aagtaacaat ttaactataa aacgtcgatt
ttatccttaa tgaaacgatt taccaccaca 540caaatttctc attttagact gcaagttttt
taaaaatttt catttctttc ttaaaactct 600gtgccgaata aaactacttt acgtaaaata
gaaaggagga aatatttatt tacacatcat 660aaaaagtctc gaggaaaatc aaaaaaacgt
cacaacaaac ataatatttt cattgaaaaa 720atcgtttcat acctatttct tttgttgtct
ctttttcatc cagtgttcag tactcactat 780aaacactgac caatataaaa ctattcgcgc
tccaaaatac cacattaaat aaaagcaact 840tcatacataa aactcgaact cataatctcc
cgttataaaa ggataccaca actttttagt 900gctctttttc aataataact ttttttttat
aaaaaaaaac taactgccag agtggaattt 960ccaactgtat ctatttgatg aaaatagcag
aaaactggtt 10005524DNAArtificial
sequencesynthetic 55gtgtactcat ctggatctgt attg
245625DNAArtificial sequencesynthetic 56ggttgaggag
cctgaatctc tgaac 25
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