Sangamo BioSciences Inc.
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Patent Application for Agrigenetics re Gene and Variations with BM1 Phenotype, Molecular Markers and Their Use (claims 46 and 47 involve ZFN)
Gene and Variations Associate with BM1 Phenotype, Molecular Markers, and their Use This disclosure concerns specific naturally-occurring mutant maize cad2 genes, which altered genes contribute to the bm1 maize phenotype in particular maize lines. In some embodiments, compositions and methods are provided that utilize a nucleic acid molecule comprising a mutant cad2 gene, and/or markers linked to a mutant cad2gene.
1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a mutant cinnamyl alcohol dehydrogenase 2 protein having at least 90% amino acid identity to SEQ ID NO:3 or having at least 90% amino acid identity to SEQ ID NO:25. 3. An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 80% identical to the nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:62, and SEQ ID NO:63. 4. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:63 and a nucleotide sequence that is at least 80% identical to SEQ ID NO:62. 5. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO:62 that encodes a truncated CAD2 protein lacking NADPH-binding and C-terminal catalytic domains. 6. An isolated nucleic acid molecule consisting of the mutant cinnamyl alcohol dehydrogenase 2 (cad2) gene of SEQ ID NO:1 or the mutant cinnamyl alcohol dehydrogenase 2 (cad2) gene of SEQ ID NO:23. 7. A mutant cinnamyl alcohol dehydrogenase 2 protein consisting of an amino acid sequence having at least 90% identity to SEQ ID NO:3 or having at least 90% identity to SEQ ID NO:25. 8. The mutant cinnamyl alcohol dehydrogenase 2 protein of claim 7, consisting of SEQ ID NO:3. 9. A method for generating a transgenic maize plant comprising introducing a premature stop codon into a CAD2 gene in the maize plant. 10. The method according to claim 9, wherein the premature stop codon is introduced by introducing an AC dinucleotide insertion into exon 3 of the CAD2 gene in the maize plant. 11. The method according to claim 9, wherein the premature stop codon is introduced by introducing an insertion into intron 1 of the CAD2 gene in the maize plant. 12. A plant comprising the nucleic acid molecule of claim 1. 13. The plant of claim 12, wherein the plant is Zea mays. 14. A method for identifying a plant comprising a mutant cinnamyl alcohol dehydrogenase 2 (cad2) gene, the method comprising: isolating nucleic acid molecules from a plant; and screening the isolated nucleic acid molecules for a nucleic acid molecule comprising: an AC di-nucleotide insertion at the location corresponding to nucleotide 3994 of the wild-type CAD2 gene, wherein presence of an AC di-nucleotide insertion at the location corresponding to nucleotide 3994 of the wild-type CAD2 gene is indicative of a mutant cad2 gene; or a transposon insertion at the location corresponding to nucleotide 2786 of the wild-type CAD2 gene, wherein presence of a transposon insertion at the location corresponding to nucleotide 2786 of the wild-type CAD2 gene is indicative of a mutant cad2 gene. 15. The method according to claim 14, wherein screening the isolated nucleic acid molecules comprises polymerase chain reaction. 16. The method according to claim 15, wherein polymerase chain reaction is performed using at least two primers that are capable of specifically hybridizing to SEQ ID NO:1. 17. The method according to claim 16, wherein the primers comprise primers selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:55. 18. The method according to claim 15, wherein polymerase chain reaction is performed using at least two primers that are capable of specifically hybridizing to SEQ ID NO:23. 19. The method according to claim 18, wherein the primers comprise primers selected from the group consisting of SEQ ID NO:47, SEQ ID NO:49, and SEQ ID NO:50. 20. A plant identified by the method according to claim 19. 21. The plant of claim 20, wherein the plant is Zea mays. 22. The plant according to claim 20, wherein the plant has a brown midrib phenotype. 23. The plant according to claim 22, wherein the Zea mays plant has at least one trait selected from the group consisting of reduced lignin, increased digestibility, and ethanol yield. 24. A method for introducing the brown midrib 1 (bm1) phenotype in maize, the method comprising: crossing a maize plant having the bm1 phenotype with a maize plant lacking the bm1 phenotype to generate F.sub.1 maize plants; using marker-assisted selection to identify an F.sub.1 maize plant comprising: an AC di-nucleotide insertion at the location corresponding to nucleotide 3994 of the wild-type CAD2 gene; or a transposon insertion at the location corresponding to nucleotide 2786 of the wild-type CAD2 gene; and propagating the identified F.sub.1 maize plant, thereby introducing the brown midrib 1 (bm1) phenotype in maize. 25. A method of producing a genetically engineered organism comprising introducing the nucleic acid molecule of claim 1 into the organism. 26. The method according to claim 25, wherein the organism is a plant. 27. The method according to claim 26, wherein the plant is Zea mays. 28. The method according to claim 27, wherein the nucleic acid molecule of claim 1 is introduced into the Zea mays plant by crossing with a Zea mays plant comprising the nucleic acid molecule of claim 1. 29. The method according to claim 25, wherein the nucleic acid molecule of claim 1 is introduced into the organism by genetic transformation. 30. The method according to claim 29, wherein the nucleic acid of claim 1 is stably integrated into the genome of the organism. 31. The method according to claim 29, wherein the organism is a plant. 32. The method according to claim 31, wherein the plant is Zea mays. 33. A method for identifying a plant comprising a mutant cinnamyl alcohol dehydrogenase 2 (cad2) gene, the method comprising: isolating nucleic acid molecules from a plant; and contacting the isolated nucleic acid molecules with means for identifying corn plants carrying a mutant cad2 gene to produce a detectable signal that is indicative of the presence of a mutant cad2 gene in the plant. 34. A method for transferring a maize mutant cinnamyl alcohol dehydrogenase 2 (cad2) gene, the method comprising: (a) analyzing with probes that are specifically hybridizable to at least one marker that is linked to the cad2 gene the genomic DNA of a first plant and the genomic DNA of a second plant; (b) sexually crossing the first and second plants to obtain a progeny population; (c) analyzing the progeny population for the presence of the at least one marker that is linked to the cad2 gene; (d) backcrossing an individual from the progeny population that comprises the at least one marker that is linked to the cad2 gene to the first or second plant to produce a next generation population; (e) determining if a member of the next generation population comprises a desired trait from the first or second plant and the cad2 gene; and (f) if no member of the next generation population comprises a desired trait from the first or second plant and the cad2 gene, repeating steps (d) and (e) until an individual is identified that comprises the desired traits from the first or second plant and the cad2 gene. 35. The method of 34, wherein individual progeny obtained in each crossing and backcrossing step is selected by CAD2 marker analysis at each generation. 36. A method for introducing a maize mutant cinnamyl alcohol dehydrogenase 2 (cad2) gene into a host organism by genetic transformation, the method comprising: analyzing the genomic DNA of a maize plant with probes that are specifically hybridizable to markers linked to the mutant cad2 gene to identify the mutant cad2 gene in the maize plant; isolating a segment of the genomic DNA of the maize plant that specifically hybridizes to probes that are specifically hybridizable to markers linked to the mutant cad2 gene; introducing the isolated segment of genomic DNA into the host organism; and analyzing the DNA of the host organism with probes that are specifically hybridizable to markers linked to the mutant cad2 gene to identify the mutant cad2 gene in the host organism. 37. A method of feeding a silage-fed animal, the method comprising: providing silage produced from the corn plant of claim 21; and feeding the animal with the silage produced from the corn plant of claim 21. 38. The method according to claim 37, wherein the silage-fed animal is a ruminant. 39. The method according to claim 38, wherein the silage-fed animal is a cow. 40. The method of claim 37, wherein the silage produced from the corn plant of claim 21 is greater than 15% of the dry matter in the animal's diet. 41. A meat product prepared from an animal fed according to the method of claim 37. 42. A beef finishing ration comprising corn silage produced from the corn plant of claim 21. 43. The beef finishing ration of claim 42, wherein the corn silage is bm1 corn silage. 44. A biofuel produced from the corn plant of claim 21. 45. A method for creating a genetically modified plant comprising a gene of interest, the method comprising: providing a nucleic acid molecule comprising a wild-type CAD2 gene and the gene of interest; introducing the nucleic acid molecule into a bm1 plant; screening the plant or its progeny for normal pigmentation; and selecting a plant having normal pigmentation, wherein the plant comprises the gene of interest. 46. The method of claim 45, wherein a zinc finger nuclease is utilized to integrate the nucleic acid molecule into the genome of the bm1 plant at the CAD2 locus. 47. A method for creating a genetically modified plant comprising a gene of interest, the method comprising: providing a nucleic acid molecule comprising a gene of interest; introducing the nucleic acid molecule into a plant homozygous for the wild-type CAD2 allele; utilizing a zinc finger nuclease to integrate the nucleic acid molecule into the genome of the plant, such that the CAD2 gene is inactivated; screening the plant or its progeny for a brown midrib phenotype; and selecting a plant having a brown midrib phenotype, wherein the selected plant comprises the gene of interest. 48. The mutant cinnamyl alcohol dehydrogenase 2 protein of claim 7, consisting of SEQ ID NO:25. CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/429,390, filed Jan. 3, 2011, the disclosure of which is hereby incorporated herein in its entirety by this reference. FIELD OF THE DISCLOSURE [0002] The present disclosure relates to a maize brown midrib (bmr) phenotype. In particular embodiments, this disclosure concerns particular altered cinnamyl alcohol dehydrogenase 2 (cad2) genes in maize, which altered genes contribute to the bm1 phenotype in some maize varieties, nucleic acid molecules comprising an altered cad2 gene, and/or protein products resulting from the translation of such nucleic acid molecules. BACKGROUND [0003] Lignins are universal components in plants that form cross-links with carbohydrates, such as hemicelluloses in the cell wall. Lignin and cellulose are the two predominant components of the plant cell wall. The plant cell wall provides a natural barrier against the extracellular environment. Many studies have demonstrated that one of the responses of plants to biotic stresses (e.g., pathogenic infection) or abiotic stresses (e.g., drought, mechanical stress, etc.) consists of reinforcement of the plant cell wall, in particular by increasing the lignin content in the plant cell wall. Many agronomical or industrial applications concern desired plant products (e.g., products used in paper production, silage production, and the production of energy, for example, in the form of biofuels), the yields of which are directly linked to the content and/or composition of lignin in the plant cell wall. [0004] Lignin polymers limit the digestibility of the fiber in the corn plant. Lignin polymers lower fiber digestion in ruminants, and the degree of lignifications may be inversely proportional to forage crop digestibility. Cherney et al. (1991) Adv. Agron. 46:157-98. Modulation of lignin content and composition may be desirable to increase the digestibility of forage. Lignin content modulation may also be desirable, e.g., to reinforce plant walls, and thereby improve resistance to stresses; or conversely to weaken the plant wall in order to facilitate the extraction of cellulose or other chemical compounds. Baucher et al. (1998) Plant Mol. Biol. 39:437-47. [0005] It is, however, difficult to know how to modify the lignin biosynthesis pathway, and to predict what the consequences of modifications will be. This is at least in part because the lignin biosynthesis pathway is a complex pathway involving a large number of enzymatic reactions. See, e.g., Dixon et al. (2001) Phytochemistry 57(7):1069-84. Possible mechanisms by which the pathway may be altered physiologically, for example, to compensate for a change introduced by a modification in the pathway, are not known. [0006] Lignin is an insoluble polymer of 3 monomers of alcohols or monolignols: p-coumaryl alcohol (H subunits), coniferyl alcohol (G subunits), and sinapyl alcohol (S subunits), that are derived from the phenylpropanoid pathway. Neish (1968) Constitution and Biosynthesis of Lignin, eds. New York, Springer Verlag 1-43. Each type of subunit can form a variety of bonds with others, and thereby constitute lignin. Other bonds may also be established with other parietal compounds (e.g., polysaccharides and proteins) so as to form a complex three-dimensional network. [0007] Steps in the complex lignin production pathway include hydroxylation, O-methylation of aromatic rings, and conversion of a carboxyl side chain to an alcohol function. The current hypothesis for the monolignol biosynthesis pathway includes successive hydroxylation and O-methylation reactions at various levels of oxidation of the side chains in a metabolic network, thereby resulting in the formation of S and G subunits. The enzymes of the network include caffeic acid 3-O-methyltransferase (COMT); hydroxyxinamate coenzyme A ligases (4CL); cytochrome P450-dependent ferulate 5-hydroxylases (F5Hs); and several isoforms of cinnamoyl CoA reductase (CCR) and of cinnamyl alcohol dehydrogenase (CAD). [0008] For several years, attempts have been made to modify the lignin content and composition of plants by over-expressing or under-expressing one or more genes of the lignin biosynthesis pathway. Anterola and Lewis (2002) Phytochemistry 61:221-94. Though various strategies have been imagined, the over-expression or under-expression of one or more enzymes in the lignin biosynthesis pathway does not always give reliable and predictable results. [0009] Another strategy consists of using, in selection schemes, mutants of a targeted gene in the lignin biosynthesis pathway. Plants containing a brown midrib (bmr) mutation exhibit altered lignin composition and digestibility. In corn, at least four independent brown midrib mutations have been identified. Kuc et al. (1968) Phytochemistry 7:1435-6. These mutations, termed "bm1, bm2, bm3, and bm4," all exhibit decreased lignin content when compared to control corn. Brown midrib corn plants are characterized by a brown pigmentation in the leaf midrib at the V4 to V6 stage and a light brown coloration of the pith after tasselling. One characterized bmr mutation is an insertion mutation in the COMT enzyme (bm3). [0010] Mature bm1 maize plants have a lignin content that is reduced by 10-20%, a slight decrease in ferulic acid esters, and a substantially reduced content (.about.40%) of p-coumaric esters and ferulic acid esters. Provan et al. (1997) J. Agric. Food 73:133-42; Barriere et al. (2004) Comptes Rendus Biologie 327:847-60. The frequency of p-hydroxyphenyl, guaiacyl, and syringyl thioacidolysis monomers is similar in bm1 and wild-type plants, showing that the bm1 mutation does not specifically affect a single type of lignin subunit. Guillaumie et al. (2007) Planta 226(1):235-50. Lignins of bm1 plants do appear to be substantially enriched in carbon-carbon inter-subunit linkages (Halpin et al. (1998) Plant J. 14(5):545-53; Barriere et al. (2004), supra), and bm1 lignins have substantial incorporation of coniferaldehyde and, to a lesser extent, of sinapaldehyde. Kim et al. (2002) J. Biol. Chem. 277:47412-9. [0011] Agriculturally important uses of corn (maize) include silage. Silage is fermented, high-moisture fodder that can be fed to ruminants. It is fermented and stored in a process called ensilage or silaging, and is usually made from corn or other grass crops, including sorghum or other cereals, using the entire green plant. Bulk silage is commonly fed to dairy cattle, while baled silage tends to be used for beef cattle, sheep, and horses. Since silage goes through a fermentation process, energy is used by fermentative bacteria to produce volatile fatty acids, such as acetate, propionate, lactate, and butyrate, which preserve the forage. The result is that the silage is lower in energy than the original forage, since the fermentative bacteria use some of the carbohydrates to produce the volatile fatty acids. Corn silage is a popular forage for ruminant animals because it is high in energy and digestibility and is easily adapted to mechanization from the stand-crop to time of feeding. Corn silage generally is slightly brown to dark green in color, and has a light, pleasant smell. [0012] The reduced lignin in brown midrib corn (bmr corn) results in silage with fiber that is more digestible than normal corn and exhibits an improved rate of biofuel conversion. Feeding bmr corn silage to lactating dairy cows has been shown to increase dry matter intake (DMI) and milk yield. Grant et al. (1995) J. Dairy Sci. 78:1970-80; Oba and Allen (2000) J. Dairy Sci. 83:1333-41; Oba and Allen (1999) J. Dairy Sci. 82:135-42. However, bmr corn silage reduced average daily gain and feed efficiency (G:F) in beef cows, compared to corn silage from a conventional corn variety. Tjardes et al. (2000) J. Anim. Sci. 78:2957-65. Brown midrib hybrid corn lines are also frequently found to be low yielding. Brown midrib hybrid corn has also typically been associated with forage lodging and lack of standability. BRIEF SUMMARY OF THE DISCLOSURE [0013] Described herein are nucleic acid molecules comprising a mutant cad2 gene that contributes to the bm1 phenotype in corn (maize). Also described are molecular markers that are linked to or that reside within a mutant maize cad2 gene. Surprisingly, the complicated pathway of lignin biosynthesis is apparently altered in the presence of a mutant cad2 gene, such that plants containing a mutant cad2 gene have lignin levels that are lower than those found in wild-type plants. The characterization of mutant cad2 genes and identification of markers linked to mutant cad2 genes may greatly facilitate the development and deployment of reduced lignin phenotypes in plant germplasms. In some embodiments, markers that are linked to or that reside within a mutant maize cad2 gene, or a mutant maize cad2 gene sequence itself, may be used to introduce a mutant maize cad2 gene into other organisms, for example, plants, yeast, and prokaryotes. [0014] In particular embodiments, a mutant cad2 gene according to the disclosure may comprise a nucleotide sequence encoding a truncated CAD2 protein. For example, a mutant cad2 gene comprising an insertion mutation in an exon (e.g., exon 3) or an intron (e.g., intron 1) of the cad2 gene may introduce a premature STOP codon that results in a shorter gene product. In some embodiments, a mutant cad2 gene according to the disclosure may comprise a naturally-occurring mutation in one or more maize lines. In some embodiments, a mutant cad2 gene is cloned from a known bmr corn variety; e.g., 515Dbm1. In other embodiments, a mutant cad2 gene is cloned from a previously unknown bmr corn variety; e.g., DASbm1. When expressed in a plant, a mutant cad2 gene according to the disclosure may result in a phenotype in the plant; for example, reduced levels of cad2 RNA in tissues of the plant, and/or reduced lignin content in tissues of the plant. [0015] Also described herein are methods of using nucleic acid molecular markers that are linked to or that reside within a mutant maize cad2 gene according to the disclosure, for example and without limitation: to identify plants having a reduced lignin phenotype; to introduce a mutant maize cad2 gene to new plant genotypes (e.g., through marker-assisted breeding or genetic transformation); to differentiate between wild-type CAD2 genes and particular mutant cad2 genes according to the disclosure; and to produce plants and plant seeds from crosses of a first plant comprising nucleic acid molecular markers that are linked to or that reside within a mutant maize cad2 gene according to the disclosure and a second plant optionally carrying a mutant maize cad2 gene. In some embodiments, a mutant maize cad2 gene is engineered into plant species other than maize. [0016] Further described are means for producing a genetically modified plant (e.g., maize) comprising a mutant maize cad2 gene, and means for identifying plants (e.g., maize) carrying a mutant maize cad2 gene. A means for producing a genetically modified plant comprising a mutant maize cad2 gene is a marker that is linked to or that resides within a mutant maize cad2 gene according to the disclosure. A means for identifying plants carrying a mutant maize cad2 gene is a probe that specifically hybridizes to a marker that is linked to or that resides within a mutant maize cad2 gene according to the disclosure. [0017] Methods are disclosed for increasing the meat quantity of a silage-fed animal, for example by increasing the gain to feed ratio (G:F) for corn silage. In some embodiments, a method for increasing the meat quantity of a silage-fed animal may comprise providing plant material obtained from maize comprising a mutant maize cad2 gene according to the disclosure, using the plant material for the production of corn silage, and incorporation of the corn silage in a finishing ration for feeding to a ruminant. Thus, meat and meat products produced from an animal fed a finishing ration according to the disclosure or according to a method the disclosure are also provided.
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