WO2012158630A1 - Sorghum maturity gene and uses thereof in modulating photoperiod sensitivity - Google Patents
Sorghum maturity gene and uses thereof in modulating photoperiod sensitivity Download PDFInfo
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- WO2012158630A1 WO2012158630A1 PCT/US2012/037809 US2012037809W WO2012158630A1 WO 2012158630 A1 WO2012158630 A1 WO 2012158630A1 US 2012037809 W US2012037809 W US 2012037809W WO 2012158630 A1 WO2012158630 A1 WO 2012158630A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
Definitions
- the invention is generally related to the field of plant genetics and molecular biology, more particularly to genes involved in plant photoperiod sensitivity, and methods for modifying photoperiod sensitivity in plants.
- Biomass yield is one of the most important attributes of a biomass or bioenergy crop designed for ligno-cellulosic conversion to biofuels or bioenergy. To maximize yield, it is essential to tailor the plants' life cycle to the agro-environments in which they are grown. The transition from vegetative to reproductive growth is a critical developmental switch and a key adaptive trait that ensures that plants set their flowers at an optimum time for pollination, seed development, and dispersal. For example, temperate environments with a long growing season allow cereal crops to exploit an extended vegetative period for resource storage. Conversely, early flowering has evolved as an adaptation to short growing seasons.
- a late or non-flowering bioenergy sorghum crop grown for biomass production will continue to accumulate biomass by building larger vegetative plants until frost or adverse environmental conditions inhibit photosynthesis. It is estimated that late/non-flowering biomass sorghum will generate more than two times the biomass accumulated by grain sorghum per acre assuming reasonable growth conditions throughout the growing season.
- compositions including the nucleic acid sequence of the sorghum Maturity gene 1 (Mai), and expression control sequences thereof are disclosed.
- the expression control sequence can be photoperiod sensitive or photoperiod insensitive.
- the compositions and methods can be used to modulating flowering in plants, particularly sorghum.
- compositions for modulating photoperiod sensitivity for flowering and other plant processes in a plant are provided. For example, methods are provided for developing genetically modified plant varieties in which flowering is accelerated, or delayed or prevented. Methods are also provided for treating a plant in order to accelerate or delay flowering in the plant. Methods and compositions for placing a polynucleotide of interest under photoperiod sensitive or photoperiod insensitive control are also disclosed.
- the compositions and methods and can be used, for example, to make photoperiod sensitive a gene that is normally or naturally photoperiod insensitive. In other embodiments, compositions and methods and can be used to make photoperiod insensitive a gene that is normally or naturally photoperiod sensitive.
- Screening methods are also provided for identifying plants for photoperiod sensitivity and chemical agents that can modify photoperiod sensitivity.
- Figure 1 is a bar graph showing frequency distribution of F 2 population of S. bicolor x S. propinquum as a function of flowering time. Also shown is a boxed line indicating average day length (hrs) over the time period. Also shown are two lines indicating the high (solid line) and low (dashed) temperature during the time period. S. propinquum and most F 2 s flowered when photoperiod was less than 12.5 hours. Segregation of the S, bicolor and S. propinquum alleles at the Mai locus imparts dichotomous phenotype when grown in a temperate environment
- Figure 2 A is a diagram mapping the 1.1 centiMorgan (cM) interval delineated by progeny testing of recombinants.
- Figure 2B is a diagram showing the % of conversion at the DNA marker loci plotted along the sorghum genome sequence (on base pair, bp, scale). The diagram also maps the relative locations of the FT gene (Sb06g012260) and SbPRR37
- Figure 3 A is a diagram illustrating two major S. bicolor haplotypes
- FIG. 3A is a physical map showing the positions of four insertion-deletion events relative to the coding region of Sb06g012260.
- Figure 3C is a diagram comparing the PRR37 alleles in S, bicolor (top) and S. propinquum (bottom). The S. propinquum allele has an "AT" insertion between 97 and 98 nucleotides after the translation starting site.
- Figure 4 is a series of pie graphs showing haplotype frequencies for the gene Sb06g012260 in sub-populations from West Africa, South Africa, Central/ East Africa, and Asia/ India.
- Figure 6 is a line graph of log p value versus Mai region (Mbp) showing the association analysis of Mai region markers and photoperiod sensitive in Sorghum bicolor based on routine application of the software TASSEL (Bradbury, et al, Bioinformatics, 23:2633-2635 (2007)), as detailed below. ( ⁇ ) single marker analysis; (e)analysis considering population structure.
- Figure 7 is a diagram showing homologs identified by BLAST of a candidate Mai gene (Sb06g012260) in sorghum, rice, and Arabidopsis genomes; and maize and sugarcane ESTs.
- BLAST a candidate Mai gene
- Sb06g012260 a candidate Mai gene in sorghum, rice, and Arabidopsis genomes
- the disclosure encompasses conventional techniques of plant breeding, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Plant Breeding: Principles and Prospects (Plant Breeding, Vol 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall, (1993); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds.
- plant is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative crop or cereal, and fruit or vegetable plant. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.
- photoperiod refers to the period of a plant's exposure to daylight every 24 hours.
- photoperiod sensitivity refers to the photoperiod that is required to induce a specific response, such as flowering. Some plants will not flower until they are exposed to day lengths that are less than a critical photoperiod (short day plants) or greater than a critical photoperiod (long day plants). In some plant species, photoperiodic control enforces long-day flowering. Therefore, a photoperiod sensitive plant can have either short-day or long-day flowering, but in both cases, the flowering is controlled by day length.
- a plant is "photoperiod insensitive” or "day neutral” if the day length does not impact when flowering occurs. In order to modulate flowering based on day length, photoperiod sensitivity can be increased.
- non-flowering plant does not flower under the agronomic conditions, regardless of the photoperiod.
- Delayed flowering refers to a plant that flowers on average at least 1 day later, including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days later, than a wild-type plant of the same species.
- non-naturally occurring plant refers to a plant that does not occur in nature without human intervention.
- Non-naturally occurring plants include transgenic plants and plants produced by non-transgenic means such as plant breeding.
- plant tissue includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
- plant part refers to a plant structure, a plant organ, or a plant tissue.
- plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
- plant organ * ' refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
- plant cell refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall.
- the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.
- plant cell culture refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
- transgenic plant refers to a plant or tree that contains recombinant genetic material not normally found in plants or trees of this type and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation.
- transgenic plant is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually). It is understood that the term transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems etc.
- construct refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism include in the 5' -3' direction, a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression.
- gene refers to a DNA sequence that encodes through its template or messenger R A a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein.
- gene also refers to a DNA sequence that encodes an RNA product.
- gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5' and 3' ends.
- orthologous genes or “orthologs” refer to genes that have a similar nucleic acid sequence because they were separated by a speciation event.
- polypeptide refers generally to peptides and proteins having more than about ten amino acids.
- the polypeptides can be "exogenous,” meaning that they are “heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell.
- isolated is meant to describe a compound of interest (e.g., nucleic acids) that is in an environment different from that in which the compound naturally occurs, e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature.
- isolated is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. Isolated nucleic acids are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components.
- nucleic acid molecule or polynucleotide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source.
- the isolated nucleic can be, for example, free of association with all components with which it is naturally associated.
- An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature.
- LD linkage disequilibrium
- Markers that are in LD do not follow Mendel's second law of independent random segregation. LD can be caused by any of several demographic or population artifacts as well as by the presence of genetic linkage between markers. However, when these artifacts are controlled and eliminated as sources of LD, then LD results directly from the fact that the loci involved are located close to each other on the same chromosome so that specific combinations of alleles for different markers (haplotypes) are inherited together. Markers that are in high LD can be assumed to be located near each other and a marker or haplotype that is in high LD with a genetic trait can be assumed to be located near the gene that affects that trait.
- locus refers to a specific position along a chromosome or DNA sequence. Depending upon context, a locus could be a gene, a marker, a chromosomal band or a specific sequence of one or more nucleotides.
- vector refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
- the vectors can be expression vectors.
- expression vector refers to a vector that includes one or more expression control sequences
- control sequence refers to a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
- Control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and the like.
- Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
- promoter refers to a regulatory nucleic acid sequence, typically located upstream (5') of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence.
- the promoters suitable for use in the constructs of this disclosure are functional in plants and in host organisms used for expressing the disclosed polynucleotides. Many plant promoters are publicly known. These include constitutive promoters, inducible promoters, tissue- and cell-specific promoters and
- a nucleic acid sequence or polynucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
- DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
- "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
- Transformed,” “transgenic,” “transfected” and “recombinant” refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
- the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule.
- extrachromosomal molecule can be auto-replicating.
- Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
- a "non- transformed,” “non-transgenic,” or “non-recombinant” host refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
- nucleic acid refers to nucleic acids normally present in the host.
- heterologous refers to elements occurring where they are not normally found.
- a promoter may be linked to a
- heterologous nucleic acid sequence e.g., a sequence that is not normally found operably linked to the promoter.
- heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number.
- a heterologous control element in a promoter sequence may be a control/ regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter.
- the term “heterologous” thus can also encompasses "exogenous” and "non-native" elements.
- percent (%) sequence identity is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
- % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D is calculated as follows:
- polypeptide refers generally to peptides and proteins having more than about ten amino acids.
- the polypeptides can be "exogenous " meaning that they are "heterologous,” i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell.
- stringent hybridization conditions mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence.
- Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared carrier DNA such as salmon sperm D A, followed by washing the hybridization support in 0.1X SSC at approximately 65°C.
- Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2000).
- Photoperiod sensitivity refers to the fact that some plants will not flower until they are exposed to day lengths that are less than a critical photoperiod (short day plants) or greater than a critical photoperiod (long day plants).
- Long day (LD) and short day (SD) plant designations refer to the day length required to induce flowering. Facultative LD or SD plants are those that show accelerated flowering in LD or SD but will eventually flower regardless of photoperiod. Most plants including sorghum must pass tlirough a juvenile stage (lasting about 14-21 days for sorghum) before they become sensitive to photoperiod.
- Sorghum is a facultative SD plant where long days inhibit flowering and short days accelerate flowering.
- the degree of flowering photoperiod sensitivity in sorghum refers to the length of the short days that are required to induce flowering.
- Different sorghum genotypes vary in their degree of photoperiod sensitivity. For example, Sorghum inbreds have been identified with photoperiod sensitivity ranging from -10.5 to -14 hours and still others that are nearly completely insensitive to photoperiod.
- a photoperiod insensitive sorghum planted in Georgia in April can flower in approximately 48-55 days; whereas a highly photoperiod sensitive sorghum planted in Georgia in April can flower in -175-180 days, or may even fail to flower at all.
- the maturity gene (Mai) contains one or more mutation or deletions in some S. bicolor genotypes such that sorghum plants containing this mutant gene are photoperiod insensitive (day-neutral). Identification of this gene allows for identification of orthologous genes in related plants. Moreover, based on this identification, methods of modulating photoperiod sensitivity in plants by modulating the expression control sequences of maturity gene in that plant are disclosed. Methods are also disclosed for modulating photoperiod sensitivity involving modulating the activity of the protein encoded by the Maturity (Mai) gene in the plant.
- compositions and methods for modifying photoperiod sensitivity in plants are provided.
- the methods can involve modulating the activity of the endogenous gene or gene(s) responsible for photoperiod sensitivity in the plant.
- the methods can involve promoting the expression of one or more endogenous gene orthologous to sorghum grain maturity gene 1 (Mai ).
- the methods can involve introducing to the plant a composition that promotes maturity gene 1 (Mai) activity in a Sorghum plant.
- Maturity gene refers to the Mai gene found in Sorghum as well as orthologous genes serving the same function in related plants.
- Sorghum has been an excellent biomass source with its high yield potential, high water use efficiency, and established production systems and is a representative plant that can be used with the disclosed methods and compositions.
- Sorghum is a genus of numerous species of grasses, some of which are raised for grain and some of which are used as fodder plants either cultivated or as part of pasture. The plants are cultivated in warmer climates worldwide.
- Sorghum is in the subfamily Panicoideae and the tribe
- Sorghum is well adapted to growth in hot, arid or semi-arid areas.
- Sorghum species include, but are not limited to Sorghum almum, Sorghum amplum, Sorghum angustwn, Sorghum arundinaceum, Sorghum bicolor, Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum
- Nucleic acid sequences for Mai genes in Sorghum bicolor and Sorghum propinquum are provided. It is understood that the skilled artisan can identify orthologous sequences in other Sorghum species for use in the present compositions and methods.
- transgenic plant having a nucleic acid molecule, or antisense constructs thereof, encoding a Ma 1 gene product, or variant, such as a codon optimized variant thereof, optionally operatively linked to an heterologous regulatory element.
- a transgenic plant characterized by high photoperiod sensitivity, low photoperiod sensitivity, or photoperiod insensitivity, wherein the cells of the plant express a nucleic acid molecule encoding an Mai gene product, or antisense construct thereof, that is operatively linked to an expression control sequence.
- the construct encodes an inhibitory nucleic acid such as siRNA or RNAi that when express down regulates the expression of Mai .
- coding sequences for a maturity gene are provided, also provided are the non-coding sequences that are known or can be identified to correspond to the coding sequences that are provided.
- a maturity gene also provided for use in the disclosed compositions and methods is the 5' untranslated region (UTR), which contains the endogenous promoter for the maturity gene.
- UTR 5' untranslated region
- the S. propinquum cultivar from which the sequences described below are derived is a short-day cultivar, that has a dominant (functional) Mai allele. Sequences for a dominant Mai gene are therefore provided. In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in short day 5.
- propinquum includes the nucleic acid sequence:
- AAATATGAAC AAATTCGTAT TATCAAAGGA AGATAGAGCA AAGATAGAGG AAGACAAAAC
- SEQ ID N0:1 Sb06g012260 - S. propinquum or functional fragment, or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:l.
- the coding sequence of the maturity Mai gene of SEQ ID NO:l, including introns, can be:
- the maturity Mai gene as it is found in short day S. propinquum includes the nucleic acid sequence:
- SEQ ID NO:3 Sb06g012260 (10.6KB) - S. propinquum
- functional fragment, or variant such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.
- Each N can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
- the coding sequence of the maturity Mai gene of SEQ ID NO:3, including mtrons, can be:
- the maturity Mai gene as it is found in short day S. propinquum includes the nucleic acid sequence:
- SEQ ID N0:5 Sb06g012260 (5.2kb) - S. propinquum or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5.
- N 1, 2, 3, 4, or 5 nucleotides in length.
- the coding sequence of the maturity Mai gene of SEQ ID NO:5, including introns, can be:
- SEQ ID N0:6 Sb06g012260 (5.2kb) - S. propinquum or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6.
- a maturity Mai protein as it is found in short-day S, propinquum can include the amino acid sequence:
- SEQ ID NO: 8 Sb06g012260 or functional fragment, or variant thereof having at least 75%, 80%», 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8.
- SEQ ID NO: 19 Sb07g008600 - S. propinq um
- a functional fragment or variant thereof having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%> sequence identity to SEQ ID NO: 19.
- the coding sequence of the maturity Mai gene of SEQ ID NO: 19, including introns, can be:
- SEQ ID NO:28 - Sb07g008600 - S. propinquum or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%» sequence identity to SEQ ID NO:28,
- the coding sequence of the maturity Mai gene of SEQ ID NO:28, without introns, can be:
- SEQ ID NO:29 - Sb07g008600 - S. propinquum or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96% » 97%, 98%, or 99% sequence identity to SEQ ID NO:29.
- the maturity Mai gene (including non-coding sequence) as it is found in short day S. propinquum includes the nucleic acid sequence:
- SEQ ID NO:20 or a functional fragment or variant thereof having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20 (Sb07g008600 - S. propinq um) .
- Each N can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
- the coding sequence of the maturity Mai gene of SEQ ID NO:20, including introns, can be:
- SEQ ID NO:30 Sb07g008600 (10.6 kb) - S. propinquum
- functional fragment or variant such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30.
- the coding sequence of the maturity Mai gene of SEQ ID NO:30, without introns, can be:
- the S. bicolor cultivar from which the sequences described below are derived are day-neutral, and have the recessive (loss of function) Mai allele. Sequences for a recessive Mai gene are therefore provided.
- the maturity Mai gene (including non-coding sequence) as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
- SEQ ID NO:9 Sb06g012260 - S. bicolor
- a variant such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.
- the coding sequence of the maturity Mai gene of SEQ ID NO: 10, including introns, can be:
- SEQ ID NO: 10 Sb06g012260 - S. bicolor
- functional fragment or variant such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.
- the coding sequence, without introns, of the maturity Mai gene as it is found in day-neutral S, bicolor can include the nucleic acid sequence:
- the maturity Mai protein as it is found in short-day - S. bicolor can include the amino acid sequence SEQ ID NO: 8, or a variant thereof having at least 95% sequence identity to SEQ ID NO: 8.
- the maturity Mai gene (including non-coding sequence) as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
- SEQ ID NO: 12 Sb06g012260 - S, bicolor
- a variant for example a codon optimized variant, thereof having at least at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.
- the coding sequence of the maturity Mai gene of SEQ ID NO:12, including introns, can be:
- SEQ ID NO: 13 Sb06g012260 - S. bicolor
- functional fragment or variant such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13.
- the maturity Mai gene (including non-coding sequence) as it is found in day-neutral S. bicolor can include the nucleic acid sequence: 1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATG TCAAACAGCC ATCAAGTCAA
- SEQ ID NO:32 - Sb07g008600 - S. bicolor or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32.
- the coding sequence, without introns, of the maturity Mai gene according to SEQ ID NO:32 as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
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Abstract
Compositions relating to the sorghum maturity gene 1 (Ma1) and expression control sequences and methods of use thereof are provided. The compositions can be used to modulate flowering and photoperiod sensitivity in a plant. For example, methods are provided for developing genetically modified plant varieties in which flowering is accelerated, delayed or prevented. Methods are provided for treating a plant in order to delay flowermg in the plant. Methods of placing a polynucleotide of interest, such a gene, under photoperiod sensitive control or photoperiod insensitive control are also provided. Screening methods are for identifying chemical agents that can modify photoperiod sensitivity are also disclosed.
Description
SORGHUM MATURITY GENE AND USES THEREOF IN MODULATING PHOTOPERIOD SENSITIVITY
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government Support under Agreement 00-35300-9215 awarded by the US Department of Agriculture. The
Government has certain rights in the invention.
FIELD OF THE INVENTION
The invention is generally related to the field of plant genetics and molecular biology, more particularly to genes involved in plant photoperiod sensitivity, and methods for modifying photoperiod sensitivity in plants.
BACKGROUND OF THE INVENTION
Biomass yield is one of the most important attributes of a biomass or bioenergy crop designed for ligno-cellulosic conversion to biofuels or bioenergy. To maximize yield, it is essential to tailor the plants' life cycle to the agro-environments in which they are grown. The transition from vegetative to reproductive growth is a critical developmental switch and a key adaptive trait that ensures that plants set their flowers at an optimum time for pollination, seed development, and dispersal. For example, temperate environments with a long growing season allow cereal crops to exploit an extended vegetative period for resource storage. Conversely, early flowering has evolved as an adaptation to short growing seasons.
For example, once grain sorghum initiates flowering, growth of the vegetative plant (stem, leaves) decreases so that carbon and nitrogen compounds can be used for grain production. As a consequence, biomass accumulation overall decreases to some extent during the reproductive phase and largely ceases once grain filling has been completed.
In contrast, a late or non-flowering bioenergy sorghum crop grown for biomass production will continue to accumulate biomass by building larger vegetative plants until frost or adverse environmental conditions inhibit photosynthesis. It is estimated that late/non-flowering biomass sorghum will generate more than two times the biomass accumulated by
grain sorghum per acre assuming reasonable growth conditions throughout the growing season.
Flowering is generally controlled by environmental factors, such as daylength. Daylength regulates flowering by a phenomenon known as photoperiod sensitivity, which allows plants to coordinate their reproduction with the environment or with other members of their species. Photoperiod sensitivity refers to the fact that some plants will not flower until they are exposed to day lengths that are less than a critical photoperiod (short day plants) or greater than a critical photoperiod (long day plants). Long day (LD) and short day (SD) plant designations refer to the day length required to induce flowering. Facultative LD or SD plants are those that show accelerated flowering in LD or SD but will eventually flower regardless of photoperiod.
Therefore, it is an object of the invention to provide a gene in sorghum responsible for genetic control of photoperiod sensitivity.
It is another object of the invention to provide late or non-flowering recombinant sorghum plants.
It is yet another object of the invention to provide methods for modifying photoperiod sensitivity in plants.
It is a further object of the invention to provide methods for imposing photoperiod sensitivity on a plant process.
SUMMARY OF THE INVENTION
Compositions including the nucleic acid sequence of the sorghum Maturity gene 1 (Mai), and expression control sequences thereof are disclosed. The expression control sequence can be photoperiod sensitive or photoperiod insensitive. The compositions and methods can be used to modulating flowering in plants, particularly sorghum.
Methods of using the compositions for modulating photoperiod sensitivity for flowering and other plant processes in a plant are provided. For example, methods are provided for developing genetically modified plant varieties in which flowering is accelerated, or delayed or prevented. Methods are also provided for treating a plant in order to accelerate or delay flowering in the plant.
Methods and compositions for placing a polynucleotide of interest under photoperiod sensitive or photoperiod insensitive control are also disclosed. The compositions and methods and can be used, for example, to make photoperiod sensitive a gene that is normally or naturally photoperiod insensitive. In other embodiments, compositions and methods and can be used to make photoperiod insensitive a gene that is normally or naturally photoperiod sensitive.
Screening methods are also provided for identifying plants for photoperiod sensitivity and chemical agents that can modify photoperiod sensitivity.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar graph showing frequency distribution of F2 population of S. bicolor x S. propinquum as a function of flowering time. Also shown is a boxed line indicating average day length (hrs) over the time period. Also shown are two lines indicating the high (solid line) and low (dashed) temperature during the time period. S. propinquum and most F2s flowered when photoperiod was less than 12.5 hours. Segregation of the S, bicolor and S. propinquum alleles at the Mai locus imparts dichotomous phenotype when grown in a temperate environment
Figure 2 A is a diagram mapping the 1.1 centiMorgan (cM) interval delineated by progeny testing of recombinants. Figure 2B is a diagram showing the % of conversion at the DNA marker loci plotted along the sorghum genome sequence (on base pair, bp, scale). The diagram also maps the relative locations of the FT gene (Sb06g012260) and SbPRR37
(Sb06g012570). The dark line at the top of the diagram indicates the span of converted regions with approximate locations of genes in the sequence shown as cross-hatches along the axis. While the terminal regions that these data exclude from consideration are physically small, they contain the majority of genes.
Figure 3 A is a diagram illustrating two major S. bicolor haplotypes
(each with two rare variants) for the gene Sb06g012260 identified from analysis of re-sequencing data. One of the haplotypes (haplotype 1) closely resembled the allele found in the short-day flowering accession of Sorghum
propinquum. Figure 3B is a physical map showing the positions of four insertion-deletion events relative to the coding region of Sb06g012260. Figure 3C is a diagram comparing the PRR37 alleles in S, bicolor (top) and S. propinquum (bottom). The S. propinquum allele has an "AT" insertion between 97 and 98 nucleotides after the translation starting site. This insertion causes frameshift shortly before the beginning of the PRR domain (arrowhead), leading to numerous nonsense mutations (arrows) and resulting in premature protein termination near the end of the PRR domain. Coding regions are shown as boxes, introns as solid horizontal lines, vertical bars indicate nucleotide substitutions between the two alleles.
Figure 4 is a series of pie graphs showing haplotype frequencies for the gene Sb06g012260 in sub-populations from West Africa, South Africa, Central/ East Africa, and Asia/ India.
Figure 5A-5C are bar graphs showing flowering (days) for individuals having haplotype 1 of Figure 3 A (empty bars) or haplotype 2 of Figure 3 A (shaded bars) for the gene Sb06g012260 in West Africa (Fig. 5 A, 2008 p=0.005; R2=0.13) and South Africa (Fig. 5B (2008), p-3.84 E-08; R2 >.33) and Fig. 5C (2007), p=0.0346; R2=O.08). These data show a statistically-significant association of the haplotypes with flowering in subpopulations in which the two haplotypes each occur at similar frequencies.
Figure 6 is a line graph of log p value versus Mai region (Mbp) showing the association analysis of Mai region markers and photoperiod sensitive in Sorghum bicolor based on routine application of the software TASSEL (Bradbury, et al, Bioinformatics, 23:2633-2635 (2007)), as detailed below. (♦) single marker analysis; (e)analysis considering population structure.
Figure 7 is a diagram showing homologs identified by BLAST of a candidate Mai gene (Sb06g012260) in sorghum, rice, and Arabidopsis genomes; and maize and sugarcane ESTs.
DETAILED DESCRIPTION OF THE INVENTION I. Definitions
Before describing the various embodiments, it is to be understood that the invention is not limited in its application to the details of
construction and the arrangement of the components set forth in the following description. Other embodiments can be practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Unless otherwise indicated, the disclosure encompasses conventional techniques of plant breeding, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Plant Breeding: Principles and Prospects (Plant Breeding, Vol 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall, (1993); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)].
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.)? Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual 3rd. edition, Cold Spring Harbor Laboratory Press.
To facilitate understanding of the disclosure, the following definitions are provided:
The term "plant" is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative crop or cereal, and fruit or vegetable plant. It also refers to a plurality of plant cells that are
largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.
The term "photoperiod" refers to the period of a plant's exposure to daylight every 24 hours.
The term "photoperiod sensitivity" refers to the photoperiod that is required to induce a specific response, such as flowering. Some plants will not flower until they are exposed to day lengths that are less than a critical photoperiod (short day plants) or greater than a critical photoperiod (long day plants). In some plant species, photoperiodic control enforces long-day flowering. Therefore, a photoperiod sensitive plant can have either short-day or long-day flowering, but in both cases, the flowering is controlled by day length.
A plant is "photoperiod insensitive" or "day neutral" if the day length does not impact when flowering occurs. In order to modulate flowering based on day length, photoperiod sensitivity can be increased.
A "non-flowering" plant does not flower under the agronomic conditions, regardless of the photoperiod.
"Delayed flowering" refers to a plant that flowers on average at least 1 day later, including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days later, than a wild-type plant of the same species.
The term "non-naturally occurring plant" refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants produced by non-transgenic means such as plant breeding.
The term "plant tissue" includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term "plant part" as used herein refers to a plant structure, a plant organ, or a plant tissue.
The term "plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
The term "plant organ*' refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
The term "plant cell" refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.
The term "plant cell culture" refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
The term "transgenic plant" refers to a plant or tree that contains recombinant genetic material not normally found in plants or trees of this type and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation. Thus, a plant that is grown from a plant cell into which recombinant DNA is introduced by
transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually). It is understood that the term transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems etc.
The term "construct" refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism include in the 5' -3' direction, a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression.
The term "gene" refers to a DNA sequence that encodes through its template or messenger R A a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term "gene" also refers to a
DNA sequence that encodes an RNA product. The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5' and 3' ends.
The term "orthologous genes" or "orthologs" refer to genes that have a similar nucleic acid sequence because they were separated by a speciation event.
As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten amino acids. The polypeptides can be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell.
The term "isolated" is meant to describe a compound of interest (e.g., nucleic acids) that is in an environment different from that in which the compound naturally occurs, e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature. "Isolated" is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. Isolated nucleic acids are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components. An "isolated" nucleic acid molecule or polynucleotide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source. The isolated nucleic can be, for example, free of association with all components with which it is naturally associated. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature.
As used herein, the term "linkage disequilibrium" or "LD" refers to the situation in which the alleles for two or more loci do not occur together in individuals sampled from a population at frequencies predicted by the product of their individual allele frequencies. Markers that are in LD do not follow Mendel's second law of independent random segregation. LD can be caused by any of several demographic or population artifacts as well as by the presence of genetic linkage between markers. However, when these artifacts are controlled and eliminated as sources of LD, then LD results
directly from the fact that the loci involved are located close to each other on the same chromosome so that specific combinations of alleles for different markers (haplotypes) are inherited together. Markers that are in high LD can be assumed to be located near each other and a marker or haplotype that is in high LD with a genetic trait can be assumed to be located near the gene that affects that trait.
As used herein, the term "locus" refers to a specific position along a chromosome or DNA sequence. Depending upon context, a locus could be a gene, a marker, a chromosomal band or a specific sequence of one or more nucleotides.
The term "vector" refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.
The term "expression vector" refers to a vector that includes one or more expression control sequences
The term "expression control sequence" refers to a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
The term "promoter" refers to a regulatory nucleic acid sequence, typically located upstream (5') of a gene or protein coding sequence that, in conjunction with various elements, is responsible for regulating the expression of the gene or protein coding sequence. The promoters suitable for use in the constructs of this disclosure are functional in plants and in host organisms used for expressing the disclosed polynucleotides. Many plant promoters are publicly known. These include constitutive promoters, inducible promoters, tissue- and cell-specific promoters and
developmentally-regulated promoters. Exemplary promoters and fusion promoters are described, e.g., in U.S. Pat. No. 6,717,034, which is herein incorporated by reference in its entirety.
A nucleic acid sequence or polynucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
"Transformed," "transgenic," "transfected" and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an
extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non- transformed," "non-transgenic," or "non-recombinant" host refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
The term "endogenous" with regard to a nucleic acid refers to nucleic acids normally present in the host.
The term "heterologous" refers to elements occurring where they are not normally found. For example, a promoter may be linked to a
heterologous nucleic acid sequence, e.g., a sequence that is not normally found operably linked to the promoter. When used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter
sequence may be a control/ regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter. The term "heterologous" thus can also encompasses "exogenous" and "non-native" elements.
The term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:
100 times the fraction W/Z,
where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.
As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten amino acids. The polypeptides can be "exogenous " meaning that they are "heterologous," i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell.
The term "stringent hybridization conditions" as used herein mean that hybridization will generally occur if there is at least 95% and preferably
at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA such as salmon sperm D A, followed by washing the hybridization support in 0.1X SSC at approximately 65°C. Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2000).
II. Compositions
Photoperiod sensitivity refers to the fact that some plants will not flower until they are exposed to day lengths that are less than a critical photoperiod (short day plants) or greater than a critical photoperiod (long day plants). Long day (LD) and short day (SD) plant designations refer to the day length required to induce flowering. Facultative LD or SD plants are those that show accelerated flowering in LD or SD but will eventually flower regardless of photoperiod. Most plants including sorghum must pass tlirough a juvenile stage (lasting about 14-21 days for sorghum) before they become sensitive to photoperiod.
In general, Sorghum is a facultative SD plant where long days inhibit flowering and short days accelerate flowering. The degree of flowering photoperiod sensitivity in sorghum refers to the length of the short days that are required to induce flowering. Different sorghum genotypes vary in their degree of photoperiod sensitivity. For example, Sorghum inbreds have been identified with photoperiod sensitivity ranging from -10.5 to -14 hours and still others that are nearly completely insensitive to photoperiod.
Flowering depends on when seeds are planted and on the latitude in which they are planted. Therefore, in some embodiments, a photoperiod insensitive sorghum planted in Georgia in April can flower in approximately 48-55 days; whereas a highly photoperiod sensitive sorghum planted in Georgia in April can flower in -175-180 days, or may even fail to flower at all.
The maturity gene (Mai) contains one or more mutation or deletions in some S. bicolor genotypes such that sorghum plants containing this mutant gene are photoperiod insensitive (day-neutral). Identification of this gene allows for identification of orthologous genes in related plants. Moreover, based on this identification, methods of modulating photoperiod sensitivity in plants by modulating the expression control sequences of maturity gene in that plant are disclosed. Methods are also disclosed for modulating photoperiod sensitivity involving modulating the activity of the protein encoded by the Maturity (Mai) gene in the plant.
A. Mai
Compositions and methods for modifying photoperiod sensitivity in plants are provided. The methods can involve modulating the activity of the endogenous gene or gene(s) responsible for photoperiod sensitivity in the plant.
For example, the methods can involve promoting the expression of one or more endogenous gene orthologous to sorghum grain maturity gene 1 (Mai ). Thus, the methods can involve introducing to the plant a composition that promotes maturity gene 1 (Mai) activity in a Sorghum plant.
The term "Maturity gene" refers to the Mai gene found in Sorghum as well as orthologous genes serving the same function in related plants.
Sorghum
Sorghum has been an excellent biomass source with its high yield potential, high water use efficiency, and established production systems and is a representative plant that can be used with the disclosed methods and compositions. Sorghum is a genus of numerous species of grasses, some of which are raised for grain and some of which are used as fodder plants either cultivated or as part of pasture. The plants are cultivated in warmer climates worldwide. Sorghum is in the subfamily Panicoideae and the tribe
Andropogoneae.
Sorghum is well adapted to growth in hot, arid or semi-arid areas.
The many subspecies are divided into four groups— grain sorghums (such as milo), grass sorghums (for pasture and hay), sweet sorghums (used to produce sorghum syrups), and broom corn (for brooms and brushes).
Sorghum species include, but are not limited to Sorghum almum, Sorghum amplum, Sorghum angustwn, Sorghum arundinaceum, Sorghum bicolor, Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum
inter jectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum timorense, Sorghum trichocladum, Sorghum versicolor, Sorghum virgatum, and Sorghum vulgar e.
Sorghum Maturity Gene 1
There are six classic maturity genes in sorghum that control flowering time temied Mal-Ma6. Therefore, in general, sorghum plants with recessive Mai -Ma6 genes (with low or no activity) flower earlier than plants with dominant or active Mal-Ma6 genes that repress flowering.
Nucleic acid sequences for Mai genes in Sorghum bicolor and Sorghum propinquum are provided. It is understood that the skilled artisan can identify orthologous sequences in other Sorghum species for use in the present compositions and methods. For example, Mai genes from Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum timorense, Sorghum trichocladum, Sorghum versicolor, Sorghum virgatum, and Sorghum vulgar e can be identified and used in the disclosed methods.
Within the species Sorghum bicolor, there are both day-neutral (photoperiod insensitive) and short-day flowering forms. The vast majority of wild members of the species are short-day, as are forms cultivated in the
tropics. Forms cultivated in temperate latitudes (such as most of the USA) for seed/grain have been selected for day-neutral mutations. Therefore, the skilled artisan can use the guidance provided by the sequence comparisons to identify variants of Mai genes that can generate a photoperiod sensitive or insensitive phenotype.
Also disclosed is a transgenic plant having a nucleic acid molecule, or antisense constructs thereof, encoding a Ma 1 gene product, or variant, such as a codon optimized variant thereof, optionally operatively linked to an heterologous regulatory element. For example, disclosed is a transgenic plant characterized by high photoperiod sensitivity, low photoperiod sensitivity, or photoperiod insensitivity, wherein the cells of the plant express a nucleic acid molecule encoding an Mai gene product, or antisense construct thereof, that is operatively linked to an expression control sequence. In some embodiments, the construct encodes an inhibitory nucleic acid such as siRNA or RNAi that when express down regulates the expression of Mai .
Nucleic Acids
Mai gene
Disclosed are polynucleotides containing a maturity gene from a sorghum plant. It is understood that where coding sequences for a maturity gene are provided, also provided are the non-coding sequences that are known or can be identified to correspond to the coding sequences that are provided. For example, where a maturity gene is provided, also provided for use in the disclosed compositions and methods is the 5' untranslated region (UTR), which contains the endogenous promoter for the maturity gene. It is understood that the skilled artisan can identify these sequences with routine skill and experimentation based on the sequences that are provided.
1. Sequences for Short Day Flowering
The S. propinquum cultivar from which the sequences described below are derived is a short-day cultivar, that has a dominant (functional) Mai allele. Sequences for a dominant Mai gene are therefore provided.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in short day 5. propinquum includes the nucleic acid sequence:
1 AAAAGAAAAG TGAGCACACC ACGACCTGTC ATCAGCTCAT GGTCAGCTCT ACAAACTTAT
61 AGATTGCATC GAGATCTAAG ACTCAGGTAC AAATCATGTC AACATCTAAT GGTTTAGAAA
121 ATGAAAAGTT TTGAGTTTCA AAATATGATA CGTGATATTA ACATTTGAAC TTTTAGCAAG
181 ATCTGAAATA AAAAATTCAA CTAGATCATG TTAACATTGA TATAATCGCT TCCAATCGCC
241 TCCCATCACT TCCGCTAGAA AACTTTTTTT CTCGATTTAA TTAATGAAAG GGTAATAACA
301 TCATTGTACA AGATTCTTTC AAACCTCAAC CCCTATCATC GACGGTGACG GCTCCCTATA
361 ACACGCACTA GTGGACGCCG GGCGGGTGGA ACCCTAAGAA GATTTAAAAA AACTTAAGAA
421 GAAGATTTTT ATCTAACTAA CTATAGTACT TATATCATAC ACTATACTAT TCAAAATATT
481 ATTTTCACAA TATGAATTT ACCCTTTTAC TCTTCATTAA AAAAATACGA AAAAAGAATC
541 ACCACGTCTC TATTTAGGGT CCTAGTCCCC ATAATTTAAG AGGCGGTGAG AGACGATGTG
601 ACGTCTATGG ACCACCGACC AAAGACACAC CTATCGTCTC CCATCGCCTT GCTTCCATCG
661 CCTCTCATCG CTTTTCATAT TCTAGATCCA GCGGCCATAG ACACACCAAT CGTTTCTCAT
721 CGCCTCTCCA ACCATTGTAA AAATATTTAT AATTTTGATA TAAAATTTGT CTTCACTTGA
781 GTTCATGCCA AAAAAATTAT ACATATTATT TTCGTGTGAG AATTTACAGA AGTGGACTCT
841 TAAGATGTCC AAATGTAAAT GACCCTATTT ATTATGAGGC GCGGATCTAT AGGCCTGACT
901 CTGAAAATGG ATTATGGATT TGAGATAATA AATTTAAGGG CCTATCTTCG CACATAACAT
961 CTATAGTTCC TAAATTTTTT TTTATTGTAG TAGTAGAACT TTTCTCCCTG TAAACCAAGT
1021 TGACGCTGGG CTTTATTTTG CGACACAGAA CACCAAATTG GTGGCTATGA ACTCTTCCAC
1081 CTGGGCAGGG AAAACGGTTT ATTATGTTTC TCTTTAATTT ATCTATCGTG GCACTATAAC
1141 ACAACATGGC TTTGCCGACA CTTCCAACTA TCGGCAAAGG GTACCTTTAC CGACACTTAA
1201 CGTCTCACGA AAGGTTTTGC CGACAATTTT CAAACAGTCG CGGTAGAAGC AGTTGGCGAA
1261 ACTTTTGCCG ACAGTTAAAG GCATCGCCGA CACATTTTCT GTAGTCAAAT GGCATACCTA
1321 CGCCGACAGT TGAACTTTCA CCGACAGTGA ACCCTTTGCC GACAGTTTGG ACCTACGCCG
1381 ACAGTTTGGA CCTTTTCCGA CAGTTGGTAT GTTAGCGAAA CCGTTTCTAG GGTGTTTCAT
1441 AAACCATGCC TTGTCCAACA GTAGAAGTGT CGGCAAAACT ATATTGCTAG GATGTAGATA
1501 CAATTTAAAT ΑΤΤΤΤΑΑΤΑΛ ATACACATCA CATTGATTGA GCAAAATCAC ATGGTCTGTT
1561 TTCACTAAAA CTGTCAGAGG TACACTCCAG TACTACCAGT ACGTCGCCCG CACAGTGGCC
1621 AAGGATTTTA CTGCTACTGT TGATTAACAT AAGCACTTGC GACTTTCCCT AAAATCTTTT
1681 ATAAAACAAC GGCCGCAATA ATATTGAACT ATTTTTTTTC TAGTACCAAA ATTAGAATTT
1741 GATCCCTCAC CTCATTACAT CCATAGTAAC ATGACCAGAT ATATATGGAC AGGATGGGAT
1801 CACTCAGCGA GCAGATACAC TGAGCGATTC ATAATCAGAT TTTTTAATTT CTTCTAGTGA
1861 AGTGGGGTTT TCCTAGTCTT TTAACATTCA AAATTTAGTA CAAACTTTCC CTAGTAAATG
1921 CCTTCTAGTA AAGATTTCCT AGTATTTTGA CTAGCGATAG TGTTTTATTA CTAATTAAAA
1981 ACATTAGAAG AACTCCATTT AGTGATTGGT TGTTTGGATT AGTCTTCTCA CGTTAGACCT
2041 ATATATGCAG GACAACTCAA GCCAGCATAA ATATATGAAA TATCTTGGTG TTTGTTTGTC
2101 TGACACAGGC AACCGCGTTT GGTATAAATG TGTTTTCTTG TTTACATTTT ACCATCTATA
2161 GTCATCTCAA TGTTATATAG TAGAGGCTTC ATGTTTGTAG TAGATAAGGT AGAGAATTGA
2221 GAATATTTTA TTTTTGTGCG ACCATCAATT TTATGTAATC TGCATTGTCT AATGCTTTAT
2281 TTGACATTTG AAACTACTTA ATTTGACAGT TATGCAGGTC CGCATGATCC TATGAAAGCA
2341 ATTAATTAGT ACGGGTAAAC TGCACTACAC AAGTTTGCTA GTACTATTCT AT ACCGAC
2401 CTGTCAATAT TACCTTAAGT TACTGATTTC AATTAGAATC TAACACATTC AGGAAAAGAA
2461 GTTTCACTAG TACAAAAATC ATTTTCGTTG GCACGTTGTT TTTTTTTTCA CAGGCAGTTC
2521 ACAATATCAT GGTGCTAGTA GAAAAATTTC AACGGGCCCA ACAAGAGAAC CGCCAGGCGG
2581 TCTTCTTAAT TCAACCGCCT GTGTAAACTT TCCATTTACA TAGGCGGCTT ACGATAAAAA
2641 CCGTGTGTAT AAATACCATT AACACAGGCA GTCGAGTTAC GACAACCGCC TGTGTAAATG
2701 TGTCTTTTTA CACAGGCGGl1 TTGTATAGAG GGCCGCCTGT GCTAATATAT TTACACAGGC
2761 TATGAGCCGC CTGTGTTAAG TCTTCTATAA ATACCCTTCG TCCACCTCCA GACAAGAACA
2821 GTTACTCCCA TGAGCTCTGC ACACTGGCGG ACCAGACGAT TCCAGTTTCC AAGGGGGGAG
2881 GTTTTGATTT TCATTTCTTT GGTGAGAAAC TTCCAAAAGG TTAGTTAGTG CCATTGATGC
294 1 ΤΑΤΊ ΪΤΤΑΑ GCGATTCTTT GGTTCAATTC TTGTATTGGA GGTGCTCTAG ATCTAGAGTT
3001 CATCATGCAT TCTTGCTTAG GGTTAGAGTT CATAGGGCAA AAAGAGAGAG ATTTAGCTAA
3061 ATTTTTATGT AAATTCATAG TAAATTGTAA AAATTAAAAA AAATAAAAAA TAAATACTTT
3121 TTAGAATTCT TGTGAGTAGA TCTATACAAT AGAGTAATGA TGAGGATATT TTGAAGTTTA
3181 TAATTTTGAT TCAGTTTTAG CTTTTCTTTT TTCAGATGAA TTAGACTTTA TAAACTCAAA
3241 CATTAAAATG TTGAAAATCA TAAAATGGCA AATAAATACT TTTTCAAATC TTTGTGCATA
3301 AATACTTCAT AGAAATCCTT GAATTATTCC TAAATTTTAT ACAATTGTTT CTTATAATTA
3361 TGAAAATGAG TTTAAACAAT TATTTAAATT CCATAAATTG TAACTCCGTA AGGTGTAGGT
3421 TTTCATCTCT GTTTAATAGA AGGAGGTTAG TATCTTAGTT AAGTCTGTTT TCGGGGGTTA
3481 TATTAGTTTT GTTTTTAGAT TGACCTACAT TAATTGTTCT TAACTAATTA CAGCTAAATA
3541 TGGAGAGGTC ATTATGGATG TACAACTTAT CAAGATTGGA CCTATCATAT GTAGTGCAGG
3601 TCCAAAAATT TATTGATGTC GCAAAGATAC ATGCTCGCAG AACAAAGGCG AAGCACATAT
3661 GTTGTCCATG CGCAGACTGC AAAAATATTA TGGTATTTGA CAATGTAGAA GCAATTACTT
3721 CCCATCTGGT TTGAAGAGGA TTTATGGAGG ACTACTTGAT TTGGACAAAA CATGGTGAGG
3781 GTAGTTTTGC ACCTTATATG CGGACAACTG ACAACACTGC AACTAACATC AATGTGGAGG
3841 GTCCAATGCC ACCTCTCAAT GAATTTCATG CTATGCCAGA TGTTAATGAA ACTCATACGT
3901 CTGATGTCAA TGAAACTCAG CATGCTAACA CAGATGTTGT TGAAGATGCA GATTTCTTAG
3961 AGGCAATAAT GAACCGTTGT GCGGATCCAT CAATATTCTT CATGAAGGGA ATGAAAGCAT
4021 TGAAGAAGGC AGCAGAGGAC ACTTTGTACG ACGAGTCAAA AGGTTGTACC AAACAATGGT
4081 CGACATTATG TGTTGTTCTT CAGTTTTTGA CGATGAAGGC TAGACATGGT TGGTCCGATG
4141 CTAGCTTCAA TGATTTCTTG CGTGTACTTG GAGACCTTCT TCCTAAGGAG AACAAAGTGC
4201 CTGCTAACAC ATACTATGCA AAGAAGCTAG TCAGTCCACT TACGATAGGT GTTGAGAAGA
4261 TCCACGCATG TAGAAATCAT TGTATTCTAT ATCGAGGTGA TCAATATAAA GACTTAGACA
4321 GTTGTCCAAA CTGTGGTGCC AGTAGGTACA AGACAAACAA AGATTTTCGG GAGGAAGAGA
4381 ATCTAGCCTC TGTTTCTACA GGGAGGAAGC GAAAGAAGAC CCAAACAAAG ACTCAACAAG
4441 ACAAGCGCTC AAAGCCTAGT AGCAATGAAG AAGTGGACTA TTATGCATTG AGAAGAGTCT
4501 CCCTATGAGC CAAAAAAGGG GACAGCAGCA GGCACAACTC TCTTTCTGAA AGGACTTGGA
4561 AAGCAGCGGA CGGCACGGCT CATTGAGCTC GAACCGTCAC AGAAAAAGGA AGCCACCGCC
4621 CAGTCAATAG AAGCCATGCC CCCATCAAAG GAAGCCCCAA GTGGCGATGT ACATATTGAA
4681 CAGCCATCAA GTCAACCATT GACCCTAAAG GATATCAGAA AGCCAACGAT TGATGATTAT
4741 GTCAATGTCC CTAGTGACTA TGTGCCCGGA AGGCCTATGC TCCAATGGAC GCTGCTCGAT
4801 TAGATTCAAT GGCTGATAAA AAGGTTTCAT GACTGGTACA TGAGAGCAGT GCATGCTAGC
4861 CTCCATGGAA TCAGAGTTGA TATACCAACA GACATGTTTG CTACTGGTAA CAAAAAAAGC
4921 AAGACATTTG TTACCTTTGA GGACATGCAC TTGTTATTGA ACTATAGGCG GCTTGACGTC
4981 CAACTCATAA CAATCTGGTG CCTGTAAGTA TCACTCATGC ACACACAATT ATTATATATT
5041 AATATGTAGT GTGAAACTCT AATATGTAGA TGTTGTCTGT AGTTTGCAAG ATCACGAGCA
5101 GATGTCATTA TTATCTGCCG GATCGATGGT CGGTTATCTG AGCCCTATCA AGTTACAAGA
5161 AAATATGAAC AAATTCGTAT TATCAAAGGA AGATAGAGCA AAGATAGAGG AAGACAAAAC
5221 ACCAGGATAA TTATGCCATC TATCTTGGTA GATCAATGCT GAGGTATAAA TATAGGGATT
5281 TTATATTGGC ACCATACAAC ATTAGGTAAG CTTGACTTCA TATACGTATT TCAAATTATC
5341 GTGTAAACAA TATACATGTG TCGCTCACTC ATTTATTCAT GCAGTGACCA TTGGATTGTT
5401 TTTTATATTT ATCCCTTCGA AGGGAAGGTG CTTGTCCTAG ACTCTTTACA TGTTCCTCCC
5461 GAGAAGTATC AACCATTCTT GGTTCAATTA GAAAGGTGAG CCAACATGAA ACCACATGCG
5521 TACTTATATA AATTAGAGTT TCAAAATAAC TTTAGTGATT TAGGTTCGAT ATCTACGGGG
5581 CATGGCGGTT TTATAAGAAA CAAAAGGGAC CTGTCGACGC TGCACGCTCA GATCCTAGGA
5641 TCCCATTGAT GATACAACAC CACTATCCGG TAAGTTTTCT GAACACATTT CATCATATAA
5701 ATAATACATA AAGCATGGCA AATTTAGAAT AATCCGTTGC TCATTATATA GTGCCACAAG
5761 CAACCACCTG GATCGGTCTA TTGTGGGTAC TATGTCTGTG AGTTTATAAG GCAGCGGGGA
5821 CGTTACGTCA AGGACAAAAA TATGGTAAAT AATATCTATG TATGAAAGTT TTCTCATTAA
5881 AGCTGCAAAA TTATATATTG AACATGTGTC AATCATGCTT TTAAACTTTA TTTTCAGCCG
5941 AAAAAGCAAG GAAAAGACGT GCCCTTTACA CCAAAGACTC TGGAAGATAT AGTAGCATAC
6001 TTGTGTGGTT TTATTATGAG AGAAATAATT TCAAGTGACA GTGCATATTT TGATCATGAG
6061 GGCGATTTAG CAAGTGATAA ATTTAGAGTG CTGACAGACA TAGCAGGTCT ARATCTGAAG
6121 CGAAACGACA TGTAAACATT GTATGGTTGT GCGGATAACA TGCATTGACG TGTATATATA
6181 TAATTTTATG GTTGATGTTT GATTTGTTTA CAATTCTATA ATATATATAT GTGGTGTATG
6241 TATGATGTTG TGTGTGTATA TATATATATA TATATATATA TATATATATA TATATATATA
6301 TATATATATA TATATAATGT TTAGCACTGT GTTTGGTGGG AAAAATTAAA ATTTGAAATA
6361 TATATAAAAA ATTATTTACA CAGACAGTGT ACGTGTCGAG CGTCGTCCTG TGCTATACAA
6421 ATACATTCTA ACAGGCGGCT CGCCTTGTCC ACCGGTCGGT TAAAAATACA TTTCCACACH
6481 GGCCTGGCTG GGAGAGCCGC CTGTGAAAAC ATAATTTTCA CAGGCGGCTC GCACAGCCCC
6541 GCCTGTACTG TGGTCCATTT TGTACTGACC CCTGGTACAG GCGGTGGGCT TGGCCGCCTG
6601 TGAAGATGCT TTTAGCACCG CCTGTAAAAA TGTTTTTTGT AGCAGTGTTT TTCTTATTAG
6661 TAGTATCTTT TATACTAATT AAGATTCAAT ¾¾¾¾¾TT0A.C CATGACATCC CCATTGCCAA
6721 GAGAATATTT CGCCGCCCCT CAAAGCAGCC AATAAGGCTT TACTAAAAAG ACTATCCACG
6781 CAGTAGAGAT TTAGTCAAAA TATTCCAATA GCAATTGTTT CCTGCCTGCT TGACCTTCGT
6841 CAGCCACTCA CTGTATAAAT ATCGCACCAC GCCCTTTGCA GGCTTACAGA GCTTGTATTA
6901 CGT&CTAACA AGGCACACAC AGTACCCTGT GTTCACCGGC CCTGCACAAA ACTCAAGCAG
6961 TTATTACTAA CATGGCGGCT AACGATTCCT TGGTTACTGC TCATGTGATA GGAGATGTCT
7021 TGGACCCCTT CTATACAACC GTTGACATGA TGATCCTATT CGATGGTACT CCTATTATCA
7081 GCGGCATGGA GTTGCGCGCT CCGGCGGTTT CTGACAGGCC AAGGGTTGAA ATTGGAGGAG
7141 ATGATTATCG AGTTGCATAT ACTCTGGTAA ACTCATGCCA TGTCAATTAA CTAGTAG TG
7201 AATTTAGATG CTGGTGGTAT CGTGGATACA TGTACTATAT GTTATGGTTG ATACATATTT
7261 GTTTAATTGA TCGCAACACC ATTTGCGGTA ACTTCAAATT ACATTCTTTC AATATATAGG
7321 TGATGGTCGA TCCTGATGCT CCTAACCCAA GCAACCCAAC CTTGAGGGAG TACTTGCACT
7381 GGTAAGAGAA ACCTATAGAC GACAATTATT GTTGTTGGCA TGTTTTGCCC ACATATACTT
7441 TGTGTGTGTA TATTTGTGCT TATGCTTCTC CATAAAATTT TGGTGTATGT CTCAAGAGAG
7501 ATAGGTATAG AGGTTAGCAG TCCTTTAAAA ATGGTTTAAT CCAGTAGTTT TTTTTCGGTC
7561 GGACTGCTCG AATTATTGTA TATATGGAGA TCACATGCTA GTAACTTTTT CAATAATTTC
7621 ATGTTTCGAG CAGGATGGTG ACTGACATCC CAGCATCAAC TGATAATACA TACGGTGAGT
7681 ACACCCCTAT TCCCATTTTG AAACAAGTAG AATGTCTATT TTTATGATTT AGTATGTTCG
7741 TGACAATAGG CTATAGCTAT TTTGAAACTT CGGGAGCATA AAATAGTACT CGATTTTGTA
7801 TAACCATAAA CACACAGCTA GCCAATCTCT ATTCATATTT ATTTTAGTTT TATTTGCCGA
7861 ACCATCCTCA ACATCATAGC CACTTGATCG ATCATCTCAA TCAGCGTTTG TATCCTTGCC
7921 CGCTTGATTA TCATCCATGG CAGTTCATAT TTTTTTTCAT TTCTTTCATG CTTGTTATAG
7981 TTTTATCTGA TGAATCCAAG ATGTTATTGA TCAATTAGTT CAGATGAGCA GTAATGCATG
8041 TTGGAGGTTT GGTAGTATAT ATACGTTCAA AATTTCACGA AATCGGTAAT TACGGTGGGA
8101 GCCAAAAAAA ATTCCAAAAT TTCGTATTAC ATTAATAATG CATGTGCTGT AGACTCATAT
8161 TTTCTATGAT TTCGATTCTG TCACCATCCT GCTCGAATAT TTAAATCATG CTAATATTTT
8221 GTTTACATCT AAATCTTTTA TAAAAATTAT AATTTATATT TGGGTTTAAC AATTTCGGGC
8281 GCGTTTAGTG AGATTGGGTA ATTTCGGAGC GAGGCCACCG GCCACACGAA AAATTNCTAT
8341 ACACGHACTA TATGTGTACA TGTACATGCA TGGCACCCTG ATAGGCTACC CCATGGGGAA
8401 AAAATTGGAA ACGGACCATT CATACGCAGT CGTGGTGCAG ACTGTGGGCC ACAATAGCAG
8461 TGTAAACATA ATTACGGTAA TCAAATACCC CATGGGACCA TATATATCAT CCACAGATCC
8521 GTACGGTGCT TCCGTGTGGA TGGTCTACAC CAGATCTTTT CCACACCATA AGGGCAGCAA
8581 TGCAGCATCA TATTCATATA TGCACTAGTG ATGTACCATT TGGCTTATAT CATATTCAAC
8641 CTAACTCCTT GGAAACATTA TGATATTCTA TTGGGTTGAA GATGTCACTA CTACAAAAAA
8701 AAATCTTATG AGAGGTGTTT TGAAAACTGC CGGAGGTGCT TAAAGGAGAC AGACGAGTTA
8761 GGACAACCGT CTCTATTAAT GTGTACTAAC TGAGGTAGTT ACCGTAACGT GCCTGACTTG
8821 ATTAACAGAT TCAACCGTCT CAGTAAAGGC CATGATTAAC CGAAACAGAT TCGAGAGTTT
8881 TCTTAAGTAG TTAAACTATT TTAATCTTCA CCGAACTTAT AGAAAATGAA AGAGCTAACA
8941 CCAAIATTTA TAAAAATAAA TTAGTATCAC TAAATACATC ACGAAATCTA TTTGGTGTTG
9001 TAGAAGTTAT CCTTTTCTAT AAAATTGATC AAATTTATGA TAACTTAGTT TTAGGAATTC
9061 ATTTATTTTA GGACAACTGA GGAAGTACAT ATTTTTTAAG TCATCCACAA AGTAGTGGAT
9121 CCAATTTATT ACATTACTCT ACTACTTCAA ACTGAACAAA AGCCTAATCC TGGTTATTTT
9181 TAGAGTGATT TTTTACAACA TCAGCAGTAG TCCAGAAAAT GGGAGGACAT TAATAAAAGT
9241 GAAAAGGAGC AGAAGAAAGA TTACGGTATT TTATTTGTGC TATTTGTTTA ACTATTGGCA
9301 GTTTGGGACC GAAATAAATA ACTGTTCGTA GCTCTATATT TGTCGATTCA AAAAGTGTAA
9361 CGATGATTTT TGTGTTTCAA AAGAAAAATA AAGAAGTGCA CCAATGATTG GATATCATAG
9421 GCTATATATG TTGGATTAAT TGCATCCAAC GTATATAGTG AAAATGCTTT TCAATCAAGT
9481 AATCTTCGAG CGGTTACCAG TTTTAATAGT TGCGAGTCGT CGTTTTTTAT GTACCCTAGG
9541 ACATATATAT CCGCATGTAG ACGATGATGA GACTAGCAAG TTTTTGAGCA
9601 AATACATAAT TATTGGATTT GCAGGCCGTG AGATGATGTG CTACGAGCCC CCTGCCCCGT
9661 CCACGGGCAT CCACCGTATG GTGCTGGTGC TATTCCAGCA GCTTGGCCGT GACACGGTGT
9721 TCGCGGCGCC GTCCAGGCGC CACAACTTCA ACACCCGTGC CTTCGCCCGC CGCTACAACC
9781 TCGGCGCGCC CGTCGCCGCC ATGTTCTTCA ACTGCCAGCG CCAGACCGGC TCCGGTGGCC
9841 CCAGGTTCAC CGGGCCCTAC ACCAGCCGAC GTCGTGCGGG CTGATGACGA CGATCGTCGT
9901 TACGTCACGT GTACCGTACA CATATATGTA TAGATATACA TGCATGCATG TTCCATGGTA
9961 TAGGATCGGT GACAAAACGT CTAATAATGT ATACACACAC ATGCATGGAA TGCATGTAAT
10021 AAGAGAATAT ATGTATAATA AGTAGGGGAG AGCATGCATA TATTGTGTAC ACGCGTCCGA
10081 TGCGTATAGC CCTTTACATT ATTGTAGTTG TAATCAGCTG TTTAAGCATT CTGCTGTGTC
10141 AGAACATGAT GCATATATAG TTTGGTGTGA GTATTGATCT AGTGGAACTC TTATCAGCCT
10201 TCAACTCTTA TCACAAGTGT AAGATATAGC TTTTATACCT TCAGGTGTCT TCCCAGTGTA
10261 CCTAGAAATG CTACAACGGT TGTATTTTAT CTATGCGCTT CACTACTGGA AACCTGAATA
10321 CTTCTGTGGA TGTCGAATTT TTCTGTGCGT TTTTTTCGAT ACACACGGAA AAATTATAAT
10381 TATTCTGTGG GTTTTAAAAT ATCCTCATAG AAAAATACAA ATACCCACAG AAAAATTATA
10441 TCATTTTTCT GTGCGTGACA ATACACTCAC AGAAAAATTA CAATTTTTGT GTGTGTTTAT
10501 ATAAAACGCA CAGAAAAAAT AATCACACAC AGAAAAATTA TAATTATTCT GTAGGTTTCT
10561 ATAAAACGCA CATAAAAAAT AAACACACAC TGAAAAATAG AACAAGCACC CTCATACTAA
10621 ATTCATATAA ACACCCATAT TTTTTTCTTT TTAATCTCTC TGTAAAACTT GTAACTAGTT
10681 TTTCCCTCTC GTACTAACTC CAAATTGGAT GATTT
(SEQ ID N0:1 Sb06g012260 - S. propinquum) or functional fragment, or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:l.
The coding sequence of the maturity Mai gene of SEQ ID NO:l, including introns, can be:
1 ATGGCGGCTA ACGATTCCTT GGTTACTGCT CATGTGATAG GAGATGTCTT GGACCCCTTC
61 TAT CAACCG TTGACATGAT GATCCTATTC GATGGTACTC CTATTATCAG CGGCATGGAG
121 TTGCGCGCTC CGGCGGTTTC TGACAGGCCA AGGGTTGAAA TTGGAGG G TGATTATCGA
181 GTTGCATATA CTCTGGTAAA CTCATGCCAT GTCAATTAAC TAGTAGTTGA ATTTAGATGC
2 1 TGGTGGTATC GTGGATACAT GTACTATATG TTATGGTTGA TACATATTTG TTTAATTGAT
301 CGCAACACCA TTTGCGGTAA CXTCAAATTA CATTCTTTCA ATATATAGGT GATGGTCGAT
361 CCTGATGCTC CTAACCCAAG CAACCCAACC TTGAGGGAGT ACTTGCACTG GTAAGAGAAA
421 CCTATAGACG ACAATTATTG TTGTTGGCAT GTTTTGCCCA CATATACTTT GTGTGTGTAT
481 ATTTGTGCTT ATGCTTCTCC ATAAAATTTT GGTGTATGTC TCAAGAGAGA TAGGTATAGA
541 GGTTAGCAGT CCTTTAAAAA TGGTTTAATC CAGTAGTTTT TTTTCGGTCG GACTGCTCGA
601 ATTATTGTAT ATATGGAGAT CACATGCTAG TAACTTTTTC AATAATTTCA TGTTTCGAGC
661 AGGATGGTGA CTGACATCCC AGCATCAACT GATAATACAT ACGGTGAGTA CACCCCTATT
721 CCCATTTTGA AACAAGTAGA ATGTCTATTT TTATGATTTA GTATGTTCGT GACAATAGGC
781 TATAGCTATT TTGAAACTTC GGGAGCATAA AATAGTACTC GATTTTGTAT AACCATAAAC
841 ACACAGCTAG CCAATCTCTA TTCATATTTA TTTTAGTTTT ATTTGCCGAA CCATCCTCAA
901 CATCATAGCC ACTTGATCGA TCATCTCAAT CAGCGTTTGT ATCCTTGCCC GCTTGATTAT
961 CATCCATGGC AGTTCATATT TTTTTTCATT TCTTTCATGC TTGTTATAGT TTTATCTGAT
1021 GAATCCAAGA TGTTATTGAT CAATTAGTTC AGATGAGCAG TAATGCATGT TGGAGGTTTG
1081 GTAGTATATA TACGTTCAAA ATTTCACGAA ATCGGTAATT ACGGTGGGAG
1141 TTCCAAAATT TCGTATTACA TTAATAATGC ATGTGCTGTA GACTCATATT TTCTATGATT
1201 TCGATTCTGT CACCATCCTG CTCGAATATT TAAATCATGC TAATATTTTG TTTACATCTA
1261 AATCTTTTAT AAAAATTATA ATTTATATTT GGGTTTAACA ATTTCGGGCG CGTTTAGTGA
1321 GATTGGGTAA TTTCGGAGCG AGGCCACCGG CCACACGAAA AATTNCTATA CACGHACTAT
1381 ATGTGTACAT GTACATGCAT GGCACCCTGA TAGGCTACCC CATGGGGAAA AAATTGGAAA
144 1 CGGACCATTC ATACGCAGTC GTGGTGCAGA CTGTGGGCCA CAATAGCAGT GTAAACATAA
1501 TTACGGTAAT CAAATACCCC ATGGGACCAT ATATATCATC CACAGATCCG TACGGTGCTT
1561 CCGTGTGGAT GGTCTACACC AGATCTTTTC CACACCATAA GGGCAGCAAT GCAGCATCAT
1621 ATTCATATAT GCACTAGTGA TGTACCATTT GGCTTATATC ATATTCAACC TAACTCCTTG
1681 GAAACATTAT GATATTCTAT TGGGTTGAAG ATGTCACTAC TACAAAAAAA AATCTTATGA
1741 GAGGTGTTTT GAAAACTGCC GGAGGTGCTT AAAGGAGACA GACGAGTTAG GACAACCGTC
1801 TCTATTAATG TGTACTAACT GAGGTAGTTA CCGTAACGTG CCTGACTTGA TTAACAGATT
1861 CAACCGTCTC AGTAAAGGCC ATGATTAACC GAAACAGATT CGAGAGTTTT CTTAAGTAGT
1921 TAAACTATTT TAATCTTCAC CGAACTTATA GAAAATGAAA GAGCTAACAC C ¾i¾ X i X" T T? \T
1981 AAAAATAAAT TAGTATCACT AAATACATCA CGAAATCTAT TTGGTGTTGT AGAAGTTATC
2041 CTTTTCTATA AAATTGATCA AATTTATGAT AACTTAGTTT TAGGAATTCA TTTATTTTAG
2101 GACAACTGAG GAAGTACATA TTTTTTAAGT CATCCACAAA GTAGTGGATC CAATTTATTA
2161 CATTACTCTA CTACTTCAAA CTGAACAAAA GCCTAATCCT GGTTATTTTT AGAGTGATTT
2221 TTTACAACAT CAGCAGTAGT CCAGAAAATG GGAGGACATT AATAAAAGTG AAAAGGAGCA
2281 GAAGAAAGAT TACGGTATTT TATTTGTGCT ATTTGTTTAA CTATTGGCAG TTTGGGACCG
2341 AAATAAATAA CTGTTCGTAG CTCTATATTT GTCGATTCAA AAAGTGTAAC GATGATTTTT
2401 GTGTTTCAAA AGAAAAATAA AGAAGTGCAC CAATGATTGG ATATCATAGG CTATATATGT
2461 TGGATTAATT GCATCCAACG TATATAGTGA AAATGCTTTT CAATCAAGTA ATCTTCGAGC
2521 GGTTACCAGT TTTAATAGTT GCGAGTCGTC GTTTTTTATG TACCCTAGGA CATATATATC
2581 CGCATGTAGA CGATGATGAG ACTAGCAAGT TTTTTTTTTT TTTTGAGCAA ATACATAATT
2641 ATTGGATTTG CAGGCCGTGA GATGATGTGC TACGAGCCCC CTGCCCCGTC CACGGGCATC
2701 CACCGTATGG TGCTGGTGCT ATTCCAGCAG CTTGGCCGTG ACACGGTGTT CGCGGCGCCG
2761 TCCAGGCGCC ACAACTTCAA CACCCGTGCC TTCGCCCGCC GCTACAACCT CGGCGCGCCC
2821 GTCGCCGCCA GTTCTTCAA CTGCCAGCGC CAGACCGGCT CCGGTGGCCC CAGGTTCACC
2881 GGGCCCTACA CCAGCCGACG TCGTGCGGGC TGA
(SEQ ID N0:2 Sb06g012260 - S. propinq um), or functional fragment, or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in short day S. propinquum includes the nucleic acid sequence:
1 CCCTGACCCT TGTTGGGCAA CATTTAGAGT CGTTAGCTTT GCAATTCTTT GGTTCCAATG
61 GATGGTTATC ATTTAGACAT ATTGGTCATG CTTAGTCAAA ACTTTATTGT TCGGCTATAA
121 ACTTTTCAGT ACTTTGTAAT AATTGGCTCG A AGATGAAG CCGGGTATAA CATATCCTTT
181 ATCTAAAAAA ATTAGTTAAC ATGAACTTC TATTCAATTC TTCATATCTC ACT GCATCT
241 TTATTGTCTA GTTAGT TTG TAGCATTGC AAAAGCATGC AACTATATAC AATGAAACGG
301 AATAAAATTT CAGCTCT TT AATTTATATT TCAAATATAG GCCAC ATAG CCATATTTCG
361 TGCTCAAGGC CACAAAATCT TGCGTACT C CCTGTTGGTA CCAAAGAGAA GACGTTATTT
421 AACTTTGTTT GACTCTTCAA TATGGTTTGA ATCAGAAAAT TAGTTAAAAG AAAAGTGAGC
481 AC CCACGAC CTGTCATCAG CTCATGGTCA GCTCTACAAA CTTATAGATT GCATCGAGAT
541 CTAAGACTCA GGTACAAATC ATGTCAACAT CTAATGGTTT AGAAAATGAA AAGTTTTGAG
601 TTTCAAAATA TGATACGTGA TATTAACATT TGAACTTTTA GCAAGATCTG AAATAAAAAA
661 TTCAACTAGA TCATGTTAAC ATTGATATAA TCGCTTCCAA TCGCCTCCCA TCACTTCCGC
721 TAGAAAACTT TTTTTCTCGA TTTAATTAAT GAAAGGGTAA TAACATCATT GTACAAGATT
781 CTTTCAAACC TCAACCCCT TCATCGACGG TGACGGCTCC CTATAACACG CACTAGTGGA
8 1 CGCCGGGCGG GTGGAACCCT AAGAAGATTT AAAAAAACTT AAGAAGAAGA TTTTTATCTA
901 ACTAACTATA GTACTTATAT CATACACTAT ACTATTC AA A TTATTTT CACAATTATG
961 AATTTACCCT TTTACTCTTC ATTAAAAAAA TACGAAAAAA GAATCACCAC GTCTCTATTT
1021 AGGGTCCTAG TCCCCA AAT TTAAGAGGCG GTGAGAGACG ATGTGACGTC TATGGACCAC
1081 CGACCAAAGA CACACCTATC GTCTCCCATC GCCTTGCTTC CATCGCCTCT CATCGCTTTT
1141 CATATTCT G ATCCAGCGGC C T GACAC CCAATCGTTT CTCATCGCCT CTCCAACCAT
1201 TG AAAAATA TTTATAATTT TGATATAAAA TTTGTCTTCA CTTGAGTTC TGCCAAAAAA
1261 ATTATACATA TTATTTTCGT GTGAGAATTT AC GAAGTGG ACTCTTAAGA TGTCCAAATG
1321 TAAATGACCC TATTTATTAT GAGGCGCGGA TCTATAGGCC TGACTCTGAA AATGGATTAT
1381 GGATTTGAGA TAATAAATTT AAGGGCCTAT CTTCGCACAT AACATCT TA GTTCCTAAAT
1 41 TTTTTTTTAT TGTAGTAGTA GAACTTTTCT CCCTGTAAAC CAAGTTGACG CTGGGCTTTA
1501 TTTTGCGACA CAGAACACCA AATTGGTGGC TATGAACTCT TCCACCTGGG CAGGGAAAAC
1561 GGTTTATTAT GTTTCTCTTT AATTTATCTA TCGTGGCACT ATAACACAAC ATGGCTTTGC
1621 CGACACTTCC AACTATCGGC AAAGGGTACC TTTACCGACA CTTAACGTCT CACGAAAGGT
1681 TTTGCCGACA ATTTTCAAAC AGTCGCGGTA GAAGCAGTTG GCGAAACTTT TGCCGACAGT
1741 TAAAGGCATC GCCGACACAT TTTCTGTAGT CAAATGGCAT ACCTACGCCG ACAGTTGAAC
1801 TTTCACCGAC AGTGAACCCT TTGCCGACAG TTTGGACCTA CGCCGACAGT TTGGACCTTT
1861 TCCGACAGTT GGTATGTTAG CGAAACCGTT TCTAGGGTGT TTCATAAACC ATGCCTTGTC
1921 CAACAGTAGA AGTGTCGGCA AAACTATATT GCTAGGATGT AGATACAATT TAAATATTTT
1981 AATAAATACA CATCACATTG ATTGAGCAAA ATCACATGGT CTGTTTTCAC TAAAACTGTC
2041 AGAGGTACAC TCCAGTACT CCAGTACGTC GCCCGCACAG TGGCCAAGGA TTTTACTGCT
2101 ACTGTTGATT AACATAAGCA CTTGCGACTT TCCCTAAAAT CTTTTA AA ACAACGGCCG 2161 CAATAATATT GAACTATTTT TTTTCTAGTA CCAAAATT G AATT GATCC CTCACCTCAT 2221 TACATCCATA GTAACATGAC CAGA ATA TGGACAGGAT GGGATCACTC AGCGAGCAGA 2281 TACACTGAGC GATTCATAAT CAGATTTTTT AATTTCTTCT AGTGAAGTGG GGTTTTCCTA 23 1 GTCTTT AAC ATTCAAAATT TAGTACAAAC TTTCCCTAGT AAATGCCTTC TAGTAAAGAT 2401 TTCCTAGTAT TTTGACTAGC GATAGTGTTT TATTACTAAT TAAAAACATT AGAAGAACTC 2 61 CATTTAGTGA TTGGTTGTTT GGATTAGTCT TCTCACGTTA GACCTATA TGCAGGACAA 2521 CTCAAGCC G CATAAATATA TGAAATATCT TGGTGTTTGT TTGTCTGACA CAGGCAACCG 2581 CGTTTGGTAT AAATGTGTTT TCTTGTTTAC ATTTTACCAT CT TAGTCAT CTCAATGTTA 2641 TAT GTAGAG GCTTCATGTT TGTAGTAGA AAGGTAGAGA ATTGAGAATA TTTTATTTTT 2701 GTGCGACCAT CAA TTTATG TAATCTGCAT TGTCTAATGC TTTATTTGAC ATTTGAAACT
2761 ACTTAATTTG ACAGTTATGC AGGTCCGCAT GATCCTATGA AAGCAATTAA TTAGTACGGG
2821 TAAACTGCAC TACACAAGTT TGCTAGTACT ATTCTATTAA CCGACCTGTC AATATTACCT
2881 TAAGTTACTG ATTTCAATTA GAATCTAACA CATTCAGGAA AAGAAGTTTC ACTAGTACAA
294 1 AAATCATTTT CGTTGGCACG TTGTTTTTTT TTTCACAGGC AGTTCACAAT ATCATGGTGC
3001 TAGTAGAAAA ATTTCAACGG GCCCAACAAG AGAACCGCCA GGCGGTCTTC TTAATTCAAC
3061 CGCCTGTGTA AACTTTCCAT TTACATAGGC GGCTTACGAT AAAAACCGTG TGTATAAATA
3121 CCATTAACAC AGGCAGTCGA GTTACGACAA CCGCCTGTGT AAATGTGTCT TTTTACACAG
318 1 GCGGTTTGTA TAGAGGGCCG CCTGTGCTAA TATATTTACA CAGGCTATGA GCCGCCTGTG
324 1 TTAAGTCTTC TATAAATACC CTTCGTCCAC CTCCAGACAA GAACAGTTAC TCCCATGAGC
3301 TCTGCACACT GGCGGACCAG ACGATTCCAG TTTCCAAGGG GGGAGGTTTT GATTTTCATT
33 61 TCTTTGGTGA GAAACTTCCA AAAGGTTAGT TAGTGCCATT GATGCTATTT TTTAAGCGAT
342 1 TCTTTGGTTC AATTCTTGTA TTGGAGGTGC TCTAGATCTA GAGTTCATCA TGCATTCTTG
3481 CTTAGGGTTA GAGTTCATAG GGCAAAAAGA GAGAGATTTA GCTAAATTTT TATGTAAATT
354 1 CATAGTAAAT TGTAAAAATT ΑΑΑΑΑΑΑΆΤΑ AAAAATAAAT ACTTTTTAGA ATTCTTGTGA
3601 GTAGATCTAT ACAATAGAG AATGATGAGG ATATTTTGAA GTTTATAATT TTGATTCAGT
3661 TTTAGCTTTT CTTTTTTCAG ATGAATTAGA CTTTATAAAC TCAAACATTA AAATGTTGAA
372 1 AATCATAAAA TGGCAAATAA ATACTTTTTC AAATCTTTGT GCATAAATAC TTCATAGAAA
3781 TCCTTGAATT ATTCCTAAAT TTTATACAAT TGTTTCTTAT AATTATGAAA ATGAGTTTAA
3841 ACAATTATTT AAATTCCATA AATTGTAACT CCGTAAGGTG TAGGTTTTCA TCTCTGTTTA
3901 ATAGAAGGAG GTTAGTATCT TAGTTAAGTC TGTTTTCGGG GGTTATATTA GTTTTGTTTT
3961 TAGATTGACC TACATTAATT GTTCTTAACT AATTACAGCT AAATATGGAG AGGTCATTAT
4021 GGATGTACAA CTTATCAAGA TTGGACCTAT CATATGTAGT GCAGGTCCAA AAATTTATTG
4081 ATGTCGCAAA GATACATGCT CGCAGAACAA AGGCGAAGCA CATATGTTGT CCATGCGCAG
41 41 ACTGCAAAAA TATTATGGTA TTTGACAATG TAGAAGCAAT TACTTCCCAT CTGGTTTGAA
4201 GAGGATTTAT GGAGGACTAC TTGATTTGGA CAAAACATGG TGAGGGTAGT TTTGCACCTT
4261 ATATGCGGAC AACTGACAAC ACTGCAACTA ACATCAATGT GGAGGGTCCA ATGCCACCTC
4 321 TCAATGAATT TCATGCTATG CCAGATGTTA ATGAAACTCA TACGTCTGAT GTCAATGAAA
4381 CTCAGCATGC TAACACAGAT GTTGTTGAAG ATGCAGATTT CT AGAGGCA ATAATGAACC
44 4 1 GTTGTGCGGA TCCATCAATA TTCTTCATGA AGGGAATGAA AGCATTGAAG AAGGCAGCAG
4501 AGGACACTTT GTACGACGAG TCAAAAGGTT GTACCAAACA ATGGTCGACA TTATGTGTTG
4 561 TTCTTCAGTT TTTGACGATG AAGGCTAGAC ATGGTTGGTC CGATGCTAGC TTCAATGATT
4 621 TCTTGCGTGT ACTTGGAGAC CTTCTTCCTA AGGAGAACAA AGTGCCTGCT AACACATACT
4681 ATGCAAAGAA GCTAGTCAGT CCACTTACGA TAGGTGTTGA GAAGATCCAC GCATGTAGAA
474 1 ATCATTGTAT TCTATATCGA GGTGATCAAT ATAAAGACTT AGACAGTTGT CCAAACTGTG
4801 GTGCCAGTAG GTACAAGACA AACAAAGATT TTCGGGAGGA AGAGAATCTA GCCTCTGTTT
4861 CTACAGGGAG GAAGCGAAAG AAGACCCAAA CAAAGACTCA ACAAGACAAG CGCTCAAAGC
4921 CTAGTAGCAA TGAAGAAGTG GACTATTATG CATTGAGAAG AGTCTCCCTA TGAGCCAAAA
498 1 AAGGGGACAG CAGCAGGCAC AACTCTCTTT CTGAAAGGAC TTGGAAAGCA GCGGACGGCA
504 1 CGGCTCATTG AGCTCGAACC GTCACAGAAA AAGGAAGCCA CCGCCCAGTC AATAGAAGCC
5101 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATA TTGAACAGCC ATCAAGTCAA
51 61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCTAGT
522 1 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TCGATTAGAT TCAATGGCTG
528 1 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTAGCCTCCA TGGAATCAGA
5341 GTTGATATAC CAACAGACAT GTTTGCTACT GGTAACAAAA AAAGCAAGAC ATTTGTTACC
5401 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
54 61 TGGTGCCTGT AAGTATCACT CATGCACACA CAATTATTAT ATATTAATAT GTAGTGTGAA
552 1 ACTCTAATAT GTAGATGTTG TCTGTAGTTT GCAAGATCAC GAGCAGATGT CATTATTATC
5581 TGCCGGATCG ATGGTCGGTT ATCTGAGCCC TATCAAGTTA CAAGAAAATA TGAACAAATT
5641 CGTATTATCA AAGGAAGATA GAGCAAAGAT AGAGGAAGAC AAAACACCAG GATAATTATG
570 1 CCATCTATCT TGGTAGATCA ATGCTGAGGT ATAAATATAG GGATTTTATA TTGGCACCAT
i¾ i¾
882 1 GAGGCCACCG GCCACACGAA AAATTCTATA CACGACTATA TGTGTACATG TACATGCATG
888 1 GCACCCTGAT AGGCTACCCC ATGGGGAAAA AATTGGAAAC GGACCATTCA TACGCAGTCG
894 1 TGGTGCAGAC TGTGGGCCAC AATAGCAGTG TAAACATAAT TACGGTAATC AAATACCCCA
9001 TGGGACCATA TATATCATCC ACAGATCCGT ACGGTGCTTC CGTGTGGATG GTCTACACCA
9061 GATCTTTTCC ACACCATAAG GGCAGCAATG CAGCATCATA TTCATATATG CACTAGTGAT
9121 GTACCATTTG GCTTATATCA TATTCAACCT AACTCCTTGG AAACATTATG ATATTCTATT
9181 GGGTTGAAGA TGTCACTACT ACAAAAAAAA ATCTTATGAG AGGTGTTTTG AAAACTGCCG
9241 GAGGTGCTTA AAGGAGACAG ACGAGTTAGG ACAACCGTCT CTATTAATGT GTACTAACTG
9301 AGGTAGTTAC CGTAACGTGC CTGACTTGAT TAACAGATTC AACCGTCTCA GTAAAGGCCA
9361 TGATTAACCG ¾¾i¾CPiG! Ί^ΐ C GAGAGTTTTC TTAAGTAGTT AAACTATTTT AATCTTCACC
942 1 GAACTTATAG AAAATGAAAG AGCTAACACC AATATTTATA AAAATAAATT AGTATCACTA
954 1 ATTTATGATA ACTTAGTTTT AGGAftTTCAT TTATTTTAGG ACAACTGAGG AAGTACATAT
9 601 TTTTTAAGTC ATCCACAAAG TAGTGGATCC AATTTATTAC ATTACTCTAC TACTTCAAAC
9661 TGAACAAAAG CCTAATCCTG GTTATTTTTA GAGTGATTTT TTACAACATC AGCAGTAGTC
9721 CAGAAAATGG GAGGACATTA ATAAAAGTGA AAAGGAGCAG AAGAAAGATT ACGGTATTTT
9781 ATTTGTGCTA TTTGTTTAAC TATTGGCAGT TTGGGACCGA AATAAATAAC TGTTCGTAGC
9841 TCTATATTTG TCGATTCGAA AGTGTAACGA TGATTTTTGT GTTTCAAAAG AAAAATAAAG
990 1 AAGTGCACCA ATGATTGGAT ATCATAGGCT ATATATGTTG GATTAATTGC ATCCAACGTA
9961 TATAGTGAAA ATGCTTTTCA ATCAAGTAAT CTTCGAGCGG TTACCAGTTT TAATAGTTGC
1002 1 GAGTCGTCGT TTTTTATGTA CCCTAGGACA TATATATCCG CATGTAGACG ATGATGAGAC
1008 1 TAGCAAGTTT TTTTTTTTTT TTGAGCAAAT ACATAATTAT TGGATTTGCA GGCCGTGAGA
10 14 1 TGATGTGCTA CGAGCCCCCT GCCCCGTCCA CGGGCATCCA CCGTATGGTG CTGGTGCTAT
10201 TCCAGCAGCT TGGCCGTGAC ACGGTGTTCG CGGCGCCGTC CAGGCGCCAC AACTTCAACA
10261 CCCGTGCCTT CGCCCGCCGC TACAACCTCG GCGCGCCCGT CGCCGCCATG TTCTTCAACT
10321 GCCAGCGCCA GACCGGCTCC GGTGGCCCCA GGTTCACCGG GCCCTACACC AGCCGACGTC
10381 GTGCGGGCTG ATGACGACGA TCGTCGTTAC GTCACGTGTA CCGTACACAT ATATGTATAG
10441 ATATACATGC ATGCATGTTC CATGGTATAG GATCGGTGAC AAAACGTCTA ATAATGTATA
10501 CACACACATG CATGGAATGC ATGTAATAAG AGAATATATG TATAATAAGT AGGGGAGAGC
10561 J T G C ' 'i^T? ¾ T TGTGTACACG CGTCCGATGC GTATAGCCCT TTACATTATT GTAGTTGTAA
1062 1 TCAG
(SEQ ID NO:3 Sb06g012260 (10.6KB) - S. propinquum), or functional fragment, or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3. Each N = can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
The coding sequence of the maturity Mai gene of SEQ ID NO:3, including mtrons, can be:
1 ATGGCGGCTA ACGATTCCTT GGTTACTGCT CATGTGATAG GAGATGTCTT GGACCCCTTC
61 TATACAACCG TTGACATGAT GATCCTATTC GATGGTACTC CTATTATCAG CGGCATGGAG
12 1 TTGCGCGCTC CGGCGGTTTC TGACAGGCCA AGGGTTGAAA TTGGAGGAGA TGATTATCGA
181 GTTGCATATA CTCTGGTAAA CTCATGCCAT GTCAATTAAC TAGTAGTTGA AT TAGATGC
241 TGGTGGTATC GTGGATACAT GTACTATATG TTATGGTTGA TACATATTTG TTTAATTGAT
301 CGCAACACCA TTTGCGGTAA CTTCAAATTA CATTCTTTCA ATATAf GGT GATGGTCGAT
361 CCTGATGCTC CTAACCCAAG CAACCCAACC TTGAGGGAGT ACTTGCACTG GTAAGAGAAA
421 CCTATAGACG ACAATTATTG TTGTTGGCAT GTTTTGCCCA CATATACTTT GTGTGTGTAT
481 ATTTGTGCTT ATGCTTCTCC ATAAAATTTT GGTGTATGTC TCAAGAGAGA TAGGTATAGA
541 GGTTAGCAGT CCTTTAAAAA TGGTTTAATC CAGTAGTTTT TTTTCGGTCG GACTGCTCGA
601 ATTATTGTAT ATATGGAGAT CACATGCTAG TAACTTTTTC AATAATTTCA TGTTTCGAGC
661 AGGATGGTGA CTGACATCCC AGCATCAACT GATAATACAT ACGGTGAGTA CACCCCTATT
721 CCCATTTTGA AACAAGTAGA ATGTCTATTT TTATGATTTA GTATGTTCGT GACAATAGGC
781 TATAGCTATT TTGAAACTTC GGGAGCATAA AATAGTACTC GATTTTGTAT AACCATAAAC
841 ACACAGCTAG CCAATCTCTA TTCATATTTA TTTTAGTTTT ATTTGCCGAA CCATCCTCAA
901 CATCATAGCC ACTTGATCGA TCATCTCAAT CAGCGTTTGT ATCCTTGCCC GCTTGATTAT
961 CATCCATGGC AGTTCATATT TTTTTTCATT TCTTTCATGC TTGTTATAGT TTTATCTGAT
1021 GAATCCAAGA TGTTATTGAT CAATTAGTTC AGATGAGCAG TAATGCATGT TGGAGGTTTG
1081 GTAGTATATA TACGTTCAAA ATTTCACGAA ATCGGTAATT ACGGTGGGAG CCAAAAAAAA
1141 TTCCAAAATT TCGTATTACA TTAATAATGC ATGTGCTGTA GACTCATATT TTCTATGATT
1201 TCGATTCTGT CACCATCCTG CTCGAATATT TAAATCATGC TAATATTTTG TTTACATCTA
1261 AATCTTTTAT AAAAATTATA ATTTATATTT GGGTTTAACA ATTTCGGGCG CGTTTAGTGA
1321 GATTGGGTAA 7TTCGGAGCG AGGCCACCGG CCACACGAAA AATTCTATAC ACGACTATAT
1381 GTGTACATGT ACATGCATGG CACCCTGATA GGCTACCCCA TGGGGAAAAA ATTGGAAACG
1441 GACCATTCAT ACGCAGTCGT GGTGCAGACT GTGGGCCACA ATAGCAGTGT AAACATAATT
1501 ACGGTAATCA AATACCCCAT GGGACCATAT ATATCATCCA CAGATCCGTA CGGTGCTTCC
1561 GTGTGGATGG TCTACACCAG ATCTTTTCCA CACCATAAGG GCAGCAATGC AGCATCATAT
1621 TCATATATGC ACTAGTGATG TACCATTTGG CTTATATCAT ATTCAACCTA ACTCCTTGGA
1681 AACATTATGA TATTCTATTG GGTTGAAGAT GTCACTACTA CAAAAAAAAA TCTTATGAGA
1741 GGTGTTTTGA AAACTGCCGG AGGTGCTTAA AGGAGACAGA CGAGTTAGGA CAACCGTCTC
1801 TATTAATGTG TACTAACTGA GGTAGTTACC GTAACGTGCC TGACTTGATT AACAGATTCA
1861 ACCGTCTCAG TAAAGGCCAT GATTAACCGA AACAGATTCG AGAGTTTTCT TAAGTAGTTA
1921 AACTATTTTA ATCTTCACCG AACTTATAGA AAATGAAAGA GCTAACACCA ATATTTATAA
1981 AAATAAATTA GTATCACTAA ATACATCACG ¾J ¾T C T ΐ Τ T" T GGTGTTGTAG AAGTTATCCT
2041 TT *3T C 1Ί¾ T ATTGATCAAA TTTATGATAA CTTAGTTTTA GGAATTCATT TATTTTAGGA
2101 CAACTGAGGA AGTACATATT TTTTAAGTCA TCCACAAAGT AGTGGATCCA ATTTATTACA
2161 TTACTCTACT ACTTCAAACT GAACAAAAGC CTAATCCTGG TTATTTTTAG AGTGATTTTT
2221 TACAACATCA GCAGTAGTCC AGAAAATGGG AGGACAT AA TAAAAGTGAA AAGGAGCAGA
2281 AGAAAGATTA CGGTATTTTA TTTGTGCTAT TTGTTTAACT ATTGGCAGTT TGGGACCGAA
2341 ATAAATAACT GTTCGTAGCT CTATATTTGT CGATTCGAAA GTGTAACGAT GATTTTTGTG
2401 TTTCAAAAGA AAAATAAAGA AGTGCACCAA TGATTGGATA TCATAGGCTA TATATGTTGG
2461 •A.T TftftT "3? CCA TCCAACGTAT ATAGTGAAAA TGCTTTTCAA TCAAGTAATC TTCGAGCGGT
2521 TACCAGTTTT AATAGTTGCG AGTCGTCGTT TTTTATGTAC CCTAGGACAT ATATATCCGC
2581 ATGTAGACGA TGATGAGACT AGCAAGTTTT Ί T" T T T" T TGAGCAAATA CATAATTATT
2641 GGATTTGCAG GCCGTGAGAT GATGTGCTAC GAGCCCCCTG CCCCGTCCAC GGGCATCCAC
2701 CGTATGGTGC TGGTGCTATT CCAGCAGCTT GGCCGTGACA CGGTGTTCGC GGCGCCGTCC
2761 AGGCGCCACA ACTTCAACAC CCGTGCCTTC GCCCGCCGCT ACAACCTCGG CGCGCCCGTC
2821 GCCGCCATGT TCTTCAACTG CCAGCGCCAG ACCGGCTCCG GTGGCCCCAG GTTCACCGGG
2881 CCCTACACCA GCCGACGTCG TGCGGGCTGA
(SEQ ID NO:4 Sb06g012260 (10.6kb) - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%», 97%, 98%, or 99% sequence identity to SEQ ID NO:4.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in short day S. propinquum includes the nucleic acid sequence:
¾ >J¾ c/
T . s X jftAiT ¾ T
ϋ\ ?¾ c
2761 TCTCAAGAGA GATAGGTATA GAGGTTAGCA GTCCTTTAAA AATGGTTTAA TCCAGTAGTT
282 1 TTTTTTCGGT CGGACTGCTC GAATTATTGT ATATATGGAG ATCACATGCT AGTAACTTTT
288 1 TCAATAATTT CATGTTTCGA GCAGGATGGT GACTGACATC CCAGCATCAA CTGATAATAC
294 1 ATACGGTGAG TACACCCCTA TTCCCATTTT GAAACAAGTA GAATGTCTAT TTTTATGATT
3001 TAGTATGTTC GTGACAATAG GCTATAGCTA TTTTGAAACT TCGGGAGCAT AAAATAGTAC
3061 TCGATTTTGT ATAACCATAA ACACACAGCT AGCCAATCTC Ί1 ¾Ώ? C ΙΑΓ 'ΛΤ T TATTTTAGTT
3121 TTATTTGCCG AACCATCCTC AACATCATAG CCACTTGATC GATCATCTCA ATCAGCGTTT
318 GTATCCTTGC CCGCTTGATT ATCATCCATG GCAGTTCATA TTTTTTTTCA TTTCTTTCAT
3241 GCTTGTTATA GTTTTATCTG ATGAATCCAA GATGTTATTG ATCAATTAGT TCAGATGAGC
3301 AGTAATGCAT GTTGGAGGTT TGGTAGTATA TATACGTTCA AAATTTCACG AAATCGGTAA
3361 TTACGGTGGG AGCCAAAAAA AATTCCAAAA TTTCGTATTA CATTAATAAT GCATGTGCTG
342 1 TAGACTCATA TTTTCTATGA TTTCGATTCT GTCACCATCC TGCTCGAATA TTTAAATCAT
348 1 GCTAATATTT TGTTTACATC TAAATCTTTT ATAAAAATTA TAATTTATAT TTGGGTTTAA
354 1 CAATTTCGGG CGCGTTTAGT GAGATTGGGT AATTTCGGAG CGAGGCCACC GGCCACACGA
3601 AAAATTCTAT ACACGACTAT ATGTGTACAT GTACATGCAT GGCACCCTGA TAGGCTACCC
3661 CATGGGGAAA AAATTGGAAA CGGACCATTC ATACGCAGTC GTGGTGCAGA CTGTGGGCCA
3721 CAATAGCAGT GTAAACATAA TTACGGTAAT CAAATACCCC ATGGGACCAT ATATATCATC
3781 CACAGATCCG TACGGTGCTT CCGTGTGGAT GG CTACACC AGATCTTTTC CACACCATAA
3841 GGGCAGCAAT GCAGCATCAT ATTCATATAT GCACTAGTGA TGTACCATTT GGCTTATATC
3901 ftT-JV T ^j Oi¾ c c TAACTCCTTG GAAACATTAT GATATTCTAT TGGGTTGAAG ATGTCACTAC
3961 AATCTTATGA GAGGTGTTTT GAAAACTGCC GGAGGTGCTT AAAGGAGACA
4021 GACGAGTTAG GACAACCGTC TCTATTAATG TGTACTAACT GAGGTAGTTA CCGTAACGTG
4081 CCTGACTTGA TTAACAGATT CAACCGTCTC AGTAAAGGCC ATGATTAACC GAAACAGATT
4 141 CGAGAGTTTT CTTAAGTAGT TAAACTATTT TAATCTTCAC CGAACTTATA GAAAATGAAA
4201 GAGCTAACAC CAATATTTAT ΆΑΑΑΑΤΑΑΑΤ TAGTATCACT AAATACATCA CGAAATCTAT
42 61 TTGGTGTTGT AGAAGTTATC CTTTTCTATA AAATTGATCA AATTTATGAT AACTTAGTTT
4 321 TAGGAATTCA TTTATTTTAG GACAACTGAG GAAGTACATA TTTTTTAAGT CATCCACAAA
4381 GTAGTGGATC CAATTTATTA CATTACTCTA CTACTTCAAA CTGAACAAAA GCCTAATCCT
44 4 1 GGTTATTTTT AGAGTGATTT TTTACAACAT CAGCAGTAGT CCAGAAAATG GGAGGACA T
4501 AATAAAAGTG AAAAGGAGCA GAAGAAAGAT TACGGTATTT TATTTGTGCT ATTTGTTTAA
4561 CTATTGGCAG TTTGGGACCG AAATAAATAA CTGTTCGTAG CTCTATATTT GTCGATTCGA
4 621 AAGTGTAACG ATGATTTTTG TGTTTCAAAA GAAAAATAAA GAAGTGCACC AATGATTGGA
4 681 TATCATAGGC TATATATGTT GGATTAATTG CATCCAACGT ATATAGTGAA AATGCTTTTC
4741 TCTTCGAGCG GTTACCAGT TTAATAGTTG CGAGTCGTCG TTTTTTATGT
4801 ACCCTAGGAC ATATATATCC GCATGTAGAC GATGATGAGA CTAGCAAGTT T TTTTTTTT
4 8 61 TTTGAGCAAA TACATAATTA TTGGATTTGC AGGCCGTGAG ATGATGTGCT ACGAGCCCCC
4 921 TGCCCCGTCC ACGGGCATCC ACCGTATGGT GCTGGTGCTA TTCCAGCAGC TTGGCCGTGA
4 981 CACGGTGTTC GCGGCGCCGT CCAGGCGCCA CAACTTCAAC ACCCGTGCCT TCGCCCGCCG
5041 CTACAACCTC GGCGCGCCCG TCGCCGCCAT GTTCTTCAAC TGCCAGCGCC AGACCGGCTC
5101 CGGTGGCCCC AGGTTCACCG GGCCCTACAC CAGCCGACGT CGTGCGGGCT GATGACGACG
5161 ATCGTCGTTA CGTCACGTGT ACCGTACACA TATATGTATA GATATACATG CATGCATG T
522 1 CCATGGTATA GGATCGGTGA CAAAACGTCT AATAATGTA
(SEQ ID N0:5 Sb06g012260 (5.2kb) - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5. N = 1, 2, 3, 4, or 5 nucleotides in length.
The coding sequence of the maturity Mai gene of SEQ ID NO:5, including introns, can be:
1 ATGGCGGCTA ACGATTCCTT GGTTACTGCT CATGTGATAG GAGATGTCTT GGACCCCTTC
61 TATACAACCG TTGACATGAT GATCCTATTC GATGGTACTC CTATTATCAG CGGCATGGAG
121 TTGCGCGCTC CGGCGGTTTC TGACAGGCCA AGGGTTGAAA TTGGAGGAGA TGATTATCGA
181 GTTGCATATA CTCTGGTAAA CTCATGCCAT GTCAATTAAC TAGTAGTTGA ATTTAGATGC
241 TGGTGGTATC GTGGATACAT GTACTATATG TTATGGTTGA TACATATTTG TTTAATTGAT
301 CGCAACACCA TTTGCGGTAA CTTCAAATTA CATTCTTTCA ATATATAGGT GATGGTCGAT
361 CCTGATGCTC CTAACCCAAG CAACCCAACC TTGAGGGAGT ACTTGCACTG GTAAGAGAAA
421 CCTATAGACG ACAATTATTG TTGTTGGCAT GTTTTGCCCA CATATACTTT GTGTGTGTAT
481 ATTTGTGCTT ATGCTTCTCC ATAAAATTTT GGTGTATGTC TCAAGAGAGA TAGGTATAGA
541 GGTTAGCAGT CCTTTAAAAA TGGTTTAATC CAGTAGTTTT TTTTCGGTCG GACTGCTCGA
601 ATTATTGTAT ATATGGAGAT CACATGCTAG TAACTTTTTC AATAATTTCA TGTTTCGAGC
661 AGGATGGTGA CTGACATCCC AGCATCAACT GATAATACAT ACGGTGAGTA CACCCCTATT
721 CCCATTTTGA AACAAGTAGA ATGTCTATTT TTATGATTTA GTATGTTCGT GACAATAGGC
781 TATAGCTATT TTGAAACTTC GGGAGCATAA AATAGTACTC GATTTTGTAT AACCATAAAC
841 ACACAGCTAG CCAATCTCTA TTCATATTTA TTTTAGTTTT ATTTGCCGAA CCATCCTCAA
901 CATCATAGCC ACTTGATCGA TCATCTCAAT CAGCGTTTGT ATCCTTGCCC GCTTGATTAT
961 CATCCATGGC AGTTCATATT TTTTTTCATT TCTTTCATGC TTGTTATAGT TTTATCTGAT
1021 GAATCCAAGA TGTTATTGAT CAATTAGTTC AGATGAGCAG TAATGCATGT TGGAGGTTTG
1081 GTAGTATATA TACGTTCAAA ATTTCACGAA ATCGGTAATT ACGGTGGGAG CCAAAAAAAA
1141 TTCCAAAATT TCGTATTACA TTAATAATGC ATGTGCTGTA GACTCATATT TTCTATGATT
1201 TCGATTCTGT CACCATCCTG CTCGAATATT TAAATCATGC TAATATTTTG TTTACATCTA
1261 AATCTTTTAT AAAAATTATA ATTTATATTT GGGTTTAACA ATTTCGGGCG CGTTTAGTGA
1321 GATTGGGTAA TTTCGGAGCG AGGCCACCGG CCACACGAAA AATTCTATAC ACGACTATAT
1381 GTGTACA GT ACATGCATGG CACCCTGATA GGCTACCCCA TGGGGAAAAA ATTGGAAACG
1441 GACCATTCAT ACGCAGTCGT GGTGCAGACT GTGGGCCACA ATAGCAGTGT AAACATAATT
1501 ACGGTAATCA AATACCCCAT GGGACCATAT ATATCATCCA CAGATCCGTA CGGTGCTTCC
1561 GTGTGGATGG TCTACACCAG ATCTTTTCCA CACCATAAGG GCAGCAATGC AGCATCATAT
1621 TCATATATGC ACTAGTGATG TACCATTTGG CTTATATCAT ATTCAACCTA ACTCCTTGGA
1681 AACATTATGA TATTCTATTG GGTTGAAGAT GTCACTACTA TCTTATGAGA
1741 GGTGTTTTGA AAACTGCCGG AGGTGCTTAA AGGAGACAGA CGAGTTAGGA CAACCGTCTC
1801 TATTAATGTG TACTAACTGA GGTAGTTACC GTAACGTGCC TGACTTGATT AACAGATTCA
1861 ACCGTCTCAG TAAAGGCCAT GATTAACCGA AACAGATTCG AGAGTTTTCT TAAGTAGTTA
1921 AACTATTTTA ATCTTCACCG AACTTATAGA AAATGAAAGA GCTAACACCA ATATTTATAA
1981 AAATAAATTA GTATCACTAA ATACATCACG AAATCTATTT GGTGTTGTAG AAGTTATCCT
2041 TTTCTATAAA ATTGATCAAA TTTATGATAA CTTAGTTTTA GGAATTCATT TATTTTAGGA
2101 CAACTGAGGA AGTACATATT TTTTAAGTCA TCCACAAAGT AGTGGATCCA ATTTATTACA
2161 TTACTCTACT ACTTCAAACT GAACAAAAGC CTAATCCTGG TTATTTTTAG AGTGATTTTT
2221 TACAACATCA GCAGTAGTCC AGAAAATGGG AGGACATTAA TAAAAGTGAA AAGGAGCAGA
2281 AGAAAGATTA CGGTATTTTA TTTGTGCTAT TTGTTTAACT ATTGGCAGTT TGGGACCGAA
2401 TTTCAAAAGA AAAATAAAGA AGTGCACCAA TGATTGGATA TCATAGGCTA TATATGTTGG
24 61 AT AATTGCA TCCAACGTAT ATAGTGAAAA TGCTTTTCAA TCAAGTAATC TTCGAGCGGT
2521 TACCAGTTT AATAGTTGCG AGTCGTCGTT TTTTATGTAC CCTAGGACAT ATATATCCGC
2581 ATGTAGACGA TGATGAGACT AGCAAGTTTT TTTTTTTTTT TGAGCAAATA CATAATTATT
2641 GGATTTGCAG GCCGTGAGAT GATGTGCTAC GAGCCCCCTG CCCCGTCCAC GGGCATCCAC
2701 CGTATGGTGC TGGTGCTATT CCAGCAGCTT GGCCGTGACA CGGTGTTCGC GGCGCCGTCC
2761 AGGCGCCACA ACTTCAACAC CCGTGCCTTC GCCCGCCGCT ACAACCTCGG CGCGCCCGTC
2821 GCCGCCATGT TCTTCAACTG CCAGCGCCAG ACCGGCTCCG GTGGCCCC G GTTCACCGGG 2881 CCCTACACCA GCCGACGTCG TGCGGGCTGA
(SEQ ID N0:6 Sb06g012260 (5.2kb) - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:6.
The coding sequence of the maturity Mai gene, without introns, as it is found in short-day S. propinquum can include the nucleic acid sequence:
1 ATGGCGGCTA ACGATTCCTT GGTTACTGCT CATGTGATAG GAGATGTCTT GGACCCCTTC
61 TATACAACCG TTGACATGAT GATCCTATTC GATGGTACTC CTATTATCAG CGGCATGGAG
121 TTGCGCGCTC CGGCGGTTTC TGACAGGCCA AGGGTTGAAA TTGGAGGAGA TGATTATCGA
181 GTTGCATATA CTCTGGTGAT GGTCGATCCT GATGCTCCTA ACCCAAGCAA CCCAACCTTG
241 AGGGAGTACT TGCACTGGAT GGTGACTGAC ATCCCAGCAT CAACTGATAA TACATACGGC
301 CGTGAGATGA TGTGCTACGA GCCCCCTGCC CCGTCCACGG GCATCCACCG TATGGTGCTG
361 GTGCTATTCC AGCAGCTTGG CCGTGACACG GTGTTCGCGG CGCCGTCCAG GCGCCACAAC
421 TTCAACACCC GTGCCTTCGC CCGCCGCTAC AACCTCGGCG CGCCCGTCGC CGCCATGTTC
481 TTCAACTGCC AGCGCCAGAC CGGCTCCGGT GGCCCCAGGT TCACCGGGCC CTACACCAGC
541 CGACGTCGTG CGGGCTGA
(SEQ ID NO:7, Sb06g012260 - S. propinquum, or fragment, or a variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7.
A maturity Mai protein as it is found in short-day S, propinquum can include the amino acid sequence:
MAANDSLVTAHVIGDVLDPFYTTVDMMILFDGTPIISGMELRAPAVSDRPRVEIGGDDYRVAYTLVHVDPDA PMPSNPTLREYLHWMVTDIPASTDNTYGREMMCYEPPAPSTG1HRMVLVLFQQLGRDTVFAAPSRRHNFNTR AFARRYNLGAPVAAMFF CQRQTGSGGPRFTGPYTSRRRAG*
(SEQ ID NO: 8, Sb06g012260) or functional fragment, or variant thereof having at least 75%, 80%», 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in short day S. propinquum includes the nucleic acid sequence:
i CACTAGTACA AAAATCATTT TCGTTGGCAC GTTGTTTTTT TTTTCACAGG CAGTTCACAA
61 TATCATGGTG CTAGTAGAAA AATTTCAACG GGCCCAACAA GAGAACCGCC AGGCGGTCTT 121 CTTAATTCAA CCGCCTGTGT AAACTTTCCA TTTACATAGG CGGCTTACGA TAAAAACCGT
181 GTGTATAAAT ACCATTAACA CAGGCAGTCG AGTTACGACA ACCGCCTGTG TAAATGTGTC 241 TTTTTACACA GGCGGTTTGT ATAGAGGGCC GCCTGTGCTA ATATATTTAC ACAGGCTATG 301 AGCCGCCTGT GTTAAGTCTT CTATAAATAC CCTTCGTCCA CCTCCAGACA AGAACAGTTA 361 CTCCCATGAG CTCTGCACAC TGGCGGACCA GACGATTCCA GTTTCCAAGG GGGGAGGTTT 421 TGATTTTCAT TTCTTTGGTG AGAAACTTCC AAAAGGTTAG TTAGTGCCAT TGATGCTATT
481 TTTTAAGCGA TTCTTTGGTT CAATTCTTGT ATTGGAGGTG CTCTAGATCT AGAGTTCATC
54 1 ATGCATTCTT GCTTAGGGTT AGAGTTCATA GGGCAAAAAG AGAGAGATTT AGCTAAATTT
601 TTATGTAAAT TCATAGTAAA TTGTAAAAAT TAAAAAAAAT AAAAAATAAA TACTTTTTAG
661 AATTCTTGTG AGTAGATCTA TACAATAGAG TAATGATGAG GATATTTTGA AGTTTATAA
721 TTTGATTCAG TTTTAGCTTT TCTTTTTTCA GATGAATTAG ACTTTATAAA CTCAAACATT
781 AAAATGTTGA AAATCATAAA ATGGCAAATA AATACTTTTT CAAATCTTTG TGCATAAATA
841 CTTCATAGAA ATCCTTGAAT TATTCCTAAA TTTTATACAA TTGTTTCTTA TAATTATGAA
901 AATGAGTTTA AACAATTATT TAAATTCCAT AAATTGTAAC TCCGTAAGGT GTAGGTTTTC
961 ATCTCTGTTT AATAGAAGGA GGTTAGTATC TTAGTTAAGT CTG TTTCGG GGGTTATATT
1021 AGTTTTGTTT TTAGATTGAC CTACATTAAT TGTTCTTAAC TAATTACAGC TAAATATGGA
1061 GAGGTCATTA TGGATGTACA ACTTATCAAG ATTGGACCTA TCATATGTAG TGCAGGTCCA
1141 AAAATTTATT GATGTCGCAA AGATACATGC TCGCAGAACA AAGGCGAAGC ACATATGTTG
1201 TCCATGCGCA GACTGCAAAA ATATTATGGT ATTTGACAAT GTAGAAGCAA TTACTTCCCA
1261 TCTGGTTTGA AGAGGATTTA TGGAGGACTA CTTGATTTGG ACAAAACATG GTGAGGGTAG
1321 TTTTGCACCT TATATGCGGA CAACTGACAA CACTGCAACT AACATCAATG TGGAGGGTCC
1381 AATGCCACCT CTCAATGAAT TTCATGC AT GCCAGATGTT AATGAAACTC ATACGTCTGA
1441 TGTCAATGAA ACTCAGCATG CTAACACAGA TGTTGTTGAA GATGCAGATT TCTTAGAGGC
1501 AATAATGAAC CGTTGTGCGG ATCCATCAAT ATTCTTCATG AAGGGAATGA AAGCATTGAA
1561 GAAGGCAGCA GAGGACACTT TGTACGACGA GTCAAAAGGT TGTACCAAAC AATGGTCGAC
1621 ATTATGTGTT GTTCTTCAGT TTTTGACGAT GAAGGCTAGA CATGGTTGGT CCGATGCTAG
1681 CTTCAATGAT TTCTTGCGTG TACTTGGAGA CCTTCTTCCT AAGGAGAACA AAGTGCCTGC
1741 TAACACATAC TATGCAAAGA AGCTAGTCAG TCCACTTACG ATAGGTGTTG AGAAGATCCA
1801 CGCATGTAGA AATCATTGTA TTCTATATCG AGGTGATCAA TATAAAGACT TAGACAGTTG
1861 TCCAAACTGT GGTGCCAGTA GGTACAAGAC AAACAAAGAT TTTCGGGAGG AAGAGAATCT
1921 AGCCTCTGTT TCTACAGGGA GGAAGCGAAA GAAGACCCAA ACAAAGACTC AACAAGACAA
1981 GCGCTCAAAG CCTAGTAGCA ATGAAGAAGT GGACTATTAT GCATTGAGAA GAGTCTCCCT
2041 ATGAGCCAAA AAAGGGGACA GCAGCAGGCA CAACTCTCTT TCTGAAAGGA CTTGGAAAGC
23.01 AGCGGACGGC ACGGCTCATT GAGCTCGAAC CGTCACAGAA AAAGGAAGCC ACCGCCCAGT
2161 CAATAGAAGC CATGCCCCCA TCAAAGGAAG CCCCAAGTGG CGATGTACAT ATTGAACAGC
2221 CATCAAGTCA ACCATTGACC CTAAAGGATA TCAGAAAGCC AACGATTGAT GATTATGTCA
2281 ATGTCCCTAG TGACTATGTG CCCGGAAGGC CTATGCTCCA ATGGACGCTG CTCGATTAGA
2341 TTCAATGGCT GATAAAAAGG TTTCATGACT GGTACATGAG AGCAGTGCAT GCTAGCCTCC
2401 ATGGAATCAG AGTTGATATA CCAACAGACA TGTTTGCTAC TGGTAACAAA AAAAGCAAGA
2461 CATTTGTTAC CTTTGAGGAC ATGCACTTGT TATTGAACTA TAGGCGGCTT GACGTCCAAC
2521 T 0 Λ T c ¾j? x CTGGTGCCTG TAAGTATCAC TCATGCACAC ACAATTATTA TATATTAATA
2581 TGTAGTGTGA AACTCTAATA TGTAGATGTT GTCTGTAGTT TGCAAGATCA CGAGCAGATG
2641 TCATTATTAT CTGCCGGATC GATGGTCGGT TATCTGAGCC CTATCAAGTT ACAAGAAAAT
2701 ATGAACAAAT TCGTATTATC AA GGAAGAT AGAGCAAAGA TAGAGGAAGA CAAAACACCA
2761 GGATAATTAT GCCATCTATC TTGGTAGATC AATGCTGAGG TATAAATATA GGGATTTTAT
2821 ATTGGCACCA TACAACATTA GGTAAGCTTG ACTTCATATA CGTATTTCAA ATTATCGTGT
2881 AAACAATATA CATGTGTCGC TCACTCATTT ATTCATGCAG TGACCATTGG ATTGTTTTTT
2941 ATATTTATCC CTTCGAAGGG AAGGTGCTTG TCCTAGACTC TTTACATGTT CCTCCCGAGA
3001 AGTATCAACC ATTCTTGGTT CAATTAGAAA GGTGAGCCAA CATGAAACCA CATGCGTACT
3061 TATATAAATT AGAGTTTCAA AATAACTTTA GTGATTTAGG TTCGATATCT ACGGGGCATG
3121 GCGGTTTTAT AAGAAACAAA AGGGACCTGT CGACGCTGCA CGCTCAGATC CTAGGATCCC
3181 ATTGATGATA CAACACCACT ATCCGGTAAG TTTTCTGAAC ACATTTCATC ATATAAATAA
3241 TACATAAAGC ATGGCAAATT TAGAATAATC CGTTGCTCAT TATATAGTGC CACAAGCAAC
3301 CACCTGGATC GGTCTATTGT GGGTACTATG TCTGTGAGTT TATAAGGCAG CGGGGACGTT
3361 ACGTCAAGGA CAAAAATATG GTAAATAATA TCTATGTATG AAAGTTTTCT CATTAAAGCT
3421 GCAAAATTAT ATATTGAACA TGTGTCAATC ATGCTTTTAA ACTTTATTTT CAGCCGAAAA
3481 AGCAAGGAAA AGACGTGCCC TT ACACCAA AGACTCTGGA AGATATAGTA GCATACTTGT
3541 GTGGTTTTAT TATGAGAGAA ATAATTTCAA GTGACAGTGC ATATTTTGAT CATGAGGGCG
3601 ATTTAGCAAG TGATAAATTT AGAGTGCTGA CAGACATAGC AGGTCTAAAT CTGAAGCGAA
3661 ACGACATGTA AACATTGTAT GGTTGTGCGG ATAACATGCA TTGACGTGTA TATATATAAT
3721 TTTATGGTTG ATGTTTGATT TGTTTACAAT TCTATAATAT ATATATGTGG TGTATGTATG
3781 ATGTTGTGTG TGTATATATA TATATATATA TATATATATA TATATATATA TATATATATA
3841 TATATATATA TAATGTTTAG CACTGTGTTT GGTGGGAAAA ΆΤΤΑΑΑΆΤΤΤ
3901 TAAAAAATTA TTTACACAGA CAGTGTACGT GTCGAGCGTC GTCCTGTGCT
3961 ATTCTAACAG GCGGCTCGCC TTGTCCACCG GTCGGTTAAA AATACATTTC CACACNGGCC
4021 TGGCTGGGAG AGCCGCCTGT GAAAACATAA TTTTCACAGG CGGCTCGCAC AGCCCCGCCT
4081 GTACTGTGGT CCATTTTGTA CTGACCCCTG GTACAGGCGG TGGGCTTGGC CGCCTGTGAA
4141 GATGCTTTTA GCACCGCCTG TAAAAATGTT TTTTGTAGCA GTGTTT
(SEQ ID NO: 19 - Sb07g008600 - S. propinq um) or a functional fragment or variant thereof having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%> sequence identity to SEQ ID NO: 19.
The coding sequence of the maturity Mai gene of SEQ ID NO: 19, including introns, can be:
1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATA TTG&ACAGCC ¾ T C i\J G C
61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAft TGTCCCTAGT
121 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TCGATTAGAT TCAATGGCTG
181 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTAGCCTCCA TGGAATCAGA
241 GTTGATATAC CAACAGACAT GTTTGCTACT GGTAACAAAA AAAGCAAGAC ATTTGTTACC
301 TTTGAGGACA TGCACTTG T ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
361 TGGTGCCTGT AAGTATCACT CATGCACACA C & ¾T V T X J X i¾ 1? jiVT 1? GTAGTGTGAA
421 ACTCTAATAT GTAGATGTTG TCTGTAGTTT GCAAGATCAC GAGCAGATGT CATTATTATC
481 TGCCGGATCG ATGGTCGGTT ATCTGAGCCC TATCAAGTTA CAAGAAAATA TGAACAAATT
541 CGTATTATCA AAGGAAGATA GAGCAAAGAT AGAGGAAGAC AAAACACCAG GATAATTATG
601 CCATCTATCT TGGTAGATCA ATGCTGAGGT ATAAATATAG GGATTTTATA TTGGCACCAT
661 ACAACATTAG GTAAGCTTGA CTTCATATAC GTATTTCAAA TTATCGTGTA AACAATATAC
721 ATGTGTCGCT CACTCATTTA TTCATGCAGT GACCATTGGA TTGTTTTTTA TATTTA CCC
781 TTCGAAGGGA AGGTGCTTGT CCTAGACTCT TTACATGTTC CTCCCGAGAA GTATCAACCA
841 TTCTTGGTTC AATTAGAAAG GTGAGCCAAC ATGAAACCAC ATGCGTACTT ATATAAATTA
901 GAGTTTCAAA ATAACTTTAG TGATTTAGGT TCGATATCTA CGGGGCATGG CGGTTTTATA
961 GGGACCTGTC GACGCTGCAC GCTCAGATCC TAGGATCCCA TTGATGATAC
1021 AACACCACTA TCCGGTAAGT TTTCTGAACA CATTTCATCA TATAAATAAT ACATAAAGCA
1081 TGGCAAATTT AGAATAATCC GTTGCTCATT ATATAGTGCC ACAAGCAACC ACCTGGATCG
1141 GTCTATTGTG GGTACTATGT CTGTGAGTTT ATAAGGCAGC GGGGACGTTA CGTCAAGGAC
1201 AAAAATATGG TAAATAATAT CTATGTATGA AAGTTTTCTC ATTAAAGCTG CAAAATTATA
1261 TATTGAACAT GTGTCAATCA TGCTTTTAAA CTTTATTTTC AGCCGAAAAA GCAAGGAAAA
1321 GACGTGCCCT TTACACCAAA GACTCTGGAA GATATAGTAG CATACTTGTG TGGTTTTATT
1381 ATGAGAGAAA TAATTTCAAG TGACAGTGCA TATTTTGATC ATGAGGGCGA TTTAGCAAGT
1441 GATAAATTTA GAGTGCTGAC AGACATAGCA GGTCTAAATC TGAAGCGAAA CGACATGTAA
(SEQ ID NO:28 - Sb07g008600 - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%» sequence identity to SEQ ID NO:28,
The coding sequence of the maturity Mai gene of SEQ ID NO:28, without introns, can be:
1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATA TTGAACAGCC ATCAAGTCAA
61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCTAGT
121 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TCGATTAGAT TCAATGGCTG
181 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTAGCCTCCA TGGAATCAGA
241 GTTGATATAC CAACAGACAT GTTTGCTACT GGTAACAAAA AAAGCAAGAC ATTTGTTACC
301 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
361 TGGTGCCTGG ACCATTGGAT TGTTTTTTAT ATTTATCCCT TCGAAGGGAA GGTGCTTGTC
421 CTAGACTCTT TACATGTTCC TCCCGAGAAG TATCAACCAT TCTTGGTTCA ATTAGAAAGG
481 GCATGGCGGT TTTATAAGAA ACAAAAGGGA CCTGTCGACG CTGCACGCTC AGATCCTAGG
541 ATCCCATTGA TGATACAACA CCACTATCCG TGCCACAAGC AACCACCTGG ATCGGTCTAT
601 TGTGGGTACT ATGTCTGTGA GTTTATAAGG CAGCGGGGAC GTTACGTCAA GGACAAAAAT
661 ATGCCGAAAA AGCAAGGAAA AGACGTGCCC TTTACACCAA AGACTCTGGA AGATATAGTA
721 GCATACTTGT GTGGTTTTAT TATGAGAGAA ATAATTTCAA GTGACAGTGC ATATTTTGAT
781 C&TGAGGGCG ATTTAGCAAG TGATAAATTT AGAGTGCTGA CAGACATAGC AGGTCTAAAT
841 CTGAAGCGAA ACGACATGTA A
(SEQ ID NO:29 - Sb07g008600 - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%», 97%, 98%, or 99% sequence identity to SEQ ID NO:29.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in short day S. propinquum includes the nucleic acid sequence:
1 CACTAGTACA AAAATCATTT TCGTTGGCAC GTTGTTTTTT TTTTCACAGG CAGTTCACAA
61 TATC&TGGTG CTAGTAGAAA AATTTCAACG GGCCCAACAA GAGAACCGCC AGGCGGTCTT
121 CTTAATTCAA CCGCCTGTGT AAACTTTCCA TTTACATAGG CGGCTTACGA TAAAAACCGT
181 GTGTATAAAT ACCATTAACA CAGGCAGTCG AGTTACGACA ACCGCCTGTG TAAATGTGTC
241 TTTTTACACA GGCGGTTTGT ATAGAGGGCC GCCTGTGCTA ATATATTTAC ACAGGCTATG
301 AGCCGCCTGT GTTAAGTCTT CTATAAATAC CCTTCGTCCA CCTCCAGACA AGAACAGTTA
361 CTCCCATGAG CTCTGCACAC TGGCGGACCA GACGATTCCA GTTTCCAAGG GGGGAGGTTT
421 TGATTTTCAT TTCTTTGGTG AGAAACTTCC AAAAGGTTAG TTAGTGCCAT TGATGCTATT
481 TTTTAAGCGA TTCTTTGGTT CAATTCTTGT ATTGGAGGTG CTCTAGATCT AGAGTTCATC
541 ATGCATTCTT GCTTAGGGTT AGAGTTCATA GGGCAAAAAG AGAGAGATTT AGCTAAATTT
601 TTATGTAAAT TCATAGTAAA TTGTAAAAAT TAAAAAAAAT AAAAAATAAA TACTTTTTAG
661 AATTCTTGTG AGTAGATCTA TACAATAGAG TAATGATGAG GATATTTTGA AGTTTATAAT
721 TTTGATTCAG TTTTAGCTTT TCTTTTTTCA GATGAATTAG ACTTTATAAA CTCAAACATT
781 AAAATGTTGA AAATCATAAA ATGGCAAATA AATACTTTTT CAAATCTTTG TGCATAAATA
841 CTTCATAGAA ATCCTTGAAT X·¾Ί TCC TTTTATACAA TTGTTTCTTA TAATTATGAA
901 AATGAGTTTA AACAATTATT TAAATTCCAT AAATTGTAAC TCCGTAAGGT GTAGGTTTTC
961 ATCTCTGTTT AATAGAAGGA GGTTAGTATC TTAGTTAAGT CTGTTTTCGG GGGTTATATT
1021 AGTTTTGTTT TTAGATTGAC CTACATTAAT TGTTCTTAAC TAATTACAGC TAAATATGGA
1081 GAGGTCATTA TGGATGTACA ACTTATCAAG ATTGGACCTA TCATATGTAG TGCAGGTCCA
1141 AAAATTTATT GATGTCGCAA AGATACATGC TCGCAGAACA AAGGCGAAGC ACATATGTTG
1201 TCCATGCGCA GACTGCAAAA ATATTATGGT ATTTGACAAT GTAGAAGCAA TTACTTCCCA
1261 TCTGGTTTGA AGAGGATTTA TGGAGGACTA CTTGATTTGG ACAAAACATG GTGAGGGT G
10
15
20
25
30
35
40
45
(SEQ ID NO:20) or a functional fragment or variant thereof having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20 (Sb07g008600 - S. propinq um) . Each N = can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
The coding sequence of the maturity Mai gene of SEQ ID NO:20, including introns, can be:
1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATA TTGAACAGCC ATCAAGTCAA
61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCTAGT
121 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TCGATTAGAT TCAATGGCTG
181 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTAGCCTCCA TGGAATCAGA
241 GTTGATATAC CAACAGACAT GTT GCTACT GGTAACAAAA AAAGCAAGAC ATTTGTTACC
301 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
361 TGGTGCCTGT AAGTATCACT CATGCACACA CAATTATTAT ATATTAATAT GTAGTGTGAA
421 ACTCTAATAT GTAGATGTTG TCTGTAGTTT GCAAGATCAC GAGCAGATGT CATTATTATC
481 TGCCGGATCG ATGGTCGGTT ATCTGAGCCC TATCAAGTTA CAAGAAAATA TGAACAAATT
541 CGTATTATCA AAGGAAGATA GAGCAAAGAT AGAGGAAGAC AAAACACCAG GATAATTATG
601 CCATCTATCT TGGTAGATCA ATGCTGAGGT ATAAATATAG GGATTTTATA TTGGCACCAT
661 ACAACATTAG GTAAGCTTGA CTTCATATAC GTATTTCAAA TTATCGTGTA AACAATATAC
721 ATGTGTCGCT CACTCATTTA TTCATGCAGT GACCATTGGA TTGTTTTTTA TATTTATCCC
781 TTCGAAGGGA AGGTGCTTGT CCTAGACTCT TTACATGTTC CTCCCGAGAA GTATCAACCA
841 TTCTTGGTTC AATTAGAAAG GTGAGCCAAC ATGAAACCAC ATGCGTACTT ATATAAATTA
901 GAGTTTCAAA ATAACTTTAG TGATTTAGGT TCGATATCTA CGGGGCATGG CGGTTTTATA
961 AGAAACAAAA GGGACCTGTC GACGCTGCAC GCTCAGATCC TAGGATCCCA TTGATGATAC
1021 AACACCACTA TCCGGTAAGT TTTCTGAACA CATTTCATCA TATAAATAAT ACATAAAGCA
1081 TGGCAAATTT AGAATAATCC GTTGCTCATT ATATAGTGCC ACAAGCAACC ACCTGGATCG
1141 GTCTATTGTG GGTACTATGT CTGTGAGTTT ATAAGGCAGC GGGGACGTTA CGTCAAGGAC
1201 AAAAATATGG TAAATAATAT CTATGTATGA AGTTTTCTCA TTAAAGCTGC AAAATTATAT
1261 ATTGAACATG TGTCAATCAT GCTTTTAAAC TTTATTTTCA GCCGAAAAAG CAAGGAAAAG
1321 ACGTGCCCTT TACACCAAAG ACTCTGGAAG ATATAGTAGC ATACTTGTGT GGTTTTATTA
1381 TGAGAGAAAT AATTTCAAGT GACAGTGCAT ATTTTGATCA TGAGGGCGAT TTAGCAAGTG
1441 ATAAATTTAG AGTGCTGACA GACATAGCAG GTCTAAATCT GAAGCGAAAC GACATGTAA
(SEQ ID NO:30 - Sb07g008600 (10.6 kb) - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30.
The coding sequence of the maturity Mai gene of SEQ ID NO:30, without introns, can be:
1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATA TTGAACAGCC ATCAAGTCAA
61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCTAGT
121 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TCGATTAGAT TCAATGGCTG
181 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTAGCCTCCA TGGAATCAGA
241 GTTGATATAC CAACAGACAT GTTTGCTACT GGTAACAAAA AAAGCAAGAC ATTTGTTACC
301 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
361 TGGTGCCTGG ACCATTGGAT TGTTTTTTAT ATTTATCCCT TCGAAGGGAA GGTGCTTGTC
(SEQ ID N0:31 - Sb07g008600 - S. propinquum) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:31.
2. Sequences for Day-Neutral Flowing
The S. bicolor cultivar from which the sequences described below are derived are day-neutral, and have the recessive (loss of function) Mai allele. Sequences for a recessive Mai gene are therefore provided.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
1561 TAGTGTTTTA TTACTAATTA AAAATATTAG AAGAACTCCA TCATTTTGGT AGTGATTGGT
1621 TGTTTGGATT AGTCTTCTCA CGTTAGACCT ATATATGCAG GACAACTCAA GCCAGCATAA
1681 ATATATGAAA TATCTTGGTG TTTGTTTGTC TGACACAGGC AACCGTGTTT GGTATAAATG
1741 TGTTTTCTTG TTTACGTTTT ACCATCTATA GTCATCTCAA TGTTTATATA GTAGAGACTT
1801 CATGTTTGTA GTAGATAAGG TAGAGAATTG AGAATATTTT ATTTTTGTGC GACCATCAAT
1861 TTTATGTAAT CTGCATTGTC TAATGCTTTA TTTGACATTT GAAACTACTT AATTTGACCG
1921 TTATGCAGGT CCGCATGATC CTATGAAAGC AATTAATTAG TACGGGTACT GCACTACACA
1981 AGTTTGCTAG TACTATTCTA TTAACCGACC TGTCAATATT ACCTTAAGTT ACTGATTTCA
2041 ATTAGAATCT AACACATTCA GGAAAAGAAG TTTTCCTTAT TAGTAGTAAC TTTTTATACT
2101 AATTAAGATT CAATAAAAAT TCACCATGAC ATCCCCATTG CCAAGAGAAT ATTTCGCCGC
2161 CCCTCAAAGC AGCCAAGGCT TTACTAAAAA GACTATCCAC GCAGTAGAGA TTTAGTCAAA
2221 ATATTCCAAT AGCAATTGTT TTCTGCCTGC TTGACCTTCG TCAGCCACTC ACTGTATAAA
2281 TATCGCACCA CGCCCTTTGC AGGCTTACAG AGCTTGTACT ACGTACTAAC AAGGCACACA
2341 CAATACCCTG TGTTCACCGG CCCTGCACAA AACTCAAGCA GTTATTACTA ACATGGCGGC
2401 TAACGATTCC TTGGTTACTG CTCATGTGAT AGGAGATGTC TTGGACCCCT TCTATACAAC
2461 CGTTGATATG ATGATCCTAT TCGATGGTAC TCCTATTATC AGCGGCATGG AGTTGCGTGC
2521 TCCGGCGGTT TCTGACAGGC CAAGGGTTGA GATTGGAGGA GATGATTATC GAGTTGCATA
2581 TACTCTGGTA AACTCATGTC ATGTCAATTA ACTAGTAGTT GAATTTAGAT GCTGGTCGTA
2641 TCGTGGATAC ATGAACTATA TGTTATGGTT GATACATATT TGTTTAATTG ATCGCAACAC
2701 CATTTGTGGT AACTTCAAAT AACATTCTTT CAATATATAG GTGATGGTCG ATCCTGATGC
2761 TCCTAACCCA AGCAACCCAA CCTTGAGGGA GTACTTGCAC TGGTAAGAGA AACCTATAGA
29 Ξ 1 CGACAATTAT TGTTGTTGGC ATGTTCTGCC CACATATACT TTGCTAGTGT GTGTATATTT
2881 GTGCTTATGC TTCTCCATAA ATTTTGGTGT ATGTCCCAAG AGAGATAGGT ATAGAGGTTA
2941 GCAGTCCTTT AAAAATGGTT TAATCCAGTA GTTTTTTTTC GGTCGGCCGG ACTGCTAGTA
3001 ACTTTCAATC ATTTCATGTT TCGAGCAGGA fGGTGACTGA CATCCCAGCA TCAACTGATA
3061 ATACATACGG TGAGATCACC CCTATTCCCA TTTTGAGACA AGTAGAATGT CTATTTTTAT
3121 GATCTAGTAT GTTCGTGACA ATAGGCTAGC TATTTTGAAA CTTCGGGAGC ATAAAATAGT
3181 ACTCGATTTT GTATAACCAT AAACACAGCT AGCCAATCTC TATTCATATT TATTTTAGTT
3241 TTATTTGCCG AACCATCCTC AACATCATAG CCACTTGATC GATCATCTCA ATCAGCGTTT
3301 GTATCCTTGC CCGCTTTGAT TATCATCCAT GACAGTTCAT ATTTTTTTTC ATTTCTTTCA
3361 TGCTTGTTAT AGTTTTATCT GATGAATCCG AGATGTTATT GATCAATTAG TTCAGATGAG
3421 CAGTAATGTA TGTTGGAGGT TTGGTAGTAT ATATACGTTC AATATTTCAC GAAATCGGTA
3481 ATTACGAAAA TCCCAAAATT TTGAATTACA TTAATAATGC ATGTGACTCA TATTTTCTAT
3541 GATTTCTATT CTGTTGCATA TCTTGTAC CAATAGATAT TTAAATCATG CTAATATTTT
3601 GTTTAGATCT AAATCTTTTA GAAAAATTAT AATTTATATT TGGG TTAAC AATTTCGGGC
3661 GCGTTTAGTG AGATTGGGTA ATTTCGGAGC GAGGCGGCCG CCGGCCACGA AAAATTCTAT
3721 ACACGACTAT ATGTGTACAT GTACATGCAT GGCACCTTGA TAGGCTACCC CGGCCCGCAT
3781 GGGGAAAAAA TTGGAAACGG ACCATTCATA CGCAGTCGTG GTGCCGACTG TGGGCCACAA
3841 TAGCAGTGTA AACATAATTA CGGTAATCAA ATACCCCGTG GGACCATATA TATCATCCAC
3901 AGATCCGTAC GGTGCTTCCG TGTGGATGGT CTACCCCAGA TCTTTTCCAC CCCATAAGGG
3961 CAGCAATGCA GCATCATATT CATATGCACT AGTGATGTAC CATTTGGCTT ATATCATATT
4021 CAACCTAACT CCTTGGAAAC ATTATGATGT TCTATTGGGG TGAAGATGTC ACTACTAAAA
4081 AAAGATCTTA TGAGAGGTGT TTTGAAAACT GCCCGAGGTG GTTAAAGGAG ACGGACGAGT
4141 TAGGACAACT GCCTCTATTA ATGTGTATTA ACCGAGGTAG TTACCGTAAC GTGCCTGACT
4201 TGATTAACAG ATTCAACCGT CTCAGTAAAG ACCATGATTA ACCGAAACGG AATCGAGAGT
4261 TTTCTCAAGT AGTTAAACTA TTTTAAACTG CACCGAACTT ATAAAAATGG TAGAGCTAAC
4321 ACCAATATTT ATAAAAATAA ATTAGTATCA CTAAATACAT CACGAAATCT ATTTGGTGTT
4381 GTAGAAGTTA TCCTTTTCTA TAAAATTGAT CAAATTTATG ATAACTTAGT TTTAGGAATT
4441 GATTTATTTT AGGACAACTA AGGAAGTACA TTTTTTAAAG TCATCCACAA AGTAGTGGAT
4501 CCAATTTATT ACATTACTCC ACTACTTCAA ACTGAACAAA AGCCTAATCC TGGTTATTTT
(SEQ ID NO:9, Sb06g012260 - S. bicolor), or a variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.
The coding sequence of the maturity Mai gene of SEQ ID NO: 10, including introns, can be:
T
1021 CAGATGAGCA GTAATGTATG TTGGAGGTTT GGTAGTATAT ATACGTTCAA TATTTCACGA
1081 AATCGGTAAT TACGAAAATC CCAAAATTTT GAATTACATT AATAA GCAT GTGACTCATA
1141 TTTTCTATGA TTTCTATTCT GTTGCATATT CTTGTACTCA ATAGATATTT AAATCATGCT
1201 AATATTTTGT TTAGATCTAA ATCTTTTAGA AAAATTATAA TTTATATTTG GGTTTAACAA
1261 TTTCGGGCGC GTTTAGTGAG ATTGGGTAAT TTCGGAGCGA GGCGGCCGCC GGCCACGAAA
1321 AATTCTATAC ACGACTATAT GTGTACATGT ACATGCATGG CACCTTGATA GGCTACCCCG
1381 GCCCGCATGG GGAAAAAATT GGAAACGGAC CATTCATACG CAGTCGTGGT GCCGACTGTG
1441 GGCCACAATA GCAGTGTAAA CATAATTACG GTAATCAAAT ACCCCGTGGG ACCATATATA
1501 TCATCCACAG ATCCGTACGG TGCTTCCGTG TGGATGGTCT ACCCCAGATC TTTTCCACCC
1561 CATAAGGGCA GCAATGCAGC ATCATATTCA TATGCACTAG TGATGTACCA TTTGGCTTAT
1621 ATCATATTCA ACCTAACTCC TTGGAAACAT TATGATGTTC TATTGGGGTG AAGATGTCAC
1681 TACTAAAAAA AGATCTTATG AGAGGTGTTT TGAAAACTGC CCGAGGTGGT TAAAGGAGAC
1741 GGACGAGTTA GGACAACTGC CTCTATTAAT GTGTATTAAC CGAGGTAGTT ACCGTAACGT
1801 GCCTGACTTG ATTAACAGAT TCAACCGTCT CAGTAAAGAC CATGATTAAC CGAAACGGAA
1861 TCGAGAGTTT TCTCAAGTAG TTAAACTATT TTAAACTGCA CCGAACTTAT AAAAATGGTA
1921 GAGCTAACAC C T i¾ TT T ¾T AAAAATAAAT TAGTATCACT AAATACATCA CGAAATCTAT
1981 TTGGTGTTGT AGAAGTTATC CTTTTCTATA AAATTGATCA AATfTATGAT AACTTAGTTT
2041 TAGGAATTGA TTTATTTTAG GACAACTAAG GAAGTACATT TTTTAAAGTC ATCCACAAAG
2101 TAGTGGATCC AATTTATTAC ATTACTCCAC TACTTCAAAC TGAACAAAAG CCTAATCCTG
2161 GTTATTTTGA GAGTGATTTT TTACAACATC AGCAGTAGTC CAGAAAATGG GAGGACATTA
2221 ATAAAAGTGA AAAGGAGCAG AAGAAAGATT ACGGTATTTT ATTTGTGCTA TTTGTTTAAC
2281 TATTGGCAGT TTGGGACCGA AAATAAATAA CTGTTCGTAG CTCTATATTT GTCCATTCGA
2341 AAGTGTAACG ATGATTATTG TGTTTCAAAA GATAAATAAA GAAGTGCACC AATGATTTGA
2401 TATCATAGGC TATATAATCC AACATGGTGA AAATGCTTTT CAATCAAGTA ATCTTCGAGC
2461 GGTTACCAGT TTTAATAGTT GCGAGTCGTC GTTTTTTATG TACCCTAGGA CATATATATA
2521 TCCGCATGTA GACGATGAGA CTAGCTAGTT TTTTTTTTTT TGAGCAAATA CATAATTATT
2581 GGATTTGCAG GCCGTGAGAT GATGTGCTAC GAGCCCCCTG CCCCGTCCAC GGGCATCCAC
2641 CGGATGGTGC TGGTGCTATT CCAGCAGCTT GGCCGTGACA CGGTGTTCGC GGCGCCGTCC
2701 AGGCGCCACA ACTTCAACAC CCGTGCCTTC GCCCGCCGCT ACAACCTCGG CGCGCCCGTC
2761 GCCGCCATGT TCTTCAACTG CCAGCGCCAG ACCGGCTCCG GTGGCCCCAG GTTCACCGGG
2821 CCCTACACCA GCCGCCGTCG TGCGGGCTGA
(SEQ ID NO: 10 Sb06g012260 - S. bicolor) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.
The coding sequence, without introns, of the maturity Mai gene as it is found in day-neutral S, bicolor can include the nucleic acid sequence:
1 ATGGCGGCTA ACGATTCCTT GGTTACTGCT CATGTGATAG GAGATGTCTT GGACCCCTTC
61 TATACAACCG TTGATATGAT GATCCTATTC GATGGTACTC CTATTATCAG CGGCATGGAG
121 TTGCGTGCTC CGGCGGTTTC TGACAGGCCA AGGGTTGAGA TTGGAGGAGA TGATTATCGA
181 GTTGCATATA CTCTGGTGAT GGTCGATCCT GATGCTCCTA ACCCAAGCAA CCCAACCTTG
241 AGGGAGTACT TGCACTGGAT GGTGACTGAC ATCCCAGCAT CAACTGATAA TACATACGGC
301 CGTGAGATGA TGTGCTACGA GCCCCCTGCC CCGTCCACGG GCATCCACCG GATGGTGCTG
361 GTGCTATTCC AGCAGCTTGG CCGTGACACG GTGTTCGCGG CGCCGTCCAG GCGCCACAAC
421 TTCAACACCC GTGCCTTCGC CCGCCGCTAC AACCTCGGCG CGCCCGTCGC CGCCATGTTC
481 TTCAACTGCC AGCGCCAGAC CGGCTCCGGT GGCCCCAGGT TCACCGGGCC CTACACCAGC
541 CGCCGTCGTG CGGGCTGA
(SEQ ID N0:11, Sb06g012260 - S. bicolor), or a variant thereof, for example a codon optimized variant, having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:l 1.
In this embodiment, the maturity Mai protein as it is found in short-day - S. bicolor can include the amino acid sequence SEQ ID NO: 8, or a variant thereof having at least 95% sequence identity to SEQ ID NO: 8.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
1 TTCCACCTGG GCAGGGAAAA CGGTTTATTA TGTTCCTCTT TAATTTATCT ATCGTGGTCT
61 GTTTTCACTA AAACTGTCAT ATTGCTACAC TCCAGTACTA CCAGTACGTC GCCCGCACAT
121 AGTGGCCAAG GATTTTACTG CTACTGTTGA TTAACATAAG CACTTGCGAC TTTCCCTAAC
181 ATCTTTTATA AAACAACGGC CGCAATAATA TTGAACTGTT TTTTTCTAGT ACCAAAAATA
241 GAATTTGATC CCTCACCTCA TTACATCCAT AGTAACATGA CCAGATATAT ATGGACAGGC
301 CGGGATCACT CGCCAGCAGA TACCCTGAGC GATTCATAAC CAGAATTTTT AATTTTTTCT
361 AGTGAAGTGG GGTTCTCCTA GTCCTTTAAC ATTCAAAATT TAGTACAAAC TTTCCTTAGT
421 AAATGTCTTC TAGTAAAGAT TTCCTAGTGT TTTGATTTGG TAGTGTTTTA TTACTAATTA
481 AAAATATTAG AAGAACTCCA TCATTTTGGT AGTGATTGGT TGTTTGGATT AGTCTTCTCA
541 CGTTAGACCT ATATATGCAG GACAACTCAA GCCAGCATAA ATATATGAAA TATCTTGGTG
601 TTTGTTTGTC TGACACAGGC AACCGTGTTT GGTATAAATG TGTTTTCTTG TTTACGTTTT
661 ACCATCTATA GTCATCTCAA TGTTTATATA GTAGAGACTT CATGTTTGTA GTAGATAAGG
721 TAGAGAATTG AGAATATTTT ATTTTTGTGC GACCATCAAT TTTATGTAAT CTGCATTGTC
781 TAATGCTTTA TTTGACATTT GAAACTACTT AATTTGACCG TTATGCAGGT CCGCATGATC
841 CTATGAAAGC AATTAATTAG TACGGGTACT GCACTACACA AGTTTGCTAG TACTATTCTA
901 TTAACCGACC TGTCAATATT ACCTTAAGTT ACTGATTTCA ATTAGAATCT AACACATTCA
961 GGAAAAGAAG TTTTCCTTAT TAGTAGTAAC TTTTTATACT AATTAAGATT CAATAAAAAT
1021 TCACCATGAC ATCCCCATTG CCAAGAGAAT ATTTCGCCGC CCCTCAAAGC AGCCAAGGCT
1081 TTACTAAAAA GACTATCCAC GCAGTAGAGA TTTAGTCAAA ATATTCCAAT AGCAATTGTT
1141 TTCTGCCTGC TTGACCTTCG TCAGCCACTC ACTGTATAAA TATCGCACCA CGCCCTTTGC
1201 AGGCTTAC&G AGCTTGTACT ACGTACTAAC AAGGCACACA CAATACCCTG TGTTCACCGG
1261 CCCTGCACAA AACTCAAGCA GTTATTACTA ACATGGCGGC TAACGATTCC TTGGTTACTG
1321 CTCATGTGAT AGGAGATGTC TTGGACCCCT TCTATACAAC CGTTGATATG ATGATCCTAT
1381 TCGATGGTAC TCCTATTATC AGCGGCATGG AGTTGCGTGC TCCGGCGGTT TCTGACAGGC
1441 CAAGGGTTGA GATTGGAGGA GATGATTATC GAGTTGCATA TACTCTGGTA AACTCATGTC
1501 ATGTCAATTA ACTAGTAGTT GAATTTAGAT GCTGGTCGTA TCGTGGATAC ¾T G C T ¾ X ¾.
1561 TGTTATGGTT GATACATATT TGTTTAATTG ATCGCAACAC CATTTGTGGT
1621 AACATTCTTT GTGATGGTCG ATCCTGATGC TCCTAACCCA AGCAACCCAA
1681 CCTTGAGGGA GTACTTGCAC TGGTAAGAGA AACCTATAGA CGACAATTAT TGTTGTTGGC
1741 ATGTTCTGCC C i\ C i¾ ' β.Ι' ¾ Ί* TTGCTAGTGT GTGTATATTT GTGCTTATGC TTCTCCATAA
1801 ATTTTGGTGT ATGTCCCAAG AGAGATAGGT ATAGAGGTTA GCAGTCCTTT AAAAATGGTT
1861 TAATCCAGTA GTTTTTTTTC GGTCGGCCGG ACTGCTAGTA ACTTTCAATC ATTTCATGTT
1921 TCGAGCAGGA TGGTGACTGA CATCCCAGCA TCAACTGATA ATACATACGG CCGTGAGATC
1981 ACCCCTATTC CCATTTTGAG ACAAGTAGAA TGTCTATTTT TATGATCTAG TATGTTCGTG
2041 ACAATAGGCT AGCTATTTTG AAACTTCGGG AGCATAAAAT AGTACTCGAT TTTGTATAAC
2101 CATAAACACA GCTAGCCAAT CTCTATTCAT GTTTTATTTG CCGAACCATC
2161 CTCAACATCA TAGCCACTTG ATCGATCATC TCAATCAGCG TTTGTATCCT TGCCCGCTTT
2221 GATTATCATC CATGACAGTT CATATTTTTT TTCATTTCTT TCATGCTTGT TATAGTTTTA
2281 TCTGATGAAT CCGAGATGTT ATTGATCAAT TAGTTCAGAT GAGCAGTAAT GTATGTTGGA
2341 GGTTTGGTAG TATATATACG TTCAATATTT CACGAAATCG GTAATTACGA AAATCCCAAA
2401 ATTTTGAATT ACATTAATAA TGCATGTGAC TCATATTTTC TATGATTTCT ATTCTGTTGC
2461 AT&TTCTTGT ACTCAATAGA TATTTAAATC ATGCTAATAT TTTGTTTAGA TCTAAATCTT
2521 TTAGAAAAAT T j¾ Ί?.ί¾ <¾ TT T TVI1 ATTTGGGTTT AACAATTTCG GGCGCGTTTA GTGAGATTGG
2581 GTAATTTCGG AGCGAGGCGG CCGCCGGCCA CGAAAAATTC TATACACGAC TATATGTGTA
2641 CATGTACATG CATGGCACCT TGATAGGCTA CCCCGGCCCG CATGGGG AA AAATTGGAAA
2701 CGGACCATTC ATACGCAGTC GTGGTGCCGA CTGTGGGCCA CAATAGCAGT GTAAACATAA
2761 TTACGGTAAT CAAATACCCC GTGGGACCAT ATATATCATC CACAGATCCG tACGGTGCTT
282 X CCGTGTGGAT GGTCTACCCC AGATCTTTTC CACCCCATAA GGGCAGCAAT GCAGCATCAT
2881 ATTCATATGC ACTAGTGATG TACCATTTGG CTTATATCAT ATTCAACCTA ACTCCTTGGA
2941 AACATTATGA TGTTCTATTG GGGTGAAGAT GTCACTACTA AAAAAAGATC TTATGAGAGG
3001 TGTTTTGAAA ACTGCCCGAG GTGGTTAAAG GAGACGGACG AGTTAGGACA ACTGCCTCTA
3061 TTAATGTGTA TTAACCGAGG TAGTTACCGT AACGTGCCTG ACTTGATTAA CAGATTCAAC
3121 CGTCTCAGTA AAGACCATGA TTAACCGAAA CGGAATCGAG AGTTTTCTCA AGTAGTTAAA
3181 CTATTTT AA CTGCACCGAA CTTATAAAAA TGGTAGAGCT AACACCAATA TTTATAAAAA
3241 TAAATTAGTA TCACTAAATA CATCACGAAA TCTATTTGGT GTTGTAGAAG TTATCCTTTT
3301 CTATAAAATT GATCAAATTT ATGATAACTf AGTTTTAGGA ATTGATTTAT TTTAGGACAA
3361 CTAAGGAAGT ACATTTTTTA AAGTCATCCA CAAAGTAGTG GATCCAATTT ATTACATTAC
3421 TCCACTACTT CAAACTGAAC AAAAGCCTAA TCCTGGTTAT TTTGAGAGTG ATTTTTTACA
3481 ACATCAGCAG TAGTCCAGAA AATGGGAGGA CATTAATAAA AGTGAAAAGG AGCAGAAGAA
3541 AGATTACGGT ATTTTATTTG TGCTATTTGT TTAACTATTG GCAGTTTGGG ACCGAAAATA
3601 AATAACTGTT CGTAGCTCTA TATTTGTCCA TTCGAAAGTG TAACGATGAT TATTGTGTTT
3661 CAAAAGATAA ATAAAGAAGT GCACCAATGA TTTGATATCA TAGGCTATAT AATCCAACAT
3721 GGTGAAAATG CTTTTCAATC AAGTAATCTT CGAGCGGTTA CCAGTTTTAA TAGTTGCGAG
3781 TCGTCGTTTT TTATGTACCC TAGGACATAT ATATATCCGC ATGTAGACGA TGAGACTAGC
3841 TAGTTTTTTT TTTTTTGAGC TTATTGGATT TGCAGGCCGT GAGATGATGT
3901 GCTACGAGCC CCCTGCCCCG TCCACGGGCA TCCACCGGAT GGTGCTGGTG CTATTCCAGC
3961 AGCTTGGCCG TGACACGGTG TTCGCGGCGC CGTCCAGGCG CCACAACTTC AACACCCGTG
4021 CCTTCGCCCG CCGCTACAAC CTCGGCGCGC CCGTCGCCGC CATGTTCTTC AACTGCCAGC
4081 GCCAGACCGG CTCCGGTGGC CCCAGGTTCA CCGGGCCCTA CACCAGCCGC CGTCGTGCGG
4141 GC GATGACG ACGATCGTCG TTACGTCACG TGTACCGTAC ATATATATGT AAGATATACA
4201 TGCATGTTCC ATGGTAAGGA TCGGTGACAA AACGTCTAAT AATGTATACA CACAXATGCA
4261 TGGAATGCAT GTAATAAGAG T i¾ Ti^T ¾ X G! T ¾. TAATAAGTAG GGGGGAGCAT GCATATATTG
4321 TACACGCGTC CGATGCGTAT ATAGCCCTAT ACATTATTGT AGTTGTAATC A
(SEQ ID NO: 12, Sb06g012260 - S, bicolor), or a variant, for example a codon optimized variant, thereof having at least at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.
The coding sequence of the maturity Mai gene of SEQ ID NO:12, including introns, can be:
1 ATGGCGGCTA ACGATTCCTT GGTTACTGCT CATGTGATAG GAGATGTCTT GGACCCCTTC
61 TAT CAACCG TTGATATGAT GATCCTATTC GATGGTACTC CTATTATCAG CGGCATGGAG
12 TTGCGTGCTC CGGGGGTfTC TGACAGGCCA AGGGTTGAGA TTGGAGGAGA TGATTATCGA
181 GTTGC ATA CTCTGGTAAA CTCATGTCAT GTCAATTAAC TAGTAGTTGA ATTTAGATGC
241 TGGTCGTATC GTGGATACAT GAACTATATG TTATGGTTGA TACATATTTG TTTAATTGAT
301 CGCAACACCA TTTGTGGTAA CTTCAAATAA CATTCTTTCA ATAT T GGT GATGGTCG T
361 CCTGATGCTC CTAACCCAAG CAACCCAACC TTGAGGGAGT ACTTGCACTG GTAAGAGAAA
421 CCTATAGACG ACAATTATTG TTGTTGGCAT GTTCTGCCCA CATATACTTT GCTAGTGTGT
481 GTATATTTGT GCTTATGCTT CTCCATAAAT TTTGGTGTAT GTCCCAAGAG AGATAGGTAT
541 AGAGGTTAGC AGTCCTTTAA AAATGGTTTA ATCCAGTAGT TTtTTTTCGG TCGGCCGGAC
601 TGCTAGTAAC TTTCAATCAT TTCATGTTTC GAGCAGGATG GTGACTGACA TCCCAGCATC
661 AACTGATAAT ACATACGGCC GTGAGATCAC CCCTATTCCC ATTTTGAGAC AAGTAGAATG
721 TCTATTTTTA TGATCTAGTA TGTTCGTGAC AATAGGCTAG CTATTTTGAA ACTTCGGGAG
781 CATAAAATAG TACTCGATTT TGTATAACCA TAAACACAGC TAGCCAATCT CTATTCATAT
841 TTATTTTAGT TTTATTTGCC GAACCATCCT GCCACTTGAT CGATCATCTC
901 AATCAGCGTT TGTATCCTTG CCCGCTTTGA TTATCATCCA TGACAGTTCA TATTTTTTTT
961 CATTTCTTTC ATGCTTGTTA TAGTTTTATC TGATGAATCC GAGATGTTAT TGATCAATTA
1021 GTTCAGATGA GCAGTAATGT ATGTTGGAGG TTTGGTAGTA TATATACGTT CAATATTTCA
1081 CGAAATCGGT AATTACGAAA ATCCCAAAAT TTTGAATTAC ATTAATAATG CATGTGACTC
1141 ATATTTTCTA TGATTTCTAT TCTGTTGCAT ATTCTTGTAC TCAATAGATA TTTAAATCAT
1201 GCTAATATTT TGTTTAGATC TAAATCTTTT AGAAAAATTA ΤΑΑΤΤΤΑΤΑΪ TTGGGTTTAA
1261 CAATTTCGGG CGCGTTTAGT GAGATTGGGT AATTTCGGAG CGAGGCGGCC GCCGGCCACG
1321 AAAAATTCTA TACACGACTA TATGTGTACA TGTACATGCA TGGCACCTTG ATAGGCTACC
1381 CCGGCCCGCA TGGGGAAAAA ATTGGAAACG GACCATTCAT ACGCAGTCGT GGTGCCGACT
1441 GTGGGCCACA ATAGCAGTGT AAACATAATT ACGGTAATCA AATACCCCGT GGGACCATAT
1501 ATATCATCCA CAGATCCGTA CGGTGCTTCC GTGTGGATGG TCTACCCCAG ATCTTTTCCA
1561 CCCCATAAGG GCAGCAATGC AGCATCATAT TCATATGCAC TAGTGATGTA CCATTTGGCT
1621 TATATCATAT TCAACCTAAC TCCTTGGAAA CATTATGATG TTCTATTGGG GTGAAGATGT
16S1 CACTACTAAA AAAAGATCTT ATGAGAGGTG TTTTGAAAAC TGCCCGAGGT GGTTAAAGGA
1741 GACGGACGAG TTAGGACAAC TGCCTCTATT AATGTGTATT AACCGAGGTA GTTACCGTAA
1801 CGTGCCTGAC TTGATTAACA GATTCAACCG TCTCAGTAAA GACCATGATT AACCGAAACG
1861 GSATCGAGAG TTTTCTCAAG TAGTTAAACT ATTTTAAACT GCACCGAACT TATAAAAATG
1921 GTAGAGCTAA CACCAATATT TATAAAAATA AATTAGTATC ACTAAATACA TCACGAAATC
1981 TATTTGGTGT TGTAGAAGTT ATCCTTTTCT ATAAAATTGA TCAAATTTAT GATAAC TAG
2041 TTTTAGGAAT TGAT7TATTT TAGGACAACT AAGGAAGTAC ATTTTTTAAA GTCATCCACA
2101 AAGTAGTGGA TCCAATTTAT TACATTACTC CACTACTTCA AACTGAACAA AAGCCTAATC
2161 CTGGTTATTT TGAGAGTGAT TTTTTACAAC ATCAGCAGTA GTCCAGAAAA TGGGAGGACA
2221 TTAATAAAAG TGAAAAGGAG CAGAAGAAAG ATTACGGTAT TTTATTTGTG CTATTTGTTT
2281 AACTATTGGC AGTTTGGGAC CGAAAATAAA TAACTGTTCG TAGCTCTATA TTTGTCCATT
2341 CGAAAGTGTA ACGATGATTA TTGTGTTTCA AAAGATAAAT AAAGAAGTGC ACCAATGATT
2401 TGATATCATA GGCTATATAA TCCAACATGG TGAAAATGCT TTTCAATCAA GTAATCTTCG
2461 AGCGGTTACC AGTTTTAATA GTTGCGAGTC GTCGTTTTTT ATGTACCCTA GGACATATAT
2521 ATATCCGCAT GTAGACGATG AGACTAGCTA GTTTTTTTTT TTTTGAGCAA ATACATAATT
2581 ATTGGATTTG CAGGCCGTGA GATGATGTGC TACGAGCCCC CTGCCCCGTC CACGGGCATC
2641 CACCGGATGG TGCTGGTGCT ATTCCAGCAG CTTGGCCGTG ACACGGTGTT CGCGGCGCCG
2701 TCCAGGCGCC CACCCGTGCC TTCGCCCGCC GCTACAACCT CGGCGCGCCC
2761 GTCGCCGCCA TGTTCTTCAA CTGCCAGCGC CAGACCGGCT CCGGTGGCCC CAGGTTCACC
2821 GGGCCCTACA CCAGCCGCCG TCGTGCGGGC TGA
(SEQ ID NO: 13 Sb06g012260 - S. bicolor) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13.
In some embodiments, the maturity Mai gene (including non-coding sequence) as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATG TCAAACAGCC ATCAAGTCAA
61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCCAGT
12 1 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TTGATAAGAT TCAATGGCCG
18 1 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTGGCCTCCA TGCAATCAGA
24 1 GTTGATATAC CAGCAAACGT GTTTGCTACT GGTAACGAAA AAAGCAAGGC ATTTGTTATC
301 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAAC TC
3 61 TGGTGTCTGT AAGTACCACT CATGCACACA CAATTATTAT TAATATGTAG TGTGAAACTC
421 TAATATGTAG ATGTTGTCTG TAGTTTGCAA GATCACGAGT AGAGGTCATT ATTATCTACC
481 GGATCAATGG TCGGTTATCT GAGCCCTATC AAGTTACAAG AAAATATGCA CAAATTTGTA
541 TTATCAAAGG AAGATAGAGC AAAGATAGAG GAAGACAAAA CACCAGAAAA AGTTGCAGAA
601 GCTATAAAAG AGTTGCAAAG GATAATTATG CCCTCTACCT TGGTAGATCA
661 ATGCTGAGGT ATAAGTATAG GGATTTTATA TTGGCACCTT AC CTTTAG GTAAGCTTGA
72 1 CTTCATATAC GTACTTCAAA TAATTATCGT GTAAACAATA TACATGTGTC GCTCACTCAT
78 1 TTATTCATGC AGTGACCATT GGATTGTTTT TTATATTTAT CCCTTCGAAA GGAAGGTGCT
841 TGTCCTAGAC TCTTTACATG TTCCTCCCGA GAAGTATCAA CCATTCTTGG TTCAATTAGA
901 AAGGTGAGCC AACATGAAAC CACATGCGTA CTTATATAAA TTAGAGTTTC AAAACAACTT
961 TAGTGATTTA TATTCGATAT CTACAGGGCA TGGCGGTTTT ATAAGAAACA AAAGGGACCG
1021 GTCGACGCCG CACGCTCAGA TCCTAGGGTG CCATTGATGA TACAACACCA CTATCCGGTA
114 1 TCCGTTGCTC ATTATATAGT GCCACAAGCA ACCATCTGGA TCGGTCTATT GTGGGTACTA
1201 TGTCTGTGAG TTTATAAGGC AGCGGGGACG TTACGTCACG GACAAAAATA TGGTAAATAA
1261 TATCTATGTA TGAAGTTTTC TCATTAAAGT TGCAAAATTA TATATTGAAC ATGTGTCAAT
132 1 CATGCTTTTA AACTTTGTTT CCAGCCAAAA AAGCAAAAAA AGGACGTGCC CTTTACACCA
1381 AAGACTCTGG AAGATATAGT AGCAGACTTG TGTGGTTTTA TTATGAGAGA AATAATTCCA
1441 AGTGACGGTG CATATTTTGA TCATGAGGGC GATTTAGCAA GTGATAAATT TAGAGTGCTG
1501 ACAGACATAG CAGGTCTAAA TCTGAAGCGA AATGACATG
(SEQ ID NO:32 - Sb07g008600 - S. bicolor) or functional fragment or variant, such as a codon optimized variant, thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32.
The coding sequence, without introns, of the maturity Mai gene according to SEQ ID NO:32 as it is found in day-neutral S. bicolor can include the nucleic acid sequence:
1 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATG TCAAACAGCC ATCAAGTCAA
61 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCCAGT
121 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TTGATAAGAT TCAATGGCCG
181 ATAAAAAGGT TTCATGACTG GTACATGAGA GCAGTGCATG CTGGCCTCCA TGCAATCAGA
241 GTTGATATAC CAGCAAACGT GTTTGCTACT GGTAACGAAA AAAGCAAGGC ATTTGTTATC
301 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
361 TGGTGTCTGG ACCATTGGAT TGTTTTTTAT ATTTATCCCT TCGAAAGGAA GGTGCTTGTC
42 1 CTAGACTCTT TACATGTTCC TCCCGAGAAG TATCAACCAT TCTTGGTTCA ATTAGAAAGG
481 GCATGGCGG TTTATAAGAA ACAAAAGGGA CCGGTCGACG CCGCACGCTC AGATCCTAGG
54 1 GTGCCATTGA TGATACAACA CCACTATCCG TGCCACAAGC AACCATCTGG ATCGGTCTAT
601 TGTGGGTACT ATGTCTGTGA GTTTATAAGG CAGCGGGGAC GTTACGTCAC GGACAAAAAT
661 ATGCCAAAAA AGCAAAAAAA GGACGTGCCC TTTACACCAA AGACTCTGGA AGATATAGTA
721 GCAGACTTGT GTGGTTTTAT TATGAGAGAA ATAATTCCAA GTGACGGTGC ATATTTTGAT
781 CATGAGGGCG ATTTAGCAAG TGATAAATTT AGAGTGCTGA CAGACATAGC AGGTCTAAAT
841 CTGAAGCGAA ATGACATGTA A
(SEQ ID NO:33, Sb07g008600 - S. bicolor), or a variant thereof, for example a codon optimized variant, having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:33.
Therefore, a maturity Mai protein as it is found in short-day S. bicolor can include the amino acid sequence:
MPPSKEAPSGDVHV QPSSQPLTLKDIRKPTIDDYVHVPSDYVPGRPMLQWTLLDKIQ PI RFHDWYMRAV HAGLHAIRVDIPANVFATGNEKSKAFVIFEDMHLLLHYRRLDVQLITI CLDHWIVFYIYPFERKVLVLDSL HVPPE YQPFLVQLERAWRFYKKQKGPVDAARSDPRVPL IQHHYPCHKQPSGSVYCGYYVCEFIRQRGRYV TD IiMPKKQKKDVPFTPKTLEDIVADLCGFIMREIIPSDGAYFDHEGDLASDKFRVLTDIAGLNLKRNDM
(SEQ ID NO:34, Sb07g008600 - S. bicolor) or functional fragment, or variant thereof having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 34.
A polynucleotide is therefore disclosed having the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 19, 20, 28, 29, 30, 31, 32, and 33. A polynucleotide having a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 19, 20, 28, 29, 30, 31, 32, or 33 is also disclosed. A
polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 1 1, 12, 13, 19, 20, 28, 29, 30, 31, 32, or 33 is also disclosed.
A polypeptide is therefore disclosed having the amino acid sequence SEQ ID NO: 8 and 34. A polypeptide having an amino acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 8 or 34 is also disclosed.
A polynucleotide that is a fragment of Mai gene is also disclosed.
Therefore, a polynucleotide having a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a fragment of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 19, 20, 28, 29, 30, 31, 32, and 33 is disclosed. The
fragment can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, or more nucleotides shorter than SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 19, 20, 28, 29, 30, 31, 32, or 33.
A polypeptide that is a fragment of the Mai protein is also disclosed having the amino acid sequence SEQ ID NO: 8 or 34. A polypeptide having an amino acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a fragment of SEQ ID NO: 8 or 34 is disclosed. The fragment can be at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids shorter than SEQ ID NO: 8 or 34.
B. Photoperiod Sensitivity Expression Control
1. Photoperiod Sensitivity
The expression control sequences of Mai are also provided for use in putting expression of other plant genes under photoperiod control. For example, the expression control sequence of the Mai gene in the short-day S. propinquum having a dominant (functional) Mai allele can be used to induce photoperiod sensitivity of other plant genes.
The day-neutral haplotype of S. bicolor is characterized by a number of insertions, deletions and polymorphisms relative to S. propinquum. The mutations in S. bicolor include three deletions in the expression control sequence (5' UTR) and one deletion in the second intron: (1) a 423 nucleotide deletion beginning with nucleotide 1,132 numbering for the first nucleotide of SEQ ID NO: 1 or nucleotide 1597 numbering from the first nucleotide of SEQ ID NO:3; (2) a 4, 186 nucleotide deletion beginning with nucleotide 2,465 from SEQ ID NO:l, or 4,231 nucleotide deletion beginning with nucleotide 2,930 numbering from the first nucleotide of SEQ ID NO:3 (3) a 3 nucleotide deletion beginning with nucleotide 6,753 numbering from the first nucleotide of SEQ ID NO: 1 , or nucleotide 7,263 numbering from the first nucleotide of SEQ ID NO:3 or nucleotide 2,024 numbering from the first nucleotide of SEQ ID NO:5; (4) a 27 nucleotide deletion beginning with nucleotide number 7,563 numbering from the first nucleotide of SEQ ID
NO:ls or nucleotide 8,073 numbering from the first nucleotide of SEQ ID NO:3, or nucleotide 2,834 numbering from the first nucleotide of SEQ ID NO:5 (Figure 3B).
Other insertions, deletions, and polymorphisms in or around S.
bicolor Mai relative to S. propinquum Mai, and their association with photoperiod sensitivity can be determined by one of skill in the art using the compositions and methods described herein. For example, additional deletions, insertions, and polymorphisms can be determined by comparing SEQ ID NO: 1, 3, or 5 of S. propinquum Mai to SEQ ID NO: 9 or 12 of S. bicolor using global sequence alignment tools. A global alignment shows an end-to-end alignment of two sequences. Tools for preparing global alignments are available in the art, for example, using EMBOSS Needle software available at ebi.ac.uk/Tools/psa/ which creates a global alignment of two sequences using the Needleman-Wunsch algorithm.
Accordingly, one or more of the Mai expression control sequences in S. propinquum that are mutated or absent from S. bicolor can be operably linked to a plant gene coding sequence to impart photoperiod sensitive (i.e., short-day) control over the plant gene coding sequence.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 AAAAGAAAAG TGAGCACACC ACGACCTGTC ATCAGCTCAT GGTCAGCTCT ACAAACTTAT
61 AGATTGCATC GAGATCTAAG ACTCAGGTAC AAATCATGTC AACATCTAAT GGTTTAGAAA
121 ATGAAAAGTT TTGAGTTTCA AAATATGATA CGTGATATTA ACATTTGAAC TTTTAGCAAG
181 ATCTGAAATA AAAAATTCAA CTAGATCATG TTAACATTGA TATAATCGCT TCCAATCGCC
241 TCCCATCACT TCCGCTAGAA AACTTTTTTT CTCGATTTAA TTAATGAAAG GGTAATAACA
301 TCATTGTACA AGATTCTTTC AAACCTCAAC CCCTATCATC GACGGTGACG GCTCCCTATA
361 ACACGCACTA GTGGACGCCG GGCGGGTGGA ACCCTAAGAA GATTTAAAAA AACTTAAGAA
421 GAAGATTTTT ATCTAACTAA CTATAGTACT TATATCATAC ACTATACTAT TCAAAATATT
481 ATTTTCACAA TTATGAATTT ACCCTTTTAC TCTTCATTAA AAAAATACGA AAAAAGAATC
541 ACCACGTCTC tATTTAGGGT CCTAGTCCCC ATAATTTAAG AGGCGGTGAG AGACGATGTG
601 ACGTCTATGG ACCACCGACC AAAGACACAC CTATCGTCTC CCATCGCCTT GCTTCCATCG
661 CCTCTCATCG CTTTTCATAT TCTAGATCCA GCGGCCATAG ACACACCAAT CGTTTCTCAT
721 CGCCTCTCCA ACCATTGTAA AAATATTTAT AATTTTGATA TAAAATTTGT CTTCACTTGA
781 GTTCATGCCA AAAAAATTAT ACATATTATT TTCGTGTGAG AATTTACAGA AGTGGACTCT
841 TAAGATGTCC AAATGTAAAT GACCCTATTT ATTATGAGGC GCGGATCTAT AGGCCTGACT
901 CTGAAAATGG ATTATGGATT TGAGATAATA AATTTAAGGG CCTATCTTCG CACATAACAT
961 CTATAGTTCC TAAATTTTTT TTTATTGTAG TAGTAGAACT TTTCTCCCTG TAAACCAAGT
1021 TGACGCTGGG CTTTATTTTG CGACACAGAA CACCAAATTG GTGGCTATGA ACTCTTCCAC
1081 CTGGGCAGGG AAAACGGTTT ATTATGTTTC TCTTTAATTT ATCTATCGTG GCACTATAAC
1141 ACAACATGGC TTTGCCGACA CTTCCAACTA TCGGCAAAGG GTACCTTTAC CGACACTTAA
1201 CGTCTCACGA AAGGTTTTGC CGACAATTTT CAAACAGTCG CGGTAGAAGC AGTTGGCGAA
1261 ACTTTTGCCG ACAGTTAAAG GCATCGCCGA CACATTTTCT GTAGTCAAAT GGCATACCTA
1321 CGCCGACAGT TGAACTTTCA CCGACAGTGA ACCCTTTGCC GACAGTTTGG ACCTACGCCG
1381 ACAGTTTGGA CCTTTTCCGA CAGTTGGTAT GTTAGCGAAA CCGTTTCTAG GGTGTTTCAT
1441 AAACCATGCC TTGTCCAACA GTAGAAGTGT CGGCAAAACT ATATTGCTAG GATGTAGATA
1501 CAATTTAAAT ATTTTAATAA ATACACATCA CATTGAT GA GCAAAATCAC ATGGTCTGTT
1561 TTCACTAAAA CTGTCAGAGG TACACTCCAG TACTACCAGT ACGTCGCCCG CACAGTGGCC
1621 AAGGATTTTA CTGCTACTGT TGATTAACAT AAGCACTTGC GACTTTCCCT AAAATCTTTT
1681 ATAAAACAAC GGCCGCAATA ATATTGAACT ATTTTTTTTC TAGTACCAAA ATTAGAATTT
1741 GATCCCTCAC CTCATTACAT CCATAGTAAC ATGACCAGAT ATATATGGAC AGGATGGGAT
1801 CACTCAGCGA GCAGATACAC TGAGCGATTC ATAATCAGAT TTTTTAATTT CTTCTAGTGA
1861 AGTGGGGTTT TCCTAGTCTT TTAACATTCA AAATTTAGTA CAAACTTTCC CTAGTAAATG
1921 CCTTCTAGTA AAGATTTCCT AGTATTTTGA CTAGCGATAG TGTTTTATTA CTAATTAAAA
1981 ACATTAGAAG AACTCCATTT AGTGATTGGT TGTTTGGATT AGTCTTCTCA CGTTAGACCT
2041 ATATATGCAG GACAACTCAA GCCAGCATAA ATATATGAAA TATCTTGGTG TTTGTTTGTC
2101 TGACACAGGC AACCGCGTTT GGTATAAATG TGTTTTCTTG TTTACATTTT ACCATCTATA
2161 GTCATCTCAA TGTTATATAG TAGAGGCTTC ATGTTTGTAG TAGATAAGGT AGAGAATTGA
2221 GAATATTTTA TTTTTGTGCG ACCATCAATT TTATGTAATC TGCATTGTCT AATGCTTTAT
2281 TTGACATTTG AAACTACTTA ATTTGACAGT TATGCAGGTC CGCATGATCC TATGAAAGCA
2341 ATTAATTAGT ACGGGTAAAC TGCACTACAC AAGTTTGCTA GTACTATTCT ATTAACCGAC
2401 CTGTCAATAT TACCTTAAGT TACTGATTTC AATTAGAATC TAACACATTC AGGAAAAGAA
2461 GTTTCACTAG TACAAAAATC ATTTTCGTTG GCACGTTGTT TTTTTTTTCA CAGGCAGTTC
2521 ACAATATCA GGTGCTAGTA GAAAAATTTC AACGGGCCCA ACAAGAGAAC CGCCAGGCGG
2581 TCTTCTTAAT TCAACCGCCT GTGTAAACTT TCCATTTACA TAGGCGGCTT ACGATAAAAA
2641 CCGTGTGTAT AAATACCATT AACACAGGCA GTCGAGTTAC GACAACCGCC TGTGTAAATG
2701 TGTCTTTTTA CACAGGCGGT TTGTATAGAG GGCCGCCTGT GCTAATATAT TTACACAGGC
2761 TATGAGCCGC CTGTGTTAAG TCTTCTATAA ATACCCTTCG TCCACCTCCA GACAAGAACA
2821 GTTACTCCCA TGAGCTCTGC ACACTGGCGG ACCAGACGAT TCCAGTTTCC AAGGGGGGAG
2681 GTTTTGATTT TCATTTCTTT GGTGAGAAAC TTCCAAAAGG TTAGTTAGTG CCATTGATGC
2941 TATTTTTTAA GCGATTCTTT GGTTCAATTC TTGTATTGGA GGTGCTCTAG ATCTAGAGTT
3001 CATCATGCAT TCTTGCTTAG GGTTAGAGTT CATAGGGCAA AAAGAGAGAG ATTTAGCTAA
3061 ATTTTTATGT AAATTCATAG TAAATTGTAA AAATTAAAAA AAATAAAAAA TAAATACTTT
3121 TTAGAATTCT TGTGAGTAGA TCTATACAAT AGAGTAATGA TGAGGATATT TTGAAGTTTA
3181 TAATTTTGAT TCAGTTTTAG CTTTTCTTTT TTCAGATGAA TTAGACTTTA TAAACTCAAA
3241 CATTAAAATG TTGAAAATCA TAAAATGGCA AATAAATACT TTTTCAAATC TTTGTGCATA
3301 AATACTTCAT AGAAATCCTT GAATTATTCC TAAATTTTAT ACAATTGTTT CTTATAATTA
3361 TGAAAATGAG TTTAAACAAT TATTTAAATT CCATAAATTG TAACTCCGTA AGGTGTAGGT
3421 TTTCATCTCT GTTTAATAGA AGGAGGTTAG TATCTTAGTT AAGTCTGTTT TCGGGGGTTA
3481 TATTAGTTTT GTTTTTAGAT TGACCTACAT TAATTGTTCT TAACTAATTA CAGCTAAATA
3541 TGGAGAGGTC ATTATGGATG TACAACTTAT CAAGATTGGA CCTATCATAT GTAGTGCAGG
3601 TCCAAAAATT TATTGATGTC GCAAAGATAC ATGCTCGCAG AACAAAGGCG AAGCACATAT
3661 GTTGTCCATG CGCAGACTGC AAAAATATTA TGGTATTTGA CAATGTAGAA GCAATTACTT
3721 CCCATCTGGT TTGAAGAGGA TTTATGGAGG ACTACTTGAT TTGGACAAAA CATGGTGAGG
3781 GTAGTTTTGC ACCTTATATG CGGACAACTG ACAACACTGC AACTAACATC AATGTGGAGG
3841 GTCCAATGCC ACCTCTCAAT GAATTTCATG CTATGCCAGA TGTTAATGAA ACTCATACGT
3901 CTGATGTCAA TGAAACTCAG CATGCTAACA CAGATGTTGT GAAGATGCA GATTTCTTAG
3961 AGGCAATAAT GAACCGTTGT GCGGATCCAT CAATATTCTT CATGAAGGGA ATGAAAGCAT
4021 TGAAGAAGGC AGCAGAGGAC ACTTTGTACG ACGAGTCAAA AGGTTGTACC AAACAATGGT
4081 CGACATTATG TGTTGTTCTT CAGTTTTTGA CGATGAAGGC TAGACATGGT TGGTCCGATG
4141 CTAGCTTCAA TGATTTCTTG CGTGTACTTG GAGACCTTCT TCCTAAGGAG AACAAAGTGC
4201 CTGCTAACAC ATACTATGCA AAGAAGCTAG TCAGTCCACT TACGATAGGT GTTGAGAAGA
4261 TCCACGCATG TAGAAATCAT TGTATTCTAT ATCGAGGTGA TCAATATAAA GACTTAGACA
4321 GTTGTCCAAA CTGTGGTGCC AGTAGGTACA AGACAAACAA AGATTTTCGG GAGGAAGAGA
4381 ATCTAGCCTC TGTTTCTACA GGGAGGAAGC GAAAGAAGAC CCAAACAAAG ACTCAACAAG
4441 ACAAGCGCTC AAAGCCTAGT AGCAATGAAG AAGTGGACTA TTATGCATTG AGAAGAGTCT
4501 CCCTATGAGC CAAAAAAGGG GACAGCAGCA GGCACAACTC TCTTTCTGAA AGGACTTGGA
4561 AAGCAGCGGA CGGCACGGCT CATTGAGCTC GAACCGTCAC AGAAAAAGGA AGCCACCGCC
4621 CAGTCAATAG AAGCCATGCC CCCATCAAAG GAAGCCCCAA GTGGCGATGT ACATATTGAA
4681 CAGCCATCAA GTCAACCATT GACCCTAAAG GATATCAGAA AGCCAACGAT TGATGATTAT
4741 GTCAATGTCC CTAGTGACTA TGTGCCCGGA AGGCCTATGC TCCAATGGAC GCTGCTCGAT
4801 TAGATTCAAT GGCTGATAAA AAGGTTTCAT GACTGGTACA TGAGAGCAGT GCATGCTAGC
4861 CTCCATGGAA TCAGAGTTGA TATACCAACA GACATGTTTG CTACTGGTAA CAAAAAAAGC
4921 AAGACATTTG TTACCTTTGA GGACATGCAC TTGTTATTGA ACTATAGGCG GCTTGACGTC
4981 CAACTCATAA CAATCTGGTG CCTGTAAGTA TCACTCATGC ACACACAATT ΑΤΤΆΤΑΤΑΤΤ
5041 AATATGTAGT GTGAAACTCT AATATGTAGA TGTTGTCTGT AGTTTGCAAG ATCACGAGCA
5101 GATGTCATTA TTATCTGCCG GATCGATGGT CGGTTATCTG AGCCCTATCA AGTTACAAGA
5161 AAATATGAAC AAATTCGTAT TATCAAAGGA AGATAGAGCA AAGATAGAGG AAGACAAAAC
5221 ACCAGGATAA TTATGCCATC TATCTTGGTA GATCAATGCT GAGGTATAAA TATAGGGATT
5281 TTATATTGGC ACCATACAAC ATTAGGTAAG CTTGACTTCA TATACGTATT TCAAATTATC
5341 GTGTAAACAA TATACATGTG TCGCTCACTC ATTTATTCAT GCAGTGACCA TTGGATTGTT
5401 TTTTATATTT ATCCCTTCGA AGGGAAGGTG CTTGTCCTAG ACTCTTTACA TGTTCCTCCC
5461 GAGAAGTATC AACCATTCTT GGTTCAATTA GAAAGGTGAG CCAACATGAA ACCACATGCG
5521 TACTTATATA AATTAGAGTT TCAAAATAAC TTTAGTGATT TAGGTTCGAT ATCTACGGGG
5581 CATGGCGGTT TTATAAGAAA CAAAAGGGAC CTGTCGACGC TGCACGCTCA GATCCTAGGA
5641 TCCCATTGAT GATACAACAC CACTATCCGG TAAGTTTTCT GAACACATTT CATCATATAA
5701 ATAATACATA AAGCATGGCA AATTTAGAAT AATCCGTTGC TCATTATATA GTGCCACAAG
5761 CAACCACCTG GATCGGTCTA TTGTGGGTAC TATGTCTGTG AGTTTATAAG GCAGCGGGGA
5821 CGTTACGTCA AGGACAAAAA TATGGTAAAT AATATCTATG TATGAAAGTT TTCTCATTAA
5881 AGCTGCAAAA TTATATATTG AACATGTGTC AATCATGCTT TTAAACTTTA TTTTCAGCCG
5941 AAAAAGCAAG GAAAAGACGT GCCCTTTACA CCAAAGACTC TGGAAGATAT AGTAGCATAC
6001 TTGTGTGGTT TTATTATGAG AGAAATAATT TCAAGTGACA GTGCATATTT TGATCATGAG
6061 GGCGATTTAG CAAGTGATAA ATTTAGAGTG CTGACAGACA TAGCAGGTCT AAATCTGAAG
6121 CGAAACGACA TGTAAACATT GTATGGTTGT GCGGATAACA TGCATTGACG TGTATATATA
6181 TAATTTTATG GTTGATGTTT GATTTGTTTA CAATTCTATA ATATATATAT GTGGTGTATG
' 6241 TATGATGTTG TGTGTGTATA TATATATATA TATATATATA TATATATATA TATATATATA
6301 TATATATATA TATATAATGT TTAGCACTGT GTTTGGTGGG AAAAATTAAA ATTTGAAATA
6361 TATATAAAAA ATTATTTACA CAGACAGTGT ACGTGTCGAG CGTCGTCCTG TGCTATACAA
6421 ATACATTCTA ACAGGCGGCT CGCCTTGTCC ACCGGTCGGT TAAAAATACA TTTCCACACN
6481 GGCCTGGCTG GGAGAGCCGC CTGTGAAAAC ATAATTTTCA CAGGCGGCTC GCACAGCCCC
6541 GCCTGTACTG TGGTCCATTT TGTACTGACC CCTGGTACAG GCGGTGGGCT TGGCCGCCTG
6601 TGAAGATGCT TTTAGCACCG CCTGTAAAAA TGTTTTTTGT AGCAGTGTTT TTCTTATTAG
6661 TAGTATCTTT TATACTAATT AAGATTCAAT AAAAATTCAC CATGACATCC CCATTGCCAA
6721 GAGAATATTT CGCCGCCCCT CAAAGCAGCC AATAAGGCTT TACTAAAAAG ACTATCCACG
6781 CAGTAGAGAT TTAGTCAAAA TATTCCAATA GCAATTGTTT CCTGCCTGCT TGACCTTCGT
6841 CAGCCACTCA CTGTATAAAT ATCGCACCAC GCCCTTTGCA GGCTTACAGA GCTTGTATTA
6901 CGTACTAACA AGGCACACAC AGTACCCTGT GTTCACCGGC CCTGCACAAA ACTCAAGCAG
6961 TTATTACTAA C
(SEQ ID N0:14) or a functional fragment or variant thereof having 75, 80,
85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 14.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 CCCTGACCCT TGTTGGGCAA CATTTAGAGT CGTTAGCTTT GCAATTCTTT GGTTCCAATG
61 GATGGTTATC ATTTAGACAT ATTGGTCATG CTTAGTCAAA ACTTTATTGT TCGGCTATAA
121 ACTTTTCAGT ACTTTGTAAT AATTGGCTCG ATAGATGAAG CCGGGTATAA CATATCCTTT
1S1 ATCTAAAAAA ATTAGTTAAC ATGAACTTCA TATTCAATTC TTCATATCTC ACTAGCATCT
241 TTATTGTCTA GTTAGTTTTG TAGCATTGCA AAAAGCATGC AACTATATAC AATGAAACGG
301 AATAAAATTT CAGCTCTA T AATTTATATT TCAAATATAG GCCACTATAG CCATATTTCG
361 TGCTCAAGGC CACAAAATCT TGCGTACTTC CCTGTTGGTA CCAAAGAGAA GACGTTATTT
421 AACTTTGTTT GACTCTTCAA TATGGTTTGA ATCAGAAAAT TAGTTAAAAG AAAAGTGAGC
481 ACACCACGAC CTGTCATCAG CTCATGGTCA GCTCTACAAA CTTATAGAT GCATCGAGAT
541 CTAAGACTCA GGTACAAATC ATGTCAACAT CTAATGGTTT AGAAAATGAA AAGTTTTGAG
601 TTTCAAAATA TGATACGTGA TATTAACATT TGAACTTTTA GCAAGATCTG AAATAAAAAA
661 TTCAACTAGA TCATGTTAAC ATTGATATAA TCGCTTCCAA TCGCCTCCCA TCACTTCCGC
721 TAGAAAACTT TTTTTCTCGA TTTAATTAAT GAAAGGGTAA TAACATCATT GTACAAGATT
781 CTTTCAAACC TCAACCCCTA TCATCGACGG TGACGGCTCC CTATAACACG CACTAGTGGA
841 CGCCGGGCGG GTGGAACCCT AAGAAGATTT AAAAAAACTT AAGAAGAAGA TTTTTATCTA
901 ACTAACTATA GTACTTATAT CATACACTAT ACTATTCAAA ATATTATTTT CACAATTATG
961 AATTTACCCT TTTACTCTTC ATTAAAAAAA TACGAAAAAA GAATCACCAC GTCTCTATTT
1021 AGGGTCCTAG TCCCCATAAT TTAAGAGGCG GTGAGAGACG ATGTGACGTC TATGGACCAC
1081 CGACCAAAGA CACACCTATC GTCTCCCATC GCCTTGCTTC CATCGCCTCT CATCGCTTTT
1141 CATATTCTAG ATCCAGCGGC CATAGACACA CCAATCGTTT CTCATCGCCT CTCCAACCAT
1201 TGTAAAAATA TTTATAATTT TGATATAAAA TTTGTCTTCA CTTGAGTTCA TGCCAAAAAA
1261 ATTATACATA TTATTTTCGT GTGAGAATTT ACAGAAGTGG ACTCTTAAGA TGTCCAAATG
1321 TAAATGACCC TATTTATTAT GAGGCGCGGA TCTATAGGCC TGACTCTGAA AATGGATTAT
1381 GGATTTGAGA TAATAAATTT AAGGGCCTAT CTTCGCACAT AACATCTATA GTTCCTAAAT
1441 TTTTTTTTAT TGTAGTAGTA GAACTTTTCT CCCTGTAAAC CAAGTTGACG CTGGGCTTTA
1S01 TTTTGCGACA CAGAACACCA AATTGGTGGC TATGAACTCT TCCACCTGGG CAGGGAAAAC
1561 GGTTTATTAT GTTTCTCTTT AATTTATCTA TCGTGGCACT A AACACAAC ATGGCTTTGC
1621 CGACACTTCC AACTATCGGC AAAGGGTACC TTTACCGACA CTTAACGTCT CACGAAAGGT
1681 TTTGCCGACA ATTTTCAAAC AGTCGCGGTA GAAGCAGTTG GCGAAACTTT TGCCGACAGT
1741 TAAAGGCATC GCCGACACAT TTTCTGTAGT CAAATGGCAT ACCTACGCCG ACAGTTGAAC
1801 TTTCACCGAC AGTGAACCCT TTGCCGACAG TTTGGACCTA CGCCGACAGT TTGGACCTTT
1861 TCCGACAGTT GGTATGTTAG CGAAACCGTT TCTAGGGTGT TTCATAAACC ATGCCTTGTC
1921 CAACAGTAGA AGTGTCGGCA AAACTATATT GCTAGGATGT AGATACAATT TAAATATT T
1981 AATAAATACA CATCACATTG ATTGAGCAAA ATCACATGGT CTGTTTTCAC TAAAACTGTC
2041 AGAGGTACAC TCCAGTACTA CCAGTACGTC GCCCGCACAG TGGCCAAGGA TTTTACTGCT
2101 ACTGTTGATT AACATAAGCA CTTGCGACTT TCCCTAAAAT CTTTTATAAA ACAACGGCCG
2161 CAATAATATT GAACTATTTT TTTTCTAGTA CCAAAATTAG AATTTGATCC CTCACCTCAT
2221 TACATCCATA GTAACATGAC CAGATATATA TGGACAGGAT GGGATCACTC AGCGAGCAGA
2281 TACACTGAGC GATTCATAAT CAGATTTTTT AATTTCTTCT AGTGAAGTGG GGTTTTCCTA
2341 GTCTTTTAAC ATTCAAAATT TAGTACAAAC TTTCCCTAGT AAATGCCTTC TAGTAAAGAT
2401 TTCCTAGTAT TTTGACTAGC GATAGTGTTT TATTACTAAT TAAAAACATT AGAAGAACTC
2461 CATTTAGTGA TTGGTTGTTT GGATTAGTCT TCTCACGTTA GACCTATATA TGCAGGACAA
2521 CTCAAGCCAG CATAAATATA TGAAATATCT TGGTGTTTGT TTGTCTGACA CAGGCAACCG
2581 CGTTTGGTAT AAATGTGTTT TCTTGTTTAC ATTTTACCAT CTATAGTCAT CTCAATGTTA
2641 TATAGTAGAG GCTTCATGTT TGTAGTAGAT AAGGTAGAGA ATTGAGAATA TTTTATTTTT
2701 GTGCGACCAT CAATTTTATG TAATCTGCAT TGTCTAATGC TTTATTTGAC ATTTGAAACT
2761 ACTTAATTTG ACAGTTATGC AGGTCCGCAT GATCCTATGA AAGCAATTAA TAGTACGGG
2821 TAAACTGCAC TACACAAGTT TGCTAGTACT ATTCTATTAA CCGACCTG C AATATTACCT
28S1 TAAGTTACTG ATTTCAATTA GAATCTAACA CATTCAGGAA AAGAAGTTTC ACTAGTACAA
2941 AAATCATTTT CGTTGGCACG TTGTTTTTTT TTTCACAGGC AGTTCACAAT ATCATGGTGC
3001 TAGTAGAAAA ATTTCAACGG GCCCAACAAG AGAACCGCCA GGCGGTCTTC TTAATTCAAC
3061 CGCCTGTGTA AACTTTCCAT TTACATAGGC GGCTTACGAT AAAAACCGTG TGTATAAATA
3121 CCATTAACAC AGGCAGTCGA GTTACGACAA CCGCCTGTGT AAATGTGTCT TTTTACACAG
3181 GCGGTTTGTA TAGAGGGCCG CCTGTGCTAA TATATTTACA CAGGCTATGA GCCGCCTGTG
3241 TTAAGTCTTC TATAAATACC CTTCGTCCAC CTCCAGACAA GAACAGTTAC TCCCATGAGC
3301 TCTGCACACT GGCGGACCAG ACGATTCCAG TTTCCAAGGG GGGAGGTTTT GATTTTCATT
3361 TCTTTGGTGA GAAACTTCCA AAAGGTTAGT TAGTGCCATT GATGCTATTT TTTAAGCGAT
3421 TCTTTGGTTC AATTCTTGTA ' TTGGAGGTGC TCTAGATCTA GAGTTCATCA TGCATTCTTG
3481 CTTAGGGTTA GAGTTCATAG GGCAAAAAGA GAGAGATTTA GCTAAATTTT TATGTAAATT
3541 CATAGTAAAT TGTAAAAATT AAAAAAAATA AAAAATAAAT ACTTTTTAGA TTCTTGTGA
3601 GTAGATCTAT ACAATAGAGT AATGATGAGG ATATTTTGAA GTTTATAATT TTGATTCAGT
3661 TTTAGCTTTT CTTTTTTCAG ATGAATTAGA CTTTATAAAC TCAAACATTA AAATGTTGAA
3721 AATCATAAAA TGGCAAATAA ATACTTTTTC AAATCTTTGT GCATAAATAC TTCATAGAAA
3781 TCCTTGAATT ATTCCTAAAT TTTATACAAT TGTTTCTTAT AATTATGAAA ATGAGTTTAA
3841 ACAATTATTT AAATTCCATA AATTGTAACT CCGTAAGGTG TAGGTTTTCA TCTCTGTTTA
3901 ATAGAAGGAG GTTAGTATCT TAGTTAAGTC TGTTTTCGGG GGTTATATTA GTTTTGTTTT
3961 TAGATTGACC TACATTAATT GTTCTTAACT AATTACAGCT AAATATGGAG AGGTCATTAT
4021 GGATGTACAA CTTATCAAGA TTGGACCTAT CATATGTAGT GCAGGTCCAA AAATTTATTG
4081 ATGTCGCAAA GATACATGCT CGCAGAACAA AGGCGAAGCA CATATGTTGT CCATGCGCAG
4141 ACTGCAAAAA TATTATGGTA TTTGACAATG TAGAAGCAAT TACTTCCCAT CTGGTTTGAA
4201 GAGGATTTAT GGAGGACTAC TTGAT TGGA CAAAACATGG TGAGGGTAGT TTGCACCTT
4261 ATATGCGGAC AACTGACAAC ACTGCAACTA ACATCAATGT GGAGGGTCCA ATGCCACCTC
4321 TCAATGAATT TCATGCTATG CCAGATGTTA ATGAAACTCA TACGTCTGAT GTCAATGAAA
4381 CTCAGCATGC TAACACAGAT GTTGTTGAAG ATGCAGATTT CTTAGAGGCA ATAATGAACC
4441 GTTGTGCGGA TCCATCAATA TTCTTCATGA AGGGAATGAA AGCATTGAAG AAGGCAGCAG
4501 AGGACACTTT GTACGACGAG TCAAAAGGTT GTACCAAACA ATGGTCGACA TTATGTGTTG
4561 TTCTTCAGTT TTTGACGATG AAGGCTAGAC ATGGTTGGTC CGATGCTAGC TTCAATGATT
4621 TCTTGCGTGT ACTTGGAGAC CTTCTTCC A AGGAGAACAA AGTGCCTGCT AACACATACT
4681 ATGCAAAGAA GCTAGTCAGT CCACTTACGA TAGGTGTTGA GAAGATCCAC GCATGTAGAA
4741 ATCATTGTAT TCTATATCGA GGTGATCAAT ATAAAGACTT AGACAGTTGT CCAAACTGTG
4801 GTGCCAGTAG GTACAAGACA AACAAAGATT TTCGGGAGGA AGAGAATCTA GCCTCTGTTT
4861 CTACAGGGAG GAAGCGAAAG AAGACCCAAA CAAAGACTCA ACAAGACAAG CGCTCAAAGC
4921 CTAGTAGCAA TGAAGAAGTG GACTATTATG CATTGAGAAG AGTCTCCCTA TGAGCCAAAA
4981 AAGGGGACAG CAGCAGGCAC AACTCTCTTT CTGAAAGGAC TTGGAAAGCA GCGGACGGCA
5041 CGGCTCATTG AGCTCGAACC GTCACAGAAA AAGGAAGCCA CCGCCCAGTC AATAGAAGCC
5101 ATGCCCCCAT CAAAGGAAGC CCCAAGTGGC GATGTACATA TTGAACAGCC ATCAAGTCAA
5161 CCATTGACCC TAAAGGATAT CAGAAAGCCA ACGATTGATG ATTATGTCAA TGTCCCTAGT
5221 GACTATGTGC CCGGAAGGCC TATGCTCCAA TGGACGCTGC TCGATTAGAT TCAATGGCTG
5281 ATAAAAAGGT T CATGACTG GTACATGAGA GCAGTGCATG CTAGCCTCCA TGGAATCAGA
5341 GTTGATATAC CAACAGACAT GTTTGCTACT GGTAACAAAA AAAGCAAGAC ATTTGTTACC
5401 TTTGAGGACA TGCACTTGTT ATTGAACTAT AGGCGGCTTG ACGTCCAACT CATAACAATC
5461 TGGTGCCTGT AAGTATCACT CATGCACACA CAATTATTAT ATATTAATAT GTAGTGTGAA
5521 ACTCTAATAT GTAGATGTTG TCTGTAGTTT GCAAGATCAC GAGCAGATGT CATTATTATC
5581 TGCCGGATCG ATGGTCGGTT ATCTGAGCCC TATCAAGTTA CAAGAAAATA TGAACAAATT
5641 CGTATTATCA AAGGAAGATA GAGCAAAGAT AGAGGAAGAC AAAACACCAG GATAATTATG
5701 CCATCTATCT TGGTAGATCA ATGCTGAGGT ATAAATATAG GGATTTTATA TTGGCACCAT
5761 ACAACATTAG GTAAGCTTGA CTTCATATAC G ATTTCAAA TTATCGTGTA AACAATATAC
5821 ATGTGTCGCT CACTCATTTA TTCATGCAGT GACCATTGGA TTGTTTTTTA TATTTA CCC
5881 TTCGAAGGGA AGGTGCTTGT CCTAGACTCT TTACATGTTC CTCCCGAGAA GTATCAACCA
5941 TTCTTGGTTC AATTAGAAAG GTGAGCCAAC ATGAAACCAC ATGCGTACTT
6001 GAGTTTCAAA ATAACTTTAG TGATTTAGGT TCGATATCTA CGGGGCATGG CGGTTTTATA
6061 AGAAACAAAA GGGACCTGTC GACGCTGCAC GCTCAGATCC TAGGATCCCA TTGATGATAC
6121 AACACCACTA TCCGGTAAGT TTTCTGAACA CATTTCATCA TATAAATAAT ACATAAAGCA
6181 TGGCAAATTT AGAATAATCC GTTGCTCATT ATATAGTGCC ACAAGCAACC ACCTGGATCG
6241 GTCTATTGTG GGTACTATGT CTGTGAGTTT ATAAGGCAGC GGGGACGTTA CGTCAAGGAC
6301 AAAAATATGG TAAATAATAT CTATGTATGA AGTTTTCTCA TTAAAGCTGC AAAATTATAT
6361 ATTGAACATG TGTCAATCAT GCTTTTAAAC TTTATTTTCA GCCGAAAAAG CAAGGAAAAG
6421 ACGTGCCCTT TACACCAAAG ACTCTGGAAG ATATAGTAGC ATACTTGTGT GGTTTTATTA
6481 TGAGAGAAAT AATTTCAAGT GACAGTGCAT ATTTTGATCA TGAGGGCGAT TTAGCAAGTG
6541 ATAAATTTAG AGTGCTGACA GACATAGCAG GTCTAAATCT GAAGCGAAAC GACATGTAAA
6601 CATTGTATGG TTGTGCGGAT AACATGCATT GACGTGTATA TATATAATTT TATGGTTGAT
6661 GTTTGATTTG TTTACAATTC TATAATATAT ATATGTGGTG TATGTATGAT GTTGTGTGTG
6721 TATATATATA TATATATATA TATATATATA TATATATATA TATATATATA TATATATATA
6781 ATGTTTAGCA CTGTGTTTGG TGGGAAAAAT TAAAATTTGA AATATATATA AAAAATTATT
6841 TACACAGACA GTGTAGTGTG AGCTGCCTGT GTAAAAATAC ATTTATACAG GCGGCTCACC
6901 TTGTCNNNNC AGGCGGTGCT AAAAGCATCT TCACAGGCGG CCAAGCCCAC CGCCTGTACC
6961 AGGGGTCAGT ACAAAATGGA CCACAGTACA GGCGGGGCTG TGCGAGCCGC CTGTGAAAAC
7021 ATAATTTTCA CAGGCGGCTC GCACAGCCCC GCCTGTACTG TGGTCCATTT TGTACTGACC
7081 CCTGGTACAG GCGGTGGGCT TGGCCGCCTG TGAAGATGCT TTTAGCACCG CCTGTAAAAA
7141 TGTTTTTTGT AGCAGTGTTT TTCTTATTAG TAGTATCTTT TATACTAATT AAGATTCAAT
7201 AAAAATTCAC CATGACATCC CCATTGCCAA GAGAATATTT CGCCGCCCCT CAAAGCAGCC
7261 AATAAGGCTT TACTAAAAAG ACTATCCACG CAGTAGAGAT TTAGTCAAAA ΊΆ T'T C C T ¾-
7321 GCAATTGTTT CCTGCCTGCT TGACCTTCGT CAGCCACTCA CTGTATAAAT ATCGCACCAC
7381 GCCCTTTGCA GGCTTACAGA GCTTGTATTA CGTACTAACA AGGCACACAC AGTACCCTGT
7441 GTTCACCGGC CCTGCACAAA ACTCAAGCAG TTATTACTAA C
(SEQ ID NO: 15) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 15. Each N = can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 CTATGCTCCA ATGGACGCTG CTCGATTAGA TTCAATGGCT GATAAAAAGG TTTCATGACT
61 GGTACATGAG AGCAGTGCAT GCTAGCCTCC ATGGAATCAG AGTTGATATA CCAACAGACA
121 TGTTTGCTAC TGGTAACAAA AAAAGCAAGA CATTTGTTAC CTTTGAGGAC ATGCACTTGT
181 TATTGAACTA TAGGCGGCTT GACGTCCAAC TCATAACAAT CTGGTGCCTG TAAGTATCAC
24 1 TCATGCACAC ACAATTATTA TATATTAATA TGTAGTGTGA AACTCTAATA TGTAGATGTT
301 GTCTGTAGTT TGCAAGATCA CGAGCAGATG CATTATTAT CTGCCGGATC GATGGTCGGT
361 TATCTGAGCC CTATCAAGTT ACAAGAAAAT ATGAACAAAT TCGTATTATC AAAGGAAGAT
421 AGAGCAAAGA TAGAGGAAGA C i¾ i ¾ . C ¾ 00 ¾ GGATAATTAT GCCATCTATC TTGGTAGATC
481 AATGCTGAGG TATAAATATA GGGATTTTAT ATTGGCACCA TACAACATTA GGTAAGCTTG
541 ACTTCATATA CGTATTTCAA ATTATCGTGT AAACAATATA CATGTGTCGC TCACTCATTT
601 ATTCATGCAG TGACCATTGG ATTGTTTTTT ATATTTATCC CTTCGAAGGG AAGGTGCTTG
661 TCCTAGACTC TTTACATGTT CCTCCCGAGA AGTATCAACC ATTCTTGGTT CAATTAGAAA
721 GGTGAGCCAA CATGAAACCA CATGCGTACT TATATAAATT AGAGTTTCAA AATAACTTTA
781 GTGATTTAGG TTCGATATCT ACGGGGCATG GCGGTTTTAT AAGAAACAAA AGGGACCTGT
841 CGACGCTGCA CGCTCAGATC CTAGGATCCC ATTGATGATA CAACACCACT &TCCGGTAAG
901 TTTTCTGAAC ACATTTCATC ATATAAATAA TACATAAAGC ATGGCAAATT TAGAATAATC
961 CGTTGCTCAT TATATAGTGC CACAAGCAAC CACCTGGATC GGTCTATTGT GGGTACTATG
1021 TCTGTGAGTT TATAAGGCAG CGGGGACGTT ACGTCAAGGA CAAAAATATG GTAAATAATA
1081 TCTATGTATG AAGTTTTCTC ATTAAAGCTG CAAAATTATA TATTGAACAT GTGTCAATCA 14 TGCTTTTAAA CTTTATTTTC AGCCGAAAAA GCAAGGAAAA GACGTGCCCT TTACACCAAA
1201 GACTCTGGAA GATATAGTAG CATACTTGTG TGGTTTTATT ATGAGAGAAA TAATTTCAAG
1261 TGACAGTGCA TATTTTGATC ATGAGGGCGA TTTAGCAAGT GATAAATTTA GAGTGCTGAC
1321 AGACATAGCA GGTCTAAATC TGAAGCGAAA CGACATGTAA ACATTGTATG GTTGTGCGGA
1381 TAACATGCAT TGACGTGTAT ATATATAATT TTATGGTTGA TGTTTGATTT GTTTACAATT
1441 CTATAATATA TATATGTGGT GTATGTATGA TGTTGTGTGT GTATATATAT ATATATATAT
1501 ATATATATAT ATATATATAT ATATATATAT ATATATATAT AATGTTTAGC ACTGTGTTTG
1561 GTGGGAAAAA TTAAAATTTG AAATATATAT AAAAAATTAT TTACACAGAC AGTGTAGTGT
1621 GAGCTGCCTG TGTAAAAATA CATTTATACA GGCGGCTCAC CTTGTNNNNN CAGGCGGTGC
1681 TAAAAGCATC TTCACAGGCG GCCAAGCCCA CCGCCTGTAC CAGGGGTCAG TACAAAATGG
1741 ACCACAGTAC AGGCGGGGCT GTGCGAGCCG CCTGTGAAAA CATAATTTTC ACAGGCGGCT
1801 CGCACAGCCC CGCCTGTACT GTGGTCCATT TTGTACTGAC CCCTGGTACA GGCGGTGGGC
1861 TTGGCCGCCT GTGAAGATGC TTTTAGCACC GCCTGTAAAA ATGTTTTTTG TAGCAGTGTT
1921 TTTCTTATTA GTAGTATCTT TTATACTAAT TAAGATTCAA TAAAAATTCA CCATGACATC
1981 CCCATTGCCA AGAGAATATT TCGCCGCCCC TCAAAGCAGC CAATAAGGCT TTACTAAAAA
2041 GACTATCCAC GCAGTAGAGA TTTAGTCAAA ATATTCCAAT AGCAATTGTT TCCTGCCTGC
2101 TTGACCTTCG TCAGCCACTC ACTGTATAAA TATCGCACCA CGCCCTTTGC AGGCTTACAG
2161 AGCTTGTATT ACGTACTAAC AAGGCACAGA CAGTACCCTG TGTTCACCGG CCCTGCACAA
2221 AACTCAAGCA GTTATTACTA AC
(SEQ ID NO: 16) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 16. Each N = can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides. In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 CACTATAACA CAACATGGCT TTGCCGACAC TTCCAACTAT CGGCAAAGGG TACCTTTACC
61 GACACTTAAC GTCTCACGAA AGGTTTTGCC GACAATTTTC AAACAGTCGC GGTAGAAGCA
121 GTTGGCGAAA CTTTTGCCGA CAGTTAAAGG CATCGCCGAC ACATTTTCTG TAGTCAAATG
181 GCATACCTAC GCCGACAGTT GAACTTTCAC CGACAGTGAA CCCTTTGCCG ACAGTTTGGA
241 CCTACGCCGA CAGTTTGGAC CTTTTCCGAC AGTTGGTATG TTAGCGAAAC CGTTTCTAGG
301 GTGTTTCATA AACCATGCCT TGTCCAACAG TAGAAGTGTC GGCAAAACTA TATTGCTAGG
361 ATGTAGATAC ΑΆΤΤΤΑΑΑΤΑ TTTTAATAAA TACACATCAC ATTGATTGAG CAAAATCACA
421 TGGTCTGTTT TCACTAAAAC TGTCAGAGGT ACACTCCAGT ACTACCAGTA CGTCGCCCGC
481 ACAGTGGCCA AGGATTTTAC TGCTACTGTT GATTAACATA AGCACTTGCG ACTTTCCCTA
541 AAATCTTTTA TAAAACAACG GCCGCAATAA TATTGAACTA TTTTTTTTCT AGTACCAAAA
601 TTAGAATTTG ATCCCTCACC TCATTACATC CATAGTAACA TGACCAGATA TATATGGACA
661 GGATGGGATC ACTCAGCGAG CAGATACACT GAGCGATTCA TAATCAGATT TTTTAATTTC
721 TTCTAGTGAA GTGGGGTTTT CCTAGTCTTT TAACATTCA AATTTAGTAC AAACTTTCCC
781 TAGTAAATGC CTTCTAGTAA AGATTTCCTA GTATTTTGAC TAGCGATAGT GTTTTATTAC
841 TAATTAAAAA CATTAGAAGA ACTCCATTTA GTGATTGGTT GTTTGGATTA GTCTTCTCAC
901 GTTAGACCTA TATATGCAGG ACAACTCAAG CCAGCATAAA TATATGAAAT ATCTTGGTGT
961 TTGTTTGTCT GACACAGGCA ACCGCGTTTG GTATAAATGT GTTTTCTTGT TTACATTTTA
1021 CCATCTATAG TCATCTCAAT GTTATATAGT AGAGGCTTCA TGTTTGTAGT AGATAAGGTA
1081 GAGAATTGAG AATATTTTAT TTTTGTGCGA CCATCAATTT TATGTAATCT GCATTGTCTA
1141 ATGCTTTATT TGACATTTGA AACTACTTAA TTTGACAGTT ATGCAGGTCC GCATGATCCT
1201 ATGAAAGCAA TTAATTAGTA CGGGTAAACT GCACTACACA AGTTTGCTAG TACTATTCTA
1261 TTAACCGACC TGTCAATATT ACCTTAAGTT ACTGATTTCA ATTAGAATCT AACACATTCA
1321 GGAAAAGAAG TTTCACTAGT ACAAAAATCA TTTTCGTTGG CACGTTGTTT TTTTTTTCAC
1381 AGGCAGTTCA CAATATCATG GTGCTAGTAG AAAAATTTCA ACGGGCCCAA CAAGAGAACC
1441 GCCAGGCGGT CTTCTTAATT CAACCGCCTG TGTAAACTTT CCATTTACAT AGGCGGCTTA
1501 CGATAAAAAC CGTGTGTATA AATACCATTA ACACAGGCAG TCGAGTTACG ACAACCGCCT
1561 GTGTAAATGT GTCTTTTTAC ACAGGCGGTT TGTATAGAGG GCCGCCTGTG CTAATATATT
1621 TACACAGGCT ATGAGCCGCC TGTGTTAAGT CTTCTATAAA TACCCTTCGT CCACCTCCAG
1681 ACAAGAACAG TTACTCCCAT GAGCTCTGCA CACTGGCGGA CCAGACGATT CCAGTTTCCA
1741 AGGGGGGAGG TTTTGATTTT CATTTCTTTG GTGAGAAACT TCCAAAAGGT TAGTTAGTGC
1801 CATTGATGCT ATTTTTTAAG CGATTCTTTG GTTCAATTCT TGTATTGGAG GTGCTCTAGA
1861 TCTAGAGTTC ATCATGCATT CTTGCTTAGG GTTAGAGTTC ATAGGGC AA AAGAGAGAGA
1921 TTTAGCTAAA TTTTTATGTA AATTCATAGT AAATTGTAAA AATTAAAAAA AATAAAAAAT
1981 AAATACTTTT TAGAATTCTT GTGAGTAGAT CTATACAATA GAGTAATGAT GAGGATATTT
2041 TGAAGTTTAT AATTTTGATT CAGTTTTAGC TTTTCTTTTT TCAGATGAAT TAGACTTTAT
2101 AAACTCAAAC ATTAAAATGT TGAAAATCAT AAAATGGCAA ATAAATACTT TTTCAAATCT
2161 TTGTGCATAA ATACTTCATA GAAATCCTTG AATTATTCCT AAATTTTATA CAATTGTTTC
2221 ΤΤΑΪΑΑΤΤΑΤ GAAAATGAGT TTAAACAATT ATTTAAATTC CATAAATTGT AACTCCGTAA
2281 GGTGTAGGTT TTCATCTCTG TTTAATAGAA GGAGGTTAGT ATCTTAGTTA AGTCTGTTTT
2341 CGGGGGTTAT ATTAGTTTTG TTTTTAGATT GACCTACATT AATTGTTCTT AACTAATTAC
2401 AGCTAAATAT GGAGAGGTCA TTATGGATGT ACAACTTATC AAGATTGGAC CTATCATATG
2461 TAGTGCAGGT CCAAAAATTT ATTGATGTCG CAAAGATACA TGCTCGCAGA ACAAAGGCGA
2521 AGCACATATG TTGTCCATGC GCAGACTGCA AAAATATTAT GGTATTTGAC AATGTAGAAG
2581 CAATTACTTC CCATCTGGTT TGAAGAGGAT TTATGGAGGA CTACTTGATT TGGACAAAAC
2641 ATGGTGAGGG TAGTTTTGCA CCTTATATGC GGACAACTGA CAAC'ACTGCA ACTAACATCA
2701 ATGTGGAGGG TCCAATGCCA CCTCTCAATG AATTTCATGC TATGCCAGAT GTTAATGAAA
2761 CTCATACGTC TGATGTCAAT GAAACTCAGC ATGCTAACAC AGATGTTGTT GAAGATGCAG
2821 ATTTCTTAGA GGCAATAATG AACCGTTGTG CGGATCCATC AATATTCTTC ATGAAGGGAA
2881 TGAAAGCATT GAAGAAGGCA GCAGAGGACA CTTTGTACGA CGAGTCAAAA GGTTGTACCA
2941 AACAATGGTC GACATTATGT GTTGTTCTTC AGTTTTTGAC GATGAAGGCT AGACATGGTT
3001 GGTCCGATGC TAGCTTCAAT GATTTCTTGC GTGTACTTGG AGACCTTCTT CCTAAGGAGA
3061 ACAAAGTGCC TGCTAACACA TACTATGCAA AGAAGCTAGT CAGTCCACTT ACGATAGGTG
3121 TTGAGAAGAT CCACGCATGT AGAAATCATT GTATTCTATA TCGAGGTGAT CAATATAAAG
3181 ACTTAGACAG TTGTCCAAAC TGTGGTGCCA GTAGGTACAA GACAAACAAA GATTTTCGGG
3241 AGGAAGAGAA TCTAGCCTCT GTTTCTACAG GGAGGAAGCG AAAGAAGACC CAAACAAAGA
3301 CTCAACAAGA CAAGCGCTCA AAGCCTAGTA GCAATGAAGA AGTGGACTAT TATGCATTGA
3361 GAAGAGTCTC CCTATGAGCC AAAAAAGGGG ACAGCAGCAG GCACAACTCT CTTTCTGAAA
3421 GGACTTGGAA AGCAGCGGAC GGCACGGCTC ATTGAGCTCG AACCGTCACA GAAAAAGGAA
3481 GCCACCGCCC AGTCAATAGA AGCCATGCCC CCATCAAAGG AAGCCCCAAG TGGCGATGTA
3541 CATATTGAAC AGCCATCAAG TCAACCATTG ACCCTAAAGG ATATCAGAAA GCCAACGATT
3601 GATGATTATG TCAATGTCCC AGTGACTAT GTGCCCGGAA GGCCTATGCT CCAATGGACG
3661 CTGCTCGATT AGATTCAATG GCTGATAAAA AGGTTTCATG ACTGGTACAT GAGAGCAGTG
3721 CATGCTAGCC TCCATGGAAT CAGAGTTGAT ATACCAACAG ACATGTTTGC TACTGGTAAC
3781 AAAAAAAGCA AGACATTTGT TACCTTTGAG GACATGCACT TGTTATTGAA CTATAGGCGG
3841 CTTGACGTCC AACTCATAAC AATCTGGTGC CTGTAAGTAT CACTCATGCA CACACAATTA
3901 TTATATATTA ATATGTAGTG TGAAACTCTA ATATGTAGAT GTTGTCTGTA GTTTGCAAGA
3961 TCACGAGCAG ATGTCATTAT TATCTGCCGG ATCGATGGTC GGTTATCTGA GCCCTATCAA
4021 GTTACAAGAA AATATGAACA AATTCGTATT ATCAAAGGAA GATAGAGCAA AGATAGAGGA
4081 AGACAAAACA CCAGGATAAT TATGCCATCT ATCTTGGTAG ATCAATGCTG AGGTATAAAT
4141 ATAGGGATTT TATATTGGCA CCATACAACA TTAGGTAAGC TTGACTTCAT ATACGTATTT
4201 CAAATTATCG TGTAAACAAT ATACATGTGT CGCTCACTCA TTTATTCATG CAGTGACCAT
4261 TGGATTGTTT TTTATATTTA TCCCTTCGAA GGGAAGGTGC TTGTCCTAGA CTCTTTACAT
4321 GTTCCTCCCG AGAAGTATCA ACCATTCTTG GTTCAATTAG AAAGGTGAGC CAACATGAAA
4381 CCACATGCGT ACTTATATAA ATTAGAGTTT CAAAATAACT TAGTGATTT AGGTTCGATA
4441 TCTACGGGGC ATGGCGGTTT TATAAGAAAC AAAAGGGACC TGTCGACGCT GCACGCTCAG
4501 ATCCTAGGAT CCCATTGATG ATACAACACC ACTATCCGGT AAGTTTTCTG AACACATTTC
4561 ATCATATAAA TAATACATAA AGCATGGCAA ATTTAGAATA ATCCGTTGCT CATTATATAG
4621 TGCCACAAGC AACCACCTGG ATCGGTCTAT TGTGGGTACT ATGTCTGTGA GTTTATAAGG
4681 CAGCGGGGAC GTTACGTCAA GGACAAAAAT ATGGTAAATA ATATCTATGT ATGAAAGTTT
4741 TCTCATTAAA GCTGCAAAAT TATATATTGA ACATGTGTCA A CATGCTTT TAAACTTTAT
4801 TTTCAGCCGA AAAAGCAAGG AAAAGACGTG CCCTTTACAC CAAAGACTCT GGAAGATATA
4861 GTAGCATACT TGTGTGGTTT TATTATGAGA GAAATAATTT CAAGTGACAG TGCATATTTT
4921 GATCATGAGG GCGATTTAGC AAGTGATAAA TTTAGAGTGC TGACAGACAT AGCAGGTCTA
4981 AATCTGAAGC GAAACGACAT GTAAACATTG TATGGTTGTG CGGATAACAT GCATTGACGT
5041 GTATATATAT AATTTTATGG TTGATGTTTG ATTTGTTTAC AATTCTATAA TATATATATG
5101 TGGTGTATGT ATGATGTTGT GTGTGTATAT ATATATATAT ATATATATAT ATATATATAT
5161 ATATATATAT ATATATATAT ATATAATGTT TAGCACTGTG TTTGGTGGGA AAAATTAAAA
5221 TTTGAAATAT ATATAAAAAA TTATTTACAC AGACAGTGTA CGTGTCGAGC GTCGTCCTGT
5281 GCTATACAAA TACATTCTAA CAGGCGGCTC GCCTTGTCCA CCGGTCGGTT AAAAATACAT
5341 TTCCACACHG GCCTGGCTGG GAGAGCCGCC TGTGAAAACA TAATTTTCAC AGGCGGCTCG
5401 CACAGCCCCG CCTGTACTGT GGTCCATTTT GTACTGACCC CTGGTACAGG CGGTGGGCTT
5461 GGCCGCCTGT GAAGATGCTT TTAGCACCGC CTGTAAAAAT GTTTTTTGTA GCAGTGTTTT
5521 TCTTATTAGT AGTATCTTTT ATACTAA TA AGATTCAATA AAAATTCACC ATGACATCCC
5581 CATTGCCAAG AGAATATTTC GCCGCCCCTC AAAGCAGCCA AT
(SEQ ID NO: 17) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 17.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 CACTATAACA CAACATGGCT TTGCCGACAC TTCCAACTAT CGGCAAAGGG TACCTTTACC
61 GACACTTAAC GTCTCACGAA AGGTTTTGCC GACAATTTTC AAACAGTCGC GGTAGAAGCA
121 GTTGGCGAAA CTTTTGCCGA CAGTTAAAGG CATCGCCGAC ACATTTTCTG TAGTCAAATG
181 GCATACCTAC GCCGACAGTT GAACTTTCAC CGACAGTGAA CCCTTTGCCG ACAGTTTGGA
241 CCTACGCCGA CAGTTTGGAC CTTTTCCGAC AGTTGGTATG TTAGCGAAAC CGTTTCTAGG
301 GTGTTTCATA AACCATGCCT TGTCCAACAG TAGAAGTGTC GGCAAAACTA TATTGCTAGG
361 ATGTAGATAC AATTTAAATA TTTTAATAAA TACACATCAC ATTGATTGAG CAAAATCACA
421 TGGTCTGTTT TCACTAAAAC TGTCAGAGGT ACACTCCAGT ACTACCAGTA CGTCGCCCGC
481 ACAGTGGCCA AGGATTTTAC TGCTACTGTT GATTAACATA AGCACTTGCG ACTTTCCCTA
541 AAATCTTTTA TAAAACAACG GCCGCAATAA TATTGAACTA TTTTTTTTCT AGTACCAAAA
60 TTAGAATTTG ATCCCTCACC TCATTACATC CATAGTAACA TGACCAGATA TATATGGACA
661 GGATGGGA C ACTCAGCGAG CAGATACACT GAGCGATTCA TAATCAGATT TTTTAATTTC
721 TTCTAGTGAA GTGGGGTTTT CCTAGTCTTT TAACATTCAA AATTTAGTAC AAACTTTCCC
781 TAGTAAATGC CTTCTAGTAA AGATTTCCTA GTATTTTGAC TAGCGATAGT GTTTTATTAC
841 AATTAAAAA CATTAGAAGA ACTCCATTTA GTGATTGGTT GTTTGGATTA GTCTTCTCAC
901 GTTAGACCTA TATATGCAGG ACMCTCAAG CCAGCATAAA TATATGAAAT ATCTTGGTGT
961 TTGTTTGTCT GACACAGGCA ACCGCGTTTG GTATAAATGT GTTTTCTTGT TTACATTTTA
1021 CCATCTATAG TCATCTCAAT GTTATATAGT AGAGGCTTCA TGTTTGTAGT AGATAAGGTA
1031 GAGAATTGAG AATATTTTAT TTTTGTGCGA CCATCAATTT TATGTAATCT GCATTGTCTA
1141 ATGCTTTATT TGACATTTGA AACTACTTAA TTTGACAGTT ATGCAGGTCC GCATGATCCT
1201 ATGAAAGCAA TTAATTAGTA CGGGTAAACT GCACTACACA AGTTTGCTAG TACTATTCTA
1261 TTAACCGACC TGTCAATATT ACCTTAAGTT ACTGATTTCA ATTAGAATCT AACACATTCA
1321 GGAAAAGAAG TTTCACTAGT ACAAAAATCA TTTTCGTTGG CACGTTGT T TTTTTTTCAC
1381 AGGCAGTTCA CAATATCATG GTGCTAGTAG AAAAATTTCA ACGGGCCCAA CAAGAGAACC
1441 GCCAGGCGGT CTTCTTAATT CAACCGCCTG TGTAAACTTT CCATTTACAT AGGCGGCTTA
1501 CGATAAAAAC CGTGTGTATA AATACCATTA ACACAGGCAG TCGAGTTACG ACAACCGCCT
1561 GTGTAAATGT GTCTTTTTAC ACAGGCGGTT TGTATAGAGG GCCGCCTGTG CTAATATATT
1621 TACACAGGCT ATGAGCCGCC TGTGTTAAGT CTTCTATAAA TACCCTTCGT CCACCTCCAG
1681 ACAAGAACAG T ACTCCCAT GAGCTCTGCA CACTGGCGGA CCAGACGATT CCAGTTTCCA
1741 AGGGGGGAGG TTTTGATTTT CATTTCTTTG GTGAGAAACT TCCAAAAGGT TAGTTAGTGC
1801 CATTGATGCT ATTTTTTAAG CGATTCTTTG GTTCAATTCT TGTATTGGAG GTGCTCTAGA
1861 TCTAGAGTTC ATCATGCATT CTTGCTTAGG GTTAGAGTTC ATAGGGCAAA AAGAGAGAGA
1921 TTTAGCTAAA TTTTTATGTA AATTCATAGT AAATTGTAAA AATTAAAAAA ΑΑΤΑΑΑΑΑΛΪ
1981 AAATACTTTT TAGAATTCTT GTGAGTAGAT CTATACAATA GAGTAATGAT GAGGATATTT
2041 TGAAGTTTAT AATTTTGATT CAGTTTTAGC TTTTCTTTTT TCAGATGAAT TAGACTTTAT
2101 AAACTCAAAC ATTAAAATGT TGAAAATCAT AAAATGGCAA ATAAATACTT TTTCAAATCT
2161 TTGTGCATAA ATACTTCATA GAAATCCTTG AATTATTCCT AAATTTTATA CAATTGTTTC
2221 TTATAATTAT GAAAATGAGT TTAAACAATT ATTTAAATTC CATAAATTGT AACTCCGTAA
2281 GGTGTAGGTT TTCATCTCTG TTTAATAGAA GGAGGTTAGT ATCTTAGTTA AGTCTGTTTT
2341 CGGGGGTTAT ATTAGTTTTG TTTTTAGATT GACCTACATT AATTGTTCTT AACTAATTAC
2401 AGCTAAATAT GGAGAGGTCA TTATGGATGT ACAACTTATC AAGATTGGAC CTATCATATG
2461 TAGTGCAGGT CCAAAAATTT ATTGATGTCG CAAAGATACA TGCTCGCAGA ACAAAGGCGA
2521 AGCACATATG T GTCCATGC GCAGACTGCA AAAATATTAT GGTATTTGAC AATGTAGAAG
2581 CAATTACTTC CCATCTGGTT TGAAGAGGAT TTATGGAGGA CTACTTGATT TGGACAAAAC
2641 ATGGTGAGGG TAGTTTTGCA CCTTATATGC GGACAACTGA CAACACTGCA ACTAACATCA
2701 ATGTGGAGGG TCCAATGCCA CCTCTCAATG AATTTCATGC TATGCCAGAT GTTAATGAAA
2761 CTCATACGTC TGATGTCAAT GAAACTCAGC ATGCTAACAC AGATGTTGTT GAAGATGCAG
2821 ATTTCTTAGA GGCAATAATG AACCGTTGTG CGGATCCATC AATATTCTTC ATGAAGGGAA
2881 TGAAAGCATT GAAGAAGGCA GCAGAGGACA CTTTGTACGA CGAGTCAAAA GGTTGTACCA
2941 AACAATGGTC GACATTATGT GTTGTTCTTC AGTTTTTGAC GATGAAGGCT AGACATGGTT
3001 GGTCCGATGC TAGCTTCAAT GATTTCTTGC GTGTACTTGG AGACCTTCTT CCTAAGGAGA
3061 ACAAAGTGCC TGCTAACACA TACTATGCAA AGAAGCTAGT CAGTCCACTT ACGATAGGTG
3121 TTGAGAAGAT CCACGCATGT AGAAATCATT GTATTCTATA TCGAGGTGAT CAATATAAAG
3181 ACTTAGACAG TTGTCCAAAC TGTGGTGCCA GTAGGTACAA GACAAACAAA GATTTTCGGG
3241 AGGAAGAGAA TCTAGCCTCT GTTTCTACAG GGAGGAAGCG AAAGAAGACC CAAACAAAGA
3301 CTCAACAAGA CAAGCGCTCA AAGCCTAGTA GCAATGAAGA AGTGGACTAT TATGCATTGA
3361 GAAGAGTCTC CCTATGAGCC AAAAAAGGGG ACAGCAGCAG GCACAACTCT CTTTCTGAAA
3421 GGACTTGGAA AGCAGCGGAC GGCACGGCTC ATTGAGCTCG AACCGTCACA GAAAAAGGAA
3481 GCCACCGCCC AGTCAATAGA AGCCATGCCC CCATCAAAGG AAGCCCCAAG TGGCGATGTA
3541 CATATTGAAC AGCCATCAAG TCAACCATTG ACCCTAAAGG ATATCAGAAA GCCAACGATT
3601 GATGATTATG TCAATGTCCC TAGTGACTAT GTGCCCGGAA GGCCTATGCT CCAATGGACG
3661 CTGCTCGATT AGATTCAATG GCTGATAAAA AGGTTTCATG ACTGGTACAT GAGAGCAGTG
3721 CATGCTAGCC TCCATGGAAT CAGAGTTGAT ATACCAACAG ACATGTTTGC TACTGGTAAC
3781 AAAAAAAGCA AGACATTTGT TACCTTTGAG GACATGCACT TGTTATTGAA CTATAGGCGG
3841 CTTGACGTCC AACTCATAAC AATCTGGTGC CTGTAAGTAT CACTCATGCA CACACAATTA
3901 TTATATATTA ATATGTAGTG TGAAACTCTA ATATGTAGAT GTTGTCTGTA GTTTGCAAGA
3961 TCACGAGCAG ATGTCATTAT TATCTGCCGG ATCGATGGTC GGTTATCTGA GCCCTATCAA
4021 GTTACAAGAA AATATGAACA AATTCGTATT ATCAAAGGAA GATAGAGCAA AGATAGAGGA
4081 AGACAAAACA CCAGGATAAT TATGCCATCT ATCTTGGTAG ATCAATGCTG AGGTATAAAT
4141 ATAGGGATTT TATATTGGCA CCATACAACA TTAGGTAAGC TTGACTTCAT ATACGTATTT
4201 CAAATTATCG TGTAAACAAT ATACATGTGT CGCTCACTCA TTTATTCATG CAGTGACCAT
4261 TGGATTGTTT TTTATATTTA TCCCTTCGAA GGGAAGGTGC TTGTCCTAGA CTGTTTACAT
4321 GTTCCTCCCG AGAAGTATCA ACCATTCTTG GTTCAATTAG AAAGGTGAGC CAACATGAAA
4381 CCACATGCGT ACTTATATAA ATTAGAGTTT TTAGTGATTT AGGTTCGATA
4441 TCTACGGGGC ATGGCGGTTT TATAAGAAAC AAAAGGGACC TGTCGACGCT GCACGCTCAG
4501 ATCCTAGGAT CCCATTGATG ATACAACACC ACTATCCGGT AAGTTTTCTG AACACATTTC
4561 ATCATATAAA TAATACATAA AGCATGGCAA ATTTAGAATA ATCCGTTGCT CATTATATAG
, 4621 TGCCACAAGC AACCACCTGG ATCGGTCTAT TGTGGGTACT ATGTCTGTGA GTTTATAAGG
4681 CAGCGGGGAC GTTACGTCAA GGACAAAAAT ATGGTAAATA ATATCTATGT ATGAAGTTTT
4741 CTCATTAAAG CTGCAAAATT ATATATTGAA CATGTGTCAA TCATGCTTTT AAACTTTATT
4801 TTCAGCCGAA AAAGCAAGGA AAAGACGTGC CCTTTACACC AAAGACTCTG GAAGATATAG
4861 TAGCATACTT GTGTGGTTTT ATTATGAGAG AAATAATTTC AAGTGACAGT GCATATTTTG
4921 ATCATGAGGG CGATTTAGCA AGTGATAAAT TTAGAGTGCT GACAGACATA GCAGGTCTAA
4981 ATCTGAAGCG AAACGACATG TAAACATTGT ATGGTTGTGC GGATAACATG CATTGACGTG
5041 TATATATATA ATTTTATGGT TGATGTTTGA TTTGTTTACA ATTCTATAAT ATATATATGT
5101 GGTGTATGTA TGATGTTGTG TGTGTATATA TATATATATA TATATATATA TATATATATA
5161 TATATATATA TATATATATA TATAATGTTT AGCACTGTGT TTGGTGGGAA AAATTAAAAT
5221 TTGAAATATA TATAAAAAAT TATTTACACA GACAGTGTAG TGTGAGCTGC CTGTGTAAAA
5281 ATACATTTAT ACAGGCGGCT CACCTTGTCN HNNCAGGCGG TGCTAAAAGC ATCTTCACAG
5341 GCGGCCAAGC CCACCGCCTG TACCAGGGGT CAGTACAAAA TGGACCACAG TACAGGCGGG
5401 GCTGTGCGAG CCGCCTGTGA AAACATAATT TTCACAGGCG GCTCGCACAG CCCCGCCTGT
5461 ACTGTGGTCC ATTTTGTACT GACCCCTGGT ACAGGCGGTG GGCTTGGCCG CCTGTGAAGA
5521 TGCTTTTAGC ACCGCCTGTA AAAATGTTTT TTGTAGCAGT GTTTTTCTTA TTAGTAGTAT
5581 CTTTTATACT AATTAAGATT CAATAAAAAT TCACCATGAC ATCCCCATTG CCAAGAGAAT
5641 ATTTCGCCGC CCCTCAAAGC AGCCAAT
(SEQ ID NO: 18) or a functional fragment or variant thereof having 75, 80,
85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 18. Eac N = can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 CACTAGTACA AAAATCATTT TCGTTGGCAC GTTGTTTTTT TTTTCACAGG CAGTTCACAA
61 TATCATGGTG CTAGTAGAAA AATTTCAACG GGCCCAACAA GAGAACCGCC AGGCGGTCTT
121 CTTAATTCAA CCGCCTGTGT AAACTTTCCA TTTACATAGG CGGCTTACGA TAAAAACCGT
181 GTGTATAAAT ACCATTAACA CAGGCAGTCG AGTTACGACA ACCGCCTGTG AAATGTGTC
241 TTTTTACACA GGCGGTTTGT ATAGAGGGCC GCCTGTGCTA ATATATTTAC ACAGGCTATG
301 AGCCGCCTGT GTTAAGTCTT CTATAAATAC CCTTCGTCCA CCTCCAGACA AGAACAGTTA
361 CTCCCATGAG CTCTGCACAC TGGCGGACCA GACGATTCCA GTTTCCAAGG GGGGAGGTTT
421 TGATTTTCAT TTCTTTGGTG AGAAACTTCC AAAAGGTTAG TTAGTGCCAT TGATGCTATT
481 TTTTAAGCGA TTCTTTGGTT CAATTCTTGT ATTGGAGGTG CTCTAGATCT AGAGTTCATC
541 ATGCATTCTT GCTTAGGGTT AGAGTTCATA GGGCAAAAAG AGAGAGATTT AGCTAAATTT
601 TTATGTAAAT TCATAGTAAA TTGTAAAAAT TAAAAAAAAT AAAAAATAAA TACTTTTTAG
661 AATTCTTGTG AGTAGATCTA TACAATAGAG TAATGATGAG GATATTTTGA AGTTTATAAT
721 TTTGATTCAG TTTTAGCTTT TCTTTTTTCA GATGAATTAG ACTTTATAAA CTCAAACATT
781 AAAATGTTGA AAATCATAAA ATGGCAAATA AATACTTTTT CAAATCTTTG TGCATAAATA
841 CTTCATAGAA ATCCTTGAAT TATTCCTAAA TTTTATACAA TTGTTTCTTA TAATTATGAA
901 AATGAGTTTA AACAATTATT TAAATTCCAT AAATTGTAAC TCCGTAAGGT GTAGGTTTTC
961 ATCTCTGTTT AATAGAAGGA GGTTAGTATC TTAGTTAAGT CTGTTTTCGG GGGTTATATT
1021 AGTTTTGTTT TTAGATTGAC CTACATTAAT TGTTCTTAAC TAATTACAGC TAAATATGGA
1081 GAGGTCATTA TGGATGTACA ACTTATCAAG ATTGGACCTA TCATATGTAG TGCAGGTCCA
1141 AAAATTTATT GATGTCGCAA AGATACATGC TCGCAGAACA AAGGCGAAGC ACATATGTTG
1201 TCCATGCGCA GACTGCAAAA ATATTATGGT ATTTGACAAT GTAGAAGCAA TTACTTCCCA
1261 TCTGGTTTGA AGAGGATTTA TGGAGGACTA CTTGATTTGG ACAAAACATG GTGAGGGTAG
1321 TTTTGCACCT TATATGCGGA CAACTGACAA CACTGCAACT AACATCAATG TGGAGGGTCC
1381 AATGCCACCT CTCAATGAAT TTCATGCTAT GCCAGATGTT AATGAAACTC ATACGTCTGA
1441 TGTCAATGAA ACTCAGCATG CTAACACAGA TGTTGTTGAA GATGCAGATT TCTTAGAGGC
1501 AATAATGAAC CGTTGTGCGG ATCCATCAAT ATTCTTCATG AAGGGAATGA AAGCATTGAA
1561 GAAGGCAGCA GAGGACACTT TGTACGACGA GTCAAAAGGT TGTACCAAAC AATGGTCGAC
1621 ATTATGTGTT GTTCTTCAGT TTTTGACGAT GAAGGCTAGA CATGGTTGGT CCGATGCTAG
1681 CTTCAATGAT TTCTTGCGTG TACTTGGAGA CCTTCTTCCT AAGGAGAACA AAGTGCCTGC
1741 TAACACATAC TATGCAAAGA AGCTAGTCAG TCCACTTACG ATAGGTGTTG AGAAGATCCA
1801 CGCATGTAGA AATCATTGTA TTCTATATCG AGGTGATCAA TATAAAGACT TAGACAGTTG
1861 TCCAAACTGT GGTGCCAGTA GGTACAAGAC AAACAAAGA.T TTTCGGGAGG AAGAGAATCT
1921 AGCCTCTGTT TCTACAGGGA GGAAGCGAAA GAAGACCCAA ACAAAGACTC AACAAGACAA
1981 GCGCTCAAAG CCTAGTAGCA ATGAAGAAGT GGACTATTAT GCATTGAGAA GAGTCTCCCT
2041 ATGAGCCAAA AAAGGGGACA GCAGCAGGCA CAACTCTCTT TCTGAAAGGA CTTGGAAAGC
2101 AGCGGACGGC ACGGCTCATT GAGCTCGAAC CGTCACAGAA AAAGGAAGCC ACCGCCCAGT
2161 CAATAGAAGC CATGCCCCCA TCAAAGGAAG CCCCAAGTGG CGATGTACAT ATTGAACAGC
2221 CATCAAGTCA ACCATTGACC CTAAAGGATA TCAGAAAGCC AACGATTGAT GATTATGTCA
2281 ATGTCCCTAG TGACTATGTG CCCGGAAGGC CTATGCTCCA ATGGACGCTG CTCGATTAGA
2341 TTCAATGGCT GATAAAAAGG TTTCATGACT GGTACATGAG AGCAGTGCAT GCTAGCCTCC
2401 ATGGAATCAG AGTTGATATA CCAACAGACA TGTTTGCTAC TGGTAACAAA AAAAGCAAGA
2461 CATTTGTTAC CTTTGAGGAC ATGCACTTGT TATTGAACTA TAGGCGGCTT GACGTCCAAC
2521 TCATAACAAT CTGGTGCCTG TAAGTATCAC TCATGCACAC ACAATTATTA TATATTAATA
2581 TGTAGTGTGA AACTCTAATA TGTAGATGTT GTCTGTAGTT TGCAAGATCA CGAGCAGATG
2641 TCATTATTAT CTGCCGGATC GATGGTCGGT TATCTGAGCC CTATCAAGTT ACAAGAAAAT
2701 ATGAACAAAT TCGTATTATC AAAGGAAGAT AGAGCAAAGA TAGAGGAAGA CAAAACACCA
2761 GGATAATTAT GCCATCTATC TTGGTAGATC AATGCTGAGG GGGATTTTAT
2821 ATTGGCACCA TACAACATTA GGTAAGCTTG ACTTCATATA CGTATTTCAA ATTATCGTGT
2881 AAACAATATA CATGTGTCGC TCACTCATTT ATTCATGCAG TGACCAT GG ATTGTTTTTT
2941 ATATTTATCC CTTCGAAGGG AAGGTGCTTG TCCTAGACTC TTTACATGTT CCTCCCGAGA
3001 AG ATCAACC ATTCTTGGTT CAATTAGAAA GGTGAGCCAA CATGAAACCA CATGCGTACT
3061 TATATAAATT AGAGTTTCAA AATAACTTTA GTGATTTAGG TTCGATATCT ACGGGGCATG
3121 GCGGTTTTAT AAGAAACAAA AGGGACCTGT CGACGCTGCA CGCTCAGATC CTAGGATCCC
3181 ATTGATGATA CAACACCACT ATCCGGTAAG TTTTCTGAAC ACATTTCATC ATATAAATAA
3241 TACATAAAGC ATGGCAAATT TAGAATAATC CGTTGCTCAT TATATAGTGC CACAAGCAAC
3301 CACCTGGATC GGTCTATTGT GGGTACTATG TCTGTGAGTT TATAAGGCAG CGGGGACGTT
3361 ACGTCAAGGA CAAAAATATG GTAAATAATA TCTATGTATG AAAGTTTTCT CATTAAAGCT
3421 GCAAAATTAT ATATTGAACA TGTGTCAATC ATGCTTTTAA ACTTTATTTT CAGCCGAAAA
3481 AGCAAGGAAA AGACGTGCCC TTTACACCAA AGACTCTGGA AGATATAGTA GCATACTTGT
3541 GTGGTTTTAT TATGAGAGAA ATAATTTCAA GTGACAGTGC ATATTTTGAT CATGAGGGCG
3601 ATTTAGCAAG TGATAAATTT AGAGTGCTGA CAGACATAGC AGGTCTAAAT CTGAAGCGAA
3661 ACGACATGTA AACATTGTAT GGTTGTGCGG ATAACATGCA TTGACGTGTA TATATATAAT
3721 TTTATGGTTG ATGTTTGATT TGTTTACAAT TCTATAATAT ATATATGTGG TGTATGTATG
3781 ATGTTGTGTG TGTATATATA TATATATATA TATATATATA TATATATATA TATATATATA
3841 TATATATATA TAATGTTTAG CACTGTGTTT GGTGGGAAAA ATTAAAATTT GAAATATATA
3901 ΤΑΑΆΑΑΑΤΤΑ TTTACACAGA CAGTGTACGT GTCGAGCGTC GTCCTGTGCT ATACAAATAC
3961 ATTCTAACAG GCGGCTCGCC TTGTCCACCG GTCGGTTAAA AATACATTTC CACACNGGCC
4021 TGGCTGGGSG AGCCGCCTGT GAAAACATAA TTTTCACAGG CGGCTCGCAC AGCCCCGCCT
4081 GTACTGTGGT CCATTTTGTA CTGACCCCTG GTACAGGCGG TGGGCTTGGC CGCCTGTGAA
4141 GATGCTTTTA GCACCGCCTG TAAAAATGTT TTTTGTAGCA GTGTTT
(SEQ ID NO: 19) or a functional fragment or variant thereof having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:19.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control has the nucleic acid sequence:
1 CACTAGTACA AAAATCATTT TCGTTGGCAC Gl*TGTT1 TT TTTTCACAGG CAGTTCACAA
61 TATCATGGTG CTAGTAGAAA AATTTCAACG GGCCCAACAA GAGAACCGCC AGGCGGTCTT
121 CTTAATTCAA CCGCC GTGT AAACTTTCCA TTTACATAGG CGGCTTACGA TAAAAACCGT
181 GTGTATAAAT ACCATTAACA CAGGCAGTCG AGTTACGACA ACCGCCTGTG TAAATGTGTC
241 TTTTTACACA GGCGGTTTGT ATAGAGGGCC GCCTGTGCTA ATATATTTAC ACAGGCTATG
301 AGCCGCCTGT GTTAAGTCTT CTATAAATAC CCTTCGTCCA CCTCCAGACA AGAACAGTTA
361 CTCCCATGAG CTCTGCACAC TGGCGGACCA GACGATTCCA GTTTCCAAGG GGGGAGGTTT
421 TGATTTTCAT TTCTTTGGTG AGAAACTTCC AAAAGGTTAG TTAGTGCCAT TGATGCTATT
481 TTTTAAGCGA TTCTTTGGTT CAATTCTTGT ATTGGAGGTG CTCTAGATCT AGAGTTCATC
541 ATGCATTCTT GCTTAGGGTT AGAGTTCATA GGGCAAAAAG AGAGAGATTT AGCTAAATTT
601 TTATGTAAAT TCATAGTAAA TTGTAAAAAT TAAAAAAAAT TACTTTTTAG
661 AATTCTTGTG AGTAGATCTA TACAATAGAG TAATGATGAG GATATTTTGA AGTTTATAAT
721 TTTGATTCAG TTTTAGCTTT TCTTTTTTCA GATGAATTAG ACTTTATAAA CTCAAACATT
781 AAAATGTTGA AAATCATAAA ATGGCAAATA AATACTTTTT CAAATCTTTG TGCATAAATA
841 CTTCATAGAA ATCCTTGAAT TATTCCTAAA TTTTATACAA TTGTTTCTTA TAATTATGAA
901 AATGAGTTTA AACAATTATT TAAATTCCAT AAATTGTAAC TCCGTAAGGT GTAGGTTTTC
961 ATCTCTGTTT AATAGAAGGA GGTTAGTATC TTAGTTAAGT CTGTTTTCGG GGGTTATATT
1021 AGTTTTGTTT TTAGATTGAC CTACATTAAT TGTTCTTAAC TAATTACAGC TAAATATGGA
1081 GAGGTCATTA TGGATGTACA ACTTATCAAG ATTGGACCTA TCATATGTAG TGCAGGTCCA
1141 GATGTCGCAA AGATACATGC TCGCAGAACA AAGGCGAAGC ACATATGTTG
1201 TCCATGCGCA GACTGCAAAA ATATTATGGT ATTTGACAAT GTAGAAGCAA TTACTTCCCA
1261 TCTGGTTTGA AGAGGATTTA TGGAGGACTA CTTGATTTGG ACAAAACATG GTGAGGGTAG
1321 TTTTGCACCT TATATGCGGA CAACTGACAA CACTGCAACT AACATCAATG TGGAGGGTCC
1381 AATGCCACCT C CAATGAAT TTCATGCTAT GCCAGATGTT AATGAAACTC ATACGTCTGA
1441 TGTCAATGAA ACTCAGCATG CTAACACAGA TGTTGTTGAA GATGCAGATT TCTTAGAGGC
1501 AATAATGAAC CGTTGTGCGG ATCCATCAAT ATTCTTCATG AAGGGAATGA AAGCATTGAA
1561 GAAGGCAGCA GAGGACACTT TGTACGACGA GTCAAAAGGT TGTACCAAAC AATGGTCGAC
1621 ATTATGTGTT GTTCT CAGT TTTTGACGAT GAAGGCTAGA CATGGTTGGT CCGATGCTAG
1681 CTTCAATGAT TTCTTGCGTG TACTTGGAGA CCTTCTTCCT AAGGAGAACA AAGTGCCTGC
1741 TAACACATAC TATGCAAAGA AGCTAGTCAG TCCACTTACG ATAGGTGTTG AGAAGATCCA
1801 CGCATGTAGA AATCATTGTA TTCTATATCG AGGTGATCAA TATAAAGACT TAGACAGTTG
1861 TCCAAACTGT GGTGCCAGTA GGTACAAGAC AAACAAAGAT TTTCGGGAGG AAGAGAATCT
1921 AGCCTCTGTT TCTACAGGGA GGAAGCGAAA GAAGACCCAA ACAAAGACTC AACAAGACAA
1981 GCGCTCAAAG CCTAGTAGCA ATGAAGAAGT GGACTATTAT GCATTGAGAA GAGTCTCCCT
2041 ATGAGCCAAA AAAGGGGACA GCAGCAGGCA CAACTCTCTT TCTGAAAGGA CTTGGAAAGC
2101 AGCGGACGGC ACGGCTCATT GAGCTCGAAC CGTCACAGAA AAAGGAAGCC ACCGCCCAGT
2161 CAATAGAAGC CATGCCCCCA TCAAAGGAAG CCCCAAGTGG CGATGTACAT ATTGAACAGC
2221 CATCAAGTCA ACCATTGACC CTAAAGGATA TCAGAAAGCC AACGATTGAT GATTATGTCA
2281 ATGTCCCTAG TGACTATGTG CCCGGAAGGC CTATGCTCCA ATGGACGCTG CTCGATTAGA
2341 TTCAATGGCT GATAAAAAGG TTTCATGACT GGTACATGAG AGCAGTGCAT GCTAGCCTCC
2401 ATGGAATCAG AGTTGATATA CCAACAGACA TGTTTGCTAC TGGTAACAAA AAAAGCAAGA
2461 CATTTGTTAC CTTTGAGGAC ATGCACTTGT TATTGAACTA TAGGCGGCTT GACGTCCAAC
2521 TCATAACAAT CTGGTGCCTG TAAGTATCAC TCATGCACAC ACAATTATTA TATATTAATA
2581 TGTAGTGTGA AACTCTAATA TGTAGATGTT GTCTGTAGTT TGCAAGATCA CGAGCAGATG
2641 TCATTATTAT CTGCCGGATC GATGGTCGGT TATCTGAGCC CTATCAAGTT ACAAGAAAAT
2701 ATGAACAAAT TCGTATTATC AAAGGAAGAT AGAGCAAAGA TAGAGGAAGA CAAAACACCA
2761 GGATAATTAT GCCATCTATC TTGGTAGATC AATGCTGAGG TATAAATATA GGGATTTTAT
2821 ATTGGCACCA TACAACATTA GGTAAGCTTG ACTTCATATA CGTATTTCAA ATTATCGTGT
2881 AAACAATATA CATGTGTCGC TCACTCATTT ATTCATGCAG TGACCATTGG ATTGTTTTTT
2941 ATATTTATCC CTTCGAAGGG AAGGTGCTTG TCCTAGACTC TTTACATGTT CCTCCCGAGA
3001 AGTATCAACC ATTCTTGGTT CAATTAGAAA GGTGAGCCAA CATGAAACCA CATGCGTACT
3061 TATATAAATT AGAGTTTCAA AATAACTTTA GTGATTTAGG TTCGATATCT ACGGGGCATG
3121 GCGGTTTTAT AAGAAACAAA AGGGACCTGT CGACGCTGCA CGCTCAGATC CTAGGATCCC
3181 ATTGATGATA CAACACCACT ATCCGGTAAG TTTTCTGAAC ACATTTCATC ATATAAATAA
3241 TACATAAAGC ATGGCAAATT TAGAATAATC CGTTGCTCAT TAT TAGTGC CACAAGCAAC
3301 CACCTGGATC GGTCTATTGT GGGTACTATG TCTGTGAGTT TATAAGGCAG CGGGGACGTT
3361 ACGTCAAGGA CAAAAATATG GTAAATAATA TCTATGTATG AAGTTTTCTC ATTAAAGCTG
3421 CAAAATTATA TATTGAACAT GTGTCAATCA TGCTTTTAAA CTTTATTTTC AGCCGAAAAA
3431 GCAAGGAAAA GACGTGCCCT TTACACCAAA GACTCTGGAA GATATAGTAG CATACTTGTG
3541 TGGTTTTATT ATGAGAGAAA TAATTTCAAG TGACAGTGCA TATTTTGATC ATGAGGGCGA
3601 TTTAGCAAGT GATAAATTTA GAGTGCTGAC AGACATAGCA GGTCTAAATC TGAAGCGAAA
3661 CGACATGTAA ACATTGTATG GTTGTGCGGA TAACATGCAT TGACGTGTAT ATATATAATT
3721 TTATGGTTGA TGTTTGATTT GTTTACAATT CTATAATATA TATATGTGGT GTATGTATGA
3781 TGTTGTGTGT GTATATATAT ATATATATAT ATATATATAT ATATATATAT ATATATATAT
3841 ATATATATAT AATGTTTAGC ACTGTGTTTG GTGGGAAAAA TTAAAATTTG AAATATATAT
3901 AAAAAATTAT TTACACAGAC AGTGTAGTGT GAGCTGCCTG TGTAAAAATA CATTTATACA
3961 GGCGGCTCAC CTTGTCNNNN CAGGCGGTGC TAAAAGCATC TTCACAGGCG GCCAAGCCCA
4021 CCGCCTGTAC CAGGGGTCAG TACA7AAATGG ACCACAGTAC AGGCGGGGCT GTGCGAGCCG
4081 CCTGTGAAAA CATAATTTTC ACAGGCGGCT CGCACAGCCC CGCCTGTACT GTGGTCCATT
4141 TTGTACTGAC CCCTGGTACA GGCGGTGGGC TTGGCCGCCT GTGAAGATGC TTTTAGCACC
4201 GCCTGTAAAA ATGTTTTTTG TAGCAGTGTT T
(SEQ ID NO:20) or a functional fragment or variant thereof having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:20. Each N = can be any nucleotide or combination of any 2, 3, 4, or 5 nucleotides.
C ACTA elements have been implicated as a mechanism of movement of genes and gene fragments in sorghum (Paterson AH et al. Nature, 457(7229):551-56 (2009)). In some embodiments, a functional Mai
expression control sequence capable of placing a target gene under photoperiod (short-day) control includes the CACTA element of SEQ ID NO: 1 or a functional fragment or variant thereof. For example, in some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control includes the nucleic acid sequence:
1 CACTATAACA CAACATGGCT TTGCCGACAC TTCCAACTAT CGGCAAAGGG TACCTTTACC 61 GACACTTAAC GTCTCACGAA AGGTTTTGCC GACAATTTTC AAACAGTCGC GGTAGAAGCA 121 GTTGGCGAAA CTTTTGCCGA CAGTTAAAGG CATCGCCGAC ACATTTTCTG TAGTCAAATG 181 GCATACCTAC GCCGACAGTT GAACTTTC C CGACAGTGAA CCCTTTGCCG ACAGTTTGGA 2 1 CCTACGCCGA CAGTTTGGAC CTTTTCCGAC AGTTGGTATG TTAGCGAAAC CGTTTCTAGG 301 GTGTTTCATA AACCATGCCT TGTCCAACAG TAGAAGTGTC GGCAAAACTA TATTGCTAGG 361 ATGTAGATAC AATTTAAATA TTTTAATAAA TACACATCAC ATTGATTGAG CAAAATCACA 421 TGG
(SEQ ID NO:21) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:21.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control includes the nucleic acid sequence:
1 TTCCACCTGG GCAGGGAAAA CGGTTTATTA TGTTCCTCTT TAATTTATCT ATCGTGGCAC
61 TATAACACAA CATGGCTTTG CCGACACTTC CAACTATCGG CAAAGGGTAC CTTTGCCGAC
121 ACTTAACGTC TCACGAAAGG TTTTGCCGAC AATTTTCAAA CAGTCGCGGT AGAAGCAGTC
181 GGCGAAACTT TTGCCGACAG TTAAAGGAGG ACACATTTTC TGTAGTCAAA TGGGCATGCC
241 TCCCGCGTTG ACTTTCACCG ACAGTGAACC CTTTGCCGAC AGTTTGGACC TACGCCGACA
301 GTTTGGATCT TTTCCGACAG TTGGTATGTT AGCGAAACCG TTTCTAGGGT GTTTCATAAA
361 CCATGCCTTG TCCAACAGTA GAAGTGTCGG C ?¾>7Vi¾J\ C Ί /ΑΤ ¾ TTGCAGATAG TAGGGTGTAG
421 ATACAATTTA AATATTTTAA TAAATACACA TCACATTGAT CGAGCAAAAT CACATGGTCT
481 GTTTTCACTA AAACTGTCAT AGGTACACTC CAGTACTACC AGTACGTCGC CCGCACATAG
54 1 TGGCCAAGGA TTTTACTGCT ACTGTTGATT AACATAAGCA CTTGCGACTT TCCCTAAAAT
601 CTTTTATAAA ACAACGGCCG CAATAATATT GAACTATTTT TGTTCTAGTA CCAAAATTAG
661 AATTTGATCC CTCACCTCAT TACATCCATA G
(SEQ ID NO:22) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:22.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control includes the nucleic acid sequence:
1 TGGCACTATA ACACAACATG GCTTTGCCGA CACTTCCAAC TATCGGCAAA GGGTACCTTT
61 GCCGACACTT AACGTCTCAC GAAAGGTTTT GCCGACAATT TTCAAACAGT CGCGGTAGAA
121 GCAGTCGGCG AAACTTTTGC CGACAGTTAA AGGAGGACAC ATTTTCTGTA GTCAAATGGG
181 CATGCCTCCC GCGTTGACTT TCACCGACAG TGAACCCTTT GCCGACAGTT TGGACCTACG
24 1 CCGACAGTTT GGATCTTTTC CGACAGTTGG TATGTTAGCG AAACCGTTTC TAGGGTGTTT
301 CATAAACCAT GCCTTGTCCA ACAGTAGAAG TGTCGGCAAA ACTATATTGC AG T GTAGG
361 GTGTAGATAC AATTTAAATA ΤΤΤΤΆΑΤΑΑΑ TACACATCAC ATTGATCGAG CAAAATCACA 4 2 1 TGG
(SEQ ID NO:23) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:23.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control includes a functional CAAT box, for example the CAAT box of SEQ ID NO: 12 or a functional fragment or variant thereof. In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod control includes the nucleic acid sequence: GCCAAT (SEQ ID NO:24) or a variant thereof, for example a consensus CAAT Box sequence such as GGCCAATCT (SEQ ID NO:25). The CAAT box of a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control is typically between 50 and 250 bases upstream of the initial transcription site.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photoperiod (short-day) control includes the nucleic acid sequence;
1 TTCTTATTAG TAGTATCTTT TATACTAATT AAGATTCAAT AAAAATTCAC CATGACATCC 61 CCATTGCCAA GAGAATATTT CGCCGCCCCT CAAAGCAGCC AATAAGGCTT TACTAAAAAG
12 1 ACTATCCACG CAGTAGAGAT TTAGTCAAAA TATTCCAATA GCAATTGTTT CCTGCCTGCT
181 TGACCTTCGT CAGCCACTCA CTGTATAAAT ATCGCACCAC GCCCTTTGCA GGCTTACAGA
241 GCTTGTATTA CGTACTAACA AGGCACACAC AGTACCCTGT GTTCACCGGC CCTGCACAAA
301 ACTCAAGCAG TTATTACTAA C
(SEQ ID NO:26) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:26.
A polynucleotide having a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a fragment of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 is also disclosed. The Mai gene in the day- neutral S. bicolor has a recessive (loss of function) Mai allele characterized by one or more mutations or deletions in the 5'UTR relative to the 5'UTR of S. propinquum that results in loss of photoperiod sensitivity. Therefore, the nucleic acids in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 can be present in short-day expression control sequences. Therefore, in
some embodiments, the photoperiod sensitive Mai expression control sequence has 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 or more of the nucleic acids in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and is capable of inducing short-day expression of a target gene.
2. Photoperiod Insensitivity
The expression control sequence of the Mai gene in the day-neutral S. bicolor having a recessive (functional) Mai allele can be used to induce photoperiod insensitivity of other plant genes. Accordingly, the Mai expression control sequences from S. bicolor can be operably linked to a plant gene coding sequence to impart photo-insensitive (i.e., day-neutral) control over the plant gene coding sequence.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photo-insensitive (day neutral) control has the nucleic acid sequence:
1 AAAAGAAAAG TGAGCACACC ACGACCTATC ATCAGCTCAT GGTCAGCTCT ACAAACTTAT
61 AGATTGCATC GAGATCTAAG ACTCAGGTAC AAATCATGTC ¾¾c ϋ τ C T¾¾ X GGTTTAGAAA
121 ATGAAAAAAG T T T Gj¾G Ί1 T Ϊ" CAAAATATGA TACTTGAAAT TAACATTTGA ACTTTTTAGC
181 AAGATCTGAA AATAAAAAAT TCAACTAAAA AATTTATAGA TCATGTTAAC ATTGATATAA
241 TCGCTTCCAA TCGCCTCCCA TCGCTTCAGC TAGAAAACTT TTTTTCTCGA TTTAATTAAT
301 TAACGTCATT GTACAAGATT CTTTCAAACC CCAACCCCTA TCATCGACGG
361 TGAGGGCTCC TATAATATGC ACTAGTGGAC GCCGGGTGGG TGGAACCTAA GAAGATTTTA
421 AAAAAAAAAT TAAGAAGAAG ATTTTTATCT AACTAACTAT ATATAGIACT TATATCATAC
481 ACTATACTAT TCAAAATATT ATTTTCACAA TTATGAATTT ACCCTTTTAC TCTTTATTAA
54 1 AAAAATATGA ATAAAGAATT ATCACGCCTC TATTTAGGGT CCTAATCCCC ATAATTTAAG
601 AGGCGATGAG AGGCGATGTG ACATCTATGG CCCACCGACC AAAGACACAA CTATCGCCTC
661 CCATCACCTT GCTTCTATCG CCTCTCATAG CTTTTCATAT TCTAGGTCCA CCGGCCATAG
721 ACACACCAAT CGCTTATCAT CGCCTTTTCC AACCATTGTA AAAATATTCA TAATTTTGAT
781 ATAAAATTTG TCTTCACTTG AGTATGGGAA AAAAATTATA CATAATGTTT TCGTGTGAGA
841 ATTTACAGGA ATGAACCCTT AAGATGTCCA AATGTAAATG ACCCTATTTA TTAAGAGGAG
901 CGGATCTATA GGCCTGGCTC TGAAAATGGA TTATGGATTG GAGATACTAA ATTTAAGGGC
961 CTATCTTCGC ACATAACATC TATAGTTCCT AAATAATTTT TTAtTGTAGT AGTAGAACTT
1021 TTCTCCCTGT AAACCATAAA CCAAGTTGAC GCTGGGCTTT ATTTTGCGAC ACAGAACACC
1081 AAATTGG£GG CTATGAACTC TTCCACCTGG GCAGGGAAAA CGGTTTATTA TGTTCCTCTT
1141 TAATTTATCT ATCGTGGTCT GTTTTCACTA AAACTGTCAT ATTGCTACAC TCCAGTACTA
1201 CCAGTACGTC GCCCGCACAT AGTGGCCAAG GATTTTACTG CTACTGTTGA TTAACATAAG
1261 CACTTGCGAC TTTCCCTAAC ATCTTTTATA AAACAACGGC CGCAATAATA TTGAACTGTT
1321 TTTTTCTAGT ■¾ C C fi>¾ ¾i¾ i P ϋ¾ GAATTTGATC CCTCACCTCA TTACATCCAT AGTAACATGA
1381 C C J GJ&T ϊ v¾ ATGGACAGGC CGGGATCACT CGCCAGCAGA TACCCTGAGC GATTCATAAC
1441 CAGAATTTTT AATTTTTTCT AGTGAAG GG GGTTCTCCTA GTCCT TAAC ATTCAAAATT
1501 TAGTACAAAC TTTCCTTAGT AAATGTCTTC TAGTASAGAT TTCCTAGTGT TTTGATTTGG
1561 TAGTGTTTTA TTACTAATTA AAAATATTAG AAGAACTCCA TCATTTTGGT AGTGATTGGT
1621 TGTTTGGATT AGTCTTCTCA CGTTAGACCT ATATATGCAG GACAACTCAA GCCAGCATAA
1681 ATATATGAAA TATCTTGGTG TTTGTTTGTC TGACACAGGC AACCGTGTTT GGTATAAATG
1741 TGTTTTCTTG TTTACGTTTT ACCATCTATA GTCATCTC&A TGTTTATATA GTAGAGACTT
1801 CATGTTTGTA GTAGATAAGG TAGAGAATTG AGAATATTTT ATTTTTGTGC GACCATCAAT
1861 TTTATGTAAT CTGCATTGTC TAATGCTTTA TTTGACATTT GAAACTACTT AATTTGACCG
1921 TTATGCAGGT CCGCATGATC CTATGAAAGC AATTAATTAG TACGGGTACT GCACTACACA
1981 AGTTTGCTAG TACTATTCTA TTAACCGACC TGTCAATATT SCCTTAAGTT ACTGATTTCA
2041 ATTAGAATCT AACACATTCA GGAAAAGAAG TTTTCCTTAT TAGTAGTAAC TTTTTATACT
2101 AATTAAGATT CAATAAAAAT TCACCATGAC ATCCCCATTG CCAAGAGAAT ATTTCGCCGC
2161 CCCTCAAAGC AGCCAAGGCT TTACTAAAAA GACTATCCAC GCAGTAGAGA TTTAGTCAAA
2221 ATATTCCAAT AGCAATTGTT TTCTGCCTGC TTGACCTTCG TCAGCCACTC ACTGTATAAA
2281 TATCGCACCA CGCCCTTTGC AGGCTTACAG AGCTTGTACT ACGTACTAAC AAGGCACACA
2341 CAATACCCTG TGTTCACCGG CCCTGCACAA AACTCAAGCA GTTATTACTA AC
(SEQ ID NO:27) or a functional fragment or variant thereof having 75, 80,
85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:27.
In some embodiments, a functional Mai expression control sequence capable of placing a target gene under photo-insensitive (day neutral) control has the nucleic acid sequence:
1 TCCTTATTAG TAGTAACTTT TTATACTAAT TAAGATTCAA TAAAAATTCA CCATGACATC
61 CCCATTGCCA AGAGAATATT TCGCCGCCCC TCAAAGCAGC CAAGGCTTTA CTAAAAAGAC 121 TATCCACGCA GTAGAGATTT AGTCAAAATA TTCCAATAGC AATTGTTTTC TGCCTGCTTG
181 ACCTTCGTCA GCCACTCACT GTATAAA T CGCACCACGC CCTTTGCAGG CTTACAGAGC
24 1 TTGTACTACG TACTAACAAG GCACACACAA TACCCTGTGT TCACCGGCCC TGCACAAAAC
301 TCAAGCAGTT ATTACTAAC
(SEQ ID NO:35) or a functional fragment or variant thereof having 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:35.
A polynucleotide having a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a fragment of SEQ ID NO:27 or 35 is also disclosed. The Mai gene in the day-neutral S. bicolor has a recessive (loss of function) Mai allele characterized by one or more mutations or deletions in the 5'UTR relative to the 5'UTR of S. propinquum that results in loss of photoperiod sensitivity. Therefore, in some embodiments, the photo- insensitive Mai expression control sequence has 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 or more of the nucleic acids in SEQ ID NO: 27 or 35, and is capable of controlling day-neutral expression of the target gene.
III. Methods of Modulating Photoperiod Sensitivity
Methods of modulating photoperiod sensitivity and flowering time in sorghum are disclosed. The methods can be used, for example, to increase high biomass production, by extending the growing period.
Methods are also disclosed for modulating photoperiod sensitivity involving operably linking the expression control sequence of a Mai gene from a photoperiod sensitive Sorghum variety or cultivar to the endogenous maturity gene in the plant. Methods are disclosed for imposing photoperiod sensitivity on other genes that are not normally controlled by photoperiod by operably linking the expression control sequence of a Mai gene from a photoperiod sensitive Sorghum variety or cultivar to the endogenous gene in the plant. Similarly, methods are also disclosed for imposing photoperiod s insensitivity on other genes that are normally controlled by photoperiod by operably linking the expression control sequence of a Mai gene from a photoperiod insensitive Sorghum variety or cultivar to the endogenous gene in the plant.
The disclosed method can involve modulating the expression or activity of a Mai gene in a plant. Activities of a gene include transcriptional activation of the gene and activities of the resulting encoded protein. The method can involve modulating the activity of a protein encoded by the Maturity gene. Activities of a protein include, for example, transcription, translation, intracellular translocation, secretion, phosphorylation by kinases, cleavage by proteases, homophilic and heterophilic binding to other proteins, ubiquitination.
In some embodiments, the method involves increasing photoperiod sensitivity in a plant. For example, in some embodiments, the method involves introducing to a plant a nucleic acid sequence that promotes photoperiod dependent expression of a functional Mai maturity gene. As a result of this method, the transgenic plant preferably has higher photoperiod sensitivity to flowering compared to control (e.g., wild-type) plant of the same species.
In some embodiments, the method involves inhibiting photoperiod sensitivity in a plant. In some embodiments, the method involves engineering
a transgenic plant to express the Mai under the control of photoperiod insensitive control sequence of Mai. As a result of this method, the transgenic plant preferably has reduced photoperiod sensitivity to flowering compared to control (e.g., wild-type) plant of the same species.
In some embodiments, the method involves engineering a transgenic plant to inhibit gene expression of the Mai gene or translation of the Mai protein. In other embodiments, the method involves introducing to the plant a composition that silences gene expression. For example, the composition can include an antisense, RNAi, dsR A, miRNA, or siRNA that targets the maturity gene in the plant and inhibits translation of the encoded protein. In still other embodiments, the method involves introducing to the plant a composition that binds to the protein encoded by the maturity gene and inhibits one or more of the protein's activities.
In some embodiments, the method involves introducing to the plant or plant cell a nucleic acid sequence that silences expression of the maturity gene in the plant. Preferably, the nucleic acid is operably linked to an expression control sequence. The expression control sequence can be a heterologous control sequence. Selection of this control sequence can be used to select the amount of gene-silencing nucleic acid expressed and therefore control photoperiod sensitivity in the plant. As a result of this method, the transgenic plant preferably has lower photoperiod sensitivity compared to control (e.g., wild-type) plant of the same species. In some embodiments, the nucleic acid can silence a polynucleotide having the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35 or a nucleic acid encoding the polypeptide of SEQ ID NO: 8 or 34, for fragments or variants thereof.
In some embodiments, photoperiod sensitivity can be modulated by elements within the nucleic acid sequence. For instance, as discussed above, wild type short day flowering sorghum contains at least four additional non- coding segments not found in day-neutral sorghum: a segment of about 400 base pairs in the 5' UTR, a segment of about 4.2 kb in the 5' UTR, a segment
of 3 base pairs in the 5' UTR, and a segment of 27 base pairs in the second intron of the coding sequence.
Methods of interfering with the non-coding segments can be used to modulate the photoperiod sensitivity of short day plants. Deleting or altering some or all of the non-coding segments or inserting additional nucleotides into the non-coding segments can be effective. Deleting, mutating, or inserting nucleotides in one or more of the Mai expression control sequences disclosed herein can decrease the photoperiod sensitivity of a gene or polynucleotide of interest. Therefore, in some embodiments deleting or mutating nucleotides in one or more of these regions of the Mai expression control sequence with shift the plant from short-day flowering to day-neutral flowering. For example, in some embodiments insertions, mutations, or deletions are introduced into a polynucleotide having SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 35 or a functional fragment, variant, or complement thereof to reduce the photoperiod sensitivity of the expression control sequence. In a preferred embodiment, mutations or deletions are introduced into a CAAT box, for example a polynucleotide having the sequence of SEQ ID NO: 23, 24, or 25 or a functional fragment, variant, or complement thereof. The insertions, mutations or deletions can shift the plant from short-day flowering to day- neutral flowering, or make the plant less photoperiod sensitive.
Inhibiting the regulatory function of the non-coding segments can also be used to modulate photoperiod sensitivity. For instance, inhibiting or preventing the interaction of one or more of the non-coding segments with another nucleic acid sequence or protein.
The additional nucleotides can be dependent or independent on a functional copy of the flowering gene. In some forms, one or more of the non-coding segments is insufficient to produce the short day trait alone. However, the combination of one or more of the non-coding segments and a functional copy of the flowering gene can result in a short day flowering plant. The non-coding segments can interact with the gene it resides within. The interaction can be non-linear. This interaction can be based on one or
more of the non-coding segments containing a gene regulatory feature that confers the short day sensing mechanism.
In some embodiments, the photoperiod sensitivity of expression control sequences disclosed herein is increased. Deleting, mutating, or inserting nucleotides in one or more of these regions of the Mai expression control sequences disclosed herein can increase the photoperiod sensitivity of a gene or polynucleotide of interest. For example, in some embodiments deleting, mutating, or inserting nucleotides in one or more of these regions of the Mai expression control sequence with shift the plant from day-neutral flowering to short-day flowering. For example, in some embodiments insertions, mutations or deletions are introduced into a polynucleotide having SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 29, 30, 31, 32, 33, 35 or a functional fragment, variant, or complement thereof to increase the photoperiod sensitivity of the control sequence. In a preferred embodiment, an insertion includes multiple copies of a C AAT box, for example a polynucleotide having the sequence of SEQ ID NO: 23, 24, or 25 or a functional fragment, variant, or complement thereof. In some embodiments the additional CAAT boxes, include, but not limited to one or more copies of SEQ ID NO: 23, 24, or 25. The inserted sequences can be added sequentially to the promoter region of the gene or polynucleotide of interest. For example, in some embodiments, one or more CAAT boxes are added beginning between about 50 and 250 nucleotides upstream of the "ATG" start site of a plant gene such as Mai . The insertions, mutations or deletions can shift the plant from day-neutral flowering to short-day flowering plants, or increase the photoperiod sensitivity of the plant.
In some embodiments, photoperiod sensitivity can be modulated by using the Mai control sequences of S. bicolor. For example, in some embodiments, the control sequences of S. bicolor, including by not limited to SEQ ID NO:27 or 35, are inserted upstream of a coding sequence of a gene of interest and cause photoperiod insensitive, or day neutral expression of the gene of interest. In some embodiments the gene of interest is Mai .
Methods of modifying the photoperiod sensitivity of Mai by replacing or supplementing the endogenous control sequences of Mai with
heterologous control sequences are also disclosed. The expression control sequences of Mai can be altered or replaced with an expression control sequence that reduces photosensitivity, but wherein expression of Mai is still photoperiod sensitive relative to Mai expression in S, bicolor. The expression control sequences of Mai can also be altered or replaced with an expression control sequence that increases photosensitivity of Mai expression relative to Mai expression in S. propinquum. For example, in some embodiments, the expression control sequence of Mai is replaced with an expression control sequence from another photoperiod sensitive gene. Cis-regulatory elements in the promoter of photoperiod-responsive genes, coordinated motifs integrating hormones and stresses to photoperiod responses, and photo-responsive genes and their promoters are known in art, and can be used to alter the photosensitivity Mai , see for example,
Mongkolsiriwatana C, i¾ et tJ (Nat. Set) 43: 164-177 (2009).
A. Recombinant Plant Gene Expression
Compositions and methods are therefore provided for operably linking plant genes to a Mai expression control sequence. Therefore, methods of imposing photoperiod sensitivity or insensitivity on a plant process are disclosed. The methods can involve producing a recombinant nucleic acid molecule that contains a plant gene responsible for the plant process operably linked to an Mai expression control sequence, for example a polynucleotide having the sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or a functional fragment or variant thereof. The plant process can be naturally photoperiod sensitive, or photoperiod insensitive. In some embodiments a photoperiod sensitive control sequence of Mai, for example SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or a functional fragment or variant thereof is operably linked to a plant gene to impart photoperiod sensitive control over the gene. In some embodiments a photoperiod insensitive control sequence of Mai, for example SEQ ID NO: 27, or a functional fragment or variant thereof is operably linked to a plant gene or coding sequence thereof to impart photoperiod sensitive control over the polypeptide encoded by the gene.
A transgenic plant or transgenic plant cell is also disclosed that has a photoperiod sensitive or msensitive plant process. These plants can contain a plant gene controlling the plant process that is operably linked to a Mai expression control sequence, for example a polynucleotide having the sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or a functional fragment or variant thereof, as described above.
Nucleic acid vectors are also disclosed that include the Mai expression control sequence, for example a polynucleotide having the sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 6, or a functional fragment or variant thereof. In some embodiments, the vectors also include an insertion site, such as a multiple cloning site, for insertion of a plant gene of interest. The insertion site can include, for example, one or more restriction enzyme digestion sites for operably linking a gene to the expression control sequence.
Methods of modifying a plant gene to be under photoperiod control are also disclosed. The method generally involves operably linking the plant gene to a functional Mai expression control sequence. The Mai sequence can in some embodiments be from any Sorghum plant variety or cultivar that is photoperiod sensitive. Likewise, the optimum conditions for photoperiod selectivity can be selected for the plant gene by selecting a Mai expression control sequence from a Sorghum variety or cultivar that flowers under the desired photoperiod conditions. Therefore, Sorghum varieties having undesirable photoperiod sensitivity can be optimized by modifying or replacing the expression control sequence of the endogenous Mai gene according to the disclosed method.
As an example, SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 contain Mai expression control sequences from a short-day cultivar of S. propinqu m, i.e., flowers when the days are short. This expression control sequence can in some embodiments be used to impose short-day photoperiodic control on other valuable plant processes.
B. Constructs and Vectors
1. Recombinant Expression of Mai
Vectors and constructs containing a Mai gene, or coding sequence, operably linked to an endogenous or heterologous expression control sequence are also disclosed. The constructs can include an expression cassette containing an Mai gene or a Mai coding, for example SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, or a nucleic acid encoding the amino acid sequence of SEQ ID NO:8 or 34. The expression sequences can be used to cause flowering in plants as described in more detail below.
2. Genes of Interest
Methods of modifying a plant gene, polynucleotide, or coding sequence to be photoperiod sensitive or insensitive are also disclosed. The method generally involves operably linking the polynucleotide to a Mai photoperiod sensitive or insensitive expression control sequence to polynucleotide or interest. The polynucleotide of interest can be a coding sequence for example a sequence encoding a polypeptide (with or without introns), or non-coding sequence such as an antisense or inhibitory nucleic acid. In some embodiments the polynucleotide includes a cDNA of a polypeptide of interest.
Plant genes and coding sequences that can be engineered to be photoperiod sensitive or insensitive are known in the art, and including, but are not limited to, those gene and coding sequences that influence traits such as germination, flowering, ripening, senescence, and combinations thereof. For example, in some embodiments it is desirable to make more or less photoperiod sensitive, genes or coding sequences that regulate or contribute to remobilization of plant constituents from vegetative tissues to harvested organs; to underground parts such as roots; rhizomes to sustain future regrowth; or combinations thereof.
3. Antisense
Mai antisense oligonucleotides are also disclosed. Mai antisense oligonucleotides can be used to delay, inhibit, or prevent expression of Mai in plants. Antisense molecules are designed to interact with a target nucleic
acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule, for example Mai coding sequences including, but not limited to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 35 or a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8 or 34. Antisense molecules are known in the art include, but are not limited to, RNA interference (RNAi) and siRNA. Methods of designing antisense molecules directed to a target sequence, for example SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 35 or a nucleic acid encoding the amino acid sequence of SEQ ID NO:8 or 34 are well also well known in the art. See for example, Elbashir, et al, Methods, 26:199-213 (2002).
The production of siRN A from a vector is more commonly done through the transcription of a short hairpin RNAs (shRNAs). Accordingly, vectors and constructs containing a nucleic acid sequence that silences Mai gene expression (e.g., siRNA, RNAi, shRNA) operably linked to a heterologous" expression control sequence are also disclosed.
4. Transformation Constructs
Transformation constructs can be engineered such that transformation of the nuclear genome and expression of transgenes from the nuclear genome occurs. Alternatively, transformation constructs can be engineered such that transformation of the plastid genome and expression of the plastid genome occurs.
An exemplary construct contains a nucleic acid sequence containing an Mai gene operatively linked in the 5' to 3' direction to a promoter that directs transcription of the nucleic acid sequence, and a 3' polyadenylation signal sequence. Typically, the construct will increase the amount of Mai in the plant by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent.
Another exemplary construct contains a nucleic acid sequence that silences Mai gene expression operatively linked in the 5' to 3' direction to a promoter that directs transcription of the nucleic acid sequence, and a 3' polyadenylation signal sequence. Typically, the transcribed nucleic acid sequence can result in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent inhibition of the Mai gene.
Another exemplary construct contains a nucleic acid sequence containing a polynucleotide of interest operatively linked in the 5' to 3' direction to a Mai expression control sequence that directs transcription of the polynucleotide, and a 3' polyadenylation signal sequence. The Mai expression control sequence can impart photoperiod sensitivity or photoperiod insensitivity to the polynucleotide of interest.
Generally, nucleic acid sequences containing an Mai gene, a Mai coding sequence, or a nucleic acid sequence that silences an Mai gene, are first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also include any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. In some embodiments the expression cassettes includes a Mai expression control sequence discussed above. These expression cassettes can then be easily transferred to the plant transformation vectors. Representative plant transformation vectors are described in plant transformation vector options available (Gene Transfer to Plants (1995), Potrykus, I. and Spangenberg, G. eds. Springer-Verlag Berlin Heidelberg New York; "Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins" (1996), Owen, M.R.L. and Pen, J. eds. John Wiley & Sons Ltd. England and Methods in Plant Molecular biology-a laboratory course manual (1995), Maliga, P., lessig, D. F., Cashmore, A. R.; Gruissem, W. and Varner, J. E. eds. Cold Spring
Laboratory Press, New York).
An additional approach is to use a vector to specifically transform the plant plastid chromosome by homologous recombination (U.S. Pat. No. 5,545,818 to McBride, et al.), in which case it is possible to take advantage of the prokaryotic nature of the plastid genome and insert a number of transgenes as an operon.
The following is a description of various components of typical expression cassettes.
1. Promoters
Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles, for all of which methods are known to those skilled in the art (Gasser & Fraley, Science 244:1293-99 (1989)). In a preferred embodiment, promoters are selected from those of plant or prokaryotic origin that are known to yield high expression in piastids. In certain embodiments the promoters are inducible. Inducible plant promoters are known in the art.
The transgenes can be inserted into an existing transcription unit (such as, but not limited to, psbA) to generate an operon. However, other insertion sites can be used to add additional expression units as well, such as existing transcription units and existing operons {e.g., atpE, accD). Such methods are described in, for example, U.S. Pat. App. Pub. 2004/0137631, which is incorporated herein by reference in its entirety. For an overview of other insertion sites used for integration of transgenes into the tobacco plastome, see Staub (Staub, J.M., "Expression of Recombinant Proteins via the Plastid Genome," in: Vinci VA, Parekh SR (eds.) Handbook of Industrial Cell Culture: Mammalian, and Plant Cells, pp. 259-278, Humana Press Inc., Totowa, NJ (2002)).
In general, the promoter can be from any class I, II or III gene. For example, any of the following plastidial promoters and/or transcription regulation elements can be used for expression in piastids. Sequences can be derived from the same species as that used for transformation. Alternatively, sequences can be derived from other species to decrease homology and to prevent homologous recombination with endogenous sequences.
For instance, the following plastidial promoters can be used for expression in plastids.
PrbcL promoter (Allison LA, Simon LD, Maliga P, EMBO J.
15:2802-2809 (1996); Shiina T, Allison L, Maliga Ps Plant Cell 10:1713- 1722 (1998));
PpsbA promoter (Agrawal GK, Kato H, Asayama M, Shirai M, Nucleic Acids Research 29:1835-1843 (2001));
Prrn 16 promoter (Svab Z, Maliga P, Proc. Natl Acad. Sci. USA 90:913-917 (1993); Allison LA, Simon LD, Maliga P, EMBO J. 15:2802- 2809 (1996));
PaccD promoter (Hajdukiewicz PTJ, Allison LA, Maliga P, EMBO J 16:4041-4048 (1997); WO 97/06250);
PclpP promoter (Hajdukiewicz PTJ, Allison LA, Maliga P, EMBO J. 16:4041-4048 (1997); WO 99/46394);
PatpB, Patpl, PpsbB promoters (Hajdukiewicz PTJ, Allison LA,
Maliga P, EMBO J. 16:4041-4048 (1997));
PrpoB promoter (Liere K, Maliga P, EMBO J. 18:249-257 (1999));
PatpB/E promoter ( apoor S, Suzuki JY, Sugiura M, Plant J. 11:327- 337 (1997)).
In addition, prokaryotic promoters (such as those from, e.g., E. coli or
Synechocystis) or synthetic promoters can also be used.
Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters known in the art may be used. For example, for constitutive expression, the CaMV 35S promoter, the rice actin promoter, or the ubiquitin promoter may be used. For example, for regulatable expression, the chemically inducible PR-1 promoter from tobacco or Arabidopsis may be used (see, e.g., U.S. Pat. No. 5,689,044 to Ryals, et al).
A suitable category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites. Preferred promoters of this kind include those described by Stanford et al. Mol Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989), Rohrmeier &
Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Moke. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).
Suitable tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis, and many of these have been cloned from both
monocotyledons and dicotyledons. A suitable promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)). A suitable promoter for root specific expression is that described by de Framond FEBS 290: 103-106 (1991); EP 0 452 269 to de Framond and a root-specific promoter is that from the T-l gene. A suitable stem specific promoter is that described in U.S. Pat. No. 5,625,136 and which drives expression of the maize trpA gene.
The promoter can be a relatively weak plant expressible promoter. Thus, the promoter can in some embodiments initiate and control
transcription of the operably linked nucleic acids about 10 to about 100 times less efficient that an optimal CaMV35S promoter. Relatively weak plant expressible promoters include the promoters or promoter regions from the opine synthase genes of Agrobacterium spp. such as the promoter or promoter region of the nopaline synthase, the promoter or promoter region of the octopine synthase, the promoter or promoter region of the mannopine synthase, the promoter or promoter region of the agropine synthase and any plant expressible promoter with comparably activity in transcription initation. Other relatively weak plant expressible promoters may be dehiscence zone selective promoters, or promoters expressed predominantly or selectively in dehiscence zone and/or valve margins of fruits, such as the promoters described in W097/13865.
Cis-regulatory elements from the promoter of photoperiod-responsive genes, coordinated motifs integrating hormones and stresses to photoperiod responses, and the promoters of photo-responsive genes such as those described in Mongkolsiriwatana C, Katsetsart J. (Nat. ScL) 43: 164-177 (2009), can also be used.
2. Transcriptional Terminators
A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of
transcription beyond the transgene and its correct polyadenylation.
Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopahne synthase terminator and the pea rbcS E9 terminator. These are used in both monocotyledonous and dicotyledonous plants.
At the extreme 3' end of the transcript, a polyadenylation signal can be engineered. A polyadenylation signal refers to any sequence that can result in polyadenylation of the mRNA in the nucleus prior to export of the mRNA to the cytosol, such as the 3' region of nopahne synthase (Bevan, ML, et al, Nucleic Acids Res. , 11, 369-385 (1983)).
3. Sequences for Expression Enhancement or
Regulation
Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes to increase their expression in transgenic plants. For example, various intron sequences such as introns of the maize Adhl gene have been shown to enhance expression, particularly in
monocotyledonous cells. In addition, a number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
4. Coding Sequence Optimization
The coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression (also referred to herein as "codon optimized") in the crop species of interest. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (see, e.g. Perlak et al., Proc, Natl. Acad. Set USA 88: 3324 (1991); and Koziel et al, Biotechnol. 11 : 194 (1993)). Therefore, in some embodiments, the disclosed nucleic acids sequences, or fragments or variants thereof, are genetically engineered for optimal expression in the crop species of interest.
5. Selectable Markers
Genetic constructs may encode a selectable marker to enable selection of plastid transformation events. There are many methods that have been described for the selection of transformed plants [for review see (Miki et al. , Journal of Biotechnology, 2004, 107, 193-232) and references incorporated within]. Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptll (U.S. Patent Nos. 5,034,322, U.S. 5,530,196), hygromycin resistance gene (U.S. Patent No. 5,668,298), the bar gene encoding resistance to
phosphinothricin (U.S. Patent No. 5,276,268), the expression of
aminoglycoside 3 "-adeny transferase (aadA) to confer spectinomycin resistance (U.S. Patent No. 5,073,675), the use of inhibition resistant 5- enolpyruvyl-3-phosphoshikimate synthetase (U.S. Patent No. 4,535,060) and methods for producing glyphosate tolerant plants (U.S. Patent No. 5,463,175; U.S. Patent No. 7,045,684). Methods of plant selection that do not use antibiotics or herbicides as a selective agent have been previously described and include expression of glucosamine-6-phosphate deaminase to inactive glucosamine in plant selection medium (U.S. Pat. No. 6,444,878) and a positive/negative system that utilizes D-amino acids (Erikson et al, Nat Biotechnol, 2004, 22, 455-8). European Patent Publication No. EP 0 530 129 A 1 describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. U.S. Patent No. 5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants. Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293). Screenable marker genes include the beta- glucuronidase gene (Jefferson et al., 1987, EMBO J. 6: 3901-3907; U.S. Patent No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Set 20: 448- 455; Pan et al, 1996, Plant Physiol 112: 893-900).
Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the
nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Mate et al. (1999), Nat
Biotechnol 17: 969-73). An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein. Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al. (2002), Nat Biotech 20: 87-90), the blue fluorescent protein, the cyan fluorescent protein, and the green fluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis and Vierstra (1998), Plant Molecular Biology 36: 521-528). A summary of fluorescent proteins can be found in Tzfira et al. (Tzfira et al. (2005), Plant Molecular Biology 57: 503-516) and Verkhusha and Lukyanov (Verkhusha, V. V. and . A. Lukyanov (2004),Nat Biotech 22: 289-296) whose references are incorporated in entirety. Improved versions of many of the fluorescent proteins have been made for various applications. Use of the improved versions of these proteins or the use of combinations of these proteins for selection of transformants will be obvious to those skilled in the art. It is also practical to simply analyze progeny from transformation events for the presence of the PHB thereby avoiding the use of any selectable marker.
For plastid transformation constructs, a preferred selectable marker is the spectinomycin-resistant allele of the plastid 16S ribosomal RNA gene (Staub JM, Maliga P, Plant Cell 4: 39-45 (1992); Svab Z, Hajdukiewicz P, Maliga P, Proc. Natl. Acad. Set USA 87: 8526-8530 (1990)). Selectable markers that have.. since been. successfully used in plastid transformation include the bacterial aadA gene that encodes aminoglycoside 3'- adenyltransferase (AadA) conferring spectinomycin and streptomycin resistance (Svab et al, Proc. Natl Acad. Set USA, 1993, 90, 913-917), nptll that encodes aminoglycoside phosphotransferase for selection on kanamycin (Carrer H, Hockenberry TN, Svab Z, Maliga P., Mol. Gen. Genet. 241 : 49-56 (1993); Lutz KA, et al, Plant J. 37: 906-913 (2004); Lutz KA, et al, Plant Physiol. 145: 1201-1210 (2007)), aphA6, another aminoglycoside phosphotransferase (Huang F-C, et al, Mol. Genet. Genomics 268: 19-27 (2002)), and chloramphenicol acetyltransferase (Li, W., et al. (2010), Plant
Mol Biol, DOI 10.1007/s 11103-010-9678-4). Another selection scheme has been reported that uses a chimeric betaine aldehyde dehydrogenase gene (BADH) capable of converting toxic betaine aldehyde to nontoxic glycine betaine (Daniell H, et al., Curr. Genet. 39: 109-116 (2001)).
5. Targeting Sequences
The disclosed vectors and constructs may further include, within the region that encodes the protein to be expressed, one or more nucleotide sequences encoding a targeting sequence. A "targeting" sequence is a nucleotide sequence that encodes an amino acid sequence or motif that directs the encoded protein to a particular cellular compartment, resulting in localization or compartmentalization of the protein. Presence of a targeting amino acid sequence in a protein typically results in translocation of all or part of the targeted protein across an organelle membrane and into the organelle interior. Alternatively, the targeting peptide may direct the targeted protein to remain embedded in the organelle membrane. The
"targeting" sequence or region of a targeted protein may contain a string of contiguous amino acids or a group of noncontiguous amino acids. The targeting sequence can be selected to direct the targeted protein to a plant organelle such as a nucleus, a microbody (e.g., a peroxisome, or a specialized version thereof, such as a glyoxysome) an endoplasmic reticulum, an endosome, a vacuole, a plasma membrane, a cell wall, a mitochondria, a chloroplast or a plastid. A chloroplast targeting sequence is any peptide sequence that can target a protein to the chloroplasts or plastids, such as the transit peptide of the small subunit of the alfalfa ribulose-biphosphate carboxylase (Khoudi, et al, Gene, 197:343-351 (1997)). A peroxisomal targeting sequence refers to any peptide sequence, either N-terminal, internal, or C-terminal, that can target a protein to the peroxisomes, such as the plant C-terminal targeting tripeptide SKL (Banjoko, A. & Trelease, R. N. Plant Physiol, 107:1201-1208 (1995); T. P. Wallace et al, "Plant
Organellular Targeting Sequences," in Plant Molecular Biology, Ed. R. Croy, BIOS Scientific Publishers Limited (1993) pp. 287-288, and peroxisomal targeting in plant is shown in M. Volokita, The Plant J., 361- 366 (1991)).
Plastid targeting sequences are known in the art and include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. Plant Mol. Biol. 30:769-780 (1996); Schnell et al. J. Biol. Chem. 266(5):3335~3342 (1991)); 5-(enolpyruvyl)shikimate-3- phosphate synthase (EPSPS) (Archer et al. J. Bioenerg. Biomemb.
22(6):789-810 (1990)); tryptophan synthase (Zhao et al. J. Biol. Chem. 270(l l):6081-6087 (1995)); plastocyanin (Lawrence et al. J. Biol. Chem. 272(33):20357-20363 (1997)); chorismate synthase (Schmidt et al. J. Biol. Chem. 268(36):27447-27457 (1993)); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. J. Biol. Chem. 263: 14996-14999 (1988)). See also Von Heijne et al. Plant Mol. Biol. Rep. 9:104-126 (1991); Clark et al. J. Biol. Chem. 264:17544-17550 (1989); Della-Cioppa et al Plant Physiol 84:965-968 (1987); Romer et al. Biochem. Biophys. Res. Commun. 196:1414-1421 (1993); and.Shah et al. Science 233:478-481 (1986). Alternative plastid targeting signals have also been described in the following: US 2008/0263728; Miras, S. et al. (2002), J Biol Chem 277(49): 47770-8; Miras, S. et al. (2007), J Biol Chem 282: 29482-29492.
6. Plants and Tissues for Transfection Both dicotyledons ("dicots") and monocotyledons ("monocots") can be used in the disclosed positive selection system. Monocot seedlings typically have one cotyledon (seed-leaf), in contrast to the two cotyledons typical of dicots. Eudicots are dicots whose pollen has three apertures (i.e. triaperturate pollen), through one of which the pollen tube emerges during pollination. Eudicots contrast with the so-called 'primitive' dicots, such as the magnolia family, which have uniaperturate pollen (i.e. with a single aperture).
Monocots include one of the large divisions of Angiosperm plants (flowering plants with seeds protected within a vessel). They are herbaceous plants with parallel veined leaves and have an embryo with a single cotyledon, as opposed to dicot plants (dicotyledonous), which have an embryo with two cotyledons. Most of the important staple crops of the world, the so-called cereals, such as wheat, barley, rice, maize, sorghum,
oats, rye and millet, are monocots. Thus, the plant can be a grass, such as wheat, barley, rice, maize, sorghum, oats, rye and millet.
The plant can therefore be a cereal crop such as wheat, oat, barley, or rice; a forage such as bahiagrass, dallisgrass, kleingrass, guineagrass, reed canary grass, orchardgrass, ricegrass, foxtail, or vetch; a legume such as soybean, lentil, or chickpea; an oilseed such as canola; a vegetable such as onion or carrot; or a specialty crop such as caraway, hemp, or sesame.
In some embodiments, the plant is a sorghum. For example, the plant can be of the species Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor, Sorghum
brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum timorense, Sorghum trichocladum, Sorghum versicolor, Sorghum virgatum, or Sorghum vulgare
In some embodiments, the plant is a miscanthus. Thus, the plant can be of the species Miscanthus floridulus, Miscanthus x. giganteus, Miscanthus sacchariflorus (Amur silver-grass), Miscanthus sinensis, Miscanthus tinctorius, or Miscanthus transmorrisonensis.
Additional representative plants useful in the compositions and methods disclosed herein include the Brass ica family including sp. napus, rapa, oleracea, nigra, carinata and juncea industrial oilseeds such as Camelina sativa, Crambe, Jatropha, castor; Arabidopsis thaliana; soybean; cottonseed; sunflower; palm; coconut; rice; safflower; peanut; mustards including Sinapis alba; sugarcane and flax.
Crops harvested as biomass, such as silage corn, alfalfa, switchgrass, or tobacco, also are useful with the methods disclosed herein. Representative tissues for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems.
IV. Methods of Making Transgenic Plants
A. Plant Transformation Techniques
The transformation of suitable agronomic plant hosts using vectors expressing transgenes can be accomplished with a variety of methods and plant tissues. Representative transformation procedures include
Agrobacterium-mediated transformation, biolistics, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, and silicon fiber-mediated transformation (U.S. Patent No. 5,464,765 to Coffee, et al; "Gene Transfer to Plants" (Potrykus, et al, eds.) Springer-Verlag Berlin Heidelberg New York (1995); "Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins" (Owen, et al, eds.) John Wiley & Sons Ltd. England (1996); and "Methods in Plant Molecular Biology: A Laboratory Course Manual" (Maliga etal. eds.) Cold Spring Laboratory Press, New York (1995)).
Plants can be transformed by a number of reported procedures (U.S.
Patent Nos. 5,015,580 to Christou, et al; 5,015,944 to Bubash; 5,024,944 to Collins, et al; 5,322,783 to Tomes et al; 5,416,011 to Hinchee et al. ;
5,169,770 to Chee et al). A number of transformation procedures have been reported for the production of transgenic maize plants including pollen transformation (U.S. Patent No. 5,629,183 to Saunders et al.), silicon fiber- mediated transformation (U.S. Patent No. 5,464,765 to Coffee et al), electroporation of protoplasts (U.S. Patent Nos. 5,231,019 Paszkowski et al; 5,472,869 to Kxzyzek et al; 5,384,253 to Krzyzek et al), gene gun (U.S. Patent Nos. 5,538,877 to Lundquist et al and 5,538,880 to Lundquist et al), and Agrobacter /wm-mediated transformation (EP 0 604 662 Al and WO 94/00977 both to Hiei Yukou et al). The Agrobacterium-mediated procedure is particularly preferred as single integration events of the transgene constructs are more readily obtained using this procedure which greatly facilitates subsequent plant breeding. Cotton can be transformed by particle bombardment (U.S. Patent Nos. 5,004,863 to Umbeck and 5,159,135 to Umbeck). Sunflower can be transformed using a combination of particle bombardment and Agrobacterium infection (EP 0486 233 A2 to Bidney, Dennis; U.S. Patent No. 5,030,572 to Power et al). Flax can be transformed
by either particle bombardment or Agrobacterium-mediated transformation. Switchgrass can be transformed using either biolistic or Agrobacterium mediated methods (Richards et al. Plant Cell Rep. 20: 48-54 (2001);
Somleva et al Crop Science 42: 2080-2087 (2002)). Methods for sugarcane transformation have also been described (Franks & Birch Aust. J. Plant Physiol. 18, 471-480 (1991); WO 2002/037951 to Elliott, Adrian, Ross et al).
Recombinase technologies which are useful in practicing the current invention include the cre-lox, FLP/FRT and Gin systems. Methods by which these technologies can be used for the purpose described herein are described for example in (U.S. Patent No. 5,527,695 to Hodges et al.; Dale and Ow, Proc. Natl Acad. Set USA, 88:10558-10562 (1991); Medberry et al, Nucleic Acids Res., 23: 485-490 (1995)).
Engineered minichromosomes can also be used to express one or more genes in plant cells. Cloned telomeric repeats introduced into cells may truncate the distal portion of a chromosome by the formation of a new telomere at the integration site. Using this method, a vector for gene transfer can be prepared by trimming off the arms of a natural plant chromosome and adding an insertion site for large inserts (Yu et al, Proc Natl AcadSci USA, 103:17331-6 (2006); Yu et al, Proc Natl AcadSci USA, 104:8924-9 (2007)). The utility of engineered minichromosome platforms has been shown using Cre//ox and FRT/FLP site-specific recombination systems on a maize minichromosome where the ability to undergo recombination was demonstrated (Yu et al, Proc Natl Acad Sci U SA, 103:17331-6 (2006); Yu et al, Proc Natl Acad Sci USA, l 04:8924-9 (2007)). Such technologies could be applied to minichromosomes, for example, to add genes to an engineered plant. Site specific recombination systems have also been demonstrated to be valuable tools for marker gene removal Kerbach, S. et al. heor. Appl. Genet. 111 :1608-1616 (2005)), gene targeting (Chawla, R et al, Plant Biotechnol .J 4:209-218 (2006); Choi, S. el al, Nucleic Acids Res., 28, El 9 (2000); Srivastava V & Ow DW, Plant Mol Biol. 46:561-566 (2001); Lyznik LA et al, Nucleic Acids Res., 21 : 969-975 (1993)) and gene conversion (Djukanovic V et al, Plant Biotechnol ., 4:345-357 (2006).
An alternative approach to chromosome engineering in plants involves in vivo assembly of autonomous plant mmichromosomes (Carlson et al, PLoS Genet., 3:1965-74 (2007). Plant cells can be transformed with centromeric sequences and screened for plants that have assembled autonomous chromosomes de novo. Useful constructs combine a selectable marker gene with genomic DNA fragments containing centromeric satellite and retroelement sequences and/or other repeats.
Another approach useful to the described invention is Engineered Trait Loci ("ETL") technology (US Patent 6,077,697; US Patent Application 2006/0143732). This system targets DNA to a heterochromatic region of plant chromosomes, such as the pericentric heterochromatin, in the short arm of acrocentric chromosomes. Targeting sequences may include ribosomal DNA (rDNA) or lambda phage DNA. The pericentric rDNA region supports stable insertion, low recombination, and high levels of gene expression. This technology is also useful for stacking of multiple traits in a plant (US Patent.Application 2006/0246586).
Zinc-finger nucleases (ZFNs) are also useful for practicing the invention in that they allow double strand DNA cleavage at specific sites in plant chromosomes such that targeted gene insertion or deletion can be performed (Shukla etaL, Nature, (2009); Townsend et al, Nature, (2009).
Following transformation by any one of the methods described above, the following procedures can, for example, be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium, regenerate the plant cells that have been transformed to produce differentiated plants, select transformed plants expressing the transgene producing the desired level of desired
polypeptide(s) in the desired tissue and cellular location.
Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-bascd techniques and techniques that do not require Agrobacterium. on-Agrobacterium techniques involve the uptake of heterologous genetic material directly by protoplasts or cells. This is accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. In each case the
transformed cells may be regenerated to whole plants using standard techniques known in the art.
Transformation of most monocotyledon species has now become somewhat routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue or organized structures, as well as Agrobacterium-mediated transformation.
Plants from transformation events are grown, propagated and bred to yield progeny with the desired trait, and seeds are obtained with the desired trait, using processes well known in the art.
B. Plastid Transformation
In another embodiment the transgene is directly transformed into the plastid genome. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513 to Maliga et al, 5,545,817 to McBride et al, and 5,545,818 to McBride et al. , in PCT application no. WO 95/16783 to
McBride et al, and in McBride et al. Proc. Natl Acad. Sci. USA 91,7301- 7305 (1994). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using bioKstics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Suitable plastids that can be transfected include, but are not limited to, chloroplasts, etioplasts, chromoplasts, leucoplasts, amyloplasts, proplastids, statoliths, elaioplasts, proteinoplasts and combinations thereof.
C. Methods for Reproducing Transgenic Plants
Following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the
transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.
In plastid transformation procedures, further rounds of regeneration of plants from explants of a transformed plant or tissue can be performed to increase the number of transgenic plastids such that the transformed plant reaches a state of homoplasmy (all plastids contain uniform plastomes containing transgene insert).
The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al Plant Cell Reports 5:81-84(1986). These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.
In some scenarios, it may be advantageous to insert a multi-gene pathway into the plant by crossing of lines containing portions of the pathway to produce hybrid plants in which the entire pathway has been reconstructed. This is especially the case when high levels of product in a seed compromises the ability of the seed to germinate or the resulting seedling to survive under normal soil growth conditions. Hybrid lines can be created by crossing a line containing one or more PHB genes with a line containing the other gene(s) needed to complete the PHB biosynthetic pathway. Use of lines that possess cytoplasmic male sterility (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52) with the appropriate maintainer and restorer lines allows these hybrid lines to be produced efficiently. Cytoplasmic male sterility systems are already available for some Brassicaceae species (Esser, K. et al., 2006, Progress in Botany, Springer Berlin Heidelberg. 67, 31-52). These Brassicaceae species can be used as gene sources to produce cytoplasmic male sterility systems for other oilseeds of interest such as Camelina.
V. Screening Methods
Methods are also provided for identifying treatments, such as chemical treatments, that can modify photoperiod sensitivity in a plant.
In some embodiments, the method involves administering a candidate agent to a transgenic plant disclosed herein and comparing the effect of the administration on photoperiod sensitivity in the plant to a control. For example, the purpose of the method can be to identify an agent that causes the transgenic plant to delay or prevent flowering.
In some embodiments, the method involves contacting cells expressing an Mai gene disclosed herein with a candidate agent, monitoring the effect of the candidate agent on Mai gene expression, and comparing the effect of the candidate agent on Mai gene expression to a control. For example, the purpose of the method can be to identify an agent that promotes Mai gene expression. In these embodiments, an increase in Mai gene expression would identify an agent that could be used to increase
photoperiod sensitivity. Likewise, the purpose of the method can be to identify an agent that inhibits Mai gene expression. In these embodiments, a decrease in Mai gene expression would identify an agent that could be used to reduce photoperiod sensitivity.
Mai gene expression can be detected using routine methods, such as immunodetection methods. The methods can be cell-based or cell-free assays. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme- Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays:
Heterogeneous and Homogeneous Systems, Handbook of Experimental
Immunology, Vol. 1 : Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods, immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIP A),
immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery /localization after photobleaching (FRAP/ FLAP).
In some embodiments, a reporter construct, such as a fiuorochrome or enzyme, is operably linked to an Mai expression control sequence. In these embodiments, the purpose of the method can be to identify an agent that modulates activation of the Mai expression control sequence by detecting the affect of a candidate agent on reporter expression.
In general, candidate agents can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the disclosed screening procedure. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compound ¾. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds.
Synthetic compound libraries are commercially available, e.g., from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
When a crude extract is found to have a desired activity, further fractionation of the positive lead can be used to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having the activity. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using animal models for diseases or conditions, such as those disclosed herein.
.. Candidate agents encompass numerous chemical classes, but are most often organic molecules, e.g., small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl orearboxyl group, for example, at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. In a further embodiment, candidate agents are peptides.
VI. Methods of Identifying Photoperiod Sensitivity Genes in Related Plants
Methods are also provided for identifying genes that control photoperiod sensitivity in other plants. Therefore, methods for identifying maturity gene orthologues in plants are provided. The methods generally involve using the gene sequences for Mai in S. bicolor or S. propinquum disclosed herein.
in preferred embodiments, the plant is closely related to Sorghum bicolor. Thus, in some embodiments, the plant is a Sorghum, Miscanthus, or Saccharum. In some embodiments, the method involves scanning the genetic sequences of a plant for genes that are orthologous to Mai .
In some embodiments, the method involves conducting a BLAST search of plant genomes for genes having the highest nucleic acid sequence identity to that of Mai in S. bicolor or S. propinquum. For example, the orthologous gene can have 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, or a nucleic acid encoding the amino acid sequence of SEQ ID NO:8 or 34, or a fragment or variant thereof.
VII. Methods of Genotyping Photoperiod Sensitive Flowering
A. Haplotypes
The sequences disclosed herein can be used to screen for photoperiod sensitive flowering in plants. For example, the genotype of one or more insertions, deletions, and polymorphisms in or around S. bicolor Mai relative to S. propinquum Mai , and can be used to phenotype a plant as photoperiod sensitive (i.e., having the S. propinquum genotype) or photoperiod insensitive (i.e., having the S. bicolor). For example, deletions, insertions, and polymorphisms can be determined by comparing SEQ ID NO: 1, 3, or 5-oiS. propinquum Mai to SEQ ID NO: 9 or 12 of S. bicolor using global sequence alignment tools, and include, but are not limited to the insertions, deletions, and polymorphisms specifically disclosed above and in Figure 3 A below.
For example, the exons of short-day S. propinquum and day neutral S. bicolor differ by five synonymous mutations: C -> T at position 47; C -> T at position 126; A -> G at position 159; T -> G at position 351; and A -> C at position 543 of SEQ ID NO:7 (S. propinquum) relative to SEQ ID NO:l 1 (S. bicolor). These single nucleotide polymorphisms (SNPs) within the Mai gene locus can serve as a haplotype for photoperiod sensitivity. As used
herein, the term "haplotype" refers to the allelic pattern of a group of
(usually contiguous) DNA markers or other polymorphic loci along an individual chromosome or double helical DNA segment.
Having three, four or five of the S. propinquum SNPs can be diagnostic of a photoperiod sensitive plant (i.e., short day flowering), while having three, four or five of the S. bicolor SNPs can be diagnostic of a photoperiod insensitive plant (i.e., day-neutral flowering). A plant is photoperiod sensitive plant (i.e., short day flowering) when it has all five S. propinquum SNPs. A plant is photoperiod insensitive plant (i.e., day-neutral flowering), when it has all five S. bicolor SNPs. For example, C:C:A:T:C relative to positions 47:126: 159:351 :543 of SEQ ID NO:7 is indicative of a photoperiod sensitive (short day flowering) plant, while T:T:G:G:C relative to positions 47:126:159:351 :543 of SEQ ID O:l l is indicative of a photoperiod insensitive (day-neutral flowering) plant.
In some embodiments, there is a correlation between the number of S. propinquum SNPs and level of photoperiod sensitivity. For example, an increasing number of S. propinquum SNPs relative to S. bicolor SNPs is correlated with increasing photoperiod sensitivity.
As described in more detail below, it is understood that genomic DNA will typically be used for determining the SNP genotype of a plant of interest. Methods of aligning sequences are known in the art, and described herein. One of skill in the art can readily identify the positions of the above- disclosed SNPs within genomic sequences, including but not limited to those disclosed herein, such as SEQ ID NO: 1, 2, 3, 4, 5, 6, 9, 10, 12, 13, or a nucleic acid encoding the amino acid sequence of SEQ ID NO: 8 or 34, or variants, fragments, homologs, or orthologs thereof, by aligning the sequence of SEQ ID NO: 7 or 11 to the genomic sequence.
Increased height naturally confers a competitive advantage in light interception. As discussed in the Examples below, favorable alleles at different genes that conferred both optimal height and flowering time to the same progeny by virtue of the suppressed recombination in this genomic region, might have become fixed more quickly than independently- segregating alleles. Accordingly, the S. propinquum haplotype of
C:C:A:T:C at positions 47:126:159:351 :543 of SEQ ID NO:7 is diagnostic of increased height relative to the S. bicolor haplotype of T:T:G:G:C at positions 47:126:159:351 :543 of SEQ ID NO: 11.
B. Methods for detecting SNPs and haplotypes
The process of determining which specific nucleotide (i.e., allele) is present at each of one or more SNP positions, such as a disclosed SNP position in the Mai gene locus, is referred to as SNP genotyping. Methods for SNP genotyping are generally known in the art (Chen et al.,
Pharmacogenomics J. , 3(2):77-96 (2003); Kwok, et al., Curr. Issues Mol. Biol. , 5(2):43-60 (2003); Shi, Am. J. Pharmacogenomics, 2(3): 197-205 (2002); and Kwok, Annu. Rev. Genomics Hum. Genet., 2:235-58 (2001)).
SNP genotyping can include the steps of collecting a biological sample from a plant, isolating genomic D A from the cells of the sample, contacting the nucleic acids with one or more primers which specifically hybridize to a region of the isolated nucleic acid containing a target SNP under conditions such that hybridization and amplification of the target nucleic acid region occurs, and determining the nucleotide present at the SNP position of interest, or, in some assays, detecting the presence or absence of an amplification product (assays can be designed so that hybridization and/or amplification will only occur if a particular SNP allele is present or absent). In some assays, the size of the amplification product is detected and compared to the length of a control sample; for example, deletions and insertions can be detected by a change in size of the amplified product compared to a normal genotype.
The neighboring sequence can be used to design SNP detection reagents such as oligonucleotide probes and primers. In some embodiment probe or primers are designed based on the cDNA of S. propinq wn (SEQ ID NO:7), or S. bicolor (SEQ ID NO: 11), In some embodiments, it may desirable for the probe or primer to bind non-coding regions of the Mai gene. Accordingly, one of skill in the art can map the above disclosed haplotype to the genomic sequence of Mai, such as SEQ ID NO:l, 2, 3, 4, 5, or 6 of S. propinquum, or SEQ ID NO: 9, 10, 12, or 13 of S. bicolor for the purpose of designing the SNP probes or primers.
UGA 1540 PCX
Common SNP genotyping methods include, but are not limited to, TaqMan assays, molecular beacon assays, nucleic acid arrays, allele- specific primer extension, allele-specific PCR, arrayed primer extension,
homogeneous primer extension assays, primer extension with detection by mass spectrometry, pyrosequencing, multiplex primer extension sorted on genetic arrays, ligation with rolling circle amplification, homogeneous ligation, multiplex ligation reaction sorted on genetic arrays, restriction- fragment length polymorphism, single base extension-tag assays, and the Invader assay. Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence . detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection.
SNPs can be scored by direct DNA sequencing. A variety of automated sequencing procedures can be utilized, including sequencing by mass spectrometry. Methods for amplifying DNA fragments and sequencing them are well known in the art.
Other .suitable methods for detecting polymorphisms include methods in which protection from cleavage agents is used to detect mismatched bases in NA/RNA or RNA/DNA duplexes (Myers et al., Science, 230: 1242 (1985); Cotton, et al, PNAS, 85:4397 (1988); and Saleeba, et al., Meth. Enzymol, 217:286-295 (1992)), comparison of the electrophoretic mobility of variant and wild type nucleic acid molecules (Orita et al., PNAS, 86:2766 (1989); Cotton, et al, Mutat. Res., 285:125-144 (1993); and Hayashi, et al., Genet. Anal. Tech. Appl, 9:73-79 (1992)), and assaying the movement of polymorphic or wild-type fragments in polyacrylamide gels containing a gradient of denaturant using denaturing gradient gel electrophoresis (DGGE) (Myers et al.s Nature, 313:495 (1985)). Sequence variations at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or chemical cleavage methods.
In one embodiment, SNP genotyping is performed using the
TaqMan® assay, which is also known as the 5' nuclease assay. The
TaqMan® assay detects the accumulation of a specific amplified product
during PCR. The TaqMan assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye. The reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye does not emit a signal. The proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter. The reporter dye and quencher dye may be at the S'-most and the 3'-most ends, respectively, or vice versa. Alternatively, the reporter dye may be at the 5'- or 3'-most end while the quencher dye is attached to an internal nucleotide, or vice versa. In yet another embodiment, both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.
During PCR, the 5' nuclease activity of D A polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target SNP-containing template which is amplified during PCR, and the probe is designed to hybridize to the target SNP site only if a particular SNP allele is present.
Another method for genotyping SNPs is the use of two
oligonucleotide probes in an OLA (U.S. Pat. No. 4,988,617). In this method, one probe hybridizes to a segment of a target nucleic acid with its 3'-most end aligned with the SNP site. A second probe hybridizes to an adjacent segment of the target nucleic acid molecule directly 3' to the first probe. The two juxtaposed probes hybridize to the target nucleic acid molecule, and are ligated in the presence of a linking agent such as a ligase if there is perfect complementarity between the 31 most nucleotide of the first probe with the SNP site. If there is a mismatch, ligation would not occur. After the reaction, the ligated probes are separated from the target nucleic acid molecule, and detected as indicators of the presence of a SNP.
Another method for SNP genotyping is based on mass spectrometry. Mass spectrometry takes advantage of the unique mass of each of the four nucleotides of DNA. SNPs can be unambiguously genotyped by mass spectrometry by measuring the differences in the mass of nucleic acids having alternative SNP alleles. MALDI-TOF {Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry technology is useful for extremely precise determinations of molecular mass, such as SNPs. Numerous approaches to SNP analysis have been developed based on mass spectrometry. Exemplary mass spectrometry-based methods of SNP genotyping include primer extension assays, which can also be utilized in combination with other approaches, such as traditional gel-based formats and microarrays.
Typically, the primer extension assay involves designing and annealing a primer to a template PCR amplicon upstream (5r) from a target SNP position. A mix of dideoxynucleotide triphosphates (ddNTPs) and/or deoxynucleotide triphosphates (dNTPs) are added to a reaction mixture containing template (e.g., a SNP-containing nucleic acid molecule which has typically been amplified, such as by PCR), primer, and DNA polymerase. Extension of the primer terminates at the first position in the template where a nucleotide complementary to one of the ddNTPs in the mix occurs. The primer can be either immediately adjacent (i.e., the nucleotide at the 3' end of the primer hybridizes to the nucleotide next to the target SNP site) or two or more nucleotides removed from the SNP position. If the primer is several nucleotides removed from the target SNP position, the only limitation is that the template sequence between the 3* end of the primer and the SNP position cannot contain a nucleotide of the same type as the one to be detected, or this will cause premature termination of the extension primer. Alternatively, if all four ddNTPs alone, with no dNTPs, are added to the reaction mixture, the primer will always be extended by only one nucleotide, corresponding to the target SNP position. In this instance, primers are designed to bind one nucleotide upstream from the SNP position (i.e., the nucleotide at the 3' end of the primer hybridizes to the nucleotide that is immediately adjacent to the target SNP site on the 5' side of the target SNP site). Extension by only one
nucleotide is preferable, as it minimizes the overall mass of the extended primer, thereby increasing the resolution of mass differences between alternative SNP nucleotides. Furthermore, mass-tagged ddNTPs can be employed in the primer extension reactions in place of unmodified ddNTPs. This increases the mass difference between primers extended with these ddNTPs, thereby providing increased sensitivity and accuracy, and is particularly useful for typing heterozygous base positions. Mass-tagging also alleviates the need for intensive sample-preparation procedures and decreases the necessary resolving power of the mass spectrometer. The extended primers can then be purified and analyzed by MALDI-TOF mass spectrometry to determine the identity of the nucleotide present at the target SNP position.
Other methods that can be used to genotype the SNPs include single- strand conformational polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE). SSCP identifies base differences by alteration in electrophoretic migration of single stranded PCR products. Single-stranded PCR products can be generated by heating or otherwise denaturing double stranded PCR products. Single-stranded nucleic acids may refold or form secondary structures that are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products are related to base-sequence differences at SNP positions. DGGE differentiates SNP alleles based on the different sequence-dependent stabilities and melting properties inherent in polymorphic DNA and the corresponding differences in electrophoretic migration patterns in a denaturing gradient gel.
Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be used to score SNPs based on the development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. If the SNP affects a restriction enzyme cleavage site, the SNP can be identified by alterations in restriction enzyme digestion patterns, and the corresponding changes in nucleic acid fragment lengths determined by gel electrophoresis.
C. SNP detection kits
Detection reagents can be developed and used to assay the disclosed SNPs individually or in combination, and such detection reagents can be readily incorporated into a kit or system format. The terms "kits" and "systems", as used herein in the context of SNP detection reagents, are intended to refer to such things as combinations of multiple SNP detection reagents, or one or more SNP detection reagents in combination with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as packaging intended for commercial sale, substrates to which SNP detection reagents are attached, electronic hardware components, etc.). SNP detection kits and systems, including but not limited to, packaged probe and primer sets (e.g., TaqMan probe/primer sets), arrays/microarrays of nucleic acid molecules, and beads that contain one or more probes, primers, or other detection reagents for detecting one or more of the disclosed SNPs are provided. The kits/systems can optionally include various electronic hardware components; for example, arrays ("DNA chips") and microfluidic systems ("lab-on-a-chip" systems) provided by various manufacturers typically comprise hardware components. Other kits/systems (e.g., probe/primer sets) may not include electronic hardware components, but may be comprised of, for example, one or more SNP detection reagents (along with, optionally, other biochemical reagents) packaged in one or more containers.
In some embodiments, a SNP detection kit typically contains one or more detection reagents and other components (e.g., a buffer, enzymes such as DNA polymerases or ligases, chain extension nucleotides such as deoxynucleotide triphosphates, and in the case of Sanger-type DNA sequencing reactions, chain terminating nucleotides, positive control sequences, negative control sequences, and the like) necessary to carry out an assay or reaction, such as amplification and/or detection of a SNP-containing nucleic acid molecule. A kit may further contain means for determining the amount of a target nucleic acid, and means for comparing the amount with a standard, and can comprise instructions for using the kit to detect the SNP- containing nucleic acid molecule of interest. In one embodiment, kits are
provided which contain the necessary reagents to carry out one or more assays to detect one or more of the disclosed SNPs. In an exemplary embodiment, SNP detection kits/systems are in the form of nucleic acid arrays, or compartmentalized kits, including microfluidic/lab-on-a-chip systems.
SNP detection kits may contain, for example, one or more probes, or pairs of probes, that hybridize to a nucleic acid molecule at or near each target SNP position. Multiple pairs of allele-specific probes may be included in the kit/system to simultaneously assay large numbers of SNPs. In some kits, the allele-specific probes are immobilized to a substrate such as an an'ay or bead.
The terms "arrays", "microarrays", and "DNA chips" are used herein interchangeably to refer to an array of distinct polynucleotides affixed to a substrate, such as glass, plastic, paper, nylon or other type of membrane, filter, chip, or any other suitable solid support. The polynucleotides can be synthesized directly on the substrate, or synthesized separate from the substrate and then affixed to the substrate.
Any number of probes, such as allele-specific probes, may be implemented in an array, and each probe or pair of probes can hybridize to a different SNP position. In the case of polynucleotide probes, they can be synthesized at designated areas (or synthesized separately and then affixed to designated areas) on a substrate using a light-directed chemical process. Each DNA chip can contain, for example, thousands to millions of individual synthetic polynucleotide probes arranged in a grid-like pattern and miniaturized. Probes can be attached to a solid support in an ordered, addressable array.
A microarray can be composed of a large number of unique, single- stranded polynucleotides, usually either synthetic antisense polynucleotides or fragments of cDNAs, fixed to a solid support. Typical polynucleotides are about 6-60 nucleotides in length, or about 15-30 nucleotides in length, or about 18-25 nucleotides in length. For certain types of microarrays or other detection kits/systems, it may be preferable to use oligonucleotides that are only about 7-20 nucleotides in length. In other types of arrays, such as
arrays used in conjunction with chemiluminescent detection technology, exemplary probe lengths can be, for example, about 15-80 nucleotides in length, or about 50-70 nucleotides in length, or about 55-65 nucleotides in length, or about 60 nucleotides in length. The microarray or detection kit can contain polynucleotides that cover the known 5' or 3' sequence of a gene/transcript or target SNP site, sequential polynucleotides that cover the full-length sequence of a gene/transcript; or unique polynucleotides selected from particular are as along the length of a target gene/transcript sequence. Polynucleotides used in the microarray or detection kit can be specific to a SNP or SNPs of interest (e.g., specific to a particular SNP allele at a target SNP site, or specific to particular SNP alleles at multiple different SNP sites).
Hybridization assays based on polynucleotide arrays rely on the differences in hybridization stability of the probes to perfectly matched and mismatched target sequence variants. For SNP genotyping, it is generally preferable that stringency conditions used in hybridization assays are high enough such that nucleic acid molecules that differ from one another at as little as a single SNP position can be differentiated. Such high stringency conditions may be preferable when using, for example, nucleic acid arrays of allele-specific probes for SNP detection. In some embodiments, the arrays are used in conjunction with chemiluminescent detection technology.
A polynucleotide probe can be synthesized on the surface of the substrate by using a chemical coupling procedure and an inkjet application apparatus, as described in PCT Publication No. WO 95/25111 . In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
Methods for using such arrays or other kits/systems, to identify SNPs and haplotypes disclosed herein in a test sample are provided. Such methods typically involve incubating a test sample of nucleic acids with an array comprising one or more probes corresponding to at least one SNP position of the present invention, and assaying for binding of a nucleic acid from the test
sample with one or more of the probes. Conditions for incubating a SNP detection reagent (or a kit/system that employs one or more such SNP detection reagents) with a test sample vary. Incubation conditions depend on such factors as the format employed in the assay, the detection methods employed, and the type and nature of the detection reagents used in the assay.
A SNP detection kit/system can include components that are used to prepare nucleic acids from a test sample for the subsequent amplification and/or detection of a SNP-containing nucleic acid molecule. Such sample preparation components can be used to produce nucleic acid extracts
(including DNA and/or RNA), proteins or membrane extracts from any bodily fluids (such as blood, serum, plasma, urine, saliva, phlegm, gastric juices, semen, tears, sweat, etc.), skin, hair, cells (especially nucleated cells), biopsies, buccal swabs or tissue specimens.
Another form of kit is a compartmentalized kit A compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include, for example, small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the test samples and reagents are not cross-contaminated, or from one container to another vessel not included in the kit, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another or to another vessel. Such containers may include, for example, one or more containers which will accept the test sample, one or more containers which contain at least one probe or other SNP detection reagent for detecting one or more of the disclosed SNPs, one or more containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and one or more containers which contain the reagents used to reveal the presence of the bound probe or other SNP detection reagents. The kit can optionally further include compartments and/or reagents for, for example, nucleic acid amplification or other enzymatic reactions such as primer extension reactions, hybridization, ligation, electrophoresis (e.g., capillary
electrophoresis), mass spectrometry, and/or laser-induced fluorescent detection. The kit may also include instructions for using the kit.
Microfluidic devices may also be used for analyzing SNPs. Such systems miniaturize and compartmentalize processes such as probe/target hybridization, nucleic acid amplification, and capillary electrophoresis reactions in a single functional device. Such microfluidic devices typically utilize detection reagents in at least one aspect of the system, and such detection reagents may be used to detect one or more of the disclosed SNPs. For genotyping SNPs, an exemplary microfluidic system may integrate, for example, nucleic acid amplification, primer extension, capillary
electrophoresis, and a detection method such as laser induced fluorescence detection.
EXAMPLES
Example 1: Genetic mapping of Mai
Materials and Methods
The methods for genetic mapping are published in Lin, et al, Genetics, 141:391-411 (1995).
Results
S. propinquum containing the Mai locus flowers later than cultivars of S. bicolor used in the U.S.A. Segregation for S, bicolor BTx623 versus S. propinquum alleles at the Mai locus imparts dichotomous phenotype when grown in a temperate environment (Lin, et al, Genetics, 141:391-411 (1995)). Interval mapping (Lander, et al, Genetics, 121:185-199 (1989)) was used to analyze an F2 population of S. bicolor BTx623, a temperate cultivated sorghum, crossed with S. propinquum, a wild tropical sorghum. As shown in Figure 1, the F2 population of S. bicolor x S. propinquum demonstrated bimodal distribution of flowering time frequency when grown in a temperate environment. Specifically, S. propinquum (189 ±1.9 days) and most F2s flowered later than S. bicolor (115.4±7.8 days) when photoperiod was less than 12.5 hours. The Mai locus alone accounts for 85.7% of phenotypic variation in flowering time (Lin, et al., Genetics, 141 :391 -411 (1995)) and mapped to chromosome 6, as later corroborated by independent
work in different germplasm (Klein, et al, The Plant Genome, 1 :S12-S26 (2008)).
By testing F3 progeny of recombinants in the region, the locus was mapped between DNA markers separated genetically by 1.1 cM (Figure 2A). The 1.1 cM region containing Mai is, physically, among the largest in the genome, with 60-fold less recombination than the genome- wide average of 0.7 mbp/cM. Spanning 34 million base-pairs (mbp), this region alone contains about 5% of sorghum genomic DNA and 1.3% (-400) of genes. QTLs for many additional traits (beyond flowering) are also closely associated with Mai , including a major dwarfing gene (Lin, et at, Genetics, 141 :391-411 (1995)). Classical literature has defined these loci as Mai and Dw2.
Exotic-converted sorghum pairs were compared in the Mai region to access recombinational information resulting from the independent conversion(s) of about 90 sorghum genotypes. "Conversion" takes 12 generations and 4 years (Stephens, et al. Crop Set, 7:396 (1967)), with one backcross followed by two generations of selfing (lacking DNA markers, this was necessary to phenotypically distinguish heterozygotes from
homozygotes for the recessive photoperiod-insensitive allele). Across the sorghum gene pool, Mai has a singularly large role in the genetic
determination of flowering. Among nine diverse exotic-converted sorghum pairs, all nine are 'converted' (introgressed with chromatin from the photoperiod-insensitive donor line) in the Mai region (Lin, et al Genetics, 141 :391-411 (1995)).
In the Mai region and any other regions that remain heterozygous, an exotic-converted pair offers about 3-4x the recombinational information than could be obtained from a single F2 or recombinant inbred genotype
(estimated using standard formulas: (AHard, Hilgardia, 24:235-278 (1956)). A set of 90 exotic-converted pairs that broadly sample sorghum diversity and BTx406, the donor of day-neutral flowering, were genotyped with 9 SSR loci distributed through the region containing Mai, with a peak introgression frequency of 84%. Haplotypes were determined and are illustrated in Figure 2C, with the dark line indicating the span of converted regions. In the region
of greatest conversion, additional genes and DNA markers were
characterized, with a peak conversion frequency of 87% for the 400 bp indel that occurs upstream (5') of the Sb06g012260 gene (Figure 2B;
Sb06g012260 itself was not characterized in this study). Frequencies of conversion at the DNA marker loci are plotted along the sorghum genome sequence, with approximate locations of genes in the sequence shown as cross-hatches along the axis. While the terminal regions that these data exclude from consideration are physically small, they contain the majority of genes.
PRR37, a candidate gene with expression patterns correlated with short-day flowering (Murphy, et al, Proceedings of the National Academy of Science of the United States of America, 108:16469-16474 (2011)), maps outside of this region, with less than 20% conversion (Figure 2B), indicating that it does not account for short-day flowering in most if any exotic sorghums.
Genes in the genomic region experiencing high frequencies of 'conversion' (introgression of day-neutral flowering) were re-sequenced in a diversity panel of 384 accessions (87% landraces, 6% wild types, 6% breeding materials and 1% advanced cultivars) phenotyped for flowering under both short-day and long-day conditions, permitting calculation of a "Photoperiod Index" (PRI) reflecting the flowering behavior of each accession (see Methods for Association Genetics). Prior data on 41 SSR markers permitted investigation of population structure and genetic diversity of the panel, providing the relatedness information needed for formal testing of associations between specific alleles and PRI (Remington, et al,
Proceedings of the National Academy of Science of the United States of America, 98:11479-11484 (2001); Thornsberry, et al, Nature Genetics, 28:286-289 (2001); Yu, et al, Nature Genetics, 38:203-208 (2005)).
Sb06g012260 was a gene discovered to be near the peak frequency of conversion. Sb06g012260 is a gene containing an 'FT' functional domain associated with regulation of flowering in Arabidopsis (Kardailsky, et al, Science, 286:1962-1965 (1999) and Oryza (Kojima, et al, Plant and Cell Physiology, 43 : 1096- 1105 (2002)). Candidate alleles of Sb06g012260 were
resequenced in a diversity panel of 384 individuals for which flowering time was known (see Example 3).
Analysis of this resequencing data identified two major haplotypes (each with two rare variants), one closely resembling the allele found in the short-day flowering accession of S. propinquwn (Figure 3 A), and the other showing greatest abundance in sorghums from South Africa, the most temperate part of the pre-Columbian range (Figure 4). Statistically- significant association of these haplotypes with PRI were found in
subpopulations in which the two haplotypes each occur at similar frequencies (Figure 4 and Figure 5 A, 5B, and 5C). Figure 5 A, 5B, 5C are based on SNPs from the coding sequence of the Mai gene. The Figures show independent analysis of each subpopulation. TASSEL association analysis including all the subpopulations and the covariance by the population structure is discussed in more detail below and shown in Tables 1 and 2.
Figures 5 A, 5B, and 5C showing flowering (days) for individuals having a short-day haplotype or a day neutral haplotype for the gene
Sb06g012260 in West Africa (Figure 5 A, 2008, p-0.005; R2=0.13) and South Africa (Figure 5B (2008), p=3.84 E-08; R =0.33; and Figure 5C (2007), p=0.0346; R2=0.08). These data also show a statistically-significant association of the haplotypes with flowering in subpopulations in which the two haplotypes each occur at similar frequencies. The most informative subpopulation, sorghums originating in the South Africa region, is the subpopulation in which the day-neutral allele (haplotype) occurs at highest frequency.
The day-neutral haplotype included four deletions: (1) a 423 base pair deletion in the 5' UTR of the Sb06g012260 (2) a -4.2 kb deletion in the 5' UTR of the Sb06g012260, (3) a three base pair deletion starting about 221 base pairs upstream of the Sb06g012260 transcription-start site, and (4) a 27 base pair deletion in the second intron; and five synonymous single nucleotide polymorphism mutations (SNPs) in the coding sequence (Figure 3B). Among the four deletions of the day-neutral haplotype, the 3-bp deletion is particularly damaging, removing from the Sb06g012260 promoter a CAAT box, an invariant DNA sequence in many eukaryotic promoters
required for sufficient transcription (Berg, et al., Biochemistry, 5 Ed.
(2002)).
Other elements of the haplotype appear likely to be associated with the phenotype by linkage drag. For example, the 423 bp insertion appears to be a CACTA transposon. CACTA elements have been implicated as a mechanism of movement of genes and gene fragments in sorghum (Paterson, et al, Nature, 457:551-556 (2009)). The element present in the short-day haplotype has a close match in the day-neutral S. bicolor BTx623 genome sequence, presumably its 'parent' element, since that hit is to an autonomous element, while the insertion into S. propinquum has lost ability to transpose. Sequence divergence between the putative 'parent' element and the insertion is 94% - using published approaches to 'date' transposon insertions
(SanMiguel, et al, Nature Genetics, 20:43-45 (1998)) suggests an 'age' of about 2 million years for the element. This suggests that the insertion may have only occurred in the S. bicolor/S. propinquum lineage, since this is much more recent than its divergence from Saccharum and other near relatives.
The approximately 4.2 kb element present in the short-day haplotype contains an inferred open reading frame found on a different chromosome of day-neutral S. bicolor BTx623 (chr. 7, Sb07g008600). Further, this element does not correspond discernibly to any gene of known function, and shows only limited similarity to two other sorghum genes, both also "putative uncharacterized proteins" (Sb03g005850; Sb08g011060). While a role in short-day flowering cannot yet be ruled out, its presence in day-neutral sorghum argues against a direct role in short-day flowering, and its mobility since the S. bicolor/S. propinquum divergence implies (as for the CACTA element) that it is likely to be an as-yet unrecognized transposon.
The remaining deletion is in the second intron.
Example 3: Association Analysis Among Mai Region Markers
Materials and Methods
A public sorghum reference germplasm set that substantially represents the spectrum of diversity in S. bicolor has been characterized with a genome-wide panel of SSRs, and phenotyped for flowering time across a number of diverse environments including some in photoperiods long enough to delay flowering of daylength-sensitive types. These data are freely available, and provide the information needed for formal testing of associations between specific alleles and phenotypes. Because it is predominantly self-pollinating with linkage disequilibrium extending over -15 kb, sorghum is an attractive system in which to employ association genetics to link DNA sequences to their phenotypic consequences.
The diversity panel was evaluated during two different planting seasons representing different day length conditions. The first planting (2007) represented short-day conditions (11.8-12.15 hrs light) and high humidity conditions, conditions under which short-day sorghums (i.e.
photoperiod sensitive) are expected to initiate flowering promptly or similar to neutral day (i.e. photoperiod insensitive). The second (2008) planting was characterized by a transition from long to short-day (13.1 -11.0 hrs) photoperiod and dry conditions, and short-day sorghums would be expected to delay flowering under these conditions.
Flowering time was recorded as the number of days required for 50% of the plants in a single row to flower (DFL50%). Photoperiod Index (PRI) of each accession was defined as the mean difference in DFL50% between the two planting seasons (i.e. PRI = DFL50%2008 - DFL50%2007).
Photoperiod sensitive accessions showed positive PRI values, while negative values identified photoperiod insensitive materials.
Results
TASSEL (Bradbury, et al, Biomformatics, 23:2633-2635 (2007)) was used to perform both linkage disequilibrium analysis and tests of association, the latter employing population structure covariates and a kinship matrix determined for the germplasm panel based on the 80 SSRs.
10 genes distributed across the target region were resequenced, in
most members of the diversity panel (excepting those for which reactions
failed, etc). TASSEL has been used to perform both linkage disequilibrium
analysis and tests of association, the latter employing population structure
covariates and a kinship matrix determined for the germplasm panel based on the existing SSRs.
The results of the resequencing is presented in Tables 1-2, and Figure
6. In partial summary, these data delimit the target region to the interval
between genes Sb06g011767 and Sb06g012520, which is 1.3Mb with 20
annotated genes. The strongest evidence is found at the -4.2 kb indel in the
Sb06g012260 gene.
Table 1 Association analysis among Mai region markers and the photoperiod by single marker analysis
Data Gene Marker df F F df df Error MS Error Rsq Model Rsq Marker
FLOW_2008_2007 SSR7 2 6.0672 0.0026 2 347 472.206 0.0338 0.0338
2.42E-
FLOW_2008_2007 SSRS 2 8.5317 04 2 345 449.7492 0.0471 0.0471
FLOWJ2008J2007 Sb06g010870 2 4.52 0.0116 2 320 483.4132 0.0275 0.0275
FLOW_2008„2007 Sb06gOH767 1 8.0108 0.0049 1 322 450.9787 0.0243 0.0243
2.94E-
FLOW_2008_2007 400bpINDEL 2 10.8095 05 2 296 454.8433 0.0681 0.0681
1.37E-
FLOW_2008J>007 4kbINDEL 2 16.5587 07 2 340 429.1795 0.0888 0.0888
Sb06g012260 I.S3E-
FLOW_200S_2007 (FT) 2 16.2049 07 2 358 437.37 0.083 0.083
1.88E-
FLOWJ2008J2007 Sb06g012520 2 13.7554 06 2 313 439.8079 0.0808 0.0808
FLOW_2008_2007 Sb06g013230 2 1.3111 0.271 2 315 473.4048 0.0083 0.0083
FLOW 2008 2007 Sb06g0138I0 2 1.4601 0.2337 2 338 474.4558 0.0086 0.0086
Table 2 Association analysis among Mai region markers and the photoperiod with the
correction of population structure (Q)
Data Gene Marker df F PF df df Error MS Error Rsq Mode) Rsq Marker
FLOW_2008_2007 SSR7 2 0.0216 0.9786 6 343 398.859 0.1933 1.02E-04
5.65E-
FLOW_2008_2007 SSR8 2 12.522 06 6 341 358.8457 0.2485 0.0552
FLOW_2008_2007 Sb06g010870 2 1.529 0.2183 6 316 405.8768 0.1937 0.0078
FLOW_2008_2007 Sb06g011767 1 4.0098 0.0461 5 318 386.0947 0.175 0.0104
FLOW_2008_2007 400bpINDEL 2 2.4506 0.088 6 292 377.6165 0.2368 0.0128
6.52E-
FLOW_2008_2007 4kbINDEL 2 7.4975 04 6 336 362.9691 0.2384 0.034
Sb06g012260
FLOW_2008_2007 (FT) 2 6.7615 0.0013 6 354 375.5924 0.2213 0.0297
FLOW_2008_2007 Sb06g012520 2 3.6981 0.0259 6 309 376.269 0.2236 0.0186
FLOW_2008_2007 Sb06g013230 2 1.9067 0.1503 6 311 375.0981 0.2242 0.0095
FLOW_2008_2007 Sb06g013810 2 6.5932 0.0016 6 334 376.9455 0.2216 0.0307
Example 3: P R37 is not Mai
As noted above, PRR37, a candidate gene with expression patterns correlated with short-day flowering (Murphy, et ah, Proceedings of the National Academy of Science of the United States of America, 108:16469- 1 474 (2011)), maps outside of this region, with less than 20% conversion (Figure 3B), indicating that it does not account for short-day flowering in most if any exotic sorghums.
Several additional lines of evidence also show that PR 37 cannot be Mai. The sorghum genotype 100M, used to discern expression patterns correlating the PR 37 candidate allele to short-day flowering (Murphy et ah, Proceedings of the National Academy of Science of the United States of America, 108:16469-16474 (2011)), also contains the short-day haplotype for Sb06g012260, which was confirmed by comparison to the short-day genotype PI209217.
Accordingly, differences in expression patterns between 100M and its near-isogenic line SM100 could be attributable either to P R37,
Sb06g012260, or other intervening genes on the introgressed segment Indeed, in short-day S. propinquwn, PR 37 contains a frameshift mutation that renders the PRR domain and much of the protein nonsensical and also causes premature termination (Figure 3C). While PRR37 cannot account for short-day flowering in most sorghums, prior work by members of the PR 37 team showed it to be in the approximate location of Ma6 (Brady, Texas A&M University, (2006)), a gene with a much smaller effect on flowering.
Homologs of the Mai candidate gene Sb06g0l2260 in sorghum (Paterson, et ah, Nature, 457:551 -56 (2009)), rice (Matsumoto, et ah,
Nature, 436:793-800 (2005)), and Arabidopsis (The Arabidopsis Genome Initiative. Nature, 408:796-815 (2000)) genomes, maize and sugarcane ESTs were identified by BLAST. The sugarcane ESTs were then translated to protein sequences. In total, 6 homologs were found in Arabidopsis (including the FT gene (Kardailsky, et ah, Science, 286(5446): 1962- 1965 (1999)), 19 in rice (including Hd3a (Kojima, et ah, Plant and Cell Physiology,
43(10): 1096-1105 (2002)) and sorghum, 26 in maize and 8 in sugarcane (Figure 7).
The candidate gene Sb06g012260 appears to have evolved as a single-gene duplication. Based on a synonymous substitution rate (Ks) of 0.43 from Sb04g008320, currently-used cereal molecular clocks suggest that this duplication occurred ~40Mya (Gaut, et ah, Proc Nat Acad Sci USA 93(19):10274-10279 (1996)). This date is more recent than the estimated divergence of rice and the sorghum/sugarcane/maize lineage, consistent with the finding that a positional ortholog was not discerned in rice. Sb04g008320 does have a rice ortholog (Os02gl3830.1) of unknown function. Other members of the sorghum gene family do have rice orthologs, and several of the sorghum family members are much more similar to rice Hd3a
(Os06g06320.1) than is the Mai candidate gene (Sb06g012260).
For Sb06g012260, a single maize ortholog, GRMZM2G019993, was identified on maize chromosome 2. Since maize has experienced a genome duplication since the divergence of the sorghum and maize lineages, the apparent presence of only one ortholog in the maize genome implies that a second duplicated copy was lost in maize. The missing homeolog would, if still present, be located on maize chrl O, at approximately 105Mb.
Independent research has suggested the possibility of a major flowering time quantitative trait locus on maize chromosome 10 (Ducrocq, et ah, Genetics, 183:1555-1563 (2009); Coles, et ah, Genetics, 184:799-812 (2010)) and the presence of numerous candidate genes including an FT homolog (ZCN19; (Chardon, et ah, Genetics, 168(4):2169-85 (2004); (Danielevskaya, et ah, Plant Physiology, 146:250-64 (2008)). In the present maize genome sequence (Schnable, et al, Science, 326(5956):! 112-15 (2009)), there are 4 maize FT genes on chromosome 10, but none at 105 Mb
(GRMZM2G338454 chrl0:5Mb; AC214791.2_FG002 chrl0:45Mb;
AC217051.3JFG006 chrl0:l 14Mb; GRMZM2G062052 chrl0:127Mb). The one of these closest to the target position
(AC217051.3_FG006 chrl 0:114Mb) is highly divergent in sequence from Sb06g012260, suggesting that it is not likely to be the ortholog.
Example 4: S. halepense has a Mutation in the Sb06g012260 Promoter
The invasive plant Sorghum halepense, or 'Johnson Grass', has adapted to day-neutral photoperiod independently of, and perhaps even more
rapidly than, breeder-improved sorghum. Sorghum halepense is a tetraploid
derived from a naturally-occurring cross between wild forms of S. bicolor
and S. propinquum (Celarier, Bull Torrey Bot Club, 85:49-62 (1958);
Paterson, et at, Proceedings of the National Academy of Sciences of the
United States of America, 92:6127-6131 (1995)). Being largely inbreeding,
its wild progenitors would have each been expected to be homozygous for
the short-day flowering Mai allele, with tetraploid S. halepense (also
inbreeding) receiving 4 copies of the allele. Among the limited sampling
available in the US National Plant Germplasm collection, two Old World
accessions ΡΪ209217 from South Africa and PI271616 from India were
confirmed to be short-day flowering - these were also both homozygous for
the short-day haplotype. However, many or all U.S. populations of S.
halepense are believed to include many members that flower in the long days of the temperate summer.
In S. halepense naturalized in the U.S., the central portion of the
short-day flowering haplotype has been largely replaced with a segment that
includes a different mutation in the Sb06g012260 promoter. The results of a
sampling of 480 plants is summarized in Table 4.
Table 4: Presence or Absence of 4 mutations in S. halepense
(% among unambiguous genotypes)
400bp 4.2kb 3bp intron
Non-ambiguous genotypes % among non-ambiguous genotypes
B: Day-neutral S. bicolor genotype; 0.47% 5.71% 0.00% 0.22%
P: Short-day S. propinquum genotype 81.63% 1.14% 10.37% 88.16%
H: S. halepense genotype 0.00% 0.00% 1.67% 0.00%
BP 17.91% 93.15% 3.68% 11.62%
BH (at least one B and one H allele) 0.00% 0.00% 0.67% 0.00%
PH 0.00% 0.00% 72.24% 0.00%
BPH (at least one allele each of B, P, and H) 0.00% 0.00% 10.70% 0.00%
Ambiguous genotypes % among total sample
PH-like (closely resembles PH) 0.00% 0.00% 29.43% 0.00%
Other 11.63% 9.59% 24.03% 5.26%
Among 480 plants sampled equally from each of five 5*. halepense populations from GA, TX (2), NE, and NJ, USA (Morrell, et al, Molecular Ecology, 14:2143-2154 (2005)), 81.6% and 88.2% of plants scorable (i.e. excluding amplification failures or ambiguous migration patterns) were homozygous for the short-day haplotype at both terminal loci (423 bp, intron indels), but only 1.1 and 10.4% at the two internal loci (4,186 and 3 nt indels) (Table 4). Only 39 bp upstream from the site of the CAAT box deletion in day-neutral S. bicolor,. 85.3% of the tetraploid S. halepense plants have at least one copy (with 1.7% being homozygous for all four copies, but noting that 1 , 2, or 3 copies cannot be distinguished in this tetraploid) of a 4 nt insertion (i.e. not found in either progenitor) that disrupts a TC-rich repeat, a cis-acting element involved in defense and stress response
(bioinformatics.psb.ugent.be/webtools/plantcare/html ). TC-rich repeats are enriched in the promoters of photoperiod-responsive genes, and photoperiod- responsiveness is thought to integrate multiple light-, hormone-, and stress- responsive elements (Mongkolsiriwatana, et al, Nat. Set, 43:164-177 (2009)). Further, 98.9% also have at least one copy of the day-neutral (deletion) allele at the 4, 186 nt indel, 5.7% being homozygous for the deletion. Finally, 15.7% of plants also carry one or more copies of the 3 nt deletion.
The adaptation of S. halepense to the temperate climate of the continental U.S.A. may predate the scientific breeding of day-neutral sorghums. Selection of day-neutral Mai alleles occurred during the first 40 years of the 20th century (Quinby, Texas A&M University Press (1974); Smith, et at, John Wiley and Sons, (2000)) while S. halepense was well- established in the U.S.A. by 1847 and of sufficient importance in 1 00 to be the subject of the first federal appropriation for weed control (McWhorter, Weed Science, 19:496 (1971)).
Sb06g012260 appears to have evolved as a single-gene duplication (Figure 7), shortly after the oryzoid (rice)— panicoid
(sorghum/sugarcane/maize) divergence. Based on a Ks of 0.43 from its nearest homolog, Sb04g008320, this duplication is an estimated 40 million years old (Gaut, et al, Proceedings of the National Academy of Sciences of
the United States of America, 93:10274-10279 (1996)), consistent with the lack of a rice ortholog. Sb04g008320 does have a rice ortholog
(Os02gl3830.1), although of unknown function.
Sb06g012260 is extensively diverged from other known floral regulators-indeed, no members of its clade have empirically-demonstrated functions (Figure 7). Other sorghum family members do have rice orthoiogs, and some resemble a rice flowering time QTL Hd3
(Os06g06320.1)(Kojima, et al, Plant and Cell Physiology, 43:1096-1105 (2002)). However, Hd3a is well over 100 million years distant from
Sb06g012260, even more than are the nearest Arabidopsis genes. One family member, Sb02g029725, locates near the likelihood peak of a second sorghum flowering QTL with a small phenotypic effect (FlrAvgBl: Lin et al 1995). Sb06g012260 has a single maize ortholog, GRMZM2G019993, on chromosome 2. Since the maize genome duplicated after its divergence with the sorghum lineage, the presence of only one maize ortholog implies that a second one was lost, from chromosome 10 at ~105Mb. Maize chromosome 10 contains a major flowering time QTL (Ducrocq, et al, Genetics,
183:1555-1563 ( 2009); Coles, et al, Genetics, 184 (2010)) and four FT homologs (Schnable, et al, Science, 326: 1112-1115 (2009)), but the nearest to 105 Mb (AC217051.3_FG006 chrl 0: 114Mb) is so divergent in sequence from Sb06g012260 that it is not considered orthologous (Figure 7).
The importance of Mai to fecundity, via flowering, may have contributed to the evolution of a 'coadapted gene complex' (Lande, Genetical Research, 26:221-235 (1975)) with cis-linkage of alleles at different loci that collectively confer an adaptive phenotype, perhaps facilitated by the recalcitrance of the region to recombination. The Mai region also holds dw2, the gene of largest effect on sorghum stature (height) (Lin, et al, Genetics, 141 :391-411 (1995)), but which can be separated from Mai by infrequent recombination (Quinby, Texas A&M University Press (1974); Lin, Texas A&M University (1998);. Increased height naturally confers a competitive advantage in light interception. Favorable alleles at different genes that conferred both optimal height and flowering time to the same progeny by virtue of the suppressed recombination in this genomic
region, might have become fixed more quickly than independently- segregating alleles. Flowering time and plant height were correlated in the diversity panel (r=0.53 in 2007, 0.73 in 2008, each significant at 0.001). While the strongest statistical association we found with plant height was at Sb06g012260 itself (p=0.007), there was also an association at Sb06g007330 (p=0.023), a putative cation efflux family protein. A putatively intervening gene, Sb06g010870, showed no association but could have recently formed alleles or be at an incorrect location, noting that this recombinationally- recalcitrant region is among the most repetitive in the sorghum genome and therefore one of the most difficult in which to assemble whole-genome shotgun sequence (Paterson AH et al. Nature, 457(7229):551-56 (2009)) .
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims
1. An isolated nucleic acid, comprising a nucleic acid sequence at least 90% identical to SEQ ID NO:l, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
2. An isolated nucleic acid, comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide consisting of the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28,
29, 30, 31 , 32, 33, 34, or a complement thereof.
3. An isolated nucleic acid, comprising a photoperiod sensitive control sequence 90% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or a complement thereof.
4. An isolated nucleic acid, comprising a photoperiod insensitive control sequence at least 90% identical to SEQ ID NO:27 or a complement thereof.
5. An isolated nucleic acid, comprising a nucleic acid sequence that hybridizes under stringent conditions to the isolated nucleic acid of claims 3 or 4.
6. A recombinant expression vector, comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1, 2, 3, , 5, 6, 9, 10, 12, 13, 28, 29,
30, 1, 32, 33, 34 or a complement thereof.
7. A recombinant expression vector, comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 7, 11, 29, 31, 33 or a complement thereof.
8. The recombinant expression vector of claim 7, further comprising a heterologous expression control sequence.
9. The recombinant expression vector of claim 8, wherein the heterologous expression control sequence comprises a constitutive promoter.
10. The recombinant expression vector of claim 8, wherein the heterologous expression control sequence comprises a tissue specific promoter.
11. A recombinant expression vector, comprising the isolated nucleic acid of claim 3, or the complement thereof, operably linked to a heterologous plant gene, wherein the nucleic acid sequence of claim 3 imposes
photoperiod sensitivity on the heterologous plant gene.
12. A recombinant expression vector, comprising the isolated nucleic acid of claim 4, or the complement thereof, operably linked to a heterologous plant gene, wherein the nucleic acid sequence of claim 3 imposes
photoperiod insensitivity on the heterologous plant gene.
13. A transgenic plant or transgenic plant cell, comprising an expression control sequence operably linked to a nucleic acid sequence that silences expression of a polynucleotide having a nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
14. The transgenic plant or plant cell of claim 13, wherein the transgenic plant has lower photoperiod sensitivity compared to non-transgenic plant of the same species.
15. The transgenic plant or plant cell of claim 13 wherein the transgenic plant or plant cell is selected from the group consisting of Brassica family, industrial oilseeds, Arabidopsis thaliaria, algae, soybean, cottonseed, sunflower, palm, coconut, rice, safflower, peanut, mustards, silage corn, alfalfa, switchgrass, miscanthus, sorghum, tobacco, sugarcane and flax.
16. The transgenic plant or plant cell of claim 15 wherein the transgenic plant or plant cell is a dicotyledon.
17. The transgenic plant or plant cell of claim 15 wherein the transgenic plant or plant cell is a monocotyledon.
18. A seed from the plant of any one of claims 13 to 17.
19. A method of delaying flowering in a plant, comprising introducing to the plant a nucleic acid sequence that silences expression of a polynucleotide having the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
20. The method of claim 19, wherein the plant is a dicotyledon.
21. The method of claim 19, wherein the plant is a monocotyledon.
22. The method of claim 19, wherein the plant has lower photoperiod sensitivity compared to a control plant of the same species.
23. A method of identifying an agent that modulates plant photoperiod sensitivity, comprising
contacting a cell containing an Mai gene with a candidate agent under conditions suitable for Mai gene expression; and
detecting the effect of the candidate agent on Mai gene expression, wherein an detectable increase or decrease in Mai gene expression is an indication that the candidate agent modulates plant photoperiod sensitivity.
24. The method of claim 23, wherein the Mai gene comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or a complement thereof.
25. A method of identifying an agent that modulates plant photoperiod sensitivity, comprising
contacting a cell containing a reporter construct operably linked to an Mai expression control sequence with a candidate agent under conditions suitable for reporter expression; and
detecting the effect of the candidate agent on reporter expression, wherein an detectable increase or decrease in reporter expression is an indication that the candidate agent modulates plant photoperiod sensitivity.
26. The method of claim 25, wherein the Mai expression control sequence comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or a complement thereof.
27. A method of imposing photoperiod sensitivity on a plant process comprising producing a recombinant nucleic acid molecule comprising a plant gene responsible for the plant process operably linked to a photoperiod sensitive Mai expression control sequence.
28. The method of claim 27, wherein the Mai expression control sequence comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or a complement thereof.
29. The method of claims 27-28 wherein the plant process is not naturally photoperiod sensitive.
30. A method of imposing photoperiod insensitivity on a plant process comprising producing a recombinant nucleic acid molecule comprising a plant gene responsible for the plant process operably linked to a photoperiod insensitive Mai expression control sequence.
31. The method of claim 30, wherein the Mai expression control sequence comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 27 or a complement thereof.
32. The method of claims 30-31 wherein the plant process is not naturally photoperiod insensitive.
33. A transgenic plant or transgenic plant cell, comprising a plant gene controlling a plant process operably linked to an Mai expression control sequence.
34. The transgenic plant or transgenic plant cell, wherein the Mai expression control sequence is a photoperiod sensitive expression control sequence.
35. The transgenic plant of claim 34, wherein the Mai expression control sequence comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or a complement thereof.
36. The transgenic plant of claims 34-35 wherein the plant process is not naturally photoperiod sensitive.
37. The transgenic plant or transgenic plant cell, wherein the Mai expression control sequence is a photoperiod insensitive expression control sequence.
38. The transgenic plant of claim 37, wherein the Mai expression control sequence comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 27 or a complement thereof.
39. The transgenic plant of claims 37-38 wherein the plant process is not naturally photoperiod insensitive.
40. A method of delaying flowering in plant comprising altering the sequence of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or variants thereof in the plant.
41. The method of claim 40, wherein the altering comprises introducing one or more nucleic acid substitutions, additions, deletions or a combination thereof in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or variants thereof.
42. A method of increasing or accelerating flowering in a plant, comprising introducing to the plant a nucleic acid sequence comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
43. A method of increasing or accelerating flowering in a plant, comprising introducing to the plant a nucleic acid sequence that increases expression of a polynucleotide having the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
44. The method of claim 43 wherein the nucleic acid sequence that increases expression is a constructively active plant promoter operably linked to a polynucleotide having at least 90% sequence identity to the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 1 1, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
45. The method of claim 43 wherein the nucleic acid sequence that increases expression is a heterologous photoperiod sensitive plant promoter operably linked to a polynucleotide having at least 90% sequence identity to the nucleic acid sequence SEQ ID NO: 1, 2, 3, 4, 5, , 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
46. A transgenic plant comprising a heterologous expression control sequence operably linked to a polynucleotide comprising a nucleic acid sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, 28, 29, 30, 31, 32, 33, 34 or a complement thereof.
47. The transgenic plant of claim 46 wherein the expression control sequence is a photoperiod sensitive plant promoter that increases photoperiod sensitive expression of the polynucleotide.
48. A transgenic plant with increased photoperiod sensitive flowering comprising a promoter with one or more CAAT boxes operatively linked to Mai , wherein the transgenic plant has increased photoperiod sensitivity relative to a non-transgenic plant of the same species.
49. The transgenic plant of claim 48 wherein Mai comprises the nucleic acid sequence of SEQ ID NO: 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, or 13.
50. The transgenic plant of claim 48 wherein the one or more boxes comprises the nucleic acid sequence of SEQ ID NO:24, 25, 26, or combinations thereof.
51. The transgenic plant of claim 48 wherein the promoter and Mai are expressed from an extrachromosomal vector.
52. The transgenic plant of claim 48 wherein the promoter and Mai are integrated into the plant's chromosome.
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| KR101960072B1 (en) * | 2017-10-30 | 2019-03-19 | 충남대학교 산학협력단 | Molecular marker for discriminating hydrogen cyanide content of sorghum line and uses thereof |
| WO2023156441A1 (en) | 2022-02-17 | 2023-08-24 | Basf Coatings Gmbh | Uv-curable coating compositions |
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| WO2016183005A1 (en) | 2015-05-12 | 2016-11-17 | E I Du Pont De Nemours And Company | Methods and compositions to alter flowering time in sorghum |
| KR101960072B1 (en) * | 2017-10-30 | 2019-03-19 | 충남대학교 산학협력단 | Molecular marker for discriminating hydrogen cyanide content of sorghum line and uses thereof |
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