# Patent

## Title
Gene and variations associate with BM1 phenotype, molecular markers, and their use

## Abstract

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.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
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
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
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.
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.
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.
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.
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).
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.
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).
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 ( ̃40%) of p-coumaric esters and ferulic acid esters. Provan et al. (1997) J. Agric. Food 73:133-42; Barriére 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; Barriére 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.
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.
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
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.
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.
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.
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.
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.
The foregoing and other features will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 includes a diagram showing the relative lengths and positions of seven overlapping 515Dbm1 cad2 PCR fragments. FIG. 1 also includes a diagram showing the relative lengths and positions of thirteen overlapping DASbm1 cad2 PCR fragments.
FIG. 2 includes a diagram showing the genomic structure of the insertion in DASbm1 cad2. Only the first two endogenous exons of cad2 are shown. A duplicated 11 bp sequence (TACTGATATCT) (SEQ ID NO: 42) in the DASbm1 transposon insertion from the first intron of CAD2 is also shown.
FIG. 3 includes an alignment of ZmCAD2 genomic sequences, using a B73 sequence from public accession AC230031. 15 SNPs and 8 insertion/deletions are identified, as well as a transposon insertion in DASbm1. An AC insertion is present only in the 515Dbm1 mutant in exon 3. Exons are shown in uppercase font. Introns and a promoter region are shown in lowercase font.
FIG. 4 includes an alignment of ZmCAD2 cDNA sequences. Shown is a DASbm1 ZmCAD2 cDNA sequence, wherein a partial sequence was amplified by primer pair CVF/CVR (underlined), and the remainder of the full-length sequence is predicted. Also shown are full-length predicted ZmCAD2 cDNA sequences from 515Dbm1 and wild-type 6XN442 (underlined). DASbm1 cDNA contains a 409 bp insertion.
FIG. 5 includes an alignment of predicted ZmCAD2 protein sequences, illustrating that CAD2 from 515Dbm1 only has 147 amino acids due to an AC insertion that creates a frame-shift and premature STOP codon. CAD2 from DASbm1 only has 48 amino acids, compared to 367 amino acids in CAD2 from 6XB442. Regions of identity are shown in bold font. The B73 CAD2 amino acid sequence is identical to that of 6XN422 CAD2, and is therefore not shown.
FIG. 6 includes data showing the relative expression levels of CAD2 RNA in wild-type, 515Dbm1, and DASbm1 corn midribs and leaves. Data is normalized to the RNA level in the midrib of wild-type corn 6XN442. Data represent the mean value of 3 plants, and error bars indicate standard deviation.
FIG. 7 includes data showing the relative amounts of total lignin in wild-type, 515Dbm1, and DASbm1 corn. Data represent the mean value of 3 plants, with five samples per plant obtained from both leaves and internodes. Error bars indicate standard deviation, and P values were generated by student's T test.
FIG. 8 includes a diagram of a KASPar assay system with primer and template sequences. Template sequences for 515Dbm1 (SEQ ID NO:46) and 6XN442 (SEQ ID NO:47) are shown.
FIG. 9 includes data showing KASPar assay bm1 allele determination. Raw fluorescence intensity data from the plate reader was analyzed using the KBioscience Laboratory Information Management System (KLIMS). A graph showing the RFU of FAM plotted against the RFU from VIC was generated, also using KLIMS. Genotype determination was performed based on the cluster separation as displayed in the cluster view.
FIG. 10 includes data showing TaqMan CAD2 allele determination. Allelic discrimination was calculated using iCycler optical system software with relative fluorescence units (RFU) of VIC displayed on the y-axis and RFU of FAM displayed on the x-axis.



SEQUENCE LISTING
The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NO:1 shows a 5924 bp genomic sequence of 515Dbm1 CAD2.
SEQ ID NO:2 shows a 1512 bp predicted cDNA sequence of 515Dbm1 CAD2.
SEQ ID NO:3 shows a 147 amino acid predicted truncated protein sequence of 515Dbm1 CAD2.
SEQ ID NO:4 shows a 5898 bp genomic sequence of 6XN442 CAD2.
SEQ ID NO:5 shows a 1510 bp predicted cDNA sequence of 6XN442 CAD2.
SEQ ID NO:6 shows a 367 amino acid predicted protein sequence of 6XN442 CAD2.
SEQ ID NO:7 shows a 5916 bp genomic sequence of B73 CAD2.
SEQ ID NO:8 shows a 1510 bp predicted cDNA sequence of B73 CAD2.
SEQ ID NOs:9-22 show forward and reverse primers used to amplify seven overlapping CAD2 fragments from both the 515Dbm1 mutant and wild-type 6XN442 corn.
SEQ ID NO:23 shows a 9417 bp genomic sequence of DASbm1 CAD2.
SEQ ID NO:24 shows a partial cDNA sequence of DASbm1 CAD2 amplified by primer pair CVF/CVR.
SEQ ID NO:25 shows a 48 amino acid predicted truncated protein sequence of 515Dbm1 CAD2.
SEQ ID NOs:26-35 show forward and reverse primers used to amplify partial CAD2 fragments from the DASbm1 mutant.
SEQ ID NOs:36 and 37 show a forward and a reverse primer used to amplify a partial CAD2 cDNA fragment.
SEQ ID NOs:38-40 show forward and reverse primers used to amplify partial CAD2 fragments from the DASbm1 mutant.
SEQ ID NO:41 shows a 3444 bp transposon insertion in DASbm1 CAD2.
SEQ ID NO:42 shows a duplicated 11 bp sequence in the DASbm1 transposon insertion from the first intron of CAD2: TACTGATATCT.
SEQ ID NOs:43-45 show primers used in a KASParTM assay to differentiate the 515Dbm1 allele from other bm1 and the wild-type CAD2 alleles.
SEQ ID NOs:46 and 47 show template sequences to which primers anneal in a KASPar assay. SEQ ID NO:46 is a genomic sequence from 515Dbm1. SEQ ID NO:47 is a genomic sequence from 6XN442.
SEQ ID NO:48 shows a probe specific for mutant DASbm1.
SEQ ID NO:49 shows a probe specific for the wild-type CAD2 allele.
SEQ ID NOs:50-52 show primers used in a KASParTM assay to differentiate the DASbm1 and wild-type CAD2 alleles.
SEQ ID NO:53 shows a probe specific for mutant 515Dbm1.
SEQ ID NO:54 shows a probe specific for the wild-type CAD2 allele.
SEQ ID NOs:55 and 56 show primers used in a KASParTM assay to differentiate the 515Dbm1 and wild-type CAD2 alleles.
SEQ ID NOs:57-62 show primers and probes used for qRT-PCR of CAD2 and controls.
SEQ ID NO:63 shows a nucleotide sequence of the first exon of CAD2.
SEQ ID NO:64 shows a nucleotide sequence of the second exon of CAD2.
SEQ ID NO:65 shows a nucleotide sequence of the third exon of CAD2.
DETAILED DESCRIPTION
I. Overview of Several Embodiments
The bm1 mutant involves a previously undescribed mutation that co-localizes with the CAD locus. Halpin et al. (1998), supra; Guillaumie et al. (2007), supra. Described herein is the identification of specific mutations within the Zea mays CAD2 coding sequence that contribute to a bm1 phenotype. Some embodiments include a mutant cad2 gene cloned from a public bm1 corn line, 515Dbm1, that contains an AC insertion in the 3rd exon, which results in a truncated CAD2 protein. Some embodiments include a mutant cad2 gene cloned from a newly discovered bmr corn line, DASbm1, that contains a transposon insertion, which results in a distinct truncated CAD2 protein. DASbm1 plants have significantly lower cad2 RNA expression levels than wild-type plants, and also exhibit reduced lignin content. Development, validation, and application of high-throughput markers linked to each specific mutation are also described.
The molecular basis of the bm1 phenotype is not as well understood as, for example, bm3. Halpin et al. (1998), supra, showed that CAD activities, protein levels, and transcript abundance, were all significantly reduced in bm1 genotypes, and a CAD gene, later identified to be CAD2, was mapped closely linked to the bm1 locus. Because some CAD protein and mRNA could be detected in mutant plants, the authors proposed that bm1 was not a null mutation of CAD2, but instead affected its expression through regulatory elements. Halpin et al. (1998), supra. Subsequent gene expression studies using microarrays showed that a large number of genes besides merely CAD2 were under-expressed in bm1 plants. Guillaumie et al. (2007), supra. This multitude of under-expressed genes included many lignin biosynthetic pathway genes and transcription factors, leading to further speculation that perhaps one of the transcription factors was the underlying gene for bm1.
More recently, nonsense mutations in the sorghum SbCAD2 gene have been shown to be the underlying mechanism for sorghum bmr mutant, bmr6, in some varieties. Saballos et al. (2009) Genetics 181:783-95; Sattler et al. (2009) Plant Physiology 150:584-95. The nonsense mutation contains a C to T substitution, which creates a premature STOP codon and results in a truncated SbCAD2 protein that lacks the NADPH-binding and C-terminal catalytic domains. Id.
In view of the foregoing, the accepted theory that the bm1 phenotype is attributable to a mutation in a transcription factor or gene regulatory element was challenged, and an investigation was conducted to determine whether a mutation in maize CAD2 could produce the bm1 phenotype. A close examination of the maize genetic/physical map in the region of the bm1 and CAD2 loci revealed that maize CAD2 is located very close to the centromere on chromosome 5. This proximity poses a significant challenge for high-resolution genetic mapping due to the known recombination suppression that surrounds centromere regions. Therefore, the success of traditional gene mapping approaches was at least unpredictable, if not entirely unlikely, and a PCR-based candidate gene cloning approach was designed to identify any specific mutations in ZmCAD2 that could be responsible for the bm1 phenotype in subject corn varieties.
Thus, in some embodiments, identification of specific cad2 mutants may be carried out using a PCR-based gene cloning approach. In some embodiments, Zea mays CAD2 may be cloned from both bm1 and wild-type germplasm to identify a specific mutation in cad2 from bm1 germplasm. In particular embodiments, an AC dinucleotide insertion mutation leading to a frame-shift is identified within bm1 germplasm. In further embodiments, a 3444 base pair transposon insertion in the first intron of CAD2 is identified within bm1 germplasm. A particular mutant cad2 gene leading to a bm1 phenotype in maize described herein comprises an AC dinucleotide insertion in the third exon of a cad2 gene cloned from bm1 variety, 515Dbm1.
A further mutant cad2 gene leading to a bm1 phenotype in maize described herein comprises a transposon insertion in the cad2 gene of a novel bm1 variety, DASbm1. The identified insertion is 3444 base pairs in length, and it is spliced into three exons (409 base pairs) that form a chimeric mRNA with CAD2. The chimeric mRNA causes a frame-shift and a premature STOP codon in the coding region that results in a truncated CAD2 protein of only 48 amino acids. This truncated protein lacks both the NADPH-binding and C-terminal catalytic domains, and is therefore most likely nonfunctional, even if it is produced in the cell.
DASbm1 plants, like 515Dbm1, have significantly reduced levels of cad2 RNA, and reduced total lignin contents. CAD2 was determined to be the underlying gene for the maize bm1 phenotype in these naturally-occurring mutants and provide a molecular basis for the observed bmr phenotype. Based on the transposon insertion in DASbm1 and the AC insertion in 515Dbm1, high-throughput KASPar and TaqMan assays for detecting these particular cad2 alleles and differentiating them from wild-type alleles have been determined and evaluated. The assays can be used for bm1 germplasm identification, for accelerating introgression of the bm1 trait, and for facilitating molecular breeding of plants having, e.g., improved silage digestibility and/or ethanol yield for biofuel. Mutated cad2 gene sequences can be used to introduce a bm1 phenotype into new maize genotypes or other crops, for example, sorghum and switch grass, using transgenic approaches. bm1 and other bmr phenotypes can also be used as visible selection markers for transgenes when combined with DAS' EXZACT Precision Technology.
High-throughput PCR markers that may be used, inter alia: to identify in an organism a cad2 mutation that contributes to a bm1 phenotype in corn; to introduce into an organism (e.g., a plant) a cad2 mutation that contributes to a bm1 phenotype in corn; and to facilitate marker-assisted breeding of bm1 corn. Thus, the development, validation and application of particular high-throughput PCR markers based on specific mutations in CAD2 (e.g., a frame-shift mutation, and a transposon insertion) are also described.
II. Abbreviations
4CL hydroxycinnamate coenzyme A ligases
ABC-transporter ATP-binding cassette transporter
AGO ARGONAUTE
APL ALTERED PHLOEM DEVELOPMENT
bmr/bm Brown midrib
bZIP basic region/leucine zipper motif
CAD cinnamyl alcohol dehydrogenase
CAD1 cinnamyl alcohol dehydrogenase 1
CAD2 cinnamyl alcohol dehydrogenase 2
CCR cinnamoyl-CoA reductase
COMT caffeic acid 3-O-methyltransferase
COV1 CONTINUOUS VASCULAR RING
DFR dihydro-flavonoid reductase
EgCAD1-type ZmCAD1 EgCAD1-type maize cinnamyl alcohol dehydrogenase 1
EST expressed sequence tag
F5H cytochrome P450-dependent ferulate 5-hydroxylases
FAM fluorophore 6-carboxyfluorescein
HCT hydroxycinnamoyl-CoA transferase 2
KLIMS KBioscience Laboratory Information Management System
LIM LIM homeodomain
MADS-box conserved MADS sequence motif (MCM1, AGAMOUS, DEFICIENS, SRF)
MP MONOPTEROUS
OMT O-methyl transferase
ORF open reading frame
PAL phenylalanine ammonia lyase
PCR polymerase chain reaction
RFU relative fluorescence units
SAD sinapyl alcohol dehydrogenase
SAMS3 S-adenosyl-methionine synthase 3
VIC® VIC® fluorophore (Applied Biosystems)
III. Terms
Backcrossing: Backcrossing methods may be used to introduce a nucleic acid sequence into plants. The backcrossing technique has been widely used for decades to introduce new traits into plants. Jensen, N., Ed. Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (non-recurrent parent) that carries a gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent, and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent plant are recovered in the converted plant, in addition to the transferred gene from the non-recurrent parent.
Corn plant: As used herein, the term “corn plant” refers to a plant of the species, Zea mays (maize).
bmr/bm corn: As used herein, the term “bm corn” (or “bmr corn”) refers to corn varieties that contain a brown midrib mutation. BM corn varieties typically exhibit a reddish brown pigmentation of the leaf midrib. BM corn is also typically characterized by lower lignin content, higher fiber digestibility, and higher dry matter intake. Non-limiting examples of BM corn varieties include bm1 corn varieties; e.g., 515Dbm1.
bm1 phenotype: As used herein, the term “bm1 phenotype” may refer to a profile of altered lignin content and/or composition that has been observed in bm1 corn. For example and without limitation, a bm1 phenotype may be characterized by one or more of the following characteristics: lignin content that is reduced by 10-20% when compared to the lignin content of a wild-type plant of the same species (Guillaumie et al. (2007), supra); a decrease in ferulic acid esters (Id.); reduced content of ferulic acid ethers (Id.); reduced content of p-coumaric esters (Marita et al. (2003) J. Agric. Food Chem. 51:1313-21); increased aldehyde levels (Id.); enrichment of lignins in carbon-carbon inter unit subunit linkages (Halpin et al. (1998), supra; Barriére et al. (2004), supra); and substantial incorporation of coniferaldehyde and/or sinapaldehyde into lignins (Kim et al. (2002), supra; and Barriére et al. (2004), supra).
Dry matter: As used herein, the term “dry matter” refers to any feedstuff, including forage.
KBiosciences Competitive Allele-Specific PCR SNP genotyping system (KASParTM): KASParTM is a commercially available homogeneous fluorescent system for determining SNP genotypes (KBiosciences Ltd., Hoddesdon, UK). A KASParTM assay comprises an SNP-specific “assay mix,” which contains three unlabelled primers, and a “reaction mix,” which contains all the other required components; for example, a universal fluorescent reporting system. In addition to these mixes, the user provides, inter alia, a FRET-capable plate reader, microtitre plate(s), and DNA samples that contain about 5 ng/μL DNA.
A typical KASParTM assay comprises the steps of: allele-specific primer design (e.g., using PrimerPickerTM, which is a free service available through the interne at the KBiosciences website); preparation of reaction mix including the allele-specific primers; admixing the reaction mix to DNA samples in a microtitre plate; thermocycling; reading the plate in a fluorescent plate reader; and plotting and scoring the fluorescent data. Data from each sample are plotted together on a 2-D graph, where the x- and y-axes correspond to FAM and VIC fluorescence values. Samples having the same SNP genotype cluster together on the plot (i.e., A/A; A/a; and a/a). More technical information about the KASPar system, including a guide of solutions to common problems, is obtainable from KBiosciences Ltd. (e.g., the KASPar SNP Genotyping System Reagent Manual).
Linked, tightly linked, and extremely tightly linked: As used herein, linkage between genes or markers may refer to the phenomenon in which genes or markers on a chromosome show a measurable probability of being passed on together to individuals in the next generation. The closer two genes or markers are to each other, the closer to (1) this probability becomes. Thus, the term “linked” may refer to one or more genes or markers that are passed together with a gene with a probability greater than 0.5 (which is expected from independent assortment where markers/genes are located on different chromosomes). When the presence of a gene contributes to a phenotype in an individual, markers that are linked to the gene may be said to be linked to the phenotype. Thus, the term “linked” may refer to a relationship between a marker and a gene, or between a marker and a phenotype.
Because the proximity of two genes or markers on a chromosome is directly related to the probability that the genes or markers will be passed together to individuals in the next generation, the term “linked” may also refer herein to one or more genes or markers that are located within about 2.0 Mb of one another on the same maize chromosome. Thus, two “linked” genes or markers may be separated by about 2.1 Mb; 2.00 Mb; about 1.95 Mb; about 1.90 Mb; about 1.85 Mb; about 1.80 Mb; about 1.75 Mb; about 1.70 Mb; about 1.65 Mb; about 1.60 Mb; about 1.55 Mb; about 1.50 Mb; about 1.45 Mb; about 1.40 Mb; about 1.35 Mb; about 1.30 Mb; about 1.25 Mb; about 1.20 Mb; about 1.15 Mb; about 1.10 Mb; about 1.05 Mb; about 1.00 Mb; about 0.95 Mb; about 0.90 Mb; about 0.85 Mb; about 0.80 Mb; about 0.75 Mb; about 0.70 Mb; about 0.65 Mb; about 0.60 Mb; about 0.55 Mb; about 0.50 Mb; about 0.45 Mb; about 0.40 Mb; about 0.35 Mb; about 0.30 Mb; about 0.25 Mb; about 0.20 Mb; about 0.15 Mb; about 0.10 Mb; about 0.05 Mb; about 0.025 Mb; and about 0.01 Mb. Particular examples of markers that may be “linked” to a bm1 phenotype in maize include nucleotide sequences on chromosome 5 of the maize genome.
As used herein, the term “tightly linked” may refer to one or more genes or markers that are located within about 0.5 Mb of one another on the same maize chromosome. Thus, two “tightly linked” genes or markers may be separated by about 0.6 Mb; about 0.55 Mb; 0.5 Mb; about 0.45 Mb; about 0.4 Mb; about 0.35 Mb; about 0.3 Mb; about 0.25 Mb; about 0.2 Mb; about 0.15 Mb; about 0.1 Mb; and about 0.05 Mb. Particular examples of markers that may be “tightly linked” to a bm1 phenotype in maize include nucleotide sequences near or within the CAD2 locus of the maize genome.
As used herein, the term “extremely tightly linked” may refer to one or more genes or markers that are located within about 100 kb of one another on the same maize chromosome. Thus, two “extremely tightly linked” genes or markers may be separated by about 125 kb; about 120 kb; about 115 kb; about 110 kb; about 105 kb; 100 kb; about 95 kb; about 90 kb; about 85 kb; about 80 kb; about 75 kb; about 70 kb; about 65 kb; about 60 kb; about 55 kb; about 50 kb; about 45 kb; about 40 kb; about 35 kb; about 30 kb; about 25 kb; about 20 kb; about 15 kb; about 10 kb; about 5 kb; and about 1 kb. Particular examples of markers that are “extremely tightly linked” to a bm1 phenotype in maize include nucleotide sequences within introns and exons of the CAD2 gene.
In view of the foregoing, it will be appreciated that markers linked to a particular gene or phenotype include those markers that are tightly linked, and those markers that are extremely tightly linked, to the gene or phenotype. Linked, tightly linked, and extremely tightly genetic markers of a bm1 phenotype may be useful in marker-assisted breeding programs to identify maize varieties having a reduced lignin content and improved digestibility, and to breed these traits into other maize varieties.
Locus: As used herein, the term “locus” refers to a position on the genome that corresponds to a measurable characteristic (e.g., a trait). An SNP locus is defined by a probe that hybridizes to DNA contained within the locus.
Marker: As used herein, a marker refers to a gene or nucleotide sequence that can be used to identify plants having a particular allele. A marker may be described as a variation at a given genomic locus. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, or “SNP”), or a long one, for example, a minisatellite/simple sequence repeat (“SSR”). A “marker allele” refers to the version of the marker that is present in a particular individual.
The term marker as used herein may refer to a cloned segment of maize chromosomal DNA (for example, nucleotide sequences near or within the CAD2 locus of the maize genome), and may also or alternatively refer to a DNA molecule that is complementary to a cloned segment of maize chromosomal DNA (for example, DNA complementary to a nucleotide sequence near or within the CAD2 locus of the maize genome). Particular examples of bm1 markers in maize include without limitation nucleic acid sequences including nucleotide 66 of the CAD2 gene; nucleic acid sequences including nucleotide 284 of the CAD2 gene; nucleic acid sequences including nucleotide 415 of the CAD2 gene; nucleic acid sequences including nucleotide 443 of the CAD2 gene; nucleic acid sequences including nucleotide 735 of the CAD2 gene; nucleic acid sequences including nucleotide 760 of the CAD2 gene; nucleic acid sequences including nucleotide 1345 of the CAD2 gene; nucleic acid sequences including nucleotide 1408 of the CAD2 gene; nucleic acid sequences including nucleotide 1585 of the CAD2 gene; nucleic acid sequences including any of nucleotides 1627-1640 and/or 1642-1648 of the CAD2 gene; nucleic acid sequences including nucleotide 2252 of the CAD2 gene; nucleic acid sequences including nucleotide 2269 of the CAD2 gene; nucleic acid sequences including nucleotide 2786 of the CAD2 gene; nucleic acid sequences including nucleotide 2966 of the CAD2 gene; nucleic acid sequences including nucleotide 3205 of the CAD2 gene; nucleic acid sequences including nucleotide 3719 of the CAD2 gene; nucleic acid sequences including nucleotide 3783 of the CAD2 gene; nucleic acid sequences including nucleotide 3798 of the CAD2 gene; nucleic acid sequences including nucleotide 3800 of the CAD2 gene; nucleic acid sequences including nucleotide 3994 of the CAD2 gene; nucleic acid sequences including nucleotide 4141 of the CAD2 gene; nucleic acid sequences including nucleotide 4338 of the CAD2 gene; nucleic acid sequences including nucleotide 4583 of the CAD2 gene; and nucleic acid sequences including nucleotide 5403 of the CAD2 gene. The foregoing markers are identified by nucleotide position in wild-type maize variety, B73.
In some embodiments, the presence of a marker in a plant may be detected through the use of a nucleic acid probe. A probe may be a DNA molecule, or an RNA molecule. RNA probes can be synthesized by means known in the art, for example, using a DNA molecule template. A probe may contain all or a portion of the nucleotide sequence of the marker and additional, contiguous nucleotide sequence from the plant genome. This is referred to herein as a “contiguous probe.” The additional, contiguous nucleotide sequence is referred to as “upstream” or “downstream” of the original marker, depending on whether the contiguous nucleotide sequence from the plant chromosome is on the 5′ or the 3′ side of the original marker, as conventionally understood. As is recognized by those of ordinary skill in the art, the process of obtaining additional, contiguous nucleotide sequence for inclusion in a marker may be repeated nearly indefinitely (limited only by the length of the chromosome), thereby identifying additional markers along the chromosome. All above-described markers may be used in some embodiments of the present invention.
An oligonucleotide probe sequence may be prepared synthetically or by cloning. Suitable cloning vectors are well-known to those of skill in the art. An oligonucleotide probe may be labeled or unlabeled. A wide variety of techniques exist for labeling nucleic acid molecules, including, for example and without limitation: radiolabeling by nick translation; random priming; tailing with terminal deoxytransferase; or the like, where the nucleotides employed are labeled, for example, with radioactive 32P. Other labels which may be used include, for example and without limitation: fluorophores (e.g., FAM and VIC); enzymes; enzyme substrates; enzyme cofactors; enzyme inhibitors; and the like. Alternatively, the use of a label that provides a detectable signal, by itself or in conjunction with other reactive agents, may be replaced by ligands to which receptors bind, where the receptors are labeled (for example, by the above-indicated labels) to provide detectable signals, either by themselves, or in conjunction with other reagents. See, e.g., Leary et al. (1983) Proc. Natl. Acad. Sci. USA 80:4045-9.
A probe may contain a nucleotide sequence that is not contiguous to that of the original marker; this probe is referred to herein as a “non-contiguous probe.” The sequence of the non-contiguous probe is located sufficiently close to the sequence of the original marker on the genome so that the non-contiguous probe is genetically linked to the same gene or trait (e.g., bm1/reduced lignin content). For example, in some embodiments, a non-contiguous probe is located within 500 kb; 450 kb; 400 kb; 350 kb; 300 kb; 250 kb; 200 kb; 150 kb; 125 kb; 100 kb; 0.9 kb; 0.8 kb; 0.7 kb; 0.6 kb; 0.5 kb; 0.4 kb; 0.3 kb; 0.2 kb; or 0.1 kb of the original marker on the maize genome.
A probe may be an exact copy of a marker to be detected. A probe may also be a nucleic acid molecule comprising, or consisting of, a nucleotide sequence which is substantially identical to a cloned segment of the subject organism's (for example, maize) chromosomal DNA. As used herein, the term “substantially identical” may refer to nucleotide sequences that are more than 85% identical. For example, a substantially identical nucleotide sequence may be 85.5%; 86%; 87%; 88%; 89%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or 99.5% identical to the reference sequence.
A probe may also be a nucleic acid molecule that is “specifically hybridizable” or “specifically complementary” to an exact copy of the marker to be detected (“DNA target”). “Specifically hybridizable” and “specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and the DNA target. A nucleic acid molecule need not be 100% complementary to its target sequence to be specifically hybridizable. A nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to non-target sequences under conditions where specific binding is desired, for example, under stringent hybridization conditions.
Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ and/or Mg++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are known to those of ordinary skill in the art, and are discussed, for example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985. Further detailed instruction and guidance with regard to the hybridization of nucleic acids may be found, for example, in Tijssen, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” in Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, N.Y., 1993; and Ausubel et al., Eds., Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, NY, 1995.
As used herein, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 50% mismatch between the hybridization molecule and the DNA target. “Stringent conditions” include further particular levels of stringency. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 50% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 20% mismatch will not hybridize; and conditions of “very high stringency” are those under which sequences with more than 10% mismatch will not hybridize.
In particular embodiments, stringent conditions can include hybridization at 60° C. in TaqMan® genotyping master mix (Applied Biosystems, Foster City, Calif., Catalog #4371355), diluted according to the manufacturer's instructions.
The following are representative, non-limiting hybridization conditions.
Very High Stringency (detects sequences that share at least 90% sequence identity): Hybridization in 5×SSC buffer at 65° C. for 16 hours; wash twice in 2×SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for 20 minutes each.
High Stringency (detects sequences that share at least 80% sequence identity): Hybridization in 5×-6×SSC buffer at 65-70° C. for 16-20 hours; wash twice in 2×SSC buffer at room temperature for 5-20 minutes each; and wash twice in 1×SSC buffer at 55-70° C. for 30 minutes each.
Moderate Stringency (detects sequences that share at least 50% sequence identity): Hybridization in 6×SSC buffer at room temperature to 55° C. for 16-20 hours; wash at least twice in 2×-3×SSC buffer at room temperature to 55° C. for 20-30 minutes each.
With respect to all probes discussed, supra, the probe may comprise additional nucleic acid sequences, for example, promoters; transcription signals; and/or vector sequences. Any of the probes discussed, supra, may be used to define additionally markers that are tightly-linked to a gene involved in a bmr phenotype (e.g., bm1), and markers thus identified may be equivalent to exemplary markers named in the present disclosure, and thus are within the scope of the invention.
Marker-assisted breeding: As used herein, the term “marker-assisted breeding” may refer to an approach to breeding directly for one or more complex traits (e.g., bm1/reduced lignin content). In current practice, plant breeders attempt to identify easily detectable traits, such as flower color, seed coat appearance, or isozyme variants that are linked to an agronomically desired trait. The plant breeders then follow the agronomic trait in the segregating, breeding populations by following the segregation of the easily detectable trait. However, there are very few of these linkage relationships available for use in plant breeding.
Marker-assisted breeding provides a time- and cost-efficient process for improvement of plant varieties. Several examples of the application of marker-assisted breeding involve the use of isozyme markers. See, e.g., Tanksley and Orton, eds. (1983) Isozymes in Plant Breeding and Genetics, Amsterdam: Elsevier. One example is an isozyme marker associated with a gene for resistance to a nematode pest in tomato. The resistance, controlled by a gene designated Mi, is located on chromosome 6 of tomato and is very tightly linked to Aps1, an acid phosphatase isozyme. Use of the Aps1 isozyme marker to indirectly select for the Mi gene provided the advantages that segregation in a population can be determined unequivocally with standard electrophoretic techniques; the isozyme marker can be scored in seedling tissue, obviating the need to maintain plants to maturity; and co-dominance of the isozyme marker alleles allows discrimination between homozygotes and heterozygotes. See Rick (1983) in Tanksley and Orton, supra.
Meat: As used herein, the term “meat” refers to animal tissue used, for example, as food. The term “meat” typically refers to skeletal muscle and associated fat, but may also refer to non-muscle organs, including lungs, livers, skin, brains, bone marrow, kidneys, testicles, intestines, etc.
Neutral detergent fiber: As used herein the term “neutral detergent fiber” (NDF) refers to a measure of slowly digested material across a wide range of feeds. NDF levels in forage increase as the plant matures. Average levels of NDF in grass silage may be approximately 55 percent DM (550 g/kg DM). The content of NDF in a total ration may be between 35-50% DM. Diets with less than 32 percent NDF may cause problems with acidosis. Diets that contain over 50 percent NDF may be restricted in their intake potential.
Nucleic acid molecule: As used herein, the term “nucleic acid molecule” may refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
Sequence identity: The term “sequence identity” or “identity,” as used herein in the context of two nucleic acid or polypeptide sequences, may refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
As used herein, the term “percentage of sequence identity” may refer to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences, and amino acid sequences) over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.
Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.
The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLASTTM; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, Md.), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLASTTM. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLASTTM (Blastn) program may be employed using the default BLOSUM62 matrix set to default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.
Single-nucleotide polymorphism: As used herein, the term “single-nucleotide polymorphism” (SNP) may refer to a DNA sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. Within a population, SNPs can be assigned a minor allele frequency that is the lowest allele frequency at a locus that is observed in a particular population. This is simply the lesser of the two allele frequencies for single-nucleotide polymorphisms. Different populations are expected to exhibit at least slightly different allele frequencies. Particular populations may exhibit significantly different allele frequencies. In some examples, markers linked to a bm1 phenotype are SNP markers.
SNPs may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence may, but will not necessarily, change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. An SNP in which both forms lead to the same polypeptide sequence is termed “synonymous” (sometimes called a silent mutation). If a different polypeptide sequence is produced, they are termed “non-synonymous.” A non-synonymous change may either be missense or nonsense, where a missense change results in a different amino acid, and a nonsense change results in a premature stop codon. SNPs that are not in protein-coding regions may still have consequences for gene splicing, transcription factor binding, or the sequence of non-coding RNA. SNPs are usually biallelic and thus easily assayed in plants and animals. Sachidanandam (2001) Nature 409:928-33.
Silage: As used herein, the term “silage” refers to a certain type of storage forage. Generally, silage is made from plants (e.g., corn plants) in a process called ensilage. During this process, plants or plant parts undergo anaerobic fermentation caused by indigenous microorganisms (e.g., one or more strains of lactic acid bacteria, for example, Lactobacillus spec.) converting sugars to acids and exhausting any oxygen present in the crop material, which depletion of oxygen preserves the forage in conjunction with bacteria-generated volatile fatty acids, such as acetate, propionate, lactate, and butyrate. Silage is widely used for feeding milk- and meat-producing animals, such as dairy cattle and beef cattle.
The term “producing silage” describes the process of how to obtain silage suitable for feeding to a meat-producing animal. Generally, silage is produced from plants, for example, corn plants, by chopping the harvested plant biomass with a forage harvester.
Fiber source: As used herein, the term “fiber source” refers to a material obtained from a plant or microbial source, which material contains edible fibers. Practical, but not limiting examples of fiber sources include, the hulls of agricultural seed products such as from soy beans, or from grains such as rice, wheat, corn, barley; the stalks from such grains (straw); vegetable/plant-based soap stocks, corn stover, which typically includes the stalks, husks and leaves from a harvested corn plant; processed component fractions of agricultural products that are enriched in fiber, for example corn gluten feed; leaf material from any plant source, and distillers dried grains with or without solubles dried thereon. Thus, in particular examples, a fiber source may include, for example, mixtures of the following: alfalfa, barley products (e.g., straw), beet pulp, soy hulls, switch grass, corn fiber, soy fiber, cocoa hulls, corn cobs, corn husks, corn stove, wheat straw, wheat chaff, rice straw, flax hulls, soy meal, corn meal, wheat germ, corn germ, shrubs, and grasses. For the purpose of clarity in the present disclosure, distillers dried grains (with or without solubles) and distillers grains (with or without solubles) contain fiber, but are not considered “fiber sources.” Distillers dried grains (with or without solubles) and distillers grains (with or without solubles) are considered “corn co-products,” as set forth below.
Corn co-product: As used herein the term “corn co-product” refers to products that remain following the wet milling or dry milling of corn. Non-limiting examples of corn co-products include corn gluten, distillers grains, distillers grains plus solubles, distiller dried grains, distillers dried grains with solubles, condensed distillers solubles, bran cake, modified distillers grains, modified distillers grain plus solubles.
Supplement: As used herein, the term “supplement” refers to any ingredient included in a feed mix to enhance the nutritional value of the feed mix. Commonly used supplements include protein (e.g., soybean meal or urea), minerals (e.g., bone meal), energy (e.g., animal fat), and vitamins.
Trait or phenotype: The terms “trait” and “phenotype” are used interchangeably herein. For the purposes of the present disclosure, a trait of particular interest is bm1/reduced lignin content.
IV. Gene and Gene Variations Associated with bm1 Phenotype, and Uses Thereof
This disclosure provides novel mutant cad2 genes that contribute to a bm1 phenotype in maize. Plants (e.g., maize) that comprise a mutant cad2 gene described herein may have cell walls with lignin levels that are lower than those found in wild-type plants of the same variety. In some embodiments, a nucleic acid molecule comprising a mutant cad2 gene according to the disclosure may be introduced into an organism, for example, to assist in the development of reduced lignin phenotypes. In particular embodiments, the organism may be a plant (e.g., maize). However, a nucleic acid molecule comprising a mutant cad2 gene according to the disclosure may be introduced into an organism that is not a plant, for example, yeast, or prokaryotes. In some embodiments, a mutant cad2 gene according to the disclosure may be used to identify plants comprising a mutant cad2 gene, or plants likely to have a bm1 or other reduced lignin phenotype. For example, the sequence of a mutant cad2 gene according to the disclosure may be used to design probes that detect the presence of a mutant cad2 gene in a plant, or in a sample prepared from a plant. In particular examples, a nucleic acid probe that hybridizes to the nucleotide sequence of a mutant cad2 gene, but not a particular wild-type CAD2 gene, is provided.
In some embodiments, the invention also includes those nucleic acid molecules that comprise a sequence that is substantially identical to a mutant cad2 gene according to the disclosure, which sequence may or may not comprise an AC dinucleotide insertion and/or a transposon insertion as described herein. For example, in some embodiments, a nucleic acid molecule may comprise a sequence that is at least about 85% identical to a mutant cad2 gene according to the disclosure. Thus, a nucleic acid molecule may comprise a sequence that is substantially identical to a mutant cad2 gene according to the disclosure that is 86%; 87%; 88%; 89%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99% or 99.5% identical to a mutant cad2 gene according to the disclosure. Such nucleic acid molecules that are substantially identical to a mutant cad2 gene according to the disclosure may be readily identified and isolated, for example, from any complete or partial genomes readily available to those of skill in the art for a variety of organisms.
Some embodiments also include functional variants of a mutant cad2 gene according to the disclosure. Functional variants of a mutant cad2 gene according to the disclosure may include, for example, a mutant cad2 gene according to the disclosure comprising one or more nucleotide substitutions, deletions, or insertions, wherein the functional variant contributes to a bm1 or other reduced lignin phenotype, as may be measured by routine techniques well-known to those of ordinary skill in the art. For example, a functional variant of a mutant cad2 gene according to the disclosure may comprise an AC dinucleotide insertion and/or a transposon insertion as described herein. The capability of a particular variant of a mutant cad2 gene according to the disclosure to reduce lignin content or contribute to a bm1 phenotype may be determined by routine introduction of the mutation or fragment into a plant, followed by routine observation of the plant for reduced lignin content or other characteristics of a bm1 phenotype. Functional variants of a mutant cad2 gene according to the disclosure may be created by site-directed mutagenesis, induced mutation, or they may occur as allelic variants (polymorphisms, e.g., SNPs).
In some embodiments, therefore, functional variants of a mutant cad2 gene according to the disclosure may comprise additional mutations of a CAD2 gene, fragments smaller than the sequence of a mutant cad2 gene according to the disclosure, and/or chimeric proteins that comprise a mutant cad2 gene according to the disclosure, which functional variants retain the properties of the mutant cad2 gene. Such mutations and fragments are therefore considered to be within the scope of the invention. One of ordinary skill in the art can readily determine whether an additional mutation or fragment of a mutant cad2 gene according to the disclosure retains the properties of the mutant cad2 gene.
In some embodiments, the ability to introduce into a plant, and detect in the plant, a mutant cad2 gene of the disclosure may facilitate marker-assisted breeding and selection of plants having a mutant cad2 gene, which plants may also be likely to have a bm1 or other reduced lignin phenotype. In particular examples, a mutant cad2 gene according to the disclosure may be introduced into a plant (e.g., by genetic transformation, or traditional breeding techniques such as crossing), and a plant comprising the mutant cad2 gene may then be selected by using a probe that detects the presence of the mutant cad2 gene in the plant. Plants into which a mutant cad2 gene of the disclosure have been introduced may be crossed with plants that may or may not also comprise a mutant cad2 gene, and progeny may be then be selected by using a probe that detects the presence of the mutant cad2 gene of the disclosure in the plant. Further crosses may be made to obtain a plant of a desired genotype.
When a nucleic acid sequence (e.g., a mutant cad2 gene according to the disclosure) is “introduced” into an organism, such as a plant, the technique or methodology used for the introduction of a nucleic acid molecule comprising the particular sequence is not essential to the invention, and may occur by any technique or methodology known to those of skill in the art. For example, a nucleic acid molecule may be introduced by direct transformation methods, such as Agrobacterium-mediated transformation of plant tissue; microprojectile bombardment; electroporation; etc. Alternatively, a nucleic acid molecule may be introduced by crossing a plant having the particular nucleotide sequence with another plant, such that progeny have the nucleotide sequence incorporated into their genome. Such breeding techniques are well-known to one skilled in the art. Marker-assisted breeding techniques, as disclosed herein, may greatly facilitate the incorporation of a mutant cad2 gene according to the disclosure through such crosses.
In embodiments wherein a mutant cad2 gene according to the disclosure is introduced to an organism, it may be desirable for the mutant cad2 gene to be introduced in such a manner that the mutant cad2 gene is operably linked to one or more regulatory sequences, for example, introduction via the use of a plasmid comprising the mutant cad2 gene according to the disclosure operably linked to the desired regulatory sequences. Regulatory sequences useful in the expression of heterologous nucleic acid sequences are well-known in the art, and include, for example and without limitation: Promoters (e.g., constitutive promoters; tissue-specific promoters; and developmental stage-specific promoters); termination sequences; enhancer sequences; subcellular targeting sequences; stabilizing or leader sequences; and introns.
A mutant cad2 gene according to the disclosure may be introduced into an organism with one or more marker genes operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection (i.e., inhibiting growth of cells that do not contain the selectable marker gene) or by positive selection (i.e., screening for the product encoded by the genetic marker). Many selectable marker genes for transformation are well known in the transformation arts and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which may be insensitive to the inhibitor. A few positive selection methods are also known in the art. Examples of marker genes suitable for use in plant cells may include, for example, and without limitation: The neomycin phosphotransferase II (nptII) gene (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803); the hygromycin phosphotransferase gene (Vanden Elzen et al. (1985) Plant Mol. Biol. 5:299); gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, and the bleomycin resistance determinant (See, e.g., Hayford et al. (1988) Plant Physiol. 86:1216; Jones et al. (1987) Mol. Gen. Genet. 210:86); Svab et al. (1990) Plant Mol. Biol. 14:197; and Hille et al. (1986) Plant Mol. Biol. 7:171); selectable marker genes that confer resistance to herbicides, such as glyphosate, glufosinate or bromoxynil (See, e.g., Comai et al. (1985) Nature 317:741-744; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; and Stalker et al. (1988) Science 242:419-423); mouse dihydrofolate reductase (Eichholtz et al. (1987) Somatic Cell Mol. Genet. 13:67); plant 5-enolpyruvylshikimate-3-phosphate synthase (Shah et al. (1986) Science 233:478); plant acetolactate synthase (Charest et al. (1990) Plant Cell Rep. 8:643).
Another class of marker genes suitable for plant transformation employs screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance, such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues, and are frequently referred to as “reporter genes,” because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase and chloramphenicol acetyltransferase. See, e.g., Jefferson (1987) Plant Mol. Biol. Rep. 5:387; Teeri et al. (1989) EMBO J. 8:343; Koncz et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:131; and DeBlock et al. (1984) EMBO J. 3:1681.
Recently, in vivo methods for visualizing GUS activity that do not require destruction of plant tissue have been made available. Molecular Probes publication 2908, Imagene GreenTM, p. 1-4, 1993; and Naleway et al. (1991) J. Cell Biol. 115:151a. Further, genes encoding Fluorescent Proteins (e.g., GFP, EGFP, EBFP, ECFP, and YFP) have been utilized as markers for gene expression in prokaryotic and eukaryotic cells. See Chalfie et al. (1994) Science 263:802. Fluorescent proteins and mutations of fluorescent proteins may be used as screenable markers.
In some embodiments, a mutant cad2 gene according to the disclosure and/or fragments or segments of a mutant cad2 gene according to the disclosure may be used to identify homologous mutant cad2 gene sequences from organisms other than maize (e.g., by sequence comparison). Sequences from organisms other than maize that are homologous to a mutant cad2 gene according to the disclosure may be identified and isolated according to well-known techniques, for example, based on their sequence homology to a mutant cad2 gene according to the disclosure.
Thus, in some embodiments, all or part of a coding sequence from a mutant cad2 gene according to the disclosure may be used as a probe which specifically hybridizes to other sequences present in a population of cloned genomic DNA fragments (i.e., a genomic library) from an organism according to routine techniques. Thus, in some embodiments, the invention includes those nucleotide sequences which specifically hybridize to a mutant cad2 gene according to the disclosure.
In further embodiments, sequences from organisms other than maize that are homologous to a mutant cad2 gene according to the disclosure may be identified and isolated by sequence comparison. For example, the complete or partial sequenced genome of an organism may be searched according to routine techniques with the sequence of a mutant cad2 gene according to the disclosure to identify genes within the genome of the organism that share a high degree of sequence identity with the mutant cad2 gene, and are therefore likely homologues of the mutant cad2 gene.
For example, all or part of a mutant cad2 gene according to the disclosure may be used as a “reference sequence.” Generally, nucleic acid sequences (e.g., cloned or genomic DNA fragments of a genomic library) that are compared to the reference sequence comprise a “comparison window,” which is a specific contiguous segment of the nucleic acid sequence. The comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window is typically at least 20 contiguous nucleotides in length, but may be 30, 40, 50, 100, or 200 nucleotides in length, or longer. To avoid a high similarity to the reference sequence due to inclusion of deletions in the polynucleotide sequence comparison window, a “gap penalty” may be introduced to be subtracted from the number of nucleotide matches.
Methods of aligning sequences for comparison are well-known in the art. The determination of percent sequence identity between any two sequences can be accomplished using available mathematical algorithms. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988), CABIOS 4:11-7; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970), J. Mol. Biol. 48:443-53; the search-for-local-alignment method of Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA 85:2444-8; the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264, and Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-7.
One of ordinary skill in the art can implement these mathematical algorithms on a computer for comparison of sequences to determine sequence identity, or to search a database comprising a plurality of sequences (e.g., an organism genome database) according to shared sequence identity with a reference sequence. Such implementations include, but are not limited to, CLUSTAL in the PC/Gene program (Intelligenetics, Mountain View, Calif.); and the ALIGN program and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, v. 10 (Accelrys Inc., San Diego, Calif.). Sequence alignments using these programs may be performed using their default parameters. Alternatively, it may be desirable to modify the default parameters in some searches (e.g., altering the value of a gap penalty). The selection of a particular computer implementation of mathematical algorithms for calculation of sequence identity and the selection of parameter values for use in a selected algorithm are within the discretion of one of skill in the art.
In some embodiments, a mutant cad2 gene according to the disclosure may be introduced into a plant comprising an additional desired gene (e.g., a mutant gene or minor allele) or phenotype by crossing. Alternatively, a mutant cad2 gene according to the disclosure may be introduced into a plant comprising an additional desired gene or phenotype, for example, by genetic transformation. In certain embodiments, a mutant cad2 gene according to the disclosure may be introduced into a plant comprising a mutation in one or more additional genes involved in lignin biosynthesis or metabolism. For example and without limitation, a mutant cad2 gene according to the disclosure may be introduced into a plant comprising a mutation in, e.g., a different CAD gene; COMT; 4CL; FSH; EgCAD1-type ZmCAD1; SAD; a chorismate mutase gene; PAL; HCT; OMT; a cytochrome P450 gene; SAMS3; CCR; DFR; a chalcone synthase gene; a laccase gene; a peroxidase gene; a ALDH gene (for example, one of six maize ALDH genes described in Skibbe et al. (2002) Plant Mol. Biol. 48:751-64); AGO; an MYB transcription factor gene (e.g., ZmMYB38; and maize APL); a LIM transcription factor gene; a maize bZIP factor gene; an MADS-box gene (e.g., ZmZAG5); an MP gene; an ABC transporter gene; a β-glucosidase gene; a glutathione S-transferase gene (e.g., ZmGST17); a nodulin MtN21-like gene; a PINOID orthologous gene; a histidine kinase (e.g., ZmHK1, ZmHK2, and ZmHK3); and/or a COV1 orthologous gene. In some embodiments, a mutant cad2 gene according to the disclosure may be introduced into a plant comprising one or more desirable trait(s) that are not involved in lignin biosynthesis or metabolism, in order to produce a plant with both a bm1 or other reduced lignin phenotype and the one or more desirable trait(s) that are not involved in lignin biosynthesis or metabolism.
It is expected that plants of these and other embodiments that contain multiple mutant genes in the lignin biosynthetic or metabolic pathways will exhibit useful novel phenotypes, for example, additional bmr phenotypes, and/or other lignin phenotypes. See, e.g., Marita et al. (2003), supra. For example, stacking the bm1 trait with the bm3 trait was observed to result in further increases in silage digestibility, and a very significant improvement in the yield of fermentable sugars in biomass hydrolysis without pre-treatment.
Some embodiments utilize means for producing a genetically modified plant comprising a mutant maize cad2 gene, and/or means for identifying plants carrying a mutant maize cad2 gene. In some examples, a means for producing a genetically modified plant comprising a mutant maize cad2 gene may be a marker that is located within a mutant maize cad2 gene according to the disclosure. In some examples, a means for identifying plants carrying a mutant maize cad2 gene may be a probe that specifically hybridizes to a marker that is located within a mutant maize cad2 gene according to the disclosure. Mutant cad2 genes according to the disclosure (with respect to a means for producing a genetically modified plant comprising a mutant maize cad2 gene, and a means for identifying plants carrying a mutant maize cad2 gene) are those mutant cad2 genes that contribute to a bm1 phenotype in either maize inbred line, 515Dbm1, or line, DASbm1. These mutant cad2 genes are characterized by either a frame shift mutation in the third exon of a CAD2 gene that results in a truncated CAD2 protein, or a 3444 base pair transposon insertion in the first intron of a CAD2 gene that results in a truncated CAD2 protein. Thus, in some embodiments, a mutant cad2 gene may comprise the first exon of the genomic ZmCAD2 gene sequence and/or the second exon of the genomic ZmCAD2 gene sequence, but may not comprise all or part of the third exon of the genomic ZmCAD2 gene sequence.
The mutant cad2 genes disclosed herein are different from previously described cad2 mutations that may be involved in bmr phenotypes. For example, a mutant cad2 gene according to the disclosure may have one or more of the following characteristics: a dinucleotide insertion in an exon of a ZmCAD2 gene (e.g., exon 3); a mutation resulting in a frame shift; a transposon insertion in an intron or exon of a ZmCAD2 gene that results in a truncated protein; a mutation resulting in a truncated ZmCAD2 enzyme; a mutation resulting in the inactivation (e.g., by removal) of the ZmCAD2 NADPH-binding and/or C-terminal catalytic domain(s); a mutation that contributes to a bm1 phenotype in inbred maize line 515Dbm1; and a mutation that contributes to a bm1 phenotype in inbred maize line DASbm1. In contrast, previously described naturally-occurring mutant cad2 genes are characterized by the insertion of a transposon in the last exon of the gene (exon 4) (U.S. Patent Application US 2010/0203196 A1); reduced expression of a full-length CAD protein (Halpin et al. (1998), supra); a point mutation leading to truncation of the Sorghum bicolor CAD2 enzyme (Saballos et al. (2009), supra; Sattler et al. (2009), supra); a point mutation in the cofactor binding site in S. bicolor CAD2 (Id.); disrupted secondary structure outside the active site in S. bicolor CAD2 (Id.); and a two-base pair adenosine insertion in a Pinus taeda L. CAD gene that results in a truncated enzyme (U.S. Pat. No. 6,921,643).
V. Molecular Markers Linked to Gene and Gene Variations Associated with a bm1 Phenotype, and Uses Thereof
Molecular markers that are linked (e.g., tightly-linked) to a mutant cad2 gene according to the disclosure are provided. A molecular marker linked to a mutant cad2 gene according to the disclosure may be described in part, by its position in a particular region on maize chromosome 5. Molecular markers that are linked to a mutant cad2 gene according to the disclosure may include SNP markers that are within, or that reside near, the mutant cad2 gene. For example, in some embodiments, a G/A SNP located at nucleotide position 5410 in the cad2 gene from bm1 maize line, 515Dbm1, and at nucleotide position 8903 in bm1 maize line, DASbm1 (or a marker equivalent thereto), may be a molecular marker that is linked to a mutant cad2 gene according to the disclosure. In some embodiments, a G/A SNP located at nucleotide position 5410 in the cad2 gene from bm1 maize line, 515Dbm1, may be a molecular marker that is linked to a mutant cad2 gene according to the disclosure.
Additional markers may be identified, for example, by determining the frequency of recombination between the additional marker and a cad2 mutation according to the disclosure, or a molecular marker that is linked to a mutant cad2 gene according to the disclosure (e.g., the G/A SNP located at nucleotide position 5410 in bm1 maize line, 515Dbm1). Such determinations my utilize an improved method of orthogonal contrasts based on the method of Mather (1931), The Measurement of Linkage in Heredity, Methuen & Co., London, followed by a test of maximum likelihood to determine a recombination frequency. Allard (1956) Hilgardia 24:235-78. If the value of the recombination frequency is less than or equal to 0.10 (i.e., 10%) in the organism (e.g., maize), then the additional marker is considered to be linked to the mutant cad2 gene, and equivalent to the particular reference marker for the purposes of use in the presently disclosed methods.
Methods of using nucleic acid molecular markers that are linked to or that reside within a mutant cad2 gene according to the disclosure to identify plants with a bm1 or other reduced lignin phenotype may result in a cost savings for plant developers, because such methods may eliminate the need to cross plants comprising a mutant cad2 gene with other plant lines, and then phenotype the progenies of the cross.
A means for producing a genetically modified plant comprising a mutant maize cad2 gene is a marker that is linked to a mutant cad2 gene according to the disclosure. Thus, a means for producing a genetically modified plant comprising a mutant maize cad2 gene includes nucleic acid sequences from maize plants, the detection of any of which nucleic acid sequences provides at least a strong indication that the plant comprising the nucleic acid sequence comprises a mutant 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. Specific hybridization of nucleic acids is a detectable signal, and a nucleic acid probe that specifically hybridizes to a marker that is linked to or that resides within a mutant maize cad2 gene according to the disclosure therefore presents a detectable signal when added to a sample obtained from a plant carrying a mutant cad2 gene according to the disclosure.
In some embodiments, linked markers flanking a mutant cad2 gene according to the disclosure in an organism's genome may be used to transfer segment(s) of donor parent DNA that unequivocally contain the mutant cad2 gene. In some embodiments, a method for using linked markers flanking a mutant cad2 gene according to the disclosure to transfer segment(s) of donor parent DNA that unequivocally contain the mutant cad2 gene may comprise analyzing the genomic DNA of two parent plants with probes that are specifically hybridizable to markers linked to the mutant cad2 gene; sexually crossing the two parental plant genotypes to obtain a progeny population, and analyzing those progeny for the presence of the markers linked to the mutant cad2 gene; backcrossing the progeny that contain the markers linked to the mutant cad2 gene to the recipient genotype to produce a first backcross population, and then continuing with a backcrossing program until a final progeny is obtained that comprises any desired trait(s) exhibited by the parent genotype and the mutant cad2 gene. In particular embodiments, individual progeny obtained in each crossing and backcrossing step are selected by linked marker analysis at each generation. In some embodiments, analysis of the genomic DNA of the two parent plants with probes that are specifically hybridizable to markers linked to the mutant cad2 gene reveals that one of the parent plants comprises fewer of the linked markers to which the probes specifically hybridize, or none of the linked markers to which the probes specifically hybridize.
In some embodiments, markers that are linked to or that reside within a mutant cad2 gene according to the disclosure, or the mutant cad2 gene sequence itself, may be used to introduce the mutant cad2 gene according to the disclosure into a maize plant by genetic transformation. In particular embodiments, the markers include without limitation the G/A SNP located at nucleotide position 5410 in bm1 maize line, 515Dbm1, and at nucleotide position 8903 in bm1 maize line, DASbm1, or a marker equivalent thereto. In some embodiments, a method for introducing a mutant cad2 gene according to the disclosure into a maize plant by genetic recombination may comprise analyzing the genomic DNA of a plant (e.g., a maize plant) with probes that are specifically hybridizable to markers linked to the mutant cad2 gene or the mutant cad2 gene itself to identify the mutant cad2 gene in the plant; isolating a segment of the genomic DNA of the plant comprising the mutant cad2 gene, for example, by extracting the genomic DNA and digesting the genomic DNA with one or more restriction endonuclease enzymes; optionally amplifying the isolated segment of DNA; introducing the isolated segment of DNA into a cell or tissue of a host maize plant; and analyzing the DNA of the host maize plant with probes that are specifically hybridizable to markers linked to the mutant cad2 gene or the mutant cad2 gene itself to identify the mutant cad3 gene in the host maize plant. In particular embodiments, the isolated segment of DNA may be introduced into the host maize plant such that it is stably integrated into the genome of the host maize plant.
In some embodiments, markers that are linked to or that reside within a mutant cad2 gene according to the disclosure, or the mutant cad2 gene sequence itself, may be used to introduce a mutant cad2 gene according to the disclosure into other organisms, for example, plants. In particular embodiments, the markers include without limitation the G/A SNP located at nucleotide position 5410 in bm1 maize line, 515Dbm1, and at nucleotide position 8903 in bm1 maize line, DASbm1, or a marker equivalent thereto. In some embodiments, a method for introducing the mutant cad2 gene according to the disclosure into an organism other than maize (e.g., soybean; alfalfa; wheat; rapeseed; rice; and sorghum) may comprise analyzing the genomic DNA of a plant other than a maize plant with probes that are specifically hybridizable to markers linked to the mutant CAD2 gene according to the disclosure or the mutant cad2 gene itself to identify the mutant cad2 gene in the plant; isolating a segment of the genomic DNA of the plant comprising the mutant cad2 gene, for example, by extracting the genomic DNA and digesting the genomic DNA with one or more restriction endonuclease enzymes; optionally amplifying the isolated segment of DNA; introducing the isolated segment of DNA into an organism other than maize; and analyzing the DNA of the organism other than maize with probes that are specifically hybridizable to markers linked to the mutant cad2 gene according to the disclosure or the mutant cad2 gene itself to identify the mutant cad2 gene in the organism. In particular embodiments, the isolated segment of DNA may be introduced into the organism such that it is stably integrated into the genome of the organism.
In some embodiments, markers that are linked to or that reside within a mutant cad2 gene according to the disclosure, or the mutant cad2 gene sequence itself, may be used to identify a plant with a bm1 or other reduced lignin phenotype. In particular embodiments, the plant is a maize plant. In some embodiments, nucleic acid molecules (e.g., genomic DNA or mRNA) may be extracted from a plant. The extracted nucleic acid molecules may then be contacted with one or more probes that are specifically hybridizable to markers linked to a mutant cad2 gene according to the disclosure or the mutant cad2 gene itself. Specific hybridization of the one or more probes to the extracted nucleic acid molecules is indicative of the presence of a bm1 or other reduced lignin phenotype in the plant.
Primer Design and Linkage Screening.
Oligonucleotide probes (e.g., primers) may be designed to specifically detect markers that are physically located in, near, or between cad2 that are linked to a bm1 mutation. In general, an oligonucleotide probe may be designed that specifically hybridizes to only one allele of a marker. In some embodiments, two oligonucleotide probes are designed to detect a bm1 marker, such that one specifically hybridizes to the bm1 allele to which the other probe does not specifically hybridize, and the other specifically hybridizes to the wild-type CAD2 allele (or different bm1 allele) to which the other probe does not specifically hybridize. As is understood by those of skill in the art, the length or composition of oligonucleotide probes for a particular marker may be varied according to established principles without rendering the probe non-specific for one allele of the marker.
In some embodiments, the oligonucleotide probes may be primers. In specific embodiments, primers may be designed to detect markers in a KASParTM genotyping assay. In particular embodiments, primers may be designed to detect markers linked to the bm1 phenotype in maize using a KASParTM genotyping assay. In these and further embodiments, the detection system may provide a high-throughput and convenient format for genotyping individuals in a mapping population, which may greatly facilitate the identification of individuals carrying a particular gene or trait, and may also greatly facilitate the implementation or execution of a marker-assisted selection program.
In specific embodiments, the oligonucleotide probes may be primers designed to detect markers in a TAQMAN® genotyping assay. This method utilizes specific primers to amplify a region of DNA containing a marker closely linked to a bm1 mutation and fluorescent labeled probes specific to the marker. The probe that specifically hybridizes to the bm1 allele may be labeled with a fluorescent dye such as FAM, while the probe that specifically hybridizes to the wild-type CAD2 allele (or different bm1 allele) may be labeled with a different fluorescent dye such, as VIC. The data is analyzed as the presence or absence of a fluorescent dye signal. The detection system may provide a high-throughput and convenient format, such as multiplexing for genotyping individuals in a mapping population, which may greatly facilitate the identification of individuals carrying a particular gene or trait, and may also greatly facilitate the implementation or execution of a marker-assisted selection program.
Thus, also described herein is a gene specific end-point TaqMan® PCR assay, generally useful for zygosity analysis of bm1 corn or putative bm1 corn. In particular embodiments, a gene specific end-point TaqMan® PCR assay may be used to analyze the zygosity of corn for a bm1 mutation.
Primers and probes for use in a gene specific end-point TaqMan® PCR assay may be designed based on a known mutation in the gene of interest. For example, primers and probes for a bm1-specific assay may be designed based on a 3444-bp insertion in the first intron of the cad2 gene. In biplex reactions, wherein oligonucleotides specific to the mutation (e.g., bm1) and the undisrupted wild-type gene (e.g., CAD2) are used in the same assay, the specific oligonucleotides will selectively amplify sequence from either or both of the mutated gene and/or the wild-type gene that is present in a genomic DNA sample.
In some embodiments, a bm1-specific assay amplifies a fragment that is unique to the 3444 bp of nucleotide sequence that was inserted into the wild-type genomic CAD2 gene. In some embodiments, a wild-type CAD2 gene-specific assay amplifies a fragment of the CAD2 gene that comprises the junction site where 3444 bp of nucleotide sequence was inserted into the wild-type CAD2 gene. In certain embodiments, a target-specific oligonucleotide probe hybridizes under high-stringency conditions to a target sequence in a genomic DNA sample between two PCR primers.
Target-specific oligonucleotides may be labeled, for example, with fluorescent dyes (e.g., FAM, VIC, and MGBNFQ), which may allow rapid quantification of a target-specific fluorescent signal. PCR products may be measured after a pre-determined number of cycles, for example, when the reaction is in the early exponential phase. Negative control samples may comprise genomic DNA from any corn variety, for example, without a bm1 mutation. Positive control samples may comprise genomic DNA from a corn variety with a bmr mutation, such as a bm1 insertion mutation in the CAD2 gene. Control hemizygous samples may comprise either genomic DNA from a corn variety predetermined to be hemizygous for a bm1 mutation; or a hemizygous sample may comprise equal proportions of negative control DNA to DNA from a corn variety predetermined to be homozygous for a bm1 mutation.
DNA may be isolated (for example, extracted, and purified) from corn plant tissue by methods known to those of skill in the art. Commercial kits for DNA isolation are available, for example, from Qiagen, Inc. In some embodiments, leaf discs from a particular plant are punched and transferred into collection tubes. The puncher may be cleaned after each sampling with 70% alcohol, rinsing in water, and drying. DNA extraction buffers may be prepared according to the manufacturer's recommendations. DNA may then be isolated using the kit according to the manufacturer's instructions. Finally, the concentration of the isolated DNA may be determined using, for example, a Quant-iTTM PicoGreen® Quantfication Kit (Invitrogen, Carlsbad, Calif.) and a spectrophotometer, or by any other suitable technique.
Once primers, probes, and genomic DNA sample(s) have been prepared or otherwise made available, a PCR reaction may be conducted to identify nucleic acid sequences of interest (e.g., sequences particular to a bmr mutation) in the genomic DNA sample(s). In particular embodiments, individual PCR reaction mixtures are prepared that contain all the reaction components, except the genomic DNA sample(s). For a biplex reaction comprising primers and gene-specific probes for bm1 mutant and wild-type CAD2 corn, the reaction mixture may comprise enzyme, reaction buffer, forward and reverse primers for the bm1 mutation, forward and reverse primers for the wild-type CAD2 gene, a gene-specific probe for the bm1 mutation, a gene-specific probe for the CAD2 gene, and water. In some PCR assay systems (e.g., TaqMan® PCR assays), enzyme and buffer may be present in a single kit component (e.g., TaqMan® genotyping master mix; Applied Biosystems, Foster City, Calif., Catalog #4371355).
Once the reaction mixture is otherwise prepared, genomic DNA sample(s) may be added, and the reaction commenced. It is not necessary to normalize the amounts of genomic DNA in a sample. However, the skilled artisan may attain best results by using genomic DNA samples with relatively equal concentrations.
In some embodiments, a PCR assay (e.g., a TagMan® PCR assay) can be set up with appropriate controls. For example, a reaction in a multi-well plate may be performed with control wells comprising: (1) negative control(s) with reagents but no DNA sample; (2) homozygous positive control(s) comprising bm1 corn genomic DNA; (3) and hemizygous positive control(s), as described above. DNA is then amplified by PCR under suitable cycle conditions. For example, in some embodiments using a GenAmp® PCR System 9700, there may be a single initial denaturation cycle at 95° C. for 15 minutes, followed by 30 cycles of denaturation (92° C. for 15 seconds) and annealing/extension (60° C. for 60 seconds). Those of skill in the art understand that PCR cycle conditions may be varied according to the practitioner's discretion, and comparable results obtained.
A PCR assay (e.g., an end-point TaqMan® PCR assay) may be used for genotype and/or zygosity analysis of bmr corn or putative bmr corn. In some embodiments, a gene-specific oligonucleotide probe may be labeled with a reporter (e.g., a fluorescent moiety). For assays using fluorometric detection, PCR reaction products may be analyzed in a spectrofluorometer (e.g., Tecan GENiosTM; Männedorf, Switzerland) using excitation and emission wavelength settings appropriate for the detection of the probe(s). For example, the fluorescent dye, FAM, may be measured with an excitation wavelength of 485 nm, and an emission wavelength of 535 nm. Alternatively, the fluorescent dye, VIC, may be measured with an excitation wavelength of 525 nm, and an emission wavelength of 560 nm.
Following the completion of the PCR reaction and probe detection, a table and distribution graph may be generated using, for example, any suitable computer graphics software. Results obtained with wild-type, hemizygous, and homozygous DNA of similar genotypic background may serve as negative and positive controls. In a segregating population, three clusters of data points may be obtained allowing the visual determination of a sample result as likely belonging to one of the segregated clusters. Alternatively, data analysis computer software may be used to calculate the probability that a sample result belongs to each segregated cluster, with the most probable cluster serving as the sample designation. When a visual determination is made, the boundary of each cluster may be arbitrary, for example, when three clusters of data points are clearly visible.
Raw fluorescence intensity data may also be analyzed directly from a plate reader using a suitable analysis package, such as KLIMSTM (KBioscience laboratory information management system). A graph with the relative fluorescence units (RFU) of a fluorescence signal generated by a specific probe for a mutant allele plotted on one axis, and the RFU of a fluorescence signal generated by a specific probe for the wild-type allele plotted on the other axis may be generated. Zygosity determinations may then be made based on the cluster separation in a graphical display of the data.
Samples that do not contain mutant genomic DNA (e.g., a bmr mutation) may only result in fluorescence readings of the wild-type PCR product. Samples containing hemizygous or homozygous mutant genomic DNA may result in RFU readings for the mutant-specific probe higher than that of a negative background control. If a sample yields no adequate results, the genomic DNA in the sample may not be of adequate quality and/or quantity, and a new DNA preparation and/or new PCR reaction should be performed. Preferably, a negative control sample containing no DNA sample shows very low detection of gene-specific probe(s). It is also preferable that known homozygous controls show only high detection of the mutant or wild-type DNA in the control, and that known hemizygous controls show both high detection of the mutant and wild-type DNA.
A “test run” of the PCR method and genotype and/or zygosity determination may be performed with all appropriate controls prior to screening of samples. Further optimization of the methods may be desirable for components that may differ among uses (e.g., method of genomic DNA preparation, Taq DNA polymerase, oligonucleotides, laboratory equipment, etc.). PCR and thermal cycling conditions may be established that amplify both mutant and/or wild-type sequences in a known genomic DNA template with acceptable levels of probe detection (e.g., acceptable RFU for fluorescently labeled oligonucleotide probes).
VI. Organisms Having Reduced CAD2 Activity and Uses Thereof
A. Organisms Having Reduced CAD2 Activity
Some embodiments of the present invention also provide an organism including a nucleic acid molecule comprising a mutant cad2 gene according to the disclosure, a nucleic acid sequence that is specifically hybridizable to a mutant cad2 gene according to the disclosure, or a functional variant of a mutant cad2 gene according to the disclosure. A suitable organism can be any suitable plant, yeast, or bacterium. By way of non-limiting example, a plant comprising the aforementioned sequences may be a plant of agronomic value, for example and without limitation: maize; soybean; alfalfa; wheat; rapeseed; rice; sorghum; beet; various vegetables including cucumber, tomato, peppers, etc.; various trees including apple, pear, peach, cherry, redwood, pine, oak, etc.; and various ornamental plants. In particular embodiments, the organism may be a plant that is used to produce silage.
Plant cells comprising a mutant cad2 gene according to the disclosure, a nucleic acid sequence that is specifically hybridizable to a mutant cad2 gene according to the disclosure, or a functional variant of a mutant cad2 gene according to the disclosure, may be cultured and kept as plant tissue culture cells, or certain plant hormones known in the art can be added to the culture media, thereby causing the plant tissue culture cells to differentiate and form a new plant variety, which new plant variety may exhibit a bm1 or other reduced lignin phenotype. Such plant culturing methods useful in these and other embodiments are routine and well-known in the art.
Some embodiments of the invention provide a virus (e.g., a bacteriophage, or plant virus) comprising a mutant cad2 gene according to the disclosure, a nucleic acid sequence that is specifically hybridizable to a mutant cad2 gene according to the disclosure, or a functional variant of a mutant cad2 gene according to the disclosure.
B. Silage Produced from Plants Having Reduced CAD2 Activity
Disclosed herein are methods for increasing the meat quantity of a silage-fed animal that use silage produced from plant varieties exhibiting reduced lignin content to improve daily gain and feed efficiency when compared to conventional corn silage in a finishing ration. Such silage can effectively replace grain corn in a beef finishing ration. In some embodiments, the method comprises providing silage produced from a plant variety comprising a mutant cad2 gene according to the disclosure, feeding the animal with the silage produced from the plant variety comprising a mutant cad2 gene according to the disclosure, and producing meat or meat products from the animal. In these and further embodiments, the silage-fed animal may be a ruminant. In particular embodiments, the silage-fed animal may be any silage-fed animal, for example, cattle, sheep, swine, horses, goats, bison, yaks, water buffalo, and deer.
In some embodiments, methods provided for increasing the meat quantity of a silage-fed animal further comprise an act selected from the group consisting of: placing the silage in a container configured for shipping, and associating indicia with the silage, wherein the indicia is capable of directing an end-user on how to administer the silage to the animal. Thus, kits comprising silage are provided, such that the kits allow an end-user to increase the meat quantity of a silage-fed animal.
Also disclosed are beef finishing rations, wherein the beef finishing ration comprises corn silage comprising a mutant cad2 gene according to the disclosure. Also disclosed are meat and meat products prepared from an animal that has been fed such silage.
Production of silage from plants (e.g., maize) having reduced CAD2 activity.
During ensilage, the cells of the plant are still alive and metabolically active, and ongoing metabolism by plant cells and microorganisms in the compressed silage forms carbon dioxide and heat by using air trapped in the ensiled plant material. Anaerobic metabolic conditions develop as the level of carbon dioxide in the silage increases. Desirable bacteria begin the fermentation process when plant respiration stops. If too much air is present, or if carbon dioxide escapes, an anaerobic condition may fail to develop. In this case, respiration may continue, and the respiring plant cells may use too much sugar and carbohydrates. This may waste nutrients needed by desirable bacteria to preserve the plant material as silage, and may yield an inferior silage. To avoid this undesirable effect, packing and covering of the silage immediately after filling may be important.
Once respiration by the plant cells ceases, acetic and lactic acids are produced by bacteria that feed on the available starches and simple sugars in the ensiled corn. To promote growth of desirable bacteria, the silage may contain a low amount of air, temperatures between 80° and 100° F., and starches and sugars for food. Fermentation may continue until the acidity of the silage is high enough to stop bacterial growth. In some examples, the desired degree of acidity is a pH of about 4.2. This degree of acidity may occur within 3 weeks after the silo is filled.
Seepage may occur if moisture in the forage is excessively high. Seepage involves the drainage of leachate (excess moisture from silage and pulp) out of the silage, which generally enters the environment as a serious pollutant. Through seepage, desirable components (e.g., nitrogenous compounds, such as protein; and minerals) of the silage may be lost. Seepage generally reaches its peak on about the fourth day after ensiling. Therefore to avoid, for example, the loss of desirable silage components from the silage, moisture content of forage going into the silo may be chosen to be sufficiently low to reduce or prevent seepage loss. However, silage that is too dry may not pack adequately, and may also exhibit a high loss of desirable components from the silage as a consequence of excessive fermentation and molding.
Plants may be ensiled at a dry matter content of about 30-40% to enable an optimal fermentation process, and to minimize losses during fermentation. To reach a dry matter content of about 30-40%, it may be desirable to let the plant material dry down in a field after mowing and before chopping with, for example, a forage harvester. When preparing silage, the grain may be harvested together with the rest of the plant. To increase the availability of nutrients in the silage for uptake in the intestinal tract of a silage-fed animal, it may be desirable to crush the grain during the chopping process.
Harvested plant material (e.g., maize plant material) may be transferred into a silo. Non-limiting examples of silos that may be useful for silage preparation include: a bunker silo, a silage heap, a concrete stave silo, or a tower silo. The plant material is compacted in the silo to remove air from the plant material, and enable anaerobic fermentation. It may be desirable to seal the silo with a plastic silage film, depending on the type of silo used. Use of a plastic cover on a trench silo, a bunker silo, or a large-diameter tower silo, may materially cut feed losses. Typically, the cover is applied immediately after the last load of plant material is packed in the silo, and the plastic covers are weighted to hold them firmly on the surface of the silage. Alternatively, the plant material may be prepared for fermentation during ensiling by baling the plant material, and wrapping the bales in silage film for sealing. On trench or bunker silos, it may be desirable to mound or crown the forage. This may facilitate drainage of rain water off the silo.
Additives may optionally be added to the plant material to improve fermentation. Examples of plant material additives that may be desirable in particular applications include microbial additives, such as Lactobacillus spp. and other inoculants; acids such as propionic acid, acetic acid or formic acid; or sugars. As will be readily understood by those of skill in the art, other methods for producing silage other than those specifically recited herein may also be used.
One advantage of silage production is that the process may have no influence on the composition, the amount, or availability of nutritive substances contained within the plant material used for producing the silage. On the contrary, purposes of the process itself are generally to both keep the quality of the plant material as it was prior to using such material for producing silage, and to preserve the positive properties of the plant material for an extended period of time. In this way, the plant material can be used as forage long after the plant material has been harvested.
Corn may be harvested for silage after the ear is well-dented, but before the leaves dry to the point that they turn brown. At this stage of growth, the ear may have accumulated most of its potential feeding value, but there may also have been little loss from the leaves and stalks. Thus, the quantity and quality of corn silage may be at its peak when the plant material is harvested during this stage. Ears usually will be well-dented when the ears are between 32-35% moisture. As time elapses after the ear has become well-dented, the feeding value of the plant material may decrease while field losses may increase. Corn harvested for silage at the milk stage (grain head releases a white liquid when opened) or dough stage (grain head begins to turn a doughy consistency) may yield less feed nutrients per acre than if it was harvested later. Plant material from corn may also ferment improperly in a silo if it is harvested too soon.
Maturity usually refers to the time when the ear has accumulated nearly all of its dry matter production potential. Temperatures during growth may influence the maturity rate of the grain, particularly during the autumn. For example, the ear's full dry matter potential may not be achieved if there are excessively cool temperatures and/or cloudy weather. Corn silage that is cut late and has brown and dead leaves and stalks may make adequate silage, but total production per acre may be sharply reduced. Significant field losses have been found when silage is made late into the fall or early winter. Also, a reduction in the amount of dry matter stored in the silo may be found with respect to silage that is cut late.
Corn that has been damaged, for example, by drought, high temperatures, blight, frost, or hail, may be salvaged for silage. However, the quality of such salvaged silage may not be as high as silage produced from undamaged corn that has reached the dent stage. The feeding value of the silage may depend upon both the state of the corn's development, and how the corn is handled after it has been damaged. Common observations of silage from immature corn include: higher moisture; fermentation in a different manner than mature corn; sour odor; and increased laxative effect. Corn that has experience from frost typically has a low carotene content. It will dry out quickly and lose leaves. Thus, it may be desirable to add water to corn that has frosted and become too dry. It may also be desirable to add water to drought corn.
It may be desirable for immature corn that has been damaged by extremely high temperatures to not be ensiled immediately. Immature, heat-damaged corn may never produce ears, but some additional stalk growth may result from delaying harvest. Additional stalk growth will result in additional feed. If corn is harvested for silage too soon after the plants have been extensively damaged by heat, the stalk may have too much moisture to produce a high-quality silage. Corn harvested too soon after extensive heat damage that has too much moisture may also lose nutrients through seepage.
Possible problems with silage made from salvaged corn include its lack of energy content due to reduced grain formation, and improper fermentation resulting from excessive dryness of the damaged plant. As is known by those of skill in the art, these problems may be corrected, at least in part, by supplementation with an additional energy source, and addition of moisture, respectively.
Corn silage may be cut into particles that are 1⁄2′′ to 3⁄4′′ in length. Particles of this size may pack more firmly, and may additionally be more palatable to silage-fed animals. Very finely cut silage that is shorter than 1⁄2′′ in length may be made with a recutter. Use of very finely cut silage increases the amount of dry matter that can be stored, e.g., in a silo. However, very finely cut silage may be less palatable to an animal that is to be fed the silage.
If silage is too dry, it may be desirable to add water, for example, to establish airtight conditions. Generally, four gallons of water may be added per ton of silage for each 1 percent desired rise in moisture content. It is understood that more or less water may be required, and measurements may be taken during the ensiling process to ensure that enough, but not too much, water is added. The water may be added as the silo is being filled. If the water is added after the silo is filled, it may seep down the silo walls, and therefore not permeate the silage mass. This seepage may cause leaching of silage nutrients, and may break the air seal and lead to improper fermentation.
High-quality silage may be made without the addition of any additives or preservatives. However, additives may be added to silage to increase one or more characteristics of the silage. For example, molasses and grain may be added to corn forage at the time of ensiling.
With large-capacity silos and high-speed filling methods, distribution and packing of silage in silos should be monitored. Improper distribution and packing may cause excessive seepage, poor fermentation, and/or losses in storage capacity. Half the capacity of a cylindrical silo is in the silo's outermost edge. For example, for a cylindrical silo that is 14′ in diameter, half its capacity is in the outermost 2′ of its diameter. If material in this outside area is packed too loosely, the capacity of the silo may be significantly reduced. Thus, tower silos may be equipped with a distributor that facilitates proper silage distribution and packing.
A loss of nutrients occurs in all silage during the ensiling process, due to the presence of living microorganisms that carry out the fermentation process. The amount of nutritional value lost during the ensiling process depends upon, inter alia, the exclusion air during filling, and the prevention of carbon dioxide loss. Carbon dioxide is necessary to arrest respiration of the ensiled plant cells; and to prevent seepage loss, undesirable fermentation, and/or spoilage due to exposure of the plant material surface. Therefore, good ensiling practices generally lead to higher quality silage with a maximal nutrient content.
Silage from plants (e.g., maize) having reduced CAD2 activity in a finishing ration.
Silage from plants having reduced CAD2 activity have a reduced lignin content when compared to wild-type plants of the same variety, and may be chopped into longer particles than normal silage, whether it is processed or not. NDF digestibility of bm1 silage may be higher than with normal silage. The composition of freshly made silage is not necessarily reflective of the composition of feed that the silage-fed animal will eat. Therefore, fermented samples may be analyzed after a period of time in the silo. For example, samples may be analyzed after at least two weeks, or at least two months, in the silo.
Once silage from plants having reduced CAD2 activity has been prepared, and the silage has been determined to be ready to be fed to an animal, the silage may be included in a finishing ration to be fed to an animal that will be used for meat, or meat product, production. In some examples, the finishing ration comprising silage may not comprise grain corn, for example, dry rolled corn, or ground corn. Typical finishing rations comprise at least about 11% protein, about 60 MCal of Net Energy, about 0.5% Calcium, about 0.35% Phosphorous, and about 0.6% Potassium. In some examples, it is an advantage that a finishing ration exhibits a higher feed efficiency (G:F). In particular examples, a finishing ration that does not comprise grain corn may result in average daily gains in an animal fed the finishing ration that are comparable to the average daily gain that would result from a normal finishing rations that uses grain corn as an energy source.
In some examples, a finishing ration is produced using silage from plants (e.g., maize) comprising a mutant cad2 gene according to the disclosure, wherein the finishing ration comprises between about 15% and about 30% silage. Thus, a finishing ration may comprise, for example, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, or 33% silage. In particular examples, a finishing ration is produced using bm1 corn silage. In some examples, a finishing ration comprising at least one fiber source is produced. Thus, a finishing ration may comprise, for example, one, two, three, four, or more than four fiber sources. In some examples, a finishing ration comprising at least one corn co-product is produced. Thus, a finishing ration may comprise, for example, one, two, three, four, or more than four corn co-products. In some examples, a finishing ration comprising less than 60% dry matter is produced. In further examples, a finishing ration comprises less than 55% dry matter. In some specific examples, a finishing ration comprises less than 50% dry matter. Thus, a finishing ration may comprise, for example, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, or 40% dry matter.
C. Stacking of a Mutant cad2 Gene with One or More Additional Trait(s)
In some embodiments, a mutant cad2 gene according to the disclosure may be introduced into a plant comprising one or more desirable trait(s), in order to produce a plant with both a bm1 or other reduced lignin phenotype, and the one or more desirable trait(s). The process of introducing a mutant cad2 gene according to the disclosure into a plant comprising one or more desirable traits (either by introduction into an existing plant line, or by introducing the mutant cad2 gene into the plant at the same time as an additional nucleic acid molecule that provides a desirable trait) is referred to herein as “stacking.” In some examples, by stacking a mutant cad2 gene with one or more desirable traits, a bm1 or other reduced lignin phenotype may be combined with the one or more desirable traits. For example, stacking of the bm1 trait with the bm3 trait may result in further increases in silage digestibility, and a very significant improvement in the yield of fermentable sugars in biomass hydrolysis without pre-treatment.
Examples of traits that may be desirable for combination with a bm1 or other reduced lignin phenotype include, without limitation: cytoplasmic male sterility; plant disease resistance genes (See, e.g., Jones et al. (1994) Science 266:789 (tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al. (1993) Science 262:1432 (tomato Pto gene for resistance to Pseudomonas syringae); and Mindrinos et al. (1994) Cell 78:1089 (RSP2 gene for resistance to Pseudomonas syringae)); a gene conferring resistance to a pest; a Bacillus thuringiensis protein, a derivative thereof, or a synthetic polypeptide modeled thereon (See, e.g., Geiser et al. (1986) Gene 48:109 (Bt δ-endotoxin gene; DNA molecules encoding δ-endotoxin genes can be purchased from American Type Culture Collection (Manassas, Va.), for example, under ATCC Accession Nos. 40098; 67136; 31995; and 31998)); a lectin (See, for example, Van Damme et al. (1994) Plant Molec. Biol. 24:25 (Clivia miniata mannose-binding lectin genes)); a vitamin-binding protein, e.g., avidin (See International PCT Publication US93/06487 (use of avidin and avidin homologues as larvicides against insect pests)); an enzyme inhibitor; a protease or proteinase inhibitor (See, e.g., Abe et al. (1987) J. Biol. Chem. 262:16793 (rice cysteine proteinase inhibitor); Huub et al. (1993) Plant Molec. Biol. 21:985 (tobacco proteinase inhibitor I; and U.S. Pat. No. 5,494,813); an amylase inhibitor (See Sumitani et al. (1993) Biosci. Biotech. Biochem. 57:1243 (Streptomyces nitrosporeus alpha-amylase inhibitor)); an insect-specific hormone or pheromone, e.g., an ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof (See, e.g., Hammock et al. (1990) Nature 344:458 (inactivator of juvenile hormone)); an insect-specific peptide or neuropeptide that disrupts the physiology of the affected pest (See, e.g., Regan (1994) J. Biol. Chem. 269:9 (insect diuretic hormone receptor); Pratt et al. (1989) Biochem. Biophys. Res. Comm. 163:1243 (allostatin from Diploptera puntata); U.S. Pat. No. 5,266,317 (insect-specific, paralytic neurotoxins)); an insect-specific venom produced in nature by a snake, a wasp, or any other organism (See, e.g., Pang et al. (1992) Gene 116:165 (a scorpion insectotoxic peptide)); an enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity; an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule, e.g., a glycolytic enzyme; a proteolytic enzyme; a lipolytic enzyme; a nuclease; a cyclase; a transaminase; an esterase; a hydrolase; a phosphatase; a kinase; a phosphorylase; a polymerase; an elastase; a chitinase; or a glucanase, whether natural or synthetic (See International PCT Publication WO 93/02197 (a callase gene); DNA molecules which contain chitinase-encoding sequences (for example, from the ATCC, under Accession Nos. 39637 and 67152); Kramer et al. (1993) Insect Biochem. Molec. Biol. 23:691 (tobacco hornworm chitinase); and Kawalleck et al. (1993) Plant Molec. Biol. 21:673 (parsley ubi4-2 polyubiquitin gene); a molecule that stimulates signal transduction (See, e.g., Botella et al. (1994) Plant Molec. Biol. 24:757 (calmodulin); and Griess et al. (1994) Plant Physiol. 104:1467 (maize calmodulin); a hydrophobic moment peptide (See, e.g., International PCT Publication WO 95/16776 (peptide derivatives of Tachyplesin which inhibit fungal plant pathogens); and International PCT Publication WO 95/18855 (synthetic antimicrobial peptides that confer disease resistance)); a membrane permease, a channel former, or a channel blocker (See, e.g., Jaynes et al. (1993) Plant Sci 89:43 (a cecropin-β lytic peptide analog to render transgenic plants resistant to Pseudomonas solanacearum); a viral-invasive protein or a complex toxin derived therefrom (See, e.g., Beachy et al. (1990) Ann. rev. Phytopathol. 28:451 (coat protein-mediated resistance against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus)); an insect-specific antibody or an immunotoxin derived therefrom (See, e.g., Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation via production of single-chain antibody fragments); a virus-specific antibody (See, e.g., Tavladoraki et al. (1993) Nature 366:469 (recombinant antibody genes for protection from virus attack)); a developmental-arrestive protein produced in nature by a pathogen or a parasite (See, e.g., Lamb et al. (1992) Bio/Technology 10:1436 (fungal endo α-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-α-1,4-D-galacturonase; Toubart et al. (1992) Plant J. 2:367 (endopolygalacturonase-inhibiting protein)); and a developmental-arrestive protein produced in nature by a plant (See, e.g., Logemann et al. (1992) Bio/Technology 10:305 (barley ribosome-inactivating gene providing increased resistance to fungal disease)).
Further examples of traits that may be desirable for combination with a bm1 or other reduced lignin phenotype include, without limitation, genes that confer resistance to a herbicide (Lee et al. (1988) EMBO J. 7:1241 (mutant ALS enzyme); Mild et al. (1990) Theor. Appl. Genet. 80:449 (mutant AHAS enzyme); U.S. Pat. Nos. 4,940,835 and 6,248,876 (mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes providing glyphosate resistance); U.S. Pat. No. 4,769,061 and ATCC accession number 39256 (aroA genes); glyphosate acetyl transferase genes (glyphosate resistance); other phosphono compounds from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes) such as those described in European application No. 0 242 246 and DeGreef et al. (1989) Bio/Technology 7:61 (glufosinate phosphinothricin acetyl transferase (PAT) genes providing glyphosate resistance); pyridinoxy or phenoxy proprionic acids and cyclohexones (glyphosate resistance); European patent application No. 0 333 033 and U.S. Pat. No. 4,975,374 (glutamine synthetase genes providing resistance to herbicides such as L-phosphinothricin); Marshall et al. (1992) Theor. Appl. Genet. 83:435 (Acc1-S1, Acc1-S2, and Acc1-S3 genes providing resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop); WO 2005012515 (GAT genes providing glyphosate resistance); WO 2005107437 (Genes conferring resistance to 2,4-D, fop and pyridyloxy auxin herbicides); and an herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) or a benzonitrile (nitrilase gene) (See, e.g., Przibila et al. (1991) Plant Cell 3:169 (mutant psbA genes); nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442; and Hayes et al. (1992) Biochem. J. 285:173 (glutathione S-transferase)).
Further examples of traits that may be desirable for combination with a bm1 or other reduced lignin phenotype include, without limitation, genes that confer or contribute to a value-added trait, for example, modified fatty acid metabolism (See, e.g., Knultzon et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:2624 (an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant)); decreased phytate content (See, e.g., Van Hartingsveldt et al. (1993) Gene 127:87 (an Aspergillus niger phytase gene enhances breakdown of phytate, adding more free phosphate to the transformed plant); and Raboy et al. (1990) Maydica 35:383 (cloning and reintroduction of DNA associated with an allele responsible for maize mutants having low levels of phytic acid)); and modified carbohydrate composition effected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch (See, e.g., Shiroza et al. (1988) J. Bacteol. 170:810 (Streptococcus mutant fructosyltransferase gene); Steinmetz et al. (1985) Mol. Gen. Genet. 20:220 (levansucrase gene); Pen et al. (1992) Bio/Technology 10:292 (α-amylase); Elliot et al. (1993) Plant Molec. Biol. 21:515 (tomato invertase genes); Sogaard et al. (1993) J. Biol. Chem. 268:22480 (barley α-amylase gene); and Fisher et al. (1993) Plant Physiol. 102:1045 (maize endosperm starch branching enzyme II)).
D. Other Uses of a Plant Having Reduced CAD2 Activity
Many agronomical or industrial applications besides the production of silage concern desired plant products, the yields of which are directly linked to the content and/or composition of lignin in the plant cell wall. Examples of such products and applications include without limitation: products used in paper production, and the production of energy, for example, in the form of biofuels. Plants comprising a mutant cad2 gene according to the disclosure may have a bm1 or other reduced lignin phenotype. As will be appreciated by those of skill in the art, plant products derived from such plants may be expected to have distinguishing characteristics from a wild-type plant of the same variety, which distinguishing characteristics may be desirable. Thus, any use of a plant comprising a mutant cad2 gene according to the disclosure that takes advantage of an expected bm1 or other reduced lignin phenotype in the plant is within the scope of the disclosure.
The following examples are provided to illustrate certain particular features and/or embodiments. The examples should not be construed to limit the disclosure to the particular features or embodiments exemplified.
EXAMPLES
Example 1
Cloning and Sequencing of CAD2 Genes from Two Bm1 Lines
Corn leaf samples from inbred lines 515Dbm1 (bm1 mutant), 6XN442 (wild-type), and DASbm1 were used. DASbm1 is a novel, naturally-occurring bmr mutant discovered in a CV5123/GR8207 genetic background. The original F1 generation from which the DASbm1 mutation originated was created in a breeding crossing block in the summer of 2003 by using GR8207 as the male, and crossing it to female CV5123. This cross was self-pollinated in the winter nursery of 2003 and the F2 ears were then bulked to generate F2 seeds. The F2 population was planted in the summer of 2004, and 60 self-pollinated ears were selected for advancement into grain testing. These 60 ears were sent to winter nursery in 2004 for self-pollination to create a go-forward S2 ear selection. The S2 ears were then planted in the 2006 summer nursery.
The discovery of a bmr mutant was made while walking the nursery looking at inbred lines. A row showing a pronounced BMR phenotype was identified. This row was ear 31 of the 60 ears selected from the CV5123/GR8207-B population, and showed very close resemblance in plant structure to the other 59 ears selected except for having the BMR trait. It could not initially be determined if the mutant was a bm3, bm1, or other mutation. S2 plants were then self-pollinated, and seeds were collected and saved. In the summer of 2007, the S3 seeds were planted, and all plants still showed the BMR trait. The S3 plants were self-pollinated to create S4 ears, and the line was noted as of possible interest for silage production. In the summer of 2008, test crosses were made to both known bm1 and bm3 inbred lines to determine the background of the bmr mutant. In the summer of 2009, the test cross seeds were grown out in a progeny test, and it was determined the mutation was a new bm1 mutation, as all plants in the progeny test with the bm1 inbred expressed the BMR trait, while the bm3 progeny showed all plants to be non-BMR. Leaf tissues were then sampled from the CV5123/GR8207 NIL, 515Dbm1 inbred, as well as heterozygous and null plants from various genetic backgrounds, and subjected to molecular genetic analysis including marker work to determine the exact nature of the DASbm1 bmr mutant.
Corn leaf samples were ground to a fine powder using the Genogrinder 2000 (SPEX CertiPrep, Metuchen, N.J.). DNA was extracted with a standard 2.5% CTAB (cetyl trimethylammonium bromide) DNA extraction protocol. Prior to PCR, DNA samples were quantified with Quant-it PicoGreen Quantification Kit (Invitrogen, Carlsbad, Calif.) according to manufacturer's instructions.
The CAD2 genomic sequence, from maize inbred line, Maize B73, was retrieved from Genbank (Genbank Accession No. AC230031). This sequence was approximately 5.9 kb in length and included a 1.7 kb promoter, four exons, three introns and a short terminator. Primers were designed based on the B73 sequence, and a PCR reaction was performed using the ABI GeneAmp PCR System 9700 (Applied Biosystems, Foster City, Calif.). Reactions contained 2.5 units of TaKaRa LA Taq (Takara Bio Inc., Shiga, Japan), 400 nM of dNTP, 200 nM of forward and reverse primer, and 30 ng of genomic DNA. The following PCR program was used: PCR started with a denaturing step of 2 minutes for 94° C.; followed by 30 cycles of 94° C. for 45 seconds, 55° C. for 45 seconds and 72° C. for 2 minutes. PCR products were visualized on a 2% E-Gel and then extracted using PURELINKTM quick gel extraction kit (Invitrogen, Carlsbad, Calif.). Purified PCR products were then sent to Eurofins MWG Operon (Huntsville, Ala.) for direct sequencing. Sequences were analyzed using Sequencher 4.8 (Ann Arbor, Mich.). Mutations were identified by comparing the CAD2 sequences amplified from bmr mutants and 6XN442 to the sequence listed for Maize B73.
Due to presence of three fairly large introns and some unusual sequence features (for example, an 82 bp TA simple repeat in intron 3), the initial attempts to amplify the CAD2 gene in one 5.9 kb fragment failed. Consequently, a series of nested PCR primers were designed (see Table 1) and used to successfully amplify seven overlapping CAD2 fragments from 515Dbm1 and the wild-type line. FIG. 1. Full-length CAD2 sequences for 515Dbm1 and the wild-type line were assembled from the seven fragments. Genomic, predicted cDNA and protein sequences for 515Dbm1 and 4XN442 are listed as SEQ ID NO: 1-6.







TABLE 1







Primer and length information for the seven overlapping genomic CAD2 PCR fragments.













Fragment





Length


Fragment
Forward Primer
Reverse Primer
(bp)





F5-R5
SEQ ID NO: 9
SEQ ID NO: 10
3327



AATGCACGATGTCAACTCTCTTGCCTATAA
AATTTTCTCATGAAAACGCTGAAACTGC



 


2933F-293
SEQ ID NO: 11
SEQ ID NO: 12
 566


3R
TGAGCACAAACGGTTAACACAC
ATTGTATCAGTTCACGGTGGTG



 


3268F-326
SEQ ID NO: 13
SEQ ID NO: 14
 426


8R
CACACCAACCACAATAATAAGCTAATACTC
AACGATCACCCCGACGCCTACC



 


3479F-347
SEQ ID NO: 15
SEQ ID NO: 16
 427


9R
CCACCGTGAACTGATACAATAACAATAAAG
ACACTGCATGCAAGATAACGCTGAAAATAA



 


3742F-374
SEQ ID NO: 17
SEQ ID NO: 18
 500


2R
TGCAACAAGAAGATCTGGTCAT
TCATACCGTACTTTCCACATCAA



 


3996F-399
SEQ ID NO: 19
SEQ ID NO: 20
 468


6R
TTTTGCGAGAAGTGGACGAGATAAGGT
AATGTTGTTGGAGGTAGGTGGTGTGGTTT



 


BM11
SEQ ID NO: 21
SEQ ID NO: 22
1364


C1-R14
GAGAAATGAGGATAACTTTCTCCATCGTT
CTATAGACGAGACAGAAGGGAACGGATTTG









A comparison of 515Dbm1 mutant cad2 with those of wild type (6XN442) and B73 revealed the presence of 15 SNPs and eight insertions/deletions. Most of these changes were located in the promoter region or introns, with the exception of an AC di-nucleotide insertion (nucleotide 3994-3995 in 515Dbm1) located in exon 3 of the bm1 mutant, and a G/A SNP (nucleotide 5410 in 515Dbm1) located in the 4th exon. See FIGS. 3 and 4. The AC insertion in 515Dbm1 creates a frame shift and a premature STOP codon 52-bp downstream of the insertion that resulted in a much shorter CAD2 protein (147 amino acids for 515Dbm1 CAD2, versus 367 amino acids for wild-type CAD2. FIG. 4. The truncated 515Dbm1 CAD2 protein is most likely nonfunctional even if it is produced, because it lacks the NADPH-binding and C-terminal catalytic domains.
The same seven primer pairs shown in Table 1 were used to attempt to amplify overlapping CAD2 fragments from DASbm1, and 6 pairs amplified the expected fragments. However, the F5/R5 pair failed to amply the expected 3327-bp fragment, or even any specific PCR products, even after multiple attempts. Therefore, additional nested PCR primers were designed (see Table 2), and used to successfully amplify the 5′ end 2757 bp of the missing 3327-bp fragment. However, the 3′ end (approximately 570 bp) were unable to be amplified using many pairs of primers and PCR conditions. With the exception the missing 570-bp gap, the amplified 5346-bp cad2 genomic sequence from DASbm1 is identical to that of B73. This was suspected to be the region that contained molecular changes for bmr phenotype.







TABLE 2







Primers for 5′ end of a missing DASbm1 genomic CAD2 PCR fragment.













Fragment





Length


Fragment
Forward Primer
Reverse Primer
(bp)





F5-149R
SEQ ID NO: 26
SEQ ID NO: 27
837



AATGCACGATGTCAACTCTCTTGCCTATAA
TGGGTTGAGAAATTGGGTAAGTGC



 


547F-547
SEQ ID NO: 28
SEQ ID NO: 29
657


R
TGCCCGGTCCAATCTTTCTTA
CGTCTTTCGAGGAGGTCTAC



 


1022F-102
SEQ ID NO: 30
SEQ ID NO: 31
626


2R
CGTTTGGTATCGTCCGAGTTGTGT
GCCGGGTAGTGCGATCTTTCTGG



 


1501F-150
SEQ ID NO: 32
SEQ ID NO: 33
600


1R
AGATCATGCTGGAAAGGTAGTAGG
TGATCGAAAATATGCCCCAAGTC



 


1955F-195
SEQ ID NO: 34
SEQ ID NO: 35
763


5R
ACGCGCCTCCTCCAGTAGTTCTCC
CAAGTTCACACGCTGCTGGGTCTG










Primers encompassing predicted exon 1 and exon 3 were designed (CVF: GTCCGAGAGGAAGGTGGTC (SEQ ID NO:36); and CVR: GGCCGTCCATCAGTGTAGA (SEQ ID NO:37)), and then used to amplify a CAD2 cDNA fragment from DASbm1, 515Dbm1 and 6XN442. As expected, a fragment of 375-bp was amplified from 515Dbm1 and 6XN442. On the other hand, the PCR product from DASbm1 was found to be significantly larger. Sequencing results revealed that cad2 cDNA from DASbm1 contains an extra 409-bp insertion between endogenous exon 1 and exon 2. FIG. 4. A BLAST search against the B73 genome suggested that the 409-bp cDNA insertion in DASbm1 is most likely the result of RNA splicing from part of a putative transposon/repetitive gene (GRMZM2G017736) located on chromosome 1 that contains several introns.
Based on the 409-bp cDNA insertion observed in DASbm1 and its likely genomic source, additional PCR primers were designed (see Table 3), and used to amplify the chimeric genomic region of CAD2 from DASbm1. FIG. 1. Sequencing results reveal that the genomic insertion is 3444-bp in length, and the total genomic cad2 amplified from DASbm1 is 9360 bp. The alignment of genomic CAD2 from DASbm1, B73, 515Dbm1, and 6XN442 is shown in FIG. 3. With the exception of the 3444-bp insertion, the remaining cad2 sequence from DASbm1 is identical to that of B73. The 3444-bp DASbm1 transposon insertion is set forth herein as SEQ ID NO:41.







TABLE 3







PCR primers for chimeric genomic region of DASbm1 CAD2.













Fragment





Length


Fragment
Forward Primer
Reverse Primer
(bp)





2687F-264
SEQ ID NO: 38
SEQ ID NO: 39
2725


3R
TATGGGTACTGCTCCTGCAC
CCTTGCAGCAGAACCACTG



 


2132F-R5
SEQ ID NO: 40
SEQ ID NO: 10
1890



AGAATATCATTAGCAGCCAGA
AATTTTCTCATGAAAACGCTGAAACTGC










Genomic Nature of the Insertion in DASbm1.
As shown in FIG. 2, the site of insertion in DASbm1 is located in the endogenous intron 1 of cad2. A closer examination of the 3444-bp insertion sequence revealed that the last 11 bases, with the sequence TACTGATATCT (SEQ ID NO:42) was a direct duplication of an 1′-bp sequence from intron 1 of cad2 located immediately upstream of the insertion. FIG. 2. These direct repeats indicate that the insertion is likely due to DNA transposon activities.
BLAST search results using the 3433-bp insertion (without the 11-bp direct repeat) against B73 genome sequences show numerous homologous hits on different chromosomes, indicating the repetitive nature of the sequence. However, only one hit on chromosome 1 (Maize B73 RefGen_v1, nucleotide positions 148086176-148089850) is 100% identical over the entire length, with the exception of a 242-bp missing gap in the DASbm1 insert. FIG. 4. Other hits are less than 100% identical, and only match part of the insert. Since CAD2 is located on chromosome 5, the insert in DASbm1 is most likely excised from its original location in chromosome 1 and re-inserted into CAD2 via transposon activities. The 242-bp missing gap may reflect the difference between B73 and DASbm1 (CV5123/GR8207) genomes, or it may be due to deletion during the transposition.
A predicted gene (GRMZM2G017736) is located within the 3433-bp insertion sequence and is apparently transcribed in normal corn plants, since quite a few corresponding full-length ESTs (e.g., EU976746 and EU976335) are found in the database. The predicted ORF in GRMZM2G017736 encodes a small protein of 167 amino acids that show high degrees of homology with many hAT (after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon) family of transposases that are usually larger proteins. Because the truncated 167 amino acid protein lacks the BED zinc finger DNA binding domain (Pfam02892) normally found in the full-length transposases, the 3433-bp fragment is more likely a Ds element that requires the presence of an Ac element elsewhere for transposition.
Once in the CAD2 locus on chromosome 5, the insertion and cad2 is a chimeric gene. The insertion is spliced into 3 exons of 409-bp and forms a chimeric mRNA with endogenous cad2 exons. FIGS. 2 and 4. The first 1158-bp fragment of the insertion are spliced together with the 5′ end 927 bp of the first intron of cad2 as a chimeric intron, the entire predicted ORF of GRMZM2G017736 is spliced as a part of a 1204-bp intron, and the last 463-bp fragment of the insertion is spliced together with the 3′ end 231 bp of the first intron of cad2 as a chimeric intron. FIG. 2.
The transposon insertion creates a premature STOP in DASbm1 cad2
As described above, the transposon insertion in DASbm1 creates a chimeric gene that is transcribed as a chimeric mRNA with an extra 409 bp between endogenous cad2 exon1 and exon 2. FIG. 4. This extra 409-bp sequence is predicted to create a frame shift and a premature STOP codon which results in a much shorter CAD2 protein of only 48 amino acids (vs. 367 amino acids for the wild-type protein). FIG. 5. Similar to the truncated CAD2 protein of 515Dbm1, the 48 amino acid DASbm1 CAD2 is most likely nonfunctional even if it is produced, because it lacks the NADPH-binding and C-terminal catalytic domains.
Example 2
CAD2 Allele Specific PCR-Based High-Throughput Assay Design and Validation
A KASParTM assay was designed based on the AC insertion in exon 3 of 515Dbm1 cad2 to distinguish the mutant allele from the wild-type allele. Thirty-two wild type inbred lines, without the bm1 mutation, as well as segregating populations containing the bm1 mutation, were tested following a protocol obtained from the manufacturer (KBioSciences, Hoddesdon, Hertfordshire, UK).
Based on the sequences surrounding the AC insertion, three oligos were designed (SEQ ID NO:43 (AC_R1); TCAGTCTCAAGAACTCACTTCTGG, SEQ ID NO:44 (AC_A1); GAAGGTGACCAAGTTCA TGCTACTGATGGACGGCCCACA and SEQ ID NO:45 (AC_A2); GAAGGTCGGAGTCAACGGATTACTGATGGACGGCCCACG) for a high-throughput KASParTM marker assay that can be used to differentiate the 515Dbm1 cad2 allele from other bm1 and wild-type CAD2 alleles. FIG. 8.
The KASParTM assay was first validated with a panel of 32 wild type inbreds and the bm1 mutant line, 515Dbm1. This analysis indicated that the non-bm1 lines did not contain the AC insertion. The assay was then validated in segregating populations of bm1 maize lines which possessed one of three genotypes: homozygous wild-type; homozygous bm1 (containing the 515Dbm1 AC insertion); and heterozygous. FIG. 9. The KASPar marker detected the bm1 AC insertion within the homozygous bm1 and heterozygous populations. Moreover, this high-throughput assay distinguished the homozygous bm1, heterozygous, and homozygous wild-type populations from one another. As such, markers such as these can be used to rapidly provide an accurate identification of zygosity for the bm1 genotype in plants suspected of containing bm1 from 515Dbm1. The assay can therefore be used for the identification of bm1 germplasm, accelerating introgression of the bm1 trait and molecular breeding using bm1 to improve digestibility of silage or increase ethanol yield.
New TaqManTM assays were developed to discriminate between all of DASbm1, 515Dbm1, and wild-type CAD2 alleles. Primers and probes that were used for DASbm1 and 515Dbm1 are listed in Table 4 and Table 5, respectively. Because DASbm1 has a large transposon insertion, separate forward primers for DASbm1 (DASbm1_F) and wild-type (Wt CAD2_F) were designed so that appropriate PCR products are amplified under standard TaqManTM assay conditions. Based on the transposon insertion in DASbm1, AC insertion in 515Dbm1, and wild-type CAD2 sequence, primers and probes for TaqManTM assays were designed using Primer Express 3.0. Primers and dual-labeled probes with FAM or VIC and Minor Grove Binding Non Fluorescence QuencherTM I (MGBNFQ) dyes were synthesized by Applied Biosystems (Foster City, Calif.). Oligos were dissolved in 1× Tris-EDTA to 100 μM. TaqManTM genotyping master mix (Applied Biosystems, Foster City, Calif., Catalog #4371355) was used for all PCR reactions. Real time PCR reactions in 25 μl volume were set up using a 96-well plate on an iCyclerTM optical system (BioRad, Hercules, Calif.) starting with 15 minutes of denaturing at 95° C. as recommended, followed by 50 cycles of 92° C. for 15 seconds, and 60° C. for 1 minute. Fluorescence signals were recorded at the end of each cycle.







TABLE 4







Primers and probes for TaqManTM assays to


discriminate DASbm1 and wild-type.








Oligonucleotide
Sequence





DASbm1_probe
6FAM-AGGCTTACTGATATCTG



(SEQ ID NO: 48)-MGBNFQ


 


Wt CAD2_probe
VIC-CTGCAGATATCAGTAGTTACG



(SEQ ID NO: 49)-MGBNFQ


 


DASbm1_F
GCCGGATTGTGACCCATTAT



(SEQ ID NO: 50)


 


DASbm1_R
CGCGACAGGCGGTAGGTA



(SEQ ID NO: 51)


 


Wt CAD2_F
GCAGCGTGTGAACTTGTAGGTAA



(SEQ ID NO: 55)
















TABLE 5







Primers and probes for TaqManTM assays to


discriminate 515Dbm1 and wild-type.








Oligonucleotide
Sequence





515Dbm1_probe
6FAM-ACGGCCCACACGC



(SEQ ID NO: 53)-MGBNFQ


 


Wt CAD2_probe
VIC-ACGGCCCACGCAGG



(SEQ ID NO: 54)-MGBNFQ


 


bm1_F
GGTCATACAACGACGTCTACACTGA



(SEQ ID NO: 55)


 


bm1_R
ATGGTGGAGGCGAATCCA



(SEQ ID NO: 56)









Results of allelic discrimination assays using the primers set forth in Tables 4 and 5 are shown in FIG. 10. Because these assays and KASParTM markers are based directly on bm1 mutations in cad2, they will provide more accurate genotyping than available indirect bm1 markers.
Example 3
Characteristics of bm1 Maize with Truncating CAD2 Mutations
Because DASbm1 represents a novel bm1 mutation, nothing is known about DASbm1, except that is has a bmr phenotype. The RNA expression levels and lignin contents in DASbm1 plants were investigated, and then compared to those of 515Dbm1 and wild-type (6XN442) plants. It was expected that 515Dbm1 and DASbm1 plants have significantly reduced CAD activities due to the mutations in the cad2 genes of these two maize lines, and the consequent premature termination of CAD2. In addition, 515Dbm1 and DASbm1 plants were hypothesized to have reduced cad2 transcript abundance.
DASbm1, 515Dbm1, and 6XN442 plants were grown in a greenhouse. Leaf tissues were harvested from four-week-old plants and midrib tissues were separated. Samples were grounded to fine powder in liquid nitrogen and extracted with Qiagen's RNeasyTM plant mini kit. SuperScriptTM III One-Step RT-PCR kit from Invitrogen and BioRad's iCyclerTM were used for quantitative RT-PCR to determine RNA expression levels. CAD2-specific oligos were designed based on the fourth exon of CAD2, and corn invertase was used as a control for the Delta-Delta CT calculation.
To compare the CAD2 RNA expression levels, the following primers/probes based on the fourth exon of CAD2 and corn invertase were designed for quantitative RT-PCR. Table 6.







TABLE 6







Primers and probes for CAD2 qRT-PCR.










Oligonucleotide
Sequence







CAD2_F
CCACATGGGCGTGAAGGTA




(SEQ ID NO: 57)



 



CAD2_R
TTCTTGGACGACGAGCTGATC




(SEQ ID NO: 58)



 



CAD2_Eprobe
6FAM-ATGGGCCACCACGTGA




(SEQ ID NO: 59)-MGBNFQ



 



INV59F
ATGGTGGAGGCGAATCCA




(SEQ ID NO: 60)



 



INV59R
TGCCGTCCGTGCCCT




(SEQ ID NO: 61)



 



INV_probe
VIC-CCGTGTACTTCTACCTGC




(SEQ ID NO: 62)-MGBNFQ










As shown in FIG. 6, midribs have much higher level of CAD2 expression than the rest of the leaf tissues without midribs regardless of genotypes The differences are 4.1, 8.0, and 3.4 fold for 6XN442, 515Dbm1, and DASbm1, respectively. Both DASbm1 and 515Dbm1 lines have significantly less cad2 expression, with an average reduction of 91% in the midrib, and 89% in the leaf, for DASbm1; and 87% in the midrib, and 93% in the leaf for 515Dbm1, when compared with wt 6XN442. However, there is no significant difference between the two bm1 mutants. FIG. 6. These results are consistent with what have been reported in the literature for maize bm1 (Halpin et al. (1998), supra), as well as sorghum mutant, bmr6 (Saballos et al. (2009), supra). The reduction in RNA expression is mostly likely due to RNA degradation mediated by nonsense mutations that create premature CAD2 proteins in the 515Dbm1 and DASbm1 mutants, a mechanism previously observed in plants and other species. Conti and Izaurralde (2005) Curr Opin Cell Biol. 17:316-25.
To compare the relative lignin contents of mutant and wild-type plants, ground leaves and internodes were subjected to FTIR spectroscopy analysis.
Fourier Transform Infrared measurements were carried out using Bruker Vertex spectrometer (Bruker Optics Inc, 19 Fortune Drive, Maiming Park, Billerica Mass. 01821), with an Attenuated Total Reflectance (ATR) Miracle sampling accessory with diamond crystal (Pike Technologies, Madison, Wis.). The spectrometer is equipped with a deuterated triglycine sulfate (DTGS) detector, operating at 4 cm−1 resolution and 0.32 cm/s mirror velocity. Two hundred and fifty-six interferograms were co-added before Fourier transformation. The instrument was allowed to purge for 5 min. with nitrogen gas (grade 1) prior to acquisition of the spectra to minimize the spectral contribution due to atmospheric carbon dioxide and water vapor. The ATR Miracle cell sampling accessory has a single-bounce diamond designed for use in the FTIR spectrometer. The depth of penetration of the infrared beam is 1.46 microns. A major advantage of this accessory is that it requires smaller sample volumes compared to the multiple-bounce HATR accessory.
Leaves (7th and 8th, 9th and 10th) and internodes (1st and 2nd, 7th and 8th, 9th and 10th) were collected from greenhouse grown corn plants just after silking, lyophilized, and ground into fine powders. A small portion of the samples were placed directly on the diamond crystal of ATR for the data acquisition. Spectra were collected in finger print region from 800-1800 cm−1. Single-beam spectra of all the samples were obtained, and spectral data are presented as the ratio of the background spectrum of air in absorbance units. After every measurement, ATR crystal was thoroughly cleaned and dried, and its spectrum was examined to ensure that sample residues from the previous acquisition were not retained on the crystal surface. All the spectra were baseline corrected and area normalized to eliminate any spectral artifacts.
The lignin contents in the samples are directly proportional to FTIR relative absorbance. As expected, the reduction in CAD2 RNA expression in both mutants results in significant reduced lignin contents. FIG. 7. On average, the total lignin content reduction was approximately 24% in DASbm1 (P=0.03) and 30% in 515Dbm1 (P=0.006) when compared to wild-type 6XN442. There was no significant difference between the two bm1 mutants (P=0.4).
bm1 mutations in maize exhibit a reddish brown pigmentation of the leaf midrib associated with significantly reduced lignin content and altered lignin composition. This phenotype is visible from V6 through later stages in newly-developed leaves and other tissues. The molecular bases of two independent bm1 mutants (DASbm1 and 515Dbm1) were identified. Based on the specific mutations, high throughput PCR assays for detecting different alleles were developed. The foregoing results demonstrate that ZmCAD2 is the underlying gene for the bm1 mutation in 515Dbm1 and DASbm1, and provide a molecular basis for the observed phenotype. Because bmr mutations are common among many monocot species, the above-described mutations and markers can also be used in other crops; for example, sorghum, sugarcane, millet, rice, and bioenergy species such as switchgrass.
Example 4
Brown Midrib1 (bm1) as a Visual Selectable Marker for Transgenes
EXZACT Precision Technology is a zinc-finger engineering technology that has been shown to precisely target any DNA sequence and accurately modify the genome through gene disruption, editing or gene addition. Based upon EXZACT Precision Technology and recent bm1 discoveries, two alternative approaches can be implemented to create a visual selectable marker to differentiate transgenic plants from their null segregants. A visual marker will enable breeders and scientists in the field to quickly identify transgenic individuals in a segregating population (T1 for example) and compare them to the nulls without detail molecular work and speed up transgenic pipeline screening process. High throughput assays are also available and can be used to confirm the visual observation if needed.
In the first representative approach, mutant bm1 maize (DASbm1 or 515Dbm1) can be used as the target germplasm for transformation. Through molecular breeding, bm1 mutation can be introduced into current transformation germplasm (B104) using CAD2-based markers. A Wt CAD2 and a gene of interest (GOI) in the same construct are targeted to the endogenous mutant cad2 locus by EXZACT Precision Technology. Because bm1 mutation is recessive, plants homozygous for GOI-WtCAD2 or heterozygous have normal pigmentation whereas null segregants have brown midrib phenotype. This also works without EXZACT Precision Technology, but transgene insertion site in the genome is random. An alternative embodiment includes engineering of one or more zinc finger protein(s) that deletes the transposon insertion in DASbm1 or the AC insertion in 515Dbm1 in the endogenous mutant cad2 locus and at the same time inserts the GOI into a nearby locus (to prevent significant segregation of GOI and bm1 marker). This eliminates the need for a Wt CAD2 in the transgenic construct.
In a second representative approach, Wt plants are used as the target germplasm for transformation. The GOI can be targeted to the endogenous Wt CAD2 locus using EXZACT Precision Technology. Site of insertion can be the same as the transposon insertion in DASbm1, AC insertion in 515Dbm1 or any position within the CAD2 locus that renders the protein nonfunctional (functional domains of CAD2 well known). In this case, plants homozygous for transgene have brown midrib phenotype whereas heterozygotes and nulls have normal pigmentation. Brown midrib and associated phenotypes are beneficial and EXZACT Precision Technology can be used to target BM1 (CAD2) and/or BM3 (Caffeic Acid O-Methyltransferase) loci.

## Claims

1. 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.

2. The method of claim 1, wherein a zinc finger nuclease is utilized to integrate the nucleic acid molecule into the genome of the bm1 plant at the CAD2 locus.

3. 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.