AU2002210243B2 - Manipulation of flowering and plant architecture - Google Patents

Description

WO 02/33091 PCT/AU01/01311 MANIPULATION OF FLOWERING AND PLANT ARCHITECTURE The present invention relates to nucleic acids or nucleic acid fragments encoding amino acid sequences for proteins involved in the control of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture, and/or inflorescence and/or flower development in plants, and the use thereof for the modification of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture, and/or inflorescence and/or flower development in plants.

Most plants have several growth phases. Following seed embryo germination, the plant apical meristem goes through a vegetative phase generating leaf primordia with axillary meristems. The axillary meristems will generate side branches or will rest dormant until apical dominance is removed.

Upon receiving appropriate signals, the apical meristem switches to reproductive development (flowering). The switch is controlled by various physiological signals and genetic pathways that will coordinate flowering. The apical meristem switched from vegetative to reproductive phase will produce an inflorescence.

Inflorescences are the flower-bearing parts of the plant. Flowers are plant structures that support gametophyte development. Inflorescences are of two basic types, determinate (where apical meristems grow indefinitely generating floral meristems from their periphery) and indeterminate (where apical meristem is transformed into a floral meristem terminating apical growth and with subsequent growth taking place from axillary meristems). Floral meristems produce a defined number of floral organ primordia in concentric whorls, which develop into sepals, petals, stamens or carpels (in the dicot flower); and into palea, lodicule, stamens and carpels (in the grass flower).

A series of homeotic genes that specify floral meristem identity and determine the fate of floral organ primordia have been identified. These genes interact to specify the basic floral organs of the dicot flower. These interactions can be summarized through the 'ABC" model of floral organ identity, where the first (outer) whorl organs (sepals) are specified by A-function genes, the second whorl organs (petals) are specified by a combination of A- and B-function WO 02/33091 PCT/AU01/01311 2 activities, third whorl organs (stamens) by a combination of B- and C-function, and fourth whorl (carpels) by a C-function activity.

Most of these genes belong to a family of transcription factors characterized by the presence of a conserved DNA-binding domain, named the MADS-box.

These genes (MADS-box genes or MADS) thus encode plant MADS-box proteins and are involved in flower and embryo development. Identified MADS-box genes in the model crucifer Arabidopsis thaliana include the meristem identity A-function genes APETALA 1 (AP1) and CAULIFLOWER (CAL); the organ identity B-function genes APETALA3 (AP3) and PISTILLATA and the organ identity C-function gene AGAMOUS (AG).

The gene CENTRORADIALIS (CEN) of snapdragon (Antirrhinum) is required to maintain an indeterminate shoot identity.

The APETALA2 (AP2) gene of Arabidopsis thaliana has a role in the specification of floral meristem identity and floral organ identity. AP2 is required for A-function organ identity. Mutations of AP2 lead to homeotic conversion of floral organs, for example conversion of sepals to leaves or to carpels, conversion of petals to stamens, absence of petals.

Other plant genes involved in pattern formation and having a putative role in vegetative and floral development have a conserved DNA-binding domain, named the Homeo-box. These genes (Homeo-box genes or HB genes) thus encode plant homeodomain proteins that may function as transcription factors in controlling downstream target genes. Mutations in HB genes may result in abnormal leaf and flower development.

It would be useful however if other genes involved in specifying floral meristem identity, involved in the fate of floral development could be identified. In this regard the inventors have identified the following new genes: LpMADS, LpCEN, LpAP2, and LpHB.

WO 02/33091 PCT/AU01/01311 3 It would be desirable to have methods of manipulating plant life cycles and growth phases eg. the transition from the vegetative to the reproductive stage, flowering processes, flowering and plant architecture and inflorescence and flower development in plants, including grass species such as ryegrasses (Lolium species) and fescues (Festuca species), thereby facilitating the production of, for example, pasture and turf grasses and pasture legumes with enhanced or shortened or modified life cycles, enhanced or reduced or otherwise modified inflorescence and flower development, inhibited flowering (eg. non-flowering), modified flowering architecture (eg. indeterminate and determinate), earlier or delayed flowering, enhanced or modified number of leaves, enhanced or reduced or otherwise modified number of reproductive shoots, enhanced persistence and improved herbage quality, enhanced seed and leaf yield, altered growth and development, leading to improved seed production, improved biomass production, improved pasture production, improved pasture quality, improved animal production and reduced environmental pollution reduced pollen allergens, reduced nitrogenous waste).

Perennial ryegrass (Lolium perenne is a key pasture grass in temperate climates throughout the world. Perennial ryegrass is also an important turf grass.

Clovers (Trifolium species) such as white clover repens), red clover (T.

pratense) and subterranean clover subterraneum), and lucerne sativa) and medics (Medicago species) are fructan-devoid, starch-accumulating key pasture legumes in temperate climates throughout the world.

While nucleic acid sequences encoding some of the enzymes involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development have been isolated for certain species of plants, there remains a need for materials useful in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development, in a wide range of plants, particularly in forage and turf grasses and legumes including ryegrasses and fescues, and for methods for their use.

WO 02/33091 PCT/AU01/01311 4 It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

In one aspect, the present invention provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for the following proteins from a ryegrass (Lolium) or fescue (Festuca) species, or functionally active fragments or variants thereof: MADS-box proteins (MADS), CENTRORADIALIS (CEN), APETALA2 (AP2), and Homeo-box proteins (HB).

The present invention also provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of proteins which are related to MADS, CEN, AP2 and HB. Such proteins are referred to herein as MADS-like, CEN-like, AP2-like, and HB-like, respectively.

The individual or simultaneous enhancement or down-regulation or otherwise manipulation of MADS-box gene activities in plants may alter flower and embryo and seed development, may for example enhance or inhibit embryo differentiation and growth, may alter flower organ identity through conversion of one floral organ in another, may lead to absence of individual floral organs, may lead to male and/or female sterility, may increase the number of specific floral organs, may enhance or inhibit and otherwise alter flowering, may enhance or delay and otherwise alter flowering in time, and may increase or otherwise alter the number of leaves made before flowering.

The enhancement or otherwise manipulation of CEN activity in plants may alter the control of phase change, may promote vegetative growth indefinitely, may delay or otherwise alter flowering in time, and may increase or otherwise alter the number of leaves made before flowering.

The down-regulation or otherwise manipulation of AP2 activity in plants may alter flower organ identity through conversion of one floral organ in another, may lead to absence of individual floral organs, may increase the number of specific floral organs, and may alter flowering architecture.

WO 02/33091 PCT/AU01/01311 The enhancement or ectopic expression or otherwise manipulation of HB activity in plants may alter the control of phase change, may promote or reduce vegetative growth, may delay or otherwise alter flowering, may alter floral organ identity, and may alter plant architecture e.g. enhanced branching, increased bushiness.

Manipulation of flowering and plant architecture has significant consequences for a wide range of applications in plant production. For example, it has applications in inducing male sterility for hybrid seed production, in changing flower architecture for enhancing value of ornamentals, in delaying flowering in forage grasses thus stopping the formation of the less digestible stems and increasing herbage quality, in altering flowering time allowing early maturing crops, in delaying vegetative phase and thus increasing biomass production, in increasing branching and thus leading to enhanced bushiness in fruit trees, in altering plant size and leading to shorter plant stature, in blocking flowering and reducing the release of allergenic pollen, etc.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass perenne).

The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.

The term "isolated" means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.

WO 02/33091 PCT/AU01/01311 6 Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term "consensus contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences.

Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or nucleic acid fragments) may be assembled into a single contiguous nucleotide sequence.

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a MADS or MADS-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 1, 3, 4, 6, 7, 9, 10, 12, 14, 15, 17, 19, 21, 23, 25, 27, 29, 54, 59, 63, 68, 72, 76, 81, 86 and 90 hereto (Sequence ID Nos: 1, 3to 11, 12, 14to 18, 19, 21 to23, 24, 26, 28 to 30, 31,33, 35, 37, 39, 41, 43, 45 to 49, 90, 92, 94, 96, 98, 100, 102, 104 and 106, respectively); (b) complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CEN or CEN-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 30, 32 and 49 hereto (Sequence ID Nos: 50, 52 to 54 and 88, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and (d) functionally active fragments and variants of the sequences recited in and In another preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an AP2 or AP2-like protein includes a nucleotide sequence selected from the group WO 02/33091 PCT/AU01/01311 7 consisting of sequences shown in Figures 33, 35, 36, and 38 hereto (Sequence ID Nos: 55, 57 to 68, 69 and 71 to 74, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an HB or HB-like protein includes a nucleotide sequence selected from the group consisting of sequences shown in Figures 39, 41, 43, 45, and 47 hereto (Sequence ID Nos: 75, 77, 79, 81 and 83 to 87, respectively); complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and By "functionally active" in relation to nucleic acids it is meant that the fragment or variant (such as an analogue, derivative or mutant) encodes a polypeptide capable of modifying control of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture, and/or inflorescence and/or flower development, in a plant. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned nucleotide sequence, more preferably at least approximately identity, most preferably at least approximately 95% identity. Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 20 nucleotides.

WO 02/33091 PCT/AU01/01311 8 The nucleic acids or nucleic acid fragments encoding at least a portion of several proteins involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development have been isolated and identified. The nucleic acids or nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art.

Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g.

po!ymerase chain reaction, ligase chain reaction).

For example, genes encoding other proteins involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture and inflorescence and flower development, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the nucleic acids or nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. Moreover, the entire sequences may be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in amplification protocols to amplify longer nucleic acids or nucleic acid fragments encoding homologous genes from DNA or RNA. For example, polymerase chain reaction may be performed on a library of WO 02/33091 PCT/AU01/01311 9 cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acids or nucleic acid fragments of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.

Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998, the entire disclosure of which is incorporated herein by reference) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments may be isolated (Ohara et al. (1989) Proc.

Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217; the entire disclosures of which are incorporated herein by reference). Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs.

In a second aspect of the present invention there is provided a substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of MADS and MADS-like, CEN and CEN-like, AP2 and AP2-like, HB and HB-like proteins; and functionally active fragments and variants thereof.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass perenne).

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated MADS or MADS-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 2, 8, 11, 13, 16, 18, 20, 22, 24, 26, 28, 55, 60, 64, 69, 73, 77, 82, 87 and 91 hereto (Sequence ID Nos: 2, 13, 20, 25, 27, 32, 34, 36, 38, 40, 42, 44, 91, 93, 95, 97, 99, 101, 103, 105 and 107, respectively); and functionally active fragments and variants thereof.

WO 02/33091 PCT/AU01/01311 In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CEN or CEN-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 31 and 50 hereto (Sequence ID Nos: 51 and 89, respectively); and functionally active fragments and variants thereof.

In another preferred embodiment of this aspect of the invention, the substantially purified or isolated AP2 or AP2-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 34 and 37 hereto (Sequence ID Nos: 56 and 70, respectively); and functionally active fragments and variants thereof.

In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated HB or MB-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 42, 44 and 46 hereto (Sequence ID Nos: 76, 78, 80 and 82, respectively); and functionally active fragments and variants thereof.

By "functionally active" in relation to polypeptides it is meant that the fragment or variant has one or more of the biological properties for the proteins MADS, MADS-like, CEN, CEN-like, AP2, AP2-like, HB and MB-like, respectively.

Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 60% identity to the relevant part of the above mentioned amino acid sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity.

Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least amino acids, more preferably at least 15 amino acids, most preferably at least amino acids.

WO 02/33091 PCT/AU01/01311 11 In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.

Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides may be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins including the amino acid sequences. These antibodies may be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest.

A genotype is the genetic constitution of an individual or group. Variations in genotype are important in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait. Such nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNP's), variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as perfect markers or candidate genes for the given trait.

Applicants have identified a number of SNP's of the nucleic acids and nucleic acid fragments of the present invention. These are indicated (marked with grey on the black background) in the figures that show multiple alignments of nucleotide sequences of nucleic acid fragments contributing to consensus contig sequences. See for example, Figures 3, 6, 9, 14, 29, 35, 38 and 47 (Sequence ID WO 02/33091 PCT/AU01/01311 12 Nos: 3 to 11, 14 to 18, 21 to 23, 28 to 30, 45 to 49, 57 to 68, 71 to 74, and 83 to 87, respectively).

Accordingly, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid or nucleic acid fragment according to the present invention, or complements or sequences antisense thereto, and functionally active fragments and variants thereof.

In a still further aspect of the present invention there is provided a method of isolating a nucleic acid or nucleic acid fragment of the present invention including a single nucleotide polymorphism (SNP), said method including sequencing nucleic acid fragments from a nucleic acid library.

The nucleic acid library may be of any suitable type and is preferably a cDNA library.

The nucleic acid or nucleic acid fragment may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction.

The sequencing may be performed by techniques known to those skilled in the art.

In a still further aspect of the present invention, there is provided use of nucleic acids or nucleic acid fragments of the present invention including SNP's, and/or nucleotide sequence information thereof, as molecular genetic markers.

In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment according to the present invention, and/or nucleotide sequence information thereof, as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, WO 02/33091 PCT/AU01/01311 13 DNA fingerprinting and in marker assisted selection, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement, e.g.

tagging QTLs for herbage quality traits, flowering intensity, flowering time, number of tillers, leafiness, bushiness, seasonal growth pattern, herbage yield, flower architecture, plant stature. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues.

In a still further aspect of the present invention there is provided a construct including a nucleic acid or nucleic acid fragment according to the present invention.

The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases may also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

In a still further aspect of the present invention there is provided a vector including a nucleic acid or nucleic acid fragment according to the present invention.

The term "vector" as used herein includes both cloning and expression vectors. Vectors are often recombinant molecules including nucleic acid molecules from several sources.

WO 02/33091 PCT/AU01/01311 14 In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid and terminator being operatively linked.

By "operatively linked" is meant that said regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment.

The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, nonchromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable, or integrative or viable in the plant cell.

The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg.

monocotyledon or dicotyledon). Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, and the rice Actin promoter.

WO 02/33091 PCT/AU01/01311 A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene.

Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos), the octopine synthase (ocs) and the rbcS genes.

The vector, in addition to the regulatory element, the nucleic acid or nucleic acid fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)].

The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.

As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.

Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

The vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, WO 02/33091 PCT/AU01/01311 16 Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as arabidopsis, tobacco, white clover, red clover, subterranean clover, alfalfa, eucalyptus, potato, sugarbeet) and gymnosperms. In a preferred embodiment, the vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), even more preferably perennial ryegrass, including forage- and turf-type cultivars. In a preferred embodiment, the vectors may be used to transform dicotyledons, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and lucerne (Medicago sativa).

Techniques for incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

Cells incorporating the vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a construct or a vector of the present invention.

WO 02/33091 PCT/AU01/01311 17 The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial ryegrass, including both forage- and turf-type cultivars. In a preferred embodiment the plant cell, plant, plant seed or other plant part is from a dicotyledon, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and lucerne (Medicago sativa).

The present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.

The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

In a further aspect of the present invention there is provided a method of modifying the control of plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture and/or flower and/or inflorescence development in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or vector according to the present invention.

By "an effective amount" it is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

WO 02/33091 PCT/AU01/01311 18 Using the methods and materials of the present invention, plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture and/or inflorescence and/or flower development may be increased, decreased or otherwise modified relative to an untransformed control plant. For example, the number of leaves produced before flowering, the number of floral organs, the number of branches, the plant stature, the number of phytomers, the number of inflorescences and/or flowers, may be increased, decreased or otherwise modified. They may be increased or otherwise modified, for example, by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be decreased or otherwise modified, for example, by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures Figure 1 shows the consensus contig nucleotide sequence of LpMADSa (Sequence ID No: 1).

Figure 2 shows the deduced amino acid sequence of LpMADSa (Sequence ID No: 2).

Figure 3 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSa (Sequence ID Nos: 3 to 11).

Figure 4 shows the consensus contig nucleotide sequence of LpMADSb (Sequence ID No: 12).

WO 02/33091 PCT/AU01/01311 19 Figure 5 shows the deduced amino acid sequence of LpMADSb (Sequence ID No: 13).

Figure 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSb (Sequence ID Nos: 14 to 18). Figure 7 shows the consensus contig nucleotide sequence of LpMADSc (Sequence ID No: 19).

Figure 8 shows the deduced amino acid sequence of LpMADSc (Sequence ID No: Figure 9 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSc (Sequence ID Nos: 21 to 23).

Figure 10 shows the nucleotide sequence of LpMADSd (Sequence ID No: 24).

Figure 11 shows the deduced amino acid sequence of LpMADSd (Sequence ID No: Figure 12 shows the consensus contig nucleotide sequence of LpMADSe (Sequence ID No: 26).

Figure 13 shows the deduced amino acid sequence of LpMADSe (Sequence ID No: 27).

Figure 14 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSe (Sequence ID Nos: 28 to Figure 15 shows the nucleotide sequence of LpMADSf (Sequence ID No: 31).

WO 02/33091 PCT/AU01/01311 Figure 16 shows the deduced amino acid sequence of LpMADSf (Sequence ID No: 32).

Figure 17 shows the nucleotide sequence of LpMADSg (Sequence ID No: 33).

Figure 18 shows the deduced amino (Sequence ID No: 34).

Figure 19 shows the nucleotide sequence Figure 20 shows the deduced amino (Sequence ID No: 36).

Figure 21 shows the nucleotide sequence 37).

Figure 22 shows the deduced amino (Sequence ID No: 38).

Figure 23 shows the nucleotide sequence 39).

Figure 24 shows the deduced amino (Sequence ID No: acid sequence of LpMADSg of LpMADSh (Sequence ID No: acid sequence of LpMADSh of LpMADSi (Sequence ID No: acid sequence of LpMADSi of LpMADSj (Sequence ID No: acid sequence of LpMADSj Figure 25 shows the nucleotide sequence of LpMADSk (Sequence ID No: 41).

Figure 26 shows the deduced amino acid sequence of LpMADSk (Sequence ID No: 42).

Figure 27 shows the consensus contig nucleotide sequence of LpMADSI (Sequence ID No: 43).

WO 02/33091 PCT/AU01/01311 21 Figure 28 shows the deduced amino acid sequence of LpMADSI (Sequence ID No: 44).

Figure 29 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpMADSI (Sequence ID Nos: 45 to 49).

Figure 30 shows the consensus contig nucleotide sequence of LpCENa (Sequence ID No: Figure 31 shows the deduced amino acid sequence of LpCENa (Sequence ID No: 51).

Figure 32 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpCENa (Sequence ID Nos: 52 to 54).

Figure 33 shows the consensus contig nucleotide sequence of LpAP2a (Sequence ID No: Figure 34 shows the deduced amino acid sequence of LpAP2a (Sequence ID No: 56).

Figure 35 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpAP2a (Sequence ID Nos: 57 to 68).

Figure 36 shows the consensus contig nucleotide sequence of LpAP2b (Sequence ID No: 69).

Figure 37 shows the deduced amino acid sequence of LpAP2b (Sequence ID No: WO 02/33091 PCT/AU01/01311 22 Figure 38 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpAP2b (Sequence ID Nos: 71 to 74).

Figure 39 shows the nucleotide sequence of LpHBa (Sequence ID No: Figure 40 shows the deduced amino acid sequence of LpHBa (Sequence ID No: 76).

Figure 41 shows the nucleotide sequence of LpHBb (Sequence ID No: 77).

Figure 42 shows the deduced amino acid sequence of LpHBb (Sequence ID No: 78).

Figure 43 shows the nucleotide sequence of LpHBc (Sequence ID No: 79).

Figure 44 shows the deduced amino acid sequence of LpHBc (Sequence ID No: Figure 45 shows the consensus contig nucleotide sequence of LpHBd (Sequence ID No: 81).

Figure 46 shows the deduced amino acid sequence of LpHBd (Sequence ID No: 82).

Figure 47 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpHBd (Sequence ID Nos: 83 to 87).

Figure 48 shows a plasmid map of the cDNA encoding perennial ryegrass LpCen.

Figure 49 shows the nucleotide sequence of perennial ryegrass LpCen cDNA (Sequence ID No: 88).

WO 02/33091 PCT/AU01/01311 23 Figure 50 shows the deduced amino acid sequence of perennial ryegrass LpCen cDNA (Sequence ID No: 89).

Figure 51 shows plasmid maps of sense and antisense constructs of LpCen in pDH51 transformation vector.

Figure 52 shows screening by Southern hybridisation for RFLPs using LpCen as a probe.

Figure 53 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS1.

Figure 54 shows the nucleotide sequence of perennial ryegrass LpMADS1 cDNA (Sequence ID No: Figure 55 shows the deduced amino acid sequence of perennial ryegrass LpMADS1 cDNA (Sequence ID No: 91).

Figure 56 shows plasmid maps of sense and antisense constructs of LpMADS1 in pDH51 transformation vector.

Figure 57 shows screening by Southern hybridisation for RFLPs using LpMADS1 as a probe.

Figure 58 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS1b.

Figure 59 shows the nucleotide sequence of perennial ryegrass LpMADS1 b cDNA (Sequence ID No: 92).

Figure 60 shows the deduced amino acid sequence of perennial ryegrass LpMADS1b cDNA (Sequence ID No: 93).

Figure 61 shows plasmid maps of sense and antisense constructs of LpMADS1b in pDH51 transformation vector.

WO 02/33091 PCT/AU01/01311 24 Figure 62 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS2-1.

Figure 63 shows the nucleotide sequence of perennial ryegrass LpMADS2- 1 cDNA (Sequence ID No: 94).

Figure 64 shows the deduced amino acid sequence of perennial ryegrass LpMADS2-1 cDNA (Sequence ID No: Figure 65 shows plasmid maps of sense and antisense constructs of LpMADS2-1 in pDH51 transformation vector.

Figure 66 shows screening by Southern hybridisation for RFLPs using LpMADS2-1 as a probe.

Figure 67 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS2-2.

Figure 68 shows the nucleotide sequence of perennial ryegrass LpMADS2- 2 cDNA (Sequence ID No: 96).

Figure 69 shows the deduced amino acid sequence of perennial ryegrass LpMADS2-2 cDNA (Sequence ID No: 97).

Figure 70 shows plasmid maps of sense and antisense constructs of LpMADS2-2 in pDH51 transformation vector.

Figure 71 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS2-3.

Figure 72 shows the nucleotide sequence of perennial ryegrass LpMADS2- 3 cDNA (Sequence ID No: 98).

Figure 73 shows the deduced amino acid sequence of perennial ryegrass LpMADS2-3 cDNA (Sequence ID No: 99).

WO 02/33091 PCT/AU01/01311 Figure 74 shows plasmid maps of sense and antisense constructs of LpMADS2-3 in pDH51 transformation vector.

Figure 75 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS3.

Figure 76 shows the nucleotide sequence of perennial ryegrass LpMADS3 cDNA (Sequence ID No: 100).

Figure 77 shows the deduced amino acid sequence of perennial ryegrass LpMADS3 cDNA (Sequence ID No: 101).

Figure 78 shows plasmid maps of sense and antisense constructs of LpMADS3 in pDH51 transformation vector.

Figure 79 shows screening by Southern hybridisation for RFLPs using LpMADS3 as a probe.

Figure 80 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS4.

Figure 81 shows the nucleotide sequence of perennial ryegrass LpMADS4 cDNA (Sequence ID No: 102).

Figure 82 shows the deduced amino acid sequence of perennial ryegrass LpMADS4 cDNA (Sequence ID No: 103).

Figure 83 shows plasmid maps of sense and antisense constructs of LpMADS4 in pDH51 transformation vector.

Figure 84 shows screening by Southern hybridisation for RFLPs using LpMADS4 as a probe.

Figure 85 shows a plasmid map of the cDNA encoding perennial ryegrass LpMADS4-2.

WO 02/33091 PCT/AU01/01311 26 Figure 86 shows the nucleotide sequence of perennial ryegrass LpMADS4- 2 cDNA (Sequence ID No: 104).

Figure 87 shows the deduced amino acid sequence of perennial ryegrass LpMADS4-2 cDNA (Sequence ID No: 105).

Figure 88 shows plasmid maps of sense and antisense constructs of LpMADS4-2 in pDH51 transformation vector.

Figure 89 shows a plasmid map of the cDNA encoding perennial ryegrass Figure 90 shows the nucleotide sequence of perennial ryegrass cDNA (Sequence ID No: 106).

Figure 91 shows the deduced amino acid sequence of perennial ryegrass cDNA (Sequence ID No: 107).

Figure 92 shows plasmid maps of sense and antisense constructs of in pDH51 transformation vector.

Figure 93 shows screening by Southern hybridisation for RFLPs using as a probe.

Figure 94 shows the regeneration of transgenic tobacco plants from direct gene transfer to protoplasts of chimeric ryegrass genes involved in flowering and plant development.

Figure 95 shows a subgrid of a microarray for the expression profiling of perennial ryegrass flowering and plant development genes. Red represents upregulated expression, green represents down-regulated expression and yellow represents no change in expression. For example, an overlay of microarray images probed with leaf blade tissues (red) and root tissues (green). Expression level is relatively expressed as up-regulated in leaf blade (red), down-regulated in leaf blade (green) and no change in expression (yellow).

WO 02/33091 PCT/AU01/01311 27 Figure 96 shows a genetic linkage map of perennial ryegrass NA6 showing map location of ryegrass genes involved in flowering and plant development.

EXAMPLE 1 Preparation of cDNA libraries, isolation and sequencing of cDNAs coding for MADS, Cen, AP2, and HB proteins from perennial ryegrass (Lolium perenne) cDNA libraries representing mRNAs from various organs and tissues of perennial ryegrass (Lolium perenne) were prepared. The characteristics of the libraries are described in Table 1.

0 TABLE1 cDNA libraries from perennial ryegrass (Lolium perenne) Library Organ/Tissue 01rg Roots from 3-4 day old light-grown seedlings 02rg Leaves from 3-4 day old light-grown seedlings 03rg Etiolated 3-4 day old dark-grown seedlings 04rg Whole etiolated seedlings (1-5 day old and 17 days old) Senescing leaves from mature plants 06rg Whole etiolated seedlings (1-5 day old and 17 days old) 07rg Roots from mature plants grown in hydroponic culture 08rg Senescent leaf tissue 09rg Whole tillers and sliced leaves 1,3, 6, 12 and 24 h after harvesting) Embryogenic suspension-cultured cells 11 rg Non-embryogenic suspension-cultured cells 12rg Whole tillers and sliced leaves 1,3, 6, 12 and 24 h after harvesting) 13rg Shoot apices including vegetative apical meristems 14rg Immature inflorescences including different stages of inflorescence meristem and inflorescence development Defatted pollen 16rg Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted) 17rg Senescing leaves and tillers 18rg Drought-stressed tillers (pseudostems from plants subjected to PEG- WO 02/33091 PCT/AU01/01311 28 simulated drought stress) 19rg Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with half-strength salts) 2, 3, 4, 5, 6, 24 and 48 h after transfer) Non-embryogenic suspension-cultured cells subjected to osmotic stress (grown in media with double-strength salts) 2, 3, 4, 5, 6, 24 and 48 h after transfer) 21 rg Drought-stressed tillers (pseudostems from plants subjected to PEGsimulated drought stress) 22rg Spikelets with open and maturing florets 23rg Mature roots (specific subtraction with leaf tissue) The cDNA libraries may be prepared by any of many methods available.

For example, total RNA may be isolated using the Trizol method (Gibco-BRL, USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following the manufacturers' instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit (Clontech, USA), cDNAs may be amplified by long distance polymerase chain reaction using the Advantage 2 PCR Enzyme system (Clontech, USA), cDNAs may be cleaned using the GeneClean spin column (Bio 101, USA), tailed and size fractionated, according to the protocol provided by Clontech. The cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega, USA) according to the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene.

Alternatively, the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA, USA). The Uni-ZAP XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript II vectors (Stratagene) using T4 DNA ligase (New WO 02/33091 PCT/AU01/01311 29 England Biolabs), followed by transfection into E. coil DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).

Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analyzed using an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2 DNA sequence analyses The cDNA clones encoding MADS, CEN, AP2 and HB proteins were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches. The cDNA sequences obtained were analysed for similarity to all publicly available DNA sequences contained in the eBioinformatics nucleotide database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the SWISS-PROT protein sequence database using BLASTx algorithm (v 2.0.1) (Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI.

The cDNA sequences obtained and identified were then used to identify additional identical and/or overlapping cDNA sequences generated using the BLASTN algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a consensus contig sequence derived from this multiple sequence alignment. The consensus contig sequence was then used as a query for a search against the WO 02/33091 PCT/AU01/01311 SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification.

EXAMPLE 3 Identification and full-length sequencing of perennial ryegrass MADS and Cen cDNAs encoding flowering and plant development proteins.

To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, a set of perennial ryegrass cDNAs encoding flowering and plant development proteins was identified and fully sequenced.

Full-length cDNAs were identified from our EST sequence database using relevant published sequences (NCBI databank) as queries for BLAST searches.

Full-length cDNAs were identified by alignment of the query and hit sequences using Sequencher (Gene Codes Corp., AnnArbor, MI 48108, USA). The original plasmid was then used to transform chemically competent XL-1 cells (prepared inhouse, CaCI 2 protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original EST sequence using Sequencher to confirm identity and one of the three clones was picked for full-length sequencing, usually the one with the best initial sequencing result.

Sequencing was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most cases the sequencing could be done from both 5' and 3' end. The sequences of the oligonucleotide primers are shown in Table 2. In some instances, however, an extended poly-A tail necessitated the sequencing of the cDNA to be completed from the 5' end.

WO 02/33091 PCT/AU01/01311 31 TABLE 2 List of primers used for sequencing of the full-length cDNAs Gene name Clone ID Sequencing Primer sequence primer LpCen O6rgl XsBO6. 1 06rgl1XsBO6.f 1 GCTATGAGAGCCCAAAGCC O6rgl XsBO6.f2 IGATCTTGGCCTCCCTGTGG LpMADS1 4rgl UsD1 0.5 1 4rgl ZsFO9.f 1 AGGCACTTGAAAGGCGAAG LpMADS2-1 l9rglAsGl2.1 l9rglAsG12.fl IACTTGAGAGATACCGCACC LpMADS3 1 3rg2CsBO7. 1 -1 3rg2CsBO7.f 1 !CACTGCTGCTACAATGCTC 1 3rg2CsB3O7.f2 GGGAGAAATCAATAGGCAAC LpMADS4-1 1 9rg2MsFO5.2 11 rg2YsGO7.f 1 ACTCACACGAAGCAAACCC LpMADS4-2 j 11 rg2YsGO7.3 11 rg2YsGO7.f 1 ACTCACACGAAGCAAACCC {l3rgl SsH07,2 l3rgl SsHO7Jf 1 GTGTGAAGGCAACCATTG Gontigs were then assembled in Sequencher. The contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRI cut site both at the 5' and 3' end.

Plasmid maps and the full cDNA sequences of perennial ryegrass Cen, MADS1, MADS1b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5 were obtained (Figures 48, 49, 53, 54, 58, 59, 62, 63, 67, 68, 71, 72, 76, 80, 81, 85, 86, 89 and EXAMPLE 4 Development of transformation vectors containing chimeric genes with Cen, MADS1, MVADS1 b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MVADS4-2 and MADS5 cDNA sequences from perennial ryegrass To alter the expression of flowering and plant development proteins Cen, MADS1, MADS1 b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5, through antisense and/or sense suppression technology and for WO 02/33091 PCT/AU01/01311 32 over-expression of these key enzymes in transgenic plants, a set of sense and antisense transformation vectors was produced.

cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) contained restriction sites for EcoRl and Xbal for directional and non-directional cloning into the target vector.

TABLE 3 List of primers used to PCIR-amplify the open reading frames Gene name Clone ID Primer Primer sequence LpCen O6rgl1XsBO6. 1 LpCenf GAATTCTAGAGATAGAGCATTCACCGTGC Lpcenr GAATTCTAGATTGGCACAACTGGAATAGC LpMADS1 l4rgi UsDlO.5 LpMADS1f GAATTCTAGAGAGGAAGAAGAAGGAGCG LpMADSl1r GAATTCTAGACATTTGATGGAATAGGAGTGG LpMADS1 b 1 4rgl1OsGO5.2 LpMADS1 bf GAATTICTAGAGACGAAGAAGAAGGAGCG LpMADSl1br GAATTCTAGATTGATGGAATAGGAGTGGAG LpMADS2-1 1 9rgl1AsGi 2.1 LpMADS2-1 f CCCGGGATCCAGCACAGGGAGAAGAAAGG LpMADS2-1 r CCCGGGATCCCCTCTCGTCGTCTGAACC LpADS2 2 -14r-gl IsHi 1.2 LpMADS2-2f GAATTCTAGAAGGAGAGGAGAGAGAGCCG LpMADS2-2r GAATTCTAGACCTCTCGTCGTCTGAACC LpMADS2-3 1 3rg2GsFO8.1 LpMADS2-3f GAATTCTAGAGAGGAAGAAGAAGGAGCG LpMADS2-3r GAATTCTAGACATTTGATGGAATAGGAGTGG LpMADS3 1 3rg2CsBO7.1 LpMADS3f GAATTCTAGAAGTGGTGCTTTCCTTGGTCG LpMADS3r GAATTCTAGAAGAACACGCATTTTATTAGC LpMADS4 1 9rg2MsFO5.2 LpMADS4-1 f CCCGGGATCCAGAGAGGAGAGGGGAAGG LpMADS4-1 r CCCGGGATCCTCCAGGTTCTCCATTCGG LpMADS4-2 11 rg2YsGO7.3 LpMADS4-2f GAATTCTAGAAGAGAGGAGAGGGOAAGGG LpMADS4-2r GAATTCTAGACTATGTCTTTGTTGTTCAGC 1 3rgl1SsHO7.2 LpMADS5f GAATTCTAGAGCTCCCACAGAAACAAGC

GAATTCTLAGABCAAAGCCTTACTTATGGG

After PCR amplification and restriction digest with the appropriate restriction enzyme (usually Xbal), the clDNA fragments were cloned into the corresponding site in pDH51, a pUCi 8-based transformation vector containing a CaMV WO 02/33091 PCT/AU01/01311 33 expression cassette. The orientation of the constructs (sense or antisense) was checked by DNA sequencing through the multi-cloning site of the vector.

Transformation vectors containing chimeric genes using full-length open reading frame cDNAs of perennial ryegrass Cen, MADS1, MADS1b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5 in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figures 51,56, 61, 65, 70, 74, 78, 83, 88 and 92).

EXAMPLE Production of transgenic tobacco plants carrying chimeric Cen, MADS1, MADS1b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and genes from perennial ryegrass A set of transgenic tobacco plants carrying chimeric Cen, MADS1, MADS1b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and cDNA genes from perennial ryegrass were produced.

pDH51-based transformation vectors with LpCen, LpMADS1, LpMADS1b, LpMADS2-1, LpMADS2-2, LpMADS2-3, LpMADS3, LpMADS4, LpMADS4-2 and cDNAs comprising the full open reading frame sequences in sense and antisense orientations under the control of the CaMV 35S promoter were generated.

Direct gene transfer experiments to tobacco protoplasts were performed using these transformation vectors.

The production of transgenic tobacco plants carrying the perennial ryegrass Cen, MADS1, MADS1b, MADS2-1, MADS2-2, MADS2-3, MADS3, MADS4, MADS4-2 and MADS5 cDNAs under the control of the constitutive CaMV promoter is described here in detail.

WO 02/33091 PCT/AU01/01311 34 Isolation of mesophyll protoplasts from tobacco shoot cultures 2-4 fully expanded leaves of a 6 week-old shoot culture were placed under sterile conditions (work in laminar flow hood, use sterilized forceps, scalpel and blades) in a 9 cm plastic culture dish containing 12 ml enzyme solution (w/v) cellulase "Onozuka" R10 and 1.0% Macerozyme® R10]. The leaves were wetted thoroughly with enzyme solution and the mid-ribs removed. The leaf halves were cut into small pieces and incubated overnight (14-18 h) at 25 0 C in the dark without shaking The protoplasts were released by gently pipetting up and down, and the suspension poured through a 100 pm stainless steel mesh sieve on a 100 ml glass beaker. The protoplast suspension was mixed gently, distributed into two 14 ml sterile plastic centrifuge tubes and carefully overlayed with 1 ml W5 solution.

After centrifugation for 5 min. at 70g (Clements Orbital 500 bench centrifuge, swing-out rotor, 400 rpm), the protoplasts were collected from the interphase and transferred to one new 14 ml centrifuge tube. 10 ml W5 solution were added, the protoplasts resuspended by gentle tilting the capped tube and pelleted as before.

The protoplasts were resuspended in 5-10 ml W5 solution and the yield determined by counting a 1:10 dilution in a haemocytometer.

Direct gene transfer to protoplasts using polyethylene glycol The protoplasts were pelleted [70g (Clements Orbital 500 bench centrifuge, 400 rpm) for 5 min.] and resuspended in transformation buffer to a density of 1.6 x 106 protoplasts/ml. Care should be taken to carry over as little as possible solution into the transformation mix. 300 pl samples of the protoplast suspension (ca. 5 x 10 s protoplasts) were aliquotted in 14 ml sterile plastic centrifuge tubes, pl of transforming DNA were added. After carefully mixing, 300 pl of PEG solution were added and mixed again by careful shaking. The transformation mix was incubated for 15 min. at room temperature with occasional shaking. 10 ml solution were gradually added, the protoplasts pelleted [70g (Clements Orbital 500 bench centrifuge, 400 rpm) for 5 min.] and the supernatant removed. The protoplasts were resuspended in 0.5 ml K3 medium and ready for cultivation WO 02/33091 PCT/AU01/01311 Culture of protoplasts, selection of transformed lines and regeneration of transgenic tobacco plants Approximately 5 x 10 5 protoplasts were placed in a 6 cm petri dish. 4.5 ml of a pre-warmed (melted and kept in a water bath at 40-45°C) 1:1 mix of K3:H medium containing 0.6% SeaPlaqueTM agarose were added and, after gentle mixing, allowed to set.

After 20-30 min the dishes were sealed with Parafilm® and the protoplasts were cultured for 24 h in darkness at 24°C, followed by 6-8 days in continuous dim light (5 pmol m" 2 s" 1 Osram L36 W/21 Lumilux white tubes), where first and multiple cell divisions occur. The agarose containing the dividing protoplasts was cut into quadrants and placed in 20 ml of A medium in a 250 ml plastic culture vessel. The corresponding selection agent was added to the final concentration of mg/I kanamycin sulphate (for npt2 expression) or 25 mg/I hygromycin B (for hph expression) or 20 mg/I phosphinotricin (for bar expression). Samples were incubated on a rotary shaker with 80 rpm and 1.25 cm throw at 24°C in continuous dim light.

Resistant colonies were first seen 3-4 weeks after protoplast plating, and after a total time of 6-8 weeks protoplast-derived resistant colonies (when 2-3 mm in diameter) were transferred onto MS morpho medium solidified with 0.6% (w/v) agarose in 12-well plates and kept for the following 1-2 weeks at 24°C in continuous dim light (5 pmol m" 2 s 1 Osram L36 W/21 Lumilux white tubes), where calli proliferated, reached a size of 8-10 mm, differentiated shoots that were rooted on MS hormone free medium leading to the recovery of transgenic tobacco plants (Table 4 and Figure 94).

WO 02/33091 WO 02/3091PC T/AU 110 1311 TABLE 4 Production of transgenic tobacco calli carrying chimeric ryegrass genes (in sense and antisense orientaion) involved in the regulation of flowering and plant development Construct Transfected Transformed Transformation protoplasts call! efficiency pDH-511-p~en 1.5 X 106S 78 5.20 x 10-5 pDH51 LpCen anti 1.5 x 106 56 3.73 x pDH51 LpMADS1 1.5 x 106 97 6.46 x 10pDH51LpMADS1 anti 1.5 X10 6 694.0x1pDH51 LpMADS1 b 1.5 x10, 67 4.470x10pDH51 LpMADS1 b anti 1.5 X10 6 48 f3.20Ox10 pDH51 LpMADS2-1 1.5 X 106 56 f3.73 x 1 pDH51 LpMAUS2-1 anti 1.5 x 10' 82 5.46 x pDH51 LpMADS2-2 1.5 x 106 52 3.46 x 10-5 pDH51 LpMADS2-2 anti 1.5 x 106 32 2.13 x 10-5 pDH51 LpMADS2-3 1.5 X 106 55 3.66x pDH51 LpMAOS2-3 anti 1.5 x 106 74 4.93 x 10- I pDH51 LpMADS3 1.5 x 106 58 3.87 pDH51 LpMADS3 anti 1.5 x 106 67 4.47 x10pDH51 LpMADS4 1.5 x 106 84 5.60 x pDH51 LpMADS4 anti 1.5 X 106 53 3.5 x pDH51 LpMADS4-2 1.5 x 106 54 3.60 x 1 0pH1LMD42anti 1.5 x 106 65 4.33 x pDH51 LpMADS5 pDH51 LpMADS5 anti 1.5 x 100 1.5 x 106 89 78 5.93 x 5.20 x WO 02/33091 PCT/AU01/01311 37 EXAMPLE 6 Genetic mapping of perennial ryegrass genes involved in flowering and plant development The cDNAs representing genes involved in flowering and plant development were amplified by PCR from their respective plasmids, gel-purified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs). RFLPs were mapped in the F, (first generation) population, NA 6 x AU 6 This population was made by crossing an individual (NA 6 from a North African ecotype with an individual (AUs) from the cultivar Aurora, which is derived from a Swiss ecotype. Genomic DNA of the 2 parents and 114 progeny was extracted using the 1 x CTAB method of Fulton et al. (1995).

Probes were screened for their ability to detect polymorphism using the DNA (10 gg) of both parents and 5 F 1 progeny restricted with the enzymes Dral, EcoRI, EcoRV or Hindlll. Hybridisations were carried out using the method of Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (Figures 52, 57, 66, 79, 84, and 93).

RFLP bands segregating within the population were scored and the data was entered into an Excel spreadsheet. Alleles showing the expected 1:1 ratio were mapped using MAPMAKER 3.0 (Lander et al. 1987). Alleles segregating from, and unique to, each parent, were mapped separately to give two different linkage maps. Markers were grouped into linkage groups at a LOD of 5.0 and ordered within each linkage group using a LOD threshold of Loci representing genes involved in flowering and plant development mapped to the linkage groups as indicated in Table 5 and in Figure 96. These gene locations can now be used as candidate genes for quantitative trait loci for flowering and plant development.

WO 02/33091 PCT/AU01/01311 38 TABLE Map locations of ryegrass genes encoding proteins involved in flowering and plant development across two genetic linkage maps of perennial ryegrass (NA6 and AU6) Probe LpAP2b Polymorphic

Y

LpCENa LpHBa LpHBb LpHBd LpMADS1/L pMADSla LpMADS3 LpMADS4-1 Mapped with Hind III Dra I EcoR V EcoR I Hind III Locus LpAP2b.1 LpAP2b.2 LpCENa LpHBa.1 LpHBa.2 LpHBb LpHBd.1 LpHBd.2 LpMADS1 LpMADS3 LpMADS4-1 Linkage group NA6 AU6 1 1 1 Y EcoR V EcoR V EcoR I EXAMPLE 7 Expression profiling of cDNAs encoding proteins involved in flowering and plant development using microarray technology cDNAs encoding proteins involved in flowering and plant development were PCR amplified and purified. The amplified products were spotted three times on each amino-silane coated glass slide (CMT-GAPS, Corning, USA) using a microarrayer MicroGrid (BioRobotics, UK). Spotting solution was also spotted in every subgrid of the microarray as negative and background controls. The duplicates were placed about 800 micron apart to prevent competitive hybridisation.

WO 02/33091 PCT/AU01/01311 39 Table 6 gives details on the tissues used to extract total RNA.

TABLE 6 List of hybridization probes used in expression profiling of perennial ryegrass genes encoding proteins involved in flowering and plant development using microarrays Hybridization probe for microarrays Organ specificity (3-month old Leaf blade plants grown hydroponically) Sheath Root Seedling grown under light 5-day old shoot condition 7-day old shoot (7LS) old shoot old root 7-day old root (7LR) old root Seedling grown under dark 5-day old shoot condition 7-day old shoot (7DS) old shoot old root 7-day old root (7DR) old root Fluorescence labelled probes were synthesis by reversed transcribing RNA and incorporating Cyanine 3 or 5 labelled dCTP. The probes were hybridised onto microarrays. In each case the experiment was repeated on two microarrays. After hybridisation for 16 hours (overnight), the microarrays were washed and scanned WO 02/33091 PCT/AU01/01311 using a confocal laser scanner (ScanArray 3000, Packard, USA). The images obtained were quantified and analysed using Imagene 4.1 and GeneSight 2.1 (BioDiscovery, USA). Data were judged as not present low expression medium expression high expression and highly expression (Table 7).

4' TABLE 7 Results of expression profiling of ryegrass genes encoding proteins involved in flowering and plant development plate row column gene id cloneid gene seq notes sheath leaf blade root 5LS 7LS 1OLS 5LR 7LR 101-1 5DS 7DS lODS 5DR 7DR Array2Vrg a 12 XnsCC2.YIGUN-7669 01 rgl Ks[BOS LpMADSa partial Array2Vrg b 1 TsfAPl-ARATt--7660 07rgl AaCll1 LpMADSa partial- Array2Vrg b 2 TsfAG1 5-BRANA7661 O7rg2GsFl 1 LpMADSa partial Array2Vrg b 3 TstAGi 5.ARATH7662 07rg I lsBO2 LpMADSa partial-- Array2Vrg b 4 TsfAG1 5-ARATH7663 07rg1 IsF01 LpMADSa partial Array2Vrg b 5 TsfAPi ARATH-7664 O7rgl UsFO6 LpMADSa partial+ Array2Vrg b 6 TatCMBl-DIACA7665 l3rglIsAO9 LpMADSa partial- Array2Vrg b 7 TsfAP1.ARATH-7666 l4rgl IsClO LpMADSa partial Array2Vrg b 8 TsfAPl-ARATH-7667 14rglVsH0l LpMAOSS partial Array2Vrg b 9 TsfAGB6ARATH-7668 l3rg2CsBD7 LpMADSb full Array2Vrg b 10 TsfAP1-ARATH-7669 l3rgl KsG07 LpMADSc partial Array2Vrg b 11 TsfAPI ARATH-7670 O7rg2BasGl2 LpMADSc partial 44 Array2Vrg b 12 TstAP1-ARATH-7671 07rg2DsAO6 LpMADSc partial -i-i Array2Vrg c 1 TstAGL2 ARATH7672 l3rgl BsFO9 LpMADSd partial Array'2Vrg c 2 TstAGL2-ARATH7673 14rgl IsHli LpMADSe full Array? Vrg c 3 TstFBP2-PETHY7674 1 3rgl FsDO6 LpMAOSI partial- Array2Vrg c 4 TstCMB1-01ACA7675 l3rg2GsFD8 LpMADSg full- Array2Vrg c 5 ZhyNHP)Q.0AEEL7676 1 7rgl QaCi 1 LpMADSh partial Array2Vrg c 6 TstCMBl-DIACA7677 19rg2MsFO5 LpMADSi full- Array2Vrg c 7 TstFBP2-PETHY7678 11 rg2YsGO7 LpMADSj full- Array2Vrg c 8 TsfAG-TOBAG-7679 13rglSsHJ7 LpMADSk full- Array2Vrg c 9 TstMAD2-PETHY7680 14rg1I2FsG1 0 LpMADSI full Array2Urg f 3 MSPS-BETVU 7618 l4rgI PsEl 2 LpMADSI lull Array2Vrg C 10 CcgCEN-ANTMA-7681 06rglOsHO7 LpCENa full 4 4- 4444 -4 -4 Array2Vrg c 11 XnsOTX1-BRARE7682 06rglWsCO9 LpAP2b partial Array2Vrg c 12 TstAFO48900--7683 1 Orgi RsCD4 LpAP2b partial 4 4 Array2Vrg d 1 1 IXnsOTX1 BRARE7684 1OrglRsEO2 LpAP2b partial +i +f-i 4-f +4- Array2Vrg- d 1 2 ITsfAHNK-HUMAN7685 17rgt PsHDl LpAP2b Ipartial I TABLE 7 (cont.) Array2Vrg d 3 TsfHT14,ARATH7686 lOrg2OsDO6 LpH~a Full Array2Vrg d 4 TsfATH1 ARATH7687 l6rgl IsF03 LpHlb Full Array2Vrg d 5 TsfHX1 AjfAlZC76BS l9rg2EsAO5 LpHlc Full- Array2Vrg d 6 TsfrlKL3-MALD07689 lOrg2HsHl2 LpHBd Full- Array2Vrg d 7 1TsfHKL3.MALDOTS90 lOrg2JsFO2 LpHBrf Full 4 Array2Vrg d 8 TsfHKL3-MALDO7691 lOrglWsDlO LpHBd full- Array2Vrg d 9 TsfHKL-3.ARATH7692 l7rglSsEO4 LpHld full Array2Vrg d 10 TsfAGL6-ARATH7693 l3rgIWsAlO LpMADSb full Array2Vrg d 11 TsfAG-TOBAC--7694 l3rglSsHO7 LpMADSk Full WO 02/33091 PCT/AU01/01311 43

REFERENCES

Feinberg, Vogelstein, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem.

132: 6-13.

Frohman et al. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl.

Acad Sci. USA 85:8998 Gish and States (1993) Identification of protein coding regions by database similarity serach. Nature Genetics 3:266-272 Lander, Green Abrahamson, Barlow, Daly, Lincoln, S.E., Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations.

Genomics 1: 174-181.

Loh, Elliott, Cwirla, Lanier, Davis, M.M. (1989). Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta chain. Science 243:217-220 Ohara, Dorit, Gilbert, W. (1989). One-sided polymerase chain reaction: The amplification of cDNA. Proc. Natl. Acad Sci USA 86:5673-5677 Sambrook, Fritsch, Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Laboratory Press Sharp, Kreis, Shewry, Gale, M.D. (1988). Location of a-amylase sequences in wheat and its relatives. Theor. Appl. Genet. 75: 286-290.

WO 02/33091 PCT/AU01/01311 44 Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

It will also be understood that the term "comprises" (or its grammatical variants) as used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

Documents cited in this specification are for reference purposes only and their inclusion is not an acknowledgement that they form part of the common general knowledge in the relevant art.

Claims (16)

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a Homeo-box protein (HB) from a Lolium species, or a functionally active fragment or variant thereof. ci S 5 2. A nucleic acid or nucleic acid fragment according to Claim 1, wherein said ci Lolium species is Lolium pernne or Lolium arundianaceum. 0 0 3. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding an HB or HB-like protein, or complementary or antisense to a nucleic acid or nucleic acid fragment encoding a HB or HB-like protein, and including a nucleotide sequence selected from the group consisting of sequences shown in Figures 39, 41, 43, 45, and 47 hereto (Sequence ID Nos: 75, 77, 79, 81 and 83 to 87, respectively); (b) complements of the sequences recited in sequences antisense to the sequences recited in and and functionally active fragments and variants of the sequences recited in and

4. A nucleic acid or nucleic acid fragment according to claim 3, wherein said functionally active fragments and variants have at least approximately 90% identity to the relevant part of the sequences recited in and and have a size of at least nucleotides. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding an HB or HB-like protein, or complementary or antisense to a nucleic acid or nucleic acid fragment encoding a HB or HB-like protein, and including a nucleotide sequence selected from the group consisting of sequence shown in Figure 41 hereto (Sequence ID No: 77); complement of the sequence recited in sequences antisense to the sequences recited in and

6. A construct including a nucleic acid or nucleic acid fragment according to any one of Claims COMS ID No: ARCS-153474 Received by IP Australia: Time 10:58 Date 2007-07-19 19/07/2007 10:57 freehills 12345878 8/15 0050037086 46

7. A vector including a nucleic acid or nucleic acid fragment according to any one of Claims

8. A vector according to Claim 7, further including a promoter and a m terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked. C- 9. A plant cell, plant, plant seed or other plant part, including a construct oaccording to claim 6 or a vector according to Claim 7 or 8. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 9 and including a construct according to claim 6 or a vector according to Claim 7 or 8.

11. A method of modifying plant life cycles and/or growth phases, flowering processes, flowering and/or plant architecture and/or flower and/or inflorescence development in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of Claims 1-5, a construct according to claim 6 and/or a vector according to Claim 7 or 8.

12. Use of a nucleic acid or nucleic acid fragment according to any one of Claims 1-5, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

13. The use according to Claim 12 wherein the molecular genetic marker is for one or more of quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and marker assisted selections.

14. The use according to Claim 12 or 13 wherein the molecular genetic marker is used in a Lolium species. The use according to any one of Claims 12 to 14 wherein the molecular genetic marker is used in forage and turf grass improvement of herbage quality traits; COMS ID No: ARCS-153474 Received by IP Australia: Time 10:58 Date 2007-07-19 19/07/2007 10:57 freehllls 12345878 9/15 005003706 C 47 0 flowering intensity; flowering time; number of tillers; leafiness; bushiness; season growth pattern; herbage yield; flower architecture or plant stature.

16. A substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid or nucleic acid fragment according to any one of Claims 1 to 0 C 17. A substantially purified or isolated polypeptide from a Lolium species, o selected from the group consisting of HB and HB-like proteins; and functionally active Ci fragments and variants thereof.

18. A polypeptide according to Claim 17, wherein said Lolium species is Lolium perenne or Lolium arundinaceum.

19. A substantially purified or isolated HB or HB-like polypeptide including an amino acid sequence selected from the group consisting of sequences shown in Figures 40, 42, 44 and 46 hereto (Sequence ID Nos: 76, 78, 80 and 82, respectively); and functionally active fragments and variants thereof.

20. A polypeptide according to claim 18, wherein said functionally active fragments and variants have at least approximately 95% identity with the recited sequences and have a size of at least 20 amino acids.

21. A substantially purified or isolated HB or HB-like polypeptide including an amino acid sequence shown in Figure 42 hereto (Sequence ID No: 78).

22. A nucleic acid or nucleic acid fragment encoding a polypeptide according to any one of claims 19 to 21.

23. A nucleic acid or nucleic acid fragment according to any one of Claims 1 substantially as hereinbefore described with reference to any one of the figures or examples. COMS ID No: ARCS-153474 Received by IP Australia: Time 10:58 Date 2007-07-19 19/07/2007 10:57 freehills 12345678 10/15 005003706 O 48 S24. A polypeptide according to any one of Claims 19 to 21 substantially as hereinbefore described with reference to any one of the figures or examples. Dated: 19 July 2007 SFreehills Patent Trade Mark Attorneys S 5 Patent Attorneys for the Applicant: Agriculture Victoria Services Pty Ltd SAgResearch Limited COMS ID No: ARCS-153474 Received by IP Australia: Time 10:58 Date 2007-07-19